U.S. patent application number 14/571091 was filed with the patent office on 2015-07-09 for bispecific, bivalent anti-vegf/anti-ang2 antibodies.
This patent application is currently assigned to HOFFMANN-LA ROCHE INC.. The applicant listed for this patent is HOFFMANN-LA ROCHE INC.. Invention is credited to Monika Baehner, Sabine Imhof-Jung, Anita Kavlie, Hubert Kettenberger, Christian Klein, Joerg Thomas Regula, Wolfgang Schaefer, Juergen Michael Schanzer, Werner Scheuer, Kay-Gunnar Stubenrauch, Markus Thomas.
Application Number | 20150191535 14/571091 |
Document ID | / |
Family ID | 42574700 |
Filed Date | 2015-07-09 |
United States Patent
Application |
20150191535 |
Kind Code |
A1 |
Baehner; Monika ; et
al. |
July 9, 2015 |
BISPECIFIC, BIVALENT ANTI-VEGF/ANTI-ANG2 ANTIBODIES
Abstract
The present invention relates to bispecific, bivalent 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.
Inventors: |
Baehner; Monika; (Muenchen,
DE) ; Imhof-Jung; Sabine; (Planegg, DE) ;
Kavlie; Anita; (Oslo, NO) ; Kettenberger; Hubert;
(Muenchen, DE) ; Klein; Christian; (Bonstetten,
CH) ; Regula; Joerg Thomas; (Muenchen, DE) ;
Schaefer; Wolfgang; (Mannhein, DE) ; Schanzer;
Juergen Michael; (Muenchen, DE) ; Scheuer;
Werner; (Penzberg, DE) ; Stubenrauch; Kay-Gunnar;
(Penzberg, DE) ; Thomas; Markus; (Penzberg,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
HOFFMANN-LA ROCHE INC. |
Little Falls |
NJ |
US |
|
|
Assignee: |
HOFFMANN-LA ROCHE INC.
Little Falls
NJ
|
Family ID: |
42574700 |
Appl. No.: |
14/571091 |
Filed: |
December 15, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13071127 |
Mar 24, 2011 |
8945552 |
|
|
14571091 |
|
|
|
|
Current U.S.
Class: |
424/136.1 ;
435/320.1; 435/328; 435/69.6; 530/387.3; 536/23.53 |
Current CPC
Class: |
C07K 16/22 20130101;
C07K 2317/52 20130101; C07K 2317/94 20130101; C07K 2317/522
20130101; C07K 2317/92 20130101; A61K 2039/505 20130101; C07K
16/468 20130101; C07K 16/3046 20130101; A61P 35/00 20180101; A61P
9/00 20180101; C07K 2317/76 20130101; C07K 16/3015 20130101; C07K
2317/31 20130101; C07K 2317/66 20130101; C07K 2317/73 20130101;
C07K 2317/56 20130101; C07K 2317/35 20130101 |
International
Class: |
C07K 16/22 20060101
C07K016/22 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 26, 2010 |
EP |
10003269.7 |
Claims
1. 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 i) said first antigen-binding site comprises as heavy chain
variable domain (VH) the amino acid sequence of SEQ ID NO: 1, and
as light chain variable domain (VL) the amino acid sequence of SEQ
ID NO: 2; and wherein ii) said second antigen-binding site
comprises as heavy chain variable domain (VH) the amino acid
sequence of SEQ ID NO: 3, and as light chain variable domain (VL)
the amino acid sequence of SEQ ID NO: 4.
2. The bispecific antibody according to claim 1 comprising a) a
heavy chain and a light chain of a first full length antibody that
specifically binds to VEGF; and b) a modified heavy chain and a
modified light chain of a full length antibody that specifically
binds to ANG-2, wherein the constant domains CL and CH1 are
replaced by each other.
3. The bispecific antibody according to claim 2 wherein a) the
heavy chain of the first full length antibody comprises the amino
acid sequence of SEQ ID NO: 7, and the light chain of the first
full length antibody comprises the amino acid sequence of SEQ ID
NO: 5, and wherein b) the modified heavy chain of the second full
length antibody comprises the amino acid sequence of SEQ ID NO: 8,
and the modified light chain of the second full length antibody
comprises the amino acid sequence of SEQ ID NO: 6.
4. The bispecific antibody according to claim 2 wherein a) the
heavy chain of the first full length antibody comprises the amino
acid sequence of SEQ ID NO: 11, and the light chain of the first
full length antibody comprises the amino acid sequence of SEQ ID
NO: 9, and wherein b) the modified heavy chain of the second full
length antibody comprises the amino acid sequence of SEQ ID NO: 12,
and the modified light chain of the second full length antibody
comprises the amino acid sequence of SEQ ID NO: 10.
5. The bispecific antibody according to claim 2, wherein a) the
heavy chain of the first full length antibody comprises the amino
acid sequence of SEQ ID NO: 15, and the light chain of the first
full length antibody comprises the amino acid sequence of SEQ ID
NO: 13, and wherein b) the modified heavy chain of the second full
length antibody comprises the amino acid sequence of SEQ ID NO: 16,
and the modified light chain of the second full length antibody
comprises the amino acid sequence of SEQ ID NO: 14.
6. A pharmaceutical composition comprising the antibody according
to any one of claims 1 to 5.
7. A method of treating a patient suffering from cancer, comprising
administering to the patient an effective amount of the antibody
according to any one of claims 1 to 5.
8. The method of claim 7, wherein the cancer is colorectal cancer,
lung cancer, breast cancer, renal cancer, ovarian cancer or
glioblastoma.
9. A method of treating a patient suffering from vascular diseases,
comprising administering to the patient an effective amount of the
antibody according to any one of claims 1 to 5.
10. A method of inhibiting angiogenesis in a patient having a
pathological condition associated with angiogenesis, comprising
administering to the patient an effective amount of the antibody
according to any one of claims 1 to 5.
11. An isolated nucleic acid encoding the bispecific antibody
according to any one of claims 1 to 5.
12. An expression vector comprising the nucleic acid according
claim 11.
13. A prokaryotic or eukaryotic host cell comprising the nucleic
acid according to claim 11.
14. A method for producing the bispecific antibody according to any
one of claims 1 to 5, comprising culturing the host cell of claim
13 so that the antibody is produced.
15. The method of claim 14, further comprising recovering said
antibody from the host cell.
16. A bispecific antibody obtained by the method of claim 14.
17. 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 the antibody comprises the amino acid sequences of SEQ ID
NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.
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,
wherein the antibody comprises the amino acid sequences of SEQ ID
NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.
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,
wherein the antibody comprises the amino acid sequences of SEQ ID
NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.
Description
RELATED APPLICATION
[0001] This application claims priority to and the benefit of
European Application No. 10003269.7 filed Mar. 26, 2010, the
contents of which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to bispecific, bivalent
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
[0003] 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). In the
case of solid tumors, the neovascularization allows the tumor cells
to acquire a growth advantage and proliferative autonomy compared
to the normal cells. Accordingly, a correlation has been observed
between density of microvessels in tumor sections and patient
survival in breast cancer as well as in several other tumors
(Weidner, N., et al., N Engl J Med. 324 (1991) 1-8; Horak, E. R.,
et al., Lancet 340 (1992) 1120-1124; and Macchiarini, P., et al.,
Lancet 340 (1992) 145-146).
VEGF and anti-VEGF Antibodies
[0004] Human vascular endothelial growth factor (VEGF/VEGF-A) (SEQ
ID No: 105) is described in e.g. Leung, D. W., et al., Science 246
(1989) 1306-9; Keck, P. J., et al., Science 246 (1989) 1309-12 and
Connolly, D. T., et al., J. Biol. Chem. 264 (1989) 20017-24. 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. VEGF shows highly
specific mitogenic activity for endothelial cells. VEGF has
important regulatory functions in the formation of new blood
vessels during embryonic vasculogenesis and in angiogenesis during
adult life (Carmeliet, P., et al., Nature, 380 (1996) 435-439;
Ferrara, N., et al., Nature, 380 (1996) 439-442; reviewed in
Ferrara, N., et al., Endocr. Rev. 18 (1997) 4-25. The significance
of the role played by VEGF has been demonstrated in studies showing
that inactivation of a single VEGF allele results in embryonic
lethality due to failed development of the vasculature (Carmeliet,
P., et al., Nature, 380 (1996) 435-439; Ferrara, N., et al.,
Nature, 380 (1996) 439-442. In addition VEGF has strong
chemoattractant activity towards monocytes, can induce the
plasminogen activator and the plasminogen activator inhibitor in
endothelial cells, and can also induce microvascular permeability.
Because of the latter activity, it is sometimes referred to as
vascular permeability factor (VPF). The isolation and properties of
VEGF have been reviewed; see Ferrara, N., et al., J. Cellular
Biochem., 47 (1991) 211-218 and Connolly, D. T., J. Cellular
Biochem., 47 (1991) 219-223. Alternative mRNA splicing of a single
VEGF gene gives rise to five isoforms of VEGF.
[0005] Anti-VEGF neutralizing antibodies suppress the growth of a
variety of human tumor cell lines in mice (Kim, K. J., et al.,
Nature 362 (1993) 841-844; Warren, S. R., et al., J. Clin. Invest.
95 (1995) 1789-1797; Borgstrom, P., et al., Cancer Res. 56 (1996)
4032-4039; and Melnyk, O., et al., Cancer Res. 56 (1996) 921-924).
WO 94/10202, WO 98/45332, WO 2005/00900 and WO 00/35956 refer to
antibodies against VEGF. Humanized monoclonal antibody bevacizumab
(sold under the trade name Avastin.RTM.) is an anti-VEGF antibody
used in tumor therapy WO 98/45331).
[0006] Ranibizumab (trade name Lucentis.RTM.) is a monoclonal
antibody fragment derived from the same parent murine antibody as
bevacizumab (Avastin). It is much smaller than the parent molecule
and has been affinity matured to provide stronger binding to VEGF-A
(WO 98/45331). It is an anti-angiogenic that has been approved to
treat the "wet" type of age-related macular degeneration (ARMD), a
common form of age-related vision loss. Another anti-VEGF antibody
is e.g. HuMab G6-31 described e.g. in US 2007/0141065.
ANG-2 and Anti-ANG-2 Antibodies
[0007] Human angiopoietin-2 (ANG-2) (alternatively abbreviated with
ANGPT2 or ANG2) (SEQ ID No: 106) is described 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 and -2 (ANG-1 (SEQ
ID No: 107) and ANG-2 (SEQ ID No: 106)) 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, 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. Ang-1 was shown to support EC survival and to promote
endothelium integrity, Davis, S., et al., Cell 87 (1996) 1161-69;
Kwak, H. J., et al., FEBS Lett 448 (1999) 249-53; Suri, C., et al.,
Science 282 (1998) 468-71; Thurston, G., et al., Science 286 (1999)
2511-2514; Thurston, G., et al., Nat. Med. 6 (2000) 460-63, whereas
ANG-2 had the opposite effect and promoted blood vessel
destabilization and regression in the absence of the survival
factors VEGF or basic fibroblast growth factor. Maisonpierre, P.
C., et al., Science 277 (1997) 55-60. However, many studies of
ANG-2 function have suggested a more complex situation. ANG-2 might
be a complex regulator of vascular remodeling that plays a role in
both vessel sprouting and vessel regression. Supporting such roles
for ANG-2, expression analyses reveal that ANG-2 is rapidly
induced, together with VEGF, in adult settings of angiogenic
sprouting, whereas ANG-2 is induced in the absence of VEGF in
settings of vascular regression. Holash, J., et al., Science 284
(1999) 1994-98; Holash, J., et al., Oncogene 18 (1999) 5356-62.
Consistent with a context-dependent role, ANG-2 specifically binds
to the same endothelial-specific receptor, Tie-2, which is
activated by Ang-1, but has context-dependent effects on its
activation. Maisonpierre, P. C., et al., Science 277 (1997)
55-60.
[0008] 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, 1., 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.
[0009] Other in vitro experiments suggested that during sustained
exposure, the effects of ANG-2 may progressively shift from that of
an antagonist to an agonist of Tie2, and at later time points, it
may contribute directly to vascular tube formation and neovessel
stabilization. Teichert-Kuliszewska, K., et al., Cardiovasc. Res.
49 (2001) 659-70. Furthermore, if ECs were cultivated on fibrin
gel, activation of Tie2 with ANG-2 was also observed, perhaps
suggesting that the action of ANG-2 could depend on EC
differentiation state. Teichert-Kuliszewska, K., et al.,
Cardiovasc. Res. 49 (2001) 659-70. In microvascular EC cultured in
a three-dimensional collagen gel, ANG-2 can also induce Tie2
activation and promote formation of capillary-like structures.
Mochizuki, Y., et al., J. Cell. Sci. 115 (2002) 175-83. Use of a
3-D spheroidal coculture as an in-vitro model of vessel maturation
demonstrated that direct contact between ECs and mesenchymal cells
abrogates responsiveness to VEGF, whereas the presence of VEGF and
ANG-2 induced sprouting. Korff, T., et al., Faseb J. 15 (2001)
447-57. Etoh, T. H. et al. demonstrated that ECs that
constitutively express Tie2, the expression of MMP-1, -9 and u-PA
were strongly upregulated by ANG-2 in the presence of VEGF. Etoh,
T., et al., Cancer Res. 61 (2001) 2145-53. With an in vivo
pupillary membrane model, Lobov, I. B. et al. showed that ANG-2 in
the presence of endogenous VEGF promotes a rapid increase in
capillary diameter, remodeling of the basal lamina, proliferation
and migration of endothelial cells, and stimulates sprouting of new
blood vessels. Lobov, I. B., et al., Proc. Natl. Acad. Sci. USA 99
(2002) 11205-10. By contrast, ANG-2 promotes endothelial cell death
and vessel regression without endogenous VEGF. Lobov, I. B., et
al., Proc. Natl. Acad. Sci. USA 99 (2002) 11205-10. Similarly, with
an in vivo tumor model, Vajkoczy, P., et al. demonstrated that
multicellular aggregates initiate vascular growth by angiogenic
sprouting via the simultaneous expression of VEGFR-2 and ANG-2 by
host and tumor endothelium. Vajkoczy, P., et al., J. Clin. Invest.
109 (2002) 777-85. This model illustrated that the established
microvasculature of growing tumors is characterized by a continuous
remodeling, putatively mediated by the expression of VEGF and ANG-2
(Vajkoczy, P., et al., J. Clin. Invest. 109 (2002) 777-85).
[0010] Knock-out mouse studies of Tie-2 and Angiopoietin-1 show
similar phenotypes and suggest that Angiopoietin-1 stimulated Tie-2
phosphorylation mediates remodeling and stabilization of developing
vessel, promoting blood vessel maturation during angiogenesis and
maintenance of endothelial cell-support cell adhesion (Dumont, D.
J., et al., Genes & Development, 8 (1994) 1897-1909; Sato, T.
N., Nature, 376 (1995) 70-74; (Thurston, G., et al., Nature
Medicine 6 (2000) 460-463). The role of Angiopoietin-1 is thought
to be conserved in the adult, where it is expressed widely and
constitutively (Hanahan, D., Science, 277 (1997) 48-50; Zagzag, D.,
et al., Exp Neurology 159 (1999) 391-400). In contrast,
Angiopoietin-2 expression is primarily limited to sites of vascular
remodeling where it is thought to block the constitutive
stabilizing or maturing function of Angiopoietin-1, allowing
vessels to revert to, and remain in, a plastic state which may be
more responsive to sprouting signals (Hanahan, D., 1997; Holash,
J., et al., Oncogene 18 (199) 5356-62; Maisonpierre, P. C., 1997).
Studies of Angiopoietin-2 expression in pathological angiogenesis
have found many tumor types to show vascular Angiopoietin-2
expression (Maisonpierre, P. C., et al., Science 277 (1997) 55-60).
Functional studies suggest Angiopoietin-2 is involved in tumor
angiogenesis and associate Angiopoietin-2 overexpression with
increased tumor growth in a mouse xenograft model (Ahmad, S. A., et
al., Cancer Res., 61 (2001) 1255-1259). Other studies have
associated Angiopoictin-2 overexpression with tumor
hypervascularity (Etoh, T., et al., Cancer Res. 61 (2001) 2145-53;
Tanaka, F., et al., Cancer Res. 62 (2002) 7124-7129).
[0011] In recent years Angiopoietin-1, Angiopoietin-2 and/or Tie-2
have been proposed as possible anti-cancer therapeutic targets. For
example U.S. Pat. No. 6,166,185, U.S. Pat. No. 5,650,490 and U.S.
Pat. No. 5,814,464 each disclose anti-Tie-2 ligand and receptor
antibodies. Studies using soluble Tie-2 were reported to decrease
the number and size of tumors in rodents (Lin, 1997; Lin 1998).
Siemeister, G., et al., Cancer Res. 59:3 (1999) 3185-91 generated
human melanoma cell lines expressing the extracellular domain of
Tie-2, injected these into nude mice and reported soluble Tie-2 to
result in significant inhibition of tumor growth and tumor
angiogenesis. Given both Angiopoietin-1 and Angiopoietin-2 bind to
Tie-2, it is unclear from these studies whether Angiopoietin-1,
Angiopoietin-2 or Tie-2 would be an attractive target for
anti-cancer therapy. However, effective anti-Angiopoietin-2 therapy
is thought to be of benefit in treating diseases such as cancer, in
which progression is dependant on aberrant angiogenesis where
blocking the process can lead to prevention of disease advancement
(Folkman, J., Nature Medicine. 1 (1995) 27-31).
[0012] In addition some groups have reported the use of antibodies
and peptides that bind to Angiopoietin-2. See, for example, U.S.
Pat. No. 6,166,185 and US 2003/10124129. WO 03/030833, WO
2006/068953, WO 03/057134 or US 2006/0122370.
[0013] Study of the effect of focal expression of Angiopoietin-2
has shown that antagonizing the Angiopoietin-1/Tie-2 signal loosens
the tight vascular structure thereby exposing ECs to activating
signals from angiogenesis inducers, e.g. VEGF (Hanahan, D.,
Science, 277 (1997) 48-50). This pro-angiogenic effect resulting
from inhibition of Angiopoietin-1 indicates that
anti-Angiopoietin-1 therapy would not be an effective anti-cancer
treatment.
[0014] ANG-2 is expressed during development at sites where blood
vessel remodeling is occurring. Maisonpierre, P. C., et al.,
Science 277 (1997) 55-60. In adult individuals, ANG-2 expression is
restricted to sites of vascular remodeling as well as in highly
vascularized tumors, including glioma, Osada, H., et al., Int. J.
Oncol. 18 (2001) 305-09); Koga, K., et al., Cancer Res. 61 (2001)
6248-54, hepatocellular carcinoma, Tanaka, S., et al., J. Clin.
Invest. 103 (1999) 341-45, gastric carcinoma, Etoh, T., et al.,
Cancer Res. 61 (2001) 2145-53; Lee, J. H., et al., Int. J. Oncol.
18 (2001) 355-61, thyroid tumor, Bunone, G., et al., Am J Pathol
155 (1999) 1967-76 non-small cell lung cancer, Wong, M. P., et al.,
Lung Cancer 29 (2000) 11-22, and cancer of colon, Ahmad, S. A., et
al., Cancer 92 (2001) 1138-43, and prostate Wurmbach, J. H., et
al., Anticancer Res. 20 (2000) 5217-20. Some tumor cells are found
to express ANG-2. For example, Tanaka, S., et al., J. Clin. Invest.
