U.S. patent application number 13/478860 was filed with the patent office on 2012-11-22 for methods of blocking tissue destruction by autoreactive t cells.
Invention is credited to Xue-Feng Bai, Yang Liu, Pan Zheng.
Application Number | 20120294884 13/478860 |
Document ID | / |
Family ID | 34923169 |
Filed Date | 2012-11-22 |
United States Patent
Application |
20120294884 |
Kind Code |
A1 |
Liu; Yang ; et al. |
November 22, 2012 |
METHODS OF BLOCKING TISSUE DESTRUCTION BY AUTOREACTIVE T CELLS
Abstract
Methods for blocking autoreactive T cell-initiated destruction
of tissues in a mammal are provided. In one embodiment, the method
comprises administering a purified CD24 polypeptide, a fusion
protein comprising such polypeptide, or a biologically active
fragment of such polypeptide to a mammalian subject who is
suspected of having or predisposed to having an autoimmune disease.
In another embodiment, anti-CD24 antibody or anti-CD24 Fab
fragments are administered to the subject. In another embodiment,
the method comprises administering a CD24 antisense molecule, an
expression vector encoding a CD24 antisense molecule, CD24 dsRNAi,
or an expression vector encoding CD24 dsRNAi to the subject. The
present invention also relates to isolated and purified CD24 fusion
proteins employed in the present methods and to transgenic mice
that express the human CD24 protein on their T cells and/or their
vascular endothelial cells but do not express murine heat shock
antigen on any cells.
Inventors: |
Liu; Yang; (Ann Arbor,
MI) ; Zheng; Pan; (Ann Arbor, MI) ; Bai;
Xue-Feng; (Columbus, OH) |
Family ID: |
34923169 |
Appl. No.: |
13/478860 |
Filed: |
May 23, 2012 |
Related U.S. Patent Documents
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Filing Date |
Patent Number |
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12774926 |
May 6, 2010 |
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13478860 |
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11129083 |
May 13, 2005 |
7744894 |
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12774926 |
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10119637 |
Apr 10, 2002 |
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11129083 |
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09822851 |
Mar 29, 2001 |
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10119637 |
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60192814 |
Mar 29, 2000 |
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Current U.S.
Class: |
424/192.1 |
Current CPC
Class: |
A61P 17/06 20180101;
C07K 14/70596 20130101; A61K 38/177 20130101; C07K 2319/30
20130101; A61K 38/00 20130101; A61P 19/02 20180101; A61K 39/0008
20130101; C07K 16/2896 20130101; A61P 25/00 20180101; C07K 2319/00
20130101; A61P 3/10 20180101; A61P 37/08 20180101; A61P 37/06
20180101 |
Class at
Publication: |
424/192.1 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61P 37/06 20060101 A61P037/06; A61P 25/00 20060101
A61P025/00 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention is supported, at least in part, by Grant No.
AI32981 from the National Institute of Health, USA. The U.S.
government has certain rights in this invention.
Claims
1.-28. (canceled)
29. A method for treating a human patient suspected of having,
known to have, or predisposed to having multiple sclerosis, said
method comprising administering to said patient a pharmaceutical
composition comprising an isolated human HSA/CD24 polypeptide
fusion and a physiologically acceptable carrier, wherein the
HSA/CD24 polypeptide fusion comprises a HSA/CD24 core region, and
wherein the HSA/CD24 polypeptide fusion is glycosylated.
30. The method of claim 29, wherein the HSA/CD24 fusion comprises
the HSA/CD24 polypeptide linked by a peptide bond to a tag, wherein
the tag consists of 5 to 300 amino acids.
Description
REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in part of U.S.
application Ser. No. 09/822,851 which was filed on Mar. 29, 2001,
and claims priority from U.S. Provisional Patent Application No.
60/192,814, filed on Mar. 29, 2000.
BACKGROUND OF THE INVENTION
[0003] The present invention relates to agents and methods for
blocking deleterious T cell mediated immune reactions. Such
reactions occur in autoimmune diseases, such as for example,
multiple sclerosis (MS), rheumatoid arthritis, systemic lupus
erythematosis, psoriasis, diabetes, and allergies. Such reactions
also occur during rejection of transplants.
[0004] In theory, autoimmune diseases can be prevented by blocking
activation of T cells and formation of autoreactive T cells.
Accordingly, there are a number of studies being conducted to
identify methods or agents that can be used to block activation of
T cells (J Clin Invest. 1995 June; 95(6):2783-9; J Med Chem. 2002
Jan. 17; 45(2):275-83). Unfortunately, since patients with
autoimmune diseases have already developed autoreactive T cells,
these methods have limited value for treatment of autoimmune
diseases. Moreover, agents that prevent systemic T cell activation
often cause serious side effects. For example, treatment with
agents that block activation of T cells can also render the patient
more susceptible to infections and cancer. Thus, it is desirable to
have new methods for treating autoimmune diseases. A method which
reduces the destruction of targeted tissues that is initiated by
autoreactive T cells is especially desirable.
SUMMARY OF THE INVENTION
[0005] The present invention provides methods for blocking or
reducing autoreactive T cell-initiated destruction of tissues in a
mammal The methods employ an agent that inhibits or reduces
interaction of the CD24 polypeptide with its functional ligand. The
CD24 polypeptide is found on the cell membrane of activated T cells
and other cell types, such as B cells, dendritic cells, epithelial
cells and vascular endothelial cells.
[0006] In one embodiment, the method comprises administering a
pharmaceutical composition comprising a biologically effective
amount of an isolated and purified polypeptide, referred to
hereinafter as the "HSA/CD24" polypeptide, a fusion protein
comprising the HSA/CD24 polypeptide polypeptide, or a biologically
active fragment of the HSA/CD24 polypeptide to a mammal in need of
the same, i.e., a mammal who is suspected of having, known to have,
or predisposed to have an autoimmune disease. As used herein,
"mammal" refers to rats, mice, cats, dogs, cows, pigs, rabbits, and
primates. Exemplary primates include monkeys, chimpanzees, and
humans. As used herein the term "HSA/CD24" refers not only to the
protein portion of the heat stable antigen (HSA) found on the
surface of mouse cells but also to the mammalian homologs of mouse
HSA. Thus, the term "HSA/CD24", as used in the present application,
encompasses the polypeptide portion of human CD24 and rat CD24, the
known human and rat homologs of mouse HSA. Preferably, the HSA/CD24
polypeptide is glycosylated. The fusion protein comprises the
HSA/CD24 polypeptide or a truncated form of the HSA/CD24 linked by
a peptide bond to a peptide or protein tag. In a preferred
embodiment, the HSA/CD24 fragment comprises the core region of the
HSA/CD24 polypeptide.
[0007] In another embodiment, the method comprises administering a
pharmaceutical composition comprising a biologically effective
amount of an anti- HSA/CD24 antibody or anti-HSA/CD24 Fab fragments
to a mammal known to have, suspected of having, or predisposed to
having an autoimmune disease.
[0008] In another aspect, the method comprises administering to the
subject an agent that reduces expression of the CD24 polypeptide in
T cells. Such methods employ agents that disrupt the function of
the CD24 gene at the genomic, transcriptional, post-transcriptional
and translational levels. In one embodiment, the agent is an
antisense molecule (referred to hereinafter as "CD24 antisense")
which reduces transcription of the CD24 gene or translation of the
CD24 gene transcript in autoreactive T cells. In another
embodiment, the agent is a double stranded RNA molecule (referred
to hereinafter as "CD24 dsRNAi") which interferes with expression
of the CD24 gene.
[0009] The present invention also relates to a method of blocking
binding of autoreactive T cells to vascular endothelial cells. In
one aspect, the method comprises contacting the vascular
endothelial cells with a sufficient amount of an HSA/CD24
polypeptide or a fragment thereof, or a fusion protein comprising
HSA/CD24 polypeptide or a fragment thereof, or anti-HSA antibodies
to inhibit interaction of the autoreactive T cells with the
vascular endothelial cells. In another aspect, the method comprises
introducing an oligonucleotide or polynucleotide that inhibits
expression of the CD24 polypeptide into the autoreactive T cell, or
the vascular endothelial cell or both. Examples of such
oligonucleotides and polynucleotides include, but are not limited
to, a CD24 antisense oligonucleotide, an expression vector
comprising a polynucleotide or nucleic acid encoding a CD24
antisense oligonucleotide, a CD24 dsRNAi, and an expression vector
comprising a polynucleotide or nucleic acid encoding a CD24
dsRNAi.
[0010] The present invention also relates to isolated and purified
HSA/CD24 fusion proteins employed in the above-described methods
and to transgenic or knock in mice that express the human CD24
protein on their T cells or their vascular endothelial cells or all
other cell types that normally express CD24 but, as a result of
targeted mutation, do not express murine HSA on any cells. Such
mice provide a unique model to test the effectiveness of drugs
designed to block or enhance the biological function of human
CD24-mediated autoimmune diseases.
BRIEF DESCRIPTION OF THE FIGURES
[0011] FIG. 1. Targeted mutations of HSA and CD28 reveal two
distinct checkpoints in the development of EAE. a. Targeted
mutations of either HSA or CD28 prevent induction of EAE. WT,
CD28(-/-) or HSA(-/-) mice were immunized with MOG peptide.
Clinical signs were scored as described in the method section. b.
Proliferative response of lymph node T cells to MOG peptides.
Draining lymph node cells from day 10-immunized mice were
stimulated with given concentrations of MOG peptide and irradiated
syngeneic naive spleen cells as antigen-presenting cells. c.
Enumeration of cytokine-producers by ELISpot. Draining lymph node
cells used in b were used as responder cells. The numbers of cells
secreting either IL2, IL4, and IFN.gamma. among 1.times.10.sup.6
lymph node cells in response to MOG peptide (AA35-55) were
presented. Data shown were means+/-SEM from three independent
experiments.
[0012] FIG. 2. Histological analysis of spinal cord of MOG
immunized WT or HSA(-/-) mice. a: The means and SEM of histological
scores of WT and HSA(-/-) mice spinal cords. Ten independent cross
sections, from cervical to sacral regions, were examined in each
spinal cord. The data are summarized from 30 spinal cord sections
from 3 mice in each group. b. Representative histology in immunized
WT mice, all sections examined contain histology lesions. c and d.
Histology sections (100.times.) of immunized HSA(-/-) mice. A
lesion-free section is presented in c, while a lesion containing
section is presented in d.
[0013] FIG. 3. Requirement for HSA expression on both T cells and
non-T host cells for the induction of EAR. Histology (63.times. for
a, b, c and the left panel of d; 200x for the right panel of d) of
spinal cords of the HSA(-/-)(a, b) or WT(c, d) recipient mice on
day 12 after adoptive transfer. Draining lymph node cells were
isolated from either WT or HSA(/-) mice after immunization, and
were stimulated with antigen and IL2 for 4 days in vitro. The
activated T cells were injected into either WT or HSA(-/-) mice
(100.times.10.sup.6 cells per mouse). EAE development was monitored
daily for clinical signs. At 12 days after transfer, recipient mice
were sacrificed and spinal cords were processed for histological
examination. No disease was observed in WT>HSA(-/-),
HSA(-/-)>WT, or HSA(-/)>HSA(-/-) recipients.
[0014] FIG. 4. Clinical scores of the adoptive transfer experiment
with 4 (WT>HSA(-/-) and HSA(-/-)>WT groups) or 5 (WT>WT
and HSA(-/-)>HSA(-/-) groups) mice per group.
[0015] FIG. 5. Transgenic expression of HSA exclusively on T cell
lineage is insufficient for EAE development. a. Phenotypes of WT,
HSA-TG, HSA(-/-), and HSATG/HSA(-/-) mice by flow cytometry using
anti-HSA and anti-CD3 mAbs. b. EAE score in WT, HSATG, HSA(-/-),
and HSATG/HSA(-/-) mice after immunization with the MOG
peptides.
