U.S. patent application number 15/437968 was filed with the patent office on 2017-08-17 for methods of use of soluble cd24 for therapy of rheumatoid arthritis.
The applicant listed for this patent is OncoImmune, Inc.. Invention is credited to Yang Liu, Wei Wu, Pan Zheng, Xincheng Zheng.
Application Number | 20170233453 15/437968 |
Document ID | / |
Family ID | 49043199 |
Filed Date | 2017-08-17 |
United States Patent
Application |
20170233453 |
Kind Code |
A1 |
Zheng; Xincheng ; et
al. |
August 17, 2017 |
METHODS OF USE OF SOLUBLE CD24 FOR THERAPY OF RHEUMATOID
ARTHRITIS
Abstract
Provided herein is a CD24 protein. The CD24 protein may include
mature human or mouse CD24, as well as a N- or C-terminally fused
portion of a mammalian immunoglobulin.
Inventors: |
Zheng; Xincheng; (Ann Arbor,
MI) ; Wu; Wei; (Washington, DC) ; Liu;
Yang; (Washington, DC) ; Zheng; Pan;
(Washington, DC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
OncoImmune, Inc. |
Rockville |
MD |
US |
|
|
Family ID: |
49043199 |
Appl. No.: |
15/437968 |
Filed: |
February 21, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14519745 |
Oct 21, 2014 |
9611309 |
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15437968 |
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14055609 |
Oct 16, 2013 |
8895022 |
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14519745 |
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13892705 |
May 13, 2013 |
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14055609 |
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13643527 |
Feb 12, 2013 |
8808697 |
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PCT/US2011/034282 |
Apr 28, 2011 |
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13892705 |
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61329078 |
Apr 28, 2010 |
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Current U.S.
Class: |
530/387.3 |
Current CPC
Class: |
C07K 14/70596 20130101;
C07K 2319/30 20130101; C07K 19/00 20130101; C07K 14/435 20130101;
C07K 14/47 20130101 |
International
Class: |
C07K 14/705 20060101
C07K014/705 |
Claims
1.-10. (canceled)
11. A method of repressing danger-associated molecular pattern
(DAMP)-mediated tissue damage in a subject undergoing hematopoietic
stem cell transplantation, comprising administering to a subject in
need thereof a CD24 protein comprising a mature human CD24
polypeptide, wherein the mature human CD24 polypeptide comprises
the amino acid sequence set forth in SEQ ID NO: 1, and wherein the
CD24 protein does not comprise an alanine or valine immediately
C-terminal to SEQ ID NO: 1.
12. The method of claim 11, wherein the subject has graft versus
host disease.
13. The method of claim 11, wherein the CD24 protein further
comprises a protein tag, wherein the protein tag is fused to the
C-terminus of the mature human CD24 or at the N-terminus of the
CD24 protein.
14. The method of claim 13, wherein the protein tag comprises a Fc
portion of a mammalian Ig protein.
15. The method of claim 14, wherein the Fc portion comprises (a) a
hinge region and CH2 and CH3 domains of the mammalian Ig protein,
wherein the Ig protein is human and wherein the Ig is selected from
the group consisting of IgG1, IgG2, IgG3, IgG4, and IgA; or (b) a
hinge region and CH3 and CH4 domains of IgM.
16. The method of claim 11, wherein the CD24 protein is
soluble.
17. The method of claim 11, wherein the CD24 protein is
glycosylated.
18. The method of claim 11, wherein the CD24 protein is produced
using a eukaryotic protein expression system.
19. The method of claim 18, wherein the expression system comprises
a vector contained in a Chinese Hamster Ovary cell line or a
replication-defective retroviral vector.
20. The method of claim 19, wherein the replication-defective
retroviral vector is stably integrated into the genome of a
eukaryotic cell.
21. The method of claim 11, wherein the CD24 protein consists of
the amino acid sequence set forth in SEQ ID NO: 1 fused at its
C-terminus to a hinge region and CH2 and CH3 domains of human IgG1
Fc.
22. The method of claim 21, wherein the hinge region and CH2 and
CH3 domains of human IgG1 Fc consist of the amino acid sequence set
forth in SEQ ID NO: 6.
Description
FIELD OF THE INVENTION
[0001] This invention relates to compositions and methods for
treating rheumatoid arthritis.
BACKGROUND OF THE INVENTION
[0002] This section provides background information which is not
necessarily prior art and a general summary of the present
disclosure which is not a comprehensive disclosure of its full
scope or all of its features.
[0003] CD24 is known as the heat-stable antigen. It is expressed as
a glycosyl-phosphatidyl-inositol (GPI)-anchored molecule and has a
wide distribution in different lineages. Because of the tendency of
CD24 to be expressed on immature cells, it has also been used as
part of stem cell markers and for lymphocyte differentiation. The
first function associated with CD24 is a costimulatory activity for
antigen-specific T cell response. In vivo studies indicated that,
as a costimulator for T cell activation in the lymphoid organ, CD24
is redundant but becomes essential in the absence of CD28. This
would not be the case for local target organs that are not as
"costimulator rich." Consistent with this notion, it has been
demonstrated that mice with a targeted mutation of CD24 are
completely resistant to induction of experimental autoimmune
encephalomyelitis (EAE).
[0004] Polymorphisms of human CD24 are associated with risk and
progression of several autoimmune diseases, including multiple
sclerosis and rheumatoid arthritis (RA). In cases of multiple
sclerosis, it has been reported that soluble CD24, consisting of
the extracellular portion of murine CD24 and human IgG1 Fc
ameliorated the clinical symptom of experimental autoimmune
diseases, the mouse model of multiple sclerosis. More recent
studies have demonstrated that CD24 interact with and represses
host response to danger-associated molecular patterns (DAMPs).
[0005] RA affects 0.5-1% of human populations. Although a number of
disease-modifying antirheumatic drugs (DMARDs) are currently
available, even the gold standard of biologic DMARDs, the
therapeutics targeting the tumor-necrosis factor alpha, lead to 50%
improvement according to American College of Rheumatology
Improvement Criteria (ACR50) in less than 50% of the patients
receiving the treatments. No cure for RA is available. It is
therefore necessary to test additional therapeutics for RA. RA is
presumed to be autoimmune diseases in the joint, although the cause
of the diseases remains largely obscure. A number of studies have
implicated T cells in the pathogenesis of rheumatoid arthritis.
More recently, it has been demonstrated that transfer of antibodies
can cause the development of inflammation of the joints of mice.
The pathology of the lesions resembles human rheumatoid
arthritis.
[0006] Animal models relevant to human RA played an important role
for the advancement of therapeutic development in DMARDs. For
example, collagen-induced arthritis in the mouse and rat were
critical for the development of therapeutics for RA. More recently,
it has been demonstrated that adaptive transfer of anti-collagen
antibodies cause robust RA-like lesion in the mice. Since
auto-antibodies are elevated in RA patients prior to the onset of
diseases, passive transfer of collagen-specific antibody is a
relevant model for human RA.
[0007] Since the pathogenesis of RA involves host response to DAMP
and since the CD24 molecule negatively regulate host response to
DAMPs, the potential of using soluble CD24 to treat RA was
investigated. The passive transfer model of RA was chosen because
of both relevance to human diseases and simplicity of experimental
designs.
SUMMARY OF THE INVENTION
[0008] Provided herein is a CD24 protein comprising a mature human
CD24 variant consisting of SEQ ID NO: 1. The CD24 protein may
further comprise a portion of a mammalian immunoglobulin (Ig),
which may be fused to the N-terminus or C-terminus of the mature
CD24. The Ig portion may be the Fc portion of a human Ig protein.
The Fc portion may consist of the hinge region and CH2 and CH3
domains of the human Ig protein, and the Ig may be IgG1, IgG2,
IgG3, IgG4, or IgA. The Fc portion may consist of the hinge region
and CH3 and CH4 regions of IgM.
[0009] The CD24 protein may be soluble, and may be glycosylated.
The CD24 protein may also be produced using a eukaryotic protein
expression system, which may comprise a vector contained in a
Chinese Hamster Ovary cell line or a replication-defective
retroviral vector. The replication-defective retroviral vector may
be stably integrated into the genome of a eukaryotic cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIGS. 1A-B. FIG. 1A shows the amino acid composition of the
CD24 fusion protein, CD24IgG1Fc (also referred to herein as CD24Fc)
(SEQ ID NO: 5). The underlined 26 amino acids are the signal
peptide of CD24 (SEQ ID NO: 4). The boxed, bold portion of the
sequence is the mature CD24 protein used in the fusion protein (SEQ
ID NO: 1). The last amino acid (A or V) that is ordinarily present
in the mature CD24 protein has been deleted from the construct to
avoid immunogenicity. The non-underlined, non-bold letters are the
sequence of IgG1 Fc, including the hinge region and CH1 and CH2
domains (SEQ ID NO: 6). FIG. 1B shows the sequence of CD24.sup.VFc
(SEQ ID NO: 7), in which the mature human CD24 protein is the
valine polymorphic variant of SEQ ID NO: 2. The various parts of
the fusion protein are marked as in FIG. 1A.
[0011] FIG. 2. Methods for purification and processing of
CD24IgG1Fc (CD24Fc) expressed from mammalian cell lines.
[0012] FIG. 3. Amino acid sequence variations between mature CD24
proteins from mouse (SEQ ID NO: 3) and human (SEQ ID NO: 2). The
potential glycosylation sites are bolded, with the N-glycosylation
sites in red.
[0013] FIGS. 4A-C. WinNonlin compartmental modeling analysis of
pharmacokenitics of CD24IgG1 (CD24Fc). The opened circles represent
the average of 3 mice, and the line is the predicted
pharmacokinetic curve. FIG. 4A. i.v. injection of 1 mg CD24IgG1.
FIG. 4B. s.c. injection of 1 mg CD24IgG1 (CD24Fc). FIG. 4C.
Comparison of the total amounts of antibody in the blood as
measured by areas under curve (AUC), half-life and maximal blood
concentration. Note that overall, the AUC and Cmax of the s.c.
injection is about 80% of i.v. injection, although the difference
is not statistically significant.
[0014] FIGS. 5A-B. CD24-Siglec G (10) interaction discriminates
between PAMP and DAMP. FIG. 5A. Host response to PAMP was
unaffected by CD24-Siglec G(10) interaction. FIG. 5B. CD24-Siglec G
(10) interaction represses host response to DAMP, possibly through
the Siglec G/10-associated SHP-1.
