U.S. patent application number 13/331891 was filed with the patent office on 2012-05-17 for chimeric vegf peptides.
This patent application is currently assigned to THE OHIO STATE UNIVERSITY RESEARCH FOUNDATION. Invention is credited to David Cohn, Pravin Kaumaya.
Application Number | 20120121626 13/331891 |
Document ID | / |
Family ID | 34860253 |
Filed Date | 2012-05-17 |
United States Patent
Application |
20120121626 |
Kind Code |
A1 |
Kaumaya; Pravin ; et
al. |
May 17, 2012 |
Chimeric VEGF Peptides
Abstract
Compositions for and methods of treating patients with
malignancies associated with overexpression of VEGF, particularly
ovarian cancer are provided herein. The compositions include but
are not limited to certain VEGF epitopes, multivalent peptides
comprising the epitopes, and chimeric peptides comprising one or
more of the epitopes and a T cell epitope.
Inventors: |
Kaumaya; Pravin;
(Westerville, OH) ; Cohn; David; (Bexley,
OH) |
Assignee: |
THE OHIO STATE UNIVERSITY RESEARCH
FOUNDATION
Columbus
OH
|
Family ID: |
34860253 |
Appl. No.: |
13/331891 |
Filed: |
December 20, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11052721 |
Feb 7, 2005 |
8080253 |
|
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13331891 |
|
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|
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60542041 |
Feb 5, 2004 |
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Current U.S.
Class: |
424/185.1 ;
530/324; 536/23.5 |
Current CPC
Class: |
A61K 39/0011 20130101;
C07K 14/475 20130101; C07K 14/005 20130101; A61K 38/00 20130101;
A61P 35/00 20180101; A61K 39/001135 20180801; C12N 2760/18422
20130101; C07K 14/12 20130101; C07K 14/52 20130101 |
Class at
Publication: |
424/185.1 ;
530/324; 536/23.5 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C07H 21/04 20060101 C07H021/04; A61P 35/00 20060101
A61P035/00; C07K 14/475 20060101 C07K014/475 |
Claims
1. A peptide comprising: a) at least one VEGF epitope selected from
the group consisting of amino acids 4 through 21 of SEQ ID NO: 1,
amino acids 24 through 38 of SEQ ID NO: 1, amino acids 76 through
96 of SEQ ID NO: 1, amino acids 126 through 143 of SEQ ID NO: 1,
amino acids 127 through 144 of SEQ ID NO: 1, amino acids 162
through 175 of SEQ ID NO: 1, amino acids 5 through 15 of SEQ ID NO:
2, amino acids 24 through 34 of SEQ ID NO: 2, amino acids 50
through 75 of SEQ ID NO: 2, amino acids 50-67 of SEQ ID NO: 2, and
amino acids 86 through 105 of SEQ ID NO: 2; b) a helper T cell
epitope; and c) a linker joining the at least one VEGF epitope to
the helper T cell epitope, wherein the linker comprises from about
2 to about 10 amino acids.
2. The peptide according to claim 1, having from about 35 to about
70 amino acids.
3. The peptide according to claim 1, wherein the helper T cell
epitope is a promiscuous Th cell epitope.
4. The peptide according to claim 1, wherein the helper T cell
epitope comprises from about 14 to about 22 amino acids.
5. The peptide according to claim 1, wherein the helper T cell
epitope comprises a sequence chosen from TT (SEQ ID NO: 3), TT1
(SEQ ID NO: 4), P2 (SEQ ID NO: 5), P30 (SEQ ID NO: 6), MVF (SEQ ID
NO: 7), HBV (SEQ ID NO: 8), and CSP (SEQ ID NO: 9).
6. The peptide according to claim 1, wherein the linker comprises
Gly-Pro-Ser-Leu (SEQ ID NO: 10).
7. An immunogenic composition comprising at least one immunogen
chosen from at least one of the peptides according to claim 1, and
at least one pharmacologically acceptable carrier.
8. A polynucleotide which encodes one of the peptides according to
claim 1.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 11/052,721, filed Feb. 7, 2005, now U.S. Pat. No. 8,080,253,
which claims priority to U.S. Provisional Application No.
60/542,041, filed Feb. 5, 2004, the entire disclosure of which is
incorporated herein by reference.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates to compositions for and
methods of treating patients with malignancies associated with
overexpression of VEGF, particularly ovarian cancer. The
compositions of the present invention include certain VEGF
epitopes, multivalent peptides comprising said epitopes, and
chimeric peptides comprising one or more of said epitopes and a T
cell epitope.
BACKGROUND
[0003] Ovarian cancer is the most lethal gynecologic malignancy,
with almost 14,000 women in the United States expected to die of
the disease in 2003 [Jemal]. Unfortunately, there is no effective
means for detection of early ovarian cancer, and as such over 75%
of cases are diagnosed when the disease has spread to the upper
abdomen or lymph nodes. Despite intensive cytotoxic chemotherapy
following radical surgery to reduce ovarian cancer volume, the
median survival of women with advanced and large-volume ovarian
cancer is under 40 months [McGuire].
[0004] Recent studies have demonstrated the critical role of
angiogenesis in tumor development and the formation of metastatic
tumor deposits. The inhibition of tumor angiogenesis has emerged as
a promising new therapeutic modality. A number of biologic
activities have been identified as being involved in this complex
process, however, vascular endothelial growth factor (VEGF) is now
known to be one of the most potent and specific pro-angiogenic
factors responsible for tumor-induced angiogenesis [Leung], and is
the most promising target for inhibition of tumor-induced
angiogenesis. VEGF is overexpressed in a number of human solid
malignancies, including ovarian cancer [Boocock, Olson]. VEGF
overexpression has also been demonstrated in women with ovarian
cancer and has been shown to be a poor prognostic factor
[Hollingsworth, Paley, Tempfer]. Thus, VEGF is a rational target
against which immunization may have a role in the treatment or
prevention of ovarian cancer.
[0005] Various strategies have been used to inhibit the function of
VEGF. These include targeting the VEGF receptor (VEGFR), using gene
therapy techniques that deliver antisense oligonucleotides, use of
soluble VEGFR, development of receptor tyrosine kinase (RTK)
inhibitors, and monoclonal antibodies (Mab) directed against VEGF
[Kim]. The most promising approach appears to be a recombinant
humanized version of a murine anti-human VEGF Mab (rhuMab VEGF,
Bevacizumab). This Mab has been tested in patients with metastatic
cancer [Gordon, Margolin]. There are, however, several
disadvantages to the use of antibody therapy. Importantly, passive
immunization strategies involve the transfer of antibody to the
patient, and immunity is short-lived as the antibodies are cleared
from the circulation. Likewise, Mabs are often immunogenic
themselves, thereby limiting their long-term use. Also, large
antibody volumes are necessary for effective sustained
immunization.
[0006] The use of vaccines to prevent or treat ovarian cancer is a
highly attractive approach because of the expected minimal side
effects of vaccine therapy. Many cancers express tumor-associated
antigens (TAA) that serve as targets for cancer vaccines.
Strategies for immunization have included whole cell vaccines,
protein and DNA vaccines, as well as peptide vaccines; each type of
antitumor vaccine has its advantages and limitations. Peptides are
an attractive anticancer vaccine in that they are safe (free of
pathogens and oncogenic potential), stable, easily constructed, and
are a cost-effective vaccine system [Dakappagari, Peoples,
Kaumaya]. Importantly, peptide vaccines lead to sustained immune
responses and memory, unlike that from passive immunization.
Limitations of peptide vaccines include the fact that unmodified
peptides are rarely immunogenic; thus rational peptide design is
imperative to the development of an effective antitumor
vaccine.
SUMMARY OF THE INVENTION
[0007] The present invention provides new compounds and
compositions for stimulating the immune system and for treating
malignancies associated with overexpression of the VEGF protein.
The compounds are immunogenic epitopes of the human VEGF protein
and human EG-VEGF protein, and chimeric and multivalent peptides
that comprise such epitopes.
[0008] The first group of compounds are referred to hereinafter
collectively as "VEGF epitopes." The VEGF epitopes comprise from
about 15 to about 50 amino acids, more preferably from 17 to 40
amino acids, most preferably from 18 to 35 amino acids. In one
aspect, the VEGF epitope shown in Table 1 below or an antigenic or
functional equivalent thereof:
TABLE-US-00001 TABLE 1 Immunogen Residues Amino Acid Sequence
Secondary Structure VEGF 126-143 KCECRPKKDRARQENPCG Turn-Helix-Turn
(of SEQ ID NO: 1) EG-VEGF 50-67 CTPLGREGEECHPGSHKV Turn-Helix-Turn
(of SEQ ID NO: 2)
[0009] In another aspect the VEGF epitope comprises amino acid 4
through amino acid 21 of human VEGF (as shown below), amino acid
24-38 of human VEGF, amino acid 127 through amino acid 144 of human
VEGF, amino acid 102 through amino acid 122 of VEGF, amino acid 162
through amino acid 175 of human VEGF, or amino acid 76 through
amino acid 96 of VEGF.
[0010] The human VEGF sequence is:
TABLE-US-00002 (SEQ ID NO: 1) 1
MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLD 60
IFQEYPDEIEYIFKPSCVPLMRCGGCSNDEGLECVPTEESNITMQIMRIKPHQGQHIGEM 120
SFLQHNKCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTHSRCKARQLELN 180
ERTCRCDKPRR. 190
[0011] In another aspect, the present VEGF epitope comprises amino
acid 5 through amino acid 15 of human EG-VEGF protein (as shown
below), amino acid 24 through amino acid 34 of human EG-VEGF, amino
acid 50 through amino acid 75 of human EG-VEGF, or amino acid 86
through amino acid 102 of human EG-VEGF.
[0012] The human EG-VEGF sequence is:
TABLE-US-00003 (SEQ ID NO: 2) 1
MRGATRVSIMLLLVTVSDCAVITGACERDVQCGAGTCCAISLWLRGLRMCTPLGREGEE
CHPGSHKVPFFRKRKHHTCPCLPNLLCSRFPDGRYRCSMDLKNINF. 105
[0013] The present invention also provides chimeric peptides,
referred to hereinafter as "chimeric VEGF peptides", which comprise
at least one of the present VEGF epitopes or an antigenic or
functional equivalent thereof. Preferably the chimeric VEGF
peptides are from about 35 to about 150, more preferably from about
35 to about 70 amino acids in length. The chimeric VEGF peptides
comprise three units. The first unit comprises at least one VEGF
epitope or an antigenic or functional equivalent thereof. The
second unit is a helper T (Th) cell epitope, preferably a
promiscuous Th cell epitope. As used herein a "promiscuous Th cell
epitope" is one that promotes release of cytokines that assist in
bypassing MHC restriction. The second unit is from about 14 to
about 22, more preferably about 15 to 21, most preferably 16 amino
acids in length. Preferably, the Th cell epitope has one of the
following amino acid sequences:
N--S--V-D-D-A-L-I--N--S-T-I--Y--S--Y--F--P--S--V, SEQ ID NO: 3,
referred to hereinafter as "TT";
P-G-I--N-G-K-A-I--H-L-V--N--N-Q-S--S-E, SEQ ID NO: 4, referred to
hereinafter as "TT1"; Q-Y--I--K-A-N--S--K--F--I-G-I-T-E-L, SEQ ID
NO: 5, referred to hereinafter as "P2";
F--N--N--F-T-V--S--F--W-L-R--V--P--K--V--S-A-S--H-L-E, SEQ ID NO:
6, referred to hereinafter as "P30";
L-S-E-I--K-G-V--I--V--H--R-L-E-G-V, SEQ ID NO: 7, referred to
hereinafter as "MVF"; F--F-L-L-T-R--I-L-T-I--P-Q-S-L-N, SEQ ID NO:
8, referred to hereinafter as "HBV";
T-C-G-V-G-V--R--V--R--S--R--V--N-A-A-N--K--K--P-E, SEQ ID NO: 9,
referred to hereinafter as "CSP".