[0016] FIG. 6. HSAIg ameliorates EAE a. Analysis of HSAIg by
SDS-PAGE. 10 .mu.g of purified HSAIg was separated by 10% reducing
(R) and non-reducing SDS-PAGE. The proteins were stained by
Comassie blue. The EAR score for control (PBS) or HSAIg-treated
mice. EAE was induced in WT mice as described in Materials and
Methods. On days 8, 10, 12, 14 and 22 after immunization, five mice
per group were injected (i.p.) with 100 .mu.g/mouse of either HSAlg
or 100 ml of PBS as control. The effect of HSAIg has been evaluated
in three independent experiments with similar results.
[0017] FIG. 7 shows the amino acid sequence, SEQ ID NO. 1, of the
mouse HSA polypeptide. The signal peptide extends from amino acid 1
through amino acid 26 of the sequence. The glycophosphatidyl (GPI)
anchor region includes and extends from amino acid 54 through amino
acid 76.
[0018] FIG. 8 shows the amino acid sequence, SEQ ID NO. 2, of the
human CD24 polypeptide. The signal peptide extends from amino acid
1 through amino acid 26 of the sequence. The glycophosphatidyl
(GPI) anchor region includes and extends from amino acid 60 through
amino acid 80.
[0019] FIG. 9 shows the amino acid sequence, SEQ ID NO. 3, of the
rat CD24 polypeptide. The signal peptide extends from amino acid 1
through amino acid 26 of the sequence. The glycophosphatidyl (GPI)
anchor region includes and extends from amino acid 57 through amino
acid 76.
[0020] FIG. 10 shows the DNA sequence, SEQ ID NO. 4, of a fusion
gene which comprises a nucleotide sequence encoding HSA fused to
the genomic sequence of human IgG1 Fc. The predicted sequence of
the cDNA, SEQ ID NO. 5, which results from splicing of the introns
IgG1 Fc sequence and the predicted amino acid sequence, SEQ ID NO.
14, are also shown in this figure. The normal font with under line
is HSA sequence, bold phase is new sequence, italics is IgG1 Fc
sequence.
[0021] FIG. 11 is a diagram of a construct for producing human CD24
gene knock-in mice. Arm 1 of the knock in construct comprises
nucleotide 2001 through nucleotide 5500 of the mouse HSA/CD14 gene,
GenBank Accession No. X72910. Arm 2 of the construct is a chimera
gene consisting of the last 256 by sequence of exon 1 of the mouse
CD24 gene, the first 240 by sequence of exon 2 of the human CD24
gene and about 3 kb of mouse CD24 sequence which comprises
remaining exon 2 sequence encoding for 3' untranslated region and
3' sequence of the mouse CD24 gene (Seq I.D. 22).
[0022] FIG. 12 is a diagram of a plasmid for producing transgenic
mice expressing human CD24 in T cells. To produce this plasmid
human CD24 coding sequence is subcloned into transgenic construct
vector p1017 Bam HI site, which is described in EMBO J. 9:
3821-3829, 1990) CD24 forward primer (CD24F.Bam): G GCC GGA TCC ATG
GGC AGA GCA ATG GTG with BamHl site 5' to ATG start codon. CD24
reverse primer (CD24R. XhoBam): G GCC GGA TCC CTC GAG TTA AGA GTA
GAG ATG CAG with Barn HI and Xho I sites 3' to TAA stop codon.
[0023] FIG. 13 is a diagram of a vector for producing mice that
express CD24 in vascular endothelial cells.
[0024] FIG. 14 shows a comparison of mouse and human CD24 cDNA
sequences, and the preferred sequences to be targeted by CD24
antisense RNA and CD24 dsRNAi . Human CD24 and mouse CD24 cDNA
sequences (Human CD24 (XM.sub.--099027) and Mouse CD24
(NM.sub.--009846)) are aligned by double blast search. The regions
with a stretch of identity that are 17 or more base pairs in length
are highlighted as preferred targets for CD24 antisense and dsRNAi
agents.
[0025] FIG. 15 is a dot plot demonstrating inhibition of CD24
expression in CHO cells using dsRNAi. Chinese hamster ovary (CHO)
cells were transiently transfected with human CD24 plasmid alone
(top), the CD24 plasmid plus dsRNAi (middle) or CD24 plasmid plus
inverted dsRNA as control (lower panel). The expression of CD24 on
cell surface was analyzed at 72 hours after transfection using
phycoerythorin-conjugated anti-human CD24 mAb (BD Pharmingen). Note
essential absence of CD24-expression in cells treated with CD24
dsRNAi.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention provides methods for blocking
destruction of tissue by autoreactive T cells in a mammalian
subject. The methods employ agents which inhibit or reduce, either
directly or indirectly, the interaction of the CD24 polypeptide
with its functional ligand. The CD 24 polypeptide is present on the
cell membrane of activated T cells and other cell types such as B
cells, dendritic cells, epithelial cells and vascular endothelial
cells.
[0027] In one embodiment, the method comprises administering a
pharmaceutical composition comprising a biologically effective
amount of an isolated and purified HSA/CD24 polypeptide or a
fragment thereof to a mammal suspected of having an autoimmune
disease. In another embodiment a fusion protein comprising the
HSA/CD24 polypeptide or fragment thereof linked by a peptide bond
to a peptide or protein tag is administered to the mammal.
Preferably, the HSA/CD24 polypeptide is glycosylated. In another
embodiment an antibody which is immunospecific for the HSA/CD24
polypeptide is administered to the mammal.
[0028] In another aspect, the method comprises administering to the
subject an agent that reduces expression of the CD24 polypeptide in
T cells. Such methods employ agents that disrupt the function of
the CD24 gene at the genomic, transcriptional, post-transcriptional
and translational levels. In this aspect, embodiments of the
present method employ antisense molecules or dsRNAi to inhibit
expression of the CD24/HSA gene and production of the CD24
polypeptide in the autoreactive T cells of the mammalian
subject
[0029] The present invention also relates to a method of treating a
human subject known to have, suspected of having, or predisposed to
having an autoimmune disease. The methods involve treating the
human subject with an agent which inhibit or reduce, either
directly or indirectly, the interaction of the CD24 polypeptide
which is present on the cell surface of the subject's cells,
including but not limited to activated T cells, with the functional
ligand of CD24. In accordance with the present invention, it is
believed that such ligands include, but are not limited to, CD24
itself, P-selectin, and very late antigen 4.
[0030] In one aspect, the therapeutic method comprises
administering a pharmaceutical composition comprising a
biologically effective amount of an isolated and purified human
CD24 polypeptide or fragment thereof, or a fusion protein
comprising such molecule, to the human subject. In another
embodiment of this therapeutic method, the pharmaceutical
composition comprises anti-human CD24 antibodies or their Fab
fragments. Preferably, the anti-human CD24 antibody is a monoclonal
antibody, more preferably a humanized monoclonal anti-human CD24
antibody. Preferably, the pharmaceutical composition is
administered after autoreactive T cells have been detected in the
human subject.
[0031] Preferably, the pharmaceutical composition is administered
by injection. The present method is useful for treating subjects
suspected of having autoimmune diseases such as for example,
multiple sclerosis (MS), rheumatoid arthritis, and
insulin-dependent diabetes mellitus. By "treating" is meant
ameliorating or tempering the severity of the condition, either
occurring or expected to occur in the future. In cases of
autoimmune demyelinating diseases of the CNS such as for example
MS, the pharmaceutical composition is administered either when
patients have clinical symptoms, or when they are in temporary
remission. Preferably, the protocol involves intravenous injection.
In the case of rheumatoid arthritis, the pharmaceutical
composition, preferably, is administered intravenously (i.v.) after
the acute symptoms are relieved by other therapeutic methods. In
the case of insulin dependent diabetes mellitus, the pharmaceutical
composition, preferably, is administered intravenously after
autoreactive T cells are detected in the peripheral blood.
[0032] Further embodiments of this therapeutic method employ
antisense molecules or dsRNAi to inhibit expression of the CD24
gene and production of CD24 polypeptide in the T cells of the human
subject.
Pharmaceutical Composition
[0033] The pharmaceutical composition comprises a biologically
effective amount of an HSA/CD24 polypeptide or a biologically
active variant thereof or alternatively a fragment of an HSA/CD24
polypeptide or a biologically active variant thereof, and
preferably a relatively inert topical carrier. Many such carriers
are routinely used and can be identified by reference to
pharmaceutical texts.
HSA Antigen
[0034] The mouse HSA antigen and the mammalian homologs thereof are
polypeptides comprising approximately 76-80 amino acids. The HSA
polypeptide and the mammalian homologs thereof are cell surface
molecules which are linked to the cell membrane via a
glycophosphatidylinositol (GPI) tail. The HSA antigen is
constitutively expressed on most hematopoietic and developing
neuronal cells. In some lymphocytes, such as for example T cells,
expression of the HSA polypeptide is induced. As shown in FIGS.
7-9, the immature forms of mouse HSA antigen, human CD24 and rat
CD24 comprise a signal sequence, a core region that is maintained
in the mature protein, and a GPI anchor region. As shown in FIG. 7,
the signal sequence of the mouse HSA antigen includes amino acid 1
through amino acid 26 of SEQ ID NO. 1; the core region includes
amino acid 27 through amino acid 53 of SEQ ID NO. 1; and the GPI
region includes amino acid 54 through amino acid 75 of SEQ ID NO.
1. As shown in FIG. 8, the signal sequence of the human CD24
antigen includes amino acid 1 through amino acid 26 of SEQ ID NO.2;
the core region includes amino acid 27 through amino acid 59 of SEQ
ID NO. 2 and the GPI region includes amino acid 60 through amino
acid 80 of SEQ ID NO. 2. As shown in FIG. 9, the signal sequence of
the rat CD24 antigen includes amino acid 1 through amino acid 26 of
SEQ ID NO.3; the core region includes amino acid 27 through amino
acid 56 of SEQ ID NO. 3 and the GPI region includes amino acid 57
through amino acid 76 of SEQ ID NO. 3. The nucleotide sequence of a
polynucleotide which encodes the human CD4 polypeptide is available
at the GenBank Accession No. AK000168. The nucleotide sequence of a
cDNA which encodes the rat CD24 polypeptide is available at GenBank
Accession No. AWK12164. The nucleotide sequence of a cDNA which
encodes mouse HSA antigen is available at GenBank Accession
M58661.
[0035] The present invention relates to novel method of using an
HSA/CD24 polyp eptide or fragment thereof to treat autoimmune
diseases. Preferably, the polypeptide or fragment is glycosylated.
In one embodiment the HSA/CD24 fragment is a truncated form of the
HSA/CD24 polypeptide which lacks a few amino acids, i.e., from 1 to
2 amino acids, at the amino terminus or carboxy terminus thereof.
In another embodiment the HSA/CD24 fragment is a polypeptide which
comprises essentially only the core region of the HSA/CD24
polypeptide, i.e. the HSA/CD24 fragment lacks most or all of the
signal peptide and most or all of the CPI anchor region. As used
herein the term HSA/CD24 polypeptide comprises all mammalian
homologs of mouse HSA , including human CD24 and rat CD24.
[0036] The HSA/CD24 polypeptide or HSA/CD24 fragment that is used
in the pharmaceutical composition is the naturally-occurring
HSA/CD24 polypeptide, a biologically active fragment of the
naturally-occurring HSA/CD24 polypeptide, a biologically active
variant of the naturally-occurring HSA/CD24 polypeptide, or a
biologically active variant of a fragment of the
naturally-occurring HSA/CD24 polypeptide The biologically active
variant of the HSA/CD24 polypeptide has an amino acid sequence
which is at least 80%, more preferably at least 93%, most
preferably at least 96% identical to the amino acid sequence of the
naturally occurring HSA/CD24 polypeptide that is present in the
mammal to whom the pharmaceutical composition is being
administered. Similarly, the biologically active variant of the
fragment of the HSA/CD24 polypeptide has an amino acid sequence
which is at least 80%, preferably at least 90%, more preferably at
least 95% identical to the amino acid sequence of the corresponding
naturally-occurring HSA/CD24 fragment. For murine HSA, alteration
in Positions 1(Asn), 4(Ser), 13(Asn), 15(Ser), 17(Ser), 21(Ser),
22(Asn), 24(Thr) and 25(Thr) of the mature peptide, i.e., the
peptide which lacks the signal peptide, may alter glycosylation and
interfere with its ability to block destruction of tissue by
autoreactive T cells. Thus, it is preferred that alternations not
be made at these sites. In the human homologue of HSA, i.e., human
CD24, 20 out of 31 amino acids are potential glycosylation
sites.
