U.S. patent application number 11/866560 was filed with the patent office on 2008-09-18 for elisa for vegf.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Johnny Gutierrez, Kyu H. Hong, Yu-Ju G. Meng.
Application Number | 20080227119 11/866560 |
Document ID | / |
Family ID | 39301650 |
Filed Date | 2008-09-18 |
United States Patent
Application |
20080227119 |
Kind Code |
A1 |
Meng; Yu-Ju G. ; et
al. |
September 18, 2008 |
ELISA for VEGF
Abstract
The vascular endothelial growth factor (VEGF) activity in a
patient's bloodstream or other biological sample can serve as a
diagnostic and prognostic index for cancer, diabetes, heart
conditions, and other pathologies. Antibody-sandwich ELISA methods
and kits for VEGF as an antigen are provided to detect types of
VEGF levels in biological samples from animal models and human
patients and can be used as a diagnostic/prognostic index.
Inventors: |
Meng; Yu-Ju G.; (Albany,
CA) ; Hong; Kyu H.; (Mountain View, CA) ;
Gutierrez; Johnny; (Johnstown, CO) |
Correspondence
Address: |
GENENTECH, INC.
1 DNA WAY
SOUTH SAN FRANCISCO
CA
94080
US
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
39301650 |
Appl. No.: |
11/866560 |
Filed: |
October 3, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60828203 |
Oct 4, 2006 |
|
|
|
Current U.S.
Class: |
435/7.21 ;
435/336; 530/387.1; 530/391.3 |
Current CPC
Class: |
G01N 33/74 20130101;
G01N 33/57496 20130101; C07K 16/22 20130101; G01N 2333/475
20130101 |
Class at
Publication: |
435/7.21 ;
530/387.1; 530/391.3; 435/336 |
International
Class: |
G01N 33/53 20060101
G01N033/53; C07K 16/18 20060101 C07K016/18; C12N 5/06 20060101
C12N005/06 |
Claims
1. A method for detecting selective vascular endothelial growth
factor (VEGF) forms (VEGF.sub.110+) in a biological sample
comprising the steps of: (a) contacting and incubating the
biological sample with a capture reagent immobilized to a solid
support, wherein the capture reagent is a antibody that recognizes
same epitope as antibody 5C3 against human VEGF, said monoclonal
antibody binding specifically to residues greater than 110 of human
VEGF; (b) separating the biological sample from the immobilized
capture reagents; (c) contacting the immobilized capture
reagent-target molecule complex with a detectable antibody that
binds to the KDR and/or FLT1 receptor binding domains of VEGF or
that binds to an epitope in VEGF1-110; and (d) measuring the level
of VEGF.sub.110+ bound to the capture reagents using a detection
means for the detectable antibody.
2. The method of claim 1, wherein the biological sample is isolated
from a human subject.
3. The method of claim 2, wherein the human subject is a vascular,
diabetic, or cancer patient and the measuring step (d) further
comprises a comparison with a standard curve to determine the level
of VEGF compared to a normal individual.
4. The method of claim 1, wherein the biological sample is tumor
lysates, plasma, serum or urine.
5. The method of claim 1, wherein the capture reagent is 5C3
monoclonal antibody.
6. The method of claim 1, wherein the immobilized capture reagents
are coated on a microtiter plate.
7. The method of claim 1, wherein the detectable antibody is
directly detectable.
8. The method of claim 7, wherein the detectable antibody is
amplified by a fluorimetric reagent.
9. The method of claim 8, wherein the detectable antibody is
biotinylated and the detection means is avidin or
streptavidin-peroxidase and 3,3',5,5'-tetramethyl benzidine.
10. The method of claim 1, wherein the detectable antibody is a
monoclonal antibody.
11. The method of claim 10, wherein the detectable antibody is a
murine monoclonal antibody.
12. The method of claim 11, wherein the immobilized monoclonal
antibody is MAb 5C3 and the detectable antibody is MAb A4.6.1.
13. An immunoassay kit for detecting VEGF.sub.110+ in a biological
sample, the kit comprising: (a) as capture reagent, an antibody
against human VEGF, wherein the monoclonal antibody binds
specifically to the residues greater than 110 of human VEGF; and
(b) as detection reagent, a detectable antibody that binds to the
KDR and/or FLT1 receptor binding domains of VEGF or that binds to
an epitope in VEGF1-110.
14. The kit of claim 13, further comprising a solid support for the
capture reagents.
15. The kit of claim 14, wherein the capture reagents are
immobilized on the solid support.
16. The kit of claim 15, wherein the capture reagents are coated on
a microtiter plate.
17. The kit of claim 16, further comprising a detection means for
the detectable antibodies.
18. The kit of claim 17, wherein the detection means is
colorimetric.
19. The kit of claim 13, further comprising purified VEGF as an
antigen standard.
20. The kit of claim 13, wherein the capture reagent antibody is
murine monoclonal antibody MAb 5C3 and the detectable antibody is
MAb A4.6.1.
21. An antibody 5C3 obtainable from (or produced by) hybridoma
5C3.1.1 deposit number PTA-7737.
22. The antibody of claim 21 conjugated to a detectable label.
23. A hybridoma 5C3.1.1 deposited under ATCC deposit number
PTA-7737.
24. An antibody that does not bind VEGF 110 and which binds to the
same epitope as the monoclonal antibody produced by the hybridoma
cell line PTA-7737.
25. The monoclonal antibody of claim 24 which is conjugated to a
detectable label.
Description
RELATED APPLICATION
[0001] This application claims priority to and the benefit of U.S.
Provisional Application Ser. No. 60/828,203, filed Oct. 4, 2006
specification of which is incorporated herein in its entirety.
FIELD OF THE INVENTION
[0002] This invention relates to immunoassays for detecting certain
populations of VEGF that can be used as diagnostic and prognostic
methods for patients with cancer, cardiovascular, or other
pathologies.
BACKGROUND
[0003] It is now well established that angiogenesis is implicated
in the pathogenesis of a variety of disorders. These include solid
tumors, intra-ocular neovascular syndromes such as proliferative
retinopathies or age-related macular degeneration (AMD), 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. 2.sup.nd Edition (Marcel Dekker, NY, 1994), pp
1625-1710). In the case of solid tumors, the neovascularization
allows the tumor cells to acquire a growth advantage and
proliferative autonomy compared to the normal cells. Accordingly, a
correlation has been observed between density of microvessels in
tumor sections and patient survival in breast cancer as well as in
several other tumors (Weidner et al. N Engl J Med 324:1-6 (1991);
Horak et al. Lancet 340:1120-1124 (1992); and Macchiarini et al.
Lancet 340:145-146 (1992)).
[0004] The search for positive regulators of angiogenesis has
yielded many candidates, including, e.g., aFGF, bFGF, TGF-.alpha.,
TGF-.beta., HGF, TNF-.alpha., angiogenin, IL-8, etc. (Folkman et
al., supra, and Klagsbrun et al., supra). Some of the negative
regulators so far identified include thrombospondin (Good et al.
Proc. Natl. Acad. Sci. USA. 87:6624-6628 (1990)), the 16-kilodalton
N-terminal fragment of prolactin (Clapp et al. Endocrinology,
133:1292-1299 (1993)), angiostatin (O'Reilly et al. Cell 79:315-328
(1994)), and endostatin (O'Reilly et al. Cell 88:277-285
(1996)).
[0005] Work done over the last several years has established the
key role of vascular endothelial growth factor (VEGF) in the
regulation of normal and abnormal angiogenesis (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 (Ferrara et al., supra).
[0006] Furthermore, VEGF has been shown to be a key mediator of
neovascularization associated with tumors and intra-ocular
disorders (Ferrara et al., 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); and Dvorak et al. Am J.
Pathol. 146:1029-1039 (1995)). Also, the concentration of VEGF in
eye fluids is highly correlated to the presence of active
proliferation of blood vessels in patients with diabetic and other
ischemia-related retinopathies (Aiello et al. N. Engl. J. Med.
331:1480-1487 (1994)). Furthermore, studies have demonstrated the
localization of VEGF in choroidal neovascular membranes in patients
affected by acute macular degeneration (AMD) (Lopez et al. Invest.
Ophtalmo. Vis. Sci. 37:855-868 (1996)).
[0007] VEGF is produced by tissues and does not have to enter the
circulation to exert its biological effect, but rather acts locally
as a paracrine regulator. A recent study by Yang et al. J. Pharm.
Exp. Ther. 284:103 (1998) found the clearance of rhVEGF.sub.165
from the circulation to be very rapid, suggesting endogenous VEGF
in the circulation is most likely the result of continual synthesis
of VEGF. In addition, several studies have tried to correlate
levels of circulating VEGF with tumor burden and have suggested
VEGF levels as a potential prognostic marker (Ferrari and
Scagliotti Eur. J. Cancer 32A:2368 (1996); Gasparini et al. J.
Natl. Cancer Inst. 89:139 (1997); Kohn Cancer 80:2219 (1997);
Baccala et al. Urology 51:327 (1998); Fujisaki et al. Am. J.
Gastroenterol. 93:249 (1998)). Clearly the ability to accurately
measure VEGF will be important to understand its potential role(s)
in many biological processes, such as maintenance of vascular
patency, menstrual cycle, ischemia, diabetes, cancer, intraocular
disorders, etc.
[0008] The literature reports widely varying concentrations of
endogenous VEGF in normal and diseased patients, ranging from
undetectable to high levels. The ability to measure endogenous VEGF
levels depends on the availability of sensitive and specific
assays. Colorimetric, chemiluminescence, and fluorometric based
enzyme-linked immunosorbent assays (ELISAs) for VEGF have been
reported. Houck et al., supra, (1992); Yeo et al. Clin. Chem. 38:71
(1992); Kondo et al. Biochim. Biophys. Acta 1221:211 (1994); Baker
et al. Obstet. Gynecol. 86:815 (1995); Hanatani et al. Biosci.
