U.S. patent application number 09/754941 was filed with the patent office on 2001-07-05 for chemokine receptor peptide vaccines for treatment and prevention of diabetes.
Invention is credited to Arimilli, Subhashini, Deshpande, Shrikant, Ferlin, Walter, Howard, Maureen.
Application Number | 20010006942 09/754941 |
Document ID | / |
Family ID | 22593363 |
Filed Date | 2001-07-05 |
United States Patent
Application |
20010006942 |
Kind Code |
A1 |
Howard, Maureen ; et
al. |
July 5, 2001 |
Chemokine receptor peptide vaccines for treatment and prevention of
diabetes
Abstract
The present invention provides immunogenic oligopeptides derived
from the chemokine receptor protein for use in compositions and
methods for the treatment, and prevention of inflammatory
responses.
Inventors: |
Howard, Maureen; (Los Altos
Hills, CA) ; Deshpande, Shrikant; (Fremont, CA)
; Ferlin, Walter; (Antibes, FR) ; Arimilli,
Subhashini; (Fremont, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Family ID: |
22593363 |
Appl. No.: |
09/754941 |
Filed: |
January 4, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09754941 |
Jan 4, 2001 |
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09164186 |
Sep 30, 1998 |
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6171590 |
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Current U.S.
Class: |
424/185.1 ;
514/17.9; 514/21.1; 514/6.9 |
Current CPC
Class: |
C07K 14/7158 20130101;
A61P 3/10 20180101; A61P 25/00 20180101; A61K 39/00 20130101; A61P
37/00 20180101 |
Class at
Publication: |
514/12 ;
424/185.1 |
International
Class: |
A61K 038/00 |
Claims
What is claimed is:
1. A method of inducing an immune response against a chemokine
receptor molecule in a patient, the method comprising administering
to the patient an immunologically effective amount of a
pharmaceutical composition comprising an adjuvant and an
immunogenic chemokine receptor polypeptide from a extracellular
region of a chemokine receptor molecule.
2. The method of claim 1, wherein the chemokine receptor is
CXCR3.
3. The method of claim 1, wherein the immunogenic peptide is
conformationally constrained.
4. The method of claim 1, wherein the immunogenic peptide is
cyclized.
5. The method of claim 1, wherein the immunogenic chemokine
receptor peptide consists of between about 10 and about 50
residues.
6. The method of claim 1, wherein the immunogenic chemokine
receptor peptide consists of between about 15 and about 30
residues.
7. The method of claim 1, wherein the immunogenic chemokine
receptor polypeptide has an amino acid sequence selected from the
group consisting of MVLEVSDHQVLNDAEVAALL, ENFSSSYDYGENESDSCCTS,
PPCPQDFSLNFDRAFLPA, DAAVQWVFGSGLCKV, SAHHDERLNATHCQYN,
FPQVGRTALRVLQLVAG, and DILMDLGALARNCGRESRVDVAKS.
8. The method of claim 1, wherein the administration is
parenteral.
9. The method of claim 1, wherein the adjuvant is alum.
10. A method of inhibiting recruitment of T cells to inflammation
site in a patient, the method comprising administering to the
patient an immunologically effective amount of a pharmaceutical
composition comprising an adjuvant and an immunogenic chemokine
receptor peptide from a extracellular region of a chemokine
receptor molecule.
11. The method of claim 10, wherein the chemokine receptor is
CXCR3.
12. The method of claim 10, wherein the immunogenic peptide is
conformationally constrained.
13. The method of claim 10, wherein the immunogenic peptide is
cyclized.
14. The method of claim 10, wherein the immunogenic chemokine
receptor peptide consists of between about 10 and about 50
residues.
15. The method of claim 10, wherein the immunogenic chemokine
receptor peptide consists of between about 15 and about 30
residues.
16. The method of claim 10, wherein the immunogenic chemokine
receptor polypeptide has an amino acid sequence selected from the
group consisting of MVLEVSDHQVLNDAEVAALL, ENFSSSYDYGENESDSCCTS,
PPCPQDFSLNFDRAFLPA, DAAVQWVFGSGLCKV, SAHHEDERLNATHCQYN,
FPQVGRTALRVLQLVAG, and DILMDLGALARNCGRESRVDVAKS.
17. The method of claim 10, wherein the inflammatory response is
associated with multiple sclerosis.
18. A pharmaceutical composition comprising an adjuvant and an
isolated immunogenic chemokine receptor polypeptide from a
extracellular region of a chemokine receptor molecule.
19. The composition of claim 18, wherein the chemokine receptor is
CXCR3.
20. The composition of claim 18, wherein the immunogenic peptide is
conformationally constrained.
21. The composition of claim 18, wherein the immunogenic peptide is
cyclized.
22. The composition of claim 18, wherein the immunogenic chemokine
receptor peptide consists of between about 10 and about 50
residues.
23. The composition of claim 18, wherein the immunogenic chemokine
receptor peptide consists of between about 15 and about 30
residues.
24. The composition of claim 18, wherein the immunogenic chemokine
receptor polypeptide has an amino acid sequence selected from the
group consisting of MVLEVSDHQVLNDAEVAALL, ENFSSSYDYGENESDSCCTS,
PPCPQDFSLNFDRAFLPA, DAAVQWVFGSGLCKV, SAHHDERLNATHCQYN,
FPQVGRTALRVLQLVAG, and DILMDLGALARNCGRESRVDVAKS.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to novel compositions and
methods for inhibiting inflammatory responses associated with
autoimmune diseases. In particular, it relates to vaccination with
peptides from the extracellular regions of chemokine receptor
molecules.
[0002] Chemokines constitute a family of small molecular weight
cytokines that are produced in inflammation and regulate leukocyte
recruitment. These molecules are ligands for seven transmembrane G
protein linked receptors that induce a signaling cascade
costimulation for T cell activation in addition to participating in
transendothelial migration of leukocytes (Oppenheim et al. Ann.
Rev. Immunol. 9:617-648 (1991), Premback et al. Nat. Med.
2:1174-1178 (1996)). Two subfamilies of chemokines, referred to as
CC and CXC, have been discovered. CC and CXC chemokines are
distinct from each other in their N terminal amino acid sequence
which starts either with cysteine-cysteine or cysteine-X-cysteine
where X is typically another L-amino acid. They are also distinct
in their binding pattern to their receptors. For example, the CC
chemokines bind to CC receptors and not to CXC receptors and vice
versa.
[0003] Different chemokines regulate the trafficking of distinct
populations of hemopoietic cells by activating specific
7-transmembrane receptors expressed by these cells (Baggiolini et
al. Adv. Immunol. 55:97-179 (1994); Gerard et al. Curr. Opin.
