U.S. patent application number 15/780285 was filed with the patent office on 2019-07-04 for anti-death receptor antibodies and methods of use thereof.
The applicant listed for this patent is GENMAB B.V.. Invention is credited to Frank BEURSKENS, Rob DE JONG, Marije OVERDIJK, Paul PARREN, David SATIJN, Janine SCHUURMAN, Kristin STRUMANE.
Application Number | 20190202926 15/780285 |
Document ID | / |
Family ID | 58796495 |
Filed Date | 2019-07-04 |
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United States Patent
Application |
20190202926 |
Kind Code |
A1 |
BEURSKENS; Frank ; et
al. |
July 4, 2019 |
ANTI-DEATH RECEPTOR ANTIBODIES AND METHODS OF USE THEREOF
Abstract
The present invention relates to monospecific or bispecific
antibody molecules that specifically bind antigens of Death
Receptors, which are members of the tumor necrosis factor (TNF)
receptor Superfamily (TNFR-SF) with an intracellular death domain.
The invention relates in particular to antibody molecules of the
IgG1 isotype having a mutation in the Fc region that enhances
clustering of IgG molecules after target binding. The invention
further relates to a combination of antibody molecules binding
different epitopes on one or more specific Death Receptors. The
invention also relates to pharmaceutical compositions containing
these molecules and the treatment of cancer using these
compositions.
Inventors: |
BEURSKENS; Frank; (Utrecht,
NL) ; OVERDIJK; Marije; (Utrecht, NL) ; DE
JONG; Rob; (Utrecht, NL) ; SATIJN; David;
(Utrecht, NL) ; STRUMANE; Kristin; (Werkhoven,
NL) ; SCHUURMAN; Janine; (Diemen, NL) ;
PARREN; Paul; (Odijk, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GENMAB B.V. |
Utrecht |
|
NL |
|
|
Family ID: |
58796495 |
Appl. No.: |
15/780285 |
Filed: |
December 1, 2016 |
PCT Filed: |
December 1, 2016 |
PCT NO: |
PCT/EP2016/079517 |
371 Date: |
May 31, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/46 20130101;
A61K 2039/507 20130101; A61K 38/00 20130101; A61K 2039/505
20130101; C07K 2317/31 20130101; C07K 2317/526 20130101; C07K
2317/73 20130101; C07K 16/2878 20130101; A61P 35/00 20180101; C07K
2317/75 20130101; C12N 15/62 20130101; C07K 2317/24 20130101; C07K
16/30 20130101; C07K 2317/52 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61P 35/00 20060101 A61P035/00; C07K 16/30 20060101
C07K016/30 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 1, 2015 |
DK |
PA 2015 00771 |
Dec 7, 2015 |
DK |
PA 2015 00787 |
Dec 7, 2015 |
DK |
PA 2015 00788 |
Nov 10, 2016 |
DK |
PA 2016 00701 |
Nov 10, 2016 |
DK |
PA 2016 00702 |
Claims
1. An antibody comprising an Fc region of a human immunoglobulin
IgG and an antigen binding region binding to a death receptor
comprising an intracellular death domain, wherein the Fc region
comprises a mutation at an amino acid position corresponding to
position E430, E345, S440, and/or Y436 in human IgG1, wherein the
positions are numbered according to EU Index.
2-4. (canceled)
5. The antibody according to claim 1, wherein the Fc region
comprises a mutation selected from the group consisting of: E430G,
E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440W, S440Y, and
Y436I.
6-8. (canceled)
9. The antibody according to claim 1, wherein the Fc region
comprises a further mutation in an amino acid position
corresponding to K439.
10. The antibody according to claim 1, wherein the Fc region
comprises a mutation at an amino acid position corresponding to
E430 and/or E345 in a human IgG1, and wherein said Fc region
comprises a further mutation at an amino acid position
corresponding to S440, with the proviso that the mutation is not
S440Y or S440W.
11. The antibody according to claim 9, wherein the further mutation
is selected from the group consisting of: K439E, K439D.
12. The antibody according to claim 10, wherein the further
mutation is selected from the group consisting of: S440K, S440R and
S440H.
13. The antibody according to claim 1, wherein the antibody further
comprises a mutation selected from K439E or S440K.
14. The antibody according to claim 1, wherein the death receptor
comprising an intracellular death domain is selected from the group
consisting of: FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR, and NGFR.
15-16. (canceled)
17. The antibody according to claim 1, wherein the antibody is an
IgG1, IgG2, IgG3, IgG4, IgE, IgD or IgM isotype.
18-19. (canceled)
20. The antibody according to claim 1, wherein the antibody is a
monoclonal antibody.
21. The antibody according to claim 1, wherein the antibody is
human, humanized or chimeric.
22. The antibody according to claim 1, wherein the antibody is
agonistic.
23. The antibody according to claim 1, wherein the antibody induces
programmed cell death in a target cell.
24-25. (canceled)
26. A multispecific antibody comprising one or more antigen binding
regions according to claim 1.
27. The multispecific antibody according to claim 26, wherein said
multispecific antibody is a bispecific antibody.
28. The bispecific antibody according to claim 27, wherein said
first antigen binding region and said second antigen binding region
binds different epitopes on one or more members of a death receptor
comprising an intracellular death domain selected from the group
consisting of: FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR and NGFR.
29. (canceled)
30. A composition comprising at least one antibody according to
claim 1 and a carrier.
31. (canceled)
32. A composition comprising a first antibody and a second
antibody, wherein both the first and second antibodies are
according to claim 1.
33. The composition according to claim 30, which comprises i) a
first antibody, wherein the Fc region comprises a first mutation at
an amino acid position corresponding to E430 or E345 in human IgG1,
and a further mutation at an amino acid position corresponding to
K439 in human IgG1, EU numbering, and ii) a second antibody,
wherein the Fc region comprises a first mutation at an amino acid
position corresponding to E430 or E345 in human IgG1, EU numbering,
and a further mutation at an amino acid position corresponding to
S440 in human IgG1.
34. The composition according to claim 33, which comprises a first
antibody wherein the further mutation is selected from the group
of: K439E and K439D, and a second antibody wherein the further
mutation is selected from the group of S440K, S440R or S440H.
35. (canceled)
36. The composition according to claim 32, wherein said first
antibody and said second antibody bind different epitopes on one or
more members of a death receptor comprising an intracellular death
domain selected form the group consisting of: such as FAS, DR4,
DR5, TNFR1, DR6, DR3, EDAR and NGFR.
37-45. (canceled)
46. A method of treating an infectious disease, autoimmune disease,
or cardiovascular anomalies comprising administering to a subject
in need thereof an effective amount of the composition of claim
30.
47. A method of treating a solid tumor and/or hematological tumor
comprising administering to a subject in need thereof an effective
amount of the composition of claim 30.
48. The method of claim 47, wherein the solid tumor is selected
from the group consisting of colorectal cancer, bladder cancer,
osteosarcoma, chondrosarcoma, breast cancer, cancers of the central
nervous system, cervical cancer, endometrium cancer, gastric
cancer, head and neck cancer, kidney cancer, liver cancer, lung
cancer, ovarian cancer, pancreatic cancer, sarcoma, and skin
cancer; and the hematological tumor is selected from the group
consisting of leukemia, lymphoma, and multiple myeloma.
49. (canceled)
50. A method of inhibiting growth of FAS, DR4, DR5, TNFR1, DR6,
DR3, EDAR or NGFR expressing tumors comprising administering to a
subject in need thereof an effective amount of the composition of
claim 30.
51. A method of inducing apoptosis in FAS, DR4, DR5, TNFR1, DR6,
DR3, EDAR or NGFR expressing tumors comprising administering to a
subject in need thereof an effective amount of the composition of
claim 30.
52. A method of treating an individual having a cancer comprising
administering to said individual an effective amount of the
antibody of claim 1.
53. The method according to claim 52 further comprising
administering an additional therapeutic agent.
54. The method according to claim 53, wherein the additional
therapeutic agent is one or more anti-cancer agent(s) selected from
the group consisting of of chemotherapeutics, kinase inhibitors,
apoptosis-modulating agents, RAS inhibitors, proteasome inhibitors,
histone deacetylase inhibitors, antibodies or antibody mimetics,
antibody-drug conjugates.
55. A kit comprising the antibody of claim 1, and instructions for
use.
56-57. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a 35 U.S.C. 371 national stage filing of
International Application No. PCT/EP2016/079517, filed Dec. 1,
2016, which claims priority to Danish Patent Application Nos. PA
2015 00771, filed Dec. 1, 2015, PA 2015 00787, filed Dec. 7, 2015,
PA 2015 00788, filed Dec. 7, 2015, PA 2016 00701, filed Nov. 10,
2016, and PA 2016 00702, filed Nov. 10, 2016. The contents of the
aforementioned applications are hereby incorporated by
reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jan. 29, 2019, is named GMI_169BUS_Sequence_Listing.txt and is
72,344 bytes in size.
FIELD OF THE INVENTION
[0003] The present invention relates to monospecific or bispecific
antibodies that specifically bind antigens of Death Receptors,
which are members of the tumor necrosis factor (TNF) receptor
Superfamily (TNFR-SF) with an intracellular death domain. The
invention relates in particular to antibody molecules of the IgG1
isotype having a mutation in the Fc region that enhances clustering
of IgG molecules after target binding. The invention further
relates to a combination of antibody molecules binding different
epitopes on one or more specific Death Receptors. The invention
also relates to pharmaceutical compositions containing these
molecules and the treatment of cancer using these compositions.
BACKGROUND OF THE INVENTION
[0004] The Death Receptors (DR) are a subset of the TNFR-SF, which
are plasma membrane receptors characterized by a cytoplasmic
sequence of .sup..about.80 amino acids known as the death domain
(DD) (Nagata et al., Cell. 1997 Feb. 7; 88(3):355-65; Ashkenzai et
al., Science. 1998 Aug. 28; 281(5381):1305-8; Locksley et al.,
Cell. 2001 Feb. 23; 104(4):487-501; Wajant Cell Death Differ. 2015
November; 22(11):1727-41). The intracellular death-domain of the
tumor necrosis factor (TNF) receptor superfamily (TNFR-SF) are
known to activate two main signalling cascades: a kinase cascade
leading to NF-kappaB and JNK activation and a caspase cascade
leading to cell death (Ashkenazi et al., Science. 1998 Aug. 28;
281(5381):1305-8). Ligand-mediated activation of death receptors
has been shown to trigger apoptosis in a variety of transformed
cell lines. Accordingly, there has been considerable efforts to
develop death receptor-targeting therapeutics for various diseases,
including agonistic antibodies. However, these efforts only
resulted in limited clinical efficacy.
[0005] Consequently, there is a need for providing improved
antibodies binding to death receptors of the tumor necrosis factor
(TNF) receptor Superfamily (TNFR-SF) with an intracellular death
domain, such as improved anti-Death Receptor antibodies for the
treatment of cancer, of infectious disease, autoimmune disease,
cardiovascular anomalies and other diseases
SUMMARY OF THE INVENTION
[0006] Surprisingly the inventors of the present invention have
found that the introduction of a specific point mutation in the Fc
region of antibodies that specifically bind antigens of Death
Receptors, which are members of the TNFR-SF comprising an
intracellular death domain significantly enhances the potency of
the antibody in vitro and in vivo by Fc.gamma.R-independent
clustering after binding of the antibody to the target on the cell
surface. Even more surprisingly the inventors have also found that
a combination of two anti-Death Receptor antibodies with a
mutations in the Fc region facilitate antibody clustering
conditional on cell surface antigen binding, resulting in the
formation of hetrohexamers and enhanced potency compared to a
combination of the two anti-Death Receptor antibodies without the
mutation.
[0007] The object of the present invention is to provide improved
anti-Death Receptor antibodies, such as anti-FAS, anti-DR4,
anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR, and anti-NGFR
antibodies, e.g. for use in the treatment of cancer. Such an
improved anti-Death Receptor antibody comprises a mutation in the
Fc domain. A further object of the present invention is to provide
an improved composition for the treatment of cancer comprising one
or more anti-Death Receptor antibodies binding to different
epitopes on Death Receptors, such as anti-FAS, anti-DR4, anti-DR5,
anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR, and anti-NGFR
antibodies. Such an improved composition as described herein
comprises at least one anti-Death Receptor antibody or the
composition comprises two anti-Death Receptor antibodies binding to
different regions on one or more Death Receptors, such as different
epitopes on one or more of the following Death Receptors selected
from the group consisting of: FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR,
and NGFR.
[0008] The present invention provides an antibody comprising an Fc
region of a human immunoglobulin IgG and an antigen binding region
binding to a Death Receptor, such as an anti-FAS, anti-DR4,
anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR, and anti-NGFR
antibody, wherein the Fc region comprises a mutation at an amino
acid corresponding to position E430, E345 or S440 in human IgG1
according to EU numbering (Edelman et al., Proc Natl Acad Sci USA.
1969 May; 63(1):78-85; Kabat et al., Sequences of Proteins of
Immunological Interest, Fifth Edition. 1991 NIH Publication No.
91-3242). Unless contradicted by the context immunoglobulin IgG has
the same meaning as IgG.
[0009] In one aspect the invention provides an antibody comprising
an Fc region of a human immunoglobulin IgG and an antigen binding
region binding to a Death Receptor comprising an intracellular
death domain, wherein the Fc region comprises a mutation at an
amino acid corresponding to position E430, E345 or S440 in human
IgG1, EU numbering.
[0010] That is, the inventors of the present invention in a first
aspect of the invention found that anti-Death Receptor antibodies,
such as anti-FAS, anti-DR4, anti-DR5, anti-TNFR1, anti-DR6,
anti-DR3, anti-EDAR, and anti-NGFR antibodies of the invention
increase apoptosis of cells expressing FAS, DR4, DR5, TNFR1, DR6,
DR3, EDAR, or NGFR, such as tumor cells compared to anti-FAS,
anti-DR4, anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR, and
anti-NGFR antibodies without a mutation at an amino acid position
corresponding to E430, E345 or 5440 of human IgG1, EU numbering.
That is, an anti-FAS, anti-DR4, anti-DR5, anti-TNFR1, anti-DR6,
anti-DR3, anti-EDAR, and anti-NGFR antibody of the present
invention is suitable for the treatment of FAS, DR4, DR5, TNFR1,
DR6, DR3, EDAR, or NGFR positive or expressing tumors. Thus the
antibodies according to the invention are suitable for treatment of
tumors which are positive for or expressing one or more antigens
consisting of the following group: FAS, DR4, DR5, TNFR1, DR6, DR3,
EDAR and NGFR.
[0011] In one embodiment of the present invention the anti-FAS,
anti-DR4, anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR, and
anti-NGFR antibody comprises an Fc region of a human immunoglobulin
IgG and an antigen binding region binding to a Death Receptor,
wherein the Fc region comprises a mutation corresponding to
position E430G or E345K in human IgG1 according to EU numbering.
Thus in one embodiment of the invention the anti-FAS, anti-DR4,
anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR, and anti-NGFR
antibody comprises an Fc region of a human immunoglobulin IgG and
an antigen binding region binding to a Death Receptor, wherein the
Fc region comprises a mutation at an E430G or E345K mutation.
[0012] In one embodiment of the present invention the anti-FAS,
anti-DR4, anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR, and
anti-NGFR antibody comprises an Fc region of a human immunoglobulin
IgG, wherein the Fc region comprises an E430G mutation.
[0013] In one embodiment of the present invention the anti-FAS,
anti-DR4, anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR, and
anti-NGFR antibody comprises an Fc region of a human immunoglobulin
IgG, wherein the Fc region comprises an E345K mutation.
[0014] In one aspect the invention provides a composition
comprising one or more anti-Death Receptor antibodies selected from
the group consisting of: anti-FAS, anti-DR4, anti-DR5, anti-TNFR1,
anti-DR6, anti-DR3, anti-EDAR, and anti-NGFR. In one embodiment the
composition comprises one or more antibodies binding to different
epitopes on FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR and NGFR. In one
embodiment the composition comprises at least a first and a second
antibody selected from the group consisting of: anti-FAS, anti-DR4,
anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR and anti-NGFR,
wherein the first antibody does not block antigen binding of the
second antibody.
[0015] In another aspect the invention provides a bispecific
antibody comprising one or more antigen binding regions binding to
FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR and NGFR. In one embodiment a
bispecific antibody of the present invention comprises a first and
a second heavy chain, wherein the first heavy chain comprises a
F405L mutation and a second heavy chain comprises a K409R mutation,
or vice versa. Thus in one embodiment a bispecific antibody
according to the present invention the bispecific antibody
comprises a first and a second heavy chain, wherein the first and
the second heavy chain comprises a mutation at an amino acid
positon corresponding to E430, E345 or S440 in human IgG1, EU
numbering and wherein the first heavy chain comprises a F405L
mutation and the second heavy chain comprises a K409R mutation.
Thus in one embodiment a bispecific antibody according to the
present invention the bispecific antibody comprises a first and a
second heavy chain, wherein the first and the second heavy chain
comprises a mutation at an amino acid position corresponding to
E430, E345 or S440 in human IgG1, EU numbering and wherein the
first heavy chain comprises a K409R mutation and the second heavy
chain comprises a F405L mutation.
[0016] In yet another aspect the invention provides a method of
treating a disease comprising administering to an individual in
need thereof an effective amount of an antibody or composition as
described herein. In one embodiment of the invention the disease is
cancer.
[0017] In another aspect of the invention the anti-FAS, anti-DR4,
anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR, or anti-NGFR
antibody, bispecific antibody or composition according to the
present invention is for use as a medicament. In one embodiment the
anti-FAS, anti-DR4, anti-DR5, anti-TNFR1, anti-DR6, anti-DR3,
anti-EDAR, or anti-NGFR antibody, bispecific antibody or
composition is for use in treatment of a disease. In one embodiment
the disease is a cancer or a tumor.
[0018] In yet another aspect the invention provides a method of
treating an individual having a cancer comprising administering to
said individual an effective amount of said antibody or composition
as described herein.
[0019] In another aspect the invention provides a kit of parts
comprising an antibody or composition according to any one of the
preceding claims, wherein said antibody or composition is in one or
more containers such as a vial.
[0020] In another aspect the invention provides the use of an
antibody or a composition as described herein for the manufacture
of a medicament for treatment of a disease. In one embodiment the
invention provides the use of an antibody or a composition as
described herein for the manufacture of a medicament for treatment
of cancer.
[0021] The antibodies and compositions described herein are
directed against or specific for human FAS, DR4, DR5, TNFR1, DR6,
DR3, EDAR or NGFR. The antibodies and compositions described
cross-react with rhesus and cynomolgus monkey FAS, DR4, DR5, TNFR1,
DR6, DR3, EDAR or NGFR. In particular, in one embodiment the
antibodies and compositions bind specifically to the extracellular
domain of FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR or NGFR. In one
particular embodiment the antibodies and compositions bind to the
same Death Receptor from the group consisting of: FAS, DR4, DR5,
TNFR1, DR6, DR3, EDAR and NGFR e.g. at non-overlapping epitopes.
That is a first antibody described herein does not block binding of
a second antibody described herein. In one particular embodiment a
composition described herein comprises a first and a second
antibody binding to FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR or NGFR
and the first antibody does not block binding of the second
antibody to FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR or NGFR.
[0022] The antibodies and compositions of the present invention can
generally be used to modulate the activity of a Death Receptor such
as FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR or NGFR. In one embodiment
the antibody or composition may trigger, activate and/or increase
or enhance the signalling that is mediated by a Death Receptor such
as FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR or NGFR. In one embodiment
the antibody or composition may have an agonistic effect on a Death
Receptor such as FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR or NGFR and
in particular trigger or increase the biological mechanisms,
responses and effects associated with FAS, DR4, DR5, TNFR1, DR6,
DR3, EDAR or NGFR, their signalling and/or the pathway in which
FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR or NGFR is involved. That is
antibodies or compositions of the present invention may induce
apoptosis or cell death in cells or tissues expressing FAS, DR4,
DR5, TNFR1, DR6, DR3, EDAR or NGFR, such as cancer cells or tumor
cells.
[0023] In one embodiment the antibodies or compositions described
herein induce, trigger, increase or enhance apoptosis, cell death
or growth arrest in cells or tissues expressing FAS, DR4, DR5,
TNFR1, DR6, DR3, EDAR or NGFR, such as cancer cells or tumor cells.
In one embodiment the antibodies or compositions described herein
are capable of binding to FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR or
NGFR on a cell surface, and in particular of binding to FAS, DR4,
DR5, TNFR1, DR6, DR3, EDAR or NGFR in such a way that the
signalling mediated by FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR or NGFR
is induced, triggered, increased or enhanced. In one embodiment the
antibodies or compositions described herein may be such that they
are capable of binding to a FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR or
NGFR in such a way that apoptosis or cell death is induced in
cancer cells or tumor cells expressing FAS, DR4, DR5, TNFR1, DR6,
DR3, EDAR or NGFR.
[0024] In one embodiment the antibodies or compositions of the
present invention induce, trigger, increase or enhance apoptosis or
cell death in cancer cells or tumor cells expressing FAS, DR4, DR5,
TNFR1, DR6, DR3, EDAR or NGFR. The increased or enhanced apoptosis
or cell death may be measured by an increase or enhanced level of
phosphatidylserine exposure on cells exposed to or treated with one
or more anti-FAS, anti-DR4, anti-DR5, anti-TNFR1, anti-DR6,
anti-DR3, anti-EDAR, and anti-NGFR antibodies of the invention.
Alternatively, the increase or enhanced apoptosis or cell death may
be measured by measuring activation of caspase 3 or caspase 7 in
cells that have been exposed to or treated with one or more
anti-DR5 antibodies of the invention. Alternatively, the increase
or enhanced apoptosis or cell death may be measured by a loss of
viability in cell cultures that have been exposed to or treated
with one or more anti-FAS, anti-DR4, anti-DR5, anti-TNFR1,
anti-DR6, anti-DR3, anti-EDAR, and anti-NGFR antibodies of the
invention, compared to untreated cell cultures. Induction of
caspase-mediated apoptosis may be assessed by demonstrating
inhibition of the loss of viability after exposure to DR5 antibody
by a caspase-inhibitor, for example ZVAD.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 shows an amino acid alignment of the four different
human IgG1 Fc allotypes identified thus far. The Fc sequence of the
IgG1m(f) (SEQ ID NO 1), IgG1m(z) (SEQ ID NO 2), IgG1m(a) (SEQ ID NO
3), IgG1m(x) (SEQ ID NO 4).
[0026] FIGS. 2A-2C show binding of DR5 antibodies with and without
hexamerization-enhancing mutation E430G or E345K to DR5-positive
COLO 205 cells. Variants of the human-mouse chimeric antibodies
(FIG. 2A) IgG1-DR5-01, (FIG. 2B) IgG1-DR5-05 and (FIG. 2C) and
bispecific antibody IgG1-DR5-01-K409R.times.IgG1-DR5-05-F405L (BsAb
DR5-01-K409R.times.DR5-05-F405L) were tested in FACS analysis for
binding to COLO 205 cells. Binding is expressed as Geometric mean
of fluorescence intensity. Anti-gp120 antibody IgG1-b12 was used as
negative control. Error bars indicate the standard deviation.
[0027] FIG. 3 shows a binding ELISA of DR4 antibodies to coated
sTRAIL-R1. Graphs represent binding of the antibody
IgG1-DR4-T1014G03-K409R with and without the E430G
hexamerization-enhancing mutation to coated sTRAIL-R1.
[0028] FIGS. 4A-4I show a viability assays with variants of
different DR5 antibodies. Introduction of the E345K (FIG. 4C),
E430G (FIGS. 4A, 4B, and 4D-4I) or E345R/E430G/S440Y (RGY) (FIG.
4D, FIG. 4I) hexamerization-enhancing mutations resulted in
enhanced killing for the different DR5 antibodies on COLO 205
(FIGS. 4A-4D) and HCT116 (FIGS. 4E-4I) colon cancer cells. Error
bars indicate standard deviation. Data are presented as
Luminescence (RLU=relative luminescence units) or as % Viable cells
calculated from the luminescence relative to samples incubated
without antibody (no kill) and samples incubated with Staurosporine
(maximal kill).
[0029] FIG. 5 shows a viability assay with variants of DR4 antibody
IgG1-DR4-T1014G03. Introduction of the E430G
hexamerization-enhancing mutation resulted in enhanced killing of
BxPC-3 human pancreatic cancer cells. Error bars indicate standard
deviation.
[0030] FIG. 6 shows a viability assay with variants of FAS antibody
IgG1-FAS-E09. Introduction of the hexamerization-enhancing triple
mutation E345R/E430G/S440Y (RGY) resulted in dose-dependent killing
of Jurkat human T lymphocytes.
[0031] FIGS. 7A-7E show that introduction of a
hexamerization-enhancing mutation resulted in enhanced induction of
killing by the antibody combination
IgG1-DR5-01-K409R+IgG1-DR5-05-F405 (FIGS. 7A and 7C) and the BsAb
DR5-01-K409R.times.DR5-05-F405 (FIGS. 7B and 7D) on both COLO 205
(FIGS. 7A and 7B) and HCT116 (FIGS. 7C-7E) colon cancer cells.
Error bars indicate standard deviation.
[0032] FIGS. 8A and 8B show the potency of the combination of the
antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G
compared to the combination without E430G mutation as measured in a
viability assay on BxPC-3 pancreatic (FIG. 8A) and HCT15 colon
cancer cells (FIG. 8B). Graphs represent mean values of duplicate
samples+/-standard deviation.
[0033] FIGS. 9A and 9B show a viability assay with repulsing and
complementary variants of IgG1-hDR5-01-G56T-E430G and
IgG1-hDR5-05-E430G. Introduction of the same repulsing mutation
(K439E or S440K) in both antibodies resulted in diminished
induction of killing of BxPC-3 pancreatic (FIG. 9A) and HCT-15
colon cancer cells (FIG. 9B). By combining the two mutations (K439E
and S440K) in both antibodies, repulsion was neutralized and
killing restored. Error bars indicate standard deviation.
[0034] FIG. 10 shows the involvement of Fc interactions in the
capacity of the antibody combination
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G with
hexamerization-enhancing mutation to induce receptor clustering on
the cell surface and induction of apoptosis. Induction of apoptosis
is inhibited by the Fc-binding peptide DCAWHLGELVWCT as shown in a
3-days viability assay on BxPC-3 human cancer cells.
[0035] FIG. 11 shows that the combination of
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G reduced the
viability of different human cancer cell lines, as determined in a
3-days viability assay. Graphs show the mean+/-standard deviation
from duplicate samples. *p<0.05, **p<0.01, ***p<0.001,
****p<0.001 (One-way ANOVA with Tukey's multiple comparisons
test). 01 is IgG1-DR5-01-K409R, 05 is IgG1-DR5-05-F405L, 01-E430G
is IgG1-DR5-01-K409R-E430G, 05-E430G is
IgG1-DR5-05-F405L-E430G.
[0036] FIG. 12 shows that introduction of a
hexamerization-enhancing mutation results in enhanced induction of
killing of HCT 116 colon cancer cells by the antibody combination
IgG1-DR5-05-F405L-E345K+IgG1-CONA-K409R-E430G and BsAb
CONA-K409R-E430G.times.DR5-05-F405L-E345K as determined in a 3-days
viability assay. Error bars indicate standard deviation. RLU:
Relative Luminescence Units.
[0037] FIGS. 13A-13C show the efficacy of the antibody combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G and BsAb
DR5-01-K409R-E430G.times.DR5-05-F405L-E430G in the presence or
absence of secondary Fc crosslinking by anti-human IgG F(ab')2 and
in comparison to DR5 antibodies IgG1-DR5-CONA and
IgG1-DR5-chTRA8-F405L in a 3-days viability assay on adherent COLO
205 (FIG. 13A) colorectal and PANC-1 (FIG. 13B) and BxPC-3 (FIG.
13C) pancreatic cancer cells. The non-target binding antibody
IgG1-b12 was included as a negative control. Graphs show the
mean+/-standard deviation from duplicate samples.
[0038] FIGS. 14A-14C show caspase-dependent programmed cell death
by the combination of humanized
IgG1-hDR5-01-E430G+IgG1-hDR5-05-E430G (01-E430G+05-E430G)
antibodies as measured in a viability assay on PANC-1 (FIGS. 14A
and 14B) and BxPC-3 (FIG. 14C) pancreatic cancer cells. ZVAD,
Z-VAD-FMK.
[0039] FIGS. 15A-15E show that cell death induction upon binding of
DR5 antibody combinations on COLO 205 colon cancer cells. COLO 205
cells were incubated with antibody sample for 5 hours (FIGS.
15A-15C) and 24 hours (FIGS. 15D and 15E). Different stages of cell
death induction were analyzed by Annexin V/PI double staining and
Active caspase-3 staining. Error bars indicate the standard
deviation of 2 duplicate samples. 01 is IgG1-DR5-01-K409R, 05 is
IgG1-DR5-05-F405L, 01-E430G is IgG1-DR5-01-K409R-E430G, 05-E430G is
IgG1-DR5-05-F405L-E430G, 01x05 is BsAb
DR5-01-K409R.times.DR5-05-F405L, 01-E430G.times.05-E430G is BsAb
DR5-01-K409R-E430G.times.DR5-05-F405L-E430G.
[0040] FIGS. 16A and 16B show the kinetics of Caspase-3/7
activation upon binding of antibody combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G (FIG. 16A) and BsAb
DR5-01-K409R-E430G.times.DR5-05-F405L-E430G (FIG. 16B) on COLO 205
colon cancer cells. COLO 205 cells were incubated with antibody for
1, 2, 5 and 24 hours. Caspase-3/7 activation was analyzed in a
homogenous luminescence assay. AU, arbitrary units. Error bars
indicate the standard deviation of duplicate samples.
[0041] FIG. 17 shows the potency of the antibody combination
IgG1-hDR5-01-K409R-E430G+IgG1-hDR5-05-F405L-E430G and of the
antibody combination IgG1-hDR5-01-E430G+IgG1-hDR5-05-E430G as
measured in a viability assay on BxPC-3 pancreatic cancer cells.
Graphs represent mean values of duplicate samples+/-standard
deviation.
[0042] FIG. 18 shows the efficacy of different ratios of
IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G
(DR5-01-E430G:DR5-05-E430G) on adherent BxPC-3 human cancer cells
as determined in a 3-days viability assay.
[0043] FIGS. 19A and 19B show the efficacy of different ratios of
IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G
(DR5-01-E430G:DR5-05-E430G) on adherent BxPC-3 (FIG. 19A) and
HCT-15 (FIG. 19B) human cancer cells as determined in a 3-days
viability assay.
[0044] FIGS. 20A and 20B the evaluation of the in vivo efficacy of
the chimeric IgG1-DR5-05-F405L with and without the
hexamerization-enhancing mutation E430G in a subcutaneous xenograft
model with COLO 205 human colon cancer cells. Tumor development
(mean & SEM) in mice treated with the indicated antibodies (5
mg/kg) is shown in time (FIG. 20A). In (FIG. 20B) the percentage of
mice with tumor sizes smaller than 750 mm3 is shown in a
Kaplan-Meier plot.
[0045] FIG. 21 shows a viability assay on Jurkat human T
lymphocytes with variants of FAS antibody IgG1-FAS-E09. In the
presence of the Fc-Fc repulsing mutation K439E or S440K, killing by
IgG1-FAS-E09 variants with hexamerization-enhancing mutations
E345R/E430G/S440Y (RGY) or E345R/E430G/Y436I (RGI) was inhibited.
RGEY: E345R/E430G/K439E/S440Y; RGIK: E345R/E430G/Y436I/S440K.
[0046] FIG. 22 shows a viability assays with DR5 antibodies
IgG1-DR5-CONA and IgG1-DR5-CONA-E430G on attached COLO 205 human
colon cancer cells. Introduction of the hexamerization-enhancing
mutation E430G resulted in induction of killing. Data are presented
as % viable cells calculated from the luminescence relative to
samples incubated without antibody (no kill) and samples incubated
with Staurosporine (maximal kill). Error bars indicate standard
deviation.
[0047] FIGS. 23A and 23B show a viability assay with DR5 antibodies
on COLO 205 human colon cancer cells. Introduction of the
hexamerization-enhancing mutation S440Y resulted in induction of
killing by the single antibodies IgG1-hDR5-01-G56T and IgG1-hDR5-05
(FIG. 23A) and increased efficacy of the antibody combination
IgG1-hDR5-01-G56T+IgG1-hDR5-05 (FIG. 23B). Data are presented as %
viable cells calculated from the luminescence relative to samples
incubated without antibody (no kill) and samples incubated with
Staurosporine (maximal kill). Error bars indicate standard
deviation.
