Note: Descriptions are shown in the official language in which they were submitted.
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'I' cell receptors containing a non-native disulfide interchain bond linked to
therapeutic agents
The present invention relates to T cell receptors (TCRs) containing a non-
native
disulphide interchain bond associated with therapeutic agents.
Back2round to the Invention
The novel TCR therapeutic combinations disclosed herein will be of use in the
treatment of autoimmune disease, organ rejection, Graft Versus Host Disease
(GVHD) and cancer. The TCR portion of the TCR therapeutic agent combinations
disclosed herein are targeting moieties.
Brief Description of the Invention
This invention makes available for the first time a dimeric TCR (dTCR) or
single-
chain TCR (scTCR) associated with a therapeutic agent, wherein said agent is
selected from IL-1, IL-la, IL-3, IL-4, IL-5, IL-6, IL-7, IL-l0, IL-11, IL-12,
IL-13,
IL-15, IL-21, IL-23, TGF-(3, IFN-y, Lymphotoxin, TNFa, Anti-CD2 antibody, Anti-
CD3 antibody, Anti-CD4 antibody, Anti-CD8 antibody, Anti-CD44 antibody, Anti-
CD45RA antibody, Anti-CD45RB antibody, Anti-CD45RO antibody, Anti-Thy 1.2
antibody, Antilymphocyte globulin, Anti-a(3TCR antibody, Anti-y8TCR antibody,
Anti-CD49a antibody, Anti-CD49b antibody, Anti-CD49c antibody, Anti-CD49d
antibody, Anti-CD49e antibody, Anti-CD49f antibody, Anti-TCR V(38 antibody,
Anti-CD16 antibody, Anti-CD28 antibody, CTLA-4-Ig, Anti-B7.2 antibody, Anti-
CD40L antibody, Anti-ICAM-1 antibody, ICAM-1, Anti-Mac antibody, Anti-LFA-1
antibody, Anti-IFN-y antibody IFN-y, IFN-yR/IgGl fusions, Anti-IL-2R
antibodies,
IL-2R antibody, IL-2 Diptheria-toxin protein, Anti-IL-12 antibody, IL-12
Antagonist
(p40), Anti-IL-1 antibody, IL-1 Antagonist, Glutamic acid decarboxylase (GAD),
Anti-GAD antibody, Viral proteins and peptides, Bacterial proteins or
peptides, A-
Galactosyl-ceramide, Calcitonin, Nicotinamide, Anti-oxidants (Vitamin E,
Probucol
analog, Probucol + deflazacoert or Aminoguanidine), Anti-Inflammatory agents
(Pentoxifylline or Rolipram), Immunomodulators (Linomide, Ling-zhi-8, D-
Glucan,
Multi-functional protein 14, Ciamexon, Cholera toxin B, Vanadate or Vitamin D3
analogue, small molecule CD80 inhibitors, Androgens, IGF-1, Immunomanipulation
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(Natural antibodies), Lupus idiotype, Lipopolysaccaride), Sulfatide, Bee
venom,
Kampo formulation, Silica, Ganglioside, Antiasialo GM-1 antibody,
Hyaluronidase,
Concanavalin A, Anti-Class I MHC antibody, or Anti-Class II MHC antibody,
Cyclosporin, FK-506, Azathioprine, Rapainycin or Deoxyspergualin, PE38
Pseudomonas exotoxin or a functional variant or fragment of any of the
foregoing,
and wherein said TCR comprises a first segment constituted by an amino acid
sequence corresponding to a TCR a chain variable domain sequence fused to the
N
terminus of an amino acid sequence corresponding to a TCR a chain constant
domain
extracellular sequence, a second segment constituted by an amino acid sequence
corresponding to a TCR (3 chain variable domain fused to the N terminus of an
arnino
acid sequence corresponding to TCR P chain constant domain extracellular
sequence,
a disulfide bond between the first and second chains, said disulfide bond
being one
which has no equivalent in native a(3T cell receptors, and in the case of said
scTCRs
further comprising a linker sequence linking the C terrninus of the first
segment to the
N terminus of the second segment, or vice versa, the length of the linker
sequence and
the position of the disulfide bond being such that the variable domain
sequences of the
first and second segments are mutually orientated substantially as in native
a(3 T cell
receptors.
Detailed Description of the Invention
The present invention provides a dimeric TCR (dTCR) or single-chain TCR
(scTCR)
associated with a tlierapeutic agent, wherein said agent selected from one of
IL-l, IL-
la, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-11, IL-12, IL-13, IL-15, IL-21, IL-
23, TGF-
(3, IFN-y, Lymphotoxin, TNFa, Anti-CD2 antibody, Anti-CD3 antibody, Anti-CD4
antibody, Anti-CD8 antibody, Anti-CD44 antibody, Anti-CD45RA antibody, Anti-
CD45RB antibody, Anti-CD45RO antibody, Anti-Thy 1.2 antibody, Antilymphocyte
globulin, Anti-a(3TCR antibody, Anti-ySTCR antibody, Anti-CD49a antibody, Anti-
CD49b antibody, Anti-CD49c antibody, Anti-CD49d antibody, Anti-CD49e antibody,
Anti-CD49f antibody, Anti-TCR V(38 antibody, Anti-CD16 antibody, Anti-CD28
antibody, CTLA-4-Ig, Anti-B7.2 antibody, Anti-CD40L antibody, Anti-ICAM-1
antibody, ICAM-1, Anti-Mac antibody, Anti-LFA-1 antibody, Anti-IFN-,y antibody
IFN-y, IFN-yR/IgGl fusions, Anti-IL-2R antibodies, IL-2R antibody, IL-2
Diptheria-
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toxin protein, Anti-IL-12 antibody, IL-12 Antagonist (p40), Anti-IL-1
antibody, IL-1
Antagonist, Glutamic acid decarboxylase (GAD), Anti-GAD antibody, Viral
proteins
and peptides, Bacterial proteins or peptides, A-Galactosyl-ceramide,
Calcitonin,
Nicotinamide, Anti-oxidants (Vitamin E, Probucol analog, Probucol +
deflazacoert or
Aminoguanidine), Anti-In.flammatory agents (Pentoxifylline or Rolipram),
Immunomodulators (Linomide, Ling-zhi-8, D-Glucan, Multi-functional protein 14,
Ciamexon, Cholera toxin B, Vanadate or Vitamin D3 analogue, small molecule
CD80
inhibitors, Androgens, IGF-1, Immunomanipulation (Natural antibodies), Lupus
idiotype, Lipopolysaccaride), Sulfatide, Bee venom, Kampo formulation, Silica,
Ganglioside, Antiasialo GM-I antibody, Hyaluronidase, Concanavalin A, Anti-
Class
I MHC antibody, or Anti-Class II MHC antibody, Cyclosporin, FK-506,
Azathioprine, Rapamycin or Deoxyspergualin, PE38 Pseudomonas exotoxin, or a
functional variant or fragment of any of the foregoing, and wherein said TCR
comprises a first segment constituted by an amino acid sequence corresponding
to a
TCR a chain variable domain sequence fused to the N terminus of an amino acid
sequence corresponding to a TCR a chain constant domain extracellular
sequence, a
second segment constituted by an amino acid sequence corresponding to a TCR (3
chain variable domain fused to the N terminus of an amino acid sequence
corresponding to TCR (3 chain constant domain extracellular sequence, a
disulfide
bond between the first and second chains, said disulfide bond being one which
has no
equivalent in native a(3 T cell receptors, and in the case of said scTCRs
further
comprising a linker sequence linking the C terminus of the first segment to
the N
terminus of the second segment, or vice versa, the length of the linker
sequence and
the position of the disulfide bond being such that the variable domain
sequences of the
first and second segments are mutually orientated substantially as in native
a(3 T cell
receptors.
As used herein the term "a dimeric TCR (dTCR) or single-chain TCR (seTCR)
associated with an therapeutic agent" is understood to refer to a TCR
covalently or
otherwise linked to an therapeutic agent. The therapeutic agent may either be
directly
linked to the TCR, or indirectly via a linker moiety.
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As used herein the term "functional variant" is understood to refer to
analogues of the
disclosed therapeutic agents which have the same therapeutic effect. For
example, as
is known to those skilled in the art, it may be possible to produce
therapeutics that
incorporate minor changes in the chemical structure or amino acid sequence
thereof
compared to those disclosed without altering the therapeutic effect of the
agents. Such
trivial variants are included in the scope of this invention.
Functional antibody fYagnaents and variants
Antibody fragments and variants/analogues which are suitable for use in the
compositions and methods described herein include, but are not limited to, the
following.
Antibody Fragments
As is known to those skilled in the art, it is possible to produce fragments
of a given
antibody wllich retain substantially the same binding characteristics as those
of the
parent antibody. The following provides details of such fragments:
Minibodies - These constructs consist of antibodies with a truncated Fc
portion. As
such they retain the complete binding domains of the antibody from which are
derived.
Fab fragments - These comprise a single immunoglobulin light chain covalently-
linked to part of an immunoglobulin heavy chain. As such, Fab fragments
coinprise a
single antigen combining site. Fab fragments are defined by the portion of an
IgG that
can be liberated by treatment with papain. Such fragments are commonly
produced
via recombinant DNA techniques. (Reeves et al., (2000) Lecture Notes on
Inznaunology (4th Edition) Published by Blackwell Science)
F(ab')2 fragments - These comprise both antigen combining sites and the hinge
region from a single antibody. F(ab')2 fragments are defined by the portion of
an IgG
that can be liberated by treatment with pepsin. Such fragments are commonly
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produced via recombinant DNA techniques. (Reeves et al., (2000) Lecture Notes
on
Inamunology (4tth Edition) Published by Blackwell Science)
Fv fragments - These comprise an immunoglobulin variable heavy domain linked
to
an immunoglobulin variable light domain. A number of Fv designs have been
produced. These include dsFvs, in which the association between the two
domains is
enhanced by an introduced disulfide bond. Alternatively, scFVs can be formed
using a
peptide linker to bind the two domains together as a single polypeptide. Fvs
constructs
containing a variable domain of a heavy or light immunoglobulin chain
associated to
the variable and constant domain of the corresponding immunoglobulin heavy or
light
chain have also been produced. FV have also been multimerised to form
diabodies
and triabodies (Maynard et al., (2000) Annu Rev Bionzed Eng 2 339-376)
NanobodiesTM - These constructs, marketed by Ablynx (Belgium), comprise
synthetic
single immunoglobulin variable heavy domain derived from a camelid (e.g. camel
or
llama) antibody.