Immunol.6:140-145 (1994)). Recent publications indicate that the
Th1 and Th2 subsets of regulatory T cells are uniquely
characterized by the chemokine receptors CXCR3 and CCR3,
respectively (Sallusto, et al. J. Exp. Med. 187:875-883 (1998);
Bonecchi, et al. J Exp. Med. 187:129-134 (1998); Qin, et al. J.
Clin Invest. 101:746-754 (1998)). Several studies have correlated
the expression of three specific chemokines, IP-10, RANTES, and
MCP-1, produced by astrocytes in the CNS with the presence of
inflammatory infiltration within this tissue during the early phase
of EAE (Ransohoff, et al. FASEB J. 7:592-600 (1993); Glabinski et
al. Am. J. Pathol. 150:617-630 (1995) Godiska, et al. J.
Neuroimmunol. 58:167-176 (1995); and Eng et al. Neurchem. Res.
21:511-525 (1996)). While all three chemokines have been shown to
be capable of recruiting T lymphocytes in certain experimental
models, IP-10 has been demonstrated to be specific for this lineage
of hemopoietic cells (Taub et al. J. Exp. Med. 177:1809-1814
(1993)); Carr, et al. Proc. Natl. Acad. Sci. USA 91:3652-3656
(1994); and Farber, J. Leukoc. Biol. 61:246-257 (1997).
MBP-immunized rats intrathecaly infused with an antisense
phosphorothioate oligonucleotide to crg-2 (the murine homologue of
human IP-10) show reduced disease clinical score of EAE (Wojcik, et
al. J. Pharmacol. Exp. Ther. 278:404-410 (1996)). In addition,
higher expression of some of the chemokine receptors such as CXCR3
on IL2 activated human T lymphocytes and not on resting T
lymphocytes has been demonstrated (Loetscher et al. J Exp. Med.
184:963-969 (1996)).
[0004] Multiple sclerosis (MS) is a T cell-dependent autoimmune
disease caused by localized demyelination in the central nervous
system (CNS), with only limited therapeutic options available to
patients. Extensive investigation has indicated that these
autoreactive T lymphocytes frequently, though not always, express
the Th1 phenotype of high level production of IFNg, IL-2 and TNFa,
with little to no IL-4, IL-5 and IL-10.
[0005] Current therapeutics for autoimmune diseases, such as MS,
involve the use of antiinflammatory agents or general
immunosuppressants. Prior art methods for controlling autoimmune
disease fail to provide a simple self-mediated method for
specifically eliminating inflammatory responses mediated by
chemokines associated with the autoimmune responses. The present
invention addresses these and other needs.
SUMMARY OF THE INVENTION
[0006] The present invention provides methods of inducing an immune
response against a chemokine receptor molecule in a patient. The
methods comprise administering to the patient an immunologically
effective amount of a pharmaceutical composition comprising an
adjuvant and an immunogenic chemokine receptor polypeptide from a
extracellular region of a chemokine receptor molecule, for example
CXCR3.
[0007] The immunogenic peptides are preferably conformationally
constrained, for example by cyclization. The length of the
immunogenic peptide is not critical to the invention. Typically,
the peptide consists of between about 10 and about 50 residues,
more often between about 15 and about 30 residues. Exemplary
immunogenic peptides of the invention include MVLEVSDHQVLNDAEVAALL,
ENFSSSYDYGENESDSCCTS, PPCPQDFSLNFDRAFLPA, DAAVQWVFGSGLCKV,
SAHHDERLNATHCQYN, FPQVGRTALRVLQLVAG, and
DILMDLGALARNCGRESRVDVAKS.
[0008] The immunogenic peptides can be administered by any of a
number of means. Typically they are administered parenterally. The
adjuvant can be, for example, alum.
[0009] In preferred embodiments, the method are used to inhibit
recruitment of T cells to inflammation sites in a patient.
Typically the inflammatory response is associated with an
autoimmune disease, such as multiple sclerosis.
[0010] The invention also provides pharmaceutical compositions
suitable for use in the above methods.
Definitions
[0011] The term "peptide" is used interchangeably with
"oligopeptide" or "polypeptide" in the present specification to
designate a series of residues, typically L-amino acids, connected
one to the other typically by peptide bonds between the
.alpha.-amino and carbonyl groups of adjacent amino acids.
[0012] The term "cyclic peptide" refers to peptides in which the
N-terminal residue is linked to the C-terminal residue either
directly or through an intermediate. Examples of links between the
two residues include disulfide bonds and thioether linkages as
described below.
[0013] An "immunogenic chemokine receptor polypeptide" of the
present invention is a polypeptide capable of eliciting an immune
response against a chemokine receptor molecule associated with
inflammation in autoimmune responses in a patient, such as multiple
sclerosis. As set forth in more detail below, the sequence of
residues in the polypeptide will be identical to or substantially
identical to a polypeptide sequence in a chemokine receptor
molecule. Thus, a polypeptide of the invention that has a sequence
"from a extracellular region of a chemokine receptor molecule" is
polypeptide that has a sequence either identical to or
substantially identical to the naturally occurring chemokine
receptor amino acid sequence of the region.
[0014] As used herein a "extracellular region" of a chemokine
receptor molecule is a region of the molecule which is exposed on
the surface of a cell expressing the native molecule. FIG. 1
provides a schematic of the extracellular domains of the human
CXCR3 molecule. This molecule has four extracellular regions
designated as SP-1, SP-2, SP-3 and SP-4, starting from the
N-terminus.
[0015] As used herein, the term "adjuvant" refers to any substance
which, when administered with or before an antigen, increases
and/or qualitatively affects the immune response against the
antigen in terms of antibody formation and/or the cell-mediated
response. Exemplary adjuvants for use in the present invention are
provided below.
[0016] The phrases "isolated" or "biologically pure" refer to
material which is substantially or essentially free from components
which normally accompany it as found in its native state. Thus, the
chemokine receptor polypeptides of this invention do not contain
materials normally associated with their in situ environment, e.g.,
other surface proteins on T cells. Even where a protein has been
isolated to a homogenous or dominant band, there are trace
contaminants in the range of 5-10% of native protein which
co-purify with the desired protein. Isolated polypeptides of this
invention do not contain such endogenous co-purified protein.
[0017] The term "residue" refers to an amino acid or amino acid
mimetic incorporated in a oligopeptide by an amide bond or amide
bond mimetic.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 shows acidification rate changes due to the binding
of anti-human CXCR3 Mab or P-10 to CXCR3-NSO transfectants.
