U.S. patent application number 15/666743 was filed with the patent office on 2017-11-16 for methods of treating autoimmune diseases.
This patent application is currently assigned to GENZYME CORPORATION. The applicant listed for this patent is GENZYME CORPORATION. Invention is credited to Johanne Kaplan, John M. McPherson.
Application Number | 20170326203 15/666743 |
Document ID | / |
Family ID | 38326221 |
Filed Date | 2017-11-16 |
United States Patent
Application |
20170326203 |
Kind Code |
A1 |
Kaplan; Johanne ; et
al. |
November 16, 2017 |
Methods of Treating Autoimmune Diseases
Abstract
Novel methods for treating patients with autoimmune diseases are
disclosed. The methods of the invention include first depleting
circulating lymphocytes in the mammal, e.g., by administering
anti-thymocyte antibody, and then, during the course of
repopulation, administering to the mammal a therapeutically
effective amount of latent TGF-.beta. and/or another agent that
promotes expansion of regulatory T cells. In certain aspects, the
disclosed process results in improved kidney function and survival
rates.
Inventors: |
Kaplan; Johanne;
(Framingham, MA) ; McPherson; John M.;
(Framingham, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GENZYME CORPORATION |
Cambridge |
MA |
US |
|
|
Assignee: |
GENZYME CORPORATION
CAMBRIDGE
MA
|
Family ID: |
38326221 |
Appl. No.: |
15/666743 |
Filed: |
August 2, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13680667 |
Nov 19, 2012 |
9744233 |
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15666743 |
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12241345 |
Sep 30, 2008 |
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13680667 |
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PCT/US2007/066416 |
Apr 11, 2007 |
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12241345 |
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60744713 |
Apr 12, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 9/12 20180101; A61P
13/02 20180101; A61K 2039/505 20130101; C07K 16/28 20130101; A61P
17/00 20180101; A61P 29/00 20180101; A61P 1/16 20180101; A61P 43/00
20180101; A61P 3/10 20180101; C07K 2317/73 20130101; A61P 9/14
20180101; A61P 19/02 20180101; A61P 37/06 20180101; A61K 39/3955
20130101; A61P 7/00 20180101; C07K 16/2809 20130101; A61K 38/1841
20130101; A61P 17/06 20180101; A61P 21/04 20180101; A61P 27/02
20180101; A61P 37/02 20180101; C07K 2317/20 20130101; A61P 25/28
20180101; A61P 37/00 20180101; C07K 16/18 20130101; A61P 25/00
20180101; C07K 16/2803 20130101; A61P 1/04 20180101; A61K 31/436
20130101; A61P 35/00 20180101; A61P 5/40 20180101; A61K 39/39541
20130101; A61K 39/39541 20130101; A61K 45/06 20130101; A61P 9/04
20180101; C07K 14/495 20130101; A61P 9/00 20180101; A61P 5/14
20180101; A61P 15/00 20180101; A61P 13/12 20180101; A61P 21/00
20180101; C07K 16/2893 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 38/18 20060101
A61K038/18; C07K 16/28 20060101 C07K016/28; C07K 16/28 20060101
C07K016/28; A61K 31/436 20060101 A61K031/436; C07K 16/28 20060101
C07K016/28; A61K 39/395 20060101 A61K039/395 |
Claims
1. A method of treating a mammal with an autoimmune disease, the
method comprising: (a) depleting circulating lymphocytes in the
mammal, (b) allowing the lymphocytes to begin repopulating, and (c)
during the repopulation phase of (b), administering to the mammal a
therapeutically effective amount of latent TGF-.beta. and/or
another agent that promotes the expansion of regulatory T
cells.
2. The method of claim 1, wherein the mammal is a human.
3. The method of claim 2, wherein the autoimmune disease is
multiple sclerosis.
4. The method of claim 1, wherein the lymphocytes depleted are
predominantly T cells.
5. The method of claim 1, wherein the lymphocytes are depleted by
administering an agent chosen from the group consisting of
anti-thymocyte antibody, anti-CD52 antibody, and anti-CD3
antibody.
6. The method of claim 5, wherein the anti-thymocyte antibody is
chosen from the group consisting of rabbit anti-thymocyte globulin
and equine anti-thymocyte globulin.
7. The method of claim 1, wherein the regulatory T cells are
CD4.sup.+ CD25.sup.+ T cells.
8. The method of claim 1, wherein latent TGF-.beta. comprises
mature TGF-.beta. and one or both of the following: (a) latency
associated peptide (LAP); and (b) latent TGF-.beta. binding protein
(LTBP).
9. The method of claim 1, wherein latent TGF-.beta. is latent
TGF-.beta.1.
10. The method of claim 1, wherein latent TGF-.beta. is
administered systemically.
11. The method of claim 1, wherein the agent that promotes
expansion of regulatory T cells is one or more agents chosen from
the group consisting of: (1) IL-10, (2) IL-4, (3) IFN-.alpha., (4)
vitamin D3, (5) dexamethasone, and (6) mycophenolate mofetil.
12. The method of claim 1, wherein the agent that promotes
expansion of regulatory T cells is rapamycin.
13. The method of claim 1, wherein the autoimmune disease is
associated with a loss of kidney function and the treatment results
in slowing of the loss of or improvement in kidney function of the
mammal.
14. The method of claim 13, wherein the slowing of loss or
improvement in kidney function is indicated by a change in systemic
blood pressure, proteinuria, albuminuria, glomerular filtration
rate, and/or renal blood flow.
15. The method of claim 13, wherein the autoimmune disease is
Goodpasture's syndrome, Wegener's syndrome, IgA nephropathy, IgM
nephropathy, or another autoimmune disease that impairs kidney
function.
16. A method of treating a mammal with an autoimmune disease
comprising: (a) administering a lymphocyte-depleting antibody to
the mammal, thereby reducing the population of peripheral blood T
cells; and (b) administering latent TGF-.beta. to the mammal in an
amount effective to slow the progression of the disease and/or
improve symptoms.
17. The method of claim 16, wherein the mammal is a human.
18. The method of claim 16, wherein the autoimmune disease is
multiple sclerosis.
19. The method of claim 16, wherein the lymphocyte-depleting
antibody is anti-thymocyte antibody, anti-CD52 antibody, or
anti-CD3 antibody.
20. The method of claim 16, wherein latent TGF-.beta. is latent
TGF-.beta.1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of U.S. application Ser. No.
13/680,667, filed Nov. 19, 2012, which is a continuation of U.S.
application Ser. No. 12/241,345, filed Sep. 30, 2008, which is a
continuation of International Application PCT/US2007/066416, filed
Apr. 11, 2007, which claims priority from U.S. Provisional
Application 60/744,713, filed Apr. 12, 2006, all of which are
hereby incorporated by reference in their entirety.
SEQUENCE LISTING
[0002] A paper copy of a sequence listing associated with this
application is being submitted herewith and is hereby incorporated
by reference in its entirety into the specification.
FIELD OF THE INVENTION
[0003] This invention relates to methods of treating autoimmune
diseases. The methods of the invention involve the use of latent
TGF-.beta. or other agents that stimulate regulatory T cells, alone
or in combination with lymphocyte-depleting agents, such as, e.g.,
anti-thymocyte globulin (ATG).
BACKGROUND OF THE INVENTION
[0004] The production of antibodies against self-antigens and/or
autoreactive T cells is a hallmark of many autoimmune diseases.
Autoantibodies and autoreactive T cells can cause severe tissue
damage (e.g., as in lupus nephritis) or loss of blood components
(e.g., as in immune thrombocytopenia purpura).
[0005] Typically, autoimmune diseases are treated with nonspecific
immunosuppressive agents, such as, e.g., cyclophosphamide,
methotrexate, azathioprine, and cyclosporine, that impede the
immune cells from attacking the organs and tissues. However,
immunosuppressive agents are often associated with significant side
effects (e.g., toxicity, the undesired suppression of the immune
system, etc.).
[0006] Due to its immunosuppressive effects, transforming growth
factor-beta (TGF-.beta.) has been suggested as a possible
therapeutic agent for certain autoimmune diseases, including
multiple sclerosis and graft-versus-host disease. Flanders et al.,
Clin. Med. Res., 1:13-20 (2003). It has also been reported as
useful to induce the generation of suppressor T cells in vitro
(see, e.g., U.S. Pat. No. 6,759,035). However, TGF-.beta. is a
pluripotent cytokine--besides having immunosuppressive properties,
it is involved in the extracellular matrix production, and other
biological processes. For a review on TGF-.beta., see, e.g.,
Cytokine Reference, eds. Oppenheim et al., Academic Press, San
Diego, Calif., 2001. Excessive or persistent expression of
TGF-.beta. plays a role in organ fibrosis (Kapanci et al., Am. J.
Resp. Crit. Care Med., 152:2163-2169 (1995); George et al., Prot.
Natl. Acad. Sci., 96:2719-12724 (1999); Kuwahara et al.,
Circulation, 106:130-135 (2002)), while systemic administration of
active TGF-.beta. has been associated with unacceptable toxicity.
In particular, in a Phase I/II clinical trial for chronic
progressive multiple sclerosis, systemic administration of active
TGF-.beta.2 resulted in unacceptable renal toxicity as evidenced by
a reduction in glomerular filtration rate. Calabresi et al.,
Neurology, 51:289-292 (1998). This result has hindered further
clinical development of therapies involving systemic administration
of active TGF-.beta.. Accordingly, the challenge of selectively
harnessing the immunosuppressive potential of TGF-.beta. without
incurring its attendant toxicities has remained. In addition, there
remains a need to develop methods of treating autoimmune diseases
that allow suppression of autoreactive immunity without undesirable
side effects.
