Note: Descriptions are shown in the official language in which they were submitted.
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METHODS FOR IDENTIFYING ANTIBODIES AND PEPTIDES USEFUL IN THE
TREATMENT OF SEPTIC SHOCK AND EXPERIMENTAL ARTHRITIS AND
USES THEREOF
FIELD OF THE INVENTION
The present invention relates to methods for identifying new peptides useful
in the
treatment of septic shock, experimental arthritis and related maladies. These
new peptides
are selected, in part, for their affinity to or mimicry of anti-Group A
mucopeptide
antibodies. The therapeutic use of the new peptides and the related antibodies
in the
treatment of septic shock are disclosed.
BACKGROUND OF THE INVENTION
Studies extending over the years have elucidated the basic structure of many
of the gram
positive and gram negative organisms. With respect to the gram positive
bacteria, the
backbone of theses organisms is the polysaccharide-peptide complex called the
mucopeptide
complex (Figure 1). The basic repeating unit is an alternating motif of
Neuraminic Acid-N-
acetyl glucosamine (NAM-NAG) molecules in which the inter-peptide bridges vary
from
organism to organism.
The gram negative bacteria share a common glycolipid commonly known as lipid
A, to
which various oligo- and polysaccharides are added depending on the bacteria.
The
common structure of lipid A is shown in Figure 2. It is well known that both
Lipid A and
the mucopeptide complex will individually elicit an extremely vigorous toxic
response when
injected into animals. The most serious of these responses is the oftentimes
massive
production of tumor necrosis factor (TNFa).
Presently, the main approach to blocking this TNF induction has been either
the
administration of antibodies directed to the TNF molecule itself or to use
soluble TNF
receptor molecules to competitively inhibit TNF binding to the target cell.
Both of these
strategies would interrupt cytokine release. Monoclonal antibodies with their
restricted
epitope specificity have been shown to be unsuccessful in this regard [Fisher
et al., Crit.
Care Med. 21:318-3272 (1994), McCioskey et al., Ann. Intern. Med. 121:1-5].
Preliminary trials with F(ab)Z anti-TNF polyclonal antibodies appear to be
more promising,
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but further trials need to be carried out before such methods are shown to be
a viable
approach.
The mechanisms for lipid A and the mucopeptide complex triggering of the
extremely toxic
response, described above, have not been elucidated, but most investigators
agree that cell
surface receptors on monocytes must be involved. The quest to identify this
receptor or
receptors is a very active field of research. Over the last several years,
Morrison and co-
workers, among others, have identified a 70 kDa protein on monocytes which
appears to
bind to lipopolysaccharides (LPS), [Dziarski, J.Biol.Cltem. 266:4719-4725
(1994), Lei et
al. , Int. Rev.lmmunol. 6:223-235 ( 1990) Lei et al. , J.Immunol. 147:1925-
1932 ( 1991 ) Rabin
et al., J.Infect.Dis. 168:135-142, (1993)]. A monoclonal antibody reactive
with this 70
kDa protein has been reported to be able to cause monocytes to produce TNF a,
which
strengthens the proposal that this 70 Kda protein might be the LPS receptor
[Morrison et
al., J.Infect.Dis. 162:1063-1068 (1990)]. Beyond this putative identification
of a receptor,
little else has been uncovered concerning this immunologically important
mechanism.
Therefore there is a need for a better understanding of the mechanisms of how
lipid A and
the mucopeptide complex induce their toxic effects. On a more practical side,
there is a
great need for alternative approaches for blocking the induction of TNFa
during septic
shock. Thus, there is a also a great need for the design of new
pharmaceuticals that can
counter-act the toxic effects of lipid A and the mucopeptide complex.
The citation of any reference herein should not be construed as an admission
that such
reference is available as "Prior Art" to the instant application.
SUMMARY OF THE INVENTION
In its broadest embodiment the present invention teaches the use of antibodies
raised against
the Group A mucopeptide for the treatment of inflammatory diseases such as
septic shock
and rheumatoid arthritis, Crohn's disease, psoriasis, and for the
identification of specific
peptides that can be used in such treatments. In certain embodiments, the
specific peptides
will serve as mimics to these antibodies. In other embodiments, the peptides
will serve as
binding partners for these antibodies.
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One aspect of the present invention is derived from the premise that there
exists a common
structural motif for gram positive and gram negative bacteria. The present
invention
exploits the ramifications of this premise and thereby introduces novel
approaches of
identifying treatments for septic shock and related diseases.
One feature of the present invention is the generation and identification of
antibodies that
bind to a common motif present on both Lipid A and the Group A mucopeptide. A
related
aspect of the present invention is the generation and characterization of
peptides that mimic
these antibodies. The peptides act by associating with LPS and the Group A
mucopeptide,
and thus inhibit these bacterial agents from binding to their receptor(s).
This, in turn, can
prevent the induction of TNFa and the onset of septic shock.
Accordingly, the present invention includes methods of obtaining such
inhibitory peptides.
In one specific embodiment the inhibitory peptides are obtained by contacting
a phage
library with permissive bacteria. The phage library contains an assortment of
phage with
each individual phage comprising a random DNA sequence that encodes specific
peptides.
The DNA sequences of the phage library are expressed and a variety of random
peptides
are generated. The random peptides are contacted with the mucopeptide complex,
and
candidate peptides are selected on the basis of their ability to bind the
mucopeptide
complex. Phage containing the candidate peptides are purified and DNA that
encode the
candidate peptides are sequenced. The amino acid sequence for the candidate
peptide can
then be deduced.
The candidate peptides are synthesized per their respective amino acid
sequences. The
synthesis can be performed, for example, by standard genetic engineering
techniques or
more preferably by solid phase peptide synthesis. The synthetic candidate
peptides are then
contacted with Lipid A, and further characterized by their ability to bind
Lipid A. In this
manner peptides that bind both Lipid A and the Group A mucopeptide can be
obtained.
The selected peptides are further contacted with the Group A mucopeptide and
an anti-
Group A mucopeptide antibody. Inhibitory peptides are chosen from selected
peptides on
the basis of their distinctive ability to inhibit the formation of that
antigen-antibody
complex.
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It should be clear that though the specific embodiment described above used a
phage library
to obtain an assortment of random peptides, this assortment also may be
generated by
chemical synthesis. In addition, the assortment of random peptides can be
generated by a
combination of these two techniques. It should also be clear that for all of
the embodiments
provided by the present invention that use an assortment of random peptides,
that either a
phage library or chemical synthesis can be utilized.
In another embodiment of the present invention, an inhibitory peptide is
further selected on
the basis of its ability to inhibit the bacterial-mediated production of tumor
necrosis factor a
(TNFa). This method includes the additional step of contacting an inhibitory
peptide with a
mononuclear cell that is challenged with either LPS or the mucopeptide. In a
preferred
embodiment of the method of identifying the inhibitory peptide, the inhibitory
peptide binds
to a common structural element present in both the Group A mucopeptide and the
LPS
molecule.
The inhibitory peptides obtained by the methods described herein are also part
of the
present invention. These inhibitory peptides have the following
characteristics: they bind to
both the Group A mucopeptide and the LPS molecule; and in addition, inhibit
the
association between an anti-Group A mucopeptide antibody and its Group A
mucopeptide
antigen.
In a preferred embodiment, the inhibitory peptide further inhibits the
bacterial-mediated
production of tumor necrosis factor in a target cell. The target cell can be a
mononuclear
cell or any other cell which secretes cytokines in response to stimulation by
the Group A
mucopeptide and/or the LPS molecule. In a more preferred embodiment the
mononuclear
cell is human.
The present invention also includes pharmaceutical compositions for treating
septic shock,
rheumatoid arthritis, Crohn's disease, psoriasis and experimental arthritis
comprising an
inhibitory peptide of the present invention and a pharmaceutically acceptable
carrier.
Methods of treating septic shock in mammals, which comprise administering to
the mammal
a therapeutically effective amount of such pharmaceutical compositions, are
included in the
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present invention. In preferred embodiments for treating septic shock or
rheumatoid
arthritis, the mammal is a human.
The present invention also includes methods of treating experimental arthritis
comprised of
administering to the subject animal a therapeutically effective amount of a
pharmaceutical
composition comprising an inhibitory peptide of the present invention and a
pharmaceutically acceptable carrier. Analogous methods of treating rheumatoid
arthritis by
administering to a human a therapeutically effective amount of the
pharmaceutical
compositions comprising an inhibitory peptide of the present invention and a
pharmaceutically acceptable carrier are contemplated by the present invention.