5X10.sup.5 CXCR3-NSO transfectant cells and untransfected
"BONZO-NSO" cells (negative control) were spotted in the
microphysiometer chambers along with agarose and 1, 5, 10 mglml
anti-CXCR3 (FIG 1a) or IP-10 (FIG 1b) was pumped for 10 min. There
is a dose dependent increase in the acidification rates with
anti-CXCR3 in NSO-CXCR3 transfectant cells. Untransfected BONZO-NSO
cells did not show any change in acidification rate changes either
with the anti-CXCR3 antibody, or IP-10. The arrow indicates the
time that ligands were added to the cells.
[0019] FIG. 2 shows the structure of the seven transmembrane
G-protein coupled human CXCR3. Only amino acids in the
extracellular domains are given
[0020] FIG. 3 is a schematic of the experiments in which peptides
vaccines of the invention were used to prevent EAE in mice.
[0021] FIG. 3 shows the results of experiments in which peptides
vaccines of the invention were used to prevent EAE in mice.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0022] The present invention provides immunogenic polypeptides
derived from chemokine receptor protein sequences for use in
compositions and methods for the treatment and prevention of
inflammatory responses. The polypeptides are capable of inducing an
immune response against chemokine receptor molecules which mediate
inflammatory responses associated with various diseases. In
preferred embodiments the polypeptides of the invention are
conformationally constrained (e.g., cyclized) and derived from
extracellular regions of human CXCR3. Immunization with the
polypeptides of the invention provides a specific immune response
to particular chemokine receptors and results in the specific
inhibition of the inflammatory responses mediated by these
molecules.
[0023] The methods can be used to treat any inflammatory response
mediated by chemokines. In particular, the methods are useful for
treatment of autoimmune diseases, such as multiple sclerosis.
Multiple sclerosis (MS), a human demyelinating disease which
afflicts 600,000 individuals worldwide, results from damage of the
myelin sheath of oligodendroglial cells in the Central Nervous
System (CNS). Although the pathogenesis and etiology of MS have not
yet been established, it is widely believed that the disease has an
immunological basis and that both genetic and environmental factors
make a contribution to the pathogenesis. The central mediator of
autoimmune attack is believed to be host CD4+T cells specific for
one or more autoantigens in the CNS, with subsequent production of
an array of tissue-destructive inflammatory mediators following
autoantigen-activation of these cells. Indeed, immunohistochemical
analysis of the focal plaques of demyelination which occur in the
brains of MS patients as a consequence of MS pathology have
revealed the presence of CD4+T cells infiltrating these
plaques.
[0024] Improved understanding of the immunopathological mechanisms
underlying MS has developed from the study of experimental models
of demyelination. The most commonly used model, experimental
allergic encephalomyelitis (EAE), is an autoimmune inflammatory
disorder of genetically susceptible mice that is mediated by
autoantigen-specific CD4+MHC class II restricted T cells. In
susceptible SJL/J mice, the disease can display a
relapsing-remitting clinical course of paralysis, which makes it an
ideal system to study the efficacy of various immunoregulatory
strategies both in the prevention and treatment of disease.
[0025] The current invention is focused not on the
cytokine-producing phenotype of autoreactive T cells in this
disease setting, but on their trafficking from the host circulation
to the site of pathology, for example within the CNS in the case of
MS. As noted above, hemopoietic cell migration is regulated by
chemokines.
[0026] In some embodiments, the invention provides peptide vaccines
against the 7-transmembrane chemokine receptor designated CXCR3.
Consistent with the chemotactic fingerprint of IP-10, CXCR3 is
expressed exclusively on activated effector T lymphocytes.
Exemplary peptides of the invention include peptides derived from
the extracellular domains of the CXCR3 protein are presented in
Table 1.
[0027] In preferred embodiments, the peptides of the invention are
cyclized. Methods for cyclizing peptides are described in detail
below. In those cases in which the peptides are cyclized by
disulfide linkages, one of skill will recognize that the peptides
will further comprise cysteine residues either within the peptide
or at each terminus.
[0028] Polypeptides suitable for use in the present invention can
be obtained in a variety of ways. Conveniently, they can be
synthesized by conventional techniques employing automatic
synthesizers, such as the Beckman, Applied Biosystems, or other
commonly available peptide synthesizers using well known protocols.
They can also be synthesized manually using techniques well known
in the art. See, e.g. Stewart and Young, Solid Phase Peptide
Synthesis, (Rockford, Ill., Pierce), 2d Ed. (1984).
[0029] Alternatively, DNA sequences which encode the particular
chemokine receptor polypeptide may be cloned and expressed to
provide the peptide. Nucleic acid molecules encoding chemokine
receptors are known in the art and sequences of such genes are
available, for instance, from GenBank (see, e.g., GenBank Accession
Nos. HSU83326 HSU97123, and AF005058).
[0030] Standard techniques can be used to screen cDNA libraries to
identify sequences encoding the desired sequences (see, Sambrook et
al., Molecular Cloning--A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, New York, 1989). Fusion proteins
(those consisting of all or part of the amino acid sequences of two
or more proteins) can be recombinantly produced. In addition, using
in vitro mutagenesis techniques, unrelated proteins can be mutated
to comprise the appropriate sequences.
[0031] Chemokine receptor proteins from a variety of natural
sources are also conveniently isolated using standard protein
purification techniques. Peptides can be purified by any of a
variety of known techniques, including, for example, reverse phase
high-performance liquid chromatography (HPLC), ion-exchange or
immunoaffinity chromatography, separation by size, or
electrophoresis (See, generally, Scopes, R., Protein Purification,
Springer-Verlag, N.Y. (1982)).
[0032] It will be understood that the immunogenic chemokine
receptor polypeptides of the present invention may be modified to
provide a variety of desired attributes, e.g., improved
pharmacological characteristics, while increasing or at least
retaining substantially all of the biological activity of the
unmodified peptide. For instance, the peptides can be modified by
extending, decreasing the amino acid sequence of the peptide.
Substitutions with different amino acids or amino acid mimetics can
also be made.
[0033] The peptides employed in the subject invention need not be
identical to peptides disclosed in the Example section, below, so
long as the subject peptides are able to induce an immune response
against the desired chemokine receptor molecule. Thus, one of skill
will recognize that a number of conservative substitutions
(described in more detail below) can be made without substantially
affecting the activity of the peptide.
[0034] Single amino acid substitutions, deletions, or insertions
can be used to determine which residues are relatively insensitive
to modification. Substitutions are preferably made with small,
relatively neutral moieties such as Ala, Gly, Pro, or similar
residues. The effect of single amino acid substitutions may also be
probed using D-amino acids. The number and types of residues which
are substituted or added depend on the spacing necessary between
essential contact points and certain functional attributes which
are sought (e.g., hydrophobicity versus hydrophilicity). Increased
immunogenicity may also be achieved by such substitutions, compared
to the parent peptide. In any event, such substitutions should
employ amino acid residues or other molecular fragments chosen to
avoid, for example, steric and charge interference which might
disrupt binding.