103 (1999) 341-45 detected ANG-2 mRNA in 10 out of 12 specimens of
human hepatocellular carcinoma (HCC). Ellis' group reported that
ANG-2 is expressed ubiquitously in tumor epithelium. Ahmad, S. A.,
et al., Cancer 92 (2001) 1138-43. Other investigators reported
similar findings. Chen, L., et al., J. Tongji Med. Univ. 21 (2001)
228-35. By detecting ANG-2 mRNA levels in archived human breast
cancer specimens, Sfiligoi, C., et al., Int. J. Cancer 103 (2003)
466-74 reported that ANG-2 mRNA is significantly associated with
auxiliary lymph node invasion, short disease-free time and poor
overall survival. Tanaka, F., et al., Cancer Res. 62 (2002) 7124-29
reviewed a total of 236 patients of non-small cell lung cancer
(NSCLC) with pathological stage-I to -IIIA, respectively. Using
immunohistochemistry, they found that 16.9% of the NSCLC patients
were ANG-2 positive. The microvessel density for ANG-2 positive
tumor is significantly higher than that of ANG-2 negative. Such an
angiogenic effect of ANG-2 was seen only when VEGF expression was
high. Moreover, positive expression of ANG-2 was a significant
factor to predict a poor postoperative survival. Tanaka, F., et
al., Cancer Res. 62 (2002) 7124-7129. However, they found no
significant correlation between Ang-1 expression and the
microvessel density. Tanaka, F., et al., Cancer Res. 62 (2002)
7124-7129. These results suggest that ANG-2 is an indicator of poor
prognosis patients with several types of cancer.
[0015] Recently, using an ANG-2 knockout mouse model, Yancopoulos'
group reported that ANG-2 is required for postnatal angiogenesis.
Gale, N. W., et al., Dev. Cell 3 (2002) 411-23. They showed that
the developmentally programmed regression of the hyaloid
vasculature in the eye does not occur in the ANG-2 knockout mice
and their retinal blood vessels fail to sprout out from the central
retinal artery. Gale, N. W., et al., Dev. Cell 3 (2002) 411-23.
They also found that deletion of ANG-2 results in profound defects
in the patterning and function of the lymphatic vasculature. Gale,
N. W., et al., Dev. Cell 3 (2002) 411-23. Genetic rescue with Ang-1
corrects the lymphatic, but not the angiogenesis defects. Gale, N.
W., et al., Dev. Cell 3 (2002) 411-23.
[0016] Peters and his colleagues reported that soluble Tie2, when
delivered either as recombinant protein or in a viral expression
vector, inhibited in vivo growth of murine mammary carcinoma and
melanoma in mouse models. Lin, P., et al., Proc. Natl. Acad. Sci.
USA 95 (1998) 8829-34; Lin, P., et al., J. Clin. Invest. 100 (1997)
2072-78. Vascular densities in the tumor tissues so treated were
greatly reduced. In addition, soluble Tie2 blocked angiogenesis in
the rat corneal stimulated by tumor cell conditioned media. Lin,
P., et al., J. Clin. Invest. 100 (1997) 2072-78. Furthermore, Isner
and his team demonstrated that addition of ANG-2 to VEGF promoted
significantly longer and more circumferential neovascularity than
VEGF alone. Asahara, T., et al., Circ. Res. 83 (1998) 233-40.
Excess soluble Tie2 receptor precluded modulation of VEGF-induced
neovascularization by ANG-2. Asahara, T., et al., Circ. Res. 83
(1998) 233-40. Siemeister, G., et al., Cancer Res. 59:3 (1999)
3185-91 showed with nude mouse xenografts that overexpression of
the extracellular ligand-binding domains of either Flt-1 or Tie2 in
the xenografts results in significant inhibition of pathway could
not be compensated by the other one, suggesting that the VEGF
receptor pathway and the Tie2 pathway should be considered as two
independent mediators essential for the process of in vivo
angiogenesis. Siemeister, G., et al., Cancer Res. 59:3 (1999)
3185-91. This is proven by a more recent publication by White, R.,
R., et al., Proc. Natl. Acad. Sci. USA 100 (2003) 5028-33. In their
study, it was demonstrated that a nuclease-resistant RNA aptamer
that specifically binds and inhibits ANG-2 significantly inhibited
neovascularization induced by bFGF in the rat corneal micropocket
angiogenesis model.
Bispecific Antibodies
[0017] A wide variety of recombinant antibody formats have been
developed in the recent past, e.g. tetravalent bispecific
antibodies by fusion of, e.g., an IgG antibody format and single
chain domains (see e.g. Coloma, M. J., et al., Nature Biotech 15
(1997) 159-163; WO 2001/077342; and Morrison, S. L., Nature Biotech
25 (2007) 1233-1234).
[0018] Also several other new formats wherein the antibody core
structure (IgA, IgD, IgE, IgG or IgM) is no longer retained such as
dia-, tria- or tetrabodies, minibodies, several single chain
formats (scFv, Bis-scFv), which are capable of binding two or more
antigens, have been developed (Holliger, P., et al., Nature Biotech
23 (2005) 1126-1136; Fischer, N., Leger, O., Pathobiology 74 (2007)
3-14; Shen, J., et al., Journal of Immunological Methods 318 (2007)
65-74; Wu, C., et al., Nature Biotech. 25 (2007) 1290-1297).
[0019] All such formats use linkers either to fuse the antibody
core (IgA, IgD, IgE, IgG or IgM) to a further binding protein (e.g.
scFv) or to fuse e.g. two Fab fragments or scFvs (Fischer, N.,
Leger, O., Pathobiology 74 (2007) 3-14). It has to be kept in mind
that one may want to retain effector functions, such as e.g.
complement-dependent cytotoxicity (CDC) or antibody dependent
cellular cytotoxicity (ADCC), which are mediated through the Fc
receptor binding, by maintaining a high degree of similarity to
naturally occurring antibodies.
[0020] In WO 2007/024715 are reported dual variable domain
immunoglobulins as engineered multivalent and multispecific binding
proteins. A process for the preparation of biologically active
antibody dimers is reported in U.S. Pat. No. 6,897,044. Multivalent
F.sub.V antibody construct having at least four variable domains
which are linked with each over via peptide linkers are reported in
U.S. Pat. No. 7,129,330. Dimeric and multimeric antigen binding
structures are reported in US 2005/0079170. Tri- or tetra-valent
monospecific antigen-binding protein comprising three or four Fab
fragments bound to each other covalently by a connecting structure,
which protein is not a natural immunoglobulin are reported in U.S.
Pat. No. 6,511,663. In WO 2006/020258 tetravalent bispecific
antibodies are reported that can be efficiently expressed in
prokaryotic and eukaryotic cells, and are useful in therapeutic and
diagnostic methods. A method of separating or preferentially
synthesizing dimers which are linked via at least one interchain
disulfide linkage from dimers which are not linked via at least one
interchain disulfide linkage from a mixture comprising the two
types of polypeptide dimers is reported in US 2005/0163782.
Bispecific tetravalent receptors are reported in U.S. Pat. No.
5,959,083. Engineered antibodies with three or more functional
antigen binding sites are reported in WO 2001/077342.
[0021] Multispecific and multivalent antigen-binding polypeptides
are reported in WO 1997/001580. WO 1992/004053 reports
homoconjugates, typically prepared from monoclonal antibodies of
the IgG class which bind to the same antigenic determinant are
covalently linked by synthetic cross-linking. Oligomeric monoclonal
antibodies with high avidity for antigen are reported in WO
1991/06305 whereby the oligomers, typically of the IgG class, are
secreted having two or more immunoglobulin monomers associated
together to form tetravalent or hexavalent IgG molecules.
Sheep-derived antibodies and engineered antibody constructs are
reported in U.S. Pat. No. 6,350,860, which can be used to treat
diseases wherein interferon gamma activity is pathogenic. In US
2005/0100543 are reported targetable constructs that are
multivalent carriers of bi-specific antibodies, i.e., each molecule
of a targetable construct can serve as a carrier of two or more
bi-specific antibodies. Genetically engineered bispecific
tetravalent antibodies are reported in WO 1995/009917. In WO
2007/109254 stabilized binding molecules that consist of or
comprise a stabilized scFv are reported.
Combination of VEGF and ANG-2 Inhibitors
[0022] WO 2007/068895 refers to a combination of an ANG-2
antagonist and a VEGF, KDR and/or FLTL antagonists. WO 2007/089445
refers to ANG-2 and VEGF inhibitor combinations.
[0023] WO 2003/106501 refers to fusion proteins binding to
Angiopoietin and containing a multimerization domain. WO
2008/132568 relates to fusion proteins binding to Angiopoietin and
VEGF. WO 2003/020906 relates to multivalent protein conjugates with
multiple ligand-binding domains of receptors.
[0024] WO 2009/136352 relates to anti-angiogenic compounds.
SUMMARY OF THE INVENTION
[0025] The invention is 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, characterized in that [0026] i) said first
antigen-binding site comprises as heavy chain variable domain (VH)
the SEQ ID NO: 1, and as light chain variable domain (VL) the SEQ
ID NO: 2; and [0027] ii) said second antigen-binding site comprises
as heavy chain variable domain (VH) the SEQ ID NO: 3, and as light
chain variable domain (VL) the SEQ ID NO: 4.
[0028] In one aspect of the invention the bispecific antibody
according to the invention is characterized in comprising [0029] a)
the heavy chain and the light chain of a first full length antibody
that specifically binds to VEGF; and [0030] b) the modified heavy
chain and modified light chain of a full length antibody that
specifically binds to ANG-2, wherein the constant domains CL and
CH1 are replaced by each other.
[0031] In one embodiment such bispecific, bivalent antibody is
characterized in comprising [0032] a) as heavy chain of the first
full length antibody the amino acid sequence of SEQ ID NO: 7, and
as light chain of the first full length antibody the amino acid
sequence of SEQ ID NO: 5, and [0033] b) as modified heavy chain of
the second full length antibody the amino acid sequence of SEQ ID
NO: 8, and as modified light chain of the second full length
antibody the amino acid sequence of SEQ ID NO: 6.
[0034] In one embodiment such bispecific, bivalent antibody is
characterized in comprising [0035] a) as heavy chain of the first
full length antibody the amino acid sequence of SEQ ID NO: 11, and
as light chain of the first full length antibody the amino acid
sequence of SEQ ID NO: 9, and [0036] b) as modified heavy chain of
the second full length antibody the amino acid sequence of SEQ ID
NO: 12, and as modified light chain of the second full length
antibody the amino acid sequence of SEQ ID NO: 10.
[0037] In one embodiment such bispecific, bivalent antibody is
characterized in comprising [0038] a) as heavy chain of the first
full length antibody the amino acid sequence of SEQ ID NO: 15, and
as light chain of the first full length antibody the amino acid
sequence of SEQ ID NO: 13, and [0039] b) as modified heavy chain of
the second full length antibody the amino acid sequence of SEQ ID
NO: 16, and as modified light chain of the second full length
antibody the amino acid sequence of SEQ ID NO: 14.
[0040] Still further aspects of the invention are a pharmaceutical
composition comprising said bispecific antibody, said composition
for the treatment of cancer, the use of said bispecific antibody
for the manufacture of a medicament for the treatment of cancer, a
method of treatment of patient suffering from cancer by
administering said bispecific antibody. to a patient in the need of
such treatment.
[0041] Still further aspects of the invention are a pharmaceutical
composition comprising said bispecific antibody, said composition
for the treatment of vascular diseases, the use of said bispecific
antibody for the manufacture of a medicament for the treatment of
vascular diseases, a method of treatment of patient suffering from
vascular diseases by administering said bispecific antibody. to a
patient in the need of such treatment.
[0042] A further aspect of the invention is a nucleic acid molecule
encoding a chain of a bispecific antibody according to the
invention.
[0043] 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.
[0044] The invention further comprises a prokaryotic or eukaryotic
host cell comprising a vector according to the invention.
[0045] 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. 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: 5, of SEQ ID NO: 6, of SEQ
ID NO: 7, and of SEQ ID NO: 8.
[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: 9, of SEQ ID NO: 10, of SEQ
ID NO: 11, and of SEQ ID NO: 12.
[0048] 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: 13, of SEQ ID NO: 14, of SEQ
ID NO: 15, and of SEQ ID NO: 16.
[0049] The bispecific, bivalent antibodies according to the
invention show benefits for human patients in need of a VEGF and
ANG-2 targeting therapy. The antibodies according to the invention
have highly valuable properties causing a benefit for a patient
suffering from such a disease, especially suffering from cancer.
The bispecific antibodies according to the invention are highly
effective in tumor growth and/or inhibition of tumor angiogenesis
or vascular diseases. The bispecific, bivalent antibodies according
to the invention The bispecific, bivalent <VEGF-ANG-2>
antibodies according to the invention show valuable
pharmacokinetic/-dynamic properties like e.g. stability, good (i.e.
slow) clearance (e.g. at low doses).
[0050] The bispecific antibodies according to the invention are
highly effective in
a) tumor growth inhibition (e.g. with the bispecific antibodies
according to the invention tumor stasis could be achieved already
at lower concentrations compared to the combination of the two
monospecific antibodies (e.g. in the Colo205 and the KPL-4 tumor
models of Example 9 and 10, tumor stasis was already achieved with
10 mg/kg XMAb1 compared to the combination of 10 mg/kg of
ANG2i-LC06+10 mg/kg of Avastin), and/or b) inhibition of tumor
angiogenesis or vascular diseases (e.g. maximal antiangiogenic
effects with the bispecific antibodies according to the invention
could already be achieved at lower concentrations compared to the
combination of the two monospecific antibodies (e.g. in the mouse
corneal angiogenesis assay of Example 8, the maximal antiangiogenic
effect was already achieved with 10 mg/kg XMAb1 compared to the
combination of 10 mg/kg of ANG2i-LC06+10 mg/kg of Avastin).
DESCRIPTION OF THE FIGURES
[0051] FIG. 1 Exemplary bivalent bispecific antibody format for
XMab examples including Knobs-into-Holes modified CH3 domains
[0052] FIG. 2a Exemplary bivalent bispecific antibody format for
OAscFab examples including Knobs-into-Holes modified CH3
domains
[0053] FIG. 2b Exemplary bivalent bispecific antibody format for
example OAscXFab1 including Knobs-into-Holes modified CH3
domains
[0054] FIG. 2c Exemplary bivalent bispecific antibody format for
examples OAscXFab2 and OAscXFab3 including Knobs-into-Holes
modified CH3 domains
[0055] FIG. 3 Simultaneous binding of <VEGF-Ang-2> XMab1 to
VEGF (1. Step) followed by binding to hAng-2 (second step)
[0056] FIG. 4 ELISA principle for quantification of binding active
mAb<Ang2/VEGF> antibodies
[0057] FIG. 5 Calibration curve of ELISA for quantification of
binding active <Ang2/VEGF> XMab1 antibodies
[0058] FIG. 6 Mouse corneal angiogenesis assay--inhibition of
vessel outgrowth from the limbus towards the VEGF gradient by
administration of a bispecific antibody according to the
invention.
[0059] FIG. 7 Mouse corneal angiogenesis assay--inhibition of
angiogenesis/vessel outgrowth from the limbus towards the VEGF
gradient by administration of a bispecific antibody according to
the invention--Comparison of the bispecific <Ang2/VEGF>
antibody XMab1, the <Ang2> Mab ANG2i-LC06 (LC06), the
<VEGF> Mab bevacizumab (Avastin) and the combination
ANG2i-LC06 and <VEGF> Mab bevacizumab (Avastin).
[0060] FIG. 8 In vivo tumor growth inhibition in mouse xenograft of
human colorectal cancer Colo205 (small tumors) by a bispecific
antibody according to the invention--Comparison of the bispecific
<Ang2/VEGF> antibody XMab1, the <Ang2> Mab ANG2i-LCO6
(LC06), the <VEGF> Mab bevacizumab (Avastin) and the
combination ANG2i-LC06 and <VEGF> Mab bevacizumab
(Avastin).
[0061] FIG. 9 In vivo tumor growth inhibition in mouse xenograft of
human colorectal cancer Colo205 (large tumors) by a bispecific
antibody according to the invention--Comparison of the bispecific
<Ang2/VEGF> antibody XMab1, the <Ang2> Mab ANG2i-LC06
(LC06), the <VEGF> Mab bevacizumab (Avastin) and the
combination ANG2i-LC06 and <VEGF> Mab bevacizumab
(Avastin).
[0062] FIG. 10 In vivo tumor growth inhibition in mouse xenograft
of human breast cancer KPL-4 (small tumors) by a bispecific
antibody according to the invention--Comparison of the bispecific
<Ang2/VEGF> antibody XMab1, the <Ang2> Mab ANG2i-LCO6
(LC06), the <VEGF> Mab bevacizumab (Avastin) and the
combination ANG2i-LC06 and <VEGF> Mab bevacizumab
(Avastin).
[0063] FIG. 11 In vivo tumor growth inhibition in mouse xenograft
of human breast cancer KPL-4 (large tumors) by a bispecific
antibody according to the invention--Comparison of the bispecific
<Ang2/VEGF> antibody XMab1, the <Ang2> Mab ANG2i-LCO6
(LC06), the <VEGF> Mab bevacizumab (Avastin) and the
combination ANG2i-LC06 and <VEGF> Mab bevacizumab
(Avastin).
[0064] FIG. 12 In vivo tumor growth inhibition in mouse xenograft
of gastric cancer N87 by a bispecific antibody according to the
invention--Comparison of the bispecific <Ang2/VEGF> antibody
XMab1, the <Ang2> Mab ANG2i-LC06 (LC06), the <VEGF> Mab
bevacizumab (Avastin) and the combination ANG2i-LC06 and
<VEGF> Mab bevacizumab (Avastin).
DETAILED DESCRIPTION OF THE INVENTION
[0065] The invention is 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. characterized in that [0066] i) said first
antigen-binding site comprises as heavy chain variable domain (VH)
the SEQ ID NO: 1, and as light chain variable domain (VL) the SEQ
ID NO: 2; and [0067] ii) said second antigen-binding site comprises
as heavy chain variable domain (VH) the SEQ ID NO: 3, and as light
chain variable domain (VL) the SEQ ID NO: 4.
[0068] In one aspect of the invention the bispecific 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
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 (see exemplary scheme in including Knobs-into-Holes
modified CH3 domains in FIG. 1). The antibodies based on this
bispecific, bivalent antibody format are named XMab in the examples
of the current invention.
[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: 7, and
as light chain of the first full length antibody the amino acid
sequence of SEQ ID NO: 5, and [0074] b) as modified heavy chain of
the second full length antibody the amino acid sequence of SEQ ID
NO: 8, and as modified light chain of the second full length
antibody the amino acid sequence of SEQ ID NO: 6.
[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: 11, and
as light chain of the first full length antibody the amino acid
sequence of SEQ ID NO: 9, and [0077] b) as modified heavy chain of
the second full length antibody the amino acid sequence of SEQ ID
NO: 12, and as modified light chain of the second full length
antibody the amino acid sequence of SEQ ID NO: 10.
[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: 15, and
as light chain of the first full length antibody the amino acid
sequence of SEQ ID NO: 13, and [0080] b) as modified heavy chain of
the second full length antibody the amino acid sequence of SEQ ID
NO: 16, and as modified light chain of the second full length
antibody the amino acid sequence of SEQ ID NO: 14.
[0081] In one embodiment such bispecific, bivalent antibody is
characterized in comprising [0082] a) as heavy chain of the first
full length antibody the amino acid sequence of SEQ ID NO: 19, and
as light chain of the first full length antibody the amino acid
sequence of SEQ ID NO: 17, and [0083] b) as modified heavy chain of
the second full length antibody the amino acid sequence of SEQ ID
NO: 20, and as modified light chain of the second full length
antibody the amino acid sequence of SEQ ID NO: 18.
