U.S. patent application number 15/907836 was filed with the patent office on 2018-10-04 for antibodies against human angiopoietin 2.
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 Ulrich Brinkmann, Remko Albert Griep, Klaus Kaluza, Anita Kavlie, Christian Klein, Joerg Thomas Regula, Werner Scheuer.
Application Number | 20180282404 15/907836 |
Document ID | / |
Family ID | 40585738 |
Filed Date | 2018-10-04 |
United States Patent
Application |
20180282404 |
Kind Code |
A1 |
Brinkmann; Ulrich ; et
al. |
October 4, 2018 |
ANTIBODIES AGAINST HUMAN ANGIOPOIETIN 2
Abstract
The present invention relates to antibodies against human
Angiopoietin 2 (anti-ANG-2 antibodies), methods for their
production, pharmaceutical compositions containing said antibodies,
and uses thereof.
Inventors: |
Brinkmann; Ulrich;
(Weilheim, DE) ; Griep; Remko Albert; (Slemmestad,
NO) ; Kaluza; Klaus; (Bad Heilbrunn, DE) ;
Kavlie; Anita; (Oslo, NO) ; Klein; Christian;
(Iffeldorf, DE) ; Regula; Joerg Thomas; (Muenchen,
DE) ; Scheuer; Werner; (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: |
40585738 |
Appl. No.: |
15/907836 |
Filed: |
February 28, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15097097 |
Apr 12, 2016 |
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15907836 |
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13693085 |
Dec 4, 2012 |
9340609 |
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15097097 |
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13358813 |
Jan 26, 2012 |
8361747 |
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13693085 |
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12635825 |
Dec 11, 2009 |
8133979 |
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13358813 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/21 20130101;
C07K 2317/565 20130101; C07K 2317/92 20130101; C07K 2317/73
20130101; A61K 39/00 20130101; A61P 9/00 20180101; A61P 9/10
20180101; C12N 15/85 20130101; C07K 2317/76 20130101; A61K 2039/505
20130101; A61P 27/02 20180101; A61P 7/00 20180101; C07K 2317/56
20130101; A61P 35/04 20180101; C07K 16/22 20130101; A61P 35/00
20180101 |
International
Class: |
C07K 16/22 20060101
C07K016/22; C12N 15/85 20060101 C12N015/85; A61K 39/00 20060101
A61K039/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 16, 2008 |
EP |
08021835.7 |
Claims
1. An isolated antibody that specifically binds to human
angiopoietin-2 (ANG-2), wherein the antibody binds to the same
epitope as an antibody comprising: (A) a heavy chain variable
domain which comprises a CDR3 region having the sequence of SEQ ID
NO: 33, a CDR2 region having the sequence of SEQ ID NO: 34 and a
CDR1 region having the sequence of SEQ ID NO: 35; and (B) a light
chain variable domain which comprises a CDR3 region having the
sequence of SEQ ID NO: 36, a CDR2 region having the sequence of SEQ
ID NO: 37 and a CDR1 region having the sequence of SEQ ID NO:
38.
2. The antibody of claim 1, wherein the antibody is a monoclonal
antibody.
3. The antibody of claim 1, wherein the antibody is a human IgG4
antibody or a human IgG1 antibody.
4. A pharmaceutical formulation comprising the antibody of any one
of claims 1 to 3.
5. An isolated antibody that specifically binds to human ANG-2,
wherein the antibody binds to the same epitope as an antibody
comprising a heavy chain comprising SEQ ID NO: 39 and a light chain
comprising SEQ ID NO: 40.
6. The antibody of claim 5, wherein the antibody is a monoclonal
antibody.
7. The antibody of claim 5, wherein the antibody is a human IgG4
antibody or a human IgG1 antibody.
8. A pharmaceutical formulation comprising the antibody of any one
of claims 5 to 7.
Description
PRIORITY TO RELATED APPLICATION(S)
[0001] This application is a continuation application of U.S.
application Ser. No. 15/097,097, filled Apr. 12, 2016, which is a
continuation of U.S. application Ser. No. 13/693,085, filed Dec. 4,
2012, now U.S. Pat. No. 9,340,609, which is a continuation of U.S.
application Ser. No. 13/358,813, filed Jan. 26, 2012, now U.S. Pat.
No. 8,361,747, which is a divisional application of U.S.
application Ser. No. 12/635,825, filed Dec. 11, 2009, now U.S. Pat.
No. 8,133,979, which claims the benefit of European Patent
Application No. 08021835.7, filed Dec. 16, 2008. The entire
contents of the above-identified applications are hereby
incorporated by reference.
SEQUENCE LISTING
[0002] This application contains a Sequence Listing which has been
submitted via EFS-Web and is hereby incorporated by reference in
its entirety. Said ASCII copy, created on Feb. 15, 2018, is named
SequenceListing.txt and is 51,915 bytes in size
FIELD OF THE INVENTION
[0003] The present invention relates to antibodies against human
Angiopoietin 2 (anti-ANG-2 antibodies), methods for their
production, pharmaceutical compositions containing said antibodies,
and uses thereof.
BACKGROUND OF THE INVENTION
[0004] Angiogenesis is implicated in the pathogenesis of a variety
of disorders which include solid tumors, intraocular neovascular
syndromes such as proliferative retinopathies or age-related
macular degeneration (AMD), rheumatoid arthritis, and psoriasis
(Folkman, J., et al., J. Biol. Chem. 267 (1992) 10931-10934;
Klagsbrun, M., et al., Annu. Rev. Physiol. 53 (1991) 217-239; and
Garner, A., Vascular diseases, In: Pathobiology of ocular disease,
A dynamic approach, Garner, A., and Klintworth, G. K. (eds.), 2nd
edition, Marcel Dekker, New York (1994), pp 1625-1710). 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).
ANG-2 and Anti-ANG-2 Antibodies
[0005] Human angiopoietin-2 (ANG-2) (alternatively abbreviated with
ANGPT2 or ANG2) (SEQ ID No: 107) 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: 108) and ANG-2 (SEQ ID No: 107) 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.
[0006] 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: Davies, S., et al., Cell, 87 (1996)
1161-1169; 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) 1161-1169; 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)
2511-14; 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.
[0007] Corneal angiogenesis assays have shown that both ANG-1 and
ANG-2 had similar effects, acting synergistically with VEGF to
promote growth of new blood vessels. Asahara, T., et al., Circ.
Res., 83, (1998) 233-40. The possibility that there was a
dose-dependent endothelial response was raised by the observation
that in vitro at high concentration, ANG-2 can also be
pro-angiogenic. Kim, I., et al., Oncogene 19 (2000) 4549-52. At
high concentration, ANG-2 acts as an apoptosis survival factor for
endothelial cells during serum deprivation apoptosis through
activation of Tie2 via PI-3 Kinase and Akt pathway. Kim, I., et
al., Oncogene 19 (2000) 4549-52.
[0008] 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., 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, M. A., et al., J Clin. Invest. 09 (2002)
777-85.
[0009] 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., Orzcogerze 18 (1999) 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 Angiopoietin-2 overexpression with
tumor hypervascularity (Etoh, T., et al., Cancer Res. 61 (2001)
2145-53; Tanaka, F., et al., Cancer Res. 62 (2002) 7124-29).
[0010] 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, P, 1997; Lin, P.,
1998). Siemester, G., et al. (1999) 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.
[0011] 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.
[0012] 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, 1997). This
pro-angiogenic effect resulting from inhibition of Angiopoietin-1
indicates that anti-Angiopoietin-1 therapy would not be an
effective anti-cancer treatment.
[0013] 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)
228-30, 235 (2001). By detecting ANG-2 mRNA levels in archived
human breast cancer specimens, Sfilogoi, 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) 7124-29. However, they
found no significant correlation between Ang-1 expression and the
microvessel density. Tanaka, F., et al., Cancer Res. 62 (2002)
7124-29. These results suggest that ANG-2 is an indicator of poor
prognosis patients with several types of cancer.
[0014] 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.
[0015] 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 (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 (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.
SUMMARY OF THE INVENTION
[0016] The present invention relates in part to an antibody which
binds specifically to human angiopoietin-2 (ANG-2), wherein said
antibody comprises, as a heavy chain variable domain CDR3 region, a
CDR3 region selected from the group consisting of: SEQ ID NO: 1,
SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 33, SEQ ID
NO: 41, and SEQ ID NO: 49.
[0017] Preferably the antibody comprises:
a) a heavy chain variable domain which comprises: a CDR3 region
selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 9,
SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 33, SEQ ID NO: 41, and SEQ
ID NO: 49; a CDR2 region selected from the group consisting of: SEQ
ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO:
34, SEQ ID NO: 42, and SEQ ID NO: 50; and a CDR1 region selected
from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID
NO: 19, SEQ ID NO: 27, SEQ ID NO: 35, SEQ ID NO: 43, and SEQ ID NO:
51; and b) the light chain variable domain which comprises: a CDR3
region selected from the group consisting of: SEQ ID NO: 4, SEQ ID
NO: 12, SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 36, SEQ ID NO: 44,
and SEQ ID NO: 52; a CDR2 region of SEQ ID NO: 5, SEQ ID NO: 13,
SEQ ID NO: 21, SEQ ID NO: 29, SEQ ID NO: 37, SEQ ID NO: 45, and SEQ
ID NO: 53; and a CDR1 region selected from the group consisting of:
SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 22, SEQ ID NO: 30, SEQ ID
NO: 38, SEQ ID NO: 46, and SEQ ID NO: 54.
[0018] Preferably the antibody comprises:
a) a heavy chain variable domain selected from the group consisting
of: SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 31, SEQ
ID NO: 39, SEQ ID NO: 47, and SEQ ID NO: 55; and b) a light chain
variable domain selected from the group consisting of: SEQ ID NO:
8, SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 40, SEQ
ID NO: 48, and SEQ ID NO: 56.
[0019] Preferably the antibody is characterized in that the
antibody does not specifically bind to Angiopoietin 1 (ANG-1).
[0020] A further embodiment of the invention is a pharmaceutical
composition comprising an antibody according to the invention.
[0021] A further embodiment of the invention is the use of an
antibody according to the invention for the manufacture of a
pharmaceutical composition.
[0022] A further embodiment of the invention is the use of an
antibody according to the invention for the prevention of
metastasis.
[0023] A further embodiment of the invention is the use of an
antibody according to the invention for the treatment of
cancer.
[0024] A further embodiment of the invention is the use of an
antibody according to the invention for the treatment of vascular
diseases.
[0025] A further embodiment of the invention is the use of an
antibody according to the invention for the treatment of
retinopathy.
[0026] A further embodiment of the invention is a nucleic acid
encoding a heavy chain variable domain and/or a light chain
variable domain of an antibody according to the invention.
[0027] The invention further provides expression vectors containing
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
such an antibody.
[0028] The invention further comprises a prokaryotic or eukaryotic
host cell comprising a vector according to the invention.
[0029] The invention further comprises a method for the production
of a recombinant human or humanized 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 antibody from said cell or the cell culture
supernatant. The invention further comprises the antibody
obtainable by such a recombinant method.
[0030] The antibodies according to the invention are especially
useful for the prevention of secondary tumors/metastasis or in the
treatment of vascular diseases such as retinopathies.
