U.S. patent application number 16/867449 was filed with the patent office on 2021-04-01 for bispecific anti-vegf/anti-ang-2 antibodies and their use in the treatment of ocular vascular diseases.
This patent application is currently assigned to Roche Glycart AG. The applicant listed for this patent is Roche Glycart AG. Invention is credited to Harald Duerr, Frank Herting, Christian Klein, Joerg Thomas Regula, Matthias Rueth, Kay-Gunnar Stubenrauch.
Application Number | 20210095013 16/867449 |
Document ID | / |
Family ID | 1000005263875 |
Filed Date | 2021-04-01 |
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United States Patent
Application |
20210095013 |
Kind Code |
A1 |
Duerr; Harald ; et
al. |
April 1, 2021 |
BISPECIFIC ANTI-VEGF/ANTI-ANG-2 ANTIBODIES AND THEIR USE IN THE
TREATMENT OF OCULAR VASCULAR DISEASES
Abstract
The present invention relates to bispecific antibody against
human vascular endothelial growth factor (VEGF/VEGF-A) and against
human angiopoietin-2 (ANG-2) of human IgG1 or IgG4 subclass with
mutations I253A, H310A, and H435A, methods for their production,
pharmaceutical compositions containing said antibodies, and uses
thereof.
Inventors: |
Duerr; Harald; (Starnberg,
DE) ; Herting; Frank; (Penzberg, DE) ; Klein;
Christian; (Bonstetten, CH) ; Regula; Joerg
Thomas; (Muenchen, DE) ; Rueth; Matthias;
(Penzberg, DE) ; Stubenrauch; Kay-Gunnar;
(Penzberg, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Roche Glycart AG |
Schlieren |
|
CH |
|
|
Assignee: |
Roche Glycart AG
Schlieren
CH
|
Family ID: |
1000005263875 |
Appl. No.: |
16/867449 |
Filed: |
May 5, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15603259 |
May 23, 2017 |
10683345 |
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16867449 |
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13940091 |
Jul 11, 2013 |
9695233 |
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15603259 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/54 20130101;
C07K 2317/33 20130101; C07K 2317/21 20130101; C07K 2317/35
20130101; C07K 2317/92 20130101; A61P 27/06 20180101; C07K 2317/565
20130101; C07K 16/22 20130101; A61K 2039/505 20130101; C07K 2317/31
20130101; C07K 2317/56 20130101; C07K 2317/52 20130101; A61P 27/02
20180101 |
International
Class: |
C07K 16/22 20060101
C07K016/22; A61P 27/02 20060101 A61P027/02; A61P 27/06 20060101
A61P027/06 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 13, 2012 |
EP |
12176299.1 |
Claims
1. A method for the reduction of the viscosity of an antibody
wherein the antibody comprises a constant heavy chain region of
human IgG1 or human IgG4 subclass(derived from human origin and)
wherein the method comprises the modification of the antibody
constant heavy chain region of human IgG1 or human IgG4 subclass
with the mutations I253A, H310A, and H435A (numbering according to
EU Index of Kabat).
2. The method of claim 1, wherein the antibody is a bispecific
antibody comprising a first antigen-binding site that specifically
binds to human VEGF and a second antigen-binding site that
specifically binds to human ANG-2, wherein i) said first
antigen-binding site specifically binding to VEGF comprises in the
heavy chain variable domain a CDR3H region of SEQ ID NO: 1, a CDR2H
region of SEQ ID NO: 2, and a CDR1H region of SEQ ID NO:3, and in
the light chain variable domain a CDR3L region of SEQ ID NO: 4, a
CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:6; and
ii) said second antigen-binding site specifically binding to ANG-2
comprises in the heavy chain variable domain a CDR3H region of SEQ
ID NO: 9, a CDR2H region of, SEQ ID NO: 10, and a CDR1H region of
SEQ ID NO: 11, and in the light chain variable domain a CDR3L
region of SEQ ID NO: 12, a CDR2L region of SEQ ID NO: 13, and a
CDR1L region of SEQ ID NO: 14, and wherein iii) the bispecific
antibody comprises a constant heavy chain region of human IgG1 or
human IgG4 subclass (derived from human origin and) comprising the
mutations I253A, H310A, and H435A (numbering according to EU Index
of Kabat).
3. The method of claim 2, wherein the bispecific antibody comprises
a constant heavy chain region of human IgG1 subclass (derived from
human origin and) comprising the mutations I253A, H310A, and H435A
(numbering according to EU Index of Kabat) and further comprising
the mutations L234A , L235A and P329G (numbering according to EU
Index of Kabat).
4. An antibody obtained by the method of any one of claims 1 to
3.
5. A bispecific antibody comprising a first antigen-binding site
that specifically binds to human VEGF and a second antigen-binding
site that specifically binds to human ANG-2, wherein i) said first
antigen-binding site specifically binding to VEGF comprises in the
heavy chain variable domain a CDR3H region of SEQ ID NO: 1, a CDR2H
region of SEQ ID NO: 2, and a CDR1H region of SEQ ID NO:3, and in
the light chain variable domain a CDR3L region of SEQ ID NO: 4, a
CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:6; and
ii) said second antigen-binding site specifically binding to ANG-2
comprises in the heavy chain variable domain a CDR3H region of SEQ
ID NO: 9, a CDR2H region of, SEQ ID NO: 10, and a CDR1H region of
SEQ ID NO: 11, and in the light chain variable domain a CDR3L
region of SEQ ID NO: 12, a CDR2L region of SEQ ID NO: 13, and a
CDR1L region of SEQ ID NO: 14, and wherein iii) the bispecific
antibody comprises a constant heavy chain region of human IgG1 or
human IgG4 subclass (derived from human origin and) comprising the
mutations I253A, H310A, and H435A (numbering according to EU Index
of Kabat)
6. The bispecific antibody according to claim 5, wherein i) said
first antigen-binding site specifically binding to VEGF comprises
as heavy chain variable domain VH an amino acid sequence of SEQ ID
NO: 7, and as light chain variable domain VL an amino acid sequence
of SEQ ID NO: 8, and ii) said second antigen-binding site
specifically binding to ANG-2 comprises as heavy chain variable
domain VH an amino acid sequence of SEQ ID NO: 15, and as light
chain variable domain VL an amino acid sequence of SEQ ID NO:
16.
7. The bispecific antibody according to any one of claims 5 to 6,
wherein the constant heavy chain region under iii) is of IgG1
subclass
8. The bispecific antibody according to claim 6, wherein the
constant heavy chain region of IgG1 subclass further comprises the
mutations L234A , L235A and P329G (numbering according to EU Index
of Kabat)
9. The bispecific antibody according to any one of claims 5 to 6,
wherein the constant heavy chain region under iii) is of IgG4
subclass
10. The bispecific antibody according to claim 9, wherein the
constant heavy chain region of IgG4 subclass further comprises the
mutations S228P and L235E (numbering according to EU Index of
Kabat)
11. The bispecific antibody according to claim 9, wherein the
constant heavy chain region of IgG4 subclass further comprises the
mutations S228P , L235E and P329G (numbering according to EU Index
of Kabat)
12. A pharmaceutical composition comprising an antibody according
to any one of claims 4 and 5 to 11.
13. A nucleic acid encoding a bispecific antibody according to any
one of claims 5 to 11.
14. Expression vector containing said nucleic acid according claim
13 capable of expressing said nucleic acid in a prokaryotic or
eukaryotic host cell.
15. A prokaryotic or eukaryotic host cell comprising a vector
according to claim 14.
16. A method for the preparation of a bispecific antibody according
to any one of claims 5 to 11 comprising the steps of a)
transforming a host cell with vectors comprising nucleic acid
molecules encoding said antibody; b) culturing the host cell under
conditions that allow synthesis of said antibody molecule; and c)
recovering said antibody molecule from said culture.
17. A bispecific antibody obtained by the method of claim 16.
18. A bispecific, bivalent antibody comprising a first
antigen-binding site that specifically binds to human VEGF and a
second antigen-binding site that specifically binds to human ANG-2,
characterized in comprising the amino acid sequences of SEQ ID NO:
25, of SEQ ID NO: 26, of SEQ ID NO: 27, and of SEQ ID NO: 28.
19. A bispecific, bivalent antibody comprising a first
antigen-binding site that specifically binds to human VEGF and a
second antigen-binding site that specifically binds to human ANG-2,
characterized in comprising the amino acid sequences of SEQ ID NO:
21, of SEQ ID NO: 22., of SEQ ID NO: 23., and of SEQ ID NO: 24.
20. A bispecific, bivalent antibody comprising a first
antigen-binding site that specifically binds to human VEGF and a
second antigen-binding site that specifically binds to human ANG-2,
characterized in comprising the amino acid sequences of SEQ ID NO:
29, of SEQ ID NO: 30, of SEQ ID NO: 31, and of SEQ ID NO: 32.
21. A method of treatment of patient suffering from ocular vascular
diseases by administering an antibody according to any one of
claims 4 and 5 to 11 to a patient in the need of such
treatment.
22. The method of claim 21, wherein the antibody is administered
via intravitreal application.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/603,259, filed May 23, 2017 which is a continuation of U.S.
application Ser. No. 13/940,091, filed Jul. 11, 2013, now U.S. Pat.
No. 9,695,233, which claims the benefit of priority under 35 USC
119(a) to European patent application number 12176299.1, filed 13
Jul. 2012, which are incorporated herein by reference in its
entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Mar. 18, 2020, is named Sequence_listing.txt and is 120,540
bytes in size.
FIELD OF THE INVENTION
[0003] The present invention relates a method for the reduction of
the viscosity of an antibody (including a bispecific antibody) of
human IgG1 or human IgG4 subclass, to bispecific antibodies against
human vascular endothelial growth factor (VEGF/VEGF-A) and against
human angiopoietin-2 (ANG-2), methods for their production,
pharmaceutical compositions containing said antibodies, and uses
thereof.
BACKGROUND OF THE INVENTION
[0004] Angiogenesis is implicated in the pathogenesis of a variety
of disorders which include solid tumors, intraocular neovascular
syndromes such as proliferative retinopathies or age-related
macular degeneration (AMD), rheumatoid arthritis, and psoriasis
(Folkman, J., et al., J. Biol. Chem. 267 (1992) 10931-10934;
Klagsbrun, M., et al., Annu. Rev. Physiol. 53 (1991) 217-239; and
Garner, A., Vascular diseases, in: Pathobiology of ocular disease,
A dynamic approach, Garner, A., and Klintworth, G. K. (eds.), 2nd
edition, Marcel Dekker, New York (1994), pp. 1625-1710).
[0005] Ranibizumab (trade name Lucentis.RTM.) is a monoclonal
antibody fragment derived from the same parent murine antibody as
bevacizumab (Avastin.RTM.). However, it has been affinity matured
to provide stronger binding to VEGF-A (WO 98/45331). It is known
that VEGF-A blocking may be related to some systemic toxicities,
therefore ranibizumab is missing an Fc part to reduce the serum
half live and consequently systemic toxicities. It is an
anti-angiogenic agent that has been approved to treat the "wet"
type of age-related macular degeneration (ARMD), a common form of
age-related vision loss.
[0006] Corneal angiogenesis assays have shown that both ANG-1 and
ANG-2 had similar effects, acting synergistically with VEGF to
promote growth of new blood vessels. Asahara, T., et al., Circ.
Res. 83 (1998) 233-40. The possibility that there was a
dose-dependent endothelial response was raised by the observation
that in vitro at high concentration, ANG-2 can also be
pro-angiogenic (Kim, I., et al., Oncogene 19 (2000) 4549-52). At
high concentration, ANG-2 acts as an apoptosis survival factor for
endothelial cells during serum deprivation apoptosis through
activation of Tie2 via PI-3 Kinase and Akt pathway (Kim, I., et
al., Oncogene 19 (2000) 4549-52).
[0007] WO 2010/040508 A9 and WO 2011/117329 relate to bispecific
anti-VEGF/anti-ANG-2 antibodies. WO 2008/132568 relates to fusion
proteins binding to growth factors. WO 2009/136352 relates to
anti-angiogenic compounds. WO 2009/080253 and WO 2011/117330
relates to bispecific bivalent antibody formats. WO 2010/069532
relates to Ang2 antibodies.
[0008] Ocular vascular diseases such as age related macular
degeneration (ARMD) and diabetic retinopathy (DR) are due to
abnormal choroidal or retinal neovascularization respectively. They
are the leading causes of visual loss in industrialized nations.
Since the retina consists of well-defined layers of neuronal,
glial, and vascular elements, relatively small disturbances such as
those seen in vascular proliferation or edema can lead to
significant loss of visual function. Inherited retinal
degenerations, such as Retinitis Pigmentosa (RP), are also
associated with vascular abnormalities, such as arteriolar
narrowing and vascular atrophy. They affect as many as 1 in 3500
individuals and are characterized by progressive night blindness,
visual field loss, optic nerve atrophy, arteriolar attenuation, and
central loss of vision often progressing to complete blindness.
[0009] Ischemic retinopathies are characterized by loss or
dysfunction of the retinal vasculature which results in a reduction
of blood flow and hypoxia. The retina responds to hypoxia by
generating signals to grow new blood vessels, but these new vessels
are usually fragile and disorganized. It is the growth of these
abnormal new vessels that creates most of the threat to vision
since they can leak, hemorrhage or lead to scarring that may end in
retinal detachment. Current treatments for ischemic retinopathies
seek to halt the growth of the pathological vessels but do not
address the underlying ischemia that drives their growth.
Furthermore, standard treatment for diabetic retinopathy, an
ischemic retinopathy that affects millions, involves destruction of
a portion of the retina with a laser in an attempt to stop new
vessel growth and preserve central vision. Strategies have been
employed to block the function of vascular endothelial growth
factor (VEGF), a major promoter of vessel growth. In the short
term, anti-VEGF therapy can improve vision, but it does not address
the underlying ischemia and in fact may exacerbate this condition
as it inhibits all vessel growth, including beneficial collaterals.
There is also the serious concern of systemic exposure of these
drugs in elderly and/or diabetic patients where new vessel growth
may be required in ischemic brains, hearts or limbs.
[0010] Typically for ocular diseases via intravitreal application
smaller antibody fragments like Fab or Fab(2) are often used as
they have a low serum half-life and the risk of systemic toxicities
is lower. However this smaller fragments typically have also lower
intravitreal half-lifes (e.g. due to the faster diffusion into
serum) and have to be dosed typically more often.
[0011] Kim et al, Molecular Vision, 15 (2009) 2803-2812 relates to
full length antibodies administered intravitreally in the eye,
wherein an IgG with FcRn binding was eliminated into the blood in
wild-type mice, whereas an IgY with no FcRn binding was not
eliminated into the blood system. Furthermore the IgG with FcRn
binding was not eliminated into the blood system in FcRn
knockdown-mice.
[0012] There is a need in the art for better means for treating and
preventing various ocular vascular diseases such as ischemic
retinopathies.
SUMMARY OF THE INVENTION
[0013] One aspect of the invention is method for the reduction of
the viscosity of an antibody wherein the antibody comprises a
constant heavy chain region of human IgG1 or human IgG4 subclass
(derived from human origin and) wherein the method comprises the
modification of the antibody constant heavy chain region of human
IgG1 or human IgG4 subclass with the mutations I253A, H310A, and
H435A (numbering according to EU Index of Kabat).
[0014] In one embodiment of the invention said method is
characterized in that the antibody is a bispecific antibody
comprising a first antigen-binding site that specifically binds to
human VEGF and a second antigen-binding site that specifically
binds to human ANG-2, wherein [0015] i) said first antigen-binding
site specifically binding to VEGF comprises in the heavy chain
variable domain a CDR3H region of SEQ ID NO: 1, a CDR2H region of
SEQ ID NO: 2, and a CDR1H region of SEQ ID NO:3, and in the light
chain variable domain a CDR3L region of SEQ ID NO: 4, a CDR2L
region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:6; and
[0016] ii) said second antigen-binding site specifically binding to
ANG-2 comprises in the heavy chain variable domain a CDR3H region
of SEQ ID NO: 9, a CDR2H region of, SEQ ID NO: 10, and a CDR1H
region of SEQ ID NO: 11, and in the light chain variable domain a
CDR3L region of SEQ ID NO: 12, a CDR2L region of SEQ ID NO: 13, and
a CDR1L region of SEQ ID NO: 14, and wherein [0017] iii) the
bispecific antibody comprises a constant heavy chain region of
human IgG1 or human IgG4 subclass (derived from human origin and)
comprising the mutations I253A, H310A, and H435A (numbering
according to EU Index of Kabat).
[0018] In one embodiment of the invention such method is
characterized in that said bispecific antibody described above
comprises a constant heavy chain region of human IgG1 subclass
(derived from human origin and) comprising the mutations I253A,
H310A, and H435A (numbering according to EU Index of Kabat) and
further comprising the mutations L234A, L235A and P329G (numbering
according to EU Index of Kabat).
[0019] One embodiment of the invention is an antibody obtained by
such method.
[0020] One embodiment of the invention is the use of the mutations
I253A, H310A, and H435A (numbering according to EU Index of Kabat)
for the reduction of the viscosity of an antibody wherein the
antibody comprises a constant heavy chain region of human IgG1 or
human IgG4 subclass(derived from human origin).
[0021] In one embodiment of the invention said use is characterized
in that the antibody is a bispecific antibody comprising a first
antigen-binding site that specifically binds to human VEGF and a
second antigen-binding site that specifically binds to human
ANG-2,
[0022] wherein [0023] i) said first antigen-binding site
specifically binding to VEGF comprises in the heavy chain variable
domain a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO:
2, and a CDR1H region of SEQ ID NO:3, and in the light chain
variable domain a CDR3L region of SEQ ID NO: 4, a CDR2L region of
SEQ ID NO:5, and a CDR1L region of SEQ ID NO:6; and [0024] ii) said
second antigen-binding site specifically binding to ANG-2 comprises
in the heavy chain variable domain a CDR3H region of SEQ ID NO: 9,
a CDR2H region of, SEQ ID NO: 10, and a CDR1H region of SEQ ID NO:
11, and in the light chain variable domain a CDR3L region of SEQ ID
NO: 12, a CDR2L region of SEQ ID NO: 13, and a CDR1L region of SEQ
ID NO: 14, and wherein [0025] iii) the bispecific antibody
comprises a constant heavy chain region of human IgG1 or human IgG4
subclass (derived from human origin and) comprising the mutations
I253A, H310A, and H435A (numbering according to EU Index of
Kabat).
[0026] In one embodiment of the invention said specific use is
characterized in that the bispecific antibody comprises a constant
heavy chain region of human IgG1 subclass (derived from human
origin and) comprising the mutations I253A, H310A, and H435A
(numbering according to EU Index of Kabat) and further comprising
the mutations L234A, L235A and P329G (numbering according to EU
Index of Kabat).
[0027] The invention is further directed to a bispecific, bivalent
antibody comprising a first antigen-binding site that specifically
binds to human VEGF and a second antigen-binding site that
specifically binds to human ANG-2, wherein [0028] i) said first
antigen-binding site specifically binding to VEGF comprises in the
heavy chain variable domain a CDR3H region of SEQ ID NO: 1, a CDR2H
region of SEQ ID NO: 2, and a CDR1H region of SEQ ID NO:3, and in
the light chain variable domain a CDR3L region of SEQ ID NO: 4, a
CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:6; and
[0029] ii) said second antigen-binding site specifically binding to
ANG-2 comprises in the heavy chain variable domain a CDR3H region
of SEQ ID NO: 9, a CDR2H region of, SEQ ID NO: 10, and a CDR1H
region of SEQ ID NO: 11, and in the light chain variable domain a
CDR3L region of SEQ ID NO: 12, a CDR2L region of SEQ ID NO: 13, and
a CDR1L region of SEQ ID NO: 14,
[0030] and wherein [0031] iii) the bispecific antibody comprises a
constant heavy chain region of human IgG1 or human IgG4 subclass
(derived from human origin and) comprising the mutations I253A,
H310A, and H435A (numbering according to EU Index of Kabat).
[0032] In one embodiment said bispecific antibody is characterized
in that [0033] i) said first antigen-binding site specifically
binding to VEGF comprises as heavy chain variable domain VH an
amino acid sequence of SEQ ID NO: 7, and as light chain variable
domain VL an amino acid sequence of SEQ ID NO: 8, and [0034] ii)
said second antigen-binding site specifically binding to ANG-2
comprises as heavy chain variable domain VH an amino acid sequence
of SEQ ID NO: 15, and as light chain variable domain VL an amino
acid sequence of SEQ ID NO: 16.
[0035] In one embodiment said bispecific antibody is characterized
in that the constant heavy chain region under iii) is of human IgG1
subclass. In one embodiment said bispecific antibody of IgG1
subclass is characterized in that the constant heavy chain region
of IgG1 subclass further comprises the mutations L234A, L235A and
P329G (numbering according to EU Index of Kabat).
[0036] In one embodiment said bispecific antibody is characterized
in that the constant heavy chain region under iii) is of human IgG4
subclass. In one embodiment said bispecific antibody of IgG4
subclass is characterized in that the constant heavy chain region
of IgG4 subclass further comprises the mutations S228P and L235E
(numbering according to EU Index of Kabat). In one embodiment said
bispecific antibody of IgG4 subclass is characterized in that the
constant heavy chain region of IgG4 subclass further comprises the
mutations 228P, L235E and P329G (numbering according to EU Index of
Kabat).
[0037] Still further aspects of the invention are a pharmaceutical
composition comprising said bispecific antibody, said
pharmaceutical composition for use in the treatment of ocular
vascular diseases, the use of said bispecific antibody for the
manufacture of a medicament for the treatment of ocular vascular
diseases, a method of treatment of patient suffering from ocular
vascular diseases by administering said bispecific antibody to a
patient in the need of such treatment. In one embodiment the
bispecific antibody or the pharmaceutical composition comprising
said bispecific antibody is administered via intravitreal
application.
[0038] A further aspect of the invention is a nucleic acid molecule
encoding a heavy and/or light chain of a bispecific antibody
according to the invention.
[0039] The invention further provides expression vectors containing
said nucleic acid according to the invention capable of expressing
said nucleic acid in a prokaryotic or eukaryotic host cell, and
host cells containing such vectors for the recombinant production
of a bispecific antibody according to the invention.
[0040] The invention further comprises a prokaryotic or eukaryotic
host cell comprising a vector according to the invention.
[0041] The invention further comprises a method for the production
of a bispecific antibody according to the invention, characterized
by expressing a nucleic acid according to the invention in a
prokaryotic or eukaryotic host cell and recovering said bispecific
antibody from said cell or the cell culture supernatant. One
embodiment is a method for the preparation of a bispecific antibody
according to the invention comprising the steps of
[0042] a) transforming a host cell with vectors comprising nucleic
acid molecules encoding said antibody;
[0043] b) culturing the host cell under conditions that allow
synthesis of said antibody molecule; and
[0044] c) recovering said antibody molecule from said culture.
[0045] The invention further comprises the antibody obtained by
such method for the production of a bispecific antibody.
[0046] Accordingly one embodiment of the invention is a bispecific,
bivalent antibody comprising a first antigen-binding site that
specifically binds to human VEGF and a second antigen-binding site
that specifically binds to human ANG-2, characterized in comprising
the amino acid sequences of SEQ ID NO: 21, of SEQ ID NO: 22, of SEQ
ID NO: 23, and of SEQ ID NO: 24.
[0047] Accordingly one embodiment of the invention is a bispecific,
bivalent antibody comprising a first antigen-binding site that
specifically binds to human VEGF and a second antigen-binding site
that specifically binds to human ANG-2, characterized in comprising
the amino acid sequences of SEQ ID NO: 25, of SEQ ID NO: 26, of SEQ
ID NO: 27, and of SEQ ID NO: 28.
[0048] The antibodies according to the invention have highly
valuable properties due to their specific modifications in the Fc
part/constant region causing a benefit for a patient suffering from
ocular vascular diseases. They show high stability in the
intravitreal environment and slow diffusion from the eye (compared
to smaller antibody fragments without a constant heavy chain
region), where the actual disease is located and treated (so
treatment schedule can potentially be improved compared to non-IgG
like antibodies like e.g. Fab and (Fab)2 fragments). Surprisingly
compared to unmodified IgG antibodies the half-life in the eye
after intravitreal application of the antibodies with the mutations
I253A, H310A, and H435A in the constant region (with no more FcRn
binding) was similar (only slightly reduced) (Tables 17a and 18a
and FIGS. 7D and 7E), whereas the diffusion from the eye into the
blood serum was similar (Table 15 and FIG. 7B). This highly
valuable as it is desired for the treatment of ocular vascular
diseases related to ANG2 and/or VEGF it to eliminate VEGF and Ang2
from the eye (e. via the transportation into the blood serum as
anti-ANG2/ANG2 antibody complex or anti-VEGF/VEGF antibody
complex). The antibodies according to the invention are cleared on
the other hand quite rapidly from serum when compared to unmodified
IgG antibodies (which is highly desired to reduce potential side
effects arising from systemic exposure).