Biotechnol. Biochem. 59:1958 (1995); Leith and Michelson Cell
Prolif. 28:415 (1995); Shifren et al. J. Clin. Endocrinol. Metab.
81:3112 (1996); Takano et al. Cancer Res. 56:2185 (1996); Toi et
al. Cancer 77:1101 (1996); Brekken et al. Cancer Res. 58:1952
(1998); Obermair et al. Br. J. Cancer 77:1870-1874 (1998); Webb et
al. Clin. Sci. 94:395-404 (1998).
[0009] For example, Houck et al., supra (1992) describe a
calorimetric ELISA that appears to have ng/ml sensitivity, which
may not be sensitive enough to detect endogenous VEGF levels. Yeo
et al., supra (1992) describe a two-site time-resolved
immunofluorometric assay, however, no VEGF was detected in normal
sera (Yeo et al. Cancer Res. 53:2912 (1993)). Baker et al., supra
(1995), using a modified version of this immunofluorometric assay,
reported detectable levels of VEGF in plasma from pregnant women,
with higher levels observed in women with preeclampsia. Similar
data in pregnant women were reported by Anthony et al. Ann. Clin.
Biochem. 34:276 (1997) using a radioimmunoassay. Hanatani et al.,
supra (1995) developed a chemiluminescent ELISA capable of
measuring circulating VEGF and report VEGF levels in sera from 30
normal individuals (male and female) from 8-36 pg/ml. Brekken et
al, supra (1998) described ELISA assays using antibodies having
binding preference to either the VEGF alone or the VEGF:Flk-1
complex.
[0010] An ELISA kit for VEGF detection is commercially available
from R&D Systems (Minneapolis, Minn.). The R&D VEGF ELISA
kit has been used in sandwich assays wherein a monoclonal antibody
is used to capture the target VEGF antigen and a polyclonal
antibody is used to detect the VEGF. Webb et al. supra (1998). See,
also, e.g., Obermair et al., supra (1998).
[0011] Keyt et al. J. Biol. Chem. 271:7788-7795 (1996); Keyt et al.
J. Biol. Chem. 271:5638 (1996); and Shifren et al., supra (1996)
also developed a calorimetric ELISA based on a dual monoclonal
antibody pair. Although this ELISA was able to detect elevated VEGF
levels in cancer patients, it lacked the sensitivity needed to
measure endogenous levels of VEGF in normal individuals. Rodriguez
et al. J. Immunol. Methods 219:45 (1998) described a two-site
fluorimetric VEGF ELISA that yields a sensitivity of 10 pg/ml VEGF
in neat plasma or serum. However, this fluorimetric assay detects
fully intact 165/165 and 165/110 species of VEGF (It has been
reported that VEGF 165/165 can be proteolytically cleaved into
three other forms: a 165/110 heterodimer, a 110/110 homodimer, and
a 55-amino-acid C-terminal fragment (Keyt et al. J. Biol. Chem.
271:7788-7795 (1996); Keck et al. Arch. Biochem. Biophys.
344:103-113 (1997)).).
[0012] Thus, there is a need to develop a diagnostic and prognostic
assay that detects higher measurable levels of VEGF in a biological
sample of an animal model or patient than existing ELISAs, and/or
can measure different isoforms of VEGF.
SUMMARY
[0013] Antibody-sandwich ELISA methods for VEGF as an antigen were
developed to detect VEGF forms in biological samples. The VEGF
ELISA provided herein is capable of detecting VEGF isoforms and
fragments of VEGF greater than 110 ("VEGF.sub.110+"). Kits thereof
are also provided.
[0014] For example, methods for detecting selective vascular
endothelial growth factor (VEGF) forms greater than 110 amino acids
(VEGF.sub.110+) in a biological sample comprise the steps of: (a)
contacting and incubating the biological sample with a capture
reagent immobilized to a solid support, wherein the capture reagent
is an antibody that recognizes same epitope as antibody 5C3 against
human VEGF, said monoclonal antibody binding specifically to
residues greater than 110 of human VEGF; (b) separating the
biological sample from the immobilized capture reagents; (c)
contacting the immobilized capture reagent-target molecule complex
with a detectable antibody that binds to the KDR and/or FLT1
receptor binding domains of VEGF; and (d) measuring the level of
VEGF.sub.110+ bound to the capture reagents using a detection means
for the detectable antibody. In certain embodiments, the detectable
antibody binds to an epitope in VEGF 1-110. In certain embodiments,
comparison ELISA can be performed to detect different types of
VEGF. In certain embodiments, the biological sample (e.g., tumor
samples or tumor lysates, plasma, serum, or urine, etc.) is
isolated from a human subject.
[0015] In one embodiment, the capture reagent is the 5C3 monoclonal
antibody. In one embodiment, the immobilized capture reagent is
coated on a microtiter plate. In certain embodiments, the
detectable antibody is a monoclonal antibody. In one embodiment,
the detectable antibody is a murine monoclonal antibody. In one
embodiment, the immobilized monoclonal antibody is MAb 5C3 and the
detectable antibody is MAb A4.6.1. In certain embodiments, the
detectable antibody is directly detectable. In one embodiment,
detectable antibody is amplified by a calorimetric reagent. In one
embodiment, the detectable antibody is biotinylated and the
detection means is avidin or streptavidin-peroxidase and
3,3',5,5'-tetramethyl benzidine.
[0016] In certain embodiments of the invention, the human subject
is a vascular, diabetic, or cancer patient and the measuring step
(d) further comprises a comparison with a standard curve to
determine the level of VEGF compared to a normal individual.
[0017] Kits are also provided. For example, an immunoassay kit for
detecting vascular endothelial growth factor (VEGF) forms greater
than 110 amino acids (VEGF.sub.110+) in a biological sample can
comprise: (a) as capture reagent, an antibody against human VEGF,
wherein the monoclonal antibody binds specifically to the residues
greater than 110 of human VEGF; and (b) as detection reagent, a
detectable antibody that binds to the KDR and/or FLT1 receptor
binding domains of VEGF. In certain embodiments, the detectable
antibody binds to an epitope in VEGF 1-110. In certain embodiments,
the kit, further comprises a solid support for the capture
reagents. For example, the capture reagents can be immobilized on
the solid support (e.g., a microtiter plate). In certain
embodiments, the kit further comprises a detection means (e.g.,
colormetric means, fluorimetric means, etc.) for the detectable
antibodies. In certain embodiments, the kit further comprises
purified VEGF as an antigen standard. In certain embodiments of the
invention, an additional VEGF ELISA or more can be provided for
comparison studies with the VEGF.sub.110+ ELISA. In one embodiment,
the kit includes a capture reagent monoclonal antibody, which is
murine monoclonal antibody MAb 5C3, and a detectable antibody,
which is MAb A4.6. 1.
[0018] In yet another embodiment, the invention provides an
anti-VEGF antibody 5C3 (obtainable from or produced by hybridoma
deposited under ATCC number PTA-7737). The invention also provides
an antibody that does not bind VEGF 1-110 and binds to the same
VEGF.sub.110+ epitope as the monoclonal antibody produced by
hybridoma cell line PTA-7737. In certain embodiments, an antibody
of the invention is conjugated to a detectable label. In one
embodiment, the hybridoma 5C3.1.1 deposited under ATCC deposit
number PTA-7737 is provided.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1, Panels A, B and C illustrates the detection of
recombinant VEGF165, VEGF121(1) (truncated, likely missing
approximately 9 amino acids from the carboxy-terminus according to
the manufacturer, R&D systems), VEGF121 (2) (from Pepro Tech),
VEGF110 (N-terminal fragment generated by plasmin digestion of
VEGF) and VEGF8-109 (artificial VEGF with amino acids 8-109 of
VEGF165) molecules by different VEGF ELISAs. (A). ELISA A using
3.5F8 for coat and biotinylated A4.6.1 for detection. (B). ELISA B
using A4.6.1 for coat and biotinylate 2E3 for detection. (C). ELISA
C using 5C3 for coat and biotinylated A4.6.1 for detection.
[0020] FIG. 2 illustrates protein blotting of VEGF produced by A673
cells, using 3.5F8 (left) or A4.6.1 (right) for probing. Samples
are VEGF purified from conditioned medium of A673 cells using an
A4.6.1 Affinity column (lane 1) and recombinant VEGF proteins
VEGF.sub.165, VEGF.sub.121 (likely missing approximately 9 amino
acids from the carboxy-terminus according to the manufacturer,
R&D systems) and VEGF.sub.8-109 produced by E. coli (lanes 2, 3
and 4, respectively).
[0021] FIG. 3 illustrates a diagram of VEGF.sub.165, VEGF.sub.121
and VEGF.sub.110 (N-terminal fragment generated by plasmin
digestion of VEGF) showing the proposed binding sites of the
antibodies used in the three VEGF ELISAs.
DETAILED DESCRIPTION
Definitions
[0022] Before describing the present invention in detail, it is to
be understood that this invention is not limited to particular
compositions or biological systems, which can, of course, vary. It
is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments only, and is not
intended to be limiting. As used in this specification and the
appended claims, the singular forms "a", "an" and "the" include
plural referents unless the content clearly dictates otherwise.
Thus, for example, reference to "a molecule" optionally includes a
combination of two or more such molecules, and the like.
[0023] The term "VEGF" as used herein refers to the 165-amino acid
vascular endothelial cell growth factor, and related 121-, 145-,
189-, and 206-amino acid vascular endothelial cell growth factors,
as described by Leung et al. Science 246:1306 (1989), Houck et al.