[0014] The third unit of the chimeric peptide joins the first and
second peptide units. The third unit is an amino acid or,
preferably, a peptide of from about 2 to about 15 amino acids, more
preferably from about 2 to about 10 amino acids, most preferably
from about 2 to about 6 amino acids in length. The most preferred
linker comprises the amino acid sequence Gly-Pro-Ser-Leu, SEQ ID
NO: 10.
[0015] The present invention also provides multivalent VEGF
peptides, which comprise a plurality, i.e., at least two of the
present VEGF epitopes or functional equivalents thereof and a Th
cell epitope. The VEGF epitopes and Th cell epitope are connected
to a core 13 sheet template. Preferably, the template comprises two
strands of alternating leucine and lysine residues, which are
connected by a linker. The linker is an amino acid or, preferably,
a peptide of from about 2 to about 15 amino acids, more preferably
from about 2 to about 10 amino acids, most preferably from about 2
to about 6 amino acids in length. The most preferred linker
comprises the amino acid sequence Gly-Pro-Ser-Leu, SEQ ID NO:
10.
[0016] The present invention also relates to an immunogenic
composition containing a mixture of VEGF epitopes, a chimeric VEGF
peptide, or a multivalent VEGF peptide and a pharmacologically
acceptable carrier. In one aspect, the carrier is a biodegradeable
microsphere. Such immunogenic compositions are useful for treating
malignancies with which overexpression of the VEGF protein is
associated.
[0017] The present invention also relates to polynucleotides which
encode at least one of the VEGF epitopes described above. Such
polynucleotides are useful for producing the epitope by recombinant
techniques. The present invention also relates to isolated
polynucleotides having a sequence which encodes a chimeric VEGF
cell peptide of the present invention. Such polynucleotides are
useful for preparing the chimeric VEGF cell peptide. Such
polynucleotides are also useful in an immunogenic composition
(e.g., DNA vaccine) for treating or preventing malignancies in
which overexpression of the VEGF protein is associated. Preferably,
such immunogenic compositions are administered intramuscularly.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 shows high-titered sera recognizing the B-cell
epitope (VEGF) and immunogen (MVF-VEGF) following active
immunization with VEGF peptides. ELISA of New Zealand white rabbit
sera against VEGF, demonstrating titers >1:500,000 at 4 weeks
following the second booster vaccination (3y+4).
[0019] FIG. 2 shows that VEGF peptide antibodies recognize rhVEGF
by ELISA. Comparison of rabbit pre-immune sera, prior to
vaccination (bar 1) and sera from rabbits vaccinated with VEGF
peptides (bars 2-4). Bar 2 and 3 represent rabbit 1 at 3 and 4
weeks after the second booster vaccination (3y+3 and 3y+4,
respectively). Bar 4 represents sera from rabbit 2 at a similar
bleed.
[0020] FIG. 3 shows a Western blot of rhVEGF blotted with (A) VEGF
peptide antibody or (B) Ab-4, a VEGF monoclonal antibody,
demonstrating recognition of the appropriate recombinant protein
dimer at 42 kDa.
[0021] FIG. 4 shows that VEGF peptide antibodies disrupt the normal
VEGF-VEGFR interaction by flow cytometry using HUVECs, presumably
through depletion of VEGF. (A) Evaluation of the positive (PC),
negative (NC) and inhibitor antibody (IC) controls of
Fluorokine.RTM. assay, and (B) the same PC and NC as in (A), and
employing either mouse or rabbit VEGF peptide antibodies, both
demonstrating disruption of the normal VEGF-VEGFR interaction. The
rabbit antibody labeled as "combo" represents rabbit VEGF peptide
antibodies following immunization with both the MVF-VEGF immunogen
as well as another immunogen not described in this
investigation.
[0022] FIG. 5 shows that VEGF peptide antibodies disrupt
angiogenesis into Matrigel.TM.. C57BL/6 mice were subcutaneously
injected with Matrigel.TM. incubated with rhVEGF with (FIG. 5A) or
without (FIG. 5B) VEGF antibodies. After 10 days, the plugs were
removed, stained, and blood vessel invasion was counted. Compared
with PBS control, addition of VEGF peptide antibodies significantly
disrupts angiogenesis in viva Magnification 40.times., stained with
Hoechst 33342.
[0023] FIG. 6 shows that VEGF peptide antibodies disrupt
angiogenesis into Matrigel.TM.. C57BL/6 mice were subcutaneously
injected with Matrigel.TM. incubated with rhVEGF, with or without
VEGF peptide antibodies. After 10 days, the plugs were removed,
stained, and blood vessel invasion was counted. Compared with PBS
control, addition of VEGF peptide antibodies significantly disrupts
angiogenesis in vivo. Each bar represents the mean (.+-.SEM) of a
group of three mice.
[0024] FIG. 7 is a graph showing rabbit anti-peptide antibodies
against immunogenic epitopes of (A) VEGF and (B) EG-VEGF, with and
without the addition of the measles virus fusion protein (MVF)
[0025] FIG. 8 shows disruption of (A) and shortening of (B) cyclic
estrous cycles as well as (C) a decrease in the number of
primordial follicles with passive immunization with anti-VEGF
antibodies in a murine model.
[0026] FIG. 9 shows that (A) Anti-VEGF and (B) anti-EG-VEGF peptide
antibodies recognize rhVEGF and rhEG-VEGF.
[0027] FIG. 10 shows Western blots of rhVEGF (A and B) blotted with
(A) anti-VEGF peptide antibody and (B) Ab-4, a monoclonal anti-VEGF
antibody. Western blot of rhEG-VEGF (C and D), blotted with (C)
rabbit anti-EG-VEGF peptide antibody and (D) rabbit
anti-VEGF/anti-peptide EG-VEGF "combination" antibody, all
demonstrating recognition of the appropriate recombinant
protein.
[0028] FIG. 11 shows the results of a Fluorokine assay for
evaluation of the functional properties of anti-VEGF peptide
antibodies. (A) Evaluation of the positive (PC), negative (NC) and
inhibitor antibody (IC) controls of Fluorokine assay, and (B) the
same PC and NC as in (A), and employing either mouse or rabbit
anti-VEGF peptide antibodies, as well as the combination
anti-VEGF/anti-EG-VEGF peptide antibody, all demonstrating
disruption of the VEGF-VEGF receptor interaction.
[0029] FIG. 12 shows injection of (A) subcutaneous matrigel and (B)
subcutaneous matrigel combined with rhVEGF, stained with
CD31:phycoerythrin conjugate antibody demonstrates increased
angiogenesis in C57/BL6 mice.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The present invention will now be described by reference to
more detailed embodiments, with occasional reference to the
accompanying drawings. This invention may, however, be embodied in
different forms and should not be construed as limited to the
embodiments set forth herein. Rather, these embodiments are
provided so that this disclosure will be thorough and complete, and
will fully convey the scope of the invention to those skilled in
the art.
[0031] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. The
terminology used in the description of the invention herein is for
describing particular embodiments only and is not intended to be
limiting of the invention. As used in the description of the
invention and the appended claims, the singular forms "a," "an,"
and "the" are intended to include the plural forms as well, unless
the context clearly indicates otherwise. All publications, patent
applications, patents, and other references mentioned herein are
expressly incorporated by reference in their entirety.
[0032] Unless otherwise indicated, all numbers expressing
quantities of ingredients, reaction conditions, and so forth used
in the specification and claims are to be understood as being
modified in all instances by the term "about." Accordingly, unless
indicated to the contrary, the numerical parameters set forth in
the following specification and attached claims are approximations
that may vary depending upon the desired properties sought to be
obtained by the present invention. At the very least, and not as an
attempt to limit the application of the doctrine of equivalents to
the scope of the claims, each numerical parameter should be
construed in light of the number of significant digits and ordinary
rounding approaches.
[0033] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the invention are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. Any numerical value, however,
inherently contains certain errors necessarily resulting from the
standard deviation found in their respective testing measurements.
Every numerical range given throughout this specification will
include every narrower numerical range that falls within such
broader numerical range, as if such narrower numerical ranges were
all expressly written herein.
[0034] Throughout this disclosure, reference will be made to
compounds according to the invention. Reference to such compounds,
in the specification and claims, includes esters and salts of such
compounds. Thus, even if not explicitly recited, such esters and
salts are contemplated, and encompassed, by reference to the
compounds themselves.
[0035] Additionally, as used herein, "peptide," "polypeptide," and
"protein," can and will be used interchangeably.
"Peptide/polypeptide/protein" may be used to refer to any of the
three, but recitations of any of the three contemplate the other
two. That is, there is no intended limit on the size of the amino
acid polymer (peptide, polypeptide, or protein), that can be
expressed using the present invention. Additionally, the recitation
of "protein" is intended to encompass enzymes, hormone, receptors,
channels, intracellular signaling molecules, and proteins with
other functions. Multimeric proteins can also be made in accordance
with the present invention.
[0036] While the naturally occurring amino acids are discussed
throughout this disclosure, non-naturally occurring amino acids, or
modified amino acids, are also contemplated and within the scope of
the invention. In fact, as used herein, "amino acid" refers to
natural amino acids, non-naturally occurring amino acids, and amino
acid analogs, all in their D and L stereoisomers. Natural amino
acids include alanine (A), arginine (R), asparagine (N), aspartic
acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine
(G), histidine (H), isoleucine (I), leucine (L), lysine (K),
methionine (M), phenylalanine (F), proline (P), serine (S),
threonine (T), tryptophan (W), tyrosine (Y), valine (V),
hydroxyproline (O and/or Hyp), isodityrosine (IDT), and
di-isodityrosine (di-IDT). Hydroxyproline, isodityrosine, and
di-isodityrosine are formed post-translationally. Use of natural
amino acids, in particular the 20 genetically encoded amino acids,
is preferred.
[0037] The present invention provides peptides that are immunogenic
epitopes of the human VEGF protein and the human EG-VEGF protein,
referred to hereinafter collectively as "VEGF epitopes".