A related aspect of the invention is a method of blocking the LPS-mediated
induction of
TNFa in a mononuclear cell, by adding an antibody prepared against pure Group
A
mucopeptide to the mononuclear cell. In preferred embodiments the mononuclear
cell is
human.
Another aspect of the present invention is derived on the premise that the
mucopeptide
complex elicits an immune response that is comparable to rheumatoid arthritis.
This aspect
of the invention includes methods of identifying an antigenic peptide. One
specific
embodiment of such a method comprises the steps of administering to an animal
a selected
peptide, then administering to the animal an amount of Group A mucopeptide
capable of
inducing experimental arthritis in said animal, and finally, identifying an
antigenic peptide
on the basis of the ability of the selected peptide to elicit a protective
antibody response
against the Group A mucopeptide induced experimental arthritis in the animal.
In one
specific embodiment of this type the animal is a rat and the amount of Group A
mucopeptide administered is in a dose equivalent to the Group A cell fragments
containing
15-30 ~g of rhamnose per gram of body weight of the rat, i. e. , a dose
equivalent to 3-6
mgs of rhamnose for a 200 gram rat. [The amount of mucopeptide is typically
expressed as
a function of the quantity of rhamnose determined on the mucopeptide, because
the
rhamnose content of the mucopeptide is relatively constant and readily
determined]. In a
preferred embodiment of this type, 125 ~,gs to 250 ugs of the selected peptide
per gram of .
body weight of the rat, i.e., a dose of 25 to 50 mgs of the selected peptide
for a 200 gram
rat, is administered to the animal prior to the administration of the Group A
mucopeptide.
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The selected peptide may be selected on the basis of its ability to bind to an
anti-Group A
mucopeptide antibody and inhibit its binding to the Group A mucopeptide. One
related
method for selecting such a peptide comprises contacting an assortment of
random peptides
with an anti-Group A mucopeptide antibody, and choosing a candidate peptide on
the basis
of the ability of the candidate peptide to bind the Group A mucopeptide
antibody;
contacting the candidate peptide with the Group A mucopeptide, and choosing
the selected
peptide on the basis of the ability of the candidate peptide not to bind the
Group A
mucopeptide. The selected peptide may then be recovered and/or otherwise
identified and
used in the screening assay described above for identifying an antigenic
peptide of the
present invention.
As for the earlier embodiments of the present invention the assortment of
random peptides
may be obtained from a phage library. In one specific embodiment the selected
peptides
are obtained by contacting a phage library with permissive bacteria. The phage
library
contains an assortment of phage with each individual phage comprising a random
DNA
sequence that encodes specific peptides. The DNA sequences of the phage
library are
expressed and a variety of random peptides are generated. The selected peptide
may be
recovered as follows: the phage encoding the selected peptide can be purified
and the DNA
that encodes the selected peptide can be sequenced. The amino acid sequence
for the
selected peptide can then be deduced from the DNA sequence. Alternatively,
synthetic
combinatorial libraries can be used.
The selected peptides can then be synthesized per their respective amino acid
sequences.
The synthesis can be performed, for example, by standard genetic engineering
techniques
or more preferably by solid phase peptide synthesis.
The antigenic peptides obtained by the methods described herein are also part
of the present
invention. These inhibitory peptides have the following characteristics: they
do not bind to
the Group A mucopeptide; they do bind to an anti-Group A mucopeptide antibody;
and they
elicit a protective antibody response in animals against Group A mucopeptide
induced
experimental arthritis. The present invention also includes pharmaceutical
compositions
comprising an antigenic peptide of the present invention and a
pharmaceutically acceptable
carrier.
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The present invention also includes methods of analyzing the ability to
prevent the onset of
experimental arthritis in an animal induced to develop experimental arthritis.
In one
embodiment the method comprises administering to the animal an antibody
prepared against
the Group A mucopeptide prior to the onset of experimental arthritis, thereby
preventing
the onset of experimental arthritis. In another embodiment a method for
preventing the
onset of experimental arthritis comprises administering to the animal a
pharmaceutical
composition comprising an antigenic peptide of the present invention and a
pharmaceutically acceptable carrier prior to the onset of experimental
arthritis.
The present invention also includes methods of treating experimental arthritis
in an animal.
In one embodiment the method comprises administering to the animal an antibody
prepared
against the Group A mucopeptide after the onset of experimental arthritis,
whereby the
experimental arthritis in said animal is ameliorated. In a related embodiment,
the present
invention includes a method of administering to the animal the pharmaceutical
composition
comprising an antigenic peptide of the present invention and a
pharmaceutically acceptable
carrier after the onset of experimental arthritis, whereby the experimental
arthritis in said
animal is ameliorated.
It should be understood that variations in the amino acid sequence of the
antigenic and
inhibitory peptides can be made in order to increase their binding affinities
to the anti-
Group A mucopeptide antibody and the Group A mucopeptide respectively. For
example,
slight variations include replacing one acidic amino acid with another, such
as replacing an
aspartate with a glutamate. More dramatic modifications are also envisioned
such as
substituting an ornithine for an arginine or even a d-amino acid for its
corresponding 1-
amino acid. All such modifications for optimizing the desired properties of
these peptides
are contemplated by the present invention.
These and other aspects of the present invention will be better appreciated by
reference to
the following drawings and Detailed Description.
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BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE I. PRIOR ART. The structure of the mucopeptide complex of the gram-
positive bacterial cell wall. Depiction of a schematic drawing of the
polysaccharide-
peptide complex (mucopeptide complex) of the cell wall of gram positive
bacteria.
FIGURE 2. PRIOR ART. The structure of the glycolipid complex of the gram-
negative bacterial cell wall. Depiction of a schematic drawing of the
glycolipid (Lipid A)
of the cell wall of gram negative bacteria.
FIGURE 3. Comparison of the structures of the mucopeptide and the glycolipid
complex. Depiction of the comparison of the structures of the peptidoglycan
(mucopeptide
complex) and Lipid A.
FIGURE 4. TNFa induction by bacterial cell wall components in the presence and
absence of antibodies prepared against the pure group A streptococcal
mucopeptide.
A three-dimensional bar graph showing a comparison of the ng of TNF alpha
produced in
human mononuclear cells (y-axis) by a single addition of 600 ng of the
mucopeptide
complex (Figure 4A) or a single addition of 20 ng of LPS (Figure 4B) in the
presence
(stippled bar) or absence (shaded bar) of antibodies prepared against the pure
Group A
streptococcal mucopeptide.
DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses novel methods and compositions for treating
septic shock,
Crohn's disease, psoriasis, and rheumatoid arthritis through the use of
antibodies raised
against the Group A mucopeptide. These antibodies may be used directly, or
indirectly by
identifying specific peptides that can be used in such treatments.
Whether or not the 70 kDa protein, discussed above, is the long sought after
LPS receptor,
the fact that the streptococcal mucopeptide complex can inhibit the
interaction of the LPS
and this 70 kDa protein is consistent with the LPS and the mucopeptide complex
having
some structural similarity. Accordingly, the present invention includes the
use of antibodies
prepared against the pure Group A streptococcal mucopeptide that are able to
block the
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induction of TNF alpha in human mononuclear cells regardless of whether they
were
stimulated by either Group A mucopeptide obtained from gram positive bacteria
or LPS
obtained from gram negative organisms (see Figure 4). As seen in Figure 3, if
one looks at
the three dimensional structures of both groups, there is a remarkable
similarity in the
backbone moieties. This similarity further suggest that a common structural
element
present in both Gram positive and Gram negative organisms is capable of
binding to a
receptor present on human mononuclear cells and inducing TNF alpha production.
The present invention describes a novel approach of using anti-mucopeptide
antibodies to
help identify novel peptides which will bind to the common structural element
present on
the Group A mucopeptide and on the LPS molecule. These peptides resemble the
binding
domains within the anti-mucopeptide antibodies and thus can block the binding
of the cell
receptor to the common structural element present in Group A mucopeptide and
LPS.
This, in turn, inhibits the induction of TNF alpha by the target cell.
Accordingly, the
present invention includes the investigation of diseases, such as septic
shock, Crohn's
disease, psoriasis and experimental arthritis (an animal model for Rheumatoid
Arthritis)
using this novel approach. It would be understood by any person with skill in
the relevant
art that such diseases are only meant to be exemplary and other diseases
having related
mechanisms can also be successfully treated by the teachings herein.
As used herein, the terms "mucopeptide complex", "Group A mucopeptide",
"peptidoglycan", and the "polysaccharide-peptide complex" are used
interchangeably and
denote an integral aspect of the cell wall of gram positive bacteria. Pieces
of this integral
aspect induce an immunological response in mammals that can include septic
shock.
