U.S. patent application number 15/780268 was filed with the patent office on 2019-05-16 for anti-dr5 antibodies and methods of use thereof.
The applicant listed for this patent is GENMAB B.V.. Invention is credited to Esther BREIJ, Marije OVERDIJK, Paul PARREN, Rik RADEMAKER, Janine SCHUURMAN, Kristin STRUMANE.
Application Number | 20190144554 15/780268 |
Document ID | / |
Family ID | 58796495 |
Filed Date | 2019-05-16 |
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United States Patent
Application |
20190144554 |
Kind Code |
A1 |
OVERDIJK; Marije ; et
al. |
May 16, 2019 |
ANTI-DR5 ANTIBODIES AND METHODS OF USE THEREOF
Abstract
The present invention relates to monospecific or bispecific
antibody molecules that specifically bind the human DR5 antigen.
The invention relates in particular to DR5-specific antibody
molecules of the IgG1 isotype having a mutation in the Fc region
that enhances clustering of IgG molecules after cell-surface
antigen binding leading to the induction of DR5 signalling,
apoptosis and cell death. The invention further relates to a
combination of antibody molecules binding different epitopes on
DR5. The invention also relates to pharmaceutical compositions
containing these molecules and the treatment of cancer using these
compositions.
Inventors: |
OVERDIJK; Marije; (Utrecht,
NL) ; STRUMANE; Kristin; (Werkhoven, NL) ;
RADEMAKER; Rik; (Copenhagen V, DK) ; BREIJ;
Esther; (Utrecht, 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.: |
15/780268 |
Filed: |
December 1, 2016 |
PCT Filed: |
December 1, 2016 |
PCT NO: |
PCT/EP2016/079518 |
371 Date: |
May 31, 2018 |
Current U.S.
Class: |
424/136.1 |
Current CPC
Class: |
C07K 16/46 20130101;
C07K 2317/52 20130101; A61K 2039/505 20130101; A61K 2039/507
20130101; A61P 35/00 20180101; C07K 2317/75 20130101; C12N 15/62
20130101; C07K 2317/31 20130101; C07K 2317/24 20130101; A61K 38/00
20130101; C07K 2317/73 20130101; C07K 16/30 20130101; C07K 2317/526
20130101; C07K 16/2878 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 16/46 20060101 C07K016/46; C12N 15/62 20060101
C12N015/62; C07K 16/30 20060101 C07K016/30; A61P 35/00 20060101
A61P035/00 |
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 a Fc region of a human immunoglobulin IgG
and an antigen binding region binding to human DR5, wherein the Fc
region comprises a mutation of an amino acid position corresponding
to E430, E345 or S440 in human IgG1, wherein the numbering is
according to the EU Index.
2-4. (canceled)
5. The antibody according to claim 1, wherein the antigen binding
region binds to an epitope on human DR5 comprising or requiring a)
one or more amino acid residues located within amino acid residues
116-138 and one or more amino acid residues located within amino
acid residues 139-166 of SEQ ID NO 46, or b) one or more amino acid
residues located within amino acid residues 79-138 of SEQ ID NO
46.
6. (canceled)
7. The antibody according to claim 1, wherein the antibody
comprises a variable heavy chain region comprising CDR1, CDR2 and
CDR3 domains and a variable light chain region comprising CDR1,
CDR2 and CDR3 domains having the amino acid sequences selected from
the group consisting of: a) SEQ ID NOs: 1, 2, 3 and SEQ ID NOs: 5,
FAS, 6, respectively; b) SEQ ID NOs: 1, 8, 3 and SEQ ID NOs: 5,
FAS, 6, respectively; c) SEQ ID NOs: 10, 2, 11 and SEQ ID NOs: 13,
RTS, 14, respectively; d) SEQ ID NOs: 16, 17, 18 and SEQ ID NOs:
21, GAS, 22, respectively; and e) the heavy and light chain CDR1,
CDR2, CDR3 sequences as defined in any one of a) to d) above having
one to five mutations or substitutions in total across said CDR
sequences.
8. The antibody according to claim 1, wherein the antigen binding
region comprises a variable heavy chain region and a variable light
chain region having the amino acid sequences selected from the
group consisting of: a) SEQ ID NO:4 and SEQ ID NO:7, respectively;
b) SEQ ID NO:9 and SEQ ID NO:7, respectively; c) SEQ ID NO:12 and
SEQ ID NO:15, respectively; d) SEQ ID NO:19 and SEQ ID NO:23,
respectively; e) SEQ ID NO:20 and SEQ ID NO:23, respectively; and
f) the heavy and light chain variable region sequences as defined
in any one of a) to e) above having one to five mutations or
substitutions in total across said sequences.
9. The antibody of claim 1, wherein the antibody is an IgG1, IgG2,
IgG3, IgG4, IgE, IgD or IgM isotype.
10-11. (canceled)
12. The antibody according to claim 1, wherein the Fc region
comprises an amino acid sequence selected from the group consisting
of: a) SEQ ID NO:29; b) SEQ ID NO:30; c) SEQ ID NO:31; d) SEQ ID
NO:32; and e) an amino acid sequence as defined in any one of a) to
d) above having one to five mutations or substitutions in total
across said sequence
13. The antibody according to claim 1, comprising a heavy chain
(HC) and a light chain (LC), wherein the LC comprises the sequence
of SEQ ID NO:39 and wherein the HC comprises a sequence selected
from the group consisting of: a) SEQ ID NO:33; b) SEQ ID NO:34; c)
SEQ ID NO:35; d) SEQ ID NO:36; e) SEQ ID NO:37; f) SEQ ID NO:38;
and g) the sequence as defined in any one of a) to f) above having
one to five mutations or substitutions in total across said
sequence.
14. The antibody according to claim 1, comprising a heavy chain
(HC) and a light chain (LC), wherein the LC comprises the sequence
of SEQ ID NO:43 and wherein the HC comprises a sequence selected
from the group consisting of: a) SEQ ID NO:40; b) SEQ ID NO:41; c)
SEQ ID NO:42; or d) the sequence as defined in any one of a) to c)
above having one to five mutations or substitutions in total across
said sequence.
15. (canceled)
16. The antibody according to claim 1, wherein the antibody is
human, humanized or chimeric.
17. The antibody according to claim 1, wherein the antibody is
agonistic.
18. The antibody according to claim 1, wherein the antibody (a)
induces programmed cell death in a target cell, such as caspase
dependent cell death:, (b) induces apoptosis in a target cell
expressing DR5; and/or (c) reduces cell viability.
19-20. (canceled)
21. A composition comprising at least one antibody according to
claim 1 and a carrier.
22. (canceled)
23. The composition according to claim 21, which comprises a first
antibody and a second antibody, wherein both the first antibody and
second antibody comprise a Fc region of a human immunoglobulin IgG
and an antigen binding region binding to human DR5, wherein the Fc
region comprises a mutation of an amino acid position corresponding
to E430, E345 or S440 in human IgG1.
24-31. (canceled)
32. A bispecific antibody comprising one or more antigen binding
regions according to claim 1.
33-38. (canceled)
39. The bispecific antibody according to claim 32, wherein (a) the
bispecific antibody comprises an Fc region comprising a first and a
second heavy chain, said first heavy chain comprises a mutation
corresponding to F405L in human IgG1 and said second heavy chain
comprises a mutation corresponding to K409R in human IgG1, or (b)
wherein the bispecific antibody comprises an Fc region comprising a
first and a second heavy chain, said first heavy chain comprises a
mutation corresponding to K409R in human IgG1 and said second heavy
chain comprises a mutation corresponding to F405L in human
IgG1.
40-43. (canceled)
44. 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
21.
45. A method of treating a solid tumors tumor and/or hematological
tumor comprising administering to a subject in need thereof an
effective amount of the composition of claim 21.
46. The method according to claim 45, 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.
47-48. (canceled)
49. A method for inducing apoptosis in DR5 expressing tumors
comprising administering to a subject in need thereof an effective
amount of the composition of claim 21.
50. (canceled)
51. A nucleic acid construct encoding an antibody according to
claim 1.
52. An expression vector comprising one or more nucleic acid
constructs according to claim 51.
53. A host cell comprising the expression vector of claim 52.
54. (canceled)
55. A method of treating cancer comprising administering to a
subject in need thereof an effective amount of the antibody of
claim 1.
56. The method according to claim further comprising administering
an additional therapeutic agent.
57. The method according to claim 56, wherein the additional
therapeutic agent is one or more anti-cancer agent(s) selected from
the group consisting of: chemotherapeutics, kinase inhibitors,
apoptosis-modulating agents, RAS inhibitors, proteasome inhibitors,
histone deacetylase inhibitors, nutraceuticals, cytokines,
antibodies or antibody mimetics, and antibody-drug conjugates.
58. A kit comprising the antibody of claim 1, and instructions for
use.
59-60. (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/EP2016/079518, filed Dec. 1,
2016, which claims the benefit of 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. 28, 2019, is named GM_169AUS_Sequence_Listing.txt and is
144,116 bytes in size.
FIELD OF THE INVENTION
[0003] The present invention relates to monospecific or bispecific
antibody molecules that specifically bind the human DR5 antigen.
The invention relates in particular to DR5 -specific antibody
molecules of the IgG1 isotype having a mutation in the Fc region
that enhances clustering of IgG molecules after cell--surface
antigen binding. The invention further relates to a combination of
antibody molecules binding different epitopes on human DR5. The
invention also relates to pharmaceutical compositions containing
these molecules and the treatment of cancer and other diseases
using these compositions.
BACKGROUND OF THE INVENTION
[0004] DR5, also known as death receptor 5, Tumor necrosis factor
receptor superfamily member 10B, TNFRSF10B, TNF-related
apoptosis-inducing ligand receptor 2, TRAIL receptor 2, TRAIL-R2
and CD262, is a cell surface receptor of the TNF receptor
superfamily that binds tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL) and mediates apoptosis. DR5 is a
single-pass type I membrane protein with three extracellular
cysteine-rich domains (CRDs), a transmembrane domain (TM) and a
cytoplasmic domain containing a death domain (DD). In the absence
of ligand, DR5 exists in the cell membrane either as monomer or as
pre-assembled complexes of two or three receptors through
interactions of the first cysteine-rich domain, also known as
pre-ligand assembly domain (PLAD) (Wassenaar et al., Proteins. 2008
Feb. 1; 70(2):333-43; Valley et al., J Biol Chem. 2012 Jun. 15;
287(25):21265-78; Sessler et al., Pharmacol Ther. 2013 November ;
140(2):186-99). A Crystal structure of TRAIL in complex with the
DR5 ectodomain showed that TRAIL binds to CRD2 and CRD3 in the
extracellular domain of DR5 in a complex containing a trimeric
receptor and a trimeric ligand (Hymowitz et al., Mol Cell. 1999
October ; 4(4):563-71). The DR5 trimers can further cluster into
higher-order receptor aggregates in lipid macrodomains, so-called
lipid rafts (Sessler et al., Pharmacol Ther. 2013 November;
140(2):186-99). In the ligand-bound conformation, the cytoplasmic
death domain-containing adaptor protein FADD associate with the
intracellular DD surface of the oligomerized DR5 molecules and
engage initiator caspases caspase-8 and caspase-10 to form the
death-inducing signaling complex (DISC).
[0005] Based on the sensitivity of cancer cells to TRAIL-mediated
apoptosis, numerous agents were developed to activate this pathway
to induce apoptosis selectively in cancer cells. Human recombinant
TRAIL (hrTRAIL), is being developed as dulanermin, and a series of
conventional (monospecific, bivalent) anti-DR5 antibodies have been
developed and tested in the clinic (reviewed in Ashkenazi et al.,
Nat Rev Drug Discov. 2008 December; 7(12):1001-12; Trivedi et al.,
Front Oncol. 2015 Apr. 2; 5:69): DR5 antibodies include lexatumumab
(HGS-ETR2), HGS-TR2J, conatumumab (AMG655), tigatuzumab (CS-1008),
drozitumab (Apomab) and LBY-135. Clinical studies with these
compounds demonstrated that DR5 antibodies were generally well
tolerated but failed to show convincing and significant clinical
benefit. Efforts to enhance the efficacy of DR5 targeting
antibodies mainly focus on (i) improving the sensitivity of cancer
cells to DR5 agonists through combination treatment, (ii)
developing biomarkers for better patient stratification, and (iii)
the development of DR5-targeting agents that activate DR5 signaling
and apoptosis-induction more effectively (reviewed in Lim et al.,
Expert Opin Ther Targets. 2015 May 25: 1-15; Twomey et al., Drug
Resist Updat. 2015 March; 19:13-21; Reddy et al., PLoS One. 2015
Sep. 17; 10(9)). Different therapeutic formats for increasing DR5
activation have been described and include oligomerization of
synthetic DR5 binding peptides, linear fusions of DR5-specific
scaffolds, nanoparticle-based delivery systems of rhTRAIL or
conatumumab and multivalent DR5 antibody-based formats (reviewed in
Holland et al., Cytokine Growth Factor Rev. 2014 Apr.;
25(2):185-93). APG880 and derivatives exist of two single chain
TRAIL receptor binding (scTRAIL-RBD) molecules (TRAIL mimics) fused
to the Fc part of a human IgG. Each scTRAIL-RBD has three receptor
binding sites resulting in a hexavalent binding mode in the fusion
protein (WO 2010/003766 A2). A prototype scTRAIL-RBD (APG350) has
been described to induce FcyR-independent antitumor efficacy in
vivo (Gieffers et al., Mol Cancer Ther, 2013. 12(12): p. 2735-47).
A tetravalent anti-DR5 antibody fragment-derived construct,
assembled by fusion of an anti-DR5 scFv fragment, human serum
albumin residues and the tetramerization domain of human p53, has
been shown to induce apoptosis more potently than the monovalent
construct (Liu et al., Biomed Pharmacother. 2015 Mar.; 70:41-5).
Nanobody molecules are single domain antibody fragments (VHH)
derived from camelid heavy chain-only antibodies, which, similarly
to scFvs, can be linked to form multivalent molecules. Preclinical
in vitro studies showed that TAS266, a tetravalent anti-DR5
Nanobody.RTM. molecule, was more potent than TRAIL or crosslinked
DR5 antibody LBY-135, which was attributed to more rapid caspase
activation kinetics (Huet et al., MAbs. 2014; 6(6):1560-70). TAS266
was also more potent in vivo than the parental murine mAb of
LBY-135. MultYbody.TM. molecules (MultYmab technology) are based on
the fusion of a homomultimerizing peptide to the Fc of one heavy
chains in an IgG heterodimer (knob into hole), making MultYbody
molecules intrinsically multivalent in solution. An anti-DR5
MultYbody was shown to induce potent killing in vitro.
Dual-affinity re-targeting (DART) molecules are covalently-linked
Fv-based diabodies. DR5 targeting tetravalent Fc DARTs comprising
either tetravalency for a single (mono-epitopic DARTs) or two DR5
epitopes (bi-epitopic DARTs) were shown to be more potent than
TRAIL and a conatumumab variant in inducing in cytotoxicity in
vitro and in vivo (Li et al., AACR Annual Meeting Apr. 20 2015,
Poster abstract #2464). Alternatively, FcyR-independent
avidity-driven DR5 hyperclustering can be mediated by a bispecific
DR5xFAP antibody (RG7386) through simultaneous binding to DR5 on
the cancer cell and to fibroblast activation protein (FAP) that is
expressed on fibroblasts in the tumor microenvironment (Friess et
al., AACR Annual Meeting Apr. 19 2015, Presentation abstract #952;
Wartha et al., Proceedings of the 105th Annual Meeting of the
American Association for Cancer Research; 2014 Apr. 5-9; San Diego,
Calif. Philadelphia (Pa.): AACR; Cancer Res 2014; 74(19
Suppl):Abstract nr 4573. doi:10.1158/1538-7445.AM2014-4573).
Finally, specific combinations of two anti-DR5 antibodies
recognizing different epitopes have shown enhanced agonistic
efficacy in vitro and in vivo compared to combinations of two
anti-DR5 antibodies recognizing overlapping or similar epitopes
(WO2014/009358).
[0006] Above described approaches show enhanced efficacy compared
to the conventional anti-DR5 antibodies in preclinical studies,
however clinical data indicate that there is still a need for
improving the DR5 agonists. Moreover, it is desirable for
antibody-based formats to preserve a pharmacokinetic (PK) as well
as other Fc-mediated effector functions of regular IgG, which
usually is not the case with antibody fragment-based constructs.
There is still a need for providing further DR5 agonists with
improved properties.
[0007] Consequently, there is a need for providing improved
anti-DR5 antibodies for the treatment of cancer, of infectious
disease, autoimmune disease, cardiovascular anomalies and other
diseases.
SUMMARY OF THE INVENTION
[0008] Surprisingly the inventors of the present invention have
found that the introduction of a specific point mutation in the Fc
region of an anti-DR5 antibody, which facilitates antibody
clustering conditional on cell-surface antigen binding independent
on secondary cross-linking, results in DR5 activation and
significantly enhances the potency of the antibody in inducing
apoptosis and cell death.
[0009] The objective of the present invention is to provide an
improved anti-DR5 antibody for use in the treatment of cancer and
other diseases. Such an improved antibody comprises a mutation in
the Fc region. A further object of the present invention is to
provide an improved composition for the treatment of cancer and
other diseases comprising one or more anti-DR5 antibodies according
the invention, e.g. wherein said antibodies bind to different
epitopes on DR5. Such an improved composition as described herein
comprises at least one anti-DR5 antibody according to the invention
and more preferably the composition comprises two anti-DR5
antibodies binding to different regions on DR5, such as different
non-competing epitopes on DR5.
[0010] The present invention provides an anti-DR5 antibody
comprising an Fc region of a human immunoglobulin IgG and an
antigen binding region binding to DR5, wherein the Fc region
comprises a mutation at an amino acid position 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).
[0011] That is, the inventors of the present invention have in a
first aspect of the invention found that anti-DR5 antibodies of the
invention comprising a mutation in the Fc region increase apoptosis
of DR5 positive cells such as tumor cells compared to anti-DR5
antibodies without a mutation at an amino acid position
corresponding to position E430, E345 or S440 in human IgG1, EU
numbering. That is, the anti-DR5 antibody of the present invention
is suitable for the treatment of DR5 positive or expressing
tumors.
[0012] In one embodiment of the present invention the anti-DR5
antibody comprises a mutation at an amino acid positon
corresponding to E430 in human IgG1 according to EU numbering,
wherein the mutation is selected form the group consisting of:
E430G, E4305, E430F and E430T.
[0013] In one embodiment of the present invention the anti-DR5
antibody comprises an Fc region of a human immunoglobulin IgG and
an antigen binding region binding to DR5, wherein the Fc region
comprises the mutation E430G (glutamic acid at position 430 into
glycine) or E345K (glutamic acid at position 345 into lysine) in
human IgG1 according to EU numbering.
[0014] In one embodiment of the present invention the anti-DR5
antibody comprises an Fc region of a human IgG1 and an antigen
binding region binding to DR5, wherein the Fc region comprises a
E430G mutation.
[0015] In one embodiment of the present invention the anti-DR5
antibody comprises a mutation at an amino acid positon
corresponding to E345 in human IgG1 according to EU numbering,
wherein the mutation is selected form the group consisting of:
E345K, E345Q, E345R and E345Y.
[0016] In one embodiment of the present invention the anti-DR5
antibody comprises an Fc region of a human IgG1 and an antigen
binding region binding to DR5, wherein the Fc region comprises an
E345K mutation.
[0017] In one embodiment of the present invention the anti-DR5
antibody comprises a mutation at an amino acid positon
corresponding to S440 in human IgG1 according to EU numbering,
wherein the mutation is selected form the group consisting of:
S440W and S440Y.
[0018] In one embodiment of the present invention the anti-DR5
antibody comprises an Fc region of a human IgG1 and an antigen
binding region binding to DR5, wherein the Fc region comprises a
S440Y mutation.
[0019] In one aspect the invention provides a composition
comprising one or more antibodies binding to DR5. In one embodiment
the composition comprises one or more antibodies binding to
different epitopes on DR5. Hereby are embodiments are provided
where the antibodies bind different epitopes or require different
amino acids within the DR5 sequence (SEQ ID NO 46) for binding to
DR5. In one embodiment the composition comprises anti-DR5
antibodies which do not compete for binding to DR5, that is in one
embodiment the anti-DR5 antibodies bind to non-overlapping
epitopes.
[0020] In another aspect the invention provides a bispecific
antibody comprising one or more antigen binding regions binding to
DR5.
[0021] In one embodiment of the present invention the bispecific
antibody comprises an Fc region comprising a first and a second
heavy chain, wherein said first and second heavy chain comprises a
mutation at an amino acid position corresponding to E430, E345 or
S440 in human IgG1, EU numbering.
[0022] In one embodiment of the present invention the bispecific
antibody comprises an Fc region comprising a first and a second
heavy chain, wherein said first heavy chain comprises a mutation
corresponding to position F405 and E430 and wherein said second
heavy chain comprises a mutation corresponding to position K409 and
E430, wherein the amino acid position is corresponding to human
IgG1 according to EU numbering.
[0023] In one embodiment of the present invention the bispecific
antibody comprises an Fc region comprising a first heavy chain with
a F405L mutation and a second heavy chain with a K409R mutation in
human IgG1 according to EU numbering. In another embodiment of the
invention the bispecific antibody comprises an Fc region comprising
a first heavy chain with a K409R mutation and a second heavy chain
with a F405L mutation in human IgG1 according to EU numbering.
[0024] 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.
[0025] In another aspect of the invention the anti-DR5 antibody,
bispecific antibody or composition according to the present
invention is for use as a medicament. In one embodiment the
anti-DR5 antibody, bispecific antibody or composition is for use in
treatment of a disease. In one embodiment the disease is a cancer
or a tumor.
[0026] 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.
[0027] In another aspect the invention provides for 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.
[0028] The anti-DR5 antibodies and compositions comprising anti-DR5
antibodies described herein are directed against or specific for
human DR5. The anti-DR5 antibodies and compositions described
herein cross-react with rhesus and cynomolgus monkey DR5. In
particular, in one embodiment of the invention the anti-DR5
antibodies and compositions bind specifically to the extracellular
domain of human DR5. In one particular embodiment of the invention
the antibodies and compositions comprising anti-DR5 antibodies bind
to human DR5 at non-overlapping epitopes. That is in one embodiment
the composition comprises at least one anti-DR5 antibody according
to the invention. In one embodiment the composition comprises a
first anti-DR5 antibody and a second anti-DR5 antibody according to
the invention. That is a first anti-DR5 antibody described herein
does not block binding of a second anti-DR5 antibody described
herein. In one particular embodiment a composition described herein
comprise a first and a second anti-DR5 antibody binding to human
DR5 and the first anti-DR5 antibody does not block binding of the
second anti-DR5 antibody to human DR5.
[0029] The anti-DR5 antibodies and compositions comprising anti-DR5
antibodies of the present invention i.e. anti-DR5 antibodies
comprising an amino acid mutation in the Fc region can generally be
used to modulate the activity of DR5. In one embodiment the
anti-DR5 antibody or composition may trigger, activate and/or
increase or enhance the signalling that is mediated by DR5. That
anti-DR5 antibodies comprising an amino acid mutation in the Fc
region according to the invention may increase or enhance the
signalling that is mediated by DR5 is to be understood as when the
signalling is compared to the same anti-DR5 antibody without said
mutation in the Fc region. In one embodiment the anti-DR5 antibody
or composition will have an agonistic effect on DR5 and in
particular trigger or increase the downstream effects of DR5. That
anti-DR5 antibodies comprising an amino acid mutation in the Fc
region according to the invention may have an agonistic effect on
DR5 is to be understood as when the agonistic effect is compared to
the DR5 ligand TRAIL or the same anti-DR5 antibody i.e. having the
same CDR sequences but without said mutation in the Fc region
according to the invention. That is anti-DR5 antibodies or
compositions of the present invention in able to induce apoptosis
or cell death in cells, tumor mass or tissues expressing DR5, such
as cancer cells or a tumor.
[0030] In one embodiment of the invention the anti-DR5 antibody or
composition described herein induce, trigger, increase or enhance
apoptosis, cell death or growth arrest in cells or tissues
expressing DR5, such as cancer cells, tumor cells or a tumor
compared to the same anti-DR5 antibody or composition without said
mutation in the Fc region. In one embodiment the anti-DR5
antibodies or compositions described herein are capable of binding
to DR5 on a cell surface, and in particular binding to DR5 in such
a way that the signalling mediated by DR5 is induced, triggered,
increased or enhanced compared to the same anti-DR5 antibody or
composition without said mutation in the Fc region. In one
embodiment the antibodies or compositions described herein may be
such that they are capable of binding to DR5 in such a way that
apoptosis or cell death is induced in cancer or tumor cells, tumors
or tissues expressing DR5.
[0031] 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 DR5. 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-DR5 antibodies of the
invention. 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-DR5 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-DR5 antibodies of the invention,
compared to untreated cell cultures. Induction of caspase-mediated
apoptosis can 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
[0032] FIG. 1 shows an amino acid alignment of the four different
human IgG1 Fc allotypes. The Fc sequence of the IgG1m(f), IgG1m(z),
IgG1m(a), IgG1m(x) is specified in SEQ ID: 29, 30, 31 and 32
respectively.
[0033] FIG. 2 shows binding of humanized (hDR5) and chimeric (DR5)
anti-DR5 antibodies to DR5-positive HCT 116 human colon cancer
cells as measured by flow cytometry on FACS. Anti-gp120 antibody
IgG1-b12 was used as a negative control. Binding is expressed as
MFI (mean fluorescence intensity). Error bars indicate the standard
deviation.
[0034] FIGS. 3A-3C show binding of anti-DR5 antibodies with and
without hexamerization-enhancing mutations E430G or E345K to
DR5-positive COLO 205 cells. Variants of the human-mouse chimeric
antibodies IgG1-DR5-01-K409R (FIG. 3A), IgG1-DR5-05-F405L (FIG. 3B)
and bispecific antibody IgG1-DR5-01-K409R.times.IgG1-DR5-05-F405L
(BsAb IgG1-DR5-01-K409.times.DR5-05-F405L) (FIG. 3C) were tested
flowcytometric analysis on FACS 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.
[0035] FIGS. 4A-4C show binding of anti-DR5 antibodies to human and
rhesus monkey DR5. Human-mouse chimeric antibodies
IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G were tested in
flowcytometric analysis on FACS for binding to (FIG. 4A)
mock-transfected CHO cells, (FIG. 4B) human DR5-transfected CHO
cells and (FIG. 4C) Rhesus macaque DR5-transfected CHO cells.
Binding is expressed as geometric mean of fluorescence intensity.
Error bars indicate the standard deviation.
[0036] FIGS. 5A-5D show (FIG. 5A) Sequence alignment of part of the
extracellular domains of human DR5 and mouse DR5 using EMBOSS
Matcher (http://www.ebi.ac.uk/Tool/psa/emboss matcher/); (.)
similar amino acid; (:) identical amino acid. (FIG. 5B) Graphical
representation of the domain-swapped DR5 extracellular domain
(white: human DR5 sequences; black: mouse DR5 sequences). Amino
acid number refer to the human sequence and domain swaps were made
based on the alignment shown in panel A. (FIG. 5C) Binding of
IgG1-hDR5-01-F405L and the isotype control antibody IgG1-b12 to a
panel of human-mouse chimeric DR5 molecules, as assessed by flow
cytometry. In each domain-swapped DR5 molecule, specific human
amino acids have been replaced by the mouse sequence, as indicated
on the x-axis. Error bars indicate the standard deviation of
duplicate samples. (FIG. 5D) Binding of IgG1-hDR5-05-F405L to a
panel of human-mouse chimeric DR5 molecules, as assessed by flow
cytometry. In each domain-swapped DR5 molecule, specific human
amino acids had been replaced by the mouse sequence, as indicated
on the x-axis. IgG1-b12 was included an isotype control antibody.
Error bars indicate the standard deviation of duplicate
samples.
[0037] FIGS. 6A and 6B show crossblock ELISA with DR5-01 and DR5-05
antibodies. Graphs represent inhibition of binding of coated
IgG1-hDR5-01-E430G (FIG. 6A) or IgG1-hDR5-05-E430G (FIG. 6B) to
soluble DR5ECD-FcHisCtag in the presence of competing antibody
IgG1-hDR5-01-E430G or IgG1-hDR5-05-E430G as measured by ELISA.
Anti-gp120 antibody IgG1-b12 (b12) was used as negative control.
DR5-01 is IgG1-hDR5-01-E430G; DR5-05 is IgG1-hDR5-05-E430G.
[0038] FIGS. 7A and 7B show a viability assay with variants of
DR5-01 and DR5-05 antibodies. Introduction of the E430G
hexamerization-enhancing mutation results in enhanced induction of
killing of DR5-positive COLO 205 (FIG. 7A) and HCT 116 (FIG. 7B)
colon cancer cells by the single human-mouse chimeric antibodies
IgG1-DR5-01-K409R and IgG1-DR5-05-F405L used alone and by the
combination thereof. Error bars indicate standard deviation.
[0039] FIGS. 8A-8C show (FIG. 8A) crossblock ELISA between
IgG1-chTRA8-F405L and IgG1-DR5-01-K409R or IgG1-DR5-05-F405L,
respectively. Combining the two non-crossblocking anti-DR5
antibodies IgG1-chTRA8-F405L-E430G and IgG1-DR5-01-K409R-E430G
(FIG. 8B) resulted in enhanced induction of killing of HCT 116
colon cancer cells (decreased EC50), whereas combining the two
crossblocking antibodies IgG1-chTRA8-F405L-E430G and
IgG1-DR5-05-F405L-E430G (FIG. 8C) did not, as determined in a
3-days viability assay. Error bars indicate standard deviation.
[0040] FIGS. 9A and 9B that introduction of a
hexamerization-enhancing mutation results in enhanced induction of
killing of HCT 116 colon cancer cells by the combination of
non-crossblocking antibodies
IgG1-DR5-05-F405L-E345K+IgG1-CONA-K409R-E430G and BsAb
IgG1-DR5-05-F405L-E345K.times.CONA-K409R-E430G. (FIG. 9A)
crossblock ELISA with IgG1-CONA-K409R and IgG1-DR5-05-F405L. (FIG.
9B) 3-days viability assay. Error bars indicate standard deviation.
RLU: Relative Luminescence Units.
[0041] FIG. 10 shows that the combination of
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G reduces the
viability of a large panel 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<0.0001 (One-way ANOVA with
Tukey's multiple comparisons test).
[0042] FIG. 11 shows the potency of the combination of humanized
IgG1-hDR5-01-K409R-E430G+IgG1-hDR5-05-F405L-E430G antibodies and of
the combination of chimeric
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G antibodies as
measured in a viability assay on BxPC-3 and PANC-1 pancreatic
cancer cell lines. Graphs represent mean values of duplicate
(BxPC-3) or triplicate (PANC-1) samples +/- standard deviation.
[0043] FIGS. 12A-12C show (FIG. 12A) Flowcytometric analysis using
FACS analysis to study the effect of mimicking deamidation in
humanized antibodies IgG1-hDR5-01-K409R and IgG1-hDR5-05-F405L on
binding to HCT 116 human colon cancer cells. Introduction of the
Asn deamidation-mimicking mutation N55D resulted in decreased
binding of IgG1-hDR5-01-K409R, but had minimal effect on binding of
IgG1-hDR5-05-F405L. (FIG. 12B) Flowcytometry analysis to study the
effect of preventing deamidation in humanized antibody DR5-01 on
binding to HCT 116 human colon cancer cells. Introduction of the
amino acid substitution G56T in IgG1-hDR5-01-E430G had no effect on
the binding of the antibody to HCT 116 cells. Binding is expressed
as Geometric mean of fluorescence intensity. (FIG. 12C) Potency of
the combination of humanized antibodies
IgG1-hDR5-01-G56T-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.
[0044] FIGS. 13A and 13B 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 results in diminished induction
of killing of BxPC-3 pancreatic (FIG. 13A) and HCT-15 colon cancer
cells (FIG. 13B). By combining the two mutations (K439E and S440K)
in both antibodies, repulsion is neutralized and killing restored.
Error bars indicate standard deviation.
[0045] FIG. 14: 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.
[0046] FIG. 15 shows the efficacy of different ratios of
combinations of IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G
(DR5-01:DR5-05) on adherent BxPC-3 human cancer cells as determined
in a 3-days viability assay.
[0047] FIGS. 16A and 16B show efficacy of different ratios of
IgG1-hDR5-01-G56T -E430G and IgG1-hDR5-05-E430G (DR5-01:DR5-05) on
adherent BxPC-3 (FIG. 16A) and HCT-15 (FIG. 16B) human cancer cells
as determined in a 3-days viability assay.
[0048] FIGS. 17A-17C show Caspase-dependent programmed cell death
by the combination of humanized
IgG1-hDR5-01-E430G+IgG1-hDR5-05-E430G antibodies as measured in a
viability assay on PANC-1 (FIGS. 17A and 17B) and BxPC-3 (FIG. 17C)
pancreatic cancer cells. 01-E430G is IgG1-hDR5-01-E430G; 05-E430G
is IgG1-hDR5-05-E430G; ZVAD is pan-caspase inhibitor
Z-VaI-AIa-DL-Asp-fluoromethylketone (Z-VAD-FMK).
[0049] FIGS. 18A-18E show cell death induction upon binding of
anti-DR5 antibody or anti-DR5 antibody combinations on COLO 205
colon cancer cells. COLO 205 cells were incubated with antibody
sample for 5 hours (FIGS. 18A-18C) and 24 hours (FIGS. 18D-18E).
Different stages of cell death induction were analyzed by Annexin
V/PI double staining and Active caspase-3 staining. Panels C and D
show Annexin V/PI double staining at 5 and 24 hours respectively.
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.
[0050] FIG. 19 shows the kinetics of Caspase-3/7 activation upon
binding of DR5 antibodies 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.
[0051] FIG. 20 shows efficacy of the combination of
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G in the presence or
absence of Fc crosslinking by F(ab').sub.2 fragments of an
anti-human IgG antibody and comparison to the anti-DR5 antibodies
IgG1-DR5-CONA and IgG1-DR5-chTRA8-F405L in a 3-days viability assay
on adherent COLO 205 colon cancer and BxPC-3 and PANC-1 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. *p<0.05, **p<0.01, ***p<0.001,
****p<0.0001 (One-way ANOVA with Bonferroni post-test for
multiple comparisons).
[0052] FIG. 21 shows the potency of the combination of humanized
IgG1-hDR5-01-K409R-E430G+IgG1-hDR5-05-F405L-E430G antibodies and of
the combination of humanized IgG1-DR5-01-E430G+IgG1-DR5-05-E430G
antibodies as measured in a viability assay on BxPC-3 pancreatic
cancer cells. Graphs represent mean values of duplicate samples +/-
standard deviation.
[0053] FIG. 22 shows the potency of the chimeric BsAb
IgG1-DR5-01-K409R-E430G.times.DR5-05-F405L-E430G antibody on
different human cancer cell lines determined in a 3-days viability
assay on adherent cells from COLO 205 colon, BxPC-3 pancreatic,
SNU-5 gastric, SK-MES-1 lung, and A375 skin cancer cell lines.
Graphs show the mean+/-standard deviation from duplicate samples.
*p<0.05, ***p<0.001, ****p<0.0001 (One-way ANOVA with
Bonferroni post-test for multiple comparisons). (01.times.05)-E430G
is BsAb IgG1-DR5-01-K409R-E430G.times.DR5-05-F405L-E430G.
[0054] FIG. 23 shows the efficacy of chimeric BsAb
IgG1-DR5-01-K409R-E430G.times.DR5-05-F405L-E430G in the presence or
absence of Fc crosslinking by F(ab').sub.2 fragments of an
anti-human IgG antibody in comparison with the anti-DR5 antibodies
IgG1-DR5-CONA and IgG1-DR5-chTRA8-F405L in a 3-days viability assay
on adherent BxPC-3 pancreatic and COLO 205 colon cancer cells. The
non-target binding antibody IgG1-b12 was included as a negative
control. Graphs show the mean+/-standard deviation from duplicate
samples. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001
(One-way ANOVA with Bonferroni post-test for multiple comparisons).
(01.times.05)-E430G is BsAb
IgG1-DR5-01-K409R-E430G.times.IgG1-DR5-05-F405L-E430G
[0055] FIGS. 24A-24E show cell death induction upon binding of
bispecific DR5 antibodies on COLO 205 colon cancer cells. COLO 205
cells were incubated with 1 .mu.g/mL antibody for 5 hours (FIGS.
24A-24C) and 24 hours (FIGS. 24D-24E). 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, 01.times.05 is BsAb
IgG1-DR5-01-K409R.times.DR5-05-F405L, 01-E430G.times.05-E430G is
BsAb IgG1-DR5-01-K409R-E430G.times.DR5-05-F405L-E430G.
[0056] FIGS. 25A-25C show evaluation of the in vivo efficacy of the
combination of the chimeric
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G antibodies in a
subcutaneous xenograft model with COLO 205 human colon cancer
cells. Tumor size (mean & SEM) in mice treated with the
indicated antibodies (5 mg/kg) is shown in time (FIG. 25A) and at
day 23 (FIG. 25B). In (FIG. 25C) the percentage of mice with tumor
sizes smaller than 750 mm.sup.3 is shown in a Kaplan-Meier
plot.
[0057] FIGS. 26A-26C show evaluation of the in vivo efficacy of
different doses of the
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G antibody
combination and comparison to IgG1-CONA in a subcutaneous COLO 205
colon cancer xenograft. Tumor size (mean & SEM) in mice treated
with the indicated antibody dose is shown in time (FIG. 26A) and on
day 16 (FIG. 26B). In (FIG. 26C) the percentage of mice with tumor
sizes smaller than 500 mm.sup.3 is shown in a Kaplan-Meier plot.
*p<0.05, ***p<0.001.
[0058] FIGS. 27A-27C show evaluation of the in vivo efficacy of
different doses of the
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G antibody
combination and comparison to IgG1-CONA-F405L in a subcutaneous
xenograft model with BxPC-3 human pancreatic cancer cells. Tumor
size in mice treated with the indicated antibodies is shown in time
(FIG. 27A, median tumor size) and at day 48 after tumor inoculation
(FIG. 27B, mean tumor size & SEM). *p<0.05, **p<0.01
(Unpaired t-test). In (FIG. 27C) the percentage of mice with tumor
sizes smaller than 500 mm.sup.3 is shown in a Kaplan-Meier
plot.
[0059] FIGS. 28A and 28B show evaluation of the in vivo efficacy of
different doses of the
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G antibody
combination and comparison to IgG1-CONA-F405L in a subcutaneous
xenograft model with A375 human skin cancer cells. Tumor size in
mice treated with the indicated antibodies is shown in time (FIG.
28A, median tumor size) and at day 29 after tumor inoculation (FIG.
28B, mean tumor size & SEM). *p<0.05, **p<0.01,
***p<0.001 (Mann Whitney test).
[0060] FIGS. 29A-29C show evaluation of the in vivo efficacy of
different doses of the
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G antibody
combination and comparison to IgG1-CONA in a subcutaneous xenograft
model with HCT-15 human colon cancer cells. Tumor size (mean &
SEM) in mice treated with the indicated antibodies is shown in time
(FIG. 29A) and at day 17 after start treatment (FIG. 29B).
****p<0.001 (Unpaired t test). In (FIG. 29C) the percentage of
mice with tumor sizes smaller than 500 mm.sup.3 is shown in a
Kaplan-Meier plot.
[0061] FIGS. 30A-30C show evaluation of the in vivo efficacy of
different doses of the
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G antibody
combination and comparison to IgG1-CONA in a subcutaneous xenograft
model with SW480 human colon cancer cells. Tumor size (mean &
SEM) in mice treated with the indicated antibodies is shown in time
(FIG. 30A) and at day 28 after start treatment (FIG. 30B).
*p<0.05, **p<0.01 (Unpaired t-test). In (FIG. 30C) the
percentage of mice with tumor sizes smaller than 500 mm.sup.3 is
shown in a Kaplan-Meier plot.
[0062] FIG. 31A-31C show evaluation of the in vivo efficacy of
different doses of the
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G antibody
combination and comparison to IgG1-CONA in a subcutaneous xenograft
model with SNU-5 human gastric cancer cells. Tumor size (mean &
SEM) in mice treated with the indicated antibodies is shown in time
(FIG. 31A) and at day 23 after start treatment (FIG. 31B).
**p<0.01, ***p<0.001 (Mann Whitney test). In (FIG. 31C) the
percentage of mice with tumor sizes smaller than 500 mm.sup.3 is
shown in a Kaplan-Meier plot.
[0063] FIGS. 32A-32C show evaluation of the in vivo efficacy of
different doses of the
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G antibody
combination and comparison to IgG1-CONA in a subcutaneous xenograft
model with SK-MES-1 human lung cancer cells. Tumor size (mean &
SEM) in mice treated with the indicated antibodies is shown in time
(FIG. 32A) and at day 14 after start treatment (FIG. 32B). In (FIG.
32C) the percentage of mice with tumor sizes smaller than 1.000
mm.sup.3 is shown in a Kaplan-Meier plot.
[0064] FIG. 33 shows binding to DR5-positive HCT 116 human colon
cancer cells by anti-DR5 antibodies IgG1-hDR5-01-G56T and
IgG1-hDR5-05 with and without the E430G mutation as measured by
flow cytometry. Anti-gp120 antibody IgG1-b12 was used as a negative
control. Binding is expressed as geometric mean fluorescence
intensity (FI). Error bars indicate the standard deviation. A
representative example of seven experiments is shown.