[0038] The VEGF epitopes and their antigenic equivalents are
capable of invoking a humoral response, which results in the
production of antibodies that are immunoreactive with the
recombinant human VEGF protein and/or human EG-VEGF protein. The
VEGF epitopes encompass peptides having one of the sequences,
referred to hereinafter as the "reference sequences", as described
above. The reference sequences were selected and scored using
computer-aided analysis using six correlates of antigenicity: (a)
the profiles of chain flexibility and mobility of individual
sequences was calculated according to Karplus and Schultz,
Naturwiss 72:212-213, 1985; (b) hydropathy profiles were generated
over a seven residue span setting and were finally smoothed with a
three residue span using the scale of Kyte and Doolittle, J. Mol.
Biol. 157:105-132, 1982; (c) hydrophilicity profiles were generated
over a 6-residue window using the program of Hopp and Woods, Proc.
Natl. Acad. Sci. USA 78:3824-3828, 1981; (d) analysis of the
exposure of an amino acid residue to water using a 1.4 .ANG. probe)
was carried out b the solvent exposure algorithm of Rose, Science
229:834-838, 1985; (e) protrusion indices that predicts portions of
proteins that are accessible and protrude into the solvent were
calculated by the method of Thornton, EMBO J. 5:409-413, 1986; (f)
the probability that a five residue sequence is antigenic was
determined by the method of Welling, FEBS Lett 188:215-218, 1985.
The basic premise is that the algorithms used in the predictions
will always locate regions that are surface-exposed on the protein
and therefore most likely to be involved in antibody binding.
[0039] Sequences were given a score of 1 to 6 based on their
respective index values and were ranked: the highest ranking
sequences had the highest individual score for the analyses
examined (6/6), and successive candidates had the next highest
score (5/6), etc. The best scoring epitopes were further ranked by
correlation with their secondary structural attributes, e.g., an
amphiphilic .alpha.-helical sequence or a 13-turn loop region are
preferred over a random coil fragment. Computer programs by Chou
and Fasman, Adv. Enzymol. Relat. Subj. Biochem. 47: 45-148, 1978
were used to predict the secondary structure (.alpha.-helix,
.beta.-strand/sheet, (3-turn/loop, random coil) and helical
amphiphilic moment. Electrostatic ion pairs and helix dipole
interaction in helical segment were also considered (e.g.,
hydrophobic/hydrophilic balance). Preferably, the
hydrophilic/hydrophobic balance is from 2/2 to 4/1.
[0040] As described herein, the VEGF cell epitopes also encompass
peptides that are antigenic and functional equivalents of the
peptides described above. Such functional equivalents have an
altered sequence in which one or more of the amino acids in the
corresponding reference sequence is substituted, or in which one or
more amino acids are deleted from or added to the reference
sequence. For example, cysteine residues may be deleted or replaced
with other amino acids to prevent formation of incorrect
intramolecular disulfide bridges upon renaturation.
[0041] While it is possible to have nonconservative amino acid
substitutions, it is preferred that, except for the substitutions
that are made to replace cysteine, the substitutions be
conservative amino acid substitutions, in which the substituted
amino acid has similar structural or chemical properties with the
corresponding amino acid in the reference sequence. By way of
example, conservative amino acid substitutions involve substitution
of one aliphatic or hydrophobic amino acids, e.g., alanine, valine,
leucine and isoleucine, with another; substitution of one
hydroxyl-containing amino acid, e.g., serine and threonine, with
another; substitution of one acidic residue, e.g., glutamic acid or
aspartic acid, with another; replacement of one amide-containing
residue, e.g., asparagine and glutamine, with another; replacement
of one aromatic residue, e.g., phenylalanine and tyrosine, with
another; replacement of one basic residue, e.g., lysine, arginine
and histidine, with another; and replacement of one small amino
acid, e.g., alanine, serine, threonine, methionine, and glycine,
with another.
[0042] Preferably, the deletions and additions are located at the
amino terminus, the carboxy terminus, or both, of one of the
sequences shown above. As a result of the alterations, the VEGF
functional epitope equivalent has an amino acid sequence which is
at least 70% identical, preferably at least 80% identical, more
preferably at least 90% identical, most preferably, at least 95%
identical to the corresponding reference sequences. Sequences which
are at least 90% identical have no more than 1 alteration, i.e.,
any combination of deletions, additions or substitutions, per 10
amino acids of the reference sequence. Percent identity is
determined by comparing the amino acid sequence of the variant with
the reference sequence using MEGALIGN project in the DNA STAR
program.
[0043] For functional equivalents that are longer than a
corresponding reference sequence, it is preferred that the
functional equivalent have a sequence which is at least 90%
identical to the reference sequence and the sequences which flank
the reference sequence in the wild-type VEGF or EG-VEGF protein. In
addition to being an antigenic equivalent of the
naturally-occurring human VEGF epitope, the functional equivalent
is also capable of raising antibodies that disrupt bind of human
VEGF or EG-VEGF to the VEGF receptor.
[0044] Preparation of Epitopes and Co-Linear Chimeric Peptides
[0045] The VEGF epitopes, chimeric VEGF peptides, and multivalent
VEGF peptides, preferably, are synthesized using commercially
available peptide synthesizers. Preferably, the chemical methods
described in Kaumaya et al., "DE NOVO" ENGINEERING OF PEPTIDE
IMMUNOGENIC AND ANTIGENIC DETERMINANTS AS POTENTIAL VACCINES, in
Peptides, Design, Synthesis and Biological Activity (1994), pp
133-164, which is specifically incorporated herein by reference,
are used.
[0046] The VEGF epitopes and chimeric peptides may also be produced
using cell-free translation systems and RNA molecules derived from
DNA constructs that encode the epitope or peptide. Alternatively,
the VEGF epitopes or chimeric peptides are made by transfecting
host cells with expression vectors that comprise a DNA sequence
that encodes the respective epitope or chimeric peptide and then
inducing expression of the polypeptide in the host cells. For
recombinant production, recombinant constructs comprising one or
more of the sequences which encode the epitope, chimeric peptide,
or a variant thereof are introduced into host cells by conventional
methods such as calcium phosphate transfection, DEAE-dextran
mediated transfection, transvection, microinjection, cationic
lipid-mediated transfection, electroporation, transduction, scrape
lading, ballistic introduction or infection.
[0047] The VEGF epitopes and chimeric peptides may be expressed in
suitable host cells, such as for example, mammalian cells, yeast,
bacteria, insect cells or other cells under the control of
appropriate promoters using conventional techniques. Suitable hosts
include, but are not limited to, E. coli, P. pastoris, Cos cells
and 293 HEK cells. Following transformation of the suitable host
strain and growth of the host strain to an appropriate cell
density, the cells are harvested by centrifugation, disrupted by
physical or chemical means, and the resulting crude extract
retained for further purification of the epitope or chimeric
peptide.
[0048] Conventional procedures for isolating recombinant proteins
from transformed host cells, such as isolation by initial
extraction from cell pellets or from cell culture medium, followed
by salting-out, and one or more chromatography steps, including
aqueous ion exchange chromatography, size exclusion chromatography
steps, and high performance liquid chromatography (HPLC), and
affinity chromatography may be used to isolate the recombinant
polypeptide. To produce glycosylated epitopes and chimeric
peptides, it is preferred that recombinant techniques be used. To
produce glycosylated epitopes and chimeric peptides which contain
the same, it is preferred that mammalian cells such as, Cos-7 and
Hep-G2 cells be employed in the recombinant techniques.
[0049] Naturally occurring variants of the VEGF epitopes above may
also be isolated by, for example, by screening an appropriate cDNA
or genomic library with a DNA sequence encoding the
polypeptide.
[0050] Identifying Functional Equivalents of the VEGF Peptide
[0051] Functional equivalents of the VEGF epitopes shown above may
generally be identified by modifying the sequence of the epitope
and then assaying the resulting polypeptide for the ability to
stimulate an immune response, e.g., production of antibodies. For
example, such assays may generally be performed by preparing a
chimeric peptide which comprises the modified polypeptide and a
promiscuous Th cell epitope, injecting the chimeric peptide into a
test animal and assaying for antibodies. Such antibodies may be
found in a variety of body fluids including sera and ascites.
Briefly, a body fluid sample is isolated from a warm-blooded
animal, such as a human, for whom it is desired to determine
whether antibodies specific for the human VEGF are present. The
body fluid is incubated with the human VEGF or EG-VEGF protein
under conditions and for a time sufficient to permit
immunocomplexes to form between the polypeptide and antibodies
specific for the protein and then assayed, preferably using an
ELISA technique. In such technique, the colorimetric change is
measured at 490 nm. Epitopes which induce production of antibodies
that exhibit a titer equal to 10,000 or greater for human VEGF or
EG-VEGF protein, are preferred. As used herein a titer of 10,000
refers to an absorbance value of 0.2 above background.
[0052] Functional equivalents of the VEGF epitope are further
identified by determining whether the peptide can be used to raise
antibodies that disrupt binding of human VEGF to the human VEGF
receptor as described in Example 1 below.
[0053] Determining Optimum Dosages for Inhibiting Agiogenesis or
Treating Cancer
[0054] In vivo methods can also be used to characterize functional
equivalents of the present epitopes or to determine optimum dosages
of the VEGF chimeric and multivalent peptides. In one aspect, a
xenograft model can be used. In this model, subcutaneous tumors are
established with an ovarian cancer (SKOV3) cell line by injecting
BALB/c athymic nude mice in the right flank with 2.times.10.sup.6
tumor cells mixed with matrigel. Tumors are allowed to reach
150-200 mm.sup.3, and then mice randomized to receive either
intraperitoneal injection of anti-VEGF or anti-EG-VEGF antibodies
or nonspecific IgG antibodies (control) every 3 days. Tumor volumes
are measured and calculated twice weekly. Tumor permeability is
assessed using the Miles assay, in which treated and control mice
undergo intracardiac injection with Evans blue solution. The
animals are then be sacrificed, photographed, and the amount of
permeability compared between treated and control mice. The
matrigel plug is removed, and vessel density examined by CD31
immunohistochemistry.
[0055] Active immunization with the VEGF and EG-VEGF peptide
vaccines is then investigated in vivo. Previous investigation has
shown that the introduction of SKOV3 ovarian cancer cells
(10.times.10.sup.6 cells in 200 .mu.L PBS) in the peritoneal cavity
of female immunodeficient mice (BALB/c nu/nu) leads to a clinical
condition identical to that seen with women with advanced ovarian
cancer, including the establishment of tumors coating the surface
of intraperitoneal organs, as well as the development of
large-volume ascites within 4 weeks of inoculation [9]. Likewise,
subcutaneous injection of SKOV3 cells (10.times.10.sup.6 cells in
50 .mu.L PBS) leads to measurable tumor within 10 days of
inoculation. As such, a combination of both subcutaneous and
intraperitoneal injection of SKOV3 cells can be investigated in
determining the in vivo efficacy of peptide immunization against
ovarian tumors.
