U.S. patent application number 17/436673 was filed with the patent office on 2022-05-12 for compositions and methods comprising engineered chimeric antigen receptor and modulator of car.
The applicant listed for this patent is AUTOLUS LIMITED. Invention is credited to Christian Itin, Vijay Peddareddigari, Martin Pule.
Application Number | 20220145325 17/436673 |
Document ID | / |
Family ID | 1000006166782 |
Filed Date | 2022-05-12 |
United States Patent
Application |
20220145325 |
Kind Code |
A1 |
Pule; Martin ; et
al. |
May 12, 2022 |
COMPOSITIONS AND METHODS COMPRISING ENGINEERED CHIMERIC ANTIGEN
RECEPTOR AND MODULATOR OF CAR
Abstract
There is provided method for making a cell composition which
comprises step of transducing a population of cells with a mixture
of at least two viral vectors, wherein at least one vector
comprises a nucleic acid sequence which encodes a chimeric antigen
receptor (CAR); and wherein at least one vector comprises a nucleic
acid encoding an activity modulator which modulates the activity of
the CAR, of a cell expressing the CAR, or of a target cell. There
is also provided a cell composition made by such a method and its
use in the treatment of diseases such as cancer.
Inventors: |
Pule; Martin; (London,
GB) ; Peddareddigari; Vijay; (London, GB) ;
Itin; Christian; (London, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AUTOLUS LIMITED |
London |
|
GB |
|
|
Family ID: |
1000006166782 |
Appl. No.: |
17/436673 |
Filed: |
March 6, 2020 |
PCT Filed: |
March 6, 2020 |
PCT NO: |
PCT/GB2020/050535 |
371 Date: |
September 7, 2021 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/71 20130101;
C07K 14/7051 20130101; C12N 15/86 20130101; A61K 48/005 20130101;
C07K 14/715 20130101; C12N 2800/40 20130101; C07K 2319/03 20130101;
C12N 2740/16043 20130101 |
International
Class: |
C12N 15/86 20060101
C12N015/86; C07K 14/725 20060101 C07K014/725; C07K 14/71 20060101
C07K014/71; C07K 14/715 20060101 C07K014/715; A61K 48/00 20060101
A61K048/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 8, 2019 |
GB |
1903237.4 |
Oct 2, 2019 |
GB |
1914216.5 |
Nov 5, 2019 |
GB |
1916077.9 |
Claims
1. A method for making a cell composition which comprises step of
transducing a population of cells with a mixture of at least two
viral vectors, wherein at least one vector comprises a nucleic acid
sequence which encodes a chimeric antigen receptor (CAR); and
wherein at least one vector comprises a nucleic acid encoding an
activity modulator which modulates the activity of the CAR, of a
cell expressing the CAR, or of a target cell.
2. A method according to claim 1, wherein the activity modulator is
a dominant negative SHP-1 or SHP-2.
3. A method according to claim 1, wherein the activity modulator is
a dominant negative transforming growth factor (TGF).beta.
receptor.
4. A method according to claim 1, wherein the activity modulator is
a constitutively active chimeric cytokine receptor.
5. A method according to claim 1, wherein in the mixture of viral
vectors at least one vector comprises a nucleic acid sequence which
encodes a dominant negative SHP-1 or SHP-2; and at least one vector
comprises a nucleic acid sequence which encodes a dominant negative
transforming growth factor (TGF).beta. receptor.
6. A method according to claim 1, wherein the mixture of viral
vectors comprises two, three, four, five or six viral vectors, at
least one of which comprises a nucleic acid sequence encoding a
CAR; and at least one of which comprises a nucleic acid sequence
encoding an activity modulator.
7. A method for making a cell composition according to claim 1
which comprises the following steps: (i) transducing a population
of cells with a mixture of at least two viral vectors; and (ii)
selecting CAR-expressing cells from the transduced cell population
from step (i).
8. A method according to claim 1, wherein each of the viral vectors
in the mixture comprises a nucleic acid sequence encoding a
CAR.
9. A viral vector composition which comprises a mixture of at least
two viral vectors, wherein at least one vector comprises a nucleic
acid sequence which encodes a chimeric antigen receptor (CAR); and
wherein at least one vector comprises a nucleic acid encoding an
activity modulator which modulates the activity of the CAR, of a
cell expressing the CAR, or of a target cell.
10. A viral vector composition according to claim 9, which
comprises a first vector and a second vector, both of which
comprise a nucleic acid sequence encoding a chimeric antigen
receptor (CAR).
11. A viral vector composition according to claim 10, wherein the
nucleic acid sequence of the first vector and the nucleic acid
sequence of the second vector encode the same CAR.
12. A viral vector composition according to claim 10 or 11, wherein
both the first vector and the second vector also comprise a nucleic
acid encoding an activity modulator which modulates the activity of
the CAR, of a cell expressing the CAR, or of a target cell.
13. A viral vector composition according to claim 12, wherein the
activity modulators are selected from: a dominant negative SHP-1 or
SHP-2; a dominant negative transforming growth factor (TGF).beta.
receptor; and a constitutively active chimeric cytokine
receptor.
14. A viral vector composition according to claim 13, wherein the
first vector comprises a nucleic acid sequence encoding a dominant
negative SHP-1 or SHP-2 and a nucleic acid sequence encoding a
dominant negative transforming growth factor (TGF).beta. receptor;
and the second vector comprises a nucleic acid sequence encoding a
constitutively active chimeric cytokine receptor.
15. A viral vector composition according to claim 11 wherein the
first and second vectors encode the same CAR and the CAR has an
antigen-binding domain which binds disialoganglioside (GD2).
16. A viral vector composition according to claim 10, wherein the
first vector and/or the second vector comprises a nucleic acid
sequence encoding a suicide gene.
17. A cell composition made by a method according to claim 1.
18. A method for treating a disease in a subject which comprises
the step of administering a cell composition according to claim 17
to the subject.
19-20. (canceled)
21. A method for determining the optimal combination of components
for a CAR-expressing cell to treat a disease, which comprises the
following steps: (i) administering a cell composition according to
claim 17 to a subject having the disease; (ii) monitoring the
patient or samples from the patient to determine which
sub-population of cells in the cell composition show the greatest
level of engraftment and/or proliferation; and (iii) analysing the
phenotype/genotype of the cells in the sub-population to ascertain
the CAR(s) and/or activity modulator(s) expressed by those cells.
Description
FIELD OF THE INVENTION
[0001] The present invention relates viral vector compositions,
their use to transduce cells and cell compositions made by such
methods.
BACKGROUND TO THE INVENTION
[0002] Tumour heterogeneity describes the observation that
different tumour cells can show distinct morphological and
phenotypic profiles, including cellular morphology, gene
expression, metabolism, motility, proliferation, and metastatic
potential.
[0003] Heterogeneity occurs between patients, between tumours
(inter-tumour heterogeneity) and within tumours (intra-tumour
heterogeneity). Multiple types of heterogeneity have been observed
between tumour cells, stemming from both genetic and non-genetic
variability.
[0004] Heterogeneity between tumour cells can be further increased
due to heterogeneity in the tumour microenvironment. Regional
differences in the tumour (e.g. availability of oxygen) impose
different selective pressures on tumour cells, leading to a wider
spectrum of dominant subclones in different spatial regions of the
tumour. The influence of microenvironment on clonal dominance is
also a likely reason for the heterogeneity between primary and
metastatic tumours seen in many patients, as well as the
inter-tumour heterogeneity observed between patients with the same
tumour type.
[0005] The heterogeneity of cancer cells introduces significant
challenges in designing effective treatment strategies.
[0006] For example, heterogeneic tumours may exhibit different
sensitivities to cytotoxic drugs among different clonal
populations. This is attributed to clonal interactions that may
inhibit or alter therapeutic efficacy.
[0007] Drug administration in heterogeneic tumours will seldom kill
all tumour cells. The initial heterogeneic tumour population may
bottleneck, such that few drug resistant cells (if any) will
survive. This allows resistant tumour populations to replicate and
grow a new tumour through the branching evolution mechanism (see
above). The resulting repopulated tumour is heterogeneic and
resistant to the initial drug therapy used. The repopulated tumour
may also return in a more aggressive manner.
[0008] Chimeric Antigen Receptors (CARs)
[0009] Chimeric antigen receptors are proteins which graft the
specificity of, for example, a monoclonal antibody (mAb) to the
effector function of a T-cell. Their usual form is that of a type I
transmembrane domain protein with an antigen recognizing amino
terminus, a spacer, a transmembrane domain all connected to a
compound endodomain which transmits T-cell survival and activation
signals (see FIG. 1A).
[0010] The most common form of these molecules are fusions of
single-chain variable fragments (scFv) derived from monoclonal
antibodies which recognize a target antigen, fused via a spacer and
a trans-membrane domain to a signalling endodomain. Such molecules
result in activation of the T-cell in response to recognition by
the scFv of its target. When T cells express such a CAR, they
recognize and kill target cells that express the target antigen.
Several CARs have been developed against tumour associated
antigens, and adoptive transfer approaches using such
CAR-expressing T cells are currently in clinical trial for the
treatment of various cancers.
[0011] Successful CAR treatment depends on expression of the target
antigen by tumour cells. In heterogenic tumours, in particular
solid cancers, antigen expression is heterogeneous, and it may not
be possible to find a single target antigen expressed by all cancer
cells.
[0012] Moreover, emerging data from CAR T-cell trials in B-cell
malignancies demonstrate that a common mechanism of resistance to
this class of therapeutics is the emergence of tumours with loss or
downregulation of the target antigen. Antigen loss or antigen-low
escape is likely to emerge as an even greater barrier to success in
solid tumours, which manifest greater heterogeneity in target
antigen expression. Potential approaches to overcome this challenge
include engineering CAR T cells to achieve multi-specificity and to
respond to lower levels of target antigen and more efficient
induction of natural antitumor immune responses as a result of
CAR-induced inflammation.
[0013] Clinical studies of CAR T-cells have established that CAR
T-cell engraftment, expansion and persistence are a pre-requisite
for clinical activity, particularly sustained responses. A key
reason for poor persistence of CAR-T cells in vivo, particularly
CAR-T cells for the treatment of solid cancers, is that the cells
struggle to overcome the hostile microenvironment of the tumour. In
particular, CAR T-cells may fail to engraft and expand within a
solid cancer tumour bed.
[0014] CAR T-cell persistence and activity can be enhanced by
administration of cytokines, or by engineering the CAR T-cell to
secrete or express cytokine, toxins or other factors. However,
these approaches have limitations: systemic administration of
cytokines can be toxic; constitutive production of cytokines may
lead to uncontrolled proliferation and transformation.
[0015] There is thus a need for alternative CAR treatment
approaches which address the problems commonly encountered with
CAR-T cell therapy, particularly bearing in mind the heterogeneity
between patients, and between tumour cells and tumour cell sites
within the same patient.
DESCRIPTION OF THE FIGURES
[0016] FIG. 1--Schematic diagram showing a classical chimeric
antigen receptors (a) Basic schema of a chimeric antigen receptor;
(b) First generation receptors; (c) Second generation receptors;
(d) Third generation receptors.
[0017] FIG. 2--Schematic diagram illustrating the mechanism of a)
T-cell activation and b) T-cell inhibition in vivo
[0018] FIG. 3--Schematic diagram illustrating the JAK-STAT
signaling pathway (activated by .alpha.-interferon).
[0019] FIG. 4--Different binding domain formats of chimeric antigen
receptors (a) Fab CAR format; (b) dAb CAR format; (c) scFv CAR
format
[0020] FIG. 5--Schematic diagram illustrating the difference in
transduced cell compositions obtained by transduction with a single
vector co-expressing two genes (A); and a mixture of two vectors,
each expressing a single gene (B). When cells are transduced with a
single vector having a bicistronic cassette, every cell which is
successfully transduced will express both transgenes at a
stoichiometry of approximately 1:1 (A). However, when cells are
transduced with multiple vectors, a much more heterogeneous
population is obtained: transduced cells may express the first
transgene alone, the second transgene alone or both transgenes. In
cells expressing both transgenes the relative level of expression
of gene A and gene B is completely variable (B).
[0021] FIG. 6--Scatter plot showing that when T cells are
transduced with a mixture of vectors (A and B), a proportion of
cells are left untransduced; a proportion of cells are transduced
with vector A alone; a proportion of cells are transduced with
vector B alone; and a proportion of cells are transduced with
vectors A and B. In this study, cells are transduced with a mixture
of vectors, one of which expresses an anti-CD19 CAR and one of
which expresses a CD22 CAR.
[0022] FIG. 7--Schematic diagram illustrating the molecules
expressed by the vectors used in the dual vector composition
described in Example 3. Vector 1 expresses a CAR with an
antigen-binding domain which binds GD2 (GD2 CAR), a constitutively
active cytokine receptor (CCR) and a sort/suicide gene (RQR8).
Vector 2 expresses the same GD2 CAR, a dominant negative SHP-2
(.DELTA.SHP2); a dominant negative transforming growth factor
(TGF).beta.II receptor (.DELTA.TGFbRII) and the same sort/suicide
gene (RQR8).
[0023] FIG. 8--Investigating the capacity of single and dual
transduced T cell populations to kill GD2-expressing (SupT1 GD2)
and non-expressing (SupT1 NT) target cells.
[0024] FIG. 9--Investigating the proliferation of single and dual
transduced T cell populations following culture in cytokine-free
complete cell culture media for 7 days without further antigen
stimulus.
[0025] FIG. 10--Investigating the capacity of single and dual
transduced T cell populations to kill GD2-expressing (SupT1 GD2)
and non-expressing (SupT1 NT) target cells in the presence or
absence of TGF.beta..
[0026] FIG. 11--Investigating cytokine production (IFN.gamma.) from
single and dual transduced T cell populations following co-culture
with GD2-expressing (SupT1 GD2) and non-expressing (SupT1 NT)
target cells in the presence or absence of TGF.beta..
[0027] FIG. 12--Results of an in vivo assay investigating the
anti-tumour activity of T cells transduced with the dual vector
composition by intravenous administration in an established
neuroblastoma xenograft model in NSG mice. 1.times.10.sup.6
CHLA-255 Fluc cells were injected i.v. into female NSG mice.
Xenografts were left to establish for 15 days until stable
engraftment was detectable by BLI. CAR-T cells were made either by
transducing cells with a single vector expressing a GD2 CAR (GD2
CAR) or by transducing cells with the dual vector composition
described in Example 3 and Illustrated in FIG. 7 (GD2 CAR+IL7
CCR/GD2 CAR+dSHP2+dTGFbRII). CAR T-cells were administered i.v. at
a dose of 3.times.10.sup.6 CAR T-cells/mouse. Quantitated
bioluminescent signal of CHLA-255 Fluc was plotted over time as
total flux (photons/s) A. Graph showing fluorescent signal over
time for mice receiving CAR-T cells expressing GD2 CAR alone (GD2
CAR); untransduced T cells (NT) or buffer alone (PBS). B Ventral
images of mice obtained on days -1, 2, 7, 10 and 14 following
administration of CAR-T cells expressing GD2 CAR alone (GD2 CAR);
untransduced T cells (NT) or buffer alone (PBS) C Graph showing
fluorescent signal over time for mice receiving cells transduced
with the dual vector composition described in Example 3 and
Illustrated in FIG. 7 (GD2 CAR+IL7 CCR/GD2 CAR+dSHP2+dTGFbRII);
untransduced T cells (NT) or buffer alone (PBS). D Ventral images
of mice obtained on days -1, 2, 7, 10 and 14 following
administration of cells transduced with the dual vector composition
described in Example 3 and Illustrated in FIG. 7 (GD2 CAR+IL7
CCR/GD2 CAR+dSHP2+dTGFbRII); untransduced T cells (NT) or buffer
alone (PBS).
[0028] FIG. 13--(A) Schematic diagram illustrating the molecules
expressed by the vectors used in the triple vector composition
"AUTO7" described in Example 6.
[0029] Vector A expresses truncated SHP2 (dSHP2); the safety switch
RQR8; an anti-PSMA CAR based on a novel humanized binder 7A12
(7A12-28z); and dominant negative TGF.beta.RII (dnTBRII).
[0030] Vector B expresses a constitutively active IL-7 receptor
(CCR_IL7).
[0031] Vector C expresses the RapaCasp9 suicide gene (RapaCasp9);
CD19 (dCD19) and an IL-12 module (flexiIL-12). "2A" is an
co-expression sequence based on the FMDV 2A peptide.
[0032] (B) An alternative arrangement for the triple vector
composition. Components are as indicated for (A) above. dNGFR is
truncated Nerve Growth Factor Receptor which is used as a marker
protein.
[0033] FIG. 14--Results of a FACS-based killing (FBK) assay
investigating the capacity of single, double and triple transduced
T cells to kill PSMA-expressing target cells. A: FBK after 24 h of
incubation using cytofluorimetry analysis to show survival of
target cells. B: Secretion of IL-2 and IFN.gamma. by CAR T-cells
measured by collecting supernatant at 24 hr from the co-cultures
described and detecting by ELISA.
[0034] AUTO7 was investigated as product of a single transduction
using the vector A. ("AUTO7/A"), or double transduction using
vectors A and B ("AUTO7/AB"), or triple transduction using vectors
A, B and C ("AUTO7/ABC"). AUTO7 was tested against a second
generation CAR developed using the same anti PSMA binder 7A12
("Parental")
[0035] SupT1 target cells were engineered to express human PSMA
antigen at different levels (SupT1-PSMAhigh, SupT1-PSMAlow) were
used as target cells. Non-engineered SupT1 cells (SupT1-NT) were
used as a negative control. CAR T-cells were co-cultured with
target cells at 1:2 effector to target ratios.
[0036] FIG. 15--Results of a FACS-based killing (FBK) assay
investigating the capacity of single, double and triple transduced
T cells to kill PSMA-expressing target cells following culture in
cytokine-free complete cell culture media. After 7 days culture in
"starvation conditions" CAR T-cells were co-cultured with
SupT1-PSMAhigh and SupT1-PSMAlow targets cells (or SupT1-NT cells
as negative control). at 1:2 and 1:8 effector to target ratios. A:
FBK after 24 h of incubation using cytofluorimetry analysis to show
survival of target cells. B: Secretion of IL-2 and IFN.gamma. by
CAR T-cells measured by collecting supernatant at 24 hr from the
co-cultures described and detecting by ELISA.
[0037] FIG. 16--Results of a FACS-based killing (FBK) assay
investigating the capacity of single, double and triple transduced
T cells to kill PSMA-expressing target cells in the presence or
absence of TGF.beta.. CAR T-cells were co-cultured with
SupT1-PSMAhigh and SupT1-PSMAlow targets for 7 days at ratio 1:2
and 1:8 (E:T) either in the presence or absence of 10 ng/ml
TGF.beta.1 (SupT1-NT were used as control). Target cell killing was
quantified by FACS and normalised to targets alone.
[0038] FIG. 17--Results of a FACS-based killing (FBK) assay
investigating the capacity of single, double and triple transduced
T cells to kill PSMA-expressing target cells following repeated
restimulation with target cells. Transduced T-cells were
co-cultured with SupT1-PSMAhigh or SupT1-PSMAlow target cells at
1:1 ratio (E:T). Every 7 days CAR T-cells were re-stimulated with
5.times.10.sup.4 SupT1 cells. Target cell killing was quantified by
FACS before each new re-stimulation.
[0039] FIG. 18--In vivo assay investigating the anti-tumour
activity of T cells transduced with the triple vector composition
by intravenous administration in a prostate cancer xenograft model
in NSG mice. 5.times.10.sup.6 PSMA positive PC3 human cell lines
were injected in the flank of female NSG mice. Xenografts were left
to establish for 3 weeks until stable engraftment was detectable by
palpation and calliper measurement. CAR T-cells were administered
i.v. at a dose of 1.times.10.sup.6 CAR T-cells/mouse. Calliper
measurement was taken 2/3 times a week. A: Data for mice receiving
cells made by single transduction using the vector A. ("AUTO7/A"),
double transduction using vectors A and B ("AUTO7/AB"), triple
transduction using vectors A, B and C ("AUTO7/ABC"); or a second
generation CAR developed using the same anti PSMA binder 7A12
("Parental"); B: Summary of data shown in FIG. 18A.
SUMMARY OF ASPECTS OF THE INVENTION
[0040] The present inventors have developed a combinatorial
approach to address the issue of tumour cell and microenvironment
heterogeneity to CAR therapies.
[0041] When cells are transduced with multiple vectors
simultaneously, the resulting product will be a mixture of cells
which are singly and combinatorially transduced. For example, if
cells are transduced with two vectors, one comprising transgene A
and one comprising transgene B, the transduced cells will be a
mixture of cells expressing A alone; B alone; and cell expressing
both A and B (FIG. 5B). For cells transduced with three vectors
each comprising a transgene, the resulting transduced cells will be
a mixture of: A alone; B alone; C alone; A and B; A and C; B and C;
and cells expressing A, B and C.
[0042] The present invention involves using such a mixture as a
therapeutic CAR-T-cell product. The use of a combinatorial product
gives in-built flexibility which enhances the product's capacity to
adapt to differences in target cells and in tumour
microenvironment.
[0043] For example, the vectors may encode a combination of
different CARs, which may vary in e.g. their antigen binding
domains and/or costimulatory domain(s).
[0044] Alternatively or in addition, one or more of the vectors may
encode an activity modulator which modulates the activity of the
CAR, of a cell expressing the CAR, or of a target cell. When the
combinatorial CAR T-cell composition is administered in vivo, the
cells will migrate to different tumour sites within the body.
Whichever sub-population of CAR-T cells expressing a particular
combination of CAR(s) and activity modulator(s) is best equipped to
survive, persist and kill target cells at that location will have a
selective advantage over the other sub-populations in the product
and will win out. In this way the CAR-T cell product can adapt to
tumour heterogeneity between patients and between sites in the same
patient.
[0045] The method can also be used to establish which combination
of vectors is optimal for generating CAR-T cells for the treatment
a particular disease or disease subtype by analysing a patient to
see which subpopulation of CAR-T cells in the patient shows the
best persistence and/or activity.
[0046] Thus, in a first aspect, the present invention provides a
method for making a cell composition which comprises step of
transducing a population of cells with a mixture of at least two
viral vectors, wherein at least one vector comprises a nucleic acid
sequence which encodes a chimeric antigen receptor (CAR).
[0047] The method of the invention may equally be applied to cells
expressing engineered T-cell receptors. Any and all of the aspects
and embodiments described below are also applicable to engineered
TCR-expressing cells.
[0048] The mixture may comprise two, three, four, five or more
viral vectors.
[0049] Two or more viral vectors in the mixture may each comprise a
CAR-encoding nucleic acid sequence. The first CAR and second CAR
may have different antigen-binding domains and/or different spacers
and/or different endodomains.
[0050] The CAR encoding nucleic acid of one or more viral vector(s)
may encode two or more CARs. For example, the nucleic acid may
encode a CAR logic gate, such as an OR gate.
[0051] The present invention provides method for making a cell
composition which comprises step of transducing a population of
cells with a mixture of at least two viral vectors, wherein at
least one vector comprises a nucleic acid sequence which encodes a
chimeric antigen receptor (CAR); and wherein at least one vector
comprises a nucleic acid encoding an activity modulator which
modulates the activity of the CAR, of a cell expressing the CAR, or
of a target cell.
[0052] The technology of the invention, insofar as it relates to
the expression of activity modulator(s) applies equally to cells
for adoptive cell therapy which do not express as CAR or engineered
TCR, such as tumour infiltrating lymphocytes (TILs). Any and all of
the aspects and embodiments described below insofar as they relate
to the expression of activity modulator(s), are also applicable
generally to therapeutic T cells such as TI Ls.
[0053] One or more viral vectors in the mixture may comprise a
nucleic acid sequence encoding both a CAR and an activity
modulator, so that a cell transduced with this vector co-expresses
the CAR and the activity modulator.
[0054] An activity modulator which modulates the activity of the
CAR may affect the balance between phosphorylation and
dephosphorylation at the CAR-expressing cell:target cell synapse.
For example, the activity modulator may comprise a kinase domain
capable of phosphorylating Immunoreceptor tyrosine-based activation
motifs (ITAMs) in the CAR endodomain.
[0055] Alternatively the activity modulator may be capable of
recruiting a kinase to be brought into proximity with the CAR,
where it can phosphorylate ITAMs in the CAR endodomain.
[0056] An activity modulator which modulates the activity of
CAR-expressing cell may be an intracellular molecule or may be
expressed at the cell surface.
[0057] In vivo, membrane-bound immunoinhibitory receptors such as
CTLA4, PD-1, LAG-3, 2B4 or BTLA 1 inhibit T cell activation. The
activity modulator may block or affect this inhibitory pathway.
[0058] The activity modulator may be an agent, such as an antibody,
which binds to an inhibitory immunoreceptor or binds to a ligand
for an inhibitory immunreceptor.
[0059] An activity modulator which blocks or reduces the inhibition
mediated by inhibitory immunoreceptors such as CTLA4, PD-1, LAG-3,
2B4 or BTLA 1 may tip the balance of
phosphorylation:dephosporylation at the T-cell:target cell synapse
in favour of phosphorylation of ITAMs, leading to T-cell
activation. For example, the activity modulator may block or reduce
the phosphorylation of ITIMs in the endodmain of inhibitory
receptor(s) or may block or reduce the dephosphorylation of ITAMs
in the CAR signalling domain by proteins such as SHP-1 and
SHP-2.
[0060] The activity modulator may be a dominant negative SHP-1 or
SHP-2.
[0061] For example, the activity modulator may be a truncated
protein which comprises an SH2 domain from a protein which binds a
phosphorylated immunoreceptor tyrosine-based inhibition motif
(ITIM), such as SHP-1 or SHP-2, but lacks a phosphatase domain.
[0062] The activity modulator may be a cytokine or chemokine such
as IL12, flexiIL-12, GM-CSF, IL7, IL15, IL21, IL2 or CCL19.
[0063] Alternatively the activity modulator may have an effect of a
cytokine/chemokine signalling pathway in the CAR-expressing
cell.
[0064] For example, the activity modulator may be a chimeric
cytokine receptor which comprises a cytokine receptor endodomain.
The exodomain may be derived from a different cytokine-receptor, or
may not be from a cytokine receptor. The exodomain may bind a
ligand, for example a tumour antigen or secreted factor. Presence
of the ligand may cause two chains of a cytokine receptor
endodomain to associate, leading to cytokine signalling.
[0065] The activity modulator may be a constitutively active
chimeric cytokine receptor. The activity modulator may comprise two
chains which dimerise, either spontaneously or in the presence of
an agent (a chemical inducer of dimerization or CID) bringing
together two cytokine receptor endodomains.
[0066] The activity modulator may affect the JAK/STAT cytokine
signalling pathway. The activity modulator may comprise an
inducible or constitutively active Signal Transducer and Activator
of Transcription (STAT) or Janus kinase (JAK).
[0067] The activity modulator may be or comprise an adhesion
molecule or a transcription factor. The transcription factor may
prevent or reduce differentiation and/or exhaustion of the
CAR-expressing cell.
[0068] The activity modulator of the present invention may modulate
TGF.beta. signalling.
[0069] For example, the activity modulator may block or reduce
TGF.beta. binding to TGF.beta. receptor; it may compete with
TGF.beta. or TG.beta.R for binding to TG.beta.R or TGF.beta.;
alternatively it may modulate the downstream TGF.beta. signalling
for example via SMADs. The activity modulator may be a dominant
negative TGF.beta. receptor.
[0070] The activity modulator of the present invention may provide
co-stimulatory signal to the T-cell.
[0071] For example, the activity modulatory may be a TNF receptor,
a chimeric TNF receptor or a TNF receptor ligand.
[0072] The activity modulator may modulate the activity of the
target cell, for example, a tumour cell.
[0073] The agent may be a toxin, a pro-drug or a pro-drug
activating compound.
[0074] The activity modulator may be an enzyme which is capable of
synthesising a small molecule when expressed or expressed in
combination in a cell. The expression of such an enzyme or
combination of enzymes in a CAR-expressing cell can confer on that
cell the capacity to synthesise a small molecule, such as a small
molecule which is toxic to a tumour cell.
[0075] Alternatively, the activity modulator may be an enzyme which
is secreted by the CAR-expressing cell. The activity modulator be
one or more enzymes which, when secreted or expressed at the cell
surface, causes depletion of a molecule extracellular to the
engineered cell which is:
[0076] (i) required by a tumour cell for survival, proliferation,
metastasis or chemoresistance, and/or [0077] (ii) detrimental to
the survival, proliferation or activity of the engineered cell.
[0078] The enzyme(s) may cause the depletion of for example an
amino acid or amino acid metabolite, a nucleobase (such as a
nucleoside or nucleotide) or a lipid.
[0079] In the method of the invention, the mixture of viral vectors
may comprise at least one vector which comprises a nucleic acid
sequence which encodes a dominant negative SHP-1 or SHP-2; and at
least one vector which comprises a nucleic acid sequence which
encodes a dominant negative transforming growth factor (TGF).beta.
receptor.
[0080] The mixture of viral vectors may comprise two, three, four,
five or six viral vectors, at least one of which comprises a
nucleic acid sequence encoding a CAR; and at least one of which
comprises a nucleic acid sequence encoding an activity
modulator.
[0081] The method may involve the following steps: [0082] (i)
transducing a population of cells with a mixture of at least two
viral vectors; and [0083] (ii) selecting CAR-expressing cells from
the transduced cell population from step (i).
[0084] Alternatively, where each of the viral vectors in the
mixture comprises a nucleic acid sequence encoding a CAR, it may
not be necessary to select or purify CAR-expressing cells from the
transduced cell population.
[0085] In a second aspect, the present invention provides a viral
vector composition. The viral vector composition may comprise a
mixture of two of more vectors. The vector composition may be
suitable for use in the method of the first aspect of the
invention.
[0086] The viral vector composition may comprise a first vector and
a second vector, both of which comprise a nucleic acid sequence
encoding a chimeric antigen receptor (CAR).
[0087] The CAR expressed by the first vector may be the same as the
CAR expressed by the second vector. For example, the CAR expressed
by the first vector may have the same antigen-binding domain as the
CAR expressed by the second vector.
[0088] The first vector and/or second vector may also express an
activity modulator which modulates the activity of the CAR, of a
cell expressing the CAR, or of a target cell. Where the first
vector and second vector both express an activity modulator, the
first vector and second vector may express a different activity
modulator or a different combination of activity modulators.
[0089] For example, the first vector and second vector may express
one or more activity modulator(s) selected from: a dominant
negative SHP-1 or SHP-2; a dominant negative transforming growth
factor (TGF).beta. receptor; and a constitutively active chimeric
cytokine receptor.
[0090] In one arrangement, the first vector may comprise a nucleic
acid sequence encoding a dominant negative SHP-1 or SHP-2 and a
nucleic acid sequence encoding a dominant negative transforming
growth factor (TGF).beta. receptor; and the second vector may
comprise a nucleic acid sequence encoding a constitutively active
chimeric cytokine receptor.
[0091] Where the first and second vectors encode the same CAR, the
CAR may have an antigen-binding domain which binds
disialoganglioside (GD2).
[0092] The first vector and/or the second vector may comprise a
nucleic acid sequence encoding a suicide gene.
[0093] The present invention also provides a cell composition made
by a method according of the invention or made by transducing a
cell with a vector composition of the invention ex vivo.
[0094] In a third aspect, the present invention provides a cell
composition made by a method according to the first aspect of the
invention or made by transducing a population of cells with a viral
vector composition of the second aspect of the invention.
[0095] In a fourth aspect, there is provided a method for treating
a disease in a subject which comprises the step of administering a
cell composition according to the third aspect of the invention to
the subject.
[0096] In a fifth aspect there is provided a cell composition
according to the third aspect of the invention for use in treating
and/or preventing a disease.
[0097] In a sixth aspect, there is provided the use of a cell
composition according to third aspect of the invention in the
manufacture of a medicament for treating and/or preventing a
disease.
[0098] In a seventh aspect, there is provided a method for
determining the optimal combination of components for a
CAR-expressing cell to treat a disease, which comprises the
following steps:
[0099] (i) administering a cell composition according to the second
aspect of the invention to a subject having the disease;
[0100] (ii) monitoring the patient or samples from the patient to
determine which sub-population of cells in the cell composition
show the greatest level of engraftment and/or proliferation;
and
[0101] (iii) analysing the phenotype/genotype of the cells in the
sub-population to ascertain the CAR(s) and/or activity modulator(s)
expressed by those cells.
Further Aspects
[0102] The present invention also provides additional aspects which
are summarised in the following numbered paragraphs:
[0103] 1. A nucleic acid construct which comprises a nucleic acid
sequence encoding a dominant negative SHP-2 and a nucleic acid
sequence encoding a dominant negative TGF.beta. receptor.
[0104] 2. A nucleic acid construct according to paragraph 1, which
has the structure:
[0105] dnSHP-coexpr-dnTGF.beta.R, or
[0106] dnTGF.beta.R-coexpr-dnSHP
[0107] in which:
[0108] dnSHP is a nucleic acid sequence encoding dominant negative
SHP-2
[0109] "coexpr" is a nucleic acid sequences enabling coexpression
of the two polypeptides as separate entities
[0110] "dnTGF.beta.R" is a nucleic acid sequence encoding a
dominant negative TGF.beta. receptor.
[0111] 3. A nucleic acid construct according to paragraph 1 which
also comprises a nucleic acid sequence encoding a CAR.
[0112] 4. A nucleic acid construct according to paragraph 3, which
has the structure:
[0113] CAR-coexpr1-dnSHP-coexpr2-dnTGF.beta.R
[0114] CAR-coexpr1-dnTGF.beta.R-coexpr2-dnSHP
[0115] dnTGF.beta.R-coexpr1-CAR-coexpr2-dnSHP
[0116] dnTGF.beta.R-coexpr1-dnSHP-coexpr2-CAR
[0117] dnSHP-coexpr1-dnTGF.beta.R-coexpr2-CAR or
[0118] dnSHP-coexpr1-CAR-coexpr2-dnTGF.beta.R
[0119] in which:
[0120] dnSHP is a nucleic acid sequence encoding dominant negative
SHP-2
[0121] "coexpr1" and "coexpr2" which may be the same or different,
are nucleic acid sequences enabling coexpression of the three
polypeptides as separate entities
[0122] "dnTGF.beta.R" is a nucleic acid sequence encoding a
dominant negative TGF.beta. receptor; and
[0123] "CAR" is a nucleic acid sequence encoding a chimeric antigen
receptor.
[0124] 5. A nucleic acid construct according to paragraph 4, which
has the structure: dnSHP-coexpr1-CAR-coexpr2-dnTGF.beta.R 6. A
nucleic acid construct according to any of paragraphs 3 to 5,
wherein the CAR binds one of the following target antigens: CD19,
CD22, BCMA, PSMA, CD79, GD2 or FCRL5.
[0125] 7. A nucleic acid construct according to paragraph 3, which
comprises a bicistronic nucleic acid sequence encoding two
CARs.
[0126] 8. A nucleic acid construct according to paragraph 7, which
has the structure:
[0127] dnSHP-coexpr1-CAR1-coexpr2-CAR2-coexpr3-dnTG.beta.R in
which:
[0128] "dnSHP" is a nucleic acid sequence encoding dominant
negative SHP-2
[0129] "coexpr1", "coexpr2" and "coexpr3" which may be the same or
different, are nucleic acid sequences enabling coexpression of the
four polypeptides as separate entities;
[0130] "CAR1" is a nucleic acid sequence encoding a first chimeric
antigen receptor;
[0131] "CAR2" is a nucleic acid sequence encoding a second chimeric
antigen receptor; and
[0132] "dnTGF.beta.R" is a nucleic acid sequence encoding a
dominant negative TGF.beta. receptor.
[0133] 9. A nucleic acid construct according to paragraph 7 or 8,
wherein one CAR binds CD19 and the other CAR binds CD22.
[0134] 10. A nucleic acid construct according to paragraph 6,
wherein the CAR binds CD19 and has an antigen-binding domain which
comprises [0135] a) a heavy chain variable region (VH) having
complementarity determining regions (CDRs) with the following
sequences:
TABLE-US-00001 [0135] CDR1 - GYAFSSS; (SEQ ID No. 1) CDR2 - YPGDED
(SEQ ID No. 2) CDR3 - SLLYGDYLDY; (SEQ ID No. 3)
and [0136] b) a light chain variable region (VL) having CDRs with
the following sequences:
TABLE-US-00002 [0136] CDR1 - SASSSVSYMH; (SEQ ID No. 4) CDR2 -
DTSKLAS (SEQ ID No. 5) CDR3 - QQWNINPLT. (SEQ ID No. 6)
[0137] 11. A nucleic acid construct according to paragraph 10,
wherein the antigen binding domain comprises a VH domain having the
sequence shown as SEQ ID No. 7 and a VL domain having the sequence
shown as SEQ ID No 8.
[0138] 12. A nucleic acid construct according to any preceding
paragraph which also comprises a nucleic acid sequence encoding a
suicide gene.
[0139] 13. A vector comprising a nucleic acid construct according
to any preceding paragraph.
[0140] 14. A kit of vectors comprising a first vector according to
paragraph 13 and a second vector comprising a nucleic acid sequence
which encodes a chimeric antigen receptor (CAR) or activity
modulator.
[0141] 15. A kit of vectors comprising a first vector which
comprises a nucleic acid sequence encoding a dominant negative
SHP-2 and a second vector which comprises a nucleic acid sequence
encoding a dominant negative TGF.beta. receptor.
[0142] 16. A kit of vectors according to paragraph 15, in which the
first vector comprises a nucleic acid sequence encoding a first CAR
and the second vector comprises a nucleic acid sequence encoding a
second CAR.
[0143] 17. A kit of vectors according to paragraph 16, wherein the
first and second CARs have the same target antigen.
[0144] 18. A kit of vectors according to paragraph 16, wherein the
first and second CARs are the same.
[0145] 19. A kit of vectors comprising a first vector according to
paragraph 12, and a second vector encoding a chimeric cytokine
receptor (CCR).
[0146] 20. A kit of vectors according to paragraph 19, in which the
first vector comprises a nucleic acid sequence encoding a first CAR
and the second vector comprises a nucleic acid sequence encoding a
second CAR.
[0147] 21. A kit of vectors according to paragraph 20, wherein the
first and second CARs have the same target antigen.
[0148] 22. A kit of vectors according to paragraph 20, wherein the
first and second CARs are the same.
[0149] 23. A kit of vectors according to any of paragraphs 14 to
22, in which the first vector comprises a nucleic acid sequence
encoding a first suicide gene and the second vector comprises a
nucleic acid sequence encoding a second suicide gene.
[0150] 24. A kit of vectors according to paragraph 23, wherein the
first and second suicide genes are triggered by the same
molecule.
[0151] 25. A kit of vectors according to paragraph 23, wherein the
first and second suicide genes are triggered by the different
molecules.
[0152] 26. A kit of vectors according to any of paragraphs 23 to
25, in which the vectors have the following structure:
[0153] Vector 1: CAR1-coexpr1-SG1-coepr2-dSHP2-coexpr3-dnTGF.beta.R
Vector 2: CAR2-coexpr4-SG2-coepr5-CCR in which:
[0154] "CAR1" is a nucleic acid sequence encoding a first chimeric
antigen receptor;
[0155] "coexpr1", "coexpr2", "coexpr3", "coexpr4", "co-expr5" which
may be the same or different, are nucleic acid sequences enabling
coexpression of the seven polypeptides as separate entities;
[0156] "SG1" is a nucleic acid sequence encoding a first suicide
gene;
[0157] "dnSHP" is a nucleic acid sequence encoding dominant
negative SHP-2;
[0158] "dnTGF.beta.R" is nucleic acid sequence encoding a dominant
negative TGF.beta. receptor;
[0159] "CAR2" is a nucleic acid sequence encoding a second chimeric
antigen receptor which may or may not be the same as CAR1;
[0160] "SG2" is a nucleic acid sequence encoding a first suicide
gene which may or may not be the same as SG1; and
[0161] "CCR" is a nucleic acid sequence encoding a chimeric
cytokine receptor.
[0162] 27. A kit of vectors according to any of paragraphs 14 to
26, which also comprises a third vector comprising a nucleic acid
sequence encoding a cytokine.
[0163] 28. A kit of vectors according to paragraph 27, wherein the
cytokine is IL-12 or Flexi-IL12
[0164] 29. A kit of vectors according to paragraph 28, in which the
vectors have the following structure:
[0165] Vector 1:
dnSHP2-coexpr1-SG1-coepr2-CAR-coexpr3-dnTGF.beta.R
[0166] Vector 2: CCR
[0167] Vector 3: SG2-coexpr4-flexiIL12
[0168] in which:
[0169] "dnSHP" is a nucleic acid sequence encoding dominant
negative SHP-2;
[0170] "coexpr1", "coexpr2", "coexpr3" and "coexpr4", which may be
the same or different, are nucleic acid sequences enabling
coexpression of the six polypeptides on Vectors 1 and 3 as separate
entities;
[0171] "SG1" is a nucleic acid sequence encoding a first suicide
gene;
[0172] "CAR" is a nucleic acid sequence encoding a chimeric antigen
receptor;
[0173] "dnTGF.beta.R" is nucleic acid sequence encoding a dominant
negative TGF.beta. receptor;
[0174] "CCR" is a nucleic acid sequence encoding a chimeric
cytokine receptor;
[0175] "SG2" is a nucleic acid sequence encoding a first suicide
gene which may or may not be the same as SG1; and
[0176] "flexiIL12" is a nucleic acid sequence encoding
flexi-IL-12.
[0177] 30. A vector composition comprising a mixture of: a vector
according to paragraph 13 and at least one other viral vector;
first and second vectors as defined in any of paragraphs 14 to 26;
or first, second and third vectors as defined in any of paragraphs
27 to 29.
[0178] 31. A method for making a cell composition which comprises
step of transducing a population of cells with a vector according
to paragraph 13, a kit of vectors according to any of paragraphs 14
to 29, or a vector composition according to paragraph 30.
[0179] 32. A cell which co-expresses dominant negative SHP-2 and
dominant negative TGF.beta. receptor.
[0180] 33. A cell according to paragraph 32, which also expresses
one or more chimeric antigen receptor(s) (CAR(s)).
[0181] 34. A cell according to paragraph 33, wherein the CAR(s)
is/are as defined in any of paragraphs 6 to 11.
[0182] 35. A cell composition made by a method according to
paragraph 31 or comprising a plurality of cells according to any of
paragraphs 32 to 34.
[0183] 36. A method for treating and/or preventing a disease, which
comprises the step of administering a cell composition according to
paragraph 35 to a subject.
[0184] 37. A method according to paragraph 36, wherein the disease
is a cancer.
