Note: Descriptions are shown in the official language in which they were submitted.
WO 99/45018
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ACITVE IMMUNIZATION AGAINST ANGIOGENESIS-ASSOCIATED
ANTIGENS
BACKGROUND OF THE INVENTION
Current immunotherapeutic approaches to inhibiting angiogenesis rely on the
passive administration of large amounts of antibodies to angiogenesis targets
such as the vitronectin receptor and vascular endothelial cell growth factor
(VEGF). Limitations of this therapeutic strategy include the difficulty of
administering large amounts of monoclonal antibodies to a patient and
maintaining a constant high level of antibodies over a long period of time in
a
patient.
The present invention overcomes these problems by using active specific
immunotherapy against angiogenesis target molecules to inhibit angiogenesis.
Such methods of immunotherapy against angiogenic molecutes include
modification of immunogens to cause an immune response against the
angiogenic molecules. Modification of the target antigen can be achieved by,
for example, conjugation of immunogenic reagents to the antigen (see US
Patent Nos. 5,334,379); haptenization of the antigen (see U.S. Patent Nos.
4,778,752 and 5,290,551 ); the use of adjuvants bound to, or administered
with,
the target antigen; binding peptide fragments to the antigen; binding the
target
antigen to MHC class I and class II restricted antigens (see U.S. Patent No.
4,478,823); changing giycosylation patterns of the target antigens (see U.S.
Patent No. 5,484,735); presenting the target antigen on antigen presenting
cells, such as dendritic cells (see U.S. Patent Nos. 5,580,563 and 5,788,963),
among other methods.
Another method where immunity is induced to an angiogenesis target molecule
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is that of using an anti-idiotypic antibody bearing the internal image of the
target
antigen. Since the mimicry of the target "self' antigen by the anti-idiotype
is
likely to be imperfect, such anti-idiotypes will break tolerance to a self
antigen,
although administration of the self antigen is not able to do so. It has been
previously shown that anti-idiotypes that bear the internal image of
carcinoembryonic antigen (CEA) can induce anti-CEA antibodies in patients
with colorectal carcinoma, while CEA itself cannot do so. Therefore, an
alternative approach to using the actual "self' angiogenesis target antigen as
a vaccine is to use an anti-idiotypic antibody that mimics the antigen, bears
an internal image of the antigen, and elicits an immune response.
An anti-idiotypic antibody, termed either an AB2 or an anti-idiotype, is one
produced in response to the presence of an antibody in an immunologically
active system. Anti-idiotypes are antibodies directed against the antigen-
combining region or variable region (known as the idiotype) of another
antibody
molecule.
The theory of idiotypic relationships and networks is based on the Jeme model
(Jeme, N.K. (1974) Ann. Immunof. {Paris) 125C: 373; Jeme, N.K. et al. (1982)
EMBO 1:234). Thus immunization with an antibody expressing a paratope
(antigen combining site) for a given antigen, should result in anti-antibodies
(anti-idiotypes}, some of which share with the antigen a complementary
structure to the paratope. Those anti-idiotypes could then possibly act as
antigens, i.e., mimic the antigen. Thus, the immune system would cant' within
it an internal image of the antigen, the anti-idiotype.
Anti-idiotypic antibodies have been studied as potential vaccines against
pathogenic organisms (Kennedy, R. C. et al., 1986, Science 232:220;
Reagan, K. J. et al., 1983, J. Virol. 48:660; McNamara, M. K. et al., 1984,
Science 226:1325; Sachs, D. L. et ai., 1982, J. Exp. Med. 155:1108) and
malignant tumors (Chen, Z. J. et al., 1991, J. Immunol. 147(3):1082; Dunn, P.
L. et al., 1987, J. Immunol. 60:181-187; Herlyn, D. et al., 1987, Proc. Natl.
Aced. Sci. USA 84:8055; Chattopadhyay, P, et al., 1991, Cancer Res.
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3
51:3183). In animal studies, murine anti-idiotypic antibodies have
demonstrated antigen-specific responses across species (xenogeneic model)
{Chapman, P. B. and A. N. Houghton, 1991, J. Clin. Invest. 88:186) and
within the same inbred species (syngeneic model) (Gaulton, G. N. et al.,
1986, J. Immunol. 137:2930; Chen, Z. J. et al., 1991, J. Immunol.
147(3):1082;).
Although anti-idiotypic antibodies of angiogenic factors have been developed
(W0/9608513, published March 21, 1996), including those for modulation of
tumor progression (Ortega, N. et al., C. R. Acad. Sci. III (FRANCE), May 1996,
319(5):411-415), there has been no disclosure prior to this invention of the
use
of anti-idiotypic antibodies mimicking angiogenic factor receptors, i.e.,
integrins,
and VEGF receptors such as basic FGF receptor, kdr, flk-1, or flt-1, to
provide
active immunity against angiogenesis. Therefore, this invention provides the
first example of such anti-idiotypes being used to prevent or inhibit
angiogenesis.