SUMMARY OF THE INVENTION
[0007] The present invention is based, in part, on the discovery
and demonstration that latent TGF-.beta. may be used to circumvent
systemic toxicity of active TGF-.beta.. Activation of latent
TGF-.beta. requires removal of the latency-associated peptide (LAP)
which can occur in vivo through a number of mechanisms including
proteolytic cleavage, exposure to reactive oxygen species, and
interactions with thrombospondin and other proteins. Murphy-Ullrich
et al., Cytokine Growth Factor Rev., 11:59-69 (2000). It is
theorized, but not relied on for the purposes of this invention,
that such conditions are likely to occur in areas of autoimmune
inflammation, such as in the kidney in lupus patients. Because the
activation of latent TGF-.beta. occurs in areas of inflammation and
tissue injury, the use of latent TGF-.beta. may avoid the toxicity
associated with systemic TGF-.beta.. Accordingly, in some
embodiments, the methods of the invention involve systemic
administration of inactive TGF-.beta. (e.g., latent TGF-.beta.) to
a mammal, whereupon the activation and/or action of TGF-.beta. is
limited to sites of inflammation and tissue damage.
[0008] The present invention is further based, in part, on the
discovery and demonstration that depletion of lymphocytes by
anti-thymocyte globulin (ATG) followed by administration of latent
TGF-.beta. is effective in improving kidney function and increasing
survival rates in a murine model of systemic lupus erythematosus.
Accordingly, in some embodiments of the invention, host lymphocytes
are depleted prior to the administration of latent TGF-.beta. so as
to yield the therapeutically desired effect of the latent
TGF-.beta. administration.
[0009] It is further theorized, but not relied on for the purposes
of this invention, that the therapeutic effect of TGF-.beta. is
achieved, in part, due to the stimulatory effects of TGF-.beta. on
the growth of regulatory T cells. Therefore, in some embodiments of
the invention, another agent that promotes the expansion of
regulatory T cells may be administered in place of, or in addition
to, latent TGF-.beta..
[0010] This invention provides methods for treating a mammal (e.g.,
a human) with an autoimmune disease, e.g., systemic lupus
erythematosus (SLE), rheumatoid arthritis (RA). In some
embodiments, the treatment results in slowing the progression of
disease and/or improvement in symptoms. The invention further
provides methods of preserving or improving kidney function in a
mammal with an autoimmune disease that impairs kidney function,
such as, e.g., SLE, Goodpasture's syndrome, Wegener's syndrome, and
Berger's disease.
[0011] In more particular embodiments, the methods of the invention
include the following steps:
[0012] (a) depleting circulating lymphocytes in a mammal,
[0013] (b) allowing the lymphocytes to begin repopulating
("repopulation phase"), and
[0014] (c) during the repopulation phase, administering to the
mammal a therapeutically effective amount of latent TGF-.beta.
and/or an agent that promotes the expansion of regulatory T
cells.
[0015] In some embodiments, the depletion of lymphocytes is
accomplished by administering anti-thymocyte antibody (e.g.,
Thymoglobulin.RTM., Atgam.TM., Fresenius.TM., and Tecelac.TM.) or
another antibody specific for an antigen(s) expressed on T
cells.
[0016] Once the circulating lymphocytes have been substantially
depleted, they are allowed to start repopulating ("repopulation
phase"). During the course of repopulation, before the complete
repopulation occurs, a therapeutically effective amount of one or
more of the following agents is administered to the mammal: (1)
latent TGF-.beta. (e.g., the latent form of any one of
TGF.beta.1-TGF.beta.3) and/or (2) one or more other agents that
promotes expansion of regulatory T cells (e.g., IL-10, IL-10 and
IL-4, IL-10 and IFN-.alpha., vitamin D3 and dexamethasone, vitamin
D3 and mycophenolate mofetil, and rapamycin).
[0017] In some embodiments, where the kidney function is
compromised due to autoimmune disease, the treatment methods result
in improvement of kidney function in the mammal (e.g., slowing the
loss thereof) as indicated by, e.g., a change in systemic blood
pressure, proteinuria, album inuria, glomerular filtration rate,
and/or renal blood flow.
[0018] The foregoing summary and the following detailed description
are exemplary and explanatory only and are not restrictive of the
invention, as claimed.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1 shows an alignment of amino acid sequences of the
precursors of human TGF-.beta.1 (SEQ ID NO:1), TGF-.beta.2 (SEQ ID
NO:2), and TGF-.beta.3 (SEQ ID NO:3). TGF-.beta.2 is shown in the
`long` alternatively spliced form in which a 28 amino acid
insertion is found in the pre-pro domain beginning at residue 119.
Conserved sequences are boxed in. Arrows indicate the sites of
proteolytic processing resulting in cleavage of the signal peptide
and of the mature C-terminal TGF-.beta.1 fragment. * refers to RGD
integrin recognition site found in the latency-associated peptide
(LAP) proteins of TGF-.beta.1 and TGF-.beta.3. + refers to cysteine
residues involved in disulfide bonds between the two monomeric LAP
proteins. # refers to a cysteine residue involved in formation of
the single disulfide bond TGF-.beta. monomers.
[0020] FIG. 2 shows the therapeutic effect of the ATG/latent
TGF-.beta.1 combination treatment on kidney function.
MRUMPJ-Tnfrs6.sup.lpr mice (a murine model of SLE) were injected
with 500 .mu.g of ATG intraperitoneally (i.p.) twice, three days
apart, with or without 4 .mu.g of latent TGF-.beta.1 in 100 .mu.l
of phosphate buffered saline (PBS). Four micrograms of latent
TGF-.beta.1 corresponds to a 1 .mu.g (.about.0.05 mg/kg) dose of
the active (mature, non-LAP-associated) portion of the molecule.
When included in a treatment, the latent TGF-.beta.1 was
administered daily for twelve days beginning eleven days after the
second ATG injection. As a negative control, SLE mice were treated
with 500 .mu.g of normal rabbit immunoglobulin (Ig) i.p. twice,
three days apart. An additional treatment group received normal
rabbit immunoglobulin and latent TGF-.beta.1 administered as above.
As a positive control, SLE mice were treated with 100 mg/kg i.p. of
cyclophosphamide in 200 .mu.l saline weekly. Proteinuria was
significantly lower in SLE mice treated with latent TGF-.beta.1 and
ATG as compared to SLE mice treated with either ATG alone, control
Ig+TGF-.beta.1, or control Ig alone. Mean total urine protein of
the combination treatment group approached the level achieved with
cyclophosphamide, a current treatment for lupus.
[0021] FIG. 3 shows the effect of the combination treatment on the
development of severe kidney disease. Mice were treated as
described above for FIG. 2. SLE mice treated with ATG and latent
TGF-.beta.1 together exhibited a decrease in the incidence of
severe proteinuria (>500 mg/dl/day) as compared to SLE mice
treated with either ATG alone, control Ig+TGF-.beta.1, or control
Ig alone.
[0022] FIG. 4 shows the effect of the combination treatment on
kidney function. Mice were treated as described above for FIG. 2.
The mean urine albumin levels were decreased in SLE mice treated
with the combination of ATG and latent TGF-.beta.1 in comparison
with SLE mice treated with either ATG alone, control
Ig+TGF-.beta.1, or control Ig alone. ATG and latent TGF-.beta.1
combination treatment resulted in mean urine albumin levels near
those achieved with cyclophosphamide treatment.
[0023] FIG. 5 shows the effect of the combination treatment on the
development of severe kidney disease. Mice were treated as
described above for FIG. 2. The percent of SLE mice having severe
albuminuria (>10 mg/dl/day) was decreased in the combination
treatment group in comparison with either ATG alone, control
Ig+TGF-.beta.1, or control Ig alone.
[0024] FIGS. 6A-6E show the effect of the combination treatment on
the development of autoantibodies. Arrows indicate start of
treatment. Mice were treated as described above for FIG. 2 and
indicated accordingly in FIGS. 6A-6E. Overall, SLE mice treated
with ATG and latent TGF-.beta.1 showed a considerable delay in the
rise of IgG anti-dsDNA antibody titers in comparison to mice
treated with either ATG alone, control Ig+TGF-.beta.1, or control
Ig alone.
[0025] FIG. 7 shows the effect of the combination treatment on
survival of SLE mice. Mice were treated as described above for FIG.
2. SLE mice treated with ATG and latent TGF-.beta.1 survived
significantly longer than SLE mice treated with either ATG alone,
control Ig+TGF-.beta.1, or control Ig alone.
[0026] FIG. 8 shows the survival data obtained in a repeat study
with MRUMPJ-Tnfrs6.sup.lpr mice treated as described above for FIG.
2. In this instance, the study was extended to 40 weeks of age (as
opposed to 24 weeks in the first study) to assess the durability of
the effect of transient treatment with ATG and latent TGF-.beta.1.
The survival benefit did in fact persist and the survival of mice
treated with ATG and latent TGF-.beta.1 was comparable to that
obtained with cyclophosphamide, the positive control (90% vs. 100%,
respectively).
[0027] FIG. 9A shows the absolute number of CD4.sup.+ CD25.sup.+
cells in cultures of splenocytes exposed to various treatments.
Splenocytes were pooled from ten MRL/lpr mice with active disease.
Six different conditions (8 wells/condition) were assayed: 1) cells
alone, 2) ATG (100 .mu.g/ml)+active TGF-.beta.1 (10 ng/ml;
Genzyme), 3) ATG alone (100 .mu.g/ml), 4) control rabbit IgG (100
.mu.g/ml)+active TGF-.beta.1 (10 ng/ml), 5) control rabbit IgG
alone (100 .mu.g/ml), and 6) active TGF-.beta.1 alone (10 ng/ml).
After five days, the replicates of each culture condition were
pooled, washed in phosphate buffered saline, counted, and stained
for FACS analysis. A sample of 5.times.10.sup.5 cells per treatment
was stained with rat anti-mouse CD4-Alexa 488 and rat anti-mouse
CD25-PerCp-Cy5.5 and analyzed by flow cytometry. An acquisition of
6,000 lymphocytes per treatment was analyzed for staining on a FACS
Calibur system (Becton Dickinson, San Diego, Calif.). Results are
expressed as the absolute number of cells of each phenotype
recovered under each culture condition (percent positive cells by
FACS.times.total number of cells recovered from the culture).