[0185] 38. A cell composition according to paragraph 35 for use in
treating and/or preventing a disease.
[0186] 39. The use of a cell according to any of paragraphs 32 to
34 in the manufacture of a medicament for treating and/or
preventing a disease.
[0187] 40. A nucleic acid construct which comprises a nucleic acid
sequence encoding a dominant negative TGF.beta. receptor; a nucleic
acid sequence encoding a IL7; and a nucleic acid sequence encoding
CCL19
[0188] 41. A nucleic acid construct according to paragraph 40,
which has the structure:
[0189] dnTGF.beta.R-coexpr1-IL7-coexpr2-CCL19;
[0190] dnTGF.beta.R-coexpr1-CCL19-coexpr2-IL7;
[0191] IL7-coexpr1-CCL19-coexpr2-dnTGF.beta.R;
[0192] IL7-coexpr1-dnTGF.beta.R-CCL19-coexpr2;
[0193] CCL19-coexpr1-IL7-coexpr2-dnTGF.beta.R; or
[0194] CCL19-coexpr1-dnTGF.beta.R-coexpr2-IL7
[0195] in which:
[0196] "dnTGF.beta.R" is a nucleic acid sequence encoding a
dominant negative TGF.beta. receptor;
[0197] "IL7" is a nucleic acid sequence encoding IL7
[0198] "CCL19" is a nucleic acid sequence encoding CCL19
[0199] "coexpr1" and "coexpr2", which may be the same or different,
are nucleic acid sequences enabling coexpression of the three
polypeptides as separate entities.
[0200] 42. A nucleic acid construct according to paragraph 40 which
also comprises a nucleic acid sequence encoding a CAR.
[0201] 43. A nucleic acid construct according to paragraph 3, which
has the structure:
[0202] CAR-coexpr1-dnTGF.beta.R-coexpr2-IL7-coexpr3-CCL19;
[0203] in which:
[0204] dnTGF.beta.R'' is a nucleic acid sequence encoding a
dominant negative TGF.beta. receptor;
[0205] "IL7" is a nucleic acid sequence encoding IL7
[0206] "CCL19" is a nucleic acid sequence encoding CCL19
[0207] "CAR" is a nucleic acid sequence encoding a chimeric antigen
receptor
[0208] "coexpr1", "coexpr2" and "coexpr3", which may be the same or
different, are nucleic acid sequences enabling coexpression of the
four polypeptides as separate entities.
[0209] 44. A nucleic acid construct according to any of paragraphs
3 to 5, wherein the CAR binds GD2.
[0210] 45. A vector comprising a nucleic acid construct according
to any of paragraphs 40 to 44.
[0211] 46. A kit of vectors comprising a first vector which
comprises a nucleic acid sequence encoding IL7 and a nucleic acid
sequence encoding CCL19; and a second vector which comprises a
nucleic acid sequence encoding a dominant negative TGF.beta.
receptor.
[0212] 47. A kit of vectors according to paragraph 46, in which
both vectors also comprise a nucleic acid sequence encoding a
chimeric antigen receptor (CAR).
[0213] 48. A kit of vectors according to paragraph 47, wherein the
CAR encoded by the first vector is the same as the CAR encoded by
the second vector.
[0214] 49. A kit of vectors according to paragraph 48, wherein the
CAR binds GD2.
[0215] 50. A method for making a cell composition which comprises
step of transducing a population of cells with a vector according
to paragraph 45 or a kit of vectors according to any of paragraphs
46 to 49.
[0216] 51. A cell which co-expresses dominant negative TGF.beta.
receptor, IL7 and CCL19.
[0217] 52. A cell according to paragraph 51, which also expresses
one or more chimeric antigen receptor(s) (CAR(s)).
[0218] 53. A cell composition made by a method according to
paragraph 46 or comprising a plurality of cells according to
paragraph 51 or 52.
[0219] 54. A method for treating and/or preventing a disease, which
comprises the step of administering a cell composition according to
paragraph 49 to a subject.
[0220] 55. A method according to paragraph 54, wherein the disease
is a cancer.
[0221] 56. A cell composition according to paragraph 53 for use in
treating and/or preventing a disease.
[0222] 57. The use of a cell according to paragraph 51 or 52 in the
manufacture of a medicament for treating and/or preventing a
disease.
[0223] In the above paragraphs and the below claims,
polypeptide-encoding elements of a nucleic acid construct or
vector, such as "dnSHP", "dnTGF.beta.R", "IL7", "CCL19" and "CAR"
may be in any order in the construct.
[0224] The following detailed description, as it relates to nucleic
acid and polypeptide sequences, polypeptide components, vectors,
cells methods etc applies equally to the aspects laid out in the
above paragraphs as to the aspects of the invention in the
claims.
DETAILED DESCRIPTION
[0225] The present invention relates to a method for making a cell
composition which comprises step of transducing a population of
cells with a mixture of at least two viral vectors.
[0226] The viral vectors may, for example, be retroviral vectors or
lentiviral vectors.
[0227] Retroviruses are double stranded RNA enveloped viruses
mainly characterized by the ability to "reverse-transcribe" their
genome from RNA to DNA. Virions measure 100-120 nm in diameter and
contain a dimeric genome of identical positive RNA strands
complexed with the nucleocapsid proteins. The genome is enclosed in
a proteic capsid that also contains enzymatic proteins, namely the
reverse transcriptase, the integrase and proteases, required for
viral infection. The matrix proteins form a layer outside the
capsid core that interacts with the envelope, a lipid bilayer
derived from the host cellular membrane, which surrounds the viral
core particle. Anchored on this bilayer, are the viral envelope
glycoproteins responsible for recognizing specific receptors on the
host cell and initiating the infection process. Envelope proteins
are formed by two subunits, the transmembrane (TM) that anchors the
protein into the lipid membrane and the surface (SU) which binds to
the cellular receptors.
[0228] Based on the genome structure, retroviruses are classified
into simple retroviruses, such as MLV and murine leukemia virus; or
complex retroviruses, such as HIV and EIAV. Retroviruses encode
four genes: gag (group specific antigen), pro (protease), pol
(polymerase) and env (envelope). The gag sequence encodes the three
main structural proteins: the matrix protein, nucleocapsid
proteins, and capsid protein. The pro sequence encodes proteases
responsible for cleaving Gag and Gag-Pol during particle assembly,
budding and maturation. The pol sequence encodes the enzymes
reverse transcriptase and integrase, the former catalyzing the
reverse transcription of the viral genome from RNA to DNA during
the infection process and the latter responsible for integrating
the proviral DNA into the host cell genome. The env sequence
encodes for both SU and TM subunits of the envelope glycoprotein.
Additionally, retroviral genome presents non-coding cis-acting
sequences such as: two LTRs (long terminal repeats), which contain
elements required to drive gene expression, reverse transcription
and integration into the host cell chromosome; a sequence named
packaging signal (.psi.) required for specific packaging of the
viral RNA into newly forming virions; and a polypurine tract (PPT)
that functions as the site for initiating the positive strand DNA
synthesis during reverse transcription. In addition to gag, pro,
pol and env, complex retroviruses, such as lentiviruses, have
accessory genes including vif, vpr, vpu, nef, tat and rev that
regulate viral gene expression, assembly of infectious particles
and modulate viral replication in infected cells.
[0229] During the process of infection, a retrovirus initially
attaches to a specific cell surface receptor. On entry into the
susceptible host cell, the retroviral RNA genome is then copied to
DNA by the virally encoded reverse transcriptase which is carried
inside the parent virus. This DNA is transported to the host cell
nucleus where it subsequently integrates into the host genome. At
this stage, it is typically referred to as the provirus. The
provirus is stable in the host chromosome during cell division and
is transcribed like other cellular proteins. The provirus encodes
the proteins and packaging machinery required to make more virus,
which can leave the cell by a process known as "budding".
[0230] When enveloped viruses, such as retrovirus and lentivirus,
bud out of the host cells, they take part of the host cell lipidic
membrane. In this way, host-cell derived membrane proteins become
part of the retroviral particle. The present invention utilises
this process in order to introduce proteins of interest into the
envelope of the viral particle.
[0231] Viral Vectors
[0232] Retroviruses and lentiviruses may be used as a vector or
delivery system for the transfer of a nucleic acid sequence, or a
plurality of nucleic acid sequences, to a target cell. The transfer
can occur in vitro, ex vivo or in vivo. When used in this fashion,
the viruses are typically called viral vectors.
[0233] Gamma-retroviral vectors, commonly designated retroviral
vectors, were the first viral vector employed in gene therapy
clinical trials in 1990 and are still one of the most used. More
recently, the interest in lentiviral vectors, derived from complex
retroviruses such as the human immunodeficiency virus (HIV), has
grown due to their ability to transduce non-dividing cells. The
most attractive features of retroviral and lentiviral vectors as
gene transfer tools include the capacity for large genetic payload
(up to 9 kb), minimal patient immune response, high transducing
efficiency in vivo and in vitro, and the ability to permanently
modify the genetic content of the target cell, sustaining a
long-term expression of the delivered gene.
[0234] The retroviral vector can be based on any suitable
retrovirus which is able to deliver genetic information to
eukaryotic cells. For example, the retroviral vector may be an
alpharetroviral vector, a gammaretroviral vector, a lentiviral
vector or a spumaretroviral vector. Such vectors have been used
extensively in gene therapy treatments and other gene delivery
applications.
[0235] The viral vector of the present invention may be a
retroviral vector, such as a gamma-retroviral vector. The viral
vector may be based on human immunodeficiency virus.
[0236] The viral vector of the present invention may be a
lentiviral vector. The vector may be based on a non-primate
lentivirus such as equine infectious anemia virus (EIAV).
[0237] Nucleic Acid Sequences and Constructs
[0238] In the mixture of viral vectors used in the method of the
present invention, each vector may comprise one or more nucleic
acid sequences. For example, one or more of the vectors in the
mixture may comprise a nucleic acid construct comprising a
plurality of nucleic acid sequences which are co-expressed. The
nucleic acid construct may, for example, be bicistronic or
tri-cistronic. The nucleic acid construct may comprise 2, 3, 4 or 5
transgenes.
[0239] The nucleic acid sequences in the nucleic acid construct may
be separated by a "coexpression" sequence which enables the two or
more polypeptides, once translated, to be expressed separately in
or on the cell.
[0240] The coexpression sequence may encode a cleavage site, such
that the nucleic acid construct produces comprises two or more
polypeptides joined by a cleavage site(s). The cleavage site may be
self-cleaving, such that when the polypeptide is produced, it is
immediately cleaved into individual polypeptides without the need
for any external cleavage activity.
[0241] The cleavage site may be any sequence which enables the two
or more polypeptides to become separated.
[0242] The term "cleavage" is used herein for convenience, but the
cleavage site may cause the polypeptides to separate into
individual entities by a mechanism other than classical cleavage.
For example, for the Foot-and-Mouth disease virus (FMDV) 2A
self-cleaving peptide (see below), various models have been
proposed for to account for the "cleavage" activity: proteolysis by
a host-cell proteinase, autoproteolysis or a translational effect
(Donnelly et al (2001) J. Gen. Virol. 82:1027-1041). The exact
mechanism of such "cleavage" is not important for the purposes of
the present invention, as long as the cleavage site, when
positioned between nucleic acid sequences which encode proteins,
causes the proteins to be expressed as separate entities.
[0243] The cleavage site may be a furin cleavage site.
[0244] Furin is an enzyme which belongs to the subtilisin-like
proprotein convertase family. The members of this family are
proprotein convertases that process latent precursor proteins into
their biologically active products. Furin is a calcium-dependent
serine endoprotease that can efficiently cleave precursor proteins
at their paired basic amino acid processing sites. Examples of
furin substrates include proparathyroid hormone, transforming
growth factor beta 1 precursor, proalbumin, pro-beta-secretase,
membrane type-1 matrix metalloproteinase, beta subunit of pro-nerve
growth factor and von Willebrand factor. Furin cleaves proteins
just downstream of a basic amino acid target sequence (canonically,
Arg-X-(Arg/Lys)-Arg' (SEQ ID No. 58)) and is enriched in the Golgi
apparatus.
[0245] The cleavage site may be a Tobacco Etch Virus (TEV) cleavage
site.
[0246] TEV protease is a highly sequence-specific cysteine protease
which is chymotrypsin-like proteases. It is very specific for its
target cleavage site and is therefore frequently used for the
controlled cleavage of fusion proteins both in vitro and in vivo.
The consensus TEV cleavage site is ENLYFQ\S (where `\` denotes the
cleaved peptide bond) (SEQ ID No. 59). Mammalian cells, such as
human cells, do not express TEV protease. Thus in embodiments in
which the present nucleic acid construct comprises a TEV cleavage
site and is expressed in a mammalian cell--exogenous TEV protease
must also expressed in the mammalian cell.
[0247] The cleavage site may encode a self-cleaving peptide.
[0248] A `self-cleaving peptide` refers to a peptide which
functions such that when the polypeptide comprising the proteins
and the self-cleaving peptide is produced, it is immediately
"cleaved" or separated into distinct and discrete first and second
polypeptides without the need for any external cleavage
activity.
[0249] The self-cleaving peptide may be a 2A self-cleaving peptide
from an aphtho- or a cardiovirus. The primary 2A/2B cleavage of the
aptho- and cardioviruses is mediated by 2A "cleaving" at its own
C-terminus. In apthoviruses, such as foot-and-mouth disease viruses
(FMDV) and equine rhinitis A virus, the 2A region is a short
section of about 18 amino acids, which, together with the
N-terminal residue of protein 2B (a conserved proline residue)
represents an autonomous element capable of mediating "cleavage" at
its own C-terminus (Donelly et al (2001) as above).
[0250] "2A-like" sequences have been found in picornaviruses other
than aptho- or cardioviruses, `picornavirus-like` insect viruses,
type C rotaviruses and repeated sequences within Trypanosoma spp
and a bacterial sequence (Donnelly et al (2001) as above).
[0251] The cleavage site may comprise the 2A-like sequence shown as
SEQ ID No. 9.
[0252] SEQ ID No. 9
[0253] RAEGRGSLLTCGDVEENPGP
[0254] The present invention provides a nucleic acid construct
which comprises a nucleic acid sequence encoding a dominant
negative SHP-1 or SHP-2 and a nucleic acid sequence encoding a
dominant negative TGF.beta.receptor.
[0255] Dominant negative SHP-1 or SHP-2 and TGF.beta. receptors are
described in more detail below.
[0256] The nucleic acid construct may have the structure:
[0257] dnSHP-coexpr-dnTGF.beta.R, or
[0258] dnTGF.beta.R-coexpr-dnSHP
[0259] in which:
[0260] dnSHP is a nucleic acid sequence encoding dominant negative
SHP-1 or SHP-2
[0261] "coexpr" is a nucleic acid sequences enabling coexpression
of the two polypeptides as separate entities
[0262] dnTGF.beta.R is a dominant negative TGF.beta. receptor.
[0263] The nucleic acid construct may also comprise a nucleic acid
sequence encoding a CAR. In which case the nucleic acid construct
may have the structure:
[0264] CAR-coexpr1-dnSHP-coexpr2-dnTG.beta.R
[0265] CAR-coexpr1-dnTG.beta.R-coexpr2-dnSHP
[0266] dnTGF.beta.R-coexpr1-CAR-coexpr2-dnSHP
[0267] dnTGF.beta.R-coexpr1-dnSHP-coexpr2-CAR
[0268] dnSHP-coexpr1-dnTG.beta.R-coexpr2-CAR or
[0269] dnSHP-coexpr1-CAR-coexpr2-dnTG.beta.R
[0270] in which:
[0271] dnSHP is a nucleic acid sequence encoding dominant negative
SHP-2
[0272] "coexpr1" and "coexpr2" which may be the same or different,
are nucleic acid sequences enabling coexpression of the three
polypeptides as separate entities
[0273] dnTGF.beta.R is a dominant negative TGF.beta. receptor;
and
[0274] CAR is a nucleic acid sequence encoding a chimeric antigen
receptor.
[0275] Suicide Gene
[0276] A nucleic acid construct may also comprise a nucleic acid
encoding a suicide gene.
[0277] Since T-cells engraft and are autonomous, a means of
selectively deleting CAR T-cells in patients is desirable. Suicide
genes are genetically encodable mechanisms which result in
selective destruction of infused T-cells in the face of
unacceptable toxicity. The earliest clinical experience with
suicide genes is with the Herpes Virus Thymidine Kinase (HSV-TK)
which renders T-cells susceptible to Ganciclovir. HSV-TK is a
highly effective suicide gene. However, pre-formed immune responses
may restrict its use to clinical settings of considerable
immunosuppression such as haploidentical stem cell transplantation.
Inducible Caspase 9 (iCasp9) is a suicide gene constructed by
replacing the activating domain of Caspase 9 with a modified
FKBP12. iCasp9 is activated by an otherwise inert small molecular
chemical inducer of dimerization (CID). iCasp9 has been recently
tested in the setting of haploidentical HSCT and can abort GvHD.
Both iCasp9 and HSV-TK are intracellular proteins, so when used as
the sole transgene, they have been co-expressed with a marker gene
to allow selection of transduced cells.
[0278] WO2016/135470 describes a suicide gene which also comprises
Caspase 9 but can be induced to dimerise using rapamycin or a
rapamycin analog.
[0279] This suicide gene, sometimes termed Rapcasp9 or Rapacasp9,
has the amino acid sequence shown as SEQ ID No. 80.
[0280] SEQ ID No. 80 (Rapcasp9)
[0281] WO2013/153391 describes a marker/suicide gene known as RQR8
which can be detected with the antibody QBEnd10 and expressing
cells lysed with the therapeutic antibody Rituximab.
[0282] The sort/suicide gene RQR8 has the amino acid sequence shown
as SEQ ID No. 79.
TABLE-US-00003 SEQ ID No. 79 (RQR8)
CPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSG
GGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW
APLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV
[0283] Including a suicide gene in one or more of the vectors in
the viral vector composition of the invention enables the selective
ablation of a proportion of transduced cells within the
subject.
[0284] For example, for two vectors A and B, transduced cells will
be a mixture of cells transduced with vector A alone, cells
transduced with vector B alone, and cells transduced with both
vectors A and B. If vector A expresses or co-expresses a suicide
gene, activating the suicide gene will cause the deletion of cells
transduced with vector A alone, or with vectors A and B, but cells
transduced with vector B alone will be spared.
[0285] This is particularly useful where one vector in the mixture
encodes a potentially dangerous or toxic gene. If a suicide gene is
included on the cassette for that vector, then in the event of an
unacceptable immunological or toxic event in the patient, cells
expressing the gene in question can be selectively deleted by
triggering the suicide gene. Cells expressing other vector
combinations which do not include the potentially dangerous
gene/suicide gene combination are spared and can continue their
therapeutic effect.
[0286] For example, a suicide gee may be included in a vector which
expresses an immunomodulatory cytokine such as IL-12 or a
constitutively active cytokine receptor (see below).
[0287] Viral Vector Composition
[0288] The present invention provides a viral vector composition
which comprises a mixture viral vectors. The composition may be
made by simply mixing two of more viral vectors. The composition
may comprise between 2 and 10 viral vectors, for example, 2, 3, 4,
5 or 6 viral vectors.
[0289] The viral vectors in the mixture may each comprise one or
more transgenes. Two or more viral vectors in the composition may
overlap in one or more transgenes. For example, two viral vectors
in the composition may comprise a nucleic acid sequence encoding
the same CAR, but may differ in the presence or type of activity
modulator(s) encoded by other nucleic acid sequences.
[0290] One or more of the viral vector(s) in the composition may
comprises a nucleic acid sequence encoding a dominant negative
SHP-1 or SHP-2. One or more viral vector(s) in the composition may
comprise a nucleic acid sequence encoding a dominant negative
TGF.beta. receptor. One or more viral vectors in the composition
may comprise a nucleic acid sequence encoding a chimeric antigen
receptor.
[0291] The viral vector composition may comprise a vector which
comprises a nucleic acid sequence encoding a dominant negative
SHP-1 or SHP-2 and a nucleic acid sequence encoding a dominant
negative TGF.beta. receptor.
[0292] The viral vector composition may comprise plurality of
vectors, each of which encode different activity modulator(s) or
activity modulator combinations.
[0293] Chimeric Antigen Receptor
[0294] In the method of the present invention at least one vector
in the mixture of viral vectors may comprise a nucleic acid
sequence which encodes a chimeric antigen receptor (CAR).
[0295] Chimeric Antigen Receptors (CARS)
[0296] CARs, which are shown schematically in FIG. 1, 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 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 CD8a 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.
[0297] Early CAR designs had endodomains derived from the
intracellular parts of either the .gamma. chain of the
Fc.epsilon.R1 or CD3.zeta.. Consequently, these first generation
receptors transmitted immunological signal 1, which was sufficient
to trigger T-cell killing of cognate target cells but failed to
fully activate the T-cell to proliferate and survive. To overcome
this limitation, compound endodomains have been constructed: fusion
of the intracellular part of a T-cell co-stimulatory molecule to
that of CD3.zeta. 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.
[0298] CAR-encoding nucleic acids may be transferred to T cells
using, for example, retroviral or lentiviral vectors to generate
cancer-specific T cells 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.
[0299] Tandem CARs (TanCARs)
[0300] Bispecific CARs, known as tandem CARs or TanCARs, have been
developed to target two or more cancer specific markers
simultaneously. In a TanCAR, the extracellular domain comprises two
antigen binding specificities in tandem, joined by a linker. The
two binding specificities (scFvs) are thus both linked to a single
transmembrane portion: one scFv being juxtaposed to the membrane
and the other being in a distal position. When a TanCAR binds
either or both of the target antigens, this results in the
transmission of an activating signal to the cell it is expressed
on.
[0301] Grada et al (2013, Mol Ther Nucleic Acids 2:e105) describes
a TanCAR which includes a CD19-specific scFv, followed by a Gly-Ser
linker and then a HER2-specific scFv. The HER2-scFv was in the
juxta-membrane position, and the CD19-scFv in the distal position.
The TanCAR was shown to induce distinct T cell reactivity against
each of the two tumour restricted antigens. This arrangement was
chosen because the respective lengths of HER2 (632 aa/125 .ANG.)
and CD19 (280aa, 65 .ANG.) lends itself to that spatial
arrangement. It was also known that the HER2 scFv bound the
distal-most 4 loops of HER2.
[0302] Antigen Binding Domain
[0303] The antigen binding domain is the portion of 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 antibody; an artificial single
binder such as a Darpin (designed ankyrin repeat protein); or a
single-chain derived from a T-cell receptor.
[0304] In a classical CAR, the antigen-binding domain comprises: a
single-chain variable fragment (scFv) derived from a monoclonal
antibody (see FIG. 4c). CARs have also been produced with domain
antibody (dAb) or VHH antigen binding domains (see FIG. 4b) or
which comprise a Fab fragment of, for example, a monoclonal
antibody (see FIG. 4a). A FabCAR comprises two chains: one having
an antibody-like light chain variable region (VL) and constant
region (CL); and one having a heavy chain variable region (VH) and
constant region (CH). One chain also comprises a transmembrane
domain and an intracellular signalling domain. Association between
the CL and CH causes assembly of the receptor.
[0305] The two chains of a Fab CAR may have the general
structure:
[0306] VH-CH-spacer-transmembrane domain-intracellular signalling
domain; and
[0307] VL-CL
[0308] or
[0309] VL-CL-spacer-transmembrane domain-intracellular signalling
domain; and
[0310] VH-CH
[0311] For Fab-type chimeric receptors, the antigen binding domain
is made up of a VH from one polypeptide chain and a VL from another
polypeptide chain.
[0312] The polypeptide chains may comprise a linker between the
VH/VL domain and the CH/CL domains. The linker may be flexible and
serve to spatially separate the VH/VL domain from the CH/CL
domain.
[0313] The antigen-binding domain of the CAR may bind a tumour
associated antigen. Various tumour associated antigens (TAA) are
known, for example as shown in the following Table 1.
TABLE-US-00004 TABLE 1 Cancer type TAA Diffuse Large B-cell
Lymphoma CD19, CD20, CD22 Breast cancer ErbB2, MUC1 AML CD13, CD33
Neuroblastoma GD2, NCAM, ALK, GD2 B-CLL CD19, CD52, CD160
Colorectal cancer Folate binding protein, CA-125 Chronic
Lymphocytic Leukaemia CD5, CD19 Glioma EGFR, Vimentin Multiple
myeloma BCMA, CD138 Renal Cell Carcinoma Carbonic anhydrase IX,
G250 Prostate cancer PSMA Bowel cancer A33
[0314] The or each CAR may bind one of the following target
antigens: CD19, CD22, BCMA, PSMA, GD2, CD79 or FCRL5.
[0315] CD19
[0316] An antigen binding domain of a CAR which binds to CD19 may
comprise a sequence derived from one of the CD19 binders shown in
Table 2.
TABLE-US-00005 TABLE 2 Binder References HD63 Pezzutto (Pezzutto,
A. et al. J. Immunol. Baltim. Md 1950 138, 2793-2799 (1987) 4g7
Meeker et al (Meeker, T. C. et al. Hybridoma 3, 305-320 (1984)
Fmc63 Nicholson et al (Nicholson, I. C. et al. Mol. Immunol. 34,
1157-1165 (1997) B43 Bejcek et al (Bejcek, B. E. et al. Cancer Res.
55, 2346-2351 (1995) SJ25C1 Bejcek et al (1995, as above) BLY3
Bejcek et al (1995, as above) B4, or re-surfaced, Roguska et al
(Roguska, M. A. et al. Protein or humanized B4 Eng. 9, 895-904
(1996) HB12b, Kansas et al (Kansas, G. S. & Tedder, T. F. J.
optimized Immunol. Baltim. Md 1950 147, and humanized 4094-4102
(1991); Yazawa et al (Yazawa et al Proc. Natl. Acad. Sci. U.S.A.
102, 15178-15183 (2005); Herbst et al (Herbst, R. et al. J.
Pharmacol. Exp. Ther. 335, 213-222 (2010)
[0317] Alternatively a CAR which binds CD19 may have an
antigen-binding domain which comprises: [0318] a) a heavy chain
variable region (VH) having complementarity determining regions
(CDRs) with the following sequences:
TABLE-US-00006 [0318] CDR1 - GYAFSSS; (SEQ ID No. 1) CDR2 - YPGDED
(SEQ ID No. 2) CDR3 - SLLYGDYLDY; (SEQ ID No. 3)
and [0319] b) a light chain variable region (VL) having CDRs with
the following sequences:
TABLE-US-00007 [0319] CDR1 - SASSSVSYMH; (SEQ ID No. 4) CDR2 -
DTSKLAS (SEQ ID No. 5) CDR3 - QQWNINPLT. (SEQ ID No. 6)
[0320] The antigen binding domain may comprise a VH domain having
the sequence shown as SEQ ID No. 7 and a VL domain having the
sequence shown as SEQ ID No 8.
TABLE-US-00008 - VH sequence SEQ ID No. 7
QVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGR
IYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSL
LYGDYLDYWGQGTTLTVSS - VL sequence SEQ ID No 8
QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDT
SKLASGVPDRFSGSGSGTSYFLTINNMEAEDAATYYCQQWNINPLTFGAG TKLELKR
[0321] CD22
[0322] A CAR which binds to CD22 may have an antigen domain derived
from m971, HA22 or BL22 as described by Haso et al. (Blood; 2013;
121(7)).
[0323] Alternatively, a CAR which binds CD22 may have an antigen
binding domain as described in United Kingdom application No.
1809773.3, such as one which comprises:
[0324] a) a heavy chain variable region (VH) having complementarity
determining regions (CDRs) with the following sequences:
TABLE-US-00009 CDR1 - NFAMA (SEQ ID No. 10) CDR2 -
SISTGGGNTYYRDSVKG (SEQ ID No. 11) CDR3 - QRNYYDGSYDYEGYTMDA; (SEQ
ID No. 12)
and
[0325] b) a light chain variable region (VL) having complementarity
determining regions (CDRs) with the following sequences:
TABLE-US-00010 CDR1 - RSSQDIGNYLT (SEQ ID No. 13) CDR2 - GAIKLED
(SEQ ID No. 14) CDR3 - LQSIQYP (SEQ ID No. 15)
[0326] The antigen binding domain of a CD22 CAR may comprise a VH
domain having the sequence shown as SEQ ID No. 16; and a VL domain
having the sequence shown as SEQ ID No. 17.
TABLE-US-00011 SEQ ID No. 16
EVQLVESGGGLVQPGRSLKLSCAASGFTFSNFAMAWVRQPPTKGLEWVAS
ISTGGGNTYYRDSVKGRFTISRDDAKNTQYLQMDSLRSEDTATYYCARQR
NYYDGSYDYEGYTMDAWGQGTSVTVSS SEQ ID No. 17
DIQMTQSPSSLSASLGDRVTITCRSSQDIGNYLTWFQQKVGRSPRRMIYG
AIKLEDGVPSRFSGSRSGSDYSLTISSLESEDVADYQCLQSIQYPFTFGS GTKLEIK
[0327] BCMA
[0328] A number of BCMA-targeted CARs are in clinical development,
including bb2121, LCAR-B38M, MCARH171, JCARH125, P-BCMA-101,
FCARH143, bb21217 and CT053.
[0329] WO2015/052538 describes a BCMA targeted CAR in which the
antigen-binding domain is derived from a proliferation inducing
ligand (APRIL) which is a natural ligand for BCMA.
[0330] UK Patent application No. 1815775.0 describes the VH and VL
domains for 14 BCMA binding domains and their use in CARs.
[0331] PSMA
[0332] T-cells expressing CARs specific for prostate-specific
membrane antigen (PSMA) are currently in clinical trial for the
treatment of prostate cancer (Junhans et al (2016) Prostate
76:1257-1270).
[0333] GD2 CARs have been developed which bind disialoganglioside
(GD2) a sialic acid-containing glycosphinolipid. Such CARs may, for
example, be based on the GD2 binder 14g2a, or huK666 as described
in WO2015/132604.
[0334] A CAR which binds GD2 may have an antigen-binding domain
which comprises:
[0335] a) a heavy chain variable region (VH) having complementarity
determining regions (CDRs) with the following sequences:
TABLE-US-00012 CDR1 - SYNIH; (SEQ ID No. 71) CDR2 -
VIWAGGSTNYNSALMS (SEQ ID No. 72) CDR3 - RSDDYSWFAY; (SEQ ID No.
73)
and
[0336] b) a light chain variable region (VL) having CDRs with the
following sequences:
TABLE-US-00013 CDR1 - RASSSVSSSYLH; (SEQ ID No. 74) CDR2 - STSNLAS
(SEQ ID No. 75) CDR3 - QQYSGYPIT. (SEQ ID No. 76)
[0337] The GD2 binding domain may comprise a VH domain having the
sequence shown as SEQ ID No. 77; and/or a VL domain having the
sequence shown as SEQ ID No 78.
TABLE-US-00014 (Humanised KM666 VH sequence) SEQ ID No. 77
QVQLQESGPGLVKPSQTLSITCTVSGFSLASYNIHWVRQPPGKGLEWLGV
IWAGGSTNYNSALMSRLTISKDNSKNQVFLKMSSLTAADTAVYYCAKRSD
DYSWFAYWGQGTLVTVSS (Humanised KM666 VH sequence) SEQ ID No. 78
ENQMTQSPSSLSASVGDRVTMTCRASSSVSSSYLHWYQQKSGKAPKVWIY
STSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYSGYPITFG QGTKVEIK
[0338] FCRL5
[0339] Commercially available monoclonal antibodies against FcRL5
are known, such as CD307e (ThermoFisher) and REA391 (Miltenyi
Biotec).
[0340] WO2016090337 describes several scFv-type antigen-binding
domains which bind FcRL5.
[0341] UK Patent application No. 1815775.0 describes anti-FCRL5
CARs.
[0342] CD79
[0343] A number of anti-CD79 antibodies have been previously
described, such as JCB117, SNB, CB3.1, 2F2 (Polatuzumab).
[0344] United Kingdom application No. 1807870.9 describes various
CD79 CARs.
[0345] Where the composition of viral vectors includes more than
one vector comprising a nucleic acid sequence encoding a CAR, the
CARs may have different antigen binding domains. The CARs may
recognise different antigens, or the CARs may bind the same antigen
but have different antigen-binding domains. CARs which bind the
same antigen but have different antigen-binding domains may bind to
different epitopes of the antigen and/or may have different
affinities and/or on or off rates.
[0346] The affinity of a CAR for the target antigen and/or its on
and off rate, can affect the capacity of a CAR to kill target
cells. For example, it is reported in US 2018/0064785 that a CAR
derived from an antibody with a fast on-rate and a fast off-rate
allows a CAR T-cell to better serially kill target cells. By giving
a patient a CAR-T cell composition which comprises a plurality of
CARs against the target antigen, the CAR with the antigen-binding
domain best suited to kill target cells in the patient or at a
particular site in the patient will receive
activation/survival/proliferation signals and will prevail. The
composition of the invention gives flexibility in this regard and
even allows CAR-T cell subpopulations having different CARs to "win
out" at different sites within the same patient.
[0347] Intracellular T Cell Signaling Domain (Endodomain)
[0348] The CAR may comprise or associate with an activating
endodomain: the signal-transmission portion of the CAR. After
antigen recognition, receptors cluster 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 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.
[0349] The endodomain of the CAR may comprise the CD28 endodomain
and OX40 and CD3-Zeta endodomain.
[0350] The endodomain may comprise:
[0351] (i) an ITAM-containing endodomain, such as the endodomain
from CD3 zeta; and/or
[0352] (ii) a co-stimulatory domain, such as the endodomain from
CD28; and/or
[0353] (iii) a domain which transmits a survival signal, for
example a TNF receptor family endodomain such as OX-40 or
4-1BB.
[0354] An endodomain which contains an ITAM motif can act as an
activation endodomain in this invention. 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 (SeQ ID NO.
60). 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 2000/063372, which relates to synthetic
signalling molecules).
[0355] A number of systems have been described in which the antigen
recognition portion of the CAR is on a separate molecule from the
signal transmission portion, such as those described in
WO015/150771; WO2016/124930 and WO2016/030691. One or more of the
viral vectors used in the method of the invention may encode such a
"split CAR". Alternatively one vector may comprise a nucleic acid
sequence encoding the antigen recognition portion and one vector
may comprise a nucleic acid sequence encoding the intracellular
signalling domain.
[0356] Where the composition of viral vectors includes more than
one vector comprising a nucleic acid sequence encoding a CAR, the
CARs may have different endodomains or different endodomain
combinations. For example, one CAR may be a second generation CAR
and one CAR may be a third generation CAR. Alternatively, both CARs
may be a second generation CAR but may have different
co-stimulatory domains. For example, different second generation
CAR signalling domains include: 41BB-CD3.zeta.; OX40-CD3.zeta. and
CD28-CD3.zeta..
[0357] Signal Peptide
[0358] One or more nucleic acid sequences in the vector composition
may encode a signal peptide so that when the CAR or activity
modulator is expressed inside a cell, the nascent protein is
directed to the endoplasmic reticulum and subsequently to the cell
surface, where it is expressed (or secreted).
[0359] The core of the signal peptide may contain a long stretch of
hydrophobic amino acids that tends to form a single alpha-helix.
The signal peptide may begin with a short positively charged
stretch of amino acids, which helps to enforce proper topology of
the polypeptide during translocation. At the end of the signal
peptide there is typically a stretch of amino acids that is
recognized and cleaved by signal peptidase. Signal peptidase may
cleave either during or after completion of translocation to
generate a free signal peptide and a mature protein. The free
signal peptides are then digested by specific proteases.
[0360] The signal peptide may be at the amino terminus of the
molecule.
[0361] A CAR may have the general formula:
[0362] Signal peptide-antigen binding domain-spacer
domain-transmembrane domain-intracellular T cell signaling domain
(endodomain).
[0363] Spacer
[0364] The CAR may 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 to the antigen binding domain to orient in different
directions to enable antigen binding.
[0365] The spacer sequence may, for example, comprise an IgG1 Fc
region, an IgG1 hinge or a CD8 stalk, or a combination thereof. The
spacer may alternatively comprise an alternative sequence which has
similar length and/or domain spacing properties as an IgG1 Fc
region, an IgG1 hinge or a CD8 stalk.
[0366] Where the composition of viral vectors includes more than
one vector comprising a nucleic acid sequence encoding a CAR, the
CARs may have different spacers.
[0367] Or Gates
[0368] A cell composition of the present invention may comprise two
or more CARs. This may be as a result of transduction with two or
more vectors, each comprising a nucleic acid sequence encoding a
CAR; or it may be as a result of transduction with a single vector
which comprises a nucleic acid construct encoding two or more
CARs.
[0369] A CAR may be used in a combination with one or more other
activatory or inhibitory chimeric antigen receptors. For example,
they may be used in combination with one or more other CARs in a
"logic-gate", a CAR combination 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. If the at least two
target antigens are arbitrarily denoted as antigen A and antigen B,
the three possible options are as follows:
[0370] "OR GATE"--T cell triggers when either antigen A or antigen
B is present on the target cell
[0371] "AND GATE"--T cell triggers only when both antigens A and B
are present on the target cell
[0372] "AND NOT GATE"--T cell triggers if antigen A is present
alone on the target cell, but not if both antigens A and B are
present on the target cell
[0373] Engineered T cells expressing these CAR combinations can be
tailored to be exquisitely specific for cancer cells, based on
their particular expression (or lack of expression) of two or more
markers.
[0374] Such "Logic Gates" are described, for example, in
WO2015/075469, WO2015/075470 and WO2015/075470.
[0375] An "OR Gate" comprises two or more activatory CARs each
directed to a distinct target antigen expressed by a target cell.
The advantage of an OR gate is that the effective targetable
antigen is increased on the target cell, as it is effectively
antigen A+antigen B. This is especially important for antigens
expressed at variable or low density on the target cell, as the
level of a single antigen may be below the threshold needed for
effective targeting by a CAR-T cell. Also, it avoids the phenomenon
of antigen escape. For example, some lymphomas and leukemias become
CD19 negative after CD19 targeting: using an OR gate which targets
CD19 in combination with another antigen provides a "back-up"
antigen, should this occur. The "back up" antigen may be CD22, as
described in WO2016/102965.
[0376] Activity Modulator
[0377] In the method of the present invention at least one vector
in the mixture of viral vectors may comprise a nucleic acid
sequence which encodes an activity modulator. When this is the
case, at least a proportion of the transduced cells in the
CAR-expressing cell composition of the invention will express one
or more activity modulator(s). An activity modulator is a molecule
made by the CAR-expressing cell which modulates the activity of the
CAR, of a cell expressing the CAR, or of a target cell.
[0378] An activity modulator may be an intracellular molecule,
expressed at the cell surface, or secreted by the CAR-expressing
cell.
[0379] Modulating the Activity of the CAR
[0380] 1. Enhancing ITAM Phosphorylation
[0381] During T cell activation in vivo (illustrated schematically
in FIG. 2a), antigen recognition by the T-cell receptor (TCR)
results in phosphorylation of Immunoreceptor tyrosine-based
activation motifs (ITAMs) on CD3.zeta.. Phosphorylated ITAMs are
recognized by the ZAP70 SH2 domains, leading to T cell
activation.
[0382] T-cell activation uses kinetic segregation to convert
antigen recognition by a TCR into downstream activation signals.
Briefly: at the ground state, the signalling components on the
T-cell membrane are in dynamic homeostasis whereby dephosphorylated
ITAMs are favoured over phosphorylated ITAMs. This is due to
greater activity of the transmembrane CD45/CD148 phosphatases over
membrane-tethered kinases such as Ick. When a T-cell engages a
target cell through a T-cell receptor (or CAR) recognition of
cognate antigen, tight immunological synapses form. This close
juxtapositioning of the T-cell and target membranes excludes
CD45/CD148 due to their large ectodomains which cannot fit into the
synapse. Segregation of a high concentration of T-cell receptor
associated ITAMs and kinases in the synapse, in the absence of
phosphatases, leads to a state whereby phosphorylated ITAMs are
favoured. ZAP70 recognizes a threshold of phosphorylated ITAMs and
propagates a T-cell activation signal.
[0383] The process is essentially the same during CAR-mediated
T-cell activation. An activating CAR comprises one or more ITAM(s)
in its intracellular signalling domain, usually because the
signalling domain comprises the endodomain of CD3.zeta.. Antigen
recognition by the CAR results in phosphorylation of the ITAM(s) in
the CAR signalling domain, causing T-cell activation.
[0384] As illustrated schematically in FIG. 2b, inhibitory
immune-receptors such as PD1 cause the dephosphorylation of
phosphorylated ITAMs. PD1 has ITIMs in its endodomain which are
recognized by the SH2 domains of molecules such as PTPN6 (SHP-1)
and SHP-2. Upon recognition, PTPN6 is recruited to the
juxta-membrane region and its phosphatase domain subsequently
de-phosphorylates ITAM domains inhibiting immune activation.
[0385] An activity modulator capable of modulating the activity of
the CAR may be capable of directly or indirectly phosphorylating
the ITAM(s) in the CAR signalling domain.
[0386] 1.1 Providing or Recruiting Kinase
[0387] For example, the activity modulator may be a membrane
targeted molecule which either comprises a kinase domain or is
capable of recruiting a separate molecule comprising a kinase
domain to the vicinity of the CAR. WO2018/193231 describes various
molecules having such a "phosphorylation amplifying
endodomain".
[0388] An activity modulator capable of directly phosphorylating
ITAMs may comprise a tyrosine kinase domain, such as a kinase
domain of a Src family kinase, examples of which include Fyn, Src,
Lck or a derivative thereof such as Lck (Y505F). The tyrosine
kinase domains of Fyn, Src, Lck and Lck (Y505) are shown below as
SEQ ID Nos. 18-21 respectively.
