U.S. patent application number 12/254655 was filed with the patent office on 2009-05-07 for truncated egf receptor.
This patent application is currently assigned to Commonwealth Scientific and Industrial Research Organisation. Invention is credited to Timothy Edward Adams, Anthony Wilkes Burgess, Teresa Anne Domagala, Thomas Charles Elleman, Thomas Peter John Garrett, Robert Nicholas Jorissen, George Oscar Lovrecz, Neil Moreton McKern, Edouard Nice, Colin Wesley WARD.
Application Number | 20090117134 12/254655 |
Document ID | / |
Family ID | 3822485 |
Filed Date | 2009-05-07 |
United States Patent
Application |
20090117134 |
Kind Code |
A1 |
WARD; Colin Wesley ; et
al. |
May 7, 2009 |
Truncated EGF Receptor
Abstract
The present invention relates to truncated EGF receptor
molecules that exhibit increased binding affinities for EGFR
ligands such as EGF and TGF1. The present invention also relates to
methods of screening for EGF receptor ligands and methods of
treatment which involve the use of these molecules.
Inventors: |
WARD; Colin Wesley;
(Carlton, AU) ; McKern; Neil Moreton; (North
Balwyn, AU) ; Lovrecz; George Oscar; (North Balwyn,
AU) ; Jorissen; Robert Nicholas; (Keysborough,
AU) ; Garrett; Thomas Peter John; (Brunswick, AU)
; Elleman; Thomas Charles; (Westmeadows, AU) ;
Burgess; Anthony Wilkes; (Camberwell, AU) ; Adams;
Timothy Edward; (Rosanna, AU) ; Domagala; Teresa
Anne; (Alexandria, AU) ; Nice; Edouard; (St.
Kilda, AU) |
Correspondence
Address: |
GREENLEE WINNER AND SULLIVAN P C
4875 PEARL EAST CIRCLE, SUITE 200
BOULDER
CO
80301
US
|
Assignee: |
Commonwealth Scientific and
Industrial Research Organisation
Campbell
AU
Ludwig institute for Cancer Research
Zurich
CH
|
Family ID: |
3822485 |
Appl. No.: |
12/254655 |
Filed: |
October 20, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11209187 |
Aug 22, 2005 |
7449559 |
|
|
12254655 |
|
|
|
|
10070007 |
Oct 10, 2002 |
6946543 |
|
|
PCT/AU01/00782 |
Jun 28, 2001 |
|
|
|
11209187 |
|
|
|
|
Current U.S.
Class: |
424/178.1 ;
514/1.1; 530/350; 530/391.7 |
Current CPC
Class: |
C07K 14/71 20130101;
A61P 17/06 20180101; A61P 35/00 20180101; A61P 43/00 20180101 |
Class at
Publication: |
424/178.1 ;
530/350; 530/391.7; 514/12 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 14/71 20060101 C07K014/71; C07K 19/00 20060101
C07K019/00; A61K 38/17 20060101 A61K038/17 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 28, 2000 |
AU |
PQ 8418 |
Claims
1. An isolated or purified truncated ErbB3 epidermal growth factor
receptor (EGFR) ectodomain comprising at least residues 1 to 491 of
ErbB3 (SEQ ID NO:3), the truncated EGFR ectodomain lacking at least
the third to seventh modules of the CR2 domain such that the
truncated EGFR ectodomain has an increased binding affinity for at
least one EGFR ligand when compared to the full length EGFR
ectodomain.
2. The truncated EGFR ectodomain of claim 1 wherein the truncated
EGFR ectodomain lacks at least the second to seventh modules of the
CR2 domain.
3. The truncated EGFR ectodomain of claim 1 wherein the truncated
EGFR ectodomain further lacks a portion of the first module of the
CR2 domain.
4. The truncated EGFR ectodomain of claim 1 wherein the truncated
EGFR ectodomain lacks residues 513-624 of ErbB3 (SEQ ID NO:3).
5. The truncated EGFR ectodomain of claim 4 wherein the truncated
EGFR ectodomain lacks residues 501-624 of ErbB3 (SEQ ID NO:3).
6. The truncated EGFR ectodomain of claim 1 wherein the truncated
EGFR ectodomain comprises residues 1-500 or residues 1-512 of ErbB3
(SEQ ID NO:3).
7. The truncated EGFR ectodomain of claim 1 wherein the truncated
EGFR ectodomain consists of residues 1-500 or residues 1-512 of
ErbB3 (SEQ ID NO:3).
8. A chimeric or fusion construct comprising a truncated EGFR
ectodomain of claim 1.
9. The chimeric or fusion construct of claim 8 wherein the
truncated EGFR ectodomain is conjugated to an immunoglobulin
constant domain.
10. The chimeric or fusion construct of claim 9 wherein the
construct comprises residues 1-500 of ErbB3 (SEQ ID NO:3) fused to
an immunoglobulin constant domain.
11. A dimer of the chimeric or fusion protein of claim 9.
12. A homodimer of the chimeric or fusion protein of claim 9.
13. A pharmaceutical composition comprising the truncated EGFR
ectodomain of claim 1 and a pharmaceutically acceptable carrier or
diluent.
14. A pharmaceutical composition comprising the chimeric or fusion
construct of claim 9 and a pharmaceutically acceptable carrier or
diluent.
15. The truncated EGFR ectodomain of claim 1 wherein the truncated
ErbB3 EGFR ectodomain has an affinity for HRG.beta. such that the
IC.sub.50 is 0.49 nM.
Description
CROSS REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/209,187, filed on Aug. 22, 2005, which is a
continuation-in-part of U.S. patent application Ser. No.
10/070,007, filed on Oct. 10, 2002, now U.S. Pat. No. 6,946,543,
which is the National Phase of International Application
PCT/AU01/00782, filed Jun. 28, 2001, designating the U.S. and
published as WO 02/00876, with a claim of priority from Australian
application no. PQ 8418, filed Jun. 28, 2000.
[0002] All of the foregoing applications, as well as all documents
cited in the foregoing applications ("application documents") and
all documents cited or referenced in the application documents are
incorporated herein by reference. Also, all documents cited in this
application ("herein cited documents") and all documents cited or
referenced in herein cited documents are incorporated herein by
reference. In addition, any manufacturer's instructions or
catalogues for any products cited or mentioned in each of the
application documents or herein cited documents are incorporated by
reference. Documents incorporated by reference into this text or
any teachings therein can be used in the practice of this
invention. Documents incorporated by reference into this text are
not admitted to be prior art.
BACKGROUND OF THE INVENTION
[0003] The present invention relates to truncated EGF receptor
molecules and to pharmaceutical compositions comprising these
molecules. The present invention also relates to methods of
screening for EGF receptor ligands and methods of treatment which
involve the use of these molecules.
[0004] The epidermal growth factor receptor (EGFR) family consists
of four distinct tyrosine kinase receptors, EGFR/HER/ErbB1,
HER2/Neu/ErbB2, HER3/ErbB3 and HER4/ErbB4 (1). These receptors are
widely expressed in epithelial, mesenchymal and neuronal tissues
and play fundamental roles during development and differentiation.
They are activated by a family of at least twelve ligands that
induce either homo- or hetero-dimerisation of the EGFR homologues.
ErbB2 is unable to bind ligand on its own but is a potent
co-receptor for all ligands when co-expressed with other members of
the EGFR/HER/ErbB family.
[0005] The EGFR is a large (1,186 residues), monomeric glycoprotein
with a single transmembrane region and a cytoplasmic tyrosine
kinase domain flanked by noncatalytic regulatory regions. Sequence
analyses have shown that the ectodomain (residues 1-621) contains
four sub-domains, here termed L1, CR1, L2 and CR2, where L and CR
are acronyms for large and Cys-rich respectively (2, 3). The L1 and
L2 domains have also been referred to as domains I and III,
respectively (4). The CR domains have been previously referred to
as domains II and IV (4), or as S1.1-S1.3 and S2.1-S2.3 where S is
an abbreviation for small (2).
[0006] Many cancer cells express constitutively active EGFR (5) or
other EGFR family members (6). Elevated levels of activated EGFR
occur in bladder, breast, lung and brain tumours. Antibodies to
EGFR can inhibit ligand activation of EGFR and the growth of many
epithelial cell lines. Patients receiving repeated doses of a
humanised chimeric anti-EGFR monoclonal antibody (Mab) showed signs
of disease stabilization. The large doses required and the cost of
production of humanised Mab is likely to limit the application of
this type of therapy. These findings indicate that the development
of EGF receptor antagonists may be attractive anticancer
agents.
SUMMARY OF THE INVENTION
[0007] The present inventors have now made the surprising finding
that the deletion of residues in the CR2 domain of the EGFR
ectodomain gives rise to a truncated ectodomain with enhanced
affinity for epidermal growth factors such as (EGF) and/or
transforming growth factor-I (TGF-.alpha.). This finding goes
against recently reported results (8) showing that deletions or
mutations in the CR2 region reduce EGFR binding affinity for
EGF.
[0008] As will be appreciated by those skilled in the art, the
truncated EGFR ectodomains of the present invention may provide
increased sensitivity in assays which screen for ligands of the EGF
receptor. Furthermore, the truncated EGFR ectodomains of the
present invention may have therapeutic potential given their high
affinity for ligand and their ability to competitively inhibit
EGF-induced proliferation responses in vitro.
[0009] Accordingly, the present invention provides a truncated EGFR
ectodomain, the truncated EGFR ectodomain lacking a substantial
portion of the CR2 domain such that the truncated EGFR ectodomain
has an increased binding affinity for at least one EGFR ligand when
compared to the full length EGFR ectodomain.
[0010] In one embodiment the present invention provides a truncated
epidermal growth factor receptor (EGFR) ectodomain comprising at
least residues 1-492 of ErbB1 or equivalent residues of another
member of the EGFR family, the truncated EGFR ectodomain lacking at
least the third to seventh modules of the CR2 domain such that the
truncated EGFR ectodomain has an increased binding affinity for at
least one EGFR ligand when compared to the full length EGFR
ectodomain.
[0011] The EGFR ligand may be, for example, amphiregulin, heparin
binding EGF, .beta.-cellulin, EGF or TGF-.alpha.. In a preferred
embodiment of the first aspect the truncated EGFR ectodomain has an
increased binding affinity for EGF and/or TGF-.alpha..
[0012] In a further preferred embodiment the truncated EGFR
ectodomain lacks at least the third to seventh modules of the CR2
domain. In a further preferred embodiment, the truncated EGFR
ectodomain lacks at least the second to seventh modules of the CR2
domain. The truncated EGFR ectodomain may further lack a portion of
the first module of the CR2 domain.
[0013] In a further preferred embodiment the truncated EGFR
ectodomain comprises at least residues 1 to 492 of ErbB1.
Preferably, the truncated EGFR ectodomain lacks residues 514-621 of
ErbB1. More preferably, the truncated EGFR ectodomain lacks
residues 502-621 of ErbB1.
[0014] In a further preferred embodiment the member of the EGFR
family is ErbB3 and the truncated EGFR comprises at least residues
1 to 491 of ErbB3. Preferably, the truncated EGFR ectodomain lacks
residues 513-624 of ErbB3. More preferably, the truncated EGFR
ectodomain lacks residues 501-624 of ErbB3.