[0065] FIG. 34 shows binding to DR5-positive HCT 116 human colon
cancer cells by anti-DR5 antibodies IgG1-hDR5-01-G56T-E430G and
IgG1-hDR5-05-E430G as measured by flow cytometry with directly
labeled antibodies. Binding is expressed as Geometric mean Alexa
647 fluorescence intensity (FI). Error bars indicate the standard
deviation.
[0066] FIGS. 35A and 35B show binding of anti-DR5 antibodies to
human and cynomolgus monkey DR5. Antibodies IgG1-hDR5-01-G56T-E430G
and IgG1-hDR5-05-E430G were tested by flow cytometry for binding to
(FIG. 35A) human DR5-transfected CHO cells and (FIG. 35B)
cynomolgus DR5-transfected CHO cells. Binding is expressed as
geometric mean of fluorescence intensity (FI). Error bars indicate
the standard deviation.
[0067] FIG. 36 shows a 3-days viability assay to show the effect of
introducing the E430G mutation in the non-crossblocking antibodies
IgG1-hDR5-01-G56T and IgG1-hDR5-05 on COLO 205 colon cancer cells.
Error bars indicate standard deviation. A representative example of
four experiments is shown.
[0068] FIGS. 37A and 37B 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. 37A) and increased efficacy of the antibody combination
IgG1-hDR5-01-G56T+IgG1-hDR5-05 (FIG. 37B). Error bars indicate
standard deviation.
[0069] FIGS. 38A and 38B show the efficacy of non-crossblocking
antibodies IgG1-DR5-CONA-E430G+IgG1-DR5-chTRA8-E430G to induce
killing of BxPC-3 human pancreatic cancer cells. (FIG. 38A)
Crossblock ELISA between IgG1-DR5-CONA-K409R (CONA) and
IgG1-DR5-chTRA8-F405L (chTRA8). (FIG. 38B) Introduction of the
E430G hexamerization-enhancing mutation resulted in enhanced
induction of killing of BxPC-3 cells by the combination of
IgG1-DR5-CONA-C49W-E430G+IgG1-DR5-chTRA8-E430G as determined in a
3-days viability assay. Error bars indicate standard deviation.
[0070] FIG. 39 shows 3-days viability assays with 133 nM human
recombinant TRAIL or 133 nM of the antibody combinations
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G (E430G) and
IgG1-hDR5-01-G56T+IgG1-hDR5-05 (WT) on different human cancer cell
lines. Graphs show the mean+/-standard deviation from duplicate
samples. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001
(One-way ANOVA with Tukey's multiple comparisons test).
[0071] FIGS. 40A and 40B show the percentage inhibition by (FIG.
40A) antibody (IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G) and
(FIG. 40B) TRAIL therapy as determined in a 3-days viability assay
screening of a cell line panel at Horizon, UK. Each data point
represents an individual cell line of the indicated human cancer
indication. Dotted lines indicate the 70% maximum response
threshold value that was set to categorize cell lines as responders
70% inhibition) and non-responders (<70% inhibition).
[0072] FIGS. 41A and 41B show the efficacy of different antibody
ratios in the combination
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G (indicated as
01-E430G:05-E430G) on adherent human (FIG. 41A) BxPC-3 pancreatic
and (FIG. 41B) HCT-15 colon cancer cells as determined in a 3-days
viability assay. Representative examples of two and three
experiments are shown for HCT-15 and BxPC-3, respectively.
[0073] FIG. 42 shows Caspase-dependent programmed cell death by the
combination of IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G
antibodies, the parental WT combination without the E430G mutation
and TRAIL as measured in a viability assay on BxPC-3 pancreatic
cancer cells. ZVAD is pan-caspase inhibitor
Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD-FMK).
[0074] FIG. 43 shows the kinetics of Caspase-3/7 activation upon
binding of the antibody combination
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G on BxPC-3 pancreatic
cancer cells, compared to the parental WT combination without the
E430G mutation and TRAIL. BxPC-3 cells were incubated with antibody
for 1, 2, 4 and 6 hours. Caspase-3/7 activation was analyzed in a
homogenous luminescence assay. RLU, relative luminescence units. A
representative example of four experiments is shown.
[0075] FIG. 44 shows efficacy of the combination of
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G in the presence or
absence of Fc crosslinking by F(ab').sub.2 fragments of an
anti-human IgG antibody and comparison to the anti-DR5 antibody
IgG1-DR5-CONA and the combination of WT antibodies
IgG1-hDR5-01-G56T+IgG1-hDR5-05 in a 3-days viability assay on
adherent HCT-15 human colon cancer and BxPC-3 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. For both cell lines, a representative example of
two experiments is shown.
[0076] FIGS. 45A-45D show the analysis of IgG1-hDR5-01-G56T-E430G
and IgG1-hDR5-05-E430G to induce complement activation upon target
cell binding on CHO cells transfected with human (FIG. 45A, FIG.
45C) or cynomolgus DR5 (FIG. 45B, FIG. 45D). (FIGS. 45A and 45B) In
vitro CDC assay with antibody concentration series in the presence
of 20% pooled normal human serum. CDC efficacy is presented as the
percentage lysis determined by the percentage propidium iodide
(PO-positive cells. (FIGS. 45C and 45D) Deposition of complement
activation products upon antibody binding in the presence of
C5-depleted serum is expressed as geometric mean of fluorescence
intensity. The IgG1-b12 mAb against HIV gp120 was used in as a
non-binding isotype control antibody.
[0077] FIGS. 46A-46E show the effect of combining the antibody
combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G with
different therapeutic agents as determined in a viability assay on
five different colon cancer cell lines. Five examples are shown
from a synergy screen of 100 compounds from different therapeutic
classes.
[0078] FIGS. 47A and 47B 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. 47A) Tumor size
(mean & SEM) in mice treated with the indicated antibodies (0.5
mg/kg) as shown in time. (FIG. 47B) Kaplan-Meier plot of tumor
progression, with a cutoff set at a tumor volume >500
mm.sup.3.
[0079] FIG. 48 shows 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 size (mean & SEM) in mice treated with the 0.5 mg/kg
antibodies is shown in time (A) and at day 21 after start treatment
(B). **P<0.0011 (Mann Whitney test). In (C) the percentage of
mice with tumor sizes smaller than 750 mm3 is shown in a
Kaplan-Meier plot.
[0080] FIGS. 49A-49C show evaluation of the in vivo efficacy of the
combination of IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-430G antibodies
in combination with 15 mg/kg paclitaxel in a subcutaneous xenograft
model with SK-MES-1 human lung cancer cells. (FIG. 49A) Tumor size
(mean & SEM) in mice treated with the indicated compounds is
shown in time. (FIG. 49B) Tumor volume per treatment group at day
16. (FIG. 49C) The percentage of mice with tumor sizes smaller than
500 mm.sup.3 is shown in a Kaplan-Meier plot.
[0081] FIGS. 50A and 50B show the clearance rate in SCID mice of 1
mg/kg i.v. administered IgG1-hDR5-01-G56T-E430G, IgG1-hDR5-05-E430G
or the combination of the two antibodies in comparison to the
parental WT antibodies without the E430G mutation. (FIG. 50A) Total
human IgG in serum samples was determined by ELISA and plotted in a
concentration versus time curve. Each data point represents the
mean+/-standard deviation of four serial diluted samples. (FIG.
50B) Clearance until day 21 after administration of the antibody
was determined following the formula D*1.000/AUC with D, injected
dose and AUC, area under the curve of the concentration-time
curve.
[0082] FIG. 51 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.
DETAILED DESCRIPTION OF THE INVENTION
[0083] 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.
[0084] As described herein, surprisingly antibodies binding to DR5
and comprising a hexamerization enhancing mutation in the Fc region
corresponding to position E430 or E345 of human IgG1 according to
EU numbering, were found to be superior at inducing apoptosis in
tumor cells expressing DR5 compared to antibodies binding DR5
without a mutation in one of the above mentioned positions.
Furthermore, compositions comprising two anti-DR5 antibodies of the
invention, which bind different epitopes on DR5, were found
superior in in vitro and in vivo studies to compositions comprising
the same anti-DR5 antibodies without the mutation. That is
compositions with two antibodies of the present invention were
superior at inducing apoptosis and/or inhibiting cell growth of
tumor cells expressing DR5 compared to compositions comprising two
DR5 antibodies without a mutation in the Fc region. By introducing
specific mutations in the Fc region, hexamerization upon target
binding on the cell surface can be enhanced, while the antibody
molecules remain monomeric in solution WO2013/004842,
WO2014/108198.
Definitions
[0085] 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).
[0086] 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.
[0087] 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.
[0088] 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.
[0089] 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.
[0090] 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.
[0091] 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.
[0092] 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.
[0093] The term "chimeric antibody", as used herein, refers to an
antibody in which both chain types i.e. heavy chain and light chain
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.
[0094] 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.
[0095] 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.
[0096] 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 29. In one embodiment of the
invention the antibody is of the IgG1m(z) allotype as defined in
SEQ ID NO 30, the IgG1m(a) allotype as defined in SEQ ID NO 31, the
IgG1m(x) allotype as defined in SEQ ID NO 32, 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).
[0097] 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.
[0098] 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.
[0099] The term "bispecific antibody" refers to an 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.
[0100] 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.RTM. molecules (Genmab A/S, WO 2011/131746), DuetMab
(Medimmune, US2014/0348839), Biclonics (Merus, WO 2013/157953),
Novlmmune (k.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
(Medlmmune), Dual Variable Domain (DVD)-Ig.TM. (Abbott, U.S. Pat.
No. 7,612,18), dual domain double head antibodies (Unilever; Sanofi
Aventis, WO20100226923), Ts2Ab (Medlmmune/AZ), BsAb (Zymogenetics),
HERCULES (Biogen Idec, US007951918), 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).
[0101] 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.
[0102] 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.
[0103] 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.
[0104] 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.
[0105] 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).
[0106] 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 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 BlAcore 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.
[0107] The term "k.sub.d" (sect), 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.
[0108] 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.
[0109] 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.
[0110] 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.
[0111] 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).
[0112] 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.
[0113] 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).
[0114] 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.
[0115] 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. In a particular embodiment,
apoptosis by one or more agonistic anti-DR5 antibodies can be
determined using methods such as, e.g., caspase-3/7 activation
assays described in examples 19, 20, 25 and 45 or
phosphatidylserine exposure described in examples 19 and 25.
Anti-DR5 antibody at a fixed concentration of e.g. 1 .mu.g/mL may
be added to adhered cells and incubated for 1 to 24 hours.
Caspase-3/7 activation can be determined by using special kits for
this purpose, such as the PE Active Caspase-3 Apoptosis Kit of BD
Pharmingen (Cat nr 550914) (example 19 and 25) or the Caspase-Glo
3/7 assay of Promega (Cat nr G8091) (examples 20 and 45).
Phosphatidylserine exposure and cell death can be determined by
using special kits for this purpose, such as the FITC Annexin V
Apoptosis Detection Kit I from BD Pharmingen (Cat nr 556547)
(examples 19 and 25).
[0116] 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.
[0117] The term "Annexin V", as used herein, refers to a protein of
the annexin group that binds phosphatidylserine (PS) on the cell
surface.
[0118] 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.
[0119] 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) as described in
examples 18 and 44. 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.sup.2C. 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).
[0120] 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-DR5
antibodies can be determined by quantifying the ATP present in the
cells as described in examples 8-18, 21-24, 38-44, 46 and 48.
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.
[0121] The term "DR5", as used herein, refers to death receptor 5,
also known as CD262 and TRAILR2, 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). In humans, the DR5 protein is
encoded by a nucleic acid sequence encoding the amino acid sequence
shown in SEQ ID NO 46, (human DR5 protein: UniprotKB/Swissprot
O14763).
[0122] 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."
[0123] The term "agonist" as used herein, refers to a molecule such
as an anti-DR5 antibody that is able to trigger a response in a
cell when bound to DR5, wherein the response may be programmed cell
death. That the anti-DR5 antibody is agonistic is to be understood
as that the antibody stimulates, activates or clusters DR5 as the
result from anti-DR5 binding to DR5. That is an agonistic anti-DR5
antibody comprising an amino acid mutation in the Fc region
according to the present invention bound to DR5 results in DR5
stimulation, clustering or activation of the same intracellular
signaling pathways as TRAIL bound to DR5. In a particular
embodiment, the agonistic activity of one or more antibodies can be
determined by incubating target cells for 3 days with an antibody
concentration dilution series (e.g. from 20,000 ng/mL to 0.05 ng/mL
final concentration in 5-fold dilutions). The antibodies may be
added directly when cells are seeded (described in examples 8, 9,
10, 39), or alternatively the cells are first allowed to adhere to
96-well flat-bottom plates before adding the antibody samples
(described in examples 11, 12, 13, 14, 15, 16, 17, 18, 21, 22, 23,
24, 38, 40, 41, 42, 43, 44, 46, 48). The agonistic activity i.e.
the agonistic effect can be quantified by measuring the amount of
viable cells using special kits for this purpose, such as the
CellTiter-Glo luminescent cell viability assay of Promega (Cat nr
G7571).
[0124] The terms "DR5 positive" and "DR5 expressing" as used
herein, refers to tissues or cell lines which show binding of a
DR5-specific antibody which can be measured with e.g. flow
cytometry or immunohistochemistry.
[0125] 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.
[0126] 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 present in the wild-type protein 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 [0127] Acidic Residues Asp (D) and Glu (E) Basic
Residues Lys (K), Arg (R), and His (H) Hydrophilic Uncharged Ser
(S), Thr (T), Asn (N), and Residues Gln (Q) Aliphatic Uncharged Gly
(G), Ala (A), Val (V), Leu (L), Residues and Ile (I) Non-polar
Uncharged Cys (C), Met (M), and Pro (P) Residues Aromatic Residues
Phe (F), Tyr (Y), and Trp (W)
Alternative Conservative Amino Acid Residue Substitution
Classes
TABLE-US-00002 [0128] 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 [0129] 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
[0130] In the context of the present invention, a substitution in a
variant is indicated as: [0131] Original amino
acid--position--substituted amino acid;
[0132] 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. 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.
[0133] 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).
[0134] 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 a/., 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).
[0135] 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).
[0136] 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.
[0137] 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).
[0138] 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:29
of at least 80%, or 85%, 90%, or at least 95%. For example, the
sequence alignments shown in FIGS. 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.
[0139] 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.
[0140] 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, NSO 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
[0141] The present invention is based, at least in part, on the
discovery that the ability of an anti-DR5 antibody to induce cell
death in a target cell expressing DR5 can be greatly enhanced by
introducing a specific mutation in the Fc region corresponding to
amino acid positions 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 DR5 and each comprising a mutation in the Fc
region may form heterohexamers and 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.
[0142] In one aspect the present invention relates to an anti-DR5
antibody comprising an Fc region of a human immunoglobulin IgG and
an antigen binding region binding to DR5, wherein the Fc region
comprises a mutation at an amino acid position corresponding to
position E430, E345 or S440 in human IgG1 according to EU
numbering.
[0143] 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.
[0144] The anti-DR5 antibody according to the present invention
comprises an Fc region comprising a first and a second heavy chain,
wherein a mutation at a position according to the present invention
corresponding to E430, E345 or S440 in human IgG1 according to EU
numbering is present in both the first and the second heavy chain,
or less preferred only be present in one of the heavy chains. In
the context of the present invention the hexamerization enhancing
mutation is an amino acid mutation at a position corresponding to
E430, E345 or S440 in human IgG1 according to EU numbering. The
hexamerixation enhancing mutation strengthens the Fc-Fc
interactions between antibodies comprising the mutation when bound
to the corresponding target on a cell surface.
[0145] By introducing specific mutations in the Fc region
corresponding to at least one of the following positions E430, E345
and S440 in human IgG1 hexamerization upon target binding on the
cell surface is enhanced, while the antibody molecules remain
monomeric in solution (WO2013/004842; WO2014/108198).
[0146] In one embodiment of the present invention the Fc region of
the anti-DR5 antibody comprises a mutation corresponding to E430G,
E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y or S440W in
human IgG1, EU numbering. Thus the anti-DR5 antibody comprises a
mutation selected from the group of: E430G, E430S, E430F, E430T,
E345K, E345Q, E345R, E345Y, S440Y and S440W in human IgG1, EU
numbering. Hereby are embodiments provided that allow for enhanced
hexamerization of antibodies upon cell-surface antigen binding. The
anti-DR5 antibody comprises an Fc region comprising a first heavy
chain and a second heavy chain, wherein one of the above mentioned
hexamerization enhancing mutations may be present in the first
and/or the second heavy chain.
[0147] In a preferred embodiment of the present invention the Fc
region comprises a mutation corresponding to E430G or E345K in
human IgG1 EU numbering. Thus the Fc region comprises a mutation
selected from E430G and E345K.
[0148] In one embodiment of the present invention the anti-DR5
antibody comprises a mutation at an amino acid positon
corresponding to E430 in human IgG1 according to EU numbering,
wherein the mutation is selected form the group consisting of:
E430G, E430S, E430F and E430T.
[0149] In one embodiment of the present invention the Fc region
comprises a mutation corresponding to E430G. Thus in one embodiment
of the present invention the Fc region comprises an E430G
mutation.
[0150] In one embodiment of the present invention the anti-DR5
antibody comprises a mutation at an amino acid positon
corresponding to E345 in human IgG1 according to EU numbering,
wherein the mutation is selected form the group consisting of:
E345K, E345Q, E345R and E345Y.
[0151] In one embodiment of the present invention the Fc region
comprises a mutation corresponding to E345K. Thus in one embodiment
of the present invention the Fc region comprises an E345K
mutation.
[0152] In one embodiment of the present invention the anti-DR5
antibody comprises a mutation at an amino acid positon
corresponding to S440 in human IgG1 according to EU numbering,
wherein the mutation is selected form the group consisting of:
S440W and S440Y.
[0153] In one embodiment of the present invention the Fc region
comprises a mutation corresponding to S440Y. Thus in one embodiment
of the present invention the Fc region comprises an S440Y
mutation.
[0154] 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. 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.
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.
[0155] The human DR5 molecule (Uniprot O14763) 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
(Uniprot O14763-2) is missing 185-213 from the extracellular
domain.
[0156] 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.
[0157] 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 Oct.; 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. Thus in one
embodiment the DR5 antibody binds to CRD2 and/or CRD3 on DR5. In
one embodiment of the invention the antibody comprises an antigen
binding region that blocks TRAIL binding to DR5. 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.
[0158] 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.
[0159] In an embodiment of the invention the antibody comprises an
antigen binding region that binds to an epitope on DR5 comprising
or requiring one or more amino acid residues located within amino
acid residues 116-138 and one or more amino acid residues located
within amino acid residues 139-166 of SEQ ID NO 46. That is the
antigen binding region binds to or requires for binding to DR5 one
or more amino acids located within positions 116-138 and one or
more amino acids located within positions 139-166. That the antigen
binding region binds to one or more amino acids comprised in a
sequence is to be understood as the antigen binding region is in
contact with or directly interacts with one or more amino acids
within the sequence. That the antigen binding region requires one
or more amino acids within a sequence means that no contact or
direct interaction between antigen binding region and one or more
amino acids in the sequence is needed, but that one or more amino
acids are required for keeping the three-dimensional structure of
the epitope.
[0160] In another preferred embodiment of the present invention the
antibody comprises an antigen binding region that binds to an
epitope on DR5 comprising or requiring one or more amino acid
residues located within amino acid residues 79-138 of SEQ ID NO
46.
[0161] In one embodiment of the invention the anti-DR5 antibody
comprises an antigen binding region comprising a variable heavy
chain (VH) region comprising CDR1, CDR2 and CDR3 domains and a
variable light chain (VL) region comprising CDR1, CDR2 and CDR3
domains having the amino acid sequences of: [0162] a) (VH) SEQ ID
NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS, 6; [0163] b) (VH) SEQ ID
NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6; [0164] c) (VH) SEQ ID
NOs 10, 2, 11 and (VL) SEQ ID NOs 13, RTS, 14; [0165] d) (VH) SEQ
ID NOs 16, 17, 18 and VL) SEQ ID NOs 21, GAS, 22 or [0166] e) the
(VH) CDR1, CDR2, CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in
any of a) to d) above having one to five mutations e.g.
substitutions in total across said six CDR sequences. [0167] That
is in one embodiment up to five mutations such as substitutions in
total are allowed across the six CDRs comprising the antigen
binding region. In some embodiments of the invention up to five
mutations e.g. substitutions such as one, two, three, four or five
mutations e.g. substitutions, are made across the three CDRs of the
VH region and no mutations are made across the CDRs of the VL
region. [0168] In other embodiments no mutations e.g. substitutions
are made across the CDRs of the VH region but up to five mutations
e.g. substitutions, such as one, two, three, four or five are found
across the CDRs of the VL region.
[0169] In one embodiment of the invention, the anti-DR5 antibody as
defined in any of the embodiments disclosed herein comprises an
antigen binding region comprising a variable heavy chain (VH)
region comprising CDR1, CDR2 and CDR3 domains and a variable light
chain (VL) region comprising CDR1, CDR2 and CDR3 domains, wherein
said VH region and said VL region has at least 75%, 80%, 85% 90%,
at least 95%, at least 97%, or at least 99% amino acid sequence
identity to the amino acid sequence as set forth in the six CDR
sequences selected from the group consisting of: [0170] a) (VH) SEQ
ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS, 6; [0171] b) (VH) SEQ
ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6; [0172] c) (VH) SEQ
ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14; and [0173] d)
(VH) SEQ ID NOs: 16, 17, 18 and VL) SEQ ID NOs: 21, GAS, 22.
[0174] In one embodiment of the invention the anti-DR5 antibody
comprises a variable heavy chain (VH) region comprising CDR1, CDR2
and CDR3 domains and a variable light chain (VL) region comprising
CDR1, CDR2 and CDR3 domains having the CDR sequences selected from
one of the groups consisting of: [0175] a) (VH) SEQ ID NOs 1, 8, 3
and (VL) SEQ ID NOs 5, FAS, 6 or [0176] b) (VH) SEQ ID NOs 10, 2,
11 and (VL) SEQ ID NOs 13, RTS, 14 or [0177] c) the (VH) CDR1, CDR2
and CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in any one of (a)
or (b) above having one to five mutations in total across said six
CDR sequences. That is in one embodiment up to five mutations such
as substitutions in total are allowed across the six CDRs
comprising the antigen binding region. In some embodiments of the
invention up to five mutations e.g. substitutions such as one, two,
three, four or five mutations e.g. substitutions, are made across
the three CDRs of the VH region and no mutations are made across
the CDRs of the VL region. In other embodiments no mutations e.g.
substitutions are made across the CDRs of the VH region but up to
five mutations e.g. substitutions, such as one, two, three, four or
five are found across the CDRs of the VL region.
[0178] In one embodiment of the invention the anti-DR5 antibody
comprises a variable heavy chain (VH) region comprising CDR1, CDR2
and CDR3 domains and a variable light chain (VL) region comprising
CDR1, CDR2 and CDR3 domains having the CDR sequences selected from
one of the groups consisting of: [0179] a) (VH) SEQ ID NOs 1, 2, 3
and (VL) SEQ ID NOs 5, FAS, 6 or [0180] b) (VH) SEQ ID NOs 10, 2,
11 and (VL) SEQ ID NOs 13, RTS, 14 or [0181] c) the (VH) CDR1, CDR2
and CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in (a) or (b)
above having up to five mutations in total across said six CDR
sequences.
[0182] That is in one embodiment up to five mutations such as
substitutions in total are allowed across the six CDRs comprising
the antigen binding region. In some embodiments of the invention up
to five mutations e.g. substitutions, such as one, two, three, four
or five mutations e.g. substitutions are made across the three CDRs
of the VH region and no mutations are made across the three CDRs or
the VL region. In other embodiments no mutations e.g. substitutions
are made across the three CDRs of the VH region but up to five
mutations e.g. substitutions are made across the six CDRs of the VL
region, wherein the mutations e.g. substitutions are conservative
or concern amino acids with similar physical or functional
properties and preferably do not modify binding affinity to
DR5.
[0183] In one embodiment of the invention, the anti-DR5 antibody as
defined in any of the embodiments disclosed herein comprises an
antigen binding region comprising a variable heavy chain (VH)
region and a variable light chain (VL) region, wherein said VH
region and said VL region has at least 75%, 80%, 85% 90%, at least
95%, at least 97%, or at least 99% amino acid sequence identity to
the amino acid sequence as set forth in the VH and VL sequences
selected from the group consisting of: [0184] a) (VH) SEQ ID NO:4
and (VL) SEQ ID NO:7; [0185] b) (VH) SEQ ID NO:9 and (VL) SEQ ID
NO:7; [0186] c) (VH) SEQ ID NO:12 and (VL) SEQ ID NO:15; [0187] d)
(VH) SEQ ID NO:19 and (VL) SEQ ID NO:23; and [0188] e) (VH) SEQ ID
NO:20 and (VL) SEQ ID NO:23.
[0189] In one embodiment of the invention the antibody comprises an
antigen binding region comprising a variable heavy chain (VH)
region and a variable light chain (VL) region having the amino acid
sequences of: [0190] a) (VH) SEQ ID NO:4 and (VL) SEQ ID NO:7;
[0191] b) (VH) SEQ ID NO:9 and (VL) SEQ ID NO:7; [0192] c) (VH) SEQ
ID NO:12 and (VL) SEQ ID NO:15; [0193] d) (VH) SEQ ID NO:19 and
(VL) SEQ ID NO:23; [0194] e) (VH) SEQ ID NO:20 and (VL) SEQ ID
NO:23 or [0195] f) the (VH) and (VL) as defined in any one of a) to
e) above having one to 10 mutations or substitutions in total
across said (VH) and (VL) sequences. That is in one embodiment up
to 10 mutations such as substitutions in total are allowed across
the VH and VL regions defined by the VH and VL sequences. In some
embodiments of the invention up to ten mutations e.g.
substitutions, such as one, two, three, four, five, six, seven,
eight, nine or ten mutations e.g. substitutions are made across the
VH or VL sequences. In one embodiment of the invention up to10
mutations or substitutions are made in the VH sequence and no
mutations are made in the VL sequence. In one embodiment of the
invention no mutations are made in the VH sequence and up to ten
mutations e.g. substitutions are made in the VL sequence. Hereby
are embodiments provided that allow for up to 10 mutations such as
substitutions across the VH and VL sequences, wherein the mutations
such as substitutions are conservative or concern amino acids with
similar physical or functional properties, thereby allowing
mutations e.g. substitutions within the VH and VL sequence without
modifying binding affinity or function of the anti-DR5
antibody.
[0196] 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.
[0197] In a preferred embodiment of the invention the antibody is
an IgG1 antibody.
[0198] 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).
[0199] In one embodiment of the present invention the antibody
comprises an Fc region comprising an amino acid sequence of the
group consisting of: [0200] a) SEQ ID NO:29; [0201] b) SEQ ID
NO:30; [0202] c) SEQ ID NO:31; [0203] d) SEQ ID NO:32 or [0204] e)
an amino acid sequence defined in any one of a) to d) having one to
five mutations e.g. substitutions in total across said sequence.
That is in one embodiment up to five mutations e.g. substitutions
in total are allowed across the Fc region. In some embodiments of
the invention up to five mutations e.g. substitutions such as one,
two, three, four or five mutations e.g. substitutions, are allowed
across the Fc region.
[0205] In one embodiment of the invention, the anti-DR5 antibody as
defined in any of the embodiments disclosed herein comprises a
heavy chain (HC) and a light chain (LC), wherein the LC comprises
the sequence of SEQ ID NO:39 and wherein the HC has at least 75%,
80%, 85%, 90%, at least 95%, at least 97%, or at least 99% amino
acid sequence identity to the amino acid sequence as set forth in
the HCs sequences selected from the group consisting of: [0206] a)
(HC) SEQ ID NO:33 ; [0207] b) (HC) SEQ ID NO:34; [0208] c) (HC) SEQ
ID NO:35; [0209] d) (HC) SEQ ID NO:36; [0210] e) (HC) SEQ ID NO:37;
and [0211] f) (HC) SEQ ID NO:38.
[0212] In one embodiment of the invention, the anti-DR5 antibody as
defined in any of the embodiments disclosed herein comprises a
heavy chain (HC) and a light chain (LC), wherein the LC has at
least 75%, 80%, 85%, 90%, at 95%, at least 97%, or at least 99%
amino acid sequence identity set forth in SEQ ID NO:39 and wherein
the HC has the amino acid sequence as set forth in the HCs
sequences selected from the group consisting of: [0213] a) (HC) SEQ
ID NO:33 ; [0214] b) (HC) SEQ ID NO:34; [0215] c) (HC) SEQ ID
NO:35; [0216] d) (HC) SEQ ID NO:36; [0217] e) (HC) SEQ ID NO:37;
and [0218] f) (HC) SEQ ID NO:38.
[0219] In one embodiment according to the invention, the antibody
comprises a heavy chain (HC) and a light chain (LC), wherein the LC
comprises the sequence of SEQ ID NO:39 and wherein the HC comprises
of one of the sequences selected from the group consisting of:
[0220] a) (HC) SEQ ID NO:33 ; [0221] b) (HC) SEQ ID NO:34; [0222]
c) (HC) SEQ ID NO:35; [0223] d) (HC) SEQ ID NO:36; [0224] e) (HC)
SEQ ID NO:37; and [0225] f) (HC) SEQ ID NO:38; or [0226] g) the
(HC) as defined in any one of a) to f) above having one to ten
mutations in total across said (HC) sequence. That is in one
embodiment up to 10 mutations such as substitutions in total are
allowed across the heavy chain defined by the heavy chain sequence.
In some embodiments of the invention up to ten mutations e.g.
substitutions, such as one, two, three, four, five, six, seven,
eight, nine or ten mutations e.g. substitutions are made across the
heavy chain sequence. Hereby are embodiments provided that allow
for up to 10 mutations such as substitutions across the heavy chain
sequence, wherein the mutations such as substitutions are
conservative or concern amino acids with similar physical or
functional properties, thereby allowing mutations or substitutions
within the heavy chain sequence without modifying binding affinity
or function of the anti-DR5 antibody.
[0227] In one embodiment of the invention, the anti-DR5 antibody as
defined in any of the embodiments disclosed herein comprises a
heavy chain (HC) and a light chain (LC), wherein the LC comprises
the sequence of SEQ ID NO:43 and wherein the HC has at least 75%,
80%, 85%, 90%, at least 95%, at least 97%, or at least 99% amino
acid sequence identity to the amino acid sequence as set forth in
the HCs sequences selected from the group consisting of: [0228] a)
(HC) SEQ ID NO:40 ; [0229] b) (HC) SEQ ID NO:41; and [0230] c) (HC)
SEQ ID NO:42.
[0231] In one embodiment of the invention, the anti-DR5 antibody as
defined in any of the embodiments disclosed herein comprises a
heavy chain (HC) and a light chain (LC), wherein the LC has at
least 75%, 80%, 85%, 90%, at 95%, at least 97%, or at least 99%
amino acid sequence identity set forth in SEQ ID NO:43 and wherein
the HC has the amino acid sequence as set forth in the HCs
sequences selected from the group consisting of: [0232] a) (HC) SEQ
ID NO:40; [0233] b) (HC) SEQ ID NO:41; and [0234] c) (HC) SEQ ID
NO:42.
[0235] In one embodiment according to the invention the antibody
comprises a heavy chain (HC) and a light chain (LC), wherein the LC
comprises the sequence of SEQ ID NO:43 and wherein the HC comprises
of one of the sequences selected from the group consisting of:
[0236] a) (HC) SEQ ID NO:40; [0237] b) (HC) SEQ ID NO:41; [0238] c)
(HC) SEQ ID NO:42; or [0239] d) the (HC) as defined in any one of
a) to c) above having one to ten mutations e.g. substitutions in
total across said (HC) sequence. [0240] That is in one embodiment
up to 10 mutations such as substitutions in total are allowed
across the heavy chain defined by the heavy chain sequence. In some
embodiments of the invention up to ten mutations e.g.
substitutions, such as one, two, three, four, five, six, seven,
eight, nine or ten mutations e.g. substitutions are made across the
heavy chain sequence. Hereby are embodiments provided that allow
for up to 10 mutations such as substitutions across the heavy chain
sequence, wherein the mutations such as substitutions are
conservative or concern amino acids with similar physical or
functional properties, thereby allowing mutations such as
substitutions within the heavy chain sequence without modifying
binding affinity or function of the anti-DR5 antibody.
[0241] In one embodiment the antibody is a human antibody, a
chimeric antibody or a humanized antibody.
[0242] In one embodiment of the present invention the anti-DR5
antibody is agonistic. That the antibody is agonistic is to be
understood as that the antibody clusters, stimulates or activates
DR5. In one embodiment, an agonistic anti-DR5 antibody of the
present invention bound to DR5 activates the same intracellular
pathways as TRAIL bound to DR5. The agonistic activity of one or
more antibodies can be determined by incubating target cells for 3
days with an antibody concentration dilution series (e.g. from
20,000 ng/mL to 0.05 ng/mL final concentration in 5-fold
dilutions). The antibodies may be added directly when cells are
seeded (described in examples 8, 9, 10, 39), or alternatively the
cells are first allowed to adhere to 96-well flat-bottom plates
before adding the antibody samples (described in examples 11, 12,
13, 14, 15, 16, 17, 18, 21, 22, 23, 24, 38, 40, 41, 42, 43, 44, 46,
48). The agonistic activity i.e. the agonistic effect can be
quantified by measuring the amount of viable cells using special
kits for this purpose, such as the CellTiter-Glo luminescent cell
viability assay of Promega (Cat nr G7571).
[0243] In one embodiment of the present invention the anti-DR5
antibody has enhanced agonistic activity. That the anti-DR5
antibody has activity is to be understood as the antibody is able
to cluster DR5 or activate at least the same intracellular pathways
as TRAIL bound to DR5. That is anti-DR5 antibody with enhanced
agonistic activity is able to induce increased level of apoptosis
or programmed cell death in a cell or tissue expressing DR5
compared to TRAIL or a wild-type IgG1 antibody against DR5.
[0244] In one embodiment of the present invention the anti-DR5
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 invention the anti-DR5 antibody induces caspase-3
and/or caspase-7 dependent cell death. In one embodiment of the
present invention the antibody induces apoptosis. Apoptosis by one
or more agonistic anti-DR5 antibodies can be determined using
methods such as, e.g., caspase-3/7 activation assays described in
examples 19, 20, 25 and 45 or phosphatidylserine exposure described
in examples 19 and 25. Anti-DR5 antibody at a fixed concentration
of e.g. 1 .mu.g/mL may be added to adhered cells and incubated for
1 to 24 hours. Caspase-3/7 activation can be determined by using
special kits for this purpose, such as the PE Active Caspase-3
Apoptosis Kit of BD Pharmingen (Cat nr 550914) (example 19 and 25)
or the Caspase-Glo 3/7 assay of Promega (Cat nr G8091) (examples 20
and 45). Phosphatidylserine exposure and cell death can be
determined by using special kits for this purpose, such as the FITC
Annexin V Apoptosis Detection Kit I from BD Pharmingen (Cat nr
556547) (examples 19 and 25).
[0245] In one embodiment of the present invention the anti-DR5
antibody induces phosphatidylserine (PS) exposure, which can be
measured by Annexin-V binding. In one embodiment of the present
invention anti-DR5 induces translocation of PS to the cell surface
of the target cell. Therefore, Annexin-V binding correlates to
programmed cell death and can be used to measure the anti-DR5
antibody's ability to induce cellular events leading to programmed
cell death.
[0246] In a preferred embodiment of the present invention the
anti-DR5 antibody induces apoptosis in a target cell expressing
DR5, such as a tumor cell.
[0247] In one embodiment of the invention the anti-DR5 antibody
reduces cell viability.
[0248] In one embodiment of the present invention the anti-DR5
antibody induces DR5 clustering. That the antibody can induce
clustering and even enhance clustering leads to activation of at
least the same intracellular signaling pathways as TRAIL bound to
DR5.
[0249] 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 DR5. 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-DR5 antibodies of the
invention. 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-DR5 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-DR5 antibodies of the invention,
compared to untreated cell cultures. Induction of caspase-mediated
apoptosis can be assessed by demonstrating inhibition of the loss
of viability after exposure to DR5 antibody by a caspase-inhibitor,
for example ZVAD.
[0250] In one embodiment of the present invention the anti-DR5
antibody engages into oligomerization such as hexamerization of
antibodies on target cells expressing DR5. Oligomerization such as
hexamerization is mediated through Fc-Fc interactions. One method
for determining this is by inhibiting Fc-Fc interactions which
indicate that antibodies oligomerizies e.g. hexamerizies. The Fc-Fc
interactions can be inhibited by a peptide binding to the
hydrophobic patch involved in Fc-Fc interactions such as
DCAWHLGELVWCT as described in example 15.
Bispecific Antibodies
[0251] In another aspect, the present invention comprises a
bispecific antibody comprising at least one antigen binding region
as described herein.
[0252] In another aspect, the present invention comprises a
bispecific antibody comprising one or more antigen binding regions
as described herein.
[0253] In one embodiment of the invention the bispecific antibody
comprises a first antigen binding region and a second antigen
binding region as defined herein.
[0254] 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 human DR5.
[0255] 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 human DR5 does not block
binding of said second antigen binding region binding to human
DR5.
[0256] In one embodiment of the present invention the bispecific
anti-DR5 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 anti-DR5
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 anti-DR5
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.
[0257] In one embodiment of the invention the bispecific antibody
comprises a first and a second antigen binding region, wherein said
first antigen binding region comprises the following six CDR
sequences, [0258] a) (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs:
5, FAS, 6 and said second antigen binding region comprises the
following six CDR sequences [0259] b) (VH) SEQ ID NOs: 10, 2, 11
and (VL) SEQ ID NOs: 13, RTS, 14, or wherein the said first antigen
binding region and said second antigen binding region comprises, c)
the six CDR sequences defined in (a) or (b) above having one to
five mutations or substitutions in total across said six CDR
sequences respectively. [0260] That is the one or more mutations or
substitutions across the six CDR sequences of the antigen binding
region do not change the binding characteristics of said first or
second antibody such as the agonistic properties, the DR5 epitope
binding and/or the ability to induce apoptosis in a target cell
expressing DR5. That is in one embodiment up to five mutations or
substitutions in total are allowed across the six CDRs comprising
the antigen binding region. In some embodiments of the invention up
to five mutations or substitutions such as one, two, three, four or
five mutations or substitutions, are made across the three CDRs of
the VH region and no mutations are made across the CDRs of the VL
region. In other embodiments no mutations or substitutions are made
across the CDRs of the VH region but up to five mutations or
substitutions, such as one, two, three, four or five are found
across the CDRs of the VL region.
[0261] In one embodiment of the invention the bispecific antibody
comprises a first and a second antigen binding region, wherein said
first antigen binding region comprises the following six CDR
sequences, [0262] a) said first antigen binding region comprises
the following six CDR sequences (VH) SEQ ID NOs: 1, 2, 3 and (VL)
SEQ ID NOs: 5, FAS, 6 and said second antigen binding region
comprises the following six CDR sequences (VH) SEQ ID NOs: 10, 2,
11 and (VL) SEQ ID NOs: 13, RTS, 14, or wherein said first antigen
binding region and said second antigen binding region comprises, b)
the six CDR sequences defined in (a) comprising one to five
mutations e.g substitutions in total across said six CDR sequences
of each first and second antigen binding region respectively.
[0263] That is the one or more mutations e.g. substitutions across
the six CDR sequences of the antigen binding region do not change
the binding characteristics of said first or second antibody such
as the agonistic properties, the DR5 epitope binding and/or the
ability to induce apoptosis in a target cell expressing DR5. That
is in one embodiment up to five mutations e.g. substitutions in
total are allowed across the six CDRs comprising the antigen
binding region. In some embodiments of the invention up to five
mutations e.g. substitutions such as one, two, three, four or five
mutations or substitutions, are made across the three CDRs of the
VH region and no mutations are made across the CDRs of the VL
region. In other embodiments no mutations e.g. substitutions are
made across the CDRs of the VH region but up to five mutations e.g.
substitutions, such as one, two, three, four or five are found
across the CDRs of the VL region.
[0264] In one embodiment of the invention the bispecific antibody
comprises a first and a second antigen binding region, wherein said
first antigen binding region comprises the following six CDR
sequences, [0265] a) (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs:
5, FAS, 6 and said second antigen binding region comprises the
following six CDR sequences [0266] b) (VH) SEQ ID NOs: 10, 2, 11
and (VL) SEQ ID NO:s 13, RTS, 14, or wherein the said first antigen
binding region and said second antigen binding region comprises, c)
the six CDR sequences defined in (a) or (b) above having one to
five mutations or substitutions in total across said six CDR
sequences respectively. That is the one or more mutations or
substitutions across the six CDR sequences of the antigen binding
region do not change the binding characteristics of said first or
second antibody such as the agonistic properties, the DR5 epitope
binding and/or the ability to induce apoptosis in a target cell
expressing DR5. That is in one embodiment up to five mutations or
substitutions in total are allowed across the six CDRs comprising
the antigen binding region. In some embodiments of the invention up
to five mutations or substitutions such as one, two, three, four or
five mutations or substitutions, are made across the three CDRs of
the VH region and no mutations are made across the CDRs of the VL
region. In other embodiments no mutations or substitutions are made
across the CDRs of the VH region but up to five mutations or
substitutions, such as one, two, three, four or five are found
across the CDRs of the VL region.