[0049] Surprisingly they also show lower viscosity (see FIG. 2)
(compared to versions without the mutations I253A, H310A, and H435A
in the constant region) and are therefore especially useful for
intravitreal application through thin needles during the treatment
of eye diseases (for such application typically thin needles are
used and high viscosity makes an appropriate application rather
difficult). The lower viscosity also allows higher concentration
formulations.
[0050] Also surprisingly the antibodies according to the invention
show a lower aggregation tendency (FIG. 4) during storage (compared
to versions without the mutations I253A, H310A, and H435A in the Fc
part) which is critical for intravitreal application in the eye (as
an aggregation in the eye can lead to complications during such
treatment). The bispecific antibodies according to the invention
show good efficacy in inhibition of vascular diseases.
[0051] In certain embodiments, the bispecific antibodies according
to the invention due to their specific modifications in the
constant region (e.g. P329G LALA) show valuable properties like no
binding Fcgamma receptors which reduces the risk of side effects
like thrombosis and/or unwanted cell death (due to e.g. ADCC).
DESCRIPTION OF THE FIGURES
[0052] FIG. 1 Scheme of concept and advantages of
<VEGF-ANG-2> IgG1 or IgG4 antibodies with AAA mutations
(mutations I253A, H310A, and H435A -numbering according to EU Index
of Kabat).
[0053] FIG. 2 Small-scale DLS-based viscosity measurement
Extrapolated viscosity at 150 mg/mL in 200 mM Arginine/Succinate,
pH 5.5 (comparison of <VEGF-ANG-2> antibodies according to
the invention VEGFang2-0016 (with AAA mutations) with a reference
VEGFang2-0015 (without such AAA mutations).
[0054] FIG. 3 DLS Aggregation depending on temperature (including
DLS aggregation onset temperature) in 20 mM His, 140 mM NaCl, pH
6.0 5 (comparison of <VEGF-ANG-2> antibodies according to the
invention VEGFang2-0016 (with AAA mutations) with a reference
VEGFang2-0015 (without such AAA mutations).
[0055] FIG. 4 7 day storage at 40.degree. C. at 100 mg/ml (Decrease
of Main and High Molecular Weight/HMW) increase) (comparison of
<VEGF-ANG-2> antibodies according to the invention
VEGFang2-0016 (with AAA mutations) which showed a lower aggregation
with a reference VEGFang2-0015 (without such AAA mutations)).
[0056] FIG. 5A FcRn steady state affinity of VEGFang2-0015 (without
AAA mutations): overlay of Biacore sensogramms at different
concentrations shows a concentration dependent binding of
VEGFang2-0015 (without AAA mutations) to FcRn.
[0057] FIG. 5B FcRn steady state affinity of A: VEGFang2-0015
(without AAA mutations): the concentration dependent binding
response curve of VEGFang2-0015 (without AAA mutations) shows
binding to FcRn.
[0058] FIG. 5C FcRn steady state affinity of VEGFang2-0016 (with
AAA mutations): overlay of Biacore sensogramms at different
concentrations shows no binding to FcRn at all concentrations.
[0059] FIG. 5D FcRn steady state affinity of VEGFang2-0016 (with
AAA mutations): the concentration dependent binding response curve
of VEGFang2-0016 (with AAA mutations) shows no binding to FcRn.
[0060] FIG. 5E FcRn steady state affinity of VEGFang2-0016 (with
AAA mutations): the concentration dependent binding response curve
of VEGFang2-0016 (with AAA mutations) shows no binding to FcRn
(Response range from -0.6 to 0.2 RU/concentration scale ranges from
0 to 0.35 M).
[0061] FIG. 6 FcgammaRIIIa interaction of VEGFang2-0015 without AAA
mutations and VEGFang2-0016 with AAA mutations measurement (both
are IgG1 subclass with P329G LALA mutations; as controls an
Anti-Dig of IgG1 subclass and a IgG4 based antibody was used).
[0062] FIG. 7A Schematic Pk-ELISA Assay Principle for determination
of concentrations of <VEGF/Ang2> bispecific antibodies in
serum and whole eye lysates.
[0063] FIG. 7B Serum concentration after intravenous application:
Comparison of compounds -VEGFang2-0015 without AAA mutations and
VEGFang2-0016 with AAA mutations.
[0064] FIG. 7C Serum concentration after intravitreal application:
Comparison of compounds -VEGFang2-0015 without AAA mutations and
VEGFang2-0016 with AAA mutations.
[0065] FIG. 7D Eye lysates concentration of VEGFang2-0016 (with AAA
mutation) in right and left eye (after intravitreal application
only into the right eye in comparison to intravenous application):
Significant concentrations could be detected only in the right eye
after intravitreal application. After intravenous application no
concentrations in eye lysates could be detected due to the low
serum half-life of VEGFang2-0016 (with AAA mutation).
[0066] FIG. 7E Eye lysates concentration of VEGFang2-0015 (without
AAA mutation) in right and left eye (after intravitreal application
only into the right eye in comparison to intravenous application):
In the right eye (and to some extent in the left eye) after
intravitreal application concentrations of VEGFang2-0015 could be
detected. This indicates the diffusion from the right eye into
serum and from there into the left eye, which can be explained by
the long half-life of VEGFang2-0015 (without AAA mutation). After
intravenous application also significant concentrations in eye
lysates of both eyes could be detected due to diffusion into the
eyes of the serum-stable VEGFang2-0015 (without AAA mutation).
DETAILED DESCRIPTION OF THE INVENTION
[0067] In one embodiment of the invention the bispecific antibody
according to the invention is bivalent.
[0068] In one aspect of the invention such bispecific, bivalent
antibody according to the invention is characterized in comprising
[0069] a) the heavy chain and the light chain of a first full
length antibody that specifically binds to VEGF; [0070] b) the
modified heavy chain and modified light chain of a second full
length antibody that specifically binds to ANG-2, wherein the
constant domains CL and CH1 are replaced by each other.
[0071] This bispecific, bivalent antibody format for the bispecific
antibody specifically binding to human vascular endothelial growth
factor (VEGF) and human angiopoietin-2 (ANG-2) is described in WO
2009/080253 (including Knobs-into-Holes modified CH3 domains). The
antibodies based on this bispecific, bivalent antibody format are
named CrossMabs.
[0072] In one embodiment such bispecific, bivalent antibody is
characterized in comprising [0073] a) as heavy chain of the first
full length antibody the amino acid sequence of SEQ ID NO: 25, and
as light chain of the first full length antibody the amino acid
sequence of SEQ ID NO: 27, and [0074] b) as modified heavy chain of
the second full length antibody the amino acid sequence of SEQ ID
NO: 26, and as modified light chain of the second full length
antibody the amino acid sequence of SEQ ID NO: 28.
[0075] In one embodiment such bispecific, bivalent antibody is
characterized in comprising [0076] a) as heavy chain of the first
full length antibody the amino acid sequence of SEQ ID NO: 21, and
as light chain of the first full length antibody the amino acid
sequence of SEQ ID NO: 23, and [0077] b) as modified heavy chain of
the second full length antibody the amino acid sequence of SEQ ID
NO: 22, and as modified light chain of the second full length
antibody the amino acid sequence of SEQ ID NO: 24.
[0078] In one embodiment such bispecific, bivalent antibody is
characterized in comprising [0079] a) as heavy chain of the first
full length antibody the amino acid sequence of SEQ ID NO: 29, and
as light chain of the first full length antibody the amino acid
sequence of SEQ ID NO: 31, and [0080] b) as modified heavy chain of
the second full length antibody the amino acid sequence of SEQ ID
NO: 30, and as modified light chain of the second full length
antibody the amino acid sequence of SEQ ID NO: 32.
[0081] Accordingly one embodiment of the invention is a bispecific,
bivalent antibody comprising a first antigen-binding site that
specifically binds to human VEGF and a second antigen-binding site
that specifically binds to human ANG-2, characterized in comprising
the amino acid sequences of SEQ ID NO: 25, of SEQ ID NO: 26, of SEQ
ID NO: 27, and of SEQ ID NO: 28.
[0082] Accordingly one embodiment of the invention is a bispecific,
bivalent antibody comprising a first antigen-binding site that
specifically binds to human VEGF and a second antigen-binding site
that specifically binds to human ANG-2, characterized in comprising
the amino acid sequences of SEQ ID NO: 21, of SEQ ID NO: 22, of SEQ
ID NO: 23, and of SEQ ID NO: 24.
[0083] Accordingly one embodiment of the invention is a bispecific,
bivalent antibody comprising a first antigen-binding site that
specifically binds to human VEGF and a second antigen-binding site
that specifically binds to human ANG-2, characterized in comprising
the amino acid sequences of SEQ ID NO: 29, of SEQ ID NO: 30, of SEQ
ID NO: 31, and of SEQ ID NO: 32.
[0084] In another aspect of the invention the bispecific antibody
according to the invention is characterized in comprising [0085] a)
the heavy chain and the light chain of a first full length antibody
that specifically binds to VEGF; [0086] b) the heavy chain and the
light chain of a second full length antibody that specifically
binds to ANG-2, wherein the N-terminus of the heavy chain is
connected to the C-terminus of the light chain via a peptide
linker.
[0087] This bispecific, bivalent antibody format for this
bispecific antibody specifically binding to human vascular
endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2)
is described in WO 2011/117330 including Knobs-into-Holes modified
CH3 domains. The antibodies based on this bispecific, bivalent
antibody format are named OAscFabs.
[0088] In one embodiment such bispecific, bivalent antibody is
characterized in comprising [0089] a) as heavy chain of the first
full length antibody the amino acid sequence of SEQ ID NO: 33, and
as light chain of the first full length antibody the amino acid
sequence of SEQ ID NO: 35, and [0090] b) as heavy chain of the
second full length antibody connected to the light chain of the
second full length antibody via a peptide linker the amino acid
sequence of SEQ ID NO: 34.
[0091] In one embodiment such bispecific, bivalent antibody is
characterized in comprising [0092] a) as heavy chain of the first
full length antibody the amino acid sequence of SEQ ID NO: 36, and
as light chain of the first full length antibody the amino acid
sequence of SEQ ID NO: 38, and [0093] b) as heavy chain of the
second full length antibody connected to the light chain of the
second full length antibody via a peptide linker the amino acid
sequence of SEQ ID NO: 37.
[0094] In one embodiment the antibody heavy chain variable domain
(VH) and the antibody light chain variable domain (VL) of the heavy
and light chain of the second full length antibody are disulfide
stabilized by introduction of a disulfide bond between the
following positions: heavy chain variable domain position 44 to
light chain variable domain position 100 (numbering always
according to EU index of Kabat (Kabat, E. A., et al., Sequences of
Proteins of Immunological Interest, 5th ed., Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)). Such further
disulfide stabilization is achieved by the introduction of a
disulfide bond between the variable domains VH and VL of the second
full length antibody heavy and light chain. Techniques to introduce
unnatural disulfide bridges for stabilization are described e.g. in
WO 94/029350, Rajagopal, V., et al, Prot. Engin. 10 (1997) 1453-59;
Kobayashi et al., Nuclear Medicine & Biology 25 (1998) 387-393;
or Schmidt, M., et al., Oncogene 18 (1999) 1711-1721.
[0095] Accordingly one embodiment of the invention is a bispecific,
bivalent antibody comprising a first antigen-binding site that
specifically binds to human VEGF and a second antigen-binding site
that specifically binds to human ANG-2, characterized in comprising
the amino acid sequences of SEQ ID NO: 33, of SEQ ID NO: 34, and of
SEQ ID NO: 35.
[0096] Accordingly one embodiment of the invention is a bispecific,
bivalent antibody comprising a first antigen-binding site that
specifically binds to human VEGF and a second antigen-binding site
that specifically binds to human ANG-2, characterized in comprising
the amino acid sequences of SEQ ID NO: 36, of SEQ ID NO: 37, and of
SEQ ID NO: 38.
[0097] In one embodiment the CH3 domains of the bispecific,
bivalent antibody according to the invention is altered by the
"knob-into-holes" technology which is described in detail with
several examples in e.g. WO 96/027011, Ridgway J. B., et al.,
Protein Eng 9 (1996) 617-621; and Merchant, A. M., et al., Nat
Biotechnol 16 (1998) 677-681. In this method the interaction
surfaces of the two CH3 domains are altered to increase the
heterodimerisation of both heavy chains containing these two CH3
domains. Each of the two CH3 domains (of the two heavy chains) can
be the "knob", while the other is the "hole". The introduction of a
disulfide bridge stabilizes the heterodimers (Merchant, A.M, et
al., Nature Biotech 16 (1998) 677-681; Atwell, S., et al. J. Mol.
Biol. 270 (1997) 26-35) and increases the yield.
[0098] In a preferred aspect of the invention all bispecific
antibodies according to the invention are characterized in that
[0099] the CH3 domain of one heavy chain and the CH3 domain of the
other heavy chain each meet at an interface which comprises an
original interface between the antibody CH3 domains;
[0100] wherein said interface is altered to promote the formation
of the bispecific antibody, wherein the alteration is characterized
in that:
[0101] a) the CH3 domain of one heavy chain is altered,
[0102] so that within the original interface the CH3 domain of one
heavy chain that meets the original interface of the CH3 domain of
the other heavy chain within the bispecific antibody,
[0103] an amino acid residue is replaced with an amino acid residue
having a larger side chain volume, thereby generating a
protuberance within the interface of the CH3 domain of one heavy
chain which is positionable in a cavity within the interface of the
CH3 domain of the other heavy chain
[0104] and
[0105] b) the CH3 domain of the other heavy chain is altered,
[0106] so that within the original interface of the second CH3
domain that meets the original interface of the first CH3 domain
within the bispecific antibody
[0107] an amino acid residue is replaced with an amino acid residue
having a smaller side chain volume, thereby generating a cavity
within the interface of the second CH3 domain within which a
protuberance within the interface of the first CH3 domain is
positionable.
[0108] Thus the antibody according to invention is preferably
characterized in that [0109] the CH3 domain of the heavy chain of
the full length antibody of a) and the CH3 domain of the heavy
chain of the full length antibody of b) each meet at an interface
which comprises an alteration in the original interface between the
antibody CH3 domains; [0110] wherein i) in the CH3 domain of one
heavy chain [0111] an amino acid residue is replaced with an amino
acid residue having a larger side chain volume, thereby generating
a protuberance within the interface of the CH3 domain of one heavy
chain which is positionable in a cavity within the interface of the
CH3 domain of the other heavy chain [0112] and wherein [0113] ii)
in the CH3 domain of the other heavy chain [0114] an amino acid
residue is replaced with an amino acid residue having a smaller
side chain volume, thereby generating a cavity within the interface
of the second CH3 domain within which a protuberance within the
interface of the first CH3 domain is positionable.
[0115] Preferably said amino acid residue having a larger side
chain volume is selected from the group consisting of arginine (R),
phenylalanine (F), tyrosine (Y), tryptophan (W).
[0116] Preferably said amino acid residue having a smaller side
chain volume is selected from the group consisting of alanine (A),
serine (S), threonine (T), valine (V).
[0117] In one aspect of the invention both CH3 domains are further
altered by the introduction of cysteine (C) as amino acid in the
corresponding positions of each CH3 domain such that a disulfide
bridge between both CH3 domains can be formed.
[0118] In one embodiment, the bispecific antibody comprises a T366W
mutation in the CH3 domain of the "knobs chain" and T366S, L368A,
Y407V mutations in the CH3 domain of the "hole chain". An
additional interchain disulfide bridge between the CH3 domains can
also be used (Merchant, A. M, et al., Nature Biotech 16 (1998)
677-681) e.g. by introducing a Y349C mutation into the CH3 domain
of the "knobs chain" and a E356C mutation or a S354C mutation into
the CH3 domain of the "hole chain".
[0119] In another embodiment, the bispecific antibody according to
the invention comprises Y349C, T366W mutations in one of the two
CH3 domains and E356C, T366S, L368A, Y407V mutations in the other
of the two CH3 domains. In a another preferred embodiment the
bispecific antibody comprises Y349C, T366W mutations in one of the
two CH3 domains and S354C, T366S, L368A, Y407V mutations in the
other of the two CH3 domains (the additional Y349C mutation in one
CH3 domain and the additional E356C or S354C mutation in the other
CH3 domain forming a interchain disulfide bridge) (numbering always
according to EU index of Kabat (Kabat, E. A., et al., Sequences of
Proteins of Immunological Interest, 5th ed., Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)). But also
other knobs-in-holes technologies as described by
[0120] EP 1 870 459 Al, can be used alternatively or additionally.
Thus another example for the bispecific antibody are R409D; K370E
mutations in the CH3 domain of the "knobs chain" and D399K; E357K
mutations in the CH3 domain of the "hole chain" (numbering always
according to EU index of Kabat (Kabat, E. A., et al., Sequences of
Proteins of Immunological Interest, 5th ed., Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)).
[0121] In another embodiment the bispecific antibody comprises a
T366W mutation in the CH3 domain of the "knobs chain" and T366S,
L368A, Y407V mutations in the CH3 domain of the "hole chain" and
additionally R409D; K370E mutations in the CH3 domain of the "knobs
chain" and D399K; E357K mutations in the CH3 domain of the "hole
chain".
[0122] In another embodiment the bispecific antibody comprises
Y349C, T366W mutations in one of the two CH3 domains and S354C,
T366S, L368A, Y407V mutations in the other of the two CH3 domains
or said trivalent, bispecific antibody comprises Y349C, T366W
mutations in one of the two CH3 domains and S354C, T366S, L368A,
Y407V mutations in the other of the two CH3 domains and
additionally R409D; K370E mutations in the CH3 domain of the "knobs
chain" and D399K; E357K mutations in the CH3 domain of the "hole
chain".
[0123] In one embodiment of the invention the bispecific antibody
according to the invention is characterized in having one or more
of the following properties (determined in assays as described in
Example 6 [0124] shows a lower serum concentration compared to
corresponding bispecific antibody without the mutations described
under iii) (96 hours after intravitreal application in mice, which
are mouse FcRn deficient, but hemizygous transgenic for human
FcRn); [0125] shows a similar (factor 0.8 to 1.2) concentration in
whole right eye lysates compared to corresponding bispecific
antibody without the mutations described under iii) (in mice, which
are mouse FcRn deficient, but hemizygous transgenic for human FcRn,
96 hours after intravitreal application in the right eye).
[0126] In one embodiment the bispecific, bivalent antibody is
characterized in comprising
[0127] a first antigen-binding site that specifically binds to
human VEGF and a second antigen-binding site that specifically
binds to human ANG-2, characterized in that [0128] i) said first
antigen-binding site comprises as heavy chain variable domain (VH)
the SEQ ID NO: 7, and as light chain variable domain (VL) the SEQ
ID NO: 8; and [0129] ii) said second antigen-binding site comprises
as heavy chain variable domain (VH) the SEQ ID NO: 15, and as light
chain variable domain (VL) the SEQ ID NO: 16; and [0130] iii) the
bispecific antibody comprises a constant heavy chain region of IgG1
or IgG4 subclass (derived from human origin and) comprising the
mutations I253A, H310A, and H435A (numbering according to EU Index
of Kabat) and having one or more of the following properties
(determined in assays as described in Example 6 [0131] shows a
lower serum concentration compared to corresponding bispecific
antibody without the mutations described under iii) (96 hours after
intravitreal application in mice, which are mouse FcRn deficient,
but hemizygous transgenic for human FcRn); [0132] shows a similar
(factor 0.8 to 1.2) concentration in whole right eye lysates
compared to corresponding bispecific antibody without the mutations
described under iii) (in mice, which are mouse FcRn deficient, but
hemizygous transgenic for human FcRn, 96 hours after intravitreal
application in the right eye).
[0133] In one embodiment the bispecific antibody is characterized
in comprising a first antigen-binding site that specifically binds
to human VEGF and a second antigen-binding site that specifically
binds to human ANG-2, characterized in that [0134] i) said first
antigen-binding site comprises as heavy chain variable domain (VH)
the SEQ ID NO: 7 with 1, 2 or 3 amino acid residue substitutions,
and as light chain variable domain (VL) the SEQ ID NO: 8 with 1, 2
or 3 amino acid residue substitutions; and [0135] ii) said second
antigen-binding site comprises as heavy chain variable domain (VH)
the SEQ ID NO: 15 with 1, 2 or 3 amino acid residue substitutions,
and as light chain variable domain (VL) the SEQ ID NO: with 1, 2 or
3 amino acid residue substitutions; and [0136] iii) the bispecific
antibody comprises a constant heavy chain region of IgG1 or IgG4
subclass (derived from human origin and) comprising the mutations
I253A, H310A, and H435A (numbering according to EU Index of
Kabat)
[0137] and having one or more of the following properties
(determined in assays as described in Example 6 [0138] shows a
lower serum concentration compared to corresponding bispecific
antibody without the mutations described under iii) (96 hours after
intravitreal application in mice, which are mouse FcRn deficient,
but hemizygous transgenic for human FcRn); [0139] shows a similar
(factor 0.8 to 1.2) concentration in whole right eye lysates
compared to corresponding bispecific antibody without the mutations
described under iii) (in mice, which are mouse FcRn deficient, but
hemizygous transgenic for human FcRn, 96 hours after intravitreal
application in the right eye).
[0140] As used herein, "antibody" refers to a binding protein that
comprises antigen-binding sites. The terms "binding site" or
"antigen-binding site" as used herein denotes the region(s) of an
antibody molecule to which a ligand actually binds. The term
"antigen-binding site" comprises an antibody heavy chain variable
domains (VH) and an antibody light chain variable domains (VL)
(pair of VH/VL)).
[0141] Antibody specificity refers to selective recognition of the
antibody for a particular epitope of an antigen. Natural
antibodies, for example, are monospecific.
[0142] "Bispecific antibodies" according to the invention are
antibodies which have two different antigen-binding specificities.
Antibodies of the present invention are specific for two different
antigens, VEGF as first antigen and ANG-2 as second antigen.
[0143] The term "monospecific" antibody as used herein denotes an
antibody that has one or more binding sites each of which bind to
the same epitope of the same antigen.
[0144] The term "valent" as used within the current application
denotes the presence of a specified number of binding sites in an
antibody molecule. As such, the terms "bivalent", "tetravalent",
and "hexavalent" denote the presence of two binding site, four
binding sites, and six binding sites, respectively, in an antibody
molecule. The bispecific antibodies according to the invention are
preferably "bivalent".
[0145] The term "VEGF" as used herein refers to human vascular
endothelial growth factor (VEGF/VEGF-A,) the 165-amino acid human
vascular endothelial cell growth factor (amino acid 27-191 of
precursor sequence of human VEGF165: SEQ
[0146] ID NO: 17; amino acids 1-26 represent the signal peptide),
and related 121, 189, and 206 vascular endothelial cell growth
factor isoforms, as described by Leung, D. W., et al., Science 246
(1989) 1306-9; Houck et al., Mol. Endocrin. 5 (1991) 1806 -1814;
Keck, P. J., et al., Science 246 (1989) 1309-12 and Connolly, D.
T., et al., J. Biol. Chem. 264 (1989) 20017-24; together with the
naturally occurring allelic and processed forms of those growth
factors. VEGF is involved in the regulation of normal and abnormal
angiogenesis and neovascularization associated with tumors and
intraocular disorders (Ferrara, N., et al., Endocr. Rev. 18 (1997)
4-25; Berkman, R. A.,et al., J. Clin. Invest. 91 (1993) 153-159;
Brown, L. F., et al., Human Pathol. 26 (1995) 86-91; Brown, L. F.,
et al., Cancer Res. 53 (1993) 4727-4735; Mattern, J., et al., Brit.
J. Cancer. 73 (1996) 931-934; and Dvorak, H. F., et al., Am. J.
Pathol. 146 (1995) 1029-1039). VEGF is a homodimeric glycoprotein
that has been isolated from several sources and includes several
isoforms. VEGF shows highly specific mitogenic activity for
endothelial cells.
[0147] The term "ANG-2" as used herein refers to human
angiopoietin-2 (ANG-2) (alternatively abbreviated with ANGPT2 or
ANG2) (SEQ ID NO: 18) which is described e.g. in Maisonpierre, P.
C., et al, Science 277 (1997) 55-60 and Cheung, A. H., et al.,
Genomics 48 (1998) 389-91. The angiopoietins-1 (SEQ ID NO: 19) and
-2 were discovered as ligands for the Ties, a family of tyrosine
kinases that is selectively expressed within the vascular
endothelium (Yancopoulos, G. D., et al., Nature 407 (2000) 242-48).
There are now four definitive members of the angiopoietin family.
Angiopoietin-3 and -4 (Ang-3 and Ang-4) may represent widely
diverged counterparts of the same gene locus in mouse and man (Kim,
I., et al., FEBS Let, 443 (1999) 353-56; Kim, I., et al., J Biol
Chem 274 (1999) 26523-28). ANG-1 and ANG-2 were originally
identified in tissue culture experiments as agonist and antagonist,
respectively (see for ANG-1: Davis, S., et al., Cell 87 (1996)
1161-69; and for ANG-2: Maisonpierre, P. C., et al., Science 277
(1997) 55-60). All of the known angiopoietins bind primarily to
Tie2 (SEQ ID NO: 20), and both Ang-1 and -2 bind to Tie2 with an
affinity of 3 nM (Kd) (Maisonpierre, P. C., et al., Science 277
(1997) 55-60).