[0084] In one embodiment such bispecific, bivalent antibody is
characterized in comprising [0085] a) as heavy chain of the first
full length antibody the amino acid sequence of SEQ ID NO: 23, and
as light chain of the first full length antibody the amino acid
sequence of SEQ ID NO: 21, and [0086] b) as modified heavy chain of
the second full length antibody the amino acid sequence of SEQ ID
NO: 24, and as modified light chain of the second full length
antibody the amino acid sequence of SEQ ID NO: 22.
[0087] In one embodiment such bispecific, bivalent antibody is
characterized in comprising [0088] a) as heavy chain of the first
full length antibody the amino acid sequence of SEQ ID NO: 27, and
as light chain of the first full length antibody the amino acid
sequence of SEQ ID NO: 25, and [0089] b) as modified heavy chain of
the second full length antibody the amino acid sequence of SEQ ID
NO: 28, and as modified light chain of the second full length
antibody the amino acid sequence of SEQ ID NO: 26.
[0090] 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: 5, of SEQ ID NO: 6, of SEQ
ID NO: 7, and of SEQ ID NO: 8.
[0091] 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: 9, of SEQ ID NO: 10, of SEQ
ID NO: 11, and of SEQ ID NO: 12.
[0092] 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: 13, of SEQ ID NO: 14, of SEQ
ID NO: 15, and of SEQ ID NO: 16.
[0093] 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: 17, of SEQ ID NO: 18, of SEQ
ID NO: 19, and of SEQ ID NO: 20.
[0094] 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.
[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: 25, of SEQ ID NO: 26, of SEQ
ID NO: 27, and of SEQ ID NO: 28.
[0096] In another aspect of the invention the bispecific antibody
according to the invention is characterized in comprising [0097] a)
the heavy chain and the light chain of a first full length antibody
that specifically binds to VEGF; [0098] 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.
[0099] An exemplary scheme of 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 shown in FIG. 2a including Knobs-into-Holes modified CH3
domains. The antibodies based on this bispecific, bivalent antibody
format are named OAscFab in the examples of the current
invention.
[0100] In one embodiment such bispecific, bivalent antibody is
characterized in comprising [0101] a) as heavy chain of the first
full length antibody the amino acid sequence of SEQ ID NO: 30, and
as light chain of the first full length antibody the amino acid
sequence of SEQ ID NO: 31, and [0102] 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: 29.
[0103] In one embodiment such bispecific, bivalent antibody is
characterized in comprising [0104] 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: 34, and [0105] 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: 32.
[0106] 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, Vol. 25 (1998)
387-393; or Schmidt, M., et al., Oncogene 18 (1999) 1711-1721.
[0107] Thus in one embodiment such bispecific, bivalent antibody is
characterized in comprising a disulfide bond between the variable
domains of the second full length antibody heavy and light chain is
between heavy chain variable domain position 44 and light chain
variable domain position 100, and comprises [0108] 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: 37, and [0109] 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: 35.
[0110] In another aspect of the invention the bispecific antibody
according to the invention is characterized in comprising [0111] a)
the heavy chain and the light chain of a first full length antibody
that specifically binds to VEGF;
[0112] b) the heavy chain and the light chain of a second full
length antibody that specifically binds to ANG-2, [0113] wherein
the N-terminus of the heavy chain is connected to the C-terminus of
the light chain via a peptide linker; and wherein the variable
domains VL and VH are replaced by each other.
[0114] An exemplary scheme of 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 shown in FIG. 2b including Knobs-into-Holes modified CH3
domains. The antibodies based on this bispecific, bivalent antibody
format are named in the examples OAscXFab1.
[0115] In one embodiment such bispecific antibody is characterized
in comprising [0116] a) as heavy chain of the first full length
antibody the SEQ ID NO: 39, and as light chain of the first full
length antibody the SEQ ID NO: 40, and [0117] 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 SEQ ID NO:
38.
[0118] In another aspect of the invention the bispecific antibody
according to the invention is characterized in comprising [0119] a)
the heavy chain and the light chain of a first full length antibody
that specifically binds to VEGF; [0120] b) the heavy chain and the
light chain of a second full length antibody that specifically
binds to ANG-2, [0121] wherein the N-terminus of the heavy chain is
connected to the C-terminus of the light chain via a peptide
linker; and wherein the constant domains CL and CH1 are replaced by
each other.
[0122] An exemplary scheme of 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 shown in FIG. 2c including Knobs-into-Holes modified CH3
domains. The antibodies based on this bispecific, bivalent antibody
format are named in the examples OAscXFab2 and OAscXFab3.
[0123] In one embodiment such bispecific antibody is characterized
in comprising [0124] a) as heavy chain of the first full length
antibody the SEQ ID NO: 42, and as light chain of the first full
length antibody the SEQ ID NO: 43, and [0125] 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 SEQ ID NO:
41.
[0126] In one embodiment such bispecific antibody is characterized
in comprising [0127] a) as heavy chain of the first full length
antibody the SEQ ID NO: 45, and as light chain of the first full
length antibody the SEQ ID NO: 46, and [0128] 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 SEQ ID NO:
44.
[0129] 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, and of
SEQ ID NO: 31.
[0130] 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: 32, of SEQ ID NO: 33, and of
SEQ ID NO: 34.
[0131] 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: 35, of SEQ ID NO: 36, and of
SEQ ID NO: 37.
[0132] 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: 38, of SEQ ID NO: 39, and of
SEQ ID NO: 40.
[0133] 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: 41, of SEQ ID NO: 42, and of
SEQ ID NO: 43.
[0134] 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: 44, of SEQ ID NO: 45, and of
SEQ ID NO: 46.
[0135] Preferably 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.
[0136] In a preferred aspect of the invention all bispecific
antibodies according to the invention are characterized in that
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; wherein said interface
is altered to promote the formation of the bispecific antibody,
wherein the alteration is characterized in that: a) the CH3 domain
of one heavy chain is altered, 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, 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 and b) the CH3
domain of the other heavy chain is altered, 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 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.
[0137] Thus the antibody according to invention is preferably
characterized in that [0138] 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; [0139] wherein i) in the CH3 domain of one
heavy chain [0140] 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 [0141] and wherein [0142] ii)
in the CH3 domain of the other heavy chain [0143] 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.
[0144] 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).
[0145] 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).
[0146] 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.
[0147] 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".
[0148] 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 EP 1 870 459 A1,
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))).
[0149] 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".
[0150] 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".
[0151] 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
Examples 3 to 7): [0152] the bispecific, bivalent antibody binds to
VEGF with a KD value of the binding affinity of 5 nM or less;
[0153] the bispecific, bivalent antibody binds to ANG-2 with a KD
value of the binding affinity of 5 nM or less; [0154] the
bispecific, bivalent antibody inhibits ANG-2-induced Tie2
phosphorylation in HEK293 cells transfected with Tie2 with an IC50
of 15 nM or less, (in one embodiment with an IC50 of 10 nM or
less); [0155] the bispecific, bivalent antibody inhibits ANG-2
binding to Tie2 with an IC50 of 20 nM or less, (in one embodiment
with an IC50 of 15 nM or less); [0156] the bispecific, bivalent
antibody inhibits VEGF binding to VEGF receptor with an IC50 of 20
nM or less, (in one embodiment with an IC50 of 15 nM or less);
[0157] the bispecific, bivalent antibody inhibits VEGF-induced
proliferation of
[0158] HUVEC cells with an with an IC50 of 10 nM or less, (in one
embodiment with an IC50 of 5 nM or less).
[0159] In one embodiment the bispecific, bivalent 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 [0160] i) said first antigen-binding
site comprises as heavy chain variable domain (VH) the SEQ ID NO:
1, and as light chain variable domain (VL) the SEQ ID NO: 2; and
[0161] ii) said second antigen-binding site comprises as heavy
chain variable domain (VH) the SEQ ID NO: 3, and as light chain
variable domain (VL) the SEQ ID NO: 4; and having one or more of
the following properties (determined in assays as described in
Examples 3 to 7): [0162] the bispecific, bivalent antibody binds to
VEGF with a KD value of the binding affinity of 5 nM or less;
[0163] the bispecific, bivalent antibody binds to ANG-2 with a KD
value of the binding affinity of 5 nM or less; [0164] the
bispecific, bivalent antibody inhibits ANG-2-induced Tie2
phosphorylation in HEK293 cells transfected with Tie2 with an 1050
of 15 nM or less, (in one embodiment with an 1050 of 10 nM or
less); [0165] the bispecific, bivalent antibody inhibits ANG-2
binding to Tie2 with an IC50 of 20 nM or less, (in one embodiment
with an 1050 of 15 nM or less); [0166] the bispecific, bivalent
antibody inhibits VEGF binding to VEGF receptor with an IC50 of 20
nM or less, (in one embodiment with an IC50 of 15 nM or less);
[0167] the bispecific, bivalent antibody inhibits VEGF-induced
proliferation of HUVEC cells with an with an IC50 of 10 nM or less,
(in one embodiment with an IC50 of 5 nM or less).
[0168] In one aspect of the invention such bispecific antibody
according to the invention is characterized in comprising [0169] a)
the heavy chain and the light chain of a first full length antibody
that specifically binds to VEGF;
[0170] b) the modified heavy chain and modified light chain of a
full length antibody that specifically binds to ANG-2, wherein the
constant domains CL and CH1 are replaced by each other;
and having one or more of the following properties (determined in
assays as described in Examples 3 to 7): [0171] the bispecific,
bivalent antibody binds to VEGF with a KD value of the binding
affinity of 5 nM or less; [0172] the bispecific, bivalent antibody
binds to ANG-2 with a KD value of the binding affinity of 5 nM or
less; [0173] the bispecific, bivalent antibody inhibits
ANG-2-induced Tie2 phosphorylation in HEK293 cells transfected with
Tie2 with an IC50 of 15 nM or less, (in one embodiment with an IC50
of 10 nM or less); [0174] the bispecific, bivalent antibody
inhibits ANG-2 binding to Tie2 with an IC50 of 20 nM or less, (in
one embodiment with an IC50 of 15 nM or less); [0175] the
bispecific, bivalent antibody inhibits VEGF binding to VEGF
receptor with an IC50 of 20 nM or less, (in one embodiment with an
IC50 of 15 nM or less); [0176] the bispecific, bivalent antibody
inhibits VEGF-induced proliferation of
[0177] HUVEC cells with an with an IC50 of 10 nM or less, (in one
embodiment with an IC50 of 5 nM or less).
[0178] In one embodiment the bispecific, bivalent 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 [0179] i) said first antigen-binding
site comprises as heavy chain variable domain (VH) the SEQ ID NO: 1
with no more than 1 amino acid residue substitutions in the CDRs,
and as light chain variable domain (VL) the SEQ ID NO: 2 with no
more than 1 amino acid residue substitutions in the CDRs; and
[0180] ii) said second antigen-binding site comprises as heavy
chain variable domain (VH) the SEQ ID NO: 3 with no more than 1
amino acid residue substitutions in the CDRs, and as light chain
variable domain (VL) the SEQ ID NO: 4 with no more than 1 amino
acid residue substitutions in the CDRs.
[0181] In one embodiment the bispecific, bivalent 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 [0182] i) said first antigen-binding
site comprises as heavy chain variable domain (VH) the SEQ ID NO: 1
with no more than 1 amino acid residue substitutions in the CDRs,
and as light chain variable domain (VL) the SEQ ID NO: 2 with no
more than 1 amino acid residue substitutions in the CDRs; and
[0183] ii) said second antigen-binding site comprises as heavy
chain variable domain (VH) the SEQ ID NO: 3 with no more than 1
amino acid residue substitutions in the CDRs, and as light chain
variable domain (VL) the SEQ ID NO: 4 with no more than 1 amino
acid residue substitutions in the CDRs; and having one or more of
the following properties (determined in assays as described in
Examples 3 to 7): [0184] the bispecific, bivalent antibody binds to
VEGF with a KD value of the binding affinity of 5 nM or less;
[0185] the bispecific, bivalent antibody binds to ANG-2 with a KD
value of the binding affinity of 5 nM or less; [0186] the
bispecific, bivalent antibody inhibits ANG-2-induced Tie2
phosphorylation in HEK293 cells transfected with Tie2 with an IC50
of 15 nM or less, (in one embodiment with an IC50 of 10 nM or
less); [0187] the bispecific, bivalent antibody inhibits ANG-2
binding to Tie2 with an IC50 of 20 nM or less, (in one embodiment
with an IC50 of 15 nM or less); [0188] the bispecific, bivalent
antibody inhibits VEGF binding to VEGF receptor with an IC50 of 20
nM or less, (in one embodiment with an IC50 of 15 nM or less);
[0189] the bispecific, bivalent antibody inhibits VEGF-induced
proliferation of HUVEC cells with an with an IC50 of 10 nM or less,
(in one embodiment with an IC50 of 5 nM or less).
[0190] In one aspect of the invention the bispecific antibody
according to the invention is characterized in comprising [0191] a)
the heavy chain and the light chain of a first full length antibody
that specifically binds to VEGF, [0192] and wherein the heavy chain
of the first full length antibody comprises the amino acid sequence
of SEQ ID NO:7 with no more than 1 amino acid residue substitutions
in the CDRs, and the light chain of the first full length antibody
comprises the amino acid sequence of SEQ ID NO: 5 with no more than
1 amino acid residue substitutions in the CDRs, and [0193] b) the
modified heavy chain and modified light chain of a full length
antibody that specifically binds to ANG-2, wherein the constant
domains CL and CH1 are replaced by each other, [0194] and wherein
the modified heavy chain of the second full length antibody
comprises the amino acid sequence of SEQ ID NO: 8 with no more than
1 amino acid residue substitutions in the CDRs, and the modified
light chain of the second full length antibody comprises the amino
acid sequence of SEQ ID NO: 6 with no more than 1 amino acid
residue substitutions in the CDRs.
[0195] In one aspect of the invention the bispecific antibody
according to the invention is characterized in comprising [0196] a)
the heavy chain and the light chain of a first full length antibody
that specifically binds to VEGF, [0197] and wherein the heavy chain
of the first full length antibody comprises the amino acid sequence
of SEQ ID NO:7 with no more than 1 amino acid residue substitutions
in the CDRs, and the light chain of the first full length antibody
comprises the amino acid sequence of SEQ ID NO: 5 with no more than
1 amino acid residue substitutions in the CDRs, and [0198] b) the
modified heavy chain and modified light chain of a full length
antibody that specifically binds to ANG-2, wherein the constant
domains CL and CH1 are replaced by each other, [0199] and wherein
the modified heavy chain of the second full length antibody
comprises the amino acid sequence of SEQ ID NO: 8 with no more than
1 amino acid residue substitutions in the CDRs, and the modified
light chain of the second full length antibody comprises the amino
acid sequence of SEQ ID NO: 6 with no more than 1 amino acid
residue substitutions in the CDRs; and having one or more of the
following properties (determined in assays as described in Examples
3 to 7): [0200] the bispecific, bivalent antibody binds to VEGF
with a KD value of the binding affinity of 5 nM or less; [0201] the
bispecific, bivalent antibody binds to ANG-2 with a KD value of the
binding affinity of 5 nM or less; [0202] the bispecific, bivalent
antibody inhibits ANG-2-induced Tie2 phosphorylation in HEK293
cells transfected with Tie2 with an 1050 of 15 nM or less, (in one
embodiment with an 1050 of 10 nM or less); [0203] the bispecific,
bivalent antibody inhibits ANG-2 binding to Tie2 with an 1050 of 20
nM or less, (in one embodiment with an 1050 of 15 nM or less);
[0204] the bispecific, bivalent antibody inhibits VEGF binding to
VEGF receptor with an 1050 of 20 nM or less, (in one embodiment
with an 1050 of 15 nM or less); [0205] the bispecific, bivalent
antibody inhibits VEGF-induced proliferation of HUVEC cells with an
with an IC50 of 10 nM or less, (in one embodiment with an 1050 of 5
nM or less).
[0206] 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).).
[0207] Antibody specificity refers to selective recognition of the
antibody for a particular epitope of an antigen. Natural
antibodies, for example, are monospecific.
[0208] "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.
[0209] 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.
[0210] 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
"bivalent".
[0211] The term "VEGF" as used herein refers to human vascular
endothelial growth factor (VEGF/VEGF-A) (SEQ ID NO: 47) which is
described e.g. in Leung, D. W., et al., Science 246 (1989) 1306-9;
Keck, P. J., et al., Science 246 (1989) 1309-12 and Connolly, D.
T., et al., J. Biol. Chem. 264 (1989) 20017-24. 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. VEGF shows highly specific mitogenic
activity for endothelial cells.
[0212] The term "ANG-2" as used herein refers to human
angiopoietin-2 (ANG-2) (alternatively abbreviated with ANGPT2 or
ANG2) (SEQ ID NO: 48) 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 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, 1., 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, 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.
[0213] 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. Also included within the scope
of the invention are functional antigen binding sites comprised of
fewer CDRs (i.e., where binding specificity is determined by three,
four or five CDRs). For example, less than a complete set of 6 CDRs
may be sufficient for binding. In some cases, a VH or a VL domain
will be sufficient.
[0214] The antibodies of the invention further comprise
immunoglobulin constant regions of one or more immunoglobulin
classes. Immunoglobulin classes include IgG, IgM, IgA, IgD, and IgE
isotypes and, in the case of IgG and IgA, their subtypes.
[0215] The terms "monoclonal antibody" or "monoclonal antibody
composition" as used herein refer to a preparation of antibody
molecules of a single amino acid composition.
[0216] 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.
[0217] 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 Clq binding and/or Fc receptor (FcR) binding.
[0218] 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 Clq 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).
[0219] The term "recombinant human 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
human antibodies have variable and constant regions in a rearranged
form. The recombinant human 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.
[0220] 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.
[0221] 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)
(includes the numbering according to the EU Index of Kabat,
(abbreviated as numbering according to Kabat herein below)).
[0222] 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 (BIAcore, GE-Healthcare Uppsala,
Sweden) (Example 3) 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, preferably 10.sup.-9 M to
10.sup.-13 mol/l.
[0223] 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.
[0224] 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.
[0225] The term "full length antibody" denotes an antibody
consisting of two "full length antibody heavy chains" and two "full
length antibody light chains" (see FIG. 1). 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.
[0226] 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.
[0227] 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).
[0228] The term "constant region derived from human origin" 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., (see 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).
[0229] Preferably the bispecific, bivalent antibodies according to
the invention have a constant region of human IgG1 subclass.
[0230] While antibodies of the IgG4 subclass show reduced Fc
receptor (Fc.gamma.RIIIa) binding, antibodies of other IgG
subclasses show binding. However Pro238, Asp265, Asp270, Asn297
(loss of Fc carbohydrate), Pro329, Leu234, Leu235, Gly236, Gly237,
Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, and His435 are
residues which, if altered, provide also reduced Fc receptor
binding (Shields, R. L., et al., J. Biol. Chem. 276 (2001)
6591-6604; Lund, J., et al., FASEB J. 9 (1995) 115-119; Morgan, A.,
et al., Immunology 86 (1995) 319-324; EP 0 307 434).
[0231] In one embodiment an antibody according to the invention has
a reduced FcR binding compared to an IgG1 antibody and the
bispecific, bivalent antibody is in regard to FcR binding of IgG4
subclass or of IgG1 subclass with a mutation in S228, L234, L235
and/or D265, and/or contains the PVA236 mutation. In one embodiment
the mutations in the bispecific bivalent antibody are in IgG4 S228P
and L235E and in IgG1 L234A and L235A.
[0232] Another aspect of the invention a bispecific, bivalent
antibody characterized in comprising [0233] a) the heavy chain and
the light chain of a first full length antibody that specifically
binds to a first antigen;
[0234] b) the heavy chain and the light chain of a second full
length antibody that specifically binds to a second antigen, [0235]
wherein the N-terminus of the heavy chain is connected to the
C-terminus of the light chain via a peptide linker; and [0236]
wherein the variable domains VL and VH or the constant domains CL
and CH1 are replaced by each other.