[0267] In one embodiment of the invention the bispecific antibody
comprises a first and a second antigen binding region wherein a)
said first antigen binding region comprises the following six CDR
sequences (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6
and said second antigen binding region comprises the following six
CDR sequences(VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NO:s 13,
RTS, 14, or wherein the said first antigen binding region and said
second antigen binding region comprises b) the six CDR sequences
defined in (a having one to five mutations or substitutions in
total across said six CDR sequences of each antigen binding region
respectively. That is the one or more mutations e.g. substitutions
across the six CDR sequences of the antigen binding region do not
change the binding characteristics of said first or second antibody
such as the agonistic properties, the DR5 epitope binding and/or
the ability to induce apoptosis in a target cell expressing DR5.
That is in one embodiment up to five mutations e.g. substitutions
in total are allowed across the six CDRs comprising the antigen
binding region. In some embodiments of the invention up to five
mutations e.g. substitutions such as one, two, three, four or five
mutations e.g. substitutions, are made across the three CDRs of the
VH region and no mutations are made across the CDRs of the VL
region. In other embodiments no mutations or substitutions are made
across the CDRs of the VH region but up to five mutations e.g.
substitutions, such as one, two, three, four or five are found
across the CDRs of the VL region.
[0268] In one embodiment of the invention the bispecific antibody
comprises a first and a second antigen binding region, wherein said
first antigen binding region comprises the following six CDR
sequences, [0269] a) (VH) SEQ ID NOs: 16, 17, 18 and (VL) SEQ ID
NOs: 21, GAS, 6 and said second antigen binding region comprises
the following six CDR sequences [0270] b) (VH) SEQ ID NOs: 10, 2,
11 and (VL) SEQ ID NOs: 13, RTS, 14, or wherein the said first
antigen binding region and said second antigen binding region
comprises, [0271] c) the six CDR sequences defined in a) or (b)
above having one to five mutations or substitutions in total across
said six CDR sequences. That is the one or more mutations or
substitutions across the six CDR sequences of the antigen binding
region do not change the binding characteristics of said first or
second antibody such as the agonistic properties, the DR5 epitope
binding and/or the ability to induce apoptosis in a target cell
expressing DR5. That is in one embodiment up to five mutations or
substitutions in total are allowed across the six CDRs comprising
the antigen binding region. In some embodiments of the invention up
to five mutations or substitutions such as one, two, three, four or
five mutations or substitutions, are made across the three CDRs of
the VH region and no mutations are made across the CDRs of the VL
region. In other embodiments no mutations or substitutions are made
across the CDRs of the VH region but up to five mutations or
substitutions, such as one, two, three, four or five are found
across the CDRs of the VL region.
[0272] In one embodiment of the invention the bispecific antibody
comprises a first and a second antigen binding region, wherein,
[0273] a) said first antigen binding region comprises the following
six CDR sequences (VH) SEQ ID NOs: 16, 17, 18 and (VL) SEQ ID NOs:
21, GAS, 6 and said second antigen binding region comprises the
following six CDR sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ
ID NOs: 13, RTS, 14, or [0274] b) said first antigen binding region
and said second antigen binding region comprises the six CDR
sequences defined in a) comprising one to five mutations e.g.
substitutions in total across said six CDR sequences of each
antigen binding region. [0275] That is the one or more mutations
e.g. substitutions across the six CDR sequences of each antigen
binding region do not change the binding characteristics of said
first or second antibody such as the agonistic properties, the DR5
epitope binding and/or the ability to induce apoptosis in a target
cell expressing DR5. That is in one embodiment up to five mutations
e.g. substitutions in total are allowed across the six CDRs
comprising the antigen binding region. In some embodiments of the
invention up to five mutations e.g. substitutions such as one, two,
three, four or five mutations e.g. substitutions, are made across
the three CDRs of the VH region and no mutations are made across
the CDRs of the VL region. In other embodiments no mutations e.g.
substitutions are made across the CDRs of the VH region but up to
five mutations e.g. substitutions, such as one, two, three, four or
five are found across the CDRs of the VL region.
[0276] In one embodiment of the invention the bispecific antibody
comprises a first and a second antigen binding region, wherein said
first antigen binding region comprises the following sequences (a)
(VH) CDR1 SEQ ID NO 1, CDR2 SEQ ID NO 8, CDR3 SEQ ID NO 3 and (VL)
CDR1 SEQ ID NO 5, CDR2 FAS, CDR3 SEQ ID NO 6, or b) the (VH) CDR1,
CDR2 and CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in (a) above
having one to five mutations in total across said six CDR sequences
and wherein said second antigen binding region comprises the
following sequences (c) (VH) CDR1 SEQ ID NO 10, CDR2 SEQ ID NO 2,
CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2 RTS, CDR3 SEQ ID
NO 14 or (d) the (VH) CDR1, CDR2 and CDR3 and (VL) CDR1, CDR2 and
CDR3 as defined in (c) above having one to five mutations in total
across said six CDR sequences.
[0277] In one embodiment of the invention the bispecific antibody
comprises a first and a second antigen binding region, wherein (a)
said first antigen binding region comprises the following sequences
(VH) CDR1 SEQ ID NO 1, CDR2 SEQ ID NO 8, CDR3 SEQ ID NO 3 and (VL)
CDR1 SEQ ID NO 5, CDR2 FAS, CDR3 SEQ ID NO 6 and said second
antigen binding region comprises the following sequences (VH) CDR1
SEQ ID NO 10, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ
ID NO 13, CDR2 RTS, CDR3 SEQ ID NO 14 or b) said first antigen
binding region or said second antigen binding region comprises one
to five mutations in total across said six CDR sequences of each
antigen binding region.
[0278] In one embodiment of the invention the bispecific antibody
comprises a first and a second antigen binding region, wherein said
first antigen binding region comprises the following sequences (a)
(VH) CDR1 SEQ ID NO 1, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 3 and (VL)
CDR1 SEQ ID NO 5, CDR2 FAS, CDR3 SEQ ID NO 6, or (b) the (VH) CDR1,
CDR2 and CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in (a) above
having one to five mutations in total across said six CDR sequences
and wherein said second antigen binding region comprises the
following sequences (c) (VH) CDR1 SEQ ID NO 10, CDR2 SEQ ID NO 2,
CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2 RTS, CDR3 SEQ ID
NO 14 or (d) the (VH) CDR1, CDR2 and CDR3 and (VL) CDR1, CDR2 and
CDR3 as defined in (c) above having one to five mutations in total
across said six CDR sequences.
[0279] In one embodiment of the invention the bispecific antibody
comprises a first and a second antigen binding region, wherein (a)
said first antigen binding region comprises the following sequences
(VH) CDR1 SEQ ID NO 1, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 3 and (VL)
CDR1 SEQ ID NO 5, CDR2 FAS, CDR3 SEQ ID NO 6 and said second
antigen binding region comprises the following sequences (VH) CDR1
SEQ ID NO 10, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ
ID NO 13, CDR2 RTS, CDR3 SEQ ID NO 14 or b) said first antigen
binding region or said second antigen binding region comprises one
to five mutations in total across said six CDR sequences of each
antigen binding region.
[0280] In one embodiment of the invention the bispecific antibody
comprises a first and a second antigen binding region, wherein said
first antigen binding region comprises the following sequences (a)
(VH) CDR1 SEQ ID NO 16, CDR2 SEQ ID NO 17, CDR3 SEQ ID NO 18 and
(VL) CDR1 SEQ ID NO 21, CDR2 GAS, CDR3 SEQ ID NO 22,or (b) the (VH)
CDR1, CDR2 and CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in (a)
above having one to five mutations in total across said six CDR
sequences and wherein said second antigen binding region comprises
the following sequences (c) (VH) CDR1 SEQ ID NO 10, CDR2 SEQ ID NO
2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2 RTS, CDR3 SEQ
ID NO 14 or (d) the (VH) CDR1, CDR2 and CDR3 and (VL) CDR1, CDR2
and CDR3 as defined in (c) above having one to five mutations in
total across said six CDR sequences.
[0281] In one embodiment of the invention the bispecific antibody
comprises a first and a second antigen binding region, wherein (a)
said first antigen binding region comprises the following sequences
(VH) CDR1 SEQ ID NO 16, CDR2 SEQ ID NO 17, CDR3 SEQ ID NO 18 and
(VL) CDR1 SEQ ID NO 21, CDR2 GAS, CDR3 SEQ ID NO 22 and said second
antigen binding region comprises the following sequences (VH) CDR1
SEQ ID NO 10, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ
ID NO 13, CDR2 RTS, CDR3 SEQ ID NO 14 or b) said first antigen
binding region or said second antigen binding region comprises one
to five mutations in total across said six CDR sequences of each
antigen binding region.
[0282] If the antibody is a bispecific antibody that comprises an
Fc region comprising a first and a second heavy chain, a mutation
according to the present invention i.e. a mutation in a position
corresponding to E430, E345 or S440 in IgG1, EU numbering, may in
principle only be present in one of the heavy chains; i.e. in
either the first or second heavy chain, although in a preferred
embodiment according to the present invention, the mutation is
present in both the first and second heavy chain of the bispecific
antibody.
[0283] 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.
[0284] 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.
[0285] 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.
[0286] 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.
[0287] 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.
[0288] 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.
[0289] 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.
[0290] 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.
[0291] In one embodiment of the invention the bispecific antibody
is comprised in a pharmaceutical composition.
Anti-DR5 Antibody Compositions
[0292] The anti-DR5 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.
[0293] In one aspect the invention relates to a composition
comprising at least one anti-DR5 antibody according to any one of
the embodiments described herein.
[0294] In one aspect the invention relates to a composition
comprising one or more anti-DR5 antibodies according to any one the
embodiments described herein. The composition may comprise one, two
or more anti-DR5 antibodies according to the invention as described
herein that are not identical, such as a combination of two
different monoclonal anti-DR5 antibodies.
[0295] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody and a second anti-DR5 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.
[0296] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody comprising a mutation in the
position corresponding to E430 in human IgG1, EU numbering and a
second anti-DR5 antibody comprising a mutation in the positon
corresponding to E430 in human IgG1, EU numbering, wherein the
first and second antibody binds different epitopes on DR5.
[0297] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody comprising a mutation in the
position corresponding to E430 in human IgG1, EU numbering and a
second anti-DR5 antibody comprising a mutation in the positon
corresponding to E430 in human IgG1, EU numbering, wherein the
first antibody does not block binding of the second antibody to
DR5.
[0298] In one embodiment of the present invention the composition
comprises a first and a second anti-DR5 antibody comprising a
mutation in a position corresponding to E430 in human IgG1, EU
numbering, such a mutation may be selected from the group
consisting of: E430G, E4305 and E430T.
[0299] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody comprising an E430G and a
second anti-DR5 antibody comprising an E430G mutation, wherein the
first and second antibody binds different epitopes on DR5.
[0300] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody and a second anti-DR5 antibody,
wherein the first anti-DR5 antibody comprises the following six CDR
sequences, [0301] a) (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs:
5, FAS, 6 and said second anti-DR5 antibody comprises the following
six CDR sequences, [0302] b) (VH) SEQ ID NOs: 10, 2, 11 and (VL)
SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5
antibody and said second anti-DR5 antibody comprises a mutation in
the position corresponding to E430.
[0303] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody and a second anti-DR5 antibody,
wherein said first anti-DR5 antibody comprises the following six
CDR sequences (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS,
6 and said second anti-DR5 antibody comprises the following six CDR
sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS,
14, and wherein the said first anti-DR5 antibody and said second
anti-DR5 antibody comprises a mutation in the position
corresponding to E430 in human IgG1, EU numbering.
[0304] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody and a second anti-DR5 antibody,
wherein the first anti-DR5 antibody comprises the following six CDR
sequences, [0305] a) (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs:
5, FAS, 6 and said second anti-DR5 antibody comprises the following
six CDR sequences, [0306] b) (VH) SEQ ID NOs: 10, 2, 11 and (VL)
SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5
antibody and said second anti-DR5 antibody comprises an E430G
mutation in the Fc region.
[0307] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody and a second anti-DR5 antibody,
wherein said first anti-DR5 antibody comprises the following six
CDR sequences (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS,
6 and said second anti-DR5 antibody comprises the following six CDR
sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS,
14, and wherein the said first anti-DR5 antibody and said first
anti-DR5 antibody and said second anti-DR5 antibody comprises an
E430G mutation in the Fc region.
[0308] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody and a second anti-DR5 antibody,
wherein the first anti-DR5 antibody comprises the following six CDR
sequences, [0309] a) (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs:
5, FAS, 6 and said second anti-DR5 antibody comprises the following
six CDR sequences, [0310] b) (VH) SEQ ID NOs: 10, 2, 11 and (VL)
SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5
antibody and said second anti-DR5 antibody comprises a mutation in
the position corresponding to E430.
[0311] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody and a second anti-DR5 antibody,
wherein said first anti-DR5 antibody comprises the following six
CDR sequences (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS,
6 and said second anti-DR5 antibody comprises the following six CDR
sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS,
14, and wherein the said first anti-DR5 antibody and said first
anti-DR5 antibody and said second anti-DR5 antibody comprises a
mutation in the position corresponding to E430 in human IgG1, EU
numbering.
[0312] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody and a second anti-DR5
antiobody, wherein the first anti-DR5 antibody comprises the
following six CDR sequences, [0313] a) (VH) SEQ ID NOs: 1, 8, 3 and
(VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody
comprises the following six CDR sequences, [0314] b) (VH) SEQ ID
NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the
said first anti-DR5 antibody and said second anti-DR5 antibody
comprises an E430G mutation in the Fc region.
[0315] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody and a second anti-DR5 antibody,
wherein said first anti-DR5 antibody comprises the following six
CDR sequences (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS,
6 and said second anti-DR5 antibody comprises the following six CDR
sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS,
14, and wherein the said first anti-DR5 antibody and said first
anti-DR5 antibody and said second anti-DR5 antibody comprises an
E430G mutation in the Fc region.
[0316] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody comprising a mutation in the
position corresponding to E345 in human IgG1, EU numbering and a
second anti-DR5 antibody comprising a mutation in the positon
corresponding to E345 in human IgG1, EU numbering, wherein the
first and second antibody binds different epitopes on DR5.
[0317] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody comprising a mutation in the
position corresponding to E345 in human IgG1, EU numbering and a
second anti-DR5 antibody comprising a mutation in the positon
corresponding to E345 in human IgG1, EU numbering, wherein the
first antibody does not block binding of the second antibody to
DR5.
[0318] In one embodiment of the present invention the composition
comprises a first and a second anti-DR5 antibody comprising a
mutation in a position corresponding to E345, such a mutation may
be selected from the group consisting of: E345K, E345Q, E345R and
E345Y.
[0319] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody comprising an E345K and a
second anti-DR5 antibody comprising an E345K mutation, wherein the
first and second antibody binds different epitopes on DR5.
[0320] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody and second anti-DR5 antibody,
wherein the first anti-DR5 antibody comprises the following six CDR
sequences, [0321] a) (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs:
5, FAS, 6 and said second anti-DR5 antibody comprises the following
six CDR sequences, [0322] b) (VH) SEQ ID NOs: 10, 2, 11 and (VL)
SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5
antibody and said second anti-DR5 antibody comprises a mutation in
the position corresponding to E345.
[0323] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody and a second anti-DR5 antibody,
wherein said first anti-DR5 antibody comprises the following six
CDR sequences (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS,
6 and said second anti-DR5 antibody comprises the following six CDR
sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS,
14, and wherein the said first anti-DR5 antibody and said second
anti-DR5 antibody comprises a mutation in the position
corresponding to E345 in human IgG1, EU numbering.
[0324] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody and a second anti-DR5 antibody,
wherein the first anti-DR5 antibody comprises the following six CDR
sequences, [0325] a) (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs:
5, FAS, 6 and said second anti-DR5 antibody comprises the following
six CDR sequences, [0326] b) (VH) SEQ ID NOs: 10, 2, 11 and (VL)
SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5
antibody and said second anti-DR5 antibody comprises an E345K
mutation in the Fc region.
[0327] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody and a second anti-DR5 antibody,
wherein said first anti-DR5 antibody comprises the following six
CDR sequences (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS,
6 and said second anti-DR5 antibody comprises the following six CDR
sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS,
14, and wherein the said first anti-DR5 antibody and said second
anti-DR5 antibody comprises an E345K mutation in the Fc region.
[0328] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody and a second anti-DR5 antibody,
wherein the first anti-DR5 antibody comprises the following six CDR
sequences, [0329] a) (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs:
5, FAS, 6 and said second anti-DR5 antibody comprises the following
six CDR sequences, [0330] b) (VH) SEQ ID NOs: 10, 2, 11 and (VL)
SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5
antibody and said second anti-DR5 antibody comprises a mutation in
the position corresponding to E345.
[0331] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody and a second anti-DR5 antibody,
wherein said first anti-DR5 antibody comprises the following six
CDR sequences (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS,
6 and said second anti-DR5 antibody comprises the following six CDR
sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS,
14, and wherein the said first anti-DR5 antibody and said second
anti-DR5 antibody comprises a mutation in the position
corresponding to E345 in human IgG1, EU numbering.
[0332] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody and a second anti-DR5 antibody,
wherein the first anti-DR5 antibody comprises the following six CDR
sequences, [0333] a) (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs:
5, FAS, 6 and said second anti-DR5 antibody comprises the following
six CDR sequences, [0334] b) (VH) SEQ ID NOs: 10, 2, 11 and (VL)
SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5
antibody and said second anti-DR5 antibody comprises an E345K
mutation in the Fc region.
[0335] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody and a second anti-DR5 antibody,
wherein said first anti-DR5 antibody comprises the following six
CDR sequences (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS,
6 and said second anti-DR5 antibody comprises the following six CDR
sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS,
14, and wherein the said first anti-DR5 antibody and said second
anti-DR5 antibody comprises an E345K mutation in the Fc region.
[0336] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody comprising a mutation in the
position corresponding to S440 in human IgG1, EU numbering and a
second anti-DR5 antibody comprising a mutation in the positon
corresponding to S440 in human IgG1, EU numbering, wherein the
first and second antibody binds different epitopes on DR5.
[0337] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody comprising a mutation in the
position corresponding to S440 in human IgG1, EU numbering and a
second anti-DR5 antibody comprising a mutation in the positon
corresponding to S440 in human IgG1, EU numbering, wherein the
first antibody does not block binding of the second antibody to
DR5.
[0338] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody comprising an S440Y and a
second anti-DR5 antibody comprising an S440Y mutation, wherein the
first and second antibody binds different epitopes on DR5.
[0339] In one embodiment of the present invention the composition
comprises a first and a second anti-DR5 antibody comprising a
mutation in a position corresponding to S440 in human IgG1, EU
numbering, such a mutation may be selected from the group
consisting of: S440W and S440Y.
[0340] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody and a second anti-DR5 antibody,
wherein the first anti-DR5 antibody comprises the following six CDR
sequences, [0341] a) (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs:
5, FAS, 6 and said second anti-DR5 antibody comprises the following
six CDR sequences, [0342] b) (VH) SEQ ID NOs: 10, 2, 11 and (VL)
SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5
antibody and said second anti-DR5 antibody comprises a mutation in
the position corresponding to S440.
[0343] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody and a second anti-DR5 antibody,
wherein said first anti-DR5 antibody comprises the following six
CDR sequences (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS,
6 and said second anti-DR5 antibody comprises the following six CDR
sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS,
14, and wherein the said first anti-DR5 antibody and said second
anti-DR5 antibody comprises a mutation in the position
corresponding to S440 in human IgG1, EU numbering.
[0344] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody and second anti-DR5 antibody,
wherein the first anti-DR5 antibody comprises the following six CDR
sequences, [0345] a) (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs:
5, FAS, 6 and said second anti-DR5 antibody comprises the following
six CDR sequences, [0346] b) (VH) SEQ ID NOs: 10, 2, 11 and (VL)
SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5
antibody and said second anti-DR5 antibody comprises an S440Y
mutation in the Fc region.
[0347] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody and a second anti-DR5 antibody,
wherein said first anti-DR5 antibody comprises the following six
CDR sequences (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS,
6 and said second anti-DR5 antibody comprises the following six CDR
sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS,
14, and wherein the said first anti-DR5 antibody and said second
anti-DR5 antibody comprises an S440Y mutation in the Fc region.
[0348] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody and a second anti-DR5 antibody,
wherein the first anti-DR5 antibody comprises the following six CDR
sequences, [0349] a) (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs:
5, FAS, 6 and said second anti-DR5 antibody comprises the following
six CDR sequences, [0350] b) (VH) SEQ ID NOs: 10, 2, 11 and (VL)
SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5
antibody and said second anti-DR5 antibody comprises a mutation in
the position corresponding to S440.
[0351] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody and a second anti-DR5 antibody,
wherein said first anti-DR5 antibody comprises the following six
CDR sequences (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS,
6 and said second anti-DR5 antibody comprises the following six CDR
sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS,
14, and wherein the said first anti-DR5 antibody and said second
anti-DR5 antibody comprises a mutation in the position
corresponding to S440 in human IgG1, EU numbering.
[0352] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody and a second anti-DR5 antibody,
wherein the first anti-DR5 antibody comprises the following six CDR
sequences, [0353] a) (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs:
5, FAS, 6 and said second anti-DR5 antibody comprises the following
six CDR sequences, [0354] b) (VH) SEQ ID NOs: 10, 2, 11 and (VL)
SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5
antibody and said second anti-DR5 antibody comprises an S440Y
mutation in the Fc region.
[0355] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody and a second anti-DR5 antibody,
wherein said first anti-DR5 antibody comprises the following six
CDR sequences (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS,
6 and said second anti-DR5 antibody comprises the following six CDR
sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS,
14, and wherein the said first anti-DR5 antibody and said second
anti-DR5 antibody comprises an S440Y mutation in the Fc region.
[0356] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody and a second anti-DR5 antibody
wherein the first and the second antibody comprises a further
hexamerization-inhibiting mutation corresponding to K439E or S440K
in human IgG1 EU numbering. In one embodiment of the present
invention the composition comprises a first and a second anti-DR5
antibody, wherein the first and second anti-DR5 antibody comprises
a hexamerization enhancing mutation in an amino acid position
corresponding to E430, E345 or S440 in human IgG1, EU numbering and
wherein the first antibody comprises a further mutation in an amino
acid position corresponding to K439 or and wherein the second
antibody comprises a further mutation in an amino acid position
corresponding to S440, with the proviso that the hexamerization
enhancing mutation is not in S440 when the further mutation is in
S440. That is in one embodiment of the present invention the
composition comprises a first and a second anti-DR5 antibody,
wherein the first anti-DR5 antibody comprises a hexamerization
enhancing mutation such as E430G and K439E, and wherein the second
anti-DR5 antibody comprises a hexamerization enhancing mutation
such as E430G and S440K. That is in one embodiment of the present
invention the composition comprises a first and a second anti-DR5
antibody, wherein the first anti-DR5 antibody comprises a
hexamerization enhancing mutation such as E345K and K439E, and
wherein the second anti-DR5 antibody comprises a hexamerization
enhancing mutation such as E345K and 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.
[0357] In one embodiment of the present invention the composition
comprises a first anti-DR5 antibody and a second anti-DR5 antibody
binding different epitopes on human DR5. In one embodiment of the
present invention the composition comprises a first anti-DR5
antibody comprising an antigen binding region that binds to an
epitope on DR5 comprising or requiring one or more amino acid
residues located within amino acid residues 116-138 and one or more
amino acid residues located within amino acid residues 139-166 of
SEQ ID NO 46 and a second anti-DR5 antibody comprising an antigen
binding region that binds to an epitope on DR5 comprising or
requiring one or more amino acid residues located within amino acid
residues 79-138 of SEQ ID NO 46.
[0358] In one embodiment of the present invention the composition
comprises said first anti-DR5 antibody binding to DR5, which does
not block binding of said second anti-DR5 antibody to DR5. That is
in one embodiment of the invention the composition comprises a
first anti-DR5 antibody binding to DR5 and a second anti-DR5
antibody binding to DR5, wherein the first and the second anti-DR5
antibody does not compete for binding to DR5. Thus it is to be
understood in the context of the present invention that a first
anti-DR5 antibody that does not block binding of a second anti-DR5
antibody may be the same as a first anti-DR5 antibody that does not
compete with a second anti-DR5 antibody.
[0359] In one embodiment of the invention, the composition
comprises a first and a second anti-DR5 antibody, wherein said
first antibody comprises a VH region and a VL region comprising six
CDR sequences, wherein the six CDR sequences in total have at least
75%, 80%, 85%, 90%, 95%, 97%, or at least 99% amino acid sequence
identity to the CDR sequences as set forth in the following : a)
(VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS, 6; and said
second antibody comprises a VH region and a VL region comprising
six CDR sequences, wherein the six CDR sequences in total have at
least 75%, 80%, 85%, 90%, 95%, 97%, or at least 99% amino acid
sequence identity to the CDR sequences as set forth in the
following; b) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13,
RTS, 14.
[0360] In one embodiment thereof the sequence identity of the six
CDR sequences in total of said first antibody and said second
antibody is at least 85%, 90%, 95%, 97%, or 99%.
[0361] In one embodiment of the present invention the composition
comprises a first and a second anti-DR5 antibody, wherein said
first antibody comprises the following six CDR sequences, [0362] a)
(VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said
second antibody comprises the following six CDR sequences, [0363]
b) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, or
wherein the said first antibody and said second antibody comprises,
[0364] c) the six CDR sequences defined in (a) or (b) above having
one to five mutations or substitutions in total across said six CDR
sequences respectively. That is the one or more mutations or
substitutions across the six CDR sequences of the antigen binding
region do not change the binding characteristics of said first or
second antibody such as the agonistic properties, the DR5 epitope
binding and/or the ability to induce apoptosis in a target cell
expressing DR5. That is in one embodiment up to five mutations or
substitutions in total are allowed across the six CDRs comprising
the antigen binding region. In some embodiments of the invention up
to five mutations or substitutions such as one, two, three, four or
five mutations or substitutions, are made across the three CDRs of
the VH region and no mutations are made across the CDRs of the VL
region. In other embodiments no mutations or substitutions are made
across the CDRs of the VH region but up to five mutations or
substitutions, such as one, two, three, four or five are found
across the CDRs of the VL region.
[0365] In one embodiment of the present invention the composition
comprises a first and a second anti-DR5 antibody, wherein [0366] a)
said first antibody comprises the following six CDR sequences (VH)
SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second
antibody comprises the following six CDR sequences (VH) SEQ ID NOs:
10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, or wherein b) the said
first antibody and said second antibody comprises the six CDR
sequences of each antibody defined in (a) or comprising one to five
mutations e.g. substitutions in total across said six CDR sequences
respectively. That is the one or more mutations e.g. substitutions
across the six CDR sequences of the antigen binding region do not
change the binding characteristics of said first or second antibody
such as the agonistic properties, the DR5 epitope binding and/or
the ability to induce apoptosis in a target cell expressing DR5.
That is in one embodiment up to five mutations e.g. substitutions
in total are allowed across the six CDRs comprising the antigen
binding region. In some embodiments of the invention up to five
mutations e.g. substitutions such as one, two, three, four or five
mutations or substitutions, are made across the three CDRs of the
VH region and no mutations are made across the CDRs of the VL
region. In other embodiments no mutations e.g. substitutions are
made across the CDRs of the VH region but up to five mutations e.g.
substitutions, such as one, two, three, four or five are found
across the CDRs of the VL region.
[0367] In one embodiment of the invention, the composition
comprises a first and a second anti-DR5 antibody, wherein said
first antibody comprises a VH region and a VL region comprising six
CDR sequences, wherein the six CDR sequences in total have at least
75%, 80%, 85%, 90%, 95%, 97%, or at least 99% amino acid sequence
identity to the CDR sequences as set forth in the following : a)
(VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS; and said
second antibody comprises a VH region and a VL region comprising
six CDR sequences, wherein the six CDR sequences in total have at
least 75%, 80%, 85%, 90%, 95%, 97%, or at least 99% amino acid
sequence identity to the CDR sequences as set forth in the
following; b) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13,
RTS, 14. In one embodiment thereof the sequence identity of the six
CDR sequences in total of said first antibody and said second
antibody is at least 85%, 90%, 95%, 97%, or 99%. [0368] In one
embodiment of the present invention the composition comprises a
first and a second anti-DR5 antibody, wherein said first antibody
comprises the following six CDR sequences, [0369] a) (VH) SEQ ID
NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second
antibody comprises the following six CDR sequences [0370] b) (VH)
SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, or wherein
the said first antibody and said second antibody comprises, [0371]
c) the six CDR sequences defined in (a) or (b) above having one to
five mutations or substitutions in total across said six CDR
sequences respectively. [0372] That is the one or more mutations or
substitutions across the six CDR sequences of the antigen binding
region do not change the binding characteristics of said first or
second antibody such as the agonistic properties, the DR5 epitope
binding and/or the ability to induce apoptosis in a target cell
expressing DR5. That is in one embodiment up to five mutations or
substitutions in total are allowed across the six CDRs comprising
the antigen binding region. In some embodiments of the invention up
to five mutations or substitutions such as one, two, three, four or
five mutations or substitutions, are made across the three CDRs of
the VH region and no mutations are made across the CDRs of the VL
region. In other embodiments no mutations or substitutions are made
across the CDRs of the VH region but up to five mutations or
substitutions, such as one, two, three, four or five are found
across the CDRs of the VL region.
[0373] In one embodiment of the present invention the composition
comprises a first and a second anti-DR5 antibody, wherein [0374] a)
said first antibody comprises the following six CDR sequences (VH)
SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second
antibody comprises the following six CDR sequences (VH) SEQ ID NOs:
10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, or wherein b) the said
first antibody and said second antibody comprises the six CDR
sequences of each antibody defined in (a) or comprising one to five
mutations e.g. substitutions in total across said six CDR sequences
respectively. That is the one or more mutations e.g. substitutions
across the six CDR sequences of the antigen binding region do not
change the binding characteristics of said first or second antibody
such as the agonistic properties, the DR5 epitope binding and/or
the ability to induce apoptosis in a target cell expressing DR5.
That is in one embodiment up to five mutations e.g. substitutions
in total are allowed across the six CDRs comprising the antigen
binding region. In some embodiments of the invention up to five
mutations e.g. substitutions such as one, two, three, four or five
mutations or substitutions, are made across the three CDRs of the
VH region and no mutations are made across the CDRs of the VL
region. In other embodiments no mutations e.g. substitutions are
made across the CDRs of the VH region but up to five mutations e.g.
substitutions, such as one, two, three, four or five are found
across the CDRs of the VL region.
[0375] In one embodiment of the invention, the composition
comprises a first and a second anti-DR5 antibody, wherein said
first antibody comprises a VH region and a VL region comprising six
CDR sequences, wherein the six CDR sequences in total have at least
75%, 80%, 85%, 90%, 95%, 97%, or at least 99% amino acid sequence
identity to the CDR sequences as set forth in the following : a)
(VH) SEQ ID NOs: 16, 17, 18 and (VL) SEQ ID NOs: 21, GAS, 6; and
said second antibody comprises a VH region and a VL region
comprising six CDR sequences, wherein the six CDR sequences in
total have at least 75%, 80%, 85%, 90%, 95%, 97%, or at least 99%
amino acid sequence identity to the CDR sequences as set forth in
the following; b) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs:
13, RTS, 14.
[0376] In one embodiment thereof the sequence identity of the six
CDR sequences in total of said first antibody and said second
antibody is at least 85%, 90%, 95%, 97%, or 99%. [0377] In one
embodiment of the present invention the composition comprises a
first and a second anti-DR5 antibody, wherein said first antibody
comprises the following six CDR sequences, [0378] a) (VH) SEQ ID
NOs: 16, 17, 18 and (VL) SEQ ID NOs: 21, GAS, 6 and said second
antibody comprises the following six CDR sequences [0379] b) (VH)
SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, or wherein
the said first antibody and said second antibody comprises, [0380]
c) the six CDR sequences defined in (a) or (b) above having one to
five mutations or substitutions in total across said six CDR
sequences. That is the one or more mutations or substitutions
across the six CDR sequences of the antigen binding region do not
change the binding characteristics of said first or second antibody
such as the agonistic properties, the DR5 epitope binding and/or
the ability to induce apoptosis in a target cell expressing DR5.
That is in one embodiment up to five mutations or substitutions in
total are allowed across the six CDRs comprising the antigen
binding region. In some embodiments of the invention up to five
mutations or substitutions such as one, two, three, four or five
mutations or substitutions, are made across the three CDRs of the
VH region and no mutations are made across the CDRs of the VL
region. In other embodiments no mutations or substitutions are made
across the CDRs of the VH region but up to five mutations or
substitutions, such as one, two, three, four or five are found
across the CDRs of the VL region.
[0381] In one embodiment of the present invention the composition
comprises a first and a second anti-DR5 antibody, wherein [0382] a)
said first antibody comprises the following six CDR sequences (VH)
SEQ ID NOs: 16, 17, 18 and (VL) SEQ ID NOs: 21, GAS, 6 and said
second antibody comprises the following six CDR sequences (VH) SEQ
ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, or wherein b)
the said first antibody and said second antibody comprises the six
CDR sequences of each antibody defined in (a) or comprising one to
five mutations e.g. substitutions in total across said six CDR
sequences respectively. That is the one or more mutations e.g.
substitutions across the six CDR sequences of the antigen binding
region do not change the binding characteristics of said first or
second antibody such as the agonistic properties, the DR5 epitope
binding and/or the ability to induce apoptosis in a target cell
expressing DR5. That is in one embodiment up to five mutations e.g.
substitutions in total are allowed across the six CDRs comprising
the antigen binding region. In some embodiments of the invention up
to five mutations e.g. substitutions such as one, two, three, four
or five mutations or substitutions, are made across the three CDRs
of the VH region and no mutations are made across the CDRs of the
VL region. In other embodiments no mutations e.g. substitutions are
made across the CDRs of the VH region but up to five mutations e.g.
substitutions, such as one, two, three, four or five are found
across the CDRs of the VL region.
[0383] In one embodiment of the present invention the composition
comprises a first and a second anti-DR5 antibody as defined in any
of the above embodiments wherein said first and second antibody
further comprises a mutation in the Fc region corresponding to
position K439 or S440 in human IgG1, EU numbering. In one
embodiment of the invention the composition comprises a first
antibody comprising a mutation corresponding to K439 such as K439E
and a second antibody comprising a mutation corresponding to S440
such as S440K. In one embodiment of the invention the composition
comprises a first antibody comprising a mutation corresponding to
S440 such as S440K and a second antibody comprising a mutation
corresponding to K439 such as K439E. Hereby embodiment are provided
wherein the composition comprises a first antibody comprising at
least two mutations such as E430G and K439E and a second antibody
comprising at least two mutations such as E430G and S440K. In
another embodiment of the present invention the composition
comprises a first antibody comprising at least two mutations such
as E345K and K439E and a second antibody comprising at least two
mutation such as E345K and S440K. Hereby are embodiments provided
that allow for hexamerization of antibodies with different
specificities.
[0384] In one embodiment of the present invention the composition
comprises a first and a second anti-DR5 antibody, wherein said
first antibody comprises the following sequences (a) (VH) CDR1 SEQ
ID NO 1, CDR2 SEQ ID NO 8, CDR3 SEQ ID NO 3 and (VL) CDR1 SEQ ID NO
5, CDR2 FAS, CDR3 SEQ ID NO 6 and said second antibody comprises
the following sequences (b) (VH) CDR1 SEQ ID NO 10, CDR2 SEQ ID NO
2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2 RTS, CDR3 SEQ
ID NO 14 or (c) the (VH) CDR1, CDR2 and CDR3 and (VL) CDR1, CDR2
and CDR3 as defined in (a) or (b) above having one to five
mutations or substitutions in total across said six CDR sequences.
That is the one or more mutations or substitutions across the six
CDR sequences of the antigen binding region do not change the
binding characteristics of said first or second antibody such as
the agonistic properties, the DR5 epitope binding and/or the
ability to induce apoptosis in a target cell expressing DR5.
[0385] In one embodiment of the present invention the composition
comprises a first and a second anti-DR5 antibody, wherein said
first and second antibody comprises the following CDR sequences (a)
said first antibody comprises the following CDR sequences (VH) CDR1
SEQ ID NO 1, CDR2 SEQ ID NO 8, CDR3 SEQ ID NO 3 and (VL) CDR1 SEQ
ID NO 5, CDR2 FAS, CDR3 SEQ ID NO 6 and said second antibody
comprises the following CDR sequences (VH) CDR1 SEQ ID NO 10, CDR2
SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2
RTS, CDR3 SEQ ID NO 14 or (b) the CDR sequences described in (a)
for each antibody comprising one to five mutations e.g.
substitutions in total across said CDR sequences for each antibody.
That is the one or more mutations e.g. substitutions across the six
CDR sequences of the antigen binding region do not change the
binding characteristics of said first or second antibody such as
the agonistic properties, the DR5 epitope binding and/or the
ability to induce apoptosis in a target cell expressing DR5.
[0386] In one embodiment of the present invention the composition
comprises a first and a second anti-DR5 antibody, wherein said
first antibody comprises the following sequences (a) (VH) CDR1 SEQ
ID NOs 1, CDR2 2, CDR3 3 and (VL) CDR1 SEQ ID NOs 5, CDR2 FAS, CDR3
6 and said second antibody comprises the following sequences (b)
(VH) CDR1 SEQ ID NOs 10, CDR2 2, CDR3 11 and (VL) SEQ ID NOs CDR1
13, CDR2 RTS, CDR3 14 or (c) the (VH) CDR1, CDR2 and CDR3 and (VL)
CDR1, CDR2 and CDR3 as defined in (a) or (b) above having one to
five mutations or substitutions in total across said six CDR
sequences. That is the one or more mutations or substitutions
across the six CDR sequences of the antigen binding region do not
change the binding characteristics of said first or second antibody
such as the agonistic properties, the DR5 epitope binding and/or
the ability to induce apoptosis in a target cell expressing
DR5.
[0387] In one embodiment of the present invention the composition
comprises a first and a second anti-DR5 antibody, wherein said
first and second antibody comprises the following CDR sequences (a)
said first antibody comprises the following CDR sequences (VH) CDR1
SEQ ID NO 1, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 3 and (VL) CDR1 SEQ
ID NO 5, CDR2 FAS, CDR3 SEQ ID NO 6 and said second antibody
comprises the following CDR sequences (VH) CDR1 SEQ ID NO 10, CDR2
SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2
RTS, CDR3 SEQ ID NO 14 or (b) the CDR sequences described in (a)
for each antibody comprising one to five mutations e.g.
substitutions in total across said CDR sequences for each antibody.
That is the one or more mutations e.g. substitutions across the six
CDR sequences of the antigen binding region do not change the
binding characteristics of said first or second antibody such as
the agonistic properties, the DR5 epitope binding and/or the
ability to induce apoptosis in a target cell expressing DR5.
[0388] In one embodiment of the present invention the composition
comprises a first and a second anti-DR5 antibody, wherein said
first antibody comprises the following sequences (a) (VH) CDR1 SEQ
ID NO 16, CDR2 SEQ ID NO 17, CDR3 SEQ ID NO 18 and (VL) CDR1 SEQ ID
NO 21, CDR2 GAS, CDR3 SEQ ID NO 22 and said second antibody
comprises the following sequences (b) (VH) CDR1 SEQ ID NO 10, CDR2
SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2
RTS, CDR3 SEQ ID NO 14 or (c) the (VH) CDR1, CDR2 and CDR3 and (VL)
CDR1, CDR2 and CDR3 as defined in (a) or (b) above having one to
five mutations or substitutions in total across said six CDR
sequences. That is the one or more mutations or substitutions
across the six CDR sequences of the antigen binding region do not
change the binding characteristics of said first or second antibody
such as the agonistic properties, the DR5 epitope binding and/or
the ability to induce apoptosis in a target cell expressing
DR5.
[0389] In one embodiment of the present invention the composition
comprises a first and a second anti-DR5 antibody, wherein said
first and second antibody comprises the following CDR sequences (a)
said first antibody comprises the following CDR sequences (VH) CDR1
SEQ ID NO 16, CDR2 SEQ ID NO 17, CDR3 SEQ ID NO 18 and (VL) CDR1
SEQ ID NO 21, CDR2 GAS, CDR3 SEQ ID NO 22 and said second antibody
comprises the following CDR sequences (VH) CDR1 SEQ ID NO 10, CDR2
SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2
RTS, CDR3 SEQ ID NO 14 or (b) the CDR sequences described in (a)
for each antibody comprising one to five mutations e.g.
substitutions in total across said CDR sequences for each antibody.
That is the one or more mutations e.g. substitutions across the six
CDR sequences of the antigen binding region do not change the
binding characteristics of said first or second antibody such as
the agonistic properties, the DR5 epitope binding and/or the
ability to induce apoptosis in a target cell expressing DR5.
[0390] In one embodiment of the invention the composition comprises
a first and a second anti-DR5 antibody, 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
1:10 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.
[0391] In one embodiment of the invention the composition comprises
a first and a second anti-DR5 antibody, wherein said first antibody
and said second antibody are present in the composition at a 1:9 to
9:1 molar ratio.
[0392] In one embodiment of the invention the composition comprises
a first and a second anti-DR5 antibody, wherein said first antibody
and said second antibody are present in the composition at
approximately a 1:1 molar ratio.
