U.S. patent application number 13/668028 was filed with the patent office on 2013-06-13 for humanized anti-egfl7 antibodies and methods using same.
This patent application is currently assigned to Genentech, Inc.. The applicant listed for this patent is Genentech, Inc.. Invention is credited to Mark S. Dennis, Jill Fredrickson, Weilan Ye.
Application Number | 20130149307 13/668028 |
Document ID | / |
Family ID | 42333507 |
Filed Date | 2013-06-13 |
United States Patent
Application |
20130149307 |
Kind Code |
A1 |
Ye; Weilan ; et al. |
June 13, 2013 |
HUMANIZED ANTI-EGFL7 ANTIBODIES AND METHODS USING SAME
Abstract
The present invention concerns antibodies to EGFL7 and the uses
of same.
Inventors: |
Ye; Weilan; (Foster City,
CA) ; Dennis; Mark S.; (San Carlos, CA) ;
Fredrickson; Jill; (Sunnyvale, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genentech, Inc.; |
South San Francisco |
CA |
US |
|
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
42333507 |
Appl. No.: |
13/668028 |
Filed: |
November 2, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12776259 |
May 7, 2010 |
8404811 |
|
|
13668028 |
|
|
|
|
61176817 |
May 8, 2009 |
|
|
|
Current U.S.
Class: |
424/136.1 ;
424/133.1; 424/158.1; 424/172.1 |
Current CPC
Class: |
A61P 5/14 20180101; A61P
9/00 20180101; A61K 31/513 20130101; A61K 31/555 20130101; C07K
2317/567 20130101; A61K 39/39558 20130101; A61P 1/04 20180101; A61P
35/00 20180101; A61P 37/04 20180101; A61K 31/56 20130101; A61P
15/00 20180101; A61P 21/00 20180101; A61P 1/08 20180101; A61K
2039/507 20130101; A61P 3/10 20180101; A61P 17/00 20180101; C07K
16/22 20130101; A61P 27/04 20180101; A61K 39/001131 20180801; C07K
16/468 20130101; A61P 9/10 20180101; A61K 38/19 20130101; A61P
31/04 20180101; C07K 2317/565 20130101; A61K 31/337 20130101; A61P
13/12 20180101; A61P 29/00 20180101; C07K 2317/24 20130101; A61P
17/06 20180101; C07K 2317/56 20130101; C07K 2317/92 20130101; A61P
19/02 20180101; A61P 43/00 20180101; A61K 31/519 20130101; A61P
11/00 20180101; A61K 39/3955 20130101; A61K 45/06 20130101; A61K
2039/505 20130101; A61P 27/02 20180101; A61P 37/06 20180101; A61K
31/282 20130101; A61P 27/06 20180101; A61K 39/0011 20130101; C07K
16/46 20130101; C07K 2317/73 20130101; A61P 31/00 20180101; A61P
3/00 20180101; C07K 16/18 20130101 |
Class at
Publication: |
424/136.1 ;
424/172.1; 424/133.1; 424/158.1 |
International
Class: |
C07K 16/18 20060101
C07K016/18; A61K 39/395 20060101 A61K039/395; A61K 45/06 20060101
A61K045/06; A61K 39/00 20060101 A61K039/00; A61K 31/519 20060101
A61K031/519; A61K 38/19 20060101 A61K038/19; A61K 31/555 20060101
A61K031/555; A61K 31/337 20060101 A61K031/337; A61K 31/513 20060101
A61K031/513; C07K 16/22 20060101 C07K016/22; A61K 31/56 20060101
A61K031/56 |
Claims
1. A method for treating a tumor, a cancer, or a cell proliferative
disorder, the method comprising administering to an individual in
need of such treatment an effective amount of an anti-EGFL7
antibody, the antibody comprising the following hypervariable
region (HVR) sequences: (i) HVR-L1 comprising
KX.sub.1SX.sub.2SX.sub.3DYX.sub.4GDSYX.sub.5S, wherein X.sub.1 is A
or R; X.sub.2 is H or Q; X.sub.3 is G or V; X.sub.4 is selected
from the group consisting of D, L, R, S, and W; and X.sub.5 is M or
V (SEQ ID NO: 210); (ii) HVR-L2 comprising
GASX.sub.1X.sub.2EX.sub.3, wherein X.sub.1 is N or Y; X.sub.2 is
selected from the group consisting of L, R and Y; and X.sub.3 is Q
or S (SEQ ID NO: 211); (iii) HVR-L3 comprising
QQNNEX.sub.1PX.sub.2T, wherein X.sub.1 is D or E; and X.sub.2 is F
or Y (SEQ ID NO: 212); (iv) HVR-H1 comprising
GX.sub.1X.sub.2X.sub.3X.sub.4TYGX.sub.5S, wherein X.sub.1 is H or
V; X.sub.2 is R or T; X.sub.3 is selected from the group consisting
of F, G, R, and S; X.sub.4 is selected from the group consisting of
D, G, R, and T; and X.sub.5 is M or Y (SEQ ID NO: 213); (v) HVR-H2
comprising
GWINX.sub.1X.sub.2SGVPTX.sub.3AX.sub.4X.sub.5X.sub.6X.sub.7X.sub.8,
wherein X.sub.1 is selected from the group consisting of I, M, T,
and W; X.sub.2 is H or R; X.sub.3 is selected from group consisting
of I, M, T, and Y; X.sub.4 is D or H; X.sub.5 is selected from
group consisting of D, M and T; X.sub.6 is F or Y; X.sub.7 is K or
S; and X.sub.8 is G or R (SEQ ID NO: 214, and (vi) HVR-H3
comprising AX.sub.1LGSX.sub.2AVDX.sub.3, wherein X.sub.1 is N or R;
X.sub.2 is selected from the group consisting of C, R, H, and Y;
and X.sub.3 is A or Y (SEQ ID NO: 215).
2. A method for treating a tumor, a cancer, or a cell proliferative
disorder, the method comprising administering to an individual in
need of such treatment an effective amount of an anti-EGFL7
antibody, the antibody comprising the following HVR sequences:
HVR-L1 comprising an amino acid sequence selected from SEQ ID NOs:
37-43, HVR-L2 comprising an amino acid sequence selected from the
group consisting of SEQ ID NOs: 44-47, HVR-L3 comprising the amino
acid sequence SEQ ID NO: 48, HVR-H1 comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs: 49-57,
HVR-H2 comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 58-73, and HVR-H3 comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs:
74-77.
3. The method of claim 1, wherein HVR-L1 comprises the amino acid
sequence KASQSVDYSGDSYMS (SEQ ID NO: 234), HVR-L2 comprises the
amino acid sequence GASYRES (SEQ ID NO: 235), HVR-L3 comprises the
amino acid sequence QQNNEEPYT (SEQ ID NO: 236), HVR-H1 comprises
the amino acid sequence GHTFTTYGMS (SEQ ID NO: 34), HVR-H2
comprises the amino acid sequence GWINTHSGVPTYADDFKG (SEQ ID NO:
35), and HVR-H3 comprises the amino acid sequence ARLGSYAVDY (SEQ
ID NO: 237).
4. The method of claim 1, wherein the heavy chain comprises the
following framework sequences: FR-H1 comprises
EX.sub.1QLVESGGGLVQPGGSLRLSCAAS, wherein X.sub.1 is I or V (SEQ ID
NO: 216); FR-H2 comprises WVRQAPGKGLEWX.sub.1, wherein X.sub.1 is
I, M, or V (SEQ ID NO: 217); FR-H3 comprises
RFTX.sub.1SX.sub.2DX.sub.3SX.sub.4X.sub.5TX.sub.6YLQMNSLRAEDTAVYX.sub.7CA-
R, wherein X.sub.1 is F or I; X.sub.2 is L or R; X.sub.3 is N or T,
X.sub.4 is selected from the group consisting of A, E, K and T;
X.sub.5 is N or S; X.sub.6 is selected from the group consisting of
A, L, M, T and V; and X.sub.7 is F or Y (SEQ ID NO: 218); and FR-H4
comprises WGQGTLVTVSS (SEQ ID NO: 219).
5. The method of claim 4, wherein the heavy chain comprises the
following framework sequences: FR-H1 comprises
EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 197); FR-H2 comprises
WVRQAPGKGLEWV (SEQ ID NO: 198); FR-H3 comprises
RFTISX.sub.1DNSKNTX.sub.2YLQMNSLRAEDTAVYYCAR, wherein X.sub.1 L or
R; X.sub.2 is selected from the group consisting of A, L, M, T and
V (SEQ ID NO: 220); and FR-H4 comprises WGQGTLVTVSS (SEQ ID NO:
200).
6. The method of claim 4, wherein the heavy chain comprises the
following framework sequences: FR-H1 comprises
EIQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 238); FR-H2 comprises
WVRQAPGKGLEWM (SEQ ID NO: 239); FR-H3 comprises
RFTISX.sub.1DNSKSTX.sub.2YLQMNSLRAEDTAVYFCAR, wherein X.sub.1 L or
R; X.sub.2 is selected from the group consisting of A, L, M, T and
V (SEQ ID NO: 240); and FR-H4 comprises WGQGTLVTVSS (SEQ ID NO:
219).
7. The method of claim 1, wherein the light chain comprises the
following framework sequences: FR-L1 comprises
DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 201), FR-L2 comprises
WYQQKPGKAPKLLIY (SEQ ID NO: 202), FR-L3 comprises
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 203), FR-L4 comprises
FGQGTKVEIK (SEQ ID NO: 221) or FGQGTKVEIKR (SEQ ID NO: 204).
8. The method of claim 1, wherein the light chain comprises the
variable domain sequence of 4F11.v17 as shown in FIG. 15 (SEQ ID
NO: 82) or the variable domain sequence of 4F11.v22 as shown in
FIG. 15 (SEQ ID NO: 83).
9. The method of claim 1, wherein the heavy chain comprises the
variable domain sequence of 4F11.v17 as shown in FIG. 16 (SEQ ID
NO: 84) or the variable domain sequence of 4F11.v22 as shown in
FIG. 16 (SEQ ID NO: 85).
10. The method of claim 1, wherein the light chain comprises the
variable domain sequence of 4F11.v17 as shown in FIG. 15 (SEQ ID
NO: 82) and the heavy chain comprises the variable domain sequence
of 4F11.v17 as shown in FIG. 16 (SEQ ID NO: 84).
11. The method of claim 1, wherein the light chain comprises the
variable domain sequence of 4F11.v22 as shown in FIG. 15 (SEQ ID
NO: 83) and the heavy chain comprises the variable domain sequence
of 4F11.v22 as shown in FIG. 16 (SEQ ID NO: 85).
12. The method of claim 1, wherein at least a portion of the
framework sequence is a human consensus framework sequence.
13. The method of claim 12, comprising human .kappa. subgroup 1
consensus framework sequence or heavy chain human subgroup III
consensus framework sequence.
14. A method for treating a tumor, a cancer, or a cell
proliferative disorder, the method comprising administering to an
individual in need of such treatment an effective amount of an
anti-EGFL7 antibody, the antibody comprising a variable domain
comprising the following HVR sequences: (i) HVR-L1 comprising
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6VX.sub.7X.sub.8X.sub.9X.sub.10I-
TYLX.sub.11, wherein X.sub.1 is selected from the group consisting
of L, Q, R, S, and T; X.sub.2 is selected from the group consisting
of P, T, and W; X.sub.3 is H or S; X.sub.4 is D or Q; X.sub.5 is G
or S; X.sub.6 is L or V; X.sub.7 is H or P; X.sub.8 is selected
from the group consisting of I, L, P, T, and Y; X.sub.9 is selected
from the group consisting of N, Q or S; X.sub.10 is selected from
the group consisting of A, G, and S; and X.sub.11 is G or H (SEQ ID
NO: 222); (ii) HVR-L2 comprising RVSNX.sub.1X.sub.2S, wherein
X.sub.1 is D or R; and X.sub.2 is selected from the group
consisting of A, G, F, I, and T (SEQ ID NO: 223); (iii) HVR-L3
comprising X.sub.1QSX.sub.2X.sub.3VPLT, wherein X.sub.1 is selected
from the group consisting of A, G, I, K, L, N, T, and V; X.sub.2 is
C or T; and X.sub.3 is F or H (SEQ ID NO: 224); (iv) HVR-H1
comprising GYX.sub.1X.sub.2X.sub.3DX.sub.4YX.sub.5N, wherein
X.sub.1 is N or T; X.sub.2 is F or V; X.sub.3 is selected from the
group consisting of I, M, R, and S; X.sub.4 is selected from the
group consisting of Y, Q, and K; and X.sub.5 is I or M (SEQ ID NO:
225); (v) HVR-H2 comprising
GDINX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6HX.sub.7X.sub.8X.sub.9X.sub-
.10X.sub.11X.sub.12X.sub.13, wherein X.sub.1 is selected from the
group consisting of A, L, N, and P; X.sub.2 is selected from the
group consisting of D, L, and R; X.sub.3 is selected from the group
consisting of G, K, N, R, S, and Y; X.sub.4 is G or S; X.sub.5 is
selected from the group consisting of G, I, K, R, S, T, and V;
X.sub.6 is selected from the group consisting of G, R, and T;
X.sub.7 is selected from the group consisting of I, V, L, and Y;
X.sub.8 is N or S; X.sub.9 is selected from the group consisting of
A, N, and Q; X.sub.10 is K or V; X.sub.11 is F or Q; X.sub.12 is K
or T; and X.sub.13 is selected from the group consisting of G, H,
R, and S (SEQ ID NO: 226); and (vi) HVR-H3 comprising
X.sub.1REGVYHX.sub.2YDDYAX.sub.3DY, wherein X.sub.1 is selected
from the group consisting of A, N, and T; X.sub.2 is D or P; and
X.sub.3 is M or W (SEQ ID NO: 227).
15. A method for treating a tumor, a cancer, or a cell
proliferative disorder, the method comprising administering to an
individual in need of such treatment an effective amount of an
anti-EGFL7 antibody, the antibody comprising the following HVR
sequences: HVR-L1 comprising an amino acid sequence selected from
SEQ ID NOs: 106-124 and 243-246, HVR-L2 comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs: 125-129,
HVR-L3 comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 130-145, HVR-H1 comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs: 146-153,
HVR-H2 comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 154-187 and 247, and HVR-H3 comprising an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 188-192.
16. The method of claim 14, wherein HVR-L1 comprises the amino acid
sequence RTSQSLVHINAITYLH (SEQ ID NO: 241), HVR-L2 comprises the
amino acid sequence RVSNRFS (SEQ ID NO: 101), HVR-L3 comprises the
amino acid sequence GQSTHVPLT (SEQ ID NO: 131), HVR-H1 comprises
the amino acid sequence GYTFIDYYMN (SEQ ID NO: 103), HVR-H2
comprises the amino acid sequence GDINLDNSGTHYNQKFKG (SEQ ID NO:
242), and HVR-H3 comprises the amino acid sequence AREGVYHDYDDYAMDY
(SEQ ID NO: 105).
17. The method of claim 14, wherein the heavy chain comprises the
following framework sequences: FR-H1 comprises
EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 197); FR-H2 comprises
WVRQAPGKGLEWX.sub.1, wherein X.sub.1 is I or V (SEQ ID NO: 228);
FR-H3 comprises
RX.sub.1TX.sub.2SX.sub.3DX.sub.4SX.sub.5X.sub.6TX.sub.7YX.sub.8QMNSLRAEDT-
AVYYC, wherein X.sub.1 is F or V; X.sub.2 is I or L; X.sub.3 is
selected from the group consisting of L, R, and V; X.sub.4 is K or
N; X.sub.5 is selected from the group consisting of K, N, R, and S;
X.sub.6 is N or S; X.sub.7 is selected from the group consisting of
A, L, and V; and X.sub.8 is L or M (SEQ ID NO: 229); and FR-H4
comprises WGQGTLVTVSS (SEQ ID NO: 200).
18. The method of claim 17, wherein the heavy chain comprises the
following framework sequences: FR-H1 comprises
EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 197); FR-H2 comprises
WVRQAPGKGLEWV (SEQ ID NO: 198); FR-H3 comprises
RFTISRDX.sub.1SKNTX.sub.2YLQMNSLRAEDTAVYYCAR, wherein X.sub.1 is N
or K; and X.sub.2 is selected from the group consisting of A, L,
and V (SEQ ID NO: 230); and FR-H4 comprises WGQGTLVTVSS (SEQ ID NO:
200).
19. The method of claim 14, wherein the light chain comprises the
following framework sequences: FR-L1 comprises
DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 201), FR-L2 comprises
WYQQKPGKAPKLLIY (SEQ ID NO: 202), FR-L3 comprises
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 203), FR-L4 comprises
FGQGTKVEIK (SEQ ID NO: 221) or FGQGTKVEIKR (SEQ ID NO: 204).
20. The method of claim 14, wherein the light chain comprises the
variable domain sequence of 18F7.v6 as shown in FIG. 27 (SEQ ID NO:
193) or the variable domain sequence of 18F7.v6k as shown in FIG.
27 (SEQ ID NO: 194).
21. The method of claim 14, wherein the heavy chain comprises the
variable domain sequence of 18F7.v6 as shown in FIG. 28 (SEQ ID NO:
195) or the variable domain sequence of 18F7.v6k as shown in FIG.
28 (SEQ ID NO: 196).
22. The method of claim 14, wherein the light chain comprises the
variable domain sequence of 18F7.v6 as shown in FIG. 27 (SEQ ID NO:
193) and the heavy chain comprises the variable domain sequence of
18F7.v6 as shown in FIG. 28 (SEQ ID NO: 195).
23. The method of claim 14, wherein the light chain comprises the
variable domain sequence of 18F7.v6k as shown in FIG. 27 (SEQ ID
NO: 194) and the heavy chain comprises the variable domain sequence
of 18F7.v6k as shown in FIG. 28 (SEQ ID NO: 196).
24. The method of claim 14, wherein at least a portion of the
framework sequence is a human consensus framework sequence.
25. The method of claim 24, comprising human .kappa. subgroup 1
consensus framework sequence or heavy chain human subgroup III
consensus framework sequence.
26. The method of claim 1 or 14, wherein said antibody is a
bispecific antibody.
27. The method of claim 26, wherein said bispecific antibody binds
to vascular endothelial growth factor (VEGF).
28. The method of claim 1 or 14, wherein the cancer is selected
from the group consisting of breast cancer, colorectal cancer, lung
cancer, esophageal cancer, bladder cancer, ovarian cancer,
pancreatic cancer, and hepatocellular carcinoma.
29. The method of claim 28, wherein the cancer is colorectal cancer
or lung cancer.
30. The method of claim 1 or 14, further comprising administering
to the individual an effective amount of a second medicament,
wherein the anti-EGFL7 antibody is a first medicament.
31. The method of claim 30, wherein the second medicament is
another antibody, a chemotherapeutic agent, a cytotoxic agent, an
anti-angiogenic agent, an immunosuppressive agent, a prodrug, a
cytokine, a cytokine antagonist, cytotoxic radiotherapy, a
corticosteroid, an anti-emetic, a cancer vaccine, an analgesic, or
a growth-inhibitory agent.
32. The method of claim 31, wherein the second medicament is an
anti-VEGF antibody.
33. The method of claim 32, where the second medicament is
bevacizumab or ranibizumab.
34. The method of claim 30, further comprising administering to the
individual an effective amount of a further medicament, wherein the
anti-EGFL7 antibody is a first medicament and an anti-VEGF antibody
is the second medicament.
35. The method of claim 34, wherein the further medicament is a
chemotherapeutic agent.
36. The method of claim 35, wherein the chemotherapeutic agent is
FOLFOX.
37. The method of claim 35, wherein the chemotherapeutic agent is
carboplatin and paclitaxel.
Description
RELATED APPLICATION
[0001] This application is a divisional of U.S. application Ser.
No. 12/776,259, filed 7 May 2010, which claims priority under 35
USC 119(e) to provisional application No. 61/176,817, filed 8 May
2009, the contents of which are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the field of
molecular biology. More specifically, the invention concerns
anti-EGFL7 antibodies, and uses of same.
BACKGROUND OF THE INVENTION
[0003] The development of a vascular supply is a fundamental
requirement for many physiological and pathological processes.
Actively growing tissues such as embryos and tumors require
adequate blood supply. They satisfy this need by producing
pro-angiogenic factors, which promote new blood vessel formation
from existing vessels via a process called angiogenesis; or from
progenitor cells through a process called vasculogenesis.
Tubulogenesis is an essential step in vascular development.
Vascular tube formation is a complex but orderly biological event
involving all or many of the following steps: a) endothelial cells
(EC) proliferate from existing ECs or differentiate from progenitor
cells; b) EC migration; c) ECs coalesce to form cord-like
structures; d) vascular cords then undergo tubulogenesis to form
vessels with a central lumen e) existing cords or vessels send out
sprouts to form secondary vessels (angiogenesis); f) primitive
vascular plexus undergo further remodeling and reshaping; and g)
peri-endothelial cells are recruited to encase the endothelial
tubes, providing maintenance and modulatory functions to the
vessels; such cells including pericytes for small capillaries,
smooth muscle cells for larger vessels, and myocardial cells in the
heart. Hanahan, D. Science 277, 48-50 (1997); Hogan, B. L. &
Kolodziej, P. A. Nature Reviews Genetics. 3, 513-23 (2002);
Lubarsky, B. & Krasnow, M. A. Cell. 112, 19-28 (2003).
[0004] It is now well established that angiogenesis, which involves
the formation of new blood vessels from preexisting endothelium, is
implicated in the pathogenesis of a variety of disorders. These
include solid tumors and metastasis, atherosclerosis, retrolental
fibroplasia, hemangiomas, chronic inflammation, intraocular
neovascular syndromes such as proliferative retinopathies, e.g.,
diabetic retinopathy, age-related macular degeneration (AMD),
neovascular glaucoma, immune rejection of transplanted corneal
tissue and other tissues, rheumatoid arthritis, and psoriasis.
Folkman et al., J. Biol. Chem., 267: 10931-10934 (1992); Klagsbrun
et al., Annu. Rev. Physiol., 53: 217-239 (1991); and Garner A.,
"Vascular diseases", In: Pathobiology of Ocular Disease. A Dynamic
Approach, Garner A., Klintworth G K, eds., 2nd Edition (Marcel
Dekker, NY, 1994), pp 1625-1710.
[0005] In the case of tumor growth, angiogenesis appears to be
crucial for the transition from hyperplasia to neoplasia, and for
providing nourishment for the growth and metastasis of the tumor.
Folkman et al., Nature, 339: 58 (1989). The neovascularization
allows the tumor cells to acquire a growth advantage and
proliferative autonomy compared to normal cells. A tumor usually
begins as a single aberrant cell which can proliferate only to a
size of a few cubic millimeters due to the distance from available
capillary beds, and it can stay `dormant` without further growth
and dissemination for a long period of time. Some tumor cells then
switch to the angiogenic phenotype to activate endothelial cells,
which proliferate and mature into new capillary blood vessels.
These newly formed blood vessels not only allow for continued
growth of the primary tumor, but also for the dissemination and
recolonization of metastatic tumor cells. Accordingly, a
correlation has been observed between density of microvessels in
tumor sections and patient survival in breast cancer as well as in
several other tumors. Weidner et al., N. Engl. J. Med, 324: 1-6
(1991); Horak et al., Lancet, 340: 1120-1124 (1992); Macchiarini et
al., Lancet, 340: 145-146 (1992). The precise mechanisms that
control the angiogenic switch is not well understood, but it is
believed that neovascularization of tumor mass results from the net
balance of a multitude of angiogenesis stimulators and inhibitors
(Folkman, 1995, Nat Med 1(1):27-31).
[0006] The process of vascular development is tightly regulated. To
date, a significant number of molecules, mostly secreted factors
produced by surrounding cells, have been shown to regulate EC
differentiation, proliferation, migration and coalescence into
cord-like structures. For example, vascular endothelial growth
factor (VEGF) has been identified as the key factor involved in
stimulating angiogenesis and in inducing vascular permeability.
Ferrara et al., Endocr. Rev., 18: 4-25 (1997). The finding that the
loss of even a single VEGF allele results in embryonic lethality
points to an irreplaceable role played by this factor in the
development and differentiation of the vascular system.
Furthermore, VEGF has been shown to be a key mediator of
neovascularization associated with tumors and intraocular
disorders. Ferrara et al., Endocr. Rev., supra. The VEGF mRNA is
overexpressed by the majority of human tumors examined. Berkman et
al., J. Clin. Invest., 91: 153-159 (1993); Brown et al., Human
Pathol., 26: 86-91 (1995); Brown et al., Cancer Res., 53: 4727-4735
(1993); Mattern et al., Brit. J. Cancer, 73: 931-934 (1996); Dvorak
et al., Am. J. Pathol., 146: 1029-1039 (1995).
[0007] Some of the steps during vessel tube formation are still
poorly defined. Particularly, little is know about how
tubulogenesis is regulated--how vascular cords progress to become
tubes, and what factors regulate this transition. In view of the
role of vasculogenesis and angiogenesis in many diseases and
disorders, it is desirable to have a means of reducing or
inhibiting one or more of the biological effects causing these
processes. All references cited herein, including patent
applications and publications, are incorporated by reference in
their entirety.
SUMMARY OF THE INVENTION
[0008] The invention is in part based on a variety of antibodies to
EGFL7. EGFL7 presents as an important and advantageous therapeutic
target, and the invention provides antibodies as therapeutic and
diagnostic agents for use in targeting pathological conditions
associated with expression and/or activity of EGFL7. Accordingly,
the invention provides methods, compositions, kits and articles of
manufacture related to EGFL7.
[0009] For example, in some embodiments, the invention provides
anti-EGFL7 antibodies.
[0010] In some embodiments, the invention provides an anti-EGFL7
antibody comprising a variable domain comprising at least one, two,
three, four or five hypervariable region (HVR) sequences selected
from the group consisting of: (i) HVR-L1 comprising
KX.sub.1SX.sub.2SX.sub.3DYX.sub.4GDSYX.sub.5S, wherein X.sub.1 is A
or R; X.sub.2 is H or Q; X.sub.3 is G or V; X.sub.4 is selected
from the group consisting of D, L, R, S, and W; and X.sub.5 is M or
V (SEQ ID NO: 210); (ii) HVR-L2 comprising
GASX.sub.1X.sub.2EX.sub.3, wherein X.sub.1 is N or Y; X.sub.2 is
selected from the group consisting of L, R and Y; and X.sub.3 is Q
or S (SEQ ID NO: 211); (iii) HVR-L3 comprising
QQNNEX.sub.1PX.sub.2T, wherein X.sub.1 is D or E; and X.sub.2 is F
or Y (SEQ ID NO: 212); (iv) HVR-H1 comprising
GX.sub.1X.sub.2X.sub.3X.sub.4TYGX.sub.5S, wherein X.sub.1 is H or
V; X.sub.2 is R or T; X.sub.3 is selected from the group consisting
of F, G, R, and S; X.sub.4 is selected from the group consisting of
D, G, R, and T; and X.sub.5 is M or Y (SEQ ID NO: 213); (v) HVR-H2
comprising
GWINX.sub.1X.sub.2SGVPTX.sub.3AX.sub.4X.sub.5X.sub.6X.sub.7X.sub.8,
wherein X.sub.1 is selected from the group consisting of I, M, T,
and W; X.sub.2 is H or R; X.sub.3 is selected from group consisting
of I, M, T, and Y; X.sub.4 is D or H; X.sub.5 is selected from
group consisting of D, M and T; X.sub.6 is F or Y; X.sub.7 is K or
S; and X.sub.8 is G or R (SEQ ID NO: 214, and (vi) HVR-H3
comprising AX.sub.1LGSX.sub.2AVDX.sub.3, wherein X.sub.1 is N or R;
X.sub.2 is selected from the group consisting of C, S, and Y; and
X.sub.3 is A or Y (SEQ ID NO: 215). In some embodiments, the
anti-EGFL7 antibody comprises all six of the aforementioned HVRs.
In some embodiments, HVR-L1 comprises an amino acid sequence
selected from SEQ ID NOs: 31 and 37-43, HVR-L2 comprises an amino
acid sequence selected from the group consisting of SEQ ID NOs: 32
and 44-47, HVR-L3 comprises an amino acid sequence selected from
the group consisting of SEQ ID NOs: 33 and 48, HVR-H1 comprises an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 34 and 49-57, HVR-H2 comprises an amino acid sequence selected
from the group consisting of SEQ ID NOs: 35 and 58-73, and HVR-H3
comprises an amino acid sequence selected from the group consisting
of SEQ ID NOs: 36 and 74-77. In some embodiments the heavy chain
comprises the following framework sequences: FR-H1 comprises
EX.sub.1QLVESGGGLVQPGGSLRLSCAAS, wherein X.sub.1 is I or V (SEQ ID
NO: 216); FR-H2 comprises WVRQAPGKGLEWX.sub.1, wherein X.sub.1 is I
or V (SEQ ID NO: 217); FR-H3 comprises
RFTX.sub.1SX.sub.2DX.sub.3SX.sub.4X.sub.5TX.sub.6YLQMNSLRAEDTAV-
YX.sub.7CAR, wherein X.sub.1 is F or I; X.sub.2 is L or R; X.sub.3
is N or T, X.sub.4 is selected from the group consisting of A, E, K
and T; X.sub.5 is N or S; X.sub.6 is selected from the group
consisting of A, L, M, T and V; and X.sub.7 is F or Y (SEQ ID NO:
218); and FR-H4 comprises WGQGTLVTVSS (SEQ ID NO: 219). In some
embodiments, the heavy chain comprises the following framework
sequences: FR-H1 comprises EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:
197); FR-H2 comprises WVRQAPGKGLEWV (SEQ ID NO: 198); FR-H3
comprises RFTISX.sub.1DNSKNTX.sub.2YLQMNSLRAEDTAVYYCAR, wherein
X.sub.1 L or R; X.sub.2 is selected from the group consisting of A,
L, M, T and V (SEQ ID NO: 220); and FR-H4 comprises WGQGTLVTVSS
(SEQ ID NO: 200). In some embodiments, the light chain comprises
the following framework sequences: FR-L1 comprises
DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 201), FR-L2 comprises
WYQQKPGKAPKLLIY (SEQ ID NO: 202), FR-L3 comprises
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 203), FR-L4 comprises
FGQGTKVEIK (SEQ ID NO: 221) or FGQGTKVEIKR (SEQ ID NO: 204). In
some embodiments, the light chain comprises the variable domain
sequence of 4F11.v17 or 4F11.v22 as shown in FIG. 15 (SEQ ID NOs:
82 and 83). In some embodiments, the heavy chain comprises the
variable domain sequence of 4F11.v17 or 4F11.v22 as shown in FIG.
16 (SEQ ID NOs: 84 and 85). In some embodiments, the invention
provides an antibody wherein the light chain comprises the variable
domain sequence of 4F11.v17 as shown in FIG. 15 (SEQ ID NO: 82) and
the heavy chain comprises the variable domain sequence of 4F11.v17
as shown in FIG. 16 (SEQ ID NO: 84). In some embodiments, the
invention provides an antibody wherein the light chain comprises
the variable domain sequence of 4F11.v22 as shown in FIG. 15 (SEQ
ID NO: 83) and the heavy chain comprises the variable domain
sequence of 4F11.v22 as shown in FIG. 16 (SEQ ID NO: 85).
[0011] In some embodiments, the invention provides a anti-EGFL7
antibody comprising a variable domain comprising at least one, two,
three, four or five HVR sequences selected from the group
consisting of: (i) HVR-L1 comprising
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6VX.sub.7X.sub.8X.sub.9X.sub.101-
TYLX.sub.11, wherein X.sub.1 is selected from the group consisting
of L, Q, R, S, and T; X.sub.2 is selected from the group consisting
of P, T, and W; X.sub.3 is H or S; X.sub.4 is D or Q; X.sub.5 is G
or S; X.sub.6 is L or V; X.sub.7 is H or P; X.sub.8 is selected
from the group consisting of I, L, P, T, and Y; X.sub.9 is selected
from the group consisting of N, Q or S; X.sub.10 is selected from
the group consisting of A, G, and S; and X.sub.11 is G or H (SEQ ID
NO: 222); (ii) HVR-L2 comprising RVSNX.sub.1X.sub.2S, wherein
X.sub.1 is D or R; and X.sub.2 is selected from the group
consisting of A, G, F, I, and T (SEQ ID NO: 223); (iii) HVR-L3
comprising X.sub.1QSX.sub.2X.sub.3VPLT, wherein X.sub.1 is selected
from the group consisting of A, G, I, K, L, N, S, T, and V; X.sub.2
is C or T; and X.sub.3 is F or H (SEQ ID NO: 224); (iv) HVR-H1
comprising GYX.sub.1X.sub.2X.sub.3DX.sub.4YX.sub.5N, wherein
X.sub.1 is N or T; X.sub.2 is F or V; X.sub.3 is selected from the
group consisting of I, M, R, and S; X.sub.4 is selected from the
group consisting of Y, Q, and K; and X.sub.5 is I or M (SEQ ID NO:
225); (v) HVR-H2 comprising
GDINX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6HX.sub.7X.sub.8X.sub.9X.sub-
.10X.sub.11X.sub.12X.sub.13, wherein X.sub.1 is selected from the
group consisting of A, L, N, and P; X.sub.2 is selected from the
group consisting of D, L, and R; X.sub.3 is selected from the group
consisting of G, K, N, R, S, and Y; X.sub.4 is G or S; X.sub.5 is
selected from the group consisting of G, I, K, R, S, T, and V;
X.sub.6 is selected from the group consisting of G, R, and T;
X.sub.7 is selected from the group consisting of I, V, and Y;
X.sub.8 is N or S; X.sub.9 is selected from the group consisting of
A, N, and Q; X.sub.10 is K or V; X.sub.11 is F or Q; X.sub.12 is K
or T; and X.sub.13 is selected from the group consisting of G, H,
R, and S (SEQ ID NO: 226), and (vi) HVR-H3 comprising
X.sub.1REGVYHX.sub.2YDDYAX.sub.3DY, wherein X.sub.1 is selected
from the group consisting of A, N, and T; X.sub.2 is D or P; and
X.sub.3 is M or W (SEQ ID NO: 227). In some embodiments, the
anti-EGFL7 antibody comprises all six of the aforementioned HVRs.
In some embodiments, HVR-L1 comprises an amino acid sequence
selected from SEQ ID NOs: 100 and 106-124, HVR-L2 comprises an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 101 and 125-129, HVR-L3 comprises an amino acid sequence
selected from the group consisting of SEQ ID NOs: 102 and 130-145,
HVR-H1 comprises an amino acid sequence selected from the group
consisting of SEQ ID NOs: 103 and 146-153, HVR-H2 comprises an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 104 and 154-187, and HVR-H3 comprises an amino acid sequence
selected from the group consisting of SEQ ID NOs: 105 and 188-192.
In some embodiments, the heavy chain comprises the following
framework sequences: FR-H1 comprises EVQLVESGGGLVQPGGSLRLSCAAS (SEQ
ID NO: 197); FR-H2 comprises WVRQAPGKGLEWX.sub.1, wherein X.sub.1
is I or V (SEQ ID NO: 228); FR-H3 comprises
RX.sub.1TX.sub.2SX.sub.3DX.sub.4SX.sub.5X.sub.6TX.sub.7YX.sub.8-
QMNSLRAEDTAVYYC, wherein X.sub.1 is F or V; X.sub.2 is I or L;
X.sub.3 is selected from the group consisting of L, R, and V;
X.sub.4 is K or N; X.sub.5 is selected from the group consisting of
K, N, R, and S; X.sub.6 is N or S; X.sub.7 is selected from the
group consisting of A, L, and V; and X.sub.8 is L or M (SEQ ID NO:
229); and FR-H4 comprises WGQGTLVTVSS (SEQ ID NO: 200). In some
embodiments, the heavy chain comprises the following framework
sequences: FR-H1 comprises EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:
197); FR-H2 comprises WVRQAPGKGLEWV (SEQ ID NO: 198); FR-H3
comprises RFTISRDX.sub.1SKNTX.sub.2YLQMNSLRAEDTAVYYCAR, wherein
X.sub.1 is N or K; and X.sub.2 is selected from the group
consisting of A, L, and V (SEQ ID NO: 230); and FR-H4 comprises
WGQGTLVTVSS (SEQ ID NO: 200). In some embodiments, the light chain
comprises the following framework sequences: FR-L1 comprises
DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 201), FR-L2 comprises
WYQQKPGKAPKLLIY (SEQ ID NO: 202), FR-L3 comprises
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 203), FR-L4 comprises
FGQGTKVEIK (SEQ ID NO: 221) or FGQGTKVEIKR (SEQ ID NO: 204). In
some embodiments, the light chain comprises the variable domain
sequence of 18F7.v6 or 18F7.v6k as shown in FIG. 27 (SEQ ID NOs:
193 and 194). In some embodiments, the heavy chain comprises the
variable domain sequence of 18F7.v6 or 18F7v6k as shown in FIG. 28
(SEQ ID NOs: 195 and 196). In some embodiments, the invention
provides an antibody wherein the light chain comprises the variable
domain sequence of 18F7.v6 as shown in FIG. 27 (SEQ ID NO: 193) and
the heavy chain comprises the variable domain sequence of 18F7.v6
as shown in FIG. 28 (SEQ ID NO: 195). In some embodiments, the
invention provides an antibody wherein the light chain comprises
the variable domain sequence of 18F7.v6k as shown in FIG. 27 (SEQ
ID NO: 194) and the heavy chain comprises the variable domain
sequence of 18F7.v6k as shown in FIG. 28 (SEQ ID NO: 196).
[0012] In some embodiments, the invention provides an antibody
where at least a portion of the framework sequence is a human
consensus framework sequence. In some embodiments, the antibody
comprises human .kappa. subgroup 1 consensus framework sequence. In
some embodiments, the antibody comprises heavy chain human subgroup
III consensus framework sequence.
[0013] In some embodiments, the invention provides an anti-EGFL7
antibody that is a bispecific antibody. In some embodiments, the
bispecific antibody binds to vascular endothelial growth factor
(VEGF), e.g. to the same VEGF epitope as bevacizumab or
ranibizumab.
[0014] In some embodiments, the invention provides a nucleic acid
encoding an antibody of the invention. In some embodiments, the
invention provides a vector comprising such a nucleic acid. In some
embodiments, the invention provides a host cell comprising the
nucleic acid or vector.
[0015] In some embodiments, the invention provides a composition
comprising an antibody of the invention. In some embodiments, the
composition comprises a carrier. In some embodiments, the
composition in a pharmaceutical composition.
[0016] In some embodiments, the invention provides a method for
making an anti-EGFL7 antibody by expressing in a suitable host cell
a vector comprising a nucleic acid encoding an antibody of the
invention and recovering the antibody. In some embodiments, the
host cell is prokaryotic. In some embodiments, the host cell is
eukaryotic.
[0017] In some embodiments, the invention provides a method for
treating a tumor, a cancer, or a cell proliferative disorder, the
method comprising administering an effective amount of an
anti-EGFL7 antibody of the invention to an individual in need of
such treatment. In some embodiments, the invention provides an
anti-EGFL7 antibody for use in the treatment of a tumor, a cancer,
or a cell proliferative disorder. In some embodiments, the cancer
is selected from the group consisting of breast cancer, colorectal
cancer, lung cancer, esophageal cancer, bladder cancer, ovarian
cancer, pancreatic cancer, and hepatocellular carcinoma. In some
embodiments, the cancer is breast cancer, colorectal cancer or lung
cancer. In some embodiments, the cell proliferative disorder is
cancer.
[0018] In some embodiments, the treatment also comprises an
effective amount of a second medicament, wherein the anti-EGFL7
antibody is a first medicament. In some embodiments, the second
medicament is another antibody, a chemotherapeutic agent, a
cytotoxic agent, an anti-angiogenic agent, an immunosuppressive
agent, a prodrug, a cytokine, a cytokine antagonist, cytotoxic
radiotherapy, a corticosteroid, an anti-emetic, a cancer vaccine,
an analgesic, or a growth-inhibitory agent. In some embodiments,
the second medicament is an anti-VEGF antibody, e.g. bevacizumab.
In some embodiments, the second medicament is administered prior to
or subsequent to the administration of the anti-EGFL7 antibody. In
some embodiments, the second medicament is administered
concurrently with the anti-EGFL7 antibody.
[0019] In some embodiments, the invention provides a method of
reducing or inhibiting angiogenesis in a subject having a
pathological condition associated with angiogenesis, comprising
administering to the subject an antibody of the invention, thereby
reducing or inhibiting angiogenesis in the subject. In some
embodiments, the invention provides an antibody of the invention
for use in the treatment of a pathological condition associated
with angiogenesis. In some embodiments, the pathological condition
is a neoplastic condition. In some embodiments, the pathological
condition in a non-neoplastic condition. In some embodiments, the
non-neoplastic condition is selected from the group consisting of
diabetic and other proliferative retinopathies, retinopathy of
prematurity, neovascular glaucoma, age-related macular
degeneration, diabetic macular edema, corneal neovascularization,
corneal graft neovascularization, retinal/choroidal
neovascularization.
[0020] In some embodiments, the invention provides a method of
enhancing efficacy of an anti-angiogenesis agent in a subject
having a pathological condition associated with angiogenesis,
comprising administering to the subject an effective amount of an
antibody of the invention in combination with the anti-angiogenesis
agent, thereby enhancing said anti-angiogenesis agent's inhibitory
activity. In some embodiments, the invention provides an antibody
of the invention for use in enhancing efficacy of an
anti-angiogenesis agent in a subject having a pathological
condition associated with angiogenesis. In some embodiments, the
pathological condition associated with angiogenesis is a tumor,
cancer or cell proliferative disorder. In some embodiments, the
pathological condition associated with angiogenesis in a
non-neoplastic condition. In some embodiments, the non-neoplastic
condition is selected from the group consisting of diabetic and
other proliferative retinopathies, retinopathy of prematurity,
neovascular glaucoma, age-related macular degeneration, diabetic
macular edema, corneal neovascularization, corneal graft
neovascularization, retinal/choroidal neovascularization. In some
embodiments, the anti-angiogenesis agent is administered prior to
or subsequent to the administration of the anti-EGFL7 antibody. In
some embodiments, the anti-angiogenesis agent is administered
concurrently with the anti-EGFL7 antibody. In some embodiments, the
anti-antigenesis agent is an anti-VEGF agent, an anti-VEGF
antibody, e.g. bevacizumab or ranibizumab.