[0148] An antigen-binding sites of the bispecific antibody of the
invention contain six complementarity determining regions (CDRs)
which contribute in varying degrees to the affinity of the binding
site for antigen. There are three heavy chain variable domain CDRs
(CDRH1, CDRH2 and CDRH3) and three light chain variable domain CDRs
(CDRL1, CDRL2 and CDRL3). The extent of CDR and framework regions
(FRs) is determined by comparison to a compiled database of amino
acid sequences in which those regions have been defined according
to variability among the sequences.
[0149] The antibodies of the invention comprise immunoglobulin
constant regions derived from human origin of one or more
immunoglobulin classes, wherein such. immunoglobulin classes
include IgG, IgM, IgA, IgD, and IgE classes and, in the case of IgG
and IgA, their subclasses, especially IgG1 and IgG4.
[0150] The terms "monoclonal antibody" or "monoclonal antibody
composition" as used herein refer to a preparation of antibody
molecules of a single amino acid composition.
[0151] The term "chimeric antibody" refers to an antibody
comprising a variable region, i.e., binding region, from one source
or species and at least a portion of a constant region derived from
a different source or species, usually prepared by recombinant DNA
techniques. Chimeric antibodies comprising a murine variable region
and a human constant region are preferred. Other preferred forms of
"chimeric antibodies" encompassed by the present invention are
those in which the constant region has been modified or changed
from that of the original antibody to generate the properties
according to the invention, especially in regard to C1q binding
and/or Fc receptor (FcR) binding. Such chimeric antibodies are also
referred to as "class-switched antibodies.". Chimeric antibodies
are the product of expressed immunoglobulin genes comprising DNA
segments encoding immunoglobulin variable regions and DNA segments
encoding immunoglobulin constant regions. Methods for producing
chimeric antibodies involve conventional recombinant DNA and gene
transfection techniques are well known in the art. See, e.g.,
Morrison, S. L., et al., Proc. Natl. Acad. Sci. USA 81 (1984)
6851-6855; U.S. Pat. No. 5,202,238 and U.S. Pat. No. 5,204,244.
[0152] The term "humanized antibody" refers to antibodies in which
the framework or "complementarity determining regions" (CDR) have
been modified to comprise the CDR of an immunoglobulin of different
specificity as compared to that of the parent immunoglobulin. In a
preferred embodiment, a murine CDR is grafted into the framework
region of a human antibody to prepare the "humanized antibody."
See, e.g., Riechmann, L., et al., Nature 332 (1988) 323-327; and
Neuberger, M. S., et al., Nature 314 (1985) 268-270. Particularly
preferred CDRs correspond to those representing sequences
recognizing the antigens noted above for chimeric antibodies. Other
forms of "humanized antibodies" encompassed by the present
invention are those in which the constant region has been
additionally modified or changed from that of the original antibody
to generate the properties according to the invention, especially
in regard to C1q binding and/or Fc receptor (FcR) binding.
[0153] The term "human antibody", as used herein, is intended to
include antibodies having variable and constant regions derived
from human germ line immunoglobulin sequences. Human antibodies are
well-known in the state of the art (van Dijk, M. A., and van de
Winkel, J. G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human
antibodies can also be produced in transgenic animals (e.g., mice)
that are capable, upon immunization, of producing a full repertoire
or a selection of human antibodies in the absence of endogenous
immunoglobulin production. Transfer of the human germ-line
immunoglobulin gene array in such germ-line mutant mice will result
in the production of human antibodies upon antigen challenge (see,
e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993)
2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258;
Brueggemann, M., et al., Year Immunol. 7 (1993) 33-40). Human
antibodies can also be produced in phage display libraries
(Hoogenboom, H. R., and Winter, G., J. Mol. Biol. 227 (1992)
381-388; Marks, J. D., et al., J. Mol. Biol. 222 (1991) 581-597).
The techniques of Cole, A., et al. and Boerner, P., et al. are also
available for the preparation of human monoclonal antibodies (Cole,
A., et al., Monoclonal Antibodies and Cancer Therapy, Liss, A. L.,
p. 77 (1985); and Boerner, P., et al., J. Immunol. 147 (1991)
86-95). As already mentioned for chimeric and humanized antibodies
according to the invention the term "human antibody" as used herein
also comprises such antibodies which are modified in the constant
region to generate the properties according to the invention,
especially in regard to C1q binding and/or FcR binding, e.g. by
"class switching" i.e. change or mutation of Fc parts (e.g. from
IgG1 to IgG4 and/or IgG1/IgG4 mutation).
[0154] The term "recombinant antibody", as used herein, is intended
to include all human antibodies that are prepared, expressed,
created or isolated by recombinant means, such as antibodies
isolated from a host cell such as a NS0 or CHO cell or from an
animal (e.g. a mouse) that is transgenic for human immunoglobulin
genes or antibodies expressed using a recombinant expression vector
transfected into a host cell. Such recombinant antibodies have
variable and constant regions in a rearranged form. The recombinant
antibodies according to the invention have been subjected to in
vivo somatic hypermutation. Thus, the amino acid sequences of the
VH and VL regions of the recombinant antibodies are sequences that,
while derived from and related to human germ line VH and VL
sequences, may not naturally exist within the human antibody germ
line repertoire in vivo.
[0155] The "variable domain" (variable domain of a light chain
(VL), variable domain of a heavy chain (VH) as used herein denotes
each of the pair of light and heavy chains which is involved
directly in binding the antibody to the antigen. The domains of
variable human light and heavy chains have the same general
structure and each domain comprises four framework (FR) regions
whose sequences are widely conserved, connected by three
"hypervariable regions" (or complementarity determining regions,
CDRs). The framework regions adopt a .beta.-sheet conformation and
the CDRs may form loops connecting the .beta.-sheet structure. The
CDRs in each chain are held in their three-dimensional structure by
the framework regions and form together with the CDRs from the
other chain the antigen binding site. The antibody heavy and light
chain CDR3 regions play a particularly important role in the
binding specificity/affinity of the antibodies according to the
invention and therefore provide a further object of the
invention.
[0156] The terms "hypervariable region" or "antigen-binding portion
of an antibody" when used herein refer to the amino acid residues
of an antibody which are responsible for antigen-binding. The
hypervariable region comprises amino acid residues from the
"complementarity determining regions" or "CDRs". "Framework" or
"FR" regions are those variable domain regions other than the
hypervariable region residues as herein defined. Therefore, the
light and heavy chains of an antibody comprise from N- to
C-terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
CDRs on each chain are separated by such framework amino acids.
Especially, CDR3 of the heavy chain is the region which contributes
most to antigen binding. CDR and FR regions are determined
according to the standard definition of Kabat, E. A., et al.,
Sequences of Proteins of Immunological Interest, 5th ed., Public
Health Service, National Institutes of Health, Bethesda, Md.
(1991).
[0157] As used herein, the term "binding" or "specifically binding"
refers to the binding of the antibody to an epitope of the antigen
(either human VEGF or human ANG-2) in an in vitro assay, preferably
in an plasmon resonance assay (BlAcore, GE-Healthcare Uppsala,
Sweden with purified wild-type antigen. The affinity of the binding
is defined by the terms ka (rate constant for the association of
the antibody from the antibody/antigen complex), k.sub.D
(dissociation constant), and K.sub.D (k.sub.D/ka). In one
embodiment binding or specifically binding means a binding affinity
(K.sub.D) of 10.sup.-8 mol/l or less, in one embodiment 10.sup.-9 M
to 10.sup.-13 mol/l.
[0158] The term "epitope" includes any polypeptide determinant
capable of specific binding to an antibody. In certain embodiments,
epitope determinant include chemically active surface groupings of
molecules such as amino acids, sugar side chains, phosphoryl, or
sulfonyl, and, in certain embodiments, may have specific three
dimensional structural characteristics, and or specific charge
characteristics. An epitope is a region of an antigen that is bound
by an antibody.
[0159] In certain embodiments, an antibody is said to specifically
bind an antigen when it preferentially recognizes its target
antigen in a complex mixture of proteins and/or macromolecules.
[0160] The term "full length antibody" denotes an antibody
consisting of two "full length antibody heavy chains" and two "full
length antibody light chains". A "full length antibody heavy chain"
is a polypeptide consisting in N-terminal to C-terminal direction
of an antibody heavy chain variable domain (VH), an antibody
constant heavy chain domain 1 (CH1), an antibody hinge region (HR),
an antibody heavy chain constant domain 2 (CH2), and an antibody
heavy chain constant domain 3 (CH3), abbreviated as
VH-CH1-HR-CH2-CH3; and optionally an antibody heavy chain constant
domain 4 (CH4) in case of an antibody of the subclass IgE.
Preferably the "full length antibody heavy chain" is a polypeptide
consisting in N-terminal to C-terminal direction of VH, CH1, HR,
CH2 and CH3. A "full length antibody light chain" is a polypeptide
consisting in N-terminal to C-terminal direction of an antibody
light chain variable domain (VL), and an antibody light chain
constant domain (CL), abbreviated as VL-CL. The antibody light
chain constant domain (CL) can be .kappa. (kappa) or .lamda.
(lambda). The two full length antibody chains are linked together
via inter-polypeptide disulfide bonds between the CL domain and the
CH1 domain and between the hinge regions of the full length
antibody heavy chains. Examples of typical full length antibodies
are natural antibodies like IgG (e.g. IgG 1 and IgG2), IgM, IgA,
IgD, and IgE. The full length antibodies according to the invention
can be from a single species e.g. human, or they can be chimerized
or humanized antibodies. The full length antibodies according to
the invention comprise two antigen binding sites each formed by a
pair of VH and VL, which both specifically bind to the same
antigen. The C-terminus of the heavy or light chain of said full
length antibody denotes the last amino acid at the C-terminus of
said heavy or light chain. The N-terminus of the heavy or light
chain of said full length antibody denotes the last amino acid at
the N-terminus of said heavy or light chain.
[0161] The term "peptide linker" as used within the invention
denotes a peptide with amino acid sequences, which is preferably of
synthetic origin. These peptides according to invention are used to
connect the C-terminus of the light chain to the N-terminus of
heavy chain of the second full length antibody (that specifically
binds to a second antigen) via a peptide linker. The peptide linker
within the second full length antibody heavy and light chain is a
peptide with an amino acid sequence with a length of at least 30
amino acids, preferably with a length of 32 to 50 amino acids. In
one the peptide linker is a peptide with an amino acid sequence
with a length of 32 to 40 amino acids. In one embodiment said
linker is (GxS)n with G=glycine, S=serine, (x=3, n=8, 9 or 10 and
m=0, 1, 2 or 3) or (x=4 and n=6, 7 or 8 and m=0, 1, 2 or 3),
preferably with x=4, n=6 or 7 and m=0, 1, 2 or 3, more preferably
with x=4, n=7 and m=2. In one embodiment said linker is
(G.sub.4S).sub.6G.sub.2.
[0162] The term "constant region" as used within the current
applications denotes the sum of the domains of an antibody other
than the variable region. The constant region is not involved
directly in binding of an antigen, but exhibits various effector
functions. Depending on the amino acid sequence of the constant
region of their heavy chains, antibodies are divided in the
classes: IgA, IgD, IgE, IgG and IgM, and several of these may be
further divided into subclasses, such as IgG1, IgG2, IgG3, and
IgG4, IgA1 and IgA2. The heavy chain constant regions that
correspond to the different classes of antibodies are called
.alpha., .delta., .epsilon., .gamma., and .mu., respectively. The
light chain constant regions which can be found in all five
antibody classes are called .kappa. (kappa) and .lamda.
(lambda).
[0163] The terms "constant region derived from human origin" or "
human constant region" as used in the current application denotes a
constant heavy chain region of a human antibody of the subclass
IgG1, IgG2, IgG3, or IgG4 and/or a constant light chain kappa or
lambda region. Such constant regions are well known in the state of
the art and e.g. described by Kabat, E. A., et al., Sequences of
Proteins of Immunological Interest, 5th ed., Public Health Service,
National Institutes of Health, Bethesda, Md. (1991) (see also e.g.
Johnson, G., and Wu, T. T., Nucleic Acids Res. 28 (2000) 214-218;
Kabat, E. A., et al., Proc. Natl. Acad. Sci. USA 72 (1975)
2785-2788). Within the application for the numbering of positions
and mutations the EU numbering system (EU Index) according to
Kabat, E. A., et al., Sequences of Proteins of Immunological
Interest, 5th ed., Public Health Service, National Institutes of
Health, Bethesda, Md. (1991) is used and referred to as "numbering
according to EU Index of Kabat".
[0164] In one embodiment the bispecific antibodies according to the
invention have a constant region of human IgG1 subclass (derived
from human IgG1 subclass) .
[0165] In one embodiment the bispecific antibodies according to the
invention have a constant region of human IgG4 subclass (derived
from human IgG1 subclass).
[0166] In one embodiment the bispecific antibody according to the
invention is of human IgG1 subclass with mutations L234A
(Leu234Ala), L235A (Leu235Ala) and P329G (Pro329Gly). Such antibody
has a reduced FcR binding (especially they show no more binding to
FcRgammaI, FcRgammaII and FcRgammaIII). This especially useful to
reduce potential side effects like e.g. thrombosis (Meyer, T., et
al., J. Thromb. Haemost. 7 (2009) 171-81). In one embodiment the
bispecific antibody according to the invention is of human IgG4
subclass with mutations S228P (Ser228Pro), L235E (Leu235Glu) and
P329G (Pro329Gly). Such antibody shows reduced FcR binding as
indicated above. While Pro329Ala mutation which was described
already removes only two third of the FcgammaRIIIa sandwich
interaction, the Pro329Gly in the antibodies according to the
invention fully imparts binding of the Fc part to FcgammaRIII. This
is especially useful as the binding to FcgammaRIII is involved in
ADCC (antibody--dependent cellular toxicity) which leads to cell
death, which may be helpful in the treatment of cancer diseases,
but which can cause serious side effect in the antibody based
treatment of other vascular or immunological diseases. So the
antibodies according to the invention of IgG1 subclass with
mutations L234A, L235A and P329G and IgG4 subclass with mutations
S228P, L235E and P329G are especially useful, as they both show no
more binding to FcRgammaI, FcRgammaII and FcRgammaIII.
[0167] The term "with (the) mutations AAA" as used herein refers
the mutations I253A (Ile253Ala), H310A (His310Ala), and H435A
(His435Ala) in the constant heavy chain region of IgG1 or IgG4,
wherein the numbering is according to the EU Index of Kabat.
[0168] The term "with (the) mutations P329G LALA" as used herein
refers to the mutations L234A (Leu234Ala), L235A (Leu235Ala) and
P329G (Pro329Gly) in the constant heavy chain region of IgG1
subclass, wherein the numbering is according to the EU Index of
Kabat. The term "with (the) mutations SPLE" as used herein refers
to the S228P (Ser228Pro) and L235E (Leu235Glu) the constant heavy
chain region of IgG4 subclass, wherein the numbering is according
to the EU Index of Kabat. The term "with (the) mutations SPLE and
P239G" as used herein refers to the S228P (Ser228Pro), L235E
(Leu235Glu) and P329G (Pro329Gly) the constant heavy chain region
of IgG4 subclass, wherein the numbering is according to the EU
Index of Kabat.
[0169] The antibody according to the invention is produced by
recombinant means. Thus, one aspect of the current invention is a
nucleic acid encoding the antibody according to the invention and a
further aspect is a cell comprising said nucleic acid encoding an
antibody according to the invention. Methods for recombinant
production are widely known in the state of the art and comprise
protein expression in prokaryotic and eukaryotic cells with
subsequent isolation of the antibody and usually purification to a
pharmaceutically acceptable purity. For the expression of the
antibodies as aforementioned in a host cell, nucleic acids encoding
the respective modified light and heavy chains are inserted into
expression vectors by standard methods. Expression is performed in
appropriate prokaryotic or eukaryotic host cells like CHO cells,
NSO cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6 cells,
yeast, or E.coli cells, and the antibody is recovered from the
cells (supernatant or cells after lysis). General methods for
recombinant production of antibodies are well-known in the state of
the art and described, for example, in the review articles of
Makrides, S. C., Protein Expr. Purif 17 (1999) 183-202; Geisse, S.,
et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, R. J., Mol.
Biotechnol. 16 (2000) 151-160; Werner, R. G., Drug Res. 48 (1998)
870-880.
[0170] Accordingly one embodiment of the invention is a method for
the preparation of a bispecific antibody according to the
invention, comprising the steps of [0171] a) transforming a host
cell with vectors comprising nucleic acid molecules encoding said
antibody; [0172] b) culturing the host cell under conditions that
allow synthesis of said antibody molecule; and [0173] c) recovering
said antibody molecule from said culture.
[0174] In one embodiment the recovering step under c includes the
use of a light chain constant domain specific capture reagent
(which e.g. specific for the kappa or the lambda constant light
chain, depending on whether a kappa or a lambda light chain in the
bispecific antibody according to invention used). In one embodiment
this light chain specific capture reagent is used in in a
bind-and-elute-mode). Examples of such light chain constant domain
specific capture reagents are e.g. KappaSelect.TM. and
LambdaFabSelect.TM. from GE Healthcare/BAC, which are based on a
highly rigid agarose base matrix that allows high flow rates and
low back pressure at large scale. They feature a ligand that binds
to the constant region of the kappa or the lambda light chain
respectively (i.e. fragments lacking the constant region of the
light chain will not bind; FIG. 1). Both are therefore capable of
binding other target molecules containing the constant region of
the light chain, for example, IgG, IgA and IgM. The ligands are
attached to the matrix via a long hydrophilic spacer arm to make it
easily available for binding to the target molecule. They are based
on a single-chain antibody fragment that is screened for either
human Ig kappa or lambda.
[0175] The bispecific antibodies are suitably separated from the
culture medium by conventional immunoglobulin purification
procedures such as, for example, protein A-Sepharose,
hydroxylapatite chromatography, gel electrophoresis, dialysis, or
affinity chromatography. DNA and RNA encoding the monoclonal
antibodies is readily isolated and sequenced using conventional
procedures. The hybridoma cells can serve as a source of such DNA
and RNA. Once isolated, the DNA may be inserted into expression
vectors, which are then transfected into host cells such as HEK 293
cells, CHO cells, or myeloma cells that do not otherwise produce
immunoglobulin protein, to obtain the synthesis of recombinant
monoclonal antibodies in the host cells.
[0176] Amino acid sequence variants (or mutants) of the bispecific
antibody are prepared by introducing appropriate nucleotide changes
into the antibody DNA, or by nucleotide synthesis. Such
modifications can be performed, however, only in a very limited
range. For example, the modifications do not alter the above
mentioned antibody characteristics such as the IgG subclass and
antigen binding, but may improve the yield of the recombinant
production, protein stability or facilitate the purification.
[0177] The term "host cell" as used in the current application
denotes any kind of cellular system which can be engineered to
generate the antibodies according to the current invention. In one
embodiment HEK293 cells and CHO cells are used as host cells. As
used herein, the expressions "cell," "cell line," and "cell
culture" are used interchangeably and all such designations include
progeny. Thus, the words "transformants" and "transformed cells"
include the primary subject cell and cultures derived therefrom
without regard for the number of transfers. It is also understood
that all progeny may not be precisely identical in DNA content, due
to deliberate or inadvertent mutations. Variant progeny that have
the same function or biological activity as screened for in the
originally transformed cell are included.
[0178] Expression in NSO cells is described by, e.g., Barnes, L.
M., et al., Cytotechnology 32 (2000) 109-123; Barnes, L. M., et
al., Biotech. Bioeng. 73 (2001) 261-270. Transient expression is
described by, e.g., Durocher, Y., et al., Nucl. Acids. Res. 30
(2002) E9. Cloning of variable domains is described by Orlandi, R.,
et al., Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837; Carter, P.,
et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and
Norderhaug, L., et al., J. Immunol. Methods 204 (1997) 77-87. A
preferred transient expression system (HEK 293) is described by
Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30 (1999)
71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996)
191-199.
[0179] The control sequences that are suitable for prokaryotes, for
example, include a promoter, optionally an operator sequence, and a
ribosome binding site. Eukaryotic cells are known to utilize
promoters, enhancers and polyadenylation signals.
[0180] A nucleic acid is "operably linked" when it is placed in a
functional relationship with another nucleic acid sequence. For
example, DNA for a pre-sequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a pre-protein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading frame. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0181] Purification of antibodies is performed in order to
eliminate cellular components or other contaminants, e.g. other
cellular nucleic acids or proteins, by standard techniques,
including alkaline/SDS treatment, CsCl banding, column
chromatography, agarose gel electrophoresis, and others well known
in the art. See Ausubel, F., et al., ed. Current Protocols in
Molecular Biology, Greene Publishing and Wiley Interscience, New
York (1987). Different methods are well established and widespread
used for protein purification, such as affinity chromatography with
microbial proteins (e.g. protein A or protein G affinity
chromatography), ion exchange chromatography (e.g. cation exchange
(carboxymethyl resins), anion exchange (amino ethyl resins) and
mixed-mode exchange), thiophilic adsorption (e.g. with
beta-mercaptoethanol and other SH ligands), hydrophobic interaction
or aromatic adsorption chromatography (e.g. with phenyl-sepharose,
aza-arenophilic resins, or m-aminophenylboronic acid), metal
chelate affinity chromatography (e.g. with Ni(II)- and
Cu(II)-affinity material), size exclusion chromatography, and
electrophoretical methods (such as gel electrophoresis, capillary
electrophoresis) (Vijayalakshmi, M. A., Appl. Biochem. Biotech. 75
(1998) 93-102).
[0182] The bispecific, bivalent antibodies according to the
invention show benefits for human patients in need of a VEGF and
ANG-2 targeting therapy.
[0183] The bivalent bispecific against human VEGF and human ANG-2
according to the current invention may have a valuable
efficacy/safety profile and may provide benefits for a patient in
the need of an anti-VEGF and anti-ANG-2 therapy.
[0184] One aspect of the invention is a pharmaceutical composition
comprising an antibody according to the invention. Another aspect
of the invention is the use of an antibody according to the
invention for the manufacture of a pharmaceutical composition. A
further aspect of the invention is a method for the manufacture of
a pharmaceutical composition comprising an antibody according to
the invention. In another aspect, the present invention provides a
composition, e.g. a pharmaceutical composition, containing an
antibody according to the present invention, formulated together
with a pharmaceutical carrier.
[0185] As used herein, "pharmaceutical carrier" includes any and
all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like that are physiologically compatible. Preferably, the carrier
is suitable for administration administered to the subject via a
local route. For example, the antibody or its composition can be
administered to the subject by intraocular application e.g. by
intraocular injection such as intravitreal injection. This can be
performed in accordance with standard procedures known in the art.
See, e.g., Ritter et al., J. Clin. Invest. 116 (2006) 3266-76;
Russelakis-Carneiro et al., Neuropathol. Appl. Neurobiol. 25 (1999)
196-206; and Wray et al., Arch. Neurol. 33 (1976) 183-5.
[0186] A composition of the present invention can be administered
by a variety of methods known in the art. As will be appreciated by
the skilled artisan, the route and/or mode of administration will
vary depending upon the desired results. To administer a compound
of the invention by certain routes of administration, it may be
necessary to coat the compound with, or co-administer the compound
with, a material to prevent its inactivation. For example, the
compound may be administered to a subject in an appropriate
carrier, for example, liposomes, or a diluent. Pharmaceutically
acceptable diluents include saline and aqueous buffer solutions.
Pharmaceutical 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.
[0187] Many possible modes of delivery can be used, including, but
not limited to intraocular application or topical application. In
one embodiment the application is intraocular and includes, but is
not limited to, subconjunctival injection, intracanieral injection,
injection into the anterior chamber via the termporal limbus,
intrastromal injection, intracorneal injection, subretinal
injection, aqueous humor injection, subtenon injection or sustained
delivery device, intravitreal injection (e.g., front, mid or back
vitreal injection). In one embodiment the application is topical
and includes, but is not limited to eye drops to the cornea.
[0188] In one embodiment the bispecific antibody or pharmaceutical
composition according to the invention is administered via
intravitreal application, e.g. via intravitreal injection. This can
be performed in accordance with standard procedures known in the
art. See, e.g., Ritter et al., J. Clin. Invest. 116 (2006) 3266-76;
Russelakis-Carneiro et al., Neuropathol. Appl. Neurobiol. 25 (1999)
196-206; and Wray et al., Arch. Neurol. 33 (1976) 183-5.
[0189] In some embodiments, therapeutic kits of the invention can
contain one or more doses of a bispecific antibody present in a
pharmaceutical composition described herein, a suitable device for
intravitreal injection of the pharmaceutical composition, and an
instruction detailing suitable subjects and protocols for carrying
out the injection. In these embodiments, the compositions are
typically administered to the subject in need of treatment via
intravitreal injection. This can be performed in accordance with
standard procedures known in the art. See, e.g., Ritter et al., J.
