U.S. patent application number 13/524325 was filed with the patent office on 2012-10-11 for antibodies directed to angiopoietin-1 and angiopoietin-2 and uses thereof.
Invention is credited to Thomas C. Boone, Jonathan D. Oliner.
Application Number | 20120258122 13/524325 |
Document ID | / |
Family ID | 40625974 |
Filed Date | 2012-10-11 |
United States Patent
Application |
20120258122 |
Kind Code |
A1 |
Boone; Thomas C. ; et
al. |
October 11, 2012 |
Antibodies Directed to Angiopoietin-1 and Angiopoietin-2 and Uses
Thereof
Abstract
Disclosed are specific binding agents, such as fully human
antibodies, that bind to angiopoietin 1 and/or angiopoietin-2. Also
disclosed are heavy chain fragments, light chain fragments, and
CDRs of the antibodies, as well as methods of making and using the
antibodies.
Inventors: |
Boone; Thomas C.; (Newbury
Park, CA) ; Oliner; Jonathan D.; (Newbury Park,
CA) |
Family ID: |
40625974 |
Appl. No.: |
13/524325 |
Filed: |
June 15, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13194854 |
Jul 29, 2011 |
8221749 |
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13524325 |
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12378993 |
Feb 19, 2009 |
8030025 |
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13194854 |
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61139361 |
Dec 19, 2008 |
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61061943 |
Jun 16, 2008 |
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61066632 |
Feb 20, 2008 |
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Current U.S.
Class: |
424/172.1 ;
435/320.1; 435/334; 435/69.6; 530/389.8; 536/23.53 |
Current CPC
Class: |
C07K 16/22 20130101;
A61P 35/00 20180101; C07K 2317/24 20130101; C07K 2317/76 20130101;
C07K 2317/92 20130101; A61K 2039/545 20130101; C07K 16/2863
20130101; C07K 2317/55 20130101; C07K 2317/73 20130101; C07K
2317/14 20130101; A61K 2039/505 20130101; C07K 2317/565 20130101;
C07K 2317/21 20130101; C07K 2317/56 20130101 |
Class at
Publication: |
424/172.1 ;
530/389.8; 536/23.53; 435/320.1; 435/334; 435/69.6 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12N 15/13 20060101 C12N015/13; A61P 35/00 20060101
A61P035/00; C12N 5/10 20060101 C12N005/10; C12P 21/02 20060101
C12P021/02; C07K 16/28 20060101 C07K016/28; C12N 15/85 20060101
C12N015/85 |
Claims
1. An isolated antibody which comprises a heavy chain variable
domain having the sequence selected from the group consisting of
SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:
5, SEQ ID NO: 6, and SEQ ID NO: 7; wherein said antibody
specifically binds to at least one of Ang1 and Ang2 ligands of Tie
2 receptor.
2. An isolated antibody which comprises a light chain variable
domain having the sequence selected from the group consisting of
SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID
NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16,
and SEQ ID NO: 17; wherein said antibody specifically binds to at
least one of Ang1 and Ang2 ligands of Tie 2 receptor.
3. An isolated antibody which comprises a heavy chain variable
domain and a light chain variable domain, wherein said heavy chain
is comprised of 3 CDRs and said light chain is comprised of 3 CDRs,
wherein the sequences of said CDRs of said antibody are selected
from the group consisting of: (a) SEQ ID NOs: 18, 26, 32 of the HC
plus SEQ ID NOs: 19, 27, 33 of the LC, (b) SEQ ID NOs: 18, 26, 34
of the HC plus SEQ ID NOs: 19, 27, 33 of the LC, (c) SEQ ID NOs:
18, 26, 35 of the HC plus SEQ ID NOs: 20, 27, 36 of the LC, (d) SEQ
ID NOs: 18, 26, 37 of the HC plus SEQ ID NOs: 19, 27, 33 of the LC,
(e) SEQ ID NOs: 18, 26, 38 of the HC plus SEQ ID NOs: 19, 27, 33 of
the LC, (f) SEQ ID NOs: 18, 26, 35 of the HC plus SEQ ID NOs: 19,
27, 33 of the LC, (g) SEQ ID NOs: 18, 26, 34 of the HC plus SEQ ID
NOs: 21, 27, 33 of the LC, (h) SEQ ID NOs: 18, 28, 39 of the HC
plus SEQ ID NOs: 19, 27, 33 of the LC, (i) SEQ ID NOs: 18, 26, 34
of the HC plus SEQ ID NOs: 22, 27, 33 of the LC, (j) SEQ ID NOs:
18, 26, 32 of the HC plus SEQ ID NOs: 22, 27, 33 of the LC, (k) SEQ
ID NOs: 18, 29, 39 of the HC plus SEQ ID NOs: 19, 27, 33 of the LC,
(l) SEQ ID NOs: 18, 26, 34 of the HC plus SEQ ID NOs: 23, 27, 33 of
the LC, (m) SEQ ID NOs: 18, 26, 35 of the HC plus SEQ ID NOs: 20,
27, 40 of the LC, (n) SEQ ID NOs: 18, 26, 32 of the HC plus SEQ ID
NOs: 21, 27, 33 of the LC, (o) SEQ ID NOs: 18, 26, 35 of the HC
plus SEQ ID NOs: 24, 27, 33 of the LC, (p) SEQ ID NOs: 18, 26, 35
of the HC plus SEQ ID NOs: 21, 27, 33 of the LC, (q) SEQ ID NOs:
18, 26, 35 of the HC plus SEQ ID NOs: 23, 27, 33 of the LC, (r) SEQ
ID NOs: 18, 26, 34 of the HC plus SEQ ID NOs: 20, 30, 33 of the LC,
(s) SEQ ID NOs: 18, 26, 34 of the HC plus SEQ ID NOs: 25, 27, 33 of
the LC, (t) SEQ ID NOs: 18, 26, 35 of the HC plus SEQ ID NOs: 20,
30, 33 of the LC, (u) SEQ ID NOs: 18, 26, 34 of the HC plus SEQ ID
NOs: 20, 27, 40 of the LC, and (v) SEQ ID NOs: 18, 26, 34 of the HC
plus SEQ ID NOs: 20, 31, 33 of the LC; wherein said antibody
specifically binds to at least one of Ang1 and Ang2 ligands of Tie
2 receptor.
4. The isolated antibody of claim 3, wherein said antibody is
selected from the group consisting of: H6L7, H5L7, H4L13, H11L7,
H10L7, H4L7, H5L6, H2L7, H5L8, H6L8, H3L7, H5L4, H4L12, H6L6, H4L2,
H4L6, H4L4, H5L11, H5L1, H4L11, H5L12, and H5L9.
5. The isolated antibody of claim 4 that is a fully human
antibody.
6. An antibody fragment of the antibody of claim 3 which comprises
a CDR region with the amino acid sequence selected from a group
consisting of: SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID
NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:32, SEQ ID NO:34, SEQ
ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:
40.
7. An isolated nucleic acid molecule encoding the antibody or the
antibody fragment of claim 1 or 6.
8. A vector containing a nucleic acid molecule of claim 7.
9. A host cell containing the vector of claim 8.
10. The host cell of claim 9 that is a CHO cell.
11. A method of making the antibody of claim 4 which comprises
expressing said antibody in a host cell.
12. The method of claim 11 wherein said host cell is a CHO
cell.
13. A pharmaceutical composition comprising any one of the
antibodies selected from the group consisting of H6L7, H5L7, H4L13,
H11L7, H10L7, H4L7, H5L6, H2L7, H5L8, H6L8, H3L7, H5L4, H4L12,
H6L6, H4L2, H4L6, H4L4, H5L11, H5L1, H4L11, H5L12, and H5L9; in
admixture with a pharmaceutically acceptable carrier therefor.
14. The pharmaceutical composition of claim 13 further comprising a
molecule selected from the group consisting of a reporter molecule,
a water soluble polymer, an antibody Fc region, and a cytotoxic
agent.
15. The pharmaceutical composition of claim 14, wherein said
pharmaceutically acceptable carrier is a pharmaceutical formulation
agent.
16. A method of inhibiting undesired angiogenesis that comprises
administering to a subject in need thereof, an effective amount of
any one of the antibodies selected from the group consisting of
H6L7, H5L7, H4L13, H11L7, H10L7, H4L7, H5L6, H2L7, H5L8, H6L8,
H3L7, H5L4, H4L12, H6L6, H4L2, H4L6, H4L4, H5L11, H5L1, H4L11,
H5L12, and H5L9.
17. The method of claim 16, wherein said undesired angiogenesis is
cancer.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
13/194,854 filed Jul. 29, 2011, now allowed, which is a divisional
of U.S. Ser. No. 12/378,993 filed Feb. 9, 2009 and now U.S. Pat.
No. 8,030,025 issued on Oct. 4, 2011, which claims the benefit of
U.S. Provisional Application Ser. No. 61/139,361 filed Dec. 19,
2008, and U.S. Provisional Application Ser. No. 61/061,943 filed
Jun. 16, 2008, and U.S. Provisional Application Ser. No. 61/066,632
filed Feb. 20, 2008, which are incorporated herein by
reference.
[0002] The present application is being filed along with a Sequence
Listing in text format. The Sequence Listing is provided as a file
entitled
A-1382-US-CNT_SeqListingAsFiledInParent02192009.sub.--12378993.t-
xt, created Feb. 19, 2009, which is 38 KB in size. The information
in the text format of the Sequence Listing is incorporated herein
by reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to specific binding agents
that recognize and bind to angiopoietins-1 (Ang-1) and/or
angiopoetin-2 (Ang-2). More specifically, the invention relates to
the production, diagnostic use, and therapeutic use of monoclonal
and polyclonal antibodies, and the antigen-binding fragments
thereof, which specifically bind Ang-1 and/or Ang-2. Aspects of the
invention also relate to hybridomas or other cell lines expressing
such antibodies. The described antibodies are useful for
diagnostics and for the treatment of diseases associated with the
activity and overproduction of Ang-1 or Ang-2.
BACKGROUND OF THE INVENTION
[0004] Angiogenesis, the formation of new blood vessels from
existing ones, is essential to many physiological and pathological
processes. Normally, angiogenesis is tightly regulated by pro- and
anti-angiogenic factors, but in the case of diseases such as
cancer, ocular neovascular diseases, arthritis, and psoriasis, the
process can go awry. Folkman, J., Nat. Med., 1:27-31 (1995).
[0005] There are a number of diseases known to be associated with
deregulated or undesired angiogenesis. Such diseases include, but
are not limited to, ocular neovascularisation, such as
retinopathies, including diabetic retinopathy, age-related macular
degeneration, psoriasis, hemangioblastoma, hemangioma,
arteriosclerosis, inflammatory disease, such as a rheumatoid or
rheumatic inflammatory disease, especially arthritis (including
rheumatoid arthritis), or other chronic inflammatory disorders,
such as chronic asthma, arterial or post-transplantational
atherosclerosis, endometriosis, and neoplastic diseases, for
example so-called solid tumors and liquid (or hematopoietic) tumors
(such as leukemias and lymphomas). Other diseases associated with
undesired angiogenesis will be apparent to those skilled in the
art.
[0006] Although many signal transduction systems have been
implicated in the regulation of angiogenesis, one of the
best-characterized and most endothelial cell-selective systems
involves the Tie-2 receptor tyrosine kinase (referred to as "Tie-2"
or "Tie-2R" (also referred to as "ORK"); murine Tie-2 is also
referred to as "tek") and its ligands, the angiopoietins (Gale, N.
W. and Yancopoulos, G. D., Genes Dev. 13:1055-1066 [1999]). There
are 4 known angiopoietins; angiopoietin-1 ("Ang-1") through
angiopoietin-4 ("Ang-4"). These angiopoietins are also referred to
as "Tie-2 ligands". (Davis, S., et al., Cell, 87:1161-1169 [1996];
Grosios, K., et al., Cytogenet Cell Genet, 84:118-120 [1999];
Holash, J., et al., Investigative Ophthalmology & Visual
Science, 42:1617-1625 [1999]; Koblizek, T. I., et al., Current
Biology, 8:529-532 [1998]; Lin, P., et al., Proc Natl Acad Sci USA,
95:8829-8834 [1998]; Maisonpierre, P. C., et al., Science,
277:55-60 [1997]; Papapetropoulos, A., et al., Lab Invest,
79:213-223 [1999]; Sato, T. N., et al., Nature, 375:70-74 [1998];
Shyu, K. G., et al., Circulation, 98:2081-2087 [1998]; Suri, C., et
al., Cell, 87:1171-1180 [1996]; Suri, C., et al., Science,
282:468-471 [1998]; Valenzuela, D. M., et al., Proceedings of the
National Academy of Sciences of the USA, 96:1904-1909 [1999];
Witzenbichler, B., et al., J Biol Chem, 273:18514-18521 [1998]).
Whereas Ang-1 binding to Tie-2 stimulates receptor phosphorylation
in cultured endothelial cells, Ang-2 has been observed to both
agonize and antagonize Tie-2 receptor phosphorylation (Davis, S.,
et al., [1996], supra; Maisonpierre, P. C., et al., [1997], supra;
Kim, I, J. H. Kim, et al., Oncogene 19(39): 4549-4552 (2000);
Teichert-Kuliszewska, K., P. C. Maisonpierre, et al.,
Cardiovascular Research 49(3): 659-70 (2001)).
[0007] The phenotypes of mouse Tie-2 and Ang-1 knockouts are
similar and suggest that Ang-1-stimulated Tie-2 phosphorylation
mediates remodeling and stabilization of developing vessels in
utero through maintenance of endothelial cell-support cell adhesion
(Dumont, D. J., et al., Genes & Development, 8:1897-1909
[1994]; Sato, T. N., et al., Nature, 376:70-74 [1995]; Sun, C., et
al., [1996], supra). The role of Ang-1 in vessel stabilization is
thought to be conserved in the adult, where it is expressed widely
and constitutively (Hanahan, D., Science, 277:48-50 [1997]; Zagzag,
D., et al., Experimental Neurology, 159:391-400 [1999]). In
contrast, Ang-2 expression is primarily limited to sites of
vascular remodeling, where it is thought to block Ang-1 function,
thereby inducing a state of vascular plasticity conducive to
angiogenesis (Hanahan, D., [1997], supra; Holash, J., et al.,
Science, 284:1994-1998 [1999]; Maisonpierre, P. C., et al., [1997],
supra).
[0008] Numerous published studies have purportedly demonstrated
vessel-selective Ang-2 expression in disease states associated with
angiogenesis. These pathological conditions include, for example,
psoriasis, macular degeneration, and cancer (Bunone, G., et al.,
American Journal of Pathology, 155:1967-1976 [1999]; Etoh, T., et
al., Cancer Research, 61:2145-2153 [2001]; Hangai, M., et al.,
Investigative Ophthalmology & Visual Science, 42:1617-1625
[2001]; Holash, J., et al., [1999] supra; Kuroda, K., et al.,
Journal of Investigative Dermatology, 116:713-720 [2001]; Otani,
A., et al., Investigative Ophthalmology & Visual Science,
40:1912-1920 [1999]; Stratmann, A., et al., American Journal of
Pathology, 153:1459-1466 [1998]; Tanaka, S., et al., J Clin Invest,
103:34-345 [1999]; Yoshida, Y., et al., International Journal of
Oncology, 15:1221-1225 [1999]; Yuan, K., et al., Journal of
Periodontal Research, 35:165-171 [2000]; Zagzag, D., et al.,
supra). Most of these studies have focused on cancer, in which many
tumor types appear to display vascular Ang-2 expression. In
contrast with its expression in pathological angiogenesis, Ang-2
expression in normal tissues is extremely limited (Maisonpierre, P.
C., et al., [1997], supra; Mezquita, J., et al., Biochemical and
Biophysical Research Communications, 260:492-498 [1999]). In the
normal adult, the three main sites of angiogenesis are the ovary,
placenta, and uterus; these are the primary tissues in normal
(i.e., non-cancerous) tissues in which Ang-2 mRNA has been
detected.
[0009] Certain functional studies suggest that Ang-2 may be
involved in tumor angiogenesis. Ahmad et al. (Cancer Res.,
61:1255-1259 [2001]) describe Ang-2 over-expression and show that
it is purportedly associated with an increase in tumor growth in a
mouse xenograft model. See also Etoh et al., supra, and Tanaka et
al., supra, wherein data is presented purportedly associating Ang-2
over expression with tumor hypervascularity. However, in contrast,
Yu et al. (Am. J. Path., 158:563-570 [2001]) report data to show
that overexpression of Ang-2 in Lewis lung carcinoma and TA3
mammary carcinoma cells purportedly prolonged the survival of mice
injected with the corresponding transfectants.
[0010] In the past few years, various publications have suggested
Ang-1, Ang-2 and Tie-2 as a possible target for anti-cancer
therapy. For example, U.S. Pat. Nos. 6,166,185, 5,650,490, and
5,814,464 each disclose the concept of anti-Tie-2 ligand antibodies
and receptor bodies. U.S. Patent App. Pub. No. 2003/0124129A1
describes certain anti-Ang 2 antibodies and their use in treatment
of cancer. Lin et al. (Proc. Natl. Acad. Sci. USA, 95:8829-8834
[1998]) injected an adenovirus expressing soluble Tie-2 into mice;
the soluble Tie-2 purportedly decreased the number and size of the
tumors developed by the mice. In a related study, Lin et al. (J.
Clin. Invest., 100:2072-2078 [1997]) injected a soluble form of
Tie-2 into rats; this compound purportedly reduced tumor size in
the rats. Siemeister et al. (Cancer Res., 59:3185-3189 [1999])
generated human melanoma cell lines expressing the extracellular
domain of Tie-2, injected these cell lines into nude mice, and
concluded that soluble Tie-2 purportedly resulted in a "significant
inhibition" of tumor growth and tumor angiogenesis.
[0011] Hence, an effective anti-Ang-2 therapy might benefit a vast
population of cancer patients because most solid tumors require
neovascularization to grow beyond 1-2 millimeters in diameter. Such
therapy might have wider application in other
angiogenesis-associated diseases as well, such as retinopathies,
arthritis, and psoriasis.
SUMMARY OF THE INVENTION
[0012] Although much evidence points to the usefulness of
inhibiting Ang2 levels in treatment of unwanted angiogenesis (or
any subset of conditions involving unwanted generation of blood
vessels, like arteriogenesis), the present state of the art does
not make clear whether the simultaneous inhibition of Ang1 would be
beneficial in such therapies and if so what degree of Ang1
inhibition, in addition to Ang2 inhibition, might prove to provide
at least an additive therapeutic effect. Accordingly, the present
invention addresses an unrecognized need to identify new agents
that specifically recognize and bind both Ang-1 and Ang-2 ligands.
The binding agents, such as the antibodies of the present
invention, have the desired activity levels in inhibiting Ang2 as
well as Ang1 that make them particularly useful in a variety of
settings such as diagnostic screening, bioassays, and therapeutic
intervention in diseases that are associated with Ang-1 and/or
Ang-2 activity, such as cancer, inflammation, and other diseases
related to undesired angiogenesis.
[0013] The various embodiments of the invention relate to targeted
binding agents that specifically bind to Ang-1 and/or Ang-2 and
therein inhibit physiological or pathological angiogenesis.
Mechanisms by which this can be achieved can include, but are not
limited to, either inhibition of binding of Ang-1 and/or Ang-2 to
the Tie1 and/or Tie2 receptor, inhibition of Ang-1 and/or Ang-2
induced Tie1 and/or Tie2 signaling, or increased clearance of Ang1
and/or Ang-2 from a patient's body, therein reducing the effective
concentration of Ang-1 and/or Ang-2.
[0014] One embodiment of the invention, the specific binding agent
is a fully human antibody that specifically binds to Ang-1 and/or
Ang-2 and prevents Ang-1 and/or Ang-2 binding to Tie1 and/or Tie2
receptors. Yet another embodiment of the invention is a fully human
monoclonal antibody that binds to Ang-1 and/or Ang-2 and also
inhibits Ang-1 and/or Ang-2 induced Tie1 and/or Tie2
phosphorylation. The antibody may bind Ang-1 and/or Ang-2 with a Kd
of less than about 100 pM, 30 pM, 20 pM, 10 pM, 5 pM or 1 pM.
Certain embodiments of the invention are antibodies of the IgG
type, e.g., IgG1, IgG2, IgG3, and IgG4.
[0015] Another embodiment of the invention provides a binding agent
such as an antibody comprising a heavy chain and a light chain,
wherein said heavy chain comprises a heavy chain variable region
selected from the group consisting of H2 (SEQ ID NO. 1); H3 (SEQ ID
NO. 2); H4(SEQ ID NO. 3); H6 (SEQ ID NO. 4); H10(SEQ ID NO. 5); H11
(SEQ ID NO. 6); H5P (SEQ ID NO. 7); and antigen binding fragments
thereof; and said light chain comprises a light chain variable
region selected from the group consisting of: L1 (SEQ ID NO. 8); L2
(SEQ ID NO. 9); L4 (SEQ ID NO. 10); L6 (SEQ ID NO. 11); L7 (SEQ ID
NO. 12); L8 (SEQ ID NO. 13); L9 (SEQ ID NO. 14); L11 (SEQ ID NO.
15); L12 (SEQ ID NO. 16); L13 (SEQ ID NO. 17); and antigen binding
fragments thereof.
[0016] The invention also provides a specific binding agent
comprising at least one peptide selected from the group consisting
of: H2 (SEQ ID NO. 1); H3 (SEQ ID NO. 2); H4(SEQ ID NO. 3); H6 (SEQ
ID NO. 4); H10(SEQ ID NO. 5); H11 (SEQ ID NO. 6); H5P (SEQ ID NO.
7); L1 (SEQ ID NO. 8); L2 (SEQ ID NO. 9); L4 (SEQ ID NO. 10); L6
(SEQ ID NO. 11); L7 (SEQ ID NO. 12); L8 (SEQ ID NO. 13); L9 (SEQ ID
NO. 14); L11 (SEQ ID NO. 15); L12 (SEQ ID NO. 16); L13 (SEQ ID NO.
17); and antigen binding fragments thereof.
[0017] It will be appreciated that the specific binding agent can
be, for example, an antibody, such as a polyclonal, monoclonal,
chimeric, humanized, or a fully human antibody. The antibody may
also be a single chain antibody. Other examples of specific binding
agents include peptibodies, such as peptibody mL4-3, avimers, other
forms of peptide molecules (such as Fc-fusion molecules and
Ab-fusion molecules (see CovX-Pfizer technology)) that contain
peptide sequences which recognize and bind to a protein target (in
this context, Ang2 and or Ang1 ligand(s)), etc.
[0018] A specific embodiment of the invention relates to
peptibodies such as mL4-3 that bind Ang1. Other embodiments of the
invention include the peptide portion of mL4-3 as well as similar
Ang1-binding peptides that can be made by addition, deletion,
and/or insertion of amino acids to and from this peptide. Similar
additions, deletions, or insertions can be made to the Fc portion
of the mL4-3 peptibody. Further alterations to the mL4-3 and
peptibodies in general are well-known in the art and taught in, for
example, WO00/24782 and WO03/057134 which are incorporated herein
by reference to the sections which describe and teach making
binding agents that contain a randomly generated peptide which
binds a desired target.
[0019] The invention further relates to a hybridoma that produces a
monoclonal antibody according to the invention, as well as a cell
lines containing (through any means such as by transfection,
transformation, electroporation) with the nucleic acid sequences
necessary to express the present specific binding agents such as
the antibodies described herein.
[0020] It will also be appreciated that the invention relates to
conjugates as described herein. The conjugate can be, for example,
a specific binding agent (such as an antibody) of the invention
conjugated to other proteinatious, carbohydrate, lipid, or mixed
moiety molecule(s).
[0021] The invention further relates to nucleic acid molecules
encoding the specific binding agents (such as an antibody) of the
invention, as well as a vector comprising such nucleic acid
molecule, as well as a host cell containing the vector.
[0022] Additionally, the invention provides a method of making a
specific binding agent comprising, (a) transforming a host cell
with at least one nucleic acid molecule encoding the specific
binding agent; (b) expressing the nucleic acid molecule in said
host cell; and (c) isolating said specific binding agent. The
invention further provides a method of making an antibody
comprising: (a) transforming a host cell with at least one nucleic
acid molecule encoding the antibody according to the invention; (b)
expressing the nucleic acid molecule in said host cell; and (c)
isolating said specific binding agent.
[0023] Further, the invention relates to a method of inhibiting
undesired angiogenesis in a mammal by administering a
therapeutically effective amount of a specific binding agent
according to the invention. The invention also provides a method of
treating cancer in a mammal by administering a therapeutically
effective amount of a specific binding agent according to the
invention.
[0024] The invention also relates to a method of inhibiting
undesired angiogenesis in a mammal comprising by administering a
therapeutically effective amount of an antibody according to the
invention. The invention additionally provides a method of treating
cancer in a mammal comprising administering a therapeutically
effective amount of antibody according to the invention.
[0025] It will be appreciated that the invention further relates to
pharmaceutical compositions comprising the specific binding agent
according to the invention and a pharmaceutically acceptable
formulation agent. The pharmaceutical composition may comprise an
antibody according to the invention and a pharmaceutically
acceptable formulation agent.
[0026] The invention provides a method of modulating or inhibiting
angiopoietin-2 activity by administering one or more specific
binding agents of the invention. The invention also provides a
method of modulating or inhibiting angiopoietin-2 activity by
administering an antibody of the invention.
[0027] The invention further relates to a method of modulating at
least one of vascular permeability or plasma leakage in a mammal
comprising administering a therapeutically effective amount of the
specific binding agent according to the invention. The invention
also relates to a method of treating at least one of ocular
neovascular disease, obesity, hemangioblastoma, hemangioma,
arteriosclerosis, inflammatory disease, inflammatory disorders,
atherosclerosis, endometriosis, neoplastic disease, bone-related
disease, or psoriasis in a mammal comprising administering a
therapeutically effective amount of a specific binding agent
according to the invention.
[0028] The invention further provides a method of modulating at
least one of vascular permeability or plasma leakage in a mammal
comprising administering a therapeutically effective amount of an
antibody according to the invention. The invention also relates to
a method of treating at least one of ocular neovascular disease,
obesity, hemangioblastoma, hemangioma, arteriosclerosis,
inflammatory disease, inflammatory disorders, atherosclerosis,
endometriosis, neoplastic disease, bone-related disease, or
psoriasis in a mammal comprising administering a therapeutically
effective amount of an antibody according to the invention.
[0029] Furthermore, the invention relates to a method of treating
cancer in a mammal comprising administering a therapeutically
effective amount of a specific binding agent according to the
invention and a chemotherapeutic agent. It will be appreciated by
those in the art that the specific binding agent and
chemotherapeutic agent need not be administered simultaneously.
[0030] The invention also provides a specific binding agent
comprising heavy chain complementarity determining region 1 (CDR 1)
of any of: SEQ ID NO. 18; The invention further relates to a
specific binding agent comprising heavy chain complementarity
determining region 2 (CDR 2) of any of: SEQ ID NO. 26; SEQ ID NO.
27; SEQ ID NO. 28; SEQ ID NO. 29; and antigen binding fragments
thereof.
[0031] The invention also relates to a specific binding agent
comprising heavy chain complementarity determining region 3 (CDR 3)
of any of: SEQ ID NO. 32; SEQ ID NO. 34; SEQ ID NO. 35; SEQ ID NO.
37; SEQ ID NO. 38; SEQ ID NO. 39); and antigen binding fragments
thereof.
[0032] The invention also provides a specific binding agent
comprising light chain complementarity determining region 1 (CDR 1)
of any of: SEQ ID NO. 19; SEQ ID NO. 20; SEQ ID NO. 21; SEQ ID NO.
22; SEQ ID NO. 23; SEQ ID NO. 24; SEQ ID NO. 25; and antigen
binding fragments thereof;
[0033] The invention further relates to a specific binding agent
comprising light chain complementarity determining region 2 (CDR 2)
of any of: SEQ ID NO. 27; SEQ ID NO. 30; SEQ ID NO. 31; and antigen
binding fragments thereof.
[0034] The invention also relates to a specific binding agent
comprising light chain complementarity determining region 3 (CDR 3)
of any of: SEQ ID NO.33; SEQ ID NO. 36; SEQ ID NO. 40; and antigen
binding fragments thereof.
[0035] Other embodiments of the invention include isolated nucleic
acid molecules encoding any of the antibodies described herein,
vectors having isolated nucleic acid molecules encoding anti-Ang-1
and/or Anti-Ang-2 antibodies or a host cell transformed with any of
such nucleic acid molecules. In addition, one embodiment of the
invention is a method of producing an anti-Ang-1 and/or anti-Ang-2
antibody by culturing host cells under conditions wherein a nucleic
acid molecule is expressed to produce the antibody followed by
recovering the antibody. It should be realized that embodiments of
the invention also include any nucleic acid molecule which encodes
an antibody or fragment of an antibody of the invention including
nucleic acid sequences optimized for increasing yields of
antibodies or fragments thereof when transfected into host cells
for antibody production.
[0036] A further embodiment herein includes a method of producing
high affinity antibodies to Ang-1 and/or Ang-2 by immunizing a
mammal with human Ang-1 or 2, or a fragment thereof, and one or
more orthologous sequences or fragments thereof.
[0037] Moreover, the invention relates to a method of detecting the
level of Ang-1 or Ang-2 in a biological sample by (a) contacting a
specific binding agent of the invention with the sample; and (b)
determining the extent of binding of the specific binding agent to
the sample. The invention also relates to a method of detecting the
level of Ang-2 in a biological sample by (a) contacting an antibody
of the invention with the sample; and (b) determining the extent of
binding of the antibody to the sample.
[0038] The invention also relates to a method of inhibiting
undesired angiogenesis in a mammal comprising administering a
therapeutically effective amount of a polypeptide or composition as
described herein. The invention also relates to a method of
modulating angiogenesis in a mammal comprising administering a
therapeutically effective amount of a polypeptide or composition as
described herein. The invention further relates to a method of
inhibiting tumor growth characterized by undesired angiogenesis in
a mammal comprising administering a therapeutically effective
amount of a polypeptide or composition as described herein.
Additionally, the invention relates to a method of treating cancer
in a mammal comprising administering a therapeutically effective
amount of a polypeptide or composition as described herein, and a
chemotherapeutic agent. The specific polypeptide or composition as
described herein and chemotherapeutic agent need not be
administered simultaneously. In a preferred embodiment, the
chemotherapeutic agent is at least one of 5-FU, CPT-11, and
Taxotere. It will be appreciated, however, that other suitable
chemotherapeutic agents and other cancer therapies can be used.
[0039] Additionally, the invention relates to a method of treating
cancer in a mammal comprising administering a therapeutically
effective amount of a polypeptide or composition as described
herein, and an anti-VEGF agent or a multikinase inhibitor (MKI). In
a preferred embodiment, the anti-VEGF agent or a multikinase
inhibitor (MKI) would be chosen from Avastin.RTM. (bevacizumab),
Lucentis.RTM. (ranibizumab), Macugen.RTM. (pegaptanib), Sutent.RTM.
(sunitinib), Nexavar.RTM. (sorafenib), motesanib diphosphate,
Zactima.RTM. (vandetanib), Recentin (AZD 2171), AG-013736
(axitinib). It will be appreciated, however, that other suitable
anti-angiogenic agents and other cancer therapies can be used.
[0040] It will be appreciated that the specific binding agents of
the invention are used to treat a number of diseases associated
with deregulated or undesired angiogenesis. Such diseases include,
but are not limited to, ocular neovascularisation, such as
retinopathies (including diabetic retinopathy and age-related
macular degeneration) psoriasis, hemangioblastoma, hemangioma,
arteriosclerosis, inflammatory disease, such as a rheumatoid or
rheumatic inflammatory disease, especially arthritis (including
rheumatoid arthritis), or other chronic inflammatory disorders,
such as chronic asthma, arterial or post-transplantational
atherosclerosis, endometriosis, and neoplastic diseases, for
example so-called solid tumors and liquid tumors (such as
leukemias). Additional diseases which can be treated by
administration of the specific binding agents will be apparent to
those skilled in the art. Such additional diseases include, but are
not limited to, obesity, vascular permeability, plasma leakage, and
bone-related disorders, including osteoporosis. Thus, the invention
further relates to methods of treating these diseases associated
with deregulated or undesired angiogenesis.
[0041] Additional embodiments of the invention include a specific
binding agent comprising at least one peptide selected from the
group consisting of: SEQ ID NO. 1; SEQ ID NO. 2; SEQ ID NO. 3; SEQ
ID NO. 4; SEQ ID NO. 5; SEQ ID NO. 6; SEQ ID NO. 7; SEQ ID NO. 8;
SEQ ID NO. 9; SEQ ID NO. 10; SEQ ID NO. 11; SEQ ID NO. 12; SEQ ID
NO. 13; SEQ ID NO. 14; SEQ ID NO. 15; SEQ ID NO. 16; SEQ ID NO. 17;
and antigen-binding fragments thereof. Also contemplated are
antibodies containing the aforementioned polypeptide sequences.
These antibodies are polyclonal, monoclonal, chimeric, humanized,
or fully human antibodies. They are single chain antibody as well
as multi-chain antibodies. Hybridomas that produce the monoclonal
antibodies are also contemplated, as well as, nucleic acid
molecules encoding the polypeptides and the antibodies, the vectors
containing these nucleic acid molecules, and the host cells, such
as CHO cells, that contain and express them. A method of making a
binding agent or an antibody of the present invention comprises
transforming a host cell with at least one nucleic acid molecule
encoding the binding agent or antibody; expressing the nucleic acid
molecule in said host cell; and isolating said specific binding
agent or antibody.
[0042] A diagnostic use of the invention includes a method of
detecting the level of angiopoietin-1 and/or angiopoietin-in a
biological sample comprising contacting an antibody or binding
agent described herein with said biological sample; and determining
the extent of binding of the antibody or binding agent to said
sample.
[0043] Amongst the specific therapeutic uses of the invention are
methods of inhibiting undesired angiogenesis (or any subset of
conditions involving unwanted generation of blood vessels, like
arteriogenesis), in a mammal comprising administering a
therapeutically effective amount of the isolated polypeptides or
the binding agents such as antibodies made therefrom. Amongst such
undesired angiogenesis (or any subset of conditions involving
unwanted generation of blood vessels, like arteriogenesis), are
cancer and inflammatory diseases in mammals. Therefore, a
pharmaceutical composition is contemplated that comprises the
isolated polypeptide, binding agent or antibody of the invention in
admixture with a pharmaceutical carrier therefore. Pharmaceutically
acceptable formulation agents, of course, are often used to prepare
such pharmaceutical compositions for administration to subjects in
need thereof.
