U.S. patent application number 10/349464 was filed with the patent office on 2003-07-17 for models of chronic and acute inflammatory diseases.
Invention is credited to Ehrhardt, Rolf, Hong, Kenneth.
Application Number | 20030135875 10/349464 |
Document ID | / |
Family ID | 22756105 |
Filed Date | 2003-07-17 |
United States Patent
Application |
20030135875 |
Kind Code |
A1 |
Ehrhardt, Rolf ; et
al. |
July 17, 2003 |
Models of chronic and acute inflammatory diseases
Abstract
Methods and compositions are provided for the creation and
screening of non-human animal models having chronic inflammation.
Immunocompromised host animals are injected with a population of
immunocompetent effector cells, depleted of CD25+ T cells. The
effector cells are tolerant of the host major histocompatibility
antigens, but reactive to at least one antigen present in the host
animal. The transferred cells are preferably stimulated and
localized by administration of an immunostimulant at a local site.
The animals are useful for a variety of screening assays and for
investigation into disease causes and pathways. A variety of
chronic inflammatory diseases may be studied with this model,
including psoriasis, rheumatoid arthritis, diabetes, inflammatory
bowel disease and multiple sclerosis.
Inventors: |
Ehrhardt, Rolf; (San
Francisco, CA) ; Hong, Kenneth; (El Cerrito,
CA) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
200 MIDDLEFIELD RD
SUITE 200
MENLO PARK
CA
94025
US
|
Family ID: |
22756105 |
Appl. No.: |
10/349464 |
Filed: |
January 21, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10349464 |
Jan 21, 2003 |
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09852448 |
May 9, 2001 |
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60203982 |
May 12, 2000 |
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Current U.S.
Class: |
800/18 ;
800/9 |
Current CPC
Class: |
G01N 33/5088 20130101;
A01K 67/0271 20130101; G01N 33/505 20130101 |
Class at
Publication: |
800/18 ;
800/9 |
International
Class: |
A01K 067/027 |
Claims
What is claimed is:
1. A non-human mammal comprising: exogenous immunocompetent
effector cells, wherein said effector cells were depleted of cells
expressing CD25 prior to introduction into said non-human mammal;
antigen presenting cells to which said immunocompetent effector
cells are tolerant and which are capable of initiating an
inflammatory response by said immunocompetent effector cells; and
inflamed tissue as a result of said inflammatory response.
2. The non-human mammal according to claim 1, wherein said
immunocompetent effector cells comprise human T cells.
3. The non-human mammal of claim 2, wherein said T cells comprise
CD4.sup.+ T cells.
4. The non-human mammal of claim 3, wherein said mammal is a
rodent.
5. The non-human mammal of claim 4, wherein said rodent is a
mouse.
6. A panel for compound testing, comprising at least mammals
according to claim 1, wherein at least one of said mammals
comprises a known immunomodulatory compound, and at least one of
said mammals comprises a test compound suspected of
immunomodulatory activity.
7. A method for inducing chronic inflammation in an non-human
mammal, the method comprising: transferring a cell population
comprising immunocompetent effector cells and lacking CD25 positive
T cells, from a donor non-human mammal to an immunocompromised
non-human mammal host, wherein said immunocompetent effector cell
population is tolerant of the host major histocompatibility
antigens but is immunoreactive with one or more antigens present in
said host; wherein said host develops chronic inflammation.
8. The method according to claim 7, wherein said immunocompetent
effector cells comprise T cells.
9. The method according to claim 8, wherein said T cells comprise
CD4+ T cells.
10. The method according to claim 9, further comprising
administering an immunostimulatory co-factor to said mammal.
11. The method according to claim 9, wherein said CD4.sup.+ T cells
are reactive to minor histocompatibility antigens present in said
host.
12. The method according to claim 10, wherein said immunostimulant
is administered at a targeted site, and said chronic inflammation
develops at said targeted site.
13. The method of claim 7, wherein said host is a rodent.
14. The method of claim 13, wherein said rodent is a scid-scid
mouse.
15. The method of claim 10, wherein said immunostimulant is a
non-replicating virus.
16. The method of claim 15, wherein said virus is an
adenovirus.
17. The method of claim 10, wherein said immunostimulant is an
immunostimulatory oligonucleotide sequence.
18. The method of claim 10, wherein said immunostimulant is a
polyclonal activating agent.