[0237] Preferably the CH3 domains of this bispecific, bivalent
antibody format 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.
For further details and embodiments see above.
[0238] Another aspect of the invention a bispecific, bivalent
antibody characterized in comprising [0239] a) the heavy chain and
the light chain of a first full length antibody that specifically
binds to a first antigen;
[0240] b) the heavy chain and the light chain of a second full
length antibody that specifically binds to a second antigen, [0241]
wherein the N-terminus of the heavy chain is connected to the
C-terminus of the light chain via a peptide linker; and [0242]
wherein the variable domains VL and VH are replaced by each
other.
[0243] An exemplary scheme of this bispecific, bivalent antibody
format is shown in FIG. 2b including Knobs-into-Holes modified CH3
domains. The antibodies based on this bispecific, bivalent antibody
format are named in the examples OAscXFab1.
[0244] In one embodiment such bispecific antibody is characterized
in comprising [0245] a) as heavy chain of the first full length
antibody the SEQ ID NO: 39, and as light chain of the first full
length antibody the SEQ ID NO: 40, and [0246] 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 SEQ ID NO:
38.
[0247] Another aspect of the invention a bispecific, bivalent
antibody characterized in comprising [0248] a) the heavy chain and
the light chain of a first full length antibody that specifically
binds to a first antigen; [0249] b) the heavy chain and the light
chain of a second full length antibody that specifically binds to a
second antigen, [0250] wherein the N-terminus of the heavy chain is
connected to the C-terminus of the light chain via a peptide
linker; and [0251] wherein the constant domains CL and CH1 are
replaced by each other.
[0252] An exemplary scheme of this bispecific, bivalent antibody
format is shown in FIG. 2c including Knobs-into-Holes modified CH3
domains. The antibodies based on this bispecific, bivalent antibody
format are named in the examples OAscXFab2 and OAscXFab3.
[0253] In one embodiment such bispecific antibody is characterized
in comprising [0254] a) as heavy chain of the first full length
antibody the SEQ ID NO: 42, and as light chain of the first full
length antibody the SEQ ID NO: 43, and [0255] 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 SEQ ID NO:
41.
[0256] In one embodiment such bispecific antibody is characterized
in comprising [0257] a) as heavy chain of the first full length
antibody the SEQ ID NO: 45, and as light chain of the first full
length antibody the SEQ ID NO: 46, and [0258] 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 SEQ ID NO:
44.
[0259] 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,
NS0 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.
[0260] Accordingly one embodiment of the invention is a method for
the preparation of a bispecific antibody according to the
invention, comprising the steps of [0261] a) transforming a host
cell with vectors comprising nucleic acid molecules encoding said
antibody; [0262] b) culturing the host cell under conditions that
allow synthesis of said antibody molecule; and [0263] c) recovering
said antibody molecule from said culture.
[0264] 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.
[0265] 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 isotype and
antigen binding, but may improve the yield of the recombinant
production, protein stability or facilitate the purification.
[0266] 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.
[0267] Expression in NS0 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.
[0268] 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.
[0269] 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.
[0270] 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).
[0271] It has now been found that the bispecific antibodies against
human VEGF and human ANG-2 according to the current invention have
valuable characteristics such as high stability and valuable
pharmacokinetic/pharmacodynamic properties like e.g. good (i.e.
slow) clearance (e.g. at low doses).
[0272] The bispecific, bivalent antibodies according to the
invention show benefits for human patients in need of a VEGF and
ANG-2 targeting therapy.
[0273] Furthermore they have biological or pharmacological activity
and show in vivo tumor growth inhibition and/or inhibition of tumor
angiogenesis.
[0274] The bispecific antibodies according to the invention are
highly effective in [0275] a) tumor growth inhibition (e.g. with
the bispecific antibodies according to the invention tumor stasis
could be achieved already at lower concentrations compared to the
combination of the two monospecific antibodies (e.g in the COLO205
and the KPL4 tumor models of Example 9 and 10, tumor stasis was
already achieved with 10 mg/kg XMAb1 compared to the combination of
10 mg/kg of ANG2i-LC06+10 mg/kg of Avastin), and/or [0276] b)
inhibition of tumor angiogenesis or vascular diseases (e.g. maximal
antiangiogenic effects with the bispecific antibodies according to
the invention could already be achieved at lower concentrations
compared to the combination of the two monospecific antibodies
(e.g. in the mouse corneal angiogenesis assay of Example 8, the
maximal antiangiogenic effect was already achieved with 10 mg/kg
XMAb1 compared to the combination of 10 mg/kg of ANG2i-LC06+10
mg/kg of Avastin).
[0277] Finally the bivalent bispecific against human VEGF and human
ANG-2 according to the current invention may have a valuable
efficacy/toxicity profile and may provide benefits for a patient in
the need of an anti-VEGF and anti-ANG-2 therapy.
[0278] 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.
[0279] One embodiment of the invention is the bispecific antibody
according to the invention for the treatment of cancer.
[0280] Another aspect of the invention is said pharmaceutical
composition for the treatment of cancer.
[0281] 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 cancer.
[0282] Another aspect of the invention is method of treatment of
patient suffering from cancer by administering an antibody
according to the invention to a patient in the need of such
treatment.
[0283] Another aspect of the invention is said pharmaceutical
composition for the prevention of metastasis.
[0284] The invention comprises the bispecific antibody according to
the invention for the prevention of metastasis.
[0285] Another aspect of the invention is the use of a bispecific
antibody according to the invention for the manufacture of a
medicament for the prevention of metastasis.
[0286] Another aspect of the invention is a method of prevention
metastasis in patient suffering from primary cancer by
administering a bispecific according to the invention to a patient
in the need of such preventative treatment.
[0287] We could show highly efficient prevention of spontaneous
metastasis/secondary tumors in vivo in a orthotopic and a
subcutaneous cancer model (see Example 9) (in contrast to
experimental model where the tumor cells are injected i.v. This is
similar to the clinical situation wherein cells disseminate from a
primary tumor and metastase to secondary organ like lung or liver
(where secondary tumors).
[0288] The term "metastasis" according to the invention refers to
the transmission of cancerous cells from the primary tumor to one
or more sites elsewhere in a patient where then secondary tumors
develop. Means to determine if a cancer has metastasized are known
in the art and include bone scan, chest X-ray, CAT scan, MRI scan,
and tumor marker tests.
[0289] The term "prevention of metastasis" or "prevention of
secondary tumors" as used herein have the same meaning and refers a
prophylactic agent against metastasis in patient suffering from
cancer in this way inhibiting or reducing a further transmission of
cancerous cells from the primary tumor to one or more sites
elsewhere in a patient. This means that the metastasis of the
primary, tumor or cancer is prevented, delayed, or reduced and thus
the development of secondary tumors is prevented, delayed, or
reduced. Preferably the metastasis i.e. secondary tumors of the
lung are prevented or reduced, which means that metastatic
transmission of cancerous cells from the primary tumor to the lung
is prevented or reduced.
[0290] 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 intravenous, intramuscular, subcutaneous,
parenteral, spinal or epidermal administration (e.g. by injection
or infusion).
[0291] 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.
[0292] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intra-arterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtrachcal,
subcutaneous, subcuticular, intra-articular, subcapsular,
subarachnoid, intraspinal, epidural and intrasternal injection and
infusion.
[0293] The term cancer as used herein refers to proliferative
diseases, such as lymphomas, lymphocytic leukemias, lung cancer,
non small cell lung (NSCL) cancer, bronchioloalviolar cell lung
cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the
head or neck, cutaneous or intraocular melanoma, uterine cancer,
ovarian cancer, rectal cancer, cancer of the anal region, stomach
cancer, gastric cancer, colon cancer, breast cancer, uterine
cancer, carcinoma of the fallopian tubes, carcinoma of the
endometrium, carcinoma of the cervix, carcinoma of the vagina,
carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus,
cancer of the small intestine, cancer of the endocrine system,
cancer of the thyroid gland, cancer of the parathyroid gland,
cancer of the adrenal gland, sarcoma of soft tissue, cancer of the
urethra, cancer of the penis, prostate cancer, cancer of the
bladder, cancer of the kidney or ureter, renal cell carcinoma,
carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer,
biliary cancer, neoplasms of the central nervous system (CNS),
spinal axis tumors, brain stem glioma, glioblastoma multiforme,
astrocytomas, schwanomas, cpendymonas, medulloblastomas,
meningiomas, squamous cell carcinomas, pituitary adenoma and Ewings
sarcoma, including refractory versions of any of the above cancers,
or a combination of one or more of the above cancers.
[0294] Another aspect of the invention is the bispecific antibody
according to the invention or said pharmaceutical composition as
anti-angiogenic agent. Such anti-angiogenic agent can be used for
the treatment of cancer, especially solid tumors, and other
vascular diseases.
[0295] One embodiment of the invention is the bispecific antibody
according to the invention for the treatment of vascular
diseases.
[0296] Another aspect of the invention is said pharmaceutical
composition for the treatment of vascular diseases.
[0297] 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 vascular diseases.
[0298] Another aspect of the invention is method of treatment of
patient suffering from vascular diseases by administering an
antibody according to the invention to a patient in the need of
such treatment.
[0299] The term "vascular diseases" includes Cancer, Inflammatory
diseases, Atherosclerosis, Ischemia, Trauma, Sepsis, COPD, Asthma,
Diabetes, AMD, Retinopathy, Stroke, Adipositas, Acute lung injury,
Hemorrhage, Vascular leak e.g. Cytokine induced, Allergy, Graves'
Disease, Hashimoto's Autoimmune Thyroiditis, Idiopathic
Thrombocytopenic Purpura, Giant Cell Arteritis, Rheumatoid
Arthritis, Systemic Lupus Erythematosus (SLE), Lupus Nephritis,
Crohn's Disease, Multiple Sclerosis, Ulcerative Colitis, especially
to 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).
[0300] These 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.
[0301] 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.
[0302] 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.
[0303] 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.
[0304] 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.
[0305] 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.
[0306] 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.
[0307] 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.
[0308] 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.
[0309] 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.
[0310] 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.
Description of the Sequence Listing (Amino Acid Sequences)
[0311] SEQ ID NO:1 variable heavy chain domain VH of <VEGF>
bevacizumab [0312] SEQ ID NO:2 variable light chain domain VL of
<VEGF> bevacizumab [0313] SEQ ID NO:3 variable heavy chain
domain VH of <ANG-2> E6Q [0314] SEQ ID NO:4 variable light
chain domain VL of <ANG-2> E6Q [0315] SEQ ID NO: 5
XMab1-<VEGF> light chain [0316] SEQ ID NO: 6
XMab1-<ANG2> light chain [0317] SEQ ID NO: 7
XMab1-<VEGF> heavy chain [0318] SEQ ID NO: 8
XMab1-<ANG2> heavy chain [0319] SEQ ID NO: 9
XMab2-<VEGF> light chain [0320] SEQ ID NO: 10
XMab2-<ANG2> light chain [0321] SEQ ID NO: 11
XMab2-<VEGF> heavy chain [0322] SEQ ID NO: 12
XMab2-<ANG2> heavy chain [0323] SEQ ID NO: 13
XMab3-<VEGF> light chain [0324] SEQ ID NO: 14
XMab3-<ANG2> light chain [0325] SEQ ID NO: 15
XMab3-<VEGF> heavy chain [0326] SEQ ID NO: 16
XMab3-<ANG2> heavy chain [0327] SEQ ID NO: 17
XMab4-<VEGF> light chain [0328] SEQ ID NO: 18
XMab4-<ANG2> light chain [0329] SEQ ID NO: 19
XMab4-<VEGF> heavy chain [0330] SEQ ID NO: 20
XMab4-<ANG2> heavy chain [0331] SEQ ID NO: 21
XMab5-<VEGF> light chain [0332] SEQ ID NO: 22
XMab5-<ANG2> light chain [0333] SEQ ID NO: 23
XMab5-<VEGF> heavy chain [0334] SEQ ID NO: 24
XMab5-<ANG2> heavy chain [0335] SEQ ID NO: 25
XMab6-<VEGF> light chain [0336] SEQ ID NO: 26
XMab6-<ANG2> light chain [0337] SEQ ID NO: 27
XMab6-<VEGF> heavy chain [0338] SEQ ID NO: 28
XMab6-<ANG2> heavy chain [0339] SEQ ID NO: 29
OAscFab1-<ANG2> peptide connected heavy chain and light chain
[0340] SEQ ID NO: 30 OAscFab1-<VEGF> heavy chain [0341] SEQ
ID NO: 31 OAscFab1-<VEGF> light chain [0342] SEQ ID NO: 32
OAscFab2-<ANG2> peptide connected heavy chain and light chain
[0343] SEQ ID NO: 33 OAscFab2-<VEGF> heavy chain [0344] SEQ
ID NO: 34 OAscFab2-<VEGF> light chain [0345] SEQ ID NO: 35
OAscFab3-<ANG2> peptide connected heavy chain and light chain
[0346] SEQ ID NO: 36 OAscFab3-<VEGF> heavy chain [0347] SEQ
ID NO: 37 OAscFab3-<VEGF> light chain [0348] SEQ ID NO:38
OAscXFab1-<ANG2> peptide connected heavy chain and light
chain [0349] SEQ ID NO: 39 OAscXFab1-<VEGF> heavy chain
[0350] SEQ ID NO: 40 OAscXFab1-<VEGF> light chain [0351] SEQ
ID NO: 41 OAscXFab2-<ANG2> peptide connected heavy chain and
light chain [0352] SEQ ID NO: 42 OAscXFab2-<VEGF> heavy chain
[0353] SEQ ID NO: 43 OAscXFab2-<VEGF> light chain [0354] SEQ
ID NO: 44 OAscXFab3-<ANG2> peptide connected heavy chain and
light chain [0355] SEQ ID NO: 45 OAscXFab3-<VEGF> heavy chain
[0356] SEQ ID NO: 46 OAscXFab3-<VEGF> light chain [0357] SEQ
ID NO: 47 Human vascular endothelial growth factor (VEGF) [0358]
SEQ ID NO: 48 Human angiopoietin-2 (ANG-2)
EXPERIMENTAL PROCEDURES
Examples
Materials & General Methods
[0359] General information regarding the nucleotide sequences of
human immunoglobulins 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)).
Recombinant DNA Techniques
[0360] 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.
Gene Synthesis
[0361] Desired gene segments can be prepared from oligonucleotides
made by chemical synthesis. The gene segments, which are flanked by
singular restriction endonuclease cleavage sites, were assembled by
annealing and ligation of oligonucleotides including PCR
amplification and subsequently cloned via the indicated restriction
sites e.g. KpnI/SacI or AscI/PacI into a pPCRScript (Stratagene)
based pGA4 cloning vector. The DNA sequences of the subcloned gene
fragments were confirmed by DNA sequencing.
[0362] Gene synthesis fragments were ordered according to given
specifications at Geneart (Regensburg, Germany). All gene segments
encoding light and heavy chains of Ang-2/VEGF bispecific antibodies
were synthesized with a 5'-end DNA sequence coding for a leader
peptide (MGWSCIILFLVATATGVHS), which targets proteins for secretion
in eukaryotic cells, and unique restriction sites at the 5' and 3'
ends of the synthesized gene. DNA sequences carrying disulfide
stabilized "knobs-into-hole" modified heavy chains were designed
with S354C and T366W mutations in the "knobs" heavy chain and
Y349C, T366S, L368A and Y407V mutations in the "hole" heavy
chain.
DNA Sequence Determination
[0363] DNA sequences were determined by double strand sequencing
performed at MediGenomix GmbH (Martinsried, Germany) or Sequiserve
GmbH (Vaterstetten, Germany).
DNA and Protein Sequence Analysis and Sequence Data Management
[0364] The GCG's (Genetics Computer Group, Madison, Wis.) 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.
Expression Vectors
[0365] For the expression of the described antibodies variants of
expression plasmids for transient expression (e.g. in HEK293 EBNA
or HEK293-F) cells or for stable expression (e.g. in CHO cells)
based either on a cDNA organization with a CMV-Intron A promoter or
on a genomic organization with a CMV promoter (e.g. FIG. 2B) were
applied.
[0366] Beside the antibody expression cassette the vectors
contained: [0367] an origin of replication which allows replication
of this plasmid in E. coli, and [0368] a .beta.-lactamase gene
which confers ampicillin resistance in E. coli.
[0369] The transcription unit of the antibody gene is composed of
the following elements: [0370] unique restriction site(s) at the 5'
end [0371] the immediate early enhancer and promoter from the human
cytomegalovirus, [0372] followed by the Intron A sequence in the
case of the cDNA organization, [0373] a 5'-untranslated region of a
human antibody gene, [0374] a immunoglobulin heavy chain signal
sequence, [0375] the human antibody chain (heavy chain, modified
heavy chain or light chain) either as cDNA or as genomic
organization with an the immunoglobulin exon-intron organization
[0376] a 3' untranslated region with a polyadenylation signal
sequence, and [0377] unique restriction site(s) at the 3' end.
[0378] For transient and stable transfections larger quantities of
the plasmids were prepared by plasmid preparation from transformed
E. coli cultures (Nucleobond AX, Macherey-Nagel).
Cell Culture Techniques
[0379] 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.
Transient Transfections in HEK293-F System
[0380] Recombinant immunoglobulin variants were expressed by
transient transfection of human embryonic kidney 293-F cells using
the FreeStyle.TM. 293 Expression System according to the
manufacturer's instruction (Invitrogen, USA). Briefly, suspension
FreeStyle.TM. 293-F cells were cultivated in FreeStyle.TM. 293
Expression medium at 37.degree. C./8% CO.sub.2 and the cells were
seeded in fresh medium at a density of 1-2.times.10.sup.6 viable
cells/ml on the day of transfection. DNA-293Fectin.TM. complexes
were prepared in Opti-MEM.RTM. I medium (Invitrogen, USA) using 325
.mu.l of 293Fectin.TM. (Invitrogen, Germany) and 250 .mu.g of heavy
and light chain plasmid DNA in a 1:1 molar ratio for a 250 ml final
transfection volume for monospecific parent antibodies.
"Knobs-into-hole" DNA-293fectin complexes with two heavy chains and
one light chain were prepared in Opti-MEM.RTM. I medium
(Invitrogen, USA) using 325 .mu.l of 293Fectin.TM. (Invitrogen,
Germany) and 250 .mu.g of "Knobs-into-hole" heavy chain 1 and 2 and
light chain plasmid DNA generally in a 1:1:1 molar ratio for a 250
ml final transfection volume (OAscFab and OAscXFab). For expression
yield optimization the ratio can be varied. XMab DNA-293fectin
complexes were prepared in Opti-MEM.RTM. I medium (Invitrogen, USA)
using 325 .mu.l of 293Fectin.TM. (Invitrogen, Germany) and 250
.mu.g of "Knobs-into-hole" heavy chain 1 and 2 and light chain
plasmid DNA in a 1:1:1:1 molar ratio for a 250 ml final
transfection volume. For expression yield optimization the ratio
can be varied. Antibody containing cell culture supernatants were
harvested 7 days after transfection by centrifugation at 14000 g
for 30 minutes and filtered through a sterile filter (0.22 .mu.m).
Supernatants were stored at -20.degree. C. until purification.
Protein Determination
[0381] The protein concentration of purified antibodies and
derivatives was determined by determining the optical density (OD)
at 280 nm, using the molar extinction coefficient calculated on the
basis of the amino acid sequence according to Pace, C. N., et. al.,
Protein Science 4 (1995) 2411-1423.