[0393] In one embodiment of the invention the composition comprises
a first and a second anti-DR5 antibody, wherein said first antibody
and said second antibody are present in the composition at a 1:1
molar ratio.
[0394] In a preferred embodiment of the invention the composition
comprises a first and a second anti-DR5 antibody, wherein said
first antibody and second antibody and/or any additional antibodies
are present in the composition at an equimolar ratio.
[0395] In one embodiment of the invention the composition is a
pharmaceutical composition.
[0396] 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.
[0397] 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)
[0398] 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).
[0399] 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.
[0400] 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.
[0401] 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. In one embodiment, the pharmaceutical composition of the
present invention is administered parenterally.
[0402] 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.
[0403] In one embodiment, the pharmaceutical composition of the
present invention is administered by intravenous or subcutaneous
injection or infusion.
[0404] 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.
[0405] 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.
[0406] 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.
[0407] 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.
[0408] 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.
[0409] 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.
[0410] 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.
[0411] 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.
[0412] 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.
[0413] 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.
[0414] 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.
[0415] 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
[0416] 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.
[0417] 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.
[0418] In another aspect, the present invention provides methods
for treating or preventing a disorder involving cells expressing
DR5 in a subject, which method comprises administration of a
therapeutically effective amount of an anti-DR5 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-DR5 antibody, a bispecific antibody or composition
according to the present invention in an amount effective to treat
or prevent the disorder.
[0419] The anti-DR5 antibodies of the present invention can be used
in the treatment or prevention of disorders involving cells
expressing DR5. For example, the antibodies may be administered to
human subjects, e.g., in vivo, to treat or prevent disorders
involving DR5-expressing cells. As used herein, the term "subject"
is typically a human to whom the anti-DR5 antibody or bispecific
antibody is administered. Subjects may for instance include human
patients having disorders that may be corrected or ameliorated by
modulating DR5 function or by killing of the DR5-expressing cell,
directly or indirectly.
[0420] In one aspect, the present invention relates to an anti-DR5
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
DR5.
[0421] In one embodiment of the present invention the composition
comprising an anti-DR5 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.
[0422] In one aspect, the present invention relates to an anti-DR5
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.
[0423] In one embodiment of the present invention the composition
comprising an anti-DR5 antibody or bispecific antibody according to
any aspect or embodiment of the invention is for use in treatment
of cancer and/or tumors.
[0424] 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.
[0425] In a particular aspect, an anti-DR5 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-DR5 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.
[0426] In one embodiment the composition comprising one or more
anti-DR5 antibodies or bispecific antibodies of the present
invention is for use in treatment of solid tumors and/or
hematological tumors
[0427] In one embodiment the composition comprising one or more
anti-DR5 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
non-small cell lung cancer (NSCLC) and small cell lung cancer
(SCLC), ovarian cancer, pancreatic cancer, including pancreatic
ductal carcinoma and pancreatic adenocarcinoma, sarcoma or skin
cancer, including malignant melanoma and non-melanoma skin
cancers.
[0428] In one embodiment of the invention the composition
comprising one or more anti-DR5 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.
[0429] In a particular embodiment of the present invention the
composition comprising one or more anti-DR5 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.
[0430] In one embodiment of the invention the composition
comprising one or more anti-DR5 antibodies or bispecific antibodies
is for use in inhibiting growth of DR5 positive or DR5 expressing
tumors or cancers.
[0431] In the present invention DR5 positive tumors or cancers are
to be understood as tumor cells and/or cancer cells expressing DR5
on the cell surface. Such DR5 expression may be detected by
immunohistochemistry, flow cytometry, imaging or other suitable
diagnostic method.
[0432] In one embodiment of the invention the composition
comprising one or more anti-DR5 antibodies or bispecific antibodies
is for use in inhibiting growth of DR5 expressing tumors or
cancers. Tumors and cancer tissues that show heterogeneous
expression of DR5 are also considered as DR5 positive tumors and
cancers.
[0433] Tumors and/or cancers may express DR5 on some tumor and/or
cancer cells and/or tissues showing DR5 expression, some tumor
and/or cancers may show over-expression or aberrant expression of
DR5, whereas other tumors and/or cancers show heterogeneous
expression of DR5. Such tumors and/or cancers may all be suitable
targets for treatment with anti-DR5 antibodies, bispecific
antibodies and compositions comprising such antibodies according to
the present invention.
[0434] In one embodiment of the invention the composition
comprising one or more anti-DR5 antibodies or bispecific antibodies
is for use in induction of apoptosis in DR5 expressing tumors.
[0435] 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-DR5 antibody,
bispecific antibody or composition according to the invention.
[0436] 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-DR5 antibody, bispecific
antibody or composition according to the invention, further
comprises administering an additional therapeutic agent to the said
individual.
[0437] 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.
[0438] In a further aspect, the invention comprises a kit of parts
comprising an anti-DR5 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.
[0439] In one embodiment of the invention the kit of parts
comprising an anti-DR5 antibody, bispecific antibody or composition
according to the invention is for simultaneous, separate or
sequential use in therapy.
[0440] In a further embodiment the present invention is for use of
an anti-DR5 antibody, bispecific antibody or a composition
according to the invention for the manufacture of a medicament for
treatment of cancer.
[0441] 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.
[0442] 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 SEQ ID Nos: 1 to 23 or 29 to 43. In one
embodiment of the present invention, the nucleic acid construct
encodes an antibody according to any embodiments disclosed
herein.
[0443] 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: 33, 34, 35,
36, 37, 38, 39, 40, 41, 42 or 43.
[0444] In another aspect, the invention relates to nucleic acids
encoding a sequence of a human, humanized or chimeric anti-DR5
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. Humanized anti-DR5
antibodies may also be denoted as "huDR5".
[0445] 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: 33 to 43
[0446] In one embodiment, the invention provides an expression
vector comprising a nucleotide sequence encoding one or more amino
acid sequences selected from the group consisting of SEQ ID NOs:
33, 34, 35,36, 37, 38, 39, 40, 41, 42 and 43, or any combination
thereof. 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: 3 and 11. In another
embodiment, the expression vector comprises a nucleotide sequence
encoding a VH amino acid sequence selected from SEQ ID NOs: 4, 9
and 12. In another embodiment, the expression vector comprises a
nucleotide sequence encoding a VL amino acid sequence selected from
SEQ ID NOs: 7, 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:58, 59, 60, 61, 62, 63, 64, 65, 66, 67 and 68.
[0447] 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.
[0448] 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 1, 793-800 (2001)), or as a
precipitated nucleic acid vector construct, such as a
CaPO.sub.4-precipitated construct (as described in for instance WO
00/46147, Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986),
Wigler et al., Cell IA, 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.
No. 5,589,466 and U.S. Pat. No. 5,973,972).
[0449] 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.
[0450] 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)).
[0451] 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.
[0452] 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, SL3-3, 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).
[0453] 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.
[0454] Such expression vectors may be used for recombinant
production of anti-DR5 antibodies. In one aspect, the anti-DR5
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.
[0455] 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 a anti-DR5 antibody, a first or a second polypeptide
described herein.
[0456] 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, NSO cells, and lymphocytic cells,
and prokaryotic cells such as E. coli and other eukaryotic hosts
such as plant cells and fungi.
[0457] The term "transfectoma", as used herein, includes
recombinant eukaryotic host cells expressing the antibody or a
target antigen, such as CHO cells, PER.C6, NSO cells, HEK-293
cells, plant cells, or fungi, including yeast cells.
[0458] In a further aspect, the invention relates to a method for
producing an antibody of the invention, said method comprising the
steps of [0459] a) culturing a hybridoma or a host cell of the
invention as described herein above, and [0460] b) retrieving
and/or purifying the antibody of the invention from the culture
media.
[0461] 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 [0462] a) culturing a host cell
comprising an expression vector comprising such a nucleotide
sequence, and [0463] b) retrieving and/or purifying the antibody
fusion protein from the culture media.
[0464] 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.
[0465] In a further aspect of the invention, the invention
comprises a host cell comprising an expression vector.
[0466] In one embodiment of the invention the host cell is a
recombinant host cell, such as a recombinant prokaryotic cell,
recombinant eukaryotic cell or recombinant microbial host cell.
TABLE-US-00004 Sequence table 1 SEQ ID NO: Name Sequence Clone SEQ
ID NO: 1 VHhDR5-01 CDR1 GFNIKDTF hDR5-01 SEQ ID NO: 2 VHhDR5-01
CDR2 IDPANGNT SEQ ID NO: 3 VHhDR5-01 CDR3 VRGLYTYYFDY SEQ ID NO: 4
VHhDR5-01 EVQLQQSGAEVVKPGA SVKLSCKASGFNIKDTFI HWVKQAPGQGLEWIG
RIDPANGNTKYDPKFQ GKATITTDTSSNTAYME LSSLRSEDTAVYYCVRGL
YTYYFDYWGQGTLVTV SS SEQ ID NO: 5 VL hDR5-01 CDR1 QSISNN VL hDR5-01
CDR2 FAS SEQ ID NO: 6 VL hDR5-01 CDR3 QQGNSWPYT SEQ ID NO: 7 VL
hDR5-01 EIVMTQSPATLSVSPGE RATLSCRASQSISNNLH WYQQKPGQAPRLLIKF
ASQSITGIPARFSGSGSG TEFTLTISSLQSEDFAVY YCQQGNSWPYTFGQG TKLEIK SEQ ID
NO: 1 VHhDR5-01-G56T GFNIKDTF hDR5-01-G56T CDR1 SEQ ID NO: 8
VHhDR5-01-G56T IDPANTNT CDR2 SEQ ID NO: 3 VHhDR5-01-G56T
VRGLYTYYFDY CDR3 SEQ ID NO: 9 VH hDR5-01-G56T EVQLQQSGAEVVKPGA
SVKLSCKASGFNIKDTFI HWVKQAPGQGLEWIG RIDPANTNTKYDPKFQG
KATITTDTSSNTAYMEL SSLRSEDTAVYYCVRGL YTYYFDYWGQGTLVTV SS SEQ ID NO:
5 VL hDR5-01-G56T QSISNN CDR1 VL hDR5-01-G56T FAS CDR2 SEQ ID NO: 6
VL hDR5-01-G56T QQGNSWPYT CDR3 SEQ ID NO: 7 VL hDR5-01-G56T
EIVMTQSPATLSVSPGE RATLSCRASQSISNNLH WYQQKPGQAPRLLIKF
ASQSITGIPARFSGSGSG TEFTLTISSLQSEDFAVY YCQQGNSWPYTFGQG TKLEIK SEQ ID
NO: 10 VH hDR5-05 CDR1 GFNIKDTH hDR5-05 SEQ ID NO: 2 VH hDR5-05
CDR2 IDPANGNT SEQ ID NO: 11 VH hDR5-05 CDR3 ARWGTNVYFAY SEQ ID NO:
12 VH hDR5-05 QVQLVQSGAEVKKPGA SVKVSCKASGFNIKDTH MHWVRQAPGQRLEWI
GRIDPANGNTEYDQKF QGRVTITVDTSASTAYM ELSSL RSEDTAVYYCAR
WGTNVYFAYWGQGTL VTVSS SEQ ID NO: 13 VL hDR5-05 CDR1 SSVSY VL
hDR5-05 CDR2 RTS SEQ ID NO: 14 VL hDR5-05 CDR3 QQYHSYPPT SEQ ID NO:
15 VL hDR5-05 DIQLTQSPSSLSASVGD RVTITCSASSSVSYMYW YQQKPGKAPKPWIYRT
SNLASGVPSRFSGSGSG TDFTLTISSLQPEDFATY YCQQYHSYPPTFGGGT KVEIK SEQ ID
NO: 16 VH CONA-CDR1 GGSISSGDYF Conatumumab IgG1-DR5-CONA SEQ ID NO:
17 VH CONA-CDR2 IHNSGTT SEQ ID NO: 18 VH CONA-CDR3 ARDRGGDYYYGMDV
SEQ ID NO: 19 VH CONA QVQLQESGPGLVKPSQ TLSLICTVSGGSISSGDY
FWSWIRQLPGKGLECIG HIHNSGTTYYNPSLKSR VTISVDTSKKQFSLRLSS
VTAADTAVYYCARDRG GDYYYGMDVWGQGTT VTVSS SEQ ID NO: 20 VH CONA-C49W
QVQLQESGPGLVKPSQ TLSLICTVSGGSISSGDY FWSWIRQLPGKGLEWI
GHIHNSGTTYYNPSLKS RVTISVDTSKKQFSLRLS SVTAADTAVYYCARDR
GGDYYYGMDVWGQG TTNITVSS SEQ ID NO: 21 VL CONA-CDR1 QGISRSY VL
CONA-CDR2 GAS SEQ ID NO: 22 VL CONA-CDR3 QQFGSSPWT SEQ ID NO: 23 VL
CONA EIVLTQSPGTLSLSPGER ATLSCRASQGISRSYLA WYQQKPGQAPSLLIYG
ASSRATGIPDRFSGSGS GTDFTLTISRLEPEDFAV YYCQQFGSSPWTFGQG TKVEIK SEQ ID
NO: 24 Human DR5 MEQRGQNAPAASGA RKRHGPGPREARGA RPGPRVPKTLVLVVA
AVLLLVSAESALITQ QDLAPQQRAAPQQK RSSPSEGLCPPGHHI SEDGRDCISCKYGQ
DYSTHWNDLLFCLR CTRCDSGEVELSPCT TTRNTVCQCEEGTFR EEDSPEMCRKCRTG
CPRGMVKVGDCTPW SDIECVHKESGTKH SGEVPAVEETVTSSP GTPASPCSLSGIIIGV
TVAAVVLIVAVFVCK SLLWKKVLPYLKGIC SGGGGDPERVDRSS QRPGAEDNVLNEIVS
ILQPTQVPEQEMEVQ EPAEPTGVNMLSPGE SEHLLEPAEAERSQR RRLLVPANEGDPTET
LRQCFDDFADLVPFD SWEPLMRKLGLMDN EIKVAKAEAAGHRDT LYTMLIKWVNKTGR
DASVHTLLDALETLG ERLAKKIEDHLLSSG KFMYLEGNADSAMS SEQ ID NO: 25 Rhesus
monkey DR5 MGQLRQSAPAASGA RKGRGPGPREARGA RPGLRVLKTLVLVVA
AARVLVSADCAPITR QSLDPQRRAAPQQK RSSPTEGLCPPGHHI SEDSRDCISCKYGQ
DYSTHWNDFLFCLR CTKCDSGEVEVNSC TTTRNTVCQCEEGTF REEDSPEICRKCRTG
CPRGMVKVKDCTPW SDIECVHKESGTKHT GEVPAVEKTVTTSPG TPASPCSLSGIIIGVI
VFVVIVVVAVIVWKT SLWKKVLPYLKGVC SGDGGDPERVDSSP QRPGAEDNALNEIVS
IVQPSQVPEQEMEV QEPAEQTDVNTLSP GESEHLLEPAKAEGP QRRGQLVPVNENDP
TETLRQCFDDFAAIV PFDAWEPLVRQLGLT NNEIKVAKAEAASSR DTLYVMLIKWVNKT
GRAASVNTLLDALET LEERLAKQKIQDRLL SSGKFMYLEDNADS ATS SEQ ID NO: 26
Murine DR5 MEPPGPSTPTASAAA RADHYTPGLRPLPKR RLLYSFALLLAVLQAV
FVPVTANPAHNRPAG LQRPEESPSRGPCLA GQYLSEGNCKPCRE GIDYTSHSNHSLDS
CILCTVCKEDKVVET RCNITTNTVCRCKPG TFEDKDSPEICQSCS NCTDGEEELTSCTPR
ENRKCVSKTAWAS WHKLGLWIGLLVPV VLLIGALLVWKTGA WRQWLLCIKRGCER
DPESANSVHSSLLD RQTSSTTNDSNHNT EPGKTQKTGKKLLVP VNGNDSADDLKFIFE
YCSDIVPFDSWNRL MRQLGLTDNQIQMV KAETLVTREALYQML LKWRHQTGRSASIN
HLLDALEAVEERDAM EKIEDYAVKSGRFTY QNAAAQPETGPGGS QCV SEQ ID NO: 27
DR5ECD-FcHistag MEQRGQNAPAASGAR KRHGPGPREARGARPG LRVPKTLVLVVAAVLLLV
SAESALITQQDLAPQQR VAPQQKRSSPSEGLCPP GHHISEDGRDCISCKYG
QDYSTHWNDLLFCLRC TRCDSGEVELSPCTTTR NTVCQCEEGTFREEDSP
EMCRKCRTGCPRGMV KVGDCTPWSDIECVHK ESGTKHSGEAPAVEETV
TSSPGTPASPCSPKSCD KTHTCPPCPAPEAEGAP SVFLFPPKPKDTLMISRT
PEVTCVVVDVSHEDPE VKFNWYVDGVEVHNA KTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYK
CKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGF
YPSDIAVEWESNGQPE
NNYKTAPPVLDSDGSFF LYSKLTVDKSRWQQGN VFSCSVMHEALHNHYT
QKSLSLSPGKHHHHHH HHEPEA SEQ ID NO: 28 DR5ECDdelHis MEQRGQNAPAASGA
RKRHGPGPREARGA RPGPRVPKTLVLVVA AVLLLVSAESALITQ QDLAPQQRAAPQQK
RSSPSEGLCPPGHHI SEDGRDCISCKYGQ DYSTHWNDLLFCLR CTRCDSGEVELSPCT
TTRNTVCQCEEGTFR EEDSPEMCRKCRTG CPRGMVKVGDCTPW SDIECVHKESGHHH HHHHH
SEQ ID NO: 29 Fc IgG1m(f) STKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEP
VTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNH
KPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRT
PEVTCVVVDVSHEDPE VKFNWYVDGVEVHNA KTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYK
CKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGF
YPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYT QKSLSLSPGK SEQ ID NO: 30 Fc IgG1m(z)
STKGPSVFPLAPSSK STSGGTAALGCLVK DYFPEPVTVSWNSG ALTSGVHTFPAVLQS
SGLYSLSSVVTVPSS SLGTQTYICNVNHKP SNTKVDKKVEPKSC DKTHTCPPCPAPELL
GGPSVFLFPPKPKDT LMISRTPEVTCVVVD VSHEDPEVKFNWYV DGVEVHNAKTKPRE
EQYNSTYRVVSVLTV LHQDWLNGKEYKCK VSNKALPAPIEKTISK AKGQPREPQVYTLPP
SREEMTKNQVSLTCL VKGFYPSDIAVEWES NGQPENNYKTTPPVL DSDGSFFLYSKLTVD
KSRWQQGNVFSCS VMHEALHNHYTQKS LSLSPGK SEQ ID NO: 31 Fc IgG1m(a)
STKGPSVFPLAPSSK STSGGTAALGCLVK DYFPEPVTVSWNSG ALTSGVHTFPAVLQS
SGLYSLSSVVTVPSS SLGTQTYICNVNHKP SNTKVDKPVEPKSC DKTHTCPPCPAPELL
GGPSVFLFPPKPKDT LMISRTPEVTCVVVD VSHEDPEVKFNWYV DGVEVHNAKTKPRE
EQYNSTYRVVSVLTV LHQDWLNGKEYKCK VSNKALPAPIEKTISK AKGQPREPQVYTLPP
SRDELTKNQVSLTCL VKGFYPSDIAVEWES NGQPENNYKTTPPVL DSDGSFFLYSKLTVD
KSRWQQGNVFSCS VMHEALHNHYTQKS LSLSPGK SEQ ID NO: 32 Fc IgG1m(x)
STKGPSVFPLAPSSK STSGGTAALGCLVK DYFPEPVTVSWNSG ALTSGVHTFPAVLQS
SGLYSLSSVVTVPSS SLGTQTYICNVNHKP SNTKVDKPVEPKSC DKTHTCPPCPAPELL
GGPSVFLFPPKPKDT LMISRTPEVTCVVVD VSHEDPEVKFNWYV DGVEVHNAKTKPRE
EQYNSTYRVVSVLTV LHQDWLNGKEYKCK VSNKALPAPIEKTISK AKGQPREPQVYTLPP
SREEMTKNQVSLTCL VKGFYPSDIAVEWES NGQPENNYKTTPPVL DSDGSFFLYSKLTVD
KSRWQQGNVFSCS VMHEGLHNHYTQKS LSLSPGK SEQ ID NO: 33 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: 34
HC-hDR5-01-E345K EVQLQQSGAEVVKPGA SVKLSCKASGFNIKDTFI
HWVKQAPGQGLEWIG RIDPANGNTKYDPKFQ GKATITTDTSSNTAYME
LSSLRSEDTAVYYCVRGL YTYYFDYWGQGTLVTV SSASTKGPSVFPLAPSSK
STSGGTAALGCLVKDYF PEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSS
VVTVPSSSLGTQTYICN VNHKPSNTKVDKRVEP KSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKT
ISKAKGQPRKPQVYTLP PSREEMTKNQVSLTCLV KGFYPSDIAVEWESNG
QPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHN HYTQKSLSLSPGK
SEQ ID NO: 35 HC-hDR5-01-E430G 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 QGNVFSCSVMHGALH NHYTQKSLSLSPGK
SEQ ID NO: 36 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: 37 HC-hDR5-01-G56T- EVQLQQSGAEVVKPGA E345K
SVKLSCKASGFNIKDTFI HWVKQAPGQGLEWIG RIDPANTNTKYDPKFQG
KATITTDTSSNTAYMEL SSLRSEDTAVYYCVRGL YTYYFDYWGQGTLVTV
SSASTKGPSVFPLAPSSK STSGGTAALGCLVKDYF PEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICN VNHKPSNTKVDKRVEP
KSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKT ISKAKGQPRKPQVYTLP PSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNG QPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHN HYTQKSLSLSPGK SEQ ID NO: 38 HC-hDR5-01-G56T-
EVQLQQSGAEVVKPGA E430G SVKLSCKASGFNIKDTFI HWVKQAPGQGLEWIG
RIDPANTNTKYDPKFQG KATITTDTSSNTAYMEL SSLRSEDTAVYYCVRGL
YTYYFDYWGQGTLVTV SSASTKGPSVFPLAPSSK STSGGTAALGCLVKDYF
PEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICN
VNHKPSNTKVDKRVEP KSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLV KGFYPSDIAVEWESNG QPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQ QGNVFSCSVMHGALH NHYTQKSLSLSPGK SEQ ID NO: 39
LC-hDR5-01 EIVMTQSPATLSVSPGE RATLSCRASQSISNNLH WYQQKPGQAPRLLIKF
ASQSITGIPARFSGSGSG TEFTLTISSLQSEDFAVY YCQQGNSWPYTFGQG
TKLEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLN NFYPREAKVQWKVDN
ALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSS PVTKSFNRGEC
SEQ ID NO: 40 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: 41 HC-hDR5-05-E345K QVQLVQSGAEVKKPGA SVKVSCKASGFNIKDTH
MHWVRQAPGQRLEWI GRIDPANGNTEYDQKF QGRVTITVDTSASTAYM
ELSSLRSEDTAVYYCAR WGTNVYFAYWGQGTL VTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPA
PELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGV
EVHNAKTKPREEQYNST YRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPI
EKTISKAKGQPRKPQVY TLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK
SEQ ID NO: 42 HC-hDR5-05-E430G 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 WQQGNVFSCSVMHG ALHNHYTQKSLSLSPGK
SEQ ID NO: 43 LC-hDR5-05 DIQLTQSPSSLSASVGD RVTITCSASSSVSYMYW
YQQKPGKAPKPWIYRT SNLASGVPSRFSGSGSG TDFTLTISSLQPEDFATY
YCQQYHSYPPTFGGGT KVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLN
NFYPREAKVQWKVDN ALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADY
EKHKVYACEVTHQGLSS PVTKSFNRGEC SEQ ID NO: 44 Human DR5, K415N
MEQRGQNAPAASGAR Human DR5, KRHGPGPREARGARPG with PRVPKTLVLVVAAVLLL
K415N mutation VSAESALITQQDLAPQQ RAAPQQKRSSPSEGLCP
PGHHISEDGRDCISCKY GQDYSTHWNDLLFCLR CTRCDSGEVELSPCTTT
RNTVCQCEEGTFREEDS PEMCRKCRTGCPRGM VKVGDCTPWSDIECVH
KESGTKHSGEVPAVEET VTSSPGTPASPCSLSGIII GVTVAAVVLIVAVFVCK
SLLWKKVLPYLKGICSG GGGDPERVDRSSQRPG AEDNVLNEIVSILQPTQ
VPEQEMEVQEPAEPTG VNMLSPGESEHLLEPAE AERSQRRRLLVPANEG
DPTETLRQCFDDFADLV PFDSWEPLMRKLGLMD NEIKVAKAEAAGHRDTL
YTMLIKWVNKTGRDAS VHTLLDALETLGERLAN QKIEDHLLSSGKFMYLE GNADSAMS SEQ
ID NO: 45 Human DR5 (natural MEQRGQNAPAASGA variant) RKRHGPGPREARGA
(Accession: RPGLRVPKTLVLVVA AAB70578) AVLLLVSAESALITQ
QDLAPQQRVAPQQK RSSPSEGLCPPGHHI SEDGRDCISCKYGQ DYSTHWNDLLFCLR
CTRCDSGEVELSPCT TTRNTVCQCEEGTFR EEDSPEMCRKCRTG CPRGMVKVGDCTPW
SDIECVHKESGTKH SGEAPAVEETVTSSP GTPASPCSLSGIIIGV TVAAVVLIVAVFVCK
SLLWKKVLPYLKGIC SGGGGDPERVDRSS QRPGAEDNVLNEIVS ILQPTQVPEQEMEVQ
EPAEPTGVNMLSPGE SEHLLEPAEAERSQR RRLLVPANEGDPTET LRQCFDDFADLVPFD
SWEPLMRKLGLMDN EIKVAKAEAAGHRDT LYTMLIKWVNKTGR DASVHTLLDALETLG
ERLAKQKIEDHLLSS GKFMYLEGNADSAM S SEQ ID NO: 46 Human DR5
MEQRGQNAPAASGA (Uniprot O14763) RKRHGPGPREARGA RPGPRVPKTLVLVVA
AVLLLVSAESALITQ QDLAPQQRAAPQQK RSSPSEGLCPPGHHI SEDGRDCISCKYGQ
DYSTHWNDLLFCLR CTRCDSGEVELSPCT TTRNTVCQCEEGTFR EEDSPEMCRKCRTG
CPRGMVKVGDCTPW SDIECVHKESGTKH SGEVPAVEETVTSSP GTPASPCSLSGIIIGV
TVAAVVLIVAVFVCK SLLWKKVLPYLKGIC SGGGGDPERVDRSS QRPGAEDNVLNEIVS
ILQPTQVPEQEMEVQ EPAEPTGVNMLSPGE SEHLLEPAEAERSQR RRLLVPANEGDPTET
LRQCFDDFADLVPFD SWEPLMRKLGLMDN EIKVAKAEAAGHRDT LYTMLIKWVNKTGR
DASVHTLLDALETLG ERLAKQKIEDHLLSS GKFMYLEGNADSAM S SEQ ID NO: 47
Human DR5del- MEQRGQNAPAASGA K386N RKRHGPGPREARGA RPGPRVPKTLVLVVA
AVLLLVSAESALITQ QDLAPQQRAAPQQK RSSPSEGLCPPGHHI SEDGRDCISCKYGQ
DYSTHWNDLLFCLR CTRCDSGEVELSPCT TTRNTVCQCEEGTFR EEDSPEMCRKCRTG
CPRGMVKVGDCTPW SDIECVHKESGIIIGV TVAAVVLIVAVFVCK SLLWKKVLPYLKGIC
SGGGGDPERVDRSS
QRPGAEDNVLNEIVS ILQPTQVPEQEMEVQ EPAEPTGVNMLSPGE SEHLLEPAEAERSQR
RRLLVPANEGDPTET LRQCFDDFADLVPFD SWEPLMRKLGLMDN EIKVAKAEAAGHRDT
LYTMLIKWVNKTGR DASVHTLLDALETLG ERLANQKIEDHLLSS GKFMYLEGNADSAM S SEQ
ID NO: 48 Cynomolgus DR5 MGQLRQSAPAASGA (NCBI RKGRGPGPREARGA
XP_005562887.1) RPGLRVLKTLVLVVA AARVLLSVSADCAPI TRQSLDPQRRAAPQ
QKRSSPTEGLCPPG HHISEDSRECISCKY GQDYSTHWNDFLFC LRCTKCDSGEVEVN
SCTTTRNTVCQCEE GTFREEDSPEICRKC RTGCPRGMVKVKDC TPWSDIECVHKESG
TKHTGEVPAVEKTVT TSPGTPASPCSLSGII IGVIVLVVIVVVAVIV WKTSLWKKVLPYLK
GVCSGGGGDPERVD SSSHSPQRPGAEDN ALNEIVSIVQPSQVP EQEMEVQEPAEQTD
VNTLSPGESEHLLEP AKAEGPQRRGQLVP VNENDPTETLRQCFD DFAAIVPFDAWEPLV
RQLGLTNNEIKVAKA EAASSRDTLYVMLIK WVNKTGRAASVNTL LDALETLEERLAKQK
IQDRLLSSGKFMYLE DNADSATS SEQ ID NO: 49 Cynomolgus DR5-
MGQLRQSAPAASGA K420N RKGRGPGPREARGA RPGLRVLKTLVLVVA AARVLLSVSADCAPI
TRQSLDPQRRAAPQ QKRSSPTEGLCPPG HHISEDSRECISCKY GQDYSTHWNDFLFC
LRCTKCDSGEVEVN SCTTTRNTVCQCEE GTFREEDSPEICRKC RTGCPRGMVKVKDC
TPWSDIECVHKESG TKHTGEVPAVEKTVT TSPGTPASPCSLSGII IGVIVLVVIVVVAVIV
WKTSLWKKVLPYLK GVCSGGGGDPERVD SSSHSPQRPGAEDN ALNEIVSIVQPSQVP
EQEMEVQEPAEQTD VNTLSPGESEHLLEP AKAEGPQRRGQLVP VNENDPTETLRQCFD
DFAAIVPFDAWEPLV RQLGLTNNEIKVAKA EAASSRDTLYVMLIK WVNKTGRAASVNTL
LDALETLEERLANQK IQDRLLSSGKFMYLE DNADSATS SEQ ID NO: 50 Cyno
DR5Mfdel- MGQLRQSAPAASGA K420N RKGRGPGPREARGA RPGLRVLKTLVLVVA
AARVLLSVSADCAPI TRQSLDPQRRAAPQ QKRSSPTEGLCPPG HHISEDSRECISCKY
GQDYSTHWNDFLFC LRCTKCDSGEVEVN SCTTTRNTVCQCEE GTFREEDSPEICRKC
RTGCPRGMVKVKDC TPWSDIECVHKESGI IIGVIVLVVIVVVAVI VWKTSLWKKVLPYL
KGVCSGGGGDPERV DSSSHSPQRPGAED NALNEIVSIVQPSQV PEQEMEVQEPAEQT
DVNTLSPGESEHLLE PAKAEGPQRRGQLV PVNENDPTETLRQCF DDFAAIVPFDAWEPL
VRQLGLTNNEIKVAK AEAASSRDTLYVMLI KWVNKTGRAASVNT LLDALETLEERLANQ
KIQDRLLSSGKFMYL EDNADSATS SEQ ID NO: 51 VH chTRA8 CDR1 GFTFSSYV SEQ
ID NO: 52 VH chTRA8 CDR2 ISSGGSYT SEQ ID NO: 53 VH chTRA8 CDR3
ARRGDSMITTDY SEQ ID NO: 54 VL chTRA8 CDR1 QDVGTA VL chTRA8 CDR2 WAS
SEQ ID NO: 55 VL chTRA8 CDR3 QQYSSYRT SEQ ID NO: 56 HC-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: 57
LC-chTRA8 DIVMTQSHKFMSTS VGDRVSITCKASQD VGTAVAWYQQKPG
QSPKLLIYWASTRH TGVPDRFTGSGSGT DFTLTISNVQSEDLA DYFCQQYSSYRTFG
GGTKLEIKRTVAAPS VFIFPPSDEQLKSGT ASVVCLLNNFYPREA KVQWKVDNALQSG
NSQESVTEQDSKDS TYSLSSTLTLSKADY EKHKVYACEVTHQG LSSPVTKSFNRGEC SEQ ID
NO: 58 Fc IgG1m(f)-E430G STKGPSVFPLAPSSK STSGGTAALGCLVK
DYFPEPVTVSWNSG ALTSGVHTFPAVLQS SGLYSLSSVVTVPSS SLGTQTYICNVNHKP
SNTKVDKRVEPKSC DKTHTCPPCPAPELL GGPSVFLFPPKPKDT LMISRTPEVTCVVVD
VSHEDPEVKFNWYV DGVEVHNAKTKPRE EQYNSTYRVVSVLTV LHQDWLNGKEYKCK
VSNKALPAPIEKTISK AKGQPREPQVYTLPP SREEMTKNQVSLTCL VKGFYPSDIAVEWES
NGQPENNYKTTPPVL DSDGSFFLYSKLTVD KSRWQQGNVFSCS VMHGALHNHYTQKS
LSLSPGK SEQ ID NO: 59 Fc IgG1m(f)-E345K STKGPSVFPLAPSSK
STSGGTAALGCLVK DYFPEPVTVSWNSG ALTSGVHTFPAVLQS SGLYSLSSVVTVPSS
SLGTQTYICNVNHKP SNTKVDKRVEPKSC DKTHTCPPCPAPELL GGPSVFLFPPKPKDT
LMISRTPEVTCVVVD VSHEDPEVKFNWYV DGVEVHNAKTKPRE EQYNSTYRVVSVLTV
LHQDWLNGKEYKCK VSNKALPAPIEKTISK AKGQPRKPQVYTLPP SREEMTKNQVSLTCL
VKGFYPSDIAVEWES NGQPENNYKTTPPVL DSDGSFFLYSKLTVD KSRWQQGNVFSCS
VMHEALHNHYTQKS LSLSPGK SEQ ID NO: 60 Fc IgG1m(f)-S440Y
STKGPSVFPLAPSSK STSGGTAALGCLVK DYFPEPVTVSWNSG ALTSGVHTFPAVLQS
SGLYSLSSVVTVPSS SLGTQTYICNVNHKP SNTKVDKRVEPKSC DKTHTCPPCPAPELL
GGPSVFLFPPKPKDT LMISRTPEVTCVVVD VSHEDPEVKFNWYV DGVEVHNAKTKPRE
EQYNSTYRVVSVLTV LHQDWLNGKEYKCK VSNKALPAPIEKTISK AKGQPREPQVYTLPP
SREEMTKNQVSLTCL VKGFYPSDIAVEWES NGQPENNYKTTPPVL DSDGSFFLYSKLTVD
KSRWQQGNVFSCS VMHEALHNHYTQKY LSLSPGK SEQ ID NO: 61 Fc
IgG1m(f)-E430G- STKGPSVFPLAPSSK K439E STSGGTAALGCLVK DYFPEPVTVSWNSG
ALTSGVHTFPAVLQS SGLYSLSSVVTVPSS SLGTQTYICNVNHKP SNTKVDKRVEPKSC
DKTHTCPPCPAPELL GGPSVFLFPPKPKDT LMISRTPEVTCVVVD
VSHEDPEVKFNWYV DGVEVHNAKTKPRE EQYNSTYRVVSVLTV LHQDWLNGKEYKCK
VSNKALPAPIEKTISK AKGQPREPQVYTLPP SREEMTKNQVSLTCL VKGFYPSDIAVEWES
NGQPENNYKTTPPVL DSDGSFFLYSKLTVD KSRWQQGNVFSCS VMHGALHNHYTQES
LSLSPGK SEQ ID NO: 62 Fc IgG1m(f)-E430G- STKGPSVFPLAPSSK S440K
STSGGTAALGCLVK DYFPEPVTVSWNSG ALTSGVHTFPAVLQS SGLYSLSSVVTVPSS
SLGTQTYICNVNHKP SNTKVDKRVEPKSC DKTHTCPPCPAPELL GGPSVFLFPPKPKDT
LMISRTPEVTCVVVD VSHEDPEVKFNWYV DGVEVHNAKTKPRE EQYNSTYRVVSVLTV
LHQDWLNGKEYKCK VSNKALPAPIEKTISK AKGQPREPQVYTLPP SREEMTKNQVSLTCL
VKGFYPSDIAVEWES NGQPENNYKTTPPVL DSDGSFFLYSKLTVD KSRWQQGNVFSCS
VMHGALHNHYTQKK LSLSPGK SEQ ID NO: 63 Fc IgG1m(f)-K409R
STKGPSVFPLAPSSK STSGGTAALGCLVK DYFPEPVTVSWNSG ALTSGVHTFPAVLQS
SGLYSLSSVVTVPSS SLGTQTYICNVNHKP SNTKVDKRVEPKSC DKTHTCPPCPAPELL
GGPSVFLFPPKPKDT LMISRTPEVTCVVVD VSHEDPEVKFNWYV DGVEVHNAKTKPRE
EQYNSTYRVVSVLTV LHQDWLNGKEYKCK VSNKALPAPIEKTISK AKGQPREPQVYTLPP
SREEMTKNQVSLTCL VKGFYPSDIAVEWES NGQPENNYKTTPPVL DSDGSFFLYSRLTVD
KSRWQQGNVFSCS VMHEALHNHYTQKS LSLSPGK SEQ ID NO: 64 Fc
IgG1m(f)-K409R- STKGPSVFPLAPSSK E345K STSGGTAALGCLVK DYFPEPVTVSWNSG
ALTSGVHTFPAVLQS SGLYSLSSVVTVPSS SLGTQTYICNVNHKP SNTKVDKRVEPKSC
DKTHTCPPCPAPELL GGPSVFLFPPKPKDT LMISRTPEVTCVVVD VSHEDPEVKFNWYV
DGVEVHNAKTKPRE EQYNSTYRVVSVLTV LHQDWLNGKEYKCK VSNKALPAPIEKTISK
AKGQPRKPQVYTLPP SREEMTKNQVSLTCL VKGFYPSDIAVEWES NGQPENNYKTTPPVL
DSDGSFFLYSRLTVD KSRWQQGNVFSCS VMHEALHNHYTQKS LSLSPGK SEQ ID NO: 65
Fc IgG1m(f)-K409R- STKGPSVFPLAPSSK E430G STSGGTAALGCLVK
DYFPEPVTVSWNSG ALTSGVHTFPAVLQS SGLYSLSSVVTVPSS SLGTQTYICNVNHKP
SNTKVDKRVEPKSC DKTHTCPPCPAPELL GGPSVFLFPPKPKDT LMISRTPEVTCVVVD
VSHEDPEVKFNWYV DGVEVHNAKTKPRE EQYNSTYRVVSVLTV LHQDWLNGKEYKCK
VSNKALPAPIEKTISK AKGQPREPQVYTLPP SREEMTKNQVSLTCL VKGFYPSDIAVEWES
NGQPENNYKTTPPVL DSDGSFFLYSRLTVD KSRWQQGNVFSCS VMHGALHNHYTQKS
LSLSPGK SEQ ID NO: 66 Fc IgG1m(f)-F405L STKGPSVFPLAPSSK
STSGGTAALGCLVK DYFPEPVTVSWNSG ALTSGVHTFPAVLQS SGLYSLSSVVTVPSS
SLGTQTYICNVNHKP SNTKVDKRVEPKSC DKTHTCPPCPAPELL GGPSVFLFPPKPKDT
LMISRTPEVTCVVVD VSHEDPEVKFNWYV DGVEVHNAKTKPRE EQYNSTYRVVSVLTV
LHQDWLNGKEYKCK VSNKALPAPIEKTISK AKGQPREPQVYTLPP SREEMTKNQVSLTCL
VKGFYPSDIAVEWES NGQPENNYKTTPPVL DSDGSFLLYSKLTVD KSRWQQGNVFSCS
VMHEALHNHYTQKS LSLSPGK SEQ ID NO: 67 Fc IgG1m(f)-F405L-
STKGPSVFPLAPSSK E345K STSGGTAALGCLVK DYFPEPVTVSWNSG ALTSGVHTFPAVLQS
SGLYSLSSVVTVPSS SLGTQTYICNVNHKP SNTKVDKRVEPKSC DKTHTCPPCPAPELL
GGPSVFLFPPKPKDT LMISRTPEVTCVVVD VSHEDPEVKFNWYV DGVEVHNAKTKPRE
EQYNSTYRVVSVLTV LHQDWLNGKEYKCK VSNKALPAPIEKTISK AKGQPRKPQVYTLPP
SREEMTKNQVSLTCL VKGFYPSDIAVEWES NGQPENNYKTTPPVL DSDGSFLLYSKLTVD
KSRWQQGNVFSCS VMHEALHNHYTQKS LSLSPGK SEQ ID NO: 68 Fc
IgG1m(f)-F405L- STKGPSVFPLAPSSK E430G STSGGTAALGCLVK DYFPEPVTVSWNSG
ALTSGVHTFPAVLQS SGLYSLSSVVTVPSS SLGTQTYICNVNHKP SNTKVDKRVEPKSC
DKTHTCPPCPAPELL GGPSVFLFPPKPKDT LMISRTPEVTCVVVD VSHEDPEVKFNWYV
DGVEVHNAKTKPRE EQYNSTYRVVSVLTV LHQDWLNGKEYKCK VSNKALPAPIEKTISK
AKGQPREPQVYTLPP SREEMTKNQVSLTCL VKGFYPSDIAVEWES NGQPENNYKTTPPVL
DSDGSFLLYSKLTVD KSRWQQGNVFSCS VMHGALHNHYTQKS LSLSPGK
EXAMPLES
Example 1
Antibody and Antigen Constructs
Expression Constructs for DR5
[0467] Codon-optimized constructs for expression of full-length DR5
proteins of human (SEQ ID NO 46), rhesus monkey (SEQ ID NO 25) and
mouse (SEQ ID NO 26) were generated based on available sequences:
human (Homo sapiens) DR5 (Genbank accession no. NP_003833,
UniprotKB/Swiss-Prot O14763-1), Rhesus monkey (Macaca mulatta) DR5
(Genbank accession no. EHH28346), murine (Mus musculus) DR5
(UniprotKB/Swiss-Prot Q9QZM4). For mapping of the binding regions
of the DR5 antibodies (as described in Example 6) the following
chimeric human/mouse DR5 constructs were made; human DR5 in which,
respectively, the following parts were replaced by the
corresponding mouse DR5 sequence (numbers refer to human sequence),
construct A aa 56-68, construct B aa 56-78, construct C aa 69-78,
construct D aa 79-115, construct E 79-138, construct F aa 97-138,
construct G aa 139-166, construct H aa 139-182, construct I aa
167-182, construct J 167-210, construct K aa 183-210. The
loss-of-function mutation K415N was introduced in the human DR5
death domain (SEQ ID NO 44). In addition, codon-optimized construct
for the extracellular domain (ECD) of human DR5 with a C-terminal
His tag were generated: DR5ECD-FcHistag (SEQ ID NO 27) and
DR5ECDdelHis (SEQ ID NO 28). All constructs contained suitable
restriction sites for cloning and an optimal Kozak (GCCGCCACC)
sequence. The constructs were cloned in the mammalian expression
vector pcDNA3.3 (Invitrogen).