19. The method of claim 18, wherein said polyclonal activating
agent is an endotoxin.
20. T he method of claim 18, wherein said polyclonal activating
agent is a superantigen.
21. The method of claim 20, wherein said superantigen is a
bacterial superantigen.
22. A method for screening a candidate therapy for efficacy in
treatment of chronic inflammation, the method comprising:
transferring a cell population comprising immunocompetent effector
cells and lacking CD25 positive T cells, from a donor non-human
mammal to an immunocompromised non-human mammal host, wherein said
immunocompetent effector cell population is tolerant of the host
major histocompatibility antigens but is immunoreactive with one or
more antigens present in said host; wherein said host develops
chronic inflammation; treating said animals with said candidate
therapy; determining the severity of disease in the presence of
said therapy, wherein a decrease in severity of disease in the
treated animals relative to control animals is indicative of
efficacy in treatment.
23. The method according to claim 22, wherein said immunocompetent
effector cells comprise T cells.
24. The method according to claim 23, wherein said T cells comprise
CD4+ T cells.
25. The method according to claim 22, further comprising
administering an immunostimulatory co-factor to said mammal.
26. The method according to claim 22, wherein said CD4.sup.+ T
cells are reactive to minor histocompatibility antigens present in
said host.
27. The method according to claim 10, wherein said immunostimulant
is administered at a targeted site, and said chronic inflammation
develops at said targeted site.
28. The method according to claim 22, wherein said candidate
therapy comprises administration of one or a combination of
candidate immunosuppressant drugs.
29. The method according to claim 22, further comprises comparison
of said disease severity to a positive control animal treated with
a known immunomodulatory compound.
Description
BACKGROUND OF THE INVENTION
[0001] Despite recent advances in genomic sequencing efforts, as
well as in the fields of pre-clinical drug screening/development
and clinical trial design, the transfer of existing "pre-clinical"
knowledge into the clinic is still very difficult. This is mainly
due to the sparse knowledge of the events that occur during the
initiation, the perpetuation and the maintenance of inflammatory
disease states in humans.
[0002] The reasons for such incomplete and often low quality
information are numerous: humans cannot intentionally be studied in
the pre-clinical phase, cell isolation is difficult from human
tissue, the starting events of an autoimmune reaction occur without
notice, and patients with autoimmune or other inflammatory diseases
may not wish to be treated as experimental subjects. As a result,
there is a lack of reliable information on which to base decisions
about clinical trials. When clinical symptoms arise and treatment
is required, rational selection from among the many potential
anti-inflammatory compounds or combinations thereof is
difficult.
[0003] In order to identify new and useful drugs, screening assays
must be able to provide biologically relevant information, so that
there is a good correlation between the information generated by
the screening assay and the pharmaceutical effectiveness of the
compound. Some of the more important features for pharmaceutical
effectiveness are specificity for the targeted cell or disease, a
lack of toxicity at relevant dosages, and specific activity of the
compound against its molecular or cellular target.
[0004] Inflammatory conditions, particularly chronic inflammatory
diseases, are of particular interest. These diseases are caused by
the action of the immune system, including the inappropriate
activation of T cells, expression of regulatory cytokines and
chemokines, loss of immune tolerance, and the like. Modulation of
the immune response varies with the specific factors produced, and
the receptors present on the responding cell.
[0005] Among these diseases are autoimmune and/or chronic
inflammatory diseases, which include multiple sclerosis and
inflammatory bowel diseases (IBD, ulcerative colitis and Crohn's
disease), colitis, diseases of the joints, such as rheumatoid
arthritis, attacks on nucleic acids, as observed with systemic
lupus erythematosus and such other diseases as psoriasis, insulin
dependent diabetes mellitus (IDDM), Sjogren's disease, myasthenia
gravis, thyroid disease, Alzheimer disease, uveitis, and
cardiovascular diseases.
[0006] The initiating step in autoimmune disease pathology is still
mysterious in many cases, particularly in humans where the diseases
are largely sporadic, and symptoms may appear years after the first
T cell launches its attack. It has therefore been difficult to
design effective therapies that prevent initiation of disease,
although there are common features in many of the later stages of
disease. Inflammation at the site of the disease is often found,
caused by the release of inflammatory cytokines by T cells and
other pro-inflammatory cells (e.g. macrophages, dendritic cells, B
cells, NK cells), and accompanied by the destruction of autologous
cells.