Clin. Invest. 116 (2006) 3266-76; Russelakis-Carneiro et al.,
Neuropathol. Appl. Neurobiol. 25 (1999) 196-206; and Wray et al.,
Arch. Neurol. 33 (1976) 183-5.
[0190] The compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of presence of microorganisms may be ensured
both by sterilization procedures, supra, and by the inclusion of
various antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol, sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents which delay absorption
such as aluminum monostearate and gelatin.
[0191] Regardless of the route of administration selected, the
compounds of the present invention, which may be used in a suitable
hydrated form, and/or the pharmaceutical compositions of the
present invention, are formulated into pharmaceutically acceptable
dosage forms by conventional methods known to those of skill in the
art.
[0192] 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, 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.
[0193] The composition must be sterile and fluid to the extent that
the composition is deliverable by syringe. In addition to water,
the carrier preferably is an isotonic buffered saline solution.
[0194] Proper fluidity can be maintained, for example, by use of
coating such as lecithin, by maintenance of required particle size
in the case of dispersion and by use of surfactants. In many cases,
it is preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol or sorbitol, and sodium chloride in
the composition.
[0195] The composition can comprise an ophthalmic depot formulation
comprising an active agent for subconjunctival administration. The
ophthalmic depot formulation comprises microparticles of
essentially pure active agent, e.g., the bispecific antibody
according to the invention. The microparticles comprising the
bispecific antibody according to the invention can be embedded in a
biocompatible pharmaceutically acceptable polymer or a lipid
encapsulating agent. The depot formulations may be adapted to
release all of substantially all the active material over an
extended period of time. The polymer or lipid matrix, if present,
may be adapted to degrade sufficiently to be transported from the
site of administration after release of all or substantially all
the active agent. The depot formulation can be liquid formulation,
comprising a pharmaceutical acceptable polymer and a dissolved or
dispersed active agent. Upon injection, the polymer forms a depot
at the injections site, e.g. by gelifying or precipitating.
[0196] Another aspect of the invention is the bispecific antibody
according to the invention for use in the treatment of ocular
vascular diseases.
[0197] One embodiment of the invention is the bispecific antibody
according to the invention for use in the treatment of ocular
vascular diseases.
[0198] Another aspect of the invention is said pharmaceutical
composition for use in the treatment of ocular vascular
diseases.
[0199] Another aspect of the invention is the use of an antibody
according to the invention for the manufacture of a medicament for
the treatment of ocular vascular disease.
[0200] Another aspect of the invention is method of treatment of
patient suffering from ocular vascular diseases by administering an
antibody according to the invention to a patient in the need of
such treatment.
[0201] The terms "ocular vascular disease" and "vascular eye
disease" are use inter changeable herein and include, but are not
limited to intraocular neovascular syndromes such as diabetic
retinopathy, diabetic macular edema--retinopathy of prematurity,
neovascular glaucoma, retinal vein occlusions, central retinal vein
occlusions, macular degeneration, age-related macular degeneration,
retinitis pigmentosa, retinal angiomatous proliferation, macular
telangectasia, ischemic retinopathy, iris neovascularization,
intraocular neovascularization, corneal neovascularization, retinal
neovascularization, choroidal neovascularization, and retinal
degeneration. (Garner, A., Vascular diseases, In: Pathobiology of
ocular disease, A dynamic approach, Garner, A., and Klintworth, G.
K., (eds.), 2nd edition, Marcel Dekker, New York (1994), pp.
1625-1710). As used herein, ocular vascular disorder refers to any
pathological conditions characterized by altered or unregulated
proliferation and invasion of new blood vessels into the structures
of ocular tissues such as the retina or cornea. In one embodiment
the ocular vascular disease is selected from the group consisting
of: wet age-related macular degeneration (wet AMD), dry age-related
macular degeneration (dry AMD), diabetic macular edema (DME),
cystoid macular edema (CME), non-proliferative diabetic retinopathy
(NPDR), proliferative diabetic retinopathy (PDR), cystoid macular
edema, vasculitis (e.g. central retinal vein occlusion),
papilloedema, retinitis, conjunctivitis, uveitis, choroiditis,
multifocal choroiditis, ocular histoplasmosis, blepharitis, dry eye
(Sjogren's disease) and other ophthalmic diseases wherein the eye
disease or disorder is associated with ocular neovascularization,
vascular leakage, and/or retinal edema. So the bispecific
antibodies according to the invention are useful in the prevention
and treatment of wet AMD, dry AMD, CME, DME, NPDR, PDR,
blepharitis, dry eye and uveitis, also preferably wet AMD, dry AMD,
blepharitis, and dry eye, also preferably CME, DME, NPDR and PDR,
also preferably blepharitis, and dry eye, in particular wet AMD and
dry AMD, and also particularly wet AMD. In some embodiments, the
ocular disease is selected from the group consisting of wet
age-related macular degeneration (wet AMD), macular edema, retinal
vein occlusions, retinopathy of prematurity, and diabetic
retinopathy.
[0202] Other diseases associated with corneal neovascularization
include, but are not limited to, epidemic keratoconjunctivitis,
Vitamin A deficiency, contact lens overwear, atopic keratitis,
superior limbic keratitis, pterygium keratitis sicca, sjogrens,
acne rosacea, phylectenulosis, syphilis, Mycobacteria infections,
lipid degeneration, chemical burns, bacterial ulcers, fungal
ulcers, Herpes simplex infections, Herpes zoster infections,
protozoan infections, Kaposi sarcoma, Mooren ulcer, Terrien's
marginal degeneration, mariginal keratolysis, rheumatoid arthritis,
systemic lupus, polyarteritis, trauma, Wegeners sarcoidosis,
Scleritis, Steven's Johnson disease, periphigoid radial keratotomy,
and corneal graph rejection.
[0203] Diseases associated with retinal/choroidal
neovascularization include, but are not limited to, diabetic
retinopathy, macular degeneration, sickle cell anemia, sarcoid,
syphilis, pseudoxanthoma elasticum, Pagets disease, vein occlusion,
artery occlusion, carotid obstructive disease, chronic
uveitis/vitritis, mycobacterial infections, Lyme's disease,
systemic lupus erythematosis, retinopathy of prematurity, retinitis
pigmentosa, retina edema (including macular edema), Eales disease,
Bechets disease, infections causing a retinitis or choroiditis,
presumed ocular histoplasmosis, Bests disease, myopia, optic pits,
Stargarts disease, pars planitis, chronic retinal detachment,
hyperviscosity syndromes, toxoplasmosis, trauma and post-laser
complications. Other diseases include, but are not limited to,
diseases associated with rubeosis (neovascularization of the angle)
and diseases caused by the abnormal proliferation of fibrovascular
or fibrous tissue including all forms of proliferative
vitreoretinopathy.
[0204] Retinopathy of prematurity (ROP) is a disease of the eye
that affects prematurely born babies. It is thought to be caused by
disorganized growth of retinal blood vessels which may result in
scarring and retinal detachment. ROP can be mild and may resolve
spontaneously, but may lead to blindness in serious cases. As such,
all preterm babies are at risk for ROP, and very low birth weight
is an additional risk factor. Both oxygen toxicity and relative
hypoxia can contribute to the development of ROP.
[0205] Macular degeneration is a medical condition predominantly
found in elderly adults in which the center of the inner lining of
the eye, known as the macula area of the retina, suffers thinning,
atrophy, and in some cases, bleeding. This can result in loss of
central vision, which entails inability to see fine details, to
read, or to recognize faces. According to the American Academy of
Ophthalmology, it is the leading cause of central vision loss
(blindness) in the United States today for those over the age of
fifty years. Although some macular dystrophies that affect younger
individuals are sometimes referred to as macular degeneration, the
term generally refers to age-related macular degeneration (AMD or
ARMD).
[0206] Age-related macular degeneration begins with characteristic
yellow deposits in the macula (central area of the retina which
provides detailed central vision, called fovea) called drusen
between the retinal pigment epithelium and the underlying choroid.
Most people with these early changes (referred to as age-related
maculopathy) have good vision. People with drusen can go on to
develop advanced AMD. The risk is considerably higher when the
drusen are large and numerous and associated with disturbance in
the pigmented cell layer under the macula. Large and soft drusen
are related to elevated cholesterol deposits and may respond to
cholesterol lowering agents or the Rheo Procedure.
[0207] Advanced AMD, which is responsible for profound vision loss,
has two forms: dry and wet. Central geographic atrophy, the dry
form of advanced AMD, results from atrophy to the retinal pigment
epithelial layer below the retina, which causes vision loss through
loss of photoreceptors (rods and cones) in the central part of the
eye. While no treatment is available for this condition, vitamin
supplements with high doses of antioxidants, lutein and zeaxanthin,
have been demonstrated by the National Eye Institute and others to
slow the progression of dry macular degeneration and in some
patients, improve visual acuity.
[0208] Retinitis pigmentosa (RP) is a group of genetic eye
conditions. In the progression of symptoms for RP, night blindness
generally precedes tunnel vision by years or even decades. Many
people with RP do not become legally blind until their 40s or 50s
and retain some sight all their life. Others go completely blind
from RP, in some cases as early as childhood. Progression of RP is
different in each case. RP is a type of hereditary retinal
dystrophy, a group of inherited disorders in which abnormalities of
the photoreceptors (rods and cones) or the retinal pigment
epithelium (RPE) of the retina lead to progressive visual loss.
Affected individuals first experience defective dark adaptation or
nyctalopia (night blindness), followed by reduction of the
peripheral visual field (known as tunnel vision) and, sometimes,
loss of central vision late in the course of the disease.
[0209] Macular edema occurs when fluid and protein deposits collect
on or under the macula of the eye, a yellow central area of the
retina, causing it to thicken and swell. The swelling may distort a
person's central vision, as the macula is near the center of the
retina at the back of the eyeball. This area holds tightly packed
cones that provide sharp, clear central vision to enable a person
to see form, color, and detail that is directly in the line of
sight. Cystoid macular edema is a type of macular edema that
includes cyst formation.
[0210] Combination Therapies: In certain embodiments the bispecific
antibody or pharmaceutical composition according to the invention
is administered alone (without an additional therapeutic agent) for
the treatment of one or more ocular vascular diseases described
herein.
[0211] In other embodiments the bispecific antibody or
pharmaceutical composition according to the invention is
administered in combination with one or more additional therapeutic
agents or methods for the treatment of one or more ocular vascular
diseases described herein.
[0212] In other embodiments, the bispecific antibody or
pharmaceutical composition according to the invention is formulated
in combination with one or more additional therapeutic agents and
administered for the treatment of one or more ocular vascular
diseases described herein.
[0213] In certain embodiments, the combination treatments provided
herein include administration the bispecific antibody or
pharmaceutical composition according to the invention is
administered sequentially with one or more additional therapeutic
agents for the treatment of one or more ocular vascular diseases
described herein.
[0214] The additional therapeutic agents include, but are not
limited to, Tryptophanyl-tRNA synthetase (TrpRS), EyeOOl (Anti-VEGF
Pegylated Aptamer), squalamine, RETAANE.RTM. (anecortave acetate
for depot suspension; Alcon, Inc.), Combretastatin A4 Prodrug
(CA4P), MACUGEN.RTM., MIFEPREX.RTM. (mifepristone-ru486), subtenon
triamcinolone acetonide, intravitreal crystalline triamcinolone
acetonide, Prinomastat (AG3340--synthetic matrix metalloproteinase
inhibitor, Pfizer), fluocinolone acetonide (including fluocinolone
intraocular implant, Bausch & Lomb/Control Delivery Systems),
VEGFR inhibitors (Sugen), VEGF-Trap (Regeneron/Aventis), VEGF
receptor tyrosine kinase inhibitors such as
4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(l-methylpiperidin-4-ylme-
thoxy)quinazoline (ZD6474),
4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)-
quinazoline (AZD2171), vatalanib (PTK787) and SU1 1248 (sunitinib),
linomide, and inhibitors of integrin v.beta.3 function and
angiostatin.
[0215] Other pharmaceutical therapies that can be sued used in
combination the bispecific antibody or pharmaceutical composition
according to the invention is administered, include, but are not
limited to, VISUDYNE.RTM. with use of a non-thermal laser, PKC 412,
Endovion (NeuroSearch A/S), neurotrophic factors, including by way
of example Glial Derived Neurotrophic Factor and Ciliary
Neurotrophic Factor, diatazem, dorzolamide, Phototrop,
9-cis-retinal, eye medication (including Echo Therapy) including
phospholine iodide or echothiophate or carbonic anhydrase
inhibitors, AE-941 (AEterna Laboratories, Inc.), Sirna-027 (Sima
Therapeutics, Inc.), pegaptanib (NeXstar Pharmaceuticals/Gilead
Sciences), neurotrophins (including, by way of example only,
NT-4/5, Genentech), Candy (Acuity Pharmaceuticals), INS-37217
(Inspire Pharmaceuticals), integrin antagonists (including those
from Jerini AG and Abbott Laboratories), EG-3306 (Ark Therapeutics
Ltd.), BDM-E (BioDiem Ltd.), thalidomide (as used, for example, by
EntreMed, Inc.), cardiotrophin-1 (Genentech), 2-methoxyestradiol
(Allergan/Oculex), DL-8234 (Toray Industries), NTC-200 (Neurotech),
tetrathiomolybdate (University of Michigan), LYN-002 (Lynkeus
Biotech), microalgal compound (Aquasearch/Albany, Mera
Pharmaceuticals), D-9120 (Celltech Group pic), ATX-S10 (Hamamatsu
Photonics), TGF-beta 2 (Genzyme/Celtrix), tyrosine kinase
inhibitors (Allergan, SUGEN, Pfizer), NX-278-L (NeXstar
Pharmaceuticals/Gilead Sciences), Opt-24 (OPTIS France SA), retinal
cell ganglion neuroprotectants (Cogent Neurosciences),
N-nitropyrazole derivatives (Texas A&M University System),
KP-102 (Krenitsky Pharmaceuticals), cyclosporin A, Timited retinal
translocation", photodynamic therapy, (including, by way of example
only, receptor-targeted PDT, Bristol-Myers Squibb, Co.; porfimer
sodium for injection with PDT; verteporfin, QLT Inc.; rostaporfin
with PDT, Miravent Medical Technologies; talaporfin sodium with
PDT, Nippon Petroleum; motexafin lutetium, Pharmacyclics, Inc.),
antisense oligonucleotides (including, by way of example, products
tested by Novagali Pharma SA and ISIS-13650, Isis Pharmaceuticals),
laser photocoagulation, drusen lasering, macular hole surgery,
macular translocation surgery, implantable miniature telescopes,
Phi-Motion Angiography (also known as Micro-Laser Therapy and
Feeder Vessel Treatment), Proton Beam Therapy, microstimulation
therapy, Retinal Detachment and Vitreous Surgery, Scleral Buckle,
Submacular Surgery, Transpupillary Thermotherapy, Photosystem I
therapy, use of RNA interference (RNAi), extracorporeal
rheopheresis (also known as membrane differential filtration and
Rheotherapy), microchip implantation, stem cell therapy, gene
replacement therapy, ribozyme gene therapy (including gene therapy
for hypoxia response element, Oxford Biomedica; Lentipak, Genetix;
PDEF gene therapy, GenVec), photoreceptor/retinal cells
transplantation (including transplantable retinal epithelial cells,
Diacrin, Inc.; retinal cell transplant, Cell Genesys, Inc.), and
acupuncture.
[0216] Any anti-angiogenic agent can be used in combination with
the bispecific antibody or pharmaceutical composition according to
the invention, including, bu not limited to, those listed by
Carmeliet and Jain, 2000, Nature 407:249-257. In certain
embodiments, the anti-angiogenic agent is another VEGF antagonist
or a VEGF receptor antagonist such as VEGF variants, soluble VEGF
receptor fragments, aptamers capable of blocking VEGF or VEGFR,
neutralizing anti-VEGFR antibodies, low molecule weight inhibitors
of VEGFR tyrosine kinases and any combinations thereof and these
include anti-VEGF aptamers (e.g. Pegaptanib), soluble recombinant
decoy receptors (e.g. VEGF Trap). In certain embodiments, the
anti-angiogenic agent is include corticosteroids, angiostatic
steroids, anecortave acetate, angiostatin, endostatin, small
interfering RNA's decreasing expression of VEGFR or VEGF ligand,
post-VEGFR blockade with tyrosine kinase inhibitors, MMP
inhibitors, IGFBP3, SDF-1 blockers, PEDF, gamma-secretase,
Delta-like ligand 4, integrin antagonists, HIF-1 alpha blockade,
protein kinase CK2 blockade, and inhibition of stem cell (i.e.
endothelial progenitor cell) homing to the site of
neovascularization using vascular endothelial cadherin (CD-144) and
stromal derived factor (SDF)-I antibodies. Small molecule RTK
inhibitors targeting VEGF receptors including PTK787 can also be
used. Agents that have activity against neovascularization that are
not necessarily anti-VEGF compounds can also be used and include
anti-inflammatory drugs, m-Tor inhibitors, rapamycin, everolismus,
temsirolismus, cyclospohne, anti-TNF agents, anti-complement
agents, and nonsteroidal antiinflammatory agents. Agents that are
neuroprotective and can potentially reduce the progression of dry
macular degeneration can also be used, such as the class of drugs
called the `neurosteroids.` These include drugs such as
dehydroepiandrosterone(DHEA)(Brand names: Prastera.TM. and
Fidelin.RTM.), dehydroepiandrosterone sulfate, and pregnenolone
sulfate. Any AMD (age-related macular degeneration) therapeutic
agent can be used in combination with the bispecific antibody or
pharmaceutical composition according to the invention, including
but not limited to verteporfin in combination with PDT, pegaptanib
sodium, zinc, or an antioxidant(s), alone or in any
combination.
[0217] The terms "subject" and "patient" are used interchangeably
and refer to mammals such as human patients and non-human primates,
as well as experimental animals such as rabbits, rats, and mice,
and other animals. Animals include all vertebrates, e.g., mammals
and non-mammals, such as dogs, cats, sheeps, cows, pigs, rabbits,
chickens, and etc. Preferred subjects for practicing the
therapeutic methods of the present invention are human. Subjects in
need of treatment include patients already suffering from an ocular
vascular disease or disorder as well as those prone to developing
the disorder.
[0218] As used herein, the expressions "cell," "cell line," and
"cell culture" are used interchangeably and all such designations
include progeny. Thus, the words "transformants" and "transformed
cells" include the primary subject cell and cultures derived
therefrom without regard for the number of transfers. It is also
understood that all progeny may not be precisely identical in DNA
content, due to deliberate or inadvertent mutations. Variant
progeny that have the same function or biological activity as
screened for in the originally transformed cell are included. Where
distinct designations are intended, it will be clear from the
context.
[0219] The term "transformation" as used herein refers to process
of transfer of a vectors/nucleic acid into a host cell. If cells
without formidable cell wall barriers are used as host cells,
transfection is carried out e.g. by the calcium phosphate
precipitation method as described by Graham, F. L., van der Eb, A.
J., Virology 52 (1973) 546-467. However, other methods for
introducing DNA into cells such as by nuclear injection or by
protoplast fusion may also be used. If prokaryotic cells or cells
which contain substantial cell wall constructions are used, e.g.
one method of transfection is calcium treatment using calcium
chloride as described by Cohen, S. N., et al., PNAS. 69 (1972)
2110-2114.
[0220] As used herein, "expression" refers to the process by which
a nucleic acid is transcribed into mRNA and/or to the process by
which the transcribed mRNA (also referred to as transcript) is
subsequently being translated into peptides, polypeptides, or
proteins. The transcripts and the encoded polypeptides are
collectively referred to as gene product. If the polynucleotide is
derived from genomic DNA, expression in a eukaryotic cell may
include splicing of the mRNA.
[0221] A "vector" is a nucleic acid molecule, in particular
self-replicating, which transfers an inserted nucleic acid molecule
into and/or between host cells. The term includes vectors that
function primarily for insertion of DNA or RNA into a cell (e.g.,
chromosomal integration), replication of vectors that function
primarily for the replication of DNA or RNA, and expression vectors
that function for transcription and/or translation of the DNA or
RNA. Also included are vectors that provide more than one of the
functions as described.
[0222] An "expression vector" is a polynucleotide which, when
introduced into an appropriate host cell, can be transcribed and
translated into a polypeptide. An "expression system" usually
refers to a suitable host cell comprised of an expression vector
that can function to yield a desired expression product.
[0223] The following examples, sequence listing and figures are
provided to aid the understanding of the present invention, the
true scope of which is set forth in the appended claims. It is
understood that modifications can be made in the procedures set
forth without departing from the spirit of the invention.