Description of the Amino Acid Sequences
TABLE-US-00001 [0031] SEQ ID NO: 1 heavy chain CDR3, <ANG-2>
Ang2i_LC06 SEQ ID NO: 2 heavy chain CDR2, <ANG-2> Ang2i_LC06
SEQ ID NO: 3 heavy chain CDR1, <ANG-2> Ang2i_LC06 SEQ ID NO:
4 light chain CDR3, <ANG-2> Ang2i_LC06 SEQ ID NO: 5 light
chain CDR2, <ANG-2> Ang2i_LC06 SEQ ID NO: 6 light chain CDR1,
<ANG-2> Ang2i_LC06 SEQ ID NO: 7 heavy chain variable domain,
<ANG-2> Ang2i_LC06 SEQ ID NO: 8 light chain variable domain,
<ANG-2> Ang2i_LC06 SEQ ID NO: 9 heavy chain CDR3,
<ANG-2> Ang2i_LC07 SEQ ID NO: 10 heavy chain CDR2,
<ANG-2> Ang2i_LC07 SEQ ID NO: 11 heavy chain CDR1,
<ANG-2> Ang2i_LC07 SEQ ID NO: 12 light chain CDR3,
<ANG-2> Ang2i_LC07 SEQ ID NO: 13 light chain CDR2,
<ANG-2> Ang2i_LC07 SEQ ID NO: 14 light chain CDR1,
<ANG-2> Ang2i_LC07 SEQ ID NO: 15 heavy chain variable domain,
<ANG-2> Ang2i_LC07 SEQ ID NO: 16 light chain variable domain,
<ANG-2> Ang2i_LC07 SEQ ID NO: 17 heavy chain CDR3,
<ANG-2> Ang2k_LC08 SEQ ID NO: 18 heavy chain CDR2,
<ANG-2> Ang2k_LC08 SEQ ID NO: 19 heavy chain CDR1,
<ANG-2> Ang2k_LC08 SEQ ID NO: 20 light chain CDR3,
<ANG-2> Ang2k_LC08 SEQ ID NO: 21 light chain CDR2,
<ANG-2> Ang2k_LC08 SEQ ID NO: 22 light chain CDR1,
<ANG-2> Ang2k_LC08 SEQ ID NO: 23 heavy chain variable domain,
<ANG-2> Ang2k_LC08 SEQ ID NO: 24 light chain variable domain,
<ANG-2> Ang2k_LC08 SEQ ID NO: 25 heavy chain CDR3,
<ANG-2> Ang2s_LC09 SEQ ID NO: 26 heavy chain CDR2,
<ANG-2> Ang2s_LC09 SEQ ID NO: 27 heavy chain CDR1,
<ANG-2> Ang2s_LC09 SEQ ID NO: 28 light chain CDR3,
<ANG-2> Ang2s_LC09 SEQ ID NO: 29 light chain CDR2,
<ANG-2> Ang2s_LC09 SEQ ID NO: 30 light chain CDR1,
<ANG-2> Ang2s_LC09 SEQ ID NO: 31 heavy chain variable domain,
<ANG-2> Ang2s_LC09 SEQ ID NO: 32 light chain variable domain,
<ANG-2> Ang2s_LC09 SEQ ID NO: 33 heavy chain CDR3,
<ANG-2> Ang2i_LC10 SEQ ID NO: 34 heavy chain CDR2,
<ANG-2> Ang2i_LC10 SEQ ID NO: 35 heavy chain CDR1,
<ANG-2> Ang2i_LC10 SEQ ID NO: 36 light chain CDR3,
<ANG-2> Ang2i_LC10 SEQ ID NO: 37 light chain CDR2,
<ANG-2> Ang2i_LC10 SEQ ID NO: 38 light chain CDR1,
<ANG-2> Ang2i_LC10 SEQ ID NO: 39 heavy chain variable domain,
<ANG-2> Ang2i_LC10 SEQ ID NO: 40 light chain variable domain,
<ANG-2> Ang2i_LC10 SEQ ID NO: 41 heavy chain CDR3,
<ANG-2> Ang2k_LC11 SEQ ID NO: 42 heavy chain CDR2,
<ANG-2> Ang2k_LC11 SEQ ID NO: 43 heavy chain CDR1,
<ANG-2> Ang2k_LC11 SEQ ID NO: 44 light chain CDR3,
<ANG-2> Ang2k_LC11 SEQ ID NO: 45 light chain CDR2,
<ANG-2> Ang2k_LC11 SEQ ID NO: 46 light chain CDR1,
<ANG-2> Ang2k_LC11 SEQ ID NO: 47 heavy chain variable domain,
<ANG-2> Ang2k_LC11 SEQ ID NO: 48 light chain variable domain,
<ANG-2> Ang2k_LC11 SEQ ID NO: 49 heavy chain CDR3,
<ANG-2> Ang2s_R3_LC03 SEQ ID NO: 50 heavy chain CDR2,
<ANG-2> Ang2s_R3_LC03 SEQ ID NO: 51 heavy chain CDR1,
<ANG-2> Ang2s_R3_LC03 SEQ ID NO: 52 light chain CDR3,
<ANG-2> Ang2s_R3_LC03 SEQ ID NO: 53 light chain CDR2,
<ANG-2> Ang2s_R3_LC03 SEQ ID NO: 54 light chain CDR1,
<ANG-2> Ang2s_R3_LC03 SEQ ID NO: 55 heavy chain variable
domain, <ANG-2> Ang2s_R3_LC03 SEQ ID NO: 56 light chain
variable domain, <ANG-2> Ang2s_R3_LC03 SEQ ID NO: 57 human
heavy chain constant region derived from IgG1 SEQ ID NO: 58 human
heavy chain constant region derived from IgG4 SEQ ID NO: 59 kappa
light chain constant region SEQ ID NO: 60 lambda light chain
constant region SEQ ID NO: 61 Human Tie-2 receptor SEQ ID NO: 62
Human angiopoietin-2 (ANG-2) with leader and His-tag SEQ ID NO: 63
Human angiopoietin-1 (ANG-1) with leader and His-tag
DESCRIPTION OF THE FIGURES
[0032] FIG. 1A: Cloning of IgGs for transient expressions into
expression vectors transient expression of Ang2i-LC06.
[0033] FIG. 1B: Cloning of IgGs for transient expressions into
expression vectors transient expression of Angi-LC08.
[0034] FIG. 2: SDS-PAGE Gel of purified anti ANG-2 antibodies
Ang2i-LC06, Ang2i-LC07 and Ang2k-LC08.
[0035] FIG. 3: Angiopoietin-Tie2 interaction ELISA.
[0036] FIG. 4: Inhibition of ANG-2 binding to Tie2 by Ang2i-LC06
and Ang2k-LC08.
[0037] FIG. 5: Inhibition of ANG-1 binding to Tie2 by Ang2i-LC06
and Ang2k-LC08.
[0038] FIG. 6: Colo205 xenograft model to test in vivo efficacy of
anti ANG-2 antibodies.
[0039] FIG. 7: KPL-4 xenograft model to test in vivo efficacy of
anti ANG-2 antibodies.
[0040] FIG. 8: ANG-1 binding via Biacore.RTM. sensogram.
[0041] FIG. 9A: Prevention of lung metastasis/secondary tumors by
the antibodies according to the invention in primary colon tumor
xenograft.
[0042] FIG. 9B: Prevention of lung metastasis/secondary tumors by
the antibodies according to the invention in primary breast
xenograft.
[0043] FIG. 10A: Inhibition of retinopathy by the antibodies
according to the invention is shown in slides of flat mounted
retinas: injected antibody.
[0044] FIG. 10B: Inhibition of retinopathy by the antibodies
according to the invention is shown in slides of flat mounted
retinas: uninjected control.
[0045] FIG. 11A: Inhibition of retinopathy by the antibodies
according to the invention: quantification of retinal flat mounts
(injected antibody versus uninjected control): human IgG as
unspecific antibody control.
[0046] FIG. 11B: Inhibition of retinopathy by the antibodies
according to the invention: quantification of retinal flat mounts
(injected antibody versus uninjected control): Ang2i-LC06.
[0047] FIG. 11C: Inhibition of retinopathy by the antibodies
according to the invention: quantification of retinal flat mounts
(injected antibody versus uninjected control): Ang2i-LC08.
[0048] FIG. 11D: Inhibition of retinopathy by the antibodies
according to the invention: quantification of retinal flat mounts
(injected antibody versus uninjected control): Avastin.
DETAILED DESCRIPTION OF THE INVENTION
[0049] The invention comprises an antibody which binds specifically
to human angiopoietin-2 (ANG-2), wherein said antibody comprises,
as a heavy chain variable domain CDR3 region, a CDR3 region
selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 9,
SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 33, SEQ ID NO: 41, and SEQ
ID NO: 49.
[0050] In one embodiment of the invention the antibody
comprises:
a) a heavy chain variable domain which comprises: a CDR3 region
selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 9,
SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 33, SEQ ID NO: 41, and SEQ
ID NO: 49; a CDR2 region selected from the group consisting of: SEQ
ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO:
34, SEQ ID NO: 42, and SEQ ID NO: 50; and a CDR1 region selected
from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID
NO: 19, SEQ ID NO: 27, SEQ ID NO: 35, SEQ ID NO: 43, and SEQ ID NO:
51; and b) a light chain variable domain which comprises: a CDR3
region selected from the group consisting of: SEQ ID NO: 4, SEQ ID
NO: 12, SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 36, SEQ ID NO: 44,
and SEQ ID NO: 52; a CDR2 region selected from the group consisting
of: SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 29, SEQ
ID NO: 37, SEQ ID NO: 45, and SEQ ID NO: 53; and a CDR1 region
selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 14,
SEQ ID NO: 22, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 46, and SEQ
ID NO: 54.
[0051] Preferably the antibody comprises:
a) a heavy chain variable domain selected from the group consisting
of: SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 31, SEQ
ID NO: 39, SEQ ID NO: 47, and SEQ ID NO: 55; and b) a light chain
variable domain selected from the group consisting of: SEQ ID NO:
8, SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 40, SEQ
ID NO: 48, and SEQ ID NO: 56.
[0052] Another embodiment of the invention is an antibody which
binds specifically to human ANG-2, which is characterized in that
the antibody is does not specifically bind to human Angiopoietin 1
(ANG-1). Typical antibodies which specifically bind to human ANG-2,
but not to human ANG-1 are e.g. Ang2s_R3_LC03, Ang2s_LC09,
Ang2i_LC06, Ang2i_LC07, and antibodies binding to the same epitope
as Ang2s_R3_LC03, Ang2s_LC09, Ang2i_LC06, Ang2i_LC07, and
Ang2i_LC10. Preferred such antibodies are those which bind to the
same epitope as Ang2i_LC06. Therefore, in one embodiment of the
invention, the antibody binds specifically to human angiopoietin-2
(ANG-2) but not to human ANG-1 binds to the same epitope as
Ang2s_R3_LC03, Ang2s_LC09, Ang2i_LC06, Ang2i_LC07, or Ang2i_LC10,
and preferably to the same epitope as Ang2i_LC06. Such antibodies
bind specifically to ANG-2, but not to ANG-1 can have improved
properties such as efficacy, less toxicity, pharmacokinetic
properties compared to ANG-2 and ANG-1 specific antibodies.
[0053] Therefore in one embodiment of the invention the antibody is
one which binds specifically to human angiopoietin-2 (ANG-2) but
not to human ANG-1 and comprises:
a) a heavy chain variable domain which comprises: a CDR3 region
selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 9,
SEQ ID NO: 25, SEQ ID NO: 33, and SEQ ID NO: 49; a CDR2 region
selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 10,
SEQ ID NO: 26, SEQ ID NO: 34, and SEQ ID NO: 50; and a CDR1 region
selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 11,
SEQ ID NO: 27, SEQ ID NO: 35, and SEQ ID NO: 51; and b) a light
chain variable domain which comprises: a CDR3 region selected from
the group consisting of: SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO:
28, SEQ ID NO: 36, and SEQ ID NO: 52; a CDR2 region selected from
the group consisting of: SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO:
29, SEQ ID NO: 37, and SEQ ID NO: 53; and a CDR1 region selected
from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID
NO: 30, SEQ ID NO: 38, and SEQ ID NO: 54.
[0054] Preferably the antibody binds specifically to human
angiopoietin-2 (ANG-2) but not to human ANG-1 and comprises:
a) a heavy chain variable domain selected from the group consisting
of: SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 31, SEQ ID NO: 39, and
SEQ ID NO: 55; and b) q light chain variable domain selected from
the group consisting of: SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO:
32, SEQ ID NO: 40, and SEQ ID NO: 56.
[0055] In one embodiment said antibody according to the invention
comprises:
a) a heavy chain variable domain which comprises a CDR3 region of
SEQ ID NO: 1 or SEQ ID NO: 9, a CDR2 region of SEQ ID NO: 2 or SEQ
ID NO: 10, and a CDR1 region of SEQ ID NO: 3 or SEQ ID NO: 11, and
b) a light chain variable domain which comprises a CDR3 region of
SEQ ID NO: 4 or SEQ ID NO: 12, a CDR2 region of SEQ ID NO: 5 or SEQ
ID NO: 13, and a CDR1 region of SEQ ID NO: 6 or SEQ ID NO: 14.
[0056] In one embodiment the antibody according to the invention
comprises:
a) a heavy chain variable domain of SEQ ID NO: 7 or SEQ ID NO: 15;
and b) a light chain variable domain of SEQ ID NO: 8 or SEQ ID NO:
16.
[0057] In one embodiment the antibody according to the invention
comprises:
a) a heavy chain variable domain which comprises a CDR3 region of
SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2, and a CDR1 region of
SEQ ID NO: 3, and b) a light chain variable domain which comprises
a CDR3 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO: 5, and a
CDR1 region of SEQ ID NO: 6.
[0058] In one embodiment the antibody according to the invention
comprises:
a) a heavy chain variable domain of SEQ ID NO: 7; and b) a light
chain variable domain of SEQ ID NO: 8.
[0059] In one embodiment the antibody according to the invention
comprises:
a) a heavy chain variable domain which comprises a CDR3 region of
SEQ ID NO: 17, a CDR2 region of SEQ ID NO: 18, and a CDR1 region of
SEQ ID NO: 19, and b) a light chain variable domain shich comprises
a CDR3 region of SEQ ID NO: 20, a CDR2 region of SEQ ID NO: 21, and
a CDR1 region of SEQ ID NO: 22.
[0060] In one embodiment the antibody according to the invention
comprises:
a) a heavy chain variable domain of SEQ ID NO: 23; and b) a light
chain variable domain of SEQ ID NO: 24.
[0061] Preferably the antibody according to the invention is of
human IgG1 subclass or is of human IgG4 subclass.
[0062] The term "antibody" encompasses the various forms of
antibody structures including but not being limited to whole
antibodies and antibody fragments, The antibody according to the
invention is preferably a humanized antibody, chimeric antibody, or
further genetically engineered antibody, as long as the
characteristic properties according to the invention are
retained.