As used herein, the terms "Lipid A", "glycolipid", "lipopolysaccharides" or
"LPS" are used
interchangeably and denote an integral aspect of the cell wall of gram
negative bacteria.
Pieces of this integral aspect induce an immunological response in mammals
that can
include septic shock.
As used herein an "inhibitory peptide" has the following characteristics: it
binds the Group
A mucopeptide; it binds the LPS molecule; and it inhibits an anti-Group A
mucopeptide
antibody from binding the Group A mucopeptide.
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As used herein an "antigenic peptide" has the following characteristics: it
does not bind to
the Group A mucopeptide; but it does bind to an anti-Group A mucopeptide
antibody; and it
elicits a protective antibody response in animals against Group A mucopeptide
induced
experimental arthritis.
5 As used herein, a "common structural element" is used interchangeably with a
"common
structural motif" and denotes a three dimensional structural similarity which
allows at least
one binding partner to selectively bind to at least two otherwise distinctive
molecules that
contain such a common structural motif with a comparable affinity.
As used herein "experimental arthritis" has clinical symptoms including
redness and
10 swelling of the paws of the animal. Such symptoms can be monitored by
observation
and/or by measurement of paw diameter. These symptoms wax and wane over tune.
An
increased paw size is indicative of arthritis.
Antibodies
According to the invention, mucopeptide prepared from natural sources, or
produced
recombinantly or by chemical synthesis, and fragments or other derivatives or
analogs
thereof, including fusion proteins, may be used as an immunogen to generate
antibodies that
recognize the mucopeptide and LPS. Similarly, LPS prepared from natural
sources, or by
chemical synthesis, and fragments or other derivatives or analogs thereof, may
be used as
an immunogen to generate antibodies that recognize both the mucopeptide and
LPS. Such
antibodies include but are not limited to polyclonal, monoclonal, chimeric,
single chain,
Fab fragments, and an Fab expression library.
A molecule is "antigenic" when it is capable of specifically interacting with
an antigen
recognition molecule of the immune system, such as an immunoglobulin
(antibody) or T
cell antigen receptor. An antigenic polypeptide contains at least about 5, and
preferably at
least about 10, amino acids. An antigenic portion of a molecule can be that
portion that is
immunodominant for antibody or T cell receptor recognition, or it can be a
portion used to
generate an antibody to the molecule by conjugating the antigenic portion to a
carrier
molecule for immunization. A molecule that is antigenic need not be itself
immunogenic,
i.e., capable of eliciting an immune response without a carrier.
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An "antibody" is any immunoglobulin, including antibodies and fragments
thereof, that
binds a specific epitope. The term encompasses polyclonal, monoclonal, and
chimeric
antibodies, the last mentioned described in further detail in U.S. Patent Nos.
4,816,397 and
4,816,567, as well as antigen binding portions of antibodies, including Fab,
F(ab')Z and
F(v) (including single chain antibodies). Accordingly, the phrase "antibody
molecule" in its
various grammatical forms as used herein contemplates both an intact
immunoglobulin
molecule and an immunologically active portion of an immunoglobulin molecule
containing
the antibody combining site. An "antibody combining site" is that structural
portion of an
antibody molecule comprised of heavy and light chain variable and
hypervariable regions
that specifically binds antigen.
Exemplary antibody molecules are intact immunoglobulin molecules,
substantially intact
immunoglobulin molecules and those portions of an immunoglobulin molecule that
contains
the paratope, including those portions known in the art as Fab, Fab', F(ab')2
and F(v),
which portions are preferred for use in the therapeutic methods described
herein.
Fab and F(ab')2 portions of antibody molecules are prepared by the proteolytic
reaction of
papain and pepsin, respectively, on substantially intact antibody molecules by
methods that
are well-known. See for example, U.S. Patent No. 4,342,566 to Theofilopolous
et al:
Fab' antibody molecule portions are also well-known and are produced from
F(ab')2
portions followed by reduction of the disulfide bonds linking the two heavy
chain portions
as with mercaptoethanol, and followed by alkylation of the resulting protein
mercaptan with
a reagent such as iodoacetamide. An antibody containing intact antibody
molecules is
preferred herein.
The phrase "monoclonal antibody" in its various grammatical forms refers to an
antibody
having only one species of antibody combining site capable of immunoreacting
with a
particular antigen. A monoclonal antibody thus typically displays a single
binding affinity
for any antigen with which it immunoreacts. A monoclonal antibody may
therefore contain
an antibody molecule having a plurality of antibody combining sites, each
immunospecific
for a different antigen; e.g., a bispecific (chimeric) monoclonal antibody.
The term "adjuvant" refers to a compound or mixture that enhances the immune
response to
an antigen. An adjuvant can serve as a tissue depot that slowly releases the
antigen and
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PCT/U598112647
also as a lymphoid system activator that non-specifically enhances the immune
response
[Hood et al., in Immunology, p. 384, Second Ed., Benjamin/Cummings, Menlo
Park,
California (1984)]. Often, a primary challenge with an antigen alone, in the
absence of an
adjuvant, will fail to elicit a humoral or cellular immune response. Adjuvants
include, but
are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant,
saponin,
mineral gels such as aluminum hydroxide, surface active substances such as
lysolecithin,
pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, keyhole
limpet
hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG
(bacille
Calmette-Guerin) and Corynebacterium parvum. Preferably, the adjuvant is
pharmaceutically acceptable.
Various procedures known in the art may be used for the production of
polyclonal
antibodies that cross-react with the mucopeptide and LPS, or fragment,
derivative or analog
thereof. For the production of antibody, various host animals can be immunized
by
injection with the mucopeptide or LPS, or a derivative (e.g., fragment or
fusion protein)
thereof, including but not limited to rabbits, mice, rats, sheep, goats, etc.
In one
embodiment, the mucopeptide, LPS or fragment thereof can be conjugated to an
immunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpet
hemocyanin
(KLH). Various adjuvants may be used to increase the immunological response,
depending
on the host species, including but not limited to Freund's (complete and
incomplete),
mineral gels such as aluminum hydroxide, surface active substances such as
lysolecithin,
pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanins,
dinitrophenol, and potentially useful human adjuvants such as BCG (bacille
Calmette-
Guerin) and Corynebacterium parvum.
For preparation of monoclonal antibodies directed toward the mucopeptide, LPS
or
fragment, analog, or derivative thereof, any technique that provides for the
production of
antibody molecules by continuous cell lines in culture may be used. These
include but are
not limited to the hybridoma technique originally developed by Kohler et al.,
Nature,
256:495-497 (1975), as well as the trioma technique, the human B-cell
hybridoma technique
[Kozbor et al., Immunology Today, 4:72 (1983)], and the EBV-hybridoma
technique to
produce human monoclonal antibodies [Cole et al. , in Monoclonal Antibodies
and Cancer
Therapy, pp. 77-96, Alan R. Liss, Inc., (1985)]. Immortal, antibody-producing
cell lines
can be created by techniques other than fusion, such as direct transformation
of B
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lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See,
e.g., M.
Schreier et al. , "Hybridoma Techniques" ( 1980); Hammerling et al. ,
"Monoclonal
Antibodies And T-cell Hybridomas" (1981); Kennett et al., "Monoclonal
Antibodies"
(1980); see also U.S. Patent Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887;
4,451,570;
4,466,917; 4,472,500; 4,491,632; and 4,493,890.
In an additional embodiment of the invention, monoclonal antibodies can be
produced in
germ-free animals utilizing recent technology (PCT/US90/02545). According to
the
invention, human antibodies may be used and can be obtained by using human
hybridomas
[Cote et al., Proc. Natl. Acad. Sci. USA, 80:2026-2030 (1983)] or by
transforming human
B cells with EBV virus in vitro (Cole et al., 1985, supra). In fact, according
to the
invention, techniques developed for the production of "chimeric antibodies"
[Morrison et
al., J. Bacteriol., 159-870 (1984); Neuberger et al., Nature, 312:604-608
(1984); Takeda
et al., Nature, 314:452-454 (1985)] by splicing the genes from a mouse
antibody molecule
specific for the mucopeptide or LPS together with genes from a human antibody
molecule
of appropriate biological activity can be used; such antibodies are within the
scope of this
invention. Such human or humanized chimeric antibodies are preferred for use
in therapy
of human diseases or disorders (described infra), since the human or humanized
antibodies
are much less likely than xenogenic antibodies to induce an immune response,
in particular
an allergic response, themselves.