TABLE-US-00001 Description of the Sequence Listing (Amino acid
sequences) SEQ ID NO: 1 heavy chain CDR3H, <VEGF> ranibizumab
SEQ ID NO: 2 heavy chain CDR2H, <VEGF> ranibizumab SEQ ID NO:
3 heavy chain CDR1H, <VEGF> ranibizumab SEQ ID NO: 4 light
chain CDR3L, <VEGF> ranibizumab SEQ ID NO: 5 light chain
CDR2L, <VEGF> ranibizumab SEQ ID NO: 6 light chain CDR1L,
<VEGF> ranibizumab SEQ ID NO: 7 heavy chain variable domain
VH, <VEGF> ranibizumab SEQ ID NO: 8 light chain variable
domain VL, <VEGF> ranibizumab SEQ ID NO: 9 heavy chain CDR3H,
<ANG-2> Ang2i_LC10 variant SEQ ID NO: 10 heavy chain CDR2H,
<ANG-2> Ang2i_LC10 variant SEQ ID NO: 11 heavy chain CDR1H,
<ANG-2> Ang2i_LC10 variant SEQ ID NO: 12 light chain CDR3L,
<ANG-2> Ang2i_LC10 variant SEQ ID NO: 13 light chain CDR2L,
<ANG-2> Ang2i_LC10 variant SEQ ID NO: 14 light chain CDR1L,
<ANG-2> Ang2i_LC10 variant SEQ ID NO: 15 heavy chain variable
domain VH, <ANG-2> Ang2i_LC10 variant SEQ ID NO: 16 light
chain variable domain VL, <ANG-2> Ang2i_LC10 variant SEQ ID
NO: 17 Human vascular endothelial growth factor (VEGF); precursor
sequence of human VEGF165 SEQ ID NO: 18 Human angiopoietin-2
(ANG-2) SEQ ID NO: 19 Human angiopoietin-1 (ANG-1) SEQ ID NO: 20
Human Tie-2 receptor SEQ ID NO 21 Heavy chain 1 of
<VEGF-ANG-2> CrossMAb IgG1 with AAA mutations (VEGFang2-0012)
SEQ ID NO 22 Heavy chain 2 of <VEGF-ANG-2> CrossMAb IgG1 with
AAA mutations (VEGFang2-0012) SEQ ID NO 23 Light chain 1 of
<VEGF-ANG-2> CrossMAb IgG1 with AAA mutations (VEGFang2-0012)
SEQ ID NO 24 Light chain 2 of <VEGF-ANG-2> CrossMAb IgG1 with
AAA mutations (VEGF-Ang2-0012) SEQ ID NO: 25 Heavy chain 1 of
<VEGF-ANG-2> CrossMAb IgG1 with AAA mutations and P329G LALA
mutations (VEGFang2-0016) SEQ ID NO: 26 Heavy chain 2 of
<VEGF-ANG-2> CrossMAb IgG1 with AAA mutations and P329G LALA
mutations (VEGFang2-0016) SEQ ID NO: 27 Light chain 1 of
<VEGF-ANG-2> CrossMAb IgG1 with AAA mutations and P329G LALA
mutations (VEGFang2-0016) SEQ ID NO: 28 Light chain 2 of
<VEGF-ANG-2> CrossMAb IgG1 with AAA mutations and P329G LALA
mutations (VEGFang2-0016) SEQ ID NO: 29 Heavy chain 1 of
<VEGF-ANG-2> CrossMAb IgG4 with AAA mutations and with SPLE
mutations SEQ ID NO: 30 Heavy chain 2 of <VEGF-ANG-2>
CrossMAb IgG4 with AAA mutations and with SPLE mutations SEQ ID NO:
31 Light chain 1 of <VEGF-ANG-2> CrossMAb IgG4 with AAA
mutations and with SPLE mutations SEQ ID NO: 32 Light chain 2 of
<VEGF-ANG-2> CrossMAb IgG4 with AAA mutations and with SPLE
mutations SEQ ID NO: 33 Heavy chain 1 of <VEGF-ANG-2> OAscFab
IgG1 with AAA mutations SEQ ID NO: 34 Heavy chain 2 of
<VEGF-ANG-2> OAscFab IgG1 with AAA mutations SEQ ID NO: 35
Light chain 1 of <VEGF-ANG-2> OAscFab IgG1 with AAA mutations
SEQ ID NO: 36 Heavy chain 1 of <VEGF-ANG-2> OAscFab IgG4 with
AAA mutations and with SPLE mutations SEQ ID NO: 37 Heavy chain 2
of <VEGF-ANG-2> OAscFab IgG4 with AAA mutations and with SPLE
mutations SEQ ID NO: 38 Light chain 1 of <VEGF-ANG-2> OAscFab
IgG4 with AAA mutations and with SPLE mutations SEQ ID NO: 39 Heavy
chain 1 of <VEGF-ANG-2> CrossMAb IgG1 wild type (without AAA
mutations) (VEGFang2-0201) SEQ ID NO: 40 Heavy chain 2 of
<VEGF-ANG-2> CrossMAb IgG1 wild type (without AAA mutations)
(VEGFang2-0201) SEQ ID NO: 41 Light chain 1 of <VEGF-ANG-2>
CrossMAb IgG1 wild type (without AAA mutations) (VEGFang2-0201) SEQ
ID NO: 42 Light chain 2 of <VEGF-ANG-2> CrossMAb IgG1 wild
type (without AAA mutations) (VEGFang2-0201) SEQ ID NO: 43 Heavy
chain 1 of <VEGF-ANG-2> CrossMAb IgG1 with P329G LALA
mutations only (without AAA mutations) (VEGFang2-0015) SEQ ID NO:
44 Heavy chain 2 of <VEGF-ANG-2> CrossMAb IgG1 with P329G
LALA mutations only (without AAA mutations) (VEGFang2-0015) SEQ ID
NO: 45 Light chain 1 of <VEGF-ANG-2> CrossMAb IgG1 with P329G
LALA mutations only (without AAA mutations) (VEGFang2-0015) SEQ ID
NO: 46 Light chain 2 of <VEGF-ANG-2> CrossMAb IgG1 with P329G
LALA mutations only (without AAA mutations) (VEGFang2-0015) SEQ ID
NO: 47 kappa light chain constant region SEQ ID NO: 48 lambda light
chain constant region SEQ ID NO: 49 heavy chain constant region
derived from human IgG1 SEQ ID NO: 50 heavy chain constant region
derived from human IgG4
[0224] In the following, embodiments of the invention are listed:
[0225] 1. A bispecific antibody comprising a first antigen-binding
site that specifically binds to human VEGF and a second
antigen-binding site that specifically binds to human ANG-2,
wherein [0226] i) said first antigen-binding site specifically
binding to VEGF comprises in the heavy chain variable domain a
CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a
CDR1H region of SEQ ID NO:3, and in the light chain variable domain
a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and
a CDR1L region of SEQ ID NO:6; and [0227] ii) said second
antigen-binding site specifically binding to ANG-2 comprises in the
heavy chain variable domain a CDR3H region of SEQ ID NO: 9, a CDR2H
region of, SEQ ID NO: 10, and a CDR1H region of SEQ ID NO: 11, and
in the light chain variable domain a CDR3L region of SEQ ID NO: 12,
a CDR2L region of SEQ ID NO: 13, and a CDR1L region of SEQ ID NO:
14, and wherein [0228] iii) the bispecific antibody comprises a
constant heavy chain region of human IgG1 or human IgG4 subclass
(derived from human origin and) comprising the mutations I253A,
H310A, and H435A (numbering according to EU Index of Kabat). [0229]
2. The bispecific antibody according to embodiment 1, wherein
[0230] i) said first antigen-binding site specifically binding to
VEGF comprises as heavy chain variable domain VH an amino acid
sequence of SEQ ID NO: 7, and as light chain variable domain VL an
amino acid sequence of SEQ ID NO: 8, and [0231] ii) said second
antigen-binding site specifically binding to ANG-2 comprises as
heavy chain variable domain VH an amino acid sequence of SEQ ID NO:
15, and as light chain variable domain VL an amino acid sequence of
SEQ ID NO: 16. [0232] 3. The bispecific antibody according to any
one of embodiments 1 to 2, wherein the constant heavy chain region
under iii) is of IgG1 subclass. [0233] 4. The bispecific antibody
according to embodiment 3, wherein the constant heavy chain region
of IgG1 subclass further comprises the mutations L234A, L235A and
P329G (numbering according to EU Index of Kabat). [0234] 5. The
bispecific antibody according to any one of embodiments 1 to 2,
wherein the constant heavy chain region under iii) is of IgG4
subclass. [0235] 6. The bispecific antibody according to embodiment
5, wherein the constant heavy chain region of IgG4 subclass further
comprises the mutations S228P and L235E (numbering according to EU
Index of Kabat). [0236] 7. The bispecific antibody according to
embodiment 5, wherein the constant heavy chain region of IgG4
subclass further comprises the mutations S228P, L235E and P329G
(numbering according to EU Index of Kabat). [0237] 8. A
pharmaceutical composition comprising an antibody according to any
one of embodiments 1 to 7. [0238] 9. The bispecific antibody
according to any one of embodiments 1 to 7 for use in the treatment
of ocular vascular diseases. [0239] 10. Use of the bispecific
antibody according to any one of embodiments 1 to 7 for the
manufacture of a medicament for the treatment of ocular vascular
diseases. [0240] 11. The bispecific antibody according to any one
of embodiments 9 or 10, wherein the antibody is administered via
intravitreal application. [0241] 12. A method of treatment of
patient suffering from ocular vascular diseases by administering an
antibody according to any one of embodiments 1 to 7 to a patient in
the need of such treatment. [0242] 13. A nucleic acid encoding a
bispecific antibody according to any one of embodiments 1 to 7.
[0243] 14. Expression vector containing said nucleic acid according
embodiment 13 capable of expressing said nucleic acid in a
prokaryotic or eukaryotic host cell. [0244] 15. A prokaryotic or
eukaryotic host cell comprising a vector according to embodiment
14. [0245] 16. A method for the preparation of a bispecific
antibody according to embodiments 1 to 7 [0246] comprising the
steps of [0247] a) transforming a host cell with vectors comprising
nucleic acid molecules encoding said antibody; [0248] b) culturing
the host cell under conditions that allow synthesis of said
antibody molecule; and [0249] c) recovering said antibody molecule
from said culture. [0250] 17. A bispecific antibody obtained by the
method of embodiment 16. [0251] 18. A bispecific, bivalent antibody
comprising a first antigen-binding site that specifically binds to
human VEGF and a second antigen-binding site that specifically
binds to human ANG-2, characterized in comprising the amino acid
sequences of SEQ ID NO: 25, of SEQ ID NO: 26, of SEQ ID NO: 27, and
of SEQ ID NO: 28. [0252] 19. A bispecific, bivalent antibody
comprising a first antigen-binding site that specifically binds to
human VEGF and a second antigen-binding site that specifically
binds to human ANG-2, characterized in comprising the amino acid
sequences of SEQ ID NO: 21, of SEQ ID NO: 22., of SEQ ID NO: 23.,
and of SEQ ID NO: 24. [0253] 20. A bispecific, bivalent antibody
comprising a first antigen-binding site that specifically binds to
human VEGF and a second antigen-binding site that specifically
binds to human ANG-2, characterized in comprising the amino acid
sequences of SEQ ID NO: 29, of SEQ ID NO: 30, of SEQ ID NO: 31, and
of SEQ ID NO: 32.
[0254] Experimental Procedures
TABLE-US-00002 TABLE 1 Bispecific antibodies and their respective
sequences Description Short Name Sequences <VEGF-ANG-2>
VEGFang2-0012 SEQ ID NO: 21, CrossMAb IgG1 with SEQ ID NO: 22, AAA
mutations SEQ ID NO: 23, SEQ ID NO: 24 <VEGF-ANG-2>
VEGFang2-0201- SEQ ID NO: 39, CrossMAb IgG1 wild type SEQ ID NO:
40, (without AAA mutations) SEQ ID NO: 41, SEQ ID NO: 42
<VEGF-ANG-2> VEGFang2-0016 SEQ ID NO: 25, CrossMAb IgG1 with
SEQ ID NO: 26, AAA mutations and SEQ ID NO: 27, P329G LALA
mutations SEQ ID NO: 28 <VEGF-ANG-2> VEGFang2-0015 SEQ ID NO:
43, CrossMAb IgG1 with SEQ ID NO: 44, P329G LALA mutations SEQ ID
NO: 45, only (without AAA SEQ ID NO: 46 mutations)
<VEGF-ANG-2> -- SEQ ID NO: 29, CrossMAb IgG4 with SEQ ID NO:
30, AAA mutations and with SEQ ID NO: 31, SPLE mutations SEQ ID NO:
32 <VEGF-ANG-2> -- SEQ ID NO: 33, OAscFab IgG1 with AAA SEQ
ID NO: 34, mutations SEQ ID NO: 35 <VEGF-ANG-2> -- SEQ ID NO:
36, OAscFab IgG4 with AAA SEQ ID NO: 37, mutations and with SPLE
SEQ ID NO: 38 mutations
[0255] Please note that the term "with (the) mutations AAA" as used
herein refers the mutations I253A (Ile253Ala), H310A (His310Ala),
and H435A (His435Ala) in the constant heavy chain region of IgG1 or
IgG4 (numbering according to EU Index of Kabat), the term "with
(the) mutations P329G LALA" as used herein refers to the mutations
L234A (Leu235Ala) , L235A (Leu234Ala) and P329G (Pro329Gly) in the
constant heavy chain region of IgG1 subclass (numbering according
to EU Index of Kabat), and the term "with (the) mutations SPLE" as
used herein refers to the S228P (Ser228Pro) and L235E (Leu235Glu)
the constant heavy chain region of IgG4 subclass (numbering
according to EU Index of Kabat).
EXAMPLES
[0256] Materials & General Methods
[0257] General information regarding the nucleotide sequences of
human immunoglobulin light and heavy chains is given in: Kabat, E.
A., et al., Sequences of Proteins of Immunological Interest, 5th
ed., Public Health Service, National Institutes of Health,
Bethesda, Md. (1991). Amino acids of antibody chains are numbered
and referred to according to EU numbering (Edelman, G. M., et al.,
Proc. Natl. Acad. Sci. USA 63 (1969) 78-85; Kabat, E. A., et al.,
Sequences of Proteins of Immunological Interest, 5th ed., Public
Health Service, National Institutes of Health, Bethesda, Md.
(1991)).
[0258] Recombinant DNA Techniques
[0259] Standard methods were used to manipulate DNA as described in
Sambrook, J. et al., Molecular Cloning: A laboratory manual; Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
The molecular biological reagents were used according to the
manufacturer's instructions.
[0260] Gene Synthesis
[0261] Desired gene segments were ordered according to given
specifications at Geneart (Regensburg, Germany).
[0262] DNA Sequence Determination
[0263] DNA sequences were determined by double strand sequencing
performed at MediGenomix GmbH (Martinsried, Germany) or Sequiserve
GmbH (Vaterstetten, Germany).
[0264] DNA and Protein Sequence Analysis and Sequence Data
Management
[0265] The GCG's (Genetics Computer Group, Madison, Wisconsin)
software package version 10.2 and Infomax's Vector NT1 Advance
suite version 8.0 was used for sequence creation, mapping,
analysis, annotation and illustration.
[0266] Expression Vectors
[0267] For the expression of the described antibodies, variants of
expression plasmids for transient expression (e.g. in HEK293-F)
cells based either on a cDNA organization with or without a
CMV-Intron A promoter or on a genomic organization with a CMV
promoter were applied.
[0268] Beside the antibody expression cassette the vectors
contained: [0269] an origin of replication which allows replication
of this plasmid in E. coli, [0270] a .beta.-lactamase gene which
confers ampicillin resistance in E. coli., and [0271] the
dihydrofolate reductase gene from Mus musculus as a selectable
marker in eukaryotic cells [0272] The transcription unit of the
antibody gene was composed of the following elements: [0273] unique
restriction site(s) at the 5' end [0274] the immediate early
enhancer and promoter from the human cytomegalovirus, [0275]
followed by the Intron A sequence in the case of the cDNA
organization, [0276] a 5'-untranslated region of a human antibody
gene, [0277] an immunoglobulin heavy chain signal sequence, [0278]
the human antibody chain (wildtype or with domain exchange) either
as cDNA or as genomic organization with the immunoglobulin
exon-intron organization [0279] a 3' untranslated region with a
polyadenylation signal sequence, and [0280] unique restriction
site(s) at the 3' end.
[0281] The fusion genes comprising the antibody chains as described
below were generated by PCR and/or gene synthesis and assembled by
known recombinant methods and techniques by connection of the
according nucleic acid segments e.g. using unique restriction sites
in the respective vectors. The subcloned nucleic acid sequences
were verified by DNA sequencing. For transient transfections larger
quantities of the plasmids were prepared by plasmid preparation
from transformed E. coli cultures (Nucleobond AX,
Macherey-Nagel).
[0282] Cell Culture Techniques
[0283] Standard cell culture techniques were used as described in
Current Protocols in Cell Biology (2000), Bonifacino, J. S., Dasso,
M., Harford, J. B., Lippincott-Schwartz, J. and Yamada, K. M.
(eds.), John Wiley & Sons, Inc.
[0284] The bispecific antibodies were expressed by transient
co-transfection of the respective expression plasmids in in HEK29-F
cells growing in suspension as described below.
Example 1
[0285] Expression and Purification
[0286] Transient Transfections in HEK293-F System
[0287] The bispecific antibodies were generated by transient
transfection with the respective plasmids (e.g. encoding the heavy
and modified heavy chain, as well as the corresponding light and
modified light chain) using the HEK293-F system (Invitrogen)
according to the manufacturer's instruction. Briefly, HEK293-F
cells (Invitrogen) growing in suspension either in a shake flask or
in a stirred fermenter in serum-free FreeStyle.TM. 293 expression
medium (Invitrogen) were transfected with a mix of the four
expression plasmids and 293fectin.TM. or fectin (Invitrogen).
[0288] For 2 L shake flask (Corning) HEK293-F cells were seeded at
a density of 1.0E*6 cells/mL in 600 mL and incubated at 120 rpm, 8%
CO2. The day after the cells were transfected at a cell density of
ca. 1.5E*6 cells/mL with ca. 42 mL mix of A) 20 mL Opti-MEM.TM.
(Invitrogen) with 600 .mu.g total plasmid DNA (1 .mu.g/mL) encoding
the heavy or modified heavy chain, respectively and the
corresponding light chain in an equimolar ratio and B) 20 ml
Opti-MEM.TM.+1.2 mL 293 fectin or fectin (2 .mu.l/mL). According to
the glucose consumption glucose solution was added during the
course of the fermentation. The supernatant containing the secreted
antibody was harvested after 5-10 days and antibodies were either
directly purified from the supernatant or the supernatant was
frozen and stored.
[0289] Purification
[0290] Bispecific antibodies were purified from cell culture
supernatants by affinity chromatography using
MabSelectSure-Sepharose.RTM. (for non_AAA mutants) (GE Healthcare,
Sweden) or kappaSelect-Agarose (for AAA mutants) (GE Healthcare,
Sweden), hydrophobic interaction chromatography using
butyl-Sepharose.RTM. (GE Healthcare, Sweden) and Superdex 200 size
exclusion (GE Healthcare, Sweden) chromatography.
[0291] Briefly, sterile filtered cell culture supernatants were
captured on a MabSelect SuRe.TM. resin equilibrated with PBS buffer
(10 mM Na.sub.2HPO.sub.4, 1 mM KH.sub.2PO.sub.4, 137 mM NaCl and
2.7 mM KC1, pH 7.4), washed with equilibration buffer and eluted
with 25 mM sodium citrate at pH 3.0. The AAA mutants were captured
on a kappaSelect resin equilibrated with 25 mM Tris, 50 mM NaCl, pH
7.2, washed with equilibration buffer and eluted with 25 mM sodium
citrate pH 2.9. The eluted protein fractions were pooled and
neutralized with 2M Tris, pH 9.0. The antibody pools were prepared
for hydrophobic interaction chromatography by adding 1.6 M ammonium
sulfate solution to a final concentration of 0.8 M ammonium sulfate
and the pH adjusted to pH 5.0 using acetic acid. After
equilibration of the butyl-Sepharose.RTM. resin with 35 mM sodium
acetate, 0.8 M ammonium sulfate, pH 5.0, the antibodies were
applied to the resin, washed with equilibration buffer and eluted
with a linear gradient to 35 mM sodium acetate pH 5.0. The
bispecific antibody containing fractions were pooled and further
purified by size exclusion chromatography using a Superdex 200
26/60 GL (GE Healthcare, Sweden) column equilibrated with 20 mM
histidine, 140 mM NaCl, pH 6.0. The bispecific antibody containing
fractions were pooled, concentrated to the required concentration
using Vivaspin ultrafiltration devices (Sartorius Stedim Biotech
S.A., France) and stored at -80.degree. C.
TABLE-US-00003 TABLE 2 Yields of bispecific <YEGF-ANG-2>
antibodies VEGFang2-0015 VEGFang2-0016 (with (without AAA mutation)
AAA mutation) Titer supernatant 64 .mu.g/ml, n.a. (2 L = 128 mg) (2
L scale) Protein A 118 mg n.a. (MabSelectSure) (~70% monomer) Kappa
Select n.a. 117 mg (~83% monomer) Butyl Sepharose 60 mg 57 mg SEC
35 mg 38 mg (>95% monomer) (>95% monomer)
[0292] Purity and antibody integrity were analyzed after each
purification step by CE-SDS using microfluidic LabChip.RTM.
technology (Caliper Life Science, USA). 5 .mu.l of protein solution
was prepared for CE-SDS analysis using the HT Protein Express
[0293] Reagent Kit according manufacturer's instructions and
analysed on LabChip.RTM. GXII system using a HT Protein Express
Chip. Data were analyzed using LabChip.RTM. GX Software.
TABLE-US-00004 TABLE 3 Removal of typical side products by
different sequential purification steps determined by CE-SDS.
Purification Step VEGFang2-0015 VEGFang2-0016 % peak area* *
analysis: CE-SDS (Caliper Labchip GXII) 3/4 1/2 3/4 1/2 (LC) mab ab
(HC)2 ab (LC)2 LC mab ab (HC)2 ab 2 LC Mab 55.7 19 10.6 9.8 3.5 0.9
-- Select Sure Kappa -- 63 13.4 3.5 6.1 5.8 7.4 Select Butyl- 81.4
1.9 2.3 8.2 3.6 1.8 76.2 1.3 0.7 8.3 7.7 5.8 Sepha- rose Super-
92.4 1.8 2.6 1.4 0.5 0.5 99 1.1 n.d. n.d. n.d. n.d. dex 200_
SEC
[0294] The aggregate content of antibody samples was analyzed by
high-performance SEC using a Superdex 200 analytical size-exclusion
column (GE Healthcare, Sweden) in 2xPBS (20 mM Na.sub.2HPO.sub.4, 2
mM KH.sub.2PO.sub.4, 274 mM NaCl and 5.4 mM KCl, pH 7.4) running
buffer at 25.degree. C. 25 .mu.g protein were injected on the
column at a flow rate of 0.75 ml/min and eluted isocratic over 50
minutes.
[0295] Analogously the <VEGF-ANG-2> bispecific antibodies
VEGFang2-0012 and VEGFang2-0201 were prepared and purified with the
following yields:
TABLE-US-00005 VEGFang2-0012 VEGFang2-0201 (with AAA mutation)
(without AAA mutation) Titer//amount -- 36 .mu.g/ml// 72 mg Scale
2.1 L 2 L Protein A -- 66 mg (MabSelectSure) (~95% monomer)
kappaSelect 43 mg -- (~65% monomer) Butyl Sepharose -- 45 mg SEC 14
mg 21 mg (>98% monomer) Yield 8.5 mg hydoxylapatite (>98%
monomer) Totatl yield 8.5 mg 21 mg (recovery) (20%) (30%)
[0296] Also the <VEGF-ANG-2> bispecific antibodies
<VEGF-ANG-2> CrossMAb IgG4 with AAA mutations and with SPLE
mutations(SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO:
32), <VEGF-ANG-2> OAscFab IgG1 with AAA mutations(SEQ ID NO:
33, SEQ ID NO: 34, SEQ ID NO: 35)and <VEGF-ANG-2> OAscFab
IgG4 with AAA mutations and with SPLE mutations(SEQ ID NO: 36, SEQ
ID NO: 37, SEQ ID NO: 38) can be prepared and purified
analogously.
Example 2
[0297] Analytics & Developability
[0298] Small-Scale DLS-Based Viscosity Measurement.
[0299] Viscosity measurement was essentially performed as described
in (He, F. et al.,
[0300] Analytical Biochemistry 399 (2009) 141-3). Briefly, samples
are concentrated to various protein concentrations in 200 mM
arginine succinate, pH 5.5, before polystyrene latex beads (300 nm
diameter) and Polysorbate 20 (0.02% v/v) are added. Samples are
transferred into an optical 384-well plate by centrifugation
through a 0.4 .mu.m filter plate and covered with paraffine oil.
The apparent diameter of the latex beads is determined by dynamic
light scattering at 25.degree. C. The viscosity of the solution can
be calculated as .eta.=.eta.0(rh/rh,0) (.eta.: viscosity; .eta.0:
viscosity of water; rh: apparent hydrodynamic radius of the latex
beads; rh,0: hydrodynamic radius of the latex beads in water.
[0301] To allow comparison of various samples at the same
concentration, viscosity-concentration data were fitted with the
Mooney equation (Equation 1) (Mooney, Colloid Sci, 1951; Monkos,
Biochem. Biophys. Acta 1997) and data interpolated accordingly.
.eta. = .eta. 0 exp ( S .PHI. 1 - K .PHI. ) Equation 1
##EQU00001##
[0302] (S: hydrodynamic interaction parameter of the protein; K:
self-crowding factor; .PHI.: volume fraction of the dissolved
protein)
[0303] Results are shown in FIG. 2: VEGFang2-0016 with AAA
mutations in the Fc part shows a lower viscosity at all measured
temperatures compared to VEGFang2-0015 without the AAA mutations in
the Fc part.
[0304] DLS Aggregation Onset Temperature
[0305] Samples are prepared at a concentration of 1 mg/mL in 20 mM
Histidine/Histidine chloride, 140 mM NaCl, pH 6.0, transferred into
an optical 384-well plate by centrifugation through a 0.4 .mu.m
filter plate and covered with paraffine oil. The hydrodynamic
radius is measured repeatedly by dynamic light scattering while the
samples are heated with a rate of 0.05.degree. C./min from
25.degree. C. to 80.degree. C. The aggregation onset temperature is
defined as the temperature at which the hydrodynamic radius starts
to increase. Results are shown in FIG. 3. In FIG. 3 the aggregation
of VEGFang2-0015 without the AAA mutations versus VEGFang2-0016
with AAA mutations in the Fc part is shown. VEGFang2-0016 showed a
aggregation onset temperature of 61.degree. C. whereas
VEGFang2-0015 without the AAA mutations showed a onset temperature
of 60.degree. C.
[0306] DLS Timecourse
[0307] Samples are prepared at a concentration of 1 mg/mL in 20 mM
Histidine/Histidine chloride, 140 mM NaCl, pH 6.0, transferred into
an optical 384-well plate by centrifugation through a 0.4 .mu.m
filter plate and covered with paraffine oil. The hydrodynamic
radius is measured repeatedly by dynamic light scattering while the
samples are kept at a constant temperature of 50.degree. C. for up
to 145 hours. In this experiment, aggregation tendencies of the
native, unfolded protein at elevated temperature would lead to an
increase of the average particle diameter over time. This DLS-based
method is very sensitive for aggregates because these contribute
over-proportionally to the scattered light intensity. Even after
145 hours at 50.degree. C. (a temperature close to the
aggregation-onset temperature, see above), an average particle
diameter increase of only less than 0.5 nm was found for both
VEGFang2-0015 and VEGFang2-0016
[0308] 7 day Storage at 40.degree. C. at 100 mg/ml (HMW
Increase)
[0309] Samples are concentrated to a final concentration of 100
mg/mL in 200 mM arginine succinate, pH 5.5, sterile filtered and
quiescently stored at 40.degree. C. for 7 days. Before and after
storage, the content of high and low molecular weight species (HMWs
and LMWs, respectively) is determined by size-exclusion
chromatography. The difference in HMW and LMW content between the
stored sample and a sample measured immediately after preparation
is reported as "HMW increase" and "LMW increase", respectively.
Results are shown in Table 4 and FIG. 4, which show that
VEGFang2-0015 (without AAA mutation) shows a higher reduction of
the main peak and a higher HMW increase compared to VEGF Ang2-0016
(with AAA mutation). Surprisingly VEGF Ang2-0016 (with AAA
mutation) showed a lower aggregation tendency compared
toVEGFang2-0015 (without AAA mutation).
TABLE-US-00006 TABLE 4 Delta Main-, HMW and LMW peaks after 7 d at
40.degree. C. delta_area % (40.degree. C.-(-80.degree. C.)) Main
Peak HMW LMW VEGFang2-0015 -3.56 2.89 0.67 (-AAA mutations)
VEGFang2-0016 -1.74 1.49 0.25 (+AAA mutations)
[0310] The functional analysis of anti-VEGF and anti-Ang2
bispecific antibodies was assessed by Surface Plasmon Resonance
(SPR) using a BlAcore.RTM. T100 or T200 instrument (GE Healthcare)
at 25.degree. C. The BIAcore.RTM. system is well established for
the study of molecule interactions. SPR-technology is based on the
measurement of the refractive index close to the surface of a gold
coated biosensor chip. Changes in the refractive index indicate
mass changes on the surface caused by the interaction of
immobilized ligand with analyte injected in solution. The mass
increases if molecules bind immobilized ligands on the surface, and
vice versa, the mass decreases in case of dissociation of the
analyte from the immobilized ligand (reflecting complex
dissociation). SPR allows a continuous real-time monitoring of
ligand/analyte binding and thus the determination of the
association rate constant (ka), the dissociation rate constant
(kd), and of the equilibrium constant (KD).