Domain Antibodies - These constructs, marketed by Domantis (Belgium), comprise
an affinity matured single immunoglobulin variable heavy domain or
immunoglobulin variable light domain.
Antibody variants and analogues
The defining functional characteristic of antibodies in the context of the
present
invention is their ability to bind specifically to a target ligand. As is
known to those
skilled in the art it is possible to engineer such binding characteristics
into a range of
other proteins. Examples of antibody variants and analogues suitable for use
in the
compositions and methods of the present invention include, but are not limited
to, the
following.
Protein scaffold-based binding polypeptides - This family of binding
constructs
comprise mutated analogues of proteins which contain native binding loops.
Examples include Affibodies, marketed by Affibody (Sweden), which are based on
a
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three-helix motif derived from one of the IgG binding domains of
Staphylococcus
aureus Protein A. Another example is provided by Evibodies, marketed by
EvoGenix
(Australia) which are based on the extracellular domains of CTLA-4into which
domains similar to antibody binding loops are grafted. A final example,
Cytokine
Traps marketed by Regeneron Pharmaceuticals (US), graft cytokine receptor
domains
into antibody scaffolds. (Nygren et al., (2000) Current Opinion in Structural
biology
7 463-469) provides a review of the uses of scaffolds for engineering novel
binding
sites in proteins. This review mentions the following proteins as sources of
scaffolds:
CP1 zinc finger, Tendamistat, Z domain (a protein A analogue), PST1, Coiled
coils,
LACI-D 1 and cytochrome b562. Other protein scaffold studies have reported the
use of
Fibronectin, Green fluorescent protein (GFP) and ankyrin repeats.
As is known to those skilled in the art antibodies or fragments, variants or
analogues
thereof can be produced which bind to various parts of a given protein ligand.
For
example, anti-CD3 antibodies can be raised to any of the polypeptide chains
from
which this complex is formed (i.e.y, 8, s, ~, and 11 CD3 chains) Antibodies
which bind
to the s CD3 chain are the preferred anti-CD3 antibodies for use in the
compositions
and methods of the present invention.
Another aspect of the invention provides a dTCR or scTCR associated with a
therapeutic agent, wherein the therapeutic agent is selected from IL-1, IL-la,
IL-3,
IL-5, IL-6, IL-7, IL-11, IL-12, TGF-0, Lymphotoxin, TNFa, Anti-CD2 antibody,
Anti-CD4 antibody, Anti-CD8 antibody, Anti-CD44 antibody, Anti-CD45RA
antibody, Anti-CD45RB antibody, Anti-CD45RO antibody, Anti-Thy 1.2 antibody,
Antilylnphocyte globulin, Anti-a(3TCR antibody, Anti-y6TCR antibody, Anti-
CD49a
antibody, Anti-CD49b antibody, Anti-CD49c antibody, Anti-CD49d antibody, Anti-
CD49e antibody,Anti-CD49f antibody, Anti-TCR V(38 antibody, Anti-CD 16
antibody, Anti-CD28 antibody, CTLA-4-Ig, Anti-B7.2 antibody, Anti-CD40L
antibody, Anti-ICAM-1 antibody, ICAM-1, Anti-Mac antibody, Anti-LFA-1
antibody, Anti-IFN-y antibody IFN-y, IFN-yR/IgGl fusions, Anti-IL-2R
antibodies,
IL-2R antibody, IL-2 Diptheria-toxin protein, Anti-IL-12 antibody, IL-12
Antagonist
(p40), Anti-IL-1 antibody, IL-1 Antagonist, Glutamic acid decarboxylase (GAD),
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Anti-GAD antibody, Viral proteins and peptides, Bacterial proteins or
peptides, A-
Galactosyl-ceramide, Calcitonin, Nicotinamide, Anti-oxidants (Vitamin E,
Probucol
analog, Probucol + deflazacoert or Aminoguanidine), Anti-Inflammatory agents
(Pentoxifylline or Rolipram), Immunomodulators (Linomide, Ling-zhi-8, D-
Glucan,
Multi-functional protein 14, Ciamexon, Cholera toxin B, Vanadate or Vitamin D3
analogue, small molecule CD80 inhibitors, Androgens, IGF-1, Immunomanipulation
(Natural antibodies), Lupus idiotype, Lipopolysaccaride), Sulfatide, Bee
venom,
Kampo formulation, Silica, Ganglioside, Antiasialo GM-1 antibody,
Hyaluronidase,
Concanavalin A, Anti-Class I MHC antibody, or Anti-Class II MHC antibody,
Cyclosporin, FK-506, Azathioprine, Rapamycin or Deoxyspergualin, or a
functional
variant or fragment of any of the foregoing.
"Anti-T cell" antibodies
One preferred group of the immunomodulatory agents of the invention are
antibodies
or functional fragrnents or variants/analogues thereof which bind epitopes
presented
only by T cells or Natural Killer (NK) cells. The following are the antibodies
which
will specifically target these cells:
Anti-CD3 antibody, Anti-CD4 antibody, Anti-CD8 antibody, Anti-a(3TCR antibody,
Anti-CD49a antibody, Anti-CD49b antibody, Anti-CD49c antibody, Anti-CD49d
antibody, Anti-CD49e antibody, Anti-CD49f antibody, Anti-ySTCR antibody, Anti-
TCR V08 antibody and Anti-CD28 antibody.
As will be known to those skilled in the art particular subsets of T cells
and/or NK
cells are targeted by the majority of the above antibodies. Only anti-CD3
antibodies
will target all NK cells and T cells.
Such antibodies, linked to a soluble TCR to form a bifunctional composition of
the
invention, will cause T cells and/or NK cells to be localised to the cells
expressing the
cognate peptide-MHC ligand for the soluble TCR. Without wishing to be limited
by
theory, the binding of these antibodies to the T cells or NK cells may cause
these cells
to be activated.
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Another aspect of the invention provides a dTCR or scTCR associated with a
therapeutic agent selected from IL-10, IL-4 or IL-13 or a functional variant
or
fragrnent of any of the foregoing.
One aspect of the invention is provided wherein the dTCR or scTCR is tissue-
specific.
In a one embodiment of the present aspect the dTCR or scTCR is specific for a
tissue
which is a target for auto-reactive T cells in autoimmune disease, organ
rejection or
Graft Versus Host Disease (GVHD). In a specific embodiment of the present
aspect
the dTCR or scTCR is islet cell-specific. The T cell clones NY8.3 (Santamaria
et al.,
J. bn7nunology (1995) 154 2494-2503) and (Nagata et al., (1995) Jlninzunologv
152
2042-2050) and G9C8 (Wong et al., JExp Med 1996) 183 67-76) are examples of
murine T cell clones that are islet cell-specific. The NY8.3 T cell clone is
specific for
a glucose-6-phosphatase catalytic subunit-related protein (IGRP)-derived
peptide
presented by the murine H2-Kd MHC and the G9C8 T cell clone is specific for an
insulin-derived peptide presented by the murine H2-Ka MHC.
A further aspect of the invention provides a dTCR or scTCR associated with a
therapeutic agent, wherein the therapeutic agent is selected from IL-15, IL-
21, IL-23,
PE3 8 Pseudomonas exotoxin, IFN-y or Anti-CD3 antibody or a functional variant
or
fragrnent of any of the foregoing.
In one aspect of the invention the TCR associated with a therapeutic agent is
a dTCR.
In an alternative aspect of the invention the TCR associated with a
therapeutic agent is
a scTCR.
There are two classes of linker that are preferred for the association of TCRs
and
therapeutic agents of the present invention. A TCR of the invention in which
the TCR
is linked by a polyalkylene glycol chain to the therapeutic agent provides one
embodiment of the present aspect. Peptidic linkers are the other class of TCR
linkers.
These two classes of linker are discussed in detail below in relation to their
use in the
formation of TCR multimers. Example 6 herein provides two examples of peptidic
linkers which may be used to form the association between the TCR and
therapeutic
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agent. As is known to those skilled in the art a variety of peptide linkers
may be
suitable to link the TCR (3 chains to the required therapeutic agents. The
following are
additional examples linker sequences which may be used for this purpose
ggcggtccg - which encodes a Gly-Gly-Pro linker.
ccc - which encodes a Pro-Gly linker including a Xmal restriction enzyme site
As mentioned above, the TCR portions of the TCR therapeutic agent combinations
disclosed herein are targeting moieties. The TCRs of the invention target TCR
ligands
such as peptide-MHC or CD1-antigen complexes. As such, it would be desirable
if
these TCR had a higher affinity and/or a slower off-rate for the TCR ligands
than
native TCRs specific for that ligand. The inventors co-ending application WO
2004/044004 details methods of producing TCR having a higher affinity and/or a
slower off-rate for the TCR ligand than native TCRs specific for that ligand.
Preferably, the affinity (KD) of the TCR for the TCR ligand is higher than 1
M,
and/or the off-rate (kOFF) is slower than 1 x 10-3 S-1. More preferably, the
affinity (KD)
of the TCR for the TCR ligand is higher than 10nM, and/or the off-rate (koff)
is slower
than 1 x 10"4 S-1. Most preferably, the affmity (KD) of the TCR for the TCR
ligand is
higher than 1nM, and/or the off-rate (koff) is slower than 1 x 10"5 S-1.
