U.S. patent application number 15/616016 was filed with the patent office on 2017-09-28 for bispecific egfr/c-met antibodies.
The applicant listed for this patent is Janssen Biotech, Inc.. Invention is credited to Mark Chiu, Sheri Moores, Joost Neijssen, Paul Parren, Janine Schuurman.
Application Number | 20170275367 15/616016 |
Document ID | / |
Family ID | 59896349 |
Filed Date | 2017-09-28 |
United States Patent
Application |
20170275367 |
Kind Code |
A1 |
Chiu; Mark ; et al. |
September 28, 2017 |
Bispecific EGFR/C-Met Antibodies
Abstract
Bispecific EGFR/c-Met antibodies and methods of making and using
the molecules.
Inventors: |
Chiu; Mark; (Spring House,
PA) ; Moores; Sheri; (Phoenixville, PA) ;
Neijssen; Joost; (Utrecht, NL) ; Parren; Paul;
(Utrecht, NL) ; Schuurman; Janine; (Utrecht,
NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Janssen Biotech, Inc. |
Horsham |
PA |
US |
|
|
Family ID: |
59896349 |
Appl. No.: |
15/616016 |
Filed: |
June 7, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15386195 |
Dec 21, 2016 |
9695242 |
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15616016 |
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14283257 |
May 21, 2014 |
9580508 |
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15386195 |
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14086588 |
Nov 21, 2013 |
9593164 |
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14283257 |
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61728912 |
Nov 21, 2012 |
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61782550 |
Mar 14, 2013 |
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61809541 |
Apr 8, 2013 |
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61864717 |
Aug 12, 2013 |
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61892797 |
Oct 18, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/4545 20130101;
A61K 31/437 20130101; A61K 31/517 20130101; C07K 2317/31 20130101;
A61K 33/24 20130101; A61K 2300/00 20130101; C07K 2317/73 20130101;
A61K 31/4545 20130101; A61K 39/3955 20130101; C07K 2318/20
20130101; A61K 31/517 20130101; C07K 2317/76 20130101; A61K 33/24
20130101; C07K 16/2863 20130101; A61K 39/395 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
31/5377 20130101; A61K 45/06 20130101; A61K 2039/505 20130101; A61K
39/395 20130101; A61K 2300/00 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 31/437 20060101 A61K031/437; A61K 31/4545 20060101
A61K031/4545; A61K 31/517 20060101 A61K031/517; A61K 31/5377
20060101 A61K031/5377; A61K 33/24 20060101 A61K033/24; A61K 39/395
20060101 A61K039/395; A61K 45/06 20060101 A61K045/06 |
Claims
1. A method of treating a subject having EGFR and/or C-Met
expressing cancer, comprising administering a therapeutically
effective amount of an isolated bispecific epidermal growth factor
receptor (EGFR)/hepatocyte growth factor receptor (c-Met) antibody
comprising a first heavy chain (HC1), a first light chain (LC1), a
second heavy chain (HC2) and a second light chain (LC2), wherein
the HC1, the LC1, the HC2 and the LC2 comprise the amino acid
sequences of SEQ ID NOs: 199, 200, 201 and 202, respectively, to a
patient in need thereof for a time sufficient to treat the
cancer.
2. The method of claim 1, wherein the cancer is associated with an
EGFR activating mutation, an EGFR gene amplification, increased
levels of circulating HGF, a c-Met activating mutation, a c-Met
gene amplification or a mutant KRAS.
3. The method of claim 2, wherein the EGFR activating mutation is
G719A, G719X (X being any amino acid), L861X (X being any amino
acid), L858R, E746K, L747S, E749Q, A750P, A755V, V765M, L858P or
T790M substitution, deletion of E746-A750, deletion of R748-P753,
insertion of Ala (A) between M766 and A767, insertion of Ser, Val
and Ala (SVA) between 5768 and V769, and insertion of Asn and Ser
(NS) between P772 and H773.
4. The method of claim 3, wherein the EGFR activating mutation is
L858R, del(E476, A750) and/or T790M substitution.
5. The method of claim 2, wherein the mutant KRAS has a G12V or
G12C substitution.
6. The method of claim 5, wherein the mutant KRAS has a G12V
substitution.
7. The method of claim 2, wherein the subject is resistant or has
acquired resistance to treatment with erlotinib, gefitinib,
afatinib, CO-1686, AZD9192 or cetuximab.
8. The method of claim 2, wherein the cancer is an epithelial cell
cancer, breast cancer, ovarian cancer, lung cancer, non-small cell
lung cancer (NSCLC), lung adenocarcinoma, small cell lung cancer,
colorectal cancer, anal cancer, prostate cancer, kidney cancer,
bladder cancer, head and neck cancer, pharynx cancer, cancer of the
nose, pancreatic cancer, skin cancer, oral cancer, cancer of the
tongue, esophageal cancer, vaginal cancer, cervical cancer, cancer
of the spleen, testicular cancer, gastric cancer, cancer of the
thymus, colon cancer, thyroid cancer, liver cancer, hepatocellular
carcinoma (HCC) or sporadic or hereditary papillary renal cell
carcinoma (PRCC).
9. The method of claim 8, wherein the subject is homozygous for
phenylalanine at position 158 of CD16 or heterozygous for valine
and phenylalanine at position 158 of CD16.
10. The method of claim 8, comprising administering a second
therapeutic agent, wherein the second therapeutic agent is a
chemotherapeutic agent or a targeted anti-cancer therapy.
11. The method of claim 10, wherein the chemotherapeutic agent is
cisplatin or vinblastine.
12. The method of claim 10, wherein the chemotherapeutic agent or
the targeted anti-cancer therapy is a tyrosine kinase inhibitor of
EGFR, c-Met, HER2, HER3, HER4 or VEGFR.
13. The method of claim 12, wherein the tyrosine kinase inhibitor
is erlotinib, gefitinib or afatinib.
14. The method of claim 10, wherein the second therapeutic agent is
administered simultaneously, sequentially or separately.
15. A method of inhibiting growth or proliferation of cells that
express EGFR and/or c-Met, comprising contacting the cells with the
bispecific EGFR/c-Met antibody comprising the HC1, the LC1, the HC2
and the LC2 of SEQ ID NOs: 199, 200, 201 and 202, respectively.
16. A method of inhibiting growth or metastasis of EGFR and/or
c-Met expressing tumor or cancer cells in a subject comprising
administering to the subject an effective amount of the bispecific
EGFR/c-Met antibody comprising the HC1, the LC1, the HC2 and the
LC2 of SEQ ID NOs: 199, 200, 201 and 202, respectively, to inhibit
the growth or metastasis of EGFR and/or c-Met expressing tumor or
cancer cells.
17. The method of claim 16, wherein the EGFR and/or c-Met
expressing tumor is an epithelial cell cancer, breast cancer,
ovarian cancer, lung cancer, non-small cell lung cancer (NSCLC),
lung adenocarcinoma, small cell lung cancer, colorectal cancer,
anal cancer, prostate cancer, kidney cancer, bladder cancer, head
and neck cancer, pharynx cancer, cancer of the nose, pancreatic
cancer, skin cancer, oral cancer, cancer of the tongue, esophageal
cancer, vaginal cancer, cervical cancer, cancer of the spleen,
testicular cancer, gastric cancer, cancer of the thymus, colon
cancer, thyroid cancer, liver cancer, hepatocellular carcinoma
(HCC) or sporadic or hereditary papillary renal cell carcinoma
(PRCC).
18. The method of claim 17, wherein the EGFR and/or c-Met
expressing tumor is associated with an EGFR activating mutation, an
EGFR gene amplification, increased levels of circulating HGF, a
c-Met activating mutation, a c-Met gene amplification or a mutant
KRAS.
19. The method of claim 18, wherein the EGFR activating mutation is
G719A, G719X (X being any amino acid), L861X (X being any amino
acid), L858R, E746K, L747S, E749Q, A750P, A755V, V765M, L858P or
T790M substitution, deletion of E746-A750, deletion of R748-P753,
insertion of Ala (A) between M766 and A767, insertion of Ser, Val
and Ala (SVA) between S768 and V769, and insertion of Asn and Ser
(NS) between P772 and H773.
20. The method of claim 19, wherein the EGFR activating mutation is
L858R, del(E476, A750) and/or T790M substitution.
21. The method of claim 18, wherein the mutant KRAS has a G12V or
G12C substitution.
22. The method of claim 21, wherein the mutant KRAS has a G12V
substitution.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 15/386,195, filed 21 Dec. 2016, currently
allowed, which is a divisional application of U.S. application Ser.
No. 14/283,257, filed 21 May 2014, now U.S. Pat. No. 9,580,508,
which is a continuation-in-part of U.S. application Ser. No.
14/086,588, filed 21 Nov. 2013, now U.S. Pat. No. 9,593,164, which
claims the benefit of U.S. Provisional Application No. 61/728,912,
filed 21 Nov. 2012, U.S. Provisional Application No. 61/782,550,
filed 14 Mar. 2013, U.S. Provisional Application No. 61/809,541,
filed 8 Apr. 2013, U.S. Provisional Application No. 61/864,717
filed 12 Aug. 2013, and U.S. Provisional Application No.
61/892,797, filed 18 Oct. 2013, the entire contents of which are
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to bispecific EGFR/c-Met
antibodies and methods of making and using the molecules.
BACKGROUND OF THE INVENTION
[0003] Epidermal growth factor receptor (EGFR, ErbBl or HER1) is a
Type I transmembrane glycoprotein of 170 kDa that is encoded by the
c-erbBl proto-oncogene. EGFR is a member of the human epidermal
growth factor receptor (HER) family of receptor tyrosine kinases
(RTK) which includes HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4).
EGFR signaling is initiated by ligand binding followed by induction
of conformational change, homodimerization or heterodimerization of
the receptor with other ErbB family members, and
trans-autophosphorylation of the receptor (Ferguson et al., Annu
Rev Biophys, 37: 353-73, 2008), which initiates signal transduction
cascades that ultimately affect a wide variety of cellular
functions, including cell proliferation and survival. Increases in
expression or kinase activity of EGFR have been linked with a range
of human cancers, making EGFR an attractive target for therapeutic
intervention (Mendelsohn et al., Oncogene 19: 6550-6565, 2000;
Grunwald et al., J Natl Cancer Inst 95: 851-67, 2003; Mendelsohn et
al., Semin Oncol 33: 369-85, 2006). Increases in both the EGFR gene
copy number and protein expression have been associated with
favorable responses to the EGFR tyrosine kinase inhibitor,
IRESSA.TM. (gefitinib), in non-small cell lung cancer (Hirsch et
al., Ann Oncol 18:752-60, 2007).
[0004] EGFR therapies include both small molecules and anti-EGFR
antibodies, approved for treatment of colorectal cancer, pancreatic
cancer, head and neck cancer, and non-small cell lung cancer
(NSCLC) (Baselga and Arteaga, J Clin Oncol 23:2445-2459 (20005;
Gill et al., J Biol Chem, 259:7755-7760, 1984; Goldstein et al.,
Clin Cancer Res, 1:131 1-1318; 1995; Prewett et al., Clin Cancer
Res, 4:2957-2966, 1998).
[0005] Efficacy of anti-EGFR therapies may depend on tumor type and
EGFR mutation/amplification status in the tumor. Side effects of
current therapeutics may include skin toxicity (De Roock et al.,
Lancet Oncol 11:753-762, 2010; Linardou et al., Nat Rev Clin Oncol,
6: 352-366, 2009; Li and Perez-Soler, Targ Oncol 4: 107-119, 2009).
EGFR tyrosine kinase inhibitors (TKI) are commonly used as 2.sup.nd
line therapies for non-small cell lung cancer (NSCLC), but often
stop working within twelve months due to resistance pathways (Riely
et al., Clin Cancer Res 12: 839-44, 2006).
[0006] c-Met encodes a transmembrane tyrosine kinase receptor. It
was first identified as a proto-oncogene in 1984 after it was found
that treatment with a carcinogen resulted in a constitutively
active fusion protein TPR-MET (Cooper et al., Nature 311:29-33,
1984). Activation of c-Met by its ligand hepatocyte growth factor
(HGF) stimulates a plethora of cell processes including growth,
motility, invasion, metastasis, epithelial-mesenchymal transition,
angiogenesis/wound healing, and tissue regeneration (Christensen et
al., Cancer Lett 225:1-26, 2005; Peters and Adjei, Nat Rev Clin
Oncol 9:314-26, 2012). c-Met is synthesized as a single chain
protein that is proteolytically cleaved into a 50 kDa alpha- and
140 kDa beta-subunits that are linked by a disulphide bond (Ma et
al., Cancer and Metastasis Reviews, 22: 309-325, 2003). c-Met is
structurally similar to other membrane receptors such as RON and
Sea. The exact stoichiometry of HGF:c-Met binding is unclear, but
it is generally believed that two HGF molecules bind to two c-Met
molecules leading to receptor dimerization and autophosphorylation
at tyrosines 1230, 1234, and 1235 (Stamos et al., The EMBO Journal
23: 2325-2335, 2004). Ligand-independent c-Met autophosphorylation
can also occur due to gene amplification, mutation or receptor
over-expression.
[0007] c-Met is frequently amplified, mutated or over-expressed in
many types of cancer including gastric, lung, colon, breast,
bladder, head and neck, ovarian, prostate, thyroid, pancreatic, and
CNS cancers. Missense mutations typically localized to the kinase
domain are commonly found in hereditary papillary renal cell
carcinomas (PRCC) and in 13% of sporadic PRCCs (Schmidt et al.,
Oncogene 18: 2343-2350, 1999). c-Met mutations localized to the
semaphorin or juxtamembrane domains of c-Met are frequently found
in gastric, head and neck, liver, ovarian, NSCLC and thyroid
cancers (Ma et al., Cancer and Metastasis Reviews, 22: 309-325,
2003; Sakakura et al., Chromosomes and Cancer, 1999. 24:299-305).
c-Met amplification has been detected in brain, colorectal,
gastric, and lung cancers, often correlating with disease
progression (Ma et al., Cancer and Metastasis Reviews, 22: 309-325,
2003). Up to 4% and 20% of non-small cell lung cancer (NSCLC) and
gastric cancers, respectively, exhibit c-Met amplification
(Sakakura et al., Chromosomes and Cancer, 1999. 24:299-305: Sierra
and Tsao, Therapeutic Advances in Medical Oncology, 3:S21-35,
2011). Even in the absence of gene amplification, c-Met
overexpression is frequently observed in lung cancer (Ichimura et
al., Jpn J Cancer Res, 87:1063-9, 1996). Moreover, in clinical
samples, nearly half of lung adenocarcinomas exhibited high levels
of c-Met and HGF, both of which correlated with enhanced tumor
growth rate, metastasis and poor prognosis (Sierra and Tsao,
Therapeutic Advances in Medical Oncology, 3:S21-35, 2011; Siegfried
et al., Ann Thorac Surg 66: 1915-8, 1998).
[0008] Nearly 60% of all tumors that become resistant to EGFR
tyrosine kinase inhibitors increase c-Met expression, amplify
c-Met, or increase c-Met only known ligand, HGF (Turke et al.,
Cancer Cell, 17:77-88, 2010), suggesting the existence of a
compensatory pathway for EGFR through c-Met. c-Met amplification
was first identified in cultured cells that became resistant to
gefitinib, an EGFR kinase inhibitor, and exhibited enhanced
survival through the Her3 pathway (Engelman et al., Science,
316:1039-43, 2007). This was further validated in clinical samples
where nine of 43 patients with acquired resistance to either
erlotinib or gefitinib exhibited c-Met amplification, compared to
only two of 62 untreated patients. Four of the nine treated
patients also acquired the EGFR activating mutation, T790M,
demonstrating simultaneous resistance pathways (Beat et al., Proc
Natl Acad Sci USA, 104:20932-7, 2007).
[0009] The individual roles of both EGFR and c-Met in cancer is
well established, making these targets attractive for combination
therapy. Both receptors signal through the same survival and
anti-apoptotic pathways (ERK and AKT); thus, inhibiting the pair in
combination may limit the potential for compensatory pathway
activation thereby improving overall efficacy. Combination
therapies targeting EGFR and c-Met are tested in clinical trials
with Tarceva.RTM. (erlotinib) in combination with anti-c-Met
monovalent antibody for NSCLC (Spigel et al., 2011 ASCO Annual
Meeting Proceedings 2011, Journal of Clinical Oncology: Chicago,
Ill. p. 7505) and Tarceva (erlotinib) in combination with ARQ-197,
a small molecule inhibitor of c-Met (Adjei et al., Oncologist,
16:788-99, 2011). Combination therapies or bispecific
anti-EGFR/c-Met molecules have been disclosed for example in: Intl.
Pat. Publ. Nos. WO2008/127710, WO2009/111691, WO2009/126834,
WO2010/039248, WO2010/115551 and U.S. Pat. Publ. No.
US2009/0042906.
[0010] Current small molecule and large molecule therapeutic
approaches to antagonize EGFR and/or c-Met signaling pathways for
therapy may be sub-optimal due to possible lack of specificity,
potential off-target activity and dose-limiting toxicity that may
be encountered with small molecule inhibitors. Typical monospecific
bivalent antibodies may result in clustering of membrane bound
receptors and unwanted activation of the downstream signaling
pathways. Monovalent antibodies having full length heavy chains
(half arms) pose significant complexity and cost to the
manufacturing process.
[0011] Accordingly, the need exists for additional monospecific and
bispecific EGFR and/or c-Met inhibitors for both therapeutic and
diagnostic purpose.
SUMMARY OF THE INVENTION
[0012] One embodiment of the invention is an isolated bispecific
epidermal growth factor receptor (EGFR)/hepatocyte growth factor
receptor (c-Met) antibody, comprising: [0013] a first heavy chain
(HC1) comprising a HC1 constant domain 3 (HC1 CH3) and a HC1
variable region 1 (VH1); [0014] a second heavy chain (HC2)
comprising a HC2 constant domain 3 (HC2 CH3) and a HC2 variable
region 2 (VH2); [0015] a first light chain (LC1) comprising a light
chain variable region 1 (VL1); and [0016] a second light chain
(LC2) comprising a light chain variable region 2 (VL2), wherein the
VH1 and the VL1 pair to form a first antigen-binding site that
specifically binds EGFR, the VH2 and the VL2 pair to form a second
antigen-binding site that specifically binds c-Met, the HC1
comprises at least one substitution in the HC1 CH3 and the HC2
comprises at least one substitution in the HC2 CH3, and the
substitution in the HC1 CH3 and the substitution in the HC2 CH3
occur at different amino acid residue positions, when residue
numbering is according to the EU index.
[0017] In other embodiments, the invention provides for bispecific
EGFR/c-Met antibodies, wherein the antibody inhibits
phosphorylation of extracellular signal-related kinases 1 and 2
(ERK1/2) in NCI-H292, NCI-H1975 or SKMES-1 cell line with an
IC.sub.50 value that is at least about 10-fold less, at least about
20-fold less, at least about 30-fold less, at least about 40-fold
less, at least about 50-fold less or at least about 60-fold less
when compared to the IC.sub.50 value of inhibition of
phosphorylation of ERK1/2 in NCI-H292, NCI-H1975 or SKMES-1 cell
lines with a mixture of a control monovalent EGFR antibody
comprising a heavy chain 3 (HC3) and a light chain 3 (LC3) and a
control monovalent c-Met antibody comprising a heavy chain 4 (HC4)
and a light chain 4 (LC4), wherein the HC3 and the HC1, the LC3 and
the LC1, the HC4 and the HC2, and the LC4 and the LC2 have
identical amino acid sequences, respectively, wherein the
phosphorylation of ERK1/2 is measured in whole cell lysates using a
sandwich immunoassay using an anti-phosphoERK1/2 antibody as a
capture antibody and an antibody binding to unphosphorylated and
phosphorylated ERK1/2 conjugated with an electrochemiluminescent
compound as a detection antibody.
[0018] In other embodiments, the invention provides for bispecific
EGFR/c-Met antibodies, wherein the antibody inhibits
phosphorylation of protein kinase B (AKT) at Ser473 in NCI-H1975
cell line with an IC.sub.50 value that is at least about 70-fold
less when compared to the IC.sub.50 value of inhibition of
phosphorylation of AKT at Ser473 in NCI-H1975 cell line with the
mixture of the control monovalent EGFR antibody comprising the HC3
and the LC3 and the control monovalent c-Met antibody comprising
the HC4 and the LC4, wherein the HC3 and the HC1, the LC3 and the
LC1, the HC4 and the HC2, and the LC4 and the LC2 have identical
amino acid sequences, respectively, wherein the phosphorylation of
AKT at Ser473 is measured in whole cell lysates using a sandwich
immunoassay using an antibody binding to unphosphorylated and
phosphorylated AKT as a capture antibody and an anti-phosphoAKT
Ser473 antibody conjugated to an electrochemiluminescent compound
as a detection antibody.
[0019] In other embodiments, the invention provides for bispecific
EGFR/c-Met antibodies that bind EGFR of SEQ ID NO: 73 at EGFR
residues K489, 1491, K467 and S492 and c-Met at residues
PEFRDSYPIKYVHAF (SEQ ID NO: 238) and FAQSKPDSAEPMDRSA (SEQ ID NO:
239).
[0020] In other embodiments, the invention provides for bispecific
EGFR/c-Met antibodies that inhibit growth of NCI-H292 or NCI-H1975
cells with an IC.sub.50 value that is at least about 300-fold less,
at least about 400-fold less, at least about 500-fold less, at
least about 600-fold less, at least about 700-fold less or at least
about 800-fold less when compared to the IC.sub.50 value of
inhibition of growth of NCI-H292 or NCI-H1975 cells with cetuximab,
when NCI-H292 or NCI-H1975 cells are grown in low attachment
conditions.
[0021] In other embodiments, the invention provides for bispecific
EGFR/c-Met antibodies that inhibit growth of HGF-expressing SKMES-1
cell tumor in SCID Beige mice with percentage (%) T/C value of at
least 500-fold less on day 36 when compared to cetuximab, when the
bispecific antibody and cetuximab are administered at 20 mg/kg
dose.
[0022] In other embodiments, the invention provides for bispecific
EGFR/c-Met antibodies wherein the HC1 CH3 comprises a K409R or a
F405L substitution and the HC2 CH3 comprises a K409R or F405L
substitution, wherein residue numbering is according to the EU
index.
[0023] In other embodiments, the invention provides for bispecific
EGFR/c-Met antibodies comprising certain heavy and light chain CDR,
VH1, VL1, VH2, VL2, HC1, LC1, HC2 and LC2 sequences.
[0024] Another embodiment of the invention is an isolated synthetic
polynucleotide encoding the HC1, the HC2, the LC1 or the LC2 of the
invention.
[0025] Another embodiment of the invention is a vector comprising
the polynucleotide of the invention.
[0026] Another embodiment of the invention is a host cell
comprising the vector of the invention.
[0027] Another embodiment of the invention is a method of producing
the isolated bispecific EGFR/c-Met antibody, comprising: [0028]
combining an isolated monospecific bivalent anti-EGFR antibody
comprising two heavy chains of SEQ ID NO: 199 and two light chains
of SEQ ID NO: 200 and an isolated monospecific bivalent anti-c-Met
antibody comprising two heavy chains of SEQ ID NO: 201 and two
light chains of SEQ ID NO: 202 in a mixture of about 1:1 molar
ratio; [0029] introducing a reducing agent into the mixture; [0030]
incubating the mixture about ninety minutes to about six hours;
[0031] removing the reducing agent; and purifying the bispecific
EGFR/c-Met antibody that comprises a first heavy chain of SEQ ID
NO: 199 and a second heavy chain of SEQ ID NO: 201, a first light
chain of SEQ ID NO: 200 and a second light chain of SEQ ID NO: 202,
wherein the first heavy chain of SEQ ID NO: 199 pairs with the
first light chain of SEQ ID NO: 200 to form the first binding
domain that specifically binds EGFR, and the second heavy chain of
SEQ ID NO: 201 pairs with the second light chain of SEQ ID NO: 202
to form the second binding domain that specifically binds
c-Met.
[0032] Another embodiment of the invention is a pharmaceutical
composition comprising the bispecific antibody of the invention and
a pharmaceutically acceptable carrier.
[0033] Another embodiment of the invention is method of treating a
subject having cancer, comprising administering a therapeutically
effective amount of the bispecific EGFR/c-Met antibody of the
invention to a patient in need thereof for a time sufficient to
treat the cancer.
[0034] Another embodiment of the invention is method of inhibiting
growth or proliferation of cells that express EGFR and/or c-Met,
comprising contacting the cells with the bispecific antibody of the
invention.
[0035] Another embodiment of the invention is method of inhibiting
growth or metastasis of EGFR and/or c-Met expressing tumor or
cancer cells in a subject comprising administering to the subject
an effective amount of the bispecific antibody of the invention to
inhibit the growth or metastasis of EGFR and/or c-Met expressing
tumor or cancer cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIGS. 1A and 1B. Amino acid alignment of the EGFR-binding
FN3 domains. The BC and FG loops are boxed at residues 22-28 and
75-86 of SEQ ID NO: 18. Some variants include thermal stability
improving L17A, N46K and E86I substitutions (residue numbering
according to Tencon SEQ ID NO: 1).
[0037] FIG. 2. Sequence alignment of the Tencon27 scaffold (SEQ ID
NO: 99) and a TCL14 library (SEQ ID NO: 100) having randomized
C-CD-F-FG alternative surface. The loop residues are boxed. Loops
and strands are indicated above the sequences.
[0038] FIG. 3. Sequence alignment of the c-Met-binding FN3 domains.
The C loop and the CD strand and the F loop and the FG strand are
boxed and span residues 29-43 and 65-81.
[0039] FIG. 4. Inhibition of c-Met phosphorylation in NCI-H292
cells pre-treated with monospecific or bispecific FN3 domain
containing molecules and stimulated with HGF is shown. Substantial
increase in the potency of the bispecific EGFR/c-Met molecule
(ECB1) was observed when compared to a monospecific c-Met-binding
FN3 domain (P114AR5P74-A5, shown as A5 in the Figure) on its own or
in combination with an EGFR-binding FN3 domain (P54AR4-83v2, shown
as 83v2 in the Figure).
[0040] FIG. 5. Inhibition of EGFR and c-Met phosphorylation in
cells pre-treated with monospecific or bispecific FN3 domain
containing molecules. In cell lines expressing high levels of EGFR,
NCI-H292 (FIG. 5A) and H596(FIG. 5B), anti-EGFR monospecific and
bispecific FN3 domain containing molecules are equally potent at
decreasing EGFR phosphorylation. In cell lines expressing low
levels of EGFR relative to c-Met, NCI-H441 (FIG. 5C), bispecific
EGFR/c-Met molecules improve the potency for inhibition of EGFR
phosphorylation compared to the monospecific EGFR-binding FN3
domain alone. In cell lines with low levels of c-Met, relative to
EGFR, NCI-H292 (FIG. 5D) and H596 (FIG. 5E), inhibition of c-Met
phosphorylation is significantly potentiated with bispecific
EGFR/c-Met molecule, compared to monospecific c-Met-binding FN3
domain only. Molecules used in the study were: bispecific ECB5
(shown as 17-A3 in the Figure), monospecific EGFR-binding FN3
domain P53A1R5-17 (shown as "17" in the Figure), bispecific
EGFR/c-Met molecule ECB3 (shown as 83-H9 in the Figure), and
monospecific c-Met binding FN3 domain P114AR7P93-H9 (shown as H9 in
the Figure).
[0041] FIG. 6. Pharmacodynamic signaling in tumors isolated from
mice dosed with bispecific EGFR/c-Met molecules for 6 h or 72 h.
All molecules significantly reduced c-Met, EGFR and ERK
phosphorylation at 6 h and 72 h, the degree if inhibition was
dependent on the affinity of the FN3 domains to EGFR and/or c-Met.
Bispecific molecules were generated by joining EGFR-binding FN3
domain with a high ("83" in the Figure is p54AR4-83v2) or medium
("17v2" in the Figure is P53A1R5-17v2) affinity to a c-Met-binding
FN3 domain with high ("A3" in the Figure is P114AR7P94-A3) or
medium ("A5" in the Figure is P114AR5P74-A5) affinity.
[0042] FIG. 7. Plasma (top) and tumor (bottom) accumulation of
bispecific EGFR/cMet molecules of variable affinities linked to an
albumin binding domain (ABD) are shown 6 h (left) and 72 h (right)
after IP dosing. Six hours after dosing, tumor accumulation is
maximal in mice dosed with a bispecific molecule harboring a medium
affinity EGFR-binding FN3 domain (17v2) or high affinity EGFR
binding domain (83v2). The bispecific molecules incorporated high
or medium affinity EGFR or c-Met binding FN3 domains as follows:
83v2-A5-ABD (ECB18; high/medium for EGFR/cMet) 83v2-A3-ABD (ECB38;
high/high) 17v2-A5 (ECB28; medium/medium) 17v2-A3-ABD (ECB39;
medium/high). In the figure, 83v2 refers to p54AR4-83v2; 17v2
refers to p53A1R5-17v2; A3 refers to p114AR7P94-A3 and A5 refers to
p114AR5P74-A5.
[0043] FIG. 8. H292-HGF tumor xenografts were implanted into SCID
Beige mice. When tumors reached an average volume of approximately
80 mm.sup.3, mice were dosed three times per week with bispecific
EGFR/c-Met molecules (25 mg/kg) or PBS vehicle. All bispecific
molecules reduced tumor growth, the tumor growth inhibition (TGI)
being dependent on the affinities of the molecules for c-Met and
EGFR (high EGFR-high cMet refers to p54AR4-83v2-p114AR7P94-A3
(ECB38); high EGFR-med cMet refers to p54AR4-83v2-p114AR5P74-A5
(ECB18); med EGFR-high cMet refers to p53A1R5-17v2-p114AR7P94-A3
(ECB39); med EGFR-med-cMet refers to p53A1R5-17-p114AR5P74-A 5
(ECB28)).
[0044] FIG. 9. H292-HGF tumor xenografts were implanted into SCID
Beige mice and they were treated with different therapies. The
anti-tumor activity of the therapies is shown (bispecific
EGFR/c-Met molecule refers to p54AR4-83v2-p114AR7P94-A3-ABD
(ECB38); the other therapies are crizotinib, erlotinib, cetuximab,
and the combination of crizotinib and erlotinib).
[0045] FIG. 10. SKMES-HGF tumor xenografts were implanted into SCID
Beige mice and the mice were treated with different therapies. The
anti-tumor activity of the therapies is shown as change in tumor
size (mm.sup.3) over time. The bispecific EGFR/c-Met antibody
EM1-mAb was dosed intraperitoneally (i.p.) twice a week at either
20 mg/kg, 5 mg/kg, or 1 mg/kg; cetuximab was dosed i.p. twice a
week at 20 mg/kg. Arrows in the figure show the administration
days. Numbers after the antibodies indicated the administered
dose.
[0046] FIG. 11. HCC827 tumor xenografts were implanted into nude
mice and the mice were treated with erlotinib or EM1-mAb at
indicated doses. EM1-mAb was dosed biweekly and erlotinib once a
day for four weeks. Arrows in the figure show the administration
days. The anti-tumor activity of the therapies is shown as change
in tumor size (mm.sup.3) over time.
[0047] FIG. 12. SNU-5 tumor xenografts were implanted into
CB17/SCID mice and the mice were treated with 10 mg/kg cetuximab or
10 mg/kg or 1 mg/kg EM1-mAb. Antibodies were dosed biweekly for
four weeks. Arrows in the figure show the administration days. The
anti-tumor activity of the therapies is shown as change in tumor
size (mm.sup.3) over time.
[0048] FIG. 13. H1975-HGF tumor xenografts were implanted into nude
mice and the mice were treated with 10 mg/kg cetuximab, 10 mg/kg
EM1-mAb, 50 mg/kg erlotinib, 15 mg/kg afatinib, or a combination of
10 mg/kg EM1-mAb and 15 mg/kg afatinib. Antibodies were dosed
biweekly and the small molecules once a day for three weeks. Arrows
in the figure show the administration days. The anti-tumor activity
of the therapies is shown as change in tumor size (mm.sup.3) over
time.
[0049] FIG. 14. HCC827-ER1 tumor xenografts were implanted into
nude mice and the mice were treated with 10 mg/kg EM1-mAb, 25 mg/kg
erlotinib, or a combination of the two. EM1-mAb was dosed biweekly
and erlotinib once a day for 19 days. Arrows in the figure show the
administration days. The anti-tumor activity of the therapies is
shown as change in tumor size (mm.sup.3) over time.
[0050] FIG. 15. Average EGFR and c-Met levels in tumor lysates
isolated from H1975 HGF tumor xenografts implanted into SCID Beige
mice after administration of a single dose of 20 mg/kg EM1-mAb.
Receptor levels are shown as % of PBS control at indicated times
post-treatment.
[0051] FIG. 16. H1975-HGF tumor xenografts were implanted into nude
mice and the mice were treated with 10 mg/kg EM1-mAb or 10 mg/kg
EM1-mAb variant IgG2 V234A/G237A/P238S/H268A/V309L/A330S/P331S
having no Fc receptor binding and lacking effector functions.
Antibodies were dosed biweekly at indicated days. The anti-tumor
activity of the therapies is shown as change in tumor size
(mm.sup.3) over time.
[0052] FIG. 17A. Correlation of EGFR surface density and inhibition
of EGFR phosphorylation by the bispecific EM-1 mAb. IC.sub.50
values for inhibition of EGFR phosphorylation were plotted vs. EGFR
molecules on the surface expressed as antibodies bound per cell
(ABC). Pearson correlation p=0.00082, r.sup.2=0.7820.
[0053] FIG. 17B. Correlation of receptor density and inhibition of
c-Met phosphorylation by the bispecific EM-1 mAb. IC.sub.50 values
for inhibition of c-Met phosphorylation were plotted vs. c-Met
molecules on the surface expressed as ABC. Pearson correlation
p<0.0001, r.sup.2=0.9336.
[0054] FIG. 18. The bispecific EGFR/c-Met EM-1 mAb induced
heterodimerization of EGFR and c-Met assessed in
.beta.-galactosidase fragment mediated complementation assay in U-2
OS cells stably expressing EGFR and c-Met .beta.-gal fusion
proteins. Control monovalent EGFR- or c-Met antibodies or parental
bivalent anti-EGFR or c-Met antibodies 2F8 or 069 had no effect.
E1-F405L-gp120-K409R ( ), M1-K409R-gp120-F405L (.quadrature.), a
combination of E1-F405L-gp120-K409R and M1-K409R-gp120-F405L (),
EM-1 bispecific mAb (.diamond-solid.), anti-cMet parental 069 mAb
(.diamond.), anti-EGFR parental 2F8mAb (.tangle-solidup.).
[0055] FIG. 19A. Inhibition of c-Met phosphorylation in H292 cells
relative to a HGF-treated control. The ratio of EGFR to c-Met
surface expression in H292 cells was 5.72. Negative control
(.diamond-solid.), E1-F405L-gp120-K409R ( ), M1-K409R-gp120-F405L
(.box-solid.), EM-1 bispecific mAb (.tangle-solidup.). The dotted
lines indicate the levels of the MSD signal from the EGF-treated
control without antibody or unstimulated control as shown in the
Figure. ug/mL indicates the antibody log concentration in
.mu.g/ml.
[0056] FIG. 19B. Inhibition of EGFR phosphorylation in H292 cells
relative to an EGFR-treated control. Negative control
(.diamond-solid.), E1-F405L-gp120-K409R ( ), M1-K409R-gp120-F405L
(.box-solid.), EM-1 bispecific mAb (.tangle-solidup.). The dotted
lines indicate the levels of the MSD signal from the EGF-treated
control without antibody or unstimulated control as shown in the
Figure. ug/mL indicates the antibody log concentration in
.mu.g/ml.
[0057] FIG. 20. Inhibition of EGFR phosphorylation in SNU-5 cells
relative to an EGF-treated control. Negative control
(.diamond-solid.), E1-F405L-gp120-K409R ( ), M1-K409R-gp120-F405L
(.box-solid.), EM-1 bispecific mAb (.tangle-solidup.). The dotted
lines indicaet the levels of the MSD signal from the negative
controls or unstimulated control as shown in the Figure. ug/mL
indicates the antibody log concentration in .mu.g/ml.
[0058] FIG. 21. Inhibition of ligand-induced receptor
phosphorylation in H1993 cells relative to an EGF-treated control.
E1-F405L-gp120-K409R ( ), M1-K409R-gp120-F405L (.box-solid.), EM-1
bispecific mAb (.tangle-solidup.), negative control
(.diamond-solid.), combination of E1-F405L-gp120-K409R and
M1-K409R-gp120-F405L (). X-axis indicates the antibody log nM
concentration. The dotted lines indicate the levels of the MSD
signal from the unstimulated (vehicle) control or EGF-treated
control without antibody.
DETAILED DESCRIPTION OF THE INVENTION
[0059] The term "fibronectin type III (FN3) domain" (FN3 domain) as
used herein refers to a domain occurring frequently in proteins
including fibronectins, tenascin, intracellular cytoskeletal
proteins, cytokine receptors and prokaryotic enzymes (Bork and
Doolittle, Proc Nat Acad Sci USA 89:8990-8994, 1992; Meinke et al.,
J Bacteriol 175:1910-1918, 1993; Watanabe et al., J Biol Chem
265:15659-15665, 1990). Exemplary FN3 domains are the 15 different
FN3 domains present in human tenascin C, the 15 different FN3
domains present in human fibronectin (FN), and non-natural
synthetic FN3 domains as described for example in U.S. Pat. Publ.
No. 2010/0216708. Individual FN3 domains are referred to by domain
number and protein name, e.g., the 3.sup.th FN3 domain of tenascin
(TN3), or the 10.sup.th FN3 domain of fibronectin (FN10).
[0060] The term "substituting" or "substituted" or `mutating" or
"mutated" as used herein refers to altering, deleting or inserting
one or more amino acids or nucleotides in a polypeptide or
polynucleotide sequence to generate a variant of that sequence.
[0061] The term "randomizing" or "randomized" or "diversified" or
"diversifying" as used herein refers to making at least one
substitution, insertion or deletion in a polynucleotide or
polypeptide sequence.
[0062] "Variant" as used herein refers to a polypeptide or a
polynucleotide that differs from a reference polypeptide or a
reference polynucleotide by one or more modifications for example,
substitutions, insertions or deletions.
[0063] The term "specifically binds" or "specific binding" as used
herein refers to the ability of an FN3 domain, a bispecific agent
that specifically binds EGFR and c-Met, or a bispecific EGFR/c-Met
antibody of the invention to bind to a predetermined antigen with a
dissociation constant (K.sub.D) of about 1.times.10.sup.-6 M or
less, for example about 1.times.10.sup.-7 M or less, about
1.times.10.sup.-8 M or less, about 1.times.10.sup.-9 M or less,
about 1.times.10.sup.-10 M or less, about 1.times.10.sup.-11 M or
less, about 1.times.10.sup.-12 M or less, or about
1.times.10.sup.-13 M or less. Typically the FN3 domain, the
bispecific agent that specifically binds EGFR and c-Met or the
bispecific EGFR/c-Met antibody of the invention binds to a
predetermined antigen (i.e. EGFR or c-Met) with a K.sub.D that is
at least ten fold less than its K.sub.D for a nonspecific antigen
(for example BSA or casein) as measured by surface plasmon
resonance using for example a Proteon Instrument (BioRad). Thus,
the bispecific EGFR/c-Met FN3 domain containing molecule, the
bispecific agent that specifically binds EGFR and c-Met or the
bispecific EGFR/c-Met antibody of the invention specifically binds
to each EGFR and c-Met with a binding affinity (K.sub.D) of at
least about 1.times.10.sup.-6 M or less, for example about
1.times.10.sup.-7 M or less, about 1.times.10.sup.-8 M or less,
about 1.times.10.sup.-9 M or less, about 1.times.10.sup.-10 M or
less, about 1.times.10.sup.-11 M or less, about 1.times.10.sup.-12
M or less, or about 1.times.10.sup.-13 M or less. The bispecific
EGFR/c-Met FN3 domain containing molecule, the bispecific agent
that specifically binds EGFR and c-Met or the bispecific EGFR/c-Met
antibody of the invention that specifically binds to a
predetermined antigen may, however, have cross-reactivity to other
related antigens, for example to the same predetermined antigen
from other species (homologs).
[0064] The term "library" refers to a collection of variants. The
library may be composed of polypeptide or polynucleotide
variants.
[0065] The term "stability" as used herein refers to the ability of
a molecule to maintain a folded state under physiological
conditions such that it retains at least one of its normal
functional activities, for example, binding to a predetermined
antigen such as EGFR or c-Met.
[0066] "Epidermal growth factor receptor" or "EGFR" as used here
refers to the human EGFR (also known as HER1 or ErbB1 (Ullrich et
al., Nature 309:418-425, 1984) having the amino acid sequence shown
in SEQ ID NO: 73 and in GenBank accession number NP_005219, as well
as naturally-occurring variants thereof. Such variants include
well-known EGFRvIII and other alternatively spliced variants (e.g.,
as identified by SwissProt Accession numbers P00533-1 (wild type;
identical to SEQ ID NO: 73 and NP_005219), P00533-2 (F404L/L405S),
P00533-3 (628-705:
CTGPGLEGCP . . . GEAPNQALLR.fwdarw.PGNESLKAML . . . SVIITASSCH and
706-1210 deleted), P00533-4 (C628S and 629-1210 deleted), variants
GlnQ98, R266, K521, 1674, G962, and P988 (Livingston et al.,
NIEHS-SNPs, environmental genome project, NIEHS ES15478), T790M,
L858R/T790M and del(E746, A750).
[0067] "EGFR ligand" as used herein encompasses all (e.g.,
physiological) ligands for EGFR, including EGF, TGF.alpha., heparin
binding EGF (HB-EGF), amphiregulin (AR), and epiregulin (EPI).
[0068] "Epidermal growth factor" (EGF) as used herein refers to the
well-known 53 amino acid human EGF having the amino acid sequence
shown in SEQ ID NO: 74.
[0069] "Hepatocyte growth factor receptor" or "c-Met" as used
herein refers to the human c-Met having the amino acid sequence
shown in SEQ ID NO: 101 or in GenBank Accession No: NP_001120972
and natural variants thereof.
[0070] "Hepatocyte growth factor" (HGF) as used herein refers to
the well-known human HGF having the amino acid sequence shown in
SEQ ID NO: 102 which is cleaved to form a dimer of an alpha and
beta chain linked by a disulfide bond.
[0071] "Blocks binding" or "inhibits binding", as used herein
interchangeably refers to the ability of the FN3 domains, the
bispecific EGFR/c-Met FN3 domain containing molecule, the
bispecific agent that specifically binds EGFR and c-Met or the
bispecific EGFR/c-Met antibody of the invention to block or inhibit
binding of the EGFR ligand such as EGF to EGFR and/or HGF to c-Met,
and encompass both partial and complete blocking/inhibition. The
blocking/inhibition of EGFR ligand such as EGF to EGFR and/or HGF
to c-Met by the FN3 domains, the bispecific EGFR/c-Met FN3 domain
containing molecule, the bispecific agent that specifically binds
EGFR and c-Met or the bispecific EGFR/c-Met antibody of the
invention reduces partially or completely the normal level of EGFR
signaling and/or c-Met signaling when compared to the EGFR ligand
binding to EGFR and/or HGF binding to c-Met without blocking or
inhibition. The FN3 domains, the bispecific EGFR/c-Met FN3 domain
containing molecule, the bispecific agent that specifically binds
EGFR and c-Met or the bispecific EGFR/c-Met antibody of the
invention "blocks binding" of the EGFR ligand such as EGF to EGFR
and/or HGF to c-Met when the inhibition is at least 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or 100%. Inhibition of binding can be
measured using well known methods, for example by measuring
inhibition of binding of biotinylated EGF on EGFR expressing A431
cells exposed to the FN3 domain, the bispecific EGFR/c-Met FN3
domain containing molecule, the bispecific agent that specifically
binds EGFR and c-Met or the bispecific EGFR/c-Met antibody of the
invention using FACS, and using methods described herein, or
measuring inhibition of binding of biotinylated HGF on c-Met
extracellular domain using well known methods and methods described
herein.
[0072] The term "EGFR signaling" refers to signal transduction
induced by EGFR ligand binding to EGFR resulting in
autophosphorylation of at least one tyrosine residue in the EGFR.
An exemplary EGFR ligand is EGF.
[0073] "Neutralizes EGFR signaling" as used herein refers to the
ability of the FN3 domains, the bispecific EGFR/c-Met FN3 domain
containing molecule, the bispecific agent that specifically binds
EGFR and c-Met or the bispecific EGFR/c-Met antibody of the
invention to inhibit EGFR signaling induced by EGFR ligand such as
EGF by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
100%.
[0074] The term "c-Met signaling" refers to signal transduction
induced by HGF binding to c-Met resulting in autophosphorylation of
at least one tyrosine residue in the c-Met. Typically at least one
tyrosine residue at positions 1230, 1234, 1235 or 1349 is
autophosphorylated upon HGF binding.
[0075] "Neutralizes c-Met signaling" as used herein refers to the
ability of the FN3 domain, the bispecific EGFR/c-Met FN3 domain
containing molecule, the bispecific agent that specifically binds
EGFR and c-Met or the bispecific EGFR/c-Met antibody of the
invention to inhibit c-Met signaling induced by HGF by at least
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
[0076] "Overexpress", "overexpressed" and "overexpressing" as used
herein interchangeably refer to a cancer or malignant cell that has
measurably higher levels of EGFR and/or c-Met on the surface
compared to a normal cell of the same tissue type. Such
overexpression may be caused by gene amplification or by increased
transcription or translation. EGFR and/or c-Met expression and
overexpression can be measured using well know assays using for
example ELISA, immunofluorescence, flow cytometry or
radioimmunoassay on live or lysed cells. Alternatively, or
additionally, levels of EGFR and/or c-Met-encoding nucleic acid
molecules may be measured in the cell for example using fluorescent
in situ hybridization, Southern blotting, or PCR techniques. EGFR
and/or c-Met is overexpressed when the level of EGFR and/or c-Met
on the surface of the cell is at least 1.5-fold higher when
compared to the normal cell.
[0077] "Tencon" as used herein refers to the synthetic fibronectin
type III (FN3) domain having the sequence shown in SEQ ID NO: 1 and
described in U.S. Pat. Publ. No. US2010/0216708.
[0078] A "cancer cell" or a "tumor cell" as used herein refers to a
cancerous, pre-cancerous or transformed cell, either in vivo, ex
vivo, and in tissue culture, that has spontaneous or induced
phenotypic changes that do not necessarily involve the uptake of
new genetic material. Although transformation can arise from
infection with a transforming virus and incorporation of new
genomic nucleic acid, or uptake of exogenous nucleic acid, it can
also arise spontaneously or following exposure to a carcinogen,
thereby mutating an endogenous gene. Transformation/cancer is
exemplified by, e.g., morphological changes, immortalization of
cells, aberrant growth control, foci formation, proliferation,
malignancy, tumor specific marker levels, invasiveness, tumor
growth or suppression in suitable animal hosts such as nude mice,
and the like, in vitro, in vivo, and ex vivo (Freshney, Culture of
Animal Cells: A Manual of Basic Technique (3rd ed. 1994)).
[0079] The term "vector" means a polynucleotide capable of being
duplicated within a biological system or that can be moved between
such systems. Vector polynucleotides typically contain elements,
such as origins of replication, polyadenylation signal or selection
markers that function to facilitate the duplication or maintenance
of these polynucleotides in a biological system. Examples of such
biological systems may include a cell, virus, animal, plant, and
reconstituted biological systems utilizing biological components
capable of duplicating a vector. The polynucleotide comprising a
vector may be DNA or RNA molecules or a hybrid of these.
[0080] The term "expression vector" means a vector that can be
utilized in a biological system or in a reconstituted biological
system to direct the translation of a polypeptide encoded by a
polynucleotide sequence present in the expression vector.
[0081] The term "polynucleotide" means a molecule comprising a
chain of nucleotides covalently linked by a sugar-phosphate
backbone or other equivalent covalent chemistry. Double and
single-stranded DNAs and RNAs are typical examples of
polynucleotides.
[0082] "Complementary DNA" or "cDNA" refers to the well-known
synthetic polynucleotide that shares the arrangement of sequence
elements found in native mature mRNA species with contiguous exons,
with the intervening introns present in genomic DNA are removed.
The codons encoding the initiator methionine may or may not be
present in cDNA. cDNA may be synthesized for example by reverse
transcription or synthetic gene assembly.
[0083] "Synthetic" or "non-natural" or "artificial" as used herein
refers to a polynucleotide or a polypeptide molecule not present in
nature.
[0084] The term "polypeptide" or "protein" means a molecule that
comprises at least two amino acid residues linked by a peptide bond
to form a polypeptide. Small polypeptides of less than about 50
amino acids may be referred to as "peptides".
[0085] The term "bispecific EGFR/c-Met molecule" or "bispecific
EGFR/c-Met FN3 domain containing molecule" as used herein refers to
a molecule comprising an EGFR binding FN3 domain and a distinct
c-Met binding FN3 domain that are covalently linked together either
directly or via a linker. An exemplary bispecific EGFR/c-Met
binding molecule comprises a first FN3 domain specifically binding
EGFR and a second FN3 domain specifically binding c-Met.
[0086] "Valent" as used herein refers to the presence of a
specified number of binding sites specific for an antigen in a
molecule. As such, the terms "monovalent", "bivalent",
"tetravalent", and "hexavalent" refer to the presence of one, two,
four and six binding sites, respectively, specific for an antigen
in a molecule.
[0087] "Mixture" as used herein refers to a sample or preparation
of two or more FN3 domains not covalently linked together. A
mixture may consist of two or more identical FN3 domains or
distinct FN3 domains Mixture as used herein also refers to a sample
or preparation of two or more monovalent antibodies that are
monovalent towards EGFR and/or monovalent towards c-Met.
[0088] The term "bispecific agent that specifically binds EGFR and
c-Met" as used herein refers to a molecule comprising a first
domain that specifically binds EGFR and a second domain that
specifically binds c-Met. An exemplary agent that specifically
binds EGFR and c-Met is a bispecific antibody. Another exemplary
bispecific agent that specifically binds EGFR and c-Met is a
molecule comprising an EGFR binding FN3 domain and a distinct c-Met
binding FN3 domain. The bispecific agent that specifically binds
EGFR and c-Met may be composed of a single polypeptide or more than
one polypeptide.
[0089] The term "bispecific anti-EGFR/c-Met antibody" or
"bispecific EGFR/c-Met antibody" as used herein refers to a
bispecific antibody having a first domain that specifically binds
EGFR and a second domain that specifically binds c-Met. The domains
specifically binding EGFR and c-Met are typically VH/VL pairs, and
the bispecific anti-EGFR/c-Met antibody is monovalent in terms of
binding to EGFR and c-Met.
[0090] The term "antibodies" as used herein is meant in a broad
sense and includes immunoglobulin molecules including polyclonal
antibodies, monoclonal antibodies including murine, human,
human-adapted, humanized and chimeric monoclonal antibodies,
antibody fragments, bispecific or multispecific antibodies,
dimeric, tetrameric or multimeric antibodies, and single chain
antibodies.
[0091] Immunoglobulins can be assigned to five major classes,
namely IgA, IgD, IgE, IgG and IgM, depending on the heavy chain
constant domain amino acid sequence. IgA and IgG are further
sub-classified as the isotypes IgA.sub.1, IgA.sub.2, IgG.sub.1,
IgG2, IgG3 and IgG4. Antibody light chains of any vertebrate
species can be assigned to one of two clearly distinct types,
namely kappa (.kappa.) and lambda (.lamda.), based on the amino
acid sequences of their constant domains.
[0092] The term "antibody fragments" refers to a portion of an
immunoglobulin molecule that retains the heavy chain and/or the
light chain antigen binding site, such as heavy chain
complementarity determining regions (HCDR) 1, 2 and 3, light chain
complementarity determining regions (LCDR) 1, 2 and 3, a heavy
chain variable region (VH), or a light chain variable region (VL).
Antibody fragments include a Fab fragment, a monovalent fragment
consisting of the VL, VH, CL and CHI domains; a F(ab).sub.2
fragment, a bivalent fragment comprising two Fab fragments linked
by a disulfide bridge at the hinge region; a Fd fragment consisting
of the VH and CHI domains; a Fv fragment consisting of the VL and
VH domains of a single arm of an antibody; a domain antibody (dAb)
fragment (Ward et al (1989) Nature 341:544-546), which consists of
a VH domain. VH and VL domains can be engineered and linked
together via a synthetic linker to form various types of single
chain antibody designs where the VH/VL domains pair
intramolecularly, or intermolecularly in those cases when the VH
and VL domains are expressed by separate single chain antibody
constructs, to form a monovalent antigen binding site, such as
single chain Fv (scFv) or diabody; described for example in PCT
Intl. Publ. Nos. WO1998/44001, WO1988/01649, WO1994/13804, and
WO1992/01047. These antibody fragments are obtained using well
known techniques known to those of skill in the art, and the
fragments are screened for utility in the same manner as are full
length antibodies.
[0093] The phrase "isolated antibody" refers to an antibody or
antibody fragment that is substantially free of other antibodies
having different antigenic specificities (e.g., an isolated
bispecific antibody specifically binding EGFR and c-Met is
substantially free of antibodies that specifically bind antigens
other than human EGFR and c-Met). An isolated antibody that
specifically binds EGFR and c-Met, however, can have
cross-reactivity to other antigens, such as orthologs of human EGFR
and/or c-Met, such as Macaca fascicularis (cynomolgus) EGFR and/or
c-Met. Moreover, an isolated antibody may be substantially free of
other cellular material and/or chemicals.
[0094] An antibody variable region consists of a "framework" region
interrupted by three "antigen binding sites". The antigen binding
sites are defined using various terms: (i) Complementarity
Determining Regions (CDRs), three in the VH (HCDR1, HCDR2, HCDR3),
and three in the VL (LCDR1, LCDR2, LCDR3), are based on sequence
variability (Wu and Kabat (1970) J Exp Med 132:211-50, 1970; Kabat
et al Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda,
Md., 1991). (ii) "Hypervariable regions", "HVR", or "HV", three in
the VH (H1, H2, H3) and three in the VL (L1, L2, L3), refer to the
regions of an antibody variable domains which are hypervariable in
structure as defined by Chothia and Lesk (Chothia and Lesk Mol Biol
196:901-17, 1987). Other terms include "IMGT-CDRs" (Lefranc et al.,
Dev Comparat Immunol 27:55-77, 2003) and "Specificity Determining
Residue Usage" (SDRU) (Almagro Mol Recognit 17:132-43, 2004). The
International ImMunoGeneTics (IMGT) database (http://www_jmgt_org)
provides a standardized numbering and definition of antigen-binding
sites. The correspondence between CDRs, HVs and IMGT delineations
is described in Lefranc et al., Dev Comparat Immunol 27:55-77,
2003.
[0095] "Chothia residues" as used herein are the antibody VL and VH
residues numbered according to Al-Lazikani (Al-Lazikani et al., J
Mol Biol 273:927-48, 1997).
[0096] "Framework" or "framework sequences" are the remaining
sequences of a variable region other than those defined to be
antigen binding sites. Because the antigen binding sites can be
defined by various terms as described above, the exact amino acid
sequence of a framework depends on how the antigen-binding site was
defined.
[0097] "Humanized antibody" refers to an antibody in which the
antigen binding sites are derived from non-human species and the
variable region frameworks are derived from human immunoglobulin
sequences. Humanized antibodies may include substitutions in the
framework regions so that the framework may not be an exact copy of
expressed human immunoglobulin or germline gene sequences.
[0098] "Human-adapted" antibodies or "human framework adapted
(HFA)" antibodies refers to humanized antibodies adapted according
to methods described in U.S. Pat. Publ. No. US2009/0118127.
Human-adapted antibodies are humanized by selecting the acceptor
human frameworks based on the maximum CDR and FR similarities,
length compatibilities and sequence similarities of CDR1 and CDR2
loops and a portion of light chain CDR3 loops.
[0099] "Human antibody" refers to an antibody having heavy and
light chain variable regions in which both the framework and the
antigen binding sites are derived from sequences of human origin.
If the antibody contains a constant region, the constant region
also is derived from sequences of human origin.
[0100] Human antibody comprises heavy or light chain variable
regions that are "derived from" sequences of human origin if the
variable regions of the antibody are obtained from a system that
uses human germline immunoglobulin or rearranged immunoglobulin
genes. Such systems include human immunoglobulin gene libraries
displayed on phage, and transgenic non-human animals such as mice
carrying human immunoglobulin loci as described herein. "Human
antibody" may contain amino acid differences when compared to the
human germline or rearranged immunoglobulin sequences due to for
example naturally occurring somatic mutations or intentional
introduction of substitutions in the framework or antigen binding
sites. Typically, "human antibody" is at least about 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or 100% identical in amino acid sequence to an
amino acid sequence encoded by a human germline or rearranged
immunoglobulin gene. In some cases, "human antibody" may contain
consensus framework sequences derived from human framework sequence
analyses, for example as described in Knappik et al., J Mol Biol
296:57-86, 2000), or synthetic HCDR3 incorporated into human
immunoglobulin gene libraries displayed on phage, for example as
described in Shi et al., J Mol Biol 397:385-96, 2010 and Intl. Pat.
Publ. No. WO2009/085462). Antibodies in which antigen binding sites
are derived from a non-human species are not included in the
definition of "human antibody".
[0101] Isolated humanized antibodies may be synthetic. Human
antibodies, while derived from human immunoglobulin sequences, may
be generated using systems such as phage display incorporating
synthetic CDRs and/or synthetic frameworks, or can be subjected to
in vitro mutagenesis to improve antibody properties, resulting in
antibodies that do not naturally exist within the human antibody
germline repertoire in vivo.
[0102] The term "recombinant antibody" as used herein, includes all
antibodies that are prepared, expressed, created or isolated by
recombinant means, such as antibodies isolated from an animal
(e.g., a mouse) that is transgenic or transchromosomal for human
immunoglobulin genes or a hybridoma prepared therefrom (described
further below), antibodies isolated from a host cell transformed to
express the antibody, antibodies isolated from a recombinant,
combinatorial antibody library, and antibodies prepared, expressed,
created or isolated by any other means that involve splicing of
human immunoglobulin gene sequences to other DNA sequences, or
antibodies that are generated in vitro using Fab arm exchange.
[0103] The term "monoclonal antibody" as used herein refers to a
preparation of antibody molecules of single molecular composition.
A monoclonal antibody composition displays a single binding
specificity and affinity for a particular epitope, or in a case of
a bispecific monoclonal antibody, a dual binding specificity to two
distinct epitopes.
[0104] The term "substantially identical" as used herein means that
the two antibody variable region amino acid sequences being
compared are identical or have "insubstantial differences".
Insubstantial differences are substitutions of 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, or 15 amino acids in an antibody variable
region sequence that do not adversely affect antibody properties
Amino acid sequences substantially identical to the variable region
sequences disclosed herein are within the scope of the invention.
In some embodiments, the sequence identity can be about 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Percent identity
can be determined for example by pairwise alignment using the
default settings of the AlignX module of Vector NTI v.9.0.0
(Invitrogen, Carlsbad, Calif.). The protein sequences of the
present invention can be used as a query sequence to perform a
search against public or patent databases to, for example, identify
related sequences. Exemplary programs used to perform such searches
are the XBLAST or BLASTP programs (http_//wwwncbi_nlm/nih_gov), or
the GenomeQuest.TM. (GenomeQuest, Westborough, Mass.) suite using
the default settings.
[0105] The term "epitope" as used herein means a portion of an
antigen to which an antibody specifically binds. Epitopes usually
consist of chemically active (such as polar, non-polar or
hydrophobic) surface groupings of moieties such as amino acids or
polysaccharide side chains and can have specific three-dimensional
structural characteristics, as well as specific charge
characteristics. An epitope can be composed of contiguous and/or
discontiguous amino acids that form a conformational spatial unit.
For a discontiguous epitope, amino acids from differing portions of
the linear sequence of the antigen come in close proximity in
3-dimensional space through the folding of the protein
molecule.
[0106] The term "in combination with" as used herein means that two
or more therapeutics can be administered to a subject together in a
mixture, concurrently as single agents or sequentially as single
agents in any order.
[0107] The numbering of amino acid residues in the antibody
constant region throughout the specification is performed according
to the EU index as described in Kabat et al., Sequences of Proteins
of Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991), unless otherwise
explicitly stated.
Compositions of Matter
[0108] The present invention provides bispecific agents that
specifically bind EGFR and c-Met. The present invention provides
polypeptides and polynucleotides encoding the bispecific agents of
the invention or complementary nucleic acids thereof, vectors, host
cells, and methods of making and using them.
Monospecific and Bispecific EGFR and/or c-Met FN3 Domain Containing
Binding Molecules
Monospecific EGFR FN3 Domain Containing Binding Molecules
[0109] The present invention provides fibronectin type III (FN3)
domains that bind specifically to epidermal growth factor receptor
(EGFR) and block binding of epidermal growth factor (EGF) to EGFR,
and thus can be widely used in therapeutic and diagnostic
applications. The present invention provides polynucleotides
encoding the FN3 domains of the invention or complementary nucleic
acids thereof, vectors, host cells, and methods of making and using
them.
[0110] The FN3 domains of the invention bind EGFR with high
affinity and inhibit EGFR signaling, and may provide a benefit in
terms of specificity and reduced off-target toxicity when compared
to small molecule EGFR inhibitors, and improved tissue penetration
when compared to conventional antibody therapeutics.
[0111] One embodiment of the invention is an isolated fibronectin
type III (FN3) domain that specifically binds epidermal growth
factor receptor (EGFR) and blocks binding of epidermal growth
factor (EGF) to EGFR.
[0112] The FN3 domains of the invention may block EGF binding to
the EGFR with an IC.sub.50 value of less than about
1.times.10.sup.-7 M, less than about 1.times.10.sup.-8 M, less than
about 1.times.10.sup.-9 M, less than about 1.times.10.sup.-10 M,
less than about 1.times.10.sup.-11 M, or less than about
1.times.10.sup.-12 M in a competition assay employing A431 cells
and detecting amount of fluorescence from bound biotinylated EGF
using streptavidin-phycoerythrin conjugate at 600 nM on A431 cells
incubated with or without the FN3 domains of the invention.
Exemplary FN3 domains may block EGF binding to the EGFR with an
IC.sub.50 value between about 1.times.10.sup.-9 M to about
1.times.10.sup.-7 M, such as EGFR binding FN3 domains having the
amino acid sequence of SEQ ID NOs: 18-29, 107-110, or 122-137. The
FN3 domains of the invention may block EGF binding to the EGFR by
at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% when
compared to binding of EGF to the EGFR in the absence of the FN3
domains of the invention using the same assay conditions.
[0113] The FN3 domain of the invention may inhibit EGFR signaling
by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% when
compared to the level of signaling in the absence of the FN3
domains of the invention using the same assay conditions.
[0114] Binding of a ligand such as EGF to EGFR stimulates receptor
dimerization, autophosphorylation, activation of the receptor's
internal, cytoplasmic tyrosine kinase domain, and initiation of
multiple signal transduction and transactivation pathways involved
in regulation of DNA synthesis (gene activation) and cell cycle
progression or division. Inhibition of EGFR signaling may result in
inhibition in one or more EGFR downstream signaling pathways and
therefore neutralizing EGFR may have various effects, including
inhibition of cell proliferation and differentiation, angiogenesis,
cell motility and metastasis.
[0115] EGFR signaling may be measured using various well know
methods, for example measuring the autophosphorylation of the
receptor at any of the tyrosines Y1068, Y1148, and Y1173 (Downward
et al., Nature 311:483-5, 1984) and/or phosphorylation of natural
or synthetic substrates. Phosphorylation can be detected using well
known methods such as an ELISA assay or a western plot using a
phosphotyrosine specific antibody. Exemplary assays can be found in
Panek et al., J Pharmacol Exp Thera 283:1433-44, 1997 and Batley et
al., Life Sci 62:143-50, 1998, and assays described herein.
[0116] In one embodiment, the FN3 domain of the invention inhibits
EGF-induced EGFR phosphorylation at EGFR residue position Tyrosine
1173 with an IC.sub.50 value of less than about 2.5.times.10.sup.-6
M, for example less than about 1.times.10.sup.-6 M, less than about
1.times.10.sup.-7 M, less than about 1.times.10.sup.-8 M, less than
about 1.times.10.sup.-9 M, less than about 1.times.10.sup.-10 M,
less than about 1.times.10.sup.-11 M, or less than about
1.times.10.sup.-12 M when measured in A431 cells using 50 ng/mL
human EGF.
[0117] In one embodiment, the FN3 domain of the invention inhibits
EGF-induced EGFR phosphorylation at EGFR residue position Tyrosine
1173 with an IC.sub.50 value between about 1.8.times.10.sup.-8 M to
about 2.5.times.10.sup.-6 M when measured in A431 cells using 50
ng/mL human EGF. Such exemplary FN3 domains are those having the
amino acid sequence of SEQ ID NOs: 18-29, 107-110, or 122-137.
[0118] In one embodiment, the FN3 domain of the invention binds
human EGFR with a dissociation constant (K.sub.D) of less than
about 1.times.10.sup.-8 M, for example less than about
1.times.10.sup.-9 M, less than about 1.times.10.sup.-10 M, less
than about 1.times.10.sup.-11 M, less than about 1.times.10.sup.-12
M, or less than about 1.times.10.sup.-13 M as determined by surface
plasmon resonance or the Kinexa method, as practiced by those of
skill in the art. In some embodiments, the FN3 domain of the
invention binds human EGFR with a K.sub.D of between about
2.times.10.sup.-10 to about 1.times.10.sup.-8 M. The affinity of a
FN3 domain for EGFR can be determined experimentally using any
suitable method. (See, for example, Berzofsky, et al.,
"Antibody-Antigen Interactions," In Fundamental Immunology, Paul,
W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby, Janis
Immunology, W. H. Freeman and Company: New York, N.Y. (1992); and
methods described herein). The measured affinity of a particular
FN3 domain-antigen interaction can vary if measured under different
conditions (e.g., osmolarity, pH). Thus, measurements of affinity
and other antigen-binding parameters (e.g., K.sub.D, K.sub.on,
K.sub.off) are preferably made with standardized solutions of
protein scaffold and antigen, and a standardized buffer, such as
the buffer described herein.
[0119] Exemplary FN3 domains of the invention that bind EGFR
include FN3 domains of SEQ ID NOs: 18-29, 107-110, or 122-137.
[0120] In one embodiment, the FN3 domain that specifically binds
EGFR comprises an amino acid sequence at least 87% identical to the
amino acid sequence of SEQ ID NO: 27.
[0121] In one embodiment, the FN3 domain that specifically binds
EGFR comprises
[0122] an FG loop comprising the sequence HNVYKDTNX.sub.9RGL (SEQ
ID NO: 179) or the sequence LGSYVFEHDVML (SEQ ID NO: 180), wherein
X.sub.9 is M or I; and
[0123] a BC loop comprising the sequence
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8 (SEQ ID
NO: 181),
[0124] wherein [0125] X.sub.1 is A, T, G or D; [0126] X.sub.2 is A,
D, Y or W; [0127] X.sub.3 is P, D or N; [0128] X.sub.4 is L or
absent; [0129] X.sub.5 is D, H, R, G, Y or W; [0130] X.sub.6 is G,
D or A; [0131] X.sub.7 is A, F, G, H or D; and [0132] X.sub.8 is Y,
F or L.
[0133] The FN3 domains of the invention that specifically bind EGFR
and inhibit autophosphorylation of EGFR may comprise as a
structural feature an FG loop comprising the sequence
HNVYKDTNX.sub.9RGL (SEQ ID NO: 179) or the sequence LGSYVFEHDVML
(SEQ ID NO: 180), wherein X.sub.9 is M or I. Such FN3 domains may
further comprise a BC loop of 8 or 9 amino acids in length and
defined by the sequence
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8 (SEQ ID
NO: 181), and inhibit EGFR autophosphorylation with an IC.sub.50
value of less than about 2.5.times.10.sup.-6 M, or with an
IC.sub.50 value of between about 1.8.times.10.sup.-8 M to about
2.5.times.10.sup.-6 M when measured in A431 cells using 50 ng/mL
human EGF.
[0134] The FN3 domains of the invention that specifically bind EGFR
and inhibit autophosphorylation of EGFR further comprise the
sequence of
TABLE-US-00001 (SEQ ID NO: 182)
LPAPKNLVVSEVTEDSLRLSWX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.s-
ub.8DSFLIQ YQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGV
HNVYKDTNX.sub.9RGLPLSAEFTT, or the sequence (SEQ ID NO: 183)
LPAPKNLVVSEVTEDSLRLSWX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.s-
ub.8DSFLIQ YQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGV
LGSYVFEHDVMLPLSAEFTT,
wherein [0135] X.sub.1 is A, T, G or D; [0136] X.sub.2 is A, D, Y
or W; [0137] X.sub.3 is P, D or N; [0138] X.sub.4 is L or absent;
[0139] X.sub.5 is D, H, R, G, Y or W; [0140] X.sub.6 is G, D or A;
[0141] X.sub.7 is A, F, G, H or D; [0142] X.sub.8 is Y, F or L; and
[0143] X.sub.9 is M or I
[0144] The EGFR binding FN3 domains can be generated and tested for
their ability to inhibit EGFR autophosphorylation using well known
methods and methods described herein.
[0145] Another embodiment of the invention is an isolated FN3
domain that specifically binds EGFR, wherein the FN3 domain
comprises the sequence shown in SEQ ID NOs: 18-29, 107-110, or
122-137.
[0146] In some embodiments, the EGFR binding FN3 domains comprise
an initiator methionine (Met) linked to the N-terminus or a
cysteine (Cys) linked to a C-terminus of a particular FN3 domain,
for example to facilitate expression and/or conjugation of
half-life extending molecules.