Expression Constructs for Antibodies
[0468] For antibody expression the VH and VL sequences, as earlier
described, of the chimeric human/mouse DR5 antibodies DR5-01 and
DR5-05 (based on EP2684896A1) and their humanized variants hDR5-01
and hDR5-05 (based on WO2014/009358) were cloned in expression
vectors (pcDNA3.3) containing the relevant constant HC and LC
regions. Desired mutations were introduced either by gene synthesis
or site directed mutagenesis.
[0469] In some of the Examples, reference antibodies against DR5
were used that have been previously described. IgG1-CONA (based on
U.S. 7,521,048 B2 and WO2010/138725) and IgG1-chTRA8 (based on
EP1506285B1 and US724442962) were cloned in the relevant antibody
expression vectors as supra.
[0470] In some of the examples the human IgG1 antibody IgG1-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
[0471] 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).
[0472] Membrane proteins were expressed in Freestyle CHO-S cells
(Life technologies), using the freestyle Max reagent, as described
by the manufacturer.
Purification and Analysis of Proteins
[0473] 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
[0474] Bispecific IgG1 antibodies were generated by
Fab-arm-exchange under controlled reducing conditions. The basis
for this method is the use of complementary CH3 domains, which
promote the formation of heterodimers under specific assay
conditions as described in WO2011/131746. The F405L and K409R (EU
numbering) mutations were introduced in anti-DR5 IgG1 antibodies to
create antibody pairs with complementary CH3 domains. The F405L
mutation was introduced in IgG1-DR5-05 and IgG1-DR5-05-E430G; the
K409R mutation was introduced in IgG1-DR5-01 and IgG1-DR5-01-E430G.
To generate bispecific antibodies, the two parental complementary
antibodies, each antibody at a final concentration of 0.5 mg/mL,
were incubated with 75 mM 2-mercaptoethylamine-HCI (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) and
IgG1-DR5-01-K409R-E430G.times.IgG1-DR5-05-F405L-E430G (BsAb
DR5-01-K409R-E430G.times.DR5-05-F405L-E430G) were generated.
[0475] The K409R mutation and/or the F405L mutation have no effect
on the antibody's binding to the corresponding antigen. That is the
K409R mutation and/or the F405L mutation have no effect of the
anti-DR5 antibody's binding to DR5.
Example 2
DR5 Expression Levels on Different Human Cancer Cell Lines
[0476] DR5 density per cell was quantified for different human
cancer cell lines by indirect immunofluorescence using QIFIKIT
(DAKO, Cat nr K0078) with mouse monoclonal antibody B-K29
(Diaclone, Cat nr 854.860.000). Cells were harvested by
trypsinization and passed through a cell strainer. Cells were
pelleted by centrifugation for 5 minutes at 1,200 rpm, washed with
PBS and resuspended at a concentration of 2.times.10.sup.6
cells/mL. The next steps were performed at 4.degree. C. 50 .mu.L of
the single cell suspensions (100,000 cells per well) were seeded in
polystyrene 96-well round-bottom plates (Greiner Bio-One, Cat nr
650101). Cells were pelleted by centrifugation for 3 minutes at
300.times.g and resuspended in 50 .mu.L antibody sample or mouse
IgG1 isotype control sample (BD/Pharmingen, Cat nr 555746) at 10
.mu.g/mL saturating concentrations. After an incubation of 30
minutes at 4.degree. C., cells were pelleted and resuspended in 150
.mu.L FACS buffer (PBS+0.1% (w/v) bovine serum albumin (BSA)+0.02%
(w/v) sodium azide). Set-up and calibration beads were added to the
plate according to the manufacturer's instructions. Cells and beads
in parallel were washed two more times with 150 .mu.L FACS buffer
and resuspended in 50 .mu.L FITC-conjugated goat-anti-mouse IgG
(1/50; DAKO, Cat nr F0479). Secondary antibody was incubated for 30
minutes at 4.degree. C. protected from light. Cells and beads were
washed twice with 150 .mu.L FACS buffer and resuspended in 150
.mu.L FACS buffer. Immunofluorescence was measured on a FACS Canto
II (BD Biosciences) by recording 10,000 events within the
population of viable cells. The Geometric mean of fluorescence
intensity of the calibration beads was used to calculate the
calibration curve that was forced to go through zero intensity and
zero concentration using GraphPad Prism software (GraphPad
Software, San Diego, Calif., USA). For each cell line, the antibody
binding capacity (ABC), an estimate for the number of DR5 molecules
expressed on the plasma membrane, was calculated using the
Geometric mean fluorescence intensity of the DR5-antibody-stained
cells, based on the equation of the calibration curve
(interpolation of unknowns from the standard curve, using GraphPad
Software). Generally, DR5 cell surface expression was low to
moderate on the cell lines assessed here. Based on these data, cell
lines were categorized according to low DR5 expression
(ABC<10,000) and moderate DR5 expression (ABC>10,000). HCT-15
(ATCC, CCL-225), HT-29 (ATCC, HTB-38) and SW480 (ATCC, CCL-228)
colon cancer, BxPC-3 (ATCC, CRL-1687), HPAF-II (ATCC, CRL-1997) and
PANC-1 (ATCC, CRL-1469) pancreatic cancer, and A549 (ATCC, CCL-185)
and SK-MES-1 (ATCC, HTB-58) lung cancer cell lines were found to
have low DR5 expression (QIFIKIT ABC range 3,081-8,411). COLO 205
(ATCC CCL-222.TM.) and HCT 116 (ATCC CCL-247) colon cancer, A375
(ATCC, CRL-1619) skin cancer and SNU-5 (ATCC, CRL-5973) gastric
cancer cell lines were found to have moderate DR5 expression
(QIFIKIT ABC range 10,777-21,262).
Example 3
Binding of Humanized DR5-01 and DR5-05 Antibodies to HCT 116
Cells
[0477] The humanized antibodies hDR5-01 and hDR5-05 are described
in patent application WO2014/009358. Binding of purified
IgG1-hDR5-01-K409R and IgG1-hDR5-05-F405L to DR5-positive HCT 116
human colon cancer cells was analyzed and compared to binding of
the chimeric antibodies IgG1-DR5-01-K409R and IgG1-DR5-05-F405L by
FACS analysis. To prepare single cell suspensions, adherent HCT 116
cells were washed twice with PBS (B. Braun; Cat nr 3623140) before
incubating with Trypsin 1.times./EDTA 0.05% for 2 minutes at
37.degree. C. 10 mL medium [McCoy's 5A medium with L-Glutamine and
HEPES (Lonza; Cat nr BE12-168F)+10% Donor Bovine Serum with Iron
(Life Technologies; Cat nr 10371-029)+100 Units Penicillin/100
Units Streptomycin (Lonza Cat nr DE17-603E)] was added before
pelleting the cells by centrifugation for 5 minutes at 1200 rpm.
Cells were resuspended in 10 mL medium, pelleted again by
centrifugation for 5 minutes at 1200 rpm, and resuspended in FACS
buffer at a concentration of 1.0.times.10.sup.6 cells/mL. The next
steps were performed at 4.degree. C. 100 .mu.L cell suspension
samples (100,000 cells per well) were seeded in polystyrene 96-well
round-bottom plates (Greiner Bio-One; Cat nr 650101) and pelleted
by centrifugation at 300.times.g for 3 minutes at 4.degree. C.
Cells were resuspended in 100 .mu.L samples of a serial dilution
antibody preparation series (range 0 to 10 .mu.g/mL in 5-fold
dilutions) and incubated for 30 minutes at 4.degree. C. Cells were
pelleted by centrifugation at 300.times.g for 3 minutes at
4.degree. C. and washed twice with 150 .mu.L FACS buffer. Cells
were incubated with 50 .mu.L secondary antibody R-phycoerythrin
(R-PE)-conjugated goat-anti-human IgG F(ab').sub.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 150 .mu.L FACS buffer, and
antibody binding was analyzed on a FACS Canto II (BD Biosciences)
by recording 10,000 events. Binding curves were analyzed using
non-linear regression analysis (sigmoidal dose-response with
variable slope) using GraphPad Prism software.
[0478] As can be seen from FIG. 2 shows that the humanized
antibodies IgG1-hDR5-01-K409R and IgG1-hDR5-05-F405L showed similar
binding curves as their corresponding chimeric antibody
IgG1-DR5-01-K409R or IgG1-DR5-05-F405L, respectively. Humanization
had no effect on the binding of the DR5 antibodies.
Example 4
Introduction of a Hexamerization-Enhancing Mutation does not Affect
Binding of Chimeric DR5-01 and DR5-05 Antibodies and Bispecific
Antibody DR5-01.times.DR5-05 to DR5-Positive Human Colon Cancer
Cells
[0479] 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 25mM 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').sub.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.
[0480] FIG. 3A 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. 3B
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. 3C 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 5
Binding of Chimeric DR5-01 and DR5-05 Antibodies to Rhesus Macaque
DR5
[0481] Binding of purified IgG1-DR5-01-K409R-E430G and
IgG1-DR5-05-F405L-E430G to CHO cells expressing Rhesus macaque DR5
or human DR5 (described in Example 1) was analyzed by FACS
analysis. One day before FACS analysis, CHO cells were transiently
transfected with a vector encoding Rhesus macaque DR5, human DR5 or
a non-coding vector (mock). To prepare single cell suspensions,
cells were washed with PBS and resuspended in FACS buffer at a
concentration of 1.0.times.10.sup.6 cells/mL. The next steps were
performed at 4.degree. C. 75 .mu.L cell suspension samples (75,000
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 10 to 0 .mu.g/mL
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').sub.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 100,000
events. Binding curves were analyzed using non-linear regression
analysis (sigmoidal dose-response with variable slope) using
GraphPad Prism software.
[0482] FIG. 4 shows that the antibodies IgG1-DR5-01-K409R-E430G and
IgG1-DR5-05-F405L-E430G showed dose-dependent binding to Rhesus
macaque DR5 expressed on CHO cells. Binding to CHO cells
transfected with human DR5 and mock-transfected CHO cell was tested
as positive and negative control, respectively. For both
IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G, EC.sub.50
values for binding to human DR5 and Rhesus macaque DR5 were in the
same range ([0.014-0.023 .mu.g/mL] and [0.051-0.066 .mu.g/mL],
respectively), indicating that IgG1-DR5-01-K409R-E430G and
IgG1-DR5-05-F405L-E430G show comparable binding to human and Rhesus
macaque DR5.
Example 6
Mapping of Binding Regions of DR5-01 and DR5-05 Antibodies on Human
DR5 using Domain-Swapped DR5 Molecules
[0483] The amino acid sequences of the extracellular domains of
human and murine DR5 show limited homology (FIG. 5 A) and the
humanized antibodies IgG1-hDR5-01-F405L and IgG1-hDR5-05-F405L do
not bind murine DR5 (FIG. 5 C, D). With the aim to identify amino
acid stretches in the human DR5 extracellular domain that are
involved in antibody binding, we developed eleven human-mouse
chimeric DR5 molecules, in which specific human DR5 domains had
been replaced by the mouse analogues (domain-swapped DR5 molecules
described in Example 1) as visualized in FIG. 5 B. The
domain-swapped DR5 variants were transiently expressed on CHO
cells. Loss of binding of the DR5 antibodies to domain-swapped DR5
molecules indicates that the swapped domain of human DR5 contains
one or more amino acids that are crucial for binding. Vice versa,
retention of binding of the DR5 antibodies to domain-swapped DR5
molecules indicates that the swapped domain of human DR5 does not
contain amino acids that are crucial for binding. For the binding
assay, 3x10.sup.6 transfected cells were washed and resuspended in
3 mL FACS buffer. 100 .mu.L cell suspension was added per well
(100.000 cells per well) of 96-well round bottom plates (Greiner
Bio-one; Cat nr 650101). The next steps were performed at 4.degree.
C. Cells were pelleted, resuspended in 50 .mu.L DR5 antibody sample
(10 .mu.g/mL final concentration) and incubated for 30 minutes at
4.degree. C. The cells were washed twice and incubated in 50 .mu.L
secondary antibody R-PE-conjugated goat-anti-human IgG F(ab').sub.2
(Jackson ImmunoResearch; Cat nr 109-116-098; 1/100) for 30 minutes
at 4.degree. C. protected from light. Cells were washed twice,
resuspended in 120 .mu.L FACS buffer, and analyzed on a FACS Canto
II (BD Biosciences). The percentage of viable PE-positive cells was
plotted using GraphPad Prism software. Surface expression was
confirmed for each domain-swapped DR5 molecule using a panel of DR5
antibodies directed against different epitopes (not shown). The
non-target binding antibody IgG1-b12 against gp120 was included as
a negative control for binding. FIG. 5 C shows that
IgG1-hDR5-01-F405L showed loss of binding to constructs E (79-138),
F (97-138), G (139-166) and H (139-182), whereas binding to
constructs A-D (covering human DR5 sequence 56-115) and I-K
(covering human DR5 sequence 167-210) was retained. Together, these
data indicate that the amino acid regions 116-138 and 139-166 each
contain one or more amino acids required for binding of
IgG1-hDR5-01-F405L to human DR5. FIG. 5 D shows that
IgG1-hDR5-05-F405L showed loss of binding to constructs D (79-115),
E (79-138) and F (97-138), whereas binding to constructs A-C
(covering human DR5 sequence 56-78) and G-K (covering human DR5
sequence 139-210) was retained. Together, these data indicate that
the amino acid region 79-138 contains one or more amino acids
required for binding of IgG1-hDR5-05-F405L to human DR5.
Example 7
Crossblock ELISA with DR5-01 and DR5-05 Antibodies
[0484] The competition between humanized DR5-01 and DR5-05
antibodies for binding to the extracellular domain of DR5 was
measured by sandwich binding assays in a sandwich enzyme-linked
immunosorbent assay (ELISA) as described in this example and by
Bio-Layer interferometry (BLI) using a ForteBio Octet.RTM. HTX
system (data not shown). For the 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-hDR5-01-E430G or
IgG1-hDR5-05-E430G) 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 (SEQ ID 27)
(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-ethyl benzothiazoline-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 EL.times.808 Absorbance Microplate Reader). FIG. 6
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-hDR5-01-E430G was not inhibited in the presence of soluble
IgG1-hDR5-05-E430G. Vice versa, binding of DR5ECD-FcHistag to
coated IgG1-hDR5-05-E430G was also not inhibited in the presence of
soluble IgG1-hDR5-01-E430G. These data indicate that
IgG1-hDR5-01-E430G and IgG1-hDR5-05-E430G did not compete with each
other for binding of DR5ECD-FcHisCtag, suggesting that they
recognize distinct epitopes in the extracellular domain of human
DR5. These data were confirmed by BLI using a classical sandwich
assay, in which IgG1-hDR5-01-F405L or IgG1-hDR5-05-F405L (20
.mu.g/ml in 10 mM Sodium Acetate pH 6.0, ForteBio Cat nr 18-1070)
were immobilized on Amine-Reactive Second Generation biosensors
(ForteBio Cat nr 18-5092). Subsequently, biosensors were incubated
with DR5ECDdelHis (SEQ ID 28) (100 nM in Sample Diluent, ForteBio
cat nr 18-1048) and binding of competing antibody (5 .mu.g/mL in
Sample Diluent) was analyzed (data not shown).
Example 8
Introduction of a Hexamerization-Enhancing Mutation Improves the
Efficacy of Cell Death Induction by DR5-01 and DR5-05 Antibodies
and of the Combination Thereof
[0485] A viability assay was performed to study the effect the
hexamerization-enhancing mutation E430G in IgG1-DR5-01-K409R and
IgG1-DR5-05-F405L on the capacity of the antibodies to kill human
colon cancer cells COLO 205 and HCT 116. The antibodies were tested
as single agent and as combinations of DR5-01 and DR5-05
antibodies. 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. 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 a serial dilution antibody preparation series
(range 0.05 to 20,000 ng/mL final concentrations in 5-fold
dilutions) was added and incubated for 3 days at 37.degree. C. In
samples that were treated with a combination of two antibodies, the
total antibody concentration in the assay was the same as in the
samples that were treated with single antibodies. As a positive
control, cells were incubated with 5 .mu.M staurosporine (Sigma
Aldrich, Cat nr S6942). 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. FIG. 7 shows the percentage viable cells,
as calculated using the following formula: % viable
cells=[(luminescence antibody sample-luminescence staurosporine
sample)/(luminescence no antibody sample-luminescence staurosporine
sample)]*100.
[0486] FIG. 7 shows that introduction of the E430G mutation
enhanced the potency of the chimeric antibodies IgG1-DR5-01-K409R
and IgG1-DR5-05-F405L in both COLO 205 (A) and HCT 116 (B) cells.
The combination of IgG1-DR5-01-K409R-E430G and
IgG1-DR5-05-F405L-E430G was more potent than either antibody alone
and more potent than the combination of the antibodies without the
E430G mutation. The combination of IgG1-DR5-01-K409R and
IgG1-DR5-05-F405L was more potent than either antibody alone. These
data show that introduction of the hexamerization-enhancing
mutation E430G resulted in enhanced induction of cell killing upon
binding of the chimeric DR5 antibodies 01 and 05, both as single
antibodies and in combination, with the combination being the most
potent.
Example 9
Combining Two Non-Crossblocking DR5 Antibodies with
Hexamerization-Enhancing Mutations Results in Enhanced Target Cell
Killing
[0487] In Example 8 it is shown that combining the two
non-crossblocking anti-DR5 antibodies IgG1-DR5-01-K409R-E430G and
IgG1-DR5-05-F405L-E430G with hexamerization enhancing mutations
resulted in enhanced killing on cancer cell lines compared to the
efficacy of the single antibodies. Here, we compare the efficacy of
two non-crossblocking versus two crossblocking anti-DR5 antibodies.
A viability assay was performed to study the capacity of the
combination of antibodies IgG1-chTRA8-F405L-E430G with either
non-crossblocking antibody IgG1-DR5-01-K409R-E430G or crossblocking
antibody IgG1-DR5-05-F405L-E430G to induce killing of HCT 116 colon
cancer cells in comparison to the single antibodies. A crossblock
ELISA for antibodies IgG1-chTRA8-F405L and IgG1-DR5-05-F405L was
performed as described in Example 7 and confirmed by a sandwich
binding assay on an Octet.RTM. HTX system (data not shown). The
viability assay on HCT 116 cells was performed as described in
Example 8 with a serial diluted antibody series ranging from
0.00005 to 20 .mu.g/mL final concentrations in 5-fold dilutions.
FIG. 8 shows that the efficacy of the single antibodies in killing
of HCT116 cells was enhanced by combining the two non-crossblocking
antibodies IgG1-chTRA8-F405L-E430G and IgG1-DR5-01-K409R-E430G
(FIG. 8 B) and not by combining the two crossblocking antibodies
IgG1-chTRA8-F405L-E430G and IgG1-DR5-05-F405L-E430G (FIG. 8 C).
Example 10
Capacity of the Combination of Non-Crossblocking Antibodies
DR5-05+CONA and Bispecific Antibody DR5-05.times.CONA with
Hexamerization-Enhancing Mutations to Induce Target Cell
Killing
[0488] A viability assay was performed to study the capacity of
another combination of two non-crossblocking antibodies
(IgG1-CONA-K409R-E430G+IgG1-DR5-05-F405L-E345K) and its bispecific
derivative BsAb IgG1-CONA-K409R-E430G.times.DR5-05-F405L-E345K to
induce killing of HCT 116 colon cancer cells in comparison to the
combination of antibodies and the bispecific antibody without
hexamerization-enhancing mutation, respectively. A crossblock ELISA
for antibodies IgG1-CONA-K409R and IgG1-DR5-05-F405L was performed
as described in Example 7 and confirmed by a sandwich binding assay
on an Octet.RTM. HTX system (data not shown). The viability assay
on HCT 116 cells was performed as described in Example 8 with a
serial diluted antibody series ranging from 0.01 to 20,000 ng/mL
final concentrations in 5-fold dilutions. FIG. 9 shows that the
combination of non-crossblocking antibodies
1gG1-CONA-K409R-E430G+IgG1-DR5-05-F405L-E345K and
BsAbIgG1-CONA-K409R-E430G.times.DR5-05-F405L-E345K with hexa
merization-enhancing mutations showed enhanced efficacy in killing
of HCT116 cells compared to these antibodies without the
hexamerization-enhancing mutations E430G or E345K.
Example 11
Capacity of the DR5-01+DR5-05 Antibody Combination with E430G
Hexamerization-Enhancing Mutation to Induce Target Cell Killing in
Different Cancer Cell Lines
[0489] A viability assay was performed to study the capacity of the
combination of human-mouse chimeric antibodies
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. Adherent 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 [COLO 205, HCT-15, SW480 and BxPC-3:
RPMI 1640 with 25 mM Hepes and L-Glutamine (Lonza Cat nr
BE12-115F)+10% DBSI (Life Technologies Cat nr 10371-029)+Pen/Strep
(Lonza Cat nr DE17-603E); HCT116 and HT-29: McCoy's5A Medium with
L-Glutamine and Hepes (Lonza, Cat nr BE12-168F)+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 6E17-605E)+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].
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.
Supernatant of the adherent cells was replaced by 150 .mu.L
antibody sample (final concentration 10 .mu.g/mL) and incubated for
3 days at 37.degree. C. As a positive control, cells were incubated
with 5 .mu.M staurosporine (Sigma Aldrich, Cat nr S6942). The
viability of the cell cultures was determined in a CellTiter-Glo
luminescent cell viability assay as described in Example 8. 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. 10). 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 chimeric 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. There was no
correlation between killing efficacy of
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G and DR5 target
expression levels (described in Example 2).
Example 12
Capacity of the Humanized DR5-01+DR5-05 Antibody Combination with
E430G Hexamerization-Enhancing Mutation to Induce Target Cell
Killing
[0490] A viability assay was performed to compare the potency of
the combination of chimeric antibodies
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G with the potency of
the combination of humanized antibodies
IgG1-hDR5-01-K409R-E430G+IgG1-hDR5-05-F405L-E430G to induce killing
of BxPC-3 and PANC-1 pancreatic cancer cells in vitro. 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. Supernatant of the adherent
cells was replaced by 150 .mu.L antibody sample of a serial
dilution antibody preparation series and incubated for 3 days at
37.degree. C. As a positive control, cells were incubated with 5
.mu.M staurosporine (Sigma Aldrich, Cat nr S6942). The viability of
the cell cultures was determined in a CellTiter-Glo luminescent
cell viability assay as described in Example 8. The combination of
the humanized antibodies with hexamerization-enhancing mutation
IgG1-hDR5-01-K409R-E430G+IgG1-hDR5-05-F405L-E430G showed similar
dose-response curves as the combination of the corresponding
chimeric antibodies IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G
(FIG. 11).
Example 13
Optimization of Antibody IgG1-hDR5-01-E430G
[0491] Amino acid sequence N55-G56 was identified as a potential
asparagine (Asn) deamidation motif in the CDR2 regions of the
IgG1-hDR5-01 and IgG1-hDR5-05 heavy chains (SEQ ID NO:2).
Deamidation at this position was mimicked by introduction of the
N55D mutation in IgG1-hDR5-01-K409R and IgG1-hDR5-05-F405L to test
the effect of deamidation on target binding.
IgG1-hDR5-01-N55D-K409R and IgG1-hDR5-05-N55D-F405L were tested for
binding to HCT 116 cells by FACS analysis as described in Example
3. FIG. 12A shows that mimicking deamidation by introduction of the
N55D mutation resulted in strongly decreased binding of
IgG1-hDR5-01-K409R on HCT 116 cells. In contrast,
IgG1-hDR5-05-F405L and IgG1-hDR5-05-N55D-F405L showed comparable
binding curves. To reduce the risk of Asn deamidation in the DR5-01
antibody, the G56T mutation was introduced in IgG1-hDR5-01-E430G
and this antibody variant was tested for binding to HCT 116 cells
by FACS analysis as described in Example 3. FIG. 12B shows that the
mutation had no effect on the binding of IgG1-hDR5-01-E430G to HCT
116 cells.
[0492] A viability assay was performed to compare the capacity of
the combination of humanized antibodies
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G with the capacity of the
combination of humanized antibodies
IgG1-hDR5-01-E430G+IgG1-hDR5-05-E430G to induce killing of BxPC-3
pancreatic cancer cells. Viability was assessed as described in
Example 11 with 1,000 cells per well and antibody concentrations
series ranging from 0.0001 to 10,000 ng/mL final concentrations in
4-fold dilutions in a total volume of 200 .mu.L. FIG. 12C shows
that introduction of the G56T mutation in IgG1-hDR5-01-E430G had no
effect on the killing efficacy of the antibody in combination with
IgG1-hDR5-05-E430G.
Example 14
Cell Death Induction by the Combination of Humanized antibodies
hDR5-01-G56T-E430G and hDR5-05-E430G Requires Fc:Fc Interactions to
Form Hexamers
[0493] To analyse 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 in 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. 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 11.
[0494] FIG. 13 shows that the combination of
IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G variants that both
harbor the same repulsion mutation (K439E or S440K) showed strongly
diminished killing efficacy in BxPC-3 (A) and HCT-15 cells (B).
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 15
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
[0495] 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 11 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 a dilution series
(range 0-100 .mu.g/mL) 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. 14). These data
indicate the involvement of Fc: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 16
Capacity of Chimeric Antibody Combination DR5-01 and DR5-05
Antibodies with E430G Hexamerization Enhancing Mutation to Induce
Cancer Cell Killing, at Different Combination Ratios
[0496] A viability assay was performed 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.
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. 50 .mu.L antibody sample with
different ratios of IgG1-DR5-01-K409R-E430G and
IgG1-DR5-05-F405L-E430G (indicated as Ratio DR5-01:DR5-05 of 100:0,
90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90 and
0:100 in serial dilution series ranging from 0.06 to 20 .mu.g/mL
final concentrations in 5-fold dilutions) was added and incubated
for 3 days at 37.degree. C. As a positive control, cells were
incubated with 5 .mu.M staurosporine (Sigma Aldrich, Cat nr S6942).
The viability of the cell cultures was determined in a
CellTiter-Glo luminescent cell viability assay as described in
Example 8. 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. 15).
Example 17
Capacity of the Combination of Humanized Antibodies DR5-01 and
DR5-05 Antibodies with E430G Hexamerization Enhancing Mutation to
Induce Cancer Cell Killing, at Different Combination Ratios
[0497] A viability assay was performed 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. Generally, the
experiments were performed as described in Example 16. Briefly,
pre-attached cells (5,000 cells per well) were incubated for 3 days
at 37.degree. C. in 150 .mu.L in polystyrene 96-well flat-bottom
plates with different ratios of IgG1-hDR5-01-G56T -E430G and
IgG1-hDR5-05-E430G (indicated in FIG. 16 as Ratio DR5-01:DR5-05 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. The viability of
the cell cultures was determined in a CellTiter-Glo luminescent
cell viability assay as described in Example 8. Killing was equally
effective at all tested antibody ratios containing both antibodies
IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G (FIG. 16).
Example 18
The Combination of Humanized DR5-01+DR5-05 Antibodies with the
E430G Hexamerization-Enhancing Mutation Induce Caspase-Dependent
Cytotoxicity
[0498] 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 8. The combination of the
humanized antibodies with hexa merization-enhancing mutation
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. 17). This was also
shown for the natural DR5 ligand TRAIL.
Example 19
Cell Death Induction upon Binding of the Combination of Chimeric
DR5-01 and DR5-05 Antibodies on COLO 205 Colon Cancer Cells, as
Assessed by Annexin V/Propidium Iodide and Active Caspase-3
Staining
[0499] 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 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.
[0500] 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.
[0501] 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.
[0502] FIG. 18 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 (A) and Active
Caspase-3-positive cells (B), compared to the negative control
antibody lgG1-b12. The percentage of Annexin V-positive/PI
-negative and Active Caspase-3 positive cells was higher in cells
treated with the combination of
1gG1-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 (C).
[0503] After 24 hours incubation, the percentage of Annexin V/PI
double-positive cells (D) 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 (E)) 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.
[0504] These data indicate that the combination of
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G induces both the
early and late stages of cell death in COLO 205 colon cancer cells,
and does so more effectively than the combination of the antibodies
without the E430G hexamerization enhancing mutation.
Example 20
Caspase-3 and -7 Activation upon Binding of the Combination of
Chimeric DR5-01 and DR5-05 Antibodies with Hexamerization-Enhancing
Mutation on COLO 205 Colon Cancer Cells
[0505] In example 19 it was described that incubation with the
combination of chimeric DR5 antibodies
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 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 (Perkin Elmer).
[0506] FIG. 19 shows that in the time course of 1, 2 to 5 hours,
caspase-3/7 activation was induced by the antibody combinations
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G and
IgG1-DR5-01-K409R+IgG1-DR5-05-F405L, and for the bispecific DR5
antibody BsAb IgG1-DR5-01-K409R-E430G.times.DR5-05-F405L-E430G.
After 24 hours, caspase-3/7 activation was almost reduced to
baseline levels for all tested DR5 antibodies. After 1 hour,
caspase-3/7 activation was already observed in cells that had been
treated with the combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G, whereas no
caspase-3/7 activation was observed in cells that had been treated
with the combination of IgG1-DR5-01-K409R+IgG1-DR5-05-F405L without
the hexamerization-enhancing mutation.
[0507] Similarly, at 2 and 5 hours, the caspase-3/7 activation
induced by the combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was stronger than
for the combination of IgG1-DR5-01-K409R+IgG1-DR5-05-F405L. These
data indicate that the combination of chimeric DR5 antibodies with
the hexamerization enhancing mutation
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G induced more rapid
and more potent Caspase-3/7 activation than the combination of
antibodies without the hexamerization enhancing mutation.
Example 21
The Potency of the Antibody Combination of Chimeric DR5-01 and
DR5-05 with the E430G Hexamerization-Enhancing Mutation is
Independent of the Presence of a Secondary Fc Crosslinker
[0508] A viability assay was performed to compare the capacity of
the antibody combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G 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, two DR5 antibodies that are known to show enhanced
killing in the presence of a secondary antibody crosslinker,
IgG1-CONA and IgG1-chTRA8-F405L, were tested in the same settings.
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. Supernatant of the adherent
cells was replaced by 150 .mu.L antibody sample (final
concentration 10 .mu.g/mL) in the absence or presence of
F(ab').sub.2 fragments of a goat-anti-human IgG antibody (1/150;
Jackson ImmunoResearch; Cat nr 109-006-098) and incubated for 3
days at 37.degree. C. As a positive control for cell killing, cells
were incubated with 5 .mu.M staurosporine (Sigma Aldrich, Cat nr
S6942). The viability of the cell cultures was determined in a
CellTiter-Glo luminescent cell viability assay as described in
Example 8. The antibody combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G induced significant
killing compared to the negative control of COLO 205, BxPC-3 and
PANC-1 cancer cells, both in presence or absence of an Fc
crosslinker (FIG. 20). In contrast, DR5 antibodies IgG1-DR5-CONA
and IgG1-DR5-chTRA8-F405L did not induce target cell killing in the
absence of an Fc crosslinker. Fc crosslinking induced killing by
IgG1-DR5-CONA and IgG1-DR5-chTRA8-F405L in COLO 205 and BxPC-3
cells, although with significantly lower potency than the antibody
combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G in
presence or absence of 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 is
independent of the presence of a secondary Fc crosslinker and that
this crosslinker-independent killing is more efficient than for
Fc-crosslinked IgG1-DR5-CONA and IgG1-DR5-chTRA8-F405L.
Example 22
Introduction of the K409R Mutation in IgG1-hDR5-01-430G and the
F405L Mutation in IgG1-hDR5-05-E430G has No Effect on the Potency
of the Combination of Humanized Antibodies
IgG1-hDR5-01-E430G+IgG1-hDR5-05-E430G
[0509] In many of the experiments described in this application,
the anti-DR5 antibodies IgG1-01 and IgG1-05 contain in the IgG Fc
domain the K409R and F405L (EU numbering index) mutation,
respectively. These mutations enable the generation of DR5
bispecific antibodies by Fab-arm-exchange reaction between
IgG1-01-K409R and IgG1-05-F405L under controlled reducing
conditions as described in WO2011/131746. Without Fab-arm exchange,
human IgG1 antibodies bearing the K409R and F405L mutations 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 IgG1-01 and IgG1-05 antibodies to induce cell death in
DR5-positive tumor cells in vitro. A viability assay was performed
to compare the capacity of the combination of humanized antibodies
IgG1-hDR5-01-K409R-E430G+IgG1-h DR5-05-F405L-E430G with the
capacity of the combination of humanized antibodies
IgG1-hDR5-01-E430G+IgG1-hDR5-05-E430G to induce killing of BxPC-3
pancreatic cancer cells. The viability assay on The BxPC-3 was
perfomed as described in Example 11 with a serial diluted antibody
series ranging from 0.001 to 20,000 ng/mL final concentrations in
4-fold dilutions. The BxPC-3 pancreatic cancer cell line showed
similar viability curves after incubation with the combination of
the humanized antibodies
IgG1-hDR5-01-K409R-E430G+IgG1-hDR5-05-F405L-E430G as with the
combination of the humanized antibodies
IgG1-hDR5-01-E430G+IgG1-hDR5-05-E430G (FIG. 21). These data
indicate that the K409R and F405L mutations had no effect on the
potency of the combination of the humanized DR5-01 and DR5-05
antibodies with E430G hexamerization enhancing mutation.
Example 23
Chimeric Bispecific Antibody
IgG1-DR5-01-K409R-E430G.times.DR5-05-F405L-E430G Induces Killing of
DR5-Positive Tumor Cells
[0510] A bispecific antibody targeting two different DR5 epitopes
was generated by Fab-arm exchange between the chimeric antibodies
IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G as described in
Example 1. A viability assay was performed as described in Example
11 to test the capacity of 10 .mu.g/mL of the chimeric BsAb
IgG1-DR5-01-K409R-E430G.times.DR5-05-F405L-E430G to induce killing
of cancer cells of different tissue origin (COLO 205 colorectal
cancer, A375 skin cancer, SK-MES-1 lung cancer, BxPC-3 pancreatic
cancer and SNU-5 gastric cancer cell lines). For all tested cell
lines, the percentage viable cells was significantly lower when
incubated with 10 .mu.g/mL of the chimeric BsAb
IgG1-DR5-01-K409R-E430G.times.DR5-05-F405L-E430G antibody compared
to the non-target binding negative control antibody IgG1-b12 (FIG.
22). These data indicate that the bispecific anti-DR5.times.DR5'
antibody with hexamerization-enhancing mutation E430G induced
killing of cancer cells of different origin, including colon,
pancreatic, gastric, lung and skin cancer, without the requirement
of a secondary crosslinker.
Example 24
The Potency of the Chimeric BsAb
IgG1-DR5-01-K409R-E430G.times.DR5-05-F4051-E430G is Independent of
the Presence of a Secondary Fc Crosslinker
[0511] A viability assay was performed to compare the potency of
the chimeric BsAb
IgG1-DR5-01-K409R-E430G.times.IgG1-DR5-05-F405L-E430G in the
absence and presence of a secondary antibody crosslinker to induce
killing of BxPC-3 pancreatic and COLO 205 colon cancer cells as
described in Example 21. For comparison, two DR5 antibodies that
are known to show enhanced killing in the presence of a secondary
antibody crosslinker, IgG1-CONA and IgG1-chTRA8-F405L, were tested
in the same setting. The chimeric BsAb
IgG1-DR5-01-K409R-E430G.times.DR5-05-F405L-E430G showed significant
killing compared to the negative control of COLO 205 and BxPC-3
cancer cells, both in presence or absence of an Fc crosslinker
(FIG. 23). In contrast, DR5 antibodies IgG1-DR5-CONA and
IgG1-DR5-chTRA8-F405L only induced killing in the presence of Fc
crosslinker
Example 25
Cell Death Induction upon Binding of the BsAb
IgG1-DR5-01-K409R-E430G.times.DR5-05-F405L-E430G on COLO 205 Colon
Cancer Cells, as Assessed by Annexin V/Propidium Iodide and Active
Caspase-3 Staining
[0512] The kinetics of cell death induction by 1 .mu.g/mL BsAb
IgG1-DR5-01-K409R-E430G.times.DR5-05-F405L-E430G on COLO 205 cells
was analyzed by Annexin V/Propidium Iodide (PI) double staining and
active caspase-3 staining as described in Example 19.
[0513] FIG. 24 shows that, after 5 hours of incubation, BsAb
IgG1-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 (A) and Active
Caspase-3-positive cells (B), compared to the negative control
antibody IgG1-b12. The percentage of Annexin V-positive/Pl-negative
and Active Caspase-3 positive cells was higher in cells that had
been treated with BsAb
IgG1-DR5-01-K409R-E430G.times.DR5-05-F405L-E430G compared to the
bispecific antibody without the E430G mutation (BsAb
IgG1-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 (C). After 24 hours incubation, the percentage of Annexin
V/PI double-positive cells (D) was enhanced in samples treated with
BsAb IgG1-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
IgG1-DR5-01-K409R-E430G.times.DR5-05-F405L-E430G was stronger
(larger increase in the percentage of Annexin V/PI double-positive
cells (E) than in samples treated with the bispecific antibody
without the E430G mutation (BsAb
IgG1-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
IgG1-DR5-01-K409R-E430G.times.DR5-05-F405L-E430G.
[0514] These data indicate that BsAB
IgG1-DR5-01-K409R-E430G.times.DR5-05-F405L-E430G induces both the
early and late stages of cell death in COLO 205 colon cancer cells,
and does so more effectively than the bispecific antibody without
the E430G hexamerization enhancing mutation.
Example 26
In Vivo Efficacy of DR5-01 and DR5-05 Antibody Variants with and
without a Hexamerization-Enhancing Mutation in a Subcutaneous COLO
205 Colon Cancer Xenograft Model
[0515] The in vivo anti-tumor efficacy of different anti-DR5
antibodies and the combination of DR5-01+DR5-05 antibodies with
hexamerization enhancing mutation was evaluated 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-17/1crHan.RTM.Hsd-Prkdcsc.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 mm.sup.3 and the mice were sorted into
groups with equal tumor size variance (Table 2 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. Three
individual mice had no detectable human IgG plasma level and were
excluded from statistical analysis (see Table 2 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.
TABLE-US-00005 TABLE 2 Treatment groups and dosing Dosing day after
# # Total antibody tumor mice analyzed Antibody dose inoculation 8
7 IgG1-DR5-01-K409R-E430G (50 .mu.g) 100 .mu.g (5 mg/kg) 6, 13
IgG1-DR5-05-F405L-E430G (50 .mu.g) 7 7 IgG1-DR5-05-F405L (100
.mu.g) 100 .mu.g (5 mg/kg) 6, 13 8 8 IgG1-DR5-01-K409R-E430G (100
.mu.g) 100 .mu.g (5 mg/kg) 6, 13 8 8 IgG1-DR5-05-F405L-E430G (100
.mu.g) 100 .mu.g (5 mg/kg) 6, 13 8 8 IgG1-CONA (100 .mu.g) 100
.mu.g (5 mg/kg) 6, 13 8 7 BsAb DR5-01-K409R .times. DR5-05-F405L
100 .mu.g (5 mg/kg) 6, 13 (100 .mu.g) 8 8 BsAb DR5-01-K409R-E430G
.times. DR5-05- 100 .mu.g (5 mg/kg) 6, 13 F405L-E430G (100 .mu.g) 8
7 IgG1-b12 (100 .mu.g) 100 .mu.g (5 mg/kg) 6, 13
[0516] FIG. 25A shows mean tumor volumes per treatment group in
time. FIG. 25B represents mean tumor volumes on day 23 after tumor
inoculation, when all groups were still intact. All anti-DR5
antibody samples inhibited tumor growth significantly compared to
the negative control antibody IgG1-b12 (non-parametric ANOVA
analysis (Kruskal-Wallis) followed by Dunn's multiple comparison
test on day 23: p<0.0001). Complete tumor abrogation was
observed for the combination of DR5-01+DR5-05 antibodies with hexa
merization-enhancing mutation
(IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G), for the
bispecific antibodies with and without hexamerization-enhancing
mutation (BsAb DR5-01-K409R.times.DR5-05-F405L and BsAb
DR5-01-K409R-E430G.times.DR5-05-F405L-E430G), and for the anti-DR5
antibodies with hexamerization-enhancing mutation
(IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G). IgG1-CONA
and IgG1-DR5-05-F405L without hexamerization-enhancing mutation
strongly inhibited tumor growth compared to IgG1-b12, but did not
result in complete tumor abrogation.