[0048] FIG. 24A shows crossblock ELISA between IgG1-DR5-CONA-K409R
and IgG1-DR5-chTRA8-F405L. (FIG. 24B) Introduction of the E430G
hexamerization-enhancing mutation resulted in enhanced induction of
killing of BxPC-3 human pancreatic cancer cells by the combination
of the non-crossblocking antibodies
IgG1-DR5-CONA-E430G+IgG1-DR5-chTRA8-E430G as determined in a 3-days
viability assay. Error bars indicate standard deviation.
[0049] FIGS. 25A-25C show the evaluation of the in vivo efficacy of
the anti-DR5 antibody concentration IgG1-hDR5-01-G56T+IgG1-hDR5-05
with and without the hexamerization-enhancing mutation E430G in a
subcutaneous xenograft model with HCT15 human colon cancer cells.
Tumor development (mean & SEM) in mice treated with the 0.5
mg/kg antibodies is shown in time (FIG. 25A) and at day 21 after
start treatment (FIG. 25B). **P<0.0011 (Mann Whitney test). In
(FIG. 25C) the percentage of mice with tumor sizes smaller than 750
mm3 is shown in a Kaplan-Meier plot.
[0050] FIGS. 26A and 26B show evaluation of the in vivo efficacy of
the antibodies IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G, both
as single agents and as a combination in comparison to the parental
antibodies without the E430G mutation in a subcutaneous xenograft
model with COLO 205 human colon cancer cells. (FIG. 26A) Tumor size
(mean & SEM) in mice treated with the indicated antibodies (0.5
mg/kg) as shown in time. (FIG. 26B) Kaplan-Meier plot of tumor
progression, with a cutoff set at a tumor volume >500
mm.sup.3.
DETAILED DESCRIPTION OF THE INVENTION
[0051] In describing the embodiments of the invention specific
terminology will be resorted to for the sake of clarity. However,
the invention is not intended to be limited to the specific terms
so selected, and it is understood that each specific term includes
all technical equivalents which operate in a similar manner to
accomplish a similar purpose.
[0052] As described herein, surprisingly it has been found that
antibodies binding to a Death Receptor such as FAS, DR4, DR5,
TNFR1, DR6, DR3, EDAR or NGFR and comprising a mutation at an amino
acid in the Fc region corresponding to position E430, E345 or S440
in human IgG1 according to EU numbering, were found to be superior
at inducing apoptosis in cancer cells expressing FAS, DR4, DR5,
TNFR1, DR6, DR3, EDAR or NGFR compared to anti-FAS, anti-DR4,
anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR or anti-NGFR
antibodies without said mutation in one of the above mentioned
positions. Furthermore, compositions comprising two or more
anti-FAS, anti-DR4, anti-DR5, anti-TNFR1, anti-DR6, anti-DR3,
anti-EDAR, and anti-NGFR antibodies of the invention, which bind
different epitopes on FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR or NGFR,
were found superior to compositions comprising the same anti-FAS,
anti-DR4, anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR or
anti-NGFR antibodies without said mutation. That is compositions
with two or more antibodies of the present invention were superior
at inducing apoptosis and/or inhibiting cell growth of cancer cells
expressing FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR or NGFR compared to
compositions comprising the two same anti-FAS, anti-DR4, anti-DR5,
anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR or anti-NGFR antibodies
without said mutation in the Fc region. It is to be understood that
in the context of this invention the same antibody is antibodies
having the identical antigen binding region. Thus the same antibody
has an identical amino acid sequence as an antibody of the present
invention, but does not have said mutation in the Fc region. By
introducing specific mutations in the Fc region, oligomerization
such as hexamerization upon target binding on the cell surface can
be enhanced, while the antibody molecules remain monomeric in
solution WO2013/004842, WO2014/108198.
Definitions
[0053] The term "immunoglobulin" as used herein, refers to a class
of structurally related glycoproteins consisting of two pairs of
polypeptide chains, one pair of light (L) low molecular weight
chains and one pair of heavy (H) chains, all four potentially
inter-connected by disulfide bonds. The structure of
immunoglobulins has been well characterized. See for instance
Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press,
N.Y. (1989)). Briefly, each heavy chain typically is comprised of a
heavy chain variable region (abbreviated herein as VH) and a heavy
chain constant region. The heavy chain constant region of IgG
antibodies typically is comprised of three domains, CH1, CH2, and
CH3. The heavy chains are inter-connected via disulfide bonds in
the so-called "hinge region". Each light chain typically is
comprised of a light chain variable region (abbreviated herein as
VL) and a light chain constant region. The light chain constant
region typically is comprised of one domain, CL. The VH and VL
regions may be further subdivided into regions of hypervariability
(or hypervariable regions which may be hypervariable in sequence
and/or form of structurally defined loops), also termed
complementarity determining regions (CDRs), interspersed with
regions that are more conserved, termed framework regions (FRs).
Each VH and VL is typically composed of three CDRs and four FRs,
arranged from amino-terminus to carboxy-terminus in the following
order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and
Lesk J. Mol. Biol. 196, 901 917 (1987)). Unless otherwise stated or
contradicted by context, reference to amino acid positions in the
present invention is according to the EU-numbering (Edelman et al.,
Proc Natl Acad Sci USA. 1969 May; 63(1):78-85; Kabat et al.,
Sequences of Proteins of Immunological Interest, Fifth Edition.
1991 NIH Publication No. 91-3242).
[0054] The term "hinge region" as used herein is intended to refer
to the hinge region of an immunoglobulin heavy chain. Thus, for
example the hinge region of a human IgG1 antibody corresponds to
amino acids 216-230 according to the EU numbering.
[0055] The term "CH2 region" or "CH2 domain" as used herein is
intended to refer the CH2 region of an immunoglobulin heavy chain.
Thus, for example the CH2 region of a human IgG1 antibody
corresponds to amino acids 231-340 according to the EU numbering.
However, the CH2 region may also be any of the other isotypes or
allotypes as described herein.
[0056] The term "CH3 region" or "CH3 domain" as used herein is
intended to refer to the CH3 region of an immunoglobulin heavy
chain. Thus, for example the CH3 region of a human IgG1 antibody
corresponds to amino acids 341-447 according to the EU numbering.
However, the CH3 region may also be any of the other isotypes or
allotypes as described herein.
[0057] The term "fragment crystallizable region", "Fc region", "Fc
fragment" or "Fc domain", which may be used interchangeably herein,
refers to an antibody region comprising, arranged from
amino-terminus to carboxy-terminus, at least a hinge region, a CH2
domain and a CH3 domain. An Fc region of an IgG1 antibody can, for
example, be generated by digestion of an IgG1 antibody with papain.
The Fc region of an antibody may mediate the binding of the
immunoglobulin to host tissues or factors, including various cells
of the immune system (such as effector cells) and components of the
complement system such as C1q, the first component in the classical
pathway of complement activation.
[0058] The term "Fab fragment" in the context of the present
invention, refers to a fragment of an immunoglobulin molecule,
which comprises the variable regions of the heavy chain and light
chain as well as the constant region of the light chain and the CH1
region of the heavy chain of an immunoglobulin. The "CH1 region"
refers e.g. to the region of a human IgG1 antibody corresponding to
amino acids 118-215 according to the EU numbering. Thus, the Fab
fragment comprises the binding region of an immunoglobulin.
[0059] The term "antibody" (Ab), as used herein refers to an
immunoglobulin molecule, a fragment of an immunoglobulin molecule,
or a derivative of either thereof. The antibody of the present
invention comprises an Fc-region of an immunoglobulin and an
antigen-binding region. The Fc region generally contains two
CH2-CH3 regions and a connecting region, e.g. a hinge region. The
variable regions of the heavy and light chains of the
immunoglobulin molecule contain a binding domain that interacts
with an antigen. The term "antibody" as used herein, also refers
to, unless otherwise specified or contradicted by the context,
polyclonal antibodies, oligoclonal antibodies, monoclonal
antibodies (such as human monoclonal antibodies), antibody
mixtures, recombinant polyclonal antibodies, chimeric antibodies,
humanized antibodies and human antibodies. An antibody as generated
can potentially possess any class or isotype.
[0060] The term "human antibody", as used herein, refers to
antibodies having variable and constant regions derived from human
germline immunoglobulin sequences. The human antibodies of the
invention may include amino acid residues not encoded by human
germline immunoglobulin sequences (e.g., mutations, insertions or
deletions introduced by random or site-specific mutagenesis in
vitro or by somatic mutation in vivo). However, the term "human
antibody", as used herein, is not intended to include antibodies in
which CDR sequences derived from the germline of another species,
such as a mouse, have been grafted onto human framework
sequences.
[0061] The term "chimeric antibody", as used herein, refers to an
antibody in which both chain types are chimeric as a result of
antibody engineering. A chimeric chain is a chain that contains a
foreign variable domain (originating from a non-human species, or
synthetic or engineered from any species including human) linked to
a constant region of human origin.
[0062] The term "humanized antibody", as used herein, refers to an
antibody in which both chain types are humanized as a result of
antibody engineering. A humanized chain is typically a chain in
which the complementarity determining regions (CDR) of the variable
domains are foreign (originating from a species other than human,
or synthetic) whereas the remainder of the chain is of human
origin. Humanization assessment is based on the resulting amino
acid sequence, and not on the methodology per se, which allows
protocols other than grafting to be used.
[0063] The term "isotype", as used herein, refers to the
immunoglobulin class (for instance IgG1, IgG2, IgG3, IgG4, IgD,
IgA1, IgA2, IgE, or IgM) that is encoded by heavy chain constant
region genes. To produce a canonical antibody, each heavy chain
isotype is to be combined with either a kappa (.kappa.) or lambda
(.lamda.) light chain.
[0064] The term "allotype", as used herein, refers to the amino
acid variation within one isotype class in the same species. The
predominant allotype of an antibody isotype varies between
ethnicity individuals. The known allotype variations within the
IgG1 isotype of the heavy chain result from 4 amino acid
substitutions in the antibody frame as illustrated in FIG. 1. In
one embodiment the antibody of the invention is of the IgG1m(f)
allotype as defined in SEQ ID NO 1. In one embodiment of the
invention the antibody is of the IgG1m(z) allotype as defined in
SEQ ID NO 2, the IgG1m(a) allotype as defined in SEQ ID NO 3, the
IgG1m(x) allotype as defined in SEQ ID NO 4, or any allotype
combination, such as IgG1m(z,a), IgG1m(z,a,x), IgG1m(f,a) (de lange
Exp Clin Immunogenet. 1989; 6(1):7-17).
[0065] The terms "monoclonal antibody", "monoclonal Ab",
"monoclonal antibody composition", "mAb", or the like, as used
herein refer to a preparation of Ab molecules of single molecular
composition. A monoclonal antibody composition displays a single
binding specificity and affinity for a particular epitope.
Accordingly, the term "human monoclonal antibody" refers to Abs
displaying a single binding specificity which have variable and
constant regions derived from human germline immunoglobulin
sequences. The human mAbs may be generated by a hybridoma which
includes a B cell obtained from a transgenic or transchromosomal
non-human animal, such as a transgenic mouse, having a genome
comprising a human heavy chain transgene repertoire and a human
light chain transgene repertoire, rearranged to produce a
functional human antibody and fused to an immortalized cell.
Alternatively, the human mAbs may be generated recombinantly.
[0066] The term "antibody mimetics" as used herein, refers to
compounds that, like antibodies, can specifically bind antigens,
but that are not structurally related to antibodies. They are
usually artificial peptides, proteins, nucleic acids or small
molecules.
[0067] The term "bispecific antibody" refers to antibody having
specificities for at least two different, typically
non-overlapping, epitopes. Such epitopes may be on the same or
different targets Examples of different classes of bispecific
antibodies comprising an Fc region include but are not limited to:
asymmetric bispecific molecules e.g. IgG-like molecules with
complementary CH3 domains and symmetric bispecific molecules e.g.
recombinant IgG-like dual targeting molecules wherein each
antigen-binding region of the molecule binds at least two different
epitopes.
[0068] Examples of bispecific molecules include but are not limited
to Triomab.RTM. (Trion Pharma/Fresenius Biotech, WO/2002/020039),
Knobs-into-Holes (Genentech, WO9850431), CrossMAbs (Roche, WO
2009/080251, WO 2009/080252, WO 2009/080253),
electrostatically-matched Fc-heterodimeric molecules (Amgen,
EP1870459 and WO2009089004; Chugai, US201000155133; Oncomed,
WO2010129304), LUZ-Y (Genentech), DIG-body, PIG-body and TIG-body
(Pharmabcine), Strand Exchange Engineered Domain body (SEEDbody)
(EMD Serono, WO2007110205), Bispecific IgG1 and IgG2 (Pfizer/Rinat,
WO11143545), Azymetric scaffold (Zymeworks/Merck, WO2012058768),
mAb-Fv (Xencor, WO2011028952), XmAb (Xencor), Bivalent bispecific
antibodies (Roche, WO2009/080254), Bispecific IgG (Eli Lilly),
DuoBody' molecules (Genmab A/S, WO 2011/131746), DuetMab
(Medimmune, US2014/0348839), Biclonics (Merus, WO 2013/157953),
NovImmune (.kappa..lamda.Bodies, WO 2012/023053), Fc.DELTA.Adp
(Regeneron, WO 2010/151792), (DT)-Ig (GSK/Domantis), Two-in-one
Antibody or Dual Action Fabs (Genentech, Adimab), mAb2 (F-Star,
WO2008003116), Zybodies.TM. (Zyngenia), CovX-body (CovX/Pfizer),
FynomAbs (Covagen/Janssen Cilag), DutaMab (Dutalys/Roche), iMab
(Medimmune), Dual Variable Domain (DVD)-Ig.TM. (Abbott, U.S. Pat.
No. 7,612,18), dual domain double head antibodies (Unilever; Sanofi
Aventis, WO20100226923), Ts2Ab (Medimmune/AZ), BsAb (Zymogenetics),
HERCULES (Biogen Idec, U.S. Ser. No. 00/795,1918), scFv-fusions
(Genentech/Roche, Novartis, Immunomedics, Changzhou Adam Biotech
Inc, CN 102250246), TvAb (Roche, WO2012025525, WO2012025530),
ScFv/Fc Fusions, SCORPION (Emergent BioSolutions/Trubion,
Zymogenetics/BMS), Interceptor (Emergent), Dual Affinity
Retargeting Technology (Fc-DART.TM.) (MacroGenics, WO2008/157379,
WO2010/080538), BEAT (Glenmark), Di-Diabody (Imclone/Eli Lilly) and
chemically crosslinked mAbs (Karmanos Cancer Center), and
covalently fused mAbs (AIMM therapeutics).
[0069] The term "full-length antibody" when used herein, refers to
an antibody (e.g., a parent or variant antibody) which contains all
heavy and light chain constant and variable domains corresponding
to those that are normally found in a wild-type antibody of that
class or isotype.
[0070] The term "oligomer" as used herein, refers to a molecule
that consists of more than one but a limited number of monomer
units (e.g. antibodies) in contrast to a polymer that, at least in
principle, consists of an unlimited number of monomers. Exemplary
oligomers are dimers, trimers, tetramers, pentamers and hexamers.
Greek prefixes are often used to designate the number of monomer
units in the oligomer, for example a tetramer being composed of
four units and a hexamer of six units. Likewise, the term
"oligomerization", as used herein, is intended to refer to a
process that converts molecules to a finite degree of
polymerization. Herein, it is observed, that antibodies and/or
other dimeric proteins comprising target-binding regions according
to the invention can form oligomers, such as hexamers, via
non-covalent association of Fc-regions after target binding, e.g.,
at a cell surface.
[0071] The term "antigen-binding region", "antigen binding region",
"binding region" or antigen binding domain, as used herein, refers
to a region of an antibody which is capable of binding to the
antigen. This binding region is typically defined by the VH and VL
domains of the antibody which may be further subdivided into
regions of hypervariability (or hypervariable regions which may be
hypervariable in sequence and/or form of structurally defined
loops), also termed complementarity determining regions (CDRs),
interspersed with regions that are more conserved, termed framework
regions (FRs). The antigen can be any molecule, such as a
polypeptide, e.g. present on a cell, bacterium, or virion or in
solution. The terms "antigen" and "target" may, unless contradicted
by the context, be used interchangeably in the context of the
present invention.
[0072] The term "target", as used herein, refers to a molecule to
which the antigen binding region of the antibody binds. The target
includes any antigen towards which the raised antibody is directed.
The term "antigen" and "target" may in relation to an antibody be
used interchangeably and constitute the same meaning and purpose
with respect to any aspect or embodiment of the present
invention.
[0073] The term "epitope" means a protein determinant capable of
specific binding to an antibody. Epitopes usually consist of
surface groupings of building blocks such as amino acids, sugar
side chains or a combination thereof and usually have specific
three-dimensional structural characteristics, as well as specific
charge characteristics. Conformational and non-conformational
epitopes are distinguished in that the binding to the former but
not the latter is lost in the presence of denaturing solvents. The
epitope may comprise amino acid residues directly involved in the
binding and other amino acid residues, which are not directly
involved in the binding, such as amino acid residues which are
effectively blocked by the specifically antigen binding peptide (in
other words, the amino acid residue is within the footprint of the
specifically antigen binding peptide).
[0074] The term "binding" as used herein refers to the binding of
an antibody to a predetermined antigen or target, typically with a
binding affinity corresponding to a K.sub.D of about 10.sup.-6 M or
less, e.g. 10.sup.-7 M or less, such as about 10.sup.-8 M or less,
such as about 10.sup.-9 M or less, about 10.sup.-10 M or less, or
about 10.sup.-11 M or even less when determined by for instance
surface plasmon resonance (SPR) technology in a BIAcore 3000
instrument using the antigen as the ligand and the antibody as the
analyte or visa versa, and binds to the predetermined antigen with
an affinity corresponding to a K.sub.D that is at least ten-fold
lower, such as at least 100 fold lower, for instance at least 1,000
fold lower, such as at least 10,000 fold lower, for instance at
least 100,000 fold lower than its affinity for binding to a
non-specific antigen (e.g., BSA, casein) other than the
predetermined antigen or a closely-related antigen. The amount with
which the affinity is lower is dependent on the K.sub.D of the
antibody, so that when the K.sub.D of the antibody is very low
(that is, the antibody is highly specific), then the degree with
which the affinity for the antigen is lower than the affinity for a
non-specific antigen may be at least 10,000 fold. The term
"K.sub.D" (M), as used herein, refers to the dissociation
equilibrium constant of a particular antibody-antigen interaction,
and is obtained by dividing k.sub.d by k.sub.a.
[0075] The term "k.sub.d" (sec.sup.-1), as used herein, refers to
the dissociation rate constant of a particular antibody-antigen
interaction. Said value is also referred to as the k.sub.off value
or off-rate.
[0076] The term "k.sub.a" (M.sup.-1.times.sec.sup.-1), as used
herein, refers to the association rate constant of a particular
antibody-antigen interaction. Said value is also referred to as the
k.sub.on value or on-rate.
[0077] The term "K.sub.A" (M.sup.-1), as used herein, refers to the
association equilibrium constant of a particular antibody-antigen
interaction and is obtained by dividing k.sub.a by k.sub.d.
[0078] As used herein, the term "affinity" is the strength of
binding of one molecule, e.g. an antibody, to another, e.g. a
target or antigen, at a single site, such as the monovalent binding
of an individual antigen binding site of an antibody to an
antigen.
[0079] As used herein, the term "avidity" refers to the combined
strength of multiple binding sites between two structures, such as
between multiple antigen binding sites of antibodies simultaneously
interacting with a target. When more than one binding interactions
are present, the two structures will only dissociate when all
binding sites dissociate, and thus, the dissociation rate will be
slower than for the individual binding sites, and thereby providing
a greater effective total binding strength (avidity) compared to
the strength of binding of the individual binding sites
(affinity).
[0080] The term "hexamerization enhancing mutation", as used
herein, refers to a mutation of an amino acid position
corresponding to E430, E345 or S440 in human IgG1 according to EU
numbering. The hexamerization enhancing mutation strengthens Fc-Fc
interactions between neighbouring IgG antibodies that are bound to
a cell surface target, resulting in enhanced hexamer formation of
the target-bound antibodies, while the antibody molecules remain
monomeric in solution as described in WO2013/004842;
WO2014/108198.
[0081] The term "repulsing mutation" or "self-repulsing mutation"
or "hexamerization-inhibiting mutation", as used herein, refers to
a mutation of an amino acid position of human IgG1 that can result
in charge repulsion between amino acids at the Fc-Fc interface,
resulting in weakening of the Fc-Fc interaction between two
adjacent Fc region containing polypeptides, and thus inhibiting
hexamerization. Examples of such a repulsing mutation in human IgG1
are K439E and S440K. The repulsion in the Fc-Fc interaction between
two adjacent Fc region containing polypeptides at the position of a
repulsing mutation can be neutralized by introduction of a second
mutation (complementary mutation) in the amino acid position that
interacts with the position harboring the first mutation. This
second mutation can be present either in the same antibody or in a
second antibody. The combination of the first and second mutation
results in neutralization of the repulsion and restoration of the
Fc-Fc interactions and thus hexamerization. Examples of such first
and second mutations are K439E (repulsing mutation) and S440K
(neutralizing the repulsion by K439E), and vice versa S440K
(repulsing mutation) and K439E (neutralizing the repulsion by
S440K).
[0082] The term "complementary mutation", as used herein, refers to
a mutation of an amino acid position in a Fc region-containing
polypeptide that relates to a first mutation in an adjacent Fc
region containing polypeptide that preferably interacts with the Fc
region-containing polypeptide containing the complementary mutation
due to the combination of the two mutations in the two adjacent Fc
region-containing polypeptides. The complementary mutation and the
related first mutation can be present either in the same antibody
(intramolecular) or in a second antibody (intermolecular). An
example of intramolecular complementary mutations is the
combination K409R and F405L that mediates preferential
heterodimerization in a bispecific antibody according to WO
2011/131746. The combination of the K439E and S440K mutations that
results in neutralization of repulsion and restoration of Fc-Fc
interactions between two adjacent Fc region containing polypeptides
and thus hexamerization is an example of complementary mutations
that can be applied both inter- and intramolecularly.
[0083] The term "apoptosis", as used herein refers to the process
of programmed cell death (PCD) that may occur in a cell.
Biochemical events lead to characteristic cell changes (morphology)
and death. These changes include blebbing, cell shrinkage,
phosphatidylserine exposure, loss of mitochondrial function,
nuclear fragmentation, chromatin condensation, caspase activation,
and chromosomal DNA fragmentation.
[0084] The term "programmed cell-death" or "PCD", as used herein
refers to the death of a cell in any form mediated by an
intracellular signaling, e.g. apoptosis, autophagy or
necroptosis.
[0085] The term "Annexin V", as used herein, refers to a protein of
the annexin group that binds phosphatidylserine (PS) on the cell
surface.
[0086] The term "caspase activation", as used herein, refers to
cleavage of inactive pro-forms of effector caspases by initiator
caspases, leading to their conversion into effector caspases, which
in turn cleave protein substrates within the cell to trigger
apoptosis.
[0087] The term "caspase-dependent programmed cell death", as used
herein refers to any form of programmed cell death mediated by
caspases. In a particular embodiment, caspase-dependent programmed
cell death by one or more agonistic anti-DR5 antibodies can be
determined by comparing the viability of a cell culture in the
presence and absence of pan-caspase inhibitor
Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD-FMK). Pan-caspase
inhibitor Z-VAD-FMK (5 .mu.M end concentration) may be added to
adhered cells in 96-well flat bottom plates and incubated for one
hour at 37.degree. C. Next, antibody concentration dilution series
(e.g. starting from e.g. 20,000 ng/mL to 0.05 ng/mL final
concentration in 5-fold dilutions) may be added and incubated for 3
days at 37.degree. C. Cell viability can be quantified using
special kits for this purpose, such as the CellTiter-Glo
luminescent cell viability assay of Promega (Cat nr G7571).
[0088] The term "cell viability", as used herein refers to the
presence of metabolically active cells. In a particular embodiment,
cell viability after incubation with one or more agonistic
anti-Death Receptor antibodies can be determined by quantifying the
ATP present in the cells. Antibody concentration dilution series
(e.g. starting from e.g. 20,000 ng/mL to 0.05 ng/mL final
concentration in 5-fold dilutions) may be added to cells in 96-well
flat bottom plates, medium may be used as negative control and 5
.mu.M staurosporine may be used as positive control for the
induction of cell death. After 3 days incubation cell viability may
be quantified using special kits for this purpose, such as the
CellTiter-Glo luminescent cell viability assay of Promega (Cat nr
G7571). The percentage viable cells can be calculated using the
following formula: % viable cells=[(luminescence antibody
sample-luminescence staurosporine sample)/(luminescence no antibody
sample-luminescence staurosporine sample)]*100.
[0089] The term "Death Receptor", as used herein, refers to a
member of the tumor necrosis factor receptor superfamily (TNFR-SF)
comprising an intracellular death domain (DD).
[0090] An intracellular death domain, as used herein, refers to the
death domain in the intracellular part of the eight members of the
TNFRSF comprising a death domain. The death domains (DDs) are
well-known protein interaction modules that belong to the death
domain superfamily (Park Apoptosis. 2011 March; 16(3):209-20).
[0091] The term DR1, as used herein, refers to death receptor 1,
also known as "TNFR1", CD120a, p55 and tumor necrosis factor
receptor superfamily member 1A (TNFRSF1A), which is a single-pass
type I membrane protein with four extracellular cysteine-rich
domains (CRD's), a transmembrane domain (TM) and a cytoplasmic
domain containing a death domain (DD) (Schall et al., Cell. 1990
Apr. 20; 61(2):361-70). Natural ligands for TNFR1 are tumor
necrosis factor alpha (TNF-alpha) and lymphotoxin-alpha (LT-alpha).
In humans, the DR1 protein is encoded by a nucleic acid sequence
encoding the amino acid sequence UniprotKB/Swissprot P19438.
[0092] The term "DR2", as used herein, refers to death receptor 2,
also known as "FAS", CD95, APO-1 and tumor necrosis factor receptor
superfamily member 6 (TNFRSF6), which is a single-pass type I
membrane protein with three extracellular cysteine-rich domains
(CRD's), a transmembrane domain (TM) and a cytoplasmic domain
containing a death domain (DD) (Lichter et al., Genomics. 1992
September; 14(1):179-80; Inazawa et al., Genomics. 1992 November;
14(3):821-2). The natural ligand for FAS is FASL (CD95L). In
humans, the DR2 protein is encoded by a nucleic acid sequence
encoding the amino acid sequence UniprotKB/Swissprot P25445.
[0093] The term "DR3", as used herein, refers to death receptor 3,
also known as APO3, Apoptosis-inducing receptor (AIR), TRAMP,
Lymphocyte-associated receptor of death (LARD), APO-3 and tumor
necrosis factor receptor superfamily member 25 (TNFRSF25), which is
a single-pass type I membrane protein with four extracellular
cysteine-rich domains (CRDs), a transmembrane domain (TM) and a
cytoplasmic domain containing a death domain (DD) (Bodmer et al.,
Immunity. 1997 January; 6(1):79-88). The natural ligand for DR3 is
TWEAK. In humans, the DR3 protein is encoded by a nucleic acid
sequence encoding the amino acid sequence UniprotKB/Swissprot
Q93038.
[0094] The term "DR4", as used herein, refers to death receptor 4,
also known as CD261, TNF-related apoptosis-inducing ligand receptor
1 (TRAILR1), APO-2 and tumor necrosis factor receptor superfamily
member 10A (TNFRSF10A), which is a single-pass type I membrane
protein with three extracellular cysteine-rich domains (CRD's), a
transmembrane domain (TM) and a cytoplasmic domain containing a
death domain (DD) (Pan et al., Science. 1997 Apr. 4;
276(5309):111-3). The natural ligand for DR4 is TRAIL. In humans,
the DR4 protein is encoded by a nucleic acid sequence encoding the
amino acid sequence UniprotKB/Swissprot 000220.
[0095] The term "DR5", as used herein, refers to death receptor 5,
also known as CD262 and TNF-related apoptosis-inducing ligand
receptor 2 (TRAILR2) and tumor necrosis factor receptor superfamily
member 1013 TNFRSF10B, which is a single-pass type I membrane
protein with three extracellular cysteine-rich domains (CRD's), a
transmembrane domain (TM) and a cytoplasmic domain containing a
death domain (DD) (Walczak et al., EMBO J. 1997 Sep. 1;
16(17):5386-97). The natural ligand for DR5 is TRAIL. In humans,
the DR5 protein is encoded by a nucleic acid sequence encoding the
amino acid sequence UniprotKB/Swissprot 014763).
[0096] The term "DR6", as used herein, refers to death receptor 6,
also known as CD358 and tumor necrosis factor receptor superfamily
member 21 (TNFRSF21), which is a single-pass type I membrane
protein with four extracellular cysteine-rich domains (CRD's), a
transmembrane domain (TM) and a cytoplasmic domain containing a
death domain (DD) (Pan et al., FEBS Lett. 1998 Jul. 24;
431(3):351-6). DR6 is activated by overexpression. A natural ligand
for DR6 is alpha-amyloid precursor protein (APP). In humans, the
DR6 protein is encoded by a nucleic acid sequence encoding the
amino acid sequence UniprotKB/Swissprot 075509.
[0097] The term "EDAR", as used herein, refers to Ectodysplasin-A
receptor, also known as Ectodermal dysplasia receptor, EDA-A1
receptor, Downless homolog, anhidrotic ectodysplasin receptor 1 and
Tumor necrosis factor receptor superfamily member EDAR, which is a
single-pass type I membrane protein with three extracellular
cysteine-rich domains (CRD's), a transmembrane domain (TM) and a
cytoplasmic domain containing a death domain (DD) (Kumar et al., J
Biol Chem. 2001 Jan. 26; 276(4):2668-77). The natural ligand for
EDAR is ectodysplasin A. In humans, the EDAR protein is encoded by
a nucleic acid sequence encoding the amino acid sequence
UniprotKB/Swissprot Q9UNE0.
[0098] The term "NGFR", as used herein, refers to nerve growth
factor receptor, also known as low-affinity nerve growth factor
receptor (LNGFR), p75NTR, CD271 and tumor necrosis factor receptor
superfamily member 16 (TNFRSF16), which is a single-pass type I
membrane protein with four extracellular cysteine-rich domains
(CRD's), a serine/threonine-rich region, a transmembrane domain
(TM) and a cytoplasmic domain containing a death domain (DD)
(Johnson et al., Cell. 1986 Nov. 21; 47(4):545-54). The natural
ligand for NGFR is nerve growth factor (NGF) that binds the
serine/threonine-rich domain in NGFR. In humans, the NGFR protein
is encoded by a nucleic acid sequence encoding the amino acid
sequence UniprotKB/Swissprot P08138.
[0099] The term "antibody binding death receptor", "anti-death
receptor antibody" death receptor-binding antibody", "death
receptor-specific antibody", "death receptor antibody" which may be
used interchangeably herein, refers to any antibody binding an
epitope on the extracellular part of a death receptor such as FAS,
DR4, DR5, TNFR1, DR6, DR3, EDAR or NGFR."
[0100] The term "antibody binding FAS", "anti-FAS antibody"
FAS-binding antibody", "FAS-specific antibody", "FAS antibody"
which may be used interchangeably herein, refers to any antibody
binding an epitope on the extracellular FAS
[0101] The term "antibody binding DR4", "anti-DR4 antibody"
DR4-binding antibody", "DR4-specific antibody", "DR4antibody" which
may be used interchangeably herein, refers to any antibody binding
an epitope on the extracellular part of DR4.
[0102] The term "antibody binding DR5", "anti-DR5 antibody"
DR5-binding antibody", "DR5-specific antibody", "DR5 antibody"
which may be used interchangeably herein, refers to any antibody
binding an epitope on the extracellular part of DR5."
[0103] The term "antibody binding TNFR1", "anti-TNFR1 antibody"
TNFR1-binding antibody", "TNFR1-specific antibody", "TNFR1
antibody" which may be used interchangeably herein, refers to any
antibody binding an epitope on the extracellular part of
TNFR1."
[0104] The term "antibody binding DR6", "anti-DR6 antibody"
DR6-binding antibody", "DR6-specific antibody", "DR6 antibody"
which may be used interchangeably herein, refers to any antibody
binding an epitope on the extracellular part of DR6."