[0044] Other methods of using the compositions of the present
invention include a method of modulating or inhibiting
angiopoietin-1 and/or angiopoietin-2 activity comprising
administering to a patient the isolated polypeptide, binding agent
or antibody described herein. Such methods of modulating or
inhibiting angiopoietin-1 and/or angiopoietin-2 activity comprise
administering to a patient the polypeptide, binding agent, or
antibody described herein. Such methods include modulating at least
one of vascular permeability or plasma leakage in a mammal
comprising administering to a mammal a therapeutically effective
amount of the isolated polypeptide, binding agent or antibody
described herein. Also included are methods of treating at least
one of ocular neovascular disease, obesity, hemangioblastoma,
hemangioma, arteriosclerosis, inflammatory disease, inflammatory
disorders, atherosclerosis, endometriosis, neoplastic disease,
bone-related disease, or psoriasis.
[0045] Also contemplated is a combotherapy (combination therapy)
method such as a method of treating cancer in a mammal comprising
administering a therapeutically effective amount of an isolated
polypeptide, binding agent or antibody described herein and a
chemotherapeutic agent. In such methods, sometimes the isolated
polypeptide, binding agent or antibody and the chemotherapeutic
agent are administered simultaneously and at other times are not,
depending upon the specific condition, regulatory approval, and the
judgement of the medical professionals.
[0046] Other types of combotherapy include a method of treating
cancer in a mammal comprising administering to a subject in need
thereof a therapeutically effective amount of an isolated
polypeptide, binding agent or antibody described herein and a
second molecule that binds a ligand to any one of the VEGF
receptors 1-3. Examples of such second molecules that bind a ligand
to any one of the VEGF receptors 1-3 are Avastin.RTM.,
Lucentis.RTM., and Macugen.RTM..
[0047] Use of the polypeptides, binding agents, or antibodies
described herein are also contemplated in combination with small
molecule agents for therapeutic administration to subjects in need
thereof. Such small molecule agents include those that modulate the
signaling of any one of the VEGF receptors 1-3 as well as those
that are multikinase inhibitors. For example, Sutent.RTM.,
Nexavar.RTM., Motesanib diphosphate, Axitinib, Zactima, AZD 2171,
Recentin, and AG-013736 are contemplated for use in combotherapy
with the polypepetides, binding agents, and antibodies described
herein.
[0048] Certain other embodiments of the invention relate to a
specific binding agent comprising CDR 1 of any of SEQ ID NO. 18;
SEQ ID NO. 19; SEQ ID NO. 20; SEQ ID NO. 21; SEQ ID NO. 22; SEQ ID
NO. 23; SEQ ID NO. 24; SEQ ID NO. 25; a specific binding agent
comprising CDR 2 of any of SEQ ID NO. 26; SEQ ID NO. 27; SEQ ID NO.
28; SEQ ID NO. 29; SEQ ID NO. 30; SEQ ID NO. 31; and a specific
binding agent comprising CDR 3 of any of SEQ ID NO. 32; SEQ ID NO.
33; SEQ ID NO. 34; SEQ ID NO. 35; SEQ ID NO. 36; SEQ ID NO. 37; SEQ
ID NO. 38; SEQ ID NO. 39; SEQ ID NO. 40. The specific binding agent
may comprise 1, 2, 3, 4, 5, or 6 CDRs.
[0049] Similarly, nucleic acid molecules encoding the
above-mentioned specific binding agents are contemplated. Also
contemplated is a method of detecting the level of angiopoietin-1
and/or angiopoietin-2 in a biological sample comprising contacting
a specific binding agent as described herein with said biological
sample; and determining the extent of binding of the specific
binding agent to said sample. Additionally, a method is
contemplated for detecting the level of angiopoietin-1 and/or
angiopoietin-2 in a biological sample comprising contacting any one
of the antibodies described herein with said biological sample; and
determining the extent of binding of the antibody to said
sample.
[0050] A further embodiment of the invention is an antibody
comprising a heavy chain and a light chain, the heavy chain
comprising a heavy chain variable region selected from the group
consisting of SEQ ID NO. 1; SEQ ID NO. 2; SEQ ID NO. 3; SEQ ID NO.
4; SEQ ID NO. 5; SEQ ID NO. 6 and, SEQ ID NO. 7; and the light
chain comprising a light chain variable region selected from the
group consisting of SEQ ID NO. 8; SEQ ID NO. 9; SEQ ID NO. 10; SEQ
ID NO. 11; SEQ ID NO. 12; SEQ ID NO. 13; SEQ ID NO. 14; SEQ ID NO.
15; SEQ ID NO. 16 and, SEQ ID NO. 17; as well as antigen binding
fragments thereof. Naturally, nucleic acid molecules encoding the
above-described antibodies and antigen-binding fragments are also
contemplated.
[0051] In another embodiment, the present invention is directed to
an isolated antibody comprising a heavy chain and a light chain,
the light chain comprising a light chain variable domain and the
heavy chain comprising a heavy chain variable domain, the heavy
chain variable domain having the sequence selected from the group
consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:
4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7; wherein the
antibody specifically binds to at least one of Ang1 and Ang2
ligands of Tie 2 receptor.
[0052] In a further embodiment, the invention is an isolated
antibody comprising a heavy chain and a light chain, the heavy
chain comprising a heavy chain variable domain and the light chain
comprising a light chain variable domain, the light chain variable
domain having the sequence selected from the group consisting of
SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID
NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16,
and SEQ ID NO: 17; wherein the antibody specifically binds to at
least one of Ang1 and Ang2 ligands of Tie 2 receptor.
[0053] In an additional embodiment, the invention is directed to an
isolated antibody comprising a heavy chain and light chain, the
heavy chain comprising a heavy chain variable domain and the light
chain comprising a light chain variable domain, wherein the heavy
chain variable domain comprises 1, 2, or 3 heavy chain CDRs
selected from the group of HC CDRs consisting of SEQ ID NOs: 18,
26, 28, 32, 34, 35, 37, 38 and, 39, and wherein the antibody
specifically binds to at least one of Ang1 and Ang2 ligands of Tie
2 receptor.
[0054] In another embodiment, the invention is directed to an
isolated antibody which comprises a light chain and a heavy chain,
wherein the light chain comprises a light chain variable domain and
the heavy chain comprises a heavy chain variable domain, wherein
the light chain variable domain comprises 1, 2, or 3, light chain
CDRs selected from the group of LC CDRs consisting of SEQ ID NOs:
19, 20, 21, 22, 23, 27, 33, 36, 40, and wherein the antibody
specifically binds to at least one of Ang1 and Ang2 ligands of Tie
2 receptor.
[0055] In a further embodiment, the invention is an isolated
antibody which comprises a heavy chain and a light chain, wherein
the heavy chain comprises a heavy chain variable domain and the
light chain comprises a light chain variable domain, wherein the
heavy chain comprises 3 heavy chain (HC) CDRs and said light chain
variable domain comprises 3 light chain (LC) CDRs, wherein the
sequences of said HC and LC CDRs of the antibody are selected from
the group consisting of:
[0056] (a) SEQ ID NOs: 18, 26, 32 of the HC plus SEQ ID NOs: 19,
27, 33 of the LC,
[0057] (b) SEQ ID NOs: 18, 26, 34 of the HC plus SEQ ID NOs: 19,
27, 33 of the LC,
[0058] (c) SEQ ID NOs: 18, 26, 35 of the HC plus SEQ ID NOs: 20,
27, 36 of the LC,
[0059] (d) SEQ ID NOs: 18, 26, 37 of the HC plus SEQ ID NOs: 19,
27, 33 of the LC,
[0060] (e) SEQ ID NOs: 18, 26, 38 of the HC plus SEQ ID NOs: 19,
27, 33 of the LC,
[0061] (f) SEQ ID NOs: 18, 26, 35 of the HC plus SEQ ID NOs: 19,
27, 33 of the LC,
[0062] (g) SEQ ID NOs: 18, 26, 34 of the HC plus SEQ ID NOs: 21,
27, 33 of the LC,
[0063] (h) SEQ ID NOs: 18, 28, 39 of the HC plus SEQ ID NOs: 19,
27, 33 of the LC,
[0064] (i) SEQ ID NOs: 18, 26, 34 of the HC plus SEQ ID NOs: 22,
27, 33 of the LC,
[0065] (j) SEQ ID NOs: 18, 26, 32 of the HC plus SEQ ID NOs: 22,
27, 33 of the LC,
[0066] (k) SEQ ID NOs: 18, 29, 39 of the HC plus SEQ ID NOs: 19,
27, 33 of the LC,
[0067] (l) SEQ ID NOs: 18, 26, 34 of the HC plus SEQ ID NOs: 23,
27, 33 of the LC,
[0068] (m) SEQ ID NOs: 18, 26, 35 of the HC plus SEQ ID NOs: 20,
27, 40 of the LC,
[0069] (n) SEQ ID NOs: 18, 26, 32 of the HC plus SEQ ID NOs: 21,
27, 33 of the LC,
[0070] (O) SEQ ID NOs: 18, 26, 35 of the HC plus SEQ ID NOs: 24,
27, 33 of the LC,
[0071] (p) SEQ ID NOs: 18, 26, 35 of the HC plus SEQ ID NOs: 21,
27, 33 of the LC,
[0072] (q) SEQ ID NOs: 18, 26, 35 of the HC plus SEQ ID NOs: 23,
27, 33 of the LC,
[0073] (r) SEQ ID NOs: 18, 26, 34 of the HC plus SEQ ID NOs: 20,
30, 33 of the LC,
[0074] (s) SEQ ID NOs: 18, 26, 34 of the HC plus SEQ ID NOs: 25,
27, 33 of the LC,
[0075] (t) SEQ ID NOs: 18, 26, 35 of the HC plus SEQ ID NOs: 20,
30, 33 of the LC,
[0076] (u) SEQ ID NOs: 18, 26, 34 of the HC plus SEQ ID NOs: 20,
27, 40 of the LC, and
[0077] (v) SEQ ID NOs: 18, 26, 34 of the HC plus SEQ ID NOs: 20,
31, 33 of the LC;
[0078] wherein the antibody specifically binds to at least one of
Ang1 and Ang2 ligands of Tie 2 receptor.
[0079] The present invention also is directed to an antibody having
a heavy chain and light chain, where the light chain has a light
chain variable domain having three LC CDRs of any one of (a)
through (v), supra, wherein the antibody specifically to at least
one of Ang1 and Ang2 ligands of Tie 2 receptor.
[0080] Additionally, the present invention also is directed to an
antibody having a heavy chain and light chain, where the heavy
chain has a heavy chain variable domain having three HC CDRs of any
one of (a) through (v), supra, wherein the antibody specifically to
at least one of Ang1 and Ang2 ligands of Tie 2 receptor.
[0081] Nucleic acid molecules encoding any of the aforementioned
antibodies and antigen-binding fragments thereof are also
contemplated. Other embodiments of this invention will be readily
apparent from the disclosure provided herewith.
BRIEF DESCRIPTION OF THE FIGURES
[0082] FIG. 1 depicts a graph of tumor size (y-axis) versus time
(x-axis) in tumor-bearing mice treated with either an anti-Ang1/2
antibody (H4L4, H4L11, or H6L7) of the invention or a highly potent
control peptibody (AMG 386) or antibody 536, compared to treatment
with an isotype control antibody. Details are described in the
Examples.
[0083] FIG. 2 depicts the tumor burden (% viable tumor [bisected
section] x tumor weight) in tumor-bearing mice treated with an
anti-Ang1/2 antibody (H4L4, H4L11, or H6L7) of the invention or a
highly potent control peptibody (AMG 386) or antibody 536 compared
to treatment with an isotype control antibody. Details are
described in the Examples.
[0084] FIG. 3 depicts the effect of H4L4, H4L11, and H6L7 of the
invention, a highly potent control peptibody (AMG 386) and antibody
536 on endothelial cell proliferation in Colo205 tumor-bearing
mice. Details are described in the Examples.
[0085] FIG. 4 depicts the H4L4 antibody dose-response relationship
in Colo205 tumor-bearing mice. Details are described in the
Examples.
[0086] FIG. 5 depicts the effect of H4L4 antibody on Colo205 tumor
burden in vivo. Details are described in the Examples.
[0087] FIG. 6 depicts the effect of the antibody H4L4 on
endothelial cell proliferation in Colo205 tumor-bearing mice.
Details are described in the Examples.
[0088] FIG. 7 depicts systemically administered mL4-3 neutralizes
Ang1-induced Tie2 phosphorylation in mouse lungs. Mice (n=3 per
group) were treated with L1-7(N) (2 mg/kg), mL4-3 (20 mg/kg) or Fc
control (20 mg/kg) daily for 23 days prior to i.v. challenge with
Ang1 or BSA. Mouse lungs were subsequently harvested, and the
levels of phosphorylated Tie2 were determined by
immunoprecipitation-Western blot analysis. Data are mean
values.+-.SE. *P=0.0005 vs Ang1 plus Fc, ANOVA with Fisher's post
hoc test.
[0089] FIG. 8 depicts pharmacologic inhibition of Ang1 during early
organogenesis alters heart development. A) Mouse embryos exposed to
300 mg/kg mL4-3 (right panel) had smaller hearts with fewer,
narrower, and more widely spaced trabeculae relative to the larger
hearts with large, wide trabeculae found in stage-matched embryos
exposed to 300 mg/kg Fc control (left panel). Representative images
are shown. B) Incidence of cardiac abnormalities in Fc- and
mL4-3-treated embryos. *P<0.0001 vs Fc, chi-square test.
[0090] FIG. 9 depicts the effect of combined Ang1 and Ang2
inhibition on the growth of Colo205 tumor xenografts. Mice (n=10
per group) were implanted with Colo205 cells, and treatment began
when tumors reached approximately 500 mm.sup.3 with Fc control (5.2
mg/kg QD), mL4-3 (3.2 mg/kg QD), L1-7(N) (2.0 mg/kg QD), L1-7(N)
combined with mL4-3 (at the same dosing regimens used in the
single-agent groups), or AMG 386 (5.6 mg/kg twice per week). One of
four representative experiments is shown. Data are mean
values.+-.SE. *P<0.0001 vs L1-7(N), RMANOVA with Scheffe post
hoc test.
[0091] FIG. 10 depicts the effect of Ang1 and Ang2 antagonism on
tumor endothelial cell proliferation, corneal angiogenesis, and
retinal angiogenesis. A) The effect of inhibition of Ang1 and Ang2
on BrdU uptake in mouse endothelial cells derived from Colo205
tumor xenografts. Tumor-bearing mice were treated for 3 days with
Fc (5.7 mg/kg QD), AMG 386 (6 mg/kg single dose), L1-7(N) (2.2
mg/kg QD), mL4-3 (3.5 mg/kg QD), or L1-7(N) combined with mL4-3 (at
the same doses and schedules used in the single-agent groups). Each
bar represents mean endothelial:total mouse cell BrdU ratios (n=3).
Data are mean values.+-.SE. *P<0.05 vs. Fc, unpaired Student's
t-test. B and C) The effect of inhibition of Ang1 and Ang2 on (B)
VEGF-induced and (C) bFGF-induced corneal angiogenesis.
Angiogenesis was induced by implanting VEGF- or bFGF-soaked nylon
discs into the corneal stroma of rats (n=8 per group). Treatment
was initiated one day prior to corneal implantation and continued
every 3 days with: Fc (60 mg/kg), L1-7(N) (5 mg/kg), mL4-3 (60
mg/kg) and L1-7(N) combined with mL4-3 (at the same dose and
schedule used in the single-agent groups). Data are mean
values.+-.SE. .sup..dagger.P<0.0001 vs Fc+VEGF (B);
.sup.#P<0.002 vs Fc+bFGF (C), ANOVA with Fisher's post hoc test.
D) Inhibition of Ang2 prevents oxygen-induced neovascularization in
the mouse retina. Starting on postnatal day P8, pups (n=5 per
group) were treated daily s.c. for nine days with Fc (200 mg/kg)
L1-7(N) (100 mg/kg) mL4-3 (100 mg/kg) or L1-7(N) combined with
mL4-3 (at the same dose and schedule used in the single-agent
groups). Data are mean values.+-.SE. .sup..sctn.P<0.0001 vs Fc,
ANOVA with Fisher's post hoc test.
[0092] FIG. 11 depicts Ang1 and Ang2 inhibitors cooperatively
suppress ovarian follicular angiogenesis. HCG was used to induce
superovulation in mice. Fc (300 mg/kg), mL4-3 (150 mg/kg), L1-7(N)
(150 mg/kg), or an mL4-3/L1-7(N) combination (150 mg/kg each)
administered s.c. (n=7-10 mice per group) were evaluated for the
ability to prevent neovascularization in ovulating follicles. Blood
vessel area was calculated from anti-CD31 immunostained sections of
individual follicles. Data are mean values.+-.SE. Two independent
experiments are shown. *P=0.005 comparing mL4-3/L1-7(N) combination
vs either single agent alone; .sup.#P<0.05 vs Fc, ANOVA with
Dunnett's post hoc test.
DETAILED DESCRIPTION OF INVENTION
[0093] The section headings are used herein for organizational
purposes only, and are not to be construed as in any way limiting
the subject matter described.
[0094] Standard techniques may be used for recombinant DNA
molecule, protein, and antibody production, as well as for tissue
culture and cell transformation. Enzymatic reactions and
purification techniques are typically performed according to the
manufacturer's specifications or as commonly accomplished in the
art using conventional procedures such as those set forth in
Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. [1989]),
or as described herein. Unless specific definitions are provided,
the nomenclature utilized in connection with, and the laboratory
procedures and techniques of analytical chemistry, synthetic
organic chemistry, and medicinal and pharmaceutical chemistry
described herein are those well known and commonly used in the art.
Standard techniques may be used for chemical syntheses, chemical
analyses, pharmaceutical preparation, formulation, and delivery,
and treatment of patients.
[0095] The terms used to describe the present invention, unless
specifically defined herein, shall have their meaning as understood
and used in the art.
[0096] It should be noted that the terms H5 and H5P are used
interchangeably and refer to the heavy chain used in various
embodiments of the invention, e.g., mAbs named as H5L7, H5L6, H5L8,
H5L4, H5L11, H5L1, H5L12, and H5L9.
[0097] The term "Ang-2" refers to the polypeptide set forth in FIG.
6 of U.S. Pat. No. 6,166,185 ("Tie-2 ligand-2"), incorporated
herein by reference, or fragments thereof as well as related
polypeptides which include allelic variants, splice variants,
derivatives, substitution, deletions, and/or insertion variants,
fusion peptides and polypeptides, and interspecies homologs. The
Ang-2 polypeptide may or may not include additional terminal
residues, e.g., leader sequences, targeting sequences, amino
terminal methionine, amino terminal methionine and lysine residues,
and/or tag or fusion proteins sequences, depending on the manner in
which it is prepared.
[0098] The term "specific binding agent" refers to a molecule,
preferably a proteinaceous molecule, that binds Ang-2 as well as
Ang-1 (and variants and derivatives thereof as defined herein) with
a greater affinity than other angiopoietins. A specific binding
agent may be a protein, peptide, nucleic acid, carbohydrate, lipid,
or small molecular weight compound which binds preferentially to
Ang-2 and Ang-1. In a preferred embodiment, the specific binding
agent according to the present invention is an antibody, such as a
polyclonal antibody, a monoclonal antibody (mAb), a chimeric
antibody, a CDR-grafted antibody, a multi-specific antibody, a
bi-specific antibody, a catalytic antibody, a humanized antibody, a
human antibody, an anti-idiotypic (anti-Id) antibody, and
antibodies that can be labeled in soluble or bound form, as well as
antigen-binding fragments, variants or derivatives thereof, either
alone or in combination with other amino acid sequences, provided
by known techniques. Such techniques include, but are not limited
to enzymatic cleavage, chemical cleavage, peptide synthesis or
recombinant techniques. The anti-Ang-2 and Ang-1 specific binding
agents of the present invention are capable of binding portions of
Ang-2 and Ang-1 that modulate, e.g., inhibit or promote, the
biological activity of Ang-2 and Ang-1 and/or other Ang-2- and
Ang-1-associated activities.
[0099] The term "polyclonal antibody" refers to a heterogeneous
mixture of antibodies that recognize and bind to different epitopes
on the same antigen. Polyclonal antibodies may be obtained from
crude serum preparations or may be purified using, for example,
antigen affinity chromatography, or Protein A/Protein G affinity
chromatography.
[0100] The term "monoclonal antibodies" refers to a collection of
antibodies encoded by the same nucleic acid molecule that are
optionally produced by a single hybridoma (or clone thereof) or
other cell line, or by a transgenic mammal such that each
monoclonal antibody will typically recognize the same epitope on
the antigen. The term "monoclonal" is not limited to any particular
method for making the antibody, nor is the term limited to
antibodies produced in a particular species, e.g., mouse, rat,
etc.
[0101] The term "chimeric antibodies" refers to antibodies in which
a portion of the heavy and/or light chain is identical with or
homologous to a corresponding sequence in an antibody derived from
a particular species or belonging to a particular antibody class or
subclass, while the remainder of the chain(s) is/are identical with
or homologous to a corresponding sequence in antibodies derived
from another species or belonging to another antibody class or
subclass. Also included are antigen-binding fragments of such
antibodies that exhibit the desired biological activity (i.e., the
ability to specifically bind Ang-2). See, U.S. Pat. No. 4,816,567
and Morrison et al., Proc Natl Acad Sci (USA), 81:6851-6855
[1985].
[0102] The term "CDR grafted antibody" refers to an antibody in
which the CDR from one antibody of a particular species or isotype
is recombinantly inserted into the framework of another antibody of
the same or different species or isotype.
[0103] The term "multi-specific antibody" refers to an antibody
having variable regions that recognize more than one epitope on one
or more antigens. A subclass of this type of antibody is a
"bi-specific antibody" which recognizes two distinct epitopes on
the same or different antigens.
[0104] "Catalytic" antibodies refers to antibodies wherein one or
more cytotoxic, or more generally one or more biologically active,
moieties are attached to the targeting binding agent.
[0105] The term "humanized antibody" refers to a specific type of
CDR-grafted antibody in which the antibody framework region is
derived from a human but each CDR is replaced with that derived
from another species, such as a murine CDR. The term "CDR" is
defined infra.
[0106] The term "fully human" antibody refers to an antibody in
which both the CDR and the framework are derived from one or more
human DNA molecules.
[0107] The term "anti-idiotype" antibody refers to any antibody
that specifically binds to another antibody that recognizes an
antigen. Production of anti-idiotype antibodies can be performed by
any of the methods described herein for production of
Ang-2-specific antibodies except that these antibodies arise from
e.g., immunization of an animal with an Ang-2-specific antibody or
Ang-2-binding fragment thereof, rather than Ang-2 polypeptide
itself or a fragment thereof.
[0108] The term "variants," as used herein, include those
polypeptides wherein amino acid residues are inserted into, deleted
from and/or substituted into the naturally occurring (or at least a
known) amino acid sequence for the binding agent. Variants of the
invention include fusion proteins as described below.
[0109] "Derivatives" include those binding agents that have been
chemically modified in some manner distinct from insertion,
deletion, or substitution variants.
[0110] "Specifically binds" refers to the ability of a specific
binding agent (such as an antibody or fragment thereof) of the
present invention to recognize and bind mature, full-length or
partial-length target polypeptide (herein Ang-2 and Ang-1), or an
ortholog thereof, such that its affinity (as determined by, e.g.,
Affinity ELISA or BIAcore assays as described herein) or its
neutralization capability (as determined by e.g., Neutralization
ELISA assays described herein, or similar assays) is at least 10
times as great, but optionally 50 times as great, 100, 250 or 500
times as great, or even at least 1000 times as great as the
affinity or neutralization capability of the same for any other
angiopoietin or other peptide or polypeptide.
[0111] The term "antigen binding domain" or "antigen binding
region" refers to that portion of the specific binding agent (such
as an antibody molecule) which contains the specific binding agent
amino acid residues (or other moieties) that interact with an
antigen and confer on the binding agent its specificity and
affinity for the antigen. In an antibody, the antigen-binding
domain is commonly referred to as the "complementarity-determining
region, or CDR."
[0112] The term "epitope" refers to that portion of any molecule
capable of being recognized by and bound by a specific binding
agent, e.g. an antibody, at one or more of the binding agent's
antigen binding regions. Epitopes usually consist of chemically
active surface groupings of molecules, such as for example, amino
acids or carbohydrate side chains, and have specific
three-dimensional structural characteristics as well as specific
charge characteristics. Epitopes as used herein may be contiguous
or non-contiguous. Moreover, epitopes may be mimetic in that they
comprise a three dimensional structure that is identical to the
epitope used to generate the antibody, yet comprise none or only
some of the amino acid residues found in the Ang-2 used to
stimulate the antibody immune response.
[0113] The term "inhibiting and/or neutralizing epitope" is an
epitope, which when bound by a specific binding agent such as an
antibody, results in the loss of (or at least the decrease in)
biological activity of the molecule, cell, or organism containing
such epitope, in vivo, in vitro, or in situ. In the context of the
present invention, the neutralizing epitope is located on or is
associated with a biologically active region of Ang-2.
Alternatively, the term "activating epitope" is an epitope, which
when bound by a specific binding agent of the invention, such as an
antibody, results in activation, or at least maintenance of a
biologically active conformation, of Ang-2.
[0114] The term "antibody fragment" refers to a peptide or
polypeptide which comprises less than a complete, intact antibody.
Complete antibodies comprise two functionally independent parts or
fragments: an antigen binding fragment known as "Fab," and a
carboxy terminal crystallizable fragment known as the "Fc"
fragment. The Fab fragment includes the first constant domain from
both the heavy and light chain (CH1 and CL1) together with the
variable regions from both the heavy and light chains that bind the
specific antigen. Each of the heavy and light chain variable
regions includes three complementarity determining regions (CDRs)
and framework amino acid residues which separate the individual
CDRs. The Fc region comprises the second and third heavy chain
constant regions (CH2 and CH3) and is involved in effector
functions such as complement activation and attack by phagocytic
cells. In some antibodies, the Fc and Fab regions are separated by
an antibody "hinge region," and depending on how the full length
antibody is proteolytically cleaved, the hinge region may be
associated with either the Fab or Fc fragment. For example,
cleavage of an antibody with the protease papain results in the
hinge region being associated with the resulting Fc fragment, while
cleavage with the protease pepsin provides a fragment wherein the
hinge is associated with both Fab fragment simultaneously. Because
the two Fab fragments are in fact covalently linked following
pepsin cleavage, the resulting fragment is termed the F(ab')2
fragment.
[0115] An Fc domain may have a relatively long serum half-life,
whereas a Fab is short-lived. [Capon et al., Nature, 337: 525-31
(1989)] When expressed as part of a fusion protein, an Fc domain
can impart longer half-life or incorporate such functions as Fc
receptor binding, Protein A binding, complement fixation and
perhaps even placental transfer into the protein to which it is
fused. The Fc region may be a naturally occurring Fc region, or may
be altered to improve certain qualities, such as therapeutic
qualities or circulation time.
[0116] The term "variable region" or "variable domain" refers to a
portion of the light and/or heavy chains of an antibody, typically
including approximately the amino-terminal 120 to 130 amino acids
in the heavy chain and about 100 to 110 amino terminal amino acids
in the light chain. The variable regions typically differ
extensively in amino acid sequence even among antibodies of the
same species. The variable region of an antibody typically
determines the binding and specificity of each particular antibody
for its particular antigen. The variability in sequence is
concentrated in those regions referred to as
complementarity-determining regions (CDRs), while the more highly
conserved regions in the variable domain are called framework
regions (FR). The CDRs of the light and heavy chains contain within
them the amino acids which are largely responsible for the direct
interaction of the antibody with antigen, however, amino acids in
the FRs can significantly affect antigen binding/recognition as
discussed herein infra.
[0117] The term "light chain" when used in reference to an antibody
collectively refers to two distinct types, called kappa (k) or
lambda (l) based on the amino acid sequence of the constant
domains.
[0118] The term "heavy chain" when used in reference to an antibody
collectively refers to five distinct types, called alpha, delta,
epsilon, gamma and mu, based on the amino acid sequence of the
heavy chain constant domain. The combination of heavy and light
chains give rise to five known classes of antibodies: IgA, IgD,
IgE, IgG and IgM, respectively, including four known subclasses of
IgG, designated as IgG.sub.1, IgG.sub.2, IgG.sub.3 and
IgG.sub.4.
[0119] The term "naturally occurring" when used in connection with
biological materials such as nucleic acid molecules, polypeptides,
host cells, and the like, refers to those which are found in nature
and not modified by a human being.
[0120] The term "isolated" when used in relation to Ang-2 or to a
specific binding agent of Ang-2 refers to a compound that is free
from at least one contaminating polypeptide or compound that is
found in its natural environment, and preferably substantially free
from any other contaminating mammalian polypeptides that would
interfere with its therapeutic or diagnostic use.
[0121] The term "mature" when used in relation to Ang-2, anti-Ang-2
antibody, or to any other proteinaceous specific binding agent of
Ang-2 refers to a peptide or a polypeptide lacking a leader or
signal sequence. When a binding agent of the invention is
expressed, for example, in a prokaryotic host cell, the "mature"
peptide or polypeptide may also include additional amino acid
residues (but still lack a leader sequence) such as an amino
terminal methionine, or one or more methionine and lysine residues.
A peptide or polypeptide produced in this manner may be utilized
with or without these additional amino acid residues having been
removed.
Specific Binding Agents and Antibodies
[0122] As used herein, the term "specific binding agent" refers to
a molecule that has specificity for recognizing and binding Ang-2
and Ang-1, as described herein. Suitable specific binding agents
include, but are not limited to, antibodies and derivatives
thereof, polypeptides, and small molecules. Suitable specific
binding agents may be prepared using methods known in the art. An
exemplary Ang-2 and Ang-1 polypeptide specific binding agent of the
present invention is capable of binding a certain portion of the
Ang-2 and Ang-1 polypeptides, and preferably modulating the
activity or function of Ang-2 and Ang-1 polypeptides.
[0123] Specific binding agents such as antibodies and antibody
fragments that specifically bind Ang-2 and Ang-1 polypeptides are
within the scope of the present invention. The antibodies may be
polyclonal including mono-specific polyclonal, monoclonal (mAbs),
recombinant, chimeric, humanized such as CDR-grafted, human, single
chain, catalytic, multi-specific and/or bi-specific, as well as
antigen-binding fragments, variants, and/or derivatives
thereof.
[0124] Polyclonal antibodies against Ang2 and Ang1 polypeptides
generally are produced in animals (e.g., rabbits, hamsters, goats,
sheep, horses, pigs, rats, gerbils, guinea pigs, mice, or any other
suitable mammal, as well as other non-mammal species) by means of
multiple subcutaneous or intraperitoneal injections of Ang-2 and/or
Ang-1 polypeptide or a fragment thereof with or without an
adjuvant. Such adjuvants include, but are not limited to, Freund's
complete and incomplete, mineral gels such as aluminum hydroxide,
and surface-active substances such as lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanin, and dinitrophenol. BCG (bacilli Calmette-Guerin) and
Corynebacterium parvum are potentially useful human adjuvants. It
may be useful to conjugate an antigen polypeptide to a carrier
protein that is immunogenic in the species to be immunized, such as
keyhole limpet hemocyanin, serum, albumin, bovine thyroglobulin, or
soybean trypsin inhibitor. Also, aggregating agents such as alum
are used to enhance the immune response. After immunization, the
animals are bled and the serum is assayed for anti-Ang-2
polypeptide antibody titer which can be determined using the assays
described herein under "Examples". Polyclonal antibodies may be
utilized in the sera from which they were detected, or may be
purified from the sera, using, for example, antigen affinity
chromatography or Protein A or G affinity chromatography.
Monoclonal antibodies directed toward Ang-2 polypeptides can be
produced using, for example but without limitation, the traditional
"hybridoma" method or the newer "phage display" technique. For
example, monoclonal antibodies of the invention may be made by the
hybridoma method as described in Kohler et al., Nature 256:495
[1975]; the human B-cell hybridoma technique [Kosbor et al.,
Immunol Today 4:72 (1983); Cote et al., Proc Natl Acad Sci (USA)
80: 2026-2030 (1983); Brodeur et al., Monoclonal Antibody
Production Techniques and Applications, pp. 51-63, Marcel
[0125] Dekker, Inc., New York, (1987)] and the EBV-hybridoma
technique [Cole et al., Monoclonal Antibodies and Cancer Therapy,
Alan R Liss Inc, New York N.Y., pp 77-96, (1985)]. Also provided by
the invention are hybridoma cell lines that produce monoclonal
antibodies reactive with Ang-2 polypeptides.
[0126] When the hybridoma technique is employed, myeloma cell lines
can be used. Such cell lines suited for use in hybridoma-producing
fusion procedures preferably are non-antibody-producing, have high
fusion efficiency, and enzyme deficiencies that render them
incapable of growing in certain selective media which support the
growth of only the desired fused cells (hybridomas). For example,
cell lines used in mouse fusions are Sp-20, P3-X63/Ag8,
P3-X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11,
MPC11-X45-GTG 1.7 and 5194/5XX0 Bul; cell lines used in rat fusions
are R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210. Other cell lines
useful for cell fusions are U-266, GM1500-GRG2, LICR-LON-HMy2 and
UC729-6. Hybridomas and other cell lines that produce monoclonal
antibodies are contemplated to be novel compositions of the present
invention.
[0127] The phage display technique may also be used to generate
monoclonal antibodies from any species. Preferably, this technique
is used to produce fully human monoclonal antibodies in which a
polynucleotide encoding a single Fab or Fv antibody fragment is
expressed on the surface of a phage particle. [Hoogenboom et al., J
Mol Biol 227: 381 (1991); Marks et al., J Mol Biol 222: 581 (1991);
see also U.S. Pat. No. 5,885,793)]. Each phage can be "screened"
using binding assays described herein to identify those antibody
fragments having affinity for Ang-2. Thus, these processes mimic
immune selection through the display of antibody fragment
repertoires on the surface of filamentous bacteriophage, and
subsequent selection of phage by their binding to Ang-2. One such
procedure is described in PCT Application No. PCT/US98/17364, filed
in the name of Adams et al., which describes the isolation of high
affinity and functional agonistic antibody fragments for MPL- and
msk-receptors using such an approach. In this approach, a complete
repertoire of human antibody genes can be created by cloning
naturally rearranged human V genes from peripheral blood
lymphocytes as previously described [Mullinax et al., Proc Natl
Acad Sci (USA) 87: 8095-8099 (1990)].