[0063] "Antibody fragments" comprise a portion of a full length
antibody, preferably the variable domain thereof, or at least the
antigen binding site thereof. Examples of antibody fragments
include diabodies, single-chain antibody molecules (scFv or scFab),
and multispecific antibodies (e.g. bispecific) formed from antibody
fragments. scFv antibodies are, e.g. described in Houston, J. S.,
Methods in Enzymol. 203 (1991) 46-88). In addition, antibody
fragments comprise single chain polypeptides having the
characteristics of a V.sub.H domain, namely being able to assemble
together with a V.sub.L domain, or of a V.sub.L domain binding to
ANG-2, namely being able to assemble together with a V.sub.H domain
to a functional antigen binding site and thereby providing the
property. ScFvs can be stabilized using e.g. a) disulfide
stabilization (see e.g. in WO 94/029350, Rajagopal, V., et al.,
Prot. Engin. (1997) 1453-59; Kobayashi, H., et al., Nuclear
Medicine & Biology, Vol. 25, (1998) 387-393; or Schmidt, M., et
al., Oncogene (1999) 18 1711-1721.) orb) stabilized frameworks
(e.g. by specific mutations of the see e.g. WO 2007/109254 specific
stabilized frameworks see e.g. U.S. Pat. No. 7,258,985, Furrer, F.,
et al., Invest. Ophthalmol. Vis. Sci. 50 (2009), pp. 771-778 or
Ottiger, M., et al., Invest. Ophthalmol. Vis. Sci. 50 (2009), pp.
779-786.
[0064] The terms "monoclonal antibody" or "monoclonal antibody
composition" as used herein refer to a preparation of antibody
molecules of a single amino acid composition.
[0065] 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.
[0066] The term "humanized antibody" refers to antibodies in which
the framework or "complementarity determining regions" (CDR) have
been modified to comprise the CDR of an immunoglobulin of different
specificity as compared to that of the parent immunoglobulin. In a
preferred embodiment, a murine CDR is grafted into the framework
region of a human antibody to prepare the "humanized antibody." See
e.g. Riechmann, L., et al., Nature 332 (1988) 323-327; and
Neuberger, M. S., et al., Nature 314 (1985) 268-270. Particularly
preferred CDRs correspond to those representing sequences
recognizing the antigens noted above for chimeric antibodies. Other
forms of "humanized antibodies" encompassed by the present
invention are those in which the constant region has been
additionally modified or changed from that of the original antibody
to generate the properties according to the invention, especially
in regard to C1q binding and/or Fc receptor (FcR) binding.
[0067] 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, S. P. C., et al., and Boerner, et al. are
also available for the preparation of human monoclonal antibodies
(Cole, S. P. C., et al., Monoclonal Antibodies and Cancer Therapy,
Liss, A. R., (1985) 77-96; and Boerner, P., et al., J. Immunol. 147
(1991) 86-95). As already mentioned for chimeric and humanized
antibodies according to the invention the term "human antibody" as
used herein also comprises such antibodies which are modified in
the constant region to generate the properties according to the
invention, especially in regard to C1q binding and/or FcR binding,
e.g. by "class switching" i.e. change or mutation of Fc parts (e.g.
from IgG1 to IgG4 and/or IgG1/IgG4 mutation.)
[0068] 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 NSO 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.
[0069] The "variable domain" (variable domain of a light chain
(V.sub.L), variable domain of a heavy chain (V.sub.H)) as used
herein denotes each of the pair of light and heavy chain domains
which are involved directly in binding the antibody to the antigen.
The variable light and heavy chain domains have the same general
structure and each domain comprises four framework (FR) regions
whose sequences are widely conserved, connected by three
"hypervariable regions" (or complementary 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's 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.
[0070] The term "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 antigen-binding portion of an
antibody comprises amino acid residues from the "complementary
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 chain
variable domains of an antibody comprise from N- to C-terminus the
domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Especially, CDR3
of the heavy chain is the region which contributes most to antigen
binding and defines the antibody's properties. 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) and/or those residues from a "hypervariable
loop."
[0071] The terms "nucleic acid" or "nucleic acid molecule", as used
herein, are intended to include DNA molecules and RNA molecules. A
nucleic acid molecule may be single-stranded or double-stranded,
but preferably is double-stranded DNA.
[0072] The term "amino acid" as used within this application
denotes the group of naturally occurring carboxy .alpha.-amino
acids comprising alanine (three letter code: ala, one letter code:
A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D),
cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E),
glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine
(leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe,
F), proline (pro, P), serine (ser, S), threonine (thr, T),
tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).
[0073] A nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid. For example, DNA
for a presequence or secretory leader is operable linked to DNA for
a polypeptide if it is expressed as a preprotein that participates
in the secretion of the polypeptide; a promoter or enhancer is
operable linked to a coding sequence if it affects the
transcription of the sequence; or a ribosome binding site is
operable 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 colinear, 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, synthetic oligonucleotide adaptors or linkers are used in
accordance with conventional practice.
[0074] 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.
[0075] As used herein, the terms "bind", "binds", and "binding"
refer to the binding of the antibody to an epitope of an antigen in
an in vitro assay, preferably in an plasmon resonance assay
(BIAcore, GE-Healthcare Uppsala, Sweden) (Example 3) with purified
wild-type ANG-2 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). 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.
[0076] Binding of the antibody to the Fc.gamma.RIII can be
investigated by a BIAcore.RTM. assay (GE-Healthcare Uppsala,
Sweden). 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).
[0077] As used herein, the term "not binding to ANG-1" denotes that
the antibody has an EC50-value above 8000 ng/ml in an in vitro
ANG-1 binding ELISA assay (according to Example 2).
[0078] 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.
[0079] The "Fc part" of an antibody is not involved directly in
binding of an antibody to an antigen, but exhibit various effector
functions. A "Fc part of an antibody" is a term well known to the
skilled artisan and defined on the basis of papain cleavage of
antibodies. Depending on the amino acid sequence of the constant
region of their heavy chains, antibodies or immunoglobulins are
divided in the classes: IgA, IgD, IgE, IgG and IgM, and several of
these may be further divided into subclasses (isotypes), e.g. IgG1,
IgG2, IgG3, and IgG4, IgA1, and IgA2. According to the heavy chain
constant regions the different classes of immunoglobulins are
called .alpha., .delta., .epsilon., .gamma., and .mu.,
respectively. The Fc part of an antibody is directly involved in
ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC
(complement-dependent cytotoxicity) based on complement activation,
C1q binding and Fc receptor binding. Complement activation (CDC) is
initiated by binding of complement factor C1q to the Fc part of
most IgG antibody subclasses. While the influence of an antibody on
the complement system is dependent on certain conditions, binding
to C1q is caused by defined binding sites in the Fc part. Such
binding sites are known in the state of the art and described e.g.
by Boakle, R. J., et al., Nature 282 (1975) 742-743, Lukas, T. J.,
et al., J. Immunol. 127 (1981) 2555-2560, Brunhouse, R., and Cebra,
J. J., Mol. Immunol. 16 (1979) 907-917, Burton, D. R., et al.,
Nature 288 (1980) 338-344, Thommesen, J. E., et al., Mol. Immunol.
37 (2000) 995-1004, Idusogie, E. E., et al., J. Immunol. 164 (2000)
4178-4184, Hezareh, M., et al., J. Virology 75 (2001) 12161-12168,
Morgan, A., et al., Immunology 86 (1995) 319-324, EP 0307434. Such
binding sites are e.g. L234, L235, D270, N297, E318, K320, K322,
P331 and P329 (numbering according to EU index of Kabat, see
below). Antibodies of subclass IgG1, IgG2 and IgG3 usually show
complement activation and C1q and C3 binding, whereas IgG4 do not
activate the complement system and do not bind C1q and C3.
[0080] The antibody according to the invention preferably comprises
a Fc part from human origin which is Fc part of a human antibody of
the subclass IgG1.
[0081] The antibody according to the invention is characterized in
that the constant chains are of human origin. Such constant chains
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). For example, a useful human heavy chain constant
region comprises an amino acid sequence of SEQ ID NO: 57 or of SEQ
ID NO: 58. For example, a useful human light chain constant region
comprises an amino acid sequence of a kappa-light chain constant
region of SEQ ID NO: 59, or of a lambda-light chain constant region
of SEQ ID NO: 60.
[0082] 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 exhibit 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).
[0083] 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. 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).
[0084] While antibodies of the IgG4 subclass show reduced Fc
receptor (Fc.gamma.RIIIa) binding, antibodies of other IgG
subclasses show strong 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).
[0085] In one embodiment an antibody according to the invention has
a reduced FcR binding compared to an IgG1 antibody and the
monospecific bivalent parent antibody is in regard to FcR binding
of IgG4 subclass or of IgG1 or IgG2 subclass with a mutation in
5228, L234, L235 and/or D265, and/or contains the PVA236 mutation.
In one embodiment the mutations in the monospecific bivalent parent
antibody are S228P, L234A, L235A, L235E and/or PVA236. In another
embodiment the mutations in the monospecific bivalent parent
antibody are in IgG4 S228P and in IgG1 L234A and L235A. Constant
heavy chain regions shown in SEQ ID NO: 57 and 58. In one
embodiment the constant heavy chain region of the monospecific
bivalent parent antibody is of SEQ ID NO: 57 with mutations L234A
and L235A. In another embodiment the constant heavy chain region of
the monospecific bivalent parent antibody is of SEQ ID NO: 58 with
mutation S228P. In another embodiment the constant light chain
region of the monospecific bivalent parent antibody is a kappa
light chain region of SEQ ID NO: 59, or a lambda light chain
constant region of SEQ ID NO: 60. In one embodiment of the
invention the constant heavy chain region of the monospecific
bivalent parent antibody is of SEQ ID NO: 57 or of SEQ ID NO: 58
with mutation S228P.
[0086] The constant region of an antibody is directly involved in
ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC
(complement-dependent cytotoxicity). Complement activation (CDC) is
initiated by binding of complement factor C1q to the constant
region of most IgG antibody subclasses. Binding of C1q to an
antibody is caused by defined protein-protein interactions at the
so called binding site. Such constant region binding sites are
known in the state of the art and described e.g. by Lukas, T. J.,
et al., J. Immunol. 127 (1981) 2555-2560; Brunhouse, R. and Cebra,
J. J., Mol. Immunol. 16 (1979) 907-917; Burton, D. R., et al.,
Nature 288 (1980) 338-344; Thommesen, J. E., et al., Mol. Immunol.
37 (2000) 995-1004; Idusogie, E. E., et al., J. Immunol. 164 (2000)
4178-4184; Hezareh, M., et al., J. Virol. 75 (2001) 12161-12168;
Morgan, A., et al., Immunology 86 (1995) 319-324; and EP 0 307 434.
Such constant region binding sites are, e.g., characterized by the
amino acids L234, L235, D270, N297, E318, K320, K322, P331, and
P329 (numbering according to EU index of Kabat).
[0087] The term "antibody-dependent cellular cytotoxicity (ADCC)"
refers to lysis of human target cells by an antibody according to
the invention in the presence of effector cells. ADCC is measured
preferably by the treatment of a preparation of CCR5 expressing
cells with an antibody according to the invention in the presence
of effector cells such as freshly isolated PBMC or purified
effector cells from buffy coats, like monocytes or natural killer
(NK) cells or a permanently growing NK cell line.
[0088] The term "complement-dependent cytotoxicity (CDC)" denotes a
process initiated by binding of complement factor C1q to the Fc
part of most IgG antibody subclasses. Binding of C1q to an antibody
is caused by defined protein-protein interactions at the so called
binding site. Such Fc part binding sites are known in the state of
the art (see above). Such Fc part binding sites are, e.g.,
characterized by the amino acids L234, L235, D270, N297, E318,
K320, K322, P331, and P329 (numbering according to EU index of
Kabat). Antibodies of subclass IgG1, IgG2, and IgG3 usually show
complement activation including C1q and C3 binding, whereas IgG4
does not activate the complement system and does not bind C1q
and/or C3.
[0089] The antibody according to the invention is produced by
recombinant means. Thus, one aspect of the current invention is a
nucleic acid encoding the antibody according to the invention and a
further aspect is a cell comprising said nucleic acid encoding an
antibody according to the invention. Methods for recombinant
production are widely known in the state of the art and comprise
protein expression in prokaryotic and eukaryotic cells with
subsequent isolation of the antibody and usually purification to a
pharmaceutically acceptable purity. For the expression of the
antibodies as aforementioned in a host cell, nucleic acids encoding
the respective modified light and heavy chains are inserted into
expression vectors by standard methods. Expression is performed in
appropriate prokaryotic or eukaryotic host cells like CHO cells,
NSO cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6 cells,
yeast, or E. coli cells, and the antibody is recovered from the
cells (supernatant or cells after lysis). General methods for
recombinant production of antibodies are well-known in the state of
the art and described, for example, in the review articles of
Makrides, S. C., Protein Expr. Purif. 17 (1999) 183-202; Geisse,
S., et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, R. J.,
Mol. Biotechnol. 16 (2000) 151-161; Werner, R. G., J. Drug Res. 48
(1998) 870-880.