Antibody Concentration Determination in Supernatants
[0382] The concentration of antibodies and derivatives in cell
culture supernatants was estimated by immunoprecipitation with
Protein A Agarose-beads (Roche). 60 .mu.L Protein A Agarose beads
are washed three times in TBS-NP40 (50 mM Tris, pH 7.5, 150 mM
NaCl, 1% Nonidet-P40). Subsequently, 1-15 mL cell culture
supernatant are applied to the Protein A Agarose beads
pre-equilibrated in TBS-NP40. After incubation for at 1 h at room
temperature the beads are washed on an Ultrafree-MC-filter column
(Amicon] once with 0.5 mL TBS-NP40, twice with 0.5 mL 2.times.
phosphate buffered saline (2.times.PBS, Roche) and briefly four
times with 0.5 mL 100 mM Na-citrate pH 5.0. Bound antibody is
eluted by addition of 35 .mu.l NuPAGE.RTM. LDS Sample Buffer
(Invitrogen). Half of the sample is combined with NuPAGE.RTM.
Sample Reducing Agent or left unreduced, respectively, and heated
for 10 min at 70.degree. C. Consequently, 20 .mu.l are applied to
an 4-12% NuPAGE.RTM. Bis-Tris SDS-PAGE (Invitrogen) (with MOPS
buffer for non-reduced SDS-PAGE and MES buffer with NuPAGE.RTM.
Antioxidant running buffer additive (Invitrogen) for reduced
SDS-PAGE) and stained with Coomassie Blue.
[0383] The concentration of antibodies and derivatives in cell
culture supernatants was measured by Protein A-HPLC chromatography.
Briefly, cell culture supernatants containing antibodies and
derivatives that bind to Protein A were applied to a HiTrap Protein
A column (GE Healthcare) in 50 mM K2HPO4, 300 mM NaCl, pH 7.3 and
eluted from the matrix with 550 mM acetic acid, pH 2.5 on a Dionex
HPLC-System. The eluted protein was quantified by UV absorbance and
integration of peak areas. A purified standard IgG1 antibody served
as a standard.
[0384] Alternatively, the concentration of antibodies and
derivatives in cell culture supernatants was measured by
Sandwich-IgG-ELISA. Briefly, StreptaWell High Bind Strepatavidin
A-96 well microtiter plates (Roche) were coated with 100 .mu.L/well
biotinylated anti-human IgG capture molecule
F(ab')2<h-Fcgamma> BI (Dianova) at 0.1 .mu.g/mL for 1 h at
room temperature or alternatively over night at 4.degree. C. and
subsequently washed three times with 200 .mu.L/well PBS, 0.05%
Tween (PBST, Sigma). 100 .mu.L/well of a dilution series in PBS
(Sigma) of the respective antibody containing cell culture
supernatants was added to the wells and incubated for 1-2 h on a
microtiterplate shaker at room temperature. The wells were washed
three times with 200 .mu.L/well PBST and bound antibody was
detected with 100 .mu.l F(ab')2<hFcgamma>POD (Dianova) at 0.1
.mu.g/mL as detection antibody for 1-2 h on a microtiterplate
shaker at room temperature. Unbound detection antibody was washed
away three times with 200 .mu.L/well PBST and the bound detection
antibody was detected by addition of 100 .mu.L ABTS/well.
Determination of absorbance was performed on a Tecan Fluor
Spectrometer at a measurement wavelength of 405 nm (reference
wavelength 492 nm).
Purification of Bispecific Antibodies
[0385] Bispecific antibodies were purified from cell culture
supernatants by affinity chromatography using Protein
A-Sepharose.TM. (GE Healthcare, Sweden) and Superdex200 size
exclusion chromatography. Briefly, sterile filtered cell culture
supernatants were applied on a HiTrap ProteinA HP (5 ml) column
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 KCl, pH 7.4). Unbound
proteins were washed out with equilibration buffer. Antibody and
antibody variants were eluted with 0.1 M citrate buffer, pH 2.8,
and the protein containing fractions were neutralized with 0.1 ml 1
M Tris, pH 8.5. Then, the eluted protein fractions were pooled,
concentrated with an Amicon Ultra centrifugal filter device (MWCO:
30 K, Millipore) to a volume of 3 ml and loaded on a Superdex200
HiLoad 120 ml 16/60 gel filtration column (GE Healthcare, Sweden)
equilibrated with 20 mM Histidin, 140 mM NaCl, pH 6.0. Fractions
containing purified bispecific antibodies with less than 5% high
molecular weight aggregates were pooled and stored as 1.0 mg/ml
aliquots at -80.degree. C.
SDS-PAGE
[0386] The NuPAGE.RTM. Pre-Cast gel system (Invitrogen) was used
according to the manufacturer's instruction. In particular, 4-20%
NuPAGE.RTM. Novex.RTM. TRIS-Glycine Pre-Cast gels and a Novex.RTM.
TRIS-Glycine SDS running buffer were used. (see e.g. FIG. 3).
Reducing of samples was achieved by adding NuPAGE.RTM. sample
reducing agent prior to running the gel.
Analytical Size Exclusion Chromatography
[0387] Size exclusion chromatography for the determination of the
aggregation and oligomeric state of antibodies was performed by
HPLC chromatography. Briefly, Protein A purified antibodies were
applied to a Tosoh TSKgel G3000SW column in 300 mM NaCl, 50 mM
KH2PO4/K2HPO4, pH 7.5 on an Agilent HPLC 1100 system or to a
Superdex 200 column (GE Healthcare) in 2.times.PBS on a Dionex
HPLC-System. The eluted protein was quantified by UV absorbance and
integration of peak areas. BioRad Gel Filtration Standard 151-1901
served as a standard. (see e.g. FIG. 4).
Mass Spectrometry
[0388] The total deglycosylated mass of crossover antibodies was
determined and confirmed via electrospray ionization mass
spectrometry (ESI-MS). Briefly, 100 .mu.g purified antibodies were
deglycosylated with 50 mU N-Glycosidase F (PNGaseF, ProZyme) in 100
mM KH2PO4/K2HPO4, pH 7 at 37.degree. C. for 12-24 h at a protein
concentration of up to 2 mg/ml and subsequently desalted via HPLC
on a Sephadex G25 column (GE Healthcare). The mass of the
respective heavy and light chains was determined by ESI-MS after
deglycosylation and reduction. In brief, 50 .mu.g antibody in 115
.mu.l were incubated with 60 .mu.l 1M TCEP and 50 .mu.l 8 M
Guanidine-hydrochloride subsequently desalted. The total mass and
the mass of the reduced heavy and light chains was determined via
ESI-MS on a Q-Star Elite MS system equipped with a NanoMate
source.
Generation of HEK293-Tie2 Cell Line
[0389] In order to determine the interference of Angiopoietin-2
antibodies with ANGPT2 stimulated Tie2 phosphorylation and binding
of ANGPT2 to Tie2 on cells a recombinant HEK293-Tie cell line was
generated. Briefly, a pcDNA3 based plasmid (RB22-pcDNA3 Topo hTie2)
coding for full-length human Tie2 (SEQ ID 108) under control of a
CMV promoter and a Neomycin resistance marker was transfected using
Fugene (Roche Applied Science) as transfection reagent into HEK293
cells (ATCC) and resistant cells were selected in DMEM 10% FCS, 500
.mu.g/ml G418. Individual clones were isolated via a cloning
cylinder, and subsequently analyzed for Tie2 expression by FACS.
Clone 22 was identified as clone with high and stable Tie2
expression even in the absence of G418 (HEK293-Tie2 clone22).
HEK293-Tie2 clone22 was subsequently used for cellular assays:
ANGPT2 induced Tie2 phosphorylation and ANGPT2 cellular ligand
binding assay.
ANGPT2 Induced Tie2 Phosphorylation Assay
[0390] Inhibition of ANGPT2 induced Tie2 phosphorylation by ANGPT2
antibodies was measured according to the following assay principle.
HEK293-Tie2 clone22 was stimulated with ANGPT2 for 5 minutes in the
absence or presence of ANGPT2 antibody and P-Tie2 was quantified by
a sandwich ELISA. Briefly, 2.times.105 HEK293-Tie2 clone 22 cells
per well were grown over night on a Poly-D-Lysine coated 96
well-microtiter plate in 100 .mu.l DMEM, 10% FCS, 500 .mu.g/ml
Geneticin. The next day a titration row of ANGPT2 antibodies was
prepared in a microtiter plate (4-fold concentrated, 75 .mu.l final
volume/well, duplicates) and mixed with 75 .mu.l of an ANGPT2
(R&D systems #623-AN] dilution (3.2 .mu.g/ml as 4-fold
concentrated solution). Antibodies and ANGPT2 were pre-incubated
for 15 min at room temperature. 100 .mu.l of the mix were added to
the HEK293-Tie2 clone 22 cells (pre-incubated for 5 min with 1 mM
NaV3O4, Sigma #S6508) and incubated for 5 min at 37.degree. C.
Subsequently, cells were washed with 200 .mu.l ice-cold PBS+1 mM
NaV3O4 per well and lysed by addition of 120 .mu.l lysis buffer (20
mM Tris, pH 8.0, 137 mM NaCl, 1% NP-40, 10% glycerol, 2 mM EDTA, 1
mM NaV3O4, 1 mM PMSF and 10 .mu.g/ml Aprotinin) per well on ice.
Cells were lysed for 30 min at 4.degree. C. on a microtiter plate
shaker and 100 .mu.l lysate were transferred directly into a p-Tie2
ELISA microtiter plate (R&D Systems, R&D #DY990) without
previous centrifugation and without total protein determination.
P-Tie2 amounts were quantified according to the manufacturer's
instructions and IC50 values for inhibition were determined using
XLfit4 analysis plug-in for Excel (Dose-response one site, model
205). IC50 values can be compared within on experiment but might
vary from experiment to experiment.
VEGF Induced HUVEC Proliferation Assay
[0391] VEGF induced HUVEC (Human Umbilical Vein Endothelial Cells,
Promocell #C-12200) proliferation was chosen to measure the
cellular function of VEGF antibodies. Briefly, 5000 HUVEC cells
(low passage number, .ltoreq.5 passages) per 96 well were incubated
in 100 .mu.l starvation medium (EBM-2 Endothelial basal medium 2,
Promocell # C-22211, 0.5% FCS, Penicilline/Streptomycine) in a
collagen I-coated BD Biocoat Collagen I 96-well microtiter plate
(BD #354407/35640 over night. Varying concentrations of antibody
were mixed with rhVEGF (30 ngl/ml final concentration, BD #354107)
and pre-incubated for 15 minutes at room temperature. Subsequently,
the mix was added to the HUVEC cells and they were incubated for 72
h at 37.degree. C., 5% CO2. On the day of analysis the plate was
equilibrated to room temperature for 30 min and cell
viability/proliferation was determined using the CellTiter-Glo.TM.
Luminescent Cell Viability Assay kit according to the manual
(Promega, # G7571/2/3). Luminescence was determined in a
spectrophotometer.
Example 1a
Expression & Purification of Bispecific, Bivalent Domain
Exchanged <VEGF-ANG-2> Antibody Molecules XMab
[0392] According the procedures described in the materials and
methods above, the bispecific, bivalent domain exchanged
<VEGF-ANG-2> antibody molecules XMab1, XMab1 and XMab3 were
expressed and purified. The VH and VL of <VEGF> part (SEQ ID
NO:1 and SEQ ID NO:2) are based on bevacizumab. The VH of
<ANG2> part (SEQ ID NO:3) was derived by a E6Q mutation (the
original amino acid glutamic acid (E) at position 6 was replaced by
glutamine (Q)) of the VH sequence of ANG2i-LC06 (which is described
in the PCT application No. PCT/EP2009/007182 (WO2010/040508)--and
which is further maturated fragment of a sequence obtained via
phage display). The VL of <ANG2> part (SEQ ID NO:4) was
derived from the VL sequences ANG2i-LC06 (see PCT application No.
PCT/EP2009/007182 (WO2010/040508)). The bispecific, bivalent domain
exchanged <VEGF-ANG-2> antibody molecules XMab1, XMab2 and
XMab3 were expressed and purified. The relevant light and heavy
chain amino acid sequences of these bispecific, bivalent antibodies
are given in SEQ ID NO: 5-8 (XMab1), in SEQ ID NO: 9-12 (XMab2),
and in SEQ ID NO: 13-16 (XMab3). For an exemplary structure see
FIG. 1.
TABLE-US-00001 Key data XMab1 XMab2 XMab3 Expression (Yield) 32
.mu.g/mL 10 .mu.g/mL 39 .mu.g/mL Purification (Yield, Prot. A. 31
mg/L, 64% 8 .mu.g/mL, 80% -- homog.)
[0393] The bispecific, bivalent <VEGF-ANG-2> antibodies
XMab4, XMab5 and XMab6 (with the relevant light and heavy chain
amino acid sequences given in SEQ ID NO: 17-20 (XMab4), in SEQ ID
NO: 21-24 (XMab5), and in SEQ ID NO: 25-28 (XMab6)) are expressed
and purified analogously.
[0394] Binding affinities and other properties were or are
determined as described.
Example 1b
Expression & Purification Bispecific, Bivalent
<VEGF-ANG-2> Antibody Molecules OAscFab
[0395] According to the procedures described in the materials and
methods above, the bispecific, bivalent <VEGF-ANG-2> antibody
molecules OAscFab1, OAscFab2, OAscFab3 were expressed and purified.
The VH and VL of <VEGF> part (SEQ ID NO:1 and SEQ ID NO:2)
are based on bevacizumab. The VH of <ANG2>E6Q part (SEQ ID
NO:3) was derived by a E6Q mutation (the original amino acid
glutamic acid (E) at position 6 was replaced by glutamine (Q)) of
the VH sequences ANG2i-LC06 (which is described in the PCT
application No. PCT/EP2009/007182 (WO2010/040508)--and which is
further maturated fragment of a sequence obtained via phage
display). The VL of <ANG2>E6Q part (SEQ ID NO:4) was derived
from the VL sequence of ANG2i-LC06 (see PCT application No.
PCT/EP2009/007182 (WO2010/040508) (WO2010/040508)). The relevant
light and heavy chain amino acid sequences of these bispecific,
bivalent antibodies are given in SEQ ID NO: 29-31 (OAscFab1), in
SEQ ID NO: 32-34 (OAscFab2), and in SEQ ID NO: 35-37 (OAscFab3).
For an exemplary structure see FIG. 2a. Expression of OAscFab1,
OAscFab1, OAscFab2 and OAscFab3 was confirmed by Western blot.
Purification of OAscFab2 and OAscFab3 led to the following
yields.
TABLE-US-00002 Protein A SEC Antibody Supernatant Yield Mono. Yield
Monomer OAscFab2 0.5 L 36.0 mg 86% 21.7 mg >95% OAscFab3 0.5 L
29.3 mg 85% 17.7 mg >95%
[0396] Binding affinities and other properties are determined as
described.
Example 1c
Expression & Purification Bispecific, Bivalent Domain Exchanged
<VEGF-ANG-2> Antibody Molecules OAscXFab
[0397] According the procedures described in the materials and
methods above, the bispecific, bivalent domain exchanged
<VEGF-ANG-2> antibody molecules OAscXFab1, OAscXFab2,
OAscXFab3, were expressed and purified. The VH and VL of
<VEGF> part (SEQ ID NO:1 and SEQ ID NO:2) are based on
bevacizumab. The VH of <ANG2>E6Q part (SEQ ID NO:3) was
derived by a E6Q mutation (the original amino acid glutamic acid
(E) at position 6 was replaced by glutamine (Q)) of the VH
sequences ANG2i-LC06 (which is described in the PCT application No.
PCT/EP2009/007182 (WO2010/040508)--and which is further maturated
fragment of a sequence obtained via phage display). The VL of
<ANG2>E6Q part (SEQ ID NO:4) was derived from the VL sequence
of ANG2i-LC06 (see PCT application No. PCT/EP2009/007182
(WO2010/040508)). The relevant light and heavy chain amino acid
sequences of these bispecific, bivalent antibodies are given in SEQ
ID NO: 38-40 (OAscXFab1), in SEQ ID NO: 41-43 (OAscXFab2), and in
SEQ ID NO: 44-46 (OAscXFab3). For an exemplary structure see FIG.
2b (OAscXFab1) and FIG. 2c (OAscXFab2, OAscXFab3). Expression was
confirmed by Western blot.
TABLE-US-00003 Key data OAscXFab1 OAscXFab2 OAscXFab3 Expression
(Yield) 23 .mu.g/mL 23 .mu.g/mL 26 .mu.g/mL
[0398] Binding affinities and other properties are determined as
described.
Example 2
Stability of Bispecific Antibodies
Denaturation Temperature (SYPRO Orange Method)
[0399] To determine the temperature at which protein denaturation
(i.e. temperature-induced loss of protein structure) occurs, a
method was used that relies a hydrophobic fluorescent dye (SYPRO
orange, Invitrogen) that exhibits strong fluorescence in
hydrophobic environments. Upon protein denaturation, hydrophobic
patches become exposed to the solvent, leading to an increased
fluorescence. At temperatures above the denaturation temperature,
fluorescence intensities decrease again, hence the temperature at
which a maximum intensity is reached is defined as the denaturation
temperature. The method is described by Ericsson, U. B., et al.,
Anal Biochem 357 (2006) 289-298 and He, F., et al., Journal of
Pharmaceutical Sciences 99 (2010) 1707-1720.
[0400] Proteins samples at a concentration of approx. 1 mg/mL in 20
mM His/HisCl, 140 mM NaCl, pH 6.0 were mixed with SYPRO orange
(5000.times. stock solution) to reach a final dilution of 1:5000. A
volume of 20 .mu.L was transferred into a 384 well-plate and
temperature-dependent fluorescence was recorded in a
LightCycler.RTM. 480 Real-Time PCR System (Roche Applied Sciences)
at a heat rate of 0.36.degree. C./min.
Aggregation Temperature by Dynamic Light Scattering (DLS)
[0401] The temperature at which thermally induced protein
aggregation occurs was determined by dynamic light scattering
(DLS). DLS yields information on the size distribution of
macromolecules in solution, derived from fluctuations of scattered
light intensities on a microsecond scale. When samples are heated
up gradually, aggregation starts at a certain temperature, giving
rise to growing particle sizes. The temperature at which particle
sizes begin to increase is defined as the aggregation temperature.
Aggregation and denaturation temperatures need not necessarily be
identical since denaturation may not necessarily be a prerequisite
for aggregation.
[0402] For aggregation temperature measurements, a DynaPro DLS
platereader (Wyatt technologies) was used. Preceding the
measurement, samples were filtered via 384-well filter plates
(Millipore MultiScreen 384-well Filtration System, 0.45 .mu.m) into
optical 384 well plates (Corning #3540). A sample volume of 35
.mu.L was used at a protein concentration of approx. 1 mg/mL in
formulation buffer (20 mM citrate, 180 mM sucrose, 20 mM arginine,
0.02% polysorbate 20). Each well was covered with 20 .mu.L paraffin
oil (Sigma) to avoid evaporation. Samples were heated from
25.degree. C. to 80.degree. C. at a rate of 0.05.degree. C./min and
DLS data were acquired continuously for a maximum number of 15
samples per run.
Aggregation Rate Per DLS
[0403] DLS is a sensitive method for detecting aggregates of
macromolecules in solution, since aggregates give rise to strong
light scattering signals. Hence, the tendency of a molecule to
aggregate can be followed over time by repeated acquisition of DLS
data. To accelerate potential aggregation to practical rates,
measurements were conducted at 50.degree. C.
[0404] Sample preparation was performed as described above. DLS
data were recorded for up to 100 hours. Aggregation rates (nm/day)
were calculated as the slope of a linear fit of average diameters
over time.
Stability in Formulation Buffer
[0405] To assess bispecific molecules for their stability with
regard to aggregation/fragmentation, samples were incubated for 3
weeks at 40.degree. C. in formulation buffer (20 mM citrate, 180 mM
sucrose, 20 mM arginine, 0.02% polysorbate 20) at a protein
concentration of approximately 1 mg/mL. A control sample was stored
for 3 weeks at -80.degree. C.