[0037] An HSA/CD24 polypeptide which is less than 100% identical to
the naturally occurring HSA/CD24 polypeptide has an altered
sequence in which one or more of the amino acids in the HSA
homologue is deleted or substituted, or one or more amino acids are
inserted into the sequence of the naturally occurring HSA/CD24
polypeptide. HSA/CD24 sequences which are at least 95% identical to
the naturally occurring HSA/CD24 sequence have no more than 5
alterations, i.e., any combination of deletions, insertions or
substitutions, per 100 amino acids of the reference sequence.
Percent identity is determined by comparing the amino acid sequence
of the altered HSA/CD24 sequence with the naturally occurring
sequence using MEGALIGN project in the DNA STAR program. Sequences
are aligned for identity calculations using the method of the
software basic local alignment search tool in the BLAST network
service (the National Center for Biotechnology Information,
Bethesda, MD) which employs the method of Altschul, S. F., Gish,
W., Miller, W., Myers, E. W. & Lipman, D. J. (1990) J. Mol.
Biol. 215, 403-410. Identities are calculated by the Align program
(DNAstar, Inc.) In all cases, internal gaps and amino acid
insertions in the candidate sequence as aligned are not ignored
when making the identity calculation.
[0038] While it is possible to have nonconservative amino acid
substitutions, it is preferred that the substitutions be
conservative amino acid substitutions, in which the substituted
amino acid has similar structural or chemical properties with the
corresponding amino acid in the reference sequence. By way of
example, conservative amino acid substitutions involve substitution
of one aliphatic or hydrophobic amino acids, e.g. alanine, valine,
leucine and isoleucine, with another; substitution of one
hydroxyl-containing amino acid, e.g. serine and threonine, with
another; substitution of one acidic residue, e.g. glutamic acid or
aspartic acid, with another; replacement of one amide-containing
residue, e.g. asparagine and glutamine, with another; replacement
of one aromatic, residue, e.g. phenylalanine and tyrosine, with
another; replacement of one basic residue, e.g. lysine, arginine
and histidine, with another; and replacement of one small amino
acid, e.g., alanine, serine, threonine, methionine, and glycine,
with another.
[0039] The biologically active fragments and variants of a
naturally-occurring HSA/CD24 have an ID.sub.50 which is comparable
to, i.e., not more than twice the value of, the ID.sub.50 of the
corresponding naturally- occurring HSA/CD24 polypeptide. The
ID.sub.50 of the HSA/CD24 variant or fragment and its corresponding
naturally-occurring polypeptide is determined by measuring the
amount of these polypeptides needed to reduce the clinical symptoms
in experimental autoimmune models, such as EAE or Type II diabetes
in NOD mouse or rat. Alternatively, one can determine the ID.sub.50
of the biologically active HSA/CD24 variant or fragment and its
corresponding naturally occurring HSA/CD24 polypeptide by an
adhesion assay or assays that measure migration of T cells through
endothelial cell monolayer in transwell culture. The amount of the
biologically active variant of the HSA/CD24 polypeptide or fragment
thereof needed to reduce binding of activated T cells to vascular
endothelial cells by at least 50%, preferably, is no greater than,
twice the amount of the corresponding naturally occurring HSA/CD24
polypeptide or fragment thereof.
[0040] The present method also employs fusion proteins comprising
an HSA/CD24 polypeptide or a biologically active fragment thereof
and a tag, i.e., or one or more amino acids, preferably from about
5 to 300 amino acids which are added to the amino terminus of, the
carboxy terminus of, or any point within the amino acid sequence of
the HSA/CD24 polypeptide or the biologically active fragment
thereof. Preferably, the HSA/CD24 polypeptide or core region
thereof is glycosylated. Typically, such additions are made to
simplify purification of an expressed recombinant form of the
corresponding HSA/CD24 polypeptide or core region thereof. Such
tags are known in the art. Representative examples of such tags
include sequences which encode a series of histidine residues, the
epitope tag FLAG, the Herpes simplex glycoprotein D,
beta-galactosidase, maltose binding protein, or glutathione
S-transferase. Preferably, the fusion protein comprises the HSA
polypeptide or a fragment thereof linked by a peptide bond to the
hinge-CH2-CH3 regions of human immunoglobin G1 ("IgG1"). The fusion
protein can be easily purified by affinity chromatography using
either anti-IgG or protein A or protein G. Since IgG is not
immunogenic in humans, the fusion protein can be administrated
repeatedly if necessary.
Methods of Preparing the HSA/CD24 Polypeptide or Fusion Protein
[0041] The HSA/CD24 polypeptides and fusion proteins may be
produced by using cell-free translation systems and RNA molecules
derived from DNA constructs that encode the polypeptide or fusion
protein. Preferably, the HSA/CD24 polypeptide or fusion protein is
made by transfecting host cells with expression vectors that
comprise a DNA sequence that encodes the respective HSA/CD24
polypeptide or fusion protein and then inducing expression of the
polypeptide in the host cells. For recombinant production,
recombinant constructs comprising one or more of the sequences
which encode the HSA/CD24 polypeptide or fusion protein are
introduced into host cells by conventional methods such as calcium
phosphate transfection, DEAE-dextran mediated transfection,
transvection, microinjection, cationic lipid-mediated transfection,
electroporation, transduction, scrape lading, ballistic
introduction or infection.
[0042] The HSA/CD24 polypeptide or fusion protein may be expressed
in suitable host cells, such as for example, mammalian cells,
yeast, insect cells or other cells under the control of appropriate
promoters using conventional techniques. Suitable hosts include,
but are not limited to, CHO, COS cells and 293 HEK cells. Following
transformation of the suitable host strain and growth of the host
strain to an appropriate cell density, the cells are harvested by
centrifugation, disrupted by physical or chemical means, and the
resulting crude extract retained for further purification of the
epitope or chimeric peptide. For obtaining properly glycosylated
forms of the protein, it is preferred that CHO cells be used.
[0043] Conventional procedures for isolating recombinant proteins
from transformed host cells, such as isolation by initial
extraction from cell pellets or from cell culture medium, followed
by salting-out, and one or more chromatography steps, including
aqueous ion exchange chromatography, size exclusion chromatography
steps, and high performance liquid chromatography (HPLC), and
affinity chromatography may be used to isolate the recombinant
polypeptide.
Carrier
[0044] The acceptable carrier is a physiologically acceptable
diluent or adjuvant. The term physiologically acceptable means a
non-toxic material that does not interfere with the effectiveness
of HSA. The characteristics of the carrier will depend on the route
of administration and particular compound or combination of
compounds in the composition. Preparation of such formulations is
within the level of skill in the art. The composition may further
contain other agents which either enhance the activity of the HSA
or complement its activity. The composition may further comprise
fillers, salts, buffers, stabilizers, solubilizers, and other
materials well known in the art.
Dosage
[0045] A biologically effective amount is an amount sufficient to
partially or completely block destruction of the targeted tissue
initiated by the autoreactive T cell or to ameliorate the
pathological effects of the autoimmune disease. The effective
amount can be achieved by one administration of the composition.
Alternatively, the effective amount is achieved by multiple
administration of the composition to the mammal.
Antibodies
[0046] The invention further provides a therapeutic method which
comprises administering a pharmaceutically effective amount of an
anti-HSA/CD24 antibody, preferably a humanized anti-HSA/CD24
antibody, to a human subject suspected of having an autoimmune
disease. The anti-HSA/CD24 antibody is immunospecific for the
HSA/CD24 polypeptide meaning the antibody has substantially greater
affinity for the HSA/CD24 polypeptide than for other polypeptides
that are found on the T cells of the mammal being treated. Various
forms of an anti- HSA/CD24 antibody may be used in this therapeutic
method. For example, the anti-HSA/CD24 antibody may be a full
length antibody (e.g., having a human immunoglobulin constant
region) or an antibody fragment (e.g. a F(ab').sub.2).
[0047] The term "antibody" as used herein encompasses monoclonal
antibodies (including full length monoclonal antibodies),
polyclonal antibodies, multispecific antibodies (e.g., bispecific
antibodies), and antibody fragments so long as they exhibit the
desired biological activity. "Antibody fragments" comprise a
portion of a full length antibody, generally the antigen binding or
variable region thereof. Examples of antibody fragments include
Fab, Fab', F(ab').sub.2, and Fv fragments.
[0048] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody preparations which typically include
different antibodies directed against different determinants
(epitopes), each monoclonal antibody is directed against a single
determinant on the antigen. The monoclonal antibodies to be used in
accordance with the present invention may be made by the hybridoma
method first described by Kohler et al., Nature 256: 495 (1975), or
may be made by recombinant DNA methods (see, e.g., U.S. Pat. No.
4,816,567). The "monoclonal antibodies" may also be isolated from
phage antibody libraries using the techniques described in Clackson
et al., Nature 352: 624-628 (1991) and Marks et al., J. Mol. Biol.
222: 581-597 (1991), for example.
[0049] The monoclonal antibodies herein include "chimeric"
antibodies (immunoglobulins) in which a portion of the heavy and/or
light chain is identical with or homologous to corresponding
sequences in antibodies derived from a particular species or
belonging to a particular antibody class or subclass, while the
remainder of the chain(s) is identical with or homologous to
corresponding sequences in antibodies derived from another species
or belonging to another antibody class or subclass, as well as
fragments of such antibodies, so long as they exhibit the desired
biological activity.
[0050] "Single-chain Fv" or "sFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. Generally, the Fv
polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the sFv to form the
desired structure for antigen binding
[0051] An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the antibody will be purified (1) to greater than 95%
by weight of antibody as determined by the Lowry method, and most
preferably more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. Isolated antibody
includes the antibody in situ within recombinant cells since at
least one component of the antibody's natural environment will not
be present. Ordinarily, however, isolated antibody will be prepared
by at least one purification step
[0052] In order to avoid potential immunogenicity of the mAbs in
human, the mAbs that have desired function are preferably
humanized. "Humanized" forms of non-human (e.g., murine) antibodies
are chimeric antibodies which contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which
hypervariable region residues of the recipient are replaced by
hypervariable region residues from a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues which are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FR
regions are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature 321:
522-525 (1986); Reichmann et al., Nature 332: 323-329 (1988); and
Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992).
[0053] Alternatively, transgenic mice with human IgV and IgC genes
may be used to produce human mAb specific for human CD24. These
mice are available from Abgenix, Inc., and Mederex, Inc, and the
art has been described fully (Nature Genetics, 1997, 15: 146).
Antisense Molecules
[0054] In certain aspects, the present therapeutic methods employ
agents which reduce or inhibit expression of the CD24 gene or
production of the CD24 polypeptide in the T cells of human subjects
known to have or suspected of having an autoimmune disease, such as
multiple sclerosis, rheumatoid arthritis, and type II diabetes.
[0055] One such agent is an antisense molecule. The antisense
molecule for CD24 is an oligomer which comprises from 20 to 200
bases, preferably less than 100 bases, and is targeted to a nucleic
acid encoding the human CD24 polypeptide, in other words, the human
CD24 gene or mRNA expressed from the human CD24 gene. The targeting
process involves determination of a site or sites within the
nucleic acid sequence of the CD24 gene or mRNA for the
oligonucleotide interaction to occur such that transcription of the
CD24 gene or translation of the CD24 mRNA will be reduced in the
human subject's T cells. Once the target site or sites have been
identified, oligonucleotides are chosen which are sufficiently
complementary to the target, i.e., hybridize sufficiently well and
with sufficient specificity, to give the desired reduction in
expression of the CD24 polypeptide. Such inhibition can be measured
in ways which are routine in the art, for example by Northern blot
assay of mRNA expression or Western blot assay of protein
expression, or flow cytometry analysis for cell surface
expression.