[0035] The substituting amino acids, however, need not be limited
to those naturally occurring in proteins, such as L-.alpha.-amino
acids, or their D-isomers. The peptides may be substituted with a
variety of moieties such as amino acid mimetics well known to those
of skill in the art.
[0036] The individual residues of the immunogenic chemokine
receptor polypeptides can be incorporated in the peptide by a
peptide bond or peptide bond mimetic. A peptide bond mimetic of the
invention includes peptide backbone modifications well known to
those skilled in the art. Such modifications include modifications
of the amide nitrogen, the .alpha.-carbon, amide carbonyl, complete
replacement of the amide bond, extensions, deletions or backbone
crosslinks. See, generally, Spatola, Chemistry and Biochemistry of
Amino Acids, Peptides and Proteins, Vol. VII (Weinstein ed., 1983).
Several peptide backbone modifications are known, these include,
.psi.[CH.sub.2S], .psi.[CH.sub.2NH], .psi.[CSNH.sub.2],
.psi.[NHCO], .psi.[COCH.sub.2] and .psi.(E) or (Z) CH=CH]. The
nomenclature used above, follows that suggested by Spatola, above.
In this context, .psi. indicates the absence of an amide bond. The
structure that replaces the amide group is specified within the
brackets.
[0037] Amino acid mimetics may also be incorporated in the
peptides. An "amino acid mimetic" as used here is a moiety other
than a naturally occurring amino acid that conformationally and
functionally serves as a substitute for an amino acid in a
polypeptide of the present invention. Such a moiety serves as a
substitute for an amino acid residue if it does not interfere with
the ability of the peptide to illicit an immune response against
the appropriate chemokine receptor molecule. Amino acid mimetics
may include non-protein amino acids, such as
.beta.-.gamma.-.delta.-amino acids, .beta.-.gamma.-.delta.-imino
acids (such as piperidine-4-carboxylic acid) as well as many
derivatives of L-.alpha.-amino acids. A number of suitable amino
acid mimetics are known to the skilled artisan, they include
cyclohexylalanine, 3-cyclohexylpropionic acid, L-adamantyl alanine,
adamantylacetic acid and the like. Peptide mimetics suitable for
peptides of the present invention are discussed by Morgan and
Gainor, (1989) Ann. Repts. Med. Chem. 24:243-252/.
[0038] As noted above, the peptides employed in the subject
invention need not be identical, but may be substantially
identical, to the corresponding sequence of the target chemokine
receptor molecule. Therefore, the peptides may be subject to
various changes, such as insertions, deletions, and substitutions,
either conservative or non-conservative, where such changes might
provide for certain advantages in their use. The polypeptides of
the invention can be modified in a number of ways so long as they
comprise a sequence substantially identical (as defined below) to a
sequence in the target region of the chemokine receptor
molecule.
[0039] Alignment and comparison of relatively short amino acid
sequences (less than about 30 residues) is typically
straightforward. Comparison of longer sequences may require more
sophisticated methods to achieve optimal alignment of two
sequences. Optimal alignment of sequences for aligning a comparison
window may be conducted by the local homology algorithm of Smith
and Waterman (1981) Adv. Appl. Math. 2:482, by the homology
alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol.
48:443, by the search for similarity method of Pearson and Lipman
(1988) Proc. Natl. Acad. Sci. (USA) 85:2444, by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package Release 7.0,
Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by
inspection, and the best alignment (i.e., resulting in the highest
percentage of sequence similarity over the comparison window)
generated by the various methods is selected.
[0040] The term "sequence identity" means that two polynucleotide
sequences are identical (i.e., on a nucleotide-by-nucleotide basis)
over a window of comparison. The term "percentage of sequence
identity" is calculated by comparing two optimally aligned
sequences over the window of comparison, determining the number of
positions at which the identical residues occurs in both sequences
to yield the number of matched positions, dividing the number of
matched positions by the total number of positions in the window of
comparison (i.e., the window size), and multiplying the result by
100 to yield the percentage of sequence identity.
[0041] As applied to polypeptides, the term "substantial identity"
means that two peptide sequences, when optimally aligned, such as
by the programs GAP or BESTFIT using default gap weights, share at
least 80 percent sequence identity, preferably at least 90 percent
sequence identity, more preferably at least 95 percent sequence
identity or more (e.g., 99 percent sequence identity). Preferably,
residue positions which are not identical differ by conservative
amino acid substitutions. Conservative amino acid substitutions
refer to the interchangeability of residues having similar side
chains. For example, a group of amino acids having aliphatic side
chains is glycine, alanine, valine, leucine, and isoleucine; a
group of amino acids having aliphatic-hydroxyl side chains is
serine and threonine; a group of amino acids having
amide-containing side chains is asparagine and glutamine; a group
of amino acids having aromatic side chains is phenylalanine,
tyrosine, and tryptophan; a group of amino acids having basic side
chains is lysine, arginine, and histidine; and a group of amino
acids having sulfur-containing side chains is cysteine and
methionine. Preferred conservative amino acids substitution groups
are: valine-leucine-isoleucine, phenylalanine-tyrosine,
lysine-arginine, alanine-valine, and asparagine-glutamine.
[0042] The polypeptides of the invention typically comprise at
least about 10 residues and more preferably at least about 15
residues from the extracellular domain of a chemokine receptor. In
certain embodiments the peptides will not exceed about 50 residues
and typically will not exceed about 30 residues. For instance, the
peptides described below consist of about 15 to about 25
residues.
[0043] In the preferred embodiments of the invention, the
immunogenic peptides are conformationally constrained. Means for
achieving this are well known in the art (see, e.g., Hruby and
Bonner in Methods in Molecular Biology, Volume 35: Peptide
Synthesis Protocols Pennington and Dunn eds (Humana Press, Totowa,
N.J., 1994). A preferred means for preparing conformationally
constrained peptides is through cyclization. Any method commonly
used to produce cyclized oligopeptides can be used to produce the
peptides of the invention. For example, in certain embodiments the
peptides will include cysteine residues at both termini, which
allow the production of cyclic peptides through disulfide linkages.
Treatment of a such a peptide with an oxidizing agent such as
oxygen, iodine or similar agent will produce a cyclic peptide which
may be further purified using chromatographic or other methods of
chemical purification. Construction of cyclic peptides can also be
accomplished through thioether linkages. For instance,
N-bromoacetyl-derivatized peptides can be reacted with
sulfhydryl-containing residues, such as cysteine. Cyclization
occurs by reaction of the free sulfhydryl of cysteine in the
peptide with the bromoacetyl group to form a thioether linkage
(Robey et al., Anal. Biochem. 177:373-7 (1989) and U.S. Pat. No.