[0517] FIG. 25C shows a Kaplan-Meier plot of tumor progression,
with a cutoff set at a tumor volume >750 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 750
mm.sup.3: p<0.001). At the end of the study (day 112), the group
of mice treated with the combination of DR5-01+DR5-05 antibodies
with hexamerization enhancing mutation
(IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G) showed
significant less mice with tumor outgrowth than the conatumumab
group (Fisher's exact contingency test p<0.01).
[0518] 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. Both DR5-01 and DR5-05
antibodies with hexamerization enhancing mutation
(IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G), the
bispecific antibodies with and without hexamerization enhancing
mutation (BsAb DR5-01-K409R.times.DR5-05-F405L and BsAb
DR5-01-K409R-E430G.times.DR5-05-F405L-E430G) and the combination of
antibodies with hexamerization-enhancing mutation
(IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G) showed better
tumor inhibition as IgG1-CONA and IgG1-DR5-05-F405L without
hexamerization-enhancing mutation.
Example 27
In Vivo Efficacy of Different Doses of the Antibody combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F4051-E430G in a Subcutaneous
COLO 205 Colon Cancer Xenograft Model
[0519] The in vivo anti-tumor efficacy of different doses
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was evaluated and
compared to an equivalent dosing of IgG1-CONA in the subcutaneous
COLO 205 human colon cancer xenograft model. Tumor cell
inoculation, mice handling, tumor outgrowth measurements and
endpoint determination were performed as described in Example 26.
At day 10, the average tumor volume was .about.400 mm.sup.3 and the
mice were sorted into groups with equal tumor size variance (Table
3 below). Mice were treated by intravenous (i.v.) injection of 40
.mu.g (2 mg/kg), 10 .mu.g (0.5 mg/kg) or 2 .mu.g (0.1 mg/kg)
antibody in 100 .mu.l PBS on day 10. Mice in the control group were
treated with 40 .mu.g (2 mg/kg) IgG1-b12. Tumors were measured
until 17 weeks after tumor inoculation.
TABLE-US-00006 TABLE 3 Treatment groups and dosing Dosing day Total
after # antibody tumor mice Antibody dose inoculation 8
IgG1-DR5-01-K409R-E430G (20 .mu.g) 40 .mu.g 10
IgG1-DR5-05-F405L-E430G (20 .mu.g) (2 mg/kg) 8
IgG1-DR5-01-K409R-E430G (5 .mu.g) 10 .mu.g 10
IgG1-DR5-05-F405L-E430G (5 .mu.g) (0.5 mg/kg) 8
IgG1-DR5-01-K409R-E430G (1 .mu.g) 2 .mu.g 10
IgG1-DR5-05-F405L-E430G (1 .mu.g) (0.1 mg/kg) 8 IgG1-CONA (40
.mu.g) 40 .mu.g 10 (2 mg/kg) 8 IgG1-CONA (10 .mu.g) 10 .mu.g 10
(0.5 mg/kg) 8 IgG1-CONA (0.1 .mu.g) 2 .mu.g 10 (0.1 mg/kg) 8
IgG1-b12 (40 .mu.g) 40 .mu.g 10 (2 mg/kg)
[0520] FIG. 26A shows mean tumor volumes per treatment group.
Treatment with a single dose of 0.5 mg/kg or 2 mg/kg of the
antibody combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G resulted in
complete tumor regression until the study was stopped on day 126.
Treatment with 0.5 mg/kg and 2 mg/kg IgG1-CONA also induced tumor
regression, but the regression was incomplete with recurring tumor
outgrowth in all mice or almost all (7/8) mice, respectively. At
0.1 mg/kg, neither IgG1-CONA nor the combination of
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G showed anti-tumor
activity. FIG. 26B shows that on day 16 after tumor inoculation,
tumor inhibition by 2 mg/kg and 0.5 mg/kg
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was significantly
better compared to an equivalent dose IgG1-CONA (unpaired
t-test).
[0521] FIG. 26C shows a Kaplan-Meier plot of tumor progression,
with a cutoff set at a tumor volume >500 mm.sup.3. At a dose of
0.5 mg/kg and 2 mg/kg, the combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G and IgG1-CONA
significantly inhibited tumor growth progression compared to the
negative control antibody IgG1-b12 (p<0.001 Mantel-Cox analysis
at tumor size cut-off 500 mm.sup.3). At a dose of 0.5 mg/kg
inhibition of tumor growth progression was significantly better for
the combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G to
an equivalent dose IgG1-CONA.
[0522] These data indicate that the combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G had stronger
anti-tumor efficacy compared to IgG1-CONA, since dosed at 2 mg/kg
the combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G
significantly reduced tumor load at day 16 compared to IgG1-CONA,
and at 0.5 mg/kg the
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G combination
significantly reduced tumor load at day 16 and prolonged
progression free survival time (tumor size cut-off 500 mm.sup.3)
compared to IgG1-CONA.
Example 28
In Vivo Efficacy of Different Doses of the Antibody Combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F4051-E430G in a Subcutaneous
BxPC-3 Pancreatic Cancer Xenograft Model
[0523] The in vivo anti-tumor efficacy of different doses
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was evaluated and
compared to an equivalent dosing of IgG1-CONA-F405L in the
subcutaneous BxPC-3 human pancreatic cancer xenograft model. At day
0, adherent cells were harvested by trypsinization.
5.times.10.sup.6 cells were injected in a volume of 100 .mu.L PBS
into the flank of 6-11 weeks old female SCID mice
(C.B-17/1crHan.RTM.Hsd-Prkdc.sup.scid; Harlan). Mice handling,
tumor outgrowth measurements and endpoint determination were
performed as described in Example 26. At day 10, the average tumor
volume was .about.250 mm.sup.3 and the mice were sorted into groups
with equal tumor size variance (Table 4 below). Mice were treated
by i.v. injection of 200 .mu.g (10 mg/kg), 40 .mu.g (2 mg/kg) or 10
.mu.g (0.5 mg/kg) antibody in 200 .mu.L PBS on day 20 and 28. Mice
in the control group were treated with 200 .mu.g (10 mg/kg)
IgG1-b12. To check for correct antibody administration, blood
samples were obtained for IgG serum determination one week after
dosing. Tumors were measured until 10 weeks after tumor
inoculation.
TABLE-US-00007 TABLE 4 Treatment groups and dosing Dosing day Total
after # antibody tumor mice Antibody per dose inoculation 8
IgG1-DR5-01-K409R-E430G (20 .mu.g) 200 .mu.g (10 20, 28
IgG1-DR5-05-F405L-E430G (20 .mu.g) mg/kg) 8 IgG1-DR5-01-K409R-E430G
(5 .mu.g) 40 .mu.g (2 20, 28 IgG1-DR5-05-F405L-E430G (5 .mu.g)
mg/kg) 8 IgG1-DR5-01-K409R-E430G (1 .mu.g) 10 .mu.g (0.5 20, 28
IgG1-DR5-05-F405L-E430G (1 .mu.g) mg/kg) 8 IgG1-CONA-F405L (40
.mu.g) 200 .mu.g (10 20, 28 mg/kg) 8 IgG1-CONA-F405L (10 .mu.g) 40
.mu.g (2 20, 28 mg/kg) 8 IgG1-CONA-F405L (0.1 .mu.g) 10 .mu.g (0.5
20, 28 mg/kg) 8 IgG1-b12 (40 .mu.g) 200 .mu.g (10 20, 28 mg/kg)
[0524] FIG. 27A shows median tumor volumes per treatment group. All
tested doses of the antibody combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor
growth compared to the negative control antibody IgG1-b12, whereas
the IgG1-CONA-F405L treatment groups did not. FIG. 27B shows that
on day 48 after tumor inoculation, tumor growth inhibition by the
combination of IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was
significantly better than equivalent doses IgG1-CONA-F405L
(unpaired t-test, p<0.05).
[0525] FIG. 27C shows a Kaplan-Meier plot of tumor progression,
with a cutoff set at a tumor volume >500 mm.sup.3. The
combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G
significantly inhibited tumor growth progression compared to the
negative control antibody IgG1-b12 and compared to IgG1-CONA-F405L
(Mantel-Cox analysis at tumor size cutoff 500 mm.sup.3:
p<0.001).
[0526] These data indicate that the combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor
growth at different doses (0.5 mg/kg, 2 mg/kg and 10 mg/kg) and
that anti-tumor efficacy was significantly better than for
equivalent doses of IgG1-CONA-F405L in an in vivo BxPC-3 human
pancreatic cancer xenograft model.
Example 29
In Vivo Efficacy of Different Doses of the Antibody Combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G in a Subcutaneous
A375 Skin Cancer Xenograft Model
[0527] The in vivo anti-tumor efficacy of different doses
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was evaluated and
compared to an equivalent dosing of IgG1-CONA-F405L in the
subcutaneous A375 human skin cancer xenograft model. At day 0,
adherent cells were harvested by trypsinization. 5.times.10.sup.6
cells were injected in a volume of 100 .mu.L PBS into the flank of
6-11 weeks old female SCID mice
(C.B-17/1crHan.RTM.Hsd-Prkdcsc.sup.scid; Harlan). Mice handling,
tumor outgrowth measurements and endpoint determination were
performed as described in Example 26. At day 19, the average tumor
volume was .about.250 mm.sup.3 and the mice were sorted into groups
with equal tumor size variance (Table 5 below). Mice were treated
by i.v. injection of 200 .mu.g (10 mg/kg), 40 .mu.g (2 mg/kg) or 10
.mu.g (0.5 mg/kg) antibody in 200 .mu.L PBS on day 19 and 26. Mice
in the control group were treated with 200 .mu.g (10 mg/kg)
IgG1-b12. To check for correct antibody administration, blood
samples were obtained for IgG serum determination one week after
dosing. Tumor volumes were analyzed until 7 weeks after tumor
inoculation.
TABLE-US-00008 TABLE 5 Treatment groups and dosing Dosing day Total
after # antibody tumor mice Antibody dose inoculation 8
IgG1-DR5-01-K409R-E430G (20 .mu.g) 200 .mu.g (10 19, 26
IgG1-DR5-05-F405L-E430G (20 .mu.g) mg/kg) 8 IgG1-DR5-01-K409R-E430G
(5 .mu.g) 40 .mu.g (2 19, 26 IgG1-DR5-05-F405L-E430G (5 .mu.g)
mg/kg) 8 IgG1-DR5-01-K409R-E430G (1 .mu.g) 10 .mu.g (0.5 19, 26
IgG1-DR5-05-F405L-E430G (1 .mu.g) mg/kg) 8 IgG1-CONA-F405L (40
.mu.g) 200 .mu.g (10 19, 26 mg/kg) 8 IgG1-CONA-F405L (10 .mu.g) 40
.mu.g (2 19, 26 mg/kg) 8 IgG1-CONA-F405L (0.1 .mu.g) 10 .mu.g (0.5
19, 26 mg/kg) 8 IgG1-b12 (40 .mu.g) 200 .mu.g (10 19, 26 mg/kg)
[0528] FIG. 28A shows median tumor volumes per treatment group. All
tested doses of the antibody combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor
growth compared to the negative control antibody IgG1-b12, whereas
the IgG1-CONA-F405L treatment groups did not. FIG. 28B shows that
on day 29 after tumor inoculation, the average tumor size in mice
treated with the combination of
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was smaller than in
mice treated with IgG1-b12 (p<0.05 for all dose levels, One-Way
ANOVA with Dunnet's correction for multiple comparisons), and that
the combination of IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G
was significantly more potent than IgG1-CONA-F405L (Mann Whitney
test, p<0.05) at equivalent doses
[0529] These data indicate that the combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor
growth at different doses (0.5 mg/kg, 2 mg/kg and 10 mg/kg) and
that anti-tumor efficacy was significantly better than for
equivalent doses of IgG1-CONA-F405L in an in vivo A375 human skin
cancer xenograft model.
Example 30
In Vivo Efficacy of Different Doses of the Antibody Combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G in a Subcutaneous
HCT-15 Colon Cancer Xenograft Model
[0530] The in vivo anti-tumor efficacy of different doses
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was evaluated and
compared to an equivalent dosing of IgG1-CONA in the subcutaneous
HCT-15 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 6-8 weeks old female BALB/c nude mice
(Shanghai Laboratory Animal Center). 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.b2 where a and b are the
long and short diameters of the tumor, respectively. Eleven days
after tumor inoculation, the mean tumor size reached 186 mm.sup.3
and mice were assigned into groups using randomized block design
and treatments were started. Mice were treated twice according to a
07D regimen by i.v. injection of 200 .mu.g (10 mg/kg), 40 .mu.g (2
mg/kg) or 10 .mu.g (0.5 mg/kg) antibody in 10 .mu.L PBS per g body
weight. Mice in the control group were treated in parallel with 200
.mu.g (10 mg/kg) IgG1-b12. After tumor inoculation, welfare of the
animals was checked daily and tumor volumes were measured twice
weekly.
TABLE-US-00009 TABLE 6 Treatment groups and dosing, Example 30
Dosing day Total after # # antibody tumor mice analyzed Antibody
dose inoculation 8 8 IgG1-DR5-01-K409R-E430G (20 .mu.g) 200 .mu.g
(10 11, 18 IgG1-DR5-05-F405L-E430G (20 .mu.g) mg/kg) 8 8
IgG1-DR5-01-K409R-E430G (5 .mu.g) 40 .mu.g (2 11, 18
IgG1-DR5-05-F405L-E430G (5 .mu.g) mg/kg) 8 8
IgG1-DR5-01-K409R-E430G (1 .mu.g) 10 .mu.g (0.5 11, 18
IgG1-DR5-05-F405L-E430G (1 .mu.g) mg/kg) 8 8 IgG1-CONA (40 .mu.g)
200 .mu.g (10 11, 18 mg/kg) 8 8 IgG1-CONA (10 .mu.g) 40 .mu.g (2
11, 18 mg/kg) 8 8 IgG1-CONA (0.1 .mu.g) 10 .mu.g (0.5 11, 18 mg/kg)
8 8 IgG1-b12 (40 .mu.g) 200 .mu.g (10 11, 18 mg/kg)
[0531] FIG. 29A shows mean tumor volumes per treatment group. All
tested doses of the antibody combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor
growth compared to the negative control antibody IgG1-b12, whereas
IgG1-CONA did not. FIG. 29B shows that on day 17 after start of
treatment, tumor growth inhibition by the combination of
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was significantly
better than equivalent doses IgG1-CONA (Unpaired t test,
p<0.05).
[0532] FIG. 29C shows a Kaplan-Meier plot of tumor progression,
with a cutoff set at a tumor volume >500 mm.sup.3. The
combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G
significantly inhibited tumor growth progression compared to
negative control antibody IgG1-b12 and compared to an equivalent
dose IgG1-CONA (Mantel-Cox analysis at tumor size cutoff 500
mm.sup.3: p<0.001).
[0533] These data indicate that the combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor
growth at different doses (0.5 mg/kg, 2 mg/kg and 10 mg/kg) and
that anti-tumor efficacy was significantly better than for
equivalent doses of IgG1-CONA in an in vivo xenograft model with
HCT-15 human colon cancer cells.
Example 31
In Vivo Efficacy of Different Doses of the Antibody Combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G in a subcutaneous
SW480 Colon Cancer Xenograft Model
[0534] The in vivo anti-tumor efficacy of different doses
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was evaluated and
compared to an equivalent dosing of IgG1-CONA in the subcutaneous
SW480 human colon cancer xenograft model at CrownBiosciences,
Taicang, China. The cells were maintained in vitro as a monolayer
culture in L-15 medium supplemented with 10% fetal bovine serum at
37.degree. C. in 100% air. Adherent cells in an exponential growth
phase were harvested by trypsin-EDTA treatment. 1.times.10.sup.7
cells were injected in a volume of 200 .mu.L PBS with Matrigel
(1:1) into the flank of 6-8 weeks old female NOD/SCID mice (Beijing
HFK Bioscience). Mouse handling and tumor volume measurements were
performed as described in Example 30. Ten days after tumor
inoculation, the mean tumor size reached 175 mm.sup.3 and mice were
assigned into groups using randomized block design and treatments
were started. Mice were treated twice according to a Q7D regimen by
i.v. injection of 200 .mu.g (10 mg/kg), 40 .mu.g (2 mg/kg) or 10
.mu.g (0.5 mg/kg) antibody in 10 .mu.L PBS per g body weight. Mice
in the control group were treated in parallel with 200 .mu.g (10
mg/kg) IgG1-b12. After tumor inoculation, welfare of the animals
was checked daily and tumor volumes were measured twice weekly.
TABLE-US-00010 TABLE 7 Treatment groups and dosing, Example 31
Dosing day after # # Total antibody tumor mice analyzed Antibody
per dose inoculation 8 8 IgG1-DR5-01-K409R-E430G (20 .mu.g) 200
.mu.g (10 10, 17 IgG1-DR5-05-F405L-E430G (20 .mu.g) mg/kg) 8 8
IgG1-DR5-01-K409R-E430G (5 .mu.g) 40 .mu.g (2 mg/kg) 10, 17
IgG1-DR5-05-F405L-E430G (5 .mu.g) 8 8 IgG1-DR5-01-K409R-E430G (1
.mu.g) 10 .mu.g (0.5 mg/kg) 10, 17 IgG1-DR5-05-F405L-E430G (1
.mu.g) 8 8 IgG1-CONA (40 .mu.g) 200 .mu.g (10 10, 17 mg/kg) 8 8
IgG1-CONA (10 .mu.g) 40 .mu.g (2 mg/kg) 10, 17 8 8 IgG1-CONA (0.1
.mu.g) 10 .mu.g (0.5 mg/kg) 10, 17 8 8 IgG1-b12 (40 .mu.g) 200
.mu.g (10 10, 17 mg/kg)
[0535] FIG. 30A shows mean tumor volumes per treatment group. All
tested doses of the antibody combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor
growth compared to the negative control antibody IgG1-b12 (10 mg/kg
p<0.0001; 2 mg/kg p<0.001; 0.5 mg/kg p<0.05). The
IgG1-CONA treatment groups were only better than IgG1-b12 at the
highest doses (10 mg/kg and 2 mg/kg p<0.01), but not at 0.5
mg/kg. FIG. 30B shows that on day 28 after start treatment, tumor
growth inhibition by the combination of
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was significantly
better than equivalent doses IgG1-CONA at 10 mg/kg and 0.5 mg/kg
(Unpaired t test, p<0.05).
[0536] FIG. 30C shows a Kaplan-Meier plot of tumor progression,
with a cutoff set at a tumor volume >500 mm.sup.3. The
combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G dosed
at 10 mg/kg significantly inhibited tumor growth progression
compared to negative control antibody IgG1-b12 and compared to an
equivalent dose IgG1-CONA (Mantel-Cox analysis at tumor size cutoff
500 mm.sup.3: p<0.001.
[0537] These data indicate that the combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor
growth at different doses (0.5 mg/kg, 2 mg/kg and 10 mg/kg) and
that anti-tumor efficacy for doses of 10 mg/kg and 0.5 mg/kg was
significantly better than for equivalent doses of IgG1-CONA in an
in vivo SW480 human colon cancer xenograft model.
Example 32
In Vivo Efficacy of Different Doses of the Antibody combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F4051-E430G in a Subcutaneous
SNU-5 Gastric Cancer Xenograft Model
[0538] The in vivo anti-tumor efficacy of different doses
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G were evaluated and
compared to an equivalent dosing of IgG1-CONA in the subcutaneous
SNU-5 human gastric cancer xenograft model at CrownBiosciences,
Taicang, China. The cells were maintained in vitro as a suspension
culture in IMDM medium supplemented with 20% fetal bovine serum at
37.degree. C. in an atmosphere of 5% CO2 in air. Cells in an
exponential growth phase were harvested and 1.times.10.sup.7 cells
were injected in a volume of 200 .mu.L PBS with Matrigel (1:1) into
the flank of 6-8 weeks old female CB17/SCIDmice (Beijing HFK
Bioscience). Mouse handling and tumor volume measurements were
performed as described in Example 30. Eight days after tumor
inoculation, the mean tumor size reached 169 mm.sup.3 and mice were
assigned into groups using randomized block design and treatments
were started. Mice were treated twice according to a Q7D regimen by
i.v. injection of 200 .mu.g (10 mg/kg), 40 .mu.g (2 mg/kg) or 10
.mu.g (0.5 mg/kg) antibody in 10 .mu.L PBS per g body weight. Mice
in the control group were treated in parallel with 200 .mu.g (10
mg/kg) IgG1-b12. After tumor inoculation, welfare of the animals
was checked daily and tumor volumes were measured twice weekly.
TABLE-US-00011 TABLE 8 Treatment groups and dosing, Example 32
Dosing day after # # Total antibody tumor mice analyzed Antibody
per dose inoculation 8 8 IgG1-DR5-01-K409R-E430G (20 .mu.g 200
.mu.g (10 8, 15 IgG1-DR5-05-F405L-E430G (20 .mu.g mg/kg) 8 8
IgG1-DR5-01-K409R-E430G (5 .mu.g 40 .mu.g (2 mg/kg) 8, 15
IgG1-DR5-05-F405L-E430G (5 .mu.g 8 8 IgG1-DR5-01-K409R-E430G (1
.mu.g 10 .mu.g (0.5 mg/kg) 8, 15 IgG1-DR5-05-F405L-E430G (1 .mu.g 8
8 IgG1-CONA (40 .mu.g 200 .mu.g (10 8, 15 mg/kg) 8 8 IgG1-CONA (10
.mu.g 40 .mu.g (2 mg/kg) 8, 15 8 8 IgG1-CONA (0.1 .mu.g 10 .mu.g
(0.5 mg/kg) 8, 15 8 8 IgG1-b12 (40 .mu.g 200 .mu.g (10 8, 15
mg/kg)
[0539] FIG. 31A shows mean tumor volumes per treatment group. All
tested doses of the antibody combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor
growth compared to the negative control antibody IgG1-b12. At the 2
mg/kg and 10 mg/kg doses, the antibody combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G resulted in
complete tumor regression that lasted over the complete study time
(7 weeks after start treatment). FIG. 31B shows that on day 23
after start treatment, tumor growth inhibition by the combination
of IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was significant
better than equivalent doses IgG1-CONA (Mann Whitney test,
p<0.05).
[0540] FIG. 31C shows a Kaplan-Meier plot of tumor progression,
with a cutoff set at a tumor volume >500 mm.sup.3. The
combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G
significantly inhibited tumor growth progression compared to
negative control antibody IgG1-b12 and compared to an equivalent
dose IgG1-CONA (Mantel-Cox analysis at tumor size cutoff 500
mm.sup.3: p<0.05).
[0541] These data indicate that the combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor
growth at different doses (0.5 mg/kg, 2 mg/kg and 10 mg/kg) and
that anti-tumor efficacy was significantly better than for
equivalent doses of IgG1-CONA in an in vivo SNU-5 human gastric
cancer xenograft model.
Example 33
In Vivo Efficacy of Different Doses of the Antibody Combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G in a Subcutaneous
SK-MES-1 Lung Cancer Xenograft Model
[0542] The in vivo anti-tumor efficacy of different doses
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was evaluated and
compared to an equivalent dosing of IgG1-CONA in the subcutaneous
SK-MES-1 human lung cancer xenograft model at CrownBiosciences,
Taicang, China. The cells were maintained in vitro as a monolayer
culture in MEM medium supplemented with 10% fetal bovine serum and
0.01 mM NEAA at 37.degree. C. in an atmosphere of 5% CO2 in air. At
day 0, 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 6-8 weeks old female
BALB/c mice (Shanghai Laboratory Animal Center). Mouse handling and
tumor volume measurements were performed as described in Example
30. Twenty-one days after tumor inoculation, the mean tumor size
reached 161 mm.sup.3 and mice were assigned into groups using
randomized block design and treatments were started. Mice were
treated twice according to a Q7D regimen by i.v. injection of 200
.mu.g (10 mg/kg), 40 .mu.g (2 mg/kg) or 10 .mu.g (0.5 mg/kg)
antibody in 10 .mu.L PBS per g body weight. Mice in the control
group were treated in parallel with 200 .mu.g (10 mg/kg) IgG1-b12.
After tumor inoculation, welfare of the animals was checked daily
and tumor volumes were measured twice weekly.
TABLE-US-00012 TABLE 9 Treatment groups and dosing, Example 33
Dosing day Total after # antibody tumor mice Antibody per dose
inoculation 8 IgG1-DR5-01-K409R-E430G (20 .mu.g) 200 .mu.g (10 21,
28 IgG1-DR5-05-F405L-E430G (20 .mu.g) mg/kg) 8
IgG1-DR5-01-K409R-E430G (5 .mu.g) 40 .mu.g (2 21, 28
IgG1-DR5-05-F405L-E430G (5 .mu.g) mg/kg) 8 IgG1-DR5-01-K409R-E430G
(1 .mu.g) 10 .mu.g (0.5 21, 28 IgG1-DR5-05-F405L-E430G (1 .mu.g)
mg/kg) 8 IgG1-CONA (40 .mu.g) 200 .mu.g (10 21, 28 mg/kg) 8
IgG1-CONA (10 .mu.g) 40 .mu.g (2 21, 28 mg/kg) 8 IgG1-CONA (0.1
.mu.g) 10 .mu.g (0.5 21, 28 mg/kg) 8 IgG1-b12 (40 .mu.g) 200 .mu.g
(10 21, 28 mg/kg)
[0543] FIG. 32A shows mean tumor volumes per treatment group. All
tested doses of the antibody combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor
growth significantly compared to the negative control antibody
IgG1-b12 (p<0.0001), whereas IgG1-CONA only had a significant
effect compared to IgG1-b12 at 10 mg/kg (p<0.01) and 2 mg/kg
(p<0.05) but not at 0.5 mg/kg (one-way ANOVA followed by
Dunnett's multiple comparisons test). FIG. 32B shows that on day 14
after start treatment, tumor growth inhibition by the combination
of IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was significant
better than equivalent doses IgG1-CONA at 2 mg/kg and 0.5 mg/kg
(unpaired t-test test, p<0.05 and p<0.01, respectively).
[0544] FIG. 32C shows a Kaplan-Meier plot of tumor progression,
with a cutoff set at a tumor volume >1.000 mm.sup.3. The
combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G
significantly inhibited tumor growth progression compared to
negative control antibody IgG1-b12 (Mantel-Cox analysis at tumor
size cutoff 1.000 mm.sup.3: p.ltoreq.0.001) and compared to an
equivalent dose IgG1-CONA at 2 mg/kg and 0.5 mg/kg (Mantel-Cox
analysis at tumor size cutoff 1.000 mm.sup.3: p<0.05).
[0545] These data indicate that the combination
IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor
growth at different doses (0.5 mg/kg, 2 mg/kg and 10 mg/kg) and
that anti-tumor efficacy was significantly better than for
equivalent doses of IgG1-CONA at 0.5 mg/kg and 2 mg/kg in an in
vivo SK-MES-1 human lung cancer xenograft model.
Example 34
DR5 Expression Levels on Different Human Cancer Cell Lines
[0546] DR5 density per cell was quantified for different human
cancer cell lines by indirect immunofluorescence using QIFIKIT with
mouse monoclonal antibody B-K29 as described in Example 2. The cell
lines were categorized according to low DR5 expression
(ABC<10,000) and moderate DR5 expression (ABC>10,000). The
human cancer cell lines SK-MEL-5 (ATCC, HTB-070) malignant
melanoma, Jurkat (ATCC, TIB-152) acute T cell leukemia and Daudi
(ATCC, CCL-231) Burkitt's lymphoma were found to have low DR5
expression (QIFIKIT ABC range 3,500-6,500). The human colorectal
carcinoma cell lines SNU-C2B (ATCC, CCL-250), LS411N (ATCC,
CRL-2159) and DLD-1 (ATCC, CCL-221) were found to have moderate DR5
expression (QIFIKIT ABC range 12,000-44,500).
Example 35
Introduction of a Hexamerization-Enhancing Mutation does not Affect
Binding of IgG1-hDR5-01-G56T and IgG1-hDR5-05 Antibodies to
DR5-Positive Human Colon Cancer Cells
[0547] Binding to human colon cancer cells HCT 116 was analyzed by
flow cytometry for purified antibody variants of IgG1-hDR5-01-G56T
and IgG1-hDR5-05 with and without the E430G mutation. Single cell
suspensions were prepared and binding was analyzed for serial
dilution antibody preparation series (range 0.0006 to 10 .mu.g/mL
final concentrations in 4-fold dilutions) as described in Example
3. After incubation with the secondary antibody, cells were washed
twice, resuspended in 100 .mu.L FACS buffer, and antibody binding
was analyzed on a BD LRSFFortessa cell analyzer (BD Biosciences).
Binding curves were analyzed using non-linear regression analysis
(sigmoidal dose-response with variable slope) using GraphPad Prism
software.
[0548] FIG. 33 shows that the antibodies IgG1-hDR5-01-G56T-E430G
and IgG1-hDR5-05-E430G showed similar dose-dependent binding to HCT
116 cells as their corresponding antibodies without the E430G
mutation. Introduction of the E430G mutation had no effect on the
binding of the DR5 antibodies. The EC50 values were calculated from
six repeat experiments as 74.4 (+/- 58.4) ng/mL for
IgG1-hDR5-01-G56T-E430G and 101.2 (+/- 52.6) ng/mL for
IgG1-hDR5-05-E430G.
Example 36
Binding of IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G as Single
Antibodies and as a Combination to DR5-Positive Human Cancer
Cells
[0549] Antibody binding to HCT 116 human cancer cells with moderate
DR5 expression was analyzed by flow cytometry for purified samples
of Alexa 647-labeled IgG1-hDR5-01-G56T-E430G and Alexa 647-labeled
IgG1-hDR5-05-E430G, both as single agents and as a combination of
the two antibodies. 1 mg/mL IgG1-hDR5-01-G56T-E430G and
IgG1-hDR5-05-E430G were labeled for 1 hour at room temperature with
a 5 molar excess of Alexa Fluor.RTM. 647 carboxylic acid,
succinimidyl ester (Molecular Probes; Cat #A-20006) in 0.1 M
NaHCO.sub.3 conjugation buffer to reach a degree of labeling of
three. Free excess Alexa 647 was removed on a PD 10 Column
(Amersham Bioscience, Cat #17-0851-01). Single cell suspensions
were prepared and binding was analyzed for serial dilution antibody
preparation series (range 0.0019 to 30 .mu.g/mL final
concentrations in 5-fold dilutions) as described in Example 3.
After antibody incubation, cells were washed twice, resuspended in
1004 FACS buffer, and antibody binding was analyzed on a BD
LRSFFortessa cell analyzer (BD Biosciences). Binding curves were
analyzed using non-linear regression analysis (sigmoidal
dose-response with variable slope) using GraphPad Prism
software.
[0550] FIG. 34 shows that both the single antibodies and the
combination of the non-crossblocking antibodies
IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G showed
dose-dependent binding on HCT 116 human cancer cells.
Example 37
Binding of Antibodies IgG1-hDR5-01-G56T-E430G and
IgG1-hDR5-05-E430G to Cynomolgus Monkey DR5
[0551] Binding of purified IgG1-hDR5-01-G56T-E430G and
IgG1-hDR5-05-E430G to CHO cells expressing the isoform short of
human DR5 or cynomolgus monkey DR5 was analyzed by flow cytometry.
Codon-optimized constructs for expression of the isoform short
human DR5 protein with death domain loss-of-function mutation K386N
(SEQ ID NO 47 based on Uniprot number O14763-2) and cynomolgus
monkey DR5 protein with deletion of amino acids 185-213 and death
domain loss-of-function mutation K420N (SEQ ID NO 50; based on NCBI
accession number XP_005562887.1) were generated as described in
Example 1. Binding to DR5-transfected CHO cells was analyzed,
generally as described in Example 5. Transfected cells were stored
in liquid nitrogen and quickly thawed at 37.degree. C. and
suspended in 10 mL medium. Cells were washed with PBS and
resuspended in FACS buffer at a concentration of 1.0.times.10.sup.6
cells/mL. 1004 cell suspension samples (100,000 cells per well)
were seeded in 96-well plates and pelleted by centrifugation at
300.times.g for 3 minutes at 4.degree. C. 25 .mu.L of serial
dilution antibody preparation series (final concentrations 0 to 20
.mu.g/mL in 6-fold dilutions) was added and incubated for 30
minutes at 4.degree. C. Next, cells were washed and incubated with
50 .mu.L secondary antibody R-PE-conjugated goat-anti-human IgG
F(ab').sub.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 50 .mu.L
FACS buffer, and antibody binding was analyzed on a BD LRSFFortessa
cell analyzer (BD Biosciences) by recording 10,000 events. Binding
curves were analyzed using non-linear regression analysis
(sigmoidal dose-response with variable slope) using GraphPad Prism
software.
[0552] FIG. 35 shows that the antibodies IgG1-hDR5-01-G56T-E430G
and IgG1-hDR5-05-E430G showed dose-dependent binding to human and
cynomolgus DR5 expressed on CHO cells. For both
IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G, EC.sub.50 values
for binding to human DR5 and cynomolgus DR5 were in the same range
based on four repeat experiments (Table 10).
TABLE-US-00013 TABLE 10 EC50 values for binding of
IgG1-hDR5-01-G561-E430G and IgG1-hDR5-05-E430G to human and
cynomolgus DR5. Based on four experiments. Human DR5- Cynomolgus
DR5- transfected CHO transfected CHO EC.sub.50 (.mu.g/mL) SD
EC.sub.50 (.mu.g/mL) SD IgG1-hDR5-01- 0.13 0.034 0.27 0.175
G56T-E430G IgG1-hDR5-05- 0.12 0.027 0.17 0.084 E430G
Example 38
Introduction of the E430G Mutation Improves the Efficacy of Cell
Death Induction by the Combination of Non-Crossblocking Antibodies
IgG1-hDR5-01-G56T+IgG1-hDR5-05
[0553] A viability assay was performed to study the effect the
hexamerization-enhancing mutation E430G in IgG1-hDR5-01-G56T and
IgG1-hDR5-05 on the capacity of the antibodies to kill human colon
cancer cells COLO 205. The antibodies with and without the E430G
mutation were tested as single agent and as combinations of the two
non-crossblocking antibodies. COLO 205 cells were harvested as
described in Example 8. 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 allowed to
adhere overnight at 37.degree. C. Subsequently, 50 .mu.L samples of
antibody concentration series (range 0.3-20,000 ng/mL final
concentration 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 (Sigma Aldrich, Cat nr S6942). The
viability of the cell cultures was determined in a CellTiter-Glo
luminescent cell viability assay as described in Example 8.
[0554] FIG. 36 shows that the combination of
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G was more potent than
either antibody alone and more potent than the combination of the
antibodies without the E430G mutation. These data show that
introduction of the hexamerization-enhancing mutation E430G
resulted in enhanced induction of cell killing upon binding of the
combination of the non-crossblocking antibodies
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G to adherent COLO 205
colon cancer cells. In contrast to the experimental setup where
antibodies were directly added when cells were seeded (Example 8),
the single antibodies IgG1-hDR5-01-G56T-E430G and
IgG1-hDR5-05-E430G did not show efficacy on COLO 205 cells in this
experiment where the cells were first allowed to adhere to the
96-wells flat-bottom plate before adding the samples.
Example 39
Introduction of Hexamerization-Enhancing Mutation S440Y Improves
the Efficacy of Anti-DR5 Antibodies to Induce Cell Death on Human
Colon Cancer Cells
[0555] 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 CellTiter-Glo luminescent cell viability assay was performed
as described in Example 8. Briefly, 100 .mu.L single cell
suspensions (5,000 cells per well) were seeded in 96-well plates
and at the same time, 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.
[0556] FIG. 37A shows that in the experimental setup where
antibodies were directly added when cells were seeded, 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. 37B).
Example 40
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
[0557] A crossblock ELISA for antibodies IgG1-DR5-CONA-K409R and
IgG1-DR5-chTRA8-F405L was performed as described in Example 7. The
K409R and F405L mutations are not relevant here and were previously
shown to have no effect on the potency of antibodies with an E430G
mutation (Example 22). FIG. 38A 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-FcHisCtag 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 indicate that IgG1-DR5-CONA-K409R and IgG1-DR5-chTRA8-F405L
did not compete with each other for binding of
DR5ECD-FcHisCtag.
[0558] 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-C49W+IgG1-DR5-chTRA-8 to kill
attached BxPC-3 human pancreatic cancer cells was studied in a
viability assay as described in Example 11. FIG. 38B shows that the
antibody combination IgG1-DR5-CONA-C49W-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 41
Capacity of the Antibody Combination IgG1-hDR5-01-G56T-E430G and
IgG1-hDR5-05-E430G to Induce Target Cell Killing in Different
Cancer Cell Lines
[0559] The efficacy of the combination of the non-crossblocking
antibodies IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G to induce
killing was analyzed on different human cancer cell lines and
compared to the parental antibody combination without the E430G
mutation and TRAIL. A viability assay on 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 was performed, essentially as described in Example 11.
Briefly, 100 .mu.L single cell suspensions (5,000 cells per well)
were seeded in 96-well plates and incubated at 37.degree. C.
overnight. 50 .mu.L of antibody sample (133 nM final concentration)
or human recombinant TRAIL/APO-2L (eBioscience, Cat nr BMS356; 133
nM final concentration) was added and incubated for 3 days at
37.degree. C.
[0560] Both TRAIL and the antibody combination
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G show killing of human
cancer target cell lines originating from different indications
(FIG. 39). Killing of the antibody combination
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G was significant compared
to the control antibody IgG1-b12 in 6 of the 11 tested cell lines.
For these responding cell lines the percentage viable cells was
significantly lower after incubation with the antibody combination
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G than after incubation
with the antibody combination without the E430G mutation. There was
no correlation between killing efficacy of
IgG1-hDR5-01-K409R-E430G+IgG1-hDR5-05-F405L-E430G and DR5 target
expression levels (described in Example 2).
Example 42
Screening of a Human Cancer Cell Line Panel for Cytotoxic Efficacy
of the Antibody Combination
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G
[0561] The activity of the antibody combination
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G was tested and compared
to the activity of TRAIL in a panel of 235 cell lines representing
14 tumor lineages: kidney, neural tissue, colorectal, gastric,
breast cancer (predominantly triple-negative breast cancer (TNBC)),
non-small cell lung cancer (NSCLC), bladder, pancreatic, ovarian,
melanoma, liver, endometrial, head and neck and small cell lung
cancer (SCLC). A 72 hour ATPlite assay (except for DLD-1 and HCT116
cell lines, for which a 120 hour assay was performed) with growth
inhibition analysis was performed in two parts at Horizon Discovery
Ltd, UK. Samples were tested as four replicates in 384-well assay
plates. Serial dilution series of antibody, starting from 0.072
.mu.M final concentration was used for all tested cell lines. For
TRAIL (Invitrogen; Cat # PHC1634) serial dilution series starting
from 0.01 .mu.M final concentration for the cell lines tested in
the first part and 0.17 .mu.M final concentration for the cell
lines tested in the second part of the screening was used.
[0562] Percentage inhibition was calculated using the formulas: If
T.gtoreq.V(0) than percentage inhibition=100*[1-(T-V(0))/(V-V(0))];
If T<V(0) than percentage inhibition=100%, with T=luminescence
of the test sample, V(0)=luminescence of the medium control sample
on day 0 and V=luminescence of the medium control sample on day 3.
Responder and non-responder cell lines were grouped by a maximum
response threshold value categorizing cell lines showing 70%
inhibition as responders and cell lines showing .ltoreq.69%
inhibition as non-responders (FIG. 40; Table 11). Responder cell
lines for both antibody (IgG1-hDR5-01-56T-E430G+IgG1-hDR5-05-E430G)
and TRAIL monotherapy were found for all tested tumor indications,
except small cell lung cancer (SCLC).
TABLE-US-00014 TABLE 11 Results for antibody
(IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G) and TRAIL
monotherapy as determined in a 3-days viability assay screening at
Horizon Discovery Ltd, UK, for a panel of cell lines representing
different human cancer indications: kidney (A), neural tissue (B),
colorectal (C), gastric (D), triple-negative breast cancer (TNBC)
(E), non- small cell lung cancer (NSCLC) (F), bladder (G),
pancreatic (H), ovarian (I), melanoma (J), liver (K), endometrial
(L), head and neck (M) and small cell lung cancer (SCLC) (N).
Tabulated are IC50 values and percentage maximal inhibition.
IgG1-hDR5-01-G56T-E430G + TRAIL IgG1-hDR5-05-E430G Max IC50 Max
IC50 Inhibition Cell Line Screening # (nM) Inhibition (%) (nM) (%)
Kidney cancer cell line screening results for antibody
(IgG1-hDR5-01-G56T- E430G + IgG1-hDR5-05-E430G) and TRAIL therapy
as determined in a 3-days viability assay screening at Horizon, UK.