[0056] Groups of 10 mice are vaccinated with the VEGF and EG-VEGF
epitopes three times at 3-week intervals. Antibody titers are
monitored by ELISA. Three weeks following the third vaccination,
mice are challenged with SKOV3 cells. Subcutaneous tumors are
measured weekly using calipers, and tumor volumes are estimated
based on the assumption that tumors are spherical. Because
intraperitoneal tumor growth and distribution cannot be measured
directly, mice are killed by anesthetic overdose approximately 4
weeks following inoculation and location of tumor, presence of
ascites, and tumor location and size recorded.
[0057] Further evidence of the anti-angiogenic effect of the
elicited anti-VEGF antibodies is suggested by inhibition of
ovulation by antibody following stimulation by gonadotropins.
Because ovulation is regulated through angiogenic stimuli [18], it
is believed that anti-angiogenic efficacy of the anti-peptide
antibodies would lead to disruption of ovulation. In this
experiment, groups of 24 day old female Sprague-Dawley rats are
immunized with anti-peptide antibodies against VEGF, EG-VEGF, both
VEGF and EG-VEGF, or nonspecific antibody (control). Four hours
following immunization, the rats are treated with 20 IU pregnant
mares' serum gonadotropin (PMSG) and 24-hours later with 20 IU
human chorionic gonadotropin (hCG), the hormonal stimulatory
sequence that leads to ovulation. After 5 days, the rats are killed
and an assessment of the efficacy of inhibition of ovulation will
be made. It is believed that in rats treated with anti-peptide
antibodies, ovulation will be efficiently inhibited and a decreased
ovarian weight and alterations in the estrous cycle and decreased
follicular growth and decreased CD31 immunostaining will be seen
compared with control animals.
[0058] Further assessment of the in vivo activity of VEGF peptide
vaccines can be conducted in a transgenic mouse model that
expresses human VEGF under the control of the rat insulin promoter
(Rip1VEGF-A) [20]. In this model, overexpression of VEGF was shown
to accelerate the onset of tumor angiogenesis, and spontaneous
tumors were shown to develop within 10 weeks (single-transgenic
mouse, RIP1Tag2) and 14 weeks (double-transgenic mouse,
Rip1Tag2/Rip1VEGF-A). In this studies, mice are immunized with VEGF
or EG-VEGF epitopes on a compressed schedule to match the
transgenic mouse tumor development (initial immunization will be at
5 weeks, with subsequent booster at 7 and 9 weeks). Mice are then
sacrificed, and tumor volumes calculated. Tumors are fixed,
embedded in OCT, snap frozen, and analyzed by H&E staining.
Vessel density can determined by CD31 immunostaining. It is
expected that VEGF peptide vaccination will lead to the prevention
or inhibition of tumor formation in this transgenic mouse model
overexpressing VEGF, and that surrogates for angiogenesis will be
decreased.
[0059] Polynucleotides
[0060] The present invention also provides isolated polynucleotides
which encode the VEGF epitopes and the chimeric peptides of the
present invention. The present polynucleotides also encompass
polynucleotides having sequences that are capable of hybridizing to
the nucleotide sequences of under stringent conditions, preferably
highly stringent conditions. Hybridization conditions are based on
the melting temperature (Tm) of the nucleic acid binding complex or
probe, as described in Berger and Kimmel (1987) Guide to Molecular
Cloning Techniques, Methods in Enzymology, vol 152, Academic Press.
The term "stringent conditions, as used herein, is the "stringency"
which occurs within a range from about Tm-5 (5.degree. below the
melting temperature of the probe) to about 20.degree. C. below Tm.
As used herein "highly stringent" conditions employ at least
0.2.times.SSC buffer and at least 65.degree. C. As recognized in
the art, stringency conditions can be attained by varying a number
of factors such as the length and nature, i.e., DNA or RNA, of the
probe; the length and nature of the target sequence, the
concentration of the salts and other components, such as formamide,
dextran sulfate, and polyethylene glycol, of the hybridization
solution. All of these factors may be varied to generate conditions
of stringency which are equivalent to the conditions listed
above.
[0061] Polynucleotides comprising sequences encoding a VEGF epitope
or a chimeric peptide of the present invention may be synthesized
in whole or in part using chemical methods or, preferably,
recombinant methods which are known in the art. Polynucleotides
which encode a VEGF may be obtained by screening a genomic library
or cDNA library with antibodies immunospecific for the to identify
clones containing such polynucleotide.
[0062] The polynucleotides are useful for producing a VEGF B
epitope. chimeric peptide, or multivalent peptide. For example, an
RNA molecule encoding a chimeric peptide is used in a cell-free
translation systems to prepare such polypeptide. Alternatively, a
DNA molecule encoding a VEGF epitope or a chimeric peptide is
introduced into an expression vector and used to transform cells.
Suitable expression vectors include for example chromosomal,
nonchromosomal and synthetic DNA sequences, e.g., derivatives of
SV40, bacterial plasmids, phage DNAs; yeast plasmids, vectors
derived from combinations of plasmids and phage DNAs, viral DNA
such as vaccinia, adenovirus, fowl pox virus, pseudorabies,
baculovirus, and retrovirus. The DNA sequence is introduced into
the expression vector by conventional procedures.
[0063] Accordingly, the present invention also relates to
recombinant constructs comprising one or more of the present
polynucleotide sequences. Suitable constructs include, for example,
vectors, such as a plasmid, phagemid, or viral vector, into which a
sequence that encodes VEGF epitope or the chimeric peptide has been
inserted. In the expression vector, the DNA sequence which encodes
the epitope or chimeric peptide is operatively linked to an
expression control sequence, i.e., a promoter, which directs mRNA
synthesis. Representative examples of such promoters, include the
LTR or SV40 promoter, the E. coli lac or trp, the phage lambda PL
promoter and other promoters known to control expression of genes
in prokaryotic or eukaryotic cells or in viruses. The expression
vector, preferably, also contains a ribosome binding site for
translation initiation and a transcription terminator. Preferably,
the recombinant expression vectors also include an origin of
replication and a selectable marker, such as for example, the
ampicillin resistance gene of E. coli to permit selection of
transformed cells, i.e., cells that are expressing the heterologous
DNA sequences. The polynucleotide sequence encoding the VEGF
epitope or the chimeric peptide is incorporated into the vector in
frame with translation initiation and termination sequences.
Preferably, the polynucleotide further encodes a signal sequence
which is operatively linked to the amino terminus of the VEGF
epitope, or chimeric peptide.
[0064] The polynucleotides encoding the VEGFF or EG-VEGF epitope or
the chimeric peptides comprising such epitopes are used to express
recombinant peptide using techniques well known in the art. Such
techniques are described in Sambrook, J. et al (1989) Molecular
Cloning A Laboratory Manual, Cold Spring Harbor Press, Plainview,
N.Y. and Ausubel, F. M. et al. (1989) Cuurent Protocols in
Molecular Biology, John Wile & Sons, New York, N.Y.
Polynucleotides encoding the VEGF or EG-VEGF epitope or the
chimeric peptides comprising such epitopes are also used to
immunize animals.
[0065] Pharmaceutical Compositions
[0066] Pharmaceutical compositions which comprise mixtures of VEGF
and/or EG-VEGF epitopes, chimeric VEGF or EG-VEGF peptides, and
multivalent VEGF- or EG-VEGF peptides or the polynucleotides which
encode the same are preferably formulated for use as a
pharmaceutical composition (e.g., an immunogenic composition or a
vaccine). Such compositions generally comprise one or more of the
present VEGF and/or EG-VEGF epitopes, one or more of the present
VEGF and/or EG-VEGF chimeric peptides, or one or more the present
VEGF or EG-VEGF multivalent peptides or the polynucleotides which
encode the same in combination with a pharmaceutically acceptable
carrier, excipient, or diluent. Such carriers will be nontoxic to
recipients at the dosages and concentrations employed.
[0067] In addition to the epitopes, multivalent peptides, and
chimeric peptides (which functions as antigens) or the
polynucleotide which encodes the same, other components, such as a
vehicle for antigen delivery and immunostimulatory substances
designed to enhance the protein's immunogenicity, are, preferably,
included in the pharmaceutical composition. Examples of vehicles
for antigen delivery include aluminum salts, water-in-oil
emulsions, biodegradable oil vehicles, oil-in-water emulsions,
biodegradable microcapsules, and liposomes. For the vaccines that
comprise the chimeric peptide, one potential vehicle for antigen
delivery is a biodegradable microsphere, which preferably is
comprised of poly (D, L-lactide-co-glycolide) (PLGA).
[0068] While any suitable carrier known to those of ordinary skill
in the art may be employed in the pharmaceutical compositions of
this invention, the type of carrier will vary depending on the mode
of administration and whether a substantial release is desired. For
parenteral administration, such as subcutaneous injection, the
carrier preferably comprises water, saline, alcohol, a fat, a wax,
or a buffer. Biodegradable microspheres (e.g., polylactic
galactide) may also be employed as carriers for the pharmaceutical
compositions of this invention. Optionally, the pharmaceutical
composition comprises an adjuvant.
[0069] The VEGF epitope mixtures, chimeric and multivalent peptides
and the polynucleotides which encode the same are useful for
enhancing or eliciting, in a subject or a cell line, a humoral
response and, preferably, a cellular immune response (e.g., the
generation of antigen-specific cytolytic T cells). As used herein,
the term "subject" refers to any warm-blooded animal, preferably a
human. A subject may be afflicted with cancer, such as ovarian
cancer, or may be normal (i.e., free of detectable disease and
infection). The pharmaceutical composition is particularly useful
for treating women who have a family history of ovarian cancer or
who have been diagnosed as having ovarian cancer.
[0070] Methods of Treatment
[0071] The present invention also provides methods of treating a
cancer which is associated with overexpression of VEGF By
"treating" is meant inhibiting or slowing or retarding the growth
of the tumor. Such cancers include ovarian cancer. The method
comprises administering a pharmaceutical composition comprising a
VEGF and/or EG-VEGF epitope mixture, one or more VEGF chimeric
peptides or one or more VEGF multivalent peptides of the present
invention to a subject. Preferably multiple intramuscular
injections, at three week intervals are used to administer the
pharmaceutical composition.
[0072] The present invention also provides methods of inhibiting
angiogenesis in rapidly growing tissues in a subject. The methods
comprise administering a mixture of VEGF and/or EG-VEGF epitopes of
the present invention, one or more chimeric VEGF and/or EG-VEGF
chimeric peptides of the present invention, one or more multivalent
VEGF and/or EG-VEGF polypeptides of the present invention or
polynucleotides that encode the same to the subject.
[0073] The peptides of this invention relate to the representative
peptides as described above, and to antigenically related variants
of these peptides. "Antigenically related variants" can be either
natural variants or artificially modified variants that
immunologically mimic the VEGF or EG-VEGF epitope described above.
Such artificially modified variants can be made by synthetic
chemistry of recombinant DNA mutagenesis techniques that are well
known to persons skilled in the art (see for example Chapter 15 of
Sambrook, et al. "Molecular Cloning a Laboratory Manual" (1989)
Cold Spring Harbor Laboratory Press). The antigenically related
variants of the peptides should have an amino acid sequence
identity of at least 75% to one of the VEGF or EG-VEGF epitopes
described above (and more preferably at least 85%, and most
preferably at least 95% identity), whilst still being capable of
immunologically mimicking the corresponding antigenic determinant
site of the human VEGF or EG-VEGF protein.