5,066,716).
[0044] Other methods of constructing cyclic peptides are known to
those skilled in the art. These include side chain-side chain, side
chain-main chain and main chain-main chain cyclizations. In
addition, linkers can be used to join the amino and carboxyl
termini of a peptide. The linker is capable of forming covalent
bonds to both the amino and carboxyl terminus. Suitable linkers are
well known to those of skill in the art and include, but are not
limited to, straight or branched-chain carbon linkers, heterocyclic
carbon linkers, or peptide linkers. The linkers may be joined to
the carboxyl and amino terminal amino acids through their side
groups (e.g., through a disulfide linkage to cysteine) or through
the alpha carbon amino and carboxyl groups of the terminal amino
acids.
[0045] For a general discussion of suitable methods for cyclization
see, Hruby and Bonner in Methods in Molecular Biology, Volume 35:
Peptide Synthesis Protocols Pennington and Dunn eds (Humana Press,
Totowa, N.J., 1994). For instance, cyclizations may include
formation of carba analogs and thioethers (Lebl et al. in Peptides
1986 Proceedings of the 19th European Peptide Symposium pp.
341-344; Robey et al., Anal Biochem. 177:373-7 (1989) and U.S. Pat.
No. 5,066,716), bis-thioethers (Mosberg et al. JACS 107:2986-2987
(1985)), azopeptides (Siemion et al. Mol. Cell. Biochem. 34:
(1991)), and other cyclic structures, such as bridging structures
(Charpentier, M., et al., J Med. Chem. 32(6):1184-1190 (1989),
Thaisrivongs, S., et al., J. Med. Chem. 34(4):127 (1991) and Ozeki,
E., et al., Int. J. Peptide Protein Res. 34:111 (1989)).
Cyclization from backbone-to-backbone positions may also be
used.
[0046] Bridging is a special type of cyclization in which distant
sites in a peptide are brought together using separate bridging
molecules or fragments. Bridging molecules may include, for
example, succinic anhydride molecules (Charpentier, B., et al.,
supra), and carboxymethylene fragments (Thaisrivongs, S., et al.,
supra). Bridging by metals can also be used (Ozeki, E., et al.,
supra).
[0047] In some embodiments, the peptides include two or more
cystine residues. The cystines can be substituted or added within
the peptide or at either terminus. The position of the cystines is
not critical so long as disulfide linkages can form between them
which allow the production of cyclic peptides. For example,
treatment of such a peptide with an oxidizing agent such as oxygen,
iodine or similar agent will produce a cyclic peptide which may be
further purified using chromatographic or other methods of chemical
purification.
[0048] In addition to use of peptides, antibodies raised against
peptides of the invention can be used to inhibit inflammatory
responses. Antibodies can be raised to the peptides of the present
invention using techniques well known to those of skill in the art.
Anti-idiotypic antibodies can also be generated. The following
discussion is presented as a general overview of the techniques
available; however, one of skill will recognize that many
variations upon the following methods are known.
[0049] A number of immunogens can be used to produce antibodies
specifically reactive with the peptides. For instance, the entire
chemokine receptor molecule or fragments containing the desired
sequence can be used. Synthetic peptides as disclosed here can be
used either in linear form or cyclized.
[0050] Methods of producing polyclonal antibodies are known to
those of skill in the art. In brief, an immunogen (antigen),
preferably a purified polypeptide, a polypeptide coupled to an
appropriate carrier (e.g., GST, keyhole limpet hemanocyanin, etc.),
or a polypeptide incorporated into an immunization vector such as a
recombinant vaccimia virus (see, U.S. Pat. No. 4,722,848) is mixed
with an adjuvant and animals are immunized with the mixture. The
animal's immune response to the immunogen preparation is monitored
by taking test bleeds and determining the titer of reactivity to
the polypeptide of interest. When appropriately high titers of
antibody to the immunogen are obtained, blood is collected from the
animal and antisera are prepared. Further fractionation of the
antisera to enrich for antibodies reactive to the polypeptide is
performed where desired (see, e.g., Coligan (1991) Current
Protocols in Immunology Wiley/Greene, N.Y.; and Harlow and Lane
(1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press,
N.Y.).
[0051] In some instances, it is desirable to prepare monoclonal
antibodies from various mammalian hosts, such as mice, rodents,
primates, humans, etc. Description of techniques for preparing such
monoclonal antibodies are found in, e.g., Stites et al. (eds.)
Basic and Clinical Immunology (4th ed.) Lange Medical Publications,
Los Altos, Calif., and references cited therein; Harlow and Lane,
Supra; Goding (1986) Monoclonal Antibodies: Principles and Practice
(2d ed.) Academic Press, New York, N.Y.; and Kohler and Milstein
(1975) Nature 256:495-497. Summarized briefly, this method proceeds
by injecting an animal with an immunogen. The animal is then
sacrificed and cells taken from its spleen, which are fused with
myeloma cells. The result is a hybrid cell or "hybridoma" that is
capable of reproducing in vitro. The population of hybridomas is
then screened to isolate individual clones, each of which secrete a
single antibody species to the immunogen. In this manner, the
individual antibody species obtained are the products of
immortalized and cloned single B cells from the immune animal
generated in response to a specific site recognized on the
immunogenic substance.
[0052] Alternative methods of immortalization include
transformation with Epstein Barr Virus, oncogenes, or retroviruses,
or other methods known in the art. Colonies arising from single
immortalized cells are screened for production of antibodies of the
desired specificity and affinity for the antigen, and yield of the
monoclonal antibodies produced by such cells is enhanced by various
techniques, including injection into the peritoneal cavity of a
vertebrate (preferably mammalian) host. Specific monoclonal and
polyclonal antibodies will usually bind with a K.sub.D of at least
about 0.1 mM, more usually at least about 50 .mu.M, and most
preferably at least about 1 .mu.M or better.
[0053] Other suitable techniques involve selection of libraries of
recombinant antibodies in phage or similar vectors (see, e.g., Huse
et al. (1989) Science 246:1275-1281; and Ward, et al. (1989) Nature
341:544-546; and Vaughan et al (1996) Nature Biotechnology,
14:309-314).
[0054] Frequently, the peptides and antibodies of the invention
will be labeled by joining, either covalently or non-covalently, a
substance which provides for a detectable signal. A wide variety of
labels and conjugation techniques are known and are reported
extensively in both the scientific and patent literature. Suitable
labels include radionucleotides, enzymes, substrates, cofactors,
inhibitors, fluorescent moieties, chemiluminescent moieties,
magnetic particles, and the like. Patents teaching the use of such
labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;
3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinant
immunoglobulins may be produced. See, Cabilly, U.S. Pat. No.
4,816,567; and Queen et al. (1989) Proc. Nat'l Acad. Sci. USA 86:
10029-10033.