[0128] Once a polynucleotide sequences are identified which encode
each chain of the full length monoclonal antibody or the Fab or Fv
fragment(s) of the invention, host cells, either eukaryotic or
prokaryotic, may be used to express the monoclonal antibody
polynucleotides using recombinant techniques well known and
routinely practiced in the art. Alternatively, transgenic animals
are produced wherein a polynucleotide encoding the desired specific
binding agent is introduced into the genome of a recipient animal,
such as, for example, a mouse, rabbit, goat, or cow, in a manner
that permits expression of the polynucleotide molecules encoding a
monoclonal antibody or other specific binding agent. In one aspect,
the polynucleotides encoding the monoclonal antibody or other
specific binding agent can be ligated to mammary-specific
regulatory sequences, and the chimeric polynucleotides can be
introduced into the germline of the target animal. The resulting
transgenic animal then produces the desired antibody in its milk
[Pollock et al., J Immunol Meth 231:147-157 (1999); Little et al.,
Immunol Today 8:364-370 (2000)]. In addition, plants may be used to
express and produce Ang-2 specific binding agents such as
monoclonal antibodies by transfecting suitable plants with the
polynucleotides encoding the monoclonal antibodies or other
specific binding agents.
[0129] In another embodiment of the present invention, a monoclonal
or polyclonal antibody or fragment thereof that is derived from
other than a human species may be "humanized" or "chimerized".
Methods for humanizing non-human antibodies are well known in the
art. (see U.S. Pat. Nos. 5,859,205, 5,585,089, and 5,693,762).
Humanization is performed, for example, using methods described in
the art [Jones et al., Nature 321: 522-525 (1986); Riechmann et
al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science
239:1534-1536 (1988)] by substituting at least a portion of, for
example a rodent, complementarity-determining region (CDRs) for the
corresponding regions of a human antibody. The invention also
provides variants and derivatives of these human antibodies as
discussed herein and well known in the art.
[0130] Also encompassed by the invention are fully human antibodies
that bind Ang-2 polypeptides, as well as, antigen-binding
fragments, variants and/or derivatives thereof. Such antibodies can
be produced using the phage display technique described above.
Alternatively, transgenic animals (e.g., mice) that are capable of
producing a repertoire of human antibodies in the absence of
endogenous immunoglobulin production can be used to generate such
antibodies. This can be accomplished by immunization of the animal
with an Ang-2 antigen or fragments thereof where the Ang-2
fragments have an amino acid sequence that is unique to Ang-2. Such
immunogens can be optionally conjugated to a carrier. See, for
example, Jakobovits et al., Proc Natl Acad Sci (USA), 90: 2551-2555
(1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggermann
et al., Year in Immuno, 7: 33 (1993). In one method, such
transgenic animals are produced by incapacitating the endogenous
loci encoding the heavy and light immunoglobulin chains therein,
and inserting loci encoding human heavy and light chain proteins
into the genome thereof. Partially modified animals, that are those
having less than the full complement of these modifications, are
then crossbred to obtain an animal having all of the desired immune
system modifications. When administered an immunogen, these
transgenic animals are capable of producing antibodies with human
variable regions, including human (rather than e.g., murine) amino
acid sequences, that are immuno-specific for the desired antigens.
See PCT application Nos., PCT/US96/05928 and PCT/US93/06926.
Additional methods are described in U.S. Pat. No. 5,545,807, PCT
application Nos. PCT/US91/245, PCT/GB89/01207, and in EP 546073B1
and EP 546073A1. Human antibodies may also be produced by the
expression of recombinant DNA in host cells or by expression in
hybridoma cells as described herein.
[0131] Transgenesis is achieved in a number of different ways. See,
for example, Bruggeman et al., Immunol Today 17:391-7 (1996). In
one approach, a minilocus is constructed such that gene segments in
a germline configuration are brought artificially close to each
other. Due to size limitations (i.e., having generally less than 30
kb), the resulting minilocus will contain a limited number of
differing gene segments, but is still capable of producing a large
repertoire of antibodies. Miniloci containing only human DNA
sequences, including promoters and enhancers are fully functional
in the transgenic mouse.
[0132] When larger number of gene segments are desired in the
transgenic animal, yeast artificial chromosomes (YACs) are
utilized. YACs can range from several hundred kilobases to 1 Mb and
are introduced into the mouse (or other appropriate animal) genome
via microinjection directly into an egg or via transfer of the YAC
into embryonic stem (ES)-cell lines. In general, YACs are
transferred into ES cells by lipofection of the purified DNA, or
yeast spheroplast fusion wherein the purified DNA is carried in
micelles and fusion is carried out in manner similar to hybridoma
fusion protocols. Selection of desired ES cells following DNA
transfer is accomplished by including on the YAC any of the
selectable markers known in the art.
[0133] As another alternative, bacteriophage P1 vectors are used
which are amplified in a bacterial E. coli host. While these
vectors generally carry less inserted DNA than a YAC, the clones
are readily grown in high enough yield to permit direct
microinjection into a mouse egg. Use of a cocktail of different P1
vectors has been shown to lead to high levels of homologous
recombination.
[0134] Once an appropriate transgenic mouse (or other appropriate
animal) has been identified, using any of the techniques known in
the art to detect serum levels of a circulating antibody (e.g.,
ELISA), the transgenic animal is crossed with a mouse in which the
endogenous Ig locus has been disrupted. The result provides progeny
wherein essentially all B cells express human antibodies.
[0135] As still another alternative, the entire animal Ig locus is
replaced with the human Ig locus, wherein the resulting animal
expresses only human antibodies. In another approach, portions of
the animal's locus are replaced with specific and corresponding
regions in the human locus. In certain cases, the animals resulting
from this procedure may express chimeric antibodies, as opposed to
fully human antibodies, depending on the nature of the replacement
in the mouse Ig locus.
[0136] Human antibodies can also be produced by exposing human
splenocytes (B or T cells) to an antigen in vitro, then
reconstituting the exposed cells in an immunocompromised mouse,
e.g. SCID or nod/SCID. See Brams et al., J Immunol, 160: 2051-2058
[1998]; Carballido et al., Nat Med, 6: 103-106 [2000]. In one
approach, engraftment of human fetal tissue into SCID mice
(SCID-hu) results in long-term hematopoiesis and human T-cell
development [McCune et al., Science 241:1532-1639 (1988); Ifversen
et al., Sem Immunol 8:243-248 (1996)]. Any humoral immune response
in these chimeric mice is completely dependent on co-development of
T-cells in the animals [Martensson et al., Immunol 83:1271-179
(1994)]. In an alternative approach, human peripheral blood
lymphocytes are transplanted intraperitoneally (or otherwise) into
SCID mice [Mosier et al., Nature 335:256-259 (1988)]. When the
transplanted cells are treated with either a priming agent, such as
Staphylococcal Enterotoxin A (SEA) [Martensson et al., Immunol 84:
224-230 (1995)], or anti-human CD40 monoclonal antibodies [Murphy
et al., Blood 86:1946-1953 (1995)], higher levels of B cell
production are detected.
[0137] Alternatively, an entirely synthetic human heavy chain
repertoire is created from unrearranged V gene segments by
assembling each human VH segment with D segments of random
nucleotides together with a human J segment [Hoogenboom et al., J
Mol Biol 227:381-388 (1992)]. Likewise, a light chain repertoire is
constructed by combining each human V segment with a J segment
[Griffiths et al., EMBO J 13:3245-3260 (1994)]. Nucleotides
encoding the complete antibody (i.e., both heavy and light chains)
are linked as a single chain Fv fragment and this polynucleotide is
ligated to a nucleotide encoding a filamentous phage minor coat
protein. When this fusion protein is expressed on the surface of
the phage, a polynucleotide encoding a specific antibody is
identified by selection using an immobilized antigen.
[0138] In still another approach, antibody fragments are assembled
as two Fab fragments by fusion of one chain to a phage protein and
secretion of the other into bacterial periplasm [Hoogenboom et al.,
Nucl Acids Res 19:4133-4137 [1991]; Barbas et al., Proc Natl Acad
Sci (USA) 88:7978-7982 (1991)].
[0139] Large-scale production of chimeric, humanized, CDR-grafted,
and fully human antibodies, or antigen-binding fragments thereof,
are typically produced by recombinant methods. Polynucleotide
molecule(s) encoding the heavy and light chains of each antibody or
antigen-binding fragments thereof, can be introduced into host
cells and expressed using materials and procedures described
herein. In a preferred embodiment, the antibodies are produced in
mammalian host cells, such as CHO cells. Details of such production
are described herein.
[0140] The specific binding agents of the present invention, such
as the antibodies, antibody fragments, and antibody derivatives of
the invention can further comprise any constant region known in the
art. The light chain constant region can be, for example, a kappa-
or lambda-type light chain constant region, e.g., a human kappa- or
lambda-type light chain constant region. The heavy chain constant
region can be, for example, an alpha-, delta-, epsilon-, gamma-, or
mu-type heavy chain constant regions, e.g., a human alpha-, delta-,
epsilon-, gamma-, or mu-type heavy chain constant region. In one
embodiment, the light or heavy chain constant region is a fragment,
derivative, variant, or mutein of a naturally occurring constant
region.
[0141] In one embodiment, the specific binding agents of the
present invention, such as the antibodies, antibody fragments, and
antibody derivatives of the invention comprise an IgG.
[0142] Techniques are known for deriving an antibody of a different
subclass or isotype from an antibody of interest, i.e., subclass
switching. Thus, IgG antibodies may be derived from an IgM
antibody, for example, and vice versa. Such techniques allow the
preparation of new antibodies that possess the antigen-binding
properties of a given antibody (the parent antibody), but also
exhibit biological properties associated with an antibody isotype
or subclass different from that of the parent antibody. Recombinant
DNA techniques may be employed. Cloned DNA encoding particular
antibody polypeptides may be employed in such procedures, e.g., DNA
encoding the constant domain of an antibody of the desired isotype.
See also Lantto et al., 2002, Methods Mol. Biol. 178:303-16.
[0143] The specific binding agents of the present invention, such
as the antibodies, antibody fragments, and antibody derivatives of
the invention may comprise the IgG1 heavy chain constant domain or
a fragment of the IgG1 heavy chain domain. The antibodies, antibody
fragments, and antibody derivatives of the invention may further
comprise the constant light chain kappa or lambda domains or a
fragment of these. Light chain constant regions and polynucleotides
encoding them are provided herein below. In another embodiment, the
antibodies, antibody fragments, and antibody derivatives of the
invention further comprise a heavy chain constant domain, or a
fragment thereof, such as the IgG2 heavy chain constant region also
shown herein below.
[0144] The nucleic acid (DNA) encoding constant heavy and constant
light chain domains, and the amino acids sequences of heavy and
light chain domains are provided herein below. Lambda variable
domains can be fused to lambda constant domains and kappa variable
domains can be fused to kappa constant domains.
TABLE-US-00001 IgG2 Heavy Constant domain DNA (SEQ ID NO: 41):
gctagcaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagcggccct-
gggctgcc
tggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacacc-
ttcccagctg
tcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacc-
tacacctgcaa
cgtagatcacaagcccagcaacaccaaggtggacaagacagttgagegcaaatgttgtgtcgagtgcccaccgt-
gcccagcac
cacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccct-
gaggtcacgtg
cgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcata-
atgccaag
acaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactg-
gctgaacgg
caaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaag-
ggcagccc
cgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcct-
ggtcaaag
gcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacct-
cccatgct
ggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtct-
tctcatgctc
cgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga
IgG2 Heavy Constant domain Protein (SEQ ID NO: 42):
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCP
APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVH
NAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKT
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
TPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Kappa Light
Constant domain DNA (SEQ ID NO: 43):
cgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgt-
tgtgtgcctgctga
ataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggag-
agtgtcacag
agcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacac-
aaagtct
acgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag
Kappa Light Constant domain Protein (SEQ ID NO: 44):
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Lambda Light
Constant domain DNA (SEQ ID NO: 45):
ggccaaccgaaagcggcgccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccac-
actggtgtgt
ctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagt-
ggagacca
ccacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaag-
tcccacag
aagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttcatag
Lambda Light Constant domain Protein (SEQ ID NO: 46):
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVET
TTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
[0145] The specific binding agents of the present invention, such
as the antibodies, antibody fragments, and antibody derivatives of
the invention include those comprising, for example, the variable
domain combinations H6L7, H5L7, H4L13, H11L7, H4L7, H10L7, H5L6,
H2L7, H5L8, H6L8, H3L7, H5L4, H4L12, H6L6, H4L2, H4L6, H4L4, H5L11,
H5L1, H4L11, H5L12, H5L9 having a desired isotype (for example,
IgA, IgG1, IgG2, IgG3, IgG4, IgM, IgE, and IgD) as well as Fab or
F(ab').sub.2 fragments thereof. Moreover, if an IgG4 is desired, it
may also be desired to introduce a point mutation in the hinge
region as described in Bloom et al., 1997, Protein Science 6:407
(incorporated by reference herein) to alleviate a tendency to form
intra-H chain disulfide bonds that can lead to heterogeneity in the
IgG4 antibodies.
Additional Useful Sequence Information
[0146] The following sequences of the IgG1, IgG2, IgG3, and IgG4
isotypes are used in combination with the variable heavy chain
sequences of the antibodies of the present invention to make a
specific desired isotype of said antibody:
TABLE-US-00002 Human IgG1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG2
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVER
KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP
EVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKC
KVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG
FYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK Human IgG3
ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVEL
KTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSC
DTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
PEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYK
CKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK
GFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQG
NIFSCSVMHEALHNRFTQKSLSLSPGK Human IgG4
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES
KYGPPCPSCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED
PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG
NVFSCSVMHEALHNHYTQKSLSLSLGK
HC Sequences of the Antibodies of the Present Invention as an
IgG2
[0147] The following sequences represent the heavy chain sequences
of the antibodies of the present invention as IgG2 isotype. The
light chain sequences remain the same, which are provided in the
Examples. The underlined sequence portions represent the IgG2
sequences:
TABLE-US-00003 H2
EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSYISSSGSTIEY
ADSVKGRETISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDILTGYGYWGQGTLVT
VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECP
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVE
VHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISK
TKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
TPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK H3
EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSYISSSGSTIQY
ADSVKGRETISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDILTGYGYWGQGTLVT
VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECP
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVE
VHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISK
TKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
TPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK H6
EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSYISSSGSTIYY
ADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDIYTGYGYWGQGTLV
TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVEC
PPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGV
EVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTIS
KTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK H10
EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSYISSSGSTIYY
ADSVKGRETISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDILTGYGLWGQGTLVT
VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTF
RVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTK
NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFELYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPGK H11
EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSYISSSGSTIYY
ADSVKGRETISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDILTGYGMWGQGTLV
TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVEC
PPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGV
EVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTIS
KTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK H4
EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSYISSSGSTIYY
ADSVKGRETISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDLLTGYGYWGQGTLV
TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVEC
PPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGV
EVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTIS
KTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK H5
EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSYISSSGSTIYY
ADSVKGRETISRDNAKNSLYLQMNSLRAEDTAVYYCARDLLDYDIWTGYGYWGQGTLV
TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVEC
PPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGV
EVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTIS
KTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Fusion Partners of Specific Binding Agents
[0148] In a further embodiment of the invention, the polypeptides
comprising the amino acid sequence variable domains of Ang-2
antibodies, such as a heavy chain variable region with an amino
acid sequence as described herein or a light chain variable region
with an amino acid sequence as described herein, may be fused at
either the N-terminus or the C-terminus to one or more domains of
an Fc region of human IgG. When constructed together with a
therapeutic protein such as the Fab of an Ang-2-specific antibody,
an Fc domain can provide longer half-life or incorporate such
functions as Fc receptor binding, Protein A binding, complement
fixation and perhaps even placental transfer. [Capon et al.,
Nature, 337: 525-531 (1989)].
[0149] In one example, the antibody hinge, CH2 and CH3 regions may
be fused at either the N-terminus or C-terminus of the specific
binding agent polypeptides such as an anti-Ang-2 Fab or Fv fragment
(obtained, e.g., from a phage display library) using methods known
to the skilled artisan. The resulting fusion protein may be
purified by use of a Protein A or Protein G affinity column.
Peptides and proteins fused to an Fc region have been found to
exhibit a substantially greater half-life in vivo than the unfused
counterpart. Also, a fusion to an Fc region allows for
dimerization/multimerization of the fusion polypeptide. The Fc
region may be a naturally occurring Fc region, or may be altered to
improve certain qualities, such as therapeutic qualities,
circulation time, decrease aggregation problems, etc. Other
examples known in the art include those wherein the Fc region,
which may be human or another species, or may be synthetic, is
fused to the N-terminus of CD30L to treat Hodgkin's Disease,
anaplastic lymphoma and T-cell leukemia (U.S. Pat. No. 5,480,981),
the Fc region is fused to the TNF receptor to treat septic shock
[Fisher et al., N Engl J Med, 334: 1697-1702 (1996)], and the Fc
region is fused to the Cd4 receptor to treat AIDS [Capon et al.,
Nature, 337: 525-31 (1989)].
[0150] Catalytic antibodies are another type of fusion molecule and
include antibodies to which one or more cytotoxic, or more
generally one or more biologically active, moieties are attached to
the specific binding agent. See, for example Rader et al., Chem Eur
J 12:2091-2095 (2000). Cytotoxic agents of this type improve
antibody-mediated cytotoxicity, and include such moieties as
cytokines that directly or indirectly stimulate cell death,
radioisotopes, chemotherapeutic drugs (including prodrugs),
bacterial toxins (ex. pseudomonas exotoxin, diphtheria toxin,
etc.), plant toxins (ex. ricin, gelonin, etc.), chemical conjugates
(e.g., maytansinoid toxins, calechaemicin, etc.), radioconjugates,
enzyme conjugates (RNase conjugates, antibody-directed
enzyme/prodrug therapy [ADEPT)]), and the like. In one aspect, the
cytotoxic agent can be "attached" to one component of a bi-specific
or multi-specific antibody by binding of this agent to one of the
alternative antigen recognition sites on the antibody. As an
alternative, protein cytotoxins can be expressed as fusion proteins
with the specific binding agent following ligation of a
polynucleotide encoding the toxin to a polynucleotide encoding the
binding agent. In still another alternative, the specific binding
agent can be covalently modified to include the desired
cytotoxin.
[0151] Examples of such fusion proteins are immunogenic
polypeptides, proteins with long circulating half lives, such as
immunoglobulin constant regions, marker proteins, proteins or
polypeptides that facilitate purification of the desired specific
binding agent polypeptide, and polypeptide sequences that promote
formation of multimeric proteins (such as leucine zipper motifs
that are useful in dimer formation/stability).
[0152] This type of insertional variant generally has all or a
substantial portion of the native molecule, linked at the N- or
C-terminus, to all or a portion of a second polypeptide. For
example, fusion proteins typically employ leader sequences from
other species to permit the recombinant expression of a protein in
a heterologous host. Another useful fusion protein includes the
addition of an immunologically active domain, such as an antibody
epitope, to facilitate purification of the fusion protein.
Inclusion of a cleavage site at or near the fusion junction will
facilitate removal of the extraneous polypeptide after
purification. Other useful fusions include linking of functional
domains, such as active sites from enzymes, glycosylation domains,
cellular targeting signals or transmembrane regions.
[0153] There are various commercially available fusion protein
expression systems that may be used in the present invention.
Particularly useful systems include but are not limited to the
glutathione-S-transferase (GST) system (Pharmacia), the maltose
binding protein system (NEB, Beverley, Mass.), the FLAG system
(IBI, New Haven, Conn.), and the 6.times.His system (Qiagen,
Chatsworth, Calif.). These systems are capable of producing
recombinant polypeptides bearing only a small number of additional
amino acids, which are unlikely to affect the antigenic ability of
the recombinant polypeptide. For example, both the FLAG system and
the 6.times.His system add only short sequences, both of which are
known to be poorly antigenic and which do not adversely affect
folding of the polypeptide to its native conformation. Another
N-terminal fusion that is contemplated to be useful is the fusion
of a Met-Lys dipeptide at the N-terminal region of the protein or
peptides. Such a fusion may produce beneficial increases in protein
expression or activity.
[0154] A particularly useful fusion construct may be one in which a
specific binding agent peptide is fused to a hapten to enhance
immunogenicity of a specific binding agent fusion construct which
is useful, for example, in the production of anti-idiotype
antibodies of the invention. Such fusion constructs to increase
immunogenicity are well known to those of skill in the art, for
example, a fusion of specific binding agent with a helper antigen
such as hsp70 or peptide sequences such as from diphtheria toxin
chain or a cytokine such as IL-2 will be useful in eliciting an
immune response. In other embodiments, fusion construct can be made
which will enhance the targeting of the antigen binding agent
compositions to a specific site or cell.
[0155] Other fusion constructs including heterologous polypeptides
with desired properties, e.g., an Ig constant region to prolong
serum half-life or an antibody or fragment thereof for targeting
also are contemplated. Other fusion systems produce polypeptide
hybrids where it is desirable to excise the fusion partner from the
desired polypeptide. In one embodiment, the fusion partner is
linked to the recombinant specific binding agent polypeptide by a
peptide sequence containing a specific recognition sequence for a
protease. Examples of suitable sequences are those recognized by
the Tobacco Etch Virus protease (Life Technologies, Gaithersburg,
Md.) or Factor Xa (New England Biolabs, Beverley, Mass.).
[0156] The invention also provides fusion polypeptides comprising
all or part of a variable domain of an Ang-2 antibody, such as a
heavy chain variable region with an amino acid sequence as
described herein or a light chain variable region with an amino
acid sequence as described herein in combination with truncated
tissue factor (tTF), a vascular targeting agent consisting of a
truncated form of a human coagulation-inducing protein that acts as
a tumor blood vessel clotting agent. The fusion of tTF to the
anti-Ang-2 antibody, or fragments thereof may facilitate the
delivery of anti-Ang-2 to target cells.
Variants of Specific Binding Agents
[0157] Variants of Specific Binding Agents of the present invention
include insertion, deletion, and/or substitution variants. In one
aspect of the invention, insertion variants are provided wherein
one or more amino acid residues supplement a specific binding agent
amino acid sequence. Insertions may be located at either or both
termini of the protein, or may be positioned within internal
regions of the specific binding agent amino acid sequence.
Insertional variants with additional residues at either or both
termini can include, for example, fusion proteins and proteins
including amino acid tags or labels. Insertion variants include
specific binding agent polypeptides wherein one or more amino acid
residues are added to a specific binding agent amino acid sequence,
or fragment thereof.
[0158] Variant products of the invention also include mature
specific binding agent products. Such specific binding agent
products have the leader or signal sequences removed, however the
resulting protein has additional amino terminal residues as
compared to wild-type Ang-2 polypeptide. The additional amino
terminal residues may be derived from another protein, or may
include one or more residues that are not identifiable as being
derived from a specific protein. Specific binding agent products
with an additional methionine residue at position -1
(Mer.sup.-1-specific binding agent) are contemplated, as are
specific binding agent products with additional methionine and
lysine residues at positions -2 and -1 (Met 2-Lys.sup.-1-specific
binding agent). Variants of specific binding agents having
additional Met, Met-Lys, Lys residues (or one or more basic
residues in general) are particularly useful for enhanced
recombinant protein production in bacterial host cells.
[0159] The invention also embraces specific binding agent variants
having additional amino acid residues that arise from use of
specific expression systems. For example, use of commercially
available vectors that express a desired polypeptide as part of
glutathione-S-transferase (GST) fusion product provides the desired
polypeptide having an additional glycine residue at amino acid
position -1 after cleavage of the GST component from the desired
polypeptide. Variants which result from expression in other vector
systems are also contemplated, including those wherein
poly-histidine tags are incorporated into the amino acid sequence,
generally at the carboxy and/or amino terminus of the sequence.
[0160] Insertional variants also include fusion proteins as
described above, wherein the amino and/or carboxy termini of the
specific binding agent-polypeptide is fused to another polypeptide,
a fragment thereof, or amino acid sequences which are not generally
recognized to be part of any specific protein sequence.
[0161] In another aspect, the invention provides deletion variants
wherein one or more amino acid residues in a specific binding agent
polypeptide are removed. Deletions can be effected at one or both
termini of the specific binding agent polypeptide, or from removal
of one or more residues within the specific binding agent amino
acid sequence. Deletion variants necessarily include all fragments
of a specific binding agent polypeptide.
[0162] Antibody fragments include those portions of the antibody
that bind to an epitope on the antigen polypeptide. Examples of
such fragments include Fab and F(ab').sub.2 fragments generated,
for example, by enzymatic or chemical cleavage of full-length
antibodies. Other binding fragments include those generated by
recombinant DNA techniques, such as the expression of recombinant
plasmids containing nucleic acid sequences encoding antibody
variable regions. The invention also embraces polypeptide fragments
of an Ang-2 binding agent wherein the fragments maintain the
ability to specifically bind an Ang-2 polypeptide. Fragments
comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 or more
consecutive amino acids of a peptide or polypeptide of the
invention are comprehended herein. Preferred polypeptide fragments
display immunological properties unique to or specific for the
antigen-binding agent so of the invention. Fragments of the
invention having the desired immunological properties can be
prepared by any of the methods well known and routinely practiced
in the art.
[0163] In still another aspect, the invention provides substitution
variants of specific binding agents of the invention. Substitution
variants are generally considered to be "similar" to the original
polypeptide or to have a certain "percent identity" to the original
polypeptide, and include those polypeptides wherein one or more
amino acid residues of a polypeptide are removed and replaced with
alternative residues. In one aspect, the substitutions are
conservative in nature, however, the invention embraces
substitutions that are also non-conservative.
[0164] Identity and similarity of related polypeptides can be
readily calculated by known methods. Such methods include, but are
not limited to, those described in Computational Molecular Biology,
Lesk, A. M., ed., Oxford University Press, New York (1988);
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York (1993); Computer Analysis of Sequence
Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana
Press, New Jersey (1994); Sequence Analysis in Molecular Biology,
von Heinje, G., Academic Press (1987); Sequence Analysis Primer,
Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York
(1991); and Carillo et al., SIAM J. Applied Math., 48:1073
(1988).
[0165] Preferred methods to determine the relatedness or percent
identity of two polypeptides are designed to give the largest match
between the sequences tested. Methods to determine identity are
described in publicly available computer programs. Preferred
computer program methods to determine identity between two
sequences include, but are not limited to, the GCG program package,
including GAP (Devereux et al., Nucl. Acid. Res., 12:387 (1984);
Genetics Computer Group, University of Wisconsin, Madison, Wis.,
BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol.,
215:403-410 (1990)). The BLASTX program is publicly available from
the National Center for Biotechnology Information (NCBI) and other
sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md.
20894; Altschul et al., supra (1990)). The well-known Smith
Waterman algorithm may also be used to determine identity.
[0166] Certain alignment schemes for aligning two amino acid
sequences may result in the matching of only a short region of the
two sequences, and this small aligned region may have very high
sequence identity even though there is no significant relationship
between the two full-length sequences. Accordingly, in certain
embodiments, the selected alignment method (GAP program) will
result in an alignment that spans at least ten percent of the full
length of the target polypeptide being compared, i.e., at least 40
contiguous amino acids where sequences of at least 400 amino acids
are being compared, 30 contiguous amino acids where sequences of at
least 300 to about 400 amino acids are being compared, at least 20
contiguous amino acids where sequences of 200 to about 300 amino
acids are being compared, and at least 10 contiguous amino acids
where sequences of about 100 to 200 amino acids are being
compared.
[0167] For example, using the computer algorithm GAP (Genetics
Computer Group, University of Wisconsin, Madison, Wis.), two
polypeptides for which the percent sequence identity is to be
determined are aligned for optimal matching of their respective
amino acids (the "matched span", as determined by the algorithm).
In certain embodiments, a gap opening penalty (which is typically
calculated as 3.times. the average diagonal; the "average diagonal"
is the average of the diagonal of the comparison matrix being used;
the "diagonal" is the score or number assigned to each perfect
amino acid match by the particular comparison matrix) and a gap
extension penalty (which is usually 1/10 times the gap opening
penalty), as well as a comparison matrix such as PAM 250 or BLOSUM
62 are used in conjunction with the algorithm. In certain
embodiments, a standard comparison matrix (see Dayhoff et al.,
Atlas of Protein Sequence and Structure, 5(3)(1978) for the PAM 250
comparison matrix; Henikoff et al., Proc. Natl. Acad. Sci. USA,
89:10915-10919 (1992) for the BLOSUM 62 comparison matrix) is also
used by the algorithm.
[0168] In certain embodiments, the parameters for a polypeptide
sequence comparison include the following:
[0169] Algorithm: Needleman et al., J. Mol. Biol., 48:443-453
(1970);
[0170] Comparison matrix: BLOSUM 62 from Henikoff et al., supra
(1992);
[0171] Gap Penalty: 12
[0172] Gap Length Penalty: 4
[0173] Threshold of Similarity: 0
[0174] The GAP program may be useful with the above parameters. In
certain embodiments, the aforementioned parameters are the default
parameters for polypeptide comparisons (along with no penalty for
end gaps) using the GAP algorithm.
[0175] In certain embodiments, the parameters for polynucleotide
molecule sequence comparisons include the following: [0176]
Algorithm: Needleman et al., supra (1970); [0177] Comparison
matrix: matches=+10, mismatch=0 [0178] Gap Penalty: 50 [0179] Gap
Length Penalty: 3
[0180] The GAP program may also be useful with the above
parameters. The aforementioned parameters are the default
parameters for polynucleotide molecule comparisons.
[0181] Other exemplary algorithms, gap opening penalties, gap
extension penalties, comparison matrices, thresholds of similarity,
etc. may be used, including those set forth in the Program Manual,
Wisconsin Package, Version 9, September, 1997. The particular
choices to be made will be apparent to those of skill in the art
and will depend on the specific comparison to be made, such as
DNA-to-DNA, protein-to-protein, protein-to-DNA; and additionally,
whether the comparison is between given pairs of sequences (in
which case GAP or BestFit are generally preferred) or between one
sequence and a large database of sequences (in which case FASTA or
BLASTA are preferred).
[0182] As used herein, the twenty conventional amino acids and
their abbreviations follow conventional usage. See Immunology--A
Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer
Associates, Sunderland, Mass. (1991)), which is incorporated herein
by reference for any purpose.
[0183] The amino acids may have either L or D stereochemistry
(except for Gly, which is neither L nor D) and the polypeptides and
compositions of the present invention may comprise a combination of
stereochemistries. However, the L stereochemistry is preferred. The
invention also provides reverse molecules wherein the amino
terminal to carboxy terminal sequence of the amino acids is
reversed. For example, the reverse of a molecule having the normal
sequence X.sub.1-X.sub.2-X.sub.3 would be X.sub.3-X.sub.2-X.sub.1.
The invention also provides retro-reverse molecules wherein, as
above, the amino terminal to carboxy terminal sequence of amino
acids is reversed and residues that are normally "L" enantiomers
are altered to the "D" stereoisomer form.
[0184] Stereoisomers (e.g., D-amino acids) of the twenty
conventional amino acids, unnatural amino acids such as .alpha.-,
.alpha.-disubstituted amino acids, N-alkyl amino acids, lactic
acid, and other unconventional amino acids may also be suitable
components for polypeptides of the present invention. Examples of
unconventional amino acids include, without limitation: aminoadipic
acid, beta-alanine, beta-aminopropionic acid, aminobutyric acid,
piperidinic acid, aminocaprioic acid, aminoheptanoic acid,
aminoisobutyric acid, aminopimelic acid, diaminobutyric acid,
desmosine, diaminopimelic acid, diaminopropionic acid,
N-ethylglycine, N-ethylaspargine, hyroxylysine, allo-hydroxylysine,
hydroxyproline, isodesmosine, allo-isoleucine, N-methylglycine,
sarcosine, N-methylisoleucine, N-methylvaline, norvaline,
norleucine, orithine, 4-hydroxyproline, .gamma.-carboxyglutamate,
.epsilon.-N,N,N-trimethyllysine, .epsilon.-N-acetyllysine,
O-phosphoserine, N-acetylserine, N-formylmethionine,
3-methylhistidine, 5-hydroxylysine, .sigma.-N-methylarginine, and
other similar amino acids and amino acids (e.g.,
4-hydroxyproline).
[0185] Similarly, unless specified otherwise, the left-hand end of
single-stranded polynucleotide sequences is the 5' end; the
left-hand direction of double-stranded polynucleotide sequences is
referred to as the 5' direction. The direction of 5' to 3' addition
of nascent RNA transcripts is referred to as the transcription
direction; sequence regions on the DNA strand having the same
sequence as the RNA and which are 5' to the 5' end of the RNA
transcript are referred to as "upstream sequences"; sequence
regions on the DNA strand having the same sequence as the RNA and
which are 3' to the 3' end of the RNA transcript are referred to as
"downstream sequences".
[0186] Conservative amino acid substitutions may encompass
non-naturally occurring amino acid residues, which are typically
incorporated by chemical peptide synthesis rather than by synthesis
in biological systems. These include peptidomimetics and other
reversed or inverted forms of amino acid moieties.
[0187] Naturally occurring residues may be divided into classes
based on common side chain properties:
[0188] 1) hydrophobic: Met, Ala, Val, Leu, Ile;
[0189] 2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0190] 3) acidic: Asp, Glu;
[0191] 4) basic: His, Lys, Arg;
[0192] 5) residues that influence chain orientation: Gly, Pro;
and
[0193] 6) aromatic: Trp, Tyr, Phe.
[0194] For example, non-conservative substitutions may involve the
exchange of a member of one of these classes for a member from
another class. Such substituted residues may be introduced into
regions of the human antibody that are homologous with non-human
antibodies, or into the non-homologous regions of the molecule.