The affinity (KD) and/or off-rate (koff) measurement can be made by any of the
known
methods. A preferred method is the Surface Plasmon Resonance (Biacore) method
of
Example 3.
In one broad aspect, the TCRs of the invention are in the form of either
single chain
TCRs (scTCRs) or dimeric TCRs (dTCRs) as described in WO 04/033685 and WO
03/020763.
A suitable scTCR form comprises a first segment constituted by an amino acid
sequence corresponding to a TCR a chain variable domain, a second segment
constituted by an amino acid sequence corresponding to a TCR P chain variable
domain sequence fused to the N terminus of an amino acid sequence
corresponding to
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a TCR (3 chain constant domain extracellular sequence, and a linker sequence
linking
the C terminus of the first segment to the N terminus of the second segment.
Alternatively the first segment may be constituted by an amino acid sequence
corresponding to a TCR (3 chain variable domain, the second segment may be
constituted by an amino acid sequence corresponding to a TCR a chain variable
domain sequence fused to the N terminus of an amino acid sequence
corresponding to
a TCR a chain constant domain extracellular sequence
More specifically the first segment rnay be constituted by an amino acid
sequence
corresponding to a TCR a chain variable domain sequence fused to the N
terminus of
an amino acid sequence corresponding to a TCR a chain constant domain
extracellular sequence, the second segment may be constituted by an amino acid
sequence corresponding to a TCR (3 chain variable domain fused to the N
terminus of
an amino acid sequence corresponding to TCR P chain constant domain
extracellular
sequence, and a disulfide bond may be provided between the first and second
chains,
said disulfide bond being one which has no equivalent in native a(3 T cell
receptors.
In the above scTCR forms, the linker sequence may link the C terminus of the
first
segment to the N terminus of the second segment, and may have the formula -
PGGG-
(SGGGG)5-P- (SEQ ID NO: 1) or -PGGG-(SGGGG)6-P- (SEQ ID NO: 2) wherein P
is proline, G is glycine and S is serine.
A suitable dTCR form of the TCRs of the present invention coinprises a first
polypeptide wherein a sequence corresponding to a TCR a chain variable domain
sequence is fused to the N terminus of a sequence corresponding to a TCR a
chain
constant domain extracellular sequence, and a second polypeptide wherein a
sequence
corresponding to a TCR P chain var-iable domain sequence fused to the N
terminus a
sequence corresponding to a TCR (3 chain constant domain extracellular
sequence, the
first and second polypeptides being linked by a disulfide bond which has no
equivalent in native a(3 T cell receptors.
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The first polypeptide may comprise a TCR a chain variable domain sequence is
fused
to the N terminus of a sequence corresponding to a TCR a chain constant domain
extracellular sequence, and a second polypeptide wherein a sequence
corresponding to
a TCR P chain variable domain sequence is fused to the N terminus a sequence
corresponding to a TCR (3 chain constant domain extracellular sequence, the
first and
second polypeptides being linked by a disulfide bond between cysteine residues
substituted for Thr 48 of exon 1 of TRAC*01 and Ser 57 of exon 1 of TRBCI*01
or
TRBC2*01 or the non-human equivalent thereof. ("TRAC" etc. nomenclature herein
as per T cell receptor Factsbook, (2001) LeFranc and LeFranc, Academic Press,
ISBN
0-12-441352-8)
The dTCR or scTCR form of the TCRs of the invention may have amino acid
sequences corresponding to human a(3 TCR extracellular constant and variable
domain sequences, and a disulfide bond may link amino acid residues of the
said
constant domain sequences, which disulfide bond has no equivalent in native
TCRs.
The disulfide bond is between cysteine residues corresponding to amino acid
residues
whose (3 carbon atoms are less than 0.6 nm apart in native TCRs, for example
between
cysteine residues substituted for Thr 48 of exon 1 of TIRAC*01 and Ser 57 of
exon 1
of TRBC1*01 or TRBC2*01 or the non-human equivalent thereof. Other sites where
cysteines can be introduced to form the disulfide bond are the following
residues in
exon 1 of TRAC*01 for the TCR a chain and TRBC1'1401 or TRBC2*01 for the TCR
(3 chain:
TCR a chain TCR (3 chain Native (3 carbon
separation (nm)
Thr 45 Ser 77 0.533
Tyr 10 Ser 17 0.359
Thr 45 Asp 59 0.560
Ser 15 Glu 15 0.59
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In addition to the non-native disulfide bond referred to above, the dTCR or
scTCR
form of the TCRs of the invention may include a disulfide bond between
residues
corresponding to those linked by a disulfide bond in native TCRs.
The dTCR or scTCR form of the TCRs of the invention preferably does not
contain a
sequence corresponding to transmembrane or cytoplasmic sequences of native
TCRs.
One embodiment of the invention provides a TCR associated with a therapeutic
agent,
wherein said therapeutic agent is a PE38 exotoxin.
PE38 exotoxin is a truncated form of a Pseudomonas exotoxin. The native
polypeptide is a 66kDa protein consisting of domains IA, II, IB and III. The
PE38
derivative consists of domain II, amino acids 380-399 of domain IB and domain
III.
As will be obvious to those skilled in the art other triuicated forms of
Pseudomonas
exotoxin may be of use in the present invention. (For example PE40).The
preferred
variant of PE38 for use in the present invention contains mutations in the
domain III
thereof such that the C-terminus amino acids are KDEL. These C-terminal
mutations
have previously been shown to increase the toxicity of the Pseudomona.s
exotoxin.
(Kreitman et al (1995) JBiochem 307 29-37)
In a preferred embodiment said TCR associated with a PE38 exotoxin comprises
the
amino acid sequences of (SEQ ID NO: 73) and (SEQ ID NO: 71). (Figures 29b and
28b respectively).
PEGylated TCR Monomers
In one particular embodiment a TCR associated with a therapeutic agent of the
invention is associated with at least one polyalkylene glycol chain(s). This
association
may be cause in a number of ways known to those skilled in the art. In a
preferred
embodiment the polyalkylene chain(s) is/are covalently linked to the TCR. In a
further embodiment the polyethylene glycol chains of the present aspect of the
invention comprise at least two polyethylene repeating units.
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1Vlultivalent TCR Complexes
One aspect of the invention provides a multivalent TCR complex comprising at
least
two TCRs associated with a therapeutic agent. In one embodiment of this
aspect, at
least two TCR molecules are linked via linker moieties to form multivalent
complexes. Such multivalent TCR complexes may be linked by either a non-
peptidic
polymer chain or a peptidic linker sequence. Preferably the complexes are
water
soluble, so the linker moiety should be selected accordingly. Furthermore, it
is
preferable that the linker moiety should be capable of attachment to defmed
positions
on the TCR molecules, so that the structural diversity of the complexes formed
is
minimised. One embodiment of the present aspect is provided by a TCR complex
of
the invention wherein the polymer chain or peptidic linker sequence extends
between
amino acid residues of each TCR which are not located in a variable region
sequence
of the TCR.
Since the complexes of the invention may be for use in medicine, the linker
moieties
should be chosen with due regard to their pharmaceutical suitability, for
example their
immunogenicity.
Examples of linker moieties which fulfil the above desirable criteria are
known in the
art, for example the art of linking antibody fragments.
There are two classes of linker that are preferred for use in the production
of
multivalent TCR molecules of the present invention. A TCR complex of the
invention
in which the TCRs are linked by a polyalkylene glycol chain provides one
embodiment of the present aspect.
The first are hydrophilic polymers such as polyalkylene glycols. The most
commonly
used of this class are based on polyethylene glycol or PEG, the structure of
which is
shown below.
HOCH2CH2O (CH2CH2O)n CH2CH2OH
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Wherein n is greater than two. However, others are based on other suitable,
optionally
substituted, polyalkylene glycols include polypropylene glycol, and copolymers
of
ethylene glycol and propylene glycol.
Such polymers may be used to treat or conjugate therapeutic agents,
particularly
polypeptide or protein therapeutics, to achieve beneficial changes to the PK
profile of
the therapeutic.. Such improvements in the PK profile of the PEG-therapeutic
conjugate are believe to result from the PEG molecule or molecules forming
a'shell'
around the therapeutic which sterically hinders the reaction with the immune
system
and reduces proteolytic degradation. (Casey et al, (2000) Tumor Targetting 4
235-
244) The size of the hydrophilic polymer used my in particular be selected on
the
basis of the intended therapeutic use of the TCR complex. Thus for example,
where
the product is intended to leave the circulation and penetrate tissue, for
example for
use in the treatment of a tumour, it may be advantageous to use low molecular
weight
polymers in the order of 5 KDa. There are numerous review papers and books
that
detail the use of PEG and similar molecules in pharmaceutical formulations.
For
example, see Harris & Zalipsky (1997) Chemistry and Biological Applications of
Polyethylene Glycol ACS Books, Washington, D.C.
The polymer used can have a linear or branched conformation. Branched PEG
molecules, or derivatives thereof, can be induced by the addition of branching
moieties including glycerol and glycerol oligomers, pentaerythritol, sorbitol
and
lysine.
Usually, the polymer will have a chemically reactive group or groups in its
structure,
for example at one or both termini, and/or on branches from the backbone, to
enable
the polymer to link to target sites in the TCR. This chemically reactive group
or
groups may be attached directly to the hydrophilic polymer, or there may be a
spacer
group/moiety between the hydrophilic polymer and the reactive chemistry as
shown
below:
Reactive chemistry-Hydrophilic polymer-Reactive chemistry
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Reactive chemistry-Spacer-Hydrophilic polymer-Spacer-Reactive chemistry
The spacer used in the formation of constructs of the type outlined above may
be any
organic moiety that is a non-reactive, chemically stable, chain, Such spacers
include,
by are not limited to the following:
-(CH2)õ- wherein n= 2 to 5
-(CH2)3NHCO(CH2)2
A multivalent TCR complex of the invention in which a divalent alkylene spacer
radical is located between the polyalkylene glycol chain and its point of
attachment to
a TCR associated with a therapeutic agent provides a further embodiment of the
present aspect.