[0055] The antibodies of this invention can also be administered to
an organism (e.g., a human patient) for therapeutic purposes (e.g.,
to inhibit an autoimmune response). Antibodies administered to an
organism other than the species in which they are raised are often
immunogenic. Thus, for example, murine antibodies administered to a
human often induce an immunologic response against the antibody
(e.g., the human anti-mouse antibody (HAMA) response) on multiple
administrations. The immunogenic properties of the antibody are
reduced by altering portions, or all, of the antibody into
characteristically human sequences thereby producing chimeric or
human antibodies, respectively.
[0056] Chimeric antibodies are immunoglobulin molecules comprising
a human and non-human portion. More specifically, the antigen
combining region (or variable region) of a humanized chimeric
antibody is derived from a non-human source (e.g., murine) and the
constant region of the chimeric antibody (which confers biological
effector function to the immunoglobulin) is derived from a human
source. The chimeric antibody should have the antigen binding
specificity of the non-human antibody molecule and the effector
function conferred by the human antibody molecule. A large number
of methods of generating chimeric antibodies are well known to
those of skill in the art (see, e.g., U.S. Pat. Nos: 5,502,167,
5,500,362, 5,491,088, 5,482,856, 5,472,693, 5,354,847, 5,292,867,
5,231,026, 5,204,244, 5,202,238, 5,169,939, 5,081,235, 5,075,431,
and 4,975,369). An alternative approach is the generation of
humanized antibodies by linking the CDR regions of non-human
antibodies to human constant regions by recombinant DNA techniques.
See Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989)
and WO 90/07861.
[0057] In one preferred embodiment, recombinant DNA vector is used
to transfect a cell line that produces an antibody against a
peptide of the invention. The novel recombinant DNA vector contains
a "replacement gene" to replace all or a portion of the gene
encoding the immunoglobulin constant region in the cell line (e.g.,
a replacement gene may encode all or a portion of a constant region
of a human immunoglobulin, or a specific immunoglobulin class), and
a "target sequence" which allows for targeted homologous
recombination with immunoglobulin sequences within the antibody
producing cell.
[0058] In another embodiment, a recombinant DNA vector is used to
transfect a cell line that produces an antibody having a desired
effector function, (e.g., a constant region of a human
immunoglobulin) in which case, the replacement gene contained in
the recombinant vector may encode all or a portion of a region of
an antibody and the target sequence contained in the recombinant
vector allows for homologous recombination and targeted gene
modification within the antibody producing cell. In either
embodiment, when only a portion of the variable or constant region
is replaced, the resulting chimeric antibody may define the same
antigen and/or have the same effector function yet be altered or
improved so that the chimeric antibody may demonstrate a greater
antigen specificity, greater affinity binding constant, increased
effector function, or increased secretion and production by the
transfected antibody producing cell line, etc.
[0059] In another embodiment, this invention provides for fully
human antibodies. Human antibodies consist entirely of
characteristically human polypeptide sequences. The human
antibodies of this invention can be produced in using a wide
variety of methods (see, e.g., Larrick et al., U.S. Pat. No.
5,001,065). In one preferred embodiment, the human antibodies of
the present invention are produced initially in trioma cells. Genes
encoding the antibodies are then cloned and expressed in other
cells, particularly, nonhuman mammalian cells. The general approach
for producing human antibodies by trioma technology has been
described by Ostberg et al. (1983), Hybridoma 2:361-367, Ostberg,
U.S. Pat. No. 4,634,664, and Engelman et al., U.S. Pat. No.
4,634,666. The antibody-producing cell lines obtained by this
method are called triomas because they are descended from three
cells; two human and one mouse. Triomas have been found to produce
antibody more stably than ordinary hybridomas made from human
cells.
[0060] Formulation and Administration
[0061] The peptides or antibodies (typically monoclonal antibodies)
of the present invention and pharmaceutical compositions thereof
are useful for administration to mammals, particularly humans, to
treat and/or prevent deleterious immune inflammatory responses,
particularly those associated with autoimmune responses. Over 30
autoimmune diseases are presently known, including myasthenia
gravis (MG), multiple sclerosis (MS), systemic lupus erythematosis
(SLE), rheumatoid arthritis (RA), insulin-dependent diabetes
mellitus (IDDM), and the like. Suitable formulations are found in
Remington's Pharmaceutical Sciences, Mack Publishing Company,
Philadelphia, PA, 17th ed. (1985).
[0062] The immunogenic peptides or antibodies of the invention are
administered prophylactically or to an individual already suffering
from the disease. The peptide compositions are administered to a
patient in an amount sufficient to elicit an effective immune
response to the chemokine receptor molecule from which the peptides
are derived. An effective immune response is one that inhibits
recruitment of T cells to sites of inflammation. An amount adequate
to accomplish this is defined as "therapeutically effective dose"
or "immunogenically effective dose." Amounts effective for this use
will depend on, e.g., the peptide composition, the manner of
administration, the stage and severity of the disease being
treated, the weight and general state of health of the patient, and
the judgment of the prescribing physician, but generally range for
the initial immunization (that is for therapeutic or prophylactic
administration) from about 0.1 mg to about 1.0 mg per 70 kilogram
patient, more commonly from about 0.5 mg to about 0.75 mg per 70 kg
of body weight. Boosting dosages are typically from about 0.1 mg to
about 0.5 mg of peptide using a boosting regimen over weeks to
months depending upon the patient's response and condition. A
suitable protocol would include injection at time 0, 4, 2, 6, 10
and 14 weeks, followed by further booster injections at 24 and 28
weeks.
[0063] It must be kept in mind that the peptides and compositions
of the present invention may generally be employed in serious
disease states, that is, life-threatening or potentially life
threatening situations. In such cases, in view of the minimization
of extraneous substances and the relative nontoxic nature of the
peptides, it is possible and may be felt desirable by the treating
physician to administer substantial excesses of these peptide
compositions.
[0064] For therapeutic use, administration should begin at the
first sign of autoimmune disease. This is followed by boosting
doses until at least symptoms are substantially abated and for a
period thereafter. In some circumstances, loading doses followed by
boosting doses may be required. The resulting immune response helps
to cure or at least partially arrest symptoms and/or complications.
Vaccine compositions containing the peptides are administered
prophylactically to a patient susceptible to or otherwise at risk
of the disease to elicit an immune response against the target
Chemokine receptor antigen.
[0065] The pharmaceutical compositions (containing either peptides
or antibodies) are intended for parenteral or oral administration.