[0105] The term "antibody binding DR3", "anti-DR3 antibody"
DR3-binding antibody", "DR3-specific antibody", "DR3 antibody"
which may be used interchangeably herein, refers to any antibody
binding an epitope on the extracellular part of DR3."
[0106] The term "antibody binding EDAR", "anti-EDAR antibody"
EDAR-binding antibody", "EDAR-specific antibody", "EDAR antibody"
which may be used interchangeably herein, refers to any antibody
binding an epitope on the extracellular part of EDAR."
[0107] The term "antibody binding NGFR", "anti-NGFR antibody"
NGFR-binding antibody", "NGFR-specific antibody", "NGFR antibody"
which may be used interchangeably herein, refers to any antibody
binding an epitope on the extracellular part of NGFR."
[0108] The term "agonist" as used herein, refers to a molecule such
as an anti-Death Receptor antibody that triggers a response in a
cell when bound to a Death Receptor, wherein the response may
beactivation of the Death Receptor. That the anti-Death Receptor
antibody is agonistic is to be understood as that the antibody
stimulates, activates or clusters of the Death Receptor as a result
of the anti-Death Receptor binding to said Death Receptor. That is
an agonistic anti-Death Receptor antibody of the present invention
bound to a Death Receptor results in Death Receptor stimulation,
clustering or activation of downstream intracellular signaling
pathways as the natural ligand bound to the Death Receptor.
[0109] A "variant" or "antibody variant" of the present invention
is an antibody molecule which comprises one or more mutations as
compared to a "parent" antibody. Exemplary parent antibody formats
include, without limitation, a wild-type antibody, a full-length
antibody or Fc-containing antibody fragment, a bispecific antibody,
a human antibody, humanized antibody, chimeric antibody or any
combination thereof.
[0110] Exemplary mutations include amino acid deletions,
insertions, and substitutions of amino acids in the parent amino
acid sequence. Amino acid substitutions may exchange a native amino
acid for another naturally-occurring amino acid, or for a
non-naturally-occurring amino acid derivative. The amino acid
substitution may be conservative or non-conservative. In the
context of the present invention, conservative substitutions may be
defined by substitutions within the classes of amino acids
reflected in one or more of the following three tables:
Amino Acid Residue Classes for Conservative Substitutions
TABLE-US-00001 [0111] Acidic Residues Asp (D) and Glu (E) Basic
Residues Lys (K), Arg (R), and His (H) Hydrophilic Uncharged
Residues Ser (S), Thr (T), Asn (N), and Gln (Q) Aliphatic Uncharged
Residues Gly (G), Ala (A), Val (V), Leu (L), and Ile (I) Non-polar
Uncharged Residues Cys (C), Met (M), and Pro (P) Aromatic Residues
Phe (F), Tyr (Y), and Trp (W)
Alternative Conservative Amino Acid Residue Substitution
Classes
TABLE-US-00002 [0112] 1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y W
Alternative Physical and Functional Classifications of Amino Acid
Residues
TABLE-US-00003 [0113] Alcohol group-containing residues S and T
Aliphatic residues I, L, V, and M Cycloalkenyl-associated residues
F, H, W, and Y Hydrophobic residues A, C, F, G, H, I, L, M, R, T,
V, W, and Y Negatively charged residues D and E Polar residues C,
D, E, H, K, N, Q, R, S, and T Positively charged residues H, K, and
R Small residues A, C, D, G, N, P, S, T, and V Very small residues
A, G, and S Residues involved in turn formation A, C, D, E, G, H,
K, N, Q, R, S, P, and T Flexible residues Q, T, K, S, G, D, E, and
R
[0114] In the context of the present invention, a substitution in a
variant is indicated as: [0115] Original amino
acid-position-substituted amino acid;
[0116] The three letter code, or one letter code, are used,
including the codes Xaa and X to indicate amino acid residue.
Accordingly, the notation "E345R" or "Glu345Arg" means, that the
variant comprises a substitution of Glutamic acid with Arginine in
the variant amino acid position corresponding to the amino acid in
position 345 in the parent antibody.
[0117] Where a position as such is not present in an antibody, but
the variant comprises an insertion of an amino acid, for example:
Position-substituted amino acid; the notation, e.g., "448E" is
used. Such notation is particular relevant in connection with
modification(s) in a series of homologous polypeptides or
antibodies. Similarly when the identity of the substitution amino
acid residues(s) is immaterial: Original amino acid-position; or
"E345". For a modification where the original amino acid(s) and/or
substituted amino acid(s) may comprise more than one, but not all
amino acid(s), the substitution of Glutamic acid for Arginine,
Lysine or Tryptophan in position 345: "Glu345Arg,Lys,Trp" or
"E345R,K,W" or "E345R/K/W" or "E345 to R, K or W" may be used
interchangeably in the context of the invention. Furthermore, the
term "a substitution" embraces a substitution into any one of the
other nineteen natural amino acids, or into other amino acids, such
as non-natural amino acids. For example, a substitution of amino
acid E in position 345 includes each of the following
substitutions: 345A, 345C, 345D, 345G, 345H, 345F, 345I, 345K,
345L, 345M, 345N, 345Q, 345R, 345S, 345T, 345V, 345W, and 345Y.
This is, by the way, equivalent to the designation 345X, wherein
the X designates any amino acid. These substitutions can also be
designated E345A, E345C, etc, or E345A,C,ect, or E345A/C/ect. The
same applies to analogy to each and every position mentioned
herein, to specifically include herein any one of such
substitutions.
[0118] For the purposes of the present invention, the sequence
identity between two amino acid sequences is determined using the
Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol.
Biol. 48: 443-453) as implemented in the Needle program of the
EMBOSS package (EMBOSS: The European Molecular Biology Open
Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277),
preferably version 5.0.0 or later. The parameters used are gap open
penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62
(EMBOSS version of BLOSUM62) substitution matrix. The output of
Needle labeled "longest identity" (obtained using the -nobrief
option) is used as the percent identity and is calculated as
follows:
(Identical Residues.times.100)/(Length of Alignment-Total Number of
Gaps in Alignment).
[0119] For the purposes of the present invention, the sequence
identity between two deoxyribonucleotide sequences is determined
using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970,
supra) as implemented in the Needle program of the EMBOSS package
(EMBOSS: The European Molecular Biology Open Software Suite, Rice
et al., 2000, supra), preferably version 5.0.0 or later. The
parameters used are gap open penalty of 10, gap extension penalty
of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4)
substitution matrix. The output of Needle labeled "longest
identity" (obtained using the -nobrief option) is used as the
percent identity and is calculated as follows:
(Identical Deoxyribonucleotides.times.100)/(Length of
Alignment-Total Number of Gaps in Alignment).
[0120] The sequence of CDR variants may differ from the sequence of
the CDR of the parent antibody sequences through mostly
conservative physical or functional amino acids substitutions at
most 5 mutations or substitutions selected from conservative,
physical or functional amino acids in total across the six CDR
sequences of the antibody binding region, such as at most 4
mutations or substitutions selected from conservative, physical or
functional amino acids, such as at most 3 mutations or
substitutions selected from conservative, physical or functional
amino acids, such as at most 2 mutations selected from
conservative, physical or functional amino acids or substitutions,
such as at most 1 mutation or substitution selected from a
conservative, physical or functional amino acid, in total across
the six CDR sequences of the antibody binding region. The
conservative, physical or functional amino acids are selected from
the 20 natural amino acids found i.e, Arg (R), His (H), Lys (K),
Asp (D), Glu (E), Ser (S), Thr (T), Asn (N), Gln (Q), Cys (C), Gly
(G), Pro (P), Ala (A), Ile (I), Leu (L), Met (M), Phe (F), Trp (W),
Tyr (Y) and Val (V).
[0121] The sequence of CDR variants may differ from the sequence of
the CDR of the parent antibody sequences through mostly
conservative, physical or functional amino acids substitutions; for
instance at least about 75%, about 80% or more, about 85% or more,
about 90% or more, (e.g., about 75-95%, such as about 92%, 93% or
94%) of the substitutions in the variant are mutations or
substitutions selected from conservative, physical or functional
amino acids residue replacements.
[0122] The conservative, physical or functional amino acids are
selected from the 20 natural amino acids found i.e, Arg (R), His
(H), Lys (K), Asp (D), Glu (E), Ser (S), Thr (T), Asn (N), Gln (Q),
Cys (C), Gly (G), Pro (P), Ala (A), Ile (I), Leu (L), Met (M), Phe
(F), Trp (W), Tyr (Y) and Val (V).
[0123] An amino acid or segment in one sequence that "corresponds
to" an amino acid or segment in another sequence is one that aligns
with the other amino acid or segment using a standard sequence
alignment program such as ALIGN, ClustalW or similar, typically at
default settings. Hence a standard sequence alignment program can
be used to identify which amino acid in an e.g. immunoglobulin
sequence corresponds to a specific amino acid in e.g. human IgG1.
Further a standard sequence alignment program can be used to
identify sequence identity e.g. a sequence identity to SEQ ID NO:1
of at least 80%, or 85%, 90%, or at least 95%. For example, the
sequence alignments shown in FIG. 1 can be used to identify any
amino acid in the Fc region of one IgG1 allotype that corresponds
to a particular amino acid in another allotype of an IgG1 Fc
sequence.
[0124] The term "vector," as used herein, refers to a nucleic acid
molecule capable of inducing transcription of a nucleic acid
segment ligated into the vector. One type of vector is a "plasmid",
which is in the form of a circular double stranded DNA loop.
Another type of vector is a viral vector, wherein the nucleic acid
segment may be ligated into the viral genome. Certain vectors are
capable of autonomous replication in a host cell into which they
are introduced (for instance bacterial vectors having a bacterial
origin of replication and episomal mammalian vectors). Other
vectors (such as non-episomal mammalian vectors) may be integrated
into the genome of a host cell upon introduction into the host
cell, and thereby are replicated along with the host genome.
Moreover, certain vectors are capable of directing the expression
of genes to which they are operatively linked. Such vectors are
referred to herein as "recombinant expression vectors" (or simply,
"expression vectors"). In general, expression vectors of utility in
recombinant DNA techniques are often in the form of plasmids. In
the present specification, "plasmid" and "vector" may be used
interchangeably as the plasmid is the most commonly used form of
vector. However, the present invention is intended to include such
other forms of expression vectors, such as viral vectors (such as
replication defective retroviruses, adenoviruses and
adeno-associated viruses), which serve equivalent functions.
[0125] The term "recombinant host cell" (or simply "host cell"), as
used herein, is intended to refer to a cell into which an
expression vector has been introduced. It should be understood that
such terms are intended to refer not only to the particular subject
cell, but also to the progeny of such a cell. Because certain
modifications may occur in succeeding generations due to either
mutation or environmental influences, such progeny may not, in
fact, be identical to the parent cell, but are still included
within the scope of the term "host cell" as used herein.
Recombinant host cells include, for example, transfectomas, such as
CHO-S cells, HEK-293F cells, Expi293F cells, PER.C6, NS0 cells, and
lymphocytic cells, and prokaryotic cells such as E. coli and other
eukaryotic hosts such as plant cells and fungi, as well as
prokaryotic cells such as E. coli.
Specific Embodiments of the Invention
[0126] The present invention is based, at least in part, on the
discovery that the ability of antibodies targeting members of the
TNFR-SF comprising an intracellular death domain, such as an
anti-FAS, anti-DR4, anti-DR5, anti-TNFR1, anti-DR6, anti-DR3,
anti-EDAR or anti-NGFR antibody to induce cell death in a target
cell expressing FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR, or NGFR can
be greatly enhanced by introducing a mutation at an amino acid in
the Fc region corresponding to amino acid position E430, E345 or
S440 in human IgG1 according to EU numbering. The invention is
further based on the surprising finding that a combination of two
antibodies binding to a first and a second epitope on FAS, DR4,
DR5, TNFR1, DR6, DR3, EDAR, or NGFR and each comprising a mutation
in the Fc region show superior induction of cell death in a target
cell compared to a combination of the two antibodies without the
mutation in the Fc region.
[0127] In one aspect the present invention relates to an antibody
comprising an Fc region of a human immunoglobulin IgG and an
antigen binding region binding to FAS, DR4, DR5, TNFR1, DR6, DR3,
EDAR, or NGFR, wherein the Fc region comprises a mutation at an
amino acid position corresponding to E430, E345 or S440 in human
IgG1 according to EU numbering.
[0128] The positions corresponding to E430, E345 and S440 in human
IgG1 according to EU numbering are located in the CH3 domain of the
Fc region.
[0129] By introducing specific mutations in the Fc domain
corresponding to at least one of the following positions E430, E345
and S440 in human IgG1 oligomerization such as hexamerization upon
target binding on the cell surface is enhanced, while the antibody
molecules remain monomeric in solution (WO2013/004842;
WO2014/108198). The hexamerization enhancing mutation strengthens
Fc-Fc interactions between neighbouring IgG antibodies that are
bound to a cell surface target, resulting in enhanced hexamer
formation of the target-bound antibodies.
[0130] In one embodiment of the present invention the Fc region of
the anti-FAS, anti-DR4, anti-DR5, anti-TNFR1, anti-DR6, anti-DR3,
anti-EDAR, and anti-NGFR antibody comprises a mutation
corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R,
E345Y, S440Y or S440W in human IgG1, EU numbering. Hereby are
embodiments provided that allow for enhanced hexamerization of
antibodies upon target binding on a cell surface.
[0131] In one embodiment of the present invention the anti-FAS,
anti-DR4, anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR, and
anti-NGFR antibody comprises a mutation at an amino acid position
corresponding to E430 in human IgG1 according to EU numbering,
wherein the mutation is selected from the group consisting of:
E430G, E430S, E430F and E430T.
[0132] In one embodiment of the present invention the anti-FAS,
anti-DR4, anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR, and
anti-NGFR antibody comprises an E430G mutation in the Fc
region.
[0133] In a preferred embodiment of the present invention the Fc
region comprises a mutation corresponding to E430G or E345K in
human IgG1 EU numbering.
[0134] In one embodiment of the present invention the anti-FAS,
anti-DR4, anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR, and
anti-NGFR antibody comprises a mutation at an amino acid position
corresponding to E345 in human IgG1 according to EU numbering,
wherein the mutation is selected from the group consisting of:
E345K, E345Q, E345R and E345Y. In one embodiment of the present
invention the anti-FAS, anti-DR4, anti-DR5, anti-TNFR1, anti-DR6,
anti-DR3, anti-EDAR, and anti-NGFR antibody comprises an E345K
mutation in the Fc region.
[0135] In one embodiment of the present invention the Fc region
comprises a mutation at an amino acid position corresponding to
position S440 in human IgG1, EU numbering where the mutation is
S440Y or S440W.
[0136] In one embodiment of the present invention the anti-FAS,
anti-DR4, anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR, and
anti-NGFR antibody comprises a mutation at an amino acid position
corresponding to S440 in human IgG1 according to EU numbering,
wherein the mutation is selected from the group consisting of:
S440Y and S440W.
[0137] In one embodiment of the present invention the Fc region
comprises a mutation corresponding to S440Y. In one embodiment of
the present invention the Fc region comprises a mutation
corresponding to E430G. In one embodiment of the present invention
the Fc region comprises a mutation corresponding to E345K.
[0138] In one embodiment of the present invention the Fc region
comprises at least a first and a second mutation at an amino acid
position corresponding to E430 and E345 in human IgG1, EU
numbering.
[0139] In one embodiment of the present invention the Fc region
further comprises a third mutation at an amino acid position
selected form the group consisting of: Y436 and S440. Hereby
embodiments are provided comprising a first, second and third
mutations which allows for enhanced Fc-Fc interactions in
solution.
[0140] In one embodiment of the invention the antibody comprises a
first, second and third mutation at an amino acid position
corresponding to E430, E345 and S440 in human IgG1, EU
numbering.
[0141] In one embodiment of the invention the antibody comprises an
Fc region wherein the first, second and third mutation at amino
acid positions corresponding to E430, E345 and S440 in human IgG1,
EU numbering, wherein the mutations are E430G, E345R, S440Y.
[0142] In one embodiment of the invention the antibody comprises a
first, second and third mutation at an amino acid position
corresponding to E430, E345 and Y436 in human IgG1, EU
numbering.
[0143] In one embodiment of the invention the antibody comprises an
Fc region wherein the first, second and third mutation at amino
acid positions corresponding to E430, E345 and Y436 in human IgG1,
EU numbering, wherein the mutations are E430G, E345R, Y436I.
[0144] In one embodiment of the present invention the Fc region
comprises a mutation at an amino acid position corresponding to
E430 and/or E345 and wherein the said Fc region comprises a further
mutation at an amino acid position corresponding to S440, with the
proviso that the mutation is not S440Y or S440W.
[0145] In one embodiment of the present invention the antibody
comprises a further mutation at an amino acid position
corresponding to one of the following positions S440 or K439 in
human IgG1, EU numbering. In one embodiment of the invention the Fc
region comprises a further mutation in a position corresponding to
S440 or K439, with the proviso that the further mutation is not in
position S440 if the hexamerization enhancing mutation is in S440.
In one embodiment of the present invention the further mutation at
an amino acid position corresponding to one of the following
positions S440 or K439 may be a hexamerization-inhibiting
mutation.
[0146] In one embodiment the Fc region comprises a further mutation
at an amino acid position corresponding to K439 in human IgG1, EU
numbering, wherein the further mutation is selected from the group
consisting of: K439E and K439D. In on embodiment the further
mutation is K439E.
[0147] In on embodiment the Fc region comprises a further mutation
at an amino acid position corresponding to S440 in human IgG1, EU
numbering, wherein the further mutation is selected from the group
consisting of: S440K, S440R and S440H. In on embodiment the further
mutation is S440K.
[0148] In one embodiment of the present invention the Fc region
comprises a further hexamerization-inhibiting mutation such as
K439E or S440K in human IgG1, EU numbering. The
hexamerization-inhibiting mutation such as K439E or S440K prevents
Fc-Fc interaction with antibodies comprising the same
hexamerization inhibiting mutation, but by combining antibodies
with a K439E mutation and antibodies with a S440K mutation the
inhibiting effect is neutralized and Fc-Fc interactions is
restored.
[0149] Antibodies comprising a mutation in a position corresponding
to E430, E345 or S440 according to the present invention and a
further mutation at an amino acid position corresponding to K439
such as a K439E mutation do not form oligomers with antibodies
comprising a further mutation at an amino acid position
corresponding to K439 such as a K439E mutation. However, antibodies
comprising hexamerization enhancing mutation in E430, E345 or S440
and a further mutation in K439 such a K439E do form oligomers with
antibodies comprising a hexamerization enhancing mutation in E430
or E345 and a further mutation in S440 such as S440K. Antibodies
comprising a mutation in a position corresponding to E430 or E345
according to the present invention and a further mutation at an
amino acid position corresponding to S440 such as a S440K mutation
do not form oligomers with antibodies comprising a further mutation
at an amino acid position corresponding to S440 such as a S440K
mutation. However, antibodies comprising hexamerization enhancing
mutation in E430 or E345 and a further mutation in S440 such a
S440K do form oligomers with antibodies comprising a hexamerization
enhancing mutation in E430 or E345 and a further mutation in K439
such as K439. In one embodiment of the present invention the Fc
region comprises a hexamerization enhancing mutation such as E430G
and a hexamerization inhibiting mutation such as K439E. In one
embodiment of the present invention the Fc region comprises a
hexamerization enhancing mutation such as E345K and a
hexamerization inhibiting mutation such as K439E. In another
embodiment of the present invention the Fc region comprises a
hexamerization enhancing mutation such as E430G and a
hexamerization inhibiting mutation such as S440K. In one embodiment
of the present invention the Fc region comprises a hexamerization
enhancing mutation such as E345K and a hexamerization inhibiting
mutation such as S440K. In one embodiment of the present invention
the Fc region comprises a hexamerization enhancing mutation such as
S440Y and a hexamerization inhibiting mutation such as K439E Hereby
embodiments are provided that allow for exclusive hexamerization
between combinations of antibodies comprising a K439E mutation and
antibodies comprising a S440K mutation.
[0150] In on embodiment the Fc region comprises a further mutation,
wherein the further mutation is selected from the group consisting
of: K439E and K439D. In on embodiment the further mutation is
K439E.
[0151] In one embodiment the Fc region comprises a further
mutation, wherein the further mutation is selected from the group
consisting of: S440K, S440R and S440H. In on embodiment the further
mutation is S440K.
[0152] The human FAS molecule is comprised of 335 amino acids in
including the signaling peptide at the first 1-25 positions,
followed by the extracellular domain at positions 26-173, a
transmembrane domain at positions 174-190 and a cytoplasmic domain
at positions 191-335. The extracellular domain is comprised of a
148 amino acid sequence.
[0153] In one embodiment the member of the death receptor
comprising an intracellular death domain is FAS.
[0154] In one embodiment of the invention the anti-FAS antibody
comprises an antigen binding region binding to an epitope within
the extracellular domain of FAS.
[0155] In one embodiment of the present invention the anti-FAS
antibody comprise an antigen binding region comprising a variable
heavy chain (VH) region and a variable light chain (VL) region
comprising the amino acid sequence of: (VH) SEQ ID NO 15: and (VL)
SEQ ID NO:16.
[0156] In one embodiment of the invention the anti-FAS antibody
comprises an Fc region comprising a mutation corresponding to E430G
or E345K in human IgG1 EU numbering.
[0157] In one embodiment of the invention the anti-FAS antibody
comprise a first, second and third mutation at an amino acid
position corresponding to E430, E345 and S440 in human IgG1, EU
numbering.
[0158] In one embodiment of the invention the anti-FAS antibody
comprises an Fc region wherein the first, second and third mutation
at amino acid positions corresponding to E430, E345 and S440 in
human IgG1, EU numbering, wherein the mutations are E430G, E345R,
S440Y.
[0159] In one embodiment of the invention the anti-FAS antibody
comprises an Fc region wherein the first, second and third mutation
at amino acid positions corresponding to E430, E345 and S440 in
human IgG1, EU numbering, wherein the mutations are first, second a
third E430G, E345R, S440Y and a further S440K mutation.
[0160] In one embodiment of the invention the anti-FAS antibody
comprises a first, second and third mutation at an amino acid
position corresponding to E430, E345 and Y436 in human IgG1, EU
numbering.
[0161] In one embodiment of the invention the anti-FAS antibody
comprises an Fc region wherein the first, second and third mutation
at amino acid positions corresponding to E430, E345 and Y436 in
human IgG1, EU numbering, wherein the mutations are E430G, E345R,
Y436I.
[0162] In one embodiment of the invention the anti-FAS antibody
comprises an Fc region wherein the first, second and third mutation
at amino acid positions corresponding to E430, E345 and Y436 in
human IgG1, EU numbering, wherein the first, second a third
mutations are E430G, E345R, Y436I and a further S440K mutation.
[0163] The human TNFR1 molecule is comprise of 455 amino acids in
including the signaling peptide at the first 1-21 positions,
followed by the extracellular domain at positions 22-211, a
transmembrane domain at positions 212-234 and a cytoplasmic domain
at positions 235-455. The extracellular domain is comprised of a
190 amino acid sequence.
[0164] In one embodiment of the invention the anti-TNFR1 antibody
comprises an antigen binding region binding to an epitope within
the extracellular domain of TNFR1.
[0165] In one embodiment of the invention the anti-TNFR1 antibody
comprises a Fc region comprising a mutation corresponding to E430G
or E345K in human IgG1 EU numbering.
[0166] The human EDAR molecule is comprise of 448 amino acids in
including the signaling peptide at the first 1-26 positions,
followed by the extracellular domain at positions 27-187, a
transmembrane domain at positions 188-208 and a cytoplasmic domain
at positions 209-448. The extracellular domain is comprised of a
161 amino acid sequence.
[0167] In one embodiment of the invention the anti-EDAR antibody
comprises an antigen binding region binding to an epitope within
the extracellular domain of EDAR.
[0168] In one embodiment of the invention the anti-EDAR antibody
comprises a Fc region comprising a mutation corresponding to E430G
or E345K in human IgG1 EU numbering.
[0169] The human NGFR molecule is comprise of 427 amino acids in
including the signaling peptide at the first 1-28 positions,
followed by the extracellular domain at positions 29-250, a
transmembrane domain at positions 251-272 and a cytoplasmic domain
at positions 273-427. The extracellular domain is comprised of a
222 amino acid sequence.
[0170] In one embodiment of the invention the anti-NGFR antibody
comprises an antigen binding region binding to an epitope within
the extracellular domain of NGFR.
[0171] In one embodiment of the invention the anti-NGFR antibody
comprises a Fc region comprising a mutation corresponding to E430G
or E345K in human IgG1 EU numbering.
[0172] The human DR3 molecule is comprise of 417 amino acids
including the signaling peptide at the first 1-24 positions,
followed by the extracellular domain at positions 25-199, a
transmembrane domain at positions 200-220 and a cytoplasmic domain
at positions 221-417. The extracellular domain is comprised of a
175 amino acid sequence.
[0173] In one embodiment of the invention the anti-DR3 antibody
comprises an antigen binding region binding to an epitope within
the extracellular domain of DR3.
[0174] In one embodiment of the invention the anti-DR3 antibody
comprises a Fc region comprising a mutation corresponding to E430G
or E345K in human IgG1 EU numbering.
[0175] The human DR4 molecule is comprise of 468 amino acids
including the signaling peptide at the first 1-23 positions,
followed by the extracellular domain at positions 24-239, a
transmembrane domain at positions 240-262 and a cytoplasmic domain
at positions 263-468. The extracellular domain is comprised of a
216 amino acid sequence.
[0176] In one embodiment of the invention the anti-DR4 antibody
comprises an antigen binding region binding to an epitope within
the extracellular domain of DR4.
[0177] In one embodiment of the invention the member of the death
receptor comprising an intracellular death domain is DR4.
[0178] In one embodiment of the present invention the anti-DR4
antibody comprise an antigen binding region comprising a variable
heavy chain (VH) region and a variable light chain (VL) region
comprising the amino acid sequence of: (VH) SEQ ID NO 13: and (VL)
SEQ ID NO:14.
[0179] In one embodiment of the invention the anti-DR4 antibody
comprises a mutation at an amino acid position corresponding to
E430 in human IgG1, EU numbering, wherein the mutation is selected
from the group consisting of: E430G, E430S, E40F and E430T. In one
embodiment of the invention the anti-DR4 antibody comprises an
E430G mutation.
[0180] In one embodiment of the invention the anti-DR4 antibody
comprises a mutation at an amino acid position corresponding to
E345 in human IgG1, EU numbering, wherein the mutation is selected
from the group consisting of: E345K E345Q, E345R and E345Y. In one
embodiment of the invention the anti-DR4 antibody comprises an
E345K mutation.
[0181] In one embodiment of the invention the anti-DR4 antibody
comprises an Fc region comprising a mutation corresponding to E430G
or E345K in human IgG1 EU numbering.
[0182] The human DR5 molecule is comprised of 440 amino acids
including a signaling peptide at the first 1-55 positions, followed
by the extracellular domain at positions 56-210, a transmembrane
domain at positions 211-231 and a cytoplasmic domain at positions
232-440. The extracellular domain is comprised of a 155 amino acid
sequence. The isoform short of DR5 is missing 185-213 from the
extracellular domain.
[0183] In one embodiment of the invention the anti-DR5 antibody
comprises an antigen binding region binding to an epitope within
the extracellular domain of DR5.
[0184] In one embodiment of the present invention the anti-DR5
antibody comprise an antigen binding region comprising a variable
heavy chain (VH) region and a variable light chain (VL) region
comprising the amino acid sequence from the group consisting
of:
a) (VH) SEQ ID NO 19: and (VL) SEQ ID NO:23,
b) (VH) SEQ ID NO 26: and (VL) SEQ ID NO:23,
c) (VH) SEQ ID NO 31: and (VL) SEQ ID NO:35 and
d) (VH) SEQ ID NO 40: and (VL) SEQ ID NO:43.
[0185] In one embodiment of the invention the anti-DR5 antibody
comprises a mutation at an amino acid position corresponding to
E430 in human IgG1, EU numbering, wherein the mutation is selected
from the group consisting of: E430G, E430S, E40F and E430T. In one
embodiment of the invention the anti-DR5 antibody comprises an
E430G mutation.
[0186] In one embodiment of the invention the anti-DR5 antibody
comprises a mutation at an amino acid position corresponding to
E345 in human IgG1, EU numbering, wherein the mutation is selected
from the group consisting of: E345K E345Q, E345R and E345Y. In one
embodiment of the invention the anti-DR5 antibody comprises an
E345K mutation.
[0187] In one embodiment of the invention the anti-DR5 antibody
comprises a Fc region comprising a mutation corresponding to E430G
or E345K in human IgG1 EU numbering.
[0188] In one embodiment of the invention the antibody comprises an
antigen binding region binding to the same binding site as TRAIL or
a binding site overlapping with the binding site of TRAIL. The
TRAIL binding motif is located in CRD2 and CRD3 based on a Crystal
structure of TRAIL in complex with the DR5 ectodomain (Hymowitz et
al., Mol Cell. 1999 October; 4(4):563-71) That is, in one
embodiment of the invention the antibody comprises an antigen
binding region binding to the same binding region on DR5 as TRAIL.
In one embodiment of the invention the antibody comprises an
antigen binding region that competes with TRAIL binding to DR5. In
one embodiment of the invention the antibody blocks TRAIL induced
mediated killing such as TRAIL induced apoptosis.
[0189] In another embodiment of the invention the antibody
comprises an antigen binding region binding to an epitope on DR5
that is different from the binding site of TRAIL. In one embodiment
of the invention the antibody comprises an antigen binding region
binding to a different binding region on DR5 than TRAIL. In one
embodiment of the invention the antibody does not block TRAIL
induced mediated killing such as TRAIL induced apoptosis.
[0190] The human DR6 molecule is comprise of 655 amino acids in
including the signaling peptide at the first 1-41 positions,
followed by the extracellular domain at positions 42-349, a
transmembrane domain at positions 350-370 and a cytoplasmic domain
at positions 371-655. The extracellular domain is comprised of a
308 amino acid sequence.
[0191] In one embodiment of the invention the anti-DR6 antibody
comprises an antigen binding region binding to an epitope within
the extracellular domain of DR6.
[0192] In one embodiment of the invention the anti-DR6 antibody
comprises a Fc region comprising a mutation corresponding to E430G
or E345K in human IgG1 EU numbering.
[0193] In one embodiment of the present invention the antibody is a
monoclonal antibody. In one embodiment of the present invention the
antibody is of the IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgD or IgM
isotype.
[0194] In a preferred embodiment of the invention the antibody is
an IgG1 antibody.
[0195] In one embodiment of the present invention the antibody is
an IgG1m(f), IgG1m(z), IgG1m(a) or an IgG1m(x) allotype, or any
allotype combination, such as IgG1m(z,a), IgG1m(z,a,x),
IgG1m(f,a).
[0196] In one embodiment the antibody is a human antibody, a
chimeric antibody or a humanized antibody.
[0197] In one embodiment of the present invention the anti-Death
Receptor antibody selected from the group consisting of: anti-FAS,
anti-DR4, anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR and
anti-NGFR is agonistic. That the antibody is agonistic is to be
understood as that the antibody clusters, stimulates or activates
the Death Receptor to which it bind at least as good as the effect
found by interaction between the Death Receptor and the natural
ligand binding to the Death Receptor, or by overexpression of the
Death Receptor. An agonistic anti-FAS antibody of the present
invention bound to FAS activates the same intracellular pathways as
FAS-Ligand bound to FAS. An agonistic anti-FAS antibody of the
present invention is able to induce apoptosis in a cell expressing
FAS.
[0198] An agonistic anti-DR4 antibody of the present invention
bound to DR4 activates the same intracellular pathways as TRAIL
bound to DR4.
[0199] An agonistic anti-DR5 antibody of the present invention
bound to DR5 activates the same intracellular pathways as TRAIL
bound to DR5.
[0200] An agonistic anti-TNFR1 antibody of the present invention
bound to TNFR1 activates the same intracellular pathways as
LT.alpha. or TNF bound to TNFR1.
[0201] An agonistic anti-DR6 antibody of the present invention
bound to DR6 activates the same intracellular pathways as DR6
overexpression or APP bound to DR6.
[0202] An agonistic anti-DR3 antibody of the present invention
bound to DR3 activates the same intracellular pathways as TWEAK
bound to DR3.