[0015] In a further preferred embodiment the member of the EGFR
family is ErbB4 and the truncated EGFR comprises at least residues
1 to 488 of ErbB4. Preferably, the truncated EGFR ectodomain lacks
residues 510-626 of ErbB4. More preferably, the truncated EGFR
ectodomain lacks residues 498-626 of ErbB4.
[0016] In a further preferred embodiment the truncated EGFR
ectodomain comprises residues 1-501 or residues 1-513 of ErbB1.
[0017] In a further preferred embodiment the truncated EGFR
ectodomain consists essentially of residues 1-501 or residues 1-513
of ErbB1.
[0018] In a further preferred embodiment the truncated EGFR
ectodomain consists of residues 1-501 or residues 1-513 of
ErbB1.
[0019] In a further preferred embodiment the member of the EGFR
family is ErbB3 and the truncated EGFR ectodomain comprises
residues 1-500 or residues 1-512 of ErbB3.
[0020] In a further preferred embodiment the member of the EGFR
family is ErbB3 and the truncated EGFR ectodomain consists
essentially of residues 1-500 or residues 1-512 of ErbB3.
[0021] In a further preferred embodiment the member of the EGFR
family is ErbB3 and the truncated EGFR ectodomain consists of
residues 1-500 or residues 1-512 of ErbB3.
[0022] In a further preferred embodiment the member of the EGFR
family is ErbB4 and the truncated EGFR ectodomain comprises
residues 1-497 or residues 1-509 of ErbB4.
[0023] In a further preferred embodiment the member of the EGFR
family is ErbB4 and the truncated EGFR ectodomain consists
essentially of residues 1-497 or residues 1-509 of ErbB4.
[0024] In a further preferred embodiment the member of the EGFR
family is ErbB4 and the truncated EGFR ectodomain consists of
residues 1-497 or residues 1-509 of ErbB4.
[0025] Further deletions or mutations may be made to the L1, CR1
and/or L2 sub-domains of the truncated EGFR ectodomain of the
present invention, provided that these further deletions or
mutations do not substantially affect the binding affinity of the
truncated EGFR ectodomain. Preferably, however, the truncated EGFR
ectodomain of the present invention comprises the L1, CR1 and L2
subdomains and the first module of the CR2 subdomain.
[0026] In a further preferred embodiment, the truncated EGFR
ectodomain has an affinity for EGF such that the K.sub.d is less
than 30 nM, more preferably less than 25 nM.
[0027] In a further preferred embodiment, the truncated EGFR
ectodomain has an affinity for TGF-.alpha. such that the K.sub.d is
less than 45 nM, more preferably less than 40 nM.
[0028] The present invention also provides a polynucleotide
encoding a truncated EGFR ectodomain of the present invention.
[0029] The present invention also provides an expression vector
comprising a polynucleotide of the present invention.
[0030] The present invention also provides a host cell comprising
an expression vector of the present invention.
[0031] The present invention also provides a method for producing a
truncated EGFR ectodomain of the present invention, the method
comprising culturing a host cell of the present invention under
conditions which allow production of the truncated EGFR ectodomain
and isolating the truncated EGFR ectodomain.
[0032] The present invention also provides a pharmaceutical
composition comprising a truncated EGFR ectodomain according to the
present invention and a pharmaceutically acceptable carrier or
diluent.
[0033] The present invention also provides a method of screening a
putative compound for the ability to modulate the activity of the
EGF receptor, the method comprising exposing the putative compound
to a truncated EGFR ectodomain according to the present invention
and monitoring the activity of the truncated EGFR ectodomain.
[0034] A suitable assay procedure may involve a competition binding
assay in a microplate format, where the putative compound is tested
for its ability to inhibit the binding of labelled ligand such as
EGF or TGF.alpha. to the truncated EGF receptor ectodomain. The
label may be a radiolabelled tag such as .sup.125I or a fluorescent
tag such as fluorescein isothiocyanate or a lanthanide ion such as
europium.
[0035] The present invention also provides a method of treating or
preventing a disease associated with signalling by a molecule of
the EGF receptor family in a subject, the method comprising
administering to the subject a truncated EGFR ectodomain of the
present invention.
[0036] Preferably, the disease associated with signalling by a
molecule of the EGF receptor family is selected from psoriasis and
tumour states comprising but not restricted to cancer of the
breast, brain, ovary, cervix, pancreas, lung, head and neck, and
melanoma, rhabdomyosarcoma, mesothelioma and glioblastoma.
[0037] The method of treatment of the present invnetion may be used
alone or in combination with other therapeutic measures. For
example, the method may be used in combination with cytotoxic
modalities, such as anti-EGFR antibodies, radiotherapy or
chemotherapy, in the treatment of tumour states.
BRIEF DESCRIPTION OF THE FIGURES
[0038] FIGS. 1A and 1B: Sequence alignment of the ectodomains of
human EGF receptor family proteins ErbB1, ErbB2, ErbB3 and
ErbB4.
[0039] FIG. 2: BIAcore analysis of the interactions between
ErbB1501 and ErbB1621 with immobilised hEGF or hTGF-.alpha.. (A):
ErbB1-501 (140, 120, 100, 80, 60 and 40 nM) was passed over
immobilised hEGF (160 RU immobilised). Samples (30 Tl) were
injected at a flow rate of 10 Tl/min. (B): ErbB1-501 was passed
over immobilised hTGF-I (132 RU immobilised). Experimental details
were as in panel A. (C): sEGFR621 (1000, 900, 800, 700, 600 and 500
nM) was passed over immobilised hEGF. (D): ErbB1-621
(concentrations as for panel C) was passed over immobilised hTGF-I.
The operating temperature was 25.degree. C. At the end of the
injection phase, dissociation was monitored with buffer alone
flowing over the sensor surface. The surface was regenerated
between samples using 10 mM HCl. The signal obtained when the
sample was passed over a parallel blank channel has been subtracted
electronically to give the specific response.
[0040] FIG. 3: Scatchard analysis of equilibrium binding data. The
dissociation constant (KD=1/KA) was calculated from the equilibrium
binding response obtained in FIG. 2 by plotting the data in
Scatchard format (Req/nC versus Req; see Experimental Procedures).
The slope of the linear fit to the data gives KA. (A): ErbB1501
versus hEGF; (B): ErbB1501 versus hTGF-I; (C): ErbB1621 versus
hEGF; (D): ErbB1621 versus hTGFI.
[0041] FIG. 4: Inhibition of EGF-stimulated cell mitogenesis by
ErbB1501. (A): The stimulation of .sup.3H-thymidine incorporation
by BaF/3ERX cells using serial dilutions of mEGF. The data was
fitted by a sigmoidal function (-) to determine the EC.sub.50. (B):
Inhibition of the mitogenic response of BaF/3ERX cells stimulated
with mEGF (207 pM) by: ErbB1501 (.quadrature.v-.quadrature.),
ErbB1621 (.quadrature..lamda.-.quadrature.) or anti-EGFR antibody
Mab528 (.quadrature..sigma.-.quadrature.). Each point was assayed
in triplicate. Error bars are shown.
[0042] FIG. 5: Covalent cross-linking of ErbB1501 dimers after
incubation with mEGF. sEGFR501 (5 .mu.M) was incubated with (+) or
without (-) mEGF (20 .mu.M) in 20 mM HEPES (pH7.4) containing 150
mM NaCl for 1 h at room temperature followed by the addition of
bis(sulfosuccinimidyl)suberate (BS3, Pierce, Rockford, Ill., USA)
to a final concentration of 0.5 mM and incubation for a further 30
min. The reaction was terminated and the degree of dimer formation
was monitored by SDS-PAGE and immunoblotting with anti-EGFR Mab528
(7) (0.5 .mu.g/ml) and horseradish-peroxidase-labelled goat
anti-mouse IgG (Bio-Rad) with detection by ECL (Amersham Pharmacia
Biotech). Analysis by non-reducing SDS-PAGE was necessary since the
antibody used to detect ErbB1501 (Mab528) is
conformation-dependent.
[0043] FIG. 6. Analysis of EGF/ErbB1501 (sEGFR501) interactions
using the analytical ultracentrifuge. (A) Sedimentation equilibrium
analysis of EGF, ErbB1501 (sEGFR501) and a mixture of EGF and
ErbB1-501 (sEGFR501). The equilibrium distributions were obtained
after centrifugation at 12,000 rpm at 20.degree. C. for 16 h.
(.quadrature.) 20 .mu.M EGF; (.smallcircle.) 10 .mu.M ErbB1501
(sEGFR501), (.DELTA.) 20 .mu.M EGF+10 .mu.M ErbB1501 (sEGFR501).
The lines of best fit drawn though the data for EGF and sEGFR501
are for single species and for molecular weight values of 6,000 and
65,600 respectively. The line drawn through the data for the
EGF/ErbB1501 (sEGFR501) mixture is the line of best-fit calculated
assuming two species with the molecular weight of the first species
fixed at 6,000 and a fitted value of 106,400 for the molecular
weight of the second species. Inset: The residual plot for the fit
of the EGF/ErbB1501 (sEGFR501) mixture. (B) Meniscus depletion
sedimentation analysis of the stoichiometry of EGF binding to
ErbB1-501 (sEGFR501). Solutions containing 5 .mu.M ErbB1-501
(sEGFR501) and different molar ratios of EGF:EGFR were spun for 16
h at 20,000 rpm and 20.degree. C. in the XLA analytical
ultracentrifuge. Under these conditions ErbB1501 (sEGFR501) and its
complexes with EGF are depleted from the meniscus leaving unbound
EGF in the supernatant. Optical density measurements at 280 nm
enable the amount of unbound EGF near the meniscus to be estimated.
(C) Data obtained for the weight-average molecular weight of the
"second" species calculated for mixtures of ErbB1501 (sEGFR501) (5
.mu.M) and EGF at the concentrations indicated under the conditions
of panel A above. The solid line corresponds to a simulated curve
based on a KD of 30 nM and a dimerisation constant of 4 .mu.M.
[0044] FIG. 7: BIAcore analysis of the binding of the Gly441Lys
ErbB1501 mutant to immobilised hEGF and hTGF-.alpha.. Purified
Gly441LyssErbB1501 (24-385 nM) was passed over immobilised
hTGF-.alpha. (Panel A) or hEGF (Panel B) using the experimental
conditions described in FIG. 2. The corresponding Scatchard
analysis, using the equilibrium binding values obtained from these
sensorgrams, is shown below (Panels C,D).
[0045] FIG. 8 .sup.EUEGF displacement from immobilized
EGFR501.sub.Fcflag. Recombinant EGFR501Fc flag protein was
immobilized by incubation in wells of a 96-well Lumitrac plate
(Nunc) that had been pre-coated with protein G. Competition binding
studies were performed with Europium-labelled EGF (Wallac) as trace
and increasing concentrations of unlabelled EGF. After incubation
overnight at 4.degree. C., the wells were washed and the bound
trace measured by time-resolved fluorescence.