Blood vessels are formed by vasculogenesis, a process during which a primary
capillary plexus is formed that is remodelled either by fusion or regression,
and
angiogenesis (also called neovascularization), a process in which vasculature
is
formed by new vessels sprouting from preexisting vessels and invading the
developing organ. (Breier et al. 1996). Angiogenesis is an important process
in the menstrual cycle in the endometrium, in pregnancy, and during neonatal
growth. Angiogenesis is also important in wound healing and in the
pathogenesis of a large variety of clinical diseases including tissue
inflammation, arthritis, tumor growth, diabetic retinopathy, and macular
degeneration by neovascularization of the retina. These clinical
manifestations
associated with angiogenesis are refer-ed to as angiogenic diseases. {Folkman
et al., Science, 235:442-447 (1987). Angiogenesis is generally absent in
healthy adult or mature tissues, although it does occur in wound healing and
in
the corpus tuteum growth cycle. See, for example, Moses et al., Science,
248:1408-1410 (1990).
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Angiogenesis is required for tumor proliferation, since tumors need an
adequate blood perfusion to obtain nutrients. Inhibiting angiogenesis by
limiting
vessel growth or selectively destroying proliferating endothelium would
natrict
tumor growth. Proposed methods of inhibiting angiogenesis include: (1 )
inhibition of release of "angiogenic molecules" such as basic-FGF (basic
fibroblast growth factor), (2) neutralization of angiogenic molecules, such as
by
use of anti-basic-FGF antibodies, and (3) inhibition of endothelial cell
response
to angiogenic stimuli. Folkman et al., Cancer Biology, 3:89-96 (1992), have
described several endothelial cell response inhibitors, including collagenase
inhibitor, angiostatic steroids, fungal-derived angiogenesis inhibitors,
platelet
factor 4, thrombospondin, arthritis drugs such as D-peniciNamine and gold
thiomalate, vitamin D3 analogs, alpha-interferon, and others that might be
used
to inhibit angiogenesis. For additional proposed inhibitors of angiogenesis,
see
Blood et al., Bioch. Biophys. Acta., 1032:89-8 (1990), Moses et al., Science
248:1408-1410 (1990), Ingber et al., Lab. Invest., 59:44-51 (1988), and U.S.
Patent Nos. 5,092,885; 5,112,946; 5,192,744; and 5,202,352. Other new
inhibitors of angiogenesis include angiostatin (O'Reilly et al., Cell 79:185-
188
(Oct. 1994)) and endostatin.
The vascular endothelium is usually quiescent and its activation is tightly
regulated during angiogenesis. Several factors have been implicated as
possible regulators of angiogenesis in vivo. These include transforming growth
factor (TGFb), acidic and basic fibroblast growth factor (aFGF and bFGF),
platelet derived growth factor (PDGF), certain integrins, and vascular
endothelial growth factor (VEGF) (Klagsbrun, M. and D'Amore. P. (1991 )
Annual Rev. Physiol. 53: 217-239).
VEGF, an endothelial cell-speck mitogen, acts as an angiogenesis inducer by
specifically promoting the proliferation of endothelial cells. VEGF is a
homodimeric glycoprotein consisting of two 23 kD subunits with structural
similarity to PDGF. Four different monomeric isoforms of VEGF exist resulting
from alternative splicing of mRNA. These include two memorane bound forms
(VEGF206 and VEGF189) and two soluble forms (VEGF165 and VEGF121 ).
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r
In all human tissues except placenta, VEGF165 is the most abundant isoform.
VEGF is expressed in embryonic tissues (Breier et al., Development (Camb.)
114:521 (1992)x, macrophages, proliferating epidermal keratinocytes during
wound healing ;Brown et al., J. Exp. Med., 176:1375 (1992)), and may be
responsible for tissue edema associated with inflammation (Ferrara et al.,
Endocr. Rev. 13:18 {1992)). In situ hybridization studies have demonstrated
high VEGF expression in a number of human tumor lines including
giioblastoma multiforme, hemangioblastoma, central nervous system
neoplasms and AIDS-associated Kaposi's sarcoma (Plate, K. et al. (1992)
Nature 359: 845-848; Plate, K. et al. (1993) Cancer Res. 53: 5822-5827;
Berkman, R. et al. (1993) J. Clin. Invest. 91: 153-159; Nakamura, S. et al.
(1992) AIDS Weekly, 13 (1 )). High levels of VEGF were also observed in
hypoxia induced angiogenesis (Shweiki, D. et al. (1992) Nature 359: 843-845).
