U.S. patent application number 17/644572 was filed with the patent office on 2022-04-14 for bi-specific antibodies for enhanced tumor selectivity and inhibition and uses thereof.
This patent application is currently assigned to Merck Patent GmbH. The applicant listed for this patent is Merck Patent GmbH. Invention is credited to Achim DOERNER, Christine Knuehl, Carolin Sellmann, Vanita D. Sood, Lars Toleikis.
Application Number | 20220111064 17/644572 |
Document ID | / |
Family ID | |
Filed Date | 2022-04-14 |
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United States Patent
Application |
20220111064 |
Kind Code |
A1 |
DOERNER; Achim ; et
al. |
April 14, 2022 |
BI-SPECIFIC ANTIBODIES FOR ENHANCED TUMOR SELECTIVITY AND
INHIBITION AND USES THEREOF
Abstract
A heterodimeric bispecific immunoglobulin molecule includes a
first Fab or scFv fragment which specifically binds to EGFR, and a
second Fab or scFv fragment which specifically binds to c-MET, and
an antibody hinge region, an antibody CH2 domain and an antibody
CH3 domain including a hybrid protein-protein interaction interface
domain. Each of the interaction interface domains is formed by an
amino acid segment of the CH3 domain of a first member and an amino
acid segment of the CH3 domain of a second member. The hybrid
protein-protein interface domain of the first chain is interacting
with the protein-protein-interface of the second chain by
homodimerization of a corresponding amino acid segment of the same
member of the immunoglobulin superfamily within interaction
domains.
Inventors: |
DOERNER; Achim; (Darmstadt,
DE) ; Toleikis; Lars; (Kleinniedesheim, DE) ;
Sood; Vanita D.; (Somerville, MA) ; Sellmann;
Carolin; (Darmstadt, DE) ; Knuehl; Christine;
(Darmstadt, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Merck Patent GmbH |
Darmstadt |
|
DE |
|
|
Assignee: |
Merck Patent GmbH
Darmstadt
DE
|
Appl. No.: |
17/644572 |
Filed: |
December 16, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15773555 |
May 3, 2018 |
11235063 |
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PCT/EP2016/001791 |
Oct 27, 2016 |
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17644572 |
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International
Class: |
A61K 47/68 20170101
A61K047/68; C07K 16/28 20060101 C07K016/28; A61P 35/00 20060101
A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 3, 2015 |
EP |
15192851.2 |
Jul 5, 2016 |
EP |
16178010.1 |
Claims
1-34. (canceled)
35. An antibody or antigen-binding fragment thereof that binds to
c-MET, comprising: a) a light chain variable region, VL, selected
from the group consisting of SEQ ID NO: 31, SEQ ID NO: 47, and SEQ
ID NO: 51, and a heavy chain variable region, VH, selected from the
group consisting of SEQ ID NO: 32 and SEQ ID NO: 48; b) a VL of SEQ
ID NO: 33, and a VH selected from the group consisting of SEQ ID
NO: 34 and SEQ ID NO: 52; or c) a humanized version of the antibody
or antigen-binding fragment thereof of a) or b).
36. The antibody or antigen-binding fragment thereof according to
claim 35, wherein said antibody or antigen-binding fragment thereof
is an immunoglobulin molecule comprising a Fab or scFv fragment
that binds to c-MET.
37. The antibody or antigen-binding fragment thereof according to
claim 35, wherein said antibody or antigen-binding fragment thereof
binds c-MET with a K.sub.D of at least 5.times.10.sup.-8 M.
38. The antibody or antigen-binding fragment thereof according to
claim 35, wherein said antibody or antigen-binding fragment thereof
is a one-armed monovalent antibody.
39. The antibody or antigen-binding fragment thereof according to
claim 35, comprising: the antibody or antigen-binding fragment of
a) or the humanized version of the antibody or antigen-binding
fragment thereof of a).
40. The antibody or antigen-binding fragment thereof according to
claim 35, comprising: the VL of SEQ ID NO: 31 and the VH of SEQ ID
NO: 32; or a humanized version of the antibody or antigen-binding
fragment thereof comprising the VL of SEQ ID NO: 31 and the VH of
SEQ ID NO: 32.
41. The antibody or antigen-binding fragment thereof according to
claim 35, comprising: the VL of SEQ ID NO: 51 and the of SEQ ID NO:
32; or a humanized version of the antibody or antigen-binding
fragment thereof comprising the VL of SEQ ID NO: 51 and the VH of
SEQ ID NO: 32.
42. The antibody or antigen-binding fragment thereof according to
claim 35, comprising: the VL of SEQ ID NO: 47 and the VH of SEQ ID
NO: 48; or a humanized version of the antibody or antigen-binding
fragment thereof comprising the VL of SEQ ID NO: 47 and the VH of
SEQ ID NO: 48.
43. The antibody or antigen-binding fragment thereof according to
claim 35, comprising: the VL of SEQ ID NO: 33 and the VH selected
from the group consisting of SEQ ID NO: 34 and SEQ ID NO: 52; or a
humanized version of the antibody or antigen-binding fragment
thereof comprising the VL of SEQ ID NO: 33 and the VH of SEQ ID NO:
34 or 52.
44. The antibody or antigen-binding fragment thereof according to
claim 35, comprising: the VL of SEQ ID NO: 33 and the VH of SEQ ID
NO: 34; or a humanized version of the antibody or antigen-binding
fragment comprising the VL of SEQ ID NO: 33 and the VH of SEQ ID
NO: 34.
45. The antibody or antigen-binding fragment thereof according to
claim 35, comprising: the VL of SEQ ID NO: 33 and the of SEQ ID NO:
52; or a humanized version of the antibody or antigen-binding
fragment thereof comprising the VL of SEQ ID NO: 33 and the VH of
SEQ ID NO: 52.
Description
FIELD OF THE INVENTION
[0001] The present invention concerns bi-specific antibodies, in
particular EGFR x c-MET bi-specific antibodies, for enhanced tumor
selectivity and inhibition, their use in the treatment of cancer
and methods of producing the same.
BACKGROUND OF THE INVENTION
[0002] Cancer cells are often characterized by an aberrant
expression of cell surface molecules, such as receptor tyrosine
kinases one of which is the epidermal growth factor receptor
(EGFR). EGFR is activated upon binding to the Epidermal Growth
Factor (EGF) and other growth factor ligands, such as TGF-.alpha.,
amphiregulin (AR), epiregulin (EP), betacelluin (BC), or HB-EGF
(Normanno et al., Gene 366 (2006) 2-16). Upon ligand-induced
dimerization and activation, several downstream signaling pathways
are triggered, including RAS/MAPK, PI3K/Akt and STAT that regulate
different cellular processes, including DNA synthesis and
proliferation. EGFR signaling is commonly found deregulated in
cancer through different mechanisms, including genetic mutations of
the receptor. Signaling properties of mutant forms of EGFR in
addition also show an altered cellular trafficking compared to wild
type EGFR, since some of the regulatory proteins that balance the
EGFR pathway present altered expression in cancer. Mutated EGFR is
for example found in non small cell lung cancer (NSCLC) and 60-80%
of colorectal cancers express a mutated EGFR.
[0003] In the advent of anti-EGFR based cancer therapy it was
hypothesized that EGFR targeted therapy would be most effective in
tumors overexpressing the protein, however studies quickly revealed
that the levels of EGFR expression were not correlated with
response to anti-EGFR antibodies, such as cetuximab (Liska Clin
Cancer Res 17(3) February 2011). Increased EGFR gene copy number,
overexpression of EGFR ligand and TP53 mutations were shown to be
associated with response to EGFR inhibitors in CRC (Khambata-Ford
et al., J Clin Oncol 2007; 25:3230-7; Moroni et al., Lancet Oncol
2005; 6:279-86; Oden-Gangloff et al., Br J Cancer 2009; 100:1330-5;
Tabernero J, J Clin Oncol. 2010 Mar. 1; 28(7):1181-9).
[0004] Side effects of current EGFR-targeted therapies targeting
EGFR overexpressing cells suffer from toxicities due to basal
expression of EGFR in tissues other than the tumor. For example,
cetuximab which is a chimeric human-murine monoclonal antibody
against EGFR, often causes skin toxicities, a phenomenon which is
also observed in EGFR therapy with gefitinib (J Eur Acad Dermatol
Venereol, 2010 April; 24(4):453-9); SpringerPlus 2013, 2:22).
[0005] Functionally, receptor tyrosine kinases also often times
also show redundancy, which will compensate for the loss of one
family member. One example is sustained ERBB3 signaling which is
observed in some cases of EGFR mutant tumors treated with gefitinib
(Science Vol. 316, 18 May 2007: p. 1039-1043). This functional
redundancy can ultimately result in acquired tumor resistance to a
therapeutic blockade of one family member (Engelmann et al, Science
316, 1039 (2007)). Acquired tumor resistance often results in
relapse during a RTK inihibitor monotherapy.
[0006] Studies revealed that intrinsic resistance to EGFR-targeted
therapy can be the result of downstream effector molecule
activation such as KRAS which is seen in 35%-40% of CRCs
(Knickelbein et al, Genes Dis. 2015 March: 2(1):4-12). Multiple
studies have now shown that KRAS mutations in CRC confer resistance
to cetuximab because of which it is recommended to limit cetuximab
therapy to patients with wild-type KRAS tumors. However, about 25%
of colorectal cancer (CRC) patients that are wild-type for KRAS,
BRAF, PIK3CA and PTEN do not respond to treatment with EGER
inhibitors (J Clin Oncol. 2010 Mar. 1; 28(7)1254-61). Molecular
analysis of the patients not responding to treatment by BEAMing
revealed an amplification of the MET gene in these patients
following treatment (Bardelli et al. Cancer Discov; 3(6): 668-73).
Upregulation of hepatocyte growth factor receptor (HGFR, c-MET)
expression and of its ligand HGF appears to be one of the major
escape routes of tumors during EGFR-targeted monotherapy. This is
also often accompanied by amplification of the gene encoding c-MET
(Engelmann et al. Science 316, 1039 (2007) Clin Cancer Res 2011;
17:472-482). In vitro experiments with gefitinib treated HCC827
cells revealed a c-MET amplification of 5-10 fold (Engelmann et al.
Science 316, 1039 (2007)).
[0007] The MET gene encodes the for hepatocyte growth factor
receptor (HGFR, c-MET), which is a heterodimeric transmembrane
receptor tyrosine kinase composed of an extracellular .alpha.-chain
and a membrane-spanning, .beta.-chain linked via disulfide bonds
and which has a single ligand, HGF, also known as scatter factor.
Structurally, c-MET comprises several conserved protein domains,
including sema, PSI (in plexins, semaphorins, integrins), 4 IPT
repeats (in immunoglobulins, plexins, transcription factors), TM
(transmembrane), JM (juxtamembrane), and TK (tyrosine kinase)
domains. Binding of HGF to MET triggers receptor dimerization and
transphosphorylation, leading to conformational changes in MET that
activate the TK domain. C-MET mediates activation of downstream
signaling pathways, including phosphoinositide 3-kinase (PI3K)/AKT,
Ras-Rac/Rho, mitogen-activated protein kinase, and phospholipase C,
that stimulate morphogenic, proliferative, and antiapoptotic
activities as well as stimulating pathways involved in cell
detachment, motility, and invasiveness.
[0008] Consistent with the role of c-MET in cell motility and
morphogenesis, metastatic lesions typically exhibit higher
expression levels of MET than primary tumors (Cipriani at al. Lung
Cancer 2009, 63:169-179). Several approaches have been pursued to
inhibit either the ligand HBF or the receptor to inhibit c-MET
signaling. For example, AMG102/Rilotumumab binds preferentially to
the mature biologically active form of HGF, interacting with the
amino-terminal portion of the .beta.-chain thereby inhibiting HGF
binding. Another monoclonal antiobody (mAb) which was explored to
inhibit HGF activity is Ficlatuzumab. Ficlatuzumab is a humanized
IgG1 antibody that binds HGF ligand with high affinity and
specificity thereby inhibiting c-MET/HGF biological activities.
[0009] Rilotumumab has been tested as monotherapy in patients
carrying recurrent glioblastomas, metastatic renal carcinomas or
ovarian cancers and in combination with chemotherapy in prostate
cancers or with antiangiogenic agents in advanced solid tumors.
Ficlatuzumab was tested both as monotherapy and in association with
EGFR inhibitors in NSCLC (Biologics 2013; 7: 61-68). However, a
phase II trial with ficlatuzumab did not reach its primary
endpoint.
[0010] Thus, despite the fact that progress has been made in the
development of both, anti-EGFR and anti-c-MET therapies, either as
monotherapy or in combination, there is a continued need for
improved anti-EGFR cancer therapies, which overcome the current
limitations of anti-EGFR based therapies and prevent c-MET-driven
tumor resistance.
SUMMARY OF THE INVENTION
[0011] The present inventors have surprisingly found that
bi-specific heterodimeric immunoglobulin molecules which bind to
both EGFR and c-MET are effective in the treatment of EGFR and
c-MET-expressing tumors.
[0012] In a first embodiment the present invention provides
heterodimeric bispecific immunoglobulin molecule which comprises
[0013] (i) a first Fab or scFv fragment which specifically binds to
EGFR, and [0014] (ii) a second Fab or scFv fragment which
specifically binds to c-MET, and [0015] (iii) an antibody hinge
region, an antibody CH2 domain and an antibody CH3 domain
comprising a hybrid protein-protein interaction interface domain
wherein each of said interaction interface domain is formed by
amino acid segments of the CH3 domain of a first member and amino
acid segments of the CH3 domain of said second member, wherein said
protein-protein interface domain of the first chain is interacting
with the protein-protein-interface of the second chain by
homodimerization of the corresponding amino acid segments of the
same member of the immunoglobulin superfamily within said
interaction domains. wherein the first or second engineered
immunoglobulin chain has the polypeptide sequence ("AG-SEED"):
GQPFRPEVHLLPPSREEMTKNQVSLTCLARGFYPX.sub.1DIAVEVVESNGQPENNYKTTP
SRQEPSQGTT TFAVTSKLTX.sub.2DKSRVVQQGNVFSCSVMHEALHNHYTQKX.sub.3ISL
(SEQ ID NO:1), wherein X.sub.1, X.sub.2 and X.sub.3 may be any
amino acid.
[0016] In one embodiment, in the heterodimeric bispecific
immunoglobulin molecule of the invention the first member of the
immunoglobulin super family is IgG and the second member is
IgA.
[0017] In one embodiment X.sub.1 is K or S, X.sub.2 is V or T, and
X.sub.3 is T or S in the heterodimeric bispecific immunoglobulin
molecule of the invention as disclosed above
[0018] In one embodiment, the first or second engineered
immunoglobulin chain of the heterodimeric bispecific immunoglobulin
molecule according to the invention has the polypeptide sequence
("GA-SEED");
GQPREPQVYTLPPPSEELALNEX1VTLTCLVKGFYPSDIAVEVVLQGSQELPREKYLTVVX2PV
X3DSD GSX4FLYSILRVX5AX6DVVKKGDTFSCSVMHEALHNHYTQKSLDR, wherein
X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5, and X.sub.6 may be any
amino acid.
[0019] According to one embodiment, X.sub.1 is L or Q, X.sub.2 is A
or T, X.sub.3 is L V, D or T; X.sub.4 is F, A, D, E, G, H, K, N, P,
Q, R, S or T; X.sub.5 is A or T, and X.sub.6 is E or D in the
inventive heterodimeric bispecific immunoglobulin molecule.
[0020] In one embodiment, the first engineered immunoglobulin chain
of the inventive heterodimeric bispecific immunoglobulin molecule
comprises the polypeptide sequence ("AG-SEED"):
GQPFRPEVHLLPPSREEMTKNQVSLTCLARGFYPKDIAVEVVESNGQPENNYKTTPSRQEP SQGTT
TFAVTSKLTVDKSRVVQQGNVFSCSVMHEALHNHYTQKTISL and the second
engineered immunoglobulin chain of the inventive heterodimeric
bispecific immunoglobulin molecule comprises the polypeptide
sequence ("GA-SEED"):
TABLE-US-00001 GQPREPQVYTLPPPSEELALNELVTLTCLVKGFYPSDIAVEWLQGSQEL
PREKYLTWAPVLDSDG SFFLYSILRVAAEDWKKGDTFSCSVMHEALHN HYTQKSLDR.
[0021] According to one embodiment, the fast engineered
immunoglobulin chain of the inventive heterodimeric bispecific
immunoglobulin molecule as disclosed above has the polypeptide
sequence ("AG-SEED"):
GQPFEPEVHTLPPSREEMTKNQVSLTCLVRGFYPSDIAVEWESNGQPENNYKTTPSRLEPS QGTT
TFAVTSKLTVDKSRVVQQGNVFSCSVNMHEALHNHYTQKSLSL and the second
engineered immunoglobulin chain of the inventive heterodimeric
bispecific immunoglobulin molecule as disclosed above has the
polypeptide sequence ("GA-SEED"):
TABLE-US-00002 GQPREPQVYTLPPPSEELALNNQVTLTCLVKGFYPSDIAVEWESNGQPE
PREKYLTWAPVLDSDG SFFLYSILRVDASRWQQGNVFSCSVMHEALHN HYTQKSLSL.
[0022] In one embodiment, the first Fab or scFv fragment of the
inventive heterodimeric bispecific immunoglobulin molecule as
disclosed above binds EGFR with an K.sub.O of at least
5.times.10.sup.-8 M.
[0023] In one embodiment, the second Fab or scFv fragment of the
inventive heterodimeric bispecific immunoglobulin molecule as
disclosed above binds c-MET with an K.sub.D of at least
5.times.10.sup.-8 M.
[0024] According to one embodiment, the first Fab or scFv fragment
of the inventive heterodimeric bispecific immunoglobulin molecule
is derived from cetuximab (C225).
[0025] In a preferred embodiment, the first Fab or scFv fragment
comprises VL and VH sequences selected from the group consisting of
SEQ ID NO: 9, SEQ ID NO:10, SEO ID NO: 11, SEQ ID NO: 12, SEQ ID
NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46.
[0026] In a preferred embodiment, the wherein the second Fab or
scFv fragment comprises VL sequences selected form the group
consisting of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID
NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29,
SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID
NO: 51
[0027] In a preferred embodiment, the VL sequences of the first Fab
or scFv fragment of the inventive heterodimeric bispecific
immunoglobulin molecule are selected the VH sequences of said
second Fab fragment are selected from the group consisting of SEQ
ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:
24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ
ID NO:34, SEQ ID NO: 48, SEQ ID NO: 50, or SEQ ID NO: 52.
[0028] According to a more preferred embodiment, the first and
second Fab or scFv fragments of the inventive heterdimeric
bispecific immunoglobulin molecule as disclosed above comprise the
amino acid sequences SEQ ID NO: 9, SEQ ID NO: 43, SEQ ID NO: 17,
SEQ ID NO:18, or SEQ ID NO: 47, SEQ ID NO: 48, or SEQ ID NO: 11,
SEQ ID NO: 44, SEQ ID NO: 31, SEQ ID NO: 51, SEQ ID NO:32, or SEQ
ID NO:45, SEQ ID NO: 46, SEQ ID NO: 29, SEQ ID NO: 49, SEQ ID NO:
30, SEQ ID NO: 50, or SEQ ID NO: 45, SEQ ID NO:46 SEQ ID NO: 33,
SEQ ID NO: 34, SEQ ID NO: 52, or SEQ ID NO: 11, SEQ ID NO: 44, SEQ
ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 52.