A multivalent TCR complex of the invention in which the polyalkylene glycol
chain
coinprises at least two polyethylene glycol repeating units provides a further
embodiment of the present aspect.
A wide variety of coupling chemistries can be used to couple polymer molecules
to
protein and peptide therapeutics. The choice of the most appropriate coupling
chemistry is largely dependant on the desired coupling site. For example N-
maleimide, Vinyl sulfone, Benzotriazole carbonate, Succinimidyl proprionate,
Succinimidyl butanoate, Thio-ester, Acetaldehyde, Acrylate, Biotin and Primary
amine coupling chemistries have been used attached to one or more of the
termini of
PEG molecules (Source: Nektar Molecular Engineering Catalogue 2003):
As stated above non-PEG based polymers also provide suitable linkers for
multimerising the TCRs of the present invention. For example, moieties
containing
maleimide termini linlced by aliphatic chains such as BMH and BMOE (Pierce,
products Nos. 22330 and 22323) can be used.
Peptidic linkers are the other class of TCR linkers. These linkers are
comprised of
chains of amino acids, and function to produce simple linkers or
multimerisation
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domains onto which TCR molecules can be attached. The biotin / streptavidin
system has previously been used to produce TCR tetramers (see WO/99/60119) for
in-vitro binding studies. However, strepavidin is a microbially-derived
polypeptide
and as such not ideally suited to use in a therapeutic.
A TCR complex of the invention in which the TCRs are linked by a peptidic
linker
derived from a human multimerisation domain provides a further embodiment of
the
present aspect. There are a number of human proteins that contain a
multimerisation
domain that could be used in the production of multivalent TCR complexes. For
example the tetramerisation domain of p53 which has been utilised to produce
tetramers of scFv antibody fragments which exhibited increased serum
persistence
and significantly reduced off-rate compared to the monomeric scFV fragment.
(Willuda et al. (2001) J. Biol. Chem. 276 (17) 14385-14392) Haemoglobin also
has a
tetramerisation domain that could potentially be used for this kind of
application.
Soluble TCRs or multivalent TCR complexes of the invention may be linked to an
enzyme capable of converting a prodrug to a drug. This allows the prodrug to
be
converted to the drug only at the site where it is required (i.e. targeted by
the sTCR).
Therapeutic Use
The invention also provides a method for delivering a therapeutic agent to a
target
cell, which method comprises contacting potential target cells with a TCR or
multivalent TCR complex in accordance with the invention under conditions to
allow
attachment of the TCR or multivalent TCR complex to the target cell, said TCR
or
multivalent TCR complex being specific for a given peptide-MHC complex.
In particular, the soluble TCR or multivalent TCR complex of the present
invention
can be used to deliver therapeutic agents to the location of cells presenting
a particular
antigen. This would be useful in many situations, for example, against tumours
or
sites of autoiminune disease. A therapeutic agent could be delivered such that
it
would exercise its effect locally but not only on the cell to which it binds.
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Thus, one particular strategy envisages iinmunostimulatory molecules linked to
TCRs
or multivalent TCR complexes according to the invention specific for tumour
antigens. For cancer treatment, the localisation in the vicinity of tumours or
metastasis
would enhance the effect of toxins or immunostimulants. Alternatively, the
soluble
TCR or multivalent TCR complex of the present invention can be used to deliver
immunoinhibitory agents to the location of cells presenting a particular
antigen related
to an autoiminune disease. For example, an Islet cell-specific TCR could be
used to
deliver an immunoinhibitory agent, such as IL- 10, IL-4 or IL- 13 or a
functional
variant or fragment of any of the foregoing to the Islet cells of a patient
suffering from
diabetes.
For vaccine delivery, the vaccine antigen could be localised in the vicinity
of antigen
presenting cells, thus enliancing the efficacy of the antigen.
It is envisaged that the administration of an interferon (IFN), such as IFN-y,
to a
patient prior to, and/or simultaneously with, the administration of the TCR
associated
with a therapeutic agent may increase levels of peptide-MHC expression on the
target
cells. This may be of particular benefit in the treatment of cancer.
Further embodiments of the invention are provided by a pharmaceutical
composition
comprising a TCR associated with a therapeutic agent or a multivalent TCR
complex
thereof together with a pharmaceutically acceptable carrier.
The invention also provides a method of treatment of cancer comprising
administering
to a subject suffering such cancer disease an effective amount of a TCR
associated
with a therapeutic agent or a multivalent TCR complex thereof. In a related
embodiment the invention provides for the use of a TCR associated with a
therapeutic
agent or a multivalent TCR complex thereof, in the preparation of a
composition for
the treatment of cancer. IL-15, IL-21 or Anti-CD3 antibody or a functional
variant or
fragment of the foregoing, are particularly preferred therapeutic agents for
use in the
treatment of cancer.
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The invention also provides a method of treatment of autoimmune disease, organ
rejection or GVHD comprising administering to a subject suffering such an
autoimmune disease, organ rejection or GVHD an effective amount of a TCR
associated with a therapeutic agent or a multivalent TCR complex thereof. In a
related
embodiment the invention provides for the use of a TCR associated with a
therapeutic
agent or a multivalent TCR complex thereof, in the preparation of a
composition for
the treatment of autoimmune disease, organ rejection or GVHD. Preferred
therapeutic
agents for use in the treatment of autoiminune disease, organ rejection or
GVHD are
IL- 10, IL-4 and IL- 13 or a functional variant or fragment of any of the
foregoing. In
another related embodiment the dTCR or scTCR of the invention is tissue-
specific. In
further related embodiment the
dTCR or scTCR is specific for a tissue which is a target for auto-reactive T
cells in
autoimmune disease, organ rejection or Graft Versus Host Disease (GVHD). In a
specific embodiment the invention provides a method of treating diabetes,
wherein the
dTCR or scTCR is islet cell-specific.
Cancers which may benefit the methods of the present invention include:
leukaemia,
head, neck, lung, breast, colon, cervical, liver, pancreatic, ovarian and
testicular)
Auto-immune diseases which may benefit the methods of the following invention
include:
Acute disseminated encephalomyelitis
Adrenal insufficiency
Allergic angiitis and granulomatosis
Amylodosis
Ankylosing spondylitis
Asthma
Autoimmune Addison's disease
Autoimmune alopecia
Autoimmune chronic active hepatitis
Autoimmune haemolytic anaemia
Autoimmune Neutrogena
Autoimmune thrombocytopenic purpura
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Behget's disease
Cerebellar degeneration
Chronic active hepatitis
Chronic inflammatory demyelinating polyradiculoneuropathy
Chronic neuropathy with monoclonal gaminopathy
Classic polyarteritis nodosa
Congenital adrenal hyperplasia
Cryopathies
Dermatitis herpetiformis
Diabetes
Eaton-Lambert myasthenic syndrome
Encephalomyelitis
Epidermolysis bullosa acquisita
Erytherna nodosa
Gluten-sensitive enteropathy
Goodpasture's syndrome
Guillain-Barre syndrome
Hashirnoto's thyroiditis
Hyperthyroidism
Idiopathic hemachromatosis
Idiopathic membranous glomerulonephritis
Isolated vasculitis of the central nervous system
Kawasaki's disease
Miniinal change renal disease
Miscellaneous vasculitides
Mixed connective tissue disease
Multifocal motor neuropathy with conduction block
Multiple sclerosis
Myasthenia gravis
Opsoclonus-myoclonus syndrome
Pemphigoid
Pemphigus
pernicious anaemia
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Polymyositis/dermatomyositis
Post-infective arthritides
Primary biliary sclerosis
Psoriasis
Reactive arthritides
Reiter's disease
Retinopathy
Rheumatoid arthritis
Sclerosing cholangitis
Sjogren's syndrome
Stiff-man syndrome
Subacute thyroiditis
Systemic lupus erytllematosis
Systeinic necrotizing vasculitides
Systemic sclerosis (scleroderma)
Takayasu's arteritis
Temporal arteritis
Thromboangiitis obliterans
Type I and type II autoil=une polyglandular syndrome
Ulcerative colitis
Uveitis
Wegener's granulomatosis
Therapeutic compositions in accordance with the invention will usually be
supplied as
part of a sterile, phaimaceutical composition which will normally include a
pharmaceutically acceptable carrier. This pharmaceutical composition may be in
any
suitable form, (depending upon the desired metliod of administering it to a
patient). It
may be provided in unit dosage form, will generally be provided in a sealed
container
and may be provided as part of a kit. Such a kit would normally (although not
necessarily) include instructions for use. It may include a plurality of said
unit dosage
forms.
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The pharmaceutical composition may be adapted for administration by any
appropriate
route, for example pareiiteral, transdermal or via inhalation, preferably a
parenteral
(including subcutaneous, intramuscular, or, most preferably intravenous)
route. Such
compositions may be prepared by any method known in the art of pharmacy, for
example by mixing the active ingredient with the carrier(s) or excipient(s)
under sterile
conditions.
Dosages of the substances of the present invention can vary between wide
limits,
depending upon the disease or disorder to be treated, the age and condition of
the
individual to be treated, etc. and a physician will ultiunately determine
appropriate
dosages to be used.
Additiorzal Aspects
A scTCR or dTCR associated with a therapeutic agent (which TCR preferably is
constituted by constant and variable sequences corresponding to human
sequences)
may be provided in substantially pure form, or as a purified or isolated
preparation. For
example, it may be provided in a form which is substantially free of otlier
proteins.
Preferred features of eac11 aspect of the invention are as for each of the
other aspects
inutatis mutandis. The prior art documents mentioned herein are incorporated
to the
fullest extent permitted by law.