According to the invention, techniques described for the production of single
chain
antibodies (U.S. Patent 4,946,778) can be adapted to produce mucopeptide/LPS-
specific
single chain antibodies. An additional embodiment of the invention utilizes
the techniques
described for the construction of Fab expression libraries [Huse et al.,
Science,
246:1275-1281 (1989)] to allow rapid and easy identification of monoclonal Fab
fragments
with the desired specificity for the mucopeptide and LPS shared epitope.
Antibody fragments which contain the idiotype of the antibody molecule can be
generated
by known techniques. For example, such fragments include but are not limited
to: the
F(ab')2 fragment which can be produced by pepsin digestion of the antibody
molecule; the
Fab' fragments which can be generated by reducing the disulfide bridges of the
F(ab')Z
fragment, and the Fab fragments which can be generated by treating the
antibody molecule
with papain and a reducing agent.
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In the production of antibodies, screening for the desired antibody can be
accomplished by
techniques known in the art, e.g., radioimmunoassay, ELISA (enzyme-linked
immunosorbent assay), "sandwich" immunoassays, immunoradiometric assays, gel
diffusion
precipitin reactions, immunodiffusion assays, in situ immunoassays (using
colloidal gold,
enzyme or radioisotope labels, for example), Western blots, precipitation
reactions,
agglutination assays (e.g., gel agglutination assays, hemagglutination
assays), complement
fixation assays, immunotluorescence assays, protein A assays, and
immunoelectrophoresis
assays, etc. In one embodiment, antibody binding is detected by detecting a
label on the
primary antibody. In another embodiment, the primary antibody is detected by
detecting
binding of a secondary antibody or reagent to the primary antibody. In a
further
embodiment, the secondary antibody is labeled. Many means are known in the art
for
detecting binding in an immunoassay and are within the scope of the present
invention. For
example, to select antibodies which recognize a specific epitope common to the
mucopeptide and LPS, one may assay generated hybridomas for a product which
binds to a
mucopeptide or LPS fragment containing such an epitope.
The foregoing antibodies can be used in methods known in the art relating to
the
localization and activity of the mucopeptide or LPS e.g., for Western
blotting, imaging the
mucopeptide or LPS in situ, measuring levels thereof in appropriate
physiological samples,
etc.
In a specific embodiment, antibodies that agonize or antagonize the activity
of the
mucopeptide and LPS can be generated. Such antibodies can be tested using the
assays
described infra for identifying ligands.
In a specific embodiment, antibodies are developed by immunizing rabbits with
synthetic
peptides predicted by the sequence of the mucopeptide. Synthetic peptides may
be
conjugated to a carrier such as KLH hemocyanin or BSA using carbodiimide and
used in
Freund's adjuvant to immunize rabbits. The expressed peptide may be prepared
in
quantity and used to immunize rabbits in Freund's adjuvant.
In another specific embodiment, the mucopeptide is used to immunize chickens,
and the
chicken anti-mucopeptide antibodies are recovered from egg yolk, e.g., by
affinity
purification on a mucopeptide-column. The affinity purified antibodies can be
selected by
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subsequent purification on a LPS-column. Preferably, chickens used in
immunization are
kept under specific pathogen free (SPF) conditions.
In another embodiment, the mucopeptide is used to immunize rabbits, and the
polyclonal
antibodies are immunopurified on a mucopeptide-column and a LPS-column prior
to further
5 use. The purified antibodies are particularly useful for semi-quantitative
assays. Panels of
monoclonal antibodies produced against the mucopeptide or LPS can be screened
for
various properties; i.e., isotype, epitope, affinity, etc. Of particular
interest are monoclonal
antibodies that neutralize the activity of the mucopeptide or LPS to bind to
the TNF
receptor. Such monoclonals can be readily identified in by the assays
described herein.
10 High affinity antibodies are particularly useful for this neutralization
process.
Preferably, the anti-modulator antibody used in the diagnostic and therapeutic
methods of
this invention is an affinity-purified polyclonal antibody. More preferably,
the antibody is a
monoclonal antibody (mAb). In addition, it is preferable for the anti-
mucopeptide or
anti-LPS molecules used herein be in the form of Fab, Fab', F(ab')2 or F(v)
portions of
15 whole antibody molecules.
Peptides
The term "peptide" is used in its broadest sense to refer to a compound of two
or more
subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may
be linked
by peptide bonds. In another embodiment, the subunit may be linked by other
the bonds,
e.g., ester, ether, etc. As used herein the term "amino acid" refers to either
natural and/or
unnatural or synthetic amino acids, including glycine and both the D or L
optical isomers,
and amino acid analogs and peptidomimetics.
Using the "phage method" [Scott and Smith, 1990, Science 249:386-390 (1990);
Cwirla, et
al., Proc. Natl. Acad. Sci., 87:6378-6382 (1990); Devlin et al., Science,
249:404-406
(1990)], very large libraries can be constructed (106-10g chemical entities).
A second
approach uses primarily chemical methods, of which the Geysen method [Geysen
et al.,
Molecular Immunology 23:709-715 ( 1986); Geysen et al. J. Immunologic Method
102:259-
274 (1987)] and the method of Fodor et al. [Science 251:767-773 (1991)] are
examples.
Furka et al. [14th International Congress of Biochemistry, Volume 5, Abstract
FR:013
(1988); Furka, Int. J. Peptide Protein Res. 37:487-493 (1991)], Houghton [U.S.
Patent No.
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16
4,631,211, issued December 1986] and Rutter et al. [U.S. Patent No. 5,010,175,
issued
April 23, 1991] describe methods to produce a mixture of peptides that can be
tested as
described herein. Such peptides can be further refined through modifications
by chemical
synthesis as described below.
In another aspect, synthetic libraries [Needels et al., Proc. Natl. Acad. Sci.
USA 90:/0700-
4 (1993); Ohlmeyer et al., Proc. Natl. Acad. Sci. USA 90:10922-10926 (1993);
Lam et al.,
International Patent Publication No. WO 92/00252; Kocis et al., International
Patent
Publication No. WO 9428028, each of which is incorporated herein by reference
in its
entirety], and the like can be used to screen for the peptides of the present
invention.
Synthetic peptides, prepared using the well known techniques of solid phase,
liquid phase,
or peptide condensation techniques, or any combination thereof, can include
natural and
unnatural amino acids. Amino acids used for peptide synthesis may be standard
Boc (N"-
amino protected N"-t-butyloxycarbonyl) amino acid resin with the standard de-
protecting,
neutralization, coupling and wash protocols of the original solid phase
procedure of
Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154), or the base-labile N"-amino
protected
9-fluorenylmethoxycarbonyl (Fmoc) amino acids first described by Carpino and
Han (1972,
J. Org. Chem. 37:3403-3409). Both Fmoc and Boc N"-amino protected amino acids
can be
obtained from Fluka, Bachem, Advanced Chemtech, Sigma, Cambridge Research
Biochemical, Bachem, or Peninsula Labs or other chemical companies familiar to
those
who practice this art. In addition, the method of the invention can be used
with other N"-
protecting groups that are familiar to those skilled in this art. Solid phase
peptide synthesis
may be accomplished by techniques familiar to those in the art and provided,
for example,
in Stewart and Young, 1984, Solid Phase Synthesis, Second Edition, Pierce
Chemical Co.,
Rockford, IL; Fields and Noble, 1990, Int. J. Pept. Protein Res. 35:161-214,
or using
automated synthesizers, such as sold by ABS. Thus, peptides of the invention
may
comprise D-amino acids, a combination of D- and L-amino acids, and various
"designer"
amino acids (e.g., ~i-methyl amino acids, Ca-methyl amino acids, and Na-methyl
amino
acids, etc.) to convey special properties. Synthetic amino acids include
ornithine for lysine,
fluorophenylalanine for phenylalanine, and norleucine for leucine or
isoleucine.
Additionally, by assigning specific amino acids at specific coupling steps, a-
helices, (3
turns, ~3 sheets, y-turns, and cyclic peptides can be generated.
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In one aspect of the invention, the peptides may comprise a special amino acid
at the C-
terminus which incorporates either a COzH or CONHz side chain to simulate a
free glycine
or a glycine-amide group. Another way to consider this special residue would
be as a D or
L amino acid analog with a side chain consisting of the linker or bond to the
bead. In one
embodiment, the pseudo-free C-terminal residue may be of the D or the L
optical
configuration; in another embodiment, a racemic mixture of D and L-isomers may
be used.
In an additional embodiment, pyroglutamate may be included as the N-terminal
residue of
the peptide.