[0029] According to a more preferred embodiment the first and
second Fab or scFv fragments of the inventive heterdimeric
bispecific immunoglobulin molecule as disclosed above comprise the
amino acid sequences SEQ ID NO: 11, SEQ ID NO: 44, SEQ ID NO: 33,
SEQ ID NO: 34, SEQ ID NO: 52, or SEQ ID NO:45, SEQ ID NO. 46, SEQ
ID NO: 29, SEQ ID NO: 49, SEQ ID NO: 30, SEQ ID NO: 50.
[0030] In one embodiment, the Fc domain of the heterodimeric
bispecific immunoglobulin molecule according to the invention
interacts with FcRn.
[0031] In one embodiment, the amino acids of the inventive
heterodimeric bispecific immunoglobulin molecule which interact
with FcRn are derived from human IgG1.
[0032] In one embodiment the inventive heterodimeric bispecific
immunoglobulin molecule as disclosed above mediates
antibody-dependent cellular cytotoxicity.
[0033] In one embodiment, the invention provides an isolated
polynucleotide encoding any of the amino acid sequences as
disclosed above.
[0034] In one embodiment, the invention provides a vector, which
comprises at least one inventive polynucleotide.
[0035] According to one embodiment, the invention provides for a
host cell which comprises at least one polynucleotide according to
the invention, or which comprises at least one vector according to
the invention.
[0036] In one embodiment, the invention provides a method for
producing a heterodimeric bispecific immunoglobulin molecule of the
invention as disclosed above, with the inventive process
comprising:
[0037] culturing a host cell according to the invention under
conditions sufficient for the heterologous expression of said
heterodimeric bispecific immunoglobulin molecule
[0038] purifying said heterodimeric bispecific immunoglobulin
molecule
[0039] In one embodiment the invention provides the heterodimeric
bispecific immunoglobulin molecule of the invention which is
obtainable by the inventive, method as disclosed above.
[0040] According to one embodiment, the heterodimeric bispecific
immunoglobulin molecule according to the invention as disclosed
above is covalently coupled to at least one linker.
[0041] In one embodiment the linker of the inventive heterodimeric
bispecific immunoglobulin molecule is coupled to a dye,
radioisotope or cytotoxin.
[0042] In one embodiment, at least one of the Fab or scFv light
chains of the inventive heterodimeric bispecific immunoglobulin
molecule is coupled to a dye, radioisotope, or cytotoxin.
[0043] In one embodiment at least one linker as disclosed above is
covalently coupled to at least one of the Fab or scFv light chains
of the inventive heterodimeric bispecific immunoglobulin molecule
as disclosed above.
[0044] According to one embodiment the inventive heterodimeric
bispecific immunoglobulin molecule comprises two linkers covalently
coupled to the Fab Of scFv light chains the heterodimeric
bispecific immunoglobulin molecule.
[0045] In one embodiment, the Fab or scFv light chains and/or the
CH3 domains and/or the CH2 domains of the inventive heterodimeric
bispecific immunoglobulin molecule are coupled to a linker, whereby
said linker is covalently coupled to a dye, radioisotope, or
cytotoxin.
[0046] According to one embodiment, the heterodimeric bispecific
immunoglobulin molecule of the invention is for use in the
treatment of cancer.
[0047] In one embodiment, the inventive heterodimeric bispecific
immunoglobulin molecule is for use in the treatment of cancer.
[0048] In one embodiment, the invention provides a composition,
which comprises the heterodimeric bispecific immunoglobulin
molecule of the invention as disclosed above and at least one
further ingredient.
[0049] In one embodiment, the invention provides a pharmaceutical
composition which comprises the inventive heterodimeric bispecific
immunoglobulin molecule above and at least one further ingredient,
or the inventive composition as disclosed above.
[0050] In one embodiment, the pharmaceutical composition of the
invention is for use in the treatment of cancer.
[0051] In one embodiment, the invention provides a method of
treating a subject in need thereof inflicted with cancer, wherein
the treatment comprises administering to said subject a
therapeutically effective amount of the inventive pharmaceutical
composition as disclosed above.
BRIEF DESCRIPTION OF THE DRAWINGS
[0052] FIG. 1: Depicted is the cellular binding on NCI-H441 cells
of two heterodimeric bispecific immunoglobulin molecules of the
invention (B10v5x225-H; CS06x225-H) and "one-armed" (oa)
heterodimeric immunoglobulin molecules. Anti-HEL anti-hen egg
lysozyme (isotype control)
[0053] FIG. 2: (A) Epitope binning results, (B) Biosensor
experiments using bio-layer interferometry (cf. Example 3)
[0054] FIG. 3: HGF displacement results
[0055] FIG. 4: ADCC experiments on A431 cells using the antibodies
as indicated.
[0056] FIG. 5: Octet analysis of one-armed heterodimeric
immunoglobulin molecule variants (either Fab or scFv). "225-L",
"225-H", "225-H" denote kinetic variants of humanized cetuximab
(hu225), "425" denotes Matuzumab
[0057] FIG. 6: Inhibition of c-MET phosphorylation in (A) NCI-H596
cells, (B) in A549 cells.
[0058] FIG. 7: Quantitative summary of the c-MET phosphorylation
inhibition (A) NCI-H596 cells, (B) A549 cells.
[0059] FIG. 8: (A) Inhibition of c-MET phosphorylation in MKN-45
cells using the immunoglobulin molecules indicated, (B) inhibition
of EGFR phosphorylation in NCI-H596 cells using the immunoglobulin
molecules as indicated.
[0060] FIG. 9: Cytotoxicity assays on A549 cells. (A) control with
no toxin conjugated, (B) assay using Fab-MMAE-CL coupled antibodies
as indicated, MMAE: monomethyl auristatin E
[0061] FIG. 10: Cytotoxicity assays on (A) EBC-1 cells,. (B)
NCI-H441 cells
[0062] FIG. 11: Cytotoxicity assay MKN-45 cells which express high
levels of c-Met and moderate levels of EGFR.
[0063] FIG. 12: Depicted is the enhanced inhibition of c-MET
phosphorylation are HGF-dependent cancer cell lines: (A) NCI-H596,
(B) KP-4.
[0064] FIG. 13: Enhanced degradation of c-MET following overnight
treatment with the inventive B10v5x225-H molecule.
[0065] FIG. 14: Internalization assay on NCI-H441 cells using the
antibodies and controls as indicated to assess the suitability of
individual constructs for their use as ADC.
[0066] FIG. 15: Depicted are the results of a cellular binding
assay using the antibody and immunoglobulin molecules
indicated.
[0067] FIG. 16: Experimental and calculated binding affinity for
computationally designed point mutants of C225. Letters in
superscript denote the following: a-The KD (nM) for wild type
(C225) and mutant mAbs was determined by surface plasmon resonance
(SPR). Where n>1, the standard deviation is given. Mutations
that improved affinity (p<0.01) are in boldface. b-Experimental
binding affinity relative to wild type (kcal/mol). c-Predicted
binding affinity relative to wild type using Rosetta. d-Predicted
change in Rosetta pair energy across the interface. e-Predicted
change hydrogen bond energy across the interface. f-Calculated
hydrogen bond energy of mutated residue side chain. g-Predicted
change in folding energy of the isolated antibody. NQ: Not
Quantifiable, very weak binding.
[0068] FIG. 17: Kinetic parameters of monovalent parental SEED
antibodies in comparison to-MET.times.EGFR bsAbs binding to soluble
c-MET and EGER extracellular domains. Kinetic constants were
determined for cetuximab and matuzumab as references. Antibodies
were captured by anti-human Fc Octet biosensors and binding
kinetics were analyzed at indicated analyte concentrations (25 to
0.8 nM or alternative, 50 to 3.1 nM). Melting temperatures (Tm)
were determined by thermal shift assays. Legend: n.d.=not
determined; KD=affinity constant, ka=association constant;
kd=dissociation constant: Tm=melting temperature; oa=one-armed.
[0069] FIG. 18: Cell surface receptor densities of human c-MET and
EGFR on several tumor cell lines from various indications.
Keratinocytes (NHEK.f-c.) were used to evaluate EGFR-related skin
toxicity and the liver cell line HepG2 for c-MET mediated liver
toxicity. Density values are presented as mean molecules per cell
of triplicates with standard deviations given in percent. Legend:
ACA=adenocarcinoma, CA=carcinoma.
[0070] FIG. 19: Inhibition of c-MET and EGFR phosphorylation by
c-Met.times.EGFR bsAbs. IC50 values were calculated upon 3PL
fitting of dose-response curves using GraphPad Prism. Standard
deviations (s.d.) were calculated for at least two independent
experiments carried out in duplicates. n=number of independent
experiments.
[0071] FIG. 20: Inhibition of c-MET and EGFR phosphorylation by
c-MET.times.EGFR bsAbs during ligand stimulation. Phosphorylated
c-MET (A) and phosphorylated EGFR (B) were quantified in A549, A431
and primary keratinocytes (NHEK) using electrochemiluminescence
assay (ECL). Cells were treated with varying concentrations of
bsAbs and a non-related isotype SEED control with subsequent
stimulation with 100 ng/ml HGF (A) or 100 ng/ml EGF (B). Triangles
indicate respective receptor phosphorylation levels for stimulated
(upwards triangle) and non-stimulated cells (downwards triangle).
Dose response curves were fitted using a 3PL model in GraphPad
Prism 5 (GraphPad Software, Inc).
[0072] FIG. 21: In vitro selectivity of c-MET.times.EGFR bsAbs in
comparison to cetuximab. (A) EBC-1 as tumor model cell line with
high to moderate c-MET and EGFR expression and T47D as epithelial
model cell line with low EGFR expression and no c-MET expression
were mixed in a ratio of 1:30. to order to distinguish the two cell
lines, EBC-1 cells were stained with the green membrane dye PKH2.
The cell mixture was incubated with 300 nM of bsAb and cetuximab
and subjected to flow cytometric analysis. Antibody binding was
detected by FITC-labeled anti-hu Fc secondary antibody.
Representative dot plots for green vs. yellow fluorescence are
shown. (B) In vitro selectivity was defined as the ratio of mean
fluorescence intensity of the EBC-1 and the T47D cell
population.
[0073] FIG. 22: Cytotoxicity of c-MET.times.EGFR bispecific SEED
antibody-drug conjugates generated by covalent, site-directed
conjugation of the tubulin inhibitor MMAE C-terminally to both
heavy chains in comparison to cetuximab as ADC and anti-hen egg
lysozyme (HEL) ADC as corresponding reference constructs.
Cytotoxicity was assessed on EGFR overexpressing tumor cells A431
(A) and MDA-MB-468 (B), on primary keratinocytes (NHEK.f-c., C) as
normal epithelial cell line, on c-MET overexpressing cells MKN45
(D) and EBC-1 (E) as well as HepG2 (F) as liver cell line. Assay
was run in duplicates in three independent experiments and curves
were fitted by sigmoidal curve fitting using GraphPad Prism 5
(GraphPad Software, Inc).
[0074] FIG. 23: Cytotoxicity of bispecific c-MET.times.EGFR ADC on
tumor cell line A431 and keratinocytes. EC.sub.50 values for A431
cells and IC.sub.50 values for keratinocytes (NHEK.f-c.) were
calculated by sigmoidal curve fitting using GraphPad Prism 5
(GraphPad Software, Inc). Asterisks indicate poor fitting results
because curves do not reach a saturating plateau at the highest
concentration (*). ED.sub.80 represents the ADC concentration at
which 80% of cells are killed in A431 cells in comparison to
untreated cells, TD.sub.20 indicates the dose at which cell
viability in keratinocytes is reduced by 20%. Two definitions for
an in vitro translational therapeutic index or therapeutic window
were calculated. The difference of IC.sub.50 and EC.sub.50 as well
as the ratio of TD.sub.20 to ED.sub.80.
[0075] FIG. 24: Analytical SE-HPLC indicates a purity >95% of
four exemplary bispecific antibodies (bsAb) following purification:
(A) B10v5x225-M, (B) B10v5x225-H, (C) CS06x225-M and (D)
CS06x225-H.
[0076] FIG. 25: Synergistic effect of CS06x225-H on inhibition of
c-MET, EGFR and AKT phosphorylation. (A) A549 cells were incubated
with 300 nM of the respective mAbs as indicated for 3 h and
stimulated with HGF and EGF. Cell lysates were subjected to Western
blotting and both phosphorylated and total EGFR, c-MET, and AKT
were detected. GAPDH was used as a loading control. (B)
Quantification of phospho-AKT levels in A549 cells after treatment
with 500 nM mAbs as well as combinations of control mAbs (500 nM
each) and stimulation with HGF and EGF. Cell lysates were subjected
electrochemiluminescence (ECL) ELISA. (C) ECL ELISA Of mAbs treated
and HGF-stimulated A549 cell lysates for phosphorylated c-MET
indicated increased potency of CS06x225-H in comparison to the
combination of oa CS06 and oa 225-H. (D) A549 cells were treated
with varying concentrations of mAbs without stimulation and lysates
were subjected to ECL ELISA detecting phosphorylated c-MET levels.
B10v5x225-M and B10v5x225-H demonstrated comparable partial agonism
to LY2875358.
[0077] FIG. 26: Internalization of bispecific antibodies (bsAbs) as
determined by flow cytometry and confocal fluorescence microscopy.
(A) Internalization was quantified by flow cytometric analysis
employing 100 nM bsAbs which were detected with anti-human
Fc-AlexaFluor488 conjugate at 37.degree. C. for 1 h in comparison
to cells incubated at 4.degree. C. Residual cell surface binding
was quenched by anti-AlexaFluor488 antibody. (B) EBC-1 cells were
incubated with 100 nM CS06x225-H and detected with anti-human
Fc-AlexaFluor488 conjugate at 37.degree. C. or 4.degree. C. Surface
staining was removed by acidic wash.
[0078] FIG. 27: Cytotoxicity of bispecific ADCs and bsAb an NHEK
after 6 days. Primary keratinocytes (NHEK) were incubated with
varying concentrations of bispecific ADC or alternatively with
bsAbs for 6 days, in order to exclude that the slow division rate
of keratinocytes in comparison to tumor cells influenced
cytotoxicity of the tubulin inhibitor MMAE. Curves were blotted
using 3PL fitting in GraphPad Prism 5 (GraphPad Software,
Inc.).
SEQUENCE LISTING
[0079] SEQ ID NO: 1 AG-SEED
[0080] SEQ ID NO: 2 AG-SEED
[0081] SEQ ID NO: 3 GA-SEED
[0082] SEQ ID NO: 4 GA-SEED
[0083] SEQ ID NO: 5 AG -SEED
[0084] SEQ ID NO: 6 GA-SEED
[0085] SEQ ID NO: 7 AG-SEED
[0086] SEQ ID NO: 8 GA-SEED
[0087] SEQ ID NO: 9 humanized C225 V.sub.L sequence
[0088] SEQ ID NO: 10 humanized C225 VL kinetic variants
[0089] SEQ ID NO: 11 humanized C225 VH sequence
[0090] SEQ ID NO: 12 humanized C225 VH kinetic variants
[0091] SEQ ID NO: 13 humanized C425 VL sequence
[0092] SEQ ID NO: 14 humanized C425 VH sequence
[0093] SEQ ID NO: 15 c-MET binder A12 VL sequence
[0094] SEQ ID NO: 16 c-MET binder A12 VH sequence
[0095] SEQ ID NO: 17 c-Met binder B10 VL sequence
[0096] SEQ ID NO: 18 c-MET binder B10 VH sequence
[0097] SEQ ID NO: 19 c-MET binder C10 VL sequence
[0098] SEQ ID NO: 20 c-MET binder C10 VH sequence
[0099] SEQ ID NO: 21 c-MET hinder E07 VL sequence
[0100] SEQ ID NO: 22 c-MET hinder E07 VH sequence
[0101] SEQ ID NO: 23 c-MET binder G02 VL sequence
[0102] SEQ ID NO: 24 c-MET binder G02 VH sequence
[0103] SEQ ID NO: 25 c-MET binder H06 VL sequence
[0104] SEQ ID NO: 26 c-MET binder H06 VH sequence
[0105] SEQ ID NO: 27 c-MET binder F03 VL sequence
[0106] SEQ ID NO: 28 c-MET binder F03 VH sequence
[0107] SEQ ID NO: 29 c-MET Binder F06 VL sequence
[0108] SEQ ID NO: 30 c-MET binder F06 VH sequence
[0109] SEQ ID NO: 31 c-MET binder B10v5 VL sequence
[0110] SEQ ID NO: 32 c-MET binder B10v5 VH sequence
[0111] SEQ ID NO: 33 c-MET binder CS06 VL sequence
[0112] SEQ ID NO: 34 c-MET binder CS06 VH sequence
[0113] SEQ ID NO: 35 glycine-serine linker
[0114] SEQ ID NO: 36 hinge 1
[0115] SEQ ID NO: 37 hinge 2
[0116] SEQ ID NO: 38 CL sequence
[0117] SEQ ID NO: 39 CH1 sequence
[0118] SEQ ID NO: 40 CH2 domain
[0119] SEQ ID NO: 41 CH3 domain (AG)
[0120] SEQ ID NO: 42 CH3 domain (GA)
[0121] SEQ ID NO: 43 humanized C225 VH S58R kinetic variant
(hu225-L)
[0122] SEQ ID NO: 44 humanized C225 VL N108Y kinetic variant
(hu225-M)
[0123] SEQ ID NO: 45 humanized C225 VH T109D kinetic variant
(hu225-H)
[0124] SEQ ID NO: 46 humanized C225 VL N109E, T116N kinetic variant
(hu225-H)
[0125] SEQ ID NO: 47 c-Met binder B10 VL variants comprising single
or multiple amino acid substitutions
[0126] SEQ ID NO: 48 c-MET binder B10 VH kinetic variant Q6E (IMGT
numbering)
[0127] SEQ ID NO: 49 c-MET binder F06 VL sequence variants
comprising single or multiple amino acid substitutions
[0128] SEQ ID NO: 50 c-Met binder F06 VL variants comprising single
or multiple amino acid substitutions
[0129] SEQ ID NO: 51 c-Met binder B10v5 VL variants comprising
single or multiple amino acid substitutions
[0130] SEQ ID NO: 52 c-Met binder CS06 H kinetic variants
DETAILED DESCRIPTION OF THE INVENTION
[0131] Although the present invention is described in detail below,
it is to be understood that this invention is not limited to the
particular methodologies, protocols and reagents described herein
as these may vary. It is also to be understood that the terminology
used herein is for the purpose of describing particular embodiments
only, and is not intended to limit the scope of the present
invention which will be limited only by the appended claims. Unless
defined otherwise, all technical and scientific terms used herein
have the same meanings as commonly understood by one of ordinary
skill in the art.
[0132] In the following, the elements of the present invention will
be described. These elements are listed with specific embodiments,
however, it should be understood that they may be combined in any
manner and in any number to create additional embodiments. The
variously described examples and preferred embodiments should not
be construed to limit the present invention to only the explicitly
described embodiments. This description should be understood to
support and encompass embodiments which combine the explicitly
described embodiments with any number of the disclosed and/or
preferred elements. Furthermore, any permutations and combinations
of all described elements in this application should be considered
disclosed by the description of the present application unless the
context indicates otherwise.
[0133] Throughout this specification and the claims which follow,
unless the context requires otherwise, the term "comprise", and
variations such as "comprises" and "comprising", will be understood
to imply the inclusion of a stated member, integer or step but not
the exclusion of any other non-stated member, integer or step. The
term "consist of" is a particular embodiment of the term
"comprise", wherein any other non-stated member, integer or step is
excluded. In the context of the present invention the term
"comprise" encompasses the term "consist of".