[0021] In some embodiments, the invention provides a method of
reducing or inhibiting perfusion and permeability of a tumor in a
subject, comprising administering to the subject an antibody of the
invention. In some embodiments, the invention provides an antibody
of the invention for use in reducing or inhibiting perfusion and
permeability of a tumor in a subject. In some embodiments, the
method or use further comprises administering an anti-angiogenesis
agent, e.g. an anti-VEGF agent (e.g. an anti-VEGF antibody such as
bevacizumab.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIG. 1 depicts amino acid sequences of EGFL7 from mouse (SEQ
ID NO: 1) and human (SEQ ID ON: 2). The locations of the EMI1,
EMI2, EGF and coiled-coiled domains are indicated. Truncated EGFL7
lacks the coiled-coiled domains. The sequence of peptides EMI1 (SEQ
ID NO: 3), EMI2 (SEQ ID NO: 4) and p2 (SEQ ID NO: 5), P4 (SEQ ID
NO: 6), p5 (SEQ ID NO: 7), and p6 (SEQ ID NO: 8) are underlined.
Peptides EMI2 and p2 are able to prevent 4F11 binding to the
truncated recombinant EGFL7 protein. Truncated huEGFL7 protein
contains positions Methionine 1--Proline 182. Truncated muEGFL7
protein contains positions Methionine 1--Proline 186. The EMI1 and
EMI 2 peptides are underlined with thick lines; the p2, p4, p5, and
p6 peptides are underlined with thin lines.
[0023] FIG. 2 depicts the amino acid sequence of the variable light
domain of the human Kappa I consensus (SEQ ID NO: 9) and 4F11.v1
(SEQ ID NO: 10). Positions are numbered according to Kabat and
hypervariable regions are boxed.
[0024] FIG. 3 depicts the amino acid sequence of the variable heavy
domain of the human subgroup III consensus (SEQ ID NO: 11) and
4F11.v1 (SEQ ID NO: 12). Positions are numbered according to Kabat
and hypervariable regions are boxed.
[0025] FIG. 4 depicts oligonucleotides used to toggle positions in
the Framework Toggle Library. The DNA sequence of 4F11.v1 and the
oligonucleotides used to generate the framework toggle are shown.
The amino acid sequences of the original and some resulting
framework toggle regions are also shown. In some cases additional
amino acid residues were also incorporated based on how the
degenerate codons were designed. Sequence identifiers are shown in
parentheses to the right of the corresponding sequence (SEQ ID NOs:
13-28).
[0026] FIG. 5 depicts results demonstrating that mu4F11 binding to
EGFL7 can be blocked by Peptide 2 (SEQ ID NO: 5), but not by
overlapping Peptides 1 or 3 (SEQ ID NOs: 29 and 30, respectively)
or a random control peptide. Cell lysates from 293 cells
transfected with hEGfl7-His were immunoprecipitated with 4F11 in
the presence or absence of different peptides. Peptide 1:
RSPGLAPARPRYA (SEQ ID NO: 29). Peptide 2: RPRYACCPGWKRT (SEQ ID NO:
5). Peptide 3: GWKRTSGLPGACG (SEQ ID NO: 30). Peptide 4: random
control peptide.
[0027] FIG. 6 depicts binding of phage displaying 4F11.v1 Fab to
truncated EGFL7 immobilized on a microtiter plate. Both samples of
4F11.v1 phage show increased binding to immobilized EGFL7 as a
function of increasing phage concentration. A control phage shows
background binding similar to levels of 4F11.v1 phage at low phage
concentrations suggesting some non-specific phage-EGFL7
interaction.
[0028] FIG. 7 depicts binding of phage displaying 4F11.v1 Fab to
EMI2 domain or p2 peptide biotinylated either through a free amino
or free thiol.
[0029] FIG. 8 depicts the abundance of residues found at each
framework position during the Framework Toggle. Amino acids
introduced at each framework position during the Framework Toggle
are listed.
[0030] FIGS. 9A & 9B depict CDR sequence changes observed in
each of the 6 "single position libraries" (SPLs) for each of the 3
frameworks. Libraries were separated by framework used (4F11.v1,
4F11.v2 and 4F11.v3). Changes obtained from each of the SPLs versus
the particular CDR sequence are highlighted. The VL and VH
sequences outside of these changes were identical to the
corresponding framework and are now shown. Sequence identifiers are
shown in parentheses to the right of the corresponding sequence
(SEQ ID NOs: 31-77). Individual sequences that appear more than
once may not always have a corresponding sequence identifier.
[0031] FIG. 10 depicts the framework and library design for the
variable light domain of limited libraries 1 and 2. The amino acid
sequence of the human Kappa I consensus (SEQ ID NO: 9) and 4F11.v1
(SEQ ID NO: 10) are shown compared to the template used for library
1 (SEQ ID NO: 78) and library 2 (SEQ ID NO: 79). Positions that
were randomized to all 20 amino acids are shown with slash through
the amino acid.
[0032] FIG. 11 depicts the framework and library design for the
variable heavy domain of limited libraries 1 and 2. The amino acid
sequence of the human subgroup III consensus (SEQ ID NO: 11) and
4F11.v1 (SEQ ID NO: 12) are shown compared to the template used for
library 1 (SEQ ID NO: 80) and library 2 (SEQ ID NO: 81). Positions
that were randomized to all 20 amino acids are shown with slash
through the amino acid.
[0033] FIG. 12 depicts the frequency of changes observed at
randomized positions in limited libraries 1 and 2. The preference
of amino acids selected at positions 53 and 54 in the light chain
and 29, 52, and 98 in the heavy chain is shown. The preference
(Sigma) for any amino acid is reported as the number of standard
deviations above a random chance occurrence of a given residue in
the library assuming a binomial distribution of amino acids.
Scoring by this method accounts for the expected codon bias and
sampling statistics when establishing a consensus.
[0034] FIGS. 13 & 14 depict inhibition of HUVEC adhesion to
immobilized human or mouse EGFL7 in vitro by humanized 4F11
variants. HUVECs (20,000 cells/well) were allowed to adhere to 96
well plates coated with 5 .mu.g/ml human or murine EGFL7 in the
presence of increasing concentrations of antibody. The number of
cells that still adhered to the plates after washing were counted
and calculated as percent of the total cells plated into each
well.
[0035] FIG. 15 depicts the amino acid sequence of the variable
light domain of the human Kappa I consensus (SEQ ID NO: 9), 4F11.v1
(SEQ ID NO: 10), 4F11.v17 (SEQ ID NO: 82), and 4F11.v22 (SEQ ID NO:
83). Positions are numbered according to Kabat and hypervariable
regions (SEQ ID NOs: 234-236) are boxed.
[0036] FIG. 16 depicts the amino acid sequence of the variable
heavy domain of the human subgroup III consensus (SEQ ID NO: 11),
4F11.v1 (SEQ ID NO: 12), 4F11.v17 (SEQ ID NO: 84), and 4F11.v22
(SEQ ID NO: 85; SEQ ID NOs: 238-240). Positions are numbered
according to Kabat and hypervariable regions (SEQ ID NOs: 34, 35,
237) are boxed.
[0037] FIG. 17 depicts the amino acid sequence of the variable
light domain of the human Kappa I consensus (SEQ ID NO: 9) and the
18F7-graft (SEQ ID NO: 86). Positions are numbered according to
Kabat and hypervariable regions are boxed.
[0038] FIG. 18 depicts the amino acid sequence of the variable
heavy domain of the human subgroup III consensus (SEQ ID NO: 11)
and the 18F7-graft (SEQ ID NO: 87). Positions are numbered
according to Kabat and hypervariable regions are boxed.
[0039] FIG. 19 depicts oligonucleotides used to toggle positions in
the Framework Toggle Library. The DNA sequence of 18F7-graft and
the oligonucleotides used to generate the framework toggle are
shown. The amino acid sequences of the original and some resulting
framework toggle regions are also shown. In some cases additional
amino acid residues were also incorporated based on how the
degenerate codons were designed. Sequence identifiers are shown in
parentheses to the right of the corresponding sequence (SEQ ID NOs:
88-99).
[0040] FIG. 20 depicts results demonstrating that mu18F7 binding to
EGFL7 can be blocked by EMI1 (SEQ ID NO: 3) or Peptide P5 (SEQ ID
NO: 7), but not by Peptides P4 or P6 (SEQ ID NOs: 6 and 8,
respectively). Chicken embryonic fibroblasts were transfected with
a plasmid containing the HA-tagged full-length human EGFL7 cDNA.
Cell lysate prepared from transfected cells was immunoprecipitated
with mu18F7 in the presence of 200-fold excess competitive
peptides. Immunoprecipitates were analyzed by western blot using an
anti-HA antibody.
[0041] FIG. 21 depicts binding of phage displaying 18F7-graft Fab
to truncated huEGFL7 immobilized on a microtiter plate. Both
samples of 18F7-graft phage show increased binding to immobilized
EGFL7 as a function of increasing phage concentration. A control
phage shows background binding similar to levels of 18F7-graft
phage at low phage concentrations suggesting some non-specific
phage-EGFL7 interaction.
[0042] FIG. 22 depicts binding of phage displaying 18F7-graft Fab
to EMI1 domain or p5 peptide biotinylated either through a free
amino or free thiol.
[0043] FIG. 23 depicts the abundance of residues found at each
framework position during the Framework Toggle. Amino acids
introduced at each framework position during the Framework Toggle
are listed.
[0044] FIGS. 24A and 24B depict CDR sequence changes observed in
each of the 6 SPLs for each of the 3 frameworks. Libraries were
separated by framework used (18F7-graft, 18F7-K, 18F7-KV, and
18F7-KA). Changes obtained from each of the SPLs versus the
particular CDR sequence are highlighted. The VL and VH sequences
outside of these changes were identical to the corresponding
framework and are now shown. Sequence identifiers are shown in
parentheses to the right of the corresponding sequence (SEQ ID NOs:
100-192). Individual sequences that appear more than once may not
always have a corresponding sequence identifier.
[0045] FIG. 25 depicts inhibition of HUVEC adhesion to immobilized
human or mouse EGFL7 in vitro by humanized 18F7 variants. HUVECs
(20,000 cells/well) were allowed to adhere to 96 well plates coated
with 5 .mu.g/ml human or murine EGFL7 in the presence of increasing
concentrations of antibody. The number of cells that still adhered
to the plates after washing were counted and calculated as percent
of the total cells plated into each well.
[0046] FIG. 26 depicts inhibition of HUVEC transwell migration.
HUVECs (50,000 cells per well) were grown for 16 hours in the top
chambers of transwell plates, and the membranes in the top chamber
were coated with 5 .mu.g/ml recombinant human EGFL7 protein.
Various concentrations of control antibody (anti-IgE) or different
variants of 18F7 were added to the culture medium in the top and
bottom chambers, whereas 20 ng/ml of recombinant human VEGF-165 was
added in the bottom wells to stimulate HUVEC migration. Cells
migrated to the undersides of the top chambers were counted and
plotted against the treatments (antibodies and concentrations).
[0047] FIG. 27 depicts the amino acid sequence of the variable
light domain of the human Kappa I consensus (SEQ ID NO: 9), 18F7.v6
(SEQ ID NO: 193), and 18F7.v6k (SEQ ID NO: 194). Positions are
numbered according to Kabat and hypervariable regions (SEQ ID NOs:
241, 101, 131) are boxed. Where sequence for 18F7.v6k is not shown
(in the second and third parts of the alignment), the corresponding
sequence is identical to the sequence for 18F7.v6.
[0048] FIG. 28 depicts the amino acid sequence of the variable
heavy domain of the human subgroup III consensus (SEQ ID NO: 11),
18F7.v6 (SEQ ID NO: 195), and 18F7.v6k (SEQ ID NO: 196). Positions
are numbered according to Kabat and hypervariable regions (SEQ ID
NOs: 103, 242, 105) are boxed. Where sequence for 18F7.v6k is not
shown (in the first and third parts of the alignment), the
corresponding sequence is identical to the sequence for
18F7.v6.
[0049] FIG. 29 depicts inhibition of H1299 xenograft tumor growth
using hu18F7.v6k alone and in combination with an anti-VEGF
antibody. HRLN female nu/nu mice were injected with
1.times.10.sup.7 H1299 human non-small cell lung cancer tumor cells
subcutaneously and allowed to develop tumors to 80-120 mm.sup.3 in
sizes. Tumor bearing mice were then randomly separated into four
groups (12 mice/group) so that the average tumor volume of each
group was at 122 mm.sup.3. These mice were then treated as: group 1
received anti-VEGF MAb (B20.4.1) at 10 mg/kg once/week; group 2
received control IgG (anti-ragweed) at 10 mg/kg once/week and
anti-EGFL7 MAb (h18F7.v6k) at 10 mg/kg twice/week; group 3 received
B20.4.1 at 10 mg/kg once/week+h18F7.v6k at 10 mg/kg twice/week;
group 4 was untreated. Tumors were measured twice/week with a
caliper and tumor volumes were calculated as "w.sup.2.times.1/2"
mm.sup.3 (w=tumor width in mm, 1=tumor length in mm). Mice were
euthanized when their tumors reached 1000 mm.sup.3 (this is defined
as "mice reached end point"). Group average (mean) tumor volumes
+/-standard errors (SEM) were plotted against time until
.gtoreq.50% of mice reached end point.
[0050] FIG. 30 depicts inhibition of LXFL 1674 xenograft tumor
growth using hu18F7.v6k alone and in combination with an anti-VEGF
antibody.
[0051] FIG. 31 depicts inhibition of neonatal trachea
vascularization using hu18F7.v6k alone and in combination with an
anti-VEGF antibody. Note: not all Ragweed control animals were
quantified since data distribution was reasonably tight in this
group.
DETAILED DESCRIPTION OF THE INVENTION
[0052] The invention provides methods, compositions, kits and
articles of manufacture for anti-EGFL7 antibodies.
[0053] Details of these methods, compositions, kits and articles of
manufacture are provided herein.
General Techniques
[0054] The techniques and procedures described or referenced herein
are generally well understood and commonly employed using
conventional methodology by those skilled in the art, such as, for
example, the widely utilized methodologies described in Sambrook et
al., Molecular Cloning: A Laboratory Manual 3rd. edition (2001)
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
CURRENT PROTOCOLS 1N MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds.,
(2003)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.):
PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G.
R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A
LABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed.
(1987)).
DEFINITIONS
[0055] An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the antibody will be purified (1) to greater than 95%
by weight of antibody as determined by the Lowry method, and most
preferably more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. Isolated antibody
includes the antibody in situ within recombinant cells since at
least one component of the antibody's natural environment will not
be present. Ordinarily, however, isolated antibody will be prepared
by at least one purification step.
[0056] An "isolated" nucleic acid molecule is a nucleic acid
molecule that is identified and separated from at least one
contaminant nucleic acid molecule with which it is ordinarily
associated in the natural source of the antibody nucleic acid. An
isolated nucleic acid molecule is other than in the form or setting
in which it is found in nature. Isolated nucleic acid molecules
therefore are distinguished from the nucleic acid molecule as it
exists in natural cells. However, an isolated nucleic acid molecule
includes a nucleic acid molecule contained in cells that ordinarily
express the antibody where, for example, the nucleic acid molecule
is in a chromosomal location different from that of natural
cells.
[0057] The term "anti-EGFL7 antibody" or "an antibody that binds to
EGFL7" refers to an antibody that is capable of binding EGFL7 with
sufficient affinity such that the antibody is useful as a
diagnostic and/or therapeutic agent in targeting EGFL7. In certain
embodiments, an antibody that binds to EGFL7 has a dissociation
constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM, .ltoreq.10 nM,
.ltoreq.1 nM, or .ltoreq.0.1 nM.
[0058] "Binding affinity" generally refers to the strength of the
sum total of noncovalent interactions between a single binding site
of a molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (Kd).
Affinity can be measured by common methods known in the art,
including those described herein. Low-affinity antibodies generally
bind antigen slowly and tend to dissociate readily, whereas
high-affinity antibodies generally bind antigen faster and tend to
remain bound longer. A variety of methods of measuring binding
affinity are known in the art, any of which can be used for
purposes of the present invention. Specific illustrative
embodiments are described in the following.
[0059] In one embodiment, the "Kd" or "Kd value" according to this
invention is measured by a radiolabeled antigen binding assay (RIA)
performed with the Fab version of an antibody of interest and its
antigen as described by the following assay that measures solution
binding affinity of Fabs for antigen by equilibrating Fab with a
minimal concentration of (.sup.125I-labeled antigen in the presence
of a titration series of unlabeled antigen, then capturing bound
antigen with an anti-Fab antibody-coated plate (Chen, et al.,
(1999) J. Mol. Biol 293:865-881). To establish conditions for the
assay, microtiter plates (Dynex) are coated overnight with 5
.mu.g/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM
sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v)
bovine serum albumin in PBS for two to five hours at room
temperature (approximately 23.degree. C.). In a non-adsorbant plate
(Nunc #269620), 100 pM or 26 pM [.sup.125I]-antigen are mixed with
serial dilutions of a Fab of interest (e.g., consistent with
assessment of an anti-VEGF antibody, Fab-12, in Presta et al.,
(1997) Cancer Res. 57:4593-4599). The Fab of interest is then
incubated overnight; however, the incubation may continue for a
longer period (e.g., 65 hours) to insure that equilibrium is
reached. Thereafter, the mixtures are transferred to the capture
plate for incubation at room temperature (e.g., for one hour). The
solution is then removed and the plate washed eight times with 0.1%
Tween.TM.-20 in PBS. When the plates have dried, 150 .mu.l/well of
scintillant (MicroScint.TM.-20; Packard) is added, and the plates
are counted on a TopCount gamma counter (Packard) for ten minutes.
Concentrations of each Fab that give less than or equal to 20% of
maximal binding are chosen for use in competitive binding assays.
According to another embodiment the Kd or Kd value is measured by
using surface plasmon resonance assays using a BIAcore.TM.-2000 or
a BIAcore.TM.-3000 (BIAcore, Inc., Piscataway, N.J.) at 25.degree.
C. with immobilized antigen CMS chips at .about.10 response units
(RU). Briefly, carboxymethylated dextran biosensor chips (CM5,
BIAcore Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8,
into 5 .mu.g/ml (-0.2 .mu.M) before injection at a flow rate of 5
.mu.l/minute to achieve approximately 10 response units (RU) of
coupled protein. Following the injection of antigen, 1M
ethanolamine is injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM)
are injected in PBS with 0.05% Tween.TM. 20 (PBST) at 25.degree. C.
at a flow rate of approximately 25 .mu.l/min. Association rates
(k.sub.on) and dissociation rates (k.sub.off) are calculated using
a simple one-to-one Langmuir binding model (BIAcore.TM. Evaluation
Software version 3.2) by simultaneous fitting the association and
dissociation sensorgram. The equilibrium dissociation constant (Kd)
is calculated as the ratio k.sub.off/k.sub.on. See, e.g., Chen, Y.,
et al., (1999) J. Mol. Biol. 293:865-881. If the on-rate exceeds
10.sup.6 M.sup.-1 S.sup.-1 by the surface plasmon resonance assay
above, then the on-rate can be determined by using a fluorescent
quenching technique that measures the increase or decrease in
fluorescence emission intensity (excitation=295 nm; emission =340
nm, 16 nm band-pass) at 25.degree. C. of a 20 nM anti-antigen
antibody (Fab form) in PBS, pH 7.2, in the presence of increasing
concentrations of antigen as measured in a spectrometer, such as a
stop-flow equipped spectrophotometer (Aviv Instruments) or a
8000-series SLM Aminco spectrophotometer (ThermoSpectronic) with a
stirred cuvette.
[0060] The term "vector," as used herein, is intended to refer to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked. One type of vector is a "plasmid",
which refers to a circular double stranded DNA loop into which
additional DNA segments may be ligated. Another type of vector is a
phage vector. Another type of vector is a viral vector, wherein
additional DNA segments may be ligated into the viral genome.
Certain vectors are capable of autonomous replication in a host
cell into which they are introduced (e.g., bacterial vectors having
a bacterial origin of replication and episomal mammalian vectors).
Other vectors (e.g., non-episomal mammalian vectors) can be
integrated into the genome of a host cell upon introduction into
the host cell, and thereby to are replicated along with the host
genome. Moreover, certain vectors are capable of directing the
expression of genes to which they are operatively linked. Such
vectors are referred to herein as "recombinant expression vectors"
(or simply, "recombinant vectors" or "expression vectors"). In
general, expression vectors of utility in recombinant DNA
techniques are often in the form of plasmids. In the present
specification, "plasmid" and "vector" may be used
interchangeably.
[0061] "Polynucleotide," or "nucleic acid," as used interchangeably
herein, refer to polymers of nucleotides of any length, and include
DNA and RNA. The nucleotides can be deoxyribonucleotides,
ribonucleotides, modified nucleotides or bases, and/or their
analogs, or any substrate that can be incorporated into a polymer
by DNA or RNA polymerase, or by a synthetic reaction. A
polynucleotide may comprise modified nucleotides, such as
methylated nucleotides and their analogs. If present, modification
to the nucleotide structure may be imparted before or after
assembly of the polymer. The sequence of nucleotides may be
interrupted by non-nucleotide components. A polynucleotide may be
further modified after synthesis, such as by conjugation with a
label. Other types of modifications include, for example, "caps",
substitution of one or more of the naturally occurring nucleotides
with an analog, internucleotide modifications such as, for example,
those with uncharged linkages (e.g., methyl phosphonates,
phosphotriesters, phosphoamidates, carbamates, etc.) and with
charged linkages (e.g., phosphorothioates, phosphorodithioates,
etc.), those containing pendant moieties, such as, for example,
proteins (e.g., nucleases, toxins, antibodies, signal peptides,
poly-L-lysine, etc.), those with intercalators (e.g., acridine,
psoralen, etc.), those containing chelators (e.g., metals,
radioactive metals, boron, oxidative metals, etc.), those
containing alkylators, those with modified linkages (e.g., alpha
anomeric nucleic acids, etc.), as well as unmodified forms of the
polynucleotide(s). Further, any of the hydroxyl groups ordinarily
present in the sugars may be replaced, for example, by phosphonate
groups, phosphate groups, protected by standard protecting groups,
or activated to prepare additional linkages to additional
nucleotides, or may be conjugated to solid or semi-solid supports.
The 5' and 3' terminal OH can be phosphorylated or substituted with
amines or organic capping group moieties of from 1 to 20 carbon
atoms. Other hydroxyls may also be derivatized to standard
protecting groups. Polynucleotides can also contain analogous forms
of ribose or deoxyribose sugars that are generally known in the
art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-
or 2'-azido-ribose, carbocyclic sugar analogs, alpha-anomeric
sugars, epimeric sugars such as arabinose, xyloses or lyxoses,
pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs
and a basic nucleoside analogs such as methyl riboside. One or more
phosphodiester linkages may be replaced by alternative linking
groups. These alternative linking groups include, but are not
limited to, embodiments wherein phosphate is replaced by P(O)S
("thioate"), P(S)S ("dithioate"), "(O)NR.sub.2 ("amidate"), P(O)R,
P(O)OR', CO or CH.sub.2 ("formacetal"), in which each R or R' is
independently H or substituted or unsubstituted alkyl (1-20 C)
optionally containing an ether (--O--) linkage, aryl, alkenyl,
cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a
polynucleotide need be identical. The preceding description applies
to all polynucleotides referred to herein, including RNA and
DNA.
[0062] "Oligonucleotide," as used herein, generally refers to
short, generally single stranded, generally synthetic
polynucleotides that are generally, but not necessarily, less than
about 200 nucleotides in length. The terms "oligonucleotide" and
"polynucleotide" are not mutually exclusive. The description above
for polynucleotides is equally and fully applicable to
oligonucleotides.
[0063] The term "EGFL7" (interchangeably termed "EGF-like-domain,
multiple 7"), as used herein, refers, unless specifically or
contextually indicated otherwise, to any native or variant (whether
native or synthetic) EGFL7 polypeptide. The term "native sequence"
specifically encompasses naturally occurring truncated or secreted
forms (e.g., an extracellular domain sequence), naturally occurring
variant forms (e.g., alternatively spliced forms) and
naturally-occurring allelic variants. The term "wild type EGFL7"
generally refers to a polypeptide comprising the amino acid
sequence of a naturally occurring EGFL7 protein. The term "wild
type EGFL7 sequence" generally refers to an amino acid sequence
found in a naturally occurring EGFL7.
[0064] The terms "antibody" and "immunoglobulin" are used
interchangeably in the broadest sense and include monoclonal
antibodies (for e.g., full length or intact monoclonal antibodies),
polyclonal antibodies, multivalent antibodies, multispecific
antibodies (e.g., bispecific antibodies so long as they exhibit the
desired biological activity) and may also include certain antibody
fragments (as described in greater detail herein). An antibody can
be human, humanized and/or affinity matured.
[0065] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
complementarity-determining regions or hypervariable regions (CDRs
or HVRs, used interchangeably herein) both in the light-chain and
the heavy-chain variable domains. The more highly conserved
portions of variable domains are called the framework (FR). The
variable domains of native heavy and light chains each comprise
four FR regions, largely adopting a (3-sheet configuration,
connected by three HVRs, which form loops connecting, and in some
cases forming part of, the (3-sheet structure. The HVRs in each
chain are held together in close proximity by the FR regions and,
with the HVRs from the other chain, contribute to the formation of
the antigen-binding site of antibodies (see Kabat et al., Sequences
of Proteins of Immunological Interest, Fifth Edition, National
Institute of Health, Bethesda, Md. (1991)). The constant domains
are not involved directly in binding an antibody to an antigen, but
exhibit various effector functions, such as participation of the
antibody in antibody-dependent cellular toxicity.
[0066] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab').sub.2 fragment that has two antigen-combining
sites and is still capable of cross-linking antigen.
[0067] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and -binding site. In a two-chain Fv
species, this region consists of a dimer of one heavy- and one
light-chain variable domain in tight, non-covalent association. In
a single-chain Fv species, one heavy- and one light-chain variable
domain can be covalently linked by a flexible peptide linker such
that the light and heavy chains can associate in a "dimeric"
structure analogous to that in a two-chain Fv species. It is in
this configuration that the three HVRs of each variable domain
interact to define an antigen-binding site on the surface of the
VH-VL dimer. Collectively, the six HVRs confer antigen-binding
specificity to the antibody. However, even a single variable domain
(or half of an Fv comprising only three HVRs specific for an
antigen) has the ability to recognize and bind antigen, although at
a lower affinity than the entire binding site.
[0068] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CH1) of the heavy chain.
Fab' fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear a free thiol group.
F(ab').sub.2 antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0069] The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called kappa (.kappa.) and lambda (.lamda.), based on the
amino acid sequences of their constant domains.
[0070] Depending on the amino acid sequence of the constant domain
of their heavy chains, immunoglobulins can be assigned to different
classes. There are five major classes of immunoglobulins: IgA, IgD,
IgE, IgG, and IgM, and several of these can be further divided into
subclasses (isotypes), e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3,
IgG.sub.4, IgA.sub.1, and IgA.sub.2. The heavy-chain constant
domains that correspond to the different classes of immunoglobulins
are called .alpha., .delta., .epsilon., .gamma., and .mu.,
respectively. The subunit structures and three-dimensional
configurations of different classes of immunoglobulins are well
known.
[0071] "Antibody fragments" comprise only a portion of an intact
antibody, wherein the portion preferably retains at least one,
preferably most or all, of the functions normally associated with
that portion when present in an intact antibody. Examples of
antibody fragments include Fab, Fab', F(ab')2, and Fv fragments;
diabodies; linear antibodies; single-chain antibody molecules; and
multispecific antibodies formed from antibody fragments. In one
embodiment, an antibody fragment comprises an antigen binding site
of the intact antibody and thus retains the ability to bind
antigen. In another embodiment, an antibody fragment, for example
one that comprises the Fc region, retains at least one of the
biological functions normally associated with the Fc region when
present in an intact antibody, such as FcRn binding, antibody half
life modulation, ADCC function and complement binding. In one
embodiment, an antibody fragment is a monovalent antibody that has
an in vivo half life substantially similar to an intact antibody.
For e.g., such an antibody fragment may comprise on antigen binding
arm linked to an Fc sequence capable of conferring in vivo
stability to the fragment.
[0072] The term "hypervariable region," "HVR," or "HV," when used
herein refers to the regions of an antibody variable domain which
are hypervariable in sequence and/or form structurally defined
loops. Generally, antibodies comprise six HVRs; three in the VH
(H1, H2, H3), and three in the VL (L1, L2, L3). In native
antibodies, H3 and L3 display the most diversity of the six HVRs,
and H3 in particular is believed to play a unique role in
conferring fine specificity to antibodies. See, e.g., Xu et al.,
Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular
Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003).
Indeed, naturally occurring camelid antibodies consisting of a
heavy chain only are functional and stable in the absence of light
chain. See, e.g., Hamers-Casterman et al., Nature 363:446-448
(1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).
[0073] A number of HVR delineations are in use and are encompassed
herein. The Kabat Complementarity Determining Regions (CDRs) are
based on sequence variability and are the most commonly used (Kabat
et al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991)). Chothia refers instead to the location of the structural
loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM
HVRs represent a compromise between the Kabat HVRs and Chothia
structural loops, and are used by Oxford Molecular's AbM antibody
modeling software. The "contact" HVRs are based on an analysis of
the available complex crystal structures. The residues from each of
these HVRs are noted below.
TABLE-US-00001 Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34
L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97
L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B
(Kabat Numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia
Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102
H96-H101 H93-H101
[0074] HVRs may comprise "extended HVRs" as follows: 24-36 or 24-34
(L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and
26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3)
in the VH. The variable domain residues are numbered according to
Kabat et al., supra, for each of these definitions.
[0075] "Framework" or "FR" residues are those variable domain
residues other than the hypervariable region residues as herein
defined.
[0076] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. In one embodiment, a humanized antibody
is a human immunoglobulin (recipient antibody) in which residues
from a HVR of the recipient are replaced by residues from a HVR of
a non-human species (donor antibody) such as mouse, rat, rabbit, or
nonhuman primate having the desired specificity, affinity, and/or
capacity. In some instances, FR residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Furthermore, humanized antibodies may comprise residues that are
not found in the recipient antibody or in the donor antibody. These
modifications may be made to further refine antibody performance.
In general, a humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin, and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally will also comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see, e.g., Jones et al.,
Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329
(1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See
also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma &
Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions
23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433
(1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.
[0077] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible mutations, e.g.,
naturally occurring mutations, that may be present in minor
amounts. Thus, the modifier "monoclonal" indicates the character of
the antibody as not being a mixture of discrete antibodies. In
certain embodiments, such a monoclonal antibody typically includes
an antibody comprising a polypeptide sequence that binds a target,
wherein the target-binding polypeptide sequence was obtained by a
process that includes the selection of a single target binding
polypeptide sequence from a plurality of polypeptide sequences. For
example, the selection process can be the selection of a unique
clone from a plurality of clones, such as a pool of hybridoma
clones, phage clones, or recombinant DNA clones. It should be
understood that a selected target binding sequence can be further
altered, for example, to improve affinity for the target, to
humanize the target binding sequence, to improve its production in
cell culture, to reduce its immunogenicity in vivo, to create a
multispecific antibody, etc., and that an antibody comprising the
altered target binding sequence is also a monoclonal antibody of
this invention. In contrast to polyclonal antibody preparations,
which typically include different antibodies directed against
different determinants (epitopes), each monoclonal antibody of a
monoclonal antibody preparation is directed against a single
determinant on an antigen. In addition to their specificity,
monoclonal antibody preparations are advantageous in that they are
typically uncontaminated by other immunoglobulins.
[0078] The modifier "monoclonal" indicates the character of the
antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by a variety of techniques, including, for
example, the hybridoma method (e.g., Kohler and Milstein, Nature,
256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995),
Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor
Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal
Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)),
recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567),
phage-display technologies (see, e.g., Clackson et al., Nature,
352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597
(1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et
al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl.
Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J.
Immunol. Methods 284(1-2): 119-132 (2004), and technologies for
producing human or human-like antibodies in animals that have parts
or all of the human immunoglobulin loci or genes encoding human
immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096;
WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad.
Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258
(1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat.
Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and
5,661,016; Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg
et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813
(1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996);
Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and
Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).
[0079] The monoclonal antibodies herein specifically include
"chimeric" antibodies in which a portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc.
Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies
include PRIMATIZED.RTM. antibodies wherein the antigen-binding
region of the antibody is derived from an antibody produced by,
e.g., immunizing macaque monkeys with the antigen of interest.
[0080] "Single-chain Fv" or "scFv" antibody fragments comprise the
VH and VL domains of antibody, wherein these domains are present in
a single polypeptide chain. Generally, the scFv polypeptide further
comprises a polypeptide linker between the VH and VL domains which
enables the scFv to form the desired structure for antigen binding.
For a review of scFv see Pluckthun, in The Pharmacology of
Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
Springer-Verlag, New York, pp. 269-315 (1994).
[0081] An "antigen" is a predetermined antigen to which an antibody
can selectively bind. The target antigen may be polypeptide,
carbohydrate, nucleic acid, lipid, hapten or other naturally
occurring or synthetic compound.
[0082] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy-chain
variable domain (VH) connected to a light-chain variable domain
(VL) in the same polypeptide chain (VH-VL). By using a linker that
is too short to allow pairing between the two domains on the same
chain, the domains are forced to pair with the complementary
domains of another chain and create two antigen-binding sites.
Diabodies are described more fully in, for example, EP 404,097; WO
93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993). Triabodies and tetrabodies are also described
in Hudson et al., Nat. Med. 9:129-134 (2003).
[0083] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human and/or has been made using any of the techniques for making
human antibodies as disclosed herein. This definition of a human
antibody specifically excludes a humanized antibody comprising
non-human antigen-binding residues. Human antibodies can be
produced using various techniques known in the art, including
phage-display libraries. Hoogenboom and Winter, J. Mol. Biol.,
227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also
available for the preparation of human monoclonal antibodies are
methods described in Cole et al., Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol.,
147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr.
Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be
prepared by administering the antigen to a transgenic animal that
has been modified to produce such antibodies in response to
antigenic challenge, but whose endogenous loci have been disabled,
e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and
6,150,584 regarding XENOMOUSE.TM. technology). See also, for
example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562
(2006) regarding human antibodies generated via a human B-cell
hybridoma technology.
[0084] The term "variable domain residue numbering as in Kabat" or
"amino acid position numbering as in Kabat," and variations
thereof, refers to the numbering system used for heavy chain
variable domains or light chain variable domains of the compilation
of antibodies in Kabat et al., supra. Using this numbering system,
the actual linear amino acid sequence may contain fewer or
additional amino acids corresponding to a shortening of, or
insertion into, a FR or HVR of the variable domain. For example, a
heavy chain variable domain may include a single amino acid insert
(residue 52a according to Kabat) after residue 52 of H2 and
inserted residues (e.g. residues 82a, 82b, and 82c, etc. according
to Kabat) after heavy chain FR residue 82. The Kabat numbering of
residues may be determined for a given antibody by alignment at
regions of homology of the sequence of the antibody with a
"standard" Kabat numbered sequence.
[0085] The Kabat numbering system is generally used when referring
to a residue in the variable domain (approximately residues 1-107
of the light chain and residues 1-113 of the heavy chain) (e.g.,
Kabat et al., Sequences of Immunological Interest. 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.
(1991)). The "EU numbering system" or "EU index" is generally used
when referring to a residue in an immunoglobulin heavy chain
constant region (e.g., the EU index reported in Kabat et al.,
supra). The "EU index as in Kabat" refers to the residue numbering
of the human IgG1 EU antibody. Unless stated otherwise herein,
references to residue numbers in the variable domain of antibodies
means residue numbering by the Kabat numbering system. Unless
stated otherwise herein, references to residue numbers in the
constant domain of antibodies means residue numbering by the EU
numbering system (e.g., see U.S. Provisional Application No.
60/640,323, Figures for EU numbering).
[0086] A "blocking" antibody or an "antagonist" antibody is one
which inhibits or reduces biological activity of the antigen it
binds. Certain blocking antibodies or antagonist antibodies
substantially or completely inhibit the biological activity of the
antigen.
[0087] The term "substantially similar" or "substantially the
same," as used herein, denotes a sufficiently high degree of
similarity between two numeric values (for example, one associated
with an antibody of the invention and the other associated with a
reference/comparator antibody), such that one of skill in the art
would consider the difference between the two values to be of
little or no biological and/or statistical significance within the
context of the biological characteristic measured by said values
(e.g., Kd values). The difference between said two values is, for
example, less than about 50%, less than about 40%, less than about
30%, less than about 20%, and/or less than about 10% as a function
of the reference/comparator value.
[0088] The phrase "substantially reduced," or "substantially
different," as used herein, denotes a sufficiently high degree of
difference between two numeric values (generally one associated
with a molecule and the other associated with a
reference/comparator molecule) such that one of skill in the art
would consider the difference between the two values to be of
statistical significance within the context of the biological
characteristic measured by said values (e.g., Kd values). The
difference between said two values is, for example, greater than
about 10%, greater than about 20%, greater than about 30%, greater
than about 40%, and/or greater than about 50% as a function of the
value for the reference/comparator molecule.
[0089] Antibody "effector functions" refer to those biological
activities attributable to the Fc region (a native sequence Fc
region or amino acid sequence variant Fc region) of an antibody,
and vary with the antibody isotype. Examples of antibody effector
functions include: Clq binding and complement dependent
cytotoxicity (CDC); Fc receptor binding; antibody-dependent
cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of
cell surface receptors (e.g. B cell receptor); and B cell
activation.
[0090] The term "Fc region" herein is used to define a C-terminal
region of an immunoglobulin heavy chain, including native sequence
Fc regions and variant Fc regions. Although the boundaries of the
Fc region of an immunoglobulin heavy chain might vary, the human
IgG heavy chain Fc region is usually defined to stretch from an
amino acid residue at position Cys226, or from Pro230, to the
carboxyl-terminus thereof. The C-terminal lysine (residue 447
according to the EU numbering system) of the Fc region may be
removed, for example, during production or purification of the
antibody, or by recombinantly engineering the nucleic acid encoding
a heavy chain of the antibody. Accordingly, a composition of intact
antibodies may comprise antibody populations with all K447 residues
removed, antibody populations with no K447 residues removed, and
antibody populations having a mixture of antibodies with and
without the K447 residue.
[0091] A "functional Fc region" possesses an "effector function" of
a native sequence Fc region. Exemplary "effector functions" include
Clq binding; CDC; Fc receptor binding; ADCC; phagocytosis; down
regulation of cell surface receptors (e.g. B cell receptor; BCR),
etc. Such effector functions generally require the Fc region to be
combined with a binding to domain (e.g., an antibody variable
domain) and can be assessed using various assays as disclosed, for
example, in definitions herein.
[0092] A "native sequence Fc region" comprises an amino acid
sequence identical to the amino acid sequence of an Fc region found
in nature. Native sequence human Fc regions include a native
sequence human IgG1 Fc region (non-A and A allotypes); native
sequence human IgG2 Fc region; native sequence human IgG3 Fc
region; and native sequence human IgG4 Fc region as well as
naturally occurring variants thereof.
[0093] A "variant Fc region" comprises an amino acid sequence which
differs from that of a native sequence Fc region by virtue of at
least one amino acid modification, preferably one or more amino
acid substitution(s). Preferably, the variant Fc region has at
least one amino acid substitution compared to a native sequence Fc
region or to the Fc region of a parent polypeptide, e.g. from about
one to about ten amino acid substitutions, and preferably from
about one to about five amino acid substitutions in a native
sequence Fc region or in the Fc region of the parent polypeptide.
The variant Fc region herein will preferably possess at least about
80% homology with a native sequence Fc region and/or with an Fc
region of a parent polypeptide, and most preferably at least about
90% homology therewith, more preferably at least about 95% homology
therewith.
[0094] "Fc receptor" or "FcR" describes a receptor that binds to
the Fc region of an antibody. In some embodiments, an FcR is a
native human FcR. In some embodiments, an FcR is one which binds an
IgG antibody (a gamma receptor) and includes receptors of the
Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII subclasses, including
allelic variants and alternatively spliced forms of those
receptors. Fc.gamma.RII receptors include Fc.gamma.RIIA (an
"activating receptor") and Fc.gamma.RIIB (an "inhibiting
receptor"), which have similar amino acid sequences that differ
primarily in the cytoplasmic domains thereof. Activating receptor
Fc.gamma.RIIA contains an immunoreceptor tyrosine-based activation
motif (ITAM) in its cytoplasmic domain. Inhibiting receptor
Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibition
motif (ITIM) in its cytoplasmic domain. (see, e.g., Daeron, Annu.
Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example,
in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et
al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab.
Clin. Med. 126:330-41 (1995). Other FcRs, including those to be
identified in the future, are encompassed by the term "FcR"
herein.
[0095] The term "Fc receptor" or "FcR" also includes the neonatal
receptor, FcRn, which is responsible for the transfer of maternal
IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim
et al., J. Immunol. 24:249 (1994)) and regulation of homeostasis of
immunoglobulins. Methods of measuring binding to FcRn are known
(see, e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997);
Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton
et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004/92219
(Hinton et al.).
[0096] Binding to human FcRn in vivo and serum half life of human
FcRn high affinity binding polypeptides can be assayed, e.g., in
transgenic mice or transfected human cell lines expressing human
FcRn, or in primates to which the polypeptides with a variant Fc
region are administered. WO 2000/42072 (Presta) describes antibody
variants with improved or diminished binding to FcRs. See also,
e.g., Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).
[0097] "Human effector cells" are leukocytes which express one or
more FcRs and perform effector functions. In certain embodiments,
the cells express at least Fc.gamma.RIII and perform ADCC effector
function(s). Examples of human leukocytes which mediate ADCC
include peripheral blood mononuclear cells (PBMC), natural killer
(NK) cells, monocytes, cytotoxic T cells, and neutrophils. The
effector cells may be isolated from a native source, e.g., from
blood.