[0007] Recent studies using murine models of experimental chronic
inflammation are defining the nature of the immunological
disturbances that initiate inflammation and destruction of specific
organs (for example, see Mombaerts et al. Cell, 1993. 75(2): p.
274-82; Tarrant et al. J Immunol, 1998. 161(1): p. 122-7; Powrie et
al. Immunity, 1994. 1: p. 553-562; Hong et al. J Immunol, 1999.
162(12): p. 7480-91; Horak, Clin Immunol Immunopathol, 1995. 76(3
Pt 2): p. S172-3; Ehrhardt et al. J Immunol, 1997. 158(2): p.
566-73; Davidson et al., J Immunol, 1998. 161(6): p. 3143-9; Kuhn
et al. Cell, 1993. 75(2): p. 263-74; Neurath et al., J Exp Med,
1995 182(5): p. 1281-90). Increased understanding of disease
promoting inflammatory cells is providing insights into the
mechanism controlling the immune responses within target
organs.
[0008] Evidence has been presented in the literature for the
involvement of different T cell subsets in the development of
disease. An important role for a distinct T cell population
including regulatory and/or suppressor T cells in maintaining the
physical integrity of organ specific immunity has been suggested by
recent several studies (Suri-Payer et al., J Immunol, 1998. 160(3):
p. 1212-8; Shevach et al., Novartis Found Symp, 1998. 215: p.
200-11). These investigators and others (Shimizu et al, J Immunol,
1999. 163(10): p. 5211-8; Itoh et al., J Immunol, 1999. 162(9): p.
5317-26; Sakaguchi et al. J Immunol, 1995. 155(3): p. 1151-64;
Takahashi et al., Int Immunol, 1998. 10(12): p. 1969-80) have
postulated that CD4+ CD25+ T cells play a crucial role in the
suppression of immune responses and one might postulate if a cell
population is transferred into an immunodeficient mouse without its
suppressor CD25+ subset, autoimmunity can occur at multiple sites
of the body. This presumes of course that autoimmune causing
effector cells are able to reach their target organ. Such an
effector cell permissive environment is probably created through
the upregulation of adhesion molecules (Berg et al., Immunol Rev,
1989. 108: p. 5-18; von Andrian et al., Proc Natl Acad Sci USA,
1991. 88(17): p. 7538-42; Berg et al., J Exp Med, 1991. 174(6): p.
1461-6; Picker et al. J Immunol, 1990. 145(10): p. 3247-55) and the
secretion of chemokines (Baggiolini, Nature, 1998. 392(6676): p.
565-8) on the affected tissues, and on endothelial cells allowing
the entrance and retention of effector cells into the tissue.
[0009] To study the regulatory effects of T cells and other
immunocompetent cells, animal models have provided a very good tool
in the past. An essential role for the study of human autoimmune
conditions was played in particular by the scid/scid
CD4.sup.+CD45Rb.sup.hi cell transfer model. Over the last decade
this model has proven to be a viable scientific tool for the study
of dysregulated immune responses, and moreover, has been proven to
be a good tool for the discovery and evaluation of treatment/drug
targets, candidates for inflammatory bowel disease and recently
psoriasis (Hong et al., supra.; Powrie et al., J Exp Med, 1996.
183(6): p. 2669-74; Schon et al., Nat Med, 1997. 3(2): p. 183-8).
Notably, not only do these animal models resemble human histology
and physiology in some ways or another, but have been helpful in
determining novel treatment strategies in humans for both
diseases.
[0010] One major disadvantage of conventional animal models is that
they are very labor-intensive and costly and thus do not permit
large throughput drug screening. Unfortunately, in vitro screening
techniques are limited in their predictive power. Thus, despite
today's advances in pre-clinical science, hard decisions must be
made without complete pre-clinical, in vivo data.
[0011] With drug discovery moving from target identification to
validations, reliable biological systems are necessary to confirm,
validate and support the recent explosion in the number of
potential new drug targets and drug compounds. The development of
robust, reproducible and scaleable animal models that
physiologically resemble human disease is very desirable; i.e.
models in which the inflammation is truly chronic in nature and the
histology that of human, and can be used as treatment models and
not only preventive ones. Such animal models must posses the
utility to rapidly advance experimental drug leads rapidly and
reliably in a semi- to high through-put fashion, leading to novel,
effective and safe therapeutics.