[0134] The terms "a" and "an" and "the" and similar reference used
in the context of describing the invention (especially in the
context of the claims) are to be construed to cover both the
singular and the plural, unless otherwise indicated herein or
clearly contradicted by context. Recitation of ranges of values
herein is merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range. Unless otherwise indicated herein, each individual value is
incorporated into the specification as if it were individually
recited herein. No language in the specification should be
construed as indicating any non-claimed element essential to the
practice of the invention.
[0135] Several documents are cited throughout the text of this
specification. Each of the documents cited herein (including all
patents, patent applications, scientific publications,
manufacturer's specifications, instructions, etc.), whether supra
or infra, are hereby incorporated by reference in their entirety.
Nothing herein is to be construed as an admission that the
invention is entitled to antedate such disclosure by virtue of
prior invention.
[0136] The described objectives are solved by the present
invention, preferably by the subject matter of the appended claims.
The inventors have surprisingly found that heterodimeric bispecific
immunoglobulin molecules according to the invention can be used to
overcome the resistance to EGFR- or c-MET-targeted monotherapies.
In addition, the inventive heterodimeric bispecific immunoglobulin
molecules have surprisingly been found to bind cells which express
one of EGFR or c-MET with a lower abundance with high
selectivity.
[0137] The described objective is solved according to a first
embodiment by the inventive heterodimeric bispecific immunoglobulin
molecule which comprises [0138] (i) a first Fab or scFv fragment
which specifically binds to EGFR, and [0139] (ii) a second Fab or
scFv fragment which specifically binds c-MET, and [0140] (iii) an
antibody hinge region, an antibody CH2 domain and an antibody CH3
domain comprising a hybrid protein-protein interaction interface
domain wherein each of said interaction interface domain is formed
by amino acid segments of the CH3 domain of a first member and
amino acid segments of the CH3 domain of said second member,
wherein said protein-protein interface domain of the first chain is
interacting with the protein-protein-interface of the second chain
by homodimerization of the corresponding amino acid segments of the
same member of the immunoglobulin superfamily within said
interaction domains,
[0141] wherein the first or second engineered immunoglobulin chain
has the polypeptide sequence ("AG-SEED"):
TABLE-US-00003 (SEQ ID NO: 1)
GQPFRPEVHLLPPSREEMTKNQVSLTCLARGFYPX.sub.1DIAVEWESNGQPE
NNYKTTPSRQEPSQGTTTFAVTSKLTX.sub.2DKSRWQQGNVFSCSVMHEALH
NHYTQKX.sub.3ISL,
[0142] wherein X.sub.1, X.sub.2 and X.sub.3 may be any amino acid.
For example, amino acids represented by X.sub.1, X.sub.2 and
X.sub.3 may each independently from each other be selected from the
group of naturally occurring amino acids. Engineered immunoglobulin
chains which are comprised in the inventive heterodimeric
bispecific immunoglobulin molecule and the respective sequences
thereof have been described in WO 2007/110205. In the inventive
heterodimeric bispecific immunoglobulin molecule the term
heterodimeric
[0143] A "heteromultimeric protein" according to the invention is a
protein molecule comprising at least a first subunit and a second
subunit, whereby each subunit contains a nonidentical domain. The
inventive heterodimeric bispecific immunoglobulin molecule
comprises two non-identical protein domains, e.g. "AG-SEED" and
"GA-SEED" which will result in a heterodimerization of the
non-identical protein domains in a ratio of 1:1. The inventive
heterodimeric bispecific immunoglobulin molecule according to a
first embodiment comprises a first Fab or scFv fragment which
specifically binds to EGFR. The term Fab fragment refers to an
antigen binding antibody fragment which can e.g. be obtained by
papain treatment of IgG type immunoglobulins, which will result in
two Fab fragment and an Fc domain. Functional aspects and pmethods
to obtain Fab fragments are described e.g. in "Applications and
Engineering of Monoclonal Antibodies" by D. J. King, CRC Press,
1998, chapter 2.4.1 Zaho et al, Protein Expression and Purification
67 (2009) 182-189; S. M. Andrew, J. A. Titus, Fragmentation of
immunoglobulin G, Curr. Protoc. Cell Biol. (2003) Unit 16.14
(Chapter 16). The inventive heterodimeric bispecific immunoglobulin
molecule may e.g. also comprise a first scFv fragment that
specifically binds to EGFR, The term "scFv" as used in the present
invention refers to a molecule comprising an antibody heavy chain
variable domain (or region; VH) and an antibody light chain
variable domain (or region; VL) connected by a linker, and lacks
constant domains, e.g. an scFv fragment according to the invention
may e.g. include binding molecules which consist of one light chain
variable domain (VL) or portion thereof, and one heavy chain
variable domain (VH) or portion thereof, wherein each variable
domain (or portion thereof) is derived from the same or different
antibodies. scFv molecules preferably comprise an linker interposed
between the VH domain and the VL domain, which may e.g. include a
peptide sequence comprised of the amino acids glycine and serine.
For example, the peptide sequence may comprise the amino acid
sequence (Gly.sub.4 Ser).sub.n, whereby n is an integer from 1-6,
e.g. n may be 1, 2, 3, 4, 5, or 6, preferably n=4 scFv molecules
and methods of obtaining them are known in the art and are
described, e.g., in U.S. Pat. No. 5,892,019, Ho et al. 1989. Gene
77:51; Bird et al. 1988 Science 242:423: Pantoliano et al. 1991.
Biochemistry 30:10117; Milenic et al. 1991. Cancer Research
51:6363; Takkinen et al. 1991. Protein Engineering 4:837.
[0144] A first Fab or scFv fragment of the inventive heterodimeric
bispecific immunoglobulin molecule specifically binds to human
epidermal growth factor receptor (EGFR). Specific binding, or any
grammatical variant thereof, refers to a binding of the first Fab
or scFv fragement with an Kd of at least 1.times.10.sup.-5 M, e.g.
1.times.10.sup.-6 M, 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.-11 M,
1.times.10.sup.-12 to EGFR. EGFR according to the invention refers
to EGFR having the sequences as provided by UniProtKB database
entry P00533, including all of its isoforms and sequence variants
(UniProtKB database entries P00533-1, P00533-2, P00533-3,
P00533-4), or any of the mutations described in Cai et al., PLoS
ONE 9(4): e95228, such as e.g. c.2126A>C, c.2155G>T,
c.2156G>C, c.2235_2249del15, c.2236_2250del15, c.2237_2251del,
c. 2239_2248ATTAAGAGGAG>C, c.2240_2257del 18, c2248G>C,
c.2303G>T, c.2573T>G, c.2582T>A, p745del_frameshift,
p.L858R, p.S768I.
[0145] The inventive heterodimeric bispecific immunoglobulin
molecule further comprises a second Fab or scFv fragment which
specifically binds to c-MET. c-MET as used herein refers to MET
Proto-Oncogene, Receptor Tyrosine Kinase (UniProtKB database antry
P08581), which may also be referred to as Hepatocyte Growth Factor
Receptor. For example, c-MET also includes sequence variants such
as those disclosed in Nat Genet. 1997 May; 16(1)68-73, e.g. c-MET
R970C (MET.sup.R970C) c-MET T992I (MET.sup.T922I), MET.sup.M1149T,
MET.sup.V1206L, MET.sup.V1238I, MET.sup.D1246N, MET.sup.Y1248C,
MET.sup.C1213V, MET.sup.D1246H, MET.sup.Y1248H, MET.sup.M1268T,
MET.sup.A320V, MET.sup.N375S. Specific binding of the second Fab or
scFv fragment to c-MET refers to a binding of the second Fab or
scFv fragement with an K.sub.d of at least 1.times.10.sup.-5 M,
e.g. 1.times.10.sup.-6 M, 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.-11 M,
1.times.10.sup.-12 to c-MET.
[0146] The inventive heterodimeric bispecific immunoglobulin
molecule according to a first embodiment of the invention further
comprises antibody hinge region, an antibody CH2 domain and an
antibody CH3. For example, there are five classes of
immunoglobulins (IgA, IgD, IgE, IgG, and IgM) all of which contain
a hinge region and which may be comprised in the inventive
heterodimeric bispecific immunoglobulin molecule. Additionally,
some of these classes of immunoglobulins have subclasses, e.g. IgG
has four subclasses (IgG1, IgG2, IgG3, and IgG4), (Alberts, B et
al., Chapter 23: The Immune System, In Molecular Biology of the
Cell, 3d Edition, Garland Publishing, Inc., New York, N.Y.), the
hinge regions of which may also be comprised in the heterodimeric
bispecific immunoglobulin molecule of the invention. The hinge
region may e.g. be divided into three regions: the upper, middle,
and lower hinge. The upper hinge is defined as the number of amino
acids between the end of the first domain of the heavy chain (CH1)
and the first cysteine forming an inter heavy chain disulfide
bridge. The middle hinge is high in proline and contains the
inter-heavy chain cysteine disulfide bridges. The tower hinge
connects the middle hinge to the CH2 domain (see e.g. Sandie, I.
and Michaelsen, T., Chapter 3: Engineering the Hinge Region to
Optimize Complement-induced Cytolysis, In Antibody Engineering: A
Practical Guide, W. H. Freeman and Co, New York, N.Y.;
Hamers-Casterman, C., Naturally Occurring Antibodies Devoid of
Light Chains, 363 Nature 446 (1993) and Terskikh, A. V.,
"Peptabody": A New Type of High Avidity Binding Protein, 94 Proc.
Natl. Acad. Sci. USA 1663 (1997)). The hinge region of the
inventive inventive heterodimeric bispecific immunoglobulin
molecule may e.g. also comprise any of the amino acid sequences of
the hinge regions disclosed in J. of Biological Chem. VOL. 280, NO.
50, pp. 41494-41503, Dec. 16, 2005.
[0147] In one embodiment, the heterodimeric bispecific
immunoglobulin molecule of the invention comprises as first member
IgG of the immunoglobulin super family and as second member IgA.
For example, the inventive heterodimeric bispecific immunoglobulin
molecule may in one embodiment comprise the hinge region according
to the amino acid sequence of SEQ ID NO: 1, or SEQ ID NO: 2. For
example, the inventive heterodimeric bispecific immunoglobulin
molecule may comprise derivatives of human IgG and IgA CH3 domains
which create complementary human strand-exchange engineered domain
(SEED) CH3 heterodimers that are composed of alternating segments
of human IgA and IgG CH3 sequences as described in Protein
Engineering, Design & Selection vol. 23 no. 4 pp. 195-202, 2010
WO 2007/110205 A1). The resulting pair of SEED CH3 domains
preferentially associates to form heterodimers when expressed in
mammalian cells. SEEDbody (Sb) fusion proteins consist of [IgG1
hinge]-CH2-[SEED CH3].
[0148] In one embodiment the heterodimeric bispecific
immunoglobulin molecule of the invention as disclosed above
comprises a first or second engineered immunoglobulin chain
("AG-SEED") which has the polypeptide sequence according to SEQ ID
NO:2 in which X.sub.1 is K or S, X.sub.2 is V or T, and X.sub.3 is
T or S. For example, the first or second engineered immunoglobulin
chain of the of the inventive heterodimeric bispecific
immunoglobulin molecule may comprise an amino acid sequence
according to SEQ ID NO: 2 in which X.sub.1 is K, X.sub.2 is V, and
X.sub.3 is S. X.sub.1 is K, X.sub.2 is V, and X.sub.3 is T, X.sub.1
is K, X.sub.2 is T, and X.sub.3 is S, X.sub.1 is K, X.sub.2 is T,
and X.sub.3 is T. X.sub.1 is S, X.sub.2 is V, and X.sub.3 S,
X.sub.1 is S, X.sub.2 is V, and X.sub.3 is T, X.sub.1 is S, X.sub.2
is T, and X.sub.3 is S, or X.sub.1 is S, X.sub.2 is T, and X.sub.3
is T.
[0149] In one embodiment the inventive heterodimeric bispecific
immunoglobulin molecule as disclosed above comprises a first or
second engineered immunoglobulin chain which has the polypeptide
sequence according to SEQ ID NO: 3 ("GA-SEED"), whereby wherein
X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5 and X.sub.6 may be any
amino acid, e.g. X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5,
X.sub.6 may be independently selected from alanine, arginine,
asparagine, aspartic acid, asparagine or aspartic acid, cysteine,
glutamic acid, glutamine, glycine, histidine, isoleucine, leucine,
lysine, methionine, phenylalanine, proline, serine, threonine,
tryptophan, tyrosine, or valine. According to a one embodiment, the
first or second engineered immunoglobulin chain of the inventive
heterodimeric bispecific immunoglobulin molecule as disclosed above
has the amino acid sequence according to SEQ ID NO:3, wherein
X.sub.1 is L or Q, X.sub.2 is A or T, X.sub.3 is L, V, D or T;
X.sub.4 is F, A, D, E, G, H, K, N, P, Q, R, S or T; X.sub.5 is A or
T, and X.sub.6 is E or D. In a preferred embodiment, the first
engineered immunoglobulin chain comprises the amino acid sequence
according to SEQ ID NO: 5 ("AG-SEED") and the second engineered
immunoglobulin chain of the inventive heterodimeric bispecific
immunoglobulin molecule as disclosed above comprises the amino acid
sequence according to SEQ ID NO: 6 ("GA-SEED").
[0150] In one embodiment the inventive heterodimeric bispecific
immunoglobulin molecule binds to EGFR as disclosed above with an
affinity of at Least K.sub.D=5.times.10.sup.-8 M, 1.times.10.sup.-9
M, 1.times.10.sup.-10 M, 1.times.10.sup.-11 M, 1.times.10.sup.-12
to EGFR. According to one embodiment the inventive heterodimeric
bispecific immunoglobulin molecule binds to c-MET as disclosed
above with an affinity of at least K.sub.D=5.times.10.sup.-8 M,
1.times.10.sup.-9 M, 1.times.10.sup.-10 M, 1.times.10.sup.-11 M,
1.times.10.sup.31 12 to c-MET. For example, the heterodimeric
bispecific immunoglobulin molecule of the invention as disclosed
above binds via a first and second Fab or scFv fragment c-MET and
EGFR with an affinity of K.sub.D=5.times.10.sup.-8 M,
1.times.10.sup.-9 M, 1.times.10.sup.-10 M, 1.times.10.sup.-11 M,
1.times.10.sup.-12 M. EGFR and c-Met may e.g. be present on a
single cell, such as a cancer cell, or e.g. to a cell, such as e.g.
cancer cell, which may be single cell, a pluarality of cells, or
tumor tissue that expresses both c-MET and EGFR. The cells may e.g.
also be in suspension, or detached from tissue and may circulate in
the blood stream of an individual, such as a human inflicted with
cancer. For example, the affinity of first and second Fab and/or
scFv fragments of the inventive heterodimeric bispecific
immunoglobulin molecule may be determined by ELISA, or surface
plasmon resonance as described in J. Biochem. Biophys. Methods 57
(2003) 213-236, Current Protocols in Protein Science (2006)
19.14.1-19.14.17.
[0151] According to one embodiment the first Fab or scFv fragment
of the heterodimeric bispecific immunoglobulin molecule of the
invention as disclosed above is derived from cetuximab (C225). For
example, the first Fab or scFv fragment of the heterodimeric
bispecific immunoglobulin molecule may comprise VL and VH sequences
of cetuximab, or e.g. VL and VH sequences of cetuximab which have
been humanized. For example, humanized as used for the inventive
heterodimeric bispecific immunoglobulin molecule refers to a
chimeric antibody or antibody fragment which contain minimal
sequence derived from non-human immunoglobulin. Humanization of a
given antibody sequence will result in a reduction of the
immunogenicity of a xenogenic antibody, such as a murine antibody,
or chimeric antibody which already comprises human sequences, for
introduction into a human, while maintaining the full antigen
binding affinity and specificity of the antibody. For example,
cetuximab is a chimeric antibody which is composed of the Fv
(variable; antigen-binding) regions of the 225 murine EGFR
monoclonal antibody specific for the N-terminal portion of human
EGFR with human IgG1 heavy and kappa light chain constant
(framework) regions.
[0152] Humanization may e.g. comprise CDR grafting technology which
involves substituting the complementarity determining regions of,
for example, a mouse antibody, into a human framework domain, e.g.,
see WO 02/22653. Strategies and methods for the resurfacing of
antibodies, and other methods for reducing immunogenicity of
antibodies within a different host, are disclosed in U.S. Pat. No.
5,639,641. Antibodies can be humanized using a variety of other
techniques including CDR-grafting (see e.g. EP 0 239 400 B1; WO
91/09967; U.S. Pat. Nos. 5,530,101; 5,585,089), veneering or
resurfacing (see e.g. EP 0 592 106; EP 0 519 596; Padlan E. A.,
1991, Molecular Immunology 28(4/5); 489-498; Studnicka G. M. et
al., 1994, Protein Engineering, 7(6): 805-814; Roguska M. A. et
al., 1994. PNAS, 91: 969-973), chain shuffling (see e.g. U.S. Pat.
No. 5,565,332), and identification of flexible residues (see e.g.
WO2009032661). Human antibodies can be made by a variety of methods
known in the art including phage display methods, such as .g. U.S.
Pat. Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; and
international patent application publication numbers WO 98/46645,
WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, WO
91/10741. Accordingly, the first Fab or scFv fragment of the
heterodimeric bispecific immunoglobulin molecule of the invention
as disclosed above may comprise VL and VH sequences according to to
any one of SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO:
12, SEQ ID NO: 43, SEQ ID NO. 44, SEQ ID NO: 45, SEQ ID NO: 46. For
example, the VL amino acid sequence of first Fab or scFv fragment
of the heterodimeric bispecific immunoglobulin molecule may
comprise the amino acid sequence according to SEQ ID NO: 9, SEQ ID
NO: 10, SEQ ID NO: 44, SEQ ID NO: 46 and VH amino acid sequences
selected from SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 11, SEQ ID
NO: 12. VL and VH sequences of the first Fab or scFv fragment as
disclosed above may e.g. comprise SEQ ID NO:43 and SEQ ID NO: 9,
SEQ ID NO: 44 and SEQ ID NO: 9, or SEQ ID NO: 45 and SEQ ID NO: 9,
or e.g. SEQ ID NO: 43 and SEQ ID NO: 9, or SEQ ID NO:45 and SEQ ID
NO: 9, or SEQ ID NO: 46 and SEQ ID NO: 11.
[0153] According to one embodiment the second Fab or scFv fragment
of the inventive heterodimeric bispecific immunoglobulin molecule
has disclosed above comprises VL sequences selected from the group
consisting of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID
NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29,
SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID
NO: 51.
[0154] In one embodiment the the second Fab or scFv fragment of the
inventive heterodimeric bispecific immunoglobulin molecule has
disclosed above comprises VH sequences selected from the group
consisting of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID
NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30,
SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 48, ,SEQ ID NO: 50, or SEQ
ID NO: 52.