Examples
The invention is further described in the following examples, which do not
limit the
scope of the invention in any way.
Reference is made in the following to the accompanying drawings in which:
Figures 1a and lb show respectively the nucleic acid sequences of the a and P
chains
of a soluble A6 TCR, mutated so as to introduce a cysteine codon. The shading
indicates the introduced cysteine codon;
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Figure 2a shows the A6 TCR a chain extracellular amino acid sequence,
including
the T48 -> C mutation (underlined) used to produce the novel disulphide inter-
chain
bond, and Figure 2b shows the A6 TCR (3 chain extracellular amino acid
sequence,
including the S57 -> C mutation (underlined) used to produce the novel
disulphide
inter-chain bond;
Figure 3a shows the A6 TCR a chain sequence including novel cysteine residue
mutated to incorporate a BamHl restriction site. Shading indicates the
mutations
introduced to form the BamHl restriction site.
Figures 3b and 3c show the DNA sequence of a and (3 chain of the JM22 TCR
mutated to include additional cysteine residues to form a non-native
disulphide bond;
Figures 4a and 4b show respectively the J1VI22 TCR a and (3 chain
extracellular amino
acid sequences produced from the DNA sequences of Figures 3b and 3c;
Figures 5a and 5b show respectively the DNA sequences of the a and (3 chains
of a
soluble AH-1.23 TCR, mutated so as to introduce a novel cysteine codon
(indicated
by shading).
Figures 6a and 6b show respectively the AH-1.23 TCR a and (3 chain
extracellular
amino acid sequences produced from the DNA sequences of Figures 5a and 5b;
Figure 7a - DNA sequence of mature human IL-10.
Figure 7b - Amino acid sequence of mature human IL-10.
Figure 8a - DNA sequence of AH1.23 TCR 0 chain containing a non-native
cysteine
involved in the formation of a novel interchain bond linked to mature human IL-
10
via a Pro-Gly linker. The introduced cysteine is indicated by shading. The DNA
sequence encoding the Pro-Gly linker is underlined.
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Figure 8b - Amino acid sequence of AH1.23 TCR (3 chain corntaining a non-
native
cysteine codon involved in the formation of a novel interchain bond linked to
mature
human IL-10 via a Pro-Gly linker. The introduced cysteine is indicated by
shading.
The Pro-Gly linlcer is underlined.
Figure 9a - DNA sequence of AH1.23 TCR (3 chain containing a non-native
cysteine
involved in the formation of a novel interchain bond linked to mature human IL-
10
via a Gly-Ser-Gly-Gly-Pro linker. The introduced cysteine is indicated by
shading.
The DNA sequence encoding the Gly-Ser-Gly-Gly-Pro linker is underlined.
Figure 9b - Amino acid sequence of AH1.23 TCR (3 chain containing a non-native
cysteine codon involved in the formation of a novel interchain bond linked to
mature
1luman IL-10 via a Gly-Ser-Gly-Gly-Pro linker. The introduced cysteine is
indicated
by shading. The Gly-Ser-Gly-Gly-Pro linker is underlined.
Figure 10a - DNA sequence of AH 1.23 TCR P chain containing a non-native
cysteine
involved in the formation of a novel interchain bond linked to mature human IL-
10
via a Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Pro linker.
The introduced cysteine is indicated by shading. The DNA sequence encoding the
Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Pro linker is
underlined.
Figure lOb - Amino acid sequence of AH1.23 TCR (3 chain containing a non-
native
cysteine codon involved in the formation of a novel interchain bond linked to
mature
human IL-10 via a Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-
Pro linker. The introduced cysteine is indicated by shading. The Gly-Ser-Gly-
Gly-
Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Pro linker is underlined.
Figure 11 a - DNA sequence of mature human IL-4.
Figure 1 1b - Amino acid sequence of mature human IL-4.
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Figure 12a - DNA sequence of AH1.23 TCR (3 chain containing a non-native
cysteine
involved in the formation of a novel interchain bond linked to mature human IL-
4 via
a Pro-Gly linker. The introduced cysteine is indicated by shading. The DNA
sequence
encoding the Pro-Gly linker is underlined.
Figure 12b - Amino acid sequence of AH1.23 TCR (3 chain containing a non-
native
cysteine codon involved in the formation of a novel interchain bond linked to
mature
human IL-4 via a Pro-Gly linker. The introduced cysteine is indicated by
shading. The
Pro-Gly linker is underlined.
Figure 13 a - DNA sequence of AH 1.23 TCR (3 chain containing a non-native
cysteine
involved in the formation of a novel interchain bond linked to mature human IL-
4 via
a Gly-Ser-Gly-Gly-Pro linker. The introduced cysteine is indicated by shading.
The
DNA sequence encoding the Gly-Ser-Gly-Gly-Pro linker is underlined.
Figure 13b - Amino acid sequence of AH1.23 TCR (3 chain containing a non-
native
cysteine codon involved in the formation of a novel interchain bond linked to
mature
human IL-4 via a Gly-Ser-Gly-Gly-Pro linker. The introduced cysteine is
indicated by
shading. The Gly-Ser-Gly-Gly-Pro linker is underlined.
Figure 14a - DNA sequence of AHl.23 TCR 0 chain containing a non-native
cysteine
involved in the fonnation of a novel interchain bond linked to mature human IL-
4 via
a Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Pro linker. 'The
introduced cysteine is indicated by shading. The DNA sequence encoding the Gly-
Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Pro linker is
underlined.
Figure 14b - Amino acid sequence of AH1.23 TCR (3 chain containing a non-
native
cysteine codon involved in the formation of a novel interchain bond linked to
mature
human IL-4 via a Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-
Pro linker. The introduced cysteine is indicated by shading. The Gly-Ser-Gly-
Gly-
Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Pro linker is underlined.
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Figure 15a - DNA sequence of mature human IL-13.
Figure 15b - Amino acid sequence of mature human IL-13.
Figure 16a - DNA sequence of AH1.23 TCR P chain containing a non-native
cysteine
involved in the formation of a novel interchain bond linked to mature human IL-
13
via a Pro-Gly linker. The introduced cysteine is indicated by shading. The DNA
sequence encoding the Pro-Gly linker is underlined.
Figure 16b - Amino acid sequence of AH1.23 TCR (3 chain containing a non-
native
cysteine codon involved in the formation of a novel interchain bond linked to
mature
human IL-13 via a Pro-Gly linker. The introduced cysteine is indicated by
shading.
The Pro-Gly linker is underlined.
Figure 17a - DNA sequence of AH1.23 TCR 0 chain containing a non-native
cysteine
involved in the formation of a novel interchain bond linked to mature human IL-
13
via a Gly-Ser-Gly-Gly-Pro linker. The introduced cysteine is indicated by
shading.
The DNA sequence encoding the Gly-Ser-Gly-Gly-Pro linker is underlined.
Figure 17b - Amino acid sequence of AH1.23 TCR (3 chain containing a non-
native
cysteine codon involved in the formation of a novel interchain bond linked to
mature
human IL-13 via a Gly-Ser-Gly-Gly-Pro linker. The introduced cysteine is
indicated
by shading. The Gly-Ser-Gly-Gly-Pro linker is underlined.
Figure 18 a - DNA sequence of AH1.23 TCR (3 chain containing a non-native
cysteine
involved in the formation of a novel interchain bond linked to mature human IL-
13
via a Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Pro linker.
The introduced cysteine is indicated by shading. The DNA sequence encoding the
Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Pro linker is
underlined.
Figure 18b - Amino acid sequence of AH1.23 TCR (3 chain containing a non-
native
cysteine codon involved in the formation of a novel interchain bond linked to
mature
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human IL-13 via a Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-
Pro linker. The introduced cysteine is indicated by shading. The Gly-Ser-Gly-
Gly-
Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Pro linker is underlined.
Figure 19 details the DNA sequence of the pEX821 plasmid.
Figure 20 provides a plasmid map of the pEX821 vector, the DNA sequence of
which
is provided by Figure 19.
Figure 21 details the DNA sequence of the pEX954 plasmid.
Figure 22 provides a plasmid map of the pEX954 plasmid, the DNA sequence of
which is provided by Figure 21.
Figure 23a details the DNA sequence encoding the higli affinity c61 NY-ESO
MTCR
beta chain and Figure 23b details the AA sequence encoded by the DNA sequence
of
Figure 23a.
Figure 24a details the DNA sequence encoding the high affinity c61 NY-ESO MTCR
beta chain linked at the C-terminus thereof via a peptide linker to IL-18.
Figure 24b
details the AA sequence of this fusion protein, the peptide linker is
underlined.
Figure 25 a details the DNA sequence encoding IL-18 pro-protein linked at the
C-
terminus thereof via a peptide linker to the high affinity c61 NY-ESO MTCR
beta
chain. The pro-IL- 18 DNA has been altered to encode a Factor X cleavage site.
Figure 25b details the AA sequence of this fusion protein, the peptide linker
is
underlined.
Figure 26a details the DNA sequence encoding the high affinity c61 NY-ESO MTCR
beta chain linked at the C-terminus thereof via a peptide linker to IL-10.
Figure 26b
details the AA sequence of this fusion protein, the peptide linker is
underlined.
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Figure 27a details the DNA sequence encoding the high affinity c61 NY-ESO MTCR
beta chain linked at the C-terminus thereof via a peptide linker to IL-13.
Figure 27b
details the AA sequence of this fusion protein, the peptide linker is
underlined.
Figure 28a details the DNA sequence encoding the high affinity c61 NY-ESO MTCR
beta chain linked at the C-terminus thereof via a peptide linker to the "KDEL"
variant
of the PE38 exotoxin. Figure 28b details the AA sequence of this fusion
protein, the
peptide linker is underlined.
Figure 29a details the DNA sequence encoding the high affinity c58 NY-ESO MTCR
alpha chain and Figure 29b details the AA sequence encoded by the DNA sequence
of
Figure 29a.