[0147] Another embodiment of the invention is an isolated
fibronectin type III (FN3) domain that specifically binds EGFR and
blocks binding of EGF to the EGFR, wherein the FN3 domain is
isolated from a library designed based on Tencon sequence of SEQ ID
NO: 1.
Monospecific c-Met FN3 Domain Containing Binding Molecules
[0148] The present invention provides fibronectin type III (FN3)
domains that bind specifically to hepatocyte growth factor receptor
(c-Met) and block binding of hepatocyte growth factor (HGF) to
c-Met, and thus can be widely used in therapeutic and diagnostic
applications. The present invention provides polynucleotides
encoding the FN3 domains of the invention or complementary nucleic
acids thereof, vectors, host cells, and methods of making and using
them.
[0149] The FN3 domains of the invention bind c-Met with high
affinity and inhibit c-Met signaling, and may provide a benefit in
terms of specificity and reduced off-target toxicity when compared
to small molecule c-Met inhibitors, and improved tissue penetration
when compared to conventional antibody therapeutics. The FN3
domains of the invention are monovalent, therefore preventing
unwanted receptor clustering and activation that may occur with
other bivalent molecules.
[0150] One embodiment of the invention is an isolated fibronectin
type III (FN3) domain that specifically binds hepatocyte growth
factor receptor (c-Met) and blocks binding of hepatocyte growth
factor (HGF) to c-Met.
[0151] The FN3 domains of the invention may block HGF binding to
c-Met with an IC.sub.50 value of less than about 1.times.10.sup.-7
M, less than about 1.times.10.sup.-8 M, less than about
1.times.10.sup.-9 M, less than about 1.times.10.sup.-10 M, less
than about 1.times.10.sup.-11 M, or less than about
1.times.10.sup.-12 M in an assay detecting inhibition of binding of
biotinylated HGF to c-Met-Fc fusion protein in the presence of the
FN3 domains of the invention. Exemplary FN3 domains may block HGF
binding to the c-Met with an IC.sub.50 value between about
2.times.10.sup.-10 M to about 6.times.10.sup.-8M. The FN3 domains
of the invention may block HGF binding to c-Met by at least 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% when compared to
binding of HGF to c-Met in the absence of the FN3 domains of the
invention using the same assay conditions.
[0152] The FN3 domain of the invention may inhibit c-Met signaling
by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% when
compared to the level of signaling in the absence of FN3 domains of
the invention using the same assay conditions.
[0153] Binding of HGF to c-Met stimulates receptor dimerization,
autophosphorylation, activation of the receptor's internal,
cytoplasmic tyrosine kinase domain, and initiation of multiple
signal transduction and transactivation pathways involved in
regulation of DNA synthesis (gene activation) and cell cycle
progression or division. Inhibition of c-Met signaling may result
in inhibition of one or more c-Met downstream signaling pathways
and therefore neutralizing c-Met may have various effects,
including inhibition of cell proliferation and differentiation,
angiogenesis, cell motility and metastasis.
[0154] c-Met signaling may be measured using various well know
methods, for example measuring the autophosphorylation of the
receptor on at least one tyrosine residues Y1230, Y1234, Y1235 or
Y1349 and/or phosphorylation of natural or synthetic substrates.
Phosphorylation may be detected, for example, using an antibody
specific for phosphotyrosine in an ELISA assay or on a western
blot. Assays for tyrosine kinase activity are described for example
in: Panek et al., J Pharmacol Exp Thera 283:1433-44, 1997 and
Batley et al., Life Sci 62:143-50, 1998, and assays described
herein.
[0155] In one embodiment, the FN3 domain of the invention inhibits
HGF-induced c-Met phosphorylation at c-Met residue position 1349
with an IC.sub.50 value of less than about 1.times.10.sup.-6 M,
less than about 1.times.10.sup.-7 M, less than about
1.times.10.sup.-8 M, less than about 1.times.10.sup.-9 M, less than
about 1.times.10.sup.-10 M, less than about 1.times.10.sup.-11 M,
or less than about 1.times.10.sup.-12 M when measured in NCI-H441
cells using 100 ng/mL recombinant human HGF.
[0156] In one embodiment, the FN3 domain of the invention inhibits
HGF-induced c-Met phosphorylation at c-Met tyrosine Y1349 with an
IC.sub.50 value between about 4.times.10.sup.-9 M to about
1.times.10.sup.-6 M when measured in NCI-H441 cells using 100 ng/mL
recombinant human HGF.
[0157] In one embodiment, the FN3 domain of the invention binds
human c-Met with an dissociation constant (K.sub.D) of equal to or
less than about 1.times.10.sup.-7 M, 1.times.10.sup.-8 M,
1.times.10.sup.-9 M, 1.times.10.sup.-10 M, 1.times.10.sup.-11M,
1.times.10.sup.-12 M, 1.times.10.sup.-13 M, 1.times.10.sup.-14 M,
or 1.times.10.sup.-15M as determined by surface plasmon resonance
or the Kinexa method, as practiced by those of skill in the art. In
some embodiments, the FN3 domain of the invention binds human c-Met
with a K.sub.D of between about 3.times.10.sup.-10 M to about
5.times.10.sup.-8 M. The affinity of a FN3 domain for c-Met may be
determined experimentally using any suitable method. (See, for
example, Berzofsky, et al., "Antibody-Antigen Interactions," In
Fundamental Immunology, Paul, W. E., Ed., Raven Press: New York,
N.Y. (1984); Kuby, Janis Immunology, W. H. Freeman and Company: New
York, N.Y. (1992); and methods described herein). The measured
affinity of a particular FN3 domain-antigen interaction can vary if
measured under different conditions (e.g., osmolarity, pH). Thus,
measurements of affinity and other antigen-binding parameters
(e.g., K.sub.D, K.sub.on, K.sub.off) are preferably made with
standardized solutions of protein scaffold and antigen, and a
standardized buffer, such as the buffer described herein.
[0158] Exemplary FN3 domains of the invention that bind c-Met
include FN3 domains having the amino acid sequence of SEQ ID NOs:
32-49 or 111-114.
[0159] In one embodiment, the FN3 domain that specifically binds
c-Met comprises an amino acid sequence at least 83% identical to
the amino acid sequence of SEQ ID NO: 41.
[0160] In one embodiment, the FN3 domain that specifically binds
c-Met comprises [0161] a C strand and a CD loop comprising the
sequence DSFX.sub.10IRYX.sub.11E [0162]
X.sub.12X.sub.13X.sub.14X.sub.15GX.sub.16 (SEQ ID NO: 184), wherein
[0163] X.sub.10 is W, F or V; [0164] X.sub.11 is D, F or L; [0165]
X.sub.12 is V, F or L; [0166] X.sub.13 is V, L or T; [0167]
X.sub.14 is V, R, G, L, T or S; [0168] X.sub.15 is G, S, A, T or K;
and [0169] X.sub.16 is E or D; and [0170] a F strand and a FG loop
comprising the sequence TEYX.sub.17VX.sub.181X.sub.19X.sub.20V
[0171] KGGX.sub.21X.sub.22SX.sub.23 (SEQ ID NO: 185), wherein
[0172] X.sub.17 is Y, W, I, V, G or A; [0173] X.sub.18 is N, T, Q
or G; [0174] X.sub.19 is L, M, N or I; [0175] X.sub.20 is G or S;
[0176] X.sub.21 is S, L, G, Y, T, R, H or K; [0177] X.sub.22 is I,
V or L; and [0178] X.sub.23 is V, T, H, I, P, Y or L.
[0179] The FN3 domains of the invention that specifically bind
c-Met and inhibit autophosphorylation of c-Met further comprises
the sequence:
TABLE-US-00002 (SEQ ID NO: 186)
LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX.sub.10IRYX.sub.11
EX.sub.12X.sub.13X.sub.14X.sub.15GX.sub.16AIVLTVPGSERSYDLTGLKPGTEYX.sub.1-
7
VX.sub.18IX.sub.19X.sub.20VKGGX.sub.21X.sub.22SX.sub.23PLSAEFTT,
wherein [0180] X.sub.10 is W, F or V; and [0181] X.sub.11 is D, F
or L; [0182] X.sub.12 is V, F or L; [0183] X.sub.13 is V, L or T;
[0184] X.sub.14 is V, R, G, L, T or S; [0185] X.sub.15 is G, S, A,
T or K; [0186] X.sub.16 is E or D; [0187] X.sub.17 is Y, W, I, V, G
or A; [0188] X.sub.18 is N, T, Q or G; [0189] X.sub.19 is L, M, N
or I; [0190] X.sub.20 is G or S; [0191] X.sub.21 is S, L, G, Y, T,
R, H or K; [0192] X.sub.22 is I, V or L; and [0193] X.sub.23 is V,
T, H, I, P, Y or L.
[0194] Another embodiment of the invention is an isolated FN3
domain that specifically binds c-Met, wherein the FN3 domain
comprises the sequence shown in SEQ ID NOs: 32-49 or 111-114.
[0195] Another embodiment of the invention is an isolated
fibronectin type III (FN3) domain that specifically binds c-Met and
blocks binding of HGF to the c-Met, wherein the FN3 domain is
isolated from a library designed based on Tencon sequence of SEQ ID
NO: 1.
Isolation of EGFR or c-Met FN3 Domains from a Library Based on
Tencon Sequence
[0196] Tencon (SEQ ID NO: 1) is a non-naturally occurring
fibronectin type III (FN3) domain designed from a consensus
sequence of fifteen FN3 domains from human tenascin-C (Jacobs et
al., Protein Engineering, Design, and Selection, 25:107-117, 2012;
U.S. Pat. Publ. No. 2010/0216708). The crystal structure of Tencon
shows six surface-exposed loops that connect seven beta-strands as
is characteristic to the FN3 domains, the beta-strands referred to
as A, B, C, D, E, F and G, and the loops referred to as AB, BC, CD,
DE, EF, and FG loops (Bork and Doolittle, Proc Natl Acad Sci USA
89:8990-8992, 1992; U.S. Pat. No. 6,673,901). These loops, or
selected residues within each loop, can be randomized in order to
construct libraries of fibronectin type III (FN3) domains that can
be used to select novel molecules that bind EGFR or c-Met. Table 1
shows positions and sequences of each loop and beta-strand in
Tencon (SEQ ID NO: 1).
[0197] Library designed based on Tencon sequence may thus have
randomized FG loop, or randomized BC and FG loops, such as
libraries TCL1 or TCL2 as described below. The Tencon BC loop is 7
amino acids long, thus 1, 2, 3, 4, 5, 6 or 7 amino acids may be
randomized in the library diversified at the BC loop and designed
based on Tencon sequence. The Tencon FG loop is 7 amino acids long,
thus 1, 2, 3, 4, 5, 6 or 7 amino acids may be randomized in the
library diversified at the FG loop and designed based on Tencon
sequence. Further diversity at loops in the Tencon libraries may be
achieved by insertion and/or deletions of residues at loops. For
example, the FG and/or BC loops may be extended by 1-22 amino
acids, or decreased by 1-3 amino acids. The FG loop in Tencon is 7
amino acids long, whereas the corresponding loop in antibody heavy
chains ranges from 4-28 residues. To provide maximum diversity, the
FG loop may be diversified in sequence as well as in length to
correspond to the antibody CDR3 length range of 4-28 residues. For
example, the FG loop can further be diversified in length by
extending the loop by additional 1, 2, 3, 4 or 5 amino acids.
[0198] Library designed based on Tencon sequence may also have
randomized alternative surfaces that form on a side of the FN3
domain and comprise two or more beta strands, and at least one
loop. One such alternative surface is formed by amino acids in the
C and the F beta-strands and the CD and the FG loops (a C-CD-F-FG
surface). A library design based on Tencon alternative C-CD-F-FG
surface and is shown in FIG. 1 and detailed generation of such
libraries is described in U.S. Pat. Publ. No. US2013/0226834.
[0199] Library designed based on Tencon sequence also includes
libraries designed based on Tencon variants, such as Tencon
variants having substitutions at residues positions 11, 14, 17, 37,
46, 73, or 86 (residue numbering corresponding to SEQ ID NO: 1),
and which variants display improved thermal stability. Exemplary
Tencon variants are described in US Pat. Publ. No. 2011/0274623,
and include Tencon27 (SEQ ID NO: 99) having substitutions E11R,
L17A, N46V and E86I when compared to Tencon of SEQ ID NO: 1.
TABLE-US-00003 TABLE 1 Tencon FN3 domain (SEQ ID NO: 1) A strand
1-12 AB loop 13-16 B strand 17-21 BC loop 22-28 C strand 29-37 CD
loop 38-43 D strand 44-50 DE loop 51-54 E strand 55-59 EF loop
60-64 F strand 65-74 FG loop 75-81 G strand 82-89
[0200] Tencon and other FN3 sequence based libraries can be
randomized at chosen residue positions using a random or defined
set of amino acids. For example, variants in the library having
random substitutions can be generated using NNK codons, which
encode all 20 naturally occurring amino acids. In other
diversification schemes, DVK codons can be used to encode amino
acids Ala, Trp, Tyr, Lys, Thr, Asn, Lys, Ser, Arg, Asp, Glu, Gly,
and Cys. Alternatively, NNS codons can be used to give rise to all
20 amino acid residues and simultaneously reducing the frequency of
stop codons. Libraries of FN3 domains with biased amino acid
distribution at positions to be diversified can be synthesized for
example using Slonomics.RTM. technology (http:_//www_sloning_com).
This technology uses a library of pre-made double stranded triplets
that act as universal building blocks sufficient for thousands of
gene synthesis processes. The triplet library represents all
possible sequence combinations necessary to build any desired DNA
molecule. The codon designations are according to the well known
IUB code.
[0201] The FN3 domains specifically binding EGFR or c-Met of the
invention can be isolated by producing the FN3 library such as the
Tencon library using cis display to ligate DNA fragments encoding
the scaffold proteins to a DNA fragment encoding RepA to generate a
pool of protein-DNA complexes formed after in vitro translation
wherein each protein is stably associated with the DNA that encodes
it (U.S. Pat. No. 7,842,476; Odegrip et al., Proc Natl Acad Sci USA
101, 2806-2810, 2004), and assaying the library for specific
binding to EGFR and/or c-Met by any method known in the art and
described in the Example Exemplary well known methods which can be
used are ELISA, sandwich immunoassays, and competitive and
non-competitive assays (see, e.g., Ausubel et al., eds, 1994,
Current Protocols in Molecular Biology, Vol. 1, John Wiley &
Sons, Inc., New York). The identified FN3 domains specifically
binding EGFR or c-Met are further characterized for their ability
to block EGFR ligand such as EGF binding to EGFR, or HGF binding to
c-Met, and for their ability to inhibit EGFR and/or c-Met signaling
using methods described herein.
[0202] The FN3 domains specifically binding to EGFR or c-Met of the
invention can be generated using any FN3 domain as a template to
generate a library and screening the library for molecules
specifically binding EGFR or c-Met using methods provided within.
Exemplar FN3 domains that can be used are the 3rd FN3 domain of
tenascin C (TN3) (SEQ ID NO: 75), Fibcon (SEQ ID NO: 76), and the
10.sup.th FN3 domain of fibronectin (FN10) (SEQ ID NO: 77).
Standard cloning and expression techniques are used to clone the
libraries into a vector or synthesize double stranded cDNA
cassettes of the library, to express, or to translate the libraries
in vitro. For example ribosome display (Hanes and Pluckthun, Proc
Natl Acad Sci USA, 94, 4937-4942, 1997), mRNA display (Roberts and
Szostak, Proc Natl Acad Sci USA, 94, 12297-12302, 1997), or other
cell-free systems (U.S. Pat. No. 5,643,768) can be used. The
libraries of the FN3 domain variants may be expressed as fusion
proteins displayed on the surface for example of any suitable
bacteriophage. Methods for displaying fusion polypeptides on the
surface of a bacteriophage are well known (U. S. Pat. Publ. No.
2011/0118144; Int. Pat. Publ. No. WO2009/085462; U.S. Pat. No.
6,969,108; U.S. Pat. No. 6,172,197; U.S. Pat. No. 5,223,409; U.S.
Pat. No. 6,582,915; U.S. Pat. No. 6,472,147).
[0203] The FN3 domains specifically binding EGFR or c-Met of the
invention can be modified to improve their properties such as
improve thermal stability and reversibility of thermal folding and
unfolding. Several methods have been applied to increase the
apparent thermal stability of proteins and enzymes, including
rational design based on comparison to highly similar thermostable
sequences, design of stabilizing disulfide bridges, mutations to
increase alpha-helix propensity, engineering of salt bridges,
alteration of the surface charge of the protein, directed
evolution, and composition of consensus sequences (Lehmann and
Wyss, Curr Opin Biotechnol, 12, 371-375, 2001). High thermal
stability may increase the yield of the expressed protein, improve
solubility or activity, decrease immunogenicity, and minimize the
need of a cold chain in manufacturing. Residues that can be
substituted to improve thermal stability of Tencon (SEQ ID NO: 1)
are residue positions 11, 14, 17, 37, 46, 73, or 86, and are
described in US Pat. Publ. No. 2011/0274623. Substitutions
corresponding to these residues can be incorporated to the FN3
domains or the bispecific FN3 domain containing molecules of the
invention.
[0204] Another embodiment of the invention is an isolated FN3
domain that specifically binds EGFR and blocks binding of EGF to
EGFR, comprising the sequence shown in SEQ ID NOs: 18-29, 107-110,
122-137, further comprising substitutions at one or more residue
positions corresponding to positions 11, 14, 17, 37, 46, 73, and 86
in Tencon (SEQ ID NO: 1).
[0205] Another embodiment of the invention is an isolated FN3
domain that specifically binds c-Met and blocks binding of HGF to
c-Met, comprising the sequence shown in SEQ ID NOs: 32-49 or
111-114, further comprising substitutions at one or more residue
positions corresponding to positions 11, 14, 17, 37, 46, 73, and 86
in Tencon (SEQ ID NO: 1).
[0206] Exemplary substitutions are substitutions E11N, E14P, L17A,
E37P, N46V, G73Y and E86I (numbering according to SEQ ID NO:
1).
[0207] In some embodiments, the FN3 domains of the invention
comprise substitutions corresponding to substitutions L17A, N46V,
and E86I in Tencon (SEQ ID NO: 1).
[0208] The FN3 domains specifically binding EGFR (FIG. 1) have an
extended FG loop when compared to Tencon (SEQ ID NO: 1). Therefore,
the residues corresponding to residues 11, 14, 17, 37, 46, 73, and
86 in Tencon (SEQ ID NO: 1) are residues 11, 14, 17, 37, 46, 73 and
91 in EGFR FN3 domains shown in FIGS. 1A and 1B except for the FN3
domain of SEQ ID NO: 24, wherein the corresponding residues are
residues 11, 14, 17, 38, 74, and 92 due to an insertion of one
amino acid in the BC loop.
[0209] Another embodiment of the invention is an isolated FN3
domain that specifically binds EGFR and blocks binding of EGF to
EGFR comprising the amino acid sequence shown in SEQ ID NOs: 18-29,
107-110, or 122-137, optionally having substitutions corresponding
to substitutions L17A, N46V, and E86I in Tencon (SEQ ID NO: 1).
[0210] Another embodiment of the invention is an isolated FN3
domain that specifically binds c-Met and blocks binding of HGF to
c-Met comprising the amino acid sequence shown in SEQ ID NOs: 32-49
or 111-114, optionally having substitutions corresponding to
substitutions L17A, N46V, and E86I in Tencon (SEQ ID NO: 1).
[0211] Measurement of protein stability and protein lability can be
viewed as the same or different aspects of protein integrity.
Proteins are sensitive or "labile" to denaturation caused by heat,
by ultraviolet or ionizing radiation, changes in the ambient
osmolarity and pH if in liquid solution, mechanical shear force
imposed by small pore-size filtration, ultraviolet radiation,
ionizing radiation, such as by gamma irradiation, chemical or heat
dehydration, or any other action or force that may cause protein
structure disruption. The stability of the molecule can be
determined using standard methods. For example, the stability of a
molecule can be determined by measuring the thermal melting ("TM")
temperature, the temperature in .degree. Celsius (.degree. C.) at
which half of the molecules become unfolded, using standard
methods. Typically, the higher the TM, the more stable the
molecule. In addition to heat, the chemical environment also
changes the ability of the protein to maintain a particular three
dimensional structure.
[0212] In one embodiment, the FN3 domains binding EGFR or c-Met of
the invention exhibit increased stability by at least 5%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, or 95% or more compared to the same domain prior to
engineering measured by the increase in the TM.
[0213] Chemical denaturation can likewise be measured by a variety
of methods. Chemical denaturants include guanidinium hydrochloride,
guanidinium thiocyanate, urea, acetone, organic solvents (DMF,
benzene, acetonitrile), salts (ammonium sulfate, lithium bromide,
lithium chloride, sodium bromide, calcium chloride, sodium
chloride); reducing agents (e.g. dithiothreitol,
beta-mercaptoethanol, dinitrothiobenzene, and hydrides, such as
sodium borohydride), non-ionic and ionic detergents, acids (e.g.
hydrochloric acid (HC1), acetic acid (CH.sub.3COOH), halogenated
acetic acids), hydrophobic molecules (e.g. phosopholipids), and
targeted denaturants. Quantitation of the extent of denaturation
can rely on loss of a functional property, such as ability to bind
a target molecule, or by physiochemical properties, such as
tendency to aggregation, exposure of formerly solvent inaccessible
residues, or disruption or formation of disulfide bonds.
[0214] In one embodiment, the FN3 domain of the invention binding
EGFR or c-Met exhibit increased stability by at least 5%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, or 95% or more compared to the same scaffold prior to
engineering, measured by using guanidinium hydrochloride as a
chemical denaturant. Increased stability can be measured as a
function of decreased tryptophan fluorescence upon treatment with
increasing concentrations of guanidine hydrochloride using well
known methods.
[0215] The FN3 domains of the invention may be generated as
monomers, dimers, or multimers, for example, as a means to increase
the valency and thus the avidity of target molecule binding, or to
generate bi- or multispecific scaffolds simultaneously binding two
or more different target molecules. The dimers and multimers may be
generated by linking monospecific, bi- or multispecific protein
scaffolds, for example, by the inclusion of an amino acid linker,
for example a linker containing poly-glycine, glycine and serine,
or alanine and proline. Exemplary linker include (GS).sub.2, (SEQ
ID NO: 78), (GGGGS).sub.5 (SEQ ID NO: 79), (AP).sub.2 (SEQ ID NO:
80), (AP).sub.5 (SEQ ID NO: 81), (AP).sub.10 (SEQ ID NO: 82),
(AP).sub.20 (SEQ ID NO: 83) and A(EAAAK).sub.5AAA (SEQ ID NO: 84),
linkers. The dimers and multimers may be linked to each other in an
N- to C-direction. The use of naturally occurring as well as
artificial peptide linkers to connect polypeptides into novel
linked fusion polypeptides is well known in the literature
(Hallewell et al., J Biol Chem 264, 5260-5268, 1989; Alfthan et
al., Protein Eng. 8, 725-731, 1995; Robinson & Sauer,
Biochemistry 35, 109-116, 1996; U.S. Pat. No. 5,856,456).
Bispecific Agents Specifically Binding EGFR and c-Met
[0216] The bispecific agents that specifically bind EGFR and c-Met
of the invention may provide a benefit in terms of specificity and
reduced off-target toxicity when compared to small molecule EGFR
and/or c-Met inhibitors. The present invention is based at least in
part on the surprising finding that the bispecific agents
specifically binding EGFR and c-Met provide a significantly
improved synergistic inhibitory effect when compared to a mixture
of EGFR-binding and c-Met-binding monospecific agents. The
molecules may be tailored to specific affinity towards both EGFR
and c-Met to maximize tumor penetration and retention. The
bispecific agents that specifically bind EGFR and c-Met provide
more efficient inhibition of EGFR and/or c-Met signaling pathways
and inhibit tumor growth more efficiently than cetuximab
(Erbitux.RTM.).
[0217] The bispecific agents specifically binding EGFR and c-Met
may be formed by any polypeptide or a multimeric polypeptide that
comprises an EGFR binding domain and a c-Met binding domain. The
EGFR and the c-Met binding domains may be an antigen binding sites
of an antibody, a VH/VL pair of an antibody, or another type of
binding molecule such as a domain based on fibronectin type III
(FN3) domain, a fibronectin type IX (FN9) domain, or any
combination thereof.
[0218] The EGFR and c-Met binding polypeptides may be derived from
existing monospecific EGFR and c-Met binding polypeptides or may be
isolated de novo.
Bispecific EGFR/c/Met FN3 Domain Containing Molecules
[0219] One embodiment of the invention is an isolated bispecific
FN3 domain containing molecule comprising a first fibronectin type
III (FN3) domain and a second FN3 domain, wherein the first FN3
domain specifically binds epidermal growth factor receptor (EGFR)
and blocks binding of epidermal growth factor (EGF) to EGFR, and
the second FN3 domain specifically binds hepatocyte growth factor
receptor (c-Met) and blocks binding of hepatocyte growth factor
(HGF) to c-Met.
[0220] The bispecific EGFR/c-Met FN3 domain containing molecules of
the invention may be generated by covalently linking any
EGFR-binding FN3 domain and any c-Met-binding FN3 domain of the
invention directly or via a linker. Therefore, the first FN3 domain
of the bispecific molecule may have characteristics as described
above for the EGFR-binding FN3 domains, and the second FN3 domain
of the bispecific molecule may have characteristics as described
above for the c-Met-binding FN3 domains.
[0221] In one embodiment, the first FN3 domain of the bispecific
EGFR/c-Met FN3 domain containing molecule inhibits EGF-induced EGFR
phosphorylation at EGFR residue Tyrosine 1173 with an IC.sub.50
value of less than about 2.5.times.10.sup.-6 M when measured in
A431 cells using 50 ng/mL human EGF, and the second FN3 domain of
the bispecific EGFR/c-Met FN3 domain containing molecule inhibits
HGF-induced c-Met phosphorylation at c-Met residue Tyrosine 1349
with an IC.sub.50 value of less than about 1.5.times.10.sup.-6 M
when measured in NCI-H441 cells using 100 ng/mL human HGF.
[0222] In another embodiment, the first FN3 domain of the
bispecific EGFR/c-Met FN3 domain containing molecule inhibits
EGF-induced EGFR phosphorylation at EGFR residue Tyrosine 1173 with
an IC.sub.50 value of between about 1.8.times.10.sup.-8 M to about
2.5.times.10.sup.-6 M when measured in A431 cells using 50 ng/mL
human EGF, and the second FN3 domain of the bispecific EGFR/c-Met
FN3 domain containing molecule inhibits HGF-induced c-Met
phosphorylation at c-Met residue Tyrosine 1349 with an IC.sub.50
value between about 4.times.10.sup.-9 M to about
1.5.times.10.sup.-6 M when measured in NCI-H441 cells using 100
ng/mL human HGF.
[0223] In another embodiment, the first FN3 domain of the
bispecific EGFR/c-Met FN3 domain containing molecule binds human
EGFR with a dissociation constant (K.sub.D) of less than about
1.times.10.sup.-8 M, and the second FN3 domain of the bispecific
EGFR/c-Met FN3 domain containing molecule binds human c-Met with a
K.sub.D of less than about 5.times.10.sup.-8 M.
[0224] In the bispecific molecule binding both EGFR and c-Met, the
first FN3 domain binds human EGFR with a K.sub.D of between about
2.times.10.sup.-10 to about 1.times.10.sup.-8 M, and the second FN3
domain binds human c-Met with a K.sub.D of between about
3.times.10.sup.-10 to about 5.times.10.sup.-8 M.
[0225] The affinity of the bispecific EGFR/c-Met molecule for EGFR
and c-Met can be determined as described above for the monospecific
molecules.
[0226] The first FN3 domain in the bispecific EGFR/c-Met molecule
of the invention may block EGF binding to EGFR with an IC.sub.50
value of between about 1.times.10.sup.-9 M to about
1.5.times.10.sup.-7 M in an assay employing A431 cells and
detecting the amount of fluorescence from bound biotinylated EGF
using streptavidin-phycoerythrin conjugate at 600 nM on A431 cells
incubated with or without the first FN3 domain. The first FN3
domain in the bispecific EGFR/c-Met molecule of the invention may
block EGF binding to the EGFR by at least 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or 100% when compared to binding of EGF to EGFR
in the absence of the first FN3 domains using the same assay
conditions.
[0227] The second FN3 domain in the bispecific EGFR/c-Met molecule
of the invention may block HGF binding to c-Met with an IC.sub.50
value of between about 2.times.10.sup.-10 M to about
6.times.10.sup.-8 M in an assay detecting inhibition of binding of
biotinylated HGF to c-Met-Fc fusion protein in the presence of the
second FN3 domain. The second FN3 domain in the bispecific
EGFR/c-Met molecule may block HGF binding to c-Met by at least 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% when compared to
binding of HGF to c-Met in the absence of the second FN3 domain
using the same assay conditions.
[0228] The bispecific EGFR/c-Met molecule of the invention may
inhibit EGFR and/or c-Met signaling by at least 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or 100% when compared to the level of
signaling in the absence of the bispecific EGFR/c-Met molecule of
the invention using the same assay conditions.
[0229] EGFR and c-Met signaling may be measured using various well
know methods as described above for the monospecific molecules.
[0230] The bispecific EGFR/c-Met molecules of the invention
comprising the first FN3 domain specifically binding EGFR and the
second FN3 domain specifically binding c-Met provide a
significantly increased synergistic inhibition of EGFR and c-Met
signaling and tumor cell proliferation when compared to the
synergistic inhibition observed by a mixture of the first and the
second FN3 domain. Synergistic inhibition can be assessed for
example by measuring inhibition of ERK phosphorylation by the
bispecific EGFR/c-Met FN3 domain containing molecules and by a
mixture of two monospecific molecules, one binding EGFR and the
other c-Met. The bispecific EGFR/c-Met molecules of the invention
may inhibit ERK phosphorylation with an at least about 100 fold
smaller, for example at least 500, 1000, 5000 or 10,000 fold
smaller IC.sub.50 value when compared to the IC.sub.50 value for a
mixture of two monospecific FN3 domains, indicating at least 100
fold increased potency for the bispecific EGFR/c-Met FN3 domain
containing molecules when compared to the mixture of two
monospecific FN3 domains. Exemplary bispecific EGFR-c-Met FN3
domain containing molecules may inhibit ERK phosphorylation with
and IC.sub.50 value of about 5.times.10.sup.-9 M or less. ERK
phosphorylation may be measured using standard methods and methods
described herein.
[0231] The bispecific EGFR/c-Met FN3 domain containing molecule of
the invention may inhibit NCI-H292 cell proliferation with an
IC.sub.50 value that is at least 30-fold less when compared to the
IC.sub.50 value of inhibition of NCI-H292 cell growth with a
mixture of the first FN3 domain and the second FN3, wherein the
cell proliferation is induced with medium containing 10% FBS
supplemented with 7.5 ng/mL HGF. The bispecific molecule of the
invention may inhibit tumor cell proliferation with an IC.sub.50
value that is about 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300,
400, 500, 600, 700, 800, or about 1000 fold less when compared to
the IC.sub.50 value of inhibition of tumor cell proliferation with
a mixture of the first FN3 domain and the second FN3 domain.
Inhibition of tumor cell proliferation may be measured using
standard methods and methods described herein.
[0232] Another embodiment of the invention is a bispecific FN3
domain containing molecule comprising a first fibronectin type III
(FN3) domain and a second FN3 domain, wherein the first FN3 domain
specifically binds epidermal growth factor receptor (EGFR) and
blocks binding of epidermal growth factor (EGF) to EGFR, and the
second FN3 domain specifically binds hepatocyte growth factor
receptor (c-Met), and blocks binding of hepatocyte growth factor
(HGF) to c-Met, wherein
[0233] the first FN3 domain comprises [0234] an FG loop comprising
the sequence HNVYKDTNX.sub.9RGL (SEQ ID NO: 179) or the sequence
LGSYVFEHDVML (SEQ ID NO: 180), wherein X.sub.9 is M or I; [0235]
and [0236] a BC loop comprising the sequence
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8(SEQ ID NO:
181), [0237] wherein [0238] X.sub.1 is A, T, G or D; [0239] X.sub.2
is A, D, Y or W; [0240] X.sub.3 is P, D or N; [0241] X.sub.4 is L
or absent; [0242] X.sub.5 is D, H, R, G, Y or W; [0243] X.sub.6 is
G, D or A; [0244] X.sub.7 is A, F, G, H or D; and [0245] X.sub.8 is
Y, F or L; and
[0246] the second FN3 domain comprises [0247] a C strand and a CD
loop comprising the sequence DSFX.sub.10IRYX.sub.11E [0248]
X.sub.12X.sub.13X.sub.14X.sub.15GX.sub.16 (SEQ ID NO: 184), wherein
[0249] X.sub.10 is W, F or V; [0250] X.sub.11 is D, F or L; [0251]
X.sub.12 is V, F or L; [0252] X.sub.13 is V, L or T; [0253]
X.sub.14 is V, R, G, L, T or S; [0254] X.sub.15 is G, S, A, T or K;
and [0255] X.sub.16 is E or D; and [0256] a F strand and a FG loop
comprising the sequence TEYX.sub.17VX.sub.18IX.sub.19X.sub.20V
[0257] KGGX.sub.21X.sub.22SX.sub.23 (SEQ ID NO: 185), wherein
[0258] X.sub.17 is Y, W, I, V, G or A; [0259] X.sub.18 is N, T, Q
or G; [0260] X.sub.19 is L, M, N or I; [0261] X.sub.20 is G or S;
[0262] X.sub.21 is S, L, G, Y, T, R, H or K; [0263] X.sub.22 is I,
V or L; and [0264] X.sub.23 is V, T, H, I, P, Y or L.
[0265] In another embodiment, the bispecific molecule comprises the
first FN3 domain that binds EGFR comprising the sequence:
TABLE-US-00004 (SEQ ID NO: 182)
LPAPKNLVVSEVTEDSLRLSWX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.s-
ub.8DSFLIQ YQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGV
HNVYKDTNX.sub.9RGLPLSAEFTT, or the sequence (SEQ ID NO: 183)
LPAPKNLVVSEVTEDSLRLSWX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.s-
ub.8DSFLIQ YQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGV
LGSYVFEHDVMLPLSAEFTT,
wherein in the SEQ ID NOs: 182 and 183; [0266] X.sub.1 is A, T, G
or D; [0267] X.sub.2 is A, D, Y or W; [0268] X.sub.3 is P, D or N;
[0269] X.sub.4 is L or absent; [0270] X.sub.5 is D, H, R, G, Y or
W; [0271] X.sub.6 is G, D or A; [0272] X.sub.7 is A, F, G, H or D;
[0273] X.sub.8 is Y, F or L; and [0274] X.sub.9 is M or I.
[0275] In another embodiment, the bispecific molecule comprises the
second FN3 domain that binds c-Met comprising the sequence
TABLE-US-00005 (SEQ ID NO: 186)
LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX.sub.10IRYX.sub.11E
X.sub.12X.sub.13X.sub.14X.sub.15GX.sub.16AIVLTVPGSERSYDLTGLKPGTEYX.sub.17-
V
X.sub.18IX.sub.19X.sub.20VKGGX.sub.21X.sub.22SX.sub.23PLSAEFTT,
wherein [0276] X.sub.10 is W, F or V; and [0277] X.sub.11 is D, F
or L; [0278] X.sub.12 is V, F or L; [0279] X.sub.13 is V, L or T;
[0280] X.sub.14 is V, R, G, L, T or S; [0281] X.sub.15 is G, S, A,
T or K; [0282] X.sub.16 is E or D; [0283] X.sub.17 is Y, W, I, V, G
or A; [0284] X.sub.18 is N, T, Q or G; [0285] X.sub.19 is L, M, N
or I; [0286] X.sub.20 is G or S; [0287] X.sub.21 is S, L, G, Y, T,
R, H or K; [0288] X.sub.22 is I, V or L; and [0289] X.sub.23 is V,
T, H, I, P, Y, T or L.
[0290] Exemplary bispecific EGFR/c-Met FN3 domain containing
molecules comprise the amino acid sequence shown in SEQ ID NOs:
50-72, 106, 118-121, or 138-167.
[0291] The bispecific EGFR/c-Met molecules of the invention
comprise certain structural characteristics associated with their
functional characteristics, such as inhibition of EGFR
autophosphorylation, such as the FG loop of the first FN3 domain
that binds EGFR comprising the sequence HNVYKDTNX.sub.9RGL (SEQ ID
NO: 179) or the sequence LGSYVFEHDVML (SEQ ID NO: 180), wherein
X.sub.9 is M or I.
[0292] In one embodiment, the bispecific EGFR/c-Met FN3 domain
containing molecules of the invention
[0293] inhibit EGF-induced EGFR phosphorylation at EGFR residues
Tyrosine 1173 with an IC.sub.50 value of less than about
8.times.10.sup.-7 M when measured in H292 cells using 50 ng/mL
human EGF;
inhibit HGF-induced c-Met phosphorylation at c-Met residue Tyrosine
1349 with an IC.sub.50 value of less than about 8.4.times.10.sup.-7
M when measured in NCI-H441 cells using 100 ng/mL human HGF;
[0294] inhibit HGF-induced NCI-H292 cell proliferation with an
IC.sub.50 value of less than about 9.5.times.10.sup.-6M wherein the
cell proliferation is induced with 10% FBS containing 7.5 ng
HGF;
[0295] bind EGFR with a K.sub.D of less than about
2.0.times.10.sup.-8 M; or
[0296] bind c-Met with a K.sub.D of less than about
2.0.times.10.sup.-8 M.
[0297] In another embodiment, the bispecific EGFR/c-Met FN3 domain
containing molecules of the invention
inhibit EGF-induced EGFR phosphorylation at EGFR residues Tyrosine
1173 with and IC.sub.50 of between about 4.2.times.10.sup.-9 M and
8.times.10.sup.-7 M when measured in H292 cells using 50 ng/mL
human EGF;
[0298] inhibit HGF-induced c-Met phosphorylation at c-Met residues
Tyrosine 1349 with an IC.sub.50 value of between about
2.4.times.10.sup.-8 M to about 8.4.times.10.sup.-7 M when measured
in NCI-H441 cells using 100 ng/mL human HGF;
[0299] inhibit HGF-induced NCI-H292 cell proliferation with an
IC.sub.50 value between about 2.3.times.10.sup.-8 M to about
9.5.times.10.sup.-6M wherein the cell proliferation is induced with
10% FBS containing 7.5 ng HGF;
[0300] bind EGFR with a K.sub.D of between about 2.times.10.sup.-10
M to about 2.0.times.10.sup.-8 M; or
[0301] bind c-Met with a K.sub.D of between about 1.times.10.sup.-9
M to about 2.0.times.10.sup.-8 M.
[0302] In one embodiment, the bispecific EGFR/c-Met molecules
comprise the EGFR-binding FN3 domain comprising the sequence
TABLE-US-00006 (SEQ ID NO: 182)
LPAPKNLVVSEVTEDSLRLSWX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.s-
ub.8DSFLIQ YQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGV
HNVYKDTNX.sub.9RGLPLSAEFTT,
wherein [0303] X.sub.1 is D; [0304] X.sub.2 is D; [0305] X.sub.3 is
P; [0306] X.sub.4 is absent; [0307] X.sub.5 is H or W; [0308]
X.sub.6 is A; [0309] X.sub.7 is F [0310] X.sub.8 is Y; and [0311]
X.sub.9 is M or I; and
[0312] the c-Met-binding FN3 domain comprising the sequence
TABLE-US-00007 (SEQ ID NO: 186)
LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX.sub.10IRYX.sub.11E
X.sub.12X.sub.13X.sub.14X.sub.15GX.sub.16AIVLTVPGSERSYDLTGLKPGTEYX.sub.17-
V
X.sub.18IX.sub.19X.sub.20VKGGX.sub.21X.sub.22SX.sub.23PLSAEFTT,
wherein [0313] X.sub.10 is W; [0314] X.sub.11 is F; [0315] X.sub.12
is F; [0316] X.sub.13 is V or L; [0317] X.sub.14 is G or S; [0318]
X.sub.15 is S or K; [0319] X.sub.16 is E or D; [0320] X.sub.17 is
V; [0321] X.sub.18 is N; [0322] X.sub.19 is L or M; [0323] X.sub.20
is G or S; [0324] X.sub.21 is S or K; [0325] X.sub.22 is I; and
[0326] X.sub.23 is P.
[0327] Exemplary bispecific EGFR/c-Met molecules are those having
the sequence shown in SEQ ID NOs: 57, 61, 62, 63, 64, 65, 66, 67
and 68.
[0328] The bispecific molecules of the invention may further
comprise substitutions at one or more residue positions in the
first FN3 domain and/or the second FN3 domain corresponding to
positions 11, 14, 17, 37, 46, 73 and 86 in Tencon (SEQ ID NO: 1) as
described above, and a substitution at position 29. Exemplary
substitutions are substitutions E11N, E14P, L17A, E37P, N46V, G73Y,
E86I and D29E (numbering according to SEQ ID NO: 1). Skilled in the
art will appreciate that other amino acids can be used for
substitutions, such as amino acids within a family of amino acids
that are related in their side chains as described infra. The
generated variants can be tested for their stability and binding to
EGFR and/or c-Met using methods herein.
[0329] In one embodiment, the bispecific EGFR/c-Met FN3 domain
containing molecule comprises the first FN3 domain that binds
specifically EGFR and the second FN3 domain that binds specifically
c-Met, wherein the first FN3 domain comprises the sequence:
TABLE-US-00008 (SEQ ID NO: 187)
LPAPKNLVVSX.sub.24VTX.sub.25DSX.sub.26RLSWDDPX.sub.27AFYX.sub.28SFLIQ
YQX.sub.29SEKVGEAIX.sub.30LTVPGSERSYDLTGLKPGTEYTVSIY
X.sub.31VHNVYKDTNX.sub.32RGLPLSAX.sub.33FTT,
wherein [0330] X.sub.24 is E, N or R; [0331] X.sub.25 is E or P;
[0332] X.sub.26 is L or A; [0333] X.sub.27 is H or W; [0334]
X.sub.28 is E or D; [0335] X.sub.29 is E or P; [0336] X.sub.30 is N
or V; [0337] X.sub.31 is G or Y; [0338] X.sub.32 is M or I; and
[0339] X.sub.33 is E or I;
[0340] and the second FN3 domain comprises the sequence:
TABLE-US-00009 (SEQ ID NO: 188)
LPAPKNLVVSX.sub.34VTX.sub.35DSX.sub.36RLSWTAPDAAFDSFWIRYF
X.sub.37FX.sub.38X.sub.39X.sub.40GX.sub.41AIX.sub.42LTVPGSERSYDLTGLKPGTEY-
V VNIX.sub.43X.sub.44VKGGX.sub.45ISPPLSAX.sub.46FTT;
wherein
X.sub.34 is E, N or R;
X.sub.35 is E or P;
X.sub.36 is L or A;
X.sub.37 is E or P;
X.sub.38 is V or L;
X.sub.39 is G or S;
X.sub.40 is S or K;
X.sub.41 is E or D;
X.sub.42 is N or V;
X.sub.43 is L or M;
X.sub.44 is G or S;
X.sub.45 is S or K; and
X.sub.46 is E or I.
[0341] In other embodiments, the bispecific EGFR/c-Met FN3 domain
containing molecule comprises the first FN3 domain comprising an
amino acid sequence at least 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence
of SEQ ID NO: 27, and the second FN3 domain comprising an amino
acid sequence at least 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino
acid sequence of SEQ ID NO: 41.
[0342] The bispecific EGFR/c-Met FN3 domain containing molecules of
the invention may be tailored to a specific affinity towards EGFR
and c-Met to maximize tumor accumulation.
[0343] Another embodiment of the invention is an isolated
bispecific FN3 domain containing molecule comprising a first
fibronectin type III (FN3) domain and a second FN3 domain, wherein
the first FN3 domain specifically binds epidermal growth factor
receptor (EGFR) and blocks binding of epidermal growth factor (EGF)
to EGFR, and the second FN3 domain specifically binds hepatocyte
growth factor receptor (c-Met), and blocks binding of hepatocyte
growth factor (HGF) to c-Met, wherein the first FN3 domain and the
second FN3 domain is isolated from a library designed based on
Tencon sequence of SEQ ID NO: 1.
[0344] The bispecific EGFR/c-Met FN3 domain containing molecule of
the invention can be generated by covalently coupling the
EGFR-binding FN3 domain and the c-Met binding FN3 domain of the
invention using well known methods. The FN3 domains may be linked
via a linker, for example a linker containing poly-glycine, glycine
and serine, or alanine and proline. Exemplary linker include
(GS).sub.2, (SEQ ID NO: 78), (GGGGS).sub.5 (SEQ ID NO: 79),
(AP).sub.2 (SEQ ID NO: 80), (AP).sub.5 (SEQ ID NO: 81), (AP).sub.10
(SEQ ID NO: 82), (AP).sub.20 (SEQ ID NO: 83), A(EAAAK).sub.5AAA
(SEQ ID NO: 84), linkers. The use of naturally occurring as well as
artificial peptide linkers to connect polypeptides into novel
linked fusion polypeptides is well known in the literature
(Hallewell et al., J Biol Chem 264, 5260-5268, 1989; Alfthan et
al., Protein Eng. 8, 725-731, 1995; Robinson & Sauer,
Biochemistry 35, 109-116, 1996; U.S. Pat. No. 5,856,456). The
bispecific EGFR/c-Met molecules of the invention may be linked
together from a C-terminus of the first FN3 domain to the
N-terminus of the second FN3 domain, or from the C-terminus of the
second FN3 domain to the N-terminus of the first FN3 domain. Any
EGFR-binding FN3 domain may be covalently linked to a c-Met-binding
FN3 domain. Exemplary EGFR-binding FN3 domains are domains having
the amino acid sequence shown in SEQ ID NOs: 18-29, 107-110, and
122-137, and exemplary c-Met binding FN3 domains are domains having
the amino acid sequence shown in SEQ ID NOs: 32-49 and 111-114. The
EGFR-binding FN3 domains to be coupled to a bispecific molecule may
additionally comprise an initiator methionine (Met) at their
N-terminus.
[0345] Variants of the bispecific EGFR/c-Met FN3 domain containing
molecules are within the scope of the invention. For example,
substitutions can be made in the bispecific EGFR/c-Met FN3 domain
containing molecule as long as the resulting variant retains
similar selectivity and potency towards EGFR and c-Met when
compared to the parent molecule. Exemplary modifications are for
example conservative substitutions that will result in variants
with similar characteristics to those of the parent molecules.
Conservative substitutions are those that take place within a
family of amino acids that are related in their side chains.
Genetically encoded amino acids can be divided into four families:
(1) acidic (aspartate, glutamate); (2) basic (lysine, arginine,
histidine); (3) nonpolar (alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine, tryptophan); and (4) uncharged
polar (glycine, asparagine, glutamine, cysteine, serine, threonine,
tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes
classified jointly as aromatic amino acids. Alternatively, the
amino acid repertoire can be grouped as (1) acidic (aspartate,
glutamate); (2) basic (lysine, arginine histidine), (3) aliphatic
(glycine, alanine, valine, leucine, isoleucine, serine, threonine),
with serine and threonine optionally be grouped separately as
aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine,
tryptophan); (5) amide (asparagine, glutamine); and (6)
sulfur-containing (cysteine and methionine) (Stryer (ed.),
Biochemistry, 2nd ed, WH Freeman and Co., 1981). Non-conservative
substitutions can be made to the bispecific EGFR/c-Met FN3 domain
containing molecule that involves substitutions of amino acid
residues between different classes of amino acids to improve
properties of the bispecific molecules. Whether a change in the
amino acid sequence of a polypeptide or fragment thereof results in
a functional homolog can be readily determined by assessing the
ability of the modified polypeptide or fragment to produce a
response in a fashion similar to the unmodified polypeptide or
fragment using the assays described herein. Peptides, polypeptides
or proteins in which more than one replacement has taken place can
readily be tested in the same manner.
[0346] The bispecific EGFR/c-Met FN3 domain containing molecules of
the invention may be generated as dimers or multimers, for example,
as a means to increase the valency and thus the avidity of target
molecule binding. The multimers may be generated by linking one or
more EGFR-binding FN3 domain and one or more c-Met-binding FN3
domain to form molecules comprising at least three individual FN3
domains that are at least bispecific for either EGFR or c-Met, for
example by the inclusion of an amino acid linker using well known
methods.
[0347] Another embodiment of the invention is a bispecific FN3
domain containing molecule comprising a first fibronectin type III
(FN3) domain and a second FN3 domain, wherein the first FN3 domain
specifically binds epidermal growth factor receptor (EGFR) and
blocks binding of epidermal growth factor (EGF) to EGFR, and the
second FN3 domain specifically binds hepatocyte growth factor
receptor (c-Met), and blocks binding of hepatocyte growth factor
(HGF) to c-Met comprising the amino acid sequence shown in SEQ ID
NOs: 50-72,106 or 138-165.
Half-Life Extending Moieties
[0348] The bispecific EGFR/c-Met FN3 domain containing molecules or
the monospecific EGFR or c-Met binding FN3 domains of the invention
may incorporate other subunits for example via covalent
interaction. In one aspect of the invention, the bispecific
EGFR/c-Met FN3 domain containing molecules of the invention further
comprise a half-life extending moiety. Exemplary half-life
extending moieties are albumin, albumin variants, albumin-binding
proteins and/or domains, transferrin and fragments and analogues
thereof, and Fc regions. An exemplary albumin-binding domain is
shown in SEQ ID NO: 117.
[0349] All or a portion of an antibody constant region may be
attached to the molecules of the invention to impart antibody-like
properties, especially those properties associated with the Fc
region, such as Fc effector functions such as C1q binding,
complement dependent cytotoxicity (CDC), Fc receptor binding,
antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis,
down regulation of cell surface receptors (e.g., B cell receptor;
BCR), and can be further modified by modifying residues in the Fc
responsible for these activities (for review; see Strohl, Curr Opin
Biotechnol. 20, 685-691, 2009).
[0350] Additional moieties may be incorporated into the bispecific
molecules of the invention such as polyethylene glycol (PEG)
molecules, such as PEG5000 or PEG20,000, fatty acids and fatty acid
esters of different chain lengths, for example laurate, myristate,
stearate, arachidate, behenate, oleate, arachidonate, octanedioic
acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic
acid, and the like, polylysine, octane, carbohydrates (dextran,
cellulose, oligo- or polysaccharides) for desired properties. These
moieties may be direct fusions with the protein scaffold coding
sequences and may be generated by standard cloning and expression
techniques. Alternatively, well known chemical coupling methods may
be used to attach the moieties to recombinantly produced molecules
of the invention.
[0351] A pegyl moiety may for example be added to the bispecific or
monospecific molecules of the invention by incorporating a cysteine
residue to the C-terminus of the molecule and attaching a pegyl
group to the cysteine using well known methods. Exemplary
bispecific molecules with the C-terminal cysteine are those having
the amino acid sequence shown in SEQ IN NO: 170-178.
[0352] Monospecific and bispecific molecules of the invention
incorporating additional moieties may be compared for functionality
by several well known assays. For example, altered properties of
monospecific and/or bispecific molecules due to incorporation of Fc
domains and/or Fc domain variants may be assayed in Fc receptor
binding assays using soluble forms of the receptors, such as the
Fc.gamma.RI, Fc.gamma.RII, Fc.gamma.RIII or FcRn receptors, or
using well known cell-based assays measuring for example ADCC or
CDC, or evaluating pharmacokinetic properties of the molecules of
the invention in in vivo models.
Polynucleotides, Vectors, Host Cells
[0353] The invention provides for nucleic acids encoding the
EGFR-binding or c-Met binding FN3 domains or the bispecific
EGFR/c-Met FN3 domain containing molecules of the invention as
isolated polynucleotides or as portions of expression vectors or as
portions of linear DNA sequences, including linear DNA sequences
used for in vitro transcription/translation, vectors compatible
with prokaryotic, eukaryotic or filamentous phage expression,
secretion and/or display of the compositions or directed mutagens
thereof. Certain exemplary polynucleotides are disclosed herein,
however, other polynucleotides which, given the degeneracy of the
genetic code or codon preferences in a given expression system,
encode the EGFR-binding or c-Met binding FN3 domains or the
bispecific EGFR/c-Met FN3 domain containing molecules of the
invention are also within the scope of the invention.
[0354] One embodiment of the invention is an isolated
polynucleotide encoding the FN3 domain specifically binding EGFR
having the amino acid sequence of SEQ ID NOs: 18-29, 107-110, or
122-137.
[0355] One embodiment of the invention is an isolated
polynucleotide comprising the polynucleotide sequence of SEQ ID
NOs: 97-98 or 168-169.
[0356] One embodiment of the invention is an isolated
polynucleotide encoding the FN3 domain specifically binding c-Met
having the amino acid sequence of the sequence shown in SEQ ID NOs:
32-49 or 111-114.
[0357] One embodiment of the invention is an isolated
polynucleotide encoding the bispecific EGFR/-c-Met FN3 domain
containing molecule having the amino acid sequence of SEQ ID NOs:
50-72, 106, 118-121 or 138-165.
[0358] One embodiment of the invention is an isolated
polynucleotide comprising the polynucleotide sequence of SEQ ID
NOs: 115-116 or 166-167.
[0359] The polynucleotides of the invention may be produced by
chemical synthesis such as solid phase polynucleotide synthesis on
an automated polynucleotide synthesizer and assembled into complete
single or double stranded molecules. Alternatively, the
polynucleotides of the invention may be produced by other
techniques such as PCR followed by routine cloning. Techniques for
producing or obtaining polynucleotides of a given known sequence
are well known in the art.
[0360] The polynucleotides of the invention may comprise at least
one non-coding sequence, such as a promoter or enhancer sequence,
intron, polyadenylation signal, a cis sequence facilitating RepA
binding, and the like. The polynucleotide sequences may also
comprise additional sequences encoding additional amino acids that
encode for example a marker or a tag sequence such as a histidine
tag or an HA tag to facilitate purification or detection of the
protein, a signal sequence, a fusion protein partner such as RepA,
Fc or bacteriophage coat protein such as pIX or pIII.
[0361] Another embodiment of the invention is a vector comprising
at least one polynucleotide of the invention. Such vectors may be
plasmid vectors, viral vectors, vectors for baculovirus expression,
transposon based vectors or any other vector suitable for
introduction of the polynucleotides of the invention into a given
organism or genetic background by any means. Such vectors may be
expression vectors comprising nucleic acid sequence elements that
can control, regulate, cause or permit expression of a polypeptide
encoded by such a vector. Such elements may comprise
transcriptional enhancer binding sites, RNA polymerase initiation
sites, ribosome binding sites, and other sites that facilitate the
expression of encoded polypeptides in a given expression system.
Such expression systems may be cell-based, or cell-free systems
well known in the art.
[0362] Another embodiment of the invention is a host cell
comprising the vector of the invention. A monospecific EGFR-binding
or c-Met binding FN3 domain or the bispecific EGFR/c-Met FN3 domain
containing molecule of the invention can be optionally produced by
a cell line, a mixed cell line, an immortalized cell or clonal
population of immortalized cells, as well known in the art. See,
e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology,
John Wiley & Sons, Inc., NY, NY (1987-2001); Sambrook, et al.,
Molecular Cloning: A Laboratory Manual, 2.sup.nd Edition, Cold
Spring Harbor, N.Y. (1989); Harlow and Lane, Antibodies, a
Laboratory Manual, Cold Spring Harbor, N.Y. (1989); Colligan, et
al., eds., Current Protocols in Immunology, John Wiley & Sons,
Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein
Science, John Wiley & Sons, NY, NY, (1997-2001).
[0363] The host cell chosen for expression may be of mammalian
origin or may be selected from COS-1, COS-7, HEK293, BHK21, CHO,
BSC-1, He G2, SP2/0, HeLa, myeloma, lymphoma, yeast, insect or
plant cells, or any derivative, immortalized or transformed cell
thereof. Alternatively, the host cell may be selected from a
species or organism incapable of glycosylating polypeptides, e.g. a
prokaryotic cell or organism, such as BL21, BL21(DE3),
BL21-GOLD(DE3), XL1-Blue, JM109, HMS174, HMS174(DE3), and any of
the natural or engineered E. coli spp, Klebsiella spp., or
Pseudomonas spp strains.
[0364] Another embodiment of the invention is a method of producing
the isolated FN3 domain specifically binding EGFR or c-Met of the
invention or the isolated bispecific EGFR/c-Met FN3 domain
containing molecule of the invention, comprising culturing the
isolated host cell of the invention under conditions such that the
isolated FN3 domain specifically binding EGFR or c-Met or the
isolated bispecific EGFR/c-Met FN3 domain containing molecule is
expressed, and purifying the domain or molecule.
[0365] The FN3 domain specifically binding EGFR or c-Met or the
isolated bispecific EGFR/c-Met FN3 domain containing molecule of
the invention can be purified from recombinant cell cultures by
well-known methods, for example by protein A purification, ammonium
sulfate or ethanol precipitation, acid extraction, anion or cation
exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography, or high
performance liquid chromatography (HPLC).
Bispecific EGFR/c-Met Antibodies
[0366] The bispecific EGFR/c-Met antibodies may be generated de
novo or may be engineered from existing monospecific anti-EGFR and
anti-c-Met antibodies.
[0367] Exemplary anti-EGFR antibodies that may be used to engineer
bispecific molecules are for example panitumumab (ABX-EGF),
nimotuzumab, necitumumab, matuzumab, and those described for
example in: U.S. Pat. No. 7,595,378, U.S. Pat. No. 7,247,301, U.S.
Pat. Publ. No. US2011/0256142, U.S. Pat. No. 5,891,996, U.S. Pat.
No. 5,212,290, U.S. Pat. No. 5,558,864, or U.S. Pat. No. 7,589,180.
For example, antibody VH domain having the amino acid sequence
shown in SEQ ID NO: 189 or 191 and antibody VL domain having the
amino acid sequences shown in SEQ ID NO: 190 or 192 may be
used.
[0368] Exemplary anti-c-Met antibodies that may be used to engineer
bispecific molecules are for example Rilotumumab, Onartuzumab,
Ficlatuzumab, and those described for example in PCT Intl. Publ.
No. WO2011/110642, US Pat. Publ. No. US2004/0166544, PCT Intl.
Publ. No. WO2005/016382, or PCT Intl. Publ. No. WO2006/015371. For
example, antibody VH domain having the amino acid sequence shown in
SEQ ID NO: 193 or 195 and antibody VL domain having the amino acid
sequences shown in SEQ ID NO: 194 or 196 may be used. The heavy and
light chain amino acid sequences of the antibodies identified by
their United States Adopted Names (USAN) is available via the
American Medical Association at http://_www_ama-assn_org or via the
CAS registry.
[0369] Monospecific EGFR and c-Met biding variable domains may be
selected de novo from for example a phage display library, where
the phage is engineered to express human immunoglobulins or
portions thereof such as Fabs, single chain antibodies (scFv), or
unpaired or paired antibody variable regions (Knappik et al., J Mol
Biol 296:57-86, 2000; Krebs et al., J Immunol Meth 254:67-84, 2001;
Vaughan et al., Nature Biotechnology 14:309-314, 1996; Sheets et
al., PITAS (USA) 95:6157-6162, 1998; Hoogenboom and Winter, J Mol
Biol 227:381, 1991; Marks et al., J Mol Biol 222:581, 1991), and
subsequently engineered into a bispecific format. The monospecific
EGFR and c-Met binding variable domains may be isolated for example
from phage display libraries expressing antibody heavy and light
chain variable regions as fusion proteins with bacteriophage pIX
coat protein as described in Shi et al (2010) J. Mol. Biol.
397:385-96 and PCT Intl. Publ. No. WO09/085462). The antibody
libraries are screened for binding to human EGFR or c-Met
extracellular domains and the obtained positive clones are further
characterized and the Fabs isolated from the clone lysates. Such
phage display methods for isolating human antibodies are
established in the art. See for example: U.S. Pat. No. 5,223,409;
U.S. Pat. No. 5,403,484; and U.S. Pat. No. 5,571,698, U.S. Pat. No.
5,427,908, U.S. Pat. No. 5,580,717, U.S. Pat. No. 5,969,108, U.S.
Pat. No. 6,172,197, U.S. Pat. No. 5,885,793; U.S. Pat. No.
6,521,404; U.S. Pat. No. 6,544,731; U.S. Pat. No. 6,555,313; U.S.
Pat. No. 6,582,915 and U.S. Pat. No. 6,593,081. The obtained de
novo variable regions binding EGFR or c-Met are engineered to
bispecific formats using the methods described herein.
Bispecific Antibody Formats
[0370] Antibodies of the present invention have two or more antigen
binding sites and are bispecific. Bispecific antibodies of the
invention include antibodies having a full length antibody
structure.
[0371] "Full length antibody" as used herein refers to an antibody
having two full length antibody heavy chains and two full length
antibody light chains. A full length antibody heavy chain (HC)
consists of well known heavy chain variable and constant domains
VH, CH1, CH2, and CH3. A full length antibody light chain (LC)
consists of well known light chain variable and constant domains VL
and CL. The full length antibody may be lacking the C-terminal
lysine (K) in either one or both heavy chains.
[0372] The term "Fab-arm" or "half molecule" refers to one heavy
chain-light chain pair that specifically binds an antigen.
[0373] Full length bispecific antibodies of the invention may be
generated for example using Fab arm exchange (or half molecule
exchange) between two monospecific bivalent antibodies by
introducing substitutions at the heavy chain CH3 interface in each
half molecule to favor heterodimer formation of two antibody half
molecules having distinct specificity either in vitro in cell-free
environment or using co-expression. The Fab arm exchange reaction
is the result of a disulfide-bond isomerization reaction and
dissociation-association of CH3 domains. The heavy-chain disulfide
bonds in the hinge regions of the parent monospecific antibodies
are reduced. The resulting free cysteines of one of the parent
monospecific antibodies form an inter heavy-chain disulfide bond
with cysteine residues of a second parent monospecific antibody
molecule and simultaneously CH3 domains of the parent antibodies
release and reform by dissociation-association. The CH3 domains of
the Fab arms may be engineered to favor heterodimerization over
homodimerization. The resulting product is a bispecific antibody
having two Fab arms or half molecules which each bind a distinct
epitope, i.e. an epitope on EGFR and an epitope on c-Met.
[0374] "Homodimerization" as used herein refers to an interaction
of two heavy chains having identical CH3 amino acid sequences.
"Homodimer" as used herein refers to an antibody having two heavy
chains with identical CH3 amino acid sequences.
[0375] "Heterodimerization" as used herein refers to an interaction
of two heavy chains having non-identical CH3 amino acid sequences.
"Heterodimer" as used herein refers to an antibody having two heavy
chains with non-identical CH3 amino acid sequences.
[0376] The "knob-in-hole" strategy (see, e.g., PCT Intl. Publ. No.
WO 2006/028936) may be used to generate full length bispecific
antibodies. Briefly, selected amino acids forming the interface of
the CH3 domains in human IgG can be mutated at positions affecting
CH3 domain interactions to promote heterodimer formation. An amino
acid with a small side chain (hole) is introduced into a heavy
chain of an antibody specifically binding a first antigen and an
amino acid with a large side chain (knob) is introduced into a
heavy chain of an antibody specifically binding a second antigen.
After co-expression of the two antibodies, a heterodimer is formed
as a result of the preferential interaction of the heavy chain with
a "hole" with the heavy chain with a "knob". Exemplary CH3
substitution pairs forming a knob and a hole are (expressed as
modified position in the first CH3 domain of the first heavy
chain/modified position in the second CH3 domain of the second
heavy chain): T366Y/F405A, T366W/F405W, F405W/Y407A, T394W/Y407T,
T394S/Y407A, T366W/T394S, F405W/T394S and
T366W/T366S_L368A_Y407V.
[0377] Other strategies such as promoting heavy chain
heterodimerization using electrostatic interactions by substituting
positively charged residues at one CH3 surface and negatively
charged residues at a second CH3 surface may be used, as described
in US Pat. Publ. No. US2010/0015133; US Pat. Publ. No.
US2009/0182127; US Pat. Publ. No. US2010/028637 or US Pat. Publ.
No. US2011/0123532. In other strategies, heterodimerization may be
promoted by following substitutions (expressed as modified position
in the first CH3 domain of the first heavy chain/modified position
in the second CH3 domain of the second heavy chain):
L351Y_F405A_Y407V/T394W, T366I_K392M_T394W/F405A_Y407V,
T366L_K392M_T394W/F405A_Y407V, L351Y_Y407A/T366A_K409F,
L351Y_Y407A/T366V_K409F, Y407A/T366A_K409F, or
T350V_L351Y_F405A_Y407V/T350V_T366L_K392L_T394W as described in
U.S. Pat. Publ. No. US2012/0149876 or U.S. Pat. Publ. No.
US2013/0195849
[0378] In addition to methods described above, bispecific
antibodies of the invention may be generated in vitro in a
cell-free environment by introducing asymmetrical mutations in the
CH3 regions of two monospecific homodimeric antibodies and forming
the bispecific heterodimeric antibody from two parent monospecific
homodimeric antibodies in reducing conditions to allow disulfide
bond isomerization according to methods described in Intl. Pat.
Publ. No. WO2011/131746. In the methods, the first monospecific
bivalent antibody (e.g., anti-c-Met antibody) and the second
monospecific bivalent antibody (e.g., anti-EGFR antibody) are
engineered to have certain substitutions at the CH3 domain that
promoter heterodimer stability; the antibodies are incubated
together under reducing conditions sufficient to allow the
cysteines in the hinge region to undergo disulfide bond
isomerization; thereby generating the bispecific antibody by Fab
arm exchange. The incubation conditions may optimally be restored
to non-reducing. Exemplary reducing agents that may be used are
2-mercaptoethylamine (2-MEA), dithiothreitol (DTT),
dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine
(TCEP), L-cysteine and beta-mercaptoethanol, preferably a reducing
agent selected from the group consisting of: 2-mercaptoethylamine,
dithiothreitol and tris(2-carboxyethyl)phosphine. For example,
incubation for at least 90 min at a temperature of at least
20.degree. C. in the presence of at least 25 mM 2-MEA or in the
presence of at least 0.5 mM dithiothreitol at a pH of from 5-8, for
example at pH of 7.0 or at pH of 7.4 may be used.
Bispecific EGFR/c-Met Antibodies
[0379] The bispecific EGFR/c-Met antibodies of the invention may
provide a benefit in terms of specificity and reduced off-target
toxicity when compared to small molecule EGFR and/or c-Met
inhibitors. The present invention is based at least in part on the
surprising finding that the bispecific EGFR/c-Met antibodies of the
invention provide a significantly improved synergistic inhibitory
effect when compared to a mixture of EGFR-binding and c-Met-binding
monospecific antibodies or published bispecific EGFR/c-Met
antibodies. Depending on the assay, the synergistic effect observed
varied between about 14- to over about 800-fold. The bispecific
EGFR/c-Met antibodies of the invention provide more efficient
inhibition of EGFR and c-Met signaling pathways and inhibit tumor
growth more efficiently than cetuximab (Erbitux.RTM.). The
bispecific EGFR/c-Met antibodies of the invention inhibit EGFR
signaling in tumors and/or tumor cell lines having EGFR activating
mutations and/or mutations in EGFR that are known to result in
resistance to treatments with tyrosine kinase inhibitors such as
gefitinib, and inhibit c-Met signaling pathway, a pathway
identified to be upregulated and to provide a compensatory
signaling upon treatment with EGFR tyrosine kinase inhibitors in
cancers such as NSCLC. The bispecific EGFR/c-Met antibodies of the
invention, in addition to directly inhibiting EGFR and c-Met
signaling, display antitumor activity through enhanced antibody
dependent cell cytotoxicity (ADCC) and degradation of the EGFR and
c-Met receptors. Contrary to the current EGFR therapies (cetuximab
and panitumumab), the bispecific EGFR/c-Met antibodies of the
invention induce, via enhanced ADCC, killing of tumor cells having
KRAS mutations.
[0380] Int. Pat. Publ. No. WO2010/115551 describes a bispecific
EGFR/c-Met antibody (BSAB01) engineered in an IgG-scFv format using
the EGFR bindingVH/VL pair of cetuximab, and the c-Met binding
VH/VL pair of an antibody 5D5 (MetMab, onartuzumab) currently in
Phase III trials. BSAB01 demonstrates approximately two-fold
(additive) increased inhibition of A431 cell proliferation when
compared to the parental antibodies (Example 7, FIG. 8b in
WO2010/115551), and a modest additive inhibition of Ovarc-8 cell
proliferation (FIG. 10a, Example 16 in WO2010/115551) when compared
to the combination of the two parental antibodies (15% vs. 10%
inhibition). Therefore, surprisingly and unexpectedly, the present
invention provides bispecific EGFR/c-Met antibodies that
demonstrate a significant synergistic effect in inhibition of EGFR
and c-Met signaling, cancer cell survival and tumor growth. By not
wishing to be bound by any theory, it is believed that the
significant synergistic effect of the bispecific antibodies of the
invention at least partially results from the epitope specificity
of both the EGFR and the c-Met binding arms, possibly resulting in
the inhibition of signaling through not only the EGFR and c-Met
homodimers but also the EGFR/HERx heterodimers.
[0381] One embodiment of the invention is an isolated bispecific
epidermal growth factor receptor (EGFR)/hepatocyte growth factor
receptor (c-Met) antibody, comprising: [0382] a) a first heavy
chain (HC1) comprising a HC1 constant domain 3 (HC1 CH3) and a HC1
variable region 1 (VH1); [0383] b) a second heavy chain (HC2)
comprising a HC2 constant domain 3 (HC2 CH3) and a HC2 variable
region 2 (VH2); [0384] c) a first light chain (LC1) comprising a
light chain variable region 1 (VL1); and a second light chain (LC2)
comprising a light chain variable region 2 (VL2), wherein the VH1
and the VL1 pair to form a first antigen-binding site that
specifically binds EGFR and the VH2 and the VL2 pair to form a
second antigen-binding site that specifically binds c-Met, wherein
the HC1 comprises at least one substitution in the HC1 CH3 and the
HC2 comprises at least one substitution in the HC2 CH3, wherein the
substitution in the HC1 CH3 and the substitution in the HC2 CH3
occur at different amino acid residue positions, when residue
numbering is according to the EU index.