[0028] FIG. 9B shows the absolute number of CD4.sup.+ CD25.sup.+
FOXP3.sup.+ cells in cultures of splenocytes treated as described
for FIG. 9A. Additionally, for intracellular detection of FOXP3,
cells stained for surface CD4/CD25 were permeabilized overnight and
stained for FOXP3.
DETAILED DESCRIPTION OF THE INVENTION
[0029] This invention provides methods of treating a mammal with an
autoimmune disease. In particular embodiments, such methods include
methods of improving kidney function in a mammal with an autoimmune
disease that compromises kidney function. In some embodiments, the
methods of the invention involve systemic administration of latent
TGF-.beta. to a mammal, wherein the activation and/or action of
TGF-.beta. is limited to sites of inflammation and tissue
damage.
[0030] In some embodiments, methods of the invention comprise the
following steps:
[0031] (a) depleting circulating lymphocytes in the mammal,
[0032] (b) allowing the lymphocytes to begin repopulating, and
[0033] (c) during the repopulation phase of (b), administering to
the mammal a therapeutically effective amount of latent TGF-.beta.
and/or an agent that promotes the expansion of regulatory T
cells.
Lymphocyte Depletion
[0034] Depletion of circulating lymphocytes can be accomplished by
administering a lymphocyte-depleting agent to the mammal or
otherwise exposing the mammal to conditions that result in a loss
of a substantial fraction of lymphoid cells (e.g., lymphocytes,
natural killer (NK) cells, monocytes, and/or dendritic cells, etc.)
in the mammal. Lymphocytes to be depleted may be T lymphocytes (T
cells) and/or T and B lymphocytes. In the depletion phase, T cell
counts are reduced by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, or more, and optionally, B lymphocyte (B cell) counts are
reduced by at least 30%, 40, 50%, 60%, 70%, 80%, 90%, 95%, or more.
In preferred embodiments, the depleted lymphocytes are
predominantly T cells, which means that the percentage of depleted
T cells is greater (e.g., 1.2-, 1.5-, 2-, 5-, 10-fold, or more)
than the percentage of depleted B cells.
[0035] The level of lymphocyte depletion can be readily assessed
by, for example, measuring the amount of peripheral blood
lymphocytes (PBLs). Lymphocyte counts can be determined using
conventional clinical laboratory techniques (e.g., by flow
cytometry). Reference values for normal PBL levels in humans are
presented in Table 1.
TABLE-US-00001 TABLE 1 Typical Mean Range Mean Range Cell Type
Marker (%) (%) (cells/.mu.l) (cells/.mu.l) Total T cells CD3 71
55-87 1,586 781-2,391 Total B cells CD19 5 1-9 277 17-537 Helper T
cells CD4 43 24-62 1,098 447-1,750 Cytotoxic cells CD8 42 19-65 836
413-1,260
[0036] In some embodiments, the lymphocyte-depleting agent is an
anti-lymphocyte antibody, e.g., anti-T cell antibodies, e.g.,
anti-thymocyte globulin (ATG), such as, e.g., Thymoglobulin.RTM.,
Atgam.TM. Fresenius.TM., and Tecelac.TM. ATG is a polyclonal
antibody directed against thymocytes. Currently marketed ATG
products are produced by injecting thymocytes from one species
(e.g., human) into another species (e.g., rabbit or horse). ATG
binds to cell surface proteins such as lymphocyte surface antigens
CD2, CD3, CD4, CD8, CD11a, CD18, CD25, HLA DR, and HLA class I
(Bourdage et al., Transplantation, 59:1194-1200 (1995)). ATG is
believed to induce immunosuppression primarily as a result of T
cell depletion (see, e.g., Bonnefoy-Bernard et al.,
Transplantation, 51:669-673 (1991)) and has been previously used
for pretreating transplant patients to reduce the risk of rejection
in the context of organ transplantation.
[0037] In addition to ATG, the lymphocyte-depleting agent consists
of or comprises a monoclonal or polyclonal antibody directed to one
or more specific lymphocyte surface antigens, e.g., anti-CD52
antibody (e.g., Campath.RTM.), anti-CD3 antibody (e.g., OKT3.RTM.),
anti-CD4 antibody (OKT.TM.), anti-CD25 (IL-2R) antibody (e.g.,
daclizumab), anti-CD5 antibody, anti-CD7 antibody, anti-TCR
antibody, anti-CD2 (e.g., Siplizumab.TM.), or an antibody against
any of other lymphocyte surface antigens specified above, etc.
[0038] In some embodiments, the lymphocyte-depleting agent is a
corticosteroid.
[0039] In some embodiments, conditions that result in depletion of
lymphocytes include exposure to gamma radiation.
[0040] A combination of any suitable agents and/or conditions to
deplete lymphocytes can be also used.
Reconstitution Phase
[0041] Following the depletion phase, the lymphocytes of the mammal
are allowed to begin repopulating by withdrawing the
lymphocyte-depleting agent or mitigating the conditions that
resulted in the loss of lymphocytes.
[0042] While in some instances, the agent of step (c) (i.e.,
TGF-.beta. or another agent that specifically stimulates regulatory
T cells) can be administered to the mammal immediately at the start
of the replenishment phase, in other cases, the agent is
administered after some repopulation has occurred. Before the step
(c) agent is administered to the mammal, the lymphocytes may be
allowed to repopulate to less than 50%, 40%, 30%, 20%, 10%, 5%, or
lower, as compared to the pre-depletion level.
[0043] In humans, lymphocytes repopulate to pre-depletion levels at
different rates depending on the depleting agent. For example, with
ATG, a complete repopulation may take two to four months, while
after treatment with Campath.TM., the repopulation may take several
years. Accordingly, in some embodiments, the length of time between
the end of the depletion phase of the lymphocytes and the
administration of step (c) agent is, for example, 0, 1, 2, 3, 4, 5,
6 days; 1, 2, 3, 4, or 5 weeks, or longer.
TGF-.beta.
[0044] In certain embodiments, the methods of the invention involve
administration of inactive TGF-.beta. which is activated after
administration. In some embodiments, inactive TGF-.beta. is
administered in the form of latent TGF-.beta.. In other
embodiments, inactive TGF-.beta. is administered in the form of a
TGF-.beta.-encoding DNA which expresses active TGF-.beta. upon
induction.
[0045] TGF-.beta. is naturally secreted in either a so-called
"small latent complex" (100 kDa) in which the biologically active
TGF-.beta. is noncovalently associated with its pro domain
("latency-associated peptide," LAP) and in a so-called "large
latent complex" (220 kDa) additionally containing latent TGF-.beta.
biding protein (LTBP). The latent forms are unable to bind to
TGF-.beta. receptors until active, i.e., mature, TGF-.beta. is
released from the complex. For a more detailed review of the latent
forms and activation process, see, e.g., Cytokine Reference, eds.
Oppenheim et al., Academic Press, San Diego, Calif., 2001, pp.
724-725. As used herein, the term "latent TGF-.beta." refers to
TGF-.beta. associated with LAP (covalently or noncovalently) and,
optionally, additionally associated with LTBP (covalently or
noncovalently). The term, therefore, refers to small and large
latent TGF-.beta. complexes. Other forms of inactive TGF-.beta.
that could be activated in the locations and at the time periods
desired would also be useful in the methods of this invention.
There are three known mammalian isoforms of TGF-.beta. (TGF-.beta.1
to TGF-.beta.3), all of which are homologous among each other
(60-80% identity). A partial listing of protein accession number
for the three mammalian isoforms is provided in Table 2; an
alignment of human TGF-.beta.s is shown in FIG. 1.
TABLE-US-00002 TABLE 2 Species TGF-.beta.1 TGF-.beta.2 TGF-.beta.3
Human PO1137 PO8112 P109600 Mouse P04202 P27090 P171125 Rat
AAD20222 AAD24484 Q07258 Porcine AAA616 AAB03850 P15203 Simian
P09533 WFMKB2
[0046] The structural and functional aspects of TGF-.beta. as well
as TGF-.beta. receptors are well known. See, e.g., Cytokine
Reference, eds. Oppenheim et al., Academic Press, San Diego,
Calif., 2001. Thus, inactivated forms of engineered TGF-.beta.s
that retain the ability to bind to one or more TGF-.beta. receptors
(TGF-.beta.RI, TGF-.beta.RII, or TGF-.beta.RIII) would also be
useful in the methods of the invention. Such inactivated forms of
engineered TGF-.beta. may contain only a partial or a mutated amino
acid sequence of the naturally occurring TGF-.beta.. For example,
inactivated forms of engineered TGF-.beta. may contain native
sequences in which conservative substitutions were made and/or
nonessential amino acids were deleted. For example, they may
comprise a sequence, which is at least 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100% identical to the 112 amino acid C-terminal
portion of SEQ ID NO:n over the entire length of this C-terminal
portion of SEQ ID NO:n, wherein n=1, 2, or 3.
Agents that Promote Regulatory T Cell Expansion
[0047] In certain embodiments, the methods of the invention involve
administration of an agent that promotes regulatory T cells
expansion. Regulatory T cells (also known as Tregs or suppressor T
cells) are cells that are capable of inhibiting the proliferation
and/or function of other lymphoid cells via contact-dependent or
contact-independent (e.g. cytokine production) mechanisms. Several
types of regulatory T cells have been described, including
.gamma..delta. T cells, Natural Killer T (NKT) cells, CD8.sup.+ T
cells, CD4.sup.+ T cells, and double negative CD4.sup.- CD8.sup.- T
cells. See, e.g., Bach et al., Immunol., 3:189-98 (2003). The
so-called "naturally occurring" regulatory T cells are CD4.sup.+
CD25.sup.+ and express the forkhead family transcription factor
FOXP3 (forkhead box p3). In addition to the FOXP3-expressing
CD4.sup.+ CD25.sup.+, a minor population of CD8.sup.+
FOXP3-expressing cells are also regulatory T cells. CD4.sup.+ Tregs
can be further divided into induced regulatory T cells that secrete
interleukin-10 (IL-10) and TGF-.beta. such as Tr1 cells and
T-helper 3 (Th3) cells. Additional surface markers for CD4.sup.+
CD25.sup.+ regulatory T cells include CD45RB, CD38, GITR, surface
TGF-.beta., CTLA4, CD103, CD134 and CD62L. For a detailed review of
various types of regulatory T cells, see, e.g., Wing et al., Scand.