[0074] For this invention "immunologically mimicking the
corresponding antigenic determinant site of the VEGF or EG-VEGF
protein is defined as a (variant) peptide being capable of inducing
antibodies that specifically recognize one of the wild-type epitope
sequences described above in the context of the whole VEGF or
EG-VEGF protein AND/OR defined as a (variant) peptide being capable
of being recognized by the same immunospecific antibody that
recognizes one of the VEGF or EG-VEGF epitopes described above in
the context of the whole VEGF or EG-VEGF protein. In the first
definition, the variant peptide should be capable of inducing such
antibodies either by itself, or in conjunction with a carrier
molecule. In the second definition, the variant peptide should be
capable of being recognized either by itself, or in conjunction
with a carrier molecule. Antigenically related variants may have
had amino acids added, inserted, substituted or deleted. Preferred
variants are those that differ from the referents by conservative
(preferably single) amino acid substitutions.
[0075] Polypeptides of the present invention can be prepared in any
suitable manner. Such polypeptides include recombinantly produced
polypeptides, synthetically produced polypeptides, or polypeptides
produced by a combination of these methods. Means for preparing
such polypeptides are well understood in the art, however examples
of the method are presented in the Examples section.
[0076] Polynucleotides of the Invention
[0077] The polynucleotides of the invention also relates to DNA
sequences that can be derived from the amino acid sequences of the
peptides and polypeptides of the invention bearing in mind the
degeneracy of codon usage. This is well known in the art, as is
knowledge of codon usage in different expression hosts which is
helpful in optimizing the recombinant expression of the peptides
and polypeptides of the invention.
[0078] The invention also provides polynucleotides which are
complementary to all the above described polynucleotides.
[0079] When the polynucleotides of the invention are used for the
recombinant production of polypeptides of the present invention,
the polynucleotide may include the coding sequence for the
polypeptide, by itself; or the coding sequence for the polypeptide
in reading frame with other coding sequences, such as those
encoding a leader or secretory sequence, a pre-, or pro- or
prepro-protein sequence, or other fusion peptide portions. For
example, a marker sequence which facilitates purification of the
fused polypeptide can be encoded. In certain preferred embodiments
of this aspect of the invention, the marker sequence is a
hexa-histidine peptide, as provided in the pQE vector (Qiagen,
Inc.) and described in Gentz et al., Proc Natl Acad Sci USA (1989)
86:821-824, or is an HA tag, or is glutathione-s-transferase. The
polynucleotide may also contain non-coding 5' and 3' sequences,
such as transcribed, non-translated sequences, splicing and
polyadenylation signals, ribosome binding sites and sequences that
stabilize mRNA.
[0080] Vectors, Host Cells, Expression
[0081] The present invention also relates to vectors which comprise
a polynucleotide or polynucleotides of the present invention, and
host cells which are genetically engineered with vectors of the
invention and to the production of peptides or polypeptides of the
invention by recombinant techniques. Cell-free translation systems
can also be employed to produce such proteins using RNAs derived
from the DNA constructs of the present invention.
[0082] For recombinant production, host cells can be genetically
engineered to incorporate expression systems or portions thereof
for polynucleotides of the present invention. Introduction of
polynucleotides into host cells can be effected by methods
described in many standard laboratory manuals, such as Davis et
al., BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook et al.,
MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989) such as calcium
phosphate transfection, DEAE-dextran mediated transfection,
transfection, microinjection, cationic lipid-mediated transfection,
electroporation, transduction, scrape loading, ballistic
introduction or infection.
[0083] Representative examples of appropriate hosts include
bacterial cells, such as meningococci, streptococci, staphylococci,
E. coli, Streptomyces and Bacillus subtilis cells; fungal cells,
such as yeast cells and Aspergillus cells; insect cells such as
Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO,
COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and
plant cells.
[0084] A great variety of expression systems can be used. Such
systems include, among others, chromosomal, episomal and
virus-derived systems, e.g., vectors derived from bacterial
plasmids, from bacteriophage, from transposons, from yeast
episomes, from insertion elements, from yeast chromosomal elements,
from viruses such as baculoviruses, papova viruses, such as SV40,
vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies
viruses and retroviruses, and vectors derived from combinations
thereof, such as those derived from plasmid and bacteriophage
genetic elements, such as cosmids and phagemids. The expression
systems may contain control regions that regulate as well as
engender expression. Generally, any system or vector suitable to
maintain, propagate or express polynucleotides to produce a
polypeptide in a host may be used. The appropriate nucleotide
sequence may be inserted into an expression system by any of a
variety of well known and routine techniques, such as, for example,
those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY
MANUAL (supra).
[0085] For secretion of the translated protein into the lumen of
the endoplasmic, reticulum, into the periplasmic space or into the
extracellular environment, appropriate secretion signals may be
incorporated into the desired polypeptide. These signals may be
endogenous to the polypeptide or they may be heterologous
signals.
[0086] Purification of Recombinantly Expressed
Peptides/Polypeptides
[0087] Peptides and polypeptides of the invention can be recovered
and purified from recombinant cell cultures by well-known methods
including ammonium sulphate or ethanol precipitation, acid
extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxyapatite
chromatography and lectin chromatography. Most preferably, high
performance liquid chromatography is employed for purification.
Well known techniques for refolding proteins may be employed to
regenerate active conformation when the polypeptide is denatured
during isolation and or purification.
[0088] Although the gene sequence of the chimeric VEGF polypeptide
in the vector can be tagged with a Histidine-tag sequence which
aids the purification of the polypeptide, it is not an essential
element to the invention, as polypeptides without the Histidine-tag
can still be purified by one of the techniques mentioned above.
[0089] Antibodies
[0090] The peptides and polypeptides of the invention, or cells
expressing them can also be used as immunogens to produce
antibodies immunospecific for the wild-type VEGF or EG-VEGF. The
term "immunospecific" means that the antibodies have substantially
greater affinity for the peptides or polypeptides of the invention
than their affinity for other related polypeptides in the prior
art.
[0091] Antibodies generated against the peptides or polypeptides
can be obtained by administering it to an animal, preferably a
nonhuman, using routine protocols in the immunization of an animal
with an antigen, the collection of the blood, the isolation of the
serum and the use of the antibodies that react with the peptide.
The serum or IgG fraction containing the antibodies may be used in
analyzing the protein. For preparation of monoclonal antibodies,
any technique which provides antibodies produced by continuous cell
line cultures can be used. Examples include the hybridoma technique
(Kohler, G. and Milstein, C., Nature (1975) 256:495-497), the
trioma technique, the human B-cell hybridoma technique (Kozbor et
al., Immunology Today (1983) 4:72) and the EBV-hybridoma technique
(Cole et al., MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96,
Alan R. Liss, Inc., 1985).
[0092] Techniques for the production of single chain antibodies
(U.S. Pat. No. 4,946,778) can also be adapted to produce single
chain antibodies to peptides or polypeptides of this invention.
Also, transgenic mice, or other organisms including other mammals,
may be used to express humanized antibodies.
[0093] The above-described antibodies may be employed to isolate or
to identify clones expressing the peptide or to purify the peptides
or polypeptides of the invention by affinity chromatography.
[0094] Vaccines
[0095] Another aspect of the invention is a vaccine composition
comprising an immunogenic amount of at least one peptide or
polypeptide of the invention. Preferably the composition should
also comprise a pharmaceutically acceptable excipient. Vaccine
preparation is generally described in Vaccine Design ("The subunit
and adjuvant approach" (eds. Powell M. F. & Newman M J). (1995)
Plenum Press New York).
[0096] Additionally, the peptides and polypeptides of the present
invention are preferably adjuvanted in the vaccine formulation of
the invention. Suitable adjuvants include an aluminum salt such as
aluminum hydroxide gel (alum) or aluminum phosphate, but may also
be a salt of calcium, iron or zinc, or may be an insoluble
suspension of acylated tyrosine, or acylated sugars, cationically
or anionically derivatized polysaccharides, or polyphosphazenes.
Other known adjuvants include CpG containing oligonucleotides. The
oligonucleotides are characterized in that the CpG dinucleotide is
unmethylated. Such oligonucleotides are well known and are
described in, for example WO96/02555.
[0097] Further preferred adjuvants are those which induce an immune
response preferentially of the TH1 type. High levels of Th1-type
cytokines tend to favor the induction of cell mediated immune
responses to the given antigen, whilst high levels of Th2-type
cytokines tend to favor the induction of humoral immune responses
to the antigen. Suitable adjuvant systems include, for example
monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl
lipid A (3D-MPL), or a combination of 3DMPL together with an
aluminum salt. CpG oligonucleotides also preferentially induce a
TH1 response. An enhanced system involves the combination of a
monophosphoryl lipid A and a saponin derivative particularly the
combination of QS21 and 313-MPL as disclosed in WO 94/00153, or a
less reactogenic composition where the QS21 is quenched with
cholesterol as disclosed in WO 96/33739. A particularly potent
adjuvant formulation involving QS21 3D-MPL & tocopherol in an
oil in water emulsion is described in WO 95/17210 and is a
preferred formulation.
[0098] Another aspect of the invention relates to a method for
inducing an immunological response in a mammal which comprises
inoculating the mammal with a peptide or polypeptide of the
invention adequate to produce antibody to inhibit angiogenesis and
to inhibit growth of tumors among others. Yet another aspect of the
invention relates to a method of inducing immunological response in
a mammal which comprises, delivering a peptide or polypeptide of
the invention via a vector directing expression of a polynucleotide
of the invention in vivo in order to induce such an immunological
response to produce antibody to protect said animal from
diseases.
[0099] A further aspect of the invention relates to an
immunological/vaccine formulation (composition) which, when
introduced into a mammalian host, induces an immunological response
in that mammal to VEGF or EG-VEGF peptide wherein the composition
comprises a polynucleotide encoding a VEGF or EG-VEGF epitope or
the VEGF or EG-VEGF epitope itself. The vaccine formulation may
further comprise a suitable carrier. The VEGF vaccine composition
is preferably administered orally, intranasally or parenterally
(including subcutaneous, intramuscular, intravenous, intradermal,
transdermal injection). Formulations suitable for parenteral
administration include aqueous and non-aqueous sterile injection
solutions which may contain anti-oxidants, buffers, bacteriostats,
and solutes which render the formulation isotonic with the blood of
the recipient; and aqueous and non-aqueous sterile suspensions
which may include suspending agents or thickening agents. The
formulations may be presented in unit-dose or multi-dose
containers, for example, sealed ampoules and vials and may be
stored in a freeze-dried condition requiring only the addition of
the sterile liquid carrier immediately prior to use. The vaccine
formulation may also include adjuvant as described above. The
dosage will depend on the specific activity of the vaccine and can
be readily determined by routine experimentation.