[0015] FIGS. 6A-B. A single injection of CD24Fc reduces clinical
score of CAIA. FIG. 6A. Diagram of experiments. BALB/c mice (8
weeks old) received mAbs on day 1 in conjunction with either
vehicle or fusion proteins. The mice were injected LPS on day 3,
and were observed daily for 3 weeks. FIG. 6B. CD24Fc reduces
clinical scores of CAIA. The fusion proteins (1 mg/mouse) or
vehicles were injected once on day 1. Clinical scores were
determined double blind. *, P<0.05; **, P<0.01; ***,
P<0.001. The effect of CD24 was reproduced in 6 independent
experiments, involving a total of 52 mice in the PBS group and 54
mice in CD24Fc group.
[0016] FIGS. 7A-B. CD24Fc reduces the levels of inflammatory
cytokines in the joint and CAIA. CAIA initiated and treated as
diagramed in FIG. 6A. The inflammatory cytokines were measured by
cytokine bead array from BD Pharmingen. FIG. 7A. Representative
FACS profile. FIG. 7B. The summary of reduced cytokines
(Mean.+-.SE) measured in the joint homogenates.
[0017] FIG. 8. CD24Fc reduces inflammation and destruction of
cartilage in the joint. On day 7, front and hind paws were
dissected from both CD24Fc treated and control mice, fixed in 4%
paraformaldehyde for 24 hours followed by decalcification with 5%
formic acid. The paws were then embedded in paraffin and the
longitudinal section were stained with H&E and Safranin O red
(Sigma-Aldrich).
[0018] FIG. 9. Therapeutic effect of CD24Fc administrated on day 5
of CAIA induction. The CAIA-induced mice were randomized into two
groups, receiving either vehicle (PBS) or CD24 Fc. The mice were
scored double blind. Representative of three independent
experiments are shown.
[0019] FIGS. 10A-B. Low doses of CD24Fc prevent development of
CAIA. FIG. 10A. Diagram of experiments. FIG. 10B. Clinical scores
of arthritis, scored double blind.
[0020] FIGS. 11A-B. Siglecg is essential for therapeutic effect of
CD24Fc, WT (FIG. 11A) and Siglecg.sup.-/- mice (FIG. 11B) received
either vehicle control or CD24Fc in conjunction of a cocktail of
anti-collagen mAbs. The clinical scores were recorded daily double
blind.
[0021] FIGS. 12A-F. Construction of CD24.sup.vFc and CD24Fc. FIG.
12A. Diagram of the fusion proteins. The polymorphic residue in
extracellular domain was deleted in CD24Fc. FIG. 12B. SDS-PAGE
analysis for the purity of the two fusion proteins. The numbers
shown are .mu.g of proteins loaded. FIG. 12C. Comparison between
CD24.sup.vFc and CD24Fc for their binding to Siglec10Fc.
Desialylated CD24Fc was used as a negative control. FIG. 12D.
Comparison between CD24Fc and CD24.sup.vFc for the therapeutic
effect in the CAIA model. CD24Fc or CD24.sup.vFc (200 .mu.g/mouse)
was injected into mice in conjunction with a cocktail of
anti-collagen antibodies on day 1. Arthritis was elicited by
treatment with LPS on day 3. The diseases were scored double blind.
Data shown in FIGS. 12C and D are means and SEM. FIGS. 12E and 12F
also compare the therapeutic effects of CD24Fc and CD24.sup.VFc, in
experiments performed similarly to the ones shown in FIG. 12D,
except that IgG1 Fc was used as a negative control. As shown in
FIG. 12E, CD24Fc reduced the RA score as early as day 4, and showed
statistically significant protection throughout the three weeks of
observation. On the other hand, as shown in FIG. 12F, CD24.sup.VFc
showed a reduction in RA score starting on day 8. Although reduced
scores were observed thereafter, the reduction did not reach
statistical significance.
[0022] FIGS. 13A-B. CD24Fc conferred protection against CIA in
DBA/1 mice. FIG. 13A. CD24Fc suppressed development of arthritis in
the CIA model. Mice received a single treatment (1 mg/mouse) on day
17 when no clinical symptoms had developed. Data shown are disease
scores among the mice that had developed arthritis with disease
scores from 3 to 8. The difference between CD24Fc and PBS group was
significant (P=0.02, Fisher's PLSD test). N=9 for vehicle and N=7
for CD24Fc group. FIG. 13B. Therapeutic effect of CD24Fc in CIA of
DBA/1 mice. The mice with clinical symptoms of scores from 3 to 8
were randomized to receive either 200 .mu.g CD24Fc or an equal
volume of control vehicle (PBS) by i.p. injection, every the other
day, five times. The mice were inspected daily to score for the
clinical symptoms for two weeks. CD24Fc significantly lessened the
clinical symptoms of arthritis when arthritis developed (P=0.02,
Fisher's PLSD test). N=6 for PBS and N=5 for CD24Fc groups.
[0023] FIGS. 14A-B. CD24Fc caused rapid recovery in mice with
ongoing chicken CIA. On day 1, 8-week old C57BL/6 mice were
immunized with 100 .mu.L of collagen-CFA emulsion (made by mixing 4
mg/ml of chick type II collagen with equal volume of CFA containing
5 mg/ml of M. tuberculosis) intradermally at the base of the tail.
On day 21, booster immunization with the same collagen-CFA emulsion
was administered intradermally 1.5 cm from the tail base. FIG. 14A.
On day 28, mice with a clinical score >3 were randomized to
receive either vehicle or CD24Fc (1 mg/mouse). The endpoint was a
reduction of score by 50% (top) or 80% (bottom). N=12 for PBS, and
N=11 for CD24Fc. FIG. 14B. Dose-dependent therapeutic effect of
CD24Fc in chicken CIA model. Details as in FIG. 14A, except that
the treatments started at the peak of disease (average score of 5.5
in both groups on day 33). Mice with a clinical score >3 were
randomized to receive 5 injections of either vehicle or CD24Fc. The
endpoint was a reduction of score by either 50% (top) or 80%
(bottom). N=11. The difference between the 100 .mu.g experimental
and the vehicle control groups was statistically significant.
[0024] FIGS. 15A-C. CD24 inhibited inflammatory cytokine production
by human macrophages. FIG. 15A. ShRNA silencing of CD24 led to
spontaneous production of TNF.alpha., IL-6 and IL-1.beta.. THP1
cells were transduced with lentiviral vectors encoding either
scrambled or two independent CD24 shRNA. The transduced cells were
differentiated into macrophages by culturing for 4 days with PMA
(15 ng/ml). After washing away PMA and nonadherent cells, the cells
were cultured for another 24 hours for measurement of inflammatory
cytokines by cytokine beads array. FIG. 15B. As in FIG. 15A, except
that the given concentration of CD24Fc or control IgG Fc was added
to macrophages in the last 24 hours. FIG. 15C. CD24Fc was more
efficient than CD24.sup.vFc in suppressing the spontaneous
production of inflammatory cytokines by CD24-silenced macrophage
cell line THP1. The data shown are as detailed in the FIG. 12
legends, except that the CD24Fc and CD24.sup.vFc are compared
side-by-side.
[0025] FIGS. 16A-C. Contribution of Siglec G to protection by
CD24Fc. FIG. 16A. CD24Fc stimulated tyrosine phosphorylation of,
and SHP-1 binding to, Siglec G. Spleen cells from CD24-deficient
mice were stimulated with either vehicle, Fc control or CD24Fc (1
.mu.g/ml) for 30 min. After lysis, the Siglec G protein was
precipitated with anti-Siglec G antisera. Siglec G phosphorylation
and its association to SHP-1 were detected by Western blot. FIG.
16B. Siglecg was essential for therapeutic effect of CD24Fc in mice
with low dose of anti-collagen antibodies. WT (FIG. 16A) and
Siglece.sup.-/- mice (FIG. 16B) received either vehicle control or
CD24Fc in conjunction of a cocktail of anti-collagen mAbs (2
mg/mouse). LPS was injected on day 3 (100 .mu.g/mouse). The
clinical scores were recorded daily double blind. Data are
representative of two experiments. FIG. 16C. Targeted mutation of
Siglecg attenuated but did not abrogate the therapeutic effect of
CD24Fc with double doses of anti-collagen antibodies. The
anti-collagen antibodies (4 mg/mouse) and CD24Fc (1 mg/mouse) were
added on day 1, while LPS (100 .mu.g/mouse) was added on day 3.
Male WT (FIG. 16A) and Siglece.sup.-/- mice (FIG. 16B) were
observed daily for clinical score. % inhibitions were calculated by
% reduction of accumulated RA score. N=5. Male mice were used at 8
weeks of age.
[0026] FIGS. 17A-B. FIGS. 17A and B show pharmacokinetic profiles
of CD24 in male and female mice at doses of 12.5, 35, and 125
mg/kg.
[0027] FIGS. 18A and B show CD24Fc serum concentrations vs. time in
cynomolgus monkeys (12.5 mg/kg dose).
DETAILED DESCRIPTION
[0028] The inventors have discovered that a soluble form of CD24 is
highly effective for treating rheumatoid arthritis. In particular,
the inventors have discovered that a variant CD24 fusion protein in
which the core of human CD24 lacks the polymorphic amino acid at
position 57 of full-length CD24 has a superior therapeutic effect
when compared with a CD24 protein which has a wild-type core CD24
sequence.
1. Definitions
[0029] The terminology used herein is for the purpose of describing
particular embodiments only and is not intended to be limiting. As
used in the specification and the appended claims, the singular
forms "a," "an" and "the" include plural referents unless the
context clearly dictates otherwise.
[0030] For recitation of numeric ranges herein, each intervening
number there between with the same degree of precision is
explicitly contemplated. For example, for the range of 6-9, the
numbers 7 and 8 are contemplated in addition to 6 and 9, and for
the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,
6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
[0031] A "peptide" or "polypeptide" is a linked sequence of amino
acids and may be natural, synthetic, or a modification or
combination of natural and synthetic.
[0032] "Substantially identical" may mean that a first and second
amino acid sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,
230, 240, 250, 260, 270, 280, 290, or 300 amino acids.