[0195] In making such changes, according to certain embodiments,
the hydropathic index of amino acids may be considered. Each amino
acid has been assigned a hydropathic index on the basis of its
hydrophobicity and charge characteristics. They are: isoleucine
(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);
cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine
(-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9);
tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate
(-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5);
lysine (-3.9); and arginine (-4.5).
[0196] The importance of the hydropathic amino acid index in
conferring interactive biological function on a protein is
understood in the art. Kyte et al., J. Mol. Biol., 157:105-131
(1982). It is known that certain amino acids may be substituted for
other amino acids having a similar hydropathic index or score and
still retain a similar biological activity. In making changes based
upon the hydropathic index, in certain embodiments, the
substitution of amino acids whose hydropathic indices are within
.+-.2 is included. In certain embodiments, those which are within
.+-.1 are included, and in certain embodiments, those within
.+-.0.5 are included.
[0197] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity, particularly where the biologically functional
protein or peptide thereby created is intended for use in
immunological embodiments, as in the present case. In certain
embodiments, the greatest local average hydrophilicity of a
protein, as governed by the hydrophilicity of its adjacent amino
acids, correlates with its immunogenicity and antigenicity, i.e.,
with a biological property of the protein.
[0198] The following hydrophilicity values have been assigned to
these amino acid residues: arginine (+3.0); lysine (+3.0);
aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3);
asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4);
proline (-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine
(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8);
isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and
tryptophan (-3.4). In making changes based upon similar
hydrophilicity values, in certain embodiments, the substitution of
amino acids whose hydrophilicity values are within .+-.2 is
included, in certain embodiments, those which are within .+-.1 are
included, and in certain embodiments, those within .+-.0.5 are
included. One may also identify epitopes from primary amino acid
sequences on the basis of hydrophilicity. These regions are also
referred to as "epitopic core regions."
[0199] Exemplary amino acid substitutions are set forth in Table
1.
TABLE-US-00004 TABLE 1 Amino Acid Substitutions Original Exemplary
Preferred Residues Substitutions Substitutions Ala Val, Leu, Ile
Val Arg Lys, Gln, Asn Lys Asn Gln, Glu, Asp Gln Asp Glu, Gln, Asn
Glu Cys Ser, Ala Ser Gln Asn, Glu, Asp Asn Glu Asp, Asn, Gln Asp
Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala,
Leu Phe, Norleucine Leu Norleucine, Ile, Ile Val, Met, Ala, Phe Lys
Arg, 1,4 Diamino-butyric Arg Acid, Gln, Asn Met Leu, Phe, Ile Leu
Phe Leu, Val, Ile, Ala, Leu Tyr Pro Ala Gly Ser Thr, Ala, Cys Thr
Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile,
Met, Leu, Phe, Leu Ala, Norleucine
[0200] A skilled artisan will be able to determine suitable
variants of the polypeptide as set forth herein using well-known
techniques. In certain embodiments, one skilled in the art may
identify suitable areas of the molecule that may be changed without
destroying activity by targeting regions not believed to be
important for activity. In certain embodiments, one can identify
residues and portions of the molecules that are conserved among
similar polypeptides. In certain embodiments, even areas that may
be important for biological activity or for structure may be
subject to conservative amino acid substitutions without destroying
the biological activity or without adversely affecting the
polypeptide structure.
[0201] Additionally, one skilled in the art can review
structure-function studies identifying residues in similar
polypeptides that are important for activity or structure. In view
of such a comparison, one can predict the importance of amino acid
residues in a protein that correspond to amino acid residues which
are important for activity or structure in similar proteins. One
skilled in the art may opt for chemically similar amino acid
substitutions for such predicted important amino acid residues.
[0202] One skilled in the art can also analyze the
three-dimensional structure and amino acid sequence in relation to
that structure in similar polypeptides. In view of such
information, one skilled in the art may predict the alignment of
amino acid residues of an antibody with respect to its three
dimensional structure. In certain embodiments, one skilled in the
art may choose not to make radical changes to amino acid residues
predicted to be on the surface of the protein, since such residues
may be involved in important interactions with other molecules.
Moreover, one skilled in the art may generate test variants
containing a single amino acid substitution at each desired amino
acid residue. The variants can then be screened using activity
assays known to those skilled in the art. Such variants could be
used to gather information about suitable variants. For example, if
one discovered that a change to a particular amino acid residue
resulted in destroyed, undesirably reduced, or unsuitable activity,
variants with such a change may be avoided. In other words, based
on information gathered from such routine experiments, one skilled
in the art can readily determine the amino acids where further
substitutions should be avoided either alone or in combination with
other mutations.
[0203] A number of scientific publications have been devoted to the
prediction of secondary structure. See Moult J., Curr. Op. in
Biotech., 7(4):422-427 (1996), Chou et al., Biochemistry,
13(2):222-245 (1974); Chou et al., Biochemistry, 113(2):211-222
(1974); Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol.,
47:45-148 (1978); Chou et al., Ann. Rev. Biochem., 47:251-276 and
Chou et al., Biophys. J., 26:367-384 (1979). Moreover, computer
programs are currently available to assist with predicting
secondary structure. One method of predicting secondary structure
is based upon homology modeling. For example, two polypeptides or
proteins which have a sequence identity of greater than 30%, or
similarity greater than 40% often have similar structural
topologies. The recent growth of the protein structural database
(PDB) has provided enhanced predictability of secondary structure,
including the potential number of folds within a polypeptide's or
protein's structure. See Holm et al., Nucl. Acid. Res.,
27(1):244-247 (1999). It has been suggested (Brenner et al., Curr.
Op. Struct. Biol., 7(3):369-376 (1997)) that there are a limited
number of folds in a given polypeptide or protein and that once a
critical number of structures have been resolved, structural
prediction will become dramatically more accurate.
[0204] Additional methods of predicting secondary structure include
"threading" (Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87
(1997); Sippl et al., Structure, 4(1):15-19 (1996)), "profile
analysis" (Bowie et al., Science, 253:164-170 (1991); Gribskov et
al., Meth. Enzym., 183:146-159 (1990); Gribskov et al., Proc. Nat.
Acad. Sci., 84(13):4355-4358 (1987)), and "evolutionary linkage"
(See Holm, supra (1999), and Brenner, supra (1997)).
[0205] In certain embodiments, antibody variants include
glycosylation variants wherein the number and/or type of
glycosylation site has been altered compared to the amino acid
sequences of the parent polypeptide. In certain embodiments,
protein variants comprise a greater or a lesser number of N-linked
glycosylation sites than the native protein. An N-linked
glycosylation site is characterized by the sequence: Asn-X-Ser or
Asn-X-Thr, wherein the amino acid residue designated as X may be
any amino acid residue except proline. The substitution of amino
acid residues to create this sequence provides a potential new site
for the addition of an N-linked carbohydrate chain. Alternatively,
substitutions which eliminate this sequence will remove an existing
N-linked carbohydrate chain. Also provided is a rearrangement of
N-linked carbohydrate chains wherein one or more N-linked
glycosylation sites (typically those that are naturally occurring)
are eliminated and one or more new N-linked sites are created.
Additional preferred antibody variants include cysteine variants
wherein one or more cysteine residues are deleted from or
substituted for another amino acid (e.g., serine) as compared to
the parent amino acid sequence. Cysteine variants may be useful
when antibodies must be refolded into a biologically active
conformation such as after the isolation of insoluble inclusion
bodies. Cysteine variants generally have fewer cysteine residues
than the native protein, and typically have an even number to
minimize interactions resulting from unpaired cysteines.
[0206] According to certain embodiments, amino acid substitutions
are those which: (1) reduce susceptibility to proteolysis, (2)
reduce susceptibility to oxidation, (3) alter binding affinity for
forming protein complexes, (4) alter binding affinities, and/or (5)
confer or modify other functional properties on such polypeptides.
According to certain embodiments, single or multiple amino acid
substitutions (in certain embodiments, conservative amino acid
substitutions) may be made in the naturally-occurring sequence (in
certain embodiments, in the portion of the polypeptide outside the
domain(s) forming intermolecular contacts). In certain embodiments,
a conservative amino acid substitution typically may not
substantially change the structural characteristics of the parent
sequence (e.g., a replacement amino acid should not tend to break a
helix that occurs in the parent sequence, or disrupt other types of
secondary structure that characterizes the parent sequence).
Examples of art-recognized polypeptide secondary and tertiary
structures are described in Proteins, Structures and Molecular
Principles (Creighton, Ed., W.H. Freeman and Company, New York
(1984)); Introduction to Protein Structure (C. Branden and J.
Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and
Thornton et at. Nature 354:105 (1991).
[0207] The specific binding agent molecules of this invention that
are polypeptide or peptide substitution variants may have up to
about ten to twelve percent of the original amino acid sequence
replaced. For antibody variants, the heavy chain may have 50, 49,
48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32,
31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15,
14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid
replaced, while the light chain may have 25, 24, 23, 22, 21, 20,
19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or
1 amino acid replaced.
Derivatives of Specific Binding Agents
[0208] The invention also provides derivatives of specific binding
agent polypeptides. Derivatives include specific binding agent
polypeptides bearing modifications other than insertion, deletion,
or substitution of amino acid residues. Preferably, the
modifications are covalent in nature, and include for example,
chemical bonding with polymers, lipids, other organic, and
inorganic moieties. Derivatives of the invention may be prepared to
increase circulating half-life of a specific binding agent
polypeptide, or may be designed to improve targeting capacity for
the polypeptide to desired cells, tissues, or organs.
[0209] The invention further embraces derivative binding agents
covalently modified to include one or more water soluble polymer
attachments such as polyethylene glycol, polyoxyethylene glycol, or
polypropylene glycol as described U.S. Pat. Nos. 4,640,835,
4,496,689, 4,301,144, 4,670,417, 4,791,192 and 4,179,337. Still
other useful polymers known in the art include
monomethoxy-polyethylene glycol, dextran, cellulose, or other
carbohydrate based polymers, poly-(N-vinyl
pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a
polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated
polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures
of these polymers. Particularly preferred are specific binding
agent products covalently modified with polyethylene glycol (PEG)
subunits. Water-soluble polymers may be bonded at specific
positions, for example at the amino terminus of the specific
binding agent products, or randomly attached to one or more side
chains of the polypeptide. The use of PEG for improving the
therapeutic capacity for specific binding agent, and for humanized
antibodies in particular, is described in U.S. Pat. No. 6,133,426
to Gonzales et al., issued Oct. 17, 2000.
Target Sites for Antibody Mutagenesis
[0210] Certain strategies can be employed to manipulate inherent
properties of an Ang-1 and/or Ang-2-specific antibody, such as the
affinity of the antibody for its target. These strategies include
the use of site-specific or random mutagenesis of the
polynucleotide molecule encoding the antibody to generate antibody
variants, followed by a screening step designed to recover antibody
variants that exhibit the desired change, e.g. increased or
decreased affinity.
[0211] The amino acid residues most commonly targeted in mutagenic
strategies are those in the CDRs. As described supra, these regions
contain the residues that actually interact with Ang-1 and/or Ang-2
and other amino acids that affect the spatial arrangement of these
residues. However, amino acids in the framework regions of the
variable domains outside the CDR regions have also been shown to
make substantial contributions to the antigen-binding properties of
the antibody, and can be targeted to manipulate such properties.
See Hudson, Curr Opin Biotech, 9:395-402 (1999) and references
therein.
[0212] Smaller and more effectively screened libraries of antibody
variants can be produced by restricting random or site-directed
mutagenesis to sites in the CDRs that correspond to areas prone to
"hyper-mutation" during the somatic affinity maturation process.
See Chowdhury and Pastan, Nature Biotech, 17: 568-572 [1999] and
references therein. The types of DNA elements known to define
hyper-mutation sites in this manner include direct and inverted
repeats, certain consensus sequences, secondary structures, and
palindromes. The consensus DNA sequences include the tetrabase
sequence Purine-G-Pyrimidine-A/T (i.e. A or G-G-C or T-A or T) and
the serine codon AGY (wherein Y can be a C or a T).
[0213] Thus, an embodiment of the present invention includes
mutagenic strategies with the goal of increasing the affinity of an
antibody for its target. These strategies include mutagenesis of
the entire variable heavy and light chain, mutagenesis of the CDR
regions only, mutagenesis of the consensus hypermutation sites
within the CDRs, mutagenesis of framework regions, or any
combination of these approaches ("mutagenesis" in this context
could be random or site-directed). Definitive delineation of the
CDR regions and identification of residues comprising the binding
site of an antibody can be accomplished though solving the
structure of the antibody in question, and the antibody-ligand
complex, through techniques known to those skilled in the art, such
as X-ray crystallography. Various methods based on analysis and
characterization of such antibody crystal structures are known to
those of skill in the art and can be employed, although not
definitive, to approximate the CDR regions. Examples of such
commonly used methods include the Kabat, Chothia, AbM and contact
definitions.
[0214] The Kabat definition is based on the sequence variability
and is the most commonly used definition to predict CDR regions.
[Johnson and Wu, Nucleic Acids Res, 28: 214-8 (2000)]. The Chothia
definition is based on the location of the structural loop regions.
[Chothia et al., J Mol Biol, 196: 901-17 (1986); Chothia et al.,
Nature, 342: 877-83 (1989)]. The AbM definition is a compromise
between the Kabat and Chothia definition. AbM is an integral suite
of programs for antibody structure modeling produced by Oxford
Molecular Group [Martin et al., Proc Natl Acad Sci (USA)
86:9268-9272 (1989); Rees, et al., ABM.TM., a computer program for
modeling variable regions of antibodies, Oxford, UK; Oxford
Molecular, Ltd.]. The AbM suite models the tertiary structure of an
antibody from primary sequencing using a combination of knowledge
databases and ab initio methods. An additional definition, known as
the contact definition, has been recently introduced. [MacCallum et
al., J Mol Biol, 5:732-45 (1996)]. This definition is based on an
analysis of the available complex crystal structures.
[0215] By convention, the CDR regions in the heavy chain are
typically referred to as H1, H2 and H3 and are numbered
sequentially in order counting from the amino terminus to the
carboxy terminus. The CDR regions in the light chain are typically
referred to as L1, L2 and L3 and are numbered sequentially in order
counting from the amino terminus to the carboxy terminus.
[0216] The CDR-H1 is approximately 10 to 12 residues in length and
typically starts 4 residues after a Cys according to the Chothia
and AbM definitions or typically 5 residues later according to the
Kabat definition. The H1 is typically followed by a Trp, typically
Trp-Val, but also Trp-Ile, or Trp-Ala. The length of H1 is
approximately 10 to 12 residues according to the AbM definition
while the Chothia definition excludes the last 4 residues.
[0217] The CDR-H2 typically starts 15 residues after the end of H1
according to the Kabat and AbM definition. The residues preceding
H2 are typically Leu-Glu-Trp-Ile-Gly but there are a number of
variations. H2 is typically followed by the amino acid sequence
Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala. According to the
Kabat definition, the length of the H2 is approximately 16 to 19
residues where the AbM definition predicts the length to be
typically 9 to 12 residues.
[0218] The CDR-H3 typically starts 33 residues after the end of H2
and is typically preceded by the amino acid sequence (typically
Cys-Ala-Arg). The H3 is typically followed by the amino acid
sequence-Gly. The length of H3 can be anywhere between 3 to 25
residues.
[0219] The CDR-L1 typically starts at approximately residue 24 and
will typically follow a Cys. The residue after the CDR-L1 is always
a Trp and will typically begin the sequence Trp-Tyr-Gln,
Trp-Leu-Gln, Trp-Phe-Gln, or Trp-Tyr-Leu. The length of CDR-L1 is
approximately 10 to 17 residues. The punitive CDR-L1 for the
antibodies of the invention follows this pattern exactly with a Cys
residue followed by 15 amino acids then Trp-Tyr-Gln.
[0220] The CDR-L2 starts approximately 16 residues after the end of
L1. It will generally follow residues Ile-Tyr, Val-Tyr, Ile-Lys or
Ile-Phe. The length of CDR-L2 is approximately 7 residues.
[0221] The CDR-L3 typically starts 33 residues after the end of L2
and typically follows a Cys. L3 is typically followed by the amino
acid sequence Phe-Gly-XXX-Gly. The length of L3 is approximately 7
to 11 residues.
[0222] Various methods for modifying antibodies have been described
in the art. For example, U.S. Pat. No. 5,530,101 (to Queen et al.,
Jun. 25, 1996) describes methods to produce humanized antibodies
wherein the sequence of the humanized immunoglobulin heavy chain
variable region framework is 65% to 95% identical to the sequence
of the donor immunoglobulin heavy chain variable region framework.
Each humanized immunoglobulin chain will usually comprise, in
addition to the CDRs, amino acids from the donor immunoglobulin
framework that are, e.g., capable of interacting with the CDRs to
affect binding affinity, such as one or more amino acids which are
immediately adjacent to a CDR in the donor immunoglobulin or those
within about 3 angstroms as predicted by molecular modeling. The
heavy and light chains may each be designed by using any one or all
of various position criteria. When combined into an intact
antibody, the humanized immunoglobulins of the present invention
will be substantially non-immunogenic in humans and retain
substantially the same affinity as the donor immunoglobulin to the
antigen, such as a protein or other compound containing an epitope.
See also, related methods in U.S. Pat. No. 5,693,761 to Queen, et
al., issued Dec. 2, 1997 ("Polynucleotides encoding improved
humanized immunoglobulins"); U.S. Pat. No. 5,693,762 to Queen, et
al., issued Dec. 2, 1997 ("Humanized Immunoglobulins"); U.S. Pat.
No. 5,585,089 to Queen, et al. issued Dec. 17, 1996 ("Humanized
Immunoglobulins").
[0223] In one example, U.S. Pat. No. 5,565,332 to Hoogenboom et al.
issued Oct. 15, 1996 ("Production of chimeric antibodies--a
combinatorial approach") describes methods for the production of
antibodies, and antibody fragments which have similar binding
specificity as a parent antibody but which have increased human
characteristics. Humanized antibodies are obtained by chain
shuffling, using, for example, phage display technology, and a
polypeptide comprising a heavy or light chain variable domain of a
non-human antibody specific for an antigen of interest is combined
with a repertoire of human complementary (light or heavy) chain
variable domains. Hybrid pairings that are specific for the antigen
of interest are identified and human chains from the selected
pairings are combined with a repertoire of human complementary
variable domains (heavy or light). In another embodiment, a
component of a CDR from a non-human antibody is combined with a
repertoire of component parts of CDRs from human antibodies. From
the resulting library of antibody polypeptide dimers, hybrids are
selected and used in a second humanizing shuffling step.
Alternatively, this second step is eliminated if the hybrid is
already of sufficient human character to be of therapeutic value.
Methods of modification to increase human character are also
described. See also Winter, FEBS Letts 430:92-92 (1998).
[0224] As another example, U.S. Pat. No. 6,054,297 to Carter et
al., issued Apr. 25, 2000 describes a method for making humanized
antibodies by substituting a CDR amino acid sequence for the
corresponding human CDR amino acid sequence and/or substituting a
FR amino acid sequence for the corresponding human FR amino acid
sequences.
[0225] As another example, U.S. Pat. No. 5,766,886 to Studnicka et
al., issued Jun. 16, 1998 ("Modified antibody variable domains")
describes methods for identifying the amino acid residues of an
antibody variable domain which may be modified without diminishing
the native affinity of the antigen binding domain while reducing
its immunogenicity with respect to a heterologous species and
methods for preparing these modified antibody variable domains
which are useful for administration to heterologous species. See
also U.S. Pat. No. 5,869,619 to Studnicka issued Feb. 9, 1999.
[0226] As discussed, modification of an antibody by any of the
methods known in the art is typically designed to achieve increased
binding affinity for an antigen and/or reduce immunogenicity of the
antibody in the recipient. In one approach, humanized antibodies
can be modified to eliminate glycosylation sites in order to
increase affinity of the antibody for its cognate antigen [Co et
al., Mol Immunol 30:1361-1367 (1993)]. Techniques such as
"reshaping," "hyperchimerization," and "veneering/resurfacing" have
produced humanized antibodies with greater therapeutic potential.
[Vaswami et al., Annals of Allergy, Asthma, & Immunol 81:105
(1998); Roguska et al., Prot Engineer 9:895-904 (1996)]. See also
U.S. Pat. No. 6,072,035 to Hardman et al., issued Jun. 6, 2000,
which describes methods for reshaping antibodies. While these
techniques diminish antibody immunogenicity by reducing the number
of foreign residues, they do not prevent anti-idiotypic and
anti-allotypic responses following repeated administration of the
antibodies. Alternatives to these methods for reducing
immunogenicity are described in Gilliland et al., J Immunol 62(6):
3663-71 (1999).
[0227] In many instances, humanizing antibodies result in a loss of
antigen binding capacity. It is therefore preferable to "back
mutate" the humanized antibody to include one or more of the amino
acid residues found in the original (most often rodent) antibody in
an attempt to restore binding affinity of the antibody. See, for
example, Saldanha et al., Mol Immunol 36:709-19 (1999).
Non-Peptide Specific Binding Agent Analogs/Protein Mimetics
[0228] Furthermore, nonpeptide specific binding agent analogs of
peptides that provide a stabilized structure or lessened
bio-degradation, are also contemplated. Specific binding agent
peptide mimetic analogs can be prepared based on a selected
inhibitory peptide by replacement of one or more residues by
nonpeptide moieties. Preferably, the nonpeptide moieties permit the
peptide to retain its natural confirmation, or stabilize a
preferred, e.g., bioactive, confirmation which retains the ability
to recognize and bind Ang-1 and/or Ang-2. In one aspect, the
resulting analog/mimetic exhibits increased binding affinity for
Ang-1 and/or Ang-2. One example of methods for preparation of
nonpeptide mimetic analogs from specific binding agent peptides is
described in Nachman et al., Regul Pept 57:359-370 (1995). If
desired, the specific binding agent peptides of the invention can
be modified, for instance, by glycosylation, amidation,
carboxylation, or phosphorylation, or by the creation of acid
addition salts, amides, esters, in particular C-terminal esters,
and N-acyl derivatives of the peptides of the invention. The
specific binding agent peptides also can be modified to create
peptide derivatives by forming covalent or noncovalent complexes
with other moieties. Covalently-bound complexes can be prepared by
linking the chemical moieties to functional groups on the side
chains of amino acids comprising the specific binding agent
peptides, or at the N- or C-terminus
[0229] In particular, it is anticipated that the specific binding
agent peptides can be conjugated to a reporter group, including,
but not limited to a radiolabel, a fluorescent label, an enzyme
(e.g., that catalyzes a colorimetric or fluorometric reaction), a
substrate, a solid matrix, or a carrier (e.g., biotin or avidin).
The invention accordingly provides a molecule comprising an
antibody molecule, wherein the molecule preferably further
comprises a reporter group selected from the group consisting of a
radiolabel, a fluorescent label, an enzyme, a substrate, a solid
matrix, and a carrier. Such labels are well known to those of skill
in the art, e.g., biotin labels are particularly contemplated. The
use of such labels is well known to those of skill in the art and
is described in, e.g., U.S. Pat. No. 3,817,837; U.S. Pat. No.
3,850,752; U.S. Pat. No. 3,996,345 and U.S. Pat. No. 4,277,437.
Other labels that will be useful include but are not limited to
radioactive labels, fluorescent labels and chemiluminescent labels.
U.S. Patents concerning use of such labels include for example U.S.
Pat. No. 3,817,837; U.S. Pat. No. 3,850,752; U.S. Pat. No.
3,939,350 and U.S. Pat. No. 3,996,345. Any of the peptides of the
present invention may comprise one, two, or more of any of these
labels.
Methods of Making Specific Binding Agents
[0230] Specific binding agents of the present invention that are
proteins can be prepared by chemical synthesis in solution or on a
solid support in accordance with conventional techniques. The
current limit for solid phase synthesis is about 85-100 amino acids
in length. However, chemical synthesis techniques can often be used
to chemically ligate a series of smaller peptides to generate full
length polypeptides. Various automatic synthesizers are
commercially available and can be used in accordance with known
protocols. See, for example, Stewart and Young, Solid Phase Peptide
Synthesis, 2d. ed., Pierce Chemical Co., (1984); Tam et al., J Am
Chem Soc, 105:6442, (1983); Merrifield, Science, 232:341-347,
(1986); and Barany and Merrifield, The Peptides, Gross and
Meienhofer, eds, Academic Press, New York, 1-284; Barany et al.,
Int. J. Peptide Protein Res., 30, 705-739 (1987); and U.S. Pat. No.
5,424,398), each incorporated herein by reference.
[0231] Solid phase peptide synthesis methods use a
copoly(styrene-divinylbenzene) containing 0.1-1.0 mM amines/g
polymer. These methods for peptide synthesis use butyloxycarbonyl
(t-BOC) or 9-fluorenylmethyloxy-carbonyl(FMOC) protection of
alpha-amino groups. Both methods involve stepwise syntheses whereby
a single amino acid is added at each step starting from the
C-terminus of the peptide (See, Coligan et al., Current Protocols
in Immunology, Wiley Interscience, 1991, Unit 9). On completion of
chemical synthesis, the synthetic peptide can be deprotected to
remove the t-BOC or FMOC amino acid blocking groups and cleaved
from the polymer by treatment with acid at reduced temperature
(e.g., liquid HF-10% anisole for about 0.25 to about 1 hour at
0.degree. C.). After evaporation of the reagents, the specific
binding agent peptides are extracted from the polymer with 1%
acetic acid solution that is then lyophilized to yield the crude
material. This can normally be purified by such techniques as gel
filtration on Sephadex G-15 using 5% acetic acid as a solvent.
Lyophilization of appropriate fractions of the column will yield
the homogeneous specific binding agent peptide or peptide
derivatives, which can then be characterized by such standard
techniques as amino acid analysis, thin layer chromatography, high
performance liquid chromatography, ultraviolet absorption
spectroscopy, molar rotation, solubility, and quantitated by the
solid phase Edman degradation.
[0232] Chemical synthesis of anti-Ang-1 and/or anti-Ang-2
antibodies, derivatives, variants, and fragments thereof, as well
as other protein-based Ang-2 binding agents permits incorporation
of non-naturally occurring amino acids into the agent.
[0233] Recombinant DNA techniques are a convenient method for
preparing full length antibodies and other large proteinaceous
specific binding agents of the present invention, or fragments
thereof. A cDNA molecule encoding the antibody or fragment may be
inserted into an expression vector, which can in turn be inserted
into a host cell for production of the antibody or fragment. It is
understood that the cDNAs encoding such antibodies may be modified
to vary from the "original" cDNA (translated from the mRNA) to
provide for codon degeneracy or to permit codon preference usage in
various host cells.
[0234] Generally, a DNA molecule encoding an antibody can be
obtained using procedures described herein in the Examples. Where
it is desirable to obtain Fab molecules or CDRs that are related to
the original antibody molecule, one can screen a suitable library
(phage display library; lymphocyte library, etc.) using standard
techniques to identify and clone related Fabs/CDRs. Probes used for
such screening may be full length or truncated Fab probes encoding
the Fab portion of the original antibody, probes against one or
more CDRs from the Fab portion of the original antibody, or other
suitable probes. Where DNA fragments are used as probes, typical
hybridization conditions are those such as set forth in Ausubel et.
al. (Current Protocols in Molecular Biology, Current Protocols
Press [1994]). After hybridization, the probed blot can be washed
at a suitable stringency, depending on such factors as probe size,
expected homology of probe to clone, the type of library being
screened, and the number of clones being screened. Examples of high
stringency screening are 0.1.times.SSC, and 0.1 percent SDS at a
temperature between 50-65.degree. C.
[0235] A variety of expression vector/host systems may be utilized
to contain and express the polynucleotide molecules encoding the
specific binding agent polypeptides of the invention. These systems
include but are not limited to microorganisms such as bacteria
transformed with recombinant bacteriophage, plasmid or cosmid DNA
expression vectors; yeast transformed with yeast expression
vectors; insect cell systems infected with virus expression vectors
(e.g., baculovirus); plant cell systems transfected with virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco
mosaic virus, TMV) or transformed with bacterial expression vectors
(e.g., Ti or pBR322 plasmid); or animal cell systems.
[0236] Mammalian cells that are useful in recombinant specific
binding agent protein productions include but are not limited to
VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS
cells (such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549,
PC12, K562 and 293 cells, as well as hybridoma cell lines as
described herein. Mammalian cells are preferred for preparation of
those specific binding agents such as antibodies and antibody
fragments that are typically glycosylated and require proper
refolding for activity. Preferred mammalian cells include CHO
cells, hybridoma cells, and myeloid cells.
[0237] Some exemplary protocols for the recombinant expression of
the specific binding agent proteins are described herein below.
[0238] The term "expression vector" refers to a plasmid, phage,
virus or vector, for expressing a polypeptide from a DNA (RNA)
sequence. An expression vector can comprise a transcriptional unit
comprising an assembly of (1) a genetic element or elements having
a regulatory role in gene expression, for example, promoters or
enhancers, (2) a structural or sequence that encodes the binding
agent which is transcribed into mRNA and translated into protein,
and (3) appropriate transcription initiation and termination
sequences. Structural units intended for use in yeast or eukaryotic
expression systems preferably include a leader sequence enabling
extracellular secretion of translated protein by a host cell.
Alternatively, where recombinant specific binding agent protein is
expressed without a leader or transport sequence, it may include an
amino terminal methionine residue. This residue may or may not be
subsequently cleaved from the expressed recombinant protein to
provide a final specific binding agent product.
[0239] For example, the specific binding agents may be
recombinantly expressed in yeast using a commercially available
expression system, e.g., the Pichia Expression System (Invitrogen,
San Diego, Calif.), following the manufacturer's instructions. This
system also relies on the pre-pro-alpha sequence to direct
secretion, but transcription of the insert is driven by the alcohol
oxidase (AOX1) promoter upon induction by methanol.
[0240] The secreted specific binding agent peptide is purified from
the yeast growth medium by, e.g., the methods used to purify the
peptide from bacterial and mammalian cell supernatants.
[0241] Alternatively, the cDNA encoding the specific binding agent
peptide may be cloned into the baculovirus expression vector
pVL1393 (PharMingen, San Diego, Calif.). This vector can be used
according to the manufacturer's directions (PharMingen) to infect
Spodoptera frugiperda cells in sF9 protein-free media and to
produce recombinant protein. The specific binding agent protein can
be purified and concentrated from the media using a
heparin-Sepharose column (Pharmacia).
[0242] Alternatively, the peptide may be expressed in an insect
system. Insect systems for protein expression are well known to
those of skill in the art. In one such system, Autographa
californica nuclear polyhedrosis virus (AcNPV) can be used as a
vector to express foreign genes in Spodoptera frugiperda cells or
in Trichoplusia larvae. The specific binding agent peptide coding
sequence can be cloned into a nonessential region of the virus,
such as the polyhedrin gene, and placed under control of the
polyhedrin promoter. Successful insertion of the specific binding
agent peptide will render the polyhedrin gene inactive and produce
recombinant virus lacking coat protein coat. The recombinant
viruses can be used to infect S. frugiperda cells or Trichoplusia
larvae in which peptide is expressed [Smith et al., J Virol 46: 584
(1983); Engelhard et al., Proc Nat Acad Sci (USA) 91: 3224-7
(1994)].
[0243] In another example, the DNA sequence encoding the specific
binding agent peptide can be amplified by PCR and cloned into an
appropriate vector for example, pGEX-3X (Pharmacia). The pGEX
vector is designed to produce a fusion protein comprising
glutathione-S-transferase (GST), encoded by the vector, and a
specific binding agent protein encoded by a DNA fragment inserted
into the vector's cloning site. The primers for the PCR can be
generated to include for example, an appropriate cleavage site.
Where the specific binding agent fusion moiety is used solely to
facilitate expression or is otherwise not desirable as an
attachment to the peptide of interest, the recombinant specific
binding agent fusion protein may then be cleaved from the GST
portion of the fusion protein. The pGEX-3X/specific binding agent
peptide construct is transformed into E. coli XL-1 Blue cells
(Stratagene, La Jolla Calif.), and individual transformants
isolated and grown. Plasmid DNA from individual transformants can
be purified and partially sequenced using an automated sequencer to
confirm the presence of the desired specific binding agent encoding
nucleic acid insert in the proper orientation.
[0244] Expression of polynucleotides encoding anti-Ang-1 and/or
anti-Ang-2 antibodies and fragments thereof using the recombinant
systems described above may result in production of antibodies or
fragments thereof that must be "re-folded" (to properly create
various disulphide bridges) in order to be biologically active.
Typical refolding procedures for such antibodies are set forth in
the Examples herein and in the following section.
[0245] Specific binding agents made in bacterial cells may be
produced as an insoluble inclusion body in the bacteria, can be
purified as follows. Host cells can be sacrificed by
centrifugation; washed in 0.15 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA;
and treated with 0.1 mg/ml lysozyme (Sigma, St. Louis, Mo.) for 15
minutes at room temperature. The lysate can be cleared by
sonication, and cell debris can be pelleted by centrifugation for
10 minutes at 12,000.times.g. The specific binding agent-containing
pellet can be resuspended in 50 mM Tris, pH 8, and 10 mM EDTA,
layered over 50% glycerol, and centrifuged for 30 min. at
6000.times.g. The pellet can be resuspended in standard phosphate
buffered saline solution (PBS) free of Mg.sup.++ and Ca.sup.++. The
specific binding agent can be further purified by fractionating the
resuspended pellet in a denaturing SDS polyacrylamide gel (Sambrook
et al., supra). The gel can be soaked in 0.4 M KCl to visualize the
protein, which can be excised and electroeluted in gel-running
buffer lacking SDS. If the GST fusion protein is produced in
bacteria, as a soluble protein, it can be purified using the GST
Purification Module (Pharmacia).
[0246] Mammalian host systems for the expression of the recombinant
protein are well known to those of skill in the art. Host cell
strains can be chosen for a particular ability to process the
expressed protein or produce certain post-translation modifications
that will be useful in providing protein activity. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation and acylation. Different host cells such as CHO, HeLa,
MDCK, 293, WI38, as well as hybridoma cell lines, and the like have
specific cellular machinery and characteristic mechanisms for such
post-translational activities and can be chosen to ensure the
correct modification and processing of the introduced, foreign
protein.