[0098] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC"
refers to a form of cytotoxicity in which secreted Ig bound onto Fc
receptors (FcRs) present on certain cytotoxic cells (e.g. NK cells,
neutrophils, and macrophages) enable these cytotoxic effector cells
to bind specifically to an antigen-bearing target cell and
subsequently kill the target cell with cytotoxins. The primary
cells for mediating ADCC, NK cells, express Fc.gamma.RIII only,
whereas monocytes express Fc.gamma.RI, Fc.gamma.RII, and
Fc.gamma.RIII. FcR expression on hematopoietic cells is summarized
in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol
9:457-92 (1991). To assess ADCC activity of a molecule of interest,
an in vitro ADCC assay, such as that described in U.S. Pat. No.
5,500,362 or 5,821,337 or U.S. Pat. No. 6,737,056 (Presta), may be
performed. Useful effector cells for such assays include PBMC and
NK cells. Alternatively, or additionally, ADCC activity of the
molecule of interest may be assessed in vivo, e.g., in an animal
model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656
(1998).
[0099] "Complement dependent cytotoxicity" or "CDC" refers to the
lysis of a target cell in the presence of complement. Activation of
the classical complement pathway is initiated by the binding of the
first component of the complement system (Clq) to antibodies (of
the to appropriate subclass), which are bound to their cognate
antigen. To assess complement activation, a CDC assay, e.g., as
described in Gazzano-Santoro et al., J. Immunol. Methods 202:163
(1996), may be performed. Polypeptide variants with altered Fc
region amino acid sequences (polypeptides with a variant Fc region)
and increased or decreased Clq binding capability are described,
e.g., in U.S. Pat. No. 6,194,551 B1 and WO 1999/51642. See also,
e.g., Idusogie et al. J. Immunol. 164: 4178-4184 (2000).
[0100] The term "Fc region-comprising antibody" refers to an
antibody that comprises an Fc region. The C-terminal lysine
(residue 447 according to the EU numbering system) of the Fc region
may be removed, for example, during purification of the antibody or
by recombinant engineering of the nucleic acid encoding the
antibody. Accordingly, a composition comprising an antibody having
an Fc region according to this invention can comprise an antibody
with K447, with all K447 removed, or a mixture of antibodies with
and without the K447 residue.
[0101] An "acceptor human framework" for the purposes herein is a
framework comprising the amino acid sequence of a VL or VH
framework derived from a human immunoglobulin framework, or from a
human consensus framework. An acceptor human framework "derived
from" a human immunoglobulin framework or human consensus framework
may comprise the same amino acid sequence thereof, or may contain
pre-existing amino acid sequence changes. Where pre-existing amino
acid changes are present, preferably no more than 5 and preferably
4 or less, or 3 or less, pre-existing amino acid changes are
present. Where pre-existing amino acid changes are present in a VH,
preferably those changes are only at three, two or one of positions
71H, 73H and 78H; for instance, the amino acid residues at those
positions may be 71A, 73T and/or 78A. In one embodiment, the VL
acceptor human framework is identical in sequence to the VL human
immunoglobulin framework sequence or human consensus framework
sequence.
[0102] A "human consensus framework" is a framework which
represents the most commonly occurring amino acid residue in a
selection of human immunoglobulin VL or VH framework sequences.
Generally, the selection of human immunoglobulin VL or VH sequences
is from a subgroup of variable domain sequences. Generally, the
subgroup of sequences is a subgroup as in Kabat et al. In one
embodiment, for the VL, the subgroup is subgroup kappa I as in
Kabat et al. In one embodiment, for the VH, the subgroup is
subgroup III as in Kabat et al.
[0103] A "VH subgroup III consensus framework" comprises the
consensus sequence obtained from the amino acid sequences in
variable heavy subgroup III of Kabat et al. In to one embodiment,
the VH subgroup III consensus framework amino acid sequence
comprises at least a portion or all of each of the following
sequences:
TABLE-US-00002 (SEQ ID NO: 197) EVQLVESGGGLVQPGGSLRLSCAAS- (SEQ ID
NO: 198) H1-WVRQAPGKGLEWV- (SEQ ID NO: 199
H2-RFTISRDNSKNTLYLQMNSLRAEDTAVYYC- (SEQ ID NO: 200)
H3-WGQGTLVTVSS.
[0104] A "VL subgroup I consensus framework" comprises the
consensus sequence obtained from the amino acid sequences in
variable light kappa subgroup I of Kabat et al. In one embodiment,
the VH subgroup I consensus framework amino acid sequence comprises
at least a portion or all of each of the following sequences:
TABLE-US-00003 (SEQ ID NO: 201) DIQMTQSPSSLSASVGDRVTITC- (SEQ ID
NO: 202) L1-WYQQKPGKAPKLLIY- (SEQ ID NO: 203)
L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC- (SEQ ID NO: 221)
L3-FGQGTKVEIK.
[0105] A "biological sample" (interchangeably termed "sample" or
"tissue or cell sample") encompasses a variety of sample types
obtained from an individual and can be used in a diagnostic or
monitoring assay. The definition encompasses blood and other liquid
samples of biological origin, solid tissue samples such as a biopsy
specimen or tissue cultures or cells derived therefrom, and the
progeny thereof. The definition also includes samples that have
been manipulated in any way after their procurement, such as by
treatment with reagents, solubilization, or enrichment for certain
components, such as proteins or polynucleotides, or embedding in a
semi-solid or solid matrix for sectioning purposes. The term
"biological sample" encompasses a clinical sample, and also
includes cells in culture, cell supernatants, cell lysates, serum,
plasma, biological fluid, and tissue samples. The source of the
biological sample may be solid tissue as from a fresh, frozen
and/or preserved organ or tissue sample or biopsy or aspirate;
blood or any blood constituents; bodily fluids such as cerebral
spinal fluid, amniotic fluid, peritoneal fluid, or interstitial
fluid; cells from any time in gestation or development of the
subject. In some embodiments, the biological sample is obtained
from a primary or metastatic tumor. The biological sample may
contain compounds which are not naturally intermixed with the
tissue in nature such as preservatives, anticoagulants, buffers,
fixatives, nutrients, antibiotics, or the like.
[0106] For the purposes herein a "section" of a tissue sample is
meant a single part or piece of a tissue sample, e.g. a thin slice
of tissue or cells cut from a tissue sample. It is understood that
multiple sections of tissue samples may be taken and subjected to
analysis according to the present invention. In some embodiments,
the same section of tissue sample is analyzed at both morphological
and molecular levels, or is analyzed with respect to both protein
and nucleic acid.
[0107] The word "label" when used herein refers to a compound or
composition which is conjugated or fused directly or indirectly to
a reagent such as a nucleic acid probe or an antibody and
facilitates detection of the reagent to which it is conjugated or
fused. The label may itself be detectable (e.g., radioisotope
labels or fluorescent labels) or, in the case of an enzymatic
label, may catalyze chemical alteration of a substrate compound or
composition which is detectable.
[0108] A "medicament" is an active drug to treat the disorder in
question or its symptoms, or side effects.
[0109] A "disorder" or "disease" is any condition that would
benefit from treatment with a substance/molecule or method of the
invention. This includes chronic and acute disorders or diseases
including those pathological conditions which predispose the mammal
to the disorder in question. Non-limiting examples of disorders to
be treated herein include malignant and benign tumors; carcinoma,
blastoma, and sarcoma.
[0110] The terms "cell proliferative disorder" and "proliferative
disorder" refer to disorders that are associated with some degree
of abnormal cell proliferation. In one embodiment, the cell
proliferative disorder is cancer.
[0111] "Tumor", as used herein, refers to all neoplastic cell
growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues. The terms "cancer",
"cancerous", "cell proliferative disorder", "proliferative
disorder" and "tumor" are not mutually exclusive as referred to
herein.
[0112] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth/proliferation. Examples of cancer
include but are not limited to, carcinoma, lymphoma, blastoma,
sarcoma, and leukemia. More particular examples of such cancers
include squamous cell cancer, small-cell lung cancer, pituitary
cancer, esophageal cancer, astrocytoma, soft tissue sarcoma,
non-small cell lung cancer, adenocarcinoma of the lung, squamous
carcinoma of the lung, cancer of the peritoneum, hepatocellular
cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma,
cervical cancer, ovarian cancer, liver cancer, bladder cancer,
hepatoma, breast cancer, colon cancer, colorectal cancer,
endometrial or uterine carcinoma, salivary gland carcinoma, kidney
cancer, liver cancer, prostate cancer, vulval cancer, thyroid
cancer, hepatic carcinoma, brain cancer, endometrial cancer, testis
cancer, cholangiocarcinoma, gallbladder carcinoma, gastric cancer,
melanoma, and various types of head and neck cancer. Dysregulation
of angiogenesis can lead to many disorders that can be treated by
compositions and methods of the invention. These disorders include
both non-neoplastic and neoplastic conditions. Neoplastics include
but are not limited those described above. Non-neoplastic disorders
include but are not limited to undesired or aberrant hypertrophy,
arthritis, rheumatoid arthritis (RA), psoriasis, psoriatic plaques,
sarcoidosis, atherosclerosis, atherosclerotic plaques, diabetic and
other proliferative retinopathies including retinopathy of
prematurity, retrolental fibroplasia, neovascular glaucoma,
age-related macular degeneration, diabetic macular edema, corneal
neovascularization, corneal graft neovascularization, corneal graft
rejection, retinal/choroidal neovascularization, neovascularization
of the angle (rubeosis), ocular neovascular disease, vascular
restenosis, arteriovenous malformations (AVM), meningioma,
hemangioma, angiofibroma, thyroid hyperplasias (including Grave's
disease), corneal and other tissue transplantation, chronic
inflammation, lung inflammation, acute lung injury/ARDS, sepsis,
primary pulmonary hypertension, malignant pulmonary effusions,
cerebral edema (e.g., associated with acute stroke/closed head
injury/trauma), synovial inflammation, pannus formation in RA,
myositis ossificans, hypertropic bone formation, osteoarthritis
(OA), refractory ascites, polycystic ovarian disease,
endometriosis, 3rd spacing of fluid diseases (pancreatitis,
compartment syndrome, burns, bowel disease), uterine fibroids,
premature labor, chronic inflammation such as IBD (Crohn's disease
and ulcerative colitis), renal allograft rejection, inflammatory
bowel disease, nephrotic syndrome, undesired or aberrant tissue
mass growth (non-cancer), hemophilic joints, hypertrophic scars,
inhibition of hair growth, Osler-Weber syndrome, pyogenic granuloma
retrolental fibroplasias, scleroderma, trachoma, vascular
adhesions, synovitis, dermatitis, preeclampsia, ascites,
pericardial effusion (such as that associated with pericarditis),
and pleural effusion.
[0113] The term "wasting" disorders (e.g., wasting syndrome,
cachexia, sarcopenia) refers to a disorder caused by undesirable
and/or unhealthy loss of weight or loss of body cell mass. In the
elderly as well as in AIDS and cancer patients, wasting disease can
result in undesired loss of body weight, including both the fat and
the fat-free compartments. Wasting diseases can be the result of
inadequate intake of food and/or metabolic changes related to
illness and/or the aging process. Cancer patients and AIDS
patients, as well as patients following extensive surgery or having
chronic infections, immunologic diseases, hyperthyroidism, Crohn's
disease, psychogenic disease, chronic heart failure or other severe
trauma, frequently suffer from wasting disease which is sometimes
also referred to as cachexia, a metabolic and, sometimes, an eating
disorder. Cachexia is additionally characterized by hypermetabolism
and hypercatabolism. Although cachexia and wasting disease are
frequently used interchangeably to refer to wasting conditions,
there is at least one body of research which differentiates
cachexia from wasting syndrome as a loss of fat-free mass, and
particularly, body cell mass (Mayer, 1999, J. Nutr. 129(1S
Suppl.):2565-259S). Sarcopenia, yet another such disorder which can
affect the aging individual, is typically characterized by loss of
muscle mass. End stage wasting disease as described above can
develop in individuals suffering from either cachexia or
sarcopenia.
[0114] As used herein, "treatment" refers to clinical intervention
in an attempt to alter the natural course of the individual or cell
being treated, and can be performed either for prophylaxis or
during the course of clinical pathology. Desirable effects of
treatment include preventing occurrence or recurrence of disease,
alleviation of symptoms, diminishment of any direct or indirect
pathological consequences of the disease, decreasing the rate of
disease progression, amelioration or palliation of the disease
state, and remission or improved prognosis. In some embodiments,
antibodies of the invention are used to delay development of a
disease or disorder.
[0115] An "anti-angiogenesis agent" or "angiogenesis inhibitor"
refers to a small molecular weight substance, a polynucleotide, a
polypeptide, an isolated protein, a recombinant protein, an
antibody, or conjugates or fusion proteins thereof, that inhibits
angiogenesis, vasculogenesis, or undesirable vascular permeability,
either directly or indirectly. For example, an anti-angiogenesis
agent is an antibody or other antagonist to an angiogenic agent as
defined above, e.g., antibodies to VEGF, antibodies to VEGF
receptors, small molecules that block VEGF receptor signaling
(e.g., PTK787/ZK2284, SU6668, SUTENT.RTM./SU11248 (sunitinib
malate), AMG706). Anti-angiogenesis agents also include native
angiogenesis inhibitors, e.g., angiostatin, endostatin, etc. See,
e.g., Klagsbrun and D'Amore, Annu. Rev. Physiol., 53:217-39 (1991);
Streit and Detmar, Oncogene, 22:3172-3179 (2003) (e.g., Table 3
listing anti-angiogenic therapy in malignant melanoma); Ferrara
& Alitalo, Nature Medicine 5(12):1359-1364 (1999); Tonini et
al., Oncogene, 22:6549-6556 (2003) (e.g., Table 2 listing
antiangiogenic factors); and, Sato Int. J. Clin. Oncol., 8:200-206
(2003) (e.g., Table 1 lists Anti-angiogenic agents used in clinical
trials).
[0116] An "individual," "subject," or "patient" is a vertebrate. In
certain embodiments, the vertebrate is a mammal. Mammals include,
but are not limited to, farm animals (such as cows), sport animals,
pets (such as cats, dogs, and horses), primates, mice and rats. In
certain embodiments, a mammal is a human.
[0117] An "effective amount" refers to an amount effective, at
dosages and for periods of time necessary, to achieve the desired
therapeutic or prophylactic result.
[0118] A "therapeutically effective amount" of a substance/molecule
of the invention, agonist or antagonist may vary according to
factors such as the disease state, age, sex, and weight of the
individual, and the ability of the substance/molecule, agonist or
antagonist to elicit a desired response in the individual. A
therapeutically effective amount is also one in which any toxic or
detrimental effects of the substance/molecule, agonist or
antagonist are outweighed by the therapeutically beneficial
effects. A "prophylactically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired prophylactic result. Typically but not necessarily,
since a prophylactic dose is used in subjects prior to or at an
earlier stage of disease, the prophylactically effective amount
will be less than the therapeutically effective amount.
[0119] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g., At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32
and radioactive isotopes of Lu), chemotherapeutic agents e.g.
methotrexate, adriamicin, vinca alkaloids (vincristine,
vinblastine, etoposide), doxorubicin, melphalan, mitomycin C,
chlorambucil, daunorubicin or other intercalating agents, enzymes
and fragments thereof such as nucleolytic enzymes, antibiotics, and
toxins such as small molecule toxins or enzymatically active toxins
of bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof, and the various antitumor or anticancer
agents disclosed below. Other cytotoxic agents are described below.
A tumoricidal agent causes destruction of tumor cells.
[0120] A "toxin" is any substance capable of having a detrimental
effect on the growth or proliferation of a cell.
[0121] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include alkylating agents such as thiotepa and cyclosphosphamide
(CYTOXAN.RTM.); alkyl sulfonates such as busulfan, improsulfan and
piposulfan; aziridines such as benzodopa, carboquone, meturedopa,
and uredopa; ethylenimines and methylamelamines including
altretamine, triethylenemelamine, triethylenephosphoramide,
triethylenethiophosphoramide and trimethylomelamine; acetogenins
(especially bullatacin and bullatacinone);
delta-9-tetrahydrocannabinol (dronabinol, MARINOL.RTM.);
beta-lapachone; lapachol; colchicines; betulinic acid; a
camptothecin (including the synthetic analogue topotecan
(HYCAMTIN.RTM.), CPT-11 (irinotecan, CAMPTOSAR.RTM.),
acetylcamptothecin, scopolectin, and 9-aminocamptothecin);
bryostatin; callystatin; CC-1065 (including its adozelesin,
carzelesin and bizelesin synthetic analogues); podophyllotoxin;
podophyllinic acid; teniposide; cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
chlorophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosoureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; antibiotics such as the
enediyne antibiotics (e.g., calicheamicin, especially calicheamicin
gamma1I and calicheamicin omegaI1 (see, e.g., Nicolaou et al.,
Angew. Chem. Intl. Ed. Engl., 33: 183-186 (1994)); CDP323, an oral
alpha-4 integrin inhibitor; dynemicin, including dynemicin A; an
esperamicin; as well as neocarzinostatin chromophore and related
chromoprotein enediyne antibiotic chromophores), aclacinomysins,
actinomycin, authramycin, azaserine, bleomycins, cactinomycin,
carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin
(including ADRIAMYCIN.RTM., morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin
HCl liposome injection (DOXIL.RTM.), liposomal doxorubicin TLC D-99
(MYOCET.RTM.), peglylated liposomal doxorubicin (CAELYX.RTM.), and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate, gemcitabine (GEMZAR.RTM.), tegafur (UFTORAL.RTM.),
capecitabine (XELODA.RTM.), an epothilone, and 5-fluorouracil
(5-FU); folic acid analogues such as denopterin, methotrexate,
pteropterin, trimetrexate; purine analogs such as fludarabine,
6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such
as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,
dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens
such as calusterone, dromostanolone propionate, epitiostanol,
mepitiostane, testolactone; anti-adrenals such as
aminoglutethimide, mitotane, trilostane; folic acid replenisher
such as frolinic acid; aceglatone; aldophosphamide glycoside;
aminolevulinic acid; eniluracil; amsacrine; bestrabucil;
bisantrene; edatraxate; defofamine; demecolcine; diaziquone;
elformithine; elliptinium acetate; an epothilone; etoglucid;
gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids
such as maytansine and ansamitocins; mitoguazone; mitoxantrone;
mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin;
losoxantrone; 2-ethylhydrazide; procarbazine; PSK.RTM.
polysaccharide complex (JHS Natural Products, Eugene, Oreg.);
razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid;
triaziquone; 2,2',2'-trichlorotriethylamine; trichothecenes
(especially T-2 toxin, verracurin A, roridin A and anguidine);
urethan; vindesine (ELDISINE.RTM., FILDESIN.RTM.); dacarbazine;
mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;
arabinoside ("Ara-C"); thiotepa; taxoid, e.g., paclitaxel
(TAXOL.RTM.), albumin-engineered nanoparticle formulation of
paclitaxel (ABRAXANE.TM.), and docetaxel (TAXOTERE.RTM.);
chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum
agents such as cisplatin, oxaliplatin (e.g., ELOXATIN.RTM.), and
carboplatin; vincas, which prevent tubulin polymerization from
forming microtubules, including vinblastine (VELBAN.RTM.),
vincristine (ONCOVIN.RTM.), vindesine (ELDISINE.RTM.,
FILDESIN.RTM.), and vinorelbine (NAVELBINE.RTM.); etoposide
(VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone;
edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase
inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such
as retinoic acid, including bexarotene (TARGRETIN.RTM.);
bisphosphonates such as clodronate (for example, BONEFOS.RTM. or
OSTAC.RTM.), etidronate (DIDROCAL.RTM.), NE-58095, zoledronic
acid/zoledronate (ZOMETA.RTM.), alendronate (FOSAMAX.RTM.),
pamidronate (AREDIA.RTM.), tiludronate (SKELID.RTM.), or
risedronate (ACTONEL.RTM.); troxacitabine (a 1,3-dioxolane
nucleoside cytosine analog); antisense oligonucleotides,
particularly those that inhibit expression of genes in signaling
pathways implicated in aberrant cell proliferation, such as, for
example, PKC-alpha, Raf, H-Ras, and epidermal growth factor
receptor (EGF-R); vaccines such as THERATOPE.RTM. vaccine and gene
therapy vaccines, for example, ALLOVECTIN.RTM. vaccine,
LEUVECTIN.RTM. vaccine, and VAXID.RTM. vaccine; topoisomerase 1
inhibitor (e.g., LURTOTECAN.RTM.); rmRH (e.g., ABARELIX.RTM.);
BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT.RTM.,
Pfizer); perifosine, COX-2 inhibitor (e.g. celecoxib or
etoricoxib), proteosome inhibitor (e.g. PS341); bortezomib
(VELCADE.RTM.); CCI-779; tipifarnib (R11577); orafenib, ABT510;
Bcl-2 inhibitor such as oblimersen sodium (GENASENSE.RTM.);
pixantrone; EGFR inhibitors (see definition below); tyrosine kinase
inhibitors (see definition below); serine-threonine kinase
inhibitors such as rapamycin (sirolimus, RAPAMUNE.RTM.);
farnesyltransferase inhibitors such as lonafarnib (SCH 6636,
SARASAR.TM.); and pharmaceutically acceptable salts, acids or
derivatives of any of the above; as well as combinations of two or
more of the above such as CHOP, an abbreviation for a combined
therapy of cyclophosphamide, doxorubicin, vincristine, and
prednisolone; and FOLFOX, an abbreviation for a treatment regimen
with oxaliplatin (ELOXATIN.TM.) combined with 5-FU and
leucovorin.
[0122] Chemotherapeutic agents as defined herein include
"anti-hormonal agents" or "endocrine therapeutics" which act to
regulate, reduce, block, or inhibit the effects of hormones that
can promote the growth of cancer. They may be hormones themselves,
including, but not limited to: anti-estrogens with mixed
agonist/antagonist profile, including, tamoxifen (NOLVADEX.RTM.),
4-hydroxytamoxifen, toremifene (FARESTON.RTM.), idoxifene,
droloxifene, raloxifene (EVISTA.RTM.), trioxifene, keoxifene, and
selective estrogen receptor modulators (SERMs) such as SERM3; pure
anti-estrogens without agonist properties, such as fulvestrant
(FASLODEX.RTM.), and EM800 (such agents may block estrogen receptor
(ER) dimerization, inhibit DNA binding, increase ER turnover,
and/or suppress ER levels); aromatase inhibitors, including
steroidal aromatase inhibitors such as formestane and exemestane
(AROMASIN.RTM.), and nonsteroidal aromatase inhibitors such as
anastrazole (ARIMIDEX.RTM.), letrozole (FEMARA.RTM.) and
aminoglutethimide, and other aromatase inhibitors include vorozole
(RIVISOR.RTM.), megestrol acetate (MEGASE.RTM.), fadrozole, and
4(5)-imidazoles; lutenizing hormone-releaseing hormone agonists,
including leuprolide (LUPRON.RTM. and ELIGARD.RTM.), goserelin,
buserelin, and tripterelin; sex steroids, including progestines
such as megestrol acetate and medroxyprogesterone acetate,
estrogens such as diethylstilbestrol and premarin, and
androgens/retinoids such as fluoxymesterone, all transretionic acid
and fenretinide; onapristone; anti-progesterones; estrogen receptor
down-regulators (ERDs); anti-androgens such as flutamide,
nilutamide and bicalutamide; and pharmaceutically acceptable salts,
acids or derivatives of any of the above; as well as combinations
of two or more of the above.
[0123] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell (such as a
cell expressing EGFL7) either in vitro or in vivo. Thus, the growth
inhibitory agent may be one which significantly reduces the
percentage of cells (such as a cell expressing EGFL7) in S phase.
Examples of growth inhibitory agents include agents that block cell
cycle progression (at a place other than S phase), such as agents
that induce G1 arrest and M-phase arrest. Classical M-phase
blockers include the vincas (vincristine and vinblastine), taxanes,
and topoisomerase II inhibitors such as doxorubicin, epirubicin,
daunorubicin, etoposide, and bleomycin. Those agents that arrest G1
also spill over into S-phase arrest, for example, DNA alkylating
agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine,
cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further
information can be found in Mendelsohn and Israel, eds., The
Molecular Basis of Cancer, Chapter 1, entitled "Cell cycle
regulation, oncogenes, and antineoplastic drugs" by Murakami et al.
(W.B. Saunders, Philadelphia, 1995), e.g., p. 13. The taxanes
(paclitaxel and docetaxel) are anticancer drugs both derived from
the yew tree. Docetaxel (TAXOTERE.RTM., Rhone-Poulenc Rorer),
derived from the European yew, is a semisynthetic analogue of
paclitaxel (TAXOL.RTM., Bristol-Myers Squibb). Paclitaxel and
docetaxel promote the assembly of microtubules from tubulin dimers
and stabilize microtubules by preventing depolymerization, which
results in the inhibition of mitosis in cells.
[0124] "Doxorubicin" is an anthracycline antibiotic. The full
chemical name of doxorubicin is
(8S-cis)-10-[(3-amino-2,3,6-trideoxy-.alpha.-L-lyxo-hexapyranosyl)oxy]-7,-
8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-napht-
hacenedione.
[0125] The term "Fc region-comprising polypeptide" refers to a
polypeptide, such as an antibody or immunoadhesin (see definitions
below), which comprises an Fc region. The C-terminal lysine
(residue 447 according to the EU numbering system) of the Fc region
may be removed, for example, during purification of the polypeptide
or by recombinant engineering the nucleic acid encoding the
polypeptide. Accordingly, a composition comprising a polypeptide
having an Fc region according to this invention can comprise
polypeptides with K447, with all K447 removed, or a mixture of
polypeptides with and without the K447 residue.
[0126] Throughout this specification and claims, the word
"comprise," or variations such as "comprises" or "comprising," will
be understood to imply the inclusion of a stated integer or group
of integers but not the exclusion of any other integer or group of
integers.
Generating Variant Antibodies Exhibiting Reduced or Absence of HAMA
Response
[0127] Reduction or elimination of a HAMA response is a significant
aspect of clinical development of suitable therapeutic agents. See,
e.g., Khaxzaeli et al., J. Natl. Cancer Inst. (1988), 80:937;
Jaffers et al., Transplantation (1986), 41:572; Shawler et al., J.
Immunol. (1985), 135:1530; Sears et al., J. Biol. Response Mod.
(1984), 3:138; Miller et al., Blood (1983), 62:988; Hakimi et al.,
J. Immunol. (1991), 147:1352; Reichmann et al., Nature (1988),
332:323; Junghans et al., Cancer Res. (1990), 50:1495. As described
herein, the invention provides antibodies that are humanized such
that HAMA response is reduced or eliminated. Variants of these
antibodies can further be obtained using routine methods known in
the art, some of which are further described below.
[0128] For example, an amino acid sequence from an antibody as
described herein can serve as a starting (parent) sequence for
diversification of the framework and/or hypervariable sequence(s).
A selected framework sequence to which a starting hypervariable
sequence is linked is referred to herein as an acceptor human
framework. While the acceptor human frameworks may be from, or
derived from, a human immunoglobulin (the VL and/or VH regions
thereof), preferably the acceptor human frameworks are from, or
derived from, a human consensus framework sequence as such
frameworks have been demonstrated to have minimal, or no,
immunogenicity in human patients.
[0129] Where the acceptor is derived from a human immunoglobulin,
one may optionally select a human framework sequence that is
selected based on its homology to the donor framework sequence by
aligning the donor framework sequence with various human framework
sequences in a collection of human framework sequences, and select
the most homologous framework sequence as the acceptor.
[0130] In one embodiment, human consensus frameworks herein are
from, or derived from, VH subgroup III and/or VL kappa subgroup I
consensus framework sequences.
[0131] Thus, the VH acceptor human framework may comprise one, two,
three or all of the following framework sequences:
FR1 comprising EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 197), FR2
comprising WVRQAPGKGLEWV (SEQ ID NO: 198), FR3 comprising FR3
comprises RFTISX.sub.1DX.sub.2SKNTX.sub.3YLQMNSLRAEDTAVYYC (SEQ ID
NO: 205), wherein X.sub.1 is A or R, X.sub.2 is T or N, and X.sub.3
is A or L, FR4 comprising WGQGTLVTVSS (SEQ ID NO: 200).
[0132] In one embodiment, the VH acceptor human framework comprises
one, two, three or all of the following framework sequences:
FR1 comprising EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 197), FR2
comprising WVRQAPGKGLEWV (SEQ ID NO: 198), FR3 comprising
RFTISADTSKNTAYLQMNSLRAEDTAVYYC (SEQ ID NO: 231),
RFTISADTSKNTAYLQMNSLRAEDTAVYYCA (SEQ ID NO: 206),
RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 207),
RFTISADTSKNTAYLQMNSLRAEDTAVYYCS (SEQ ID NO: 208), or
RFTISADTSKNTAYLQMNSLRAEDTAVYYCSR (SEQ ID NO: 209)
[0133] FR4 comprising WGQGTLVTVSS (SEQ ID NO: 200).
[0134] The VL acceptor human framework may comprise one, two, three
or all of the following framework sequences:
FR1 comprising DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 201), FR2
comprising WYQQKPGKAPKLLIY (SEQ ID NO: 202), FR3 comprising
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 203), FR4 comprising
FGQGTKVEIK (SEQ ID NO: 221).
[0135] While the acceptor may be identical in sequence to the human
framework sequence selected, whether that is from a human
immunoglobulin or a human consensus framework, the present
invention contemplates that the acceptor sequence may comprise
pre-existing amino acid substitutions relative to the human
immunoglobulin sequence or human consensus framework sequence.
These pre-existing substitutions are preferably minimal; usually
four, three, two or one amino acid differences only relative to the
human immunoglobulin sequence or consensus framework sequence.
[0136] Hypervariable region residues of the non-human antibody are
incorporated into the VL and/or VH acceptor human frameworks. For
example, one may incorporate residues corresponding to the Kabat
CDR residues, the Chothia hypervariable loop residues, the Abm
residues, and/or contact residues. Optionally, the extended
hypervariable region residues as follows are incorporated: 24-34
(L1), 50-56 (L2) and 89-97 (L3), 26-35 (H1), 50-65 or 49-65 (H2)
and 93-102, 94-102, or 95-102 (H3).
[0137] While "incorporation" of hypervariable region residues is
discussed herein, it will be appreciated that this can be achieved
in various ways, for example, nucleic acid encoding the desired
amino acid sequence can be generated by mutating nucleic acid
encoding the mouse variable domain sequence so that the framework
residues thereof are changed to acceptor human framework residues,
or by mutating nucleic acid encoding the human variable domain
sequence so that the hypervariable domain residues are changed to
non-human residues, or by synthesizing nucleic acid encoding the
desired sequence, etc.
[0138] In the examples herein, hypervariable region-grafted
variants were generated by Kunkel mutagenesis of nucleic acid
encoding the human acceptor sequences, using a separate
oligonucleotide for each hypervariable region. Kunkel et al.,
Methods Enzymol. 154:367-382 (1987). Appropriate changes can be
introduced within the framework and/or hypervariable region, using
routine techniques, to correct and re-establish proper
hypervariable region-antigen interactions.
[0139] Phage(mid) display (also referred to herein as phage display
in some contexts) can to be used as a convenient and fast method
for generating and screening many different potential variant
antibodies in a library generated by sequence randomization.
However, other methods for making and screening altered antibodies
are available to the skilled person.
[0140] Phage(mid) display technology has provided a powerful tool
for generating and selecting novel proteins which bind to a ligand,
such as an antigen. Using the techniques of phage(mid) display
allows the generation of large libraries of protein variants which
can be rapidly sorted for those sequences that bind to a target
molecule with high affinity. Nucleic acids encoding variant
polypeptides are generally fused to a nucleic acid sequence
encoding a viral coat protein, such as the gene III protein or the
gene VIII protein. Monovalent phagemid display systems where the
nucleic acid sequence encoding the protein or polypeptide is fused
to a nucleic acid sequence encoding a portion of the gene III
protein have been developed. (Bass, S., Proteins, 8:309 (1990);
Lowman and Wells, Methods: A Companion to Methods in Enzymology,
3:205 (1991)). In a monovalent phagemid display system, the gene
fusion is expressed at low levels and wild type gene III proteins
are also expressed so that infectivity of the particles is
retained. Methods of generating peptide libraries and screening
those libraries have been disclosed in many patents (e.g. U.S. Pat.
No. 5,723,286, U.S. Pat. No. 5,432,018, U.S. Pat. No. 5,580,717,
U.S. Pat. No. 5,427,908 and U.S. Pat. No. 5,498,530).
[0141] Libraries of antibodies or antigen binding polypeptides have
been prepared in a number of ways including by altering a single
gene by inserting random DNA sequences or by cloning a family of
related genes. Methods for displaying antibodies or antigen binding
fragments using phage(mid) display have been described in U.S. Pat.
Nos. 5,750,373, 5,733,743, 5,837,242, 5,969,108, 6,172,197,
5,580,717, and 5,658,727. The library is then screened for
expression of antibodies or antigen binding proteins with the
desired characteristics.
[0142] Methods of substituting an amino acid of choice into a
template nucleic acid are well established in the art, some of
which are described herein. For example, hypervariable region
residues can be substituted using the Kunkel method. See, e.g.,
Kunkel et al., Methods Enzymol. 154:367-382 (1987).
[0143] The sequence of oligonucleotides includes one or more of the
designed codon sets for the hypervariable region residues to be
altered. A codon set is a set of different nucleotide triplet
sequences used to encode desired variant amino acids. Codon sets
can be represented using symbols to designate particular
nucleotides or equimolar mixtures of nucleotides as shown in below
according to the IUB code.
[0144] IUB Codes [0145] G Guanine [0146] A Adenine [0147] T Thymine
[0148] C Cytosine [0149] R (A or G) [0150] Y (C or T) [0151] M (A
or C) [0152] K (G or T) [0153] S(C or G) [0154] W (A or T) [0155] H
(A or C or T) [0156] B (C or G or T) [0157] V (A or C or G) [0158]
D (A or G or T) [0159] N (A or C or G or T)
[0160] For example, in the codon set DVK, D can be nucleotides A or
G or T; V can be A or G or C; and K can be G or T. This codon set
can present 18 different codons and can encode amino acids Ala,
Trp, Tyr, Lys, Thr, Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys.
[0161] Oligonucleotide or primer sets can be synthesized using
standard methods. A set of oligonucleotides can be synthesized, for
example, by solid phase synthesis, containing sequences that
represent all possible combinations of nucleotide triplets provided
by the codon set and that will encode the desired group of amino
acids. Synthesis of oligonucleotides with selected nucleotide
"degeneracy" at certain positions is well known in that art. Such
sets of nucleotides having certain codon sets can be synthesized
using commercial nucleic acid synthesizers (available from, for
example, Applied Biosystems, Foster City, Calif.), or can be
obtained commercially (for example, from Life Technologies,
Rockville, Md.). Therefore, a set of oligonucleotides synthesized
having a particular codon set will typically include a plurality of
oligonucleotides with different sequences, the differences
established by the codon set within the overall sequence.
Oligonucleotides, as used according to the invention, have
sequences that allow for hybridization to a variable domain nucleic
acid template and also can include restriction enzyme sites for
cloning purposes.
[0162] In one method, nucleic acid sequences encoding variant amino
acids can be created by oligonucleotide-mediated mutagenesis. This
technique is well known in the art as described by Zoller et al.
Nucleic Acids Res. 10:6487-6504 (1987). Briefly, nucleic acid
sequences encoding variant amino acids are created by hybridizing
an oligonucleotide set encoding the desired codon sets to a DNA
template, where the template is the single-stranded form of the
plasmid containing a variable region nucleic acid template
sequence. After hybridization, DNA polymerase is used to synthesize
an entire second complementary strand of the template that will
thus incorporate the oligonucleotide primer, and will contain the
codon sets as provided by the oligonucleotide set.
[0163] Generally, oligonucleotides of at least 25 nucleotides in
length are used. An optimal oligonucleotide will have 12 to 15
nucleotides that are completely complementary to the template on
either side of the nucleotide(s) coding for the mutation(s). This
ensures that the oligonucleotide will hybridize properly to the
single-stranded DNA template molecule. The oligonucleotides are
readily synthesized using techniques known in the art such as that
described by Crea et al., Proc. Nat'l. Acad. Sci. USA, 75:5765
(1978).
[0164] The DNA template is generated by those vectors that are
either derived from bacteriophage M13 vectors (the commercially
available M13 mp 18 and M13 mp 19 vectors are suitable), or those
vectors that contain a single-stranded phage origin of replication
as described by Viera et al., Meth. Enzymol., 153:3 (1987). Thus,
the DNA that is to be mutated can be inserted into one of these
vectors in order to generate single-stranded template. Production
of the single-stranded template is described in sections 4.21-4.41
of Sambrook et al., above.
[0165] To alter the native DNA sequence, the oligonucleotide is
hybridized to the single stranded template under suitable
hybridization conditions. A DNA polymerizing enzyme, usually T7 DNA
polymerase or the Klenow fragment of DNA polymerase I, is then
added to synthesize the complementary strand of the template using
the oligonucleotide as a primer for synthesis. A heteroduplex
molecule is thus formed such that one strand of DNA encodes the
mutated form of gene 1, and the other strand (the original
template) encodes the native, unaltered sequence of gene 1. This
heteroduplex molecule is then transformed into a suitable host
cell, usually a prokaryote such as E. coli JM101. After growing the
cells, they are plated onto agarose plates and screened using the
oligonucleotide primer radiolabelled with a 32-Phosphate to
identify the bacterial colonies that contain the mutated DNA.
[0166] The method described immediately above may be modified such
that a homoduplex molecule is created wherein both strands of the
plasmid contain the mutation(s). The modifications are as follows:
The single stranded oligonucleotide is annealed to the
single-stranded template as described above. A mixture of three
deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine
(dGTP), and deoxyribothymidine (dTT), is combined with a modified
thiodeoxyribocytosine called dCTP-(aS) (which can be obtained from
Amersham). This mixture is added to the template-oligonucleotide
complex. Upon addition of DNA polymerase to this mixture, a strand
of DNA identical to the template except for the mutated bases is
generated. In addition, this new strand of DNA will contain
dCTP-(aS) instead of dCTP, which serves to protect it from
restriction endonuclease digestion. After the template strand of
the double-stranded heteroduplex is nicked with an appropriate
restriction enzyme, the template strand can be digested with ExoI11
nuclease or another appropriate nuclease past the region that
contains the site(s) to be mutagenized. The reaction is then
stopped to leave a molecule that is only partially single-stranded.
A complete double-stranded DNA homoduplex is then formed using DNA
polymerase in the presence of all four deoxyribonucleotide
triphosphates, ATP, and DNA ligase. This homoduplex molecule can
then be transformed into a suitable host cell.
[0167] As indicated previously the sequence of the oligonucleotide
set is of sufficient length to hybridize to the template nucleic
acid and may also, but does not necessarily, contain restriction
sites. The DNA template can be generated by those vectors that are
either derived from bacteriophage M13 vectors or vectors that
contain a single-stranded phage origin of replication as described
by Viera et al. Meth. Enzymol., 153:3 (1987). Thus, the DNA that is
to be mutated must be inserted into one of these vectors in order
to generate single-stranded template. Production of the
single-stranded template is described in sections 4.21-4.41 of
Sambrook et al., supra.
[0168] According to another method, a library can be generated by
providing upstream and downstream oligonucleotide sets, each set
having a plurality of oligonucleotides with different sequences,
the different sequences established by the codon sets provided
within the sequence of the oligonucleotides. The upstream and
downstream oligonucleotide sets, along with a variable domain
template nucleic acid sequence, can be used in a polymerase chain
reaction to generate a "library" of PCR products. The PCR products
can be referred to as "nucleic acid cassettes", as they can be
fused with other related or unrelated nucleic acid sequences, for
example, viral coat proteins and dimerization domains, using
established molecular biology techniques.
[0169] The sequence of the PCR primers includes one or more of the
designed codon sets for the solvent accessible and highly diverse
positions in a hypervariable region. As described above, a codon
set is a set of different nucleotide triplet sequences used to
encode desired variant amino acids.
[0170] Antibody selectants that meet the desired criteria, as
selected through appropriate screening/selection steps can be
isolated and cloned using standard recombinant techniques.
Antibody Fragments
[0171] The present invention encompasses antibody fragments.
Antibody fragments may be generated by traditional means, such as
enzymatic digestion, or by recombinant techniques. In certain
circumstances there are advantages of using antibody fragments,
rather than whole antibodies. The smaller size of the fragments
allows for rapid clearance, and may lead to improved access to
solid tumors. For a review of certain antibody fragments, see
Hudson et al. (2003) Nat. Med. 9:129-134.
[0172] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., Journal of Biochemical and Biophysical Methods 24:107-117
(1992); and Brennan et al., Science, 229:81 (1985)). However, these
fragments can now be produced directly by recombinant host cells.
Fab, Fv and ScFv antibody fragments can all be expressed in and
secreted from E. coli, thus allowing the facile production of large
amounts of these fragments. Antibody fragments can be isolated from
the antibody phage libraries discussed above. Alternatively,
Fab'-SH fragments can be directly recovered from E. coli and
chemically coupled to form F(ab').sub.2 fragments (Carter et al.,
Bio/Technology 10:163-167 (1992)). According to another approach,
F(ab').sub.2 fragments can be isolated directly from recombinant
host cell culture. Fab and F(ab').sub.2 fragment with increased in
vivo half-life comprising salvage receptor binding epitope residues
are described in U.S. Pat. No. 5,869,046. Other techniques for the
production of antibody fragments will be apparent to the skilled
practitioner. In certain embodiments, an antibody is a single chain
Fv fragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and
5,587,458. Fv and scFv are the only species with intact combining
sites that are devoid of constant regions; thus, they may be
suitable for reduced nonspecific binding during in vivo use. scFv
fusion proteins may be constructed to yield fusion of an effector
protein at either the amino or the carboxy terminus of an scFv. See
Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment
may also be a "linear antibody", e.g., as described in U.S. Pat.
No. 5,641,870, for example. Such linear antibodies may be
monospecific or bispecific.