[0385] In some embodiments described herein, the bispecific
EGFR/c-Met antibody inhibits phosphorylation of extracellular
signal-related kinases 1 and 2 (ERK1/2) in NCI-H292, NCI-H1975 or
SKMES-1 cell line with an IC.sub.50 value that is at least about
10-fold less, at least about 20-fold less, at least about 30-fold
less, at least about 40-fold less, at least about 50-fold less or
at least about 60-fold less when compared to the IC.sub.50 value of
inhibition of phosphorylation of ERK1/2 in NCI-H292, NCI-H1975 or
SKMES-1 cell line with a mixture of a control monovalent EGFR
antibody comprising a heavy chain 3 (HC3) and a light chain 3 (LC3)
and a control monovalent c-Met antibody comprising a heavy chain 4
(HC4) and a light chain 4 (LC4), wherein the HC3 and the HC1, the
LC3 and the LC1, the HC4 and the HC2, and the LC4 and the LC2 have
identical amino acid sequences, respectively, and the
phosphorylation of ERK1/2 is measured in whole cell lysates using a
sandwich immunoassay using an anti-phosphoERK1/2 antibody as a
capture antibody and an antibody binding to unphosphorylated and
phosphorylated ERK1/2 conjugated with an electrochemiluminescent
compound as a detection antibody. The bispecific EGFR/c-Met
antibodies of the invention provide a synergistic more pronounced
inhibition of EGFR and c-Met signaling when compared to the
combination of monospecific EGFR antibodies and monospecific c-Met
antibodies, when inhibition is assessed by inhibition of ERK1/2
phosphorylation. Such exemplary bispecific EGFR/c-Met antibody is
the antibody EM1-mAb of the invention.
[0386] "Control monospecific EGFR antibody" as used herein refers
to an antibody that has a first Fab arm that binds EGFR that is
identical in amino acid sequence to the EGFR-binding Fab arm of the
bispecific EGFR/c-Met antibody to be tested, and has a second Fab
arm that is "inert" and binds an unrelated/irrelevant antigen,
human immunodeficiency virus (HIV) gp120. The second Fab arm has a
light chain having the sequence of SEQ ID NO: 209 and a heavy chain
having the sequence of SEQ ID NO: 198 in instances when the EGFR
binding Fab arm in the bispecific EGFR/c-Met antibody to be tested
comprises the F405L substitution. The second Fab arm has a light
chain having the sequence of SEQ ID NO: 209 and a heavy chain
having the sequence of SEQ ID NO: 197 in instances when the EGFR
binding Fab arm in the bispecific EGFR/c-Met antibody to be tested
comprises the K409R substitution.
[0387] "Control monospecific c-Met antibody" as used herein refers
to an antibody that has a first Fab arm that binds c-Met that is
identical in amino acid sequence to the c-Met-binding Fab arm of
the bispecific EGFR/c-Met antibody to be tested, and has a second
Fab arm that is "inert" and binds the unrelated/irrelevant antigen
HIV gp120. The second Fab Fab arm has a light chain having the
sequence of SEQ ID NO: 209 and a heavy chain having the sequence of
SEQ ID NO: 198 in instances when the c-Met binding Fab arm in the
bispecific EGFR/c-Met antibody to be tested comprises the F405L
substitution. The second inert Fab arm has a light chain having the
sequence of SEQ ID NO: 209 and a heavy chain having the sequence of
SEQ ID NO: 197 in instances when the c-Met binding Fab arm in the
bispecific EGFR/c-Met antibody to be tested comprises the K409R
substitution.
[0388] In some embodiments described herein, the bispecific
EGFR/c-Met antibody inhibits phosphorylation of ERK1/2 with an
IC.sub.50 value of about 2.times.10.sup.-9 M or less, about
1.times.10.sup.-9 M or less, or about 1.times.10.sup.-10 M or
less.
[0389] In some embodiments described herein, ERK1 is phosphorylated
at residues Thr202 and Tyr204, and ERK2 is phosphorylated at
residues Thr185 and Tyr197.
[0390] In some embodiments described herein, the bispecific
EGFR/c-Met antibody inhibits phosphorylation of protein kinase B
(AKT) at Ser473 in NCI-H1975 cell line with an IC.sub.50 value that
is at least about 70-fold less when compared to the IC.sub.50 value
of inhibition of phosphorylation of AKT at Ser473 in NCI-H1975 cell
line with the mixture of the control monovalent EGFR antibody
comprising the HC3 and the LC3 and the control monovalent c-Met
antibody comprising the HC4 and the LC4, wherein the HC3 and the
HC1, the LC3 and the LC1, the HC4 and the HC2, and the LC4 and the
LC2 have identical amino acid sequences, respectively, wherein the
phosphorylation of AKT at Ser473 is measured in whole cell lysates
using a sandwich immunoassay using an antibody binding to
unphosphorylated and phosphorylated AKT as a capture antibody and
an anti-phosphoAKT Ser473 antibody conjugated to an
electrochemiluminescent compound as a detection antibody.
[0391] In some embodiments described herein, the bispecific
EGFR/c-Met antibody inhibits phosphorylation of protein kinase B
(AKT) at Thr308 in NCI-H1975 cell line with an IC.sub.50 value that
is at least about 100-fold less when compared to the IC.sub.50
value of inhibition of phosphorylation of AKT at Thr308 in
NCI-H1975 cell line with the mixture of the control monovalent EGFR
antibody comprising the HC3 and the LC3 and the control monovalent
c-Met antibody comprising the HC4 and the LC4, wherein the HC3 and
the HC1, the LC3 and the LC1, the HC4 and the HC2, and the LC4 and
the LC2 have identical amino acid sequences, respectively, wherein
the phosphorylation of AKT at Thr308 is measured in whole cell
lysates using a sandwich immunoassay using an antibody binding to
unphosphorylated and phosphorylated AKT as a capture antibody and
an anti-phosphoAKT Thr308 antibody conjugated to an
electrochemiluminescent compound as a detection antibody.
[0392] The bispecific EGFR/c-Met antibodies of the invention
provide a synergistic more pronounced inhibition of EGFR and c-Met
signaling when compared to the combination of monospecific EGFR
antibodies and monospecific c-Met antibodies, when inhibition is
assessed by inhibition of AKT phosphorylation. Such exemplary
bispecific EGFR/c-Met antibody is the antibody EM1-mAb of the
invention.
[0393] In some embodiments described herein, the bispecific
EGFR/c-Met antibody inhibits phosphorylation of AKT at Ser473 or at
Thr308 with and IC.sub.50 value of about 1.times.10.sup.-9 M or
less.
[0394] In some embodiments described herein, the bispecific
EGFR/c-Met antibody binds EGFR of SEQ ID NO: 73 at EGFR residues
K489, 1491, K467 and 5492 and c-Met at residues PEFRDSYPIKYVHAF
(SEQ ID NO: 238) and FAQSKPDSAEPMDRSA (SEQ ID NO: 239). Such an
exemplary bispecific antibody is the EM1-mAb. The bispecific EM-1
antibody binds EGFR and c-Met at distinct epitopes when compared to
the antibody BSABO1 as described above and in Int. Pat. Publ. No.
WO2010/115551. The parental EGFR binding arm (cetuximab) of BSABO1
binds EGFR amino acid residues R353, Q384, Q408, H409, F412, 5418,
5440, K443, K465, 1467, 5468, and N473 in mature EGFR,
corresponding to residues R367, Q408, Q432, H433, F436, 5442, 5464,
K467, K489, 1491, S492 and N497 of full length EGFR of SEQ ID NO:
73 (Li et al., Cancer Cell 7:301-311, 2005). The parental c-Met
binding arm of BSAB01 (mAb 5D5) binds c-Met residues 325-340
PGAQLARQIGASLNDD (SEQ ID NO: 240). Epitope mapping of the EGFR
binding parental antibody (2F8) of the EM1-mAb is described in US.
Pat. Publ. No. US2011/0256142A1. Cetuximab and the parental 2F8
antibody bind partially overlapping but distinct epitopes.
[0395] Epitope mapping can be done using standard methods. For
example, when the structures of both individual components are
known, in silico protein-protein docking can be carried out to
identify compatible sites of interaction. Hydrogen-deuterium (H/D)
exchange can be carried out with the antigen and antibody complex
to map regions on the antigen that may be bound by the antibody.
Segment and point mutagenesis of the antigen can be used to locate
amino acids important for antibody binding.
[0396] In some embodiments described herein, the bispecific
EGFR/c-Met antibody neutralizes EGFR and c-Met signaling.
[0397] The bispecific EGFR/c-Met antibody of the invention may
neutralize EGFR and c-Met signaling by at least 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or 100% when compared to the level of
signaling in the absence of the bispecific EGFR/c-Met molecule of
the invention using the same assay conditions.
[0398] Binding of a ligand such as EGF to EGFR stimulates receptor
dimerization, autophosphorylation, activation of the receptor's
internal, cytoplasmic tyrosine kinase domain, and initiation of
multiple signal transduction and transactivation pathways involved
in regulation of DNA synthesis (gene activation) and cell cycle
progression or division. Neutralization of EGFR signaling may
result in inhibition in one or more EGFR downstream signaling
pathways and therefore neutralizing EGFR may have various effects,
including inhibition of cell proliferation and differentiation,
angiogenesis, cell motility and metastasis, and inhibition of
downstream signaling pathways.
[0399] EGFR signaling and neutralization of EGFR signaling may be
measured using various well know methods, for example measuring the
autophosphorylation of the receptor at any of the tyrosines Y1068,
Y1148, and Y1173 (Downward et al., Nature 311:483-5, 1984) and/or
phosphorylation of natural or synthetic substrates, and inhibition
of autophosphorylation and/or phosphorylation of natural or
synthetic substrates by the bispecific antibodies of the invention.
Phosphorylation can be detected using well known methods such as an
ELISA assay or a western plot using a phosphotyrosine specific
antibody. Exemplary assays can be found in Panek et al., J
Pharmacol Exp Thera 283:1433-44, 1997 and Batley et al., Life Sci
62:143-50, 1998, and as described herein.
[0400] Binding of HGF to c-Met stimulates receptor dimerization,
autophosphorylation, activation of the receptor's cytoplasmic
tyrosine kinase domain, and initiation of multiple signal
transduction and transactivation pathways involved in regulation of
DNA synthesis (gene activation) and cell cycle progression or
division. Inhibition of c-Met signaling may result in inhibition in
one or more c-Met downstream signaling pathways and therefore
neutralizing c-Met may have various effects, including inhibition
of cell proliferation and differentiation, angiogenesis, cell
motility and metastasis.
[0401] c-Met signaling and neutralization of c-Met signaling may be
measured using various well know methods, for example measuring the
autophosphorylation of the receptor on at least one tyrosine
residues Y1230, Y1234, Y1235 or Y1349, and/or phosphorylation of
natural or synthetic substrates. Phosphorylation can be detected,
for example, using an antibody specific for phosphotyrosine in an
ELISA assay or on a western blot. Exemplary assays can be found in
Panek et al., J Pharmacol Exp Thera 283:1433-44, 1997 and Batley et
al., Life Sci 62:143-50, 1998, and as described herein.
[0402] EGFR and c-Met signaling may be measured using various well
know methods as described herein, such as measuring inhibition of
ERK1/2 and AKT phosphorylation. Inhibition of ERK1 phosphorylation
at Thr202 and Tyr204 and ERK2 phosphorylation at Thr185 and Tyr187
and inhibition of AKT at Ser473 or Thr308 can be measured for
example in NCI-H1975 cell lysates utilizing a sandwich assay with
capture antibody coated on solid support, and the detection
antibody conjugated with an electrochemiluminescent compound such
as Meso Scale Discover (MSD) SULFO-TAG label, followed by detection
of the signal with a plate reader.
[0403] In some embodiments described herein, the bispecific
EGFR/c-Met antibody inhibits growth of NCI-H292 or NCI-H1975 cells
with an IC.sub.50 value that is at least about 300-fold less, at
least about 400-fold less, at least about 500-fold less, at least
about 600-fold less, at least about 700-fold less or at least about
800-fold less when compared to the IC.sub.50 value of inhibition of
growth of NCI-H292 or NCI-H1975 cells with cetuximab, when NCI-H292
or NCI-H1975 cells are grown in low attachment conditions.
[0404] Inhibition of cell growth may be assessed by known methods.
For example, the cells may be plated in plates coated with
hydrogels or biomimetic polymers (for example Ultra Low Attachment
plates by Corning) to prevent or reduce cell attachment, and the
effect of antibodies on 7.5 ng/mL HGF-induced cell growth can be
assessed by measuring percent cell viability after incubation for
72 hours using standard methods.
[0405] The bispecific EGFR/c-Met antibodies of the invention
provide a synergistic more pronounced inhibition of EGFR and/or
c-Met expressing cancer cells when compared to the combination of
monospecific EGFR antibodies and monospecific c-Met antibodies and
to the standard of care cetuximab. Such an exemplary bispecific
EGFR/c-Met antibody is the antibody EM1-mAb of the invention. The
bispecific EGFR/c-Met antibodies of the invention inhibit cancer
cells that express the wild type EGFR and the wild type c-Met, and
also cancer cells that express the EGFR L858R/T790M mutant, which
mutation is identified to contribute to resistance to treatments
with small molecule tyrosine kinase inhibitors (TKIs) such as
gefitinib. Therefore the bispecific EGFR/c-Met antibodies of the
invention may provide a benefit in a broader patient population
when compared to cetuximab and TKIs.
[0406] In some embodiments described herein, the bispecific
EGFR/c-Met antibody inhibits growth of HGF-expressing SKMES-1 cell
tumor in SCID Beige mice with a percentage (%) T/C value of at
least 500-fold less on day 36 when compared to cetuximab, when the
bispecific antibody and cetuximab are administered at 20 mg/kg
dose.
[0407] Tumor xenograft models using SCID Beige mice are well known.
SKMES-1 cells may be engineered to express human HGF using standard
methods. Typically, SCID Beige mice may be subcutaneously
inoculated with SKMES-1 cells expressing human HGF embedded in
extracellular matrix such as Culturex in the dorsal flank of each
animal One week after implantation, mice may be stratified into
groups with equivalent tumor volumes, and thereafter dosed for
example three times per week with the bispecific EGFR/c-Met
antibodies of the invention, control or benchmark antibodies or
small molecules. Tumor volumes may be recorded twice weekly, and
tumor growth inhibition (TGI) may be observed by calculating the
percentage (%) T/C value. The % T/C value is indicative of
anti-tumor efficacy. T and C are the mean volumes of the treated
and control groups, respectively, on a given day.
[0408] The bispecific EGFR/c-Met antibodies of the invention
provide a significantly improved efficacy in in vivo tumor killing
when compared to the standard of care cetuximab, and therefore may
provide a benefit in a patient population when compared to
cetuximab.
[0409] In some embodiments described herein, the bispecific
EGFR/c-Met antibody heterodimerizes EGFR and c-Met on the cell
surface. While not wishing to be bound by any particular theory, it
is believed that the binding of the antibody of the invention to
EGFR homodimers, c-Met homodimers, and EGFR/c-Met heterodimers
results in simultaneous inhibition of ligand mediated signaling
downstream of EGFR homodimers, c-Met homodimers and EGFR/c-Met
heterodimers, providing synergistic inhibition when compared to the
combination of monospecific anti-EGFR and anti-c-Met
antibodies.
[0410] In some embodiments described herein, the bispecific
EGFR/c-Met antibody comprises the HC1 and the HC2 of IgG1, IgG2,
IgG3 or IgG4 isotype.
[0411] In some embodiments described herein, the bispecific
EGFR/c-Met antibody comprises the HC1 and the HC2 of IgG1
isotype.
[0412] In some embodiments described herein, the bispecific
EGFR/c-Met antibody HC1 CH3 comprises at least one, two, three,
four, five, six, seven or eight substitutions and the HC2 CH3
comprises at least one, two, three, four, five, six, seven or eight
substitutions at residue positions 350, 366, 368, 370, 399, 405,
407 or 409, when residue numbering is according to the EU
index.
[0413] In some embodiments described herein, the bispecific
EGFR/c-Met antibody HC1 CH3 comprises at least one, two, three or
four substitutions and the HC2 CH3 comprises at least one, two,
three or four substitutions at residue positions 350, 370, 405 or
409, when residue numbering is according to the EU index.
[0414] Antibody domains and numbering are well known. Two CH3
domains (or CH3 regions) are non-identical when they differ with at
least one amino acid substitution from each other. An IgG1 CH3
region typically consists of residues 341-446 on IgG1 (residue
numbering according to the EU index). An exemplary IgG1 constant
region is shown in SEQ ID NO: 203. The CH3 domain spans residues
224-329 of SEQ ID NO: 203, and correspond to residues 341-446
according to EU index.
[0415] In some embodiments described herein, the bispecific
EGFR/c-Met antibody HC1 CH3 comprises at least one substitution and
the HC2 CH3 comprises at least one substitution at residue
positions 405 or 409, when residue numbering is according to the EU
index.
[0416] In some embodiments described herein, the bispecific
EGFR/c-Met antibody HC1 CH3 comprises a K409R or a F405L
substitution and the HC2 CH3 comprises a K409R or a F405L
substitution, wherein residue numbering is according to the EU
index.
[0417] In some embodiments described herein, the bispecific
EGFR/c-Met antibody HC1 CH3 comprises the F405L substitution and
the HC2 CH3 comprises the K409R substitution.
[0418] In some embodiments described herein, the HC1 CH3 and the
HC2 CH3 substitutions are substitutions at position 366, 368, 370,
399, 405, 407 or 409 (numbering according to the EU index). These
positions correspond to linear residue positions 248, 250, 252,
281, 287, 289 and 291, respectively, in a heavy chain constant
region of SEQ ID NO: 203 and 204.
[0419] In some embodiments described herein, the HC1 CH3 position
409 has an amino acid substitution other than Lys, Leu or Met and
the HC2 CH3 position 405 has an amino acid substitution other than
Phe.
[0420] In some embodiments described herein, the HC1 CH3 position
405 has an amino acid substitution other than Phe and the HC2 CH3
position 409 has an amino acid substitution other than Lys, Leu or
Met.
[0421] In some embodiments described herein, the HC1 CH3 position
409 has an amino acid substitution other than Lys, Leu or Met and
the HC2 CH3 position 405 has an amino acid substitution other than
Phe, Arg or Gly.
[0422] In some embodiments described herein, the HC1 CH3 position
405 has an amino acid substitution other than Phe, Arg or Gly and
the HC2 CH3 position 409 has an amino acid substitution other than
Lys, Leu or Met
[0423] In some embodiments described herein, the HC1 CH3 has Phe at
position 405 and an amino acid other than Lys, Leu or Met at
position 409 and the HC2 CH3 has an amino acid other than Phe at
position 405 and a Lys at position 409.
[0424] In some embodiments described herein, the HC1 CH3 has an
amino acid other than Phe at position 405 and Lys at position 409
and the HC2 CH3 has Phe at position 405 and an amino acid other
than Lys, Leu or Met at position 409.
[0425] In some embodiments described herein, the HC1 CH3 has Phe at
position 405 and an amino acid other than Lys, Leu or Met at
position 409 and the HC2 CH3 has a substitution other than Phe, Arg
or Gly at position 405 and Lys at position 409.
[0426] In some embodiments described herein, the HC1 CH3 has a
substitution other than Phe, Arg or Gly at position 405 and Lys at
position 409 and the HC2 CH3 has Phe at position 405 and an amino
acid other than Lys, Leu or Met at position 409.
[0427] In some embodiments described herein, the HC1 CH3 has Phe at
position 405 and an amino acid other than Lys, Leu or Met at
position 409 and the HC2 CH3 has Leu at position 405 and Lys at
position 409.
[0428] In some embodiments described herein, the HC1 CH3 has Leu at
position 405 and Lys at position 409 and the HC2 CH3 has Phe at
position 405 and an amino acid other than Lys, Leu or Met at
position 409.
[0429] In some embodiments described herein, the HC1 CH3 has Phe at
position 405 and aArg at position 409 and the HC2 CH3 has an amino
acid other than Phe, Arg or Gly at position 405 and Lys at position
409.
[0430] In some embodiments described herein, the HC1 CH3 has an
amino acid other than Phe, Arg or Gly at position 405 and Lys at
position 409 and the HC2 CH3 has Phe at position 405 and Arg at
position 409.
[0431] In some embodiments described herein, the HC1 CH3 has Phe at
position 405 and Arg at position 409 and the HC2 CH3 has Leu at
position 405 and Lys at position 409.
[0432] In some embodiments described herein, the HC1 CH3 has Leu at
position 405 and Lys at position 409 and the HC2 CH3 has Phe at
position 405 and Arg at position 409.
[0433] In some embodiments described herein, the HC1 CH3 has Phe at
position 405 and Lys at position 409 and the HC2 CH3 has Leu at
position 405 and aArg at position 409.
[0434] In some embodiments described herein, the HC1 CH3 has Leu at
position 405 and aArg at position 409 and the HC2 CH3 has Phe at
position 405 and Lys at position 409.
[0435] In some embodiments described herein, the HC1 CH3 has an
amino acid other than Lys, Leu or Met at position 409 and the HC2
CH3 has Lys at position 409, Thr at position 370 and Leu at
position 405.
[0436] In some embodiments described herein, the HC1 CH3 has Lys at
position 409, Thr at position 370 and Leu at position 405 and the
HC2 CH3 has an amino acid other than Lys, Leu or Met at position
409.
[0437] In some embodiments described herein, the HC1 CH3 has Arg at
position 409 and the HC2 CH3 has Lys at position 409, Thr at
position 370 and Leu at position 405.
[0438] In some embodiments described herein, the HC1 CH3 has Lys at
position 409, Thr at position 370 and Leu at position 405 and the
HC2 CH3 has Arg at position 409.
[0439] In some embodiments described herein, the HC1 CH3 has Lys at
position 370, Phe at position 405 and aArg at position 409 and the
HC2 CH3 has Lys at position 409, Thr at position 370 and Leu at
position 405.
[0440] In some embodiments described herein, the HC1 CH3 has Lys at
position 409, Thr at position 370 and Leu at position 405 and the
HC2 CH3 has Lys at position 370, Phe at position 405 and Arg at
position 409.
[0441] In some embodiments described herein, the HC1 CH3 has an
amino acid other than Lys, Leu or Met at position 409 and the HC2
CH3 has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg,
Ser or Thr at position 407.
[0442] In some embodiments described herein, the HC1 CH3 has an
amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr
at position 407 and the HC2 CH3 has an amino acid other than Lys,
Leu or Met at position 409.
[0443] In some embodiments described herein, the HC1 CH3 has an
amino acid other than Lys, Leu or Met at position 409 and the HC2
CH3 has Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position
407.
[0444] In some embodiments described herein, the HC1 CH3 has Ala,
Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407 and the
HC2 CH3 has an amino acid other than Lys, Leu or Met at position
409.
[0445] In some embodiments described herein, the HC1 CH3 has an
amino acid other than Lys, Leu or Met at position 409 and the HC2
CH3 has Gly, Leu, Met, Asn or Trp at position 407.
[0446] In some embodiments described herein, the HC1 CH3 has Gly,
Leu, Met, Asn or Trp at position 407 and the HC2 CH3 has an amino
acid other than Lys, Leu or Met at position 409.
[0447] In some embodiments described herein, the HC1 CH3 has Tyr at
position 407 and an amino acid other than Lys, Leu or Met at
position 409 and the HC2 CH3 has an amino acid other than Tyr, Asp,
Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407 and Lys at
position 409.
[0448] In some embodiments described herein, the HC1 CH3 has an
amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr
at position 407 and Lys at position 409 and the HC2 CH3 has Tyr at
position 407 and an amino acid other than Lys, Leu or Met at
position 409.
[0449] In some embodiments described herein, the HC1 CH3 has Tyr at
position 407 and an amino acid other than Lys, Leu or Met at
position 409 and the HC2 CH3 has Ala, Gly, His, Ile, Leu, Met, Asn,
Val or Trp at position 407 and Lys at position 409.
[0450] In some embodiments described herein, the HC1 CH3 has Ala,
Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407 and Lys at
position 409 and the HC2 CH3 has Tyr at position 407 and an amino
acid other than Lys, Leu or Met at position 409.
[0451] In some embodiments described herein, the HC1 CH3 has Tyr at
position 407 and an amino acid other than Lys, Leu or Met at
position 409 and the HC2 CH3 has Gly, Leu, Met, Asn or Trp at
position 407 and Lys at position 409.
[0452] In some embodiments described herein, the HC1 CH3 has Gly,
Leu, Met, Asn or Trp at position 407 and Lys at position 409 and
the HC2 CH3 has Tyr at position 407 and an amino acid other than
Lys, Leu or Met at position 409.
[0453] In some embodiments described herein, the HC1 CH3 has Tyr at
position 407 and Arg at position 409 and the HC2 CH3 has an amino
acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at
position 407 and Lys at position 409.
[0454] In some embodiments described herein, the HC1 CH3 has an
amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr
at position 407 and Lys at position 409 and the HC2 CH3 has Tyr at
position 407 and Arg at position 409.
[0455] In some embodiments described herein, the HC1 CH3 has Tyr at
position 407 and Arg at position 409 and the HC2 CH3 has Ala, Gly,
His, Ile, Leu, Met, Asn, Val or Trp at position 407 and Lys at
position 409.
[0456] In some embodiments described herein, the HC1 CH3 has Ala,
Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407 and Lys at
position 409 and the HC2 CH3 has Tyr at position 407 and Arg at
position 409.
[0457] In some embodiments described herein, the HC1 CH3 has Tyr at
position 407 and Arg at position 409 and the HC2 CH3 has Gly, Leu,
Met, Asn or Trp at position 407 and Lys at position 409.
[0458] In some embodiments described herein, the HC1 CH3 has Gly,
Leu, Met, Asn or Trp at position 407 and Lys at position 409 and
the HC2 CH3 has Tyr at position 407 and Arg at position 409.
[0459] In some embodiments described herein, the HC1 CH3 has an
amino acid other than Lys, Leu or Met at position 409, and the HC2
CH3 has (i) an amino acid other than Phe, Leu and Met at position
368, or (ii) a Trp at position 370, or (iii) an amino acid other
than Asp, Cys, Pro, Glu or Gln at position 399.
[0460] In some embodiments described herein, the HC1 CH3 has (i) an
amino acid other than Phe, Leu and Met at position 368, or (ii) a
Trp at position 370, or (iii) an amino acid other than Asp, Cys,
Pro, Glu or Gln at position 399 and the HC2 CH3 has an amino acid
other than Lys, Leu or Met at position 409.
[0461] In some embodiments described herein, the HC1 CH3 has Arg,
Ala, His or Gly at position 409, and the HC2 CH3 has (i) Lys, Gln,
Ala, Asp, Glu, Gly, His, Ile, Asn, Arg, Ser, Thr, Val, or Trp at
position 368, or (ii) Trp at position 370, or (iii) Ala, Gly, Ile,
Leu, Met, Asn, Ser, Thr, Trp, Phe, His, Lys, Arg or Tyr at position
399.
[0462] In some embodiments described herein, the HC1 CH3 has (i)
Lys, Gln, Ala, Asp, Glu, Gly, His, Ile, Asn, Arg, Ser, Thr, Val, or
Trp at position 368, or (ii) Trp at position 370, or (iii) Ala,
Gly, Ile, Leu, Met, Asn, Ser, Thr, Trp, Phe, His, Lys, Arg or Tyr
at position 399 and the HC2 CH3 has Arg, Ala, His or Gly at
position 409.
[0463] In some embodiments described herein, the HC1 CH3 has Arg at
position 409, and the HC2 CH3 has (i) Asp, Glu, Gly, Asn, Arg, Ser,
Thr, Val, or Trp at position 368, or (ii) Trp at position 370, or
(iii) Phe, His, Lys, Arg or Tyr at position 399.
[0464] In some embodiments described herein, the HC1 CH3 has (i)
Asp, Glu, Gly, Asn, Arg, Ser, Thr, Val, or Trp at position 368, or
(ii) Trp at position 370, or (iii) Phe, His, Lys, Arg or Tyr at
position 399 and the HC2 CH3 has Arg at position 409.
[0465] In some embodiments described herein, the HC1 CH3 comprises
a K409R substitution or a F405L substitution and the HC2 CH3
comprises a K409R substitution or a F405L substitution, wherein the
residue numbering is according to the EU index.
[0466] In some embodiments described herein, the HC1 CH3 comprises
the F405L substitution and the HC2 CH3 comprises the K409R
substitution.
[0467] Substitutions are typically made at the DNA level to a
molecule such as the constant domain of the antibody using standard
methods.
[0468] In some embodiments described herein, the bispecific
EGFR/c-Met antibody comprises the VH1 and the VL1, wherein
[0469] the VH1 comprises the heavy chain complementarity
determining region (HCDR) 1 (HCDR1), HCDR 2 (HCDR2) and HCDR 3
(HCDR3) amino acid sequences of SEQ ID NOs: 210, 211 and 212,
respectively; and
[0470] the VL1 comprises the light chain complementarity
determining region (LCDR) 1 (LCDR1), LCDR 2 (LCDR2) and LCDR 3
(LCDR3) amino acid sequences of SEQ ID NOs: 213, 214 and 215,
respectively.
[0471] In some embodiments described herein, the bispecific
EGFR/c-Met antibody comprises the VH2 and the VL2, wherein
[0472] the VH2 comprises the HCDR1, the HCDR2, and the HCDR3 amino
acid sequences of SEQ ID NOs: 216, 217 and 218, respectively;
and
[0473] the VL2 comprises the LCDR1, the LCDR2 and the LCDR3 amino
acid sequences of SEQ ID NOs: 219, 220 and 221, respectively.
[0474] In some embodiments described herein, the bispecific
EGFR/c-Met antibody comprises the VH1, the VL1, the VH2 and the VL2
amino acid sequences of SEQ ID NOs: 189, 190, 193 and 194,
respectively.
[0475] In some embodiments described herein, the bispecific
EGFR/c-Met antibody comprises the HC1, the LC1, the HC2 and the LC2
amino acid sequences of SEQ ID NOs: 199, 200, 201 and 202,
respectively, optionally having a C-terminal lysine removed from
the HC1, the HC2, or both the HC1 and the HC2.
[0476] In some embodiments described herein, the bispecific
EGFR/c-Met antibody comprises the VH1 and the VL1, wherein
[0477] the VH1 comprises the HCDR1, the HCDR2, and the HCDR3 amino
acid sequences of SEQ ID NOs: 222, 223 and 224, respectively;
and
[0478] the VL1 comprises the LCDR1, the LCDR2 and the LCDR3 amino
acid sequences of SEQ ID NOs: 225, 226 and 227, respectively.
[0479] In some embodiments described herein, the bispecific
EGFR/c-Met antibody comprises the VH2 and the VL2, wherein
[0480] the VH2 comprises the HCDR1, the HCDR2, and the HCDR3 amino
acid sequences of SEQ ID NOs: 228, 229 and 230, respectively;
and
[0481] the VL2 comprises the LCDR1, the LCDR2 and the LCDR3 amino
acid sequences of SEQ ID NOs: 231, 232 and 233, respectively.
[0482] In some embodiments described herein, the bispecific
EGFR/c-Met antibody comprises the VH1, the VL1, the VH2 and the VL2
amino acid sequences of SEQ ID NOs: 191, 192, 195 and 196,
respectively.
[0483] In some embodiments described herein, the bispecific
EGFR/c-Met antibody comprises the HC1, the LC1, the HC2 and the LC2
amino acid sequences of SEQ ID NOs: 234, 235, 236 and 237,
respectively, optionally having the C-terminal lysine removed from
the HC1, the HC2, or both the HC1 and the HC2.
[0484] In some embodiments described herein, the bispecific
EGFR/c-Met antibodies may block EGF binding to the EGFR and HGF
binding to c-Met with an IC.sub.50 value of less than about
1.times.10.sup.-8 M, less than about 1.times.10.sup.-9 M, less than
about 1.times.10.sup.-10 M, less than about 1.times.10.sup.-11 M,
or less than about 1.times.10.sup.-12 M in a competition assay
employing recombinant human EGFR or recombinant human c-Met
extracellular domains coated on plates and incubated with or
without the bispecific EGFR/c-Met antibodies of the invention. The
bispecific EGFR/c-Met antibodies described herein may block EGF
binding to EGFR and HGF binding to c-Met by at least 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or 100% when compared to binding of
EGF to the EGFR and HGF binding to c-Met in the absence of the
bispecific EGFR/c-Met antibodies of the invention described herein
using the same assay conditions.
[0485] In some embodiments described herein, the bispecific
EGFR/c-Met antibody comprises the HC1, LC1, HC2 and LC2, wherein
the HC1, the LC1, the HC2 and the LC2 are encoded by synthetic
polynucleotides comprising the sequence of SEQ ID NOs: 205, 206,
207 and 208, respectively.
[0486] The bispecific EGFR/c-Met antibodies of the invention may be
generated using techniques described herein, such as utilizing CH3
engineering and generating the antibodies using in vitro Fab arm
exchange. An exemplary bispecific antibody may be generated from
two monospecific antibodies by combining about 1-20 mg/mL of each
antibody at a 1:1 molar ratio in PBS at pH 7.0-7.4 in a buffer
having a final concentration of 75 mM 2-mercaptoethanolamine
(2-MEA), incubating for 2-6 hours at 25-37.degree. C., followed by
removal of 2-MEA via dialysis, diafiltration, tangential flow
filtration, and spinned cell filtration. The yield of the
bispecific antibody may be more than about 80%, more than about
90%, more than about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99%.
[0487] Some embodiments described herein provide for methods of
producing the isolated bispecific EGFR/c-Met antibody,
comprising:
[0488] combining an isolated monospecific bivalent anti-EGFR
antibody comprising two heavy chains of SEQ ID NO: 199 and two
light chains of SEQ ID NO: 200 and an isolated monospecific
bivalent anti-c-Met antibody comprising two heavy chains of SEQ ID
NO: 201 and two light chains of SEQ ID NO: 202 in a mixture of
about 1:1 molar ratio;
[0489] introducing a reducing agent into the mixture;
[0490] incubating the mixture about ninety minutes to about six
hours;
[0491] removing the reducing agent; and
[0492] purifying the bispecific EGFR/c-Met antibody that comprises
a first heavy chain of SEQ ID NO: 199 and a second heavy chain of
SEQ ID NO: 201, a first light chain of SEQ ID NO: 200 and a second
light chain of SEQ ID NO: 202, wherein the first heavy chain of SEQ
ID NO: 199 pairs with the first light chain of SEQ ID NO: 200 to
form the first binding domain that specifically binds EGFR, and the
second heavy chain of SEQ ID NO: 201 pairs with the second light
chain of SEQ ID NO: 202 to form the second binding domain that
specifically binds c-Met.
[0493] In some embodiments described herein, the reducing agent is
2-mercaptoethanolamine (2-MEA).
[0494] In some embodiments described herein, 2-MEA is present at a
concentration of about 25 mM to about 75 mM.
[0495] In some embodiments described herein, the incubating step is
performed at a temperature of about 25.degree. C. to about
37.degree. C.
[0496] Some embodiments described herein provide for an isolated
bispecific EGFR/-c-Met antibody comprising a HC1, a LC1, a HC2 and
a LC2, wherein the HC1 comprises the sequence of SEQ ID NO: 199,
the LC1 comprises the sequence of SEQ ID NO: 200, the HC2 comprises
the sequence of SEQ ID NO: 201, and the LC2 comprises the sequence
of SEQ ID NO: 202, wherein the HC1, the LC1, the HC2 and/or the LC2
further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or
15 conservative amino acid substitutions.
[0497] Some embodiments described herein provide for an isolated
bispecific EGFR/-c-Met antibody comprising the HC1, the LC1, the
HC2 and the LC2, wherein the HC1 comprises the sequence of SEQ ID
NO: 234, the LC1 comprises the sequence of SEQ ID NO: 235, the HC2
comprises the sequence of SEQ ID NO: 236, and the LC2 comprises the
sequence of SEQ ID NO: 237, wherein the HC1, the LC1, the HC2
and/or the LC2 further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14 or 15 conservative amino acid substitutions.
[0498] Bispecific EGFR/c-Met antibodies whose HC1, LC1, HC2 and LC2
amino acid sequences differ insubstantially from those antibodies
disclosed herein are encompassed within the scope of the invention.
Typically, this involves one or more conservative amino acid
substitutions with an amino acid having similar charge,
hydrophobic, or stereochemical characteristics in the
antigen-binding sites or in the frameworks without adversely
altering the properties of the antibody. Conservative substitutions
may also be made to improve antibody properties, for example
stability or affinity. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, or 15 amino acid substitutions may be made for example to the
VH1, the VL1, the VH2 and/or the VL2. For example, a "conservative
amino acid substitution" may involve a substitution of a native
amino acid residue with a nonnative residue such that there is
little or no effect on the polarity or charge of the amino acid
residue at that position. Furthermore, any native residue in the
polypeptide may also be substituted with alanine, as has been
previously described for alanine scanning mutagenesis (MacLennan et
al., Acta Physiol Scand Suppl 643:55-67, 1998; Sasaki et al., Adv
Biophys 35:1-24, 1998). Desired amino acid substitutions may be
determined by those skilled in the art at the time such
substitutions are desired. For example, amino acid substitutions
can be used to identify important residues of the molecule
sequence, or to increase or decrease the affinity of the molecules
described herein. Exemplary conservative amino acid substitutions
are described supra.
[0499] Amino acid substitutions may be done for example by PCR
mutagenesis (U.S. Pat. No. 4,683,195). Libraries of variants may be
generated using well known methods, for example using random (NNK)
or non-random codons, for example DVK codons, which encode 11 amino
acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp) and
screening the libraries for variants with desired properties.
[0500] In some embodiments described herein, amino acid
substitutions can be made to the constant region of the antibody.
For example different IgG1 allotypes can be used in the bispecific
EGFR/c-Met antibodies of the invention, such as well known G1m17
allotype, G1m3 allotype or G1m1 allotype, or a combination
thereof.
[0501] In some embodiments described herein, pharmacokinetic
properties of the bispecific EGFR/c-Met antibodies may be enhanced
by substitutions in the Fc domain that modulate antibody half-life.
In some embodiments described herein, the bispecific EGFR/c-Met
antibody comprises a substitution M252Y/S254T/T256E in the HC1
and/or the HC2, wherein residue numbering is according to the EU
index. M252Y/S254T/T256E substitutions have been show to increase
antibody half-life (Dall'Acqua et al., J Biol Chem 281:23514-24,
2006).
[0502] The bispecific EGFR/c-Met antibodies having conservative
substitutions and/or additional substitutions in their Fc region
are tested for their characteristics using the methods described
herein.
[0503] In some embodiment described herein, immune effector
properties of the bispecific EGFR/c-Met antibodies may be enhanced
or silenced through Fc modifications by techniques known to those
skilled in the art. For example, Fc effector functions such as C1q
binding, complement dependent cytotoxicity (CDC),
antibody-dependent cell-mediated cytotoxicity (ADCC),
antibody-dependent cell-mediated phagocytosis (ADCP), down
regulation of cell surface receptors (e.g., B cell receptor; BCR),
etc. may be provided and/or controlled by modifying residues in the
Fc responsible for these activities.
[0504] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC"
refers to a cell-mediated reaction in which non-specific cytotoxic
cells that express Fc receptors (FcRs) (e.g. Natural Killer (NK)
cells, neutrophils, and macrophages) recognize bound antibody on a
target cell and subsequently cause lysis of the target cell.
[0505] The ability of monoclonal antibodies to induce ADCC can be
enhanced by engineering their oligosaccharide component. Human IgG1
or IgG3 are N-glycosylated at Asn297 with the majority of the
glycans in the well known biantennary G0, G0F, G1, G1F, G2 or G2F
forms. Antibodies produced by non-engineered CHO cells typically
have a glycan fucose content of about at least 85%. The removal of
the core fucose from the biantennary complex-type oligosaccharides
attached to the Fc regions enhances the ADCC of antibodies via
improved Fc.gamma.RIIIa binding without altering antigen binding or
CDC activity. Such mAbs can be achieved using different methods
reported to lead to the successful expression of relatively high
defucosylated antibodies bearing the biantennary complex-type of Fc
oligosaccharides such as control of culture osmolality (Konno et
al., Cytotechnology 64(:249-65, 2012), application of a variant CHO
line Lec13 as the host cell line (Shields et al., J Biol Chem
277:26733-26740, 2002), application of a variant CHO line EB66 as
the host cell line (Olivier et al., MAbs; 2(4), 2010; Epub ahead of
print; PMID:20562582), application of a rat hybridoma cell line
YB2/0 as the host cell line (Shinkawa et al., J Biol Chem
278:3466-3473, 2003), introduction of small interfering RNA
specifically against the .alpha. 1,6-fucosyltrasferase (FUT8) gene
(Mori et al., Biotechnol Bioeng88:901-908, 2004), or coexpression
of .beta.-1,4-N-acetylglucosaminyltransferase III and Golgi
.alpha.-mannosidase II or a potent alpha-mannosidase I inhibitor,
kifunensine (Ferrara et al., J Biol Chem281:5032-5036, 2006,
Ferrara et al., Biotechnol Bioeng 93:851-861, 2006; Xhou et al.,
Biotechnol Bioeng 99:652-65, 2008).
[0506] In some embodiments described herein, ADCC elicited by the
bispecific EGFR/c-Met antibodies may also be enhanced by certain
substitutions in the antibody Fc. Exemplary substitutions are for
example substitutions at amino acid positions 256, 290, 298, 312,
356, 330, 333, 334, 360, 378 or 430 (residue numbering according to
the EU index) as described in U.S. Pat. No. 6,737,056.
[0507] In some embodiments described herein, the bispecific
EGFR/c-Met antibody of the invention has a biantennary glycan
structure with fucose content of about between 1% to about 15%, for
example 15%, 14%, 13%, 12%, 11% 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%
or 1% In some embodiments, the bispecific EGFR/c-Met antibody has a
glycan structure with fucose content of about 50%, 40%, 45%, 40%,
35%, 30%, 25%, or 20%.
[0508] "Fucose content" means the amount of the fucose
monosaccharide within the sugar chain at Asn297. The relative
amount of fucose is the percentage of fucose-containing structures
related to all glycostructures. These may be characterized and
quantified by multiple methods, for example: 1) using MALDI-TOF of
N-glycosidase F treated sample (e.g. complex, hybrid and oligo- and
high-mannose structures) as described in Int Pat. Publ. No.
WO2008/077546 2); 2) by enzymatic release of the Asn297 glycans
with subsequent derivatization and detection/quantitation by HPLC
(UPLC) with fluorescence detection and/or HPLC-MS (UPLC-MS); 3)
intact protein analysis of the native or reduced mAb, with or
without treatment of the Asn297 glycans with Endo S or other enzyme
that cleaves between the first and the second GlcNAc
monosaccharides, leaving the fucose attached to the first GlcNAc;
4) digestion of the mAb to constituent peptides by enzymatic
digestion (e.g., trypsin or endopeptidase Lys-C), and subsequent
separation, detection and quantitation by HPLC-MS (UPLC-MS); 5)
Separation of the mAb oligosaccharides from the mAb protein by
specific enzymatic deglycosylation with PNGase F at Asn 297. The
oligosaccharides thus released can be labeled with a fluorophore,
separated and identified by various complementary techniques which
allow: fine characterization of the glycan structures by
matrix-assisted laser desorption ionization (MALDI) mass
spectrometry by comparison of the experimental masses with the
theoretical masses, determination of the degree of sialylation by
ion exchange HPLC (GlycoSep C), separation and quantification of
the oligosaccharide forms according to hydrophilicity criteria by
normal-phase HPLC (GlycoSep N), and separation and quantification
of the oligosaccharides by high performance capillary
electrophoresis-laser induced fluorescence (HPCE-LIF).
[0509] "Low fucose" or "low fucose content" as used in the
application refers to antibodies with fucose content of about
between 1%-15%.
[0510] "Normal fucose" or `normal fucose content" as used herein
refers to antibodies with fucose content of about over 50%,
typically about over 80% or over 85%.
[0511] Some embodiments of the invention provide a synthetic
nucleic acid encoding the heavy chains and the light chains of the
bispecific EGFR/c-Met binding antibodies of the invention as
described herein as isolated polynucleotides or as portions of
expression vectors or as portions of linear DNA sequences,
including linear DNA sequences used for in vitro
transcription/translation, vectors compatible with prokaryotic,
eukaryotic or filamentous phage expression, secretion and/or
display of the compositions or directed mutagens thereof.
[0512] Some embodiments of the invention provide an isolated
polynucleotide comprising the polynucleotide sequence of SEQ ID
NOs: 205, 206, 207 or 208.
[0513] The polynucleotides of the invention may be produced by
chemical synthesis such as solid phase polynucleotide synthesis on
an automated polynucleotide synthesizer and assembled into complete
single or double stranded molecules. Alternatively, the
polynucleotides of the invention may be produced by other
techniques such as PCR followed by routine cloning. Techniques for
producing or obtaining polynucleotides of a given known sequence
are well known in the art.
[0514] The polynucleotides of the invention may comprise at least
one non-coding sequence, such as a promoter or enhancer sequence,
intron, polyadenylation signal, a cis sequence facilitating RepA
binding, and the like. The polynucleotide sequences may also
comprise additional sequences encoding additional amino acids that
encode for example a marker or a tag sequence such as a histidine
tag or an HA tag to facilitate purification or detection of the
protein, a signal sequence, a fusion protein partner such as RepA,
Fc or bacteriophage coat protein such as pIX or pIII.
[0515] Some embodiments described herein provide for a vector
comprising the polynucleotide of the invention. Such vectors may be
plasmid vectors, viral vectors, vectors for baculovirus expression,
transposon based vectors or any other vector suitable for
introduction of the polynucleotide of the invention into a given
organism or genetic background by any means. For example,
polynucleotides encoding heavy and light chains of the bispecific
antibodies of the invention may be inserted into expression
vectors. The light and heavy chains may be cloned in the same or
different expression vectors. The DNA segments encoding
immunoglobulin chains may be operably linked to control sequences
in the expression vector(s) that ensure the expression of
immunoglobulin polypeptides. Such control sequences include signal
sequences, promoters (e.g. naturally associated or heterologous
promoters), enhancer elements, and transcription termination
sequences, and may be chosen to be compatible with the host cell
chosen to express the antibody. Once the vector has been
incorporated into the appropriate host, the host may be maintained
under conditions suitable for high level expression of the proteins
encoded by the incorporated synthetic polynucleotides.
[0516] Suitable expression vectors are typically replicable in the
host organisms either as episomes or as an integral part of the
host chromosomal DNA. Commonly, expression vectors contain
selection markers such as ampicillin-resistance,
hygromycin-resistance, tetracycline resistance, kanamycin
resistance or neomycin resistance to permit detection of those
cells transformed with the desired DNA sequences.
[0517] Some embodiments described herein provide for a host cell
comprising the vector of the invention. The term "host cell" refers
to a cell into which a vector has been introduced. It is understood
that the term host cell is intended to refer not only to the
particular subject cell but to the progeny of such a cell. Because
certain modifications may occur in succeeding generations due to
either mutation or environmental influences, such progeny may not
be identical to the parent cell, but are still included within the
scope of the term "host cell" as used herein. Such host cells may
be eukaryotic cells, prokaryotic cells, plant cells or archeal
cells.
[0518] Exemplary eukaryotic cells may be of mammalian, insect,
avian or other animal origins. Mammalian eukaryotic cells include
immortalized cell lines such as hybridomas or myeloma cell lines
such as SP2/0 (American Type Culture Collection (ATCC), Manassas,
Va., CRL-1581), NS0 (European Collection of Cell Cultures (ECACC),
Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646)
and Ag653 (ATCC CRL-1580) murine cell lines. An exemplary human
myeloma cell line is U266 (ATTC CRL-TIB-196). Other useful cell
lines include those derived from Chinese Hamster Ovary (CHO) cells
such as CHO-K1SV (Lonza Biologics, Walkersville, Md.), CHO-K1 (ATCC
CRL-61) or DG44.
Uses of Bispecific EGFR/c-Met FN3 Domain Containing Molecules,
Bispecific EGFR/-c-Met Antibodies and EGFR-Binding or c-Met Binding
FN3 Domains of the Invention
[0519] The bispecific EGFR/c-Met FN3 domain containing molecules,
the EGFR binding FN3 domains, the c-Met binding FN3 domains or the
bispecific EGFR-c-Met antibodies of the invention may be used to
diagnose, monitor, modulate, treat, alleviate, help prevent the
incidence of, or reduce the symptoms of human disease or specific
pathologies in cells, tissues, organs, fluid, or, generally, a
host. The methods of the invention may be used to treat an animal
patient belonging to any classification. Examples of such animals
include mammals such as humans, rodents, dogs, cats and
farm/domestic animals.
[0520] One aspect of the invention is a method for inhibiting
growth or proliferation of cells that express EGFR and/or c-Met,
comprising contacting the cells with the isolated bispecific
EGFR/c-Met FN3 domain containing molecule, the EGFR binding FN3
domain, the c-Met binding FN3 domain or the bispecific EGFR/c-Met
antibody of the invention.
[0521] Another aspect of the invention is a method for inhibiting
growth or metastasis of EGFR and/or c-Met-expressing tumor or
cancer cells in a subject comprising administering to the subject
an effective amount of the isolated bispecific EGFR/c-Met FN3
domain containing molecule, the EGFR binding FN3 domain, the c-Met
binding FN3 domain or the bispecific EGFR/c-Met antibody of the
invention so that the growth or metastasis of EGFR- and/or
c-Met-expressing tumor or cancer cell is inhibited.
[0522] Another aspect of the invention is a method of treating a
subject having cancer, comprising administering a therapeutically
effective amount of the isolated bispecific EGFR/c-Met FN3 domain
containing molecule, the EGFR binding FN3 domain, the c-Met binding
FN3 domain or the bispecific EGFR/c-Met antibody of the invention
to a patient in need thereof for a time sufficient to treat the
cancer.
[0523] The bispecific EGFR/c-Met FN3 domain containing molecule,
the EGFR binding FN3 domain, the c-Met binding FN3 domain or the
bispecific EGFR/c-Met antibodies of the invention may be used for
treatment of any disease or disorder characterized by abnormal
activation or production of EGFR, c-Met, EGF, soluble EGFR, soluble
c-Met or other EGFR ligand or HGF, or disorder related to EGFR or
c-Met expression, which may or may not involve malignancy or
cancer, where abnormal activation and/or production of EGFR, c-Met,
EGF or other EGFR ligand, or HGF is occurring in cells or tissues
of a subject having, or predisposed to, the disease or
disorder.
[0524] The FN3 domains that specifically bind c-Met and block
binding of HGF to c-Met of the invention may be for treatment of
tumors, including cancers and benign tumors. Cancers that are
amenable to treatment by the c-Met binding FN3 domains of the
invention include those that overexpress c-Met. Exemplary cancers
that are amenable to treatment by the FN3 domains of the invention
include epithelial cell cancers, breast cancer, ovarian cancer,
lung cancer, colorectal cancer, anal cancer, prostate cancer,
kidney cancer, bladder cancer, head and neck cancer, gastric
cancer, ovarian cancer, pancreatic cancer, skin cancer, oral
cancer, esophageal cancer, vaginal cancer, cervical cancer, cancer
of the spleen, testicular cancer, and cancer of the thymus.
[0525] The FN3 domains that specifically bind EGFR and blocks
binding of EGF to the EGFR of the invention may be used for
treatment of tumors, including cancers and benign tumors. Cancers
that are amenable to treatment by the FN3 domains of the invention
include those that overexpress EGFR or variants. Exemplary cancers
that are amenable to treatment by the FN3 domains of the invention
include epithelial cell cancers, breast cancer, ovarian cancer,
lung cancer, colorectal cancer, anal cancer, prostate cancer,
kidney cancer, bladder cancer, head and neck cancer, ovarian
cancer, pancreatic cancer, skin cancer, oral cancer, esophageal
cancer, vaginal cancer, cervical cancer, cancer of the spleen,
testicular cancer, and cancer of the thymus. The bispecific
EGFR/c-Met FN3 domain containing molecules or the bispecific
EGFR/c-Met antibodies of the invention may be used for treatment of
tumors, including cancers and benign tumors. Exemplary cancers that
are amenable to treatment by the bispecific EGFR/c-Met FN3 domain
containing molecule or the bispecific EGFR/c-Met antibody of the
invention include those that over-express EGFR and/or c-Met,
cancers associated with elevated EGFR activity and/or expression
levels (such as, for example, an EGFR activating mutation, an EGFR
gene amplification, or ligand mediated EGFR activation) and
elevated c-Met activity and/or expression levels (such as, for
example, a c-Met activating mutation, a c-Met gene amplification,
or HGF mediated c-Met activation).
[0526] Exemplary EGFR activating mutations that may be associated
with cancer include point mutations, deletion mutations, insertion
mutations, inversions or gene amplifications that lead to an
increase in at least one biological activity of EGFR, such as
elevated tyrosine kinase activity, formation of receptor homodimers
and heterodimers, enhanced ligand binding etc. Mutations can be
located in any portion of an EGFR gene or regulatory region
associated with an EGFR gene and include mutations in exon 18, 19,
20 or 21 or mutations in the kinase domain. Exemplary activating
EGFR mutations are G719A, L861X (X being any amino acid), L858R,
E746K, L747S, E749Q, A750P, A755V, V765M, L858P or T790M
substitutions, deletion of E746-A750, deletion of R748-P753,
insertion of Ala between M766 and A767, insertion of SVA (Ser, Val,
Ala) between 5768 and V769, and insertion of NS (Asn, Ser) between
P772 and H773. Other examples of EGFR activating mutations are
known in the art (see e.g., U.S. Pat. Publ. No. US2005/0272083).
Information about EGFR and other ErbB receptors including receptor
homo- and hetero-dimers, receptor ligands, autophosphorylation
sites, and signaling molecules involved in ErbB mediated signaling
is known in the art (see e.g., Hynes and Lane, Nature Reviews
Cancer 5: 341-354, 2005).
[0527] Exemplary c-Met activating mutations include point
mutations, deletion mutations, insertion mutations, inversions or
gene amplifications that lead to an increase in at least one
biological activity of a c-Met protein, such as elevated tyrosine
kinase activity, formation of receptor homodimers and heterodimers,
enhanced ligand binding etc. Mutations can be located in any
portion of the c-Met gene or regulatory regions associated with the
gene, such as mutations in the kinase domain of c-Met. Exemplary
c-Met activating mutations are mutations at residue positions N375,
V13, V923, R175, V136, L229, S323, R988, S1058/T1010 and E168.
Methods for detecting EGFR and c-Met mutations or gene
amplifications are well known.
[0528] Exemplary cancers that are amenable to treatment by the
bispecific molecules of the invention such as the bispecific
EGFR/c-Met antibodies of the invention include epithelial cell
cancers, breast cancer, ovarian cancer, lung cancer, non-small cell
lung cancer (NSCLC), lung adenocarcinoma, small cell lung cancer,
colorectal cancer, anal cancer, prostate cancer, kidney cancer,
bladder cancer, head and neck cancer, pharynx cancer, cancer of the
nose, pancreatic cancer, skin cancer, oral cancer, cancer of the
tongue, esophageal cancer, vaginal cancer, cervical cancer, cancer
of the spleen, testicular cancer, gastric cancer, cancer of the
thymus, colon cancer, thyroid cancer, liver cancer (hepatocellular
carcinoma (HCC)) or sporadic or hereditary papillary renal cell
carcinoma (PRCC).
[0529] Another aspect of the invention is a method of treating a
subject having cancer, comprising administering a therapeutically
effective amount of the isolated bispecific EGFR/c-Met antibody of
the invention to a patient in need thereof for a time sufficient to
treat the cancer, wherein the subject is homozygous for
phenylalanine at position 158 of CD16 (Fc.gamma.RIIIa-158F/F
genotype) or heterozygous for valine and phenylalanine at position
158 of CD16 (Fc.gamma.RIIIa-158F/V genotype). CD16 is also known as
the Fc gamma receptor Ma (Fc.gamma.RIIIa) or the low affinity
immunoglobulin gamma Fc region receptor III-A isoform.
Valine/phenylalanine (V/F) polymorphism at Fc.gamma.RIIIa protein
residue position 158 has been shown to affect Fc.gamma.RIIIa
affinity to human IgG. Receptor with Fc.gamma.RIIIa-158F/F or
Fc.gamma.RIIIa-158F/V polymorphisms demonstrates reduced Fc
engagement and therefore reduced ADCC when compared to the
Fc.gamma.RIIIa-158V/V. The lack of or low amount of fucose on human
N-linked oligosaccharides improves the ability of the antibodies to
induce ADCC due to improved binding of the antibodies to human
Fc.gamma.RIIIa (CD16) (Shields et al., J Biol Chem 277:26733-40,
2002). The antibodies of the invention have reduced fucose content
of about between 1% to about 10%. In some embodiments, the
bispecific EGFR/c-Met antibody has a glycan structure with fucose
content of about 50%, 40%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%,
9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%. Therefore, the antibodies of
the invention may be more efficacious in the treatment of patients
with Fc.gamma.RIIIa-158F/F or Fc.gamma.RIIIa-158F/V genotypes.
Patients can be analyzed for their Fc.gamma.RIIIa polymorphism
using routine methods.
[0530] In some methods described herein, the antibodies of the
invention may be used to treat a subject having cancer that is
resistant or has acquired resistance to treatment with one or more
EGFR inhibitors. Exemplary EGFR inhibitors for which cancer may
acquire resistance are anti-EGFR antibodies cetuximab
(Erbitux.RTM.), panitumumab (Vectibix.RTM.), matuzumab,
nimotuzumab, small molecule EGFR inhibitors Tarceva.RTM.
(erlotinib), IRESSA (gefitinib), EKB-569 (pelitinib, irreversible
EGFR TKI), pan-ErbB and other receptor tyrosine kinase inhibitors,
lapatinib (EGFR and HER2 inhibitor), pelitinib (EGFR and HER2
inhibitor), vandetanib (ZD6474, ZACTIMA.TM., EGFR, VEGFR2 and RET
TKI), PF00299804 (dacomitinib, irreversible pan-ErbB TKI), CI-1033
(irreversible pan-erbB TKI), afatinib (BIBW2992, irreversible
pan-ErbB TKI), AV-412 (dual EGFR and ErbB2 inhibitor), EXEL-7647
(EGFR, ErbB2, GEVGR and EphB4 inhibitor), CO-1686 (irreversible
mutant-selective EGFR TKI), AZD9291 (irreversible mutant-selective
EGFR TKI), and HKI-272 (neratinib, irreversible EGFR/ErbB2
inhibitor). The methods described herein may be used to treat
cancer that is resistant to treatment with gefitinib, erlotinib,
afatinib, CO-1686, AZD9291 and/or cetuximab. An exemplary antibody
that can be used is EM1-mAb.
[0531] Another aspect of the invention is a method of treating a
subject having cancer, comprising administering a therapeutically
effective amount of the isolated bispecific EGFR/c-Met antibody of
the invention to a patient in need thereof for a time sufficient to
treat the cancer, wherein the subject is resistant or has acquired
resistance to treatment with erlotinib, gefitinib, afatinib,
CO-1686, AZD9291 or cetuximab.
[0532] Various qualitative and/or quantitative methods may be used
to determine if a subject is resistant, has developed or is
susceptible to developing a resistance to treatment with an EGFR
inhibitor. Symptoms that may be associated with resistance to an
EGFR inhibitor include, for example, a decline or plateau of the
well-being of the patient, an increase in the size of a tumor,
arrested or slowed decline in growth of a tumor, and/or the spread
of cancerous cells in the body from one location to other organs,
tissues or cells. Re-establishment or worsening of various symptoms
associated with cancer may also be an indication that a subject has
developed or is susceptible to developing resistance to EGFR
inhibitors, such as anorexia, cognitive dysfunction, depression,
dyspnea, fatigue, hormonal disturbances, neutropenia, pain,
peripheral neuropathy, and sexual dysfunction. The symptoms
associated with cancer may vary according to the type of cancer.
For example, symptoms associated with cervical cancer may include
abnormal bleeding, unusual heavy vaginal discharge, pelvic pain
that is not related to the normal menstrual cycle, bladder pain or
pain during urination, and bleeding between regular menstrual
periods, after sexual intercourse, douching, or pelvic exam.
Symptoms associated with lung cancer may include persistent cough,
coughing up blood, shortness of breath, wheezing chest pain, loss
of appetite, losing weight without trying and fatigue. Symptoms for
liver cancer may include loss of appetite and weight, abdominal
pain, especially in the upper right part of abdomen that may extend
into the back and shoulder, nausea and vomiting, general weakness
and fatigue, an enlarged liver, abdominal swelling (ascites), and a
yellow discoloration of the skin and the whites of eyes (jaundice).
One skilled in oncology may readily identify symptoms associated
with a particular cancer type.
[0533] Others means to determine if a subject has developed a
resistance to an EGFR inhibitor include examining EGFR
phosphorylation, ERK1/2 phosphorylation and/or AKT phosphorylation
in cancer cells, where increased phosphorylation may be indicative
that the subject has developed or is susceptible to developing
resistance to an EGFR inhibitor. Methods of determining EGFR,
ERK1/2 and/or AKT phosphorylation are well known and described
herein. Identification of a subject who has developed a resistance
to an EGFR inhibitor may involve detection of elevated c-Met
expression levels or elevated c-Met activity, for example, arising
from increased levels of circulating HGF, an activating mutation of
the c-Met gene or a c-Met gene amplification.
[0534] Another embodiment of the invention is a method of treating
NSCLC in a patient having an NSCLC tumor or tumor metastasis having
an activating EGFR mutation or EGFR gene amplification, comprising
administering to the patient a therapeutically effective amount of
the bispecific EGFR/c-Met antibody of the invention.
[0535] The bispecific EGFR/c-Met antibodies of the invention can be
used to treat non-small cell lung cancer (NSCLC), which includes
squamous cell carcinoma, adenocarcinoma, and large cell carcinoma.
In some embodiments, cells of the NSCLC have an epithelial
phenotype. In some embodiments, the NSCLC has acquired resistance
to treatment with one or more EGFR inhibitors.
[0536] In NSCLC, specific mutations in the EGFR gene are associated
with high response rates (70-80%) to EGFR tyrosine kinase
inhibitors (EGFR-TKIs). A 5 amino acid deletion in exon 19 or the
point mutation L858R in EGFR are associated with EGFR-TKI
sensitivity (Nakata and Gotoh, Expert Opin Ther Targets 16:771-781,
2012). These mutations result in a ligand-independent activation of
the EGFR kinase activity. Activating EGFR mutations occur in 10-30%
of NSCLC patients and are significantly more common in East Asians,
women, never smokers, and patients with adenocarcinoma histology
(Janne and Johnson Clin Cancer Res 12(14 Suppl): 4416s-4420s,
2006). EGFR gene amplification is also strongly correlated with
response after EGFR-TKI treatment (Cappuzzo et al., J Natl Cancer
Inst 97:643-55, 2005).
[0537] Although the majority of NSCLC patients with EGFR mutations
initially respond to EGFR TKI therapy, virtually all acquire
resistance that prevents a durable response. 50-60% of patients
acquire resistance due to a second-site point mutation in the
kinase domain of EGFR (T790M). Nearly 60% of all tumors that become
resistant to EGFR tyrosine kinase inhibitors increase c-Met
expression, amplify the c-Met gene, or increase its only known
ligand, HGF (Turke et al., Cancer Cell, 17:77-88, 2010).
[0538] Another embodiments of the invention is a method of treating
patient having cancer, comprising administering a therapeutically
effective amount of the bispecific EGFR/c-Met antibody of the
invention to a patient in need thereof for a time sufficient to
treat the cancer, wherein the cancer is associated with an EGFR
activating mutation, an EGFR gene amplification, increased levels
of circulating HGF, a c-Met activating mutation, a c-Met gene
amplification or a mutant KRAS.
[0539] In some embodiments the EGFR activating mutation is G719A,
G719X (X being any amino acid), L861X (X being any amino acid),
L858R, E746K, L747S, E749Q, A750P, A755V, V765M, L858P or T790M
substitution, deletion of E746-A750, deletion of R748-P753,
insertion of Ala (A) between M766 and A767, insertion of Ser, Val
and Ala (SVA) between 5768 and V769, and insertion of Asn and Ser
(NS) between P772 and H773.
[0540] Another embodiments of the invention is a method of treating
patient having cancer, comprising administering a therapeutically
effective amount of the bispecific EGFR/c-Met antibody of the
invention to a patient in need thereof for a time sufficient to
treat the cancer, wherein the cancer is associated with an EGFR
mutation L858R, T790M or deletion of residues E746-A750 (del(E746,
A750)), EGFR amplification or c-Met amplification.
[0541] In some embodiments, the cancer is associated with wild type
EGFR and wild type c-Met.
[0542] In some embodiments, the cancer is associated with wild type
EGFR and c-Met amplification.
[0543] In some embodiments, the cancer is associated with EGFR
L858R and T790M mutations and wild type c-Met.
[0544] In some embodiments, the cancer is associated with EGFR
deletion del(E764, A750) and wild type c-Met.
[0545] In some embodiments, the cancer is associated with EGFR
deletion del(E764, A750) and c-Met amplification.
[0546] In some embodiments, the cancer is associated with EGFR
deletion del(E764, A750), EGFR amplification and c-Met
amplification.
[0547] In some embodiments, the patient has a NSCLC associated with
EGFR L858R and T790M mutations and wild type c-Met.
[0548] In some embodiments, the patient has a NSCLC associated with
EGFR amplification and wild type c-Met.
[0549] In some embodiments, the patient has a NSCLC associated with
EGFR amplification and c-Met amplification.
[0550] In some embodiments, the patient has a NSCLC associated with
EGFR deletion del(E764, A750) and wild type c-Met.
[0551] In some embodiments, the patient has a NSCLC associated with
EGFR deletion del(E764, A750) and c-Met amplification.
[0552] In some embodiments, the patients are treated with the
EM1-mAb of the invention. The EM1-mAb of the invention shows
efficacy in in vivo tumor animal models, when the tumors are
associated with L858R, T790M, del(E746, A750) EGFR, EGFR
amplification, wild type c-Met and/or c-Met amplification.
Amplification of EGFR or c-Met may be evaluated by standard
methods, for example by determining the copy number of the EGFR or
c-Met gene by southern blotting, FISH, or comparative genomic
hybridization (CGH).
[0553] Another embodiments of the invention is a method of treating
patient having cancer, comprising administering a therapeutically
effective amount of the bispecific EGFR/c-Met antibody of the
invention to a patient in need thereof for a time sufficient to
treat the cancer, wherein the cancer is associated with EGFR
mutations L858R, T790M or deletion of residues E746-A750 (del(E746,
A750)), EGFR amplification or c-Met amplification, and mutant
KRAS.
[0554] In some embodiments, the mutant KRAS has a G12V
substitution. KRAS belongs to the family of RAS proto-oncogenes
encoding guanosine triphosphatases (GTPases), and mediates EGFR
signal transduction downstream of the receptor. Tumors with
proto-oncogenic KRAS mutations such as the activating G12V or G12C
mutation would therefore not be expected to be treatable by EGFR
antibodies. Clinical studies with anti-EGFR antibodies cetuximab or
panitumumab demonstrated that patients with KRAS-mutated colorectal
tumors do not respond to these agents (Van Cutsem et al., N Eng J
Med 360:1408-1417, 2009; Lievre et al., J Clin Oncol 26:374-379,
2008; Amado et al., J Clin Oncol 26:1626-1634m 2008). The
bispecific EGFR/c-Met antibodies of the invention mediate KRAS
mutant cell line killing via effective ADCC, and therefore,
contrary to the current anti-EGFR therapies, may be efficacious in
treatment of patients whose cancer is associated with KRAS
activating mutations. Such exemplary antibody is the EM1-mAb.
[0555] The terms "treat" or "treatment" refers to both therapeutic
treatment and prophylactic or preventative measures, wherein the
object is to prevent or slow down (lessen) an undesired
physiological change or disorder, such as the development or spread
of cancer. For purposes of this invention, beneficial or desired
clinical results include, but are not limited to, alleviation of
symptoms, diminishment of extent of disease, stabilized (i.e., not
worsening) state of disease, delay or slowing of disease
progression, amelioration or palliation of the disease state, and
remission (whether partial or total), whether detectable or
undetectable. "Treatment" can also mean prolonging survival as
compared to expected survival if not receiving treatment. Those in
need of treatment include those already with the condition or
disorder as well as those prone to have the condition or disorder
or those in which the condition or disorder is to be prevented.
[0556] A "therapeutically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
a desired therapeutic result. A therapeutically effective amount of
the bispecific EGFR/c-Met antibody of the invention may vary
according to factors such as the disease state, age, sex, and
weight of the individual, and the ability of the bispecific
EGFR/c-Met antibody of the invention to elicit a desired response
in the individual. Exemplary indicators of an effective EGFR/c-Met
therapeutic that may decline or abate in association with
resistance include, for example, improved well-being of the
patient, decrease or shrinkage of the size of a tumor, arrested or
slowed growth of a tumor, and/or absence of metastasis of cancer
cells to other locations in the body.