[0203] An agonistic anti-EDAR antibody of the present invention
bound to EDAR activates the same intracellular pathways as
ectodysplasin A bound to EDAR.
[0204] An agonistic anti-NGFR antibody of the present invention
bound to NGFR activates the same intracellular pathways as NGF
bound to NGFR.
[0205] In one embodiment of the present invention the anti-Death
Receptor antibody, such as anti-FAS, anti-DR4, anti-DR5,
anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR or anti-NGFR antibody has
enhanced agonistic activity. That the anti-FAS, anti-DR4, anti-DR5,
anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR or anti-NGFR antibody has
enhanced agonistic activity is to be understood as the antibody is
able to cluster the FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR, or NGFR
receptor or activate the same intracellular pathways as the natural
ligand bound to FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR, or NGFR, but
at an enhanced level. That is an anti-FAS, anti-DR4, anti-DR5,
anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR or anti-NGFR antibody of
the invention, i.e having a mutation in the Fc region according to
the invention, with enhanced agonistic activity is able to induce
increased level of apoptosis or programmed cell death in a cell or
tissue expressing the FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR, or NGFR
receptor compared to the natural ligand or the same antibody
without said mutation binding to the receptor.
[0206] Thus it is to be understood in the context of the present
invention that the enhanced agonistic activity of an antibody
according to the invention i.e. comprising an amino acid mutation
at a position corresponding to E430, E345 or S440 in human IgG1, EU
numbering, may be evaluated by comparing the antibody according to
the invention with the same antibody without said mutation. In the
context of the present invention the same antibody is to be
understood as an antibody having the identical amino acid sequence
as the antibody according to the invention, but without said
mutation.
[0207] In one embodiment of the present invention the anti-Death
Receptor receptor antibody, such as anti-FAS, anti-DR4, anti-DR5,
anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR or anti-NGFR antibody
induces programmed cell death in a target cell. In one embodiment
of the present invention the anti-DR5 antibody induces
caspase-dependent cell death. Caspase-dependent cell death may be
induced by activation of caspase-3 and/or caspase-7. In one
embodiment of the present invention the antibody induces
apoptosis.
[0208] In one embodiment of the present invention the anti-Death
Receptor antibody, such as anti-FAS, anti-DR4, anti-DR5,
anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR or anti-NGFR antibody
induces phosphatidylserine (PS) exposure, which can be measured by
Annexin-V binding. Therefore, Annexin-V binding correlates to
programmed cell death and can be used to measure the anti-Death
Receptor antibody, such as anti-FAS, anti-DR4, anti-DR5,
anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR or anti-NGFR antibody's
ability to induce cellular events leading to programmed cell
death.
[0209] In a preferred embodiment of the present invention the
anti-Death Receptor antibody, such as anti-FAS, anti-DR4, anti-DR5,
anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR or anti-NGFR antibody
induces apoptosis in a target cell expressing the Death Receptor
such as FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR, or NGFR, such as a
tumor cell.
[0210] In one embodiment of the invention the anti-Death Receptor
antibody, such as anti-FAS, anti-DR4, anti-DR5, anti-TNFR1,
anti-DR6, anti-DR3, anti-EDAR or anti-NGFR antibody reduces cell
viability.
[0211] In one embodiment the present invention the anti-Death
Receptor antibody, such as anti-FAS, anti-DR4, anti-DR5,
anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR or anti-NGFR antibody
induces clustering of the Death Receptor such as FAS, DR4, DR5,
TNFR1, DR6, DR3, EDAR, or NGFR. That the antibody can induce
clustering and even enhance clustering leads to activation of the
same intracellular signaling pathways as the natural ligand bound
to one of the following group of targets FAS, DR4, DR5, TNFR1, DR6,
DR3, EDAR, or NGFR.
[0212] In one embodiment the antibodies or compositions of the
present invention induce, trigger, increase or enhance apoptosis or
cell death in cancer cells or cancer tissues expressing one or more
Death Receptors such as FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR and/or
NGFR. The increased or enhanced apoptosis or cell death can be
measured by an increase or enhanced level of phosphatidylserine
exposure on cells exposed to or treated with one or more anti-FAS,
anti-DR4, anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR
and/or anti-NGFR antibodies of the invention. Thus it is to be
understood in the context of the present invention that induced,
triggered, increased or enhanced apoptosis or cell death of an
antibody according to the invention i.e. comprising an amino acid
mutation at a position corresponding to E430, E345 or S440 in human
IgG1, EU numbering, may be evaluated by comparing the antibody
according to the invention with the same antibody without said
mutation. In the context of the present invention the same antibody
is to be understood as an antibody having the identical amino acid
sequence as the antibody according to the invention, but without
said mutation.
[0213] Alternatively, the increase or enhanced apoptosis or cell
death can be measured by measuring activation of caspase 3 or
caspase 7 in cells that have been exposed to or treated with one or
more anti-FAS, anti-DR4, anti-DR5, anti-TNFR1, anti-DR6, anti-DR3,
anti-EDAR and/or anti-NGFR antibodies of the invention.
Alternatively, the increase or enhanced apoptosis or cell death can
be measured by a loss of viability in cell cultures that have been
exposed to or treated with one or more anti-FAS, anti-DR4,
anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR and/or
anti-NGFR antibodies of the invention, compared to untreated cell
cultures, in which the loss of viability can be inhibited by a
caspase-inhibitor, for example ZVAD.
[0214] In one embodiment of the present invention the anti-FAS,
anti-DR4, anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR or
anti-NGFR antibody induces hexamerization of antibodies on target
cells expressing FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR or NGFR.
Bispecific Antibodies
[0215] In another aspect, the present invention relates to a
bispecific antibody comprising at least one antigen binding region
binding a death receptor e.g. FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR
or NGFR as described herein.
[0216] In another aspect, the present invention comprises a
bispecific antibody comprising one or more antigen binding regions
binding a death receptor e.g. FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR
or NGFR as described herein.
[0217] In one embodiment of the invention the bispecific antibody
comprises a first antigen binding region and a second antigen
binding region binding a death receptor as defined herein.
[0218] In one embodiment of the invention the bispecific antibody
comprises a first and a second antigen binding region, wherein said
first antigen binding region and said second antigen binding region
bind different epitopes on the same death receptor.
[0219] In one embodiment of the present invention the bispecific
antibody comprises a first and a second Fc region, wherein the
first and/or second Fc region comprises a mutation of an amino acid
position corresponding to E430, E345 or S440 in human IgG1, EU
numbering according to the invention. In one embodiment of the
present invention the bispecific anti-DR5 antibody comprises a
first and a second Fc region, wherein the first and second Fc
region comprises a mutation of an amino acid position corresponding
to E430, E345 or S440 in human IgG1, EU numbering. In one
embodiment of the present invention the bispecific antibody
comprises a first and a second Fc region, wherein the first Fc
region comprises a mutation of an amino acid position corresponding
to E430, E345 or S440 in human IgG1, EU numbering. In one
embodiment of the present invention the bispecific antibody
comprises a first and a second Fc region, wherein the second Fc
region comprises a mutation of an amino acid position corresponding
to E430, E345 or S440 in human IgG1, EU numbering.
[0220] In one embodiment of the invention the bispecific antibody
comprises a first and a second antigen binding region, wherein said
first antigen binding region binding to a death receptor selected
from the following group FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR and
NGFR does not block binding of said second antigen binding region
binding a death receptor selected from the following group FAS,
DR4, DR5, TNFR1, DR6, DR3, EDAR and NGFR, wherein the first and the
second antigen binding region does not bind to the same death
receptor.
[0221] In a particular embodiment the antibody may be bispecific
antibody such as the heterodimeric protein described in WO
11/131746, which is hereby incorporated herein by reference.
[0222] In one embodiment, the antibody is a bispecific antibody
which comprises a first heavy chain comprising a first Fc region of
an immunoglobulin and a first antigen-binding region, and a second
heavy chain comprising a second Fc region of an immunoglobulin and
a second antigen-binding region, wherein the first and second
antigen-binding regions bind different epitopes on the same antigen
or on different antigens.
[0223] In a further embodiment said first heavy chain comprising a
first Fc region comprises a further amino acid substitution at a
position selected from those corresponding to K409, T366, L368,
K370, D399, F405, and Y407 in the Fc region of a human IgG1 heavy
chain; and wherein said second heavy chain comprising a second Fc
region comprises a further amino acid substitution at a position
selected from those corresponding to F405, T366, L368, K370, D399,
Y407, and K409 in the Fc region of a human IgG1 heavy chain, and
wherein said further amino acid substitution in the first heavy
chain comprising a first Fc region is different from the said
further amino acid substitution in the second heavy chain
comprising a second Fc region.
[0224] In a further embodiment said first heavy chain comprising a
first Fc region comprises an amino acid substitution at a position
corresponding to K409 in the Fc-region of a human IgG1 heavy chain;
and said second heavy chain comprising a second Fc region comprises
an amino acid substitution at a position corresponding to F405 in
the Fc-region of a human IgG1 heavy chain.
[0225] In one embodiment of the invention the bispecific antibody
comprises introducing a first and second Fc region comprising a
mutation in at least one amino acid residue selected from those
corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311,
D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1
heavy chain, with the proviso that the mutation in S440 is S440Y or
S440W.
[0226] In a further embodiment the mutation in the first and second
Fc region in at least one amino acid residue selected from those
corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311,
D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1
heavy chain, with the proviso that the mutation in S440 is S440Y or
S440W, may be in the same amino acid residue position or a
different position. In a further embodiment it may be the same or a
different mutation in the same amino acid residue position in the
first and second Fc region.
[0227] In another embodiment the bispecific antibody comprises a
first or second CH2-CH3 region comprising a mutation in at least
one amino acid residue selected from those corresponding to E345,
E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436,
and K447 in the Fc-region of a human IgG1 heavy chain, with the
proviso that the mutation in S440 is S440Y or S440W.
[0228] In one embodiment of the invention the bispecific antibody
comprises a first and a second heavy chain, wherein said first
heavy chain comprises a mutation corresponding to F405L in human
IgG1 according to EU numbering and said second heavy chain
comprises a mutation corresponding to K409R in human IgG1 according
EU numbering.
[0229] In one embodiment of the invention the bispecific antibody
is comprised in a pharmaceutical composition.
Anti-Death Receptor Antibody Compositions
[0230] The anti-death receptor antibodies i.e. anti-FAS, anti-DR4,
anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR or anti-NGFR
antibodies such as monoclonal antibodies or bispecific antibodies
according to any aspect or embodiment of the present invention may
be comprised in a composition, such as a pharmaceutical
composition, diagnostic composition or any other composition.
[0231] In one aspect the invention relates to a composition
comprising at least one anti-death receptor antibody according to
any one of the embodiments described herein.
[0232] In one aspect the invention relates to a composition
comprising one or more anti-death receptor antibodies according to
any one the embodiments described herein. The composition may
comprise one, two or more anti-death domain receptor antibodies
according to the invention as described herein that are not
identical, such as a combination of two different monoclonal
anti-death domain receptor antibodies.
[0233] In one embodiment of the present invention the composition
comprises a first anti-death receptor antibody and a second
anti-death receptor antibody as described herein. That is in one
embodiment of the present invention the composition comprises a
first antibody as described herein and a second antibody as
described herein, wherein the first and the second antibody are not
identical. That is in one embodiment of the present invention the
composition comprises a first antibody selected from the group
consisting of: anti-FAS, anti-DR4, anti-DR5, anti-TNFR1, anti-DR6,
anti-DR3, anti-EDAR and anti-NGFR antibody as described herein and
a second antibody selected from the group consisting of: anti-FAS,
anti-DR4, anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR and
anti-NGFR antibody, wherein the first and the second antibody do
not bind to the same antigen or epitope. Hereby antibody
compositions are described wherein the first and second antibody is
not identical.
[0234] In one embodiment of the present invention the composition
comprises a first anti-death receptor antibody and a second
anti-death receptor antibody as described herein i.e. having a
mutation in an amino acid corresponding to position E430, E345 or
S440 in human IgG1, EU numbering.
[0235] In one embodiment of the present invention the mutation in
an amino acid corresponding to position E430 in human IgG1, EU
numbering is selected from the group consisting of: E430G, E430S,
E430F and E430T.
[0236] In one embodiment of the present invention the mutation in
an amino acid corresponding to position E345 in human IgG1, EU
numbering is selected from the group consisting of: E345K, E345Q,
E345R and E345Y.
[0237] In one embodiment of the present invention the mutation in
an amino acid corresponding to position S440 in human IgG1, EU
numbering is selected from the group consisting of: S440W and
S440Y
[0238] In one embodiment of the present invention the composition
comprises a first anti-FAS antibody comprising a mutation at an
amino acid position corresponding to E430, E345 or S440 in human
IgG1, EU numbering and a second antibody comprising a mutation at
an amino acid position corresponding to E430, E345 or S440 in human
IgG1, EU numbering, wherein the second antibody is selected from
the group consisting of:
a) anti-DR4 antibody; b) anti-DR5 antibody; c) anti-TNFR1 antibody;
d) anti-DR6 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e) anti-NGFR antibody.
[0239] In one embodiment of the present invention the first and
second antibody comprises a mutation at an amino acid position,
wherein said amino acid position is the same. In one embodiment of
the present invention the first and second antibody comprises a
mutation at an amino acid position, wherein said amino acid
position is not the same. Thus in one embodiment of the present
invention the first and antibody comprises a mutation at an amino
acid position, wherein said amino acid position in said first and
second antibody is different.
[0240] In one embodiment of the present invention the composition
comprises a first anti-FAS antibody comprising a mutation at an
amino acid position corresponding to E430 in human IgG1, EU
numbering, and a second antibody comprising a mutation at an amino
acid position corresponding to E430, in human IgG1, EU numbering,
wherein the second antibody is selected from the group consisting
of:
a) anti-DR4 antibody; b) anti-DR5 antibody; c) anti-TNFR1 antibody;
d) anti-DR6 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e) anti-NGFR antibody.
[0241] In one embodiment of the present invention the composition
comprises a first anti-FAS antibody comprising a E430G mutation and
a second antibody comprising a E430G mutation, wherein the second
antibody is selected from the group consisting of:
a) anti-DR4 antibody; b) anti-DR5 antibody; c) anti-TNFR1 antibody;
d) anti-DR6 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e] anti-NGFR antibody.
[0242] In one embodiment of the present invention the composition
comprises a first anti-FAS antibody comprising a mutation at an
amino acid position corresponding to E345 in human IgG1, EU
numbering, and a second antibody comprising a mutation at an amino
acid position corresponding to E345, in human IgG1, EU numbering,
wherein the second antibody is selected from the group consisting
of:
a) anti-DR4 antibody; b) anti-DR5 antibody; c) anti-TNFR1 antibody;
d) anti-DR6 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e) anti-NGFR antibody.
[0243] In one embodiment of the present invention the composition
comprises a first anti-FAS antibody comprising a E345K mutation in
the Fc region, and a second antibody comprising a E345K mutation in
the Fc region, wherein the second, wherein the second antibody is
selected from the group consisting of:
a) anti-DR4 antibody; b) anti-DR5 antibody; c) anti-TNFR1 antibody;
d) anti-DR6 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e) anti-NGFR antibody.
[0244] In one embodiment of the present invention the composition
comprises a first anti-DR4 antibody comprising a mutation at an
amino acid position corresponding to E430, E345 or S440 in human
IgG1, EU numbering and a second antibody comprising a mutation at
an amino acid position corresponding to E430, E345 or S440 in human
IgG1, EU numbering, wherein the second antibody is selected from
the group consisting of:
a) anti-FAS antibody; b) anti-DR5 antibody; c) anti-TNFR1 antibody;
d) anti-DR6 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e) anti-NGFR antibody.
[0245] In one embodiment of the present invention the composition
comprises a first anti-DR4 antibody comprising a mutation at an
amino acid position corresponding to E430 in human IgG1, EU
numbering, and a second antibody comprising a mutation at an amino
acid position corresponding to E430, in human IgG1, EU numbering,
wherein the second antibody is selected from the group consisting
of:
a) anti-FAS antibody; b) anti-DR5 antibody; c) anti-TNFR1 antibody;
d) anti-DR6 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e) anti-NGFR antibody.
[0246] In one embodiment of the present invention the composition
comprises a first anti-DR4 antibody comprising a mutation at an
amino acid position corresponding to E345 in human IgG1, EU
numbering, and a second antibody comprising a mutation at an amino
acid position corresponding to E345, in human IgG1, EU numbering,
wherein the second antibody is selected from the group consisting
of:
a) anti-FAS antibody; b) anti-DR5 antibody; c) anti-TNFR1 antibody;
d) anti-DR6 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e) anti-NGFR antibody.
[0247] In one embodiment of the present invention the composition
comprises a first anti-DR4 antibody comprising an E345K mutation in
the Fc region and a second antibody comprising an E345K mutation in
the Fc region, wherein the second antibody is selected from the
group consisting of:
a) anti-FAS antibody; b) anti-DR5 antibody; c) anti-TNFR1 antibody;
d) anti-DR6 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e) anti-NGFR antibody.
[0248] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody comprising a mutation at an
amino acid position corresponding to E430, E345 or S440 in human
IgG1, EU numbering and a second antibody comprising a mutation at
an amino acid position corresponding to E430, E345 or S440 in human
IgG1, EU numbering, wherein the second antibody is selected from
the group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-TNFR1 antibody;
d) anti-DR6 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e) anti-NGFR antibody.
[0249] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody comprising a mutation at an
amino acid position corresponding to E430 in human IgG1, EU
numbering, and a second antibody comprising a mutation at an amino
acid position corresponding to E430, in human IgG1, EU numbering,
wherein the second antibody is selected from the group consisting
of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-TNFR1 antibody;
d) anti-DR6 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e) anti-NGFR antibody.
[0250] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody comprising a mutation at an
amino acid position corresponding to E345 in human IgG1, EU
numbering, and a second antibody comprising a mutation at an amino
acid position corresponding to E345, in human IgG1, EU numbering,
wherein the second antibody is selected from the group consisting
of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-TNFR1 antibody;
d) anti-DR6 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e) anti-NGFR antibody.
[0251] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody comprising an E430G mutation in
the Fc region and a second antibody comprising an E430G mutation in
the Fc region, wherein the second antibody is selected from the
group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-TNFR1 antibody;
d) anti-DR6 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e) anti-NGFR antibody.
[0252] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody comprising an E345K mutation in
the Fc region and a second antibody comprising an E345K mutation in
the Fc region, wherein the second antibody is selected from the
group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-TNFR1 antibody;
d) anti-DR6 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e] anti-NGFR antibody.
[0253] In one embodiment of the present invention the composition
comprises a first anti-TNFR1 antibody comprising a mutation at an
amino acid position corresponding to E430, E345 or S440 in human
IgG1, EU numbering and a second antibody comprising a mutation at
an amino acid position corresponding to E430, E345 or S440 in human
IgG1, EU numbering, wherein the second antibody is selected from
the group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-DR5 antibody;
d) anti-DR6 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e) anti-NGFR antibody.
[0254] In one embodiment of the present invention the composition
comprises a first anti-TNFR1 antibody comprising an E430G mutation
in the Fc region and a second antibody comprising an E430G mutation
in the Fc region, wherein the second antibody is selected from the
group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-DR5 antibody;
d) anti-DR6 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e] anti-NGFR antibody.
[0255] In one embodiment of the present invention the composition
comprises a first anti-TNFR1 antibody comprising an E345K mutation
in the Fc region and a second antibody comprising a E345K mutation
in the Fc region, wherein the second antibody is selected from the
group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-DR5 antibody;
d) anti-DR6 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e] anti-NGFR antibody.
[0256] In one embodiment of the present invention the composition
comprises a first anti-DR6 antibody comprising a mutation at an
amino acid position corresponding to E430, E345 or S440 in human
IgG1, EU numbering and a second antibody comprising a mutation at
an amino acid position corresponding to E430, E345 or S440 in human
IgG1, EU numbering, wherein the second antibody is selected from
the group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-TNFR1 antibody;
d) anti-DR5 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e) anti-NGFR antibody.
[0257] In one embodiment of the present invention the composition
comprises a first anti-DR6 antibody comprising an E430G mutation in
the Fc region and a second antibody comprising an E430G mutation in
the Fc region, wherein the second antibody is selected from the
group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-TNFR1 antibody;
d) anti-DR5 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e] anti-NGFR antibody.
[0258] In one embodiment of the present invention the composition
comprises a first anti-DR6 antibody comprising an E345K mutation in
the Fc region and a second antibody comprising an E345K mutation in
the Fc region, wherein the second antibody is selected from the
group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-TNFR1 antibody;
d) anti-DR5 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e] anti-NGFR antibody.
[0259] In one embodiment of the present invention the composition
comprises a first anti-DR3 antibody comprising a mutation at an
amino acid position corresponding to E430, E345 or S440 in human
IgG1, EU numbering and a second antibody comprising a mutation at
an amino acid position corresponding to E430, E345 or S440 in human
IgG1, EU numbering, wherein the second antibody is selected from
the group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-TNFR1 antibody;
d) anti-DR5 antibody; c) anti-DR6 antibody; d) anti-EDAR antibody
and e) anti-NGFR antibody.
[0260] In one embodiment of the present invention the composition
comprises a first anti-DR3 antibody comprising an E430G mutation in
the Fc region and a second antibody comprising an E430G mutation in
the Fc region, wherein the second antibody is selected from the
group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-TNFR1 antibody;
d) anti-DR5 antibody; c) anti-DR6 antibody; d) anti-EDAR antibody
and e] anti-NGFR antibody.
[0261] In one embodiment of the present invention the composition
comprises a first anti-DR3 antibody comprising an E345K mutation in
the Fc region and a second antibody comprising an E345K mutation in
the Fc region, wherein the second antibody is selected from the
group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-TNFR1 antibody;
d) anti-DR5 antibody; c) anti-DR6 antibody; d) anti-EDAR antibody
and e] anti-NGFR antibody.
[0262] In one embodiment of the present invention the composition
comprises a first anti-EDAR antibody comprising a mutation at an
amino acid position corresponding to E430, E345 or S440 in human
IgG1, EU numbering and a second antibody comprising a mutation at
an amino acid position corresponding to E430, E345 or S440 in human
IgG1, EU numbering, wherein the second antibody is selected from
the group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-TNFR1 antibody;
d) anti-DR5 antibody; c) anti-DR6 antibody; d) anti-DR3 antibody
and e] anti-NGFR antibody.
[0263] In one embodiment of the present invention the composition
comprises a first anti-EDAR antibody comprising an E430G mutation
in the Fc region and a second antibody comprising an E430G mutation
in the Fc region, wherein the second antibody is selected from the
group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-TNFR1 antibody;
d) anti-DR5 antibody; c) anti-DR6 antibody; d) anti-DR3 antibody
and e] anti-NGFR antibody.
[0264] In one embodiment of the present invention the composition
comprises a first anti-EDAR antibody comprising an E345K mutation
in the Fc region and a second antibody comprising an E345K mutation
in the Fc region, wherein the second antibody is selected from the
group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-TNFR1 antibody;
d) anti-DR5 antibody; c) anti-DR6 antibody; d) anti-DR3 antibody
and e) anti-NGFR antibody.
[0265] In one embodiment of the present invention the composition
comprises a first anti-NGFR antibody comprising a mutation at an
amino acid position corresponding to E430, E345 or S440 in human
IgG1, EU numbering and a second antibody comprising a mutation at
an amino acid position corresponding to E430, E345 or S440 in human
IgG1, EU numbering, wherein the second antibody is selected from
the group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-TNFR1 antibody;
d) anti-DR5 antibody; c) anti-DR6 antibody; d) anti-DR3 antibody
and e] anti-EDAR antibody.
[0266] In one embodiment of the present invention the composition
comprises a first anti-NGFR antibody comprising an E430G mutation
in the Fc region and a second antibody comprising an E430G mutation
in the Fc region, wherein the second antibody is selected from the
group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-TNFR1 antibody;
d) anti-DR5 antibody; c) anti-DR6 antibody; d) anti-DR3 antibody
and e] anti-EDAR antibody.
[0267] In one embodiment of the present invention the composition
comprises a first anti-NGFR antibody comprising an E345K mutation
in the Fc region and a second antibody comprising an E345K mutation
in the Fc region, wherein the second antibody is selected from the
group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-TNFR1 antibody;
d) anti-DR5 antibody; c) anti-DR6 antibody; d) anti-DR3 antibody
and e] anti-EDAR antibody.
[0268] The hexamerization-inhibiting mutation such as K439E or
S440K prevents Fc-Fc interaction with antibodies comprising the
same hexamerization-inhibiting mutation, but by combining
antibodies with a K439E mutation and antibodies with a S440K
mutation the inhibiting effect is neutralized and Fc-Fc
interactions is restored.
[0269] In one embodiment of the present invention the composition
comprises a first antibody selected from the group consisting of;
anti-FAS, anti-DR4, anti-DR5, anti-TNFR1, anti-DR6, anti-DR3,
anti-EDAR and anti-NGFR antibody and a second selected form the
group consisting of; anti-FAS, anti-DR4, anti-DR5, anti-TNFR1,
anti-DR6, anti-DR3, anti-EDAR and anti-NGFR antibody wherein the
first and the second antibody comprises a further
hexamerization-inhibiting mutation corresponding to K439E or S440K
in human IgG1 EU numbering.
[0270] In one embodiment of the present invention the composition
comprises a first and a second antibody selected from the group
consisting of; anti-FAS, anti-DR4, anti-DR5, anti-TNFR1, anti-DR6,
anti-DR3, anti-EDAR and anti-NGFR antibody, wherein the first
antibody comprises a hexamerization enhancing mutation such as
E430G and an hexamerization inhibiting mutation such as K439E, and
wherein the second antibody comprises a hexamerization enhancing
mutation such as E430G and an hexamerization inhibiting mutation
such S440K. Hereby embodiments are provided that allow compositions
wherein hexamerization exclusively occur between combinations of
antibodies comprising a K439E mutation and antibodies comprising a
S440K mutation thereby allowing for hexamerization between
antibodies with different binding specificities.
[0271] In one embodiment of the present invention the composition
comprises a first and a second antibody selected from the group
consisting of; anti-FAS, anti-DR4, anti-DR5, anti-TNFR1, anti-DR6,
anti-DR3, anti-EDAR and anti-NGFR antibody, wherein the first
antibody comprises a hexamerization enhancing mutation such as
E345K and an hexamerization inhibiting mutation such K439E, and
wherein the second antibody comprises a hexamerization enhancing
mutation such as E345K and an hexamerization inhibiting mutation
such S440K. Hereby are embodiments provided that allow compositions
wherein hexamerization exclusively occur between combinations of
antibodies comprising a K439E mutation and antibodies comprising a
S440K mutation thereby allowing for hexamerization between
antibodies with different binding specificities.
[0272] In one embodiment of the present invention the composition
comprises a first anti-FAS antibody comprising an E430G and a K439E
mutation in the Fc region and a second antibody comprising a E430G
and a S440K mutation in the Fc region, wherein the second antibody
is selected from the group consisting of:
a) anti-DR4 antibody; b) anti-DR5 antibody; c) anti-TNFR1 antibody;
d) anti-DR6 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e] anti-NGFR antibody.
[0273] In one embodiment of the present invention the composition
comprises a first anti-FAS antibody comprising an E345K and a K439E
mutation in the Fc region and a second antibody comprising an E345K
and a S440K mutation in the Fc region, wherein the second antibody
is selected from the group consisting of:
a) anti-DR4 antibody; b) anti-DR5 antibody; c) anti-TNFR1 antibody;
d) anti-DR6 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e) anti-NGFR antibody.
[0274] In one embodiment of the present invention the composition
comprises a first anti-DR4 antibody comprising an E430G and a K439E
mutation in the Fc region and a second antibody comprising an E430G
and a S440K mutation in the Fc region, wherein the second antibody
is selected from the group consisting of:
a) anti-FAS antibody; b) anti-DR5 antibody; c) anti-TNFR1 antibody;
d) anti-DR6 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e) anti-NGFR antibody.
[0275] In one embodiment of the present invention the composition
comprises a first anti-DR4 antibody comprising an E345K and a K439E
mutation in the Fc region and a second antibody comprising an E345K
and a S440K mutation in the Fc region, wherein the second antibody
is selected from the group consisting of:
a) anti-FAS antibody; b) anti-DR5 antibody; c) anti-TNFR1 antibody;
d) anti-DR6 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e] anti-NGFR antibody.
[0276] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody comprising an E430G and a K439E
mutation in the Fc region and a second antibody comprising an E430G
and a S440K mutation in the Fc region, wherein the second antibody
is selected from the group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-TNFR1 antibody;
d) anti-DR6 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e) anti-NGFR antibody.
[0277] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody comprising an E345K and a K439E
mutation in the Fc region and a second antibody comprising an E345K
and a S440K mutation in the Fc region, wherein the second antibody
is selected from the group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-TNFR1 antibody;
d) anti-DR6 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e] anti-NGFR antibody.
[0278] In one embodiment of the present invention the composition
comprises a first anti-TNFR1 antibody comprising an E430G and a
K439E mutation in the Fc region and a second antibody comprising an
E430G and a S440K mutation in the Fc region, wherein the second
antibody is selected from the group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-DR5 antibody;
d) anti-DR6 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e] anti-NGFR antibody.
[0279] In one embodiment of the present invention the composition
comprises a first anti-TNFR1 antibody comprising an E345K and a
K439E mutation in the Fc region and a second antibody comprising an
E345K and a S440K mutation in the Fc region, wherein the second
antibody is selected from the group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-DR5 antibody;
d) anti-DR6 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e] anti-NGFR antibody.
[0280] In one embodiment of the present invention the composition
comprises a first anti-DR6 antibody comprising an E430G and a K439E
mutation in the Fc region and a second antibody comprising an E430G
and a S440K mutation in the Fc region, wherein the second antibody
is selected from the group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-TNFR1 antibody;
d) anti-DR5 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e) anti-NGFR antibody.
[0281] In one embodiment of the present invention the composition
comprises a first anti-DR6 antibody comprising an E345K and a K439E
mutation in the Fc region and a second antibody comprising an E345K
and a S440K mutation in the Fc region, wherein the second antibody
is selected from the group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-TNFR1 antibody;
d) anti-DR5 antibody; c) anti-DR3 antibody; d) anti-EDAR antibody
and e) anti-NGFR antibody.
[0282] In one embodiment of the present invention the composition
comprises a first anti-DR3 antibody comprising an E430G and a K439E
mutation in the Fc region and a second antibody comprising an E430G
and a S440K mutation in the Fc region, wherein the second antibody
is selected from the group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-TNFR1 antibody;
d) anti-DR5 antibody; c) anti-DR6 antibody; d) anti-EDAR antibody
and e] anti-NGFR antibody.
[0283] In one embodiment of the present invention the composition
comprises a first anti-DR3 antibody comprising an E345K and a K439E
mutation in the Fc region and a second antibody comprising an E345K
and a S440K mutation in the Fc region, wherein the second antibody
is selected from the group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-TNFR1 antibody;
d) anti-DR5 antibody; c) anti-DR6 antibody; d) anti-EDAR antibody
and e] anti-NGFR antibody.
[0284] In one embodiment of the present invention the composition
comprises a first anti-EDAR antibody comprising an E430G and a
K439E mutation in the Fc region and a second antibody comprising an
E430G and a S440K mutation in the Fc region, wherein the second
antibody is selected from the group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-TNFR1 antibody;
d) anti-DR5 antibody; c) anti-DR6 antibody; d) anti-DR3 antibody
and e) anti-NGFR antibody.
[0285] In one embodiment of the present invention the composition
comprises a first anti-EDAR antibody comprising an E345K and a
K439E mutation in the Fc region and a second antibody comprising an
E345K and a S440K mutation in the Fc region, wherein the second
antibody is selected from the group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-TNFR1 antibody;
d) anti-DR5 antibody; c) anti-DR6 antibody; d) anti-DR3 antibody
and e) anti-NGFR antibody.
[0286] In one embodiment of the present invention the composition
comprises a first anti-NGFR antibody comprising an E430G and a
K439E mutation in the Fc region and a second antibody comprising an
E430G and a S440K mutation in the Fc region, wherein the second
antibody is selected from the group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-TNFR1 antibody;
d) anti-DR5 antibody; c) anti-DR6 antibody; d) anti-DR3 antibody
and e) anti-EDAR antibody.