In a further embodiment, subunits of peptides that confer useful chemical and
structural
properties will be chosen. For example, peptides comprising D-amino acids will
be
resistant to L-amino acid-specific proteases in vivo. In addition, the present
invention
envisions preparing peptides that have more well defined structural
properties, and the use
of peptidomimetics, and peptidomimetic bonds, such as ester bonds, to prepare
peptides
with novel properties. In another embodiment, a peptide may be generated that
incorporates a reduced peptide bond, i.e., R,-CHZ-NH-R2, where R, and RZ are
amino acid
residues or sequences. A reduced peptide bond may be introduced as a dipeptide
subunit.
Such a molecule would be resistant to peptide bond hydrolysis, e.g., protease
activity.
Such peptides would provide ligands with unique function and activity, such as
extended
half lives in vivo due to resistance to metabolic breakdown, or protease
activity.
Furthermore, it is well known that in certain systems constrained peptides
show enhanced
functional activity (Hruby, 1982, Life Sciences 31:189-199; Hruby et al.,
1990, Biochem J.
268:249-262); the present invention provides a method to produce a constrained
peptide
that incorporates random sequences at all other positions.
1. Constrained and cyclic peptides.
A constrained, cyclic or rigidized peptide may be prepared synthetically,
provided that in at
least two positions in the sequence of the peptide an amino acid or amino acid
analog is
inserted that provides a chemical functional group capable of crosslinking to
constrain,
cyclise or rigidize the peptide after treatment to form the cross-link.
Cyclization will be
favored when a turn-inducing amino acid is incorporated. Examples of amino
acids capable
of crosslinking a peptide are cysteine to form disulfides, aspartic acid to
form a lactone or a
lactam, and a chelator such as y-carboxyl-glutamic acid (Gla) (Bachem) to
chelate a
transition metal and form a cross-link. Protected y-carboxyl glutamic acid may
be prepared
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18
by modifying the synthesis described by Zee-Cheng and Olson ( 1980, Biophys.
Biochem.
Res. Conunun. 94:1128-1132). A peptide in which the peptide sequence comprises
at least
two amino acids capable of crosslinking may be treated, e.g., by oxidation of
cysteine
residues to form a disulfide or addition of a metal ion to form a chelate, so
as to cross-link
the peptide and form a constrained, cyclic or rigidized peptide.
2. Non-classical amino acids that induce conformational constraints.
The following non-classical amino acids may be incorporated in the peptide in
order to
introduce particular conformational motifs: 1,2,3,4-tetrahydroisoquinoline-3-
carboxylate
(Kazmierski et al., 1991, J. Am. Chem. Soc. 113:2275-2283); (2S,3S)-methyl-
phenylalanine, (2S,3R)-methyl-phenylalanine, (2R,3S)-methyl-phenylalanine and
(2R,3R)-
methyl-phenylalanine (Kazmierski and Hruby, 1991, Tetrahedron Lett.}; 2-
aminotetrahydronaphthalene-2-carboxylic acid (Landis, 1989, Ph.D. Thesis,
University of
Arizona); hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et al.,
1989, J.
Takeda Res. Labs. 43:53-76); ~3-carboline (D and L) (Kazmierski, 1988, Ph.D.
Thesis,
University of Arizona); HIC (histidine isoquinoline carboxylic acid) (Zechel
et al., 1991,
Int. J. Pep. Protein Res. 43); and HIC (histidine cyclic urea)
(Dharanipragada).
The following amino acid analogs and peptidomimetics may be incorporated into
a peptide
to induce or favor specific secondary structures: LL-Acp (LL-3-amino-2-
propenidone-6-
carboxylic acid), a ~i-turn inducing dipeptide analog (Kemp et al., 1985, J.
Org. Chem.
50:5834-5838); ~i-sheet inducing analogs (Kemp et al., 1988, Tetrahedron Lett.
29:5081-
5082); ~i-turn inducing analogs (Kemp et al., 1988, Tetrahedron Lett. 29:5057-
5060);
«-helix inducing analogs (Kemp et al. , 1988, Tetrahedron Lett. 29:4935-4938);
y-turn
inducing analogs (Kemp et al., 1989, J. Org. Chem. 54:109:1IS); and analogs
provided by
the following references: Nagai and Sato, 1985, Tetrahedron Lett. 26:647-650;
DiMaio et
al., 1989, J. Chem. Soc. Perkin Trans. p. 1687; also a Gly-Ala turn analog
(Kahn et al.,
1989, Tetrahedron Lett. 30:2317); amide bond isostere (Jones et al., 1988,
Tetrahedron
Lett. 29:3853-3856); tretrazol (Zabrocki et al., 1988, J. Am. Chem. Soc.
110:5875-5880);
DTC (Samanen et al., 1990, Int. J. Protein Pep. Res. 35:501:509); and analogs
taught in
Olson et al., 1990, J. Am. Chem. Sci. 112:323-333 and Garvey et al., 1990, J.
Org.
Chem. 56:436. Conformationally restricted mimetics of beta turns and beta
bulges, and
peptides containing them, are described in U.S. Patent No. 5,440,013, issued
August 8,
1995 to Kahn.
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19
3. Derivatized and modified peptides.
The present invention further provides for modification or derivatization of a
peptide of the
invention. Modifications of peptides are well known to one of ordinary skill,
and include
phosphorylation, carboxymethylation, and acylation. Modifications may be
effected by
chemical or enzymatic means.
In another aspect, glycosylated or fatty acylated peptide derivatives may be
prepared.
Preparation of glycosylated or fatty acylated peptides is well known in the
art as
exemplified by the following references:
1. Garg and Jeanloz, 1985, in Advances in Carbohydrate Chemistry and
Biochemistry, Vol. 43, Academic Press.
2. Kunz, 1987, in Ang. Chem. Int. Ed. English 26:294-308.
3. Horvat et al., 1988, Int. J. Pept. Protein Res. 31:499-507.
4. Bardaji et al., 1990, Ang. Chem. Int. Ed. English, 23:231.
5. Toth et al., 1990, in Peptides: Chemistry, Structure and Biology, Rivier
and Marshal, eds., ESCOM Publ., Leiden, pp. 1078-1079.
6. Torres et al. , 1989, Experientia 45:574-576.
7. Torres et al., 1989, EMBO J. 8:2925-2932.
8. Hordever and Musiol, 1990, in Peptides: Chemistry, Structure and Biology,
loc. cit., pp. 811-812.
9. Zee-Cheng and Olson, 1989, Biochem. Biophys. Res. Commun. 94:1128-
1132.
10. Marki et al., 1977, Helv. Chem. Acta., 60:807.
11. Fuju et al. 1987, J. Chem. Soc. Chem. Commun., pp. 163-164.
12. Ponsati et al., 1990, Peptides 1990, Giralt and Andreu, eds., ESCOM
Publ., pp. 238-240.
13. Fuji et al., 1987, 1988, Peptides: Chemistry and Biology, Marshall, ed.,
ESCOM Publ., Leiden, pp. 217-219.
There are two major classes of peptide-carbohydrate linkages. First, ether
bonds join the
serine or threonine hydroxyl to a hydroxyl of the sugar. Second, amide bonds
join
glutamate or aspartate carboxyl groups to an amino group on the sugar. In
particular,
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references 1 and 2, supra, teach methods of preparing peptide-carbohydrate
ethers and
amides. Acetai and ketal bonds may also bind carbohydrate to peptide.
Fatty acyl peptide derivatives may also be prepared. For example, and not by
way of
5 limitation, a free amino group (N-terminal or lysyl) may be acylated, e.g.,
myristoylated.
In another embodiment an amino acid comprising an aliphatic side chain of the
structure -
(CHZ)"CH3 may be incorporated in the peptide. This and other peptide-fatty
acid conjugates
suitable for use in the present invention are disclosed in U.K. Patent GB-
8809162.4,
International Patent Application PCT/AU89/00166, and reference 5, supra.
10 Genetic E~ineering of the Peptides
In accordance with the present invention there may be employed conventional
molecular
biology, microbiology, and recombinant DNA techniques within the skill of the
art. Such
techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch
& Maniatis,
Molecular Cloning: A Laboratory Manual, Second Edition ( 1989) Cold Spring
Harbor
15 Laboratory Press, Cold Spring Harbor, New York (herein "Sambrook et al. ,
1989"); DNA
Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985);
Oligonucleotide
Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization [B.D. Hames & S.J.
Higgins
eds. (1985)]; Transcription And Translation (B.D. Hames & S.J. Higgins, eds.
(1984)];
Animal Cell Culture [R.I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes
[IRL
20 Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984);
F.M. Ausubel
et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.
(1994).