Administration/Pharmaceutical Compositions
[0557] The invention provides for pharmaceutical compositions
comprising the bispecific EGFR/c-Met antibody of the invention and
a pharmaceutically acceptable carrier. For therapeutic use, the
bispecific EGFR/c-Met FN3 domain containing molecules, the
EGFR-binding FN3 domains, the c-Met-binding FN3 domains or the
bispecific EGFR/c-Met antibodies of the invention may be prepared
as pharmaceutical compositions containing an effective amount of
the domain, molecule or antibody as an active ingredient in a
pharmaceutically acceptable carrier. The term "carrier" refers to a
diluent, adjuvant, excipient, or vehicle with which the active
compound is administered. Such vehicles may be liquids, such as
water and oils, including those of petroleum, animal, vegetable or
synthetic origin, such as peanut oil, soybean oil, mineral oil,
sesame oil and the like. For example, 0.4% saline and 0.3% glycine
can be used. These solutions are sterile and generally free of
particulate matter. They may be sterilized by conventional,
well-known sterilization techniques (e.g., filtration). The
compositions may contain pharmaceutically acceptable auxiliary
substances as required to approximate physiological conditions such
as pH adjusting and buffering agents, stabilizing, thickening,
lubricating and coloring agents, etc. The concentration of the
molecules or antibodies of the invention in such pharmaceutical
formulation may vary widely, i.e., from less than about 0.5%,
usually to at least about 1% to as much as 15 or 20% by weight and
will be selected primarily based on required dose, fluid volumes,
viscosities, etc., according to the particular mode of
administration selected. Suitable vehicles and formulations,
inclusive of other human proteins, e.g., human serum albumin, are
described, for example, in e.g. Remington: The Science and Practice
of Pharmacy, 21.sup.st Edition, Troy, D. B. ed., Lipincott Williams
and Wilkins, Philadelphia, Pa. 2006, Part 5, Pharmaceutical
Manufacturing pp 691-1092, See especially pp. 958-989.
[0558] The mode of administration for therapeutic use of the
bispecific EGFR/c-Met FN3 domain containing molecules, the
EGFR-binding FN3 domains, the c-Met-binding FN3 domains or the
bispecific EGFR/c-Met antibodies of the invention may be any
suitable route that delivers the agent to the host, such as
parenteral administration, e.g., intradermal, intramuscular,
intraperitoneal, intravenous or subcutaneous, pulmonary,
transmucosal (oral, intranasal, intravaginal, rectal), using a
formulation in a tablet, capsule, solution, powder, gel, particle;
and contained in a syringe, an implanted device, osmotic pump,
cartridge, micropump; or other means appreciated by the skilled
artisan, as well known in the art. Site specific administration may
be achieved by for example intrarticular, intrabronchial,
intraabdominal, intracapsular, intracartilaginous, intracavitary,
intracelial, intracerebellar, intracerebroventricular, intracolic,
intracervical, intragastric, intrahepatic, intracardial,
intraosteal, intrapelvic, intrapericardiac, intraperitoneal,
intrapleural, intraprostatic, intrapulmonary, intrarectal,
intrarenal, intraretinal, intraspinal, intrasynovial,
intrathoracic, intrauterine, intravascular, intravesical,
intralesional, vaginal, rectal, buccal, sublingual, intranasal, or
transdermal delivery.
[0559] Thus, a pharmaceutical composition of the invention for
intramuscular injection may be prepared to contain 1 ml sterile
buffered water, and between about 1 ng to about 100 mg/kg, e.g.
about 50 ng to about 30 mg/kg or more preferably, about 5 mg to
about 25 mg/kg, of the bispecific EGFR/c-Met FN3 domain containing
molecules, the EGFR-binding FN3 domains or the c-Met-binding FN3
domains of the invention.
[0560] The bispecific EGFR/c-Met antibodies of the invention may be
administered to a patient by any suitable route, for example
parentally by intravenous (IV) infusion or bolus injection,
intramuscularly or subcutaneously or intraperitoneally. IV infusion
can be given over as little as 15 minutes, but more often for 30
minutes, 60 minutes, 90 minutes or even 2 or 3 hours. The
bispecific EGFR/c-Met antibodies of the invention may also be
injected directly into the site of disease (e.g., the tumor
itself). The dose given to a patient having a cancer is sufficient
to alleviate or at least partially arrest the disease being treated
("therapeutically effective amount") and may be sometimes 0.1 to 10
mg/kg body weight, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
mg/kg, but may even higher, for example 15, 20, 30, 40, 50, 60, 70,
80, 90 or 100 mg/kg. A fixed unit dose may also be given, for
example, 50, 100, 200, 500 or 1000 mg, or the dose may be based on
the patient's surface area, e.g., 400, 300, 250, 200, or 100
mg/m.sup.2. Usually between 1 and 8 doses, (e.g., 1, 2, 3, 4, 5, 6,
7 or 8) may be administered to treat cancer, but 10, 12, 20 or more
doses may be given. Administration of the bispecific EGFR/c-Met
antibody of the invention may be repeated after one day, two days,
three days, four days, five days, six days, one week, two weeks,
three weeks, one month, five weeks, six weeks, seven weeks, two
months, three months, four months, five months, six months or
longer. Repeated courses of treatment are also possible, as is
chronic administration. The repeated administration may be at the
same dose or at a different dose.
[0561] For example, a pharmaceutical composition comprising the
bispecific EGFR/c-Met antibody of the invention for intravenous
infusion may be made up to contain about 200 ml of sterile Ringer's
solution, and about 8 mg to about 2400 mg, about 400 mg to about
1600 mg, or about 400 mg to about 800 mg of the bispecific
EGFR/c-Met antibody for administration to a 80 kg patient. Methods
for preparing parenterally administrable compositions are well
known and are described in more detail in, for example,
"Remington's Pharmaceutical Science", 15th ed., Mack Publishing
Company, Easton, Pa.
[0562] The bispecific EGFR/c-Met FN3 domain containing molecules,
the EGFR-binding FN3 domains, the c-Met-binding FN3 domains or the
bispecific EGFR/c-Met antibodies of the invention may be
lyophilized for storage and reconstituted in a suitable carrier
prior to use. This technique has been shown to be effective with
conventional protein preparations and well known lyophilization and
reconstitution techniques can be employed.
[0563] The bispecific EGFR/c-Met FN3 domain containing molecules,
the EGFR-binding FN3 domains, the c-Met-binding FN3 domains or the
bispecific EGFR/c-Met antibodies of the invention may be
administered in combination with a second therapeutic agent
simultaneously, sequentially or separately. The second therapeutic
agent may be a chemotherapeutic agent or a targeted anti-cancer
therapy.
[0564] The bispecific EGFR/c-Met antibody may be administered
together with any one or more of the chemotherapeutic drugs or
other anti-cancer therapeutics known to those of skill in the art.
Chemotherapeutic agents are chemical compounds useful in the
treatment of cancer and include growth inhibitory agents or other
cytotoxic agents and include alkylating agents, anti-metabolites,
anti-microtubule inhibitors, topoisomerase inhibitors, receptor
tyrosine kinase inhibitors, angiogenesis inhibitors and the like.
Examples of chemotherapeutic agents include alkylating agents such
as thiotepa and cyclosphosphamide (CYTOXAN.RTM.); alkyl sulfonates
such as busulfan, improsulfan and piposulfan; aziridines such as
benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
trietylenephosphoramide, triethylenethiophosphaoramide and
trimethylolomelamine; nitrogen mustards such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, ranimustine; antibiotics such as
aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,
cactinomycin, calicheamicin, carabicin, carminomycin,
carzinophilin, chromomycins, dactinomycin, daunorubicin,
detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin,
esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic
acid, nogalamycin, olivomycins, peplomycin, potfiromycin,
puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin,
tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such
as methotrexate and 5-FU; folic acid analogues such as denopterin,
methotrexate, pteropterin, trimetrexate; purine analogues such as
fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine
analogues such as ancitabine, azacitidine, 6-azauridine, carmofur,
cytarabine, dideoxyuridine, doxifluridine, enocitabine,
floxuridine; androgens such as calusterone, dromostanolone
propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals
such as aminoglutethimide, mitotane, trilostane; folic acid
replenisher such as frolinic acid; aceglatone; aldophosphamide
glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene;
edatraxate; defofamine; demecolcine; diaziquone; elfornithine;
elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol;
nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid;
2-ethylhydrazide; procarbazine; PSK.RTM.; razoxane; sizofiran;
spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; urethan; vindesine; dacarbazine;
mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;
arabinoside ("Ara-C"); cyclophosphamide; thiotepa; members of
taxoid or taxane family, such as paclitaxel (TAXOL.RTM.docetaxel
(TAXOTERE.RTM.) and analogues thereof; chlorambucil; gemcitabine;
6-thioguanine; mercaptopurine; methotrexate; platinum analogues
such as cisplatin and carboplatin; vinblastine; platinum; etoposide
(VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;
vinorelbine; navelbine; novantrone; teniposide; daunomycin;
aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor
RFS 2000; difluoromethylornithine (DMFO); retinoic acid;
esperamicins; capecitabine; inhibitors of receptor tyrosine kinases
and/or angiogenesis, including sorafenib (NEXAVAR.RTM.), sunitinib
(SUTENT.RTM.), pazopanib (VOTRIENT.TM.), toceranib (PALLADIA.TM.),
vandetanib (ZACTIMA.TM.), cediranib (RECENTIN.RTM.), regorafenib
(BAY 73-4506), axitinib (AG013736), lestaurtinib (CEP-701),
erlotinib (TARCEVA.RTM.), gefitinib (IRESSA.TM.), BIBW 2992
(TOVOK.TM.), lapatinib (TYKERB.RTM.), neratinib (HKI-272), and the
like, and pharmaceutically acceptable salts, acids or derivatives
of any of the above. Also included in this definition are
anti-hormonal agents that act to regulate or inhibit hormone action
on tumors such as anti-estrogens including for example tamoxifen,
raloxifene, aromatase inhibiting 4(5)-imidazoles,
4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone,
and toremifene (FARESTON.RTM.); and anti-androgens such as
flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and
pharmaceutically acceptable salts, acids or derivatives of any of
the above. Other conventional cytotoxic chemical compounds as those
disclosed in Wiemann et al., 1985, in Medical Oncology (Calabresi
et aL, eds.), Chapter 10, McMillan Publishing, are also applicable
to the methods of the present invention.
[0565] Exemplary agents that may be used in combination with the
bispecific EGFR/c-Met FN3 domain containing molecules, the
EGFR-binding FN3 domains, the c-Met-binding FN3 domains or the
bispecific EGFR/c-Met antibodies of the invention include tyrosine
kinase inhibitors and targeted anti-cancer therapies such as
Iressa.RTM. (gefitinib) and Tarceva (erlotinib) and other
antagonists of HER2, HER3, HER4 or VEGF. Exemplary HER2 antagonists
include CP-724-714, HERCEPTIN.TM. (trastuzumab), OMNITARG.TM.
(pertuzumab), TAK-165, lapatinib (EGFR and HER2 inhibitor), and
GW-282974. Exemplary HER3 antagonists include anti-Her3 antibodies
(see e.g., U.S. Pat. Publ. No. US2004/0197332). Exemplary HER4
antagonists include anti-HER4 siRNAs (see e.g., Maatta et al., Mol
Biol Cell 17: 67-79, 2006. An exemplary VEGF antagonist is
Bevacizumab (Avastin.TM.).
[0566] When a small molecule is used in combination with the
bispecific EGFR/c-Met antibody of the invention, it is typically
administered more often, preferably once a day, but 2, 3, 4 or more
times per day is also possible, as is every two days, weekly or at
some other interval. Small molecule drugs are often taken orally
but parenteral administration is also possible, e.g., by IV
infusion or bolus injection or subcutaneously or intramuscularly.
Doses of small molecule drugs may typically be from 10 to 1000 mg,
or about 100, 150, 200 or 250 mg.
[0567] When the bispecific EGFR/c-Met antibody of the invention is
administered in combination with a second therapeutic agent, the
combination may take place over any convenient timeframe. For
example, the bispecific EGFR/c-Met antibody and the second
therapeutic agent may be administered to a patient on the same day,
and even in the same intravenous infusion. However, the bispecific
EGFR/c-Met antibody and the second therapeutic agent may also be
administered on alternating days or alternating weeks, fortnights
or months, and so on. In some methods, the bispecific EGFR/c-Met
antibody and the second therapeutic agent are administered with
sufficient proximity in time that they are simultaneously present
(e.g., in the serum) at detectable levels in the patient being
treated. In some methods, an entire course of treatment of the
bispecific EGFR/c-Met antibody consisting of a number of doses over
a time period is followed or preceded by a course of treatment of
the second therapeutic agent also consisting of a number of doses.
In some methods, treatment with the bispecific EGFR/c-Met antibody
administered second is begun if the patient has resistance or
develops resistance to the second therapeutic agent administered
initially. The patient may receive only a single course or multiple
courses of treatment with one or both the bispecific EGFR/c-Met
antibody and the second therapeutic agent. A recovery period of 1,
2 or several days or weeks may be used between administration of
the bispecific EGFR/c-Met antibody and the second therapeutic
agent. When a suitable treatment regimen has already been
established for the second therapeutic agent, that regimen may be
used in combination with the bispecific EGFR/c-Met antibody of the
invention. For example, Tarceva.RTM. (erlotinib) is taken as a 100
mg or 150 mg pill once a day, and Iressa.RTM. (gefitinib) is taken
as 250 mg tablet daily.
[0568] The bispecific EGFR/c-Met antibody, optionally in
combination with the second therapeutic agent may be administered
together with any form of radiation therapy including external beam
radiation, intensity modulated radiation therapy (IMRT) and any
form of radiosurgery including Gamma Knife, Cyberknife, Linac, and
interstitial radiation (e.g. implanted radioactive seeds, GliaSite
balloon), and/or with surgery. Combination with radiation therapy
can be especially appropriate for head and neck cancer and brain
tumors.
[0569] While having described the invention in general terms, the
embodiments of the invention will be further disclosed in the
following examples that should not be construed as limiting the
scope of the claims.
Example 1. Construction of Tencon Libraries
[0570] Tencon (SEQ ID NO: 1) is an immunoglobulin-like scaffold,
fibronectin type III (FN3) domain, designed from a consensus
sequence of fifteen FN3 domains from human tenascin-C (Jacobs et
al., Protein Engineering, Design, and Selection, 25:107-117, 2012;
U.S. Pat. Publ. No. 2010/0216708). The crystal structure of Tencon
shows six surface-exposed loops that connect seven beta-strands.
These loops, or selected residues within each loop, can be
randomized in order to construct libraries of fibronectin type III
(FN3) domains that can be used to select novel molecules that bind
to specific targets.
Tencon:
LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLK
PGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO 1):
Construction of TCL1 Library
[0571] A library designed to randomize only the FG loop of Tencon
(SEQ ID NO: 1), TCL1, was constructed for use with the cis-display
system (Jacobs et al., Protein Engineering, Design, and Selection,
25:107-117, 2012). In this system, a single-strand DNA
incorporating sequences for a Tac promoter, Tencon library coding
sequence, RepA coding sequence, cis-element, and ori element is
produced. Upon expression in an in vitro transcription/translation
system, a complex is produced of the Tencon-RepA fusion protein
bound in cis to the DNA from which it is encoded. Complexes that
bind to a target molecule are then isolated and amplified by
polymerase chain reaction (PCR), as described below.
[0572] Construction of the TCL1 library for use with cis-display
was achieved by successive rounds of PCR to produce the final
linear, double-stranded DNA molecules in two halves; the 5'
fragment contains the promoter and Tencon sequences, while the 3'
fragment contains the repA gene and the cis- and ori elements.
These two halves are combined by restriction digest in order to
produce the entire construct. The TCL1 library was designed to
incorporate random amino acids only in the FG loop of Tencon,
KGGHRSN (SEQ ID NO: 86). NNS codons were used in the construction
of this library, resulting in the possible incorporation of all 20
amino acids and one stop codon into the FG loop. The TCL1 library
contains six separate sub-libraries, each having a different
randomized FG loop length, from 7 to 12 residues, in order to
further increase diversity. Design of Tencon-based libraries are
shown in Table 2.
TABLE-US-00010 TABLE 2 Library BC Loop Design FG Loop Design WT
Tencon TAPDAAFD* KGGHRSN** TCL1 TAPDAAFD* XXXXXXX XXXXXXXX
XXXXXXXXX XXXXXXXXXX XXXXXXXXXXX XXXXXXXXXXXX TCL2 ########
#####S## *TAPDAAFD: residues 22-28 of SEQ ID NO: 1; **KGGHRSN: SEQ
ID NO: 86 X refers to degenerate amino acids encoded by NNS codons.
# refers to the "designed distribution of amino acids" described in
the text.
[0573] To construct the TCL1 library, successive rounds of PCR were
performed to append the Tac promoter, build degeneracy into the FG
loop, and add necessary restriction sites for final assembly.
First, a DNA sequence containing the promoter sequence and Tencon
sequence 5' of the FG loop was generated by PCR in two steps. DNA
corresponding to the full Tencon gene sequence was used as a PCR
template with primers POP2220 (SEQID NO: 2) and TC5' to FG (SEQID
NO: 3). The resulting PCR product from this reaction was used as a
template for the next round of PCR amplification with primers
130mer (SEQID NO: 4) and Tc5' to FG to complete the appending of
the 5' and promoter sequences to Tencon. Next, diversity was
introduced into the FG loop by amplifying the DNA product produced
in the first step with forward primer POP2222 (SEQID NO: 5), and
reverse primers TCF7 (SEQID NO: 6), TCF8 (SEQID NO: 7), TCF9 (SEQID
NO: 8), TCF10 (SEQID NO: 9), TCF11 (SEQID N NO: 10), or TCF12
(SEQID NO: 11), which contain degenerate nucleotides. At least
eight 100 .mu.L PCR reactions were performed for each sub-library
to minimize PCR cycles and maximize the diversity of the library.
At least 5 .mu.g of this PCR product were gel-purified and used in
a subsequent PCR step, with primers POP2222 (SEQ ID NO: 5) and
POP2234 (SEQID NO: 12), resulting in the attachment of a
6.times.His tag and NotI restriction site to the 3' end of the
Tencon sequence. This PCR reaction was carried out using only
fifteen PCR cycles and at least 500 ng of template DNA. The
resulting PCR product was gel-purified, digested with NotI
restriction enzyme, and purified by Qiagen column.
[0574] The 3' fragment of the library is a constant DNA sequence
containing elements for display, including a PspOMI restriction
site, the coding region of the repA gene, and the cis- and ori
elements. PCR reactions were performed using a plasmid (pCR4Blunt)
(Invitrogen) containing this DNA fragment with M13 Forward and M13
Reverse primers. The resulting PCR products were digested by PspOMI
overnight and gel-purified. To ligate the 5' portion of library DNA
to the 3' DNA containing the repA gene, 2 pmol of 5' DNA were
ligated to an equal molar amount of 3' repA DNA in the presence of
NotI and PspOMI enzymes and T4 ligase. After overnight ligation at
37.degree. C., a small portion of the ligated DNA was run on a gel
to check ligation efficiency. The ligated library product was split
into twelve PCR amplifications and a 12-cycle PCR reaction was run
with primer pair POP2250 (SEQID NO: 13) and DidLigRev (SEQID NO:
14). The DNA yield for each sub-library of TCL1 library ranged from
32-34 .mu.g.
[0575] To assess the quality of the library, a small portion of the
working library was amplified with primers Tcon5new2 (SEQID NO: 15)
and Tcon6 (SEQID NO: 16), and was cloned into a modified pET vector
via ligase-independent cloning. The plasmid DNA was transformed
into BL21-GOLD (DE3) competent cells (Stratagene) and 96 randomly
picked colonies were sequenced using a T7 promoter primer. No
duplicate sequences were found. Overall, approximately 70-85% of
clones had a complete promoter and Tencon coding sequence without
frame-shift mutation. The functional sequence rate, which excludes
clones with STOP codons, was between 59% and 80%.
Construction of TCL2 Library
[0576] TCL2 library was constructed in which both the BC and the FG
loops of Tencon were randomized and the distribution of amino acids
at each position was strictly controlled. Table 3 shows the amino
acid distribution at desired loop positions in the TCL2 library.
The designed amino acid distribution had two aims. First, the
library was biased toward residues that were predicted to be
structurally important for Tencon folding and stability based on
analysis of the Tencon crystal structure and/or from homology
modeling. For example, position 29 was fixed to be only a subset of
hydrophobic amino acids, as this residue was buried in the
hydrophobic core of the Tencon fold. A second layer of design
included biasing the amino acid distribution toward that of
residues preferentially found in the heavy chain HCDR3 of
antibodies, to efficiently produce high-affinity binders (Birtalan
et al., J Mol Biol 377:1518-28, 2008; Olson et al., Protein Sci
16:476-84, 2007). Towards this goal, the "designed distribution" of
Table 3 refers to the distribution as follows: 6% alanine, 6%
arginine, 3.9% asparagine, 7.5% aspartic acid, 2.5% glutamic acid,
1.5% glutamine, 15% glycine, 2.3% histidine, 2.5% isoleucine, 5%
leucine, 1.5% lysine, 2.5% phenylalanine, 4% proline, 10% serine,
4.5% threonine, 4% tryptophan, 17.3% tyrosine, and 4% valine. This
distribution is devoid of methionine, cysteine, and STOP
codons.
TABLE-US-00011 TABLE 3 Residue Position* WT residues Distribution
in the TCL2 library 22 T designed distribution 23 A designed
distribution 24 P 50% P + designed distribution 25 D designed
distribution 26 A 20% A + 20% G + designed distribution 27 A
designed distribution 28 F 20% F, 20% I, 20% L, 20% V, 20% Y 29 D
33% D, 33% E, 33% T 75 K designed distribution 76 G designed
distribution 77 G designed distribution 78 H designed distribution
79 R designed distribution 80 S 100% S 81 N designed distribution
82 P 50% P + designed distribution *residue numbering is based on
Tencon sequence of SEQ ID NO: 1
[0577] The 5' fragment of the TCL2 library contained the promoter
and the coding region of Tencon (SEQ ID NO: 1), which was
chemically synthesized as a library pool (Sloning Biotechnology).
This pool of DNA contained at least 1.times.10.sup.11 different
members. At the end of the fragment, a BsaI restriction site was
included in the design for ligation to RepA.
[0578] The 3' fragment of the library was a constant DNA sequence
containing elements for display including a 6.times.His tag, the
coding region of the repA gene, and the cis-element. The DNA was
prepared by PCR reaction using an existing DNA template (above),
and primers LS1008 (SEQID NO: 17) and DidLigRev (SEQID NO: 14). To
assemble the complete TCL2 library, a total of 1 .mu.g of
BsaI-digested 5' Tencon library DNA was ligated to 3.5 .mu.g of the
3' fragment that was prepared by restriction digestion with the
same enzyme. After overnight ligation, the DNA was purified by
Qiagen column and the DNA was quantified by measuring absorbance at
260 nm. The ligated library product was amplified by a 12-cycle PCR
reaction with primer pair POP2250 (SEQID NO: 13) and DidLigRev
(SEQID NO: 14). A total of 72 reactions were performed, each
containing 50 ng of ligated DNA products as a template. The total
yield of TCL2 working library DNA was about 100 .mu.g. A small
portion of the working library was sub-cloned and sequenced, as
described above for library TCL1. No duplicate sequences were
found. About 80% of the sequences contained complete promoter and
Tencon coding sequences with no frame-shift mutations.
Construction of TCL14 Library
[0579] The top (BC, DE, and FG) and the bottom (AB, CD, and EF)
loops, e.g., the reported binding surfaces in the FN3 domains are
separated by the beta-strands that form the center of the FN3
structure. Alternative surfaces residing on the two "sides" of the
FN3 domains having different shapes than the surfaces formed by
loops only are formed at one side of the FN3 domain by two
anti-parallel beta-strands, the C and the F beta-strands, and the
CD and FG loops, and is herein called the C-CD-F-FG surface.
[0580] A library randomizing an alternative surface of Tencon was
generated by randomizing select surface exposed residues of the C
and F strands, as well as portions of the CD and FG loops as shown
in FIG. 1. A Tencon variant, Tencon27 (SEQ ID NO: 99) having
following substitutions when compared to Tencon (SEQ ID NO: 1) was
used to generate the library; E11R L17A, N46V, E861. A full
description of the methods used to construct this library is
described in US. Pat. Publ. No. US2013/0226834
Example 2: Selection of Fibronectin Type III (FN3) Domains that
Bind EGFR and Inhibit EGF Binding
Library Screening
[0581] Cis-display was used to select EGFR binding domains from the
TCL1 and TCL2 libraries. A recombinant human extracellular domain
of EGFR fused to an IgG1 Fc (R&D Systems) was biotinylated
using standard methods and used for panning (residues 25-645 of
full length EGFR of SEQ ID NO: 73). For in vitro transcription and
translation (ITT), 2-6 .mu.g of library DNA were incubated with 0.1
mM complete amino acids, 1X S30 premix components, and 30 .mu.L of
S30 extract (Promega) in a total volume of 100 .mu.L and incubated
at 30.degree. C. After 1 hour, 450 .mu.L of blocking solution (PBS
pH 7.4, supplemented with 2% bovine serum albumin, 100 .mu.g/mL
herring sperm DNA, and 1 mg/mL heparin) were added and the reaction
was incubated on ice for 15 minutes. EGFR-Fc:EGF complexes were
assembled at molar ratios of 1:1 and 10:1 EGFR to EGF by mixing
recombinant human EGF (R&D Systems) with biotinylated
recombinant EGFR-Fc in blocking solution for 1 hour at room
temperature. For binding, 500 .mu.L of blocked ITT reactions were
mixed with 100 .mu.L of EGFR-Fc:EGF complexes and incubated for 1
hour at room temperature, after which bound complexes were pulled
down with magnetic neutravidin or streptavidin beads (Seradyne).
Unbound library members were removed by successive washes with PBST
and PBS. After washing, DNA was eluted from the bound complexes by
heating to 65.degree. C. for 10 minutes, amplified by PCR, and
attached to a DNA fragment encoding RepA by restriction digestion
and ligation for further rounds of panning High affinity binders
were isolated by successively lowering the concentration of target
EGFR-Fc during each round from 200 nM to 50 nM and increasing the
washing stringency. In rounds 4 and 5, unbound and weakly bound FN3
domains were removed by washing in the presence of a 10-fold molar
excess of non-biotinylated EGFR-Fc overnight in PBS.
[0582] Following panning, selected FN3 domains were amplified by
PCR using oligonucleotides Tcon5new2 (SEQID NO: 15) and Tcon6
(SEQID NO: 16), subcloned into a pET vector modified to include a
ligase independent cloning site, and transformed into BL21-GOLD
(DE3) (Stratagene) cells for soluble expression in E. coli using
standard molecular biology techniques. A gene sequence encoding a
C-terminal poly-histidine tag was added to each FN3 domain to
enable purification and detection. Cultures were grown to an
optical density of 0.6-0.8 in 2YT medium supplemented with 100
.mu.g/mL carbenicillin in 1-mL 96-well blocks at 37.degree. C.
before the addition of IPTG to 1 mM, at which point the temperature
was reduced to 30.degree. C. Cells were harvested approximately 16
hours later by centrifugation and frozen at -20.degree. C. Cell
lysis was achieved by incubating each pellet in 0.6 mL of
BugBuster.RTM. HT lysis buffer (Novagen EMD Biosciences) with
shaking at room temperature for 45 minutes.
Selection of FN3 Domains that Bind EGFR on Cells
[0583] To assess the ability of different FN3 domains to bind EGFR
in a more physiological context, their ability to bind A431 cells
was measured. A431 cells (American Type Culture Collection, cat.
#CRL-1555) over-express EGFR with .about.2.times.10.sup.6 receptors
per cell. Cells were plated at 5,000/well in opaque black 96-well
plates and allowed to attach overnight at 37.degree. C., in a
humidified 5% CO.sub.2 atmosphere. FN3 domain-expressing bacterial
lysates were diluted 1,000-fold into FACS stain buffer (Becton
Dickinson) and incubated for 1 hour at room temperature in
triplicate plates. Lysates were removed and cells were washed 3
times with 150 .mu.L/well of FACS stain buffer. Cells were
incubated with 50 .mu.L/well of anti-penta His-Alexa488 antibody
conjugate (Qiagen) diluted 1:100 in FACS stain buffer for 20
minutes at room temperature. Cells were washed 3 times with 150
.mu.L/well of FACS stain buffer, after which wells were filled with
100 .mu.L of FACS stain buffer and read for fluorescence at 488 nm
using an Acumen eX3 reader. Bacterial lysates containing FN3
domains were screened for their ability to bind A431 cells (1320
crude bacterial lysates for TCL1 and TCL2 libraries) and 516
positive clones were identified, where binding was .gtoreq.10-fold
over the background signal. 300 lysates from the TCL14 library were
screened for binding, resulting in 58 unique FN3 domain sequences
that bind to EGFR.
Selection of FN3 Domains that Inhibit EGF Binding to EGFR on
Cells
[0584] To better characterize the mechanism of EGFR binding, the
ability of various identified FN3 domain clones to bind EGFR in an
EGF-competitive manner was measured using A431 cells and run in
parallel with the A431 binding assay screen. A431 cells were plated
at 5,000/well in opaque black 96-well plates and allowed to attach
overnight at 37.degree. C. in a humidified 5% CO.sub.2 atmosphere.
Cells were incubated with 50 .mu.L/well of 1:1,000 diluted
bacterial lysate for 1 hour at room temperature in triplicate
plates. Biotinylated EGF (Invitrogen, cat. #E-3477) was added to
each well for a final concentration of 30 ng/mL and incubated for
10 minutes at room temperature. Cells were washed 3 times with 150
.mu.L/well of FACS stain buffer. Cells were incubated with 50
.mu.L/well of streptavidin-phycoerythrin conjugate (Invitrogen)
diluted 1:100 in FACS stain buffer for 20 minutes at room
temperature. Cells were washed 3 times with 150 .mu.L/well of FACS
stain buffer, after which wells were filled with 100 .mu.L of FACS
stain buffer and read for fluorescence at 600 nm using an Acumen
eX3 reader.
[0585] Bacterial lysates containing the FN3 domains were screened
in the EGF competition assay described above. 1320 crude bacterial
lysates from TCL1 and TCL2 libraries were screened resulting in 451
positive clones that inhibited EGF binding by >50%.
Expression and Purification of Identified FN3 Domains Binding
EGFR
[0586] His-tagged FN3 domains were purified from clarified E. coli
lysates with His MultiTrap.TM. HP plates (GE Healthcare) and eluted
in buffer containing 20 mM sodium phosphate, 500 mM sodium
chloride, and 250 mM imidazole at pH 7.4. Purified samples were
exchanged into PBS pH 7.4 for analysis using PD MultiTrap.TM. G-25
plates (GE Healthcare).
Size Exclusion Chromatography Analysis
[0587] Size exclusion chromatography was used to determine the
aggregation state of the FN3 domains binding EGFR. Aliquots (10
.mu.L) of each purified FN3 domain were injected onto a Superdex 75
5/150 column (GE Healthcare) at a flow rate of 0.3 mL/min in a
mobile phase of PBS pH 7.4. Elution from the column was monitored
by absorbance at 280 nm. FN3 domains that exhibited high levels of
aggregation by SEC were excluded from further analysis.
Off-Rate of Selected EGFR-Binding FN3 Domains from EGFR-Fc
[0588] Select EGFR-binding FN3 domains were screened to identify
those with slow off-rates (k.sub.off) in binding to EGFR-Fc on a
ProteOn XPR-36 instrument (Bio-Rad) to facilitate selection of high
affinity binders. Goat anti-human Fc IgG (R&D systems), at a
concentration of 5 .mu.g/mL, was directly immobilized via amine
coupling (at pH 5.0) on all 6 ligand channels in horizontal
orientation on the chip with a flow rate of 30 .mu.L/min in PBS
containing 0.005% Tween-20. The immobilization densities averaged
about 1500 Response Units (RU) with less than 5% variation among
different channels. EGFR-Fc was captured on the anti-human Fc IgG
surface to a density around 600 RU in vertical ligand orientation.
All tested FN3 domains were normalized to a concentration of 1
.mu.M and tested for their binding in horizontal orientation. All 6
analyte channels were used for the FN3 domains to maximize
screening throughput. The dissociation phase was monitored for 10
minutes at a flow rate of 100 .mu.L/min. The inter-spot binding
signals were used as references to monitor non-specific binding
between analytes and the immobilized IgG surface, and were
subtracted from all binding responses. The processed binding data
were locally fit to a 1:1 simple Langmuir binding model to extract
the k.sub.off for each FN3 domain binding to captured EGFR-Fc.
Inhibition of EGF-Stimulated EGFR Phosphorylation
[0589] Purified EGFR-binding FN3 domains were tested for their
ability to inhibit EGF-stimulated phosphorylation of EGFR in A431
cells at a single concentration. EGFR phosphorylation was monitored
using the EGFR phospho(Tyr1173) kit (Meso Scale Discovery). Cells
were plated at 20,000/well in clear 96-well tissue culture-treated
plates (Nunc) in 100 .mu.L/well of RPMI medium (Gibco) containing
GlutaMAX.TM. with 10% fetal bovine serum (FBS) (Gibco) and allowed
to attach overnight at 37.degree. C. in a humidified 5% CO.sub.2
atmosphere. Culture medium was removed completely and cells were
starved overnight in 100 .mu.L/well of medium containing no FBS at
37.degree. C. in a humidified 5% CO.sub.2 atmosphere. Cells were
then treated with 100 .mu.L/well of pre-warmed (37.degree. C.)
starvation medium containing EGFR-binding FN3 domains at a
concentration of 2 .mu.M for 1 hour at 37.degree. C. in a
humidified 5% CO.sub.2 atmosphere. Controls were treated with
starvation medium only. Cells were stimulated by the addition and
gentle mixing of 100 .mu.L/well of pre-warmed (37.degree. C.)
starvation medium containing 100 ng/mL recombinant human EGF
(R&D Systems, cat. #236-EG), for final concentrations of 50
ng/mL EGF and 1 .mu.M EGFR-binding FN3 domain, and incubation at
37.degree. C., 5% CO.sub.2 for 15 minutes. One set of control wells
was left un-stimulated as negative controls. Medium was completely
removed and cells were lysed with 100 .mu.L/well of Complete Lysis
Buffer (Meso Scale Discovery) for 10 minutes at room temperature
with shaking, as per the manufacturer's instructions. Assay plates
configured for measuring EGFR phosphorylated on tyrosine 1173 (Meso
Scale Discovery) were blocked with the provided blocking solution
as per the manufacturer's instructions at room temperature for
1.5-2 hours. Plates were then washed 4 times with 200 .mu.L/well of
1X Tris Wash Buffer (Meso Scale Discovery). Aliquots of cell lysate
(30 .mu.L/well) were transferred to assay plates, which were
covered with plate sealing film (VWR) and incubated at room
temperature with shaking for 1 hour. Assay plates were washed 4
times with 200 .mu.L/well of Tris Wash Buffer, after which 25 .mu.L
of ice-cold Detection Antibody Solution (Meso Scale Discovery) were
added to each well, being careful not to introduce bubbles. Plates
were incubated at room temperature with shaking for 1 hour,
followed by 4 washes with 200 .mu.L/well of Tris Wash Buffer.
Signals were detected by addition of 150 .mu.L/well of Read Buffer
(Meso Scale Discovery) and reading on a SECTOR.RTM. Imager 6000
instrument (Meso Scale Discovery) using manufacturer-installed
assay-specific default settings. Percent inhibition of the
EGF-stimulated positive control signal was calculated for each
EGFR-binding FN3 domain.
[0590] Inhibition of EGF-stimulated EGFR phosphorylation was
measured for 232 identified clones from the TCL1 and TCL2
libraries. 22 of these clones inhibited EGFR phosphorylation by
.gtoreq.50% at 1 .mu.M concentration. After removal of clones that
either expressed poorly or were judged to be multimeric by size
exclusion chromatography, nine clones were carried forward for
further biological characterization. The BC and FG loop sequences
of these clones are shown in Table 4. Eight of the nine selected
clones had a common FG loop sequence (HNVYKDTNMRGL; SEQ ID NO: 95)
and areas of significant similarity were seen between several
clones in their BC loop sequences.
TABLE-US-00012 TABLE 4 FN3 Domain BC Loop FG Loop SEQ ID SEQ ID SEQ
ID Clone ID NO: Sequence NO: Sequence NO: P53A1R5- 18 ADPHGFYD 87
HNVYKDTN 95 17 MRGL P54AR4- 19 TYDRDGYD 88 HNVYKDTN 95 17 MRGL
P54AR4- 20 WDPFSFYD 89 HNVYKDTN 95 47 MRGL P54AR4- 21 DDPRGFYE 90
HNVYKDTN 95 48 MRGL P54AR4- 22 TWPYADLD 91 HNVYKDTN 95 73 MRGL
P54AR4- 23 GYNGDHFD 92 HNVYKDTN 95 74 MRGL P54AR4- 24 DYDLGVYD 93
HNVYKDTN 95 81 MRGL P54AR4- 25 DDPWDFYE 94 HNVYKDTN 95 83 MRGL
P54CR4- 26 TAPDAAFD 85 LGSYVFEH 96 31 DVM
Example 3: Characterization of EGFR-Binding FN3 Domains that
Inhibit EGF Binding
Large-Scale Expression, Purification, and Endotoxin Removal
[0591] The FN3 domains shown in Table 4 were scaled up to provide
more material for detailed characterization. An overnight culture
containing each EGFR-binding FN3 domain variant was used to
inoculate 0.8 L of Terrific broth medium supplemented with 100
.mu.g/mL ampicillin at a 1/80 dilution of overnight culture into
fresh medium, and incubated with shaking at 37.degree. C. The
culture was induced when the optical density at 600 nm reached
.about.1.2-1.5 by addition of IPTG to a final concentration of 1 mM
and the temperature was reduced to 30.degree. C. After 4 hours,
cells were collected by centrifugation and the cell pellet was
stored at -80.degree. C. until needed.
[0592] For cell lysis, the thawed pellet was resuspended in 1X
BugBuster.RTM. supplemented with 25 U/mL Benzonase.RTM.
(Sigma-Aldrich) and 1 kU/mL rLysozyme.TM. (Novagen EMD Biosciences)
at a ratio of 5 mL of BugBuster.RTM. per gram of pellet. Lysis
proceeded for 1 hour at room temperature with gentle agitation,
followed by centrifugation at 56,000.times.g for 50 minutes at
4.degree. C. The supernatant was collected and filtered through a
0.2 .mu.m filter, then loaded on to a 5-mL HisTrap FF column
pre-equilibrated with Buffer A (50 mM Tris-HCl pH 7.5, 500 mM NaCl,
10 mM imidazole) using a GE Healthcare AKTAexplorer 100s
chromatography system. The column was washed with 20 column volumes
of Buffer A and further washed with 16% Buffer B (50 mM Tris-HCl
pH7.5, 500 mM NaCl, 250 mM imidazole) for 6 column volumes. The FN3
domains were eluted with 50% B for 10 column volumes, followed by a
gradient from 50-100% B over 6 column volumes. Fractions containing
the FN3 domain protein were pooled, concentrated using a Millipore
10K MWCO concentrator, and filtered before loading onto a
HiLoad.TM. 16/60 Superdex.TM. 75 column (GE Healthcare)
pre-equilibrated with PBS. The protein monomer peak eluting from
the size exclusion column was retained.
[0593] Endotoxins were removed using a batch approach with
ActiClean Etox resin (Sterogene Bioseparations). Prior to endotoxin
removal, the resin was pre-treated with 1 N NaOH for 2 hours at
37.degree. C. (or overnight at 4.degree. C.) and washed extensively
with PBS until the pH had stabilized to .about.7 as measured with
pH indicator paper. The purified protein was filtered through a 0.2
.mu.m filter before adding to 1 mL of Etox resin at a ratio of 10
mL of protein to 1 mL of resin. The binding of endotoxin to resin
was allowed to proceed at room temperature for at least 2 hours
with gentle rotation. The resin was removed by centrifugation at
500.times.g for 2 minutes and the protein supernatant was retained.
Endotoxin levels were measured using EndoSafe.RTM.-PTS.TM.
cartridges and analyzed on an EndoSafe.RTM.-MCS reader (Charles
River). If endotoxin levels were above 5 EU/mg after the first Etox
treatment, the above procedure was repeated until endotoxin levels
were decreased to .gtoreq.5 EU/mg. In cases where the endotoxin
level was above 5 EU/mg and stabilized after two consecutive
treatments with Etox, anion exchange or hydrophobic interaction
chromatography conditions were established for the protein to
remove the remaining endotoxins.
Affinity Determination of Selected EGFR-Binding FN3 Domains to
EGFR-Fc (EGFR-Fc Affinity)
[0594] Binding affinity of selected EGFR-binding FN3 domains to
recombinant EGFR extracellular domain was further characterized by
surface Plasmon resonance methods using a Proteon Instrument
(BioRad). The assay set-up (chip preparation, EGFR-Fc capture) was
similar to that described above for off-rate analysis. Selected
EGFR binding FN3 domains were tested at 1 .mu.M concentration in
3-fold dilution series in the horizontal orientation. A buffer
sample was also injected to monitor the baseline stability. The
dissociation phase for all concentrations of each EGFR-binding FN3
domain was monitored at a flow rate of 100 .mu.L/min for 30 minutes
(for those with k.sub.off.about.10.sup.-2 s.sup.-1 from off-rate
screening), or 1 hour (for those with k.sub.off.about.10.sup.-3
s.sup.-1 or slower). Two sets of reference data were subtracted
from the response data: 1) the inter-spot signals to correct for
the non-specific interactions between the EGFR-binding FN3 domain
and the immobilized IgG surface; 2) the buffer channel signals to
correct for baseline drifting due to the dissociation of captured
EGFR-Fc surface over time. The processed binding data at all
concentrations for each FN3 domain were globally fit to a 1:1
simple Langmuir binding model to extract estimates of the kinetic
(k.sub.on, k.sub.off) and affinity (K.sub.D) constants. Table 5
shows the kinetic constants for each of the constructs, with the
affinity varying from 200 pM to 9.6 nM.
Binding of Selected EGFR-Binding FN3 Domains to EGFR on Cells
("A431 Cell Binding Assay")
[0595] A431 cells were plated at 5,000/well in opaque black 96-well
plates and allowed to attach overnight at 37.degree. C., in a
humidified 5% CO.sub.2 atmosphere. Purified EGFR-binding FN3
domains (1.5 nM to 30 .mu.M) were added to the cells (in 50 uL) for
1 hour at room temperature in triplicate plates. Supernatant was
removed and cells were washed 3 times with 150 .mu.L/well of FACS
stain buffer. Cells were incubated with 50 .mu.L/well of anti-penta
His-Alexa488 antibody conjugate (Qiagen) diluted 1:100 in FACS
stain buffer for 20 minutes at room temperature. Cells were washed
3 times with 150 .mu.L/well of FACS stain buffer, after which wells
were filled with 100 .mu.L of FACS stain buffer and read for
fluorescence at 488 nm using an Acumen eX3 reader. Data were
plotted as raw fluorescence signal against the logarithm of the FN3
domain molar concentration and fitted to a sigmoidal dose-response
curve with variable slope using GraphPad Prism 4 (GraphPad
Software) to calculate EC.sub.50 values. Table 5 reports the
EC.sub.50 for each of the constructs ranging from 2.2 nM to >20
.mu.M.
Inhibition of EGF Binding to EGFR on Cells Using Selected
EGFR-Binding FN3 Domains (A431 Cell EGF Competition Assay)
[0596] A431 cells were plated at 5,000/well in opaque black 96-well
plates and allowed to attach overnight at 37.degree. C., in a
humidified 5% CO.sub.2 atmosphere. Purified EGFR-binding FN3
domains (1.5 nM to 30 .mu.M) were added to the cells (50
.mu.L/well) for 1 hour at room temperature in triplicate plates.
Biotinylated EGF (Invitrogen, Cat #: E-3477) was added to each well
to give a final concentration of 30 ng/mL and incubated for 10
minutes at room temperature. Cells were washed 3 times with 150
.mu.L/well of FACS stain buffer. Cells were incubated with 50
.mu.L/well of streptavidin-phycoerythrin conjugate (Invitrogen)
diluted 1:100 in FACS stain buffer for 20 minutes at room
temperature. Cells were washed 3 times with 150 .mu.L/well of FACS
stain buffer, after which wells were filled with 100 .mu.L of FACS
stain buffer and read for fluorescence at 600 nm using an Acumen
eX3 reader. Data were plotted as the raw fluorescence signal
against the logarithm of FN3 domain molar concentration and fitted
to a sigmoidal dose-response curve with variable slope using
GraphPad Prism 4 (GraphPad Software) to calculate IC.sub.50 values.
Table 5 reports the IC.sub.50 values ranging from 1.8 nM to 121
nM.
Inhibition of EGF-Stimulated EGFR Phosphorylation (Phoshpo EGFR
Assay)
[0597] Select FN3 domains that significantly inhibited
EGF-stimulated EGFR phosphorylation were assessed more completely
by measuring IC.sub.50 values for inhibition. Inhibition of
EGF-stimulated EGFR phosphorylation was assessed at varying FN3
domain concentrations (0.5 nM to 10 04) as described above in
"inhibition of EGF stimulated EGFR phosphorylation". Data were
plotted as electrochemiluminescence signal against the logarithm of
the FN3 domain molar concentration and IC.sub.50 values were
determined by fitting data to a sigmoidal dose response with
variable slope using GraphPad Prism 4 (GraphPad Software). Table 5
shows the IC.sub.50 values which ranged from 18 nM to >2.5
.mu.m.
Inhibition of Human Tumor Cell Growth (NCI-H292 Growth and NCI-H322
Growth Assay)
[0598] Inhibition of EGFR-dependent cell growth was assessed by
measuring viability of the EGFR over-expressing human tumor cell
lines, NCI-H292 and NCI-H322 (American Type Culture Collection,
cat. #CRL-1848 & #CRL-5806, respectively), following exposure
to EGFR-binding FN3 domains. Cells were plated at 500 cells/well
(NCI-H292) or 1,000 cells/well (NCI-H322) in opaque white 96-well
tissue culture-treated plates (Nunc) in 100 .mu.L/well of RPMI
medium (Gibco) containing GlutaMAX.TM. and 10 mM HEPES,
supplemented with 10% heat inactivated fetal bovine serum (Gibco)
and 1% penicillin/streptomycin (Gibco), and allowed to attach
overnight at 37.degree. C. in a humidified 5% CO.sub.2 atmosphere.
Cells were treated by addition of 5 .mu.L/well of
phosphate-buffered saline (PBS) containing a concentration range of
EGFR-binding FN3 domains. Controls were treated with 5 .mu.L/well
of PBS only or 25 mM ethylenediaminetetraacetic acid in PBS. Cells
were incubated at 37.degree. C., 5% CO.sub.2 for 120 hours. Viable
cells were detected by addition of 75 .mu.L/well of
CellTiter-Glo.RTM. reagent (Promega), followed by mixing on a plate
shaker for 2 minutes, and incubation in the dark at room
temperature for a further 10 minutes. Plates were read on a
SpectraMax M5 plate reader (Molecular Devices) set to luminescence
mode, with a read time of 0.5 seconds/well against a blank of
medium only. Data were plotted as a percentage of PBS-treated cell
growth against the logarithm of FN3 domain molar concentration.
IC.sub.50 values were determined by fitting data to the equation
for a sigmoidal dose response with variable slope using GraphPad
Prism 4 (GraphPad Software). Table 5 shows IC.sub.50 values ranging
from 5.9 nM to 1.15 .mu.M and 9.2 nM to >3.1 .mu.M, using the
NCI-H292 and NCI-H322 cells respectively. Table 5 shows the summary
of biological properties of EGFR-binding FN3 domains for each
assay.
TABLE-US-00013 TABLE 5 A431 EGFR- Cell A431 Phospho- NCI- NCI- FN3
SEQ Fc Binding Cell EGF EGFR H292 H322 Domain ID Affinity EC.sub.50
Competition IC.sub.50 Growth Growth Clone ID NO: (nM) (nM)
IC.sub.50 (nM) (nM) IC.sub.50 (nM) IC.sub.50 (nM) P53AlR5-17 18
1.89 4.0 9.8 >2500 86 65 P54AR4-17 19 9.62 16 21 184 ND ND
P54AR4-47 20 2.51 8.6 7.1 295 44 39 P54AR4-48 21 7.78 12 9.8 170 ND
ND P54AR4-73 22 0.197 9.4 4.6 141 83 73 P54AR4-74 23 ND 77 ND ND ND
ND P54AR4-81 24 ND 84 121 ND ND ND P54AR4-83 25 0.255 2.2 1.8 18
5.9 9.2 P54CR4-31 26 0.383 >20000 55 179 1150 >3073
Example 4: Engineering of EGFR-Binding FN3 Domains
[0599] A subset of the EGFR binding FN3 domains was engineered to
increase the conformational stability of each molecule. The
mutations L17A, N46V, and E861 which have been shown to improve FN3
domain stability (described in US Pat. Publ. No. US2011/0274623)
were incorporated into clones P54AR4-83, P54CR4-31, and P54AR4-37
by DNA synthesis. The new mutants, P54AR5-83v2, P54CR431-v2, and
P54AR4-37v2 were expressed and purified as described above.
Differential scanning calorimetry in PBS was used to assess the
stability of each mutant in order to compare it to that of the
corresponding parent molecule. Table 6 shows that each variant
molecule was stabilized significantly, with an average increase in
the T.sub.m of 18.5.degree. C.
TABLE-US-00014 TABLE 6 FN3 domain Clone SEQ ID NO: T.sub.m
(.degree. C.) P54AR4-83 25 50.6 P54AR4-83v2 27 69.8 P54CR4-31 26
60.9 P54CR4-31v2 28 78.9 P54AR4-37 22 45.9 P54AR4-37v2 29 64.2
Example 5: Selection of Fibronectin Type III (FN3) Domains that
Bind c-Met and Inhibit HGF Binding
[0600] Panning on Human c-Met
[0601] The TCL14 library was screened against biotinylated-human
c-Met extracellular domain (bt-c-Met) to identify FN3 domains
capable of specifically binding c-Met. For selections, 3 .mu.g of
TCL14 library was in vitro transcribed and translated (IVTT) in E.
Coli S30 Linear Extract (Promega, Madison, Wis.) and the expressed
library blocked with Cis Block (2% BSA (Sigma-Aldrich, St. Louis,
Mo.), 100 .mu.g/ml Herring Sperm DNA (Promega), 1 mg/mL heparin
(Sigma-Aldrich)). For selections, bt-c-Met was added at
concentrations of 400 nM (Round 1), 200 nM (Rounds 2 and 3) and 100
nM (Rounds 4 and 5). Bound library members were recovered using
neutravidin magnetic beads (Thermo Fisher, Rockford, Ill.) (Rounds
1, 3, and 5) or streptavidin magnetic beads (Promega) (Rounds 2 and
4) and unbound library members were removed by washing the beads
5-14 times with 500 uL PBS-T followed by 2 washes with 500 .mu.L
PBS.
[0602] Additional selection rounds were performed to identify FN3
domains molecules with improved affinities. Briefly, outputs from
round 5 were prepared as described above and subjected to
additional iterative rounds of selection with the following
changes: incubation with bt-c-Met was decreased from 1 hour to 15
minutes and bead capture was decreased from 20 minutes to 15
minutes, bt-c-Met decreased to 25 nM (Rounds 6 and 7) or 2.5 nM
(Rounds 8 and 9), and an additional 1 hour wash was performed in
the presence of an excess of non-biotinylated c-Met. The goal of
these changes was to simultaneously select for binders with a
potentially faster on-rate and a slower off-rate yielding a
substantially lower K.sub.D.
[0603] Rounds 5, 7 and 9 outputs were PCR cloned into a modified
pET15 vector (EMD Biosciences, Gibbstown, N.J.) containing a ligase
independent cloning site (pET15-LIC) using TCON6 (SEQID No. 30) and
TCONS E86I short (SEQID No. 31) primers, and the proteins were
expressed as C-terminal His6-tagged proteins after transformations
and IPTG induction (1 mM final, 30.degree. C. for 16 hours) using
standard protocols. The cells were harvested by centrifugation and
subsequently lysed with Bugbuster HT (EMD Biosciences) supplemented
with 0.2 mg/mL Chicken Egg White Lysozyme (Sigma-Aldrich). The
bacterial lysates were clarified by centrifugation and the
supernatants were transferred to new 96 deep-well plates.
Screening for FN3 Domains that Inhibit HGF Binding to c-Met
[0604] FN3 domains present in E. coli lysates were screened for
their ability to inhibit HGF binding to purified c-Met
extracellular domain in a biochemical format. Recombinant human
c-Met Fc chimera (0.5 .mu.g/mL in PBS, 100 .mu.L/well) was coated
on 96-well White Maxisorp Plates (Nunc) and incubated overnight at
4.degree. C. The plates were washed two times with 300 .mu.l/well
of Tris-buffered saline with 0.05% Tween 20 (TBS-T, Sigma-Aldrich)
on a Biotek plate washer. Assay plates were blocked with
StartingBlock T20 (200 .mu.L/well, Thermo Fisher Scientific,
Rockland, Ill.) for 1 hour at room temperature (RT) with shaking
and again washed twice with 300 .mu.l of TBS-T. FN3 domain lysates
were diluted in StartingBlock T20 (from 1:10 to 1:100,000) using
the Hamilton STARplus robotics system. Lysates (50 .mu.L/well) were
incubated on assay plates for 1 hour at RT with shaking. Without
washing the plates, bt-HGF (1 .mu.g/mL in StartingBlock T20, 50
biotinylated) was added to the plate for 30 min at RT while
shaking. Control wells containing Tencon27 lysates received either
Starting Block T20 or diluted bt-HGF. Plates were then washed four
times with 300 .mu.l/well of TBS-T and incubated with 100
.mu.l/well of Streptavidin-HRP (1:2000 in TBS-T, Jackson
Immunoresearch, West Grove, Pa.) for 30-40 minutes at RT with
shaking. Again the plates were washed four times with TBS-T. To
develop signal, POD Chemiluminescence Substrate (50 .mu.L/well,
Roche Diagnostics, Indianapolis, Ind.), prepared according to
manufacturer's instructions, was added to the plate and within
approximately 3 minutes luminescence was read on the Molecular
Devices M5 using SoftMax Pro. Percent inhibition was determined
using the following calculation: 100-((RLU.sub.sample-Mean
RLU.sub.No bt-HGF control)/(Mean RLU.sub.bt-HGF control-Mean
RLU.sub.No bt-HGF control)*100). Percent inhibition values of 50%
or greater were considered hits.
High-Throughput Expression and Purification of FN3 Domains
[0605] His-tagged FN3 domains were purified from clarified E. coli
lysates with His MultiTrap.TM. HP plates (GE Healthcare) and eluted
in buffer containing 20 mM sodium phosphate, 500 mM sodium
chloride, and 250 mM imidazole at pH 7.4. Purified samples were
exchanged into PBS pH 7.4 for analysis using PD MultiTrap.TM. G-25
plates (GE Healthcare).
IC.sub.50 Determination of Inhibition of HGF Binding to c-Met
[0606] Select FN3 domains were further characterized in the HGF
competition assay. Dose response curves for purified FN3 domains
were generated utilizing the assay described above (starting
concentrations of 5 .mu.M). Percent inhibition values were
calculated. The data were plotted as % inhibition against the
logarithm of FN3 domain molar concentrations and IC.sub.50 values
were determined by fitting data to a sigmoidal dose response with
variable slope using GraphPad Prism 4.
[0607] 35 unique sequences were identified from Round 5 to exhibit
activity at dilutions of 1:10, with IC.sub.50 values ranging from
0.5 to 1500 nM. Round 7 yielded 39 unique sequences with activity
at dilutions of 1:100 and IC.sub.50 values ranging from 0.16 to 2.9
nM. 66 unique sequences were identified from Round 9, where hits
were defined as being active at dilutions of 1:1000. IC.sub.50
values as low as 0.2 nM were observed in Round 9 (Table 8).
Affinity Determination of Selected c-Met-Binding FN3 Domains to
c-Met-Fc (EGFR-Fc Affinity)
[0608] Affinities were determined for select c-Met binding FN3
domains as is described in Example 3 for affinity determination for
selected EGFR-binding FN3 domains, except that c-Met-Fc was used in
the assays.
Example 6: Characterization of FN3 Domains that Bind c-Met and
Inhibit HGF Binding
[0609] FN3 domains were expressed and purified as described above
in Example 2. Size exclusion chromatography and kinetic analysis
was done as described above in Examples 1 and 2, respectively.
Table 7 shows the sequences of the C-strand, CD loop, F-strand, and
FG loop, and a SEQ ID NO: for the entire amino acid sequence for
each domain.
TABLE-US-00015 TABLE 7 Clone SEQ ID C CD F FG Name NO: loop strand
loop strand P114AR5P74-A5 32 FDSFW VVVGGE TEYYV KGGSISV IRYDE NILGV
P114AR5P75-E9 33 FDSFF FLRSGE TEYWV KGGLVST IRYDE TILGV
P114AR7P92-F3 34 FDSFW FLGSGE TEYIV KGGSISH IRYFE NIMGV
P114AR7P92-F6 35 FDSFW FLGSGE TEYVV KGGGLSV IRYFE NILGV
P114AR7P92-G8 36 FDSFV FLGSGE TEYVV KGGYISI IRYFE QILGV
P114AR7P92-H5 37 FDSFW FLLGGE TEYVV KGGTVSP IRYLE QIMGV
P114AR7P93-D11 38 FDSFW FLGSGE TEYVV KGGYISY IRYFE GINGV
P114AR7P93-G8 39 FDSFW FLGSGE TEYGV KGGRVST IRYFE TINGV
P114AR7P93-H9 40 FDSFW FLGSGE TEYVV KGGHISL IRYFE QIIGV
P114AR7P94-A3 41 FDSFW FLGSGE TEYVV KGGKISP IRYFE NIMGV
P114AR7P94-E5 42 FDSFW FLGSGE TEYAV KGGRVSV IRYFE NIMGV
P114AR7P95-B9 43 FDSFW FLGSGE TEYVV KGGSISV IRYFE QILGV
P114AR7P95-D3 44 FDSFW FLGSGE TEYVV KGGSISY IRYFE NIMGV
P114AR7P95-D4 45 FDSFW FLGSGE TEYVV KGGYISI IRYFE QILGV
P114AR7P95-E3 46 FDSFW FLGSGE TEYVV KGGTVSP IRYFE QIMGV
P114AR7P95-F10 47 FDSFW FTTAGE TEYVV KGGSISP IRYFE NIMGV
P114AR7P95-G7 48 FDSFW LLSTGE TEYVV KGGSISP IRYFE NIMGV
P114AR7P95-H8 49 FDSFW FVSKGE TEYVV KGGSISP IRYFE NIMGV C loop
residues correspond to residues 28-37 of indicated SEQ ID NO CD
strand residues correspond to residues 38-43 of indicated SEQ ID NO
F loop residues correspond to residues 65-74 of indicated SEQ ID NO
FG strand residues correspond to residues 75-81 of indicated SEQ ID
NO
Binding of Selected c-Met-Binding FN3 Domains to c-Met on Cells
("H441 Cell Binding Assay")
[0610] NCI-H441 cells (Cat # HTB-174, American Type Culture
Collection, Manassas, Va.) were plated at 20,000 cells per well in
Poly-D-lysine coated black clear bottom 96-well plates (BD
Biosciences, San Jose, Calif.) and allowed to attach overnight at
37.degree. C., 5% CO.sub.2. Purified FN3 domains (50 .mu.L/well; 0
to 1000 nM) were added to the cells for 1 hour at 4.degree. C. in
duplicate plates. Supernatant was removed and cells were washed
three times with FACS stain buffer (150 .mu.L/well, BD Biosciences,
cat #554657). Cells were incubated with biotinylated-anti HIS
antibody (diluted 1:160 in FACS stain buffer, 50 .mu.L/well,
R&D Systems, cat # BAM050) for 30 minutes at 4.degree. C. Cells
were washed three times with FACS stain buffer (150 .mu.L/well),
after which wells were incubated with anti-mouse IgG1-Alexa 488
conjugated antibody (diluted 1:80 in FACS stain buffer, 50
.mu.L/well, Life Technologies, cat # A21121) for 30 minutes at
4.degree. C. Cells were washed three times with FACS stain buffer
(150 .mu.L/well) and left in FACS stain buffer (50 .mu.L/well).
Total fluorescence was determined using an Acumen eX3 reader. Data
were plotted as raw fluorescence signal against the logarithm of
the FN3 domain molar concentration and fitted to a sigmoidal
dose-response curve with variable slope using GraphPad Prism 4
(GraphPad Software) to calculate EC.sub.50 values. FN3 domains were
found to exhibit a range of binding activities, with EC.sub.50
values between 1.4 nM and 22.0 nM, as shown in Table 8.
Inhibition of HGF-Stimulated c-Met Phosphorylation
[0611] Purified FN3 domains were tested for their ability to
inhibit HGF-stimulated phosphorylation of c-Met in NCI-H441, using
the c-Met phospho(Tyr1349) kit from Meso Scale Discovery
(Gaithersburg, Md.). Cells were plated at 20,000/well in clear
96-well tissue culture-treated plates in 100 .mu.L/well of RPMI
medium (containing Glutamax and HEPES, Life Technologies) with 10%
fetal bovine serum (FBS; Life Technologies) and allowed to attach
overnight at 37.degree. C., 5% CO.sub.2. Culture medium was removed
completely and cells were starved overnight in serum-free RPMI
medium (100 .mu.L/well) at 37.degree. C., 5% CO.sub.2. Cells were
then replenished with fresh serum-free RPMI medium (100 .mu.L/well)
containing FN3 domains at a concentration of 20 .mu.M and below for
1 hour at 37.degree. C., 5% CO.sub.2. Controls were treated with
medium only. Cells were stimulated with 100 ng/mL recombinant human
HGF (100 .mu.L/well, R&D Systems cat #294-HGN) and incubated at
37.degree. C., 5% CO.sub.2 for 15 minutes. One set of control wells
was left un-stimulated as negative controls. Medium was then
completely removed and cells were lysed with Complete Lysis Buffer
(50 .mu.L/well, Meso Scale Discovery) for 10 minutes at RT with
shaking, as per manufacturer's instructions. Assay plates
configured for measuring phosphorylated c-Met were blocked with the
provided blocking solution as per the manufacturer's instructions
at room temperature for 1 hour. Plates were then washed three times
with Tris Wash Buffer (200 .mu.L/well, Meso Scale Discovery). Cell
lysates (30 .mu.L/well) were transferred to assay plates, and
incubated at RT with shaking for 1 hour. Assay plates were then
washed four times with Tris Wash Buffer, after which ice-cold
Detection Antibody Solution (25 .mu.L/well, Meso Scale Discovery)
was added to each well for 1 hr at RT with shaking. Plates were
again rinsed four times with Tris Wash Buffer. Signals were
detected by addition of 150 Read Buffer (150 .mu.L/well, Meso Scale
Discovery) and reading on a SECTOR.RTM. Imager 6000 instrument
(Meso Scale Discovery) using manufacturer-installed assay-specific
default settings. Data were plotted as electrochemiluminescence
signal against the logarithm of FN3 domain molar concentration and
IC.sub.50 values were determined by fitting data to a sigmoidal
dose response with variable slope using GraphPad Prism 4. FN3
domains were found to inhibit phosphorylated c-Met with IC.sub.50
values ranging from 4.6 nM to 1415 nM as shown in Table 8.
Inhibition of Human Tumor Cell Growth or Viability
[0612] Inhibition of c-Met-dependent cell growth was assessed by
measuring viability of U87-MG cells (American Type Culture
Collection, cat # HTB-14), following exposure to c-Met-binding FN3
domains Cells were plated at 8000 cells per well in opaque white
96-well tissue culture-treated plates (Nunc) in 100 .mu.L/well of
RPMI medium, supplemented with 10% FBS and allowed to attach
overnight at 37.degree. C., 5% CO.sub.2. Twenty-four hours after
plating, medium was aspirated and cells were replenished with
serum-free RPMI medium. Twenty-four hours after serum starvation,
cells were treated by addition of serum-free medium containing
c-Met-binding FN3 domains (30 .mu.L/well). Cells were incubated at
37.degree. C., 5% CO.sub.2 for 72 hours. Viable cells were detected
by addition of 100 .mu.L/well of CellTiter-Glo.RTM. reagent
(Promega), followed by mixing on a plate shaker for 10 minutes.
Plates were read on a SpectraMax M5 plate reader (Molecular
Devices) set to luminescence mode, with a read time of 0.5
seconds/well. Data were plotted as raw luminescence units (RLU)
against the logarithm of FN3 domain molar concentration. IC.sub.50
values were determined by fitting data to an equation for a
sigmoidal dose response with variable slope using GraphPad Prism 4.
Table 8 reports IC.sub.50 values ranging from 1 nM to >1000 nM.
Characteristics of the c-Met binding FN3 domains are summarized in
Table 8.
TABLE-US-00016 TABLE 8 pMet Inhbibition inhibition of Clone HGF
H441 Cell in Proliferation SEQ Affinity competition binding cells
of U87-MG ID (Kd, IC.sub.50 (EC.sub.50, (IC.sub.50, cells
(IC.sub.50, Name NO: nM) (nM) nM) nM) nM) P114AR5P74-A5 32 10.1 5.2
18.7 1078 464.4 P114AR5P75-E9 33 45.8 51.9 ND 1415 1193.9
P114AR7P92-F3 34 0.4 0.2 1.5 8.3 2.7 P114AR7P92-F6 35 3.1 2.2 4.9
165.3 350.5 P114AR7P92-G8 36 1.0 1.6 5.9 155.3 123.9 P114AR7P92-H5
37 11.6 ND 22.0 766.4 672.3 P114AR7P93-D11 38 ND ND 2.3 16 14.4
P114AR7P93-G8 39 6.9 1 3.8 459.5 103.5 P114AR7P93-H9 40 3.3 2.9
12.9 288.2 269.9 P114AR7P94-A3 41 0.4 0.2 1.4 5 9.3 P114AR7P94-E5
42 4.2 0.7 3.4 124.3 195.6 P114AR7P95-B9 43 0.5 0.3 ND 9.8 17.4
P114AR7P95-D3 44 0.3 0.2 1.5 4.6 1.7 P114AR7P95-D4 45 0.4 ND 1.4
19.5 19.4 P114AR7P95-E3 46 1.5 ND 3.2 204.6 209.2 P114AR7P95-F10 47
4.2 1.4 4.4 187.6 129.7 P114AR7P95-G7 48 20.0 ND 11.3 659.3 692
P114AR7P95-H8 49 3.7 ND 4.1 209.8 280.7
Thermal Stability of c-Met-Binding FN3 Domains Differential
Scanning Calorimetry in PBS was Used to Assess the Stability of
Each FN3 Domain. Results of the Experiment are Shown in Table
9.
TABLE-US-00017 TABLE 9 Thermal Clone Stability Name SEQ ID NO: (Tm,
C.) P114AR5P74-A5 32 74.1 P114AR5P75-E9 33 ND P114AR7P92-F3 34 81.5
P114AR7P92-F6 35 76.8 P114AR7P92-G8 36 90.9 P114AR7P92-H5 37 87
P114AR7P93-D11 38 ND P114AR7P93-G8 39 76.8 P114AR7P93-H9 40 88.2
P114AR7P94-A3 41 86.2 P114AR7P94-E5 42 80 P114AR7P95-B9 43 86.3
P114AR7P95-D3 44 82 P114AR7P95-D4 45 85.3 P114AR7P95-E3 46 94.2
P114AR7P95-F10 47 85.2 P114AR7P95-G7 48 87.2 P114AR7P95-H8 49
83
Example 7. Generation and Characterization of Bispecific
Anti-EGFR/c-Met Molecules
Generation of Bispecific EGFR/c-Met Molecules
[0613] Numerous combinations of the EGFR and c-Met-binding FN3
domains described in Examples 1-6 were joined into bispecific
molecules capable of binding to both EGFR and c-Met. Additionally,
EGFR-binding FN3 domains having amino acid sequences shown in SEQ
ID NOs: 107-110 and c-Met binding FN3 domains having amino acid
sequences shown in SEQ ID NOs: 111-114 were made and joined into
bispecific molecules. Synthetic genes were created to encode for
the amino acid sequences described in SEQ ID NOs: 50-72 and 106
(Table 10) such that the following format was maintained:
EGFR-binding FN3 domain followed by a peptide linker followed by a
c-Met-binding FN3 domain. A poly-histidine tag was incorporated at
the C-terminus to aid purification. In addition to those molecules
described in Table 10, the linker between the two FN3 domains was
varied according to length, sequence composition and structure
according to those listed in Table 11. It is envisioned that a
number of other linkers could be used to link such FN3 domains
Bispecific EGFR/c-Met molecules were expressed and purified from E.
coli as described for monospecific EGFR or c-Met FN3 domains using
IMAC and gel filtration chromatography steps.