TABLE-US-00015 Tyrosine kinase domain Fyn (SEQ ID NO: 18)
LQLIKRLGNGQFGEVWMGTWNGNTKVAIKTLKPGTMSPESFLEEAQIMKK
LKHDKLVQLYAVVSEEPIYIVTEYMNKGSLLDFLKDGEGRALKLPNLVDM
AAQVAAGMAYIERMNYIHRDLRSANILVGNGLICKIADFGLARLIEDNEY
TARQGAKFPIKWTAPERALYGRFTIKSDVWSFGILLTELVTKGRVPYPGM
NNREVLEQVERGYRMPCPQDCPISLHELMIHCWKKDPEERPTFEYLQSFL EDYF Tyrosine
kinase domain of Src (SEQ ID NO: 19)
LRLEVKLGQGCFGEVWMGTWNGTTRVAIKTLKPGTMSPEAFLQEAQVMKK
LRHEKLVQLYAVVSEEPIYIVTEYMSKGSLLDFLKGETGKYLRLPQLVDM
AAQIASGMAYVERMNYVHRDLRAANILVGENLVCKVADFGLARLIEDNEY
TARQGAKFPIKWTAPEAALYGRFTIKSDVWSFGILLTELTTKGRVPYPGM
VNREVLDQVERGYRMPCPPECPESLHDLMCQCWRKEPEERPTFEYLQAFL EDYF Tyrosine
kinase domain of Lck (SEQ ID NO: 20):
LKLVERLGAGQFGEVWMGYYNGHTKVAVKSLKQGSMSPDAFLAEANLMKQ
LQHQRLVRLYAVVTQEPIYIITEYMENGSLVDFLKTPSGIKLTINKLLDM
AAQIAEGMAFIEERNYIHRDLRAANILVSDTLSCKIADFGLARLIEDNEY
TAREGAKFPIKWTAPEAINYGTFTIKSDVWSFGILLTEIVTHGRIPYPGM
TNPEVIQNLERGYRMVRPDNCPEELYQLMRLCWKERPEDRPTFDYLRSVL EDFF Tyrosine
kinase domain of Lck_Y505F (SEQ ID NO: 21)
LKLVERLGAGQFGEVWMGYYNGHTKVAVRSLKQGSMSPDAFLAEANLMKQ
LQHQRLVRLYAVVTQEPIYIITEYMENGSLVDFLKTPSGIKLTINKLLDM
AAQIAEGMAFIEERNYIHRDLRAANILVSDTLSCKIADFGLARLIEDNEY
TAREGAKFPIKWTAPEAINYGTFTIKSDVWSFGILLTEIVTHGRIPYPGM
TNPEVIQNLERGYRMVRPDNCPEELYQLMRLCWKERPEDRPTFDYLRSVL EDFF
[0389] An activity modulator capable of indirectly phosphorylating
ITAMs may comprise the intracellular domain of CD4 or CD8
coreceptor.
[0390] As mentioned above, during T-cell activation, the ITAMs of
CD3 (or the CAR) are phosphorylated by a Lck and then bound by
ZAP70. After ZAP70 binds to CD3, co-receptors CD4 or CD8 become
associated with the TCR/CD3 complex and bind to the major
compatibility complex (MHC). CD4/CD8 co-receptor association with
the complex stabilises the TCR-MHC peptide (MHCp) interaction and
the recruited/free Lck continues the phosphorylation of CD3
elements, ZAP70, as well as many other downstream targets.
[0391] An activity modulator comprising the cytoplasmic tail of CD4
and CD8 will amplify the signal generated by the CAR by recruiting
Lck, which is essential for activating many molecular components of
the signaling cascade of an activated T cell. The sequences of the
intracellular domain of human CD4 and CD8 are shown below as SEQ ID
No. 22 and 23
TABLE-US-00016 Cytoplasmic tail of CD4 (SEQ ID NO: 22)
CVRCRHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTCSPI Cytoplasmic tail of CD8
(SEQ ID NO: 23) LYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV
[0392] An activity modulator which modulated the activity of the
CAR may be membrane-tethered. In this respect, such an activity
modulator may comprise a transmembrane domain or a myristoylation
sequence.
[0393] Modulating the Activity of the CAR-T Cell
[0394] 1. Checkpoint Inhibition
[0395] An activity modulator capable of modulating the activity of
the CAR-expressing cell may block or reduce the inhibition of
CAR-mediated T-cell activation mediated by inhibitory
immunoreceptors such as CTLA4, PD-1, LAG-3, 2B4 or BTLA 1 (as
mentioned above and illustrated schematically in FIG. 2b).
[0396] The activity modulator may be an agent, such as an antibody,
which binds to an inhibitory immunoreceptor or binds to a ligand
for an inhibitory immunreceptor. The activity modulator may bind to
CTLA4, PD-1, LAG-3, 2B4 or BTLA 1, or bind to a ligand for CTLA4,
PD-1, LAG-3, 2B4 or BTLA 1.
[0397] PD-1/PD-L1
[0398] In the cancer disease state, the interaction of PD-L1 on the
tumour cells with PD-1 on a T-cell reduces T-cell activation, as
described above, thus hampering the immune system in its efforts to
attack the tumour cells. Use of an inhibitor that blocks the
interaction of PD-L1 with the PD-1 receptor can prevent the cancer
from evading the immune system in this way. Several PD-1 and PD-L1
inhibitors are being trialled within the clinic for use in advanced
melanoma, non-small cell lung cancer, renal cell carcinoma, bladder
cancer and Hodgkin lymphoma, amongst other cancer types. Some such
inhibitors are now approved, including the PD1 inhibitors Nivolumab
and Pembrolizumab and the PD-L1 inhibitors Atezolizumab, Avelumab
and Durvalumab.
[0399] CTLA4
[0400] CTLA4 is a member of the immunoglobulin superfamily that is
expressed by activated T cells and transmits an inhibitory signal
to T cells. CTLA4 is homologous to the T-cell co-stimulatory
protein, CD28, and both molecules bind to CD80 and CD86, also
called B7-1 and B7-2 respectively, on antigen-presenting cells.
CTLA-4 binds CD80 and CD86 with greater affinity and avidity than
CD28 thus enabling it to outcompete CD28 for its ligands. CTLA4
transmits an inhibitory signal to T cells, whereas CD28 transmits a
stimulatory signal.
[0401] Antagonistic antibodies against CTLA4 include ipilimumab and
tremelimumab.
[0402] LAG-3
[0403] Lymphocyte-activation gene 3, also known as LAG-3 and CD223,
is an immune checkpoint receptor with diverse biologic effects on
T-cell function.
[0404] Antibodies to LAG3 include relatlimab, which currently in
phase 1 clinical testing and a number of others in preclinical
development. LAG-3 may be a better checkpoint inhibitor target than
CTLA-4 or PD-1 since antibodies to these two checkpoints only
activate effector T cells, and do not inhibit Treg activity,
whereas an antagonist LAG-3 antibody can both activate T effector
cells (by downregulating the LAG-3 inhibiting signal into
pre-activated LAG-3+ cells) and inhibit induced (i.e.
antigen-specific) Treg suppressive activity. Combination therapies
are also ongoing involving LAG-3 antibodies and CTLA-4 or PD-1
antibodies.
[0405] 1.2 Dominant Negative SHP
[0406] An activity modulator which blocks or reduces the inhibition
mediated by inhibitory immunoreceptors such as CTLA4, PD-1, LAG-3,
2B4 or BTLA 1 may tip the balance of
phosphorylation:dephosporylation at the T-cell:target cell synapse
in favour of phosphorylation of ITAMs, leading to T-cell
activation. For example, the activity modulator may block or reduce
the phosphorylation of ITIMs in the endodmain of inhibitory
receptor(s) or may block or reduce the dephosphorylation of ITAMs
in the CAR signalling domain by proteins such as SHP-1 and
SHP-2.
[0407] WO2016/193696 describes various different types of protein
capable of modulating the balance of
phosphorylation:dephosporylation at the T-cell:target cell synapse.
For example, the activity modulator may comprise a truncated form
of SHP-1 or SHP-2 which comprises one or both SH2 domains, but
lacks the phosphatase domain. When expressed in a CAR-T cell, these
molecules act as dominant negative versions of wild-type SHP-1 and
SHP-2 and compete with the endogenous molecule for binding to
phosphorylated ITIMs.
[0408] The activity modulator may be 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. The truncated protein may comprise one or both
SHP-1 SH2 domain(s) but lack the SHP-1 phosphatase domain.
Alternatively the truncated protein may comprise one or both SHP-2
SH2 domain(s) but lack the SHP-2 phosphatase domain.
[0409] SHP-1
[0410] Src homology region 2 domain-containing phosphatase-1
(SHP-1) is a member of the protein tyrosine phosphatase family. It
is also known as PTPN6.
[0411] The N-terminal region of SHP-1 contains two tandem SH2
domains which mediate the interaction of SHP-1 and its substrates.
The C-terminal region contains a tyrosine-protein phosphatase
domain.
[0412] SHP-1 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.
[0413] Human SHP-1 protein has the UniProtKB accession number
P29350.
[0414] An activity modulator may comprise or consist of the SHP-1
tandem SH2 domain which is shown below as SEQ ID NO: 24.
TABLE-US-00017 SHP-1 SH2 complete domain (SEQ ID NO: 24)
MVRWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQV
THIRIQNSGDFYDLYGGEKFATLTELVEYYTQQQGVLQDRDGTIIHLKY
PLNCSDPTSERWYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVL
SVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKK
TGIEEASGAFVYLRQPYY
[0415] SHP-1 has two SH2 domains at the N-terminal end of the
sequence, at residues 4-100 and 110-213. An activity modulator may
comprise one or both of the sequences shown as SEQ ID No. 25 and
26.
TABLE-US-00018 SHP-1 SH2 1 (SEQ ID NO: 25)
WFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVTHI
RIQNSGDFYDLYGGEKFATLTELVEYYTQQQGVLQDRDGTIIHLKYPL SHP-2 SH2 2 (SEQ
ID No. 26) WYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGP
GSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFV YLRQPY
[0416] The activity modulator may comprise a variant of SEQ ID NO:
24, 25 or 26 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 SHP-1.
[0417] SHP-2
[0418] SHP-2, also known as PTPN11, PTP-1D and PTP-2C 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.
[0419] Human SHP-2 has the UniProtKB accession number P35235-1.
[0420] An activity modulator may comprise or consist of the SHP-1
tandem SH2 domain which is shown below as SEQ ID NO: 29. SHP-1 has
two SH2 domains at the N-terminal end of the sequence, at residues
6-102 and 112-216. An activity modulator may comprise one or both
of the sequences shown as SEQ ID No. 27 and 28.
TABLE-US-00019 SHP-2 first SH2 domain (SEQ ID NO: 27)
WFHPNITGVEAENLLLTRGVDGSFLARPSKSNPGDFTLSVRRNGAVTHI
KIQNTGDYYDLYGGEKFATLAELVQYYMEHHGQLKEKNGDVIELKYPL SHP-2 second SH2
domain (SEQ ID No. 28)
WFHGHLSGKEAEKLLTEKGKHGSFLVRESQSHPGDFVLSVRTGDDKGES
NDGKSKVTHVMIRCQELKYDVGGGERFDSLTDLVEHYKKNPMVETLGTV LQLKQPL SHP-2
both SH2 domains (SEQ ID No. 29)
WFHPNITGVEAENLLLTRGVDGSFLARPSKSNPGDFTLSVRRNGAVTHI
KIQNTGDYYDLYGGEKFATLAELVQYYMEHHGQLKEKNGDVIELKYPLN
CADPTSERWFHGHLSGKEAEKLLTEKGKHGSFLVRESQSHPGDFVLSVR
TGDDKGESNDGKSKVTHVMIRCQELKYDVGGGERFDSLTDLVEHYKKNP
MVETLGTVLQLKQPL
[0421] The activity modulator may comprise a variant of SEQ ID NO:
27, 28 or 29 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 SHP-2.
[0422] 3. Cytokines and Cytokine Signalling
[0423] An activity modulator may be a cytokine or chemokine. A
cytokine may modulate the activity of the CAR-expressing cell
and/or modulate the tumour microenvironment.
[0424] The activity modulator may be a cytokine or chemokine be
selected from: IL12, flexiIL12, GM-CSF, IL7, IL15, IL21, IL2 and
CCL19. In particular, the agent may be IL-7 or IL-12.
[0425] IL-7 is a cytokine important for B and T cell development.
IL-7 stimulates the differentiation of multipotent (pluripotent)
hematopoietic stem cells into lymphoid progenitor cells and
stimulates proliferation of all cells in the lymphoid lineage (B
cells, T cells and NK cells.
[0426] Il-7 and the hepatocyte growth factor (HGF) form a
heterodimer that functions as a pre-pro-B cell growth-stimulating
factor. This cytokine is found to be a cofactor for V(D)J
rearrangement of the T cell receptor beta (TCR ) during early T
cell development. The amino acid sequence of human 11-7 is
available from UniProt (Accession No. P13232)
[0427] Interleukin 12 (IL-12) is a potent immunomodulatory cytokine
of particular interest for modulating the tumour microenvironment
redirecting the immune response against cancer. IL-12 is
systemically toxic therefore methods for producing IL-12 locally
are of interest. PCT/GB2018/052204 describes a construct where
production of an immunomodulatory cytokine such as IL-12 is under
the control of a promoter which is activated in the presence of an
environmental metabolite, such as kynurenine. Selective production
of IL-12 in the presence of an metabolite such as kynurenine
enables local production of IL-12 by the CAR- or TCR-expressing
cell, only when it is present in the tumour microenvironment.
[0428] Alternatively, the immunomodulatory cytokine may be placed
downstream of a frame-slip motif or a translational readthrough
motif. This provides a means of controlling cytokine expression and
reducing the level of expression of cytokine relative to the
CAR.
[0429] A frame-slip motif (FSM) may comprise a repeat of uracil,
thymine or guanine bases, such as the sequence UUUUUUU (SEQ ID No.
61).
[0430] A frame-slip motif may also comprise a stop codon. For
example, a FSM may comprise one of the following sequences:
TABLE-US-00020 (SEQ ID NO. 62) UUUUUUUGA (SEQ ID NO. 63) UUUUUUUAG
(SEQ ID NO. 64) UUUUUUUAA.
[0431] A translational readthrough motif (TRM) may comprise the
sequence STOP-CUAG or STOP-CAAUUA, in which "STOP" is a stop codon.
For example, a translational readthrough motif may comprise one of
the following sequences:
TABLE-US-00021 (SEQ ID No. 65) UGA-CUAG (SEQ ID No. 66) UAG-CUAG
(SEQ ID No. 67) UAA-CUAG (SEQ ID No. 68) UGA-CAAUUA (SEQ ID No. 69)
UAG-CAAUUA (SEQ ID No. 70) UAA-CAAUUA
[0432] IL-12 is a heterodimeric cytokine encoded by two separate
genes, IL-12A (p35) and IL-12B (p40). The active heterodimer
(referred to as `p70`), is formed following protein synthesis. The
activity modulator may be "flexi-IL12", which is a fusion between
the IL-12.alpha. and IL-12.beta. subunits, joined by a linker. A
suitable flexi-IL-12 sequence is shown below as SEQ ID No. 81.
TABLE-US-00022 (a flexi-IL-12 sequence) SEQ ID No. 81
MWIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVL
GSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILK
DQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQG
VTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVH
KLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTP
HSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRY
YSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPLATPDPGMFPCLHHSQ
NLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELT
KNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDSKMYQVEFKT
MNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFY
KTKIKLCILLHAFRIRAVTIDRVMSYLNAS
[0433] In SEQ ID No. 81, the serine-glycine linker is in bold and
underlined.
[0434] The activity modulator is the present invention may be a
cytokine which is selectively expressed depending the presence of
an environmental metabolite in the microenvironment of the cell.
The environmental metabolite may activate the aryl hydrocarbon
receptor (AHR). The environmental metabolite may be a tryptophan
metabolite such as is kynurenine.
[0435] Alternatively, the agent may affect the expression or
activity of a cytokine or chemokine. For example, the agent may be
a dominant negative version of a cytokine or chemokine. A dominant
negative version may, for example, be a mutated or truncated
version of the cytokine/chemokine which binds to the receptor and
competes with the wild-type cytokine/chemokine but does not trigger
cytokine/chemokine signalling.
[0436] For example, the agent may be a dominant negative version of
a cytokine receptor or chemokine receptor. A dominant negative
version may, for example, be a mutated or truncated version of the
cytokine/chemokine receptor which binds to the cytokine blocking
its binding to the wild-type cytokine/chemokine receptor.
[0437] Alternatively, the agent may be an antibody or antibody
fragment which blocks or otherwise modulates a cytokine or
chemokine signalling pathway.
[0438] The activity modulator may be a chimeric cytokine receptor
which comprises a cytokine receptor endodomain.
[0439] The activity modulator may comprise the exodomain from an
immunoinhibitory cytokine, such as IL-4, fused to the endodomain
from a cytokine such as IL-7 which enhances T-cell proliferation
(Leen et al (2014) Mol. Ther. 22:1211-1220).
[0440] The activity modulator may be a chemokine such as CCL19.
Chemokine (C-C motif) ligand 19 (CCL19) is a small cytokine
belonging to the CC chemokine family that is also known as EBI1
ligand chemokine (ELC) and macrophage inflammatory protein-3-beta
(MIP-3-beta). CCL19 elicits its effects on its target cells by
binding to the chemokine receptor chemokine receptor CCR7. It
attracts certain cells of the immune system, including dendritic
cells and antigen-engaged B cells and CCR7+ central-memory T-Cells.
The amino acid sequence for human CCL19 is available from UniProt
(Accession number Q99731).
[0441] 3.1 Chimeric Cytokine Receptors
[0442] Alternatively, the activity modulator may comprise a
non-cytokine receptor exodomain. WO2017/029512 describes chimeric
cytokine receptors (CCR) comprising: an exodomain which binds to a
ligand selected from a tumour secreted factor, a chemokine and a
cell-surface antigen; and a cytokine receptor endodomain.
[0443] The chimeric cytokine receptor may comprise two
polypeptides: [0444] (i) a first polypeptide which comprises:
[0445] (a) a first antigen-binding domain which binds a first
epitope of the ligand [0446] (b) a first chain of the cytokine
receptor endodomain; and [0447] (ii) a second polypeptide which
comprises: [0448] (a) a second antigen-binding domain which binds a
second epitope of the ligand (b) a second chain of the
cytokine-receptor endodomain.
[0449] Alternatively the chimeric cytokine receptor which comprises
two polypeptides: [0450] (i) a first polypeptide which comprises:
[0451] (a) a heavy chain variable domain (VH) [0452] (b) a first
chain of the cytokine receptor endodomain; and [0453] (ii) a second
polypeptide which comprises: [0454] (a) a light chain variable
domain (VL) [0455] (b) a second chain of the cytokine-receptor
endodomain.
[0456] For example, the cytokine receptor endodomain may
comprise:
[0457] (i) IL-2 receptor .beta.-chain endodomain
[0458] (ii) IL-7 receptor .alpha.-chain endodomain;
[0459] (iii) IL-15 receptor .alpha.-chain endodomain; or
[0460] (iv) common .gamma.-chain receptor endodomain.
[0461] The cytokine receptor endodomain may comprise (i), (ii) or
(iii); and (iv).
[0462] The cytokine receptor endodomain may comprise the
.alpha.-chain endodomain and the .beta.-chain endodomain from
granulocyte-macrophage colony-stimulating factor receptor
(GMCSF-R)
[0463] The ligand may be a tumour secreted factor, for example a
tumour secreted factor selected from: prostate-specific antigen
(PSA), carcinoembryonic antigen (CEA), vascular endothelial growth
factor (VEGF) and CA125.
[0464] The ligand may be a chemokine, for example a chemokine
selected from chemokine selected from: CXCL12, CCL2, CCL4, CCL5 and
CCL22.
[0465] The ligand may be a cell-surface molecule, such as a
transmembrane protein. The ligand may be, for example, CD22.
[0466] Constitutively Active Chimeric Cytokine Receptors
[0467] The activity modulator may be a constitutively active
chimeric cytokine receptor. The activity modulator may comprise two
chains which dimerise, either spontaneously or in the presence of
an agent (a chemical inducer of dimerization or CID) bringing
together two cytokine receptor endodomains.
[0468] The activity modulator may therefore comprise a dimerization
domain; and a cytokine receptor endodomain.
[0469] Dimerisation may occur spontaneously, in which case the
chimeric transmembrane protein will be constitutively active.
Alternatively, dimerization may occur only in the presence of a
chemical inducer of dimerization (CID) in which case the
transmembrane protein only causes cytokine-type signalling in the
presence of the CID.
[0470] Suitable dimerization domains and CIDs are described in
WO2015/150771, the contents of which are hereby incorporated by
reference.
[0471] For example, one dimerization domain may comprise the
rapamycin binding domain of FK-binding protein 12 (FKBP12), the
other may comprise the FKBP12-Rapamycin Binding (FRB) domain of
mTOR; and the CID may be rapamycin or a derivative thereof.
[0472] One dimerization domain may comprise the FK506 (Tacrolimus)
binding domain of FK-binding protein 12 (FKBP12) and the other
dimerization domain may comprise the cyclosporin binding domain of
cylcophilin A; and the CID may be an FK506/cyclosporin fusion or a
derivative thereof.
[0473] One dimerization domain may comprise an oestrogen-binding
domain (EBD) and the other dimerization domain may comprise a
streptavidin binding domain; and the CID may be an estrone/biotin
fusion protein or a derivative thereof.
[0474] One dimerization domain may comprise a
glucocorticoid-binding domain (GBD) and the other dimerization
domain may comprise a dihydrofolate reductase (DHFR) binding
domain; and the CID may be a dexamethasone/methotrexate fusion
protein or a derivative thereof.
[0475] One dimerization domain may comprise an 06-alkylguanine-DNA
alkyltransferase (AGT) binding domain and the other dimerization
domain may comprise a dihydrofolate reductase (DHFR) binding
domain; and the CID may be an 06-benzylguanine
derivative/methotrexate fusion protein or a derivative thereof.
[0476] One dimerization domain may comprise a retinoic acid
receptor domain and the other dimerization domain may comprise an
ecodysone receptor domain; and the CID may be RSL1 or a derivative
thereof.
[0477] Where the dimerization domain spontaneously heterodimerizes,
it may be based on the dimerization domain of an antibody. In
particular it may comprise the dimerization portion of a heavy
chain constant domain (CH) and a light chain constant domain (CL).
The "dimerization portion" of a constant domain is the part of the
sequence which forms the inter-chain disulphide bond.
[0478] The chimeric cytokine receptor may comprise the Fab portion
of an antibody as exodomain. In this respect, the chimeric antigen
may comprise two polypeptides: [0479] (i) a first polypeptide which
comprises: [0480] (a) a heavy chain constant domain (CH) [0481] (b)
a first chain of the cytokine receptor endodomain; and [0482] (ii)
a second polypeptide which comprises: [0483] (a) a light chain
constant domain (CL) [0484] (b) a second chain of the
cytokine-receptor endodomain.
[0485] The cytokine receptor endodomain may comprise: [0486] (i)
IL-2 receptor .beta.-chain endodomain [0487] (ii) IL-7 receptor
.alpha.-chain endodomain; or [0488] (iii) IL-15 receptor
.alpha.-chain endodomain; and/or [0489] (iv) common .gamma.-chain
receptor endodomain.
[0490] The cytokine receptor endodomain may comprise the
.alpha.-chain endodomain and the .beta.-chain endodomain from
granulocyte-macrophage colony-stimulating factor receptor
(GMCSF-R)
[0491] A constitutively active CCR having an IL-2, IL-7 or GM-CSF
receptor endodomain may have one of the following structures:
[0492] Fab_CCR_IL2:
HuLightKappa-IL2RgTM-IL2RgEndo-2A-HuCH1-IL2bTM-IL2RbENDO
[0493] Fab_CCR_I L7:
HuLightKappa-IL2RgTM-IL2RgEndo-2A-HuCH1-IL7RaTM-IL7RaENDO
[0494] Fab_CCR_GMCSF:
[0495]
HuLightKappa-GMCSFRbTM-GMCSFRbEndo-2A-HuCH1-GMCSFRaTM-GMCSFRaENDO
[0496] In which:
[0497] HuLightKappa is a human light kappa chain
[0498] IL2RgTM is a transmembrane domain from human IL2R common
gamma chain
[0499] IL2RgEndo is an endodomain derived from human IL2R common
gamma chain
[0500] 2A is a sequence enabling the co-expression of the two
polypeptides, which may be a self cleaving peptide such as a 2A
peptide
[0501] HuCH1 is a human CH1
[0502] IL2bTM is a transmembrane domain from human IL-2R beta
[0503] IL2RbENDO is an endodomain from human IL2R beta
[0504] IL7RaTM is a transmembrane domain from human IL-7R alpha
[0505] IL7RaENDO is an endodomain from human IL-7R alpha
[0506] GMCSFRbTM is a transmembrane domain from Human GM-CSFR
common beta chain
[0507] GMCSFRbEndo is an endodomain from GM-CSFR common beta
chain
[0508] GMCSFRaTM is a transmembrane domain from Human GF-CSFR
alpha
[0509] GMCSFRaENDO is an endodomain Derived from Human GM-CSFR
alpha
[0510] The sequences for the components for making a constitutively
active cytokine receptor as shown below as SEQ ID NO. 30 to 43.
TABLE-US-00023 (Human Light Kappa Chain) SEQ ID No. 30
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC (Human
Hinge) SEQ ID No. 31 EPKSCDKTHTCPPCPKDPK (Human CH1) SEQ ID No. 32
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV (Transmembrane
domain from human IL2R common gamma chain): SEQ ID No. 33
VVISVGSMGLIISLLCVYFWL (Transmembrane domain from human IL-2R beta)
SEQ ID No. 34 IPWLGHLLVGLSGAFGFIILVYLLI (Transmembrane domain from
human IL-7R alpha) SEQ ID No. 36 PILLTISILSFFSVALLVILACVLW
(Transmembrane domain from Human GF-CSFR alpha) SEQ ID No. 37
NLGSVYIYVLLIVGTLVCGIVLGFLF (Transmembrane domain from Human GM-CSFR
common beta chain) SEQ ID No. 38 VLALIVIFLTIAVLLAL (Endodomain from
human IL2R common gamma chain) SEQ ID No. 39
ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLV
SEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET (Endodomain from human IL-2R
beta) SEQ ID No. 40
NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFS
PGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQG
YFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLS
GEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQE
RVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVS
FPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV (Endodomain from human
IL-7R alpha) SEQ ID No. 41
KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDD
IQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFG
RDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTT
NSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNEQ (Endodomain
Derived from Human GM-CSFR alpha) SEQ ID No. 42
KRFLRIQRLFPPVPQIKDKLNDNEVEDEIIWEEFTPEEGKGYREEVLTV KEIT (Endodomain
from GM-CSFR common beta chain) SEQ ID No. 43
RFCGIYGYRLRRKWEEKIPNPSKSHLFQNGSAELWPPGSMSAFTSGSPP
HQGPWGSRFPELEGVFPVGFGDSEVSPLTIEDPKHVCDPPSGPDTTPAA
SDLPTEQPPSPQPGPPAASHTPEKQASSFDFNGPYLGPPHSRSLPDILG
QPEPPQEGGSQKSPPPGSLEYLCLPAGGQVQLVPLAQAMGPGQAVEVER
RPSQGAAGSPSLESGGGPAPPALGPRVGGQDQKDSPVAIPMSSGDTEDP
GVASGYVSSADLVFTPNSGASSVSLVPSLGLPSDQTPSLCPGLASGPPG
APGPVKSGFEGYVELPPIEGRSPRSPRNNPVPPEAKSPVLNPGERPADV
SPTSPQPEGLLVLQQVGDYCFLPGLGPGPLSLRSKPSSPGPGPEIKNLD
QAFQVKKPPGQAVPQVPVIQLFKALKQQDYLSLPPWEVNKPGEVC
[0511] The activity modulator may comprise a variant of one or more
of SEQ ID NO: 30 to 43 having at least 80, 85, 90, 95, 98 or 99%
sequence identity, provided that the variant sequence has the
required properties. For example, a variant CH or CL sequence
should retain the capacity to dimerise with a CL/CH
containing-chain. A variant chain from a cytokine receptor
endodomain should retain the capacity to trigger cytokine-mediated
signalling when coupled with the reciprocal chain for that cytokine
receptor.
[0512] 3.3 JAK/STAT
[0513] Signal Transducer and Activator of Transcription (STAT)
molecules are a family of transcription factors that are involved
in cytokine-mediated signal transduction. STAT transcription
factors are recruited to the cytoplasmic region of cell surface
receptors and are activated via phosphorylation. Once activated,
they dimerize to form an activated STAT molecule comprising a first
polypeptide and a second polypeptide, and translocate into the cell
nucleus where they influence gene expression. They play a role in
regulating cell growth processes and cell differentiation. The
JAK-STAT signalling pathway consists of three main components: (1)
a receptor which penetrates the cell membrane (2) Janus kinase
(JAK), which is bound to the receptor and (3) Signal Transducer and
Activator of Transcription (STAT), which carries the signal into
the nucleus and DNA (see FIG. 3).
[0514] It is possible to enhance engraftment and persistence of
CAR-expressing cells by including in the cell a constitutively
active or inducible active JAK or STAT molecule. International
Patent Application No. PCT/GB2018/052583 describes various
alternative arrangements for Constitutively active STAT molecules
(FIG. 4 of International Patent Application No. PCT/GB2018/052583)
and inducible STAT molecules (FIG. 5 of International Patent
Application No. PCT/GB2018/052583).
[0515] As described in International Patent Application No.
PCT/GB2018/052583, a constitutively active JAK molecule may be made
by expressing two JAK polypeptides which spontaneously dimerise or
are linked by a linker, as described below for constitutively
active STAT molecules. Alternatively, constitutively active JAK may
be expressed which comprises a gain-of-function mutation.
[0516] The activity modulator may be a constitutively active or an
inducible Signal Transducer and Activator of Transcription (STAT)
molecule.
[0517] The STAT molecule of the cell may a first STAT polypeptide
comprising a first dimerizing domain (DD) and a second STAT
polypeptide comprising a second DD, which binds to the first
DD.
[0518] The first and second DDs of the STAT molecule of the cell
may, for example, comprise leucine zipper domains; or a heavy chain
constant region and a light chain constant region.
[0519] An inducible STAT molecule of the cell may be inducibly
active in the presence of an agent which causes dimerization of the
first DD and second DD of the STAT molecule, thereby inducing
activation of the STAT molecule. For example, the first DD may
comprise FRB, the second DD may comprise FKBP12 and the agent may
be rapamycin.
[0520] Alternatively, the STAT molecule of the cell may be
inducibly inactive in the presence of an agent which causes
dissociation of the first DD and second DD of the STAT molecule,
thereby inducing non-activation of the STAT molecule. The first DD
may comprise TetRB and the second DD may comprise TiP and the agent
may be tetracycline, doxycycline or minocycline.
[0521] A constitutively active STAT may comprise a Gain of Function
(GOF) mutation or may comprise a first STAT polypeptide and a
second STAT polypeptide linked by a linker sequence.
[0522] The cell may comprise a membrane-tethering molecule
comprising a tethering domain and a first binding domain (BD), and
a constitutively active STAT molecule which comprises a second BD
which binds specifically to the first BD. Binding of the first and
second BD may be disrupted by the presence of an agent, such that
in the presence of the agent the constitutively active STAT
molecule dissociates from the membrane-tethering molecule, so that
the constitutively active STAT molecule is free to translocate to
the nucleus.
[0523] The first and second DD of the STAT molecule of the cell; or
the first BD of the membrane-tethering molecule of the cell and
second BD of the STAT molecule of the cell may comprise a Tet
Repressor Protein (TetR) and a Transcription Inducing Peptide
(TiP), respectively; and the agent may be tetracycline, doxycycline
or minocycline.
[0524] The cell may comprise a) a CAR and a constitutively active
STAT molecule joined by a STAT release domain and b) a STAT release
molecule which releases the constitutively active STAT molecule
from the CAR at the STAT release domain only upon recognition of a
target antigen specific to the CAR, such that upon release, the
constitutively active STAT molecule is free to translocate to the
nucleus.
[0525] The STAT release molecule may comprise a CAR targeting
domain, for example which binds to a phosphorylated immunoreceptors
tyrosine based activation motif (ITAM). For example, the CAR
targeting domain may comprise one or more ZAP70 SH2 domains.
[0526] The STAT release domain of the cell of the present invention
may comprise a protease cleavage site, and the STAT release
molecule of the cell may comprise a protease domain, such that upon
recognition of a target antigen of the CAR, the protease domain
cleaves at the protease cleavage site, releasing the STAT
molecule.
[0527] 4. Adhesion Molecule
[0528] Cell adhesion molecules (CAMs) are proteins located on the
cell surface involved in binding with other cells or with the
extracellular matrix (ECM) in cell adhesion.
[0529] These proteins are typically transmembrane receptors and are
composed of three domains: an intracellular domain that interacts
with the cytoskeleton, a transmembrane domain, and an extracellular
domain that interacts either with other CAMs of the same kind
(homophilic binding) or with other CAMs or the extracellular matrix
(heterophilic binding).
[0530] Most CAMs belong to four protein families: Ig
(immunoglobulin) superfamily (IgSF CAMs), the integrins, the
cadherins, and the selectins.
[0531] The activity modulator of the present invention may be or
comprise an adhesion molecule which modulates CAR- or
TCR-expressing cell activity.
[0532] 5. Transcription Factor
[0533] The agent of the invention may be or comprise a
transcription factor which modulates activity of the CAR- or
TCR-expressing cell.
[0534] A transcription factor is a protein which controls the rate
of transcription of genetic information from DNA to messenger RNA,
by binding to a specific DNA sequence and regulate the expression
of a gene which comprises or is adjacent to that sequence.
[0535] Transcription factors work by promoting (as an activator),
or blocking (as a repressor) the recruitment of RNA polymerase.
[0536] Transcription factors contain at least one DNA-binding
domain (DBD), which attaches to either an enhancer or promoter
region of DNA. Depending on the transcription factor, the
transcription of the adjacent gene is either up- or down-regulated.
Transcription factors also contain a trans-activating domain (TAD),
which has binding sites for other proteins such as transcription
coregulators.
[0537] Transcription factors use a variety of mechanisms for the
regulation of gene expression, including stabilizing or blocking
the binding of RNA polymerase to DNA, or catalyzing the acetylation
or deacetylation of histone proteins. The transcription factor may
have histone acetyltransferase (HAT) activity, which acetylates
histone proteins, weakening the association of DNA with histones
and making the DNA more accessible to transcription, thereby
up-regulating transcription. Alternatively the transcription factor
may have histone deacetylase (HDAC) activity, which deacetylates
histone proteins, strengthening the association of DNA with
histones and making the DNA less accessible to transcription,
thereby down-regulating transcription. Another mechanism by which
they may function is by recruiting coactivator or corepressor
proteins to the transcription factor DNA complex.
[0538] The transcription may be constitutively active or
conditionally active, i.e. requiring activation.
[0539] The transcription factor may be naturally occurring or
artificial.
[0540] 5.1 Transcriptional Reprogramming
[0541] In order for a CAR-T cell to be effective, it is important
that it persists and expands in vivo and resists overly rapid
differentiation and exhaustion. CAR T-cell persistence and
engraftment is related to the proportion of naive, central memory
and T-stem-cell memory T-cells administered.
[0542] WO2018/115865 describes a cell which co-expresses a chimeric
antigen receptor (CAR) and a transcription factor. Expression of
the transcription factor may prevent or reduce differentiation
and/or exhaustion of the cell in vitro and/or in vivo. By
co-expressing the CAR with a transcription factor in a cell, it is
possible to prevent or reduce differentiation and/or exhaustion of
the cell. This results in a greater proportion of naive, central
memory and stem-cell memory cells in the cell composition for
immunotherapy, and more effective persistence and expansion of the
cells in vivo.
[0543] The activity modulator of the present invention may be a
transcription factor. The transcription factor may prevent or
reduce differentiation and/or exhaustion of the cell.
[0544] The transcription factor may be an effector memory
repressor, such as BLIMP-1
[0545] Alternatively, the transcription factor may be a central
memory repressor such as BCL6 or Bach2.
[0546] The transcription factor may be or comprise Bach2 or a
modified version of Bach2 which has reduced or removed capacity to
be phosphorylated by ALK. For example, modified Bach2 may comprise
a mutation at one or more of the following positions: Ser-535,
Ser-509, Ser-520.
[0547] The transcription factor may be FOXO1, EOM ES, Runx3 or CBF
beta.
[0548] 6. Modulating TGF.beta. Signalling
[0549] Engineered cells face hostile microenvironments which limit
adoptive immunotherapy. One of the main inhibitory mechanisms
within the tumour microenvironment is transforming growth factor
beta (TGF.beta.). The TGF.beta. signalling pathway has a pivotal
role in the regulatory signalling that controls a variety of
cellular processes. TGF.beta. play also a central role in T cell
homeostasis and control of cellular function. Particularly,
TGF.beta. signalling is linked to an immuno-depressed state of the
T-cells, with reduced proliferation and activation. TGF.beta.
expression is associated with the immunosuppressive
microenvironment of tumour.
[0550] A variety of cancerous tumour cells are known to produce
TGF.beta. directly. In addition to the TGF.beta. production by
cancerous cells, TGF.beta. can be produced by the wide variety of
non-cancerous cells present at the tumour site such as
tumour-associated T cells, natural killer (NK) cells, macrophages,
epithelial cells and stromal cells.
[0551] The transforming growth factor beta receptors are a
superfamily of serine/threonine kinase receptors. These receptors
bind members of the TGF.beta. superfamily of growth factor and
cytokine signalling proteins. There are five type II receptors
(which are activatory receptors) and seven type I receptors (which
are signalling propagating receptors).
[0552] Auxiliary co-receptors (also known as type III receptors)
also exist. Each subfamily of the TGF.beta. superfamily of ligands
binds to type I and type II receptors.
[0553] The three transforming growth factors have many activities.
TGF.beta.1 and 2 are implicated in cancer, where they may stimulate
the cancer stem cell, increase fibrosis/desmoplastic reactions and
suppress immune recognition of the tumour.
[0554] TGF.beta.1, 2 and 3 signal via binding to receptors
T.beta.RII and then association to T.beta.RI and in the case of
TGF.beta.2 also to T.beta.RIII. This leads to subsequent signalling
through SMADs via T.beta.RI.
[0555] TGF.beta.s are typically secreted in the pre-pro-form. The
"pre" is the N-terminal signal peptide which is cleaved off upon
entry into the endoplasmic reticulum (ER). The "pro" is cleaved in
the ER but remains covalently linked and forms a cage around the
TGF.beta. called the Latency Associated Peptide (LAP). The cage
opens in response to various proteases including thrombin and
metalloproteases amongst others. The C-terminal region becomes the
mature TGF.beta. molecule following its release from the pro-region
by proteolytic cleavage. The mature TGF.beta. protein dimerizes to
produce an active homodimer.
[0556] The TGF.beta. homodimer interacts with a LAP derived form
the N-terminal region of the TGF.beta. gene product, forming a
complex called Small Latent Complex (SLC). This complex remains in
the cell until it is bound by another protein, an extracellular
matrix (ECM) protein called Latent TGF.beta. binding protein (LTBP)
which together forms a complex called the large latent complex
(LLC). LLC is secreted to the ECM. TGF.beta. is released from this
complex to a biologically active form by several classes of
proteases including metalloproteases and thrombin.
[0557] The activity modulator of the present invention may modulate
TGF.beta. signalling.
[0558] For example, the activity modulator may block or reduce
TGF.beta. binding to TGF.beta. receptor; it may compete with
TGF.beta. or TG.beta.R for binding to TG.beta.R or TGF.beta.;
alternatively it may modulate the downstream TGF.beta. signalling
for example via SMADs.
[0559] The activity modulator may be an agent such an antibody
which binds TGF.beta. or TGF.beta. receptor.
[0560] Fresolimumab is a immunomodulatory antibody which blocks
TGF.beta.1-3. Fresolimumab has been tested in metastatic melanoma
and high-grade glioma. This showed some effectiveness in the
enhancement of a tumour-specific immune response but failed to
eradicate the tumour. Other antibodies which bind TGF.beta. include
Lerdelimumab and Metelimumab.
[0561] Bedinger et al (2016) describes various human monoclonal
antibodies that neutralize multiple TGF.beta. isoforms (MAbs 8(2):
389-404)
[0562] Alternatively the activity modulator may be a recombinant
Fc-fusion proteins containing the soluble ectodomain of either
T.beta.R11 (T.beta.RII-Fc) or the type Ill receptor, betaglycan.
Soluble T.beta.RII and soluble T.beta.RIII (.beta.glycan) function
as decoy receptors preventing binding of TGF-.beta..
[0563] 6.1 Dominant Negative TGF.beta.
[0564] The activity modulator may be a secreted factor which is
capable of binding a transforming growth factor beta receptor
(T.beta.R) and disrupting its interaction with transforming growth
factor beta (TGF.beta.).
[0565] The activity modulator may be a dominant negative
TGF.beta..
[0566] "Dominant negative TGF.beta." or dnTGF.beta. as used herein
means that the secreted factor TGF.beta. acts antagonistically to
the wild-type TGF.beta..
[0567] The dominant negative TGF.beta. inhibits signalling induced
by wild-type TGF.beta. and thus neutralise its biological
effects.
[0568] The activity modulator may be a mutant TGF.beta..
[0569] The mature protein of wild-type TGF.beta.2 is shown below as
in SEQ ID NO: 44.
TABLE-US-00024 (SEQ ID NO: 44)
ALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACP
YLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKI
EQLSNMIVKSCKCS.
[0570] A mutant TGF.beta. may comprise one or more mutations at
amino acid residues W30, W32, L101, L51, Q67 and Y6 when the amino
acid number is determined by alignment with SEQ ID NO: 44 and
wherein:
[0571] amino acid residue 30 is mutated to N, R, K, D, Q, L, S, P,
V, I, G, C, T, A or E; and/or
[0572] amino acid residue 32 is mutated to A; and/or
[0573] amino acid residue 101 is mutated to A, E; and/or
[0574] amino acid residue 51 is mutated to Q, W, Y, A; and/or
[0575] amino acid residue 67 is mutated to H, F, Y, W, Y;
and/or
[0576] amino acid residue 6 is mutated to A or a variant
thereof.
[0577] Alternatively the activity modulator may comprise a
truncated TGF.beta. polypeptide such as monomeric TGF.beta.. Kim et
al (2017) describe an engineered TGF.beta. monomer that functions
as a dominant negative to block TGF.beta. signalling (J. Biol.
Chem. doi: 10.1074/jbc.M116.768754).
[0578] Truncated TGF.beta. may lack the heel helix .alpha.3, a
structural motif essential for binding the TGF.beta. type I
receptor (T.beta.RI) but dispensable for binding T.beta.RII.
[0579] The amino acid sequence of a TGF.beta. monomer is set forth
in SEQ ID NO: 45. SEQ ID NO: 45 comprises a signal peptide and a
latency associated peptide (LAP).
TABLE-US-00025 (SEQ ID NO: 45)
MHYCVLSAFLILHLVTVALSLSTCSTLDMDQFMRKRIEAIRGQILSKLK
LTSPPEDYPEPEEVPPEVISIYNSTRDLLQEKASRRAAACERERSDEEY
YAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKA
EFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEG
EWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEE
LEARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQ
QTNRRKKRALDAAYCFRNVQDNCCLRPLYIDFRKDLGWKWIHEPKGYNA
NFCAGACPYRASKSPSCVSQDLEPLTIVYYVGRKPKVEQLSNMIVKSCK CS.