The biological response of VEGF is mediated through its high affinity VEGF
receptors which are selectively expressed on endothelial cells during
embryogenesis (Millauer, B., et al. (1993) Cell 72: 835-846) and during tumor
formation. VEGF receptors typically are class III receptor type tyrosine
kinases
characterized by having several, typically 5 or 7, immunoglobulin-like loops
in
their amino-terminal extracellular receptor ligand-binding domains (Kaipainen
et
al., J. Exp. Med. 178:2077-2088 (1993)). The other two regions include a
transmembrane region and a carboxy terminal intracellular catalytic domain
interrupted by an insertion of hydrophilic interkinase sequences of variable
lengths, called the kinase insert domain (Terman et al., Oncogene 6:1677-1683
(1991 ). VEGF receptors include flt-1, sequenced by Shibuya M. et al.,
Oncogene 5, 519-524 (1990); KDR, described in PCTIUS92/01300, filed
February 20, 1992, and in Terman et al., Oncogene 6:1677-1683 (1991 ); and
tlk-9, sequenced by Matthews W. et al. Proc. Natl. Acad. Sci. USA, 88:9026-
9030 (1991 ). KDR is the human form of flk-1.
High levels of flk-1 are expressed by endothelial cells that infiltrate
gliomas
(Plate, K. et al., (1992) Nature 359: 845-848). Flk-1 levels are specifically
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6
upregulated by VEGF produced by human glioblastomas (Plate, K. et al.
(1993) Cancer Res. 53: 5822-5827). The finding of high levels of flk-1
expression in glioblastoma associated endothelial cells (GAEC) indicates that
receptor activity is probably induced during tumor formation since flk-1
transcripts are barely detectable in normal brain endothelial cells. This
upregulation is confined to the vascular endothelial cells in close proximity
to
the tumor. Blocking VEGF activity with neutralizing anti-VEGF monoclonal
antibodies (mAbs) resulted in an inhibition of the growth of human tumor
xenografts in nude mice (Kim, K. et al. (1993) Nature 362: 841-844),
indicating
a direct role for VEGF in tumor-related angiogenesis.
Integrins are a class of cellular receptors known to bind extracellular matrix
proteins, and therefore mediate cell-cell and cell-extracellular matrix
interactions, called cell adhesion events. The integrin receptors constitute a
family of proteins with shared structural characteristics of non-covalent
heterodimeric glycoprotein complexes formed of a and p subunits.
Angiogenesis in tissues has been shown to require integrin av~i3, and
inhibitors
of avp3 have been shown to inhibit angiogenesis (PCT Int'l. Application No.
PCTlUS95/03035, filed March 9, 1995). However, the use of anti-idiotypic
antibodies mimicking any integrins, or any other angiogenesis targets, to
inhibit
angiogenesis has not been previously demonstrated.
Although angiogenesis related receptors are upregulated in tumor infiltrated
vascular endothelial cells, the expression of these receptors is low in normal
cells that are not associated with angiogenesis. Therefore, such normal cells
would not be affected by inducing an immune response to such receptors to
inhibit angiogenesis, and therefore to inhibit tumor growth.
An object of this invention is to provide a method of inhibiting an unwanted
angiogenic condition, such as tumor angiogenesis, fieumatoid arthritis,
diabetic
retinopathy and psoriasis, by inducing an immune response in the subject
against an angiogenic molecule. Another object of this invention is to provide
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immunogens that are capable of inducing an immune response in a subject
against an angiogenic molecule.
SUMMARY OF THE INVENTION
The present invention provides a method of inhibiting an unwanted angiogenic
condition in a mammal comprising treating the mammal with an effective
amount of an immunogen that causes an immune response against a
molecule that induces angiogenesis in the mammal.
The present invention also provides an immunogen that mimics a mammalian
angiogenic molecule wherein the immunogen is not native to the mammal.
The invention also provides a method of inhibiting an unwanted angiogenic
condition in a mammal comprising treating the mammal with an effective
amount of a vector containing DNA that expresses an immunogen that
causes an immune response against a molecule that induces angiogenesis in
the mammal.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method of inhibiting an unwanted angiogenic
condition in a mammal. The method comprises treating the mammal with an
effective amount of an immunogen that causes an immune response against
a molecule that induces or regulates angiogenesis in the mammal. The
unwanted angiogenic condition may be tumor growth, arthritis, macular
degeneration, psoriasis, or any other pathological angiogenic condition. The
animal is preferably a mammal, which may be a human or an animal typically
used for experimentation, such as mice, rats or rabbits.
In addition to providing the methods of the invention, the present invention
also provides the immunogens used in these methods. The immunogens of
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the invention unexpectedly induce an effective immune response when
properly presented to the immune system. The immune response preferably
inhibits, i.e. prevents, slows or stops, angiogenesis, and therefore inhibits
or
eliminates the pathological condition associated with angiogenesis, such as
growth of tumors.
The immunogens of the invention may be any angiogenic molecule associated
with the process of angiogenesis, such as, but not limited to vascular
endothelial growth factor (VEGF), angiopoietin-1, angiopoietin-2, and basic
fibroblast growth factor (bFGF). Further, the immunogen may be receptors
associated with the process of angiogenesis, for example, flk-1, flt-1, and
KDR;
or integrins such as the vitronectin receptor av(i3; or vascular endothelial
cadherins (VE-Cadherin-1 and VE-Cadherin-2); TIE-1, TIE-2lTek. However,
these are examples of angiogenic molecules and receptors, and any
angiogenic molecule or molecular target involved in angiogenesis may be
utilized in the invention. The immunogen may be obtained from any animal or
may be synthetically derived, providing it is substantially the same as that
produced by the animal. The immunogen may also be introduced to any
animal as naked DNA, or as a nucleic acid formulation, or as a plasmid
containing DNA, which provide for the expression of a full length protein or a
fragment thereof. (See U.S. Patent Nos. 5,589,466 and 5,630,796.) The
immunogen may be a fragment of an antigen, epitope or antigenic determinant.