[0247] A number of selection systems can be used to recover the
cells that have been transformed for recombinant protein
production. Such selection systems include, but are not limited to,
HSV thymidine kinase, hypoxanthine-guanine
phosphoribosyltransferase and adenine phosphoribosyltransferase
genes, in tk-, hgprt- or aprt-cells, respectively. Also,
anti-metabolite resistance can be used as the basis of selection
for DHFR which confers resistance to methotrexate; gpt which
confers resistance to mycophenolic acid; neo which confers
resistance to the aminoglycoside G418 and confers resistance to
chlorsulfuron; and hygro which that confers resistance to
hygromycin. Additional selectable genes that may be useful include
trpB, which allows cells to utilize indole in place of tryptophan,
or hisD, which allows cells to utilize histinol in place of
histidine. Markers that give a visual indication for identification
of transformants include anthocyanins, .beta.-glucuronidase and its
substrate, GUS, and luciferase and its substrate, luciferin.
Purification and Refolding of Specific Binding Agents
[0248] In some cases, the specific binding agents produced using
procedures described above may need to be "refolded" and oxidized
into a proper tertiary structure and generating di-sulfide linkages
in order to be biologically active. Refolding can be accomplished
using a number of procedures well known in the art. Such methods
include, for example, exposing the solubilized polypeptide agent to
a pH usually above 7 in the presence of a chaotropic agent. The
selection of chaotrope is similar to the choices used for inclusion
body solubilization, however a chaotrope is typically used at a
lower concentration. An exemplary chaotropic agent is guanidine. In
most cases, the refolding/oxidation solution will also contain a
reducing agent plus its oxidized form in a specific ratio to
generate a particular redox potential which allows for dusykfide
shuffling to occur for the formation of cysteine bridges. Some
commonly used redox couples include cysteine/cystamine,
glutathione/dithiobisGSH, cupric chloride, dithiothreitol
DTT/dithiane DTT, and 2-mercaptoethanol (bME)/dithio-bME. In many
instances, a co-solvent may be used to increase the efficiency of
the refolding. Commonly used cosolvents include glycerol,
polyethylene glycol of various molecular weights, and arginine.
[0249] It will be desirable to purify specific binding agent
proteins or variants thereof of the present invention. Protein
purification techniques are well known to those of skill in the
art. These techniques involve, at one level, the crude
fractionation of the polypeptide and non-polypeptide fractions.
Having separated the specific binding agent polypeptide from other
proteins, the polypeptide of interest can be further purified using
chromatographic and electrophoretic techniques to achieve partial
or complete purification (or purification to homogeneity).
Analytical methods particularly suited to the preparation of a pure
specific binding agent peptide are ion-exchange chromatography,
exclusion chromatography; polyacrylamide gel electrophoresis;
isoelectric focusing. A particularly efficient method of purifying
peptides is fast protein liquid chromatography or even HPLC.
[0250] Certain aspects of the present invention concerns the
purification, and in particular embodiments, the substantial
purification, of an encoded specific binding agent protein or
peptide. The term "purified specific binding agent protein or
peptide" as used herein, is intended to refer to a composition,
isolatable from other components, wherein the specific binding
agent protein or peptide is purified to any degree relative to its
naturally-obtainable state. A purified specific binding agent
protein or peptide therefore also refers to a specific binding
agent protein or peptide, free from the environment in which it may
naturally occur.
[0251] Generally, "purified" will refer to a specific binding agent
composition that has been subjected to fractionation to remove
various other components, and which composition substantially
retains its expressed biological activity. Where the term
"substantially purified" is used, this designation will refer to a
specific binding agent composition in which the specific binding
agent protein or peptide forms the major component of the
composition, such as constituting about 50%, about 60%, about 70%,
about 80%, about 90%, about 95% or more of the proteins in the
composition.
[0252] Various methods for quantifying the degree of purification
of the specific binding agent will be known to those of skill in
the art in light of the present disclosure. These include, for
example, determining the specific binding activity of an active
fraction, or assessing the amount of specific binding agent
polypeptides within a fraction by SDS/PAGE analysis. A preferred
method for assessing the purity of a specific binding agent
fraction is to calculate the binding activity of the fraction, to
compare it to the binding activity of the initial extract, and to
thus calculate the degree of purification, herein assessed by a
"-fold purification number." The actual units used to represent the
amount of binding activity will, of course, be dependent upon the
particular assay technique chosen to follow the purification and
whether or not the expressed specific binding agent protein or
peptide exhibits a detectable binding activity.
[0253] Various techniques suitable for use in specific binding
agent protein purification will be well known to those of skill in
the art. These include, for example, precipitation with ammonium
sulphate, PEG, antibodies (immunoprecipitation) and the like or by
heat denaturation, followed by centrifugation; chromatography steps
such as affinity chromatography (e.g., Protein-A-Sepharose), ion
exchange, gel filtration, reverse phase, hydroxylapatite and
affinity chromatography; isoelectric focusing; gel electrophoresis;
and combinations of such and other techniques. As is generally
known in the art, it is believed that the order of conducting the
various purification steps may be changed, or that certain steps
may be omitted, and still result in a suitable method for the
preparation of a substantially purified specific binding agent.
[0254] There is no general requirement that the specific binding
agent always be provided in its most purified state. Indeed, it is
contemplated that less substantially specific binding agent
products will have utility in certain embodiments. Partial
purification may be accomplished by using fewer purification steps
in combination, or by utilizing different forms of the same general
purification scheme. For example, it is appreciated that a
cation-exchange column chromatography performed utilizing an HPLC
apparatus will generally result in a greater "-fold" purification
than the same technique utilizing a low-pressure chromatography
system. Methods exhibiting a lower degree of relative purification
may have advantages in total recovery of specific binding agent
protein product, or in maintaining binding activity of an expressed
specific binding agent protein.
[0255] It is known that the migration of a polypeptide can vary,
sometimes significantly, with different conditions of SDS/PAGE
[Capaldi et al., Biochem Biophys/Res Comm, 76: 425 (1977)]. It will
therefore be appreciated that under differing electrophoresis
conditions, the apparent molecular weights of purified or partially
purified specific binding agent expression products may vary.
Binding Assays
[0256] Immunological binding assays typically utilize a capture
agent to bind specifically to and often immobilize the analyte
target antigen. The capture agent is a moiety that specifically
binds to the analyte. In one embodiment of the present invention,
the capture agent is an antibody or fragment thereof that
specifically binds Ang-2 and/or Ang-1. These immunological binding
assays are well known in the art [see, Asai, ed., Methods in Cell
Biology, Vol. 37, Antibodies in Cell Biology, Academic Press, Inc.,
New York (1993)].
[0257] Immunological binding assays frequently utilize a labeling
agent that will signal the existence of the bound complex formed by
the capture agent and antigen. The labeling agent can be one of the
molecules comprising the bound complex; i.e. it can be labeled
specific binding agent or a labeled anti-specific binding agent
antibody. Alternatively, the labeling agent can be a third
molecule, commonly another antibody, which binds to the bound
complex. The labeling agent can be, for example, an anti-specific
binding agent antibody bearing a label. The second antibody,
specific for the bound complex, may lack a label, but can be bound
by a fourth molecule specific to the species of antibodies which
the second antibody is a member of. For example, the second
antibody can be modified with a detectable moiety, such as biotin,
which can then be bound by a fourth molecule, such as
enzyme-labeled streptavidin. Other proteins capable of specifically
binding immunoglobulin constant regions, such as protein A or
protein G may also be used as the labeling agent. These binding
proteins are normal constituents of the cell walls of streptococcal
bacteria and exhibit a strong non-immunogenic reactivity with
immunoglobulin constant regions from a variety of species [see,
generally Akerstrom, J Immunol, 135:2589-2542 (1985); and Chaubert,
Mod Pathol, 10:585-591 (1997)].
[0258] Throughout the assays, incubation and/or washing steps may
be required after each combination of reagents. Incubation steps
can vary from about 5 seconds to several hours, preferably from
about 5 minutes to about 24 hours. However, the incubation time
will depend upon the assay format, analyte, volume of solution,
concentrations, and the like. Usually, the assays will be carried
out at ambient temperature, although they can be conducted over a
range of temperatures.
A. Non-Competitive Binding Assays:
[0259] Immunological binding assays can be of the non-competitive
type. These assays have an amount of captured analyte that is
directly measured. For example, in one preferred "sandwich" assay,
the capture agent (antibody) can be bound directly to a solid
substrate where it is immobilized. These immobilized antibodies
then capture (bind to) antigen present in the test sample. The
protein thus immobilized is then bound to a labeling agent, such as
a second antibody having a label. In another preferred "sandwich"
assay, the second antibody lacks a label, but can be bound by a
labeled antibody specific for antibodies of the species from which
the second antibody is derived. The second antibody also can be
modified with a detectable moiety, such as biotin, to which a third
labeled molecule can specifically bind, such as streptavidin. [See,
Harlow and Lane, Antibodies, A Laboratory Manual, Ch 14, Cold
Spring Harbor Laboratory, NY (1988), incorporated herein by
reference].
B. Competitive Binding Assays:
[0260] Immunological binding assays can be of the competitive type.
The amount of analyte present in the sample is measure indirectly
by measuring the amount of an added analyte displaced, or competed
away, from a capture agent by the analyte present in the sample. In
one preferred competitive binding assay, a known amount of analyte,
usually labeled, is added to the sample and the sample is then
contacted with an antibody (the capture agent). The amount of
labeled analyze bound to the antibody is inversely proportional to
the concentration of analyte present in the sample. (See, Harlow
and Lane, Antibodies, A Laboratory Manual, Ch 14, pp. 579-583,
supra).
[0261] In another preferred competitive binding assay, the antibody
is immobilized on a solid substrate. The amount of protein bound to
the antibody may be determined either by measuring the amount of
protein present in a protein/antibody complex, or alternatively by
measuring the amount of remaining uncomplexed protein. The amount
of protein may be detected by providing a labeled protein. See,
Harlow and Lane, Antibodies, A Laboratory Manual, Ch 14,
supra).
[0262] Yet another preferred competitive binding assay, hapten
inhibition is utilized. Here, a known analyte is immobilized on a
solid substrate. A known amount of antibody is added to the sample,
and the sample is contacted with the immobilized analyte. The
amount of antibody bound to the immobilized analyte is inversely
proportional to the amount of analyte present in the sample. The
amount of immobilized antibody may be detected by detecting either
the immobilized fraction of antibody or the fraction that remains
in solution. Detection may be direct where the antibody is labeled
or indirect by the subsequent addition of a labeled moiety that
specifically binds to the antibody as described above.
C. Utilization of Competitive Binding Assays:
[0263] The competitive binding assays can be used for
cross-reactivity determinations to permit a skilled artisan to
determine if a protein or enzyme complex which is recognized by a
specific binding agent of the invention is the desired protein and
not a cross-reacting molecule or to determine whether the antibody
is specific for the antigen and does not bind unrelated antigens.
In assays of this type, antigen can be immobilized to a solid
support and an unknown protein mixture is added to the assay, which
will compete with the binding of the specific binding agents to the
immobilized protein. The competing molecule also binds one or more
antigens unrelated to the antigen. The ability of the proteins to
compete with the binding of the specific binding agents antibodies
to the immobilized antigen is compared to the binding by the same
protein that was immobilized to the solid support to determine the
cross-reactivity of the protein mix.
D. Other Binding Assays:
[0264] The present invention also provides Western blot methods to
detect or quantify the presence of Ang-1 and/or Ang-2 in a sample.
The technique generally comprises separating sample proteins by gel
electrophoresis on the basis of molecular weight and transferring
the proteins to a suitable solid support, such as nitrocellulose
filter, a nylon filter, or derivatized nylon filter. The sample is
incubated with antibodies or fragments thereof that specifically
bind Ang-1 and/or Ang-2 and the resulting complex is detected.
These antibodies may be directly labeled or alternatively may be
subsequently detected using labeled antibodies that specifically
bind to the antibody.
[0265] Binding assays to detect those Ang-1 and/or Ang-2 specific
binding agents that disrupt Ang-2 binding to its receptor are set
forth in the Examples herein.
Diagnostic Assays
[0266] The antibodies or antigen-binding fragments thereof of
present invention are useful for the diagnosis of conditions or
diseases characterized by expression of Ang-1 and/or Ang-2 or
subunits, or in assays to monitor patients being treated with
inducers of Ang-1 and/or Ang-2, its fragments, agonists or
inhibitors of Ang-1 and/or Ang-2 activity. Diagnostic assays for
Ang-1 and/or Ang-2 include methods utilizing a specific binding
agent and a label to detect Ang-1 and/or Ang-2 in human body fluids
or extracts of cells or tissues. The specific binding agents of the
present invention can be used with or without modification. In a
preferred diagnostic assay, the specific binding agents will be
labeled by attaching, e.g., a label or a reporter molecule. A wide
variety of labels and reporter molecules are known, some of which
have been already described herein. In particular, the present
invention is useful for diagnosis of human disease. A variety of
protocols for measuring Ang-1 and/or Ang-2 proteins using either
polyclonal or monoclonal antibodies specific for the respective
protein are known in the art. Examples include enzyme-linked
immunosorbent assay (ELISA), radioimmunoassay (RIA) and
fluorescence activated cell sorting (FACS). A two-site,
monoclonal-based immunoassay utilizing monoclonal antibodies
reactive to two non-interfering epitopes on Ang-1 and/or Ang-2 is
preferred, but a competitive binding assay can be employed. These
assays are described, for example, in Maddox et al., J Exp Med,
158:1211 [1983].
[0267] In order to provide a basis for diagnosis, normal or
standard values for human Ang-1 and/or Ang-2 expression are usually
established. This determination can be accomplished by combining
body fluids or cell extracts from normal subjects, preferably
human, with a specific binding agent, for example, an antibody, to
Ang-1 and/or Ang-2, under conditions suitable for complex formation
that are well known in the art. The amount of standard complex
formation can be quantified by comparing the binding of the
specific binding agents to known quantities of Ang-1 and/or Ang-2
protein, with both control and disease samples. Then, standard
values obtained from normal samples can be compared with values
obtained from samples from subjects potentially affected by
disease. Deviation between standard and subject values suggests a
role for Ang-1 and/or Ang-2 in the disease state.
[0268] For diagnostic applications, in certain embodiments,
specific binding agents typically will be labeled with a detectable
moiety. The detectable moiety can be any one that is capable of
producing, either directly or indirectly, a detectable signal. For
example, the detectable moiety may be a radioisotope, such as
.sup.3H, .sup.14C, .sup.32P, .sup.35S, or .sup.125I, a fluorescent
or chemiluminescent compound, such as fluorescein isothiocyanate,
rhodamine, or luciferin; or an enzyme, such as alkaline
phosphatase, .beta.-galactosidase, or horseradish peroxidase [Bayer
et al., Meth Enz, 184: 138-163, (1990)].
Diseases
[0269] The present invention provides a specific binding agent that
binds to Ang-1 and/or Ang-2 that is useful for the treatment of
human diseases and pathological conditions. Agents that modulate
Ang-1 and/or Ang-2 binding activity, or other cellular activity,
may be used in combination with other therapeutic agents to enhance
their therapeutic effects or decrease potential side effects.
[0270] In one aspect, the present invention provides reagents and
methods useful for treating diseases and conditions characterized
by undesirable or aberrant levels of Ang-1 and/or Ang-2 activity in
a cell. These diseases include cancers, and other
hyperproliferative conditions, such as hyperplasia, psoriasis,
contact dermatitis, immunological disorders, and infertility.
[0271] The present invention also provides methods of treating
cancer in an animal, including humans, comprising administering to
the animal an effective amount of a specific binding agent that
inhibits or decreases Ang-1 and/or Ang-2 activity. The invention is
further directed to methods of inhibiting cancer cell growth,
including processes of cellular proliferation, invasiveness, and
metastasis in biological systems. Methods include use of a compound
of the invention as an inhibitor of cancer cell growth. Preferably,
the methods are employed to inhibit or reduce cancer cell growth,
invasiveness, metastasis, or tumor incidence in living animals,
such as mammals. Methods of the invention are also readily
adaptable for use in assay systems, e.g., assaying cancer cell
growth and properties thereof, as well as identifying compounds
that affect cancer cell growth.
[0272] The cancers treatable by methods of the present invention
preferably occur in mammals. Mammals include, for example, humans
and other primates, as well as pet or companion animals such as
dogs and cats, laboratory animals such as rats, mice and rabbits,
and farm animals such as horses, pigs, sheep, and cattle.
[0273] Tumors or neoplasms include growths of tissue cells in which
the multiplication of the cells is uncontrolled and progressive.
Some such growths are benign, but others are termed malignant and
may lead to death of the organism. Malignant neoplasms or cancers
are distinguished from benign growths in that, in addition to
exhibiting aggressive cellular proliferation, they may invade
surrounding tissues and metastasize. Moreover, malignant neoplasms
are characterized in that they show a greater loss of
differentiation (greater dedifferentiation), and of their
organization relative to one another and their surrounding tissues.
This property is also called "anaplasia."
[0274] Neoplasms treatable by the present invention also include
solid tumors, i.e., carcinomas and sarcomas. Carcinomas include
those malignant neoplasms derived from epithelial cells that
infiltrate (invade) the surrounding tissues and give rise to
metastases. Adenocarcinomas are carcinomas derived from glandular
tissue, or which form recognizable glandular structures. Another
broad category or cancers includes sarcomas, which are tumors whose
cells are embedded in a fibrillar or homogeneous substance like
embryonic connective tissue. The invention also enables treatment
of cancers of the myeloid or lymphoid systems, including leukemias,
lymphomas and other cancers that typically do not present as a
tumor mass, but are distributed in the vascular or lymphoreticular
systems.
[0275] The type of cancer or tumor cells amenable to treatment
according to the invention include, for example, ACTH-producing
tumor, acute lymphocytic leukemia, acute nonlymphocytic leukemia,
cancer of the adrenal cortex, bladder cancer, brain cancer, breast
cancer, cervical cancer, chronic lymphocytic leukemia, chronic
myelocytic leukemia, colorectal cancer, cutaneous T-cell lymphoma,
endometrial cancer, esophageal cancer, Ewing's sarcoma, gallbladder
cancer, hairy cell leukemia, head and neck cancer, Hodgkin's
lymphoma, Kaposi's sarcoma, kidney cancer, liver cancer, lung
cancer (small and non-small cell), malignant peritoneal effusion,
malignant pleural effusion, melanoma, mesothelioma, multiple
myeloma, neuroblastoma, glioma, non-Hodgkin's lymphoma,
osteosarcoma, ovarian cancer, ovarian (germ cell) cancer,
pancreatic cancer, penile cancer, prostate cancer, retinoblastoma,
skin cancer, soft tissue sarcoma, squamous cell carcinomas, stomach
cancer, testicular cancer, thyroid cancer, trophoblastic neoplasms,
uterine cancer, vaginal cancer, cancer of the vulva, and Wilms'
tumor.
[0276] The invention is particularly illustrated herein in
reference to treatment of certain types of experimentally defined
cancers. In these illustrative treatments, standard
state-of-the-art in vitro and in vivo models have been used. These
methods can be used to identify agents that can be expected to be
efficacious in in vivo treatment regimens. However, it will be
understood that the method of the invention is not limited to the
treatment of these tumor types, but extends to any solid tumor
derived from any organ system. Cancers whose invasiveness or
metastasis is associated with Ang-2 expression or activity are
especially susceptible to being inhibited or even induced to
regress by means of the invention.
[0277] The invention can also be practiced by including with a
specific binding agent of the invention, such as an antibody, in
combination with another anti-cancer chemotherapeutic agent, such
as any conventional chemotherapeutic agent. The combination of a
specific binding agent with such other agents can potentiate the
chemotherapeutic protocol. Numerous chemotherapeutic protocols will
present themselves in the mind of the skilled practitioner as being
capable of incorporation into the method of the invention. Any
chemotherapeutic agent can be used, including alkylating agents,
antimetabolites, hormones and antagonists, radioisotopes, as well
as natural products. For example, the compound of the invention can
be administered with antibiotics such as doxorubicin and other
anthracycline analogs, nitrogen mustards such as cyclophosphamide,
pyrimidine analogs such as 5-fluorouracil, cisplatin, hydroxyurea,
taxol and its natural and synthetic derivatives, and the like. As
another example, in the case of mixed tumors, such as
adenocarcinoma of the breast, where the tumors include
gonadotropin-dependent and gonadotropin-independent cells, the
compound can be administered in conjunction with leuprolide or
goserelin (synthetic peptide analogs of LH-RH). Other
antineoplastic protocols include the use of a tetracycline compound
with another treatment modality, e.g., surgery, radiation, etc.,
also referred to herein as "adjunct antineoplastic modalities."
Thus, the method of the invention can be employed with such
conventional regimens with the benefit of reducing side effects and
enhancing efficacy.
[0278] The present invention thus provides compositions and methods
useful for the treatment of a wide variety of cancers, including
solid tumors and leukemias. Types of cancer that may be treated
include, but are not limited to: adenocarcinoma of the breast,
prostate, and colon; all forms of bronchogenic carcinoma of the
lung; myeloid; melanoma; hepatoma; neuroblastoma; papilloma;
apudoma; choristoma; branchioma; malignant carcinoid syndrome;
carcinoid heart disease; carcinoma (e.g., Walker, basal cell,
basosquamous, Brown-Pearce, ductal, Ehrlich tumor, Krebs 2, merkel
cell, mucinous, non-small cell lung, oat cell, papillary,
scirrhous, bronchiolar, bronchogenic, squamous cell, and
transitional cell); histiocytic disorders; leukemia; histiocytosis
malignant; Hodgkin's disease; immunoproliferative small lung cell
carcinoma; non-Hodgkin's lymphoma; plasmacytoma;
reticuloendotheliosis; melanoma; chondroblastoma; chondroma;
chondrosarcoma; fibroma; fibrosarcoma; giant cell tumors;
histiocytoma; lipoma; liposarcoma; mesothelioma; myxoma;
myxosarcoma; osteoma; osteosarcoma; chordoma; craniopharyngioma;
dysgerminoma; hamartoma; mesenchymoma; mesonephroma; myosarcoma;
ameloblastoma; cementoma; odontoma; teratoma; thymoma; tophoblastic
tumor. Further, the following types of cancers may also be treated:
adenoma; cholangioma; cholesteatoma; cyclindroma;
cystadenocarcinoma; cystadenoma; granulosa cell tumor;
gynandroblastoma; hepatoma; hidradenoma; islet cell tumor; Leydig
cell tumor; papilloma; Sertoli cell tumor; theca cell tumor;
leiomyoma; leiomyosarcoma; myoblastoma; myoma; myosarcoma;
rhabdomyoma; rhabdomyosarcoma; ependymoma; ganglioneuroma; glioma;
medulloblastoma; meningioma; neurilemmoma; neuroblastoma;
neuroepithelioma; neurofibroma; neuroma; paraganglioma;
paraganglioma nonchromaffin; angiokeratoma; angiolymphoid
hyperplasia with eosinophilia; angioma sclerosing; angiomatosis;
glomangioma; hemangioendothelioma; hemangioma; hemangiopericytoma;
hemangiosarcoma; lymphangioma; lymphangiomyoma; lymphangiosarcoma;
pinealoma; carcinosarcoma; chondrosarcoma; cystosarcoma phyllodes;
fibrosarcoma; hemangiosarcoma; leiomyosarcoma; leukosarcoma;
liposarcoma; lymphangiosarcoma; myosarcoma; myxosarcoma; ovarian
carcinoma; rhabdomyosarcoma; sarcoma; neoplasms; nerofibromatosis;
and cervical dysplasia.
[0279] Another aspect of the present invention is using the
materials and methods of the present invention to prevent and/or
treat any hyperproliferative condition of the skin including
psoriasis and contact dermatitis or other hyperproliferative
diseases. It has been demonstrated that patients with psoriasis and
contact dermatitis have elevated Ang-2 activity within these
lesions [Ogoshi et al., J. Inv. Dermatol., 110:818-23 (1998)].
Preferably, specific binding agents specific for Ang-2 will be used
in combination with other pharmaceutical agents to treat humans
that express these clinical symptoms. The specific binding agents
can be delivered using any of the various carriers through routes
of administration described herein and others that are well known
to those of skill in the art.
[0280] Other aspects of the present invention include treating
various retinopathies (including diabetic retinopathy and
age-related macular degeneration) in which angiogenesis is
involved, as well as disorders/diseases of the female reproductive
tract such as endometriosis, uterine fibroids, and other such
conditions associated with dysfunctional vascular proliferation
(including endometrial microvascular growth) during the female
reproductive cycle.
[0281] Still another aspect of the present invention relates to
treating abnormal vascular growth including cerebral arteriovenous
malformations (AVMs) gastrointestinal mucosal injury and repair,
ulceration of the gastroduodenal mucosa in patients with a history
of peptic ulcer disease, including ischemia resulting from stroke,
a wide spectrum of pulmonary vascular disorders in liver disease
and portal hypertension in patients with nonhepatic portal
hypertension.
[0282] Another aspect of present invention is the prevention of
cancers utilizing the compositions and methods provided by the
present invention. Such reagents will include specific binding
agents against Ang-2.
Pharmaceutical Compositions
[0283] Pharmaceutical compositions of Ang-1 and/or Ang-2 specific
binding agents are within the scope of the present invention.
Pharmaceutical compositions comprising antibodies are described in
detail in, for example, U.S. Pat. No. 6,171,586, to Lam et al.,
issued Jan. 9, 2001. Such compositions comprise a therapeutically
or prophylactically effective amount of a specific binding agent,
such as an antibody, or a fragment, variant, derivative or fusion
thereof as described herein, in admixture with a pharmaceutically
acceptable agent. In a preferred embodiment, pharmaceutical
compositions comprise antagonist specific binding agents that
modulate partially or completely at least one biological activity
of Ang-1 and/or Ang-2 in admixture with a pharmaceutically
acceptable agent. Typically, the specific binding agents will be
sufficiently purified for administration to an animal.
[0284] The pharmaceutical composition may contain formulation
materials for modifying, maintaining or preserving, for example,
the pH, osmolarity, viscosity, clarity, color, isotonicity, odor,
sterility, stability, rate of dissolution or release, adsorption or
penetration of the composition. Suitable formulation materials
include, but are not limited to, amino acids (such as glycine,
glutamine, asparagine, arginine or lysine); antimicrobials;
antioxidants (such as ascorbic acid, sodium sulfite or sodium
hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl,
citrates, phosphates, other organic acids); bulking agents (such as
mannitol or glycine), chelating agents [such as ethylenediamine
tetraacetic acid (EDTA)]; complexing agents (such as caffeine,
polyvinylpyrrolidone, beta-cyclodextrin or
hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;
disaccharides and other carbohydrates (such as glucose, mannose, or
dextrins); proteins (such as serum albumin, gelatin or
immunoglobulins); coloring; flavoring and diluting agents;
emulsifying agents; hydrophilic polymers (such as
polyvinylpyrrolidone); low molecular weight polypeptides;
salt-forming counter ions (such as sodium); preservatives (such as
benzalkonium chloride, benzoic acid, salicylic acid, thimerosal,
phenethyl alcohol, methylparaben, propylparaben, chlorhexidine,
sorbic acid or hydrogen peroxide); solvents (such as glycerin,
propylene glycol or polyethylene glycol); sugar alcohols (such as
mannitol or sorbitol); suspending agents; surfactants or wetting
agents (such as pluronics, PEG, sorbitan esters, polysorbates such
as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin,
cholesterol, tyloxapal); stability enhancing agents (sucrose or
sorbitol); tonicity enhancing agents (such as alkali metal halides
(preferably sodium or potassium chloride, mannitol sorbitol);
delivery vehicles; diluents; excipients and/or pharmaceutical
adjuvants. (Remington's Pharmaceutical Sciences, 18th Edition, A.
R. Gennaro, ed., Mack Publishing Company, 1990).
[0285] The optimal pharmaceutical composition will be determined by
one skilled in the art depending upon, for example, the intended
route of administration, delivery format, and desired dosage. See
for example, Remington's Pharmaceutical Sciences, supra. Such
compositions may influence the physical state, stability, rate of
in vivo release, and rate of in vivo clearance of the specific
binding agent.
[0286] The primary vehicle or carrier in a pharmaceutical
composition may be either aqueous or non-aqueous in nature. For
example, a suitable vehicle or carrier may be water for injection,
physiological saline solution or artificial cerebrospinal fluid,
possibly supplemented with other materials common in compositions
for parenteral administration. Neutral buffered saline or saline
mixed with serum albumin are further exemplary vehicles. Other
exemplary pharmaceutical compositions comprise Tris buffer of about
pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may
further include sorbitol or a suitable substitute therefore. In one
embodiment of the present invention, binding agent compositions may
be prepared for storage by mixing the selected composition having
the desired degree of purity with optional formulation agents
(Remington's Pharmaceutical Sciences, supra) in the form of a
lyophilized cake or an aqueous solution. Further, the binding agent
product may be formulated as a lyophilizate using appropriate
excipients such as sucrose.
[0287] The pharmaceutical compositions can be selected for
parenteral delivery. Alternatively, the compositions may be
selected for inhalation or for enteral delivery such as orally,
aurally, opthalmically, rectally, or vaginally. The preparation of
such pharmaceutically acceptable compositions is within the skill
of the art.
[0288] The formulation components are present in concentrations
that are acceptable to the site of administration. For example,
buffers are used to maintain the composition at physiological pH or
at slightly lower pH, typically within a pH range of from about 5
to about 8.
[0289] When parenteral administration is contemplated, the
therapeutic compositions for use in this invention may be in the
form of a pyrogen-free, parenterally acceptable aqueous solution
comprising the desired specific binding agent in a pharmaceutically
acceptable vehicle. A particularly suitable vehicle for parenteral
injection is sterile distilled water in which a binding agent is
formulated as a sterile, isotonic solution, properly preserved. Yet
another preparation can involve the formulation of the desired
molecule with an agent, such as injectable microspheres,
bio-erodible particles, polymeric compounds (polylactic acid,
polyglycolic acid), beads, or liposomes, that provides for the
controlled or sustained release of the product which may then be
delivered via a depot injection. Hyaluronic acid may also be used,
and this may have the effect of promoting sustained duration in the
circulation. Other suitable means for the introduction of the
desired molecule include implantable drug delivery devices.
[0290] In another aspect, pharmaceutical formulations suitable for
parenteral administration may be formulated in aqueous solutions,
preferably in physiologically compatible buffers such as Hanks'
solution, ringer's solution, or physiologically buffered saline.
Aqueous injection suspensions may contain substances that increase
the viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. Additionally, suspensions of the
active compounds may be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils, such as sesame oil, or synthetic fatty acid esters, such as
ethyl oleate, triglycerides, or liposomes. Non-lipid polycationic
amino polymers may also be used for delivery. Optionally, the
suspension may also contain suitable stabilizers or agents to
increase the solubility of the compounds and allow for the
preparation of highly concentrated solutions.
[0291] In another embodiment, a pharmaceutical composition may be
formulated for inhalation. For example, a binding agent may be
formulated as a dry powder for inhalation. Polypeptide or nucleic
acid molecule inhalation solutions may also be formulated with a
propellant for aerosol delivery. In yet another embodiment,
solutions may be nebulized. Pulmonary administration is further
described in PCT Application No. PCT/US94/001875, which describes
pulmonary delivery of chemically modified proteins.
[0292] It is also contemplated that certain formulations may be
administered orally. In one embodiment of the present invention,
binding agent molecules that are administered in this fashion can
be formulated with or without those carriers customarily used in
the compounding of solid dosage forms such as tablets and capsules.
For example, a capsule may be designed to release the active
portion of the formulation at the point in the gastrointestinal
tract when bioavailability is maximized and pre-systemic
degradation is minimized. Additional agents can be included to
facilitate absorption of the binding agent molecule. Diluents,
flavorings, low melting point waxes, vegetable oils, lubricants,
suspending agents, tablet disintegrating agents, and binders may
also be employed.
[0293] Pharmaceutical compositions for oral administration can also
be formulated using pharmaceutically acceptable carriers well known
in the art in dosages suitable for oral administration. Such
carriers enable the pharmaceutical compositions to be formulated as
tablets, pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for ingestion by the patient.
[0294] Pharmaceutical preparations for oral use can be obtained
through combining active compounds with solid excipient and
processing the resultant mixture of granules (optionally, after
grinding) to obtain tablets or dragee cores. Suitable auxiliaries
can be added, if desired. Suitable excipients include carbohydrate
or protein fillers, such as sugars, including lactose, sucrose,
mannitol, and sorbitol; starch from corn, wheat, rice, potato, or
other plants; cellulose, such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose;
gums, including arabic and tragacanth; and proteins, such as
gelatin and collagen. If desired, disintegrating or solubilizing
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, and alginic acid or a salt thereof, such as
sodium alginate.
[0295] Dragee cores may be used in conjunction with suitable
coatings, such as concentrated sugar solutions, which may also
contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and suitable organic solvents or solvent mixtures. Dyestuffs or
pigments may be added to the tablets or dragee coatings for product
identification or to characterize the quantity of active compound,
i.e., dosage.
[0296] Pharmaceutical preparations that can be used orally also
include push-fit capsules made of gelatin, as well as soft, sealed
capsules made of gelatin and a coating, such as glycerol or
sorbitol. Push-fit capsules can contain active ingredients mixed
with fillers or binders, such as lactose or starches, lubricants,
such as talc or magnesium stearate, and, optionally, stabilizers.
In soft capsules, the active compounds may be dissolved or
suspended in suitable liquids, such as fatty oils, liquid, or
liquid polyethylene glycol with or without stabilizers.
[0297] Another pharmaceutical composition may involve an effective
quantity of binding agent in a mixture with non-toxic excipients
that are suitable for the manufacture of tablets. By dissolving the
tablets in sterile water, or other appropriate vehicle, solutions
can be prepared in unit dose form. Suitable excipients include, but
are not limited to, inert diluents, such as calcium carbonate,
sodium carbonate or bicarbonate, lactose, or calcium phosphate; or
binding agents, such as starch, gelatin, or acacia; or lubricating
agents such as magnesium stearate, stearic acid, or talc.
[0298] Additional pharmaceutical compositions will be evident to
those skilled in the art, including formulations involving binding
agent molecules in sustained- or controlled-delivery formulations.