Preferably, the pharmaceutical compositions are administered
parenterally, e.g., subcutaneously, intradernally, or
intramuscularly. Thus, the invention provides compositions for
parenteral administration which comprise a solution of the
immunogenic peptides dissolved or suspended in an acceptable
carrier, preferably an aqueous carrier. A variety of aqueous
carriers may be used, e.g., water, buffered water, 0.4% saline,
0.3% glycine, hyaluronic acid and the like. These compositions may
be sterilized by conventional, well known sterilization techniques,
or may be sterile filtered. The resulting aqueous solutions may be
packaged for use as is, or lyophilized, the lyophilized preparation
being combined with a sterile solution prior to administration. The
compositions may contain pharmaceutically acceptable auxiliary
substances as required to approximate physiological conditions,
such as buffering agents, tonicity adjusting agents, wetting agents
and the like, for example, sodium acetate, sodium lactate, sodium
chloride, potassium chloride, calcium chloride, sorbitan
monolaurate, triethanolamine oleate, etc.
[0066] For solid compositions, conventional nontoxic solid carriers
may be used which include, for example, pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharin,
talcum, cellulose, glucose, sucrose, magnesium carbonate, and the
like. For oral administration, a pharmaceutically acceptable
nontoxic composition is formed by incorporating any of the normally
employed excipients, such as those carriers previously listed, and
generally 10-95% of active ingredient, that is, one or more
peptides of the invention, and more preferably at a concentration
of 25%-75%.
[0067] As noted above, the peptide compositions are intended to
induce an immune response to the peptides. Thus, compositions and
methods of administration suitable for maximizing the immune
response are preferred. For instance, peptides may be introduced
into a host, including humans, linked to a carrier or as a
homopolymer or heteropolymer of active peptide units.
Alternatively, the a "cocktail" of polypeptides can be used. A
mixture of more than one polypeptide has the advantage of increased
immunological reaction and, where different peptides are used to
make up the polymer, the additional ability to induce antibodies to
a number of epitopes. For instance, polypeptides comprising
sequences from extracellular regions of .alpha. and .beta. chains
may be used in combination. Useful carriers are well known in the
art, and include, e.g., KLH, thyroglobulin, albumins such as human
serum albumin, tetanus toxoid, polyamino acids such as
poly(lysine:glutamic acid), influenza, hepatitis B virus core
protein, hepatitis B virus recombinant vaccine and the like.
[0068] The use of more than one polypeptide is particularly useful
to enhance the immune response against polypeptides of the
invention. As demonstrated below, although the polypeptides may be
derived from self Chemokine receptor molecules expressed in the
patient, they can induce an immune response. In some instances, the
immune response to the self polypeptide may not be sufficiently
strong. In these instances, it may be necessary to break tolerance
to the polypeptide. The compositions may comprise one or more of
the foreign polypeptides that are sufficiently similar to the self
polypeptides to induce an immune response against both the foreign
and self polypeptides (see, Mamula et al. J. Immunol. 149:789-795
(1992). Suitable proteins include synthetic polypeptides designed
for this purpose or polypeptide sequences from homologous proteins
from natural sources, such as proteins encoded by a different
allele at the same locus as the self polypeptide.
[0069] The compositions also include an adjuvant. As used here,
number of adjuvants are well known to one skilled in the art.
Suitable adjuvants include incomplete Freund's adjuvant, alum,
aluminum phosphate, aluminum hydroxide,
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),
N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred
to as nor-MDP),
N-acetylmuramyl-Lalanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipa-
lmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A,
referred to as MTP-PE), and RIBI, which contains three components
extracted from bacteria, monophosphoryl lipid A, trehalose
dimycolate and cell wall skeleton (NPL+TDM+CWS) in a 2%
squalene/Tween 80 emulsion. The effectiveness of an adjuvant may be
determined by measuring the amount of antibodies directed against
the immunogenic peptide.
[0070] A particularly useful adjuvant and immunization schedule are
described in Kwak et al. New Eng. J Med. 327-1209-1215 (1992). The
immunological adjuvant described there comprises 5% (wt/vol)
squalene, 2.5% Pluronic L121 polymer and 0.2 % polysorbate in
phosphate buffered saline.
[0071] The concentration of immunogenic peptides of the invention
in the pharmaceutical formulations can vary widely, i.e. from less
than about 0.1%, usually at or at least about 2% to as much as 20%
to 50% or more by weight, and will be selected primarily by fluid
volumes, viscosities, etc., in accordance with the particular mode
of administration selected.
[0072] The peptides of the invention can also be expressed by
attenuated viral hosts, such as vaccinia or fowlpox. This approach
involves the use of vaccinia virus as a vector to express
nucleotide sequences that encode the peptides of the invention.
Upon introduction into a host, the recombinant vaccinia virus
expresses the immunogenic peptide, and thereby elicits an immune
response. Vaccinia vectors and methods useful in immunization
protocols are described in, e.g., U.S. Pat. No. 4,722,848. Another
vector is BCG (Bacille Calnette Guerin). BCG vectors are described
in Stover et al. (Nature 351:456-460 (1991)). A wide variety of
other vectors useful for therapeutic administration or immunization
of the peptides of the invention, e.g., Salmonella typhi vectors
and the like, will be apparent to those skilled in the art from the
description herein.
[0073] The DNA encoding one or more of the peptides of the
invention can also be administered to the patient. This approach is
described, for instance, in Wolff et. al., Science 247:1465-1468
(1990) as well as U.S. Pat. Nos. 5,580,859 and 5,589,466.
[0074] In order to enhance serum half-life, the peptides may also
be encapsulated, introduced into the lumen of liposomes, prepared
as a colloid, or other conventional techniques may be employed
which provide an extended serum half-life of the peptides. A
variety of methods are available for preparing liposomes, as
described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467
(1980), U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028.
[0075] The peptides or antibodies of the invention can also be used
for diagnostic purposes. For instance, peptides can be used to
screen for autoantibodies to ensure that the vaccination has been
effective. Antibodies can be used to detect the presence of
particular Chemokine receptor molecules associated with
disease.
[0076] The following examples are offered by way of illustration,
not by way of limitation.
EXAMPLE 1
[0077] This example describes a reproducible bioassay for CXCR3
activation.
[0078] A stable line transfectant of NSO-1 cells expressing the
cDNA for human CXCR3 was prepared according to standard techniques.
The surface expression of hu-CXCR3 on the transfectants compared to
untransfected parent NSO-1 cells was confirmed by FACS staining
using mouse anti-human CXCR3 monoclonal antibody (R&D
systems).
[0079] To establish a bioassay for CXCR3 activation, the CXCR3
transfectant cells were cultured with either human IP-10, or the
mouse anti-human CXCR3 antibody, and the physiological response of
the cells was measured using a microphysiometer. This machine
measures changes in the pH of the extracellular medium of the cell
cultures which result from ligand receptor binding on the cell
surface. These extracellular acidification rate measurements have
previously been used as markers of antigen specific T cell
activation and T cell epitope identification. The current assay is
used to provide a biological read out for the identification of
chemokine or chemokine receptor peptides involved in binding to IP-
10 or agonistic anti-CXCR3 antibody.