[0056] "Hybridization", as used herein means hydrogen bonding, also
known as Watson-Crick base pairing, between complementary bases,
usually on opposite nucleic acid strands or two regions of a
nucleic acid strand. "Specifically hybridizable" and
"complementary" are terms which are used to indicate a sufficient
degree of complementarity such that stable and specific binding
occurs between the DNA or RNA target and the oligonucleotide. It is
understood that an oligonucleotide need not be 100% complementary
to its target nucleic acid sequence to be specifically
hybridizable. An oligonucleotide is specifically hybridizable when
binding of the oligonucleotide to the target interferes with the
normal function of the target molecule to cause a loss of utility,
and there is a sufficient degree of complementarity to avoid
non-specific binding of the oligonucleotide to non-target sequences
under conditions in which specific binding is desired, i.e., under
physiological conditions in the case of in vivo assays or
therapeutic treatment or, in the case of in vitro assays, under
conditions in which the assays are conducted. Affinity of an
oligonucleotide for its target (in this case a nucleic acid
encoding CD24 polypeptide) is routinely determined by measuring the
Tm of an oligonucleotide/target pair, which is the temperature at
which the oligonucleotide and target dissociate; dissociation is
detected spectrophotometrically. The higher the Tm, the greater the
affinity of the oligonucleotide for the target.
[0057] In the context of this therapeutic method, the term
"oligonucleotide" refers to an oligomer or polymer of nucleotide or
nucleoside monomers consisting of naturally occurring bases, sugars
and intersugar (backbone) linkages. The term "oligonucleotide" also
includes oligomers comprising non-naturally occurring monomers, or
portions thereof, which function similarly. Modifications may be on
one or more bases, sugars, or backbone linkages, or combinations of
these; such modifications are well known in the art. Modified or
substituted oligonucleotides are often preferred over native forms
because of properties such as, for example, enhanced cellular
uptake and increased stability in the presence of nucleases. A
number of nucleotide and nucleoside modifications have been shown
to make the oligonucleotide into which they are incorporated more
resistant to nuclease digestion than the native
oligodeoxynucleotide. Nuclease resistance is routinely measured by
incubating oligonucleotides with cellular extracts or isolated
nuclease solutions and measuring the extent of intact
oligonucleotide remaining over time, usually by gel
electrophoresis. Oligonucleotides which have been modified to
enhance their nuclease resistance survive intact for a longer time
than unmodified oligonucleotides. A variety of oligonucleotide
modifications have been demonstrated to enhance or confer nuclease
resistance. In some cases, oligonucleotide modifications which
enhance target binding affinity are also, independently, able to
enhance nuclease resistance. A discussion of antisense
oligonucleotides and some desirable modifications can be found in
De Mesmaeker et al. Acc. Chem. Res. 1995, 28, 366-374.
[0058] Specific examples of some oligonucleotides contemplated for
the present method include those containing modified backbones, for
example, phosphorothioates, phosphotriesters, methyl phosphonates,
short chain alkyl or cycloalkyl intersugar linkages or short chain
heteroatomic or heterocyclic intersugar linkages. The
oligonucleotides may be chimeric oligonucleotides. "Chimeric
oligonucleotides" as used herein mean oligonucleotides which
contain two or more chemically distinct regions, each made up of at
least one nucleotide. These oligonucleotides typically contain at
least one region of modified nucleotides that confers one or more
beneficial properties (such as, for example, increased nuclease
resistance, increased uptake into cells, increased binding affinity
for the RNA target) and a region that is a substrate for RNase H
cleavage. In one embodiment, a chimeric oligonucleotide comprises
at least one region modified to increase target binding affinity,
and, usually, a region that acts as a substrate for RNAse H. RNAse
H is a cellular endonuclease that cleaves the RNA strand of RNA:DNA
duplexes; activation of this enzyme therefore results in cleavage
of the RNA target, and thus can greatly enhance the efficiency of
antisense inhibition. Cleavage of the RNA target can be routinely
demonstrated by gel electrophoresis.
[0059] Alternatively, an expression vector comprising a
polynucleotide or nucleic acid encoding an antisense
oligonucleotide targeted to nucleic acids that encode the CD24
polypeptide are introduced into the subject's T cells. The CD24
antisense encoding nucleic acid is operatively linked to a
promoter. A "promoter" is a sequence that directs the binding of
RNA polymerase and thereby promotes RNA synthesis, which in the
present method is synthesis of the antisense oligonucleotide.
Operatively linked is understood to mean that the CD24 antisense
encoding sequence is joined to the promoter region such that the
promoter is oriented 5' to the CD24 antisense encoding sequence and
is of an appropriate distance from the transcription start site, so
that the transcription of the polynucleotide which encodes the CD24
antisense oligonucleotide will be dependent on or controlled by the
promoter sequence. The arts of restriction enzyme digestion and
nucleic acid ligation to be used in construction of the CD24
antisense encoding polynucleotide-promoter construct are well known
in the art as exemplified by Maniatis et al., Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor, N.Y., 1982, (incorporated
herein by reference). Many examples of constitutive promoters have
been described in the art such as those isolated from
cytomegalovirus early gene, murine MHC class I, actin, etc. An
example of T-cell specific promoter is the lck promoter (EMBO J. 9:
3821-3829,1990), which we have shown to be able to drive T
cell-specific expression (Eur. J. Immunol 27:2524-2528, 1997) . An
example of vascular cell specific vector is described by Sato et
al. (Proc. Natl. Acad Sci USA, 94:3058-63(1997)
dsRNAi
[0060] Another agent for reducing or inhibiting expression of the
CD24 gene and production of the polypeptide in the T cells of human
subjects is a double stranded oligonucleotide or polynucleotide
known as dsRNAi. One strand of the dsRNA comprises a CD24 sense
sequence; while the other strand comprises a CD24 antisense
sequence. Preferably, the dsRNAi further comprises a linker
connecting the antisense sequence to the sense sequence.
Preferably, the CD24 sense and anti-sense sequences are from 19-30
bases in length. The linker is at least 10 bases in length, and
preferably, from 10-20 bases in length. The CD24 dsRNAi prevents
accumulation of CD24 mRNA in the transformed cells, most likely
through a post-transcription gene silencing method known in the art
as double-stranded RNA interferences.
[0061] dsRNAi can be synthesized using standard techniques. For
example single-stranded RNA corresponding to the sense CD24
sequence, and single stranded RNA corresponding to the antisense
CD24 sequence can be synthesized according to methods known in the
art. The single stranded RNAs can then be annealed in vitro by
methods known in the art, to produce the dsRNA. To increase the
stability of the dsRNAi, several nucleotide with de-oxyl-nucleotide
can be incorporated at the 3' of the oligonucleotides.
[0062] Alternatively, an expression vector comprising a
polynucleotide or nucleic acid encoding CD24 dsRNAi is introduced
into the subject's T cells or, the subject's vascular endothelial
cells, or both. Such polynucleotide comprises a sequence which
encodes a sense CD24 RNA coding sequence and an antisense CD24 RNA
coding sequence and a linker sequence which links the sense CD24
RNA coding sequence to the antisense CD24 RNA coding sequence. The
expression vector further comprises a promoter operatively linked
to the CD24 dsRNAi coding sequence.
Targeting of the CD24 Antisense and dsRNAi Molecules
[0063] In accordance with the present method, targeted regions of
the CD24 gene and CD24 mRNA include not only the coding region for
the CD24 polypeptide, but also the 5'-untranslated region, the
3'-untranslated region, the 5' cap region, intron regions and
intron/exon or splice junction regions of the targeted nucleic
acid. The functions of messenger RNA to be interfered with include
all vital functions such as translocation of the RNA to the site
for protein translation, actual translation of protein from the
RNA, splicing or maturation of the RNA and possibly even
independent catalytic activity which may be engaged in by the RNA.
The overall effect of such interference with the CD24 mRNA function
is to cause interference with expression of the CD24
polypeptide.
[0064] Although numerous areas can be targeted for anti-sense and
dsRNAi molecules, there is a significant advantage to target areas
in which the sequence is completely conserved between CD24 in mouse
and man. In this way, the CD24 anti-sense and dsRNAi molecules can
be screened for both efficacy and toxicity in preclinical models
before they are used for human clinical trials. A comparison
between human and mouse CD24 cDNA sequence, as listed in FIG. 14,
revealed 8 areas within the 2.1 kb areas of human CD24 that can be
as targets for antisense and dsRNAi molecules.
Delivery of the CD24 Antisense and dsRNAi Molecules
[0065] Single-stranded CD24 anti-sense oligonucleotides and dsRNAi
molecules can be introduced into the subject's cells, including but
not limited to T cells, vascular endothelial cells, or both. The
molecules are introduced into the cells either ex vivo or in vivo.
"Ex vivo" means that these molecules are introduced into the T
cells or endothelial cells outside the body of the subject from
whom the T cells or endothelial cells are obtained. The cells are
then re-introduced back into the subject. For in vivo delivery to
these target cells, the CD24 antisense and dsRNAi molecules are
introduced into the subject by injection. Preferably, the injection
is intravenous, or intralesional injection (as in the case of
rheumatoid arthritis).
[0066] Delivery of the CD24 antisense or CD24 dsRNAi encoding
polynucleotide-promoter construct into the subject may be either
direct, in which case the subject is directly exposed to the
construct or construct-carrying vector, or indirect, in which case
cells are first transformed with the construct in vitro, then
transplanted into the patient. The latter method is referred to as
cell-based gene-therapy.
[0067] A retroviral vector may be used to deliver the CD24
antisense and CD24 dsRNAi encoding construct (see Miller et al.,
1993, Meth. Enzymol. 217:581-599). Retroviral vectors have been
modified to delete retroviral sequences that are not necessary for
packaging of the viral genome and are maintained in infected cells
by integration into genomic sites upon cell division. More detail
about retroviral vectors can be found in Boesen et al. (1994)
Biotherapy 6:291-302,: Clowes et al, (1994) J. Clin. Invest.
93:644-651; Kiem et al. (1994) Blood 83:1467-1473; Salmons and
Gunzberg (1993) Human Gene Therapy 4:129-141; and Grossman and
Wilson (1993) Curr. Opin. in Genetics and Devel. 3:110-114.
[0068] A lentiviral vector (Science. 1996 Apr 12;272(5259):263-7.)
can also be used to deliver genes that encode the antisense drug
either in vivo or to ex vivo cells. Unlike a typical retroviral
vector, the lentiviral vector can be used to deliver gene to
non-dividing cells.
[0069] Alternatively, liposomes may be employed to deliver the CD24
antisense and dsRNAi encoding constructs to target tissues using
methods known in the art. The liposomes may be constructed to
contain a targeting moiety or ligand, such as an antigen, an
antibody, or a virus on their surface to facilitate delivery to the
appropriate tissue. For example, liposomes prepared with
ultraviolet (UV) inactivated Hemagglutinating Virus of Japan (HVJ)
may be used to deliver DNA to selected tissues (Morishita, et al.).
The liposomes may also be surface-coated with phospholipid
-polyethyleneglycol conjugates, to extend blood circulation time
and allow for greater targeting via the bloodstream. Liposomes of
this type are well known. A variety of liposome have been described
in the art to deliver double-stranded nucleotide or naked DNA into
cells, both for ex vivo cells, or for in vivo delivery.
[0070] Receptor-mediated endocytic pathways for the uptake of DNA
may permit the targeted delivery of the CD24 antisense and dsRNAi
encoding constructs to specific cell types in vivo.
Receptor-mediated methods of polynucleotide delivery in vivo
involve the generation of complexes between vectors and specific
polypeptide ligands that can be recognized by receptors on the cell
surface.