[0155] According to one embodiment, the first and second Fab or
scFv fragments of the heterodimeric bispecific immunoglobulin
molecule of the invention as disclosed above comprise the amino
acid sequences selected from SEQ ID NO: 9, SEQ ID NO: 43, SEQ ID
NO: 17, SEQ ID NO: 18, or SEQ ID NO: 47, SEQ ID NO: 48, (e.g. which
may be comprised in the inventive molecule "225-LxB10"), or SEQ ID
NO: 11, SEQ ID NO: 44, SEQ ID NO: 31, SEQ ID NO: 51, SEQ ID NO:32
(e.g. which may be comprised in the inventive molecule
"225-MxB10v5"), or SEQ ID NO:45, SEQ ID NO: 46, SEQ ID NO: 29, SEQ
ID NO: 49, SEQ ID NO: 30, SEQ ID NO: 50, (e.g. which may be
comprised in the inventive molecule "225-HxF06"), or SEQ ID NO: 45,
SEQ ID NO: 46, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 52 (e.g.
which may be comprised in the inventive molecule "225-HxCS06"), or
SEQ ID NO: 11, SEQ ID NO: 44, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID
NO: 52 (e.g. which may be comprised in the inventive molecule
"225-MxCS06").
[0156] According to one embodiment the first and second Fab or scFv
fragments of the heterodimeric bispecific immunoglobulin molecule
of the invention as disclosed above comprise the amino acid
sequences according to SEQ ID NO: 11, SEQ ID NO: 44, SEQ ID NO: 33,
SEQ ID NO: 34, SEQ ID NO; 52 (e.g. corresponding to the inventive
molecule "225-MxCS06"), or SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO:
29, SEQ ID NO: 49, SEQ ID NO: 30, SEQ ID NO: 50 (e.g. corresponding
to the inventive molecule "225-HxCS06").
[0157] In one embodiment the inventive the Fc domain of the
heterodimeric bispecific immunoglobulin molecule interacts with the
neonatal Fc receptor (FcRn). FcRn is a major histocompatibility
complex class I-like heterodimer composed of the soluble light
chain .beta.2-microglobulin (.beta.2m) and a membrane-bound heavy
chain. Crystal structure analysis revealed that the human FcRn
(hFcRn) binds to the CH2-CH3 hinge region of both heavy chains of
the Fc homodimer of an IgG, resulting in a 2:1 stoichiometry. The
interaction between FcRn and Fc is mainly stabilized by salt
bridges between anionic FcRn residues and histidine residues of the
IgG, which are protonated at acidic pH. Site-directed mutagenesis
studies and crystal structure analysis of the FcRn/IgG Fc complex
show that the Fc amino acid residues at positions 252-256 in the
CH2 domains and at 310, 433, 434, and 435 in the CH3 domains are at
the core or in close proximity to the FcRn interaction site, and
that the conserved histidine residues H310 and possibly H435 are
responsible for the pH dependence (see e.g. mAbs 6:4, 928-942,
July/August 2014; Nature Reviews Immunology 7, 715-725 (September
2007)). For example, the inventive heterodimeric bispecific
immunoglobulin molecule may interact with the FcRn via salt bridges
as disclosed above, or may interact with FcRn by salt bridges that
involve other amino acids of both AG-SEED and GA-SEED, thereby
protecting the inventive heterodimeric bispecific immunoglobulin
molecule from degradation and extending its serum half-life.
Extended half-life of the inventive heterodimeric bispecific
immunoglobulin molecule may e.g. be employed to minimize adverse
reactions caused by high doses of the inventive heterodimeric
bispecific immunoglobulin molecule if administered to an individual
e.g. by i v. or i.m. application, which will e.g. also result in a
decreased frequency of injection of the inventive heterodimeric
bispecific immunoglobulin molecule. This will e.g. also reduce the
financial burden on an individual which may be in need of a
treatment with the inventive heterodimeric bispecific
immunoglobulin molecule. For example, sequence variants or the
AG-SEED and GA-SEED may be used to reduce the interaction of the
inventive heterodimeric bispecific immunoglobulin molecule with
FcRn thereby shortening its serum half-life. Sequence variants e.g.
include those disclosed above, AG-SEED with X.sub.1, X.sub.2 and
X.sub.3 representing any amino acid, or e.g. preferably an AG-SEED
in which X.sub.1 is K or S, X.sub.2 is V or T, and X.sub.3 is T or
S, e.g. a GA-SEED as disclosed above wherein X.sub.1, X.sub.2,
X.sub.3, X.sub.4, X.sub.5 and X.sub.6 may be any amino acid. It may
e.g. be preferred that in the GA-SEED X.sub.1 is L or Q, X.sub.2 is
A or T, X.sub.3 is L, V, D or T, X.sub.4 is F, A, D, E, G, H, K, N,
P, Q, R, S or T; X.sub.5 is A or T, and X.sub.6 is E or D.
[0158] In one embodiment, the amino acids of the inventive
heterodimeric bispecific immunoglobulin molecule as disclosed above
which interact with FcRn are derived from IgG1, preferably human
IgG1. For example, the amino acids which interact with FcRn
comprise those of wildtype IgG1 as disclosed above, e.g. Fc amino
acid residues at positions 252-256 in the CH2 domains and at 310,
433, 434, and 435 in the CH3 domains are at the core or in close
proximity to the FcRn interaction site, whereby the conserved
histidine residues H310 and possibly H435 may e.g. confer for the
pH dependence of the interaction between the inventive
heterodimeric immunoglobulin molecule and FcRn.
[0159] In one embodiment the inventive heterodimeric bispecific
immunoglobulin molecule as disclosed above mediates
antibody-dependent cellular cytotoxicity. For example, the
inventive heterodimeric bispecific immunoglobulin molecule induces
ADCC when bound to EGFR and c-MET expressed on the surface of the
same cell cell, or e.g. when bound to two cells, one of which
expresses EGFR and the second one of which expresses c-MET, whereby
e.g. EGFR and c-Met are as defined above. Binding of the
heterodimeric bispecific immunoglobulin molecule of the invention
to EGFR and c-Met present on the same cell or on two individual
cells, but preferably one the same cell, is as disclosed above. The
term ADCC (antibody dependent cell cytotoxicity) as used for the
inventive heterodimeric bispecific immunoglobulin molecule refers
to a mechanism of cell-mediated immune defense whereby an effector
cell of the immune system actively lyses a target cell, whose
membrane-surface antigens have been bound by specific antibodies.
ADCC is mediated by e.g. the binding of CD16 (FcyRIII) expressed on
NK cells to the Fc domain of antibodies (see e.g. Clynes et al.
(2000) Nature Medicine 6, 443-446). ADCC may e.g. be improved by
amino acid substitutions in the Fc domain which affect the binding
of the Fc domain to CD16. For example. Shields et al. (J Biol Chem
9(2), 6591-6604 (2001)) showed that amino acid substitutions at
positions 298, 333, and/or 334 of the Fc region (EU numbering of
residues) improve ADCC. Alternatively, increased Fc receptor
binding and effector function may e.g. be obtained by altering the
glycosylation of the Fc region. The two complex biantennary
oligosaccharides attached to Asn 297 of the Fc domain are typically
buried between the CH2 domains, forming extensive contacts with the
polypeptide backbone, and their presence is essential for the
antibody to mediate effector functions including ADCC (Lifely et
al., Glycobiology 5, 813-822 (1995); Jefferis et al., Immunol Rev
163, 59-76 (1998); Wright and Morrison, Trends Biotechnol 15, 26-32
(1997)), Overexpression of e.g.
.beta.(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a
glycosyltransferase catalyzing the formation of bisected
oligosaccharides, significantly increases the in vitro ADCC
activity of antibodies. Thus overexpression of e.g. of GnTIII in
cell lines used for the production of the inventive heterodimeric
bispecific immunoglobulin molecule, may result in inventive fusion
proteins enriched in bisected oligosaccharides, which are generally
also non-fucosylated and may exhibit increased ADCC.
[0160] In one embodiment the invention provides an isolated
polynucleotide when encodes at least one of the amino acid
sequences according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,
SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:
8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ
ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID
NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO: 21,
SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID
SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID
NO: 30, SEQ ID NO. 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34,
SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID
NO: 39, SEQ ID NO:40, SEQ ID NO: 41, SEQ ID NO:42, SEQ ID NO: 43,
SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:
48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO:51, SEQ ID NO: 52 of
the inventive bispecific heterodimeric immunoglobulin molecule. For
example, the isolated polynucleotide of the invention may encode at
least one, e.g. one, two, three, four, five, six, seven, eight,
nine or ten of the amino acid sequences as disclosed above. For
example, in one embodiment the isolated polynucleotide comprises
polynucleotides which encode at least one of the amino acid
sequences according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,
SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO. 7, SEQ ID NO:
8, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ
ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO: 47,
SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO:51, SEQ ID
NO: 52 of the inventive bispecific heterodimeric immunoglobulin
molecule. For example the isolated polynucleotide of the invention
may comprise polynucleotides which encode amino acid sequences
according to (225M, CS06) SEQ ID NO: 11, SEQ ID NO: 44, SEQ ID NO:
33, SEQ ID NO: 34, SEQ ID NO: 52, or (225H, CS06) SEQ ID NO: 45,
SEQ ID NO: 46, SEQ ID NO: 29, SEQ ID NO: 49, SEQ ID NO: 30, SEQ ID
NO: 50. For example, in one embodiment the isolated polynucleotide
according to the invention may e.g. comprise polynucleotides
encoding the amino acid sequences according to SEQ ID NO: 31, SEQ
ID NO: 32, SEQ ID NO: 43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46.
In one embodiment the polynucleotide according to the invention
e.g. comprises polynucleotides which encode the amino according to
SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO:45, SEQ ID NO:46. For
example, in one embodiment the inventive polynucleotide encodes
amino acid sequences according to SEQ ID NO: 31, SEQ ID NO: 32, SEQ
ID NO:45, SEQ ID NO:46. In one embodiment, the polynucleotide
according to the invention comprises polynucleotides which encode
the amino acid sequences selected from SEQ ID NO: 9, SEQ ID NO: 43,
SEQ ID NO: 17, SEQ ID NO:18, or SEQ ID NO: 47, SEQ ID NO: 48 (e.g.
which may be comprised in the inventive molecule "225-LxB10"), or
SEQ ID NO: 11, SEQ ID NO: 44, SEQ ID NO: 31, SEQ ID NO: 51, SEQ
NO:32 (e.g. which may be comprised in the inventive molecule
"225-MxB10v5"), or SEQ ID NO:45, SEQ ID NO: 46, SEQ ID NO: 29, SEQ
ID NO: 49, SEQ ID NO: 30, SEQ ID NO: 50, (e.g. which may be
comprised in the inventive molecule "225-HxF06"), or SEQ ID NO: 45,
SEQ ID NO: 46, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 52 (e.g.
which may be comprised in the inventive molecule "225-HxCS06"), or
SEQ ID NO: 11, SEQ ID NO: 44, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID
NO: 52 (e.g. which may be comprised in the inventive molecule
"225-MxCS06"), or SEQ ID NO: 11, SEQ ID NO: 44, SEQ ID NO: 33, SEQ
ID NO: 34, SEQ ID NO: 52 (e.g. corresponding to the inventive
molecule "225-MxCS06"), or SEQ ID NO:45, SEQ ID NO: 46, SEQ ID NO:
29, SEQ ID NO: 49, SEQ ID NO: 30, SEQ ID NO: 50 (e.g. corresponding
to the inventive molecule "225-HxCS06"), or (225M, CS06) SEQ ID NO:
11, SEQ ID NO: 44, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 52, or
(225H, CS06) SEQ ID NO:45, SEQ ID NO: 46, SEQ ID NO: 29, SEQ ID NO:
49, SEQ ID NO: 30, SEQ ID NO 50, or (e.g. corresponding inventive
molecule "225M, B10v5") SEQ ID NO: 11, SEQ ID NO: 44, SEQ ID NO:
31, SEQ ID NO: 51, SEQ ID NO:32, or (e.g. corresponding inventive
molecule "225H, CS06") SEQ ID NO:45, SEQ ID NO: 46, SEQ ID NO: 29,
SEQ ID NO: 49, SEQ ID NO: 30, SEQ ID NO: 50. For example, the
nucleotide sequence of each of the above amino acid sequences of
the invention may be obtained by translation using web-based tools,
such as "Translate tool" (http://web.expasy.org/translate/) and may
e.g. be codon-optimized accordance with the intended expression
system or host (see e.g. Trends Mol Med. 2014 November;
20(11):604-13; Genome Res. 2007 April; , 17(4):401-4). For example,
the polynucleotides encoding the amino acid sequences as disclosed
above may be comprised on individual polynucleotides, each of which
is considered a polynucleotide according to the invention, or e.g.
the polynucleotide according to the invention may comprise
polynucleotides encoding two of the amino acid sequences as
disclosed above e.g. SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO:
33, SEQ ID NO: 34, or SEQ ID NO:45, SEQ ID NO:46, or SEQ ID NO: 29,
SEQ ID NO: 30, or SEQ ID NO: 11, SEQ ID NO: 9, or SEQ ID NO 49, SEQ
ID NO: 50. The polynucleotides according to the invention as
disclosed above may e.g. be used for the production of the
inventive bispecific heterodimeric immunoglobulin molecule, e.g. by
heterologous expression in a suitable host, or host cell.
[0161] The term "isolated" as used with the polynucleotides
according to the invention refers to polynucleotides which are
separated from e.g. constituents, cellular and otherwise, in which
the polynucleotide are normally associated with in nature, e.g. the
isolated polynucleotide is at least 80%, 90%, 95% pure by weight,
devoid or contaminating constituents. For example, isolated
polynucleotides of the invention may refer to a DNA molecule that
is separated from sequences with which it is immediately contiguous
(in the 5' and 3' directions) in the naturally occurring genome of
the organism from which it was derived. For example, the "isolated
polynucleotide" may comprise a DNA molecule inserted into a vector,
such as a plasmid or virus vector, or integrated into the genomic
DNA of a procaryote or eucaryote.
[0162] In one embodiment the present invention provides a vector
which comprises at least one polynucleotide according to the
invention as disclosed above. The tem vector or expression vector
according to the invention refers to a nucleic acid molecule
capable of extra-chromosomal replication. Preferred vectors are
those capable of autonomous replication and expression of nucleic
acids to which they are linked. Vectors capable of directing the
expression of genes to which they are operatively linked are
referred to herein as "expression vectors". In general, expression
vectors of utility in recombinant DNA techniques are often in the
form of "plasmids" which refer generally to circular double
stranded DNA loops which, in their vector form are not bound to the
chromosome. Nucleic acid sequences necessary for expression of the
heterodimeric bispecific immunoglobulin molecule in eukaryotic
cells comprise e.g. at least one promoter, and enhancers,
termination and polyadenylation signals as well as a selectable
marker, such as e.g. an antibiotic resistance. Expression vectors
which may be used for expression of the inventive heterodimeric
bispecific immunoglobulin molecule may e.g. comprise pCMV, pcDNA,
p4X3, p4X4, p4X5, p4X6, pVL1392, pVL1393, pACYC177, PRS420, or if
viral based vector systems are to be used e.g. pBABEpuro, pWPXL,
pXP-derived vectors may e.g. comprise pCMV, pcDNA, p4X3, p4X4,
p4X5, p4X6, pVL1392, pVL1393, pACYC177, PRS420, or if viral based
vector systems are to be used e.g. pBABEpuro, pWPXL, pXP-derived
vectors.
[0163] In one embodiment, present invention provides a host cell
which comprises the polynucleotide sequence or vector as disclosed
above, e.g. a polynucleotide or vector or expression vector which
comprises at least one coding sequence for the inventive
heterodimeric bispecific immunoglobulin molecule as disclosed
above. For example, a host cell for use in the invention may be a
yeast cell, insect cell or mammalian cell. For example, the host
cell of the invention may be an insect cell selected from Sf9,
Sf21, S2, Hi5, or BTI-TN-5B1-4 cells, or e.g. the host cell of the
invention may be a yeast cell selected from Saccharomnyces
cerevislae, Hansenula polymorpha, Schizosaccharomyces pombe,
Schwanniomyces occidentalis, Kluyveromyceslactis, Yarrowia
lipolytica and Pichia pastoris, or e.g. the host cell of the
invention may be a mammalian cell selected from HEK293, HEK293T,
HEK293E, HEK 293F, NS0, per.C6, MCF-7, HeLa, Cos-1, Cos-7, PC-12,
3T3, Vero, vero-76, PC3, U87, SAOS-2, LNCAP, DU145, A431, A549,
B35, H1299, HUVEC, Jurkat, MDA-MB-231, MDA-MB-468, MDA-MB-435,
Caco-2, CHO, CHO-K1, CHO-B11, CHO-DG44, BHK, AGE1.HN, Namalwa,
WI-38, MRC-5, HepG2, L-929, RAB-9, SIRC, RK13, 11B11, 1D3, 2.402,
A-10, B-35, C-6, F4/80, IEC-18, L2, MH1C1, NRK, NRK-49F, NRK-52E,
RMC, CV-1, BT, MDBK, CPAE, MDCK.1, MDCK.2, and D-17.
[0164] In one embodiment the invention provides a method for
producing the heterodimeric bispecific immunoglobulin molecule of
the invention as disclosed above, whereby the inventive method
comprises the steps of culturing a host cell according to the
invention as disclosed above under conditions sufficient for the
heterologous expression of said heterodimeric bispecific
immunoglobulin molecule and purifying said heterodimeric bispecific
immunoglobulin molecule. For example, host cells of the invention
may be allowed to grow in DMEM containing 10% FBS, and were
incubated at 37.degree. C. in 10% CO.sub.2 or e.g. in protein-free
culture medium to aid in the subsequent isolation and purification,
or e.g. in Grace's insect medium, express Five .RTM. SFM (Life
Technologies), or High Five.RTM. medium (Life Technologies), YNM
medium, YPD broth, or e.g. PichiaPink (Life technologies). For
example, expression of the inventive, heterodimeric bispecific
immunoglobulin molecule in mammalian cells may be done according to
the method as described in Methods Mol Biol. 2012; 907:341-58.
Insect cells may e.g. also be used for the expression of the
inventive heterodimeric bispecific immunoglobulin molecule such as
e.g. Drosophila S2 cells as described in Journal of Immunological
Methods 318 (2007) 37-46. Yeast cells, for example, may also be
used for the expression of the inventive heterodimeric bispecific
immunoglobulin molecule, such as Pichia pastoris as described in
Appl Microbiol Biotechnol. 2014 December; 98(24):10023-39, or
Biotechnol Lett. 2015 July; 37(7):1347-54.
[0165] The host cells of the invention may e.g. be allowed to grow
between 12-408 h, e.g. for about 12 to about 400 h, e.g. between 14
h, 16 h, 18 h, 20 h, 24 h, 36 h, 48 h, 72 h, 96 h to about 120 h,
144 h, 168 h, 192, 216 h, 240 h, 264 h, 288 h, 312 h, 336 h, 360 h,
384 h, 408 h. Subsequently, the inventive vNAR or inventive fusion
protein may be isolated and purified. For example, the
heterodimeric bispecific immunoglobulin molecule of the invention
may be purified and isolated by chromatography, e.g. ion-exchange
chromatography, size-exclusion chromatography, ammonium sulfate
precipitation, or ultrafiltration. For example, the inventive
heterodimeric bispecific immunoglobulin molecule may also comprise
a signal sequence, which refers to an amino acid sequence which is
capable of initiating the passage of a polypeptide, to which it is
operably linked, e.g. by a peptide bond, into the endoplasmic
reticulum (ER) of a host cell. The signal peptide is generally
cleaved off by an endopeptidase (e.g. a specific ER-located signal
peptidase) to release the (mature) polypeptide. The length of a
signal peptide is typically in the range from about 10 to about 40
amino acids.