[0406] Size exclusion chromatography for the quantification of
aggregates and low-molecular weight (LMW) species was performed by
HPLC. An amount of 25-100 .mu.g of protein was applied to a Tosoh
TSKgel G3000SWXL column in 300 mM NaCl, 50 mM potassium phosphate,
pH 7.5 on an Ultimate3000 HPLC system (Dionex). The eluted protein
was quantified by UV absorbance at 280 nm.
Results
TABLE-US-00004 [0407] Stability of XMab1 Method (SEQ ID NO: 5-8)
Denaturation temperature (SYPRO orange 71.degree. C. method)
Aggregation temperature by Dynamic 65.degree. C. Light Scattering
(DLS) Aggregation rate per DLS 0.04 nm/day Stability in formulation
buffer .DELTA.HMW: 0.6 area % (difference between 40.degree. C. and
-80.degree. C. after 3 .DELTA.LMW: 0.5 area % weeks storage)
.DELTA.Monomer: -1.2 area %
Example 3
Binding Properties of Bispecific Antibody <VEGF-Ang-2>
[0408] A) Binding properties characterized by Surface Plasmon
Resonance (SPR) Analysis
[0409] Simultaneous binding of both antigens was confirmed by
applying Surface Plasmon Resonance (SPR) using a BIAcore T100
instrument (GE Healthcare Biosciences AB, Uppsala, Sweden). VEGF
was immobilized to a CM5 Sensorchip using standard amine coupling
chemistry. In a first step, <VEGF-Ang-2> XMAb was injected at
a concentration of 10 .mu.g/ml in HBS buffer (10 mM HEPES, 150 mM
NaCl, 0.05% Tween 20, pH 7.4) at 25.degree. C. After binding of the
antibody to the immobilized VEGF, hAng-2 was injected at 10
.mu.g/ml in a second step (FIG. 3)
[0410] In a further experiment the affinity and binding kinetics of
<VEGF-Ang-2> XMab were determined. Briefly,
goat<hIgG-Fcgamma> polyclonal antibodies were immobilized on
a CM4 chip via amine coupling for presentation of the bispecific
antibody against Ang-2 and VEGF. Binding was measured in HBS buffer
at 25.degree. C. or 37.degree. C. Purified Ang-2-His (R&D
systems or in house purified) or VEGF (R&D systems or in house
purified) was added in various concentrations between 0.37 nM and
30 nM or between 3.7 nM and 200 nM in solution. Association was
measured by an injection of 3 minutes; dissociation was measured by
washing the chip surface with HBS buffer for 10 minutes and a KD
value was estimated using a 1:1 Langmuir binding model. Due to
heterogenity of the Ang-2 preparation no 1:1 binding could be
observed. Therefore KD values are apparent values. The determined
affinity of <VEGF-Ang-2> XMab to VEGF was extremely high, the
calculated off-rate was out of Biacore specifications even at
37.degree. C. In Table 1 the binding constants for both antigens
are summarized.
TABLE-US-00005 TABLE 1 Kinetic parameters of <VEGF-Ang-2>
XMab1 binding to Ang-2 and VEGF Apparent ka Apparent kd Apparent KD
Analyte (1/Ms) (1/s) (M) Ang-2 2.7E+06 6.3E-04 2.4E-10 VEGF 1.2E+05
<1E-06 <1E-10
B) Assay for Quantification of Binding Active Bispecific
<Ang2/VEGF> XMab1
[0411] Additionally to the SPR analysis, an ELISA was established
to quantify the amount of binding active bispecific
mAb<Ang2/VEGF> antibodies. In this assay, hAng2 is directly
coated to the wells of a maxisorp microtiter plate (MTP) in the
first step. Meanwhile, the samples/reference standards
(mAb<Ang2/VEGF>) were pre-incubated in the wells of another
MTP with digoxigenylated VEGF. After pre-incubation and coating,
excess of unbound Ang2 was removed by washing the Ang2 coated MTP.
The pre-incubated mixture of <Ang2/VEGF> and VEGF-Dig was
then transferred to the hAng2 coated MTP and incubated. After
incubation, the excess of pre-incubation solution was removed by
washing followed by incubation with a horse-radish peroxidase
labeled anti-digoxigenin antibody. The antibody-enzyme conjugate
catalyzes the color reaction of the ABTS.RTM. substrate. The signal
was measured by ELISA reader at 405 nm wavelength (reference
wavelength: 490 nm ([405/490] nm)). Absorbance values of each
sample were determined in duplicates. (A scheme exemplifying this
test system is shown in FIG. 4 and Calibration curve of ELISA for
quantification is shown in FIG. 5)
Example 4
Tie2 Phosphorylation
[0412] In order to confirm that the anti-ANGPT2 related activities
are retained in the bispecific bivalent <VEGF-ANGPT2>
antibody XMAb1 Tie2 phosphorylation assay was performed. The
efficacy of XMAb1 was determined in the ANGPT2 stimulated Tie2
phosphorylation assay as described above.
[0413] It was shown that XMAb1 interferes with ANGPT2 stimulated
Tie2 phosphorylation in the ANGPT2 stimulated Tie2 phosphorylation
assay as described above. IC50 for XMAb1 was 7.4 nM+/-2.3.
Example 5
Inhibition of huANG-2 Binding to Tie-2 (ELISA)
[0414] The interaction ELISA was performed on 384 well microtiter
plates (MicroCoat, DE, Cat. No. 464718) at RT. After each
incubation step plates were washed 3 times with PBST. ELISA plates
were coated with 5 .mu.g/ml Tie-2 protein for 1 hour (h).
Thereafter the wells were blocked with PBS supplemented with 0.2%
Tween-20 and 2% BSA (Roche Diagnostics GmbH, DE) for 1 h. Dilutions
of purified bispecific Xmab antibodies in PBS were incubated
together with 0.2 .mu.g/ml huAngiopoietin-2 (R&D Systems, UK,
Cat. No. 623-AN) for 1 h at RT. After washing a mixture of 0.5
.mu.g/ml biotinylated anti-Angiopoietin-2 clone BAM0981 (R&D
Systems, UK) and 1:3000 diluted streptavidin HRP (Roche Diagnostics
GmbH, DE, Cat. No. 11089153001) was added for 1 h. Thereafter the
plates were washed 3 times with PBST. Plates are developed with
freshly prepared ABTS reagent (Roche Diagnostics GmbH, DE, buffer
#204 530 001, tablets #11 112 422 001) for 30 minutes at RT.
Absorbance was measured at 405 nm and the 1050 was determined.
XMab1 showed an inhibition of ANG-2 binding to Tie-2 with an IC50
of 12 nM.
Example 6
Inhibition of hVEGF Binding to hVEGF Receptor (ELISA)
[0415] The test was performed on 384 well microtiter plates
(MicroCoat, DE, Cat. No. 464718) at RT. After each incubation step
plates were washed 3 times with PBST. At the beginning, plates were
coated with 1 .mu.g/ml hVEGF-R protein (R&D Systems, UK, Cat.
No. 321-FL) for 1 hour (h). Thereafter the wells were blocked with
PBS supplemented with 0.2% Tween-20 and 2% BSA (Roche Diagnostics
GmbH, DE) for 1 h. Dilutions of purified bispecific XMab antibodies
in PBS were incubated together with 0.15 .mu.g/ml huVEGF121
(R&D Systems, UK, Cat. No. 298-VS) for 1 h at RT. After washing
a mixture of 0.5 .mu.g/ml anti VEGF clone Mab923 (R&D Systems,
UK) and 1:2000 horse radish peroxidase (HRP)-conjugated F(ab')2
anti mouse IgG (GE Healthcare, UK, Cat. No. NA9310V) was added for
1 h. Thereafter the plates were washed 6 times with PBST. Plates
were developed with freshly prepared ABTS reagent (Roche
Diagnostics GmbH, DE, buffer #204 530 001, tablets #11 112 422 001)
for 30 minutes at RT. Absorbance was measured at 405 nm and the
1050 was determined. XMab1 showed an inhibition of Inhibition of
VEGF binding to VEGF Receptor with an IC50 of 10 nM.
Example 7
HUVEC Proliferation
[0416] In order to confirm that the anti-VEGF related activities
are retained in the bispecific bivalent <VEGF-ANG2> antibody
XMAb1 VEGF-induced HUVEC proliferation assay was performed. It was
shown that XMAb1 interferes with VEGF-induced HUVEC proliferation
in a comparable manner as bevacizumab in the VEGF-induced HUVEC
proliferation assay as described above. XMAb1 interferes in a
concentration dependent manner with VEGF-induced HUVEC
proliferation comparable to the parental antibody bevacizumab
(Avastin). IC50 was 1.1 nM for bevacizumab and 2.3 nM for XMAb.
Example 8
Mouse Cornea Micropocket Angiogenesis Assay
[0417] 8 to 10 weeks old female Balb/c mice were purchased from
Charles River, Sulzfeld, Germany. The protocol was 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 were 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 was implanted and the surface of the implantation area was
smoothened. Discs were incubated in corresponding growth factor or
in vehicle for at least 30 min. After 3, 5 and 7 days (or
alternatively only after 3 days), eyes were photographed and
vascular response was measured. The assay was quantified by
calculating the percentage of the area of new vessels per total
area of the cornea.
[0418] The discs were 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 was monitored over time on day 3, 5 and 7. One
day prior to disc implantation the antibodies (<Ang-2/VEGF>
XMAb1, <hVEGF> Avastin (bevacizumab)) were administered
intravenously at a dose of 10 mg/kg for Avastin and XMAb1. Animals
in the control group received vehicle. The application volume was
10 ml/kg.
[0419] To test the effect of XMAb1 on VEGF-induced angiogenesis in
vivo, we performed the mouse corneal angiogenesis assay. In this
assay a VEGF soaked Nylaflo 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. Our results demonstrate that systemic administration of
the XMAb1 (10 mg/kg) almost completely inhibited the outgrowth of
the vessel from the limbus towards the VEGF gradient from study day
3 to 5 (FIG. 6). In a further experiment, direct comparison studies
were performed. The discs were loaded with 300 ng VEGF or with PBS
as a control and implanted for 3 days. The outgrowth of vessels
from the limbus to the disc was monitored over time on day 3. One
day prior to disc implantation the antibodies (bispecific
<Ang-2/VEGF> antibody XMAb1, parent <VEGF> antibody
bevacizumab (Avastin), parent <Ang-2> antibody ANG2i-LC06,
and the combination of <VEGF> antibody bevacizumab (Avastin)
and <Ang-2> antibody ANG2i-LC06) were administered
intravenously at a dose of 10 mg/kg for bevacizumab (Avastin), 10
mg/kg for XMAb1, 10 mg/kg for bevacizumab (Avastin), and 10 mg/kg
for ANG2i-LC06. The combination of bevacizumab (Avastin) and
ANG2i-LC06 was administered with 10 mg/kg for bevacizumab (Avastin)
and 10 mg/kg for ANG2i-LC06. Animals in the control group received
vehicle. The application volume was 10 ml/kg.
[0420] Our results (see FIG. 7 and Table below) demonstrate that
systemic administration of the XMAb1 (10 mg/kg) almost completely
inhibited the outgrowth of the vessel from the limbus towards the
VEGF gradient at study day 3 comparable to the combination of
bevacizumab and ANG2i-LC06. Anti-Ang-2 monotherapy in contrast only
slightly inhibited VEGF-induced angiogenesis (FIG. 7). The maximum
effect could already be achieved at lower concentrations of 10
mg/kg XMAb1 compared to the combination of 10 mg/kg of
ANG2i-LC06+10 mg/kg of bevacizumab (Avastin).
TABLE-US-00006 TABLE Percent inhibition of VEGF-induced
angiogenesis on day 3 in a Mouse cornea micropocket angiogenesis
assay % inhibition of VEGF-induced angiogenesis day 3 VEGF (300 ng)
0 ANG2i-LC06 38 (10 mg/kg) Bevacizumab (Avastin) 83 (10 mg/kg)
XMab1 (10 mg/kg) 96 Ang2i-LC06 (10 mg/kg) + 95 bevacizumab
(Avastin) (10 mg/kg)
Example 9
In Vivo Efficacy of Bispecific Antibody <VEGF-ANG-2> Antibody
in Colo205 Xenograft Model in Scid Beige Mice
Cell Lines and Culture Conditions:
[0421] Colo205 human colorectal cancer cells (ATCC No. CCL-222).
Tumor cell line were routinely cultured in RPMI 1640 medium (PAA,
Laboratories, Austria) supplemented with 10% fetal bovine serum
(PAA Laboratories, Austria) and 2 mM L-glutamine, at 37.degree. C.
in a water-saturated atmosphere at 5% CO.sub.2. Passage 2-5 is used
for transplantation.
Animals:
[0422] Female SCID beige mice; age 4-5 weeks at arrival (purchased
from Charles River Germany) were maintained under
specific-pathogen-free condition with daily cycles of 12 h light/12
h darkness according to committed guidelines (GV-Solas; Felasa;
TierschG). Experimental study protocol was reviewed and approved by
local government. After arrival animals were maintained in the
quarantine part of the animal facility for one week to get
accustomed to new environment and for observation. Continuous
health monitoring is carried out on regular basis. Diet food
(Provimi Kliba 3337) and water (acidified pH 2.5-3) were provided
ad libitum. Age of mice at start of the study is about 10
weeks.
Tumor Cell Injection:
[0423] At the day of injection, tumor cells were harvested
(trypsin-EDTA) from culture flasks (Greiner) and transferred into
50 ml culture medium, washed once and resuspended in PBS. After an
additional washing step with PBS and filtration (cell strainer;
Falcon 100 .mu.m) the final cell titer was adjusted to
2.5.times.10.sup.7/ml. Tumor cell suspension was carefully mixed
with transfer pipette to avoid cell aggregation. After this, cell
suspension was filled into a 1.0 ml tuberculin syringe (Braun
Melsungen) using a wide needle (1.10.times.40 mm); for injection
needle size is changed (0.45.times.25 mm) and for every injection a
new needle was used. Anesthesia was performed using a Stephens
inhalation unit for small animals with preincubation chamber
(plexiglas), individual mouse nose-mask (silicon) and not flammable
or explosive anesthesia compound Isoflurane (cp-pharma) in a closed
circulation system. Two days before injection coat of the animals
was shaved and for cell injection skin of anaesthetized animals was
carefully lifted up with an anatomic forceps and 100 .mu.l cell
suspension (=2.5.times.10.sup.6 cells) was injected subcutaneously
in the right flank of the animals.
Treatment of Animals
[0424] Treatment of animals started at day of randomization at a
mean tumor volume of .about.100 mm3, respectively. Mice were
treated once weekly i.p. with the different compounds as indicated
in following table.
TABLE-US-00007 No of Route/Mode of animals Compound Dose (mg/kg)
administration 10 Xolair 10 i.p. once weekly 10 <VEGF>
Avastin 10 i.p. once weekly 10 <ANG-2> Ang2i- 10 i.p. once
weekly LC06 10 Ang2i-LC06 + 10 i.p. once weekly Avastin 10 i.p.
once weekly 10 XMAb1 10 i.p. once weekly
Monitoring:
[0425] Animals were controlled 2.times. per week for their health
status. Body weights were documented 2.times. per week after cell
injection. The tumor dimensions were measured by caliper beginning
on the staging day and subsequently 2 times per week during the
whole treatment period. Tumor volume was calculated according to
NCI protocol (Tumor weight=1/2ab.sup.2, where "a" and "b" are the
long and the short diameters of the tumor, respectively).
Termination criteria were the critical tumor mass (up to 1.7 g or
O>1.5 cm), body weight loss more than 20% from baseline, tumor
ulceration or poor general condition of the animals.
[0426] The results (see (FIG. 8) show that the bispecific bivalent
<VEGF-ANG-2> antibody XMAb1 showed a higher tumor growth
inhibition in xenograft tumor model Colo205 in Scid beige mice
compared to the treatment with monospecific antibodies. The
efficacy of the combination of ANG2i-LC06 and bevacizumab showed
comparable results to the XMAb1. Maximal efficacy of XMAb1 was
already reached with 10 mg/kg.
[0427] In a second experiment the effect of XMAb1 on bigger tumors
was analyzed.
Treatment of Animals Treatment of animals started at day of
randomization at a mean tumor volume of .about.400 mm3,
respectively. Mice were treated once weekly i.p. with the different
compounds as indicated in following table.
TABLE-US-00008 No of Route/Mode of animals Compound Dose (mg/kg)
administration 10 Xolair 10 i.p. once weekly 10 <VEGF>
Avastin 10 i.p. once weekly 10 <ANG-2> Ang2i- 10 i.p. once
weekly LC06 10 Ang2i-LC06 + 10 i.p. once weekly Avastin 10 i.p.
once weekly 10 XMAb1 10 i.p. once weekly
Monitoring:
[0428] Animals were controlled 2.times. per week for their health
status. Body weights were documented 2.times. per week after cell
injection. The tumor dimensions were measured by caliper beginning
on the staging day and subsequently 2 times per week during the
whole treatment period. Tumor volume is calculated according to NCI
protocol (Tumor weight=1/2ab.sup.2, where "a" and "b" are the long
and the short diameters of the tumor, respectively). Termination
criteria were the critical tumor mass (up to 1.7 g or O>1.5 cm),
body weight loss more than 20% from baseline, tumor ulceration or
poor general condition of the animals.
[0429] The results (see FIG. 9) show that the bispecific bivalent
<VEGF-ANG-2> antibody XMAb1 showed a higher tumor growth
inhibition in xenograft tumor model Colo205 in Scid beige mice
compared to the treatment with monospecific antibodies which showed
no efficacy in big tumors compared to the control. The efficacy of
the combination of ANG2i-LC06 and bevacizumab showed comparable
results to the XMAb1. Maximal efficacy of XMAb1 was already reached
with 10 m g/kg.
[0430] Taken together the results demonstrate that independent of
the tumor size XMAb1 shows superior efficacy compared to the
treatment with monospecific antibodies.
[0431] Tumor stasis in these models could already be achieved at
lower concentrations of 10 mg/kg XMAb1 compared to the combination
of 10 mg/kg of ANG2i-LC06+10 mg/kg of Avastin.
Example 10
In Vivo Efficacy of Bispecific Antibody <VEGF-ANG-2> Antibody
in Orthotopic KPL-4 Xenograft Model in Scid Beige Mice
[0432] Tumor Cell Line
[0433] The human breast cancer cell line KPL-4 ((Kurebayashi, J.,
et al., Br. J. Cancer 79 (1999) 707-17)) has been established from
the malignant pleural effusion of a breast cancer patient with an
inflammatory skin metastasis. Tumor cells were routinely cultured
in DMEM medium (PAN Biotech, Germany) supplemented with 10% fetal
bovine serum (PAN Biotech, Germany) and 2 mM L-glutamine (PAN
Biotech, Germany) at 37.degree. C. in a water-saturated atmosphere
at 5% CO.sub.2. Culture passage was performed with trypsin/EDTA
1.times. (PAN) splitting three times/week.
Mice
[0434] After arrival, female SCID beige mice (age 10-12 weeks; body
weight 18-20 g) Charles River, Sulzfeld, Germany) were maintained
in the quarantine part of the AALAAC approved animal facility for
one week to get them accustomed to the new environment and for
observation. Continuous health monitoring was carried out. The mice
were kept under SPF-conditions according to the international
guidelines (GV-Solas; Felasa; TierschG) with daily cycles of 12 h
light/12 h darkness. Diet food (Kliba Provimi 3347) and water
(filtered) were provided ad libitum. Experimental study protocol
was reviewed and approved by the local government (Regierung von
Oberbayern; registration no. 211.2531.2-22/2003).