SUMMARY OF THE INVENTION
[0012] Models are provided for chronic inflammatory diseases. The
models are useful for testing and screening of biologically active
agents for the treatment of chronic and acute inflammatory disease.
A cell population comprising immunocompetent effector cells, which
lacks CD25.sup.+ suppressor T cells, is transferred into a cellular
environment that lacks CD25.sup.+ suppressor T cells but contains a
T cell antigen. Preferably, an immunostimulant and/or
immunomodulatory co-factor and/or T cell antigen is introduced at a
targeted site or organ after the T cell introduction to enhance T
cell response and homing. Animals develop acute and chronic
inflammatory responses at the targeted site, and provide a useful
model for the development of inflammation, and for drug/gene
screening in the prevention and treatment of chronic inflammatory
disease in humans.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a graph depicting disease penetration.
[0014] FIGS. 2A and 2B are graphs depicting the disease scalability
and distribution. Each symbol represents a single ear measurement.
(n=10).
[0015] FIG. 3 is a graph demonstrating the chronicity of the
induced disease. Error bars=SEM. A group of animals are considered
diseased if average ear thickness >25 .mu.m.
[0016] FIG. 4 is a graph illustrating the effects of a co-factor in
disease induction. The disease induction protocol was modified to
examine animals with and without LPS co-injection.
[0017] FIG. 5 shows the effects of anti-IL-12 mAb treatment.
[0018] FIG. 6 is a graph depicting antibody screening against the
inflammation model of the invention.
[0019] FIG. 7 is a graph depicting the effect of anti-CD43
monoclonal antibody in the inflammation model of the invention.
[0020] FIG. 8 is a graph depicting the effect of an oral compound
on disease progression.
[0021] FIG. 9 shows the effect of cyclosporin on disease
progression.
[0022] FIG. 10 shows the effect of different co-factors on disease
induction.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0023] Non-human animal models for chronic inflammatory disease are
provided. The animals are particularly suited as models for T cell
mediated autoimmune diseases, such as multiple sclerosis, insulin
dependent diabetes mellitus, rheumatoid arthritis, and the
like.
[0024] An immunocompromised host animal is injected with a
population of immune cells depleted of CD25.sup.+ cells, from a
donor animal of the same or related species. The transferred cells
may comprise T cells, natural killer (NK) cells, monocytes, etc.
The cell population may be further selected to enrich for T cell
types of interest. For example, the calls may be selected to have
the phenotype CD4.sup.+CD25.sup.-. The CD25.sup.+ population
contains suppressive T cells that act to down-regulate the T cell
response. By transferring a population of CD25.sup.- cells, the
immune responsiveness of the cells is increased.
[0025] The host and donor animals are matched, and/or tolerant at
the host major histocompatibility antigens, e.g., are of the same
MHC haplotype (MHC matched) but the transferred T cells are
responsive to at least one antigen present in the recipient, e.g.
are mismatched at one or more minor antigens, or otherwise
responsive to the presence of a T cell antigen. For example, a
mismatch at minor histocompatibility loci provides the antigenic
immune stimulation for development of chronic inflammation. In a
preferred embodiment, an immunostimulant/modulatory co-factor is
administered at a targeted site, in order to localize the effector
T cells.
[0026] These animals provide a useful model for the specific
pathogenic requirements of Th1 promoting cytokines and cells. By
providing a more accurate model for the human disease, potential
therapeutics can be evaluated in the animal model for safety and
efficacy prior to clinical trials. In addition to screening
candidate pharmaceutical agents, the subject animals are useful in
determining the role of "triggering" agents in development of
disease, the role of specific T cell subsets and cytokines, and the
role of specific antigens in activation and maintenance of
inflammatory T cells.
[0027] Immunocompromised mammalian hosts suitable for implantation
and having the desired immune incapacity exist or can be created.
The significant factor is that the immunocompromised host is
incapable of mounting an immune response against the introduced
pathogenic effector T cells. Preferred host animals will lack CD25+
T cells. Of particular interest are small mammals, e.g. rabbits,
gerbils, hamsters, guinea pigs, etc., particularly rodents, e.g.
mouse and rat, which are immunocompromised due to a genetic defect
that results in an inability to undergo germline DNA rearrangement
at the loci encoding immunoglobulins and T-cell antigen receptors
or to a genetic defect in thymus development (nu/nu).