The immunogen used in the invention may be a small molecule not native to
the mammal or a nucleic acid molecule not native to the mammal. Such
small molecules or nucleic acids may be synthesized or isolated from an
organism other than the mammal. The immunogen of the invention may also
be a peptide molecule, or peptidomimetic, capable of illiciting an immune
response against a molecules involved in angiogenesis. Such peptide
molecules and peptidomimetics may be synthesized or isolated from an
organism other than the mammal. Methods for screening small molecules,
nuclic acid molecules, peptide molecules and peptidomimetics are well-known
in the art. (See, for example, J. Biomolecular Screening, 1 (1 ), pg 27-31,
1996.)
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The immunogen used in the method of the invention may be an antigen that
is native to the mammal, and is modified to improve immunogenicity. The
term "native" is defined herein as meaning autologous or homologous to an
animal. In other words, the native antigens of the invention are "self'
proteins
and therefore typically non-immunogenic in the animal from which they are
derived. The antigen may be purified or substantially purified. The
immunogens may also be not native, meaning foreign, to a mammal of the
invention, such as small molecules or nucleic acid molecules that are not
native
to the mammal.
The immunogens of the invention are modified in various ways known in the
art, such as by conjugating or genetically fusing the immunogen to an
immunogenic reagent. Conjugation or fusion of the immunogen to an
immunogenic reagent can stimulate an immune response to the immunogen.
The conjugates and fused molecules of this invention can be prepared by any
of the known methods for coupling or fusing antigens to carriers or fusion
molecules. The conjugates may also be prepared recombinantly as fusion
proteins by methods well-known in the art. The preferred method of
conjugation is covalent coupling whereby the antigen is bound directly to the
immunogenic reagent. Preferred immunogenic reagents include
polysaccharides (U.S. Patent No. 5,623,057), and peptidoglycans (U.S. Patent
No. 5,153,173). These U.S. Patents, as well as all such patents presented in
the instant specification, are herein incorporated by reference.
Another method of modifying the immunogens of the invention is for them to be
bound or genetically fused with a cytokine, lymphokine, hormone or growth
factor (U.S. Patent No. 5,334,379). Examples of such molecules include, but
are not limited to, interferons, GM-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6
and IL-7
(U.S. Patent No. 5,334,379). As stated above, these U.S. Patents are herein
incorporated by reference.
Another method of modifying the immunogens of the invention is haptenization
(chemically linking) of the antigen. A hapten is a substance having the
ability
to, when coupled with a protein, elicit an immune response. The immunogen of
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the invention can itself be haptenized , or can be bound to hapten-modfied
proteins. (U.S. Patent Nos. 4,778,752 and 5,290,551 ).
An additional method of modifying the immunogens of the invention is
glycosylation of the antigens or glycosyiation of the carrier molecules of the
antigens (see U.S. Patent Nos. 5,484,735 and 4,629,692).
Furthermore, peptidomimetic compounds, i.e., compounds which mimic the
activity of peptides, may be used in modification of the immunogens of the
invention (U.S. Patent Nos. 5,386,011 and 5,153,173). Additionally,
peptidomimetics, which are immunogenic and illicit an immune response
against molecules involved in angiogenesis are themselves useful as
immunogens in the invention.
Modification of the immunogens of the invention by bonding the immunogen
with a Major Histocompatibility Complex (MHC) antigen, forming a complex that
is also useful in this invention. (U.S. Patent No. 4,478,823). The source of
such MHC antigens and the methods of bonding the immunogens of the
invention to the MHC antigens are detailed in the cited U.S. Patent No.
4,478,823, which is herein incorporated by reference.
PREPARATION OF IMMUNOGENS
Many of the angiogenic protein molecules of the invention are known, and may
be obtained in natural or recombinant form by known methods. Newly
identified, as well as molecules not yet identified may also be obtained and
used as part of the invention. Methods of obtaining such molecules include
isolating the angiogenic protein directly from cells; isolating or
synthesizing DNA
encoding the protein and using the DNA to produce recombinant protein; and
synthesizing the protein chemically from individual amino acids.
The entire angiogenic molecule gene or fragments of the gene may, for
example, be isolated by using the known DNA sequence to construct
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11
oligonucieotide probes. To do so, DNA restriction fragments are identfied by
Southern hybridization using labelled oligonucleotide probes derived from the
known sequence.