[0090] The antibodies according to the invention 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.
[0091] Amino acid sequence variants (or mutants) of the antibody
according to the invention 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, e.g. as described above. 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.
[0092] 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.
[0093] Expression in NSO cells is described by, e.g., Barnes, L.
M., et al., Cytotechnology 32 (2000) 109-123; Barnes, L. M., et
al., Biotech. Bioeng. 73 (2001) 261-270. Transient expression is
described by, e.g., Durocher, Y., et al., Nucl. Acids. Res. 30
(2002) E9. Cloning of variable domains is described by Orlandi, R.,
et al., Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837; Carter, P.,
et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and
Norderhaug, L., et al., J. Immunol. Methods 204 (1997) 77-87. A
preferred transient expression system (HEK 293) is described by
Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30 (1999)
71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996)
191-199.
[0094] 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.
[0095] 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.
[0096] 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).
[0097] The invention comprises a method for the treatment of a
patient in need of therapy, characterized by administering to the
patient a therapeutically effective amount of an antibody according
to the invention.
[0098] The invention comprises the use of an antibody according to
the invention for therapy.
[0099] The invention comprises the use of an antibody according to
the invention for the preparation of a medicament for the
prevention of metastasis.
[0100] The invention comprises the use of an antibody according to
the invention for the preparation of a medicament for the treatment
of cancer.
[0101] 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.
[0102] Another aspect of the invention is said pharmaceutical
composition for the prevention of metastasis.
[0103] Another aspect of the invention is an antibody according to
the invention for the prevention of metastasis.
[0104] Another aspect of the invention is the use of an antibody
according to the invention for the manufacture of a medicament for
the prevention of metastasis.
[0105] Another aspect of the invention is a method of prevention
metastasis in patient suffering from primary cancer by
administering an antibody according to the invention to a patient
in the need of such preventative treatment.
[0106] We could show higly efficient prevention of spotanouenes
metastasis/secondary tumors in vivo in a orthotopic and a
subcutanoues 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).
[0107] 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.
[0108] 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
relapsed HER2 positive 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 developement of secondary tunmors is 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.
[0109] Another aspect of the invention is said pharmaceutical
composition for the treatment of cancer.
[0110] Another aspect of the invention is an antibody according to
the invention for the treatment of cancer.
[0111] 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.
[0112] 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.
[0113] 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).
[0114] 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.
[0115] 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, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular, sub
arachnoid, intraspinal, epi dural and intrasternal injection and
infusion.
[0116] 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, ependymonas, 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.
[0117] Another aspect of the invention is 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.
[0118] 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.
[0119] Another aspect of the invention is an antibody according to
the invention for the treatment of vascular diseases.
[0120] A preferred embodiment is an antibody according to the
invention for the treatment of retinopathy.
[0121] A preferred embodiment is the use of an antibody according
to the invention for the manufacture of a medicament for the
treatment of retinopathy
[0122] 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.
[0123] 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).
[0124] 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.
[0125] 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.
[0126] 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.
[0127] 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.
[0128] 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.
[0129] 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.
[0130] 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., and van der Eb,
Virology 52 (1973) 456-467. 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, F. N, et al, PNAS. 69 (1972) 7110ff.
[0131] 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.
[0132] 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.
[0133] 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.
[0134] 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.
Experimental Procedure 1
Materials & General Methods
[0135] 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
[0136] 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
[0137] Desired gene segments were 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. Gene synthesis
fragments were ordered according to given specifications at Geneart
(Regensburg, Germany).
DNA Sequence Determination
[0138] 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
[0139] The GCG's (Genetics Computer Group, Madison, Wis.) software
package version 10.2 and Infomax's Vector NT1.RTM. Advance suite
version 8.0 was used for sequence creation, mapping, analysis,
annotation and illustration.
Expression Vectors
[0140] 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. 1) were
applied.
[0141] Beside the antibody expression cassette the vectors
contained: [0142] an origin of replication which allows replication
of this plasmid in E. coli, and [0143] a .beta.-lactamase gene
which confers ampicillin resistance in E. coli.
[0144] The transcription unit of the antibody gene is composed of
the following elements: [0145] unique restriction site(s) at the 5'
end [0146] the immediate early enhancer and promoter from the human
cytomegalovirus, [0147] followed by the Intron A sequence in the
case of the cDNA organization, [0148] a 5'-untranslated region of a
human antibody gene, [0149] a immunoglobulin heavy chain signal
sequence, [0150] 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
[0151] a 3' untranslated region with a polyadenylation signal
sequence, and [0152] unique restriction site(s) at the 3' end.
[0153] The fusion genes comprising the heavy chain sequences of the
selected antibody as described below were generated by PCR and/or
gene synthesis and assembled with known recombinant methods and
techniques by connection of the according nucleic acid segments
e.g. using unique NsiI and EcoRI sites in the genomic heavy chain
vectors. The subcloned nucleic acid sequences were verified by DNA
sequencing. 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
[0154] 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
[0155] Antibodies were generated by transient transfection of the
two plasmids encoding the heavy or modified heavy chain,
respectively and the corresponding light chain using the HEK293-F
system (Invitrogen) according to the manufacturer's instruction.
Briefly, HEK293-F cells (Invitrogen) growing in suspension either
in a shake flask or in a stirred fermenter in serumfree
FreeStyle.TM. 293 expression medium (Invitrogen) were transfected
with a mix of the two respective expression plasmids and
293Fectin.TM. or fectin (Invitrogen). For e.g. 2 L shake flask
(Corning) HEK293-F cells were seeded at a density of 1.0E*6
cells/mL in 600 mL and incubated at 120 rpm, 8% CO2. The day after
the cells were transfected at a cell density of ca. 1.5E*6 cells/mL
with ca. 42 mL mix of A) 20 mL Opti-MEM.RTM. (Invitrogen) with 600
.mu.g total plasmid DNA (1 .mu.g/mL) encoding the heavy or modified
heavy chain, respectively and the corresponding light chain in an
equimolar ratio and B) 20 ml Opti-MEM.RTM.+1.2 mL 293Fectin.TM. or
fectin (2 .mu.l/mL). According to the glucose consumption glucose
solution was added during the course of the fermentation. The
supernatant containing the secreted antibody was harvested after
5-10 days and antibodies were either directly purified from the
supernatant or the supernatant was frozen and stored.
Protein Determination
[0156] 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
[0157] 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.RTM.-MC-filter
column (Amicon.RTM.) 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.RTM. Blue.
[0158] 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.TM.
Protein A column (GE Healthcare) in 50 mM K2HPO4, 300 mM NaCl, pH
7.3 and eluted from the matrix with 50 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.
[0159] Alternatively, the concentration of antibodies and
derivatives in cell culture supernatants was measured by
Sandwich-IgG-ELISA. Briefly, StreptaWell.RTM. High Bind
Streptavidin A-96 well microtiter plates (Roche) were coated with
100 .mu.L/well biotinylated anti-human IgG capture molecule
F(ab')2<h-Fc.gamma.> 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.TM. (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).
Protein Purification
[0160] Proteins were purified from filtered cell culture
supernatants referring to standard protocols. In brief, antibodies
were applied to a Protein A Sepharose column (GE Healthcare) and
washed with PBS. Elution of antibodies was achieved at acidic pH
followed by immediate neutralization of the sample. Aggregated
protein was separated from monomeric antibodies by size exclusion
chromatography (Superdex.RTM. 200, GE Healthcare) in 20 mM
Histidine, 140 mM NaCl pH 6.0. Monomeric antibody fractions were
pooled, concentrated if required using e.g. a MILLIPORE Amicon.RTM.
Ultra (30 MWCO) centrifugal concentrator and stored at -80.degree.
C. Part of the samples were provided for subsequent protein
analytics and analytical characterization e.g. by SDS-PAGE, size
exclusion chromatography, mass spectrometry and Endotoxin
determination (see FIG. 2).
SDS-PAGE
[0161] 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. 1).
Reducing of samples was achieved by adding NuPAGE.RTM. sample
reducing agent prior to running the gel.
Analytical Size Exclusion Chromatography
[0162] 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.RTM. G3000SW column in 300 mM NaCl, 50 mM
KH2PO4/K2HPO4, pH 7.5 on an Dionex HPLC system or to a
Superdex.RTM. 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.
Mass Spectrometry
[0163] The total deglycosylated mass of 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 1 M TCEP and 50 .mu.l 8 M
Guanidinium-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.RTM. Elite MS system equipped with a
NanoMate.RTM. source.
ANG-1 and ANG-2 Binding ELISA
[0164] The binding properties of antibodies directed against ANGPTs
(Angiopoietin 1 or 2) were evaluated in an ELISA assay with
full-length Angiopoietin-2-His protein (R&D Systems #623-AN/CF
or in house produced material) or Angiopoietin-1-His (R&D
systems #923-AN). Therefore 96 well plates (Falcon.TM. polystyrene
clear enhanced microtiter plates or Nunc Maxisorp.RTM.) were coated
with 100 .mu.l 1 .mu.g/mL recombinant human Angiopoietin-1 or
Angiopoietin-2 (carrier-free) in PBS (Sigma) for 2 h at room
temperature or over night at 4.degree. C. The wells were washed
three times with 300 .mu.l PBST (0.2% Tween.RTM. 20) and blocked
with 200 .mu.l 2% BSA 0.1% Tween.RTM. 20 for 30 min at room
temperature and subsequently washed three times with 300 .mu.l
PBST. 100 .mu.L/well of a dilution series (40 pM-0.01 pM) of
purified test antibody against <ANG-2> and as a reference
Mab536 (Oliner, J., et al., Cancer Cell. Nov. 6 (2004) 507-16, US
2006/0122370) in PBS was added to the wells and incubated for 1 h
on a microtiterplate shaker at room temperature. The wells were
washed three times with 300 .mu.l PBST (0.2% Tween.RTM. 20) and
bound antibody was detected with 100 .mu.L/well 0.1 .mu.g/ml F(ab')
<hk>POD (Biozol Cat. No. 206005) in 2% BSA 0.1% Tween.RTM. 20
as detection antibody for 1 h on a microtiterplate shaker at room
temperature. Unbound detection antibody was washed away three times
with 300 .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).
ANG-2 Binding BIACORE
[0165] Binding of the antibodies to the antigen e.g. human ANG-2
were investigated by surface plasmon resonance using a BIACORE T100
instrument (GE Healthcare Biosciences AB, Uppsala, Sweden).
Briefly, for affinity measurements goat<hIgG-Fcgamma>
polyclonal antibodies were immobilized on a CM4 chip via amine
coupling for presentation of the antibodies against human ANG-2.
Binding was measured in HBS buffer (HBS-P (10 mM HEPES, 150 mM
NaCl, 0.05% Tween.RTM. 20, ph 7.4), 25.degree. C. Purified
ANG-2-His (R&D systems or in house purified) was added in
various concentrations between 0.41 nM and 200 nM in solution.
Association was measured by an ANG-2-injection of 3 minutes;
dissociation was measured by washing the chip surface with HBS
buffer for 5 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; KD values are thus
only relative estimations. Negative control data (e.g. buffer
curves) were subtracted from sample curves for correction of system
intrinsic baseline drift and for noise signal reduction.
Biacore.RTM. T100 Evaluation Software version 1.1.1 was used for
analysis of sensorgrams and for calculation of affinity data.
Alternatively, Ang-2 could be captured with a capture level of
2000-1700 RU via a PentaHisAntibody (PentaHis-Ab BSA-free, Qiagen
No. 34660) that was immobilized on a CM5 chip via amine coupling
(BSA-free) (see below).
Inhibition of huANG-2 Binding to Tie-2 (ELISA)
[0166] 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 0.5 .mu.g/ml Tie-2 protein (R&D Systems, UK,
Cat. No. 313-TI) for at least 2 hours (h). Thereafter the wells
were blocked with PBS supplemented with 0.2% Tween.RTM. 20 and 2%
BSA (Roche Diagnostics GmbH, DE) for 1 h. Dilutions of purified
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 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.
Inhibition of huANG-1 Binding to Tie-2 (ELISA)
[0167] The interaction ELISA was performed on 384 well microtiter
plates (MaxiSorp.RTM. Nunc#442768) at RT. After each incubation
step plates were washed 3 times with PBST. ELISA plates were coated
with 0.5 .mu.g/ml Tie-2 protein (R&D Systems, UK, Cat. No.
313-TI or in house produced material) for at least 2 hours (h).
Thereafter the wells were blocked with PBS supplemented with 0.2%
Tween.RTM. 20 and 2% BSA (Roche Diagnostics GmbH, DE) for 1 h.
Dilutions of purified antibodies in PBS were incubated together
with 0.2 .mu.g/ml huAngiopoietin-1 (R&D Systems #923-AN/CF or
in house produced material) for 1 h at RT. After washing a mixture
of 0.5 .mu.g/ml biotinylated anti-Angiopoietin-1 clone (R&D
Systems #BAF923) and 1:3000 diluted streptavidin HRP (Roche
Diagnostics GmbH, DE, Cat. No. 11089153001) 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.