[0287] In one embodiment of the present invention the composition
comprises a first anti-NGFR antibody comprising an E345K and a
K439E mutation in the Fc region and a second antibody comprising an
E345K and a S440K mutation in the Fc region, wherein the second
antibody is selected from the group consisting of:
a) anti-FAS antibody; b) anti-DR4 antibody; c) anti-TNFR1 antibody;
d) anti-DR5 antibody; c) anti-DR6 antibody; d) anti-DR3 antibody
and e) anti-EDAR antibody.
[0288] Hereby are embodiments provided that allow compositions
wherein hexamerization exclusively occur between combinations of
antibodies comprising a K439E mutation and antibodies comprising a
S440K mutation thereby allowing for hexamerization between
antibodies with different binding specificities.
[0289] In one embodiment of the present invention the composition
comprises a first anti-death receptor antibody and a second
anti-death receptor antibody binding different epitopes on the same
death receptor.
[0290] In one embodiment of the present invention the composition
comprises said first anti-death receptor antibody binding to a
death receptor, which does not block binding of said second
anti-death receptor antibody, when the first and the second
anti-death receptor antibody bind to the same target. That is in
one embodiment of the invention the composition comprises a first
anti-death receptor antibody and a second anti-death receptor
antibody, wherein the first and the second antibody does not
compete for binding to the death domain receptor.
[0291] In one embodiment of the invention the composition comprises
a first and a second anti-death receptor antibody selected from the
following group anti-FAS, anti-DR4, anti-DR5, anti-TNFR1, anti-DR6,
anti-DR3, anti-EDAR and anti-NGFR, wherein said first antibody and
said second antibody are present in the composition at a 1:49 to
49:1 molar ratio, such as 1:1 molar ratio, a 1:2 molar ratio, a 1:3
molar ratio, a 1:4 molar ratio, a 1:5 molar ratio, a 1:6 molar
ratio, a 1:7 molar ratio, a 1:8 molar ratio, a 1:9 molar ratio, a
1:5 molar ratio, a 1:5 molar ratio, a 1:5 molar ratio, a 1:10 molar
ratio, a 1:15 molar ratio, a 1:20 molar ratio, a 1:25 molar ratio,
a 1:30 molar ratio, a 1:35 molar ratio, a 1:40 molar ratio, a 1:45
molar ratio a 1:50 molar ratio, a 50:1 molar ratio, a 45:1 molar
ratio, a 40:1 molar ratio, a 35:1 molar ratio, a 30:1 molar ratio a
25:1 molar ratio, a 20:1 molar ratio, a 15:1 molar ratio, a 10:1
molar ratio, a 9:1 molar ratio, a 8:1 molar ratio, a 7:1 molar
ratio, a 6:1 molar ratio, a 5:1 molar ratio, a 4:1 molar ratio, a
3:1 molar ratio, a 2:1 molar ratio.
[0292] In one embodiment of the invention the composition comprises
a first and a second antibody, wherein said first antibody and said
second antibody are present in the composition at a 1:9 to 9:1
molar ratio.
[0293] In one embodiment of the invention the composition comprises
a first and a second anti-death receptor antibody, wherein said
first antibody and said second antibody are present in the
composition at approximately a 1:1 molar ratio.
[0294] In one embodiment of the invention the composition comprises
a first and a second anti-death receptor antibody, wherein said
first antibody and said second antibody are present in the
composition at a 1:1 molar ratio.
[0295] In a preferred embodiment of the invention the composition
comprises a first and a second anti-death receptor antibody,
wherein said first antibody and second antibody and/or any
additional antibodies are present in the composition at an
equimolar ratio.
[0296] In one embodiment of the invention the composition is a
pharmaceutical composition.
[0297] In one embodiment of the invention the bispecific antibody
is comprised in a pharmaceutical composition.
[0298] Pharmaceutical compositions of the present invention may
comprise antibodies such as monoclonal antibodies or bispecific
antibodies according to any aspect or embodiment of the present
invention.
[0299] The pharmaceutical compositions may be formulated with
pharmaceutically acceptable carriers or diluents as well as any
other known adjuvants and excipients in accordance with
conventional techniques such as those disclosed in (Rowe et al.,
Handbook of Pharmaceutical Excipients, 2012 June, ISBN
9780857110275)
[0300] The pharmaceutically acceptable carriers or diluents as well
as any other known adjuvants and excipients should be suitable for
the antibody or bispecific antibody of the present invention and
the chosen mode of administration. Suitability for carriers and
other components of pharmaceutical compositions is determined based
on the lack of significant negative impact on the desired
biological properties of the chosen compound or pharmaceutical
composition of the present invention (e.g., less than a substantial
impact (10% or less relative inhibition, 5% or less relative
inhibition, etc.) upon antigen binding).
[0301] A pharmaceutical composition of the present invention may
also include diluents, fillers, salts, buffers, detergents (e. g.,
a nonionic detergent, such as Tween-20 or Tween-80), stabilizers
(e.g., sugars or protein-free amino acids), preservatives, tissue
fixatives, solubilizers, and/or other materials suitable for
inclusion in a pharmaceutical composition. The actual dosage levels
of the active ingredients in the pharmaceutical compositions of the
present invention may be varied so as to obtain an amount of the
active ingredient which is effective to achieve the desired
therapeutic response for a particular patient, composition, and
mode of administration, without being toxic to the patient. The
selected dosage level will depend upon a variety of pharmacokinetic
factors including the activity of the particular compositions of
the present invention employed, or the amide thereof, the route of
administration, the time of administration, the rate of excretion
of the particular compound being employed, the duration of the
treatment, other drugs, compounds and/or materials used in
combination with the particular compositions employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the
medical arts.
[0302] The pharmaceutical composition may be administered by any
suitable route and mode. Suitable routes of administering a
compound of the present invention in vivo and in vitro are well
known in the art and may be selected by those of ordinary skill in
the art.
[0303] In one embodiment, the pharmaceutical composition of the
present invention is administered parenterally.
[0304] The terms "parenteral administration" and "administered
parenterally" as used herein refers to modes of administration
other than enteral and topical administration, usually by
injection, and include epidermal, intravenous, intramuscular,
intra-arterial, intrathecal, intracapsular, intra-orbital,
intracardiac, intradermal, intraperitoneal, intratendinous,
transtracheal, subcutaneous, subcuticular, intra-articular,
subcapsular, subarachnoid, intraspinal, intracranial,
intrathoracic, epidural and intrasternal injection and
infusion.
[0305] In one embodiment, the pharmaceutical composition of the
present invention is administered by intravenous or subcutaneous
injection or infusion.
[0306] In one embodiment of the present invention the
pharmaceutical composition comprises one or more antibodies
according to the invention such as monoclonal antibodies or
bispecific antibodies together with a pharmaceutical carrier.
[0307] Pharmaceutically acceptable carriers include any and all
suitable solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonicity agents, antioxidants and
absorption-delaying agents, and the like that are physiologically
compatible with a compound of the present invention.
[0308] Examples of suitable aqueous and non-aqueous carriers which
may be employed in the pharmaceutical compositions of the present
invention include water, saline, phosphate-buffered saline,
ethanol, dextrose, polyols (such as glycerol, propylene glycol,
polyethylene glycol, and the like), and suitable mixtures thereof,
vegetable oils, such as olive oil, corn oil, peanut oil, cottonseed
oil, and sesame oil, carboxymethyl cellulose colloidal solutions,
tragacanth gum and injectable organic esters, such as ethyl oleate,
and/or various buffers. Other carriers are well known in the
pharmaceutical arts.
[0309] Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. The use
of such media and agents for pharmaceutically active substances is
known in the art. Except insofar as any conventional media or agent
is incompatible with the active compound, use thereof in the
pharmaceutical compositions of the present invention is
contemplated.
[0310] Proper fluidity may be maintained, for example, by the use
of coating materials, such as lecithin, by the maintenance of the
required particle size in the case of dispersions, and by the use
of surfactants.
[0311] Pharmaceutical compositions of the present invention may
also comprise pharmaceutically acceptable antioxidants for instance
(1) water-soluble antioxidants, such as ascorbic acid, cysteine
hydrochloride, sodium bisulfate, sodium metabisulfite, sodium
sulfite and the like; (2) oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and (3) metal-chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0312] Pharmaceutical compositions of the present invention may
also comprise isotonicity agents, such as sugars, polyalcohols,
such as mannitol, sorbitol, glycerol or sodium chloride in the
compositions.
[0313] The pharmaceutical compositions of the present invention may
also contain one or more adjuvants appropriate for the chosen route
of administration such as preservatives, wetting agents,
emulsifying agents, dispersing agents, preservatives or buffers,
which may enhance the shelf life or effectiveness of the
pharmaceutical composition. The compounds of the present invention
may be prepared with carriers that will protect the compound
against rapid release, such as a controlled release formulation,
including implants, transdermal patches, and micro-encapsulated
delivery systems. Such carriers may include gelatin, glyceryl
monostearate, glyceryl distearate, biodegradable, biocompatible
polymers such as ethylene vinyl acetate, polyanhydrides,
polyglycolic acid, collagen, poly-ortho-esters, and polylactic acid
alone or with a wax, or other materials well known in the art.
Methods for the preparation of such formulations are generally
known to those skilled in the art. In one embodiment, the compounds
of the present invention may be formulated to ensure proper
distribution in vivo. Pharmaceutically acceptable carriers for
parenteral administration include sterile aqueous solutions or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. The use of such
media and agents for pharmaceutically active substances is known in
the art. Except insofar as any conventional media or agent is
incompatible with the active compound, use thereof in the
pharmaceutical compositions of the present invention is
contemplated. Other active or therapeutic compounds may also be
incorporated into the compositions.
[0314] Pharmaceutical compositions for injection or infusion must
typically be sterile and stable under the conditions of manufacture
and storage. The composition may be formulated as a solution,
micro-emulsion, liposome, or other ordered structure suitable to
high drug concentration. The carrier may be an aqueous or a
non-aqueous solvent or dispersion medium containing for instance
water, ethanol, polyols (such as glycerol, propylene glycol,
polyethylene glycol, and the like), and suitable mixtures thereof,
vegetable oils, such as olive oil, and injectable organic esters,
such as ethyl oleate. The proper fluidity may be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. In many cases, it will be preferable
to include isotonic agents, for example, sugars, polyalcohols such
as glycerol, mannitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions
may be brought about by including in the composition an agent that
delays absorption, for example, monostearate salts and gelatin.
Sterile injectable solutions may be prepared by incorporating the
active compound in the required amount in an appropriate solvent
with one or a combination of ingredients e.g. as enumerated above,
as required, followed by sterilization microfiltration. Generally,
dispersions are prepared by incorporating the active compound into
a sterile vehicle that contains a basic dispersion medium and the
required other ingredients e.g. from those enumerated above. In the
case of sterile powders for the preparation of sterile injectable
solutions, examples of methods of preparation are vacuum-drying and
freeze-drying (lyophilization) that yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0315] Sterile injectable solutions may be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by sterilization
microfiltration. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions,
examples of methods of preparation are vacuum-drying and
freeze-drying (lyophilization) that yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0316] The pharmaceutical composition of the present invention may
contain one or more monoclonal antibodies or one or more bispecific
antibodies of the present invention, a combination of an antibody
or a bispecific antibody according to the invention with another
therapeutic compound, or a combination of compounds of the present
invention.
Therapeutic Applications
[0317] The antibodies such as monoclonal antibodies, bispecific
antibodies or compositions according to any aspect or embodiment of
the present invention may be used as a medicament, i.e. for
therapeutic applications.
[0318] In one embodiment of the present invention the composition
comprises one or more antibodies according to the invention such as
monoclonal antibodies or bispecific antibodies for use as a
medicament.
[0319] In another aspect, the present invention provides methods
for treating or preventing a disorder involving cells expressing a
death receptor such as FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR or NGFR
in a subject, which method comprises administration of a
therapeutically effective amount of an anti-FAS, anti-DR4,
anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR, and anti-NGFR
antibody, bispecific antibody or a composition comprising one or
more antibodies of the present invention to a subject in need
thereof. The method typically involves administering to a subject
in need thereof an anti-FAS, anti-DR4, anti-DR5, anti-TNFR1,
anti-DR6, anti-DR3, anti-EDAR, and anti-NGFR antibody, a bispecific
antibody or composition according to the present invention in an
amount effective to treat or prevent the disorder.
[0320] The anti-death receptor antibodies of the present invention
can be used in the treatment or prevention of disorders involving
cells expressing the death receptor. For example, the antibodies
may be administered to human subjects, e.g., in vivo, to treat or
prevent disorders involving FAS-expressing cells, DR4-expressing
cells, DR5-expressing cells, TNFR1-expressing cells, DR6-expressing
cells, DR3-expressing cells, EDAR-expressing cells or
NGFR-expressing cells. As used herein, the term "subject" is
typically a human to whom the anti-FAS, anti-DR4, anti-DR5,
anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR or anti-NGFR antibody or
bispecific antibody is administered. Subjects may for instance
include human patients having disorders that may be corrected or
ameliorated by modulating FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR or
NGFR function or by killing of the FAS-expressing cells,
DR4-expressing cells, DR5-expressing cells, TNFR1-expressing cells,
DR6-expressing cells, DR3-expressing cells, EDAR-expressing cells
or NGFR-expressing cells, directly or indirectly.
[0321] In one aspect, the present invention relates to an
anti-death receptor antibody, bispecific antibody or composition as
defined in any aspect or embodiment herein, for use in treatment or
to ameliorate symptoms of a disease or disorder involving cells
expressing one or more death receptors i.e. FAS-expressing cells,
DR4-expressing cells, DR5-expressing cells, TNFR1-expressing cells,
DR6-expressing cells, DR3-expressing cells, EDAR-expressing cells
or NGFR-expressing cells. In some diseases or disorders the cells
express more than one death receptor. That is in some diseases or
disorders the cells expresses any combination of the following
group of death receptors FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR and
NGFR.
[0322] In one embodiment of the present invention the composition
comprising an anti-death receptor i.e. an anti-FAS, anti-DR4,
anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR or anti-NGFR
antibody or bispecific antibody according to any aspect or
embodiment as disclosed herein, for use in treatment of infectious
disease, autoimmune disease or cardiovascular anomalies.
[0323] In one aspect, the present invention relates to an
anti-death receptor i.e. an anti-FAS, anti-DR4, anti-DR5,
anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR or anti-NGFR antibody,
bispecific antibody or composition as defined in any aspect or
embodiment herein, for use in treatment or to ameliorate symptoms
of cancer and/or tumors.
[0324] In one embodiment of the present invention the composition
comprising an anti-death receptor i.e. an anti-FAS, anti-DR4,
anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR or anti-NGFR
antibody or bispecific antibody according to any aspect or
embodiment of the invention is for use in treatment of cancer
and/or tumors.
[0325] The term "cancer" refers to or describes the physiological
condition in mammals such as humans that is typically characterized
by unregulated growth. Most cancers belong to one of two larger
groups of cancers i.e., solid tumors and hematological tumors.
[0326] In a particular aspect, an anti-death receptor i.e. an
anti-FAS, anti-DR4, anti-DR5, anti-TNFR1, anti-DR6, anti-DR3,
anti-EDAR or anti-NGFR antibody, bispecific antibody or composition
is administered prophylactically in order to reduce the risk of
developing cancer, delay the onset of an event in cancer
progression or reduce the risk of recurrence when a cancer is in
remission and/or a primary tumor has been surgically removed. In
the latter case, the anti-death receptor i.e. an anti-FAS,
anti-DR4, anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR or
anti-NGFR antibody, bispecific antibody or composition could, for
example, be administered in association with (i.e., before, during,
or after) the surgery. Prophylactic administration may also be
useful in patients where it is difficult to locate a tumor that is
believed to be present due to other biological factors.
[0327] In one embodiment the composition comprising one or more
anti-death receptor i.e. an anti-FAS, anti-DR4, anti-DR5,
anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR or anti-NGFR antibodies
or bispecific antibodies of the present invention is for use in
treatment of solid tumors and/or hematological tumors
[0328] In one embodiment the composition comprising one or more
anti-death receptor i.e. an anti-FAS, anti-DR4, anti-DR5,
anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR or anti-NGFR antibodies
or bispecific antibodies of the present invention is for use in
treatment of solid tumors such as, colorectal cancer, including
colorectal carcinoma and colorectal adenocarcinoma, bladder cancer,
osteosarcoma, chondrosarcoma, breast cancer, including
triple-negative breast cancer, cancers of the central nervous
system, including glioblastoma, astrocytoma, neuroblastoma, neural
fibrosarcoma, neuroendocrine tumors, cervical cancer, endometrium
cancer, gastric cancer, including gastric adenocarcinoma, head and
neck cancer, kidney cancer, liver cancer, including hepatocellular
carcinoma, lung cancer, including NSCLC and SCLC, ovarian cancer,
pancreatic cancer, including pancreatic ductal carcinoma and
pancreatic adenocarcinoma, sarcoma or skin cancer, including
malignant melanoma and non-melanoma skin cancers.
[0329] In one embodiment of the invention the composition
comprising one or more anti-death receptor i.e. an anti-FAS,
anti-DR4, anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR or
anti-NGFR antibodies or bispecific antibodies is for use in
treatment of hematological tumors such as, leukemia, including
chronic lymphocytic leukemia and myeloid leukemia, including acute
myeloid leukemia and chronic myeloid leukemia, lymphoma, including
Non-Hodgkin lymphoma or multiple myeloma, including Hodgkin
Lymphoma, and including myelodysplastic syndromes.
[0330] In a particular embodiment of the present invention the
composition comprising one or more anti-death receptor i.e. an
anti-FAS, anti-DR4, anti-DR5, anti-TNFR1, anti-DR6, anti-DR3,
anti-EDAR or anti-NGFR antibodies or bispecific antibodies is for
use in treatment of a cancer selected from the following group of
cancers; bladder cancer, bone cancer, colorectal cancer, sarcoma,
endometrium cancer, fibroblast cancer, gastric cancer, head and
neck cancer, kidney cancer, leukemia, liver cancer, lung cancer,
lymphoma, muscle cancer, neural tissue cancer, ovary cancer,
pancreas cancer and skin cancer.
[0331] In one embodiment of the invention the composition
comprising one or more anti-death receptor i.e. an anti-FAS,
anti-DR4, anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR or
anti-NGFR antibodies or bispecific antibodies is for use in
inhibiting growth of FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR or NGFR
positive or FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR or NGFR expressing
tumors or cancers.
[0332] In the present invention FAS, DR4, DR5, TNFR1, DR6, DR3,
EDAR or NGFR positive tumors or cancers are to be understood as
tumor cells and/or cancer cells expressing DR5 on the cell surface.
Such FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR or NGFR expression may be
detected by immunohistochemistry, flow cytometry or other suitable
diagnostic method.
[0333] In one embodiment of the invention the composition
comprising one or more anti-FAS, anti-DR4, anti-DR5, anti-TNFR1,
anti-DR6, anti-DR3, anti-EDAR or anti-NGFR antibodies or bispecific
antibodies is for use in inhibiting growth of FAS, DR4, DR5, TNFR1,
DR6, DR3, EDAR or NGFR tumors or cancers. Tumors and cancer tissues
that show heterogenous expression of FAS, DR4, DR5, TNFR1, DR6,
DR3, EDAR or NGFR are also considered as FAS, DR4, DR5, TNFR1, DR6,
DR3, EDAR or NGFR positive tumors and cancers.
[0334] Tumors and/or cancers may express FAS, DR4, DR5, TNFR1, DR6,
DR3, EDAR or NGFR on some tumor and/or cancer cells and/or tissues
showing FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR or NGFR expression,
some tumor and/or cancers may show over-expression or aberrant
expression of FAS, DR4, DR5, TNFR1, DR6, DR3, EDAR or NGFR, whereas
other tumors and/or cancers show heterogeneous expression of FAS,
DR4, DR5, TNFR1, DR6, DR3, EDAR or NGFR. Such tumors and/or cancers
may all be suitable targets for treatment with anti-FAS, anti-DR4,
anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR or anti-NGFR
antibodies, bispecific antibodies and compositions comprising such
antibodies according to the present invention.
[0335] In one embodiment of the invention the composition
comprising one or more anti-FAS, anti-DR4, anti-DR5, anti-TNFR1,
anti-DR6, anti-DR3, anti-EDAR or anti-NGFR antibodies or bispecific
antibodies is for use in induction of apoptosis in FAS-expressing
tumors, DR4-expressing tumors, DR5-expressing tumors,
TNFR1-expressing tumors, DR6-expressing tumors, DR3-expressing
tumors, EDAR-expressing tumors, or NGFR-expressing tumors. In one
embodiment the tumor is expressing a one or more death receptors,
that is a combination of two death receptors, a combination of
three death receptors, a combination of four death receptors, a
combination of five death receptors, a combination of six death
receptors, a combination of seven death receptors, a combination of
eight death receptors.
[0336] Another aspect of the present invention comprises a method
of treating an individual having a cancer comprising administering
to said individual an effective amount of an anti-FAS, anti-DR4,
anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR or anti-NGFR
antibody, bispecific antibody or composition according to the
invention.
[0337] In one embodiment of the invention the method of treating an
individual having a cancer comprising administering to said
individual an effective amount of an anti-FAS, anti-DR4, anti-DR5,
anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR or anti-NGFR antibody,
bispecific antibody or composition according to the invention,
further comprises administering an additional therapeutic agent to
the said individual.
[0338] In one embodiment of the invention the additional
therapeutic agent is a single agent or a combination of agents
comprising an agent or regimen selected from the group
chemotherapeutics (including but not limited to paclitaxel,
temozolomide, cisplatin, carboplatin, oxaliplatin, irinotecan,
doxorubicin, gemcitabine, 5-fluorouracil, pemetrexed), kinase
inhibitors (including but not limited to sorafenib, sunitinib or
everolimus), apoptosis-modulating agents (including but not limited
to recombinant human TRAIL or birinapant), RAS inhibitors,
proteasome inhibitors (including but not limited to bortezomib),
histon deacetylase inhibitors (including but not limited to
vorinostat), nutraceuticals, cytokines (including but not limited
to IFN-.gamma.), antibodies or antibody mimetics (including but not
limited to anti-TF, anti-AXL, anti-EGFR, anti-IGF-1R, anti-VEGF,
anti-CD20, anti-CD38, anti-HER2, anti-PD-1, anti-PD-L1, anti-CTLA4,
anti-CD40, anti-CD137, anti-GITR, anti-VISTA (or other
immunomodulatory targets) antibodies and antibody mimetics), and
antibody-drug conjugates such as brentuximab vedotin, trastuzumab
emtansine, HuMax-TF-ADC or HuMax-AXL-ADC.
[0339] In a further aspect, the invention comprises a kit of parts
comprising an anti-FAS, anti-DR4, anti-DR5, anti-TNFR1, anti-DR6,
anti-DR3, anti-EDAR or anti-NGFR antibody, bispecific antibody or
composition according to the, wherein said antibody, bispecific
antibody or composition is in one or more containers such as one or
more vials.
[0340] In one embodiment of the invention the kit of parts
comprising an anti-FAS, anti-DR4, anti-DR5, anti-TNFR1, anti-DR6,
anti-DR3, anti-EDAR or anti-NGFR antibody, bispecific antibody or
composition according to the invention is for simultaneous,
separate or sequential use in therapy.
[0341] In a further embodiment the present invention is for use of
an anti-FAS, anti-DR4, anti-DR5, anti-TNFR1, anti-DR6, anti-DR3,
anti-EDAR or anti-NGFR antibody, bispecific antibody or a
composition according to the invention for the manufacture of a
medicament for treatment of cancer.
[0342] When describing the embodiments of the present invention,
the combinations and permutations of all possible embodiments have
not been explicitly described. Nevertheless, the mere fact that
certain measures are recited in mutually different dependent claims
or described in different embodiments does not indicate that a
combination of these measures cannot be used to advantage. The
present invention envisages all possible combinations and
permutations of the described embodiments.
[0343] In another aspect of the present invention, the invention
comprises a nucleic acid construct encoding an antibody according
to amino acid sequences set forth in table 1. That is in one
embodiment, the present invention comprises, a nucleic acid
construct encoding an antibody corresponding to the amino acid
sequences set forth in table 1. In one embodiment of the present
invention, the nucleic acid construct encodes an antibody according
to any embodiments disclosed herein.
[0344] In a further aspect, the present invention relates to a
nucleic acid encoding an antibody according to the present
invention, wherein the Fc region comprises a mutation of an amino
acids position corresponding to E430, E345 or S440 in a human IgG1,
EU numbering. It is further contemplated that the nucleic acid
encoding an antibody according to the invention comprises the amino
acid substitutions in the specific amino acid positions herein
described. Thus, in one embodiment, the nucleic acid encodes an
antibody having the sequence according to SEQ ID NO: 1 to 50.
[0345] In another aspect, the invention relates to nucleic acids
encoding a sequence of a human, humanized or chimeric anti-FAS,
anti-DR4, anti-DR5, anti-TNFR1, anti-DR6, anti-DR3, anti-EDAR or
anti-NGFR antibody for use in the invention, to expression vectors
encoding the sequences of such an antibody, to host cells
comprising such expression vectors, to hybridomas which produce
such antibodies, and to methods of producing such an antibody by
culturing such host cells or hybridomas under appropriate
conditions whereby the antibody is produced and, optionally,
retrieved.
[0346] In one embodiment, the invention provides an expression
vector comprising a nucleotide sequence encoding one or more of the
amino acid sequence according to SEQ ID Nos: 1 to 51.
[0347] In another embodiment, the expression vector comprises a
nucleotide sequence encoding any one or more of the VH CDR3 amino
acid sequences selected from SEQ ID NOs: 18, 22, 19, 30, 39 and 46.
In another embodiment, the expression vector comprises a nucleotide
sequence encoding a VH amino acid sequence selected from SEQ ID
NOs: 13, 15, 19, 26, 31 and 40. In another embodiment, the
expression vector comprises a nucleotide sequence encoding a VL
amino acid sequence selected from SEQ ID NOs: 14, 16, 23, 35, 43
and 15. In another embodiment, the expression vector comprises a
nucleotide sequence encoding the constant region of a human
antibody light chain, of a human antibody heavy chain, or both. In
another embodiment, the expression vector comprising a nucleotide
sequence encoding the constant region of a human antibody heavy
chain of selected from the group consisting of: SEQ ID NOs: 20, 27,
32 and 47.
[0348] In a particular embodiment, the expression vector comprises
a nucleotide sequence encoding a variant of one or more of the
above amino acid sequences, said variant having at most 25 amino
acid modifications, such as at most 20, such as at most 15, 14, 13,
12, or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4,
3, 2, or 1 amino acid modifications, such as deletions or
insertions, preferably substitutions, such as conservative
substitutions, or at least 80% identity to any of said sequences,
such as at least 85% identity or 90% identity or 95% identity, such
as 96% identity or 97% identity or 98% identity or 99% identity to
any of the afore-mentioned amino acid sequences.
[0349] An expression vector in the context of the present invention
may be any suitable vector, including chromosomal, non-chromosomal,
and synthetic nucleic acid vectors (a nucleic acid sequence
comprising a suitable set of expression control elements). Examples
of such vectors include derivatives of SV40, bacterial plasmids,
phage DNA, baculovirus, yeast plasmids, vectors derived from
combinations of plasmids and phage DNA, and viral nucleic acid (RNA
or DNA) vectors. In one embodiment, a humanized CD3
antibody-encoding nucleic acid is comprised in a naked DNA or RNA
vector, including, for example, a linear expression element (as
described in for instance Sykes and Johnston, Nat Biotech 17,
355-59 (1997)), a compacted nucleic acid vector (as described in
for instance U.S. Pat. No. 6,077,835 and/or WO 00/70087), a plasmid
vector such as pBR322, pUC 19/18, or pUC 118/119, a "midge"
minimally-sized nucleic acid vector (as described in for instance
Schakowski et al., Mol Ther 3, 793-800 (2001)), or as a
precipitated nucleic acid vector construct, such as a
CaPO.sub.4.sup.--precipitated construct (as described in for
instance WO 00/46147, Benvenisty and Reshef, PNAS USA 83, 9551-55
(1986), Wigler et al., Cell 14, 725 (1978), and Coraro and Pearson,
Somatic Cell Genetics 7, 603 (1981)). Such nucleic acid vectors and
the usage thereof are well known in the art (see for instance U.S.
Pat. Nos. 5,589,466 and 5,973,972). In one embodiment, the vector
is suitable for expression of the humanized anti-DR5 antibody, in a
bacterial cell. Examples of such vectors include expression vectors
such as BlueScript (Stratagene), pIN vectors (Van Heeke &
Schuster, J Biol Chem 264, 5503-5509 (1989)), pET vectors (Novagen,
Madison Wis.) and the like.
[0350] An expression vector may also or alternatively be a vector
suitable for expression in a yeast system. Any vector suitable for
expression in a yeast system may be employed. Suitable vectors
include, for example, vectors comprising constitutive or inducible
promoters such as alpha factor, alcohol oxidase and PGH (reviewed
in: F. Ausubel et al., ed. Current Protocols in Molecular Biology,
Greene Publishing and Wiley InterScience New York (1987), and Grant
et al., Methods in Enzymol 153, 516-544 (1987)).
[0351] A nucleic acid and/or vector may also comprise a nucleic
acid sequence encoding a secretion/localization sequence, which can
target a polypeptide, such as a nascent polypeptide chain, to the
periplasmic space or into cell culture media. Such sequences are
known in the art, and include secretion leader or signal peptides,
organelle-targeting sequences (e.g., nuclear localization
sequences, ER retention signals, mitochondrial transit sequences,
chloroplast transit sequences), membrane localization/anchor
sequences (e.g., stop transfer sequences, GPI anchor sequences),
and the like.
[0352] In an expression vector of the invention, anti-DR5
antibody-encoding nucleic acids and the first and the second
polypeptides nucleic acids may comprise or be associated with any
suitable promoter, enhancer, and other expression-facilitating
elements. Examples of such elements include strong expression
promoters (e.g., human CMV IE promoter/enhancer as well as RSV,
SV40, 5L3-3, MMTV, and HIV LTR promoters), effective poly (A)
termination sequences, an origin of replication for plasmid product
in E. coli, an antibiotic resistance gene as selectable marker,
and/or a convenient cloning site (e.g., a polylinker). Nucleic
acids may also comprise an inducible promoter as opposed to a
constitutive promoter such as CMV IE (the skilled artisan will
recognize that such terms are actually descriptors of a degree of
gene expression under certain conditions).
[0353] In one embodiment, the anti-DR5 antibody-encoding expression
is positioned in and/or delivered to the host cell or host animal
via a viral vector.
[0354] Such expression vectors may be used for recombinant
production of anti-FAS, anti-DR4, anti-DR5, anti-TNFR1, anti-DR6,
anti-DR3, anti-EDAR or anti-NGFR antibodies.
[0355] In one aspect, the anti-FAS, anti-DR4, anti-DR5, anti-TNFR1,
anti-DR6, anti-DR3, anti-EDAR or anti-NGFR antibodies of any aspect
or embodiment described herein are provided by use of recombinant
eukaryotic or prokaryotic host cell which produces the antibody.
Accordingly, the invention provides a recombinant eukaryotic or
prokaryotic host cell, such as a transfectoma, which produces an
anti-DR5 antibody as defined herein. Examples of host cells include
yeast, bacterial and mammalian cells, such as CHO or HEK-293 cells.
For example, in one embodiment, the host cell comprises a nucleic
acid stably integrated into the cellular genome that comprises a
sequence coding for expression of a anti-DR5 antibody described
herein. In one embodiment, the host cell comprises a nucleic acid
stably integrated into the cellular genome that comprise a sequence
coding for expression of a first or a second polypeptide described
herein. In another embodiment, the host cell comprises a
non-integrated nucleic acid, such as a plasmid, cosmid, phagemid,
or linear expression element, which comprises a sequence coding for
expression of an anti-FAS, anti-DR4, anti-DR5, anti-TNFR1,
anti-DR6, anti-DR3, anti-EDAR or anti-NGFR antibody, a first or a
second polypeptide described herein.
[0356] The term "recombinant host cell" (or simply "host cell"), as
used herein, is intended to refer to a cell into which an
expression vector has been introduced. It should be understood that
such terms are intended to refer not only to the particular subject
cell, but also to the progeny of such a cell. Because certain
modifications may occur in succeeding generations due to either
mutation or environmental influences, such progeny may not, in
fact, be identical to the parent cell, but are still included
within the scope of the term "host cell" as used herein.