J. Immunol., 62(1):1 (2005); Jonuleit et al., J. Immunol.,
171:6323-6327 (2003); Horwitz et al., J. Leukocyte Biol.,
74:471-478 (2003).
[0048] Accordingly, in some embodiments, the regulatory T cells
that are being stimulated include one or more of the following
groups: (1) regulatory T cells that express IL-10; (2) regulatory T
cells that express TGF-.beta. (including Tr1 cells and Th3 cells);
(3) CD4.sup.+ CD25.sup.+ cells (including cells having additional
markers CD45RB.sup.+, CD38.sup.+, GITR, surface TGF-.beta., CTLA-4,
CD103, CD134 and/or CD62L); (4) FOXP3-expressing T cells (including
CD8.sup.+ cells and CD4.sup.+ cells); (5) .gamma..delta. T cells;
(6) NK T cells; and (7) double negative CD4.sup.- CD8.sup.- T
cells.
[0049] TGF-.beta., in addition to its direct immunosuppressive
activity, may also be capable of stimulating regulatory T cells.
Gorelik and Flavell, Nature Reviews Immunology, 2:46-53 (2002);
Chen et al., J. Exp. Med., 198:1875-1886 (2003); Marie et al., J.
Exp. Med., 7:1061-1067 (2005); Huber et al., J. Immunol.,
173:6526-6531 (2004).
[0050] Examples of agents, other than TGF-.beta., that promote
regulatory T cell expansion include: (1) IL-10; (2) IL-10 and IL-4;
(3) IL-10 and IFN-.alpha.; (4) vitamin D3 and dexamethasone; (5)
vitamin D3 and mycophenolate mofetil, and (6) rapamycin. (See,
e.g., Barrat et al., J. Exp. Med., 195:603-616 (2002); Jonuleit et
al., J. Immunol., 171:6323-6327 (2003); Gregori et al., J Immunol.,
167:1945-1953 (2001); Battaglia et al., Blood, 105:4743-4748
(2005).)
[0051] In some embodiments, an increase of, e.g., at least 10%,
20%, 30%, 40%, 50%, 100%, or more in the expansion of regulatory T
cells in the presence of an agent as opposed to its absence is
considered indicative of the agent's capacity to promote regulatory
T cells expansion. TGF-.beta. and other agents can be assayed for
their capacity to promote regulatory T cell expansion using routine
methods. Examples of some of the more frequently used in vitro
assays include the following:
[0052] (1) flow cytometry analysis, wherein co-expression of CD4,
CD25, and/or FOXP3, and/or CD62L, and/or GITR, and/or CTLA4, and/or
surface TGF-.beta., and/or CD103, and/or CD134 is used as
indication of a regulatory T cell phenotype (see, e.g., Jonuleit,
supra);
[0053] (2) inhibition of T cell proliferation in a co-culture
system as described in, e.g., Chen et al., J. Exp. Med.,
198:1875-1886 (2003). (In this assay, regulatory T cells are added
to responder T cells and the co-culture is stimulated with anti-CD3
or allogeneic lymphocytes. In the presence of regulatory T cells,
the responder T cells become unable to proliferate in response to
these stimuli. The degree of proliferation is typically measured by
tritiated thymidine incorporation.); and
[0054] (3) cytokine profiling as described in, e.g., Barrat, supra,
and Jonuleit, supra. (In this assay, a supernatant from cultured
regulatory T cells is analyzed for the presence of the
immunosuppressive cytokines such as, e.g., IL-10 and TGF-.beta.,
known to be produced by regulatory T cells.)
Uses
[0055] The methods of the invention can be used to treat a mammal
that has an autoimmune disease such as, e.g., systemic lupus
erythematosus (SLE) and autoimmune rheumatoid arthritis (RA).
Examples of mammals include humans or other primates (e.g.,
chimpanzees), rodents (e.g., mice, rats, or guinea pigs), rabbits,
cats, dogs, horses, cows, and pigs. In some of the subjects
afflicted, the treatment is expected to result in inhibiting the
progression of disease and/or improvement in symptoms.
[0056] Examples of additional autoimmune diseases include
insulin-dependent diabetes mellitus (IDDM; type I diabetes),
inflammatory bowel disease (IBD), graft-versus-host disease (GVHD),
celiac disease, autoimmune thyroid disease, Sjogren's syndrome,
autoimmune gastritis, autoimmune hepatitis, cutaneous autoimmune
diseases, autoimmune dilated cardiomyopathy, multiple sclerosis
(MS), myasthenia gravis (MG), vasculitis (e.g., Takayasu's
arteritis and Wegener's granulomatosis), autoimmune diseases of the
muscle, autoimmune diseases of the testis, autoimmune ovarian
disease, autoimmune uveitis, Graves' disease, psoriasis, ankylosing
spondylitis, Addison disease, Hashimoto thyroiditis, idiopathic
thrombocytopenic purpura, and vitiligo.
[0057] The methods of the invention are expected to slow the
progression of autoimmune disease, improve at least some symptoms,
and/or increase survival. For example, the methods of the invention
may result in a reduction in the levels of autoantibodies, B cells
producing autoantibodies, and/or autoreactive T cells. The
reduction in any of these parameters can be, for example, at least
10%, 20%, 30%, 50%, 70% or more as compared to pretreatment
levels.
[0058] The invention further provides methods of preserving or
improving kidney function in a mammal with an autoimmune disease
that compromises kidney function. Examples of autoimmune diseases
that may compromise kidney function include SLE (e.g., lupus
nephritis), Goodpasture's syndrome, Wegener's granulomatosis
(Wegener's syndrome), Berger's disease (IgA nephropathy), and IgM
nephropathy. In some of the patients afflicted with such diseases,
the treatment is expected to result in improvement of kidney
function (e.g., slowing the loss of, preserving, or improving the
same) as indicated by, e.g., a change in systemic blood pressure,
proteinuria, albuminuria, glomerular filtration rate, and/or renal
blood flow.
[0059] The term "renal function" refers to the ability of a kidney
to perform its physiological functions such as pressure filtration,
selective reabsorption, tubular secretion, and/or systemic blood
pressure regulation. Methods for assessing renal function are well
known in the art and include, but are not limited to, measurements
of blood systemic and glomerular capillary pressure, proteinuria,
albuminuria, microscopic and macroscopic hematuria, serum
creatinine level (e.g., one formula for estimating renal function
in humans equates a creatinine level of 2.0 mg/dl to 50% of normal
kidney function and 4.0 mg/dl to 25%), decline in the glomerular
filtration rate (GFR) (e.g., as indicated by the rate of creatinine
clearance, or using inulin assays), and degree of tubular
damage.
[0060] For a detailed review of renal function and related disease
states, see The Kidney: Physiology and Pathophysiology, eds. Seldin
et al., 3.sup.rd ed., Lippincott, Williams & Wilkins
Publishers, 2000. Normally, less than 0.15 g of protein is excreted
into the urine in a 24-hour period. Almost all types of kidney
disease cause mild (up to 500 mg per day) to moderate (up to 4 g
per day) protein leakage into the urine. The normal concentration
of albumin in the urine is less than 1.0 mg/dl. Generally, 30-300
mg/dl urinary albumin is considered microalbuminuria, and greater
than 300 mg/dl is considered macroalbuminuria. The normal values of
serum creatinine are 0.6-1.5 mg/dl for men and 0.6-1.1 mg/dl for
women. The relationship between creatinine levels, renal function,
and the stage of renal disease is shown in Table 3.
TABLE-US-00003 TABLE 3 Estimated Creatinine Reduction of Level
(mg/dl) Renal Function Stage of Renal Disease 0.6-1.5 Up to 25%
Reduced or diminished renal reserve >1.5 >50% Renal
insufficiency 4.8 75% Renal failure 10 90% End-stage renal
disease
[0061] Therefore, the methods of the invention may be useful in
patients having an autoimmune disease with reduced or diminished
renal reserve, renal insufficiency, renal failure, or end-stage
renal disease. For example, methods of the invention may be used in
patient with microalbuminuria, macroalbuminuria, and/or proteinuria
levels over 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 g or more per a
24-hour period, and/or serum creatinine levels of about 1.0, 1.5,
2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0, 8.0, 9.0, 10
mg/dl or higher.
[0062] In some embodiments, the methods of the invention reduce the
amount of protein secreted in the urine (proteinuria), amount of
albumin secreted in the urine (albuminuria), and/or the patient's
serum creatinine levels by at least 10%, 20%, 30%, 40%, 50%, 60%,
70%, or more, relative to control subjects. In other embodiments,
the methods of the invention slow the loss of renal function by at
least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or more, relative to
control subjects. Nonlimiting illustrative methods for assessing
renal function are described herein and, for example, in WO
01/66140.