[0046] FIG. 9 Relative affinities of full-length ErbB4
ectodomain-Fc fusion homodimers and ErbB2/4 full-length ectodomain
heterodimers for .beta.-heregulin. Human 293T fibroblasts were
transiently transfected with a mammalian expression vector encoding
the full-length ErbB4 ectodomain-Fc fusion alone, or with vectors
encoding both full-length ErbB2 and ErbB4 ectodomain-Fc fusion
proteins. Supernatants were harvested and recombinant receptors
immobilized by incubation in wells of a 96-well Lumitrac plate
(Nunc) that had been pre-coated with protein G. Competition binding
studies were performed with Europium-labelled betacellulin (BTC) as
trace and increasing concentrations of unlabelled .beta.-heregulin
(Sigma). After incubation overnight at 4.degree. C., the wells were
washed and the bound trace measured by time-resolved
fluorescence.
[0047] FIG. 10 Relative affinities of truncated ErbB3 ectodomain-Fc
and ErbB4 ectodomain-Fc fusion protein homodimers for
.beta.-heregulin. Human 293T fibroblasts were transiently
transfected with a mammalian expression vector encoding either the
truncated ErbB3 ectodomain-Fc fusion protein, or with a vector
encoding the truncated ErbB4 ectodomain-Fc fusion protein.
Supernatants were harvested and recombinant receptors immobilized
by incubation in wells of a 96-well Lumitrac plate (Nunc) that had
been pre-coated with protein G. Competition binding studies were
performed with Europium-labelled .beta.-heregulin
(.sup.EuHRG.beta.) as trace and increasing concentrations of
unlabelled HRG.beta. (Sigma). After incubation overnight at
4.degree. C., the wells were washed and the bound trace measured by
time-resolved fluorescence.
DETAILED DESCRIPTION OF THE INVENTION
[0048] When used herein the term "EGFR" is intended to encompass
members of the EGF receptor family such as ErbB1 (also known as the
EGF receptor), ErbB2, ErbB3 and ErbB4. In general, EGF receptor
family molecules show similar domain arrangements and share
significant sequence identity, preferably at least 40%
identity.
[0049] When used herein, the phrase "full length EGFR ectodomain"
refers to the ectodomain consisting of residues 1-621 of ErbB1 or
equivalent residues of other members of the EGF receptor family.
The amino acid sequence of the full length ectodomain has been
previously described (13). The full length ectodomain contains four
sub-domains, referred to as L1, CR1, L2 and CR2, where L and CR are
acronyms for large and cys-rich respectively. FIGS. 1A and 1B show
a sequence alignment of the full length ectodomains of ErbB1,
ErbB2, ErbB3 and ErbB4.
[0050] The CR2 sub-domain of ErbB1, ErbB3 and ErbB4 consists of the
following seven modules joined by linkers of 2 or 3 amino acid
residues and bounded by cysteine residues as follows:
TABLE-US-00001 ErbB1 ErbB2 ErbB3 ErbB4 First module 482-499 490-507
481-498 478-495 Second module 502-511 510-519 501-510 498-507 Third
module 515-531 523-539 514-530 511-527 Fourth module 534-555
542-563 533-554 530-552 Fifth module 558-567 566-575 557-566
555-564 Sixth module 571-593 579-602 570-591 568-589 Seventh module
596-612 605-621 594-610 592-608
[0051] The results presented herein show that deletions in the CR2
region of the EGFR ectodomain unexpectedly increase binding
affinity of the ectodomain for EGF and/or TGF-.alpha.. In light of
this information, a person skilled in the art would be able to
readily generate a number of candidate truncated ectodomains and
screen these candidates for increased ligand affinity and for
therapeutic potential.
[0052] For example, truncated ectodomains may be prepared by
recombinant DNA technology as described herein or as described
previously (8). Alternatively, truncated ectodomains may be
prepared by subjecting the full length ectodomain or full length
receptor to limited proteolysis as described previously (9).
[0053] Binding affinity and inhibitor potency may be measured for
candidate truncated ectodomains using biosensor technology.
[0054] Truncated EGFR ectodomains of the invention may be in a
substantially isolated form. It will be understood that the protein
may be mixed with carriers or diluents which will not interfere
with the intended purpose of the protein and still be regarded as
substantially isolated. A truncated ectodomain of the invention may
also be in a substantially purified form, in which case it will
generally comprise the protein in a preparation in which more than
90%, e.g. 95%, 98% or 99% of the protein in the preparation is a
protein of the invention.
[0055] In the context of the present invention, the amino acid
sequence of the truncated EGFR ectodomain may be modified provided
that the modification does not adversely affect the binding
affinity of the truncated ectodomain for at least one EGFR ligand.
For example, modified ectodomains may be constructed by making
various substitutions of residues or sequences or deleting terminal
or internal residues or sequences not needed for binding activity.
Generally, substitutions should be made conservatively; i.e., the
most preferred substitute amino acids are those having
physiochemical characteristics resembling those of the residue to
be replaced. Similarly, when a deletion or insertion strategy is
adopted, the potential effect of the deletion or insertion on
biological activity should be considered. In order to preserve the
biological activity of the truncated ectodomains, deletions and
substitutions will preferably result in homologous or
conservatively substituted sequences, meaning that a given residue
is replaced by a biologically similar residue. Examples of
conservative substitutions include substitution of one aliphatic
residue for another, such as Ile, Val, Leu, Met or Ala for one
another, or substitutions of one polar residue for another, such as
between Lys and Arg; Glu and Asp; or Gln and Asn. Other such
conservative substitutions, for example, substitutions of entire
regions having similar hydrophobicity characteristics, are well
known. Moreover, particular amino acid differences between human,
murine and other mammalian EGFRs is suggestive of additional
conservative substitutions that may be made without altering the
essential biological characteristics of the truncated EGFR
ectodomains.
[0056] Modifications encompassed by the present invention also
include various structural forms of the primary protein which
retain binding affinities. Due to the presence of ionizable amino
and carboxyl groups, for example, a truncated ectodomain may be in
the form of acidic or basic salts, or may be in neutral form.
Individual amino acid residues may also be modified by oxidation or
reduction.
[0057] The primary amino acid structure may be modified by forming
covalent or aggregative conjugates with other chemical moieties,
such as glycosyl groups, lipids, phosphate, acetyl groups and the
like, or by creating amino acid sequence mutants. Other
modifications within the scope of this invention include covalent
or aggregative conjugates of the truncated ectodomain with other
proteins or polypeptides, such as by synthesis in recombinant
culture as N-terminal or C-terminal fusions. For example, the
conjugated polypeptide may be a signal (or leader) polypeptide
sequence at the N-terminal region of the protein which
co-translationally or post-translationally directs transfer of the
protein from its site of synthesis to its site of function inside
or outside of the cell membrane or wall (e.g., the yeast I-factor
leader). Truncated EGFR ectodomain fusions may comprise peptides
added to facilitate purification or identification of the truncated
ectodomain (e.g., poly-His) or to enhance stability or delivery of
the ectodomain in vivo.
[0058] The truncated EGFR ectodomains of the present invention may
also be fused to the constant domain of an immunoglobulin molecule.
For example, a recombinant chimeric antibody molecule may be
produced having truncated EGFR ectodomain sequences substituted for
the variable domains of either or both of the immunoglobulin
molecule heavy and light chains and having unmodified constant
region domains. This may result in a single chimeric antibody
molecule having truncated EGFR ectodomains displayed bivalently.
Such polyvalent forms of the truncated EGFR ectodomain may have
enhanced binding affinity for EGFR ligands. Details relating to the
construction of such chimeric antibody molecules are disclosed in
WO 89/09622 and EP 315062.
[0059] As TGF.alpha. exists as a membrane bound form, the truncated
ectodomains of the present invention may be used to target
compounds to cancer cells. Accordingly, truncated EGFR ectodomain
fusions may comprise compounds useful in the diagnosis or treatment
of cancer cells such as drugs, isotopes or toxins.
[0060] Truncated EGFR ectodomain derivatives may also be obtained
by cross-linking agents, such as M-maleimidobenzoyl succinimide
ester and N-hydroxysuccinimide, at cysteine and lysine residues.
The truncated ectodomains may also be covalently bound through
reactive side groups to various insoluble substrates, such as
cyanogen bromide-activated, bisoxirane-activated,
carbonyldiimidazole-activated or tosyl-activated agarose
structures, or by adsorbing to polyolefin surfaces (with or without
glutaraldehyde cross-linking). Once bound to a substrate, the
truncated ectodomain may be used to selectively bind (for purpose
of assay or purification) anti-EGFR antibodies or EGF.
[0061] It may also be desirable to use derivatives of the
ectodomains of the invention that are conformationally constrained.
Conformational constraint refers to the stability and preferred
conformation of the three-dimensional shape assumed by a peptide.
Conformational constraints include local constraints, involving
restricting the conformational mobility of a single residue in a
peptide; regional constraints, involving restricting the
conformational mobility of a group of residues, which residues may
form some secondary structural unit; and global constraints,
involving the entire peptide structure.
[0062] It will be appreciated that the truncated EGFR ectodomains
of the present invention may be used as immunogens, reagents in
receptor-based immunoassays, or as binding agents for affinity
purification procedures of EGF or other binding ligands.
[0063] Truncated EGFR ectodomains may be tested for their ability
to modulate receptor activity using a cell-based assay
incorporating a stably transfected, EGF-responsive reporter gene
(10). The assay addresses the ability of EGF to activate the
reporter gene in the presence of novel ligands. It offers a rapid
(results within 6-8 hours of hormone exposure), high-throughput
(assay can be conducted in a 96-well format for automated counting)
analysis using an extremely sensitive detection system
(chemiluminescence). Once candidate compounds have been identified,
their ability to antagonise signal transduction via the EGF-R can
be assessed using a number of routine in vitro cellular assays such
as inhibition of EGF-mediated cell proliferation. Ultimately, the
efficiency of truncated EGFR ectodomains as tumour therapeutics may
be tested in vitro in animals bearing tumour isografts and
xenografts as described (11, 12).
[0064] Truncated EGFR ectodomains of the invention (and substances
identified by the assay methods of the invention) may preferably be
combined with various components to produce compositions of the
invention. Preferably the compositions are combined with a
pharmaceutically acceptable carrier or diluent to produce a
pharmaceutical composition (which may be for human or animal use).
Suitable carriers and diluents include isotonic saline solutions,
for example phosphate-buffered saline. The compositions may be
formulated, for example, for parenteral, intramuscular,
intravenous, subcutaneous, intraocular, oral or transdermal
administration. Typically, each protein may be administered at a
dose of from 0.01 to 30 mg/kg body weight, preferably from 0.1 to
10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
[0065] The routes of administration and dosages described are
intended only as a guide since a skilled practitioner will be able
to determine readily the optimum route of administration and dosage
for any particular patient and condition.
[0066] In view of the ability of the truncated EGFR ectodomains of
the present invention to bind strongly to EGFR ligands, the
truncated ectodomains will be useful in diagnostic assays for EGFR
ligands, as well as in raising antibodies to the EGFR for use in
diagnosis and therapy. In addition, purified truncated EGFR
ectodomains may be used directly in therapy to bind or scavenge
EGFR ligands, thereby providing means for regulating the activities
of these ligands. In particular, truncated EGFR ectodomains of the
present invention may be administered for the purpose of inhibiting
EGF-dependent responses.
[0067] Throughout this specification the word "comprise", or
variations such as "comprises" or "comprising", will be understood
to imply the inclusion of a stated element, integer or step, or
group of elements, integers or steps, but not the exclusion of any
other element, integer or step, or group of elements, integers or
steps.