Humanized Antibodies
[0173] The invention encompasses humanized antibodies. Various
methods for humanizing non-human antibodies are known in the art.
For example, a humanized antibody can have one or more amino acid
residues introduced into it from a source which is non-human. These
non-human amino acid residues are often referred to as "import"
residues, which are typically taken from an "import" variable
domain. Humanization can be essentially performed following the
method of Winter and co-workers (Jones et al. (1986) Nature
321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen
et al. (1988) Science 239:1534-1536), by substituting hypervariable
region sequences for the corresponding sequences of a human
antibody. Accordingly, such "humanized" antibodies are chimeric
antibodies (U.S. Pat. No. 4,816,567) wherein substantially less
than an intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some
hypervariable region residues and possibly some FR residues are
substituted by residues from analogous sites in rodent
antibodies.
[0174] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies can be important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the
rodent is then accepted as the human framework for the humanized
antibody. See, e.g., Sims et al. (1993) J. Immunol. 151:2296;
Chothia et al. (1987) J. Mol. Biol. 196:901. Another method uses a
particular framework derived from the consensus sequence of all
human antibodies of a particular subgroup of light or heavy chains.
The same framework may be used for several different humanized
antibodies. See, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci.
USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623.
[0175] It is further generally desirable that antibodies be
humanized with retention of high affinity for the antigen and other
favorable biological properties. To achieve this goal, according to
one method, humanized antibodies are prepared by a process of
analysis of the parental sequences and various conceptual humanized
products using three-dimensional models of the parental and
humanized sequences. Three-dimensional immunoglobulin models are
commonly available and are familiar to those skilled in the art.
Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
hypervariable region residues are directly and most substantially
involved in influencing antigen binding.
Human Antibodies
[0176] Human antibodies of the invention can be constructed by
combining Fv clone variable domain sequence(s) selected from
human-derived phage display libraries with known human constant
domain sequences(s) as described above. Alternatively, human
monoclonal antibodies of the invention can be made by the hybridoma
method. Human myeloma and mouse-human heteromyeloma cell lines for
the production of human monoclonal antibodies have been described,
for example, by Kozbor J. Immunol., 133: 3001 (1984); Brodeur et
al., Monoclonal Antibody Production Techniques and Applications,
pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et
al., J. Immunol., 147: 86 (1991).
[0177] It is now possible to produce transgenic animals (e.g. mice)
that are capable, upon immunization, of producing a full repertoire
of human antibodies in the absence of endogenous immunoglobulin
production. For example, it has been described that the homozygous
deletion of the antibody heavy-chain joining region (JH) gene in
chimeric and germ-line mutant mice results in complete inhibition
of endogenous antibody production. Transfer of the human germ-line
immunoglobulin gene array in such germ-line mutant mice will result
in the production of human antibodies upon antigen challenge. See,
e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551
(1993); Jakobovits et al., Nature, 362: 255 (1993); Bruggermann et
al., Year in Immunol., 7: 33 (1993).
[0178] Gene shuffling can also be used to derive human antibodies
from non-human, e.g. rodent, antibodies, where the human antibody
has similar affinities and specificities to the starting non-human
antibody. According to this method, which is also called "epitope
imprinting", either the heavy or light chain variable region of a
non-human antibody fragment obtained by phage display techniques as
described herein is replaced with a repertoire of human V domain
genes, creating a population of non-human chain/human chain scFv or
Fab chimeras. Selection with antigen results in isolation of a
non-human to chain/human chain chimeric scFv or Fab wherein the
human chain restores the antigen binding site destroyed upon
removal of the corresponding non-human chain in the primary phage
display clone, i.e. the epitope governs (imprints) the choice of
the human chain partner. When the process is repeated in order to
replace the remaining non-human chain, a human antibody is obtained
(see PCT WO 93/06213 published Apr. 1, 1993). Unlike traditional
humanization of non-human antibodies by HVR grafting, this
technique provides completely human antibodies, which have no FR or
HVR residues of non-human origin.
Bispecific Antibodies
[0179] Bispecific antibodies are monoclonal antibodies that have
binding specificities for at least two different antigens. In
certain embodiments, bispecific antibodies are human or humanized
antibodies. In certain embodiments, one of the binding
specificities is for EGFL7 and the other is for any other antigen.
In certain embodiments, the other antigen is vascular endothelial
growth factor (VEGF), e.g. the epitope bound by the antibodies
bevacizumab and ranibizumab. In certain embodiments, the bispecific
antibody has a first arm comprising the HVR sequences of an
antibody of the invention and a second arm comprising the HVR
sequences of bevacizumab or ranibizumab. In certain embodiments,
the bispecific antibody comprises the VH and VL sequences of
bevacizumab or ranibizumab. In certain embodiments, bispecific
antibodies may bind to two different epitopes of EGFL7. Bispecific
antibodies may also be used to localize cytotoxic agents to cells
which express EGFL7. These antibodies possess a EGFL7-binding arm
and an arm which binds a cytotoxic agent, such as, e.g., saporin,
anti-interferon-.alpha., vinca alkaloid, ricin A chain,
methotrexate or radioactive isotope hapten. Bispecific antibodies
can be prepared as full length antibodies or antibody fragments
(e.g. F(ab').sub.2 bispecific antibodies).
[0180] Methods for making bispecific antibodies are known in the
art. Traditionally, the recombinant production of bispecific
antibodies is based on the co-expression of two immunoglobulin
heavy chain-light chain pairs, where the two heavy chains have
different specificities (Milstein and Cuello, Nature, 305: 537
(1983)). Because of the random assortment of immunoglobulin heavy
and light chains, these hybridomas (quadromas) produce a potential
mixture of 10 different antibody molecules, of which only one has
the correct bispecific structure. The purification of the correct
molecule, which is usually done by affinity chromatography steps,
is rather cumbersome, and the product yields are low. Similar
procedures are disclosed in WO 93/08829 published May 13, 1993, and
in Traunecker et al., EMBO J., 10: 3655 (1991).
[0181] According to a different approach, antibody variable domains
with the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences. The
fusion, for example, is with an immunoglobulin heavy chain constant
domain, comprising at least part of the hinge, CH2, and CH3
regions. In certain embodiments, the first heavy-chain constant
region (CH1), containing the site necessary for light chain
binding, is present in at least one of the fusions. DNAs encoding
the immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression
vectors, and are co-transfected into a suitable host organism. This
provides for great flexibility in adjusting the mutual proportions
of the three polypeptide fragments in embodiments when unequal
ratios of the three polypeptide chains used in the construction
provide the optimum yields. It is, however, possible to insert the
coding sequences for two or all three polypeptide chains in one
expression vector when the expression of at least two polypeptide
chains in equal ratios results in high yields or when the ratios
are of no particular significance.
[0182] In one embodiment of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with
a first binding specificity in one arm, and a hybrid immunoglobulin
heavy chain-light chain pair (providing a second binding
specificity) in the other arm. It was found that this asymmetric
structure facilitates the separation of the desired bispecific
compound from unwanted immunoglobulin chain combinations, as the
presence of an immunoglobulin light chain in only one half of the
bispecific molecule provides for a facile way of separation. This
approach is disclosed in WO 94/04690. For further details of
generating bispecific antibodies see, for example, Suresh et al.,
Methods in Enzymology, 121:210 (1986).
[0183] According to another approach, the interface between a pair
of antibody molecules can be engineered to maximize the percentage
of heterodimers which are recovered from recombinant cell culture.
The interface comprises at least a part of the C.sub.H3 domain of
an antibody constant domain. In this method, one or more small
amino acid side chains from the interface of the first antibody
molecule are replaced with larger side chains (e.g. tyrosine or
tryptophan). Compensatory "cavities" of identical or similar size
to the large side chain(s) are created on the interface of the
second antibody molecule by replacing large amino acid side chains
with smaller ones (e.g. alanine or threonine). This provides a
mechanism for increasing the yield of the heterodimer over other
unwanted end-products such as homodimers.
[0184] Bispecific antibodies include cross-linked or
"heteroconjugate" antibodies. For example, one of the antibodies in
the heteroconjugate can be coupled to avidin, the other to biotin.
Such antibodies have, for example, been proposed to target immune
system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for
treatment of HIV infection (WO 91/00360, WO 92/00373, and EP
03089). Heteroconjugate antibodies may be made using any convenient
cross-linking method. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
[0185] Techniques for generating bispecific antibodies from
antibody fragments have also been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. Brennan et al., Science, 229: 81 (1985) describe a
procedure wherein intact antibodies are proteolytically cleaved to
generate F(ab').sub.2 fragments. These fragments are reduced in the
presence of the dithiol complexing agent sodium arsenite to
stabilize vicinal dithiols and prevent intermolecular disulfide
formation. The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes.
[0186] Recent progress has facilitated the direct recovery of
Fab'-SH fragments from E. coli, which can be chemically coupled to
form bispecific antibodies. Shalaby et al., J. Exp. Med., 175:
217-225 (1992) describe the production of a fully humanized
bispecific antibody F(ab').sub.2 molecule. Each Fab' fragment was
separately secreted from E. coli and subjected to directed chemical
coupling in vitro to form the bispecific antibody. The bispecific
antibody thus formed was able to bind to cells overexpressing the
HER2 receptor and normal human T cells, as well as trigger the
lytic activity of human cytotoxic lymphocytes against human breast
tumor targets.
[0187] Various techniques for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al., J. Immunol.,
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers. The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain (VH) connected to a light-chain
variable domain (VL) by a linker which is too short to allow
pairing between the two domains on the same chain. Accordingly, the
VH and VL domains of one fragment are forced to pair with the
complementary VL and VH domains of another fragment, thereby
forming two antigen-binding sites. Another strategy for making
bispecific antibody fragments by the use of single-chain Fv (sFv)
dimers has also been reported. See Gruber et al., J. Immunol.,
152:5368 (1994).
[0188] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al. J.
Immunol. 147: 60 (1991).
Multivalent Antibodies
[0189] A multivalent antibody may be internalized (and/or
catabolized) faster than a bivalent antibody by a cell expressing
an antigen to which the antibodies bind. The antibodies of the
present invention can be multivalent antibodies (which are other
than of the IgM class) with three or more antigen binding sites
(e.g. tetravalent antibodies), which can be readily produced by
recombinant expression of nucleic acid encoding the polypeptide
chains of the antibody. The multivalent antibody can comprise a
dimerization domain and three or more antigen binding sites. In
certain embodiments, the dimerization domain comprises (or consists
of) an Fc region or a hinge region. In this scenario, the antibody
will comprise an Fc region and three or more antigen binding sites
amino-terminal to the Fc region. In certain embodiments, a
multivalent antibody comprises (or consists of) three to about
eight antigen binding sites. In one such embodiment, a multivalent
antibody comprises (or consists of) four antigen binding sites. The
multivalent antibody comprises at least one polypeptide chain (for
example, two polypeptide chains), wherein the polypeptide chain(s)
comprise two or more variable domains. For instance, the
polypeptide chain(s) may comprise VD1-(X.sub.1)
n-VD2-(X.sub.2)n-Fc, wherein VD1 is a first variable domain, VD2 is
a second variable domain, Fc is one polypeptide chain of an Fc
region, X.sub.1 and X.sub.2 represent an amino acid or polypeptide,
and n is 0 or 1. For instance, the polypeptide chain(s) may
comprise: VH-CH1-flexible linker-VH-CH1-Fc region chain; or
VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein may
further comprise at least two (for example, four) light chain
variable domain polypeptides. The multivalent antibody herein may,
for instance, comprise from about two to about eight light chain
variable domain polypeptides. The light chain variable domain
polypeptides contemplated here comprise a light chain variable
domain and, optionally, further comprise a CL domain.
Single-Domain Antibodies
[0190] In some embodiments, an antibody of the invention is a
single-domain antibody. A single-domain antibody is a single
polyeptide chain comprising all or a portion of the heavy chain
variable domain or all or a portion of the light chain variable
domain of an antibody. In certain embodiments, a single-domain
antibody is a human single-domain antibody (Domantis, Inc.,
Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1). In one
embodiment, a single-domain antibody consists of all or a portion
of the heavy chain variable domain of an antibody.
Antibody Variants
[0191] In some embodiments, amino acid sequence modification(s) of
the antibodies described herein are contemplated. For example, it
may be desirable to improve the binding affinity and/or other
biological properties of the antibody. Amino acid sequence variants
of the antibody may be prepared by introducing appropriate changes
into the nucleotide sequence encoding the antibody, or by peptide
synthesis. Such modifications include, for example, deletions from,
and/or insertions into and/or substitutions of, residues within the
amino acid sequences of the antibody. Any combination of deletion,
insertion, and substitution can be made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics. The amino acid alterations may be introduced in
the subject antibody amino acid sequence at the time that sequence
is made.
[0192] A useful method for identification of certain residues or
regions of the antibody that are preferred locations for
mutagenesis is called "alanine scanning mutagenesis" as described
by Cunningham and Wells (1989) Science, 244:1081-1085. Here, a
residue or group of target residues are identified (e.g., charged
residues such as arg, asp, his, lys, and glu) and replaced by a
neutral or negatively charged amino acid (e.g., alanine or
polyalanine) to affect the interaction of the amino acids with
antigen. Those amino acid locations demonstrating functional
sensitivity to the substitutions then are refined by introducing
further or other variants at, or for, the sites of substitution.
Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se need
not be predetermined. For example, to analyze the performance of a
mutation at a given site, ala scanning or random mutagenesis is
conducted at the target codon or region and the expressed
immunoglobulins are screened for the desired activity.
[0193] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue. Other insertional variants of the
antibody molecule include the fusion to the N- or C-terminus of the
antibody to an enzyme (e.g. for ADEPT) or a polypeptide which
increases the serum half-life of the antibody.
[0194] In certain embodiments, an antibody of the invention is
altered to increase or decrease the extent to which the antibody is
glycosylated. Glycosylation of polypeptides is typically either
N-linked or O-linked. N-linked refers to the attachment of a
carbohydrate moiety to the side chain of an asparagine residue. The
tripeptide sequences asparagine-X-serine and
asparagine-X-threonine, where X is any amino acid except proline,
are the recognition sequences for enzymatic attachment of the
carbohydrate moiety to the asparagine side chain. Thus, the
presence of either of these tripeptide sequences in a polypeptide
creates a potential glycosylation site. O-linked glycosylation
refers to the attachment of one of the sugars N-aceylgalactosamine,
galactose, or xylose to a hydroxyamino acid, most commonly serine
or threonine, although 5-hydroxyproline or 5-hydroxylysine may also
be used.
[0195] Addition or deletion of glycosylation sites to the antibody
is conveniently accomplished by altering the amino acid sequence
such that one or more of the above-described tripeptide sequences
(for N-linked glycosylation sites) is created or removed. The
alteration may also be made by the addition, deletion, or
substitution of one or more serine or threonine residues to the
sequence of the original antibody (for O-linked glycosylation
sites).
[0196] Where the antibody comprises an Fc region, the carbohydrate
attached thereto may be altered. Native antibodies produced by
mammalian cells typically comprise a branched, biantennary
oligosaccharide that is generally attached by an N-linkage to
Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al.
(1997) TIBTECH 15:26-32. The oligosaccharide may include various
carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc),
galactose, and sialic acid, as well as a fucose attached to a
GlcNAc in the "stem" of the biantennary oligosaccharide structure.
In some embodiments, modifications of the oligosaccharide in an
antibody of the invention may be made in order to create antibody
variants with certain improved properties.
[0197] For example, antibody variants are provided having a
carbohydrate structure that lacks fucose attached (directly or
indirectly) to an Fc region. Such variants may have improved ADCC
function. See, e.g., US Patent Publication Nos. US 2003/0157108
(Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd).
Examples of publications related to "defucosylated" or
"fucose-deficient" antibody variants include: US 2003/0157108; WO
2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US
2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US
2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO
2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol.
Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng.
87: 614 (2004). Examples of cell lines capable of producing
defucosylated antibodies include Lec13 CHO cells deficient in
protein fucosylation (Ripka et al. Arch. Biochem. Biophys.
249:533-545 (1986); US Pat Appl No US 2003/0157108 A1, Presta, L;
and WO 2004/056312 A1, Adams et al., especially at Example 11), and
knockout cell lines, such as alpha-1,6-fucosyltransferase gene,
FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech.
Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng.,
94(4):680-688 (2006); and WO2003/085107).
[0198] Antibodies variants are further provided with bisected
oligosaccharides, e.g., in which a biantennary oligosaccharide
attached to the Fc region of the antibody is bisected by GlcNAc.
Such antibody variants may have reduced fucosylation and/or
improved ADCC function. Examples of such antibody variants are
described, e.g., in WO 2003/011878 (Jean-Mairet et al.); U.S. Pat.
No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.).
Antibody variants with at least one galactose residue in the
oligosaccharide attached to the Fc region are also provided. Such
antibody variants may have improved CDC function. Such antibody
variants are described, e.g., in WO 1997/30087 (Patel et al.); WO
1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
[0199] In certain embodiments, an antibody variant comprises an Fc
region with one or more amino acid substitutions which further
improve ADCC, for example, substitutions at positions 298, 333,
and/or 334 of the Fc region (Eu numbering of residues). Such
substitutions may occur in combination with any of the variations
described above.
[0200] In certain embodiments, the invention contemplates an
antibody variant that possesses some but not all effector
functions, which make it a desirable candidate for many
applications in which the half life of the antibody in vivo is
important yet certain effector functions (such as complement and
ADCC) are unnecessary or deleterious. In certain embodiments, the
Fc activities of the antibody are measured to ensure that only the
desired properties are maintained. In vitro and/or in vivo
cytotoxicity assays can be conducted to confirm the
reduction/depletion of CDC and/or ADCC activities. For example, Fc
receptor (FcR) binding assays can be conducted to ensure that the
antibody lacks Fc.gamma.R binding (hence likely lacking ADCC
activity), but retains FcRn binding ability. The primary cells for
mediating ADCC, NK cells, express Fc.gamma.RIII only, whereas
monocytes express Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII. FcR
expression on hematopoietic cells is summarized in Table 3 on page
464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991).
Non-limiting examples of in vitro assays to assess ADCC activity of
a molecule of interest is described in U.S. Pat. No. 5,500,362
(see, e.g. Hellstrom, I., et al. Proc. Nat'l Acad. Sci. USA
83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad.
Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see
Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).
Alternatively, non-radioactive assays methods may be employed (see,
for example, ACTI.TM. non-radioactive cytotoxicity assay for flow
cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox
96.RTM. non-radioactive cytotoxicity assay (Promega, Madison,
Wis.). Useful effector cells for such assays include peripheral
blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest may be assessed in vivo, e.g., in a animal model such as
that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA
95:652-656 (1998). Clq binding assays may also be carried out to
confirm that the antibody is unable to bind Clq and hence lacks CDC
activity. To assess complement activation, a CDC assay may be
performed (see, for example, Gazzano-Santoro et al., J. Immunol.
Methods 202:163 (1996); Cragg, M. S. et al., Blood 101:1045-1052
(2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743
(2004)). FcRn binding and in vivo clearance/half life
determinations can also be performed using methods known in the art
(see, for example, Petkova, S. B. et al., Int'l. Immunol.
18(12):1759-1769 (2006)).
[0201] Other antibody variants having one or more amino acid
substitutions are provided. Sites of interest for substitutional
mutagenesis include the hypervariable regions, but FR alterations
are also contemplated. Conservative substitutions are shown in
Table 1 under the heading of "preferred substitutions." More
substantial changes, denominated "exemplary substitutions" are
provided in the "Amino Acid Substitution Table", or as further
described below in reference to amino acid classes. Amino acid
substitutions may be introduced into an antibody of interest and
the products screened, e.g., for a desired activity, such as
improved antigen binding, decreased immunogenicity, improved ADCC
or CDC, etc.
TABLE-US-00004 Amino Acid Substitution Table Original Exemplary
Preferred Residue Substitutions Substitutions Ala (A) Val; Leu; Ile
Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln
Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu
(E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile
(I) Leu; Val; Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine;
Ile; Val; Ile Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu;
Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala
Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr
(Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala;
Norleucine
[0202] Modifications in the biological properties of an antibody
may be accomplished by selecting substitutions that affect (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Amino acids may be grouped
according to similarities in the properties of their side chains
(in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth
Publishers, New York (1975)): [0203] (1) non-polar: Ala (A), Val
(V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M) [0204]
(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y),
Asn (N), Gln (Q) [0205] (3) acidic: Asp (D), Glu (E) [0206] (4)
basic: Lys (K), Arg (R), His (H)
[0207] Alternatively, naturally occurring residues may be divided
into groups based on common side-chain properties: [0208] (1)
hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; [0209] (2)
neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; [0210] (3) acidic:
Asp, Glu; [0211] (4) basic: His, Lys, Arg; [0212] (5) residues that
influence chain orientation: Gly, Pro; [0213] (6) aromatic: Trp,
Tyr, Phe.
[0214] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class. Such substituted
residues also may be introduced into the conservative substitution
sites or, into the remaining (non-conserved) sites.
[0215] One type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody (e.g. a
humanized or human antibody). Generally, the resulting variant(s)
selected for further development will have modified (e.g.,
improved) biological properties relative to the parent antibody
from which they are generated. An exemplary substitutional variant
is an affinity matured antibody, which may be conveniently
generated using phage display-based affinity maturation techniques.
Briefly, several hypervariable region sites (e.g. 6-7 sites) are
mutated to generate all possible amino acid substitutions at each
site. The antibodies thus generated are displayed from filamentous
phage particles as fusions to at least part of a phage coat protein
(e.g., the gene III product of M13) packaged within each particle.
The phage-displayed variants are then screened for their biological
activity (e.g. binding affinity). In order to identify candidate
hypervariable region sites for modification, scanning mutagenesis
(e.g., alanine scanning) can be performed to identify hypervariable
region residues contributing significantly to antigen binding.
Alternatively, or additionally, it may be beneficial to analyze a
crystal structure of the antigen-antibody complex to identify
contact points between the antibody and antigen. Such contact
residues and neighboring residues are candidates for substitution
according to techniques known in the art, including those
elaborated herein. Once such variants are generated, the panel of
variants is subjected to screening using techniques known in the
art, including those described herein, and variants with superior
properties in one or more relevant assays may be selected for
further development.
[0216] Nucleic acid molecules encoding amino acid sequence variants
of the antibody are prepared by a variety of methods known in the
art. These methods include, but are not limited to, isolation from
a natural source (in the case of naturally occurring amino acid
sequence variants) or preparation by oligonucleotide-mediated (or
site-directed) mutagenesis, PCR mutagenesis, and cassette
mutagenesis of an earlier prepared variant or a non-variant version
of the antibody.
[0217] It may be desirable to introduce one or more amino acid
modifications in an Fc region of antibodies of the invention,
thereby generating an Fc region variant. The Fc region variant may
comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3
or IgG4 Fc region) comprising an amino acid modification (e.g. a
substitution) at one or more amino acid positions including that of
a hinge cysteine.
[0218] In accordance with this description and the teachings of the
art, it is contemplated that in some embodiments, an antibody of
the invention may comprise one or more alterations as compared to
the wild type counterpart antibody, e.g. in the Fc region. These
antibodies would nonetheless retain substantially the same
characteristics required for therapeutic utility as compared to
their wild type counterpart. For example, it is thought that
certain alterations can be made in the Fc region that would result
in altered (i.e., either improved or diminished) C1q binding and/or
Complement Dependent Cytotoxicity (CDC), e.g., as described in
WO99/51642. See also Duncan & Winter, Nature 322:738-40 (1988);
U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO94/29351
concerning other examples of Fc region variants. WO00/42072
(Presta) and WO 2004/056312 (Lowman) describe antibody variants
with improved or diminished binding to FcRs. See, also, Shields et
al. J. Biol. Chem. 9(2): 6591-6604 (2001). Antibodies with
increased half lives and improved binding to the neonatal Fc
receptor (FcRn), which is responsible for the transfer of maternal
IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim
et al., J. Immunol. 24:249 (1994)), are described in
US2005/0014934A1 (Hinton et al.). These antibodies comprise an Fc
region with one or more substitutions therein which improve binding
of the Fc region to FcRn. Polypeptide variants with altered Fc
region amino acid sequences and increased or decreased Clq binding
capability are described in U.S. Pat. No. 6,194,551B1, WO99/51642.
See, also, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).
[0219] In another aspect, the invention provides antibodies
comprising modifications in the interface of Fc polypeptides
comprising the Fc region, wherein the modifications facilitate
and/or promote heterodimerization. These modifications comprise
introduction of a protuberance into a first Fc polypeptide and a
cavity into a second Fc polypeptide, wherein the protuberance is
positionable in the cavity so as to promote complexing of the first
and second Fc polypeptides. Methods of generating antibodies with
these modifications are known in the art, e.g., as described in
U.S. Pat. No. 5,731,168.
[0220] In yet another aspect, it may be desirable to create
cysteine engineered antibodies, e.g., "thioMAbs," in which one or
more residues of an antibody are substituted with cysteine
residues. In particular embodiments, the substituted residues occur
at accessible sites of the antibody. By substituting those residues
with cysteine, reactive thiol groups are thereby positioned at
accessible sites of the antibody and may be used to conjugate the
antibody to other moieties, such as drug moieties or linker-drug
moieties, as described further herein. In certain embodiments, any
one or more of the following residues may be substituted with
cysteine: V205 (Kabat numbering) of the light chain; A118 (EU
numbering) of the heavy chain; and S400 (EU numbering) of the heavy
chain Fc region.
Antibody Derivatives
[0221] The antibodies of the present invention can be further
modified to contain additional nonproteinaceous moieties that are
known in the art and readily available. Preferably, the moieties
suitable for derivatization of the antibody are water soluble
polymers. Non-limiting examples of water soluble polymers include,
but are not limited to, polyethylene glycol (PEG), copolymers of
ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,
polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,
poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer,
polyaminoacids (either homopolymers or random copolymers), and
dextran or poly(n-vinyl pyrrolidone)polyethylene glycol,
propropylene glycol homopolymers, prolypropylene oxide/ethylene
oxide co-polymers, polyoxyethylated polyols (e.g., glycerol),
polyvinyl alcohol, and mixtures thereof. Polyethylene glycol
propionaldehyde may have advantages in manufacturing due to its
stability in water. The polymer may be of any molecular weight, and
may be branched or unbranched. The number of polymers attached to
the antibody may vary, and if more than one polymer are attached,
they can be the same or different molecules. In general, the number
and/or type of polymers used for derivatization can be determined
based on considerations including, but not limited to, the
particular properties or functions of the antibody to be improved,
whether the antibody derivative will be used in a therapy under
defined conditions, etc.
[0222] In another embodiment, conjugates of an antibody and
nonproteinaceous moiety that may be selectively heated by exposure
to radiation are provided. In one embodiment, the nonproteinaceous
moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA
102: 11600-11605 (2005)). The radiation may be of any wavelength,
and includes, but is not limited to, wavelengths that do not harm
ordinary cells, but which heat the nonproteinaceous moiety to a
temperature at which cells proximal to the
antibody-nonproteinaceous moiety are killed.
Activity Assays
[0223] The antibodies of the present invention can be characterized
for their physical/chemical properties and biological functions by
various assays known in the art.
[0224] In one aspect, assays are provided for identifying
anti-EGFL7 antibodies thereof having biological activity.
Biological activity may include, e.g., the modulation of one or
more aspects of EGFL7-associated effects, including but not limited
to EGFL7 binding, EGFL7-mediated protection of endothelial cells
under hypoxic stress, and the ability of EGFL7 to mediate
endothelial cell adhesion.
[0225] In certain embodiments of the invention, the immunoglobulins
produced herein are analyzed for their biological activity. In some
embodiments, the immunoglobulins of the present invention are
tested for their antigen binding activity. The antigen binding
assays that are known in the art and can be used herein include
without limitation any direct or competitive binding assays using
techniques such as western blots, radioimmunoassays, ELISA (enzyme
linked immunosorbent assay), "sandwich" immunoassays,
immunoprecipitation assays, fluorescent immunoassays, and protein A
immunoassays. An illustrative antigen binding assay is provided
below in the Examples section.
[0226] The purified antibodies can be further characterized by a
series of assays including, but not limited to, N-terminal
sequencing, amino acid analysis, non-denaturing size exclusion high
pressure liquid chromatography (HPLC), mass spectrometry, ion
exchange chromatography and papain digestion.
[0227] In some embodiments, the present invention contemplates
altered antibodies that possess some but not all effector
functions, which make it a desired candidate for many applications
in which the half life of the antibody in vivo is important yet
certain effector functions (such as complement and ADCC) are
unnecessary or deleterious. In certain embodiments, the Fc
activities of the produced immunoglobulin are measured to ensure
that only the desired properties are maintained. In vitro and/or in
vivo cytotoxicity assays can be conducted to confirm the
reduction/depletion of CDC and/or ADCC activities. For example, Fc
receptor (FcR) binding assays can be conducted to ensure that the
antibody lacks Fc.gamma.R binding (hence likely lacking ADCC
activity), but retains FcRn binding ability. The primary cells for
mediating ADCC, NK cells, express Fc.gamma.RIII only, whereas
monocytes express Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII. FcR
expression on hematopoietic cells is summarized in Table 3 on page
464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). An
example of an in vitro assay to assess ADCC activity of a molecule
of interest is described in U.S. Pat. No. 5,500,362 or 5,821,337.
Useful effector cells for such assays include peripheral blood
mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest may be assessed in vivo, e.g., in a animal model such as
that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998). Clq
binding assays may also be carried out to confirm that the antibody
is unable to bind Clq and hence lacks CDC activity. To assess
complement activation, a CDC assay, e.g. as described in
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be
to performed. FcRn binding and in vivo clearance/half life
determinations can also be performed using methods known in the
art.
[0228] In some embodiments, the invention provides altered
antibodies that possess increased effector functions and/or
increased half-life.
Vectors, Host Cells and Recombinant Methods
[0229] For recombinant production of an antibody of the invention,
the nucleic acid encoding it is isolated and inserted into a
replicable vector for further cloning (amplification of the DNA) or
for expression. DNA encoding the antibody is readily isolated and
sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the antibody). Many
vectors are available. The choice of vector depends in part on the
host cell to be used. Generally, preferred host cells are of either
prokaryotic or eukaryotic (generally mammalian) origin. It will be
appreciated that constant regions of any isotype can be used for
this purpose, including IgG, IgM, IgA, IgD, and IgE constant
regions, and that such constant regions can be obtained from any
human or animal species.
[0230] a. Generating Antibodies Using Prokaryotic Host Cells:
[0231] i. Vector Construction
[0232] Polynucleotide sequences encoding polypeptide components of
the antibody of the invention can be obtained using standard
recombinant techniques. Desired polynucleotide sequences may be
isolated and sequenced from antibody producing cells such as
hybridoma cells. Alternatively, polynucleotides can be synthesized
using nucleotide synthesizer or PCR techniques. Once obtained,
sequences encoding the polypeptides are inserted into a recombinant
vector capable of replicating and expressing heterologous
polynucleotides in prokaryotic hosts. Many vectors that are
available and known in the art can be used for the purpose of the
present invention. Selection of an appropriate vector will depend
mainly on the size of the nucleic acids to be inserted into the
vector and the particular host cell to be transformed with the
vector. Each vector contains various components, depending on its
function (amplification or expression of heterologous
polynucleotide, or both) and its compatibility with the particular
host cell in which it resides. The vector components generally
include, but are not limited to: an origin of replication, a
selection marker gene, a promoter, a ribosome binding site (RBS), a
signal sequence, the heterologous nucleic acid insert and a
transcription termination sequence.
[0233] In general, plasmid vectors containing replicon and control
sequences which are derived from species compatible with the host
cell are used in connection with these hosts. The vector ordinarily
carries a replication site, as well as marking sequences which are
capable of providing phenotypic selection in transformed cells. For
example, E. coli is typically transformed using pBR322, a plasmid
derived from an E. coli species. pBR322 contains genes encoding
ampicillin (Amp) and tetracycline (Tet) resistance and thus
provides easy means for identifying transformed cells. pBR322, its
derivatives, or other microbial plasmids or bacteriophage may also
contain, or be modified to contain, promoters which can be used by
the microbial organism for expression of endogenous proteins.
Examples of pBR322 derivatives used for expression of particular
antibodies are described in detail in Carter et al., U.S. Pat. No.
5,648,237.
[0234] In addition, phage vectors containing replicon and control
sequences that are compatible with the host microorganism can be
used as transforming vectors in connection with these hosts. For
example, bacteriophage such as .lamda.GEM.TM.-11 may be utilized in
making a recombinant vector which can be used to transform
susceptible host cells such as E. coli LE392.
[0235] The expression vector of the invention may comprise two or
more promoter-cistron pairs, encoding each of the polypeptide
components. A promoter is an untranslated regulatory sequence
located upstream (5') to a cistron that modulates its expression.
Prokaryotic promoters typically fall into two classes, inducible
and constitutive. Inducible promoter is a promoter that initiates
increased levels of transcription of the cistron under its control
in response to changes in the culture condition, e.g. the presence
or absence of a nutrient or a change in temperature.
[0236] A large number of promoters recognized by a variety of
potential host cells are well known. The selected promoter can be
operably linked to cistron DNA encoding the light or heavy chain by
removing the promoter from the source DNA via restriction enzyme
digestion and inserting the isolated promoter sequence into the
vector of the invention. Both the native promoter sequence and many
heterologous promoters may be used to direct amplification and/or
expression of the target genes. In some embodiments, heterologous
promoters are utilized, as they generally permit greater
transcription and higher yields of expressed target gene as
compared to the native target polypeptide promoter.
[0237] Promoters suitable for use with prokaryotic hosts include
the PhoA promoter, the (3-galactamase and lactose promoter systems,
a tryptophan (trp) promoter system and hybrid promoters such as the
tac or the trc promoter. However, other promoters that are
functional in bacteria (such as other known bacterial or phage
promoters) are suitable as well. Their nucleotide sequences have
been published, thereby enabling a skilled worker operably to
ligate them to cistrons encoding the target light and heavy chains
(Siebenlist et al. (1980) Cell 20: 269) using linkers or adaptors
to supply any required restriction sites.
[0238] In one aspect of the invention, each cistron within the
recombinant vector comprises a secretion signal sequence component
that directs translocation of the expressed polypeptides across a
membrane. In general, the signal sequence may be a component of the
vector, or it may be a part of the target polypeptide DNA that is
inserted into the vector. The signal sequence selected for the
purpose of this invention should be one that is recognized and
processed (i.e. cleaved by a signal peptidase) by the host cell.
For prokaryotic host cells that do not recognize and process the
signal sequences native to the heterologous polypeptides, the
signal sequence is substituted by a prokaryotic signal sequence
selected, for example, from the group consisting of the alkaline
phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II
(STII) leaders, LamB, PhoE, PelB, OmpA and MBP. In one embodiment
of the invention, the signal sequences used in both cistrons of the
expression system are STII signal sequences or variants
thereof.
[0239] In another aspect, the production of the immunoglobulins
according to the invention can occur in the cytoplasm of the host
cell, and therefore does not require the presence of secretion
signal sequences within each cistron. In that regard,
immunoglobulin light and heavy chains are expressed, folded and
assembled to form functional immunoglobulins within the cytoplasm.
Certain host strains (e.g., the E. coli trxB-strains) provide
cytoplasm conditions that are favorable for disulfide bond
formation, thereby permitting proper folding and assembly of
expressed protein subunits. Proba and Pluckthun Gene, 159:203
(1995).
[0240] Prokaryotic host cells suitable for expressing antibodies of
the invention include Archaebacteria and Eubacteria, such as
Gram-negative or Gram-positive organisms. Examples of useful
bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B.
subtilis), Enterobacteria, Pseudomonas species (e.g., P.
aeruginosa), Salmonella typhimurium, Serratia marcescans,
Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or
Paracoccus. In one embodiment, gram-negative cells are used. In one
embodiment, E. coli cells are used as hosts for the invention.
Examples of E. coli strains include strain W3110 (Bachmann,
Cellular and Molecular Biology, vol. 2 (Washington, D.C.: American
Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No.
27,325) and derivatives thereof, including strain 33D3 having
genotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8
.DELTA.ompT.DELTA.(nmpc-fepE) degP41 kanR (U.S. Pat. No.
5,639,635). Other strains and derivatives thereof, such as E. coli
294 (ATCC.RTM. 31,446), E. coli B, E. coliX 1776 (ATCC.RTM. 31,537)
and E. coli RV308(ATCC.RTM. 31,608) are also suitable. These
examples are illustrative rather than limiting. Methods for
constructing derivatives of any of the above-mentioned bacteria
having defined genotypes are known in the art and described in, for
example, Bass et al., Proteins, 8:309-314 (1990). It is generally
necessary to select the appropriate bacteria taking into
consideration replicability of the replicon in the cells of a
bacterium. For example, E. coli, Serratia, or Salmonella species
can be suitably used as the host when well known plasmids such as
pBR322, pBR325, pACYC177, or pKN410 are used to supply the
replicon. Typically the host cell should secrete minimal amounts of
proteolytic enzymes, and additional protease inhibitors may
desirably be incorporated in the cell culture.
ii. Antibody Production
[0241] Host cells are transformed with the above-described
expression vectors and cultured in conventional nutrient media
modified as appropriate for inducing promoters, selecting
transformants, or amplifying the genes encoding the desired
sequences.
[0242] Transformation means introducing DNA into the prokaryotic
host so that the DNA is replicable, either as an extrachromosomal
element or by chromosomal integrant. Depending on the host cell
used, transformation is done using standard techniques appropriate
to such cells. The calcium treatment employing calcium chloride is
generally used for bacterial cells that contain substantial
cell-wall barriers. Another method for transformation employs
polyethylene glycol/DMSO. Yet another technique used is
electroporation.
[0243] Prokaryotic cells used to produce the polypeptides of the
invention are grown in media known in the art and suitable for
culture of the selected host cells. Examples of suitable media
include luria broth (LB) plus necessary nutrient supplements. In
some embodiments, the media also contains a selection agent, chosen
based on the construction of the expression vector, to selectively
permit growth of prokaryotic cells containing the expression
vector. For example, ampicillin is added to media for growth of
cells expressing ampicillin resistant gene.
[0244] Any necessary supplements besides carbon, nitrogen, and
inorganic phosphate sources may also be included at appropriate
concentrations introduced alone or as a mixture with another
supplement or medium such as a complex nitrogen source. Optionally
the culture medium may contain one or more reducing agents selected
from the group consisting of glutathione, cysteine, cystamine,
thioglycollate, dithioerythritol and dithiothreitol. The
prokaryotic host cells are cultured at suitable temperatures. For
E. coli growth, for example, the preferred temperature ranges from
about 20.degree. C. to about 39.degree. C., more preferably from
about 25.degree. C. to about 37.degree. C., even more preferably at
about 30.degree. C. The pH of the medium may be any pH ranging from
about 5 to about 9, depending mainly on the host organism. For E.
coli, the pH is preferably from about 6.8 to about 7.4, and more
preferably about 7.0.
[0245] If an inducible promoter is used in the expression vector of
the invention, protein expression is induced under conditions
suitable for the activation of the promoter. In one aspect of the
invention, PhoA promoters are used for controlling transcription of
the polypeptides. Accordingly, the transformed host cells are
cultured in a phosphate-limiting medium for induction. Preferably,
the phosphate-limiting medium is the C.R.A.P medium (see, e.g.,
Simmons et al., J. Immunol. Methods (2002), 263:133-147). A variety
of other inducers may be used, according to the vector construct
employed, as is known in the art.
[0246] In one embodiment, the expressed polypeptides of the present
invention are secreted into and recovered from the periplasm of the
host cells. Protein recovery typically involves disrupting the
microorganism, generally by such means as osmotic shock, sonication
or lysis. Once cells are disrupted, cell debris or whole cells may
be removed by centrifugation or filtration. The proteins may be
further purified, for example, by affinity resin chromatography.
Alternatively, proteins can be transported into the culture media
and isolated therein. Cells may be removed from the culture and the
culture supernatant being filtered and concentrated for further
purification of the proteins produced. The expressed polypeptides
can be further isolated and identified using commonly known methods
such as polyacrylamide gel electrophoresis (PAGE) and Western blot
assay.
[0247] In one aspect of the invention, antibody production is
conducted in large quantity by a fermentation process. Various
large-scale fed-batch fermentation procedures are available for
production of recombinant proteins. Large-scale fermentations have
at least 1000 liters of capacity, preferably about 1,000 to 100,000
liters of capacity. These fermentors use agitator impellers to
distribute oxygen and nutrients, especially glucose (the preferred
carbon/energy source). Small scale fermentation refers generally to
fermentation in a fermentor that is no more than approximately 100
liters in volumetric capacity, and can range from about 1 liter to
about 100 liters.
[0248] In a fermentation process, induction of protein expression
is typically initiated after to the cells have been grown under
suitable conditions to a desired density, e.g., an OD550 of about
180-220, at which stage the cells are in the early stationary
phase. A variety of inducers may be used, according to the vector
construct employed, as is known in the art and described above.
Cells may be grown for shorter periods prior to induction. Cells
are usually induced for about 12-50 hours, although longer or
shorter induction time may be used.
[0249] To improve the production yield and quality of the
polypeptides of the invention, various fermentation conditions can
be modified. For example, to improve the proper assembly and
folding of the secreted antibody polypeptides, additional vectors
overexpressing chaperone proteins, such as Dsb proteins (DsbA,
DsbB, DsbC, DsbD and or DsbG) or FkpA (a peptidylprolyl
cis,trans-isomerase with chaperone activity) can be used to
co-transform the host prokaryotic cells. The chaperone proteins
have been demonstrated to facilitate the proper folding and
solubility of heterologous proteins produced in bacterial host
cells. Chen et al. (1999) J Bio Chem 274:19601-19605; Georgiou et
al., U.S. Pat. No. 6,083,715; Georgiou et al., U.S. Pat. No.
6,027,888; Bothmann and Pluckthun (2000) J. Biol. Chem.
275:17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem.
275:17106-17113; Arie et al. (2001) Mol. Microbiol. 39:199-210.