[0166] In one embodiment the invention provides a heterodimeric
bispecific immunoglobulin molecule according to the invention as
disclosed above which is obtainable by the inventive method as
disclosed above. For example, the heterodimeric bispecific
immunoglobulin molecule of the invention as disclosed above may be
produced by the inventive method as disclosed above and
isolated.
[0167] In one embodiment the heterodimeric bispecific
immunoglobulin molecule of the invention as disclosed above is
covalently coupled to at least one linker. The term "linker" or
"linker peptide" refers to a synthetic or artifical amino acid
sequence that connects or links two molecules, such as e.g. two
polypeptide sequences that link two polypeptide domains, or e.g. a
protein and a cytostatic drug, or toxin. The term "synthetic" or
"artifical" as used in the present invention refers to amino acid
sequences that are not naturally occurring. The linker which is
covalently bound to the heterodimeric bispecific immunoglobulin
molecule of the invention is cleavable or non-cleavable. The term
"cleavable" as used in the present invention refers to linkers
which may be cleaved by proteases, acids, or by reduction of a
disulfide body (e.g. glutathion-mediated or glutathion sensitive).
For example, cleavable linkers may comprise valine-citrulline
linkers, hydrazone linkers, or disulfide linkers. Non-cleavable
linkers which may e.g. be covalently bound to the amino
donor-comprising substrate of the invention comprise
maleimidocaproyllinker to MMAF (mc-MMAF),
N-maleimidomethylcyclohexane-1-carboxylate MCC), or
mercapto-acetamidocaproyl linkers. For example, the linkers which
are covalently coupled to the inventive heterodimeric bispecific
immunoglobulin molecule may also include linkers as described in WO
2010/138719, or e.g. those described in WO 2014/093379.
[0168] In one embodiment the linker of the heterodimeric bispecific
immunoglobulin molecule of the invention as disclosed above is
coupled to a dye, radioisotope, or cytotoxin, The term "coupled" as
used for the linker as disclosed above refers to the fact that the
dye, radioisotope or cytoxin may e.g. be non-covalently via e.g.
ionic, or hydrophobic interactions, or covalently attached to the
linker molecule as disclosed above. For example, the linker may
comprise streptavidin and the dye, radioisotope or cytotoxin may be
covalently bound to biotin. For example, the dye which may be
covalently linked or coupled to the inventive heterodimeric
bispecific immunoglobulin molecule may also be a fluorophore, such
as e.g. 1,8-ANS, 4-methylumbelliferone, 7-amino-4-methylcoumarin,
7-hydroxy-4-methylcoumarin, Acridine, Alexa Fluor 350.TM., Alexa
Fluor 405.TM., AMCA, AMCA-X, ATTO Rho6G, ATTO Rho11, ATTO Rho12,
ATTO Rho13, ATTO Rho14, ATTO Rho101, Pacific Blue, Alexa Fluor
430.TM., Alexa Fluor 480.TM., Alexa Fluor 488.TM., BODIPY 492/515,
Alexa Fluor 532.TM., Alexa Fluor 546.TM., Alexa Fluor 555.TM.,
Alexa Fluor 594.TM., BODIPY 505/515, Cy2, cyQUANT GR, Fluo-3,
Fluo-4, GFP (EGFP), mHoneydew, Oregon Green.TM. 488, Oregon
Green.TM. 514, EYFP, DsRed, DsRed2, dTomato, Cy3.5, Phycoerythrin
(PE), Rhodamine Red, mTangerine, mStrawberry, mOrange, mBanana,
Tetramethylrhodamine (TRITC), R-Phycoerythrin, ROX, DyLight 594,
Calcium Crimson, Alexa Fluor 594.TM., Alexa Fluor 610.TM., Texas
Red, mCherry, mKate, Alexa Fluor 660.TM., Alexa Fluor 680.TM.
allophycocyanin, DRAQ-5, carboxynaphthofluorescein, C7, DyLight
750, Cellvue NIR780, DM-NERF, Eosin, Erythrosin, Fluorescein, FAM,
Hydroxycoumarin, IRDyes (IRD40, IRD 700, IRD 800), JOE, Lissamine
rhodamine B, Marina Blue, Methoxy coumarin, Naphtho fluorescein,
PyMPO, 5-carboxy-4',5'-dichloro-2',7'-dimethoxy fluorescein, 5-
carboxy-2',4',5',7'-tetrachlorofluorescein, 5-carboxyfluorescein,
5-carboxyrhodamine, 6- carboxyrhodamine, 6-carboxytetramethyl
amino, Cascade Blue, Cy2, Cy3, Cy5,6-FAM, dansyl chloride, HEX,
6-JOE, NBD (7-nitrobenz-2-oxa-1,3-diazole), Oregon Green 488,
Oregon Green 500, Oregon Green 514, Pacific Blue, phthalic acid,
terephthalic add, isophthalic acid, cresyl fast violet, cresyl blue
violet, brilliant cresyl blue, para-aminobenzoic acid, erythrosine,
phthalocyanines, azomethines, cyanines, xanthines,
succinylfluoresceins, rare earth metal cryptales, europium
trisbipyridine diamine, a europium cryptate or chelate, diamine,
dicyanins, or La Jolla blue dye. Dyes which may be used in the
invention may e.g. also include quantum dots. The term quantum dot
as used in the present invention refers to a single spherical
nanocrystal of semiconductor material where the radius of the
nanocrystal is less than or equal to the size of the exciton Bohr
radius for that semiconductor material (the value for the exciton
Bohr radius can be calculated from data found in handbooks
containing information on semiconductor properties, such as the CRC
Handbook of Chemistry and Physics, 83rd ed., Lide, David R.
(Editor), CRC Press, Boca Raton, Fla. (2002)). Quantum dots are
known in the art, as they are described in references, such as
Weller, Angew. Chem. Int. Ed. Engl. 32: 41-53 (1993), Alivisatos,
J. Phys. Chem. 100: 13226-13239 (1996), and Alivisatos, Science
271: 933-937 (1996), Quantum dots may e.g. be from about 1 nm to
about 1000 nm diameter, e.g. 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60
nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm,
350 nm, 400 nm, 450 nm, or 500 nm, preferably at least about 2 nm
to about 50 nm, more preferably QDs are at least about 2 nm to
about 20 nm in diameter (for example about 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nm). QDs are
characterized by their substantially uniform nanometer size,
frequently exhibiting approximately a 10% to 15% polydispersion or
range in size. A QD is capable of emitting electromagnetic
radiation upon excitation (i.e., the QD is photoluminescent) and
includes a "core" of one or more first semiconductor materials, and
may be surrounded by a "shell:" of a second semiconductor material,
A QD core surrounded by a semiconductor shell is referred to as a
"core/shell" QD. The surrounding "shell" material will preferably
have a bandgap energy that is larger than the bandgap energy of the
core material and may be chosen to have an atomic spacing close to
that of the "core" substrate. The core and/or the shell can be a
semiconductor material including, but not limited to, those of the
groups II-VI (ZnS, ZnSe, ZnTe, US, CdSe, CdTe, HgS, HgSe, HgTe,
MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe,
and the like) and III-V (GaN, GaP, GaAs, GaSb, InN, InP, InAs,
InSb, and the like) and IV (Ge, Si, and the like) material, PbS,
PbSe, and an alloy or a mixture thereof. Preferred shell materials
include ZnS. Quantum dots may be coupled to the inventive linker,
enzyme, or protein by any method known in the art such as e.g. the
methods disclosed Nanotechnology 2011 Dec. 9; 22(49):494006;
Colloids and Surfaces B: Biointerfaces 84 (2011) 360-368. For
example, the linker as disclosed above may be covalently bound or
coupled to a radioisotope such as e.g. .sup.47Ca, .sup.14C,
.sup.137Cs, .sup.57Co, .sup.60Co, .sup.87CU, .sup.87Ga, .sup.123I,
.sup.125I, .sup.129I, .sup.131I, .sup.32P, .sup.75Se, .sup.85Sr,
.sup.35S, .sup.201Th, .sup.3H, preferably, the radioisotopes are
incorporated into a further moelcule, such as e.g. a chelator.
Typical chelators that may e.g. be used as a further molecule
covalently bound to the amino donor-comprising substrate of the
invention are DPTA, EDTA (Ethylenediamine-tetraacetic acid), EGTA
(Ethyleneglycol-O,O'-bis(2-aminoethyl-N,N,N',N'-tetraacetic acid,
NTA (Nitrilotriacetic acid), HEDTA
(N-(2-Hydroxyethyl)-ethylenediaminee-N,N',N'-triaceticacid), DTPA
(2-[Bis[2-[bis(carboxymethyl)amino]-ethyl]amino]acetic acid), or
DOTA (1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic
acid).
[0169] For example, the linker may be covalently coupled to a
cytotoxin, which may e.g. also be referred to as "payload" (see
e.g. Perez et al. Drug Discovery Today Vol 19 (7), July 2014).
Cytotoxins which are e.g. suited for covalent attachment to linker
molecules may be grouped into two main classes: The first class
includes cytotoxins which disrupt microtubule assembly and the
second class cytotoxins which target DNA structure. Accordingly,
cytotoxins which may e.g. be covalently coupled to the linker as
disclosed above include doxorubicin, calicheamicin, auristatin,
maytansine duoarmycin and analogs thereof, .alpha.-amaitin,
tubulysin and analogs thereof. Methods for covalently coupling or
attaching cytotoxins to linkers are known in the art and may e.g.
be done according to the method disclosed in Mol. Pharmaceutics
2015, 12, 1813-1835.
[0170] In one embodiment the at least one linker as disclosed above
is covalently coupled to at least one Fab or scFv light chain (VL)
of the inventive heterodimeric bispecific immunoglobulin molecule.
Accordingly at least one light chain, e.g. one or two light chains
of the inventive heterodimeric bispecific immunoglobulin molecule
may be coupled to a linker as disclosed above. For example,
covalent coupling may be done by introducing, one or more, e.g. 2,
3, or 4, 5 or 6, additional cysteine residues into the scFv
molecule, mainly at the C-terminus, which allow conjugation to
sulfhydryl-reactive reagents as disclosed in e.g. Marty et al.
Protein Expression and Purification 21, 156-154 (2001); Nataranja,
A et al., Bioconjugate Chem. 16, 113-121; Krimner et al. Protein
Eng., Des. Sel. 19, 461-470; Albrecht et al. Bioconjugate Chem. 15,
16-26), Cysteine residues can e.g. also be alkylated by reacting
them with .alpha.-haloketones or Michael acceptors, such as
maleimide derivates. Alternatively, the modification of lysine
residues may e.g. be utilized which is the oldest and and most
straightforward method for labeling proteins via the primary lysine
amino groups. The .epsilon.-amino group of lysine within the
protein of interest can be readily reacted with activated esters,
sulfonyl chlorides, isocyanates and isothiocyanates to result in
the corresponding amides, sulfonamides, ureas and thioureas (see
e.g. Takaoka at el., Angew. Chem. Int. Ed. 2013, 52, 4088-4106).
Further examples for bioconjugation include the conjugation of
fluorescent proteins, dyes, or the tethering with functional
molecules, e.g. PEGs, porphyrins, peptides, peptide nucleic acids,
and drugs (Takaoka et al., Angew. Chem. Int. Ed. 2013, 52,
4088-4106).
[0171] For example, enzyme-mediated conjugation may also be applied
for covalently coupling the linker as disclosed above to the
inventive heterodimeric bispecific immunoglobulin molecule. For
example, WO 2014/001325 A1 discloses the use of sortase A for
site-specific bioconjugation to Fc regions of an antibody. Sortase
A (SrtA) is a bacterial integral membrane protein first described
in Staphylococcus aureus. SrtA catalyzes a transpeptidation
reaction anchoring proteins to the bacterial cell wall. Upon
recognition of a sorting signal LPXTG, (X=D, E, A, N, Q, or K) a
catalytic cysteine cleaves the peptide bond between residues T and
G which results in the formation of a thioacyl intermediate. This
thioacyl intermediate subsequently then can reacts with an
amino-terminal glycine acting as a nucleophile. SrtA accepts
N-terminal (oligo)glycine as a nucleophiles, creating a new peptide
bond between two molecules. SrtA functions at physiological
conditions and has been used for bioconjugation reactions to label
proteins with e.g. biotin, or to functionalize a HER2-specific
recombinant Fab with the plant cytotoxin gelonin (see e.g. Popp et
al. (2011) Angew Chemie Int. Ed. 50: 5024-5032 Kornberger et al
(2014) mAbs 6 (2): 354-366). Typically, target proteins such as
e.g. the VL and VH chains of the first and/or second Fab or scFv
fragments as disclosed above, are labeled carboxyterminally with
the LPXTG motif followed by a purification tag such that the
SrtA-mediated transpeptidation removes the purification tag and
generates the labeled protein.
[0172] In one embodiment the heterodimeric bispecific
immunoglobulin molecule according to the invention as disclosed
above comprises two linkers covalently coupled to the Fab or scFv
light chains of said heterodimeric bispecific immunoglobulin
molecule. For example, the linkers may be coupled to the light
chain of the VL chain of the first Fab or scFv fragment of the
inventive the heterodimeric bispecific immunoglobulin molecule
which specifically binds to EGFR as disclosed above and e.g. to the
VL chain of the second Fab or scFv fragment of the inventive the
heterodimeric bispecific immunoglobulin molecule which specifically
binds to c-MET as disclosed above.
[0173] In one embodiment the Fab or scFv light chains and/or the
CH3 domains and/or the CH2 domains of the heterodimeric bispecific
immunoglobulin molecule of the invention as disclosed above are
covalently coupled to a linker, whereby said linker is covalently
coupled to a dye, radioisotope, or cytotoxin as disclosed above.
For example, the VL chains of the first and second Fab or scFv
fragment may be covalently coupled to a linker as disclosed above,
whereby the linker is further coupled to a dye radioisotope or
cytotoxin as disclosed above, or both engineered CH3 domains of the
inventive heterodimeric bispecific immunoglobulin molecule as
disclosed above ("AG-SEED", "GA-SEED") may be covalently coupled to
a linker as disclosed above, or e.g. the CH2 domains of the
heterodimeric bispecific immunoglobulin molecule of the invention
as disclosed above, may each be covalently coupled to a linker as
disclosed above. Studies with anti-CD30 monoclonal
antibody--auristatin E (MMAE) conjugates have shown that ADCs with
a antibody:drug stoichiometry of 1:2-1:4 are most effective, with a
ratio of 1:4 being most preferable (see e.g. Hamblett et al.
Clinical Cancer Research (2004) Vol. 10, 7063-7070). Thus, the
inventive heterodimeric bispecific immunoglobulin molecule as
disclosed above may e.g. comprises 2, 3, or 4 linker molecules
which are covalently coupled to the inventive heterodimeric
bispecific immunoglobulin molecule, whereby each linker is
preferably coupled to a cytotoxin as disclosed above, e.g. the VL
chains and the VH chains of the heterodimeric bispecific
immunoglobulin molecule of the invention may be coupled to a
cytotoxin via a linker as disclosed above. For example, the VH
chains and the CH3 domains of the inventive heterodimeric
bispecific immunoglobulin molecule as disclosed above may be
covalently coupled to a linker, whereby each linker is further
coupled to a cytotoxin. Alternatively, the VL chains of the first
and second Fab or scFv fragment and the CH3 or CH2 domains of the
inventive heterodimeric bispecific immunoglobulin molecule as
disclosed above may be covalently coupled to a linker which is
further coupled to a cytotoxin as disclosed above.
[0174] In one embodiment the heterodimeric bispecific
immunoglobulin molecule according to the invention as disclosed
above is for use in the treatment of cancer. The term "cancer" as
used in the present invention refers to a variety of conditions
caused by the abnormal, uncontrolled growth of cells, e.g. cells
capable of causing cancer, referred to as "cancer cells", possess
characteristic properties such as uncontrolled proliferation,
immortality, metastatic potential, rapid growth and proliferation
rate, and/or certain typical morphological features. Cancer cells
may e.g. be in the form of a tumor, but such cells may also exist
singly within a subject, or may be a non-tumorigenic cancer cell.
The term cancer as used in the context of the inventive method of
treatment may e.g. refer to prostate cancer, breast cancer, adrenal
cancer, leukemia, lymphoma, myeloma, bone and connective tissue
sarcoma, brain tumors, thyroid cancer, pancreatic cancer, pituitary
cancer, eye cancer, vaginal cancer, vulvar cancer, cervical cancer,
uterine cancer, ovarian cancer, esophageal cancer, stomach cancer,
colon cancer, rectal cancer, liver cancer, gallbladder cancer,
cholangiocarcinoma, lung cancer, testicular cancer, penal cancer,
oral cancer, skin cancer, kidney cancers, Wilms' tumor and bladder
cancer, metastatic (mCRC), non-resctable liver metastases, squamous
cell carcinoma of the head and neck, non-small cell lung cancer
(NSCLC), head and neck squamous cell carcinoma (HNSCC), Merkel cell
carcinoma.