The term "sequence similarity" in all its grammatical forms refers to the
degree of identity
or correspondence between nucleic acid or amino acid sequences of proteins
that do not
share a common evolutionary origin (see Reeck et al., supra). However, in
common usage
and in the instant application, the term "homologous," when modified with an
adverb such
as "highly," refers to sequence similarity and not a common evolutionary
origin.
In a specific embodiment, two DNA sequences are "substantially homologous" or
"substantially similar" when at least about SO to (preferably at least about
75 % , and most
preferably at least about 90 or 95 % ) of the nucleotides match over the
defined length of the
DNA sequences.
i
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21
Similarly, in a particular embodiment, two amino acid sequences are
"substantially
homologous" or "substantially similar" when greater than 50% of the amino
acids are
identical, or greater than about 80% are similar (functionally identical).
Preferably, the
similar or homologous sequences are identified by alignment using, for
example, the GCG
(Genetics Computer Group, Program Manual for the GCG Package, Version 7,
Madison,
Wisconsin) pileup program.
The term "corresponding to" is used herein to refer similar or homologous
sequences,
whether the exact position is identical or different from the molecule to
which the similarity
or homology is measured. Thus, the term "corresponding to" refers to the
sequence
similarity, and not the numbering of the amino acid residues or nucleotide
bases.
Identification of a specific DNA encoding a particularly tong peptide of the
present
invention may be accomplished in a number of ways. For example, if an amount
of a
portion of the DNA or a fragment thereof, is available and can be purified and
labeled, the
generated DNA fragments may be screened by nucleic acid hybridization to a
labeled probe
(Benton and Davis, 1977, Science 196:180; Grunstein and Hogness, 1975, Proc.
Natl.
Acad. Sci. U.S.A. 72:3961). For example, a set of oligonucleotides
corresponding to the
amino acid sequence of the peptide can be prepared and used as probes for DNA.
Those
DNA fragments with substantial homology to the probe will hybridize. The
greater the
degree of homology, the more stringent hybridization conditions can be used.
Expression of Antigenic and Inhibitory Peptides and Their Analoes
The nucleotide sequence coding for a desired peptide, derivative or analog
thereof, or a
functionally active derivative, can be inserted into an appropriate expression
vector, i.e., a
vector which contains the necessary elements for the transcription and
translation of the
inserted peptide-coding sequence. Such elements are termed herein a
"promoter." Thus,
the nucleic acid encoding a peptide of the invention is operationally
associated with a
promoter in an expression vector of the invention. An expression vector also
preferably
includes a replication origin.
The necessary transcriptional and translational signals can be provided on a
recombinant
expression vector.
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Potential host-vector systems include but are not limited to mammalian cell
systems infected
with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems
infected with virus
(e.g., baculovirus); microorganisms such as yeast containing yeast vectors; or
bacteria
transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The
expression
elements of vectors vary in their strengths and specificities. Depending on
the host-vector
system utilized, any one of a number of suitable transcription and translation
elements may
be used.
The cell into which the vector containing the peptide DNA is cultured in an
appropriate cell
culture medium under conditions that provide for expression of the peptide by
the cell.
Any of a number of methods for the insertion of DNA fragments into a such a
vector may
be used to construct expression vectors containing a nucleic acid consisting
of appropriate
transcriptional/translational control signals and the peptide coding
sequences. These
methods may include in vitro DNA and synthetic techniques.
Expression of peptide may be controlled by any promoter/enhancer element known
in the
art, but these regulatory elements must be functional in the host selected for
expression.
Promoters which may be used to control the peptide nucleic acid expression
include, but are
not limited to, the SV40 early promoter region (Benoist and Chambon, 1981,
Nature
290:304-310), the promoter contained in the 3' long terminal repeat of Rous
sarcoma virus
(Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine kinase
promoter (Wagner
et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory
sequences of the
metallothionein gene (Brinster et al., 1982, Nature 296:39-42); prokaryotic
expression
vectors such as the ~-lactamase promoter (Villa-Kamaroff, et al., 1978, Proc.
Natl. Acad.
Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoer, et al., 1983, Proc.
Natl. Acad.
Sci. U.S.A. 80:21-25); see also "Useful proteins from recombinant bacteria" in
Scientific
American, 1980, 242:74-94; promoter elements from yeast or other fungi such as
the Gal 4
promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol
kinase)
promoter, alkaline phosphatase promoter; and the animal transcriptional
control regions,
which exhibit tissue specificity and have been utilized in transgenic animals.
Expression vectors containing a nucleic acid encoding an antigenic or
inhibitory peptide of
the invention can be identified by four general approaches: (a) PCR
amplification of the
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23
desired plasmid DNA or specific mRNA, (b) nucleic acid hybridization, (c)
presence or
absence of selection marker gene functions, and (d) expression of inserted
sequences. In
the first approach, the nucleic acids can be amplified by PCR to provide for
detection of the
amplified product. In the second approach, the presence of a foreign gene
inserted in an
expression vector can be detected by nucleic acid hybridization using probes
comprising
sequences that are homologous to an inserted marker gene. In the third
approach, the
recombinant vector/host system can be identified and selected based upon the
presence or
absence of certain "selection marker" gene functions (e.g., ~i-galactosidase
activity,
thymidine kinase activity, resistance to antibiotics, transformation
phenotype, occlusion
body formation in baculovirus, etc.) caused by the insertion of foreign genes
in the vector.
In another example, if the nucleic acid encoding the peptide is inserted
within the "selection
marker" gene sequence of the vector, recombinants containing the peptide
insert can be
identified by the absence of the selection marker gene function. In the fourth
approach,
recombinant expression vectors can be identified by assaying for the activity,
biochemical,
or immunological characteristics of the peptide expressed by the recombinant,
by the
methods described herein.
A wide variety of host/expression vector combinations may be employed in
expressing the
DNA sequences of this invention. Useful expression vectors, for example, may
consist of
segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable
vectors include derivatives of SV40 and known bacterial plasmids, e.g., E.
coli plasmids
col El, pCRI, pBR322, pMal-C2, pET, pGEX (Smith et al., 1988, Gene 67:31-40),
pMB9
and their derivatives, plasmids such as RP4; phage DNAS, e.g., the numerous
derivatives
of phage ~,, e.g., NM989, and other phage DNA, e.g., M13 and filamentous
single
stranded phage DNA; yeast plasmids such as the 2~. plasmid or derivatives
thereof; vectors
useful in eukaryotic cells, such as vectors useful in insect or mammalian
cells; vectors
derived from combinations of plasmids and phage DNAs, such as plasmids that
have been
modified to employ phage DNA or other expression control sequences; and the
like.
For example, in a baculovirus expression systems, both non-fusion transfer
vectors, such as
but not limited to pVL941 (BamHl cloning site; Summers), pVL1393 (BamHl, SmaI,
XbaI, EcoRl, NotI, XmaIII, BgIII, and PstI cloning site; Invitrogen), pVL1392
(BgIII, PstI,
NotI, XmaIII, EcoRI, XbaI, SmaI, and BamHl cloning site; Summers and
Invitrogen), and
pBlueBacIII (BamHl, BgIII, PstI, NcoI, and HindIII cloning site, with
blue/white
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24
recombinant screening possible; Invitrogen), and fusion transfer vectors, such
as but not
limited to pAc700 (BamHl and KpnI cloning site, in which the BamHl recognition
site
begins with the initiation codon; Summers), pAc701 and pAc702 (same as pAc700,
with
different reading frames), pAc360 (BamH 1 cloning site 36 base pairs
downstream of a
polyhedron initiation codon; Invitrogen(195)), and pBIueBacHisA, B, C (three
different
reading frames, with BamHl, BgIII, PstI, NcoI, and HindIII cloning site, an N-
terminal
peptide for ProBond purification, and blue/white recombinant screening of
plaques;
Invitrogen (220)) can be used.
Once a particular synthetic DNA molecule is identified and isolated, several
methods
known in the art may be used to propagate it. Once a suitable host system and
growth
conditions are established, recombinant expression vectors can be propagated
and prepared
in quantity. As previously explained, the expression vectors which can be used
include, but
are not limited to, the following vectors or their derivatives: human or
animal viruses such
as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast
vectors;
bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNA vectors, to
name but a
few.
Vectors are introduced into the desired host cells by methods known in the
art, e.g. ,
transfection, electroporation, microinjection, transduction, cell fusion, DEAE
dextran,
calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene
gun, or a
DNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem. 267:963-
967; Wu and
Wu, 1988, J. Biol. Chem. 263:14621-14624; Hartmut et al., Canadian Patent
Application
No. 2,012,311, filed March 15, 1990).