Recombinant host cells include, for example, transfectomas, such as
CHO cells, HEK-293 cells, PER.C6, NS0 cells, and lymphocytic cells,
and prokaryotic cells such as E. coli and other eukaryotic hosts
such as plant cells and fungi.
[0357] The term "transfectoma", as used herein, includes
recombinant eukaryotic host cells expressing the antibody or a
target antigen, such as CHO cells, PER.C6, NS0 cells, HEK-293
cells, plant cells, or fungi, including yeast cells.
[0358] In a further aspect, the invention relates to a method for
producing an antibody of the invention, said method comprising the
steps of
a) culturing a hybridoma or a host cell of the invention as
described herein above, and b) retrieving and/or purifying the
antibody of the invention from the culture media.
[0359] In a further aspect, the nucleotide sequence encoding a
sequence of an antibody further encodes a second moiety, such as a
therapeutic polypeptide. Exemplary therapeutic antibodies are
described elsewhere herein. In one embodiment, the invention
relates to a method for producing an antibody fusion protein, said
method comprising the steps of
a) culturing a host cell comprising an expression vector comprising
such a nucleotide sequence, and b) retrieving and/or purifying the
antibody fusion protein from the culture media.
[0360] In one aspect of the present invention, the invention
comprises an expression vector comprising on or more nucleic acid
constructs encoding an antibody according to any embodiment
disclosed herein.
[0361] In a further aspect of the invention, the invention
comprises a host cell comprising an expression vector.
TABLE-US-00004 Sequence Table 1 SEQ ID NO: Name Sequence Clone SEQ
ID NO: 1 Fc IgG1m (f) ASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPE
PVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVN
HKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNA KTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSR
EEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGN VFSCSVMHEALHNHYT QKSLSLSPGK SEQ ID NO: 2 Fc
IgG1m(z) ASTKGPSVFPLAPSS KSTSGGTAALGCLV KDYFPEPVTVSWNS
GALTSGVHTFPAVLQ SSGLYSLSSVVTVPS SSLGTQTYICNVNHK PSNTKVDKKVEPKS
CDKTHTCPPCPAPEL LGGPSVFLFPPKPKD TLMISRTPEVTCVVV DVSHEDPEVKFNWY
VDGVEVHNAKTKPR EEQYNSTYRVVSVLT VLHQDWLNGKEYKC KVSNKALPAPIEKTIS
KAKGQPREPQVYTLP PSREEMTKNQVSLT CLVKGFYPSDIAVEW ESNGQPENNYKTTPP
VLDSDGSFFLYSKLT VDKSRWQQGNVFS CSVMHEALHNHYTQ KSLSLSPGK SEQ ID NO: 3
Fc IgG1m(a) ASTKGPSVFPLAPSS KSTSGGTAALGCLV KDYFPEPVTVSWNS
GALTSGVHTFPAVLQ SSGLYSLSSVVTVPS SSLGTQTYICNVNHK PSNTKVDKPVEPKSC
DKTHTCPPCPAPELL GGPSVFLFPPKPKDT LMISRTPEVTCVVVD VSHEDPEVKFNWYV
DGVEVHNAKTKPRE EQYNSTYRVVSVLTV LHQDWLNGKEYKCK VSNKALPAPIEKTISK
AKGQPREPQVYTLPP SRDELTKNQVSLTCL VKGFYPSDIAVEWES NGQPENNYKTTPPVL
DSDGSFFLYSKLTVD KSRWQQGNVFSCS VMHEALHNHYTQKS LSLSPGK SEQ ID NO: 4
Fc IgG1m(x) ASTKGPSVFPLAPSS KSTSGGTAALGCLV KDYFPEPVTVSWNS
GALTSGVHTFPAVLQ SSGLYSLSSVVTVPS SSLGTQTYICNVNHK PSNTKVDKPVEPKSC
DKTHTCPPCPAPELL GGPSVFLFPPKPKDT LMISRTPEVTCVVVD VSHEDPEVKFNWYV
DGVEVHNAKTKPRE EQYNSTYRVVSVLTV LHQDWLNGKEYKCK VSNKALPAPIEKTISK
AKGQPREPQVYTLPP SREEMTKNQVSLTCL VKGFYPSDIAVEWES NGQPENNYKTTPPVL
DSDGSFFLYSKLTVD KSRWQQGNVFSCS VMHEGLHNHYTQKS LSLSPGK SEQ ID NO: 5
Fc IgG1m(f)-E430G ASTKGPSVFPLAPSS KSTSGGTAALGCLV KDYFPEPVTVSWNS
GALTSGVHTFPAVLQ SSGLYSLSSVVTVPS SSLGTQTYICNVNHK PSNTKVDKRVEPKS
CDKTHTCPPCPAPEL LGGPSVFLFPPKPKD TLMISRTPEVTCVVV DVSHEDPEVKFNWY
VDGVEVHNAKTKPR EEQYNSTYRVVSVLT VLHQDWLNGKEYKC KVSNKALPAPIEKTIS
KAKGQPREPQVYTLP PSREEMTKNQVSLT CLVKGFYPSDIAVEW ESNGQPENNYKTTPP
VLDSDGSFFLYSKLT VDKSRWQQGNVFS CSVMHGALHNHYTQ KSLSLSPGK SEQ ID NO: 6
Fc IgG1m(f)-E345K ASTKGPSVFPLAPSS KSTSGGTAALGCLV KDYFPEPVTVSWNS
GALTSGVHTFPAVLQ SSGLYSLSSVVTVPS SSLGTQTYICNVNHK PSNTKVDKRVEPKS
CDKTHTCPPCPAPEL LGGPSVFLFPPKPKD TLMISRTPEVTCVVV DVSHEDPEVKFNWY
VDGVEVHNAKTKPR EEQYNSTYRVVSVLT VLHQDWLNGKEYKC KVSNKALPAPIEKTIS
KAKGQPRKPQVYTL PPSREEMTKNQVSLT CLVKGFYPSDIAVEW ESNGQPENNYKTTPP
VLDSDGSFFLYSKLT VDKSRWQQGNVFS CSVMHEALHNHYTQ KSLSLSPGK SEQ ID NO: 7
Fc IgG1m(f)-S440Y ASTKGPSVFPLAPSS KSTSGGTAALGCLV KDYFPEPVTVSWNS
GALTSGVHTFPAVLQ SSGLYSLSSVVTVPS SSLGTQTYICNVNHK PSNTKVDKRVEPKS
CDKTHTCPPCPAPEL LGGPSVFLFPPKPKD TLMISRTPEVTCVVV DVSHEDPEVKFNWY
VDGVEVHNAKTKPR EEQYNSTYRVVSVLT VLHQDWLNGKEYKC KVSNKALPAPIEKTIS
KAKGQPREPQVYTLP PSREEMTKNQVSLT CLVKGFYPSDIAVEW ESNGQPENNYKTTPP
VLDSDGSFFLYSKLT VDKSRWQQGNVFS CSVMHEALHNHYTQ KYLSLSPGK SEQ ID NO: 8
Fc IgG1m(f)-F405L ASTKGPSVFPLAPSS KSTSGGTAALGCLV KDYFPEPVTVSWNS
GALTSGVHTFPAVLQ SSGLYSLSSVVTVPS SSLGTQTYICNVNHK PSNTKVDKRVEPKS
CDKTHTCPPCPAPEL LGGPSVFLFPPKPKD TLMISRTPEVTCVVV DVSHEDPEVKFNWY
VDGVEVHNAKTKPR EEQYNSTYRVVSVLT VLHQDWLNGKEYKC KVSNKALPAPIEKTIS
KAKGQPREPQVYTLP PSREEMTKNQVSLT CLVKGFYPSDIAVEW ESNGQPENNYKTTPP
VLDSDGSFLLYSKLT VDKSRWQQGNVFS CSVMHEALHNHYTQ KSLSLSPGK SEQ ID NO: 9
Fc IgG1m(f)-K409R ASTKGPSVFPLAPSS KSTSGGTAALGCLV KDYFPEPVTVSWNS
GALTSGVHTFPAVLQ SSGLYSLSSVVTVPS SSLGTQTYICNVNHK PSNTKVDKRVEPKS
CDKTHTCPPCPAPEL LGGPSVFLFPPKPKD TLMISRTPEVTCVVV DVSHEDPEVKFNWY
VDGVEVHNAKTKPR EEQYNSTYRVVSVLT VLHQDWLNGKEYKC KVSNKALPAPIEKTIS
KAKGQPREPQVYTLP PSREEMTKNQVSLT CLVKGFYPSDIAVEW ESNGQPENNYKTTPP
VLDSDGSFFLYSRLT VDKSRWQQGNVFS CSVMHEALHNHYTQ KSLSLSPGK SEQ ID NO:
10 Fc IgG1m(f)-K439E ASTKGPSVFPLAPSS KSTSGGTAALGCLV KDYFPEPVTVSWNS
GALTSGVHTFPAVLQ SSGLYSLSSVVTVPS SSLGTQTYICNVNHK PSNTKVDKRVEPKS
CDKTHTCPPCPAPEL LGGPSVFLFPPKPKD TLMISRTPEVTCVVV DVSHEDPEVKFNWY
VDGVEVHNAKTKPR EEQYNSTYRVVSVLT VLHQDWLNGKEYKC KVSNKALPAPIEKTIS
KAKGQPREPQVYTLP PSREEMTKNQVSLT CLVKGFYPSDIAVEW ESNGQPENNYKTTPP
VLDSDGSFFLYSKLT VDKSRWQQGNVFS CSVMHEALHNHYTQ ESLSLSPGK SEQ ID NO:
11 Fc IgG1m(f)-5440K ASTKGPSVFPLAPSS KSTSGGTAALGCLV KDYFPEPVTVSWNS
GALTSGVHTFPAVLQ SSGLYSLSSVVTVPS SSLGTQTYICNVNHK PSNTKVDKRVEPKS
CDKTHTCPPCPAPEL LGGPSVFLFPPKPKD
TLMISRTPEVTCVVV DVSHEDPEVKFNWY VDGVEVHNAKTKPR EEQYNSTYRVVSVLT
VLHQDWLNGKEYKC KVSNKALPAPIEKTIS KAKGQPREPQVYTLP PSREEMTKNQVSLT
CLVKGFYPSDIAVEW ESNGQPENNYKTTPP VLDSDGSFFLYSKLT VDKSRWQQGNVFS
CSVMHEALHNHYTQ KKLSLSPGK SEQ ID NO: 12 Fc IgG1m(f)-Y436I
ASTKGPSVFPLAPSS KSTSGGTAALGCLV KDYFPEPVTVSWNS GALTSGVHTFPAVLQ
SSGLYSLSSVVTVPS SSLGTQTYICNVNHK PSNTKVDKRVEPKS CDKTHTCPPCPAPEL
LGGPSVFLFPPKPKD TLMISRTPEVTCVVV DVSHEDPEVKFNWY VDGVEVHNAKTKPR
EEQYNSTYRVVSVLT VLHQDWLNGKEYKC KVSNKALPAPIEKTIS KAKGQPREPQVYTLP
PSREEMTKNQVSLT CLVKGFYPSDIAVEW ESNGQPENNYKTTPP VLDSDGSFFLYSKLT
VDKSRWQQGNVFS CSVMHEALHNHITQ KSLSLSPGK SEQ ID NO: 13 VH
DR4-T1014G03 EVQLVQSGAEVKMP GASVKLSCRVSGDT FTAYFIHWVRQAPG
QGLEWMGWFNPISG TAGSAEKFRGRVAM TRDTSISTAYMELNR LTFDDTAVYYCARQH
RGNTFDPWGQGTLV TVSS SEQ ID NO: 14 VL DR4-T1014G03 QSALTQPASVSGSP
GQSITISCTGTSSDI GAYKYVSWYQQHPG KAPKLVIYEVSNRPS GVSSRFSGSKSGQT
ASLTISGLQADDEAD YYCNSYQGYNTWVF GGGTKVTVLG SEQ ID NO: 15 VH FAS-E09
QLQLQESGPGLVKP SETLSLTCTVSGASI SANSYYGVWVRQSP GKGLEWVGSIAYRG
NSNSGSTYYNPSLKS RATVSVDTSKNQVS LRLTSVTAADTALYY CARRQLLDDGTGYQ
WAAFDVWGQGTMV TVSS SEQ ID NO: 16 VL FAS-E09 QSVLTQPPSVSEAPR
QTVTISCSGNSFNIG RYPVNWYQQLPGKA PKLLIYYNNLRFSGV SDRFSGSKSGTSAS
LAIRDLLSEDEADYY CSTWDDTLKGWVF GGGTKVTVL SEQ ID NO: 17 VH hDR5-01
CDR1 GFNIKDTF hDR5-01 SEQ ID NO: 2 VH hDR5-01 CDR2 IDPANGNT SEQ ID
NO: 18 VH hDR5-01 CDR3 VRGLYTYYFDY SEQ ID NO: 19 VH hDR5-01
EVQLQQSGAEVVKPGA SVKLSCKASGFNIKDTFI HWVKQAPGQGLEWIG
RIDPANGNTKYDPKFQ GKATITTDTSSNTAYME LSSLRSEDTAVYYCVRGL
YTYYFDYWGQGTLVTV SS SEQ ID NO: 20 HC-hDR5-01 EVQLQQSGAEVVKPGA
SVKLSCKASGFNIKDTFI HWVKQAPGQGLEWIG RIDPANGNTKYDPKFQ
GKATITTDTSSNTAYME LSSLRSEDTAVYYCVRGL YTYYFDYWGQGTLVTV
SSASTKGPSVFPLAPSSK STSGGTAALGCLVKDYF PEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICN VNHKPSNTKVDKRVEP
KSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNG QPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHN HYTQKSLSLSPGK SEQ ID NO: 21 VL hDR5-01 CDR1 QSISNN
VL hDR5-01 CDR2 FAS SEQ ID NO: 22 VL hDR5-01 CDR3 QQGNSWPYT SEQ ID
NO: 23 VL hDR5-01 EIVMTQSPATLSVSPGE RATLSCRASQSISNNLH
WYQQKPGQAPRLLIKF ASQSITGIPARFSGSGSG TEFTLTISSLQSEDFAVY
YCQQGNSWPYTFGQG TKLEIK SEQ ID NO: 24 LC-hDR5-01 EIVMTQSPATLSVSPGE
RATLSCRASQSISNNLH WYQQKPGQAPRLLIKF ASQSITGIPARFSGSGSG
TEFTLTISSLQSEDFAVY YCQQGNSWPYTFGQG TKLEIKRTVAAPSVFIFPP
SDEQLKSGTASVVCLLN NFYPREAKVQWKVDN ALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSS PVTKSFNRGEC SEQ ID NO: 17 VH
hDR5-01-G56T GFNIKDTF hDR5-01-G56T CDR1 SEQ ID NO: 25 VH
hDR5-01-G56T IDPANTNT CDR2 SEQ ID NO: 19 VH hDR5-01-G56T
VRGLYTYYFDY CDR3 SEQ ID NO: 26 VH hDR5-01-G56T EVQLQQSGAEVVKPGA
SVKLSCKASGFNIKDTFI HWVKQAPGQGLEWIG RIDPANTNTKYDPKFQG
KATITTDTSSNTAYMEL SSLRSEDTAVYYCVRGL YTYYFDYWGQGTLVTV SS SEQ ID NO:
27 HC-hDR5-01-G56T EVQLQQSGAEVVKPGA SVKLSCKASGFNIKDTFI
HWVKQAPGQGLEWIG RIDPANTNTKYDPKFQG KATITTDTSSNTAYMEL
SSLRSEDTAVYYCVRGL YTYYFDYWGQGTLVTV SSASTKGPSVFPLAPSSK
STSGGTAALGCLVKDYF PEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSS
VVTVPSSSLGTQTYICN VNHKPSNTKVDKRVEP KSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKT
ISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLV KGFYPSDIAVEWESNG
QPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHN HYTQKSLSLSPGK
SEQ ID NO: 21 VL hDR5-01-G56T QSISNN CDR1 VL hDR5-01-G56T FAS CDR2
SEQ ID NO: 22 VL hDR5-01-G56T QQGNSWPYT CDR3 SEQ ID NO: 23 VL
hDR5-01-G56T EIVMTQSPATLSVSPGE RATLSCRASQSISNNLH WYQQKPGQAPRLLIKF
ASQSITGIPARFSGSGSG TEFTLTISSLQSEDFAVY YCQQGNSWPYTFGQG TKLEIK SEQ ID
NO: 28 VH hDR5-05 CDR1 GFNIKDTH hDR5-05 SEQ ID NO: 29 VH hDR5-05
CDR2 IDPANGNT SEQ ID NO: 30 VH hDR5-05 CDR3 ARWGTNVYFAY SEQ ID NO:
31 VH hDR5-05 QVQLVQSGAEVKKPGA SVKVSCKASGFNIKDTH MHWVRQAPGQRLEWI
GRIDPANGNTEYDQKF QGRVTITVDTSASTAYM ELSSLRSEDTAVYYCAR
WGTNVYFAYWGQGTL VTVSS SEQ ID NO: 32 HC-hDR5-05 QVQLVQSGAEVKKPGA
SVKVSCKASGFNIKDTH MHWVRQAPGQRLEWI GRIDPANGNTEYDQKF
QGRVTITVDTSASTAYM ELSSLRSEDTAVYYCAR WGTNVYFAYWGQGTL
VTVSSASTKGPSVFPLA PSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKR
VEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDV
SHEDPEVKFNWYVDGV EVHNAKTKPREEQYNST YRVVSVLTVLHQDWLN
GKEYKCKVSNKALPAPI EKTISKAKGQPREPQVY TLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWES NGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK SEQ ID NO: 33 VL
hDR5-05 CDR1 SSVSY VL hDR5-05 CDR2 RTS SEQ ID NO: 34 VL hDR5-05
CDR3 QQYHSYPPT SEQ ID NO: 35 VL hDR5-05 DIQLTQSPSSLSASVGD
RVTITCSASSSVSYMYW YQQKPGKAPKPWIYRT SNLASGVPSRFSGSGSG
TDFTLTISSLQPEDFATY YCQQYHSYPPTFGGGT KVEIK SEQ ID NO: 36 LC-hDR5-05
DIQLTQSPSSLSASVGD RVTITCSASSSVSYMYW YQQKPGKAPKPWIYRT
SNLASGVPSRFSGSGSG TDFTLTISSLQPEDFATY YCQQYHSYPPTFGGGT
KVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLN NFYPREAKVQWKVDN
ALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSS PVTKSFNRGEC
SEQ ID NO: 37 VH CONA-CDR1 GGSISSGDYF Conatumumab IgG1-DR5-CONA SEQ
ID NO: 38 VH CONA-CDR2 IHNSGTT SEQ ID NO: 39 VH CONA-CDR3
ARDRGGDYYYGMDV SEQ ID NO: 40 VH CONA QVQLQESGPGLVKPSQ
TLSLTCTVSGGSISSGDY FWSWIRQLPGKGLECIG HIHNSGTTYYNPSLKSR
VTISVDTSKKQFSLRLSS VTAADTAVYYCARDRG GDYYYGMDVWGQGTT VTVSS SEQ ID
NO: 41 VL CONA-CDR1 QGISRSY VL CONA-CDR2 GAS SEQ ID NO: 42 VL
CONA-CDR3 QQFGSSPWT SEQ ID NO: 43 VL CONA EIVLTQSPGTLSLSPGER
ATLSCRASQGISRSYLA WYQQKPGQAPSLLIYG ASSRATGIPDRFSGSGS
GTDFTLTISRLEPEDFAV YYCQQFGSSPWTFGQG TKVEIK SEQ ID NO: 44 VH
DR5-chTRA8 CDR1 GFTFSSYV SEQ ID NO: 45 VH DR5-chTRA8 CDR2 ISSGGSYT
SEQ ID NO: 46 VH DR5-chTRA8 CDR3 ARRGDSMITTDY SEQ ID NO: 47
HC-DR5-chTRA8 EVMLVESGGGLVKP GGSLKLSCAASGFT FSSYVMSWVRQTPE
KRLEWVATISSGGS YTYYPDSVKGRFTIS RDNAKNTLYLQMSS LRSEDTAMYYCARR
GDSMITTDYWGQG TTLTVSSASTKGPSV FPLAPSSKSTSGGTA ALGCLVKDYFPEPVT
VSWNSGALTSGVHT FPAVLQSSGLYSLSS VVTVPSSSLGTQTYI CNVNHKPSNTKVDK
RVEPKSCDKTHTCPP CPAPELLGGPSVFLF PPKPKDTLMISRTPE VTCVVVDVSHEDPE
VKFNWYVDGVEVHN AKTKPREEQYNSTYR VVSVLTVLHQDWLN GKEYKCKVSNKALPA
PIEKTISKAKGQPRE PQVYTLPPSREEMTK NQVSLTCLVKGFYPS DIAVEWESNGQPEN
NYKTTPPVLDSDGSF FLYSKLTVDKSRWQ QGNVFSCSVMHEAL HNHYTQKSLSLSPG K SEQ
ID NO: 48 VL DR5-chTRA8 CDR1 QDVGTA SEQ ID NO: VL DR5-chTRA8 CDR2
WAS SEQ ID NO: 49 VL DR5-chTRA8 CDR3 QQYSSYRT SEQ ID NO: 50
LC-DR5-chTRA8 DIVMTQSHKFMSTS VGDRVSITCKASQD VGTAVAWYQQKPG
QSPKLLIYWASTRH TGVPDRFTGSGSGT DFTLTISNVQSEDLA DYFCQQYSSYRTFG
GGTKLEIKRTVAAPS VFIFPPSDEQLKSGT ASVVCLLNNFYPREA KVQWKVDNALQSG
NSQESVTEQDSKDS TYSLSSTLTLSKADY EKHKVYACEVTHQG LSSPVTKSFNRGEC
EXAMPLES
Example 1: Antibodies and Antigens
[0362] Expression constructs for antibodies For antibody expression
variable heavy (VH) chain and Variable light (VL) chain sequences
were cloned in pcDNA3.3 expression vectors containing IgG1 heavy
chain (HC) and light chain (LC) constant regions. Desired mutations
were introduced either by gene synthesis or site directed
mutagenesis. Antibodies mentioned in this application have VH and
VL sequences derived from previously described chimeric human/mouse
DR5 antibodies DR5-01 and DR5-05 (based on EP2684896A1), humanized
DR5 antibodies hDR5-01 and hDR5-05 (based on WO2014/009358),
IgG1-CONA (based on U.S. Pat. No. 7,521,048 B2 and WO2010/138725),
IgG1-chTRA8 (based on EP1506285B1 and U.S. Pat. No. 7,244,429B2),
IgG1-DR5-H48-2 (based on US 2004 0214235 A1), IgG1-DR4-T1014G03
(based on U.S. Pat. No. 7,361,341), and IgG1-FAS-E09 (based on
Chodorge et al., Cell Death Differ. 2012 July; 19(7): 1187-1195).
In some of the examples the human IgG1 antibody b12, a
gp120-specific antibody was used as a negative control (Barbas et
al., J Mol Biol. 1993 Apr. 5; 230(3):812-23).
Transient Expression
[0363] Antibodies were expressed as IgG1,K. Plasmid DNA mixtures
encoding both heavy and light chains of antibodies were transiently
transfected in Expi293F cells (Life technologies, USA) using
293fectin (Life technologies) essentially as described by Vink et
al. (Vink et al., Methods, 65 (1), 5-10 2014).
Purification and Analysis of Proteins
[0364] Antibodies were purified by immobilized protein G
chromatography. His-tagged recombinant protein was purified by
immobilized metal affinity chromatography. Protein batches were
analyzed by a number of bioanalytical assays including SDS-PAGE,
size exclusion chromatography and measurement of endotoxin
levels.
Generation of Bispecific Antibodies
[0365] Bispecific IgG1 antibodies were generated by
Fab-arm-exchange under controlled reducing conditions. The basis
for this method is the use of complimentary CH3 domains, which
promote the formation of heterodimers under specific assay
conditions as described in WO2011/131746 (Labrijn et al., Proc Natl
Acad Sci USA. 2013 Mar. 26; 110(13):5145-50). To create antibody
pairs with complementary CH3 domains, the F405L mutation (EU
numbering) was introduced in IgG1-DR5-05, IgG1-DR5-05-E430G and
IgG1-DR5-05-E345K; and the K409R mutation was introduced in
IgG1-DR5-01, IgG1-DR5-01-E430G, IgG1-DR5-01-E345K and
IgG1-CONA-E430G. To generate bispecific antibodies, two parental
complementary antibodies, each antibody at a final concentration of
0.5 mg/mL, were incubated with 75 mM 2-mercaptoethylamine-HCl
(2-MEA) in a total volume of 100 .mu.L TE at 31.degree. C. for 5
hours. The reduction reaction was stopped by removing the reducing
agent 2-MEA using spin columns (Microcon centrifugal filters, 30 k,
Millipore) according to the manufacturer's protocol. In this way
the bispecific antibodies IgG1-DR5-01-K409R.times.IgG1-DR5-05-F405L
(BsAb DR5-01-K409R.times.DR5-05-F405L),
IgG1-DR5-01-K409R-E430G.times.IgG1-DR5-05-F405L-E430G (BsAb
DR5-01-K409R-E430G.times.DR5-05-F405L-E430G),
IgG1-DR5-01-K409R-E345K.times.IgG1-DR5-05-F405L-E345K (BsAb
DR5-01-K409R-E345K.times.DR5-05-F405L-E345K) and
IgG1-DR5-CONA-K409R-E430G.times.IgG1-DR5-05-F405L-E345K (BsAb
DR5-CONA-K409R-E430G.times.DR5-05-F405L-E345K) were generated.
Example 2: Introduction of a Hexamerization-Enhancing Mutation does
not Affect Binding of IgG1-DR5-01-K409R, IgG1-DR5-05-F405L and
Bispecific Antibody IgG1-DR5-01-K409R.times.DR5-05-F405L to
DR5-Positive Human Colon Cancer Cells
[0366] Binding of purified antibody variants of IgG1-DR5-01-K409R,
IgG1-DR5-05-F405L and bispecific antibody
IgG1-DR5-01-K409R.times.IgG1-DR5-05-F405L (BsAb
DR5-01-K409R.times.DR5-05-F405L) with and without a
hexamerization-enhancing mutation (E430G or E345K) to human colon
cancer cells COLO 205 was analyzed by FACS analysis. Cells were
harvested by pooling the culture supernatant containing
non-adherent cells and trypsinized adherent COLO 205 cells. Cells
were centrifuged for 5 minutes at 1,200 rpm and resuspended in 10
mL culture medium [RPMI 1640 with 25 mM Hepes and L-Glutamine
(Lonza Cat nr BE12-115F)+10% Donor Bovine Serum with Iron (Life
Technologies Cat nr 10371-029)+50 Units Penicillin/50 Units
Streptomycin (Lonza Cat nr DE17-603E)]. Cells were counted,
centrifuged again and resuspended in FACS buffer at a concentration
of 0.3.times.10.sup.6 cells/mL. The next steps were performed at
4.degree. C. 100 .mu.L cell suspension samples (30,000 cells per
well) were seeded in polystyrene 96-well round-bottom plates and
pelleted by centrifugation at 300.times.g for 3 minutes at
4.degree. C. Cells were resuspended in 50 .mu.L samples of a serial
dilution antibody preparation series (range 0 to 10 .mu.g/mL final
concentrations in 5-fold dilutions) and incubated for 30 minutes at
4.degree. C. Plates were centrifuged at 300.times.g for 3 minutes
at 4.degree. C. and cells were washed twice with 150 .mu.L FACS
buffer. Cells were incubated with 50 .mu.L secondary antibody
R-PE-conjugated goat-anti-human IgG F(ab')2 (Jackson
ImmunoResearch; Cat nr 109-116-098; 1/100) for 30 minutes at
4.degree. C. protected from light. Cells were washed twice with 150
.mu.L FACS buffer, resuspended in 100 .mu.L FACS buffer, and
antibody binding was analyzed on a FACS Canto II (BD Biosciences)
by recording 5,000 events. Binding curves were analyzed using
non-linear regression analysis (sigmoidal dose-response with
variable slope) using GraphPad Prism software.
[0367] FIG. 2A shows that the antibodies IgG1-DR5-01-K409R-E430G
and IgG1-DR5-01-K409R-E345K showed similar dose-dependent binding
to human colon cancer cells COLO 205 as IgG1-DR5-01-K409R. FIG. 2B
shows that the antibodies IgG1-DR5-05-F405L-E430G and
IgG1-DR5-05-F405L-E345K showed similar dose-dependent binding to
COLO 205 cells as IgG1-DR5-05-F405L. FIG. 2C shows that BsAb
DR5-01-K409R-E430G.times.DR5-05-F405L-E430G and BsAb
DR5-01-K409R-E345K.times.DR5-05-F405L-E345K showed similar
dose-dependent binding to COLO 205 cells as BsAb
DR5-01-K409R.times.DR5-05-F405L. These data indicate that
introduction of the hexamerization-enhancing mutations E430G or
E345K did not affect binding of antibodies IgG1-DR5-01-K409R,
IgG1-DR5-05-F405L and BsAb DR5-01-K409R.times.DR5-05-F405L on
DR5-positive COLO 205 cells.
Example 3: Introduction of a Hexamerization-Enhancing Mutation does
not Affect Binding of DR4 Antibody to Soluble Human DR4
[0368] Binding of purified antibody variants of IgG1-DR4-T1014G03
with and without hexamerization-enhancing mutation E430G to coated
human soluble DR4 was analyzed in a sandwich enzyme-linked
immunosorbent assay (ELISA). 96-well flat bottom ELISA plates
(Greiner bio-one; Cat nr 655092) were coated overnight at 4.degree.
C. with 2 .mu.g/mL sTRAIL-R1 (Peprotech cat nr 310-18) in 100 .mu.L
PBS. The wells were washed three times with PBST [PBS with 0.05%
Tween-20 (Sigma-Aldrich; Cat nr 63158)]. The wells were blocked by
adding 200 .mu.L PBSA [PBS with 1% Bovine Serum Albumin (BSA; Roche
Cat #10735086001)] and incubated for 1 hour at room temperature
while shaking. The wells were washed three times with PBST. Next,
antibody samples of IgG1-DR4-T1014G03-K409R or
IgG1-DR4-T1014G03-K409R-E430G (range 0 to 2,000 ng/mL final
concentrations in 3-fold dilutions) were added in a total volume of
100 .mu.L PBSTA (PBST with 0.2% BSA) and incubated for 1.5 hour at
room temperature while shaking. After washing three times with
PBST, wells were incubated on an ELISA shaker with 100 .mu.L
Horseradish Peroxidase (HRP)-conjugated goat anti-human IgG
Fc.gamma. antibody (Jackson ImmunoResearch; Cat nr. 109-035-098;
1:10.000) in PBSTA for 1.5 hour at room temperature. After washing
three times with PBST, the reaction was visualized through an
incubation with 100 .mu.L 2,2'-azino-bis
(3-ethylbenzothiazoline-6-sulfonic acid [ABTS (Roche; Cat nr
11112597001)] at RT protected from light. Fluorescence at 405 nm
was measured on an ELISA reader (BioTek ELx808 Absorbance
Microplate Reader).
[0369] FIG. 3 shows that the antibodies IgG1-DR4-T1014G3-K409R and
IgG1-DR4-T1014G3-K409R-E430G showed similar dose-dependent binding
to coated soluble receptor, indicating that introduction of the
hexamerization-enhancing mutation E430G did not affect binding of
the antibody to its target.