Exatnple 1 - Design ofprimers and mutagenesis ofA6 Tax TCR a and,Q chains
For mutating A6 Tax threonine 48 of exon 1 in TRAC*Olto cysteine, the
following
primers were designed (mutation shown in lower case):
5'-C ACA GAC AAA tgT GTG CTA GAC AT (SEQ ID NO: 3)
5'-AT GTC TAG CAC Aca TTT GTC TGT G (SEQ ID NO: 4)
For inutating A6 Tax serine 57 of exon 1 in both TRBC1*01 and TRBC2*01 to
cysteine, the following priiners were designed (mutation shown in lower case):
5'-C AGT GGG GTC tGC ACA GAC CC (SEQ ID NO: 5)
5'-GG GTC TGT GCa GAC CCC ACT G (SEQ ID NO: 6)
PCR nzutagenesis:
Expression plasmids containing the genes for the A6 Tax TCR a or (3 chain were
mutated using the a-chain primers or the (3-chain primers respectively, as
follows.
100 ng of plasmid was mixed with 5 l 10 mM dNTP, 25 l lOxPfu-buffer
(Stratagene), 10 units Pfu polymerase (Stratagene) and the final volume was
adjusted
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to 240 l with H20. 48 l of this mix was supplemented with primers diluted to
give
a final concentration of 0.2 M in 50 l final reaction volume. After an
initial
denaturation step of 30 seconds at 95 C, the reaction mixture was subjected to
15
rounds of denaturation (95 C, 30 sec.), annealing (55 C, 60 sec.), and
elongation
(73 C, 8 min.) in a Hybaid PCR express PCR machine. The product was then
digested for 5 hours at 37 C with 10 units of Dpnl restriction enzyme (New
England
Biolabs). 10 l of the digested reaction was transformed into competent XL1-
Blue
bacteria and grown for 18 hours at 37 C. A single colony was picked and grown
over
night in 5 ml TYP + ampicillin (16 g/l Bacto-Tryptone, 16 g/l Yeast Extract, 5
g/l
NaCI, 2.5 g/l K2HPO4, 100 mg/l Ampicillin). Plasmid DNA was purified on a
Qiagen
mini-prep column according to the manufacturer's instructions and the sequence
was
verified by automated sequencing. The respective mutated nucleic acid and
amino
acid sequences are shown in Figures la and 2a for the a chain and Figures lb
and 2b
for the (3 chain.
Exainple 2 - Expression, refolding and purification of soluble TCR
The expression plasmids containing the mutated a-chain and (3-chain
respectively
were transformed separately into E.coli strain BL21pLysS, and single
ampicillin-
resistant colonies were grown at 37 C in TYP (ampicillin 100 g/ml) medium to
OD600 of 0.4 before inducing protein expression with 0.5mM IPTG. Cells were
harvested three hours post-induction by centrifugation for 30 minutes at
4000rpm in a
Beckman J-6B. Cell pellets were re-suspended in a buffer containing 50mM Tris-
HCI, 25% (w/v) sucrose, 1mM NaEDTA, 0.1% (w/v) NaAzide, 10mM DTT, pH 8Ø
After an overnight freeze-thaw step, re-suspended cells were sonicated in 1
minute
bursts for a total of around 10 minutes in a Milsonix XL2020 sonicator using a
standard 12mm diameter probe. Inclusion body pellets were recovered by
centrifugation for 30 minutes at 13000rpm in a Beckman J2-21 centrifuge. Three
detergent washes were then carried out to remove cell debris and membrane
components. Each time the inclusion body pellet was homogenised in a Triton
buffer
(50mM Tris-HCI, 0.5% Triton-X100, 200mM NaCI, 10mM NaEDTA, 0.1% (w/v)
NaAzide, 2mM DTT, pH 8.0) before being pelleted by centrifugation for 15
minutes
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at 13000rpm in a Beckman J2-21. Detergent and salt was then removed by a
similar
wash in the following buffer: 50mM Tris-HCI, 1mM NaEDTA, 0.1% (w/v) NaAzide,
2mM DTT, pH 8Ø Finally, the inclusion bodies were divided into 30 mg
aliquots
and frozen at -70 C. Inclusion body protein yield was quantitated by
solubilising with
6M guanidine-HCl and measurement with a Bradford dye-binding assay (PerBio).
Denaturation of soluble TCRs; 30mg of the solubilised TCR 0-chain inclusion
body
and 60mg of the solubilised TCR a-chain inclusion body was thawed from frozen
stocks. The inclusion bodies were diluted to a final concentration of 5mg/ml
in 6M
guanidine solution, and DTT (2M stock) was added to a final concentration of
10mM.
The mixture was incubated at 37 C for 30 min.
Ref lding of soluble TCRs: 1 L refolding buffer was stirred vigorously at 5 C
3 C.
The redox couple (2-mercaptoethylamine and cystamine (to final concentrations
of
6.6inM and 3.7mM, respectively) were added approximately 5 minutes before
addition of the denatured TCR chains. The protein was then allowed to refold
for
approximately 5 hours 15 minutes with stirring at 5 C :L 3 C.
Dialysis of refolded soluble TCRs: The refolded TCR was dialysed in Spectrapor
1
membrane (Spectrum; Product No. 132670) against 10 L 10 mM Tris pH 8.1 at 5 C
~
3 C for 18-20 hours. After this time, the dialysis buffer was changed to fresh
10 mM
Tris pH 8.1 (10 L) and dialysis was continued at 5 C 3 C for another 20-22
hours.
Example 3 - BIAcore surface plasmon resonance characterisation of sTCR binding
to
specific pMHC
A surface plasmon resonance biosensor (BIAcore 3000TM ) was used to analyse
the
binding of a sTCR to its peptide-MHC ligand. This was facilitated by producing
single pMHC complexes (described below) which were immobilised to a
streptavidin-
coated binding surface in a semi-oriented fashion, allowing efficient testing
of the
binding of a soluble T-cell receptor to up to four different pMHC (immobilised
on
separate flow cells) simultaneously. Manual injection of HLA complex allows
the
precise level of immobilised class I molecules to be manipulated easily.
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Such immobilised complexes are capable of binding both T-cell receptors and
the
coreceptor CD8aa, both of which may be injected in the soluble phase. Specific
binding of TCR is obtained even at low concentrations (at least 40 g/ml),
implying
the TCR is relatively stable. The pMHC binding properties of sTCR are observed
to
be qualitatively and quantitatively similar if sTCR is used either in the
soluble or
immobilised phase. This is an important control for partial activity of
soluble species
and also suggests that biotinylated pMHC complexes are biologically as active
as
non-biotinylated complexes.
Biotinylated class I HLA-A2 - peptide complexes were refolded in vitro from
bacterially-expressed inclusion bodies containing the constituent subunit
proteins and
synthetic peptide, followed by purification and in vitro enzymatic
biotinylation
(O'Callaghan et al. (1999) Anal. Bioehem. 266: 9-15). HLA-heavy chain was
expressed with a C-terminal biotinylation tag which replaces the transmembrane
and
cytoplasrnic domains of the protein in an appropriate construct. Inclusion
body
expression levels of -75 mg/litre bacterial culture were obtained. The HLA
light-
chain or (32-microglobulin was also expressed as inclusion bodies in E.coli
from an
appropriate construct, at a level of -500 mg/litre bacterial culture.
E. coli cells were lysed and inclusion bodies are purified to approximately
80% purity.
Protein from inclusion bodies was denatured in 6 M guanidine-HCI, 50 mM Tris
pH
8.1, 100 naM NaCI, 10 mM DTT, 10 mM EDTA, and was refolded at a concentration
of 30 mg/litre heavy chain, 30 mg/litre (32in into 0.4 M L-Arginine-HCI, 100
mM Tris
pH 8.1, 3.7 m1VI cystamine, mM cysteamine, 4 mg/ml peptide (e.g. tax 11-19),
by
addition of a single pulse of denatured protein into refold buffer at < 5 C.
Refolding
was allowed to reach completion at 4 C for at least 1 hour.
Buffer was exchanged by dialysis in 10 volumes of 10 mM Tris pH 8.1. Two
changes
of buffer were necessary to reduce the ionic strength of the solution
sufficiently. The
protein solution was then filtered through a 1.5 m cellulose acetate filter
and loaded
onto a POROS 50HQ anion exchange column (8 ml bed voluine). Protein was eluted
with a linear 0-500 mM NaCl gradient. HLA-A2-peptide complex eluted at
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approximately 250 mM NaCI, and peak fractions were collected, a cocktail of
protease inhibitors (Calbiochem) was added and the fractions were chilled on
ice.
Biotinylation tagged HLA complexes were buffer exchanged into 10 mM Tris pH
8.1,
5 mM NaCI using a Pharmacia fast desalting column equilibrated in the same
buffer.
Immediately upon elution, the protein-containing fractions were chilled on ice
and
protease inhibitor cocktail (Calbiochem) was added. Biotinylation reagents
were then
added: 1 mM biotin, 5 mM ATP (buffered to pH 8), 7.5 mM MgC12, and 5 g/ml
BirA enzyme (purified according to O'Callaghan et al. (1999) Anal. Biochem.
266: 9-
15). The mixture was then allowed to incubate at room temperature overnight.
Biotinylated HLA complexes were purified using gel filtration chromatography.
A
Pharmacia Superdex 75 HR 10/30 column was pre-equilibrated with filtered PBS
and
1 ml of the biotinylation reaction mixture was loaded and the column was
developed
with PBS at 0.5 ml/min. Biotinylated HLA coinplexes eluted as a single peak at
approximately 15 ml. Fractions containing protein were pooled, chilled on ice,
and
protease inhibitor cocktail was added. Protein concentration was determined
using a
Coomassie-binding assay (PerBio) and aliquots of biotinylated HLA complexes
were
stored frozen at -20 C. Streptavidin was immobilised by standard amine
coupling
methods.