A704 1 0.475 99.7 3.443 77.1 A498 2 1.223 98.9 0.079 96.1 G-401 2
0.509 94.1 0.068 76.6 CAL-54 1 1.533 91.7 0.268 71.6 ACHN 2 0.843
89.9 0.356 32.4 CAKI-2 1 1.565 87.3 5.7 769-P 2 0.957 57.7 1.941
39.4 G-402 2 0.605 50.4 0.173 15.0 786-0 2 0.005 7.7 287.593 2.6
Neural tissue cancer cell line screening results for antibody
(IgG1-hDR5-01-G56T- E430G + IgG1-hDR5-05-E430G) and TRAIL therapy
as determined in a 3-days viability assay screening at Horizon, UK.
A172 2 0.888 100.0 0.029 98.2 SF295 2 0.764 99.2 0.023 87.5 SF126 2
0.713 98.9 0.013 85.6 H4 2 0.459 98.8 0.007 98.9 YH-13 2 1.248 94.4
0.317 39.5 U-87 MG 2 1.784 87.8 0.053 8.0 DBTRG-05MG 2 0.971 46.8
0.087 36.0 KNS-81 2 13.008 30.6 0.013 -6.6 SNB-75 2 3.225 14.2
105.178 9.6 NMC-G1 2 0.005 13.8 17.591 15.2 Colorectal cancer cell
line screening results for antibody (IgG1-hDR5-01-G56T- E430G +
IgG1-hDR5-05-E430G) and TRAIL therapy as determined in a 3-days
viability assay screening at Horizon, UK. CL-11 1 0.620 100.0 1.318
85.9 GP2D 1 0.738 100.0 0.003 100.0 HT-115 1 2.101 100.0 0.107 99.7
SNU-1197 1 1.076 100.0 0.053 100.0 COLO-205 1 0.360 99.9 2.269 83.5
COLO-206F 1 0.200 99.9 0.146 99.2 CL-34 1 0.380 99.8 0.024 98.8
HRT-186 1 0.433 99.3 9.240 52.5 HCT-15 1 0.813 98.8 0.129 98.6
SNU-407 1 0.836 98.5 0.098 96.1 MDST8 1 1.035 93.6 1.190 58.3
COLO-201 1 0.568 93.6 0.168 89.1 HT55 1 1.025 91.0 0.110 76.4
SNU-175 1 1.813 90.1 0.122 96.2 HCT-116_ARID1A (Q456*/Q456*) 1
0.235 86.2 10.502 50.2 DLD-1 1 0.938 83.1 3.954 55.1 SNU283 1 5.628
81.9 40.3 CL-40 1 1.555 79.9 1.975 57.3 HCT-116_KRAS (+/-) 1 0.855
77.7 0.253 88.7 SW837 1 1.399 76.6 0.940 83.8 LOVO 1 4.512 75.7
44.5 LS-411N 1 3.549 73.1 26.9 HT-29 1 2.966 65.5 12.2 SNU1033 1
4.446 60.9 10.057 51.7 SW480 1 1.093 60.2 2.037 55.2 COLO-678 1
3.670 58.6 10.1 DLD-1_BRCA2 (-/-) 1 1.826 58.3 0.516 60.0
HCT-116_PIK3A (+/-) KO mt H1047R 1 0.756 57.8 1.251 59.8 C2BBe1 1
49.5 8.8 SNU-C2B 1 43.0 0.713 71.3 SW1116 1 42.4 23.1
HCT-116_PAR-007 1 29.0 14.2 HCT-116_TP53 (-/-) 1 19.6 17.2 SW1417 1
19.1 12.2 RKO 1 14.5 10.5 HCT-116_PTEN (-/-) 1 11.9 38.0 COL0320DM
1 10.1 8.0 COLO-320 1 9.9 3.9 Gastric cancer cell line screening
results for antibody (IgG1-hDR5-01-G56T- E430G +
IgG1-hDR5-05-E430G) and TRAIL therapy as determined in a 3-days
viability assay screening at Horizon, UK. SNU-620 1 0.809 100.0
0.045 99.9 SNU-668 1 0.370 99.9 0.041 99.6 SNU-719 1 1.483 98.9
1.132 85.4 SNU-601 1 0.520 98.6 0.284 85.2 NUGC-3 1 0.671 96.7 38.7
GSU 1 0.169 96.4 0.454 81.6 SNU5 1 0.729 95.9 0.109 91.2 SNU-216 1
5.484 84.0 49.6 NCC-StC-K140 1 1.059 77.8 9.8 KE-97 1 1.175 57.2
23.4 LMSU 1 3.563 56.6 9.0 RERF-GC-1B 1 45.1 14.0 MKN1 1 36.8 10.5
SH-10-TC 1 31.6 34.9 ECC12 1 22.7 27.4 GSS 1 11.8 15.4 ECC10 1 8.3
14.0 Breast cancer cell line screening results for antibody
(IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G) and TRAIL therapy as
determined in a 3-days viability assay screening at Horizon, UK.
SUM159PT* 2 0.569 99.2 0.033 98.6 DU-4475* 2 1.102 94.5 0.079 87.0
HCC1806* 2 2.678 92.5 0.050 97.8 BT-549* 2 1.021 83.3 0.285 44.0
BT-20* 2 2.843 82.8 0.089 48.9 HCC1187* 2 1.521 82.8 0.066 99.4
MDA-MB-436* 2 0.762 77.7 0.053 76.3 HCC38* 2 0.903 70.6 0.080 96.9
HCC70* 2 18.703 69.3 346.839 3.6 HMC-1-8* 2 0.714 67.7 0.232 70.0
MDA-MB-231* 1 1.409 60.2 0.110 53.3 SK-BR-3 2 1.757 40.4 0.518 38.1
MDA-MB-468* 2 3.772 33.3 0.647 89.4 HCC1937* 2 8.548 28.0 0.647
28.5 T47D 2 2.557 17.1 346.732 8.4 MDA-MB-453* 2 0.723 13.0 34.643
20.7 MCF7 2 19.492 10.2 0.055 21.7 Non-small-cell lung cancer
(NSCLC) cell line screening results for antibody (IgG1-
hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G) and TRAIL therapy as
determined in a 3-days viability assay screening at Horizon, UK.
DV-90 1 0.215 100.0 1.189 96.1 NCI-H820 1 0.857 99.7 0.023 100.0
LCLC-97TM1 1 1.202 96.6 4.6 COR-L23-CPR 1 0.707 96.0 5.482 58.4
LOU-NH91 1 0.529 94.8 5.382 63.0 LCLC-103H 1 0.548 92.2 8.018 54.2
T3M-10 1 1.058 90.1 0.495 83.7 LU-99 1 1.879 81.9 6.3 HOP-62 1
0.899 81.7 55.6 EPLC-272H 1 1.233 79.7 51.7 LUDLU-1 1 2.196 77.9
0.475 91.1 RERF-LC-KJ 1 2.771 71.4 0.633 68.9 LXF-289 1 3.582 62.7
12.7 COR-L105 1 2.990 57.5 35.7 LC-1sq 1 7.710 56.6 0.781 70.1
NCI-H460 1 15.034 53.3 0.853 86.1 LC1F 1 50.3 0.451 74.3 SW1573 1
46.1 13.9 LU-65 1 38.9 32.4 HLC-1 1 36.8 32.6 VMRC-LCD 1 21.6 19.2
LK-2 1 19.0 5.6 Calu-1 1 13.0 22.0 CAL-12T 1 10.4 11.9 COLO-699 1
7.7 2.8 BEN 1 7.5 8.1 Bladder cancer cell line screening results
for antibody (IgG1-hDR5-01-G56T- E430G + IgG1-hDR5-05-E430G) and
TRAIL therapy as determined in a 3-days viability assay screening
at Horizon, UK. 5637 2 0.828 99.3 0.060 99.1 SW780 1 0.421 98.5
0.016 97.9 RT-112 2 3.520 96.1 0.325 50.9 RT4 2 4.638 95.5 0.244
91.5 UM-UC-3 1 0.906 94.4 0.005 99.4 TCCSUP 2 1.367 69.6 0.048 51.5
T-24 2 1.300 63.0 0.166 20.5 HT-1197 2 0.782 40.9 0.167 31.8 SCaBER
1 3.768 29.6 0.082 25.8 J82 1 68.272 15.9 33.567 11.5 HT-1376 2
67.114 15.3 159.873 10.6 Pancreatic cancer cell line screening
results for antibody (IgG1-hDR5-01-G56T- E430G +
IgG1-hDR5-05-E430G) and TRAIL therapy as determined in a 3-days
viability assay screening at Horizon, UK. HuP-T3 1 0.728 91.8 0.223
88.4 PSN1 1 0.655 91.6 0.205 86.9 Panc 02.13 1 2.288 85.9 1.905
60.5 BxPC-3 1 0.448 83.9 46.5 KP-4 1 1.853 80.0 23.6 CFPAC-1 1
13.635 57.2 13.2 HPAF-II 1 9.896 56.9 13.3 KP-2 1 10.251 54.2 3.9
KLM-1 1 41.0 12.7 KP-3 1 37.3 23.5 CAPAN-2 1 20.6 4.8 PK-1 1 6.4
0.548 88.3 Ovary cancer cell line screening results for antibody
(IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G) and TRAIL therapy as
determined in a 3-days viability assay screening at Horizon, UK.
SNU119 1 0.681 99.3 0.082 83.7 59M 1 0.846 98.6 0.049 98.5 JHOM-2B
1 8.294 82.0 21.7 COV434 1 1.093 80.4 0.395 73.1 OVCAR-5 1 2.731
79.0 1.120 70.6 OVK18 1 0.865 73.2 0.230 80.0 JHOM-1 1 1.835 68.8
0.596 58.1 COV644 1 3.375 68.2 0.271 74.9 MCAS 1 13.877 57.2 56.779
48.4 JHOS-4 1 73.734 49.6 12.5 OV7 1 48.3 20.4 COV504 1 19.0 11.7
OVTOKO 1 18.9 28.0 OVISE 1 13.5 4.0 KURAMOCHI 1 10.1 13.4 JHOC-5 1
8.5 18.4 Melanoma cell line screening results for antibody
(IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G) and TRAIL therapy as
determined in a 3-days viability assay screening at Horizon, UK.
COLO-679 1 0.524 99.5 24.9 COLO-783 1 0.503 98.2 0.224 80.9
COLO-800 1 0.365 95.7 33.5 Hs 294T 1 0.595 94.1 0.019 91.1 RVH-421
1 0.577 91.3 22.5 MEL-HO 1 0.760 89.2 16.7 WM-266-4 1 1.257 80.3
42.5 COLO858 1 0.567 68.1 12.6 MEL-JUSO 1 1.349 67.6 7.6 COLO-818 1
1.061 64.8 7.8 IGR-39 1 0.813 63.9 20.1 IGR-1 1 1.066 60.1 23.3
IGR-37 1 9.359 54.7 18.0 COLO-849 1 51.9 17.6 A375 1 51.2 47.1 Hs
936.T 1 41.0 20.7 SK-MEL-30 1 12.5 6.1 IPC-298 1 11.2 5.7 HMCB 1
7.0 1.0 Liver cancer cell line screening results for antibody
(IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G) and TRAIL therapy as
determined in a 3-days viability assay screening at Horizon, UK.
SNU-878 1 0.709 99.5 36.2
SNU-308 1 1.521 98.8 0.937 72.4 HuH-28 1 0.903 96.7 17.5 SNU-478 1
2.097 82.2 0.516 83.3 HLE 1 0.315 81.9 8.360 56.0 SNU-869 1 1.842
68.8 2.951 58.6 Li-7 1 1.614 65.9 20.4 HuCCT1 1 8.034 55.6 7.5
SNU-1196 1 44.8 44.3 HUH-6-clone5 1 42.3 20.2 SNU-1079 1 40.3 24.9
HuH-1 1 30.7 47.7 RH-41 1 10.1 3.0 SNU-761 1 9.1 7.1 Endometrial
cancer cell line screening results for antibody (IgG1-hDR5-01-G56T-
E430G + IgG1-hDR5-05-E430G) and TRAIL therapy as determined in a
3-days viability assay screening at Horizon, UK. HEC-265 1 0.399
100.0 0.021 100.0 MES-SA 1 0.510 100.0 0.107 92.6 JHUEM-2 1 0.613
89.6 0.165 66.4 RL95-2 1 1.649 88.0 0.155 97.7 SNG-II 1 1.049 79.3
1.028 77.2 JHUEM-3 1 46.2 18.4 TEN 1 43.2 1.205 70.0 HEC-1-A 1 39.6
22.8 HEC-108 1 32.8 1.479 64.9 MFE-296 1 22.7 8.7 COLO-684 1 14.3
16.6 SK-UT-1 1 13.4 10.9 HEC-1 1 13.2 8.1 MFE-280 1 11.4 11.0
HEC-50B 1 5.5 16.6 Head and neck cancer cell line screening results
for antibody (IgG1-hDR5-01- G56T-E430G + IgG1-hDR5-05-E430G) and
TRAIL therapy as determined in a 3-days viability assay screening
at Horizon, UK. YD-15 1 1.369 100.0 0.114 100.0 TE-4 1 0.879 99.9
0.107 81.0 KYM-1 1 0.438 99.4 30.2 FTC-238 1 0.426 93.8 3.922 86.7
KYSE-70 1 1.346 79.2 46.0 TE-10 1 3.602 68.0 24.4 TE-6 1 7.502 61.9
3.923 55.3 TE-9 1 1.139 60.3 0.300 73.6 TE-1 1 3.051 58.3 23.0 BICR
31 1 4.670 52.6 38.9 KYSE-410 1 51.0 10.6 CJM 1 48.8 12.8 BICR 22 1
42.0 13.6 KYSE-30 1 38.0 8.0 SCC-15 1 34.8 19.4 TE-8 1 33.4 39.6
PE-CA-PJ34-cl C12 1 28.9 4.7 EC-GI-10 1 25.0 29.6 TE-5 1 23.0 11.6
HSC-4 1 12.8 0.5 YD-8 1 10.8 3.4 KYSE-270 1 7.1 5.4 BICR 18 1 -0.1
3.4 Small cell lung cancer (SCLC) cancer cell line screening
results for antibody (IgG1- hDR5-01-G56T-E430G +
IgG1-hDR5-05-E430G) and TRAIL therapy as determined in a 3-days
viability assay screening at Horizon, UK. LU-134-A 1 47.7 28.0
IST-SL2 1 24.4 21.0 NCI-H69 1 23.2 14.5 NCI-H345 1 19.2 16.4 LU-139
1 18.7 10.0 SHP-77 1 9.2 9.1 NCI-H446 1 7.3 11.7 LU-135 1 6.0 9.7
*TNBC
Example 43
Capacity of Antibody Combination
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G to Induce Cancer Cell
Killing at Different Combination Ratios
[0563] A viability assay was performed to study the capacity of the
antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G to
induce killing of BxPC-3 pancreatic cancer cells and HCT-15 colon
cancer cells, when combined at different ratios of
IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G. Antibody ratios of
1:0, 9:1, 3:1, 1:1, 1:3, 1:9 and 0:1 in serial dilution series
(ranging from 0.006 to 20 .mu.g/mL final concentrations in 5-fold
dilutions) were tested in a CellTiter-Glo luminescent cell
viability assay as described in Example 16.
[0564] At 20 .mu.g/mL, 4 .mu.g/mL and 0.8 .mu.g/mL total antibody
concentrations, killing of BxPC-3 (FIG. 41A) and HCT-15 (FIG. 41B)
cells was equally effective at all tested antibody ratios
containing both antibodies IgG1-hDR5-01-G56T-E430G and
IgG1-hDR5-05-E430G. In contrast, the single antibodies (ratios 1:0
and 0:1) did not induce killing. At 0.16 .mu.g/mL total antibody
concentrations, the tested combinations of
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G induced killing,
although to a lesser extent than the higher antibody concentrations
and efficacy is impacted by the using different ratios.
Example 44
The Antibody Combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G
Induce Caspase-Dependent Programmed Cell Death
[0565] A viability assay was performed to compare the cytotoxicity
of the combination of antibody variants of IgG1-hDR5-01-G56T and
IgG1-hDR5-05 with and without the hexamerization-enhancing mutation
E430G in the presence and absence of a caspase inhibitor. A
CellTiter-Glo luminescent cell viability assay with serial dilution
series of antibody or TRAIL samples (range 0.002 to 133 nM final
concentrations in 4-fold dilutions) was performed as described in
Example 18.
[0566] The killing of BxPC-3 cells was inhibited in the presence of
pan-caspase inhibitor Z-VAD-FMK for TRAIL and the antibody
combinations IgG1-hDR5-01-G56T+IgG1-hDR5-05 and
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G (FIG. 42). These data
indicate that, like TRAIL, the antibody combinations
IgG1-hDR5-01-G56T+IgG1-hDR5-05 and
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G induced
caspase-dependent programmed cell death.
Example 45
Caspase-3 and -7 Activation upon Binding of the Antibody
Combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G on Human
Cancer Cells
[0567] Caspase-3/7 activation was measured in time using the
Caspase-Glo 3/7 assay, essentially as described in Example 20.
Briefly, cells were harvested by trypsinization, passed through a
cell strainer, pelleted by centrifugation for 5 minutes at 1,200
rpm and resuspended in culture medium at a concentration of
1.6.times.10.sup.5 cells/mL. 25 .mu.L of the single cell
suspensions (4,000 cells per well) were seeded in 384-wells culture
plates (Perkin Elmer, Cat nr 6007680) and incubated overnight at
37.degree. C. 25 .mu.L sample was added (26.6 nM final
concentrations) and incubated for 1, 2, 4 and 6 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 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).
[0568] In the time course of 1, 2, 4 to 6 hours, both TRAIL and the
antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G
induced more rapid and more potent caspase-3/7 activation on BxPC-3
cells compared to the WT antibody combination
IgG1-hDR5-01-G56T+IgG1-hDR5-05 without the hexamerization enhancing
mutation (FIG. 43).
Example 46
The In Vitro Potency of the Antibody Combination
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G does not Require the
Presence of a Secondary Fc Crosslinker
[0569] A viability assay was performed to compare the capacity of
the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G
to induce killing of human HCT-15 colon cancer cells and BxPC-3
pancreatic cancer cells in the absence and presence of a secondary
antibody crosslinker. IgG1-DR5-CONA, which is known to show
enhanced killing in the presence of a secondary antibody
crosslinker, was tested in the same assay for comparison. A
viability assay in absence and presence of secondary crosslinker
was performed, essentially as described in Example 21. Briefly, 100
.mu.the single cell suspensions (5,000 cells per well) were seeded
in 96-well plates and incubated overnight at 37.degree. C. 50 .mu.L
antibody sample (final concentration 4 .mu.g/mL) in the absence or
presence of F(ab').sub.2 fragments of a goat-anti-human IgG
antibody and incubated for 3 days at 37.degree. C. As a positive
control for cell killing, 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
Example 8.
[0570] The combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G
induced potent killing in BxPC-3 and HCT15 cells, and cytotoxicity
was not further enhanced in the presence of a secondary crosslinker
(FIG. 44). In contrast, the efficacy of IgG1-DR5-CONA and the wild
type antibody combination IgG1-hDR5-01-G56T+IgG1-hDR5-05 was
enhanced by the presence of a secondary crosslinker in both BxPC-3
and HCT15. These data indicate that killing of BxPC-3 and HCT15
cancer cells by the antibody combination
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G is independent of the
presence of a secondary Fc crosslinker.
Example 47
Complement Activation upon Binding of IgG1-hDR5-01-G56T-E430G and
IgG1-hDR5-05-E430G to CHO Cells Transiently Transfected with Either
Human or Cynomolgus DR5
[0571] To analyze the capacity of the antibodies
IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G to activate
complement, an in vitro complement-dependent cytotoxicity (CDC)
assay and deposition of complement component C3c was measured on
CHO cells that were transiently transfected with the isoform short
of either human or monkey DR5. The DR5 constructs harbored the
K386N (human) or K420N (cynomolgus monkey) mutation in their death
domain to prevent killing by the induction of apoptosis upon
binding of the agonistic antibodies. Transient transfections of CHO
cells with human or monkey (Macaca fascicularis) DR5 were performed
as described in Example 1.
[0572] For the CDC assay, 0.1.times.10.sup.6 cells were
pre-incubated in polystyrene round-bottom 96-well plates (Greiner
bio-one Cat #650101) with concentration series of purified
antibodies in a total volume of 80 .mu.L for 15 min on a shaker at
RT. Next, 20 .mu.L normal human serum (NHS; Cat #M0008 Sanquin,
Amsterdam, The Netherlands) was added as a source of complement and
incubated in a 37.degree. C. incubator for 45 min (20% final NHS
concentration; 0.003-10.0 .mu.g/mL final antibody concentrations in
3-fold dilutions). The reaction was stopped by putting the plates
on ice before pelleting the cells by centrifugation and replacing
the supernatant by 30 .mu.L of 2 .mu.g/mL propidium iodide solution
(PI; Sigma Aldrich, Zwijnaarde, The Netherlands). The percentage of
PI-positive cells was determined by flow cytrometry on an
Intellicyt iQue.TM. screener (Westburg). The data were analyzed
using best-fit values of a non-linear dose-response fit using
log-transformed concentrations in GraphPad PRISM 5.
[0573] For the analysis of C3b deposition, 0.1.times.10.sup.6 cells
were pre-incubated in round-bottom 96-well plates with
concentration series of purified antibodies (0.003-10.0 .mu.g/mL
final antibody concentrations in 3-fold dilutions) in a total
volume of 80 .mu.L for 15 min on a shaker at RT. Next, 20 .mu.L
C5-depleted serum (Quidel; Cat # A501) was added as a source of
complement and incubated in a 37.degree. C. incubator for 45 min
(20% final NHS concentration). Cells were pelleted and subsequently
incubated with 50 .mu.L FITC-labeled polyclonal rabbit-anti-human
C3c complement (Dako; Cat #F0201; 2 .mu.g/mL) in FACS buffer for 30
minutes at 4.degree. C. Cells were washed twice with FACS buffer
and resuspended in 30 .mu.L FACS buffer. The C3b-deposition on
cells was determined by flow cytrometry on an Intellicyt iQue.TM.
screener (Westburg). The data were analyzed using best-fit values
of a non-linear dose-response fit using log-transformed
concentrations in GraphPad PRISM 5.
[0574] Both complement-dependent killing (FIG. 45A-B) and C3b
deposition (FIG. 45C-D) on DR5-transfected CHO cells was observed
for IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G with
dose-response curves for both the single antibodies and for the
combination. These data indicate that the intrinsic capacity of the
IgG1 antibodies IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G to
induce complement activation upon target binding on the cell
surface was preserved for both the single antibodies
IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G and the combination
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G.
Example 48
Drug Combination Screen Analysis for Efficacy Enhancement of the
Antibody Combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G
with a Panel of Compounds on Human Colon Cancer Cell Lines
[0575] In order to identify clinically relevant compounds that
display synergistic inhibitory effects in combination with the
antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G,
100 compounds representing different therapeutic classes were
screened for potential synergy in colon cancer cell lines. A 72
hour (for LS-411N, SNU-C2B and SW480) or 120 hour (for DLD-1 and
HCT 116) ATPlite assay with growth inhibition analysis was
performed in a 6x6 optimized combination matrix in 384-well assay
plates at Horizon Discovery Ltd, UK. All samples were tested in
four replicates. Percentage growth inhibition was calculated using
the formulas: If T.gtoreq.V(0) than percentage growth
inhibition=100*[1-(T-V(0))/(V-V(0))]; If T<V(0) than percentage
growth inhibition=100*[1-(T-V(0))/V(0)], with T=luminescence of the
test sample, V(0)=luminescence of the medium control sample on day
0 and V=luminescence of the medium control sample on day 3. In
order to identify synergistic effects, mean self-cross activity was
determined for each therapeutic class using representative
compounds. To measure combination effects in excess of Loewe
additivity, Horizon Discovery Ltd has devised a scalar measure to
characterize the strength of synergistic interaction termed the
Synergy Score. The Synergy Score equation integrates the
experimentally-observed activity volume at each point in the matrix
in excess of a model surface numerically derived from the activity
of the component agents using the Loewe model for additivity.
Additional terms in the Synergy Score equation are used to
normalize for various dilution factors used for individual agents
and to allow for comparison of synergy scores across an entire
experiment. The inclusion of positive inhibition gating or an Idata
multiplier removes noise near the zero effect level, and biases
results for synergistic interactions that occur at high activity
levels. The Synergy Score (S) was calculated using the formula:
S=log fx log fy .SIGMA. max(0,Idata)(Idata-ILoewe) with
f.sub.x,y=dilution factors used for each single agent. Synergy
Scores greater than the mean self-cross plus 3.sigma. were
considered candidate synergies at the 99% confidence levels.
[0576] Table 12 shows the Synergy Scores for all 100 tested
compounds. Synergy with the antibody combination
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G was observed for one or
more cell lines with compounds from the different therapeutic
classes, including chemotherapeutics (including cytoskeletal
regulators and DNA/RNA damaging agents), kinase inhibitors, PI3K
pathway inhibitors, RAS inhibitors, apoptosis-modulating agents,
proteasome inhibitors, epigenetic modulators (including HDAC
inhibitors) and others. FIG. 46 shows five examples of the growth
inhibition effect of tested compounds in combination with the
antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G.
Birinapant (FIG. 46C), oxaliplatin (FIG. 46A), irinotecan (FIG.
46B) and paclitaxel (FIG. 46E) are examples that enhanced the
effect of IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G, while
baricitinib (FIG. 46D) is an example that showed no effect on the
activity of IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G.
Example 49
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
[0577] 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 26. 3.times.10.sup.6 cells were injected in a volume of 100
.mu.L PBS into the flank of 5-8 weeks old female SCID mice
(C.B-17/1crHan.RTM.Hsd-Prkdcsc.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-00015 TABLE 13 Treatment groups and dosing Dosing day
Total after # # antibody tumor mice analyzed Antibody dose
inoculation 8 8 IgG1-hDR5-01-G56T-E430G 0.5 mg/kg 9 8 8
IgG1-hDR5-05-E430G 0.5 mg/kg 9 8 8 IgG1-hDR5-01-G56T-E430G 0.5
mg/kg 9 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
[0578] FIG. 47A 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. 47B 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).
Example 50
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
[0579] 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
0.5 mg/kg antibody (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.
[0580] FIG. 48A 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. At day 21 the average tumor size in
mice treated with the combination
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G was significantly
smaller than in mice treated with an equivalent dose
IgG1-hDR5-01-G56T+IgG1-hDR5-05 (Mann Whitney test: P<0.0011)
(FIG. 48B). FIG. 48C shows a Kaplan-Meier plot of tumor
progression, with a cutoff set at a tumor volume >750 mm.sup.3.
Tumor outgrowth in mice treated with the combination
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G was significantly later
than in mice treated with an equivalent dose
IgG1-hDR5-01-G56T+IgG1-hDR5-05.
[0581] These data indicate 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 51
In Vivo Efficacy of IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G in
Combination with Paclitaxel in a Subcutaneous SK-MES-1 Human Lung
Cancer Xenograft Model
[0582] The in vivo anti-tumor efficacy of
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G was evaluated in
combination with paclitaxel in the subcutaneous SK-MES-1 human lung
cancer xenograft model at CrownBiosciences, Taicang, China. Cell
culturing, tumor cell inoculation, mice handling, tumor outgrowth
measurements and endpoint determination were performed as described
in Example 33. 21 days after tumor inoculation, the mean tumor size
reached 167 mm.sup.3 and mice were assigned into groups using
randomized block design and treatments were started. Mice were
treated twice according to a 07D regimen by i.v. injections of 2
mg/kg antibody and 15 mg/kg paclitaxel both dosed in 10 .mu.L PBS
per g body weight as indicated in Table 14.
TABLE-US-00016 TABLE 14 Treatment groups and dosing, Example 53
Dosing day # Total after mice Compound per dose randomization 8
IgG1-hDR5-01-G56T-E430G 2 mg/kg 0, 7 IgG1-hDR5-05-E430G 8
Paclitaxel 15 mg/kg 0, 7 8 IgG1-hDR5-01-G56T-E430G 2 mg/kg 0, 7
IgG1-hDR5-05-E430G antibody + 15 Paclitaxel mg/kg paclitaxel 8
IgG1-b12 2 mg/kg 0, 7
[0583] FIG. 49A shows mean tumor volumes per treatment group.
Antibody treatment alone (2 mg/kg
IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G) or 2 mg/kg antibody
treatment in combination with 15 mg/kg paclitaxel or 15 mg/kg
paclitaxel alone all demonstrated anti-tumor efficacy compared to
IgG1-b12. FIG. 49B shows tumor volume per treatment group at day
16. In all treatment groups, tumor load was significantly lower
compared to IgG1-b12 (Mann-Whitney test, p<0.01). FIG. 49C shows
a Kaplan-Meier plot of tumor progression, with a cutoff set at a
tumor volume >500 mm.sup.3. The combination of 15 mg/kg
paclitaxel with 2 mg/kg IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G
antibody significantly prolonged progression-free survival compared
to paclitaxel or antibody alone (Gehan-Breslow-Wilcoxon test, tumor
size cut-off 500 mm.sup.3: p<0.05).
Example 52
Pharmacokinetic (PK) Analysis of IgG1-hDR5-01-G56T-E430G and
IgG1-hDR5-5 05-E430G
[0584] The clearance rate of IgG1-hDR5-01-G56T-E430G and
IgG1-hDR5-05-E430G was studied in a PK experiment in SCID mice for
the single compounds and for the combination of the two antibodies
in comparison to the parental antibodies without the E430G
mutation.
[0585] 7-10 weeks old female SCID (C.B-17/IcrHan@Hsd-Prkdc<scid,
Harlan) mice (3 mice per group) were injected intravenously with 20
.mu.g antibody (1 mg/kg) in a 200 .mu.L injection volume. 50-100
.mu.L blood samples were collected from the saphenous vein at 10
minutes, 4 hours, 1 day, 2 days, 7 days, 14 days and 21 days after
antibody administration. Blood was collected into
heparin-containing vials and centrifuged for 5 minutes at 10,000 g.
Plasma samples were diluted 1:20 for the four first time points
(154 sample in 2854 PBSA (PBS supplemented with 0.2% bovine serum
albumin (BSA)) and 1:10 for the last two time points (304 sample in
27 .mu.L PBSA) and stored at -20.degree. C. until determination of
antibody concentrations.
[0586] Total human IgG concentrations were determined using a
sandwich ELISA. Mouse anti-human IgG-kappa mAb clone MH16 (CLB
Sanquin, Cat # #M1268) was used as capturing antibody and coated in
100 .mu.L overnight at 4.degree. C. to 96-well Microlon ELISA
plates (Greiner, Germany) at a concentration of 2 .mu.g/mL in PBS.
Plates were blocked by incubating on a plate shaker for 1 h at RT
with PBSA. After washing, 100 .mu.L of serial diluted plasma
samples (range 0.037-1 .mu.g/mL in 3-fold dilutions) were added and
incubated on a plate shaker for 1 h at RT. Plates were washed three
times with 300 .mu.L PBST (PBS supplemented with 0.05% Tween 20)
and subsequently incubated on a plate shaker for 1 h at RT with 100
.mu.L peroxidase-labeled goat anti-human IgG immunoglobulin
(#109-035-098, Jackson, West Grace, Pa.; 1:10.000 in PBST
supplemented with 0.2% BSA). Plates were washed again three times
with 300 .mu.L PBST before incubation for 15 minutes at RT with 100
.mu.L substrate 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic
acid) [ABTS; Roche, Cat #11112 422001; 1 tablet in 50 mL ABTS
buffer (Roche, Cat #11112 597001)] protected from light. The
reaction was stopped by adding 100 .mu.L 2% oxalic acid and
incubation for 10 minutes at RT. Absorbance was measured in a
microplate reader (Biotek, Winooski, Vt.) at 405 nm. Concentration
was calculated by using the injected material as a reference curve.
As a plate control, purified human IgG1 (The binding site, Cat
#BP078) was included. Human IgG concentrations (in .mu.g/mL) were
plotted (FIG. 50A) and Area under the curve (AUC) was calculated
using Graphpad prism 6.0. Clearance until the last day of blood
sampling (day 21) was determined by the formula D*1.000/AUC, in
which D is the dose of injection (1 mg/kg) (FIG. 50B).
[0587] No difference in the plasma clearance rate was observed
between IgG1-hDR5-01-G56T-E430G or IgG1-hDR5-05-E430G and their
parental antibodies without the E430G mutation, both when injected
as single agents or as the combinations of those (FIG. 50).
Example 53
Anti-DR5 Antibody IgG1-DR5-CONA with a Hexamerization-Enhancing
Mutation E430G is able to Kill Human Colon Cancer Cells
[0588] 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 8. 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 8. 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.
[0589] FIG. 51 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.