[0580] The activity modulator may have an amino acid sequence set
forth in SEQ ID NO: 45 or a variant thereof. The variant TGF.beta.
monomer may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence
identity to SEQ ID NO: 45, provided that the polypeptide provides a
monomer which is capable of binding a transforming growth factor
beta receptor (T.beta.R) and disrupting its interaction with
transforming growth factor beta (TGF.beta.).
[0581] 6.2 Dominant Negative TGF.beta. Receptor
[0582] The active TGF.beta. receptor (T.beta.R) is a
hetero-tetramer, composed by two TGF.beta..pi.receptor I (T.beta.I)
and two TGF.beta. receptor II (T.beta.RII). TGF.beta.1 is secreted
in a latent form and is activated by multiple mechanisms. Once
activated it forms a complex with the T.beta.RII T.beta.RI that
phosphorylates and activates T.beta.RI.
[0583] The activity modulator may be a dominant negative TGF.beta.
receptor. A dominant negative TGF.beta. receptor may lack the
kinase domain.
[0584] For example, the activity modulator may comprise or consist
of the sequence shown as SEQ ID No. 46, which is a monomeric
version of TGF receptor II
TABLE-US-00026 (dn TGF.beta. RII) SEQ ID No. 46
TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNC
SITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPK
CIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTG
ISLLPPLGVAISVIIIFYCYRVNRQQKLSS
[0585] A dominant-negative TGF-.beta.RII (dnTGF-.beta.RII) has been
reported to enhance PSMA targeted CAR-T cell proliferation,
cytokine secretion, resistance to exhaustion, long-term in vivo
persistence, and the induction of tumour eradication in aggressive
human prostate cancer mouse models (Kloss et al (2018) Mol. Ther.
26:1855-1866).
[0586] 6.3 SMADs
[0587] As mentioned above, the active TGF.beta. receptor (T.beta.R)
is a hetero-tetramer, composed by two TGF.beta. receptor I
(T.beta.RI) and two TGF.beta. receptor II (T.beta.RII). Signaling
is initiated when activated TGF-.beta. binds to transforming growth
factor-.beta. receptor-2 (T.beta.RII) with high affinity. This
binding requires the participation of the transforming growth
factor-.beta. receptor-3 (T.beta.RIII), also known as .beta.glycan,
which causes a conformational change in T.beta.RII that facilitates
ligand-receptor binding TGF-.beta. receptor-1/ALK-5 (T.beta.R1), a
serine/threonine kinase, is then recruited to the
TGF-.beta./T.beta.RII complex and initiates signaling by
phosphorylating SMAD2 and SMAD3, which belong to the
receptor-regulated family of SMAD proteins. Phosphorylated SMAD2
and SMAD3 combine to form a heteromeric complex with SMAD4 that
translocates to the cell nucleus to interact with various
transcriptional factors that ultimately leads to the cellular
response
[0588] SMAD proteins are intracellular transcription factors for
conveying extracellular signal from membrane to the nucleus upon
the activation of TGF.beta.. Three types of SMAD have been
identified: a receptor-regulated SMAD s (R-SMAD) including SMAD 2
and SMAD 3 and a common-mediator SMAD (co-SMAD) including SMAD 4
only. Finally, an inhibitory SMAD s (I-SMAD) including SMAD 6 and
SMAD 7.
[0589] SMAD proteins consist of two globular domains connected by a
linker region. The main function of the SMAD N-terminal domain, or
"Mad homology 1" (MH1) domain, is to bind DNA. The C-terminal
domain, or MH2 domain, mediates protein-protein interaction with
numerous regulator and effector proteins, including the T.beta.R
receptors, certain cytoplasmic anchor proteins, lineage-specific
DNA-binding cofactors, and chromatin modifiers. In the presence of
TGF.beta., R-SMAD is phosphorylated by TGF.beta. receptor. This
phosphorylation targets two serine residues in the SMAD C terminus
sequence, pSer-X-pSer, creating an acidic tail that drives the
formation of SMAD transcriptional complexes. Missense mutation of
two conserved amino acid in the N terminal portion of the MH2
domain has been identified in patient with colorectal cancer. These
two mutations cause the acquisition of a dominant negative
behaviour that counteract the activity of the WT SMAD protein.
[0590] The activity modulator may be a SMAD signalling inhibitors
such as Galunisertib which has been tested as a monotherapy or in
combination with alkylating agents, Lomustine or temozolamide for
glioblastoma and other combinations.
[0591] Alternatively the activity modulator may be a dominant
negative version of the signal transduced SMAD 2 and SMAD 3 and
SMAD4 expressing only the MH2 domain. The activity modulator may
be: i) MH2, ii) MH2 truncated, missing the last 24 aa and iii)
truncated SMAD 2_MH2-linker-SMAD 3_MH2. These dominant negatives
compete with the wild type SMAD protein for the receptor-docking
domains and for the binding with partner proteins thus reducing or
blocking TGF.beta. signalling.
[0592] A dnSMAD may be selected from one or more of SMAD2, SMAD3
and or SMAD4. The dnSMAD lacks a functional MH1 domain.
[0593] The MH1 domain is a conserved MAD homology domain of at the
N terminus of a SMAD protein. The MH1 domain is capable of DNA
binding and negatively regulates the functions of the MH2
domain.
[0594] The MH2 domain is a conserved MAD homology domain of at the
C terminus of a SMAD protein. The MH2 domain contain a central
.beta.-sandwich with a conserved loop-helix is capable of binding
phospho-serine residues. The MH2 domain mediates protein:protein
interactions with regulator and effector proteins, including the
T.beta.R receptors, cytoplasmic anchor proteins, lineage-specific
DNA-binding cofactor and chromatin modifiers.
[0595] The activity modulator may comprise or consist essentially
of or consists of a wild-type MH2 domain from SMAD2, SMAD3 and
SMAD4. The amino acids sequences for these MH2 domains are shown
below as SEQ ID Nos 47 to 49.
TABLE-US-00027 (SEQ ID NO: 47-MH2 domain of SMAD2)
WCSIAYYELNQRVGETFHASQPSLTVDGFTDPSNSERFCLGLLSNVNRN
ATVEMTRRHIGRGVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPA
TVCKIPPGCNLKIFNNQEFAALLAQSVNQGFEAVYQLTRMCTIRMSFVK
GWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQMGSPSVRCSSMS (SEQ ID NO: 48-MH2
domain of SMAD3) WCSISYYELNQRVGETFHASQPSMTVDGFTDPSNSERFCLGLLSNVNRN
AAVELTRRHIGRGVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPA
TVCKIPPGCNLKIFNNQEFAALLAQSVNQGFEAVYQLTRMCTIRMSFVK
GWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQMGSPSIRCSSVS (SEQ ID NO: 49-MH2
domain of SMAD4) WCSIAYFEMDVQVGETFKVPSSCPIVTVDGYVDPSGGDRFCLGQLSNVH
RTEAIERARLHIGKGVQLECKGEGDVWVRCLSDHAVFVQSYYLDREAGR
APGDAVHKIYPSAYIKVFDLRQCHRQMQQQAATAQAAAAAQAAAVAGNI
PGPGSVGGIAPAISLSAAAGIGVDDLRRLCILRMSFVKGWGPDYPRQSI
KETPCWIElHLHRALQLLDEVLHTMPIADPQPLD
[0596] The activity modulator may comprise or consist essentially
of a truncated version of one of the MH2 domains outlined above,
with a deletion of up to 24 amino acids from the C terminus of the
wild type MH2 domain.
[0597] The activity modulator may comprises a mutation in the MH2
domain which increases the binding affinity of the dnSMAD for a
phosphorylated TGF.beta. receptor. The activity modulator may
consists essentially of or consists of: a MH2 domain of a SMAD4
polypeptide comprising a mutation R497H, K507Q and/or R515G where
the amino acid numbering corresponds to the numbering set forth in
SEQ ID NO: 49; an MH2 domain of SMAD3 comprising a mutation K378R
and/or K314R where the amino acid numbering corresponds to the
numbering set forth in SEQ ID NO:48.
[0598] The activity modulator may be a chimeric dnSMAD which
comprises at least two dnSMAD polypeptides as defined above. The
dnSMAD polypeptides of the chimeric dnSMAD may be connected by a
linker domain.
[0599] The amino acid sequence of a chimeric dnSMAD comprising a
dnSMAD2 polypeptide and a dnSMAD3 polypeptide is shown below as SEQ
ID No. 50.
TABLE-US-00028 (SEQ ID NO: 50)
WCSIAYYELNQRVGETFHASQPSLTVDGFTDPSNSERFCLGLLSNVNRN
ATVEMTRRHIGRGVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPA
TVCKIPPGCNLKIFNNQEFAALLAQSVNQGFEAVYQLTRMCTIRMSFVK
GWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQMLEYSGGGSGGGSL
EWCSISYYELNQRVGETFHASQPSMTVDGFTDPSNSERFCLGLLSNVNR
NAAVELTRRHIGRGVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHP
ATVCKIPPGCNLKIFNNQEFAALLAQSVNQGFEAVYQLTRMCTIRMSFV
KGWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQM
[0600] A dnSMAD or chimeric dnSMAD may comprise a sequence shown
as: SEQ ID NO: 47 to 50; or a variant having at least 80%
(preferably at least 85%, at least 90%, at least 95%, at least 97%,
or at least 99%) sequence identity to SEQ ID NO: 47 to 50 provided
that the variant sequence retains the capacity to compete with the
wild type SMAD protein for the receptor-docking domains and for the
binding with partner proteins and reduces or blocks TG.beta.
signalling.
[0601] 7. Metabolic Enzymes
[0602] The activity modulator may be a metabolic enzyme such as
AMP-activated protein kinase (AMPK) or Isocitrate dehydrogenase
(IDH)
[0603] AMPK plays a role in cellular energy homeostasis, largely to
activate glucose and fatty acid uptake and oxidation when cellular
energy is low.
[0604] AMPK is a heterotrimeric protein complex formed by a, 13,
and .gamma. subunits. Each of these three subunits takes on a
specific role in both the stability and activity of AMPK.
Specifically, the .gamma. subunit includes four particular
Cystathionine beta synthase (CBS) domains giving AMPK its ability
to sensitively detect shifts in the AMP:ATP ratio. The four CBS
domains create two binding sites for AMP commonly referred to as
Bateman domains. Binding of one AMP to a Bateman domain
cooperatively increases the binding affinity of the second AMP to
the other Bateman domain. As AMP binds both Bateman domains the
.gamma. subunit undergoes a conformational change which exposes the
catalytic domain found on the a subunit. It is in this catalytic
domain where AMPK becomes activated when phosphorylation takes
place at threonine-172 by an upstream AMPK kinase (AMPKK). The
.alpha., .beta., and .gamma. subunits can also be found in
different isoforms: the .gamma. subunit can exist as either the
.gamma.1, .gamma.2 or .gamma.3 isoform; the .beta. subunit can
exist as either the .beta.1 or .beta.2 isoform; and the a subunit
can exist as either the .alpha.1 or .alpha.2 isoform.
[0605] The following human genes encode AMPK subunits:
[0606] .alpha.--PRKAA1, PRKAA2
[0607] .beta.--PRKAB1, PRKAB2
[0608] .gamma.--PRKAG1, PRKAG2, PRKAG3
[0609] The activity modulator may comprise one or more AMPK
subunits. The activity modulator may comprise .alpha., .beta., and
.gamma. subunits from AMPK.
[0610] IDH catalyzes the oxidative decarboxylation of isocitrate,
producing alpha-ketoglutarate (.alpha.-ketoglutarate) and CO.sub.2.
This is a two-step process, which involves oxidation of isocitrate
(a secondary alcohol) to oxalosuccinate (a ketone), followed by the
decarboxylation of the carboxyl group beta to the ketone, forming
alpha-ketoglutarate. In humans, IDH exists in three isoforms: IDH3
catalyzes the third step of the citric acid cycle while converting
NAD+ to NADH in the mitochondria. The isoforms IDH1 and IDH2
catalyze the same reaction outside the context of the citric acid
cycle and use NADP+ as a cofactor instead of NAD+. They localize to
the cytosol as well as the mitochondrion and peroxisome.
[0611] The activity modulator may IHD1, IHD2 or IHD3. The amino
acid sequences for human IDH1, 2 and 3 are on the NCBI database
with the following accession numbers: CAG38738.1 (IDH1);
NP_002159.2 (IDH2, isoform 1); NP_001276839.1 (IDH2, isoform 2);
NP_001277043.1 (IDH2, isoform 3); NP_689993.4 (IDH3, isoform 1);
NP_001274178.1 (IDH3, isoform 2); NP_001339753.1 (IDH3, isoform
3).
[0612] 8. Co-Stimulatory Signals
[0613] The activity modulator of the present invention may provide
co-stimulatory signal to the T-cell.
[0614] For example, the activity modulatory may be a TNF receptor,
a chimeric TNF receptor or a TNF receptor ligand.
[0615] TNF-family co-stimulatory molecules provide survival and
expansion signals for T-cells during their ontogeny. These TNF
receptors (TNFRs) signal via TNF receptor associated factor (TRAF)
second messengers.
[0616] TNFRSF9 (4-1BB), TNFRSF4 (OX40), TNFRSF5 (CD40) and TNFRSF14
(GITR) transmit survival signals to T-cells. TNFRSF7 (CD27) and
TNFRSF14 (HVEM) are expressed by naive T-cells. The expression of
OX40 and 4-1BB is induced in response to antigen stimulation, these
TNFRs have been proposed to be markers of effector T cells.
Although CD27 and GITR can be constitutively expressed by
conventional T cells, their expression is also strongly upregulated
following T-cell activation, possibly in parallel with the
upregulation of OX40 and 4-1BB expression.
[0617] The induction or upregulation of OX40, 4-1BB and GITR
expression occurs within 24 hours following the recognition of
antigen by and activation of naive T cells, and much more rapidly
by memory T cells; the expression of these receptors can last for
several hours or even days.
[0618] The TNF receptor TNFRSF35/Death receptor 3 (D3R) is
activated by TL1A which is upregulated by inflamed tissue
transiently and this interaction appears to be important for the
late stage of T-cell activity after an established immune
response.
[0619] CD40 is not expressed by T-cells, but CD40L is and
CD40/CD40L is particularly important for B-cell differentiation and
expansion.
[0620] TNFRSF11A (RANK) is not expressed by T-cells, but the
RANK/RANK-L pathway is important to immune development as well as
being a key pathway for osteoclast activity and is active during
bone metastasis.
[0621] TNFRSF12A (Fn14) is not expressed by T-cells, but is
expressed along with its ligand TWEAK in damaged or inflamed
tissues and most cancers.
[0622] 8.1 Chimeric TNF Receptors
[0623] International Patent Application No. PCT/GB2018/053629
describes chimeric TNF receptors which comprise (a) a binding
domain which is capable of binding a TNFR ligand; and (b) a TNFR
signalling domain.
[0624] The presence of a chimeric TNFR enables the tight temporal
and/or spatial control of TNFR signalling to be decoupled in order
to provide improved survival signals for engineered cells, for
example CAR T cells. The chimeric TNFR may compensate for the lack
of a complete physiological immune response in a tumour
microenvironment. The chimeric TNFR may be constructed such that
the antigen-binding domain is engaged, and thus a required
co-stimulatory signal induced, in the tumour microenvironment.
[0625] The antigen-binding domain of the chimeric TNFR may comprise
the ligand binding domain of a TNFR. For example, the
antigen-binding domain may comprise the ligand binding domain of
D3R, HVEM, CD27, CD40, RANK or Fn14.
[0626] The signalling domain of the chimeric TNFR may be an
activating signalling domain, such as one which is capable of
signalling via TNFR-associated factors (TRAFs). For example, the
activating signalling domain may comprise the signalling portion of
the 4-1BB, OX40, or GITR endodomain.
[0627] The activity modulator may be a HVEM-41BB chimera, a
CD27-41BB chimera, a RANK-41BB chimera or an Fn14-41BB chimera.
Examples of suitable amino acid sequences for these chimeric TNF
receptors are shown below as SEQ ID No. 51 to 54, in which the
ectotomain is shown in normal text, the transmembrane domain in
bold and the 41BB endodomain in italics.
TABLE-US-00029 (HVEM-41BB) SEQ ID No. 51
MEPPGDWGPPPWRSTPRTDVLRLVLYLTFLGAPCYAPALPSCKEDEYPV
GSECCPKCSPGYRVKEACGELTGTVCEPCPPGTYIAHLNGLSKCLQCQM
CDPAMGLRASRNCSRTENAVCGCSPGHFCIVQDGDHCAACRAYATSSPG
QRVQKGGTESQDTLCQNCPPGTFSPNGTLEECQHQTKCSWLVTKAGAGT
SSSHWVIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFM
RPVQTTQEEDGCSCRFPEEEEGGCEL (CD27-41BB) SEQ ID No. 52
MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFL
VKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTI
TANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPY
VSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRIISFF
LALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDG CSCRFPEEEEGGCEL
(RANK-41BB) SEQ ID No. 53
MAPRARRRRPLFALLLLCALLARLQVALQIAPPCTSEKHYEHLGRCCNK
CEPGKYMSSKCTTTSDSVCLPCGPDEYLDSWNEEDKCLLHKVCDTGKAL
VAVVAGNSTTPRRCACTAGYHWSQDCECCRRNTECAPGLGAQHPLQLNK
DTVCKPCLAGYFSDAFSSTDKCRPWTNCTFLGKRVEHHGTEKSDAVCSS
SLPARKPPNEPHVYLPIISFFLALTSTALLFLLFFLTLRFSVVKRGRKK
LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (Fn14-41BB) SEQ ID No. 54
MARGSLRRLLRLLVLGLWLALLRSVAGEQAPGTAPCSRGSSWSADLDKC
MDCASCRARPHSDFCLGCAAAPPAPFRLLWPIISFFLALTSTALLFLLF
FLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC EL
[0628] 8.2 TNF Receptor Ligands
[0629] The TNF-related cytokines (TNF family ligands) are type II
transmembrane proteins (intracellular N-terminus) with a short
cytoplasmic tail (15 to 25 residues in length) and a larger
extracellular region (.about.50 amino acids) containing the
signature TNF homology domain where the receptor binding sites are
located.
[0630] A summary of TNFRs and their ligands is provided in Table
3.
TABLE-US-00030 TABLE 3 Protein (member #) Synonyms Gene Ligand(s)
Tumor necrosis CD120a TNFRSF1A TNF-alpha factor receptor 1
(cachectin) Tumor necrosis CD120b TNFRSF1B TNF-alpha factor
receptor 2 (cachectin) Lymphotoxin beta CD18 LTBR Lymphotoxin
receptor beta (TNF-C) OX40 CD134 TNFRSF4 OX40L CD40 Bp50 CD40 CD154
Fas receptor Apo-1, CD95 FAS FasL Decoy receptor 3 TR6, M68
TNFRSF6B FasL, LIGHT, TL1A CD27 S152, Tp55 CD27 CD70, Siva CD30
Ki-1 TNFRSF8 CD153 4-1BB CD137 TNFRSF9 4-1BB ligand Death receptor
4 TRAILR1, Apo-2, TNFRSF10A TRAIL CD261 Death receptor 5 TRAILR2,
CD262 TNFRSF10B TRAIL Decoy receptor 1 TRAILR3, LIT, TNFRSF10C
TRAIL TRID, CD263 Decoy receptor 2 TRAILR4, TNFRSF10D TRAIL TRUNDD,
CD264 RANK CD265 TNFRSF11A RANKL Osteoprotegerin OCIF, TR1
TNFRSF11B TWEAK receptor Fn14, CD266 TNFRSF12A TWEAK TACI IGAD2,
CD267 TNFRSF13B APRIL, BAFF, CAMLG BAFF receptor CD268 TNFRSF13C
BAFF Herpesvirus entry ATAR, TR2, TNFRSF14 LIGHT mediator CD270
Nerve growth factor p75NTR, CD271 NGFR NGF, BDNF, receptor NT-3,
NT-4 B-cell maturation TNFRSF13A, TNFRSF17 BAFF antigen CD269
Glucocorticoid- AITR, CD357 TNFRSF18 GITR ligand induced TNFR-
related TROY TAJ, TRADE TNFRSF19 unknown Death receptor 6 CD358
TNFRSF21 Death receptor 3 Apo-3, TRAMP, TNFRSF25 TL1A LARD, WS-1
Ectodysplasin A2 XEDAR EDA2R EDA-A2 receptor
[0631] The activity modulator may be or comprise a TNF receptor
ligand such as CD40L (CD154), OX40L (CD134) or 41BBL. The amino
acid sequences for these proteins are shown below as SEQ ID No. 55
to 57
TABLE-US-00031 (CD40L) SEQ ID No. 55
MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRR
LDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDI
MLNKEETKKENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYT
MSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLK
SPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPS QVSHGTGFTSFGLLKL
(OX40L) SEQ ID NO. 56
MERVQPLEENVGNAARPRFERNKLLLVASVIQGLGLLLCFTYICLHFSA
LQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCD
GFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKD
KVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL (41BBL) SEQ ID NO. 57
MEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFL
ACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQ
NVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQL
ELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSA
FGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIP AGLPSPRSE
[0632] Chimeric RTK
[0633] Receptor Protein-Tyrosine Kinase (RPTKs) constitute a family
of intracellular signal regulators that mediate embryonic
development, cell growth, metabolism, and immune function. RPTKs
are often dysregulated in cancer, driving proliferation.
[0634] The proliferative capacity of CAR T-cells is inconsistent
between different binders and structures, and the characteristics
bestowing maximum proliferative incentive are elusive. CAR T-cells
are exposed to the hostile tumour microenvironment, and
incorporation of RPTK in a dimer format can provide a proliferative
advantage to the CAR T-cells. Expression of an RTK which is capable
of signalling in the absence of cognate ligand breaks the tumour
immune-depriving milieu in order to improve the CAR T-cell survival
signal.
[0635] RTKs signal through ligand-induced
dimerization/oligomerisation, which leads to auto-phosphorylation
of tyrosine residues in the kinase domain activation loop of their
cytoplasmic tail. Ligand-mediated oligomerisation of the RTK leads
to a two-step activation, the increase in catalytic activity and
the creation of docking sites for downstream signalling
proteins.
[0636] Typically RTKs Homodimerize in Order to Signal.
[0637] The RTK auto-phosphorylation may occur in cis or in trans.
The phosphorylated tyrosine residues constitute docking sites for
numerous SH2-containing signalling molecules. Generally, all RTKs
signal through common downstream signal protein such as: PI3
kinase, Ras-Raf-MAPK, JNK, and PLC.gamma.. The signalling is
mediated by the JAK-STAT pathway.
[0638] The activity modulator may be a receptor tyrosine kinase
(RTK) which is capable of signalling in the absence of cognate
ligand. Such RTK are described in GB patent application No.
1803079.1.
[0639] The RTK may be over-expressed and/or comprise a mutation
such that it is capable of signaling in the absence of cognate
ligand.
[0640] The RTK may be a chimeric RTK. The chimeric RTK may comprise
an ectodomain or an endodomain which mediates dimerization or
oligomerization of the chimeric RTK.
[0641] The domain which mediates dimerization or oligomerization of
the chimeric RTK may comprise a disulphide bond, for example it may
be or comprise a hinge domain.
[0642] Alternatively, the domain which mediates dimerization or
oligomerization of the chimeric RTK may comprise a chemically
operable dimerization or oligomerization domain. Dimerization or
oligomerization of the chimeric RTK may inducible by an agent.
[0643] A summary of human RTK subfamilies and RTKs is shown in
Table 4.
TABLE-US-00032 TABLE 4 RTK RTK RTK Family RTK Family RTK Family RTK
Epidermal EGFR Fibroblast FGFR-1 Related RYK growth factor growth
to receptor factor receptor (EGFR) receptor tyrosine (FGFR) kinase
(RYK) ERBB2 FGFR-2 Discoidin DDR1 ERBB3 FGFR-3 domain DDR2 receptor
family (DDR) ERBB4 FGFR-4 RET RET Insulin INSR KLG/CCK CCK4
Leukocyte LTK Receptor IGF-1R Erythro- EPHA1 receptor ALK poietin-
tyrosine producing kinase human (LTK) IRR hepato- EPHA2 Muscle-
MUSK cellular Specific receptors Kinase (EPHR) (MUSK) Platelet
PDGFR-.alpha. EPHA3 NGFR TRKA derived PDGFR-.beta. EPHA4 TRKB
growth factor CSF-1R EPHA5 TRKC receptor (PDGFR) KIT/SCFR EPHA6
Hepatocyte MET KLK2/FLT3 EPHA7 growth RON factor receptor (HGFR)
Vascular VEGFR1 EPHA8 LMR AATYK endothelial VEGFR2 EPHB1 AATYK2
growth factor VEGFR3 EPHB2 AATYK3 receptor (VEGFR) TIE TIE EPHB3
Tyrosine- AXL TEK EPHB4 protein MER Receptor ROR1 EPHB5 kinase
TYRO3 tyrosine receptor kinase- (AXL) like orphan ROR2 EPHB6
Unknown RTK106 receptors (ROR) ROS ROS
[0644] Illustrative UniProt Accession Numbers and associated amino
acid sequences of the human RTKs shown in Table 2 are provided in
Table 5.
TABLE-US-00033 TABLE 5 RTK UniProt Accession Number EGFR P00533
ERBB2 P04626 ERBB3 P21860 ERBB4 Q15303 INSR P06213 IGF-1R P08069
IRR P14616 PDGFR-.alpha. P16234 PDGFR-.beta. P09619 CSF-1R P07333
KIT/SCFR P10721 KLK2/FLT3 P20151 VEGFR1 P17948 VEGFR2 P35968 VEGFR3
P35916 TIE P35590 TEK Q02763 ROR1 Q01973 ROR2 Q01974 ROS P08922
FGFR-1 P11362 FGFR-2 P21802 FGFR-3 P22607 FGFR-4 P22455 CCK4 Q13308
EPHA1 P21709 EPHA2 P29317 EPHA3 P29320 EPHA4 P54764 EPHA5 P54756
EPHA6 Q9UF33 EPHA7 Q15375 EPHA8 P29322 EPHB1 P54762 EPHB2 P29323
EPHB3 P54753 EPHB4 P54760 EPHB5 EPHB6 O15197 RYK P34925 DDR1 Q08345
DDR2 Q16832 RET P07949 LTK P29376 ALK Q9UM73 MUSK O15146 TRKA
P04629 TRKB Q16620 TRKC Q16288 MET P08581 RON Q04912 AATYK Q6ZMQ8
AATYK2 Q8IWU2 AATYK3 Q5XJV6 AXL P30530 MER Q12866 TYRO3 Q06418
RTK106
[0645] Modulating Target Cell Activity
[0646] The activity modulator may modulate the activity of the
target cell, for example, the tumour cell.
[0647] For example, the agent may be a toxin, such as a toxin which
is toxic to tumour cells. For example, the agent may be diphtheria
toxin, Pseudomonas toxin or Shigella toxin.
[0648] Alternatively the activity modulator may be a pro-drug or a
pro-drug activating compound. The activity modulator may be a
pro-drug activating enzyme.
[0649] Prodrugs are widely used in the targeted delivery of
cytotoxic compounds to cancer cells. Prodrugs are inactive or less
active derivatives of drug molecules which undergo enzymatic or
chemical transformation to regenerate the active forms.
[0650] In targeted cancer therapy, conventional chemotherapeutic
agents, which lack intrinsic target specificity, are rationally
modified to focus and redirect their cytotoxicity to tumor cells.
The usefulness of many conventional, nonspecific chemotherapeutic
agents, such as doxorubicin, paclitaxel, camptothecan, cisplatin,
and their derivatives have been significantly extended by
modification into prodrugs, particularly those harboring
cell-targeting moieties.
[0651] The activity modulator may be an enzyme which activates a
specific inactive substrate (prodrug), which is administered
separately, to a cytotoxic product. The activity modulator may be
or comprise cytosine deaminase (CD) which converts the prodrug
5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), whose downstream
antimetabolites lead to a so-called "thymineless death".
Alternatively the activity modulator may be or comprise a cytosine
deaminase/uracil phospho-ribosyltransferase fusion (CD/UPRT;
encoded by the Fcy::Fur gene) which has also been used to generate
the 5-FU-based antimetabolites. Other antimetabolite prodrugs
include the nucleoside analogs such as acyclovir and ganciclovir,
which are activated to their active triphosphate using recombinant
thymidylate kinase, as well as 6-methyl-2'-deoxyriboside and
2-fluoro-2'-deoxyadenosine, which are converted by purine
nucleoside phosphorylase to 6'-methylpurine and 2-fluoroadenine,
respectively. Human deoxycytidine kinase (DCK) and thymidylate
kinase (tmpk) are capable of mono-phosphorylating a range of
(non-physiologic) prodrugs such as gemcitabine (dFdC),
bromovinyl-deoxyuridine (BVdU), cytarabine (AraC), and
3'-azido-3'-deoxythymidine (AZT) monophosphate. A chimeric fusion
of DCK with uridine monophosphate kinase (DCK::UMK) has also been
developed to directly activate gemcitabine to its cytotoxic
diphosphate metabolite (dFdCDP) in pancreatic carcinoma. There are
also "designer" prodrugs in which a chemotherapeutic agent is
derivatized to a substrate for a specific activating enzyme.
Examples include phenoxyacetamide conjugates of doxorubicin and
melphalan that are hydrolyzed by penicillin-V amidase, a
dipiperidinyl conjugate of etoposide (VP-16) that is hydrolyzed by
a recombinant carboxylesterase, and a cephalosporin conjugate of
5-FU designed for hydrolysis by .beta.-lactamase.
[0652] The activity modulator may be an enzyme capable of cleaving
a prodrug-conjugate for example, a pro-drug conjugate comprising a
toxin. Like prodrug conjugates, many targeted toxins consist of a
targeting moiety (e.g., an antibody, in the case of immunotoxins),
a cleavable linker, and a drug (cytotoxic enzyme). Moxetumomab
pasudotox, consists of a truncated exotoxin A from Pseudomonas
aeruginosa in which the native receptor-binding domain (located in
the N-terminal 250 residues) has been replaced with a single-chain
variable fragment targeting the cell-surface CD22 antigen.
Cytotoxic activity is conferred entirely by the C-terminal segment
(residues 405 to 613, termed PE3). As is, this conjugate is an
inactive toxin: cytotoxic activation requires cleavage by the
protease furin during endocytosis between residues 279 and 280.
Thus, moxetumomab pasudotox is functionally a targeted prodrug
conjugate in which residues 251 to 364 from exotoxin A (domain II)
serves as a linker whose cleavage releases the cytotoxic PE3.
[0653] 1. Biosynthetic CAR T Cell
[0654] The activity modulator may be an enzyme which is capable of
synthesising a small molecule when expressed or expressed in
combination in a cell.
[0655] International patent application No. PCT/GB2018/053262
describes engineered cells which encode a transgenic synthetic
biology pathway that enables the engineered cell to produce a small
molecule, in particular a therapeutic small molecule. The
engineered cell may comprises; (i) a chimeric antigen receptor
(CAR) or a transgenic T-cell receptor (TCR); and (ii) one or more
engineered polynucleotides which encode one or more enzymes which
are capable of synthesising a therapeutic small molecule when
expressed in combination in the cell.
[0656] There may be, for example one, two, three four or five
enzymes. The one or more enzymes may be encoded in one or more open
reading frames. The one or more enzymes may be encoded in a single
open reading frame. Suitably, each enzyme may be separated by a
cleavage site. The cleavage site may be a self-cleavage site, such
as a sequence encoding a FMD-2A like peptide.
[0657] The therapeutic small molecule may, for example be a
cytotoxic molecule; a cytostatic molecule; an agent which is
capable of inducing differentiation of the tumour; or a
proinflammatory molecule. In particular, the small molecule may be
violacein or a derivative thereof, or geraniol.
[0658] Target Cell Microenvironment Modulating Agent
[0659] The activity modulator may be an agent which modulates the
environment of the target cell, for example, the tumour cell.
[0660] For example, the agent may be a cytokine such as IL-7 or
IL-12 or a chemokine such as CCL19 as discussed above.
Alternatively, the agent may affect the expression or activity of a
cytokine or chemokine as discussed above.
[0661] 1. CAR-T Cell Secreting Enzymes
[0662] The immune microenvironment contains small molecule
metabolites and nutrients which can alter the balance between the
tumour survival and/or progression and an immune response.
Modulation of the microenvironment may alter the balance in favour
of the immune response and/or can improve the activity or efficacy
of adaptive immune therapy, such as the efficacy of engineered
cells expressing a CAR or transgenic TCR.
[0663] An activity modulatory in connection with the present
invention may modulate the level of one or more metabolites or
nutrients in the tumour microenvironment, skewing the balance in
favour of immune cells such as T-cells involved in an immune
response and/or away from tumour cells.
[0664] For example, the activity modulator be one or more enzymes
which, when secreted or expressed at the cell surface, causes
depletion of a molecule extracellular to the engineered cell which
is:
[0665] (i) required by a tumour cell for survival, proliferation,
metastasis or chemoresistance, and/or [0666] (ii) detrimental to
the survival, proliferation or activity of the engineered cell.
[0667] United Kingdom application No. 1820443.8 describes
engineered cells which secrete such enzymes or express them at the
cell surface.
[0668] The enzyme(s) may cause the depletion of an amino acid or
amino acid metabolite, a nucleobase (such as a nucleoside or
nucleotide) or a lipid.
[0669] Where the activity modulator causes the depletion of a
nutrient or metabolite which is needed for tumour cell growth or
survival, the immune cell (e.g. CAR-T cell) may be engineered to
survive in the absence of the molecule in the extracellular
environment. For example, the cell may be engineered to synthesise
the molecule or a precursor thereof intracellularly.
[0670] Cell Composition
[0671] The present invention also provides a cell composition made
by the method of the present invention.
[0672] The invention provides cell composition made by transduction
of cells with a plurality of viral vectors such that the
composition comprises a mixture of untransduced cells, singly
transduced cells and combinatorially transduced cells.
[0673] At least one vector in the mixture of viral vectors used in
the method of the present invention comprises a nucleic acid
sequence encoding a CAR. The cell composition may therefore
comprise a mixture of singly and combinatorially transduced
CAR-expressing cells.
[0674] "Combinatorially transduced" means that the cell is
transduced with at least two viral vectors. For example, if cells
are transduced with two vectors, one comprising transgene A and one
comprising transgene B, the transduced cells will be a mixture of
cells expressing A alone; B alone; and cell expressing both A and
B. In this situation, cells expressing A and B are combinatorially
transduced.
[0675] For cells transduced with three vectors each comprising a
transgene, the resulting transduced cells will be a mixture of: A
alone; B alone; C alone; A and B; A and C; B and C; and cells
expressing A, B and C. In this situation, the three
sub-populations, expressing A and B; A and C; B and C; and cells
expressing A, B and C are combinatorially transduced.
[0676] The cell composition comprises a plurality of
sub-populations derived by transduction with different vector
combinations in the mixture of viral vectors.
[0677] The cell composition may comprise cytolytic immune cells
such as a T cells and/or or NK cells.
[0678] T cells or T lymphocytes are a type of lymphocyte that play
a central role in cell-mediated immunity. They can be distinguished
from other lymphocytes, such as B cells and natural killer cells
(NK cells), by the presence of a T-cell receptor (TCR) on the cell
surface. There are various types of T cell, as summarised
below.
[0679] 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.
[0680] 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.
[0681] Memory T cells are a subset of antigen-specific T cells that
persist long-term after an infection has resolved. They quickly
expand to large numbers of effector T cells upon re-exposure to
their cognate antigen, thus providing the immune system with
"memory" against past infections. Memory T cells comprise three
subtypes: central memory T cells (TCM cells) and two types of
effector memory T cells (TEM cells and TEMRA cells). Memory cells
may be either CD4+ or CD8+. Memory T cells typically express the
cell surface protein CD45RO.
[0682] 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.
[0683] Two major classes of CD4+ Treg cells have been
described--naturally occurring Treg cells and adaptive Treg
cells.
[0684] 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.
[0685] Adaptive Treg cells (also known as Tr1 cells or Th3 cells)
may originate during a normal immune response.
[0686] Natural Killer cells (or NK cells) form part of the innate
immune system. NK cells provide rapid responses to innate signals
from virally infected cells in an MHC independent manner
[0687] 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.
[0688] The cells of the invention may be any of the cell types
mentioned above.
[0689] The cells to be transduced with a method of the invention
may be derived from a blood sample, for example from a
leukapheresate. The cells may be or comprise peripheral blood
mononuclear cells (PBMCs).
[0690] Cells 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).
[0691] Alternatively, cells may be derived from ex vivo
differentiation of inducible progenitor cells or embryonic
progenitor cells to, for example, T or NK cells. Alternatively, an
immortalized T-cell line which retains its lytic function and could
act as a therapeutic may be used.
[0692] The cells may be activated and/or expanded prior to being
transduced with nucleic acid encoding the molecules providing the
chimeric polypeptide according to the first aspect of the
invention, for example by treatment with an anti-CD3 monoclonal
antibody.
[0693] After transduction, the cells may then by purified, for
example, selected on the basis of expression of the CAR. It may be
desirable to select cells on the basis of CAR expression when there
may be a sub-population of cells post-transduction which express an
activity modulator in the absence of a CAR. Where each of the
vectors in the mixture of viral vectors comprises a nucleic acid
sequence encoding a CAR, it may not be necessary to purify or sort
cells on the basis of CAR-expression, as it should not be possible
for any cells to express an activity modulator in the absence of a
CAR.
[0694] Pharmaceutical Composition
[0695] The cell composition of the present invention, comprising a
mixture of singly and combinatorially transduced CAR-expressing
cells, may be administered to a patient as a pharmaceutical
composition.
[0696] 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.
[0697] Method of Treatment
[0698] The present invention provides a method for treating a
disease which comprises the step of administering the a cell
composition of the present invention (for example in a
pharmaceutical composition as described above) to a subject.
[0699] A method for treating a disease relates to the therapeutic
use of the cell composition of the present invention. The cell
composition may be administered to a subject having an existing
disease or condition in order to lessen, reduce or improve at least
one symptom associated with the disease and/or to slow down, reduce
or block the progression of the disease.
[0700] The method for preventing a disease relates to the
prophylactic use of the cell composition of the present invention.
The cell composition may be administered to a subject who has not
yet contracted the disease and/or who is not showing any symptoms
of the disease to prevent or impair the cause of the disease or to
reduce or prevent development of at least one symptom associated
with the disease. The subject may have a predisposition for, or be
thought to be at risk of developing, the disease.
[0701] The method may involve the steps of: [0702] (i) isolating a
cell-containing sample;
[0703] (ii) transducing the such cells with a mixture of at least
two viral vectors; [0704] (iii) administering the cells from (ii)
to a subject.
[0705] The present invention also provides a cell composition of
the present invention for use in treating and/or preventing a
disease.
[0706] The invention also relates to the use of a cell composition
of the present invention in the manufacture of a medicament for the
treatment of a disease.
[0707] The disease to be treated by the methods of the present
invention may be a cancerous disease, such as bladder cancer,
breast cancer, colon cancer, endometrial cancer, kidney cancer
(renal cell), leukaemia, lung cancer, melanoma, non-Hodgkin
lymphoma, pancreatic cancer, prostate cancer and thyroid
cancer.
[0708] The disease may be Multiple Myeloma (MM), B-cell Acute
Lymphoblastic Leukaemia (B-ALL), Chronic Lymphocytic Leukaemia
(CLL), Neuroblastoma, T-cell acute Lymphoblastic Leukaema (T-ALL)
or diffuse large B-cell lymphoma (DLBCL).
[0709] The disease may be a plasma cell disorder such as
plasmacytoma, plasma cell leukemia, multiple myeloma,
macroglobulinemia, amyloidosis, Waldenstrom's macroglobulinemia,
solitary bone plasmacytoma, extramedullary plasmacytoma,
osteosclerotic myeloma, heavy chain diseases, monoclonal gammopathy
of undetermined significance or smoldering multiple myeloma.
[0710] The cells of the composition of the present invention may be
capable of killing target cells, such as cancer cells. The target
cell may be characterised by the presence of a tumour secreted
ligand or chemokine ligand in the vicinity of the target cell. The
target cell may be characterised by the presence of a soluble
ligand together with the expression of a tumour-associated antigen
(TAA) at the target cell surface.
[0711] Different supopulations of cells within the composition of
the invention may have different levels of ability to kill target
cells, both between different patients having the same disease and
within a patient at different disease sites (e.g. tumour
sites).
[0712] Results-Based Engineering of Car-Expressing Cells
[0713] The present invention also provides a method for determining
the optimal combination of components for a CAR-expressing cell to
treat a disease.
[0714] As explained above, the cell composition of the invention
comprises a mixture of CAR-expressing cells, some of which are
single transduced and some of which are combinatorially transduced.
The number of different subpopulations will depend on the number of
viral vectors in the mixture used for transduction. For three
vectors A, B and C, there should be seven populations of transduced
cells: those transduced with: A alone; B alone; C alone; A and B; A
and C; B and C; and A, B and C. For four vectors, A, B, C and D,
there should be 15 different subpopulations: A alone; B alone; C
alone; D alone; A and B; A and C; A and D; B and C; B and D; C and
D; A, B and C; A, B and D; A, C and D; B, C and D; and A, B, C and
D.
[0715] If each vector in the composition comprises a nucleic acid
encoding a CAR and/or a nucleic acid encoding an activity
modulator, then the cell composition will be a mixture
subpopulations of transduced cells each expressing a different
combination of CAR(s) and activity modulator(s).
[0716] For example, the mixture of viral vectors may express one or
more CAR(s) and a plurality of different activity modulators.
CAR-expressing cells made by transduction of cells with the mixture
of vectors will express a plurality of different combinations of
activity modulator(s) which may give the CAR-expressing cell
different properties in terms of target cell killing, survival,
engraftment, resistance to checkpoint inhibition and/or resistance
to the hostile tumour microenvironment. In vivo, one subpopulation
of cells expressing a particular combination of CAR(s) and activity
modulators will be best suited to the particular conditions within
the pateient or within a particular site in the patient. That
subpopulation will receive the most effective activation, survival
and/or proliferation signal and will "win out" from other
subpopulations in the cell composition.
[0717] By analysing CAR-expressing cells in a patient following
administration, it is possible to work out which subpopulation is
best survive, engraft and kill target cells. By analysing the
phenotype or genotype of this subpopulation, it is then possible to
work out which combination of vectors the subpopulation were
successfully transduced with during the making of the cell
composition.