Generation of HEK293-Tie2 Cell Line
[0168] 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 61) under control of a
CMV promoter and a Neomycin resistance marker was transfected using
FuGENE.RTM. (Roche Applied Science) as transfection reagent into
HEK293 cells (ATCC.RTM.) 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
[0169] 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.
ANGPT1 Induced Tie2 Phosphorylation Assay
[0170] Inhibition of ANGPT1 induced Tie2 phosphorylation by ANGPT1
antibodies was measured according to the following assay principle.
HEK293-Tie2 clone22 was stimulated with ANGPT1 for 5 minutes in the
absence or presence of ANGPT1 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 ANGPT1 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 ANGPT1
(R&D systems #923-AN] dilution (0.8 .mu.g/ml as 4-fold
concentrated solution). Antibodies and ANGPT1 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.
Example 1
Expression & Purification of Monoclonal <ANG-2>
Antibodies Ang2i-LC06, Ang2i-LC07 and Ang2k-LC08
[0171] Light and heavy chains of the corresponding antibodies
Ang2i-LC06, Ang2i-LC07 and Ang2k-LC08 were constructed in
expression vectors as described above. The heavy chain and the
kappa light was cloned in a genomic expression cassette, whereas
the lambda light chain was cloned as cDNA with intron A (FIG. 1B).
The plasmids were amplified in E. coli, purified, and subsequently
transfected for transient expression of recombinant proteins in
HEK293-F cells (utilizing Invitrogen's FreeStyle.TM. 293 system).
After 7 days, HEK 293-F cell supernatants were harvested, filtered
and the antibodies were purified by protein A and size exclusion
chromatography. Homogeneity of all antibodies was confirmed by
SDS-PAGE under non reducing and reducing conditions and analytical
size exclusion chromatography. Under reducing conditions (FIG. 1),
polypeptide heavy chains of <ANG-2> antibodies showed upon
SDS-PAGE apparent molecular sizes of ca. 50 kDa analogous to the
calculated molecular weights, polypeptide light chains showed
apparent molecular masses of 25 kDa according to their predicted
size. Mass spectrometry confirmed the identity of the purified
antibodies. Expression levels of all constructs were analyzed by
Protein A HPLC.
[0172] Size exclusion chromatography analysis of the purified. All
antibodies were prepared and analytically characterized analogously
to the procedure described. The SEC data of the corresponding
antibodies were summarized in the table below.
TABLE-US-00002 Antibody Theoretical Experimental SEC (%) chain mass
(Da) mass (Da) main peak <ANG-2> HC 50343 50325 (pyro-Glu)
99.7% Ang-2i_LC07 LC 22738 22720 (pyro-Glu) <ANG-2> HC 50343
50325 (pyro-Glu) 99.8% Ang-2i_LC06 LC 22620 22605 (pyro-Glu)
<ANG-2> HC 49544 49527 (pyro-Glu) 99.8% Ang-2k_LC08 LC 22685
22667 (pyro-Glu)
Example 2
ELISA Binding Assay to Human ANG-1 and to Human ANG-2
[0173] The binding of <ANG-2> antibodies Ang2i-LC06,
Ang2i-LC07 and Ang2k-LC08 to human ANG-1 and human ANG-2 was
determined in an ANG-1 or ANG-2 binding ELISA as described above.
Briefly, the ELISA-type assay is based on the immobilization of
human wild-type Angiopoietin-1 or -2 in a microtiter plate. Binding
of an antibody directed against the immobilized ANG-1 or ANG-2 is
measured via an <human Fe> (anti-IgG) antibody with a POD
conjugate. A dilution series of the <ANG-2> antibody allows
determining an EC50 concentration. As a reference the human
anti-ANG-2 antibody <ANG-2> antibody Mab536 (Oliner et al.,
Cancer Cell. Nov. 6 (2004) 507-16, US 2006/0122370) was used. The
determined EC50 concentrations are summarized in the table
below.
TABLE-US-00003 hANG-1 binding hANG-2 binding Antibody EC50 EC50
<ANG-2> 2538 ng/mL 133 ng/mL MAb536 <ANG-2> >8000
ng/mL 84 ng/mL Ang2i-LC06 <ANG-2> >8000 ng/mL 3006 ng/mL
Ang2i-LC07 <ANG-2> 4044 ng/mL 105 ng/mL Ang2i-LC08
[0174] All antibodies binds specifically to ANG-2. MAb536 and
Ang2k-LC08 show also specific binding towards ANG-1, whereas
Ang2i-LC06 and Ang2i-LC07 do not specifically bind to ANG-1 as they
have an EC50-value of above 8000 ng/ml (detection limit).
Example 3
Binding to ANG-2 Via Biacore
[0175] The affinity for binding to human ANGPT2 was examined with a
Biacore.RTM. assay as describes above. Briefly, is this assay a
capturing antibody (anti-Fc) is immobilized to the surface of the
Biacore.RTM. chip, which captures and presents the corresponding
antibody (for example Ang2i-LC06). The ligand (here ANGPT2) is
captured from solution. The affinity for this interaction is
determined with the assumption of a 1:1 interaction. Details of
this experiment can be found in the general methods section. The
affinities determined for ANGPT2-binding (KD) are summarized in the
table below.
TABLE-US-00004 Average Experiment 1 Experiment 2 (from 1 + 2) KD kd
t.sub.(1/2)diss KD kd t.sub.(1/2)diss KD t.sub.(1/2)diss hAng-2
(pM) (1/s) (min) (pM) (1/s) (min) (pM) (min) Ang2i- 11 7.16E-05 161
21 1.14E-04 102 16 132 LC06 Ang2k- 16 1.61E-04 72 27 2.28E-04 51 22
61 LC08 MAb536 29 1.44E-04 80 29 1.25E-04 92 29 86 The antibodies
Ang2i-LC06 and Ang2k bind with high affinity to ANGPT2.
Example 4
Neutralization of ANGPT1/2-Tie2 Interaction (Human)
[0176] Blocking of human ANGPT1/2/human Tie2 interaction was shown
by receptor interaction ELISA. 384-well Maxisorp.RTM. plates (Nunc)
were coated with 0.5 .mu.g/ml human Tie2 (R&D Systems, UK, Cat.
No. 313-TI or in house produced material) for 2 h at room
temperature and blocked with PBS supplemented with 0.2% Tween.RTM.
20 and 2% BSA (Roche Diagnostics GmbH, DE) for 1 h at room
temperature under shaking. In the meantime, Dilutions of purified
antibodies in PBS were incubated together with 0.2 .mu.g/ml
huAngiopoietin-1/2 (R&D Systems #923-AN/CF, R&D Systems,
UK, Cat. No. 623-AN or in house produced material) for 1 h at RT.
After washing a mixture of 0.5 .mu.g/ml biotinylated
anti-Angiopoietin-1/2 clone (R&D Systems #BAF923, 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 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.
[0177] The obtained inhibitory concentrations are summarized in the
following table.
TABLE-US-00005 ANGPT1/Tie2 ANGPT2/Tie2 Antibody interaction ELISA
interaction ELISA Ang2i-LC06 >100 nM 0.1 nM Ang2k-LC08 11 nM
0.17 nM MAb536 n.d. 0.15 nM
[0178] The table above shows different selectivity profiles for the
two antibodies Ang2i-LC06 and Ang2k-LC08. Ang2i-LC06 is ANGPT2
selective, whereas Ang2k-LC08 is ANGPT1/2 cross reactive in
inhibition for ANGPT1/2 Tie2 interaction.
Example 5
Tie2 Phosphorylation
[0179] The ability of the identified ANGPT2 antibodies to interfere
with ANGPT2 and ANGPT1 mediated Tie2 phosphorylation was examined
in the ANGPT2 and ANGPT1 induced Tie2 phosphorylation assays as
described above. A schematic representation of the assay setup is
depicted in FIG. 3.
[0180] Both antibodies Ang2i-LC06 and Ang2k-LC08 showed a
dose-dependent interference with ANGPT2 stimulated Tie2
phosphorylation as shown in FIG. 4 with comparable IC50 values.
Ang2i-LC06 interfered with ANGPT2 stimulated Tie2 phosphorylation
with a IC50 value of approx. 508 ng/ml and Ang2k-LC08 interfered
with ANGPT2 stimulated Tie2 phosphorylation with a IC50 value of
approx. 499 ng/ml. In contrast, only Ang2k-LC08 interfered with
ANGPT1 stimulated Tie2 phosphorylation with a IC50 value of approx.
391 ng/ml whereas Ang2i-LC06 did not interfere with ANGPT2
stimulated Tie2 phosphorylation in the same tested concentration
range (FIG. 5).
Example 6: In Vivo Efficacy
Effect of Anti ANGPT Antibodies on Colo205 Xenograft Growth
[0181] In vivo efficacy of <ANGPT2> antibodies Ang2i-LC06 and
Ang2k-LC08 in comparison to <ANGPT2> Mab536 in staged
subcutaneous Colo205 xenograft model
[0182] The purified Ang2i-LC06 and Ang2k-LC08 antibodies were
compared to the antibody Mab536 in the staged subcutaneous Colo205
xenograft model (Ang2_PZ_Colo205_006) in female SCID beige
mice.
[0183] Antibodies: Mab536 was provided as frozen stock solution
(c=4.5 mg/mL), Ang2i-LC06 and Ang2k-LC08 were provided as frozen
stock solution (c=1 mg/mL) in 20 mM Histidine, 140 mM NaCl, pH 6.0.
Antibody solution was diluted appropriately in PBS from stock prior
injections where required and PBS was applied as vehicle. The
humanized IgG1 anti-IgE antibody Xolair.RTM. (omalizumab) served as
positive control and was bought from a pharmacy.
[0184] Cell lines and culture conditions: Colo205 human colorectal
cancer cells were originally obtained from ATCC.RTM. and after
expansion deposited in the Roche Penzberg internal cell bank. Tumor
cell line was 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% CO2. Passage 3 was used for
transplantation.
[0185] Animals: Female SCID beige mice (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 was 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 was about 12-14 weeks.
[0186] Monitoring: Animals were controlled daily for clinical
symptoms and detection of adverse effects. For monitoring
throughout the experiment body weight of animals was documented and
tumor volume was measured by caliper after staging.
[0187] Tumor cell injection: At day of injection Colo205 cells were
centrifuged, washed once and resuspended in PBS. After an
additional washing with PBS cell concentration and cell size were
determined using a cell counter and analyzer system (Vi-CELL,
Beckman Coulter). For injection of Colo205 cells, the final titer
was adjusted to 5.0.times.10E7 cells/ml, viability ca. 90%.
Subsequently 100 .mu.l of this suspension corresponding to 2.5*106
cells per animal was injected s.c. into the right flank of the
mice.
[0188] Treatment of animals: Animal treatment started at day of
randomisation, 16 days after cell transplantation (study
Ang2_PZ_Colo205_006) at a mean tumor volume of 178 mm3.
[0189] Dose schedule of study Ang2_PZ_Colo205_006:
TABLE-US-00006 Cumulative No of Dose Route/Mode of No of dose Group
animals Compound (mg/kg) administration treatments (mg/kg) 1 10
Vehicle i.p. once weekly 5 2 10 Xolair .RTM. 10 i.p. once weekly 5
50 3 10 Ang2i- 10 i.p. once weekly 5 50 LC06 5 10 Ang2k- 10 i.p.
once weekly 5 50 LC08 6 10 MAB536 10 i.p. once weekly 5 50
[0190] Tumor growth inhibition until Day 50 is shown in FIG. 6. The
data show that the ANGPT2 selective antibody Ang2i-LC06 was the
most active antibody (Tumor control ration (TCR) value 0.39).
Ang2i-LC06 was more efficacious in inhibiting tumor growth than
antibody MAb536 (TCR value 0.47) and the ANGPT2 selective, ANGPT1
cross-reactive antibody Ang2k-LC08 (TCR value 0.46).
Effect of Anti ANGPT Antibodies on KPL-4 Xenograft Growth
[0191] In vivo efficacy of <ANGPT2> antibodies Ang2i-LC06 and
Ang2k-LC08 in comparison to <ANGPT2> Mab536 in staged
orthotopic KPL-4 xenograft model. The purified Ang2i-LC06 and
Ang2k-LC08 antibodies were compared to the antibody Mab536 in the
staged orthotopic KPL-4 xenograft model (Ang2_PZ_KPL-4_002) in
female SCID beige mice.
[0192] Antibodies: Mab536 was provided as frozen stock solution
(c=4.5 mg/mL), Ang2i-LC06 and Ang2k-LC08 were provided as frozen
stock solution (c=1 mg/mL) in 20 mM Histidine, 140 mM NaCl, pH 6.0.
Antibody solution was diluted appropriately in PBS from stock prior
injections where required and PBS was applied as vehicle.
[0193] Cell lines and culture conditions: KPL-4 human breast cancer
cells were originally established from the malignant pleural
effusion of a breast cancer patient with an inflammatory skin
metastasis. KPL-4 cells were kindly provided by Prof. J.