[0100] Yet another aspect relates to an immunological/vaccine
formulation which comprises the polynucleotide of the invention.
Such techniques are known in the art, see for example Wolff et al.,
Science, (1990) 247: 1465-8.
EXAMPLES
[0101] Exemplary methods are described below, although methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of the present peptides,
compositions and methods. All publications and other references
mentioned herein are incorporated by reference in their entirety.
The materials, methods, and examples are illustrative only and not
intended to be limiting.
Example 1
Abstract
[0102] VEGF epitopes were identified using a computer-aided
analysis employing specific correlates for antigenicity. These
epitopes were synthesized, purified, and combined the measles virus
fusion (MVF) protein, a T-cell epitope. VEGF peptide antibodies
were elicited in mice and rabbits following primary and booster
vaccination. ELISA and Western blotting determined antibody
specificity. VEGF peptide antibody function was assessed by
Fluorokine.RTM. VEGF receptor interaction assay. Vascular invasion,
a marker of angiogenesis, was determined by injecting C57BL/6 mice
subcutaneously with Matrigel.TM. (a mouse sarcoma-derived basement
membrane) incubated with and without recombinant human VEGF protein
in the presence or absence of VEGF peptide antibodies and mouse
VEGF monoclonal antibodies. Angiogenesis into the Matrigel.TM.
assessed with Hoechst staining, and angiogenesis was determined
quantitatively by counting blood vessels that invaded into the
Matrigel.TM. Differences in relative angiogenesis were compared
with Student's t-test.
[0103] VEGF peptides from the predicted antigenic region elicited
high-titer antibodies in mice and rabbits (1:500,000 at 3y+4).
These antibodies were demonstrated to be specific for rhVEGF by
ELISA and Western blot analysis. In vitro, the VEGF peptide
antibodies led to a significant disruption of the normal
interaction between VEGF and the VEGF receptor compared with
control by the Fluorokine.RTM. assay. In vivo, the Matrigel.TM.
invasion assay revealed that VEGF peptide antibodies led to a
profound quantitative decrease in blood vessel invasion into the
matrix compared with control (vascular invasion of VEGF alone=118.2
versus VEGF+VEGF peptide antibodies=26.4, P=0.005).
[0104] Conclusion: VEGF peptide antibodies elicited from antigenic
regions of VEGF are immunogenic, specific, and anti-angiogenic.
Thus, active immunization with a VEGF peptide vaccine is expected
to be a biologically relevant treatment in women with epithelial
ovarian cancer.
[0105] Materials and Methods
[0106] VEGF Epitope Selection
[0107] The selection of candidate VEGF B-cell epitopes was
performed using computer-aided analysis using specific correlates
for antigenicity (Peptide Companion, PeptiSearch), employing the
profiles of chain flexibility and motility, hydropathy, protrusion
indices, and antigenicity [Kaumaya 1994]. Sequences were given a
score of 1 to 6 based on their respective index values and were
ranked. The best scoring epitopes were further ranked by
correlation with their secondary structural attributes, where an
amphiphilic alpha-helical sequence or a beta-turn loop is preferred
over random coil fragments. Finally, consideration was given to the
individual amino acid sequence. Electrostatic ion pairs and helix
dipole interaction in helical segment were also considered
(hydrophobic/hydrophilic balance). The sequences receiving the
highest scores were selected for further investigation. Our group
has evaluated a number of antigenic peptides containing T-cell
epitopes derived from non-human sources that have been identified
to be "promiscuous" in their recognition in association with many
MHC molecules and their capacity to elicit T.sub.H responses
[Kaumaya 1993]. Measles virus fusion (MVF) protein sequence 288-302
was chosen as the promiscuous epitope to overcome the challenge of
tolerance and MHC polymorphism. The MVF epitope was linearly joined
to the VEGF epitope by a four-residue linker (GPSL) (SEQ ID NO: 10)
on a peptide synthesizer. The glycine and proline residues in the
linker potentiate a beta-turn in the oligopeptide, whereas the
serine favors hydrogen bonds with the free HN of the backbone. The
flexible nature of the linker allows for independent folding of the
T- and B-cell epitopes. Peptides were purified by reverse-phase
HPLC to ensure >95% purity. The identity of the peptides was
performed by matrix-assisted LASER desorption ionization-time of
flight spectrometry (MALDI-TOF).
[0108] Vaccination and Elicitation of VEGF Peptide Antibodies
[0109] To generate VEGF antibodies, New Zealand white rabbits
(Charles River Laboratories, Inc., Wilmington, Mass.) and BALB/c
mice (Harlan, Indianapolis, Ind.) were immunized subcutaneously at
multiple sites with a total of 1 mg of each peptide emulsified in a
Squaline/Arlacel vehicle containing nor-MDP (N-acetyl-glucosamine-3
yl-acetyl L-alanyl-D-isoglutamine). Subsequent booster injections
were given at 3 weeks (secondary immunization, 2y) and 6 weeks
(tertiary immunization, 3y) after primary (1y) immunization. Rabbit
and mouse sera were collected weekly, and complement was
inactivated by heating to 56.degree. C. High-titered sera were
purified on a protein A/G-agarose column (Pierce, Rockford, Ill.),
and eluted antibodies were concentrated and exchanged in PBS using
Mr 100,000 cutoff centrifuge filter units (Millipore, Bedford,
Mass.). Antibody concentration was quantified by ELISA.
[0110] Characterization of VEGF Peptide Antibodies
[0111] Western blot analysis was undertaken to determine whether
the VEGF peptide antibodies recognize the VEGF protein. Proteins,
including rhVEGF, were resolved by 15% SDS-PAGE, transferred to
nitrocellulose, and probed with VEGF peptide antibodies or a mouse
VEGF monoclonal antibody (Ab-4, Neoprobe, Inc., Fremont, Calif.).
Protein transfer was monitored with prestained molecular weight
standards. Immunoreactive bands were detected by enhanced
chemiluminescence (Pierce Biotechnology, Inc., Rockford, Ill.)
using horseradish peroxidase-conjugated goat anti-rabbit
immunoglobulins.
[0112] In an effort to determine the effect of our VEGF peptide
antibodies on the interaction of VEGF with the VEGF receptor, we
employed the Fluorokine.RTM. assay (R&D Systems, Minneapolis,
Minn.). In brief, 5.times.10.sup.5HUVECs were washed and incubated
with the biotinylated rhVEGF that in turn binds to the cells via
the VEGFR. The cells are then directly incubated with
avidin-fluorescein, which attaches to the receptor-bound
biotinylated VEGF. Unbound biotinylated cytokine participates in an
amplification reaction with the bound cytokine that results in an
enhanced signal without compromising specificity. Cells expressing
the VEGFR are fluorescently stained, with the intensity of staining
proportional to the density of the receptors. Relative receptor
density is then determined by flow cytometry. Through this
experiment, we initially standardized the flow cytometry component
of the assay, in which different cell populations were identified
using the supplied positive (rhVEGF) and negative (no stimulation)
controls, as well as with an inhibitory antibody provided with the
kit. Following standardization, various concentrations of mouse or
rabbit VEGF peptide antibodies were used instead of the supplied
inhibitory antibody to determine the proportional VEGFR
density.
[0113] Determination of Anti-Angiogenic Properties of VEGF Peptide
Antibodies
[0114] Analysis of blood vessel invasion was determined in a mouse
model employing Matrigel.TM., a solubilized basement membrane
matrix extracted from EHS mouse sarcoma [Passiniti]. Matrigel.TM.
is a liquid at 4.degree. C., but polymerizes at 4.degree. C., thus
allowing for its removal from an animal host for analysis. Sets of
5 female C57BL/6 mice (Harlan, Indianapolis, Ind.) were injected
subcutaneously with a total volume of 500 .mu.L including
Matrigel.TM., various concentrations of rhVEGF, and various
concentrations of antibody (either VEGF monoclonal antibody
(MAB293, R&D Systems, Minneapolis, Minn.) or VEGF peptide
antibodies). After 10 days, the mice were sacrificed. The
Matrigel.TM. plugs were then removed, sectioned, and stained with
hematoxylin and eosin and the nuclear stain Hoechst 33342. Invasion
into the plug was determined using an inverted fluorescent
microscope at 40.times. magnification. Blood vessels at the
periphery of the plug were identified and counted in a
circumferential manner around the plug, and counted using
computer-aided analysis in a blinded fashion. Statistical
comparisons between groups were made using the Student's
t-test.
[0115] Results
[0116] Computer-aided analysis of candidate B-cell epitopes of VEGF
was used to select residues 126-143 of SEQ ID NO: 1
(KCECRPKKDRARQENPCG), which correlates with a secondary structure
of turn-helix-turn, as being potentially immunogenic and antigenic.
This epitope was linearly joined to the promiscuous T-cell epitope
of the measles virus protein (MVF) with the four-residue GPSL (SEQ
ID NO: 10) linker on a peptide synthesizer. Peptides were purified,
and their identity confirmed by MALDI-TOF. Rabbits and mice were
immunized subcutaneously with the MVF-VEGF immunogen, and sera was
obtained and purified. High VEGF peptide antibody titers (1:500,000
at 4 weeks following tertiary immunization, 3y+4) were identified
by ELISA as demonstrated in rabbits in FIG. 1.
[0117] ELISA (FIG. 2) and Western blot (FIG. 3A) demonstrate that
VEGF peptide antibodies recognize the rhVEGF protein. Ab-4, a
monoclonal antibody against VEGF, was used as a positive control in
the Western blot, and confirmed the expected rhVEGF protein
homodimer at 42 kDa (FIG. 3B).
[0118] Following demonstration of the antigenic and immunogenic
properties of the VEGF peptide antibodies, the functional
properties of the antibodies were evaluated. These VEGF peptide
antibodies were demonstrated to significantly disrupt the normal
interaction between VEGF and the VEGF receptor (VEGFR) as
determined by the Fluorokine.RTM. assay (FIG. 4). In this
experiment, the addition of the VEGF peptide antibodies leads to
binding of VEGF, thus leading to a decrease in the normal
VEGF-VEGFR interaction.
[0119] We went on to determine whether these VEGF peptide
antibodies had anti-angiogenic properties as a result of inhibition
of VEGF function. When compared with subcutaneous plugs of
Matrigel.TM. incubated with rhVEGF prior to injection in C57BL/6
mice, plugs concurrently incubated with rhVEGF and VEGF peptide
antibodies demonstrated significantly decreased angiogenesis into
the Matrigel.TM. (P=0.005, FIGS. 5 and 6). The anti-angiogenic
properties of the VEGF peptide antibodies were equivalent to that
of the VEGF monoclonal antibody used as a positive control
(P.dbd.NS comparing VEGF peptide antibodies to VEGF monoclonal
antibodies, data not shown).
[0120] Discussion
[0121] We demonstrate that with rational peptide design employing
VEGF B-cell epitopes, VEGF-specific autoantibodies are elicited.
These antibodies recognized the full length protein from which the
peptide was designed, and inhibit the expected protein function.