[0175] In one embodiment the invention provides a composition which
comprises the heterodimeric bispecific immunoglobulin molecule of
the invention as disclosed above and at least one further
ingredient. For example, the inventive composition may comprise the
heterodimeric bispecific immunoglobulin molecule of the invention
as disclosed above and one or more of water, buffer, stabilizer,
salt, sugar, preservative (e.g. benzalkonium chloride), lipids,
anti-oxidants, carboxylic acids, polyethylene, glycol (PEG). For
example, the buffer or buffer solution may have a pH from about 5
to about 9, e.g. from about pH 5 to about pH 6, or from about pH 6
to about pH 7, or from about pH 8 to about pH 9, or from about 5.1,
5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.5,
6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 to
about 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 and may e.g.
comprise sodium acetate, histidine, citrate, succinate or phosphate
buffers. For example, sodium acetate, histidine, citrate, succinate
or phosphate may be present in the composition according to the
invention in a concentration of from about 10 mM, 15 mM, 20 mM, 25
mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM to about 60 mM, 70 mM, 80 mM,
90 mM, 100 mM, 125 mM, 150 mM. For example, the buffer solutions as
disclosed above may be combined with a preservative such as
benzalkonium chloride to stabilize the inventive heterodimeric
bispecific immunoglobulin molecule as disclosed above. Other
ingredients may e.g. include, polyethylene, glycol with an average
molecular weights of 200-4000 Dalton, e.g. 300, 400, 500 600, 700,
800, 900, 1000, 1500, 1750, 2000, 2250, 2500, 3000, 3500 Dalton and
its derivatives. Polyethylene glycol derivatives may e.g. also be
used and may e.g. include polyethylene glycol monolaurate,
polyethylene glycol mono-oleate and polyethylene glycol
monopalmitate. For example, the composition according to the
invention may comprise the inventive heterodimeric bispecific
immunoglobulin molecule as disclosed above in aqueous or
lyophilized form and at least one further chemotherapeutic agent,
wherein the agent is selected from the group comprising
capecitabine, 5-fluoro-2'-deoxyuiridine, irinotecan,
6-mercaptopurine (6-MP), cladribine, clofarabine, cytarabine,
floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate,
bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin,
topotecan, teniposide, colcemid, colchicine, pemetrexed,
pentostatin, thioguanine; leucovorin, cisplatin, carboplatin,
oxaliplatin, or a combination of 5-FU, leucovorin, a combination of
5-fluorouracil/folinic acid (5-FU/FA), a combination of
5-fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), a
combination of 5-FU, leucovorin, oxaliplatin (FOLFOX), or a
combination of 5-FU, leucovorin, and irinotecan (FOLFIRI), or a
combination of leucovorin, 5-FU oxaliplatin, and irinotecan
(FOLFOXIRI), or a combination of Capecitabine and oxaliplatin
(CapeOx). in one embodiment the present invention provides a
pharmaceutical composition which comprises the heterodimeric
bispecific immunoglobulin molecule of the invention as disclosed
above and at least one further ingredient, or which comprises the
inventive composition as disclosed above. For example, the
pharmaceutical composition of the invention may comprise the
heterodimeric bispecific immunoglobulin molecule of the invention
as disclosed above in a concentration from about 10 mg/ml, 20
mg/ml, 25 mg/ml, 30 mg/ml, 35 mg/ml, 45 mg/ml, 50 mg/ml, 55 mg/ml,
60 mg/ml to about 70 mg/ml, 75 mg/ml, 80 mg/ml, 100 mg/ml, 112
mg/ml, 125 mg/ml, 150 mg/ml, 175 mg/ml, 200 mg/ml, or e.g. from
about 10 mg/ml to about 20 mg/ml, 25 mg/ml, 30 mg/ml, 35 mg/ml, 40
mg/ml, 45 mg/ml, 50 mg/ml, 55 mg/ml, 60 mg/ml to about 70 mg/ml, 75
mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 112 mg/ml, 125 mg/ml, 150
mg/ml, 175 mg/ml, 200 mg/ml, or e.g. 20 mg/ml, 25 mg/ml, 30 mg/ml,
35 mg/ml, 40 mg/ml, 45 mg/ml, 50 mg/ml, 55 mg/ml, 60 mg/ml, to
about 70 mg/ml, 75 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 112 mg/ml,
125 mg/ml, 150 mg/ml, 175 mg/ml, 200 mg/ml, and e.g. an aqueous
buffer as disclosed above. The inventive pharmaceutical composition
as disclosed above, may e.g. also comprise surfactants such e.g.
anionic surfactants such as e.g. a mixture of sodium alkyl
sulfates, cationic surfactants, such as e.g. quaternary ammonium
and pyridinium cationic surfactants, or non-ionic surfactants, such
as e.g. Sorbitan esters, polysorbates, e.g. Polysorbat 20
(Polyoxyethylen-(20)-sorbitanmonolaurat), Polysorbat 21
(Polyoxyethylen-(4)-sorbitanmonolaurat), Polysorbat 40
(Polyoxyethylen-(20)-sorbitanmonopalmitat), Polysorbat 60
(Polyoxyethylen-(20)-sorbitan-monostearat), Polysorbat 61
(Polyoxyethylen-(4)-sorbitanmonostearat), Polysorbat 65
(Polyoxyethylen-(20)-sorbitantristearat), Polysorbat 80
(Polyoxyethylen-(20)-sorbitanmonooleat), Polysorbat 81
(Polyoxyethylen-(5)-sorbitanmonooleat) Polysorbat 85
(Polyoxyethylen-(20)-sorbitantrioleat), Polysorbat 120
(Polyoxyethylen-(20)-sorbitanmonoisostearat), poloxamers e.g.
poloxamer 105, poloxarner 108, poloxamer 122, poloxamer 124,
poloxamer 105 benzoate. Perservatives which may be comprised in the
pharmaceutical composition according to the invention may be
benzalkonium chlorid in a concentration of 0.004% to 0.01%. For
example, the inventive pharmaceutical composition may be formulated
by use of conventional techniques as discrete dosage forms, such as
capsules, a solution or a suspension in an aqueous liquid or a
non-aqueous liquid; or as an oil-in-water liquid emulsion or a
water-in-oil emulsion and as a bolus; together with suitable
pharmaceutically acceptable carrier.
[0176] In one embodiment the pharmaceutical composition of the
invention as disclosed above is for use in the treatment of cancer.
For example, the inventive pharmaceutical composition as disclosed
above for use in the treatment of cancer may be administered to a
person inflicted with cancer.
[0177] In one embodiment the invention provides a method of
treatment which comprises administering to a subject a
therapeutically effective amount of the inventive pharmaceutical
composition as disclosed above. For example, the inventive method
of treatment may comprise administering a person in need thereof
afflicted with cancer as disclosed above from about 0.001 mg/kg to
about 50 mg/kg of the inventive pharmaceutical composition, or from
about 0.005 mg/kg to about 45 mg/kg, or from about 0.01 mg/kg to
about 40 mg/kg, or from about 0.05 mg/kg to about 35 mg/kg, or from
about 0.1 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 1.5 mg/kg, 2
mg/kg, 2.5 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8
mg/kg, 9 mg/kg, 10 mg/kg, 12.5 mg/kg, 15 mg/kg, 17.5 mg/kg, 20
mg/kg, 22.5 mg/kg, 25 mg/kg to about 26 mg/kg, 27 mg/kg, 28 mg/kg,
29 mg/kg, 30 mg/kg, 32.5 mg/kg, 35 mg/kg, 37.5 mg/kg, 40 mg/kg,
42.5 mg/kg, 45 mg/kg. As used the term "mg/kg" refers to mg of the
inventive pharmaceutical composition/kg body weight in the present
invention. For example, a pharmaceutically effective amount of the
inventive pharmaceutical composition may be administered to an
individual inflicted with cancer. The pharmaceutically effective
amount depends on the individual, the type of cancer to be treated,
the body weight and age of the individual, the level of the disease
or the administration route.
EXAMPLES
Example 1: Generation of Anti c-Met and Anti-EGFR Binders
[0178] Generation of c-MET Binders
[0179] Panning of naive phage display antibody gene libraries
HAL718 against human c-MET was performed according to Hust and
colleagues. 36; 37 Briefly, after pre-selection with panning buffer
(1% skim milk powder, 1% BSA, 0.05% Tween.RTM.20 in PBS) in
maxisorp 96 well plates (Nunc), scFv displaying phages were
selected on 1 .mu.g immobilized c-MET-Fc (R&D Systems,
358-MT/CF) or c-MET SEMA domain (produced in house) and eluted with
trypsin. After two to three rounds of panning, c-MET specific
binders were enriched and screened by capture c-MET ELISA of
produced scFv.
[0180] For affinity maturation, (a) error prone PCR for variable
domains was performed using the GeneMorph II Random Mutagenesis
Kits (Agilent Technologies) according to the manufacturer's
instruction, (b) randomization of complementary-determining region
three of the heavy chain (CDR-H3) ordered by GeneArt applying a
parsimonious mutagenesis strategy 70, and (c) light chain shuffling
using the diversity of the HAL7/8 were conducted. Panning was
carried out using phage display and yeast display for F06 and B10,
respectively. For clone F06, an off-rate screening strategy was
applied by stringent washing (ten times) with 100 .mu.l panning
buffer per well as well as adding soluble c-MET for competition
(starting in the second round). CS06 was based on rational
combination of abundant mutations from approach (a) and (b), B10v5
was derived from approach (c) using yeast surface display as
described in e.g. Biotechnol. Bioeng. 2009 103: 1192-201; Protein
Eng Des Sel 2010; 23: 155-9.
Generation of Anti-EGFR Binders
[0181] The structure of C225 bound to the extracellular domain of
EGFR 42 was optimized with the Rosetta Protein Structure and Design
program (version 2.3.0) using a fixed backbone protocol and side
chain optimization to minimize the energy of the starting model for
design according to the Rosetta energy function. Interfacial water
molecules observed in the crystal structure were retained during
the minimization, but not during subsequent design calculations.
Thirty-seven residues at or near the antibody-antigen interface
were selected for a saturating, in silico point mutagenesis. At
each of these residues 19 variants were created (wild type and 18
mutations, no cysteine) optimizing the rotamer of the mutated
residue while keeping the backbone fixed. Using these preliminary
models, neighbour residues were identified as any residue with at
least 3 heavy atoms within 5.5 .ANG. of a heavy atom on the design
residue. The rotamer of the mutated residue and its neighbours were
optimized using the standard Rosetta score function (a linear
combination of terms including a Lennard-Jones potential, an
orientation dependent hydrogen bonding potential an implicit
solvation model and statistical terms that capture
backbone-dependent amino acid and rotamer preferences. The
hydrophobic substitutions will be described elsewhere. The polar
substitutions were filtered to only those variants with
improvements of at least 0.5 Rosetta energy units in either the
orientation-dependent hydrogen bonding score or the pair potential
relative to the repacked native to select improved variants. The
three affinity enhancing point substitutions were combined into a
triple mutant, and this was repacked and scored by Rosetta as
described above for the point mutants. The affinity of the selected
variants was measured in vitro by surface plasmon resonance. The
variants were also transferred to the hu225 scFv and the affinities
in this context were verified by biolayer interferometry.
Example 2: Expression and Purification of Bispecific
c-MET.times.EGFR SEEDbodies
[0182] Several combinations of EGFR and c-MET antibody fragments
according to the invention as disclosed herein were joined to
bispecific antibodies using the SEED-technology. Bispecific
c-MET.times.EGFR SEEDbodies were expressed by transient
transfection of Expi293F.TM. cells (human embryonic kidney cells)
according to the manufacturer's instruction of the transfection kit
(Invitrogen). Briefly, suspension Expi293F.TM. cells were cultured
in Expi293F.TM. expression medium (Invitrogen) at 37.degree. C., 5%
CO.sub.2 and 180 rpm. On the day of the transfection, cells were
seeded in fresh medium with a density of final 2.times.10.sup.6
viable cells/ml. DNA-ExpiFectamine.TM.293 reagent mixture diluted
in Opti-MEM.RTM. I medium (Invitrogen) was added to the cells. 16 h
post transfection, ExpiFectamine.TM.293 transfection enhancer 1 and
2 were added. Cell supernatants containing secreted antibodies were
harvested 5 days after transfection by centrifugation at
4,300.times.g, 4.degree. C. and 20 min and filtration through 0.22
.mu.m Stericup or Steriflip devices (Millipore). Small scale
productions were performed in a volume of 25 ml and purification
was carried out with PROSEP.RTM. A centrifugal Protein A columns
(Millipore, #P36486) according to manufacturers' instructions
followed by dialysis to PBS pH 7.4 using Pur-A-Lyzer.TM. Dialysis
Kit (Sigma-Aldrich).
[0183] Large scale productions were performed in an expression
volume of 200 ml. Supernatants were purified by affinity
chromatography (5 ml HiTrap MabSelect SuRe, GE Healthcare) on an
AKTA Explorer 100 (GE Healthcare) with subsequent preparative size
exclusion chromatography (HiLoad 26/60Superdex 200 pg, GE
Healthcare). Protein concentrations were determined by UV A280
spectroscopy and purity was analyzed by gel electrophoresis with
4%/8% NuPAGE BisTris gels (Life technologies) and coomassie
staining as well as analytical size exclusion high performance
liquid chromatography (TSK Super SW3000, Tosoh). Endotoxin levels
were assessed by Limulus amebocyte lysate Endosafe.RTM. PTS
cartridges and Endosafe.RTM. PTS reader (Charles River).
[0184] Antibody VH and sequences for humanized oa 5D5 (MetMAb,
onartuzumab), LY2875358 (LA480_vC8H241, emibetuzumab), and h224G11
(ABT-700) were derived from publicly available information (e.g.
U.S. Pat. Nos. 6,214,344B1, 8,398,974 B2, 0,273,060A1). Sequences
were cloned in mammalian expression vectors containing constant
IgG1 light and heavy chain fragments except in case of oa 5D5
knob-into-hole technology was applied (e.g. as disclosed in Protein
Eng 1996; 9: 617-21). All anti-c-MET reference antibodies as well
as cetuximab (C225, Erbitux) and matuzumab were produced in-house
(Merck) in HEK293E cells using standard transfection and
purification procedure e.g. as described above.
Example 3 : Binding of Bispecific c-MET.times.EGFR Antibodies to
c-MET and EGER on Cells
[0185] Bispecific c-MET.times.EGFR antibodies, one-armed
(monovalent) control antibodies (anti-c-MET and anti-EGFR) as well
as a non-related isotype control (anti-hen egg lysozyme, anti-HEL)
were tested for their binding to c-MET and EGFR expressing NCI-H441
cells (as e.g. shown in FIG. 1, FIG. 15). NCI-H441 cells were
detached with trypsin, centrifuged at 250.times.g for 10 min at
4.degree. C. and resuspended in FACS buffer (1% BSA 1.times.PBS).
Cells were transferred to in 96 well round bottom plates at a
density of 1.times.10.sup.5 cells/well on ice. Purified
c-MET.times.EGFR bispecific antibodies (0.02-200 mM) were added in
FACS buffer in triplicates for 1 h on ice. Cells were centrifuged
for 1000.times.g for 5 min at 4.degree. C. and washed 3 times with
100 .mu.l FACS buffer. Cells were incubated with 500 ng/well
Fluorescein (FITC)-conjugated goat anti-human Fc gamma fragment IgG
specific antibody (Jackson ImmunoResearch) diluted in FACS buffer
on ice for 1 h. Cells were washed again 3 times with 100 .mu.l FACS
buffer. For counter staining of non-viable cells, centrifuged were
resuspended in 200 .mu.l propidium iodide solution (Invitrogen)
diluted in FACS buffer (1:200). Cell were analyzed for fluorescence
at 488 nm using a Guava easyCyte HT cytometer (Millipore). Data
were plotted as mean fluorescence intensity (raw fluorescence
substracted by background, e.g. non-stained cell control) against
the logarithm of the bispecific antibody concentration and fitted
to a sigmoidal dose-response curve with variable slope using
GraphPad Prism 4 (GraphPad Software).
Example 4: Epitope Binning of c-MET Binders Using Bio-Layer
Interferometry (BLI)
[0186] An epitope binning experiment was carried out with c-MET
antibodies which were used in the bispecific antibodies and
compared to reference antibodies from the literature (MetMAb,
Emibetuzumab, h224G11). Biosensor experiments using bio-layer
interferometry were performed on an Octet Red platform (Forte Bio)
equipped with anti-human Fc(AHC) biosensor tips (Forte Bio). All
data were collected at 30.degree. C. in kinetics buffer (PBS pH
7.4, 0.1% BSA, 0.02% Tween-20. Human c-MET ECD-His (HGFR,
hepatocyte growth factor receptor extracellular domain) was
produced and purified in-house. Biosensor tips were equilibrated 30
sec in PBS. Then, 25 nM for bivalent IgGs and 50 nM for monovalent
one-armed antibodies in PBS were immobilized on biosensor tips for
200 sec as primary antibody. Tips were quenched with 400 nM of a
non-related control antibody (anti-hen egg lysozyme, anti-HEL SEED,
diluted in PBS) to minimize subsequent binding of secondary
antibodies to biosensor tips. Following acquisition of a baseline
in kinetics buffer for 60 sec, human c-MET-ECD was subjected to
immobilized primary antibodies for 600 sec. Afterwards,
interactions of secondary anti-c-MET antibodies to c-MET-ECD bound
to immobilized primary antibodies was analyzed for 600 sec Analysis
of secondary antibody binding was analyzed visually by
distinguishing simultaneous binding characterized by a higher
binding rate [nM] compared to a non-related isotype control
(anti-HEL SEED). The results of the epitope binning are depicted in
FIG. 2A.
Example 5: HGF Competition ELISA Assay/HGF Displacement by
Monoclonal Antibodies
[0187] Competition of recombinant human HGF (Hepatocyte growth
factor, R&D Systems, 294-HGN/CF) with antibody binding to
recombinant human c-MET ECD (HGFR extracellular domain, Hepatocyte
growth factor receptor) was detected by ELISA using HGF in solid
phase, Recombinant human HGF (1.255 pmol) was immobilized on 96
well Maxisorp plates (Thermo Scientific) overnight at 4.degree. C.
After blocking plates with 2% BSA, biotinylated recombinant human
c-MET ECD (1.13 pmol) pre-incubated with serial dilutions of
antibodies (200 nM to 0.2 nM) were added to plates. Binding was
revealed using HRP-conjugated strepatvidin (Merck Millipore) and
TMB substrate and sulfuric acid (1 step UltraTMB ELISA solution).
Resulting absorbance for c-MET ECD binding to HGF without addition
of anti-c-MET directed antibody was defined as 100% HGF binding.
Anti-HEL (hen egg lysozyme) was used as an unrelated isotype
control antibody. Data were plotted as % HGF binding against the
logarithm of the antibody concentration and fitted to a sigmoidal
dose-response curve with variable slope using GraphPad Prism 4
(GraphPad Software). The results of the displacement are depicted
in FIG. 3.
Example 6: Cell Titer Glow Assay
[0188] Cell viability was quantified using the cell titer glow
assay (Promega) and was performed according to the manufacturer's
instructions. Briefly, cells were detached and seeded in the inner
wells of opaque white tissue culture treated 96 well plates
(Perkin&Elmer). The seeding cell number ranged from 8,000 to
15,000 viable cells per well depending on the cell line in 80 .mu.l
per well. Cells were allowed to attach at least three hours in a
humidified chamber at 37.degree. C., 5% CO2. Then, cells were
treated with antibodies in duplicates which were diluted in cell
line specific medium (ranging from 60 to 0.01 nM final). Depending
on the assay, Fab-toxin conjugates were added in a threefold molar
excess (Fab-toxin from Moradec, MMAE or DMSA). After 72 hours,
viability of cells was detected by adding 100 .mu.l per well of
CellTiter-Glo.RTM. reagent (Promega) with subsequent mixing on a
plate shaker for two minutes at 350 rpm and 10 min incubation in
the dark at room temperature. Luminescence was measured at a
Synergy 5 (Biotek) with a read time of 0.5 seconds per well
(sensitivity: 170). Background luminescence in wells with only
medium with the CellTiter-Glo.RTM. reagent (Promega) was
subtracted. Data were plotted as percentage of untreated cell
viability against the logarithm of antibody concentration and
fitted to a sigmoidal dose-response curve with variable slope using
Graph pad Prism 4 (GraphPad Software).
Example 7: ADC Generation and Antibody Dependent Cellular
Cytotoxicity
ADC Generation
[0189] Sortase mediated site-directed conjugation of
valine-citrulline (vc)-monomethyl auristatin E (MMAE) to antibody
Fc was performed as described elsewhere (see e.g. ACS Chem.Biol.
2015; 10: 2158-65). Briefly, antibodies or the inventive
heterodimeric bispecific immunoglobulin molecules carrying enzyme
recognition site C-terminally on both heavy chains were generated,
transfected and purified by affinity chromatography. Then, one
equivalent of antibody was incubated with 11 equivalents of
substrate-vc-MMAE conjugate in the presence of 5 .mu.M Sortase and
5 mM CaCl2 in reaction buffer (50 mM Tris, 150 mM NaCl, pH 7.5) for
30 min at 22.degree. C. The reaction was stopped with 10 mM EDTA as
calcium ion chelator. The resulting ADC was purified by size
exclusion chromatography.
Antibody Dependent Cellular Cytotoxicity.
[0190] Capability of the antibodies to induce ADCC was assessed
using fire ADCC Reporter Bioassay Core Kit (Promega) according to
the manufacturer's instruction. Briefly, target cells (A431 cells)
were detached and seeded into the inner wells of opaque white
tissue culture treated 96 well plates (Perkin&Elmer) with a
cell density of 12,500 viable cells per well (100 .mu.l), A431
cells were cultured in ADCC buffer containing RPMI 1640 medium
(Gibco) supplement with 4% low IgG fetal bovine serum (FBS, Gibco).
Cells were allowed to attach overnight in a humidified chamber at
37.degree. C., 5% CO2. The next day, medium was removed and cells
were treated with 25 .mu.l antibodies per well diluted in ADCC
buffer (final concentrations ranging from 5 to 0.0016 nM).