[0068] Throughout this specification, the term "consisting
essentially of" is intended to exclude elements which would
materially affect the properties of the claimed composition.
[0069] The invention will now be described in further detail with
reference to the following non-limiting Examples.
EXPERIMENTAL DETAILS
Materials and Methods
[0070] Construction of plasmids used for the expression of
truncated forms of hErbB1 ectodomain. The plasmid pErbB1, used in
the construction of truncated hErbB1 cDNAs, comprises nucleotides
167-3970 of hErbB1 (13) in the multiple cloning site of plasmid
pUC18. Coding is in the opposite sense to the LacZ .alpha. peptide,
and the insertion is downstream of the XbaI site of pUC18. This
plasmid was used later in excision of the truncated constructs for
insertion into the mammalian expression vector pEE14 (14).
[0071] Construction of pErbB1476. An initial plasmid containing
nucleotides 167-3150 of hErbB1 was constructed by ligation of a
XbaI/NsiI fragment from pEGFR and XbaI/PstI-cut pBluescript KS+.
From this plasmid, a 4 kbp fragment BbsI/BglII fragment (containing
all of pBluescript KS+ and nucleotides 167-1150 and 2951-3150 of
EGFR) and a 528 bp BbsI/PvuII fragment (nucleotides 1151-1679) were
ligated with a 70 bp PCR-derived PvuII and BglII fragment, encoding
amino acids 474-476 of hEGFR, an enterokinase cleavage site and a
c-myc epitope tag to facilitate purification. The 70 bp PCR
cassette was produced using a similar previous construct (15) as
template. A plasmid for mammalian cell transfection, pErbB1476, was
constructed from this plasmid by ligation of a 1.6 kbp XbaI/EcoRV
fragment with XbaI/SmaI-cut pEE14.
[0072] Construction of pErbB1501 and pErbB1513. In each
construction PCR was used with three oligonucleotides to produce a
fragment of hEGFR cDNA (nucleotides 1121 to 1760 or 1121 to 1797
respectively), followed by a sequence encoding an enterokinase
cleavage site, a c-myc epitope tag and a termination codon. The
upstream primer in PCR corresponded to an arbitrary choice of
nucleotides 1121-1140 of hEGFR cDNA, while two overlapping
downstream primers were used to construct additional sequence
adjacent to nucleotide 1760 or 1797 respectively. The PCR products
were cloned using the pCR-Script vector (Stratagene). In each case
this allowed an ApaI fragment harbouring the newly constructed
sequence beginning at nucleotide 1738 of hErbB1, to be excised for
subsequent insertion into the large ApaI fragment of PEGFR (which
included the entire pUC18 sequence with hErbB1 cDNA to nucleotide
1737), in order to prepare a plasmid encoding a truncated hEGFR
with XbaI restriction sites adjacent to the coding sequence. From
these pUC18-based plasmids the fragments harbouring the truncated
hErbB1 cDNAs were excised by XbaI digestion, and inserted into
plasmid pEE14 at the XbaI site to prepare plasmids pErbB1501 and
pErbB1513 respectively for mammalian cell transfection.
[0073] Mutagenesis. The 1.7 kbp fragment harbouring the truncated
hErbB1 cDNA of pEGFR501 was introduced into M13 mp18 (16) for
mutagenesis. Oligonucleotide-directed in vitro mutagenesis, using
the USB-T7 Gen.TM. in vitro mutagenesis kit, was employed to
produce three single site mutants of the truncated human sErbB1501
ectodomain, with residues Glu367, Gly441 and Glu472 respectively
mutated to Lys to match the corresponding residues in chicken ErbB1
(4). Clones incorporating the mutations were identified by colony
hybridisation (17) using .sup.32P-labelled mutagenic
oligonucleotide as a probe, and the mutations were confirmed by DNA
sequence analysis (18). Vehicles for mammalian cell expression were
generated for each mutant by excising the 1.7 kbp fragment
harbouring the mutated sErbB1501 cDNA from M13 RF-DNA by XbaI
digestion and inserting it into plasmid pEE14 (16) at the XbaI
site.
[0074] Vector assembly for production of ErbB1, ErbB3 and ErbB4 Fc
fusion proteins. DNA templates encoding a number of full-length and
truncated ectodomain fragments for ErbB1, ErbB3 and ErbB4, fused in
frame with a genomic fragment coding for the human IgG1 Fc region
together with an N-terminal spacer peptide and a C-terminal epitope
tag, were generated using standard molecular techniques. DNA
templates were assembled in plasmid expression vectors, enabling
transcription and the subsequent translation of the encoded fusion
protein following transfection into mammalian cells.
[0075] Cell Culture, DNA Transfection and Protein Analysis. For
transient transfection assays, human 293T fibroblasts maintained in
DMEM plus 10% fetal calf serum (FCS) were transfected with plasmid
DNA using FuGENE (Roche Molecular Biochemicals, Sydney, NSW)
according to the manufacturer's instructions. Supernatants were
harvested 48 h after transfection, and cell lysates were prepared
in NP-40 lysis buffer. To characterise secreted EGFR mutants,
aliquots of supernatant and lysate were immunoprecipitated with a
monoclonal antibody (9E10) to the c-myc tag, or with Mab 225
(HB-8508, American Type Culture Collection), a conformationally
dependent monoclonal antibody recognising the extracellular domain
of the hErbB1 (19). Immune complexes were collected on Protein
A-Sepharose beads (Zymed Laboratories, Bioscientific Pty. Ltd.,
Gymea, NSW), fractionated by SDS polyacrylamide gel electrophoresis
(10% gel) and transferred to nitrocellulose membranes. Truncated
hEGFR ectodomains and mutants were identified by probing membranes
with horseradish peroxidase (HRP)-conjugated Mab9E10 (Roche),
followed by chemilumiscent detection with Pierce Super Signal
substrate.
[0076] Stable cell lines expressing ErbB1501 were established in
the Lec8 mutant cell line from CHOK (7) using glutamine synthetase
as a selectable marker (15). Supernatants from methionine
sulfoximine (MSX)-resistant cell colonies were screened for
secreted receptor by biosensor analysis (see below) or by dot
blotting onto nitrocellulose and probing with HRP-Mab9E10. A single
cell line was selected for cloning by limiting dilution.
[0077] Lec8 cells expressing ErbB1501 protein were cultured in a
Celligen Plus bioreactor (New Brunswick Scientific, New Jersey,
USA) using 70 g Fibra-Cell Disks carriers with 1.7 L working
volume. Continuous perfusion culture using glutamine-free DMEM/F12
medium supplemented with non-essential amino acids, nucleosides and
10% FCS was maintained for 6 weeks. Selection pressure was
maintained with 25 .mu.M MSX for the duration of the fermentation.
Perfusion rate was adjusted as required to ensure a residual
glucose level of 1.0-1.5 g/L, with a corresponding lactate
concentration of 2.0-2.3 g/L.
[0078] HEK 293T cells were transfected with plasmid DNA encoding
ErbB1, ErbB3 and ErbB4 Fc fusion proteins complexed with FuGENE
(Boehringer) 24 hours after seeding into six-well plates. Twenty
four hours later, the culture medium was replaced with serum-free
medium, and supernatant harvested 24-48 hours later.
[0079] Purification of Truncated EGFR Ectodomains. For biosensor
and AUC analyses, conditioned medium containing the sEGFR truncated
proteins (4 L) was adjusted to pH 8.0 with Tris-HCl (Sigma)
containing sodium azide (0.02% (w/v)) (TBSA), and particulates
removed by centrifugation prior to recovery of c-myc-tagged protein
by affinity purification at 4.degree. C. on a column of monoclonal
antibody 9E10 covalently-bound to agarose, using peptide elution
(15). Eluted protein was further purified by size exclusion
chromatography on Superdex 200 (HR10/30, Amersham Pharmacia
Biotech) at room temperature using TBSA buffer at a flow rate of
0.8 ml/min. Protein was detected by absorbance at 280 nm.
[0080] BIAcore Binding Assays. Protein-protein interactions were
monitored in real time on an instrumental optical biosensor using
surface plasmon resonance detection (BIAcore 2000 or 3000, BIAcore,
Uppsala, Sweden). Recombinant hEGF or hTGF-I (Gropep, Adelaide,
Australia) were purified immediately prior to immobilisation by
micropreparative RP-HPLC using a SMART system (Amersham Pharmacia
Biotech) as described previously (20). The proteins were
immobilised onto the biosensor surface using amine coupling
chemistry (N-hydroxysuccinimide and
N-ethyl-N'-dimethylaminopropyl-carbodiimide) at a flow rate of 4
.mu.l/min. Typically 100-200 RU were immobilised equivalent to
0.1-0.2 ng/mm.sup.2 (20). Automated targeting of immobilisation
levels was achieved using the BIAcore 3.1 control software
(21).
[0081] Prior to analysis, ErbB1621 (23), ErbB1501 and the ErbB1501
mutant samples were characterised by micropreparative size
exclusion chromatography (Superose 12 3.2/30, Amersham Pharmacia
Biotech) to ensure size homogeneity (20) and pooled fractions were
diluted in BIAcore buffer (HBS: 10 mM Hepes pH 7.4 containing 3.4
mM EDTA, 0.15 mM NaCl and 0.005% (v/v) Tween 20) to the appropriate
concentration. Typically, samples (30 .mu.l) at concentrations of
10-1000 nM were injected sequentially over the sensor surfaces at a
flow rate of 5 or 10 .mu.l/min. Following completion of the
injection phase, dissociation was monitored in BIAcore buffer at
the same flow rate. The sensor surface and sample blocks were
maintained at 25.degree. C. Bound receptor was eluted, and the
surface regenerated between injections, using 40 .mu.l of 10 mM
HCl. This treatment did not denature hEGF or hTGF-.alpha.
immobilised onto the sensor surface, as shown by equivalent signals
on re-injection of receptor.
[0082] Kinetic rate constants (ka, kd) were determined using the
BIAevaluation 3.02 software (BIAcore,
http//www.biacore.com/products/eval3.html) as described previously
(22), or by global analysis using CLAMP (23, 24). Equilibrium
binding constants (KA, KD) were determined by direct non-linear
least squares analysis of the binding data using an equation
defining steady state equilibrium (KA*Conc*Rmax/(KA*Conc*n);
BIAevaluation 3.1). The data was also plotted in Scatchard format
(Req/nC versus Req, where Req is the biosensor response at
equilibrium, n is the valency and C is the concentration) (25).
[0083] Analytical Ultracentrifugation. Experiments were performed
using a Beckman XL-A analytical ultracentrifuge (Beckman Coulter,
Inc., Fullerton, Calif.) equipped with absorption optics, using an
An60-Ti rotor with cells containing quartz windows, as described
previously (23). Centrifugation experiments were conducted at
20.degree. C. using a sample volume of 100 .mu.l. Equilibrium
sedimentation distributions obtained at 12,000 and 20,000 rpm, were
monitored at 280 or 290 nm and analysed using the program SEDEQ1B
(26). The partial specific volume of EGF was taken as 0.71 ml/g
(23).