[0071] For general reviews of the methods of in vivo polynucleotide
delivery (also referred to as gene therapy), see Goldspiel et al
(1993) Clinical Pharmacy 12:488-505; Wu and Wu (1991) Biotherapy
3:87-95; Tolstoshev (1993) Ann. Rev. Pharmacol. Toxicol.
32:573-596; Mulligan, (1993) Science 260:926-932; and Morgan and
Anderson (1993) Ann. Rev. Biochem. 62:191-217; May, 1993, TIBTECH
11(5):155-215. Methods commonly known in the art of recombinant DNA
technology which can be used are described in Ausubel et al. (eds.)
(1993) Current Protocols in Molecular Biology, John Wiley &
Sons, NY; and Kriegler (1990) Gene Transfer and Expression, A
Laboratory Manual, Stockton Press, NY.
Transgenic and Knock-In Mouse Models to Test the Effect of CD24
Blockers In Vivo
[0072] Since the major cell type in the CNS that expresses HSA is
the brain vascular endothelial cells, transgenic vectors that give
specific expression of human CD24 in both T cells and vascular
endothelial cells are used to prepare the transgenic mice. In one
preferred embodiment T cell-specific expression is achieved using a
transgenic vector comprised of human CD24 open reading frame and
the proximal lck promoter and vascular endothelial cell specific
expression is achieved using a transgenic vector comprised of human
CD24 open-reading frame and the Tie II promoter, as described in
Proc. Natl. Acad Sci USA, 94:3058-63(1997). To avoid interference
by the endogenous HSA, the transgenic vector is injected into the
fertilized embryos from mice with a targeted mutation of mouse CD24
as described in J. Exp. Med. 185: 251-262, 1997. Alternatively, the
transgenic mice expressing the CD24 gene can be bred to the CD24
(-/-) mice to avoid expression of endogenous CD24. The tissue
specificity of the transgene expression is verified with anti-CD24
mAb, which is available from Pharmingen (San Diego, Calif.), by
flow cytometry and immunohistochemistry according to established
procedure.
[0073] Alternatively, human CD24 knock-in mice can be developed to
screen for therapeutic agents targeted at the human CD24 antigen.
The major advantage of the knock-in mice is that all the cells that
express murine CD24 can be rendered to express human CD24 genes,
and as such, the knock-in mice are more relevant for testing the
efficacy and safety of drugs targeted at the human CD24 antigens.
Since mouse and human CD24 protein have identical amino acid
sequence in the signal peptide region (encoded) regions, the CD24
knock-in mouse can be made by replacing only the coding region in
exon 2. Moreover, since a major portion of the coding region
encodes for GPI-cleavage signal peptide that will be removed from
the mature protein, replacement of the mouse HSA/CD24 with human
CD24 can be achieved simply by replacing the 81 by region that
encode for the core region of mature mouse CD24 protein with a 96
by region that encodes for the core region of mature human CD24
protein. Replacement of some human CD24 protein amino acids with
murine counterpart may be tolerated if such replacement does not
change the binding activity of these molecules to anti-human CD24
antibodies, and functional ligands of human CD24 protein. For
convenience of cloning, the replacement can be significantly larger
than proposed region. Once the construct is produced, it is used to
transfect mouse embryonic stem (ES) cells, to select for
transfectants in which at least one allele of the CD24 gene is
replaced the construct through homologous recombination. The
recombinant alleles can be screened by PCR and/or Southern blot
according the established procedures. The recombinant ES cells are
tested for functionality of the recombinant allele. Once this is
verified, the ES cells are used to produce chimera mice. Further
breeding yield mice that are homozygous for the knock-in alleles,
which express human CD24 gene on cells that had been programmed to
express CD24.
[0074] The transgenic and knock-in mice produced as described above
are used to screen drugs targeted at the human CD24 molecules. One
example is to screen for drugs which inhibit or ameliorate
autoimmune conditions such as multiple sclerosis and diabetes. The
most suitable murine model for multiple sclerosis is EAE, which is
induced by immunizing mice with MOG according to a established
procedure.
[0075] The preferred method for testing drugs targeted at CD24 for
diabetes is to breed the human CD24 transgenic mice with non-obese
diabetic (NOD) mice to cross the transgene to NOD background. The
drugs targeted at HSA or its homologue are administrated at
approximately 2-3 weeks to determine their 1D50 in the reduction of
insulitis and spontaneous diabetes.
Blocking Binding of Autoreactive T Cells to Endotheial Cells In
Vitro or In Vivo
[0076] The present invention also relates to a method of blocking
binding of autoreactive T cells to endothelial cells, in vitro or
in vivo. In one aspect, the method comprises contacting the
endothelial cells with a sufficient amount of HSA or a fusion
protein comprising HSA to inhibit interaction of the autoreactive T
cells with HSA molecules present on the surface of the endothelial
cells. The cells may be in vitro, i.e., in tissue culture, or in
vivo, i.e., in the body of a mammal. Blocking interaction between
the endothelial and T cells in vitro is achieved by adding the
protein to a chamber that contains both T cells and endothelial
cells. The amount of T cells bound to a monolayer of endothelial
cells in the presence or absence of HSA protein is quantified
either by counting the number of cells attached, or by other
methods to quantify the number of T cells that were labeled prior
to adding to the monolayer.
[0077] Interaction between the endothelial and autoreactive T cells
in vivo is inhibited by injecting the protein intravenously. To
quantify the extent of inhibition fluorescent labeled T cells are
administered to an animal and the rolling of T cells along the
blood vessel is measured using established procedures known in the
art.
[0078] In another aspect, interaction of autoreactive T cells with
vascular endothelial cells is blocked or reduced by inhibiting or
reducing expression of the CD24 polypeptide in the autoreactive T
cells, the endothelial cells or both. Expression of the CD 24
polypeptide in the target cells is accomplished by introducing CD24
antisense oligonucleotides or CD24 dsRNAi into the target cells, or
alternatively, transfecting these cells with a polynucleotide which
encodes the CD24 antisense oligonucleotide or CD24 dsRNAi. As used
herein, transfect, refers to introduction of a polynucleotide into
the cell where the polynucleotide may be incorporated into the
genome of the cell, converted into an autonomous replicon, or
transiently expressed. The transfection can be in vivo or ex vivo.
"Ex vivo transfection" means that transfection occurs outside the
body of the subject from whom the target cells were obtained. "In
vivo transfection" means transfection of the target cells within
the body of the subject.
[0079] All references cited herein are specifically incorporated
herein in their entirety.
EXAMPLES
[0080] The following examples are for illustration only and are not
intended to limit the scope of the invention.
Example 1
Treatment of Animals with Experimental Autoimmune Encephalomyelitis
with HSAIg
Methods
[0081] Mice Wild type C57BL/6 mice (WT) were purchased from the
National Cancer Institute (Bethesda, Md.). Mice homozygous for the
disrupted HSA (produced with ES cells from C57BL/6 mice) (18) (24)
or CD28 (25) (backcrossed to C57BL/6 for more than 8 generations)
locus have been described before and are maintained at the animal
facilities of the Ohio State University Medical Center. HSA
transgenic mice (HSATG) have been described previously (See Zhou,
Q., Wu, Y., Nielsen, P. J., and Liu, Y. 1997. Homotypic interaction
of the heat-stable antigen is not responsible for its
co-stimulatory activity for T cell clonal expansion. Eur J Immunol.
27: 2524-2528, which is specifically incorporated herein by
reference.) and have been backcrossed to C57BL/6j background for
more than 5 generations. Mice with HSA exclusively expressed on the
T cell lineage (HSATG/HSA(-/-)) were generated by crossing HSATG
with the HSA(-/-) mice.
[0082] Induction and clinical evaluation of EAE The immunogen, MOG
peptide 35-55 of rat origin (MEVGWYRSPFSRVVHLYRNGK), was
synthesized by Research Genetics, Inc. (Huntsville, Ala., USA). The
purity of the peptide was >90%. Mice of 8-12 wks of age were
immunized subcutaneously with 200 .mu.g MOG peptide in complete
Freund's Adjuvant (400 .mu.g of Mycobacterium tuberculosis per ml)
in a total volume of 100 .mu.l. They received 200 .mu.g of Pertusis
toxin (List Biological, Campbell, Calif.) in 200 .mu.l PBS in the
tail vein immediately after the immunization, and again 48 hours
later. The mice were observed every other day and scored on a scale
of 0-5 with gradations of 0.5 for intermediate scores: 0, no
clinical signs; 1, loss of tail tone; 2, wobbly gait; 3, hind limb
paralysis; 4, hind and fore limb paralysis; 5, death. T cell
proliferation assay Draining lymph node cells were isolated 10 days
after immunization. 5.times.10.sup.5 cells/well were stimulated
with given concentrations of MOG peptide in the presence
6.times.10.sup.5 cells/well of irradiated (2,000 rad) syngeneic
splenocytes for 60 hours. The cultures were pulsed with
.sup.3H-thymidine (1 .mu.Ci/well; ICN Pharmaceuticals Inc., Costa
Mesa, Calif. USA) for another 12 hours, and incorporation of
3H-thymidine was measured in a liquid scintillation P-plate
counter.
[0083] ELISpot assay to evaluate frequencies of T cells that
produce IFN-.gamma., IL-2 and IL-4 upon restimulation with MOG
peptide in vitro The antibody pairs and the procedures have been
described (20), except that the MOG peptide was used for
stimulation at 10 .mu.g/ml. The numbers presented are those of
cytokine producers per million of draining lymph node cells.
Histology
[0084] Mice were sacrificed by CO.sub.2 inhalation. Spinal cords
were removed by insufflation and fixed in 10% formalin/PBS.
Paraffin sections were prepared and stained with hematoxylin and
eosin. Neurological lesions were graded on each of the 10 cross
sections per spinal cord, according the following criteria: 0, no
infiltrate; 1, 3 or less focal meningeal infiltrates; 2, more than
3 focal meningeal infiltrates; 3, up to 5 perivascular infiltrate
foci in the parenchyma with involvement of less than 5% of the
white matter; 4, 5-10 perivascular foci in the parenchyma or
invasions involving 5-25% the white matter; 5, more than 10
perivascular foci or diffuse infiltration involving more than 25%
of the white matter.
Passive Transfer of EAE
[0085] Groups of 8-10 WT and HSA(-/-) mice were immunized with 200
.mu.g of MOG peptide subcutaneously. At 10 days after immunization,
draining lymph nodes were harvested and stimulated at
4.times.10.sup.6/m1 in Click's EHAA medium supplemented. with 15%
fetal calf sera, 5% IL-2 supematant, and 50 .mu.g/ml of MOG peptide
for 4 days. 1.times.10.sup.8 cells were injected i.p. into each
recipient mouse that had been .gamma.-irradiated (550 rad) 1 h
earlier.
Preparation of Fusion Protein and Treatment of EAE z
[0086] The HSA fragment encoding the signal peptide and the mature
protein sequence were amplified by PCR, using GGA AAG CTT ATG GGC
AGA GC, SEQ ID NO.:6, as forward primer, CGA GAT CTC TGG TGG TAG CG
, SEQ ID NO.:7, as reverse primer, and HSA cDNA as template. The
PCR products were digested with Hind III and Bgi II enzymes and
were ligated to Hind III and Xba I-digested pCDM8 vector
(Invitrogen, San Diego) and a Xba I and Barn HI-treated DNA
fragment encoding human IgG1 Fc, which were amplified by PCR using
CAG GGA TCC CGA GGG TGA GTA CTA AGC TAG CTT CAG CGC TCC TGC CTG,
SEQ ID NO.:7, as forward primer and CTT CGA CCA GTC TAG AAG CAT CCT
CGT GCG ACC GCG AGA GC, SEQ ID NO.:8, as reverse primer, and DNA
from human peripheral blood as template. The construct was verified
by DNA sequencing and was used to transfect the Chinese Hamster
Ovary cell line. The cells that secreted HSAIg fusion protein were
amplified in DMEM containing 5% fetal calf serum until confluence.