Altemativeiy, DNA encoding the angiogenic molecule may be synthesized from
individual nucleotides. Known methods for synthesizing DNA include preparing
overlapping double-stranded oligonucleotides, filling in the gaps, and
ligating
the ends together.
The DNA prepared as described above may be amplified by polymerase chain
reaction (PCR). Alternatively, the DNA may be amplfied by insertion into a
cloning vector, which is transfected into a suitable host cell, from which the
DNA may be recovered. See, generally, Sambrook et al, "Molecular Cloning,"
Second Edition, Cold Spring Harbor Laboratory Press (1987).
Recombinant methods well known in the art may be used for preparing the
protein. Briefly, the angiogenic molecule-encoding DNA is inserted into an
expression vector, which is transfected into a suitable host. The DNA is
expressed, and the protein is harvested. See Sambrook et al., Id.
Equivalents of the angiogenic protein may also be used in the invention. Such
equivalents include analogs that induce an immune response comparable to
that of the protein. In addition, such equivalents are immunologically cross-
reactive with their con-esponding protein. The equivalent may, for example, be
a fragment of the protein, or a substitution, addition or deletion mutant of
the
protein.
The angiogenic protein fragment preferably contains sufficient amino acid
residues to define an epitope of the antigen. The fragment may, for example,
be a minigene encoding only the epitope. Methods for isolating and identifying
immunogenic fragments from known immunogenic proteins are described by
Salfeld et al. in J. Viral. 63, 798-808 (1989) and by Isola et al. in J.
Viral. 63,
2325-2334 (1989).
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If the fragment defines a suitable epitope, but is too short to be
immunogenic, it
_ may be conjugated to a carrier molecule. Some suitable carrier molecules
include keyhole limpet hemocyanin, Ig sequences, TrpE, and human or bovine
serum albumen. Conjugation may be carried out by methods known in the art.
One such method is to combine a cysteine residue of the fragment with a
cysteine residue on the carrier molecule.
Equivalent proteins have equivalent amino acid sequences. An amino acid
sequence that is substantially the same as another sequence, but that differs
from the other sequence by one or more substitutions, additions and/or
deletions, is considered to be an equivalent sequence. Preferably, less than
25%, more preferably less than 10%, and most preferably less than 5% of the
number of amino acid residues in a sequence are substituted for, added to, or
deleted from the proteins of the invention.
For example, it is known to substitute amino acids in a sequence with
equivalent amino acids. Groups of amino acids generally considered to be
equivalent are:
(a) Ala(A) Ser(S) Thr(T) Pro(P) Gly(G);
(b) Asn{N) Asp(D) Glu(E) Gln(Q);
(c) His(H) Arg{R) Lys(K);
(d) Met(M) Leu(L) Ile(I) Val(V); and
(e) Phe(F) Tyr(Y) Trp(W).
The present invention also provides anti-idiotypic antibodies or fragments
thereof that mimic angiogenic molecules. An "antibody" in accordance with the
present specification is defined broadly as a protein that specifically binds
to
an epitope. The antibodies may be polyclonal, or, preferably, monoclonal. The
angiogenic molecule may be any molecule involved in the process of
angiogenesis, such as, but not limited to, vitronectin, vascular endothelial
growth factor (VEGF), and basic fibroblast growth factor (bFGF), flt-1, flk-1,
KDR, angiopoietin 1 and 2, VE-Cadherin 1 and 2, TIE-1 and TIE-?JTEK.
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The modified immunogens and anti-idiotypic antibodies of the invention elicit
an
immune response in an animal against angiogenesis. The animal is preferably
a mammal, such as a rabbit, rat, or mouse. Preferably, the animal is a
human. An immune response means production of antibodies, i.e. humoral,
and/or a cell-mediated response, such as a T-cell response including helper
and cytotoxic T cell responses.
The anti-idiotypic antibody of the invention is directed against any antibody,
which is itself directed against antigenic determinants of receptors to
angiogenic molecules, or the angiogenic molecule itself. Such antibodies are
known in the art, such as the anti-flk-1 antibody, DC101, described in
PCT/US95/01678, filed February 10, 1995; or anti-av~i3 antibody, i.e. LM609
monoclonal antibody described in PCT Int'I. Application No. PCTIUS95103035,
filed March 9, 1995. Such angiogenic molecules, such as VEGF, are also
known. Further, anti-receptor and anti-angiogenic molecule antibodies may
be obtained by methods known in the art such as those described below.
PREPARATION OP ANTIBODIES
The polydonal and monoclonal antibodies of the invention may be produced by
methods known in the art. These methods include the immunological method
described by Kohler and Milstein in Nature 256, 495-497 ( s 975) and Campbell
in "Monoclonal Antibody Technology, The Production and Characterization of
Rodent and Human Hybridomas" in Burdon et al., Eds., Laboratory Techniques
in Biochemistry and Molecular Biology, Volume 13, Elsevier science
Publishers, Amsterdam (9985); as well as by the recombinant DNA method
described by Huse et al in Science 246, 1275-1281 (1989).