[0084] Chemical Cross Linking. Chemically cross-linked ErbB1501
dimers were generated by the incubation of sEGFR501 (5 .mu.M) with
mEGF (20 .mu.M) in 20 mM HEPES pH7.4 containing 150 mM NaCl for 1 h
at room temperature followed by the addition of
bis(sulfosuccinimidyl)suberate (BS3, Pierce, Rockford, Ill., USA)
to a final concentration of 0.5 mM and incubation for a further 30
min. The reaction was terminated by the addition of Tris-HCl buffer
(pH 7.5) to a final concentration of 10 mM. Monomer-dimer
separation was achieved on Novex non-reducing SDS-PAGE gels (10%).
Proteins were transferred onto poly(vinyl difluoride) (PVDF)
membranes (Bio-Rad, Hercules, Calif., USA) and identified by
incubation with anti-EGFR Mab528 (19) (0.5 .mu.g/ml) followed by
horseradish-peroxidase labelled goat anti-mouse IgG (Bio-Rad) and
ECL detection (Amersham Pharmacia Biotech).
[0085] Cell Proliferation Assays. BaF/3ERX cells, a cell line
derived from BaF/3 cells transfected with human EGFR (obtained from
Ludwig Institute for Cancer Research, Melbourne) were washed three
times to remove residual IL-3 and resuspended in RPMI 1640+10% FCS.
Cells were seeded into 96 well plates using a Biomek 2000 robotic
autosampler (Beckman) at 2.times.10.sup.4 cells per 200 .mu.l and
incubated for 4 h at 37.degree. C. in 10% CO.sub.2. Appropriate
concentrations of ErbB1501 or ErbB1621 or the anti-EGFR monoclonal
antibody Mab528, were added to the first titration point and
titrated in two-fold dilutions across the 96 well plate in
duplicate with or without a constant amount of mEGF (207 pM).
.sup.3H-Thymidine (0.5 .mu.Ci/well) was added and the plates were
incubated for 20 h at 37.degree. C. in 5% CO.sub.2. The cells were
then lysed in 0.5 M NaOH at room temperature for 30 min before
harvesting onto nitrocellulose filter mats using an automatic
harvester (Tomtec, Conn., USA). The mats were dried in a microwave,
placed in a plastic counting bag and scintillant (10 ml) was added.
.sup.3H-Thymidine incorporation was determined using an automated
beta counter (1205 Betaplate, Wallac, Finland).
[0086] Ligand binding assays. The wells of a 96-well Lumitrac 600
plate (Greiner) were coated with protein G (2 ug/ml in 10 mM sodium
citrate buffer, pH 9.6; Sigma). The wells were subsequently blocked
with 0.5% ovalbumin/Tris-buffered saline (TBS). Culture supernatant
from cell transfectants was added to the wells and incubated
overnight at 4.degree. C. to allow the binding of Fc fusion
proteins to protein G. Wells were washed and a cocktail of a fixed
concentration of Europium (Eu)-labelled ligand (EGF or HRG.beta.,
depending on the fusion protein), together with varying
concentrations of unlabelled competitor ligand, added to each well
in ligand-binding buffer. After incubation at room temperature,
wells were aspirated, washed in TBS plus 0.05% Tween-20, and
Enhancement Solution (Perkin-Elmer) added to each well for 20
minutes. Samples were then assayed for time-resolved fluorescence
(TRF) using a Wallac Victor2 1420 Multilabel Counter.
Example 1
Production and Purification of Truncated EGFR Ectodomains
[0087] Preliminary analysis of conditioned media from cells
transiently expressing ErbB1476, ErbB1501 and ErbB1513 showed that
only the latter two truncated receptors gave detectable binding to
hEGF immobilised on the BIAcore biosensor. Stably transfected Lec8
cells expressing ErbB1501, were generated and used to produce
truncated receptor protein at a yield of .about.1.8 mg/L of
fermentation medium for physical-chemical characterisation.
[0088] ErbB1501 purified from a Mab9E10 anti-c-myc peptide affinity
column using peptide elution showed a single symmetrical peak on
size exclusion chromatography (apparent molecular mass of .about.80
kDa) and migrated as a single band of .about.70 kDa on SDS-PAGE
under reducing conditions. ErbB1501 gave a unique expected
sequence, LEEKKVXQGT (13) on N-terminal amino acid sequence
analysis, the X at cycle 7 being due to the presence of a
disulphide-bonded cysteine residue at that position. The apparent
molecular mass of approximately 70 kDa on SDS-PAGE is due to the
residual glycosylation reported for the glycosylation defective Lec
8 cells (33) since the calculated mass of human ErbB1501
apo-protein is .about.57.5 kDa. There are eight potential N-linked
glycosylation sites in ErbB1501 (13) and incubation of ErbB1501
with peptide-N-glycosidase (PNG'ase) at 37.degree. C. resulted in
the generation of a major band migrating on SDS-PAGE with an
apparent molecular mass of 57-58 kDa (data not shown). We have
shown previously using BIAcore analysis that removal of
carbohydrate using PNGase does not affect binding of sEGFR621 to
the immobilised ligand, in agreement with the concept that
glycosylation is required for correct processing but not for
biological activity. All subsequent experiments were carried out
using the .about.70 kDa ErbB1501.
Example 2
Affinity Binding of sEGFR501
[0089] The BIAcore biosensor was used to determine both the rate
and equilibrium binding constants for the interaction between
ErbB1501 and hEGF or hTGF-.alpha.. Full length ectodomain
(ErbB1621) was used as a positive control for the surface
reactivity, since this interaction has been studied in detail
previously (23, 27).
[0090] Representative sensorgrams for the interaction between
ErbB1501 or ErbB1621 and hEGF or TGF-.alpha. are shown in FIG. 2.
Visual inspection revealed that the curves approached equilibrium
over the concentration ranges tested. Additionally, the
hTGF-.alpha. sensorgrams appeared to show more rapid, and virtually
complete, dissociation. Thermodynamic analysis of the equilibrium
binding data in Scatchard format (FIG. 3) indicated KD values of 30
and 47 nM (correlation coefficient R=0.993 and 0.999 respectively)
for the interactions between ErbB1501 and immobilised hEGF or
hTGF-.alpha. and 412 and 961 nM (R=0.997 and 0.999 respectively)
for the corresponding interactions with ErbB1621. The values
obtained by Scatchard transformation were also confirmed by direct
non-linear least squares analysis of the binding data (data not
shown) using an equation defining steady state equilibrium
(KA*Conc*Rmax/(KA*Conc*n); BIAevaluation 3.1). Using this analysis,
KD values of 32 and 46 nM were calculated for the interaction
between ErbB1501 and immobilised hEGF and hTGF-.alpha. respectively
and 570 and 959 nM for the interaction between full-length
ectodomain (ErbB1621) and immobilised hEGF and hTGF-.alpha.. The
values obtained with ErbB1621 were in good agreement with those
reported previously (23), confirming the surface viability.
[0091] The individual rate constants were determined from those
parts of the curves where first order kinetics appeared to be
operative (27, 28), and the corresponding dissociation constants
calculated (Table 1). Again, there was good agreement between the
KD values calculated in this manner and those obtained from the
equilibrium binding data. It is interesting to note that the
binding curves obtained with both ErbB1501 and ErbB1621 for
hTGF-.alpha. appeared to be better fitted to a 1:1 model than the
corresponding data for the hEGF surface (as suggested by the
virtually complete dissociation).
TABLE-US-00002 TABLE 1 Comparative kinetic data for ligand binding
by truncated and full-length EGFR ectodomains. Interaction k.sub.a
(M.sup.-1s.sup.-1) .times. 10.sup.-5 k.sub.d (s.sup.-1) K.sub.D
(nM) ErbB1501/EGF 10-17 0.02 13-21 ErbB1501/TGF-.alpha. 9.3-10.5
0.04 35-40 ErbB1621/EGF 2.9-4.8 0.08 180-300 ErbB1621/TGF-.alpha.
0.7-1.0 0.08 840-1320
Example 3
Antagonist Activity of ErbB1501
[0092] The observation that ErbB1501 bound EGF with high affinity
prompted us to test whether ErbB1501 would act as a competitive
inhibitor for the mitogenic stimulation of EGFR in a cell-based
assay using the BaF/3ERX cell line. This cell line responds to mEGF
with an EC50 of approximately 30 pM (FIG. 4A). The competition
assay (FIG. 4B) used a constant concentration of mEGF (207 pM),
which causes maximal stimulation (FIG. 4A), and varying levels
(0.00045-0.5 .mu.M) of ErbB1501, ErbB1621 or the neutralising
anti-EGFR monoclonal antibody Mab528 raised against epidermal
growth factor receptors on a human epidermoid carcinoma cell line,
A431 (19). This antibody has been shown to prevent the growth of
A431 cell xenografts, bearing high numbers of EGF receptors, in
nude mice. The ErbB1501 (IC50=0.02 .mu.M) was almost 10 fold more
potent than the full-length ectodomain (IC50=0.15 .mu.M) and
approximately 3-fold more potent than the Mab528 anti-EGFR
monoclonal antibody (IC50=0.06 .mu.M).
Example 4
Dimerisation of sEGFR501
[0093] Chemical cross-linking revealed that ErbB1501 formed dimeric
complexes in the presence of ligand. In the presence of 20 .mu.M
mEGF, a single high molecular weight species (apparent Mr 180,000
Da) was formed after chemical cross-linking which was not
detectable when the cross-linking was attempted in the absence of
ligand (FIG. 5). Western blotting was employed to confirm the
authenticity of the bands observed, but similar data were obtained
with silver or Coomassie blue staining. In addition, size exclusion
chromatographic analysis of the reaction mixture, using a TSK
G2000SW column developed with a mobile phase of PBS at a flow rate
of 0.25 ml/min, showed a peak of apparent Mr 158,000 which
corresponded to dimer (data not shown). Similar results have
previously been obtained with ErbB1621.
[0094] Analytical ultracentrifugation showed that the EGF binding
sites on ErbB1501 were saturated at an equimolar ratio of ligand
and receptor leading to the formation of a 2:2 EGF/ErbB1501 complex
(FIG. 6). The data for 20 .TM. EGF alone (FIG. 6A) indicate a
single solute of molecular weight 5,980 Da, in good agreement with
the value calculated from the amino acid composition (6,040 Da).
The molecular weight (65,600 Da) and partial specific volume (0.71
ml/g) determined for 10 .TM. ErbB1501 alone was calculated from the
sedimentation equilibrium distribution (FIG. 6A) and is based on
the known amino acid composition and a calculated value of 12%
(w/w) for the carbohydrate composition.
[0095] Sedimentation equilibrium data for a mixture of EGF (20
.mu.M) and ErbB1501 (10 .mu.M) was analyzed assuming two species
(FIG. 6A). The molecular weight of the first species was fixed at
the value obtained for free EGF (6,000 Da) with the molecular
weight and weight fraction of the second species used as fitting
parameters. Under these conditions the molecular weight of the
second species provides a good approximation to the weight-average
molecular weight of ErbB1501 and its complexes. The best-fit value
showed a complex of weight-average MW 106,400 Da, higher than
predicted for a 1:1 complex (71,600 Da) and more consistent with
the formation of a significant proportion of dimeric 2:2
EGF/ErbB1501 complex (see below).