[0250] To minimize proteolysis of expressed heterologous proteins
(especially those that are proteolytically sensitive), certain host
strains deficient for proteolytic enzymes can be used for the
present invention. For example, host cell strains may be modified
to effect genetic mutation(s) in the genes encoding known bacterial
proteases such as Protease III, OmpT, DegP, Tsp, Protease I,
Protease Mi, Protease V, Protease VI and combinations thereof. Some
E. coli protease-deficient strains are available and described in,
for example, Joly et al. (1998), supra; Georgiou et al., U.S. Pat.
No. 5,264,365; Georgiou et al., U.S. Pat. No. 5,508,192; Hara et
al., Microbial Drug Resistance, 2:63-72 (1996).
[0251] In one embodiment, E. coli strains deficient for proteolytic
enzymes and transformed with plasmids overexpressing one or more
chaperone proteins are used as host cells in the expression system
of the invention.
[0252] iii. Antibody Purification
[0253] Standard protein purification methods known in the art can
be employed. The following procedures are exemplary of suitable
purification procedures: fractionation on immunoaffinity or
ion-exchange columns, ethanol precipitation, reverse phase HPLC,
chromatography on silica or on a cation-exchange resin such as
DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation,
and gel filtration using, for example, Sephadex.RTM. G-75.
[0254] In one aspect, Protein A immobilized on a solid phase is
used for immunoaffinity purification of the full length antibody
products of the invention. Protein A is a 41 kD cell wall protein
from Staphylococcus aureas which binds with a high affinity to the
Fc region of antibodies. Lindmark et al (1983) J. Immunol. Meth.
62:1-13. The solid phase to which Protein A is immobilized is
preferably a column comprising a glass or silica surface, more
preferably a controlled pore glass column or a silicic acid column.
In some applications, the column has been coated with a reagent,
such as glycerol, in an attempt to prevent nonspecific adherence of
contaminants.
[0255] As the first step of purification, the preparation derived
from the cell culture as described above is applied onto the
Protein A immobilized solid phase to allow specific binding of the
antibody of interest to Protein A. The solid phase is then washed
to remove contaminants non-specifically bound to the solid phase.
Finally the antibody of interest is recovered from the solid phase
by elution.
[0256] b. Generating Antibodies Using Eukaryotic Host Cells:
[0257] The vector components generally include, but are not limited
to, one or more of the following: a signal sequence, an origin of
replication, one or more marker genes, an enhancer element, a
promoter, and a transcription termination sequence.
[0258] (i) Signal Sequence Component
[0259] A vector for use in a eukaryotic host cell may also contain
a signal sequence or other polypeptide having a specific cleavage
site at the N-terminus of the mature protein or polypeptide of
interest. The heterologous signal sequence selected preferably is
one that is recognized and processed (i.e., cleaved by a signal
peptidase) by the host cell. In mammalian cell expression,
mammalian signal sequences as well as viral secretory leaders, for
example, the herpes simplex gD signal, are available.
[0260] The DNA for such precursor region is ligated in reading
frame to DNA encoding the antibody.
[0261] (ii) Origin of Replication
[0262] Generally, an origin of replication component is not needed
for mammalian expression vectors. For example, the SV40 origin may
typically be used only because it contains the early promoter.
[0263] (iii) Selection Gene Component
[0264] Expression and cloning vectors may contain a selection gene,
also termed a selectable marker. Typical selection genes encode
proteins that (a) confer resistance to antibiotics or other toxins,
e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)
complement auxotrophic deficiencies, where relevant, or (c) supply
critical nutrients not available from complex media.
[0265] One example of a selection scheme utilizes a drug to arrest
growth of a host cell. Those cells that are successfully
transformed with a heterologous gene produce a protein conferring
drug resistance and thus survive the selection regimen. Examples of
such dominant selection use the drugs neomycin, mycophenolic acid
and hygromycin.
[0266] Another example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the antibody nucleic acid, such as DHFR, thymidine
kinase, metallothionein-I and -II, preferably primate
metallothionein genes, adenosine deaminase, ornithine
decarboxylase, etc.
[0267] For example, cells transformed with the DHFR selection gene
are first identified by culturing all of the transformants in a
culture medium that contains methotrexate (Mtx), a competitive
antagonist of DHFR. An appropriate host cell when wild-type DHFR is
employed is the Chinese hamster ovary (CHO) cell line deficient in
DHFR activity (e.g., ATCC.RTM. CRL-9096).
[0268] Alternatively, host cells (particularly wild-type hosts that
contain endogenous DHFR) transformed or co-transformed with DNA
sequences encoding an antibody, wild-type DHFR protein, and another
selectable marker such as aminoglycoside 3'-phosphotransferase
(APH) can be selected by cell growth in medium containing a
selection agent for the selectable marker such as an
aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See
U.S. Pat. No. 4,965,199.
[0269] (iv) Promoter Component
[0270] Expression and cloning vectors usually contain a promoter
that is recognized by the host organism and is operably linked to
the antibody polypeptide nucleic acid. Promoter sequences are known
for eukaryotes. Virtually alleukaryotic genes have an AT-rich
region located approximately 25 to 30 bases upstream from the site
where transcription is initiated. Another sequence found 70 to 80
bases upstream from the start of transcription of many genes is a
CNCAAT (SEQ ID NO: 232) region where N may be any nucleotide. At
the 3' end of most eukaryotic genes is an AATAAA (SEQ ID NO: 233)
sequence that may be the signal for addition of the poly A tail to
the 3' end of the coding sequence. All of these sequences are
suitably inserted into eukaryotic expression vectors.
[0271] Antibody polypeptide transcription from vectors in mammalian
host cells is controlled, for example, by promoters obtained from
the genomes of viruses such as polyoma virus, fowlpox virus,
adenovirus (such as Adenovirus 2), bovine papilloma virus, avian
sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and
Simian Virus 40 (SV40), from heterologous mammalian promoters,
e.g., the actin promoter or an immunoglobulin promoter, from
heat-shock promoters, provided such promoters are compatible with
the host cell systems.
[0272] The early and late promoters of the SV40 virus are
conveniently obtained as an SV40 restriction fragment that also
contains the SV40 viral origin of replication. The immediate early
promoter of the human cytomegalovirus is conveniently obtained as a
HindIII E restriction fragment. A system for expressing DNA in
mammalian hosts using the bovine papilloma virus as a vector is
disclosed in U.S. Pat. No. 4,419,446. A modification of this system
is described in U.S. Pat. No. 4,601,978. Alternatively, the Rous
Sarcoma Virus long terminal repeat can be used as the promoter.
[0273] (v) Enhancer Element Component
[0274] Transcription of DNA encoding the antibody polypeptide of
this invention by higher eukaryotes is often increased by inserting
an enhancer sequence into the vector. Many enhancer sequences are
now known from mammalian genes (globin, elastase, albumin,
.alpha.-fetoprotein, and insulin). Typically, however, one will use
an enhancer from a eukaryotic cell virus. Examples include the SV40
enhancer on the late side of the replication origin (bp 100-270),
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and adenovirus
enhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing
elements for activation of eukaryotic promoters. The enhancer may
be spliced into the vector at a position 5' or 3' to the antibody
polypeptide-encoding sequence, but is preferably located at a site
5' from the promoter.
[0275] (vi) Transcription Termination Component
[0276] Expression vectors used in eukaryotic host cells will
typically also contain sequences necessary for the termination of
transcription and for stabilizing the mRNA. Such sequences are
commonly available from the 5' and, occasionally 3', untranslated
regions of eukaryotic or viral DNAs or cDNAs. These regions contain
nucleotide segments transcribed as polyadenylated fragments in the
untranslated portion of the mRNA encoding an antibody. One useful
transcription termination component is the bovine growth hormone
polyadenylation region. See WO94/11026 and the expression vector
disclosed therein.
[0277] (vii) Selection and Transformation of Host Cells
[0278] Suitable host cells for cloning or expressing the DNA in the
vectors herein include higher eukaryote cells described herein,
including vertebrate host cells. Propagation of vertebrate cells in
culture (tissue culture) has become a routine procedure. Examples
of useful mammalian host cell lines are monkey kidney CV1 line
transformed by SV40 (COS-7, ATCC.RTM. CRL 1651); human embryonic
kidney line (293 or 293 cells subcloned for growth in suspension
culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster
kidney cells (BHK, ATCC.RTM. CCL 10); Chinese hamster ovary
cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216
(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251
(1980)); monkey kidney cells (CV1 ATCC.RTM. CCL 70); African green
monkey kidney cells (VERO-76, ATCC.RTM. CRL-1587); human cervical
carcinoma cells (HELA, ATCC.RTM. CCL 2); canine kidney cells (MDCK,
ATCC.RTM. CCL 34); buffalo rat liver cells (BRL 3A, ATCC.RTM. CRL
1442); human lung cells (W138, ATCC.RTM. CCL 75); human liver cells
(Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC.RTM.
CCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68
(1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep
G2).
[0279] Host cells are transformed with the above-described
expression or cloning vectors for antibody production and cultured
in conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants, or amplifying the genes
encoding the desired sequences.
[0280] (viii) Culturing the Host Cells
[0281] The host cells used to produce an antibody of this invention
may be cultured in a variety of media. Commercially available media
such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM),
(Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium
((DMEM), Sigma) are suitable for culturing the host cells. In
addition, any of the media described in Ham et al., Meth. Enz.
58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S.
Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469;
WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used as
culture media for the host cells. Any of these media may be
supplemented as necessary with hormones and/or other growth factors
(such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleotides (such as adenosine and
thymidine), antibiotics (such as GENTAMYCINT.TM. drug), trace
elements (defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The culture conditions, such as
temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
[0282] (ix) Purification of Antibody
[0283] When using recombinant techniques, the antibody can be
produced intracellularly, or directly secreted into the medium. If
the antibody is produced intracellularly, as a first step, the
particulate debris, either host cells or lysed fragments, are
removed, for example, by centrifugation or ultrafiltration. Where
the antibody is secreted into the medium, supernatants from such
expression systems are generally first concentrated using a
commercially available protein concentration filter, for example,
an Amicon or Millipore Pellicon ultrafiltration unit. A protease
inhibitor such as PMSF may be included in any of the foregoing
steps to inhibit proteolysis and antibiotics may be included to
prevent the growth of adventitious contaminants.
[0284] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being the preferred purification technique.
The suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc domain that is present
in the antibody. Protein A can be used to purify antibodies that
are based on human .gamma.1, .gamma.2, or .gamma.4 heavy chains
(Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is
recommended for all mouse isotypes and for human .gamma.3 (Guss et
al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity
ligand is attached is most often agarose, but other matrices are
available. Mechanically stable matrices such as controlled pore
glass or poly(styrenedivinyl)benzene allow for faster flow rates
and shorter processing times than can be achieved with agarose.
Where the antibody comprises a CH3 domain, the Bakerbond ABX.TM.
resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.
Other techniques for protein purification such as fractionation on
an ion-exchange column, ethanol precipitation, Reverse Phase HPLC,
chromatography on silica, chromatography on heparin SEPHAROSE.TM.
chromatography on an anion or cation exchange resin (such as a
polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium
sulfate precipitation are also available depending on the antibody
to be recovered.
[0285] Following any preliminary purification step(s), the mixture
comprising the antibody of interest and contaminants may be
subjected to low pH hydrophobic interaction chromatography using an
elution buffer at a pH between about 2.5-4.5, preferably performed
at low salt concentrations (e.g., from about 0-0.25M salt).
[0286] Immunoconjugates
[0287] The invention also provides immunoconjugates
(interchangeably referred to as "antibody-drug conjugates," or
"ADCs") comprising an antibody conjugated to one or more cytotoxic
agents, such as a chemotherapeutic agent, a drug, a growth
inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically
active toxin of bacterial, fungal, plant, or animal origin, or
fragments thereof), or a radioactive isotope (i.e., a
radioconjugate).
[0288] Immunoconjugates have been used for the local delivery of
cytotoxic agents, i.e., drugs that kill or inhibit the growth or
proliferation of cells, in the treatment of cancer (Lambert, J.
(2005) Curr. Opinion in Pharmacology 5:543-549; Wu et al (2005)
Nature Biotechnology 23(9):1137-1146; Payne, G. (2003) i 3:207-212;
Syrigos and Epenetos (1999) Anticancer Research 19:605-614;
Niculescu-Duvaz and Springer (1997) Adv. Drug Deliv. Rev.
26:151-172; U.S. Pat. No. 4,975,278). Immunoconjugates allow for
the targeted delivery of a drug moiety to a tumor, and
intracellular accumulation therein, where systemic administration
of unconjugated drugs may result in unacceptable levels of toxicity
to normal cells as well as the tumor cells sought to be eliminated
(Baldwin et al., Lancet (Mar. 15, 1986) pp. 603-05; Thorpe (1985)
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review," in Monoclonal Antibodies '84: Biological And Clinical
Applications (A. Pinchera et al., eds) pp. 475-506. Both polyclonal
antibodies and monoclonal antibodies have been reported as useful
in these strategies (Rowland et al., (1986) Cancer Immunol.
Immunother. 21:183-87). Drugs used in these methods include
daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et
al., (1986) supra). Toxins used in antibody-toxin conjugates
include bacterial toxins such as diphtheria toxin, plant toxins
such as ricin, small molecule toxins such as geldanamycin (Mandler
et al (2000) J. Nat. Cancer Inst. 92(19):1573-1581; Mandler et al
(2000) Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et
al (2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP
1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA
93:8618-8623), and calicheamicin (Lode et al (1998) Cancer Res.
58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342). The toxins
may exert their cytotoxic effects by mechanisms including tubulin
binding, DNA binding, or topoisomerase inhibition. Some cytotoxic
drugs tend to be inactive or less active when conjugated to large
antibodies or protein receptor ligands.
[0289] ZEVALIN.RTM. (ibritumomab tiuxetan, Biogen/Idec) is an
antibody-radioisotope conjugate composed of a murine IgG1 kappa
monoclonal antibody directed against the CD20 antigen found on the
surface of normal and malignant B lymphocytes and 111In or 90Y
radioisotope bound by a thiourea linker-chelator (Wiseman et al
(2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al (2002)
Blood 99(12):4336-42; Witzig et al (2002) J. Clin. Oncol.
20(10):2453-63; Witzig et al (2002) J. Clin. Oncol.
20(15):3262-69). Although ZEVALIN has activity against B-cell
non-Hodgkin's Lymphoma (NHL), administration results in severe and
prolonged cytopenias in most patients. MYLOTARG.TM. (gemtuzumab
ozogamicin, Wyeth Pharmaceuticals), an antibody-drug conjugate
composed of a huCD33 antibody linked to calicheamicin, was approved
in 2000 for the treatment of acute myeloid leukemia by injection
(Drugs of the Future (2000) 25(7):686; U.S. Pat. Nos. 4,970,198;
5,079,233; 5,585,089; 5,606,040; 5,693,762; 5,739,116; 5,767,285;
5,773,001). Cantuzumab mertansine (Immunogen, Inc.), an
antibody-drug conjugate composed of the huC242 antibody linked via
the disulfide linker SPP to the maytansinoid drug moiety, DM1, is
advancing into Phase II trials for the treatment of cancers that
express CanAg, such as colon, pancreatic, gastric, and other
cancers. MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen
Inc.), an antibody-drug conjugate composed of the anti-prostate
specific membrane antigen (PSMA) monoclonal antibody linked to the
maytansinoid drug moiety, DM1, is under development for the
potential treatment of prostate tumors. The auristatin peptides,
auristatin E (AE) and monomethylauristatin (MMAE), synthetic
analogs of dolastatin, were conjugated to chimeric monoclonal
antibodies cBR96 (specific to Lewis Y on carcinomas) and cAC10
(specific to CD30 on hematological malignancies) (Doronina et al
(2003) Nature Biotechnol. 21(7):778-784) and are under therapeutic
development.
[0290] In certain embodiments, an immunoconjugate comprises an
antibody and a chemotherapeutic agent or other toxin.
Chemotherapeutic agents useful in the generation of
immunoconjugates are described herein (e.g., above). Enzymatically
active toxins and fragments thereof that can be used include
diphtheria A chain, nonbinding active fragments of diphtheria
toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A
chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites
fordii proteins, dianthin proteins, Phytolaca americana proteins
(PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin,
crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin,
restrictocin, phenomycin, enomycin, and the tricothecenes. See,
e.g., WO 93/21232 published Oct. 28, 1993. A variety of
radionuclides are available for the production of radioconjugated
antibodies. Examples include .sup.212Bi, .sup.131I, .sup.131In,
.sup.90Y, and .sup.186Re. Conjugates of the antibody and cytotoxic
agent are made using a variety of bifunctional protein-coupling
agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate
(SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters
(such as dimethyl adipimidate HCl), active esters (such as
disuccinimidyl suberate), aldehydes (such as glutaraldehyde),
bis-azido compounds (such as bis (p-azidobenzoyl)hexanediamine),
bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
toluene 2,6-diisocyanate), and bis-active fluorine compounds (such
as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin
immunotoxin can be prepared as described in Vitetta et al.,
Science, 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026.
[0291] Conjugates of an antibody and one or more small molecule
toxins, such as a calicheamicin, maytansinoids, dolastatins,
aurostatins, a trichothecene, and CC1065, and the derivatives of
these toxins that have toxin activity, are also contemplated
herein.
[0292] Maytansine and Maytansinoids
[0293] In some embodiments, the immunoconjugate comprises an
antibody (full length or fragments) conjugated to one or more
maytansinoid molecules.
[0294] Maytansinoids are mitototic inhibitors which act by
inhibiting tubulin polymerization. Maytansine was first isolated
from the east African shrub Maytenus serrata (U.S. Pat. No.
3,896,111). Subsequently, it was discovered that certain microbes
also produce maytansinoids, such as maytansinol and C-3 maytansinol
esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and
derivatives and analogues thereof are disclosed, for example, in
U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608;
4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428;
4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650;
4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533.
[0295] Maytansinoid drug moieties are attractive drug moieties in
antibody drug conjugates because they are: (i) relatively
accessible to prepare by fermentation or chemical modification,
derivatization of fermentation products, (ii) amenable to
derivatization with functional groups suitable for conjugation
through the non-disulfide linkers to antibodies, (iii) stable in
plasma, and (iv) effective against a variety of tumor cell
lines.
[0296] Immunoconjugates containing maytansinoids, methods of making
same, and their therapeutic use are disclosed, for example, in U.S.
Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1.
Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996)
described immunoconjugates comprising a maytansinoid designated DM1
linked to the monoclonal antibody C242 directed against human
colorectal cancer. The conjugate was found to be highly cytotoxic
towards cultured colon cancer cells, and showed antitumor activity
in an in vivo tumor growth assay. Chari et al., Cancer Research
52:127-131 (1992) describe immunoconjugates in which a maytansinoid
was conjugated via a disulfide linker to the murine antibody A7
binding to an antigen on human colon cancer cell lines, or to
another murine monoclonal antibody TA.1 that binds the HER-2/neu
oncogene. The cytotoxicity of the TA.1-maytansinoid conjugate was
tested in vitro on the human breast cancer cell line SK-BR-3, which
expresses 3.times.10.sub.5 HER-2 surface antigens per cell. The
drug conjugate achieved a degree of cytotoxicity similar to the
free maytansinoid drug, which could be increased by increasing the
number of maytansinoid molecules per antibody molecule. The
A7-maytansinoid conjugate showed low systemic cytotoxicity in
mice.
[0297] Antibody-maytansinoid conjugates are prepared by chemically
linking an antibody to a maytansinoid molecule without
significantly diminishing the biological activity of either the
antibody or the maytansinoid molecule. See, e.g., U.S. Pat. No.
5,208,020. An average of 3-4 maytansinoid molecules conjugated per
antibody molecule has shown efficacy in enhancing cytotoxicity of
target cells without negatively affecting the function or
solubility of the antibody, although even one molecule of
toxin/antibody would be expected to enhance cytotoxicity over the
use of naked antibody. Maytansinoids are well known in the art and
can be synthesized by known techniques or isolated from natural
sources. Suitable maytansinoids are disclosed, for example, in U.S.
Pat. No. 5,208,020 and in the other patents and nonpatent
publications referred to hereinabove. Preferred maytansinoids are
maytansinol and maytansinol analogues modified in the aromatic ring
or at other positions of the maytansinol molecule, such as various
maytansinol esters.
[0298] There are many linking groups known in the art for making
antibody-maytansinoid conjugates, including, for example, those
disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1,
Chari et al., Cancer Research 52:127-131 (1992), and U.S. patent
application Ser. No. 10/960,602, filed Oct. 8, 2004.
Antibody-maytansinoid conjugates comprising the linker component
SMCC may be prepared as disclosed in U.S. patent application Ser.
No. 10/960,602, filed Oct. 8, 2004. The linking groups include
disulfide groups, thioether groups, acid labile groups, photolabile
groups, peptidase labile groups, or esterase labile groups, as
disclosed in the above-identified patents, disulfide and thioether
groups being preferred. Additional linking groups are described and
exemplified herein.
[0299] Conjugates of the antibody and maytansinoid may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
Particularly preferred coupling agents include
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et
al., Biochem. J. 173:723-737 (1978)) and
N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a
disulfide linkage.
[0300] The linker may be attached to the maytansinoid molecule at
various positions, depending on the type of the link. For example,
an ester linkage may be formed by reaction with a hydroxyl group
using conventional coupling techniques. The reaction may occur at
the C-3 position having a hydroxyl group, the C-14 position
modified with hydroxymethyl, the C-15 position modified with a
hydroxyl group, and the C-20 position having a hydroxyl group. In a
preferred embodiment, the linkage is formed at the C-3 position of
maytansinol or a maytansinol analogue.
[0301] Auristatins and Dolastatins
[0302] In some embodiments, the immunoconjugate comprises an
antibody conjugated to dolastatins or dolostatin peptidic analogs
and derivatives, the auristatins (U.S. Pat. Nos. 5,635,483;
5,780,588). Dolastatins and auristatins have been shown to
interfere with microtubule dynamics, GTP hydrolysis, and nuclear
and cellular division (Woyke et al (2001) Antimicrob. Agents and
Chemother. 45(12):3580-3584) and have anticancer (U.S. Pat. No.
5,663,149) and antifungal activity (Pettit et al (1998) Antimicrob.
Agents Chemother. 42:2961-2965). The dolastatin or auristatin drug
moiety may be attached to the antibody through the N (amino)
terminus or the C (carboxyl) terminus of the peptidic drug moiety
(WO 02/088172).
[0303] Exemplary auristatin embodiments include the N-terminus
linked monomethylauristatin drug moieties DE and DF, disclosed in
"Monomethylvaline Compounds Capable of Conjugation to Ligands",
U.S. Ser. No. 10/983,340, filed Nov. 5, 2004.
[0304] Typically, peptide-based drug moieties can be prepared by
forming a peptide bond between two or more amino acids and/or
peptide fragments. Such peptide bonds can be prepared, for example,
according to the liquid phase synthesis method (see E. Schroder and
K. Lubke, "The Peptides", volume 1, pp 76-136, 1965, Academic
Press) that is well known in the field of peptide chemistry. The
auristatin/dolastatin drug moieties may be prepared according to
the methods of: U.S. Pat. No. 5,635,483; U.S. Pat. No. 5,780,588;
Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al
(1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R., et al.
Synthesis, 1996, 719-725; and Pettit et al (1996) J. Chem. Soc.
Perkin Trans. 1 5:859-863. See also Doronina (2003) Nat Biotechnol
21(7):778-784; "Monomethylvaline Compounds Capable of Conjugation
to Ligands", U.S. Ser. No. 10/983,340, filed Nov. 5, 2004
(disclosing, e.g., linkers and methods of preparing
monomethylvaline compounds such as MMAE and MMAF conjugated to
linkers).
[0305] Calicheamicin
[0306] In other embodiments, the immunoconjugate comprises an
antibody conjugated to one or more calicheamicin molecules. The
calicheamicin family of antibiotics are capable of producing
double-stranded DNA breaks at sub-picomolar concentrations. For the
preparation of conjugates of the calicheamicin family, see U.S.
Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701,
5,770,710, 5,773,001, 5,877,296 (all to American Cyanamid Company).
Structural analogues of calicheamicin which may be used include,
but are not limited to, .gamma.1I, .alpha.2I, .alpha.3I,
N-acetyl-.gamma.1I, PSAG and .theta.I1 (Hinman et al., Cancer
Research 53:3336-3342 (1993), Lode et al., Cancer Research
58:2925-2928 (1998) and the aforementioned U.S. patents to American
Cyanamid). Another anti-tumor drug that the antibody can be
conjugated is QFA which is an antifolate. Both calicheamicin and
QFA have intracellular sites of action and do not readily cross the
plasma membrane. Therefore, cellular uptake of these agents through
antibody mediated internalization greatly enhances their cytotoxic
effects.
[0307] Other Cytotoxic Agents
[0308] Other antitumor agents that can be conjugated to the
antibodies include BCNU, streptozoicin, vincristine and
5-fluorouracil, the family of agents known collectively LL-E33288
complex described in U.S. Pat. Nos. 5,053,394, 5,770,710, as well
as esperamicins (U.S. Pat. No. 5,877,296).
[0309] Enzymatically active toxins and fragments thereof which can
be used include diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and to PAP-S), momordica charantia
inhibitor, curcin, crotin, sapaonaria officinalis inhibitor,
gelonin, mitogellin, restrictocin, phenomycin, enomycin and the
tricothecenes. See, for example, WO 93/21232 published Oct. 28,
1993.
[0310] The present invention further contemplates an
immunoconjugate formed between an antibody and a compound with
nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease
such as a deoxyribonuclease; DNase).
[0311] For selective destruction of the tumor, the antibody may
comprise a highly radioactive atom. A variety of radioactive
isotopes are available for the production of radioconjugated
antibodies. Examples include At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32,
Pb.sup.212 and radioactive isotopes of Lu. When the conjugate is
used for detection, it may comprise a radioactive atom for
scintigraphic studies, for example tc99m or I123, or a spin label
for nuclear magnetic resonance (NMR) imaging (also known as
magnetic resonance imaging, mri), such as iodine-123 again,
iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15,
oxygen-17, gadolinium, manganese or iron.
[0312] The radio- or other labels may be incorporated in the
conjugate in known ways. For example, the peptide may be
biosynthesized or may be synthesized by chemical amino acid
synthesis using suitable amino acid precursors involving, for
example, fluorine-19 in place of hydrogen. Labels such as
tc.sup.99m or I.sup.123, Re.sup.186, Re.sup.188 and In.sup.111 can
be attached via a cysteine residue in the peptide. Yttrium-90 can
be attached via a lysine residue. The IODOGEN method (Fraker et al
(1978) Biochem. Biophys. Res. Commun. 80: 49-57) can be used to
incorporate iodine-123. "Monoclonal Antibodies in
Immunoscintigraphy" (Chatal, CRC Press 1989) describes other
methods in detail.
[0313] Conjugates of the antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as his (p-azidobenzoyl)hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026. The linker may be
a to "cleavable linker" facilitating release of the cytotoxic drug
in the cell. For example, an acid-labile linker,
peptidase-sensitive linker, photolabile linker, dimethyl linker or
disulfide-containing linker (Chari et al., Cancer Research
52:127-131 (1992); U.S. Pat. No. 5,208,020) may be used.
[0314] The compounds expressly contemplate, but are not limited to,
ADC prepared with cross-linker reagents: BMPS, EMCS, GMBS, HBVS,
LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS,
sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and
sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which
are commercially available (e.g., from Pierce Biotechnology, Inc.,
Rockford, Ill., U.S.A). See pages 467-498, 2003-2004 Applications
Handbook and Catalog.
[0315] Preparation of Antibody Drug Conjugates
[0316] In the antibody drug conjugates (ADC), an antibody (Ab) is
conjugated to one or more drug moieties (D), e.g. about 1 to about
20 drug moieties per antibody, through a linker (L). The ADC of
Formula I may be prepared by several routes, employing organic
chemistry reactions, conditions, and reagents known to those
skilled in the art, including: (1) reaction of a nucleophilic group
of an antibody with a bivalent linker reagent, to form Ab-L, via a
covalent bond, followed by reaction with a drug moiety D; and (2)
reaction of a nucleophilic group of a drug moiety with a bivalent
linker reagent, to form D-L, via a covalent bond, followed by
reaction with the nucleophilic group of an antibody. Additional
methods for preparing ADC are described herein.
Ab-(L-D).sub.p I
[0317] The linker may be composed of one or more linker components.
Exemplary linker components include 6-maleimidocaproyl ("MC"),
maleimidopropanoyl ("MP"), valine-citrulline ("val-cit"),
alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl
("PAB"), N-Succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"),
N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate
("SMCC"), and N-Succinimidyl (4-iodo-acetyl)aminobenzoate ("SIAB").
Additional linker components are known in the art and some are
described herein. See also "Monomethylvaline Compounds Capable of
Conjugation to Ligands", U.S. Ser. No. 10/983,340, filed Nov. 5,
2004.
[0318] In some embodiments, the linker may comprise amino acid
residues. Exemplary amino acid linker components include a
dipeptide, a tripeptide, a tetrapeptide or a pentapeptide.
Exemplary dipeptides include: valine-citrulline (vc or val-cit),
alanine-phenylalanine (af or ala-phe). Exemplary tripeptides
include: glycine-valine-citrulline (gly-val-cit) and
glycine-glycine-glycine (gly-gly-gly). Amino acid residues which
comprise an amino acid linker component include those occurring
naturally, as well as minor amino acids and non-naturally occurring
amino acid analogs, such as citrulline. Amino acid linker
components can be designed and optimized in their selectivity for
enzymatic cleavage by a particular enzymes, for example, a
tumor-associated protease, cathepsin B, C and D, or a plasmin
protease.
[0319] Nucleophilic groups on antibodies include, but are not
limited to: (i) N-terminal amine groups, (ii) side chain amine
groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine,
and (iv) sugar hydroxyl or amino groups where the antibody is
glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic
and capable of reacting to form covalent bonds with electrophilic
groups on linker moieties and linker reagents including: (i) active
esters such as NHS esters, HOBt esters, haloformates, and acid
halides; (ii) alkyl and benzyl halides such as haloacetamides;
(iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain
antibodies have reducible interchain disulfides, i.e. cysteine
bridges. Antibodies may be made reactive for conjugation with
linker reagents by treatment with a reducing agent such as DTT
(dithiothreitol). Each cysteine bridge will thus form,
theoretically, two reactive thiol nucleophiles. Additional
nucleophilic groups can be introduced into antibodies through the
reaction of lysines with 2-iminothiolane (Traut's reagent)
resulting in conversion of an amine into a thiol. Reactive thiol
groups may be introduced into the antibody (or fragment thereof) by
introducing one, two, three, four, or more cysteine residues (e.g.,
preparing mutant antibodies comprising one or more non-native
cysteine amino acid residues).
[0320] Antibody drug conjugates may also be produced by
modification of the antibody to introduce electrophilic moieties,
which can react with nucleophilic substituents on the linker
reagent or drug. The sugars of glycosylated antibodies may be
oxidized, e.g. with periodate oxidizing reagents, to form aldehyde
or ketone groups which may react with the amine group of linker
reagents or drug moieties. The resulting imine Schiff base groups
may form a stable linkage, or may be reduced, e.g. by borohydride
reagents to form stable amine linkages. In one embodiment, reaction
of the carbohydrate portion of a glycosylated antibody with either
glactose oxidase or sodium meta-periodate may yield carbonyl
(aldehyde and ketone) groups in the protein that can react with
appropriate groups on the drug (Hermanson, Bioconjugate
Techniques). In another embodiment, proteins containing N-terminal
serine or threonine residues can react with sodium meta-periodate,
resulting in production of an aldehyde in place of the first amino
acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146;
U.S. Pat. No. 5,362,852). Such aldehyde can be reacted with a drug
moiety or linker nucleophile.
[0321] Likewise, nucleophilic groups on a drug moiety include, but
are not limited to: amine, thiol, hydroxyl, hydrazide, oxime,
hydrazine, thiosemicarbazone, hydrazine carboxylate, and
arylhydrazide groups capable of reacting to form covalent bonds
with electrophilic groups on linker moieties and linker reagents
including: (i) active esters such as NHS esters, HOBt esters,
haloformates, and acid halides; (ii) alkyl and benzyl halides such
as haloacetamides; (iii) aldehydes, ketones, carboxyl, and
maleimide groups.
[0322] Alternatively, a fusion protein comprising the antibody and
cytotoxic agent may be made, e.g., by recombinant techniques or
peptide synthesis. The length of DNA may comprise respective
regions encoding the two portions of the conjugate either adjacent
one another or separated by a region encoding a linker peptide
which does not destroy the desired properties of the conjugate.
[0323] In yet another embodiment, the antibody may be conjugated to
a "receptor" (such streptavidin) for utilization in tumor
pre-targeting wherein the antibody-receptor conjugate is
administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g., avidin) which is conjugated to
a cytotoxic agent (e.g., a radionucleotide).
Pharmaceutical Formulations
[0324] Therapeutic formulations comprising an antibody of the
invention are prepared for storage by mixing the antibody having
the desired degree of purity with optional physiologically
acceptable carriers, excipients or stabilizers (Remington: The
Science and Practice of Pharmacy 20th edition (2000)), in the form
of aqueous solutions, lyophilized or other dried formulations.
Acceptable carriers, excipients, or stabilizers are nontoxic to
recipients at the dosages and concentrations employed, and include
buffers such as phosphate, citrate, histidine and other organic
acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g., Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
[0325] The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect each other. Such molecules are suitably present in
combination in amounts that are effective for the purpose
intended.
[0326] The active ingredients may also be entrapped in microcapsule
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsule and poly-(methylmethacylate) microcapsule,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington: The Science and Practice of Pharmacy 20th edition
(2000).
[0327] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0328] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the
immunoglobulin of the invention, which matrices are in the form of
shaped articles, e.g., films, or microcapsule. Examples of
sustained-release matrices include polyesters, hydrogels (for
example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated immunoglobulins remain
in the body for a long time, they may denature or aggregate as a
result of exposure to moisture at 37.degree. C., resulting in a
loss of biological activity and possible changes in immunogenicity.
Rational strategies can be devised for stabilization depending on
the mechanism involved. For example, if the aggregation mechanism
is discovered to be intermolecular S--S bond formation through
thio-disulfide interchange, stabilization may be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
Uses
[0329] An antibody of the present invention may be used in, for
example, in vitro, ex vivo and in vivo therapeutic methods.
[0330] The invention provides methods and compositions useful for
modulating disease states associated with expression and/or
activity of EGFL7, such as increased expression and/or activity or
undesired expression and/or activity, said methods comprising
administration of an effective dose of an anti-EGFL7 antibody to an
individual in need of such treatment.
[0331] In one aspect, the invention provides methods for treating
or preventing a tumor, a cancer, and/or a cell proliferative
disorder, the methods comprising administering an effective amount
of an anti-EGFL7 antibody to an individual in need of such
treatment.
[0332] In one aspect, the invention provides methods for inhibiting
angiogenesis, the methods comprising administering an effective
amount of an anti-EGFL7 antibody to an individual in need of such
treatment.
[0333] In one aspect, the invention provides methods for enhancing
the efficacy of another anti-angiogenic agent, the methods
comprising administering an effective amount of an anti-EGFL7
antibody to an individual in need of such treatment. In some
embodiments, the individual has a tumor, a cancer, and/or a cell
proliferative disorder. In some embodiments, the other
anti-angiogenic agent targets VEGF, e.g. an anti-VEGF antibody.
[0334] It is understood that any suitable anti-EGFL7 antibody may
be used in methods of treatment, including monoclonal and/or
polyclonal antibodies, a human antibody, a chimeric antibody, an
affinity-matured antibody, a humanized antibody, and/or an antibody
fragment. In some embodiments, any anti-EGFL7 antibody described
herein is used for treatment.
[0335] In any of the methods herein, one may administer to the
subject or patient along with the antibody herein an effective
amount of a second medicament (where the antibody herein is a first
medicament), which is another active agent that can treat the
condition in the subject that requires treatment. For instance, an
antibody of the invention may be co-administered with another
antibody, chemotherapeutic agent(s) (including cocktails of
chemotherapeutic agents), anti-angiogenic agent(s),
immunosuppressive agents(s), cytokine(s), cytokine antagonist(s),
and/or growth-inhibitory agent(s). The type of such second
medicament depends on various factors, including the type of
disorder, the severity of the disease, the condition and age of the
patient, the type and dose of first medicament employed, etc.
[0336] Where an antibody of the invention inhibits tumor growth,
for example, it may be particularly desirable to combine it with
one or more other therapeutic agents that also inhibit tumor
growth. For instance, an antibody of the invention may be combined
with an anti-angiogenic agent, such as an anti-VEGF antibody (e.g.,
AVASTIN.RTM.) and/or anti-ErbB antibodies (e.g. HERCEPTIN.RTM.
trastuzumab anti-HER2 antibody or an anti-HER2 antibody that binds
to Domain II of HER2, such as OMNITARG.TM. pertuzumab anti-HER2
antibody) in a treatment scheme, e.g. in treating any of the
disease described herein, including colorectal cancer, lung cancer,
hepatocellular carcinoma, breast cancer and/or pancreatic cancer.
In some instances, the previous combinations may be accomplished
using a bispecific antibody. Alternatively, or additionally, the
patient may receive combined radiation therapy (e.g. external beam
irradiation or therapy with a radioactive labeled agent, such as an
antibody). Such combined therapies noted above include combined
administration (where the two or more agents are included in the
same or separate formulations), and separate administration, in
which case, administration of the antibody of the invention can
occur prior to, and/or following, administration of the adjunct
therapy or therapies. In addition, combining an antibody of this
invention with a relatively non-cytotoxic agent such as another
biologic molecule, e.g., another antibody is expected to reduce
cytotoxicity versus combining the antibody with a chemotherapeutic
agent of other agent that is highly toxic to cells.
[0337] Treatment with a combination of the antibody herein with one
or more second medicaments preferably results in an improvement in
the signs or symptoms of cancer. For instance, such therapy may
result in an improvement in survival (overall survival and/or
progression-free survival) relative to a patient treated with the
second medicament only (e.g., a chemotherapeutic agent only),
and/or may result in an objective response *(partial or complete,
preferably complete). Moreover, treatment with the combination of
an antibody herein and one or more second medicament(s) preferably
results in an additive, and more preferably synergistic (or greater
than additive), therapeutic benefit to the patient. Preferably, in
this combination method the timing between at least one
administration of the second medicament and at least one
administration of the antibody herein is about one month or less,
more preferably, about two weeks or less.
[0338] For treatment of cancers, the second medicament is
preferably another antibody, chemotherapeutic agent (including
cocktails of chemotherapeutic agents), anti-angiogenic agent,
immunosuppressive agent, prodrug, cytokine, cytokine antagonist,
cytotoxic radiotherapy, corticosteroid, anti-emetic, cancer
vaccine, analgesic, anti-vascular agent, and/or growth-inhibitory
agent. The cytotoxic agent includes an agent interacting with DNA,
the antimetabolites, the topoisomerase I or II inhibitors, or the
spindle inhibitor or stabilizer agents (e.g., preferably vinca
alkaloid, more preferably selected from vinblastine,
deoxyvinblastine, vincristine, vindesine, vinorelbine, vinepidine,
vinfosiltine, vinzolidine and vinfunine), or any agent used in
chemotherapy such as 5-FU, a taxane, doxorubicin, or
dexamethasone.
[0339] In some embodiments, the second medicament is another
antibody used to treat cancers such as those directed against the
extracellular domain of the HER2/neu receptor, e.g., trastuzumab,
or one of its functional fragments, pan-HER inhibitor, a Src
inhibitor, a MEK inhibitor, or an EGFR inhibitor (e.g., an
anti-EGFR antibody (such as one inhibiting the tyrosine kinase
activity of the EGFR), which is preferably the mouse monoclonal
antibody 225, its mouse-man chimeric derivative C225, or a
humanized antibody derived from this antibody 225 or derived
natural agents, dianilinophthalimides, pyrazolo- or
pyrrolopyridopyrimidines, quinazilines, gefitinib, erlotinib,
cetuximab, ABX-EFG, canertinib, EKB-569 and PKI-166), or
dual-EGFR/HER-2 inhibitor such as lapatanib. Additional second
medicaments include alemtuzumab (CAMPATH.TM.), FavID (IDKLH), CD20
antibodies with altered glycosylation, such as GA-101/GLYCARTT.TM.,
oblimersen (GENASENSE.TM.), thalidomide and analogs thereof, such
as lenalidomide (REVLIMIDT.TM.), imatinib, sorafenib, ofatumumab
(HUMAX-CD20.TM.), anti-CD40 antibody, e.g. SGN-40, and anti-CD-80
antibody, e.g. galiximab.
[0340] The anti-emetic agent is preferably ondansetron
hydrochloride, granisetron hydrochloride, metroclopramide,
domperidone, haloperidol, cyclizine, lorazepam, prochlorperazine,
dexamethasone, levomepromazine, or tropisetron. The vaccine is
preferably GM-CSF DNA and cell-based vaccines, dendritic cell
vaccine, recombinant viral vaccines, heat shock protein (HSP)
vaccines, allogeneic or autologous tumor vaccines. The analgesic
agent preferably is ibuprofen, naproxen, choline magnesium
trisalicylate, or oxycodone hydrochloride. The anti-vascular agent
preferably is bevacizumab, or rhuMAb-VEGF. Further second
medicaments include anti-proliferative agents such a farnesyl
protein transferase inhibitors, anti-VEGF inhibitors, p53
inhibitors, or PDGFR inhibitors. The second medicament herein
includes also biologic-targeted therapy such as treatment with
antibodies as well as small-molecule-targeted therapy, for example,
against certain receptors.