Example 3
[0311] Binding to VEGF, Ang2, FcgammaR and FcRn
[0312] VEGF Isoforms Kinetic Affinity Including Assessment of
Species-Crossreactivity
[0313] Around 12000 resonance units (RU) of the capturing system
(10 .mu.g/ml goat anti human F(ab)'.sub.2; Order Code: 28958325; GE
Healthcare Bio-Sciences AB, Sweden) were coupled on a CMS chip (GE
Healthcare BR-1005-30) at pH 5.0 by using an amine coupling kit
supplied by the GE Healthcare. The sample and system buffer was
PBS-T (10 mM phosphate buffered saline including 0.05% Tween20) pH
7.4. The flow cell was set to 25.degree. C.--and the sample block
set to 12.degree. C.--and primed with running buffer twice. The
bispecific antibody was captured by injecting a 50 nM solution for
30 sec at a flow of 5 .mu.l/min. Association was measured by
injection of human hVEGF121, mouse mVEGF120 or rat rVEGF164 in
various concentrations in solution for 300 sec at a flow of 30
.mu.l/min starting with 300 nM in 1:3 dilutions. The dissociation
phase was monitored for up to 1200 sec and triggered by switching
from the sample solution to running buffer. The surface was
regenerated by 60 sec washing with a Glycine pH 2.1 solution at a
flow rate of 30 .mu.l/min. Bulk refractive index differences were
corrected by subtracting the response obtained from a goat anti
human F(ab').sub.2 surface. Blank injections are also subtracted
(=double referencing). For calculation of apparent K.sub.D and
other kinetic parameters the Langmuir 1:1 model was used. Results
are shown in Table 5.
[0314] Ang2 Solution Affinity Including Assessment of
Species-Crossreactivity
[0315] Solution affinity measures the affinity of an interaction by
determining the concentration of free interaction partners in an
equilibrium mixture. The solution affinity assay involves the
mixing of an <VEGF-ANG-2> bispecific antibody, kept at a
constant concentration, with a ligand (=Ang2) at varying
concentrations. Maximum possible resonance units (e.g. 17000
resonance units (RU)) of an antibody was immobilized on the CMS
chip (GE Healthcare BR-1005-30) surface at pH 5.0 using an amine
coupling kit supplied by the GE Healthcare. The sample and system
buffer was HBS-P pH 7.4. Flow cell was set to 25.degree. C. and
sample block to 12.degree. C. and primed with running buffer twice.
To generate a calibration curve increasing concentrations of Ang2
were injected into a BlAcore flowcell containing the immobilized
VEGF-ANG-2> bispecific antibody. The amount of bound Ang2 was
determined as resonance units (RU) and plotted against the
concentration. Solutions of each ligand (11 concentrations from 0
to 200 nM for the VEGF-ANG-2> bispecific antibody) were
incubated with 10 nM Ang2 and allowed to reach equilibrium at room
temperature. Free Ang2 concentrations were determined from
calibration curve generated before and after measuring the response
of solutions with known amounts of Ang2. A 4-parameter fit was set
with XLfit4 (IDBS Software) using Model 201 using free Ang2
concentration as y-axis and used concentration of antibody for
inhibition as x-axis. The affinity was calculated by determining
the inflection point of this curve. The surface was regenerated by
one time 30 sec washing with a 0.85% H.sub.3PO.sub.4 solution at a
flow rate of 30 .mu.l/min. Bulk refractive index differences were
corrected by subtracting the response obtained from a blank-coupled
surface. Results are shown in Table 6.
[0316] FeRn Steady State Affinity
[0317] For FcRn measurement a steady state affinity was used to
compare bispecific antibodies against each other. Human FcRn was
diluted into coupling buffer (10 .mu.g/ml, Na-Acetate pH5.0) and
immobilized on a C1-Chip (GE Healthcare BR-1005-35) by targeted
immobilization procedure using a BlAcore wizard to a final response
of 200 RU. Flow cell was set to 25.degree. C. and sample block to
12.degree. C. and primed with running buffer twice. The sample and
system buffer was PBS-T (10 mM phosphate buffered saline including
0.05% Tween20) pH 6.0. To assess different IgG concentrations for
each antibody, a concentration of 62.5 nM, 125 nM and 250 nM, 500
nM was prepared. Flow rate was set to 30 .mu.l/min and the
different samples were injected consecutively onto the chip surface
choosing 180 sec association time. The surface was regenerated by
injected PBS-T pH 8 for 60 sec at a flow rate of 30 .mu.l/min. Bulk
refractive index differences were corrected by subtracting the
response obtained from a blank surface. Buffer injections are also
subtracted (=double referencing). For calculation of steady state
affinity the method from the Bia-Evaluation software was used.
Briefly, the RU values (RU max) were plotted against the analysed
concentrations, yielding a dose-response curve. Based on a
2-parametric fit, the upper asymptote is calculated, allowing the
determination of the half-maximal RU value and hence the affinity.
Results are shown in FIG. 5 and Table 7. Analogously the affinity
to cyno, mouse and rabbit FcRn can be determined.
[0318] FcgammaRIIIa Measurement
[0319] For FcgammaRIIIa measurement a direct binding assay was
used. Around 3000 resonance units (RU) of the capturing system (1
.mu.g/ml Penta-His; Quiagen) were coupled on a CMS chip (GE
Healthcare BR-1005-30) at pH 5.0 by using an amine coupling kit
supplied by the GE Healthcare. The sample and system buffer was
HBS-P+pH 7.4. The flow cell was set to 25.degree. C.--and sample
block to 12.degree. C.--and primed with running buffer twice. The
FcgammaRIIIa -His-receptor was captured by injecting a 100 nM
solution for 60 sec at a flow of 5 .mu.l/min. Binding was measured
by injection of 100 nM of bispecific antibody or monospecific
control antibodies (anti-Dig for IgG1 subclass and an IgG4 subclass
antibody) for 180 sec at a flow of 30 .mu.l/. The surface was
regenerated by 120 sec washing with Glycine pH 2.5 solution at a
flow rate of 30 .mu.l/min. Because FcgammaRIIIa binding differs
from the Langmuir 1:1 model, only binding/no binding was determined
with this assay. In a similar manner FcgammaRIa , and FcgammaRIIa
binding can be determined. Results are shown in FIG. 6, where it
follows that by introduction of the mutations P329G LALA no more
binding to FcgammaRIIIa could be detected.
[0320] Assessment of Independent VEGF- and Ang2-Binding to the
<VEGF-ANG-2> Bispecific Antibodies
[0321] Around 3500 resonance units (RU) of the capturing system (10
.mu.g/ml goat anti human IgG; GE Healthcare Bio-Sciences AB,
Sweden) were coupled on a CM4 chip (GE Healthcare BR-1005-34) at pH
5.0 by using an amine coupling kit supplied by the GE Healthcare.
The sample and system buffer was PBS-T (10 mM phosphate buffered
saline including 0.05% Tween20) pH 7.4. The temperature of the flow
cell was set to 25.degree. C. and of the sample block to 12.degree.
C. Before capturing, the flow cell was primed with running buffer
twice.
[0322] The bispecific antibody was captured by injecting a 10 nM
solution for 60 sec at a flow of 5 .mu.l/min. Independent binding
of each ligand to the bispecific antibody was analysed by
determining the active binding capacity for each ligand, either
added sequentially or simultaneously (flow of 30 .mu.l/min): [0323]
1. Injection of human VEGF with a concentration of 200 nM for 180
sec (identifies the single binding of the antigen). [0324] 2.
Injection of human Ang2 with a concentration of 100 nM for 180 sec
(identifies single binding of the antigen). [0325] 3. Injection of
human VEGF with a concentration of 200 nM for 180 sec followed by
an additional injection of human Ang2 with a concentration of 100
nM for 180 sec (identifies binding of Ang2 in the presence of
VEGF). [0326] 4. Injection of human Ang2 with a concentration of
100 nM for 180 sec followed by an additional injection of human
VEGF with a concentration of 200 nM (identifies binding of VEGF in
the presence of Ang2). [0327] 5. Co-Injection of human VEGF with a
concentration of 200 nM and of human Ang2 with a concentration of
100 nM for 180 sec (identifies the binding of VEGF and of Ang2 at
the same time).
[0328] The surface was regenerated by 60 sec washing with a 3 m
MgCl2 solution at a flow rate of 30 .mu.l/min. Bulk refractive
index differences were corrected by subtracting the response
obtained from a goat anti human IgG surface.
[0329] The bispecific antibody is able to bind both antigens mutual
independently if the resulting final signal of the approaches 3, 4
& 5 equals or is similar to the sum of the individual final
signals of the approaches 1 and 2. Results are shown in Table 9,
where both antibodies VEGFang2-0016, VEGFang2-0012 are shown to be
able to bind mutual independently to VEGF and ANG2
[0330] Assessment of Simultaneous VEGF- and Ang2-binding to the
<VEGF-ANG-2> Bispecific Antibodies
[0331] First, around 1600 resonance units (RU) of VEGF (20
.mu.g/ml) were coupled on a CM4 chip (GE Healthcare BR-1005-34) at
pH 5.0 by using an amine coupling kit supplied by the GE
Healthcare. The sample and system buffer was PBS-T (10 mM phosphate
buffered saline including 0.05% Tween 20) pH 7.4. Flow cell was set
to 25.degree. C. and sample block to 12.degree. C. and primed with
running buffer twice. Second, 50 nM solution of the bispecific
antibody was injected for 180 sec at a flow of 30 .mu.l/min. Third,
hAng-2 was injected for 180 sec at a flow of 30 .mu.l/min. The
binding response of hAng-2 depends from the amount of the
bispecific antibody bound to VEGF and shows simultaneous binding.
The surface was regenerated by 60 sec washing with a 0.85% H3PO4
solution at a flow rate of 30 .mu.l/min. Simultaneous binding is
shown by an additional specific binding signal of hAng2 to the
previous VEGF bound <VEGF-ANG-2> bispecific antibodies. For
both bispecific antibodies VEGFang2-0015 and VEGFang2-0016
simultaneous VEGF- and Ang2-binding to the <VEGF-ANG-2>
bispecific antibodies could be detected (data not shown).
TABLE-US-00007 TABLE 5 Results: Kinetic affinities to VEGF isoforms
from different species VEGFang2- VEGFang2- VEGFang2- VEGFang2-
0015- 0016- 0012- 0201- apparent apparent apparent apparent
affinity affinity affinity affinity Human VEGF .ltoreq.1 pM
.ltoreq.1 pM .ltoreq.1 pM .ltoreq.1 pM 121 (out of (out of (out of
(out of Biacore Biacore Biacore Biacore specification)
specification) specification) specification) mouseVEGF no binding
no binding no binding no binding 120 Rat VEGF 13 nM 14 nM 24 nM 35
nM 164
TABLE-US-00008 TABLE 6 Results: Solution affinities to Ang2
VEGFang2- VEGFang2- VEGFang2- VEGFang2-- 0015 KD 0016 KD 0012 KD
0201 KD [nM] [nM] [nM] [nM] humanAng2 8 20 20 tbd cynoAng2 5 13 10
tbd mouseAng2 8 13 8 tbd rabbitAng2 4 11 8 tbd
TABLE-US-00009 TABLE 7 Results: Affinity to FcRn of
<VEGF-ANG-2> bispecific antibodies VEGFang2- VEGFang2-
VEGFang2- VEGFang2-- 0015 0016 0012 0201 [affinity] [affinity]
[affinity] [affinity] Human 0.8 .mu.M no binding no binding 0.8
.mu.M FcRn Cyno 0.9 .mu.M no binding no binding 1.0 .mu.M FcRn
Mouse 0.2 .mu.M no binding no binding 0.2 .mu.M FcRn
TABLE-US-00010 TABLE 8 Results Binding to FcgammaRI-IIIa VEGFang2-
VEGFang2- VEGFang2- VEGFang2- 0015 0016 0012 0201 FcyRIa No binding
No binding Binding Binding FcyRIIa No binding No binding No binding
Binding FcyRIIIa No binding No binding No binding Binding
TABLE-US-00011 TABLE 9 Results: Independent binding of VEGF-and
Ang2 to <VEGF-ANG-2> bispecific antibodies 3) first 4) first
5) VEGF Ang2 Coinjection then then Ang2 + 1) Ang2 2) VEGF Ang2 VEGF
VEGF [RUmax] [RUmax] [RUmax] [RUmax] [RUmax] VEGFang2- 174 50 211
211 211 0016 VEGFang2- 143 43 178 177 178 0012
Example 4
[0332] Mass Spectrometry
[0333] This section describes the characterization of
<VEGF-ANG-2> bispecific antibodies with emphasis on the
correct assembly. The expected primary structures were confirmed by
electrospray ionization mass spectrometry (ESI-MS) of the
deglycosylated, and intact or IdeS-digested (IgG-degrading enzyme
of S. pyogenes) <VEGF-ANG-2> bispecific antibodies. The
IdeS-digestion was performed with 100 .mu.g purified antibody
incubated with 2 .mu.g IdeS protease (Roche) in 100 mmol/L
NaH.sub.2PO.sub.4/Na.sub.2HPO.sub.4, pH 7.1 at 37.degree. C. for 5
h. Subsequently, the antibodies were deglycosylated with
N-Glycosidase F, Neuraminidase and O-glycosidase (Roche) in 100
mmol/L NaH.sub.2PO.sub.4/Na.sub.2HPO.sub.4, pH 7.1 at 37.degree. C.
for up to 16 h at a protein concentration of 1 mg/ml and
subsequently desalted via HPLC on a Sephadex G25 column (GE
Healthcare). The total mass was determined via ESI-MS on a maXis 4G
UHR-QTOF MS system (Bruker Daltonik) equipped with a TriVersa
NanoMate.RTM. source (Advion).
[0334] The masses obtained for the IdeS-digested, deglycosylated
(Table 10), or intact, deglycosylated (Table 11) molecules
correspond to the predicted masses deduced from the amino acid
sequences for the <VEGF-ANG-2> bispecific antibodies
consisting of two different light chains LC.sub.Ang2 and
LC.sub.Lucentis, and two different heavy chains HC.sub.Ang2 and
HC.sub.Lucentis.
TABLE-US-00012 TABLE 10 Masses of the deglycosylated and
IdeS-digested bispecific <VEGF/ANG2> antibodies VEGFang2-0201
(without AAA mutation) andVEGFang2-0012 (with AAA mutation) F(ab')2
of the VEGF- Deglycosylated Fc of ANG-2> bispecific the
VEGF-ANG-2> antibody bispecific antibody Predicted Observed
Predicted Observed Average Average Average Average Mass Mass Mass
Mass Sample [Da] [Da] [Da] [Da] VEGFang2- 99360.8 99360.7 47439.2
47430.1 0201 VEGFang2- 99360.8 99361.1 47087.7 47082.0 0012
TABLE-US-00013 TABLE 11 Masses of the deglycosylated
<VEGF/ANG2> antibodies VEGFang2-0016 (with AAA mutation) and
VEGFang2-0015 (without AAA mutation) Deglycosylated VEGF-ANG-2>
bispecific antibody Predicted Observed Average Average Mass Mass
[Da] [Da] VEGFang2- 146156.9 146161.2 0016 VEGFang2- 146505.3
146509.4 0015
Example 5
[0335] Fc-Rn Chromatography
[0336] Coupling to Streptavidin Sepharose:
[0337] One gram streptavidin Sepharose.RTM. (GE Healthcare) was
added to the biotinylated and dialyzed receptor and incubated for
two hours with shaking. The receptor derivatized sepharose was
filled in a 1 ml XK column (GE Healthcare).
[0338] Chromatography Using the FcRn Affinity Column:
[0339] Conditions:
[0340] column dimensions: 50 mm.times.5 mm
[0341] bed height: 5 cm
[0342] loading: 50 .mu.g sample
[0343] equilibration buffer: 20 mM MES, with 150 mM NaCl, adjusted
to pH 5.5
[0344] elution buffer: 20 mM Tris/HC1, with 150 mM NaCl, adjusted
to pH 8.8
[0345] elution: 7.5 CV equilibration buffer, in 30 CV to 100%
elution buffer, 10 CV elution buffer
[0346] Hu FcRn Affinity Column Chromatography
[0347] In the following table retention times of <VEGF-ANG-2>
bispecific antibodies on affinity columns comprising human FcRn are
given. Data were obtained using the conditions above. In the
following Table retention times of <VEGF-ANG-2> bispecific
antibodies on human FcRn are given.
TABLE-US-00014 TABLE 12 Results: retention times of
<VEGF-ANG-2> bispecific antibodies retention time antibody
[min] VEGFAng2-0015 78.5 (without AAA mutation) VEGFAng2-0201 78.9
(without AAA mutation) VEGFAng2-0012 2.7 (Void-peak) (with AAA
mutation) VEGFAng2-0016 2.7 (Void-peak) (with AAA mutation)
Example 6
[0348] Pharmacokinetic(PK) Properties
[0349] PK Data with Fc-Rn Mice Transgenic for Human FcRn
[0350] In Life Phase
[0351] The study included female C57BL/6J mice (background); mouse
FcRn deficient, but hemizygous transgenic for human FcRn (huFcRn,
line 276-/tg)
[0352] Part 1
[0353] All mice were injected once intravitreally into the right
eye with 2 .mu.L/animal of the appropriate solution (i.e. 21 .mu.g
compound/animal (VEGFAng2-0015 (without AAA mutation) or 23.6 .mu.g
compound/animal (VEGFAng2-0016 (with AAA mutation).
[0354] Mice were allocated to 2 groups with 6 animals each. Blood
samples are taken from group 1 at 2, 24 and 96 hours and from group
2 at 7, 48 and 168 hours after dosing.
[0355] Injection into the vitreous of the right mouse eye was
performed by using the NanoFil Microsyringe system for nanoliter
injection from World Precision
[0356] Instruments, Inc., Berlin, Germany. Mice were anesthetized
with 2.5% Isoflurane and for visualization of the mouse eye a Leica
MZFL 3 microscope with a 40 fold magnification and a ring-light
with a Leica KL 2500 LCD lightning was used. Subsequently, 2 .mu.L
of the compound were injected using a 35-gauge needle.
[0357] Blood was collected via the retrobulbar venous plexus of the
contralateral eye from each animal for the determination of the
compound levels in serum.
[0358] Serum samples of at least 50 .mu.l were obtained from blood
after 1 hour at RT by centrifugation (9300 xg) at 4.degree. C. for
3 min. Serum samples were frozen directly after centrifugation and
stored frozen at -80.degree. C. until analysis. Treated eyes of the
animals of group 1 were isolated 96 hours after treatment and of
the animals of group 2 168 hours after treatment. Samples were
stored frozen at -80.degree. C. until analysis.
[0359] Part 2
[0360] All mice were injected once intravenously via the tail vein
with 200 .mu.L/animal of the appropriate solution (i.e. 21 .mu.g
compound/animal (VEGFAng2-0015 (without AAA mutation) or 23.6 .mu.g
compound/animal (VEGFAng2-0016 (with AAA mutation).
[0361] Mice were allocated to 2 groups with 5 animals each. Blood
samples are taken from group 1 at 1, 24 and 96 hours and from group
2 at 7, 48 and 168 hours after dosing. Blood was collected via the
retrobulbar venous plexus from each animal for the determination of
the compound levels in serum.
[0362] Serum samples of at least 50 .mu.l were obtained from blood
after 1 hour at RT by centrifugation (9300 xg) at 4.degree. C. for
3 min. Serum samples were frozen directly after centrifugation and
stored frozen at -80.degree. C. until analysis.
[0363] Preparation of Whole Eye Lysates (Mice)
[0364] The eye lysates were gained by physico-chemical
disintegration of the whole eye from laboratory animals. For
mechanical disruption, each eye was transferred into a 1.5-mL micro
vial with conical bottom. After freeze and thawing, the eyes were
washed with 1 mL cell washing buffer once (Bio-Rad, Bio-Plex Cell
Lysis Kit, Cat. No. 171-304011). In the following step, 5004 of
freshly prepared cell lysis buffer were added and the eyes were
grinded using a 1.5 mL tissue grinding pestle (Kimble Chase, 1.5 mL
pestle, Art. No. 749521-1500). The mixture was then frozen and
thawed five times and grinded again. To separate lysate from
remaining tissue the samples were centrifuged for 4 min at 4500 g.
After centrifuging the supernatant was collected and stored at
-20.degree. C. until further analysis in the quantification
ELISA.
[0365] Analysis
[0366] The concentrations of the <VEGF/ANG2> antibodies in
mice serum and eye lysates were determined with an enzyme linked
immunosorbent assay (ELISA)
[0367] For quantification of <VEGF/ANG2> antibodies in mouse
serum samples and eye lysates, a standard solid-phase serial
sandwich immunoassay with biotinylated and digoxigenated monoclonal
antibodies used as capture and detection antibodies was performed.
To verify the integrity of the bispecifity of the analyte the
biotinylated capture antibody recognizes the anti-VEGF-binding site
whereas the digoxigenated detection antibody will bind to the
anti-Ang2 binding site of the analyte. The bound immune complex of
capture antibody, analyte and detection antibody on the solid phase
of the streptavidin coated micro titer plate (SA-MTP) is then
detected with a horseradish-peroxidase coupled to an
anti-digoxigenin antibody. After washing unbound material from the
SA-MTP and addition of ABTS-substrate, the gained signal is
proportional to the amount of analyte bound on the solid phase of
the SA-MTP. Quantification is then done by converting the measured
signals of the samples into concentrations referring to calibrators
analyzed in parallel.
[0368] In a first step the SA-MTP was coated with 100 .mu.L/well of
biotinylated capture antibody solution
(mAb<Id<VEGF>>M-2.45.51-IgG-Bi(DDS)) with a
concentration of 1 .mu.g/mL for one hour at 500 rpm on a
MTP-shaker. Meanwhile calibrators, QC-samples and samples were
prepared. Calibrators and QC-samples are diluted to 2% serum
matrix; samples were diluted until the signals were within the
linear range of the calibrators.
[0369] After coating the SA-MTP with capture antibody, the plate
was washed three times with washing buffer and 300 .mu.L/well.
Subsequently 100 .mu.l/well of the calibrators, QC-samples and
samples were pipetted on the SA-MTP and incubated again for one
hour at 500 rpm. The analyte was now bound with its anti-VEGF
binding site via the capture antibody to the solid phase of the
SA-MTP. After incubation and removal of unbound analyte by washing
the plate 100 .mu.L/well of the first detection antibody
(mAb<Id-<Ang2>>M-2.6.81-IgG-Dig(XOSu)) with a
concentration of 250 ng/mL was added to the SA-MTP. Again, the
plate was incubated for one hour at 500 rpm on a shaker. After
washing, 100 .mu.L/well of the second detection antibody
(pAb<Digoxigenin>S-Fab-POD (poly)) at a concentration of 50
mU/mL was added to the wells of the SA-MTP and the plate was
incubated again for one hour at 500 rpm. After a final washing step
to remove excess of detection antibody, 100 .mu.L/well substrate
(ABTS) is added. The antibody-enzyme conjugate catalyzes the color
reaction of the ABTS.RTM. substrate. The signal was then measured
by an ELISA reader at 405 nm wavelength (reference wavelength: 490
nm ([405/490] nm)).
[0370] Pharmacokinetic Evaluation
[0371] The pharmacokinetic parameters were calculated by
non-compartmental analysis, using the pharmacokinetic evaluation
program WinNonlin.TM. (Pharsight), version 5.2.1.