The antibody may be prepared in any mammal, including mice, rats, rabbits,
goats and humans. The antibody may be a member of one of the following
immunoglobulin classes: IgG, IgM, IgA, IgD, or IgE, and the subclasses
thereof.
In one embodiment of the invention, a panel of syngeneic anti-idiotypic
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monoclonal antibodies is developed using DC101 mAb and anti-av(i3 mAb,
which are both monoclonal antibodies to angiogenic receptors. An anti-
idiotypic
monoclonal antibody bearing the internal image of the determinants recognized
by the DC101 monoclonal antibody and anti-av~i3 monoclonal antibody is
identified by determining which anti-idiotypic monoclonal antibody can induce
anti-avi3s mAb and anti-Flk-1 antibodies in the test animal.
Further, one embodiment of the invention provides a cell which produces the
anti-idiotypic antibody of the invention. This cell may be any cell, including
genetically engineered bacterial cells such as E. coli cells containing DNA to
produce the antibody as well as the more typical mammalian cells such as B
cells hybridized with murine myeloma cell lines using standard fusion
procedures (Keamey, J. F. et al., 1981, Eur. J. Immunol. 11:877). Methods
for producing hybridomas, which have the capacity to produce a monoclonal
antibody, are well known in the art. (see Niman et al. Proc. Natl. Acad. Sci.
USA 80:4949-4953 (1983) and Galfre et al., Meth. Enzymol., 73:3-46 (1981)).
Functional Eauivalents of Antibodies
The invention also includes functional equivalents of the antibodies described
in
this specification. Functional equivalents have binding characteristics
comparable to those of the antibodies, and include, for example, chimerized,
humanized and single chain antibodies as well as fragments thereof.
Diabodies may also be functional equivalents of the antibodies of this
invention.
Methods of producing such functional equivalents are disclosed in PCT
Application No. WO 93121319, European Patent Application No. EPO 239,400;
PCT Application Wo 89/09622; European Patent Application No. 338,745; and
European Patent Application EPO 332,424.
Functional equivalents include polypeptides with amino acid sequences
substantially the same as the amino acid sequence of the variable or
hypervariable regions of the antibodies of the invention. "Substantially the
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same" amino acid sequence is defined herein as a sequence with at least 70%
percent homology to an amino acid sequence of an antibody of the invention,
as determined by the FASTA search method in accordance with Pearson and
t_ipman, Proc. Natl. Acad. Sci. USA 85, 2444-2448 (1988).
Chimerized antibodies preferably have constant regions derived substantially
or
exclusively from human antibody constant regions and variable regions derived
substantially or exclusively from the sequence of the variable region from a
mammal other than a human.
Humanized antibodies preferably have constant regions and variable regions
other than the complementarity determining regions (CDRs) derived
substantially or exclusively from the corresponding human antibody regions and
CDRs derived substantially or exclusively from a mammal other than a human.
Suitable mammals other than a human include any mammal from which
monoclonal antibodies may be made, such as a rabbit, rat, mouse, horse, goat,
or primate.
Single chain antibodies or Fv fragments are polypeptides which consist of the
V
region of the heavy chain of the antibody linked to the V region of the light
chain
with or without an interconnecting linker. This comprises the entire antibody
combining site, and is the minimal antibody binding site. These chains may be
produced in bacteria.
Functional equivalents further include fragments of antibodies that have the
same or binding characteristics comparable to those of the whole antibody.
Such fragments may contain one or both Fab fragments or the F(ab')2
fragment. Preferably the antibody fragments contain all six cornplementarity
determining regions of the whole antibody, although fragments containing fewer
than all of such regions, such as three, four or five CDRs, may also be
functional.
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Diabodies are examples of additional functional equivalents. A diabody is an
antibody fragment which has two antigen binding sites and can be a bivalent or
bispec~c fragment. Bispecific diabodies are heterodimers of two 'crossover'
scFv fragments in which the variable light and variable heavy domains of the
two antibodies are present on different polypeptide chains. (Carter and
Merchant, Current Opinions in Biotechnology (8):449-454, 1997.)
Further, the functional equivalents may be or may combine members of any
one of the following imrnunogiobulin classes: IgG, IgM, IgA, IgD, or IgE, and
the
subclasses thereof.
Gene Theraav and Vector Deliverv
Intracellularly expressed antibodies, referred to as "intrabodies" can be
designed to bind and inactivate target molecules inside cells. The genes
encoding can be expressed intracellularly. The specific and high-affinity
binding properties of antibodies, combined with their ability to be stably
expressed in precise intracellular locations inside mammalian cells, provides
molecules for gene therapy applications. (Marasco, W., Gene Ther (4) 1, p11-
5, 1997).
Genes encoding immunogens not native to the mammal may be introduced
into mammalian cells, particularly endothelial cells, by methods known in the
art. Such methods have been described, for example, in Mulligan, et al., U.S.
patent 5,674,722. The methods described in Mulligan, et al., U.S. patent
5,674,722 for preparing vectors useful for introducing genes into mammalian
cells, particularly endothelial cells, are incorporated herein by reference.