Techniques for formulating a variety of other sustained- or
controlled-delivery means, such as liposome carriers, bio-erodible
microparticles or porous beads and depot injections, are also known
to those skilled in the art. See for example, PCT/US93/00829 that
describes controlled release of porous polymeric microparticles for
the delivery of pharmaceutical compositions. Additional examples of
sustained-release preparations include semipermeable polymer
matrices in the form of shaped articles, e.g. films, or
microcapsules. Sustained release matrices may include polyesters,
hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP 58,481),
copolymers of L-glutamic acid and gamma ethyl-L-glutamate [Sidman
et al., Biopolymers, 22:547-556 (1983)], poly
(2-hydroxyethyl-methacrylate) [Langer et al., J Biomed Mater Res,
15:167-277, (1981)] and [Langer et al., Chem Tech, 12:98-105
(1982)], ethylene vinyl acetate (Langer et al., supra) or
poly-D(-)-3-hydroxybutyric acid (EP 133,988). Sustained-release
compositions also include liposomes, which can be prepared by any
of several methods known in the art. See e.g., Eppstein et al.,
Proc Natl Acad Sci (USA), 82:3688-3692 (1985); EP 36,676; EP
88,046; EP 143,949.
[0299] The pharmaceutical composition to be used for in vivo
administration typically must be sterile. This may be accomplished
by filtration through sterile filtration membranes. Where the
composition is lyophilized, sterilization using this method may be
conducted either prior to or following lyophilization and
reconstitution. The composition for parenteral administration may
be stored in lyophilized form or in solution. In addition,
parenteral compositions generally are placed into a container
having a sterile access port, for example, an intravenous solution
bag or vial having a stopper pierceable by a hypodermic injection
needle.
[0300] Once the pharmaceutical composition has been formulated, it
may be stored in sterile vials as a solution, suspension, gel,
emulsion, solid, or a dehydrated or lyophilized powder. Such
formulations may be stored either in a ready-to-use form or in a
form (e.g., lyophilized) requiring reconstitution prior to
administration.
[0301] In a specific embodiment, the present invention is directed
to kits for producing a single-dose administration unit. The kits
may each contain both a first container having a dried protein and
a second container having an aqueous formulation. Also included
within the scope of this invention are kits containing single and
multi-chambered pre-filled syringes (e.g., liquid syringes and
lyosyringes).
[0302] An effective amount of a pharmaceutical composition to be
employed therapeutically will depend, for example, upon the
therapeutic context and objectives. One skilled in the art will
appreciate that the appropriate dosage levels for treatment will
thus vary depending, in part, upon the molecule delivered, the
indication for which the binding agent molecule is being used, the
route of administration, and the size (body weight, body surface or
organ size) and condition (the age and general health) of the
patient. Accordingly, the clinician may titer the dosage and modify
the route of administration to obtain the optimal therapeutic
effect. A typical dosage may range from about 0.1 mg/kg to up to
about 100 mg/kg or more, depending on the factors mentioned above.
In other embodiments, the dosage may range from 0.1 mg/kg up to
about 100 mg/kg; or 1 mg/kg up to about 100 mg/kg; or 5 mg/kg up to
about 100 mg/kg.
[0303] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays or in animal
models such as mice, rats, rabbits, dogs, or pigs. An animal model
may also be used to determine the appropriate concentration range
and route of administration. Such information can then be used to
determine useful doses and routes for administration in humans.
[0304] The exact dosage will be determined in light of factors
related to the subject requiring treatment. Dosage and
administration are adjusted to provide sufficient levels of the
active compound or to maintain the desired effect. Factors that may
be taken into account include the severity of the disease state,
the general health of the subject, the age, weight, and gender of
the subject, time and frequency of administration, drug
combination(s), reaction sensitivities, and response to therapy.
Long-acting pharmaceutical compositions may be administered every 3
to 4 days, every week, or biweekly depending on the half-life and
clearance rate of the particular formulation.
[0305] The frequency of dosing will depend upon the pharmacokinetic
parameters of the binding agent molecule in the formulation used.
Typically, a composition is administered until a dosage is reached
that achieves the desired effect. The composition may therefore be
administered as a single dose, or as multiple doses (at the same or
different concentrations/dosages) over time, or as a continuous
infusion. Further refinement of the appropriate dosage is routinely
made. Appropriate dosages may be ascertained through use of
appropriate dose-response data.
[0306] The route of administration of the pharmaceutical
composition is in accord with known methods, e.g. orally, through
injection by intravenous, intraperitoneal, intracerebral
(intra-parenchymal), intracerebroventricular, intramuscular,
intra-ocular, intraarterial, intraportal, intralesional routes,
intramedullary, intrathecal, intraventricular, transdermal,
subcutaneous, intraperitoneal, intranasal, enteral, topical,
sublingual, urethral, vaginal, or rectal means, by sustained
release systems or by implantation devices. Where desired, the
compositions may be administered by bolus injection or continuously
by infusion, or by implantation device.
[0307] Alternatively or additionally, the composition may be
administered locally via implantation of a membrane, sponge, or
another appropriate material on to which the desired molecule has
been absorbed or encapsulated. Where an implantation device is
used, the device may be implanted into any suitable tissue or
organ, and delivery of the desired molecule may be via diffusion,
timed-release bolus, or continuous administration.
[0308] In some cases, it may be desirable to use pharmaceutical
compositions in an ex vivo manner. In such instances, cells,
tissues, or organs that have been removed from the patient are
exposed to the pharmaceutical compositions after which the cells,
tissues and/or organs are subsequently implanted back into the
patient.
[0309] In other cases, a binding agent which is a polypeptide can
be delivered by implanting certain cells that have been genetically
engineered, using methods such as those described herein, to
express and secrete the polypeptide. Such cells may be animal or
human cells, and may be autologous, heterologous, or xenogeneic.
Optionally, the cells may be immortalized. In order to decrease the
chance of an immunological response, the cells may be encapsulated
to avoid infiltration of surrounding tissues. The encapsulation
materials are typically biocompatible, semi-permeable polymeric
enclosures or membranes that allow the release of the protein
product(s) but prevent the destruction of the cells by the
patient's immune system or by other detrimental factors from the
surrounding tissues.
Combination Therapy
[0310] Specific binding agents of the invention can be utilized in
combination with other therapeutic in the treatment of Ang-1 and/or
Ang-2 pathologies. These other therapeutics include, for example
radiation treatment, chemotherapeutic agents, as well as other
growth factors or inhibitors.
[0311] Chemotherapy treatment can employ anti-neoplastic agents
including, for example, alkylating agents including: nitrogen
mustards, such as mechlorethamine, cyclophosphamide, ifosfamide,
melphalan and chlorambucil; nitrosoureas, such as carmustine
(BCNU), lomustine (CCNU), and semustine (methyl-CCNU);
ethylenimines/methylmelamine such as thriethylenemelamine (TEM),
triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM,
altretamine); alkyl sulfonates such as busulfan; triazines such as
dacarbazine (DTIC); antimetabolites including folic acid analogs
such as methotrexate and trimetrexate, pyrimidine analogs such as
5-fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine
arabinoside (AraC, cytarabine), 5-azacytidine,
2,2'-difluorodeoxycytidine, purine analogs such as
6-mercaptopurine, 6-thioguanine, azathioprine, 2'-deoxycoformycin
(pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine
phosphate, and 2-chlorodeoxyadenosine (cladribine, 2-CdA); natural
products including antimitotic drugs such as paclitaxel, vinca
alkaloids including vinblastine (VLB), vincristine, and
vinorelbine, taxotere, estramustine, and estramustine phosphate;
ppipodophylotoxins such as etoposide and teniposide; antibiotics
such as actimomycin D, daunomycin (rubidomycin), doxorubicin,
mitoxantrone, idarubicin, bleomycins, plicamycin (mithramycin),
mitomycinC, and actinomycin; enzymes such as L-asparaginase;
biological response modifiers such as interferon-alpha, IL-2, G-CSF
and GM-CSF; miscellaneous agents including platinium coordination
complexes such as cisplatin and carboplatin, anthracenediones such
as mitoxantrone, substituted urea such as hydroxyurea,
methylhydrazine derivatives including N-methylhydrazine (MIH) and
procarbazine, adrenocortical suppressants such as mitotane
(o,p'-DDD) and aminoglutethimide; hormones and antagonists
including adrenocorticosteroid antagonists such as prednisone and
equivalents, dexamethasone and aminoglutethimide; progestin such as
hydroxyprogesterone caproate, medroxyprogesterone acetate and
megestrol acetate; estrogen such as diethylstilbestrol and ethinyl
estradiol equivalents; antiestrogen such as tamoxifen; androgens
including testosterone propionate and fluoxymesterone/equivalents;
antiandrogens such as flutamide, gonadotropin-releasing hormone
analogs and leuprolide; and non-steroidal antiandrogens such as
flutamide.
[0312] Combination therapy can done in conjunction with the growth
factors listed below or with agents that are designed to inhibit
the growth factors listed below. The growth factors include
cytokines, lymphokines, growth factors, or other hematopoietic
factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,
IL-16, IL-17, IL-18, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF, GM-CSF,
thrombopoietin, stem cell factor, and erythropoietin. Other are
compositions can include known angiopoietins, for example Ang-1,
-2, -4, -Y, and/or the human Ang-like polypeptide, and/or vascular
endothelial growth factor (VEGF). Growth factors include
angiogenin, bone morphogenic protein-1, bone morphogenic protein-2,
bone morphogenic protein-3, bone morphogenic protein-4, bone
morphogenic protein-5, bone morphogenic protein-6, bone morphogenic
protein-7, bone morphogenic protein-8, bone morphogenic protein-9,
bone morphogenic protein-10, bone morphogenic protein-11, bone
morphogenic protein-12, bone morphogenic protein-13, bone
morphogenic protein-14, bone morphogenic protein-15, bone
morphogenic protein receptor-IA, bone morphogenic protein receptor
IB, brain derived neurotrophic factor, ciliary neutrophic factor,
ciliary neutrophic factor receptor, cytokine-induced neutrophil
chemotactic factor-1, cytokine-induced neutrophil, chemotactic
factor-2, cytokine-induced neutrophil chemotactic factor-2,
endothelial cell growth factor, endothelin-1, epidermal growth
factor, epithelial-derived neutrophil attractant, fibroblast growth
factor-4, fibroblast growth factor-5, fibroblast growth factor-6,
fibroblast growth factor-7, fibroblast growth factor-8, fibroblast
growth factor-8b, fibroblast growth factor-8c, fibroblast growth
factor-9, fibroblast growth factor-10, fibroblast growth factor
acidic, fibroblast growth factor basic, glial cell line-derived
neutrophic factor receptor-1, glial cell line-derived neutrophic
factor receptor-2, growth related protein, growth related
protein-2, growth related protein-2, growth related protein-3,
heparin binding epidermal growth factor, hepatocyte growth factor,
hepatocyte growth factor receptor, insulin-like growth factor I,
insulin-like growth factor receptor, insulin-like growth factor II,
insulin-like growth factor binding protein, keratinocyte growth
factor, leukemia inhibitory factor, leukemia inhibitory factor
receptor-1, nerve growth factor nerve growth factor receptor,
neurotrophin-3, neurotrophin-4, placenta growth factor, placenta
growth factor-2, platelet-derived endothelial cell growth factor,
platelet derived growth factor, platelet derived growth factor A
chain, platelet derived growth factor AA, platelet derived growth
factor AB, platelet derived growth factor B chain, platelet derived
growth factor BB, platelet derived growth factor receptor-1,
platelet derived growth factor receptor-2, pre-B cell growth
stimulating factor, stem cell factor, stem cell factor receptor,
transforming growth factor-1, transforming growth factor-2,
transforming growth factor-3, transforming growth factor-1.2,
transforming growth factor-4, transforming growth factor-5, latent
transforming growth factor-1, transforming growth factor binding
protein I, transforming growth factor binding protein II,
transforming growth factor binding protein III, tumor necrosis
factor receptor type I, tumor necrosis factor receptor type II,
urokinase-type plasminogen activator receptor, vascular endothelial
growth factor, and chimeric proteins and biologically or
immunologically active fragments thereof.
[0313] Combination therapy can also be achieved with a specific
binding agent of the present invention, such as an antibody, in
combination with an apoptotic inducer such as a specific binding
agent (e.g., an agonistic antibody or TRAIL ligand) that induces
apoptosis via the DR4 (TRAIL R-1) and/or the DR5 (TRAIL R-2)
receptor. Examples of such specific binding agents are provided in
WO 2007/027713, incorporated herein by reference, which discloses
agonistic antibodies that induce apoptosis via the DR5
receptor.
Immunotherapeutics
[0314] Immunotherapeutics generally rely on the use of immune
effector cells and molecules to target and destroy cancer cells.
The immune effectors may be, for example an antibody of the present
invention that recognizes some marker on the surface of a target
cell. The antibody alone may serve as an effector of therapy or it
may recruit other cells to actually effect cell killing. The
antibody may also be conjugated to a drug or toxin
(chemotherapeutic, radionuclide, ricin A chain, cholera toxin,
pertussis toxin, etc.) and thus may merely serve as a targeting
agent.
[0315] According to the present invention, mutant forms of Ang-1
and/or Ang-2 may be targeted by immunotherapy either antibodies or
antibody conjugates of the invention. It is particularly
contemplated that the antibody compositions of the invention may be
used in a combined therapy approach in conjunction with Ang-2
targeted therapy.
[0316] Passive immunotherapy has proved to be particularly
effective against a number of cancers. See, for example, WO
98/39027.
[0317] The following examples are intended for illustration
purposes only, and should not be construed as limiting the scope of
the invention in any way.
Example 1
Generation of Affinity Matured Antibodies Against Ang2 by Phage
Display
Overall Strategy
[0318] CDR randomization was employed to enhance the activity of
Ang2 antibody, similar to previous approaches (Chen Y et al., 1999
J Mol Biol (293)865-881; Yelton D E et al., 1995 J Immunol (155)
1994-2004; Yang W-P et al., 1995 J Mol Biol (254) 392-403).
Briefly, the variable regions of Ang2 antibody 536 were cloned into
the TargetQuest modified pCES-1 vector (Dyax Corp, de Haard H J et
al 1999 J Biol Chem (274) 18218-30). All CDR regions were targeted
for randomization of each CDR residue by mutagenesis using NNK
containing oligonucleotides. After the mutagenesis reaction, phage
clones were interrogated for each position using phage ELISA to
identify beneficial mutations (for methods see WO 2004/046306,WO
2003/03057134, and US 2003/0099647 A1, for general phage antibody
refs., Marks J D et al., 1991 J Mol Biol (222) 581-597; Hoogenboom
H R et al 1992 J Mol Biol (227) 381-388; Griffiths A D et al., 1993
EMBO J. (12) 725-734; Vaughan T P et al., 1996 Nat Biotechnol (14)
309-314). Clones with beneficial mutations were converted to full
antibodies. Heavy chain clones were paired with light chain clones
and resulting IgG was tested for neutralization activity. Top 22
clones were characterized further.
A. Ab536 Fab Template Construction
[0319] The variable regions of 536 antibody were cloned into pCES-1
vector using standard molecular biology techniques (Molecular
Cloning: A Laboratory Manual, 3.sup.rd Edition Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 2001). The heavy chain
variable and full length light chain fragments were generated by
PCR using the following oligonucleotides:
TABLE-US-00005 Heavy chain reverse: CCGCTGTGCCCCCAGAGGTGC Heavy
chain forward: ttttttccatggccgaggtccagctggtgcagtc Light chain
reverse: TTTTTTGGCGCGCCTTATTAACACTCTCCCCTGTTGAAGCT Light chain
forward: ttttttgtgcacttgacattgtgatgactcagtct
[0320] The variable region of heavy chain was inserted between the
restriction sites, NcoI and BstEII. The full length light chain was
inserted between restriction sites, ApaLI and AscI. The resulting
construct was used as a template for CDR randomization.
B. CDR Mutations
[0321] Antibody 536 as a Fab in vector pCES-1 was affinity matured
by a step-wise site-directed mutagenesis using oligonucleotides
bearing NNK (N=ATCG; K=GT) codons for each of the CDR positions.
The QuikChange Site-Directed Mutagenesis Kit (Stratagene #200518-5)
was used following the manufacturer recommended protocol. To
identify phages with enhanced binding to Ang2, phage ELISA
performed with biotinylated human Ang2 protein coated at 2 ug/ml in
PBS onto the 96 well Maxisorp plates (NUNC). Briefly, after
blocking with 2% milk in PBS, overnight phage culture that were
grown with helper phage was incubated and bound phages were
detected with anti-M13 antibodies conjugated with HRP (Pharmacia).
Luminescence signal was compared relative to parental 536 Fab, and
clones with superior signal were selected for further analysis.
C. IgG Conversion of Phage Fab
[0322] After phage ELISA and sequence analysis, 95 clones each from
light chain and heavy chain mutagenesis with enhanced binding
against Ang2 were selected and converted into IgG. Briefly, the
variable regions of each clone were PCR amplified using a pair of
primers. Primer sequences for LC were CTG CTG CTG TGG CTG AGA GGT
GCG CGC TGT GAT ATT GTG ATG ACT CAG TCT CCA CTC TCC and AAA AAA CGT
ACG TTT GAT CTC CAG CTT GGT CC. Primers for HC were
TTTTTTTTGCGCGCTGTGAGGTCCAGCTGGTGCAGTC and AAAAAAGGCACTA
GAGACGGTGACCAGGGTTCC. After digesting with BssHII and BsiWI for LC,
and BssHII and BsmBI for HC, the variable regions were inserted
into pcDNA3 vectors containing VK1 leader sequence and constant
sequence of human Kappa and human IgG1 using standard molecular
biology techniques. Each ligation mixture was transformed into two
96 well plates of XL10 gold competent cells (Stratagene), and the
transformation mixture were grown overnight for plasmid prep the
next day. Resulting DNA were paired with relative parental 536 LC
or HC DNA, and transiently transfected into 293T cells in OPTI-MEM
using Fugene6. After 7 days, the media from transfected cells were
collected, and IgG concentration was quantified by Lance assay
using anti-human IgG antibody (Fc specific) labeled with europium
and anti-human IgG antibody labeled with APC (Perkin Elmer).
D. Selection of IgG Clones
[0323] Conditioned media that contain IgG were tested in HTRF
neutralizing assay for its inhibitory effect of Tie-2 interaction
with either Ang1 or Ang2. From initial screening, 15LC clones and
11HC clones that showed improved activity were picked. The DNA of
selected clones were prepared and confirmed by sequencing. Then the
combination of each LC and HC mutants, along with 536 parental
clone, were transfected into two 96 plates seeded with 293T cells.
Conditioned media were collected, and analyzed for the IgG
concentration and inhibitory effect in Tie2 neutralizing assay.
From this combination, 22 clones that contain single mutation in
each LC and HC were selected for further analysis.
E. Expression and Purification of Human Affinity Matured Ang2
Antibodies in CHO Cells
[0324] CS-9 cells used for transfection of the anti-Ang2 IgG
expression plasmid(s) are a serum-free suspension CHO cell line.
They were derived by gradually adapting DXB-11 CHO cells to grow in
serum-free medium as described in Rasmussen et al, 1998 (Rasmussen,
B., Davis, R., Thomas, J., Reddy, P. 1998. Isolation,
characterization and recombinant protein expression in Veggie-CHO:
A serum-free CHO host cell line. Cytotechnology. 28: 31-42). DXB-11
cells are a DHFR-deficient mutant derivative from CHO-K1 cells.
(Chasin and Urlaub, 1983; Urlaub and Chasin. 1980. Isolation of
Chinese hamster cell mutants deficient in dihydrofolate reductase
activity. Proc. Natl. Acad. Sci. USA 77, 4216-4220.; Chasin L. A.,
Graf, L., Ellis, N., Lanzberg, M., Urlaub, G. 1982. Gene
amplification in dihydro folate reductase deficient mutants.
Schimke, R. T. (Ed.) Gene amplification; Cold Spring Harbor, N.Y.
Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., p161-166).
CHO-K1 is an epithelioid cell line originally isolated from the
Chinese hamster ovary (Kao and Puck. Genetics of somatic mammalian
cells. VII. Induction and isolation of nutritional mutants in
Chinese hamster cells. Proc. Nat. Acad. Sci. 60: 1275-1281,
1968).
[0325] To derive the CS-9 host cell line, DXB-11 cells were grown
in media with gradual reduction in serum over 100 passages to
obtain serum-free-adapted cells referred to as SF-CHO (Rasmussen et
al, 1998). The SF-CHO cells were subsequently sub-cloned by
limiting dilution cloning and individual clones were evaluated. The
CS-9 clone was selected as the host cell line for expression of
recombinant proteins and banked in serum-free medium. The bank was
tested for adventious agents and sterility and found to be free of
viral, mycoplamsa and microbial agents. The host cell line, CS-9,
is a DHFR deficient CHO cell line auxotrophic for glycine,
hypoxanthine and thymidine (GHT). The plasmids pDC323 and pDC324
each encode a portion of the DHFR cDNA and the 2 plasmids must
complement each other to express a functional DHFR molecule by
association of the 2 DHFR fragments in vivo.
[0326] The following twenty-two antibodies, each consisted of two
heavy chains and 2 light (kappa or lambda) chains as designated in
the following Table 2.
TABLE-US-00006 TABLE 2 Antibody Heavy Antibody Light Antibody*
Chain Chain H6L7 H6 HC L7 LC H5L7 H5 HC L7 LC H4L13 H4 HC L13 LC
H11L7 H11 HC L7 LC H10L7 H10 HC L7 LC H4L7 H4 HC L7 LC H5L6 H5 HC
L6 LC H2L7 H2 HC L7 LC H5L8 H5 HC L8 LC H6L8 H6 HC L8 LC H3L7 H3 HC
L7 LC H5L4 H5 HC L4 LC H4L12 H4 HC L12 LC H6L6 H6 HC L6 LC H4L2 H4
HC L2 LC H4L6 H4 HC L6 LC H4L4 H4 HC L4 LC H5L11 H5 HC L11 LC H5L1
H5 HC L1 LC H4L11 H4 HC L11 LC H5L12 H5 HC L12 LC H5L9 H5 HC L9 LC
*Tested for binding to hAng-2, mAng-2, and hAng-1 as described
herein.
Tables 3 and 4 set forth the sequences and SEQ ID NOs. of the heavy
and light (kappa and lambda) chains of the 22 anti-Ang-1 and/or
anti-Ang-2 antibodies converted from phage to full length IgG1
antibodies. The complementarity-determining regions (CDRs) of the
monoclonal antibodies were predicted using the VBASE database which
uses the technique described by Kabat et al in: Sequences of
Proteins of Immunological Interest (NIH Publication No. 91-3242;
U.S. Dept. Health and Human Services, 5.sup.th ed.). Fab regions
were aligned to sequences in the database with the closest germline
sequence and then visually compared with such sequences. The CDRs
for each variable region (heavy or light chain), both residue and
sequences are set forth in Table 5.
TABLE-US-00007 TABLE 3 Heavy Chain Variable Regions Antibody HC
Sequence 536 HC EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGK (Ref)
GLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA
EDTAVYYCARDLLDYDILTGYGYWGQGTLVTVSS H2
EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGK (SEQ ID NO. 1)
GLEWVSYISSSGSTIEYADSVKGRFTISRDNAKNSLYLQMNSLRA
EDTAVYYCARDLLDYDILTGYGYWGQGTLVTVSS H3
EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGK (SEQ ID NO. 2)
GLEWVSYISSSGSTIQYADSVKGRFTISRDNAKNSLYLQMNSLRA
EDTAVYYCARDLLDYDILTGYGYWGQGTLVTVSS H4
EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGK (SEQ ID NO. 3)
GLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA
EDTAVYYCARDLLDYDLLTGYGYWGQGTLVTVSS H6
EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGK (SEQ ID NO. 4)
GLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA
EDTAVYYCARDLLDYDIYTGYGYWGQGTLVTVSS H10
EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGK (SEQ ID NO 5)
GLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA
EDTAVYYCARDLLDYDILTGYGLWGQGTLVTVSS H11
EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGK (SEQ ID NO. 6)
GLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA
EDTAVYYCARDLLDYDILTGYGMWGQGTLVTVSS H5P
EVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGK (SEQ ID NO. 7)
GLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA
EDTAVYYCARDLLDYDIWTGYGYWGQGTLVTVSS
TABLE-US-00008 TABLE 4 Light Chain Variable Regions Antibody LC
Sequence 536 kappa DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKP
(Ref) GQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGV
YYCMQGTHWPPTFGQGTKLEIK L1
DIVMTQSPLSLPVTPGEPASISCRSIQSLLQSNGYNYLDWYLQKP (SEQ ID NO. 8)
GQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGV
YYCMQGTHWPPTFGQGTKLEIK L2
DIVMTQSPLSLPVTPGEPASISCRSSQSLLLSNGYNYLDWYLQKP (SEQ ID NO. 9)
GQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGV
YYCMQGTHWPPTFGQGTKLEIK L4
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSHGYNYLDWYLQKP (SEQ ID NO. 10)
GQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGV
YYCMQGTHWPPTFGQGTKLEIK L6
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSVGYNYLDWYLQKP (SEQ ID NO. 11)
GQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGV
YYCMQGTHWPPTFGQGTKLEIK L7
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNFLDWYLQKP (SEQ ID NO. 12)
GQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGV
YYCMQGTHWPPTFGQGTKLEIK L8
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNMLDWYLQKP (SEQ ID NO. 13)
GQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGV
YYCMQGTHWPPTFGQGTKLEIK L9
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKP (SEQ ID NO. 14)
GQSPQLLIYAGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGV
YYCMQGTHWPPTFGQGTKLEIK L11
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKP (SEQ ID NO. 15)
GQSPQLLIYLGSDRASGVPDRFSGSGSGTDFTLKISRVEAEDVGV
YYCMQGTHWPPTFGQGTKLEIK L12
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKP (SEQ ID NO. 16)
GQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGV
YYCMQATHWPPTFGQGTKLEIK L13
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKP (SEQ ID NO. 17)
GQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGV
YYCMQVTHWPPTFGQGTKLEIK
TABLE-US-00009 TABLE 5 Complementarity-Determining Regions (CDRs)
of Heavy Chains (HC) and Light Chains (LC) of Ang-1 and/or Ang-2
Antibodies; Residues and Sequence CDR 1 CDR 2 CDR 3 Antibody
Residues Sequence Residues Sequence Residues Sequence Ab 536 31-35
SYGMH 50-66 YISSSGSTIYYADSVKG 99-111 DLLDYDILTGYGY HC Ab 536 24-39
RSSQSLLHSNGYNYLD 55-61 LGSNRAS 94-102 MQGTHWPPT LC H6L7 HC 31-35
SYGMH 50-66 YISSSGSTIYYADSVKG 99-111 DLLDYDI TGYGY (SEQ ID NO. 18)
(SEQ ID NO. 26) (SEQ ID NO. 32) H6L7 LC 24-39 RSSQSLLHSNGYN LD
55-61 LGSNRAS 94-102 MQGTHWPPT (SEQ ID NO. 19) (SEQ ID NO. 27) (SEQ
ID NO. 33) H5L7 HC 31-35 SYGMH 50-66 YISSSGSTIYYADSVKG 99-111
DLLDYDI TGYGY (SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 34) H5L7
LC 24-39 RSSQSLLHSNGYN LD 55-61 LGSNRAS 94-102 MQGTHWPPT (SEQ ID
NO. 19) (SEQ ID NO. 27) (SEQ ID NO. 33) H4L13 31-35 SYGMH 50-66
YISSSGSTIYYADSVKG 99-111 DLLDYD LTGYGY HC (SEQ ID NO. 18) (SEQ ID
NO. 26) (SEQ ID NO. 35) H4L13 24-39 RSSQSLLHSNGYNYLD 55-61 LGSNRAS
94-102 MQ THWPPT LC (SEQ ID NO. 20) (SEQ ID NO. 27) (SEQ ID NO. 36)
H11L7 31-35 SYGMH 50-66 YISSSGSTIYYADSVKG 99-111 DLLDYDILTG GY HC
(SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 37) H11L7 24-39
RSSQSLLHSNGYN LD 55-61 LGSNRAS 94-102 MQGTHWPPT LC (SEQ ID NO. 19)
(SEQ ID NO. 27) (SEQ ID NO. 33) H10L7 31-35 SYGMH 50-66
YISSSGSTIYYADSVKG 99-111 DLLDYDILTG GY HC (SEQ ID NO. 18) (SEQ ID
NO. 26) (SEQ ID NO. 38) H10L7 24-39 RSSQSLLHSNGYN LD 55-61 LGSNRAS
94-102 MQGTHWPPT LC (SEQ ID NO. 19) (SEQ ID NO. 27) (SEQ ID NO. 33)
H4L7 HC 31-35 SYGMH 50-66 YISSSGSTIYYADSVKG 99-111 DLLDYD LTGYGY
(SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 35) H4L7 LC 24-39
RSSQSLLHSNGYN LD 55-61 LGSNRAS 94-102 MQGTHWPPT (SEQ ID NO. 19)
(SEQ ID NO. 27) (SEQ ID NO. 33) H5L6 HC 31-35 SYGMH 50-66
YISSSGSTIYYADSVKG 99-111 DLLDYDI TGYGY (SEQ ID NO. 18) (SEQ ID NO.
26) (SEQ ID NO. 34) H5L6 LC 24-39 RSSQSLLHS GYNYLD 55-61 LGSNRAS
94-102 MQGTHWPPT (SEQ ID NO. 21) (SEQ ID NO. 27) (SEQ ID NO. 33)
H2L7 HC 31-35 SYGMH 50-66 YISSSGSTI YADSVKG 99-111 DLLDYDILTGYGY
(SEQ ID NO. 18) (SEQ ID NO. 28) (SEQ ID N0. 39) H2L7 LC 24-39
RSSQSLLHSNGYN LD 55-61 LGSNRAS 94-102 MQGTHWPPT (SEQ ID NO. 19)
(SEQ ID NO. 27) (SEQ ID NO. 33) H5L8 HC 31-35 SYGMH 50-66
YISSSGSTIYYADSVKG 99-111 DLLDYDI TGYGY (SEQ ID NO. 18) (SEQ ID NO.
26) (SEQ ID NO. 34) H5L8 LC 24-39 RSSQSLLHSNGYN LD 55-61 LGSNRAS
94-102 MQGTHWPPT (SEQ ID NO. 22) (SEQ ID NO. 27) (SEQ ID NO. 33)
H6L8 HC 31-35 SYGMH 50-66 YISSSGSTIYYADSVKG 99-111 DLLDYDI TGYGY
(SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 32) H6L8 LC 24-39
RSSQSLLHSNGYN LD 55-61 LGSNRAS 94-102 MQGTHWPPT (SEQ ID NO. 22)
(SEQ ID NO. 27) (SEQ ID NO. 33) H3L7 HC 31-35 SYGMH 50-66 YISSSGSTI
YADSVKG 99-111 DLLDYDILTGYGY (SEQ ID NO. 18) (SEQ ID NO. 29) (SEQ
ID N0. 39) H3L7 LC 24-39 RSSQSLLHSNGYN LD 55-61 LGSNRAS 94-102
MQGTHWPPT (SEQ ID NO. 19) (SEQ ID NO. 27) (SEQ ID NO. 33) H5L4 HC
31-35 SYGMH 50-66 YISSSGSTIYYADSVKG 99-111 DLLDYDI TGYGY (SEQ ID
NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 34) H5L4 LC 24-39 RSSQSLLHS
GYNYLD 55-61 LGSNRAS 94-102 MQGTHWPPT (SEQ ID NO. 23) (SEQ ID NO.
27) (SEQ ID NO. 33) H4L12 31-35 SYGMH 50-66 YISSSGSTIYYADSVKG
99-111 DLLDYD LTGYGY HC (SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO.
35) H4L12 24-39 RSSQSLLHSNGYNYLD 55-61 LGSNRAS 94-102 MQ THWPPT LC
(SEQ ID NO. 20) (SEQ ID NO. 27) (SEQ ID NO. 40) H6L6 HC 31-35 SYGMH
50-66 YISSSGSTIYYADSVKG 99-111 DLLDYDI TGYGY (SEQ ID NO. 18) (SEQ
ID NO. 26) (SEQ ID NO. 32) H6L6 LC 24-39 RSSQSLLHS GYNYLD 55-61
LGSNRAS 94-102 MQGTHWPPT (SEQ ID NO. 21) (SEQ ID NO. 27) (SEQ ID
NO. 33) H4L2 HC 31-35 SYGMH 50-66 YISSSGSTIYYADSVKG 99-111 DLLDYD
LTGYGY (SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 35) H4L2 LC
24-39 RSSQSLL SNGYNYLD 55-61 LGSNRAS 94-102 MQGTHWPPT (SEQ ID NO.
24) (SEQ ID NO. 27) (SEQ ID NO. 33) H4L6 HC 31-35 SYGMH 50-66
YISSSGSTIYYADSVKG 99-111 DLLDYD LTGYGY (SEQ ID NO. 18) (SEQ ID NO.
26) (SEQ ID NO. 35) H4L6 LC 24-39 RSSQSLLHS GYNYLD 55-61 LGSNRAS
94-102 MQGTHWPPT (SEQ ID NO. 21) (SEQ ID NO. 27) (SEQ ID NO. 33)
H4L4 HC 31-35 SYGMH 50-66 YISSSGSTIYYADSVKG 99-111 DLLDYD LTGYGY
(SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 35) H4L4 LC 24-39
RSSQSLLHS GYNYLD 55-61 LGSNRAS 94-102 MQGTHWPPT (SEQ ID NO. 23)
(SEQ ID NO. 27) (SEQ ID NO. 33) H5L11 31-35 SYGMH 50-66
YISSSGSTIYYADSVKG 99-111 DLLDYDI TGYGY HC (SEQ ID NO. 18) (SEQ ID
NO. 26) (SEQ ID NO. 34) H5L11 24-39 RSSQSLLHSNGYNYLD 55-61 LGS RAS
94-102 MQGTHWPPT LC (SEQ ID NO. 20) (SEQ ID NO. 30) (SEQ ID NO. 33)
H5L1 HC 31-35 SYGMH 50-66 YISSSGSTIYYADSVKG 99-111 DLLDYDI TGYGY
(SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 34) H5L1 LC 24-39 RS
QSLL SNGYNYLD 55-61 LGSNRAS 94-102 MQGTHWPPT (SEQ ID NO. 25) (SEQ
ID NO. 27) (SEQ ID NO. 33) H4L11 31-35 SYGMH 50-66
YISSSGSTIYYADSVKG 99-111 DLLDYD LTGYGY HC (SEQ ID NO. 18) (SEQ ID
NO. 26) (SEQ ID NO. 35) H4L11 24-39 RSSQSLLHSNGYNYLD 55-61 LGS RAS
94-102 MQGTHWPPT LC (SEQ ID NO. 20) (SEQ ID NO. 30) (SEQ ID NO. 33)
H5L12 31-35 SYGMH 50-66 YISSSGSTIYYADSVKG 99-111 DLLDYDI TGYGY HC
(SEQ ID NO. 18) (SEQ ID NO. 26) (SEQ ID NO. 34) H5L12 24-39
RSSQSLLHSNGYNYLD 55-61 LGSNRAS 94-102 MQ THWPPT LC (SEQ ID NO. 20)
(SEQ ID NO. 27) (SEQ ID NO. 40) H5L9 HC 31-35 SYGMH 50-66
YISSSGSTIYYADSVKG 99-111 DLLDYDI TGYGY (SEQ ID NO. 18) (SEQ ID NO.