[0033] "Treatment" or "treating," when referring to protection of
an animal from a disease, means preventing, suppressing,
repressing, or completely eliminating the disease. Preventing the
disease involves administering a composition of the present
invention to an animal prior to onset of the disease. Suppressing
the disease involves administering a composition of the present
invention to an animal after induction of the disease but before
its clinical appearance. Repressing the disease involves
administering a composition of the present invention to an animal
after clinical appearance of the disease.
[0034] A "variant" may mean means a peptide or polypeptide that
differs in amino acid sequence by the insertion, deletion, or
conservative substitution of amino acids, but retain at least one
biological activity. Representative examples of "biological
activity" include the ability to bind to a toll-like receptor and
to be bound by a specific antibody. Variant may also mean a protein
with an amino acid sequence that is substantially identical to a
referenced protein with an amino acid sequence that retains at
least one biological activity. A conservative substitution of an
amino acid, i.e., replacing an amino acid with a different amino
acid of similar properties (e.g., hydrophilicity, degree and
distribution of charged regions) is recognized in the art as
typically involving a minor change. These minor changes can be
identified, in part, by considering the hydropathic index of amino
acids, as understood in the art. Kyte et al., J. Mol. Biol.
157:105-132 (1982). The hydropathic index of an amino acid is based
on a consideration of its hydrophobicity and charge. It is known in
the art that amino acids of similar hydropathic indexes can be
substituted and still retain protein function. In one aspect, amino
acids having hydropathic indexes of .+-.2 are substituted. The
hydrophilicity of amino acids can also be used to reveal
substitutions that would result in proteins retaining biological
function. A consideration of the hydrophilicity of amino acids in
the context of a peptide permits calculation of the greatest local
average hydrophilicity of that peptide, a useful measure that has
been reported to correlate well with antigenicity and
immunogenicity. U.S. Pat. No. 4,554,101, incorporated fully herein
by reference. Substitution of amino acids having similar
hydrophilicity values can result in peptides retaining biological
activity, for example immunogenicity, as is understood in the art.
Substitutions may be performed with amino acids having
hydrophilicity values within .+-.2 of each other. Both the
hyrophobicity index and the hydrophilicity value of amino acids are
influenced by the particular side chain of that amino acid.
Consistent with that observation, amino acid substitutions that are
compatible with biological function are understood to depend on the
relative similarity of the amino acids, and particularly the side
chains of those amino acids, as revealed by the hydrophobicity,
hydrophilicity, charge, size, and other properties.
2. CD24
[0035] Provided herein is a CD24 protein, which may have the amino
sequence of mature human CD24, which may be
SETTTGTSSNSSQSTSNSGLAPNPTNATTK (SEQ ID NO: 1) or
SETTTGTSSNSSQSTSNSGLAPNPTNATTK(V/A) (SEQ ID NO: 2), or mouse CD24,
which may be NQTSVAPFPGNQNISASPNPTNATTRG (SEQ ID NO: 3), or a
variant thereof. The CD24 may be soluble. The CD24 may further
comprise a N-terminal signal peptide, which may have the amino acid
sequence MGRAMVARLGLGLLLLALLLPTQIYS (SEQ ID NO: 4). The CD24 may
also have an amino acid sequence described in FIG. 1 or 3. The CD24
may exist in one of two allelic forms, such that the C-terminal
amino acid of the mature human CD24 may be a valine or an alanine.
The C-terminal valine or alanine may be immunogenic and may be
omitted from the CD24 to reduce its immunogenicity. The difference
between the two alleles may affect the risk of autoimmune diseases,
including multiple sclerosis and RA. Nevertheless, since the two
allelic forms affect the expression levels of membrane-bounded
form, the variation should not affect the function of CD24.
[0036] Despite considerable sequence variations in the amino acid
sequence of the mature CD24 proteins from mouse and human, they are
functionally equivalents in interaction with the danger-associated
molecular patterns (DAMP). Since host response to DAMP is
considered important for the pathogenesis of RA, the mouse and
human CD24 may be functionally equivalent in treating RA. As a
result of sequence conservation between mouse and human CD24
primarily in the C-terminus and in the abundance of glycosylation
sites, significant variations in the mature CD24 proteins may be
tolerated in using the CD24 to treat RA, especially if those
variations do not affect the conserved residues in the C-terminus
or do not affect the glycosylation sites from either mouse or human
CD24.
[0037] a. Fusion
[0038] The CD24 may be fused at its N- or C-terminal end to a
portion of a mammalian Ig protein, which may be human or mouse. The
portion may be a Fc region of the Ig protein. The Fc region may
comprise the hinge region and CH2 and CH3 domains of the Ig
protein. The Ig protein may be human IgG1, IgG2, IgG3, IgG4, IgM,
or IgA. The Fc portion may comprise SEQ ID NO: 6. The Ig protein
may also be IgM, and the Fc portion may comprise the hinge region
and CH3 and CH4 domains of IgM. The CD24 may also be fused at its
N- or C-terminus to a protein tag, which may be GST, His, or FLAG.
Methods for making fusion proteins and purifying fusion proteins
are well known in the art.
[0039] b. Production
[0040] The CD24 may be heavily glycosylated, and may be involved in
functions of CD24 such as costimulation and interaction with
danger-associated molecular patterns. The CD24 may be prepared
using a eukaryotic expression system. The expression system may
entail expression from a vector in mammalian cells, such as Chinese
Hamster Ovary (CHO) cells. The system may also be a viral vector,
such as a replication-defective retroviral vector that may be used
to infect eukaryotic cells. The CD24 may also be produced from a
stable cell line that expresses CD24 from a vector or a portion of
a vector that has been integrated into the cellular genome. The
stable cell line may express CD24 from an integrated
replication-defective retroviral vector. The expression system may
be GPEx.TM..
3. Method of Treatment
[0041] The CD24 may be used to treat rheumatoid arthritis. The CD24
may be administered to a subject in need thereof. The subject may
be a mammal such as a human.
[0042] a. Combined CD24 Therapy
[0043] The CD24 may be combined with another drug, such as a
disease-modifying antirheumatic drug (DMARD). The drug may be a
nonsteriod anti-inflammatory drug (NSAID), which may be a propionic
acid derivative, an acetic acid derivative, an enolic acid
derivative, a fenamic acid derivative, or a selective Cox2
inhibitor. The drug may also be a corticosteroid or Methotrexate.
The drug may be a biologic, which may be a TNF-.alpha. antagonist
such as an anti-TNF-.alpha. antibody or a fusion protein that binds
to TNF-.alpha. (Enbrel), an anti-CD20 mAb, an antagonist of
costimulatory molecule CD80 and CD86 such as a monoclonal antibody
or a fusion protein (CTLA4Ig) that binds to the two molecules, or
an antagonist for a receptor of either IL-1 or IL-6. The CD24 and
the other drug may be administrated together or sequentially.
[0044] b. Pharmaceutical Composition
[0045] The CD24 may be contained in a pharmaceutical composition,
which may comprise a solvent, which may keep the CD24 stable over
an extended period. The solvent may be PBS, which may keep the CD24
stable for at least 36 months at -20.degree. C.
(-15.about.-25.degree. C.). The solvent may be capable of
accommodating the CD24 in combination with the other drug.
[0046] c. Dosage
[0047] The dose to be used for human may ultimately be determined
through a clinical trial to determine a dose with acceptable
toxicity and clinical efficacy. The initial clinical dose for human
may be estimated through pharmacokinetics and toxicity studies in
rodents and non-human primates. The dose of CD24 may be 0.01 mg/kg
to 1000 mg/Kg, and may be 1 to 500 mg/kg, depending on the severity
of disease being treated and the route of administration.
[0048] d. Administration
[0049] The route of administration of the pharmaceutical
composition may be parenteral. Parenteral administration includes,
but is not limited to, intravenous, intraarterial, intraperitoneal,
subcutaneous, intramuscular, intrathecal, intraarticular and direct
injection into affected joints. For veterinary use, the agent may
be administered as a suitably acceptable formulation in accordance
with normal veterinary practice. The veterinarian can readily
determine the dosing regimen and route of administration that is
most appropriate for a particular animal. The pharmaceutical
composition may be administered to a human patient, cat, dog, large
animal, or an avian.
[0050] The CD24 may be administered simultaneously or
metronomically with other treatments. The term "simultaneous" or
"simultaneously" as used herein, means that the CD24 and other
treatment be administered within 48 hours, preferably 24 hours,
more preferably 12 hours, yet more preferably 6 hours, and most
preferably 3 hours or less, of each other. The term
"metronomically" as used herein means the administration of the
agent at times different from the other treatment and at a certain
frequency relative to repeat administration.
[0051] The CD24 may be administered at any point prior to another
treatment including about 120 hr, 118 hr, 116 hr, 114 hr, 112 hr,
110 hr, 108 hr, 106 hr, 104 hr, 102 hr, 100 hr, 98 hr, 96 hr, 94
hr, 92 hr, 90 hr, 88 hr, 86 hr, 84 hr, 82 hr, 80 hr, 78 hr, 76 hr,
74 hr, 72 hr, 70 hr, 68 hr, 66 hr, 64 hr, 62 hr, 60 hr, 58 hr, 56
hr, 54 hr, 52 hr, 50 hr, 48 hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 hr,
36 hr, 34 hr, 32 hr, 30 hr, 28 hr, 26 hr, 24 hr, 22 hr, 20 hr, 18
hr, 16 hr, 14 hr, 12 hr, 10 hr, 8 hr, 6 hr, 4 hr, 3 hr, 2 hr, 1 hr,
55 mins., 50 mins., 45 mins., 40 mins., 35 mins., 30 mins., 25
mins., 20 mins., 15 mins, 10 mins, 9 mins, 8 mins, 7 mins., 6
mins., 5 mins., 4 mins., 3 mins, 2 mins, and 1 mins. The CD24 may
be administered at any point prior to a second treatment of the
CD24 including about 120 hr, 118 hr, 116 hr, 114 hr, 112 hr, 110
hr, 108 hr, 106 hr, 104 hr, 102 hr, 100 hr, 98 hr, 96 hr, 94 hr, 92
hr, 90 hr, 88 hr, 86 hr, 84 hr, 82 hr, 80 hr, 78 hr, 76 hr, 74 hr,
72 hr, 70 hr, 68 hr, 66 hr, 64 hr, 62 hr, 60 hr, 58 hr, 56 hr, 54
hr, 52 hr, 50 hr, 48 hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 hr, 36 hr,
34 hr, 32 hr, 30 hr, 28 hr, 26 hr, 24 hr, 22 hr, 20 hr, 18 hr, 16
hr, 14 hr, 12 hr, 10 hr, 8 hr, 6 hr, 4 hr, 3 hr, 2 hr, 1 hr, 55
mins., 50 mins., 45 mins., 40 mins., 35 mins., 30 mins., 25 mins.,
20 mins., 15 mins., 10 mins., 9 mins., 8 mins., 7 mins., 6 mins., 5
mins., 4 mins., 3 mins, 2 mins, and 1 mins.