The cell monolayers were washed with serum-free medium and cultured
in optimal M medium for 72 hours. The supernatants were collected
and the HSAIg was purified using a protein G column according to
the manufacturer's protocol. The purity of the protein was verified
by SDS PAGE.
Results
[0087] To test if HSA is essential for the development of EAE, we
immunized C57BL/6 wild-type (WT), and HSA- or CD28-deficient mice
with myelin oligodendrocyte glycoprotein (MOG) peptide AA35-55 in
conjunction with complete Freund's adjuvant and pertusis toxin. As
shown in FIG. 1a, wild-type mice developed acute EAE within two
weeks of peptide immunization, while those with targeted mutation
of either HSA or CD28 were completely resistant to EAE induction.
Interestingly, while targeted mutation of CD28 ablated induction of
MOG-specific T cells, as revealed by proliferative response of
draining lymph node cells, that of HSA had little effect on
peptide-specific T cell proliferation (Fig. lb). Moreover, the
frequencies of antigen-specific, IL2-, IL4-, and IFNy-producing
cells were not altered in HSA(-/-) mice (FIG. 1c). The anti-MOG
peptide IgG responses were also detected in HSA-deficient mice
(data not shown). The differential effects of HSA and CD28
mutations on T cell priming reveal that these genes mediate two
distinct checkpoints in the development of EAE: CD28 controls
induction of auto-reactive T cells, while HSA determines their
pathogenicity.
[0088] Histological analysis of MOG-peptide immunized WT and
HSA-confirms the clinical scores. The histological scores were
summarized in FIG. 2a, while representative histology sections were
presented FIG. 2b-d. As shown in FIG. 2b, active immunization with
MOG peptide induces multiple neurological lesions in the wild-type
mice, characterized by multiple lesions with extensive invasion of
parenchyma. In contrast, the spinal cords of HSA-KO mice are either
devoid of any lesion (FIG. 2c), or with one or two low grade
lesions involving meninges (FIG. 2d).
[0089] We adoptively transferred activated draining lymph node
cells to WT and HSA-deficient recipients. As shown in FIGS. 3 and
4, WT T cells induced severe EAE in WT recipients within 8 days of
adoptive transfer. Interestingly, none of the HSA-deficient
recipients developed EAE. Thus HSA expression on T cells alone
appears insufficient for EAE development. Moreover, T cells from
HSA-deficient mice failed to induce disease regardless of HSA gene
status in the recipient, which indicates that HSA expression on T
cells is necessary for EAE development. These results strongly
suggest that HSA must be expressed on both host cells and
auto-reactive T cells in order to induce EAE.
[0090] To substantiate these observations, we produced mice that
expressed HSA exclusively on T cells. We have previously reported
the transgenic mice in which expression of HSA was under the
control of the lck proximal promoter (HSATG) (22). For this study,
We crossed the HSA transgene to HSA-deficient mice to produce mice
that expressed HSA exclusively on T cells (FIG. 5a). To test if HSA
expression on the T cell lineage is sufficient for EAE development,
we immunized WT, HSA-TG, HSA(-/-) and HSATG HSA(-/-) mice with MOG.
As shown in FIG. 5b, wild-type and HSATG mice developed EAR with
essentially identical kinetics, which indicates that transgenic
expression of HSA on T cells does not prevent the production and
effector function of self-reactive T cells. Nevertheless, much like
HSA (-/-) mice, the mice with exclusive HSA-expression on the T
cell lineage failed to develop EAE. These results demonstrated
clearly that HSA expression on T cell lineage alone is insufficient
for EAE development.
[0091] The fact that HSA may be a critical checkpoint after
activation of self-reactive T cells suggests a novel approach in
treating autoimmune neurological diseases. Since an anti-HSA mAb
was toxic in the EAE model to address this issue (Data not shown),
we produced a fusion protein between the extracellular domain of
HSA and the Fc portion of human IgG1, to block the HSA-mediated
interactions. As shown in FIG. 6a, the fusion protein has an
apparent molecular weight of about 10010 under non-reducing
SDS-PAGE. After reduction, it migrated as a 50 kD band. We treated
mice starting at 8-10 days after immunization with MOG peptide,
when MOG-specific T cells response had already expanded in the
local lymph nodes. As shown in FIG. 6b, HSAIg drastically
ameliorated EAE. All HSAIg-treated mice recovered substantially
earlier than did the control mice. Since MOG-reactive T cells had
been activated prior to HSAIg administration, the clinical signs in
the treated group may reflect the fact that some autoreactive T
cells had already migrated into the central nervous system.
[0092] HSAIg, a fusion protein consisting of the extracellular
domain of mouse HAS and the Fc portion of immunoglobulin,
drastically ameliorates the clinical sign of EAE even when
administrated after self-reactive T cells had been expanded. Thus,
identification of HSA as a novel checkpoint, even after activation
and expansion of self-reactive T cells, provides a novel approach
for immunotherapy of autoimmune neurological diseases, such as
multiple sclerosis.
Example 2
Production Human CD24Ig Fusion Protein
[0093] Fragments of the human CD24 polypeptides lacking the GPI
anchor region are fused with human Ig constant region to form
CD24-Ig fusion protein. In one embodiment the CD24 polypeptide
fragment comprises the signal peptide. In another embodiment the
CD24 polypeptide fragment lacks the signal peptide. The fragment of
the human CD24 coding sequence is subcloned into vector pIg (from
Novagen) Hind III and BamHI sites. Suitable primers useful in
subcloning include, but are not limited to, CD24 forward primer
(CD24F.H3): G GCC AAG CTT ATG GGC AGA GCA ATG GTG, SEQ ID NO.:9,
with Hind III site 5' to ATG start codon. CD24-Ig reverse primer
(CD24Rig.Bm): GG CCG GAT CCA CTT ACC TGT CGC CTT GGT GGT GGC ATT,
SEQ ID NO.10, with Barn HI site and the SD sequence (A CTT ACC TGT,
SEQ ID NO.:11) next to 3' end of TTKA (direct sequence: ACC ACC AAG
GCG, SEQ ID NO.:12) in Human CD24. The construct is transfected
into CHO cells, and the CD24Ig is secreted into the tissue culture
medium. CD24Ig is purified by affinity chromatography using a
Protein G column. The clone compresses CD24 signal peptide, CD24
core peptide and the IgG/Fc portion, but lacks the GPI anchor
signaling region.
Example 3
Production of Anti-Human CD24 mAb that Blocks Autoreactive T
Cells-Initiated Tissue Destruction
[0094] Human CD24 coding sequence is subcloned into vector pCDM8
(from Invitrogen) Hind III and Xho I sites. CD24 forward primer
(CD24F.H3): G GCC AAG CTT ATG GGC AGA GCA ATG GTG with Hind III
site 5' to ATG start codon. CD24 reverse primer (CD24R. Xho): A TCC
CTC GAG TTA AGA GTA GAG ATG CAG with Xho I site 3' to TAA stop
codon. The CD24 cDNA is transfected into murine 3T3 cells. The 3T3
cell lines that stably express human CD24 molecules are used to
immunize syngeneic mice. After 2-3 immunization, spleen cells are
fused with myeloma AgX865, after selection with HAT medium the
supernatants are screened for anti-human CD24 mAbs. The antibodies
are tested for their ability to block both adhesion of human T
cells to human endothelial cells in vitro, and their ability to
block human CD24-mediated T cell trafficking to target tissues,
such as the pancreas and the central nervous system using the
transgenic model detailed below.
Example 4
Testing Putative Inhibitors of Multiple Sclerosis with CD24
Transgenic and Knock-In Mice
[0095] The immunogen, MOG peptide 35-55 of rat origin
(MEVGWYRSPFSRVVHLYRNGK, SEQ ID NO.:13), is available from Research
Genetics, Inc. (Huntsville, Ala., USA). Mice of 8-12 wks of age are
immunized subcutaneously with 200 .mu.g MOG peptide in complete
Freund's Adjuvant (400 .mu.g of Mycobacterium tuberculosis per ml)
in a total volume of 100 .mu.l. They receive 200 .mu.g of Pertusis
toxin (List Biological, Campbell, Calif.) in 200 Id PBS in the tail
vein immediately after the immunization, and again 48 hours later.
The mice are observed every other day and scored on a scale of 0-5
with gradations of 0.5 for intermediate scores: 0, no clinical
signs; 1, loss of tail tone; 2, wobbly gait; 3, hind limb
paralysis; 4, hind and fore limb paralysis; 5, death. The putative
inhibitory molecules are injected at 1 week after immunization.
Those that substantially reduce the clinical score of EAE are
selected for further testing.
Example 5
Generation of Human CD24 Gene Knock-in Mice
[0096] The basic strategy used to produce CD24 gene knock-in mice
is to replace part of murine CD24 gene exon 2 sequence with that of
human CD24 sequence. We took advantage of the fact that signal
peptide, encoded by exon 1 of mouse and human CD24 gene, are
identical between mouse and human CD24. We therefore replaced only
part of the mouse exon 2 sequence with that of 240 by of human
CD24. The construct with the desired sequence is shown in FIG.
13.
[0097] As shown in FIG. 13, arm 1 of the construct comprised of a
2.7 kb fragment of mouse CD24 gene, cloned from 129RI ES cells (Seq
ID. 20). The arm 2 of the construct is a chimera gene consisting of
the last 256 by sequence of CD24 exon 1, first 240 by human CD24
exon 2 sequence and about 3 kb of mouse CD25 sequence comprising of
both remaining exon 2 sequence encoding for 3' untranslated region
and 3' sequence of the CD24 gene Seq I.D. 22). The construct is
used to transfect ES cells. The recombinants are screened by
procedures established in the art, including PCR and Southern blot.
The ES cells with the illustrated knock-in alleles are transfected
with plasmid encoding Cre recombinase that recognize the lox P
sequence. Since ES cells expression CD24 gene, as revealed by cell
surface flow cytometry, the functionality of the knock-in alleles
can be confirmed by cell surface expression of human CD24. The ES
cells with the capacity to express human CD24 are used to produce
chimera mice by blastocyte injection according to technique known
in the art. Mice with germ-line transmission are produced by
breeding the chimera mice.
Example 6
Inhibition of CD24 Expression by dsRNAi Technology
[0098] Mouse and human CD24 genes are highly homologous. It is
therefore possible to select regions that are identical between
mouse and human CD24 as target for dsRNAi drug. An alignment
between Human CD24 (XM.sub.--099027) and Mouse CD24
(NM.sub.--009846) is shown in FIG. 14. Eight regions with a stretch
of identical nucleotide that is 17 by or longer are highlighted and
as preferred target sequences. Although identity between mouse and
human is not an essential feature of the dsRNAi molecule, targeting
the dsRNAi to identical regions provides a dsRNAi which can be used
to inhibit expression of both mouse and human CD24 genes. As a
result, preclinical small rodent models can be used to screen for
the efficacy of dsRNAi molecule in animal disease models, in
addition to cell culture.
[0099] CHO cells transfected with either mouse or human CD24 cDNA
are transfected with dsRNAi, produced by in vitro annealing.
Briefly, both sense and antisense RNA corresponding to nt. 46-64
(+1 as translation starting site) of mouse and human CD24 gene plus
two thymidine were synthesized by a commercial vendors. The
sequence of the two strands are as follows: CD24-46/64 iRNA.F:
5'-CUG GCA CUG CUC CUA CCC ATT-3' (seq ID. 16), and CD24-46/64
iRNA.R: 5'-UGG GUA GGA GCA GUG CCA GTT-3' (seq ID 17). Control
oligonucleotides were designed based the inverted sequence, as
follows invCD24-46/64 iRNA.F: 5'-ACC CAU CCU CGU CAC GGU C TT-'
(seq ID. 18) invCD24-46/64 iRNA.R: 5'-GAC CGU GAC GAG GAU GGG
UTT-3' (seq ID 19). For annealing of siRNA, 20 uM single strands
will be incubated in annealing buffer (100 mM KOAc, 30 mM HEPES at
p17.4, 2 mM MgAc) for 1 min at 94 degree followed by lh at 37
degree and resulting dsRNA. The resulting dsRNAi is used to
transfect CHO cells. At 48 hours after transfection, the cells are
analyzed for CD24 expression by flow cytometry.