26) (SEQ ID NO. 34) H5L9 LC 24-39 RSSQSLLHSNGYNYLD 55-61 GSNRAS
94-102 MQGTHWPPT (SEQ ID NO. 20) (SEQ ID NO. 31) (SEQ ID NO.
33)
Example 2
Molecular Assays to Evaluate Ang-2 Antibodies
[0327] Molecular assays (Affinity ELISA, Neutralization ELISA and
BIAcore) were developed to assess direct antibody binding to Ang-2
and related family members (for example, Ang-1), and the effect of
antibodies on the Ang-2:Tie2 interaction. These in vitro and
cell-based assays are described as follows.
A. Affinity ELISA
[0328] For the initial screening of candidate anti-Ang-2
antibodies, purified human Ang-2 (R and D Systems, Inc; catalog
number 623-AN; Ang-2 is provided as a mixture of 2 truncated
versions) or murine Ang-2 polypeptide (prepared as described above)
were used. For confirmatory binding assays, human Ang-2 was
obtained from conditioned media of human 293T cells transfected
with full length human Ang-2 DNA and cultured in serum free DMEM
containing about 50 micrograms per ml of bovine serum albumin
(BSA).
[0329] Using microtiter plates, approximately 100 microliters per
well of Ang-2 was added to each well and the plates were incubated
about 2 hours, after which the plates were washed with phosphate
buffered saline (PBS) containing about 0.1 percent Tween-20 four
times. The wells were then blocked using about 250 microliters per
well of about 5 percent BSA in PBS, and the plates were incubated
at room temperature for about 2 hours. After incubation, excess
blocking solution was discarded, and about 100 microliters of
candidate anti-Ang-2 antibody was added to each well in a dilution
series starting at a concentration of about 40 nanomolar and then
serially diluting 4-fold in PBS containing about 1 percent BSA. The
plates were then incubated overnight at room temperature. After
incubation, plates were washed with PBS containing about 0.1
percent Tween-20. Washing was repeated four additional times, after
which about 100 microliters per well of goat anti-human IgG(Fc)-HRP
(Pierce Chemical Co., catalog #31416) previously diluted 1:5000 in
PBS containing 1 percent BSA (bovine serum albumin) was added.
Plates were incubated approximately 1 hour at room temperature.
Plates were then washed five times in PBS containing about 0.1
percent Tween-20, after which about 100 microliters per well of TMB
(3,3',5,5'-Tetramethylbenzidine Liquid Substrate System; Sigma
chemical Company, St. Louis, Mo., catalog number T8665) substrate
was added and plates were incubated about 5-15 minutes until blue
color developed. Absorbance was then read in a spectrophotomer at
about 370 nm.
B. Neutralization ELISA
[0330] Microtiter plates to which human Ang-2 polypeptide was bound
were prepared as described for the Affinity ELISA. Candidate
anti-Ang-2 antibodies were prepared in serial dilutions as
described for the Affinity ELISA above in a solution of PBS
containing about 1 percent BSA and about 1 nM Tie2 (provided as a
Tie2-Fc molecule where the Tie2 portion contains only the soluble
extracellular portion of the molecule; R and D Systems, catalog
number 313-TI). After about 100 microliters of the antibody/Tie2
solution was added to each well, the plates were incubated
overnight at room temperature, and then washed five times in PBS
containing about 0.1 percent Tween-20. After washing, about 100
microliters per well of anti-Tie2 antibody (Pharmingen Inc.,
catalog #557039) was added to a final concentration of about 1
microgram per ml and the plates were incubated about 1 hour at room
temperature then washed five time in PBS containing about 0.1
percent Tween-20. Next, about 100 microliters per well of goat
anti-mouse-IgG-HRP (Pierce Chemical CO., catalog #31432) was added
at a dilution of 1:10,000 in PBS containing about 1 percent BSA.
Plates were incubated at room temperature for about 1 hour, after
which they were washed five times with PBS containing about 0.1
percent Tween-20. About 100 microliters per well of TMB substrate
(described above) was then added and color was allowed to develop.
Absorbance was then read in a spectrophotomer at 370 nm.
C. Affinity BIAcore
[0331] An affinity analysis of each candidate Ang-2 antibody was
performed on a BIAcore.RTM.2000 (Biacore, Inc., Piscataway, N.J.)
with PBS and 0.005 percent P20 surfactant (BIAcore, Inc.) as
running buffer. Recombinant Protein G (Repligen, Needham, Mass.)
was immobilized to a research grade CM5 sensor chip (Biacore, Inc.)
via primary amine groups using the Amine Coupling Kit (Biacore,
Inc.) according to the manufacturer's suggested protocol.
[0332] Binding assays were carried out by first attaching about 100
Ru of each candidate anti-Ang-2 antibody to the immobilized Protein
G, after which various concentrations (0-100 nM) of huAng-2 or
mAng-2 were then injected over the bound antibody surface at a flow
rate of about 50 ul/min for about 3 minutes. Antibody binding
kinetics including k.sub.a (association rate constant), k.sub.d
(dissociation rate constant) and K.sub.D (dissociation equilibrium
constant) were determined using the BIA evaluation 3.1 computer
program (BIAcore, Inc.). Lower dissociation equilibrium constants
indicated greater affinity of the antibody for Ang-2.
[0333] All twenty two of the antibodies and a negative control IgG1
(referred to as RDB1) were tested using affinity and neutralization
ELISA (as described in Example 3 above) as well as the BIAcore
neutralization assay to determine their affinity, neutralization,
and specificity capabilities. The results are set forth below
(Table 2) and were calculated using standard procedures. Three
antibodies, H6L7, H4L4 and H4L11 were evaluated for IC50
neutralizing concentrations against human and murine Ang-1 and
Ang-2., using the ELISA analysis described above. All three
antibodies were shown to crossreact with mouse, rabbit, and
cynomolgus monkey Ang1 and Ang2, exhibiting similar potencies
across angiopoietin orthologs. The results are reported in the
following Table 3.
D. HTRF hAng-1 and hAng-2 Antibody IC50s and IC90s
[0334] Equal volume of 1.6 nM Streptavidin-Europium (SA-EU) and 8
nM Biotinylated angiopoietin 2 (in-house) or Biotinylated
agiopoietin 1 (R&D Cat# BAF923) were mixed and incubated at
room temperature for 30 minutes in the dark with rotation in a 15
ml conical tube (Fisher 352096). Then 50 ul of the above
SA-EU/Biotinylated Ang 2(1) mixture was added to each well on a
Mixing Plate (Costar 3356). To the Mixing Plate, 50 ul of serial
diluted Ang1 and Ang2 antibody at 4.times. final concentrations
were added to each well. The Mixing Plate was then incubated at
room temperature for 1 hour on a shaker in the dark. On an Assay
Plate (Costar 3356), 20 ul of 10 nM huTie-2-Fc-APC (Prozyme Custom
Lot# DF99-048) was added to each well. Then 20 ul of the mixture
from each well on the Mixing Plate was transferred to each well on
the Assay Plate. The Assay Plate was incubated at room temperature
for 2 hours in the dark with rotation. Then the Assay Plate was
read on RUBYstar plate reader (BMG labtechnologies, INC). All the
reagents in the assay were diluted with HTRF buffer (50 mM Tris
HCl, 100 mM NaCl, 0.1% BSA and 0.05% Tween 20). IC50 and IC90 were
calculated with GRAFIT 5.0.
TABLE-US-00010 TABLE 6 Biochemical Potency of Antibodies Against
hAng1 and hAng2 IC50 IC90 IC50 IC90 hAng1 hAng1 IC90/ hAng2 hAng2
IC90/ Antibody IC50 IC50 H6L7 0.06 0.49 8.0 0.06 0.19 3.3 H5L7 0.07
0.42 6.3 0.07 0.23 3.6 H4L13 0.15 1.6 11 0.06 0.19 3.2 H11L7 0.15
1.2 8.1 0.06 0.20 3.2 H10L7 0.15 2.2 14 0.06 0.19 3.4 H4L7 0.23 2.8
12 0.06 0.22 3.5 H5L6 0.32 3.6 11 0.07 0.23 3.4 H2L7 0.33 3.6 11
0.06 0.20 3.3 H5L8 0.37 3.6 10 0.07 0.21 3.1 H6L8 0.57 7.7 13 0.05
0.19 3.6 H3L7 0.58 7.1 12 0.06 0.23 4.0 H5L4 0.60 11 19 0.07 0.21
2.8 H4L12 0.63 8.7 14 0.06 0.21 3.4 H6L6 0.66 10 16 0.06 0.20 3.4
H4L2 0.66 6.8 10 0.06 0.19 3.1 H4L6 0.74 15 20 0.06 0.20 3.2 H4L4
0.87 8.2 9.4 0.06 0.16 2.7 H5L11 0.97 18 18 0.08 0.25 3.3 H5L1 1.7
24 15 0.06 0.29 4.4 AMG 386* 2.6 106 41 0.03 0.13 4.1 AMG 386* 3.9
278 71 0.03 0.15 4.4 H4L11 7.3 107 15 0.05 0.17 3.4 H5L12 14 159 11
0.07 0.31 4.4 H5L9 18 181 10 0.19 1.57 8.5 *Peptibody. indicates
data missing or illegible when filed
Example 3
Molecular Characterization of Angiopoietin Antibodies
[0335] Four of the fully human IgG2 antibodies (Ab536, H4L4, H6L7,
and H4L11) with potent hAng2 inhibitory activity and a range of
hAng1 inhibitory activities were selected for further studies. All
4 antibodies were shown to crossreact with mouse, rabbit, and
cynomolgus monkey Ang1 and Ang2, exhibiting similar potencies
across angiopoietin orthologs (Tables 7 and 8).
TABLE-US-00011 TABLE 7 Biochemical Potency of Angiopoietin
Antibodies Against Ang2 Orthologs Human Ang2 Cyno Ang2 Murine Ang2
Rabbit Ang2 Clone IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM) H6L7 0.22
0.19 0.13 0.15 H4L4 0.22 0.24 0.15 0.15 AMG 386 0.12 0.17 0.10 0.10
H4L11 0.21 0.16 0.12 0.12 536 LC1 0.27 0.19 0.14 0.20
ELISA measuring neutralization of ligand/receptor interaction.
TABLE-US-00012 TABLE 8 Biochemical Potency of Angiopoietin
Antibodies Against Ang1 Orthologs Human Ang1 Cyno Ang1 Murine Ang1
Rabbit Ang1 Clone IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM) H6L7 0.12
0.19 0.12 0.18 H4L4 3.2 4.0 3.3 2.8 AMG 386 1.2 2.9 2.7 4.3 H4L11
17 14 7.7 16 536 LC1 515 531 305 502
ELISA measuring neutralization of ligand/receptor interaction.
Example 4
Activity of Angiopoietin Antibodies in Colo205 Tumor Xenografts
[0336] Three antibodies (H6L7, H4L4 and H4L11) were evaluated in
the Colo205 human colorectal carcinoma xenograft model. For each
study group, mice were injected subcutaneously on the right flank
with 2.times.106 cells in Matrigel.TM.. Ten animals with average
tumor volume of 300 mm3 were randomly assigned to each experimental
group. The animals were injected IP twice per week, beginning on
day 17 post implantation, with 300 .mu.g of the
angiopoietin-targeted antibodies or isotype control antibody. AMG
386 at the optimum biological dose (OBD) in this model of 14 .mu.g
(SC) twice weekly was included as a positive control and antibody
536LC1 was included at 300 .mu.g twice weekly. Body weight and
tumor size were measured twice weekly.
[0337] As shown in FIG. 1, all three antibodies significantly
inhibited tumor growth compared to treatment with an isotype
control antibody (p<0.0001). Treatment with H6L7 and H4L4,
resulted in significantly greater inhibition of tumor growth
compared to 536LC1. Data represents mean.+-.SEM. At the end of the
experiment, tumors were harvested, fixed in zinc-formalin and
paraffin embedded. Histological sections of tumor were stained with
hematoxylin. The viable tumor fraction was then estimated, using
RGB thresholding and automated pixel counting, from a 1.times.
digital image of the entire tumor cross-section. Viable tumor
burden was calculated as the viable fraction multiplied by the
terminal tumor weight. Data represents mean.+-.SEM (n=10). FIG. 2
demonstrates that antibodies H6L7, H4L4 and H4L11 also
significantly reduced tumor burden relative to control
(p<0.0001), suggesting that the volume-based tumor measurements
underestimated the anti-tumor effect of the antibodies.
[0338] Histopathology was performed on tumors and normal tissues
from the mice in the study shown in FIGS. 1 and 2. Treatment of
xenograft-bearing nude mice with the angiopoietin inhibitors (AMG
386, 536LC1, H4L4, H4L11, or H6L7) did not elicit adverse anatomic
effects in non-target tissues.
Example 5
Effect of Anti-Ang-1 and/or Ang-2 Antibodies on Endothelial Cell
Proliferation
[0339] In a parallel experiment, animals with approximately 400
mm.sup.3 tumors were treated with 536LC1 (AKA LC1), H4L11, H4L4,
H6L7, AMG 386 or control IgG2 for 72 hrs. Seventeen hours prior to
sacrifice, animals were implanted with osmotic minipumps containing
3 mg/mL BrdU. Upon sacrifice, endothelial cells were isolated from
the Colo205 tumor-bearing mice and were analyzed by flow cytometry
to assess proliferation. Dissociated cells were stained with
anti-mouse CD45-FITC and CD31-PE antibodies, followed by fixation
and staining with anti-BrdU-alexa647 antibodies. Data represents
mean.+-.SEM (n=5). As shown in FIG. 3, treatment with all
antibodies significantly reduced the percentage of BrdU positive
cells (p<0.002 compared to IgG2 control). These data are
consistent with an anti-angiogenic therapeutic mechanism whereby
the angiopoietin-targeted antibodies inhibit tumor endothelial cell
proliferation in vivo.
Example 6
Dose Titration of H4L4 in Colo205 Tumor Xenografts
[0340] The antibody H4L4 was selected for more extensive analysis
exploring the dose dependency of H4L4-mediated tumor growth
inhibition. The animals were injected with H4L4 IP twice-weekly
beginning on day 14 at doses ranging from 3 .mu.g to 300 .mu.g. AMG
386 at the optimum biological dose of 14 .mu.g (SC) twice weekly
was included as a positive control. As shown in FIG. 4, all doses
of H4L4 significantly inhibited tumor growth and viable tumor
burden (p<0.0001), with an OBD of .about.30 .mu.g in the viable
tumor burden analysis (FIG. 5).
Example 7
Effect of H4L4 on Colo205 Tumor Endothelial Cell Proliferation In
Vivo
[0341] In a parallel experiment, Colo205 tumor-bearing mice with
tumors of approximately 450 mm.sup.3 were treated with a single
dose of H4L4, AMG 386 or control IgG2 for 72 hours and then
analyzed as in FIG. 3. As shown in FIG. 6, treatment with H4L4
significantly inhibited endothelial cell proliferation in a
dose-dependent manner, with an OBD of 30 .mu.g.
Example 8
Pharmacokinetics of H4L4, H6L7, H4L11, and 536LC1 in Mice, Rats and
Cynomologus monkey
[0342] The pharmacokinetics (PK) of H4L4, H6L7, H4L11, and 536LC1
have been characterized in CD-1 mice after single-dose intravenous
(IV) or intraperitoneal (IP) administration. The PK of H4L4 and
H6L7 was also characterized in Sprague-Dawley rats and cynomolgus
monkeys after single-dose IV administration.
[0343] After single-dose IV or IP administration to mice, H4L4
exposure appeared to increase approximately dose-proportionally in
the dose range of 0.1 to 10 mg/kg (Table 4). The overall mean
terminal half-life (t.sub.1/2,z), clearance (CL), and volume of
distribution at steady-state (V.sub.ss) was 207 hrs, 0.43 mL/hr/kg,
and 128 mL/kg, respectively. The bioavailability (% F) after IP
administration was greater than 90% for all dose groups. In
contrast, H6L7, H4L11, and 536LC1 exhibited nonlinear PK in mice
with exposure increasing greater than dose proportionally from 0.1
to 10 mg/kg. The exposure of H6L7 in rats and monkeys also
increased greater than dose-proportionally after a single IV dose
of 0.1 to 10 mg/kg.
[0344] In contrast to its linear PK profile in mice, H4L4 exhibited
nonlinear rat and monkey PK. The mean residence time (MRT) in rats
ranged from 57 to 217 hours; the CL ranged from 0.3 to 1.4
mL/hr/kg; the V.sub.ss ranged from 57 to 68 mL/kg. In monkeys, the
MRT ranged from 40 to 163 hours; the CL ranged from 0.4 to 1.9
mL/hr/kg; the V.sub.ss ranged from 49 to 75 mL/kg.
[0345] The PK of H4L4 was also assessed in nude mice bearing
Colo205 tumor xenografts in a pharmacology study at 3, 10, 30, 100
or 300 .mu.g dose/mouse, administered IP twice weekly for 4 weeks.
Serum H4L4 exposure increased approximately dose proportionally as
assessed by serum trough concentrations. The PK of H4L4 in nude
mice was similar to that observed in CD-1 mice, and PK did not
appear to change over time.
TABLE-US-00013 TABLE 9 PK Parameters of H4L4 and H6L7 in
Preclinical Species Mouse Rat Monkey 0.1 1 10 0.1 1 10 0.1 1 10
H4L4 Dose (mg/kg) t.sub.1/2,z (hr) 196 180 244 42.5 66.7 213 36.2
35.6 80.1 MRT (hr) 259 273 420 57.4 107 217 40.1 52.0 163 CL
(mL/hr/kg) 0.666 0.386 0.249 1.39 0.552 0.319 1.89 0.933 0.414
V.sub.ss (mL/kg) 173 105 105 66.4 57.0 68.5 74.9 48.6 67.4 V.sub.0
(mL/kg) 71.0 46.6 46.7 43.3 34.5 36.1 48.2 40.8 44.0 H6L7 Dose
(mg/kg) t.sub.1/2,z (hr) 8.27 99.0 82.8 9.43 25.8 92.9 10.4 32.6
65.0 MRT (hr) 11.6 54.3 263 13.2 41.2 158 15.1 51.8 158 CL
(mL/hr/kg) 19.4 2.00 0.349 3.31 1.02 0.331 2.81 0.824 0.358
V.sub.ss (mL/kg) 224 109 91.8 43.3 41.3 51.9 41.3 42.2 55.2 V.sub.0
(mL/kg) 90.9 58.4 56.6 35.4 35.3 38.1 40.0 37.7 43.1
Example 9
Angiopoietin-1 Neutralization Mediates Context-Dependent
Suppression of Angiogenesis and Tumor Growth
[0346] While Angiopoietin-2 (Ang2) is a key mediator of postnatal
angiogenesis, the role of Angiopoietin-1 (Ang1) in this setting is
less clear. To investigate the postnatal function of Ang1, we have
developed potent and selective peptibodies (peptide-Fc fusion
proteins) that inhibit the interaction between Ang1 and its
receptor, Tie2. We show that selective Ang1 antagonism has no
independent effect in models of angiogenesis-associated diseases
(cancer and diabetic retinopathy), although it can induce ovarian
atrophy in normal juvenile rats and inhibit ovarian follicular
angiogenesis in a hormone-induced ovulation model. Surprisingly,
the activity of Ang1 inhibitors appears to be unmasked in some
disease models when combined with Ang2 inhibitors. Dual inhibition
of Ang1 and Ang2 cooperatively suppresses ovarian follicular
angiogenesis and tumor xenograft growth; however, Ang1 inhibition
fails to augment the activity of Ang2 inhibition in suppressing
tumor endothelial cell proliferation, corneal angiogenesis, and
oxygen-induced retinal angiogenesis. In no case was Ang1 inhibition
shown to 1) confer superior activity to that of Ang2 inhibition or
dual Ang1/Ang2 inhibition or 2) antagonize the effects of Ang2
inhibition. These results imply that Ang1 plays a context-dependent
role in promoting postnatal angiogenesis and
angiogenesis-associated pathology. Ang1 plays an important role in
developmental angiogenesis, but its function in postnatal
neovascularization is less clear. Ang1 has been shown to mediate
both pro- and anti-angiogenic effects in various postnatal
settings. To investigate the function of Ang1 by inhibiting
endogenous Ang1. To that end, we have developed Ang1-neutralizing
peptibodies and tested them alone or in combination with Ang2
inhibitors in preclinical models of postnatal angiogenesis.
[0347] We generated Ang1-neutralizing peptibodies to investigate
the functional role of Ang1 in angiogenesis. Phage display peptide
libraries were panned to identify peptides that bound Ang1, but not
Ang2. The resulting clones were converted into peptibodies by
expressing the peptides in E. coli as fusions to the Fc portion of
human IgG1. Peptibodies were then screened by enzyme-linked
immunosorbent assay (ELISA) and homogeneous time-resolved
fluorescence (HTRF) assays for their ability to neutralize the
interaction between Tie2 and angiopoietins. One of these
peptibodies was affinity-matured to increase its ability to
antagonize Ang1, and a resultant peptibody, mL4-3, was chosen for
the studies herein. mL4-3 exhibited similar potency against several
Ang1 orthologs, and it displayed>40,000-fold selectivity over
Ang2 (Tables 10 and 11). Also shown in Table 10 are two previously
described peptibodies: AMG 386 [also known as 2xCon4 (C)] and
L1-7(N). L1-7(N) is a very potent and selective Ang2 inhibitor, and
AMG 386 is a dual inhibitor of Ang1 and Ang2. The pharmacokinetic
profiles of mL4-3 in rodents were acceptable for daily to weekly
s.c. dosing (Table 3).
[0348] mL4-3 can be used as a reagent for interrogating Ang1
function in vivo. To assess whether mL4-3 was capable of
selectively sequestering Ang1 in vivo, mL4-3, L1-7(N), and Fc were
administered s.c. to mice, followed by an i.v. challenge with
recombinant Ang1. Ang1 induced Tie2 phosphorylation in mouse lung
endothelium (approximately 5-fold), an effect that could be
prevented by mL4-3, but not by L1-7(N) or Fc (FIG. 7).
[0349] Next, we wanted to determine whether mL4-3 could neutralize
endogenous Ang1 in a setting in which Ang1 was known to play a
physiologically relevant role. Developmental genetic knockout
studies have shown that Ang1 deletion reduces cardiac size and
endocardial folding in embryos. In an attempt to replicate this
phenotype pharmacologically, mL4-3 was administered to pregnant
mice in early and middle gestation. Embryos were harvested at
embryonic day 12.5, the time at which lethality was observed in
Ang1-null mouse embryos. Pharmacokinetic assessment of mouse embryo
lysates demonstrated a mean mL4-3 trough level of 3.0 .mu.g/g of
tissue, confirming that mL4-3 was capable of crossing the placenta.
Histological analysis revealed reduced cardiac size and
trabeculation, similar to, but less dramatic than that observed in
Ang1-null embryos (FIG. 8). The less pronounced phenotype of the
mL4-3 treated embryos may be a consequence of suboptimal embryonic
mL4-3 exposures and incomplete Ang1 sequestration. Nonetheless,
mL4-3 clearly induces embryonic cardiac defects that phenocopy
those of Ang1 genetic knockout mice, confirming the utility of
mL4-3 as a reagent for investigating Ang1 function in vivo.
[0350] Ang1 antagonism augments Ang2 antagonism in suppressing
tumor growth. In a previous report, we demonstrated that
systemically administered L1-7(N) and AMG 386 were capable of
inhibiting the growth of Colo205 tumor xenografts implanted into
nude mice. In that study, the antitumor effects of AMG 386 were
modestly superior to those of L1-7(N) (P=0.006). To confirm that
dual Ang1/Ang2 inhibition confers better tumor growth suppression
than Ang2 inhibition alone, a similar experiment was performed, but
this time groups treated with mL4-3 or a combination of mL4-3 and
L1-7(N) were also tested (FIG. 9). The AMG 386 treatment group and
the mL4-3/L1-7(N) combination treatment group showed comparable
antitumor efficacy; moreover, both groups exhibited efficacy
superior to that mediated by either L1-7(N) or mL4-3 alone. In
fact, mL4-3 had no discernable single-agent effect on tumor growth,
implying that combining Ang2 antagonism with Ang1 antagonism may
have unmasked the antitumor effect of Ang1 inhibition. Additional
replicates of these experiments confirmed that AMG 386 and the
mL4-3/L1-7(N) combination mediated greater tumor growth suppression
than L1-7(N) alone (data not shown). However, in a minority of
instances, these differences did not reach statistical
significance, perhaps reflecting the subtle nature of the
incremental advantage conferred by dual Ang1/Ang2 inhibition over
selective Ang2 inhibition. Selective Ang1 inhibition had no
antitumor effect on its own in any of the experiments in which it
was tested (FIG. 9 and data not shown).
[0351] Ang2 antagonism, but not Ang1 antagonism, inhibits tumor
endothelial cell proliferation, corneal angiogenesis, and retinal
angiogenesis. We previously showed that dual Ang1/Ang2 inhibition
was capable of suppressing Colo205 tumor endothelial cell
proliferation in vivo. To investigate whether this effect was
conferred through Ang1 inhibition, Ang2 inhibition, or a
combination of the two, Colo205 tumor-bearing mice were treated
with mL4-3, L1-7(N), mL4-3/L1-7(N), or AMG 386. As with the tumor
volume readout described in the previous section, mL4-3 had no
single-agent effect on tumor endothelial cell proliferation, while
L1-7(N) was inhibitory (FIG. 10A). Curiously, however, dual
Ang1/Ang2 inhibition conferred no greater effect on endothelial
cell proliferation than Ang2 inhibition alone (FIG. 10A), an
observation that has been repeatedly reproduced (data not shown)
and stands in contrast to the apparently cooperative effects of
combined Ang1/Ang2 inhibition on Colo205 tumor growth. This
dissimilarity implies that repression of endothelial cell
proliferation is only one component underlying the tumor growth
inhibition mediated by angiopoietin antagonism.
[0352] These agents were next tested in two models of ocular
angiogenesis, one involving the cornea and the other involving the
retina. The cornea is normally avascular, but pathological
angiogenesis can occur in the cornea secondary to conditions such
as keratitis and corneal transplant rejection. VEGF- and basic
fibroblast growth factor (bFGF)-induced models of corneal
angiogenesis were used to test the roles of Ang1 and Ang2
antagonism in neovessel formation. As observed with endothelial
cell proliferation, corneal angiogenesis appeared to be dependent
on Ang2, but not on Ang1 (FIGS. 10B and 10C). The same conclusion
could be drawn from evaluation of these angiopoietin-antagonizing
peptibodies in a Tie2-dependent retinal model of angiogenesis in
which neovascularization was induced by changes in ambient oxygen
tension (FIG. 10D). Thus, in three preclinical settings
(endothelial cell proliferation, corneal angiogenesis, and retinal
angiogenesis), Ang2 inhibition dramatically suppressed neovessel
formation, while Ang1 inhibition had no effect alone or in
combination with Ang2 inhibition.
[0353] Selective inhibition of Ang1 or Ang2 induces ovarian
atrophy, but not epiphyseal plate thickening. To assess the effects
of angiopoietin inhibition in normal animals, rats were treated
systemically with mL4-3, L1-7(N), or AMG 386 for one month. AMG
386, like VEGF antagonists, has been observed to induce epiphyseal
plate thickening and ovarian atrophy, effects considered to be
mechanism-based consequences of antiangiogenic therapy. In the
present study, AMG 386 provoked epiphyseal plate thickening in all
treated animals, while, remarkably, L1-7(N) and mL4-3 failed to
alter epiphyseal morphology in any rats (Table 13). Thus, induction
of epiphyseal plate thickening appears to require inhibition of
both Ang1 and Ang2. In striking contrast, all three peptibodies
produced ovarian atrophy at similar incidence rates, indicating
that selective inhibition of Ang1 or Ang2 is sufficient to induce
ovarian atrophy.
[0354] Ang1 and Ang2 Inhibitors Cooperatively Suppress Ovarian
Follicular Angiogenesis. To better understand the effects of
angiopoietin inhibition on the ovary, we employed a hormone-induced
model of ovarian follicular angiogenesis that allowed controlled
assessment of neovascularization in mice that had never previously
ovulated. In this model, pregnant mare serum (PMS) and human
chorionic gonadotropin (HCG) were used to induce rapid,
synchronized ovulation in multiple follicles (FIG. 11). Mice were
treated systemically with Fc control, mL4-3, L1-7(N), or an
mL4-3/L1-7(N) combination to determine the effects of these agents
on neovessel formation in transforming Graafian follicles. Two
identically-designed replicates of this experiment were performed
on different days, and remarkably, both yielded almost identical
activity profiles with respect to percentage inhibition of blood
vessel area (replicate 1, replicate 2): L1-7(N) (8%, 11%), mL4-3
(15%, 14%), mL4-3/L1-7(N) (24%, 26%). All single-agent and
combination peptibody groups, with the exception of the L1-7(N)
group in Experiment 1, mediated statistically significant
inhibition of angiogenesis relative to the Fc control (P<0.05)
(FIG. 11). Thus, both inhibition of ovarian angiogenesis and
induction of ovarian atrophy could be elicited by inhibiting Ang1,
Ang2, or both, consistent with the notion that the observed ovarian
atrophy was a consequence of failed neovessel development.
[0355] We demonstrate that Ang1 inhibition plays a
context-dependent role in the suppression of angiogenesis in
preclinical disease models and in normal animals. In utero,
pharmacologic Ang1 inhibition partially phenocopied the genetic
ablation of Ang1, consistent with the important role of Ang1 in
developmental angiogenesis. Postnatally, selective Ang1 antagonism
inhibited ovarian angiogenesis and induced ovarian atrophy, effects
that could also be achieved by inhibiting Ang2 alone or Ang1 plus
Ang2 together. However, in postnatal disease models, Ang1
inhibition had little effect on its own, although its biological
activity appeared to be unmasked in some settings when combined
with Ang2 suppression. The mechanism underlying the differential
dependency on Ang1 in these settings remains to be determined
[0356] The ovary, by virtue of its role in reproductive cycling, is
one of the few organs that undergoes normal angiogenesis in adults.
Based on the ovarian expression patterns of Ang1 and Ang2 in
hormone-induced ovulating rats, it has been proposed that Ang2
plays an early role in vessel invasion, and Ang1 plays a later role
to mature the newly-formed vessels. Under this hypothesis, Ang2 and
Ang1 perform opposing functions, where Ang2 initially displaces
Ang1 from Tie2, resulting in vessel destabilization and
angiogenesis. This state of plasticity is subsequently reversed
when Ang1 ousts Ang2 from the receptor to re-establish vascular
quiescence and stability. In conflict with this model, the data
from the current study imply that Ang1 and Ang2 both play
pro-angiogenic roles in the ovary.
[0357] In the Colo205 tumor xenograft model, antagonism of Ang1 and
Ang2 mediated greater tumor suppression than was achieved by
inhibiting Ang1 or Ang2 individually, indicating that this model is
dependent on both angiopoietins. However, in the same model, only
Ang2 inhibition was capable of down-modulating tumor endothelial
cell proliferation, suggesting that Ang1 is not involved in this
function. What accounts for the different dependencies of these two
endpoints on Ang1? One possibility is that Ang1 inhibition has a
direct effect on tumor cells. This seems unlikely, however, given
that AMG 386, a dual inhibitor of Ang1 and Ang2, has no effect on
the in vitro growth of cultured Colo205 tumor cells. A second
possibility is that Ang1 antagonism plays an anti-angiogenic role
that is not conferred through inhibition of endothelial cell
proliferation, but instead through mechanisms that might impact
functions such as endothelial cell migration or invasion. This
explanation could be applicable if Ang1 and Ang2 mediated
qualitatively or quantitatively different signals through Tie2, or
if Ang1 signaled through additional receptors that were not
responsive to Ang2. A third possibility is that Ang1 signals
through Tie2 on non-endothelial cells, such as Tie2-expressing
monocytes (TEMs). TEMs are recruited to tumors, where they cluster
around neovessels. Selective ablation of TEMs in tumor-bearing mice
suppresses tumor angiogenesis and inhibits tumor growth, and it has
been postulated that TEMs promote tumor angiogenesis by providing
paracrine signals that stimulate neovessels. Perhaps Ang1
stimulates TEMs to release pro-angiogenic cytokines other than the
angiopoietins. In such a setting, inhibition of Ang1 could have an
indirect anti-neovascular effect that might complement the direct
anti-angiogenic effect of Ang2 suppression.
[0358] In contrast to the subtle and context-dependent effects of
Ang1 inhibition, Ang2 inhibition frequently mediated effects that
were equivalent or nearly equivalent to those conferred by combined
antagonism of Ang1 and Ang2, implying that Ang2 may be the dominant
angiopoietin involved in postnatal angiogenesis. Ang1 appears to be
the dominant angiopoietin involved in prenatal angiogenesis,
suggesting a shift in the dependency on these two factors around
the time of birth. Our inhibitors do not antagonize Ang4, but the
functional relevance of this factor is unclear, given its
lung-restricted expression pattern.
[0359] Ang1 and Ang2 have been shown to play both similar and
opposing functional roles in various in vitro and in vivo systems.