[0028] Presently available hosts include mice that have been
genetically engineered by transgenic disruption to lack the
recombinase function associated with RAG-1 and/or RAG-2 (e.g.
commercially available TIM.TM. RAG-2 transgenic), to lack Class I
and/or Class II MHC antigens (e.g. the commercially available C1D
and C2D transgenic strains), or to lack expression of the Bcl-2
proto-oncogene. Of particular interest are mice that have a
homozygous mutation at the scid locus, causing a severe combined
immunodeficiency which is manifested by a lack of functionally
recombined immunoglobulin and T-cell receptor genes. The scid/scid
mutation is available or may be bred into a number of different
genetic backgrounds, e.g. CB.17, ICR (outbred), C3H, BALB/c,
C57BI/6, AKR, BA, B10, 129, etc. Other mice which are useful as
recipients are NOD scid/scid; SGB scid/scid, bh/bh; CB.17 scid/hr;
NIH-3 bg/nu/xid and META nu/nu. Transgenic mice, rats and pigs are
available which lack functional B cells and T cells due to a
homozygous disruption in the CD3.sub..epsilon. gene.
Immunocompromised rats include HsdHan:RNU-rnu; HsdHan:RNU-rnu/+;
HsdHan:NZNU-rnu; HsdHan,:NZNU-rnu/+; LEW/HanHsd-rnu;
LEW/HanHsd-rnu/+; WAG/HanHsd-rnu and WAG/HanHsd-rnu/+.
[0029] Generally, the host will be at least about four weeks old.
For example, mice are often used at about 4 to 12 weeks of age. The
mammalian host will be grown in conventional ways. Depending on the
degree of immunocompromised status of the mammalian host, it may be
protected to varying degrees from infection. An aseptic environment
is indicated. Prophylactic antibiosis may be used for protection
from infection. Alternatively, it may be satisfactory to isolate
the potential hosts from other animals in gnotobiotic environments
after cesarean derivation. The feeding and maintenance of the host
will for the most part follow gnotobiotic techniques.
[0030] The major histocompatibility locus haplotype of the host
animal is determined either through conventional typing methods,
e.g. where outbred animals are used, or from known information
concerning the genetic characteristics of the animal. In mice, the
genes of the major histocompatibility locus (MHC) have been very
well characterized. The MHC region is comprised of a number of
genes, of which at least five contribute to acute graft rejection
and graft vs. host disease. The specific MHC genes of interest
include the class I antigens: H2-K, H2-D, and H2-L; and the class
II antigens: H2 I region, which includes H2-Aa, Ab, BI, Ea, Eb,
Eb2, Ob, and Pb. Specific information on the haplotype of most
known mouse strains may be found in Klein et al. (1983)
Immunogenetics 17(6):553-96.
[0031] The immunocompromised host animals are injected with a cell
population comprising immunocompetent T cells, and lacking
CD25.sup.+ cells. Conveniently, a cell population is depleted by
reagents specific for CD25 (negative selection), e.g. anti-CD25
antibodies, by flow cytometry, magnetic bead depletion, etc.
Alternatively, T cell populations naturally deficient in CD25
expression, or deficient through gene targeting from CD25 knockout
mice may be used.
[0032] The T cells may be from an allogeneic or xenogeneic donor,
and are tolerant to the major histocompatibility antigens of the
recipient, but immunoreactive with an antigen present in the
recipient, e.g. a T call antigen provided by viral infection of the
recipient, chronic infection with a bacterial or protozoan
pathogen, sustained release of an antigenic compound, the presence
of one or more minor histocompatibility antigens of the recipient,
etc. By tolerant is meant that when mixed with appropriate cells
(e.g., irradiated lymphocytes) from the recipient, the donor T
cells proliferate to a substantially lesser extent (e.g., <about
10% to 25%) than in an analogous mixed lymphocyte reaction between
MHC mismatched cells.
[0033] In contrast to the MHC locus, there are many minor
histocompatibility antigen loci dispersed throughout the genome.
Minor antigens generally result from the presentation of cellular
proteins on the surface of cells in conjunction with self MHC.