Afterwards, recombinant Jurkat cells (Promega) were added which
function as effector cells (360 .mu.l effector cells diluted in 3.6
ml ADCC buffer, 25 .mu.l per well). After six hours of incubation
in a humidified chamber at 37.degree. C., CO2, 75 .mu.l of Bio Glo
Luciferase Substrate (Promega), which was equilibrated at room
temperature, was added per well. After ten minutes of incubation at
room temperature protected from light, luminescence was measured at
a Synergy 5 (Biotek) with a read time of 0.5 seconds per well
(sensitivity: 170), Background luminescence in wells with only
medium was subtracted. Relative luminescence units were plotted
against the logarithm of antibody concentration and fitted to a
sigmoidal dose-response curve with variable slope using GraphPad
Prism 4 (GraphPad Software).
Example 8: Receptor Phosphorylation Assay
[0191] To assess the effect of binding of the inventive
heterodimeric bispecific immunoglobulin molecules on c-MET and
EGFR-mediated signaling phosphorylation levels of both c-MET and
EGFR were determined by c-MET or EGFR capture
electrochemiluminescence (ECL) ELISA (MSD assay). All reagents were
obtained from Meso Scale Discovery and prepared according to the
manufacturer's instructions. Briefly, cells were plated in 96-well
tissue culture plates (Sigma-Aldrich) one day before treatment,
serum starved and treated with serially diluted antibodies (0-167
nM in starvation medium) for 1 h at 37.degree. C., 5% CO2. Upon
stimulation with either 100 ng/ml HGF and/or EGF (both R&D
Systems) for 5 min at 37.degree. C., cells were lysed with ice-cold
lysis buffer supplemented with protease and phosphatase inhibitors
(Calbiochem). High bind 96-well plates including electrodes (Meso
Scale Discovery) were coated with capture anti-total c-MET (Cell
Signaling Technologies) or anti-total EGFR antibodies (Abcam)
followed by blocking with 3% Block A in PBS supplemented with 0.05%
Tween.RTM.20. After incubation with cell lysates, detection was
carried out with anti-phospho c-MET (Cell Signaling Technologies),
anti-phospho-tyrosine antibodies (R&D Systems) and by the
supplier recommended detection substances. Measurements were
performed with the SECTOR.RTM. Imager 6000 (Meso Scale Discovery).
For quantification of phospho-AKT levels, the Phospho(Ser473)/Total
AKT Assay Whole Cell Lyate Kit (Meso Scale Discovery) was used.
Dose response curves were plotted as the logarithm of antibody
concentration versus ECL signal. IC.sub.50 values were calculated
by a 3PL fitting model using GraphPad Prism 5 (GraphPad Software,
Inc.). Data from at least two experiments were used to calculate
mean IC.sub.50.+-.standard deviation (s.d.), see e.g. FIG. 20, FIG.
25 (A).
Example 9: Quantification of Cell Surface Receptor Density
[0192] Receptor surface expression levels on selected cell lines
were determined using the QFIKIT (Dako K0078) employing flow
cytometry, the results of which are shown in FIG. 18. Briefly, five
populations of calibration beads presenting different numbers et
mouse mAb molecules on their surfaces were used as a calibration
standard, 1.5.times.105 cells/well were labeled with primary mouse
anti-EGFR (ab187287, Abcam) and mouse anti-c-MET antibodies
(MAB3582, R&D Systems) at saturating doses (5 .mu.g/ml). Then,
beads and cells were stained with secondary goat anti-mouse Fc
F(ab').sub.2 FITC conjugate (10 .mu.g/ml, Jackson Immune Research)
and were subjected to flow cytometry measurement using a Guava
easyCyte HT cytometer (Millipore). Beads and cells were measured on
the same day using the same settings. Based on a calibration line
for fluorescence of beads versus bead surface density, antigen cell
surface densities for c-MET and EGFR were calculated.
Example 10: Internalization Assay
[0193] Internalization of the inventive heterodimeric bispecific
immunoglobulin molecules was either determined by flow cytometry
using an anti-Alexa Fluor 488 quenching antibody or by confocal
microscopy applying pH stripping. For flow cytometry, cells
(1.times.105) were incubated with 100 nM bsAbs followed by Alexa
Fluor 488 conjugated anti human Fc (Fc.gamma. specific, Jackson
Immuno Research). After washing with FACS buffer, cells were
incubated at either 37.degree. C. or 4.degree. C. for 1 h allowing
internalization. Afterwards, residual surface binding of bsAb was
quenched by anti-Alexa Fluor 488 IgG (Life Technologies) and cells
were fixated with 4% (w/v) formaldehyde (Calbiochem) and subjected
to flow cytometric analysis. Internalization was calculated as
following:
rel . .times. interalization .times. [ % ] = ( 37 .times. .degree.
.times. .times. C . .times. with .times. .times. quench ) - ( 4
.times. .degree. .times. .times. C . .times. with .times. .times.
quench ) ( 37 .times. .degree. .times. .times. C . .times. without
.times. .times. quench ) .times. 100 ##EQU00001##
[0194] For fluorescence microscopy, cells (3.times.10.sup.5) were
grown on glass coverslips (Menzel Glaser) placed in 6 well plates.
Two days later, cells were kept on ice and treated with 100 nM
bsAbs followed by detection with Alexa Fluor 488 conjugated anti
human Fc Fab fragment. After washing with 1% BSA in PBS, cells were
incubated in respective medium at either 37.degree. C. or 4.degree.
C. for 1 h allowing internalization. By addition of ice-cold low pH
buffer (50 mM glycine, 150 mM NaCl, pH 2.7 adjusted with HCl),
residual bsAbs on the cell surface were removed. Finally, cells
were fixated with 4% (w/v) formaldehyde and mounted on object
slides with ProLong Diamond Antifade Mountant with DAPI (Life
Technologies). Analysis was carried out with a Leica TCS SPS
confocal microscope equipped with a 100.times.objective (Leica
Microsystems).
Example 11: Cell Culture
[0195] Human cancer cell lines which were used according to the
present invention were obtained from the American Type Culture
Collection (A431, A549, MDA-MB-468, NCI-H1975, NCI-H441, NCI-H596),
the Riken Biorescourse Center Cell Bank (EBC-1, KP-4), Lipha
(HepG2), and German Collection of Microorganims and Cell Cultures
(MKN45) and maintained according to standard culture conditions
(37.degree. C., 5% CO.sub.2, 95% humidity) using recommended media
formulations, A549 and A431 were cultivated in Dulbecco's Modified
Eagle's Medium (DMEM, Life Technologies) containing 10% Fetal
Bovine Serum (FBS, Life Technologies). MDA-MB-468, NCI-H1975,
HepG2, and MKN45 were maintained in RPMI-1640 (Life Technologies)
supplemented with 10% FBS, 2 mM L-glutamine and 1 mM sodium
pyruvate (both Life Technologies). NCI-H441, NCI-H596 were
cultivated in RPMI-1640 with 10% FBS, 2 mM L-glutamine, 1 mM sodium
pyruvate, 2.5 g/L D(+)-glucose (Sigma-Aldrich) and 10 mM HEPES
(Life Technologies). KP-4 cells were cultivated in DMEM/F-12 with
10% FBS. EBC-1 cells were maintained in Minimal Essential Medium
(MEM) with 10% FBS and 2 mM L-glutamine. NHEK.f.-c, (PromoCell,
#C-12007) were obtained from PromoCell and propagated in
recommended keratinocyte growth medium with supplements (PromoCell,
#C-20111) and with the DetachKit (PromoCell, #C-41210) for cell
detachment. Expi293F.TM. cells were purchased from Life
Technologies and cultivated in corresponding Expi293 expression
medium. All cell lines were shown to be sterile, certified
mycoplasma-free, and never exceeded passage 20.
Example 12 Surface Plasmon Resonance
[0196] Affinity and kinetic parameters of in silico designed C225
variants was verified by surface plasmon resonance. Computationally
guided substitutions were introduced into the wild-type C225 using
the QuikChangeII kit (Stratagene) with mutagenic primers. The
variant antibodies were expressed in HEK-293-6E cells. Cleared
supernatant was purified by affinity chromatography using protein
A. The antibody concentration was determined by absorbance at 280
nm, and the purity was verified by SDS-PAGE analysis. Surface
plasmon resonance was performed on a Biacore A-100 (GE Healthcare).
CM5 chips were coupled with goat anti-human IgG antibody (Jackson
ImmunoResearch, Inc., 109-005-098) and used to capture the
wild-type C225 or designed variants. Human EGFR (extracellular
domain, R&D Systems, 1095-ER) was used as analyte. The affinity
was determined by titrating the analyte from 0 to 40 nM and
determining kinetic rate constants using the BiaEvaluation software
to fit the association and dissociation phases using a 1:1 Langmuir
binding model. The KD was determined as the ratio of the kinetic
constants.
Example 13: Thermal Shift Assay
[0197] Thermal stability of the inventive heterodimeric bispecific
immunoglobulin molecules, as well as of controls (C225 (cetuximab),
matuzumab and "one-armed" (oa) constructs) was measured using a
StepOnePlus Real-Time PCR System (Life Technologies) according to
the manufacturer's instructions, the results of which are shown in
FIG. 17 and the corresponding description. Briefly, 1 .mu.M protein
was mixed with a 20 fold excess of SYPRO Orange (Life Technologies)
in PBS pH 7.4. Melting curves were recorded from 25.degree. C. to
99.degree. C. with an increment of 1.degree. C./60 s. Data were
analyzed with the Protein Thermal Shift.TM. Software (Life
technologies) by calculating the maximum of the second derivative
curve.
Sequence CWU 1
1
541106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATUREhu225 VL kinetic
variantsMOD_RES(35)..(35)Any amino acidMOD_RES(75)..(75)Any amino
acidMOD_RES(103)..(103)Any amino acid 1Gly Gln Pro Phe Arg Pro Glu
Val His Leu Leu Pro Pro Ser Arg Glu1 5 10 15Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Ala Arg Gly Phe 20 25 30Tyr Pro Xaa Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45Asn Asn Tyr Lys
Thr Thr Pro Ser Arg Gln Glu Pro Ser Gln Gly Thr 50 55 60Thr Thr Phe
Ala Val Thr Ser Lys Leu Thr Xaa Asp Lys Ser Arg Trp65 70 75 80Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 85 90
95Asn His Tyr Thr Gln Lys Xaa Ile Ser Leu 100 1052106PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideMISC_FEATUREAG-SEEDMOD_RES(35)..(35)Lys or
SerMOD_RES(75)..(75)Val or ThrMOD_RES(103)..(103)Thr or Ser 2Gly
Gln Pro Phe Arg Pro Glu Val His Leu Leu Pro Pro Ser Arg Glu1 5 10
15Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Ala Arg Gly Phe
20 25 30Tyr Pro Xaa Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu 35 40 45Asn Asn Tyr Lys Thr Thr Pro Ser Arg Gln Glu Pro Ser Gln
Gly Thr 50 55 60Thr Thr Phe Ala Val Thr Ser Lys Leu Thr Xaa Asp Lys
Ser Arg Trp65 70 75 80Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His 85 90 95Asn His Tyr Thr Gln Lys Xaa Ile Ser Leu
100 1053106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATUREGA-SEEDMOD_RES(23)..(23)Any amino
acidMOD_RES(58)..(58)Any amino acidMOD_RES(61)..(61)Any amino
acidMOD_RES(67)..(67)Any amino acidMOD_RES(76)..(76)Any amino
acidMOD_RES(78)..(78)Any amino acid 3Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Pro Ser Glu1 5 10 15Glu Leu Ala Leu Asn Glu
Xaa Val Thr Leu Thr Cys Leu Val Lys Gly 20 25 30Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Leu Gln Gly Ser Gln Glu 35 40 45Leu Pro Arg Glu
Lys Tyr Leu Thr Trp Xaa Pro Val Xaa Asp Ser Asp 50 55 60Gly Ser Xaa
Phe Leu Tyr Ser Ile Leu Arg Val Xaa Ala Xaa Asp Trp65 70 75 80Lys
Lys Gly Asp Thr Phe Ser Cys Ser Val Met His Glu Ala Leu His 85 90
95Asn His Tyr Thr Gln Lys Ser Leu Asp Arg 100 1054106PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideMISC_FEATUREGA-SEEDMOD_RES(23)..(23)Leu or
GlnMOD_RES(58)..(58)Ala or ThrMOD_RES(61)..(61)Leu, Val, Asp or
ThrMOD_RES(67)..(67)Phe, Ala, Asp, Glu, Gly, His, Lys, Asn, Pro,
Gln, Arg, Ser or ThrMOD_RES(76)..(76)Ala or ThrMOD_RES(78)..(78)Glu
or Asp 4Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Pro Ser
Glu1 5 10 15Glu Leu Ala Leu Asn Glu Xaa Val Thr Leu Thr Cys Leu Val
Lys Gly 20 25 30Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Leu Gln Gly
Ser Gln Glu 35 40 45Leu Pro Arg Glu Lys Tyr Leu Thr Trp Xaa Pro Val
Xaa Asp Ser Asp 50 55 60Gly Ser Xaa Phe Leu Tyr Ser Ile Leu Arg Val
Xaa Ala Xaa Asp Trp65 70 75 80Lys Lys Gly Asp Thr Phe Ser Cys Ser
Val Met His Glu Ala Leu His 85 90 95Asn His Tyr Thr Gln Lys Ser Leu
Asp Arg 100 1055106PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptideMISC_FEATUREAG-SEED 5Gly Gln Pro Phe
Arg Pro Glu Val His Leu Leu Pro Pro Ser Arg Glu1 5 10 15Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Ala Arg Gly Phe 20 25 30Tyr Pro
Lys Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45Asn
Asn Tyr Lys Thr Thr Pro Ser Arg Gln Glu Pro Ser Gln Gly Thr 50 55
60Thr Thr Phe Ala Val Thr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp65
70 75 80Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His 85 90 95Asn His Tyr Thr Gln Lys Thr Ile Ser Leu 100
1056106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATUREGA-SEED 6Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Pro Ser Glu1 5 10 15Glu Leu Ala Leu Asn
Glu Leu Val Thr Leu Thr Cys Leu Val Lys Gly 20 25 30Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Leu Gln Gly Ser Gln Glu 35 40 45Leu Pro Arg
Glu Lys Tyr Leu Thr Trp Ala Pro Val Leu Asp Ser Asp 50 55 60Gly Ser
Phe Phe Leu Tyr Ser Ile Leu Arg Val Ala Ala Glu Asp Trp65 70 75
80Lys Lys Gly Asp Thr Phe Ser Cys Ser Val Met His Glu Ala Leu His
85 90 95Asn His Tyr Thr Gln Lys Ser Leu Asp Arg 100
1057106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATUREAG-SEED 7Gly Gln Pro Phe Glu Pro
Glu Val His Thr Leu Pro Pro Ser Arg Glu1 5 10 15Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Arg Gly Phe 20 25 30Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45Asn Asn Tyr
Lys Thr Thr Pro Ser Arg Leu Glu Pro Ser Gln Gly Thr 50 55 60Thr Thr
Phe Ala Val Thr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp65 70 75
80Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
85 90 95Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 100
1058106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATUREGA-SEED 8Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Pro Ser Glu1 5 10 15Glu Leu Ala Leu Asn
Asn Gln Val Thr Leu Thr Cys Leu Val Lys Gly 20 25 30Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 35 40 45Glu Pro Arg
Glu Lys Tyr Leu Thr Trp Ala Pro Val Leu Asp Ser Asp 50 55 60Gly Ser
Phe Phe Leu Tyr Ser Ile Leu Arg Val Asp Ala Ser Arg Trp65 70 75
80Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
85 90 95Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 100
1059107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATUREhu225 VL sequence 9Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn 20 25 30Ile His
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Lys
Tyr Ala Ser Glu Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro
Thr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10510107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATUREhu225 kinetic
variantsMOD_RES(1)..(1)Asp or LeuMOD_RES(27)..(27)Gln, Met or
ArgMOD_RES(31)..(31)Thr or ValMOD_RES(32)..(32)Asn, Arg, Phe or
MetMOD_RES(49)..(49)Lys or MetMOD_RES(50)..(50)Tyr or
TrpMOD_RES(92)..(92)Asn, Arg, Ser, Tyr, or MetMOD_RES(93)..(93)Asn
or GluMOD_RES(96)..(96)Thr or Asn 10Xaa Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Xaa Ser Ile Gly Xaa Xaa 20 25 30Ile His Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Xaa Xaa Ala Ser Glu
Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Tyr Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Val Ala Thr Tyr Tyr Cys Gln Gln Asn Xaa Xaa Trp Pro Xaa 85 90 95Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 10511119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideMISC_FEATUREhu225 VH sequence 11Glu Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30Gly Val His Trp
Met Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Val Ile
Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr 50 55 60Ser Arg
Val Thr Ile Thr Ser Asp Lys Ser Thr Ser Thr Ala Tyr Met65 70 75
80Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln
Gly 100 105 110Thr Leu Val Thr Val Ser Ser 11512119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideMISC_FEATUREhu225 VH kinetic
variantsMOD_RES(28)..(28)Ser or TrpMOD_RES(53)..(53)Ser, Ala, Glu,
His, Gln or ArgMOD_RES(56)..(56)Asn, Ile, Leu, Met, Lys or
ArgMOD_RES(58)..(58)Asp, Glu, Gln or ArgMOD_RES(74)..(74)Ser or
TrpMOD_RES(100)..(100)Thr, Phe, Trp or AspMOD_RES(101)..(101)Tyr or
TrpMOD_RES(103)..(103)Asp or Glu 12Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Phe Xaa Leu Thr Asn Tyr 20 25 30Gly Val His Trp Met Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Val Ile Trp Xaa
Gly Gly Xaa Thr Xaa Tyr Asn Thr Pro Phe Thr 50 55 60Ser Arg Val Thr
Ile Thr Ser Asp Lys Xaa Thr Ser Thr Ala Tyr Met65 70 75 80Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg
Ala Leu Xaa Xaa Tyr Xaa Tyr Glu Phe Ala Tyr Trp Gly Gln Gly 100 105
110Thr Leu Val Thr Val Ser Ser 11513107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideMISC_FEATUREhu425 VL sequence 13Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Ser Ala Ser Ser Ser Val Thr Tyr Met 20 25 30Tyr Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr Ser
Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser
Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu65 70 75
80Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser His Ile Phe Thr
85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
10514121PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATUREhu425 VH sequence 14Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser His 20 25 30Trp
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45Gly Glu Phe Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Glu Lys Phe
50 55 60Lys Ser Lys Ala Thr Met Thr Val Asp Thr Ser Thr Asn Thr Ala
Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Ser Arg Asp Tyr Asp Tyr Asp Gly Arg Tyr Phe
Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
12015115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATUREc-MET binder A12 VL sequence 15Gln
Ala Gly Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln1 5 10
15Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Ala Arg Lys Ser Val
20 25 30His Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Val Leu Val Val
Tyr 35 40 45Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser
Gly Ser 50 55 60Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val
Glu Ala Gly65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp
Ser Ser Ser Asp Gln 85 90 95Leu Tyr Val Phe Gly Thr Gly Thr Lys Val
Thr Val Leu Gly Gln Pro 100 105 110Lys Ala Gly
11516127PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATUREc-MET binder A12 VH sequence 16Gln
Val Gln Leu Gln Gln Ser Gly Ala Gly Leu Leu Lys Pro Ser Glu1 5 10
15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr
20 25 30Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
Ile 35 40 45Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser
Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln
Phe Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Phe Cys Ala 85 90 95Arg Gly Val Pro Tyr Tyr Tyr Gly Ser Gly
Arg Tyr Gly Asp Gly Asn 100 105 110Trp Phe Asp Pro Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 115 120 12517116PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideMISC_FEATUREc-MET binder B10 VL sequence 17Gln Ser Val
Leu Thr Gln Pro Pro Ser Thr Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val
Thr Ile Ser Cys Phe Gly Ser Ser Ser Asn Val Gly Val Asn 20 25 30Thr
Val Asn Trp Tyr Arg Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45Ile Tyr Asp Asn Asn Leu Arg Pro Ser Gly Val Pro Glu Arg Phe Ser