[0341] Many anti-angiogenic agents have been identified and are
known in the art, including those listed herein, e.g., listed under
Definitions, and by, e.g., Carmeliet and Jain, Nature 407:249-257
(2000); Ferrara et al., Nature Reviews: Drug Discovery, 3:391-400
(2004); and Sato Int. J. Clin. Oncol., 8:200-206 (2003). See also,
US Patent Application US20030055006. In one embodiment, an
anti-EGFL7 antibody is used in combination with an anti-VEGF
neutralizing antibody (or fragment) and/or another VEGF antagonist
or a VEGF receptor antagonist including, but not limited to, for
example, soluble VEGF receptor (e.g., VEGFR-1, VEGFR-2, VEGFR-3,
neuropilins (e.g., NRP1, NRP2)) fragments, aptamers capable of
blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, low
molecule weight inhibitors of VEGFR tyrosine kinases (RTK),
antisense strategies for VEGF, ribozymes against VEGF or VEGF
receptors, antagonist variants of VEGF; and any combinations
thereof. Alternatively, or additionally, two or more angiogenesis
inhibitors may optionally be co-administered to the patient in
addition to VEGF antagonist and other agent. In certain embodiment,
one or more additional therapeutic agents, e.g., anti-cancer
agents, can be administered in combination with anti-EGFL7
antibody, the VEGF antagonist, and an anti-angiogenesis agent.
[0342] Chemotherapeutic agents useful herein are described supra,
e.g., in the definition of "chemotherapeutic agent".
[0343] Such second medicaments may be administered within 48 hours
after the antibodies herein are administered, or within 24 hours,
or within 12 hours, or within 3-12 hours after said agent, or may
be administered over a pre-selected period of time, which is
preferably about 1 to 2 days. Further, the dose of such agent may
be sub-therapeutic.
[0344] The antibodies herein can be administered concurrently,
sequentially, or alternating with the second medicament or upon
non-responsiveness with other therapy. Thus, the combined
administration of a second medicament includes co-administration
(concurrent administration), using separate formulations or a
single pharmaceutical formulation, and consecutive administration
in either order, wherein preferably there is a time period while
both (or all) medicaments simultaneously exert their biological
activities. All these second medicaments may be used in combination
with each other or by themselves with the first medicament, so that
the express "second medicament" as used herein does not mean it is
the only medicament besides the first medicament, respectively.
Thus, the second medicament need not be one medicament, but may
constitute or comprise more than one such drug.
[0345] These second medicaments as set forth herein are generally
used in the same dosages and with administration routes as the
first medicaments, or about from 1 to 99% of the dosages of the
first medicaments. If such second medicaments are used at all,
preferably, they are used in lower amounts than if the first
medicament were not present, especially in subsequent dosings
beyond the initial dosing with the first medicament, so as to
eliminate or reduce side effects caused thereby.
[0346] The invention also provides methods and compositions for
inhibiting or preventing relapse tumor growth or relapse cancer
cell growth. Relapse tumor growth or relapse cancer cell growth is
used to describe a condition in which patients undergoing or
treated with one or more currently available therapies (e.g.,
cancer therapies, such as chemotherapy, radiation therapy, surgery,
hormonal therapy and/or biological therapy/immunotherapy, anti-VEGF
antibody therapy, particularly a standard therapeutic regimen for
the particular cancer) is not clinically adequate to treat the
patients or the patients are no longer receiving any beneficial
effect from the therapy such that these patients need additional
effective therapy. As used herein, the phrase can also refer to a
condition of the "non-responsive/refractory" patient, e.g., which
describe patients who respond to therapy yet suffer from side
effects, develop resistance, do not respond to the therapy, do not
respond satisfactorily to the therapy, etc. In various embodiments,
a cancer is relapse tumor growth or relapse cancer cell growth
where the number of cancer cells has not been significantly
reduced, or has increased, or tumor size has not been significantly
reduced, or has increased, or fails any further reduction in size
or in number of cancer cells. The determination of whether the
cancer cells are relapse tumor growth or relapse cancer cell growth
can be made either in vivo or in vitro by any method known in the
art for assaying the effectiveness of treatment on cancer cells,
using the art-accepted meanings of "relapse" or "refractory" or
"non-responsive" in such a context. A tumor resistant to anti-VEGF
treatment is an example of a relapse tumor growth.
[0347] The invention provides methods of blocking or reducing
relapse tumor growth or relapse cancer cell growth in a subject by
administering anti-EGFL7 antibody to block or reduce the relapse
tumor growth or relapse cancer cell growth in subject. In certain
embodiments, the antagonist can be administered subsequent to the
other cancer therapeutic. In certain embodiments, the anti-EGFL7
antibody is administered simultaneously with cancer therapy.
Alternatively, or additionally, the anti-EGFL7 antibody therapy
alternates with another cancer therapy, which can be performed in
any order. The invention also encompasses methods for administering
one or more inhibitory antibodies to prevent the onset or
recurrence of cancer in patients predisposed to having cancer.
Generally, the subject was or is concurrently undergoing cancer
therapy. In one embodiment, the cancer therapy is treatment with an
anti-angiogenesis agent, e.g., a VEGF antagonist. The
anti-angiogenesis agent includes those known in the art and those
found under the Definitions herein. In one embodiment, the
anti-angiogenesis agent is an anti-VEGF neutralizing antibody or
fragment (e.g., humanized A4.6.1, AVASTIN.RTM. (Genentech, South
San Francisco, Calif.), Y0317, M4, G6, B20, 2C3, etc.). See, e.g.,
U.S. Pat. Nos. 6,582,959, 6,884,879, 6,703,020; WO98/45332; WO
96/30046; WO94/10202; EP 0666868B1; US Patent Applications
20030206899, 20030190317, 20030203409, and 20050112126; Popkov et
al., Journal of Immunological Methods 288:149-164 (2004); and,
WO2005012359. Additional agents can be administered in combination
with VEGF antagonist and an anti-EGFL7 antibody for blocking or
reducing relapse tumor growth or relapse cancer cell growth.
[0348] The antibodies of the invention (and adjunct therapeutic
agent) is/are administered by any suitable means, including
parenteral, subcutaneous, intraperitoneal, intrapulmonary, and
intranasal, and, if desired for local treatment, intralesional
administration. Parenteral infusions include intramuscular,
intravenous, intraarterial, intraperitoneal, or subcutaneous
administration. In addition, the antibodies are suitably
administered by pulse infusion, particularly with declining doses
of the antibody. Dosing can be by any suitable route, e.g. by
injections, such as intravenous or subcutaneous injections,
depending in part on whether the administration is brief or
chronic.
[0349] The location of the binding target of an antibody of the
invention may be taken into consideration in preparation and
administration of the antibody. When the binding target is an
intracellular molecule, certain embodiments of the invention
provide for the antibody or antigen-binding fragment thereof to be
introduced into the cell where the binding target is located. In
one embodiment, an antibody of the invention can be expressed
intracellularly as an intrabody. The term "intrabody," as used
herein, refers to an antibody or antigen-binding portion thereof
that is expressed intracellularly and that is capable of
selectively binding to a target molecule, as described, e.g., in
Marasco, Gene Therapy 4: 11-15 (1997); Kontermann, Methods 34:
163-170 (2004); U.S. Pat. Nos. 6,004,940 and 6,329,173; U.S. Patent
Application Publication No. 2003/0104402, and PCT Publication No.
WO2003/077945. See also, for example, WO96/07321 published Mar. 14,
1996, concerning the use of gene therapy to generate intracellular
antibodies.
[0350] Intracellular expression of an intrabody may be effected by
introducing a nucleic acid encoding the desired antibody or
antigen-binding portion thereof (lacking the wild-type leader
sequence and secretory signals normally associated with the gene
encoding that antibody or antigen-binding fragment) into a target
cell. One or more nucleic acids encoding all or a portion of an
antibody of the invention can be delivered to a target cell, such
that one or more intrabodies are expressed which are capable of
binding to an intracellular target polypeptide and modulating the
activity of the target polypeptide. Any standard method of
introducing nucleic acids into a cell may be used, including, but
not limited to, microinjection, ballistic injection,
electroporation, calcium phosphate precipitation, liposomes, and
transfection with retroviral, adenoviral, adeno-associated viral
and vaccinia vectors carrying the nucleic acid of interest.
[0351] In certain embodiments, nucleic acid (optionally contained
in a vector) may be introduced into a patient's cells by in vivo
and ex vivo methods. In one example of in vivo delivery, nucleic
acid is injected directly into the patient, e.g., at the site where
therapeutic intervention is required. In a further example of in
vivo delivery, nucleic acid is introduced into a cell using
transfection with viral vectors (such as adenovirus, Herpes simplex
I virus, or adeno-associated virus) and lipid-based systems (useful
lipids for lipid-mediated transfer of the gene are DOTMA, DOPE and
DC-Chol, for example). For review of certain gene marking and gene
therapy protocols, see Anderson et al., Science 256:808-813 (1992),
and WO 93/25673 and the references cited therein. In an example of
ex vivo treatment, a patient's cells are removed, nucleic acid is
introduced into those isolated cells, and the modified cells are
administered to the patient either directly or, for example,
encapsulated within porous membranes which are implanted into the
patient (see, e.g., U.S. Pat. Nos. 4,892,538 and 5,283,187). A
commonly used vector for ex vivo delivery of a nucleic acid is a
retroviral vector.
[0352] In another embodiment, internalizing antibodies are
provided. Antibodies can possess certain characteristics that
enhance delivery of antibodies into cells, or can be modified to
possess such characteristics. Techniques for achieving this are
known in the art. For example, cationization of an antibody is
known to facilitate its uptake into cells (see, e.g., U.S. Pat. No.
6,703,019). Lipofections or liposomes can also be used to deliver
the antibody into cells. Where antibody fragments are used, the
smallest inhibitory fragment that specifically binds to the target
protein may be advantageous. For example, based upon the
variable-region sequences of an antibody, peptide molecules can be
designed that retain the ability to bind the target protein
sequence. Such peptides can be synthesized chemically and/or
produced by recombinant DNA technology. See, e.g., Marasco et al.,
Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993).
[0353] Entry of antibodies into target cells can be enhanced by
other methods known in the art. For example, certain sequences,
such as those derived from HIV Tat or the Antennapedia homeodomain
protein are able to direct efficient uptake of heterologous
proteins across cell membranes. See, e.g., Chen et al., Proc. Natl.
Acad. Sci. USA (1999), 96:4325-4329.
[0354] When the binding target of an antibody is located in the
brain, certain embodiments of the invention provide for the
antibody to traverse the blood-brain barrier. Several art-known
approaches exist for transporting molecules across the blood-brain
barrier, including, but not limited to, physical methods,
lipid-based methods, stem cell-based methods, and receptor and
channel-based methods.
[0355] Physical methods of transporting an antibody across the
blood-brain barrier include, but are not limited to, circumventing
the blood-brain barrier entirely, or by creating openings in the
blood-brain barrier. Circumvention methods include, but are not
limited to, direct injection into the brain (see, e.g.,
Papanastassiou et al., Gene Therapy 9: 398-406 (2002)),
interstitial infusion/convection-enhanced delivery (see, e.g., Bobo
et al., Proc. Natl. Acad. Sci. USA 91: 2076-2080 (1994)), and
implanting a delivery device in the brain (see, e.g., Gill et al.,
Nature Med. 9: 589-595 (2003); and Gliadel Wafers.TM., Guildford
Pharmaceutical). Methods of creating openings in the barrier
include, but are not limited to, ultrasound (see, e.g., U.S. Patent
Publication No. 2002/0038086), osmotic pressure (e.g., by
administration of hypertonic mannitol (Neuwelt, E. A., Implication
of the Blood-Brain Barrier and its Manipulation, Vols 1 & 2,
Plenum Press, N.Y. (1989)), permeabilization by, e.g., bradykinin
or permeabilizer A-7 (see, e.g., U.S. Pat. Nos. 5,112,596,
5,268,164, 5,506,206, and 5,686,416), and transfection of neurons
that straddle the blood-brain barrier with vectors containing genes
encoding the antibody (see, e.g., U.S. Patent Publication No.
2003/0083299).
[0356] Lipid-based methods of transporting an antibody across the
blood-brain barrier include, but are not limited to, encapsulating
the antibody in liposomes that are coupled to antibody binding
fragments that bind to receptors on the vascular endothelium of the
blood-brain barrier (see, e.g., U.S. Patent Application Publication
No. 20020025313), and coating the antibody in low-density
lipoprotein particles (see, e.g., U.S. Patent Application
Publication No. 20040204354) or apolipoprotein E (see, e.g., U.S.
Patent Application Publication No. 20040131692).
[0357] Stem-cell based methods of transporting an antibody across
the blood-brain barrier entail genetically engineering neural
progenitor cells (NPCs) to express the antibody of interest and
then implanting the stem cells into the brain of the individual to
be treated. See Behrstock et al. (2005) Gene Ther. 15 Dec. 2005
advanced online publication (reporting that NPCs genetically
engineered to express the neurotrophic factor GDNF reduced symptoms
of Parkinson disease when implanted into the brains of rodent and
primate models).
[0358] Receptor and channel-based methods of transporting an
antibody across the blood-brain barrier include, but are not
limited to, using glucocorticoid blockers to increase permeability
of the blood-brain barrier (see, e.g., U.S. Patent Application
Publication Nos. 2002/0065259, 2003/0162695, and 2005/0124533);
activating potassium channels (see, e.g., U.S. Patent Application
Publication No. 2005/0089473), inhibiting ABC drug transporters
(see, e.g., U.S. Patent Application Publication No. 2003/0073713);
coating antibodies with a transferrin and modulating activity of
the one or more transferrin receptors (see, e.g., U.S. Patent
Application Publication No. 2003/0129186), and cationizing the
antibodies (see, e.g., U.S. Pat. No. 5,004,697).
[0359] Antibodies of the invention would be formulated, dosed, and
administered in a fashion consistent with good medical practice.
Factors for consideration in this context include the particular
disorder being treated, the particular mammal being treated, the
clinical condition of the individual patient, the cause of the
disorder, the site of delivery of the agent, the method of
administration, the scheduling of administration, and other factors
known to medical practitioners. The antibody need not be, but is
optionally formulated with one or more agents currently used to
prevent or treat the disorder in question. The effective amount of
such other agents depends on the amount of antibody present in the
formulation, the type of disorder or treatment, and other factors
discussed above. These are generally used in the same dosages and
with administration routes as described herein, or about from 1 to
99% of the dosages described herein, or in any dosage and by any
route that is empirically/clinically determined to be
appropriate.
[0360] For the prevention or treatment of disease, the appropriate
dosage of an antibody of the invention (when used alone or in
combination with one or more other additional therapeutic agents)
will depend on the type of disease to be treated, the type of
antibody, the severity and course of the disease, whether the
antibody is administered for preventive or therapeutic purposes,
previous therapy, the patient's clinical history and response to
the antibody, and the discretion of the attending physician. The
antibody is suitably administered to the patient at one time or
over a series of treatments. Depending on the type and severity of
the disease, about 1 .mu.g/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg)
of antibody can be an initial candidate dosage for administration
to the patient, whether, for example, by one or more separate
administrations, or by continuous infusion. One typical daily
dosage might range from about 1 .mu.g/kg to 100 mg/kg or more,
depending on the factors mentioned above. For repeated
administrations over several days or longer, depending on the
condition, the treatment would generally be sustained until a
desired suppression of disease symptoms occurs. One exemplary
dosage of the antibody would be in the range from about 0.05 mg/kg
to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0
mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be
administered to the patient. Such doses may be administered
intermittently, e.g. every week or every three weeks (e.g. such
that the patient receives from about two to about twenty, or e.g.
about six doses of the antibody). An initial higher loading dose,
followed by one or more lower doses may be administered. An
exemplary dosing regimen comprises administering an initial loading
dose of about 4 mg/kg, followed by a weekly maintenance dose of
about 2 mg/kg of the antibody. However, other dosage regimens may
be useful. The progress of this therapy is easily monitored by
conventional techniques and assays.
[0361] Diagnostic Methods and Methods of Detection
[0362] The anti-EGFL7 antibodies of the invention are useful in
assays detecting EGFL7 expression (such as diagnostic or prognostic
assays) in specific cells or tissues wherein the antibodies are
labeled as described below and/or are immobilized on an insoluble
matrix.
[0363] In another aspect, the invention provides methods for
detection of EGFL7, the methods comprising detecting
EGFL7-anti-EGFL7 antibody complex in the sample. The term
"detection" as used herein includes qualitative and/or quantitative
detection (measuring levels) with or without reference to a
control.
[0364] In another aspect, the invention provides any of the
anti-EGFL7 antibodies described herein, wherein the anti-EGFL7
antibody comprises a detectable label.
[0365] In another aspect, the invention provides a complex of any
of the anti-EGFL7 antibodies described herein and EGFL7. In some
embodiments, the complex is in vivo or in vitro. In some
embodiments, the complex comprises a cancer cell. In some
embodiments, the anti-EGFL7 antibody is detectably labeled.
[0366] Anti-EGFL7 antibodies (e.g., any of the EGFL7 antibodies
described herein) can be used for the detection of EGFL7 in any one
of a number of well known detection assay methods.
[0367] For example, a biological sample may be assayed for EGFL7 by
obtaining the sample from a desired source, admixing the sample
with anti-EGFL7 antibody to allow the antibody to form
antibody/EGFL7 complex with any EGFL7 present in the mixture, and
detecting any antibody/EGFL7 complex present in the mixture. The
biological sample may be prepared for assay by methods known in the
art which are suitable for the particular sample. The methods of
admixing the sample with antibodies and the methods of detecting
antibody/EGFL7 complex are chosen according to the type of assay
used. Such assays include immunohistochemistry, competitive and
sandwich assays, and steric inhibition assays. For sample
preparation, a tissue or cell sample from a mammal (typically a
human patient) may be used. Examples of samples include, but are
not limited to, cancer cells such as colon, breast, prostate,
ovary, lung, stomach, pancreas, lymphoma, and leukemia cancer
cells. EGFL7 may also be measured in serum. The sample can be
obtained by a variety of procedures known in the art including, but
not limited to surgical excision, aspiration or biopsy. The tissue
may be fresh or frozen. In one embodiment, the sample is fixed and
embedded in paraffin or the like. The tissue sample may be fixed
(i.e. preserved) by conventional methodology (See e.g., "Manual of
Histological Staining Method of the Armed Forces Institute of
Pathology," 3r.sup.d edition (1960) Lee G. Luna, H T (ASCP) Editor,
The Blakston Division McGraw-Hill Book Company, New York; The Armed
Forces Institute of Pathology Advanced Laboratory Methods in
Histology and Pathology (1994) Ulreka V. Mikel, Editor, Armed
Forces Institute of Pathology, American Registry of Pathology,
Washington, D.C.). One of ordinary skill in the art will appreciate
that the choice of a fixative is determined by the purpose for
which the sample is to be histologically stained or otherwise
analyzed. One of ordinary skill in the art will also appreciate
that the length of fixation depends upon the size of the tissue
sample and the fixative used. By way of example, neutral buffered
formalin, Bouin's or paraformaldehyde, may be used to fix a sample.
Generally, the sample is first fixed and is then dehydrated through
an ascending series of alcohols, infiltrated and embedded with
paraffin or other sectioning media so that the tissue sample may be
sectioned. Alternatively, one may section the tissue and fix the
sections obtained. By way of example, the tissue sample may be
embedded and processed in paraffin by conventional methodology (See
e.g., "Manual of Histological Staining Method of the Armed Forces
Institute of Pathology", supra). Examples of paraffin that may be
used include, but are not limited to, Paraplast, Broloid, and
Tissuemay. Once the tissue sample is embedded, the sample may be
sectioned by a microtome or the like (See e.g., "Manual of
Histological Staining Method of the Armed Forces Institute of
Pathology", supra). By way of example for this procedure, sections
may range from about three microns to about five microns in
thickness. Once sectioned, the sections may be attached to slides
by several standard methods. Examples of slide adhesives include,
but are not limited to, silane, gelatin, poly-L-lysine and the
like. By way of example, the paraffin embedded sections may be
attached to positively charged slides and/or slides coated with
poly-L-lysine. If paraffin has been used as the embedding material,
the tissue sections are generally deparaffinized and rehydrated to
water. The tissue sections may be deparaffinized by several
conventional standard methodologies. For example, xylenes and a
gradually descending series of alcohols may be used (See e.g.,
"Manual of Histological Staining Method of the Armed Forces
Institute of Pathology", supra). Alternatively, commercially
available deparaffinizing non-organic agents such as Hemo-De7 (CMS,
Houston, Tex.) may be used.
[0368] Analytical methods for EGFL7 all use one or more of the
following reagents: labeled EGFL7 analogue, immobilized EGFL7
analogue, labeled anti-EGFL7 antibody, immobilized anti-EGFL7
antibody and steric conjugates. The labeled reagents also are known
as "tracers."
[0369] The label used is any detectable functionality that does not
interfere with the binding of EGFL7 and anti-EGFL7 antibody.
Numerous labels are known for use in immunoassay, examples
including moieties that may be detected directly, such as
fluorochrome, chemiluminescent, and radioactive labels, as well as
moieties, such as enzymes, that must be reacted or derivatized to
be detected.
[0370] The label used is any detectable functionality that does not
interfere with the binding of EGFL7 and anti-EGFL7 antibody.
Numerous labels are known for use in immunoassay, examples
including moieties that may be detected directly, such as
fluorochrome, chemiluminescent, and radioactive labels, as well as
moieties, such as enzymes, that must be reacted or derivatized to
be detected. Examples of such labels include the radioisotopes
.sup.32P, .sup.14C, .sup.125I, .sup.3H, and .sup.131I, fluorophores
such as rare earth chelates or fluorescein and its derivatives,
rhodamine and its derivatives, dansyl, umbelliferone,
luceriferases, e.g., firefly luciferase and bacterial luciferase
(U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,
horseradish peroxidase (HRP), alkaline phosphatase,
.beta.-galactosidase, glucoamylase, lysozyme, saccharide oxidases,
e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate
dehydrogenase, heterocyclic oxidases such as uricase and xanthine
oxidase, coupled with an enzyme that employs hydrogen peroxide to
oxidize a dye precursor such as HRP, lactoperoxidase, or
microperoxidase, biotin/avidin, spin labels, bacteriophage labels,
stable free radicals, and the like.
[0371] Conventional methods are available to bind these labels
covalently to proteins or to polypeptides. For instance, coupling
agents such as dialdehydes, carbodiimides, dimaleimides,
bis-imidates, bis-diazotized benzidine, and the like may be used to
tag the antibodies with the above-described fluorescent,
chemiluminescent, and enzyme labels. See, for example, U.S. Pat.
Nos. 3,940,475 (fluorimetry) and 3,645,090 (enzymes); Hunter et
al., Nature, 144: 945 (1962); David et al., Biochemistry, 13:
1014-1021 (1974); Pain et al., J. Immunol. Methods, 40: 219-230
(1981); and Nygren, J. Histochem. and Cytochem., 30: 407-412
(1982). Preferred labels herein are enzymes such as horseradish
peroxidase and alkaline phosphatase. The conjugation of such label,
including the enzymes, to the antibody is a standard manipulative
procedure for one of ordinary skill in immunoassay techniques. See,
for example, O'Sullivan et al., "Methods for the Preparation of
Enzyme-antibody Conjugates for Use in Enzyme Immunoassay," in
Methods in Enzymology, ed. J. J. Langone and H. Van Vunakis, Vol.
73 (Academic Press, New York, N.Y., 1981), pp. 147-166.
[0372] Immobilization of reagents is required for certain assay
methods. Immobilization entails separating the anti-EGFL7 antibody
from any EGFL7 that remains free in solution. This conventionally
is accomplished by either insolubilizing the anti-EGFL7 antibody or
EGFL7 analogue before the assay procedure, as by adsorption to a
water-insoluble matrix or surface (Bennich et al., U.S. Pat. No.
3,720,760), by covalent coupling (for example, using glutaraldehyde
cross-linking), or by insolubilizing the anti-EGFL7 antibody or
EGFL7 analogue afterward, e.g., by immunoprecipitation.
[0373] The expression of proteins in a sample may be examined using
immunohistochemistry and staining protocols. Immunohistochemical
staining of tissue sections has been shown to be a reliable method
of assessing or detecting presence of proteins in a sample.
Immunohistochemistry ("IHC") techniques utilize an antibody to
probe and visualize cellular antigens in situ, generally by
chromogenic or fluorescent methods. For sample preparation, a
tissue or cell sample from a mammal (typically a human patient) may
be used. The sample can be obtained by a variety of procedures
known in the art including, but not limited to surgical excision,
aspiration or biopsy. The tissue may be fresh or frozen. In one
embodiment, the sample is fixed and embedded in paraffin or the
like. The tissue sample may be fixed (i.e. preserved) by
conventional methodology. One of ordinary skill in the art will
appreciate that the choice of a fixative is determined by the
purpose for which the sample is to be histologically stained or
otherwise analyzed. One of ordinary skill in the art will also
appreciate that the length of fixation depends upon the size of the
tissue sample and the fixative used.
[0374] IHC may be performed in combination with additional
techniques such as morphological staining and/or fluorescence
in-situ hybridization. Two general methods of IHC are available;
direct and indirect assays. According to the first assay, binding
of antibody to the target antigen (e.g., EGFL7) is determined
directly. This direct assay uses a labeled reagent, such as a
fluorescent tag or an enzyme-labeled primary antibody, which can be
visualized without further antibody interaction. In a typical
indirect assay, unconjugated primary antibody binds to the antigen
and then a labeled secondary antibody binds to the primary
antibody. Where the secondary antibody is conjugated to an
enzymatic label, a chromogenic or fluorogenic substrate is added to
provide visualization of the antigen. Signal amplification occurs
because several secondary antibodies may react with different
epitopes on the primary antibody.
[0375] The primary and/or secondary antibody used for
immunohistochemistry typically will be labeled with a detectable
moiety. Numerous labels are available.
[0376] Aside from the sample preparation procedures discussed
above, further treatment of the tissue section prior to, during or
following IHC may be desired. For example, epitope retrieval
methods, such as heating the tissue sample in citrate buffer may be
carried out (see, e.g., Leong et al. Appl. Immunohistochem.
4(3):201 (1996)).
[0377] Following an optional blocking step, the tissue section is
exposed to primary antibody for a sufficient period of time and
under suitable conditions such that the primary antibody binds to
the target protein antigen in the tissue sample. Appropriate
conditions for achieving this can be determined by routine
experimentation. The extent of binding of antibody to the sample is
determined by using any one of the detectable labels discussed
above. Preferably, the label is an enzymatic label (e.g. HRPO)
which catalyzes a chemical alteration of the chromogenic substrate
such as 3,3'-diaminobenzidine chromogen. Preferably the enzymatic
label is conjugated to antibody which binds specifically to the
primary antibody (e.g. the primary antibody is rabbit polyclonal
antibody and secondary antibody is goat anti-rabbit antibody).
[0378] Specimens thus prepared may be mounted and coverslipped.
Slide evaluation is then determined, e.g. using a microscope, and
staining intensity criteria, routinely used in the art, may be
employed.
[0379] Other assay methods, known as competitive or sandwich
assays, are well established and widely used in the commercial
diagnostics industry.
[0380] Competitive assays rely on the ability of a tracer EGFL7
analogue to compete with the test sample EGFL7 for a limited number
of anti-EGFL7 antibody antigen-binding sites. The anti-EGFL7
antibody generally is insolubilized before or after the competition
and then the tracer and EGFL7 bound to the anti-EGFL7 antibody are
separated from the unbound tracer and EGFL7. This separation is
accomplished by decanting (where the binding partner was
preinsolubilized) or by centrifuging (where the binding partner was
precipitated after the competitive reaction). The amount of test
sample EGFL7 is inversely proportional to the amount of bound
tracer as measured by the amount of marker substance. Dose-response
curves with known amounts of EGFL7 are prepared and compared with
the test results to quantitatively determine the amount of EGFL7
present in the test sample. These assays are called ELISA systems
when enzymes are used as the detectable markers.
[0381] Another species of competitive assay, called a "homogeneous"
assay, does not require a phase separation. Here, a conjugate of an
enzyme with the EGFL7 is prepared and used such that when
anti-EGFL7 antibody binds to the EGFL7 the presence of the
anti-EGFL7 antibody modifies the enzyme activity. In this case, the
EGFL7 or its immunologically active fragments are conjugated with a
bifunctional organic bridge to an enzyme such as peroxidase.
Conjugates are selected for use with anti-EGFL7 antibody so that
binding of the anti-EGFL7 antibody inhibits or potentiates the
enzyme activity of the label. This method per se is widely
practiced under the name of EMIT.
[0382] Steric conjugates are used in steric hindrance methods for
homogeneous assay. These conjugates are synthesized by covalently
linking a low-molecular-weight hapten to a small EGFL7 fragment so
that antibody to hapten is substantially unable to bind the
conjugate at the same time as anti-EGFL7 antibody. Under this assay
procedure the EGFL7 present in the test sample will bind anti-EGFL7
antibody, thereby allowing anti-hapten to bind the conjugate,
resulting in a change in the character of the conjugate hapten,
e.g., a change in fluorescence when the hapten is a
fluorophore.
[0383] Sandwich assays particularly are useful for the
determination of EGFL7 or anti-EGFL7 antibodies. In sequential
sandwich assays an immobilized anti-EGFL7 antibody is used to
adsorb test sample EGFL7, the test sample is removed as by washing,
the bound EGFL7 is used to adsorb a second, labeled anti-EGFL7
antibody and bound material is then separated from residual tracer.
The amount of bound tracer is directly proportional to test sample
EGFL7. In "simultaneous" sandwich assays the test sample is not
separated before adding the labeled anti-EGFL7. A sequential
sandwich assay using an anti-EGFL7 monoclonal antibody as one
antibody and a polyclonal anti-EGFL7 antibody as the other is
useful in testing samples for EGFL7.
[0384] The foregoing are merely exemplary detection assays for
EGFL7. Other methods now or hereafter developed that use anti-EGFL7
antibody for the determination of EGFL7 are included within the
scope hereof, including the bioassays described herein.
[0385] In one aspect, the invention provides methods to detect
(e.g., presence or absence of or amount) a polynucleotide(s) (e.g.,
EGFL7 polynucleotides) in a biological sample from an individual,
such as a human subject. A variety of methods for detecting
polynucleotides can be employed and include, for example, RT-PCR,
taqman, amplification methods, polynucleotide microarray, and the
like.
[0386] Methods for the detection of polynucleotides (such as mRNA)
are well known and include, for example, hybridization assays using
complementary DNA probes (such as in situ hybridization using
labeled EGFL7 riboprobes), Northern blot and related techniques,
and various nucleic acid amplification assays (such as RT-PCR using
complementary primers specific for EGFL7, and other amplification
type detection methods, such as, for example, branched DNA, SPIA,
Ribo-SPIA, SISBA, TMA and the like).
[0387] Biological samples from mammals can be conveniently assayed
for, e.g., EGFL7 mRNAs using Northern, dot blot or PCR analysis.
For example, RT-PCR assays such as quantitative PCR assays are well
known in the art. In an illustrative embodiment of the invention, a
method for detecting EGFL7 mRNA in a biological sample comprises
producing cDNA from the sample by reverse transcription using at
least one primer; amplifying the cDNA so produced using an EGFL7
polynucleotide as sense and antisense primers to amplify EGFL7
cDNAs therein; and detecting the presence or absence of the
amplified EGFL7 cDNA. In addition, such methods can include one or
more steps that allow one to determine the amount (levels) of EGFL7
mRNA in a biological sample (e.g. by simultaneously examining the
levels a comparative control mRNA sequence of a housekeeping gene
such as an actin family member). Optionally, the sequence of the
amplified EGFL7 cDNA can be determined.
[0388] Probes and/or primers may be labeled with a detectable
marker, such as, for example, a radioisotope, fluorescent compound,
bioluminescent compound, a chemiluminescent compound, metal
chelator or enzyme. Such probes and primers can be used to detect
the presence of EGFL7 polynucleotides in a sample and as a means
for detecting a cell expressing EGFL7 proteins. As will be
understood by the skilled artisan, a great many different primers
and probes may be prepared (e.g., based on the sequences provided
in herein) and used effectively to amplify, clone and/or determine
the presence or absence of and/or amount of EGFL7 mRNAs.
[0389] Optional methods of the invention include protocols
comprising detection of polynucleotides, such as EGFL7
polynucleotide, in a tissue or cell sample using microarray
technologies. For example, using nucleic acid microarrays, test and
control mRNA samples from test and control tissue samples are
reverse transcribed and labeled to generate cDNA probes. The probes
are then hybridized to an array of nucleic acids immobilized on a
solid support. The array is configured such that the sequence and
position of each member of the array is known. For example, a
selection of genes that have potential to be expressed in certain
disease states may be arrayed on a solid support. Hybridization of
a labeled probe with a particular array member indicates that the
sample from which the probe was derived expresses that gene.
Differential gene expression analysis of disease tissue can provide
valuable information. Microarray technology utilizes nucleic acid
hybridization techniques and computing technology to evaluate the
mRNA expression profile of thousands of genes within a single
experiment. (see, e.g., WO 01/75166 published Oct. 11, 2001; (See,
for example, U.S. Pat. No. 5,700,637, U.S. Pat. No. 5,445,934, and
U.S. Pat. No. 5,807,522, Lockart, Nature Biotechnology,
14:1675-1680 (1996); Cheung, V. G. et al., Nature Genetics
21(Suppl):15-19 (1999) for a discussion of array fabrication). DNA
microarrays are miniature arrays containing gene fragments that are
either synthesized directly onto or spotted onto glass or other
substrates. Thousands of genes are usually represented in a single
array. A typical microarray experiment involves the following
steps: 1. preparation of fluorescently labeled target from RNA
isolated from the sample, 2. hybridization of the labeled target to
the microarray, 3. washing, staining, and scanning of the array, 4.
analysis of the scanned image and 5. generation of gene expression
profiles. Currently two main types of DNA microarrays are being
used: oligonucleotide (usually 25 to 70 mers) arrays and gene
expression arrays containing PCR products prepared from cDNAs. In
forming an array, oligonucleotides can be either prefabricated and
spotted to the surface or directly synthesized on to the surface
(in situ).
[0390] The Affymetrix GeneChip.RTM. system is a commercially
available microarray system which comprises arrays fabricated by
direct synthesis of oligonucleotides on a glass surface. Probe/Gene
Arrays: Oligonucleotides, usually 25 mers, are directly synthesized
onto a glass wafer by a combination of semiconductor-based
photolithography and solid phase chemical synthesis technologies.
Each array contains up to 400,000 different oligos and each oligo
is present in millions of copies. Since oligonucleotide probes are
synthesized in known locations on the array, the hybridization
patterns and signal intensities can be interpreted in terms of gene
identity and relative expression levels by the Affymetrix
Microarray Suite software. Each gene is represented on the array by
a series of different oligonucleotide probes. Each probe pair
consists of a perfect match oligonucleotide and a mismatch
oligonucleotide. The perfect match probe has a sequence exactly
complimentary to the particular gene and thus measures the
expression of the gene. The mismatch probe differs from the perfect
match probe by a single base substitution at the center base
position, disturbing the binding of the target gene transcript.
This helps to determine the background and nonspecific
hybridization that contributes to the signal measured for the
perfect match oligo. The Microarray Suite software subtracts the
hybridization intensities of the mismatch probes from those of the
perfect match probes to determine the absolute or specific
intensity value for each probe set. Probes are chosen based on
current information from GenBank.RTM. and other nucleotide
repositories. The sequences are believed to recognize unique
regions of the 3' end of the gene. A GeneChip.RTM. Hybridization
Oven ("rotisserie" oven) is used to carry out the hybridization of
up to 64 arrays at one time. The fluidics station performs washing
and staining of the probe arrays. It is completely automated and
contains four modules, with each module holding one probe array.
Each module is controlled independently through Microarray Suite
software using preprogrammed fluidics protocols. The scanner is a
confocal laser fluorescence scanner which measures fluorescence
intensity emitted by the labeled cRNA bound to the probe arrays.
The computer workstation with Microarray Suite software controls
the fluidics station and the scanner. Microarray Suite software can
control up to eight fluidics stations using preprogrammed
hybridization, wash, and stain protocols for the probe array. The
software also acquires and converts hybridization intensity data
into a presence/absence call for each gene using appropriate
algorithms. Finally, the software detects changes in gene
expression between experiments by comparison analysis and formats
the output into .txt files, which can be used with other software
programs for further data analysis.
[0391] In some embodiments, the treatment is for a cancer selected
from the group consisting of colorectal cancer, lung cancer,
ovarian cancer, pituitary cancer, pancreatic cancer, mammary
fibroadenoma, prostate cancer, head and neck squamous cell
carcinoma, soft tissue sarcoma, breast cancer, neuroblastomas,
melanoma, breast carcinoma, gastric cancer, colorectal cancer
(CRC), epithelial carcinomas, brain cancer, endometrial cancer,
testis cancer, cholangiocarcinoma, gallbladder carcinoma, and
hepatocellular carcinoma.
[0392] Biological samples are described herein, e.g., in the
definition of Biological Sample. In some embodiment, the biological
sample is serum or of a tumor.
Articles of Manufacture
[0393] In another aspect of the invention, an article of
manufacture containing materials useful for the treatment,
prevention and/or diagnosis of the disorders described above is
provided. The article of manufacture comprises a container and a
label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, syringes,
etc. The containers may be formed from a variety of materials such
as glass or plastic. The container holds a composition which is by
itself or when combined with another composition(s) effective for
treating, preventing and/or diagnosing the condition and may have a
sterile access port (for example the container may be an
intravenous solution bag or a vial having a stopper pierceable by a
hypodermic injection needle). At least one active agent in the
composition is an antibody of the invention. The label or package
insert indicates that the composition is used for treating the
condition of choice, such as cancer. Moreover, the article of
manufacture may comprise (a) a first container with a composition
contained therein, wherein the composition comprises an antibody of
the invention; and (b) a second container with a composition
contained therein. The article of manufacture in this embodiment of
the invention may further comprise a package insert indicating that
the first and second antibody compositions can be used to treat a
particular condition, e.g. cancer. Alternatively, or additionally,
the article of manufacture may further comprise a second (or third)
container comprising a pharmaceutically-acceptable buffer, such as
bacteriostatic water for injection (BWFI), phosphate-buffered
saline, Ringer's solution and dextrose solution. It may further
include other materials desirable from a commercial and user
standpoint, including other buffers, diluents, filters, needles,
and syringes.
[0394] The following are examples of the methods and compositions
of the invention. It is understood that various other embodiments
may be practiced, given the general description provided above.
EXAMPLES
[0395] Commercially available reagents referred to in the Examples
were used according to manufacturer's instructions unless otherwise
indicated.
Example 1
Generation of Humanized mu4F11 Antibodies
[0396] This example demonstrates the humanization of the murine
antibody 4F11 (mu4F11) directed against EGFL7. Residue numbers are
according to Kabat (Kabat et al., Sequences of proteins of
immunological interest, 5th Ed., Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)). Single letter amino
acid abbreviations are used.
Materials and Methods
[0397] Full length human EGFL7 and a truncated form of human EGFL7
(residues 1-182) containing the EMI and 2 EGF domains (lacking the
2 coiled-coiled domains) were expressed in CHO cells and purified
by conventional means (FIG. 1). Peptides containing the 4F11
epitope on EGFL7 called p2 (RPRYACCPGWKRT; SEQ ID NO: 5) and EMI2
(PARPRYACCPGWKRTSGLPGACGAAICQPP; SEQ ID NO: 4) were made
synthetically.
[0398] A hybridoma expressing the murine antibody 4F11 was obtained
by immunizing
[0399] Egf17 knockout mice with recombinant full length human EGFL7
protein expressed in E. coli and refolded. Antibodies were screened
by ELISA using recombinant human or murine EGFL7 coated plates. A
panel of function blocking antibodies were identified by their
ability to block HUVEC adhesion to EGFL7 coated plates. Several
antibodies were identified as cross-species function blocking
antibodies, including one designated 4F11 (see co-owned
International Patent Application WO 2007/106915, filed 16 Mar. 2007
and published 20 Sep. 2007).
[0400] Cloning of Murine 4F11 Variable Domains and Generation of a
Chimeric 4F11 Antibody--
[0401] Total RNA was extracted from hybridoma cells producing 4F11
using standard methods. The variable light (VL) and variable heavy
(VH) domains were amplified using RT-PCR with degenerate primers to
the heavy and light chains. The forward primers were specific for
the N-terminal amino acid sequence of the VL and VH regions.
Respectively, the LC and HC reverse primers were designed to anneal
to a region in the constant light (CL) and constant heavy domain 1
(CH1), which are highly conserved across species. Amplified VL and
VH were cloned into mammalian expression vectors to generate a
chimeric antibody ch4F11. The polynucleotide sequence of the
inserts was determined using routine sequencing methods.
[0402] Direct Hypervariable Region Grafts onto Acceptor Human
Consensus Framework--
[0403] The phagemid used for this work is a monovalent Fab-g3
display vector and consists of 2 open reading frames under control
of a single phoA promoter. The first open reading frame consists of
the stII signal sequence fused to the VL and CH1 domains of the
acceptor light chain and the second consists of the stII signal
sequence fused to the VH and CH1 domains of the acceptor heavy
chain followed by the minor phage coat protein P3.
[0404] To make the CDR grafts, hypervariable regions from mu4F11
were grafted into the huKI and huI11 consensus acceptor frameworks
to generate the direct CDR-graft (4F11.v1) (FIGS. 2 and 3). In the
VL domain the following regions were grafted to the human consensus
acceptor: positions 24-34 (L1; SEQ ID NO: 31), 50-56 (L2; SEQ ID
NO: 32) and 89-97 (L3; SEQ ID NO: 33). In the VH domain, positions
26-35 (H1; SEQ ID NO: 34), 49-65 (H2; SEQ ID NO: 35) and 95-102
(H3; SEQ ID NO: 36) were grafted. MacCallum et al. (MacCallum et
al. J. Mol. Biol. 262: 732-745 (1996)) have analyzed antibody and
antigen complex crystal structures and found position 49 of the
heavy chain to be part of the contact region thus it seems
reasonable to include this position in the definition of CDR-H2
when humanizing antibodies.
[0405] 4F11.v1 was generated by Kunkel mutagenesis as both a Fab
displayed on phage and as an IgG using separate oligonucleotides
for each hypervariable region. Correct clones were identified by
DNA sequencing.
[0406] Framework Toggle--
[0407] To identify framework positions important for binding, a
framework toggle phage library was generated to offer either the
murine or human amino acid at position 4 in VL, and positions 2,
48, 69, 71, 73, 75, 76, 78 and 91 in VH. These positions were
diversified as outlined in FIG. 4, by Kunkel mutagenesis using 5
oligonucleotides to mutate 4F11.v1 that was used as a template.