[0718] This information may be used to design a homogenous
CAR-expressing cell composition, in which every transduced cells
expresses the most successful combination of CAR(s) and activity
modulator(s).
[0719] The method may comprise the following steps: [0720] (i)
administering a cell composition to a subject having the disease;
[0721] (ii) monitoring the patient or sample(s) from the patient to
determine which sub-population of cells in the cell composition
show the greatest level of survival, engraftment, proliferation
activation and/or target cell killing; and [0722] (iii) analysing
the phenotype/genotype of the cells in the sub-population to
ascertain the CAR(s) and/or activity modulator(s) expressed by
those cells.
[0723] There is also provided a method for producing a
CAR-expressing cell composition for use in the treatment of a
disease which comprises the step of determining the optimal
combination of components for a CAR-expressing cell to treat a
disease by a method described above, then transducing cells with a
single vector expressing the identified combination of components.
The resulting cell composition will be homogeneous in the sense
that all CAR-expressing cells will express the same combination of
components.
[0724] It is also possible to transducing cells with two or more
vectors expressing the identified combination of components, then
sort for cells which express all identified components in order to
arrive at a homogeneous CAR-expressing cell composition.
[0725] The present invention also provides a cell composition made
by such a method.
[0726] The invention will now be further described by way of
Examples, which are meant to serve to assist one of ordinary skill
in the art in carrying out the invention and are not intended in
any way to limit the scope of the invention.
EXAMPLES
Example 1--Generation of a CAR-T Cell Composition Transduced with
Multiple Vectors
[0727] Lentiviral vectors were generated expressing either a) the
second-generation anti-CD19 CAR described in WO2016/139487 which
comprises an anti-CD19 antigen binding domain, a CD8 stalk spacer
and transmembrane domain, and a compound 4-1BB-CD3.zeta. endodomain
under the control of a PGK promoter
(pCCL.PGK.aCD19cat-CD8STK-41BBZ); or b) an anti-CD22 CAR having
CDRs shown as SEQ ID No. 10-15 above, a CD8 stalk spacer and a
second generation endodomain comprising CD3.zeta. and a 4-1BB
costimulatory domain, under the control of an EF1.alpha. promoter
(pCCL.EF1a.aCD22_9A8-1-64_LH_scFv-CD8STK-41BBz).
[0728] Two separate lentiviral supernatants were produced and mixed
1:1 at an MOI of 2.5+2.5. The pattern of expression of the two CARs
was investigated by flow cytometry using an anti-idiotype antibody
to stain for expression of the anti-CD19 CAR and soluble CD22 to
stain for expression of the CD22 CAR. The results are shown in FIG.
6. Post-transduction with the lentiviral composition, the cells are
a mixture of untransduced cells (46.5%); cells expressing the
anti-CD19 CAR alone (23.1%); cells expressing the anti-CD22 CAR
alone (11.1%) and cells expressing both the CD19 and CD22 CARs
(19.3%).
Example 2--Expression of CAR(s) in Combination with Enhancement
Module(s) in a Combinatorial Fashion by Transduction with Multiple
Vectors
[0729] Vectors are constructed which contain a second generation
anti-CD19 CAR (Fmc63-41BBz) in combination with a marker gene and
one of the following enhancement modules, which are predicted to
display activity under specific conditions:
[0730] dnSHP2--A truncated version of SHP2 which has the SH2
domains but lacks the phosphatase domain. The sequence is shown
above as SEQ ID No. 29. The truncated protein acts as a dominant
negative and competes with the wild-type proteins for binding to
phosphorylated ITIM on inhibitory immunoreceptors such as PD1.
[0731] dnTGFBRII--A dominant negative TGF.beta. receptor which
lacks the kinase domain. The sequence is shown above as SEQ ID No.
46. The dnTGFBRII competes with wild-type TGF-.beta. receptor for
binding to TGF-.beta., downregulating TG.beta.-mediated
signalling.
[0732] CCR--A constitutively active chimeric cytokine receptor
having an IL-2, IL-7 or GM-CSF receptor endodomain, as described
above. The sequences for components of for such constitutively
active CCRs are given above as SEQ ID No. 30-43. The present of
such a receptor causes constitutive cytokine signalling (i.e. in
the absence of the relevant cytokine).
[0733] Retroviral vectors are constructed containing the following
cassettes, in which GFP, mKate and RQR8 are marker genes:
[0734] Vector A--CAR+dnSHP+GFP
[0735] Vector B--CAR+dnTBRII+mKate
[0736] Vector C--CAR+CCR+RQR8
[0737] T cells are transduced with a mix of two retroviruses
encoding two of the above vectors in a 1:1 mixture at an MOI of
2.5:2.5. The resulting mixed population contains T cells expressing
the first vector OR the second vector OR both vectors together.
Transduction is detected and measured by flow cytometry measuring
the expression of the marker genes. For example, for cells
transduced with a mixture of vectors A and B, transduction is
detected and measured using the marker genes GFP and mKate.
[0738] The transduced T cell population is labelled with the dye
Cell Trace Violet (CTV), a fluorescent dye which is hydrolysed and
retained within the cell. It is excited by the 405 nm (violet)
laser and fluorescence can be detected in the pacific blue channel.
The T-cells are resuspended at 2.times.10.sup.6 cells per ml in
PBS, and 1 ul/ml of 5 mM CTV is added. The T-cells are incubated
with the CTV for 20 minutes at 37.degree. C. Subsequently, the
cells are quenched by adding 5V of complete media. After a 5
minutes incubation, the T-cells are washed and resuspended in 2 ml
of complete media. An additional 10 minute incubation at room
temperature allows the occurrence of acetate hydrolysis and
retention of the dye.
[0739] Labelled T-cells are co-cultured with CD-19 expressing
target cells for four or seven days. In order to investigate the
function of the dnSHP2 enhancement module in vector mixtures
comprising Vector A, target cells are used which co-express the
target antigen and PDL1. In order to investigate the function of
the dnTBRII enhancement module in vector mixtures comprising vector
B, T-cells are co-cultured with target cells in the presence of
soluble TGF-.beta..
[0740] The assay is carried out in a 96-well plate in 0.2 ml total
volume using 5.times.10.sup.4 transduced T-cells per well and an
equal number of target cells (ratio 1:1). At the day four or day 7
time point, the T-cells are analysed by flow cytometry to measure
the dilution of the CTV which occurs as the T-cells divide. The
number of T-cells present at the end of the co-culture is
calculated, and expressed as a fold of proliferation compared to
the input number of T cells.
[0741] Preferential expansion in the module corresponding to the
immunosuppression exerted by the target cells is assessed by a
decrease in cell trace violet dye in that population and by an
increase in the marker corresponding to that population.
Example 3--Generation of an Anti-GD2-CAR T-Cell Product with
Enhancement Modules by Dual Transduction with Two Separate
Retroviral Vectors
[0742] Background: Neuroblastoma is the most common extracranial
solid cancer in children with poor long-term survival in those with
high-risk disease.
[0743] A currently ongoing phase I clinical study of GD2-targeted
CART for refractory/relapsed neuroblastoma (NCT02761915) shows
activity against disseminated disease without inducing on
target/off tumor toxicity. However, CART persistence was limited
and clinical activity transient and incomplete.
[0744] Building on the GD2 CAR used in this study, we have
developed a next generation T-cell product candidate termed
AUTO6NG. The AUTO6NG product consists of 3 distinct populations of
GD2-targeted CAR T-cells, produced by dual transduction of T-cells
with two separate retroviral vectors. The first vector directs the
expression of a GD2-targeting CAR, co-expressed with a
constitutively signalling IL7 cytokine receptor (IL7R_CCR) or a
constitutively signalling IL2 cytokine receptor (IL2R_CCR) (product
A), while the second vector is a tri-cistronic retroviral vector
encoding the same GD2 CAR, co-expressed with dominant negative
TGFbRII (dnTGFbRII) and truncated SHP2 (dSHP2) (product B). dSHP2
confers resistance to inhibitory signals such as those from
PD1.
[0745] The vector design is illustrated schematically in FIG.
7.
[0746] The GD2 CAR is as described in WO2015/132604, with an
antigen-binding domain with a VH domain having the sequence shown
as SEQ ID No. 77 and a VL domain having the sequence shown as SEQ
ID No. 78.
[0747] The constitutively signalling IL2 and IL7 cytokine receptors
are as described in WO2017/029512, The IL2 CCR comprises a
comprising a first polypeptide having an IL-2 receptor .beta.-chain
endodomain (SEQ ID No. 40) and a second polypeptide comprising a
common .gamma.-chain receptor endodomain (SEQ ID No. 39); whereas
the IL7 CCR comprises a first polypeptide having an IL-7 receptor
.alpha.-chain endodomain (SEQ ID No. 41) and a second polypeptide
comprising a common .gamma.-chain receptor endodomain (SEQ ID No.
39).
[0748] The sort/suicide gene RQR8 is as described in WO2013/153391
and has the sequence shown as SEQ ID No. 79.
[0749] The dominant negative TGFbRII (dnTGFbRII) has the sequence
shown above as SEQ ID No. 46.
[0750] The truncated SHP2 (dSHP2) has the sequence shown above as
SEQ ID No. 29.
Example 4--Investigating the Cytotoxic Capacity of Single and Dual
Transduced Cells and the Function of Various Vector-Expressed
Elements In Vitro
[0751] Human T-cells were either dual transduced with both vectors
yielding a mix of product A/B/A+B (AUTO6NG) or single transduced
with each vector individually giving raise to product A or B.
Controls included non-transduced cells (NT) and cells expressing
GD2 CAR alone.
[0752] i) Cytotoxicity Assay
[0753] The various effector cell types were co-cultured with
GD2-expressing SupT1 target cells (SupT1 GD2) or control,
non-transduced target cells (SupT1 NT) for 72 hours and the
percentage of target cell lysis was analysed by flow cytometry. The
results are shown in FIG. 8. All CAR-expressing effector cells were
capable of killing GD2-expressing target cells. T-cells transduced
with the dual vector composition (product A/B/A+B) were highly
potent in cytotoxicity assays against GD2 positive tumour cell
lines with no differences observed compared with single transduced
CAR T-cells (product A or B).
[0754] ii) Validation of the CCR
[0755] The various transduced CAR T-cells described above and
control NT T-cells were labelled with Cell Trace Violet (CTV) and
cultured in cytokine-free complete cell culture media for 7 days
without further antigen stimulus. In vitro persistence was
quantified as percent proliferating cells which have diluted the
CTV dye and absolute CAR T-cell count after 7 days. The results are
shown in FIG. 9. T cells transduced with product A, expressing
either the constitutively signalling IL7 cytokine receptor
(IL7R_CCR) or the constitutively signalling IL2 cytokine receptor
(IL2R_CCR); or transduced with product A+B showed increased
proliferation compared with untransduced cells (NT), cells
transduced with a vector expressing GD2 CAR alone, or cells
transduced with the vector expressing product B alone (GD2
CAR+dSHP2+dTGFbRII). Expression of either the IL2 or IL7R_CCR
conferred exogenous-cytokine-independent viability and homeostatic
proliferation of modified T-cells, without causing autonomous
T-cell growth.
[0756] iii) Validation of the dTGFbRII
[0757] The various transduced CAR T-cells described above and
control NT T-cells were co-cultured with GD2-expressing SupT1
target cells (SupT1 GD2) or control, non-transduced target cells
(SupT1 NT) at a 1:2 or 1:8 E:T ratio, for 7 days, in the presence
or absence of 10 ng/ml TGF.beta.. Killing of target cells was
analysed by flow cytometry and secretion of IFN.gamma. was analysed
by ELISA. The results are shown in FIGS. 10 and 11 respectively.
CAR T cells transduced with product B, expressing dnTGFbRII; or
transduced with product A+B, showed resistance to
TGF.beta.-mediated inhibition of target cell killing compared with
CAR-T cells expressing GD2 CAR alone, or cells transduced with the
vector expressing either product A alone (GD2 CAR+IL2 CCR or GD2
CAR+IL7 CCR). CAR T cells transduced with product B or product A+B,
showed restoration of IFN.gamma. secretion in the presence of
TGF.beta. to a level comparable to that observed in the absence of
TGF.beta.. By contrast, IFN.gamma. secretion by CAR-T cells
expressing GD2 CAR alone, or cells transduced with the vector
expressing either product A alone, was significantly inhibited by
the presence of TGF.beta.. T-cells expressing dnTGFbRII therefore
proved resistant to TGF.beta.-mediated immunosuppression in
vitro.
Example 5--Investigating Anti-Tumour Activity of Dual-Transduced
CAR-T Cell Product In Vivo in an Xenograft Model of
Neuroblastoma
[0758] An in vivo assay was used to investigate the anti-tumour
activity of T cells transduced with a dual vector composition by
intravenous administration in an established neuroblastoma
xenograft model in NSG mice. Ten- to 14-week-old female NSG mice
were intravenously injected with 1 million Firefly luciferase
expressing CHLA-255 cells (CHLA-255 FFluc). Xenografts were left to
establish for 15 days until stable engraftment was detectable by
BLI. CAR-T cells were made either by transducing cells with a
single vector expressing a GD2 CAR (GD2 CAR) or by transducing
cells with the dual vector composition described in Example 3 and
Illustrated in FIG. 7 (GD2 CAR+IL7 CCR/GD2 CAR+dSHP2+dTGFbRII).
Mice were injected intravenously with 10.times.10.sup.6 CAR T-cells
(50% transduction efficiency), 3.times.10.sup.6 CAR T-cells (50%
transduction efficiency), 20.times.10.sup.6 NT T-cells (total
T-cells equivalent to 10.times.10.sup.6 CAR T-cell dose) or PBS.
Fourteen days later, tumour growth was assessed by bi-weekly
bioluminescent imaging.
[0759] The results are shown in FIG. 12. Intravenous delivery of
CAR T cells expressing a simple GD2 CAR alone had no significant
effect on tumour growth (FIGS. 12 A and B). By contrast,
intravenous delivery of CAR T cells transduced with the dual vector
composition at both the 3.times.10.sup.6 and 10.times.10.sup.6
doses exhibited potent anti-tumour activity and extended survival
in NSG mice with established tumour burden (FIGS. 12 C and D).
Example 6--Generation of an Anti-PSMA CAR T-Cell Product with
Enhancement Modules by Triple Transduction with Three Separate
Retroviral Vectors
[0760] The present inventors developed a
combinatorial/multi-modular CAR T therapeutic for the treatment of
prostate cancer ("AUTO7") which consists of several functional
modules: 1) an anti-PSMA CAR having a second generation CD28-CD3z
compound endodomain, (7A12-28z); 2) a safety switch, RapaCasp9
which is described in WO2016/135470 and has the sequence shown as
SEQ ID No. 80 above; 3) dominant negative TG.beta.RII to induce
TGF.beta.1 resistance (dnTBRII) having the sequence shown as SEQ ID
No. 46 above; 4) truncated SHP2 (dnSHP2) for PD1/PD-L1 pathway
inhibition having the sequence shown as SEQ ID No. 29 above; 5) a
constitutively active IL7 receptor (CCR_IL7) to induce
proliferation as described above; 6) a sort-suicide gene RQR8
having the sequence shown as SEQ ID No. 79; and 7) an
ultra-low-secreting IL-12 (flexiIL-12) for lymphocyte
recruitment/activation which is positioned downstream of a
stop-skip sequence (SS) which has the sequence shown as SEQ ID No.
81. The vector design is shown in FIG. 13A.
[0761] The first vector (A) encodes dnSHP2, the suicide gene RQR8,
the CAR and dnTBRII; the second vector (B) encodes the
constitutively active IL7 receptor; the third vector (C) encodes a
second suicide gene: Rapcasp9; and flexi-IL12.
[0762] Having different suicide genes on vector A and vector C
provides flexibility. If a CAR-related toxicity is observed in the
patient, then it is possible to selectively remove cells expressing
the CAR in the usual way treating the patient with Rituximab, which
triggers apoptosis of these cells via the RQR8 sort/suicide gene.
If, however, a toxicity is observed which is thought to be related
to IL-12 secretion, then cells can be selectively destroyed which
are transduced with vector C by adding rapamycin or a rapamycin
analogue to trigger apoptosis via the RapaCasp9 suicide gene. As
the therapeutic product comprises cells transduced with a mixture
of three vectors, it will be a combinatorial product, with distinct
subpopulations of cells transduced with all the various
combinations of one, two or all three vectors. This means that some
CAR-expressing cells should survive triggering of the RapaCasp9
suicide gene. For example, cell transduced with vector A alone or
the combination of vectors A and B will not express RapaCasp9 and
should be unaffected by treatment with rapamycin. This means that
it is possible to "modify" the therapeutic product in vivo and
selectively delete the IL-12 expressing cells, while leaving a
proportion of CAR-expressing cells to maintain the CAR-mediated
anti-tumour effect.
[0763] AUTO7 was investigated as product of a single transduction
using the vector A. ("AUTO7/A"), or double transduction using
vectors A and B ("AUTO7/AB"), or triple transduction using vectors
A, B and C ("AUTO7/ABC"). AUTO7 was tested against a second
generation CAR developed using the same anti-PSMA binder 7A12
("Parental").
Example 7--Investigating the Cytotoxic Capacity of Single, Dual and
Triple Transduced Cells and the Function of Various
Vector-Expressed Elements In Vitro
[0764] The capacity of the T-cells transduced with the single, dual
and triple vector combinations described above to kill target cells
was investigated using a FACS-based killing assay. SupT1 cells
engineered to express human PSMA antigen at different levels
(SupT1-PSMAhigh, SupT1-PSMAlow) were used as target cells.
Non-engineered SupT1 cells (SupT1-NT) were used as a negative
control. CAR T-cells were co-cultured with target cells at 1:2
effector to target ratios. FBK was assayed after 24 h of incubation
by cytofluorimetry analysis and the results are shown in FIG. 14A.
Secretion of IL-2 and IFN.gamma. by CAR T-cells was measured by
collecting supernatant at 24 hr from the co-cultures described and
detected by ELISA and the results are shown in FIGS. 14B and C
respectively.
[0765] It was found that cell compositions produced by single
transduction using the vector A. ("AUTO7/A"), double transduction
using vectors A and B ("AUTO7/AB"), or triple transduction using
vectors A, B and C ("AUTO7/ABC") were all highly potent in
cytotoxicity assays against PSMA positive tumour cell lines.
Cytotoxicity and cytokine release were comparable to those observed
with a second generation CAR developed using the same anti PSMA
binder 7A12 ("Parental") which was used as a CAR alone control.
[0766] In order to investigate the capacity of the single, dual and
triple transduced AUTO7 T cells to kill target cells following
culture in the absence of IL-2, transduced T-cells were cultured in
starvation conditions with media without the supplement of IL2 for
7 days. After 7 days CAR T-cells were counted and plated together
with SupT1-PSMAhigh and SupT1-PSMAlow targets cells (or SupT1-NT
cells as negative control). CAR T-cells were co-cultured with
target cells at 1:2 and 1:8 effector to target ratios. FBK was
assayed after 24 h of incubation by cytofluorimetry analysis (FIG.
15A). Secretion of IL-2 and IFN.gamma. by CAR T-cells was measured
by collecting supernatant at 24 hr from the co-cultures described
and detected by ELISA (FIG. 15B).
[0767] It was found that, although cells expressing the control CAR
and cells transduced with vector A alone showed in inhibition in
cytotoxicity and cytokine release following culture under
starvation conditions, this effect was less pronounced with cells
transduced with the dual or triple vector combination (vectors A+B
or A, B+C). Vector B comprises a gene expressing a constitutively
active cytokine receptor: the IL7R_CCR module. Expression of this
module confers cytokine independent viability and proliferation,
without interfering with the cytotoxicity; since after starvation,
cells transduced with the AUTO7/AB or AUTO7/ABC were shown to be
still efficient at killing PSMA expressing target cells.
[0768] In order to investigate the capacity of the single, dual and
triple transduced AUTO7 T cells to kill target cells in the
presence of TGF.beta., transduced T-cells were co-cultured with
SupT1-PSMAhigh and SupT1-PSMAlow targets for 7 days at ratio 1:2
and 1:8 (E:T) either in the presence or absence of 10 ng/ml
TGF.beta.1 (SupT1-NT were used as control). Target cell killing was
quantified by FACS and normalised to targets alone. The results are
shown in FIG. 16.
[0769] At a 1:8 E:T ratio, killing of target cells by T cells
expressing the CAR alone control was inhibited by the addition of
TGF.beta.1 to the culture medium. This inhibitory effect was
reduced for cells transduced with any of the vector combinations A;
A+B; or A, B+C. Vector A encodes the dnTGF.beta.RII element which
has been previously shown to block TGF.beta.-mediated inhibition of
T-cell signalling.
[0770] In order to investigate the capacity of the single, dual and
triple transduced AUTO7 T cells to kill target cells following
repeated exposure to target antigen, transduced cells were
co-cultured with SupT1-PSMAhigh or SupT1-PSMAlow target cells at
1:1 ratio (E:T) and every 7 days CAR T-cells were re-stimulated
with 5.times.10.sup.4 SupT1 cells. Target cell killing was
quantified by FACS before each new re-stimulation. The results are
shown in FIG. 17.
[0771] After two restimulation events (week 2) cells transduced
with the vector combination A+B or A+B+C showed much better killing
of target cells than CAR-alone or Vector A-transduced cells. After
three restimulation events (week 3) cells transduced with the
vector combination A+B+C showed the best killing of target
cells.
Example 8--Investigating the Cytotoxic Capacity of Single, Dual and
Triple Transduced Cells and the Function of Various
Vector-Expressed Elements In Vivo
[0772] An in vivo assay was used to investigate the anti-tumour
activity of T cells transduced with a single, dual or triple vector
composition by intravenous administration in an established
xenograft model in NSG mice.
[0773] Female NSG mice were injected with 5.times.10.sup.6 PSMA
positive PC3 human cell lines in the flank. Xenografts were left to
establish for 3 weeks until stable engraftment was detectable by
palpation and calliper measurement. CAR T-cells were administered
i.v. at a dose of 1.times.10.sup.6 CAR T-cells/mouse. Calliper
measurement was taken 2/3 times a week. The results are shown in
FIG. 18.
[0774] Mice treated with T-cells expressing the CAR alone
(Parenteral) showed slowed tumour growth than those receiving
non-transduced cells, but the tumour growth was not controlled.
Mice receiving T cells transduced with vector A, which encodes CAR,
dnSHP2 and dnTGF.beta.RII, showed an initial growth in tumour
(September 9) followed by a reduction in tumour size (September
23). At the end of the study, some resumption of tumour growth was
observed (October 7).
[0775] Mice receiving T cells transduced with vector A+B showed an
initial growth in tumour (September 9-16), but then the tumour size
reduced and this effect continued for the duration of the study.
Triple transduced AUTO7 CAR T cells completely eradicated tumour
with no signs of toxicity. The vector combinations, especially A+B
and A+B+C show a significant improvement over CAR alone in reducing
tumour growth and promoting survival of mice in a xenograft
model.
[0776] The results demonstrate the feasibility and efficacy of the
multi-modular AUTO7 product. Addition of IL7R_CCR, ss_fIL12,
dnTGF.beta.RII and dSHP2 modules to the anti-PSMA CAR product
enhanced T cell functions by extending persistence, proliferation,
activation and resistance to TGF.beta.1 and PD1/PDL1 driven immune
inhibition.
[0777] Methodology
[0778] Binder Generation
[0779] PSMA-binder was generated by CDR grafting of an anti-PSMA
antibody derived from genetically vaccinated rats.
[0780] Cell Lines
[0781] PC3 cells, SupT1 cell lines (NT and PSMA+) were cultured in
RPMI-1640 Medium supplemented with 10% fetal bovine serum (FBS) and
1% GlutaMAX. T-cells were isolated from peripheral blood
mononuclear cells (PBMCs) and maintained in RPMI-1640 Medium
supplemented with 10% FBS, 1% GlutaMAX and 100 U/mL IL-2.
[0782] Transduction
[0783] Retrovirus was generated by transiently transfecting HEK293T
cells using GeneJuice with RDF plasmid (RD114 envelope), gag/pol
plasmid and CAR plasmid. Retroviral viral supernatant was harvested
at 48 and 72 hours. T cells were stimulated using 0.5 .mu.g/mL of
anti-CD3 and anti-CD28 antibodies in T175 TC-treated flasks and
maintained in 100 U/mL IL-2. Non-TC treated six-well plates were
coated with Retronectin and incubated at 4.degree. C. for 24 hours
prior to T cell transduction. A total of 3 ml of viral
supernatant/supernatants was plated prior to the addition of 1 ml
of activated T cells at a concentration of 1.times.10.sup.6
cells/ml, 100 U/mL of IL-2 was then added and centrifuged at
1000.times.g for 40 minutes at room temperature and incubated at
37.degree. C. and 5% CO2 for 2-3 days.
[0784] Human T-cells were: [0785] triple transduced with vector A.,
B. and C. yielding a mix of product (AUTO7/ABC) [0786] dual
transduced with vector A. and B. yielding a mix of product
(AUTO7/AB) [0787] single transduced with vector A. yielding a
single product (AUTO7/A)
[0788] Cytotoxicity Assay
[0789] CAR T-cells were co-cultured with SupT1-NT and SupT1-PSMA at
an effector to target ratio (E:T ratio) of 1:2 or 1:8 a TC-treated
96-well plate. Readouts were taken 24 hours post co-culture by
staining with anti-CD3-PeCy7 and Qben10-APC to differentiate
effector T-cells and target cells, SYTOX Blue7-AAD dead cell stain
was used to exclude dead cells. Cytotoxicity readouts were accessed
by flow cytometry.
[0790] Cytotoxicity Assay in Presence of TGF
[0791] CAR T-cells were co-cultured with SupT1-NT and
SupT1-PSMAhigh and SupT1-PSMAlow at an effector to target ratio
(E:T ratio) of 1:2 or 1:8 a TC-treated 96-well plate. TGF.beta.1
was added at a concentration of 10 ng/ml on day 0, and cytotoxicity
readouts were accessed by flow cytometry on day 7.
[0792] In Vitro Re-Stimulation Assay
[0793] CAR T-cells were co-cultured with SupT1-PSMAhigh or
SupT1-PSMAlow target cells at 1:1 ratio (E:T). Every 7 days CAR
T-cells were re-stimulated with 5.times.10.sup.4 SupT1 cells.
Cytotoxicity was assessed by FBK assay as describe above, and
target:effectors ratio after re-stimulation was ascended by
cytofluorimetric analisis. Supernatants were collected to assess
cytokine release.
[0794] Cytokine ELISA
[0795] Human IL-2 ELISA MAX.TM. Deluxe and Human IFN-.gamma. ELISA
MAX.TM. Deluxe kits were used to access the levels of cytokine
secreted into co-culture supernatants taken from Cytotoxicity
Assay.
[0796] In Vivo Experiment
[0797] 5.times.10.sup.6 PSMA positive PC3 human cell lines were
injected in the flank of female NSG mice. Xenografts were left to
establish for 3 weeks until stable engraftment was detectable by
palpation and calliper measurement. Human PBMC were created by
single, double or triple transduction. CAR T-cells were
administered i.v. at a dose of 1.times.10.sup.6 CAR T-cells/mouse.
Calliper measurement was taken 2/3 times a week and followed up for
until the end of the animal protocol.
[0798] All publications mentioned in the above specification are
herein incorporated by reference. Various modifications and
variations of the described methods and system of the invention
will be apparent to those skilled in the art without departing from
the scope and spirit of the invention. Although the invention has
been described in connection with specific preferred embodiments,
it should be understood that the invention as claimed should not be
unduly limited to such specific embodiments. Indeed, various
modifications of the described modes for carrying out the invention
which are obvious to those skilled in molecular biology or related
fields are intended to be within the scope of the following claims.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 81 <210> SEQ ID NO 1 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: heavy chain variable region
(VH) complementarity determining region (CDR) CDR1 <400>
SEQUENCE: 1 Gly Tyr Ala Phe Ser Ser Ser 1 5 <210> SEQ ID NO 2
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: VH CDR, CDR2 <400> SEQUENCE: 2 Tyr Pro Gly Asp
Glu Asp 1 5 <210> SEQ ID NO 3 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: VH CDR, CDR3
<400> SEQUENCE: 3 Ser Leu Leu Tyr Gly Asp Tyr Leu Asp Tyr 1 5
10 <210> SEQ ID NO 4 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: light chain variable region (VL)
CDR, CDR1 <400> SEQUENCE: 4 Ser Ala Ser Ser Ser Val Ser Tyr
Met His 1 5 10 <210> SEQ ID NO 5 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: VL CDR, CDR2
<400> SEQUENCE: 5 Asp Thr Ser Lys Leu Ala Ser 1 5 <210>
SEQ ID NO 6 <211> LENGTH: 9 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: VL CDR, CDR3 <400> SEQUENCE: 6 Gln Gln Trp
Asn Ile Asn Pro Leu Thr 1 5 <210> SEQ ID NO 7 <211>
LENGTH: 119 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: VH
domain sequence <400> SEQUENCE: 7 Gln Val Gln Leu Gln Gln Ser
Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser
Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Ser 20 25 30 Trp Met Asn
Trp Val Lys Gln Arg Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly
Arg Ile Tyr Pro Gly Asp Glu Asp Thr Asn Tyr Ser Gly Lys Phe 50 55
60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr
65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr
Phe Cys 85 90 95 Ala Arg Ser Leu Leu Tyr Gly Asp Tyr Leu Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115
<210> SEQ ID NO 8 <211> LENGTH: 107 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: VL domain sequence <400>
SEQUENCE: 8 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser
Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser
Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser
Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly
Val Pro Asp Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr
Phe Leu Thr Ile Asn Asn Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr
Tyr Tyr Cys Gln Gln Trp Asn Ile Asn Pro Leu Thr 85 90 95 Phe Gly
Ala Gly Thr Lys Leu Glu Leu Lys Arg 100 105 <210> SEQ ID NO 9
<211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM:
Foot-and-mouth disease virus <400> SEQUENCE: 9 Arg Ala Glu
Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu 1 5 10 15 Asn
Pro Gly Pro 20 <210> SEQ ID NO 10 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: VH CDR, CDR1
<400> SEQUENCE: 10 Asn Phe Ala Met Ala 1 5 <210> SEQ ID
NO 11 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: VH CDR, CDR2 <400> SEQUENCE: 11 Ser Ile
Ser Thr Gly Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val Lys 1 5 10 15
Gly <210> SEQ ID NO 12 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: VH CDR, CDR3 <400>
SEQUENCE: 12 Gln Arg Asn Tyr Tyr Asp Gly Ser Tyr Asp Tyr Glu Gly
Tyr Thr Met 1 5 10 15 Asp Ala <210> SEQ ID NO 13 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: VL
CDR, CDR1 <400> SEQUENCE: 13 Arg Ser Ser Gln Asp Ile Gly Asn
Tyr Leu Thr 1 5 10 <210> SEQ ID NO 14 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: VL CDR, CDR2
<400> SEQUENCE: 14 Gly Ala Ile Lys Leu Glu Asp 1 5
<210> SEQ ID NO 15 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: VL CDR, CDR3 <400> SEQUENCE:
15 Leu Gln Ser Ile Gln Tyr Pro 1 5 <210> SEQ ID NO 16
<211> LENGTH: 127 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: VH domain sequence <400> SEQUENCE: 16 Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe 20
25 30 Ala Met Ala Trp Val Arg Gln Pro Pro Thr Lys Gly Leu Glu Trp
Val 35 40 45 Ala Ser Ile Ser Thr Gly Gly Gly Asn Thr Tyr Tyr Arg
Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala
Lys Asn Thr Gln Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Ser Glu
Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Gln Arg Asn Tyr Tyr
Asp Gly Ser Tyr Asp Tyr Glu Gly Tyr 100 105 110 Thr Met Asp Ala Trp
Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 125 <210> SEQ
ID NO 17 <211> LENGTH: 107 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: VL domain sequence <400> SEQUENCE: 17 Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Asp Ile Gly Asn Tyr
20 25 30 Leu Thr Trp Phe Gln Gln Lys Val Gly Arg Ser Pro Arg Arg
Met Ile 35 40 45 Tyr Gly Ala Ile Lys Leu Glu Asp Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr
Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Val Ala Asp Tyr Gln Cys