Administration
According to the invention, the component or components of a therapeutic
composition of
the invention may be introduced parenterally, transmucosally, e.g. , orally,
nasally, or
rectally, or transdermally. Preferably, administration is parenteral, e.g. ,
via intravenous
injection, and also including, but is not limited to, infra-arteriole,
intramuscular,
intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial
administration.
In another embodiment, the therapeutic compound can be delivered in a vesicle,
in
particular a liposome [see Langer, Science 249:1527-1533 (1990); Treat et al.,
in
CA 02295440 1999-12-16
WO 98/57657 PCT/US98/12647
Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler
(eds.), Liss: New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-
327; see
generally ibid.]. To reduce its systemic side effects, this may be a preferred
method for
introducing the peptides and antibodies of the present invention.
5 In yet another embodiment, the therapeutic compound can be delivered in a
controlled
release system. For example, the peptides may be administered using
intravenous infusion,
an implantable osmotic pump, a transdermal patch, Iiposomes, or other modes of
administration. In one embodiment, a pump may be used [see Langer, supra;
Sefton, CRC
Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980);
Saudek et
10 al., N. Engl. J. Med. 321:574 (1989)]. In another embodiment, polymeric
materials can be
used [see Medical Applications of Controlled Release, Langer and Wise (eds.),
CRC Press:
Boca Raton, Florida { 1974); Controlled Drug Bioavailability, Drug Product
Design and
Performance, Smolen and Ball (eds.), Wiley: New York (1984); Ranger and
Peppas, J.
Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al.,
Science 228:190
15 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J.
Neurosurg. 71:105
( 1989)] . In yet another embodiment, a controlled release system can be
placed in proximity
of the therapeutic target, i. e. , the brain, thus requiring only a fraction
of the systemic dose
[see, e.g., Goodson, in Medical Applications of Controlled Release, supra,
vol. 2, pp. 115-
138 (1984)].
20 Other controlled release systems are discussed in the review by Langer
[Science 249:1527-
1533 ( 1990)] .
In a further aspect, recombinant cells that have been transformed with nucleic
acids
encoding either the antigenic or inhibitory peptides of the present invention
and express
high levels of these peptides can be transplanted in a subject in need of
these peptides.
25 Preferably autologous cells transformed with these peptides are
transplanted to avoid
rejection; alternatively, technology is available to shield non-autologous
cells that produce
soluble factors within a polymer matrix that prevents immune recognition and
rejection.
Thus, the therapeutic peptides of the present invention can be delivered by
intravenous,
intraarterial, intraperitoneal, intramuscular, or subcutaneous routes of
administration.
Alternatively, these peptides properly formulated, can be administered by
nasal or oral
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26
administration. A constant supply of these peptides can be ensured by
providing a
therapeutically effective dose (i. e. , a dose effective to induce metabolic
changes in a
subject) at the necessary intervals, e.g., daily, every 12 hours, etc. These
parameters will
depend on the severity of the disease condition being treated, other actions,
the weight, age,
S and sex of the subject, and other criteria, which can be readily determined
according to
standard good medical practice by those of skill in the art.
A subject in whom administration of these peptides is an effective therapeutic
regiment is
preferably a human, but can be any animal. Thus, as can be readily appreciated
by one of
ordinary skill in the art, the methods and pharmaceutical compositions of the
present
invention are particularly suited to administration to any animal,
particularly a mammal,
and including, but by no means limited to, domestic animals, such as feline or
canine
subjects, farm animals, such as but not limited to bovine, equine, caprine,
ovine, and
porcine subjects, wild animals (whether in the wild or in a zoological
garden), research
animals, such as mice, rats, rabbits, goats, sheep, pigs, dogs, cats, etc. ,
avian species, such
as chickens, turkeys, songbirds, etc., i.e., for veterinary medical use.
Nasal Delivery. Nasal delivery of the pharmaceutical compositions containing
the
inhibitory or antigenic peptides (or derivative thereof) is also contemplated.
Nasal delivery
allows the passage of the peptide to the blood stream directly after
administering the
therapeutic product to the nose, without the necessity for deposition of the
product in the
lung. Formulations for nasal delivery include those with dextran or
cyclodextran.
For nasal administration, a useful device is a small, hard bottle to which a
metered dose
sprayer is attached. In one embodiment, the metered dose is delivered by
drawing the
solution of peptides into a chamber of defined volume, which chamber has an
aperture
dimensioned to aerosolize and aerosol formulation by forming a spray when a
liquid in the
chamber is compressed. The chamber is compressed to administer the peptides.
In a
specific embodiment, the chamber is a piston arrangement. Such devices are
commercially
available.
Alternatively, a plastic squeeze bottle with an aperture or opening
dimensioned to
aerosolize an aerosol formulation by forming a spray when squeezed. The
opening is
usually found in the top of the bottle, and the top is generally tapered to
partially fit in the
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27
nasal passages for efficient administration of the aerosol formulation.
Preferably, the nasal
inhaler will provide a metered amount of the aerosol formulation, for
administration of a
measured dose of the drug.
The term "mucosal penetration enhancer" refers to a reagent that increases the
rate or
facility of transmucosal penetration of ketamine, such as but not limited to,
a bile salt, fatty
acid, surfactant or alcohol. In specific embodiments, the permeation enhancer
can be
sodium cholate, sodium dodecyl sulphate, sodium deoxycholate,
taurodeoxycholate, sodium
glycocholate, dimethylsulfoxide or ethanol. Suitable penetration enhancers
also include
glycyrrhetinic acid (U.S. Patent No. 5,112,804 to Kowarski) and polysorbate-
80, the latter
preferably in combination with an non-ionic surfactant such as nonoxynol-9,
Iaureth-9,
poloxamer-124, octoxynol-9, or lauramide-DEA (European Patent EP 0 242 643 B1
by
Stoltz).
Various and numerous methods are known in the art for transdermal
administration of a
drug, e.g., via a transdermal patch. Transdermal patches are described in for
example,
U.S. Patent No. 5,407,713, issued April 18, 1995 to Rolando et al.; U.S.
Patent No.
5,352,456, issued October 4, 1004 to Fallon et al.; U.S. Patent No. 5,332,213
issued
August 9, 1994 to D'Angelo et al.; U.S. Patent No. 5,336,168, issued August 9,
1994 to
Sibalis; U.S. Patent No. 5,290,561, issued March 1, 1994 to Farhadieh et al.;
U.S. Patent
No. 5,254,346, issued October 19, 1993 to Tucker et al.; U.S. Patent No.
5,164,189,
issued November 17, 1992 to Berger et al.; U.S. Patent No. 5,163,899, issued
November
17, 1992 to Sibalis; U.S. Patent Nos. 5,088,977 and 5,087,240, both issued
February 18,
1992 to Sibalis; U.S. Patent No. 5,008,110, issued April 16, 1991 to Benecke
et al.; and
U.S. Patent No. 4,921,475, issued May 1, 1990 to Sibalis, the disclosure of
each of which
is incorporated herein by reference in its entirety.
It can be readily appreciated that a transdermal route of administration may
be enhanced by
use of a dermal penetration enhancer, e.g., such as enhancers described in
U.S. Patent No.
5,164,189 (supra), U.S. Patent No. 5,008,110 (supra), and U.S. Patent No.
4,879,119,
issued November 7, 1989 to Aruga et al., the disclosure of each of which is
incorporated
herein by reference in its entirety.
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The present invention may be better understood by reference to the following
non-limiting
Examples, which are provided as exemplary of the invention.
EXAMPLES
MATERIALS AND METHODS
Protocol for affinity purified anti-mucopeptide antibodies.
The anti-mucopeptide antibody was affinity purified in the following manner.
Five ml of
rabbit anti-mucopeptide antibody was first passed over a protein A sepharose
column and
the antibody fraction binding to the column was eluted in a 0.05 M sodium
Acetate buffer
pH 2.5. The volume of the eluant was adjusted back to the original volume of
the serum (5
ml) and the pH was adjusted to pH 7.2 with SN NAOH. 5 mg of streptococcal
mucopeptide (dry weight) was then mixed with 0.5 ml of anti-mucopeptide
purified
antibody and then rotated gently at 37°C for 2-3 hours. The sample was
stored overnight
in the cold at 4°C. The following day, the sample was centrifuged at
14000 RPM for 15-
minutes. The supernatant was removed and the pellet was resuspended in 0.5 ml
of anti-
15 mucopeptide purified antibody and the incubation procedure was repeated.
This procedure
was repeated once more for the third time.