Mol. Endocrin. 5:1806 (1991), and Neufeld et al., supra, together
with the naturally occurring allelic and processed forms of those
growth factors. See also, e.g., FIG. 1A and B of U.S. Pat. No.
6,057,428. Active VEGF fragment can be released from ECM-bound VEGF
by plasmin cleavage, generating the first 110 amino acids (see,
e.g., Keyt B A, et al.,: The carboxyl-terminal domain (111-165) of
vascular endothelial growth factor is critical for its mitogenic
potency. J Biol Chem. 271: 7788-7795 (1996)). "VEGF.sub.110+" as
used herein refers to VEGF fragments that are greater than 110
amino acids (from the N-terminal), but do not include the first 110
amino acids or smaller fragments (e.g., VEGF.sub.8-109).
[0024] The term "detecting" is used in the broadest sense to
include both qualitative and quantitative measurements of a target
molecule. In one aspect, the detecting method as described herein
is used to identify the mere presence of VEGF.sub.110+ or VEGF in a
biological sample. In another aspect, the method is used to test
whether VEGF.sub.110+ or VEGF in a sample is at a detectable level.
In yet another aspect, the method can be used to quantify the
amount of VEGF.sub.110+ or VEGF in a sample and further to compare
the VEGF.sub.110+ or VEGF levels from different samples.
[0025] The term "biological sample" refers to a body sample from
any animal, but preferably is from a mammal, more preferably from a
human. In certain embodiments, such biological sample is from a
vascular, diabetic, or cancer patient. Such samples include
biological fluids such as serum, plasma, vitreous fluid, lymph
fluid, synovial fluid, follicular fluid, seminal fluid, amniotic
fluid, milk, whole blood, urine, cerebro-spinal fluid, saliva,
sputum, tears, perspiration, mucus, tumor lysates, and tissue
culture medium, as well as tissue extracts such as homogenized
tissue, tumor tissue, and cellular extracts. In certain
embodiments, the sample is a body sample from any animal, in one
embodiment it is from a mammal, in one embodiment from a human
subject. In one embodiment, such biological sample is from clinical
patients.
[0026] The term "detectable antibody" refers to an antibody that is
capable of being detected either directly through a label amplified
by a detection means, or indirectly through, e.g., another antibody
that is labeled. For direct labeling, the antibody is typically
conjugated to a moiety that is detectable by some means. In one
embodiment, the detectable antibody is biotinylated antibody.
[0027] The term "detection means" refers to a moiety or technique
used to detect the presence of the detectable antibody in the ELISA
herein and includes detection agents that amplify the immobilized
label such as label captured onto a microtiter plate. In one
embodiment, the detection means is a colorimetric detection agent
such as avidin or streptavidin-HRP.
[0028] The term "capture reagent" refers to a reagent capable of
binding and capturing a target molecule in a sample such that under
suitable condition, the capture reagent-target molecule complex can
be separated from the rest of the sample. Typically, the capture
reagent is immobilized or immobilizable. In a sandwich immunoassay,
the capture reagent is preferably an antibody or a mixture of
different antibodies against a target antigen.
[0029] The term "antibody" herein is used in the broadest sense and
specifically covers intact monoclonal antibodies, polyclonal
antibodies, multispecific antibodies (e.g. bispecific antibodies)
formed from at least two intact antibodies, and antibody fragments
so long as they exhibit the desired biological activity.
[0030] "Antibody fragments" comprise a portion of an intact
antibody, preferably comprising the antigen-binding or variable
region thereof. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2, and Fv fragments; diabodies; linear antibodies;
single-chain antibody molecules; and multispecific antibodies
formed from antibody fragments.
[0031] For the purposes herein, an "intact antibody" is one
comprising heavy- and light-chain variable domains as well as an Fc
region.
[0032] "Native antibodies" are usually heterotetrameric
glycoproteins of about 150,000 daltons, composed of two identical
light (L) chains and two identical heavy (H) chains. Each light
chain is linked to a heavy chain by one covalent disulfide bond,
while the number of disulfide linkages varies among the heavy
chains of different immunoglobulin isotypes. Each heavy and light
chain also has regularly spaced intrachain disulfide bridges. Each
heavy chain has at one end a variable domain (V.sub.H) followed by
a number of constant domains. Each light chain has a variable
domain at one end (V.sub.L) and a constant domain at its other end;
the constant domain of the light chain is aligned with the first
constant domain of the heavy chain, and the light-chain variable
domain is aligned with the variable domain of the heavy chain.
Particular amino acid residues are believed to form an interface
between the light-chain and heavy-chain variable domains.
[0033] 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 naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody preparations that typically include different
antibodies directed against different determinants (epitopes), each
monoclonal antibody is directed against a single determinant on the
antigen. In addition to their specificity, the monoclonal
antibodies are advantageous in that they are synthesized by the
hybridoma culture, uncontaminated by other immunoglobulins. 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 the hybridoma method first described by Kohler et al.,
Nature, 256:495 (1975), or may be made by recombinant DNA methods
(see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies"
may also be isolated from phage antibody libraries using the
techniques described in Clackson et al. Nature 352:624-628 (1991)
and Marks et al., J. Mol. Biol., 222:581-597 (1991), for
example.
[0034] The monoclonal antibodies herein specifically include
"chimeric" antibodies (immunoglobulins) 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 (U.S. Pat. No. 4,816,567; Morrison et
al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric
antibodies of interest herein include "primatized" antibodies
comprising variable-domain antigen-binding sequences derived from a
non-human primate (e.g. Old World Monkey, such as baboon, rhesus or
cynomolgus monkey) and human constant-region sequences (U.S. Pat.
No. 5,693,780).
[0035] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or non-human primate having
the desired specificity, affinity, and capacity. In some instances,
framework region (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 are made further to refine antibody performance. In
general, the 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 also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see 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). In one
embodiment, a humanized 5C3 antibody is provided and used the
methods provided herein.
[0036] 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
hypervariable regions in both the light-chain and the heavy-chain
variable domains. The more highly conserved portions of variable
domains are called the framework regions (FRs). The variable
domains of native heavy and light chains each comprise four FRs,
largely adopting a sheet configuration, connected by three
hypervariable regions, which form loops connecting, and in some
cases forming part of, the .beta.-sheet structure. The
hypervariable regions in each chain are held together in close
proximity by the FRs and, with the hypervariable regions 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, 5.sup.th Ed. Public Health Service,
National Institutes 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
cytotoxicity (ADCC).
[0037] 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-binding sites
and is still capable of cross-linking antigen.
[0038] "Fv" is the minimum antibody fragment that contains a
complete antigen-recognition and antigen-binding site. This region
consists of a dimer of one heavy-chain and one light-chain variable
domain in tight, non-covalent association. It is in this
configuration that the three hypervariable regions of each variable
domain interact to define an antigen-binding site on the surface of
the V.sub.H-V.sub.L dimer. Collectively, the six hypervariable
regions confer antigen-binding specificity to the antibody.
However, even a single variable domain (or half of an Fv comprising
only three hypervariable regions specific for an antigen) has the
ability to recognize and bind antigen, although at a lower affinity
than the entire binding site.
[0039] 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 at least one free thiol
group. F(ab').sub.2 antibody fragments originally were produced as
pairs of Fab' fragments that have hinge cysteines between them.
Other chemical couplings of antibody fragments are also known.
[0040] 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.
[0041] Depending on the amino acid sequence of the constant domain
of their heavy chains, antibodies can be assigned to different
classes. There are five major classes of intact antibodies: IgA,
IgD, IgE, IgG, and IgM, and several of these may be further divided
into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and
IgA2. The heavy-chain constant domains that correspond to the
different classes of antibodies are called .alpha., .delta.,
.epsilon., .gamma. and .mu., respectively. The subunit structures
and three-dimensional configurations of different classes of
immunoglobulins are well known.
[0042] "Single-chain Fv" or "scFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. Preferably, the Fv
polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains that 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).
[0043] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody that are responsible for
antigen binding. The hypervariable region comprises amino acid
residues from a "complementarity-determining region" or "CDR" (e.g.
residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light-chain
variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the
heavy-chain variable domain; Kabat et al., Sequences of Proteins of
Immunological Interest, 5.sup.th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)) and/or those
residues from a "hypervariable loop" (e.g. residues 26-32 (L1),
50-52 (L2) and 91-96 (L3) in the light-chain variable domain and
26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy-chain variable
domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
"Framework" or "FR" residues are those variable domain residues
other than the hypervariable region residues as herein defined.
[0044] "Mammal" for purposes of treatment refers to any animal
classified as a mammal, including humans, domestic, and farm
animals, and zoo, sports, or pet animals, such as dogs, horses,
cats, sheep, pigs, cows, etc. Preferably, the mammal is human.