Leu Gln Ser Ile Gln Tyr Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr
Lys Leu Glu Ile Lys 100 105 <210> SEQ ID NO 18 <211>
LENGTH: 254 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
tyrosine kinase domain of Fyn <400> SEQUENCE: 18 Leu Gln Leu
Ile Lys Arg Leu Gly Asn Gly Gln Phe Gly Glu Val Trp 1 5 10 15 Met
Gly Thr Trp Asn Gly Asn Thr Lys Val Ala Ile Lys Thr Leu Lys 20 25
30 Pro Gly Thr Met Ser Pro Glu Ser Phe Leu Glu Glu Ala Gln Ile Met
35 40 45 Lys Lys Leu Lys His Asp Lys Leu Val Gln Leu Tyr Ala Val
Val Ser 50 55 60 Glu Glu Pro Ile Tyr Ile Val Thr Glu Tyr Met Asn
Lys Gly Ser Leu 65 70 75 80 Leu Asp Phe Leu Lys Asp Gly Glu Gly Arg
Ala Leu Lys Leu Pro Asn 85 90 95 Leu Val Asp Met Ala Ala Gln Val
Ala Ala Gly Met Ala Tyr Ile Glu 100 105 110 Arg Met Asn Tyr Ile His
Arg Asp Leu Arg Ser Ala Asn Ile Leu Val 115 120 125 Gly Asn Gly Leu
Ile Cys Lys Ile Ala Asp Phe Gly Leu Ala Arg Leu 130 135 140 Ile Glu
Asp Asn Glu Tyr Thr Ala Arg Gln Gly Ala Lys Phe Pro Ile 145 150 155
160 Lys Trp Thr Ala Pro Glu Arg Ala Leu Tyr Gly Arg Phe Thr Ile Lys
165 170 175 Ser Asp Val Trp Ser Phe Gly Ile Leu Leu Thr Glu Leu Val
Thr Lys 180 185 190 Gly Arg Val Pro Tyr Pro Gly Met Asn Asn Arg Glu
Val Leu Glu Gln 195 200 205 Val Glu Arg Gly Tyr Arg Met Pro Cys Pro
Gln Asp Cys Pro Ile Ser 210 215 220 Leu His Glu Leu Met Ile His Cys
Trp Lys Lys Asp Pro Glu Glu Arg 225 230 235 240 Pro Thr Phe Glu Tyr
Leu Gln Ser Phe Leu Glu Asp Tyr Phe 245 250 <210> SEQ ID NO
19 <211> LENGTH: 254 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: tyrosine kinase domain of Src <400>
SEQUENCE: 19 Leu Arg Leu Glu Val Lys Leu Gly Gln Gly Cys Phe Gly
Glu Val Trp 1 5 10 15 Met Gly Thr Trp Asn Gly Thr Thr Arg Val Ala
Ile Lys Thr Leu Lys 20 25 30 Pro Gly Thr Met Ser Pro Glu Ala Phe
Leu Gln Glu Ala Gln Val Met 35 40 45 Lys Lys Leu Arg His Glu Lys
Leu Val Gln Leu Tyr Ala Val Val Ser 50 55 60 Glu Glu Pro Ile Tyr
Ile Val Thr Glu Tyr Met Ser Lys Gly Ser Leu 65 70 75 80 Leu Asp Phe
Leu Lys Gly Glu Thr Gly Lys Tyr Leu Arg Leu Pro Gln 85 90 95 Leu
Val Asp Met Ala Ala Gln Ile Ala Ser Gly Met Ala Tyr Val Glu 100 105
110 Arg Met Asn Tyr Val His Arg Asp Leu Arg Ala Ala Asn Ile Leu Val
115 120 125 Gly Glu Asn Leu Val Cys Lys Val Ala Asp Phe Gly Leu Ala
Arg Leu 130 135 140 Ile Glu Asp Asn Glu Tyr Thr Ala Arg Gln Gly Ala
Lys Phe Pro Ile 145 150 155 160 Lys Trp Thr Ala Pro Glu Ala Ala Leu
Tyr Gly Arg Phe Thr Ile Lys 165 170 175 Ser Asp Val Trp Ser Phe Gly
Ile Leu Leu Thr Glu Leu Thr Thr Lys 180 185 190 Gly Arg Val Pro Tyr
Pro Gly Met Val Asn Arg Glu Val Leu Asp Gln 195 200 205 Val Glu Arg
Gly Tyr Arg Met Pro Cys Pro Pro Glu Cys Pro Glu Ser 210 215 220 Leu
His Asp Leu Met Cys Gln Cys Trp Arg Lys Glu Pro Glu Glu Arg 225 230
235 240 Pro Thr Phe Glu Tyr Leu Gln Ala Phe Leu Glu Asp Tyr Phe 245
250 <210> SEQ ID NO 20 <211> LENGTH: 254 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: tyrosine kinase domain of
Lck <400> SEQUENCE: 20 Leu Lys Leu Val Glu Arg Leu Gly Ala
Gly Gln Phe Gly Glu Val Trp 1 5 10 15 Met Gly Tyr Tyr Asn Gly His
Thr Lys Val Ala Val Lys Ser Leu Lys 20 25 30 Gln Gly Ser Met Ser
Pro Asp Ala Phe Leu Ala Glu Ala Asn Leu Met 35 40 45 Lys Gln Leu
Gln His Gln Arg Leu Val Arg Leu Tyr Ala Val Val Thr 50 55 60 Gln
Glu Pro Ile Tyr Ile Ile Thr Glu Tyr Met Glu Asn Gly Ser Leu 65 70
75 80 Val Asp Phe Leu Lys Thr Pro Ser Gly Ile Lys Leu Thr Ile Asn
Lys 85 90 95 Leu Leu Asp Met Ala Ala Gln Ile Ala Glu Gly Met Ala
Phe Ile Glu 100 105 110 Glu Arg Asn Tyr Ile His Arg Asp Leu Arg Ala
Ala Asn Ile Leu Val 115 120 125 Ser Asp Thr Leu Ser Cys Lys Ile Ala
Asp Phe Gly Leu Ala Arg Leu 130 135 140 Ile Glu Asp Asn Glu Tyr Thr
Ala Arg Glu Gly Ala Lys Phe Pro Ile 145 150 155 160 Lys Trp Thr Ala
Pro Glu Ala Ile Asn Tyr Gly Thr Phe Thr Ile Lys 165 170 175 Ser Asp
Val Trp Ser Phe Gly Ile Leu Leu Thr Glu Ile Val Thr His 180 185 190
Gly Arg Ile Pro Tyr Pro Gly Met Thr Asn Pro Glu Val Ile Gln Asn 195
200 205 Leu Glu Arg Gly Tyr Arg Met Val Arg Pro Asp Asn Cys Pro Glu
Glu 210 215 220 Leu Tyr Gln Leu Met Arg Leu Cys Trp Lys Glu Arg Pro
Glu Asp Arg 225 230 235 240 Pro Thr Phe Asp Tyr Leu Arg Ser Val Leu
Glu Asp Phe Phe 245 250 <210> SEQ ID NO 21 <211>
LENGTH: 254 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
tyrosine kinase domain of Lck_Y505F <400> SEQUENCE: 21 Leu
Lys Leu Val Glu Arg Leu Gly Ala Gly Gln Phe Gly Glu Val Trp 1 5 10
15 Met Gly Tyr Tyr Asn Gly His Thr Lys Val Ala Val Arg Ser Leu Lys
20 25 30 Gln Gly Ser Met Ser Pro Asp Ala Phe Leu Ala Glu Ala Asn
Leu Met 35 40 45 Lys Gln Leu Gln His Gln Arg Leu Val Arg Leu Tyr
Ala Val Val Thr 50 55 60 Gln Glu Pro Ile Tyr Ile Ile Thr Glu Tyr
Met Glu Asn Gly Ser Leu 65 70 75 80 Val Asp Phe Leu Lys Thr Pro Ser
Gly Ile Lys Leu Thr Ile Asn Lys 85 90 95 Leu Leu Asp Met Ala Ala
Gln Ile Ala Glu Gly Met Ala Phe Ile Glu 100 105 110 Glu Arg Asn Tyr
Ile His Arg Asp Leu Arg Ala Ala Asn Ile Leu Val 115 120 125 Ser Asp
Thr Leu Ser Cys Lys Ile Ala Asp Phe Gly Leu Ala Arg Leu 130 135 140
Ile Glu Asp Asn Glu Tyr Thr Ala Arg Glu Gly Ala Lys Phe Pro Ile 145
150 155 160 Lys Trp Thr Ala Pro Glu Ala Ile Asn Tyr Gly Thr Phe Thr
Ile Lys 165 170 175 Ser Asp Val Trp Ser Phe Gly Ile Leu Leu Thr Glu
Ile Val Thr His 180 185 190 Gly Arg Ile Pro Tyr Pro Gly Met Thr Asn
Pro Glu Val Ile Gln Asn 195 200 205 Leu Glu Arg Gly Tyr Arg Met Val
Arg Pro Asp Asn Cys Pro Glu Glu 210 215 220 Leu Tyr Gln Leu Met Arg
Leu Cys Trp Lys Glu Arg Pro Glu Asp Arg 225 230 235 240 Pro Thr Phe
Asp Tyr Leu Arg Ser Val Leu Glu Asp Phe Phe 245 250 <210> SEQ
ID NO 22 <211> LENGTH: 40 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cytoplasmic tail of CD4 <400> SEQUENCE: 22
Cys Val Arg Cys Arg His Arg Arg Arg Gln Ala Glu Arg Met Ser Gln 1 5
10 15 Ile Lys Arg Leu Leu Ser Glu Lys Lys Thr Cys Gln Cys Pro His
Arg 20 25 30 Phe Gln Lys Thr Cys Ser Pro Ile 35 40 <210> SEQ
ID NO 23 <211> LENGTH: 32 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cytoplasmic tail of CD8 <400> SEQUENCE: 23
Leu Tyr Cys Asn His Arg Asn Arg Arg Arg Val Cys Lys Cys Pro Arg 1 5
10 15 Pro Val Val Lys Ser Gly Asp Lys Pro Ser Leu Ser Ala Arg Tyr
Val 20 25 30 <210> SEQ ID NO 24 <211> LENGTH: 214
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: SHP-1 SH2
complete domain <400> SEQUENCE: 24 Met Val Arg Trp Phe His
Arg Asp Leu Ser Gly Leu Asp Ala Glu Thr 1 5 10 15 Leu Leu Lys Gly
Arg Gly Val His Gly Ser Phe Leu Ala Arg Pro Ser 20 25 30 Arg Lys
Asn Gln Gly Asp Phe Ser Leu Ser Val Arg Val Gly Asp Gln 35 40 45
Val Thr His Ile Arg Ile Gln Asn Ser Gly Asp Phe Tyr Asp Leu Tyr 50
55 60 Gly Gly Glu Lys Phe Ala Thr Leu Thr Glu Leu Val Glu Tyr Tyr
Thr 65 70 75 80 Gln Gln Gln Gly Val Leu Gln Asp Arg Asp Gly Thr Ile
Ile His Leu 85 90 95 Lys Tyr Pro Leu Asn Cys Ser Asp Pro Thr Ser
Glu Arg Trp Tyr His 100 105 110 Gly His Met Ser Gly Gly Gln Ala Glu
Thr Leu Leu Gln Ala Lys Gly 115 120 125 Glu Pro Trp Thr Phe Leu Val
Arg Glu Ser Leu Ser Gln Pro Gly Asp 130 135 140 Phe Val Leu Ser Val
Leu Ser Asp Gln Pro Lys Ala Gly Pro Gly Ser 145 150 155 160 Pro Leu
Arg Val Thr His Ile Lys Val Met Cys Glu Gly Gly Arg Tyr 165 170 175
Thr Val Gly Gly Leu Glu Thr Phe Asp Ser Leu Thr Asp Leu Val Glu 180
185 190 His Phe Lys Lys Thr Gly Ile Glu Glu Ala Ser Gly Ala Phe Val
Tyr 195 200 205 Leu Arg Gln Pro Tyr Tyr 210 <210> SEQ ID NO
25 <211> LENGTH: 97 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: SHP-1 SH2 1 <400> SEQUENCE: 25 Trp Phe His
Arg Asp Leu Ser Gly Leu Asp Ala Glu Thr Leu Leu Lys 1 5 10 15 Gly
Arg Gly Val His Gly Ser Phe Leu Ala Arg Pro Ser Arg Lys Asn 20 25
30 Gln Gly Asp Phe Ser Leu Ser Val Arg Val Gly Asp Gln Val Thr His
35 40 45 Ile Arg Ile Gln Asn Ser Gly Asp Phe Tyr Asp Leu Tyr Gly
Gly Glu 50 55 60 Lys Phe Ala Thr Leu Thr Glu Leu Val Glu Tyr Tyr
Thr Gln Gln Gln 65 70 75 80 Gly Val Leu Gln Asp Arg Asp Gly Thr Ile
Ile His Leu Lys Tyr Pro 85 90 95 Leu <210> SEQ ID NO 26
<211> LENGTH: 104 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SHP-1 SH2 2 <400> SEQUENCE: 26 Trp Tyr His Gly
His Met Ser Gly Gly Gln Ala Glu Thr Leu Leu Gln 1 5 10 15 Ala Lys
Gly Glu Pro Trp Thr Phe Leu Val Arg Glu Ser Leu Ser Gln 20 25 30
Pro Gly Asp Phe Val Leu Ser Val Leu Ser Asp Gln Pro Lys Ala Gly 35
40 45 Pro Gly Ser Pro Leu Arg Val Thr His Ile Lys Val Met Cys Glu
Gly 50 55 60 Gly Arg Tyr Thr Val Gly Gly Leu Glu Thr Phe Asp Ser
Leu Thr Asp 65 70 75 80 Leu Val Glu His Phe Lys Lys Thr Gly Ile Glu
Glu Ala Ser Gly Ala 85 90 95 Phe Val Tyr Leu Arg Gln Pro Tyr 100
<210> SEQ ID NO 27 <211> LENGTH: 97 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SHP-2 first SH2 domain <400>
SEQUENCE: 27 Trp Phe His Pro Asn Ile Thr Gly Val Glu Ala Glu Asn
Leu Leu Leu 1 5 10 15 Thr Arg Gly Val Asp Gly Ser Phe Leu Ala Arg
Pro Ser Lys Ser Asn 20 25 30 Pro Gly Asp Phe Thr Leu Ser Val Arg
Arg Asn Gly Ala Val Thr His 35 40 45 Ile Lys Ile Gln Asn Thr Gly
Asp Tyr Tyr Asp Leu Tyr Gly Gly Glu 50 55 60 Lys Phe Ala Thr Leu
Ala Glu Leu Val Gln Tyr Tyr Met Glu His His 65 70 75 80 Gly Gln Leu
Lys Glu Lys Asn Gly Asp Val Ile Glu Leu Lys Tyr Pro 85 90 95 Leu
<210> SEQ ID NO 28 <211> LENGTH: 105 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SHP-2 second SH2 domain <400>
SEQUENCE: 28 Trp Phe His Gly His Leu Ser Gly Lys Glu Ala Glu Lys
Leu Leu Thr 1 5 10 15 Glu Lys Gly Lys His Gly Ser Phe Leu Val Arg
Glu Ser Gln Ser His 20 25 30 Pro Gly Asp Phe Val Leu Ser Val Arg
Thr Gly Asp Asp Lys Gly Glu 35 40 45 Ser Asn Asp Gly Lys Ser Lys
Val Thr His Val Met Ile Arg Cys Gln 50 55 60 Glu Leu Lys Tyr Asp
Val Gly Gly Gly Glu Arg Phe Asp Ser Leu Thr 65 70 75 80 Asp Leu Val
Glu His Tyr Lys Lys Asn Pro Met Val Glu Thr Leu Gly 85 90 95 Thr
Val Leu Gln Leu Lys Gln Pro Leu 100 105 <210> SEQ ID NO 29
<211> LENGTH: 211 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SHP-2 both SH2 domains <400> SEQUENCE: 29 Trp
Phe His Pro Asn Ile Thr Gly Val Glu Ala Glu Asn Leu Leu Leu 1 5 10
15 Thr Arg Gly Val Asp Gly Ser Phe Leu Ala Arg Pro Ser Lys Ser Asn
20 25 30 Pro Gly Asp Phe Thr Leu Ser Val Arg Arg Asn Gly Ala Val
Thr His 35 40 45 Ile Lys Ile Gln Asn Thr Gly Asp Tyr Tyr Asp Leu
Tyr Gly Gly Glu 50 55 60 Lys Phe Ala Thr Leu Ala Glu Leu Val Gln
Tyr Tyr Met Glu His His 65 70 75 80 Gly Gln Leu Lys Glu Lys Asn Gly
Asp Val Ile Glu Leu Lys Tyr Pro 85 90 95 Leu Asn Cys Ala Asp Pro
Thr Ser Glu Arg Trp Phe His Gly His Leu 100 105 110 Ser Gly Lys Glu
Ala Glu Lys Leu Leu Thr Glu Lys Gly Lys His Gly 115 120 125 Ser Phe
Leu Val Arg Glu Ser Gln Ser His Pro Gly Asp Phe Val Leu 130 135 140
Ser Val Arg Thr Gly Asp Asp Lys Gly Glu Ser Asn Asp Gly Lys Ser 145
150 155 160 Lys Val Thr His Val Met Ile Arg Cys Gln Glu Leu Lys Tyr
Asp Val 165 170 175 Gly Gly Gly Glu Arg Phe Asp Ser Leu Thr Asp Leu
Val Glu His Tyr 180 185 190 Lys Lys Asn Pro Met Val Glu Thr Leu Gly
Thr Val Leu Gln Leu Lys 195 200 205 Gln Pro Leu 210 <210> SEQ
ID NO 30 <211> LENGTH: 107 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Human Light Kappa Chain <400> SEQUENCE: 30
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5
10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser
Phe Asn Arg Gly Glu Cys 100 105 <210> SEQ ID NO 31
<211> LENGTH: 19 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Human Hinge <400> SEQUENCE: 31 Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Lys 1 5 10 15 Asp Pro
Lys <210> SEQ ID NO 32 <211> LENGTH: 97 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Human CH1 <400>
SEQUENCE: 32 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser 1 5 10 15 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe 20 25 30 Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly 35 40 45 Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu 50 55 60 Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr 65 70 75 80 Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg 85 90 95 Val
<210> SEQ ID NO 33 <211> LENGTH: 21 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Transmembrane domain from human IL2R
common gamma chain <400> SEQUENCE: 33 Val Val Ile Ser Val Gly
Ser Met Gly Leu Ile Ile Ser Leu Leu Cys 1 5 10 15 Val Tyr Phe Trp
Leu 20 <210> SEQ ID NO 34 <211> LENGTH: 25 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Transmembrane domain from
human IL-2R beta <400> SEQUENCE: 34 Ile Pro Trp Leu Gly His
Leu Leu Val Gly Leu Ser Gly Ala Phe Gly 1 5 10 15 Phe Ile Ile Leu
Val Tyr Leu Leu Ile 20 25 <210> SEQ ID NO 35 <400>
SEQUENCE: 35 000 <210> SEQ ID NO 36 <211> LENGTH: 25
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Transmembrane
domain from human IL-7R alpha <400> SEQUENCE: 36 Pro Ile Leu
Leu Thr Ile Ser Ile Leu Ser Phe Phe Ser Val Ala Leu 1 5 10 15 Leu
Val Ile Leu Ala Cys Val Leu Trp 20 25 <210> SEQ ID NO 37
<211> LENGTH: 26 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Transmembrane domain from Human GF-CSFR alpha
<400> SEQUENCE: 37 Asn Leu Gly Ser Val Tyr Ile Tyr Val Leu
Leu Ile Val Gly Thr Leu 1 5 10 15 Val Cys Gly Ile Val Leu Gly Phe
Leu Phe 20 25 <210> SEQ ID NO 38 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Transmembrane
domain from Human GM-CSFR common beta chain <400> SEQUENCE:
38 Val Leu Ala Leu Ile Val Ile Phe Leu Thr Ile Ala Val Leu Leu Ala
1 5 10 15 Leu <210> SEQ ID NO 39 <211> LENGTH: 86
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Endodomain from
human IL2R common gamma chain <400> SEQUENCE: 39 Glu Arg Thr
Met Pro Arg Ile Pro Thr Leu Lys Asn Leu Glu Asp Leu 1 5 10 15 Val
Thr Glu Tyr His Gly Asn Phe Ser Ala Trp Ser Gly Val Ser Lys 20 25
30 Gly Leu Ala Glu Ser Leu Gln Pro Asp Tyr Ser Glu Arg Leu Cys Leu
35 40 45 Val Ser Glu Ile Pro Pro Lys Gly Gly Ala Leu Gly Glu Gly
Pro Gly 50 55 60 Ala Ser Pro Cys Asn Gln His Ser Pro Tyr Trp Ala
Pro Pro Cys Tyr 65 70 75 80 Thr Leu Lys Pro Glu Thr 85 <210>
SEQ ID NO 40 <211> LENGTH: 286 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Endodomain from human IL-2R beta
<400> SEQUENCE: 40 Asn Cys Arg Asn Thr Gly Pro Trp Leu Lys
Lys Val Leu Lys Cys Asn 1 5 10 15 Thr Pro Asp Pro Ser Lys Phe Phe
Ser Gln Leu Ser Ser Glu His Gly 20 25 30 Gly Asp Val Gln Lys Trp
Leu Ser Ser Pro Phe Pro Ser Ser Ser Phe 35 40 45 Ser Pro Gly Gly
Leu Ala Pro Glu Ile Ser Pro Leu Glu Val Leu Glu 50 55 60 Arg Asp
Lys Val Thr Gln Leu Leu Leu Gln Gln Asp Lys Val Pro Glu 65 70 75 80
Pro Ala Ser Leu Ser Ser Asn His Ser Leu Thr Ser Cys Phe Thr Asn 85
90 95 Gln Gly Tyr Phe Phe Phe His Leu Pro Asp Ala Leu Glu Ile Glu
Ala 100 105 110 Cys Gln Val Tyr Phe Thr Tyr Asp Pro Tyr Ser Glu Glu
Asp Pro Asp 115 120 125 Glu Gly Val Ala Gly Ala Pro Thr Gly Ser Ser
Pro Gln Pro Leu Gln 130 135 140 Pro Leu Ser Gly Glu Asp Asp Ala Tyr
Cys Thr Phe Pro Ser Arg Asp 145 150 155 160 Asp Leu Leu Leu Phe Ser
Pro Ser Leu Leu Gly Gly Pro Ser Pro Pro 165 170 175 Ser Thr Ala Pro
Gly Gly Ser Gly Ala Gly Glu Glu Arg Met Pro Pro 180 185 190 Ser Leu
Gln Glu Arg Val Pro Arg Asp Trp Asp Pro Gln Pro Leu Gly 195 200 205
Pro Pro Thr Pro Gly Val Pro Asp Leu Val Asp Phe Gln Pro Pro Pro 210
215 220 Glu Leu Val Leu Arg Glu Ala Gly Glu Glu Val Pro Asp Ala Gly
Pro 225 230 235 240 Arg Glu Gly Val Ser Phe Pro Trp Ser Arg Pro Pro
Gly Gln Gly Glu 245 250 255 Phe Arg Ala Leu Asn Ala Arg Leu Pro Leu
Asn Thr Asp Ala Tyr Leu 260 265 270 Ser Leu Gln Glu Leu Gln Gly Gln
Asp Pro Thr His Leu Val 275 280 285 <210> SEQ ID NO 41
<211> LENGTH: 196 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Endodomain from human IL-7R alpha <400>
SEQUENCE: 41 Lys Lys Arg Ile Lys Pro Ile Val Trp Pro Ser Leu Pro
Asp His Lys 1 5 10 15 Lys Thr Leu Glu His Leu Cys Lys Lys Pro Arg
Lys Asn Leu Asn Val 20 25 30 Ser Phe Asn Pro Glu Ser Phe Leu Asp
Cys Gln Ile His Arg Val Asp 35 40 45 Asp Ile Gln Ala Arg Asp Glu
Val Glu Gly Phe Leu Gln Asp Thr Phe 50 55 60 Pro Gln Gln Leu Glu
Glu Ser Glu Lys Gln Arg Leu Gly Gly Asp Val 65 70 75 80 Gln Ser Pro
Asn Cys Pro Ser Glu Asp Val Val Ile Thr Pro Glu Ser 85 90 95 Phe
Gly Arg Asp Ser Ser Leu Thr Cys Leu Ala Gly Asn Val Ser Ala 100 105
110 Cys Asp Ala Pro Ile Leu Ser Ser Ser Arg Ser Leu Asp Cys Arg Glu
115 120 125 Ser Gly Lys Asn Gly Pro His Val Tyr Gln Asp Leu Leu Leu
Ser Leu 130 135 140 Gly Thr Thr Asn Ser Thr Leu Pro Pro Pro Phe Ser
Leu Gln Ser Gly 145 150 155 160 Ile Leu Thr Leu Asn Pro Val Ala Gln
Gly Gln Pro Ile Leu Thr Ser 165 170 175 Leu Gly Ser Asn Gln Glu Glu
Ala Tyr Val Thr Met Ser Ser Phe Tyr 180 185 190 Gln Asn Glu Gln 195
<210> SEQ ID NO 42 <211> LENGTH: 54 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Endodomain Derived from Human
GM-CSFR alpha <400> SEQUENCE: 42 Lys Arg Phe Leu Arg Ile Gln
Arg Leu Phe Pro Pro Val Pro Gln Ile 1 5 10 15 Lys Asp Lys Leu Asn
Asp Asn His Glu Val Glu Asp Glu Ile Ile Trp 20 25 30 Glu Glu Phe
Thr Pro Glu Glu Gly Lys Gly Tyr Arg Glu Glu Val Leu 35 40 45 Thr
Val Lys Glu Ile Thr 50 <210> SEQ ID NO 43 <211> LENGTH:
437 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Endodomain from
GM-CSFR common beta chain <400> SEQUENCE: 43 Arg Phe Cys Gly
Ile Tyr Gly Tyr Arg Leu Arg Arg Lys Trp Glu Glu 1 5 10 15 Lys Ile
Pro Asn Pro Ser Lys Ser His Leu Phe Gln Asn Gly Ser Ala 20 25 30
Glu Leu Trp Pro Pro Gly Ser Met Ser Ala Phe Thr Ser Gly Ser Pro 35
40 45 Pro His Gln Gly Pro Trp Gly Ser Arg Phe Pro Glu Leu Glu Gly
Val 50 55 60 Phe Pro Val Gly Phe Gly Asp Ser Glu Val Ser Pro Leu
Thr Ile Glu 65 70 75 80 Asp Pro Lys His Val Cys Asp Pro Pro Ser Gly
Pro Asp Thr Thr Pro 85 90 95 Ala Ala Ser Asp Leu Pro Thr Glu Gln
Pro Pro Ser Pro Gln Pro Gly 100 105 110 Pro Pro Ala Ala Ser His Thr
Pro Glu Lys Gln Ala Ser Ser Phe Asp 115 120 125 Phe Asn Gly Pro Tyr
Leu Gly Pro Pro His Ser Arg Ser Leu Pro Asp 130 135 140 Ile Leu Gly
Gln Pro Glu Pro Pro Gln Glu Gly Gly Ser Gln Lys Ser 145 150 155 160
Pro Pro Pro Gly Ser Leu Glu Tyr Leu Cys Leu Pro Ala Gly Gly Gln 165
170 175 Val Gln Leu Val Pro Leu Ala Gln Ala Met Gly Pro Gly Gln Ala
Val 180 185 190 Glu Val Glu Arg Arg Pro Ser Gln Gly Ala Ala Gly Ser
Pro Ser Leu 195 200 205 Glu Ser Gly Gly Gly Pro Ala Pro Pro Ala Leu
Gly Pro Arg Val Gly 210 215 220 Gly Gln Asp Gln Lys Asp Ser Pro Val
Ala Ile Pro Met Ser Ser Gly 225 230 235 240 Asp Thr Glu Asp Pro Gly
Val Ala Ser Gly Tyr Val Ser Ser Ala Asp 245 250 255 Leu Val Phe Thr
Pro Asn Ser Gly Ala Ser Ser Val Ser Leu Val Pro 260 265 270 Ser Leu
Gly Leu Pro Ser Asp Gln Thr Pro Ser Leu Cys Pro Gly Leu 275 280 285
Ala Ser Gly Pro Pro Gly Ala Pro Gly Pro Val Lys Ser Gly Phe Glu 290
295 300 Gly Tyr Val Glu Leu Pro Pro Ile Glu Gly Arg Ser Pro Arg Ser
Pro 305 310 315 320 Arg Asn Asn Pro Val Pro Pro Glu Ala Lys Ser Pro
Val Leu Asn Pro 325 330 335 Gly Glu Arg Pro Ala Asp Val Ser Pro Thr
Ser Pro Gln Pro Glu Gly 340 345 350 Leu Leu Val Leu Gln Gln Val Gly
Asp Tyr Cys Phe Leu Pro Gly Leu 355 360 365 Gly Pro Gly Pro Leu Ser
Leu Arg Ser Lys Pro Ser Ser Pro Gly Pro 370 375 380 Gly Pro Glu Ile
Lys Asn Leu Asp Gln Ala Phe Gln Val Lys Lys Pro 385 390 395 400 Pro
Gly Gln Ala Val Pro Gln Val Pro Val Ile Gln Leu Phe Lys Ala 405 410
415 Leu Lys Gln Gln Asp Tyr Leu Ser Leu Pro Pro Trp Glu Val Asn Lys
420 425 430 Pro Gly Glu Val Cys 435 <210> SEQ ID NO 44
<211> LENGTH: 112 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: mature protein of wild-type TGFbeta2 <400>
SEQUENCE: 44 Ala Leu Asp Ala Ala Tyr Cys Phe Arg Asn Val Gln Asp
Asn Cys Cys 1 5 10 15 Leu Arg Pro Leu Tyr Ile Asp Phe Lys Arg Asp
Leu Gly Trp Lys Trp 20 25 30 Ile His Glu Pro Lys Gly Tyr Asn Ala
Asn Phe Cys Ala Gly Ala Cys 35 40 45 Pro Tyr Leu Trp Ser Ser Asp
Thr Gln His Ser Arg Val Leu Ser Leu 50 55 60 Tyr Asn Thr Ile Asn
Pro Glu Ala Ser Ala Ser Pro Cys Cys Val Ser 65 70 75 80 Gln Asp Leu
Glu Pro Leu Thr Ile Leu Tyr Tyr Ile Gly Lys Thr Pro 85 90 95 Lys
Ile Glu Gln Leu Ser Asn Met Ile Val Lys Ser Cys Lys Cys Ser 100 105
110 <210> SEQ ID NO 45 <211> LENGTH: 394 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: TGFbeta monomer sequence
<400> SEQUENCE: 45 Met His Tyr Cys Val Leu Ser Ala Phe Leu
Ile Leu His Leu Val Thr 1 5 10 15 Val Ala Leu Ser Leu Ser Thr Cys
Ser Thr Leu Asp Met Asp Gln Phe 20 25 30 Met Arg Lys Arg Ile Glu
Ala Ile Arg Gly Gln Ile Leu Ser Lys Leu 35 40 45 Lys Leu Thr Ser
Pro Pro Glu Asp Tyr Pro Glu Pro Glu Glu Val Pro 50 55 60 Pro Glu
Val Ile Ser Ile Tyr Asn Ser Thr Arg Asp Leu Leu Gln Glu 65 70 75 80
Lys Ala Ser Arg Arg Ala Ala Ala Cys Glu Arg Glu Arg Ser Asp Glu 85
90 95 Glu Tyr Tyr Ala Lys Glu Val Tyr Lys Ile Asp Met Pro Pro Phe
Phe 100 105 110 Pro Ser Glu Asn Ala Ile Pro Pro Thr Phe Tyr Arg Pro
Tyr Phe Arg 115 120 125 Ile Val Arg Phe Asp Val Ser Ala Met Glu Lys
Asn Ala Ser Asn Leu 130 135 140 Val Lys Ala Glu Phe Arg Val Phe Arg
Leu Gln Asn Pro Lys Ala Arg 145 150 155 160 Val Pro Glu Gln Arg Ile
Glu Leu Tyr Gln Ile Leu Lys Ser Lys Asp 165 170 175 Leu Thr Ser Pro
Thr Gln Arg Tyr Ile Asp Ser Lys Val Val Lys Thr 180 185 190 Arg Ala
Glu Gly Glu Trp Leu Ser Phe Asp Val Thr Asp Ala Val His 195 200 205
Glu Trp Leu His His Lys Asp Arg Asn Leu Gly Phe Lys Ile Ser Leu 210
215 220 His Cys Pro Cys Cys Thr Phe Val Pro Ser Asn Asn Tyr Ile Ile
Pro 225 230 235 240 Asn Lys Ser Glu Glu Leu Glu Ala Arg Phe Ala Gly
Ile Asp Gly Thr 245 250 255 Ser Thr Tyr Thr Ser Gly Asp Gln Lys Thr
Ile Lys Ser Thr Arg Lys 260 265 270 Lys Asn Ser Gly Lys Thr Pro His
Leu Leu Leu Met Leu Leu Pro Ser 275 280 285 Tyr Arg Leu Glu Ser Gln
Gln Thr Asn Arg Arg Lys Lys Arg Ala Leu 290 295 300 Asp Ala Ala Tyr
Cys Phe Arg Asn Val Gln Asp Asn Cys Cys Leu Arg 305 310 315 320 Pro
Leu Tyr Ile Asp Phe Arg Lys Asp Leu Gly Trp Lys Trp Ile His 325 330
335 Glu Pro Lys Gly Tyr Asn Ala Asn Phe Cys Ala Gly Ala Cys Pro Tyr
340 345 350 Arg Ala Ser Lys Ser Pro Ser Cys Val Ser Gln Asp Leu Glu
Pro Leu 355 360 365 Thr Ile Val Tyr Tyr Val Gly Arg Lys Pro Lys Val
Glu Gln Leu Ser 370 375 380 Asn Met Ile Val Lys Ser Cys Lys Cys Ser
385 390 <210> SEQ ID NO 46 <211> LENGTH: 177
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION:
dominant-negative TGFbeta RII (dnTGFbeta RII) <400> SEQUENCE:
46 Thr Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val
1 5 10 15 Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys
Phe Cys 20 25 30 Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser
Cys Met Ser Asn 35 40 45 Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro
Gln Glu Val Cys Val Ala 50 55 60 Val Trp Arg Lys Asn Asp Glu Asn
Ile Thr Leu Glu Thr Val Cys His 65 70 75 80 Asp Pro Lys Leu Pro Tyr
His Asp Phe Ile Leu Glu Asp Ala Ala Ser 85 90 95 Pro Lys Cys Ile
Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe 100 105 110 Met Cys
Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser 115 120 125
Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu Leu Leu Val Ile Phe Gln 130
135 140 Val Thr Gly Ile Ser Leu Leu Pro Pro Leu Gly Val Ala Ile Ser
Val 145 150 155 160 Ile Ile Ile Phe Tyr Cys Tyr Arg Val Asn Arg Gln
Gln Lys Leu Ser 165 170 175 Ser <210> SEQ ID NO 47
<211> LENGTH: 194 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: MH2 domain of SMAD2 <400> SEQUENCE: 47 Trp Cys
Ser Ile Ala Tyr Tyr Glu Leu Asn Gln Arg Val Gly Glu Thr 1 5 10 15
Phe His Ala Ser Gln Pro Ser Leu Thr Val Asp Gly Phe Thr Asp Pro 20
25 30 Ser Asn Ser Glu Arg Phe Cys Leu Gly Leu Leu Ser Asn Val Asn
Arg 35 40 45 Asn Ala Thr Val Glu Met Thr Arg Arg His Ile Gly Arg
Gly Val Arg 50 55 60 Leu Tyr Tyr Ile Gly Gly Glu Val Phe Ala Glu
Cys Leu Ser Asp Ser 65 70 75 80 Ala Ile Phe Val Gln Ser Pro Asn Cys
Asn Gln Arg Tyr Gly Trp His 85 90 95 Pro Ala Thr Val Cys Lys Ile
Pro Pro Gly Cys Asn Leu Lys Ile Phe 100 105 110 Asn Asn Gln Glu Phe
Ala Ala Leu Leu Ala Gln Ser Val Asn Gln Gly 115 120 125 Phe Glu Ala
Val Tyr Gln Leu Thr Arg Met Cys Thr Ile Arg Met Ser 130 135 140 Phe
Val Lys Gly Trp Gly Ala Glu Tyr Arg Arg Gln Thr Val Thr Ser 145 150
155 160 Thr Pro Cys Trp Ile Glu Leu His Leu Asn Gly Pro Leu Gln Trp
Leu 165 170 175 Asp Lys Val Leu Thr Gln Met Gly Ser Pro Ser Val Arg
Cys Ser Ser 180 185 190 Met Ser <210> SEQ ID NO 48
<211> LENGTH: 194 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: MH2 domain of SMAD3 <400> SEQUENCE: 48 Trp Cys
Ser Ile Ser Tyr Tyr Glu Leu Asn Gln Arg Val Gly Glu Thr 1 5 10 15
Phe His Ala Ser Gln Pro Ser Met Thr Val Asp Gly Phe Thr Asp Pro 20
25 30 Ser Asn Ser Glu Arg Phe Cys Leu Gly Leu Leu Ser Asn Val Asn
Arg 35 40 45 Asn Ala Ala Val Glu Leu Thr Arg Arg His Ile Gly Arg
Gly Val Arg 50 55 60 Leu Tyr Tyr Ile Gly Gly Glu Val Phe Ala Glu
Cys Leu Ser Asp Ser 65 70 75 80 Ala Ile Phe Val Gln Ser Pro Asn Cys
Asn Gln Arg Tyr Gly Trp His 85 90 95 Pro Ala Thr Val Cys Lys Ile
Pro Pro Gly Cys Asn Leu Lys Ile Phe 100 105 110 Asn Asn Gln Glu Phe
Ala Ala Leu Leu Ala Gln Ser Val Asn Gln Gly 115 120 125 Phe Glu Ala
Val Tyr Gln Leu Thr Arg Met Cys Thr Ile Arg Met Ser 130 135 140 Phe
Val Lys Gly Trp Gly Ala Glu Tyr Arg Arg Gln Thr Val Thr Ser 145 150
155 160 Thr Pro Cys Trp Ile Glu Leu His Leu Asn Gly Pro Leu Gln Trp
Leu 165 170 175 Asp Lys Val Leu Thr Gln Met Gly Ser Pro Ser Ile Arg
Cys Ser Ser 180 185 190 Val Ser <210> SEQ ID NO 49
<211> LENGTH: 230 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: MH2 domain of SMAD4 <400> SEQUENCE: 49 Trp Cys
Ser Ile Ala Tyr Phe Glu Met Asp Val Gln Val Gly Glu Thr 1 5 10 15
Phe Lys Val Pro Ser Ser Cys Pro Ile Val Thr Val Asp Gly Tyr Val 20
25 30 Asp Pro Ser Gly Gly Asp Arg Phe Cys Leu Gly Gln Leu Ser Asn
Val 35 40 45 His Arg Thr Glu Ala Ile Glu Arg Ala Arg Leu His Ile
Gly Lys Gly 50 55 60 Val Gln Leu Glu Cys Lys Gly Glu Gly Asp Val
Trp Val Arg Cys Leu 65 70 75 80 Ser Asp His Ala Val Phe Val Gln Ser
Tyr Tyr Leu Asp Arg Glu Ala 85 90 95 Gly Arg Ala Pro Gly Asp Ala
Val His Lys Ile Tyr Pro Ser Ala Tyr 100 105 110 Ile Lys Val Phe Asp
Leu Arg Gln Cys His Arg Gln Met Gln Gln Gln 115 120 125 Ala Ala Thr
Ala Gln Ala Ala Ala Ala Ala Gln Ala Ala Ala Val Ala 130 135 140 Gly
Asn Ile Pro Gly Pro Gly Ser Val Gly Gly Ile Ala Pro Ala Ile 145 150
155 160 Ser Leu Ser Ala Ala Ala Gly Ile Gly Val Asp Asp Leu Arg Arg
Leu 165 170 175 Cys Ile Leu Arg Met Ser Phe Val Lys Gly Trp Gly Pro
Asp Tyr Pro 180 185 190 Arg Gln Ser Ile Lys Glu Thr Pro Cys Trp Ile
Glu Ile His Leu His 195 200 205 Arg Ala Leu Gln Leu Leu Asp Glu Val
Leu His Thr Met Pro Ile Ala 210 215 220 Asp Pro Gln Pro Leu Asp 225
230 <210> SEQ ID NO 50 <211> LENGTH: 380 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: chimeric dnSMAD comprising
a dnSMAD2 polypeptide and a dnSMAD3 polypeptide <400>
SEQUENCE: 50 Trp Cys Ser Ile Ala Tyr Tyr Glu Leu Asn Gln Arg Val
Gly Glu Thr 1 5 10 15 Phe His Ala Ser Gln Pro Ser Leu Thr Val Asp
Gly Phe Thr Asp Pro 20 25 30 Ser Asn Ser Glu Arg Phe Cys Leu Gly
Leu Leu Ser Asn Val Asn Arg 35 40 45 Asn Ala Thr Val Glu Met Thr
Arg Arg His Ile Gly Arg Gly Val Arg 50 55 60 Leu Tyr Tyr Ile Gly
Gly Glu Val Phe Ala Glu Cys Leu Ser Asp Ser 65 70 75 80 Ala Ile Phe
Val Gln Ser Pro Asn Cys Asn Gln Arg Tyr Gly Trp His 85 90 95 Pro
Ala Thr Val Cys Lys Ile Pro Pro Gly Cys Asn Leu Lys Ile Phe 100 105
110 Asn Asn Gln Glu Phe Ala Ala Leu Leu Ala Gln Ser Val Asn Gln Gly
115 120 125 Phe Glu Ala Val Tyr Gln Leu Thr Arg Met Cys Thr Ile Arg
Met Ser 130 135 140 Phe Val Lys Gly Trp Gly Ala Glu Tyr Arg Arg Gln
Thr Val Thr Ser 145 150 155 160 Thr Pro Cys Trp Ile Glu Leu His Leu
Asn Gly Pro Leu Gln Trp Leu 165 170 175 Asp Lys Val Leu Thr Gln Met
Leu Glu Tyr Ser Gly Gly Gly Ser Gly 180 185 190 Gly Gly Ser Leu Glu
Trp Cys Ser Ile Ser Tyr Tyr Glu Leu Asn Gln 195 200 205 Arg Val Gly
Glu Thr Phe His Ala Ser Gln Pro Ser Met Thr Val Asp 210 215 220 Gly
Phe Thr Asp Pro Ser Asn Ser Glu Arg Phe Cys Leu Gly Leu Leu 225 230
235 240 Ser Asn Val Asn Arg Asn Ala Ala Val Glu Leu Thr Arg Arg His
Ile 245 250 255 Gly Arg Gly Val Arg Leu Tyr Tyr Ile Gly Gly Glu Val
Phe Ala Glu 260 265 270 Cys Leu Ser Asp Ser Ala Ile Phe Val Gln Ser
Pro Asn Cys Asn Gln 275 280 285 Arg Tyr Gly Trp His Pro Ala Thr Val
Cys Lys Ile Pro Pro Gly Cys 290 295 300 Asn Leu Lys Ile Phe Asn Asn
Gln Glu Phe Ala Ala Leu Leu Ala Gln 305 310 315 320 Ser Val Asn Gln
Gly Phe Glu Ala Val Tyr Gln Leu Thr Arg Met Cys 325 330 335 Thr Ile
Arg Met Ser Phe Val Lys Gly Trp Gly Ala Glu Tyr Arg Arg 340 345 350
Gln Thr Val Thr Ser Thr Pro Cys Trp Ile Glu Leu His Leu Asn Gly 355
360 365 Pro Leu Gln Trp Leu Asp Lys Val Leu Thr Gln Met 370 375 380
<210> SEQ ID NO 51 <211> LENGTH: 271 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: chimeric TNF receptor HVEM-41BB
<400> SEQUENCE: 51 Met Glu Pro Pro Gly Asp Trp Gly Pro Pro
Pro Trp Arg Ser Thr Pro 1 5 10 15 Arg Thr Asp Val Leu Arg Leu Val
Leu Tyr Leu Thr Phe Leu Gly Ala 20 25 30 Pro Cys Tyr Ala Pro Ala
Leu Pro Ser Cys Lys Glu Asp Glu Tyr Pro 35 40 45 Val Gly Ser Glu
Cys Cys Pro Lys Cys Ser Pro Gly Tyr Arg Val Lys 50 55 60 Glu Ala
Cys Gly Glu Leu Thr Gly Thr Val Cys Glu Pro Cys Pro Pro 65 70 75 80
Gly Thr Tyr Ile Ala His Leu Asn Gly Leu Ser Lys Cys Leu Gln Cys 85
90 95 Gln Met Cys Asp Pro Ala Met Gly Leu Arg Ala Ser Arg Asn Cys
Ser 100 105 110 Arg Thr Glu Asn Ala Val Cys Gly Cys Ser Pro Gly His
Phe Cys Ile 115 120 125 Val Gln Asp Gly Asp His Cys Ala Ala Cys Arg
Ala Tyr Ala Thr Ser 130 135 140 Ser Pro Gly Gln Arg Val Gln Lys Gly
Gly Thr Glu Ser Gln Asp Thr 145 150 155 160 Leu Cys Gln Asn Cys Pro
Pro Gly Thr Phe Ser Pro Asn Gly Thr Leu 165 170 175 Glu Glu Cys Gln
His Gln Thr Lys Cys Ser Trp Leu Val Thr Lys Ala 180 185 190 Gly Ala
Gly Thr Ser Ser Ser His Trp Val Ile Ile Ser Phe Phe Leu 195 200 205
Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu 210
215 220 Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile
Phe 225 230 235 240 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln
Glu Glu Asp Gly 245 250 255 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu
Gly Gly Cys Glu Leu 260 265 270 <210> SEQ ID NO 52
<211> LENGTH: 260 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: chimeric TNF receptor CD27-41BB <400> SEQUENCE:
52 Met Ala Arg Pro His Pro Trp Trp Leu Cys Val Leu Gly Thr Leu Val
1 5 10 15 Gly Leu Ser Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg
His Tyr 20 25 30 Trp Ala Gln Gly Lys Leu Cys Cys Gln Met Cys Glu
Pro Gly Thr Phe 35 40 45 Leu Val Lys Asp Cys Asp Gln His Arg Lys
Ala Ala Gln Cys Asp Pro 50 55 60 Cys Ile Pro Gly Val Ser Phe Ser
Pro Asp His His Thr Arg Pro His 65 70 75 80 Cys Glu Ser Cys Arg His
Cys Asn Ser Gly Leu Leu Val Arg Asn Cys 85 90 95 Thr Ile Thr Ala
Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys 100 105 110 Arg Asp
Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu 115 120 125
Thr Ala Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His 130
135 140 Leu Pro Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His
Met 145 150 155 160 Gln Thr Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg
Thr Leu Ser Thr 165 170 175 His Trp Pro Pro Gln Arg Ser Leu Cys Ser
Ser Asp Phe Ile Arg Ile 180 185 190 Ile Ser Phe Phe Leu Ala Leu Thr
Ser Thr Ala Leu Leu Phe Leu Leu 195 200 205 Phe Phe Leu Thr Leu Arg
Phe Ser Val Val Lys Arg Gly Arg Lys Lys 210 215 220 Leu Leu Tyr Ile
Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr 225 230 235 240 Gln
Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly 245 250
255 Gly Cys Glu Leu 260 <210> SEQ ID NO 53 <211>
LENGTH: 281 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
chimeric TNF receptor RANK-41BB <400> SEQUENCE: 53 Met Ala
Pro Arg Ala Arg Arg Arg Arg Pro Leu Phe Ala Leu Leu Leu 1 5 10 15
Leu Cys Ala Leu Leu Ala Arg Leu Gln Val Ala Leu Gln Ile Ala Pro 20
25 30 Pro Cys Thr Ser Glu Lys His Tyr Glu His Leu Gly Arg Cys Cys
Asn 35 40 45 Lys Cys Glu Pro Gly Lys Tyr Met Ser Ser Lys Cys Thr
Thr Thr Ser 50 55 60 Asp Ser Val Cys Leu Pro Cys Gly Pro Asp Glu
Tyr Leu Asp Ser Trp 65 70 75 80 Asn Glu Glu Asp Lys Cys Leu Leu His
Lys Val Cys Asp Thr Gly Lys 85 90 95 Ala Leu Val Ala Val Val Ala
Gly Asn Ser Thr Thr Pro Arg Arg Cys 100 105 110 Ala Cys Thr Ala Gly
Tyr His Trp Ser Gln Asp Cys Glu Cys Cys Arg 115 120 125 Arg Asn Thr
Glu Cys Ala Pro Gly Leu Gly Ala Gln His Pro Leu Gln 130 135 140 Leu
Asn Lys Asp Thr Val Cys Lys Pro Cys Leu Ala Gly Tyr Phe Ser 145 150
155 160 Asp Ala Phe Ser Ser Thr Asp Lys Cys Arg Pro Trp Thr Asn Cys
Thr 165 170 175 Phe Leu Gly Lys Arg Val Glu His His Gly Thr Glu Lys
Ser Asp Ala 180 185 190 Val Cys Ser Ser Ser Leu Pro Ala Arg Lys Pro
Pro Asn Glu Pro His 195 200 205 Val Tyr Leu Pro Ile Ile Ser Phe Phe
Leu Ala Leu Thr Ser Thr Ala 210 215 220 Leu Leu Phe Leu Leu Phe Phe
Leu Thr Leu Arg Phe Ser Val Val Lys 225 230 235 240 Arg Gly Arg Lys
Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg 245 250 255 Pro Val
Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro 260 265 270
Glu Glu Glu Glu Gly Gly Cys Glu Leu 275 280 <210> SEQ ID NO
54 <211> LENGTH: 149 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: chimeric TNF receptor Fn14-41BB <400>
SEQUENCE: 54 Met Ala Arg Gly Ser Leu Arg Arg Leu Leu Arg Leu Leu
Val Leu Gly 1 5 10 15 Leu Trp Leu Ala Leu Leu Arg Ser Val Ala Gly
Glu Gln Ala Pro Gly 20 25 30 Thr Ala Pro Cys Ser Arg Gly Ser Ser
Trp Ser Ala Asp Leu Asp Lys 35 40 45 Cys Met Asp Cys Ala Ser Cys
Arg Ala Arg Pro His Ser Asp Phe Cys 50 55 60 Leu Gly Cys Ala Ala
Ala Pro Pro Ala Pro Phe Arg Leu Leu Trp Pro 65 70 75 80 Ile Ile Ser
Phe Phe Leu Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu 85 90 95 Leu
Phe Phe Leu Thr Leu Arg Phe Ser Val Val Lys Arg Gly Arg Lys 100 105
110 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr
115 120 125 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu
Glu Glu 130 135 140 Gly Gly Cys Glu Leu 145 <210> SEQ ID NO
55 <211> LENGTH: 261 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: TNF receptor ligand CD40L <400> SEQUENCE:
55 Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly
1 5 10 15 Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val
Phe Leu 20 25 30 Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val
Tyr Leu His Arg 35 40 45 Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn
Leu His Glu Asp Phe Val 50 55 60 Phe Met Lys Thr Ile Gln Arg Cys
Asn Thr Gly Glu Arg Ser Leu Ser 65 70 75 80 Leu Leu Asn Cys Glu Glu
Ile Lys Ser Gln Phe Glu Gly Phe Val Lys 85 90 95 Asp Ile Met Leu
Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu 100 105 110 Met Gln
Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser 115 120 125
Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly 130
135 140 Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys
Gln 145 150 155 160 Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr
Ala Gln Val Thr 165 170 175 Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln
Ala Pro Phe Ile Ala Ser 180 185 190 Leu Cys Leu Lys Ser Pro Gly Arg
Phe Glu Arg Ile Leu Leu Arg Ala 195 200 205 Ala Asn Thr His Ser Ser
Ala Lys Pro Cys Gly Gln Gln Ser Ile His 210 215 220 Leu Gly Gly Val
Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn 225 230 235 240 Val
Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe 245 250
255 Gly Leu Leu Lys Leu 260 <210> SEQ ID NO 56 <211>
LENGTH: 183 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: TNF
receptor ligand OX40L <400> SEQUENCE: 56 Met Glu Arg Val Gln
Pro Leu Glu Glu Asn Val Gly Asn Ala Ala Arg 1 5 10 15 Pro Arg Phe
Glu Arg Asn Lys Leu Leu Leu Val Ala Ser Val Ile Gln 20 25 30 Gly
Leu Gly Leu Leu Leu Cys Phe Thr Tyr Ile Cys Leu His Phe Ser 35 40
45 Ala Leu Gln Val Ser His Arg Tyr Pro Arg Ile Gln Ser Ile Lys Val
50 55 60 Gln Phe Thr Glu Tyr Lys Lys Glu Lys Gly Phe Ile Leu Thr
Ser Gln 65 70 75 80 Lys Glu Asp Glu Ile Met Lys Val Gln Asn Asn Ser
Val Ile Ile Asn 85 90 95 Cys Asp Gly Phe Tyr Leu Ile Ser Leu Lys
Gly Tyr Phe Ser Gln Glu 100 105 110 Val Asn Ile Ser Leu His Tyr Gln
Lys Asp Glu Glu Pro Leu Phe Gln 115 120 125 Leu Lys Lys Val Arg Ser
Val Asn Ser Leu Met Val Ala Ser Leu Thr 130 135 140 Tyr Lys Asp Lys
Val Tyr Leu Asn Val Thr Thr Asp Asn Thr Ser Leu 145 150 155 160 Asp
Asp Phe His Val Asn Gly Gly Glu Leu Ile Leu Ile His Gln Asn 165 170
175 Pro Gly Glu Phe Cys Val Leu 180 <210> SEQ ID NO 57
<211> LENGTH: 254 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: TNF receptor ligand 41BBL <400> SEQUENCE: 57 Met
Glu Tyr Ala Ser Asp Ala Ser Leu Asp Pro Glu Ala Pro Trp Pro 1 5 10
15 Pro Ala Pro Arg Ala Arg Ala Cys Arg Val Leu Pro Trp Ala Leu Val
20 25 30 Ala Gly Leu Leu Leu Leu Leu Leu Leu Ala Ala Ala Cys Ala
Val Phe 35 40 45 Leu Ala Cys Pro Trp Ala Val Ser Gly Ala Arg Ala
Ser Pro Gly Ser 50 55 60 Ala Ala Ser Pro Arg Leu Arg Glu Gly Pro
Glu Leu Ser Pro Asp Asp 65 70 75 80 Pro Ala Gly Leu Leu Asp Leu Arg
Gln Gly Met Phe Ala Gln Leu Val 85 90 95 Ala Gln Asn Val Leu Leu
Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp 100 105 110 Pro Gly Leu Ala
Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu 115 120 125 Asp Thr
Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe 130 135 140
Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser 145
150 155 160 Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala
Gly Ala 165 170 175 Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala
Ser Ser Glu Ala 180 185 190 Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg
Leu Leu His Leu Ser Ala 195 200 205 Gly Gln Arg Leu Gly Val His Leu
His Thr Glu Ala Arg Ala Arg His 210 215 220 Ala Trp Gln Leu Thr Gln
Gly Ala Thr Val Leu Gly Leu Phe Arg Val 225 230 235 240 Thr Pro Glu
Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu 245 250 <210> SEQ
ID NO 58 <211> LENGTH: 4 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: basic amino acid furin target sequence
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (2)..(2) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (3)..(3) <223>
OTHER INFORMATION: Xaa may be Arg or Lys <400> SEQUENCE: 58
Arg Xaa Xaa Arg 1 <210> SEQ ID NO 59 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: consensus
Tobacco Etch Virus (TEV) cleavage site <400> SEQUENCE: 59 Glu
Asn Leu Tyr Phe Gln Ser 1 5 <210> SEQ ID NO 60 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: ITAM
(immunoreceptor tyrosine-based activation motif) <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(2)..(3) <223> OTHER INFORMATION: Xaa can be any naturally
occurring amino acid <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (4)..(4) <223> OTHER
INFORMATION: Xaa may be Leu or Ile <400> SEQUENCE: 60 Tyr Xaa
Xaa Xaa 1 <210> SEQ ID NO 61 <211> LENGTH: 7
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: frame-slip
motif (FSM) <400> SEQUENCE: 61 uuuuuuu 7 <210> SEQ ID
NO 62 <211> LENGTH: 9 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: frame-slip motif (FSM) comprising a stop codon
<400> SEQUENCE: 62 uuuuuuuga 9 <210> SEQ ID NO 63
<211> LENGTH: 9 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: FSM comprising a stop codon <400> SEQUENCE: 63
uuuuuuuag 9 <210> SEQ ID NO 64 <211> LENGTH: 9
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: FSM comprising
a stop codon <400> SEQUENCE: 64 uuuuuuuaa 9 <210> SEQ
ID NO 65 <211> LENGTH: 7 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: translational readthrough motif (TRM) comprising
a stop codon <400> SEQUENCE: 65 ugacuag 7 <210> SEQ ID
NO 66 <211> LENGTH: 7 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: TRM comprising a stop codon <400>
SEQUENCE: 66 uagcuag 7 <210> SEQ ID NO 67 <211> LENGTH:
7 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: TRM comprising
a stop codon <400> SEQUENCE: 67 uaacuag 7 <210> SEQ ID
NO 68 <211> LENGTH: 9 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: TRM comprising a stop codon <400>
SEQUENCE: 68 ugacaauua 9 <210> SEQ ID NO 69 <211>
LENGTH: 9 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: TRM
comprising a stop codon <400> SEQUENCE: 69 uagcaauua 9
<210> SEQ ID NO 70 <211> LENGTH: 9 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: TRM comprising a stop codon
<400> SEQUENCE: 70 uaacaauua 9 <210> SEQ ID NO 71
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: VH CDR, CDR1 <400> SEQUENCE: 71 Ser Tyr Asn Ile
His 1 5 <210> SEQ ID NO 72 <211> LENGTH: 16 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: VH CDR, CDR2 <400>
SEQUENCE: 72 Val Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala
Leu Met Ser 1 5 10 15 <210> SEQ ID NO 73 <211> LENGTH:
10 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: VH CDR, CDR3
<400> SEQUENCE: 73 Arg Ser Asp Asp Tyr Ser Trp Phe Ala Tyr 1
5 10 <210> SEQ ID NO 74 <211> LENGTH: 12 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: VL CDR, CDR1 <400>
SEQUENCE: 74 Arg Ala Ser Ser Ser Val Ser Ser Ser Tyr Leu His 1 5 10
<210> SEQ ID NO 75 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: VL CDR, CDR2 <400> SEQUENCE:
75 Ser Thr Ser Asn Leu Ala Ser 1 5 <210> SEQ ID NO 76
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: VL CDR, CDR3 <400> SEQUENCE: 76 Gln Gln Tyr Ser
Gly Tyr Pro Ile Thr 1 5 <210> SEQ ID NO 77 <211>
LENGTH: 118 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
humanised KM666 VH sequence <400> SEQUENCE: 77 Gln Val Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr
Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ala Ser Tyr 20 25
30 Asn Ile His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45 Gly Val Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala
Leu Met 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn
Gln Val Phe Leu 65 70 75 80 Lys Met Ser Ser Leu Thr Ala Ala Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Arg Ser Asp Asp Tyr Ser Trp
Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser
115 <210> SEQ ID NO 78 <211> LENGTH: 108 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: humanised KM666 VL sequence
<400> SEQUENCE: 78 Glu Asn Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Met Thr Cys Arg
Ala Ser Ser Ser Val Ser Ser Ser 20 25 30 Tyr Leu His Trp Tyr Gln
Gln Lys Ser Gly Lys Ala Pro Lys Val Trp 35 40 45 Ile Tyr Ser Thr
Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Gly Tyr Pro 85
90 95 Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
<210> SEQ ID NO 79 <211> LENGTH: 136 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: sort/suicide gene RQR8 <400>
SEQUENCE: 79 Cys Pro Tyr Ser Asn Pro Ser Leu Cys Ser Gly Gly Gly
Gly Ser Glu 1 5 10 15 Leu Pro Thr Gln Gly Thr Phe Ser Asn Val Ser
Thr Asn Val Ser Pro 20 25 30 Ala Lys Pro Thr Thr Thr Ala Cys Pro
Tyr Ser Asn Pro Ser Leu Cys 35 40 45 Ser Gly Gly Gly Gly Ser Pro
Ala Pro Arg Pro Pro Thr Pro Ala Pro 50 55 60 Thr Ile Ala Ser Gln
Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro 65 70 75 80 Ala Ala Gly
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 85 90 95 Ile
Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 100 105
110 Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn Arg Arg Arg Val
115 120 125 Cys Lys Cys Pro Arg Pro Val Val 130 135 <210> SEQ
ID NO 80 <211> LENGTH: 517 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: suicide gene Rapcasp9 <400> SEQUENCE: 80
Met Ala Ser Arg Ile Leu Trp His Glu Met Trp His Glu Gly Leu Glu 1 5
10 15 Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met
Phe 20 25 30 Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
Pro Gln Thr 35 40 45 Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly
Arg Asp Leu Met Glu 50 55 60 Ala Gln Glu Trp Cys Arg Lys Tyr Met
Lys Ser Gly Asn Val Lys Asp 65 70 75 80 Leu Leu Gln Ala Trp Asp Leu
Tyr Tyr His Val Phe Arg Arg Ile Ser 85 90 95 Lys Leu Glu Tyr Ser
Gly Gly Gly Ser Leu Glu Gly Val Gln Val Glu 100 105 110 Thr Ile Ser
Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr 115 120 125 Cys
Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp 130 135
140 Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln
145 150 155 160 Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met
Ser Val Gly 165 170 175 Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr
Ala Tyr Gly Ala Thr 180 185 190 Gly His Pro Gly Ile Ile Pro Pro His
Ala Thr Leu Val Phe Asp Val 195 200 205 Glu Leu Leu Lys Leu Glu Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Gly Gly Gly Gly
Ser Gly Val Asp Gly Phe Gly Asp Val Gly Ala 225 230 235 240 Leu Glu
Ser Leu Arg Gly Asn Ala Asp Leu Ala Tyr Ile Leu Ser Met 245 250 255
Glu Pro Cys Gly His Cys Leu Ile Ile Asn Asn Val Asn Phe Cys Arg 260
265 270 Glu Ser Gly Leu Arg Thr Arg Thr Gly Ser Asn Ile Asp Cys Glu
Lys 275 280 285 Leu Arg Arg Arg Phe Ser Ser Leu His Phe Met Val Glu
Val Lys Gly 290 295 300 Asp Leu Thr Ala Lys Lys Met Val Leu Ala Leu
Leu Glu Leu Ala Gln 305 310 315 320 Gln Asp His Gly Ala Leu Asp Cys
Cys Val Val Val Ile Leu Ser His 325 330 335 Gly Cys Gln Ala Ser His
Leu Gln Phe Pro Gly Ala Val Tyr Gly Thr 340 345 350 Asp Gly Cys Pro
Val Ser Val Glu Lys Ile Val Asn Ile Phe Asn Gly 355 360 365 Thr Ser
Cys Pro Ser Leu Gly Gly Lys Pro Lys Leu Phe Phe Ile Gln 370 375 380
Ala Cys Gly Gly Glu Gln Lys Asp His Gly Phe Glu Val Ala Ser Thr 385
390 395 400 Ser Pro Glu Asp Glu Ser Pro Gly Ser Asn Pro Glu Pro Asp
Ala Thr 405 410 415 Pro Phe Gln Glu Gly Leu Arg Thr Phe Asp Gln Leu
Asp Ala Ile Ser 420 425 430 Ser Leu Pro Thr Pro Ser Asp Ile Phe Val
Ser Tyr Ser Thr Phe Pro 435 440 445 Gly Phe Val Ser Trp Arg Asp Pro
Lys Ser Gly Ser Trp Tyr Val Glu 450 455 460 Thr Leu Asp Asp Ile Phe
Glu Gln Trp Ala His Ser Glu Asp Leu Gln 465 470 475 480 Ser Leu Leu
Leu Arg Val Ala Asn Ala Val Ser Val Lys Gly Ile Tyr 485 490 495 Lys
Gln Met Pro Gly Cys Phe Asn Phe Leu Arg Lys Lys Leu Phe Phe 500 505
510 Lys Thr Ser Ala Ser 515 <210> SEQ ID NO 81 <211>
LENGTH: 520 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: a
flexi-IL-12 sequence <400> SEQUENCE: 81 Met Trp Ile Trp Glu
Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp 1 5 10 15 Trp Tyr Pro
Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr 20 25 30 Pro
Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val 35 40
45 Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly Asp
50 55 60 Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser
His Ser 65 70 75 80 Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp
Ser Thr Asp Ile 85 90 95 Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys
Thr Phe Leu Arg Cys Glu 100 105 110 Ala Lys Asn Tyr Ser Gly Arg Phe
Thr Cys Trp Trp Leu Thr Thr Ile 115 120 125 Ser Thr Asp Leu Thr Phe
Ser Val Lys Ser Ser Arg Gly Ser Ser Asp 130 135 140 Pro Gln Gly Val
Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val 145 150 155 160 Arg
Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp 165 170
175 Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val
180 185 190 Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser
Phe Phe 195 200 205 Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn
Leu Gln Leu Lys 210 215 220 Pro Leu Lys Asn Ser Arg Gln Val Glu Val
Ser Trp Glu Tyr Pro Asp 225 230 235 240 Thr Trp Ser Thr Pro His Ser
Tyr Phe Ser Leu Thr Phe Cys Val Gln 245 250 255 Val Gln Gly Lys Ser
Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp 260 265 270 Lys Thr Ser
Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val 275 280 285 Arg
Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser 290 295
300 Val Pro Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
305 310 315 320 Gly Gly Ser Arg Asn Leu Pro Leu Ala Thr Pro Asp Pro
Gly Met Phe 325 330 335 Pro Cys Leu His His Ser Gln Asn Leu Leu Arg
Ala Val Ser Asn Met 340 345 350 Leu Gln Lys Ala Arg Gln Thr Leu Glu
Phe Tyr Pro Cys Thr Ser Glu 355 360 365 Glu Ile Asp His Glu Asp Ile
Thr Lys Asp Lys Thr Ser Thr Val Glu 370 375 380 Ala Cys Leu Pro Leu
Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser 385 390 395 400 Arg Glu
Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys 405 410 415
Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Ser 420
425 430 Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu
Met 435 440 445 Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu
Ala Val Ile 450 455 460 Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser
Glu Thr Val Pro Gln 465 470 475 480 Lys Ser Ser Leu Glu Glu Pro Asp
Phe Tyr Lys Thr Lys Ile Lys Leu 485 490 495 Cys Ile Leu Leu His Ala
Phe Arg Ile Arg Ala Val Thr Ile Asp Arg 500 505 510 Val Met Ser Tyr
Leu Asn Ala Ser 515 520
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 81 <210>
SEQ ID NO 1 <211> LENGTH: 7 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: heavy chain variable region (VH) complementarity
determining region (CDR) CDR1 <400> SEQUENCE: 1 Gly Tyr Ala
Phe Ser Ser Ser 1 5 <210> SEQ ID NO 2 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: VH CDR, CDR2
<400> SEQUENCE: 2 Tyr Pro Gly Asp Glu Asp 1 5 <210> SEQ
ID NO 3 <211> LENGTH: 10 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: VH CDR, CDR3 <400> SEQUENCE: 3 Ser Leu Leu
Tyr Gly Asp Tyr Leu Asp Tyr 1 5 10 <210> SEQ ID NO 4
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: light chain variable region (VL) CDR, CDR1 <400>
SEQUENCE: 4 Ser Ala Ser Ser Ser Val Ser Tyr Met His 1 5 10
<210> SEQ ID NO 5 <211> LENGTH: 7 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: VL CDR, CDR2 <400> SEQUENCE: 5
Asp Thr Ser Lys Leu Ala Ser 1 5 <210> SEQ ID NO 6 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: VL
CDR, CDR3 <400> SEQUENCE: 6 Gln Gln Trp Asn Ile Asn Pro Leu
Thr 1 5 <210> SEQ ID NO 7 <211> LENGTH: 119 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: VH domain sequence
<400> SEQUENCE: 7 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser
Gly Tyr Ala Phe Ser Ser Ser 20 25 30 Trp Met Asn Trp Val Lys Gln
Arg Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Tyr Pro
Gly Asp Glu Asp Thr Asn Tyr Ser Gly Lys Phe 50 55 60 Lys Asp Lys
Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr 65 70 75 80 Met
Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90
95 Ala Arg Ser Leu Leu Tyr Gly Asp Tyr Leu Asp Tyr Trp Gly Gln Gly
100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> SEQ ID NO 8
<211> LENGTH: 107 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: VL domain sequence <400> SEQUENCE: 8 Gln Ile Val
Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu
Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25
30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr
35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Asp Arg Phe Ser
Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Phe Leu Thr Ile Asn Asn
Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
Asn Ile Asn Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu
Leu Lys Arg 100 105 <210> SEQ ID NO 9 <211> LENGTH: 20
<212> TYPE: PRT <213> ORGANISM: Foot-and-mouth disease
virus <400> SEQUENCE: 9 Arg Ala Glu Gly Arg Gly Ser Leu Leu
Thr Cys Gly Asp Val Glu Glu 1 5 10 15 Asn Pro Gly Pro 20
<210> SEQ ID NO 10 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: VH CDR, CDR1 <400> SEQUENCE:
10 Asn Phe Ala Met Ala 1 5 <210> SEQ ID NO 11 <211>
LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: VH
CDR, CDR2 <400> SEQUENCE: 11 Ser Ile Ser Thr Gly Gly Gly Asn
Thr Tyr Tyr Arg Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ ID NO
12 <211> LENGTH: 18 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: VH CDR, CDR3 <400> SEQUENCE: 12 Gln Arg
Asn Tyr Tyr Asp Gly Ser Tyr Asp Tyr Glu Gly Tyr Thr Met 1 5 10 15
Asp Ala <210> SEQ ID NO 13 <211> LENGTH: 11 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: VL CDR, CDR1 <400>
SEQUENCE: 13 Arg Ser Ser Gln Asp Ile Gly Asn Tyr Leu Thr 1 5 10
<210> SEQ ID NO 14 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: VL CDR, CDR2 <400> SEQUENCE:
14 Gly Ala Ile Lys Leu Glu Asp 1 5 <210> SEQ ID NO 15
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: VL CDR, CDR3 <400> SEQUENCE: 15 Leu Gln Ser Ile
Gln Tyr Pro 1 5 <210> SEQ ID NO 16 <211> LENGTH: 127
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: VH domain
sequence <400> SEQUENCE: 16 Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Lys Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe 20 25 30 Ala Met Ala Trp
Val Arg Gln Pro Pro Thr Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser
Ile Ser Thr Gly Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Thr Gln Tyr 65
70 75 80 Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Gln Arg Asn Tyr Tyr Asp Gly Ser Tyr Asp
Tyr Glu Gly Tyr 100 105 110 Thr Met Asp Ala Trp Gly Gln Gly Thr Ser
Val Thr Val Ser Ser 115 120 125 <210> SEQ ID NO 17
<211> LENGTH: 107 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: VL domain sequence <400> SEQUENCE: 17 Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Asp Ile Gly Asn Tyr 20
25 30 Leu Thr Trp Phe Gln Gln Lys Val Gly Arg Ser Pro Arg Arg Met
Ile 35 40 45 Tyr Gly Ala Ile Lys Leu Glu Asp Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile
Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Val Ala Asp Tyr Gln Cys Leu
Gln Ser Ile Gln Tyr Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys
Leu Glu Ile Lys 100 105 <210> SEQ ID NO 18 <211>
LENGTH: 254 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
tyrosine kinase domain of Fyn <400> SEQUENCE: 18 Leu Gln Leu
Ile Lys Arg Leu Gly Asn Gly Gln Phe Gly Glu Val Trp 1 5 10 15 Met
Gly Thr Trp Asn Gly Asn Thr Lys Val Ala Ile Lys Thr Leu Lys 20 25
30 Pro Gly Thr Met Ser Pro Glu Ser Phe Leu Glu Glu Ala Gln Ile Met
35 40 45 Lys Lys Leu Lys His Asp Lys Leu Val Gln Leu Tyr Ala Val
Val Ser 50 55 60 Glu Glu Pro Ile Tyr Ile Val Thr Glu Tyr Met Asn
Lys Gly Ser Leu 65 70 75 80 Leu Asp Phe Leu Lys Asp Gly Glu Gly Arg
Ala Leu Lys Leu Pro Asn 85 90 95 Leu Val Asp Met Ala Ala Gln Val
Ala Ala Gly Met Ala Tyr Ile Glu 100 105 110 Arg Met Asn Tyr Ile His
Arg Asp Leu Arg Ser Ala Asn Ile Leu Val 115 120 125 Gly Asn Gly Leu
Ile Cys Lys Ile Ala Asp Phe Gly Leu Ala Arg Leu 130 135 140 Ile Glu
Asp Asn Glu Tyr Thr Ala Arg Gln Gly Ala Lys Phe Pro Ile 145 150 155
160 Lys Trp Thr Ala Pro Glu Arg Ala Leu Tyr Gly Arg Phe Thr Ile Lys
165 170 175 Ser Asp Val Trp Ser Phe Gly Ile Leu Leu Thr Glu Leu Val
Thr Lys 180 185 190 Gly Arg Val Pro Tyr Pro Gly Met Asn Asn Arg Glu
Val Leu Glu Gln 195 200 205 Val Glu Arg Gly Tyr Arg Met Pro Cys Pro
Gln Asp Cys Pro Ile Ser 210 215 220 Leu His Glu Leu Met Ile His Cys
Trp Lys Lys Asp Pro Glu Glu Arg 225 230 235 240 Pro Thr Phe Glu Tyr
Leu Gln Ser Phe Leu Glu Asp Tyr Phe 245 250 <210> SEQ ID NO
19 <211> LENGTH: 254 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: tyrosine kinase domain of Src <400>
SEQUENCE: 19 Leu Arg Leu Glu Val Lys Leu Gly Gln Gly Cys Phe Gly
Glu Val Trp 1 5 10 15 Met Gly Thr Trp Asn Gly Thr Thr Arg Val Ala
Ile Lys Thr Leu Lys 20 25 30 Pro Gly Thr Met Ser Pro Glu Ala Phe
Leu Gln Glu Ala Gln Val Met 35 40 45 Lys Lys Leu Arg His Glu Lys
Leu Val Gln Leu Tyr Ala Val Val Ser 50 55 60 Glu Glu Pro Ile Tyr
Ile Val Thr Glu Tyr Met Ser Lys Gly Ser Leu 65 70 75 80 Leu Asp Phe
Leu Lys Gly Glu Thr Gly Lys Tyr Leu Arg Leu Pro Gln 85 90 95 Leu
Val Asp Met Ala Ala Gln Ile Ala Ser Gly Met Ala Tyr Val Glu 100 105
110 Arg Met Asn Tyr Val His Arg Asp Leu Arg Ala Ala Asn Ile Leu Val
115 120 125 Gly Glu Asn Leu Val Cys Lys Val Ala Asp Phe Gly Leu Ala
Arg Leu 130 135 140 Ile Glu Asp Asn Glu Tyr Thr Ala Arg Gln Gly Ala
Lys Phe Pro Ile 145 150 155 160 Lys Trp Thr Ala Pro Glu Ala Ala Leu
Tyr Gly Arg Phe Thr Ile Lys 165 170 175 Ser Asp Val Trp Ser Phe Gly
Ile Leu Leu Thr Glu Leu Thr Thr Lys 180 185 190 Gly Arg Val Pro Tyr
Pro Gly Met Val Asn Arg Glu Val Leu Asp Gln 195 200 205 Val Glu Arg
Gly Tyr Arg Met Pro Cys Pro Pro Glu Cys Pro Glu Ser 210 215 220 Leu
His Asp Leu Met Cys Gln Cys Trp Arg Lys Glu Pro Glu Glu Arg 225 230
235 240 Pro Thr Phe Glu Tyr Leu Gln Ala Phe Leu Glu Asp Tyr Phe 245
250 <210> SEQ ID NO 20 <211> LENGTH: 254 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: tyrosine kinase domain of
Lck <400> SEQUENCE: 20 Leu Lys Leu Val Glu Arg Leu Gly Ala
Gly Gln Phe Gly Glu Val Trp 1 5 10 15 Met Gly Tyr Tyr Asn Gly His
Thr Lys Val Ala Val Lys Ser Leu Lys 20 25 30 Gln Gly Ser Met Ser
Pro Asp Ala Phe Leu Ala Glu Ala Asn Leu Met 35 40 45 Lys Gln Leu
Gln His Gln Arg Leu Val Arg Leu Tyr Ala Val Val Thr 50 55 60 Gln
Glu Pro Ile Tyr Ile Ile Thr Glu Tyr Met Glu Asn Gly Ser Leu 65 70
75 80 Val Asp Phe Leu Lys Thr Pro Ser Gly Ile Lys Leu Thr Ile Asn
Lys 85 90 95 Leu Leu Asp Met Ala Ala Gln Ile Ala Glu Gly Met Ala
Phe Ile Glu 100 105 110 Glu Arg Asn Tyr Ile His Arg Asp Leu Arg Ala
Ala Asn Ile Leu Val 115 120 125 Ser Asp Thr Leu Ser Cys Lys Ile Ala
Asp Phe Gly Leu Ala Arg Leu 130 135 140 Ile Glu Asp Asn Glu Tyr Thr
Ala Arg Glu Gly Ala Lys Phe Pro Ile 145 150 155 160 Lys Trp Thr Ala
Pro Glu Ala Ile Asn Tyr Gly Thr Phe Thr Ile Lys 165 170 175 Ser Asp
Val Trp Ser Phe Gly Ile Leu Leu Thr Glu Ile Val Thr His 180 185 190
Gly Arg Ile Pro Tyr Pro Gly Met Thr Asn Pro Glu Val Ile Gln Asn 195
200 205 Leu Glu Arg Gly Tyr Arg Met Val Arg Pro Asp Asn Cys Pro Glu
Glu 210 215 220 Leu Tyr Gln Leu Met Arg Leu Cys Trp Lys Glu Arg Pro
Glu Asp Arg 225 230 235 240 Pro Thr Phe Asp Tyr Leu Arg Ser Val Leu
Glu Asp Phe Phe 245 250 <210> SEQ ID NO 21 <211>
LENGTH: 254 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
tyrosine kinase domain of Lck_Y505F <400> SEQUENCE: 21 Leu
Lys Leu Val Glu Arg Leu Gly Ala Gly Gln Phe Gly Glu Val Trp 1 5 10
15 Met Gly Tyr Tyr Asn Gly His Thr Lys Val Ala Val Arg Ser Leu Lys
20 25 30 Gln Gly Ser Met Ser Pro Asp Ala Phe Leu Ala Glu Ala Asn
Leu Met
35 40 45 Lys Gln Leu Gln His Gln Arg Leu Val Arg Leu Tyr Ala Val
Val Thr 50 55 60 Gln Glu Pro Ile Tyr Ile Ile Thr Glu Tyr Met Glu
Asn Gly Ser Leu 65 70 75 80 Val Asp Phe Leu Lys Thr Pro Ser Gly Ile
Lys Leu Thr Ile Asn Lys 85 90 95 Leu Leu Asp Met Ala Ala Gln Ile
Ala Glu Gly Met Ala Phe Ile Glu 100 105 110 Glu Arg Asn Tyr Ile His
Arg Asp Leu Arg Ala Ala Asn Ile Leu Val 115 120 125 Ser Asp Thr Leu
Ser Cys Lys Ile Ala Asp Phe Gly Leu Ala Arg Leu 130 135 140 Ile Glu
Asp Asn Glu Tyr Thr Ala Arg Glu Gly Ala Lys Phe Pro Ile 145 150 155
160 Lys Trp Thr Ala Pro Glu Ala Ile Asn Tyr Gly Thr Phe Thr Ile Lys
165 170 175 Ser Asp Val Trp Ser Phe Gly Ile Leu Leu Thr Glu Ile Val
Thr His 180 185 190 Gly Arg Ile Pro Tyr Pro Gly Met Thr Asn Pro Glu
Val Ile Gln Asn 195 200 205 Leu Glu Arg Gly Tyr Arg Met Val Arg Pro
Asp Asn Cys Pro Glu Glu 210 215 220 Leu Tyr Gln Leu Met Arg Leu Cys
Trp Lys Glu Arg Pro Glu Asp Arg 225 230 235 240 Pro Thr Phe Asp Tyr
Leu Arg Ser Val Leu Glu Asp Phe Phe 245 250 <210> SEQ ID NO
22 <211> LENGTH: 40 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cytoplasmic tail of CD4 <400> SEQUENCE: 22
Cys Val Arg Cys Arg His Arg Arg Arg Gln Ala Glu Arg Met Ser Gln 1 5
10 15 Ile Lys Arg Leu Leu Ser Glu Lys Lys Thr Cys Gln Cys Pro His
Arg 20 25 30 Phe Gln Lys Thr Cys Ser Pro Ile 35 40 <210> SEQ
ID NO 23 <211> LENGTH: 32 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cytoplasmic tail of CD8 <400> SEQUENCE: 23
Leu Tyr Cys Asn His Arg Asn Arg Arg Arg Val Cys Lys Cys Pro Arg 1 5
10 15 Pro Val Val Lys Ser Gly Asp Lys Pro Ser Leu Ser Ala Arg Tyr
Val 20 25 30 <210> SEQ ID NO 24 <211> LENGTH: 214
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: SHP-1 SH2
complete domain <400> SEQUENCE: 24 Met Val Arg Trp Phe His
Arg Asp Leu Ser Gly Leu Asp Ala Glu Thr 1 5 10 15 Leu Leu Lys Gly
Arg Gly Val His Gly Ser Phe Leu Ala Arg Pro Ser 20 25 30 Arg Lys
Asn Gln Gly Asp Phe Ser Leu Ser Val Arg Val Gly Asp Gln 35 40 45
Val Thr His Ile Arg Ile Gln Asn Ser Gly Asp Phe Tyr Asp Leu Tyr 50
55 60 Gly Gly Glu Lys Phe Ala Thr Leu Thr Glu Leu Val Glu Tyr Tyr
Thr 65 70 75 80 Gln Gln Gln Gly Val Leu Gln Asp Arg Asp Gly Thr Ile
Ile His Leu 85 90 95 Lys Tyr Pro Leu Asn Cys Ser Asp Pro Thr Ser
Glu Arg Trp Tyr His 100 105 110 Gly His Met Ser Gly Gly Gln Ala Glu
Thr Leu Leu Gln Ala Lys Gly 115 120 125 Glu Pro Trp Thr Phe Leu Val
Arg Glu Ser Leu Ser Gln Pro Gly Asp 130 135 140 Phe Val Leu Ser Val
Leu Ser Asp Gln Pro Lys Ala Gly Pro Gly Ser 145 150 155 160 Pro Leu
Arg Val Thr His Ile Lys Val Met Cys Glu Gly Gly Arg Tyr 165 170 175
Thr Val Gly Gly Leu Glu Thr Phe Asp Ser Leu Thr Asp Leu Val Glu 180
185 190 His Phe Lys Lys Thr Gly Ile Glu Glu Ala Ser Gly Ala Phe Val
Tyr 195 200 205 Leu Arg Gln Pro Tyr Tyr 210 <210> SEQ ID NO
25 <211> LENGTH: 97 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: SHP-1 SH2 1 <400> SEQUENCE: 25 Trp Phe His
Arg Asp Leu Ser Gly Leu Asp Ala Glu Thr Leu Leu Lys 1 5 10 15 Gly
Arg Gly Val His Gly Ser Phe Leu Ala Arg Pro Ser Arg Lys Asn 20 25
30 Gln Gly Asp Phe Ser Leu Ser Val Arg Val Gly Asp Gln Val Thr His
35 40 45 Ile Arg Ile Gln Asn Ser Gly Asp Phe Tyr Asp Leu Tyr Gly
Gly Glu 50 55 60 Lys Phe Ala Thr Leu Thr Glu Leu Val Glu Tyr Tyr
Thr Gln Gln Gln 65 70 75 80 Gly Val Leu Gln Asp Arg Asp Gly Thr Ile
Ile His Leu Lys Tyr Pro 85 90 95 Leu <210> SEQ ID NO 26
<211> LENGTH: 104 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SHP-1 SH2 2 <400> SEQUENCE: 26 Trp Tyr His Gly
His Met Ser Gly Gly Gln Ala Glu Thr Leu Leu Gln 1 5 10 15 Ala Lys
Gly Glu Pro Trp Thr Phe Leu Val Arg Glu Ser Leu Ser Gln 20 25 30
Pro Gly Asp Phe Val Leu Ser Val Leu Ser Asp Gln Pro Lys Ala Gly 35
40 45 Pro Gly Ser Pro Leu Arg Val Thr His Ile Lys Val Met Cys Glu
Gly 50 55 60 Gly Arg Tyr Thr Val Gly Gly Leu Glu Thr Phe Asp Ser
Leu Thr Asp 65 70 75 80 Leu Val Glu His Phe Lys Lys Thr Gly Ile Glu
Glu Ala Ser Gly Ala 85 90 95 Phe Val Tyr Leu Arg Gln Pro Tyr 100
<210> SEQ ID NO 27 <211> LENGTH: 97 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SHP-2 first SH2 domain <400>
SEQUENCE: 27 Trp Phe His Pro Asn Ile Thr Gly Val Glu Ala Glu Asn
Leu Leu Leu 1 5 10 15 Thr Arg Gly Val Asp Gly Ser Phe Leu Ala Arg
Pro Ser Lys Ser Asn 20 25 30 Pro Gly Asp Phe Thr Leu Ser Val Arg
Arg Asn Gly Ala Val Thr His 35 40 45 Ile Lys Ile Gln Asn Thr Gly
Asp Tyr Tyr Asp Leu Tyr Gly Gly Glu 50 55 60 Lys Phe Ala Thr Leu
Ala Glu Leu Val Gln Tyr Tyr Met Glu His His 65 70 75 80 Gly Gln Leu
Lys Glu Lys Asn Gly Asp Val Ile Glu Leu Lys Tyr Pro 85 90 95 Leu
<210> SEQ ID NO 28 <211> LENGTH: 105 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SHP-2 second SH2 domain <400>
SEQUENCE: 28 Trp Phe His Gly His Leu Ser Gly Lys Glu Ala Glu Lys
Leu Leu Thr 1 5 10 15 Glu Lys Gly Lys His Gly Ser Phe Leu Val Arg
Glu Ser Gln Ser His 20 25 30 Pro Gly Asp Phe Val Leu Ser Val Arg
Thr Gly Asp Asp Lys Gly Glu 35 40 45 Ser Asn Asp Gly Lys Ser Lys
Val Thr His Val Met Ile Arg Cys Gln 50 55 60 Glu Leu Lys Tyr Asp
Val Gly Gly Gly Glu Arg Phe Asp Ser Leu Thr 65 70 75 80 Asp Leu Val
Glu His Tyr Lys Lys Asn Pro Met Val Glu Thr Leu Gly 85 90 95 Thr
Val Leu Gln Leu Lys Gln Pro Leu 100 105
<210> SEQ ID NO 29 <211> LENGTH: 211 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SHP-2 both SH2 domains <400>
SEQUENCE: 29 Trp Phe His Pro Asn Ile Thr Gly Val Glu Ala Glu Asn
Leu Leu Leu 1 5 10 15 Thr Arg Gly Val Asp Gly Ser Phe Leu Ala Arg
Pro Ser Lys Ser Asn 20 25 30 Pro Gly Asp Phe Thr Leu Ser Val Arg
Arg Asn Gly Ala Val Thr His 35 40 45 Ile Lys Ile Gln Asn Thr Gly
Asp Tyr Tyr Asp Leu Tyr Gly Gly Glu 50 55 60 Lys Phe Ala Thr Leu
Ala Glu Leu Val Gln Tyr Tyr Met Glu His His 65 70 75 80 Gly Gln Leu
Lys Glu Lys Asn Gly Asp Val Ile Glu Leu Lys Tyr Pro 85 90 95 Leu
Asn Cys Ala Asp Pro Thr Ser Glu Arg Trp Phe His Gly His Leu 100 105
110 Ser Gly Lys Glu Ala Glu Lys Leu Leu Thr Glu Lys Gly Lys His Gly
115 120 125 Ser Phe Leu Val Arg Glu Ser Gln Ser His Pro Gly Asp Phe
Val Leu 130 135 140 Ser Val Arg Thr Gly Asp Asp Lys Gly Glu Ser Asn
Asp Gly Lys Ser 145 150 155 160 Lys Val Thr His Val Met Ile Arg Cys
Gln Glu Leu Lys Tyr Asp Val 165 170 175 Gly Gly Gly Glu Arg Phe Asp
Ser Leu Thr Asp Leu Val Glu His Tyr 180 185 190 Lys Lys Asn Pro Met
Val Glu Thr Leu Gly Thr Val Leu Gln Leu Lys 195 200 205 Gln Pro Leu
210 <210> SEQ ID NO 30 <211> LENGTH: 107 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Human Light Kappa Chain
<400> SEQUENCE: 30 Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85
90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105
<210> SEQ ID NO 31 <211> LENGTH: 19 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Human Hinge <400> SEQUENCE: 31
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Lys 1 5
10 15 Asp Pro Lys <210> SEQ ID NO 32 <211> LENGTH: 97
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Human CH1
<400> SEQUENCE: 32 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys Ser 1 5 10 15 Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe 20 25 30 Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 35 40 45 Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 50 55 60 Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr 65 70 75 80
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg 85
90 95 Val <210> SEQ ID NO 33 <211> LENGTH: 21
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Transmembrane
domain from human IL2R common gamma chain <400> SEQUENCE: 33
Val Val Ile Ser Val Gly Ser Met Gly Leu Ile Ile Ser Leu Leu Cys 1 5
10 15 Val Tyr Phe Trp Leu 20 <210> SEQ ID NO 34 <211>
LENGTH: 25 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Transmembrane domain from human IL-2R beta <400> SEQUENCE: 34
Ile Pro Trp Leu Gly His Leu Leu Val Gly Leu Ser Gly Ala Phe Gly 1 5
10 15 Phe Ile Ile Leu Val Tyr Leu Leu Ile 20 25 <210> SEQ ID
NO 35 <400> SEQUENCE: 35 000 <210> SEQ ID NO 36
<211> LENGTH: 25 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Transmembrane domain from human IL-7R alpha
<400> SEQUENCE: 36 Pro Ile Leu Leu Thr Ile Ser Ile Leu Ser
Phe Phe Ser Val Ala Leu 1 5 10 15 Leu Val Ile Leu Ala Cys Val Leu
Trp 20 25 <210> SEQ ID NO 37 <211> LENGTH: 26
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Transmembrane
domain from Human GF-CSFR alpha <400> SEQUENCE: 37 Asn Leu
Gly Ser Val Tyr Ile Tyr Val Leu Leu Ile Val Gly Thr Leu 1 5 10 15
Val Cys Gly Ile Val Leu Gly Phe Leu Phe 20 25 <210> SEQ ID NO
38 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Transmembrane domain from Human GM-CSFR common
beta chain <400> SEQUENCE: 38 Val Leu Ala Leu Ile Val Ile Phe
Leu Thr Ile Ala Val Leu Leu Ala 1 5 10 15 Leu <210> SEQ ID NO
39 <211> LENGTH: 86 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Endodomain from human IL2R common gamma chain
<400> SEQUENCE: 39 Glu Arg Thr Met Pro Arg Ile Pro Thr Leu
Lys Asn Leu Glu Asp Leu 1 5 10 15 Val Thr Glu Tyr His Gly Asn Phe
Ser Ala Trp Ser Gly Val Ser Lys 20 25 30 Gly Leu Ala Glu Ser Leu
Gln Pro Asp Tyr Ser Glu Arg Leu Cys Leu 35 40 45 Val Ser Glu Ile
Pro Pro Lys Gly Gly Ala Leu Gly Glu Gly Pro Gly 50 55 60 Ala Ser
Pro Cys Asn Gln His Ser Pro Tyr Trp Ala Pro Pro Cys Tyr 65 70 75 80
Thr Leu Lys Pro Glu Thr 85 <210> SEQ ID NO 40 <211>
LENGTH: 286
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Endodomain from
human IL-2R beta <400> SEQUENCE: 40 Asn Cys Arg Asn Thr Gly
Pro Trp Leu Lys Lys Val Leu Lys Cys Asn 1 5 10 15 Thr Pro Asp Pro
Ser Lys Phe Phe Ser Gln Leu Ser Ser Glu His Gly 20 25 30 Gly Asp
Val Gln Lys Trp Leu Ser Ser Pro Phe Pro Ser Ser Ser Phe 35 40 45
Ser Pro Gly Gly Leu Ala Pro Glu Ile Ser Pro Leu Glu Val Leu Glu 50
55 60 Arg Asp Lys Val Thr Gln Leu Leu Leu Gln Gln Asp Lys Val Pro
Glu 65 70 75 80 Pro Ala Ser Leu Ser Ser Asn His Ser Leu Thr Ser Cys
Phe Thr Asn 85 90 95 Gln Gly Tyr Phe Phe Phe His Leu Pro Asp Ala
Leu Glu Ile Glu Ala 100 105 110 Cys Gln Val Tyr Phe Thr Tyr Asp Pro
Tyr Ser Glu Glu Asp Pro Asp 115 120 125 Glu Gly Val Ala Gly Ala Pro