[0052] The CD24 may be administered at any point after another
treatment including about 1 min, 2 mins., 3 mins., 4 mins., 5
mins., 6 mins., 7 mins., 8 mins., 9 mins., 10 mins., 15 mins., 20
mins., 25 mins., 30 mins., 35 mins., 40 mins., 45 mins., 50 mins.,
55 mins., 1 hr, 2 hr, 3 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, 14 hr,
16 hr, 18 hr, 20 hr, 22 hr, 24 hr, 26 hr, 28 hr, 30 hr, 32 hr, 34
hr, 36 hr, 38 hr, 40 hr, 42 hr, 44 hr, 46 hr, 48 hr, 50 hr, 52 hr,
54 hr, 56 hr, 58 hr, 60 hr, 62 hr, 64 hr, 66 hr, 68 hr, 70 hr, 72
hr, 74 hr, 76 hr, 78 hr, 80 hr, 82 hr, 84 hr, 86 hr, 88 hr, 90 hr,
92 hr, 94 hr, 96 hr, 98 hr, 100 hr, 102 hr, 104 hr, 106 hr, 108 hr,
110 hr, 112 hr, 114 hr, 116 hr, 118 hr, and 120 hr. The CD24 may be
administered at any point prior after a previous CD24 treatment
including about 120 hr, 118 hr, 116 hr, 114 hr, 112 hr, 110 hr, 108
hr, 106 hr, 104 hr, 102 hr, 100 hr, 98 hr, 96 hr, 94 hr, 92 hr, 90
hr, 88 hr, 86 hr, 84 hr, 82 hr, 80 hr, 78 hr, 76 hr, 74 hr, 72 hr,
70 hr, 68 hr, 66 hr, 64 hr, 62 hr, 60 hr, 58 hr, 56 hr, 54 hr, 52
hr, 50 hr, 48 hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 hr, 36 hr, 34 hr,
32 hr, 30 hr, 28 hr, 26 hr, 24 hr, 22 hr, 20 hr, 18 hr, 16 hr, 14
hr, 12 hr, 10 hr, 8 hr, 6 hr, 4 hr, 3 hr, 2 hr, 1 hr, 55 mins., 50
mins., 45 mins., 40 mins., 35 mins., 30 mins., 25 mins., 20 mins.,
15 mins., 10 mins., 9 mins., 8 mins., 7 mins., 6 mins., 5 mins., 4
mins., 3 mins, 2 mins, and 1 mins.
[0053] The following examples are provided to illustrate the
methods of the invention and are by no means to limit the use of
the methods.
Example 1
Soluble CD24 Proteins
[0054] The extracellular domain of CD24 was fused to IgG1 Fc. The
amino acid composition of the CD24 fusion protein is provided in
FIG. 1. A replication-defective retroviral vector that drives
expression of the CD24Ig fusion protein was then generated. The
GPEx.TM. (an acronym for gene product expression) system offers
several important advantages, the most important of which is the,
on average, >1000 insertions/cell but with only 1
copy/insertion. Moreover, since the retrovirus preferentially
inserts into the transcriptional active locus, the GPEx.TM.
resulted in a high level of expression of the targeted protein.
Stable cell lines that produce a high yield of CD24Ig were
generated. In addition 45 grams of GLP grade products and
.about.100 grams of cGMP grade products were produced. The methods
used for downstream processing of media harvested from the
bioreactor are summarized in the flow chart below (FIG. 2).
Harvest Clarification
[0055] The bioreactor culture media was clarified using Cuno 60M02
Maximizer depth filters followed by a Millipore Opticap 0.22 um
filter. The filtrate was collected into a sterile collection bag.
Samples were obtained for CD24-Fc yield quantitation by ELISA.
Protein A Capture
[0056] The clarified media was passed over a column of Protein A
resin (GE Healthcare MabSelect) at a concentration not exceeding 16
g/L of resin (based on ELISA) and a contact time of 4 minutes. The
column was washed with the equilibration buffer (50 mM Tris+0.15M
NaCl pH7.5), then with 10 mM sodium citrate/citric acid pH 6.0 for
5cvs. Bound CD24Ig was eluted from the column using 10 mM sodium
citrate/citric acid pH 3.5
Viral Inactivation
[0057] The Protein A eluate fraction was immediately brought to pH
3.0 with the addition of 2M Hydrochloric acid and held at this pH
for 30 minutes at ambient temperature. It was then brought to pH
5.0 with the addition of 1M Tris base, and filtered to clarity
using a 0.65 um glass fiber filter (Sartorius Sartopure GF2) and
0.2 um (Sartorius Sartopore 2) into a sterile collection bag.
SP-Sepharose Chromatography
[0058] The viral inactivated material was applied to a column of
SP-Sepharose (GE Healthcare) at a concentration not exceeding 25
g/L of resin (based on A280 nm of 1.22=1 mg/mL) and a linear flow
rate of 250 cm/hr. The column was washed with the equilibration
buffer (10 mM sodium citrate/citric acid pH 5.0) and bound CD24Ig
was eluted from the column using 10 mM sodium citrate/citric
acid+0.2M NaCl pH5.0. The effluent was collected into a sterile
collection bag.
Mustang Q Chromatography
[0059] The SP-Sepharose elute was adjusted to pH 7.5 by the
addition of 1M Tris base and diluted with WFI to reduce the
conductivity. The diluted material was applied to a Mustang Q
filter (Pall) at a concentration not exceeding 0.5 g/L of resin
(based on A280 nm of 1.22=1 mg/mL) and at a flow rate of 5 column
volumes/minute. The filter was washed with the equilibration buffer
(10 mM Tris pH 7.5) and the CD24-Fc is contained in the flow
through and is collected into a sterile collection bag.
Viral Filtration
[0060] The Mustang Q flow through was then filtered at a constant
pressure of 30 psi through a 0.2 mM filter and a Millipore NFP
viral filter (nominal pore size 20 nm) and was collected into a
sterile collection bag.
Concentration and Final Formulation
[0061] The product was concentrated and diafiltered using a 10 kDa
ultrafiltration membrane (Millipore Prep/Scale) into a 10 mM sodium
phosphate, 150 mM sodium chloride pH 7.2 at approximately 10 mg/mL
final concentration as determined by absorbance at 280 nm.
Analytical samples were drawn from the bulk whilst in a biosafety
cabinet. Labeling was performed and the samples were delivered to
QC for testing while the bulk aliquots were stored at 2-8.degree.
C. pending release.
Viral Clearance Studies
[0062] The viral clearance validation was performed at Cardinal
Health, NC, on samples prepared at CHM. Qualified scientists from
Gala Biotech performed the chromatography and filtration steps in
the Cardinal Health Viral Validation facility with the assistance
of Cardinal Health personnel. The scale down procedure was
developed from the 200 L scale process. Two viruses were chosen to
be used in this study. The first was Xenotropic murine Leukemia
virus (XMuLv), which is an enveloped RNA virus of 80-130 nm in size
from the Retroviridae viral family. The second was Porcine
Parvovirus (PPV), which is a nonenveloped DNA virus of 18-26 nm in
size. This is considered a robust virus, and was expected to
demonstrate a much lower viral reduction through the purification
protocol than the XMuLv.
Example 2
CD24 Pharmacokinetics
[0063] 1 mg of CD24IgG1 (CD24Fc) was injected into naive C57BL/6
mice and collected blood samples at different timepoints (5 min, 1
hr, 4 hrs, 24 hrs, 48 hrs, 7 days, 14 days and 21 days) with 3 mice
in each timepoint. The sera were diluted 1:100 and the levels of
CD24Ig was detected using a sandwich ELISA using purified
anti-human CD24 (3.3 .mu.g/ml) as the capturing antibody and
peroxidase conjugated goat anti-human IgG Fc (5 .mu.g/ml) as the
detecting antibodies. As shown in FIG. 4a. The decay curve of
CD24Ig revealed a typical biphase decay of the protein. The first
biodistribution phase had a half life of 12.4 hours. The second
phase follows a model of first-order elimination from the central
compartment. The half life for the second phase was 9.54 days,
which is similar to that of antibodies in vivo. These data suggest
that the fusion protein is very stable in the blood stream. In
another study in which the fusion protein was injected
subcutaneously, an almost identical half life of 9.52 days was
observed (FIG. 4b). More importantly, while it took approximately
48 hours for the CD24Ig to reach peak levels in the blood, the
total amount of the fusion protein in the blood, as measured by
AUC, was substantially the same by either route of injection. Thus,
from therapeutic point of view, different route of injection should
not affect the therapeutic effect of the drug. This observation
greatly simplified the experimental design for primate toxicity and
clinical trials.
Example 3
CD24 for Treating RA
[0064] For decades, it has been assumed that RA is predominantly a
T-cell mediated autoimmune diseases. In the last two decades, there
is a reawaking on the possible role for antibodies and B
lymphocytes in RA pathogenesis. Thus, in addition or rheumatoid
factors, a host of autoreactive antibodies have been found in RA
patients, although it has not been definitively addressed in human.
However, several lines of evidence have demonstrated that in the
mouse models, antibodies specific for either ubiquitous or tissue
specific antigens are sufficient to cause RA symptoms. For
instance, antibodies from the K/B.times.N TCR transgenic mice were
found to be fully capable of transferring RA-like diseases in the
new host. Likewise, a cocktail for 4 anti-collagen antibodies is
now widely used to induce RA in the mouse. This model is now called
CAIA, for collagen antibody-induced arthritis.