[0045] The terms "cancer", "cancerous", and "malignant" refer to or
describe the physiological condition in mammals that is typically
characterized by unregulated cell growth. Examples of cancer
include but are not limited to, carcinoma including adenocarcinoma,
lymphoma, blastoma, melanoma, sarcoma, and leukemia. More
particular examples of such cancers include squamous cell cancer,
lung cancer (including small-cell lung cancer, non-small cell lung
cancer, adenocarcinoma of the lung, and squamous carcinoma of the
lung), cancer of the peritoneum, hepatocellular cancer, gastric or
stomach cancer (including gastrointestinal cancer),
gastrointestinal stromal cancer, pancreatic cancer, glioblastoma,
cervical cancer, ovarian cancer, liver cancer (e.g., hepatic
carcinoma and hepatoma), bladder cancer, hepatoma, breast cancer,
colon cancer, colorectal cancer, rectal cancer, endometrial or
uterine carcinoma, salivary gland carcinoma, kidney or renal
cancer, liver cancer, prostate cancer, vulval cancer, thyroid
cancer, basal cell carcinoma, testicular cancer, esophageal cancer,
hepatic carcinoma, soft-tissue sarcoma, kaposi's sarcoma, carcinoid
carcinoma, mesothelioma, multiple myeloma, and various types of
head and neck cancer, as well as B-cell lymphoma (including low
grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic
(SL) NHL; intermediate grade/follicular NHL; intermediate grade
diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic
NHL; high grade small non-cleaved cell NHL; bulky disease NHL;
mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's
Macroglobulinemia); Hodgkin's lymphoma, chronic lymphocytic
leukemia (CLL), acute lymphoblastic leukemia (ALL), Hairy cell
leukemia; chronic myeloblastic leukemia, and post-transplant
lymphoproliferative disorder (PTLD), as well as abnormal vascular
proliferation associated with phakomatoses, edema (such as that
associated with brain tumors), and Meigs' syndrome.
[0046] The phrases "vascular" and "cardiovascular" are used
interchangeably and describe patients with indications that
stimulate angiogenesis and/or cardiovascularization, and those that
inhibit angiogenesis and/or cardiovascularization. Such disorders
include, for example, arterial disease, such as atherosclerosis,
hypertension, inflammatory vasculitis, Reynaud' s disease and
Reynaud's phenomenon, aneurysms, and arterial restenosis; venous
and lymphatic disorders such as thrombophlebitis, lymphangitis, and
lymphedema; and other vascular disorders such as peripheral
vascular disease, AMD, cancer such as vascular tumors, e.g.,
hemangioma (capillary and cavernous), glomus tumors,
telangiectasia, bacillary angiomatosis, hemangioendothelioma,
angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma,
and lymphangiosarcoma, tumor angiogenesis, trauma such as wounds,
burns, and other injured tissue, implant fixation, scarring,
ischemia reperfusion injury, rheumatoid arthritis, cerebrovascular
disease, renal diseases such as acute renal failure, and
osteoporosis. This would also include angina, myocardial
infarctions such as acute myocardial infarctions, cardiac
hypertrophy, and heart failure such as congestive heart failure
(CHF).
[0047] The term "diabetes" refers to a progressive disease of
carbohydrate metabolism involving inadequate production or
utilization of insulin and is characterized by hyperglycemia and
glycosuria. This term includes all forms of diabetes, such as type
I and type II diabetes and insulin-resistant diabetes, such as
Mendenhall's Syndrome, Werner Syndrome, leprechaunism, lipoatrophic
diabetes, and other lipoatrophies.
[0048] The term "affinity purified" refers to purifying a substance
by eluting it through an affinity chromatography column.
ELISA
[0049] Vascular endothelial growth factor (VEGF) is a homodimeric
glycoprotein and is a key angiogenic factor of blood vessel
formation during development and in pathological angiogenesis
associated with tumors. Expression of VEGF is potentiated in
response to hypoxia, and potentially other factors such as growth
factors, hormones and oncogenes. (see, e.g., Ferrara N: Vascular
endothelial growth factor: Basic science and clinical progress.
Endocrine Reviews 25: 581-611 (2004)). The human VEGF gene has
eight exons separated by introns. Alternative RNA splicing results
in the generation of at least four major isoforms having 121, 165,
189 and 206 amino acids in monomer (see, e.g., Houck K A, et al.,:
The vascular endothelial growth factor family: identification of a
fourth molecular species and characterization of alternative
splicing of RNA. Mol Endocrinol 5: 1806-1814 (1991); and, Tischer
E, et al.,: The human gene for vascular endothelial growth factor.
Multiple protein forms are encoded through alternative exon
splicing. J Biol Chem 266: 11947-11954 (1991)). Less frequent
isoforms including those having 145 (see, e.g., Poltorak Z., et
al.,: VEGF145, a secreted vascular endothelial growth factor
isoform that binds to extracellular matrix. J Biol Chem 272:
7151-7158 (1997)) and 183 (see, e.g., Jingjing L, et al.,: Human
Muller cells express VEGF183, a novel spliced variant of vascular
endothelial growth factor. Invest Ophthalmol Vis Sci 40:752-759
(1999)) amino acids in monomer have also been reported. All VEGF
isoforms bind two receptor tyrosine kinases, VEGFR-1 (see, e.g., De
Vries C, et al.,: The fms-like tyrosine kinase, a receptor for
vascular endothelial growth factor. Science 255:989-991 (1992)) and
VEGFR-2 (see, e.g., Terman B I, et al.,: Identification of a new
endothelial cell growth factor receptor tyrosine kinase. Oncogene
6:1677-1683 (1991)). VEGF.sub.165 also interacts with neuropilin
(see, e.g., Soker S. et al.,: Neuropilin-1 is expressed by
endothelial and tumor cells as an isoform-specific receptor for
vascular endothelial growth factor. Cell 92: 735-745 (1998)).
VEGF.sub.189 and VEGF.sub.206 bind to heparin with a high affinity
and are mostly sequestered in the extracellular matrix (ECM).
VEGF.sub.165 binds to heparin with intermediate affinity and is
partially soluble and partially bound to cell surface and ECM.
VEGF.sub.121 does not bind to heparin and is freely soluble.
VEGF.sub.121 and VEGF.sub.165 were found to be the most dominantly
expressed variants in breast and ovarian cancer tumor specimens and
cell lines by reverse transcription-PCR analysis, while
VEGF.sub.206 expression was not detected. VEGF.sub.183 and
VEG.sub.189 expression was found to be non-detectable or at low
levels in the cell lines and were detected in some of the tumor
specimens (see, e.g., Stimpfl M, et al.,: Vascular Endothelial
growth factor splice variants and their prognostic value in breast
and ovarian cancer. Clinical Cancer Research 8: 2253-2259
(2002)).
[0050] Active VEGF fragment can be released from ECM-bound VEGF by
plasmin cleavage, generating the first 110 amino acids (see, e.g.,
Keyt B A, et al., The carboxyl-terminal domain (111-165) of
vascular endothelial growth factor is critical for its mitogenic
potency. J Biol Chem. 271: 7788-7795 (1996)). This could be a
mechanism to locally regulate the bioavailability of VEGF during
physiological and pathological processes of angiogenesis. See,
e.g., Houck K A, et al. Dual regulation of vascular endothelial
growth factor bioavailability by genetic and proteolytic
mechanisms. J Biol Chem 1992; 267:26031-26037 (1992); Keyt B A, et
al. The carboxy-terminal domain (111-165) of vascular endothelial
growth factor is critical for its mitogenic potency. J Biol Chem.
271:7788-7795 (1996); and, Roth D, et al. Plasmin modulates
vascular endothelial growth factor-A-mediated angiogenesis during
wound repair. Am Pathology 168: 670-684. (10-12) (2006). However,
VEGF.sub.110 concentrations in biological samples have not been
reported. Active VEGF fragments may also be released from ECM-bound
VEGF by matrix metalloproteinase (MMP) cleavage. This is supported
by the finding of degraded VEGF fragments with amino acids
additional to 1-110 in ascites from ovarian cancer patients. Both
plasmin and MMP3 were detected in the ascites. See, e.g., Lee S,
Shahla M J, et al. Processing of VEGF-A by matrix
metalloproteinases regulates bioavailability and vascular
patterning in tumors. J Cell Biology 169:681-691 (2005).
[0051] Enzyme-linked immunosorbent assays (ELISAs) for various
antigens include those based on colorimetry, chemiluminescence, and
fluorometry. ELISAs have been successfully applied in the
determination of low amounts of drugs and other antigenic
components in plasma and urine samples, involve no extraction
steps, and are simple to carry out. The assay described herein is
an ELISA that utilizes antibodies as capture reagents and
detectable antibodies for VEGF and VEGF.sub.110+. In certain
embodiments, the ELISA is cell-based. In the first step of the
assay the biological sample suspected of containing VEGF or
containing VEGF.sub.110+ is contacted and incubated with the
capture (or coat) antibodies so that the capture antibodies capture
or bind to the VEGF or VEGF.sub.110+ so that it can be detected in
a detection step. The detection step involves use of the detectable
antibody, which, when contacted with any of the bound VEGF or
VEGF.sub.110+ binds to the protein of interest, if present, and a
detection means is used to detect the label on the antibody and
hence the presence or amount of VEGF or VEGF.sub.110+ present. This
ELISA can be compared with an ELISA that recognizes total VEGF
(e.g., U.S. Pat. No. 6,855,508; those described herein, and those
known in the art) or isoforms of VEGF to determine the type of VEGF
present.
[0052] For example, in certain embodiments, the assay utilizes the
following steps.
[0053] First Step
[0054] In the first step of the assay herein, the biological sample
is contacted and incubated with the immobilized capture (or coat)
reagent, which is an anti-VEGF monoclonal antibody. This antibody
may be from any species, but preferably the monoclonal antibody is
a murine or rat monoclonal antibody, more preferably murine, and
most preferably MAb 5C3 derived from the hybridoma identified
herein. Hence, in a specific preferred embodiment, the immobilized
monoclonal antibody is a murine monoclonal antibody, most
preferably MAb 5C3. Immobilization conventionally is accomplished
by insolubilizing the capture reagent either before the assay
procedure, as by adsorption to a water-insoluble matrix or surface
(U.S. Pat. No. 3,720,760) or non-covalent or covalent coupling (for
example, using glutaraldehyde or carbodiimide cross-linking, with
or without prior activation of the support with, e.g., nitric acid
and a reducing agent as described in U.S. Pat. No. 3,645,852 or in
Rotmans et al. J. Immunol. Methods 57:87-98 (1983)), or afterward,
e.g., by immunoprecipitation.