The interactions between A6 Tax sTCR containing a novel inter-chain bond and
its
ligand/ MHC complex or an irrelevant HLA-peptide combination, the production
of
which is described above, were analysed on a BlAcore 3000TM surface plasmon
resonance (SPR) biosensor. SPR measures changes in refractive index expressed
in
response units (RU) near a sensor surface within a small flow cell, a
principle that can
be used to detect receptor ligand interactions and to analyse their affinity
and kinetic
parameters. The probe flow cells were prepared by immobilising the individual
HLA-
peptide complexes in separate flow cells via binding between the biotin cross
linked
onto (32m and streptavidin which have been chemically cross linked to the
activated
surface of the flow cells. The assay was then performed by passing sTCR over
the
surfaces of the different flow cells at a constant flow rate, measuring the
SPR
response in doing so. Initially, the specificity of the interaction was
verified by
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passing sTCR at a constant flow rate of 5 1 min-1 over two different
surfaces; one
coated with -5000 RU of specific peptide-HLA complex, the second coated with
-5000 RU of non-specific peptide-HLA complex. Injections of soluble sTCR at
constant flow rate and different concentrations over the peptide-HLA complex
were
used to define the background resonance. The values of these control
measurements
were subtracted from the values obtained with specific peptide-HLA complex and
used to calculate binding affinities expressed as the dissociation constant,
Kd (Price &
Dwek, Principles and Problems in Physical Chemistry for Biochemists (2 a
Edition)
1979, Clarendon Press, Oxford).
The Kd value obtained (1.8 M) is close to that reported for the interaction
between
A6 Tax sTCR without the novel di-sulphide bond and pMHC (0.91 M - Ding et al,
1999, Inzmunity 11:45-56).
Exanaple 4- Production of soluble JM22 TCR c ntaining a novel disulphide bond.
The (3 chain of the soluble A6 TCR prepared in Example 1 contains in the
native
sequence a BglII restriction site (AAGCTT) suitable for use as a ligation
site.
PCR mutagenesis was carried as detailed below to introduce a BamHl restriction
site
(GGATCC) into the a chain of soluble A6 TCR, 5' of the novel cysteine codon.
The
sequence described in Figure 1 a was used as a template for this mutagenesis.
The
following primers were used:
IBamxI I
5'-ATATCCAGAACCCgGAtCCTGCCGTGTA- 3'(SEQ ID NO: 7)
5' -TACACGGCAGGAaTCcGGGTTCTGGATAT-3' (SEQ IDNO: 8)
100 ng of plasmid was mixed with 5 l 10 mM dNTP, 25 l l OxPfu-buffer
(Stratagene), 10 units Pfu polymerase (Stratagerxe) and the final volume was
adjusted
to 240 l with H2O. 48 l of this mix was supplemented with primers diluted to
give
a final concentration of 0.2 M in 50 l final reaction volume. After an
initial
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denaturation step of 30 seconds at 95 C, the reaction mixture was subjected to
15
rounds of denaturation (95 C, 30 sec.), annealing (55 C, 60 sec.), and
elongation
(73 C, 8 min.) in a Hybaid PCR express PCR machine. The product was then
digested for 5 hours at 37 C with 10 units of Dpnl restriction enzyme (New
England
Biolabs). 10 l of the digested reaction was transformed into competent XL1 -
Blue
bacteria and grown for 18 hours at 37 C. A single colony was picked and grown
over
night in 5 ml TYP + ampicillin (16 g/1 Bacto-Tryptone, 16 g/l Yeast Extract, 5
g/l
NaCI, 2.5 g/1 K2HPO4, 100 mg/1 Ainpicillin). Plasmid DNA was purified on a
Qiagen
mini-prep colunm according to the manufacturer's instructions and the sequence
was
verified by automated sequencing. The mutations introduced into the a chain
were
"silent", therefore the amino acid sequence of this chain remained unchanged
from
that detailed in Figure 2a. The DNA sequence for the mutated a chain is shown
in
Figure 3a.
In order to produce a soluble JM22 TCR incorporating a novel disulphide bond,
A6
TCR plasmids containing the a chain BamHl and (3 chain Bg11I restriction sites
were
used as templates. The following primers were used:
I Nde1 1
5'-GGAGATATACATATGCAACTACTAGAACAA-3'(SEQ IDN :9)
5'-TACACGGCAGGATCCGGGTTCTGGATATT- 3'(SEQ ID NO : 10)
BamHII
~Nde1 ~
5 ' -GGAGATATACATATGGTGGATGGTGGAATC-3 ' (SEQ ID NO: 11)
5'-CCCAAGCTTAGTCTGCTCTACCCCAGGCCTCGGC-3'(SEQ ID NO: 12)
1 sglII1
JM22 TCR a and (3-chain constructs were obtained by PCR cloning as follows.
PCR reactions were performed using the primers as shown above, and templates
containing the JM22 TCR chains. The PCR products were restriction digested
with
the relevant restriction enzymes, and cloned into pGMT7 to obtain expression
plasmids. The sequence of the plasmid inserts were confirmed by automated DNA
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sequencing. Figures 3b and 3c show the DNA sequence of the mutated a and (3
cliains
of the JM22 TCR respectively, and Figures 4a and 4b show the resulting amino
acid
sequences.
The respective TCR chains were expressed, co-refolded and purified as
described in
Exainples 1 and 2.
A Biacore analysis of the binding of the JM22 TCR to pMHC was carried out as
described in Example 3. The Kd of this disulphide-linked TCR for the HLA-flu
complex was determined to be 7.9 0.51 M
Example S- Production of soluble AH-1.23 TCR containing a novel disulphide
intey--
chain bond
cDNA encoding AH-1.23 TCR was isolated from T cells supplied by Hill Gaston
(Medical School, Addenbrooke's Hospital, Cambridge) according to known
techniques. cDNA encoding NY-ESO TCR was produced by treatment of the mRNA
with reverse transcriptase.
In order to produce a soluble AH-1.23 TCR incorporating a novel disulphide
bond,
TCR plasmids containing the a chain BamHI and (3 chain BglII restriction sites
were
used as a framework as described in Example 4. The following primers were
used:
I Ndel 1
5'-GGGAAGCTTACATATGAAGGAGGTGGAGCAGAATTCTGG-3'(SEQID NO: 13)
5'-TACACGGCAGGATCCGGGTTCTGGATATT- 3'(SEQ ID NO: 14)
BamHil
Ndei
5'-TTGGAATTCACATATGGGCGTCATGCAGAA.CCCAAGACAC-3
(SEQ ID NO: 15)
5' -CCCAAGCTTAGTCTGCTCTACCCCAGGCCTCGGC-3' (SEQ ID NO: 16)
lBglIII
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AH-1.23 TCR a and (3-chain constructs were obtained by PCR cloning as follows.
PCR reactions were performed using the primers as shown above, and templates
containing the AH-1.23 TCR chains. The PCR products were restriction digested
with the relevant restriction enzymes, and cloned into pGMT7 to obtain
expression
plasmids. The sequence of the plasmid inserts were confirmed by automated DNA
sequencing. Figures 5a and 5b show the DNA sequence of the mutated a and (3
chains
of the AH-1.23 TCR respectively, and Figures 6a and 6b show the resulting
amino
acid sequences.
The respective TCR chains were expressed, co-refolded and purified as
described in
Example 2.
Exanaple 6- Production of a soluble AH-1.213 TCR - IL-1 D fusion protein.
Synthetic genes including the mature human IL-10 DNA sequence detailed in
Figure
7a and one of a number of DNA extensions at the 5' end of the IL- 10 DNA
sequence
can then be produced. The 5' DNA extensions are linker sequences used to
attach the
IL-10 DNA to that encoding the AH1.23 TCR (3 chain.
Linker sequences:
ccc - which encodes a Pro-Gly linker including a Xmal restriction enzyme site
ggatccggcggtccg - (SEQ ID NO: 17) which encodes a Gly-Ser-Gly-Gly-Pro (SEQ ID
NO: 18) linker including a BamHl restriction enzyme site.
ggatccggtgggggcggaagtggaggcagcggtg.g_atccggcggtccg - (SEQ ID NO:19) which
encodes a Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Pro
(SEQ ID NO: 20) linker including two BamHl restriction enzyme sites.
One of the above synthetic genes is then sub-cloned into the pGMT7 plasmid
containing the AH1.23 TCR P chain, produced as described in Example 5 to form
a
DNA sequence encoding the TCR (3 chain-linker-IL-10 fusion protein.
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The DNA and amino acid sequence of the AH1.23 TCR (3 chain - Pro-Gly - IL- 10
fusion is detailed in Figures 8a and 8b respectively.
The DNA and amino acid sequence of the AH1.23 TCR (3 chain - Gly-Ser-Gly-Gly-
Pro (SEQ ID NO: 18) - IL-10 fusion is detailed in Figures 9a and 9b
respectively.
The DNA and amino acid sequence of the AH1.23 TCR P chain - Gly-Ser-Gly-Gly-
Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly -Pro- (SEQ ID NO: 20) IL- 10
fusion
is detailed in Figures 10a and 10b respectively.
These AH1.23 TCR (3 chain - Linker- IL-10 fusion proteins are then refolded
with
the AH1.23 TCR a chain using the methods detailed in Example 2 to produce the
coinplete soluble AH1.23 TCR-IL-10 fusion protein.
The methods detailed above describe the production of a soluble a(3TCR onto
which
an IL-10 monomer is attached to the C-terminus of the TCR P chain. IL-10 is
often
found in the form of a homodimer. Therefore, it may be advantageous to
dimerise the
IL-10 polypeptide attached to the soluble AH1.23 TCR. This can be achieved in
a
number of ways. For example, a single-chain version of the mature form human
IL-10
homodimer can be fused to the TCR 0 prior to refolding with the TCR a chain.