[0065] Genetic analyses of CAIA model indicate critical roles for
complement. Although other possibilities exist, these requirements
suggest potential involvement of antibody-mediated tissue damage in
the pathogenesis of RA. The linkage between tissue damage and
inflammation is a long-standing observation in immunology. Nearly
two decades ago, Matzinger proposed what was popularly called
danger theory. In essence, she argued that the immune system is
turned on when it senses the dangers in the host. Although the
nature of danger was not well defined at the time, it has been
determined that necrosis is associated with the release of
intracellular components such as HMGB1 and Heat-shock proteins,
which were called DAMP, for danger-associated molecular patterns.
DAMP were found to promote production of inflammatory cytokines and
autoimmune diseases. In animal models, inhibitors of HMGB1 and
HSP90 were found to ameliorate RA. The involvement of DAMP raised
the prospect that negative regulation for host response to DAMP can
be explored for RA therapy.
CD24-Siglec 10 Interaction in Host Response to Tissue Injuries
[0066] Using acetaminophen-induced liver necrosis and ensuring
inflammation, we observed that through interaction Siglec G, CD24
provides a powerful negative regulation for host response to tissue
injuries. CD24 is a GPI anchored molecules that is broadly
expressed in hematopoietic cells and other tissue stem cells.
Genetic analysis of a variety of autoimmune disease in human,
including multiple sclerosis, systemic lupus erythromatosus, RA,
and giant cell arthritis, showed significant association between
CD24 polymorphism and risk of autoimmune diseases. Siglec G is a
member of I-lectin family, defined by their ability to recognize
sialic acid containing structure. Siglec G recognized sialic acid
containing structure on CD24 and negatively regulates production of
inflammatory cytokines by dendritic cells. In terms of its ability
to interact with CD24, human Siglec 10 and mouse Siglec G are
functionally equivalent. However, it is unclear if there is a
one-to-one correlation between mouse and human homologues. Although
the mechanism remains to be full elucidated, it is plausible that
SiglecG-associated SHP1 may be involved in the negative regulation.
These data, reported in Science recently, leads to a new model in
which CD24-Siglec G/10 interaction may play a critical in
discrimination pathogen-associated molecular pattern (PAMP) from
DAMP (FIG. 5).
[0067] At least two overlapping mechanisms may explain the function
of CD24. First, by binding to a variety of DAMP, CD24 may trap the
inflammatory stimuli to prevent their interaction with TLR or RAGE.
This notion is supported by observations that CD24 is associated
with several DAMP molecules, including HSP70, 90, HMGB1 and
nucleolin. Second, perhaps after associated with DAMP, CD24 may
stimulate signaling by Siglec G. Both mechanisms may act in concert
as mice with targeted mutation of either gene mounted much stronger
inflammatory response. In fact, DC cultured from bone marrow from
either CD24-/- or Siglec G-/- mice produced much higher
inflammatory cytokines when stimulated with either HMGB1, HSP70, or
HSP90. In contrast, no effect were found in their response to PAMP,
such as LPS and PolyI:C. These data not only provided a mechanism
for the innate immune system to distinguish pathogen from tissue
injury, but also suggest that CD24 and Siglec G as potential
therapeutic targets for diseases associated with tissue
injuries.
Therapeutic Effect of CD24Fc on Collagen-Antibody-Induced
Arthritis
[0068] Given the suspected role for innate immunity to tissue
injury in the pathogenesis of RA and the role for CD24-Siglec G/10
pathway in negatively regulate such response, the possibility of
stimulating this pathway to treat RA was explored. Pathogenesis of
essentially all autoimmune diseases involves induction of immune
response to autoantigen and autoimmune destruction. The autoimmune
destructive phase was focused on, based the novel function of
CD24-Siglec G interaction. Therefore, for the preliminary analysis,
collagen antibody-induced arthritis model was adopted to evaluate
potential therapeutic effect.
[0069] As shown in FIG. 6a, the CAIA was induced on 8 weeks old
BALB/c mice by i.v. injection of a cocktail of 4 anti-collagen mAbs
(MD Biosciences, St. Paul, Minn.) at 2 mg/mouse on day 1, and i.p.
injection of 100 .mu.g/mouse of LPS (MD Bioscience) on day 3. The
mice were treated on day 1 with either 1 mg CD24Fc or equal volume
of 1.times.PBS vehicle as negative control. As shown in FIG. 6b, in
comparison with vehicle control, CD24Fc provided highly significant
therapeutic effects.
[0070] To understand the mechanism by which CD24Fc reduces
arthritis in this model, cytokines were measured from homogenized
joints of CD24Fc treated mice or PBS control group, and measured
the supernatant of 200 .mu.g tissue homogenates by cytokine beads
array. A typical example is shown in FIG. 8a, while the summary
data are shown in FIG. 7b. These data demonstrated that
systematically administrated CD24 reduces the levels of multiple
inflammatory cytokines including TNF-.alpha., IL-6, MCP-1(CCL2) and
IL-1.beta..
[0071] The effect of CD24Fc is substantiated by histological
analysis of the synovial joints of CAIA mice, as presented in FIG.
8. On day 7 after induction of arthritis, H&E staining
demonstrated that the joint synoviums in the PBS group are heavily
infiltrated with inflammatory cells including neutrophil,
macrophage, and lymphocytes (FIG. 8a). This was much reduced in the
CD24Fc treated mice (FIG. 8b). In addition, sever cartilage damages
were revealed by the loss of safranin O red staining in PBS-treated
(FIG. 8c) mice, but not CD24Fc-treated group (FIG. 8d).
[0072] To determine whether mice, CD24Fc have therapeutic effect on
ongoing RA, treatment was started at either 5 or 7 days after
induction of RA. As shown in FIG. 9, significant reduction of RA
score was observed as soon as two days after CD24Fc treatment. The
therapeutic effect lasted for the remaining period of observation
even without additional treatment. These data further strengthen
the therapeutic potential of CD24Fc on ongoing diseases.
[0073] In order to estimate the therapeutic doses of CD24Fc in
human, CD24Fc was titrated through a wide range of doses. As shown
in FIG. 10, as little as 2 microgram/mice is sufficient to have
statistically significant therapeutic effect.
Siglecg-Dependent Therapeutic Effect of CD24Fc
[0074] To determine whether CD24Fc protect mice by interacting with
Siglec G, we determined if the therapeutic effect depends on the
Siglecg gene. Since the Siglecg-deficient mice were produced with
ES cells from C57BL/6 mice, we used WT C57BL/6 mice as control. As
shown in FIG. 11a, since the B6 mice are known to be less
susceptible to the CAIA, the overall disease score is lower than
that observed in the BALB/c mice. Nevertheless, a single injection
of the CD24Fc essentially wiped out the clinical signs in the WT
mice. Importantly, even though the disease is less severe in the
Siglecg-deficient mice, CD24Fc had no therapeutic effect.
Therefore, the therapeutic effect of CD24Fc is strictly dependent
on the Siglecg gene.
[0075] Taken together, the data described herein demonstrates high
therapeutic efficacy of CD24Fc for CAIA. Given our extensive data
on safety, stability and our successful manufacture of CD24Fc all
point to great potential of the fusion protein as a therapeutic for
RA.
Example 4
Toxicity and Pharmacokinetics of CD24
[0076] Toxicity
[0077] A study of CD24Fc in mice was performed in order to evaluate
the toxicity and systemic exposure of CD24Fc when intravenously
administered to mice once weekly for four weeks, and the
reversibility of toxicity following a four-week recovery period. A
total of 320 male and female mice were assigned to Groups 1-4 for
evaluation of the toxicity (20/sex/group) and reversibility
(20/sex/group). All of these mice were treated intravenously with
either PBS buffer or CD24Fc at doses of 12.5, 35, or 125 mg/kg once
weekly for 4 weeks. A total of 360 mice were assigned to Groups 5-8
for evaluation of the systemic exposure of CD24Fc following the
first dose (3/sex in Group 5 and 27/sex/group in Groups 6-8) and
the last dose (6/sex in Group 5 and 30/sex/group in Groups 6-8).
All of these mice were treated intravenously with either PBS buffer
or CD24Fc at doses of 12.5, 35, or 125 mg/kg once on Day 1 or once
weekly for 4 weeks.
[0078] No morbidity or mortality was found in mice treated with
either PBS buffer or CD24Fc, with the exception that one male mouse
treated with 35 mg/kg of CD24Fc in Group 7 was found dead on Day
21. This death occurred in 1/680 of mice and did not appear to be
associated with the treatment. No treatment-related changes were
noted in clinical observations, body weights, food consumption,
ophthalmology, hematology, coagulation, clinical chemistry, organ
weights, and organ-to-body or to-brain weight ratios. No
treatment-related macroscopic and microscopic findings were
observed in all examined animals.
[0079] Anti-CD24Fc antibody was detected in 10/30 of mice on Day 30
and 12/30 of mice on Day 57, which appeared to be dose dependent
with no difference between male and female mice. A higher systemic
exposure following the first dose and last dose was observed in
male animals compared to female animals. An approximately linear
increase in AUC values was observed when mice were treated with
increasing doses of CD24Fc from 12.5 mg/kg to 125 mg/kg. An
accumulation of CD24Fc in mice was observed after dosing once
weekly for four weeks.
[0080] In summary, no observed adverse effects were noted in mice
treated with CD24Fc at doses of 12.5, 35 or 125 mg/kg once weekly
for 4 weeks with a 4 week recovery period. The NOAEL was considered
to be equal to or greater than 125 mg/kg.
[0081] Another study of CD24Fc was performed in cynomolgus monkeys
to examine the pharmacokinetics and potential toxicity, acquired
immunity, and immunogenicity following administration of CD24Fc
administered by intravenous infusion once weekly for four weeks,
and to evaluate recovery from any effects of the test article over
a dose-free period of at least 10 weeks. The test article, CD24Fc
(Lot No. FP004.12-06097-001), and the control article, 1.times.
phosphate-buffered saline (PBS), pH 7.2 (Lot No. 654446), were
supplied as a clear, colorless, preformulated aqueous solution and
as a preformulated aqueous solution, respectively. The test article
formulation was used as received for dose administration.
[0082] Forty experimentally naive cynomolgus monkeys (20 males and
20 females), 2.9 to 4.7 years of age for the males and 2.9 to 5.0
years of age for the females, and weighing 2.1 to 3.2 kg for the
males and 2.2 to 2.9 kg for the females at the outset (Day -1) of
the study, were assigned to dose groups as shown in Table 2
below.