The inability to draw consistent conclusions in this regard across
multiple publications may be in part a consequence of the different
conditions under which the question was examined. These differences
include evaluation of 1) in vitro versus in vivo systems, 2)
prenatal versus postnatal angiogenesis, 3) varying vascular beds,
4) pathological versus normal angiogenesis, and 5) gain-of-function
versus loss-of-function experimental designs. This final difference
may be particularly important, as the addition of exogenous factors
to a model system may be a less physiologically relevant means to
elucidate function than removal of endogenous factors. Perhaps the
most informative published experiments in this regard are those in
which Ang1 and Ang2 have been genetically deleted in the germline
of rodents. These studies provide significant insight into the
developmental roles of Ang1 and Ang2. However, it is more difficult
to genetically examine the postnatal in vivo function of Ang1 and
Ang2 without the availability of conditional knockout systems; the
constitutive Ang1 knockout mouse dies in utero (as does the
constitutive Ang2 knockout on some strain backgrounds), and the
postnatal phenotype of surviving Ang2 knockout mice may be
influenced by residual effects of developmental gene deletion. By
using pharmacologic Ang1 and Ang2 inhibitors to examine the
postnatal roles of Ang1 and Ang2 in vivo, we have circumvented
these issues. The results of the current study imply that Ang1 and
Ang2 do not functionally oppose one another in postnatal systems,
and in some cases, they appear to act cooperatively.
[0360] Pathological angiogenesis is associated with altered
angiopoietin levels in a number of diseases, including cancer,
diabetic retinopathy, macular degeneration, rheumatoid arthritis,
osteoarthritis, and psoriasis. Angiopoietin-targeted interventions
in these therapeutic indications may provide clinical benefit. The
data presented herein suggest that, in some settings, combined
inhibition of Ang1 and Ang2 may provide superior therapeutic
efficacy to that mediated by targeting Ang2 alone.
Methods
Phage Display Selection of Ang1-Binding Peptides.
[0361] Three filamentous phage libraries, TN8-IX (5.times.10.sup.9
independent transformants), TN12-I (1.4.times.10.sup.9 independent
transformants), and Linear (2.3.times.10.sup.9 independent
transformants) (Dyax Corp., Cambridge, Mass.), were used to select
for Ang1-binding phage. After negative selection on empty
streptavidin Dynabeads (Invitrogen Corporation, Carlsbad, Calif.)
blocked with 2% bovine serum albumin (BSA) or beads loaded with
biotinylated Ang2 (R&D Systems, Inc., Minneapolis, Minn.),
remaining phage were incubated with beads loaded with biotinylated
Ang1 (R&D Systems, Inc.). After extensive washing, the phage
from each round of selection were eluted in a nonspecific manner
using 100 mM triethylamine solution (Sigma-Aldrich Inc., St. Louis,
Mo.). The eluted phage were amplified in E. coli strain XL-1 Blue
MRF', purified by precipitation, and then used for the next round
of selection.
[0362] After three rounds of selection, individual phage clones
were isolated and analyzed by phage ELISA and DNA sequencing.
Briefly, Ang1 protein was coated on 96-well Maxisorp plates (Nunc
brand, Thermo Fisher Scientific, Rochester, N.Y.) and blocked with
PBST (PBS with 0.05% Tween-20) containing 4% dry milk. Phage
supernatants were incubated in the wells and bound phage were
detected with an HRP-conjugated anti-M13 antibody (Amersham
Pharmacia Biotech, Piscataway, N.J.). To check cross-reactivity to
Ang2 or streptavidin, control plates were set up in a similar
fashion. ELISA results and DNA sequencing data were used as
criteria for selecting peptide sequences to express in a peptibody
format. Peptibodies were evaluated in an HTRF assay, and several
were chosen for affinity maturation.
[0363] Peptide affinity maturation was performed by generating and
panning nucleotide-doped phage display libraries. Libraries with
over 1.times.10.sup.9 independent transformants were obtained.
These focused libraries were panned by a procedure similar to that
used for panning the primary libraries.
Peptibody Expression and Purification.
[0364] Peptibody mL4-3 was expressed and purified as described in
Oliner, J., et al. 2004., Cancer Cell 6:507-516. The amino acid
sequence of mL4-3 is as follows, where Fc in bold italics denotes
the human IgG1 Fc sequence as described previously in Oliner, J.,
et al. 2004., Cancer Cell 6:507-516:
TABLE-US-00014 (SEQ ID NO: 47)
MREWTEEMQVIFDAMMFGPRNDRGGSGSATGSGSTASSGSGSATHREWTE
EMQVIFDAMMFGPRNDRGGGGG-
[0365] The amino acid sequence of the Fc portion of the peptibody
mL4-3 is as follows (from amino terminus to carboxyl terminus):
TABLE-US-00015 (SEQ ID NO: 48)
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK
Angiopoietin: Tie2 Neutralization HTRF Assay.
[0366] Europium-labeled streptavidin (LANCE reagent, PerkinElmer
Inc., Boston, Mass.) and biotinylated human Ang1 (R&D Systems,
Inc.) or Ang2 were mixed in HTRF buffer (50 mM Tris-HCl, pH 7.5,
100 mM NaCl, 0.05% Tween 20, 0.1% BSA) and incubated at room
temperature in the dark for 30 minutes on a shaker. Equal volumes
of the above mixture and serially diluted peptibodies or Fc were
mixed and incubated for 1 hour at room temperature. Equal volumes
of allophycocyanin-conjugated Tie2-Fc (Tie2-APC) (Prozyme, San
Leandro, Calif.) and the above mixture were mixed and incubated for
2 hours at room temperature. The final concentrations of reagents
in the assay were 4 nM europium-streptavidin, 2 nM biotinylated
Ang1 or Ang2, and 5 nM Tie2-APC. Peptibodies were serially diluted
from 10,000 nM to 0.5 nM or 100 nM to 0.005 nM to generate full
titration curves. Neutralization of angiopoietin:Tie2 interaction
was measured by the diminishing energy transfer between APC and
europium and was quantified using a Rubystar plate reader (BMG
Labtechnologies, Offenberg, Germany). The potency of
angipoietin/Tie2 neutralization was determined by calculating the
percentage inhibition of each peptibody dilution in reference to
the maximum (no angiopoietin in the assay mixture) and minimum
inhibition (no peptibody in the assay mixture) controls. IC.sub.50
values were calculated by plotting percentage inhibition using
XLfit4, where fit=A+((B-A)/(1+((C/X) D))) (IDBS, Guildford,
UK).
Angiopoietin:Tie2 Neutralization ELISA.
[0367] Ninety-six-well microtiter plates were coated with a panel
of recombinant angiopoietins in 293T cell conditioned media
(DMEM/50 ug/ml BSA) at 37.degree. C. for 1 hour. The conditioned
media were used at angiopoietin concentrations that conferred 70%
of maximally achievable binding to 1 nM hTie2-Fc (Recombinant
hTie2-Fc, Catalog #313-TI, R&D Systems Inc.). Plates were
washed three times with PBS/0.1% Tween-20 and then block for 2
hours at room temperature with PBS/5% BSA. The blocking solution
was removed without washing the plates. mL4-3 or Fc serially
diluted in a solution of 1 nM Tie2-Fc/1% BSA/PBS was added to the
angiopoietin-coated plates, which were incubated overnight at room
temperature and then washed with PBS/1% Tween-20. A mouse-derived
anti-Tie2 antibody (Catalog #557039, BD Pharmingen Inc., San Jose,
Calif.) was added to each well at a final concentration of 1 ug/ml
and incubated for 1 hour at room temperature. Plates were then
washed 3 times with PBS/0.1% Tween-20. Goat anti-mouse-IgG-HRP
(Horseradish peroxidase-conjugated goat anti-mouse antibody,
Catalog #31432, Pierce, Rockford, Ill.) was added at a dilution of
1:10,000 in PBS/1% BSA to each well and the plates were incubated
for 1 hour at room temperature. Plates were washed three times with
PBS/0.1% Tween-20 before TMB substrate (SureBlue Reserve TMB,
Catalog#53-00002, KPL, Gaithersburg, Md.) was added and optical
density at 650 nM was measured on a plate reader (SpectraMax,
Molecular Devices, Sunnyvale, Calif.). The degree of
angiopoietin:Tie2 neutralization (IC.sub.50) was determined by
comparison against a Tie2 standard curve (the binding activity of
serially diluted Tie2 in the absence of competitor) using
XLfit.
Animal Studies.
[0368] All procedures were approved by the Amgen Animal Care and
Use Committee and met Association for Assessment and Accreditation
of Laboratory Animal Care standards.
Pharmacokinetic Assessment.
[0369] Three CD-1 mice received a single s.c. injection of 3.2
mg/kg of mL4-3, and two Sprague-Dawley rats received a single i.v.
injection of 10 mg/kg of mL4-3. Blood samples were collected up to
274 hours from the mice and 336 hours from the rats for serum
pharmacokinetic assessment. mL4-3 concentrations in serum samples
from each species were measured by an enzyme-linked immunosorbent
assay (ELISA). Polystyrene 96-well plates were coated with human
Ang1, followed by incubation with mL4-3-containing serum samples.
After washing away any unbound substances, a horseradish
peroxidase-labeled monoclonal mouse anti-IgG1 antibody was added to
the wells. Following a wash step to remove any unbound monoclonal
antibody, TMB-peroxidase substrate was added to the wells. The
optical density units measured at 450-650 nm were converted to
concentrations by comparing to a concurrently analyzed standard
curve.
[0370] Pharmacokinetic parameters were calculated by
noncompartmental analysis of the individual serum
concentration-time data (WinNonlin Professional, version 3.3;
Pharsight Corp, Mountain View, Calif.). Terminal phase half-life
(t.sub.1/2) was calculated as t.sub.1/2=ln(2)/.lamda..sub.z, in
which .lamda..sub.z is the first-order terminal phase elimination
rate constant estimated via linear regression of the terminal
log-linear decay phase. Area under the serum concentration-time
curve (AUC.sub.0-last) was estimated by the linear/log trapezoidal
method from time 0 to the time of the last quantifiable
concentration (C.sub.last). AUC.sub.0-inf was estimated from time 0
to infinity as
AUC.sub.0-inf=AUC.sub.0-last+C.sub.last/.lamda..sub.z.
AUC.sub.0-inf values were normalized to a 1 mg/kg dose.
[0371] Because the pharmacokinetic properties of mL4-3, L1-7, and
AMG 386 were dissimilar, the dose levels and schedules of each
agent were chosen, where possible, to achieve equimolar serum
steady-state C.sub.min concentrations within pharmacology
studies.
Administration of mL4-3 to Pregnant Mice. Two groups of six 129/SV
female mice were impregnated by C57BL/6 males. Pregnant females
were dosed with 300 mg/kg Fc control or mL4-3 by s.c.
administration on gestational days E4.5, E7.5 and E11.5.
Conceptuses (embryos and placentae) were removed on day E12.5,
evaluated for gross abnormalities, and fixed by immersion in
IHC-zinc (mL4-3-treated, n=10; Fc control-treated, n=10) or Bouin's
solution (mL4-3-treated, n=5; Fc control-treated, n=6).
Paraffin-embedded tissues were step-sectioned at 50-1.1m intervals
through the heart (embryos in both longitudinal and transverse
orientation) and the middle of the placenta. Serial sections from
each interval were stained with hematoxylin and eosin (H&E) or
stained with a conventional indirect immunohistochemistry procedure
using polyclonal anti-CD31 (rat anti-mouse monoclonal MEC 13.3, BD
Biosciences Pharmingen, San Diego, Calif.) to specifically label
blood vessels. Criteria for scoring changes were established by
evaluating sections with a foreknowledge of the treatment.
Subsequently, lesion severity was graded rapidly using a tiered
scale (minimal, mild, moderate, or marked) and a blinded analytical
paradigm. These ordinal pathology data were analyzed using the
Chi-square test contained in the JMP statistical software package
(v.5.1; SAS Institute Inc., Cary, N.C.). An embryo from each
pregnant mother collected on day E12.5 was analyzed by ELISA using
human Ang1 as a capture reagent and horseradish peroxidase-labeled
monoclonal mouse anti-IgG1 antibody as a detection reagent.
Tie2 Phosphorylation Assay.
[0372] The effect of the selective angiopoietin inhibitors on
Ang1-induced Tie2 phosphorylation in mouse lungs was performed as
described in Hodous, B. L., et al. 2007., J. Med. Chem.
50:611-626). Briefly, CD-1 nude mice (Charles River Laboratories,
Wilmington, Mass.) were treated s.c. once daily for 23 days with Fc
control (20 mg/kg), mL4-3 (20 mg/kg) or L1-7(N) (2 mg/kg). Mice
(n=3 per group) were then administered 12 .mu.g by i.v. injection
of recombinant Ang1 (R&D Systems Inc.). Fifteen minutes later,
mouse lungs were harvested, and the levels of phosphorylated Tie2
were determined by immunoprecipitation-Western blot analysis.
Statistical analysis was performed using analysis of variance
(ANOVA) followed by Fisher's post hoc test using StatView 5.0.1
software (SAS Institute Inc.). Results are expressed as
mean.+-.standard error (SE).
Tumor Xenograft Models.
[0373] Eight- to 10-week old female CD1 nude mice (Charles River
Laboratories) were used in all experiments. Mice were injected s.c.
with 2.times.10.sup.6 Colo205 cells in one-third volume Matrigel
(BD Biosciences, San Jose, Calif.). Peptibodies or Fc control were
administered by s.c. injection once tumors were established. AMG
386 was dosed twice per week; the other peptibodies and Fc control
were dosed once daily. Where necessary, Fc control protein was
added to the treatment groups to match the total amount of protein
delivered in the combination group (5.2 mg/kg). Tumor measurements
and body weights were recorded twice per week. All tumor studies
were performed in a blinded fashion. Tumor volume was calculated as
length.times.width.times.height in mm.sup.3 Results are expressed
as mean.+-.SE. Statistical analysis was performed using repeated
measures analysis of variance followed by a Scheffe post hoc test
using StatView 5.0.1 software (SAS Institute Inc.).
Tumor Endothelial Cell Proliferation Assay.
[0374] Tumor endothelial cell proliferation was assayed as
previously described (Oliner, J., et al. 2004., Cancer Cell
6:507-516). Briefly, Colo205 tumor-bearing mice were treated
systemically with peptibodies for 72 hours and implanted with
osmotic pumps containing 3 mg/mL BrdU 16 hours prior to euthanasia.
Tumors were harvested, dissociated, fixed, and stained to allow
determination of BrdU incorporation in tumor endothelial cells.
Statistical analysis was performed using an unpaired t-test.
Corneal Angiogenesis Model.
[0375] VEGF and bFGF-induced angiogenesis studies were performed in
female CD rats (n=8 per group) as described in Coxon, A., et al.
Arthritis Rheum 46:2604-2612, 2002. Inhibition of interleukin-1 but
not tumor necrosis factor suppresses neovascularization in rat
models of corneal angiogenesis and adjuvant arthritis. Treatment
with Fc (60 mg/kg), L1-7(N) (5 mg/kg), mL4-3 (60 mg/kg), or the
combination of L1-7(N) and mL4-3 (at the same doses used in the
single-agent groups) was initiated on the day prior to surgery and
continued on day 3 and day 6. On day 8 the study was terminated and
the corneas were photographed, as described (Oliner, J., et al.
2004., Cancer Cell 6:507-516). For each corneal image, the number
of blood vessels intersecting the midpoint between the implanted
disc and the limbus was counted. All evaluations were performed in
a blinded fashion. Statistical significance was assessed by ANOVA
followed by Fisher's post hoc test.
Retinal Neovascularization.
[0376] Ischemic retinopathy was produced in C57BL/6J mice using the
method described by Smith et al., Invest. Ophthalmol V is Sci 35:
101-111, 1994. Postnatal day seven (P7) pups and their mothers were
placed in a hyperoxic chamber (75.+-.0.5% oxygen) for 5 days and
then returned to room air for an additional 5 days (n=7 pups per
group). Chamber temperature was maintained between 20.degree. C.
and 22.degree. C., and oxygen was constantly controlled by an
oxygen control unit (ProOx Model P110 coupled to an oxygen sensor
Model E702 Biospherix Ltd, Redfield, N.Y.). One cage with P7 pups
remained at room air (normoxia condition). Fc control (200 mg/kg),
mL4-3 (100 mg/kg), L1-7(N) (100 mg/kg), or mL4-3/L1-7(N)
combination (100 mg/kg each) was administered s.c. once daily for
nine days starting on P8. From P8 to P11 the injections were
administered using ports to gain access into the chamber. On P17
the pups were sacrificed and their eyes removed and fixed using
Davidson's fixative. The eyes were then processed into paraffin
using standard methods. Step sections were cut parallel to the
optical axis at 100-.mu.m intervals. The blocks were completely
through-sectioned, resulting in 15 or 16 sections per eye. All
sections were stained with H&E. Of the 15 or 16 slides in the
step-section series, the middle 10 consecutive slides were used in
the analyses, bracketing either side of the optical axis. For each
section, the number of vascular nuclei (both endothelial and
pericyte nuclei) that were on the vitreous side of the inner
limiting membrane were counted. The individual slide counts were
recorded and all ten section counts summed for each animal. Five
mice in each study group were counted. All counts were performed in
a blinded fashion, without knowledge of treatment conditions.
Statistical analysis was performed by ANOVA followed by Fisher's
post hoc test.
Ovarian Follicular Angiogenesis.
[0377] Superovulation was induced in study mice using standard
methodology. Briefly, four-week old female C57BL/6J mice were
injected with 5-7 IU PMS, effectively resetting the estrus cycle.
Forty-eight hours later, the mice were injected with 5 IU of HCG to
induce superovulation. The females were then faux-bred and remained
on study for 24 hours. Study mice were treated with peptibodies
twice per day. Dosing commenced at the time of the initial PMS
injection and continued for two consecutive days, with the fourth
dose given concurrently with the HCG injection. Mice were
euthanized 48 hours following the HCG injection. Right and left
ovaries were removed and immersion-fixed in cold zinc tris
solution. After 48 hours, ovaries were transferred to 70% ethanol
and processed to paraffin using standard methods. Two sequential
sections were cut from each ovary pair and individually stained
either with H&E or immunostained for vascular endothelium
(CD31, rat anti-mouse monoclonal MEC 13.3, BD Biosciences
Pharmingen) using DAB as the chromogen. Additionally, the anti-CD31
IHC sections were lightly counterstained with hematoxylin. The
individual follicles selected for analysis were identified based on
transformational state. This was determined by treatment-blind
inspection of the H&E sections under low power. Corresponding
images of ten transformed follicles per animal, where feasible,
were then captured at 10.times. objective magnification from the
anti-CD31 immunostained sections. The follicle section area was
delineated as a ROI, and the CD31-positive area fraction was
determined via RGB thresholding using MetaMorph image analysis
software (MetaMorph v6.1, UIC, Downingtown, Pa.). Statistical
analysis was performed by ANOVA followed by Dunnett's post hoc
test.
Evaluation of Normal Tissues in Treated Rats.
[0378] Peptibodies were evaluated in Sprague-Dawley rats (Charles
River Laboratories) for effects on normal tissues. Animals received
300 mg/kg of AMG 386, L1-7(N) or mL4-3 IV twice weekly for 28 days
(n=10 animals per group). At scheduled necropsy, a full tissue set
was sectioned, stained, and observed for microscopic changes.
TABLE-US-00016 TABLE 10 Peptibodies competitively inhibit
angiopoietin: Tie2 interactions hAng1 hAng2 Agent IC.sub.50 (nM)
IC.sub.50 (nM) L1-7(N) >10,000 0.064 mL4-3 0.022 3085 AMG 386
6.2 0.029 Fc >10,000 >10,000 h, human
TABLE-US-00017 TABLE 11 mL4-3 selectively neutralizes Ang1:Tie2
interactions hAng1 mAng1 rAng1 cAng1 hAng2 mAng2 cAng2 IC.sub.50
IC.sub.50 IC.sub.50 IC.sub.50 IC.sub.50 IC.sub.50 IC.sub.50 Agent
(nM) (nM) (nM) (nM) (nM) (nM) (nM) mL4-3 0.045 0.033 0.061 0.039
1876 >10,000 1890 Fc >10,000 >10,000 >10,000 >10,000
>10,000 >10,000 >10,000 h, human; m, mouse; r, rabbit; c,
cynomolgus monkey
TABLE-US-00018 TABLE 12 Mean pharmacokinetic parameters of
angiopoietin inhibitors in mice and rats Mouse Rat Dose-normalized
Dose-normalized t.sub.1/2 AUC.sub.0-inf t.sub.1/2 AUC.sub.0-inf
Agent (hr) (.mu.M hr/mg/kg) (hr) (.mu.M hr/mg/kg) mL4-3 45 5.0 42
3.6 L1-7(N).sup.a 56 7.0 47 4.6 AMG 386.sup.a 97 15 85 8.7
.sup.aAdapted from Oliner et al (18).
TABLE-US-00019 TABLE 13 Selective inhibition of Ang1 or Ang2
induces ovarian atrophy, but not epiphyseal plate thickening
Epiphyseal plate Epiphyseal plate Agent thickening (males)
thickening (females) Ovarian atrophy AMG 386 10 10 8 L1-7(N) 0 0 8
mL4-3 0 0 6 n = 10 per group
Sequence CWU 1
1
481122PRTHomo sapiens 1Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val
Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Tyr Ile Ser Ser Ser Gly Ser
Thr Ile Glu Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Leu
Leu Asp Tyr Asp Ile Leu Thr Gly Tyr Gly Tyr Trp 100 105 110Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 115 1202122PRTHomo sapiens 2Glu Val
Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Gln Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Asp Leu Leu Asp Tyr Asp Ile Leu Thr
Gly Tyr Gly Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 1203122PRTHomo sapiens 3Glu Val Gln Leu Val Gln Ser Gly Gly
Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Tyr Ile Ser Ser Ser
Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Asp Leu Leu Asp Tyr Asp Leu Leu Thr Gly Tyr Gly Tyr Trp 100 105
110Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 1204122PRTHomo
sapiens 4Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro
Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Leu Leu Asp Tyr
Asp Ile Tyr Thr Gly Tyr Gly Tyr Trp 100 105 110Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 115 1205122PRTHomo sapiens 5Glu Val Gln Leu Val
Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Tyr
Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asp Leu Leu Asp Tyr Asp Ile Leu Thr Gly Tyr Gly Leu
Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
1206122PRTHomo sapiens 6Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val
Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Tyr Ile Ser Ser Ser Gly Ser
Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Leu
Leu Asp Tyr Asp Ile Leu Thr Gly Tyr Gly Met Trp 100 105 110Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 115 1207122PRTHomo sapiens 7Glu Val
Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Asp Leu Leu Asp Tyr Asp Ile Trp Thr
Gly Tyr Gly Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 1208112PRTHomo sapiens 8Asp Ile Val Met Thr Gln Ser Pro Leu
Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg
Ser Ile Gln Ser Leu Leu Gln Ser 20 25 30Asn Gly Tyr Asn Tyr Leu Asp
Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr
Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val
Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly 85 90 95Thr His
Trp Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
1109112PRTHomo sapiens 9Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu
Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ser Leu Leu Leu Ser 20 25 30Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr
Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Leu Gly
Ser Asn Arg Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala
Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly 85 90 95Thr His Trp Pro
Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
11010112PRTHomo sapiens 10Asp Ile Val Met Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser
Ser Gln Ser Leu Leu His Ser 20 25 30His Gly Tyr Asn Tyr Leu Asp Trp
Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Leu
Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu
Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly 85 90 95Thr His Trp
Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
11011112PRTHomo sapiens 11Asp Ile Val Met Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser
Ser Gln Ser Leu Leu His Ser 20 25 30Val Gly Tyr Asn Tyr Leu Asp Trp
Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Leu
Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu
Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly 85 90 95Thr His Trp
Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
11012112PRTHomo sapiens 12Asp Ile Val Met Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser
Ser Gln Ser Leu Leu His Ser 20 25 30Asn Gly Tyr Asn Phe Leu Asp Trp
Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Leu
Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu
Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly 85 90 95Thr His Trp
Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
11013112PRTHomo sapiens 13Asp Ile Val Met Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser
Ser Gln Ser Leu Leu His Ser 20 25 30Asn Gly Tyr Asn Met Leu Asp Trp
Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Leu
Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu
Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly 85 90 95Thr His Trp
Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
11014112PRTHomo sapiens 14Asp Ile Val Met Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser
Ser Gln Ser Leu Leu His Ser 20 25 30Asn Gly Tyr Asn Tyr Leu Asp Trp
Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Ala
Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu
Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly 85 90 95Thr His Trp
Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
11015112PRTHomo sapiens 15Asp Ile Val Met Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser
Ser Gln Ser Leu Leu His Ser 20 25 30Asn Gly Tyr Asn Tyr Leu Asp Trp
Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Leu
Gly Ser Asp Arg Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu
Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly 85 90 95Thr His Trp
Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
11016112PRTHomo sapiens 16Asp Ile Val Met Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser
Ser Gln Ser Leu Leu His Ser 20 25 30Asn Gly Tyr Asn Tyr Leu Asp Trp
Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Leu
Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu
Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95Thr His Trp
Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
11017112PRTHomo sapiens 17Asp Ile Val Met Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser
Ser Gln Ser Leu Leu His Ser 20 25 30Asn Gly Tyr Asn Tyr Leu Asp Trp
Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Leu
Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu
Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Val 85 90 95Thr His Trp
Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
110185PRTHomo sapiens 18Ser Tyr Gly Met His1 51916PRTHomo sapiens
19Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Phe Leu Asp1
5 10 152016PRTHomo sapiens 20Arg Ser Ser Gln Ser Leu Leu His Ser
Asn Gly Tyr Asn Tyr Leu Asp1 5 10 152116PRTHomo sapiens 21Arg Ser
Ser Gln Ser Leu Leu His Ser Val Gly Tyr Asn Tyr Leu Asp1 5 10
152216PRTHomo sapiens 22Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly
Tyr Asn Met Leu Asp1 5 10 152316PRTHomo sapiens 23Arg Ser Ser Gln
Ser Leu Leu His Ser His Gly Tyr Asn Tyr Leu Asp1 5 10 152416PRTHomo
sapiens 24Arg Ser Ser Gln Ser Leu Leu Leu Ser Asn Gly Tyr Asn Tyr
Leu Asp1 5 10 152516PRTHomo sapiens 25Arg Ser Ile Gln Ser Leu Leu
Gln Ser Asn Gly Tyr Asn Tyr Leu Asp1 5 10 152617PRTHomo sapiens
26Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val Lys1
5 10 15Gly277PRTHomo sapiens 27Leu Gly Ser Asn Arg Ala Ser1
52817PRTHomo sapiens 28Tyr Ile Ser Ser Ser Gly Ser Thr Ile Glu Tyr
Ala Asp Ser Val Lys1 5 10 15Gly2917PRTHomo sapiens 29Tyr Ile Ser
Ser Ser Gly Ser Thr Ile Gln Tyr Ala Asp Ser Val Lys1 5 10
15Gly307PRTHomo sapiens 30Leu Gly Ser Asp Arg Ala Ser1 5317PRTHomo
sapiens 31Ala Gly Ser Asn Arg Ala Ser1 53213PRTHomo sapiens 32Asp
Leu Leu Asp Tyr Asp Ile Tyr Thr Gly Tyr Gly Tyr1 5 10339PRTHomo
sapiens 33Met Gln Gly Thr His Trp Pro Pro Thr1 53413PRTHomo sapiens
34Asp Leu Leu Asp Tyr Asp Ile Trp Thr Gly Tyr Gly Tyr1 5
103513PRTHomo sapiens 35Asp Leu Leu Asp Tyr Asp Leu Leu Thr Gly Tyr
Gly Tyr1 5 10369PRTHomo sapiens 36Met Gln Val Thr His Trp Pro Pro
Thr1 53713PRTHomo sapiens 37Asp Leu Leu Asp Tyr Asp Ile Leu Thr Gly
Met Gly Tyr1 5 103813PRTHomo sapiens 38Asp Leu Leu Asp Tyr Asp Ile
Leu Thr Gly Leu Gly Tyr1 5 103913PRTHomo sapiens 39Asp Leu Leu Asp
Tyr Asp Ile Leu Thr Gly Tyr Gly Tyr1 5 10409PRTHomo sapiens 40Met
Gln Ala Thr His Trp Pro Pro Thr1 541981DNAHomo sapiensCDS(1)..(981)
41gct agc acc aag ggc cca tcg gtc ttc ccc ctg gcg ccc tgc tcc agg
48Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1
5 10 15agc acc tcc gag agc aca gcg gcc ctg ggc tgc ctg gtc aag gac
tac 96Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gct ctg
acc agc 144Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45ggc gtg cac acc ttc cca gct gtc cta cag tcc tca gga
ctc tac tcc 192Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60ctc agc
agc gtg gtg acc gtg ccc tcc agc aac ttc ggc acc cag acc 240Leu Ser
Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr65 70 75
80tac acc tgc aac gta gat cac aag ccc agc aac acc aag gtg gac aag
288Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95aca gtt gag cgc aaa tgt tgt gtc gag tgc cca ccg tgc cca gca
cca 336Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala
Pro 100 105 110cct gtg gca gga ccg tca gtc ttc ctc ttc ccc cca aaa
ccc aag gac 384Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp 115 120 125acc ctc atg atc tcc cgg acc cct gag gtc acg
tgc gtg gtg gtg gac 432Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp 130 135 140gtg agc cac gaa gac ccc gag gtc cag
ttc aac tgg tac gtg gac ggc 480Val Ser His Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly145 150 155 160gtg gag gtg cat aat gcc
aag aca aag cca cgg gag gag cag ttc aac 528Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175agc acg ttc cgt
gtg gtc agc gtc ctc acc gtt gtg cac cag gac tgg 576Ser Thr Phe Arg
Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190ctg aac
ggc aag gag tac aag tgc aag gtc tcc aac aaa ggc ctc cca 624Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200
205gcc ccc atc gag aaa acc atc tcc aaa acc aaa ggg cag ccc cga gaa
672Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu
210 215 220cca cag gtg tac acc ctg ccc cca tcc cgg gag gag atg acc
aag aac 720Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn225 230 235 240cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc
tac ccc agc gac atc 768Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile 245 250 255gcc gtg gag tgg gag agc aat ggg cag
ccg gag aac aac tac aag acc 816Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr 260 265 270aca cct ccc atg ctg gac tcc
gac ggc tcc ttc ttc ctc tac agc aag 864Thr Pro Pro Met Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285ctc acc gtg gac aag
agc agg tgg cag cag ggg aac gtc ttc tca tgc 912Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300tcc gtg atg
cat gag gct ctg cac aac cac tac acg cag aag agc ctc 960Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu305 310 315
320tcc ctg tct ccg ggt aaa tga 981Ser Leu Ser Pro Gly Lys
32542326PRTHomo sapiens 42Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr
Val Pro Ser Ser Asn Phe Gly Thr Gln Thr65 70 75 80Tyr Thr Cys Asn
Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Thr Val Glu
Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110Pro
Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120
125Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
130 135 140Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val
Asp Gly145 150 155 160Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Phe Asn 165 170 175Ser Thr Phe Arg Val Val Ser Val Leu
Thr Val Val His Gln Asp Trp 180 185 190Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205Ala Pro Ile Glu Lys
Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn225 230 235
240Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
245 250 255Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr 260 265 270Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys 275 280 285Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys 290 295 300Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu305 310 315 320Ser Leu Ser Pro Gly
Lys 32543324DNAHomo sapiensCDS(1)..(324) 43cgt acg gtg gct gca cca
tct gtc ttc atc ttc ccg cca tct gat gag 48Arg Thr Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15cag ttg aaa tct gga
act gcc tct gtt gtg tgc ctg ctg aat aac ttc 96Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30tat ccc aga gag
gcc aaa gta cag tgg aag gtg gat aac gcc ctc caa 144Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45tcg ggt aac
tcc cag gag agt gtc aca gag cag gac agc aag gac agc 192Ser Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60acc tac
agc ctc agc agc acc ctg acg ctg agc aaa gca gac tac gag 240Thr Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75
80aaa cac aaa gtc tac gcc tgc gaa gtc acc cat cag ggc ctg agc tcg
288Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95ccc gtc aca aag agc ttc aac agg gga gag tgt tag 324Pro Val
Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 10544107PRTHomo sapiens
44Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1
5 10 15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys 100 10545321DNAHomo sapiensCDS(1)..(321) 45ggc caa ccg aaa
gcg gcg ccc tcg gtc act ctg ttc ccg ccc tcc tct 48Gly Gln Pro Lys
Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser1 5 10 15gag gag ctt
caa gcc aac aag gcc aca ctg gtg tgt ctc ata agt gac 96Glu Glu Leu
Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30ttc tac
ccg gga gcc gtg aca gtg gcc tgg aag gca gat agc agc ccc 144Phe Tyr
Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 35 40 45gtc
aag gcg gga gtg gag acc acc aca ccc tcc aaa caa agc aac aac 192Val
Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 50 55
60aag tac gcg gcc agc agc tat ctg agc ctg acg cct gag cag tgg aag
240Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp
Lys65 70 75 80tcc cac aga agc tac agc tgc cag gtc acg cat gaa ggg
agc acc gtg 288Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly
Ser Thr Val 85 90 95gag aag aca gtg gcc cct aca gaa tgt tca tag
321Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 10546106PRTHomo
sapiens 46Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro
Ser Ser1 5 10 15Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu
Ile Ser Asp 20 25 30Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala
Asp Ser Ser Pro 35 40 45Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser
Lys Gln Ser Asn Asn 50 55 60Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu
Thr Pro Glu Gln Trp Lys65 70 75 80Ser His Arg Ser Tyr Ser Cys Gln
Val Thr His Glu Gly Ser Thr Val 85 90 95Glu Lys Thr Val Ala Pro Thr
Glu Cys Ser 100 1054772PRTHomo sapiens 47Met Arg Glu Trp Thr Glu
Glu Met Gln Val Ile Phe Asp Ala Met Met1 5 10 15Phe Gly Pro Arg Asn
Asp Arg Gly Gly Ser Gly Ser Ala Thr Gly Ser 20 25 30Gly Ser Thr Ala
Ser Ser Gly Ser Gly Ser Ala Thr His Arg Glu Trp 35 40 45Thr Glu Glu
Met Gln Val Ile Phe Asp Ala Met Met Phe Gly Pro Arg 50 55 60Asn Asp
Arg Gly Gly Gly Gly Gly65 7048227PRTHomo sapiens 48Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55
60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65
70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser 130 135 140Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200
205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220Pro Gly Lys225
* * * * *