[0055] The solid phase used for immobilization may be any inert
support or carrier that is essentially water insoluble and useful
in immunometric assays, including supports in the form of, e.g.,
surfaces, particles, porous matrices, etc. Examples of commonly
used supports include small sheets, Sephadex, polyvinyl chloride,
plastic beads, and assay plates or test tubes manufactured from
polyethylene, polypropylene, polystyrene, and the like including
96-well microtiter plates, as well as particulate materials such as
filter paper, agarose, cross-linked dextran, and other
polysaccharides. Alternatively, reactive water-insoluble matrices
such as cyanogen bromide-activated carbohydrates and the reactive
substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016;
4,195,128; 4,247,642; 4,229,537; and 4,330,440 are suitably
employed for capture reagent immobilization. In one embodiment the
immobilized capture reagent is coated on a microtiter plate, and in
particular the preferred solid phase used is a multi-well
microtiter plate that can be used to analyze several samples at one
time, e.g., a microtest 96-well ELISA plate such as that sold as
Nune Maxisorb or Immulon. In certain embodiments, the plate is a
MICROTEST.TM. or MAXISORP.TM. 96-well ELISA plate such as that sold
as NUNC MAXISORB.TM. or IMMULON.TM..
[0056] The solid phase is coated with the capture reagent as
defined above, which may be linked by a non-covalent or covalent
interaction or physical linkage as desired. Techniques for
attachment include those described in U.S. Pat. No. 4,376,110 and
the references cited therein. If covalent, the plate or other solid
phase is incubated with a cross-linking agent together with the
capture reagent under conditions well known in the art, e.g., such
as for 1 hour at room temperature.
[0057] Commonly used cross-linking agents for attaching the capture
reagent to the solid phase substrate include, e.g.,
1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxy-succinimide esters, for example, esters with
4-azido-salicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis-(succinimidyl-propionate), and bifunctional
maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents
such as methyl-3-[(p-azidophenyl)-dithio]pro-pioimi-date yield
photoactivatable intermediates capable of forming cross-links in
the presence of light.
[0058] If 96-well plates are utilized, they are typically coated
with the capture reagent (typically diluted in a buffer such as
0.05 M sodium carbonate by incubation for at least about 10 hours,
more preferably at least overnight, at temperatures of about
4-20.degree. C., or about 4-8.degree. C., and at a pH of about
8-12, or about pH 9-10, or about pH 9.6). If shorter coating times
are desired, one can coat, e.g., 96-well plates at room temperature
for two hours. The plates may be stacked and coated long in advance
of the assay itself, and then the assay can be carried out
simultaneously on several samples in a manual, semi-automatic, or
automatic fashion, such as by using robotics.
[0059] The coated plates are then typically treated with a blocking
agent that binds non-specifically to and saturates the binding
sites to prevent unwanted binding of the free ligand to the excess
sites on the wells of the plate. Examples of appropriate blocking
agents for this purpose include, e.g., gelatin, bovine serum
albumin, egg albumin, casein, and non-fat milk. The blocking
treatment typically takes place under conditions of ambient
temperatures for about 1-4 hours, preferably about 1 to 3 hours, or
overnight at 0-4.degree. C.
[0060] After coating and blocking, the VEGF standard.(purified
VEGF) or the biological sample to be analyzed, appropriately
diluted, is added to the immobilized phase. The preferred dilution
rate is about 1-15%, preferably about 10%, by volume. Buffers that
may be used for dilution for this purpose include (a) PBS
containing 0.5% BSA, 0.05% TWEEN .sub.20.TM. detergent (P20), 0.05%
PROCLIN.TM. 300 antibiotic, 5 mM EDTA, 0.25% Chaps surfactant, 0.2%
beta-gamma globulin, and 0.35M NaCl, pH 7.4; (b) PBS containing
0.5% bovine serum albumin, 0.05% polysorbate 20, 5 mM EDTA, 0.25%
CHAPS, 0.2% bovine .gamma.-globulins, and 0.35 M NaCl; pH 7.4 (c)
PBS containing 0.5% BSA, 0.05% polysorbate 20 (P20), and 0.05%
PROCLIN.TM. 300, pH 7; (d) PBS containing0.5% BSA,0.05% P20, 0.05%
PROCLIN.TM. 300, 5 mMEDTA, and 0.35 M NaCl, pH 6.35; (e) PBS
containing 0.5% BSA, 0.05% P20, 0.05% PROCLIN.TM. 300, 5 mM EDTA,
0.2% beta-gamma globulin, and 0.35 M NaCl, pH 7.4; and (f) PBS
containing 0.5% BSA, 0.05% P20, 0.05% PROCLIN.TM. 300, 5 mM EDTA,
0.25% Chaps, and 0.35 M NaCl, pH 7.4. PROCLIN.TM. 300 acts as a
preservative, and TWEEN 20.TM. acts as a detergent to eliminate
non-specific binding.
[0061] While the concentration of the capture reagents will
generally be determined by the concentration range of interest of
the VEGF taking any necessary dilution of the biological sample
into account, the final concentration of the capture reagent will
normally be determined empirically to maximize the sensitivity of
the assay over the range of interest.
[0062] The conditions for incubation of sample and immobilized
capture reagent are selected to maximize sensitivity of the assay
and to minimize dissociation. Preferably, the incubation is
accomplished at fairly constant temperatures, ranging from about 0C
to about 40.degree. C., preferably from about 20 to 25 .degree. C.
The time for incubation depends primarily on the temperature, being
generally no greater than about 10 hours to avoid an insensitive
assay. Preferably, the incubation time is from about 0.5 to 3
hours, and more preferably 1.5-3 hours at room temperature to
maximize binding of free VEGF.sub.110+ or VEGF to capture reagents.
The duration of incubation may be longer if a protease inhibitor is
added to prevent proteases in the biological fluid from degrading
the VEGF.
[0063] At this stage, the pH of the incubation mixture will
ordinarily be in the range of about 4-9.5, preferably in the range
of about 6-9, more preferably about 7-8, and most preferably the pH
of the assay (ELISA) diluent is pH 7.4. The pH of the incubation
buffer is chosen to maintain a significant level of specific
binding of the capture reagent to the VEGF.sub.110+ or VEGF being
captured. Various buffers may be employed to achieve and maintain
the desired pH during this step, including borate, phosphate,
carbonate, Tris-HCl or Tris-phosphate, acetate, barbital, and the
like. The particular buffer employed is not critical to the
invention, but in individual assays one buffer may be preferred
over another.
[0064] Second Step
[0065] In the second step of the assay method herein, which is
optional, the biological sample is separated (preferably by
washing) from the immobilized capture reagent to remove uncaptured
molecules. The solution used for washing is generally a buffer
("washing buffer") with a pH determined using the considerations
and buffers described above for the incubation step, with a
preferable pH range of about 6-9. The washing may be done three or
more times. The temperature of washing is generally from
refrigerator to. moderate temperatures, with a constant temperature
maintained during the assay period, typically from about
0-40.degree. C., more preferably about 4-30.degree. C. For example,
the wash buffer can be placed in ice at 4.degree. C. in a reservoir
before the washing, and a plate washer can be utilized for this
step. A cross-linking agent or other suitable agent may also be
added at this stage to allow the now-bound VEGF.sub.110+ or VEGF to
be covalently attached to the capture reagent if there is any
concern that the captured VEGF.sub.110+ or VEGF may dissociate to
some extent in the subsequent steps.
[0066] Third Step
[0067] In the next step, the immobilized capture reagent is
contacted with detectable antibodies, preferably at a temperature
of about 20-40.degree. C., more preferably about 20-25.degree. C.,
with the exact temperature and time for contacting the two being
dependent primarily on the detection means employed. For example,
when strepatavidin-peroxidase and 3,3' ,5,5'-tetramethyl benzidine
are used as the means for detection, e.g., in one embodiment, the
contacting is carried out (e.g., about 1 hour or more) to amplify
the signal to the maximum. Preferably a molar excess of an antibody
with respect to the maximum concentration of free VEGF.sub.110+ or
VEGF expected (as described above) is added to the plate after it
is washed. This antibody is directly or indirectly detectable.
While the detectable antibody may be a polyclonal or monoclonal
antibody, e.g., in certain embodiments, it is a monoclonal
antibody, in one embodiment murine, and in one embodiment MAb A4.6.
1. Also, the detectable antibody can be directly detectable, and in
one embodiment has a colorimetric label, and in another embodiment
has a flurometric label. More preferably, the detectable antibody
is biotinylated and the detection means is avidin or streptavidin-
peroxidase and 3,3',5,5'-tetramethyl benzidine. The readout of the
detection means can be fluorimetric or calorimetric. The affinity
of the antibody must be sufficiently high that small amounts of the
free VEGF.sub.110+ or VEGF can be detected, but not so high that it
causes the VEGF.sub.110+ or VEGF to be pulled from the capture
reagents.
[0068] Fourth Step
[0069] In the last step of the assay method, the level of free VEGF
that is now bound to the capture reagent is measured using a
detection means for the detectable antibody. If the biological
sample is from a vascular, diabetic, or cancer patient, the
measuring step preferably comprises comparing the reaction that
occurs as a result of the above three steps with a standard curve
to determine the level of VEGF.sub.110+ or VEGF compared to a
normal individual, or preferably comprises comparing the reaction
that occurs as a result of the above three steps with an another
VEGF ELISA recognizing different isoforms or total VEGF to
determine the level of the types of VEGF when the ELISAs are
compared, and optionally compared to a normal individual.