[0083] All animals were dosed via 1-hour intravenous infusion once
weekly for 4 consecutive weeks. The first day of dosing was
designated Day 1 (04 and 5 Sep. 2009, Sets A and B, respectively).
The animals were evaluated for changes in clinical signs
(evaluations of morbidity and/or mortality [twice daily] and cage
side observations [once daily], body weight (Weeks -2 and -1, and
weekly thereafter), electrocardiography (prestudy and Day 28),
ophthalmology (prestudy and Day 28), and clinical pathology indices
(including serum chemistry, hematology, and coagulation (three
prestudy and Days 28 and 98), and urinalysis [via cystocentesis on
Days 28 and 98]). Blood samples were collected for toxicokinetic
analysis predose, 15 minutes, and 6, 24, and 72 hours in relation
to dose administration on Days 1 and 22, predose on Day 8, and on
Day 29 (at the same time of day as Day 22 end of infusion). Blood
samples for flow cytometry were collected prestudy (three time
points), and on Days 28 and 98. Blood samples for anti-drug
antibodies (ADA) were collected prestudy and on Days 28 and 98. To
evaluate the T-cell dependent antibody response (TDAR), the animals
were immunized with a 1:1 emulsion of keyhole limpet hemocyanin
(KLH) and Incomplete Freund's Adjuvant (IFA) injected
intramuscularly on the thigh to achieve a dose of 750 mg/animal on
Day 10, and to recovery animals on Day 84. Blood samples for
anti-KLH response were collected prestudy, and on Days 20, 24, 28,
94, and 98. Twenty four (24) animals (3/sex/group) were euthanized
one week after the last dose. The remaining 16 animals
(2/sex/group) were continued on the study without further dosing,
and euthanized on Day 98 (76 days after the last dose). At
termination, a full necropsy was conducted on all animals, and
tissues were collected, preserved, processed, and examined
microscopically by a Study Pathologist certified by the American
College of Veterinary Pathologists (ACVP). At recovery, a full
necropsy was conducted on all animals, and tissues were collected,
preserved, processed, and selected tissues (based on clinical
pathology and gross observations) were examined microscopically by
a study pathologist certified by the ACVP.
[0084] The results showed that CD24Fc administered via 1-hour
intravenous infusion once weekly for 4 consecutive weeks at 12.5
mg/kg/week (Group 2), 35 mg/kg/week (Group 3), or 125 mg/kg/week
(Group 4) was generally well tolerated in male and female
cynomolgus monkeys through a 4-week dosing period, followed by a 10
week recovery period. There were no CD24Fc-related changes in
clinical observations, food consumption, body weights,
electrocardiographic measurements, ophthalmic examinations, serum
chemistry (one exception noted below), coagulation, urinalysis
parameters, macroscopic findings, or organ weights or organ weight
ratios. There were no CD24Fc-related changes in peripheral blood
mononuclear cell subset counts obtained by flow cytometry, or
anti-KLH immunoglobulin G (IgG) responses to KLH challenge. There
was a possible approximately 2-fold increase in ALP related to
CD24Fc for one male administered 35 mg/kg/week at Day 98 compared
to predose and Day 28 values. There was also a possible
CD24Fc-related slight decrease in hemoglobin at Day 28 for 35
mg/kg/week males. The magnitude and/or nature of these changes was
not considered adverse in this study. On Day 28, one male
administered 35 mg/kg/week had an approximate 13% decrease in
hemoglobin concentration. On Day 98 (76 days after the last dose),
changes in hematology for this animal included a 21% decrease in
hemoglobin, a 16% increase in red blood cell counts, a 21% decrease
in mean cell volume (MCV), a 32% decrease in mean cell hemoglobin
(MCH), a 14% decrease in mean cell hemoglobin concentration (MCHC),
a 24% increase in red cell distribution width (RDW), and an
approximately 2-fold increase in platelet and reticulocyte counts
compared to prestudy (Day -2) levels. Day 98 blood smear analysis
documented hypochromic red blood cells, microcytosis, anisocytosis,
schistocytes, poikilocytes, and spherocytes, as well as an apparent
increase in platelet numbers. These hematology changes are not
considered typical findings in cynomolgus monkeys. However, no
corresponding histopathology abnormalities were identified, and
these hematology findings were not observed in any other animals in
the mid-dose (35 mg/kg/week) or high-dose (125 mg/kg/week)
groups.
[0085] At Day 29 terminal necropsy, there were microscopic findings
of fibrin thrombi in hepatic sinusoids and multifocal glial nodules
in the brain of one female administered 125 mg/kg/week. The changes
were mild and occurred only in one animal, but they were not
typical background findings. More extensive independent studies
established that the lesion was also observed in placebo samples
and therefore was not drug-related. At recovery necropsy,
histopathologic analysis of the following subset of tissues was
performed based on clinical pathology or gross findings: all
tissues except for mammary glands from a male administered 35
mg/kg/week, the duodenum from another male administered 35
mg/kg/week, and testes from one male administered 125 mg/kg/week.
No microscopic findings were found related to the administration of
CD24Fc.
[0086] CD24Fc was detected in serum samples from all dosed animals.
No measurable amount of CD24Fc was detected in control or predose
serum samples. Incurred sample reanalysis (ISR) was performed, and
the results met the acceptance criteria. Serum was screened for the
presence of anti-CD24Fc antibody. Seven animals out of the 40 total
were identified as positive at the prestudy time point (17.5%).
This rate of positive samples prior to test article administration
is similar to the rate observed in the validation (12.5%) which
demonstrated a subpopulation of animals with pre-existing
anti-CD24Fc antibodies. Three animals were identified as positive
during the dosing period of the study. The validation experiment
also demonstrated that 10 .mu.g/mL of CD24Fc inhibited the
measurement of anti-CD24Fc antibodies in reference to the positive
control, and all dose groups at the Day 29 toxicokinetic time point
had concentrations greater than 10 .mu.g/mL measured in serum. Only
one animal (125 mg/kg/week dose group) screened positive for
anti-CD24Fc antibodies within the recovery interval.
[0087] Based on the hematology findings, the
no-observed-adverse-effect level (NOAEL) for CD24Fc was considered
to be 12.5 mg/kg/week under the conditions of this study when
administered once weekly for four weeks.
[0088] Pharmacokinetics of CD24 in Mice and Monkeys
[0089] The pharmacokinetics of CD24Fc were examined in the mouse
and cynomolgus monkeys. These animal PK studies were carried out by
two preclinical contract research organizations. The mouse study
was performed by JOINN Laboratory at Beijing, China, while the
cynomolgus monkey study was carried out by Charles River Laboratory
at Reno, Nev., USA. Both studies were in compliance with the Good
Laboratory Protocol (GLP) and the final reports were audited. As an
independent report, the WinNonLin analysis of pharmacokinetic
characterizations of CD24Fc in this report were performed by either
JOINN Laboratory at Beijing, China (PK in mouse) or OncoImmune Inc.
(PK in cynomolgus monkey).
[0090] In the mouse PK study, CD24Fc was intravenously administered
to mice once weekly for four weeks. A total of 360 mice were
assigned to Groups 1-4 for evaluation of the systemic exposure of
CD24Fc following the first dose (3/sex in Group 1 and 27/sex/group
in Groups 2-4) and the last dose (6/sex in Group 1 and 30/sex/group
in Groups 2-4.). All of these mice were treated intravenously with
either PBS buffer or CD24Fc at doses of 12.5, 35, or 125 mg/kg once
weekly for 4 weeks. Blood samples were collected at each time point
from 3 mice/sex/group in Groups 1-4 via orbital vein for
determination of the serum concentrations of CD24Fc. For Group 1,
blood samples were collected prior to dosing on Days 1 and 29 and
at 4 hours after dosing on Day 29. For Groups 2-4, blood samples
were collected at 5 and 15 minutes; and 1, 4, 8, 24, 48, 72, and
168 hours following dosing on Days 1 and 29; and 366 hours
following dosing on Day 29.
[0091] The summarized results of the mouse serum concentration of
CD24Fc following the first dose and last dose are included in Table
1, and the WinNonlin analysis of serum CD24Fc concentration vs.
time points is included in FIG. 17.
TABLE-US-00001 TABLE 1 Pharmacokinetic parameters of CD24Fc in mice
Female Male unit 125 mg/kg 35 mg/kg 12.5 mg/kg 125 mg/kg 35 mg/kg
12.5 mg/kg First dose parameters T.sub.1/2 h 46.36 59.18 63.93
48.51 70.57 63.00 C.sub.max mg/ml 3.17 0.66 0.32 2.13 0.99 0.54
AUC.sub.(0-168 h) h * mg/ml 39.04 8.65 5.85 44.00 19.99 7.55
AUC.sub.inf h * mg/ml 43.53 11.84 8.00 49.10 26.13 10.18 Vd ml/kg
192.06 252.33 144.09 178.18 136.39 111.55 Cl ml/h/kg 2.87 2.96 1.56
2.55 1.34 1.23 MRT h 39.08 67.85 56.68 39.58 61.09 66.21 C.sub.max
ratio 10.04 2.09 1.00 3.92 1.83 1.00 AUC ratio 5.44 1.48 1.00 4.82
2.57 1.00 Last dose parameters t.sub.1/2 h 87.51 131.86 128.63
75.02 167.27 58.90 C.sub.max mg/ml 3.61 0.40 0.14 5.07 0.76 0.65
AUC.sub.(0-336 h) h * mg/ml 177.00 21.35 7.68 251.18 88.20 35.75
AUC.sub.inf h * mg/ml 187.95 26.41 9.07 258.93 112.49 36.36 Vd
ml/kg 83.96 252.16 255.74 52.25 75.08 29.22 Cl ml/h/kg 0.67 1.33
1.38 0.48 0.31 0.34 MRT h 91.64 121.30 106.00 77.98 106.29 70.30
C.sub.max ratio 25.09 2.81 1.00 7.86 1.17 1.00 AUC ratio 20.72 2.91
1.00 7.12 3.09 1.00 Faccu 4.32 2.23 1.13 5.27 4.31 3.57
[0092] The summarized results of the monkey serum concentrations of
CD24Fc following the single dose are included in Table 2, and the
WinNonlin analysis of serum CD24Fc concentration vs. time points is
included in FIG. 18.