Methods of Administration
[0063] In the methods of the invention, "administration" is not
limited to any particular delivery system and may include, without
limitation, parenteral (including subcutaneous, intravenous,
intramedullary, intraarticular, intramuscular, or intraperitoneal
injection), rectal, topical, transdermal, or oral (for example, in
capsules, suspensions, or tablets). Administration to an individual
may occur in a single dose or in repeat administrations, and in any
of a variety of physiologically acceptable salt forms, and/or with
an acceptable pharmaceutical carrier and/or additive as part of a
pharmaceutical composition. Physiologically acceptable salt forms
and standard pharmaceutical formulation techniques and excipients
are well known to persons skilled in the art (see, e.g.,
Physicians' Desk Reference (PDR.RTM.) 2005, 59.sup.th ed., Medical
Economics Company, 2004; and Remington: The Science and Practice of
Pharmacy, eds. Gennado et al. 21th ed., Lippincott, Williams &
Wilkins, 2005).
[0064] Latent TGF-.beta. can also be administered by means of gene
therapy (i.e., by administering a TGF-.beta.-encoding DNA in an
appropriate vector), for example, as described in Kitani et al., J.
Exp. Med., 192(1):41-52 (2000).
[0065] The appropriate effective doses for the latent TGF-.beta.,
agents promoting Tregs, and lymphocyte depleting agents will be
chosen by a treating clinician and will range roughly from 0.01
.mu.g/kg to 25 mg/kg, from 0.1 .mu.g/kg to 10 mg/kg, from 1
.mu.g/kg to 1 mg/kg, 10 .mu.g/kg to 1 mg/kg, from 10 .mu.g/kg to
100 .mu.g/kg, from 100 .mu.g/kg to 1 mg/kg, and from 500 .mu.g/kg
to 5 mg/kg. Additionally, specific dosages indicated in the
Examples or in the PDR.RTM. 2005 and later editions may be used to
arrive at the desired dosage. For example, the currently approved
uses of Thymoglobulin.RTM. in the United States include
transplantation (from 1 mg/kg to 2.5 mg/kg for 2-14 days) and
aplastic anemia (from 2.5 mg/kg to 3.5 mg/kg for 5 days).
[0066] Effective dosages achieved in one animal may be converted
for use in another animal, including humans, using conversion
factors known in the art. See, e.g., Freireich et al., Cancer
Chemother. Reports, 50(4):219-244 (1966) and Table 4 for equivalent
surface area dosage factors. Examples of autoimmune disease models
and appropriate methods can be found in, e.g., Cohen et al. (eds.)
Autoimmune Disease Models, Academic Press, 2005.
TABLE-US-00004 TABLE 4 From: Mouse Rat Monkey Dog Human To: (20 g)
(150 g) (3.5 kg) (8 kg) (60 kg) Mouse 1 0.5 0.25 0.17 0.08 Rat 2 1
0.5 0.25 0.14 Monkey 4 2 1 0.6 0.33 Dog 6 4 1.7 1 0.5 Human 12 7 3
2 1
[0067] The following Examples are provided for illustrative
purposes and are not intended to be limiting.
EXAMPLES
Potency Assessment of Activated TGF-.beta.1
[0068] Recombinant human latent TGF-.beta.1 was produced in CHO
cells (Genzyme, Framingham, Mass.). Disruption of LAP from latent
TGF-.beta.1 was achieved through acidification. LAP-TGF-.beta.1 was
diluted to 200 ng/mL in assay medium (DMEM plus non-essential amino
acids, L-glutamine, pen-strep, and 10% FBS). Five hundred
microliters of the diluted sample was activated by adding 100 .mu.L
of 1N HCl and incubating at room temperature for 20 minutes. The
sample was subsequently neutralized with 100 .mu.L of 1.2 N
NaOH/0.5 M HEPES.
[0069] The activated TGF-.beta.1 sample was analyzed using the A549
Cell Potency Assay and the activity assessed in comparison to a
human recombinant TGF-.beta.2 (Genzyme, Framingham, Mass.) control.
The A549 potency assay is based on the TGF-.beta.1-induced release
of IL-11 by the human lung epithelial cell line, A549 and is
described in Wang et al., Am. J. Physiol., 276:L175-L185 (1999).
IL-11 release from the A549 cells in response to TGF-.beta.1 was
measured using an ELISA procedure (R&D Systems, Minneapolis,
Minn.).
Murine Lupus Model
[0070] Animals and Reagents--
[0071] Female MRL/lpr mice were obtained from the Jackson
Laboratory (Bar Harbor, Me.) and were received at 5-6 weeks of age.
ATG was generated by the immunization of rabbits with Balb/c mouse
thymocytes as follows. Rabbits were immunized subcutaneoulsy with
5.times.10.sup.7 fresh thymocytes on day 0 and boosted
intravenously with 5.times.10.sup.7 fresh thymocytes on day 14.
Serum collected on days 20, 22, and 25 was pooled and the IgG
fraction was isolated by chromatography and sodium sulfate
precipitation. A commercial preparation of IgG from naive rabbits
was used as a negative control (Sigma, St. Louis, Mo.). Recombinant
human latent TGF-.beta.1 was produced in CHO cells (Genzyme,
Framingham, Mass.). Cyclophosphamide was purchased from VWR
Scientific Products (West Chester, Pa.).
[0072] Treatment--
[0073] Animals were monitored for proteinuria, albuminuria, and
titers of IgG antibodies to double-stranded DNA (dsDNA) every three
weeks (see below). Therapeutic treatment was initiated when animals
started developing antibodies to dsDNA and/or elevated proteinuria
at 12-13 weeks of age. Treatment with ATG or control rabbit IgG
consisted of two intraperitoneal (i.p.) injections of 500 .mu.g
(.about.25 mg/kg) delivered three days apart (days 0 and 3). Latent
TGF-.beta.1 was given from days 14-25 as twelve daily i.p.
injections of 4 .mu.g per mouse. A 4 .mu.g dose of latent
TGF-.beta.1 corresponds to a 1 .mu.g (.about.0.05 mg/kg) dose of
the active (mature, non-LAP-associated) portion of the molecule.
Cyclophosphamide was used as a positive control and was delivered
i.p. weekly at a dose of 100 mg/kg from 12-13 weeks of age until
the end of the study at 24-25 weeks of age. The treatment groups
consisted of control rabbit IgG, control rabbit IgG+latent
TGF-.beta.1, ATG, ATG+latent TGF-.beta.1, or cyclophosphamide with
ten animals per group.
[0074] Proteinuria and Albuminuria--
[0075] Levels of protein in the urine of individual mice were
measured using a colorimetric assay designed to measure total
protein concentration. Levels of albumin in the urine were assessed
with a quantitative ELISA assay.
[0076] A 24-hour urine collection was performed every three weeks
by placing mice into individual metabolic cages. Proteinuria was
measured using the Microprotein-PR.TM. kit from Sigma (St. Louis,
Mo.) according to manufacturer's instructions. Briefly, urine was
added to a reagent solution containing pyrogallol red-molybdate
complex. The mixture was incubated at 37.degree. C. for ten minutes
to allow for binding of the reagent to basic amino groups on
proteins leading to a shift in absorbance at 600 nM. The increase
in optical density (O.D.) at 600 nM is directly proportional to
protein concentration and a reference standard was used to
calculate the protein concentration of test samples according to
the following formula:
OD sample OD standard .times. Conc standard .times. Dilution = Conc
sample ( mg / dl ) ##EQU00001##
[0077] Levels of albumin in the urine were assessed using an
indirect competitive ELISA kit, according to manufacturer's
instructions (Albuwell M from Exocell Inc, Philadelphia, Pa.).
Briefly, serial two-fold dilutions of urine samples were added to
duplicate wells of an ELISA plate coated with mouse albumin. Rabbit
anti-mouse albumin antibody was then added to the wells allowing
for competition between binding of the antibody to albumin in the
sample and albumin attached to the well. This was followed by the
addition of horseradish peroxidase (HRP)-conjugated anti-rabbit
immunoglobulin and HRP substrate to detect the amount of rabbit
anti-mouse albumin antibody bound to the well. The O.D. at 450 nm
was inversely proportional to the logarithm of the amount of
albumin in the urine sample. The albumin concentration in the urine
samples was derived from a standard curve obtained with known
concentrations of murine albumin.
[0078] Anti-dsDNA ELISA--
[0079] Titers of IgG antibodies to dsDNA in serum samples from
individual mice were measured by ELISA.
[0080] Serum samples from individual mice were collected every
three weeks. Titers of antibodies to dsDNA were assessed by ELISA.
Mouse double-stranded DNA (The Jackson Laboratory) was digested
with S1 nuclease (Invitrogen, Carlsbad, Calif.) to remove any
single-stranded DNA and was then used to coat the wells of a
96-well ELISA plate (100 .mu.l/well of 1 .mu.g/ml dsDNA) overnight
at 4.degree. C. The plates were pretreated with 0.01% protamine
sulfate in water (150 .mu.l/well for 90 minutes at room
temperature) to facilitate adhesion of the DNA. After coating, the
plates were incubated with 2.5% BSA blocking buffer for one hour at
37.degree. C. and washed. One hundred microliters of serial
two-fold dilutions of serum were then added to duplicate wells and
incubated at 37.degree. C. for one hour. The plates were washed and
HRP-conjugated goat anti-mouse IgG (Pierce, Rockford, Ill.) was
added to detect antibodies bound to dsDNA (37.degree. C. for one
hour). After washing, HRP substrate was added for 30 minutes at
room temperature and the O.D. of the colorimetric product was read
at 490 nM with a reference wavelength of 650 nM on a dual
wavelength plate reader (Molecular Devices, Sunnyvale, Calif.). The
antibody titer was defined as the reciprocal of the dilution of
serum giving an O.D. greater or equal to 0.1. Normal mouse serum
was used as a negative control (titer <200, the lowest dilution
tested) and serum from aged MRL/lpr lupus mice was used as a
positive control (titer of 6400-25600).
[0081] Histology--
[0082] Kidneys were collected for histological analysis at the time
of scheduled sacrifice or during the course of the study from
moribund animals that required euthanasia. The kidneys were sliced
longitudinally and were fixed in neutral buffered formalin.