[0100] As shown in FIG. 15, transient transfection lead to
expression of CD24 on about 7% of the CHO cells. Inverted dsRNA
reduced expression of CD24 some what, although significant number
of CHO cells (2%) still express high level of CD24. Importantly,
the expression of CD24 is completely abrogated when the CHO cells
are co-transfected with dsRNAi corresponding to human/mouse CD24
sequence. These results revealed that the dsRNAi can be used to
inhibit expression of CD24.
Sequence CWU 1
1
33176PRTMus musculus 1Met Gly Arg Ala Met Val Ala Arg Leu Gly Leu
Gly Leu Leu Leu Leu1 5 10 15Ala Leu Leu Leu Pro Thr Gln Ile Tyr Cys
Asn Gln Thr Ser Val Ala 20 25 30Pro Phe Pro Gly Asn Gln Asn Ile Ser
Ala Ser Pro Asn Pro Ser Asn 35 40 45Ala Thr Thr Arg Gly Gly Gly Ser
Ser Leu Gln Ser Thr Ala Gly Leu 50 55 60Leu Ala Leu Ser Leu Ser Leu
Leu His Leu Tyr Cys65 70 75280PRTHomo sapiens 2Met Gly Arg Ala Met
Val Ala Arg Leu Gly Leu Gly Leu Leu Leu Leu1 5 10 15Ala Leu Leu Leu
Pro Thr Gln Ile Tyr Ser Ser Glu Thr Thr Thr Gly 20 25 30Thr Ser Ser
Asn Ser Ser Gln Ser Thr Ser Asn Ser Gly Leu Ala Pro 35 40 45Asn Pro
Thr Asn Ala Thr Thr Lys Val Ala Gly Gly Ala Leu Gln Ser 50 55 60Thr
Ala Ser Leu Phe Val Val Ser Leu Ser Leu Leu His Leu Tyr Ser65 70 75
80376PRTRattus norvegicus 3Met Gly Arg Ala Met Val Val Arg Leu Gly
Leu Gly Leu Leu Leu Leu1 5 10 15Ala Leu Leu Leu Pro Thr Gln Ile Tyr
Cys Asn Gln Thr Ser Val Ala 20 25 30Pro Phe Ser Gly Asn Gln Ser Ile
Ser Ala Ala Pro Asn Pro Thr Asn 35 40 45Ala Thr Thr Arg Ser Gly Cys
Ser Ser Leu Gln Ser Thr Ala Gly Leu 50 55 60Leu Ala Leu Ser Leu Ser
Leu Leu His Leu Tyr Cys65 70 7541494DNAArtificial SequenceSynthetic
fusion gene 4atgggcagag cgatgggggc caggctaggg ctggggttgc tgcttctggc
actgctccta 60cccacgcaga tttactgcaa ccaaacatct gttgcaccgt ttcccggtaa
ccagaatatt 120tctgcttccc caaatccaag taacgctacc accagagatc
ccgagggtga gtactaagct 180agcttcagcg ctcctgcctg gacgcatccc
ggctatgcag ccccagtcca gggcagcaag 240gcaggccccg tctgcctctt
cacccggagc ctctgcccgc cccactcatg ctcagggaga 300gggtcttctg
gctttttccc aggctctggg caggcacagg ctaggtgccc ctaacccagg
360ccctgcacac aaaggggcag gtgctgggct cagacctgcc aagagccata
tccgggagga 420ccctgcccct gacctaagcc caccccaaag gccaaactct
ccactccctc agccggacac 480cttctctcct cccagattcc agtaactccc
aatcttctct ctgcagagcc caaatcttgt 540gacaaaactc acacatgccc
accgtgccca ggtaagccag cccaggcctc gccctccagc 600tcaaggcggg
acaggtgccc tagagtagcc tgcatccagg gacaggcccc agccgggtgc
660tgacacgtcc acctccatct cttcctcagc acctgaactc ctggggggac
cgtcagtctt 720cctcttcccc ccaaaaccca aggacaccct catgatctcc
cggacccctg aggtcacatg 780cgtggtggtg gacgtgagcc acgaagaccc
tgaggtcaag ttcaactggt acgtggacgg 840cgtggaggtg cataatgcca
agacaaagcc gcgggaggag cagtacaaca gcacgtaccg 900ggtggtcagc
gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg
960caaggtctcc aacaaagccc tcccagcccc catcgagaaa accatctcca
aagccaaagg 1020tgggacccgt ggggtgcgag ggccacatgg acagaggccg
gctcggccca ccctctgccc 1080tgagagtgac cgctgtacca acctctgtcc
tacagggcag ccccgagaac cacaggtgta 1140caccctgccc ccatcccggg
atgagctgac caagaaccag gtcagcctga cctgcctggt 1200caaaggcttc
tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa
1260caactacaag accacgcctc ccgtgctgga ctccgacggc tccttcttcc
tctacagcaa 1320gctcaccgtg gacaagagca ggtggcagca ggggaacgtc
ttctcatgct ccgtgatgca 1380tgaggctctg cacaaccact acacgcagaa
gagcctctcc ctgtctccgg gtaaatgagt 1440gcgacggccg gcaagccccg
ctccccgggc tctcgcggtc gcacgaggat gctt 14945864DNAArtificial
SequenceSynthetic fusion gene 5atgggcagag cgatgggggc caggctaggg
ctggggttgc tgcttctggc actgctccta 60cccacgcaga tttactgcaa ccaaacatct
gttgcaccgt ttcccggtaa ccagaatatt 120tctgcttccc caaatccaag
taacgctacc accagagatc ccgaggagcc caaatcttgt 180gacaaaactc
acacatgccc accgtgccca ggcacctgaa ctcctggggg gaccgtcagt
240cttcctcttc cccccaaaac ccaaggacac cctcatgatc tcccggaccc
ctgaggtcac 300atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc
aagttcaact ggtacgtgga 360cggcgtggag gtgcataatg ccaagacaaa
gccgcgggag gagcagtaca acagcacgta 420ccgggtggtc agcgtcctca
ccgtcctgca ccaggactgg ctgaatggca aggagtacaa 480gtgcaaggtc
tccaacaaag ccctcccagc ccccatcgag aaaaccatct ccaaagccaa
540aggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga
gctgaccaag 600aaccaggtca gcctgacctg cctggtcaaa ggcttctatc
ccagcgacat cgccgtggag 660tgggagagca atgggcagcc ggagaacaac
tacaagacca cgcctcccgt gctggactcc 720gacggctcct tcttcctcta
cagcaagctc accgtggaca agagcaggtg gcagcagggg 780aacgtcttct
catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc
840ctctccctgt ctccgggtaa atga 864620DNAMus musculus 6ggaaagctta
tgggcagagc 20720DNAMus musculus 7cgagatctct ggtggtagcg 20841DNAHomo
sapiens 8cttcgaccag tctagaagca tcctcgtgcg accgcgagag c
41928DNAArtificial SequenceSynthetic primer 9ggccaagctt atgggcagag
caatggtg 281038DNAArtificial SequenceSynthetic primer 10ggccggatcc
acttacctgt cgccttggtg gtggcatt 381110DNAHomo sapiens 11acttacctgt
101212DNAHomalozoon vermiculare 12accaccaagg cg 121321PRTRattus
norvegicus 13Met Glu Val Gly Trp Tyr Arg Ser Pro Phe Ser Arg Val
Val His Leu1 5 10 15Tyr Arg Asn Gly Lys 2014287PRTArtificial
SequenceSynthetic fusion protein 14Met Gly Arg Ala Met Gly Ala Arg
Leu Gly Leu Gly Leu Leu Leu Leu1 5 10 15Ala Leu Leu Leu Pro Thr Gln
Ile Tyr Cys Asn Gln Thr Ser Val Ala 20 25 30Pro Phe Pro Gly Asn Gln
Asn Ile Ser Ala Ser Pro Asn Pro Ser Asn 35 40 45Ala Thr Thr Arg Asp
Pro Glu Glu Pro Lys Ser Cys Asp Lys Thr His 50 55 60Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val65 70 75 80Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 85 90 95Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 100 105
110Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
115 120 125Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser 130 135 140Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys145 150 155 160Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile 165 170 175Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro 180 185 190Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 195 200 205Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 210 215 220Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser225 230
235 240Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg 245 250 255Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu 260 265 270His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 275 280 2851548DNAHomo sapiens 15cagggatccc
gagggtgagt actaagctag cttcagcgct cctgcctg 481628DNAArtificial
SequenceSynthetic primer 16atccctcgag ttaagagtag agatgcag
281721DNAArtificial SequenceSynthetic oligonucleotide 17uggguaggag
cagugccagt t 211821DNAArtificial SequenceSynthetic oligonucleotide
18acccauccuc gucacgguct t 211921DNAArtificial SequenceSynthetic
oligonucleotide 19gaccgugacg aggaugggut t 212021DNAArtificial
SequenceSynthetic oligonucleotide 20cuggcacugc uccuacccat t
212128DNAArtificial SequenceSynthetic primer 21ggccggatcc
atgggcagag caatggtg 28223475DNAArtificial SequenceSynthetic
Chimeric Gene Construct 22ttcaggaatt cagaatttga aatgcggcta
agagaacaat gtggggaaaa aagagtctta 60gggcggatca gggactggag agtgtaattc
agtggtggag cgtgaatact tagtgtccct 120aagaccctgt gtttggcttc
tacctgcagc tacagagact attatctgct tgagtgttat 180tacagagtgc
aaatggagat agctgtggat ggttctgaaa tttatctgtg atttcttttt
240ccttatttat tttagattta ctagatttac tcgagtgaaa caacaactgg
aacttcaagt 300aactcctccc agagtacttc caacactggg ttggccccaa
atccaactaa tgccaccacc 360aaggcggctg gtggtgccct gcagtcaaca
gccagtctct tcgtggtctc actctctctt 420ctgcatctct actcttaaga
gactcaggcc aagaaacgtc ttctaaactt ccccatcttc 480taaacccaat
ccaaatggcg tctggaagtc caatgaagtc caatgtgatc aggaagaaac
540aggtccacct cgaattggct gttaccatat ctcaacagaa aacacggaga
attcgaaatt 600cgacgggatt aaaggacgcg tgaaaggttt gagagaagag
agatgccgct attgaatctg 660ctggagtttt acatcccaag atgaagacag
cattcagaat tgatgtgatt tccttgaatg 720tggcttagga aatgtggaca
cttaaaactc tcacttgaaa ttgggcacag gtttgatgta 780gagataagga
cggggtgcgg aatggagacc cattttgtca ttgattcatc tgaccgataa
840ggccatagtg cagttaggtg atattcgaag cttctttgat gctctttatg
tatatgttgg 900aaggaactac caggcgttgc tttaaattcc caatgtgttg
tttcgttact actaatttaa 960taccgtaagc tctaggtaaa gttccatgtt
gttgaactct gactgttctc tttggaattg 1020aacgttttgc atcctcctcc
tgtggcttta ggtctgacat tgtatttgac ctttactagt 1080aattaacatg
tgccaggcaa tggtggattg gaacccatcc ccaagtccag ccaccactga
1140ataaatctga tttcaaaagt caaacagtag acatttccca ttgtcgtttc