The immunogen may be presented to the immune system by a vehicle. For
example, the immunogen may be present on the surface of an antigen
presenting cell, such as a dendritic cell, or combined with a pharmaceutically
acceptable carrier or adjuvant.
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Antigen presenting cells are generally eukaryotic cells with major
histocompatibility complex (MHC), either Class I or Class It, gene products at
their cell surface. For the purposes of this spec~cation, antigen presenting
cells also include recombinant eucaryotic cells, such as peripheral blood
cells,
and recombinant bacterial cells. Some examples of antigen presenting cells as
defined by this specification include dendritic cells, macrophages that are
preferably MHC Class II positive, monocytes that are preferably MHC Class II
positive, and lymphocytes. (Also see U.S. Patent No. 5,597,563).
In one embodiment of the subject invention, the antigen presenting cell is a
recombinant eucaryotic cell that expresses exogenous DNA encoding the
antigen of the invention. The recombinant eucaryotic cell may be prepared in
vivo or in vitro.
Suitable cloning/expression vectors for inserting DNA into eucaryotic cells
include well-known derivatives of SV-40, adenovirus, cytomegalovirus (CMV),
and retrovirus-derived DNA sequences. Any such vectors, when coupled with
vectors derived from a combination of plasmids and phage DNA, i.e. shuttle
vectors, allow for the cloning and/or expression of protein coding sequences
in
both procaryotic and eucaryotic cells.
Other eucaryotic expression vectors are known in the art, e.g., P.J. Southern
and P. Berg, J. Mol. Appl. Genet. 1, 327-341 (1982); S. Subramani et al, Mol.
Cell. Biol. 1, 854-864 (1981 ); R.J. Kaufmann and P.A. Sharp, "Amplification
And Expression Of Sequences Cotransfected with A Modular Dihydrofoiate
Reductase Complementary DNA Gene," J. Mol. Biol. 159, 601-621 (1982); R.J.
Kaufmann and P.A. Sharp, Mol. Cell. Biol. 159, 601-664 (1982); S.I. Scahill et
al, "Expression and Characterization of the Product of a Human Immune
Interferon DNA Gene in Chinese Hamster Ovary Cells," Proc. Nab. Acad. Sci.
USA 80, 4654-4659 (1983); G. Urlaub and L.A. Chasin, Proc. Natl. Acad. Sci.
USA 77, 4216-4220, (1980).
The immunogens of the invention may also be presented to the immune
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system on the surface of recombinant bacterial cells. A suitable recombinant
backerial cell is an aviruient strain of Mycobacterium bovis, such as baalle
Calmette-Guerin (BCG), or an avirulent strain of Salmonella, such as S.
typhimurium. The recombinant bacterial cells may be prepared by cloning DNA
comprising the active portion of the antigen in an avirufent strain, as is
known in
the art; see, for example, Curtiss et al., Vaccine 6_, 155-160 (1988) and
Galan et
al., Gene 94, 29-35 (1990) for preparing recombinant Salmonella and Stover,
C.K. et al., Vaccines 91, Cold Spring Harbor Laboratory Press, pp. 393-398
(1991 ) for preparing recombinant BCG.
Cloning vectors may comprise segments of chromosomal, non-chromosomal
and synthetic DNA sequences. Some suitable prokaryotic cloning vectors
include plasmids from E. coli, such as colE1, pCR1, pBR322, pMB9, pUC,
pKSM, and RP4. Prokaryotic vectors also include derivatives of phage DNA
such as M13, fd, and other filamentous single-stranded DNA phages.
Vectors for expressing proteins in bacteria, especially E. coli, are also
known.
Such vectors include the pK233 (or any of the tac family of plasmids), T7, and
lambda P~. Examples of vectors that express fusion proteins are PATH vectors
described by Dieckmann and Tzagoloff in J. Biol. Chem. 260, 1513-1520
(1985). These vectors contain DNA sequences that encode anthranilate
synthetase (TrpE) followed by a polylinker at the carboxy terminus. Other
expression vector systems are based on beta-galactosidase (pEX); lambda P~;
maltose binding protein (pMAL); glutathione S-transferase (pGST) - see Gene
67, 31 (1988) and Peptide Research 3, 167 (1990).
The expression vectors useful in the present invention contain at least one
expression control sequence that is operatively linked to the DNA sequence or
fragment to be expressed. The control sequence is inserted in the vector in
order to control and to regulate the expression of the cloned DNA sequence.
Examples of useful expression control sequences are the iac system, the trp
system, the tac system, the trc system, major operator and promoter regions of
phage lambda, the control region of fd coat protein, and promoters derived
from
polyoma, adenovirus, retrovirus, and simian virus, e.g., the early and late
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promoters of SV40, and other sequences known to control the expression of
genes in prokaryotic or eukaryotic cells and their viruses or combinations
thereof.
The immunogens of the invention may also be combined with a suitable
medium. Suitable media include pharmaceutically acceptable carriers, such
as phosphate buffered saline solution, liposomes and emulsions.