Sections of approximately 5 .mu.m were stained with hematoxylin and
eosin (H&E) and periodic acid-Schiff (PAS) stains. The slides
were scored by pathologist for glomerular morphology, interstitial
inflammation, and protein casts according to the scoring systems
described in Tables 5A and 5B.
TABLE-US-00005 TABLE 5A Glomeruli 0 No significant lesions
(comparable to WHO class I) 1 Minimal to mild disease,
characterized by mesangial deposits (comparable to WHO class IIA) 2
Mild to moderate disease, characterized by hypercellularity with or
without mesangial deposits (comparable to WHO class IIB) 3 Moderate
to severe disease, characterized by mesangioproliferative
glomerulopathy and "wire loop" capillaries with or without
fibrinoid necrosis of capillary loops, rupture of Bowman's capsule,
and periglomerular inflammation and fibrosis ("crescent" formation)
affecting less than 25% of the glomeruli. Focal synechiation of
glomerular tuft to the Bowman's capsular epithelium is often
present and may be the only prominent finding; if synechiation is
the only finding, a score of 3 will be assigned if less than 75% of
the glomerular tufts are affected. 4 Moderate to severe disease
with same characteristics as score 3, but affecting 25-50% of the
glomerular tufts 5 Severe disease with same characteristics as
score 3, but affecting 50-75% of the glomerular tufts 6 Severe
disease with same characteristics as score 3, but affecting greater
than 75% of the glomerular tufts Scores 3-6 divide WHO scores III
and IV into four sub-scores
TABLE-US-00006 TABLE 5B Interstitial Inflammation 0 No significant
lesions 1 Minimal to mild inflammation and fibrosis 2 Mild to
moderate inflammation and fibrosis 3 Moderate to severe
inflammation and fibrosis 4 Severe and diffuse inflammation and
fibrosis Scores 0-4, based on density of chronic inflammation
(lymphocytes, plasma cells, and macrophages) with fibrosis within
the interstitium and surrounding renal blood vessels
[0083] Statistics--
[0084] Statistical analysis was conducted using Tukey's multiple
comparison tests to determine whether significant differences
existed between treatment groups. P values equal to or less than
0.05 were accepted as statistically significant.
[0085] Proteinuria and Albuminuria--
[0086] Treatment with ATG or latent TGF-.beta.1 alone (control
Ig+TGF.beta.1) largely failed to inhibit the development of
proteinuria (FIG. 2), although by the end of the study the
incidence of severe proteinuria (>500 mg/dl/day) was slightly
reduced in these single agent treatment groups compared to the mice
treated with control Ig (60-67% vs. 90%, respectively) (FIG. 3). In
contrast, treatment with a combination of ATG and latent
TGF-.beta.1 resulted in marked inhibition in the incidence (30% vs.
90%) and severity of proteinuria, suggesting a synergistic effect
between these two agents (FIGS. 2 and 3).
[0087] The observed reduction in the levels of total protein in the
urine of mice treated with the combination of ATG and latent
TGF-.beta.1 was also reflected in the measurements of urine albumin
levels (FIGS. 4 and 5). ELISA quantitation indicated that the
incidence and severity of album inuria was considerably reduced in
mice treated with the combined therapy as compared either the
negative control Ig group or the single agent therapy groups (ATG,
control Ig+TGF.beta.1).
[0088] Antibodies to dsDNA--
[0089] The majority of mice in the negative control group (normal
rabbit Ig) as well as the latent TGF-.beta.1+control Ig and
ATG-treated groups, gradually developed rising titers of IgG
antibodies against dsDNA with comparable kinetics. By comparison,
there was a considerable delay in the rise of anti-dsDNA titers in
the group treated with the combination of ATG and latent
TGF-.beta.1 (FIG. 6). Deposition of the immune complexes
(DNA-anti-DNA complexes) in the glomeruli is believed to play an
important role in the inflammation and renal pathology
characteristic of lupus. However, the apparent inhibition in the
development of antibodies to dsDNA in the combination treatment
group could not entirely account for the preservation of kidney
function, as there was a poor correlation between titers of
antibodies and degree of proteinuria at the end of the study.
[0090] Survival--
[0091] Dosing with ATG and/or latent TGF-.beta.1 was well tolerated
and did not give rise to any obvious adverse events. All of the
deaths, except for one animal in the latent TGF-.beta.1+control Ig
group, were associated with very high levels of proteinuria and
presumably resulted from kidney failure. All of the treatment
groups showed an overall improvement in survival when compared to
the negative control rabbit Ig-treated group (FIG. 7). The highest
degree of survival (100%) was seen in the cyclophosphamide and the
ATG/latent TGF-.beta.1 combination treatment groups, followed by
the ATG (90%) and latent TGF-.beta.1+control Ig (70%) treatment
groups. The negative control Ig group had only a 40% survival rate
by the end of the study.
[0092] Histology--
[0093] The results of the histological analyses are presented in
Table 6. Mice treated with ATG and latent TGF-.beta.1 exhibited
lesser degrees of glomerulopathy compared to control mice or mice
that received either ATG alone or latent TGF-.beta.1 and control
Ig. These histologic findings correlated with clinical findings of
decreased proteinuria/albuminuria and improved survival in the
combination-treated animals.
[0094] A minimal decrease in inflammation scores was noted in
groups treated with ATG, latent TGF-.beta.1+control Ig and the
combination of ATG and latent TGF-.beta.1 in comparison to the
group treated with control rabbit IgG alone.
TABLE-US-00007 TABLE 6 Treatment Group Glomeruli Score Inflammation
Score Control Rabbit IgG 4.3 .+-. 1.4 2.8 .+-. 0.4 Latent
TGF-.beta.1 3.1 .+-. 0.8 2.6 .+-. 0.5 ATG 2.7 .+-. 0.5 2.4 .+-. 0.5
ATG + Latent TGF-.beta.1 2.3 .+-. 0.7 2.7 .+-. 0.5 Cyclophosphamide
1.0 .+-. 0.7 1.3 .+-. 0.5
[0095] A repeat study was performed with MRUMPJ-Tnfrs6.sup.lpr mice
following the same treatment regimen as described above. In this
instance, the study was extended to 40 weeks of age (as opposed to
24 weeks in the first study) to assess the durability of the effect
of transient treatment with ATG+latent TGF-.beta.1. The results
showed a long-term survival benefit. A 90% survival rate was
observed in mice treated with ATG and latent TGF-.beta.1 as
compared to 30% survival in mice receiving control rabbit Ig, and
10% survival in the group treated with ATG. This compares favorably
with cyclophosphamide which provided 100% survival but required
chronic weekly injections as opposed to a one time transient course
of treatment with ATG+latent TGF-.beta.1 (FIG. 8).
[0096] A similar study was conducted in NZB/NZWF1 mice, another
model of spontaneous lupus. The same treatment regimen was used
and, under these conditions, there was no statistically significant
effect of treatment with ATG and latent TGF-.beta.1 or either agent
alone, on the course or severity of disease. Due to differences in
the characteristics and kinetics of disease between the two models,
it is likely that the treatment regimen needs to be optimized for
the NZB/NZWF1 strain.
[0097] To investigate the mechanism of action underlying the
activity of ATG+TGF-.beta.1, spleen cells from MRL/lpr lupus mice
were cultured in vitro with ATG+/-TGF-.beta.1 and the cells
recovered were analyzed by FACS for the presence of Tregs. Pooled
spleen cells from ten MRL/lpr mice with active disease (.about.25
weeks old) were resuspended at 2.times.10.sup.6 cells/ml in
serum-free AIM-V medium (Gibco, Grand Island, N.Y.) supplemented
with 100 U/ml penicillin, 100 .mu.g/ml streptomycin and 2 mM
glutamine. The cells were cultured in 24-well plates containing 2
ml cells/well under six different conditions (8 wells/condition):
1) cells alone, 2) ATG (100 .mu.g/ml)+active TGF-.beta.1 (10 ng/ml;
Genzyme), 3) ATG alone (100 .mu.g/ml), 4) control rabbit IgG (100
.mu.g/ml)+active TGF-.beta.1 (10 ng/ml), 5) control rabbit IgG
alone (100 .mu.g/ml), and 6) active TGF-.beta.1 alone (10 ng/ml).
Active TGF-.beta.1 was used to mimic the activation process that
would normally occur in vivo. The cells were incubated for five
days at 37.degree. C. and 5% CO.sub.2. Cells from each culture
condition were then pooled, washed in phosphate buffered saline,
counted, and stained for FACS analysis. A total of 5.times.10.sup.5
cells per sample were stained with rat anti-mouse CD4-Alexa 488
(Cat. No. 557667; BD Pharmingen, San Diego, Calif.) and rat
anti-mouse CD25-PerCp-Cy5.5 (Cat. No. 551071; BD Pharmingen). For
intracellular detection of FOXP3, cells stained for surface
CD4/CD25 were permeabilized overnight and stained using the
eBioscience (San Diego, Calif.) FOXP3 staining kit (Cat. No.
72-5775) according to manufacturer's instructions. An acquisition
of 6,000 lymphocytes per treatment was analyzed for staining on a
FACS Calibur system (Becton Dickinson, San Diego, Calif.). Results
are expressed as the absolute number of cells of each phenotype
recovered under each culture condition (percent positive cells by
FACS.times.total number of cells recovered from the culture).
[0098] As shown in FIG. 9, the number of CD4.sup.+ CD25.sup.+ T
cells recovered was the greatest in cultures containing
ATG+TGF-.beta.1. Regulatory T cells typically express a CD4.sup.+
CD25.sup.+ phenotype but activated T cells can also exhibit this
phenotype. Additional FOXP3 staining provides further evidence of a
Treg phenotype and the results obtained confirmed that treatment
with ATG+TGF-.beta.1 produced the greatest number of CD4.sup.+
CD25.sup.+ FOXP3.sup.+ Tregs. Treatment with ATG alone also
appeared to lead to a slight increase in this population (compared
to cells alone) which was enhanced by the addition of TGF-.beta.1.