[0372] Results: A) Serum Concentrations
[0373] Results for serum concentrations are shown in Tables 13 to
16 and FIG. 7B to 7C
TABLE-US-00015 TABLE 13 VEGFAng2-0015 (without AAA mutation):
Comparison of serum concentrations after intravitreal and
intravenous application Serum concentration Serum concentration
after intravitreal after intravenous application application
Average conc. Average conc. ID [.mu.g/mL] [.mu.g/mL] 1 h 17.7 2 h
9.8 7 h 10.4 12.1 24 h 6.4 8.3 48 h 6.5 6.9 96 h 3.4 4.1 168 h 2.9
2.7
TABLE-US-00016 TABLE 14 VEGFAng2-0016 (with AAA mutation):
Comparison of serum concentrations after intravitreal and
intravenous application Serum concentration Serum concentration
after intravitreal after intravenous application application
Average conc. Average conc. ID [.mu.g/mL] [.mu.g/mL] 1 h 18.4 2 h
7.0 7 h 8.7 10.0 24 h 2.2 3.3 48 h 1.0 1.0 96 h 0.1 0.1 168 h 0.0
0.0
TABLE-US-00017 TABLE 15 VEGFang2-0015 (without AAA mutation) and
VEGFang2-0016 (with AAA mutation): Comparison of serum
concentrations after intravitreal application) VEGFang2-0015
VEGFang2-0016 (without AAA mutation) (with AAA mutation) Average
conc. Average conc. ID [.mu.g/mL] [.mu.g/mL] 2 h 9.8 7.0 7 h 10.4
8.7 24 h 6.4 2.2 48 h 6.5 1.0 96 h 3.4 0.1 168 h 2.9 0.0
TABLE-US-00018 TABLE 16 VEGFang2-0015 (without AAA mutation) and
VEGFang2-0016 (with AAA mutation): Comparison of serum
concentrations after intravenous application VEGFang2-0015
VEGFang2-0016 (without AAA mutation) (with AAA mutation) Average
conc. Average conc. ID [.mu.g/mL] [.mu.g/mL] 1 h 17.7 18.4 7 h 12.1
10.0 24 h 8.3 3.3 48 h 6.9 1.0 96 h 4.1 0.1 168 h 2.7 0.0
[0374] Results: B) Concentrations in Eye-Lysates of Left and Right
Eyes
[0375] Results for concentrations in eye lysates are shown in
Tables 17 to 18 and FIGS. 7D to 7E
TABLE-US-00019 TABLE 17 a: Concentrations of VEGFang2-0015 (without
AAA mutation) in eye lysates after intra vitreal application into
right eye Mean conc. values from n = 6 mice ID mean conc. [ng/mL]
96 h Left eye 8.7 Right eye 46.1 168 h Left eye 4.3 Right eye t
12.9 b: Concentrations of VEGFang2-0015 (without AAA mutation) in
eye lysates after intravenous application Mean conc. values from n
= 5 mice ID mean conc. [ng/mL] 96 h Left eye 4.2 Right eye 7.5 168
h Left eye 3.4 Right eye 6.1
TABLE-US-00020 TABLE 18 a: Concentrations of VEGFang2-0016 (with
AAA mutation) in eye lysates after intra vitreal application into
right eye Mean conc. values from n = 5 mice ID mean conc. [ng/mL]
96 h Left eye 0.3 Right eye 34.5 168 h Left eye 0.1 Right eye 9.0
b: Concentrations of VEGFang2-0016 (with AAA mutation) in eye
lysates after intravenous application Mean conc. values from n = 5
mice ID mean conc. [ng/mL] 96 h Left eye 0.0 Right eye 0.1 168 h
Left eye 0.0 Right eye 0.1
[0376] Summary of Results:
[0377] After intravitreal application the bispecific
<VEGF/ANG2> antibody according to the invention VEGFang2-0016
(with AAA mutation) shows similar concentrations (after 96 and 168
hours) in the eye lysates as compared to the bispecific
<VEGF/ANG2> antibody without AAA mutation VEGFang2-0015.
[0378] Also after intravitreal application the bispecific
<VEGF/ANG2> antibody according to the invention VEGFang2-0016
(with AAA mutation) shows in addition a faster clearance and
shorter half-life in the serum as compared to the bispecific
<VEGF/ANG2> antibody without AAA mutation VEGFang2-0015.
Example 7
[0379] Mouse Cornea Micropocket Angiogenesis Assay
[0380] To test the anti-angiogenic effect bispecific
<VEGF/ANG2> antibody with the respective anti-VEGF VH and VL
of SEQ ID NO: 7 and 8 and the anti-ANG2 VH and VL of SEQ ID NO: 15
and 16 on VEGF-induced angiogenesis in vivo, we perform the mouse
corneal angiogenesis assay. In this assay a VEGF soaked
Nylaflo.RTM. disc is implanted into a pocket of the avascular
cornea at a fixed distance to the limbal vessels. Vessels
immediately grow into the cornea towards the developing VEGF
gradient. 8 to 10 weeks old female Balb/c mice were purchased from
Charles River, Sulzfeld, Germany. The protocol is modified
according to the method described by Rogers, M. S., et al., Nat.
Protoc. 2 (2007) 2545-2550. Briefly, micropockets with a width of
about 500 .mu.m are prepared under a microscope at approximately 1
mm from the limbus to the top of the cornea using a surgical blade
and sharp tweezers in the anesthetized mouse. The disc
(Nylaflo.RTM., Pall Corporation, Michigan) with a diameter of 0.6
mm is implanted and the surface of the implantation area was
smoothened. Discs are incubated in corresponding growth factor or
in vehicle for at least 30 min. After 3, 5 and 7 days (or
alternatively only after 3, 5 or 7 days) , eyes are photographed
and vascular response is measured. The assay is quantified by
calculating the percentage of the area of new vessels per total
area of the cornea.
[0381] The discs are loaded with 300 ng VEGF or with PBS as a
control and implanted for 7 days. The outgrowth of vessels from the
limbus to the disc is monitored over time on day 3, 5 and/or 7. One
day prior to disc implantation the antibodies are administered
intravenously at a dose of 10 mg/kg (due to the intravenous
application the serum-stable VEGFang2-0015 (without AAA mutation)
which only differs from VEGFang2-0016 by the AAA mutation and has
the same anti-VEGF and anti-ANG2 VHs and VLs to mediate efficacy,
is used as surrogate) for testing the anti-angiogenic effect on
VEGF-induced angiogenesis in vivo. Animals in the control group
receive vehicle. The application volume is 10 ml/kg.
Sequence CWU 1
1
50114PRTArtificialheavy chain CDR3H, <VEGF>ranibizumab 1Tyr
Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val1 5
10217PRTArtificialheavy chain CDR2H, <VEGF>ranibizumab 2Trp
Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe Lys1 5 10
15Arg35PRTArtificialheavy chain CDR1H, <VEGF>ranibizumab 3His
Tyr Gly Met Asn1 549PRTArtificiallight chain CDR3L,
<VEGF>ranibizumab 4Gln Gln Tyr Ser Thr Val Pro Trp Thr1
557PRTArtificiallight chain CDR2L, <VEGF>ranibizumab 5Phe Thr
Ser Ser Leu His Ser1 5611PRTArtificiallight chain CDR1L,
<VEGF>ranibizumab 6Ser Ala Ser Gln Asp Ile Ser Asn Tyr Leu
Asn1 5 107123PRTArtificialheavy chain variable domain VH,
<VEGF>ranibizumab 7Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Tyr Asp Phe Thr His Tyr 20 25 30Gly Met Asn Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Trp Ile Asn Thr Tyr Thr
Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60Lys Arg Arg Phe Thr Phe
Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Tyr
Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val 100 105 110Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
1208107PRTArtificiallight chain variable domain VL,
<VEGF>ranibizumab 8Asp 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 Gln Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Val Leu Ile 35 40 45Tyr Phe Thr Ser Ser Leu His
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys 100 105920PRTArtificialheavy chain
CDR3H, <ANG-2> Ang2i_LC10 variant 9Ser Pro Asn Pro Tyr Tyr
Tyr Asp Ser Ser Gly Tyr Tyr Tyr Pro Gly1 5 10 15Ala Phe Asp Ile
201017PRTArtificialheavy chain CDR2H, <ANG-2> Ang2i_LC10
variant 10Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys
Phe Gln1 5 10 15Gly115PRTArtificialheavy chain CDR1H, <ANG-2>
Ang2i_LC10 variant 11Gly Tyr Tyr Met His1 51211PRTArtificiallight
chain CDR3L, <ANG-2> Ang2i_LC10 variant 12Gln Val Trp Asp Ser
Ser Ser Asp His Trp Val1 5 10137PRTArtificiallight chain CDR2L,
<ANG-2> Ang2i_LC10 variant 13Asp Asp Ser Asp Arg Pro Ser1
51411PRTArtificiallight chain CDR1L, <ANG-2> Ang2i_LC10
variant 14Gly Gly Asn Asn Ile Gly Ser Lys Ser Val His1 5
1015129PRTArtificialheavy chain variable domain VH, <ANG-2>
Ang2i_LC10 variant 15Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Ser Gly
Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr
Arg Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Arg
Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Pro
Asn Pro Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr Tyr 100 105 110Pro Gly
Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser 115 120
125Ser16110PRTArtificiallight chain variable domain VL,
<ANG-2> Ang2i_LC10 variant 16Ser Tyr Val Leu Thr Gln Pro Pro
Ser Val Ser Val Ala Pro Gly Gln1 5 10 15Thr Ala Arg Ile Thr Cys Gly
Gly Asn Asn Ile Gly Ser Lys Ser Val 20 25 30His Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45Asp Asp Ser Asp Arg
Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60Asn Ser Gly Asn
Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly65 70 75 80Asp Glu
Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95Trp
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105
11017191PRTHomo sapiens 17Met Asn Phe Leu Leu Ser Trp Val His Trp
Ser Leu Ala Leu Leu Leu1 5 10 15Tyr Leu His His Ala Lys Trp Ser Gln
Ala Ala Pro Met Ala Glu Gly 20 25 30Gly Gly Gln Asn His His Glu Val
Val Lys Phe Met Asp Val Tyr Gln 35 40 45Arg Ser Tyr Cys His Pro Ile
Glu Thr Leu Val Asp Ile Phe Gln Glu 50 55 60Tyr Pro Asp Glu Ile Glu
Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu65 70 75 80Met Arg Cys Gly
Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro 85 90 95Thr Glu Glu
Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His 100 105 110Gln
Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys 115 120
125Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Asn Pro Cys Gly
130 135 140Pro Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln Asp Pro
Gln Thr145 150 155 160Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg
Cys Lys Ala Arg Gln 165 170 175Leu Glu Leu Asn Glu Arg Thr Cys Arg
Cys Asp Lys Pro Arg Arg 180 185 19018496PRTHomo sapiens 18Met Trp
Gln Ile Val Phe Phe Thr Leu Ser Cys Asp Leu Val Leu Ala1 5 10 15Ala
Ala Tyr Asn Asn Phe Arg Lys Ser Met Asp Ser Ile Gly Lys Lys 20 25
30Gln Tyr Gln Val Gln His Gly Ser Cys Ser Tyr Thr Phe Leu Leu Pro
35 40 45Glu Met Asp Asn Cys Arg Ser Ser Ser Ser Pro Tyr Val Ser Asn
Ala 50 55 60Val Gln Arg Asp Ala Pro Leu Glu Tyr Asp Asp Ser Val Gln
Arg Leu65 70 75 80Gln Val Leu Glu Asn Ile Met Glu Asn Asn Thr Gln
Trp Leu Met Lys 85 90 95Leu Glu Asn Tyr Ile Gln Asp Asn Met Lys Lys
Glu Met Val Glu Ile 100 105 110Gln Gln Asn Ala Val Gln Asn Gln Thr
Ala Val Met Ile Glu Ile Gly 115 120 125Thr Asn Leu Leu Asn Gln Thr
Ala Glu Gln Thr Arg Lys Leu Thr Asp 130 135 140Val Glu Ala Gln Val
Leu Asn Gln Thr Thr Arg Leu Glu Leu Gln Leu145 150 155 160Leu Glu
His Ser Leu Ser Thr Asn Lys Leu Glu Lys Gln Ile Leu Asp 165 170
175Gln Thr Ser Glu Ile Asn Lys Leu Gln Asp Lys Asn Ser Phe Leu Glu
180 185 190Lys Lys Val Leu Ala Met Glu Asp Lys His Ile Ile Gln Leu
Gln Ser 195 200 205Ile Lys Glu Glu Lys Asp Gln Leu Gln Val Leu Val
Ser Lys Gln Asn 210 215 220Ser Ile Ile Glu Glu Leu Glu Lys Lys Ile
Val Thr Ala Thr Val Asn225 230 235 240Asn Ser Val Leu Gln Lys Gln
Gln His Asp Leu Met Glu Thr Val Asn 245 250 255Asn Leu Leu Thr Met
Met Ser Thr Ser Asn Ser Ala Lys Asp Pro Thr 260 265 270Val Ala Lys
Glu Glu Gln Ile Ser Phe Arg Asp Cys Ala Glu Val Phe 275 280 285Lys
Ser Gly His Thr Thr Asn Gly Ile Tyr Thr Leu Thr Phe Pro Asn 290 295
300Ser Thr Glu Glu Ile Lys Ala Tyr Cys Asp Met Glu Ala Gly Gly
Gly305 310 315 320Gly Trp Thr Ile Ile Gln Arg Arg Glu Asp Gly Ser
Val Asp Phe Gln 325 330 335Arg Thr Trp Lys Glu Tyr Lys Val Gly Phe
Gly Asn Pro Ser Gly Glu 340 345 350Tyr Trp Leu Gly Asn Glu Phe Val
Ser Gln Leu Thr Asn Gln Gln Arg 355 360 365Tyr Val Leu Lys Ile His
Leu Lys Asp Trp Glu Gly Asn Glu Ala Tyr 370 375 380Ser Leu Tyr Glu
His Phe Tyr Leu Ser Ser Glu Glu Leu Asn Tyr Arg385 390 395 400Ile
His Leu Lys Gly Leu Thr Gly Thr Ala Gly Lys Ile Ser Ser Ile 405 410
415Ser Gln Pro Gly Asn Asp Phe Ser Thr Lys Asp Gly Asp Asn Asp Lys
420 425 430Cys Ile Cys Lys Cys Ser Gln Met Leu Thr Gly Gly Trp Trp
Phe Asp 435 440 445Ala Cys Gly Pro Ser Asn Leu Asn Gly Met Tyr Tyr
Pro Gln Arg Gln 450 455 460Asn Thr Asn Lys Phe Asn Gly Ile Lys Trp
Tyr Tyr Trp Lys Gly Ser465 470 475 480Gly Tyr Ser Leu Lys Ala Thr
Thr Met Met Ile Arg Pro Ala Asp Phe 485 490 49519498PRTHomo sapiens
19Met Thr Val Phe Leu Ser Phe Ala Phe Leu Ala Ala Ile Leu Thr His1
5 10 15Ile Gly Cys Ser Asn Gln Arg Arg Ser Pro Glu Asn Ser Gly Arg
Arg 20 25 30Tyr Asn Arg Ile Gln His Gly Gln Cys Ala Tyr Thr Phe Ile
Leu Pro 35 40 45Glu His Asp Gly Asn Cys Arg Glu Ser Thr Thr Asp Gln
Tyr Asn Thr 50 55 60Asn Ala Leu Gln Arg Asp Ala Pro His Val Glu Pro
Asp Phe Ser Ser65 70 75 80Gln Lys Leu Gln His Leu Glu His Val Met
Glu Asn Tyr Thr Gln Trp 85 90 95Leu Gln Lys Leu Glu Asn Tyr Ile Val
Glu Asn Met Lys Ser Glu Met 100 105 110Ala Gln Ile Gln Gln Asn Ala
Val Gln Asn His Thr Ala Thr Met Leu 115 120 125Glu Ile Gly Thr Ser
Leu Leu Ser Gln Thr Ala Glu Gln Thr Arg Lys 130 135 140Leu Thr Asp
Val Glu Thr Gln Val Leu Asn Gln Thr Ser Arg Leu Glu145 150 155
160Ile Gln Leu Leu Glu Asn Ser Leu Ser Thr Tyr Lys Leu Glu Lys Gln
165 170 175Leu Leu Gln Gln Thr Asn Glu Ile Leu Lys Ile His Glu Lys
Asn Ser 180 185 190Leu Leu Glu His Lys Ile Leu Glu Met Glu Gly Lys
His Lys Glu Glu 195 200 205Leu Asp Thr Leu Lys Glu Glu Lys Glu Asn
Leu Gln Gly Leu Val Thr 210 215 220Arg Gln Thr Tyr Ile Ile Gln Glu
Leu Glu Lys Gln Leu Asn Arg Ala225 230 235 240Thr Thr Asn Asn Ser
Val Leu Gln Lys Gln Gln Leu Glu Leu Met Asp 245 250 255Thr Val His
Asn Leu Val Asn Leu Cys Thr Lys Glu Gly Val Leu Leu 260 265 270Lys
Gly Gly Lys Arg Glu Glu Glu Lys Pro Phe Arg Asp Cys Ala Asp 275 280
285Val Tyr Gln Ala Gly Phe Asn Lys Ser Gly Ile Tyr Thr Ile Tyr Ile
290 295 300Asn Asn Met Pro Glu Pro Lys Lys Val Phe Cys Asn Met Asp
Val Asn305 310 315 320Gly Gly Gly Trp Thr Val Ile Gln His Arg Glu
Asp Gly Ser Leu Asp 325 330 335Phe Gln Arg Gly Trp Lys Glu Tyr Lys
Met Gly Phe Gly Asn Pro Ser 340 345 350Gly Glu Tyr Trp Leu Gly Asn
Glu Phe Ile Phe Ala Ile Thr Ser Gln 355 360 365Arg Gln Tyr Met Leu
Arg Ile Glu Leu Met Asp Trp Glu Gly Asn Arg 370 375 380Ala Tyr Ser
Gln Tyr Asp Arg Phe His Ile Gly Asn Glu Lys Gln Asn385 390 395
400Tyr Arg Leu Tyr Leu Lys Gly His Thr Gly Thr Ala Gly Lys Gln Ser
405 410 415Ser Leu Ile Leu His Gly Ala Asp Phe Ser Thr Lys Asp Ala
Asp Asn 420 425 430Asp Asn Cys Met Cys Lys Cys Ala Leu Met Leu Thr
Gly Gly Trp Trp 435 440 445Phe Asp Ala Cys Gly Pro Ser Asn Leu Asn
Gly Met Phe Tyr Thr Ala 450 455 460Gly Gln Asn His Gly Lys Leu Asn
Gly Ile Lys Trp His Tyr Phe Lys465 470 475 480Gly Pro Ser Tyr Ser
Leu Arg Ser Thr Thr Met Met Ile Arg Pro Leu 485 490 495Asp
Phe201124PRTHomo sapiens 20Met Asp Ser Leu Ala Ser Leu Val Leu Cys
Gly Val Ser Leu Leu Leu1 5 10 15Ser Gly Thr Val Glu Gly Ala Met Asp
Leu Ile Leu Ile Asn Ser Leu 20 25 30Pro Leu Val Ser Asp Ala Glu Thr
Ser Leu Thr Cys Ile Ala Ser Gly 35 40 45Trp Arg Pro His Glu Pro Ile
Thr Ile Gly Arg Asp Phe Glu Ala Leu 50 55 60Met Asn Gln His Gln Asp
Pro Leu Glu Val Thr Gln Asp Val Thr Arg65 70 75 80Glu Trp Ala Lys
Lys Val Val Trp Lys Arg Glu Lys Ala Ser Lys Ile 85 90 95Asn Gly Ala
Tyr Phe Cys Glu Gly Arg Val Arg Gly Glu Ala Ile Arg 100 105 110Ile
Arg Thr Met Lys Met Arg Gln Gln Ala Ser Phe Leu Pro Ala Thr 115 120
125Leu Thr Met Thr Val Asp Lys Gly Asp Asn Val Asn Ile Ser Phe Lys
130 135 140Lys Val Leu Ile Lys Glu Glu Asp Ala Val Ile Tyr Lys Asn
Gly Ser145 150 155 160Phe Ile His Ser Val Pro Arg His Glu Val Pro
Asp Ile Leu Glu Val 165 170 175His Leu Pro His Ala Gln Pro Gln Asp
Ala Gly Val Tyr Ser Ala Arg 180 185 190Tyr Ile Gly Gly Asn Leu Phe
Thr Ser Ala Phe Thr Arg Leu Ile Val 195 200 205Arg Arg Cys Glu Ala
Gln Lys Trp Gly Pro Glu Cys Asn His Leu Cys 210 215 220Thr Ala Cys
Met Asn Asn Gly Val Cys His Glu Asp Thr Gly Glu Cys225 230 235
240Ile Cys Pro Pro Gly Phe Met Gly Arg Thr Cys Glu Lys Ala Cys Glu
245 250 255Leu His Thr Phe Gly Arg Thr Cys Lys Glu Arg Cys Ser Gly
Gln Glu 260 265 270Gly Cys Lys Ser Tyr Val Phe Cys Leu Pro Asp Pro
Tyr Gly Cys Ser 275 280 285Cys Ala Thr Gly Trp Lys Gly Leu Gln Cys
Asn Glu Ala Cys His Pro 290 295 300Gly Phe Tyr Gly Pro Asp Cys Lys
Leu Arg Cys Ser Cys Asn Asn Gly305 310 315 320Glu Met Cys Asp Arg
Phe Gln Gly Cys Leu Cys Ser Pro Gly Trp Gln 325 330 335Gly Leu Gln
Cys Glu Arg Glu Gly Ile Pro Arg Met Thr Pro Lys Ile 340 345 350Val
Asp Leu Pro Asp His Ile Glu Val Asn Ser Gly Lys Phe Asn Pro 355 360
365Ile Cys Lys Ala Ser Gly Trp Pro Leu Pro Thr Asn Glu Glu Met Thr
370 375 380Leu Val Lys Pro Asp Gly Thr Val Leu His Pro Lys Asp Phe
Asn His385 390 395 400Thr Asp His Phe Ser Val Ala Ile Phe Thr Ile
His Arg Ile Leu Pro 405 410 415Pro Asp Ser Gly Val Trp Val Cys Ser
Val Asn Thr Val Ala Gly Met 420 425 430Val Glu Lys Pro Phe Asn Ile
Ser Val Lys Val Leu Pro Lys Pro Leu 435 440 445Asn Ala Pro Asn Val
Ile Asp Thr Gly His Asn Phe Ala Val Ile Asn 450 455 460Ile Ser Ser
Glu Pro Tyr Phe Gly Asp Gly Pro Ile Lys Ser Lys Lys465 470 475
480Leu Leu Tyr Lys Pro Val Asn His Tyr Glu Ala Trp Gln His Ile Gln
485 490 495Val Thr Asn Glu Ile Val Thr Leu Asn Tyr Leu Glu Pro Arg
Thr Glu 500 505 510Tyr Glu Leu Cys Val Gln Leu Val Arg Arg Gly Glu
Gly Gly
Glu Gly 515 520 525His Pro Gly Pro Val Arg Arg Phe Thr Thr Ala Ser
Ile Gly Leu Pro 530 535 540Pro Pro Arg Gly Leu Asn Leu Leu Pro Lys
Ser Gln Thr Thr Leu Asn545 550 555 560Leu Thr Trp Gln Pro Ile Phe
Pro Ser Ser Glu Asp Asp Phe Tyr Val 565 570 575Glu Val Glu Arg Arg
Ser Val Gln Lys Ser Asp Gln Gln Asn Ile Lys 580 585 590Val Pro Gly
Asn Leu Thr Ser Val Leu Leu Asn Asn Leu His Pro Arg 595 600 605Glu
Gln Tyr Val Val Arg Ala Arg Val Asn Thr Lys Ala Gln Gly Glu 610 615
620Trp Ser Glu Asp Leu Thr Ala Trp Thr Leu Ser Asp Ile Leu Pro
Pro625 630 635 640Gln Pro Glu Asn Ile Lys Ile Ser Asn Ile Thr His
Ser Ser Ala Val 645 650 655Ile Ser Trp Thr Ile Leu Asp Gly Tyr Ser
Ile Ser Ser Ile Thr Ile 660 665 670Arg Tyr Lys Val Gln Gly Lys Asn
Glu Asp Gln His Val Asp Val Lys 675 680 685Ile Lys Asn Ala Thr Ile
Thr Gln Tyr Gln Leu Lys Gly Leu Glu Pro 690 695 700Glu Thr Ala Tyr
Gln Val Asp Ile Phe Ala Glu Asn Asn Ile Gly Ser705 710 715 720Ser
Asn Pro Ala Phe Ser His Glu Leu Val Thr Leu Pro Glu Ser Gln 725 730
735Ala Pro Ala Asp Leu Gly Gly Gly Lys Met Leu Leu Ile Ala Ile Leu
740 745 750Gly Ser Ala Gly Met Thr Cys Leu Thr Val Leu Leu Ala Phe
Leu Ile 755 760 765Ile Leu Gln Leu Lys Arg Ala Asn Val Gln Arg Arg
Met Ala Gln Ala 770 775 780Phe Gln Asn Val Arg Glu Glu Pro Ala Val
Gln Phe Asn Ser Gly Thr785 790 795 800Leu Ala Leu Asn Arg Lys Val
Lys Asn Asn Pro Asp Pro Thr Ile Tyr 805 810 815Pro Val Leu Asp Trp
Asn Asp Ile Lys Phe Gln Asp Val Ile Gly Glu 820 825 830Gly Asn Phe
Gly Gln Val Leu Lys Ala Arg Ile Lys Lys Asp Gly Leu 835 840 845Arg
Met Asp Ala Ala Ile Lys Arg Met Lys Glu Tyr Ala Ser Lys Asp 850 855
860Asp His Arg Asp Phe Ala Gly Glu Leu Glu Val Leu Cys Lys Leu
Gly865 870 875 880His His Pro Asn Ile Ile Asn Leu Leu Gly Ala Cys
Glu His Arg Gly 885 890 895Tyr Leu Tyr Leu Ala Ile Glu Tyr Ala Pro
His Gly Asn Leu Leu Asp 900 905 910Phe Leu Arg Lys Ser Arg Val Leu
Glu Thr Asp Pro Ala Phe Ala Ile 915 920 925Ala Asn Ser Thr Ala Ser
Thr Leu Ser Ser Gln Gln Leu Leu His Phe 930 935 940Ala Ala Asp Val
Ala Arg Gly Met Asp Tyr Leu Ser Gln Lys Gln Phe945 950 955 960Ile
His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Gly Glu Asn Tyr 965 970
975Val Ala Lys Ile Ala Asp Phe Gly Leu Ser Arg Gly Gln Glu Val Tyr
980 985 990Val Lys Lys Thr Met Gly Arg Leu Pro Val Arg Trp Met Ala
Ile Glu 995 1000 1005Ser Leu Asn Tyr Ser Val Tyr Thr Thr Asn Ser
Asp Val Trp Ser 1010 1015 1020Tyr Gly Val Leu Leu Trp Glu Ile Val
Ser Leu Gly Gly Thr Pro 1025 1030 1035Tyr Cys Gly Met Thr Cys Ala
Glu Leu Tyr Glu Lys Leu Pro Gln 1040 1045 1050Gly Tyr Arg Leu Glu
Lys Pro Leu Asn Cys Asp Asp Glu Val Tyr 1055 1060 1065Asp Leu Met
Arg Gln Cys Trp Arg Glu Lys Pro Tyr Glu Arg Pro 1070 1075 1080Ser
Phe Ala Gln Ile Leu Val Ser Leu Asn Arg Met Leu Glu Glu 1085 1090
1095Arg Lys Thr Tyr Val Asn Thr Thr Leu Tyr Glu Lys Phe Thr Tyr
1100 1105 1110Ala Gly Ile Asp Cys Ser Ala Glu Glu Ala Ala 1115
112021453PRTArtificialHeavy chain 1 of <VEGF-ANG-2> CrossMAb
IgG1 with AAA mutations (VEGFang2-0012) 21Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr 20 25 30Gly Met Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Trp Ile
Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60Lys Arg
Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp
Val 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly 115 120 125Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly 130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val145 150 155 160Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200
205Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
210 215 220Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu225 230 235 240Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr 245 250 255Leu Met Ala Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val 260 265 270Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu Ala Gln Asp Trp Leu305 310 315
320Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
325 330 335Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro 340 345 350Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu
Thr Lys Asn Gln 355 360 365Val Ser Leu Trp Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala 370 375 380Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr385 390 395 400Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430Val
Met His Glu Ala Leu His Asn Ala Tyr Thr Gln Lys Ser Leu Ser 435 440
445Leu Ser Pro Gly Lys 45022463PRTArtificialHeavy chain 2 of
<VEGF-ANG-2> CrossMAb IgG1 with AAA mutations (VEGFang2-0012)
22Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly
Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr
Asp Ser Ser Gly Tyr Tyr Tyr 100 105 110Pro Gly Ala Phe Asp Ile Trp
Gly Gln Gly Thr Met Val Thr Val Ser 115 120 125Ser Ala Ser Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140Glu Gln Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn145 150 155
160Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
165 170 175Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp 180 185 190Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr 195 200 205Glu Lys His Lys Val Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser 210 215 220Ser Pro Val Thr Lys Ser Phe Asn
Arg Gly Glu Cys Asp Lys Thr His225 230 235 240Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 245 250 255Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ala Ser Arg Thr 260 265 270Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 275 280
285Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
290 295 300Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser305 310 315 320Val Leu Thr Val Leu Ala Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys 325 330 335Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile 340 345 350Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Cys Thr Leu Pro 355 360 365Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala 370 375 380Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn385 390 395
400Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
405 410 415Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys
Ser Arg 420 425 430Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu 435 440 445His Asn Ala Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 450 455 46023214PRTArtificialLight chain 1 of
<VEGF-ANG-2> CrossMAb IgG1 with AAA mutations (VEGFang2-0012)
23Asp 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 Gln Asp Ile Ser Asn
Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val
Leu Ile 35 40 45Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Tyr Ser Thr Val Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys Val 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
21024213PRTArtificialLight chain 2 of <VEGF-ANG-2> CrossMAb
IgG1 with AAA mutations (VEGF-Ang2-0012) 24Ser Tyr Val Leu Thr Gln
Pro Pro Ser Val Ser Val Ala Pro Gly Gln1 5 10 15Thr Ala Arg Ile Thr
Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val 20 25 30His Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45Asp Asp Ser
Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60Asn Ser
Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly65 70 75
80Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His
85 90 95Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala
Ser 100 105 110Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr 115 120 125Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro 130 135 140Glu Pro Val Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val145 150 155 160His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 165 170 175Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile 180 185 190Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val 195 200
205Glu Pro Lys Ser Cys 21025453PRTArtificialHeavy chain 1 of
<VEGF-ANG-2> CrossMAb IgG1 with AAA mutations and P329G LALA
mutations (VEGFang2-0016) 25Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Tyr Asp Phe Thr His Tyr 20 25 30Gly Met Asn Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Trp Ile Asn Thr Tyr Thr
Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60Lys Arg Arg Phe Thr Phe
Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Tyr
Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val 100 105 110Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120
125Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val145 150 155 160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe 165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220Ser Cys Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala225 230 235
240Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
245 250 255Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val 260 265 270Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val 275 280 285Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser 290 295 300Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu Ala Gln Asp Trp Leu305 310 315 320Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala 325 330 335Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350Gln
Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln 355 360
365Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
370 375 380Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr385 390 395 400Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu 405 410 415Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser 420 425 430Val Met His Glu Ala Leu His
Asn Ala Tyr Thr Gln Lys Ser Leu Ser 435 440 445Leu Ser Pro Gly Lys
45026463PRTArtificialHeavy
chain 2 of <VEGF-ANG-2> CrossMAb IgG1 with AAA mutations and
P329G LALA mutations (VEGFang2-0016) 26Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn
Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg
Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75 80Met
Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr Tyr
100 105 110Pro Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr
Val Ser 115 120 125Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp 130 135 140Glu Gln Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn145 150 155 160Phe Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175Gln Ser Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190Ser Thr Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205Glu
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215
220Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Asp Lys Thr
His225 230 235 240Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly
Gly Pro Ser Val 245 250 255Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ala Ser Arg Thr 260 265 270Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu 275 280 285Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys 290 295 300Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser305 310 315 320Val
Leu Thr Val Leu Ala Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 325 330
335Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile
340 345 350Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr
Leu Pro 355 360 365Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
Leu Ser Cys Ala 370 375 380Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn385 390 395 400Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser 405 410 415Asp Gly Ser Phe Phe
Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg 420 425 430Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 435 440 445His
Asn Ala Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455
46027214PRTArtificialLight chain 1 of <VEGF-ANG-2> CrossMAb
IgG1 with AAA mutations and P329G LALA mutations (VEGFang2-0016)
27Asp 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 Gln Asp Ile Ser Asn
Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val
Leu Ile 35 40 45Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Tyr Ser Thr Val Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys Val 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