[0070] Antibody Production
[0071] Polyclonal antibodies to the VEGF generally are raised in
animals by multiple subcutaneous (sc) or intraperitoneal (ip)
injections of the VEGF and an adjuvant. It may be useful to
conjugate the VEGF or a fragment containing the target amino acid
sequence to a protein that is immunogenic in the species to be
immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine
thyroglobulin, or soybean trypsin inhibitor using a bifunctional or
derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide
ester (conjugation through cysteine residues), N-hydroxysuccinimide
(through lysine residues), glutaraldehyde, succinic anhydride,
SOC12, or R1N .dbd.C.dbd.NR, where R and R1 are different alkyl
groups.
[0072] The antibodies used as the coat or detectable antibodies may
be obtained from any convenient vertebrate source, such as murine,
primate, lagomorpha, goat, rabbit, rat, chicken, bovine, ovine,
equine, canine, feline, or porcine. Chimeric or humanized
antibodies may also be employed, as described, e.g., in U.S. Pat.
No. 4,816,567; Morrison et al. Proc. Natl. Acad. Sci. USA 81:6851
(1984); Neuberger et al. Nature 312: 604 (1984); Takeda et al.
Nature 314:452 (1985); and WO 98/45331 published Oct. 15, 1998, as
well as in those additional references set forth above.
[0073] Animals may be immunized against the immunogenic conjugates
or derivatives by combining 1 mg or 1 .mu.g of conjugate (for
rabbits or mice, respectively) with 3 volumes of Freund's complete
adjuvant and injecting the solution intradermally at multiple
sites. One month later the animals are boosted with 1/5 to 1/10 the
original amount of conjugate in Freund's incomplete adjuvant by
subcutaneous injection at multiple sites. 7 to 14 days later
animals are bled and the serum is assayed for anti-VEGF titer.
Animals are boosted until the titer plateaus. Preferably, the
animal is boosted with the conjugate of VEGF, but conjugated to a
different protein and/or through a different cross-linking agent.
Conjugates also can be made in recombinant cell culture as protein
fusions. Also, aggregating agents such as alum are used to enhance
the immune response. Methods for the production of polyclonal
antibodies are described in numerous immunology textbooks, such as
Davis et al. Microbiology, 3.sup.rd Edition, (Harper & Row, New
York, N.Y., 1980).
[0074] Monoclonal antibodies are prepared by recovering spleen
cells from immunized animals and immortalizing the cells in
conventional fashion, e.g. by fusion with myeloma cells or by
Epstein-Barr virus transformation, and screening for clones
expressing the desired antibody. See, e.g., Kohler and Milstein
Eur. J. Immunol. 6:511 (1976). Monoclonal antibodies, or the
antigen-binding region of a monoclonal antibody, such as Fab or
(Fab).sub.2 fragments, may alternatively be produced by recombinant
methods.
[0075] Examples of suitable antibodies include those already
utilized in known RIAs for the protein in question, e.g., those
antibodies directed against VEGF as described in the references
given in the introduction herein.
[0076] In certain embodiments, an anti-VEGF antibody 5C3, which is
obtainable from or produced by hybridoma deposited under ATCC
number PTA-7737, is used, optionally with another anti-VEGF
antibody, A4.6. 1. The invention also provides an antibody that
does not bind VEGF 1-110 and binds to the same VEGF.sub.110+
epitope as the monoclonal antibody produced by hybridoma cell line
PTA-7737. A hybridoma 5C3.1.1 deposited under ATCC deposit number
PTA-7737 is provided.
[0077] Detection
[0078] The antibody added to the immobilized capture reagents will
be either directly labeled, or detected indirectly by addition,
after washing off of excess first antibody, of a molar excess of a
second, labeled antibody directed against IgG of the animal species
of the first antibody. In the latter, indirect assay, labeled
antisera against the first antibody are added to the sample so as
to produce the labeled antibody in situ.
[0079] The label used for either the first or second antibody is
any detectable functionality that does not interfere with the
binding of free VEGF.sub.110+ or VEGF to the antibody. Examples of
suitable labels are those numerous labels known for use in
immunoassay, including moieties that may be detected directly, such
as fluorochrome, chemiluminscent, 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, biotin/streptavidin,
biotin/Streptavidin-.beta.-galactosidase with MUG, spin labels,
bacteriophage labels, stable free radicals, and the like. As noted
above, the fluorimetric detection is one example.
[0080] Conventional methods are available to bind these labels
covalently to proteins or 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). In certain
embodiments, labels herein are fluorescent to increase
amplification and sensitivity to 8 pg/ml, more preferably biotin
with streptavidin-.beta.-galactosidase and MUG for amplifying the
signal. In certain embodiments, a calorimetric label is used, e.g.,
where the detectable antibody is biotinylated and the detection
means is avidin or streptavidin- peroxidase and
3,3',5,5'-tetramethyl benzidine.
[0081] 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.
[0082] Following the addition of last labeled antibody, the amount
of bound antibody is determined by removing excess unbound labeled
antibody through washing and then measuring the amount of the
attached label using a detection method appropriate to the label,
and correlating the measured amount with the amount of free
VEGF.sub.110+ or VEGF in the biological sample. For example, in the
case of enzymes, the amount of color developed and measured will be
a direct measurement of the amount of VEGF.sub.110+ or VEGF
present. Specifically, if HRP is the label, the color is detected
using the substrate 3,3',5,5'-tetramethyl benzidine at 450 nm
absorbance.
[0083] In one example, after an enzyme-labeled second antibody
directed against the first unlabeled antibody is washed from the
immobilized phase, color or chemiluminiscence is developed and
measured by incubating the immobilized capture reagent with a
substrate of the enzyme. Then the amount of free VEGF.sub.110+ or
VEGF concentration is calculated by comparing with the color or
chemiluminescence generated by the standard VEGF run in
parallel.
[0084] Kits
[0085] As a matter of convenience, the assay method of this
invention can be provided in the form of a kit. Such a kit is a
packaged combination including the basic elements of: [0086] (a)
capture reagent comprised of the monoclonal antibody against human
VEGF molecule, wherein the monoclonal antibody recognizes
VEGF.sub.110+; and [0087] (b) detection reagents comprised of
detectable (labeled or unlabeled) antibodies that bind to the KDR
and FLT1 receptor binding domains of VEGF. These basic elements are
defined hereinabove. In certain embodiment, the detection reagents
comprise a detectable antibody(ies) that bind to epitope of
VEGF1-110.
[0088] Preferably, the kit further comprises a solid support for
the capture reagents, which may be provided as a separate element
or on which the capture reagents are already immobilized. Hence,
the capture antibodies in the kit may be immobilized on a solid
support, or they may be immobilized on such support that is
included with the kit or provided separately from the kit.
[0089] Preferably, the capture reagents are coated on a microtiter
plate. The detection reagent may be labeled antibodies detected
directly or unlabeled antibodies that are detected by labeled
antibodies directed against the unlabeled antibodies raised in a
different species. Where the label is an enzyme, the kit will
ordinarily include substrates and cofactors required by the enzyme,
and where the label is a fluorophore, a dye precursor that provides
the detectable chromophore. Where the detection reagent is
unlabeled, the kit may further comprise a detection means for the
detectable antibodies, such as the labeled antibodies directed to
the unlabeled antibodies, preferably in a fluorimetric-detected
format. Where the label is an enzyme, the kit will ordinarily
include substrates and cofactors required by the enzyme, where the
label is a fluorophore, a dye precursor that provides the
detectable chromophore, and where the label is biotin, an avidin
such as avidin, streptavidin, or streptavidin conjugated to HRP or
.beta.-galactosidase with MUG.
[0090] In one specific embodiment, the capture reagent is
monoclonal antibody, preferably rodent, more preferably murine or
rat, still more preferably murine, and most preferably MAb 5C3.
Also in certain embodiments, the detectable antibody is a
biotinylated monoclonal antibody, the monoclonal antibody is
rodent, more preferably murine or rat, still more preferably
murine, yet still more preferably MAb A4.6. 1. In certain
embodiments, the capture reagent is immobilized in this kit.
[0091] In certain embodiments, the kit can contain multiple ELISA
for comparison studies as described herein for detecting various
forms of VEGF and VEGF.sub.110+.
[0092] The kit also typically contains instructions for carrying
out the assay, and/or VEGF as an antigen standard (e.g., purified
VEGF, preferably recombinantly produced VEGF, and VEGF110), as well
as other additives such as stabilizers, washing and incubation
buffers, and the like.
[0093] Examples of standards for VEGF are recombinant human VEGF
produced in mammalian cells available from Genentech, Inc., South
San Francisco, Calif., and from those companies and processes
described herein.
[0094] The components of the kit will be provided in predetermined
ratios, with the relative amounts of the various reagents suitably
varied to provide for concentrations in solution of the reagents
that substantially maximize the sensitivity of the assay.
Particularly, the reagents may be provided as dry powders, usually
lyophilized, including excipients, which on dissolution will
provide for a reagent solution having the appropriate concentration
for combining with the sample to be tested.
Deposit of Materials
[0095] The following material has been deposited with the American
Type Culture Collection, 10801 University Boulevard, Manassas, Va.
20110-2209, USA (ATCC): 5C3.1.1 was deposited with the ATCC under
accession number PTA-7737 deposited on Jul. 19, 2006.