Kurebayashi (Kawasaki Medical School, Kurashiki, Japan). 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% CO2. Culture passage was performed
with trypsin/EDTA 1.times. (PAN) splitting three times/week.
[0194] Animals: Female SCID beige mice (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 was 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 was about 12 weeks.
[0195] Monitoring: Animals were controlled daily for clinical
symptoms and detection of adverse effects. For monitoring
throughout the experiment body weight of animals was documented and
tumor volume was measured by caliper after staging.
[0196] Tumor cell injection: 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.TM.; 100 .mu.m) the final
cell titer was adjusted to 1.5.times.108/ml. Tumor cell suspension
was carefully mixed with transfer pipette to avoid cell
aggregation. Anesthesia was performed using a Stephens's 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 (3*106/animal) 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 30 G.times.1/2'' needle.
[0197] Treatment of animals started at day of randomization with
tumors ranging from 60-180 mm 3.35 days after cell transplantation
(study Ang2_PZ_KPL-4_002) at a mean tumor volume of ca. 90 mm3.
[0198] Dose schedule of study Ang2_PZ_KPL-4_002:
TABLE-US-00007 Cumulative No of Dose Route/Mode of No of dose Group
animals Compound (mg/kg) administration treatments (mg/kg) 1 10
Vehicle i.p. once weekly 5 2 10 Xolair .RTM. 10 i.p. once weekly 5
50 3 10 Ang2i- 10 i.p. once weekly 5 50 LC06 5 10 Ang2k- 10 i.p.
once weekly 5 50 LC08 6 10 MAB536 10 i.p. once weekly 5 50
[0199] Tumor growth inhibition until day 64 is shown in FIG. 7. The
data show that the ANGPT2 selective antibody Ang2i-LC06 was the
most active antibody (TCR value 0.55) in the KPL-4 model.
Ang2i-LC06 was more efficacious in inhibiting tumor growth than
antibody MAb536 (TCR value 0.57) and the ANGPT2 selective, ANGPT1
cross-reactive antibody Ang2k-LC08 (TCR value 0.57).
Example 7
Binding to ANG-1 Via Biacore
[0200] The affinity for binding to human ANG-1 was examined with a
Biacore.RTM. assay: huAng-1 was immobilized on a CM5 biosensorchip
using amine-coupling chemistry. The protein was injected for 20 min
in sodium acetate pH 4.5 at 10 .mu.g/ml at a flow rate of 5
.mu.l/min. This resulted in a surface density of appr. 20000 RU. On
the reference flow cell BSA was immobilized under the same
conditions. The antibodies were diluted in HBS-P to 100 nM and
injected for 3 min (association phase). After washing with running
buffer for 3 min (dissociation phase), the surface was regenerated
by injecting 10 mM sodium hydroxide for 1 min at 5 .mu.l/min.
Results are shown in FIG. 8: Ang2k_LC08 had a halftime of complex
dissociation of approximately 50s, Ang2i_LC06 of appr. 5s and
Ang2i_LC10 showed no binding to ANG-1.
Example 8
Prevention of Metastasis/Secondary Tumors In Vivo in Bearing
Primary Tumors
[0201] a) Prevention of Metastasis/Secondary in Mice Xenografted
with Primary Colo205 Tumors Cell Lines and Culture Conditions:
[0202] Colo205 human colorectal cancer cells were originally
obtained from ATCC.RTM. and after expansion deposited in the Roche
Penzberg internal cell bank. Tumor cell line was 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 3 was used for transplantation.
Animals:
[0203] 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 was 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 was about 10
weeks.
Tumor Cell Injection:
[0204] At the day of injection, Colo205 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.TM. 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 was 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
were 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. Tumor growth of the primary
tumors was monitored (data not shown)
Monitoring of Secondary Tumors e.g. in the Lung by Quantification
of Human Alu Sequences
[0205] At study termination (day 103) lungs were collected from
animals of all groups. Briefly, samples are transferred immediately
into fluid nitrogen. In a further step total DNA was isolated from
the samples with MagNA Pure.RTM. LC Instrument according to
manufacturer's instructions. Human Alu specific primers were chosen
for selective amplification of Alu sequences by quantitative PCR
(LightCycler.RTM. instrument). (T. Schneider et. al., Clin. Exp.
Metas. 2002; 19: 571-582).
Treatment of Animals
[0206] Treatment of animals with Avastin.RTM. (10 mg/kg i.p. once
weekly) was started 14 days after cell transplantation (study
Ang2_PZ_Colo205_008) at a mean tumor volume of 340 mm.sup.3. After
7 weeks mice were randomized for subsequent secondary treatment
starting at day 51 with compounds listed in table below. Secondary
treatment starting at day 51 of Study Ang2_PZ_Colo205_008.
TABLE-US-00008 Cumulative No of Dose Route/Mode of No of dose Group
animals Compound (mg/kg) administration treatments (mg/kg) 10
Avastin .RTM. 10 mg/kg i.p. once weekly 11 110 10 LC06 + 10 mg/kg
i.p. once weekly 6 60 Avastin .RTM. 10 mg/kg i.p. once weekly 11
110 10 LC06 10 mg/kg i.p. once weekly 6 60
Results of Prevention of Metastasis/Secondary Tumors (in the Lung)
are Listed in the Table Below and Shown in FIG. 9A
TABLE-US-00009 [0207] TABLE 1 Quantification of human ALU DNA in
the lungs of mice originally bearing primary Colo205 tumors, after
treatment with different antibodies Avastin Avastin .RTM. +
Ang2i-LC06 Ang2i_LC06 101 0.0264 201 0.0042 301 0.0047 102 5.6740
202 0.0044 302 0.0055 103 0.0307 203 0.0065 303 0.0050 104 0.0203
204 0.0081 304 0.0064 105 0.0215 205 0.0063 305 0.0062 106 0.0338
206 0.0061 306 0.0066 107 0.0075 207 0.0053 307 0.0250 108 0.0113
208 0.0506 308 0.0062 109 0.0087 209 0.0065 309 0.0067 110 0.0587
210 0.0160 310 0.0064 mean 0.5893 0.0114 0.0079 median 0.0240
0.0064 0.0063
[0208] Results show a clearly improved prevention of secondary
tumors/metastasis by ANG2i-LC06 compared with Avastin.RTM.
b) Prevention of Metastasis/Secondary in Mice Xenografted with
Primary KPL-4 Tumors Tumor Cell Line
[0209] The human breast cancer cell line KPL-4 (kindly provided by
Prof. J. Kurebayashi) has been established from the malignant
pleural effusion of a breast cancer patient with an inflammatory
skin metastasis. Tumor cells are 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 is performed with trypsin/EDTA 1.times. (PAN)
splitting three times/week.
Mice
[0210] 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 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
[0211] 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.TM. 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 30 G.times.1/2'' needle. Tumor growth of the primary
tumors was monitored (data not shown)
Monitoring of Secondary Tumors e.g. in the Lung by Quantification
of Human Alu Sequences
[0212] At study termination (day 103) lungs were collected from
animals of all groups. Briefly, samples are transferred immediately
into fluid nitrogen. In a further step total DNA was isolated from
the samples with MagNA Pure.RTM. LC Instrument according to
manufacturer's instructions. Human Alu specific primers were chosen
for selective amplification of Alu sequences by quantitative PCR
(LightCycler.RTM. instrument). (T. Schneider et. al., Clin. Exp.
Metas. 2002; 19: 571-582)
Treatment of Animals
[0213] Treatment of animals was started 35 days after cell
transplantation at a mean tumor volume of 60-160 mm.sup.3.
Compounds and dose schedule is listed in the table below.
TABLE-US-00010 Cumulative No of Dose Route/Mode of No of dose Group
animals Compound (mg/kg) administration treatments (mg/kg) 10
Vehicle i.p. twice weekly 5 10 Xolair .RTM. 10 i.p. twice weekly 5
50 10 Ang2i_LC06 10 i.p. once weekly 4 40 10 Ang2i_LC07 10 i.p.
once weekly 4 40 10 Ang2k_LC08 10 i.p. once weekly 4 40
Results of prevention of metastasis/secondary tumors (in the lung)
are listed in the table below and shown in FIG. 9B
TABLE-US-00011 TABLE 2 Quantification of human ALU DNA in the lungs
of mice originally bearing primary KPL4 tumors, after treatment
with different antibodies Vehicle Xolair .RTM. Ang2i_LC06
Ang2i_LC07 Ang2i_LC08 101 0.0098 201 0.0157 401 0.0273 501 0.0069
102 0.0090 202 0.0516 302 0.0076 402 0.0060 502 0.0261 103 0.0119
203 0.0108 303 0.0413 403 0.0046 503 0.0067 104 0.0405 204 0.0148
304 0.0042 404 0.0164 504 0.0044 205 0.0020 305 0.0041 405 0.0040
505 0.0039 106 0.0381 206 0.0340 306 0.0093 406 0.0044 506 0.0051
107 0.0281 207 0.0141 307 0.0038 407 0.0060 507 0.0037 208 0.0422
308 0.0044 408 0.0174 508 0.0037 109 0.0121 209 0.0227 309 0.0036
409 0.0314 509 0.0051 110 0.0143 210 0.0383 310 0.0094 410 0.0083
540 0.0200 median 0.0132 0.0192 0.0044 0.0072 0.0051 mean 0.0205
0.0246 0.0098 0.0126 0.0086
Results show a very efficient prevention of secondary
tumors/metastasis by ANG2i-LC06, ANG2i-LC07, ANG2k-LC08.
Example 9
Effects in the Treatment of Retinopathy
Methods
[0214] C57/Bl6 pups are cross fostered to CD1 nursing dams and are
exposed to 75% oxygen from P7 to P12 (PRO-OX 110 chamber oxygen
controller, Biospherix Ltd, Redfield, N.Y.) which induces vessel
obliteration and cessation of capillaries in the centre of the
retina. The pups and nursing dams are placed in normal air leading
to relative hypoxia and the induction of neovascularisation. On
P13, pups were anaesthetised using isofluorane (5% induction, 3%
maintenance combined with 1.5% oxygen) and the eye was exposed and
1 .mu.l intraocular injections using a Nanofil syringe fitted with
a 35 gauge needle (WPI, Sarasota, Fla.) into the left eye was
performed. On P17, both eyes were dissected, fixed in 4%
paraformaldehyde for 4 h at 4.degree. C. and retinas were
dissected. Retinas were permeabilised in PBS containing 0.5% Triton
X-100 and 1% bovine serum albumin, stained with 20 .mu.g/ml
biotinylated isolectin B4 (Sigma Aldrich, Gillingham, UK) in PBS pH
6.8, 1% Triton-X100, 0.1 mM CaCl.sub.2, 0.1 mM MgCl.sub.2, followed
by 20 .mu.g/ml ALEXA 488-streptavidin (Molecular Probes, Eugene,
Oreg.) and flat mounted in Vectashield.RTM. (Vector Laboratories,
Burlingame, Calif.). Retinas were imaged using a Nikon
epi-fluorescence microscope at 4.times. magnification.
Quantification of neovascular and ischaemic areas were performed in
a blinded fashion using Photoshop.RTM. CS3 along with Image J (NIH)
and expressed as percentage of total retinal area
(=normal+ischaemic+neovascular).
Results
[0215] FIG. 10A show representative flat mounted retinas with the
retinal vasculature visualised by isolectin staining. The centre
ischemic areas induce neovascularisation and re-growth of the
retinal vessels by upregulation of angiogenic inducers. The
neovascular front is hyperproliferative leading to tortuous vessels
in an irregular vessel pattern. The most outer areas contain the
normal unaffected vessels. Quantification of retinal flat mounts
showed that inhibition of VEGF with Avastin.RTM. reduced retinal
neovascularisation (see FIG. 10B, uninjected 36.7.+-.1.8% to
injected 22.4.+-.3.0%) as expected. Inhibition of Ang2 using
antibodies LC06 or LC08 also led to a reduction in
neovascularisation (31.5.+-.1.1% to 18.8.+-.1.3% and 34.0.+-.3.1%
to 25.4.+-.3.4%). Control injection of human Ig G had no effect on
neovascularisation (see FIG. 10B, 38.3.+-.1.1% to 38.3.+-.0.8%).