[0408] Randomization of the Hypervariable Regions
[0409] Sequence diversity was introduced at positions in the
hypervariable regions using Kunkel mutagenesis. To generate a
single position library (SPL), each position in each hypervariable
region was individually randomized to all possible 20 amino acids
using oligonucleotides encoding NNS. This resulted in 76
sub-libraries, each having a diversity of 20 that were combined
into a pooled "single position library" (SPL) encompassing variants
with a single mutation located within one of the hypervariable
regions. The six templates used for mutagenesis had one stop codon
(TAA) in the middle of CDR L1, L2, L3 H1, H2 or H3 to avoid
reselecting the wild type sequence. When generating the SPL, the
oligonucleotides used to introduce diversity also repaired the stop
codon in the corresponding template.
[0410] For the limited libraries, 4 oligonucleotides were
incorporated simultaneously which repaired the stop codons (TAA)
and introduced NNS at positions 53 and 54 in CDR-L2, 29 in CDR-H1,
52 in CDR-H2 and 98 in CDR-H3.
[0411] Generation of Phage Libraries
[0412] Oligonucleotides designed to introduce diversity into
framework positions or hypervariable regions as outlined above,
were phosphorylated separately in 20 .mu.l reactions containing 660
ng of oligonucleotide, 50 mM Tris pH 7.5, 10 mM MgCl.sub.2, 1 mM
ATP, 20 mM DTT, and 5 U polynucleotide kinase for 1 h at 37.degree.
C.
[0413] To generate the framework toggle or limited libraries all
phosphorylated oligonucleotides directed to introduce diversity
were added simultaneously to the mutagenesis reaction. For the SPL,
76 individual Kunkel mutagenesis reactions were performed in a
96-well PCR plate. From the phosphorylated oligonucleotides
reactions (above), 2 .mu.l was added to 300 ng Kunkel template
containing the corresponding stop codon in 50 mM Tris pH 7.5, 10 mM
MgCl.sub.2 in a final volume of 10 .mu.l. The mixture was annealed
at 90.degree. C. for 2 min, 50.degree. C. for 5 min and then cooled
on ice. The annealed template was then filled in by adding 0.5
.mu.l 10 mM ATP, 0.5 .mu.l 10 mM dNTPs (10 mM each of dATP, dCTP,
dGTP and dTTP), 1 .mu.l 100 mM DTT, 1 .mu.l 10.times.TM buffer (0.5
M Tris pH 7.5, 0.1 M MgCl.sub.2), 80 U T4 ligase, and 4 U T7
polymerase in a total volume of 20 .mu.l for 2 h at room
temperature. These filled-in and ligated products were then each
transformed into XL1-Blue cells, grown in 0.5 ml of 2YT containing
5 .mu.g/ml of tetracycline and M13/KO7 helper phage (MOI 10) for 2
hr at 37.degree. C. and then pooled and transferred to 500 ml 2YT
containing 50 .mu.g/ml carbenicillin and grown 16 h at 37.degree.
C.
[0414] Phage Selections--
[0415] Multiple forms of antigen were used for phage selections.
Full length or truncated EGFL7 (5 .mu.g/ml) were immobilized in 50
mM sodium bicarbonate pH 9.6 on MaxiSorp.TM. microtiter plates
(Nunc) overnight at 4.degree. C. EMI2 and p2 peptides were also
biotinylated either through their free cysteine (using maleimide
PEO.sub.2-biotin; Pierce) or through the free amine on their amino
terminus (using NHS-LC-biotin, Pierce). For biotinylation
reactions, a 2-fold molar excess of biotin reagent was used in PBS.
Biotinylated EMI2 and p2 peptides were captured on NeutrAvidin.RTM.
(2 .mu.g/ml) that had been immobilized in 50 mM sodium bicarbonate
pH 9.6 on MaxiSorp.TM. microtiter plates (Nunc) overnight at
4.degree. C. All plates were blocked for at least 1 h using
Blocker.TM. Casein (Pierce).
[0416] Phage were harvested from the culture supernatant and
suspended in PBS containing 5% powdered milk and 0.05% Tween.TM. 20
(PBSBT). Following addition of the phage library and a 1 hr
incubation, microtiter wells were washed extensively with PBS
containing 0.05% Tween.TM. 20 (PBST) and bound phage were eluted by
incubating the wells with 20 mM HCl, 500 mM KCl for 30 min. Eluted
phage were neutralized with 1 M Tris, pH 8 and amplified using
XL1-Blue cells and M13/KO7 helper phage and grown overnight at
37.degree. C. in 2YT, 50 .mu.g/ml carbenicillin. The titers of
phage eluted from a target containing well were compared to titers
of phage recovered from a non-target containing well to assess
enrichment.
[0417] Selection stringency was increased by both capturing phage
that bound to decreasing concentrations of biotinylated p2 peptide
in solution followed by capture on NeutrAvidin.RTM. for 10 min (on
rate selection) and by increasing the washing time and temperature
to allow weak binding phage to be washed away (off rate
selection).
[0418] IgG Production--
[0419] For screening purposes, IgG variants were initially produced
in 293 cells. Vectors coding for VL and VH (25 .mu.g) were
transfected into 293 cells using the FuGENE.RTM. system. 500 .mu.l
of FuGENE.RTM. was mixed with 4.5 ml of DMEM media containing no
FBS and incubated at room temperature for 5 min. Each chain (25
.mu.g) was added to this mixture and incubated at room temperature
for 20 min and then transferred to five T-150 flasks for
transfection overnight at 37.degree. C. in 5% CO.sub.2. The
following day the media containing the transfection mixture was
removed and replaced with 23 ml PS04 media with 0.1 ml/L trace
elements (A0934) and 10 mg/L insulin (A0940). Cells were incubated
for an additional 5 days after which the media was harvested at
1000 rpm for 5 min and sterile filtered using a 0.22 .mu.m low
protein-binding filter. Samples could be stored at 4.degree. C.
after addition of 2.5 ml 0.1% PMSF for every 125 ml of media.
[0420] Affinity Determinations
[0421] Affinity determinations were performed by surface plasmon
resonance using a BIAcore.TM.-2000. Truncated EGFL7 or p2 peptide
was immobilized (approximately 50-200 RU) in 10 mM sodium acetate
pH 4.8 on a CMS sensor chip. Purified IgG variants were injected
(using a 2-fold serial dilution of 0.5 to 1000 nM in PBST) at a
flow rate of 30 .mu.L/min. Each sample was analyzed with 3-minute
association and 3.5-minute disassociation. After each injection the
chip was regenerated using 10 mM glycine pH 1.7.
[0422] Binding response was corrected by subtracting a control flow
cell from IgG variant flow cells. A 1:1 Languir model of
simultaneous fitting of k.sub.on and k.sub.off was used for
kinetics analysis.
[0423] Cell Adhesion Assay--
[0424] Mouse EGFL7 (mEGFL7-CB-His) or human EGFL7 (EGFL7-CB-His)
were coated on microtiter plates at 5 .mu.g/ml in sodium carbonate
buffer 0/N at 4.degree. C. then blocked with 1% BSA in PBS.
Anti-EGFL7 antibodies were added (0.01 .mu.g/ml to 100 .mu.g/ml),
followed by the addition of 20,000 Human Umbilical Vein Endothelial
Cells (HUVEC)/well in appropriate cell growth medium (EGM Lonza).
Control cells were seeded in wells without antibody to calculate
100% of seeded cells. Each antibody concentration was tested in
triplicate. The plates were spun down for 5 min at 140 g to
synchronize contact of cells with substrate and then incubated in
CO.sub.2 incubator for 30 min and washed 3 times with PBS. Cells
that adhered to the plates were counted using CyQUANT.RTM. buffer
(Molecular Probes) and calculated as percent of the total cells
plated. The percentages of cells that bound to the plate were
plotted against the concentrations of each antibody.
Results and Discussion
[0425] Humanization of 4F11--
[0426] The human acceptor framework used for humanization of mu4F11
is based on the consensus human kappa I VL domain and the consensus
human subgroup III VH domain. Each complimentarity determining
region (CDR) for mu4F11 was identified and grafted into the human
acceptor framework to generate a CDR graft (4F11.v1) that could be
displayed as an Fab on phage (FIGS. 2 and 3).
[0427] Antigen Evaluation for Phage Selection--
[0428] The 4F11 epitope on EGFL7 was mapped to the second EMI
domain and more specifically to peptide p2 using a competition
Western blot analysis (FIGS. 1 and 5). Phage displaying 4F11.v1
bound to immobilized full length and truncated EGFL7, but
significant non-specific phage binding was also observed using a
control phage (FIG. 6). For this reason, the p2 and EMI2 peptides
that block 4F11 binding to truncated EGFL7 were used for phage
selections. The peptides were biotinylated either through their
free cysteine to generate p2S and EMI2c (using maleimide
PEO.sub.2-biotin; Pierce) or through the free amine on their amino
terminus to generate p2N and EMI2.n (using NHS-LC-biotin, Pierce).
To assess binding, biotinylated peptides were captured in
microtiter wells coated with NeutrAvidin.RTM.. Phage displaying
4F11.v1 were used to assess binding to captured biotinylated EMI2
and p2 peptides. 4F11.v1 phage bound to best to p2S (FIG. 7). The
amount of phage captured was greatest when a concentration of 50 nM
biotinylated peptide was used for binding to the NeutrAvidin.RTM.
coated well.
[0429] Screening Framework Positions--
[0430] The framework toggle library was panned for 4 rounds of
selection on immobilized biotinylated p2S peptide. DNA sequence
analysis of 96 clones from the last round was used to evaluate the
amino acid importance, based on abundance, at each toggled position
(FIG. 8). Amino acid abundance prior to and after 4 rounds of
selection suggested the replacement of L78 with Thr (L78T) or Val
(L78V) might lead to improved binding.
[0431] Screening CDR Positions--
[0432] In order to identify further improvements, Single to
Position Libraries were generated using 3 frameworks: the initial
CDR graft (4F11.v1), 4F11.v1 with L78T (4F11.v2), and 4F11.v1 with
L78V (4F11.v3). For each SPL, each position in each CDR was
individually randomized to all possible amino acids (a total of 76
libraries, each containing 20 members, pooled into one SPL for each
framework). Six 4F11.v1 DNA templates (containing stop codons in
the appropriate CDRs) were used to generate the three SPLs. The
framework change at positions 78 in VH was introduced during SPL
generation by using mutagenic oligonucleotides coding for the
appropriate framework change (L78V or L78V). Thus, framework and
individual CDR positions were mutated simultaneously. The SPLs were
panned on soluble p2S peptide that was captured using immobilized
NeutrAvidin.RTM. as outlined in Table 1:
TABLE-US-00005 TABLE 1 SPL Phage Selection Conditions Kon Selection
Koff Selection Antigen Binding Time Excess Peptide Capture Round 1
Immobilized 1 hour None -- p2S (50 nM on NeutrAvidin .RTM.) Round 2
Immobilized 1 hour None -- p2S (50 nM on NeurAvidin) Round 3 20 nM
p2S in 30 min 3 hours 10 min solution Round 4 5 nM p2S in 30 min
4.5 hours; 37.degree. C. 10 min solution Round 5 10 nM p2S in 20
min 70 hours; 37.degree. C. 10 min solution
Selection stringency was gradually increased by decreasing the
concentration of p2S peptide, reducing the time allowed for binding
and increasing the wash time and temperature. The highest phage
recovery during the last 3 rounds of selection was observed with
SPLs based on 4F11.v2 and 4F11.v3.
[0433] Clones from the last round were picked for DNA sequence
analysis. Individual sequence changes were identified in each CDR
(FIGS. 9A and 9B). The most abundant SPL clones had changes in VL
at position N53Y in CDR-L2. Changes that appeared frequently and in
more than one SPL were incorporated into 4F11.v3. These variants
(.v4 through .v12), the 4F11-graft (.v1) and changes to the VH
framework (4F11.v2 and 4F11.v3), were expressed as IgG, purified
and tested for binding to immobilized p2S by Biacore and using the
Cell Adhesion Assay (Table 2). The weaker inhibition of cell
adhesion observed for 4F11.v6 and 4F11.v10 compared to mu4F11
indicated further affinity improvements were desirable.
TABLE-US-00006 TABLE 2 Humanized 4F11 Variants Expressed as IgG
Hu4F11 Binding to p2S (Biacore .TM.) Cell Adhesion Assay Variant
Light Chain Heavy Chain ka (1/Ms) kd (1/s) KD (nM) Variant/ch4F11
(fold) chimera 2.21E+05 7.51E-4 3.44 1.00 .v1 Graft Graft 3.42E+04
3.31E-03 96.8 .v2 Graft Graft + 78T 5.35E+04 2.91E-03 54.4 .v3
Graft Graft + 78V 8.45E+04 2.92E-03 34.6 .v4 Graft + L1: D28R Graft
+ 78V 2.31E+05 3.14E-03 13.6 .v5 Graft + L1: M33V Graft + 78V
7.11E+04 2.42E-03 34.0 .v6 Graft + L2: N53Y Graft + 78V 1.23E+05
2.40E-03 19.5 4.01 .v7 Graft + L2: L54R Graft + 78V ND .v8 Graft +
L3: Y96F Graft + 78V ND .v9 Graft Graft + 78V + 2.64E+05 2.30E-03
9.0 H1: F29R .v10 Graft + L2: N53Y Graft + 78V + 7.20E+04 1.97E-03
27.4 4.38 H3: S98Y .v11 Graft Graft + 78V + ND H3: S98Y .v12 Graft
Graft + 78V + 1.96E+05 2.18E-03 11.1 H2: T52aI
[0434] Reassessment of Framework Positions--
[0435] Additional framework changes were incorporated in sets.
Variants were expressed as IgG (variants 13 through 16) and tested
by Biacore.TM. as outlined in Table 3:
TABLE-US-00007 TABLE 3 Humanized 4F11 Framework Variants Expressed
as IgG Binding to p2S (Biacore .TM.) Hu4F11 Light Heavy ka kd KD
Variant Chain Chain (1/Ms) (1/s) (nM) .v13 Graft + Graft + 1.49E+05
1.86E-03 12.5 M4L R71L, L78A .v14 Graft + Graft + 1.41E+05 9.84E-04
7.0 M4L V21, V48M, I69F, R71L, N73T, K75A, N76S, L78A, Y91F .v15
Graft Graft + 9.05E+04 2.76E-03 30.5 R71L, L78A .v16 Graft Graft +
9.81E+04 1.65E-03 16.8 V2I, V48M, I69F, R71L, N73T, K75A, N76S,
L78A, Y91F ch4F11 2.15E+05 7.92E-04 3.7
Of these, 4F11.v14 had the best affinity relative to mu4F11.
[0436] Limited Libraries--
[0437] Two limited libraries were generated based on 4F11.v13 as a
template. Library 1 contained the same framework changes as
4F11.V13 (LC:M4L and HC: R71L, L78A) while library 2 contained 2
additional changes in the heavy chain: N76S and Y91F (FIGS. 10 and
11). CDR changes in L1 and L3 (D28S and D94E) were also
incorporated into both libraries. Diversification was limited to
positions where changes had been previously observed following
selection of the SPLs (FIGS. 10 and 11). These positions (53 and 54
in the light chain and 29, 52, 98 in the heavy chain) were
diversified to include all twenty amino acids and panned against
immobilized p2S peptide that was captured using immobilized
NeutrAvidin as outlined in Table 4:
TABLE-US-00008 TABLE 4 Limited Library Phage Selection Conditions
Kon Selection Koff Selection Antigen Binding Time Excess Peptide
Capture Round 1 Immobilized 1 hour None -- b-p2S (50 nM on
NeutrAvidin .RTM.) Round 2 Immobilized 1 hour None -- b-p2S (50 nM
on NeurAvidin .RTM.) Round 3 50 nM b-p2S in 60 min 30 min, r.t. 10
min solution Round 4 10 nM b-p2S in 30 min 1 hr; 37.degree. C. 5
min solution Round 5 10 nM b-p2S in 15 min 2 hr; 37.degree. C. 5
min solution
[0438] The preference for different amino acid residues at the
randomized positions for each library is plotted in FIG. 12. In
light chain, both libraries preferentially selected 53Y and 54R,
while in the heavy chain, there was a preference for 29F and 52T.
In CDR-H3 of the heavy chain, 3 amino acids were selected at
position 98 (98Y, 98H, and 98R) in both libraries. To identify the
best combination of these changes, variants 17 through 26 (Table 5)
were constructed, expressed and characterized as IgG. Several
variants had affinities as good or better than ch4F11. Variants
were further characterized in the cell migration assay using human
or mouse EGFL7 (Table 5 and FIGS. 13 and 14).
TABLE-US-00009 TABLE 5 Humanized 4F11 Variants Expressed as IgG
Binding to p2S Cell Adhesion Assay Light Chain Heavy Chain (Biacore
.TM.) (human EGFL7) Hu4F11 Position Position VH Framework Fold
change in KD Variant/ch4F11 Variant 53 54 29 52 98 used
(variant/chimera) (fold) ch4F11 N L F T S 1 1.00 .v17 Y R F T Y
Library 1 0.93 0.86 .v18 Y R F T R Library 1 1.81 1.86 .v19 Y R G T
R Library 1 1.43 12.87 .v20 Y R F T H Library 1 1.79 0.70 .v21 Y L
R T R Library 1 1.31 3.12 .v22 Y R F T Y Library 2 1 0.81 .v23 Y R
F T R Library 2 3.88 nd .v24 Y R G T R Library 2 0.48 2.52 .v25 Y R
F T H Library 2 0.56 1.82 .v26 Y L R T R Library 2 0.66 1.38
[0439] The VL and VH domains from hu4F11.v17 and 4F11.v22 compared
to mu4F11 and 4F11.v1 are shown in FIGS. 15 and 16,
respectively.
Example 2
Generation of Humanized mu18F7 Antibodies
[0440] This example demonstrates the humanization of the murine
antibody 18F7 (mu18F7) directed against EGFL7. Residue numbers are
according to Kabat (Kabat et al., Sequences of proteins of
immunological interest, 5th Ed., Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)). Single letter amino
acid abbreviations are used.
Materials and Methods
[0441] Full length human EGFL7 and a truncated form of human EGFL7
(residues 1-182) containing the EMI and 2 EGF domains (lacking the
2 coiled-coiled domains) were expressed in CHO cells and purified
by conventional means (FIG. 1). Peptides containing the 18F7
epitope on EGFL7 called p5 (RACSTYRTIYRTA; SEQ ID NO: 7) and EMI'
(LTTCDGHRACSTYRTIYRTAYRRSPG; SEQ ID NO: 3) were made
synthetically.
[0442] A hybridoma expressing the murine antibody 18F7 was obtained
by immunizing Egf17 to knockout mice with recombinant full length
human EGFL7 protein expressed in E. coli and refolded. Antibodies
were screened by ELISA using recombinant human or murine EGFL7
coated plates. A panel of function blocking antibodies were
identified by their ability to block HUVEC adhesion to EGFL7 coated
plates. Several antibodies were identified as cross-species
function blocking antibodies, including one designated 18F7 (see
co-owned International Patent Application WO 2007/106915, filed 16
Mar. 2007 and published 20 Sep. 2007).
[0443] Cloning of Murine 18F7 Variable Domains and Generation of a
Chimeric 18F7 Antibody--
[0444] Total RNA was extracted from hybridoma cells producing 18F7
using standard methods. The variable light (VL) and variable heavy
(VH) domains were amplified using RT-PCR with degenerate primers to
the heavy and light chains. The forward primers were specific for
the N-terminal amino acid sequence of the VL and VH regions.
Respectively, the LC and HC reverse primers were designed to anneal
to a region in the constant light (CL) and constant heavy domain 1
(CH1), which are highly conserved across species. Amplified VL and
VH were cloned into mammalian expression vectors to generate a
chimeric antibody ch18F7. The polynucleotide sequence of the
inserts was determined using routine sequencing methods.
[0445] Direct Hypervariable Region Grafts onto the Acceptor Human
Consensus Framework--
[0446] The phagemid used for this work is a monovalent Fab-g3
display vector and consists of 2 open reading frames under control
of a single phoA promoter. The first open reading frame consists of
the stII signal sequence fused to the VL and CH1 domains of the
acceptor light chain and the second consists of the stII signal
sequence fused to the VH and CH1 domains of the acceptor heavy
chain followed by the minor phage coat protein P3.
[0447] To make the CDR grafts, hypervariable regions from mu18F7
were grafted into the huKI and huI11 consensus acceptor frameworks
to generate the direct CDR-graft (18F7-graft) (FIGS. 17 and 18). In
the VL domain the following regions were grafted to the human
consensus acceptor: positions 24-34 (L1; SEQ ID NO: 100), 50-56
(L2; SEQ ID NO: 101) and 89-97 (L3; SEQ ID NO: 102). In the VH
domain, positions 26-35 (H1; SEQ ID NO: 103), 49-65 (H2; SEQ ID NO:
104) and 95-102 (H3; SEQ ID NO: 105) were grafted. MacCallum et al.
(MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)) have analyzed
antibody and antigen complex crystal structures and found position
49 of the heavy chain to be part of the contact region thus it
seems reasonable to include this position in the definition of
CDR-H2 when humanizing antibodies.
[0448] The 18F7-graft was generated by Kunkel mutagenesis as both a
Fab displayed on phage and as an IgG using separate
oligonucleotides for each hypervariable region. Correct clones were
identified by DNA sequencing.
[0449] Framework Toggle--
[0450] To identify framework positions important for binding, a
framework toggle phage library was generated to offer either the
murine or human amino acid at positions 87 in VL, and positions 48,
67, 69, 71, 73, 75, 76, 78 and 80 in VH. These positions were
diversified as outlined in FIG. 19, by Kunkel mutagenesis using 3
oligonucleotides to mutate the 18F7-graft that was used as a
template.
[0451] Randomization of the Hypervariable Regions--
[0452] Full sequence diversity was introduced separately at each
position in the hypervariable regions of the 18F7-graft using
Kunkel mutagenesis to generate single position libraries that were
pooled together. Each position in each hypervariable region of
18F7-graft was fully randomized to all possible 20 amino acids
using oligonucleotides encoding NNS at the respective positions.
Multiple libraries were made each consisting of 20 members having a
single position located within one of the hypervariable regions
fully randomized. To cover each position in the hypervariable
regions, 76 libraries of this type were generated and combined into
a pooled "single position library" (SPL) encompassing single
mutations located throughout each hypervariable position. A stop
codon (TAA) was introduced in the middle of each CDR to avoid
reselecting the wild type CDR grafted sequence. This was
accomplished by Kunkel mutagenesis and resulted in 6 different
templates for the 18F7-graft--each with a stop codon introduced
into a different CDR. When generating the library, the
oligonucleotides used to introduce diversity also repaired the stop
codon in the corresponding template.
[0453] Generation of Phage Libraries--
[0454] Oligonucleotides designed to introduce diversity into
framework positions or each hypervariable region as outlined above,
were phosphorylated separately in 20 .mu.l reactions containing 660
ng of oligonucleotide, 50 mM Tris pH 7.5, 10 mM MgCl.sub.2, 1 mM
ATP, 20 mM DTT, and 5 U polynucleotide kinase for 1 h at 37.degree.
C.
[0455] To generate the framework toggle library all 3
phosphorylated oligonucleotides directed to introduce diversity
were added simultaneously to the mutagenesis reaction. For the SPL,
76 individual Kunkel mutagenesis reactions were performed in a
96-well PCR plate. From the phosphorylated oligonucleotides
reactions (above), 2 .mu.l was added to 300 ng Kunkel template
containing the corresponding stop codon in 50 mM Tris pH 7.5, 10 mM
MgCl.sub.2 in a final volume of 10 .mu.l. The mixture was annealed
at 90.degree. C. for 2 min, 50.degree. C. for 5 min and then cooled
on ice. The annealed template was then filled in by adding 0.5
.mu.l 10 mM ATP, 0.5 .mu.l 10 mM dNTPs (10 mM each of dATP, dCTP,
dGTP and dTTP), 1 .mu.l 100 mM DTT, 1 .mu.l 10.times.TM buffer (0.5
M Tris pH 7.5, 0.1 M MgCl.sub.2), 80 U T4 ligase, and 4 U T7
polymerase in a total volume of 20 .mu.l for 2 h at room
temperature. These filled-in and ligated products were then each
transformed into XL1-Blue cells, grown in 0.5 ml of 2YT containing
5 .mu.g/ml of tetracycline and M13/KO7 helper phage (MOI 10) for 2
hr at 37.degree. C. and then pooled and transferred to 500 ml 2YT
containing 50 .mu.g/ml carbenicillin and grown 16 h at 37.degree.
C.
[0456] Phage Selections--
[0457] Multiple forms of antigen were used for phage selections.
Full length or truncated EGFL7 (5 .mu.g/ml) were immobilized in 50
mM sodium bicarbonate pH 9.6 on MaxiSorp.TM. microtiter plates
(Nunc) overnight at 4.degree. C. EMI1 and p5 peptides were also
biotinylated either through their free cysteine (using maleimide
PEO.sub.2-biotin; Pierce) or through the free amine on their amino
terminus (using NHS-LC-biotin, Pierce). For biotinylation
reactions, a 2-fold molar excess of biotin reagent was used in PBS.
Biotinylated EMI1 and p5 peptides were captured on NeutrAvidin.RTM.
(2 .mu.g/ml) that had been immobilized in 50 mM sodium bicarbonate
pH 9.6 on MaxiSorp.TM. microtiter plates (Nunc) overnight at
4.degree. C. All plates were blocked for at least 1 h using
Blocker.TM. Casein (Pierce).
[0458] Phage were harvested from the culture supernatant and
suspended in PBS containing 5% powdered milk and 0.05% Tween.TM. 20
(PBSBT). Following addition of the phage library and a 1 hr
incubation, microtiter wells were washed extensively with PBS
containing 0.05% Tween.TM. 20 (PBST) and bound phage were eluted by
incubating the wells with 20 mM HCl, 500 mM KCl for 30 min. Eluted
phage were neutralized with 1 M Tris, pH 8 and amplified using
XL1-Blue cells and M13/KO7 helper phage and grown overnight at
37.degree. C. in 2YT, 50 .mu.g/ml carbenicillin. The titers of
phage eluted from a target containing well were compared to titers
of phage recovered from a non-target containing well to assess
enrichment.
[0459] Selection stringency was increased by both capturing phage
that bound to decreasing concentrations of biotinylated p5 peptide
in solution followed by capture on NeutrAvidin.RTM. for 10 min (on
rate selection) and by increasing the washing time and temperature
to allow weak binding phage to be washed away (off rate
selection).
[0460] IgG Production--
[0461] For screening purposes, IgG variants were initially produced
in 293 cells. Vectors coding for VL and VH (25 .mu.g) were
transfected into 293 cells using the FuGENE.RTM. system. 500 .mu.l
of FuGENE.RTM. was mixed with 4.5 ml of DMEM media containing no
FBS and incubated at room temperature for 5 min. Each chain (25
.mu.g) was added to this mixture and incubated at room temperature
for 20 min and then transferred to five T-150 flasks for
transfection overnight at 37.degree. C. in 5% CO.sub.2. The
following day the media containing the transfection mixture was
removed and replaced with 23 ml PS04 media with 0.1 ml/L trace
elements (A0934) and 10 mg/L insulin (A0940). Cells were incubated
for an additional 5 days after which the media was harvested at
1000 rpm for 5 min and sterile filtered using a 0.22 .mu.m low
protein-binding filter. Samples could be stored at 4.degree. C.
after addition of 2.5 ml 0.1% PMSF for every 125 ml of media.
[0462] Affinity Determinations--
[0463] Affinity determinations were performed by surface plasmon
resonance using a BIAcore.TM.-2000. Truncated EGFL7 or p5 peptide
was immobilized (approximately 50-200 RU) in 10 mM sodium acetate
pH 4.8 on a CMS sensor chip. Purified IgG variants were injected
(using a 2-fold serial dilution of 0.5 to 1000 nM in PBST) at a
flow rate of 30 .mu.L/min. Each sample was analyzed with 3-minute
association and 3.5-minute disassociation. After each injection the
chip was regenerated using 10 mM glycine pH 1.7.
[0464] Binding response was corrected by subtracting a control flow
cell from IgG variant flow cells. A 1:1 Languir model of
simultaneous fitting of k.sub.on and k.sub.off was used for
kinetics analysis.
Results and Discussion
[0465] Humanization of 18F7--
[0466] The human acceptor framework used for humanization of mu18F7
is based on the consensus human kappa I VL domain and the consensus
human subgroup III VH domain. Each CDR for mu18F7 was identified
and grafted into the human acceptor framework to generate a CDR
graft that could be displayed as an Fab on phage (FIGS. 17 and
18).
[0467] Antigen Evaluation for phage selection--
[0468] The 18F7 epitope on EGFL7 was mapped to the first EMI domain
and more specifically to peptide p5 using a competition Western
blot analysis (FIGS. 1 and 20). Phage displaying the 18F7-graft
bound to immobilized full length and truncated EGFL7, but
significant non-specific phage binding was also observed using a
control phage (FIG. 21). For this reason, the p5 and EMI1 peptides
that block 18F7 binding to truncated EGFL7 were used for phage
selections. The peptides were biotinylated either through their
free cysteine to generate p5c and EMI1c (using maleimide
PEO.sub.2-biotin; Pierce) or through the free amine on their amino
terminus to generate p5n and EMI1n (using NHS-LC-biotin, Pierce).
To assess binding, biotinylated peptides were captured in
microtiter wells coated with NeutrAvidin.RTM.. Following 2 rounds
of selection on immobilized truncated EGFL7, the framework toggle
library phage pool was used to assess binding to captured
biotinylated EMI1 and p5 peptides. The phage pool bound to p5n and
EMI1n, but not to p5c or EMI1c (FIG. 22). The amount of phage
captured was greatest when a concentration of 50 nM biotinylated
peptide was used for binding to the NeutrAvidin.RTM. coated
well.
[0469] After selection for 2 rounds against immobilized truncated
EGFL7, the framework toggle library was further panned for 2 rounds
of selection on immobilized biotinylated p5n peptide. DNA sequence
analysis of 96 clones from the last round was used to evaluate the
amino acid importance, based on abundance, at each toggled position
(FIG. 23). Amino acid abundance prior to and after 4 rounds of
selection suggested the changes N73K and L78A lead to improved
binding. Although less prominent, L78V and V481 also do so and L78V
was further studied.
[0470] SPLs were explored in an effort to identify further
improvements using 4 frameworks derived from the framework toggle
library results. The 4 frameworks included the initial CDR graft
(18F7-graft), 18F7-graft with N73K (18F7.v2), 18F7-graft with N73K
and L78A (18F7.v3), and 18F7-graft with N73K and L78V (18F7.v4).
For each framework, an SPL was generated where each position in
each CDR was individually randomized to all possible amino acids (a
total of 76 libraries, each containing 20 members, pooled into one
SPL). Six 18F7-graft DNA templates (containing stop codons in the
appropriate CDRs) were used to generate all four SPLs. Framework
changes at positions 73 and 78 in VH were introduced during SPL
generation by using mutagenic oligonucleotides coding for the
appropriate framework changes. Thus, framework and individual CDR
positions were mutated simultaneously. The four SPLs were panned
for 2 rounds against immobilized truncated EGFL7 followed by 3
rounds of selection on soluble biotinylated p5n peptide that was
captured using immobilized NeutrAvidin.RTM. as outlined in Table
6:
TABLE-US-00010 TABLE 6 SPL Phage Selection Conditions Kon Selection
Koff Selection Antigen Binding Time Excess Peptide Capture Round 1
Immobilized 1 hour None -- b-p5N (50 nM on NeutrAvidin .RTM.) Round
2 Immobilized 1 hour None -- b-p5N (50 nM on NeurAvidin .RTM.)
Round 3 20 nM b-p5N 30 min 3 hours 10 min Round 4 5 nM b-p5N 30 min
4.5 hours; 37.degree. C. 10 min Round 5 10 nM b-p5N 20 min 70
hours; 37.degree. C. 10 min
Selection stringency was gradually increased by decreasing the
concentration of biotinylated p5n peptide, reducing the time
allowed for binding and increasing the wash time and temperature.
The highest phage recovery during the last 3 rounds of selection
was observed with SPLs based on 18F7.v3 and 18F7.v4.
[0471] Clones from the last round were picked for DNA sequence
analysis. Individual sequence changes were identified in each CDR
(FIGS. 24A and 24B). The most abundant SP library clones had
changes in VL at position S89. Changes that appeared frequently and
in more than one SP library were incorporated into 18F7.v3. These
variants (.v5 through .v10), the 18F7-graft (.v1) and changes to
the VH framework (.v2, .v3 and .v4), were expressed as IgG for
further analysis by Biacore.TM. (Tables 7 and 8).
TABLE-US-00011 TABLE 7 18F7-graft Variants Expressed as IgG
hu18F7grafted Light Heavy variants Chain Chain .v1 Graft Graft .v2
Graft 73K .v3 Graft 73K & 78A .v4 Graft 73K & 78V .v5
L2:F55G 73K & 78A .v6 L3:S89G 73K & 78A .v7 L3:S89A 73K
& 78A .v8 L3:S89V 73K & 78A .v9 Graft 73K & 78A &
H1:Y32K .v10 Graft 73K & 78A & H3:D100P
TABLE-US-00012 TABLE 8 Biacore .TM. Analysis of 18F7-graft Variants
Biacore Analysis of 18F7 Variants Over mEGFL7-CB at 37 C. 37 C.
over immobolized P5 ka (1/Ms/1e5) kd (1/s*1e4) KD (nM) ka
(1/Ms/1e5) kd (1/s*1e4) KD (nM) Chimeric 18F7 n = 5 0.36 .+-. 0.16
0.96 .+-. 0.78 3.17 .+-. 3.48 Chimeric 18F7 n = 5 1.09 .+-. 0.30
5.44 .+-. 0.49 5.51 .+-. 2.46 18F7 V1 0.97 2.74 2.82 18F7 V1 3.17
20 6.3 18F7 V2 1.10 1.98 1.81 18F7 V2 4.58 11.9 2.59 18F7 V3 0.55
0.905 1.64 18F7 V3 2.03 8.58 4.22 18F7 V4 0.50 2.08 4.15 18F7 V4
1.82 13.2 7.27 18F7 V5 0.73 1.17 1.61 18F7 V5 3.30 8.92 2.7 18F7 V6
0.50 0.204 0.408 18F7 V6 n = 3 1.21 .+-. 0.14 3.89 .+-. 0.71 3.21
.+-. 0.49 18F7 V7 0.70 0.958 1.38 18F7 V7 2.51 7.18 2.87 18F7 V8
0.98 1.99 2.04 18F7 V8 4.72 10.2 2.16 18F7 V9 0.59 0.932 1.58 18F7
V9 2.28 7.63 3.35 18F7 V10 1.60 1.95 1.22 18F7 V10 8.25 8.11 0.982
18F7 V6A n = 2 0.55 .+-. 0.01 4.04 .+-. 0.00 7.29 .+-. 0.17 18F7
V6A n = 2 3.73 .+-. 0.91 13.70 .+-. 1.41 3.74 .+-. 0.52 18F7 V6B n
= 2 0.79 .+-. 0.25 1.74 .+-. 1.75 2.68 .+-. 3.06 18F7 V6B n = 2
1.96 .+-. 0.25 6.38 .+-. 1.82 3.22 .+-. 0.52 18F7 V6C n = 2 0.36
.+-. 0.06 0.81 .+-. 0.36 2.40 .+-. 1.42 18F7 V6C n = 2 1.24 .+-.
0.28 5.02 .+-. 0.31 4.13 .+-. 0.66 18F7 V6D 1.03 7.91 7.65 18F7 V6D
n = 2 3.13 .+-. 1.89 23.25 .+-. 5.59 9.77 .+-. 7.69 18F7 V6E 0.48
4.74 9.9 18F7 V6E 3.03 15.4 5.1 18F7 V6I 0.23 4.88 21.3 18F7 V6I
1.90 31.6 16.7 18F7 V6J 0.21 0.164 6.8 18F7 V6J 1.08 6.07 5.6 18F7
V6K 0.28 0.454 1.6 18F7 V6K 1.27 6.62 5.2 Biacore .TM. analysis of
these 10 variants indicated all bound quite well to immobilized
mEGFL7 or the p5 peptide. 18F7.v6 had the slowest dissociation rate
(kd) and was effective in the HUVEC adhesion assay (FIG. 25).
[0472] Polishing of 18F7.v6--
[0473] Potential iso-aspartic acid forming sites (Asn-Gly) in
CDR-L1 (N28,G29) and CDR-H2 (N54, G55) in 18F7.v6 were eliminated
by testing alternative amino acids at these positions (Table
9).
TABLE-US-00013 TABLE 9 Variants Tested to Eliminate Potential
Iso-aspartic Acid Forming Sites Light Chain Heavy Chain hu18F7.v6
variants (N28 & G29) (N54 & G55) .v6A L1:SG .v6 (SEQ ID NO:
243) (SEQ ID NO: 104) .v6B .v6 H2:SG (SEQ ID NO: 100) (SEQ ID NO:
177) .v6C .v6 H2:NS (SEQ ID NO: 100) (SEQ ID NO: 247) .v6D L1:SG
H2:SG (SEQ ID NO: 243) (SEQ ID NO: 177) .v6E L1:SG H2:NS (SEQ ID
NO: 243) (SEQ ID NO: 247) .v6I L1:QG H2:NS (SEQ ID NO: 244) (SEQ ID
NO: 247) .v6J L1:NS H2:NS (SEQ ID NO: 245) (SEQ ID NO: 247) .v6K
L1:NA H2:NS (SEQ ID NO: 246) (SEQ ID NO: 247)
[0474] An SG sequence in H2 (.v6B and .v6D) expressed poorly, while
this sequence in L1 (.v6A) to increased the dissociation rate. In
contrast, an NS sequence in H2 (.v6C) had little affect on the
kinetics (Tables 8 and 9). Of the other changes sampled in L1 in
conjunction with H2:NS (.v6C), the changes NS (.v6J) and NA (.v6K)
had a minimal affect on the dissociation rate (Table 8 and 9, FIG.
25). These changes can be used to improve the stability of 18F7.v6
while still maintaining desired biological properties compared to
murine 18F7 (FIGS. 25 and 26). The VL and VH domains from
hu18F7.v6K are shown in FIGS. 27 and 28, respectively.
Example 3
Treatment of Tumor-Bearing Mice or Neonatal Mice with Humanized
Anti-EGFL7 Antibody
[0475] We investigated the ability of humanized anti-EGFL7
antibodies of the invention (alone and in combination with
anti-VEGF therapy) to inhibit angiogenesis and/or tumor growth in a
variety of models. We observed anti-EGFL7 antibodies enhance the
efficacy of anti-VEGF therapy.
[0476] RRLN female nu/nu mice were injected subcutaneously with
1.times.10.sup.7 H1299 human non-small cell lung cancer tumor cells
and allowed to develop tumors to 80-120 mm.sup.3. to Tumor-bearing
mice were then randomly separated into four groups (12 mice each)
so that the average tumor size in each group was 122 mm.sup.3.
These mice were then treated as follows: Group 1: intraperitoneal
(ip) injection of anti-VEGF antibody (B20-4.1; WO2005/012359 and
WO2005/044853) once per week at 10 mg/kg; Group 2: ip injection of
a negative control antibody anti-ragweed IgG once per week at 10
mg/kg and anti-EGFL7 antibody (hu18F7.v6K) twice per week at 10
mg/kg; Group 3: ip injection of B20-4.1 once per week at 10 mg/kg
and hu18F7.v6K twice per week at 10 mg/kg; Group 4: no treatment.
Tumors were measured twice per week with a caliper and tumor
volumes were calculated as (w.sup.2.times.l)/2 (w=tumor width in
mm, l=tumor length in mm). Mice were euthanized when their tumors
reached 1000 mm.sup.3 (defined as "mice reached end point"). Group
average tumor volumes +/-SEM were plotted against time until
.gtoreq.50% of the mice reached end point. The data from this
experiment showed that treatment with neither B20.4-1 nor
hu18F7.v6K alone significantly reduced tumor growth over untreated
control, although treatment with hu18F7.v6K exhibited a trend
toward reduced growth (FIG. 29). In contrast, treatment with both
B20.4-1 and hu18F7.v6K significantly inhibited tumor growth (FIG.
29).
[0477] Balb-c nude mice were injected subcutaneously in the right
flank with 5.times.10.sup.6 HM7 carcinoma cells in 0.1 ml
Matrigel.TM.. When mean tumor size reached 80-150 mm.sup.3, animals
were separated into 4 groups of 10 mice each so that the average
tumor sizes in all the groups were roughly equal and treated as
follows: Group 1: ip injection of anti-ragweed IgG twice per week
at 5 mg/kg; Group 2: ip injection of B20-4.1.1 (PCT/US2008/013248)
twice per week at 5 mg/kg; Group 3: ip injection of anti-EGFL7
antibody (hu18F7.v6k) twice per week at 10 mg/kg; Group 4: ip
injection of B20-4.1.1 twice per week at 5 mg/kg and hu18F7.v6k
twice per week at 10 mg/kg. Tumors were measured twice per week
with a caliper and the width and length recorded. Mice were
euthanized when tumors were greater than 1000 mm.sup.3 or when
tumor growth or ulceration interfered with animal health. We
observed that tumors in Groups 1 and 3 had similar growth rates,
whereas tumors in Group 2 grew more slowly than those in Group 1
and tumors in Group 4 exhibited a negative growth.
[0478] Balb-c nude mice were injected subcutaneously in the right
flank with primary human large cell lung cancer tumor explants
(LXFL 1674). The experiment comprised a reference group (Group 1)
dosed with human IgG (hIgG) and murine IgG (mIgG) control
antibodies, Group 2 that received hu18F7.v6k and mIgG, Group 3 that
received B20-4.1.1 and hIgG, and Group 4 that received hu18F7.v6K
and B20-4.1.1. All treatments were given twice weekly ip with
hu18F7.v6k (or hIgG) given at 10 mg/kg/dose four hours prior to B20
to (or mIgG) administered at 5 mg/kg/dose. Mice were sacrificed
individually when tumor volume exceeded 2000 mm.sup.3. Groups were
evaluated for efficacy as long as more than 50% of Group 1 control
mice were alive (day 14). Group size at the start of dosing was 10
mice bearing one tumor of 5-10 mm in diameter each per mouse. The
advantage of the combination therapy over the B20-4.1.1 monotherapy
was statistically significant (FIG. 30).