Thr Gly Ser Ser Pro Gln Pro Leu Gln 130 135 140 Pro Leu Ser Gly Glu
Asp Asp Ala Tyr Cys Thr Phe Pro Ser Arg Asp 145 150 155 160 Asp Leu
Leu Leu Phe Ser Pro Ser Leu Leu Gly Gly Pro Ser Pro Pro 165 170 175
Ser Thr Ala Pro Gly Gly Ser Gly Ala Gly Glu Glu Arg Met Pro Pro 180
185 190 Ser Leu Gln Glu Arg Val Pro Arg Asp Trp Asp Pro Gln Pro Leu
Gly 195 200 205 Pro Pro Thr Pro Gly Val Pro Asp Leu Val Asp Phe Gln
Pro Pro Pro 210 215 220 Glu Leu Val Leu Arg Glu Ala Gly Glu Glu Val
Pro Asp Ala Gly Pro 225 230 235 240 Arg Glu Gly Val Ser Phe Pro Trp
Ser Arg Pro Pro Gly Gln Gly Glu 245 250 255 Phe Arg Ala Leu Asn Ala
Arg Leu Pro Leu Asn Thr Asp Ala Tyr Leu 260 265 270 Ser Leu Gln Glu
Leu Gln Gly Gln Asp Pro Thr His Leu Val 275 280 285 <210> SEQ
ID NO 41 <211> LENGTH: 196 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Endodomain from human IL-7R alpha <400>
SEQUENCE: 41 Lys Lys Arg Ile Lys Pro Ile Val Trp Pro Ser Leu Pro
Asp His Lys 1 5 10 15 Lys Thr Leu Glu His Leu Cys Lys Lys Pro Arg
Lys Asn Leu Asn Val 20 25 30 Ser Phe Asn Pro Glu Ser Phe Leu Asp
Cys Gln Ile His Arg Val Asp 35 40 45 Asp Ile Gln Ala Arg Asp Glu
Val Glu Gly Phe Leu Gln Asp Thr Phe 50 55 60 Pro Gln Gln Leu Glu
Glu Ser Glu Lys Gln Arg Leu Gly Gly Asp Val 65 70 75 80 Gln Ser Pro
Asn Cys Pro Ser Glu Asp Val Val Ile Thr Pro Glu Ser 85 90 95 Phe
Gly Arg Asp Ser Ser Leu Thr Cys Leu Ala Gly Asn Val Ser Ala 100 105
110 Cys Asp Ala Pro Ile Leu Ser Ser Ser Arg Ser Leu Asp Cys Arg Glu
115 120 125 Ser Gly Lys Asn Gly Pro His Val Tyr Gln Asp Leu Leu Leu
Ser Leu 130 135 140 Gly Thr Thr Asn Ser Thr Leu Pro Pro Pro Phe Ser
Leu Gln Ser Gly 145 150 155 160 Ile Leu Thr Leu Asn Pro Val Ala Gln
Gly Gln Pro Ile Leu Thr Ser 165 170 175 Leu Gly Ser Asn Gln Glu Glu
Ala Tyr Val Thr Met Ser Ser Phe Tyr 180 185 190 Gln Asn Glu Gln 195
<210> SEQ ID NO 42 <211> LENGTH: 54 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Endodomain Derived from Human
GM-CSFR alpha <400> SEQUENCE: 42 Lys Arg Phe Leu Arg Ile Gln
Arg Leu Phe Pro Pro Val Pro Gln Ile 1 5 10 15 Lys Asp Lys Leu Asn
Asp Asn His Glu Val Glu Asp Glu Ile Ile Trp 20 25 30 Glu Glu Phe
Thr Pro Glu Glu Gly Lys Gly Tyr Arg Glu Glu Val Leu 35 40 45 Thr
Val Lys Glu Ile Thr 50 <210> SEQ ID NO 43 <211> LENGTH:
437 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Endodomain from
GM-CSFR common beta chain <400> SEQUENCE: 43 Arg Phe Cys Gly
Ile Tyr Gly Tyr Arg Leu Arg Arg Lys Trp Glu Glu 1 5 10 15 Lys Ile
Pro Asn Pro Ser Lys Ser His Leu Phe Gln Asn Gly Ser Ala 20 25 30
Glu Leu Trp Pro Pro Gly Ser Met Ser Ala Phe Thr Ser Gly Ser Pro 35
40 45 Pro His Gln Gly Pro Trp Gly Ser Arg Phe Pro Glu Leu Glu Gly
Val 50 55 60 Phe Pro Val Gly Phe Gly Asp Ser Glu Val Ser Pro Leu
Thr Ile Glu 65 70 75 80 Asp Pro Lys His Val Cys Asp Pro Pro Ser Gly
Pro Asp Thr Thr Pro 85 90 95 Ala Ala Ser Asp Leu Pro Thr Glu Gln
Pro Pro Ser Pro Gln Pro Gly 100 105 110 Pro Pro Ala Ala Ser His Thr
Pro Glu Lys Gln Ala Ser Ser Phe Asp 115 120 125 Phe Asn Gly Pro Tyr
Leu Gly Pro Pro His Ser Arg Ser Leu Pro Asp 130 135 140 Ile Leu Gly
Gln Pro Glu Pro Pro Gln Glu Gly Gly Ser Gln Lys Ser 145 150 155 160
Pro Pro Pro Gly Ser Leu Glu Tyr Leu Cys Leu Pro Ala Gly Gly Gln 165
170 175 Val Gln Leu Val Pro Leu Ala Gln Ala Met Gly Pro Gly Gln Ala
Val 180 185 190 Glu Val Glu Arg Arg Pro Ser Gln Gly Ala Ala Gly Ser
Pro Ser Leu 195 200 205 Glu Ser Gly Gly Gly Pro Ala Pro Pro Ala Leu
Gly Pro Arg Val Gly 210 215 220 Gly Gln Asp Gln Lys Asp Ser Pro Val
Ala Ile Pro Met Ser Ser Gly 225 230 235 240 Asp Thr Glu Asp Pro Gly
Val Ala Ser Gly Tyr Val Ser Ser Ala Asp 245 250 255 Leu Val Phe Thr
Pro Asn Ser Gly Ala Ser Ser Val Ser Leu Val Pro 260 265 270 Ser Leu
Gly Leu Pro Ser Asp Gln Thr Pro Ser Leu Cys Pro Gly Leu 275 280 285
Ala Ser Gly Pro Pro Gly Ala Pro Gly Pro Val Lys Ser Gly Phe Glu 290
295 300 Gly Tyr Val Glu Leu Pro Pro Ile Glu Gly Arg Ser Pro Arg Ser
Pro 305 310 315 320 Arg Asn Asn Pro Val Pro Pro Glu Ala Lys Ser Pro
Val Leu Asn Pro 325 330 335 Gly Glu Arg Pro Ala Asp Val Ser Pro Thr
Ser Pro Gln Pro Glu Gly 340 345 350 Leu Leu Val Leu Gln Gln Val Gly
Asp Tyr Cys Phe Leu Pro Gly Leu 355 360 365 Gly Pro Gly Pro Leu Ser
Leu Arg Ser Lys Pro Ser Ser Pro Gly Pro 370 375 380 Gly Pro Glu Ile
Lys Asn Leu Asp Gln Ala Phe Gln Val Lys Lys Pro 385 390 395 400 Pro
Gly Gln Ala Val Pro Gln Val Pro Val Ile Gln Leu Phe Lys Ala 405 410
415 Leu Lys Gln Gln Asp Tyr Leu Ser Leu Pro Pro Trp Glu Val Asn Lys
420 425 430 Pro Gly Glu Val Cys 435 <210> SEQ ID NO 44
<211> LENGTH: 112 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: mature protein of wild-type TGFbeta2 <400>
SEQUENCE: 44 Ala Leu Asp Ala Ala Tyr Cys Phe Arg Asn Val Gln Asp
Asn Cys Cys 1 5 10 15 Leu Arg Pro Leu Tyr Ile Asp Phe Lys Arg Asp
Leu Gly Trp Lys Trp 20 25 30 Ile His Glu Pro Lys Gly Tyr Asn Ala
Asn Phe Cys Ala Gly Ala Cys 35 40 45 Pro Tyr Leu Trp Ser Ser Asp
Thr Gln His Ser Arg Val Leu Ser Leu 50 55 60 Tyr Asn Thr Ile Asn
Pro Glu Ala Ser Ala Ser Pro Cys Cys Val Ser 65 70 75 80
Gln Asp Leu Glu Pro Leu Thr Ile Leu Tyr Tyr Ile Gly Lys Thr Pro 85
90 95 Lys Ile Glu Gln Leu Ser Asn Met Ile Val Lys Ser Cys Lys Cys
Ser 100 105 110 <210> SEQ ID NO 45 <211> LENGTH: 394
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: TGFbeta monomer
sequence <400> SEQUENCE: 45 Met His Tyr Cys Val Leu Ser Ala
Phe Leu Ile Leu His Leu Val Thr 1 5 10 15 Val Ala Leu Ser Leu Ser
Thr Cys Ser Thr Leu Asp Met Asp Gln Phe 20 25 30 Met Arg Lys Arg
Ile Glu Ala Ile Arg Gly Gln Ile Leu Ser Lys Leu 35 40 45 Lys Leu
Thr Ser Pro Pro Glu Asp Tyr Pro Glu Pro Glu Glu Val Pro 50 55 60
Pro Glu Val Ile Ser Ile Tyr Asn Ser Thr Arg Asp Leu Leu Gln Glu 65
70 75 80 Lys Ala Ser Arg Arg Ala Ala Ala Cys Glu Arg Glu Arg Ser
Asp Glu 85 90 95 Glu Tyr Tyr Ala Lys Glu Val Tyr Lys Ile Asp Met
Pro Pro Phe Phe 100 105 110 Pro Ser Glu Asn Ala Ile Pro Pro Thr Phe
Tyr Arg Pro Tyr Phe Arg 115 120 125 Ile Val Arg Phe Asp Val Ser Ala
Met Glu Lys Asn Ala Ser Asn Leu 130 135 140 Val Lys Ala Glu Phe Arg
Val Phe Arg Leu Gln Asn Pro Lys Ala Arg 145 150 155 160 Val Pro Glu
Gln Arg Ile Glu Leu Tyr Gln Ile Leu Lys Ser Lys Asp 165 170 175 Leu
Thr Ser Pro Thr Gln Arg Tyr Ile Asp Ser Lys Val Val Lys Thr 180 185
190 Arg Ala Glu Gly Glu Trp Leu Ser Phe Asp Val Thr Asp Ala Val His
195 200 205 Glu Trp Leu His His Lys Asp Arg Asn Leu Gly Phe Lys Ile
Ser Leu 210 215 220 His Cys Pro Cys Cys Thr Phe Val Pro Ser Asn Asn
Tyr Ile Ile Pro 225 230 235 240 Asn Lys Ser Glu Glu Leu Glu Ala Arg
Phe Ala Gly Ile Asp Gly Thr 245 250 255 Ser Thr Tyr Thr Ser Gly Asp
Gln Lys Thr Ile Lys Ser Thr Arg Lys 260 265 270 Lys Asn Ser Gly Lys
Thr Pro His Leu Leu Leu Met Leu Leu Pro Ser 275 280 285 Tyr Arg Leu
Glu Ser Gln Gln Thr Asn Arg Arg Lys Lys Arg Ala Leu 290 295 300 Asp
Ala Ala Tyr Cys Phe Arg Asn Val Gln Asp Asn Cys Cys Leu Arg 305 310
315 320 Pro Leu Tyr Ile Asp Phe Arg Lys Asp Leu Gly Trp Lys Trp Ile
His 325 330 335 Glu Pro Lys Gly Tyr Asn Ala Asn Phe Cys Ala Gly Ala
Cys Pro Tyr 340 345 350 Arg Ala Ser Lys Ser Pro Ser Cys Val Ser Gln
Asp Leu Glu Pro Leu 355 360 365 Thr Ile Val Tyr Tyr Val Gly Arg Lys
Pro Lys Val Glu Gln Leu Ser 370 375 380 Asn Met Ile Val Lys Ser Cys
Lys Cys Ser 385 390 <210> SEQ ID NO 46 <211> LENGTH:
177 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION:
dominant-negative TGFbeta RII (dnTGFbeta RII) <400> SEQUENCE:
46 Thr Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val
1 5 10 15 Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys
Phe Cys 20 25 30 Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser
Cys Met Ser Asn 35 40 45 Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro
Gln Glu Val Cys Val Ala 50 55 60 Val Trp Arg Lys Asn Asp Glu Asn
Ile Thr Leu Glu Thr Val Cys His 65 70 75 80 Asp Pro Lys Leu Pro Tyr
His Asp Phe Ile Leu Glu Asp Ala Ala Ser 85 90 95 Pro Lys Cys Ile
Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe 100 105 110 Met Cys
Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser 115 120 125
Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu Leu Leu Val Ile Phe Gln 130
135 140 Val Thr Gly Ile Ser Leu Leu Pro Pro Leu Gly Val Ala Ile Ser
Val 145 150 155 160 Ile Ile Ile Phe Tyr Cys Tyr Arg Val Asn Arg Gln
Gln Lys Leu Ser 165 170 175 Ser <210> SEQ ID NO 47
<211> LENGTH: 194 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: MH2 domain of SMAD2 <400> SEQUENCE: 47 Trp Cys
Ser Ile Ala Tyr Tyr Glu Leu Asn Gln Arg Val Gly Glu Thr 1 5 10 15
Phe His Ala Ser Gln Pro Ser Leu Thr Val Asp Gly Phe Thr Asp Pro 20
25 30 Ser Asn Ser Glu Arg Phe Cys Leu Gly Leu Leu Ser Asn Val Asn
Arg 35 40 45 Asn Ala Thr Val Glu Met Thr Arg Arg His Ile Gly Arg
Gly Val Arg 50 55 60 Leu Tyr Tyr Ile Gly Gly Glu Val Phe Ala Glu
Cys Leu Ser Asp Ser 65 70 75 80 Ala Ile Phe Val Gln Ser Pro Asn Cys
Asn Gln Arg Tyr Gly Trp His 85 90 95 Pro Ala Thr Val Cys Lys Ile
Pro Pro Gly Cys Asn Leu Lys Ile Phe 100 105 110 Asn Asn Gln Glu Phe
Ala Ala Leu Leu Ala Gln Ser Val Asn Gln Gly 115 120 125 Phe Glu Ala
Val Tyr Gln Leu Thr Arg Met Cys Thr Ile Arg Met Ser 130 135 140 Phe
Val Lys Gly Trp Gly Ala Glu Tyr Arg Arg Gln Thr Val Thr Ser 145 150
155 160 Thr Pro Cys Trp Ile Glu Leu His Leu Asn Gly Pro Leu Gln Trp
Leu 165 170 175 Asp Lys Val Leu Thr Gln Met Gly Ser Pro Ser Val Arg
Cys Ser Ser 180 185 190 Met Ser <210> SEQ ID NO 48
<211> LENGTH: 194 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: MH2 domain of SMAD3 <400> SEQUENCE: 48 Trp Cys
Ser Ile Ser Tyr Tyr Glu Leu Asn Gln Arg Val Gly Glu Thr 1 5 10 15
Phe His Ala Ser Gln Pro Ser Met Thr Val Asp Gly Phe Thr Asp Pro 20
25 30 Ser Asn Ser Glu Arg Phe Cys Leu Gly Leu Leu Ser Asn Val Asn
Arg 35 40 45 Asn Ala Ala Val Glu Leu Thr Arg Arg His Ile Gly Arg
Gly Val Arg 50 55 60 Leu Tyr Tyr Ile Gly Gly Glu Val Phe Ala Glu
Cys Leu Ser Asp Ser 65 70 75 80 Ala Ile Phe Val Gln Ser Pro Asn Cys
Asn Gln Arg Tyr Gly Trp His 85 90 95 Pro Ala Thr Val Cys Lys Ile
Pro Pro Gly Cys Asn Leu Lys Ile Phe 100 105 110 Asn Asn Gln Glu Phe
Ala Ala Leu Leu Ala Gln Ser Val Asn Gln Gly 115 120 125 Phe Glu Ala
Val Tyr Gln Leu Thr Arg Met Cys Thr Ile Arg Met Ser 130 135 140 Phe
Val Lys Gly Trp Gly Ala Glu Tyr Arg Arg Gln Thr Val Thr Ser 145 150
155 160 Thr Pro Cys Trp Ile Glu Leu His Leu Asn Gly Pro Leu Gln Trp
Leu 165 170 175 Asp Lys Val Leu Thr Gln Met Gly Ser Pro Ser Ile Arg
Cys Ser Ser 180 185 190 Val Ser <210> SEQ ID NO 49
<211> LENGTH: 230 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: MH2 domain of SMAD4 <400> SEQUENCE: 49 Trp Cys
Ser Ile Ala Tyr Phe Glu Met Asp Val Gln Val Gly Glu Thr 1 5 10 15
Phe Lys Val Pro Ser Ser Cys Pro Ile Val Thr Val Asp Gly Tyr Val 20
25 30 Asp Pro Ser Gly Gly Asp Arg Phe Cys Leu Gly Gln Leu Ser Asn
Val
35 40 45 His Arg Thr Glu Ala Ile Glu Arg Ala Arg Leu His Ile Gly
Lys Gly 50 55 60 Val Gln Leu Glu Cys Lys Gly Glu Gly Asp Val Trp
Val Arg Cys Leu 65 70 75 80 Ser Asp His Ala Val Phe Val Gln Ser Tyr
Tyr Leu Asp Arg Glu Ala 85 90 95 Gly Arg Ala Pro Gly Asp Ala Val
His Lys Ile Tyr Pro Ser Ala Tyr 100 105 110 Ile Lys Val Phe Asp Leu
Arg Gln Cys His Arg Gln Met Gln Gln Gln 115 120 125 Ala Ala Thr Ala
Gln Ala Ala Ala Ala Ala Gln Ala Ala Ala Val Ala 130 135 140 Gly Asn
Ile Pro Gly Pro Gly Ser Val Gly Gly Ile Ala Pro Ala Ile 145 150 155
160 Ser Leu Ser Ala Ala Ala Gly Ile Gly Val Asp Asp Leu Arg Arg Leu
165 170 175 Cys Ile Leu Arg Met Ser Phe Val Lys Gly Trp Gly Pro Asp
Tyr Pro 180 185 190 Arg Gln Ser Ile Lys Glu Thr Pro Cys Trp Ile Glu
Ile His Leu His 195 200 205 Arg Ala Leu Gln Leu Leu Asp Glu Val Leu
His Thr Met Pro Ile Ala 210 215 220 Asp Pro Gln Pro Leu Asp 225 230
<210> SEQ ID NO 50 <211> LENGTH: 380 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: chimeric dnSMAD comprising a dnSMAD2
polypeptide and a dnSMAD3 polypeptide <400> SEQUENCE: 50 Trp
Cys Ser Ile Ala Tyr Tyr Glu Leu Asn Gln Arg Val Gly Glu Thr 1 5 10
15 Phe His Ala Ser Gln Pro Ser Leu Thr Val Asp Gly Phe Thr Asp Pro
20 25 30 Ser Asn Ser Glu Arg Phe Cys Leu Gly Leu Leu Ser Asn Val
Asn Arg 35 40 45 Asn Ala Thr Val Glu Met Thr Arg Arg His Ile Gly
Arg Gly Val Arg 50 55 60 Leu Tyr Tyr Ile Gly Gly Glu Val Phe Ala
Glu Cys Leu Ser Asp Ser 65 70 75 80 Ala Ile Phe Val Gln Ser Pro Asn
Cys Asn Gln Arg Tyr Gly Trp His 85 90 95 Pro Ala Thr Val Cys Lys
Ile Pro Pro Gly Cys Asn Leu Lys Ile Phe 100 105 110 Asn Asn Gln Glu
Phe Ala Ala Leu Leu Ala Gln Ser Val Asn Gln Gly 115 120 125 Phe Glu
Ala Val Tyr Gln Leu Thr Arg Met Cys Thr Ile Arg Met Ser 130 135 140
Phe Val Lys Gly Trp Gly Ala Glu Tyr Arg Arg Gln Thr Val Thr Ser 145
150 155 160 Thr Pro Cys Trp Ile Glu Leu His Leu Asn Gly Pro Leu Gln
Trp Leu 165 170 175 Asp Lys Val Leu Thr Gln Met Leu Glu Tyr Ser Gly
Gly Gly Ser Gly 180 185 190 Gly Gly Ser Leu Glu Trp Cys Ser Ile Ser
Tyr Tyr Glu Leu Asn Gln 195 200 205 Arg Val Gly Glu Thr Phe His Ala
Ser Gln Pro Ser Met Thr Val Asp 210 215 220 Gly Phe Thr Asp Pro Ser
Asn Ser Glu Arg Phe Cys Leu Gly Leu Leu 225 230 235 240 Ser Asn Val
Asn Arg Asn Ala Ala Val Glu Leu Thr Arg Arg His Ile 245 250 255 Gly
Arg Gly Val Arg Leu Tyr Tyr Ile Gly Gly Glu Val Phe Ala Glu 260 265
270 Cys Leu Ser Asp Ser Ala Ile Phe Val Gln Ser Pro Asn Cys Asn Gln
275 280 285 Arg Tyr Gly Trp His Pro Ala Thr Val Cys Lys Ile Pro Pro
Gly Cys 290 295 300 Asn Leu Lys Ile Phe Asn Asn Gln Glu Phe Ala Ala
Leu Leu Ala Gln 305 310 315 320 Ser Val Asn Gln Gly Phe Glu Ala Val
Tyr Gln Leu Thr Arg Met Cys 325 330 335 Thr Ile Arg Met Ser Phe Val
Lys Gly Trp Gly Ala Glu Tyr Arg Arg 340 345 350 Gln Thr Val Thr Ser
Thr Pro Cys Trp Ile Glu Leu His Leu Asn Gly 355 360 365 Pro Leu Gln
Trp Leu Asp Lys Val Leu Thr Gln Met 370 375 380 <210> SEQ ID
NO 51 <211> LENGTH: 271 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: chimeric TNF receptor HVEM-41BB <400>
SEQUENCE: 51 Met Glu Pro Pro Gly Asp Trp Gly Pro Pro Pro Trp Arg
Ser Thr Pro 1 5 10 15 Arg Thr Asp Val Leu Arg Leu Val Leu Tyr Leu
Thr Phe Leu Gly Ala 20 25 30 Pro Cys Tyr Ala Pro Ala Leu Pro Ser
Cys Lys Glu Asp Glu Tyr Pro 35 40 45 Val Gly Ser Glu Cys Cys Pro
Lys Cys Ser Pro Gly Tyr Arg Val Lys 50 55 60 Glu Ala Cys Gly Glu
Leu Thr Gly Thr Val Cys Glu Pro Cys Pro Pro 65 70 75 80 Gly Thr Tyr
Ile Ala His Leu Asn Gly Leu Ser Lys Cys Leu Gln Cys 85 90 95 Gln
Met Cys Asp Pro Ala Met Gly Leu Arg Ala Ser Arg Asn Cys Ser 100 105
110 Arg Thr Glu Asn Ala Val Cys Gly Cys Ser Pro Gly His Phe Cys Ile
115 120 125 Val Gln Asp Gly Asp His Cys Ala Ala Cys Arg Ala Tyr Ala
Thr Ser 130 135 140 Ser Pro Gly Gln Arg Val Gln Lys Gly Gly Thr Glu
Ser Gln Asp Thr 145 150 155 160 Leu Cys Gln Asn Cys Pro Pro Gly Thr
Phe Ser Pro Asn Gly Thr Leu 165 170 175 Glu Glu Cys Gln His Gln Thr
Lys Cys Ser Trp Leu Val Thr Lys Ala 180 185 190 Gly Ala Gly Thr Ser
Ser Ser His Trp Val Ile Ile Ser Phe Phe Leu 195 200 205 Ala Leu Thr
Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu 210 215 220 Arg
Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 225 230
235 240 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp
Gly 245 250 255 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys
Glu Leu 260 265 270 <210> SEQ ID NO 52 <211> LENGTH:
260 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: chimeric TNF
receptor CD27-41BB <400> SEQUENCE: 52 Met Ala Arg Pro His Pro
Trp Trp Leu Cys Val Leu Gly Thr Leu Val 1 5 10 15 Gly Leu Ser Ala
Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr 20 25 30 Trp Ala
Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe 35 40 45
Leu Val Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro 50
55 60 Cys Ile Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro
His 65 70 75 80 Cys Glu Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val
Arg Asn Cys 85 90 95 Thr Ile Thr Ala Asn Ala Glu Cys Ala Cys Arg
Asn Gly Trp Gln Cys 100 105 110 Arg Asp Lys Glu Cys Thr Glu Cys Asp
Pro Leu Pro Asn Pro Ser Leu 115 120 125 Thr Ala Arg Ser Ser Gln Ala
Leu Ser Pro His Pro Gln Pro Thr His 130 135 140 Leu Pro Tyr Val Ser
Glu Met Leu Glu Ala Arg Thr Ala Gly His Met 145 150 155 160 Gln Thr
Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr 165 170 175
His Trp Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg Ile 180
185 190 Ile Ser Phe Phe Leu Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu
Leu 195 200 205 Phe Phe Leu Thr Leu Arg Phe Ser Val Val Lys Arg Gly
Arg Lys Lys 210 215 220 Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg
Pro Val Gln Thr Thr 225 230 235 240 Gln Glu Glu Asp Gly Cys Ser Cys
Arg Phe Pro Glu Glu Glu Glu Gly 245 250 255 Gly Cys Glu Leu 260
<210> SEQ ID NO 53 <211> LENGTH: 281 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: chimeric TNF receptor RANK-41BB
<400> SEQUENCE: 53 Met Ala Pro Arg Ala Arg Arg Arg Arg Pro
Leu Phe Ala Leu Leu Leu 1 5 10 15 Leu Cys Ala Leu Leu Ala Arg Leu
Gln Val Ala Leu Gln Ile Ala Pro 20 25 30 Pro Cys Thr Ser Glu Lys
His Tyr Glu His Leu Gly Arg Cys Cys Asn 35 40 45 Lys Cys Glu Pro
Gly Lys Tyr Met Ser Ser Lys Cys Thr Thr Thr Ser 50 55 60 Asp Ser
Val Cys Leu Pro Cys Gly Pro Asp Glu Tyr Leu Asp Ser Trp 65 70 75 80
Asn Glu Glu Asp Lys Cys Leu Leu His Lys Val Cys Asp Thr Gly Lys 85
90 95 Ala Leu Val Ala Val Val Ala Gly Asn Ser Thr Thr Pro Arg Arg
Cys 100 105 110 Ala Cys Thr Ala Gly Tyr His Trp Ser Gln Asp Cys Glu
Cys Cys Arg 115 120 125 Arg Asn Thr Glu Cys Ala Pro Gly Leu Gly Ala
Gln His Pro Leu Gln 130 135 140 Leu Asn Lys Asp Thr Val Cys Lys Pro
Cys Leu Ala Gly Tyr Phe Ser 145 150 155 160 Asp Ala Phe Ser Ser Thr
Asp Lys Cys Arg Pro Trp Thr Asn Cys Thr 165 170 175 Phe Leu Gly Lys
Arg Val Glu His His Gly Thr Glu Lys Ser Asp Ala 180 185 190 Val Cys
Ser Ser Ser Leu Pro Ala Arg Lys Pro Pro Asn Glu Pro His 195 200 205
Val Tyr Leu Pro Ile Ile Ser Phe Phe Leu Ala Leu Thr Ser Thr Ala 210
215 220 Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu Arg Phe Ser Val Val
Lys 225 230 235 240 Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
Pro Phe Met Arg 245 250 255 Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
Cys Ser Cys Arg Phe Pro 260 265 270 Glu Glu Glu Glu Gly Gly Cys Glu
Leu 275 280 <210> SEQ ID NO 54 <211> LENGTH: 149
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: chimeric TNF
receptor Fn14-41BB <400> SEQUENCE: 54 Met Ala Arg Gly Ser Leu
Arg Arg Leu Leu Arg Leu Leu Val Leu Gly 1 5 10 15 Leu Trp Leu Ala
Leu Leu Arg Ser Val Ala Gly Glu Gln Ala Pro Gly 20 25 30 Thr Ala
Pro Cys Ser Arg Gly Ser Ser Trp Ser Ala Asp Leu Asp Lys 35 40 45
Cys Met Asp Cys Ala Ser Cys Arg Ala Arg Pro His Ser Asp Phe Cys 50
55 60 Leu Gly Cys Ala Ala Ala Pro Pro Ala Pro Phe Arg Leu Leu Trp
Pro 65 70 75 80 Ile Ile Ser Phe Phe Leu Ala Leu Thr Ser Thr Ala Leu
Leu Phe Leu 85 90 95 Leu Phe Phe Leu Thr Leu Arg Phe Ser Val Val
Lys Arg Gly Arg Lys 100 105 110 Lys Leu Leu Tyr Ile Phe Lys Gln Pro
Phe Met Arg Pro Val Gln Thr 115 120 125 Thr Gln Glu Glu Asp Gly Cys
Ser Cys Arg Phe Pro Glu Glu Glu Glu 130 135 140 Gly Gly Cys Glu Leu
145 <210> SEQ ID NO 55 <211> LENGTH: 261 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: TNF receptor ligand CD40L
<400> SEQUENCE: 55 Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro
Arg Ser Ala Ala Thr Gly 1 5 10 15 Leu Pro Ile Ser Met Lys Ile Phe
Met Tyr Leu Leu Thr Val Phe Leu 20 25 30 Ile Thr Gln Met Ile Gly
Ser Ala Leu Phe Ala Val Tyr Leu His Arg 35 40 45 Arg Leu Asp Lys
Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val 50 55 60 Phe Met
Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser 65 70 75 80
Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys 85
90 95 Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe
Glu 100 105 110 Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His
Val Ile Ser 115 120 125 Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln
Trp Ala Glu Lys Gly 130 135 140 Tyr Tyr Thr Met Ser Asn Asn Leu Val
Thr Leu Glu Asn Gly Lys Gln 145 150 155 160 Leu Thr Val Lys Arg Gln
Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr 165 170 175 Phe Cys Ser Asn
Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser 180 185 190 Leu Cys
Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala 195 200 205
Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His 210
215 220 Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val
Asn 225 230 235 240 Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly
Phe Thr Ser Phe 245 250 255 Gly Leu Leu Lys Leu 260 <210> SEQ
ID NO 56 <211> LENGTH: 183 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: TNF receptor ligand OX40L <400> SEQUENCE:
56 Met Glu Arg Val Gln Pro Leu Glu Glu Asn Val Gly Asn Ala Ala Arg
1 5 10 15 Pro Arg Phe Glu Arg Asn Lys Leu Leu Leu Val Ala Ser Val
Ile Gln 20 25 30 Gly Leu Gly Leu Leu Leu Cys Phe Thr Tyr Ile Cys
Leu His Phe Ser 35 40 45 Ala Leu Gln Val Ser His Arg Tyr Pro Arg
Ile Gln Ser Ile Lys Val 50 55 60 Gln Phe Thr Glu Tyr Lys Lys Glu
Lys Gly Phe Ile Leu Thr Ser Gln 65 70 75 80 Lys Glu Asp Glu Ile Met
Lys Val Gln Asn Asn Ser Val Ile Ile Asn 85 90 95 Cys Asp Gly Phe
Tyr Leu Ile Ser Leu Lys Gly Tyr Phe Ser Gln Glu 100 105 110 Val Asn
Ile Ser Leu His Tyr Gln Lys Asp Glu Glu Pro Leu Phe Gln 115 120 125
Leu Lys Lys Val Arg Ser Val Asn Ser Leu Met Val Ala Ser Leu Thr 130
135 140 Tyr Lys Asp Lys Val Tyr Leu Asn Val Thr Thr Asp Asn Thr Ser
Leu 145 150 155 160 Asp Asp Phe His Val Asn Gly Gly Glu Leu Ile Leu
Ile His Gln Asn 165 170 175 Pro Gly Glu Phe Cys Val Leu 180
<210> SEQ ID NO 57 <211> LENGTH: 254 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: TNF receptor ligand 41BBL
<400> SEQUENCE: 57 Met Glu Tyr Ala Ser Asp Ala Ser Leu Asp
Pro Glu Ala Pro Trp Pro 1 5 10 15 Pro Ala Pro Arg Ala Arg Ala Cys
Arg Val Leu Pro Trp Ala Leu Val 20 25 30 Ala Gly Leu Leu Leu Leu
Leu Leu Leu Ala Ala Ala Cys Ala Val Phe 35 40 45 Leu Ala Cys Pro
Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser 50 55 60 Ala Ala
Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp 65 70 75 80
Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val 85
90 95 Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser
Asp 100 105 110 Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser
Tyr Lys Glu 115 120 125 Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly
Val Tyr Tyr Val Phe 130 135 140 Phe Gln Leu Glu Leu Arg Arg Val Val
Ala Gly Glu Gly Ser Gly Ser 145 150 155 160 Val Ser Leu Ala Leu His
Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala 165 170 175 Ala Ala Leu Ala
Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala 180 185 190 Arg Asn
Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala 195 200
205
Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His 210
215 220 Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg
Val 225 230 235 240 Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg
Ser Glu 245 250 <210> SEQ ID NO 58 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: basic amino
acid furin target sequence <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (2)..(2) <223>
OTHER INFORMATION: Xaa can be any naturally occurring amino acid
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (3)..(3) <223> OTHER INFORMATION: Xaa may be Arg or
Lys <400> SEQUENCE: 58 Arg Xaa Xaa Arg 1 <210> SEQ ID
NO 59 <211> LENGTH: 7 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: consensus Tobacco Etch Virus (TEV) cleavage site
<400> SEQUENCE: 59 Glu Asn Leu Tyr Phe Gln Ser 1 5
<210> SEQ ID NO 60 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: ITAM (immunoreceptor tyrosine-based
activation motif) <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (2)..(3) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (4)..(4) <223> OTHER INFORMATION: Xaa may be Leu or
Ile <400> SEQUENCE: 60 Tyr Xaa Xaa Xaa 1 <210> SEQ ID
NO 61 <211> LENGTH: 7 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: frame-slip motif (FSM) <400> SEQUENCE: 61
uuuuuuu 7 <210> SEQ ID NO 62 <211> LENGTH: 9
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: frame-slip
motif (FSM) comprising a stop codon <400> SEQUENCE: 62
uuuuuuuga 9 <210> SEQ ID NO 63 <211> LENGTH: 9
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: FSM comprising
a stop codon <400> SEQUENCE: 63 uuuuuuuag 9 <210> SEQ
ID NO 64 <211> LENGTH: 9 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: FSM comprising a stop codon <400>
SEQUENCE: 64 uuuuuuuaa 9 <210> SEQ ID NO 65 <211>
LENGTH: 7 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
translational readthrough motif (TRM) comprising a stop codon
<400> SEQUENCE: 65 ugacuag 7 <210> SEQ ID NO 66
<211> LENGTH: 7 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: TRM comprising a stop codon <400> SEQUENCE: 66
uagcuag 7 <210> SEQ ID NO 67 <211> LENGTH: 7
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: TRM comprising
a stop codon <400> SEQUENCE: 67 uaacuag 7 <210> SEQ ID
NO 68 <211> LENGTH: 9 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: TRM comprising a stop codon <400>
SEQUENCE: 68 ugacaauua 9 <210> SEQ ID NO 69 <211>
LENGTH: 9 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: TRM
comprising a stop codon <400> SEQUENCE: 69 uagcaauua 9
<210> SEQ ID NO 70 <211> LENGTH: 9 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: TRM comprising a stop codon
<400> SEQUENCE: 70 uaacaauua 9 <210> SEQ ID NO 71
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: VH CDR, CDR1 <400> SEQUENCE: 71 Ser Tyr Asn Ile
His 1 5 <210> SEQ ID NO 72 <211> LENGTH: 16 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: VH CDR, CDR2 <400>
SEQUENCE: 72 Val Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala
Leu Met Ser 1 5 10 15 <210> SEQ ID NO 73 <211> LENGTH:
10 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: VH CDR, CDR3
<400> SEQUENCE: 73 Arg Ser Asp Asp Tyr Ser Trp Phe Ala Tyr 1
5 10 <210> SEQ ID NO 74 <211> LENGTH: 12 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: VL CDR, CDR1 <400>
SEQUENCE: 74 Arg Ala Ser Ser Ser Val Ser Ser Ser Tyr Leu His 1 5 10
<210> SEQ ID NO 75 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE:
<223> OTHER INFORMATION: VL CDR, CDR2 <400> SEQUENCE:
75 Ser Thr Ser Asn Leu Ala Ser 1 5 <210> SEQ ID NO 76
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: VL CDR, CDR3 <400> SEQUENCE: 76 Gln Gln Tyr Ser
Gly Tyr Pro Ile Thr 1 5 <210> SEQ ID NO 77 <211>
LENGTH: 118 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
humanised KM666 VH sequence <400> SEQUENCE: 77 Gln Val Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr
Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ala Ser Tyr 20 25
30 Asn Ile His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45 Gly Val Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala
Leu Met 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn
Gln Val Phe Leu 65 70 75 80 Lys Met Ser Ser Leu Thr Ala Ala Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Arg Ser Asp Asp Tyr Ser Trp
Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser
115 <210> SEQ ID NO 78 <211> LENGTH: 108 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: humanised KM666 VL sequence
<400> SEQUENCE: 78 Glu Asn Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Met Thr Cys Arg
Ala Ser Ser Ser Val Ser Ser Ser 20 25 30 Tyr Leu His Trp Tyr Gln
Gln Lys Ser Gly Lys Ala Pro Lys Val Trp 35 40 45 Ile Tyr Ser Thr
Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Gly Tyr Pro 85
90 95 Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
<210> SEQ ID NO 79 <211> LENGTH: 136 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: sort/suicide gene RQR8 <400>
SEQUENCE: 79 Cys Pro Tyr Ser Asn Pro Ser Leu Cys Ser Gly Gly Gly
Gly Ser Glu 1 5 10 15 Leu Pro Thr Gln Gly Thr Phe Ser Asn Val Ser
Thr Asn Val Ser Pro 20 25 30 Ala Lys Pro Thr Thr Thr Ala Cys Pro
Tyr Ser Asn Pro Ser Leu Cys 35 40 45 Ser Gly Gly Gly Gly Ser Pro
Ala Pro Arg Pro Pro Thr Pro Ala Pro 50 55 60 Thr Ile Ala Ser Gln
Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro 65 70 75 80 Ala Ala Gly
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 85 90 95 Ile
Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 100 105
110 Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn Arg Arg Arg Val
115 120 125 Cys Lys Cys Pro Arg Pro Val Val 130 135 <210> SEQ
ID NO 80 <211> LENGTH: 517 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: suicide gene Rapcasp9 <400> SEQUENCE: 80
Met Ala Ser Arg Ile Leu Trp His Glu Met Trp His Glu Gly Leu Glu 1 5
10 15 Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met
Phe 20 25 30 Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
Pro Gln Thr 35 40 45 Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly
Arg Asp Leu Met Glu 50 55 60 Ala Gln Glu Trp Cys Arg Lys Tyr Met
Lys Ser Gly Asn Val Lys Asp 65 70 75 80 Leu Leu Gln Ala Trp Asp Leu
Tyr Tyr His Val Phe Arg Arg Ile Ser 85 90 95 Lys Leu Glu Tyr Ser
Gly Gly Gly Ser Leu Glu Gly Val Gln Val Glu 100 105 110 Thr Ile Ser
Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr 115 120 125 Cys
Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp 130 135
140 Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln
145 150 155 160 Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met
Ser Val Gly 165 170 175 Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr
Ala Tyr Gly Ala Thr 180 185 190 Gly His Pro Gly Ile Ile Pro Pro His
Ala Thr Leu Val Phe Asp Val 195 200 205 Glu Leu Leu Lys Leu Glu Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Gly Gly Gly Gly
Ser Gly Val Asp Gly Phe Gly Asp Val Gly Ala 225 230 235 240 Leu Glu
Ser Leu Arg Gly Asn Ala Asp Leu Ala Tyr Ile Leu Ser Met 245 250 255
Glu Pro Cys Gly His Cys Leu Ile Ile Asn Asn Val Asn Phe Cys Arg 260
265 270 Glu Ser Gly Leu Arg Thr Arg Thr Gly Ser Asn Ile Asp Cys Glu
Lys 275 280 285 Leu Arg Arg Arg Phe Ser Ser Leu His Phe Met Val Glu
Val Lys Gly 290 295 300 Asp Leu Thr Ala Lys Lys Met Val Leu Ala Leu
Leu Glu Leu Ala Gln 305 310 315 320 Gln Asp His Gly Ala Leu Asp Cys
Cys Val Val Val Ile Leu Ser His 325 330 335 Gly Cys Gln Ala Ser His
Leu Gln Phe Pro Gly Ala Val Tyr Gly Thr 340 345 350 Asp Gly Cys Pro
Val Ser Val Glu Lys Ile Val Asn Ile Phe Asn Gly 355 360 365 Thr Ser
Cys Pro Ser Leu Gly Gly Lys Pro Lys Leu Phe Phe Ile Gln 370 375 380
Ala Cys Gly Gly Glu Gln Lys Asp His Gly Phe Glu Val Ala Ser Thr 385
390 395 400 Ser Pro Glu Asp Glu Ser Pro Gly Ser Asn Pro Glu Pro Asp
Ala Thr 405 410 415 Pro Phe Gln Glu Gly Leu Arg Thr Phe Asp Gln Leu
Asp Ala Ile Ser 420 425 430 Ser Leu Pro Thr Pro Ser Asp Ile Phe Val
Ser Tyr Ser Thr Phe Pro 435 440 445 Gly Phe Val Ser Trp Arg Asp Pro
Lys Ser Gly Ser Trp Tyr Val Glu 450 455 460 Thr Leu Asp Asp Ile Phe
Glu Gln Trp Ala His Ser Glu Asp Leu Gln 465 470 475 480 Ser Leu Leu
Leu Arg Val Ala Asn Ala Val Ser Val Lys Gly Ile Tyr 485 490 495 Lys
Gln Met Pro Gly Cys Phe Asn Phe Leu Arg Lys Lys Leu Phe Phe 500 505
510 Lys Thr Ser Ala Ser 515 <210> SEQ ID NO 81 <211>
LENGTH: 520 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: a
flexi-IL-12 sequence <400> SEQUENCE: 81 Met Trp Ile Trp Glu
Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp 1 5 10 15 Trp Tyr Pro
Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr 20 25 30 Pro
Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val 35 40
45 Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly
Asp
50 55 60 Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser
His Ser 65 70 75 80 Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp
Ser Thr Asp Ile 85 90 95 Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys
Thr Phe Leu Arg Cys Glu 100 105 110 Ala Lys Asn Tyr Ser Gly Arg Phe
Thr Cys Trp Trp Leu Thr Thr Ile 115 120 125 Ser Thr Asp Leu Thr Phe
Ser Val Lys Ser Ser Arg Gly Ser Ser Asp 130 135 140 Pro Gln Gly Val
Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val 145 150 155 160 Arg
Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp 165 170
175 Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val
180 185 190 Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser
Phe Phe 195 200 205 Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn
Leu Gln Leu Lys 210 215 220 Pro Leu Lys Asn Ser Arg Gln Val Glu Val
Ser Trp Glu Tyr Pro Asp 225 230 235 240 Thr Trp Ser Thr Pro His Ser
Tyr Phe Ser Leu Thr Phe Cys Val Gln 245 250 255 Val Gln Gly Lys Ser
Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp 260 265 270 Lys Thr Ser
Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val 275 280 285 Arg
Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser 290 295
300 Val Pro Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
305 310 315 320 Gly Gly Ser Arg Asn Leu Pro Leu Ala Thr Pro Asp Pro
Gly Met Phe 325 330 335 Pro Cys Leu His His Ser Gln Asn Leu Leu Arg
Ala Val Ser Asn Met 340 345 350 Leu Gln Lys Ala Arg Gln Thr Leu Glu
Phe Tyr Pro Cys Thr Ser Glu 355 360 365 Glu Ile Asp His Glu Asp Ile
Thr Lys Asp Lys Thr Ser Thr Val Glu 370 375 380 Ala Cys Leu Pro Leu
Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser 385 390 395 400 Arg Glu
Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys 405 410 415
Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Ser 420
425 430 Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu
Met 435 440 445 Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu
Ala Val Ile 450 455 460 Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser
Glu Thr Val Pro Gln 465 470 475 480 Lys Ser Ser Leu Glu Glu Pro Asp
Phe Tyr Lys Thr Lys Ile Lys Leu 485 490 495 Cys Ile Leu Leu His Ala
Phe Arg Ile Arg Ala Val Thr Ile Asp Arg 500 505 510 Val Met Ser Tyr
Leu Asn Ala Ser 515 520
* * * * *