50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu
Gln65 70 75 80Ala Glu Asp Glu Gly Asp Tyr Tyr Cys Gln Ser Tyr Asp
Ser Ser Leu 85 90 95Ser Asp Val Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu Gly Gln 100 105 110Pro Lys Ala Gly 11518117PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideMISC_FEATUREc-MET binder B10 VH sequence 18Glu Val Gln
Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Lys Asp Arg Arg Ile Thr His Thr Tyr Trp
Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
11519114PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATUREc-MET binder C10 VL sequence 19Gln
Ser Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys1 5 10
15Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val
20 25 30His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val
Tyr 35 40 45Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser
Gly Ser 50 55 60Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val
Glu Ala Gly65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp
Ser Ser Ser Asp Leu 85 90 95Trp Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu Gly Gln Pro Lys 100 105 110Ala Gly20123PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideMISC_FEATUREc-MET binder C10 VH sequence 20Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe
50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala
Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Ser Leu Asn Phe Pro Asp Ile Ala Val Ala Gly
Tyr Gly Asp Tyr 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 12021117PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptideMISC_FEATUREc-MET binder E07 VL
sequence 21Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Leu
Gly Gln1 5 10 15Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val
Gly Gly Tyr 20 25 30Asp Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys
Ala Pro Gln Leu 35 40 45Met Ile Tyr Asp Val Thr Ser Arg Pro Ser Glu
Val Ser Asn Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser
Leu Ala Ile Thr Gly Leu65 70 75 80Gln Ala Asp Asp Glu Ala Asp Tyr
Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95Ser Thr Leu Val Val Phe Gly
Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110Gln Pro Lys Ala Gly
11522118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATUREc-MET binder E07 VH sequence 22Gln
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly1 5 10
15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Gly Ser Ile Ser Ser Ser
20 25 30Asn Trp Trp Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu
Trp 35 40 45Ile Gly Glu Ile Tyr His Ser Gly Ser Thr Asn Tyr Asn Pro
Ser Leu 50 55 60Lys Ser Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn
Gln Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Gly Ser Gly Tyr Asp Phe Asp
Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser
11523116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATUREc-MET binder G02 VL sequence 23Gln
Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln1 5 10
15Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr
20 25 30Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Glu Ala Pro Lys
Leu 35 40 45Met Ile Tyr Glu Val Ser Lys Arg Pro Ser Gly Val Pro Asp
Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile
Ser Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Trp
Ser Tyr Ala Gly Ser 85 90 95Tyr Thr Tyr Val Phe Gly Ala Gly Thr Lys
Val Ser Val Leu Gly Gln 100 105 110Pro Lys Ala Gly
11524119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATUREc-MET binder G02 VH sequence 24Glu
Val Gln Leu Val Glu Thr Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp
Ser Ala 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Lys Met Gly Tyr Gly Thr Gly Ala Phe
Asp Ile Trp Gly Gln Gly 100 105 110Thr Met Val Thr Val Ser Ser
11525117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATUREc-MET binder H06 VL sequence 25Gln
Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10
15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn
20 25 30Tyr Val Tyr Trp Tyr Gln His Leu Pro Gly Thr Ala Pro Lys Leu
Leu 35 40 45Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg
Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser
Gly Leu Gln65 70 75 80Ser Glu Asp Glu Gly Asp Tyr Tyr Cys Ala Ser
Trp Asp Asp Asn Leu 85 90 95Asn Ala His Trp Val Phe Gly Gly Gly Thr
Lys Leu Thr Val Val Ser 100 105 110Gln Pro Lys Ala Gly
11526119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATUREc-MET binder H06 VH sequence 26Gln
Val Gln Leu Gln Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Leu
20 25 30Asp Ile Asn Trp Val Arg Gln Ala Ser Gly Gln Gly Leu Glu Trp
Met 35 40 45Gly Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln
Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Ser Thr Ser Val Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Glu Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asn Arg Pro Glu Thr Gly Asp Phe
Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11527117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATUREc-MET binder F03 VL sequence 27Leu
Pro Val Leu Thr Gln Pro His Ser Val Ser Gly Ser Pro Gly Lys1 5 10
15Thr Val Thr Ile Ser Cys Thr Gly Ser Ser Asp Tyr Ile Ala Ser Asn
20 25 30Tyr Val His Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Thr
Val 35 40 45Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg
Phe Ser 50 55 60Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr
Ile Ser Gly65 70 75 80Leu Gln Thr Glu Asp Glu Ala Asp Tyr Tyr Cys
Gln Ser Tyr Asp Ser 85 90 95Ser Asn His Val Val Phe Gly Gly Gly Thr
Lys Leu Thr Val Val Gly 100 105 110Gln Pro Lys Ala Gly
11528124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATUREc-MET binder F03 VH sequence 28Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr
20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Leu 35 40 45Ala Phe Ile Arg His Asp Gly Gly Asn Asn Phe Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr65 70 75 80Met Gln Met Ser Ser Leu Arg Pro Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Lys Asp Phe Ala Met Thr Gln Trp Leu
Pro Glu Arg Gly Met Asp 100 105 110Val Trp Gly Gln Gly Thr Thr Val
Thr Val Ser Ser 115 12029117PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptideMISC_FEATUREc-MET binder
F06 VL sequence 29Gln Leu Val Leu Thr Gln Ser Pro Ser Val Ser Val
Ala Pro Gly Lys1 5 10 15Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile
Arg Asn Val Gly Val 20 25 30His Trp Tyr Gln Lys Lys Pro Gly Gln Ala
Pro Ile Leu Val Val Tyr 35 40 45Asp Asp Asp Asp Arg Pro Ser Gly Val
Pro Glu Arg Phe Ser Gly Ser 50 55 60Asn Ser Gly Asn Thr Ala Thr Leu
Thr Ile Ser Arg Val Glu Ala Gly65 70 75 80Asp Glu Ala Asp Tyr Tyr
Cys Gln Val Trp Asp Ser Ala Thr Asp Gln 85 90 95Ala Arg Gln Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110Pro Lys Ala
Gly Cys 11530124PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptideMISC_FEATUREc-MET binder F06 VH
sequence 30Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser1 5 10 15Ser Ala Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe
Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn
Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu
Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gln Arg Gly Tyr
Asp Tyr Tyr Tyr Tyr Tyr Gly Met Asp 100 105 110Val Trp Gly Gln Gly
Thr Thr Val Thr Val Ser Ser 115 12031114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideMISC_FEATUREc-MET binder B10v5 VL sequence 31Glu Pro Val
Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Glu1 5 10 15Thr Ala
Thr Ile Pro Cys Gly Gly Asp Ser Leu Gly Ser Lys Ile Val 20 25 30His
Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Leu Leu Val Val Tyr 35 40
45Asp Asp Ala Ala Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60Lys Ser Gly Thr Thr Ala Thr Leu Thr Ile Ser Ser Val Glu Ala
Gly65 70 75 80Asp Glu Ala Asp Tyr Phe Cys Gln Val Tyr Asp Tyr His
Ser Asp Val 85 90 95Glu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
Gly Gln Pro Lys 100 105 110Ala Ala32117PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideMISC_FEATUREc-MET binder B10v5 VH sequence 32Glu Val Gln
Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Lys Asp Arg Arg Ile Thr His Thr Tyr Trp Gly
Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
11533114PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATUREc-MET binder CS06 VL sequence
33Gln Leu Val Leu Thr Gln Ser Pro Ser Val Ser Val Ala Pro Gly Lys1
5 10 15Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Arg Asn Val Gly
Val 20 25 30His Trp Tyr Gln Lys Lys Pro Gly Gln Ala Pro Ile Leu Val
Val Tyr 35 40 45Asp Asp Asp Asp Arg Pro Ser Gly Val Pro Glu Arg Phe
Ser Gly Ser 50 55 60Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg
Val Glu Ala Gly65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp
Asp Ser Ala Thr Asp Gln 85 90 95Arg Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly Gln Pro Lys 100 105 110Ala Gly34124PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideMISC_FEATUREc-MET binder CS06 VH sequence 34Gln Val Gln
Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Ala
Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Asn 20 25 30Ala
Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Ile Tyr Ala Gln Lys Phe
50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala
Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Asp Gln Arg Gly Tyr Tyr Tyr Tyr Tyr Tyr
Tyr Gly Met Asp 100 105 110Val Trp Gly Gln Gly Thr Thr Val Thr Val
Ser Ser 115 1203520PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideMISC_FEATUREglycine-serine linker 35Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10
15Gly Gly Gly Ser 203615PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMISC_FEATUREhinge 1 36Glu Pro
Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro1 5 10
153715PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMISC_FEATUREhinge 2 37Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro1 5 10 153899PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideMISC_FEATURECL sequence 38Pro Ser Val Thr Leu Phe Pro
Pro Ser Ser Glu Glu Leu Gln Ala Asn1 5 10 15Lys Ala Thr Leu Val Cys
Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val 20 25 30Thr Val Ala Trp Lys
Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu 35 40 45Thr Thr Thr Pro
Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser 50 55 60Tyr Leu Ser
Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser65 70 75 80Cys
Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro 85 90
95Thr Glu Cys3998PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptideMISC_FEATURECH1 sequence 39Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg
Val40110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATURECH2 domain 40Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5 10 15Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His65 70 75
80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 100
105 11041110PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptideMISC_FEATURECH3 domain (AG) 41Gly Gln
Pro Phe Arg Pro Glu Val His Leu Leu Pro Pro Ser Arg Glu1 5 10 15Glu
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Ala Arg Gly Phe 20 25
30Tyr Pro Lys Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
35 40 45Asn Asn Tyr Lys Thr Thr Pro Ser Arg Gln Glu Pro Ser Gln Gly
Thr 50 55 60Thr Thr Phe Ala Val Thr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp65 70 75 80Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His 85 90 95Asn His Tyr Thr Gln Lys Thr Ile Ser Leu Ser
Pro Gly Lys 100 105 11042110PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptideMISC_FEATURECH3 domain
(GA) 42Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Pro Ser
Glu1 5 10 15Glu Leu Ala Leu Asn Glu Leu Val Thr Leu Thr Cys Leu Val
Lys Gly 20 25 30Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Leu Gln Gly
Ser Gln Glu 35 40 45Leu Pro Arg Glu Lys Tyr Leu Thr Trp Ala Pro Val
Leu Asp Ser Asp 50 55 60Gly Ser Phe Phe Leu Tyr Ser Ile Leu Arg Val
Ala Ala Glu Asp Trp65 70 75 80Lys Lys Gly Asp Thr Phe Ser Cys Ser
Val Met His Glu Ala Leu His 85 90 95Asn His Tyr Thr Gln Lys Ser Leu
Asp Arg Ser Pro Gly Lys 100 105 11043119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideMISC_FEATUREhumanized C225 VH kinetic variant S58R (IMGT
numbering) hu225-L 43Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Phe Ser Leu Thr Asn Tyr 20 25 30Gly Val His Trp Met Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Val Ile Trp Arg Gly Gly Asn
Thr Asp Tyr Asn Thr Pro Phe Thr 50 55 60Ser Arg Val Thr Ile Thr Ser
Asp Lys Ser Thr Ser Thr Ala Tyr Met65 70 75 80Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Ala Leu Thr
Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly 100 105 110Thr Leu
Val Thr Val Ser Ser 11544107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptideMISC_FEATUREhumanized C225
VL kinetic variant N108Y (IMGT numbering) hu225-M 44Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn 20 25 30Ile His
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Lys
Tyr Ala Ser Glu Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Asn Tyr Asn Trp Pro
Thr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10545119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATUREhumanized c225 VH T109D kinetic
variant (hu225-H) 45Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe
Ser Leu Thr Asn Tyr 20 25 30Gly Val His Trp Met Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45Gly Val Ile Trp Ser Gly Gly Asn Thr
Asp Tyr Asn Thr Pro Phe Thr 50 55 60Ser Arg Val Thr Ile Thr Ser Asp
Lys Ser Thr Ser Thr Ala Tyr Met65 70 75 80Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Ala Leu Asp Tyr
Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val
Thr Val Ser Ser 11546107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptideMISC_FEATUREhumanized C225
VL N109E, T116N kinetic variant (hu225-H) 46Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn 20 25 30Ile His Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Lys Tyr Ala
Ser Glu Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Asn Asn Glu Trp Pro Asn
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10547116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATUREc-Met binder BIO VL variants
comprising single or multiple amino acid substitutions selected
from V3A, TllV, T14S, R18S, R43Q, L45P, E74D, T85N, S86T, A90T,
T92S G100A (numbering according to IMGT
numbering)MOD_RES(3)..(3)Val or AlaMOD_RES(10)..(10)Thr or
ValMOD_RES(13)..(13)Thr or SerMOD_RES(17)..(17)Arg or
SerMOD_RES(38)..(38)Arg or GlnMOD_RES(40)..(40)Leu or
ProMOD_RES(61)..(61)Glu or AspMOD_RES(70)..(70)Thr or
AsnMOD_RES(71)..(71)Ser or ThrMOD_RES(75)..(75)Ala or
ThrMOD_RES(77)..(77)Thr or SerMOD_RES(85)..(85)Gly or Ala 47Gln Ser
Xaa Leu Thr Gln Pro Pro Ser Xaa Ser Gly Xaa Pro Gly Gln1 5 10 15Xaa
Val Thr Ile Ser Cys Phe Gly Ser Ser Ser Asn Val Gly Val Asn 20 25
30Thr Val Asn Trp Tyr Xaa Gln Xaa Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45Ile Tyr Asp Asn Asn Leu Arg Pro Ser Gly Val Pro Xaa Arg Phe
Ser 50 55 60Gly Ser Lys Ser Gly Xaa Xaa Ala Ser Leu Xaa Ile Xaa Gly
Leu Gln65 70 75 80Ala Glu Asp Glu Xaa Asp Tyr Tyr Cys Gln Ser Tyr
Asp Ser Ser Leu 85 90 95Ser Asp Val Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly Gln 100 105 110Pro Lys Ala Gly
11548117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATUREc-MET binder B10 VH kinetic
variant Q6E (IMGT numbering)MOD_RES(6)..(6)Gln or Glu 48Glu Val Gln
Leu Val Xaa Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Lys Asp Arg Arg Ile Thr His Thr Tyr Trp Gly
Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
11549115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATUREC-MET binder F06 VL sequence
variants comprising single or multiple amino acid substitutions
selected from Q1S, L2Y, S7P, K44Q, I51V, V71I (numbering acc. To
IMGT numbering)MOD_RES(1)..(1)Gln or SerMOD_RES(2)..(2)Leu or
TyrMOD_RES(7)..(7)Ser or ProMOD_RES(37)..(37)Lys or
GlnMOD_RES(44)..(44)Ile or ValMOD_RES(57)..(57)Val or Ile 49Xaa Xaa
Val Leu Thr Gln Xaa Pro Ser Val Ser Val Ala Pro Gly Lys1 5 10 15Thr
Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Arg Asn Val Gly Val 20 25
30His Trp Tyr Gln Xaa Lys Pro Gly Gln Ala Pro Xaa Leu Val Val Tyr
35 40 45Asp Asp Asp Asp Arg Pro Ser Gly Xaa Pro Glu Arg Phe Ser Gly
Ser 50 55 60Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu
Ala Gly65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser
Ala Thr Asp Gln 85 90 95Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu Gly Gln Pro Lys 100 105 110Ala Gly Cys 11550124PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideMISC_FEATUREc-MET binder F06 VH variants comprising
single or multiple amino acid substitutions selected from Q5V,
A19V, M115I, M115L, M115V, M115A, M115F (numbering according to
IMGT numbering)MOD_RES(5)..(5)Gln or ValMOD_RES(18)..(18)Ala or
ValMOD_RES(111)..(111)Met, Ile, Leu, Val, Ala or Phe 50Gln Val Gln
Leu Xaa Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Xaa
Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala
Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe
50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala
Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Asp Gln Arg Gly Tyr Asp Tyr Tyr Tyr Tyr
Tyr Gly Xaa Asp 100 105 110Val Trp Gly Gln Gly Thr Thr Val Thr Val
Ser Ser 115 12051114PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptideMISC_FEATUREc-MET binder B10v5 VL
variants comprising single or multiple amino acid substitutions
selected from E1S, P2Y, E17Q, T20R, P22T, R45K, L51V, T85N, S93R,
F103Y (IMGT numbering)MOD_RES(1)..(1)Glu or SerMOD_RES(2)..(2)Pro
or TyrMOD_RES(16)..(16)Glu or GlnMOD_RES(19)..(19)Thr or
ArgMOD_RES(21)..(21)Pro or ThrMOD_RES(38)..(38)Arg or
LysMOD_RES(44)..(44)Leu or ValMOD_RES(68)..(68)Thr or
AsnMOD_RES(76)..(76)Ser or ArgMOD_RES(86)..(86)Phe or Tyr 51Xaa Xaa
Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Xaa1 5 10 15Thr
Ala Xaa Ile Xaa Cys Gly Gly Asp Ser Leu Gly Ser Lys Ile Val 20 25
30His Trp Tyr Gln Gln Xaa Pro Gly Gln Ala Pro Xaa Leu Val Val Tyr
35 40 45Asp Asp Ala Ala Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly
Ser 50 55 60Lys Ser Gly Xaa Thr Ala Thr Leu Thr Ile Ser Xaa Val Glu
Ala Gly65 70 75 80Asp Glu Ala Asp Tyr Xaa Cys Gln Val Tyr Asp Tyr
His Ser Asp Val 85 90 95Glu Val Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu Gly Gln Pro Lys 100 105 110Ala Ala52124PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideMISC_FEATUREc-MET binder CS06 VH kinetic variants
comprising including single, double, triple or quadruple
combination of the following listed mutations Q3R, Y37N, N66I,
G110S, D111.1Y, Y111.2D, S126Y (IMGT numbering)MOD_RES(3)..(3)Gln
or ArgMOD_RES(32)..(32)Asn or TyrMOD_RES(59)..(59)Asn or
IleMOD_RES(102)..(102)Ser or GlyMOD_RES(104)..(105)Tyr or
AspMOD_RES(123)..(123)Tyr or Ser 52Gln Val Xaa Leu Gln Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Ala Lys Val Ser Cys Lys
Ala Ser Gly Gly Thr Phe Ser Ser Xaa 20 25 30Ala Ile Ser Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Pro
Ile Phe Gly Thr Ala Xaa Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Asp Gln Arg Xaa Tyr Xaa Xaa Tyr Tyr Tyr Tyr Gly Met Asp 100 105
110Val Trp Gly Gln Gly Thr Thr Val Thr Val Xaa Ser 115
120535PRTUnknownDescription of Unknown SrtA signal motif
sequenceMOD_RES(3)..(3)Asp, Glu, Ala, Asn, Gln or Lys 53Leu Pro Xaa
Thr Gly1 5545PRTUnknownDescription of Unknown SrtA signal motif
sequenceMOD_RES(3)..(3)Any amino acid 54Leu Pro Xaa Thr Gly1 5
* * * * *
References