[0479] We also tested the humanized anti-EGFL7 antibody hu18F7.v6k
and its parental antibody murine 18F7 in a murine neonatal organ
angiogenesis assay. Newborn mice were injected with antibodies at
days 1 and 3 afer birth and organs were harvested on day 5. The
vasculatures in multiple organs were stained with a vascular
endothelial cell marker CD31 and vascular densities were
quantified. The groups were: Group 1 which received 15 mg/ml
anti-ragweed antibody (n=3/experiment), Group 2 which received 5
mg/ml anti-VEGF antibody (G6.31; WO2005/012359 and WO2005/044853;
n=3/experiment) and 10 mg/ml ragweed antibody, Group 3 which
received 5 mg/ml G6.31 and 10 mg/ml murine 18F7 (n=4/experiment),
and Group 4 which received 5 mg/ml G6.31 and 10 mg/ml hu18F7.v6k
(n=4/experiment). The pooled results from three independent
experiments are shown in FIG. 31, which demonstrated that the
anti-VEGF antibody 66.31 has significant anti-angiogenesis
activity, and the combination of G6.31 with either hu18F7.v6k or
murine 18F7 significantly enhanced the activity of G6.31. Similar
anti-angiogenesis activites were observed in the intestinal villus
vasculature. These results confirm that hu18F7.v6k and murine 18F7
have similar anti-angiogenesis activities in this model.
Example 4
Inhibition of Tumor Perfusion and Permeability by Anti-EGFL7
Antibodies in Human Subjects
[0480] We conducted dynamic contrast-magnetic resonance imaging
(DCE-MRI) assessments on human subjects who had been administered
with two cycles of 3 mg/kg or 15 mg/kg hu18F7.v6k to explore
changes in tumor vasculature in response to the antibody. DCE-MRI
is an imaging modality that allows for the functional analysis of
tumor microcirculation. Changes in vascular parameters such as
V.sub.e, the fractional extravascular and extracellular leakage
volume, and K.sub.trans, the volume transfer constant, reflect
changes in tumor perfusion and permeability. Two baseline
pre-treatment scans were obtained approximately 5-7 days apart (but
at least 24 hours apart) prior to dosing in Cycle 1 (for example,
Day -1 and Day -7 relative to administration of antibody).
Post-treatment scans were obtained on Day 15 of Cycle 1 and on Day
8 of Cycle 2 (.+-.2 days to allow for scheduling difficulties).
Evaluable metastatic lesions had to measure .gtoreq.3 cm in the
liver, or .gtoreq.2 cm elsewhere in at least one dimension. In
addition to DCE-MRI acquisition sequences, other MRI acquisition
sequences, such as diffusion-weighted imaging and T1- and
T2-weighted imaging were acquired during the same image acquisition
visit for each subject. As shown in Table 10, we observed that
treatment with anti-EGFL7 antibodies reduced K.sub.rans in some
solid tumors by up to approximately 40%.
TABLE-US-00014 TABLE 10 Median K.sub.trans Patent Identifier, liver
tumor volume, and hu18F7.v6k Cycle 1, Cycle 2, dosage Base 1 Base 2
Day 15 Day 8 3301, 82 cc ND 0.021 0.0171 ND (3 mg/kg) 3503, 7 cc ND
0.0542 0.0435 0.0327 (15 mg/kg) 3505, 11 cc 0.0278 0.0288 0.0239
0.019 (15 mg/kg) 3505, 142 cc 0.0239 0.022 0.0229 0.02 (15 mg/kg)
3902, 235 cc 0.022 0.019 0.02 0.022 (3 mg/kg)
Sequence CWU 1
1
2471278PRTMus sp. 1Met Gln Thr Met Trp Gly Ser Gly Glu Leu Leu Val
Ala Trp Phe Leu 1 5 10 15 Val Leu Ala Ala Asp Gly Thr Thr Glu His
Val Tyr Arg Pro Ser Arg 20 25 30 Arg Val Cys Thr Val Gly Ile Ser
Gly Gly Ser Ile Ser Glu Thr Phe 35 40 45 Val Gln Arg Val Tyr Gln
Pro Tyr Leu Thr Thr Cys Asp Gly His Arg 50 55 60 Ala Cys Ser Thr
Tyr Arg Thr Ile Tyr Arg Thr Ala Tyr Arg Arg Ser 65 70 75 80 Pro Gly
Val Thr Pro Ala Arg Pro Arg Tyr Ala Cys Cys Pro Gly Trp 85 90 95
Lys Arg Thr Ser Gly Leu Pro Gly Ala Cys Gly Ala Ala Ile Cys Gln 100
105 110 Pro Pro Cys Gly Asn Gly Gly Ser Cys Ile Arg Pro Gly His Cys
Arg 115 120 125 Cys Pro Val Gly Trp Gln Gly Asp Thr Cys Gln Thr Asp
Val Asp Glu 130 135 140 Cys Ser Thr Gly Glu Ala Ser Cys Pro Gln Arg
Cys Val Asn Thr Val 145 150 155 160 Gly Ser Tyr Trp Cys Gln Gly Trp
Glu Gly Gln Ser Pro Ser Ala Asp 165 170 175 Gly Thr Arg Cys Leu Ser
Lys Glu Gly Pro Ser Pro Val Ala Pro Asn 180 185 190 Pro Thr Ala Gly
Val Asp Ser Met Ala Arg Glu Glu Val Tyr Arg Leu 195 200 205 Gln Ala
Arg Val Asp Val Leu Glu Gln Lys Leu Gln Leu Val Leu Ala 210 215 220
Pro Leu His Ser Leu Ala Ser Arg Ser Thr Glu His Gly Leu Gln Asp 225
230 235 240 Pro Gly Ser Leu Leu Ala His Ser Phe Gln Gln Leu Asp Arg
Ile Asp 245 250 255 Ser Leu Ser Glu Gln Val Ser Phe Leu Glu Glu His
Leu Gly Ser Cys 260 265 270 Ser Cys Lys Lys Asp Leu 275 2274PRTHomo
sapiens 2Met Arg Gly Ser Gln Glu Val Leu Leu Met Trp Leu Leu Val
Leu Ala 1 5 10 15 Val Gly Gly Thr Glu His Ala Tyr Arg Pro Gly Arg
Arg Val Cys Ala 20 25 30 Val Arg Ala His Gly Asp Pro Val Ser Glu
Ser Phe Val Gln Arg Val 35 40 45 Tyr Gln Pro Phe Leu Thr Thr Cys
Asp Gly His Arg Ala Cys Ser Thr 50 55 60 Tyr Arg Thr Ile Tyr Arg
Thr Ala Tyr Arg Arg Ser Pro Gly Leu Ala 65 70 75 80 Pro Ala Arg Pro
Arg Tyr Ala Cys Cys Pro Gly Trp Lys Arg Thr Ser 85 90 95 Gly Leu
Pro Gly Ala Cys Gly Ala Ala Ile Cys Gln Pro Pro Cys Arg 100 105 110
Asn Gly Gly Ser Cys Val Gln Pro Gly Arg Cys Arg Cys Pro Ala Gly 115
120 125 Trp Arg Gly Asp Thr Cys Gln Ser Asp Val Asp Glu Cys Ser Ala
Arg 130 135 140 Arg Gly Gly Cys Pro Gln Arg Cys Ile Asn Thr Ala Gly
Ser Tyr Trp 145 150 155 160 Cys Gln Cys Trp Glu Gly His Ser Leu Ser
Ala Asp Gly Thr Leu Cys 165 170 175 Val Pro Lys Gly Gly Pro Pro Arg
Val Ala Pro Asn Pro Thr Gly Val 180 185 190 Asp Ser Ala Met Lys Glu
Glu Val Gln Arg Leu Gln Ser Arg Val Asp 195 200 205 Leu Leu Glu Glu
Lys Leu Gln Leu Val Leu Ala Pro Leu His Ser Leu 210 215 220 Ala Ser
Gln Ala Leu Glu His Gly Leu Pro Asp Pro Gly Ser Leu Leu 225 230 235
240 Val His Ser Phe Gln Gln Leu Gly Arg Ile Asp Ser Leu Ser Glu Gln
245 250 255 Ile Ser Phe Leu Glu Glu Gln Leu Gly Ser Cys Ser Cys Lys
Lys Asp 260 265 270 Ser Gly 326PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 3Leu Thr Thr Cys Asp Gly His
Arg Ala Cys Ser Thr Tyr Arg Thr Ile 1 5 10 15 Tyr Arg Thr Ala Tyr
Arg Arg Ser Pro Gly 20 25 430PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 4Pro Ala Arg Pro Arg Tyr
Ala Cys Cys Pro Gly Trp Lys Arg Thr Ser 1 5 10 15 Gly Leu Pro Gly
Ala Cys Gly Ala Ala Ile Cys Gln Pro Pro 20 25 30 513PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 5Arg
Pro Arg Tyr Ala Cys Cys Pro Gly Trp Lys Arg Thr 1 5 10
613PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Leu Thr Thr Cys Asp Gly His Arg Ala Cys Ser Thr
Tyr 1 5 10 713PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 7Arg Ala Cys Ser Thr Tyr Arg Thr Ile Tyr
Arg Thr Ala 1 5 10 813PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 8Arg Thr Ala Tyr Arg Arg Ser
Pro Gly Val Thr Pro Ala 1 5 10 9108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
9Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn
Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Tyr Asn Ser Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 105 10112PRTHomo sapiens 10Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20
25 30 Gly Asp Ser Tyr Met Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro 35 40 45 Lys Leu Leu Ile Tyr Gly Ala Ser Asn Leu Glu Ser Gly
Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Asn Asn 85 90 95 Glu Asp Pro Tyr Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 11113PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
11Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Val Ile Ser Gly Asp Gly Gly Ser Thr Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Phe Asp
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser
12117PRTHomo sapiens 12Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly His Thr Phe Thr Thr Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr
His Ser Gly Val Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Leu Gly Ser Ser Ala Val Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110 Val Thr Val Ser Ser 115 1339DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 13gcc tat gca gat atc cag atg acc cag tcc ccg agc
tcc 39Ala Tyr Ala Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 1 5 10
1413PRTArtificial SequenceSynthetic Construct 14Ala Tyr Ala Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser 1 5 10 1510PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 15Asp
Ile Val Leu Thr Gln Ser Pro Ala Ser 1 5 10 1639DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 16gcctatcgag atatccagmt cacccagtcc ccgagctcc
391739DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 17aca aac gcg tac gct gag gtt cag ctg gtg
gag tct ggc 39Thr Asn Ala Tyr Ala Glu Val Gln Leu Val Glu Ser Gly 1
5 10 1813PRTArtificial SequenceSynthetic Construct 18Thr Asn Ala
Tyr Ala Glu Val Gln Leu Val Glu Ser Gly 1 5 10 198PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 19Gln
Ile Gln Leu Val Gln Ser Gly 1 5 2039DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 20acaaacgcgt acgctgagrt ccagctggtg gagtctggc
392136DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 21ggt aag ggc ctg gaa tgg gtt gca agg att
tat cct 36Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro 1 5 10
2212PRTArtificial SequenceSynthetic Construct 22Gly Lys Gly Leu Glu
Trp Val Ala Arg Ile Tyr Pro 1 5 10 2312PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 23Gly
Lys Gly Leu Lys Trp Met Gly Trp Ile Asn Thr 1 5 10
2436DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 24ggtaagggcc tggaatggrt ggcaaggatt tatcct
3625102DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 25gtc aag ggc cgt ttc act ata agc cgc gac
aac tcc aaa aac aca ctg 48Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu 1 5 10 15 tac cta caa atg aac agc gag gac
act gcc gtc tat tat tgt agc cgc 96Tyr Leu Gln Met Asn Ser Glu Asp
Thr Ala Val Tyr Tyr Cys Ser Arg 20 25 30 tgg gga 102Trp Gly
2634PRTArtificial SequenceSynthetic Construct 26Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 1 5 10 15 Tyr Leu Gln
Met Asn Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg 20 25 30 Trp
Gly 2734PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 27Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr
Ser Ala Ser Thr Ala 1 5 10 15 His Leu Gln Ile Asn Asn Glu Asp Thr
Ala Thr Tyr Phe Cys Ala Arg 20 25 30 Leu Gly 28102DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
28gtcaagggcc gtttcactwt cagcckcgac amctccrmaa rcacarygta cctacaaatg
60aacagcgagg acactgccgt ctattwctgt gcgcgtctgg gt
1022913PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 29Arg Ser Pro Gly Leu Ala Pro Ala Arg Pro Arg Tyr
Ala 1 5 10 3013PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 30Gly Trp Lys Arg Thr Ser Gly Leu Pro
Gly Ala Cys Gly 1 5 10 3115PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 31Lys Ala Ser Gln Ser Val Asp
Tyr Asp Gly Asp Ser Tyr Met Ser 1 5 10 15 327PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 32Gly
Ala Ser Asn Leu Glu Ser 1 5 339PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 33Gln Gln Asn Asn Glu Asp Pro
Tyr Thr 1 5 3410PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 34Gly His Thr Phe Thr Thr Tyr Gly Met
Ser 1 5 10 3518PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 35Gly Trp Ile Asn Thr His Ser Gly Val
Pro Thr Tyr Ala Asp Asp Phe 1 5 10 15 Lys Gly 3610PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 36Ala
Arg Leu Gly Ser Ser Ala Val Asp Tyr 1 5 10 3715PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 37Lys
Arg Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Met Ser 1 5 10 15
3815PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 38Lys Ala Ser His Ser Val Asp Tyr Asp Gly Asp Ser
Tyr Met Ser 1 5 10 15 3915PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 39Lys Ala Ser Gln Ser Gly Asp
Tyr Asp Gly Asp Ser Tyr Met Ser 1 5 10 15 4015PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 40Lys
Ala Ser Gln Ser Val Asp Tyr Arg Gly Asp Ser Tyr Met Ser 1 5 10 15
4115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 41Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser
Tyr Val Ser 1 5 10 15 4215PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 42Lys Ala Ser Gln Ser Val Asp
Tyr Leu Gly Asp Ser Tyr Met Ser 1 5 10 15 4315PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 43Lys
Ala Ser Gln Ser Val Asp Tyr Trp Gly Asp Ser Tyr Met Ser 1 5 10 15
447PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 44Gly Ala Ser Tyr Leu Glu Ser 1 5
457PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 45Gly Ala Ser Asn Arg Glu Ser 1 5
467PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 46Gly Ala Ser Asn Tyr Glu Ser 1 5
477PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 47Gly Ala Ser Asn Leu Glu Gln 1 5
489PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 48Gln Gln Asn Asn Glu Asp Pro Phe Thr 1 5
4910PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 49Gly His Thr Gly Thr Thr Tyr Gly Met Ser 1 5 10
5010PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 50Gly His Thr Phe Thr Thr Tyr Gly Tyr Ser 1 5 10
5110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 51Gly His Thr Phe Asp Thr Tyr Gly Met Ser 1 5 10
5210PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 52Gly His Thr Phe Arg Thr Tyr Gly Met Ser 1 5 10
5310PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 53Gly Val Thr Phe Thr Thr Tyr Gly Met Ser 1 5 10
5410PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 54Gly
His Arg Phe Thr Thr Tyr Gly Met Ser 1 5 10 5510PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 55Gly
His Thr Phe Gly Thr Tyr Gly Met Ser 1 5 10 5610PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 56Gly
His Thr Arg Thr Thr Tyr Gly Met Ser 1 5 10 5710PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 57Gly
His Thr Ser Thr Thr Tyr Gly Met Ser 1 5 10 5818PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 58Gly
Trp Ile Asn Trp His Ser Gly Val Pro Thr Tyr Ala Asp Asp Phe 1 5 10
15 Lys Gly 5918PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 59Gly Trp Ile Asn Met His Ser Gly Val
Pro Thr Tyr Ala Asp Asp Phe 1 5 10 15 Lys Gly 6018PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 60Gly
Trp Ile Asn Thr His Ser Gly Val Pro Thr Met Ala Asp Asp Phe 1 5 10
15 Lys Gly 6118PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 61Gly Trp Ile Asn Thr His Ser Gly Val
Pro Thr Tyr Ala His Asp Phe 1 5 10 15 Lys Gly 6218PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 62Gly
Trp Ile Asn Thr His Ser Gly Val Pro Thr Tyr Ala Asp Xaa Phe 1 5 10
15 Lys Gly 6318PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 63Gly Trp Ile Asn Ile His Ser Gly Val
Pro Thr Tyr Ala Asp Asp Phe 1 5 10 15 Lys Gly 6418PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 64Gly
Trp Ile Asn Trp His Ser Gly Val Pro Thr Tyr Ala Asp Asp Phe 1 5 10
15 Lys Gly 6518PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 65Gly Trp Ile Asn Thr Arg Ser Gly Val
Pro Thr Tyr Ala Asp Asp Phe 1 5 10 15 Lys Gly 6618PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 66Gly
Trp Ile Asn Thr His Ser Gly Val Pro Thr Ile Ala Asp Asp Phe 1 5 10
15 Lys Gly 6718PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 67Gly Trp Ile Asn Thr His Ser Gly Val
Pro Thr Tyr Ala Asp Asp Phe 1 5 10 15 Ser Gly 6818PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 68Gly
Trp Ile Asn Thr His Ser Gly Val Pro Thr Thr Ala Asp Asp Phe 1 5 10
15 Lys Gly 6918PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 69Gly Trp Ile Asn Thr His Ser Gly Val
Pro Thr Tyr Ala Asp Thr Phe 1 5 10 15 Lys Gly 7018PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 70Gly
Trp Ile Asn Ile His Ser Gly Val Pro Thr Tyr Ala Asp Asp Phe 1 5 10
15 Lys Gly 7118PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 71Gly Trp Ile Asn Thr His Ser Gly Val
Pro Thr Tyr Ala Asp Met Phe 1 5 10 15 Lys Gly 7218PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 72Gly
Trp Ile Asn Thr His Ser Gly Val Pro Thr Tyr Ala Asp Asp Tyr 1 5 10
15 Lys Gly 7318PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 73Gly Trp Ile Asn Thr His Ser Gly Val
Pro Thr Tyr Ala Asp Asp Phe 1 5 10 15 Lys Arg 7410PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 74Ala
Asn Leu Gly Ser Ser Ala Val Asp Tyr 1 5 10 7510PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 75Ala
Arg Leu Gly Ser Cys Ala Val Asp Tyr 1 5 10 7610PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 76Ala
Arg Leu Gly Ser Tyr Ala Val Asp Tyr 1 5 10 7710PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 77Ala
Arg Leu Gly Ser Ser Ala Val Asp Ala 1 5 10 78112PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
78Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Asp Tyr
Ser 20 25 30 Gly Asp Ser Tyr Met Ser Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Gly Ala Ser Asn Leu Glu
Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90 95 Glu Glu Pro Tyr Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110
79112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 79Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala
Ser Gln Ser Val Asp Tyr Ser 20 25 30 Gly Asp Ser Tyr Met Ser Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr
Gly Ala Ser Asn Leu Glu Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90
95 Glu Glu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 110 80117PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 80Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly His Thr Phe Thr Thr Tyr 20 25 30 Gly Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp
Ile Asn Thr His Ser Gly Val Pro Thr Tyr Ala Asp Asp Phe 50 55 60
Lys Gly Arg Phe Thr Ile Ser Leu Asp Asn Ser Lys Asn Thr Ala Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Leu Gly Ser Ser Ala Val Asp Tyr Trp Gly
Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
81117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 81Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly His Thr Phe Thr Thr Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr
His Ser Gly Val Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Leu Asp Asn Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90
95 Ala Arg Leu Gly Ser Ser Ala Val Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110 Val Thr Val Ser Ser 115 82112PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
82Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Asp Tyr
Ser 20 25 30 Gly Asp Ser Tyr Met Ser Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Gly Ala Ser Tyr Arg Glu
Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90 95 Glu Glu Pro Tyr Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110
83112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 83Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala
Ser Gln Ser Val Asp Tyr Ser 20 25 30 Gly Asp Ser Tyr Met Ser Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr
Gly Ala Ser Tyr Arg Glu Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90
95 Glu Glu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 110 84117PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 84Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly His Thr Phe Thr Thr Tyr 20 25 30 Gly Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp
Ile Asn Thr His Ser Gly Val Pro Thr Tyr Ala Asp Asp Phe 50 55 60
Lys Gly Arg Phe Thr Ile Ser Leu Asp Asn Ser Lys Asn Thr Ala Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Leu Gly Ser Tyr Ala Val Asp Tyr Trp Gly
Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
85117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 85Glu Ile Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly His Thr Phe Thr Thr Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr
His Ser Gly Val Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Leu Asp Asn Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90
95 Ala Arg Leu Gly Ser Tyr Ala Val Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110 Val Thr Val Ser Ser 115 86113PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
86Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Leu Val His
Ile 20 25 30 Asn Gly Ile Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro
Gly Lys Ala 35 40 45 Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg
Phe Ser Gly Val Pro 50 55 60 Ser Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln Pro Glu Asp
Phe Ala Thr Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Val Pro Leu
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg
87123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 87Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Tyr Thr Phe Ile Asp Tyr 20 25 30 Tyr Met Asn Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Asp Ile Asn Leu
Asp Asn Gly Gly Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Glu Gly Val Tyr His Asp Tyr Asp Asp Tyr Ala Met Asp Tyr
100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
8813PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 88Glu Asp Phe Ala Thr Tyr Tyr Cys Ser Gln Ser Thr
His 1 5 10 8913PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 89Glu Asp Leu Gly Val Tyr Phe Cys Ser
Gln Ser Thr His 1 5 10 9039DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 90gaagacttcg
caacttatta ctgtagccag agcacccac 399139DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 91gaagacttcg caacttattw ctgtagccag agcacccac
399213PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 92Gly Lys Gly Leu Glu Trp Val Gly Asp Ile Asn Leu
Asp 1 5 10 9313PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 93Gly Lys Ser Leu Glu Trp Ile Gly Asp
Ile Asn Leu Asp 1 5 10 9439DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 94ggtaagggcc
tggaatgggt tggtgatatc aacctggat 399539DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 95ggtaagggcc tggaatggrt cggtgatatc aacctggat
399626PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 96Gln Lys Phe Lys Gly Arg Phe Thr Ile Ser Arg Asp
Thr Ser Lys Asn 1 5 10 15 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg
20 25 9726PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 97Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp
Lys Ser Ser Ser 1 5 10 15 Thr Ala Tyr Met Glu Leu His Ser Leu Thr
20 25 9878DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 98cagaaattca aaggtcgttt cactataagc
cgcgacacct ccaaaaacac actgtaccta 60caaatgaaca gcttaaga
789978DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 99cagaaattca aaggtcgtky cactmtcagc
sktgacamgt ccarsarcac asygtacmtg 60caaatgaaca gcttaaga
7810016PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 100Arg Thr Ser Gln Ser Leu Val His Ile Asn Gly
Ile Thr Tyr Leu His 1 5 10 15 1017PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 101Arg Val Ser Asn Arg Phe
Ser 1 5 1029PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 102Ser Gln Ser Thr His Val Pro Leu Thr 1
5 10310PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 103Gly Tyr Thr Phe Ile Asp Tyr Tyr Met Asn 1 5 10
10418PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 104Gly Asp Ile Asn Leu Asp Asn Gly Gly Thr His
Tyr Asn Gln Lys Phe 1 5 10 15 Lys Gly 10516PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 105Ala
Arg Glu Gly Val Tyr His Asp Tyr Asp Asp Tyr Ala Met Asp Tyr 1 5 10
15 10616PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 106Gln Thr Ser Gln Ser Leu Val His Ile Asn Gly
Ile Thr Tyr Leu His 1 5 10 15 10716PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 107Arg
Thr Ser Gln Ser Leu Val His Tyr Asn Gly Ile Thr Tyr Leu His 1 5 10
15 10816PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 108Arg Thr Ser Gln Ser Leu Val Pro Ile Asn Gly
Ile Thr Tyr Leu His 1 5 10 15 10916PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 109Ser
Thr Ser Gln Ser Leu Val His Ile Asn Gly Ile Thr Tyr Leu His 1 5 10
15 11016PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 110Arg Thr Ser Gln Ser Leu Val His Leu Asn Gly
Ile Thr Tyr Leu His 1 5 10 15 11116PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 111Leu
Thr Ser Gln Ser Leu Val His Ile Asn Gly Ile Thr Tyr Leu His 1 5 10
15 11216PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 112Arg Trp Ser Gln Ser Leu Val His Ile Asn Gly
Ile Thr Tyr Leu His 1 5 10 15 11316PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 113Arg
Pro Ser Gln Ser Leu Val His Ile Asn Gly Ile Thr Tyr Leu His 1 5 10
15 11416PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 114Arg Thr His Gln Ser Leu Val His Ile Asn Gly
Ile Thr Tyr Leu His 1 5 10 15 11516PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 115Arg
Thr Ser Gln Ser Val Val His Ile Asn Gly Ile Thr Tyr Leu His 1 5 10
15 11616PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 116Arg Thr Ser Gln Ser Leu Val His Thr Asn Gly
Ile Thr Tyr Leu His 1 5 10 15 11716PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 117Arg
Thr Ser Gln Ser Leu Val His Leu Asn Gly Ile Thr Tyr Leu His 1 5 10
15 11816PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 118Arg Thr Ser Gln Ser Leu Val His Pro Asn Gly
Ile Thr Tyr Leu His 1 5 10 15 11916PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 119Arg
Thr Ser Gln Ser Leu Val His Ile Asn Gly Ile Thr Tyr Leu Gly 1 5 10
15 12016PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 120Thr Thr Ser Gln Ser Leu Val His Ile Asn Gly
Ile Thr Tyr Leu His 1 5 10 15 12116PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 121Leu
Thr Ser Gln Ser Leu Val His Ile Asn Gly Ile Thr Tyr Leu His 1 5 10
15 12216PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 122Arg Thr Ser Asp Ser Leu Val His Ile Asn Gly
Ile Thr Tyr Leu His 1 5 10 15 12316PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 123Arg
Thr Ser Gln Gly Leu Val His Ile Asn Gly Ile Thr Tyr Leu His 1 5 10
15 12416PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 124Arg Thr Ser Gln Ser Leu Val His Tyr Asn Gly
Ile Thr Tyr Leu His 1 5 10 15 1257PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 125Arg Val Ser Asn Asp Phe
Ser 1 5 1267PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 126Arg Val Ser Asn Arg Ile Ser 1 5
1277PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 127Arg Val Ser Asn Arg Thr Ser 1 5
1287PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 128Arg Val Ser Asn Arg Gly Ser 1 5
1297PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 129Arg Val Ser Asn Arg Ala Ser 1 5
1309PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 130Ala Gln Ser Thr His Val Pro Leu Thr 1 5
1319PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 131Gly Gln Ser Thr His Val Pro Leu Thr 1 5
1329PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 132Leu Gln Ser Thr His Val Pro Leu Thr 1 5
1339PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 133Ser Gln Ser Cys His Val Pro Leu Thr 1 5
1349PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 134Ser Gln Ser Thr Phe Val Pro Leu Thr 1 5
1359PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 135Ala Gln Ser Thr His Val Pro Leu Thr 1 5
1369PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 136Gly Gln Ser Thr His Val Pro Leu Thr 1 5
1379PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 137Leu Gln Ser Thr His Val Pro Leu Thr 1 5
1389PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 138Ala Gln Ser Thr His Val Pro Leu Thr 1 5
1399PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 139Asn Gln Ser Thr His Val Pro Leu Thr 1 5
1409PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 140Ala Gln Ser Thr His Val Pro Leu Thr 1 5
1419PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 141Ile Gln Ser Thr His Val Pro Leu Thr 1 5
1429PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 142Leu Gln Ser Thr His Val Pro Leu Thr 1 5
1439PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 143Val Gln Ser Thr His Val Pro Leu Thr 1 5
1449PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 144Thr Gln Ser Thr His Val Pro Leu Thr 1 5
1459PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 145Lys Gln Ser Thr His Val Pro Leu Thr 1 5
14610PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 146Gly Tyr Thr Val Ile Asp Tyr Tyr Met Asn 1 5 10
14710PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 147Gly Tyr Thr Phe Ile Asp Tyr Tyr Ile Asn 1 5 10
14810PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 148Gly Tyr Asn Phe Ile Asp Tyr Tyr Met Asn 1 5 10
14910PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 149Gly Tyr Thr Phe Met Asp Tyr Tyr Met Asn 1 5 10
15010PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 150Gly Tyr Thr Phe Arg Asp Tyr Tyr Met Asn 1 5 10
15110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 151Gly Tyr Thr Phe Ser Asp Tyr Tyr Met Asn 1 5 10
15210PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 152Gly Tyr Thr Phe Ile Asp Gln Tyr Met Asn 1 5 10
15310PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 153Gly Tyr Thr Phe Ile Asp Lys Tyr Met Asn 1 5 10
15418PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 154Gly Asp Ile Asn Leu Asp Gly Gly Gly Thr His
Tyr Asn Gln Lys Phe 1 5 10 15 Lys Gly 15518PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 155Gly
Asp Ile Asn Leu Asp Asn Gly Lys Thr His Tyr Asn Gln Lys Phe 1 5 10
15 Lys Gly 15618PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 156Gly Asp Ile Asn Leu Leu Asn Gly Gly
Thr His Tyr Asn Gln Lys Phe 1 5 10 15 Lys Gly 15718PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 157Gly
Asp Ile Asn Leu Asp Asn Gly Arg Thr His Tyr Asn Gln Lys Phe 1 5 10
15 Lys Gly 15818PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 158Gly Asp Ile Asn Leu Asp Asn Gly Ile
Thr His Tyr Asn Gln Lys Phe 1 5 10 15 Lys Gly 15918PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 159Gly
Asp Ile Asn Leu Asp Asn Gly Gly Gly His Tyr Asn Gln Lys Phe 1 5 10
15 Lys Gly 16018PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 160Gly Asp Ile Asn Leu Asp Asn Gly Gly
Thr His Tyr Ser Gln Lys Phe 1 5 10 15 Lys Gly 16118PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 161Gly
Asp Ile Asn Leu Asp Asn Gly Gly Thr His Tyr Asn Asn Lys Phe 1 5 10
15 Lys Gly 16218PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 162Gly Asp Ile Asn Leu Asp Asn Gly Gly
Thr His Tyr Asn Gln Lys Gln 1 5 10 15 Lys Gly 16318PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 163Gly
Asp Ile Asn Leu Asp Asn Gly Gly Thr His Tyr Asn Gln Lys Phe 1 5 10
15 Thr Gly 16418PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 164Gly Asp Ile Asn Leu Asp Asn Gly Gly
Thr His Tyr Asn Gln Lys Phe 1 5 10 15 Lys His 16518PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 165Gly
Asp Ile Asn Leu Asp Asn Gly Gly Thr His Tyr Asn Gln Lys Phe 1 5 10
15 Lys Ser 16618PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 166Gly Asp Ile Asn Ala Asp Asn Gly Gly
Thr His Tyr Asn Gln Lys Phe 1 5 10 15 Lys Gly 16718PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 167Gly
Asp Ile Asn Leu Asp Asn Gly Thr Thr His Tyr Asn Gln Lys Phe 1 5 10
15 Lys Gly 16818PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 168Gly Asp Ile Asn Leu Asp Asn Gly Gly
Thr His Tyr Asn Ala Lys Phe 1 5 10 15 Lys Gly 16918PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 169Gly
Asp Ile Asn Leu Asp Asn Gly Gly Thr His Tyr Asn Asn Lys Phe 1 5 10
15 Lys Gly 17018PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 170Gly Asp Ile Asn Leu Asp Asn Gly Gly
Thr His Tyr Asn Gln Val Phe 1 5 10 15 Lys Gly 17118PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 171Gly
Asp Ile Asn Leu Asp Asn Gly Gly Thr His Tyr Asn Gln Lys Phe 1 5 10
15 Lys Arg 17218PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 172Gly Asp Ile Asn Leu Asp Arg Gly Gly
Thr His Tyr Asn Gln Lys Phe 1 5 10 15 Lys Gly 17318PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 173Gly
Asp Ile Asn Asn Asp Asn Gly Gly Thr His Tyr Asn Gln Lys Phe 1 5 10
15 Lys Gly 17418PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 174Gly Asp Ile Asn Pro Asp Asn Gly Gly
Thr His Tyr Asn Gln Lys Phe 1 5 10 15 Lys Gly 17518PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 175Gly
Asp Ile Asn Leu Arg Asn Gly Gly Thr His Tyr Asn Gln Lys Phe 1 5 10
15 Lys Gly 17618PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 176Gly Asp Ile Asn Leu Asp Tyr Gly Gly
Thr His Tyr Asn Gln Lys Phe 1 5 10 15 Lys Gly 17718PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 177Gly
Asp Ile Asn Leu Asp Ser Gly Gly Thr His Tyr Asn Gln Lys Phe 1 5 10
15 Lys Gly 17818PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 178Gly Asp Ile Asn Leu Asp Arg Gly Gly
Thr His Tyr Asn Gln Lys Phe 1 5 10 15 Lys Gly 17918PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 179Gly
Asp Ile Asn Leu Asp Lys Gly Gly Thr His Tyr Asn Gln Lys Phe 1 5 10
15 Lys Gly 18018PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 180Gly Asp Ile Asn Leu Asp Asn Gly Val
Thr His Tyr Asn Gln Lys Phe 1 5 10 15 Lys Gly 18118PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 181Gly
Asp Ile Asn Leu Asp Asn Gly Ser Thr His Tyr Asn Gln Lys Phe 1 5 10
15 Lys Gly 18218PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 182Gly Asp Ile Asn Leu Asp Asn Gly Gly
Arg His Tyr Asn Gln Lys Phe 1 5 10 15 Lys Gly 18318PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 183Gly
Asp Ile Asn Leu Asp Asn Gly Gly Thr His Val Asn Gln Lys Phe 1 5 10
15 Lys Gly 18418PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 184Gly Asp Ile Asn Leu Asp Asn Gly Gly
Thr His Ile Asn Gln Lys Phe 1 5 10 15 Lys Gly 18518PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 185Gly
Asp Ile Asn Leu Asp Asn Gly Gly Thr His Leu Asn Gln Lys Phe 1 5 10
15 Lys Gly 18618PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 186Gly Asp Ile Asn Leu Asp Asn Gly Gly
Thr His Tyr Asn Gln Lys Phe 1 5 10 15 Lys Arg 18718PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 187Gly
Asp Ile Asn Leu Asp Asn Gly Gly Thr His Tyr Asn Gln Lys Phe 1 5 10
15 Lys Ser 18816PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 188Asn Arg Glu Gly Val Tyr His Asp Tyr
Asp Asp Tyr Ala Met Asp Tyr 1 5 10 15 18916PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 189Thr
Arg Glu Gly Val Tyr His Asp Tyr Asp Asp Tyr Ala Met Asp Tyr 1 5 10
15 19016PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 190Ala Arg Glu Gly Val Tyr His Pro Tyr Asp Asp
Tyr Ala Met Asp Tyr 1 5 10 15 19116PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 191Ala
Arg Glu Gly Val Tyr His Pro Tyr Asp Asp Tyr Ala Met Asp Tyr 1 5 10
15 19216PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 192Ala Arg Glu Gly Val Tyr His Asp Tyr Asp Asp
Tyr Ala Trp Asp Tyr 1 5 10 15 193113PRTHomo sapiens 193Asp Ile Gln
Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Thr Ser Gln Ser Leu Val His Ile 20 25 30 Asn
Gly Ile Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala 35 40
45 Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile 65 70 75 80 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
Cys Gly Gln Ser 85 90 95 Thr His Val Pro Leu Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 110 Arg 194113PRTHomo sapiens
194Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Leu Val
His Ile 20 25 30 Asn Ala Ile Thr Tyr Leu His Trp Tyr Gln Gln Lys
Pro Gly Lys Ala 35 40 45 Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn
Arg Phe Ser Gly Val Pro 50 55 60 Ser Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln Pro Glu
Asp Phe Ala Thr Tyr Tyr Cys Gly Gln Ser 85 90 95 Thr His Val Pro
Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg
195123PRTHomo sapiens 195Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Tyr Thr Phe Ile Asp Tyr 20 25 30 Tyr Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Asp Ile Asn
Leu Asp Asn Gly Gly Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr Ala Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Glu Gly Val Tyr His Asp Tyr Asp Asp Tyr Ala Met Asp
Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
196123PRTHomo sapiens 196Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Tyr Thr Phe Ile Asp Tyr 20 25 30 Tyr Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Asp Ile Asn
Leu Asp Asn Ser Gly Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr Ala Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Glu Gly Val Tyr His Asp Tyr Asp Asp Tyr Ala Met Asp
Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
19725PRTHomo sapiens 197Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
20 25 19813PRTHomo sapiens 198Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 1 5 10 19930PRTHomo sapiens 199Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 30 20011PRTHomo
sapiens 200Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10
20123PRTHomo sapiens 201Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20
20215PRTHomo sapiens 202Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile Tyr 1 5 10 15 20332PRTHomo sapiens 203Gly Val Pro Ser
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 30
20411PRTHomo sapiens 204Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
1 5 10 20530PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 205Arg Phe Thr Ile Ser Xaa Asp Xaa
Ser Lys Asn Thr Xaa Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 30 20631PRTHomo sapiens
206Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln
1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
Ala 20 25 30 20732PRTHomo sapiens 207Arg Phe Thr Ile Ser Ala Asp
Thr Ser Lys Asn Thr Ala Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 20831PRTHomo
sapiens 208Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Ser 20 25 30 20932PRTHomo sapiens 209Arg Phe Thr Ile Ser
Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 1 5 10 15 Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg 20 25 30
21015PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 210Lys Xaa Ser Xaa Ser Xaa Asp Tyr Xaa Gly Asp
Ser Tyr Xaa Ser 1 5 10 15 2117PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 211Gly Ala Ser Xaa Xaa Glu
Xaa 1 5 2129PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 212Gln Gln Asn Asn Glu Xaa Pro Xaa Thr 1
5 21310PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 213Gly Xaa Xaa Xaa Xaa Thr Tyr Gly Xaa Ser 1 5 10
21418PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 214Gly Trp Ile Asn Xaa Xaa Ser Gly Val Pro Thr
Xaa Ala Xaa Xaa Xaa 1 5 10 15 Xaa Xaa 21510PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 215Ala
Xaa Leu Gly Ser Xaa Ala Val Asp Xaa 1 5 10 21625PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 216Glu
Xaa Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 21713PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 217Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Xaa 1 5 10
21832PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 218Arg Phe Thr Xaa Ser Xaa Asp Xaa Ser Xaa
Xaa Thr Xaa Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Xaa Cys Ala Arg 20 25 30 21911PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 219Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 22032PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
220Arg Phe Thr Ile Ser Xaa Asp Asn Ser Lys Asn Thr Xaa Tyr Leu Gln
1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
Ala Arg 20 25 30 22110PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 221Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys 1 5 10 22216PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 222Xaa Xaa Xaa Xaa Xaa Xaa
Val Xaa Xaa Xaa Xaa Ile Thr Tyr Leu Xaa 1 5 10 15 2237PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 223Arg
Val Ser Asn Xaa Xaa Ser 1 5 2249PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 224Xaa Gln Ser Xaa Xaa Val
Pro Leu Thr 1 5 22510PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 225Gly Tyr Xaa Xaa Xaa Asp
Xaa Tyr Xaa Asn 1 5 10 22618PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 226Gly Asp Ile Asn Xaa Xaa
Xaa Xaa Xaa Xaa His Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa
22716PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 227Xaa Arg Glu Gly Val Tyr His Xaa Tyr Asp Asp
Tyr Ala Xaa Asp Tyr 1 5 10 15 22813PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 228Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Xaa 1 5 10
22930PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 229Arg Xaa Thr Xaa Ser Xaa Asp Xaa Ser Xaa
Xaa Thr Xaa Tyr Xaa Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 20 25 30 23032PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
230Arg Phe Thr Ile Ser Arg Asp Xaa Ser Lys Asn Thr Xaa Tyr Leu Gln
1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
Ala Arg 20 25 30 23130PRTHomo sapiens 231Arg Phe Thr Ile Ser Ala
Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 30
2326DNAUnknownDescription of Unknown Eukaryotic sequence 232cncaat
62336DNAUnknownDescription of Unknown Eukaryotic sequence 233aataaa
623415PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 234Lys Ala Ser Gln Ser Val Asp Tyr Ser Gly Asp
Ser Tyr Met Ser 1 5 10 15 2357PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 235Gly Ala Ser Tyr Arg Glu
Ser 1 5 2369PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 236Gln Gln Asn Asn Glu Glu Pro Tyr Thr 1
5 23710PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 237Ala Arg Leu Gly Ser Tyr Ala Val Asp Tyr 1 5 10
23825PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 238Glu Ile Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
20 25 23913PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 239Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Met 1 5 10 24032PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 240Arg Phe Thr Ile Ser Leu Asp Asn Ser
Lys Ser Thr Ala Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Phe Cys Ala Arg 20 25 30 24116PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 241Arg
Thr Ser Gln Ser Leu Val His Ile Asn Ala Ile Thr Tyr Leu His 1 5 10
15 24218PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 242Gly Asp Ile Asn Leu Asp Asn Ser Gly Thr His
Tyr Asn Gln Lys Phe 1 5 10 15 Lys Gly 24316PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 243Arg
Thr Ser Gln Ser Leu Val His Ile Ser Gly Ile Thr Tyr Leu His 1 5 10
15 24416PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 244Arg Thr Ser Gln Ser Leu Val His Ile Gln Gly
Ile Thr Tyr Leu His 1 5 10 15 24516PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 245Arg
Thr Ser Gln Ser Leu Val His Ile Asn Ser Ile Thr Tyr Leu His 1 5 10
15 24616PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 246Arg Thr Ser Gln Ser Leu Val His Ile Asn Ala
Ile Thr Tyr Leu His 1 5 10 15 24718PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 247Gly
Asp Ile Asn Leu Asp Asn Ser Gly Thr His Tyr Asn Gln Lys Phe 1 5 10
15 Lys Gly
* * * * *