[0096] High-speed meniscus depletion experiments were performed to
determine the molar ratio required for saturation of ErbB1501 with
EGF (FIG. 6B). A solution of ErbB1501 (5 .mu.M) was titrated with
EGF to determine the molar ratio at which free EGF is detectable at
the meniscus. The results show that this occurs above 5 .mu.M EGF,
implying an equimolar ratio is required for saturation of the EGF
binding site(s) on ErbB1501. These data, taken together with the
observed weight average molecular weight of the EGF/ErbB1501
complex obtained from the equilibrium analysis (FIG. 6A), confirm
that the stoichiometry of the EGF/ErbB1501 dimeric species is 2:2
not 2:1.
[0097] Sedimentation equilibrium was used for the analysis of data
obtained for ErbB1501 (5 .mu.M) in the presence of a range of EGF
concentrations (FIG. 6C). The weight average molecular weight
obtained for the "second" species increases rapidly as the ratio of
EGF/ErbB1501 is increased to 1:1 and then tends to plateau around
approximately 108,000 at ratios above 2:1 (FIG. 6C). The data in
FIG. 6A could also be fitted assuming a mixture of 1:1 and 2:2
complexes with weight fractions of the monomeric and dimeric
ErbB1501 complexes of 57% and 31% respectively. Similar data was
obtained with ErbB1621 (23).
Example 5
ErbB1501-441 Mutant Binding Studies
[0098] Biosensor analysis was also used to analyse the binding of
the transiently expressed sEGFR501 mutants to both immobilised hEGF
and hTGF-.alpha. surfaces. The presence of the mutant proteins in
culture supernatants from transfected cell lines was demonstrated
by both immunoblotting with the anti-EGFR monoclonal antibody, Mab
528, and biosensor analysis using Mab 528 immobilised on the
surface. Culture supernatants from all cell lines showed
demonstrable binding to the Mab surface (441>472=wt>367).
[0099] In preliminary experiments, the Glu367Lys mutant and the
Glu472Lys mutant showed similar binding characteristics to sEGFR501
when passed over the hEGF sensor surface. The Gly441 Lys mutant
showed much reduced binding, even though the Mab528 surface had
indicated that the Gly441 Lys mutant was present at higher
concentrations than sEGFR501. Interestingly, when the same samples
were passed over the parallel hTGF-.alpha. sensor surface the
Gly441 Lys mutant now showed the highest binding, whilst the
binding of the Glu367Lys mutant the Glu472Lys mutant and wild type
ErbB1501 were again similar but lower.
[0100] For full biosensor analysis the mutant proteins present in
the conditioned media from transient transfected 293T fibroblasts
were concentrated and purified by a combination of affinity
purification using the 9E10 monoclonal antibody and size exclusion
chromatography on Superdex 200 and Superose 12. The sensorgrams
obtained with the immobilised hEGF and hTGF-.alpha. surfaces (160
and 132 RU immobilised respectively) are shown in FIGS. 7A, 7B. As
we had observed in the preliminary experiments, whilst the binding
characteristics of the Glu367Lys and Glu472Lys mutants were
essentially undistinguishable from those of ErbB1501 shown in FIG.
2 the Gly441Lys mutant again showed preferential binding to the
hTGF-.alpha. surface (FIGS. 7A, 7B). Scatchard analysis of the
equilibrium binding data (FIGS. 7C, 7D) indicated that whilst
binding to the TGF-.alpha. surface was similar to that observed
with ErbB1501 (KD=77 nM, correlation coefficient R=0.999), the
reactivity of the Gly441Lys mutant towards the EGF surface was now
considerably reduced (KD=455 nM, R=0.995). Similar values (78 nM
and 469 nM) were obtained by direct non-linear least squares
analysis of the binding data using the equation defining steady
state equilibrium.
[0101] Kinetic analysis of the binding data (Table 2) indicated
that the interaction with the immobilised TGF-.alpha. could be
described by an association rate constant (ka) of
5.2-6.9.times.10-5M-1 s-1 and a dissociation rate constant (kd) of
0.025s-1 giving a KD=kd/ka of 36-44 nM. The corresponding
interaction with EGF was described by a ka of 1.9-2.3.times.10-5M-1
s-1 and a significantly faster kd of 0.103 s-1 giving a KD=kd/ka of
442-545 nM, in good agreement with the results observed from the
thermodynamic analysis.
TABLE-US-00003 TABLE 2 Kinetic analysis of the binding of Gly441Lys
sErbB1501 to immobilised hEGF and hTGF-.alpha.. Interaction k.sub.a
(M.sup.-1s.sup.-1) .times. 10.sup.-5 k.sub.d (s.sup.-1) K.sub.D
(nM) TGF-.alpha. 5.2-6.9 0.025 36-48 EGF 1.9-2.3 0.103 442-545
Example 6
ErbB1501.Fc Fusion Protein--Ligand Binding Studies
[0102] A previous published study had demonstrated that the
full-length extracellular domain of ErbB1, expressed as an Fc
fusion protein homodimer, bound a number of EGF ligands with IC50
values ranging from 1-10 nM (29), while the corresponding
heterodimer with ErbB2 showed at best a two-fold increase in
affinity. Using a monolabelled Eu-EGF (Wallac) as trace, we show
that the truncated form of the extracellular domain of the EGF
receptor, expressed as an Fc fusion homodimer, binds EGF ligand
with 10-fold higher affinity than that reported for the full-length
ErbB1 ectodomain-Fc fusion protein (30) (see FIG. 8).
Example 7
ErbB3500.Fc and ErbB4497.Fc Fusion Proteins--Ligand Binding
Studies
[0103] ErbB3 and ErbB4 Fc constructs, incorporating the
corresponding EGF receptor domains used in ErbB1 501.Fc, were
assembled and analysed following transient transfection. In
preliminary studies, the full-length erbB4 ectodomain Fc fusion
protein, expressed as a homodimer and with ErbB2 ectodomain Fc
fusion protein as a heterodimer, was used to validate the data of
Fitzpatrick et. al. (30). As demonstrated in FIG. 9, the
full-length receptor Fc homodimer exhibits an IC.sub.50 value for
heregulin .beta. (HRG.beta.) of 18 nM, which is in good agreement
with that previously reported (5.1 nM; (29)) allowing for
differences in trace ligand preparation. We also find that
co-expression of ErbB2 has dramatic affect on the IC.sub.50 value
for ligand, resulting in an increase by two orders of magnitude.
Jones et. al. (29) report an increase in IC.sub.50 of 200-fold
using the heterodimer receptors; again, this probably reflects
differences in assay format.
[0104] The IC.sub.50 values obtained using the truncated forms of
ErbB3 and ErbB4 as homodimer Fc fusion proteins are shown in FIG.
10. While ErbB3 500. Fc has an IC.sub.50 of 0.49 nM for HRG.beta.,
the value obtained for ErbB4 497. Fc (0.057 nM) approaches that
obtained for the full-length ectodomain ErbB2/4 Fc heterodimer
reported by Genentech (0.025 nM; (30)).
Discussion
[0105] The characteristics of truncated versions of EGFR
ectodomains (ErbB1 501, ErbB3 500 and ErbB4 497) that bind hEGF,
TGF-.alpha. (ErbB1 501) and HRG.beta. (ErbB3 500 and ErbB4 497)
with high affinity are described herein. The KD values of 13-21 nM
for hEGF binding to ErbB1501 are similar to those, (15-30 nM), seen
with chemically cross-linked dimers of full-length EGFR ectodomain
and are 10 to 25-fold higher than the values generally reported for
soluble, full-length EGFR ectodomain derived from either A431
tumour cells, transfected Sf9 insect cells or CHO cells.
[0106] ErbB1501, which lacks most of (6 of 7 modules of) CR2,
exhibits ligand-induced receptor dimerisation indicating that the
regions responsible for dimerisation are unlikely to include CR2.
It also confirms that membrane anchoring is not required for the
generation of high affinity dimers in contrast to the situation
with ErbB2/ErbB3 heterodimers and neuregulin. The
ultracentrifugation analyses showed that the binding sites on ErbB1
501 were saturated, and the extent of dimerisation began to
plateau, at molar ratios greater than of 1:1 (FIG. 5C), even at the
relatively low concentration of ErbB1 501 of 5M (320 .mu.g/ml).
This compares favourably with the small angle X-ray scattering data
and our previous analytical ultracentrifugation analyses that
showed that ErbB1 621 dimerisation, induced by EGF or TGF-.alpha.
binding, reached a maximum when the ratio of EGF/ErbB1 was 1:1.
[0107] It is envisaged that the truncated constructs of the present
invention will have therapeutic potential given their high affinity
for ligand and their ability to competitively inhibit EGF-induced
proliferation responses in a model cell system. The inhibition
shown by ErbB1 501, for example, was greater than that achieved in
the same assay with a neutralising monoclonal antibody raised
against the receptor (Mab528), chimeric forms of which (C225) are
currently in clinical trials.
[0108] ErbB1 501 was also employed to investigate the residue
responsible for the differential binding between hTGF-.alpha. and
hEGF observed with chicken EGFR (9). These data demonstrate that
the Lys442 in chicken EGFR, which corresponds to Gly441 in hEGFR,
is the residue responsible for discriminating between hTGF-.alpha.
and hEGF binding.
[0109] A number of studies have described the ligand-binding
characteristics of isoforms of the ectodomains of different EGF
receptor family members, either alone or as Fc fusion proteins.
Full-length ectodomain homodimers of ErbB3 and ErbB4, fused to the
human IgG1 Fc domain, bind their respective ligands with a range of
affinities (IC.sub.50 values 1-1000 nM;). However,
heterodimerisation with the corresponding ErbB2 ectodomain Fc
fusion protein is required before very high affinity binding (<1
nM) of the sort observed for cell-surface heterodimers is achieved
(30). Our present studies indicate that truncated ectodomain
fragments of ErbB1, 3 and 4, expressed as Fc fusion protein
homodimers, bind ligand with very high affinity (<1 nM). In the
example of ErbB4, the affinity approaches that reported for the
heterodimer of the full length ectodomain Fc fusion proteins of
ErbB2 and ErbB4 (30).
[0110] 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
apparent to those skilled in molecular biology or related fields
are intended to be within the scope of the invention.
REFERENCES
[0111] 1. Burgess A W and Thumwood C M. (1994) Pathology 26,
453-463 [0112] 2. Bajaj, M., Waterfield, M. D., Schlessinger, J.,
Taylor, W. R. and Blundell, T. (1987) Biochim. Biophys. Acta. 916,
220-226 [0113] 3. Ward, C. W., Hoyne, P. A. and Flegg, R. H. (1995)
Proteins-Struct. Funct. Genet. 22, 141-153 [0114] 4. Lax, I.,
Johnson, A., Howk, R., Sap, J., Bellot, F., Winkler, M., Ullrich,
A., Vennstrom, B., Schlessinger, J., and Givol, D. (1988) Molec.
Cellul. Biol. 8, 1970-1978 [0115] 5. Sandgreen, E. P., Luettke, N.