Sequence CWU 1
1
63120PRTArtificialheavy chain CDR3, <ANG-2>Ang2i_LC06 1Ser
Pro Asn Pro Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr Tyr Pro Gly 1 5 10
15 Ala Phe Asp Ile 20 217PRTArtificialheavy chain CDR2,
<ANG-2>Ang2i_LC06 2Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn
Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly 35PRTArtificialheavy chain
CDR1, <ANG-2>Ang2i_LC06 3Gly Tyr Tyr Met His 1 5
411PRTArtificiallight chain CDR3, <ANG-2>Ang2i_LC06 4Gln Val
Trp Asp Ser Ser Ser Asp His Tyr Val 1 5 10 57PRTArtificiallight
chain CDR2, <ANG-2>Ang2i_LC06 5Asp Asp Ser Asp Arg Pro Ser 1
5 611PRTArtificiallight chain CDR1, <ANG-2>Ang2i_LC06 6Gly
Gly Asn Asn Ile Gly Ser Lys Ser Val His 1 5 10
7129PRTArtificialheavy chain variable domain,
<ANG-2>Ang2i_LC06 7Gln Val Gln Leu Val Glu 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 8110PRTArtificiallight chain variable
domain, <ANG-2>Ang2i_LC06 8Gln 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 100 105 110 920PRTArtificialheavy chain CDR3,
<ANG-2>Ang2i_LC07 9Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser Ser
Gly Tyr Tyr Tyr Pro Gly 1 5 10 15 Ala Phe Asp Ile 20
1017PRTArtificialheavy chain CDR2, <ANG-2>Ang2i_LC07 10Trp
Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln 1 5 10
15 Gly 115PRTArtificialheavy chain CDR1, <ANG-2>Ang2i_LC07
11Gly Tyr Tyr Met His 1 5 1211PRTArtificiallight chain CDR3,
<ANG-2>Ang2i_LC07 12Gln Val Trp Asp Ser Asp Ser Asp Gln Gly
Val 1 5 10 137PRTArtificiallight chain CDR2,
<ANG-2>Ang2i_LC07 13Asp Asp Ser Glu Arg Pro Ser 1 5
1411PRTArtificiallight chain CDR1, <ANG-2>Ang2i_LC07 14Gly
Gly Asn Phe Ile Gly Gly Lys Ser Val His 1 5 10
15129PRTArtificialheavy chain variable domain,
<ANG-2>Ang2i_LC07 15Gln Val Gln Leu Val Glu 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 16110PRTArtificiallight chain variable
domain, <ANG-2>Ang2i_LC07 16Gln Pro Val Leu Thr Gln Ser Pro
Ser Val Ser Val Ala Pro Gly Glu 1 5 10 15 Thr Ala Arg Val Ala Cys
Gly Gly Asn Phe Ile Gly Gly Lys Ser Val 20 25 30 His Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Asp Asp
Ser Glu Arg Pro Ser Gly Ile Pro Glu Arg Ile Ser Gly Ser 50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Ile Ile Thr Arg Ala Glu Ala Gly 65
70 75 80 Asp Glu Ala Asp Tyr His Cys Gln Val Trp Asp Ser Asp Ser
Asp Gln 85 90 95 Gly Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu
Gly Gln 100 105 110 1715PRTArtificialheavy chain CDR3,
<ANG-2>Ang2k_LC08 17Pro Thr Leu Asp Ile Tyr Met Gly Tyr Tyr
Tyr Gly Met Asp Val 1 5 10 15 1817PRTArtificialheavy chain CDR2,
<ANG-2>Ang2k_LC08 18Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr
Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 195PRTArtificialheavy chain
CDR1, <ANG-2>Ang2k_LC08 19Ser Tyr Gly Met His 1 5
2011PRTArtificiallight chain CDR3, <ANG-2>Ang2k_LC08 20Ala
Ala Trp Asp Asp Ser Leu Asn Gly Pro Val 1 5 10
217PRTArtificiallight chain CDR2, <ANG-2>Ang2k_LC08 21Asn Asn
Asp Gln Arg Pro Ser 1 5 2213PRTArtificiallight chain CDR1,
<ANG-2>Ang2k_LC08 22Ser Gly Phe Ala Ser Asn Ile Gly Ser Asn
Ser Val Asn 1 5 10 23124PRTArtificialheavy chain variable domain,
<ANG-2> Ang2k_LC08 23Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 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 Pro Thr Leu Asp Ile Tyr Met Gly Tyr Tyr Tyr Gly Met
Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
120 24112PRTArtificiallight chain variable domain, <ANG-2>
Ang2k_LC08 24Gln Pro Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Ala
Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Phe Ala Ser
Asn Ile Gly Ser Asn 20 25 30 Ser Val Asn Trp Tyr Gln Gln Val Pro
Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asn Asn Asp Gln Arg
Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Arg Ser Gly
Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp
Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Asn
Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105
110 257PRTArtificialheavy chain CDR3, <ANG-2> Ang2s_LC09
25Asp Leu Gly Tyr Asp Tyr Val 1 5 2619PRTArtificialheavy chain
CDR2, <ANG-2> Ang2s_LC09 26Arg Ile Lys Ser Lys Thr Asp Gly
Gly Thr Thr Asp Tyr Ala Ala Pro 1 5 10 15 Val Lys Gly
275PRTArtificialheavy chain CDR1, <ANG-2> Ang2s_LC09 27Asn
Ala Trp Met Ser 1 5 287PRTArtificiallight chain CDR3, <ANG-2>
Ang2s_LC09 28Met Gln Ala Leu Gln Ile Pro 1 5 297PRTArtificiallight
chain CDR2, <ANG-2> Ang2s_LC09 29Leu Gly Ser Asn Arg Ala Ser
1 5 3016PRTArtificiallight chain CDR1, <ANG-2> Ang2s_LC09
30Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp 1
5 10 15 31119PRTArtificialheavy chain variable domain,
<ANG-2> Ang2s_LC09 31Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Lys
Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60 Pro Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85
90 95 Tyr Cys Thr Thr Asp Leu Gly Tyr Asp Tyr Val Trp Gly Ser Pro
Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
32109PRTArtificiallight chain variable domain, <ANG-2>
Ang2s_LC09 32Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro
Ala Ser Ile 1 5 10 15 Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
Asn Gly Tyr Asn Tyr 20 25 30 Leu Asp Trp Tyr Leu Gln Lys Pro Gly
Gln Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Leu Gly Ser Asn Arg Ala
Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala 65 70 75 80 Glu Asp Val
Gly Val Tyr Tyr Cys Met Gln Ala Leu Gln Ile Pro Phe 85 90 95 Thr
Phe Gly Pro Gly Thr Lys Val Thr Val Leu Arg Thr 100 105
3320PRTArtificialheavy chain CDR3, <ANG-2> Ang2i_LC10 33Ser
Pro Asn Pro Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr Tyr Pro Gly 1 5 10
15 Ala Phe Asp Ile 20 3417PRTArtificialheavy chain CDR2,
<ANG-2> Ang2i_LC10 34Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn
Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly 355PRTArtificialheavy chain
CDR1, <ANG-2> Ang2i_LC10 35Gly Tyr Tyr Met His 1 5
3611PRTArtificiallight chain CDR3, <ANG-2> Ang2i_LC10 36Gln
Val Trp Asp Ser Ser Ser Asp His Trp Val 1 5 10
377PRTArtificiallight chain CDR2, <ANG-2> Ang2i_LC10 37Asp
Asp Ser Asp Arg Pro Ser 1 5 3811PRTArtificiallight chain CDR1,
<ANG-2> Ang2i_LC10 38Gly Gly Asn Asn Ile Gly Ser Lys Ser Val
His 1 5 10 39129PRTArtificialheavy chain variable domain,
<ANG-2> Ang2i_LC10 39Gln Val Gln Leu Val Glu 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 40105PRTArtificiallight chain variable
domain, <ANG-2> Ang2i_LC10 40Gln Pro Pro Ser Val Ser Val Ala
Pro Gly Gln Thr Ala Arg Ile Thr 1 5 10 15 Cys Gly Gly Asn Asn Ile
Gly Ser Lys Ser Val His Trp Tyr Gln Gln 20 25 30 Lys Pro Gly Gln
Ala Pro Val Leu Val Val Tyr Asp Asp Ser Asp Arg 35 40 45 Pro Ser
Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr 50 55 60
Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr 65
70 75 80 Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His Trp Val Phe
Gly Gly 85 90 95 Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105
4115PRTArtificialheavy chain CDR3, <ANG-2> Ang2k_LC11 41Pro
Thr Leu Asp Ile Tyr Met Gly Tyr Tyr Tyr Gly Met Asp Val 1 5 10 15
4217PRTArtificialheavy chain CDR2, <ANG-2> Ang2k_LC11 42Val
Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10
15 Gly 435PRTArtificialheavy chain CDR1, <ANG-2> Ang2k_LC11
43Ser Tyr Gly Met His 1 5 4412PRTArtificiallight chain CDR3,
<ANG-2> Ang2k_LC11 44Gln Val Trp Asp Ser Ser Ser Asp His Pro
Gly Val 1 5 10 457PRTArtificiallight chain CDR2, <ANG-2>
Ang2k_LC11 45Asp Asp Ser Asp Arg Pro Ser 1 5 4611PRTArtificiallight
chain CDR1, <ANG-2> Ang2k_LC11 46Gly Gly Asn Asn Ile Gly Ser
Lys Ser Val His 1 5 10 47124PRTArtificialheavy chain variable
domain, <ANG-2> Ang2k_LC11 47Gln Val Gln Leu Val Glu Ser Gly
Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val
Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 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 Pro Thr Leu Asp Ile Tyr Met Gly Tyr Tyr
Tyr Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val
Ser Ser 115 120 48106PRTArtificiallight chain variable domain,
<ANG-2> Ang2k_LC11misc_feature(98)..(98)Xaa can be any
naturally occurring amino acidmisc_feature(102)..(102)Xaa can be
any naturally occurring amino acid 48Gln Pro Pro Ser Val Ser Val
Ala Pro Gly Gln Thr Ala Arg Ile Thr 1 5 10 15 Cys Gly Gly Asn Asn
Ile Gly Ser Lys Ser Val His Trp Tyr Gln Gln 20 25 30 Lys Pro Gly
Gln Ala Pro Val Leu Val Val Tyr Asp Asp Ser Asp Arg 35 40 45 Pro
Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr 50 55
60 Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr
65 70 75 80 Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His Pro Gly Val
Phe Gly 85 90 95 Gly Xaa Thr Lys Leu Xaa Val Leu Gly Gln 100 105
497PRTArtificialheavy chain CDR3, <ANG-2> Ang2s_R3_LC03 49Asp
Leu Gly Tyr Asp Tyr Val 1 5 5019PRTArtificialheavy chain CDR2,
<ANG-2> Ang2s_R3_LC03 50Arg Ile Lys Ser Lys Thr Asp Gly Gly
Thr Thr Asp Tyr Ala Ala Pro 1 5 10 15 Val Lys Gly
515PRTArtificialheavy chain CDR1, <ANG-2> Ang2s_R3_LC03 51Asn
Ala Trp Met Ser 1 5
5210PRTArtificiallight chain CDR3, <ANG-2> Ang2s_R3_LC03
52Gln Gln Tyr Asp Asn Leu Pro Met Tyr Thr 1 5 10
537PRTArtificiallight chain CDR2, <ANG-2> Ang2s_R3_LC03 53His
Ala Ser Asn Leu Glu Thr 1 5 5411PRTArtificiallight chain CDR1,
<ANG-2> Ang2s_R3_LC03 54Gln Ala Ser Gln Asp Ile Ser Asn Arg
Leu Asn 1 5 10 55118PRTArtificialheavy chain variable domain,
<ANG-2> Ang2s_R3_LC03 55Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile
Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60 Pro
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70
75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val
Tyr 85 90 95 Tyr Cys Thr Thr Asp Leu Gly Tyr Asp Tyr Val Trp Gly
Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115