TABLE-US-00002 TABLE 2 Pharmacokinetic parameters of CD24Fc in
cynomolgus monkeys PK Parameters unit 35 mg/kg 12.5 mg/kg t1/2 h
148.39 188.29 Cmax mg/ml 0.89 0.25 AUC (0-t) h * mg/ml 67.09 17.02
AUCinf h * mg/ml 67.31 20.06 Vd ml/kg 108.21 153.10 Cl ml/h/kg 0.52
0.62 MRT h 204.22 151.07 Cmax ratio 3.56 1.00 AUC ratio 3.36
1.00
Example 5
A Variant CD24 has Improved Activity Over Wild-Type CD24
[0093] This example shows that a CD24 polypeptide containing human
CD24 missing the polymorphic amino acid at position 57 (SEQ ID NO:
1) (CD24Fc) is more effective for treating RA than a CD24
polypeptide containing wild-type CD24 (SEQ ID NO: 2)
(CD24.sup.VFc).
[0094] Methods
[0095] Antibodies, Fusion Proteins and Other Materials
[0096] CD24Fc and CD24vFc were manufactured by OncoImmune, Inc.;
Bovine type II collagen, Catalog No. 20022, Chondrex Inc., Redmond,
Wash.; a cocktail of 4 anti-collagen mAbs Catolog No. CIA-MAB-50
for BALB/c and CIA-MAB-2C for C57BL/6 mice, MD Bioproducts, St.
Paul, Minn.; Chick type II collagen, Catalog No. 20011, Chondrex
Inc., Redmond, Wash.; Complete Freund's adjuvant: Catalog No. 7008,
Chondrex Inc., Redmond, Wash., with heat-killed M. tuberculosis H37
Ra (non-viable) at concentration of 1 mg/ml; Complete Freund's
adjuvant: Catalog No. 7023, Chondrex Inc., Redmond, Wash., with
heat-killed M. tuberculosis H37 Ra (non-viable) at concentration of
5 mg/ml; Incomplete Freund's adjuvant: Catalog No. 7002, Chondrex
Inc., Redmond, Wash.; Lipopolysaccharide (LPS), Catalog No. 9028,
Chondrex Inc., Redmond, Wash., from E. coli 0111:B4; Cytometric
Bead Array (CBA) Mouse Inflammation Kit, Catalog No. 552364, BD
Biosciences, San Jose, Calif.; Cytometric Bead Array (CBA) Human
Inflammatory Cytokines Kit, Catalog No. 551811, BD Biosciences, San
Jose, Calif.
[0097] Experimental Animals
[0098] BALB/cAnNCr (01B05, NCI) and C57BL/6NCr (01055, NCI) mice,
male, 7 weeks old, were purchased from the National Cancer
Institute (NCI) at Frederick, Md. DBA/1J (000670, JAX) mice, male,
7 weeks old, were received from Jackson Laboratories. All mice were
quarantined for 7 days prior to immunization. During quarantine,
the animals were examined for general health and acceptability for
use in this study. Individual animals were identified by ear mark.
Animal cages were identified by study number, animal number, and
group number. To minimize cage variation, different treatments were
given to individual mouse in the same cages and scored in a
double-blind protocol.
[0099] CAIA Model
[0100] BALB/c mice (8 weeks old) received mAbs (2 mg/mouse) on day
1 in conjunction with either vehicle or fusion proteins. Mice
received LPS (100 .mu.g/mouse) on day 3, and were observed daily
for 3 weeks. The fusion proteins (0.2 or 1 mg/mouse) or vehicles
were injected once on day 1. In the C57BL/6 mice, the dose of
anti-collagen antibodies was either 2 mg/mouse or 4 mg/mouse.
[0101] CIA Models
[0102] CIA in DBA/1 mice. On day 1, 8-week old DBA/1 mice were
immunized with 100 .mu.L of collagen-CFA emulsion (made by mixing 2
mg/ml of bovine type II collagen with equal volume of CFA
containing 1 mg/ml of M. tuberculosis) subcutaneously at the base
of the tail. On day 10, mice were booster-immunized with 60 .mu.L
of collagen-IFA emulsion (made by mixing 2 mg/ml of collagen with
equal volume of IFA) subcutaneously 1.5 cm from the tail base.
Treatments were initiated either before or after the development of
symptom of arthritis.
[0103] CIA in C57BL/6 mice. On day 1, 8-week old C57BL/6 mice were
immunized with 100 .mu.L of collagen-CFA emulsion (made by mixing 4
mg/ml of chick type II collagen with equal volume of CFA containing
5 mg/ml of M. tuberculosis) intradermally at the base of the tail.
On day 21, booster immunization with the same collagen-CFA emulsion
was administered intradermally 1.5 cm from the tail base. On day
28, mice with clinical symptoms were randomized to receive either
vehicle or CD24Fc.
[0104] Treatment in RA Models
[0105] The scoring of arthritis was based on the following
scale.
[0106] 0, normal; 1, mild, but definite redness and swelling of the
ankle or wrist, or apparent redness and swelling limited to
individual digits, regardless of the number of affected digits; 2,
moderate redness and swelling of ankle of wrist; 3, severe redness
and swelling of the entire paw including digits; 4, maximally
inflamed limb with involvement of multiple joints. A combined score
of 4 limbs in a mouse was reported as the disease score for the
mouse.
[0107] Prophylactic treatment in CAIA was initiated at the same
time as the anti-collagen antibodies.
[0108] Prophylactic model in CIA model in DBA/1 mice: On day 17,
the immunized mice were randomly divided into two groups and were
treated with vehicle (PBS) or given doses of CD24Fc. The mice were
observed double blind for three weeks.
[0109] Therapeutic CIA in DBA/1 model with ongoing diseases were
initiated at either 25 days after the first immunization using mice
with clinical scores from 3 to 8.
[0110] Therapeutic CIA in the C57BL/6 mice was initiated on day 28
or using only those mice with a clinical score from 3 to 8.
[0111] Statistical Methods
[0112] Group means and standard deviation values (when deemed
appropriate) were calculated for all numerical data obtained. The
difference between CD24Fc and control mice was statistically
analyzed with Fisher PLSD Test, or t-test for pairwise
comparisons.
[0113] Results
[0114] Therapeutic Effect of Non-Polymorphic CD24Fc on
Collage-Antibody-Induced Arthritis
[0115] Human mature CD24 exists in two allelic forms, in which
either a valine or alanine is present at the C-terminus (position
57 of the CD24 amino acid sequence). Fusion proteins including
either allelic form may provoke anti-drug antibodies in some RA
patients. Thus, in order to avoid immunogenicity, it was tested
whether the polymorphic residue can be removed from CD24, while
maintaining regulatory function of CD24. As diagrammed in FIG. 12A,
two fusion proteins were created, one with the entire extracellular
domain of CD24.sup.V allele (CD24.sup.VFc) (the mature CD24
sequence having SEQ ID NO: 2), while the other had a one amino acid
deletion at the C-terminus of the resulting mature CD24 (CD24Fc)
(the mature CD24 sequence having SEQ ID NO: 1). Both forms were
expressed and purified to a similar degree (FIG. 12B).
[0116] To determine whether the valine on CD24 is required for
CD24-Siglec 10 interaction, their binding of CD24Fc and
CD24.sup.VFc to a Siglec 10Fc fusion protein was compared. As shown
in FIG. 12C, CD24Fc interacted with Siglec 10Fc in a dose-dependent
manner. The interaction depended on sialic acid on CD24Fc as
pre-treatment of CD24Fc with sialidase prevented the binding.
Surprisingly, while CD24.sup.VFc also interacted with Siglec 10Fc,
the interaction was significantly weaker than that of
CD24-Fc-Siglec 10Fc. The CAIA was induced in 8 week-old BALB/c mice
by i.v. injection of a cocktail of 4 anti-collagen mAbs in
conjunction with CD24.sup.VFc, CD24Fc, human IgG1Fc or equal volume
of 1.times.PBS vehicle as negative control. As shown in FIG. 12D,
in comparison with vehicle control, CD24Fc provided highly
significant therapeutic effects. Surprisingly, CD24.sup.VFc was far
less effective, with activity not very different from the negative
control. In experiments similar to the ones shown in FIG. 12D,
FIGS. 12E and 12F also show that CD24Fc is more effective than
CD24.sup.VFc, although unlike in the experiments in FIG. 12D,
CD24.sup.VFc did have more activity than the negative control.
Comparisons of the therapeutic effects of CD24Fc and CD24.sup.VFC
indicate that deleting the polymorphic amino acid residue is not
only likely to remove a potential issue of immunogenicity, but it
also significantly increases the anti-inflammatory activity of the
CD24 protein. Since the Fc portion was identical in the two
constructs, the therapeutic effect is largely attributable to CD24
function. Since Fc protein often exacerbated arthritis (data not
shown), vehicle controls were used for in vivo studies to avoid
inducing a confounding effect.
[0117] Therapeutic Effect of CD24Fc in Collagen-Induced Arthritis
(CIA) Models
[0118] Since CAIA primarily reflects joint inflammation initiated
by antibody-induced tissue injuries, and since RA involves both
adaptive and innate immune-mediated destruction that can be better
reflected in CIA setting, two CIA models were used to study to
potential therapeutic effect of the CD24Fc. First, the prophylactic
effect of CD24Fc were tested in the DBA/1 mouse. As shown in FIG.
13A, treatment with a single dose of CD24Fc prior to the
development of clinical symptoms substantially reduced subsequent
disease scores (P=0.02). To determine whether CD24Fc confers
therapeutic effect for ongoing CIA in the DBA/1 mice, the treatment
was initiated when the mice had arthritis scores from 3 to 8. The
CD24Fc (200 .mu.g/mouse) or vehicle was delivered every other day
for 5 times. As shown in FIG. 13B, a clear reduction of arthritis
score was observed as early as after two treatments. Significant
reduction of clinical symptoms was observed in the CD24Fc group
(P=0.02).
[0119] It has been reported that chicken collagen induces severe
arthritis in C57BL/6 background. Therefore, this model was used to
substantiate the therapeutic effect of CD24Fc. Since a significant
variation in disease score was observed within the same group, 50%
and 80% reductions of disease scores were used as the endpoints of
the study. The percentage of mice that reached either therapeutic
endpoint over a three week period was compared. As shown in FIG.