C., Palmiter, R. D., Brinster, R. L., Lee, D. C. (1990) Cell 61,
1121-1135 [0116] 6. Hines, N. E. (1993) Semin. Cancer Biol. 4,
19-26 [0117] 7. Stanley, P. (1989) Molec. Cellul. Biol. 9, 377-383
[0118] 8. Saxon, M. L. and Lee, D. C. (1999) J. Biol. Chem. 274,
28356-28362 [0119] 9. Kohda, D., Odaka, M., Lax, I., Kawasaki, H.,
Suzuki, K., Ullrich, A., Schlessinger, J. and Inagaki, F. (1993) J.
Biol. Chem. 268, 1976-1981 [0120] 10. Souriau, C., Fort, P., Roux,
P., Hartley, O., Lefranc, M-P., Weill, M., 1997, Nucleic Acids Res.
25:1585-1590 [0121] 11. Rockwell, P., O'Connor, W. J., King, K.,
Goldstein, N. I., Zhang, L. M., Stein, C. A., 1997, Proc Natl Acad
Sci USA 94:6523-6528; [0122] 12. Prewett, M., Rothman, M., Waksal,
H., Feldman, M., Bander, N. H., Hicklin, D. J., 1998 Clin Cancer
Res 4:2957-2966 [0123] 13. Ullrich, A., Coussens, L., Hayflick, J.
S., Dull, T. J., Gray, A., Tam, A. W., Lee, J., Yarden, Y.,
Libermann, T. A., Schlessinger, J., Downard, J., Mayes, E. L. V.,
Whittle, N., Waterfield, M. D. and Seeburg, P. H. (1984) Nature
309, 418-425 [0124] 14. Bebbington, C. R. and Hentschel, C. C. G.
(1987) in DNA Cloning (Glover, D., ed.), Vol. III, pp. 163-188, IRL
Press, Oxford, U. K. [0125] 15. McKern, N. M., Lou, M., Frenkel, M.
J., Verkuylen, A., Bentley, J. D., Lovrecz, G. O., Ivancic, N.,
Elleman, T. C., Garrett, T. P. J., Cosgrove L. & Ward, C. W.
(1997) Protein Sci. 6, 2663-2666 [0126] 16 Norrander, J., Kempe, T.
and Messing, J. (1983) Gene 26, 101-106 [0127] 17. Carter, P.
(1987) Methods Enzymol. 154, 382-403 [0128] 18. Sanger, F.,
Nicklen, S, and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U.S.A.
74, 5463-5467 [0129] 19. Gill, G. N., Kawamoto, T., Cochet, C., Le,
A., Sato, J. D., Masui, H., McLeod, C., and Mendelsohn, J. (1984)
J. Biol. Chem. 259, 7755-7760 [0130] 20. Nice, E., Lackmann, M.,
Smyth, F., Fabri, L., Burgess, A. W. (1994) J. Chromatogr. A 660,
169-185 [0131] 21. Catimel B, Domagala T, Nerrie M, Weinstock J,
White S, Abud H, Heath J and Nice E, (1999) Prot. Pept. Lett. 6,
319-340 [0132] 22. Catimel, B., Nerrie, M., Lee, F. T., Scott, A.
M., Ritter, G., Welt, S., Old, L. J., Burgess, A. W. and Nice, E.
C. (1997) J. Chromatogr. 776, 15-30. [0133] 23. Domagala, T.,
Konstantopoulos, N., Smyth, F., Jorissen, R. N., Fabri, L.,
Geleick, D., Lax, I., Schlessinger, J., Sawyer, W., Howlett, G. J.,
Burgess, A. W. and Nice, E. C. (2000) Growth Factors 18, 11-29
[0134] 24. Morton, T. A. and Myszka, D. G. (1998) Methods Enzymol.
295, 268-294. [0135] 25. Hammacher, A., Simpson, R. J. and Nice, E.
C. (1996) 271, 5464-5473 [0136] 26. Minton, A. P. (1994) In Modern
Analytical Ultracentrifugation: Acquisition and Interpretation of
Data for Biological and Synhtetic Polymer systems. Schuster, T. M.
and Laue, T. M. eds, Birkhauser, Boston, p 81 [0137] 27. De
Crescenzo, G., Grothe, S., Lortie, R., Debanne, M. T. and
O'Connor-McCourt M. (2000) Biochemistry, 9466-9476 [0138] 28. Nice
E. C. and Catimel B. (1999) BioEssays 21, 339-352 [0139] 29. Jones,
J. T. et. al. (1999) FEBS Letters 447:227-231 [0140] 30.
Fitzpatrick, V. D. et. al. (1998) FEBS Letters 431:102-106
Sequence CWU 1
1
41621PRTHomo sapiens 1Leu Glu Glu Lys Lys Val Cys Gln Gly Thr Ser
Asn Lys Leu Thr Gln1 5 10 15Leu Gly Thr Phe Glu Asp His Phe Leu Ser
Leu Gln Arg Met Phe Asn 20 25 30Asn Cys Glu Val Val Leu Gly Asn Leu
Glu Ile Thr Tyr Val Gln Arg 35 40 45Asn Tyr Asp Leu Ser Phe Leu Lys
Thr Ile Gln Glu Val Ala Gly Tyr 50 55 60Val Leu Ile Ala Leu Asn Thr
Val Glu Arg Ile Pro Leu Glu Asn Leu65 70 75 80Gln Ile Ile Arg Gly
Asn Met Tyr Tyr Glu Asn Ser Tyr Ala Leu Ala 85 90 95Val Leu Ser Asn
Tyr Asp Ala Asn Lys Thr Gly Leu Lys Glu Leu Pro 100 105 110Met Arg
Asn Leu Gln Glu Ile Leu His Gly Ala Val Arg Phe Ser Asn 115 120
125Asn Pro Ala Leu Cys Asn Val Glu Ser Ile Gln Trp Arg Asp Ile Val
130 135 140Ser Ser Asp Phe Leu Ser Asn Met Ser Met Asp Phe Gln Asn
His Leu145 150 155 160Gly Ser Cys Gln Lys Cys Asp Pro Ser Cys Pro
Asn Gly Ser Cys Trp 165 170 175Gly Ala Gly Glu Glu Asn Cys Gln Lys
Leu Thr Lys Ile Ile Cys Ala 180 185 190Gln Gln Cys Ser Gly Arg Cys
Arg Gly Lys Ser Pro Ser Asp Cys Cys 195 200 205His Asn Gln Cys Ala
Ala Gly Cys Thr Gly Pro Arg Glu Ser Asp Cys 210 215 220Leu Val Cys
Arg Lys Phe Arg Asp Glu Ala Thr Cys Lys Asp Thr Cys225 230 235
240Pro Pro Leu Met Leu Tyr Asn Pro Thr Thr Tyr Gln Met Asp Val Asn
245 250 255Pro Glu Gly Lys Tyr Ser Phe Gly Ala Thr Cys Val Lys Lys
Cys Pro 260 265 270Arg Asn Tyr Val Val Thr Asp His Gly Ser Cys Val
Arg Ala Cys Gly 275 280 285Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly
Val Arg Lys Cys Lys Lys 290 295 300Cys Glu Gly Pro Cys Arg Lys Val
Cys Asn Gly Ile Gly Ile Gly Glu305 310 315 320Phe Lys Asp Ser Leu
Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys 325 330 335Asn Cys Thr
Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe 340 345 350Arg
Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu 355 360
365Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln
370 375 380Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn
Leu Glu385 390 395 400Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln
Phe Ser Leu Ala Val 405 410 415Val Ser Leu Asn Ile Thr Ser Leu Gly
Leu Arg Ser Leu Lys Glu Ile 420 425 430Ser Asp Gly Asp Val Ile Ile
Ser Gly Asn Lys Asn Leu Cys Tyr Ala 435 440 445Asn Thr Ile Asn Trp
Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr 450 455 460Lys Ile Ile
Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln465 470 475
480Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro
485 490 495Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu
Cys Val 500 505 510Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu
Phe Val Glu Asn 515 520 525Ser Glu Cys Ile Gln Cys His Pro Glu Cys
Leu Pro Gln Ala Met Asn 530 535 540Ile Thr Cys Thr Gly Arg Gly Pro
Asp Asn Cys Ile Gln Cys Ala His545 550 555 560Tyr Ile Asp Gly Pro
His Cys Val Lys Thr Cys Pro Ala Gly Val Met 565 570 575Gly Glu Asn
Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val 580 585 590Cys
His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly 595 600
605Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser 610 615
6202631PRTHomo sapiens 2Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys
Leu Arg Leu Pro Ala1 5 10 15Ser Pro Glu Thr His Leu Asp Met Leu Arg
His Leu Tyr Gln Gly Cys 20 25 30Gln Val Val Gln Gly Asn Leu Glu Leu
Thr Tyr Leu Pro Thr Asn Ala 35 40 45Ser Leu Ser Phe Leu Gln Asp Ile
Gln Glu Val Gln Gly Tyr Val Leu 50 55 60Ile Ala His Asn Gln Val Arg
Gln Val Pro Leu Gln Arg Leu Arg Ile65 70 75 80Val Arg Gly Thr Gln
Leu Phe Glu Asp Asn Tyr Ala Leu Ala Val Leu 85 90 95Asp Asn Gly Asp
Pro Leu Asn Asn Thr Thr Pro Val Thr Gly Ala Ser 100 105 110Pro Gly
Gly Leu Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu Ile Leu 115 120
125Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr Gln Asp
130 135 140Thr Ile Leu Trp Lys Asp Ile Phe His Lys Asn Asn Gln Leu
Ala Leu145 150 155 160Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys
His Pro Cys Ser Pro 165 170 175Met Cys Lys Gly Ser Arg Cys Trp Gly
Glu Ser Ser Glu Asp Cys Gln 180 185 190Ser Leu Thr Arg Thr Val Cys
Ala Gly Gly Cys Ala Arg Cys Lys Gly 195 200 205Pro Leu Pro Thr Asp
Cys Cys His Glu Gln Cys Ala Ala Gly Cys Thr 210 215 220Gly Pro Lys
His Ser Asp Cys Leu Ala Cys Leu His Phe Asn His Ser225 230 235
240Gly Ile Cys Glu Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp
245 250 255Thr Phe Glu Ser Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe
Gly Ala 260 265 270Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu Ser
Thr Asp Val Gly 275 280 285Ser Cys Thr Leu Val Cys Pro Leu His Asn