Immunotherapy for cancer treatment has evolved substantially over
the past decades. Previously, patients were treated with
nonspecific immune stimulants, whereas currently therapy is focused
on identifying specific tumor-associated antigens (TAAs) as targets
for immunotherapy. Tumor-specific immunotherapy can be categorized
into passive, where antibodies are targeted directly to tumor
cells, and active, where vaccination with peptides, tumor cells,
tumor cell lysates, carbohydrates, gene constructs, and
anti-idiotpye antibodies that mimic TAAs are employed in a host
that mounts a specific immune response.
[0122] Historically, active immunization with peptides has had
limited efficacy because of their limited immunogenicity.
Antibodies elicited in animals by immunization with synthetic
peptides have generally been shown to have low affinity for the
native protein, partly because antibody recognition sites are
usually conformational, and the peptide immunogens lacked defined
structure in solution. The genetically restricted stimulatory
activity of peptides was also a major obstacle to developing
vaccine approaches for use in an outbred human population
[Dulofeut]. Covalent conjugation of B-cell epitope peptides to
large carrier molecules was sometimes used to address this problem
but often resulted in hypersensitivity, conformational changes,
appearance of undefined structures, loss of epitopes, inappropriate
presentation of epitopes, and batch-to-batch conjugate variability.
We have addressed several of these issues in our rational approach
to subunit peptide vaccine design [Dakappagari].
[0123] Our strategy involved de novo design of topographic
determinants that focused on preserving the native protein sequence
while facilitating folding of the peptide into a stable
conformation that mimics the native protein structure [Kobs,
Kaumaya 1990]. We have demonstrated the effectiveness of
incorporating promiscuous T-helper epitopes derived from nonhuman
molecules into these constructs to overcome human MHC genetic
polymorphism [Kaumaya 1993]. Our previous work in a variety of
model systems has demonstrated that this approach can elicit
high-titer antibodies that recognize native protein in an outbred
population, and is confirmed in this investigation of VEGF
epitopes.
[0124] Importantly, subunit peptide vaccines can focus immune
responses to biologically active epitopes. The need for
epitope-based vaccines stems from the fact that tolerance to
self-antigens, such as VEGF, may limit a functional immune response
to whole protein-based vaccines due to activation of suppressor T
cells that maintain tolerance to host antigens or alternate
regulatory mechanisms [Sakaguchi]. The capacity to narrowly focus
the immune response is of particular relevance to VEGF, where
interaction of the antibody with specific sites has the potential
of inhibiting growth. In contrast to passive therapy, the
continuous availability of tumor-targeting antibodies can be
ensured at low cost.
[0125] Previous investigators have developed similar strategies of
anti-VEGF cancer therapy. Interest in VEGF as a model antigen to
explore immunogene therapy has been demonstrated through the
construction of a plasmid DNA encoding Xenopus homologous VEGF
[Wei]. This group determined that immunogene tumor therapy with
this vaccine led to the development of VEGF-specific antibodies
that were anti-angiogenic and inhibited tumor formation.
Importantly, treatment of mice with the immunogene led to no
significant toxic effects. In other work, vaccination with
dendritic cells transfected with VEGF mRNA has been demonstrated to
lead to cytotoxic T lymphocyte (CTL) responses, to the disruption
of angiogenesis, and to antitumor efficacy without significant
morbidity or mortality in vivo in a murine model [Nair]. Thus,
previous work has demonstrated the feasibility of active
immunization using VEGF as a TAA.
[0126] Limitations of this investigation are the fact that the
antigen chosen for investigation, VEGF, is ubiquitously expressed
in normal and pathologic conditions, and its inhibition may lead to
potentially serious biologic consequences. Although fetal
development is strongly controlled by angiogenesis, only
reproduction, wound healing and cancer are controlled by
angiogenesis in the adult host. As such, we believe that the
relative control and expression of VEGF overexpression in
malignancy would lead to an acceptable therapeutic ratio in the
treatment of solid tumors. This is supported by previous
investigation of other methods of decreasing the effects of VEGF
(i.e. through DNA vaccines or inhibition of the VEGFR) that failed
to demonstrate significant toxicity.
[0127] Most women with ovarian cancer are diagnosed with advanced
disease, and despite the majority obtaining a complete clinical
response following induction chemotherapy, 80% will recur and
succumb to their disease. This scenario suggests that microscopic
residual disease after initial therapy is responsible for disease
recurrence. For this reason, a current clinical research focus in
the treatment of ovarian cancer is the consideration of maintenance
chemotherapy. Here, following initial treatment, patients achieving
a complete clinical response have been demonstrated to have a
better disease-free survival when a prolonged course of treatment
is initiated immediately [Markman].
[0128] Interestingly, investigation of the role of active
immunization with the anti-idiotype antibody ACA125 (which imitates
the tumor-associated antigen CA125 in ovarian cancer) as a
maintenance chemotherapy in ovarian cancer has demonstrated a
positive impact on overall survival [Wagner]. Thus, active
immunization as maintenance chemotherapy to prevent symptomatic
recurrence of ovarian cancer is an attractive concept. Angiogenesis
has been demonstrated to influence cancer growth variably at
different stages of malignant proliferation. Importantly,
premalignant neoplastic conditions and small malignant tumors are
thought to grow under the direct influence of endothelial mitogens
such as VEGF, whereas larger malignant tumors may grow and
metastasize independent of angiogenic factors [Hanahan and
Folkman]. The concept that angiogenic factors control early tumor
growth has been applied to the clinical management of ovarian
cancer. Current research efforts are directed at investigating
chemotherapy agents that may act as anti-angiogenic, cytostatic
agents. These compounds, such as tamoxifen and thalidomide, are
being evaluated in women with early recurrent, asymptomatic ovarian
cancer to determine if anti-angiogenic therapy may prevent the
development of clinically significant, symptomatic disease. As
such, anti-angiogenic therapy with active immunization using VEGF
epitopes could serve as a rational maintenance therapy that could
significantly impact the treatment of ovarian cancer.
[0129] From this investigation, we demonstrate that rational design
of peptide vaccines against VEGF leads to elicitation of
high-titered VEGF peptide antibodies that are specific and
anti-angiogenic.
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stage III and stage IV ovarian cancer. N Engl Med 1996; 334:1-6.
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N. Vascular endothelial growth factor is a secreted angiogenic
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McLaren J, Barker P J, Wright K A, Twentyman P R, Smith S K.
Expression of vascular endothelial growth factor and its receptors
flt and KDR in ovarian carcinoma. J Natl Cancer Inst 1995;
87:506-16. [0135] 6. Hollingsworth H C, Kohn E C, Steinberg S M,
Rothenberg M L, Merino M J. Tumor angiogenesis in advanced stage
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J, Staskus K A, Gebhard K, Mohanraj D, Twiggs L B, Carson L F,
Ramakrishnan S. Vascular endothelial growth factor expression in
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Vascular endothelial growth factor serum concentrations in ovarian
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Winer J, Armanini M, Gillett N, Phillips H S, Ferrara N. Inhibition
of vascular endothelial growth factor-induced angiogenesis
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10. Gordon M S, Margloin K, Talpaz M, Sledge G W Jr, Holmgren E,
Benjamin R, Stalter S, Shak S, Adelman D. Phase I safety and
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endothelial growth factor in patients with advanced cancer. J Clin
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E, Gaudreault J, Fyfe G, Adelman B, Stalter S, Breed J. Phase Ib
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safety data. J Clin Oncol 2001:19:851-6. [0141] 12. Dakappagari N,
Douglas D B, Triozzi P L, Stevens V C, Kaumaya P T P. Prevention of
mammary tumors with a chimeric HER-2 B-cell epitope peptide
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Goedegebuure P S, Smith R, Linehan D C, Yoshino I, Eberlein T J.
Breast and ovarian cancer-specific cytotoxic T lymphocytes
recognize the same HER2/neu-derived peptide. Proc Natl Acad Sci USA
1995; 92:432-6. [0143] 14. Kaumaya P T P, Kobs-Conrad S, DiGeorge A
M, Stevens V. De novo engineering of protein immunogenic and
antigenic determinants. Peptides 1994; 9:133-64. [0144] 15. Kaumaya
P T, Kobs-Conrad S, Seo Y H, Lee H, Van Buskirk A M, Feng N,
Sheridan J F, Stevens V. Peptide vaccines incorporating a
"promiscuous" T-cell epitope bypass certain haplotype restricted
immune responses and provide broad spectrum immunogenicity. J Mol
Recognit 1993; 6:81-94. [0145] 16. Passaniti A, Taylor R M, Pili R,
Guo Y, Long P V, Haney J A, Pauly R R, Grant D S, Martin G R. A
simple, quantitative method for assessing angiogenesis and
antiangiogenic agents using reconstituted basement membrane,
heparin, and fibroblast growth factor. Lab Invest 1992; 67:519-28.
[0146] 17. Deulofeut H, Iglesias A, Mikael N, Bing D H, Awdeh Z,
Yunis J, Marcus-Bagley D, Kruskall M S, Alper C A, Yunis E J.
Cellular recognition and HLA restriction of a midsequence HBsAg
peptide in hepatitis B vaccinated individuals. Mol Immunol 1993;
30:941-948. [0147] 18. Kobs-Conrad S, Lee H, DiGeorge A M, Kaumaya
P T. Engineered topographic determinants with .alpha..beta.,
.beta..alpha..beta., and .beta..alpha..beta..alpha. topologies show
high affinity binding to native protein antigen (lactate
dehydrogenase-C4). J Biol Chem 1993; 268:25285-25295. [0148] 19.
Kaumaya P T, Berndt K D, Heidorn D B, Trewhella J, Kezdy F J,
Goldberg E. Synthesis and biophysical characterization of
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.alpha..alpha.topology. Biochemistry 1990; 29:13-23. [0149] 20.
Sakaguchi S. Regulatory T cells: key controllers of immunologic
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J, Yang L, Zhao X, Tian L, Lu Y, Shu J M, Lu C J, Niu T, Kang B,
Mao Y Q, Liu F, Wen Y J, Lei S, Luo F, Zhou L Q, Peng F, Jiang Y,
Liu J Y, Zhou H, Wang Q R, He Q M, Xiao F, Lou Y Y, Xie X J, Li Q,
Wu Y, Ding Z Y, Hu B, Hu M, Zhang W. Immunogene therapy of tumors
with vaccine based on Xenopus homologous vascular endothelial
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98:11545-50. [0151] 22. Nair S, Boczkowski D, Moeller B, Dewhirst
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J, Kowalski J, Foster J, Hass P, Zhang Z, Dillard-Telm L, Frantz G,
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Ferrara N. Identification of an angiogenic mitogen selective for
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Example 2
Selection of VEGF and EG-VEGF Epitopes
[0156] The selection of candidate VEGF and EG-VEGF B-cell epitopes
has been performed using computer-aided analysis using specific
correlates for antigenicity employing the profiles of chain
flexibility and motility, hydropathy, protrusion indices, and
antigenicity. Sequences were given a score of 1 to 6 based on their
respective index values and were ranked. The best scoring epitopes
were further ranked by correlation with their secondary structural
attributes, where an amphiphilic alpha-helical sequence or a
beta-turn loop is preferred over random coil fragments. Finally,
consideration was given to the individual amino acid sequence.