14A, CD24Fc accelerated recovery of mice after severe clinical
signs had developed. To test the therapeutic effect at the peak of
diseases, another week was allowed to pass for mice to reach peak
clinical score, at which point mice were treated with repeated
injections of either 100 or 50 .mu.g/mouse (once every other day
for a total of 5 injections). As shown in FIG. 14B, a transient
increase of recovery was achieved with 50 .mu.g/mouse/injection.
However, a sustained recovery was achieved with only 100
.mu.g/mouse/injection. These data demonstrate a dose-dependent
therapeutic effect even when the drug was administrated at the peak
of diseases.
[0120] CD24Fc Inhibits Production of Inflammatory Cytokines by a
Human Macrophage Cell Line with shRNA Silencing of CD24
[0121] To determine whether CD24 regulates production of
inflammatory cytokines in human cell line, CD24 was silenced in
human THP1 cell line and then differentiation into macrophage was
induced by treating the cells with PMA. As shown in FIG. 15A, CD24
silencing substantially increased production of TNF.alpha.,
IL-1.beta., and IL-6. These data demonstrate an essential role for
endogenous human CD24 in production of inflammatory cytokines.
Importantly, CD24Fc strongly inhibited production of TNF.alpha., as
well as IL-1.beta. and IL-6 (FIG. 15B). Consistent with the
therapeutic effect in vivo, CD24.sup.VFc was approximately 10-fold
less effective in inhibiting the production of inflammatory
cytokines in the macrophage cell line (FIG. 15C).
[0122] CD24Fc Confers Protection by Signaling Through Siglec G
[0123] It has been reported that CD24Fc interacts with Siglec G in
mice and Siglec 10 in humans. To determine whether CD24Fc signals
through Siglec G, spleen cells from CD24.sup.-/- mice were
incubated with either vehicle, Fc or CD24Fc for 30 min, and
tyrosine phosphorylation and SHP-1 binding to Siglec G were
measured. As shown in FIG. 16A, CD24Fc strongly stimulated tyrosine
phosphorylation of Siglec G. Correspondingly, the amount of SHP1
co-precipitated with Siglec G was dramatically increased. These
results demonstrate that CD24Fc is capable of signaling through
Siglec G.
[0124] To test the significance of CD24Fc signaling through Siglec
G in therapy of RA, WT mice and Siglec G-deficient mice were
compared for their response to CD24Fc. As shown in FIG. 16B, when
lower doses of anti-collagen antibodies were used, disease was less
severe, yet the protection was completely dependent on the Siglecg
gene. However, with increased doses of anti-collagen antibodies,
the protective effect was only partially dependent on Siglecg, as
the protection was still obvious, albeit less pronounced (52% in KO
vs 72% in WT mice) (FIG. 16C). These data demonstrate that while
CD24Fc can signal through Siglec G to confer protection against
CAIA, an additional mechanism exists that allows CD24Fc to protect
against RA in the absence of Siglec G.
[0125] Discussion
[0126] Taken together, the results demonstrate that CD24Fc has
potent therapeutic effects in three mouse RA models, including a
CAIA and two CIA models. The efficacies in multiple models
indicates that CD24Fc has a therapeutic effect among RA in humans
with different underlying pathogenesis. For decades, it has been
assumed that RA is predominantly a T-cell mediated autoimmune
diseases. In the last two decades, there has been a re-awaking on
the possible role for antibodies and B lymphocytes in RA
pathogenesis. Thus, in addition to rheumatoid factors, a host of
auto-antibodies have been found in RA patients. Several lines of
evidence have demonstrated that in the mouse models, antibodies
specific for either ubiquitous or tissue specific antigens are
sufficient to cause RA symptoms. For instance, antibodies from
K/B.times.N TCR transgenic mice were found to be fully capable of
transferring RA-like diseases in the new host. Likewise, a cocktail
of 4 anti-collagen antibodies is now widely used to induce RA in
mice. Genetic analyses of the CAIA model indicate critical roles
for complement. Although other possibilities exist, these
requirements suggest potential involvement of antibody-mediated
tissue damage in the pathogenesis of RA. The efficacy of CD24Fc in
this model demonstrates that the fusion protein may be useful for
antibody-mediated destruction phase in RA patients.
[0127] The CIA model is commonly used for RA as it can mimic both
induction and effector function of both adaptive and innate
immunity. Two CIA models were used to validate the therapeutic
effect of CD24Fc. Bovine collagen-induced RA in DBA/1 mice is the
most commonly used model. The above data show that CD24Fc reduced
the disease score either before or after onset of disease in this
model. One drawback of the bovine CIA model is that only relatively
small numbers of strains are susceptible. In particular, C57BL/6
mice, which are commonly used for genetic studies are resistant.
More recently, a protocol has been developed to induce CIA in
C57BL/6 mice using chicken collagen. As shown above, CD24Fc
accelerated recovery of arthritis induced by chicken collagen. The
fact that the CD24Fc confers protection in multiple models
demonstrates the robustness of its therapeutic effect.
[0128] An important issue relating to drug development is mechanism
of action. Since it has been reported that CD24Fc binds to both
Siglec G and human Siglec 10, the significance of this interaction
was evaluated. In vitro, as shown above, CD24Fc signals through
Siglec G and triggers tyrosine phosphorylation. In vivo, as shown
above, CD24Fc works at least in part through Siglec G. These
results demonstrate that it is plausible that CD24Fc protects
against RA through strengthening the Siglec G-mediated protection
against innate immunity to DAMPs. To date, no RA drug has been
developed by fortifying the negative regulation over innate
response to DAMPs. Therefore, CD24Fc and other fusion proteins
containing variant CD24 missing the polymorphic A/V amino acid (SEQ
ID NO: 1) represents a new class of therapeutics for RA. This
approach may be preferable to antibodies targeting individual DAMPs
or inflammatory cytokins. Since multiple DAMPs are released during
autoimmune destruction, targeting individual DAMP may be less
effective than targeting a broad-spectrum regulator such as
CD24-Siglec G pathway. Nevertheless, it should be pointed out that
the data in this example demonstrate that the protection is not
completely dependent on signaling through Siglec G. At least two
additional mechanisms can be invoked. First, by binding to DAMPs,
CD24 may reduce the amounts of DAMPs available for their agonist
receptors, such as RAGE, TLR. Second, since CD24 is heterogeneously
glycosylated, it may bind to other members of Siglecs to confer
negative regulation.
[0129] Conclusion
[0130] A fusion protein comprising a non-polymorphic extracellular
domain of human CD24 (comprising SEQ ID NO: 1) protects mice
against arthritis initiated by either anti-collagen antibodies or
immunization of collagen. The protection is at least partially
dependent on its interaction with Siglec G. The data demonstrate
the potential of harnessing the negative regulation of innate
immunity to tissue injuries. Unexpectedly, the non-polymorphic
variant of CD24 is superior to wild-type CD24 in suppressing
inflammatory cytokine production and protecting mice against RA.
Sequence CWU 1
1
7130PRTHomo sapiens 1Ser Glu Thr Thr Thr Gly Thr Ser Ser Asn Ser
Ser Gln Ser Thr Ser 1 5 10 15 Asn Ser Gly Leu Ala Pro Asn Pro Thr
Asn Ala Thr Thr Lys 20 25 30 231PRTHomo sapiensXaa(31)..(31)Val or
Ala 2Ser Glu Thr Thr Thr Gly Thr Ser Ser Asn Ser Ser Gln Ser Thr
Ser 1 5 10 15 Asn Ser Gly Leu Ala Pro Asn Pro Thr Asn Ala Thr Thr
Lys Xaa 20 25 30 327PRTMus musculus 3Asn Gln Thr Ser Val Ala Pro
Phe Pro Gly Asn Gln Asn Ile Ser Ala 1 5 10 15 Ser Pro Asn Pro Thr
Asn Ala Thr Thr Arg Gly 20 25 426PRTHomo sapiens 4Met Gly Arg Ala
Met Val Ala Arg Leu Gly Leu Gly Leu Leu Leu Leu 1 5 10 15 Ala Leu
Leu Leu Pro Thr Gln Ile Tyr Ser 20 25 5287PRTArtificial
SequenceHuman CD24 IgG1 Fc fusion 5Met Gly Arg Ala Met Val Ala Arg
Leu Gly Leu Gly Leu Leu Leu Leu 1 5 10 15 Ala Leu Leu Leu Pro Thr
Gln Ile Tyr Ser Ser Glu Thr Thr Thr Gly 20 25 30 Thr Ser Ser Asn
Ser Ser Gln Ser Thr Ser Asn Ser Gly Leu Ala Pro 35 40 45 Asn Pro
Thr Asn Ala Thr Thr Lys Pro Lys Ser Cys Asp Lys Thr His 50 55 60
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 65
70 75 80 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr 85 90 95 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp Pro Glu 100 105 110 Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys 115 120 125 Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser 130 135 140 Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 145 150 155 160 Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 165 170 175 Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 180 185
190 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
195 200 205 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn 210 215 220 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser 225 230 235 240 Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg 245 250 255 Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu 260 265 270 His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 275 280 285 6231PRTHomo
sapiens 6Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro 1 5 10 15 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys 20 25 30 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val 35 40 45 Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp 50 55 60 Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 65 70 75 80 Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 85 90 95 Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 100 105 110
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 115
120 125 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys 130 135 140 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp 145 150 155 160 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys 165 170 175 Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser 180 185 190 Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 195 200 205 Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 210 215 220 Leu Ser
Leu Ser Pro Gly Lys 225 230 7288PRTArtificialCD24VFc 7Met Gly Arg
Ala Met Val Ala Arg Leu Gly Leu Gly Leu Leu Leu Leu 1 5 10 15 Ala
Leu Leu Leu Pro Thr Gln Ile Tyr Ser Ser Glu Thr Thr Thr Gly 20 25
30 Thr Ser Ser Asn Ser Ser Gln Ser Thr Ser Asn Ser Gly Leu Ala Pro
35 40 45 Asn Pro Thr Asn Ala Thr Thr Lys Val Pro Lys Ser Cys Asp
Lys Thr 50 55 60 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser 65 70 75 80 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg 85 90 95 Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp Pro 100 105 110 Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala 115 120 125 Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 130 135 140 Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 145 150 155
160 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
165 170 175 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu 180 185 190 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys 195 200 205 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser 210 215 220 Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp 225 230 235 240 Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 245 250 255 Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 260 265 270 Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 275 280
285
* * * * *