56110PRTArtificiallight chain variable domain, <ANG-2>
Ang2s_R3_LC03 56Asp Ile Gln Val Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln
Asp Ile Ser Asn Arg 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr His Ala Ser Asn Leu Glu
Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile
Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro Met 85 90 95 Tyr
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr 100 105 110
57330PRTHomo sapiens 57Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90
95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215
220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 325 330 58327PRTHomo sapiens 58Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10
15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr
Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145
150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265
270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325
59107PRTHomo sapiens 59Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90
95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 60104PRTHomo
sapiens 60Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser
Glu Glu 1 5 10 15 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile
Ser Asp Phe Tyr 20 25 30 Pro Gly Ala Val Thr Val Ala Trp Lys Ala
Asp Ser Ser Pro Val Lys 35 40 45 Ala Gly Val Glu Thr Thr Thr Pro
Ser Lys Gln Ser Asn Asn Lys Tyr 50 55 60 Ala Ala Ser Ser Tyr Leu
Ser Leu Thr Pro Glu Gln Trp Lys Ser His 65 70 75 80 Arg Ser Tyr Ser
Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 85 90 95 Thr Val
Ala Pro Thr Glu Cys Ser 100 611124PRTHomo sapiens 61Met Asp Ser Leu
Ala Ser Leu Val Leu Cys Gly Val Ser Leu Leu Leu 1 5 10 15 Ser Gly
Thr Val Glu Gly Ala Met Asp Leu Ile Leu Ile Asn Ser Leu 20 25 30
Pro Leu Val Ser Asp Ala Glu Thr Ser Leu Thr Cys Ile Ala Ser Gly 35
40 45 Trp Arg Pro His Glu Pro Ile Thr Ile Gly Arg Asp Phe Glu Ala
Leu 50 55 60 Met Asn Gln His Gln Asp Pro Leu Glu Val Thr Gln Asp
Val Thr Arg 65 70 75 80 Glu Trp Ala Lys Lys Val Val Trp Lys Arg Glu
Lys Ala Ser Lys Ile 85 90 95 Asn Gly Ala Tyr Phe Cys Glu Gly Arg
Val Arg Gly Glu Ala Ile Arg 100 105 110 Ile Arg Thr Met Lys Met Arg
Gln Gln Ala Ser Phe Leu Pro Ala Thr 115 120 125 Leu Thr Met Thr Val
Asp Lys Gly Asp Asn Val Asn Ile Ser Phe Lys 130 135 140 Lys Val Leu
Ile Lys Glu Glu Asp Ala Val Ile Tyr Lys Asn Gly Ser 145 150 155 160
Phe Ile His Ser Val Pro Arg His Glu Val Pro Asp Ile Leu Glu Val 165
170 175 His Leu Pro His Ala Gln Pro Gln Asp Ala Gly Val Tyr Ser Ala
Arg 180 185 190 Tyr Ile Gly Gly Asn Leu Phe Thr Ser Ala Phe Thr Arg
Leu Ile Val 195 200 205 Arg Arg Cys Glu Ala Gln Lys Trp Gly Pro Glu
Cys Asn His Leu Cys 210 215 220 Thr Ala Cys Met Asn Asn Gly Val Cys
His Glu Asp Thr Gly Glu Cys 225 230 235 240 Ile Cys Pro Pro Gly Phe
Met Gly Arg Thr Cys Glu Lys Ala Cys Glu 245 250 255 Leu His Thr Phe
Gly Arg Thr Cys Lys Glu Arg Cys Ser Gly Gln Glu 260 265 270 Gly Cys
Lys Ser Tyr Val Phe Cys Leu Pro Asp Pro Tyr Gly Cys Ser 275 280 285
Cys Ala Thr Gly Trp Lys Gly Leu Gln Cys Asn Glu Ala Cys His Pro 290
295 300 Gly Phe Tyr Gly Pro Asp Cys Lys Leu Arg Cys Ser Cys Asn Asn
Gly 305 310 315 320 Glu Met Cys Asp Arg Phe Gln Gly Cys Leu Cys Ser
Pro Gly Trp Gln 325 330 335 Gly Leu Gln Cys Glu Arg Glu Gly Ile Pro
Arg Met Thr Pro Lys Ile 340 345 350 Val Asp Leu Pro Asp His Ile Glu
Val Asn Ser Gly Lys Phe Asn Pro 355 360 365 Ile Cys Lys Ala Ser Gly
Trp Pro Leu Pro Thr Asn Glu Glu Met Thr 370 375 380 Leu Val Lys Pro
Asp Gly Thr Val Leu His Pro Lys Asp Phe Asn His 385 390 395 400 Thr
Asp His Phe Ser Val Ala Ile Phe Thr Ile His Arg Ile Leu Pro 405 410
415 Pro Asp Ser Gly Val Trp Val Cys Ser Val Asn Thr Val Ala Gly Met
420 425 430 Val Glu Lys Pro Phe Asn Ile Ser Val Lys Val Leu Pro Lys
Pro Leu 435 440 445 Asn Ala Pro Asn Val Ile Asp Thr Gly His Asn Phe
Ala Val Ile Asn 450 455 460 Ile Ser Ser Glu Pro Tyr Phe Gly Asp Gly
Pro Ile Lys Ser Lys Lys 465 470 475 480 Leu Leu Tyr Lys Pro Val Asn
His Tyr Glu Ala Trp Gln His Ile Gln 485 490 495 Val Thr Asn Glu Ile
Val Thr Leu Asn Tyr Leu Glu Pro Arg Thr Glu 500 505 510 Tyr Glu Leu
Cys Val Gln Leu Val Arg Arg Gly Glu Gly Gly Glu Gly 515 520 525 His
Pro Gly Pro Val Arg Arg Phe Thr Thr Ala Ser Ile Gly Leu Pro 530 535
540 Pro Pro Arg Gly Leu Asn Leu Leu Pro Lys Ser Gln Thr Thr Leu Asn
545 550 555 560 Leu Thr Trp Gln Pro Ile Phe Pro Ser Ser Glu Asp Asp
Phe Tyr Val 565 570 575 Glu Val Glu Arg Arg Ser Val Gln Lys Ser Asp
Gln Gln Asn Ile Lys 580 585 590 Val Pro Gly Asn Leu Thr Ser Val Leu
Leu Asn Asn Leu His Pro Arg 595 600 605 Glu Gln Tyr Val Val Arg Ala
Arg Val Asn Thr Lys Ala Gln Gly Glu 610 615 620 Trp Ser Glu Asp Leu
Thr Ala Trp Thr Leu Ser Asp Ile Leu Pro Pro 625 630 635 640 Gln Pro
Glu Asn Ile Lys Ile Ser Asn Ile Thr His Ser Ser Ala Val 645 650 655
Ile Ser Trp Thr Ile Leu Asp Gly Tyr Ser Ile Ser Ser Ile Thr Ile 660
665 670 Arg Tyr Lys Val Gln Gly Lys Asn Glu Asp Gln His Val Asp Val
Lys 675 680 685 Ile Lys Asn Ala Thr Ile Thr Gln Tyr Gln Leu Lys Gly
Leu Glu Pro 690 695 700 Glu Thr Ala Tyr Gln Val Asp Ile Phe Ala Glu
Asn Asn Ile Gly Ser 705 710 715 720 Ser Asn Pro Ala Phe Ser His Glu
Leu Val Thr Leu Pro Glu Ser Gln 725 730 735 Ala Pro Ala Asp Leu Gly
Gly Gly Lys Met Leu Leu Ile Ala Ile Leu 740 745 750 Gly Ser Ala Gly
Met Thr Cys Leu Thr Val Leu Leu Ala Phe Leu Ile 755 760 765 Ile Leu
Gln Leu Lys Arg Ala Asn Val Gln Arg Arg Met Ala Gln Ala 770 775 780
Phe Gln Asn Val Arg Glu Glu Pro Ala Val Gln Phe Asn Ser Gly Thr 785
790 795 800 Leu Ala Leu Asn Arg Lys Val Lys Asn Asn Pro Asp Pro Thr
Ile Tyr 805 810 815 Pro Val Leu Asp Trp Asn Asp Ile Lys Phe Gln Asp
Val Ile Gly Glu 820 825 830 Gly Asn Phe Gly Gln Val Leu Lys Ala Arg
Ile Lys Lys Asp Gly Leu 835 840 845 Arg Met Asp Ala Ala Ile Lys Arg
Met Lys Glu Tyr Ala Ser Lys Asp 850 855 860 Asp His Arg Asp Phe Ala
Gly Glu Leu Glu Val Leu Cys Lys Leu Gly 865 870 875 880 His His Pro
Asn Ile Ile Asn Leu Leu Gly Ala Cys Glu His Arg Gly 885 890 895 Tyr
Leu Tyr Leu Ala Ile Glu Tyr Ala Pro His Gly Asn Leu Leu Asp 900 905
910 Phe Leu Arg Lys Ser Arg Val Leu Glu Thr Asp Pro Ala Phe Ala Ile
915 920 925 Ala Asn Ser Thr Ala Ser Thr Leu Ser Ser Gln Gln Leu Leu
His Phe 930 935 940 Ala Ala Asp Val Ala Arg Gly Met Asp Tyr Leu Ser
Gln Lys Gln Phe 945 950 955 960 Ile His Arg Asp Leu Ala Ala Arg Asn
Ile Leu Val Gly Glu Asn Tyr 965 970 975 Val Ala Lys Ile Ala Asp Phe
Gly Leu Ser Arg Gly Gln Glu Val Tyr 980 985 990 Val Lys Lys Thr Met
Gly Arg Leu Pro Val Arg Trp Met Ala Ile Glu 995 1000 1005 Ser Leu
Asn Tyr Ser Val Tyr Thr Thr Asn Ser Asp Val Trp Ser 1010 1015 1020
Tyr Gly Val Leu Leu Trp Glu Ile Val Ser Leu Gly Gly Thr Pro 1025
1030 1035 Tyr Cys Gly Met Thr Cys Ala Glu Leu Tyr Glu Lys Leu Pro
Gln 1040 1045 1050 Gly Tyr Arg Leu Glu Lys Pro Leu Asn Cys Asp Asp
Glu Val Tyr 1055 1060 1065 Asp Leu Met Arg Gln Cys Trp Arg Glu Lys
Pro Tyr Glu Arg Pro 1070 1075 1080 Ser Phe Ala Gln Ile Leu Val Ser
Leu Asn Arg Met Leu Glu Glu 1085 1090 1095 Arg Lys Thr Tyr Val Asn
Thr Thr Leu Tyr Glu Lys Phe Thr Tyr 1100 1105
1110 Ala Gly Ile Asp Cys Ser Ala Glu Glu Ala Ala 1115 1120
62504PRTArtificialHuman angiopoietin-2 (ANG-2) with leader and
His-tag 62Met 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 63506PRTArtificialHuman
angiopoietin-1 (ANG-1) with leader and His-tag 63Met Thr Val Phe
Leu Ser Phe Ala Phe Leu Ala Ala Ile Leu Thr His 1 5 10 15 Ile Gly
Cys Ser Asn Gln Arg Arg Ser Pro Glu Asn Ser Gly Arg Arg 20 25 30
Tyr Asn Arg Ile Gln His Gly Gln Cys Ala Tyr Thr Phe Ile Leu Pro 35
40 45 Glu His Asp Gly Asn Cys Arg Glu Ser Thr Thr Asp Gln Tyr Asn
Thr 50 55 60 Asn Ala Leu Gln Arg Asp Ala Pro His Val Glu Pro Asp
Phe Ser Ser 65 70 75 80 Gln Lys Leu Gln His Leu Glu His Val Met Glu
Asn Tyr Thr Gln Trp 85 90 95 Leu Gln Lys Leu Glu Asn Tyr Ile Val
Glu Asn Met Lys Ser Glu Met 100 105 110 Ala Gln Ile Gln Gln Asn Ala
Val Gln Asn His Thr Ala Thr Met Leu 115 120 125 Glu Ile Gly Thr Ser
Leu Leu Ser Gln Thr Ala Glu Gln Thr Arg Lys 130 135 140 Leu Thr Asp
Val Glu Thr Gln Val Leu Asn Gln Thr Ser Arg Leu Glu 145 150 155 160
Ile Gln Leu Leu Glu Asn Ser Leu Ser Thr Tyr Lys Leu Glu Lys Gln 165
170 175 Leu Leu Gln Gln Thr Asn Glu Ile Leu Lys Ile His Glu Lys Asn
Ser 180 185 190 Leu Leu Glu His Lys Ile Leu Glu Met Glu Gly Lys His
Lys Glu Glu 195 200 205 Leu Asp Thr Leu Lys Glu Glu Lys Glu Asn Leu
Gln Gly Leu Val Thr 210 215 220 Arg Gln Thr Tyr Ile Ile Gln Glu Leu
Glu Lys Gln Leu Asn Arg Ala 225 230 235 240 Thr Thr Asn Asn Ser Val
Leu Gln Lys Gln Gln Leu Glu Leu Met Asp 245 250 255 Thr Val His Asn
Leu Val Asn Leu Cys Thr Lys Glu Gly Val Leu Leu 260 265 270 Lys Gly
Gly Lys Arg Glu Glu Glu Lys Pro Phe Arg Asp Cys Ala Asp 275 280 285
Val Tyr Gln Ala Gly Phe Asn Lys Ser Gly Ile Tyr Thr Ile Tyr Ile 290
295 300 Asn Asn Met Pro Glu Pro Lys Lys Val Phe Cys Asn Met Asp Val
Asn 305 310 315 320 Gly Gly Gly Trp Thr Val Ile Gln His Arg Glu Asp
Gly Ser Leu Asp 325 330 335 Phe Gln Arg Gly Trp Lys Glu Tyr Lys Met
Gly Phe Gly Asn Pro Ser 340 345 350 Gly Glu Tyr Trp Leu Gly Asn Glu
Phe Ile Phe Ala Ile Thr Ser Gln 355 360 365 Arg Gln Tyr Met Leu Arg
Ile Glu Leu Met Asp Trp Glu Gly Asn Arg 370 375 380 Ala Tyr Ser Gln
Tyr Asp Arg Phe His Ile Gly Asn Glu Lys Gln Asn 385 390 395 400 Tyr
Arg Leu Tyr Leu Lys Gly His Thr Gly Thr Ala Gly Lys Gln Ser 405 410
415 Ser Leu Ile Leu His Gly Ala Asp Phe Ser Thr Lys Asp Ala Asp Asn
420 425 430 Asp Asn Cys Met Cys Lys Cys Ala Leu Met Leu Thr Gly Gly
Trp Trp 435 440 445 Phe Asp Ala Cys Gly Pro Ser Asn Leu Asn Gly Met
Phe Tyr Thr Ala 450 455 460 Gly Gln Asn His Gly Lys Leu Asn Gly Ile
Lys Trp His Tyr Phe Lys 465 470 475 480 Gly Pro Ser Tyr Ser Leu Arg
Ser Thr Thr Met Met Ile Arg Pro Leu 485 490 495 Asp Phe Ser Gly His
His His His His His 500 505
* * * * *