tcactcacca 1200caagcaccaa attcactaga gtacactggt tccagagagc
agaatcatgt tggccttggc 1260taatttcaaa atgctgtctt ttactttggt
atatgttgag ggcttttcat aatttaaagt 1320gtgttctgtt agcaaggcaa
aaattatgag tcttaattct acaggcaaat atgcaaagga 1380gccaaaactg
taaacccagc atttgggatg tgaagactgg aagctaactc tcattgaatt
1440cacaaagtct tttatacgat ttctgtacat actttttttt tttttaagag
aaaaacaaac 1500ggtggatcag aatagccacg tttggaatac tttggttatc
cattcatatt tttagatagt 1560tattggtcct gtgcctgaaa gggggcttgg
ttctaccgta agtttttcca atttccttga 1620tatacacata ccttctaaaa
cctagacatt tcctgaaaaa aatcttttgt tcgcatggtc 1680acacactgat
gcttacccgt acagtagtct tgataaccag agtcattttc tccatcttta
1740gaaaccttcc tgggaagaag gagagctcac agacccgaag ctactgtgtg
tgtgaatgaa 1800cactcccctt gcctcacacc tgaatgctgt acatctattt
gattgtaaat tgtgtttgtg 1860tatttatgct ttgattcata gtaacttctc
atgttatgga attgatttgc attgaacaca 1920aactgtaaat aaaagaaaga
aatggcggag agagcagtct gttgaattta tttacttact 1980ttttaaaaag
acttatttat tttatgtatg tgagtatatc gaagttgttt tcagacacac
2040cagaagaggg tatcagatgg ttgtgagcca ctgtgtggtt gctgggaatt
gaactcagga 2100cctctagaag ggtagccagt gctcttaacc actgagccat
ctctccagcc cccagtctgt 2160tgaatttaaa gtgtttcttg agcaataatt
atgggtgatc atggctgtta agggatatat 2220cttgttctac taactagaac
attacatgct gtctattttt gaaaggccag ctagcagcag 2280gtttggtttc
ctcccaaagc tgctcccccc cttccaagtg ctgggaataa aggcgtgtgc
2340ctccacgcct gtctctagtt gacatcttta agcttttaag gttgtacacc
tacttgctca 2400gcaactgaga gccagctgtg tgccaaggta cccatgactg
atgaagttgg ctggggagag 2460agtctttgag atgagaggtc tctggtttgc
caggcagggc tcttaggaca acaccagcag 2520ggcagggctc tgggaccaca
gattgagaac ccacaatggc cttgaacctt agacctgaat 2580gacaggtgtt
ggtgggagaa catgagcgga aattttcgtg gaatgaacag cttctaagtc
2640acctctactt tctcttaccg gcccagaggt ctacacctca ctttggtttt
ctaaattggc 2700tctcccctgc tttttccata tatcaaacac attcctggat
tcctaacatc tttactgtga 2760ttcagggacc accagaaagg gcaggctgga
aactgctgtt cttaggcaga gttccataag 2820aaacctcagg tctacccttt
aagacttaga tgatctggag ctctcttcaa tgatgtctac 2880agattgccct
ccccgctgca ccccactccg cagccatatg aagtatacta ggttggtgtg
2940ggggtaactg agaactactt attgacatgt aaactggtca ccacagttca
ttgtctcagc 3000atgttttgtc tccagatgaa caatagcctc tctctagtag
agaagtgtct tgcacacaaa 3060cagaaacatt tcccagaagt gccagtgtcg
ttcattcatc ctactttggt ttaagtgtct 3120gattgttttt tgttttggaa
actgtctcat tctgtatgta gcctaggctg gtctcaaact 3180tagggtggtc
ttacctcagt ctcctgagcc ccgggcttgt gtcgtcacac ccagcttttc
3240tgtgttttgt tttttggttt tgttttgttt tgaaacaggg ttttgctatg
tgacttaggc 3300atactatgta gcctgggctg gccttgaact catggacatc
tgcctctgtt tcctgagagc 3360tagagttaca gatgtgtgtc acttatgttc
actcttagta tcctgtgatt tatgttagat 3420attactgaaa attattacta
aatcttgtca gttgtagata cgatgggaga atgta 34752334DNAArtificial
SequenceSynthetic primer 23ggccggatcc ctcgagttaa gagtagagat gcag
3424154DNAHomo sapiens 24gaagctactg tgtgtgtgaa tgaacactct
tttgctttat tccagaatgc tgtacatcta 60ttttggattg tatattgtgt ttgtgtattt
acgctttgat tcatagtaac ttcttatgga 120attgatttgc attgaacaca
aactgtaaat aaaa 15425158DNAMus musculus 25gaagctactg tgtgtgtgaa
tgaacactcc ccttgcctca cacctgaatg ctgtacatct 60atttgattgt aaattgtgtt
tgtgtattta tgctttgatt catagtaact tctcatgtta 120tggaattgat
ttgcattgaa cacaaactgt aaataaaa 15826327DNAHomo
sapiensmodified_base(109)..(116)Variable base 26aggcaaaaat
gtaaaggagt caaaactaca aatcaagtat ttgggaagtg aagactggaa 60gctaatttgc
attaaattca caaactttta tactctttct gtatatacnn nnnnnncttt
120aaaaaacaac tatggatcag aatagccaca tttggaatac tttttgttat
cagtcaatat 180ttttagatag ttagaacctg gtcctaagcc taaaagtggg
cttgattctg cagtaaatct 240tttacaactg cctcgaaaca cagaaacctt
tttaaaaata gacactcccc gaagtctttt 300gttcgcatgg tcacacactg atgctta
32727273DNAMus musculus 27aggcaaatat gcaaaggagc caaaactgta
aacccagcat ttgggatgtg aagactggaa 60tttaagagaa aaacaaacgg tggatcagaa
tagccacgtt tggaatactt tggttatcca 120ttcatatttt tagatagtta
ttggtcctgt gcctgaaagg gggcttggtt ctaccgtaag 180tttttccaat
ttccttgata tacacatacc ttctaaaacc tagacatttc ctgaaaaaaa
240tcttttgttc gcatggtcac acactgatgc tta 2732839DNAHomo sapiens
28gctgctgctg ctggcactgc tcctacccac gcagattta 392939DNAMus musculus
29gttgctgctt ctggcactgc tcctacccac gcagattta 3930262DNAMus musculus
30ttcaggaatt cagaatttga aatgcggcta agagaacaat gtggggaaaa aagagtctta
60gggcggatca gggactggag agtgtaattc agtggtggag cgtgaatact tagtgtccct
120aagaccctgt gtttggcttc tacctgcagc tacagagact attatctgct
tgagtgttat 180tacagagtgc aaatggagat agctgtggat ggttctgaaa
tttatctgtg atttcttttt 240ccttatttat tttagattta ct 26231253DNAHomo
sapiens 31agatttactc gagtgaaaca acaactggaa cttcaagtaa ctcctcccag
agtacttcca 60acactgggtt ggccccaaat ccaactaatg ccaccaccaa ggcggctggt
ggtgccctgc 120agtcaacagc cagtctcttc gtggtctcac tctctcttct
gcatctctac tcttaagaga 180ctcaggccaa gaaacgtctt ctaaacttcc
ccatcttcta aacccaatcc aaatggcgtc 240tggaagtcca atg 253321587DNAMus
musculus 32aagtccaatg tgatcaggaa gaaacaggtc cacctcgaat tggctgttac
catatctcaa 60cagaaaacac ggagaattcg aaattcgacg ggattaaagg acgcgtgaaa
ggtttgagag 120aagagagatg ccgctattga atctgctgga gttttacatc
ccaagatgaa gacagcattc 180agaattgatg tgatttcctt gaatgtggct
taggaaatgt ggacacttaa aactctcact 240tgaaattggg cacaggtttg
atgtagagat aaggacgggg tgcggaatgg agacccattt 300tgtcattgat
tcatctgacc gataaggcca tagtgcagtt aggtgatatt cgaagcttct
360ttgatgctct ttatgtatat gttggaagga actaccaggc gttgctttaa
attcccaatg 420tgttgtttcg ttactactaa tttaataccg taagctctag
gtaaagttcc atgttgttga 480actctgactg ttctctttgg aattgaacgt
tttgcatcct cctcctgtgg ctttaggtct 540gacattgtat ttgaccttta
ctagtaatta acatgtgcca ggcaatggtg gattggaacc 600catccccaag
tccagccacc actgaataaa tctgatttca aaagtcaaac agtagacatt
660tcccattgtc gtttctcact caccacaagc accaaattca ctagagtaca
ctggttccag 720agagcagaat catgttggcc ttggctaatt tcaaaatgct
gtcttttact ttggtatatg 780ttgagggctt ttcataattt aaagtgtgtt
ctgttagcaa ggcaaaaatt atgagtctta 840attctacagg caaatatgca
aaggagccaa aactgtaaac ccagcatttg ggatgtgaag 900actggaagct
aactctcatt gaattcacaa agtcttttat acgatttctg tacatacttt
960tttttttttt aagagaaaaa caaacggtgg atcagaatag ccacgtttgg
aatactttgg 1020ttatccattc atatttttag atagttattg gtcctgtgcc
tgaaaggggg cttggttcta 1080ccgtaagttt ttccaatttc cttgatatac
acataccttc taaaacctag acatttcctg 1140aaaaaaatct tttgttcgca
tggtcacaca ctgatgctta cccgtacagt agtcttgata 1200accagagtca
ttttctccat ctttagaaac cttcctggga agaaggagag ctcacagacc
1260cgaagctact gtgtgtgtga atgaacactc cccttgcctc acacctgaat
gctgtacatc 1320tatttgattg taaattgtgt ttgtgtattt atgctttgat
tcatagtaac ttctcatgtt 1380atggaattga tttgcattga acacaaactg
taaataaaag aaagaaatgg cggagagagc 1440agtctgttga atttatttac
ttacttttta aaaagactta tttattttat gtatgtgagt 1500atatcgaagt
tgttttcaga cacaccagaa gagggtatca gatggttgtg agccactgtg
1560tggttgctgg gaattgaact caggacc 1587331373DNAMus musculus
33tctagaaggg tagccagtgc tcttaaccac tgagccatct ctccagcccc cagtctgttg
60aatttaaagt gtttcttgag caataattat gggtgatcat ggctgttaag ggatatatct
120tgttctacta actagaacat tacatgctgt ctatttttga aaggccagct
agcagcaggt 180ttggtttcct cccaaagctg ctccccccct tccaagtgct
gggaataaag gcgtgtgcct 240ccacgcctgt ctctagttga catctttaag
cttttaaggt tgtacaccta cttgctcagc 300aactgagagc cagctgtgtg
ccaaggtacc catgactgat gaagttggct ggggagagag 360tctttgagat
gagaggtctc tggtttgcca ggcagggctc ttaggacaac accagcaggg
420cagggctctg ggaccacaga ttgagaaccc acaatggcct tgaaccttag
acctgaatga 480caggtgttgg tgggagaaca tgagcggaaa ttttcgtgga
atgaacagct tctaagtcac 540ctctactttc tcttaccggc ccagaggtct
acacctcact
ttggttttct aaattggctc 600tcccctgctt tttccatata tcaaacacat
tcctggattc ctaacatctt tactgtgatt 660cagggaccac cagaaagggc
aggctggaaa ctgctgttct taggcagagt tccataagaa 720acctcaggtc
taccctttaa gacttagatg atctggagct ctcttcaatg atgtctacag
780attgccctcc ccgctgcacc ccactccgca gccatatgaa gtatactagg
ttggtgtggg 840ggtaactgag aactacttat tgacatgtaa actggtcacc
acagttcatt gtctcagcat 900gttttgtctc cagatgaaca atagcctctc
tctagtagag aagtgtcttg cacacaaaca 960gaaacatttc ccagaagtgc
cagtgtcgtt cattcatcct actttggttt aagtgtctga 1020ttgttttttg
ttttggaaac tgtctcattc tgtatgtagc ctaggctggt ctcaaactta
1080gggtggtctt acctcagtct cctgagcccc gggcttgtgt cgtcacaccc
agcttttctg 1140tgttttgttt tttggttttg ttttgttttg aaacagggtt
ttgctatgtg acttaggcat 1200actatgtagc ctgggctggc cttgaactca
tggacatctg cctctgtttc ctgagagcta 1260gagttacaga tgtgtgtcac
ttatgttcac tcttagtatc ctgtgattta tgttagatat 1320tactgaaaat
tattactaaa tcttgtcagt tgtagatacg atgggagaat gta 1373
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