[0080] The experiments indicated that human IP-10 and anti-CXCR3
antibody both triggered substantial acidification rate changes in
the CXCR3 transfectants, as shown in FIG. 1. Importantly, the same
ligands induce no change in acidification rate of untransfected
NSO-1 cells (FIG. 1).
EXAMPLE 2
[0081] This example describes synthesis of human CXCR3 derived
peptides.
[0082] A schematic representation of the surface portion of human
CXCR3 is given in FIG. 2. The receptor has 4 surface portions, 4
intracellular portions and seven transmembrane portions. The
surface portions were designated starting from N-terminus as SP-1,
SP-2, SP-3 and SP-4.
[0083] The peptides shown in Table 1 derived from these four
surface portions were prepared by solid phase peptide synthesis.
The names of the peptides are based on the surface portion of CXCR3
from which they were derived. For example, SP-1-1 means this
peptide was derived from the first portion of the CXCR3
protein.
1TABLE 1 Peptide Name Sequence SP-1-1 MVLEVSDHQVLNDAEVAALL-NH2
SP-1-2 ENFSSSYDYGENESDSCCTS-NH2 SP-1-3 PPCPQDFSLNFDRAFLPA-NH2
SP-2-1 DAAVQWVFGSGLCKV-NH2 SP-3-1 SAHHDERLNATHCQYN-NH2 SP-3-2
FPQVGRTALRVLQLVAG-NH2 SP-4-1 DILMDLGALARNCGRESRVDVAKS-NH2
[0084] The ability of these receptor-derived peptides to bind
anti-CXCR3 antibody was evaluated using a standard ELISA format.
The peptides were dissolved in 0.1 M bicarbonate buffer and coated
on a 96 well ELISA plate overnight. The excess peptides were
removed and nonspecific binding sites in the wells were blocked by
0.1 % bovine serum albumin. Anti-CXCR3 antibody (0.5 .mu.g/well)
was added to these wells and incubated for 2 hours. Excess antibody
was removed by washing with PBS. HRP conjugated goat anti-mouse
antibody was used as secondary antibody for detection. Two of the
seven receptor-derived peptides, namely SP-1-3 and SP-4-1, showed
substantial binding to anti-CXCR3 antibody. Further support for the
conclusion that anti-CXCR3 antibody bound two of the seven
receptor-derived peptides was provided by FACS analysis of the same
interactions. These studies revealed that the receptor-derived
peptide SP-4-1 potently blocked binding of anti-CXCR3 antibody to
the CXCR3 cell line transfectant. The receptor-derived peptide
SP-1-3 provided partial inhibition of the binding of the antibody
to CXCR3 transfectants. In contrast, an antibody nonbinding
receptor-derived peptide, SP-2-1, failed to inhibit binding to
CXCR3 transfectants. The data described here collectively
demonstrate that anti-CXCR3 antibody can bind to two separate
peptide portions of the CXCR3 extracellular domain.
[0085] EXAMPLE 3
[0086] This example demonstrates that peptide vaccines of the
invention can be used to prevent EAE in mice.
[0087] Protocols for animal experiments:
[0088] SJL mice (6-8 weeks old) were obtained from Jackson
Laboratories. They were kept in quarantine for two weeks. These
mice get EAE when immunized with a peptide from proteolipid protein
(PLP). The peptide sequence used for the immunization of these mice
is PLP 139-151 and is amidated at the C terminus
(HSLGWLGHPDKF-NH2). For the experiments, induction of the disease
is considered Day 0. Three weeks before the induction of the
disease, the mice were vaccinated with Human CXCR3S-P4-1 peptide
mixed with complete Freund's Adjuvant.
[0089] Preparation of peptide CFA emulsion for treatment
[0090] 4mg of human CXCR3 SP-4-1 peptide was dissolved in 1 ml of
phosphate buffered saline pH 7.4 (PBS). One ml of CFA obtained from
VWR (Difco, Adjuvant Complete H37RA) was added to the peptide
solution and the mixture was sonicated for 5 seconds using a fine
tip sonicator. The emulsion was taken in 1 ml syringe (needed 2
syringes) fitted with a 25 gauge needle. Each mouse was given 100
.mu.l of the emulsin under each flank near the hind legs by
subcutaneous injection (total volume per mouse=200 .mu.l, total
peptide per mouse=200 .mu.g).
[0091] Preparation of CFA alone:
[0092] One ml of PBS and 1 ml of CFA were mixed and sonicated for 5
seconds and the emulsin is drawn into a 1 ml syringe fitted with a
25 gauge needle. Each mouse was given 100 .mu.l of this emulsion
under each flank near hind legs by subcutaneous injection (total
volume of CFA per mouse=200 .mu.l).
[0093] Induction of Disease:
[0094] To an aqueous solution of peptide (4 mg per ml in PBS) equal
volume of CFA was added and the mixture was sonicated for 5
seconds. The emulsion was drawn in a 1 ml syringe fitted with a 25
gauge needle and subcutaneously injected (200 .mu.l total per
mouse) in mice at the foot pad and back.
[0095] Testing the antibody response to SP4-1 peptide
[0096] The mice were bled at week 0 and week 5 and serum was tested
by ELISA for the presence of antibodies against the human CXCR3
SP-4-1 peptide. Briefly, the SP4-1 peptide dissolved in 0.1 M
sodium bicarbonate buffer was plated on a 96-well ELISA plate
overnight. The nonspecific binding sites on the plate were coated
with 0.1% bovine serum albumin solution in PBS. The wells were
washed and serum (diluted in PBS) was added to the wells and
incubated at room temperature for 1.5 hours. The wells were then
washed and HRP conjugated anti-mouse immunoglobulin antibody was
used to detect the presence of anti-SP4-1 antibodies). The results
of this ELISA clearly indicate that the SP4-1 treated mice show an
antibody response against this peptide.
[0097] Results of EAE study
[0098] Sp4-1 was administered with CFA at 21 days and 14 days
before induction of EAE as described above (FIG. 3). The results
are shown in FIG. 4. There it can be seen that while most of the
untreated and CFA treated mice showed clinical symptoms of EAE, 7
out of 8 mice treated with SP4-1 in CFA showed no clinical
symptoms.
[0099] The above examples are provided to illustrate the invention
but not to limit its scope. Other variants of the invention will be
readily apparent to one of ordinary skill in the art and are
encompassed by the appended claims. All publications, patents, and
patent applications cited herein are hereby incorporated by
reference.
* * * * *