TABLE-US-00018 TABLE 10 Bispecifcic EGFR/c- EGFR-binding
cMET-binding Met molecule FN3 comain FN3 domain Linker Clone SEQ ID
SEQ ID SEQ ID SEQ ID ID NO: Clone ID NO: Clone ID NO: Sequence NO:
ECB1 50 P54AR4-83v2 27 P114AR5P74-A5 32 (GGGGS).sub.4 79 ECB2 51
P54AR4-83v2 27 P114AR7P94-A3 41 (GGGGS).sub.4 79 ECB3 52
P54AR4-83v2 27 P114AR7P93-H9 40 (GGGGS).sub.4 79 ECB4 53
P54AR4-83v2 27 P114AR5P75-E9 33 (GGGGS).sub.4 79 ECB5 54
P53A1R5-17v2 107 P114AR7P94-A3 41 (GGGGS).sub.4 79 ECB6 55
P53A1R5-17v2 107 P114AR7P93-H9 40 (GGGGS).sub.4 79 ECB7 56
P53A1R5-17v2 107 P114AR5P75-E9 33 (GGGGS).sub.4 79 ECB15 57
P54AR4-83v2 27 P114AR7P94-A3 41 (AP).sub.5 81 ECB27 58 P54AR4-83v2
27 P114AR5P74-A5 32 (AP).sub.5 81 ECB60 59 P53A1R5-17v2 107
P114AR7P94-A3 41 (AP).sub.5 81 ECB37 60 P53A1R5-17v2 107
P114AR5P74-A5 32 (AP).sub.5 81 ECB94 61 P54AR4-83v22 108
P114AR7P94-A3v22 111 (AP).sub.5 81 ECB95 62 P54AR4-83v22 108
P114AR9P121-A6v2 112 (AP).sub.5 81 ECB96 63 P54AR4-83v22 108
P114AR9P122-A7v2 113 (AP).sub.5 81 ECB97 64 P54AR4-83v22 108
P114AR7P95-C5v2 114 (AP).sub.5 81 ECB106 65 P54AR4-83v23 109
P114AR7P94-A3v22 111 (AP).sub.5 81 ECB107 66 P54AR4-83v23 109
P114AR9P121-A6v2 112 (AP).sub.5 81 ECB108 67 P54AR4-83v23 109
P114AR9P122-A7v2 113 (AP).sub.5 81 ECB109 68 P54AR4-83v23 109
P114AR7P95-C5v2 114 (AP).sub.5 81 ECB118 69 P53A1R5-17v22 110
P114AR7P94-A3v22 111 (AP).sub.5 81 ECB119 70 P53A1R5-17v22 110
P114AR9P121-A6v2 112 (AP).sub.5 81 ECB120 71 P53A1R5-17v22 110
P114AR9P122-A7v2 113 (AP).sub.5 81 ECB121 72 P53A1R5-17v22 110
P114AR7P95-C5v2 114 (AP).sub.5 81 ECB91 106 P54AR4-83v22 108
P114AR7P95-C5v2 114 (AP).sub.5 81 ECB18 118 P54AR4-83v2 27
P114AR5P74-A5 32 (AP).sub.5 81 ECB28 119 P53A1R5-17v2 107
P114AR5P74-A5 32 (AP).sub.5 81 ECB38 120 P54AR4-83v2 27
P114AR7P94-A3 41 (AP).sub.5 81 ECB39 121 P53A1R5-17v2 107
P114AR7P94-A3 41 (AP).sub.5 81
TABLE-US-00019 TABLE 11 SEQ ID Linker length in Linker NO: amino
acids Structure GS 78 2 Disordered GGGGS 105 5 Disordered
(GGGGS).sub.4 79 20 Disordered (AP).sub.2 80 4 Rigid (AP).sub.5 81
5 Rigid (AP).sub.10 82 20 Rigid (AP).sub.20 83 40 Rigid
A(EAAAK).sub.5AAA 84 29 .alpha.-helical
Bispecific EGFR/c-Met Molecules Enhance Potency Compared to
Monospecific Molecules Alone, Suggesting Avidity
[0614] NCI-H292 cells were plated in 96 well plates in RPMI medium
containing 10% FBS. 24 hours later, medium was replaced with serum
free RPMI. 24 hours after serum starvation, cells were treated with
varying concentrations of FN3 domains: either a high affinity
monospecific EGFR FN3 domain (P54AR4-83v2), a weak affinity
monospecific c-Met FN3 domain (P114AR5P74-A5), the mixture of the
two monospecific EGFR and c-Met FN3 domains, or a bispecific
EGFR/c-Met molecules comprised of the low affinity c-Met FN3 domain
linked to the high affinity EGFR FN3 domain (ECB1). Cells were
treated for 1 h with the monospecific or bispecific molecules and
then stimulated with EGF, HGF, or a combination of EGF and HGF for
15 minutes at 37.degree. C., 5% CO.sub.2. Cells were lysed with MSD
Lysis Buffer and cell signaling was assessed using appropriate MSD
Assay plates, according to manufacturer's instructions, as
described above.
[0615] The low affinity c-Met FN3 domain inhibited phosphorylation
of c-Met with an IC.sub.50 of 610 nM (FIG. 4). As expected the EGFR
FN3 domain was not able to inhibit c-Met phosphorylation and the
mixture of the mono-specific molecules looked identical to the
c-Met FN3 domain alone. However, the bi-specific EGFR/c-Met
molecule inhibited phosphorylation of c-Met with an IC.sub.50 of 1
nM (FIG. 4), providing more than a 2-log shift in improving potency
relative to the c-Met monospecific alone.
[0616] The potential for the bispecific EGFR/c-Met molecule to
enhance the inhibition of c-Met and/or EGFR phosphorylation through
an avidity effect was evaluated in multiple cell types with
variable c-Met and EGFR densities and ratios (FIG. 5). NCI-H292,
NCI-H441, or NCI-H596 cells were plated in 96 well plates in RPMI
medium containing 10% FBS. 24 hours later, medium was replaced with
serum free RPMI. 24 hours after serum starvation, cells were
treated with varying concentrations of either monospecific
EGFR-binding FN3 domain, monospecific c-Met FN3 domain, or a
bispecific EGFR/c-Met molecule (ECB5, comprised of P53A1R5-17v2 and
P114AR7P94-A3). Cells were treated for 1 h with the monospecific or
bispecific molecules and then stimulated with EGF, HGF, or a
combination of EGF and HGF for 15 minutes at 37.degree. C., 5%
CO.sub.2. Cells were lysed with MSD Lysis Buffer and cell signaling
was assessed using appropriate MSD Assay plates, according to
manufacturer's instructions, as described above.
[0617] FIG. 5 (A-C) shows the inhibition of EGFR using a
monospecific EGFR-binding FN3 domain compared to a bispecific
EGFR/cMet molecule in three different cell lines. To assess avidity
in an EGFR phosphorylation assay, a medium affinity EGFR-binding
FN3 domain (1.9 nM) (P53A1R5-17v2) was compared to a bispecific
EGFR/c-Met molecule containing the same EGFR-binding FN3 domain
linked to a high-affinity c-Met-binding FN3 domain (0.4 nM)
(P114AR7P94-A3). In NCI-H292 and H596 cells, inhibition of
phosphorylation of EGFR was comparable for the monospecific and
bispecific molecules (FIGS. 5A and 5B), likely because these cell
lines have a high ratio of EGFR to c-Met receptors. To test this
theory, inhibition of EGFR phosphorylation was evaluated in
NCI-H441 cells which exhibit more c-Met receptors than EGFR.
Treatment of NCI-H441 cells with the bispecific EGFR/c-Met molecule
decreased the IC.sub.50 for inhibition of EGFR phosphorylation
compared to the monospecific EGFR-binding FN3 domain by 30-fold
(FIG. 5C).
[0618] The potential for enhanced potency with a bi-specific
EGFR/c-Met molecule was evaluated in a c-Met phosphorylation assay
using a molecule with a high affinity to EGFR (0.26 nM) and medium
affinity to c-Met (10.1 nM). In both NCI-H292 and NCI-H596 cells,
the inhibition of phosphorylation of c-Met was enhanced with the
bispecific molecule compared to the monospecific c-Met-binding FN3
domain, by 134 and 1012 fold, respectively (FIGS. 3D and 3E).
[0619] It was verified that the enhanced potency for inhibition of
EGFR and c-Met phosphorylation with the bispecific EGFR/c-Met
molecules translated into an enhanced inhibition of signaling and
proliferation. For these experiments, the mixture of FN3
EGFR-binding and c-Met-binding FN3 domains was compared to a
bispecific EGFR/c-Met molecule. As described in Tables 12 and 13,
the IC.sub.50 values for ERK phosphorylation (Table 12) and
proliferation of NCI-H292 cells (Table 13) were decreased when
cells were treated with the bispecific EGFR/c-Met molecule compared
to the mixture of the monospecific binders. The IC.sub.50 for
inhibition of ERK phosphorylation for the bi-specific EGFR/c-Met
molecule was 143-fold lower relative to the mixture of the two
monospecific EGFR and c-Met FN3 domains, showing the effect of
avidity to the potency of the molecules in this assay. In Table 12,
the monospecific EGFR- and c-Met binding FN3 domains do not fully
inhibit activity and therefore the IC.sub.50 values shown should be
considered lower limits. The proliferation assay was completed
using different combinations EGFR and c-Met binding FN3 domains
either as a mixture or linked in a bispecific format. The IC.sub.50
for inhibition of proliferation for the bispecific EGFR/c-Met
molecule was 34-236-fold lower relative to the mixture of the
monospecific parent EGFR or c-Met binding FN3 domains. This
confirmed that the avidity effect observed at the level of the
receptors (FIG. 4 and FIG. 5) translates into an improvement in
inhibiting cell signaling (Table 12) and cell proliferation (Table
13).
TABLE-US-00020 TABLE 12 Specificity of the FN3-domain IC.sub.50
(nM) (ERK molecule Clone name Type phosphorylation) EGFR
P54AR4-83v2 monospecific >10,000 c-Met P114AR5P74-A5
monospecific 2366 EGFR or c-Met P54AR4-83v2 + mixture of 798.4
P114AR5P74-A5 monospecific molecules EGFR and c-Met ECB1 bispecific
5.6
TABLE-US-00021 TABLE 13 EGFR- IC.sub.50 for Fold increase binding
mixture of IC.sub.50 for in IC.sub.50 for FN3 c-Met binding
monospecific bispecific mixture of domain FN3 domain molecules
molecule monospecific/ (affinity) (affinity) (nM) (nM) bispecific
P54AR4- P114ARP94-A3 36.5 1.04 35 83v2 (0.4 nM) (0.26 nM) P54AR4-
P114AR7P93- 274.5 8.05 34 83v2 H9 (3.3 nM) (0.26 nM) P54AR4-
P114AR5P74- 1719 7.29 236 83v2 A5 (10.1 nM) (0.26 nM)
In Vivo Tumor Xenografts: PK/PD
[0620] In order to determine efficacy of the monospecific and
bispecific FN3 domain molecules in vivo, tumor cells were
engineered to secrete human HGF (murine HGF does not bind to human
c-Met). Human HGF was stably expressed in NCI-H292 cells using
lentiviral infection (Lentiviral DNA vector expressing human HGF
(Accession #X.sub.16322) and lentiviral packaging kit from
Genecopoeia). After infection, HGF-expressing cells were selected
with 4 .mu.g/mL puromycin (Invitrogen). Human HGF protein was
detected in the conditioned medium of pooled cells using assay
plates from MesoScale Discovery.
[0621] SCID Beige mice were subcutaneously inoculated with NCI-H292
cells expressing human HGF (2.0.times.10.sup.6 cells in Cultrex
(Trevigen) in a volume of 200 .mu.L) on the dorsal flank of each
animal Tumor measurements were taken twice weekly until tumor
volumes ranged between 150-250 mm.sup.3. Mice were then given a
single i.p. dose of bispecific EGFR/c-Met molecules (linked to an
albumin binding domain to increase half-life) or PBS vehicle. At 6
h or 72 h after dosing, tumors were extracted and immediately
frozen in liquid nitrogen. Blood samples were collected via cardiac
puncture into 3.8% citrate containing protease inhibitors
Immediately after collection, the blood samples were centrifuged
and the resulting plasma was transferred to sample tubes and stored
at -80.degree. C. Tumors were weighed, cut into small pieces, and
lysed in Lysing Matrix A tubes (LMA) containing RIPA buffer with
HALT protease/phosphatase inhibitors (Pierce), 50 mM sodium
fluoride (Sigma), 2 mM activated sodium orthovanadate (Sigma), and
1 mM PMSF (MesoScale Discovery). Lysates were removed from LMA
matrix and centrifuged to remove insoluble protein. The soluble
tumor protein was quantified with a BCA protein assay and diluted
to equivalent protein levels in tumor lysis buffer. Phosphorylated
c-Met, EGFR and ERK were measured using assay plates from MesoScale
Discovery (according to Manufacturer's protocol and as described
above).
[0622] FIG. 6 shows the results of the experiments. Each bispecific
EGFR/c-Met molecule significantly reduced the levels of
phosphorylated c-Met, EGFR, and ERK at both 6 h and 72 h. The data
presented in FIG. 6 show the importance of inhibiting both c-Met
and EGFR simultaneously and how the affinity of the bispecific
EGFR/c-Met molecule for each receptor plays a role in inhibition of
downstream ERK. The molecules containing the high affinity
EGFR-binding FN3 domains (P54AR4-83v2; shown as "8" in the Figure,
K.sub.D=0.26 nM) inhibited phosphorylation of EGFR to a larger
extent compared to those containing the medium affinity
EGFR-binding FN3 domains (P53A1R5-17v2; shown as "17" in the figure
K.sub.D=1.9 nM) at both 6 h and 72 h. All four bispecific molecules
tested completely inhibited phosphorylation of ERK at the 6 hour
time point, regardless of affinity. At the 72 hour time point, the
molecules containing the tight affinity c-Met-binding domain
(P114AR7P94-A3; shown as "A3" in the figure K.sub.D=0.4 nM)
significantly inhibited phosphorylation of ERK compared to the
medium affinity c-Met-binding FN3 domain (P114AR5P74-A5; shown as
"A5" in the Figure; K.sub.D=10.1 nM; FIG. 6).
[0623] The concentration of each bispecific EGFR/c-Met molecule was
measured at 6 and 72 hours after dosing in the blood and in the
tumor (FIG. 7). Interestingly, the bispecific molecule with the
medium affinity EGFR-binding domain (P53A1R5-17v2; K.sub.D=1.9 nM)
but high affinity c-Met-binding FN3 domain (P114AR7P94-A3;
K.sub.D=0.4 nM) had significantly more tumor accumulation at 6
hours relative to the other molecules, while the difference is
diminished by 72 hours. It can be hypothesized that cells outside
the tumor have lower levels of both EGFR and c-Met surface
expression and therefore the medium affinity EGFR molecule doesn't
bind to normal tissue as tightly compared to the higher affinity
EGFR-binding FN3 domain. Therefore there is more free medium
affinity EGFR-binding FN3 domain available to bind in the tumor.
Therefore, identifying the appropriate affinities to each receptor
may allow for identification of a therapeutic with decreased
systemic toxicities and increased tumor accumulation.
Tumor Efficacy Studies with Bispecific EGFR/c-Met Molecules
[0624] SCID Beige mice were subcutaneously inoculated with NCI-H292
cells expressing human HGF (2.0.times.10.sup.6 cells in Cultrex
(Trevigen) in 200 .mu.L) in the dorsal flank of each animal One
week after implantation, mice were stratified into groups with
equivalent tumor volumes (mean tumor volume=77.9+/-1.7 mm.sup.3)
Mice were dosed three times per week with the bispecific molecules
and tumor volumes were recorded twice weekly. Tumor growth
inhibition (TGI) was observed with four different bispecific
molecules, with variable affinities for c-Met and EGFR. FIG. 8
shows the benefit of inhibiting both c-Met and EGFR as a delay in
tumor growth was observed in the mice treated with molecules
containing the high affinity EGFR-binding FN3 domain compared to
the medium affinity EGFR-binding FN3 domain when the c-Met-binding
FN3 domain was medium affinity (open vs. closed triangles,
P54AR4-83v2-P114AR5P74-A5 compared to P53A1R5-17-P114AR5P74-A5). In
addition, the data shows the importance of having a high affinity
c-Met-binding FN3 domain as bispecific molecules containing either
the high or medium affinity EGFR-binding FN3 domain but high
affinity c-Met-binding FN3 domain showed the most efficacy (dotted
gray and black lines, P54AR4-83v2-P114AR7P94-A3 and
P53A1R5-17v2-P114AR7P94-A3).
Efficacy of Bispecific Molecule and Other Inhibitors of EGFR and
c-Met
[0625] The in vivo therapeutic efficacies of a bispecific
EGFR/c-Met molecule (ECB38) and the small molecule inhibitors
crizotinib (c-Met inhibitor) and erlotinib (EGFR inhibitor),
cetuximab (anti-EGFR antibody), each as a single agent, and the
combination of crizotinib and erlotinib were evaluated in the
treatment of subcutaneous H292-HGF human lung cancer xenograft
model in SCID/Beige mice.
[0626] The H292-HGF cells were maintained in vitro in RPMI1640
medium supplemented with fetal bovine serum (10% v/v), and
L-glutamine (2 mM) at 37.degree. C. in an atmosphere of 5% CO.sub.2
in air. The cells were routinely subcultured twice weekly by
trypsin-EDTA treatment. The cells growing in an exponential growth
phase were harvested and counted for tumor inoculation.
[0627] Each mouse was inoculated subcutaneously at the right flank
region with H292-HGF tumor cells (2.times.10.sup.6) in 0.1 ml of
PBS with Cultrex (1:1) for tumor development. The treatments were
started when the mean tumor size reached 139 mm.sup.3. The test
article administration and the animal numbers in each study group
were shown in the following experimental design table. The date of
tumor cell inoculation was denoted as day 0. Table 14 shows the
treatment groups.
TABLE-US-00022 TABLE 14 Dose Dosing Planned Actual Group N
Treatment (mg/kg) Route Schedule Schedule 1 10 Vehicle 0 i.p. QD
.times. 3 QD .times. 3 Control weeks weeks 2 10 bispecific 25 i.p.
3 times/ 3 times/ EGFR/ week .times. week .times. c-Met 3 weeks 3
weeks molecule 3 10 crizotinib 50 p.o. QD .times. 3 QD .times. 17
weeks days 4 10 erlotinib 50 p.o. QD .times. 2 QD .times. 3 weeks
weeks 5 10 crizotinib 50 p.o. QD .times. 3 QD .times. 3 weeks weeks
6 10 cetuximab 1 mg/mouse i.p. Q4d*6 Q4d*6 N: animal number; p.o.:
oral administration; i.p.: intraperitoneal injection 3 times/week:
doses were given on days 1, 3 and 5 of the week. QD: once daily
Q4d: once every four days; the interval of the combination of
crizotinib and erlotinib was 0.5 hrs; dosing volume was adjusted
based on body weight (10 l/g); a: dosing was not given on day 14
post grouping.
Before commencement of treatment, all animals were weighed and the
tumor volumes were measured. Since the tumor volume can affect the
effectiveness of any given treatment, mice were assigned into
groups using randomized block design based upon their tumor
volumes. This ensures that all the groups are comparable at the
baseline. The randomized block design was used to assign
experimental animals to groups. First, the experimental animals
were divided into homogeneous blocks according to their initial
tumor volume. Secondly, within each block, randomization of
experimental animals to treatments was conducted. Using randomized
block design to assign experimental animals ensured that each
animal had the same probability of being assigned to a given
treatment and therefore systematic error was reduced.
[0628] At the time of routine monitoring, the animals were checked
for any effects of tumor growth and treatments on normal behavior,
such as mobility, visual estimation of food and water consumption,
body weight gain/loss (body weights were measured twice weekly),
eye/hair matting and any other abnormal effect.
[0629] The endpoint was whether tumor growth can be delayed or
tumor bearing mice can be cured. Tumor size was measured twice
weekly in two dimensions using a caliper, and the volume was
expressed in mm.sup.3 using the formula: V=0.5 a.times.b.sup.2
where a and b are the long and short diameters of the tumor,
respectively. The tumor size was then used for calculations of both
T-C and T/C values. T-C was calculated with T as the time (in days)
required for the mean tumor size of the treatment group to reach
1000 mm.sup.3, and C was the time (in days) for the mean tumor size
of the control group to reach the same size. The T/C value (in
percent) was an indication of antitumor efficacy; T and C were the
mean tumor volume of the treated and control groups, respectively,
on a given day. Complete tumor regression (CR) is defined as tumors
that are reduced to below the limit of palpation (62.5 mm.sup.3).
Partial tumor regression (PR) is defined as tumors that are reduced
from initial tumor volume. A minimum duration of CR or PR in 3 or
more successive tumor measurements is required for a CP or PR to be
considered durable.
[0630] Animals for which the body weight loss exceeded 20%, or for
which the mean tumor size of the group exceeds 2000 mm.sup.3 were
euthanized. The study was terminated after two weeks of observation
after the final dose.
[0631] Summary statistics, including mean and the standard error of
the mean (SEM), are provided for the tumor volume of each group at
each time point are shown in Table 15. Statistical analyses of
difference in tumor volume among the groups were evaluated using a
one-way ANOVA followed by individual comparisons using Games-Howell
(equal variance not assumed). All data were analyzed using SPSS
18.0. p<0.05 was considered to be statistically significant.
TABLE-US-00023 TABLE 15 Tumor volume (mm.sup.3)a bispecific
crizotinib; EGFR/c-Met erlotinib at molecule at crizotinib at
erlotinib at 50 mg/kg; cetuximab at 1 Days Vehicle 25 mg/kg 50
mg/kg 50 mg/kg 50 mg/kg mg/mouse 7 139 .+-. 7 137 .+-. 7 140 .+-. 9
141 .+-. 8 139 .+-. 8 139 .+-. 10 9 230 .+-. 20 142 .+-. 7 217 .+-.
20 201 .+-. 19 134 .+-. 9 168 .+-. 13 13 516 .+-. 45 83 .+-. 6 547
.+-. 43 392 .+-. 46 109 .+-. 10 212 .+-. 20 16 808 .+-. 104 44 .+-.
7 914 .+-. 92 560 .+-. 70 127 .+-. 15 252 .+-. 28 20 1280 .+-. 209
30 .+-. 6 1438 .+-. 239 872 .+-. 136 214 .+-. 30 371 .+-. 48 23
1758 .+-. 259 23 .+-. 7 2102 .+-. 298 1122 .+-. 202 265 .+-. 40 485
.+-. 61 27 2264 .+-. 318 21 .+-. 5 -- 1419 .+-. 577 266 .+-. 42 640
.+-. 82 30 -- 23 .+-. 6 -- 1516 .+-. 623 482 .+-. 61 869 .+-.
100
[0632] The mean tumor size of the vehicle treated group (Group 1)
reached 1,758 mm.sup.3 at day 23 after tumor inoculation. Treatment
with the bispecific EGFR/c-Met molecule at 25 mg/kg dose level
(Group 2) led to complete tumor regression (CR) in all mice which
were durable in >3 successive tumor measurements (average TV=23
mm.sup.3, T/C value=1%, p=0.004 compared with the vehicle group at
day 23).
[0633] Treatment with crizotinib as a single agent at 50 mg/kg dose
level (Group 3) showed no antitumor activity; the mean tumor size
was 2,102 mm.sup.3 at day 23 (T/C value=120%, p=0.944 compared with
the vehicle group).
[0634] Treatment with erlotinib as a single agent at 50 mg/kg
dosing level (Group 4) showed minor antitumor activity, but no
significant difference was found compared with the vehicle group;
the mean tumor size was 1,122 mm.sup.3 at day 23 (T/C value=64%,
p=0.429 compared with the vehicle group), with 4 days of tumor
growth delay at tumor size of 1,000 mm.sup.3 compared with the
vehicle group.
[0635] The combination of crizotinib (50 mg/kg, Group 5) and
erlotinib (50 mg/kg, Group 5) showed significant antitumor
activity; the mean tumor size was 265 mm.sup.3 at day 23 (T/C=15%;
p=0.008), with 17 days of tumor growth delay at tumor size of 1,000
mm.sup.3 compared with the vehicle group.
[0636] Cetuximab at 1 mg/mouse dosing level as a single agent
(Group 6) showed significant antitumor activities; the mean tumor
size was 485 mm.sup.3 at day 23 (T/C=28%; p=0.018), with 17 days of
tumor growth delay at tumor size of 1,000 mm.sup.3 compared with
the vehicle group. FIG. 15 and Table 16 show the anti-tumor
activities of the various therapies.
TABLE-US-00024 TABLE 16 Tumor Size T-C (mm.sup.3) at (days) at
Treatment day 23 T/C (%) 1000 mm.sup.3 P value Vehicle 1758 .+-.
259 -- -- -- bispecific EGFR/c-Met 23 .+-. 7 1 -- 0.004 molecule
(25 mg/kg) crizotinib (50 mg/kg) 2102 .+-. 298 120 -1 0.944
erlotinib (50 mg/kg) 1122 .+-. 202 64 4 0.429 crizotinib +
erlotinib 265 .+-. 40 15 17 0.008 (50 mg/kg + 50 mg/kg) cetuximab
(1 mg/ 485 .+-. 61 28 17 0.018 mouse)
[0637] Medium to severe body weight loss was observed in the
vehicle group which might be due to the increasing tumor burden; 3
mice died and 1 mouse were euthanized when BWL>20% by day 23.
Slight toxicity of the bispecific EGFR/c-Met molecule was observed
in Group 2; 3 mice were euthanized when BWL>20% during the
treatment period; the body weight was gradually recovered when the
treatment was withdrawn during the 2 weeks of observation period.
More severe body weight loss was observed in the crizotinib or
erlotinib monotherapy group compared to the vehicle group,
suggesting the treatment related toxicity. The combination of
crizotinib and erlotinib was generally tolerated during the dosing
phase, but severe body weight loss was observed at the end of the
study, which might be due to the resumption of the fast tumor
growth during the non-treatment period. The monotherapy of
cetuximab was well tolerated in the study; body weight loss was
only observed at the end of the study due to the resume of the
tumor growth.
[0638] In summary, the bispecific EGFR/c-Met molecule at 25 mg/kg
(3 times/week.times.3 weeks) produced a complete response in
H292-HGF human lung cancer xenograft model in SCID/Beige mice. The
treatment was tolerated in 7 out of 10 mice, and resulted in severe
body weight loss in 3 out of 10 mice. FIG. 9 shows the impact of
the various therapies on tumor size during the time points after
treatment.
Example 8: Half-Life Extension of the Bispecific EGFR/c-Met
Molecules
[0639] Numerous methods have been described to reduce kidney
filtration and thus extend the serum half-life of proteins
including modification with polyethylene glycol (PEG) or other
polymers, binding to albumin, fusion to protein domains which bind
to albumin or other serum proteins, genetic fusion to albumin,
fusion to IgG Fc domains, and fusion to long, unstructured amino
acid sequences.
[0640] Bispecific EGFR/c-Met molecules were modified with PEG in
order to increase the hydrodynamic radius by incorporating a free
cysteine at the C-terminus of the molecule. Most commonly, the free
thiol group of the cysteine residue is used to attach PEG molecules
that are functionalized with maleimide or iodoacetemide groups
using standard methods. Various forms of PEG can be used to modify
the protein including linear PEG of 1000, 2000, 5000, 10,000,
20,000, or 40,000 kDa. Branched PEG molecules of these molecular
weights can also be used for modification. PEG groups may also be
attached through primary amines in the bispecific EGFR/c-Met
molecules in some instances.
[0641] In addition to PEGylation, the half-life of bispecific
EGFR/c-Met molecules was extended by producing these proteins as
fusion molecules with a naturally occurring 3-helix bundle serum
albumin binding domain (ABD) or a consensus albumin binding domain
(ABDCon). These protein domains were linked to the C-terminus of
the c-Met-binding FN3 domain via any of the linkers described in
Table 12. The ABD or ABDCon domain may also be placed between the
EGFR-binding FN3 domain and the c-Met binding FN3 domain in the
primary sequence.
Example 9: Characterization of Select Bispecific EGFR/c-Met
Molecules
[0642] Select bispecific EGFR/c-Met molecules were characterized
for their affinity to both EGFR and c-Met, their ability to inhibit
EGFR and c-Met autophosphorylation, and their effect on
proliferation of HGF cells. Binding affinity of the bispecific
EGFR/c-Met molecules to recombinant EGFR and/or c-Met extracellular
domain was further evaluated by surface Plasmon resonance methods
using a Proteon Instrument (BioRad) according to protocol described
in Example 3. Results of the characterization are shown in Table
17.
TABLE-US-00025 TABLE 17 H292-HGF pMet H292 Proliferation inhibition
pEGFR inhibition in K.sub.D K.sub.D in H441 inhibition in
HGF-induced (EGFR, (c-Met, cells H292 cells H292 cells nM) nM)
(IC.sub.50, nM) (IC.sub.50, nM) (IC.sub.50, nM) ECB15 0.2 2.6 n/a
4.2 23 ECB94 1 4.3 53.8 5.1 29.6 ECB95 1.1 6.2 178.8 13.6 383.4
ECB96 1.6 22.1 835.4 24.7 9480 ECB97 1.3 1.7 24.2 16.6 31.0 ECB106
16.7 5.1 53.3 367.4 484.5 ECB107 16.9 9 29.9 812.3 2637 ECB108 15.3
25.5 126.2 814.4 11372 ECB109 17.3 2.1 26 432 573.6
Example 10. Generation of Bispecific EGFR/cMet Antibodies
[0643] Several monospecific EGFR and c-Met antibodies were
expressed as IgG1, kappa, having Fc substitutions K409R or F405L
(numbering according to the EU index) in their Fc regions. The
monospecific antibodies were expressed in two CHO cell lines, one
cell line having reduced fucosylation ability resulting in
antibodies with 1-15% fucose content in the antibody polysaccharide
chain.
[0644] The monospecific antibodies were purified using standard
methods using a Protein A column (HiTrap MabSelect SuRe column).
After elution, the pools were dialyzed into D-PBS, pH 7.2
[0645] Bispecific EGFR/c-Met antibodies were generated by combining
a monospecific EGFR mAb and a monospecific c-Met mAb in in vitro
Fab arm exchange (as described in WO2011/131746). Briefly, at about
1-20 mg/ml at a molar ratio of 1:1 of each antibody in PBS, pH
7-7.4 and 75 mM 2-mercaptoethanolamine (2-MEA) was mixed together
and incubated at 25-37.degree. C. for 2-6 h, followed by removal of
the 2-MEA via dialysis, diafiltration, tangential flow filtration
and/or spinned cell filtration using standard methods.
[0646] Several monospecific anti-EGFR antibodies and anti-c-Met
antibodies were combined in matrix in in vitro Fab arm exchange to
generate bispecific antibodies that were subsequently characterized
further. The generated bispecific antibodies were ranked using a
four step strategy using assays as follows: Step 1: binding to
NCI-H441, NCI-H1975 and A549 cells in a FACS assay. Step 2:
inhibition of pMet phosphorylation in A549 cells. Step 3:
inhibition of proliferation in NCI-H1975, KP4 and NCI-H441 cells.
Step 4: inhibition of EGFR phosphorylation in A549 and SNU-5 cells.
Noteworthy, the characteristics of the parental antibodies were not
preserved in the bispecific antibody. For example, the presence of
certain EGFR binding arms in the bispecific antibody resulted in a
loss or reduced inhibition, or enhanced c-Met phosphorylation.
Based on the characterization studies select pairs were chosen.
[0647] A monospecific bivalent anti-EGFR antibody E1-K409R was
generated comprising the VH and VL regions of an anti-EGFR antibody
2F8 having the VH of SEQ ID NO: 189 and the VL of SEQ ID NO: 190
(antibody 2F8 is described in Int. Pat. Publ. No. WO2002/100348)
and an IgG1 constant region with a K409R substitution.
[0648] A monospecific bivalent anti-EGFR antibody E1-F405L was
generated comprising the VH and VL regions of an anti-EGFR antibody
2F8 having the VH of SEQ ID NO: 189 and the VL of SEQ ID NO: 190
(antibody 2F8 is described in Int. Pat. Publ. No. WO2002/100348)
and an IgG1 constant region with a F405L substitution.
[0649] A monospecific bivalent anti-EGFR antibody E2-K409R was
generated comprising the VH and VL regions of an anti-EGFR antibody
018 having the VH of SEQ ID NO: 191 and the VL of SEQ ID NO: 192
(antibody 018 is described in Int. Pat. Publ. No. WO2009/030239)
and an IgG1 constant region with a K409R substitution.
[0650] A monospecific bivalent anti-EGFR antibody E2-F405L was
generated comprising the VH and VL regions of an anti-EGFR antibody
018 having the VH of SEQ ID NO: 191 and the VL of SEQ ID NO: 192
(antibody 018 is described in Int. Pat. Publ. No. WO2009/030239)
and an IgG1 constant region with a F405L substitution.
[0651] A monospecific bivalent anti-c-Met antibody M1-K409R was
generated comprising the VH and VL regions of an anti-c-Met
antibody 069 having the VH of SEQ ID NO: 193 and the VL of SEQ ID
NO: 194 (antibody 069 is described in WO2011/110642) and an IgG1
constant region with a K409R substitution.
[0652] A monospecific bivalent anti-c-Met antibody M1-F405L was
generated comprising the VH and VL regions of an anti-c-Met
antibody 069 having the VH of SEQ ID NO: 193 and the VL of SEQ ID
NO: 194 (antibody 069 is described in WO2011/110642) and an IgG1
constant region with a F405L substitution.
[0653] A monospecific anti-c-Met antibody M2-K409R was generated
comprising the VH and VL regions of an anti-c-Met antibody 058
having the VH of SEQ ID NO: 195 and the VL of SEQ ID NO: 196
(antibody 058 is described in WO2011/110642) and an IgG1 constant
region with a K409R substitution.
[0654] A monospecific anti-c-Met antibody M2-F405L was generated
comprising the VH and VL regions of an anti-c-Met antibody 058
having the VH of SEQ ID NO: 195 and the VL of SEQ ID NO: 196
(antibody 058 is described in WO2011/110642) and an IgG1 constant
region with a F405L substitution.
[0655] The VH, VL, HC and LC sequences of the antibodies are shown
below:
TABLE-US-00026 >SEQ ID NO: 189 EGFR mAb E1 VH
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVA
VIWDDGSYKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
DGITMVRGVMKDYFDYWGQGTLVTVSS >SEQ ID NO: 190 EGFR mAb E1 VL
AIQLTQSPSSLSASVGDRVTITCRASQDISSALVWYQQKPGKAPKLLIY
DASSLESGVPSRFSGSESGTDFTLTISSLQPEDFATYYCQQFNSYPLTF GGGTKVEIK >SEQ
ID NO: 191 EGFR mAb E2 VH EVQLVESGGG LVQPGGSLRL SCAASGFTFS
SYWMNWVRQA PGKGLEWVAN IKKDGSEKYY VDSVKGRFTI SRDNAKNSLY LQMNSLRAED
TAVYYCARDL GWGWGWYFDL WGRGTLVTVSS >SEQ ID NO: 192 EGFR mAb E2 VL
EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA
RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPPTFGQ GTKVEIK >SEQ ID NO:
193 cMet mAb M1 VH
QVQLVQSGAEVKKPGASVKVSCETSGYTFTSYGISWVRQAPGHGLEWMG
WISAYNGYTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR
DLRGTNYFDYWGQGTLVTVSS >SEQ ID NO: 194 cMet mAb M1 VL
DIQMTQSPSSVSASVGDRVTITCRASQGISNWLAWFQHKPGKAPKLLIY
AASSLLSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFP-IT FGQGTRLEIK
>SEQ ID NO: 195 cMet mAb M2 VH
EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYYMYWVRQTPEKRLEWVA
TISDDGSYTYYPDSVKGRFTISRDNAKNNLYLQMSSLKSEDTAMYYCAR
EGLYYYGSGSYYNQDYWGQGTLVTVSS >SEQ ID NO: 196 cMet mAb M2 VL
QLTQSPSSLSASVGDRVTITCRASQGLSSALAWYRQKPGKAPKLLIYDA
SSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFTSYPQITFG QGTRLEIK >SEQ
ID NO: 199 EM1-mAb H1 (anti-EGFR, 405L)
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVA
VIWDDGSYKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
DGITMVRGVMKDYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV
PSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN
AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT
ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK
>SEQ ID NO: 200 EM-1 mAb L1
AIQLTQSPSSLSASVGDRVTITCRASQDISSALVWYQQKPGKAPKLLIY
DASSLESGVPSRFSGSESGTDFTLTISSLQPEDFATYYCQQFNSYPLTF
GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV
THQGLSSPVTKSFNRGEC >SEQ ID NO: 201 EM-1 mAb H2 (K409R,
anti-cMet) QVQLVQSGAEVKKPGASVKVSCETSGYTFTSYGISWVRQAPGHGLEWMG
WISAYNGYTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR
DLRGTNYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG
TQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK >SEQ
ID NO: 202 EM-1 mAb L2
DIQMTQSPSSVSASVGDRVTITCRASQGISNWLAWFQHKPGKAPKLLIY
AASSLLSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPITF
GQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV
THQGLSSPVTKSFNRGEC >SEQ ID NO: 234 E2 mAb HC1 (EGFR-F405L)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMNWVRQAPGKGLEWVA
NIKKDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR
DLGWGWGWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG
CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK
>SEQ ID NO: 235 E2 mAb LC1 (EGFR)
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY
DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTF
GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV
THQGLSSPVTKSFNRGEC >SEQ ID NO: 236 E2 mAb HC2 (c-Met-K409R)
EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYYMYWVRQTPEKRLEWVA
TISDDGSYTYYPDSVKGRFTISRDNAKNNLYLQMSSLKSEDTAMYYCAR
EGLYYYGSGSYYNQDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV
PSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN
AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT
ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK
>SEQ ID NO: 237 E2 mAb LC2 (cMet)
QLTQSPSSLSASVGDRVTITCRASQGLSSALAWYRQKPGKAPKLLIYDA
SSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFTSYPQITFG
QGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
HQGLSSPVTKSFNRGEC
[0656] The generated monospecific anti-EGFR and c-Met antibodies
were mixed for in vitro Fab arm exchange in matrix and
characterized in various assays. The bispecific antibody EM1-mAb
comprises the EGFR binding arm of mAb E1-F405L and the c-Met
binding arm of mAb M1-K409R. The bispecific antibody EM2-mAb
comprises the EGFR binding arm of mAb E2-F405L and the c-Met
binding arm of mAb M2-K409R. The bispecific antibody EM3-mAb
comprises the EGFR binding arm of mAb E1-K409R and the c-Met
binding arm of mAb M1-F405L. The bispecific antibody EM4-mAb
comprises the EGFR binding arm of mAb E2-K409R and the c-Met
binding arm of mAb M2-F405L. EM1-mAb and EM3-mAb had comparable
characteristics.
[0657] The bispecific EM1-mAb was cultured in a CHO cell line
having reduced fucosylation ability of glycoproteins, and hence
have a fucosyl content of about 1-15%. The removal of the core
fucose from the biantennary complex-type oligosaccharides attached
to the Fc regions significantly enhances the ADCC of antibodies via
improved Fc.gamma.RIIIa binding without altering antigen binding or
CDC activity. Such mAbs can be achieved using different methods
reported to lead to the successful expression of relatively high
defucosylated therapeutic antibodies bearing the biantennary
complex-type of Fc oligosaccharides and are described supra.
Example 11. Purification of Bispecific EGFR/c-Met Antibodies
[0658] The bispecific EM1-mAb was further purified after the in
vitro Fab-arm exchange using hydrophobic interaction chromatography
to minimize residual parental c-Met and EGFR antibodies using
standard methods.
Example 12. Characterization of Bispecific EGFR/c-Met
Antibodies
[0659] The EGFR/c-Met bispecific antibody EM1-mAb was tested in
various assays for its characteristics including inhibition of
EGF-stimulated EGFR phosphorylation, HGF-stimulated c-Met
phosphorylation, ERK1/2 phosphorylation, AKT phosphorylation,
inhibition of ligand binding and cell viability. The
characteristics of the EM1-mAb was compared to control monovalent
EGFR- or c-Met binding antibodies, and to known EGFR inhibitors
such as erlotinib (CAS 183321-74-6; tyrosine kinase inhibitor) and
cetuximab (CAS 205923-56-4).
[0660] As the parent antibodies of the EM-1 mAb antibodies are
bivalent, control monovalent EGFR and c-Met antibodies were
generated in a bispecific format combined with a Fab arm that binds
to an unrelated/irrelevant antigen to accurately compare the
synergy and avidity of a bispecific EM-1 mAb in comparison to a
mixture of corresponding control monovalent molecules.
[0661] To generate the control monovalent EGFR and c-Met
antibodies, a monospecific anti-HIV gp120 antibody gp120-K409R was
generated comprising heavy chain of SEQ ID NO: 198 and a light
chain of SEQ ID NO: 209. A monospecific anti-HIV gp120 antibody
gp120-F405L was generated comprising the heavy chain of SEQ ID NO:
197 and the light chain of SEQ ID NO: 209.
[0662] The control monovalent anti-EGFR mAb E1-F405L-gp120-K409R
was generated by in vitro Fab arm exchange between E1-F405L and
gp120-K409R, and the control monovalent anti-cMet mAb
M1-K409R-gp120-F405L was generated by in vitro Fab-arm exchange
between M1-K409R and gp120-F405L and purified as described
earlier.
[0663] The following cell lines were used in characterization of
the bispecific antibodies: NCI-H292 (American Type Culture
Collection (ATCC), Cat. No. CRL-1848), NCI-H1975 (ATCC Cat. No.
CRL-5908), SKMES-1 (ATCC Cat. No. HTB-58), A431 (ATCC Cat. No.
CRL-1555), NCI-H441 (ATCC Cat. No. HTB-174), NCI-H3255 (DCTD
tumor/cell line repository, NCI, Frederick, NCI-Navy Medical
oncology Cell Line supplement. J Cell Biochem suppl 24, 1996; Tracy
S. cancer Res 64:7241-4, 2004; Shimamura T. cancer Res 65:6401-8,
2005) and HCC-827 (ATCC Cat. No. CRL-2868). NCI-H292 and SKMES-1
cells express both wild type EGFR and wild type c-Met. NCI-3255
expresses mutant L858R EGFR and displays EGFR and c-Met
amplification. H1975 expresses mutant L858R/T790M EGFR and wild
type c-Met. HCC-827 expresses A (E746, A750) EGFR and displays EGFR
amplification. Cell line NCI-H292, NCI-H975, NCI-H441 and NCI-H3255
are interchangeably referred to as H292, H975, H441 and H3255,
respectively, in the specification.
Binding of Bispecific EGFR/cMet Antibodies to EGFR and c-Met on
Cells (A431 Cell Binding Assay)
[0664] The bispecific EGFR/c-Met antibody EM1-mAb was tested for
binding to EGFR and c-Met on cells using protocol described in
Example 3 ("A431 Cell Binding Assay") and Example 6 ("H441 Cell
Binding Assay"). Cetuximab and a control antibody monovalent
towards EGFR E1-F405L-gp120-K409R were used as controls for the
A431 cells. Cetuximab had an EC.sub.50 value of 5.0 nM. Table 18
shows the EC.sub.50 values for binding. EM1-mAb demonstrated a
1.9-fold (A431 cells) and 2.3-fold (H441 cells) decrease in binding
when compared to the bivalent monospecific parental control
antibodies. Cetuximab was comparable to the bivalent parental
control antibodies. EM1-mAb displays higher EC.sub.50 binding
values than the values for the parental mAbs due to the monovalent
binding of EGFR and c-Met. EM1-mAb has similar binding EC.sub.50
values as the single arm E1/inert arm and E2/inert arm bispecific
monovalent mAbs.
TABLE-US-00027 TABLE 18 EC.sub.50 (nM) binding to cells
E1-F405L-gp120-K409R (A431 cells) or M1- K409R-gp120-F405L EM1-mAb
Parental mAbs (H441 cells) A431 (assay for 9.6 .+-. 3 5.1 .+-. 0.3
10.1 .+-. 0.6 EGFR binding) H441 (assay for 1.5 .+-. 0.7 0.65 .+-.
0.1 1.0 .+-. 0.3 c-Met binding)
Inhibition of Ligand Binding to the Receptor
[0665] The bispecific antibodies were tested for their ability to
block binding of EGF to EGFR extracellular domain and HGF to c-Met
extracellular domain in an ELISA assay. Recombinant human EGF R-Fc
(R&D Systems, Cat #: 344-ER-050) or human HGF (R&D Systems,
Cat #: 294-HGN-025/CF) was coated onto MSD HighBind plates (Meso
Scale Discovery, Gaithersburg, Md.) for 2 hr at room temperature.
MSD Blocker A buffer (Meso Scale Discovery, Gaithersburg, Md.) was
added to each well and incubated for 2 hr at room temperature.
Plates were washed three times with 0.1 M HEPES buffer, pH 7.4,
followed by the addition of a mixture of either fluorescently
labeled (MSD) EGF or biotinylated HGF proteins with different
concentrations of antibodies. Ruthenium-labeled EGF protein was
incubated for 30 min at RT with increasing concentrations of
different antibodies, from 1 nM to 4 .mu.M. After 2-hour incubation
with gentle shaking at room temperature, the plates were washed 3
times with 0.1M HEPES buffer (pH 7.4). MSD Read Buffer T was
diluted and dispensed and the signals were analyzed with a SECTOR
Imager 6000. The HGF inhibition assays were performed as the
EGF/EGFR inhibition assays except that 10 nM of biotinylated HGF
was incubated for 30 min at RT with increasing concentrations of
different antibodies, from 1 nM to 2 .mu.M.
[0666] EM1-mAb inhibited EGF binding to EGFR with an IC.sub.50
value of 10.7 nM.+-.1.2 in the presence of 50 nM EGF and with an
IC.sub.50 value of 10.6.+-.1.5 nM in the presence of 80 nM EGF. The
parental bivalent antibody inhibited EGF binding to EGFR with an
IC.sub.50 value of 0.14.+-.1.5 nM in the presence of 50 nM EGF and
with an IC.sub.50 value of 1.7.+-.1.4 nM in the presence of 80 nM
EGF. EM1 mAb had a weaker inhibition of EGF binding to the EGFR
extracellular domain because of the monovalent binding of EM1 mAb
as compared to the parental bivalent mAb.
[0667] EM1-mAb inhibited HGF binding to c-Met with an IC.sub.50
value of 29.9.+-.1.5 nM. The parental bivalent antibody inhibited
HGF binding to c-Met with and IC.sub.50 of 14.8.+-.1.6 nM. EM1 mAb
had a weaker inhibition of HGF binding to the cMet extracellular
domain because of the monovalent binding of EM1-mAb as compared to
the parental bivalent mAb.
Inhibition of EGF-Stimulated EGFR Phosphorylation and
HGF-Stimulated c-Met Phosphorylation
[0668] Antibodies were tested to determine IC.sub.50 values for
inhibition of EGFR and c-Met phosphorylation. Inhibition of
EGF-stimulated EGFR phosphorylation and HGF-stimulated c-Met
phosphorylation were assessed at varying antibody concentrations
(0.035-700 nM final) as described in Example 2 ("Inhibition of
EGF-Stimulated EGFR Phosphorylation") and Example 6 ("Inhibition of
HGF-Stimulated c-Met Phosphorylation"). In some experiments, both
EGF and HGF were added to the cells so the same cell lysate could
be used to detect both EGFR and c-Met phosphorylation.
[0669] The control anti-EGFR mAb E1-F405L-gp120-K409R monovalent
for EGFR and the parental bivalent anti-EGFR antibody with low
fucose content were used as control antibodies. Table 19 shows the
IC.sub.50 values of the assays.
TABLE-US-00028 TABLE 19 pEGFR (IC.sub.50, nM) pMet (IC.sub.50, nM)
Cell line Antibody H292 H1975 H292 H1975 EM1-mAb 8.6-29 1.5
0.55-0.83 0.64 E1-F405L-gp120-K409R 10.9-13.1 ND 0.7-4.sup. ND
Parental EGFR 1.5 ND No effect ND (F405L)mAb* *Antibody had low
fucose content
Enhanced Inhibition of pERK and pAKT with EM1-mAb Compared to
Mixture of Monovalent Antibodies (mAb pERK Assay) (mAb pAKT
Assay)
[0670] The potential for enhanced potency with a bispecific
EGFR/c-Met antibody was evaluated by assessing mAb effects on pERK
and pAKT downstream signaling. For these experiments, the mixture
of monovalent control EGFR and monovalent control c-Met antibodies
was compared to the bispecific EM1-mAb. Cells were plated in clear
96-well tissue culture-treated plates (Nunc) in 100 .mu.L/well of
RPMI medium containing GlutaMAX and 25 mM Hepes (Invitrogen),
supplemented with 1 mM sodium pyruvate (Gibco), 0.1 mM NEAA
(Gibco), 10% heat inactivated fetal bovine serum (Gibco), and 7.5
ng/mL HGF (R&D Systems cat #294-HGN) and allowed to attach
overnight at 37.degree. C. in a humidified 5% CO.sub.2 atmosphere.
Cells were not serum-starved. Cells were treated for 30 min (pERK
assay) or 1 hour (pAkt assay) with varying concentrations (0.11-700
nM final) of monovalent control antibodies or EM1-mAb.
[0671] Cells were assessed for pERK or pAKT levels using the
following kits and according to manufacturer's instructions from
Meso Scale Discovery: Phospho-ERK1/2 (Thr202/Tyr204; Thr185/Tyr187)
Assay Whole Cell Lysate Kit (cat# K151DWD, Phospho-Akt (Ser473)
Assay Whole Cell Lysate Kit (cat#K151CAD), Phospho-Akt (Thr308)
Assay Whole Cell Lysate Kit (cat#K151DYD). For the pERK assay,
cells were lysed, and whole cell lysates were added to plates
coated with anti-phospho-ERK1/2 antibody (recognizing ERK1
phosphorylated at residues Thr202 and Tyr204 and ERK2
phosphorylated at residues Thr185 and Tyr187), and phosphorylated
ERK1/2 was detected with anti-total ERK1/2 antibody conjugated with
MSD SULFO-TAG.TM. reagent. For the pAKT Ser473 assay, the capture
antibody was anti-totalAKT antibody and the detection antibody
anti-pAKT Ser473 antibody conjugated with MSD SULFO-TAG.TM.
reagent. For the pAKT Thr308 assay, the capture antibody was
anti-totalAKT antibody and the detection antibody anti-pAKT Thr308
antibody conjugated with MSD SULFO-TAG.TM. reagent.
[0672] Plates were read on a SECTOR.RTM. Imager 6000 instrument
(Meso Scale Discovery) using manufacturer-installed assay-specific
default settings. Data were plotted as electrochemiluminescence
signal against the logarithm of antibody concentration and
IC.sub.50 values were determined by fitting the data to a sigmoidal
dose response with variable slope using GraphPad Prism 5 software.
NCI-H292, H1975 and SKMES-1 cell lines were used in these
assays.
[0673] The IC.sub.50 for inhibition of ERK phosphorylation by the
bispecific EM1-mAb was about 14-63 fold lower relative to the
mixture of the two monovalent control antibodies, depending on a
cell line tested (Table 20). The improved potency of the EM1-mAb
compared to the mixture of two monovalent control antibodies
suggests a cooperative or avidity effect due to enhanced binding of
EM1-mAb to these cell lines. The IC.sub.50 for inhibition of Ser475
(pAKTS475) and Thr308 (pAKTT308) AKT phosphorylation in NCI-H1975
cell line was about 75-fold and 122-fold lower, respectively, when
compared to the mixture of the two monovalent control antibodies
(Table 21). The improved potency of the EM1-mAb compared to the
mixture of two monovalent control antibodies suggests a cooperative
or avidity effect due to enhanced binding of EM1-mAb to these cell
lines. Thus, the bispecific nature of the EM1-mAb resulted in an
enhanced effect on downstream signaling effectors.
TABLE-US-00029 TABLE 20 IC.sub.50 (nM) pERK Antibody Fold change
Mixture of E1- bispecific vs. F405L-gp120- mixture of two K409R and
M1- monovalent Bispecific K409R-gp120- control Cell line EM1-mAb
F405L antibodies H292 0.64 34.94 55 H1975 1.67 106 63 SKMES-1 0.54
7.63 14
TABLE-US-00030 TABLE 21 IC.sub.50 (nM) IC.sub.50 (nM) Antibody
pAKTS473 pAKTT308 Bispecific EM1-mAb 0.87 0.96 Mixture of
E1-F405L-gp120- 65 117 K409R and M1-K409R-gp120- F405L Fold change
mixture of two 75 122 monovalent vs. bispecific
Inhibition of Human Tumor Cell Growth or Viability by
Antibodies
[0674] Inhibition of c-Met-dependent cell growth was assessed by
measuring viability of various tumor cells following exposure to
the bispecific EM1-mAb. NCI-H292, SKMES-1, NCI-H1975 and NCI-H3255
cells were used in the studies.
[0675] Cells were cultured in standard 2D and low attachment
formats. Erlotinib and cetuximab were used as controls. Table 22
summarizes the IC.sub.50 values for the assay.
Inhibition of Human Tumor Cell Growth or Viability by
Antibodies--Standard 2D Format
[0676] The inhibition of cell growth was assessed by measuring the
viability of NCI-H292 and NCI-H1975 following exposure to
antibodies in two formats. For the standard 2D format cells were
plated in opaque white 96-well tissue culture-treated plates
(PerkinElmer) in RPMI medium containing GlutaMAX and 25 mM Hepes
(Invitrogen), supplemented with 1 mM sodium pyruvate (Gibco), 0.1
mM NEAA (Gibco), and 10% heat inactivated fetal bovine serum
(Gibco), and allowed to attach overnight at 37.degree. C., 5%
CO.sub.2. Cells were treated with varying concentrations of
antibodies (0.035-700 nM final), along with HGF (7.5 ng/mL, R&D
Systems cat #294-HGF), then incubated at 37.degree. C., 5% CO.sub.2
for 72 hours. Some wells were left untreated with either HGF or
antibodies as controls. Viable cells were detected using
CellTiter-Glo.RTM. reagent (Promega), and data were analyzed as
described in Example 3 in "Inhibition of Human Tumor Cell Growth
(NCI-H292 growth and NCI-H322 growth assay)".
Inhibition of Human Tumor Cell Growth or Viability by
Antibodies--Low Attachment Format
[0677] To assess survival in low attachment conditions, cells were
plated in Ultra Low Attachment 96-well plates (Corning Costar) in
50 .mu.L/well of RPMI medium (Invitrogen) containing GlutaMAX and
25 mM Hepes, supplemented with 1 mM sodium pyruvate (Gibco), 0.1 mM
NEAA (Gibco), and 10% heat inactivated fetal bovine serum (Gibco),
and allowed to attach overnight at 37.degree. C., 5% CO.sub.2.
Cells were treated with varying concentrations of antibodies
(0.035-700 nM final), along with HGF (7.5 ng/mL, R&D Systems
cat#294-HGN), then incubated at 37.degree. C., 5% CO.sub.2 for 72
hours. Some wells were left untreated with either HGF or antibodies
as controls. Viable cells were detected using CellTiter-Glo.RTM.
reagent (Promega), and data were analyzed as described above in
"Inhibition of Human Tumor Cell Growth (NCI-H292 growth and
NCI-H322 growth assay)" in Example 3, except that lysates were
transferred to opaque white 96-well tissue culture-treated plates
(PerkinElmer) prior to reading luminescence.
[0678] In the standard 2D culture, EM1-mAb inhibited NCI-H292
growth with an IC.sub.50 of 31 nM, and in low attachment conditions
with an IC.sub.50 of 0.64 nM. EM-1 mAb inhibited NCI-H1975 cell
growth with an IC.sub.50 of >700 nM and 0.8-1.35 nM in standard
2D and low attachment culture, respectively. In NCI-H292 cells
expressing both wild type EGFR and cMet, EM1-mAb had over 22 fold
improved potency in the standard 2D and about 330-fold improved
potency in the low attachment culture conditions when compared to
cetuximab. In NCI-H1975 cell, which express L858R, T790M EGFR
mutant and wild type cMet, EM-1 mAb had at least a 518-fold
improved potency when compared to cetuximab in low attachment
culture conditions. Table 22 shows the summary of the assays.
TABLE-US-00031 TABLE 22 EM1- Cetux- EM1- mAb imab mAb Low Cetuximab
Low Standard attach- Standard attach- 2D culture ment 2D culture
ment EGFR cMet IC.sub.50 IC.sub.50 IC.sub.50 IC.sub.50 Cell line
state state (nM) (nM) (nM) (nM) NCI-H292 WT WT 31 0.64 >700 212
NCI-H1975 L858R, WT >700 0.8-1.35 >700 >700 T790M
Combination of Erlotinib and EM1-mAb is Efficient in Inhibition of
Growth of EGFR Mutant Cell Lines
[0679] The inhibition of cell growth by the combination of
erlotinib plus EM1-mAb was evaluated in both standard 2D culture
conditions and in the low attachment format. NCI-H3255 and HCC-827
cells were plated as described above in "Inhibition of Human Tumor
Cell Growth or Viability by Antibodies". HGF (7.5 ng/mL, R&D
Systems cat #294-HGN) was added to cells along with treatment with
antibodies. Cells were treated with varying concentrations of
antibodies (0.11-700 nM final), or erlotinib (0.46-3000 nM final),
or the combination of erlotinib plus antibody, using increasing
amounts of each in a fixed ratio (e.g. lowest concentration of
combination=lowest concentration of antibody (0.11 nM)+lowest
concentration of erlotinib (0.46 nM)). Some wells were left
untreated with either HGF or antibodies as controls. Cells were
incubated at 37.degree. C., 5% CO.sub.2 for 72 hours, then viable
cells were detected using CellTiter-Glo.RTM. reagent (Promega), and
data were analyzed as described above in "Inhibition of Human Tumor
Cell Growth (NCI-H292 growth and NCI-H322 growth assay)". Table 23
summarizes the results of the experiment. In the table, the
IC.sub.50 values for the combinations are relative to either the
antibody, or erlotinib, depending on what is indicated in
parentheses.
[0680] In both NCI-H3255 and HCC-827 cells (EGFR mutant cell lines)
the addition of EM1-mAb to erlotinib both increased the potency of
inhibition of cell viability and was more effective resulting in
fewer viable cells overall. In the NCI-H3255 cells using standard
2D conditions, the IC.sub.50 for erlotinib alone was 122 nM,
whereas the combination was 49 nM. Similarly, in HCC-827 cells, the
IC.sub.50 for erlotinib alone was 27 nM, whereas the combination
was 15 nM. Also, the combination of erlotinib plus EM1-mAb was more
effective than the combination of erlotinib plus cetuximab. Thus,
in the presence of HGF, addition of EM1-mAb increased the
effectiveness of erlotinib in this assay.
[0681] NCI-H3255 cells express L858R mutant EGFR and amplified
cMet. HCC-827 cells express EGFR mutants with deletions at amino
acid positions 746 and 750 and wild type c-Met. EM1-mAb has
stronger effects in the viability of HCC-827 and NCI-3255 in the
presence of erlotinib than erlotinib alone in either standard or
low attachment cultures.
TABLE-US-00032 TABLE 23 EM1 mAb + erlotinib Samples erlotinib
IC.sub.50 (nM) IC.sub.50 (nM) NCI-H3255, standard 2D culture 49.0
122 NCI-H3255, low attachment culture 10.6 47.1 HCC-827, standard
2D culture 14.6 27.4 HCC-827, low attachment culture 3.5 9.5
Example 13. Antibody Mediated Cellular Cytotoxicity (ADCC) of
EM1-mAb in In Vitro Cell Lines
[0682] ADCC assays were performed as previously described (Scallon
et al., Mol Immunol 44:1524-1534 2007). Briefly, PBMCs were
purified from human blood by Ficoll gradients and used as effector
cells for ADCC assays. NCI-H292, NCI-H1975 or NCI-H441 cells were
used as target cells with a ratio of 1 target cell to 50 effector
cells. Target cells were pre-labeled with BATDA (PerkinElmer) for
20 minutes at 37.degree. C., washed twice and resuspended in DMEM,
10% heat-inactivated FBS, 2 mM L-glutamine (all from Invitrogen).
Target (1.times.10.sup.4 cells) and effector cells
(0.5.times.10.sup.6 cells) were combined and 100 .mu.l of cells
were added to the wells of 96-well U-bottom plates. An additional
100 .mu.l was added with or without wild type and
protease-resistant mAb constructs. All samples were performed in
duplicate. The plates were centrifuged at 200 g for 3 minutes,
incubated at 37.degree. C. for 2 hours, and then centrifuged again
at 200 g for 3 minutes. A total of 20 .mu.l of supernatant was
removed per well and cell lysis was measured by the addition of 200
.mu.l of the DELPHIA Europium-based reagent (PerkinElmer).
Fluorescence was measured using an Envision 2101 Multilabel Reader
(PerkinElmer). Data were normalized to maximal cytotoxicity with
0.67% Triton X-100 (Sigma Aldrich) and minimal control determined
by spontaneous release of BATDA from target cells in the absence of
any antibody using the following equation: (experimental
release-spontaneous release)/(maximal release-spontaneous
release).times.100%. Data were fit to a sigmoidal dose-response
model using GraphPad Prism v5.
[0683] The ADCC results for the EM1 mAbs and comparators are
summarized in the Table 24 (NCI-H292 cells), Table 25 (NCI-H1975
cells) and Table 26 (NCI-H441 cells) and Table 27 (NCI-H1993 cells)
list the EC.sub.50 values and maximum lysis achieved. NCI-H292
cells express wild type (WT) EGFR, WT c-Met, and WT KRAS; NCI-H1975
cells express mutant EGFR (L858R T790M), WT cMet and WT KRAS;
NCI-H441 express WT EGFR, WT cMet, and mutant KRAS (G12V), and
NCI-H1993 cells express WT EGFR, amplified cMet, WT KRAS. KRAS is
also known as GTPase KRas and as V-Ki-ras2 Kirsten rat sarcoma
viral oncogene homolog.
[0684] The EM1-mAb has higher potency of ADCC responses than
cetuximab and the normal fucose version of EM1-mAb as indicated by
having lower EC.sub.50 values. The EM1 mAb has higher efficacy in
terms of maximum lysis achieved than cetuximab and the normal
fucose bispecific mAb. From profiles of on Tables 24-27, the EM-1
mAb has ADCC activity on cells that have mutant and WT EGFR, WT
with normal and amplified levels of cMet, and WT and mutant
KRAS.
TABLE-US-00033 TABLE 24 Potency (EC.sub.50 Efficacy (maximum mAb
.mu.g/ml) R.sup.2 lysis achieved) EM1 mAb 0.0058 0.93 19% Anti-EGFR
x cMet 0.22 0.85 13% normal fucose bispecific mAb Cetuximab 0.0064
0.94 12%
TABLE-US-00034 TABLE 25 Potency (EC.sub.50 Efficacy (maximum mAb
.mu.g/ml) R.sup.2 lysis achieved) EM1 mAb 0.022 0.91 19% Anti-EGFR
x cMet 1.8 0.79 13% normal fucose bispecific mAb Cetuximab 0.029
0.70 11%
TABLE-US-00035 TABLE 26 Potency (EC.sub.50 Efficacy (maximum mAb
.mu.g/ml) R.sup.2 lysis achieved) EM1 mAb 0.022 0.97 24% Anti-EGFR
x cMet 0.52 0.87 7.9% normal fucose bispecific mAb Cetuximab 0.013
0.85 15%
TABLE-US-00036 TABLE 27 Potency (EC.sub.50 Efficacy (maximum mAb
.mu.g/ml) R.sup.2 lysis achieved) EM1 mAb 0.0013 0.95 27% Anti-EGFR
x cMet 0.054 0.87 17% normal fucose bispecific mAb Cetuximab 0.0042
0.76 21%
Example 14. Tumor Efficacy Studies with the EM1-mAb
[0685] The efficacy of the EM1 mAb against tumor growth was
conducted as described in Example 7 "Tumor efficacy studies with
bispecific EGFR/c-Met molecules". In brief, NCI-H292-HGF cells were
implanted subcutaneously (s.c.) with Cultrex at 2.times.10.sup.6
into female SCID Beige mice The mice were stratified by tumor
volume 7 days after implant into 5 Groups with 10 mice per group.
The dosing began after the starting mean tumor volume per group
ranged from 62-66 mm.sup.3 (small tumors). PBS or therapeutic were
dosed intraperitoneally (i.p.) 2 times per week.
[0686] The evaluation of the efficacy also employed SKMES-HGF, a
human squamous cell carcinoma that was transfected with human HGF
(hepatic growth factor). These cells were implanted s.c. at
10.times.10.sup.6 into female SCID Beige mice These mice were
stratified by tumor volume 12 days after implant into 5 groups with
8 mice per group. The first study began with starting mean tumor
volume per group ranged from 98-101 mm.sup.3 (large tumors). PBS or
therapeutic mAbs were-dosed i.p. 2.times./week for 4 weeks. In the
larger sized tumor study, the mice that were stratified after the
tumor volumes were about 200-300 mm.sup.3 by splitting into 2
groups. These mice were then treated with either cetuximab (20
mg/kg) or EM1-mAb (20 mg/kg), i.p., 2.times./week (3 weeks).
[0687] The summary of the data is shown in Table 28. FIG. 10 shows
the efficacy of the molecules over time. EM1-mAb has an improved
tumor suppression profile when compared to cetuximab in H292-HGF
small tumor model and in SKMES-HGF small and large tumor
models.
TABLE-US-00037 TABLE 28 Dosing at Partial Complete Sample and time
Cell line mg per kg regression regression EM1 at day 35 H292-HGF
small 20 10/10 10/10 tumor 5 10/10 10/10 1 0/10 0/10 Cetuximab at
day H292-HGF small 20 0/10 0/10 35 tumor EM1 at day 67 SKMES - HGF
20 0/8 8/8 small tumor 5 1/8 6/8 1 2/8 4/8 Cetuximab at day 20 0/8
6/8 67 EM1 at day 70 SKMES - HGF 20 4/7 3/7 Cetuximab at day large
tumor 20 0/7 0/7 35
Table 29 shows the tumor sizes in treatment groups from the
SKMES-HGF tumors, and table 30 shows the anti-tumor activity.
[0688] EM1-mAb inhibited tumor growth in the SKMES-HGF model 97% or
more at multiple doses down to 1 mg/kg. While initially cetuximab
was very effective (88% TGI at 20 mg/kg), after dosing ended the
cetuximab-treated tumors grew back. In contrast, the tumors treated
with EM1-mAb at either 5 or 20 mg/kg did not grow back over the
course of the study (>2 months).
TABLE-US-00038 TABLE 29 Tumor volume (mm.sup.3) bispecific
bispecific bispecific EM1 at EM1 at EM1 at Cetuximab at Days
Vehicle 20 mg/kg 5 mg/kg 1 mg/kg 20 mg/kg 1 99 .+-. 6 99 .+-. 7 101
.+-. 6 101 .+-. 6 98 .+-. 5 8 146 .+-. 14 48 .+-. 10 49 .+-. 9 49
.+-. 10 60 .+-. 8 15 192 .+-. 21 9 .+-. 1 22 .+-. 10 41 .+-. 13 44
.+-. 23 22 326 .+-. 43 3 .+-. 2 17 .+-. 12 33 .+-. 15 42 .+-. 23 29
577 .+-. 55 2 .+-. 1 15 .+-. 9 38 .+-. 17 85 .+-. 60 36 994 .+-.
114 0.2 .+-. 0.1 13 .+-. 9 26 .+-. 14 125 .+-. 62 50 -- 0.04 .+-.
0.04 10 .+-. 7 18 .+-. 9 423 .+-. 115 57 -- 0.1 .+-. 0.2 3 .+-. 2
21 .+-. 10 650 .+-. 116 67 -- 0 .+-. 0 8 .+-. 7 34 .+-. 22 1257
.+-. 151
TABLE-US-00039 TABLE 30 Tumor Size T-C (mm.sup.3)a at (days) at
Treatment day 36 T/C (%) 1000 mm.sup.3 P value Vehicle 994 .+-. 114
-- -- -- bispecific EM1 at 0.19 .+-. 0.12 0.02 -- 20 mg/kg
bispecific EM1 at 13 .+-. 9 1.3 -- 5 mg/kg bispecific EM1 at 26
.+-. 14 2.6 -- 1 mg/kg Cetuximab 125 .+-. 62 13 31 (20 mg/kg)
Example 15. Inhibition of Cell Migration with EM1-mAb In Vitro
Method
[0689] Effect of the EM-mAb and the control monovalent antibodies
on inhibition of tumor cell migration was assessed in NIH-1650
cells. EGFR mutant cell line H1650 (Lung Bronchioloalveolar
carcinoma cells harboring an exon 19 mutation [deletion E746,
A7501]) was cultured in tissue culture flasks under normal culture
conditions (37.degree. C., 5% CO.sub.2, 95% humidity). All media
and supplementation were as suggested by the supplier of the cells
(American Type Culture Collection, Manassas, Va., USA).
[0690] Spheroids were generated by plating H1650 lung tumor cells
at 10,000 cells/well into "U" bottom Ultra Low Adherence (ULA)
96-well plates (Corning, Tewksbury, USA) at 200 .mu.l/well. These
plates stimulate spontaneous formation of a single spheroid of
cells within 24 hours (upon incubation at 37.degree. C., 5%
CO.sub.2) and the spheroids were grown for four days under normal
culture conditions.
[0691] Round bottom 96-well plates (BD Bioscience) were coated with
0.1% gelatin (EMD Millipore, Billerica, USA) in sterile water for 1
h at 37.degree. C. For compound evaluation studies, day 4 10,000
cell tumor spheroids (H1650 and NCI-H1975) were transferred to the
coated round bottom plates and treated with the EM1-mAb, the
control monovalent anti-EGFR mAb E1-F405L-120-K409R having low
fucose content, the control monovalent anti-cMet mAb
M1-K409R-gp120-F405L having low fucose content, and a combination
of the two monovalent antibodies E1-F405L-120-K409R and
M1-K409R-gp120-F405L (produced in low fucose) in a dilution series
with 20 ng/ml of HGF (R&D systems). Controls were treated with
vehicle which was IgG.sub.1 kappa isotype control (concentration
equal to highest drug-treated cells). Effects of compounds were
analyzed at 48 hrs by measuring the area covered by migrating cells
using bright field images in a fully automated Operetta high
content imaging system (Perkin Elmer) with a 2.times. objective.
Inhibition of cell migration (total area) due to treatment effect
was assessed by normalizing data by dividing by media only control
to create a percentage cell migration to control. Thus, a value
less than 1 would be inhibitory to cell migration.
Results
[0692] The EM1-mAb demonstrated potent synergistic inhibition of
cell migration in H1650 (L858R EGFR mutant) and H1975 (L858R/T790M
EGFR mutant) cells when compared to a combination of the control
monovalent anti-EGRF and anti-c-Met antibodies E1-F405L-120-K409R
and M1-K409R-120-F405L. In H1650 cells, the six highest
concentrations of the EM1-mAb significantly inhibited cell
migration (p<0.001) compared to the isotype control. The
EC.sub.50 value for the EM1-mAb was 0.23 nM, whereas the EC.sub.50
value for the combination of the monospecific control antibodies
was 4.39 nM. The EM1-mAb therefore was about 19 fold more efficient
in inhibiting H1650 cell migration when compared to the combination
of the monovalent control antibodies. The level of cell migration
inhibition of EM1-mAb was superior to the combination of
monospecific control mAbs for H1650 and H1975 cells. Table 31 shows
the EC.sub.50 values for the assay.