These results support the hypothesis that treatment with
ATG+TGF-.beta.1 can promote the expansion of Tregs and that such
cells may provide a therapeutic benefit under conditions of
autoimmunity.
Murine Model of Arthritis
[0099] The effect of ATG+/-TGF-.beta.1 was tested in a
collagen-induced arthritis mouse model. To induce disease, DBA/1
mice (Jackson Laboratory) were immunized on day 0 with bovine type
II collagen (Cat. No. 2002-2, Chondrex) in complete Freund's
adjuvant in a 100 .mu.l total volume at the base of the tail. A
booster immunization with collagen in incomplete Freund's adjuvant
was given on day 22. Treatment with ATG or control rabbit IgG
consisted of two intraperitoneal (i.p.) injections of 500 .mu.g
(.about.25 mg/kg) delivered three days apart (days 23 and 26).
Latent TGF-.beta.1 was given from days 28-37 as ten daily i.p.
injections of 4 .mu.g per mouse. A 4 .mu.g dose of latent
TGF-.beta.1 corresponds to a 1 .mu.g (.about.0.05 mg/kg) dose of
the active (mature, non-LAP-associated) portion of the molecule.
The treatment groups included (1) control rabbit IgG, (2) control
rabbit IgG+latent TGF-.beta.1, (3) ATG, and (4) ATG+latent
TGF-.beta.1, with ten animals per group. Starting on day 21,
individual mice were examined and scored for clinical signs of
disease 2-3 times per week. The arthritic score scale is defined in
Table 7.
TABLE-US-00008 TABLE 7 Severity Score Gross Pathology 0 No evidence
of erythema and swelling 1 Erythema and mild swelling confined to
the mid-foot (tarsals) or ankle joint 2 Erythema and mild swelling
extending from the ankle to the mid foot 3 Erythema and moderate
swelling extending from the ankle to the metatarsal joints 4
Erythema and severe swelling encompass the ankle, foot, and digits
Each paw is assessed individually and the arthritic score for a
given mouse is the sum of the scores for all paws (maximum score of
16)
[0100] Treatment with ATG alone resulted in a reduction in disease
scores and the addition of latent TGF-.beta.1 did not appear to
provide an additional benefit under the conditions tested. The
results are shown in Table 8.
TABLE-US-00009 TABLE 8 Arthritic Score (Mean .+-. SEM) Rabbit Ig +
ATG + Day Rabbit Ig Latent TGF-.beta.1 ATG Latent TGF-.beta.1 22
0.0 .+-. 0.0 0.0 .+-. 0.0 0.0 .+-. 0.0 0.0 .+-. 0.0 26 2.7 .+-. 2.3
3.7 .+-. 2.5 2.8 .+-. 2.6 5.0 .+-. 3.5 29 10.0 .+-. 4.2 10.1 .+-.
4.3 3.4 .+-. 4.9 5.8 .+-. 5.3 32 10.5 .+-. 4.4 11.6 .+-. 3.0 5.5
.+-. 6.0 6.6 .+-. 5.1 35 10.5 .+-. 4.4 12.0 .+-. 2.7 6.5 .+-. 5.9
7.6 .+-. 4.3 39 10.9 .+-. 3.9 12.9 .+-. 2.0 7.0 .+-. 5.5 8.1 .+-.
3.3 41 11.0 .+-. 4.1 12.5 .+-. 2.8 7.3 .+-. 5.2 8.8 .+-. 3.6 43
11.7 .+-. 3.7 13.4 .+-. 1.8 7.5 .+-. 5.4 8.9 .+-. 3.8 46 11.5 .+-.
4.5 13.4 .+-. 2.8 9.5 .+-. 5.1 9.1 .+-. 3.5 50 12.7 .+-. 3.0 13.5
.+-. 2.1 9.5 .+-. 4.9 9.0 .+-. 3.7 53 12.4 .+-. 4.0 13.8 .+-. 2.1
9.6 .+-. 4.8 9.3 .+-. 3.7 57 12.4 .+-. 4.0 13.8 .+-. 2.1 9.6 .+-.
4.8 9.3 .+-. 3.7
[0101] The collagen-induced arthritis is a short-term animal model,
in which the treatment takes place on a timescale of weeks, versus
months for the lupus model. This shorter timescale might be
insufficient to observe the benefit added by administering
TGF-.beta.1 with the ATG, which was seen in the lupus model. Thus,
different dosing regimens or further testing of additional animal
models may show benefits of combined administration of ATG and
latent TGF-6.
Murine Model of Uveitis
[0102] The effect of ATG+/-TGF-.beta.1 was tested in a mouse model
of uveitis. To induce disease, B10.RIII mice (Jackson Laboratory)
were immunized subcutaneously on day 0 with 100 .mu.g of amino
acids 161-180 of human interphotoreceptor retinoid binding protein
(IRBP.sub.161-180) (custom synthesis, New England Peptide) in
complete Freund's adjuvant at two sites (between shoulder blades
and in pelvic region). Starting on day 10, funduscopic examinations
were performed on individual mice and a disease score was assigned.
To perform the examination, the eyes of mice were dilated using one
or two drops of Mydriacyl.TM. 1% (Cat. No. 1120, JA Webster) and
rested in a darkened room for approximately five minutes. Mice were
manually restrained and the retinas of both eyes visualized using
an indirect ophthalmoscope with a 78 diopter lens. The eyes were
scored for inflammation using a progressive scoring system between
0 and 5, as described in Table 9.
TABLE-US-00010 TABLE 9 Score Gross Pathology 0 Normal retina 1
Vascular inflammation proximal to the optic nerve 2 >10
inflammatory lesions confined to one quadrant of the eye 3 >10
inflammatory lesions in more than one quadrant of the eye 4
Inflammatory lesions are contiguous 5 Retinal detachment
[0103] Treatment with ATG or control rabbit IgG was initiated at
disease onset (score of 1) and consisted of two i.p. injections of
500 .mu.g (.about.25 mg/kg) delivered four days apart (days 10 and
14). Latent TGF-.beta.1 was given from days 15-27 as thirteen daily
i.p. injections of 4 .mu.g per mouse. A 4 .mu.g dose of latent
TGF-.beta.1 corresponds to a 1 .mu.g (.about.0.05 mg/kg) dose of
the active (mature, non-LAP-associated) portion of the molecule.
The treatment groups included (1) phosphate buffered saline (PBS)
control (2) control rabbit IgG, (3) control rabbit IgG+latent
TGF-.beta.1, (4) ATG, and (5) ATG+latent TGF-.beta.1, with six
animals per group. Treatment with ATG alone resulted in a reduction
in disease scores and the addition of latent TGF-.beta.1 did not
appear to provide an additional benefit under the conditions
tested. The results are shown in Table 10.
TABLE-US-00011 TABLE 10 Uveitis Score (Mean .+-. SEM) Rabbit Ig +
ATG + Day PBS Rabbit Ig Latent TGF-.beta.1 ATG Latent TGF-.beta.1 8
0.00 .+-. 0.00 0.00 .+-. 0.00 0.00 .+-. 0.00 0.00 .+-. 0.00 0.00
.+-. 0.00 10 1.25 .+-. 0.16 1.22 .+-. 0.15 1.29 .+-. 0.18 1.13 .+-.
0.13 1.13 .+-. 0.23 14 4.00 .+-. 0.00 3.17 .+-. 0.32 3.67 .+-. 0.22
2.10 .+-. 0.48 2.17 .+-. 0.34 17 3.81 .+-. 0.10 3.00 .+-. 0.35 3.58
.+-. 0.29 1.75 .+-. 0.37 2.00 .+-. 0.37 21 3.81 .+-. 0.10 2.58 .+-.
0.34 3.33 .+-. 0.33 1.92 .+-. 0.42 2.08 .+-. 0.31 24 3.94 .+-. 0.06
2.58 .+-. 0.42 3.50 .+-. 0.34 2.00 .+-. 0.41 1.92 .+-. 0.36 29 3.75
.+-. 0.11 2.27 .+-. 0.47 3.33 .+-. 0.33 2.08 .+-. 0.42 2.00 .+-.
0.40 35 3.88 .+-. 0.09 2.25 .+-. 0.46 3.33 .+-. 0.40 1.92 .+-. 0.40
2.00 .+-. 0.35 46 3.56 .+-. 0.13 2.09 .+-. 0.41 3.25 .+-. 0.39 2.00
.+-. 0.44 1.92 .+-. 0.40 56 3.69 .+-. 0.12 2.17 .+-. 0.44 3.25 .+-.
0.39 1.67 .+-. 0.38 1.75 .+-. 0.25
[0104] The uveitis model is a short-term animal model, in which the
treatment takes place on a timescale of weeks, versus months for
the lupus model. This shorter timescale might be insufficient to
observe the benefit added by administering TGF-.beta.1 with the
ATG, which was seen in the lupus model. Thus, different dosing
regimens or further testing of additional animal models may show
benefits of combined administration of ATG and latent
TGF-.beta..
[0105] All publications, patents, patent applications, and
biological sequences cited in this disclosure are incorporated by
reference in their entirety.