21028213PRTArtificialLight chain 2 of <VEGF-ANG-2> CrossMAb
IgG1 with AAA mutations and P329G LALA mutations (VEGFang2-0016)
28Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln1
5 10 15Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser
Val 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val
Val Tyr 35 40 45Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe
Ser Gly Ser 50 55 60Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg
Val Glu Ala Gly65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp
Asp Ser Ser Ser Asp His 85 90 95Trp Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Ser Ser Ala Ser 100 105 110Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr 115 120 125Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 130 135 140Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val145 150 155
160His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
165 170 175Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
Tyr Ile 180 185 190Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val 195 200 205Glu Pro Lys Ser Cys
21029450PRTArtificialHeavy chain 1 of <VEGF-ANG-2> CrossMAb
IgG4 with AAA mutations and with SPLE mutations 29Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr 20 25 30Gly Met
Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly
Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55
60Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe
Asp Val 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly 115 120 125Pro Ser Val Phe Pro Leu Ala Pro Cys Ser
Arg Ser Thr Ser Glu Ser 130 135 140Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val145 150 155 160Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190Thr
Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val 195 200
205Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys
210 215 220Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu
Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ala 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser Gln Glu 260 265 270Asp Pro Glu Val Gln Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 290 295 300Val Val Ser
Val Leu Thr Val Leu Ala Gln Asp Trp Leu Asn Gly Lys305 310 315
320Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Cys 340 345 350Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
Gln Val Ser Leu 355 360 365Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly
Ser Phe Phe Leu Val Ser Arg Leu Thr Val Asp 405 410 415Lys Ser Arg
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu
Ala Leu His Asn Ala Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440
445Gly Lys 45030460PRTArtificialHeavy chain 2 of <VEGF-ANG-2>
CrossMAb IgG4 with AAA mutations and with SPLE mutations 30Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25
30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys
Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr
Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser
Ser Gly Tyr Tyr Tyr 100 105 110Pro Gly Ala Phe Asp Ile Trp Gly Gln
Gly Thr Met Val Thr Val Ser 115 120 125Ser Ala Ser Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140Glu Gln Leu Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn145 150 155 160Phe Tyr
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170
175Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
180 185 190Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr 195 200 205Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser 210 215 220Ser Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys Pro Pro Cys Pro225 230 235 240Pro Cys Pro Ala Pro Glu Phe
Glu Gly Gly Pro Ser Val Phe Leu Phe 245 250 255Pro Pro Lys Pro Lys
Asp Thr Leu Met Ala Ser Arg Thr Pro Glu Val 260 265 270Thr Cys Val
Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 275 280 285Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 290 295
300Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr305 310 315 320Val Leu Ala Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val 325 330 335Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
Lys Thr Ile Ser Lys Ala 340 345 350Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Cys Gln 355 360 365Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Trp Cys Leu Val Lys Gly 370 375 380Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro385 390 395 400Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 405 410
415Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
420 425 430Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn Ala 435 440 445Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
450 455 46031214PRTArtificialLight chain 1 of <VEGF-ANG-2>
CrossMAb IgG4 with AAA mutations and with SPLE mutations 31Asp 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 Gln Asp Ile Ser Asn Tyr 20 25
30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile
35 40 45Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser
Thr Val Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys Val 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
21032213PRTArtificialLight chain 2 of <VEGF-ANG-2> CrossMAb
IgG4 with AAA mutations and with SPLE mutations 32Ser Tyr Val Leu
Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln1 5 10 15Thr Ala Arg
Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val 20 25 30His Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45Asp
Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55
60Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly65
70 75 80Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp
His 85 90 95Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser
Ala Ser 100 105 110Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys
Ser Arg Ser Thr 115 120 125Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro 130 135 140Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val145 150 155 160His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 165 170 175Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr 180 185 190Cys
Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val 195 200
205Glu Ser Lys Tyr Gly 21033453PRTArtificialHeavy chain 1 of
<VEGF-ANG-2> OAscFab IgG1 with AAA mutations 33Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr 20 25 30Gly
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe
50 55 60Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp
Tyr Phe Asp Val 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly
115 120 125Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly 130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val145 150 155 160Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe 165 170 175Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu225 230
235 240Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr 245 250 255Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val 260 265 270Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val 275 280 285Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser 290 295 300Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu Ala Gln Asp Trp Leu305 310 315 320Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345
350Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
355 360 365Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala 370 375 380Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr385 390 395 400Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Val Ser Lys Leu 405 410 415Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430Val Met His Glu Ala
Leu His Asn Ala Tyr Thr Gln Lys Ser Leu Ser 435 440 445Leu Ser Pro
Gly Lys 45034705PRTArtificialHeavy chain 2 of <VEGF-ANG-2>
OAscFab IgG1 with AAA mutations 34Ser Tyr Val Leu Thr Gln Pro Pro
Ser Val Ser Val Ala Pro Gly Gln1 5 10 15Thr Ala Arg Ile Thr Cys Gly
Gly Asn Asn Ile Gly Ser Lys Ser Val 20 25 30His Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45Asp Asp Ser Asp Arg
Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60Asn Ser Gly Asn
Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly65 70 75 80Asp Glu
Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95Trp
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105
110Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln
115 120 125Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr
Pro Gly 130 135 140Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro
Val Lys Ala Gly145 150 155 160Val Glu Thr Thr Thr Pro Ser Lys Gln
Ser Asn Asn Lys Tyr Ala Ala 165 170 175Ser Ser Tyr Leu Ser Leu Thr
Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190Tyr Ser Cys Gln Val
Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205Ala Pro Thr
Glu Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 210 215 220Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly225 230
235 240Gly Gly Gly Ser Gly Gly Gln Val Gln Leu Val Glu Ser Gly Ala
Glu 245 250 255Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys
Ala Ser Gly 260 265 270Tyr Thr Phe Thr Gly Tyr Tyr Met His Trp Val
Arg Gln Ala Pro Gly 275 280 285Gln Gly Leu Glu Trp Met Gly Trp Ile
Asn Pro Asn Ser Gly Gly Thr 290 295 300Asn Tyr Ala Gln Lys Phe Gln
Gly Arg Val Thr Met Thr Arg Asp Thr305 310 315 320Ser Ile Ser Thr
Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp 325 330 335Thr Ala
Val Tyr Tyr Cys Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp 340 345
350Ser Ser Gly Tyr Tyr Tyr Pro Gly Ala Phe Asp Ile Trp Gly Gln Gly
355 360 365Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe 370 375 380Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu385 390 395 400Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp 405 410 415Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu 420 425 430Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 435 440 445Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 450 455 460Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys465 470
475 480Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro 485 490 495Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ala Ser 500 505 510Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp 515 520 525Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn 530 535 540Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val545 550 555 560Val Ser Val Leu
Thr Val Leu Ala Gln Asp Trp Leu Asn Gly Lys Glu 565 570 575Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 580 585
590Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
595 600 605Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
Leu Trp 610 615 620Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu625 630 635 640Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu 645 650 655Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys 660 665 670Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu 675 680 685Ala Leu His
Asn Ala Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 690 695
700Lys70535214PRTArtificialLight chain 1 of <VEGF-ANG-2>
OAscFab IgG1 with AAA mutations 35Asp Ile Gln Leu Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45Tyr Phe Thr Ser Ser
Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp 85 90 95Thr
Phe Gly Gln Gly Thr Lys Val 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 21036450PRTArtificialHeavy chain 1 of
<VEGF-ANG-2> OAscFab IgG4 with AAA mutations and with SPLE
mutations 36Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe
Thr His Tyr 20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr
Tyr Ala Ala Asp Phe 50 55 60Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr
Ser Lys Ser Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Tyr Pro Tyr Tyr Tyr
Gly Thr Ser His Trp Tyr Phe Asp Val 100 105 110Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125Pro Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 130 135 140Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val145 150
155 160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe 165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr
Tyr Thr Cys Asn Val 195 200 205Asp His Lys Pro Ser Asn Thr Lys Val
Asp Lys Arg Val Glu Ser Lys 210 215 220Tyr Gly Pro Pro Cys Pro Pro
Cys Pro Ala Pro Glu Phe Glu Gly Gly225 230 235 240Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ala 245 250 255Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 260 265
270Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
Tyr Arg 290 295 300Val Val Ser Val Leu Thr Val Leu Ala Gln Asp Trp
Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys
Gly Leu Pro Ser Ser Ile Glu 325 330 335Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Cys 340 345 350Thr Leu Pro Pro Ser
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365Ser Cys Ala
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val385 390
395 400Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Arg Leu Thr Val
Asp 405 410 415Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
Val Met His 420 425 430Glu Ala Leu His Asn Ala Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Leu 435 440 445Gly Lys 45037702PRTArtificialHeavy
chain 2 of <VEGF-ANG-2> OAscFab IgG4 with AAA mutations and
with SPLE mutations 37Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser
Val Ala Pro Gly Gln1 5 10 15Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn
Ile Gly Ser Lys Ser Val 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Val Leu Val Val Tyr 35 40 45Asp Asp Ser Asp Arg Pro Ser Gly
Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60Asn Ser Gly Asn Thr Ala Thr
Leu Thr Ile Ser Arg Val Glu Ala Gly65 70 75 80Asp Glu Ala Asp Tyr
Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95Trp Val Phe Gly
Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110Ala Ala
Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120
125Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly
130 135 140Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys
Ala Gly145 150 155 160Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn
Asn Lys Tyr Ala Ala 165 170 175Ser Ser Tyr Leu Ser Leu Thr Pro Glu
Gln Trp Lys Ser His Arg Ser 180 185 190Tyr Ser Cys Gln Val Thr His
Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205Ala Pro Thr Glu Cys
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 210 215 220Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly225 230 235
240Gly Gly Gly Ser Gly Gly Gln Val Gln Leu Val Glu Ser Gly Ala Glu
245 250 255Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala
Ser Gly 260 265 270Tyr Thr Phe Thr Gly Tyr Tyr Met His Trp Val Arg
Gln Ala Pro Gly 275 280 285Gln Gly Leu Glu Trp Met Gly Trp Ile Asn
Pro Asn Ser Gly Gly Thr 290 295 300Asn Tyr Ala Gln Lys Phe Gln Gly
Arg Val Thr Met Thr Arg Asp Thr305 310 315 320Ser Ile Ser Thr Ala
Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp 325 330 335Thr Ala Val
Tyr Tyr Cys Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp 340 345 350Ser
Ser Gly Tyr Tyr Tyr Pro Gly Ala Phe Asp Ile Trp Gly Gln Gly 355 360
365Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
370 375 380Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
Ala Leu385 390 395 400Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp 405 410 415Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu 420 425 430Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser 435 440 445Ser Ser Leu Gly Thr
Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 450 455 460Ser Asn Thr
Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro465 470 475
480Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe
485 490 495Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ala Ser Arg
Thr Pro 500 505 510Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
Asp Pro Glu Val 515 520 525Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr 530 535 540Lys Pro Arg Glu Glu Gln Phe Asn
Ser Thr Tyr Arg Val Val Ser Val545 550 555 560Leu Thr Val Leu Ala
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 565 570 575Lys Val Ser
Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 580 585 590Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 595 600
605Cys Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val
610 615 620Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly625 630 635 640Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp 645 650 655Gly Ser Phe Phe Leu Tyr Ser Arg Leu
Thr Val Asp Lys Ser Arg Trp 660 665 670Gln Glu Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His 675 680 685Asn Ala Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Leu Gly Lys 690 695
70038214PRTArtificialLight chain 1 of <VEGF-ANG-2> OAscFab
IgG4 with AAA mutations and with SPLE mutations 38Asp 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 Gln Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45Tyr Phe Thr
Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp
85 90 95Thr Phe Gly Gln Gly Thr Lys Val 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 21039453PRTArtificialHeavy chain 1 of
<VEGF-ANG-2> CrossMAb IgG1 wild type (without AAA mutations)
(VEGFang2-0201) 39Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr
Asp Phe Thr His Tyr 20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Gly Trp Ile Asn Thr Tyr Thr Gly Glu
Pro Thr Tyr Ala Ala Asp Phe 50 55 60Lys Arg Arg Phe Thr Phe Ser Leu
Asp Thr Ser Lys Ser Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Tyr Pro Tyr
Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val 100 105 110Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135
140Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val145 150 155 160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe 165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu225 230 235 240Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250
255Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
260 265 270Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val 275 280 285Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser 290 295 300Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu305 310 315 320Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350Gln Val Tyr
Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365Val
Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375
380Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr385 390 395 400Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu 405 410 415Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser 420 425 430Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser 435 440 445Leu Ser Pro Gly Lys
45040463PRTArtificialHeavy chain 2 of <VEGF-ANG-2> CrossMAb
IgG1 wild type (without AAA mutations) (VEGFang2-0201) 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 Tyr Thr Phe Thr Gly Tyr 20 25
30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys
Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr
Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser
Ser Gly Tyr Tyr Tyr 100 105 110Pro Gly Ala Phe Asp Ile Trp Gly Gln
Gly Thr Met Val Thr Val Ser 115 120 125Ser Ala Ser Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140Glu Gln Leu Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn145 150 155 160Phe Tyr
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170
175Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
180 185 190Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr 195 200 205Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser 210 215 220Ser Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys Asp Lys Thr His225 230 235 240Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val 245 250 255Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 260 265 270Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 275 280 285Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 290 295
300Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser305 310 315 320Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys 325 330 335Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile 340 345 350Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Cys Thr Leu Pro 355 360 365Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Ser Cys Ala 370 375 380Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn385 390 395 400Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 405 410
415Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg
420 425 430Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu 435 440 445His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 450 455 46041214PRTArtificialLight chain 1 of
<VEGF-ANG-2> CrossMAb IgG1 wild type ( without AAA mutations)
(VEGFang2-0201) 41Asp 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 Gln
Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Val Leu Ile 35 40 45Tyr Phe Thr Ser Ser Leu His Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp 85 90 95Thr Phe Gly Gln Gly
Thr Lys Val 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
21042213PRTArtificialLight chain 2 of <VEGF-ANG-2> CrossMAb
IgG1 wild type (without AAA mutations) (VEGFang2-0201) 42Ser Tyr
Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln1 5 10 15Thr
Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val 20 25
30His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr
35 40 45Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly
Ser 50 55 60Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu
Ala Gly65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser
Ser Ser Asp His 85 90 95Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu Ser Ser Ala Ser 100 105 110Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys Ser Thr 115 120 125Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro 130 135 140Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val145 150 155 160His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 165 170
175Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
180 185 190Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val 195 200 205Glu Pro Lys Ser Cys 21043453PRTArtificialHeavy
chain 1 of <VEGF-ANG-2> CrossMAb IgG1 with P329G LALA
mutations only (without AAA mutations) (VEGFang2-0015) 43Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr 20 25
30Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp
Phe 50 55 60Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr
Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His
Trp Tyr Phe Asp Val 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly 115 120 125Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val145 150 155 160Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170
175Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
180 185 190Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val 195 200 205Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys 210 215 220Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Ala225 230 235 240Ala Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295
300Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu305 310 315 320Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Gly Ala 325 330 335Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro 340 345 350Gln Val Tyr Thr Leu Pro Pro Cys
Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365Val Ser Leu Trp Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr385 390 395 400Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410
415Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
420 425 430Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser 435 440 445Leu Ser Pro Gly Lys 45044463PRTArtificialHeavy
chain 2 of <VEGF-ANG-2> CrossMAb IgG1 with P329G LALA
mutations only (without AAA mutations) (VEGFang2-0015) 44Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25
30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys
Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr
Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser
Ser Gly Tyr Tyr Tyr 100 105 110Pro Gly Ala Phe Asp Ile Trp Gly Gln
Gly Thr Met Val Thr Val Ser 115 120 125Ser Ala Ser Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140Glu Gln Leu Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn145 150 155 160Phe Tyr
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170
175Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
180 185 190Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr 195 200 205Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser 210 215 220Ser Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys Asp Lys Thr His225 230 235 240Thr Cys Pro Pro Cys Pro Ala
Pro Glu Ala Ala Gly Gly Pro Ser Val 245 250 255Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 260 265 270Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 275 280 285Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 290 295
300Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser305 310 315 320Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys 325 330 335Cys Lys Val Ser Asn Lys Ala Leu Gly Ala
Pro Ile Glu Lys Thr Ile 340 345 350Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Cys Thr Leu Pro 355 360 365Pro Ser Arg Asp Glu Leu
Thr Lys Asn
Gln Val Ser Leu Ser Cys Ala 370 375 380Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn385 390 395 400Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 405 410 415Asp Gly
Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg 420 425
430Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
435 440 445His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 450 455 46045214PRTArtificialLight chain 1 of
<VEGF-ANG-2> CrossMAb IgG1 with P329G LALA mutations only
(without AAA mutations) (VEGFang2-0015) 45Asp 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 Gln Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45Tyr Phe Thr
Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp
85 90 95Thr Phe Gly Gln Gly Thr Lys Val 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 21046213PRTArtificialLight chain 2 of
<VEGF-ANG-2> CrossMAb IgG1 with P329G LALA mutations only
(without AAA mutations) (VEGFang2-0015) 46Ser Tyr Val Leu Thr Gln
Pro Pro Ser Val Ser Val Ala Pro Gly Gln1 5 10 15Thr Ala Arg Ile Thr
Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val 20 25 30His Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45Asp Asp Ser
Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60Asn Ser
Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly65 70 75
80Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His
85 90 95Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala
Ser 100 105 110Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr 115 120 125Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro 130 135 140Glu Pro Val Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val145 150 155 160His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 165 170 175Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile 180 185 190Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val 195 200
205Glu Pro Lys Ser Cys 21047107PRTHomo sapiens 47Arg Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55
60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65
70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100
10548105PRThomo sapiens 48Gln Pro Lys Ala Ala Pro Ser Val Thr Leu
Phe Pro Pro Ser Ser Glu1 5 10 15Glu Leu Gln Ala Asn Lys Ala Thr Leu
Val Cys Leu Ile Ser Asp Phe 20 25 30Tyr Pro Gly Ala Val Thr Val Ala
Trp Lys Ala Asp Ser Ser Pro Val 35 40 45Lys Ala Gly Val Glu Thr Thr
Thr Pro Ser Lys Gln Ser Asn Asn Lys 50 55 60Tyr Ala Ala Ser Ser Tyr
Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser65 70 75 80His Arg Ser Tyr
Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 85 90 95Lys Thr Val
Ala Pro Thr Glu Cys Ser 100 10549330PRTHomo sapiens 49Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40
45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310
315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 33050327PRTHomo
sapiens 50Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys
Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Lys Thr65 70 75 80Tyr Thr Cys Asn Val Asp His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Ser Lys Tyr Gly
Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110Glu Phe Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140Asp
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp145 150
155 160Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe 165 170 175Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp 180 185 190Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Gly Leu 195 200 205Pro Ser Ser Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg 210 215 220Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Gln Glu Glu Met Thr Lys225 230 235 240Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265
270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
Phe Ser 290 295 300Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser305 310 315 320Leu Ser Leu Ser Leu Gly Lys 325
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