Therefore, virtually any protein that is expressed by the host,
processed and presented in the context of MHC antigens, and is
polymorphic between host and donor, can serve as a minor
histocompatibility antigen. Where there is a persistent or chronic
infection, epitopes relating to the infectious agent can serve as
minor histocompatibility antigens. It has been suggested that some
cutaneous antigens may serve as a trigger for chronic inflammatory
disease (e.g. H-40, described by Forman et al. (1984) J. Exp. Med.
159:1724-1740; and other antigens described by Chang et al. (1994)
P.N.A.S. 91:9282-9286; or Menssen et al. (1995) J. Immunol.
155:4078-4083). The subject animals are valuable models for
determining the role of specific genetic loci in contributing to
the development of inflammatory disease. Such screening may utilize
animals that are mismatched only at the loci of interest, and then
determining whether the difference is sufficient for induction of
disease.
[0034] There are a number of suitable animals to use as the source
of T cells. In most cases the donor and recipient will be of the
same species, although for some purposes xenogeneic donors may be
used. In one embodiment of the invention, the donor is allogeneic
but is matched at the MHC locus. For example, congenic mouse and
rat strains are available that are isogenic at the MHC locus, but
have a different genetic background. Alternatively, a parental
strain may be used as a donor, while an F1 animal acts as
recipient, e.g. a BALB/c donor into a BALB/c.times.C57bl/6
recipient. Alternatively, syngenic cells can be used in the
presence of other exogenous T cell antigen(s) in the host
environment, e.g. proteins, peptides, endotoxins, superantigens,
and the like. Alternatively, CB57/BL6 mice can be used as donors,
and the donor cells can be transferred into RAG-1.TM. and/or
RAG-2.TM. deficient mice.
[0035] Alternatively, one may use a chimeric animal as the source
of donor cells. For example, one can create a chimera by
transferring hematopoietic stem cells (HSC) into a recipient, where
the HSC are of a different genotype than the recipient. The HSC
then differentiate into T cells which are "educated" in the thymus,
and so are restricted to the recipient MHC type. These cells from
the chimera can then be harvested and used in the subject methods,
because they are both tolerant and restricted to the MHC type of
the thymus. It will be understood by one of skill in the art that
the thymic MHC in this example must be compatible with the ultimate
recipient animal. This procedure can also be used to create
xenogeneic chimeras (see for example, U.S. Pat. No. 5,625,127),
allowing the use of human cells in the subject methods.
[0036] The injected cell population comprises immunocompetent T
cells, and may also comprise other CD25 negative hematopoietic
cells, including macrophages, B cells, monocytes, etc. T cells are
conveniently isolated from secondary immune organs, e.g. spleen,
lymph node, thymus, etc. For example, an unfractionated suspension
of spleen cells, lymph node, etc. can be depleted of CD25.sup.+
cells and injected into the animal. Cells may also be isolated from
peripheral blood, cord blood, apheresis product, etc. Cell
populations may be enriched for various cell fractions of interest,
e.g. by density gradient, elutriation, cell sorting, etc. In one
embodiment of the invention, the population is selected for CD4
positive cells, which enriches for T helper cells. In another
embodiment of the invention, the cell population is depleted of
hematopoietic and lymphoid progenitor cells, as known in the art,
in order to decrease the possibility of de novo T cell maturation
in the host animal.
[0037] In another embodiment, the CD25 depleted cell population is
pre-incubated with antigen presenting cells, which may be
syngeneic, allogeneic, xenogeneic, usually comprising an exogenous
antigen to which the CD25 depleted population is responsive; having
mismatches at minor MHC loci; and the like. Optionally,
pro-inflammatory factors, e.g. lymphokines, endotoxins,
superantigens etc.; or antibodies against suppressor factors, e.g.
TGF-.beta. or IL-10, etc. are present. The cells are incubated for
a period of time sufficient to induce an immune response, and are
then introduced into a normal non-immunocompromised or
immunocompromised host. In another embodiment, whole cells are
incubated with pro-inflammatory cytokines that down-regulate CD25
expression on T cells and then are introduced into the host
environment, e.g. non-immunocompromised or immunocompromised,
syngenic or minor-haplotype mismatched.