Sequence CWU 1
1
6818PRTArtificial SequenceSynthetic 1Gly Phe Asn Ile Lys Asp Thr
Phe1 528PRTArtificial SequenceSynthetic 2Ile Asp Pro Ala Asn Gly
Asn Thr1 5311PRTArtificial SequenceSynthetic 3Val Arg Gly Leu Tyr
Thr Tyr Tyr Phe Asp Tyr1 5 104118PRTArtificial SequenceSynthetic
4Glu 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
11556PRTArtificial SequenceSynthetic 5Gln Ser Ile Ser Asn Asn1
569PRTArtificial SequenceSynthetic 6Gln Gln Gly Asn Ser Trp Pro Tyr
Thr1 57107PRTArtificial SequenceSynthetic 7Glu 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
10588PRTArtificial SequenceSynthetic 8Ile Asp Pro Ala Asn Thr Asn
Thr1 59118PRTArtificial SequenceSynthetic 9Glu 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 115108PRTArtificial
SequenceSynthetic 10Gly Phe Asn Ile Lys Asp Thr His1
51111PRTArtificial SequenceSynthetic 11Ala Arg Trp Gly Thr Asn Val
Tyr Phe Ala Tyr1 5 1012118PRTArtificial SequenceSynthetic 12Gln 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
115135PRTArtificial SequenceSynthetic 13Ser Ser Val Ser Tyr1
5149PRTArtificial SequenceSynthetic 14Gln Gln Tyr His Ser Tyr Pro
Pro Thr1 515106PRTArtificial SequenceSynthetic 15Asp 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
1051610PRTArtificial SequenceSynthetic 16Gly Gly Ser Ile Ser Ser
Gly Asp Tyr Phe1 5 10177PRTArtificial SequenceSynthetic 17Ile His
Asn Ser Gly Thr Thr1 51814PRTArtificial SequenceSynthetic 18Ala Arg
Asp Arg Gly Gly Asp Tyr Tyr Tyr Gly Met Asp Val1 5
1019122PRTArtificial SequenceSynthetic 19Gln 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
12020122PRTArtificial SequenceSynthetic 20Gln 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 45Trp 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
120217PRTArtificial SequenceSynthetic 21Gln Gly Ile Ser Arg Ser
Tyr1 5229PRTArtificial SequenceSynthetic 22Gln Gln Phe Gly Ser Ser
Pro Trp Thr1 523108PRTArtificial SequenceSynthetic 23Glu 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 10524439PRTHomo Sapiens 24Met Glu Gln Arg Gly Gln Asn Ala Pro
Ala Ala Ser Gly Ala Arg Lys1 5 10 15Arg His Gly Pro Gly Pro Arg Glu
Ala Arg Gly Ala Arg Pro Gly Pro 20 25 30Arg Val Pro Lys Thr Leu Val
Leu Val Val Ala Ala Val Leu Leu Leu 35 40 45Val Ser Ala Glu Ser Ala
Leu Ile Thr Gln Gln Asp Leu Ala Pro Gln 50 55 60Gln Arg Ala Ala Pro
Gln Gln Lys Arg Ser Ser Pro Ser Glu Gly Leu65 70 75 80Cys Pro Pro
Gly His His Ile Ser Glu Asp Gly Arg Asp Cys Ile Ser 85 90 95Cys Lys
Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn Asp Leu Leu Phe 100 105
110Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu Val Glu Leu Ser Pro
115 120 125Cys Thr Thr Thr Arg Asn Thr Val Cys Gln Cys Glu Glu Gly
Thr Phe 130 135 140Arg Glu Glu Asp Ser Pro Glu Met Cys Arg Lys Cys
Arg Thr Gly Cys145 150 155 160Pro Arg Gly Met Val Lys Val Gly Asp
Cys Thr Pro Trp Ser Asp Ile 165 170 175Glu Cys Val His Lys Glu Ser
Gly Thr Lys His Ser Gly Glu Val Pro 180 185 190Ala Val Glu Glu Thr
Val Thr Ser Ser Pro Gly Thr Pro Ala Ser Pro 195 200 205Cys Ser Leu
Ser Gly Ile Ile Ile Gly Val Thr Val Ala Ala Val Val 210 215 220Leu
Ile Val Ala Val Phe Val Cys Lys Ser Leu Leu Trp Lys Lys Val225 230
235 240Leu Pro Tyr Leu Lys Gly Ile Cys Ser Gly Gly Gly Gly Asp Pro
Glu 245 250 255Arg Val Asp Arg Ser Ser Gln Arg Pro Gly Ala Glu Asp
Asn Val Leu 260 265 270Asn Glu Ile Val Ser Ile Leu Gln Pro Thr Gln
Val Pro Glu Gln Glu 275 280 285Met Glu Val Gln Glu Pro Ala Glu Pro
Thr Gly Val Asn Met Leu Ser 290 295 300Pro Gly Glu Ser Glu His Leu
Leu Glu Pro Ala Glu Ala Glu Arg Ser305 310 315 320Gln Arg Arg Arg
Leu Leu Val Pro Ala Asn Glu Gly Asp Pro Thr Glu 325 330 335Thr Leu
Arg Gln Cys Phe Asp Asp Phe Ala Asp Leu Val Pro Phe Asp 340 345
350Ser Trp Glu Pro Leu Met Arg Lys Leu Gly Leu Met Asp Asn Glu Ile
355 360 365Lys Val Ala Lys Ala Glu Ala Ala Gly His Arg Asp Thr Leu
Tyr Thr 370 375 380Met Leu Ile Lys Trp Val Asn Lys Thr Gly Arg Asp
Ala Ser Val His385 390 395 400Thr Leu Leu Asp Ala Leu Glu Thr Leu
Gly Glu Arg Leu Ala Lys Lys 405 410 415Ile Glu Asp His Leu Leu Ser
Ser Gly Lys Phe Met Tyr Leu Glu Gly 420 425 430Asn Ala Asp Ser Ala
Met Ser 43525440PRTMacaca mulatta 25Met Gly Gln Leu Arg Gln Ser Ala
Pro Ala Ala Ser Gly Ala Arg Lys1 5 10 15Gly Arg Gly Pro Gly Pro Arg
Glu Ala Arg Gly Ala Arg Pro Gly Leu 20 25 30Arg Val Leu Lys Thr Leu
Val Leu Val Val Ala Ala Ala Arg Val Leu 35 40 45Val Ser Ala Asp Cys
Ala Pro Ile Thr Arg Gln Ser Leu Asp Pro Gln 50 55 60Arg Arg Ala Ala
Pro Gln Gln Lys Arg Ser Ser Pro Thr Glu Gly Leu65 70 75 80Cys Pro
Pro Gly His His Ile Ser Glu Asp Ser Arg Asp Cys Ile Ser 85 90 95Cys
Lys Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn Asp Phe Leu Phe 100 105
110Cys Leu Arg Cys Thr Lys Cys Asp Ser Gly Glu Val Glu Val Asn Ser
115 120 125Cys Thr Thr Thr Arg Asn Thr Val Cys Gln Cys Glu Glu Gly
Thr Phe 130 135 140Arg Glu Glu Asp Ser Pro Glu Ile Cys Arg Lys Cys
Arg Thr Gly Cys145 150 155 160Pro Arg Gly Met Val Lys Val Lys Asp
Cys Thr Pro Trp Ser Asp Ile 165 170 175Glu Cys Val His Lys Glu Ser
Gly Thr Lys His Thr Gly Glu Val Pro 180 185 190Ala Val Glu Lys Thr
Val Thr Thr Ser Pro Gly Thr Pro Ala Ser Pro 195 200 205Cys Ser Leu
Ser Gly Ile Ile Ile Gly Val Ile Val Phe Val Val Ile 210 215 220Val
Val Val Ala Val Ile Val Trp Lys Thr Ser Leu Trp Lys Lys Val225 230
235 240Leu Pro Tyr Leu Lys Gly Val Cys Ser Gly Asp Gly Gly Asp Pro
Glu 245 250 255Arg Val Asp Ser Ser Pro Gln Arg Pro Gly Ala Glu Asp
Asn Ala Leu 260 265 270Asn Glu Ile Val Ser Ile Val Gln Pro Ser Gln
Val Pro Glu Gln Glu 275 280 285Met Glu Val Gln Glu Pro Ala Glu Gln
Thr Asp Val Asn Thr Leu Ser 290 295 300Pro Gly Glu Ser Glu His Leu
Leu Glu Pro Ala Lys Ala Glu Gly Pro305 310 315 320Gln Arg Arg Gly
Gln Leu Val Pro Val Asn Glu Asn Asp Pro Thr Glu 325 330 335Thr Leu
Arg Gln Cys Phe Asp Asp Phe Ala Ala Ile Val Pro Phe Asp 340 345
350Ala Trp Glu Pro Leu Val Arg Gln Leu Gly Leu Thr Asn Asn Glu Ile
355 360 365Lys Val Ala Lys Ala Glu Ala Ala Ser Ser Arg Asp Thr Leu
Tyr Val 370 375 380Met Leu Ile Lys Trp Val Asn Lys Thr Gly Arg Ala
Ala Ser Val Asn385 390 395 400Thr Leu Leu Asp Ala Leu Glu Thr Leu
Glu Glu Arg Leu Ala Lys Gln 405 410 415Lys Ile Gln Asp Arg Leu Leu
Ser Ser Gly Lys Phe Met Tyr Leu Glu 420 425 430Asp Asn Ala Asp Ser
Ala Thr Ser 435 44026381PRTMus Musculus 26Met Glu Pro Pro Gly Pro
Ser Thr Pro Thr Ala Ser Ala Ala Ala Arg1 5 10 15Ala Asp His Tyr Thr
Pro Gly Leu Arg Pro Leu Pro Lys Arg Arg Leu 20 25 30Leu Tyr Ser Phe
Ala Leu Leu Leu Ala Val Leu Gln Ala Val Phe Val 35 40 45Pro Val Thr
Ala Asn Pro Ala His Asn Arg Pro Ala Gly Leu Gln Arg 50 55 60Pro Glu
Glu Ser Pro Ser Arg Gly Pro Cys Leu Ala Gly Gln Tyr Leu65 70 75
80Ser Glu Gly Asn Cys Lys Pro Cys Arg Glu Gly Ile Asp Tyr Thr Ser
85 90 95His Ser Asn His Ser Leu Asp Ser Cys Ile Leu Cys Thr Val Cys
Lys 100 105 110Glu Asp Lys Val Val Glu Thr Arg Cys Asn Ile Thr Thr
Asn Thr Val 115 120 125Cys Arg Cys Lys Pro Gly Thr Phe Glu Asp Lys
Asp Ser Pro Glu Ile 130 135 140Cys Gln Ser Cys Ser Asn Cys Thr Asp
Gly Glu Glu Glu Leu Thr Ser145 150 155 160Cys Thr Pro Arg Glu Asn
Arg Lys Cys Val Ser Lys Thr Ala Trp Ala 165 170 175Ser Trp His Lys
Leu Gly Leu Trp Ile Gly Leu Leu Val Pro Val Val 180 185 190Leu Leu
Ile Gly Ala Leu Leu Val Trp Lys Thr Gly Ala Trp Arg Gln 195 200
205Trp Leu Leu Cys Ile Lys Arg Gly Cys Glu Arg Asp Pro Glu Ser Ala
210 215 220Asn Ser Val His Ser Ser Leu Leu Asp Arg Gln Thr Ser Ser
Thr Thr225 230 235 240Asn Asp Ser Asn His Asn Thr Glu Pro Gly Lys
Thr Gln Lys Thr Gly 245 250 255Lys Lys Leu Leu Val Pro Val Asn Gly
Asn Asp Ser Ala Asp Asp Leu 260 265 270Lys Phe Ile Phe Glu Tyr Cys
Ser Asp Ile Val Pro Phe Asp Ser Trp 275 280 285Asn Arg Leu Met Arg
Gln Leu Gly Leu Thr Asp Asn Gln Ile Gln Met 290 295 300Val Lys Ala
Glu Thr Leu Val Thr Arg Glu Ala Leu Tyr Gln Met Leu305 310 315
320Leu Lys Trp Arg His Gln Thr Gly Arg Ser Ala Ser Ile Asn His Leu
325 330 335Leu Asp Ala Leu Glu Ala Val Glu Glu Arg Asp Ala Met Glu
Lys Ile 340 345 350Glu Asp Tyr Ala Val Lys Ser Gly Arg Phe Thr Tyr
Gln Asn Ala Ala 355 360 365Ala Gln Pro Glu Thr Gly Pro Gly Gly Ser
Gln Cys Val 370 375 38027453PRTArtificial SequenceSynthetic 27Met
Glu Gln Arg Gly Gln Asn Ala Pro Ala Ala Ser Gly Ala Arg Lys1 5 10
15Arg His Gly Pro Gly Pro Arg Glu Ala Arg Gly Ala Arg Pro Gly Leu
20 25 30Arg Val Pro Lys Thr Leu Val Leu Val Val Ala Ala Val Leu Leu
Leu 35 40 45Val Ser Ala Glu Ser
Ala Leu Ile Thr Gln Gln Asp Leu Ala Pro Gln 50 55 60Gln Arg Val Ala
Pro Gln Gln Lys Arg Ser Ser Pro Ser Glu Gly Leu65 70 75 80Cys Pro
Pro Gly His His Ile Ser Glu Asp Gly Arg Asp Cys Ile Ser 85 90 95Cys
Lys Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn Asp Leu Leu Phe 100 105
110Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu Val Glu Leu Ser Pro
115 120 125Cys Thr Thr Thr Arg Asn Thr Val Cys Gln Cys Glu Glu Gly
Thr Phe 130 135 140Arg Glu Glu Asp Ser Pro Glu Met Cys Arg Lys Cys
Arg Thr Gly Cys145 150 155 160Pro Arg Gly Met Val Lys Val Gly Asp
Cys Thr Pro Trp Ser Asp Ile 165 170 175Glu Cys Val His Lys Glu Ser
Gly Thr Lys His Ser Gly Glu Ala Pro 180 185 190Ala Val Glu Glu Thr
Val Thr Ser Ser Pro Gly Thr Pro Ala Ser Pro 195 200 205Cys Ser Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 210 215 220Ala
Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys225 230
235 240Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val 245 250 255Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr 260 265 270Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu 275 280 285Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His 290 295 300Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys305 310 315 320Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 325 330 335Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 340 345
350Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
355 360 365Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn 370 375 380Tyr Lys Thr Ala Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu385 390 395 400Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val 405 410 415Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln 420 425 430Lys Ser Leu Ser Leu
Ser Pro Gly Lys His His His His His His His 435 440 445His Glu Pro
Glu Ala 45028192PRTArtificial SequenceSynthetic 28Met Glu Gln Arg
Gly Gln Asn Ala Pro Ala Ala Ser Gly Ala Arg Lys1 5 10 15Arg His Gly
Pro Gly Pro Arg Glu Ala Arg Gly Ala Arg Pro Gly Pro 20 25 30Arg Val
Pro Lys Thr Leu Val Leu Val Val Ala Ala Val Leu Leu Leu 35 40 45Val
Ser Ala Glu Ser Ala Leu Ile Thr Gln Gln Asp Leu Ala Pro Gln 50 55
60Gln Arg Ala Ala Pro Gln Gln Lys Arg Ser Ser Pro Ser Glu Gly Leu65
70 75 80Cys Pro Pro Gly His His Ile Ser Glu Asp Gly Arg Asp Cys Ile
Ser 85 90 95Cys Lys Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn Asp Leu
Leu Phe 100 105 110Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu Val
Glu Leu Ser Pro 115 120 125Cys Thr Thr Thr Arg Asn Thr Val Cys Gln
Cys Glu Glu Gly Thr Phe 130 135 140Arg Glu Glu Asp Ser Pro Glu Met
Cys Arg Lys Cys Arg Thr Gly Cys145 150 155 160Pro Arg Gly Met Val
Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile 165 170 175Glu Cys Val
His Lys Glu Ser Gly His His His His His His His His 180 185
19029329PRTHomo Sapiens 29Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys Ser1 5 10 15Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe 20 25 30Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly 35 40 45Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu 50 55 60Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr65 70 75 80Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg 85 90 95Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 100 105 110Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 115 120
125Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
130 135 140Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr145 150 155 160Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu 165 170 175Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His 180 185 190Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys 195 200 205Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 210 215 220Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met225 230 235
240Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
245 250 255Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn 260 265 270Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu 275 280 285Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val 290 295 300Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln305 310 315 320Lys Ser Leu Ser Leu
Ser Pro Gly Lys 32530329PRTHomo Sapiens 30Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser1 5 10 15Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 20 25 30Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 35 40 45Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 50 55 60Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr65 70 75
80Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
85 90 95Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro 100 105 110Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys 115 120 125Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val 130 135 140Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr145 150 155 160Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu 165 170 175Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 180 185 190Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 195 200
205Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
210 215 220Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met225 230 235 240Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro 245 250 255Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn 260 265 270Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu 275 280 285Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 290 295 300Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln305 310 315
320Lys Ser Leu Ser Leu Ser Pro Gly Lys 32531329PRTHomo Sapiens
31Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser1
5 10 15Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe 20 25 30Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly 35 40 45Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu 50 55 60Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr Tyr65 70 75 80Ile Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys Pro 85 90 95Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro 100 105 110Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys 115 120 125Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 130 135 140Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr145 150 155
160Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
165 170 175Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His 180 185 190Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys 195 200 205Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln 210 215 220Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu225 230 235 240Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 245 250 255Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 260 265 270Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 275 280
285Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
290 295 300Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln305 310 315 320Lys Ser Leu Ser Leu Ser Pro Gly Lys
32532329PRTHomo Sapiens 32Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys Ser1 5 10 15Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe 20 25 30Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly 35 40 45Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu 50 55 60Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr65 70 75 80Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Pro 85 90 95Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 100 105 110Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 115 120
125Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
130 135 140Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr145 150 155 160Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu 165 170 175Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His 180 185 190Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys 195 200 205Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 210 215 220Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met225 230 235
240Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
245 250 255Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn 260 265 270Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu 275 280 285Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val 290 295 300Phe Ser Cys Ser Val Met His Glu
Gly Leu His Asn His Tyr Thr Gln305 310 315 320Lys Ser Leu Ser Leu
Ser Pro Gly Lys 32533448PRTArtificial SequenceSynthetic 33Glu 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 44534448PRTArtificial SequenceSynthetic 34Glu 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 Lys 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
44535448PRTArtificial SequenceSynthetic 35Glu 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 Gly Ala 420 425 430Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
44536448PRTArtificial SequenceSynthetic 36Glu 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
44537448PRTArtificial SequenceSynthetic 37Glu 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 Lys 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
44538448PRTArtificial SequenceSynthetic 38Glu 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 Gly Ala 420 425 430Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
44539214PRTArtificial SequenceSynthetic 39Glu 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 21040448PRTArtificial SequenceSynthetic
40Gln 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 44541448PRTArtificial SequenceSynthetic 41Gln 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 Lys 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
44542448PRTArtificial SequenceSynthetic 42Gln 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 Gly Ala 420 425 430Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
44543213PRTArtificial SequenceSynthetic 43Asp 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 21044440PRTHomo Sapiens 44Met Glu Gln Arg
Gly Gln Asn Ala Pro Ala Ala Ser Gly Ala Arg Lys1 5 10 15Arg His Gly
Pro Gly Pro Arg Glu Ala Arg Gly Ala Arg Pro Gly Pro 20 25 30Arg Val
Pro Lys Thr Leu Val Leu Val Val Ala Ala Val Leu Leu Leu 35 40 45Val
Ser Ala Glu Ser Ala Leu Ile Thr Gln Gln Asp Leu Ala Pro Gln 50 55
60Gln Arg Ala Ala Pro Gln Gln Lys Arg Ser Ser Pro Ser Glu Gly Leu65
70 75 80Cys Pro Pro Gly His His Ile Ser Glu Asp Gly Arg Asp Cys Ile
Ser 85 90 95Cys Lys Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn Asp Leu
Leu Phe 100 105 110Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu Val
Glu Leu Ser Pro 115 120 125Cys Thr Thr Thr Arg Asn Thr Val Cys Gln
Cys Glu Glu Gly Thr Phe 130 135 140Arg Glu Glu Asp Ser Pro Glu Met
Cys Arg Lys Cys Arg Thr Gly Cys145 150 155 160Pro Arg Gly Met Val
Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile 165 170 175Glu Cys Val
His Lys Glu Ser Gly Thr Lys His Ser Gly Glu Val Pro 180 185 190Ala
Val Glu Glu Thr Val Thr Ser Ser Pro Gly Thr Pro Ala Ser Pro 195 200
205Cys Ser Leu Ser Gly Ile Ile Ile Gly Val Thr Val Ala Ala Val Val
210 215 220Leu Ile Val Ala Val Phe Val Cys Lys Ser Leu Leu Trp Lys
Lys Val225 230 235 240Leu Pro Tyr Leu Lys Gly Ile Cys Ser Gly Gly
Gly Gly Asp Pro Glu 245 250 255Arg Val Asp Arg Ser Ser Gln Arg Pro
Gly Ala Glu Asp Asn Val Leu 260 265 270Asn Glu Ile Val Ser Ile Leu
Gln Pro Thr Gln Val Pro Glu Gln Glu 275 280 285Met Glu Val Gln Glu
Pro Ala Glu Pro Thr Gly Val Asn Met Leu Ser 290 295 300Pro Gly Glu
Ser Glu His Leu Leu Glu Pro Ala Glu Ala Glu Arg Ser305 310 315
320Gln Arg Arg Arg Leu Leu Val Pro Ala Asn Glu Gly Asp Pro Thr Glu
325 330 335Thr Leu Arg Gln Cys Phe Asp Asp Phe Ala Asp Leu Val Pro
Phe Asp 340 345 350Ser Trp Glu Pro Leu Met Arg Lys Leu Gly Leu Met
Asp Asn Glu Ile 355 360 365Lys Val Ala Lys Ala Glu Ala Ala Gly His
Arg Asp Thr Leu Tyr Thr 370 375 380Met Leu Ile Lys Trp Val Asn Lys
Thr Gly Arg Asp Ala Ser Val His385 390 395 400Thr Leu Leu Asp Ala
Leu Glu Thr Leu Gly Glu Arg Leu Ala Asn Gln 405 410 415Lys Ile Glu
Asp His Leu Leu Ser Ser Gly Lys Phe Met Tyr Leu Glu 420 425 430Gly
Asn Ala Asp Ser Ala Met Ser 435 44045440PRTHomo Sapiens 45Met Glu
Gln Arg Gly Gln Asn Ala Pro Ala Ala Ser Gly Ala Arg Lys1 5 10 15Arg
His Gly Pro Gly Pro Arg Glu Ala Arg Gly Ala Arg Pro Gly Leu 20 25
30Arg Val Pro Lys Thr Leu Val Leu Val Val Ala Ala Val Leu Leu Leu
35 40 45Val Ser Ala Glu Ser Ala Leu Ile Thr Gln Gln Asp Leu Ala Pro
Gln 50 55 60Gln Arg Val Ala Pro Gln Gln Lys Arg Ser Ser Pro Ser Glu
Gly Leu65 70 75 80Cys Pro Pro Gly His His Ile Ser Glu Asp Gly Arg
Asp Cys Ile Ser 85 90 95Cys Lys Tyr Gly Gln Asp Tyr Ser Thr His Trp
Asn Asp Leu Leu Phe 100 105 110Cys Leu Arg Cys Thr Arg Cys Asp Ser
Gly Glu Val Glu Leu Ser Pro 115 120 125Cys Thr Thr Thr Arg Asn Thr
Val Cys Gln Cys Glu Glu Gly Thr Phe 130 135 140Arg Glu Glu Asp Ser
Pro Glu Met Cys Arg Lys Cys Arg Thr Gly Cys145 150 155 160Pro Arg
Gly Met Val Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile 165 170
175Glu Cys Val His Lys Glu Ser Gly Thr Lys His Ser Gly Glu Ala Pro
180 185 190Ala Val Glu Glu Thr Val Thr Ser Ser Pro Gly Thr Pro Ala
Ser Pro 195 200 205Cys Ser Leu Ser Gly Ile Ile Ile Gly Val Thr Val
Ala Ala Val Val 210 215 220Leu Ile Val Ala Val Phe Val Cys Lys Ser
Leu Leu Trp Lys Lys Val225 230 235 240Leu Pro Tyr Leu Lys Gly Ile
Cys Ser Gly Gly Gly Gly Asp Pro Glu 245 250 255Arg Val Asp Arg Ser
Ser Gln Arg Pro Gly Ala Glu Asp Asn Val Leu 260 265 270Asn Glu Ile
Val Ser Ile Leu Gln Pro Thr Gln Val Pro Glu Gln Glu 275 280 285Met
Glu Val Gln Glu Pro Ala Glu Pro Thr Gly Val Asn Met Leu Ser 290 295
300Pro Gly Glu Ser Glu His Leu Leu Glu Pro Ala Glu Ala Glu Arg
Ser305 310 315 320Gln Arg Arg Arg Leu Leu Val Pro Ala Asn Glu Gly
Asp Pro Thr Glu 325 330 335Thr Leu Arg Gln Cys Phe Asp Asp Phe Ala
Asp Leu Val Pro Phe Asp 340 345 350Ser Trp Glu Pro Leu Met Arg Lys
Leu Gly Leu Met Asp Asn Glu Ile 355 360 365Lys Val Ala Lys Ala Glu
Ala Ala Gly His Arg Asp Thr Leu Tyr Thr 370 375 380Met Leu Ile Lys
Trp Val Asn Lys Thr Gly Arg Asp Ala Ser Val His385 390 395 400Thr
Leu Leu Asp Ala Leu Glu Thr Leu Gly Glu Arg Leu Ala Lys Gln 405 410
415Lys Ile Glu Asp His Leu Leu Ser Ser Gly Lys Phe Met Tyr Leu Glu
420 425 430Gly Asn Ala Asp Ser Ala Met Ser 435 44046440PRTHomo
Sapiens 46Met Glu Gln Arg Gly Gln Asn Ala Pro Ala Ala Ser Gly Ala
Arg Lys1 5 10 15Arg His Gly Pro Gly Pro Arg Glu Ala Arg Gly Ala Arg
Pro Gly Pro 20 25 30Arg Val Pro Lys Thr Leu Val Leu Val Val Ala Ala
Val Leu Leu Leu 35 40 45Val Ser Ala Glu Ser Ala Leu Ile Thr Gln Gln
Asp Leu Ala Pro Gln 50 55 60Gln Arg Ala Ala Pro Gln Gln Lys Arg Ser
Ser Pro Ser Glu Gly Leu65 70 75 80Cys Pro Pro Gly His His Ile Ser
Glu Asp Gly Arg Asp Cys Ile Ser 85 90 95Cys Lys Tyr Gly Gln Asp Tyr
Ser Thr His Trp Asn
Asp Leu Leu Phe 100 105 110Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly
Glu Val Glu Leu Ser Pro 115 120 125Cys Thr Thr Thr Arg Asn Thr Val
Cys Gln Cys Glu Glu Gly Thr Phe 130 135 140Arg Glu Glu Asp Ser Pro
Glu Met Cys Arg Lys Cys Arg Thr Gly Cys145 150 155 160Pro Arg Gly
Met Val Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile 165 170 175Glu
Cys Val His Lys Glu Ser Gly Thr Lys His Ser Gly Glu Val Pro 180 185
190Ala Val Glu Glu Thr Val Thr Ser Ser Pro Gly Thr Pro Ala Ser Pro
195 200 205Cys Ser Leu Ser Gly Ile Ile Ile Gly Val Thr Val Ala Ala
Val Val 210 215 220Leu Ile Val Ala Val Phe Val Cys Lys Ser Leu Leu
Trp Lys Lys Val225 230 235 240Leu Pro Tyr Leu Lys Gly Ile Cys Ser
Gly Gly Gly Gly Asp Pro Glu 245 250 255Arg Val Asp Arg Ser Ser Gln
Arg Pro Gly Ala Glu Asp Asn Val Leu 260 265 270Asn Glu Ile Val Ser
Ile Leu Gln Pro Thr Gln Val Pro Glu Gln Glu 275 280 285Met Glu Val
Gln Glu Pro Ala Glu Pro Thr Gly Val Asn Met Leu Ser 290 295 300Pro
Gly Glu Ser Glu His Leu Leu Glu Pro Ala Glu Ala Glu Arg Ser305 310
315 320Gln Arg Arg Arg Leu Leu Val Pro Ala Asn Glu Gly Asp Pro Thr
Glu 325 330 335Thr Leu Arg Gln Cys Phe Asp Asp Phe Ala Asp Leu Val
Pro Phe Asp 340 345 350Ser Trp Glu Pro Leu Met Arg Lys Leu Gly Leu
Met Asp Asn Glu Ile 355 360 365Lys Val Ala Lys Ala Glu Ala Ala Gly
His Arg Asp Thr Leu Tyr Thr 370 375 380Met Leu Ile Lys Trp Val Asn
Lys Thr Gly Arg Asp Ala Ser Val His385 390 395 400Thr Leu Leu Asp
Ala Leu Glu Thr Leu Gly Glu Arg Leu Ala Lys Gln 405 410 415Lys Ile
Glu Asp His Leu Leu Ser Ser Gly Lys Phe Met Tyr Leu Glu 420 425
430Gly Asn Ala Asp Ser Ala Met Ser 435 44047411PRTHomo Sapiens
47Met Glu Gln Arg Gly Gln Asn Ala Pro Ala Ala Ser Gly Ala Arg Lys1
5 10 15Arg His Gly Pro Gly Pro Arg Glu Ala Arg Gly Ala Arg Pro Gly
Pro 20 25 30Arg Val Pro Lys Thr Leu Val Leu Val Val Ala Ala Val Leu
Leu Leu 35 40 45Val Ser Ala Glu Ser Ala Leu Ile Thr Gln Gln Asp Leu
Ala Pro Gln 50 55 60Gln Arg Ala Ala Pro Gln Gln Lys Arg Ser Ser Pro
Ser Glu Gly Leu65 70 75 80Cys Pro Pro Gly His His Ile Ser Glu Asp
Gly Arg Asp Cys Ile Ser 85 90 95Cys Lys Tyr Gly Gln Asp Tyr Ser Thr
His Trp Asn Asp Leu Leu Phe 100 105 110Cys Leu Arg Cys Thr Arg Cys
Asp Ser Gly Glu Val Glu Leu Ser Pro 115 120 125Cys Thr Thr Thr Arg
Asn Thr Val Cys Gln Cys Glu Glu Gly Thr Phe 130 135 140Arg Glu Glu
Asp Ser Pro Glu Met Cys Arg Lys Cys Arg Thr Gly Cys145 150 155
160Pro Arg Gly Met Val Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile
165 170 175Glu Cys Val His Lys Glu Ser Gly Ile Ile Ile Gly Val Thr
Val Ala 180 185 190Ala Val Val Leu Ile Val Ala Val Phe Val Cys Lys
Ser Leu Leu Trp 195 200 205Lys Lys Val Leu Pro Tyr Leu Lys Gly Ile
Cys Ser Gly Gly Gly Gly 210 215 220Asp Pro Glu Arg Val Asp Arg Ser
Ser Gln Arg Pro Gly Ala Glu Asp225 230 235 240Asn Val Leu Asn Glu
Ile Val Ser Ile Leu Gln Pro Thr Gln Val Pro 245 250 255Glu Gln Glu
Met Glu Val Gln Glu Pro Ala Glu Pro Thr Gly Val Asn 260 265 270Met
Leu Ser Pro Gly Glu Ser Glu His Leu Leu Glu Pro Ala Glu Ala 275 280
285Glu Arg Ser Gln Arg Arg Arg Leu Leu Val Pro Ala Asn Glu Gly Asp
290 295 300Pro Thr Glu Thr Leu Arg Gln Cys Phe Asp Asp Phe Ala Asp
Leu Val305 310 315 320Pro Phe Asp Ser Trp Glu Pro Leu Met Arg Lys
Leu Gly Leu Met Asp 325 330 335Asn Glu Ile Lys Val Ala Lys Ala Glu
Ala Ala Gly His Arg Asp Thr 340 345 350Leu Tyr Thr Met Leu Ile Lys
Trp Val Asn Lys Thr Gly Arg Asp Ala 355 360 365Ser Val His Thr Leu
Leu Asp Ala Leu Glu Thr Leu Gly Glu Arg Leu 370 375 380Ala Asn Gln
Lys Ile Glu Asp His Leu Leu Ser Ser Gly Lys Phe Met385 390 395
400Tyr Leu Glu Gly Asn Ala Asp Ser Ala Met Ser 405
41048445PRTMacaca fascicularis 48Met Gly Gln Leu Arg Gln Ser Ala
Pro Ala Ala Ser Gly Ala Arg Lys1 5 10 15Gly Arg Gly Pro Gly Pro Arg
Glu Ala Arg Gly Ala Arg Pro Gly Leu 20 25 30Arg Val Leu Lys Thr Leu
Val Leu Val Val Ala Ala Ala Arg Val Leu 35 40 45Leu Ser Val Ser Ala
Asp Cys Ala Pro Ile Thr Arg Gln Ser Leu Asp 50 55 60Pro Gln Arg Arg
Ala Ala Pro Gln Gln Lys Arg Ser Ser Pro Thr Glu65 70 75 80Gly Leu
Cys Pro Pro Gly His His Ile Ser Glu Asp Ser Arg Glu Cys 85 90 95Ile
Ser Cys Lys Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn Asp Phe 100 105
110Leu Phe Cys Leu Arg Cys Thr Lys Cys Asp Ser Gly Glu Val Glu Val
115 120 125Asn Ser Cys Thr Thr Thr Arg Asn Thr Val Cys Gln Cys Glu
Glu Gly 130 135 140Thr Phe Arg Glu Glu Asp Ser Pro Glu Ile Cys Arg
Lys Cys Arg Thr145 150 155 160Gly Cys Pro Arg Gly Met Val Lys Val
Lys Asp Cys Thr Pro Trp Ser 165 170 175Asp Ile Glu Cys Val His Lys
Glu Ser Gly Thr Lys His Thr Gly Glu 180 185 190Val Pro Ala Val Glu
Lys Thr Val Thr Thr Ser Pro Gly Thr Pro Ala 195 200 205Ser Pro Cys
Ser Leu Ser Gly Ile Ile Ile Gly Val Ile Val Leu Val 210 215 220Val
Ile Val Val Val Ala Val Ile Val Trp Lys Thr Ser Leu Trp Lys225 230
235 240Lys Val Leu Pro Tyr Leu Lys Gly Val Cys Ser Gly Gly Gly Gly
Asp 245 250 255Pro Glu Arg Val Asp Ser Ser Ser His Ser Pro Gln Arg
Pro Gly Ala 260 265 270Glu Asp Asn Ala Leu Asn Glu Ile Val Ser Ile
Val Gln Pro Ser Gln 275 280 285Val Pro Glu Gln Glu Met Glu Val Gln
Glu Pro Ala Glu Gln Thr Asp 290 295 300Val Asn Thr Leu Ser Pro Gly
Glu Ser Glu His Leu Leu Glu Pro Ala305 310 315 320Lys Ala Glu Gly
Pro Gln Arg Arg Gly Gln Leu Val Pro Val Asn Glu 325 330 335Asn Asp
Pro Thr Glu Thr Leu Arg Gln Cys Phe Asp Asp Phe Ala Ala 340 345
350Ile Val Pro Phe Asp Ala Trp Glu Pro Leu Val Arg Gln Leu Gly Leu
355 360 365Thr Asn Asn Glu Ile Lys Val Ala Lys Ala Glu Ala Ala Ser
Ser Arg 370 375 380Asp Thr Leu Tyr Val Met Leu Ile Lys Trp Val Asn
Lys Thr Gly Arg385 390 395 400Ala Ala Ser Val Asn Thr Leu Leu Asp
Ala Leu Glu Thr Leu Glu Glu 405 410 415Arg Leu Ala Lys Gln Lys Ile
Gln Asp Arg Leu Leu Ser Ser Gly Lys 420 425 430Phe Met Tyr Leu Glu
Asp Asn Ala Asp Ser Ala Thr Ser 435 440 44549445PRTMacaca
fascicularis 49Met Gly Gln Leu Arg Gln Ser Ala Pro Ala Ala Ser Gly
Ala Arg Lys1 5 10 15Gly Arg Gly Pro Gly Pro Arg Glu Ala Arg Gly Ala
Arg Pro Gly Leu 20 25 30Arg Val Leu Lys Thr Leu Val Leu Val Val Ala
Ala Ala Arg Val Leu 35 40 45Leu Ser Val Ser Ala Asp Cys Ala Pro Ile
Thr Arg Gln Ser Leu Asp 50 55 60Pro Gln Arg Arg Ala Ala Pro Gln Gln
Lys Arg Ser Ser Pro Thr Glu65 70 75 80Gly Leu Cys Pro Pro Gly His
His Ile Ser Glu Asp Ser Arg Glu Cys 85 90 95Ile Ser Cys Lys Tyr Gly
Gln Asp Tyr Ser Thr His Trp Asn Asp Phe 100 105 110Leu Phe Cys Leu
Arg Cys Thr Lys Cys Asp Ser Gly Glu Val Glu Val 115 120 125Asn Ser
Cys Thr Thr Thr Arg Asn Thr Val Cys Gln Cys Glu Glu Gly 130 135
140Thr Phe Arg Glu Glu Asp Ser Pro Glu Ile Cys Arg Lys Cys Arg
Thr145 150 155 160Gly Cys Pro Arg Gly Met Val Lys Val Lys Asp Cys
Thr Pro Trp Ser 165 170 175Asp Ile Glu Cys Val His Lys Glu Ser Gly
Thr Lys His Thr Gly Glu 180 185 190Val Pro Ala Val Glu Lys Thr Val
Thr Thr Ser Pro Gly Thr Pro Ala 195 200 205Ser Pro Cys Ser Leu Ser
Gly Ile Ile Ile Gly Val Ile Val Leu Val 210 215 220Val Ile Val Val
Val Ala Val Ile Val Trp Lys Thr Ser Leu Trp Lys225 230 235 240Lys
Val Leu Pro Tyr Leu Lys Gly Val Cys Ser Gly Gly Gly Gly Asp 245 250
255Pro Glu Arg Val Asp Ser Ser Ser His Ser Pro Gln Arg Pro Gly Ala
260 265 270Glu Asp Asn Ala Leu Asn Glu Ile Val Ser Ile Val Gln Pro
Ser Gln 275 280 285Val Pro Glu Gln Glu Met Glu Val Gln Glu Pro Ala
Glu Gln Thr Asp 290 295 300Val Asn Thr Leu Ser Pro Gly Glu Ser Glu
His Leu Leu Glu Pro Ala305 310 315 320Lys Ala Glu Gly Pro Gln Arg
Arg Gly Gln Leu Val Pro Val Asn Glu 325 330 335Asn Asp Pro Thr Glu
Thr Leu Arg Gln Cys Phe Asp Asp Phe Ala Ala 340 345 350Ile Val Pro
Phe Asp Ala Trp Glu Pro Leu Val Arg Gln Leu Gly Leu 355 360 365Thr
Asn Asn Glu Ile Lys Val Ala Lys Ala Glu Ala Ala Ser Ser Arg 370 375
380Asp Thr Leu Tyr Val Met Leu Ile Lys Trp Val Asn Lys Thr Gly
Arg385 390 395 400Ala Ala Ser Val Asn Thr Leu Leu Asp Ala Leu Glu
Thr Leu Glu Glu 405 410 415Arg Leu Ala Asn Gln Lys Ile Gln Asp Arg
Leu Leu Ser Ser Gly Lys 420 425 430Phe Met Tyr Leu Glu Asp Asn Ala
Asp Ser Ala Thr Ser 435 440 44550416PRTMacaca fascicularis 50Met
Gly Gln Leu Arg Gln Ser Ala Pro Ala Ala Ser Gly Ala Arg Lys1 5 10
15Gly Arg Gly Pro Gly Pro Arg Glu Ala Arg Gly Ala Arg Pro Gly Leu
20 25 30Arg Val Leu Lys Thr Leu Val Leu Val Val Ala Ala Ala Arg Val
Leu 35 40 45Leu Ser Val Ser Ala Asp Cys Ala Pro Ile Thr Arg Gln Ser
Leu Asp 50 55 60Pro Gln Arg Arg Ala Ala Pro Gln Gln Lys Arg Ser Ser
Pro Thr Glu65 70 75 80Gly Leu Cys Pro Pro Gly His His Ile Ser Glu
Asp Ser Arg Glu Cys 85 90 95Ile Ser Cys Lys Tyr Gly Gln Asp Tyr Ser
Thr His Trp Asn Asp Phe 100 105 110Leu Phe Cys Leu Arg Cys Thr Lys
Cys Asp Ser Gly Glu Val Glu Val 115 120 125Asn Ser Cys Thr Thr Thr
Arg Asn Thr Val Cys Gln Cys Glu Glu Gly 130 135 140Thr Phe Arg Glu
Glu Asp Ser Pro Glu Ile Cys Arg Lys Cys Arg Thr145 150 155 160Gly
Cys Pro Arg Gly Met Val Lys Val Lys Asp Cys Thr Pro Trp Ser 165 170
175Asp Ile Glu Cys Val His Lys Glu Ser Gly Ile Ile Ile Gly Val Ile
180 185 190Val Leu Val Val Ile Val Val Val Ala Val Ile Val Trp Lys
Thr Ser 195 200 205Leu Trp Lys Lys Val Leu Pro Tyr Leu Lys Gly Val
Cys Ser Gly Gly 210 215 220Gly Gly Asp Pro Glu Arg Val Asp Ser Ser
Ser His Ser Pro Gln Arg225 230 235 240Pro Gly Ala Glu Asp Asn Ala
Leu Asn Glu Ile Val Ser Ile Val Gln 245 250 255Pro Ser Gln Val Pro
Glu Gln Glu Met Glu Val Gln Glu Pro Ala Glu 260 265 270Gln Thr Asp
Val Asn Thr Leu Ser Pro Gly Glu Ser Glu His Leu Leu 275 280 285Glu
Pro Ala Lys Ala Glu Gly Pro Gln Arg Arg Gly Gln Leu Val Pro 290 295
300Val Asn Glu Asn Asp Pro Thr Glu Thr Leu Arg Gln Cys Phe Asp
Asp305 310 315 320Phe Ala Ala Ile Val Pro Phe Asp Ala Trp Glu Pro
Leu Val Arg Gln 325 330 335Leu Gly Leu Thr Asn Asn Glu Ile Lys Val
Ala Lys Ala Glu Ala Ala 340 345 350Ser Ser Arg Asp Thr Leu Tyr Val
Met Leu Ile Lys Trp Val Asn Lys 355 360 365Thr Gly Arg Ala Ala Ser
Val Asn Thr Leu Leu Asp Ala Leu Glu Thr 370 375 380Leu Glu Glu Arg
Leu Ala Asn Gln Lys Ile Gln Asp Arg Leu Leu Ser385 390 395 400Ser
Gly Lys Phe Met Tyr Leu Glu Asp Asn Ala Asp Ser Ala Thr Ser 405 410
415518PRTArtificial SequenceSynthetic 51Gly Phe Thr Phe Ser Ser Tyr
Val1 5528PRTArtificial SequenceSynthetic 52Ile Ser Ser Gly Gly Ser
Tyr Thr1 55312PRTArtificial SequenceSynthetic 53Ala Arg Arg Gly Asp
Ser Met Ile Thr Thr Asp Tyr1 5 10546PRTArtificial SequenceSynthetic
54Gln Asp Val Gly Thr Ala1 5558PRTArtificial SequenceSynthetic
55Gln Gln Tyr Ser Ser Tyr Arg Thr1 556449PRTArtificial
SequenceSynthetic 56Glu 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
445Lys57213PRTArtificial SequenceSynthetic 57Asp 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 21058329PRTArtificial SequenceSynthetic
58Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser1
5 10 15Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe 20 25 30Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly 35 40 45Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu 50 55 60Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr Tyr65 70 75 80Ile Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys Arg 85 90 95Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro 100 105 110Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys 115 120 125Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 130 135 140Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr145 150 155
160Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
165 170 175Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His 180 185 190Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys 195 200 205Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln 210 215 220Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met225 230 235 240Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 245 250 255Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 260 265 270Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 275 280
285Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
290 295 300Phe Ser Cys Ser Val Met His Gly Ala Leu His Asn His Tyr
Thr Gln305 310 315 320Lys Ser Leu Ser Leu Ser Pro Gly Lys
32559329PRTArtificial SequenceSynthetic 59Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser1 5 10 15Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 20 25 30Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 35 40 45Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 50 55 60Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr65 70 75
80Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg
85 90 95Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro 100 105 110Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys 115 120 125Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val 130 135 140Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr145 150 155 160Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu 165 170 175Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 180 185 190Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 195 200
205Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
210 215 220Pro Arg Lys Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met225 230 235 240Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro 245 250 255Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn 260 265 270Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu 275 280 285Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 290 295 300Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln305 310 315
320Lys Ser Leu Ser Leu Ser Pro Gly Lys 32560329PRTArtificial
SequenceSynthetic 60Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser1 5 10 15Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe 20 25 30Pro Glu Pro Val Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly 35 40 45Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu 50 55 60Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr65 70 75 80Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg 85 90 95Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 100 105 110Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 115 120 125Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 130 135
140Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr145 150 155 160Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu 165 170 175Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His 180 185 190Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys 195 200 205Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 210 215 220Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met225 230 235 240Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 245 250
255Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
260 265 270Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu 275 280 285Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val 290 295 300Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln305 310 315 320Lys Tyr Leu Ser Leu Ser Pro
Gly Lys 32561329PRTArtificial SequenceSynthetic 61Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser1 5 10 15Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 20 25 30Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 35 40 45Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 50 55
60Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr65
70 75 80Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Arg 85 90 95Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys Pro 100 105 110Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys 115 120 125Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val 130 135 140Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr145 150 155 160Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 165 170 175Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 180 185 190Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 195 200
205Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
210 215 220Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met225 230 235 240Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro 245 250 255Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn 260 265 270Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu 275 280 285Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 290 295 300Phe Ser Cys
Ser Val Met His Gly Ala Leu His Asn His Tyr Thr Gln305 310 315
320Glu Ser Leu Ser Leu Ser Pro Gly Lys 32562329PRTArtificial
SequenceSynthetic 62Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser1 5 10 15Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe 20 25 30Pro Glu Pro Val Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly 35 40 45Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu 50 55 60Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr65 70 75 80Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg 85 90 95Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 100 105 110Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 115 120 125Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 130 135
140Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr145 150 155 160Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu 165 170 175Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His 180 185 190Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys 195 200 205Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 210 215 220Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met225 230 235 240Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 245 250
255Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
260 265 270Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu 275 280 285Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val 290 295 300Phe Ser Cys Ser Val Met His Gly Ala Leu
His Asn His Tyr Thr Gln305 310 315 320Lys Lys Leu Ser Leu Ser Pro
Gly Lys 32563329PRTArtificial SequenceSynthetic 63Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser1 5 10 15Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 20 25 30Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 35 40 45Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 50 55
60Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr65
70 75 80Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Arg 85 90 95Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys Pro 100 105 110Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys 115 120 125Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val 130 135 140Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr145 150 155 160Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 165 170 175Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 180 185 190Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 195 200
205Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
210 215 220Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met225 230 235 240Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro 245 250 255Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn 260 265 270Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu 275 280 285Tyr Ser Arg Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 290 295 300Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln305 310 315
320Lys Ser Leu Ser Leu Ser Pro Gly Lys 32564329PRTArtificial
SequenceSynthetic 64Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser1 5 10 15Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe 20 25 30Pro Glu Pro Val Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly 35 40 45Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu 50 55 60Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr65 70 75 80Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg 85 90 95Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 100 105 110Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 115 120 125Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 130 135
140Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr145
150 155 160Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu 165 170 175Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His 180 185 190Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys 195 200 205Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln 210 215 220Pro Arg Lys Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met225 230 235 240Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 245 250 255Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 260 265
270Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
275 280 285Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val 290 295 300Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln305 310 315 320Lys Ser Leu Ser Leu Ser Pro Gly Lys
32565329PRTArtificial SequenceSynthetic 65Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser1 5 10 15Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 20 25 30Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 35 40 45Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 50 55 60Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr65 70 75
80Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg
85 90 95Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro 100 105 110Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys 115 120 125Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val 130 135 140Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr145 150 155 160Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu 165 170 175Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 180 185 190Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 195 200
205Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
210 215 220Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met225 230 235 240Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro 245 250 255Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn 260 265 270Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu 275 280 285Tyr Ser Arg Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 290 295 300Phe Ser Cys
Ser Val Met His Gly Ala Leu His Asn His Tyr Thr Gln305 310 315
320Lys Ser Leu Ser Leu Ser Pro Gly Lys 32566329PRTArtificial
SequenceSynthetic 66Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser1 5 10 15Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe 20 25 30Pro Glu Pro Val Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly 35 40 45Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu 50 55 60Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr65 70 75 80Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg 85 90 95Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 100 105 110Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 115 120 125Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 130 135
140Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr145 150 155 160Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu 165 170 175Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His 180 185 190Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys 195 200 205Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 210 215 220Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met225 230 235 240Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 245 250
255Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
260 265 270Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Leu Leu 275 280 285Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val 290 295 300Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln305 310 315 320Lys Ser Leu Ser Leu Ser Pro
Gly Lys 32567329PRTArtificial SequenceSynthetic 67Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser1 5 10 15Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 20 25 30Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 35 40 45Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 50 55
60Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr65
70 75 80Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Arg 85 90 95Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys Pro 100 105 110Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys 115 120 125Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val 130 135 140Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr145 150 155 160Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 165 170 175Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 180 185 190Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 195 200
205Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
210 215 220Pro Arg Lys Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met225 230 235 240Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro 245 250 255Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn 260 265 270Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Leu Leu 275 280 285Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 290 295 300Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln305 310 315
320Lys Ser Leu Ser Leu Ser Pro Gly Lys 32568329PRTArtificial
SequenceSynthetic 68Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser1 5 10 15Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe 20 25 30Pro Glu Pro Val Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly 35 40 45Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu 50 55 60Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr65 70 75 80Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg 85 90 95Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 100 105 110Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 115 120 125Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 130 135
140Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr145 150 155 160Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu 165 170 175Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His 180 185 190Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys 195 200 205Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 210 215 220Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met225 230 235 240Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 245 250
255Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
260 265 270Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Leu Leu 275 280 285Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val 290 295 300Phe Ser Cys Ser Val Met His Gly Ala Leu
His Asn His Tyr Thr Gln305 310 315 320Lys Ser Leu Ser Leu Ser Pro
Gly Lys 325
* * * * *
References