Tumor Cell Injection
[0435] At the day of injection tumor cells were harvested
(trypsin-EDTA) from culture flasks (Greiner TriFlask) and
transferred into 50 ml culture medium, washed once and resuspended
in PBS. After an additional washing step with PBS and filtration
(cell strainer; Falcon O 100 .mu.m) the final cell titer was
adjusted to 1.5.times.10.sup.8/ml. Tumor cell suspension was
carefully mixed with transfer pipette to avoid cell aggregation.
Anesthesia is performed using a Stephens inhalation unit for small
animals with preincubation chamber (plexiglas), individual mouse
nose-mask (silicon) and not flammable or explosive anesthesia
compound Isoflurane (Pharmacia-Upjohn, Germany) in a closed
circulation system. Two days before injection coat of the animals
were shaved. For i.m.f.p. injection cells were injected
orthotopically at a volume of 20 .mu.l into the right penultimate
inguinal mammary fat pad of each anesthetized mouse. For the
orthotopic implantation, the cell suspension was injected through
the skin under the nipple using a using a Hamilton microliter
syringe and a 30G.times.1/2'' needle.
Treatment of Animals
[0436] Treatment of animals started at day of randomization at a
mean tumor volume of .about.80 mm3, respectively. Mice were treated
once weekly i.p. with the different compounds as indicated in
following table.
TABLE-US-00009 No of Route/Mode of animals Compound Dose (mg/kg)
administration 10 Xolair 10 i.p. once weekly 10 <VEGF>
Avastin 10 i.p. once weekly 10 <ANG-2> Ang2i- 10 i.p. once
weekly LC06 10 Ang2i-LC06 + 10 i.p. once weekly Avastin 10 i.p.
once weekly 10 XMAb1 10 i.p. once weekly
Monitoring of Tumor Growth
[0437] Animals were controlled 2.times. per week for their health
status. Body weights were documented 2.times. per week after cell
injection. The tumor dimensions were measured by caliper on the
staging day, at beginning of treatment period 2 times per week.
Tumor volume was calculated according to NCI protocol (B. Teicher;
Anticancer drug development guide, Humana Press, 1997, Chapter 5,
page 92) (Tumor weight=1/2ab.sup.2, where "a" and "b" are the long
and the short diameters of the tumor, respectively).
[0438] Termination criteria were the critical tumor mass (up to 1.7
g or O>1.5 cm), body weight loss more than 20% from baseline,
tumor ulceration or poor general condition of the animals.
[0439] The results (see FIG. 10) show that the bispecific bivalent
<VEGF-ANG-2> antibody XMAb1 showed a higher tumor growth
inhibition in xenograft tumor model Colo205 in Scid beige mice
compared to the treatment with monospecific antibodies. The
efficacy of the combination of ANG2i-LC06 and bevacizumab showed
comparable results to the XMAb1. Maximal efficacy of XMAb1 was
already reached with 10 mg/kg.
[0440] In a second experiment the effect of XMAb1 on bigger tumors
was analyzed.
Treatment of Animals
[0441] Treatment of animals started at day of randomization at a
mean tumor volume of .about.160 mm3, respectively. Mice were
treated once weekly i.p. with the different compounds as indicated
in following table.
TABLE-US-00010 No of Route/Mode of animals Compound Dose (mg/kg)
administration 10 Xolair 10 i.p. once weekly 10 <VEGF>
Avastin 10 i.p. once weekly 10 <ANG-2> Ang2i- 10 i.p. once
weekly LC06 10 Ang2i-LC06 + 10 i.p. once weekly Avastin 10 i.p.
once weekly 10 XMAb1 10 i.p. once weekly
Monitoring:
[0442] Animals were controlled 2.times. per week for their health
status. Body weights were documented 2.times. per week after cell
injection. The tumor dimensions were measured by caliper on the
staging day, at beginning of treatment period 2 times per week.
Tumor volume was calculated according to NCI protocol (B. Teicher;
Anticancer drug development guide, Humana Press, 1997, Chapter 5,
page 92) (Tumor weight=1/2ab.sup.2, where "a" and "b" are the long
and the short diameters of the tumor, respectively).
[0443] Termination criteria were the critical tumor mass (up to 1.7
g or O>1.5 cm), body weight loss more than 20% from baseline,
tumor ulceration or poor general condition of the animals.
[0444] The results (see FIG. 11) show that the bispecific bivalent
<VEGF-ANG-2> antibody XMAb1 showed a higher tumor growth
inhibition in xenograft tumor model Colo205 in Scid beige mice
compared to the treatment with monospecific antibodies. The
efficacy of the combination of ANG2i-LC06 and bevacizumab showed
comparable results to the XMAb1. Maximal efficacy of XMAb1 was
already reached with 10 mg/kg.
[0445] Taken together the results demonstrate that independent of
the tumor size XMAb1 shows superior efficacy compared to the
treatment with monospecific antibodies.
[0446] Tumor stasis in these models could be already achieved at
lower concentrations of 10 mg/kg XMAb1 compared to the combination
of 10 mg/kg of Ang2i-LC06+10 mg/kg of Avastin.
Example 11
Effect of Treatment with XMAb1 on Micro-Vessel Density in s.c.
Colo205 Xenograft
[0447] Vascular density is assessed by counting all vessels of a
tumor slide. Vessels were labeled with fluorescent anti-mouse CD34
antibody (clone MEC14.7) on paraffin-embedded sections. Vessels
were quantified and microvessel density is calculated as vessels
per mm.sup.2. All results were expressed as mean.+-.SEM. To define
significant differences of experimental groups, Dunnetts-Method was
used. p<0.05 was considered as statistically significant. The
results show that total intratumoral MVD was decreased in treated
tumors. Treatment with ANG2i-LC06 reduced MVD by 29%, bevacizumab
by </=0%, bevacizumab+ANG2i-LC06 by 15% and XMAb1 by 28%.
Example 12
In Vivo Efficacy of Bispecific Antibody <VEGF-ANG-2> Antibody
in s.c. N87 Xenograft Model in Scid Beige Mice
[0448] Tumor Cell Line
[0449] The human gastric cancer cell line N87 cancer cells (NCI-N87
(ATCC No. CRL 5822)). Tumor cells were routinely cultured in
RPMI1640 supplemented with 10% fetal bovine serum (PAN Biotech,
Germany) and 2 mM L-glutamine (PAN Biotech, Germany) at 37.degree.
C. in a water-saturated atmosphere at 5% CO.sub.2. Culture passage
was performed with trypsin/EDTA 1.times. (PAN) splitting three
times/week.
Mice
[0450] After arrival, female SCID beige mice (age 10-12 weeks; body
weight 18-20 g) Charles River, Sulzfeld, Germany) were maintained
in the quarantine part of the AALAAC approved animal facility for
one week to get them accustomed to the new environment and for
observation. Continuous health monitoring was carried out. The mice
were kept under SPF-conditions according to the international
guidelines (GV-Solas; Felasa; TierschG) with daily cycles of 12 h
light/12 h darkness. Diet food (Kliba Provimi 3347) and water
(filtered) were provided ad libitum. Experimental study protocol
was reviewed and approved by the local government (Regierung von
Oberbayern; registration no. 211.2531.2-22/2003).
Tumor Cell Injection
[0451] At the day of cell injection, cells were harvested from
culture flasks (Greiner T 75), transferred into 50 ml culture
medium, washed once and resuspended in PBS. After an additional
washing with PBS the cell concentration was measured with a
Vi-Cell.TM. (Cell Viability Analyzer, Beckman Coulter, Madison,
Wis., U.S.A.). The tumor cell suspension (PBS) was mixed carefully
(to reduce cell aggregation) and kept on ice. The cell suspension
was filled into a 1.0 ml syringe. For injection, a needle size of
0.45.times.25 mm was used. To generate primary tumors,
5.times.10.sup.6 N87 tumor cells in a volume of 100 .mu.l PBS were
injected subcutaneously into the right flank of each mouse.
Treatment of Animals
[0452] Treatment of animals started at day of randomization at a
mean tumor volume of .about.130 mm3, respectively. Mice are treated
once weekly i.p. with the different compounds as indicated in
following table.
TABLE-US-00011 No of Route/Mode of animals Compound Dose (mg/kg)
administration 10 Xolair 10 i.p. once weekly 10 <VEGF>
Avastin 10 i.p. once weekly 10 <ANG-2> Ang2i- 10 i.p. once
weekly LC06 10 XMAb1 10 i.p. once weekly
Monitoring of Tumor Growth
[0453] Animals were controlled 1.times. per week for their health
status. Body weights were documented 1.times. per week after cell
injection. The tumor dimensions were measured by caliper on the
staging day, at beginning of treatment period once per week. Tumor
volume was calculated according to NCI protocol (B. Teicher;
Anticancer drug development guide, Humana Press, 1997, Chapter 5,
page 92) (Tumor weight =1/2ab.sup.2, where "a" and are the long and
the short diameters of the tumor, respectively).
[0454] Termination criteria were the critical tumor mass (up to 1.7
g or O>1.5 cm), body weight loss more than 20% from baseline,
tumor ulceration or poor general condition of the animals.
[0455] The results show that the bispecific bivalent
<VEGF-ANG-2> antibody XMAb1 showed a higher tumor growth
inhibition in xenograft tumor model Colo205 in Scid beige mice
compared to the treatment with monospecific antibodies (FIG. 12).
Sequence CWU 1
1
491123PRTArtificialvariable heavy chain domain VH of <VEGF>
bevacizumab 1Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr
Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr
Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr
Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val 100 105
110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
2107PRTArtificialvariable light chain domain VL of <VEGF>
bevacizumab 2Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln
Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Val Leu Ile 35 40 45 Tyr Phe Thr Ser Ser Leu His
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp 85 90 95 Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
3128PRTArtificialvariable heavy chain domain VH of <ANG-2>
E6Q 3Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly
Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr
Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg
Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser
Pro Asn Pro Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr Tyr 100 105 110 Pro
Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser 115 120
125 4108PRTArtificialvariable light chain domain VL of <
ANG-2> E6Q 4Gln Pro Gly Leu Thr Gln Pro Pro Ser Val Ser Val Ala
Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile
Gly Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser
Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr
Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala
Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 Tyr
Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu 100 105
5214PRTArtificialXMab1 -<VEGF> light chain 5Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35
40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Ser Thr Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165
170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
6213PRTArtificialXMab1 -<ANG2> light chain 6Gln Pro Gly Leu
Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala
Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val 20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35
40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly
Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val
Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp
Ser Ser Ser Asp His 85 90 95 Tyr Val Phe Gly Thr Gly Thr Lys Val
Thr Val Leu Ser Ser Ala Ser 100 105 110 Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr 115 120 125 Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 130 135 140 Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 145 150 155 160
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 165
170 175 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile 180 185 190 Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val 195 200 205 Glu Pro Lys Ser Cys 210
7453PRTArtificialXMab1 -<VEGF> heavy chain 7Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp
Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser
Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro His Tyr Tyr Gly Ser
Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165
170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290
295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Cys
Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Trp Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410
415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 8463PRTArtificialXMab1
-<ANG2> heavy chain 8Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn
Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly
Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr
Tyr 100 105 110 Pro Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val
Thr Val Ser 115 120 125 Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210
215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Asp Lys Thr
His 225 230 235 240 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val 245 250 255 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr 260 265 270 Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu 275 280 285 Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys 290 295 300 Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 305 310 315 320 Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 325 330
335 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
340 345 350 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr
Leu Pro 355 360 365 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
Leu Ser Cys Ala 370 375 380 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn 385 390 395 400 Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser 405 410 415 Asp Gly Ser Phe Phe
Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg 420 425 430 Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 435 440 445 His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 460
9214PRTArtificialXMab2 -<VEGF> light chain 9Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35
40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Ser Thr Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165
170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
10212PRTArtificialXMab2 -<VEGF> heavy chain 10Gln Pro Gly Leu
Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala
Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val 20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35
40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly
Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val
Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp
Ser Ser Ser Asp His 85 90 95 Tyr Val Phe Gly Thr Gly Thr Lys Val
Thr Val Leu Gly Ala Ser Thr 100 105 110 Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser 115 120 125 Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 130 135 140 Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 145 150 155 160
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 165
170 175 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys 180 185 190 Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu 195 200 205 Pro Lys Ser Cys 210
11453PRTArtificialXMab2 -<VEGF> heavy chain 11Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp
Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser
Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro His Tyr Tyr Gly Ser
Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165
170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290
295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Cys
Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Trp Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410
415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 12461PRTArtificialXMab2
-<ANG2> heavy chain 12Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn
Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly
Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr
Tyr 100 105 110 Pro Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val
Thr Val Ser 115 120 125 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu
Phe Pro Pro Ser Ser 130 135 140 Glu Glu Leu Gln Ala Asn Lys Ala Thr
Leu Val Cys Leu Ile Ser Asp 145 150 155 160 Phe Tyr Pro Gly Ala Val
Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 165 170 175 Val Lys Ala Gly
Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 180 185 190 Lys Tyr
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 195 200 205
Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 210
215 220 Glu Lys Thr Val Ala Pro Thr Glu Cys Ser Asp Lys Thr His Thr
Cys 225 230 235 240 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu 245 250 255 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu 260 265 270 Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys 275 280 285 Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys 290 295 300 Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 305 310 315 320 Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 325 330
335 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
340 345 350 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro
Pro Ser 355 360 365 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser
Cys Ala Val Lys 370 375 380 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln 385 390 395 400 Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly 405 410 415 Ser Phe Phe Leu Val
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 420 425 430 Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 435 440 445 His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 460
13214PRTArtificialXMab3 -<VEGF> light chain 13Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35
40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Ser Thr Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165
170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
14213PRTArtificialXMab3-<ANG2> light chain 14Gln Pro Gly Leu
Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala
Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val 20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35
40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly
Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val
Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp
Ser Ser Ser Asp His 85 90 95 Tyr Val Phe Gly Thr Gly Thr Lys Val
Thr Val Leu Ser Ser Ala Ser 100 105 110 Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr 115 120 125 Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 130 135 140 Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 145 150 155 160
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 165
170 175 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile 180 185 190 Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val 195 200 205 Glu Pro Lys Ser Cys 210
15453PRTArtificialXMab3 -<VEGF> heavy chain 15Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp
Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser
Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro His Tyr Tyr Gly Ser
Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165
170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290
295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Cys Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Ser Cys Ala
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu 405 410
415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 16463PRTArtificialXMab3
-<ANG2> heavy chain 16Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn
Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly
Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr
Tyr 100 105 110 Pro Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val
Thr Val Ser 115 120 125 Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210
215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Asp Lys Thr
His 225 230 235 240 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val 245 250 255 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr 260 265 270 Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu 275 280 285 Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys 290 295 300 Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 305 310 315 320 Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 325 330
335 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
340 345 350 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro 355 360 365 Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
Leu Trp Cys Leu 370 375 380 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn 385 390 395 400 Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser 405 410 415 Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 420 425 430 Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 435 440 445 His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 450 455 460 17214PRTArtificialXMab4 -<VEGF> light
chain 17Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile
Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Val Leu Ile 35 40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp 85 90 95 Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu
Cys 210 18212PRTArtificialXMab4-<ANG2> light chain 18Gln Pro
Gly Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15
Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val 20
25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val
Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe
Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser
Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val
Trp Asp Ser Ser Ser Asp His 85 90 95 Tyr Val Phe Gly Thr Gly Thr
Lys Val Thr Val Leu Gly Ala Ser Thr 100 105 110 Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 115 120 125 Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 130 135 140 Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 145 150
155 160 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser 165 170 175 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys 180 185 190 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu 195 200 205 Pro Lys Ser Cys 210
19453PRTArtificialXMab4 -<VEGF> heavy chain 19Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp
Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser
Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro His Tyr Tyr Gly Ser
Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165
170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290
295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Cys Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Ser Cys Ala
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu 405 410
415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 20461PRTArtificialXMab4
-<ANG2> heavy chain 20Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn
Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly
Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr
Tyr 100 105 110 Pro Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val
Thr Val Ser 115 120 125 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu
Phe Pro Pro Ser Ser 130 135 140 Glu Glu Leu Gln Ala Asn Lys Ala Thr
Leu Val Cys Leu Ile Ser Asp 145 150 155 160 Phe Tyr Pro Gly Ala Val
Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 165 170 175 Val Lys Ala Gly
Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 180 185 190 Lys Tyr
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 195 200 205
Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 210
215 220 Glu Lys Thr Val Ala Pro Thr Glu Cys Ser Asp Lys Thr His Thr
Cys 225 230 235 240 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu 245 250 255 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu 260 265 270 Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys 275 280 285 Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys 290 295 300 Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 305 310 315 320 Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 325 330
335 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
340 345 350 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Cys 355 360 365 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp
Cys Leu Val Lys 370 375 380 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln 385 390 395 400 Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly 405 410 415 Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 420 425 430 Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 435 440 445 His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 460
21214PRTArtificialXMab5 -<VEGF> light chain 21Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35
40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Ser Thr Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165
170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
22212PRTArtificialXMab5 -<ANG2> light chain 22Gln Pro Gly Leu
Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala
Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val 20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35
40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly
Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val
Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp
Ser Ser Ser Asp His 85 90 95 Tyr Val Phe Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr 100 105 110 Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser 115 120 125 Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 130 135 140 Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 145 150 155 160
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 165
170 175 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys 180 185 190 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu 195 200 205 Pro Lys Ser Cys 210 23453PRTArtificialXMab5
-<VEGF> heavy chain 23Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn
Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg
Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp
Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210
215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330
335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
340 345 350 Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys
Asn Gln 355 360 365 Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu
Ser Pro Gly Lys 450 24461PRTArtificialXMab5 -<ANG2> heavy
chain 24Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn
Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val
Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu
Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr Tyr 100
105 110 Pro Gly Ala Phe Asp Ile Trp Gly Gly Gly Thr Lys Val Thr Val
Leu 115 120 125 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro
Pro Ser Ser 130 135 140 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val
Cys Leu Ile Ser Asp 145 150 155 160 Phe Tyr Pro Gly Ala Val Thr Val
Ala Trp Lys Ala Asp Ser Ser Pro 165 170 175 Val Lys Ala Gly Val Glu
Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 180 185 190 Lys Tyr Ala Ala
Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 195 200 205 Ser His
Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 210 215 220
Glu Lys Thr Val Ala Pro Thr Glu Cys Ser Asp Lys Thr His Thr Cys 225
230 235 240 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
Phe Leu 245 250 255 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu 260 265 270 Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys 275 280 285 Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys 290 295 300 Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu 305 310 315 320 Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 325 330 335 Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 340 345
350 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser
355 360 365 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala
Val Lys 370 375 380 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln 385 390 395 400 Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly 405 410 415 Ser Phe Phe Leu Val Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln 420 425 430 Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn 435 440 445 His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 460
25214PRTArtificialXMab6 -<VEGF> light chain 25Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35
40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Ser Thr Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165
170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
26212PRTArtificialXMab6 -<ANG2> light chain 26Gln Pro Gly Leu
Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala
Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val 20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35
40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly
Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val
Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp
Ser Ser Ser Asp His 85 90 95 Tyr Val Phe Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr 100 105 110 Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser 115 120 125 Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 130 135 140 Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 145 150 155 160
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 165
170 175 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys 180 185 190 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu 195 200 205 Pro Lys Ser Cys 210 27453PRTArtificialXMab6
-<VEGF> heavy chain 27Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn
Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg
Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp
Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210
215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330
335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
340 345 350 Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln 355 360 365 Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Val Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu
Ser Pro Gly Lys 450 28461PRTArtificialXMab6 -<ANG2> heavy
chain 28Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly
Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr
Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg
Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser
Pro Asn Pro Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr Tyr 100 105 110 Pro
Gly Ala Phe Asp Ile Trp Gly Gly Gly Thr Lys Val Thr Val Leu 115 120
125 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser
130 135 140 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile
Ser Asp 145 150 155 160 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys
Ala Asp Ser Ser Pro 165 170 175 Val Lys Ala Gly Val Glu Thr Thr Thr
Pro Ser Lys Gln Ser Asn Asn 180 185 190 Lys Tyr Ala Ala Ser Ser Tyr
Leu Ser Leu Thr Pro Glu Gln Trp Lys 195 200 205 Ser His Arg Ser Tyr
Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 210 215 220 Glu Lys Thr
Val Ala Pro Thr Glu Cys Ser Asp Lys Thr His Thr Cys 225 230 235 240
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 245
250 255 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu 260 265 270 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys 275 280 285 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys 290 295 300 Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu 305 310 315 320 Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 325 330 335 Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 340 345 350 Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys 355 360 365
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys 370
375 380 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln 385 390 395 400 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly 405 410 415 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln 420 425 430 Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn 435 440 445 His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 450 455 460 29706PRTArtificialOAscFab1
-<ANG2> peptide connected heavy chain and light chain 29Gln
Pro Gly Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10
15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val
20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val
Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg
Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile
Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln
Val Trp Asp Ser Ser Ser Asp His 85 90 95 Tyr Val Phe Gly Thr Gly
Thr Lys Val Thr Val Leu Arg Thr Val Ala 100 105 110 Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145
150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys Gly
Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 225 230 235 240 Gly Gly Gly
Gly Ser Gly Gly Gln Val Gln Leu Val Gln Ser Gly Ala 245 250 255 Glu
Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser 260 265
270 Gly Tyr Thr Phe Thr Gly Tyr Tyr Met His Trp Val Arg Gln Ala Pro
275 280 285 Gly Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Pro Asn Ser
Gly Gly 290 295 300 Thr Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr
Met Thr Arg Asp 305 310 315 320 Thr Ser Ile Ser Thr Ala Tyr Met Glu
Leu Ser Arg Leu Arg Ser Asp 325 330 335 Asp Thr Ala Val Tyr Tyr Cys
Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr 340 345 350 Asp Ser Ser Gly Tyr
Tyr Tyr Pro Gly Ala Phe Asp Ile Trp Gly Gln 355 360 365 Gly Thr Met
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 370 375 380 Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 385 390
395 400 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser 405 410 415 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val 420 425 430 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro 435 440 445 Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys 450 455 460 Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys Ser Cys Asp 465 470 475 480 Lys Thr His Thr
Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly 485 490 495 Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile 500 505 510 Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu 515 520 525 Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 530 535 540 Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 545 550 555
560 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
565 570 575 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu 580 585 590 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr 595 600 605 Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser Leu 610 615 620 Trp Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp 625 630 635 640 Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 645 650 655 Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 660 665 670 Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 675 680
685 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
690 695 700 Gly Lys 705 30453PRTArtificialOAscFab1 -<VEGF>
heavy chain 30Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr
Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr
Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr
Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val 100 105
110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230
235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350
Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355
360 365 Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Val Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly
Lys 450 31214PRTArtificialOAscFab1 -<VEGF> light chain 31Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val
Leu Ile 35 40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Ser Thr Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145
150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
32705PRTArtificialOAscFab2 -<ANG2> peptide connected heavy
chain and light chain 32Gln Pro Gly Leu Thr Gln Pro Pro Ser Val Ser
Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn
Asn Ile Gly Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg
Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly
Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp
Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90
95 Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln Pro Lys
100 105 110 Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu
Leu Gln 115 120 125 Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp
Phe Tyr Pro Gly 130 135 140 Ala Val Thr Val Ala Trp Lys Ala Asp Ser
Ser Pro Val Lys Ala Gly 145 150 155 160 Val Glu Thr Thr Thr Pro Ser
Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175 Ser Ser Tyr Leu Ser
Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190 Tyr Ser Cys
Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205 Ala
Pro Thr Glu Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 210 215
220 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
225 230 235 240 Gly Gly Gly Ser Gly Gly Gln Val Gln Leu Val Gln Ser
Gly Ala Glu 245 250 255 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser
Cys Lys Ala Ser Gly 260 265 270 Tyr Thr Phe Thr Gly Tyr Tyr Met His
Trp Val Arg Gln Ala Pro Gly 275 280 285 Gln Gly Leu Glu Trp Met Gly
Trp Ile Asn Pro Asn Ser Gly Gly Thr 290 295 300 Asn Tyr Ala Gln Lys
Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr 305 310 315 320 Ser Ile
Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp 325 330 335
Thr Ala Val Tyr Tyr Cys Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp 340
345 350 Ser Ser Gly Tyr Tyr Tyr Pro Gly Ala Phe Asp Ile Trp Gly Gln
Gly 355 360 365 Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe 370 375 380 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala Leu 385 390 395 400 Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp 405 410 415 Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu 420 425 430 Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 435 440 445 Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 450 455 460
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 465
470 475 480 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro 485 490 495 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser 500 505 510 Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp 515 520 525 Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn 530 535 540 Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 545 550 555 560 Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 565 570 575 Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 580 585
590 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
595 600 605 Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
Leu Trp 610 615 620 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu 625 630 635 640 Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu 645 650 655 Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys 660 665 670 Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu 675 680 685 Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 690 695 700 Lys
705 33453PRTArtificialOAscFab2 -<VEGF> heavy chain 33Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20
25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala
Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser
Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro His Tyr Tyr
Gly Ser Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150
155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275
280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Cys Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Ser
Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395
400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu
405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450
34214PRTArtificialOAscFab2 -<VEGF> light chain 34Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25
30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile
35 40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Tyr Ser Thr Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155
160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
35705PRTArtificialOAscFab3 -<ANG2> peptide connected heavy
chain and light chain 35Gln Pro Gly Leu Thr Gln Pro Pro Ser Val Ser
Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn
Asn Ile Gly Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg
Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly
Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp
Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90
95 Tyr Val Phe Gly Cys Gly Thr Lys Val Thr Val Leu Gly Gln Pro Lys
100 105 110 Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu
Leu Gln 115 120 125 Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp
Phe Tyr Pro Gly 130 135 140 Ala Val Thr Val Ala Trp Lys Ala Asp Ser
Ser Pro Val Lys Ala Gly 145 150 155 160 Val Glu Thr Thr Thr Pro Ser
Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175 Ser Ser Tyr Leu Ser
Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190 Tyr Ser Cys
Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205 Ala
Pro Thr Glu Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 210 215
220 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
225 230 235 240 Gly Gly Gly Ser Gly Gly Gln Val Gln Leu Val Gln Ser
Gly Ala Glu 245 250 255 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser
Cys Lys Ala Ser Gly 260 265 270 Tyr Thr Phe Thr Gly Tyr Tyr Met His
Trp Val Arg Gln Ala Pro Gly 275 280 285 Gln Cys Leu Glu Trp Met Gly
Trp Ile Asn Pro Asn Ser Gly Gly Thr 290 295 300 Asn Tyr Ala Gln Lys
Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr 305 310 315 320 Ser Ile
Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp 325 330 335
Thr Ala Val Tyr Tyr Cys Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp 340
345 350 Ser Ser Gly Tyr Tyr Tyr Pro Gly Ala Phe Asp Ile Trp Gly Gln
Gly 355 360 365 Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe 370 375 380 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala Leu 385 390 395 400 Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp 405 410 415 Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu 420 425 430 Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 435 440 445 Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 450 455 460
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 465
470 475 480 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro 485 490 495 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser 500 505 510 Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp 515 520 525 Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn 530 535 540 Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 545 550 555 560 Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 565 570 575 Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 580 585
590 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
595 600 605 Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
Leu Trp 610 615 620 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu 625 630 635 640 Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu 645 650 655 Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys 660 665 670 Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu 675 680 685 Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 690 695 700 Lys
705 36453PRTArtificialOAscFab3 -<VEGF> heavy chain 36Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20
25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala
Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser
Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro His Tyr Tyr
Gly Ser Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150
155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275
280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Cys Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Ser
Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395
400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu
405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450
37214PRTArtificialOAscFab3 -<VEGF> light chain 37Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25
30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile
35 40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Tyr Ser Thr Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155
160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
38708PRTArtificialOAscXFab1 -<ANG2> peptide connected heavy
chain and light chain 38Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro
Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg
Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr Tyr
100 105 110 Pro Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr
Val Ser 115 120 125 Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215
220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly
225 230 235 240 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser 245 250 255 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gln Pro Gly Leu 260 265 270 Thr Gln Pro Pro Ser Val Ser Val Ala
Pro Gly Gln Thr Ala Arg Ile 275 280 285 Thr Cys Gly Gly Asn Asn Ile
Gly Ser Lys Ser Val His Trp Tyr Gln 290 295 300 Gln Lys Pro Gly Gln
Ala Pro Val Leu Val Val Tyr Asp Asp Ser Asp 305 310 315 320 Arg Pro
Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn 325 330 335
Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala Asp 340
345 350 Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His Tyr Val Phe
Gly 355 360 365 Thr Gly Thr Lys Val Thr Val Leu Ser Ser Ala Ser Thr
Lys Gly Pro 370 375 380 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr 385 390 395 400 Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr 405 410 415 Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 420 425 430 Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 435 440 445 Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 450 455 460
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 465
470 475 480 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu 485 490 495 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu 500 505 510 Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser 515 520 525 His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu 530 535 540 Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 545 550 555 560 Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 565 570 575 Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 580 585
590 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
595 600 605 Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn
Gln Val 610 615 620 Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val 625 630 635 640 Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro 645 650 655 Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr 660 665 670 Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 675 680 685 Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 690 695 700 Ser
Pro Gly Lys 705 39453PRTArtificialOAscXFab1 -<VEGF> heavy
chain 39Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe
Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu
Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser
Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr
Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val 100 105 110 Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120
125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe 165 170 175 Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195
200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315
320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro 340 345 350 Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln 355 360 365 Val Ser Leu Ser Cys Ala Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu 405 410 415 Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440
445 Leu Ser Pro Gly Lys 450 40214PRTArtificialOAscXFab1
-<VEGF> light chain 40Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser
Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45 Tyr Phe Thr Ser
Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp 85
90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205
Phe Asn Arg Gly Glu Cys 210 41708PRTArtificialOAscXFab2
-<ANG2> peptide connected heavy chain and light chain 41Gln
Pro Gly Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10
15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val
20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val
Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg
Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile
Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln
Val Trp Asp Ser Ser Ser Asp His 85 90 95 Tyr Val Phe Gly Thr Gly
Thr Lys Val Thr Val Leu Ser Ser Ala Ser 100 105 110 Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 115 120 125 Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 130 135 140
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 145
150 155 160 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser 165 170 175 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile 180 185 190 Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val 195 200 205 Glu Pro Lys Ser Cys Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly 210 215 220 Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 225 230 235 240 Gly Gly Ser
Gly Gly Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 245 250 255 Lys
Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 260 265
270 Thr Phe Thr Gly Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln
275 280 285 Gly Leu Glu Trp Met Gly Trp Ile Asn Pro Asn Ser Gly Gly
Thr Asn 290 295 300 Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr
Arg Asp Thr Ser 305 310 315 320 Ile Ser Thr Ala Tyr Met Glu Leu Ser
Arg Leu Arg Ser Asp Asp Thr 325 330 335 Ala Val Tyr Tyr Cys Ala Arg
Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser 340 345 350 Ser Gly Tyr Tyr Tyr
Pro Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr 355 360 365 Met Val Thr
Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile 370 375 380 Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val 385 390
395 400 Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp
Lys 405 410 415 Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser
Val Thr Glu 420 425 430 Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu 435 440 445 Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr Ala Cys Glu Val Thr 450 455 460 His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu 465 470 475 480 Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 485 490 495 Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 500 505 510
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 515
520 525 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu 530 535 540 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr 545 550 555 560 Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn 565 570 575 Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro 580 585 590 Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 595 600 605 Val Tyr Thr Leu
Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val 610 615 620 Ser Leu
Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 625 630 635
640 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
645 650 655 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr 660 665 670 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val 675 680 685 Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu 690 695 700 Ser Pro Gly Lys 705
42453PRTArtificialOAscXFab2 -<VEGF> heavy chain 42Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25
30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala
Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys
Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro His Tyr Tyr Gly
Ser Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155
160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280
285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Cys Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Ser Cys
Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu 405
410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450
43214PRTArtificialOAscXFab2 -<VEGF> light chain 43Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25
30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile
35 40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Tyr Ser Thr Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155
160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
44705PRTArtificialOAscXFab3 -<ANG2> peptide connected heavy
chain and light chain 44Gln Pro Gly Leu Thr Gln Pro Pro Ser Val Ser
Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn
Asn Ile Gly Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg
Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly
Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp
Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90
95 Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Ala Ser Thr
100 105 110 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser 115 120 125 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu 130 135 140 Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His 145 150 155 160 Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser 165 170 175 Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 180 185 190 Asn Val Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 195 200 205 Pro
Lys Ser Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 210 215
220 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240 Gly Ser Gly Gly Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys 245 250 255 Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr 260 265 270 Phe Thr Gly Tyr Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly 275 280 285 Leu Glu Trp Met Gly Trp Ile
Asn Pro Asn Ser Gly Gly Thr Asn Tyr 290 295 300 Ala Gln Lys Phe Gln
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile 305 310 315 320 Ser Thr
Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala 325 330 335
Val Tyr Tyr Cys Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser Ser 340
345 350 Gly Tyr Tyr Tyr Pro Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr
Met 355 360 365 Val Thr Val Ser Gly Gln Pro Lys Ala Ala Pro Ser Val
Thr Leu Phe 370 375 380 Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn
Lys
Ala Thr Leu Val Cys 385 390 395 400 Leu Ile Ser Asp Phe Tyr Pro Gly
Ala Val Thr Val Ala Trp Lys Ala 405 410 415 Asp Ser Ser Pro Val Lys
Ala Gly Val Glu Thr Thr Thr Pro Ser Lys 420 425 430 Gln Ser Asn Asn
Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro 435 440 445 Glu Gln
Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu 450 455 460
Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser Asp Lys 465
470 475 480 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro 485 490 495 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser 500 505 510 Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp 515 520 525 Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn 530 535 540 Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 545 550 555 560 Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 565 570 575 Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 580 585
590 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
595 600 605 Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
Leu Trp 610 615 620 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu 625 630 635 640 Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu 645 650 655 Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys 660 665 670 Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu 675 680 685 Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 690 695 700 Lys
705 45453PRTArtificialOAscXFab3 -<VEGF> heavy chain 45Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20
25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala
Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser
Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro His Tyr Tyr
Gly Ser Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150
155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275
280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Cys Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Ser
Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395
400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu
405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450
46214PRTArtificialOAscXFab3 -<VEGF> light chain 46Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25
30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile
35 40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Tyr Ser Thr Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155
160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
47191PRTHomo sapiens 47Met Asn Phe Leu Leu Ser Trp Val His Trp Ser
Leu Ala Leu Leu Leu 1 5 10 15 Tyr Leu His His Ala Lys Trp Ser Gln
Ala Ala Pro Met Ala Glu Gly 20 25 30 Gly Gly Gln Asn His His Glu
Val Val Lys Phe Met Asp Val Tyr Gln 35 40 45 Arg Ser Tyr Cys His
Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu 50 55 60 Tyr Pro Asp
Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu 65 70 75 80 Met
Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro 85 90
95 Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His
100 105 110 Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn
Lys Cys 115 120 125 Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu
Asn Pro Cys Gly 130 135 140 Pro Cys Ser Glu Arg Arg Lys His Leu Phe
Val Gln Asp Pro Gln Thr 145 150 155 160 Cys Lys Cys Ser Cys Lys Asn
Thr Asp Ser Arg Cys Lys Ala Arg Gln 165 170 175 Leu Glu Leu Asn Glu
Arg Thr Cys Arg Cys Asp Lys Pro Arg Arg 180 185 190
48504PRTArtificialHuman angiopoietin-2 (ANG-2) with leader and
His-tag 48Met Trp Gln Ile Val Phe Phe Thr Leu Ser Cys Asp Leu Val
Leu Ala 1 5 10 15 Ala Ala Tyr Asn Asn Phe Arg Lys Ser Met Asp Ser
Ile Gly Lys Lys 20 25 30 Gln Tyr Gln Val Gln His Gly Ser Cys Ser
Tyr Thr Phe Leu Leu Pro 35 40 45 Glu Met Asp Asn Cys Arg Ser Ser
Ser Ser Pro Tyr Val Ser Asn Ala 50 55 60 Val Gln Arg Asp Ala Pro
Leu Glu Tyr Asp Asp Ser Val Gln Arg Leu 65 70 75 80 Gln Val Leu Glu
Asn Ile Met Glu Asn Asn Thr Gln Trp Leu Met Lys 85 90 95 Leu Glu
Asn Tyr Ile Gln Asp Asn Met Lys Lys Glu Met Val Glu Ile 100 105 110
Gln Gln Asn Ala Val Gln Asn Gln Thr Ala Val Met Ile Glu Ile Gly 115
120 125 Thr Asn Leu Leu Asn Gln Thr Ala Glu Gln Thr Arg Lys Leu Thr
Asp 130 135 140 Val Glu Ala Gln Val Leu Asn Gln Thr Thr Arg Leu Glu
Leu Gln Leu 145 150 155 160 Leu Glu His Ser Leu Ser Thr Asn Lys Leu
Glu Lys Gln Ile Leu Asp 165 170 175 Gln Thr Ser Glu Ile Asn Lys Leu
Gln Asp Lys Asn Ser Phe Leu Glu 180 185 190 Lys Lys Val Leu Ala Met
Glu Asp Lys His Ile Ile Gln Leu Gln Ser 195 200 205 Ile Lys Glu Glu
Lys Asp Gln Leu Gln Val Leu Val Ser Lys Gln Asn 210 215 220 Ser Ile
Ile Glu Glu Leu Glu Lys Lys Ile Val Thr Ala Thr Val Asn 225 230 235
240 Asn Ser Val Leu Gln Lys Gln Gln His Asp Leu Met Glu Thr Val Asn
245 250 255 Asn Leu Leu Thr Met Met Ser Thr Ser Asn Ser Ala Lys Asp
Pro Thr 260 265 270 Val Ala Lys Glu Glu Gln Ile Ser Phe Arg Asp Cys
Ala Glu Val Phe 275 280 285 Lys Ser Gly His Thr Thr Asn Gly Ile Tyr
Thr Leu Thr Phe Pro Asn 290 295 300 Ser Thr Glu Glu Ile Lys Ala Tyr
Cys Asp Met Glu Ala Gly Gly Gly 305 310 315 320 Gly Trp Thr Ile Ile
Gln Arg Arg Glu Asp Gly Ser Val Asp Phe Gln 325 330 335 Arg Thr Trp
Lys Glu Tyr Lys Val Gly Phe Gly Asn Pro Ser Gly Glu 340 345 350 Tyr
Trp Leu Gly Asn Glu Phe Val Ser Gln Leu Thr Asn Gln Gln Arg 355 360
365 Tyr Val Leu Lys Ile His Leu Lys Asp Trp Glu Gly Asn Glu Ala Tyr
370 375 380 Ser Leu Tyr Glu His Phe Tyr Leu Ser Ser Glu Glu Leu Asn
Tyr Arg 385 390 395 400 Ile His Leu Lys Gly Leu Thr Gly Thr Ala Gly
Lys Ile Ser Ser Ile 405 410 415 Ser Gln Pro Gly Asn Asp Phe Ser Thr
Lys Asp Gly Asp Asn Asp Lys 420 425 430 Cys Ile Cys Lys Cys Ser Gln
Met Leu Thr Gly Gly Trp Trp Phe Asp 435 440 445 Ala Cys Gly Pro Ser
Asn Leu Asn Gly Met Tyr Tyr Pro Gln Arg Gln 450 455 460 Asn Thr Asn
Lys Phe Asn Gly Ile Lys Trp Tyr Tyr Trp Lys Gly Ser 465 470 475 480
Gly Tyr Ser Leu Lys Ala Thr Thr Met Met Ile Arg Pro Ala Asp Phe 485
490 495 Ser Gly His His His His His His 500 4919PRTArtificialleader
peptide 49Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala
Thr Gly 1 5 10 15 Val His Ser
* * * * *