TABLE-US-00040 TABLE 31 H1650 H1975 EC.sub.50 Inhibition at
Inhibition at Samples (nM) 30 nM 30 nM EM1-mAb 0.23 64% 38% Mixture
of E1-F405L-gp120-K409R* 4.39 59% 20% and M1-K409R-gp120-F405L*
E1-F405L-gp120-K409R* 5.44 15% 7% M1-K409R-gp120-F405L* 7.36 43 10%
*antibodies have low fucose content
Example 16. Epitope Mapping of Anti-c-Met Antibody 069 and 5D5
[0693] The anti-c-Met mAb 069 binding epitope was mapped using the
linear and constrained CLIPS peptide technology. The peptides
scanned the SEMA, PSI, and Ig domains of human cMet. The linear and
CLIPS peptides were synthesized using the amino acid sequence of
the aforementioned cMet using standard Fmoc chemistry and
deprotected using trifluoric acid with scavengers. The constrained
peptides were synthesized on chemical scaffolds in order to
reconstruct conformational epitopes using Chemically linked
Peptides on Scaffolds (CLIPS) Technology (Timmerman et al., J Mol
Recognition 20:283, 2007). The linear and constrained peptides were
coupled to PEPSCAN cards and screened using a PEPSCAN based ELISA
(Slootstra et al., Molecular Diversity 1, 87-96, 1996). The
anti-c-Met mab 069 binding epitope is a discontinuous epitope
consisting of c-Met amino acids 239-253 PEFRDSYPIKYVHAF (SEQ ID NO:
238) and 346-361 FAQSKPDSAEPMDRSA (SEQ ID NO: 239). c-Met amino
acid sequence is shown in SEQ ID NO: 201.
[0694] Similar methods were used to map mAb 5D5 (MetMab,
Onartuzumab) epitope. mAb 5D5 binds c-Met residues 325-340
PGAQLARQIGASLNDD (SEQ ID NO: 240).
Example 17. In Vivo Tumor Efficacy Studies with EM1-mAb
[0695] The efficacy of EM1 mAb against tumor growth was conducted
as described in Example 7 "Tumor efficacy studies with bispecific
EGFR/c-Met molecules" and Example 14 employing additional tumor
cell lines with EGFR mutation or EGFR and/or c-Met amplifications.
In brief, SNU-5, H1975, HCC827 cells, H1975 cells expressing human
HGF, or a clone of HCC827 cells selected for its increased
resistance to erlotinib (HCC827-ER1 cells) were implanted
subcutaneously (s.c.) into female nude mice, except that SNU-5
cells were implanted in CR17/SCID mice. Mice were dosed
intraperitoneally with PBS or EM1-mAb, cetuximab (CAS 205923-56-4),
erlotinib (CAS 183321-74-6), afatinib (CAS 439081-18-2), or a
combination of EM-1 mAb and afatinib and EM-1 mAb and erlotinib at
indicated dosage and schedule shown in Table 32. Antitumor efficacy
was measured as % TGI (tumor growth inhibition) calculated as 100-%
T/C (T=mean tumor size of the treatment group; C=mean tumor size of
the control group on a given day as described in Example 7).
[0696] In tumors with primary EGFR activating mutations (no
resistance to EGFR TKIs): (HCC827 tumor, EGFR del(E746, A750)),
EM1-mAb dosed 10 mg/kg inhibited tumor growth by 82%. Erlotinib was
similarly effective in this model, as was the combination of
erlotinib and EM1-mAb. FIG. 11 shows efficacy of the therapeutics
over time in the HCC827 tumor model.
[0697] In tumors with wild type EGFR and c-Met gene amplification
(gastric cancer model SNU-5), EM1-mAb showed antitumor activity
with full tumor regression (98% TGI, at day 34 p<0.01, compared
to vehicle using one-way ANOVA followed by individual comparisons
using Games-Howell). Antitumor activity of anti-EGFR mAb cetuximab
was less, 49% at day 34, in this model. FIG. 12 shows the efficacy
of the therapeutics over time in the SNU-5 model.
[0698] EM1-mAb was tested in a NSCLC model containing primary EGFR
activating mutation and the T790M EGFR mutation which renders
tumors resistant to 1.sup.st generation EGFR TKIs (H1975 model).
EM1-mAb inhibited tumor growth with a 57% TGI in the H1975 cell
line model implanted in nude mice (p<0.0001, compared to PBS
vehicle using Logrank analysis with Prism 3.03). As expected,
erlotinib was not effective in this model with the T790M mutation.
Afatinib was equally effective as the EM1-mAb (57% TGI). Cetuximab
and the combination of EM1-mAb with afatinib were the most
effective, regressing tumors with 91% and 96% tumor growth
inhibition, respectively, (p<0.0001 for both cetuximab compared
to PBS and EM1-mAb+afatinib compared to the PBS+afatinib vehicles
group using Logrank analysis with Prism 3.03). c-Met signaling
pathways are not activated in this model as the mouse HGF does not
bind to human c-Met.
[0699] EM1-mAb was tested in several models that were engineered to
express human HGF using a lentiviral transduction system. This
allows modeling of ligand activation of the c-Met pathway in vivo
because mouse HGF does not activate the human c-Met on the
implanted human tumor cells. Results with SKMES-HGF model are shown
in Example 14 and FIG. 10, and the % TGI summarized in Table 32.
EM1-mAb inhibited tumor growth in the H1975-HGF model 71%
(p<0.0001, compared to PBS vehicle using Logrank analysis with
Prism 3.03). Afatinib, erlotinib and cetuximab were less
efficacious in this model. The combination of EM1-mAb and afatinib
was very effective (96% TGI, p<0.0001, compared to the
PBS+afatinib vehicles group using Logrank analysis with Prism
3.03). FIG. 13 shows the efficacy of the molecules over time in the
H1975-HGF model. Erlotinib, afatinib and cetuximab thus lose their
antitumor efficacy in tumor models in which c-Met pathway is
activated.
[0700] EM1-mAb was tested in a tumor model characterized by primary
EGFR activating mutation and increased resistance to 1.sup.st
generation EGFR TKI (erlotinib) due to c-Met gene amplification
(HCC827-ER1 model). EM1-mAb dosed at 10 mg/kg partially regressed
HCC827-ER1 tumors implanted with 86% TGI at day 25, and was more
efficacious than erlotinib alone (65% TGI at day 25). Combination
of EM1-mAb and erlotinib did not further improve efficacy. FIG. 14
shows the efficacy of the molecules over time.
[0701] EM1-mAb thus demonstrates efficacy in tumor models with wild
type EGFR, with primary activating EGFR mutations, with the EGFR
mutation T790M associated with resistance to EGFR therapeutics, as
well as in models where c-Met is activated in either a
ligand-dependent (autocrine HGF expression) or ligand-independent
(c-Met gene amplification) manner Combination of EM1-mAb with
erlotinib or afatinib may improve efficacy in some tumor
models.
TABLE-US-00041 TABLE 32 % TGI (day Treatment of study); (dose in
mg/kg), compared to Tumor Type EGFR cMet schedule PBS vehicle
SKMES-HGF WT WT EM1-mAb (20), 100 (36) lung BIWx4wk squamous
cetuximab (20), 88 (36) BIWx4wk SNU-5 WT AMP EM1-mAb (10), 98 (34)
gastric BIWx4wk cetuximab (10), 49 (34) BIWx4wk H1975 L858R; WT
EM1-mAb (10), 57 (18) NSCLC T790M BIWx3wk cetuximab (10), 91 (18)
BIWx3wk erlotinib (50), QDx21d 9 (18) afatinib (15), QDx21d 57 (18)
EM1-mAb (10), 96 (18) BIWx3wk + afatinib (15), QDx21d H1975-HGF
L858R; WT EM1-mAb (10), 71 (16) NSCLC T790M BIWx3wk cetuximab (10),
42 (16) BIWx3wk erlotinib (50), QDx21d 20 (16) afatinib (15),
QDx21d 29 (16) EM1-mAb (10), 96 (16) BIWx3wk + afatinib (15),
QDx21d HCC827 del (E746, WT EM1-mAb (10), 82 (35) NSCLC A750);
BIWx4wk AMP erlotinib (25), QDx28d 79 (35) EM1-mAb (10), 78 (35)
BIWx3wk + erlotinib (25), QDx28d HCC827-ER1 del (E746, AMP EM1-mAb
(10), 86 (25) NSCLC A750); BIWx4wk AMP erlotinib (25), QDx28d 65
(25) EM1-mAb (10), 87 (25) BIWx3wk + erlotinib (25), QDx28d BIW =
biweekly QD = once per day WT = wild type AMP = amplified
Example 18. EM1 mAb Induced Degradation of EGFR and c-Met In
Vivo
[0702] To demonstrate engagement of both EGFR and c-Met by EM1-mAb
in the tumor, samples were taken from H1975-HGF tumors at various
times after a single dose of 20 mg/kg EM1-mAb Tumor lysates were
prepared, normalized to total protein, and samples run on SDS-PAGE
gels. Gels were transferred to nitrocellulose and Western blotted
for either EGFR (Mouse (mAb) Anti-human EGFR (EGF-R2); Santa Cruz
Biotechnology, Cat# sc-73511) or c-Met (Mouse (mAb) Anti-human Met
(L41G3); Cell Signaling Technology, Cat#3148). EGFR levels were
normalized to GAPDH; c-Met levels were normalized to actin. The
levels of receptors from EM1-mAb treated tumors were compared to
those of PBS-treated tumors to get % total receptor. EM1-mAb
treatment decreased the total EGFR and cMet receptor levels in
H1975-HGF tumors to between 20% to 60% of control, depending on the
time point analyzed. FIG. 15 shows the average receptor levels
compared to PBS over time. pEGFR, pc-Met and pAKT were also
decreased at 72 hours after the single dose of EM1.
Example 19. Anti-Tumor Activity Comparing IgG.sub.1 and
IgG.sub.2.sigma. Variant Isoforms of EGFR/c-Met Bispecific
Antibodies
[0703] To better understand the contribution of effector function
to the efficacy observed in the H1975-HGF model, a comparison was
performed between EM1-mAb and a variant of EM1-mAb having an IgG2
Fc with effector silencing substitutions
V234A/G237A/P238S/H268A/V309L/A330S/P331S on IgG2 (substitutions
described in Intl. Pat. Appl. No. WO2011/066501) (numbering
according to the EU index). An IgG2 antibody with
V234A/G237A/P238S/H268A/V309L/A330S/P331S substitutions does not
interact with Fc receptors or effector cells (such as NK cells and
macrophages). Any loss of activity observed with the IgG2
V234A/G237A/P238S/H268A/V309L/A330S/P331S variant of the EM1-mAb
may thus represent antitumor activity contributed by
effector-mediated mechanisms such as ADCC and/or ADCP. After 32 day
post tumor cell implant in the H1975-HGF model described above,
there is an indication of loss of antitumor activity with the IgG2
V234A/G237A/P238S/H268A/V309L/A330S/P331S variant of the EM1-mAb
when compared to the parental EM1-mAb, suggesting that
effector-mediated mechanisms contribute to the function of EM-1
mAb. FIG. 16 shows the antitumor activity of the molecules.
Example 20. EGFR and c-Met Receptor Densities on Cells Correlate
with EM1-mAb Efficacy In Vitro
[0704] The role of EGFR and c-Met cell-surface densities in in
vitro efficacy of EM-1 mAb, particularly in the context of
heterogeneous cancer cell populations, was studied. In vitro
efficacy of EM-1 mAb was assessed by its ability to inhibit EGFR
and/or c-Met receptor phosphorylation as described in Example 12
"Inhibition of EGF-Stimulated EGFR Phosphorylation and
HGF-Stimulated c-Met Phosphorylation".
[0705] EGFR and c-Met expression levels were quantified in a panel
of tumor cell lines using Quantitative Flow Cytometry (QFCM) using
the control monovalent antibodies E1-F405L-gp120-K409R or
M1-K409R-gp120-F405L, EM-1 mAb or commercially available anti-EGFR
and c-Met antibodies. Tumor cell lines and their EGFR and c-Met
genotypes used in the study are shown in Table 33.
TABLE-US-00042 TABLE 33 EGFR cMet Cell Geno- Ampli- Geno- Ampli-
line Origin type fied* type fied* H292 Lung WT N WT N mucoepider-
moid SKMES-1 Lung WT N WT N squamous HCC827 Lung adeno del (E746, Y
(19) WT N A750) H1975 Lung adeno L858R, T790M N WT N H3255 Lung
adeno L858R Y (12) WT N H1650 Lung del (E746, Y (4) WT N Bronchi-
A750) oloalveolar HCC4006 Lung adeno del (L747, Y (5) WT N S752)
HCC2935 Lung adeno del (E746, N WT N A750) H820 Lung adeno del
(E746, UNK WT Y (4-6) A750), T790M H1993 Lung adeno WT UNK WT Y
(UNK) SNU-5 Stomach WT N WT Y (9) adeno WT: wild-type;; del:
deletion mutant; Lung adeno: Lung adenocarcinoma, UNK: Unknown, Y:
Yes, N: No *Value in parenthesis indicates the number of gene
copies per cell
Cell Culture
[0706] Tumor cell lines were cultured in 150 cm.sup.2 tissue
culture flasks under standard culture conditions (37.degree. C., 5%
CO.sub.2, 95% humidity). Media formulations used were as
recommended by the supplier of the cells (ATCC, Manassas, Va.,
USA). All cells were supplemented with 0.1 mM Non-Essential Amino
Acids (Life Technologies) and 1 mM Sodium Pyruvate (Life
Technologies) unless otherwise indicated. Media were routinely
changed two to three times weekly. When subconfluent, cell
monolayers were passaged using Accutase (Sigma-Aldrich, St Louis,
USA). Cells were harvested for receptor quantitation at
approximately 80% confluence and passage number was never allowed
to exceed p15. To dissociate cells for quantitation analysis,
enzyme-free Cellstripper (Corning) was used to avoid proteolysis of
surface antigens.
Conjugation of Antibodies with R-Phycoerythrin (RPE)
[0707] Antibodies were conjugated to RPE (Prozyme) using
heterobifunctional chemistry. The R-PE was activated using
sulfo-SMCC (Pierce) for 60 minutes. Activated R-PE was separated
from free sulfo-SMCC by gel filtration chromatography. The
antibodies were reduced using dithiothreitol (DTT, Sigma) for 30
minutes and separated from free DTT by gel filtration
chromatography. The activated R-PE was covalently coupled to the
reduced antibodies for 90 minutes. The reaction was quenched with
N-Ethylmaleimide (Fluka) for 20 minutes. The RPE-conjugated
antibodies were purified using a size-exclusion Tosoh TSKgel
G3000SW column in 100 mM sodium phosphate, 100 mM sodium sulfate,
0.05% sodium azide, pH 6.5 on an AKTA explorer FPLC system. The RPE
conjugated antibodies were stored at 2-8.degree. C. in light
occlusive containers. For analytical size-exclusion chromatography
the pooled fractions were applied to a Tosoh TSKgel G3000SWxl
column in 100 mM sodium phosphate, 100 mM sodium sulfate, 0.05%
sodium azide, pH6.5. The samples were quantified by UV absorbance
at 280 nm.
Flow Cytometry Staining
[0708] Immediately following dissociation the cells were assessed
for viability by exclusion of 0.4% Trypan Blue (Life Technologies,
Carlsbad, USA). Cell number and viability were calculated using a
C-chip hemocytometer (Incyto, Covington, USA). Cells were
resuspended at 1.times.10.sup.6 cells/mL in BSA Stain Buffer (BD
Biosciences, San Jose, USA), at which point strict adherence to
4.degree. C. was followed to minimize receptor internalization.
Fixation of cells was avoided due to the potential effects of
paraformaldehyde on antibody dissociation rates and/or epitope
integrity. FcRs (though not present on these cells) were blocked
with 5 .mu.l per test of Human TruStain FcX.TM. (BioLegend, San
Diego, USA) for 30 minutes at 4.degree. C. 1.times.10.sup.5 cells
per well were transferred to 96-well V-bottom microplates (Greiner,
Monroe, USA). Cells were incubated on ice for 1-2 hours with serial
dilutions of either in-house R-phycoerythrin labeled control
monovalent anti-EGFR mAb E1-F405L-gp120-K409R or control monovalent
anti-c-Met mAb M1-K409R-120-F405L or the bispecific EM1 mAb at
empirically determined saturating concentrations. Commercial
antibodies used in these studies included RPE-conjugated anti-human
HGFR/cMet (R&D systems, Minneapolis, USA), and anti-human EGFR
(BioLegend, San Diego, USA). Isotype matched controls included
RPE-conjugated mouse IgG1 Isotype control, (R&D systems,
Minneapolis, USA) and mouse IgG2bk isotype control (BioLegend, San
Diego, USA). These were included at the highest concentration used
for the respective receptor specific mAb. Cells were washed two
times with 150 .mu.L of BSA Stain Buffer and resuspended in 250
.mu.L of BSA Stain Buffer containing 1:50 diluted DRAQ7 live/dead
stain (Cell Signaling Technology, Danvers, USA). Single stain
controls for RPE and DRAQ7 were included. Samples were read on
either a BD FACSCaiibur (BD Biosciences) or Miltenyi MACSQUant flow
cytometer (Miltenyi Biotec, Auburn, USA). The data collection
channels for PE/DRAQ7 were: FL2/FL4 (FACSCalibur) and B2/P4
(MACSQuant). Live cells were gated according to DRAQ7 exclusion and
the geometric mean fluorescence intensity was determined for
>5,000 live events, collected at a low rate. FlowJo vX software
(FlowJo, Ashland, USA) was used for analysis.
Determination of Antibody Binding Capacity (ABC)
[0709] Receptor density values were reported as antibodies bound
per cell (ABC). ABC values were derived from standard. curves
generated with commercial microspheres coated with defined amounts
of a spectrally-matched fluorochrome, QuantiBRITE.TM. PE Beads,
[0710] To calculate ABC values, geometric means for the four
QuantiBRITE singlet bead populations were derived in FlowJo. Linear
regression was used to create a standard curve in GraphPad Prism
(GraphPad Software, La Jolla, USA) of the log [# of RPE
molecules/bead] versus the log [MFI]. R.sup.2 values were typically
.gtoreq.0.99. ABC values for the RPE-mAbs were then interpolated
from the QuantiBRITE standard curve. ABC values presented here are
the specific antibody binding sites (sABC) calculated by
subtracting the ABC values from an anti-RSV isotype-matched control
from the ABC of the EGFR or cMet mAbs.
[0711] The observed EGFR and c-Met densities using the antibodies
E1-F405L-gp120-K409R or M1-K409R-gp120-F405L in a panel of tumor
cell lines is shown in Table 34. The experiments were done in
duplicates in cell lines where S.E.M. is indicated in table, and as
single point m cell lines with no S.E.M. indicated.
TABLE-US-00043 TABLE 34 c-Met density EGFR density Cell Line Mean
S.E.M. Mean S.E.M. H1975 90867 5649 65523 1954 HCC827 204217 9413
419194 5874 H1650 58618 5374 101889 n.d. H3255 126692 46221 714987
n.d. H820 192226 4738 111323 1409 HCC2935 44768 n.d. 78669 n.d.
HCC4006 59616 n.d. 49339 n.d. H1993 564704 n.d. 340404 n.d. H292
63915 n.d. 365617 n.d. SKMES-1 47469 n.d. 174532 n.d. SNU-5 504140
n.d. 118721 n.d.
[0712] The IC.sub.50 values of the EM-1 mAb for inhibition of
phosphorylation of EGFR and c-Met was determined as described in
Example 12. The potency of inhibition of receptor phosphorylation
by the EM-1 mAb was inversely proportional to the receptor density
for both EGFR (FIG. 17A) and c-Met (FIG. 17B). The correlation of
inhibition of receptor phosphorylation to receptor quantitation was
statistically significant with Pearson correlation p=0.00082,
(r.sup.2=0.7820) for EGFR and p<0.0001 (r.sup.2=0.9336) for
c-Met.
Example 21. EM-1 Bispecific EGFR/c-Met mAb Heterodimerizes EGFR and
c-Met on the Cell Surface
[0713] The effect of the EM-1 mAb on EGFR and c-Met
heterodimerization was assessed as a mechanism for the observed
synergistic inhibition with the mAb.
Heterodimerization Assay Using Enzyme Fragment Complementation
[0714] For the EGFR/c-Met heterodimerization assay, complementary
inactive fragments of .beta.-galactosidase (.beta.-gal) were fused
to the C-terminus of c-Met and EGFR. Stably transfected human
osteosarcoma U-2 OS cell line (ATCC HTB-96.TM.) was generated
expressing both constructs, and enzyme fragment complementation was
carried out essentially as described in Wehrman et al., Proc Natl
Acad Sci USA 103: 19063-8, 2006. Induced heterodimerization of EGFR
and c-Met leads to association of the complementary .beta.-gal
fragments regenerating fully functional enzyme whose activity can
be monitored using a chemiluminescent substrate.
[0715] The generated cells were incubated with 12 point 1:3
serially diluted titrations of the control monovalent anti-EGFR
antibody E1-F405L-gp120-K409R, the control monovalent anti-c-Met
antibody M1-K409R-120-F405L, the combination of
E1-F405L-gp120-K409R and M1-K409R-gp120-F405L, EM-1 mAb, anti-cMet
parental mAb 069 or anti-EGFR parental mAb 2F8.
[0716] EM1 mAb produced a dose-dependent increase in
chemiluminescence in the engineered U-2 OS cells indicating the EM1
mAb induced heterodimerization of EGFR and c-Met (FIG. 18). Neither
the control monovalent antibodies nor parental bivalent antibodies
induced heterodimerization of EGFR and c-Met. Thus, the synergism
observed with the EM1 mAb may result from its ability to
heterodimerize EGFR and c-Met, and inhibit downstream signaling of
the heterodimer.
Example 22. The EM-1 Bispecific EGFR/c-Met mAb Synergistically
Inhibits EGFR or c-Met Phosphorylation
[0717] The potency of the bispecific anti-EGFR/c-Met EM-1 mAb to
block ligand-induced receptor phosphorylation relative to the
control monovalent anti-EGFR mAb E1-F405L-120-K409R or anti-c-Met
mAb M1-K409R-120-F405L, or a combination of the two was evaluated
in H292, H1993 and SNU-5 cell lines having different ratios of EGFR
to c-Met surface expression. The specific ABC values for EGFR and
c-Met in H292, H1993, and SNU-5 cells are reported in Table 33.
Inhibition of EGFR and c-Met phosphorylation was assessed as
described in Example 12.
[0718] The ratio of surface EGFR to c-Met was 5.72 in H292, 0.6 in
H1993 and 0.2 in SNU-5 cell line.
[0719] FIG. 19 shows the inhibition of ligand-induced c-Met (FIG.
19A) or EGFR (FIG. 19B) phosphorylation in H292 cells. The EM-1 mAb
inhibited c-Met phosphorylation with a 5-fold lower IC.sub.50 value
(IC.sub.50 0.80.+-.0.11 nM) when compared to the control
M1-K409R-gp120-F405L antibody (IC.sub.50 4.05.+-.0.21 nM) or a
combination of M1-K409R-gp120-F405L and E1-F405L-gp120-K409R. The
enhanced potency of the EM-1 mAb in inhibiting c-Met
phosphorylation may be a result of induced heterodimerization of
EGFR and c-Met due to high EGFR expression in this cell line. The
IC.sub.50 values for pEGFR inhibition were similar with the EM-1
mAb and the control monovalent E1-F405L-gp120-K409R. Thus, the
higher surface EGFR levels in this cell line drive synergistic
inhibition of c-Met signaling by the EM1 mAb. There was no change
in the potency of the EM-1 mAb in blocking EGFR receptor
phosphorylation relative to the E1-F405L-120-K409R alone.
[0720] FIG. 20 shows the inhibition of ligand-induced EGFR
phosphorylation in SNU-5 cells. The ratio of EGFR to c-Met ABC in
SNU-5 was 0.2.
[0721] EM1 mAb inhibited EGFR phosphorylation more potently
(IC.sub.50 12.2 nM) when compared to the E1-F405L-gp120-K409R (no
IC.sub.50 values was obtainable from the titration curve). Thus,
the higher surface c-Met expression levels in this cell line drive
synergistic inhibition of EGFR signaling by the EM1 mAb. There was
no change in the potency of the EM1 mAb in blocking c-Met
phosphorylation relative to M1-K409R-120-F405L alone.
[0722] FIG. 21 shows the inhibition of ligand-induced EGFR
phosphorylation in H1993 cells. The ratio of EGFR to c-Met ABC in
H1993 was 0.6.
[0723] EM1 mAb inhibited EGFR phosphorylation more potently when
compared to E1-F405L-120-K409R. Thus, the higher surface c-Met
expression levels also in this cell line drive synergistic
inhibition of EGFR signaling by the EM1 mAb. As in SNU-5 cell line,
there was no change in the potency of the EM-1 mAb in blocking
c-Met phosphorylation relative to M1-K409R-gp120-F405L alone.
Sequence CWU 1
1
240189PRTArtificial SequenceTencon FN3 scaffold 1Leu Pro Ala Pro
Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser 1 5 10 15 Leu Arg
Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Leu 20 25 30
Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Asn Leu Thr 35
40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro
Gly 50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val Lys Gly Gly
His Arg Ser 65 70 75 80 Asn Pro Leu Ser Ala Glu Phe Thr Thr 85
258DNAArtificial Sequenceprimer 2ggaaacagga tctaccatgc tgccggcgcc
gaaaaacctg gttgtttctg aagttacc 58321DNAArtificial Sequenceprimer
3aacaccgtag atagaaacgg t 214131DNAArtificial Sequenceprimer
4cggcggttag aacgcggcta caattaatac ataaccccat ccccctgttg acaattaatc
60atcggctcgt ataatgtgtg gaattgtgag cggataacaa tttcacacag gaaacaggat
120ctaccatgct g 131521DNAArtificial Sequenceprimer 5cggcggttag
aacgcggcta c 21666DNAArtificial
Sequenceprimermisc_feature(26)..(27)n is a, c, g, or
tmisc_feature(29)..(30)n is a, c, g, or tmisc_feature(32)..(33)n is
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tmisc_feature(38)..(39)n is a, c, g, or tmisc_feature(41)..(42)n is
a, c, g, or tmisc_feature(44)..(45)n is a, c, g, or t 6ggtggtgaat
tccgcagaca gcggsnnsnn snnsnnsnns nnsnnaacac cgtagataga 60aacggt
66769DNAArtificial Sequenceprimermisc_feature(26)..(27)n is a, c,
g, or tmisc_feature(29)..(30)n is a, c, g, or
tmisc_feature(32)..(33)n is a, c, g, or tmisc_feature(35)..(36)n is
a, c, g, or tmisc_feature(38)..(39)n is a, c, g, or
tmisc_feature(41)..(42)n is a, c, g, or tmisc_feature(44)..(45)n is
a, c, g, or tmisc_feature(47)..(48)n is a, c, g, or t 7ggtggtgaat
tccgcagaca gcggsnnsnn snnsnnsnns nnsnnsnnaa caccgtagat 60agaaacggt
69872DNAArtificial Sequenceprimermisc_feature(26)..(27)n is a, c,
g, or tmisc_feature(29)..(30)n is a, c, g, or
tmisc_feature(32)..(33)n is a, c, g, or tmisc_feature(35)..(36)n is
a, c, g, or tmisc_feature(38)..(39)n is a, c, g, or
tmisc_feature(41)..(42)n is a, c, g, or tmisc_feature(44)..(45)n is
a, c, g, or tmisc_feature(47)..(48)n is a, c, g, or
tmisc_feature(50)..(51)n is a, c, g, or t 8ggtggtgaat tccgcagaca
gcggsnnsnn snnsnnsnns nnsnnsnnsn naacaccgta 60gatagaaacg gt
72975DNAArtificial Sequenceprimermisc_feature(26)..(27)n is a, c,
g, or tmisc_feature(29)..(30)n is a, c, g, or
tmisc_feature(32)..(33)n is a, c, g, or tmisc_feature(35)..(36)n is
a, c, g, or tmisc_feature(38)..(39)n is a, c, g, or
tmisc_feature(41)..(42)n is a, c, g, or tmisc_feature(44)..(45)n is
a, c, g, or tmisc_feature(47)..(48)n is a, c, g, or
tmisc_feature(50)..(51)n is a, c, g, or tmisc_feature(53)..(54)n is
a, c, g, or t 9ggtggtgaat tccgcagaca gcggsnnsnn snnsnnsnns
nnsnnsnnsn nsnnaacacc 60gtagatagaa acggt 751080DNAArtificial
Sequenceprimermisc_feature(28)..(29)n is a, c, g, or
tmisc_feature(31)..(32)n is a, c, g, or tmisc_feature(34)..(35)n is
a, c, g, or tmisc_feature(37)..(38)n is a, c, g, or
tmisc_feature(40)..(41)n is a, c, g, or tmisc_feature(43)..(44)n is
a, c, g, or tmisc_feature(46)..(47)n is a, c, g, or
tmisc_feature(49)..(50)n is a, c, g, or tmisc_feature(52)..(53)n is
a, c, g, or tmisc_feature(55)..(56)n is a, c, g, or
tmisc_feature(58)..(59)n is a, c, g, or t 10rmggtggtga attccgcaga
cagcggsnns nnsnnsnnsn nsnnsnnsnn snnsnnsnna 60acaccgtaga tagaaacggt
801181DNAArtificial Sequenceprimermisc_feature(26)..(27)n is a, c,
g, or tmisc_feature(29)..(30)n is a, c, g, or
tmisc_feature(32)..(33)n is a, c, g, or tmisc_feature(35)..(36)n is
a, c, g, or tmisc_feature(38)..(39)n is a, c, g, or
tmisc_feature(41)..(42)n is a, c, g, or tmisc_feature(44)..(45)n is
a, c, g, or tmisc_feature(47)..(48)n is a, c, g, or
tmisc_feature(50)..(51)n is a, c, g, or tmisc_feature(53)..(54)n is
a, c, g, or tmisc_feature(56)..(57)n is a, c, g, or
tmisc_feature(59)..(60)n is a, c, g, or t 11ggtggtgaat tccgcagaca
gcggsnnsnn snnsnnsnns nnsnnsnnsn nsnnsnnsnn 60aacaccgtag atagaaacgg
t 811281DNAArtificial Sequenceprimer 12aagatcagtt gcggccgcta
gactagaacc gctgccatgg tgatggtgat ggtgaccgcc 60ggtggtgaat tccgcagaca
g 811330DNAArtificial Sequenceprimer 13cggcggttag aacgcggcta
caattaatac 301422DNAArtificial Sequenceprimer 14catgattacg
ccaagctcag aa 221565DNAArtificial Sequenceprimer 15gagccgccgc
caccggttta atggtgatgg tgatggtgac caccggtggt gaattccgca 60gacag
651637DNAArtificial Sequenceprimer 16aagaaggaga accggtatgc
tgccggcgcc gaaaaac 371789DNAArtificial Sequenceprimer 17tttgggaagc
ttctaggtct cggcggtcac catcaccatc accatggcag cggttctagt 60ctagcggccc
caactgatct tcaccaaac 891894PRTArtificial SequenceEGFR binding FN3
domain 18Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu
Asp Ser 1 5 10 15 Leu Arg Leu Ser Trp Ala Asp Pro His Gly Phe Tyr
Asp Ser Phe Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly
Glu Ala Ile Asn Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr
Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Thr Val Ser
Ile Tyr Gly Val His Asn Val Tyr Lys Asp 65 70 75 80 Thr Asn Met Arg
Gly Leu Pro Leu Ser Ala Glu Phe Thr Thr 85 90 1994PRTArtificial
SequenceEGFR binding FN3 domain 19Leu Pro Ala Pro Lys Asn Leu Val
Val Ser Glu Val Thr Glu Asp Ser 1 5 10 15 Leu Arg Leu Ser Trp Thr
Tyr Asp Arg Asp Gly Tyr Asp Ser Phe Leu 20 25 30 Ile Gln Tyr Gln
Glu Ser Glu Lys Val Gly Glu Ala Ile Asn Leu Thr 35 40 45 Val Pro
Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60
Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys Asp 65
70 75 80 Thr Asn Met Arg Gly Leu Pro Leu Ser Ala Glu Phe Thr Thr 85
90 2094PRTArtificial SequenceEGFR binding FN3 domain 20Leu Pro Ala
Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser 1 5 10 15 Leu
Arg Leu Ser Trp Gly Tyr Asn Gly Asp His Phe Asp Ser Phe Leu 20 25
30 Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Asn Leu Thr
35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys
Pro Gly 50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn
Val Tyr Lys Asp 65 70 75 80 Thr Asn Met Arg Gly Leu Pro Leu Ser Ala
Glu Phe Thr Thr 85 90 2194PRTArtificial SequenceEGFR binding FN3
domain 21Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu
Asp Ser 1 5 10 15 Leu Arg Leu Ser Trp Asp Asp Pro Arg Gly Phe Tyr
Glu Ser Phe Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly
Glu Ala Ile Asn Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr
Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Thr Val Ser
Ile Tyr Gly Val His Asn Val Tyr Lys Asp 65 70 75 80 Thr Asn Met Arg
Gly Leu Pro Leu Ser Ala Glu Phe Thr Thr 85 90 2294PRTArtificial
SequenceEGFR binding FN3 domain 22Leu Pro Ala Pro Lys Asn Leu Val
Val Ser Glu Val Thr Glu Asp Ser 1 5 10 15 Leu Arg Leu Ser Trp Thr
Trp Pro Tyr Ala Asp Leu Asp Ser Phe Leu 20 25 30 Ile Gln Tyr Gln
Glu Ser Glu Lys Val Gly Glu Ala Ile Asn Leu Thr 35 40 45 Val Pro
Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60
Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys Asp 65
70 75 80 Thr Asn Met Arg Gly Leu Pro Leu Ser Ala Glu Phe Thr Thr 85
90 2394PRTArtificial SequenceEGFR binding FN3 domain 23Leu Pro Ala
Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser 1 5 10 15 Leu
Arg Leu Ser Trp Gly Tyr Asn Gly Asp His Phe Asp Ser Phe Leu 20 25
30 Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Asn Leu Thr
35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys
Pro Gly 50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn
Val Tyr Lys Asp 65 70 75 80 Thr Asn Met Arg Gly Leu Pro Leu Ser Ala
Glu Phe Thr Thr 85 90 2495PRTArtificial SequenceEGFR binding FN3
domain 24Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu
Asp Ser 1 5 10 15 Leu Arg Leu Ser Trp Asp Tyr Asp Leu Gly Val Tyr
Phe Asp Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val
Gly Glu Ala Ile Asn Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser
Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val
Ser Ile Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Met
Arg Gly Leu Pro Leu Ser Ala Glu Phe Thr Thr 85 90 95
2594PRTArtificial SequenceEGFR binding FN3 domain 25Leu Pro Ala Pro
Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser 1 5 10 15 Leu Arg
Leu Ser Trp Asp Asp Pro Trp Ala Phe Tyr Glu Ser Phe Leu 20 25 30
Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Asn Leu Thr 35
40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro
Gly 50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val
Tyr Lys Asp 65 70 75 80 Thr Asn Met Arg Gly Leu Pro Leu Ser Ala Glu
Phe Thr Thr 85 90 2694PRTArtificial SequenceEGFR binding FN3 domain
26Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser 1
5 10 15 Leu Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe
Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile
Asn Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr
Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr Gly
Val Leu Gly Ser Tyr Val Phe 65 70 75 80 Glu His Asp Val Met Leu Pro
Leu Ser Ala Glu Phe Thr Thr 85 90 2794PRTArtificial SequenceEGFR
binding FN3 domain 27Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu
Val Thr Glu Asp Ser 1 5 10 15 Ala Arg Leu Ser Trp Asp Asp Pro Trp
Ala Phe Tyr Glu Ser Phe Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu
Lys Val Gly Glu Ala Ile Val Leu Thr 35 40 45 Val Pro Gly Ser Glu
Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr
Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys Asp 65 70 75 80 Thr
Asn Met Arg Gly Leu Pro Leu Ser Ala Ile Phe Thr Thr 85 90
2894PRTArtificial SequenceEGFR binding FN3 domain 28Leu Pro Ala Pro
Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser 1 5 10 15 Ala Arg
Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Leu 20 25 30
Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu Thr 35
40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro
Gly 50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val Leu Gly Ser
Tyr Val Phe 65 70 75 80 Glu His Asp Val Met Leu Pro Leu Ser Ala Ile
Phe Thr Thr 85 90 2994PRTArtificial SequenceEGFR binding FN3 domain
29Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser 1
5 10 15 Leu Arg Leu Ser Trp Thr Trp Pro Tyr Ala Asp Leu Asp Ser Phe
Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile
Asn Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr
Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr Gly
Val His Asn Val Tyr Lys Asp 65 70 75 80 Thr Asn Met Arg Gly Leu Pro
Leu Ser Ala Glu Phe Thr Thr 85 90 3037PRTArtificial Sequenceprimer
30Ala Ala Gly Ala Ala Gly Gly Ala Gly Ala Ala Cys Cys Gly Gly Thr 1
5 10 15 Ala Thr Gly Cys Thr Gly Cys Cys Gly Gly Cys Gly Cys Cys Gly
Ala 20 25 30 Ala Ala Ala Ala Cys 35 3165PRTArtificial
Sequenceprimer 31Gly Ala Gly Cys Cys Gly Cys Cys Gly Cys Cys Ala
Cys Cys Gly Gly 1 5 10 15 Thr Thr Thr Ala Ala Thr Gly Gly Thr Gly
Ala Thr Gly Gly Thr Gly 20 25 30 Ala Thr Gly Gly Thr Gly Ala Cys
Cys Ala Cys Cys Gly Gly Thr Gly 35 40 45 Gly Thr Gly Ala Ala Gly
Ala Thr Cys Gly Cys Ala Gly Ala Cys Ala 50 55 60 Gly 65
3289PRTArtificial Sequencec-Met binding FN3 domain 32Leu Pro Ala
Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp Ser 1 5 10 15 Ala
Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Trp 20 25
30 Ile Arg Tyr Asp Glu Val Val Val Gly Gly Glu Ala Ile Val Leu Thr
35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys
Pro Gly 50 55 60 Thr Glu Tyr Tyr Val Asn Ile Leu Gly Val Lys Gly
Gly Ser Ile Ser 65 70 75 80 Val Pro Leu Ser Ala Ile Phe Thr Thr 85
3389PRTArtificial Sequencec-Met binding FN3 domain 33Leu Pro Ala
Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp Ser 1 5 10 15 Ala
Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Phe 20 25
30 Ile Arg Tyr Asp Glu Phe Leu Arg Ser Gly Glu Ala Ile Val Leu Thr
35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys
Pro Gly 50 55 60 Thr Glu Tyr Trp Val Thr Ile Leu Gly Val Lys Gly
Gly Leu Val Ser 65 70 75 80 Thr Pro Leu Ser Ala Ile Phe Thr Thr 85
3489PRTArtificial Sequencec-Met binding FN3 domain 34Leu Pro Ala
Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp Ser 1 5 10 15 Ala
Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Trp 20 25
30 Ile Arg Tyr Phe Glu Phe Leu Gly Ser Gly Glu Ala Ile Val Leu
Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu
Lys Pro Gly 50 55 60 Thr Glu Tyr Ile Val Asn Ile Met Gly Val Lys
Gly Gly Ser Ile Ser 65 70 75 80 His Pro Leu Ser Ala Ile Phe Thr Thr
85 3589PRTArtificial Sequencec-Met binding FN3 domain 35Leu Pro Ala
Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp Ser 1 5 10 15 Ala
Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Trp 20 25
30 Ile Arg Tyr Phe Glu Phe Leu Gly Ser Gly Glu Ala Ile Val Leu Thr
35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys
Pro Gly 50 55 60 Thr Glu Tyr Val Val Asn Ile Leu Gly Val Lys Gly
Gly Gly Leu Ser 65 70 75 80 Val Pro Leu Ser Ala Ile Phe Thr Thr 85
3689PRTArtificial Sequencec-Met binding FN3 domain 36Leu Pro Ala
Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp Ser 1 5 10 15 Ala
Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Val 20 25
30 Ile Arg Tyr Phe Glu Phe Leu Gly Ser Gly Glu Ala Ile Val Leu Thr
35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys
Pro Gly 50 55 60 Thr Glu Tyr Val Val Gln Ile Leu Gly Val Lys Gly
Gly Tyr Ile Ser 65 70 75 80 Ile Pro Leu Ser Ala Ile Phe Thr Thr 85
3789PRTArtificial Sequencec-Met binding FN3 domain 37Leu Pro Ala
Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp Ser 1 5 10 15 Ala
Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Trp 20 25
30 Ile Arg Tyr Leu Glu Phe Leu Leu Gly Gly Glu Ala Ile Val Leu Thr
35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys
Pro Gly 50 55 60 Thr Glu Tyr Val Val Gln Ile Met Gly Val Lys Gly
Gly Thr Val Ser 65 70 75 80 Pro Pro Leu Ser Ala Ile Phe Thr Thr 85
3889PRTArtificial Sequencec-Met binding FN3 domain 38Leu Pro Ala
Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp Ser 1 5 10 15 Ala
Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Trp 20 25
30 Ile Arg Tyr Phe Glu Phe Leu Gly Ser Gly Glu Ala Ile Val Leu Thr
35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys
Pro Gly 50 55 60 Thr Glu Tyr Val Val Gly Ile Asn Gly Val Lys Gly
Gly Tyr Ile Ser 65 70 75 80 Tyr Pro Leu Ser Ala Ile Phe Thr Thr 85
3989PRTArtificial Sequencec-Met binding FN3 domain 39Leu Pro Ala
Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp Ser 1 5 10 15 Ala
Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Trp 20 25
30 Ile Arg Tyr Phe Glu Phe Leu Gly Ser Gly Glu Ala Ile Val Leu Thr
35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Asp Leu Lys
Pro Gly 50 55 60 Thr Glu Tyr Gly Val Thr Ile Asn Gly Val Lys Gly
Gly Arg Val Ser 65 70 75 80 Thr Pro Leu Ser Ala Ile Phe Thr Thr 85
4089PRTArtificial Sequencec-Met binding FN3 domain 40Leu Pro Ala
Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp Ser 1 5 10 15 Ala
Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Trp 20 25
30 Ile Arg Tyr Phe Glu Phe Leu Gly Ser Gly Glu Ala Ile Val Leu Thr
35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys
Pro Gly 50 55 60 Thr Glu Tyr Val Val Gln Ile Ile Gly Val Lys Gly
Gly His Ile Ser 65 70 75 80 Leu Pro Leu Ser Ala Ile Phe Thr Thr 85
4189PRTArtificial Sequencec-Met binding FN3 domain 41Leu Pro Ala
Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp Ser 1 5 10 15 Ala
Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Trp 20 25
30 Ile Arg Tyr Phe Glu Phe Leu Gly Ser Gly Glu Ala Ile Val Leu Thr
35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys
Pro Gly 50 55 60 Thr Glu Tyr Val Val Asn Ile Met Gly Val Lys Gly
Gly Lys Ile Ser 65 70 75 80 Pro Pro Leu Ser Ala Ile Phe Thr Thr 85
4289PRTArtificial Sequencec-Met binding FN3 domain 42Leu Pro Ala
Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp Ser 1 5 10 15 Ala
Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Trp 20 25
30 Ile Arg Tyr Phe Glu Phe Leu Gly Ser Gly Glu Ala Ile Val Leu Thr
35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys
Pro Gly 50 55 60 Thr Glu Tyr Ala Val Asn Ile Met Gly Val Lys Gly
Gly Arg Val Ser 65 70 75 80 Val Pro Leu Ser Ala Ile Phe Thr Thr 85
4389PRTArtificial Sequencec-Met binding FN3 domain 43Leu Pro Ala
Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp Ser 1 5 10 15 Ala
Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Trp 20 25
30 Ile Arg Tyr Phe Glu Phe Leu Gly Ser Gly Glu Ala Ile Val Leu Thr
35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys
Pro Gly 50 55 60 Thr Glu Tyr Val Val Gln Ile Leu Gly Val Lys Gly
Gly Ser Ile Ser 65 70 75 80 Val Pro Leu Ser Ala Ile Phe Thr Thr 85
4489PRTArtificial Sequencec-Met binding FN3 domain 44Leu Pro Ala
Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp Ser 1 5 10 15 Ala
Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Trp 20 25
30 Ile Arg Tyr Phe Glu Phe Leu Gly Ser Gly Glu Ala Ile Val Leu Thr
35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys
Pro Gly 50 55 60 Thr Glu Tyr Val Val Asn Ile Met Gly Val Lys Gly
Gly Ser Ile Ser 65 70 75 80 Tyr Pro Leu Ser Ala Ile Phe Thr Thr 85
4589PRTArtificial Sequencec-Met binding FN3 domain 45Leu Pro Ala
Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp Ser 1 5 10 15 Ala
Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Trp 20 25
30 Ile Arg Tyr Phe Glu Phe Leu Gly Ser Gly Glu Ala Ile Val Leu Thr
35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys
Pro Gly 50 55 60 Thr Glu Tyr Val Val Gln Ile Leu Gly Val Lys Gly
Gly Tyr Ile Ser 65 70 75 80 Ile Pro Leu Ser Ala Ile Phe Thr Thr 85
4689PRTArtificial Sequencec-Met binding FN3 domain 46Leu Pro Ala
Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp Ser 1 5 10 15 Ala
Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Trp 20 25
30 Ile Arg Tyr Phe Glu Phe Leu Gly Ser Gly Glu Ala Ile Val Leu Thr
35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys
Pro Gly 50 55 60 Thr Glu Tyr Val Val Gln Ile Met Gly Val Lys Gly
Gly Thr Val Ser 65 70 75 80 Pro Pro Leu Ser Ala Ile Phe Thr Thr 85
4789PRTArtificial Sequencec-Met binding FN3 domain 47Leu Pro Ala
Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp Ser 1 5 10 15 Ala
Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Trp 20 25
30 Ile Arg Tyr Phe Glu Phe Thr Thr Ala Gly Glu Ala Ile Val Leu Thr
35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys
Pro Gly 50 55 60 Thr Glu Tyr Val Val Asn Ile Met Gly Val Lys Gly
Gly Ser Ile Ser 65 70 75 80 Pro Pro Leu Ser Ala Ile Phe Thr Thr 85
4889PRTArtificial Sequencec-Met binding FN3 domain 48Leu Pro Ala
Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp Ser 1 5 10 15 Ala
Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Trp 20 25
30 Ile Arg Tyr Phe Glu Leu Leu Ser Thr Gly Glu Ala Ile Val Leu Thr
35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys
Pro Gly 50 55 60 Thr Glu Tyr Val Val Asn Ile Met Gly Val Lys Gly
Gly Ser Ile Ser 65 70 75 80 Pro Pro Leu Ser Ala Ile Phe Thr Thr 85
4989PRTArtificial Sequencec-Met binding FN3 domain 49Leu Pro Ala
Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp Ser 1 5 10 15 Ala
Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Trp 20 25
30 Ile Arg Tyr Phe Glu Phe Val Ser Lys Gly Glu Ala Ile Val Leu Thr
35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys
Pro Gly 50 55 60 Thr Glu Tyr Val Val Asn Ile Met Gly Val Lys Gly
Gly Ser Ile Ser 65 70 75 80 Pro Pro Leu Ser Ala Ile Phe Thr Thr 85
50205PRTArtificial Sequencebispecific EGFR/cMet binding molecule
50Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp 1
5 10 15 Ser Ala Arg Leu Ser Trp Asp Asp Pro Trp Ala Phe Tyr Glu Ser
Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala
Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu
Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile Tyr
Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Met Arg Gly Leu
Pro Leu Ser Ala Ile Phe Thr Thr Gly 85 90 95 Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 100 105 110 Gly Gly Ser
Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val 115 120 125 Thr
Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe 130 135
140 Asp Ser Phe Trp Ile Arg Tyr Asp Glu Val Val Val Gly Gly Glu Ala
145 150 155 160 Ile Val Leu Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp
Leu Thr Gly 165 170 175 Leu Lys Pro Gly Thr Glu Tyr Tyr Val Asn Ile
Leu Gly Val Lys Gly 180 185 190 Gly Ser Ile Ser Val Pro Leu Ser Ala
Ile Phe Thr Thr 195 200 205 51204PRTArtificial Sequencebispecific
EGFR/cMet binding molecule 51Met Leu Pro Ala Pro Lys Asn Leu Val
Val Ser Glu Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Asp
Asp Pro Trp Ala Phe Tyr Glu Ser Phe 20 25 30 Leu Ile Gln Tyr Gln
Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu 35 40 45 Thr Val Pro
Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly
Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys 65 70
75 80 Asp Thr Asn Met Arg Gly Leu Pro Leu Ser Ala Ile Phe Thr Thr
Gly 85 90 95 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly 100 105 110 Gly Gly Ser Leu Pro Ala Pro Lys Asn Leu Val
Val Ser Arg Val Thr 115 120 125 Glu Asp Ser Ala Arg Leu Ser Trp Thr
Ala Pro Asp Ala Ala Phe Asp 130 135 140 Ser Phe Trp Ile Arg Tyr Phe
Glu Phe Leu Gly Ser Gly Glu Ala Ile 145 150 155 160 Val Leu Thr Val
Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu 165 170 175 Lys Pro
Gly Thr Glu Tyr Val Val Asn Ile Met Gly Val Lys Gly Gly 180 185 190
Lys Ile Ser Pro Pro Leu Ser Ala Ile Phe Thr Thr 195 200
52205PRTArtificial Sequencebispecific EGFR/cMet binding molecule
52Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp 1
5 10 15 Ser Ala Arg Leu Ser Trp Asp Asp Pro Trp Ala Phe Tyr Glu Ser
Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala
Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu
Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile Tyr
Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Met Arg Gly Leu
Pro Leu Ser Ala Ile Phe Thr Thr Gly 85 90 95 Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 100 105 110 Gly Gly Ser
Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val 115 120 125 Thr
Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe 130 135
140 Asp Ser Phe Trp Ile Arg Tyr Phe Glu Phe Leu Gly Ser Gly Glu Ala
145 150 155 160 Ile Val Leu Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp
Leu Thr Gly 165 170 175 Leu Lys Pro Gly Thr Glu Tyr Val Val Gln Ile
Ile Gly Val Lys Gly 180 185 190 Gly His Ile Ser Leu Pro Leu Ser Ala
Ile Phe Thr Thr 195 200 205 53205PRTArtificial Sequencebispecific
EGFR/cMet binding molecule 53Met Leu Pro Ala Pro Lys Asn Leu Val
Val Ser Glu Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Asp
Asp Pro Trp Ala Phe Tyr Glu Ser Phe 20 25 30 Leu Ile Gln Tyr Gln
Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu 35 40 45 Thr Val Pro
Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly
Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys 65 70
75 80 Asp Thr Asn Met Arg Gly Leu Pro Leu Ser Ala Ile Phe Thr Thr
Gly 85 90 95 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly 100 105 110 Gly Gly Ser Met Leu Pro Ala Pro Lys Asn Leu
Val Val Ser Arg Val 115 120 125 Thr Glu Asp Ser Ala Arg Leu Ser Trp
Thr Ala Pro Asp Ala Ala Phe 130 135 140 Asp Ser Phe Phe Ile Arg Tyr
Asp Glu Phe Leu Arg Ser Gly Glu Ala 145 150 155
160 Ile Val Leu Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly
165 170 175 Leu Lys Pro Gly Thr Glu Tyr Trp Val Thr Ile Leu Gly Val
Lys Gly 180 185 190 Gly Leu Val Ser Thr Pro Leu Ser Ala Ile Phe Thr
Thr 195 200 205 54205PRTArtificial Sequencebispecific EGFR/cMet
binding molecule 54Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu
Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Ala Asp Pro His
Gly Phe Tyr Asp Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu
Lys Val Gly Glu Ala Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu
Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr
Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr
Asn Met Arg Gly Leu Pro Leu Ser Ala Ile Phe Thr Thr Gly 85 90 95
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 100
105 110 Gly Gly Ser Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg
Val 115 120 125 Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala Pro Asp
Ala Ala Phe 130 135 140 Asp Ser Phe Trp Ile Arg Tyr Phe Glu Phe Leu
Gly Ser Gly Glu Ala 145 150 155 160 Ile Val Leu Thr Val Pro Gly Ser
Glu Arg Ser Tyr Asp Leu Thr Gly 165 170 175 Leu Lys Pro Gly Thr Glu
Tyr Val Val Asn Ile Met Gly Val Lys Gly 180 185 190 Gly Lys Ile Ser
Pro Pro Leu Ser Ala Ile Phe Thr Thr 195 200 205 55205PRTArtificial
Sequencebispecific EGFR/cMet binding molecule 55Met Leu Pro Ala Pro
Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp 1 5 10 15 Ser Ala Arg
Leu Ser Trp Ala Asp Pro His Gly Phe Tyr Asp Ser Phe 20 25 30 Leu
Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu 35 40
45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro
50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val
Tyr Lys 65 70 75 80 Asp Thr Asn Met Arg Gly Leu Pro Leu Ser Ala Ile
Phe Thr Thr Gly 85 90 95 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly 100 105 110 Gly Gly Ser Met Leu Pro Ala Pro
Lys Asn Leu Val Val Ser Arg Val 115 120 125 Thr Glu Asp Ser Ala Arg
Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe 130 135 140 Asp Ser Phe Trp
Ile Arg Tyr Phe Glu Phe Leu Gly Ser Gly Glu Ala 145 150 155 160 Ile
Val Leu Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly 165 170
175 Leu Lys Pro Gly Thr Glu Tyr Val Val Gln Ile Ile Gly Val Lys Gly
180 185 190 Gly His Ile Ser Leu Pro Leu Ser Ala Ile Phe Thr Thr 195
200 205 56205PRTArtificial Sequencebispecific EGFR/cMet binding
molecule 56Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr
Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Ala Asp Pro His Gly Phe
Tyr Asp Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val
Gly Glu Ala Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser
Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val
Ser Ile Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Met
Arg Gly Leu Pro Leu Ser Ala Ile Phe Thr Thr Gly 85 90 95 Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 100 105 110
Gly Gly Ser Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val 115
120 125 Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala
Phe 130 135 140 Asp Ser Phe Trp Ile Arg Tyr Phe Glu Phe Leu Gly Ser
Gly Glu Ala 145 150 155 160 Ile Val Leu Thr Val Pro Gly Ser Glu Arg
Ser Tyr Asp Leu Thr Gly 165 170 175 Leu Lys Pro Gly Thr Glu Tyr Val
Val Gln Ile Ile Gly Val Lys Gly 180 185 190 Gly His Ile Ser Leu Pro
Leu Ser Ala Ile Phe Thr Thr 195 200 205 57194PRTArtificial
Sequencebispecific EGFR/cMet binding molecule 57Met Leu Pro Ala Pro
Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp 1 5 10 15 Ser Ala Arg
Leu Ser Trp Asp Asp Pro Trp Ala Phe Tyr Glu Ser Phe 20 25 30 Leu
Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu 35 40
45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro
50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val
Tyr Lys 65 70 75 80 Asp Thr Asn Met Arg Gly Leu Pro Leu Ser Ala Ile
Phe Thr Thr Ala 85 90 95 Pro Ala Pro Ala Pro Ala Pro Ala Pro Leu
Pro Ala Pro Lys Asn Leu 100 105 110 Val Val Ser Arg Val Thr Glu Asp
Ser Ala Arg Leu Ser Trp Thr Ala 115 120 125 Pro Asp Ala Ala Phe Asp
Ser Phe Trp Ile Arg Tyr Phe Glu Phe Leu 130 135 140 Gly Ser Gly Glu
Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg Ser 145 150 155 160 Tyr
Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr Val Val Asn Ile 165 170
175 Met Gly Val Lys Gly Gly Lys Ile Ser Pro Pro Leu Ser Ala Ile Phe
180 185 190 Thr Thr 58194PRTArtificial Sequencebispecific EGFR/cMet
binding molecule 58Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu
Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Asp Asp Pro Trp
Ala Phe Tyr Glu Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu
Lys Val Gly Glu Ala Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu
Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr
Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr
Asn Met Arg Gly Leu Pro Leu Ser Ala Ile Phe Thr Thr Ala 85 90 95
Pro Ala Pro Ala Pro Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu 100
105 110 Val Val Ser Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr
Ala 115 120 125 Pro Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Asp
Glu Val Val 130 135 140 Val Gly Gly Glu Ala Ile Val Leu Thr Val Pro
Gly Ser Glu Arg Ser 145 150 155 160 Tyr Asp Leu Thr Gly Leu Lys Pro
Gly Thr Glu Tyr Tyr Val Asn Ile 165 170 175 Leu Gly Val Lys Gly Gly
Ser Ile Ser Val Pro Leu Ser Ala Ile Phe 180 185 190 Thr Thr
59195PRTArtificial Sequencebispecific EGFR/cMet binding molecule
59Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp 1
5 10 15 Ser Ala Arg Leu Ser Trp Ala Asp Pro His Gly Phe Tyr Asp Ser
Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala
Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu
Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile Tyr
Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Met Arg Gly Leu
Pro Leu Ser Ala Ile Phe Thr Thr Ala 85 90 95 Pro Ala Pro Ala Pro
Ala Pro Ala Pro Met Leu Pro Ala Pro Lys Asn 100 105 110 Leu Val Val
Ser Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr 115 120 125 Ala
Pro Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu Phe 130 135
140 Leu Gly Ser Gly Glu Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg
145 150 155 160 Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr
Val Val Asn 165 170 175 Ile Met Gly Val Lys Gly Gly Lys Ile Ser Pro
Pro Leu Ser Ala Ile 180 185 190 Phe Thr Thr 195 60194PRTArtificial
Sequencebispecific EGFR/cMet binding molecule 60Met Leu Pro Ala Pro
Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp 1 5 10 15 Ser Ala Arg
Leu Ser Trp Ala Asp Pro His Gly Phe Tyr Asp Ser Phe 20 25 30 Leu
Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu 35 40
45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro
50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val
Tyr Lys 65 70 75 80 Asp Thr Asn Met Arg Gly Leu Pro Leu Ser Ala Ile
Phe Thr Thr Ala 85 90 95 Pro Ala Pro Ala Pro Ala Pro Ala Pro Leu
Pro Ala Pro Lys Asn Leu 100 105 110 Val Val Ser Arg Val Thr Glu Asp
Ser Ala Arg Leu Ser Trp Thr Ala 115 120 125 Pro Asp Ala Ala Phe Asp
Ser Phe Trp Ile Arg Tyr Asp Glu Val Val 130 135 140 Val Gly Gly Glu
Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg Ser 145 150 155 160 Tyr
Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr Tyr Val Asn Ile 165 170
175 Leu Gly Val Lys Gly Gly Ser Ile Ser Val Pro Leu Ser Ala Ile Phe
180 185 190 Thr Thr 61194PRTArtificial Sequencebispecific EGFR/cMet
binding molecule 61Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu
Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Asp Asp Pro Trp
Ala Phe Tyr Glu Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu
Lys Val Gly Glu Ala Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu
Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr
Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr
Asn Ile Arg Gly Leu Pro Leu Ser Ala Ile Phe Thr Thr Ala 85 90 95
Pro Ala Pro Ala Pro Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu 100
105 110 Val Val Ser Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr
Ala 115 120 125 Pro Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe
Glu Phe Leu 130 135 140 Gly Ser Gly Glu Ala Ile Val Leu Thr Val Pro
Gly Ser Glu Arg Ser 145 150 155 160 Tyr Asp Leu Thr Gly Leu Lys Pro
Gly Thr Glu Tyr Val Val Asn Ile 165 170 175 Leu Gly Val Lys Gly Gly
Lys Ile Ser Pro Pro Leu Ser Ala Ile Phe 180 185 190 Thr Thr
62194PRTArtificial Sequencebispecific EGFR/cMet binding molecule
62Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp 1
5 10 15 Ser Ala Arg Leu Ser Trp Asp Asp Pro Trp Ala Phe Tyr Glu Ser
Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala
Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu
Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile Tyr
Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Ile Arg Gly Leu
Pro Leu Ser Ala Ile Phe Thr Thr Ala 85 90 95 Pro Ala Pro Ala Pro
Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu 100 105 110 Val Val Ser
Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala 115 120 125 Pro
Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu Phe Val 130 135
140 Gly Ser Gly Glu Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg Ser
145 150 155 160 Tyr Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr Val
Val Asn Ile 165 170 175 Leu Gly Val Lys Gly Gly Ser Ile Ser Pro Pro
Leu Ser Ala Ile Phe 180 185 190 Thr Thr 63194PRTArtificial
Sequencebispecific EGFR/cMet binding molecule 63Met Leu Pro Ala Pro
Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp 1 5 10 15 Ser Ala Arg
Leu Ser Trp Asp Asp Pro Trp Ala Phe Tyr Glu Ser Phe 20 25 30 Leu
Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu 35 40
45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro
50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val
Tyr Lys 65 70 75 80 Asp Thr Asn Ile Arg Gly Leu Pro Leu Ser Ala Ile
Phe Thr Thr Ala 85 90 95 Pro Ala Pro Ala Pro Ala Pro Ala Pro Leu
Pro Ala Pro Lys Asn Leu 100 105 110 Val Val Ser Arg Val Thr Glu Asp
Ser Ala Arg Leu Ser Trp Thr Ala 115 120 125 Pro Asp Ala Ala Phe Asp
Ser Phe Trp Ile Arg Tyr Phe Glu Phe Val 130 135 140 Ser Lys Gly Asp
Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg Ser 145 150 155 160 Tyr
Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr Val Val Asn Ile 165 170
175 Leu Gly Val Lys Gly Gly Ser Ile Ser Pro Pro Leu Ser Ala Ile Phe
180 185 190 Thr Thr 64194PRTArtificial Sequencebispecific EGFR/cMet
binding molecule 64Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu
Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Asp Asp Pro Trp
Ala Phe Tyr Glu Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu
Lys Val Gly Glu Ala Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu
Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr
Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr
Asn Ile Arg Gly Leu Pro Leu Ser Ala Ile Phe Thr Thr Ala 85 90 95
Pro Ala Pro Ala Pro Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu 100
105 110 Val Val Ser Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr
Ala 115 120 125 Pro Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe
Glu Phe Leu 130 135 140 Gly Ser Gly Glu Ala Ile Val Leu Thr Val Pro
Gly Ser Glu Arg Ser 145 150 155 160 Tyr Asp Leu Thr Gly Leu Lys Pro
Gly Thr Glu Tyr Val Val Asn Ile 165 170 175 Leu Ser Val Lys Gly Gly
Ser Ile Ser Pro Pro Leu Ser Ala Ile Phe 180 185
190 Thr Thr 65194PRTArtificial Sequencebispecific EGFR/cMet binding
molecule 65Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr
Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Asp Asp Pro His Ala Phe
Tyr Glu Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val
Gly Glu Ala Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser
Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val
Ser Ile Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Ile
Arg Gly Leu Pro Leu Ser Ala Ile Phe Thr Thr Ala 85 90 95 Pro Ala
Pro Ala Pro Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu 100 105 110
Val Val Ser Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala 115
120 125 Pro Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu Phe
Leu 130 135 140 Gly Ser Gly Glu Ala Ile Val Leu Thr Val Pro Gly Ser
Glu Arg Ser 145 150 155 160 Tyr Asp Leu Thr Gly Leu Lys Pro Gly Thr
Glu Tyr Val Val Asn Ile 165 170 175 Leu Gly Val Lys Gly Gly Lys Ile
Ser Pro Pro Leu Ser Ala Ile Phe 180 185 190 Thr Thr
66194PRTArtificial Sequencebispecific EGFR/cMet binding molecule
66Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp 1
5 10 15 Ser Ala Arg Leu Ser Trp Asp Asp Pro His Ala Phe Tyr Glu Ser
Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala
Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu
Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile Tyr
Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Ile Arg Gly Leu
Pro Leu Ser Ala Ile Phe Thr Thr Ala 85 90 95 Pro Ala Pro Ala Pro
Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu 100 105 110 Val Val Ser
Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala 115 120 125 Pro
Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu Phe Val 130 135
140 Gly Ser Gly Glu Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg Ser
145 150 155 160 Tyr Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr Val
Val Asn Ile 165 170 175 Leu Gly Val Lys Gly Gly Ser Ile Ser Pro Pro
Leu Ser Ala Ile Phe 180 185 190 Thr Thr 67194PRTArtificial
Sequencebispecific EGFR/cMet binding molecule 67Met Leu Pro Ala Pro
Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp 1 5 10 15 Ser Ala Arg
Leu Ser Trp Asp Asp Pro His Ala Phe Tyr Glu Ser Phe 20 25 30 Leu
Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu 35 40
45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro
50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val
Tyr Lys 65 70 75 80 Asp Thr Asn Ile Arg Gly Leu Pro Leu Ser Ala Ile
Phe Thr Thr Ala 85 90 95 Pro Ala Pro Ala Pro Ala Pro Ala Pro Leu
Pro Ala Pro Lys Asn Leu 100 105 110 Val Val Ser Arg Val Thr Glu Asp
Ser Ala Arg Leu Ser Trp Thr Ala 115 120 125 Pro Asp Ala Ala Phe Asp
Ser Phe Trp Ile Arg Tyr Phe Glu Phe Val 130 135 140 Ser Lys Gly Asp
Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg Ser 145 150 155 160 Tyr
Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr Val Val Asn Ile 165 170
175 Leu Gly Val Lys Gly Gly Ser Ile Ser Pro Pro Leu Ser Ala Ile Phe
180 185 190 Thr Thr 68194PRTArtificial Sequencebispecific EGFR/cMet
binding molecule 68Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu
Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Asp Asp Pro His
Ala Phe Tyr Glu Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu
Lys Val Gly Glu Ala Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu
Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr
Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr
Asn Ile Arg Gly Leu Pro Leu Ser Ala Ile Phe Thr Thr Ala 85 90 95
Pro Ala Pro Ala Pro Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu 100
105 110 Val Val Ser Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr
Ala 115 120 125 Pro Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe
Glu Phe Leu 130 135 140 Gly Ser Gly Glu Ala Ile Val Leu Thr Val Pro
Gly Ser Glu Arg Ser 145 150 155 160 Tyr Asp Leu Thr Gly Leu Lys Pro
Gly Thr Glu Tyr Val Val Asn Ile 165 170 175 Leu Ser Val Lys Gly Gly
Ser Ile Ser Pro Pro Leu Ser Ala Ile Phe 180 185 190 Thr Thr
69194PRTArtificial Sequencebispecific EGFR/cMet binding molecule
69Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp 1
5 10 15 Ser Ala Arg Leu Ser Trp Ala Asp Pro His Gly Phe Tyr Asp Ser
Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala
Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu
Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile Tyr
Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Ile Arg Gly Leu
Pro Leu Ser Ala Ile Phe Thr Thr Ala 85 90 95 Pro Ala Pro Ala Pro
Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu 100 105 110 Val Val Ser
Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala 115 120 125 Pro
Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu Phe Leu 130 135
140 Gly Ser Gly Glu Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg Ser
145 150 155 160 Tyr Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr Val
Val Asn Ile 165 170 175 Leu Gly Val Lys Gly Gly Lys Ile Ser Pro Pro
Leu Ser Ala Ile Phe 180 185 190 Thr Thr 70194PRTArtificial
Sequencebispecific EGFR/cMet binding molecule 70Met Leu Pro Ala Pro
Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp 1 5 10 15 Ser Ala Arg
Leu Ser Trp Ala Asp Pro His Gly Phe Tyr Asp Ser Phe 20 25 30 Leu
Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu 35 40
45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro
50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val
Tyr Lys 65 70 75 80 Asp Thr Asn Ile Arg Gly Leu Pro Leu Ser Ala Ile
Phe Thr Thr Ala 85 90 95 Pro Ala Pro Ala Pro Ala Pro Ala Pro Leu
Pro Ala Pro Lys Asn Leu 100 105 110 Val Val Ser Arg Val Thr Glu Asp
Ser Ala Arg Leu Ser Trp Thr Ala 115 120 125 Pro Asp Ala Ala Phe Asp
Ser Phe Trp Ile Arg Tyr Phe Glu Phe Val 130 135 140 Gly Ser Gly Glu
Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg Ser 145 150 155 160 Tyr
Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr Val Val Asn Ile 165 170
175 Leu Gly Val Lys Gly Gly Ser Ile Ser Pro Pro Leu Ser Ala Ile Phe
180 185 190 Thr Thr 71194PRTArtificial Sequencebispecific EGFR/cMet
binding molecule 71Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu
Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Ala Asp Pro His
Gly Phe Tyr Asp Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu
Lys Val Gly Glu Ala Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu
Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr
Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr
Asn Ile Arg Gly Leu Pro Leu Ser Ala Ile Phe Thr Thr Ala 85 90 95
Pro Ala Pro Ala Pro Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu 100
105 110 Val Val Ser Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr
Ala 115 120 125 Pro Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe
Glu Phe Val 130 135 140 Ser Lys Gly Asp Ala Ile Val Leu Thr Val Pro
Gly Ser Glu Arg Ser 145 150 155 160 Tyr Asp Leu Thr Gly Leu Lys Pro
Gly Thr Glu Tyr Val Val Asn Ile 165 170 175 Leu Gly Val Lys Gly Gly
Ser Ile Ser Pro Pro Leu Ser Ala Ile Phe 180 185 190 Thr Thr
72194PRTArtificial Sequencebispecific EGFR/cMet binding molecule
72Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp 1
5 10 15 Ser Ala Arg Leu Ser Trp Ala Asp Pro His Gly Phe Tyr Asp Ser
Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala
Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu
Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile Tyr
Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Ile Arg Gly Leu
Pro Leu Ser Ala Ile Phe Thr Thr Ala 85 90 95 Pro Ala Pro Ala Pro
Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu 100 105 110 Val Val Ser
Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala 115 120 125 Pro
Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu Phe Leu 130 135
140 Gly Ser Gly Glu Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg Ser
145 150 155 160 Tyr Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr Val
Val Asn Ile 165 170 175 Leu Ser Val Lys Gly Gly Ser Ile Ser Pro Pro
Leu Ser Ala Ile Phe 180 185 190 Thr Thr 731210PRTHOMO SAPIENS 73Met
Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala 1 5 10
15 Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln
20 25 30 Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp
His Phe 35 40 45 Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val
Val Leu Gly Asn 50 55 60 Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr
Asp Leu Ser Phe Leu Lys 65 70 75 80 Thr Ile Gln Glu Val Ala Gly Tyr
Val Leu Ile Ala Leu Asn Thr Val 85 90 95 Glu Arg Ile Pro Leu Glu
Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr 100 105 110 Tyr Glu Asn Ser
Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn 115 120 125 Lys Thr
Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu 130 135 140
His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu 145
150 155 160 Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser
Asn Met 165 170 175 Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln
Lys Cys Asp Pro 180 185 190 Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala
Gly Glu Glu Asn Cys Gln 195 200 205 Lys Leu Thr Lys Ile Ile Cys Ala
Gln Gln Cys Ser Gly Arg Cys Arg 210 215 220 Gly Lys Ser Pro Ser Asp
Cys Cys His Asn Gln Cys Ala Ala Gly Cys 225 230 235 240 Thr Gly Pro
Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp 245 250 255 Glu
Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro 260 265
270 Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly
275 280 285 Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr
Asp His 290 295 300 Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr
Glu Met Glu Glu 305 310 315 320 Asp Gly Val Arg Lys Cys Lys Lys Cys
Glu Gly Pro Cys Arg Lys Val 325 330 335 Cys Asn Gly Ile Gly Ile Gly
Glu Phe Lys Asp Ser Leu Ser Ile Asn 340 345 350 Ala Thr Asn Ile Lys
His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp 355 360 365 Leu His Ile
Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr 370 375 380 Pro
Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu 385 390
395 400 Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr
Asp 405 410 415 Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg
Thr Lys Gln 420 425 430 His Gly Gln Phe Ser Leu Ala Val Val Ser Leu
Asn Ile Thr Ser Leu 435 440 445 Gly Leu Arg Ser Leu Lys Glu Ile Ser
Asp Gly Asp Val Ile Ile Ser 450 455 460 Gly Asn Lys Asn Leu Cys Tyr
Ala Asn Thr Ile Asn Trp Lys Lys Leu 465 470 475 480 Phe Gly Thr Ser
Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu 485 490 495 Asn Ser
Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro 500 505 510
Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn 515
520 525 Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu
Gly 530 535 540 Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln
Cys His Pro 545 550 555 560 Glu Cys Leu Pro Gln Ala Met Asn Ile Thr
Cys Thr Gly Arg Gly Pro 565 570 575 Asp Asn Cys Ile Gln Cys Ala His
Tyr Ile Asp Gly Pro His Cys Val 580 585 590 Lys Thr Cys Pro Ala Gly
Val Met Gly Glu Asn Asn Thr Leu Val Trp 595 600 605 Lys Tyr Ala Asp
Ala Gly His Val Cys His Leu Cys His Pro Asn Cys 610 615 620 Thr Tyr
Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly 625 630 635
640 Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu
645 650 655 Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg
Arg His 660 665 670 Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln
Glu Arg Glu Leu 675
680 685 Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu
Leu 690 695 700 Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val
Leu Gly Ser 705 710 715 720 Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu
Trp Ile Pro Glu Gly Glu 725 730 735 Lys Val Lys Ile Pro Val Ala Ile
Lys Glu Leu Arg Glu Ala Thr Ser 740 745 750 Pro Lys Ala Asn Lys Glu
Ile Leu Asp Glu Ala Tyr Val Met Ala Ser 755 760 765 Val Asp Asn Pro
His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser 770 775 780 Thr Val
Gln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp 785 790 795
800 Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn
805 810 815 Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp
Arg Arg 820 825 830 Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu
Val Lys Thr Pro 835 840 845 Gln His Val Lys Ile Thr Asp Phe Gly Leu
Ala Lys Leu Leu Gly Ala 850 855 860 Glu Glu Lys Glu Tyr His Ala Glu
Gly Gly Lys Val Pro Ile Lys Trp 865 870 875 880 Met Ala Leu Glu Ser
Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp 885 890 895 Val Trp Ser
Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser 900 905 910 Lys
Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu 915 920
925 Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr
930 935 940 Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg
Pro Lys 945 950 955 960 Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met
Ala Arg Asp Pro Gln 965 970 975 Arg Tyr Leu Val Ile Gln Gly Asp Glu
Arg Met His Leu Pro Ser Pro 980 985 990 Thr Asp Ser Asn Phe Tyr Arg
Ala Leu Met Asp Glu Glu Asp Met Asp 995 1000 1005 Asp Val Val Asp
Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe 1010 1015 1020 Phe Ser
Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu 1025 1030 1035
Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn 1040
1045 1050 Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln
Arg 1055 1060 1065 Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp
Ser Ile Asp 1070 1075 1080 Asp Thr Phe Leu Pro Val Pro Glu Tyr Ile
Asn Gln Ser Val Pro 1085 1090 1095 Lys Arg Pro Ala Gly Ser Val Gln
Asn Pro Val Tyr His Asn Gln 1100 1105 1110 Pro Leu Asn Pro Ala Pro
Ser Arg Asp Pro His Tyr Gln Asp Pro 1115 1120 1125 His Ser Thr Ala
Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln 1130 1135 1140 Pro Thr
Cys Val Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala 1145 1150 1155
Gln Lys Gly Ser His Gln Ile Ser Leu Asp Asn Pro Asp Tyr Gln 1160
1165 1170 Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn Gly Ile Phe
Lys 1175 1180 1185 Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val
Ala Pro Gln 1190 1195 1200 Ser Ser Glu Phe Ile Gly Ala 1205 1210
7453PRTHOMO SAPIENS 74Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp
Gly Tyr Cys Leu His 1 5 10 15 Asp Gly Val Cys Met Tyr Ile Glu Ala
Leu Asp Lys Tyr Ala Cys Asn 20 25 30 Cys Val Val Gly Tyr Ile Gly
Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45 Trp Trp Glu Leu Arg 50
752201PRTHomo sapiens 75Met Gly Ala Met Thr Gln Leu Leu Ala Gly Val
Phe Leu Ala Phe Leu 1 5 10 15 Ala Leu Ala Thr Glu Gly Gly Val Leu
Lys Lys Val Ile Arg His Lys 20 25 30 Arg Gln Ser Gly Val Asn Ala
Thr Leu Pro Glu Glu Asn Gln Pro Val 35 40 45 Val Phe Asn His Val
Tyr Asn Ile Lys Leu Pro Val Gly Ser Gln Cys 50 55 60 Ser Val Asp
Leu Glu Ser Ala Ser Gly Glu Lys Asp Leu Ala Pro Pro 65 70 75 80 Ser
Glu Pro Ser Glu Ser Phe Gln Glu His Thr Val Asp Gly Glu Asn 85 90
95 Gln Ile Val Phe Thr His Arg Ile Asn Ile Pro Arg Arg Ala Cys Gly
100 105 110 Cys Ala Ala Ala Pro Asp Val Lys Glu Leu Leu Ser Arg Leu
Glu Glu 115 120 125 Leu Glu Asn Leu Val Ser Ser Leu Arg Glu Gln Cys
Thr Ala Gly Ala 130 135 140 Gly Cys Cys Leu Gln Pro Ala Thr Gly Arg
Leu Asp Thr Arg Pro Phe 145 150 155 160 Cys Ser Gly Arg Gly Asn Phe
Ser Thr Glu Gly Cys Gly Cys Val Cys 165 170 175 Glu Pro Gly Trp Lys
Gly Pro Asn Cys Ser Glu Pro Glu Cys Pro Gly 180 185 190 Asn Cys His
Leu Arg Gly Arg Cys Ile Asp Gly Gln Cys Ile Cys Asp 195 200 205 Asp
Gly Phe Thr Gly Glu Asp Cys Ser Gln Leu Ala Cys Pro Ser Asp 210 215
220 Cys Asn Asp Gln Gly Lys Cys Val Asn Gly Val Cys Ile Cys Phe Glu
225 230 235 240 Gly Tyr Ala Gly Ala Asp Cys Ser Arg Glu Ile Cys Pro
Val Pro Cys 245 250 255 Ser Glu Glu His Gly Thr Cys Val Asp Gly Leu
Cys Val Cys His Asp 260 265 270 Gly Phe Ala Gly Asp Asp Cys Asn Lys
Pro Leu Cys Leu Asn Asn Cys 275 280 285 Tyr Asn Arg Gly Arg Cys Val
Glu Asn Glu Cys Val Cys Asp Glu Gly 290 295 300 Phe Thr Gly Glu Asp
Cys Ser Glu Leu Ile Cys Pro Asn Asp Cys Phe 305 310 315 320 Asp Arg
Gly Arg Cys Ile Asn Gly Thr Cys Tyr Cys Glu Glu Gly Phe 325 330 335
Thr Gly Glu Asp Cys Gly Lys Pro Thr Cys Pro His Ala Cys His Thr 340
345 350 Gln Gly Arg Cys Glu Glu Gly Gln Cys Val Cys Asp Glu Gly Phe
Ala 355 360 365 Gly Val Asp Cys Ser Glu Lys Arg Cys Pro Ala Asp Cys
His Asn Arg 370 375 380 Gly Arg Cys Val Asp Gly Arg Cys Glu Cys Asp
Asp Gly Phe Thr Gly 385 390 395 400 Ala Asp Cys Gly Glu Leu Lys Cys
Pro Asn Gly Cys Ser Gly His Gly 405 410 415 Arg Cys Val Asn Gly Gln
Cys Val Cys Asp Glu Gly Tyr Thr Gly Glu 420 425 430 Asp Cys Ser Gln
Leu Arg Cys Pro Asn Asp Cys His Ser Arg Gly Arg 435 440 445 Cys Val
Glu Gly Lys Cys Val Cys Glu Gln Gly Phe Lys Gly Tyr Asp 450 455 460
Cys Ser Asp Met Ser Cys Pro Asn Asp Cys His Gln His Gly Arg Cys 465
470 475 480 Val Asn Gly Met Cys Val Cys Asp Asp Gly Tyr Thr Gly Glu
Asp Cys 485 490 495 Arg Asp Arg Gln Cys Pro Arg Asp Cys Ser Asn Arg
Gly Leu Cys Val 500 505 510 Asp Gly Gln Cys Val Cys Glu Asp Gly Phe
Thr Gly Pro Asp Cys Ala 515 520 525 Glu Leu Ser Cys Pro Asn Asp Cys
His Gly Gln Gly Arg Cys Val Asn 530 535 540 Gly Gln Cys Val Cys His
Glu Gly Phe Met Gly Lys Asp Cys Lys Glu 545 550 555 560 Gln Arg Cys
Pro Ser Asp Cys His Gly Gln Gly Arg Cys Val Asp Gly 565 570 575 Gln
Cys Ile Cys His Glu Gly Phe Thr Gly Leu Asp Cys Gly Gln His 580 585
590 Ser Cys Pro Ser Asp Cys Asn Asn Leu Gly Gln Cys Val Ser Gly Arg
595 600 605 Cys Ile Cys Asn Glu Gly Tyr Ser Gly Glu Asp Cys Ser Glu
Val Ser 610 615 620 Pro Pro Lys Asp Leu Val Val Thr Glu Val Thr Glu
Glu Thr Val Asn 625 630 635 640 Leu Ala Trp Asp Asn Glu Met Arg Val
Thr Glu Tyr Leu Val Val Tyr 645 650 655 Thr Pro Thr His Glu Gly Gly
Leu Glu Met Gln Phe Arg Val Pro Gly 660 665 670 Asp Gln Thr Ser Thr
Ile Ile Gln Glu Leu Glu Pro Gly Val Glu Tyr 675 680 685 Phe Ile Arg
Val Phe Ala Ile Leu Glu Asn Lys Lys Ser Ile Pro Val 690 695 700 Ser
Ala Arg Val Ala Thr Tyr Leu Pro Ala Pro Glu Gly Leu Lys Phe 705 710
715 720 Lys Ser Ile Lys Glu Thr Ser Val Glu Val Glu Trp Asp Pro Leu
Asp 725 730 735 Ile Ala Phe Glu Thr Trp Glu Ile Ile Phe Arg Asn Met
Asn Lys Glu 740 745 750 Asp Glu Gly Glu Ile Thr Lys Ser Leu Arg Arg
Pro Glu Thr Ser Tyr 755 760 765 Arg Gln Thr Gly Leu Ala Pro Gly Gln
Glu Tyr Glu Ile Ser Leu His 770 775 780 Ile Val Lys Asn Asn Thr Arg
Gly Pro Gly Leu Lys Arg Val Thr Thr 785 790 795 800 Thr Arg Leu Asp
Ala Pro Ser Gln Ile Glu Val Lys Asp Val Thr Asp 805 810 815 Thr Thr
Ala Leu Ile Thr Trp Phe Lys Pro Leu Ala Glu Ile Asp Gly 820 825 830
Ile Glu Leu Thr Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr 835
840 845 Ile Asp Leu Thr Glu Asp Glu Asn Gln Tyr Ser Ile Gly Asn Leu
Lys 850 855 860 Pro Asp Thr Glu Tyr Glu Val Ser Leu Ile Ser Arg Arg
Gly Asp Met 865 870 875 880 Ser Ser Asn Pro Ala Lys Glu Thr Phe Thr
Thr Gly Leu Asp Ala Pro 885 890 895 Arg Asn Leu Arg Arg Val Ser Gln
Thr Asp Asn Ser Ile Thr Leu Glu 900 905 910 Trp Arg Asn Gly Lys Ala
Ala Ile Asp Ser Tyr Arg Ile Lys Tyr Ala 915 920 925 Pro Ile Ser Gly
Gly Asp His Ala Glu Val Asp Val Pro Lys Ser Gln 930 935 940 Gln Ala
Thr Thr Lys Thr Thr Leu Thr Gly Leu Arg Pro Gly Thr Glu 945 950 955
960 Tyr Gly Ile Gly Val Ser Ala Val Lys Glu Asp Lys Glu Ser Asn Pro
965 970 975 Ala Thr Ile Asn Ala Ala Thr Glu Leu Asp Thr Pro Lys Asp
Leu Gln 980 985 990 Val Ser Glu Thr Ala Glu Thr Ser Leu Thr Leu Leu
Trp Lys Thr Pro 995 1000 1005 Leu Ala Lys Phe Asp Arg Tyr Arg Leu
Asn Tyr Ser Leu Pro Thr 1010 1015 1020 Gly Gln Trp Val Gly Val Gln
Leu Pro Arg Asn Thr Thr Ser Tyr 1025 1030 1035 Val Leu Arg Gly Leu
Glu Pro Gly Gln Glu Tyr Asn Val Leu Leu 1040 1045 1050 Thr Ala Glu
Lys Gly Arg His Lys Ser Lys Pro Ala Arg Val Lys 1055 1060 1065 Ala
Ser Thr Glu Gln Ala Pro Glu Leu Glu Asn Leu Thr Val Thr 1070 1075
1080 Glu Val Gly Trp Asp Gly Leu Arg Leu Asn Trp Thr Ala Ala Asp
1085 1090 1095 Gln Ala Tyr Glu His Phe Ile Ile Gln Val Gln Glu Ala
Asn Lys 1100 1105 1110 Val Glu Ala Ala Arg Asn Leu Thr Val Pro Gly
Ser Leu Arg Ala 1115 1120 1125 Val Asp Ile Pro Gly Leu Lys Ala Ala
Thr Pro Tyr Thr Val Ser 1130 1135 1140 Ile Tyr Gly Val Ile Gln Gly
Tyr Arg Thr Pro Val Leu Ser Ala 1145 1150 1155 Glu Ala Ser Thr Gly
Glu Thr Pro Asn Leu Gly Glu Val Val Val 1160 1165 1170 Ala Glu Val
Gly Trp Asp Ala Leu Lys Leu Asn Trp Thr Ala Pro 1175 1180 1185 Glu
Gly Ala Tyr Glu Tyr Phe Phe Ile Gln Val Gln Glu Ala Asp 1190 1195
1200 Thr Val Glu Ala Ala Gln Asn Leu Thr Val Pro Gly Gly Leu Arg
1205 1210 1215 Ser Thr Asp Leu Pro Gly Leu Lys Ala Ala Thr His Tyr
Thr Ile 1220 1225 1230 Thr Ile Arg Gly Val Thr Gln Asp Phe Ser Thr
Thr Pro Leu Ser 1235 1240 1245 Val Glu Val Leu Thr Glu Glu Val Pro
Asp Met Gly Asn Leu Thr 1250 1255 1260 Val Thr Glu Val Ser Trp Asp
Ala Leu Arg Leu Asn Trp Thr Thr 1265 1270 1275 Pro Asp Gly Thr Tyr
Asp Gln Phe Thr Ile Gln Val Gln Glu Ala 1280 1285 1290 Asp Gln Val
Glu Glu Ala His Asn Leu Thr Val Pro Gly Ser Leu 1295 1300 1305 Arg
Ser Met Glu Ile Pro Gly Leu Arg Ala Gly Thr Pro Tyr Thr 1310 1315
1320 Val Thr Leu His Gly Glu Val Arg Gly His Ser Thr Arg Pro Leu
1325 1330 1335 Ala Val Glu Val Val Thr Glu Asp Leu Pro Gln Leu Gly
Asp Leu 1340 1345 1350 Ala Val Ser Glu Val Gly Trp Asp Gly Leu Arg
Leu Asn Trp Thr 1355 1360 1365 Ala Ala Asp Asn Ala Tyr Glu His Phe
Val Ile Gln Val Gln Glu 1370 1375 1380 Val Asn Lys Val Glu Ala Ala
Gln Asn Leu Thr Leu Pro Gly Ser 1385 1390 1395 Leu Arg Ala Val Asp
Ile Pro Gly Leu Glu Ala Ala Thr Pro Tyr 1400 1405 1410 Arg Val Ser
Ile Tyr Gly Val Ile Arg Gly Tyr Arg Thr Pro Val 1415 1420 1425 Leu
Ser Ala Glu Ala Ser Thr Ala Lys Glu Pro Glu Ile Gly Asn 1430 1435
1440 Leu Asn Val Ser Asp Ile Thr Pro Glu Ser Phe Asn Leu Ser Trp
1445 1450 1455 Met Ala Thr Asp Gly Ile Phe Glu Thr Phe Thr Ile Glu
Ile Ile 1460 1465 1470 Asp Ser Asn Arg Leu Leu Glu Thr Val Glu Tyr
Asn Ile Ser Gly 1475 1480 1485 Ala Glu Arg Thr Ala His Ile Ser Gly
Leu Pro Pro Ser Thr Asp 1490 1495 1500 Phe Ile Val Tyr Leu Ser Gly
Leu Ala Pro Ser Ile Arg Thr Lys 1505 1510 1515 Thr Ile Ser Ala Thr
Ala Thr Thr Glu Ala Leu Pro Leu Leu Glu 1520 1525 1530 Asn Leu Thr
Ile Ser Asp Ile Asn Pro Tyr Gly Phe Thr Val Ser 1535 1540 1545 Trp
Met Ala Ser Glu Asn Ala Phe Asp Ser Phe Leu Val Thr Val 1550 1555
1560 Val Asp Ser Gly Lys Leu Leu Asp Pro Gln Glu Phe Thr Leu Ser
1565 1570 1575 Gly Thr Gln Arg Lys Leu Glu Leu Arg Gly Leu Ile Thr
Gly Ile 1580 1585 1590 Gly Tyr Glu Val Met Val Ser Gly Phe Thr Gln
Gly His Gln Thr 1595 1600 1605 Lys Pro Leu Arg Ala Glu Ile Val Thr
Glu Ala Glu Pro Glu Val 1610 1615 1620 Asp Asn Leu Leu Val Ser Asp
Ala Thr Pro Asp Gly Phe Arg Leu 1625 1630 1635 Ser Trp Thr Ala Asp
Glu Gly Val Phe Asp Asn Phe Val Leu Lys 1640 1645 1650 Ile Arg Asp
Thr Lys Lys Gln Ser Glu Pro Leu Glu Ile Thr Leu 1655 1660 1665 Leu
Ala Pro Glu Arg Thr Arg Asp Ile Thr Gly Leu Arg Glu Ala
1670 1675 1680 Thr Glu Tyr Glu Ile Glu Leu Tyr Gly Ile Ser Lys Gly
Arg Arg 1685 1690 1695 Ser Gln Thr Val Ser Ala Ile Ala Thr Thr Ala
Met Gly Ser Pro 1700 1705 1710 Lys Glu Val Ile Phe Ser Asp Ile Thr
Glu Asn Ser Ala Thr Val 1715 1720 1725 Ser Trp Arg Ala Pro Thr Ala
Gln Val Glu Ser Phe Arg Ile Thr 1730 1735 1740 Tyr Val Pro Ile Thr
Gly Gly Thr Pro Ser Met Val Thr Val Asp 1745 1750 1755 Gly Thr Lys
Thr Gln Thr Arg Leu Val Lys Leu Ile Pro Gly Val 1760 1765 1770 Glu
Tyr Leu Val Ser Ile Ile Ala Met Lys Gly Phe Glu Glu Ser 1775 1780
1785 Glu Pro Val Ser Gly Ser Phe Thr Thr Ala Leu Asp Gly Pro Ser
1790 1795 1800 Gly Leu Val Thr Ala Asn Ile Thr Asp Ser Glu Ala Leu
Ala Arg 1805 1810 1815 Trp Gln Pro Ala Ile Ala Thr Val Asp Ser Tyr
Val Ile Ser Tyr 1820 1825 1830 Thr Gly Glu Lys Val Pro Glu Ile Thr
Arg Thr Val Ser Gly Asn 1835 1840 1845 Thr Val Glu Tyr Ala Leu Thr
Asp Leu Glu Pro Ala Thr Glu Tyr 1850 1855 1860 Thr Leu Arg Ile Phe
Ala Glu Lys Gly Pro Gln Lys Ser Ser Thr 1865 1870 1875 Ile Thr Ala
Lys Phe Thr Thr Asp Leu Asp Ser Pro Arg Asp Leu 1880 1885 1890 Thr
Ala Thr Glu Val Gln Ser Glu Thr Ala Leu Leu Thr Trp Arg 1895 1900
1905 Pro Pro Arg Ala Ser Val Thr Gly Tyr Leu Leu Val Tyr Glu Ser
1910 1915 1920 Val Asp Gly Thr Val Lys Glu Val Ile Val Gly Pro Asp
Thr Thr 1925 1930 1935 Ser Tyr Ser Leu Ala Asp Leu Ser Pro Ser Thr
His Tyr Thr Ala 1940 1945 1950 Lys Ile Gln Ala Leu Asn Gly Pro Leu
Arg Ser Asn Met Ile Gln 1955 1960 1965 Thr Ile Phe Thr Thr Ile Gly
Leu Leu Tyr Pro Phe Pro Lys Asp 1970 1975 1980 Cys Ser Gln Ala Met
Leu Asn Gly Asp Thr Thr Ser Gly Leu Tyr 1985 1990 1995 Thr Ile Tyr
Leu Asn Gly Asp Lys Ala Glu Ala Leu Glu Val Phe 2000 2005 2010 Cys
Asp Met Thr Ser Asp Gly Gly Gly Trp Ile Val Phe Leu Arg 2015 2020
2025 Arg Lys Asn Gly Arg Glu Asn Phe Tyr Gln Asn Trp Lys Ala Tyr
2030 2035 2040 Ala Ala Gly Phe Gly Asp Arg Arg Glu Glu Phe Trp Leu
Gly Leu 2045 2050 2055 Asp Asn Leu Asn Lys Ile Thr Ala Gln Gly Gln
Tyr Glu Leu Arg 2060 2065 2070 Val Asp Leu Arg Asp His Gly Glu Thr
Ala Phe Ala Val Tyr Asp 2075 2080 2085 Lys Phe Ser Val Gly Asp Ala
Lys Thr Arg Tyr Lys Leu Lys Val 2090 2095 2100 Glu Gly Tyr Ser Gly
Thr Ala Gly Asp Ser Met Ala Tyr His Asn 2105 2110 2115 Gly Arg Ser
Phe Ser Thr Phe Asp Lys Asp Thr Asp Ser Ala Ile 2120 2125 2130 Thr
Asn Cys Ala Leu Ser Tyr Lys Gly Ala Phe Trp Tyr Arg Asn 2135 2140
2145 Cys His Arg Val Asn Leu Met Gly Arg Tyr Gly Asp Asn Asn His
2150 2155 2160 Ser Gln Gly Val Asn Trp Phe His Trp Lys Gly His Glu
His Ser 2165 2170 2175 Ile Gln Phe Ala Glu Met Lys Leu Arg Pro Ser
Asn Phe Arg Asn 2180 2185 2190 Leu Glu Gly Arg Arg Lys Arg Ala 2195
2200 7689PRTArtificial SequenceFibcon FN3 domain 76Leu Asp Ala Pro
Thr Asp Leu Gln Val Thr Asn Val Thr Asp Thr Ser 1 5 10 15 Ile Thr
Val Ser Trp Thr Pro Pro Ser Ala Thr Ile Thr Gly Tyr Arg 20 25 30
Ile Thr Tyr Thr Pro Ser Asn Gly Pro Gly Glu Pro Lys Glu Leu Thr 35
40 45 Val Pro Pro Ser Ser Thr Ser Val Thr Ile Thr Gly Leu Thr Pro
Gly 50 55 60 Val Glu Tyr Val Val Ser Leu Tyr Ala Leu Lys Asp Asn
Gln Glu Ser 65 70 75 80 Pro Pro Leu Val Gly Thr Gln Thr Thr 85
7794PRTHomo sapiens 77Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr Pro Thr 1 5 10 15 Ser Leu Leu Ile Ser Trp Asp Ala Pro
Ala Val Thr Val Arg Tyr Tyr 20 25 30 Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45 Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60 Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg Gly Asp 65 70 75 80 Ser
Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr 85 90
784PRTArtificial Sequencelinker 78Gly Ser Gly Ser 1
7925PRTArtificial Sequencelinker 79Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly
Gly Gly Ser 20 25 804PRTArtificial Sequencelinker 80Ala Pro Ala Pro
1 8110PRTArtificial Sequencelinker 81Ala Pro Ala Pro Ala Pro Ala
Pro Ala Pro 1 5 10 8220PRTArtificial Sequencelinker 82Ala Pro Ala
Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro 1 5 10 15 Ala
Pro Ala Pro 20 8340PRTArtificial Sequencelinker 83Ala Pro Ala Pro
Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro 1 5 10 15 Ala Pro
Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro 20 25 30
Ala Pro Ala Pro Ala Pro Ala Pro 35 40 8429PRTArtificial
Sequencelinker 84Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu
Ala Ala Ala Lys 1 5 10 15 Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys
Ala Ala Ala 20 25 858PRTArtificial Sequencetencon BC loop 85Thr Ala
Pro Asp Ala Ala Phe Asp 1 5 867PRTArtificial Sequencetencon FG loop
86Lys Gly Gly His Arg Ser Asn 1 5 878PRTArtificial SequenceEGFR
binding FN3 domain BC loop 87Ala Asp Pro His Gly Phe Tyr Asp 1 5
888PRTArtificial SequenceEGFR binding FN3 domain BC loop 88Thr Tyr
Asp Arg Asp Gly Tyr Asp 1 5 898PRTArtificial SequenceEGFR binding
FN3 domain BC loop 89Trp Asp Pro Phe Ser Phe Tyr Asp 1 5
908PRTArtificial SequenceEGFR binding FN3 domain BC loop 90Asp Asp
Pro Arg Gly Phe Tyr Glu 1 5 918PRTArtificial SequenceEGFR binding
FN3 domain BC loop 91Thr Trp Pro Tyr Ala Asp Leu Asp 1 5
928PRTArtificial SequenceEGFR binding FN3 domain BC loop 92Gly Tyr
Asn Gly Asp His Phe Asp 1 5 938PRTArtificial SequenceEGFR binding
FN3 domain BC loop 93Asp Tyr Asp Leu Gly Val Tyr Asp 1 5
948PRTArtificial SequenceEGFR binding FN3 domain BC loop 94Asp Asp
Pro Trp Asp Phe Tyr Glu 1 5 9512PRTArtificial SequenceEGFR binding
FN3 domain FG loop 95His Asn Val Tyr Lys Asp Thr Asn Met Arg Gly
Leu 1 5 10 9611PRTArtificial SequenceEGFR binding FN3 domain FG
loop 96Leu Gly Ser Tyr Val Phe Glu His Asp Val Met 1 5 10
97285DNAArtificial SequenceEGFR binding FN3 domain from ECB97;
P54AR4-83V22 97atgttgccag cgccgaagaa cctggtagtt agcgaggtta
ctgaggacag cgcgcgtctg 60agctgggacg atccgtgggc gttctacgag agctttctga
tccagtatca agagagcgag 120aaagtcggtg aagcgattgt gctgaccgtc
ccgggctccg agcgttccta cgacctgacc 180ggtttgaagc cgggtaccga
gtatacggtg agcatctacg gtgttcacaa tgtctataag 240gacactaata
tccgcggtct gcctctgagc gccattttca ccacc 28598285DNAArtificial
SequenceEGFR binding FN3 domain from ECB15; P54AR4-83V2
98atgctgccag cccctaagaa tctggtcgtg agcgaagtaa ccgaggacag cgcccgcctg
60agctgggacg acccgtgggc gttctatgag tctttcctga ttcagtatca agaaagcgaa
120aaagttggcg aagcgatcgt cctgaccgtc ccgggtagcg agcgctccta
cgatctgacc 180ggcctgaaac cgggtacgga gtacacggtg tccatttacg
gtgttcacaa tgtgtataaa 240gacaccaaca tgcgtggcct gccgctgtcg
gcgattttca ccacc 2859989PRTArtificial Sequencetencon27 FN3 domain
99Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp Ser 1
5 10 15 Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe
Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile
Val Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr
Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr Gly
Val Lys Gly Gly His Arg Ser 65 70 75 80 Asn Pro Leu Ser Ala Ile Phe
Thr Thr 85 10089PRTArtificial SequenceTCL14
libraryMISC_FEATURE(32)..(32)Xaa is any amino
acidMISC_FEATURE(34)..(34)Xaa is any amino
acidMISC_FEATURE(36)..(36)Xaa is any amino
acidMISC_FEATURE(38)..(38)Xaa is any amino
acidMISC_FEATURE(39)..(39)Xaa is any amino
acidMISC_FEATURE(40)..(40)Xaa is any amino
acidMISC_FEATURE(41)..(41)Xaa is any amino
acidMISC_FEATURE(68)..(68)Xaa is any amino
acidMISC_FEATURE(70)..(70)Xaa is any amino
acidMISC_FEATURE(72)..(72)Xaa is any amino
acidMISC_FEATURE(78)..(78)Xaa is any amino
acidMISC_FEATURE(79)..(79)Xaa is any amino
acidMISC_FEATURE(81)..(81)Xaa is any amino acid 100Leu Pro Ala Pro
Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp Ser 1 5 10 15 Ala Arg
Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Xaa 20 25 30
Ile Xaa Tyr Xaa Glu Xaa Xaa Xaa Xaa Gly Glu Ala Ile Val Leu Thr 35
40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro
Gly 50 55 60 Thr Glu Tyr Xaa Val Xaa Ile Xaa Gly Val Lys Gly Gly
Xaa Xaa Ser 65 70 75 80 Xaa Pro Leu Ser Ala Ile Phe Thr Thr 85
1011408PRTHOMO SAPIENS 101Met Lys Ala Pro Ala Val Leu Ala Pro Gly
Ile Leu Val Leu Leu Phe 1 5 10 15 Thr Leu Val Gln Arg Ser Asn Gly
Glu Cys Lys Glu Ala Leu Ala Lys 20 25 30 Ser Glu Met Asn Val Asn
Met Lys Tyr Gln Leu Pro Asn Phe Thr Ala 35 40 45 Glu Thr Pro Ile
Gln Asn Val Ile Leu His Glu His His Ile Phe Leu 50 55 60 Gly Ala
Thr Asn Tyr Ile Tyr Val Leu Asn Glu Glu Asp Leu Gln Lys 65 70 75 80
Val Ala Glu Tyr Lys Thr Gly Pro Val Leu Glu His Pro Asp Cys Phe 85
90 95 Pro Cys Gln Asp Cys Ser Ser Lys Ala Asn Leu Ser Gly Gly Val
Trp 100 105 110 Lys Asp Asn Ile Asn Met Ala Leu Val Val Asp Thr Tyr
Tyr Asp Asp 115 120 125 Gln Leu Ile Ser Cys Gly Ser Val Asn Arg Gly
Thr Cys Gln Arg His 130 135 140 Val Phe Pro His Asn His Thr Ala Asp
Ile Gln Ser Glu Val His Cys 145 150 155 160 Ile Phe Ser Pro Gln Ile
Glu Glu Pro Ser Gln Cys Pro Asp Cys Val 165 170 175 Val Ser Ala Leu
Gly Ala Lys Val Leu Ser Ser Val Lys Asp Arg Phe 180 185 190 Ile Asn
Phe Phe Val Gly Asn Thr Ile Asn Ser Ser Tyr Phe Pro Asp 195 200 205
His Pro Leu His Ser Ile Ser Val Arg Arg Leu Lys Glu Thr Lys Asp 210
215 220 Gly Phe Met Phe Leu Thr Asp Gln Ser Tyr Ile Asp Val Leu Pro
Glu 225 230 235 240 Phe Arg Asp Ser Tyr Pro Ile Lys Tyr Val His Ala
Phe Glu Ser Asn 245 250 255 Asn Phe Ile Tyr Phe Leu Thr Val Gln Arg
Glu Thr Leu Asp Ala Gln 260 265 270 Thr Phe His Thr Arg Ile Ile Arg
Phe Cys Ser Ile Asn Ser Gly Leu 275 280 285 His Ser Tyr Met Glu Met
Pro Leu Glu Cys Ile Leu Thr Glu Lys Arg 290 295 300 Lys Lys Arg Ser
Thr Lys Lys Glu Val Phe Asn Ile Leu Gln Ala Ala 305 310 315 320 Tyr
Val Ser Lys Pro Gly Ala Gln Leu Ala Arg Gln Ile Gly Ala Ser 325 330
335 Leu Asn Asp Asp Ile Leu Phe Gly Val Phe Ala Gln Ser Lys Pro Asp
340 345 350 Ser Ala Glu Pro Met Asp Arg Ser Ala Met Cys Ala Phe Pro
Ile Lys 355 360 365 Tyr Val Asn Asp Phe Phe Asn Lys Ile Val Asn Lys
Asn Asn Val Arg 370 375 380 Cys Leu Gln His Phe Tyr Gly Pro Asn His
Glu His Cys Phe Asn Arg 385 390 395 400 Thr Leu Leu Arg Asn Ser Ser
Gly Cys Glu Ala Arg Arg Asp Glu Tyr 405 410 415 Arg Thr Glu Phe Thr
Thr Ala Leu Gln Arg Val Asp Leu Phe Met Gly 420 425 430 Gln Phe Ser
Glu Val Leu Leu Thr Ser Ile Ser Thr Phe Ile Lys Gly 435 440 445 Asp
Leu Thr Ile Ala Asn Leu Gly Thr Ser Glu Gly Arg Phe Met Gln 450 455
460 Val Val Val Ser Arg Ser Gly Pro Ser Thr Pro His Val Asn Phe Leu
465 470 475 480 Leu Asp Ser His Pro Val Ser Pro Glu Val Ile Val Glu
His Thr Leu 485 490 495 Asn Gln Asn Gly Tyr Thr Leu Val Ile Thr Gly
Lys Lys Ile Thr Lys 500 505 510 Ile Pro Leu Asn Gly Leu Gly Cys Arg
His Phe Gln Ser Cys Ser Gln 515 520 525 Cys Leu Ser Ala Pro Pro Phe
Val Gln Cys Gly Trp Cys His Asp Lys 530 535 540 Cys Val Arg Ser Glu
Glu Cys Leu Ser Gly Thr Trp Thr Gln Gln Ile 545 550 555 560 Cys Leu
Pro Ala Ile Tyr Lys Val Phe Pro Asn Ser Ala Pro Leu Glu 565 570 575
Gly Gly Thr Arg Leu Thr Ile Cys Gly Trp Asp Phe Gly Phe Arg Arg 580
585 590 Asn Asn Lys Phe Asp Leu Lys Lys Thr Arg Val Leu Leu Gly Asn
Glu 595 600 605 Ser Cys Thr Leu Thr Leu Ser Glu Ser Thr Met Asn Thr
Leu Lys Cys 610 615 620 Thr Val Gly Pro Ala Met Asn Lys His Phe Asn
Met Ser Ile Ile Ile 625 630 635 640 Ser Asn Gly His Gly Thr Thr Gln
Tyr Ser Thr Phe Ser Tyr Val Asp 645 650 655 Pro Val Ile Thr Ser Ile
Ser Pro Lys Tyr Gly Pro Met Ala Gly Gly 660 665 670 Thr Leu Leu Thr
Leu Thr Gly Asn Tyr Leu Asn Ser Gly Asn Ser Arg 675 680 685 His Ile
Ser Ile Gly Gly Lys Thr Cys Thr Leu Lys Ser Val Ser Asn 690 695 700
Ser Ile Leu Glu Cys Tyr Thr Pro Ala Gln Thr Ile Ser Thr Glu Phe 705
710 715 720 Ala Val Lys Leu Lys Ile Asp Leu Ala Asn Arg Glu Thr Ser
Ile Phe 725 730 735 Ser Tyr Arg Glu Asp Pro Ile Val Tyr Glu Ile His
Pro Thr Lys Ser 740 745 750 Phe Ile Ser Thr Trp Trp Lys Glu Pro Leu
Asn Ile Val Ser Phe Leu 755 760 765 Phe Cys Phe Ala Ser Gly Gly Ser
Thr Ile Thr Gly Val Gly Lys Asn 770 775 780 Leu Asn Ser Val Ser Val
Pro Arg Met Val Ile Asn Val His Glu Ala 785 790 795 800 Gly Arg Asn
Phe Thr Val Ala Cys Gln His Arg Ser Asn Ser Glu Ile 805 810 815 Ile
Cys Cys Thr
Thr Pro Ser Leu Gln Gln Leu Asn Leu Gln Leu Pro 820 825 830 Leu Lys
Thr Lys Ala Phe Phe Met Leu Asp Gly Ile Leu Ser Lys Tyr 835 840 845
Phe Asp Leu Ile Tyr Val His Asn Pro Val Phe Lys Pro Phe Glu Lys 850
855 860 Pro Val Met Ile Ser Met Gly Asn Glu Asn Val Leu Glu Ile Lys
Gly 865 870 875 880 Asn Asp Ile Asp Pro Glu Ala Val Lys Gly Glu Val
Leu Lys Val Gly 885 890 895 Asn Lys Ser Cys Glu Asn Ile His Leu His
Ser Glu Ala Val Leu Cys 900 905 910 Thr Val Pro Asn Asp Leu Leu Lys
Leu Asn Ser Glu Leu Asn Ile Glu 915 920 925 Trp Lys Gln Ala Ile Ser
Ser Thr Val Leu Gly Lys Val Ile Val Gln 930 935 940 Pro Asp Gln Asn
Phe Thr Gly Leu Ile Ala Gly Val Val Ser Ile Ser 945 950 955 960 Thr
Ala Leu Leu Leu Leu Leu Gly Phe Phe Leu Trp Leu Lys Lys Arg 965 970
975 Lys Gln Ile Lys Asp Leu Gly Ser Glu Leu Val Arg Tyr Asp Ala Arg
980 985 990 Val His Thr Pro His Leu Asp Arg Leu Val Ser Ala Arg Ser
Val Ser 995 1000 1005 Pro Thr Thr Glu Met Val Ser Asn Glu Ser Val
Asp Tyr Arg Ala 1010 1015 1020 Thr Phe Pro Glu Asp Gln Phe Pro Asn
Ser Ser Gln Asn Gly Ser 1025 1030 1035 Cys Arg Gln Val Gln Tyr Pro
Leu Thr Asp Met Ser Pro Ile Leu 1040 1045 1050 Thr Ser Gly Asp Ser
Asp Ile Ser Ser Pro Leu Leu Gln Asn Thr 1055 1060 1065 Val His Ile
Asp Leu Ser Ala Leu Asn Pro Glu Leu Val Gln Ala 1070 1075 1080 Val
Gln His Val Val Ile Gly Pro Ser Ser Leu Ile Val His Phe 1085 1090
1095 Asn Glu Val Ile Gly Arg Gly His Phe Gly Cys Val Tyr His Gly
1100 1105 1110 Thr Leu Leu Asp Asn Asp Gly Lys Lys Ile His Cys Ala
Val Lys 1115 1120 1125 Ser Leu Asn Arg Ile Thr Asp Ile Gly Glu Val
Ser Gln Phe Leu 1130 1135 1140 Thr Glu Gly Ile Ile Met Lys Asp Phe
Ser His Pro Asn Val Leu 1145 1150 1155 Ser Leu Leu Gly Ile Cys Leu
Arg Ser Glu Gly Ser Pro Leu Val 1160 1165 1170 Val Leu Pro Tyr Met
Lys His Gly Asp Leu Arg Asn Phe Ile Arg 1175 1180 1185 Asn Glu Thr
His Asn Pro Thr Val Lys Asp Leu Ile Gly Phe Gly 1190 1195 1200 Leu
Gln Val Ala Lys Gly Met Lys Tyr Leu Ala Ser Lys Lys Phe 1205 1210
1215 Val His Arg Asp Leu Ala Ala Arg Asn Cys Met Leu Asp Glu Lys
1220 1225 1230 Phe Thr Val Lys Val Ala Asp Phe Gly Leu Ala Arg Asp
Met Tyr 1235 1240 1245 Asp Lys Glu Tyr Tyr Ser Val His Asn Lys Thr
Gly Ala Lys Leu 1250 1255 1260 Pro Val Lys Trp Met Ala Leu Glu Ser
Leu Gln Thr Gln Lys Phe 1265 1270 1275 Thr Thr Lys Ser Asp Val Trp
Ser Phe Gly Val Leu Leu Trp Glu 1280 1285 1290 Leu Met Thr Arg Gly
Ala Pro Pro Tyr Pro Asp Val Asn Thr Phe 1295 1300 1305 Asp Ile Thr
Val Tyr Leu Leu Gln Gly Arg Arg Leu Leu Gln Pro 1310 1315 1320 Glu
Tyr Cys Pro Asp Pro Leu Tyr Glu Val Met Leu Lys Cys Trp 1325 1330
1335 His Pro Lys Ala Glu Met Arg Pro Ser Phe Ser Glu Leu Val Ser
1340 1345 1350 Arg Ile Ser Ala Ile Phe Ser Thr Phe Ile Gly Glu His
Tyr Val 1355 1360 1365 His Val Asn Ala Thr Tyr Val Asn Val Lys Cys
Val Ala Pro Tyr 1370 1375 1380 Pro Ser Leu Leu Ser Ser Glu Asp Asn
Ala Asp Asp Glu Val Asp 1385 1390 1395 Thr Arg Pro Ala Ser Phe Trp
Glu Thr Ser 1400 1405 102697PRTHOMO SAPIENS 102Gln Arg Lys Arg Arg
Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys 1 5 10 15 Thr Thr Leu
Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys 20 25 30 Val
Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly 35 40
45 Leu Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln
50 55 60 Cys Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys
Lys Glu 65 70 75 80 Phe Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp
Tyr Ile Arg Asn 85 90 95 Cys Ile Ile Gly Lys Gly Arg Ser Tyr Lys
Gly Thr Val Ser Ile Thr 100 105 110 Lys Ser Gly Ile Lys Cys Gln Pro
Trp Ser Ser Met Ile Pro His Glu 115 120 125 His Ser Phe Leu Pro Ser
Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn 130 135 140 Tyr Cys Arg Asn
Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr 145 150 155 160 Ser
Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser 165 170
175 Glu Val Glu Cys Met Thr Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met
180 185 190 Asp His Thr Glu Ser Gly Lys Ile Cys Gln Arg Trp Asp His
Gln Thr 195 200 205 Pro His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro
Asp Lys Gly Phe 210 215 220 Asp Asp Asn Tyr Cys Arg Asn Pro Asp Gly
Gln Pro Arg Pro Trp Cys 225 230 235 240 Tyr Thr Leu Asp Pro His Thr
Arg Trp Glu Tyr Cys Ala Ile Lys Thr 245 250 255 Cys Ala Asp Asn Thr
Met Asn Asp Thr Asp Val Pro Leu Glu Thr Thr 260 265 270 Glu Cys Ile
Gln Gly Gln Gly Glu Gly Tyr Arg Gly Thr Val Asn Thr 275 280 285 Ile
Trp Asn Gly Ile Pro Cys Gln Arg Trp Asp Ser Gln Tyr Pro His 290 295
300 Glu His Asp Met Thr Pro Glu Asn Phe Lys Cys Lys Asp Leu Arg Glu
305 310 315 320 Asn Tyr Cys Arg Asn Pro Asp Gly Ser Glu Ser Pro Trp
Cys Phe Thr 325 330 335 Thr Asp Pro Asn Ile Arg Val Gly Tyr Cys Ser
Gln Ile Pro Asn Cys 340 345 350 Asp Met Ser His Gly Gln Asp Cys Tyr
Arg Gly Asn Gly Lys Asn Tyr 355 360 365 Met Gly Asn Leu Ser Gln Thr
Arg Ser Gly Leu Thr Cys Ser Met Trp 370 375 380 Asp Lys Asn Met Glu
Asp Leu His Arg His Ile Phe Trp Glu Pro Asp 385 390 395 400 Ala Ser
Lys Leu Asn Glu Asn Tyr Cys Arg Asn Pro Asp Asp Asp Ala 405 410 415
His Gly Pro Trp Cys Tyr Thr Gly Asn Pro Leu Ile Pro Trp Asp Tyr 420
425 430 Cys Pro Ile Ser Arg Cys Glu Gly Asp Thr Thr Pro Thr Ile Val
Asn 435 440 445 Leu Asp His Pro Val Ile Ser Cys Ala Lys Thr Lys Gln
Leu Arg Val 450 455 460 Val Asn Gly Ile Pro Thr Arg Thr Asn Ile Gly
Trp Met Val Ser Leu 465 470 475 480 Arg Tyr Arg Asn Lys His Ile Cys
Gly Gly Ser Leu Ile Lys Glu Ser 485 490 495 Trp Val Leu Thr Ala Arg
Gln Cys Phe Pro Ser Arg Asp Leu Lys Asp 500 505 510 Tyr Glu Ala Trp
Leu Gly Ile His Asp Val His Gly Arg Gly Asp Glu 515 520 525 Lys Cys
Lys Gln Val Leu Asn Val Ser Gln Leu Val Tyr Gly Pro Glu 530 535 540
Gly Ser Asp Leu Val Leu Met Lys Leu Ala Arg Pro Ala Val Leu Asp 545
550 555 560 Asp Phe Val Ser Thr Ile Asp Leu Pro Asn Tyr Gly Cys Thr
Ile Pro 565 570 575 Glu Lys Thr Ser Cys Ser Val Tyr Gly Trp Gly Tyr
Thr Gly Leu Ile 580 585 590 Asn Tyr Asp Gly Leu Leu Arg Val Ala His
Leu Tyr Ile Met Gly Asn 595 600 605 Glu Lys Cys Ser Gln His His Arg
Gly Lys Val Thr Leu Asn Glu Ser 610 615 620 Glu Ile Cys Ala Gly Ala
Glu Lys Ile Gly Ser Gly Pro Cys Glu Gly 625 630 635 640 Asp Tyr Gly
Gly Pro Leu Val Cys Glu Gln His Lys Met Arg Met Val 645 650 655 Leu
Gly Val Ile Val Pro Gly Arg Gly Cys Ala Ile Pro Asn Arg Pro 660 665
670 Gly Ile Phe Val Arg Val Ala Tyr Tyr Ala Lys Trp Ile His Lys Ile
675 680 685 Ile Leu Thr Tyr Lys Val Pro Gln Ser 690 695
103276DNAArtificial Sequencec-Met binding FN3 domain 103ctgccggctc
cgaagaactt ggtggtgagc cgtgttaccg aagatagcgc acgcctgagc 60tggacggcac
cggatgcggc gttcgatagc ttctggattc gctattttga gtttctgggt
120agcggtgagg caattgttct gacggtgccg ggctctgaac gctcctacga
tttgaccggt 180ctgaaaccgg gcaccgagta tgtggtgaac attctgagcg
ttaagggcgg tagcatcagc 240ccaccgctga gcgcgatctt cacgactggt ggttgc
276104267DNAArtificial Sequencec-Met binding FN3 domain
104ctgccggcac cgaagaacct ggttgtcagc cgtgtgaccg aggatagcgc
acgtttgagc 60tggaccgctc cggatgcagc ctttgacagc ttctggattc gttactttga
atttctgggt 120agcggtgagg cgatcgttct gacggtgccg ggctctgaac
gcagctatga tttgacgggc 180ctgaagccgg gtactgagta cgtggttaac
atcatgggcg ttaagggtgg taaaatcagc 240ccgccattgt ccgcgatctt taccacg
2671055PRTArtificial Sequencelinker 105Gly Gly Gly Gly Ser 1 5
106194PRTArtificial SequenceBispecific EGFR/c-Met binding molecule
106Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp
1 5 10 15 Ser Ala Arg Leu Ser Trp Asp Asp Pro Trp Ala Phe Tyr Glu
Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu
Ala Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp
Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile
Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Ile Arg Gly
Leu Pro Leu Ser Ala Ile Phe Thr Thr Ala 85 90 95 Pro Ala Pro Ala
Pro Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu 100 105 110 Val Val
Ser Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala 115 120 125
Pro Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu Phe Leu 130
135 140 Gly Ser Gly Glu Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg
Ser 145 150 155 160 Tyr Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr
Val Val Asn Ile 165 170 175 Leu Ser Val Lys Gly Gly Ser Ile Ser Pro
Pro Leu Ser Ala Ile Phe 180 185 190 Thr Thr 10794PRTArtificial
SequenceEGFR binding FN3 domain 107Leu Pro Ala Pro Lys Asn Leu Val
Val Ser Glu Val Thr Glu Asp Ser 1 5 10 15 Ala Arg Leu Ser Trp Ala
Asp Pro His Gly Phe Tyr Asp Ser Phe Leu 20 25 30 Ile Gln Tyr Gln
Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu Thr 35 40 45 Val Pro
Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60
Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys Asp 65
70 75 80 Thr Asn Met Arg Gly Leu Pro Leu Ser Ala Ile Phe Thr Thr 85
90 10894PRTArtificial SequenceEGFR binding FN3 domain 108Leu Pro
Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser 1 5 10 15
Ala Arg Leu Ser Trp Asp Asp Pro Trp Ala Phe Tyr Glu Ser Phe Leu 20
25 30 Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu
Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu
Lys Pro Gly 50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His
Asn Val Tyr Lys Asp 65 70 75 80 Thr Asn Ile Arg Gly Leu Pro Leu Ser
Ala Ile Phe Thr Thr 85 90 10994PRTArtificial SequenceEGFR binding
FN3 domain 109Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr
Glu Asp Ser 1 5 10 15 Ala Arg Leu Ser Trp Asp Asp Pro His Ala Phe
Tyr Glu Ser Phe Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu Lys Val
Gly Glu Ala Ile Val Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser
Tyr Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Thr Val
Ser Ile Tyr Gly Val His Asn Val Tyr Lys Asp 65 70 75 80 Thr Asn Ile
Arg Gly Leu Pro Leu Ser Ala Ile Phe Thr Thr 85 90
11094PRTArtificial SequenceEGFR binding FN3 domain 110Leu Pro Ala
Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser 1 5 10 15 Ala
Arg Leu Ser Trp Ala Asp Pro His Gly Phe Tyr Asp Ser Phe Leu 20 25
30 Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu Thr
35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys
Pro Gly 50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn
Val Tyr Lys Asp 65 70 75 80 Thr Asn Ile Arg Gly Leu Pro Leu Ser Ala
Ile Phe Thr Thr 85 90 11189PRTArtificial Sequencec-Met binding FN3
domain 111Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu
Asp Ser 1 5 10 15 Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe
Asp Ser Phe Trp 20 25 30 Ile Arg Tyr Phe Glu Phe Leu Gly Ser Gly
Glu Ala Ile Val Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr
Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Val Val Asn
Ile Leu Gly Val Lys Gly Gly Lys Ile Ser 65 70 75 80 Pro Pro Leu Ser
Ala Ile Phe Thr Thr 85 11289PRTArtificial Sequencec-Met binding FN3
domain 112Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu
Asp Ser 1 5 10 15 Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe
Asp Ser Phe Trp 20 25 30 Ile Arg Tyr Phe Glu Phe Val Gly Ser Gly
Glu Ala Ile Val Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr
Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Val Val Asn
Ile Leu Gly Val Lys Gly Gly Ser Ile Ser 65 70 75 80 Pro Pro Leu Ser
Ala Ile Phe Thr Thr 85 11389PRTArtificial Sequencec-Met binding FN3
domain 113Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu
Asp Ser 1 5 10 15 Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe
Asp Ser Phe Trp 20 25 30 Ile Arg Tyr Phe Glu Phe Val Ser Lys Gly
Asp Ala Ile Val Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr
Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Val Val Asn
Ile Leu Gly Val Lys Gly Gly
Ser Ile Ser 65 70 75 80 Pro Pro Leu Ser Ala Ile Phe Thr Thr 85
11489PRTArtificial Sequencec-Met binding FN3 domain 114Leu Pro Ala
Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp Ser 1 5 10 15 Ala
Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Trp 20 25
30 Ile Arg Tyr Phe Glu Phe Leu Gly Ser Gly Glu Ala Ile Val Leu Thr
35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys
Pro Gly 50 55 60 Thr Glu Tyr Val Val Asn Ile Leu Ser Val Lys Gly
Gly Ser Ile Ser 65 70 75 80 Pro Pro Leu Ser Ala Ile Phe Thr Thr 85
115591DNAArtificial SequenceBispecific EGFR/c-Met binding molecule
115atgttgccag cgccgaagaa cctggtagtt agcgaggtta ctgaggacag
cgcgcgtctg 60agctgggacg atccgtgggc gttctacgag agctttctga tccagtatca
agagagcgag 120aaagtcggtg aagcgattgt gctgaccgtc ccgggctccg
agcgttccta cgacctgacc 180ggtttgaagc cgggtaccga gtatacggtg
agcatctacg gtgttcacaa tgtctataag 240gacactaata tccgcggtct
gcctctgagc gccattttca ccaccgcacc ggcaccggct 300ccggctcctg
ccccgctgcc ggctccgaag aacttggtgg tgagccgtgt taccgaagat
360agcgcacgcc tgagctggac ggcaccggat gcggcgttcg atagcttctg
gattcgctat 420tttgagtttc tgggtagcgg tgaggcaatt gttctgacgg
tgccgggctc tgaacgctcc 480tacgatttga ccggtctgaa accgggcacc
gagtatgtgg tgaacattct gagcgttaag 540ggcggtagca tcagcccacc
gctgagcgcg atcttcacga ctggtggttg c 591116582DNAArtificial
SequenceBispecific EGFR/c-Met binding molecule 116atgctgccag
cccctaagaa tctggtcgtg agcgaagtaa ccgaggacag cgcccgcctg 60agctgggacg
acccgtgggc gttctatgag tctttcctga ttcagtatca agaaagcgaa
120aaagttggcg aagcgatcgt cctgaccgtc ccgggtagcg agcgctccta
cgatctgacc 180ggcctgaaac cgggtacgga gtacacggtg tccatttacg
gtgttcacaa tgtgtataaa 240gacaccaaca tgcgtggcct gccgctgtcg
gcgattttca ccaccgcgcc tgcgccagcg 300cctgcaccgg ctccgctgcc
ggcaccgaag aacctggttg tcagccgtgt gaccgaggat 360agcgcacgtt
tgagctggac cgctccggat gcagcctttg acagcttctg gattcgttac
420tttgaatttc tgggtagcgg tgaggcgatc gttctgacgg tgccgggctc
tgaacgcagc 480tatgatttga cgggcctgaa gccgggtact gagtacgtgg
ttaacatcat gggcgttaag 540ggtggtaaaa tcagcccgcc attgtccgcg
atctttacca cg 58211751PRTArtificial Sequencealbumin binding domain
117Thr Ile Asp Glu Trp Leu Leu Lys Glu Ala Lys Glu Lys Ala Ile Glu
1 5 10 15 Glu Leu Lys Lys Ala Gly Ile Thr Ser Asp Tyr Tyr Phe Asp
Leu Ile 20 25 30 Asn Lys Ala Lys Thr Val Glu Gly Val Asn Ala Leu
Lys Asp Glu Ile 35 40 45 Leu Lys Ala 50 118250PRTArtificial
SequenceBispecific EGFR/c-Met binding molecule 118Met Leu Pro Ala
Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp 1 5 10 15 Ser Ala
Arg Leu Ser Trp Asp Asp Pro Trp Ala Phe Tyr Glu Ser Phe 20 25 30
Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu 35
40 45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys
Pro 50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn
Val Tyr Lys 65 70 75 80 Asp Thr Asn Met Arg Gly Leu Pro Leu Ser Ala
Ile Phe Thr Thr Ala 85 90 95 Pro Ala Pro Ala Pro Ala Pro Ala Pro
Leu Pro Ala Pro Lys Asn Leu 100 105 110 Val Val Ser Arg Val Thr Glu
Asp Ser Ala Arg Leu Ser Trp Thr Ala 115 120 125 Pro Asp Ala Ala Phe
Asp Ser Phe Trp Ile Arg Tyr Asp Glu Val Val 130 135 140 Val Gly Gly
Glu Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg Ser 145 150 155 160
Tyr Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr Tyr Val Asn Ile 165
170 175 Leu Gly Val Lys Gly Gly Ser Ile Ser Val Pro Leu Ser Ala Ile
Phe 180 185 190 Thr Thr Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Leu
Ala Glu Ala 195 200 205 Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr
Gly Val Ser Asp Tyr 210 215 220 Tyr Lys Asn Leu Ile Asn Asn Ala Lys
Thr Val Glu Gly Val Lys Ala 225 230 235 240 Leu Leu Asp Glu Ile Leu
Ala Ala Leu Pro 245 250 119250PRTArtificial SequenceBispecific
EGFR/c-Met binding molecule 119Met Leu Pro Ala Pro Lys Asn Leu Val
Val Ser Glu Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Ala
Asp Pro His Gly Phe Tyr Asp Ser Phe 20 25 30 Leu Ile Gln Tyr Gln
Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu 35 40 45 Thr Val Pro
Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly
Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys 65 70
75 80 Asp Thr Asn Met Arg Gly Leu Pro Leu Ser Ala Ile Phe Thr Thr
Ala 85 90 95 Pro Ala Pro Ala Pro Ala Pro Ala Pro Leu Pro Ala Pro
Lys Asn Leu 100 105 110 Val Val Ser Arg Val Thr Glu Asp Ser Ala Arg
Leu Ser Trp Thr Ala 115 120 125 Pro Asp Ala Ala Phe Asp Ser Phe Trp
Ile Arg Tyr Asp Glu Val Val 130 135 140 Val Gly Gly Glu Ala Ile Val
Leu Thr Val Pro Gly Ser Glu Arg Ser 145 150 155 160 Tyr Asp Leu Thr
Gly Leu Lys Pro Gly Thr Glu Tyr Tyr Val Asn Ile 165 170 175 Leu Gly
Val Lys Gly Gly Ser Ile Ser Val Pro Leu Ser Ala Ile Phe 180 185 190
Thr Thr Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Leu Ala Glu Ala 195
200 205 Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp
Tyr 210 215 220 Tyr Lys Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly
Val Lys Ala 225 230 235 240 Leu Leu Asp Glu Ile Leu Ala Ala Leu Pro
245 250 120250PRTArtificial SequenceBispecific EGFR/c-Met binding
molecule 120Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr
Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Asp Asp Pro Trp Ala Phe
Tyr Glu Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val
Gly Glu Ala Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser
Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val
Ser Ile Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Met
Arg Gly Leu Pro Leu Ser Ala Ile Phe Thr Thr Ala 85 90 95 Pro Ala
Pro Ala Pro Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu 100 105 110
Val Val Ser Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala 115
120 125 Pro Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu Phe
Leu 130 135 140 Gly Ser Gly Glu Ala Ile Val Leu Thr Val Pro Gly Ser
Glu Arg Ser 145 150 155 160 Tyr Asp Leu Thr Gly Leu Lys Pro Gly Thr
Glu Tyr Val Val Asn Ile 165 170 175 Met Gly Val Lys Gly Gly Lys Ile
Ser Pro Pro Leu Ser Ala Ile Phe 180 185 190 Thr Thr Ala Pro Ala Pro
Ala Pro Ala Pro Ala Pro Leu Ala Glu Ala 195 200 205 Lys Val Leu Ala
Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr 210 215 220 Tyr Lys
Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala 225 230 235
240 Leu Leu Asp Glu Ile Leu Ala Ala Leu Pro 245 250
121250PRTArtificial SequenceBispecific EGFR/c-Met binding molecule
121Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp
1 5 10 15 Ser Ala Arg Leu Ser Trp Ala Asp Pro His Gly Phe Tyr Asp
Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu
Ala Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp
Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile
Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Met Arg Gly
Leu Pro Leu Ser Ala Ile Phe Thr Thr Ala 85 90 95 Pro Ala Pro Ala
Pro Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu 100 105 110 Val Val
Ser Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala 115 120 125
Pro Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu Phe Leu 130
135 140 Gly Ser Gly Glu Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg
Ser 145 150 155 160 Tyr Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr
Val Val Asn Ile 165 170 175 Met Gly Val Lys Gly Gly Lys Ile Ser Pro
Pro Leu Ser Ala Ile Phe 180 185 190 Thr Thr Ala Pro Ala Pro Ala Pro
Ala Pro Ala Pro Leu Ala Glu Ala 195 200 205 Lys Val Leu Ala Asn Arg
Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr 210 215 220 Tyr Lys Asn Leu
Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala 225 230 235 240 Leu
Leu Asp Glu Ile Leu Ala Ala Leu Pro 245 250 12295PRTArtificial
SequenceEGFR binding FN3 domain 122Met Leu Pro Ala Pro Lys Asn Leu
Val Val Ser Glu Val Thr Glu Asp 1 5 10 15 Ser Leu Arg Leu Ser Trp
Ala Asp Pro His Gly Phe Tyr Asp Ser Phe 20 25 30 Leu Ile Gln Tyr
Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Asn Leu 35 40 45 Thr Val
Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60
Gly Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys 65
70 75 80 Asp Thr Asn Met Arg Gly Leu Pro Leu Ser Ala Glu Phe Thr
Thr 85 90 95 12395PRTArtificial SequenceEGFR binding FN3 domain
123Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp
1 5 10 15 Ser Leu Arg Leu Ser Trp Thr Tyr Asp Arg Asp Gly Tyr Asp
Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu
Ala Ile Asn Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp
Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile
Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Met Arg Gly
Leu Pro Leu Ser Ala Glu Phe Thr Thr 85 90 95 12495PRTArtificial
SequenceEGFR binding FN3 domain 124Met Leu Pro Ala Pro Lys Asn Leu
Val Val Ser Glu Val Thr Glu Asp 1 5 10 15 Ser Leu Arg Leu Ser Trp
Gly Tyr Asn Gly Asp His Phe Asp Ser Phe 20 25 30 Leu Ile Gln Tyr
Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Asn Leu 35 40 45 Thr Val
Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60
Gly Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys 65
70 75 80 Asp Thr Asn Met Arg Gly Leu Pro Leu Ser Ala Glu Phe Thr
Thr 85 90 95 12595PRTArtificial SequenceEGFR binding FN3 domain
125Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp
1 5 10 15 Ser Leu Arg Leu Ser Trp Asp Asp Pro Arg Gly Phe Tyr Glu
Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu
Ala Ile Asn Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp
Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile
Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Met Arg Gly
Leu Pro Leu Ser Ala Glu Phe Thr Thr 85 90 95 12695PRTArtificial
SequenceEGFR binding FN3 domain 126Met Leu Pro Ala Pro Lys Asn Leu
Val Val Ser Glu Val Thr Glu Asp 1 5 10 15 Ser Leu Arg Leu Ser Trp
Thr Trp Pro Tyr Ala Asp Leu Asp Ser Phe 20 25 30 Leu Ile Gln Tyr
Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Asn Leu 35 40 45 Thr Val
Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60
Gly Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys 65
70 75 80 Asp Thr Asn Met Arg Gly Leu Pro Leu Ser Ala Glu Phe Thr
Thr 85 90 95 12795PRTArtificial SequenceEGFR binding FN3 domain
127Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp
1 5 10 15 Ser Leu Arg Leu Ser Trp Gly Tyr Asn Gly Asp His Phe Asp
Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu
Ala Ile Asn Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp
Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile
Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Met Arg Gly
Leu Pro Leu Ser Ala Glu Phe Thr Thr 85 90 95 12896PRTArtificial
SequenceEGFR binding FN3 domain 128Met Leu Pro Ala Pro Lys Asn Leu
Val Val Ser Glu Val Thr Glu Asp 1 5 10 15 Ser Leu Arg Leu Ser Trp
Asp Tyr Asp Leu Gly Val Tyr Phe Asp Ser 20 25 30 Phe Leu Ile Gln
Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Asn 35 40 45 Leu Thr
Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys 50 55 60
Pro Gly Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr 65
70 75 80 Lys Asp Thr Asn Met Arg Gly Leu Pro Leu Ser Ala Glu Phe
Thr Thr 85 90 95 12995PRTArtificial SequenceEGFR binding FN3 domain
129Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp
1 5 10 15 Ser Leu Arg Leu Ser Trp Asp Asp Pro Trp Ala Phe Tyr Glu
Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu
Ala Ile Asn Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp
Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile
Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Met Arg Gly
Leu Pro Leu Ser Ala Glu Phe Thr Thr 85 90 95 13095PRTArtificial
SequenceEGFR binding FN3 domain 130Met Leu Pro Ala Pro Lys Asn Leu
Val Val Ser Glu Val Thr Glu Asp 1 5 10 15 Ser Leu Arg Leu Ser Trp
Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe 20 25 30 Leu Ile Gln Tyr
Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Asn Leu 35 40 45 Thr Val
Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55
60
Gly Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val Leu Gly Ser Tyr Val 65
70 75 80 Phe Glu His Asp Val Met Leu Pro Leu Ser Ala Glu Phe Thr
Thr 85 90 95 13195PRTArtificial SequenceEGFR binding FN3 domain
131Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp
1 5 10 15 Ser Ala Arg Leu Ser Trp Asp Asp Pro Trp Ala Phe Tyr Glu
Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu
Ala Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp
Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile
Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Met Arg Gly
Leu Pro Leu Ser Ala Ile Phe Thr Thr 85 90 95 13295PRTArtificial
SequenceEGFR binding FN3 domain 132Met Leu Pro Ala Pro Lys Asn Leu
Val Val Ser Glu Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp
Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe 20 25 30 Leu Ile Gln Tyr
Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu 35 40 45 Thr Val
Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60
Gly Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val Leu Gly Ser Tyr Val 65
70 75 80 Phe Glu His Asp Val Met Leu Pro Leu Ser Ala Ile Phe Thr
Thr 85 90 95 13395PRTArtificial SequenceEGFR binding FN3 domain
133Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp
1 5 10 15 Ser Leu Arg Leu Ser Trp Thr Trp Pro Tyr Ala Asp Leu Asp
Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu
Ala Ile Asn Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp
Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile
Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Met Arg Gly
Leu Pro Leu Ser Ala Glu Phe Thr Thr 85 90 95 13495PRTArtificial
SequenceEGFR binding FN3 domain 134Met Leu Pro Ala Pro Lys Asn Leu
Val Val Ser Glu Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp
Ala Asp Pro His Gly Phe Tyr Asp Ser Phe 20 25 30 Leu Ile Gln Tyr
Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu 35 40 45 Thr Val
Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60
Gly Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys 65
70 75 80 Asp Thr Asn Met Arg Gly Leu Pro Leu Ser Ala Ile Phe Thr
Thr 85 90 95 13595PRTArtificial SequenceEGFR binding FN3 domain
135Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp
1 5 10 15 Ser Ala Arg Leu Ser Trp Asp Asp Pro Trp Ala Phe Tyr Glu
Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu
Ala Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp
Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile
Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Ile Arg Gly
Leu Pro Leu Ser Ala Ile Phe Thr Thr 85 90 95 13695PRTArtificial
SequenceEGFR binding FN3 domain 136Met Leu Pro Ala Pro Lys Asn Leu
Val Val Ser Glu Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp
Asp Asp Pro His Ala Phe Tyr Glu Ser Phe 20 25 30 Leu Ile Gln Tyr
Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu 35 40 45 Thr Val
Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60
Gly Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys 65
70 75 80 Asp Thr Asn Ile Arg Gly Leu Pro Leu Ser Ala Ile Phe Thr
Thr 85 90 95 13795PRTArtificial SequenceEGFR binding FN3 domain
137Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp
1 5 10 15 Ser Ala Arg Leu Ser Trp Ala Asp Pro His Gly Phe Tyr Asp
Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu
Ala Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp
Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile
Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Ile Arg Gly
Leu Pro Leu Ser Ala Ile Phe Thr Thr 85 90 95 138204PRTArtificial
SequenceBispecific EGFR/c-Met binding domain 138Leu Pro Ala Pro Lys
Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser 1 5 10 15 Ala Arg Leu
Ser Trp Asp Asp Pro Trp Ala Phe Tyr Glu Ser Phe Leu 20 25 30 Ile
Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu Thr 35 40
45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly
50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr
Lys Asp 65 70 75 80 Thr Asn Met Arg Gly Leu Pro Leu Ser Ala Ile Phe
Thr Thr Gly Gly 85 90 95 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly 100 105 110 Gly Ser Met Leu Pro Ala Pro Lys
Asn Leu Val Val Ser Arg Val Thr 115 120 125 Glu Asp Ser Ala Arg Leu
Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp 130 135 140 Ser Phe Trp Ile
Arg Tyr Asp Glu Val Val Val Gly Gly Glu Ala Ile 145 150 155 160 Val
Leu Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu 165 170
175 Lys Pro Gly Thr Glu Tyr Tyr Val Asn Ile Leu Gly Val Lys Gly Gly
180 185 190 Ser Ile Ser Val Pro Leu Ser Ala Ile Phe Thr Thr 195 200
139203PRTArtificial SequenceBispecific EGFR/c-Met binding domain
139Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser
1 5 10 15 Ala Arg Leu Ser Trp Asp Asp Pro Trp Ala Phe Tyr Glu Ser
Phe Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala
Ile Val Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu
Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr
Gly Val His Asn Val Tyr Lys Asp 65 70 75 80 Thr Asn Met Arg Gly Leu
Pro Leu Ser Ala Ile Phe Thr Thr Gly Gly 85 90 95 Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 100 105 110 Gly Ser
Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu 115 120 125
Asp Ser Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser 130
135 140 Phe Trp Ile Arg Tyr Phe Glu Phe Leu Gly Ser Gly Glu Ala Ile
Val 145 150 155 160 Leu Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu
Thr Gly Leu Lys 165 170 175 Pro Gly Thr Glu Tyr Val Val Asn Ile Met
Gly Val Lys Gly Gly Lys 180 185 190 Ile Ser Pro Pro Leu Ser Ala Ile
Phe Thr Thr 195 200 140204PRTArtificial SequenceBispecific
EGFR/c-Met binding domain 140Leu Pro Ala Pro Lys Asn Leu Val Val
Ser Glu Val Thr Glu Asp Ser 1 5 10 15 Ala Arg Leu Ser Trp Asp Asp
Pro Trp Ala Phe Tyr Glu Ser Phe Leu 20 25 30 Ile Gln Tyr Gln Glu
Ser Glu Lys Val Gly Glu Ala Ile Val Leu Thr 35 40 45 Val Pro Gly
Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60 Thr
Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys Asp 65 70
75 80 Thr Asn Met Arg Gly Leu Pro Leu Ser Ala Ile Phe Thr Thr Gly
Gly 85 90 95 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly 100 105 110 Gly Ser Met Leu Pro Ala Pro Lys Asn Leu Val
Val Ser Arg Val Thr 115 120 125 Glu Asp Ser Ala Arg Leu Ser Trp Thr
Ala Pro Asp Ala Ala Phe Asp 130 135 140 Ser Phe Trp Ile Arg Tyr Phe
Glu Phe Leu Gly Ser Gly Glu Ala Ile 145 150 155 160 Val Leu Thr Val
Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu 165 170 175 Lys Pro
Gly Thr Glu Tyr Val Val Gln Ile Ile Gly Val Lys Gly Gly 180 185 190
His Ile Ser Leu Pro Leu Ser Ala Ile Phe Thr Thr 195 200
141204PRTArtificial SequenceBispecific EGFR/c-Met binding domain
141Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser
1 5 10 15 Ala Arg Leu Ser Trp Asp Asp Pro Trp Ala Phe Tyr Glu Ser
Phe Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala
Ile Val Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu
Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr
Gly Val His Asn Val Tyr Lys Asp 65 70 75 80 Thr Asn Met Arg Gly Leu
Pro Leu Ser Ala Ile Phe Thr Thr Gly Gly 85 90 95 Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 100 105 110 Gly Ser
Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr 115 120 125
Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp 130
135 140 Ser Phe Phe Ile Arg Tyr Asp Glu Phe Leu Arg Ser Gly Glu Ala
Ile 145 150 155 160 Val Leu Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp
Leu Thr Gly Leu 165 170 175 Lys Pro Gly Thr Glu Tyr Trp Val Thr Ile
Leu Gly Val Lys Gly Gly 180 185 190 Leu Val Ser Thr Pro Leu Ser Ala
Ile Phe Thr Thr 195 200 142204PRTArtificial SequenceBispecific
EGFR/c-Met binding domain 142Leu Pro Ala Pro Lys Asn Leu Val Val
Ser Glu Val Thr Glu Asp Ser 1 5 10 15 Ala Arg Leu Ser Trp Ala Asp
Pro His Gly Phe Tyr Asp Ser Phe Leu 20 25 30 Ile Gln Tyr Gln Glu
Ser Glu Lys Val Gly Glu Ala Ile Val Leu Thr 35 40 45 Val Pro Gly
Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60 Thr
Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys Asp 65 70
75 80 Thr Asn Met Arg Gly Leu Pro Leu Ser Ala Ile Phe Thr Thr Gly
Gly 85 90 95 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly 100 105 110 Gly Ser Met Leu Pro Ala Pro Lys Asn Leu Val
Val Ser Arg Val Thr 115 120 125 Glu Asp Ser Ala Arg Leu Ser Trp Thr
Ala Pro Asp Ala Ala Phe Asp 130 135 140 Ser Phe Trp Ile Arg Tyr Phe
Glu Phe Leu Gly Ser Gly Glu Ala Ile 145 150 155 160 Val Leu Thr Val
Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu 165 170 175 Lys Pro
Gly Thr Glu Tyr Val Val Asn Ile Met Gly Val Lys Gly Gly 180 185 190
Lys Ile Ser Pro Pro Leu Ser Ala Ile Phe Thr Thr 195 200
143204PRTArtificial SequenceBispecific EGFR/c-Met binding domain
143Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser
1 5 10 15 Ala Arg Leu Ser Trp Ala Asp Pro His Gly Phe Tyr Asp Ser
Phe Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala
Ile Val Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu
Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr
Gly Val His Asn Val Tyr Lys Asp 65 70 75 80 Thr Asn Met Arg Gly Leu
Pro Leu Ser Ala Ile Phe Thr Thr Gly Gly 85 90 95 Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 100 105 110 Gly Ser
Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr 115 120 125
Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp 130
135 140 Ser Phe Trp Ile Arg Tyr Phe Glu Phe Leu Gly Ser Gly Glu Ala
Ile 145 150 155 160 Val Leu Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp
Leu Thr Gly Leu 165 170 175 Lys Pro Gly Thr Glu Tyr Val Val Gln Ile
Ile Gly Val Lys Gly Gly 180 185 190 His Ile Ser Leu Pro Leu Ser Ala
Ile Phe Thr Thr 195 200 144204PRTArtificial SequenceBispecific
EGFR/c-Met binding domain 144Leu Pro Ala Pro Lys Asn Leu Val Val
Ser Glu Val Thr Glu Asp Ser 1 5 10 15 Ala Arg Leu Ser Trp Ala Asp
Pro His Gly Phe Tyr Asp Ser Phe Leu 20 25 30 Ile Gln Tyr Gln Glu
Ser Glu Lys Val Gly Glu Ala Ile Val Leu Thr 35 40 45 Val Pro Gly
Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60 Thr
Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys Asp 65 70
75 80 Thr Asn Met Arg Gly Leu Pro Leu Ser Ala Ile Phe Thr Thr Gly
Gly 85 90 95 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly 100 105 110 Gly Ser Met Leu Pro Ala Pro Lys Asn Leu Val
Val Ser Arg Val Thr 115 120 125 Glu Asp Ser Ala Arg Leu Ser Trp Thr
Ala Pro Asp Ala Ala Phe Asp 130 135 140 Ser Phe Trp Ile Arg Tyr Phe
Glu Phe Leu Gly Ser Gly Glu Ala Ile 145 150 155 160 Val Leu Thr Val
Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu 165 170 175 Lys Pro
Gly Thr Glu Tyr Val Val Gln Ile Ile Gly Val Lys Gly Gly 180 185 190
His Ile Ser Leu Pro Leu Ser Ala Ile Phe Thr Thr 195 200
145193PRTArtificial SequenceBispecific EGFR/c-Met binding domain
145Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser
1 5 10 15 Ala Arg Leu Ser Trp Asp Asp Pro Trp Ala Phe Tyr Glu Ser
Phe Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala
Ile Val Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu
Thr Gly
Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val
His Asn Val Tyr Lys Asp 65 70 75 80 Thr Asn Met Arg Gly Leu Pro Leu
Ser Ala Ile Phe Thr Thr Ala Pro 85 90 95 Ala Pro Ala Pro Ala Pro
Ala Pro Leu Pro Ala Pro Lys Asn Leu Val 100 105 110 Val Ser Arg Val
Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala Pro 115 120 125 Asp Ala
Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu Phe Leu Gly 130 135 140
Ser Gly Glu Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg Ser Tyr 145
150 155 160 Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr Val Val Asn
Ile Met 165 170 175 Gly Val Lys Gly Gly Lys Ile Ser Pro Pro Leu Ser
Ala Ile Phe Thr 180 185 190 Thr 146193PRTArtificial
SequenceBispecific EGFR/c-Met binding domain 146Leu Pro Ala Pro Lys
Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser 1 5 10 15 Ala Arg Leu
Ser Trp Asp Asp Pro Trp Ala Phe Tyr Glu Ser Phe Leu 20 25 30 Ile
Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu Thr 35 40
45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly
50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr
Lys Asp 65 70 75 80 Thr Asn Met Arg Gly Leu Pro Leu Ser Ala Ile Phe
Thr Thr Ala Pro 85 90 95 Ala Pro Ala Pro Ala Pro Ala Pro Leu Pro
Ala Pro Lys Asn Leu Val 100 105 110 Val Ser Arg Val Thr Glu Asp Ser
Ala Arg Leu Ser Trp Thr Ala Pro 115 120 125 Asp Ala Ala Phe Asp Ser
Phe Trp Ile Arg Tyr Asp Glu Val Val Val 130 135 140 Gly Gly Glu Ala
Ile Val Leu Thr Val Pro Gly Ser Glu Arg Ser Tyr 145 150 155 160 Asp
Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr Tyr Val Asn Ile Leu 165 170
175 Gly Val Lys Gly Gly Ser Ile Ser Val Pro Leu Ser Ala Ile Phe Thr
180 185 190 Thr 147194PRTArtificial SequenceBispecific EGFR/c-Met
binding domain 147Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val
Thr Glu Asp Ser 1 5 10 15 Ala Arg Leu Ser Trp Ala Asp Pro His Gly
Phe Tyr Asp Ser Phe Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu Lys
Val Gly Glu Ala Ile Val Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg
Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Thr
Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys Asp 65 70 75 80 Thr Asn
Met Arg Gly Leu Pro Leu Ser Ala Ile Phe Thr Thr Ala Pro 85 90 95
Ala Pro Ala Pro Ala Pro Ala Pro Met Leu Pro Ala Pro Lys Asn Leu 100
105 110 Val Val Ser Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr
Ala 115 120 125 Pro Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe
Glu Phe Leu 130 135 140 Gly Ser Gly Glu Ala Ile Val Leu Thr Val Pro
Gly Ser Glu Arg Ser 145 150 155 160 Tyr Asp Leu Thr Gly Leu Lys Pro
Gly Thr Glu Tyr Val Val Asn Ile 165 170 175 Met Gly Val Lys Gly Gly
Lys Ile Ser Pro Pro Leu Ser Ala Ile Phe 180 185 190 Thr Thr
148193PRTArtificial SequenceBispecific EGFR/c-Met binding domain
148Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser
1 5 10 15 Ala Arg Leu Ser Trp Ala Asp Pro His Gly Phe Tyr Asp Ser
Phe Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala
Ile Val Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu
Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr
Gly Val His Asn Val Tyr Lys Asp 65 70 75 80 Thr Asn Met Arg Gly Leu
Pro Leu Ser Ala Ile Phe Thr Thr Ala Pro 85 90 95 Ala Pro Ala Pro
Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu Val 100 105 110 Val Ser
Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala Pro 115 120 125
Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Asp Glu Val Val Val 130
135 140 Gly Gly Glu Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg Ser
Tyr 145 150 155 160 Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr Tyr
Val Asn Ile Leu 165 170 175 Gly Val Lys Gly Gly Ser Ile Ser Val Pro
Leu Ser Ala Ile Phe Thr 180 185 190 Thr 149193PRTArtificial
SequenceBispecific EGFR/c-Met binding domain 149Leu Pro Ala Pro Lys
Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser 1 5 10 15 Ala Arg Leu
Ser Trp Asp Asp Pro Trp Ala Phe Tyr Glu Ser Phe Leu 20 25 30 Ile
Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu Thr 35 40
45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly
50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr
Lys Asp 65 70 75 80 Thr Asn Ile Arg Gly Leu Pro Leu Ser Ala Ile Phe
Thr Thr Ala Pro 85 90 95 Ala Pro Ala Pro Ala Pro Ala Pro Leu Pro
Ala Pro Lys Asn Leu Val 100 105 110 Val Ser Arg Val Thr Glu Asp Ser
Ala Arg Leu Ser Trp Thr Ala Pro 115 120 125 Asp Ala Ala Phe Asp Ser
Phe Trp Ile Arg Tyr Phe Glu Phe Leu Gly 130 135 140 Ser Gly Glu Ala
Ile Val Leu Thr Val Pro Gly Ser Glu Arg Ser Tyr 145 150 155 160 Asp
Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr Val Val Asn Ile Leu 165 170
175 Gly Val Lys Gly Gly Lys Ile Ser Pro Pro Leu Ser Ala Ile Phe Thr
180 185 190 Thr 150193PRTArtificial SequenceBispecific EGFR/c-Met
binding domain 150Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val
Thr Glu Asp Ser 1 5 10 15 Ala Arg Leu Ser Trp Asp Asp Pro Trp Ala
Phe Tyr Glu Ser Phe Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu Lys
Val Gly Glu Ala Ile Val Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg
Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Thr
Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys Asp 65 70 75 80 Thr Asn
Ile Arg Gly Leu Pro Leu Ser Ala Ile Phe Thr Thr Ala Pro 85 90 95
Ala Pro Ala Pro Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu Val 100
105 110 Val Ser Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala
Pro 115 120 125 Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu
Phe Val Gly 130 135 140 Ser Gly Glu Ala Ile Val Leu Thr Val Pro Gly
Ser Glu Arg Ser Tyr 145 150 155 160 Asp Leu Thr Gly Leu Lys Pro Gly
Thr Glu Tyr Val Val Asn Ile Leu 165 170 175 Gly Val Lys Gly Gly Ser
Ile Ser Pro Pro Leu Ser Ala Ile Phe Thr 180 185 190 Thr
151193PRTArtificial SequenceBispecific EGFR/c-Met binding domain
151Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser
1 5 10 15 Ala Arg Leu Ser Trp Asp Asp Pro Trp Ala Phe Tyr Glu Ser
Phe Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala
Ile Val Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu
Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr
Gly Val His Asn Val Tyr Lys Asp 65 70 75 80 Thr Asn Ile Arg Gly Leu
Pro Leu Ser Ala Ile Phe Thr Thr Ala Pro 85 90 95 Ala Pro Ala Pro
Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu Val 100 105 110 Val Ser
Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala Pro 115 120 125
Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu Phe Val Ser 130
135 140 Lys Gly Asp Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg Ser
Tyr 145 150 155 160 Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr Val
Val Asn Ile Leu 165 170 175 Gly Val Lys Gly Gly Ser Ile Ser Pro Pro
Leu Ser Ala Ile Phe Thr 180 185 190 Thr 152193PRTArtificial
SequenceBispecific EGFR/c-Met binding domain 152Leu Pro Ala Pro Lys
Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser 1 5 10 15 Ala Arg Leu
Ser Trp Asp Asp Pro Trp Ala Phe Tyr Glu Ser Phe Leu 20 25 30 Ile
Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu Thr 35 40
45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly
50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr
Lys Asp 65 70 75 80 Thr Asn Ile Arg Gly Leu Pro Leu Ser Ala Ile Phe
Thr Thr Ala Pro 85 90 95 Ala Pro Ala Pro Ala Pro Ala Pro Leu Pro
Ala Pro Lys Asn Leu Val 100 105 110 Val Ser Arg Val Thr Glu Asp Ser
Ala Arg Leu Ser Trp Thr Ala Pro 115 120 125 Asp Ala Ala Phe Asp Ser
Phe Trp Ile Arg Tyr Phe Glu Phe Leu Gly 130 135 140 Ser Gly Glu Ala
Ile Val Leu Thr Val Pro Gly Ser Glu Arg Ser Tyr 145 150 155 160 Asp
Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr Val Val Asn Ile Leu 165 170
175 Ser Val Lys Gly Gly Ser Ile Ser Pro Pro Leu Ser Ala Ile Phe Thr
180 185 190 Thr 153193PRTArtificial SequenceBispecific EGFR/c-Met
binding domain 153Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val
Thr Glu Asp Ser 1 5 10 15 Ala Arg Leu Ser Trp Asp Asp Pro His Ala
Phe Tyr Glu Ser Phe Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu Lys
Val Gly Glu Ala Ile Val Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg
Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Thr
Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys Asp 65 70 75 80 Thr Asn
Ile Arg Gly Leu Pro Leu Ser Ala Ile Phe Thr Thr Ala Pro 85 90 95
Ala Pro Ala Pro Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu Val 100
105 110 Val Ser Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala
Pro 115 120 125 Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu
Phe Leu Gly 130 135 140 Ser Gly Glu Ala Ile Val Leu Thr Val Pro Gly
Ser Glu Arg Ser Tyr 145 150 155 160 Asp Leu Thr Gly Leu Lys Pro Gly
Thr Glu Tyr Val Val Asn Ile Leu 165 170 175 Gly Val Lys Gly Gly Lys
Ile Ser Pro Pro Leu Ser Ala Ile Phe Thr 180 185 190 Thr
154193PRTArtificial SequenceBispecific EGFR/c-Met binding domain
154Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser
1 5 10 15 Ala Arg Leu Ser Trp Asp Asp Pro His Ala Phe Tyr Glu Ser
Phe Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala
Ile Val Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu
Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr
Gly Val His Asn Val Tyr Lys Asp 65 70 75 80 Thr Asn Ile Arg Gly Leu
Pro Leu Ser Ala Ile Phe Thr Thr Ala Pro 85 90 95 Ala Pro Ala Pro
Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu Val 100 105 110 Val Ser
Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala Pro 115 120 125
Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu Phe Val Gly 130
135 140 Ser Gly Glu Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg Ser
Tyr 145 150 155 160 Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr Val
Val Asn Ile Leu 165 170 175 Gly Val Lys Gly Gly Ser Ile Ser Pro Pro
Leu Ser Ala Ile Phe Thr 180 185 190 Thr 155193PRTArtificial
SequenceBispecific EGFR/c-Met binding domain 155Leu Pro Ala Pro Lys
Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser 1 5 10 15 Ala Arg Leu
Ser Trp Asp Asp Pro His Ala Phe Tyr Glu Ser Phe Leu 20 25 30 Ile
Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu Thr 35 40
45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly
50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr
Lys Asp 65 70 75 80 Thr Asn Ile Arg Gly Leu Pro Leu Ser Ala Ile Phe
Thr Thr Ala Pro 85 90 95 Ala Pro Ala Pro Ala Pro Ala Pro Leu Pro
Ala Pro Lys Asn Leu Val 100 105 110 Val Ser Arg Val Thr Glu Asp Ser
Ala Arg Leu Ser Trp Thr Ala Pro 115 120 125 Asp Ala Ala Phe Asp Ser
Phe Trp Ile Arg Tyr Phe Glu Phe Val Ser 130 135 140 Lys Gly Asp Ala
Ile Val Leu Thr Val Pro Gly Ser Glu Arg Ser Tyr 145 150 155 160 Asp
Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr Val Val Asn Ile Leu 165 170
175 Gly Val Lys Gly Gly Ser Ile Ser Pro Pro Leu Ser Ala Ile Phe Thr
180 185 190 Thr 156193PRTArtificial SequenceBispecific EGFR/c-Met
binding domain 156Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val
Thr Glu Asp Ser 1 5 10 15 Ala Arg Leu Ser Trp Asp Asp Pro His Ala
Phe Tyr Glu Ser Phe Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu Lys
Val Gly Glu Ala Ile Val Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg
Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Thr
Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys Asp 65 70 75 80 Thr Asn
Ile Arg Gly Leu Pro Leu Ser Ala Ile Phe Thr Thr Ala Pro 85 90 95
Ala Pro Ala Pro Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu Val 100
105 110 Val Ser Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala
Pro 115 120 125 Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu
Phe Leu Gly 130
135 140 Ser Gly Glu Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg Ser
Tyr 145 150 155 160 Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr Val
Val Asn Ile Leu 165 170 175 Ser Val Lys Gly Gly Ser Ile Ser Pro Pro
Leu Ser Ala Ile Phe Thr 180 185 190 Thr 157193PRTArtificial
SequenceBispecific EGFR/c-Met binding domain 157Leu Pro Ala Pro Lys
Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser 1 5 10 15 Ala Arg Leu
Ser Trp Ala Asp Pro His Gly Phe Tyr Asp Ser Phe Leu 20 25 30 Ile
Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu Thr 35 40
45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly
50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr
Lys Asp 65 70 75 80 Thr Asn Ile Arg Gly Leu Pro Leu Ser Ala Ile Phe
Thr Thr Ala Pro 85 90 95 Ala Pro Ala Pro Ala Pro Ala Pro Leu Pro
Ala Pro Lys Asn Leu Val 100 105 110 Val Ser Arg Val Thr Glu Asp Ser
Ala Arg Leu Ser Trp Thr Ala Pro 115 120 125 Asp Ala Ala Phe Asp Ser
Phe Trp Ile Arg Tyr Phe Glu Phe Leu Gly 130 135 140 Ser Gly Glu Ala
Ile Val Leu Thr Val Pro Gly Ser Glu Arg Ser Tyr 145 150 155 160 Asp
Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr Val Val Asn Ile Leu 165 170
175 Gly Val Lys Gly Gly Lys Ile Ser Pro Pro Leu Ser Ala Ile Phe Thr
180 185 190 Thr 158193PRTArtificial SequenceBispecific EGFR/c-Met
binding domain 158Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val
Thr Glu Asp Ser 1 5 10 15 Ala Arg Leu Ser Trp Ala Asp Pro His Gly
Phe Tyr Asp Ser Phe Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu Lys
Val Gly Glu Ala Ile Val Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg
Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Thr
Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys Asp 65 70 75 80 Thr Asn
Ile Arg Gly Leu Pro Leu Ser Ala Ile Phe Thr Thr Ala Pro 85 90 95
Ala Pro Ala Pro Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu Val 100
105 110 Val Ser Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala
Pro 115 120 125 Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu
Phe Val Gly 130 135 140 Ser Gly Glu Ala Ile Val Leu Thr Val Pro Gly
Ser Glu Arg Ser Tyr 145 150 155 160 Asp Leu Thr Gly Leu Lys Pro Gly
Thr Glu Tyr Val Val Asn Ile Leu 165 170 175 Gly Val Lys Gly Gly Ser
Ile Ser Pro Pro Leu Ser Ala Ile Phe Thr 180 185 190 Thr
159193PRTArtificial SequenceBispecific EGFR/c-Met binding domain
159Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser
1 5 10 15 Ala Arg Leu Ser Trp Ala Asp Pro His Gly Phe Tyr Asp Ser
Phe Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala
Ile Val Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu
Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr
Gly Val His Asn Val Tyr Lys Asp 65 70 75 80 Thr Asn Ile Arg Gly Leu
Pro Leu Ser Ala Ile Phe Thr Thr Ala Pro 85 90 95 Ala Pro Ala Pro
Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu Val 100 105 110 Val Ser
Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala Pro 115 120 125
Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu Phe Val Ser 130
135 140 Lys Gly Asp Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg Ser
Tyr 145 150 155 160 Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr Val
Val Asn Ile Leu 165 170 175 Gly Val Lys Gly Gly Ser Ile Ser Pro Pro
Leu Ser Ala Ile Phe Thr 180 185 190 Thr 160193PRTArtificial
SequenceBispecific EGFR/c-Met binding domain 160Leu Pro Ala Pro Lys
Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser 1 5 10 15 Ala Arg Leu
Ser Trp Ala Asp Pro His Gly Phe Tyr Asp Ser Phe Leu 20 25 30 Ile
Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu Thr 35 40
45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly
50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr
Lys Asp 65 70 75 80 Thr Asn Ile Arg Gly Leu Pro Leu Ser Ala Ile Phe
Thr Thr Ala Pro 85 90 95 Ala Pro Ala Pro Ala Pro Ala Pro Leu Pro
Ala Pro Lys Asn Leu Val 100 105 110 Val Ser Arg Val Thr Glu Asp Ser
Ala Arg Leu Ser Trp Thr Ala Pro 115 120 125 Asp Ala Ala Phe Asp Ser
Phe Trp Ile Arg Tyr Phe Glu Phe Leu Gly 130 135 140 Ser Gly Glu Ala
Ile Val Leu Thr Val Pro Gly Ser Glu Arg Ser Tyr 145 150 155 160 Asp
Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr Val Val Asn Ile Leu 165 170
175 Ser Val Lys Gly Gly Ser Ile Ser Pro Pro Leu Ser Ala Ile Phe Thr
180 185 190 Thr 161193PRTArtificial SequenceBispecific EGFR/c-Met
binding domain 161Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val
Thr Glu Asp Ser 1 5 10 15 Ala Arg Leu Ser Trp Asp Asp Pro Trp Ala
Phe Tyr Glu Ser Phe Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu Lys
Val Gly Glu Ala Ile Val Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg
Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Thr
Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys Asp 65 70 75 80 Thr Asn
Ile Arg Gly Leu Pro Leu Ser Ala Ile Phe Thr Thr Ala Pro 85 90 95
Ala Pro Ala Pro Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu Val 100
105 110 Val Ser Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala
Pro 115 120 125 Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu
Phe Leu Gly 130 135 140 Ser Gly Glu Ala Ile Val Leu Thr Val Pro Gly
Ser Glu Arg Ser Tyr 145 150 155 160 Asp Leu Thr Gly Leu Lys Pro Gly
Thr Glu Tyr Val Val Asn Ile Leu 165 170 175 Ser Val Lys Gly Gly Ser
Ile Ser Pro Pro Leu Ser Ala Ile Phe Thr 180 185 190 Thr
162249PRTArtificial SequenceBispecific EGFR/c-Met binding domain
162Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser
1 5 10 15 Ala Arg Leu Ser Trp Asp Asp Pro Trp Ala Phe Tyr Glu Ser
Phe Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala
Ile Val Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu
Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr
Gly Val His Asn Val Tyr Lys Asp 65 70 75 80 Thr Asn Met Arg Gly Leu
Pro Leu Ser Ala Ile Phe Thr Thr Ala Pro 85 90 95 Ala Pro Ala Pro
Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu Val 100 105 110 Val Ser
Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala Pro 115 120 125
Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Asp Glu Val Val Val 130
135 140 Gly Gly Glu Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg Ser
Tyr 145 150 155 160 Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr Tyr
Val Asn Ile Leu 165 170 175 Gly Val Lys Gly Gly Ser Ile Ser Val Pro
Leu Ser Ala Ile Phe Thr 180 185 190 Thr Ala Pro Ala Pro Ala Pro Ala
Pro Ala Pro Leu Ala Glu Ala Lys 195 200 205 Val Leu Ala Asn Arg Glu
Leu Asp Lys Tyr Gly Val Ser Asp Tyr Tyr 210 215 220 Lys Asn Leu Ile
Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala Leu 225 230 235 240 Leu
Asp Glu Ile Leu Ala Ala Leu Pro 245 163249PRTArtificial
SequenceBispecific EGFR/c-Met binding domain 163Leu Pro Ala Pro Lys
Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser 1 5 10 15 Ala Arg Leu
Ser Trp Ala Asp Pro His Gly Phe Tyr Asp Ser Phe Leu 20 25 30 Ile
Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu Thr 35 40
45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly
50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr
Lys Asp 65 70 75 80 Thr Asn Met Arg Gly Leu Pro Leu Ser Ala Ile Phe
Thr Thr Ala Pro 85 90 95 Ala Pro Ala Pro Ala Pro Ala Pro Leu Pro
Ala Pro Lys Asn Leu Val 100 105 110 Val Ser Arg Val Thr Glu Asp Ser
Ala Arg Leu Ser Trp Thr Ala Pro 115 120 125 Asp Ala Ala Phe Asp Ser
Phe Trp Ile Arg Tyr Asp Glu Val Val Val 130 135 140 Gly Gly Glu Ala
Ile Val Leu Thr Val Pro Gly Ser Glu Arg Ser Tyr 145 150 155 160 Asp
Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr Tyr Val Asn Ile Leu 165 170
175 Gly Val Lys Gly Gly Ser Ile Ser Val Pro Leu Ser Ala Ile Phe Thr
180 185 190 Thr Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Leu Ala Glu
Ala Lys 195 200 205 Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val
Ser Asp Tyr Tyr 210 215 220 Lys Asn Leu Ile Asn Asn Ala Lys Thr Val
Glu Gly Val Lys Ala Leu 225 230 235 240 Leu Asp Glu Ile Leu Ala Ala
Leu Pro 245 164249PRTArtificial SequenceBispecific EGFR/c-Met
binding domain 164Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val
Thr Glu Asp Ser 1 5 10 15 Ala Arg Leu Ser Trp Asp Asp Pro Trp Ala
Phe Tyr Glu Ser Phe Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu Lys
Val Gly Glu Ala Ile Val Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg
Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Thr
Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys Asp 65 70 75 80 Thr Asn
Met Arg Gly Leu Pro Leu Ser Ala Ile Phe Thr Thr Ala Pro 85 90 95
Ala Pro Ala Pro Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu Val 100
105 110 Val Ser Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala
Pro 115 120 125 Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu
Phe Leu Gly 130 135 140 Ser Gly Glu Ala Ile Val Leu Thr Val Pro Gly
Ser Glu Arg Ser Tyr 145 150 155 160 Asp Leu Thr Gly Leu Lys Pro Gly
Thr Glu Tyr Val Val Asn Ile Met 165 170 175 Gly Val Lys Gly Gly Lys
Ile Ser Pro Pro Leu Ser Ala Ile Phe Thr 180 185 190 Thr Ala Pro Ala
Pro Ala Pro Ala Pro Ala Pro Leu Ala Glu Ala Lys 195 200 205 Val Leu
Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr Tyr 210 215 220
Lys Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala Leu 225
230 235 240 Leu Asp Glu Ile Leu Ala Ala Leu Pro 245
165249PRTArtificial SequenceBispecific EGFR/c-Met binding domain
165Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser
1 5 10 15 Ala Arg Leu Ser Trp Ala Asp Pro His Gly Phe Tyr Asp Ser
Phe Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala
Ile Val Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu
Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr
Gly Val His Asn Val Tyr Lys Asp 65 70 75 80 Thr Asn Met Arg Gly Leu
Pro Leu Ser Ala Ile Phe Thr Thr Ala Pro 85 90 95 Ala Pro Ala Pro
Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu Val 100 105 110 Val Ser
Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala Pro 115 120 125
Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu Phe Leu Gly 130
135 140 Ser Gly Glu Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg Ser
Tyr 145 150 155 160 Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr Val
Val Asn Ile Met 165 170 175 Gly Val Lys Gly Gly Lys Ile Ser Pro Pro
Leu Ser Ala Ile Phe Thr 180 185 190 Thr Ala Pro Ala Pro Ala Pro Ala
Pro Ala Pro Leu Ala Glu Ala Lys 195 200 205 Val Leu Ala Asn Arg Glu
Leu Asp Lys Tyr Gly Val Ser Asp Tyr Tyr 210 215 220 Lys Asn Leu Ile
Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala Leu 225 230 235 240 Leu
Asp Glu Ile Leu Ala Ala Leu Pro 245 166588DNAArtificial
SequenceBispecific EGFR/c-Met binding domain 166ttgccagcgc
cgaagaacct ggtagttagc gaggttactg aggacagcgc gcgtctgagc 60tgggacgatc
cgtgggcgtt ctacgagagc tttctgatcc agtatcaaga gagcgagaaa
120gtcggtgaag cgattgtgct gaccgtcccg ggctccgagc gttcctacga
cctgaccggt 180ttgaagccgg gtaccgagta tacggtgagc atctacggtg
ttcacaatgt ctataaggac 240actaatatcc gcggtctgcc tctgagcgcc
attttcacca ccgcaccggc accggctccg 300gctcctgccc cgctgccggc
tccgaagaac ttggtggtga gccgtgttac cgaagatagc 360gcacgcctga
gctggacggc accggatgcg gcgttcgata gcttctggat tcgctatttt
420gagtttctgg gtagcggtga ggcaattgtt ctgacggtgc cgggctctga
acgctcctac 480gatttgaccg gtctgaaacc gggcaccgag tatgtggtga
acattctgag cgttaagggc 540ggtagcatca gcccaccgct gagcgcgatc
ttcacgactg gtggttgc 588167579DNAArtificial SequenceBispecific
EGFR/c-Met binding domain 167ctgccagccc ctaagaatct ggtcgtgagc
gaagtaaccg aggacagcgc ccgcctgagc 60tgggacgacc cgtgggcgtt ctatgagtct
ttcctgattc agtatcaaga aagcgaaaaa 120gttggcgaag cgatcgtcct
gaccgtcccg ggtagcgagc gctcctacga tctgaccggc 180ctgaaaccgg
gtacggagta cacggtgtcc atttacggtg ttcacaatgt gtataaagac
240accaacatgc gtggcctgcc gctgtcggcg attttcacca ccgcgcctgc
gccagcgcct 300gcaccggctc cgctgccggc accgaagaac ctggttgtca
gccgtgtgac cgaggatagc 360gcacgtttga gctggaccgc tccggatgca
gcctttgaca gcttctggat tcgttacttt 420gaatttctgg gtagcggtga
ggcgatcgtt ctgacggtgc cgggctctga acgcagctat 480gatttgacgg
gcctgaagcc gggtactgag tacgtggtta acatcatggg cgttaagggt
540ggtaaaatca gcccgccatt gtccgcgatc tttaccacg
579168282DNAArtificial SequenceEGFR binding FN3 domain from
ECB97
168ttgccagcgc cgaagaacct ggtagttagc gaggttactg aggacagcgc
gcgtctgagc 60tgggacgatc cgtgggcgtt ctacgagagc tttctgatcc agtatcaaga
gagcgagaaa 120gtcggtgaag cgattgtgct gaccgtcccg ggctccgagc
gttcctacga cctgaccggt 180ttgaagccgg gtaccgagta tacggtgagc
atctacggtg ttcacaatgt ctataaggac 240actaatatcc gcggtctgcc
tctgagcgcc attttcacca cc 282169282DNAArtificial SequenceEGFR
binding FN3 domain from ECB15 169ctgccagccc ctaagaatct ggtcgtgagc
gaagtaaccg aggacagcgc ccgcctgagc 60tgggacgacc cgtgggcgtt ctatgagtct
ttcctgattc agtatcaaga aagcgaaaaa 120gttggcgaag cgatcgtcct
gaccgtcccg ggtagcgagc gctcctacga tctgaccggc 180ctgaaaccgg
gtacggagta cacggtgtcc atttacggtg ttcacaatgt gtataaagac
240accaacatgc gtggcctgcc gctgtcggcg attttcacca cc
282170195PRTArtificial SequenceBispecific EGFR/c-Met binding
molecule 170Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr
Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Asp Asp Pro Trp Ala Phe
Tyr Glu Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val
Gly Glu Ala Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser
Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val
Ser Ile Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Ile
Arg Gly Leu Pro Leu Ser Ala Ile Phe Thr Thr Ala 85 90 95 Pro Ala
Pro Ala Pro Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu 100 105 110
Val Val Ser Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala 115
120 125 Pro Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu Phe
Leu 130 135 140 Gly Ser Gly Glu Ala Ile Val Leu Thr Val Pro Gly Ser
Glu Arg Ser 145 150 155 160 Tyr Asp Leu Thr Gly Leu Lys Pro Gly Thr
Glu Tyr Val Val Asn Ile 165 170 175 Leu Gly Val Lys Gly Gly Lys Ile
Ser Pro Pro Leu Ser Ala Ile Phe 180 185 190 Thr Thr Cys 195
171195PRTArtificial SequenceBispecific EGFR/c-Met binding molecule
171Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp
1 5 10 15 Ser Ala Arg Leu Ser Trp Asp Asp Pro Trp Ala Phe Tyr Glu
Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu
Ala Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp
Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile
Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Ile Arg Gly
Leu Pro Leu Ser Ala Ile Phe Thr Thr Ala 85 90 95 Pro Ala Pro Ala
Pro Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu 100 105 110 Val Val
Ser Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala 115 120 125
Pro Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu Phe Val 130
135 140 Gly Ser Gly Glu Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg
Ser 145 150 155 160 Tyr Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr
Val Val Asn Ile 165 170 175 Leu Gly Val Lys Gly Gly Ser Ile Ser Pro
Pro Leu Ser Ala Ile Phe 180 185 190 Thr Thr Cys 195
172195PRTArtificial SequenceBispecific EGFR/c-Met binding molecule
172Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp
1 5 10 15 Ser Ala Arg Leu Ser Trp Asp Asp Pro Trp Ala Phe Tyr Glu
Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu
Ala Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp
Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile
Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Ile Arg Gly
Leu Pro Leu Ser Ala Ile Phe Thr Thr Ala 85 90 95 Pro Ala Pro Ala
Pro Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu 100 105 110 Val Val
Ser Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala 115 120 125
Pro Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu Phe Val 130
135 140 Ser Lys Gly Asp Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg
Ser 145 150 155 160 Tyr Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr
Val Val Asn Ile 165 170 175 Leu Gly Val Lys Gly Gly Ser Ile Ser Pro
Pro Leu Ser Ala Ile Phe 180 185 190 Thr Thr Cys 195
173195PRTArtificial SequenceBispecific EGFR/c-Met binding molecule
173Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp
1 5 10 15 Ser Ala Arg Leu Ser Trp Asp Asp Pro Trp Ala Phe Tyr Glu
Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu
Ala Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp
Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile
Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Ile Arg Gly
Leu Pro Leu Ser Ala Ile Phe Thr Thr Ala 85 90 95 Pro Ala Pro Ala
Pro Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu 100 105 110 Val Val
Ser Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala 115 120 125
Pro Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu Phe Leu 130
135 140 Gly Ser Gly Glu Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg
Ser 145 150 155 160 Tyr Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr
Val Val Asn Ile 165 170 175 Leu Ser Val Lys Gly Gly Ser Ile Ser Pro
Pro Leu Ser Ala Ile Phe 180 185 190 Thr Thr Cys 195
174195PRTArtificial SequenceBispecific EGFR/c-Met binding molecule
174Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp
1 5 10 15 Ser Ala Arg Leu Ser Trp Asp Asp Pro His Ala Phe Tyr Glu
Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu
Ala Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp
Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile
Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Ile Arg Gly
Leu Pro Leu Ser Ala Ile Phe Thr Thr Ala 85 90 95 Pro Ala Pro Ala
Pro Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu 100 105 110 Val Val
Ser Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala 115 120 125
Pro Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu Phe Leu 130
135 140 Gly Ser Gly Glu Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg
Ser 145 150 155 160 Tyr Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr
Val Val Asn Ile 165 170 175 Leu Gly Val Lys Gly Gly Lys Ile Ser Pro
Pro Leu Ser Ala Ile Phe 180 185 190 Thr Thr Cys 195
175195PRTArtificial SequenceBispecific EGFR/c-Met binding molecule
175Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp
1 5 10 15 Ser Ala Arg Leu Ser Trp Asp Asp Pro His Ala Phe Tyr Glu
Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu
Ala Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp
Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile
Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Ile Arg Gly
Leu Pro Leu Ser Ala Ile Phe Thr Thr Ala 85 90 95 Pro Ala Pro Ala
Pro Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu 100 105 110 Val Val
Ser Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala 115 120 125
Pro Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu Phe Val 130
135 140 Gly Ser Gly Glu Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg
Ser 145 150 155 160 Tyr Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr
Val Val Asn Ile 165 170 175 Leu Gly Val Lys Gly Gly Ser Ile Ser Pro
Pro Leu Ser Ala Ile Phe 180 185 190 Thr Thr Cys 195
176195PRTArtificial SequenceBispecific EGFR/c-Met binding molecule
176Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp
1 5 10 15 Ser Ala Arg Leu Ser Trp Asp Asp Pro His Ala Phe Tyr Glu
Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu
Ala Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp
Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile
Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Ile Arg Gly
Leu Pro Leu Ser Ala Ile Phe Thr Thr Ala 85 90 95 Pro Ala Pro Ala
Pro Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu 100 105 110 Val Val
Ser Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala 115 120 125
Pro Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu Phe Val 130
135 140 Ser Lys Gly Asp Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg
Ser 145 150 155 160 Tyr Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr
Val Val Asn Ile 165 170 175 Leu Gly Val Lys Gly Gly Ser Ile Ser Pro
Pro Leu Ser Ala Ile Phe 180 185 190 Thr Thr Cys 195
177195PRTArtificial SequenceBispecific EGFR/c-Met binding molecule
177Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp
1 5 10 15 Ser Ala Arg Leu Ser Trp Asp Asp Pro His Ala Phe Tyr Glu
Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu
Ala Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp
Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile
Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Ile Arg Gly
Leu Pro Leu Ser Ala Ile Phe Thr Thr Ala 85 90 95 Pro Ala Pro Ala
Pro Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu 100 105 110 Val Val
Ser Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala 115 120 125
Pro Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu Phe Leu 130
135 140 Gly Ser Gly Glu Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg
Ser 145 150 155 160 Tyr Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr
Val Val Asn Ile 165 170 175 Leu Ser Val Lys Gly Gly Ser Ile Ser Pro
Pro Leu Ser Ala Ile Phe 180 185 190 Thr Thr Cys 195
178195PRTArtificial SequenceBispecific EGFR/c-Met binding molecule
178Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp
1 5 10 15 Ser Ala Arg Leu Ser Trp Asp Asp Pro Trp Ala Phe Tyr Glu
Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu
Ala Ile Val Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp
Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile
Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp Thr Asn Ile Arg Gly
Leu Pro Leu Ser Ala Ile Phe Thr Thr Ala 85 90 95 Pro Ala Pro Ala
Pro Ala Pro Ala Pro Leu Pro Ala Pro Lys Asn Leu 100 105 110 Val Val
Ser Arg Val Thr Glu Asp Ser Ala Arg Leu Ser Trp Thr Ala 115 120 125
Pro Asp Ala Ala Phe Asp Ser Phe Trp Ile Arg Tyr Phe Glu Phe Leu 130
135 140 Gly Ser Gly Glu Ala Ile Val Leu Thr Val Pro Gly Ser Glu Arg
Ser 145 150 155 160 Tyr Asp Leu Thr Gly Leu Lys Pro Gly Thr Glu Tyr
Val Val Asn Ile 165 170 175 Leu Ser Val Lys Gly Gly Ser Ile Ser Pro
Pro Leu Ser Ala Ile Phe 180 185 190 Thr Thr Cys 195
17912PRTArtificial SequenceConsensus FG loop of EGFR bindiing FN3
domainVARIANT(9)..(9)Xaa may be Met or Ile 179His Asn Val Tyr Lys
Asp Thr Asn Xaa Arg Gly Leu 1 5 10 18012PRTArtificial SequenceGF
loop of EGFR binding FN3 domain 180Leu Gly Ser Tyr Val Phe Glu His
Asp Val Met Leu 1 5 10 1818PRTArtificial SequenceConsensus BC loop
of EGFR binding FN3 domainVARIANT(1)..(1)Xaa may be Ala, Thr, Gly
or AspVARIANT(2)..(2)Xaa may be Ala, Asp, Tyr or
TrpVARIANT(3)..(3)Xaa may be Pro, Asp or AsnVARIANT(4)..(4)Xaa may
be Leu or absentVARIANT(5)..(5)Xaa may be Asp, His, Arg, Gly, Tyr
or TrpVARIANT(6)..(6)Xaa may be Glu, Asp or AlaVARIANT(7)..(7)Xaa
may be Ala, Phe, Gly, His or AspVARIANT(8)..(8)Xaa may be Tyr, Phe
or Leu 181Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 18295PRTArtificial
SequenceEGFR binding FN3 domain consensus
sequenceVARIANT(22)..(22)Xaa may be Ala, Thr, Gly or
AspVARIANT(23)..(23)Xaa may be Ala, Asp, Tyr or
TrpVARIANT(24)..(24)Xaa may be Pro, Asp or AsnVARIANT(25)..(25)Xaa
may be Leu or absentVARIANT(26)..(26)Xaa may be Asp, His, Arg, Gly,
Tyr or TrpVARIANT(27)..(27)Xaa may be Gly, Asp or
AlaVARIANT(28)..(28)Xaa may be Ala, Phe, Gly, His or
AspVARIANT(29)..(29)Xaa may be Tyr, Phe or LeuVARIANT(84)..(84)Xaa
may be Met or Ile 182Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu
Val Thr Glu Asp Ser 1 5 10 15 Leu Arg Leu Ser Trp Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Asp Ser Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser
Glu Lys Val Gly Glu Ala Ile Asn Leu 35 40 45 Thr Val Pro Gly Ser
Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu
Tyr Thr Val Ser Ile Tyr Gly Val His Asn Val Tyr Lys 65 70 75 80 Asp
Thr Asn Xaa Arg Gly Leu Pro Leu Ser Ala Glu Phe Thr Thr 85 90 95
18395PRTArtificial SequenceEGFR binding FN3 domain consensus
sequenceVARIANT(22)..(22)Xaa may be Ala, Thr, Gly or
AspVARIANT(23)..(23)Xaa may be Ala, Asp, Tyr or
TrpVARIANT(24)..(24)Xaa may be Pro, Asp or AsnVARIANT(25)..(25)Xaa
may be Leu or absentVARIANT(26)..(26)Xaa may be Asp, His, Arg, Gly,
Tyr or TrpVARIANT(27)..(27)Xaa may be Gly, Asp or
AlaVARIANT(28)..(28)Xaa may be Ala, Phe, Gly, His or
AspVARIANT(29)..(29)Xaa may be Tyr, Phe or Leu 183Leu Pro Ala Pro
Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser 1
5 10 15 Leu Arg Leu Ser Trp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Ser
Phe 20 25 30 Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala
Ile Asn Leu 35 40 45 Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu
Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Thr Val Ser Ile Tyr
Gly Val Leu Gly Ser Tyr Val 65 70 75 80 Phe Glu His Asp Val Met Leu
Pro Leu Ser Ala Glu Phe Thr Thr 85 90 95 18415PRTArtificial
Sequencec-Met binding FN3 domain C strand and CD loop consensus
sequenceVARIANT(4)..(4)Xaa is Trp, Phe or ValVARIANT(8)..(8)Xaa is
Asp, Phe or LeuVARIANT(10)..(10)Xaa is Val, Phe or
LeuVARIANT(11)..(11)Xaa is Val, Leu or ThrVARIANT(12)..(12)Xaa is
Val, Arg, Gly, Leu, Thr or SerVARIANT(13)..(13)Xaa is Gly, Ser,
Ala, Thr or LysVARIANT(15)..(15)Xaa is Glu or Asp 184Asp Ser Phe
Xaa Ile Arg Tyr Xaa Glu Xaa Xaa Xaa Xaa Gly Xaa 1 5 10 15
18517PRTArtificial Sequencec-Met binding FN3 domain F strand and FG
loop consensus sequenceVARIANT(4)..(4)Xaa is Tyr, Trp, Ile, Val,
Gly or AlaVARIANT(6)..(6)Xaa is Asn, Thr, Qln or
GlyVARIANT(8)..(8)Xaa is Leu, Met, Asn or IleVARIANT(9)..(9)Xaa is
Gly or SerVARIANT(14)..(14)Xaa is Ser, Leu, Gly, Tyr, Thr, Arg, His
or LysVARIANT(15)..(15)Xaa is Ile, Val or LeuVARIANT(17)..(17)Xaa
is Val, Thr, His, Ile, Pro, Tyr or Leu 185Thr Glu Tyr Xaa Val Xaa
Ile Xaa Xaa Val Lys Gly Gly Xaa Xaa Ser 1 5 10 15 Xaa
18689PRTArtificial Sequencec-Met binding FN3 domain consensus
sequenceVARIANT(32)..(32)Xaa is Trp, Phe or ValVARIANT(36)..(36)Xaa
is Asp, Phe or LeuVARIANT(38)..(38)Xaa is Val, Phe or
LeuVARIANT(39)..(39)Xaa is Val, Leu or ThrVARIANT(40)..(40)Xaa is
Val, Arg, Gly, Leu, Thr or SerVARIANT(41)..(41)Xaa is Gly, Ser,
Ala, Thr or LysVARIANT(43)..(43)Xaa is Glu or
AspVARIANT(68)..(68)Xaa is Tyr, Trp, Ile, Val, Gly or
AlaVARIANT(70)..(70)Xaa is Asn, Thr, Qln or GlyVARIANT(72)..(72)Xaa
is Leu, Met, Asn or IleVARIANT(73)..(73)Xaa is Gly or
SerVARIANT(78)..(78)Xaa is Ser, Leu, Gly, Tyr, Thr, Arg, His or
LysVARIANT(79)..(79)Xaa is Ile, Val or LeuVARIANT(81)..(81)Xaa is
Val, Thr, His, Ile, Pro, Tyr or Leu 186Leu Pro Ala Pro Lys Asn Leu
Val Val Ser Arg Val Thr Glu Asp Ser 1 5 10 15 Ala Arg Leu Ser Trp
Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Xaa 20 25 30 Ile Arg Tyr
Xaa Glu Xaa Xaa Xaa Xaa Gly Xaa Ala Ile Val Leu Thr 35 40 45 Val
Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly 50 55
60 Thr Glu Tyr Xaa Val Xaa Ile Xaa Xaa Val Lys Gly Gly Xaa Xaa Ser
65 70 75 80 Xaa Pro Leu Ser Ala Glu Phe Thr Thr 85
18794PRTArtificial SequenceEGFR consensus FN3 domain of bispecific
EGFR/c-Met moleculeVARIANT(11)..(11)Xaa is Glu, Asn or
ArgVARIANT(14)..(14)Xaa is Glu or ProVARIANT(17)..(17)Xaa is Leu or
AlaVARIANT(25)..(25)Xaa is His or TrpVARIANT(29)..(29)Xaa is Glu or
AspVARIANT(37)..(37)Xaa is Glu or ProVARIANT(46)..(46)Xaa is Asn or
ValVARIANT(73)..(73)Xaa is Gly or TyrVARIANT(83)..(83)Xaa is Met or
IleVARIANT(91)..(91)Xaa is Glu or Ile 187Leu Pro Ala Pro Lys Asn
Leu Val Val Ser Xaa Val Thr Xaa Asp Ser 1 5 10 15 Xaa Arg Leu Ser
Trp Asp Asp Pro Xaa Ala Phe Tyr Xaa Ser Phe Leu 20 25 30 Ile Gln
Tyr Gln Xaa Ser Glu Lys Val Gly Glu Ala Ile Xaa Leu Thr 35 40 45
Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly 50
55 60 Thr Glu Tyr Thr Val Ser Ile Tyr Xaa Val His Asn Val Tyr Lys
Asp 65 70 75 80 Thr Asn Xaa Arg Gly Leu Pro Leu Ser Ala Xaa Phe Thr
Thr 85 90 18889PRTArtificial Sequencec-Met consensus FN3 domain of
bispecific EGFR/c-Met moleculeVARIANT(11)..(11)Xaa is Glu, Asn or
ArgVARIANT(14)..(14)Xaa is Glu or ProVARIANT(17)..(17)Xaa is Leu or
AlaVARIANT(37)..(37)Xaa is Glu or ProVARIANT(39)..(39)Xaa is Val or
LeuVARIANT(40)..(40)Xaa is Gly or SerVARIANT(41)..(41)Xaa is Ser or
LysVARIANT(43)..(43)Xaa is Glu or AspVARIANT(46)..(46)Xaa is Asn or
ValVARIANT(72)..(72)Xaa is Leu or MetVARIANT(73)..(73)Xaa is Gly or
SerVARIANT(78)..(78)Xaa is Ser or LysVARIANT(86)..(86)Xaa is Glu or
Ile 188Leu Pro Ala Pro Lys Asn Leu Val Val Ser Xaa Val Thr Xaa Asp
Ser 1 5 10 15 Xaa Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp
Ser Phe Trp 20 25 30 Ile Arg Tyr Phe Xaa Phe Xaa Xaa Xaa Gly Xaa
Ala Ile Xaa Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp
Leu Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Val Val Asn Ile
Xaa Xaa Val Lys Gly Gly Xaa Ile Ser 65 70 75 80 Pro Pro Leu Ser Ala
Xaa Phe Thr Thr 85 189125PRTHomo Sapiens 189Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Gly Met
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Val Ile Trp Asp Asp Gly Ser Tyr Lys Tyr Tyr Gly Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Ile Thr Met Val Arg Gly Val
Met Lys Asp Tyr Phe 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 120 125 190107PRTHomo Sapiens 190Ala Ile Gln
Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala 20 25
30 Leu Val Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Glu Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Phe Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys 100 105 191121PRTHomo Sapiens 191Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ala Asn Ile Lys Lys Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Gly Trp Gly Trp Gly Trp
Tyr Phe Asp Leu Trp Gly 100 105 110 Arg Gly Thr Leu Val Thr Val Ser
Ser 115 120 192107PRTHomo Sapiens 192Glu Ile Val Leu Thr Gln Ser
Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr
Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp
Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105 193119PRTHomo Sapiens 193Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Glu
Thr Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val
Arg Gln Ala Pro Gly His Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile
Ser Ala Tyr Asn Gly Tyr Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln
Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70
75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Asp Leu Arg Gly Thr Asn Tyr Phe Asp Tyr Trp
Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
194107PRTHomo Sapiens 194Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Gly Ile Ser Asn Trp 20 25 30 Leu Ala Trp Phe Gln His
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser
Ser Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Ile 85
90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105
195125PRTHomo Sapiens 195Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Tyr Trp Val Arg
Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser
Asp Asp Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr 65 70 75 80
Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85
90 95 Ala Arg Glu Gly Leu Tyr Tyr Tyr Gly Ser Gly Ser Tyr Tyr Asn
Gln 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125 196106PRTHomo Sapiens 196Gln Leu Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly Asp Arg 1 5 10 15 Val Thr Ile Thr Cys
Arg Ala Ser Gln Gly Leu Ser Ser Ala Leu Ala 20 25 30 Trp Tyr Arg
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp 35 40 45 Ala
Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 50 55
60 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp
65 70 75 80 Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Ser Tyr Pro Gln
Ile Thr 85 90 95 Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105
197457PRTHomo Sapiens 197Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Gln Ala
Ser Gly Tyr Arg Phe Ser Asn Phe 20 25 30 Val Ile His Trp Val Arg
Gln Ala Pro Gly Gln Arg Phe Glu Trp Met 35 40 45 Gly Trp Ile Asn
Pro Tyr Asn Gly Asn Lys Glu Phe Ser Ala Lys Phe 50 55 60 Gln Asp
Arg Val Thr Phe Thr Ala Asp Thr Ser Ala Asn Thr Ala Tyr 65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Ala Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Val Gly Pro Tyr Ser Trp Asp Asp Ser Pro Gln Asp Asn
Tyr 100 105 110 Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Ile Val
Ser Ser Ala 115 120 125 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser 130 135 140 Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe 145 150 155 160 Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 165 170 175 Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 180 185 190 Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr 195 200 205
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg 210
215 220 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro 225 230 235 240 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys 245 250 255 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val 260 265 270 Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr 275 280 285 Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu 290 295 300 Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 305 310 315 320 Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 325 330
335 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
340 345 350 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met 355 360 365 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro 370 375 380 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn 385 390 395 400 Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Leu Leu 405 410 415 Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 420 425 430 Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 435 440 445 Lys
Ser Leu Ser Leu Ser Pro Gly Lys 450 455 198457PRTHomo Sapiens
198Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15 Ser Val Lys Val Ser Cys Gln Ala Ser Gly Tyr Arg Phe Ser
Asn Phe 20 25 30 Val Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg
Phe Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Tyr Asn Gly Asn Lys
Glu Phe Ser Ala Lys Phe 50 55 60 Gln Asp Arg Val Thr Phe Thr Ala
Asp Thr Ser Ala Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu
Arg Ser Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Gly
Pro Tyr Ser Trp Asp Asp Ser Pro Gln Asp Asn Tyr 100 105 110 Tyr Met
Asp Val Trp Gly Lys Gly Thr Thr Val Ile Val Ser Ser Ala 115 120 125
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser 130
135 140 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe 145 150 155 160 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly 165 170 175 Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu 180 185 190 Ser Ser Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr 195 200 205 Ile Cys Asn Val Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg 210 215 220 Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 225 230 235 240
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 245
250 255 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val 260 265 270 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr 275 280 285 Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu 290 295 300 Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His 305 310 315 320 Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 325 330 335 Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 340 345 350 Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 355 360 365
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 370
375 380 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn 385 390 395 400 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu 405 410 415 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val 420 425 430 Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln 435 440 445 Lys Ser Leu Ser Leu Ser
Pro Gly Lys 450 455 199455PRTHomo Sapiens 199Gln Val Gln Leu Val
Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Gly
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ala Val Ile Trp Asp Asp Gly Ser Tyr Lys Tyr Tyr Gly Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Ile Thr Met Val Arg Gly
Val Met Lys Asp Tyr Phe 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170
175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Arg Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295
300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Leu Leu Tyr Ser 405 410 415
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420
425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys 450 455 200214PRTHomo
Sapiens 200Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp
Ile Ser Ser Ala 20 25 30 Leu Val Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Glu Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu 85 90 95 Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115
120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly
Glu Cys 210 201449PRTHomo Sapiens 201Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser
Cys Glu Thr Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser
Trp Val Arg Gln Ala Pro Gly His Gly Leu Glu Trp Met 35 40 45 Gly
Trp Ile Ser Ala Tyr Asn Gly Tyr Thr Asn Tyr Ala Gln Lys Leu 50 55
60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Asp Leu Arg Gly Thr Asn Tyr Phe Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185
190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys
Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310
315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435
440 445 Lys 202214PRTHomo Sapiens 202Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Trp 20 25 30 Leu Ala Trp
Phe Gln His Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr
Ala Ala Ser Ser Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe
Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg
Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185
190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205 Phe Asn Arg Gly Glu Cys 210 203330PRTHomo Sapiens
203Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130
135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250
255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe 275 280 285 Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 325 330 204330PRTHomo Sapiens 204Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40
45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Leu 275 280 285 Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
2051368DNAHomo Sapiens 205caggtgcagc tggtcgagag cggcggaggg
gtggtgcagc ccggcagaag cctgaggctg 60tcctgcgccg ccagcggctt caccttcagc
acctacggca tgcactgggt gcggcaggcc 120ccaggcaagg gcctggagtg
ggtggccgtg atctgggacg acggcagcta caagtactac 180ggcgacagcg
tgaagggcag gttcaccatc agcagggaca acagcaagaa caccctgtac
240ctgcagatga acagcctgag ggccgaggac accgccgtgt actactgcgc
cagggacggc 300atcaccatgg tgcggggcgt gatgaaggac tacttcgact
actggggcca gggcaccctg 360gtgaccgtga gcagcgccag caccaagggc
ccaagcgtgt tccccctggc ccccagcagc 420aagagcacca gcggcggcac
agccgccctg ggctgcctgg tgaaggacta cttccccgag 480ccagtgaccg
tgtcctggaa ctctggcgcc ctgacctccg gcgtgcacac cttccccgcc
540gtgctgcaga gcagcggcct gtacagcctg agcagcgtgg tgaccgtgcc
cagcagcagc 600ctgggcaccc agacctacat ctgcaacgtg aaccacaagc
ccagcaacac caaggtggac 660aagagagtgg agcccaagag ctgcgacaag
acccacacct gccccccctg cccagcccca 720gagctgctgg gcggacccag
cgtgttcctg ttccccccca agcccaagga caccctgatg 780atcagcagga
cccccgaggt gacctgcgtg gtggtggacg tgagccacga ggacccagag
840gtgaagttca actggtacgt ggacggcgtg gaggtgcaca acgccaagac
caagcccaga 900gaggagcagt acaacagcac ctacagggtg gtgtccgtgc
tgaccgtgct gcaccaggac 960tggctgaacg gcaaggaata caagtgcaag
gtctccaaca aggccctgcc agcccccatc 1020gaaaagacca tcagcaaggc
caagggccag ccacgggagc cccaggtgta caccctgccc 1080cccagccggg
aggagatgac caagaaccag gtgtccctga cctgtctggt gaagggcttc
1140taccccagcg acatcgccgt ggagtgggag agcaacggcc agcccgagaa
caactacaag 1200accacccccc cagtgctgga cagcgacggc agcttcctcc
tgtacagcaa gctgaccgtg 1260gacaagtcca ggtggcagca gggcaacgtg
ttcagctgca gcgtgatgca cgaggccctg 1320cacaaccact acacccagaa
gtccctgagc ctgagccccg gcaaatga 1368206642DNAHomo Sapiens
206atccagctga cccagagccc cagcagcctg agcgccagcg tgggcgaccg
ggtgaccatc 60acctgccggg ccagccagga catcagcagc gccctggtct ggtatcagca
gaagcccggc 120aaggccccca agctgctgat ctacgacgcc agctccctgg
aaagcggcgt gcccagccgg 180ttcagcggca gcgagagcgg caccgacttc
accctgacca tcagcagcct gcagcccgag 240gacttcgcca cctactactg
ccagcagttc aacagctacc ccctgacctt tggcggcgga 300acaaaggtgg
agatcaagcg tacggtggcc gctcccagcg tgttcatctt cccccccagc
360gacgagcagc tgaagagcgg caccgccagc gtggtgtgcc tgctgaacaa
cttctacccc 420cgggaggcca aggtgcagtg gaaggtggac aacgccctgc
agagcggcaa cagccaggag 480agcgtcaccg agcaggacag caaggactcc
acctacagcc tgagcagcac cctgaccctg 540tccaaggccg actacgagaa
gcacaaggtg tacgcctgcg aggtgaccca ccagggcctg 600tccagccccg
tgaccaagag cttcaacagg ggcgagtgct ga 6422071350DNAHomo Sapiens
207caggttcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc
agtgaaggtc 60tcctgcgaga cttctggtta cacctttacc agctatggta tcagctgggt
gcgacaggcc 120cctggacacg ggcttgagtg gatgggatgg atcagcgctt
acaatggtta cacaaactat 180gcacagaagc tccagggcag ggtcaccatg
accacagaca catccacgag cacagcctac 240atggagctga ggagcctgag
atctgacgac acggccgtgt attactgtgc gagagatctg 300agaggaacta
actactttga ctactggggc cagggaaccc tggtcaccgt ctcctcagcc
360tccaccaagg gcccaagcgt gttccctctg gcccccagca gcaagagcac
atctggcgga 420acagccgccc tgggctgcct ggtgaaagac tacttccccg
agcccgtgac cgtgtcctgg 480aactctggcg ccctgaccag cggcgtgcac
acctttccag ccgtgctgca gagcagcggc 540ctgtacagcc tgtccagcgt
ggtgaccgtg cccagcagct ccctgggcac ccagacctac 600atctgcaacg
tgaaccacaa gcccagcaac accaaggtgg acaagcgggt ggaacccaag
660agctgcgaca agacccacac ctgtcccccc tgccctgccc ctgaactgct
gggcggaccc 720tccgtgttcc tgttcccccc aaagcccaag gacaccctga
tgatcagccg gacccccgaa 780gtgacctgcg tggtggtgga cgtgtcccac
gaggaccctg aagtgaagtt caattggtac 840gtggacggcg tggaagtgca
caacgccaag accaagccca gagaggaaca gtacaacagc 900acctaccggg
tggtgtccgt gctgacagtg ctgcaccagg actggctgaa cggcaaagag
960tacaagtgca aggtctccaa caaggccctg cctgctccca tcgagaaaac
catcagcaag 1020gccaagggcc agccccgcga gcctcaggtg tacacactgc
ctcccagccg ggaagagatg 1080accaagaacc aggtgtccct gacctgtctg
gtgaaaggct tctaccccag cgatatcgcc 1140gtggaatggg agagcaacgg
acagcccgag aacaactaca agaccacccc ccctgtgctg 1200gacagcgacg
gctccttctt cctgtactct cggctgaccg tggacaagag ccggtggcag
1260cagggaaacg tgttcagctg cagcgtgatg cacgaggccc tgcacaacca
ctacacccag 1320aagtccctga gcctgagccc cgggaagtga 1350208645DNAHomo
Sapiens 208gacatccaga tgacccagtc cccctcctcc gtgtccgcct ctgtgggcga
cagagtgacc 60atcacctgtc gggcctccca gggcatctcc aactggctgg cctggttcca
gcacaagccc 120ggcaaggccc ccaagctgct gatctacgcc gcctcctccc
tgctgtccgg cgtgccctcc 180agattctccg gctctggctc cggcaccgac
ttcaccctga ccatctccag cctgcagccc 240gaggacttcg ccacctacta
ctgccagcag gccaactcct tccccatcac cttcggccag 300ggcacccggc
tggaaatcaa gcgtacggtg gccgctccca gcgtgttcat cttccccccc
360agcgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa
caacttctac 420ccccgggagg ccaaggtgca gtggaaggtg gacaacgccc
tgcagagcgg caacagccag 480gagagcgtca ccgagcagga cagcaaggac
tccacctaca gcctgagcag caccctgacc 540ctgtccaagg ccgactacga
gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 600ctgtccagcc
ccgtgaccaa gagcttcaac aggggcgagt gctga 645209215PRTHomo Sapiens
209Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15 Glu Arg Ala Thr Phe Ser Cys Arg Ser Ser His Ser Ile Arg
Ser Arg 20 25 30 Arg Val Ala Trp Tyr Gln His Lys Pro Gly Gln Ala
Pro Arg Leu Val 35 40 45 Ile His Gly Val Ser Asn Arg Ala Ser Gly
Ile Ser Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Thr Arg Val Glu 65 70 75 80 Pro Glu Asp Phe Ala Leu
Tyr Tyr Cys Gln Val Tyr Gly Ala Ser Ser 85 90 95 Tyr Thr Phe Gly
Gln Gly Thr Lys Leu Glu Arg Lys Arg Thr Val Ala 100 105 110 Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130
135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu
Cys 210 215 2105PRTHomo Sapiens 210Thr Tyr Gly Met His 1 5
21117PRTHomo Sapiens 211Val Ile Trp Asp Asp Gly Ser Tyr Lys Tyr Tyr
Gly Asp Ser Val Lys 1 5 10 15 Gly 21216PRTHomo Sapiens 212Asp Gly
Ile Thr Met Val Arg Gly Val Met Lys Asp Tyr Phe Asp Tyr 1 5 10 15
21311PRTHomo Sapiens 213Arg Ala Ser Gln Asp Ile Ser Ser Ala Leu Val
1 5 10 2147PRTHomo Sapiens 214Asp Ala Ser Ser Leu Glu Ser 1 5
2159PRTHomo Sapiens 215Gln Gln Phe Asn Ser Tyr Pro Leu Thr 1 5
2165PRTHomo Sapiens 216Ser Tyr Gly Ile Ser 1 5 21717PRTHomo Sapiens
217Trp Ile Ser Ala Tyr Asn Gly Tyr Thr Asn Tyr Ala Gln Lys Leu Gln
1 5 10 15 Gly 21810PRTHomo Sapiens 218Asp Leu Arg Gly Thr Asn Tyr
Phe Asp Tyr 1 5 10 21911PRTHomo Sapiens 219Arg Ala Ser Gln Gly Ile
Ser Asn Trp Leu Ala 1 5 10 2207PRTHomo Sapiens 220Ala Ala Ser Ser
Leu Leu Ser 1 5 2219PRTHomo Sapiens 221Gln Gln Ala Asn Ser Phe Pro
Ile Thr 1 5 2225PRTHomo Sapiens 222Ser Tyr Trp Met Asn 1 5
22317PRTHomo Sapiens 223Asn Ile Lys Lys Asp Gly Ser Glu Lys Tyr Tyr
Val Asp Ser Val Lys 1 5 10 15 Gly 22412PRTHomo Sapiens 224Asp Leu
Gly Trp Gly Trp Gly Trp Tyr Phe Asp Leu 1 5 10 22511PRTHomo Sapiens
225Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 2267PRTHomo
Sapiens 226Asp Ala Ser Asn Arg Ala Thr 1 5 2279PRTHomo Sapiens
227Gln Gln Arg Ser Asn Trp Pro Pro Thr 1 5 2285PRTHomo Sapiens
228Asp Tyr Tyr Met Tyr 1 5 22917PRTHomo Sapiens 229Thr Ile Ser Asp
Asp Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys 1 5 10 15 Gly
23016PRTHomo Sapiens 230Glu Gly Leu Tyr Tyr Tyr Gly Ser Gly Ser Tyr
Tyr Asn Gln Asp Tyr 1 5 10 15 23111PRTHomo Sapiens 231Arg Ala Ser
Gln Gly Leu Ser Ser Ala Leu Ala 1 5 10 2327PRTHomo Sapiens 232Asp
Ala Ser Ser Leu Glu Ser 1 5 23310PRTHomo Sapiens 233Gln Gln Phe Thr
Ser Tyr Pro Gln Ile Thr 1 5 10 234451PRTHomo Sapiens 234Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ala Asn Ile Lys Lys Asp Gly Ser Glu Lys Tyr Tyr Val Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Gly Trp Gly Trp
Gly Trp Tyr Phe Asp Leu Trp Gly 100 105 110 Arg Gly Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155
160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg
Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280
285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Leu Leu Tyr Ser Lys Leu Thr Val 405
410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser 435 440 445 Pro Gly Lys 450 235214PRTHomo Sapiens
235Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser
Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile
Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr
Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130
135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys
210 236455PRTHomo Sapiens 236Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Tyr Trp Val
Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile
Ser Asp Asp Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr 65 70
75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95 Ala Arg Glu Gly Leu Tyr Tyr Tyr Gly Ser Gly Ser Tyr
Tyr Asn Gln 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195
200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val
Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro 225 230 235 240 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315
320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Arg Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440
445 Leu Ser Leu Ser Pro Gly Lys 450 455 237213PRTHomo Sapiens
237Gln Leu Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg 1 5 10 15 Val Thr
Ile Thr Cys Arg Ala Ser Gln Gly Leu Ser Ser Ala Leu Ala 20 25 30
Trp Tyr Arg Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp 35
40 45 Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
Gly 50 55 60 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro Glu Asp 65 70 75 80 Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Ser
Tyr Pro Gln Ile Thr 85 90 95 Phe Gly Gln Gly Thr Arg Leu Glu Ile
Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165
170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 23815PRTHomo
Sapiens 238Pro Glu Phe Arg Asp Ser Tyr Pro Ile Lys Tyr Val His Ala
Phe 1 5 10 15 23916PRTHomo Sapiens 239Phe Ala Gln Ser Lys Pro Asp
Ser Ala Glu Pro Met Asp Arg Ser Ala 1 5 10 15 24016PRTHomo Sapiens
240Pro Gly Ala Gln Leu Ala Arg Gln Ile Gly Ala Ser Leu Asn Asp Asp
1 5 10 15
* * * * *
References