TABLE-US-00001 Hybridoma ATCC Accession No. Deposit Date 5C3.1.1
PTA-7737 Jul. 19, 2006 A4.6.1 HB10709 Mar. 29, 1991
[0096] The deposit was made under the provisions of the Budapest
Treaty on the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure and the
Regulations thereunder (Budapest Treaty). This assures maintenance
of a viable culture of the deposit for 30 years from the date of
deposit. The deposits will be made available by ATCC under the
terms of the Budapest Treaty, and subject to an agreement between
Genentech, Inc. and ATCC, which assures permanent and unrestricted
availability of the progeny of the culture of the deposit to the
public upon issuance of the pertinent U.S. patent or upon laying
open to the public of any U.S. or foreign patent application,
whichever comes first, and assures availability of the progeny to
one determined by the U.S. Commissioner of Patents and Trademarks
to be entitled thereto according to 35 USC .sctn. 122 and the
Commissioner's rules pursuant thereto (including 37 CFR .sctn. 1.14
with particular reference to 886 OG 638).
[0097] The assignee of the present application has agreed that if a
culture of the materials on deposit should die or be lost or
destroyed when cultivated under suitable conditions, the materials
will be promptly replaced on notification with another of the same.
Availability of the deposited material is not to be construed as a
license to practice the invention in contravention of the rights
granted under the authority of any government in accordance with
its patent laws.
[0098] The specification is considered to be sufficient to enable
one skilled in the art to practice the invention. The invention is
not to be limited in scope by the construct deposited, since the
deposited embodiment is intended as a single illustration of
certain aspects of the invention and any constructs that are
functionally equivalent are within the scope of the invention. The
deposit of material herein does not constitute an admission that
the written description is inadequate to enable the practice of any
aspect of the invention, including the best more thereof, nor is it
to be construed as limiting the scope of the claims to the specific
illustrations that it represents. Indeed, various modifications of
the invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
[0099] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
EXAMPLES
Example 1
[0100] Vascular endothelial growth factor (VEGF), which is
expressed as different isoforms due to alternative RNA splicing, is
known to play a key role in tumor angiogenesis. We measured the
concentrations of VEGF.sub.165 and total VEGF and evaluated the
relative amount of VEGF.sub.110, which is an active fragment
generated by plasmin digestion of VEGF. ELISA A (VEGF165-206 ELISA)
detects VEGF.sub.165 and longer isoforms but not VEGF.sub.121.
ELISA B (VEGF110-206 ELISA) detects VEGF.sub.165 and isoforms,
VEGF.sub.121 and VEGF.sub.110. ELISA C (VEGF121-206 ELISA) detects
VEGF.sub.165 and longer isoforms, VEGF.sub.121 and VEGF fragments
with molecular weight larger than VEGF.sub.110 but not VEGF.sub.110
(referred to herein as "VEGF.sub.110+").
[0101] Materials and Methods
[0102] Reagents and cells: Recombinant VEGF.sub.165 (Genentech),
VEGF.sub.121 (PeproTech, Rocky Hill, N.J.), VEGF.sub.8-109
(consisting of amino acids 8-109 of VEGF.sub.165) and truncated
VEGF.sub.121 (R&D Systems, Minneapolis, Minn.) were produced in
E. coli. Truncated VEGF.sub.121 has an intact N-terminus by mass
spectrometry but has a mass of 26 KDa, consistent with truncation
of approximately nine amino acids from the carboxy-terminus
according to the manufacturer. It migrated between VEGF.sub.110 and
VEGF.sub.121 when analyzed by SDS-PAGE under reducing conditions.
VEGF.sub.110 was prepared by plasmin digestion of VEGF.sub.165
(Keyt B A, et al.,: The carboxyl-terminal domain (111-165) of
vascular endothelial growth factor is critical for its mitogenic
potency. J Biol Chem. 271: 7788-7795 (1996)). The molecular weight
measured by mass spectrometry was 25390, matching the theoretical
mass of 25389. The concentration was determined using bicinchorinic
acid method (Pierce, Rockford, Ill.). Molecular weights used for
concentration calculation of VEGF.sub.8-109, VEGF.sub.121 and
VEGF.sub.165 were 23.8, 28.9 and 38.2 KDa, respectively. Monoclonal
anti-VEGF antibodies A4.6.1, 3.5F8, 2E3 and 5C3 were generated by
immunizing mice with VEGF.sub.165 produced in CHO cells (Kim K J,
et al.,: The vascular endothelial growth factor proteins:
Identification of biologically relevant regions by neutralizing
monoclonal antibodies. Growth Factors 7: 53-64 (1992)). Breast cell
lines SK-BR-3, BT-474, T-47D and MCF-7 as well as ovarian cell
lines ES-2, OVCAR-3 and SK-OV-3 (American Type Culture Collection,
Rockville, Md.) were grown in RPMI, 2 mM L-glutamine and 10% FBS
(except 20% for OVC-AR-3) in a humidified 5% CO.sub.2 incubator at
37.degree. C.
[0103] Purification of VEGF in conditioned media of A673 cells:
A673 cells (American Type Culture Collection,) were grown in 50:50
F12/DMEM, 2 mM L-glutamine and 5% FBS to 60% confluency and then in
serum free medium (Genentech) till confluency. VEGF was purified
from the supernatants using an A4.6. 1-Sepharose column that was
prepared with CNBr activated Sepharose (Amersham Biosciences,
Piscataway, N.J.). The column eluate and recombinant VEGF controls
(0.2 .mu.g per lane) were run on a 18% Tris-Glycine gels
(Invitrogen, Carlsbad, Calif.) under reducing conditions and were
blotted unto nitrocellulose. The blot was blocked with 0.5 M
Tris-HCl, pH 7.5, 1.5 M NaCl, 50 mM EDTA, 0.5% Trition100
containing 3% bovine serum albumin and probed with 200 ng/ml of
3.5F8 or A4.6.1 followed by 2 ng/ml of goat anti-mouse Fc-HRP
(Jackson ImmunoResearch). Signals were developed using SuperSignal
West Dura (Pierce) and recorded on X-ray film.
[0104] VEGF ELISAs for measuring VEGF concentrations
[0105] ELISA A (VEGF165-206 ELISA). Unless otherwise mentioned, a
fluorometric ELISA A was used for measuring VEGF in samples. The
fluorimetric ELISA A used 3.5F8 for coat and biotinylated A4.6.1
followed by streptavidin-.beta. galactosidase for detection and
4-methylumbelliferyl-.beta.-D galactoside as the substrate
(Rodriguez C R, et al.,: A sensitive fluorometric enzyme-linked
immunosorbent assay that measures vascular endothelial growth
factor 165 in human plasma. J Immunol Methods 219: 45-55 (1998)).
The VEGF.sub.165 standards were 1-128 pg/mL, or 0.026-3.35 pM. The
colorimetric ELISA A used 3.5F8 for coat and biotinylated A4.6.1
for detection, following the protocol used for the ELISA C
described below. The VEGF.sub.165 standards were 1.6-200 pg/mL.
[0106] ELISA B (VEGF110-206 ELISA) (previously named VEGF121-206
ELISA, Konecny G E, et al.,: Association between HER-2/neu and
Vascular Endothelial Growth Factor Expression Predicts Clinical
Outcome in Primary Breast Cancer Patients. Clinical Cancer
Research, 10: 1706-1716 (2004)): MaxiSorp 96-well microwell plates
were coated with 0.5 .mu.g/ml antibody A4.6.1 in 50 mM carbonate
buffer, pH 9.6 at 100 .mu.l/well at 4.degree. C. overnight. Plates
were washed after this step and between the subsequent room
temperature incubation steps with PBS, pH 7.4, containing 0.05%
polysorbate 20. Plates were blocked with 0.5% bovine serum albumin,
10 ppm Proclin.TM. 300 (Supelco, Bellefonte, Pa.) in PBS (150
.mu.l/well) for 1 h. VEGF standards (1.56-200 pg/ml VEGF.sub.165 or
0.0409-5.24 pM VEGF in twofold serial dilution) and serially
diluted samples (minimum 1:10 dilution) in twofold or threefold
serial dilution in PBS, pH 7.4, containing 0.5% bovine serum
albumin, 0.05% polysorbate 20, 5 mM EDTA, 0.25% CHAPS, 0.2% bovine
.gamma.-globulins (Sigma, St. Louis, Mo.) and 0.35 M NaCl (sample
buffer) were added to the plates (100 .mu.l/well) and incubated for
2 h. Bound VEGF was detected by incubating biotinylated 2E3 (or
another antibody that binds to a receptor binding domain of VEGF)
on the plates for 1 h followed by streptavidin-HRP (Amersham,
Copenhagen, Denmark) for 30 min, biotinyl-tyramide (ELAST ELISA
amplification System, Perkin Elmer Life Sciences Inc., MA) for 15
min and streptavidin-HRP for 30 min. The substrate TMB
(3,3',5,5'-tetramethyl benzidine) (Kirkegaard & Perry
Laboratories) was added and the reaction was stopped by adding 1 M
phosphoric acid. Absorbance was read at 450 nm on a Titertek
stacker reader (ICN, Costa Mesa, Calif.). The titration curves were
fit using a four-parameter regression curve-fitting program
(KaleidaGraph, Synergy software, Reading, Pa.). Data points which
fell in the range of the standard curve were used for calculating
the putative VEGF concentrations in the samples. The recovery of
1.56-200 pg/ml VEGF.sub.165 in 10% human EDTA plasma (Golden West
Biologicals Inc., Temecula, Calif.) was 92-120% after subtracting
the putative 2.1 pg/ml endogenous VEGF in the 10% plasma used for
this study.