Sequence CWU 1
1
31391PRTHomo sapiens 1Met Pro Pro Ser Gly Leu Arg Leu Leu Pro Leu
Leu Leu Pro Leu Leu 1 5 10 15 Trp Leu Leu Val Leu Thr Pro Gly Pro
Pro Ala Ala Gly Leu Ser Thr 20 25 30 Cys Lys Thr Ile Asp Met Glu
Leu Val Lys Arg Lys Arg Ile Glu Ala 35 40 45 Ile Arg Gly Gln Ile
Leu Ser Lys Leu Arg Ile Ala Ser Pro Pro Ser 50 55 60 Gln Gly Glu
Val Pro Pro Gly Pro Leu Pro Glu Ala Val Leu Ala Leu 65 70 75 80 Tyr
Asn Ser Thr Arg Asp Arg Val Ala Gly Glu Ser Ala Glu Pro Glu 85 90
95 Pro Glu Pro Glu Ala Asp Tyr Tyr Ala Lys Glu Val Thr Arg Val Leu
100 105 110 Met Val Glu Thr His Asn Glu Ile Tyr Asp Lys Phe Lys Gln
Ser Thr 115 120 125 His Ser Ile Tyr Met Phe Phe Asn Thr Ser Glu Leu
Arg Glu Ala Val 130 135 140 Pro Glu Pro Val Leu Leu Ser Arg Ala Glu
Leu Arg Leu Leu Arg Arg 145 150 155 160 Leu Lys Leu Lys Val Glu Gln
His Val Glu Leu Tyr Gln Lys Tyr Ser 165 170 175 Asn Asn Ser Trp Arg
Tyr Leu Ser Asn Arg Leu Leu Ala Pro Ser Asp 180 185 190 Ser Pro Glu
Trp Leu Ser Phe Asp Val Thr Gly Val Val Arg Gln Trp 195 200 205 Leu
Ser Arg Gly Gly Glu Ile Glu Gly Phe Arg Ile Ser Ala His Cys 210 215
220 Ser Cys Asp Ser Arg Asp Asn Thr Leu Gln Val Asp Ile Asn Gly Phe
225 230 235 240 Thr Thr Gly Arg Arg Gly Asp Leu Thr Ala Ile His Gly
Met Asn Arg 245 250 255 Pro Phe Leu Leu Leu Met Ala Thr Pro Leu Glu
Arg Ala Gln His Leu 260 265 270 Gln Ser Ser Arg His Arg Arg Ala Leu
Asp Thr Asn Tyr Cys Phe Ser 275 280 285 Ser Thr Glu Lys Asn Cys Cys
Val Arg Gln Leu Tyr Ile Asp Phe Arg 290 295 300 Lys Asp Leu Gly Trp
Lys Trp Ile His Glu Pro Lys Gly Tyr His Ala 305 310 315 320 Asn Phe
Cys Leu Gly Pro Cys Pro Tyr Ile Trp Ser Ile Asp Thr Gln 325 330 335
Tyr Ser Lys Val Leu Ala Leu Tyr Asn Gln His Asn Pro Gly Ala Ser 340
345 350 Ala Ala Pro Cys Cys Val Pro Gln Ala Leu Glu Pro Leu Pro Ile
Val 355 360 365 Tyr Tyr Val Gly Arg Lys Pro Lys Val Glu Gln Leu Ser
Asn Met Ile 370 375 380 Val Arg Ser Cys Lys Cys Ser 385 390
2442PRTHomo sapiens 2Met His Tyr Cys Val Leu Ser Ala Phe Leu Ile
Leu His Leu Val Thr 1 5 10 15 Val Ala Leu Ser Leu Ser Thr Cys Ser
Thr Leu Asp Met Asp Gln Phe 20 25 30 Met Arg Lys Arg Ile Glu Ala
Arg Ile Gly Gln Ile Leu Ser Lys Leu 35 40 45 Lys Ile Thr Ser Pro
Pro Glu Asp Tyr Pro Glu Pro Glu Glu Val Pro 50 55 60 Pro Glu Val
Ile Ser Ile Tyr Asn Ser Thr Arg Asp Leu Leu Gln Glu 65 70 75 80 Lys
Ala Ser Arg Arg Ala Ala Ala Cys Glu Arg Glu Arg Ser Asp Glu 85 90
95 Glu Tyr Tyr Ala Lys Glu Val Tyr Lys Ile Asp Met Pro Pro Phe Phe
100 105 110 Pro Ser Glu Thr Val Cys Pro Val Val Thr Thr Pro Ser Gly
Ser Val 115 120 125 Gly Ser Leu Cys Ser Arg Gln Ser Gln Val Leu Cys
Gly Tyr Leu Asp 130 135 140 Ala Ile Pro Pro Thr Phe Tyr Arg Pro Tyr
Phe Arg Ile Val Arg Phe 145 150 155 160 Asp Val Ser Ala Met Glu Lys
Asn Ala Ser Asn Leu Val Lys Ala Glu 165 170 175 Phe Arg Val Phe Arg
Leu Gln Asn Pro Lys Ala Arg Val Pro Glu Gln 180 185 190 Arg Ile Glu
Leu Tyr Gln Ile Leu Lys Ser Lys Asp Leu Thr Ser Pro 195 200 205 Thr
Gln Arg Tyr Ile Asp Ser Lys Val Val Lys Thr Arg Ala Glu Gly 210 215
220 Glu Trp Leu Ser Phe Asp Val Thr Asp Ala Val His Glu Trp Leu His
225 230 235 240 His Lys Asp Arg Asn Leu Gly Phe Lys Ile Ser Leu His
Cys Pro Cys 245 250 255 Cys Thr Phe Val Pro Ser Asn Asn Tyr Ile Ile
Pro Asn Lys Ser Glu 260 265 270 Glu Leu Glu Ala Arg Phe Ala Gly Ile
Asp Gly Thr Ser Thr Tyr Thr 275 280 285 Ser Gly Asp Gln Lys Thr Ile
Lys Ser Thr Arg Lys Lys Asn Ser Gly 290 295 300 Lys Thr Pro His Leu
Leu Leu Met Leu Leu Pro Ser Tyr Arg Leu Glu 305 310 315 320 Ser Gln
Gln Thr Asn Arg Arg Lys Lys Arg Ala Leu Asp Ala Ala Tyr 325 330 335
Cys Phe Arg Asn Val Gln Asp Asn Cys Cys Leu Arg Pro Leu Tyr Ile 340
345 350 Asp Phe Lys Arg Asp Leu Gly Trp Lys Trp Ile His Glu Pro Lys
Gly 355 360 365 Tyr Asn Ala Asn Phe Cys Ala Gly Ala Cys Pro Tyr Leu
Trp Ser Ser 370 375 380 Asp Thr Gln His Ser Arg Val Leu Ser Leu Tyr
Asn Thr Ile Asn Pro 385 390 395 400 Glu Ala Ser Ala Ser Pro Cys Cys
Val Ser Gln Asp Leu Glu Pro Leu 405 410 415 Thr Ile Leu Tyr Tyr Ile
Gly Lys Thr Pro Lys Ile Glu Gln Leu Ser 420 425 430 Asn Met Ile Val
Lys Ser Cys Lys Cys Ser 435 440 3412PRTHomo sapiens 3Met Lys Met
His Leu Gln Arg Ala Leu Val Val Leu Ala Leu Leu Asn 1 5 10 15 Phe
Ala Thr Val Ser Leu Ser Leu Ser Thr Cys Thr Thr Leu Asp Phe 20 25
30 Gly His Ile Lys Lys Lys Arg Val Glu Ala Ile Arg Gly Gln Ile Leu
35 40 45 Ser Lys Leu Arg Ile Thr Ser Pro Pro Glu Pro Thr Val Met
Thr His 50 55 60 Val Pro Tyr Gln Val Leu Ala Leu Tyr Asn Ser Thr
Arg Glu Leu Leu 65 70 75 80 Glu Glu Met His Gly Glu Arg Glu Glu Gly
Cys Thr Gln Glu Asn Thr 85 90 95 Glu Ser Glu Tyr Tyr Ala Lys Glu
Ile His Lys Phe Asp Met Ile Gln 100 105 110 Gly Leu Ala Glu His Asn
Glu Leu Ala Val Cys Pro Lys Gly Ile Thr 115 120 125 Ser Lys Val Phe
Arg Phe Asn Val Ser Ser Val Glu Lys Asn Arg Thr 130 135 140 Asn Leu
Phe Arg Ala Glu Phe Arg Val Leu Arg Val Pro Asn Pro Ser 145 150 155
160 Ser Lys Arg Asn Glu Gln Arg Ile Glu Leu Phe Gln Ile Leu Arg Pro
165 170 175 Asp Glu His Ile Ala Lys Gln Arg Tyr Ile Gly Gly Lys Asn
Leu Pro 180 185 190 Thr Arg Gly Thr Ala Glu Trp Leu Ser Phe Asp Val
Thr Asp Thr Val 195 200 205 Arg Glu Trp Leu Leu Arg Arg Glu Ser Asn
Leu Gly Leu Glu Ile Ser 210 215 220 Ile His Cys Pro Cys His Thr Phe
Gln Pro Asn Gly Asp Ile Leu Glu 225 230 235 240 Asn Ile His Glu Val
Met Glu Ile Lys Phe Lys Gly Val Asp Asn Glu 245 250 255 Asp Asp His
Gly Arg Gly Asp Leu Gly Arg Leu Lys Lys Gln Lys Asp 260 265 270 His
His Asn Pro His Leu Ile Leu Met Met Ile Pro Pro His Arg Leu 275 280
285 Asp Asn Pro Gly Gln Gly Gly Gln Arg Lys Lys Arg Ala Leu Asp Thr
290 295 300 Asn Tyr Cys Phe Arg Asn Leu Glu Glu Asn Cys Cys Val Arg
Pro Leu 305 310 315 320 Tyr Ile Asp Glu Arg Gln Asp Leu Gly Trp Lys
Trp Val His Glu Pro 325 330 335 Lys Gly Tyr Tyr Ala Asn Phe Cys Ser
Gly Pro Cys Pro Tyr Leu Arg 340 345 350 Ser Ala Asp Thr Thr His Ser
Thr Val Leu Gly Leu Tyr Asn Thr Leu 355 360 365 Asn Pro Glu Ala Ser
Ala Ser Pro Cys Cys Val Pro Gln Asp Leu Glu 370 375 380 Pro Leu Thr
Ile Leu Tyr Tyr Val Gly Arg Thr Pro Lys Val Glu Gln 385 390 395 400
Leu Ser Asn Met Val Val Lys Ser Cys Lys Cys Ser 405 410
* * * * *