Example 4: Introduction of a Hexamerization-Enhancing Mutation
Improves the Efficacy of Cell Death Induction by DR5 Antibodies
[0370] Viability assays were performed to study the effect of
introducing hexamerization-enhancing mutation E345K or E430G in
different DR5 antibodies to induce killing of human colon cancer
cells COLO 205 or HCT116. COLO 205 cells were harvested by pooling
the culture supernatant containing non-adherent cells and
trypsinized adherent cells. HCT 116 cells were harvested by
trypsinization. Cells were passed through a cell strainer, pelleted
by centrifugation for 5 minutes at 1,200 rpm and resuspended in
culture medium at a concentration of 0.5.times.10.sup.5 cells/mL
[COLO 205: RPMI 1640 with 25 mM Hepes and L-Glutamine (Lonza Cat nr
BE12-115F)+10% Donor Bovine Serum with Iron (DBSI; Life
Technologies Cat nr 10371-029)+50 Units Penicillin/50 Units
Streptomycin (Pen/Strep; Lonza Cat nr DE17-603E); HCT 116:
McCoy's5A Medium with L-Glutamine and Hepes (Lonza, Cat nr
BE12-168F)+10% DBSI+Pen/Strep]. 100 .mu.L of the single cell
suspension (5,000 cells per well) was seeded in polystyrene 96-well
flat-bottom plates (Greiner Bio-One, Cat nr 655182). 50 .mu.L of
serial dilution antibody preparation series (range 0.05 to 20,000
ng/mL final concentrations in 5-fold dilutions) were added and
incubated for 3 days at 37.degree. C. The viability of the cultured
cells was determined in a CellTiter-Glo luminescent cell viability
assay (Promega, Cat nr G7571) that quantifies the ATP present,
which is an indicator of metabolically active cells. From the kit,
20 .mu.L luciferin solution reagent was added per well and mixed by
shaking the plate for 2 minutes at 500 rpm. Next, plates were
incubated for 1.5 hours at 37.degree. C. 100 .mu.L supernatant was
transferred to a white OptiPlate-96 (Perkin Elmer, Cat nr 6005299)
and luminescence was measured on an EnVision Multilabel Reader
(PerkinElmer). Data were analyzed and plotted using non-linear
regression (sigmoidal dose-response with variable slope) using
GraphPad Prism software. When samples with 5 .mu.M staurosporine
(Sigma Aldrich, Cat nr S6942) were included as positive control,
the percentage viable cells was calculated using the following
formula: % viable cells=[(luminescence antibody sample-luminescence
staurosporine sample)/(luminescence no antibody sample-luminescence
staurosporine sample)]*100. For the experiments where no
staurosporine control sample was included, data are presented as
Luminescence.
[0371] E345K variants of IgG1-DR5-01-K409R and IgG1-DR5-05-F405L
were tested on COLO 205. E430G-variants of IgG1-DR5-01-K409R,
IgG1-DR5-05-F405L and IgG1-CONA-K409R were tested on both COLO 205
and HCT116 cells. IgG1-CONA was also tested as RGY-variant, a
triple mutant E345K/E430G/S440Y that occurs as hexamer in solution
(Diebolder et al., Science. 2014 Mar. 14; 343(6176):1260-3).
IgG1-H48-2-F405L and IgG1-DR5-chTRA8-F405L were tested as
E430G-variant on HCT116 cells. FIG. 4 shows that introduction of
the hexamerization-enhancing mutations enhanced the potency of the
different DR5 antibodies in COLO 205 and HCT 116 colon cancer
cells.
Example 5: Introduction of a Hexamerization-Enhancing Mutation
Improves the Efficacy of a DR4 Antibody to Induce Cell Death
[0372] A viability assay was performed to study the effect of
introducing hexamerization-enhancing mutation E430G in DR4 antibody
IgG1-DR4-T1014G03-K409R to induce killing of BxPC-3 human
pancreatic cancer cells. Cells were harvested by trypsinization and
passed through a cell strainer. Cells were pelleted by
centrifugation for 5 minutes at 1,200 rpm and resuspended in
culture medium at a concentration of 0.5.times.10.sup.5 cells/mL
[RPMI 1640 with 25 mM Hepes and L-Glutamine (Lonza Cat nr
BE12-115F)+10% DBSI+Pen/Strep]. 100 .mu.L of the single cell
suspensions (5,000 cells per well) were seeded in polystyrene
96-well flat-bottom plates (Greiner Bio-One, Cat nr 655182) and
incubated overnight at 37.degree. C. 50 .mu.L of serial dilution
antibody preparation series (range 0.0006 to 40 .mu.g/mL final
concentrations in 4-fold dilutions) were added and incubated for 3
days at 37.degree. C. As a negative and positive control, cells
were incubated without antibody or with 5 .mu.M staurosporine,
respectively. The viability of the cultured cells was determined in
a CellTiter-Glo luminescent cell viability assay as described in
Example 4. FIG. 5 shows that introduction of the
hexamerization-enhancing mutation E430G enabled the DR4 antibody
IgG1-DR4-T1014G03-K409R-E430G to induce dose-dependent killing of
BxPC-3 pancreatic cancer cells, whereas the antibody without the
E430G mutation was unable to induce killing at the tested antibody
concentrations.
Example 6: Introduction of Hexamerization-Enhancing Mutations
Improves the Efficacy of Cell Death Induction by a FAS Antibody
[0373] A viability assays was performed to study the effect of
introducing hexamerization-enhancing mutations E345K/E430G/S440Y
(RGY) in the FAS antibody IgG1-FAS-E09 to induce killing of Jurkat
human T lymphocytes (ATTC TIB-152.TM.). Jurkat cells were harvested
and resuspended in culture medium at a concentration of
0.3.times.10.sup.6 cells/mL (RPMI 1640 with 25 mM Hepes and
L-Glutamine+10% Cosmic Calf Serum (CCS, Perbio Cat nr
SH30087.03)+Pen/Strep). 100 .mu.L of the single cell suspension
(30,000 cells per well) was seeded in polystyrene 96-well
flat-bottom plates (Greiner Bio-One, Cat nr 655182). 50 .mu.L of
serial dilution antibody preparation series (range 0.005 to 10,000
ng/mL final concentrations in 5-fold dilutions) were added and
incubated for 3 days at 37.degree. C. The viability of the cultured
cells was determined by TOPRO-3 iodine. TOPRO-3 binds to DNA but
cannot pass intact plasma and nuclear membranes and will therefore
only stain dying cells that have decreased membrane integrity.
Cells were resuspended and transferred to a U-bottom 96-Wells plate
(Greiner, Cat nr 650101). Cells were pelleted by centrifugation for
3 minutes at 300.times.g and washed with 150 .mu.L FACS buffer.
Cells were pelleted by centrifugation for 3 minutes at 300.times.g
and resuspended in 100 .mu.L FACS buffer supplemented with TOPRO-3
iodine (1:1,000; final concentration 1 .mu.M; Life Technologies,
Cat nr T3605). TOPRO-3 staining was analyzed on a FACS Canto II (BD
Biosciences) by recording 20,000 events. Data were analyzed and
plotted using non-linear regression (sigmoidal dose-response with
variable slope) using GraphPad Prism software. FIG. 6 shows the
percentage viable cells, as calculated from the percentage
TOPRO-3-negative cells. Introduction of the
hexamerization-enhancing mutations RGY enabled the FAS antibody
IgG1-FAS-E09 to induce dose-dependent killing of Jurkat human T
lymphocytes, whereas the antibody without the E345R/E430G/S440Y
triple mutation was unable to induce killing at the tested antibody
concentrations.
Example 7: Introduction of Hexamerization-Enhancing Mutations
Improves the Efficacy of Cell Death Induction by the Antibody
Combination IgG1-DR5-01-K409R+IgG1-DR5-05-F405L and by the BsAb
DR5-01-K409R.times.DR5-05-F405L
[0374] The effect of the hexamerization-enhancing mutation E345K or
E430G on the capacity of the antibody combination
IgG1-DR5-01-K409R+IgG1-DR5-05-F405L to kill human colon cancer
cells COLO 205 and HCT116 was studied in a viability assay as
described in Example 4. Also the effect of introducing the E345K or
E430G mutation in the BsAb DR5-01-K409R.times.DR5-05-F405L was
tested on COLO 205 or HCT116. FIG. 7 shows that the antibody
combinations IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G and
IgG1-DR5-01-K409R-E345K+IgG1-DR5-05-F405L-E345K showed enhanced
potency compared to the antibody combination
IgG1-DR5-01-K409R+IgG1-DR5-05-F405L without the E345K or E430G
hexamerization-enhancing mutation on both COLO 205 (FIG. 7 A) and
HCT116 cells (FIG. 7 C). The BsAb
DR5-01-K409R-E430G.times.DR5-05-F405L-E430G also showed enhanced
potency compared to the BsAb DR5-01-K409R.times.DR5-05-F405L
without the E430G hexamerization-enhancing mutation on both COLO
205 (FIG. 7 B) and HCT116 cells (FIG. 7 D). The BsAb
DR5-01-K409R-E345K.times.DR5-05-F405L-E345K showed enhanced potency
compared to the BsAb DR5-01-K409R.times.DR5-05-F405L without the
E430G hexamerization-enhancing mutation on HCT116 cells (FIG. 7
E)
Example 8: Introduction of a Hexamerization-Enhancing Mutation
Improves the Efficacy of Cell Death Induction by the Combination of
IgG1-hDR5-01-G56T+IgG1-hDR5-05 Antibodies
[0375] The effect of the hexamerization-enhancing mutation E430G on
the capacity of the antibody combination
IgG1-hDR5-01-G56T+IgG1-hDR5-05 to kill HCT15 colon and BxPC-3
pancreatic cancer cells was studied in a viability assay. Cells
were harvested by trypsinization and passed through a cell
strainer. Cells were pelleted by centrifugation for 5 minutes at
1,200 rpm and resuspended in culture medium at a concentration of
0.5.times.10.sup.5 cells/mL (RPMI 1640 with 25 mM Hepes and
L-Glutamine (Lonza Cat nr BE12-115F)+10% DBSI+Pen/Strep). 100 .mu.L
of the single cell suspensions (5,000 cells per well) were seeded
in polystyrene 96-well flat-bottom plates (Greiner Bio-One, Cat nr
655182) and incubated overnight at 37.degree. C. 50 .mu.L antibody
samples of serial dilution antibody preparation series (range 0.3
to 20,000 ng/mL final concentrations in 4-fold dilutions) were
added and incubated for 3 days at 37.degree. C. As negative and
positive control, cells were incubated without antibody and with 5
.mu.M staurosporine, respectively. The viability of the cell
cultures was determined in a CellTiter-Glo luminescent cell
viability assay as described in Example 4. FIG. 8 shows that the
antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G
showed dose-dependent killing on both BxPC-3 (FIG. 8A) and HCT15
cells (FIG. 8B), whereas the antibody combination without the E430G
hexamerization-enhancing mutation induced little to no killing at
the tested antibody concentrations.
Example 9: Cell Death Induction by the Antibody Combination
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G Requires Fc-Fc
Interactions to Form Hexamers
[0376] To analyze the requirement of antibody hexamer formation by
IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G to induce cell
death, we made use of the self-repulsing mutations K439E and S440K
(Diebolder et al., Science. 2014 Mar. 14; 343(6176):1260-3). The Fc
repulsion between antibodies that is introduced by the presence of
either K439E or S440K in one IgG1 antibody or a combination of
antibodies results in inhibition of hexamerization, even in the
presence of a hexamerization enhancing mutation such as E345K or
E430G (WO2013/0044842). The repulsion by the K439E and S440K
mutations is neutralized by combining both mutations in a mixture
of two antibodies each harboring one or the other mutation,
resulting in restoration of the Fc-Fc interactions and
hexamerization.
[0377] For both IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G,
variants with either the K439E or S440K mutation were generated and
tested in all different combinations. A viability assay was
performed with serial dilution antibody preparation series ranging
from 0.3 to 20,000 ng/mL total concentrations in 4-fold dilutions
on BxPC-3 pancreatic and HCT-15 colon cancer cells as described in
Example 4.
[0378] FIG. 9 shows that the antibody combinations with
IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G variants harboring
both the same repulsion mutation (K439E or S440K) showed strongly
diminished killing efficacy in BxPC-3 (FIG. 9A) and HCT-15 cells
(FIG. 9B). Killing efficacy was restored when repulsion was
neutralized by combining two antibodies each having one of the
complementary mutations K439E or S440K. These data indicate that
hexamerization by Fc-Fc interactions is required for the induction
of cell death by IgG1-hDR5-01-G56T-E430G and
IgG1-hDR5-05-E430G.
Example 10: Antibody Fc-Fc Interactions are Involved in DR5
Clustering and Induction of Apoptosis by the Antibody Combination
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G with Hexamerization
Enhancing Mutations
[0379] To test the involvement of Fc-Fc-mediated antibody
hexamerization in the induction of cell death by the antibody
combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G, we made use
of the 13-residue peptide DCAWHLGELVWCT (DeLano et al., Science
2000 Feb. 18; 287(5456):1279-83) that binds the Fc in a region
containing the core amino acids in the hydrophobic patch that are
involved in Fc-Fc interactions (Diebolder et al., Science. 2014
Mar. 14; 343(6176):1260-3). A viability assay on BxPC-3 cells was
performed as described in Example 4 for the antibody combination
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G in presence or absence
of the DCAWHLGELVWCT peptide. Briefly, after overnight incubation
of the cells at 37.degree. C., culture medium was removed and
replaced by 100 .mu.L culture medium containing serial diluted
peptide concentrations (range 0 to 100 .mu.g/mL final
concentrations) of the Fc-binding DCAWHLGELVWCT peptide, a
non-specific control peptide GWTVFQKRLDGSV, or no peptide. Next, 50
.mu.L antibody samples (833 ng/mL final concentration) were added
and incubated for 3 days at 37.degree. C. The capacity of the
antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G to
induce killing of BxPC-3 cells was strongly inhibited by 100
.mu.g/mL Fc-binding DCAWHLGELVWCT peptide (FIG. 10). These data
indicate the involvement of Fc interactions in the capacity of the
antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G
with hexamerization-enhancing mutations to induce DR5 clustering on
the cell surface of cancer cells and induction of apoptosis.
Example 11: Capacity of the Antibody Combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F4051-E430G to Induce Target
Cell Killing in Different Cancer Cell Lines
[0380] A viability assay was performed to study the capacity of the
antibody combination IgG1-DR5-01-K409R+IgG1-DR5-05-F405L with and
without the hexamerization-enhancing mutation E430G to induce
killing of COLO 205, HCT-15, HCT 116, HT-29 and SW480 colon cancer,
BxPC-3, HPAF-II and PANC-1 pancreatic cancer, SNU-5 gastric cancer,
A549 and SK-MES-1 lung cancer, and A375 skin cancer cells. The
assay was performed as described in Example 4, with the exception
that here a fixed antibody concentration of 10 .mu.g/mL was used.
Medium compositions of cell lines not previously described are as
follows: SW480: RPMI 1640 with 25 mM Hepes and L-Glutamine+10%
DBSI+Pen/Strep; HT-29: McCoy's5A Medium with L-Glutamine and
Hepes+10% DBSI+Pen/Strep; HPAF-II and SK-MES-1: Eagle's Minimum
Essential Medium (EMEM, ATCC Cat nr 30-2003)+10% DBSI+Pen/Strep;
PANC-1 and A375: DMEM 4.5 g/L Glucose without L-Gln with HEPES
(Lonza Cat nr LO BE12-709F)+10% DBSI+1 mM L-Glutamine (Lonza Cat nr
BE17-605E)+Pen/Strep; SNU-5: IMDM (Lonza Cat nr BE12-722F)+10%
DBSI+Pen/Strep; A549: F-12K Medium (ATCC Cat nr 30-2004)+10% DBSI+1
mM L-Glutamine+Pen/Strep).
[0381] For all tested cell lines, the percentage viable cells was
significant lower after incubation with 10 .mu.g/mL of the antibody
combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G than
after incubation with the non-target binding negative control
antibody IgG1-b12 (FIG. 11). In all but two of the tested cell
lines, the efficacy of the antibody combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was significant
better than for the combination IgG1-DR5-01-K409R+IgG1-DR5-05-F405L
without hexamerization-enhancing mutation. These data indicate that
the combination of DR5 antibodies with hexamerization-enhancing
mutations IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was very
effective in killing of cancer target cells of different origin,
including colon, pancreatic, gastric, lung and skin cancer, without
the requirement of a secondary cross-linking agent.
Example 12: Introduction of Hexamerization-Enhancing Mutations
Improves the Efficacy of Cell Death Induction by the Antibody
Combination IgG1-CONA-K409R+IgG1-DR5-05-F405L and BsAb
CONA-K409R.times.DR5-05-F405L
[0382] The effect of the hexamerization-enhancing mutations on the
capacity of the antibody combination IgG1-DR5-01-K409R+IgG1-CONA
and BsAb CONA-K409R.times.DR5-05-F405L to kill HCT116 colon cancer
cells was studied in a viability assay as described in Example 4.
FIG. 12 shows that the antibody combination
IgG1-CONA-K409R-E430G+IgG1-DR5-05-F405L-E345K and BsAb
CONA-K409R-E430G.times.DR5-05-F405L-E345K with
hexamerization-enhancing mutations showed enhanced efficacy in
killing of HCT116 cells compared to the combination and bispecific
antibody without the hexamerization-enhancing mutations E430G or
E345K.
Example 13: The Potency of Antibody Combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F4051-E430G and BsAb
DR5-01-K409R-E430G.times.DR5-05-F4051-E430G with
Hexamerization-Enhancing Mutations is Independent of Fc.gamma.R
Binding by a Secondary Crosslinker
[0383] A viability assay was performed to compare the capacity of
the antibody combinations with hexamerization mutation in the
absence and presence of secondary antibody crosslinker to induce
killing of COLO 205 colorectal and BxPC-3 and PANC-1 pancreatic
cancer cells. For comparison, the DR5 antibodies IgG1-CONA and
IgG1-chTRA8-F405L that are known to require a secondary antibody
crosslinker to induce killing, were tested in the same settings.
The viability assay was performed as described in Example 4 in the
absence or presence of goat-anti-human IgG F(ab')2 (1/150; Jackson
ImmunoResearch; Cat nr 109-006-098). DR5 antibodies IgG1-CONA and
IgG1-chTRA8-F405L did not induce target cell killing in the absence
of an Fc crosslinker (FIG. 13). Fc crosslinking induced killing by
IgG1-DR5-CONA and IgG1-DR5-chTRA8-F405L in COLO 205 and BxPC-3
cells The antibody combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G and BsAb
DR5-01-K409R-E430G.times.DR5-05-F405L-E430G induced significant
killing compared to the negative control, both in presence or
absence of a secondary Fc crosslinker. These data indicate that
killing of COLO 205, BxPC-3 and PANC-1 cancer cells by the antibody
combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G and
BsAb DR5-01-K409R-E430G.times.DR5-05-F405L-E430G is independent of
Fc.gamma.R-mediated binding by a secondary Fc crosslinker and that
this crosslinker-independent killing is more efficient than for
Fc.gamma.R-crosslinked IgG1-DR5-CONA and IgG1-DR5-chTRA8-F405L.
Example 14: The Antibody Combination
IgG1-hDR5-01-E430G+IgG1-hDR5-05-E430G with the E430G
Hexamerization-Enhancing Mutation Induce Caspase-Dependent
Cytotoxicity
[0384] A viability assay was performed to compare the cytotoxicity
of the combination of humanized antibodies
IgG1-hDR5-01-E430G+IgG1-hDR5-05-E430G in the presence and absence
of a caspase inhibitor. PANC-1 and BxPC3 pancreatic cancer cells
were harvested by trypsinization and passed through a cell
strainer. Cells were pelleted by centrifugation for 5 minutes at
1,200 rpm and resuspended in culture medium at a concentration of
0.5.times.10.sup.5 cells/mL. 100 .mu.L of the single cell
suspensions (5,000 cells per well) were seeded in polystyrene
96-well flat-bottom plates (Greiner Bio-One, Cat nr 655182) and
incubated overnight at 37.degree. C. 25 .mu.L pan-caspase inhibitor
Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD-FMK, 5 .mu.M end
concentration in 150 .mu.L, Bachem, Cat nr 4026865.0005) was added
to the cell cultures and incubated for one hour at 37.degree. C.
before adding 25 .mu.L antibody sample of a serial dilution
antibody preparation series (range 1 to 20 .mu.g/mL final
concentrations in 4-fold dilutions) and further incubation for 3
days at 37.degree. C. As a positive control, cells were incubated
with 5 .mu.M staurosporine (Sigma Aldrich, Cat nr S6942).
Recombinant human TRAIL/APO-2L (eBioscience, Cat nr BMS356) was
used at 6 .mu.g/mL final concentration. The viability of the cell
cultures was determined in a CellTiter-Glo luminescent cell
viability assay as described in Example 4. The antibody combination
with hexamerization-enhancing mutations
IgG1-hDR5-01-E430G+IgG1-hDR5-05-E430G was unable to reduce the
viability of PANC-1 and BxPC3 pancreatic cancer cells in presence
of the pan-caspase inhibitor Z-VAD-FMK, indicating that the
combination of IgG1-hDR5-01-E430G+IgG1-hDR5-05-E430G induced
caspase-dependent programmed cell death (FIG. 14). This was also
shown for the natural DR5 ligand TRAIL.
Example 15: Cell Death Induction Upon Binding of the Antibody
Combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F4051-E430G and
BsAb DR5-01-K409R-E430G.times.DR5-05-F4051-E430G on COLO 205 Colon
Cancer Cells, as Assessed by Annexin V/Propidium Iodide and Active
Caspase-3 Staining
[0385] The kinetics of cell death induction was analyzed by Annexin
V/Propidium Iodide (PI) double staining and active caspase-3
staining. Annexin-V binds phosphatidylserine that is exposed on the
cell surface after initiation of programmed cell death, which is a
reversible process. PI is a dye that intercalates into
double-stranded DNA and RNA when it enters cells. Because PI cannot
pass intact plasma and nuclear membranes, it will not stain living
cells but only enter and stain dying cells that have decreased
membrane integrity. Due to these characteristics, the Annexin V/PI
double staining can be applied to discriminate between initiation
(Annexin V-positive/PI-negative) and irreversible (Annexin
V-positive/PI-positive) programmed cell death. Caspase-3 is
activated by both the extrinsic death receptor-induced and
intrinsic mitochondrial cell death pathways. Therefore, active
caspase-3 is also a marker for initiation of the death cascade. The
induction of cell death upon binding of the combination of
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G and BsAb
DR5-01-K409R-E430G.times.DR5-05-F405L-E430G was analyzed in the
DR5-positive COLO 205 colon cancer cells. Cells were harvested by
pooling the culture supernatant containing non-adherent cells and
trypsinized adherent cells. Cells were passed through a cell
strainer, pelleted by centrifugation for 5 minutes at 1,200 rpm and
resuspended in culture medium at a concentration of
0.2.times.10.sup.6 cells/mL. 500 .mu.L of the single cell
suspensions (100,000 cells per well) were seeded in 24-wells
flat-bottom culture plates (Greiner Bio-One, Cat nr 662160) and
incubated for 16 hours at 37.degree. C. 500 .mu.L antibody sample
was added (1 .mu.g antibody final concentration) and incubated for
5 hours or 24 hours at 37.degree. C. As a positive control, cells
were incubated with 5 .mu.M staurosporine (Sigma Aldrich, Cat nr
S6942). Cells were washed once with 250 .mu.L 1.times.PBS. Adherent
cells were harvested by incubating with 100 .mu.L 0.05% trypsin for
10 minutes at 37.degree. C. 200 .mu.L medium was added to the
trypsinized cells and cells were transferred to a 96-wells
round-bottom FACS plate (Greiner Bio-One, Cat nr 650101) and pooled
with the non-adherent cells. Cells were pelleted by centrifugation
for 5 minutes at 1,200 rpm, resuspended in 200 .mu.L ice cold PBS
and divided into two samples of 100 .mu.L in 96-Wells round-bottom
FACS plates for the Annexin V/PI and active caspase-3 staining,
respectively.
[0386] Annexin V/PI double staining was performed using the FITC
Annexin V Apoptosis Detection Kit I (BD Pharmingen, Cat nr 556547).
Cells were washed once with ice cold PBS and incubated in 50 .mu.L
Annexin V/PI Staining Solution (Annexin V-FITC 1:00 and PI 1:25)
for 15 minutes at 4.degree. C. Cells were washed with 100 .mu.L
Binding Buffer, resuspended in 20 .mu.L Binding Buffer and
fluorescence was measured on an iQue Screener (IntelliCyt) within 1
hour. Data were analyzed and plotted using GraphPad Prism
software.
[0387] Active caspase-3 staining was performed using the PE Active
Caspase-3 Apoptosis Kit (BD Pharmingen, Cat nr 550914). Cells were
washed once with ice cold PBS, resuspended in 100 .mu.L
Cytofix/Cytoperm Fixation and Permeabilization Solution and
incubated for 20 minutes on ice. Cells were pelleted at room
temperature, washed twice with 100 .mu.L 1.times. Perm/Wash Buffer
and resuspended in 100 .mu.L PE Rabbit Anti-Active Caspase-3 (1:10)
for an incubation of 30 minutes at room temperature. Cells were
pelleted at room temperature, washed once with 100 .mu.L 1.times.
Perm/Wash Buffer and resuspended in 20 .mu.L 1.times. Perm/Wash
Buffer. Fluorescence was measured on an iQue Screener. Data were
analyzed and plotted using GraphPad Prism software.
[0388] FIG. 15 shows that, after 5 hours of incubation, the
combination of the chimeric antibodies
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G efficiently induced
the early stages of cell death as indicated by an increase in the
percentage of Annexin V-positive/PI-negative (FIG. 15A) and Active
Caspase-3-positive cells (FIG. 15B), compared to the negative
control antibody IgG1-b12. The percentage of Annexin
V-positive/PI-negative and Active Caspase-3 positive cells was
higher in cells treated with the combination of
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G compared to the
combination of the DR5 antibodies without the E430G mutation
(IgG1-DR5-01-K409R+IgG1-DR5-05-F405L) or any of the single
antibodies. At the 5 hour time point, the percentage of AnnexinV/PI
double-positive cells was comparable to background levels in all
samples (FIG. 15C).
[0389] BsAb DR5-01-K409R-E430G.times.DR5-05-F405L-E430G efficiently
induced the early stages of cell death as indicated by an increase
in the percentage of Annexin V-positive/PI-negative (FIG. 15A) and
Active Caspase-3-positive cells (FIG. 15B) after 5 hours
incubation, compared to the negative control antibody IgG1-b12. The
percentage of Annexin V-positive/PI-negative and Active Caspase-3
positive cells was higher in cells that had been treated with BsAb
DR5-01-K409R-E430G.times.DR5-05-F405L-E430G compared to the
bispecific antibody without the E430G mutation (BsAb
DR5-01-K409R.times.DR5-05-F405L) or any of the monospecific
antibodies. At the 5 hour time point, the percentage of AnnexinV/PI
double positive cells was comparable to background levels in all
samples (FIG. 15C).
[0390] After 24 hours incubation, the percentage of Annexin V/PI
double-positive cells (FIG. 15D) was enhanced in samples treated
with IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G, indicating
that the cells had entered the irreversible stages of cell death.
Also at this stage, the effect of the combination of
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was stronger
(larger increase in the percentage of Annexin V/PI double-positive
cells (FIG. 15E)) than in samples treated with a combination of DR5
antibodies without the E430G mutation
(IgG1-DR5-01-K409R+IgG1-DR5-05-F405L) or any of the single
antibodies. At the same time point, the percentage of Active
Caspase 3 positive cells was highest in cells treated with
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G.
[0391] After 24 hours incubation, the percentage of Annexin V/PI
double-positive cells (FIG. 15D) was enhanced in samples treated
with BsAb DR5-01-K409R-E430G.times.DR5-05-F405L-E430G, indicating
that the cells had entered the irreversible stages of cell death.
Also at this stage, the effect of BsAb
DR5-01-K409R-E430G.times.DR5-05-F405L-E430G was stronger (larger
increase in the percentage of Annexin V/PI double-positive cells
(FIG. 15E) than in samples treated with the bispecific antibody
without the E430G mutation (BsAb DR5-01-K409R.times.DR5-05-F405L)
or any of the monospecific antibodies. At the same time point, the
percentage of Active Caspase 3 positive cells was highest in cells
treated with BsAb DR5-01-K409R-E430G.times.DR5-05-F405L-E430G.
[0392] These data indicate that the antibody combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G and BsAB
DR5-01-K409R-E430G.times.DR5-05-F405L-E430G induced both the early
and late stages of cell death in COLO 205 colon cancer cells, and
did so more effectively than the antibody combination and BsAb
without the E430G hexamerization enhancing mutation.
Example 16: Caspase-3 and -7 Activation Upon Binding of the
Antibody Combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F4051-E430G or BsAb
DR5-01-K409R-E430G.times.DR5-05-F4051-E430G with
Hexamerization-Enhancing Mutation on COLO 205 Colon Cancer
Cells
[0393] In Example 15 it was described that incubation with the
antibody combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G induced caspase-3
activation in COLO 205 colon cancer cells. The percentage of active
caspase-3-positive cells was higher after 5 hours than after 24
hours of incubation with the antibody combination. In this example,
Caspase-3/7 activation was measured in time using the Caspase-Glo
3/7 assay (Promega, Cat nr G8091), in which a substrate with the
Caspase-3/7 recognition motif DEVD releases aminoluciferin, a
substrate of luciferase, upon cleavage. Cells were harvested by
pooling the culture supernatant containing non-adherent cells and
trypsinized adherent COLO 205. Cells were passed through a cell
strainer, pelleted by centrifugation for 5 minutes at 1,200 rpm and
resuspended in culture medium at a concentration of
0.8.times.10.sup.5 cells/mL. 25 .mu.L of the single cell
suspensions (2,000 cells per well) were seeded in 384-wells culture
plates (Perkin Elmer, Cat nr 6007680) and incubated for 16 hours at
37.degree. C. 25 .mu.L antibody sample was added (1 .mu.g antibody
final concentration) and incubated for 1, 2, 5 and 24 hours at
37.degree. C. Plates were removed from the incubator to let the
temperature decrease till room temperature. Cells were pelleted by
centrifugation for three minutes at 300.times.g. 25 .mu.L
supernatant was removed and replaced by 25 .mu.L Caspase-Glo 3/7
Substrate. After mixing by shaking for one minute at 500 rpm, the
plates were incubated for one hour at room temperature.
Luminescence was measured on an EnVision Multilabel Reader
(PerkinElmer). In the time course of 1, 2 to 5 hours, the antibody
combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G showed
faster and stronger induction of caspase-3/7 activation than the
combination of IgG1-DR5-01-K409R+IgG1-DR5-05-F405L without the
hexamerization-enhancing mutation (FIG. 16A). Similarly BsAb
DR5-01-K409R-E430G.times.DR5-05-F405L-E430G showed faster and
stronger induction of caspase-3/7 activation than BsAb
DR5-01-K409R.times.DR5-05-F405L without the
hexamerization-enhancing mutation (FIG. 16B). After 24 hours,
caspase-3/7 activation was almost reduced to baseline levels for
all tested DR5 antibodies.
Example 17: Introduction of the K409R or F405L Mutation has No
Effect on the Potency of Antibodies with a Hexamerization-Enhancing
Mutation
[0394] In many of the experiments described in this application,
the anti-death receptor antibodies contain in the IgG Fc domain the
K409R or F405L (EU numbering) mutation. These mutations enable the
generation of bispecific death receptor antibodies by
Fab-arm-exchange reaction between a K409R-containing IgG1 and a
F405L-containing IgG1 under controlled reducing conditions as
described in WO2011/131746. Without Fab-arm exchange, human IgG1
antibodies bearing the K409R or F405L mutation are thought to show
the same functional characteristics as wild type human IgG1
(Labrijn et al., Proc Natl Acad Sci USA. 2013 Mar. 26;
110(13):5145-50). Here we show that the presence of the K409R or
F405L mutations has no effect on the capacity of the combination of
the parental IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G
antibodies to induce cell death in tumor cells in vitro. A
viability assay was performed as described in Example 5 to compare
the capacity of the antibody combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G with the capacity
of the antibody combination IgG1-hDR5-01-E430G+IgG1-hDR5-05-E430G
to induce killing of BxPC-3 pancreatic cancer cells.
[0395] The BxPC-3 pancreatic cancer cell line showed similar
viability curves after incubation with the antibody combination
IgG1-hDR5-01-K409R-E430G+IgG1-hDR5-05-F405L-E430G as with the
antibody combination IgG1-hDR5-01-E430G+IgG1-hDR5-05-E430G (FIG.
17). These data indicate that the K409R and F405L mutations had no
effect on the potency of the combination of the antibodies with
E430G hexamerization enhancing mutation.