The immunogens may also be combined with pharmaceutically acceptable
adjuvants that may enhance the immune response, such as muramyl
peptides, lymphokines, such as interferon, interieukin-1 and interleukin-6, or
bacterial adjuvants. The adjuvant may comprise suitable particles onto which
the immunogen is adsorbed, such as aluminum oxide particles. These
compositions containing adjuvants may be prepared as is known in the art.
An example of a bacterial adjuvant is BCG. When used as an antigen
presenting cell as described above, recombinant BCG may additionally act as
its own adjuvant. In this case, additional adjuvant may not be needed although
one or more additional adjuvants may optionally be present. When used in its
natural (non-recombinant) state, BCG acts solely as an adjuvant by being
combined with the immunogen or anti-idiotypic antibody, resulting in a form
that
induces an effective immune response.
The immunogen or immunogen composition may be administered to a
mammal by methods known in the art. Such methods include, for example,
intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or
intradermal administration.
Assays for Determining the Level of Antibody to Anti-Receptor Antibody
or Receptor in Cells
The level of anti-receptor antibody or to receptor in a sample may be
determined by assays known in the art using the anti-idiotypic antibody of the
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invention. The results of these assays may be used for diagnostic purposes.
The target antibody or antigen may be immobilized on a support either
- indirectly by using an anti-target antibody or directly to the support.
Since the
anti-idiotypic antibody mimics the receptor and will compete with and thereby
inhibit the ability of the receptor to bind with anti-receptor antibodies, a
competitive assay may be used to measure the concentration of receptor or
anti-receptor antibodies by correlating the level of anti-idiotypic antibody
binding to the concentration of receptor or anti-receptor antibodies.
A variety of assays are available for detecting proteins with labeled
antibodies. In a one-step assay, the target molecule, if it is present, is
immobilized and incubated with a labeled anti-idiotypic antibody. The labeled
anti-idiotypic antibody binds to the immobilized target molecule. After
washing to remove unbound molecules, the sample is assayed for the
presence of the label.
In a two-step assay, immobilized target molecule is incubated with an
unlabeled anti-idiotypic antibody. The target molecule-unlabeled anti-
idiotypic
antibody complex, if present, is then bound to a second, labeled antibody that
is specific for the unlabeled antibody. The sample is washed and assayed for
the presence of the label, as described above.
The choice of marker used to label the antibodies will vary depending upon
the application. However, the choice of marker is readily determinable to one
skilled in the art. The labeled antibodies may be polycional or monoclonal. In
a preferred embodiment, the antibody is monoclonal.
Purification Methods
The invention also provides a method of purifying anti-receptor antibodies in
a
sample comprising contacting the sample with an anti-idiotypic antibody of the
invention, isolating the complex between anti-receptor antibodies from the
sample and the anti-idiotypic antibody, and recovering the anti-receptor
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antibodies from the complex. The anti-idiotypic antibody is preferably
immobilized.
Experimental Examples
Dendritic cells (DC): DC were isolated using the following protocol. Bone
marrow from C57BL6 mice was depleted of red blood cells by treatment in
0.5% amonium chloride, followed by treatment with a combination of
monoclonal antibodies specific for CD4, CD8, la, granulocytes and then with
rabbit complement. The remaining cells were cultured in RPMI 1640
containing 10% FBS as well as GM-CSF and IL-4. Three days later,
nonadherent cells were discarded and adherent cells cultured for 4 more
days. Adherent cells were then transferred to a new plate and cultured for
another 3 days before harvesting for use as antigen presenting cells.
Pulsing of DC with Flk-1AP antigen: DC were washed twice in serum-free
AIMV media and incubated overnight in AIMV with 100uglml of an affinity-
purified fusion protein of Flk-1 and alkaline phosphatase (Flk-1AP). The cells
were then washed twice in AIMV before being used for vaccination.
Vaccination of mice with DC pulsed with Flk-1AP: Lewis Lung carcinoma
metastasis model was used to assess the antitumor effect of vaccination with
DC pulsed with Flk-1AP. Briefly, C57BL6 mice were injected either
intravenously or intraperiteneally, with each mouse receiving 1 x 105 Flk-1 AP
pulsed DC at Day 0. Seven days later, each mouse was inoculated intra-
footpad with 1x 105 D122 cells. Visible tumors were surgically removed at
Day 10. During this period, mice received two boost vaccinations every ten
days. At Day 60, mice were sacrificed and lungs removed for weighing and
assessment of metastases. As a control, a group of mice were vaccinated
with DC alone.
Experimental Results
Inhibition of tumor metastases: Mice vaccinated with DC pulsed with Flk-
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1AP developed a significantly smaller number of tumors in the lungs
compared to the groups vaccinated with DC alone. Comparably, the average
- weight of the lungs from the Flk-AP group was significantly lower than the
controls. In both instances, the number of tumors present and the weight of
the lungs of mice vaccinated with DC pulsed with Flk-1AP was less than half
of the control group.