Gln Glu Val Thr Ala Glu 290 295 300Asp Gly Thr Gln Arg Cys Glu Lys
Cys Ser Lys Pro Cys Ala Arg Val305 310 315 320Cys Tyr Gly Leu Gly
Met Glu His Leu Arg Glu Val Arg Ala Val Thr 325 330 335Ser Ala Asn
Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe Gly Ser 340 345 350Leu
Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr 355 360
365Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe Glu Thr Leu Glu Glu
370 375 380Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro Asp Ser Leu
Pro Asp385 390 395 400Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg
Gly Arg Ile Leu His 405 410 415Asn Gly Ala Tyr Ser Leu Thr Leu Gln
Gly Leu Gly Ile Ser Trp Leu 420 425 430Gly Leu Arg Ser Leu Arg Glu
Leu Gly Ser Gly Leu Ala Leu Ile His 435 440 445His Asn Thr His Leu
Cys Phe Val His Thr Val Pro Trp Asp Gln Leu 450 455 460Phe Arg Asn
Pro His Gln Ala Leu Leu His Thr Ala Asn Arg Pro Glu465 470 475
480Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His Gln Leu Cys Ala Arg
485 490 495Gly His Cys Trp Gly Pro Gly Pro Thr Gln Cys Val Asn Cys
Ser Gln 500 505 510Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys Arg
Val Leu Gln Gly 515 520 525Leu Pro Arg Glu Tyr Val Asn Ala Arg His
Cys Leu Pro Cys His Pro 530 535 540Glu Cys Gln Pro Gln Asn Gly Ser
Val Thr Cys Phe Gly Pro Glu Ala545 550 555 560Asp Gln Cys Val Ala
Cys Ala His Tyr Lys Asp Pro Pro Phe Cys Val 565 570 575Ala Arg Cys
Pro Ser Gly Val Lys Pro Asp Leu Ser Tyr Met Pro Ile 580 585 590Trp
Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln Pro Cys Pro Ile Asn 595 600
605Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys Gly Cys Pro Ala Glu
610 615 620Gln Arg Ala Ser Pro Leu Thr625 6303624PRTHomo sapiens
3Ser Glu Val Gly Asn Ser Gln Ala Val Cys Pro Gly Thr Leu Asn Gly1 5
10 15Leu Ser Val Thr Gly Asp Ala Glu Asn Gln Tyr Gln Thr Leu Tyr
Lys 20 25 30Leu Tyr Glu Arg Cys Glu Val Val Met Gly Asn Leu Glu Ile
Val Leu 35 40 45Thr Gly His Asn Ala Asp Leu Ser Phe Leu Gln Trp Ile
Arg Glu Val 50 55 60Thr Gly Tyr Val Leu Val Ala Met Asn Glu Phe Ser
Thr Leu Pro Leu65 70 75 80Pro Asn Leu Arg Val Val Arg Gly Thr Gln
Val Tyr Asp Gly Lys Phe 85 90 95Ala Ile Phe Val Met Leu Asn Tyr Asn
Thr Asn Ser Ser His Ala Leu 100 105 110Arg Gln Leu Arg Leu Thr Gln
Leu Thr Glu Ile Leu Ser Gly Gly Val 115 120 125Tyr Ile Glu Lys Asn
Asp Lys Leu Cys His Met Asp Thr Ile Asp Trp 130 135 140Arg Asp Ile
Val Arg Asp Arg Asp Ala Glu Ile Val Val Lys Asp Asn145 150 155
160Gly Arg Ser Cys Pro Pro Cys His Glu Val Cys Lys Gly Arg Cys Trp
165 170 175Gly Pro Gly Ser Glu Asp Cys Gln Thr Leu Thr Lys Thr Ile
Cys Ala 180 185 190Pro Gln Cys Asn Gly His Cys Phe Gly Pro Asn Pro
Asn Gln Cys Cys 195 200 205His Asp Glu Cys Ala Gly Gly Cys Ser Gly
Pro Gln Asp Thr Asp Cys 210 215 220Phe Ala Cys Arg His Phe Asn Asp
Ser Gly Ala Cys Val Pro Arg Cys225 230 235 240Pro Gln Pro Leu Val
Tyr Asn Lys Leu Thr Phe Gln Leu Glu Pro Asn 245 250 255Pro His Thr
Lys Tyr Gln Tyr Gly Gly Val Cys Val Ala Ser Cys Pro 260 265 270His
Asn Phe Val Val Asp Gln Thr Ser Cys Val Arg Ala Cys Pro Pro 275 280
285Asp Lys Met Glu Val Asp Lys Asn Gly Leu Lys Met Cys Glu Pro Cys
290 295 300Gly Gly Leu Cys Pro Lys Ala Cys Glu Gly Thr Gly Ser Gly
Ser Arg305 310 315 320Phe Gln Thr Val Asp Ser Ser Asn Ile Asp Gly
Phe Val Asn Cys Thr 325 330 335Lys Ile Leu Gly Asn Leu Asp Phe Leu
Ile Thr Gly Leu Asn Gly Asp 340 345 350Pro Trp His Lys Ile Pro Ala
Leu Asp Pro Glu Lys Leu Asn Val Phe 355 360 365Arg Thr Val Arg Glu
Ile Thr Gly Tyr Leu Asn Ile Gln Ser Trp Pro 370 375 380Pro His Met
His Asn Phe Ser Val Phe Ser Asn Leu Thr Thr Ile Gly385 390 395
400Gly Arg Ser Leu Tyr Asn Arg Gly Phe Ser Leu Leu Ile Met Lys Asn
405 410 415Leu Asn Val Thr Ser Leu Gly Phe Arg Ser Leu Lys Glu Ile
Ser Ala 420 425 430Gly Arg Ile Tyr Ile Ser Ala Asn Arg Gln Leu Cys
Tyr His His Ser 435 440 445Leu Asn Trp Thr Lys Val Leu Arg Gly Pro
Thr Glu Glu Arg Leu Asp 450 455 460Ile Lys His Asn Arg Pro Arg Arg
Asp Cys Val Ala Glu Gly Lys Val465 470 475 480Cys Asp Pro Leu Cys
Ser Ser Gly Gly Cys Trp Gly Pro Gly Pro Gly 485 490 495Gln Cys Leu
Ser Cys Arg Asn Tyr Ser Arg Gly Gly Val Cys Val Thr 500 505 510His
Cys Asn Phe Leu Asn Gly Glu Pro Arg Glu Phe Ala His Glu Ala 515 520
525Glu Cys Phe Ser Cys His Pro Glu Cys Gln Pro Met Glu Gly Thr Ala
530 535 540Thr Cys Asn Gly Ser Gly Ser Asp Thr Cys Ala Gln Cys Ala
His Phe545 550 555 560Arg Asp Gly Pro His Cys Val Ser Ser Cys Pro
His Gly Val Leu Gly 565 570 575Ala Lys Gly Pro Ile Tyr Lys Tyr Pro
Asp Val Gln Asn Glu Cys Arg 580 585 590Pro Cys His Glu Asn Cys Thr
Gln Gly Cys Lys Gly Pro Glu Leu Gln 595 600 605Asp Cys Leu Gly Gln
Thr Leu Val Leu Ile Gly Lys Thr His Leu Thr 610 615 6204626PRTHomo
sapiens 4Gln Ser Val Cys Ala Gly Thr Glu Asn Lys Leu Ser Ser Leu
Ser Asp1 5 10 15Leu Glu Gln Gln Tyr Arg Ala Leu Arg Lys Tyr Tyr Glu
Asn Cys Glu 20 25 30Val Val Met Gly Asn Leu Glu Ile Thr Ser Ile Glu
His Asn Arg Asp 35 40 45Leu Ser Phe Leu Arg Ser Val Arg Glu Val Thr
Gly Tyr Val Leu Val 50 55 60Ala Leu Asn Gln Phe Arg Tyr Leu Pro Leu
Glu Asn Leu Arg Ile Ile65 70 75 80Arg Gly Thr Lys Leu Tyr Glu Asp
Arg Tyr Ala Leu Ala Ile Phe Leu 85 90 95Asn Tyr Arg Lys Asp Gly Asn
Phe Gly Leu Gln Glu Leu Gly Leu Lys 100 105 110Asn Leu Thr Glu Ile
Leu Asn Gly Gly Val Tyr Val Asp Gln Asn Lys 115 120 125Phe Leu Cys
Tyr Ala Asp Thr Ile His Trp Gln Asp Ile Val Arg Asn 130 135 140Pro
Trp Pro Ser Asn Leu Thr Leu Val Ser Thr Asn Gly Ser Ser Gly145 150
155 160Cys Gly Arg Cys His Lys Ser Cys Thr Gly Arg Cys Trp Gly Pro
Thr 165 170 175Glu Asn His Cys Gln Thr Leu Thr Arg Thr Val Cys Ala
Glu Gln Cys 180 185 190Asp Gly Arg Cys Tyr Gly Pro Tyr Val Ser Asp
Cys Cys His Arg Glu 195 200 205Cys Ala Gly Gly Cys Ser Gly Pro Lys
Asp Thr Asp Cys Phe Ala Cys 210 215 220Met Asn Phe Asn Asp Ser Gly
Ala Cys Val Thr Gln Cys Pro Gln Thr225 230 235 240Phe Val Tyr Asn
Pro Thr Thr Phe Gln Leu Glu His Asn Phe Asn Ala 245 250 255Lys Tyr
Thr Tyr Gly Ala Phe Cys Val Lys Lys Cys Pro His Asn Phe 260 265
270Val Val Asp Ser Ser Ser Cys Val Arg Ala Cys Pro Ser Ser Lys Met
275 280 285Glu Val Glu Glu Asn Gly Ile Lys Met Cys Lys Pro Cys Thr
Asp Ile 290 295 300Cys Pro Lys Ala Cys Asp Gly Ile Gly Thr Gly Ser
Leu Met Ser Ala305 310 315 320Gln Thr Val Asp Ser Ser Asn Ile Asp
Lys Phe Ile Asn Cys Thr Lys 325 330 335Ile Asn Gly Asn Leu Ile Phe
Leu Val Thr Gly Ile His Gly Asp Pro 340 345 350Tyr Asn Ala Ile Glu
Ala Ile Asp Pro Glu Lys Leu Asn Val Phe Arg 355 360 365Thr Val Arg
Glu Ile Thr Gly Phe Leu Asn Ile Gln Ser Trp Pro Pro 370 375 380Asn
Met Thr Asp Phe Ser Val Phe Ser Asn Leu Val Thr Ile Gly Gly385 390
395 400Arg Val Leu Tyr Ser Gly Leu Ser Leu Leu Ile Leu Lys Gln Gln
Gly 405 410 415Ile Thr Ser Leu Gln Phe Gln Ser Leu Lys Glu Ile Ser
Ala Gly Asn 420 425 430Ile Tyr Ile Thr Asp Asn Ser Asn Leu Cys Tyr
Tyr His Thr Ile Asn 435 440 445Trp Thr Thr Leu Phe Ser Thr Ile Asn
Gln Arg Ile Val Ile Arg Asp 450 455 460Asn Arg Lys Ala Glu Asn Cys
Thr Ala Glu Gly Met Val Cys Asn His465 470 475 480Leu Cys Ser Ser
Asp Gly Cys Trp Gly Pro Gly Pro Asp Gln Cys Leu 485 490 495Ser Cys
Arg Arg Phe Ser Arg Gly Arg Ile Cys Ile Glu Ser Cys Asn 500 505
510Leu Tyr Asp Gly Glu Phe Arg Glu Phe Glu Asn Gly Ser Ile Cys Val
515 520 525Glu Cys Asp Pro Gln Cys Glu Lys Met Glu Asp Gly Leu Leu
Thr Cys 530 535 540His Gly Pro Gly Pro Asp Asn Cys Thr Lys Cys Ser
His Phe Lys Asp545 550 555 560Gly Pro Asn Cys Val Glu Lys Cys Pro
Asp Gly Leu Gln Gly Ala Asn 565 570 575Ser Phe Ile Phe Lys Tyr Ala
Asp Pro Asp Arg Glu Cys His Pro Cys 580 585 590His Pro Asn Cys
Thr
Gln Gly Cys Asn Gly Pro Thr Ser His Asp Cys 595 600 605Ile Tyr Tyr
Pro Trp Thr Gly His Ser Thr Leu Pro Gln His Ala Arg 610 615 620Thr
Pro625
* * * * *