Example 18: Cancer Cell Kill Capacity of Different Antibody Ratios
in the Combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F4051-E430G
[0396] A viability assay was performed as described in Example 5 to
study the capacity of the antibody combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G to induce killing
of BxPC-3 pancreatic cancer cells, when combined at different
ratios of IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G. The
antibodies were combined at different ratios of
IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G, indicated as
Ratio DR5-01-E430G:DR5-05-E430G of 100:0, 90:10, 80:20, 70:30,
60:40, 50:50, 40:60, 30:70, 20:80, 10:90 and 0:100. At 20 .mu.g/mL
and 4 .mu.g/mL total antibody concentrations, killing was equally
effective at all tested antibody ratios containing both antibodies
IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G. At 0.8
.mu.g/mL and 0.16 .mu.g/mL total antibody concentrations, all
tested antibody ratios containing both antibodies
IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G induced killing
(FIG. 18).
Example 19: Cancer Cell Kill Capacity of Different Antibody Ratios
in the Combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G
[0397] A viability assay was performed as described in Example 5 to
study the capacity of the antibody combination
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G to induce killing of
BxPC-3 pancreatic and HCT-15 colon cancer cells, when combined at
different antibody ratios (indicated in FIG. 19 as Ratio
DR5-01-E430G:DR5-05-E430G of 100:0, 98:2, 96:4, 94:6, 92:8, 90:10,
50:50, 10:90, 8:92, 6:94, 4:96, 2:98 and 0:100) at final antibody
concentrations of 10 .mu.g/mL for BxPC-3 and 20 .mu.g/mL for
HCT-15. Killing was equally effective at all tested antibody ratios
containing both antibodies IgG1-hDR5-01-G56T-E430G and
IgG1-hDR5-05-E430G (FIG. 19).
Example 20: Effect of a Hexamerization-Enhancing Mutation on the In
Vivo Efficacy of an Anti-DR5 Antibody in a Subcutaneous COLO 205
Colon Cancer Xenograft Model
[0398] The in vivo anti-tumor efficacy of IgG1-DR5-05-F405L-E430G
was compared to that of IgG1-DR5-05-F405L without a
hexamerization-enhancing mutation in a subcutaneous model with COLO
205 human colon cancer cells. At day 0, cells were harvested by
pooling the culture supernatant containing non-adherent cells and
trypsinized adherent cells. 3.times.10.sup.6 cells were injected in
a volume of 200 .mu.L PBS into the flank of 6-11 weeks old female
SCID mice (C.B-17/IcrHan.RTM.Hsd-Prkdc.sup.scid; Harlan). All
experiments and animal handlings were approved by the local
authorities, and were conducted according to all applicable
international, national and local laws and guidelines. Tumor
development was monitored at least twice per week by caliper
(PLEXX) measurement as 0.52.times.(length).times.(width).sup.2.
Tumors were measured until an endpoint tumor volume of 1,500
mm.sup.3, until tumors showed ulcerations, until serious clinical
signs were observed, or until tumor growth blocked movements of the
mouse. At day 6, the average tumor volume was .about.200 mm3 and
the mice were sorted into groups with equal tumor size variance
(Table below). Mice were treated by intraperitoneal (i.p.)
injection of 100 .mu.g antibody in 200 .mu.L PBS on day 6 and 13 (5
mg/kg per dose). To check for correct antibody administration,
blood samples were obtained for IgG serum determination three days
after the first dose. One individual mouse had no detectable human
IgG plasma level and was excluded from statistical analysis (Table
below). For the other mice, human antibody plasma concentrations
were according to the expectations when assuming a 2-compartment
model with Vcen=50 mL/kg, Vs=100 mL/kg and an elimination half-life
of 11.6 days (data not shown). Tumors were measured until 16 weeks
after tumor inoculation.
Treatment Groups and Dosing
TABLE-US-00005 [0399] Dosing day # # after tumor mice analyzed
Antibody Total antibody dose inoculation 7 7 IgG1-DR5-05- 100 .mu.g
(5 mg/kg) 6, 13 F405L (100 .mu.g) 8 8 IgG1-DR5-05- 100 .mu.g (5
mg/kg) 6, 13 F405L-E430G (100 .mu.g) 8 7 IgG1-b12 100 .mu.g (5
mg/kg) 6, 13 (100 .mu.g)
[0400] FIG. 20A shows mean tumor volumes per treatment group in
time. Complete tumor abrogation was observed for the anti-DR5
antibody with hexamerization-enhancing mutation
(IgG1-DR5-05-F405L-E430G). In contrast, IgG1-DR5-05-F405L without
hexamerization-enhancing mutation strongly inhibited tumor growth
compared to IgG1-b12, but did not result in complete tumor
abrogation.
[0401] FIG. 20B shows a Kaplan-Meier plot of tumor progression,
with a cutoff set at a tumor volume >750 mm3. Compared to mice
treated with negative control antibody IgG1-b12, tumor outgrowth
was significantly delayed in the groups treated with anti-DR5
antibodies (Mantel-Cox analysis at tumor size cut-off 750 mm3:
p<0.001). At the end of the study (day 112), the group of mice
treated with IgG1-DR5-05-F405L-E430G showed significant less mice
with tumor outgrowth than the IgG1-DR5-05-F405L without
hexamerization-enhancing mutation group (p<0.001).
[0402] These data show that introduction of the E430G
hexamerization-enhancing mutation in IgG1-DR5-05-F405L resulted in
enhanced tumor inhibition in the subcutaneous COLO 205 colon cancer
tumor model compared to IgG1-DR5-05-F405L without the
hexamerization-enhancing mutation.
Example 21: Cell Death Induction by IgG1-FAS-E09 Variants with
Hexamerization-Enhancing Mutations
[0403] Introduction of the hexamerization-enhancing mutations
E345R/E430G/S440Y enabled the FAS antibody IgG1-FAS-E09 to induce
dose-dependent killing of Jurkat human T lymphocytes as described
in Example 6. To analyze the requirement of antibody Fc-Fc
interactions by hexamerized IgG1-FAS-E09 variants to induce cell
death, we made use of the self-repulsing mutations K439E and S440K
in combination with the hexamerization-enhancing mutations
E345R/E430G/S440Y (RGY) and E345R/E430G/Y4361(RGI), respectively
(WO2014006217).
[0404] A viability assay on Jurkat human T lymphocytes was
performed, essentially as described in Example 6. Briefly, 19,200
cells in 100 .mu.L per well were seeded in 96-well plates. 50 .mu.L
of serial dilution antibody preparation series (range 0.0006 to 10
.mu.g/mL final concentrations in 6-fold dilutions) were added and
incubated for 4 days at 37.degree. C. The viability of the cultured
cells was determined by TOPRO-3 iodine as described in Example 6.
TOPRO-3 staining was analyzed by flow cytometry on a BD LSRFORTESSA
cell analyzer (BD Biosciences). Data were analyzed and plotted
using non-linear regression (sigmoidal dose-response with variable
slope) using GraphPad Prism software. FIG. 21 shows the percentage
viable cells, as calculated from the percentage TOPRO-3-negative
cells. Introduction of the hexamerization-enhancing mutations RGY
enabled the FAS antibody IgG1-FAS-E09 to induce dose-dependent
killing of Jurkat human T lymphocytes. Killing by IgG1-FAS-E09-RGY
was inhibited by presence of the Fc-Fc repulsion mutation K439E in
IgG1-FAS-E09-RGEY. Also IgG1-FAS-E09-RGIK, containing the repulsion
mutation S440K did not induce killing of Jurkat cells. Killing
efficacy was restored when Fc-Fc repulsion was neutralized by
combining the two antibodies IgG1-FAS-E09-RGEY and
IgG1-FAS-E09-RGIK, each having one of the complementary mutations
K439E or S440K. These data illustrate that hexamerization by Fc-Fc
interactions is required for the induction of cell death by
IgG1-FAS-E09 variants with the hexamerization mutation RGY or
RGI.
Example 22: Anti-DR5 Antibody IgG1-DR5-CONA with a
Hexamerization-Enhancing Mutation E430G is Able to Kill Human Colon
Cancer Cells
[0405] The present study illustrate the ability of the anti-DR5
antibody IgG1-DR5-CONA with the hexamerization-enhancing mutation
E430G to kill attached human colon cancer cells COLO 205. COLO 205
cells were harvested as described in Example 4. 100 .mu.L of the
single cell suspensions (5,000 cells per well) were seeded in
96-well flat-bottom plates and incubated overnight at 37.degree. C.
50 .mu.L samples of antibody concentration series (range 0.04 to 10
.mu.g/mL final concentrations in 4-fold dilutions) were added and
incubated for 3 days at 37.degree. C. As a positive control, cells
were incubated with 5 .mu.M staurosporine. The viability of the
cell cultures was determined in a CellTiter-Glo luminescent cell
viability assay as described in Example 4. Luminescence was
measured on an EnVision Multilabel Reader (PerkinElmer). Data were
analyzed and plotted using non-linear regression (sigmoidal
dose-response with variable slope) using GraphPad Prism software.
The percentage viable cells was calculated using the following
formula: % viable cells=[(luminescence antibody sample-luminescence
staurosporine sample)/(luminescence no antibody sample-luminescence
staurosporine sample)]*100.
[0406] FIG. 22 shows that introduction of the
hexamerization-enhancing mutation E430G resulted in dose-dependent
killing by IgG1-DR5-CONA-E430G, whereas the parental wild type
antibody IgG1-DR5-CONA was not able to kill attached COLO 205 colon
cancer cells.
Example 23: Introduction of Hexamerization-Enhancing Mutation S440Y
Improves the Efficacy of Anti-DR5 Antibodies to Induce Cell Death
on Human Colon Cancer Cells
[0407] The effect of the hexamerization-enhancing mutation S440Y on
the capacity of the single antibodies and the combination of
IgG1-hDR5-01-G56T and IgG1-hDR5-05 to kill COLO 205 human colon
cancer cells was studied in a viability assay. Cells were harvested
and a viability assay was performed as described in Example 4.
Briefly, 100 .mu.L single cell suspensions (5,000 cells per well)
were seeded in 96-well plates. 50 .mu.L of serial dilution antibody
preparation series (range 0.0003 to 20 .mu.g/mL final
concentrations in 4-fold dilutions) were added and incubated for 3
days at 37.degree. C. The viability of the cultured cells was
determined in a CellTiter-Glo luminescent cell viability assay as
described in Example 4. Luminescence data were analyzed as
described in Example 22.
[0408] FIG. 23A shows that introduction of the
hexamerization-enhancing mutation S440Y resulted in dose-dependent
killing by the single antibodies IgG1-hDR5-01-G56T-S440Y and
IgG1-hDR5-05-S440Y, whereas the parental wild type antibodies
IgG1-hDR5-01-G56T and IgG1-hDR5-05 were not able to kill COLO 205
colon cancer cells. Also the efficacy of the combination of
IgG1-hDR5-01-G56T+IgG1-hDR5-05 was improved by introduction of the
S440Y mutation in both antibodies, represented by the decreased
EC50 (FIG. 23B).
Example 24: Introduction of the Hexamerization-Enhancing Mutation
E430G Improves the Efficacy of Cell Death Induction by the
Combination of Anti-DR5 Antibodies
IgG1-DR5-CONA+IgG1-DR5-chTRA8
[0409] The competition between IgG1-DR5-CONA-K409R and
IgG1-DR5-chTRA8-F405L for binding to the extracellular domain of
DR5 was measured by sandwich binding assays in a sandwich
enzyme-linked immunosorbent assay (ELISA). 96-well flat bottom
ELISA plates (Greiner bio-one; Cat nr 655092) were coated overnight
at 4.degree. C. with 2 .mu.g/mL DR5 antibody (IgG1-DR5-CONA-K409R
or IgG1-DR5-chTRA8-F405L) in 100 .mu.L PBS. The wells were blocked
by adding 200 .mu.L PBSA [PBS/1% Bovine Serum Albumin (BSA; Roche
Cat #10735086001)] and incubated for 1 hour at room temperature.
The wells were washed three times with PBST [PBS/0.05% Tween-20
(Sigma-Aldrich; Cat nr 63158)]. Next, DR5ECD-FcHistag (0.2 .mu.g/mL
final concentration) and competing antibody (1 .mu.g/mL final
concentration) were added in a total volume of 100 .mu.L PBSTA
(PBST/0.2% BSA) and incubated for 1 hour at room temperature while
shaking. After washing three times with PBST, wells were incubated
on an ELISA shaker with 100 .mu.L biotinylated anti-His tag
antibody (R&D Systems; Cat nr BAM050; 1:2.000) in PBSTA for one
hour at room temperature. After washing three times with PBST,
wells were incubated with streptavidin-labelled Poly-HRP (Sanquin;
Cat nr M2032; 1:10.000) in PBSTA for 20 minutes at room temperature
on an ELISA shaker. After washing three times with PBST, the
reaction was visualized through an incubation with 100 .mu.L
2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid [ABTS
(Roche; Cat nr 11112597001)] for 30 minutes at RT protected from
light. The substrate reaction was stopped by adding an equal volume
of 2% oxalic acid. Fluorescence at 405 nm was measured on an ELISA
reader (BioTek ELx808 Absorbance Microplate Reader).
[0410] FIG. 24A shows binding competition expressed as percentage
inhibition of DR5ECD-FcHisCtag binding to coated antibody in
presence of competing antibody, relative to binding of
DR5ECD-FcHisCtag in absence of competing antibody (%
inhibition=100-[(binding in presence of competing antibody/binding
in absence of competing antibody)]*100). Binding of DR5ECD-FcHistag
to coated IgG1-DR5-CONA-K409R was not inhibited in the presence of
soluble IgG1-DR5-chTRA8-F405L. Vice versa, binding of
DR5ECD-FcHistag to coated IgG1-DR5-chTRA8-F405L was also not
inhibited in the presence of soluble IgG1-DR5-CONA-K409R. These
data illustrate that IgG1-DR5-CONA-K409R and IgG1-DR5-chTRA8-E430G
did not compete with each other for binding of
DR5ECD-FcHisCtag.
[0411] Next, the effect of the hexamerization-enhancing mutation
E430G on the capacity of the combination of the non-crossblocking
anti-DR5 antibodies IgG1-DR5-CONA+IgG1-DR5-chTRA-8 to kill attached
BxPC-3 human pancreatic cancer cells was studied in a viability
assay as described in Example 5. FIG. 24 shows that the antibody
combination IgG1-DR5-CONA-E430G+IgG1-DR5-chTRA8-E430G with
hexamerization-enhancing mutations showed increased dose-dependent
killing of BxPC-3 cells compared to the combination of the parental
antibodies without the E430G hexamerization-enhancing mutation.
Example 25: Effect of a Hexamerization-Enhancing Mutation on the In
Vivo Efficacy of the Combination of Anti-DR5 Antibodies
IgG1-hDR5-01-G56T+IgG1-hDR5-05 in a Subcutaneous HCT15 Colon Cancer
Xenograft Model
[0412] The in vivo anti-tumor efficacy of the anti-DR5 antibody
combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G was compared
to that of IgG1-hDR5-01-G56T+IgG1-hDR5-05 without the E430G
hexamerization-enhancing mutation in the subcutaneous HCT15 human
colon cancer xenograft model at CrownBiosciences, Taicang, China.
The cells were maintained in vitro as a monolayer culture in
RPMI-1640 medium supplemented with 10% fetal bovine serum at
37.degree. C. in an atmosphere of 5% CO2 in air. Adherent cells in
an exponential growth phase were harvested by trypsin-EDTA
treatment. 5.times.10.sup.6 cells were injected in a volume of 100
.mu.L PBS into the flank of 7-9 weeks old female BALB/c nude mice.
The care and use of animals during the study were conducted in
accordance with the regulations of the Association for Assessment
and Accreditation of Laboratory Animal Care (AAALAC). Tumor volumes
were measured twice weekly in two dimensions using a caliper, and
the volume was expressed in mm.sup.3 using the formula: V=0.5
a.times.b.sup.2 where a and b are the long and short diameters of
the tumor, respectively. Mice were assigned into groups using
randomized block design and treatments were started when the mean
tumor size reached 161 mm.sup.3 (8 mice per group). Mice were
treated three times according to a Q7D regimen by i.v. injection of
10 .mu.g antibody (0.5 mg/kg, i.e. 0.25 mg/kg of each antibody in
the combination). Mice in the control group were treated in
parallel with 0.5 mg/kg IgG1-b12.
[0413] FIG. 25A shows mean tumor volumes per treatment group. The
antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G
showed better tumor growth inhibition than
IgG1-hDR5-01-G56T+IgG1-hDR5-05. FIG. 25B shows tumor volume per
treatment group at day 21. The combination
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G inhibited tumor growth
progression significantly better than an equivalent dose
IgG1-hDR5-01-G56T+IgG1-hDR5-05 (Mann Whitney test (P<0.0011)).
FIG. 25C shows a Kaplan-Meier plot of tumor progression, with a
cutoff set at a tumor volume >750 mm.sup.3. The combination
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G inhibited tumor growth
progression better than an equivalent dose
IgG1-hDR5-01-G56T+IgG1-hDR5-05.
[0414] These data illustrate that introduction of the E430G
hexamerization-enhancing mutation in the anti-DR5 antibody
combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G
resulted in enhanced tumor growth inhibition in an in vivo
xenograft model with HCT15 human colon cancer cells.
Example 26: In Vivo Efficacy of the Anti-DR5 Antibodies
IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G in a Subcutaneous
COLO 205 Colon Cancer Xenograft Model
[0415] The in vivo anti-tumor efficacy of antibodies
IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G was evaluated for
the single antibodies and the combination of both antibodies and
compared to the parental antibodies without the E430G mutation in
the subcutaneous COLO 205 human colon cancer xenograft model. Tumor
cell inoculation, mice handling, tumor outgrowth measurements and
endpoint determination were performed, essentially as described in
Example 20. 3.times.10.sup.6 cells were injected in a volume of 100
1.11 PBS into the flank of 5-8 weeks old female SCID mice
(C.B-17/IcrHan*Hsd-Prkdc.sup.scid; Harlan). At day 9, the average
tumor volume was measured and the mice were sorted into groups with
equal tumor size variance. Mice were treated by intravenous (i.v.)
injection of 10 .mu.g (0.5 mg/kg) antibody in 200 .mu.L PBS on day
9. Mice in the control group were treated with 10 .mu.g (0.5 mg/kg)
IgG1-b12.
TABLE-US-00006 TABLE 2 Treatment groups and dosing Dosing day after
# # Total antibody tumor mice analyzed Antibody dose inoculation 8
8 IgG1-hDR5-01-G56T- 0.5 mg/kg 9 E430G 8 8 IgG1-hDR5-05-E430G 0.5
mg/kg 9 8 8 IgG1-hDR5-01-G56T- 0.5 mg/kg 9 E430G IgG1-hDR5-05-E430G
8 8 IgG1-hDR5-01-G56T 0.5 mg/kg 9 8 8 IgG1-hDR5-05 0.5 mg/kg 9 8 8
IgG1-hDR5-01-G56T 0.5 mg/kg 9 IgG1-hDR5-05 8 8 IgG1-b12 0.5 mg/kg
9
[0416] FIG. 26A shows mean tumor volumes per treatment group in
time. Introduction of the E430G mutation in the single antibodies
IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G resulted in enhanced
inhibition of tumor growth compared to the parental antibodies
without the E430G mutation. Treatment with the antibody
combinations induced complete tumor regression, both for
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G and for the combination
of parental antibodies without the E430G mutation. At day 19 the
average tumor size in all groups treated with DR5-antibodies was
significantly smaller than in animals treated with the negative
control antibody IgG1-b12 (Mann Whitney test (P<0.001))(data not
shown). FIG. 26B shows a Kaplan-Meier plot of tumor progression,
with a cutoff set at a tumor volume >500 mm.sup.3. Compared to
mice treated with negative control antibody IgG1-b12, tumor
outgrowth was significantly delayed in all groups treated with
anti-DR5 antibodies (Mantel-Cox analysis at tumor size cut-off 500
mm.sup.3: p<0.0001). Mice treated with the single antibodies
IgG1-hDR5-01-G56T and IgG1-hDR5-05 without the
hexamerization-enhancing mutation E430G showed tumor outgrowth
significantly earlier compared to the mice treated with the other
tested anti-DR5 antibodies ((Mantel-Cox analysis at tumor size
cut-off 500 mm.sup.3: p<0.0001).
Sequence CWU 1
1
501330PRTHomo Sapiens 1Ala 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 Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120
125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235
240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 325 3302330PRTHomo Sapiens 2Ala 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 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 3303330PRTHomo
Sapiens 3Ala 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 95Pro Val Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150
155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235 240Leu Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265
270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 325 3304330PRTHomo Sapiens 4Ala 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 95Pro Val
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105
110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230
235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met
His Glu Gly Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 325 3305330PRTArtificial SequenceSynthetic
5Ala 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 Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155
160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300Val Phe Ser Cys Ser Val Met His Gly Ala Leu His Asn His
Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
3306330PRTArtificial SequenceSynthetic 6Ala 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 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215
220Gln Pro Arg Lys Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 3307330PRTArtificial
SequenceSynthetic 7Ala 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 Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250
255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr305 310 315 320Gln Lys Tyr Leu Ser Leu Ser
Pro Gly Lys 325 3308330PRTArtificial SequenceSynthetic 8Ala 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 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg
Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Leu 275 280 285Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 3309330PRTArtificial
SequenceSynthetic 9Ala 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 Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250
255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe 275 280 285Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 325 33010330PRTArtificial SequenceSynthetic 10Ala 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 Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr305 310 315 320Gln Glu Ser Leu Ser Leu Ser Pro Gly Lys 325
33011330PRTArtificial SequenceSynthetic 11Ala 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 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Lys Leu Ser Leu Ser Pro Gly Lys 325
33012330PRTArtificial SequenceSynthetic 12Ala 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 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Ile Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
33013118PRTArtificial SequenceSynthetic 13Glu Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Met Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser
Cys Arg Val Ser Gly Asp Thr Phe Thr Ala Tyr 20 25 30Phe Ile His Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Phe
Asn Pro Ile Ser Gly Thr Ala Gly Ser Ala Glu Lys Phe 50 55 60Arg Gly
Arg Val Ala Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75
80Met Glu Leu Asn Arg Leu Thr Phe Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gln His Arg Gly Asn Thr Phe Asp Pro Trp Gly Gln Gly
Thr 100 105 110Leu Val Thr Val Ser Ser 11514111PRTArtificial
SequenceSynthetic 14Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly
Ser Pro Gly Gln1 5 10 15Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser
Asp Ile Gly Ala Tyr 20 25 30Lys Tyr Val Ser Trp Tyr Gln Gln His Pro
Gly Lys Ala Pro Lys Leu 35 40 45Val Ile Tyr Glu Val Ser Asn Arg Pro
Ser Gly Val Ser Ser Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Gln Thr
Ala Ser Leu Thr Ile Ser Gly Leu65 70 75 80Gln Ala Asp Asp Glu Ala
Asp Tyr Tyr Cys Asn Ser Tyr Gln Gly Tyr 85 90 95Asn Thr Trp Val Phe
Gly Gly Gly Thr Lys Val Thr Val Leu Gly 100 105
11015132PRTArtificial SequenceSynthetic 15Gln Leu Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr
Cys Thr Val Ser Gly Ala Ser Ile Ser Ala Asn 20 25 30Ser Tyr Tyr Gly
Val Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu 35 40 45Trp Val Gly
Ser Ile Ala Tyr Arg Gly Asn Ser Asn Ser Gly Ser Thr 50 55 60Tyr Tyr
Asn Pro Ser Leu Lys Ser Arg Ala Thr Val Ser Val Asp Thr65 70 75
80Ser Lys Asn Gln Val Ser Leu Arg Leu Thr Ser Val Thr Ala Ala Asp
85 90 95Thr Ala Leu Tyr Tyr Cys Ala Arg Arg Gln Leu Leu Asp Asp Gly
Thr 100 105 110Gly Tyr Gln Trp Ala Ala Phe Asp Val Trp Gly Gln Gly
Thr Met Val 115 120 125Thr Val Ser Ser 13016110PRTArtificial
SequenceSynthetic 16Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Glu
Ala Pro Arg Gln1 5 10 15Thr Val Thr Ile Ser Cys Ser Gly Asn Ser Phe
Asn Ile Gly Arg Tyr 20 25 30Pro Val Asn Trp Tyr Gln Gln Leu Pro Gly
Lys Ala Pro Lys Leu Leu 35 40 45Ile Tyr Tyr Asn Asn Leu Arg Phe Ser
Gly Val Ser Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala
Ser Leu Ala Ile Arg Asp Leu Leu65 70 75 80Ser Glu Asp Glu Ala Asp
Tyr Tyr Cys Ser Thr Trp Asp Asp Thr Leu 85 90 95Lys Gly Trp Val Phe
Gly Gly Gly Thr Lys Val Thr Val Leu 100 105 110178PRTArtificial
SequenceSynthetic 17Gly Phe Asn Ile Lys Asp Thr Phe1
5188PRTArtificial SequenceSynthetic 18Ile Asp Pro Ala Asn Gly Asn
Thr1 51911PRTArtificial SequenceSynthetic 19Val Arg Gly Leu Tyr Thr
Tyr Tyr Phe Asp Tyr1 5 1020118PRTArtificial SequenceSynthetic 20Glu
Val Gln Leu Gln Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala1 5 10
15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30Phe Ile His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp
Ile 35 40 45Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro
Lys Phe 50 55 60Gln Gly Lys Ala Thr Ile Thr Thr Asp Thr Ser Ser Asn
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Val Arg Gly Leu Tyr Thr Tyr Tyr Phe Asp
Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser
11521448PRTArtificial SequenceSynthetic 21Glu Val Gln Leu Gln Gln
Ser Gly Ala Glu Val Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser
Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Phe Ile His Trp
Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Arg Ile
Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60Gln Gly
Lys Ala Thr Ile Thr Thr Asp Thr Ser Ser Asn Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Val Arg Gly Leu Tyr Thr Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly
Thr 100 105 110Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro 115 120 125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro 260 265 270Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val
Glu Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315 320Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345
350Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser 405 410 415Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala 420 425 430Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
445226PRTArtificial SequenceSynthetic 22Gln Ser Ile Ser Asn Asn1
5239PRTArtificial SequenceSynthetic 23Gln Gln Gly Asn Ser Trp Pro
Tyr Thr1 524107PRTArtificial SequenceSynthetic 24Glu Ile Val Met
Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30Leu His
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Lys
Phe Ala Ser Gln Ser Ile Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser65
70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Asn Ser Trp Pro
Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
10525214PRTArtificial SequenceSynthetic 25Glu Ile Val Met Thr Gln
Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30Leu His Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Lys Phe Ala
Ser Gln Ser Ile Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser65 70 75
80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Asn Ser Trp Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 210268PRTArtificial SequenceSynthetic
26Ile Asp Pro Ala Asn Thr Asn Thr1 527118PRTArtificial
SequenceSynthetic 27Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Val
Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Phe
Asn Ile Lys Asp Thr 20 25 30Phe Ile His Trp Val Lys Gln Ala Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45Gly Arg Ile Asp Pro Ala Asn Thr Asn
Thr Lys Tyr Asp Pro Lys Phe 50 55 60Gln Gly Lys Ala Thr Ile Thr Thr
Asp Thr Ser Ser Asn Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Arg Gly Leu Tyr
Thr Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr
Val Ser Ser 11528448PRTArtificial SequenceSynthetic 28Glu Val Gln
Leu Gln Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala1 5 10 15Ser Val
Lys Leu Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Phe
Ile His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45Gly Arg Ile Asp Pro Ala Asn Thr Asn Thr Lys Tyr Asp Pro Lys Phe
50 55 60Gln Gly Lys Ala Thr Ile Thr Thr Asp Thr Ser Ser Asn Thr Ala
Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Val Arg Gly Leu Tyr Thr Tyr Tyr Phe Asp Tyr Trp
Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185
190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp
Lys Thr 210 215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro 260 265 270Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310
315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr 325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu 340 345 350Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425
430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445298PRTArtificial SequenceSynthetic 29Gly Phe Asn Ile Lys
Asp Thr His1 53011PRTArtificial SequenceSynthetic 30Ala Arg Trp Gly
Thr Asn Val Tyr Phe Ala Tyr1 5 1031118PRTArtificial
SequenceSynthetic 31Gln 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
Asn Ile Lys Asp Thr 20 25 30His Met His Trp Val Arg Gln Ala Pro Gly
Gln Arg Leu Glu Trp Ile 35 40 45Gly Arg Ile Asp Pro Ala Asn Gly Asn
Thr Glu Tyr Asp Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Val
Asp Thr Ser Ala 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 Trp Gly Thr
Asn Val Tyr Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr
Val Ser Ser 11532448PRTArtificial SequenceSynthetic 32Gln 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 Asn Ile Lys Asp Thr 20 25 30His
Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40
45Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Glu Tyr Asp Gln Lys Phe
50 55 60Gln Gly Arg Val Thr Ile Thr Val Asp Thr Ser Ala 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 Trp Gly Thr Asn Val Tyr Phe Ala Tyr Trp
Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185
190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp
Lys Thr 210 215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro 260 265 270Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310
315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr 325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu 340 345 350Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425
430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445335PRTArtificial SequenceSynthetic 33Ser Ser Val Ser
Tyr1 5349PRTArtificial SequenceSynthetic 34Gln Gln Tyr His Ser Tyr
Pro Pro Thr1 535106PRTArtificial SequenceSynthetic 35Asp Ile Gln
Leu 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 Ser Tyr Met 20 25 30Tyr
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ile Tyr 35 40
45Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
Glu65 70 75 80Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr His Ser Tyr
Pro Pro Thr 85 90 95Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
10536213PRTArtificial SequenceSynthetic 36Asp Ile Gln Leu 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 Ser Tyr Met 20 25 30Tyr Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ile Tyr 35 40 45Arg Thr Ser
Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu65 70 75
80Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr His Ser Tyr Pro Pro Thr
85 90 95Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
Pro 100 105 110Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly Thr 115 120 125Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala Lys 130 135 140Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln Glu145 150 155 160Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200
205Asn Arg Gly Glu Cys 2103710PRTArtificial SequenceSynthetic 37Gly
Gly Ser Ile Ser Ser Gly Asp Tyr Phe1 5 10387PRTArtificial
SequenceSynthetic 38Ile His Asn Ser Gly Thr Thr1 53914PRTArtificial
SequenceSynthetic 39Ala Arg Asp Arg Gly Gly Asp Tyr Tyr Tyr Gly Met
Asp Val1 5 1040122PRTArtificial SequenceSynthetic 40Gln Val Gln Leu
Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30Asp Tyr
Phe Trp Ser Trp Ile Arg Gln Leu Pro Gly Lys Gly Leu Glu 35 40 45Cys
Ile Gly His Ile His Asn Ser Gly Thr Thr Tyr Tyr Asn Pro Ser 50 55
60Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Lys Gln Phe65
70 75 80Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
Tyr 85 90 95Cys Ala Arg Asp Arg Gly Gly Asp Tyr Tyr Tyr Gly Met Asp
Val Trp 100 105 110Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
120417PRTArtificial SequenceSynthetic 41Gln Gly Ile Ser Arg Ser
Tyr1 5429PRTArtificial SequenceSynthetic 42Gln Gln Phe Gly Ser Ser
Pro Trp Thr1 543108PRTArtificial SequenceSynthetic 43Glu Ile Val
Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg
Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Ile Ser Arg Ser 20 25 30Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Ser Leu Leu 35 40
45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu
Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Gly
Ser Ser Pro 85 90 95Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105448PRTArtificial SequenceSynthetic 44Gly Phe Thr Phe Ser Ser
Tyr Val1 5458PRTArtificial SequenceSynthetic 45Ile Ser Ser Gly Gly
Ser Tyr Thr1 54612PRTArtificial SequenceSynthetic 46Ala Arg Arg Gly
Asp Ser Met Ile Thr Thr Asp Tyr1 5 1047449PRTArtificial
SequenceSynthetic 47Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val
Lys Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Ser Tyr 20 25 30Val Met Ser Trp Val Arg Gln Thr Pro Glu
Lys Arg Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Ser Gly Gly Ser Tyr
Thr Tyr Tyr Pro Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95Ala Arg Arg Gly Asp Ser Met Ile Thr Thr Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200
205Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys
210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315
320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr 340 345 350Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445Lys486PRTArtificial SequenceSynthetic 48Gln Asp Val Gly Thr Ala1
5498PRTArtificial SequenceSynthetic 49Gln Gln Tyr Ser Ser Tyr Arg
Thr1 550213PRTArtificial SequenceSynthetic 50Asp Ile Val Met Thr
Gln Ser His Lys Phe Met Ser Thr Ser Val Gly1 5 10 15Asp Arg Val Ser
Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30Val Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Trp
Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser65 70 75
80Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Arg Thr
85 90 95Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
Pro 100 105 110Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly Thr 115 120 125Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala Lys 130 135 140Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln Glu145 150 155 160Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200
205Asn Arg Gly Glu Cys 210
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