[0107] ELISA C (VEGF121-206 ELISA): Microwell plates were coated
with 1 .mu.g/ml anti-VEGF 5C3 antibody and blocked as described
above. VEGF standards (4.00-512 pg/ml VEGF.sub.165 or 0.105-13.4 pM
VEGF in 2-fold serial dilution) and serially diluted samples in
sample buffer were added to the plates. The plates were incubated
for 2 h. Bound VEGF was detected by adding biotinylated A4.6.1
followed by streptavidin-HRP and TMB as the substrate. Plates were
read and data were analyzed as described above. The recovery of
4.00-512 pg/ml VEGF.sub.165 in 10% plasma was 77-101% after
subtracting 1.6 pg/ml putative endogenous VEGF in the 10% plasma
used for this study.
[0108] Results and Discussion
[0109] VEGF ELISAs: The previously described ELISA A uses 3.5F8 for
coat and biotinylated A4.6.1 for detection (Rodriguez C R, et al.,:
A sensitive fluorometric enzyme-linked immunosorbent assay that
measures vascular endothelial growth factor 165 in human plasma. J
Immunol Methods 219: 45-55, 1998). It detects VEGF165
(VEGF.sub.165) but not VEGF121(1) (VEGF.sub.121(1)), which is from
R&D systems and missing approximately 9 amino acids from the
carboxy-terminus, and VEGF121(2) (VEGF.sub.121(2)), which is from
PeproTech (FIG. 1A). 3.5F8 binds VEGF.sub.165 but not VEGF.sub.121
by BIAcore. A4.6. 1 binds to the receptor binding domain (Kim K J,
et al.,: The vascular endothelial growth factor proteins:
Identification of biologically relevant regions by neutralizing
monoclonal antibodies. Growth Factors 7: 53-64, 1992) that is
present in all isoforms and in VEGF.sub.110. 3.5F8 likely binds
near amino acids 116 and 118, which are not present in
VEGF.sub.121. 5C3 likely binds near amino acids 111-113, which are
not present in VEGF.sub.110 (FIG. 3). ELISA A can likely detect
VEGF isoforms which contained VEGF.sub.165 sequences including
VEGF.sub.183, VEGF.sub.189 and VEGF.sub.206 (see, e.g., Stimpfl M,
et al.,: Vascular Endothelial growth factor splice variants and
their prognostic value in breast and ovarian cancer. Clinical
Cancer Research 8: 2253-2259, 2002). ELISA B (previously named
VEGF121-206 ELISA, Konecny G E, et al., Association between
HER-2/neu and Vascular Endothelial Growth Factor Expression
Predicts Clinical Outcome in Primary Breast Cancer Patients.
Clinical Cancer Research, 10: 1706-1716, 2004) uses A4.6.1 for coat
and biotinylated 2E3 for detection. A4.6.1 and 2E3 bind to the
receptor binding domain that is present in all three molecules.
See, e.g., Kim K J, et al. The vascular endothelial growth factor
proteins: Identification of biologically relevant regions by
neutralizing monoclonal antibodies. Growth Factors 7:53-64 (1992);
and, Muller Y A, et al. Vascular endothelial growth factor: Crystal
structure and functional mapping of the kinase domain receptor
binding site. Proc Natl Acad Sci USA 94:7192-7197 (1997). Other
antibodies that bind in these regions can also be used. This ELISA
detects VEGF.sub.165, VEGF.sub.121, truncated VEGF.sub.121 (missing
approximately 9 amino acids from the carboxy-terminus),
VEGF.sub.110 and VEGF.sub.8-109 equally well (FIG. 1B). This ELISA
can detect total VEGF, including fragments larger than VEGF.sub.110
generated by matrix metalloproteinase digestion. ELISA C, described
herein, which uses 5C3 for coat and biotinylated A4.6.1 for
detection, detects VEGF.sub.165, VEGF.sub.121, and truncated
VEGF.sub.121 equally well but does not detect VEGF.sub.110 or
VEGF.sub.8-109 (FIG. 1, C). 5C3 binds VEGF.sub.121 but not
VEGF.sub.8-109 by BIAcore. This ELISA can detect all the VEGF
molecules detected by the VEGF.sub.110-206 except VEGF.sub.110 and
smaller fragments.
[0110] The sensitivities of ELISA A, ELISA B and ELISA C were 10,
16 and 40 pg/ml VEGF.sub.165 (or 0.26, 0.41 and 1.05 pM for
different VEGF isoforms and fragments) for VEGF in samples using a
minimum 1:10 dilution, respectively. ELISA B and ELISA C were
reproducible (Table 1 & 2). ELISA B and ELISA C were specific
to VEGF (VEGF-A). VEGF-B, VEGF-C and VEGF-D at concentrations up to
50 ng/ml only gave background signals. Insulin-like growth factor
1, growth hormone, recombinant nerve growth factor, tumor necrosis
factor (Genentech), platelet-derived growth factor AB, placenta
growth factor, transforming growth factor .beta.1 (R&D Systems)
(up to 200 ng/ml) only gave background signals. Heparin (Leo
Laboratories, Bucks, UK and Dublin, Ireland) (up to 100 U/ml) did
not have a significant effect on the assay.
TABLE-US-00002 TABLE 1 ELISA B (VEGF.sub.110-206 ELISA): The
standard range was 1.56-200 pg/ml VEGF.sub.165 (0.0409-5.24 pM
VEGF) in buffer. The OD ratio of 1.56 pg/ml standard relative to
the blank was 1.37 .+-. 0.11. CV is coefficient of variation. Mean
Control.sup.a (pg/ml) Inter % CV Intra % CV Low 3.07 17.7 13.5
Middle 38.0 9.50 6.54 High 127 9.11 6.95 .sup.aThe middle and high
controls were made by spiking recombinant VEGF.sub.165 into human
EDTA plasma. The low control was made by spiking VEGF.sub.165 into
70% plasma since plasma contained endogenous VEGF. Controls were
diluted 1:10 and assayed in duplicate in 34 independent assays.
TABLE-US-00003 TABLE 2 ELISA C (VEGF.sub.121-206 ELISA). The
standard range was 4.00-512 pg/ml VEGF.sub.165 (0.105-13.4 pM
VEGF). The OD ratio of 4 pg/ml standard relative to the blank was
2.72 .+-. 0.37. CV is coefficient of variation. Mean Control.sup.a
(pg/ml) Inter % CV Intra % CV Low 3.28 20.6 8.35 Middle 11.7 6.56
2.39 High 56.5 2.57 1.37 .sup.aThe controls were made by spiking
recombinant VEGF.sub.165 into human EDTA plasma. They were diluted
1:10 and assayed in duplicate in 15 independent assays.
[0111] VEGF in conditioned media of cell lines: Conditioned media
from six stable CHO clones transfected with VEGF.sub.165 cDNA (Meng
et al., 2000) were measured by the three ELISAs, which used
non-glycosylated VEGF produced in E. coli as standard. Glycosylated
recombinant VEGF.sub.165 in conditioned media from six stable CHO
clones gave very similar concentrations in the three ELISAs.
Concentrations measured by ELISA B were 28, 63, 64, 43, 3.8 and 3.2
nM, respectively. Ratios of VEGF concentrations measured by ELISA A
and ELISA C compared to those by ELISA B were 0.90.+-.0.08 and
1.08.+-.0.10, respectively. Therefore, the three ELISAs quantitated
glycosylated VEGF equally well and there was little proteolysis of
VEGF.sub.165 under the culture conditions.
[0112] VEGF concentrations in A673 cell conditioned medium measured
by ELISA A, ELISA B and ELISA C were 0.15, 0.29 and 0.24 nM VEGF,
respectively. The concentration measured by ELISA A was lower,
indicating VEGF.sub.121 was present. When VEGF was purified from
conditioned medium using an A4.6.1 affinity column and analyzed by
protein blotting, two bands, likely glycosylated and
non-glycosylated VEGF.sub.165 were detected by 3.5F8. The lower
band had the same mobility as the purified VEGF.sub.165 produced in
E. coli (FIG. 2, left). N-glycanase treatment converted the upper
band to the lower band. Two additional lower molecular weight
bands, likely glycosylated (partially overlapping with the putative
non-glycosylated VEGF.sub.165 band) and non-glycosylated
VEGF.sub.121 were detected by A4.6.1 (FIG. 2, right). The lower
band had the same mobility as the purified VEGF.sub.121 produced in
E. coli and N-glycanase treatment converted the upper band to the
lower band.
[0113] VEGF concentrations in conditioned media from breast cell
lines SK-BR-3, BT-474, T-47D and MCF-7 measured by ELISA B were
3.6, 16, 13, and 13 pM, respectively. Ratios of VEGF concentrations
measured by ELISA A to those by ELISA B were 0.49, 0.42, 0.43 and
0.38 (or 49%, 42%, 43% or 38%), respectively, in agreement with 43,
35, 40 and 41% of VEGF.sub.165 expression in these respective cell
lines (Stimpfl M, et al.,: Vascular Endothelial growth factor
splice variants and their prognostic value in breast and ovarian
cancer. Clinical Cancer Research 8: 2253-2259, 2002). Ratios of
VEGF concentrations measured by ELISA C to those by ELISA B were
1.1-1.2 for these cell lines, indicating that little VEGF.sub.110
was present. VEGF concentrations in conditioned media from ovarian
cell lines ES-2, OVCAR-3 and SK-OV-3 measured by ELISA B were 32,
11 and 20 pM, respectively. Ratios of VEGF concentrations measured
by ELISA A to those by ELISA B were 0.24, 0.20, and 0.32 (or 24%,
20% and 32%), respectively, compared to 38, 42 and 24% of
VEGF.sub.165 expression in these respective cell lines (Stimpfl et
al., supra). Ratios of VEGF concentrations measured by ELISA C to
those by ELISA B were 0.64-0.79 for these cell lines, indicating
VEGF.sub.110 (or smaller fragments) may be present.
* * * * *