[0038] Inflammatory diseases Can also be transferred from one
animal expressing disease to another naive animal by extracting
effector cells from the diseased animal and injecting them into
multiple naive animals. In another embodiment, a secondary transfer
is performed, where whole spleen or lymph node cells from a primary
host that was previously treated with a CD25 depleted population,
as described above, are transferred into a secondary host. The
primary host may be diseased or not-diseased. The cells from the
primary host may be unfractionated spleen, lymph node, etc., or may
be depleted of CD25 positive cells. Effector cells can be found in
secondary lymphoid tissue, especially spleen but also draining
lymph node, and the actual diseased organ tissue.
[0039] Separation of the desired cells for engraftment will
generally use affinity separation to provide a substantially CD25
negative population, usually comprising after separation not more
than about 5% CD25.sup.+ cells, more usually not more than about 3%
CD25.sup.+ cells, and may be less than about 1% CD25.sup.+ cells.
Techniques for affinity separation may include magnetic separation,
using antibody-coated magnetic beads, affinity chromatography,
cytotoxic agents joined to a monoclonal antibody or used in
conjunction with a monoclonal antibody, e.g. complement and
cytotoxins, and "panning" with antibody attached to a solid matrix,
e.g. plate, or other convenient technique. Techniques providing
accurate separation include fluorescence activated cell sorters,
which can have varying degrees of sophistication, such as multiple
color channels, low angle and obtuse light scattering detecting
channels, impedance channels, etc. The cells may be selected
against dead cells by employing dyes associated with dead cells
(propidium iodide, LDS). Any technique may be employed which is not
unduly detrimental to the viability of the selected cells.
[0040] The affinity reagents may be specific receptors or ligands
for the cell surface molecules indicated above. In addition to
antibody reagents, peptide-MHC antigen and T cell receptor pairs
may be used; peptide ligands and receptor; ligand and receptor
molecules, and the like. Antibodies and T cell receptors may be
monoclonal or polyclonal, and may be produced by transgenic
animals, immunized animals, immortalized human or animal B-cells,
cells transfected with DNA vectors encoding the antibody or T cell
receptor, etc. The details of the preparation of antibodies and
their suitability for use as specific binding agents are well-known
to those skilled in the art.
[0041] Of particular interest is the use of antibodies as affinity
reagents. Conveniently, these antibodies are conjugated with a
label for use in separation or used in conjunction with a labeled
second antibody that binds to them. Labels include magnetic beads,
which allow for direct separation; biotin, which can be bound to
avidin or streptavidin bound to a support; fluorochromes, which can
be used with a fluorescence activated cell sorter; or the like, to
allow for ease of separation of the particular cell type.
Fluorochromes that find use include phycobiliproteins, e.g.
phycoerythrin and allophycocyanins, fluorescein and Texas red.
[0042] The antibodies are added to a suspension of lymphocytes, and
incubated for a period of time sufficient to bind the available
cell surface antigens. The incubation will usually be at least
about 5 minutes and usually less than about 30 minutes. It is
desirable to have a sufficient concentration of antibodies in the
reaction mixture so that the efficiency of the separation is not
limited by lack of antibody. The appropriate concentration is
determined by titration. The medium in which the cells are
separated will be any medium which maintains the viability of the
cells and binding of antibody. A preferred medium is phosphate
buffered saline containing from 0.1 to 0.5% BSA. Various media are
commercially available and may be used according to the nature of
the cells, including Dulbecco's Modified Eagle Medium (DMEM),
Hank's Basic Salt Solution (HBSS), Dulbecco's phosphate buffered
saline (DPBS), RPMI, Iscove's medium, PBS with 5 mM EDTA, etc.,
frequently supplemented with fetal calf serum, BSA, HSA, etc.
[0043] The labeled cells are then separated as to the expression of
CD25, and optionally CD4. The separated cells may be collected in
any appropriate medium that maintains the viability of the cells,
usually having a cushion of serum at the bottom of the collection
tube. Various media are commercially available and may be used
according to the nature of the cells, including DMEM, HBSS, DPBS,
RPMI, Iscove's medium, etc., frequently supplemented with fetal
calf serum.
[0044] Compositions enriched for the desired T cells are achieved
in this manner. 90% of CD25pos T cells are depleted in the final
CD25negative T cell population. The enriched cell population may be
used immediately, or may be frozen at liquid nitrogen temperatures
and stored for long periods of time, being thawed for use when
needed. The frozen cells will usually be stored in 10% DMSO, 10-90%
FCS, 40% RPMI 1640 or other medium. Once thawed, the calls may
optionally be expanded by use of growth factors or stromal cells
associated with T cell proliferation and differentiation.