Alternatively, mature form human IL-10 can be added in solution to either the
TCR (3
Chain-IL-10 fusion proteins formed as described above prior to refolding with
the
soluble TCR a chain, or to the refolded a(3TCR-IL-10 fusion proteins.
Alternatively,
an additional IL-10 molecule can be added to the TCR a chain as a fusion
protein
using the methods described in this example for the production of the TCR P
chain -
IL- 10 fusion protein. The two TCR chain-IL- 10 fusion proteins can then be re-
folded
together using the methods described in Example 2. Finally, complexes
comprising
two TCR, each containing a single IL-10 polypeptide linked to the TCR P chain,
may
be formed by homo-dimerisation of the IL-10 polypeptides. This would result in
the
formation of a complex of the following type:
a(3TCR-IL-10 homodimer-a(3TCR
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Exafnple 7- Production of soluble AH-1.23 TCR - IL-4 and AH-1.23 TCR - IL-13
fusion proteins.
The methods detailed in Example 6 can also be used to produce fusion proteins
containing the soluble AH-1.23 TCR linked to other polypeptides.
Synthetic genes including the mature human IL-4 DNA sequence detailed in
Figure
11a and one of the 5' DNA extension sequences listed in Example 6 can be
constructed and sub-cloned into the pGMT7 plasmid containing the AH1.23 TCR (3
chain, produced as described in Example 5 to form a DNA sequence encoding the
TCR (3 chain-linker- IL-4 fusion proteins.
The DNA and amino acid sequence of the AH1.23 TCR (3 chain - Pro-Gly - IL-4
fusion is detailed in Figures 12a and 12b respectively.
The DNA and amino acid sequence of the AH1.23 TCR (3 chain - Gly-Ser-Gly-Gly-
Pro- (SEQ ID NO: 18) - IL-4 fusion is detailed in Figures 13a and 13b
respectively.
The DNA and amino acid sequence of the AH1.23 TCR (3 chain - Gly-Ser-Gly-Gly-
Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Pro-(SEQ ID NO: 20)- IL-4 fusion
is detailed in Figures 14a and 14b respectively.
Synthetic genes including the mature human IL-13 DNA sequence detailed in
Figure
15a and one of the 5' DNA extension sequences listed in Example 6 can be
constructed and sub-cloned into the pGMT7 plasmid containing the AH1.23 TCR (3
chain, produced as described in Example 5 to form a DNA sequence encoding the
TCR (3 chain-linker- IL-4 fusion proteins.
The DNA and amino acid sequence of the AH1.23 TCR 0 chain - Pro-Gly - IL-13
fusion is detailed in Figures 16a and 16b respectively.
The DNA and amino acid sequence of the AH1.23 TCR 0 chain - Gly-Ser-Gly-Gly-
Pro (SEQ ID NO: 18)- IL-13 fusion is detailed in Figures 17a and 17b
respectively.
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The DNA and amino acid sequence of the AH1.23 TCR 0 chain - Gly-Ser-Gly-Gly-
Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Pro- (SEQ ID NO: 20)- IL-13 fusion
is detailed in Figures 18a and 18b respectively.
These AH1.23 TCR (3 chain - Linker- interleukin fusion proteins are then
refolded
with the AH1.23 TCR a chain using the metllods detailed in Example 2 to
produce the
complete soluble AH1.23 TCR-interleukin fusion protein.
Example 10 - Tlzynaidine incorporation assay fof assessing the ability of
AH1.23
TCR-IL-10 fusion proteins to cause Mast cell proliferation.
5 x106 cells of the D36 murine mast cell line which proliferates in response
to huinan
IL-10 in the presence of IL-4 are cultured in RPMI 1640 medium.
A range (0, 0.01, 0.1, 0.5 and 1 M of the AH1.23 TCR-IL-10 fusion protein
prepared
as described in Example 9 and IL-4 are added to the above culture. (Schlaak et
al.,
(1994) J ITnnaunological Methods 168 49-54)
1.85 MBq / ml of H3 Thymidine is then added to 1 x 105 cells of the above
culture in a
96 well plate. These cultures are then incubated for a further 8 hours at 37
C, 5% C02.
The cells are harvested using a cell-harvester, and the level of thymidine
incorporation
into the cells is measured using a Top Count (3 counter.
A reduction in thymidine incorporation into the D36 cells in the presence of
the
AH1.23 TCR-IL-10 fusion protein, compared to that seen in the absence of the
fusion
protein indicates that the IL-10 part of the fusion protein is active and
causing D36
mast cell proliferation.
Exarnple 11 Prepar=ation of high affinity NY-ES MTCR - tlaerapeutic agent
fusion
proteins.
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Synthetic genes comprising the DNA sequence encoding the soluble high affinity
c6l
NY-ESO TCR (3 chain detailed in Figure 23a linked via a DNA sequence encoding
a
peptide linker to DNA encoding a number of imunoinodulaotory agents were
synthesised:
There are a nuinber of companies that provide a suitable DNA service, such as
Geneart (Germany)
Figure 24a details the DNA sequence encoding the high affinity c61 NY-ESO MTCR
beta chain linked at the C-terminus thereof via a peptide linker to IL-18.
Figure 24b
details the AA sequence of this fusion protein, the peptide linker is
underlined.
Figure 25a details the DNA sequence encoding IL-18 pro-protein linked at the C-
terminus thereof via a peptide linker to the high affinity c61 NY-ESO MTCR
beta
chain. The pro-IL-18 DNA sequence has been altered to encode a Factor X
cleavage
site which facilitates post translation removal of the amino acids within the
pro-
sequence. Figure 25b details the AA sequence of this fusion protein, the
peptide
linker is underlined.
Figure 26a details the DNA sequence encoding the high affinity c61 NY-ESO MTCR
beta chain linked at the C-terminus thereof via a peptide linker to IL-10.
Figure 26b
details the AA sequence of this fusion protein, the peptide linker is
underlined.
Figure 27a details the DNA sequence encoding the high affinity c61 NY-ESO MTCR
beta chain linked at the C-terminus tliereof via a peptide linker to IL-13.
Figure 27b
details the AA sequence of this fusion protein, the peptide linker is
underlined.
Figure 28a details the DNA sequence encoding the high affinity c61 NY-ESO MTCR
beta chain linked at the C-terminus thereof via a peptide linker to the "KDEL"
variant
of the PE38 exotoxin. Figure 28b details the AA sequence of this fusion
protein the
peptide linleer is underlined.
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The DNA sequences above c61 NY-ESO TCR beta chain can be ligated into the
pEX821 vector. (See Figures 19 and 20 for the DNA sequence and plasmid map of
this vector respectively)
Disulfide-linked a(3TCR-therapeutic agents are then produced following the
methods
substantially as described in Example 2. Briefly, DNA encoding the high
affinity c61
NY-ESO alpha chain detailed in Figure 29a is synthesised and ligated into the
pEX954 vector. (See Figures 21 and 22 for the DNA sequence and plasmid map of
this vector respectively) The TCR beta chain fusion proteins described above
are then
refolded in the presence of the c61 NY-ESO TCR alpha chain.
Figure 29a details the DNA sequence encoding the high affinity c58 NY-ESO MTCR
alpha chain and Figure 29b details the AA sequence encoded by the DNA sequence
of
Figure 29a.
Exanzple 12 MTCR-PE-38 fusion protein cytotoxicity assay.
1x10"6 of the required target cells (e.g. SK-MEL tumour cells or J82 cells)
were
suspended in 10 ml RPMI media + 10% fetal calf serum (FCS). If required the
target
cells were then pulsed with 10 M of cognate peptide for 2 hours at 37 C. The
samples were then washed three times in RPMI + 10% FCS, centrifuging at 1200
rpm
for 5 min in between each wash. The washed cells were then re-counted and re-
suspended in the appropriate volume of RPMI + 10% FCS media to provide a final
cell density of 2 x 105 cells/ml.
The MTCR-PE38 fusion proteins prepared as described in Example 11 were diluted
in
RPMI media + 10% FCS to a final concentration of 2 x 10-6 M to provide a
working
standard. This working standard was then used to prepare a set of serial
dilutions.
Preparation of experinaental and control samples in microtitre plate wells:
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Experimental sample wells were filled with 50 l mTCR-PE38 in media and 50 l
cells in medium. To produce a total volume of 100 l in 96 well flat bottom
white
opaque walled plates (Nunc 136101). The mTCR-PE38 serial dilutions prepared
above were used to provide a range of mTCR-PE38 concentrations in these wells.
Control sample wells were prepared using either 100 l of cells (cell-only
controls) or
100 l of mTCR-PE38 and media (effector-only controls).
The experimental and control samples were then incubated at 37 C, 5% CO2 for
48 or
96 hours. The nuinber of viable cells remaining in each well was then assessed
using a
CellTiter-Glo'E'Luminescent assay (Promega Cat No: G7572) following the
manufacturers instructions.
Results
Figures 30a and 30b demonstrate that the NY-ESO+ SK-MEL 37 and Mel 624 tumour
cell lines can be killed by the 1G4 MTCR-PE38 fusion protein.
EC50 values for the effect of the 1G4 MTCR-PE38 fusion protein on the SK-MEL
37
and Mel 624 tumour cell lines after 48 hours incubation of 5.9 x 10-9 and 1 x
10-8 M
respectively were calculated from the data presented in Figure 30a.
EC50 values for the effect of the 1G4 MTCR-PE38 fusion protein on the SK-MEL
37
and Mel 624 tumour cell lines after 96 hours incubation of 5.7 x 10-9 and 2.1
x 10-8 M
respectively were calculated from the data presented in Figure 30b.
The results provided by Figures 30a and 30b both demonstrate that pulsing the
J82
target cells with the cognate SLLMWITQC NY-ESO peptide leads to more efficient
killing of these cells by the NY-ESO TCR-PE38 construct compared to that
observed
with unpulsed J82 target cells.