Electrostatic ion pairs and helix dipole interaction in helical
segment were also considered (hydrophobic/hydrophilic balance). The
sequences receiving the highest scores were selected for further
investigation. Table 1 lists the sequences and secondary structure
for VEGF and EG-VEGF epitopes selected for investigation.
[0157] Our group has evaluated a number of antigenic peptides
containing T-cell epitopes derived from non-human sources that have
been identified to be "promiscuous" in their recognition in
association with many MHC molecules and their capacity to elicit
T.sub.H responses. Measles virus fusion (MVF) protein sequence
288-302 was chosen as the promiscuous epitope to overcome the
challenge of tolerance and MHC polymorphism. The MVF epitope was
linearly joined to the VEGF or EG-VEGF epitope by a four-residue
linker (GPSL) (SEQ ID NO: 10) on a peptide synthesizer. The glycine
and proline residues in the linker potentiate a beta-turn in the
oligopeptide, whereas the serine will favor hydrogen bonds with the
free HN of the backbone. The flexible nature of the linker allows
for independent folding of the T- and B-cell epitopes. Peptides
were purified by reverse-phase HPLC to ensure >95% purity. The
identity of the peptides was performed by matrix-assisted LASER
desorption ionization-time of flight spectrometry.
[0158] To generate anti-VEGF and anti-EG-VEGF antibodies, rabbits
were immunized subcutaneously at multiple sites with a total of 1
mg of each peptide emulsified in a Squaline/Arlacel vehicle
containing nor-MDP (N-acetyl-glucosamine-3 yl-acetyl
L-alanyl-D-isoglutamine). Subsequent booster injections were given
at 3 and 6 weeks after primary immunization. Rabbit sera was
obtained weekly and purified, and quantified by ELISA (FIG. 7).
[0159] Following the purification of anti-VEGF and anti-EG-VEGF
antibodies, we went on to determine whether the elicited antibodies
had anti-angiogenic properties. Previous data demonstrates that
ovarian function is tightly regulated through angiogenic stimuli,
and VEGF has been shown to be important in the recruitment and
selection of follicles. We thus hypothesized that if the antibodies
were functioning as anti-angiogenic molecules, then inhibition of
follicle selection and growth and estrous cycle disruption would be
expected with neutralization of VEGF.
[0160] Thirteen female C57BL/6 mice were injected intraperitoneally
with 25 .mu.g of purified anti-VEGF antibody every 3 days for 15
days. Estrous cycles were monitored by obtaining daily vaginal
smears, and the estrous cycles of treated mice were compared with
control mice injected with nonspecific IgG antibodies. In keeping
with the anti-angiogenic effect of elicited anti-VEGF antibodies, a
significant disruption of the established estrous cycle was seen
beginning day 3 following immunization (FIG. 8A). The average
length of the estrous cycles was also significantly decreased by
over 66% (FIG. 8B). Primordial follicle growth was assessed at day
3 and day 7 following immunization by harvesting the ovaries of
euthanized mice. A significant and persistent reduction in the
number of primordial follicles (p<0.05 for both comparisons) was
demonstrated in mice treated with anti-VEGF antibodies, suggesting
potent anti-angiogenic efficacy of the VEGF antibodies (FIG.
8C).
[0161] Collectively, these data demonstrated that immunogenic
epitopes of VEGF and EG-VEGF can be identified and synthesized, and
immunization leads to production of anti-VEGF and anti-EG-VEGF
antibodies. Such antibodies are biologically active, and function
as anti-angiogenic molecules.
[0162] Determination of Immunogenicity of Anti-VEGF and
Anti-EG-VEGF Antibodies Elicited from Active Immunization with
Peptide Vaccines
[0163] ELISA demonstrated anti-VEGF and anti-EG-VEGF peptide
antibodies to recognize the respective recombinant human VEGF (FIG.
9A) or EG-VEGF protein (FIG. 9B). In this experiment, recombinant
proteins were used to coat ELISA plates, and the appropriate
anti-peptide antibody was used.
[0164] Furthermore, we demonstrated that the anti-VEGF and
anti-EG-VEGF peptide antibodies recognize rhVEGF by Western blot,
with the resultant bands in the expected location (42 kD, dimer)
for VEGF blotted with anti-VEGF peptide antibody (FIG. 10A) and
VEGF monoclonal antibody (NeoMarkers Ab-4, FIG. 10B). Likewise, a
Western blot with rhEG-VEGF blotted with anti-EG-VEGF peptide
antibodies (FIG. 100) and a combination anti-VEGF/anti-EG-VEGF
peptide antibody (FIG. 10D) resulted in a band in the expected
location (22 kD, dimer) for rhEG-VEGF.
[0165] Assessment of in Vitro Biologic Effect of anti-VEGF and
anti-EG-VEGF Peptide Antibodies
[0166] Following demonstration of the antigenic and immunogenic
properties of our anti-peptide antibodies, we went on to evaluate
the functional properties of these molecules. In an effort to
determine the effect of our anti-VEGF peptide antibodies on the
interaction of VEGF with the VEGF receptor, we used the Fluorokine
assay. Briefly, washed cells are incubated with the biotinylated
cytokine that in turn binds to the cells via specific cell surface
receptors. The cells are then directly incubated with
avidin-fluorescein, which attaches to the receptor bound
biotinylated cytokine. Unbound biotinylated cytokine participates
in an amplification reaction with the bound cytokine that results
in an enhanced signal without compromising specificity.
[0167] Cells expressing the specific cytokine receptors are
fluorescently stained, with the intensity of staining proportional
to the density of the receptors. Relative receptor density is then
determined by flow cytometric analysis using 488 nm wavelength
laser excitation. Through this experiment, we initially
standardized the flow cytometry component of the assay (FIG. 11A),
in which different cell populations were identified using the kit
supplied positive and negative controls. Following standardization,
we used HUVEC cells that proliferate under the influence of VEGF to
determine the efficacy of binding of our anti-peptide antibodies to
VEGF, resulting in disruption of the normal VEGF-VEGF receptor
interaction. As can be seen in FIG. 11B, a shift in population
toward decreased receptor density is demonstrated when anti-VEGF
peptide antibodies are used, suggesting a disruption in the normal
VEGF-VEGF receptor interaction.
[0168] Assessment of Anti-Angiogenic In Vivo Biologic Effect of
Anti-VEGF Peptide Vaccines
[0169] Although we have demonstrated the interaction between VEGF
and our anti-peptide VEGF antibodies, it is still important to
determine whether the effect of this interaction leads to a
modification of angiogenic properties of VEGF. To determine the
anti-angiogenic properties of our antibodies, we injected
subcutaneous matrigel (basement membrane generated from EHS
sarcoma) into immunocompetent C57/BL6 mice, with or without rhVEGF.
The matrigel was removed after 7 days, and cryostat sections were
cut and stained with a conjugate of CD31 (which binds to
endothelial cells) and phycoerythrin. Angiogenesis was
qualitatively and quantitatively determined through fluorescence
confocal microscopy. The addition of rhVEGF to the matrigel led to
a significant increase in angiogenesis relative to control.
[0170] The specification is most thoroughly understood in light of
the teachings of the references cited within the specification, all
of which are hereby incorporated by reference in their entirety.
The embodiments within the specification provide an illustration of
embodiments of the invention and should not be construed to limit
the scope of the invention. The skilled artisan recognizes that
many other embodiments are encompassed by the claimed invention and
that it is intended that the specification and examples be
considered as exemplary only, with a true scope and spirit of the
invention being indicated by the following claims.
Sequence CWU 1
1
101190PRTHomo sapiens 1Met Asn Phe Leu Leu Ser Trp Val His Trp Ser
Leu Ala Leu Leu Leu1 5 10 15Tyr Leu His His Ala Lys Trp Ser Gln Ala
Ala Pro Met Ala Glu Gly 20 25 30Gly Gly Gln Asn His His Glu Val Val
Lys Phe Met Asp Val Tyr Gln 35 40 45Arg Ser Tyr Cys His Pro Ile Glu
Thr Leu Asp Ile Phe Gln Glu Tyr 50 55 60Pro Asp Glu Ile Glu Tyr Ile
Phe Lys Pro Ser Cys Val Pro Leu Met65 70 75 80Arg Cys Gly Gly Cys
Ser Asn Asp Glu Gly Leu Glu Cys Val Pro Thr 85 90 95Glu Glu Ser Asn
Ile Thr Met Gln Ile Met Arg Ile Lys Pro His Gln 100 105 110Gly Gln
His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys Glu 115 120
125Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Asn Pro Cys Gly Pro
130 135 140Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln Asp Pro Gln
Thr Cys145 150 155 160Lys Cys Ser Cys Lys Asn Thr His Ser Arg Cys
Lys Ala Arg Gln Leu 165 170 175Glu Leu Asn Glu Arg Thr Cys Arg Cys
Asp Lys Pro Arg Arg 180 185 1902105PRTHomo sapiens 2Met Arg Gly Ala
Thr Arg Val Ser Ile Met Leu Leu Leu Val Thr Val1 5 10 15Ser Asp Cys
Ala Val Ile Thr Gly Ala Cys Glu Arg Asp Val Gln Cys 20 25 30Gly Ala
Gly Thr Cys Cys Ala Ile Ser Leu Trp Leu Arg Gly Leu Arg 35 40 45Met
Cys Thr Pro Leu Gly Arg Glu Gly Glu Glu Cys His Pro Gly Ser 50 55
60His Lys Val Pro Phe Phe Arg Lys Arg Lys His His Thr Cys Pro Cys65
70 75 80Leu Pro Asn Leu Leu Cys Ser Arg Phe Pro Asp Gly Arg Tyr Arg
Cys 85 90 95Ser Met Asp Leu Lys Asn Ile Asn Phe 100
105319PRTClostridium tetani 3Asn Ser Val Asp Asp Ala Leu Ile Asn
Ser Thr Ile Tyr Ser Tyr Phe1 5 10 15Pro Ser Val417PRTClostridium
tetani 4Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn Gln Ser
Ser1 5 10 15Glu515PRTClostridium tetani 5Gln Tyr Ile Lys Ala Asn
Ser Lys Phe Ile Gly Ile Thr Glu Leu1 5 10 15621PRTClostridium
tetani 6Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val
Ser1 5 10 15Ala Ser His Leu Glu 20715PRTMeasles virus 7Leu Ser Glu
Ile Lys Gly Val Ile Val His Arg Leu Glu Gly Val1 5 10
15815PRTHepatitis B virus 8Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile
Pro Gln Ser Leu Asn1 5 10 15920PRTPlasmodium vivax 9Thr Cys Gly Val
Gly Val Arg Val Arg Ser Arg Val Asn Ala Ala Asn1 5 10 15Lys Lys Pro
Glu 20104PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide linker 10Gly Pro Ser Leu1
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