Following these absorptions, the pellet was resuspended in 1 ml of 0.05 M
sodium acetate
buffer pH 2.5 and rotated gently at room temperature for 1 hour and then
centrifuged as
described above. The pH of the supernatant was brought back to pH 7.2 by the
addition of
20 SN NAOH and then tested with an ELISA assay using a preparation of
sonicated
mucopeptide at a concentration of lug/well. The ELISA titer was compared to
the original
non-absorbed antibody and the volume adjusted so that the absorbed antibody
has the same
titer as the original non-absorbed anti-mucopeptide antibody.
The biological activity of the affinity purified antibody was then tested
using human
mononuclear cell preparations as described above. Serial dilutions of the
affinity purified
anti-mucopeptide antibody was then added to the wells followed by the addition
of different
concentrations of LPS and streptococcal cell wall mucopeptide preparations
starting at a
concentration of 100 ~g/ml. Control samples included cells in which the
antibody wasn't
added. The cell suspension was incubated for 24 hrs at 37°C in a COZ
incubator and then
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following the subsequent centrifugation, the supernatants were harvested and
tested for
TNF alpha production. As an additional control phytohemagglutinin-stimulated
cells were
tested to rule out the possibility that non-specific inhibitors were contained
in the affinity
purified antibody preparation. The TNF alpha assays were carried out with a
standard
capture ELISA kit from Endogen (Cambridge, MA).
EXAMPLE 1
Identifvine peptides useful for the treatment of septic shock
The observation that the anti-mucopeptide antibody blocks the induction of TNF
production
by both mucopeptide and LPS suggests that there is a common epitope in both
structures.
This, in turn, further suggests that binding domains on some anti-mucopeptide
antibodies
have the right sequence to bind to both of these compounds. These binding
sequences are
located in the following manner. Phage libraries have been constructed which
when
infected into host E. coli produce random peptide sequences of approximately
10 to 15
amino acids [Parmley and Smith, Gene 73:305-318 (1988), Scott and Smith,
Science
249:386-249 (1990)]. Specifically, the phage library can be mixed in low
dilutions with
permissive E, coli in low melting point LB agar which is then poured on top of
LB agar
plates. After incubating the plates at 37°C for a period of time, small
clear plaques in a
lawn of E. coli will form which represents active phage growth and lysis of
the E. coli. A
representative of these phages can be absorbed to nylon filters by placing dry
filters onto
the agar plates. The filters can be marked for orientation, removed, and
placed in washing
solutions to block any remaining absorbent sites. The filters can then be
placed in a
solution containing radioactive mucopeptide complex. After a specified
incubation period,
the filters can be thoroughly washed and developed for autoradiography.
Plagues
containing the phage that bind to the mucopeptide complex can be identified.
These phages
can be further cloned and then retested for their ability to bind to the
mucopeptide complex
as before. Once the phages have been purified, the binding sequence contained
within the
phage can be determined by standard DNA sequencing techniques. Once the DNA
sequence is known, synthetic peptides can be generated which represents these
sequences.
These peptides can be tested for their ability to: (1) bind the mucopeptide
complex, (2) bind
to lipid A, and (3) inhibit the anti-mucopeptide antibodies.
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A single peptide with strong positive results indicates a single dominant
binding peptide.
Isolation of multiple peptides indicates that the peptides have a common
sequence, e.g. the
phage library has generated the exact same peptide more than once, or several
difference
peptides have a similar reactivity.
5
The peptides) shown positive by ELISA, can be synthesized and assayed to test
whether it
(they) can block the anti-mucopeptide effect on TNF induction by either LPS or
mucopeptide. The effective peptides) can be synthesized in large quantities
for use in
animal models of shock and eventually in humans to prevent septic shock. It
should be
10 emphasized that synthetic peptide production is relatively non-labor
intensive, easily
manufactured, quality controlled and thus, large quantities of the desired
product can be
produced quite cheaply. Similar combinations of mass produced synthetic
peptides have
recently been used in trials of a malaria vaccine with great success
[Patarroyo, Vaccine
10:175-178 (1990)] and shown to be quite safe for use in humans.
15 EXAMPLE 2
The use of anti-Group A mucopevtide antibodies and peptides in the treatment
of
experimental arthritis.
The disease rheumatoid arthritis is a disease of unknown etiology but presumed
to be an
autoimmune disease involving the target organ, the joints. While many
experimental
20 models of arthritis have been proposed (see Table 1), the streptococcal
induced model is
preferred for the following reasons. First, it is a single dose of a sonicated
cell wall
preparation which, unlike the other models, induces arthritis which then waxes
and wanes
for over half the life of the animal. Secondly, the end result bears a
striking resemblance to
the human disease. Finally, it is cell mediated and the model can be
transferred to naive
25 recipients by passive administration of donor lymphocytes without the
streptococcal antigen
being present. TNF plays an important role in disease induction and in an
experimental
model the disease can be blocked by injecting soluble TNF receptor molecules
just after
initiation of the disease.
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Table 1
Features of Rheumatoid Arthritis and Animal Models of Arthritis
RheumatoidStreptococcalAdjuvant Collagen
Arthritis Arthritis ArthritisArthritis
Clinical Features
Recurrent + + - _
Joint Distribution+ + + + -
Nodules I + I + I - _
Pathological Features
Monocyte Infiltrates+ + + +
Pannus + + + + /-
Cellular Features
Ia Expression + + ND +
Serological Features
Rheumatoid Factor+ ? - ND
Complement Deposition+ + ND ND
Anti II Collagen + ? + ND
T ~TII ;mA;~..,ro~ L__- ~
rl...v :r 1.....
_..-.
------- ---_ __ __.... ..... ......., uvwiuaaaaa.u.
Since the inciting agent is primarily the streptococcal mucopeptide-
polysaccharide complex,
the approach to the model proceeds by two routes of investigation. The first
approach
induces the experimental arthritis in rats. At selected times, either before
or after the onset
of arthritis, animals are injected with the anti-mucopeptide antibody. If the
injection is
before the onset of the disease, the anti-mucopeptide antibody protects
against disease
induction. If injected after the onset of disease, it ameliorates the symptoms
and severity of
disease. Since the antibody can confer passive protection, the peptides
described above can
prevent the onset of the induced arthritis when injected concurrently with the
mucopeptide
complex by LV. or LP.
A second approach is based on a interesting observation made previously in the
model
[Janusz et al., J.Exp.Med. 160:1360-1366 (1984)). If one enzymatically digests
the
mucopeptide-polysaccharide complex with enzymes (phage lysin, mutanolysin,
etc.) into
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smaller pieces and injects these fragments, the recipient animal, after a
period of time, is no
longer susceptible to the induction of arthritis by the mucopeptide complex.
Thus, these
injected fragments seem to protect against induction of classical arthritis by
the larger
complex. This protection can be mediated through eliciting anti-mucopeptide
antibodies
directed against the responsible epitope which induces the arthritis.
These experiments can be extended to show that this protection can be
passively transferred
to a naive animal by antibodies, either by injecting the available anti-
mucopeptide
antibodies or those antibodies elicited after injecting enzymatically derived
fragments of the
mucopeptide complex. After determining that protection from induced arthritic
disease can
be mediated through antibodies, these antibodies can be used to identify
peptide mimitopes
of the mucopeptide fragments which will elicit a similar protective antibody
response.
Using the random peptide phage libraries discussed herein, the protective
antibodies can be
used to identify peptides which mimic the structure of the fragments of the
mucopeptide
complex. These peptides have the following characteristics: (1) they react
with the anti-
mucopeptide antibodies, (2) they do not bind to the mucopeptide complex, and
(3) they do
not elicit a protective antibody response in animals against mucopeptide
complex induced
arthritic disease. A similar strategy has been successfully used to find
peptides that mimic
the structure of Group C polysaccharide capsular antigens of Neisseria
mengitidis
[Westerink and Giardina, Microb.Pathog. 12:19-26 (I992)]. These peptides can
be used to
induce a protective antibody response to experimental arthritis.
The various forms of human arthritis (Crohn's disease, psoriatic arthritis,
etc.) all appear to
have a common antigen which is driving the etiology. The identification of a
common
epitope or mimitope to all these antigens, can lead to the amelioration or
prevention of the
manifestations of a number of these arthrides.
The present invention is not to be limited in scope by the specific
embodiments describe
herein. Indeed, various modifications of the invention in addition to those
described herein
will become apparent to those skilled in the art from the foregoing
description and the
accompanying figures. Such modifications are intended to fall within the scope
of the
appended claims.
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It is further to be understood that all base sizes or amino acid sizes, and
all molecular
weight or molecular mass values, given for nucleic acids or polypeptides are
approximate,
and are provided for description.
All documents cited above, are herein incorporated by reference in their
entireties.