[0045] The population of purified T cells are injected into the
immunocompromised recipient. Routes of administration include
systemic injection, e.g. intravascular, subcutaneous, or
intraperitoneal injection. Where the recipient animal is a mouse,
the number of cells injected will usually be at least about
0.5.times.10.sup.5 and not more than about 5.times.10.sup.5, more
usually at least about 1.times.10.sup.5, preferably between about
3.times.10.sup.5 and 4.times.10.sup.5. Where the recipient animal
is a larger animal, the number of cells will be increased
accordingly.
[0046] Preferably, after transfer of the T cell population, a
localized immunostimulant and/or immunomodulating co-factor is
delivered in order to facilitate localization, retention and
replication of the effector, disease causing T cells. An
immunostimulant or immunomodulator can be any agent that can
contribute or induce either directly or indirectly inflammation
through the release of cytokines, lymphokines and the upregulation
of adhesion molecules. To accomplish this, the co-factor is
administered, generally at a localized site, following transfer of
the T cells. The timing of administration is varied depending on
the desired effect, but is generally performed from 1 day to 1 week
after T cell transfer. Many immunostimulants are known in the art,
including LPS and endotoxins in small doses, alpha interferons,
interleukin-1, modified tumor necrosis factor, CD40 ligand, poly
IC, virus, etc.
[0047] In one embodiment of the invention, the immunostimulatory
co-factor is a virus or viral vector, e.g. adenovirus, vaccinia,
HSV, SV40, and AAV, etc. The immuno-stimulatory effect may be
provided by the viral coat proteins present on the virus particles,
and/or by viral proteins or other genes expressed upon infection of
the target cell. Live virus is not required for the co-factor
effect, killed virus or vector encoding viral proteins are also
suitable. Suitable systems are disclosed, for example, in
Fisher-Hoch et al., PNAS 86:317-321, 1989; Flexner et al., Ann.
N.Y. Acad. Sci. 569:86-103, 1989; Flexner et al., Vaccine 8:17-21,
1990; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO
89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO
91/02805; Berkner, Biotechniques 6:616-627, 1988; Rosenfeld et al.,
Science 252:431-434, 1991; Kolls et al., PNAS 91:215-219, 1994;
Kass-Eisler et al., PNAS 90:11498-11502, 1993; Guzman et al.,
Circulation 88:2838-2848, 1993; and Guzman et al., Cir. Res.
73:1202-1207, 1993. Techniques for incorporating DNA into such
expression systems are well known to those of ordinary skill in the
art. The DNA may also be "naked," as described, for example, in
published PCT application WO 90/11092, and Ulmer et al., Science
259:1745-1749, 1993. The uptake of naked DNA may be increased by
coating the DNA onto biodegradable beads, which are efficiently
transported into the cells. In addition to viral genes, vectors and
viruses can be modified to encode immunomodulatory genes, e.g.
IL-2, IL-12, CD40, IFN-gamma, GM-CSF, TNF-alpha, etc.
[0048] The immunostimulant is administered to the host in the
manner conventional for the particular composition, generally as a
single unit dose in buffered saline, optionally combined with an
adjuvant formulation, where booster doses, typically one to several
weeks later, may additionally be delivered enterally or
parenterally, e.g., subcutaneously, cutaneously, intramuscularly,
intradermally, intravenously, intraarterially, intraperitoneally,
intranasally, orally, intraheart, intrapancreas, intraarticular,
etc. Localization can be achieved by administration at the targeted
site, use of sustained release implants, delivery in the form of
non-diffusible particles, and the like, as known in the art.
[0049] In one embodiment of the invention, the immunostimulant is a
polyclonal activating agent, which may include endotoxins, e.g.
lipopolysaccharide (LPS); and superantigens (exotoxins) (see Herman
et al. (1991) Annu Rev Immunol 9:745-72). Endotoxin primarily
interacts with CD14 receptors on macrophages, while superantigens
preferentially activate T cells. Both cell types are thus triggered
to release pro-inflammatory cytokines. Superantigens (SAgs) are
presented by major histocompatibility complex (MHC) class II
molecules and interact with a large number of T cells expressing
specific T cell receptor V beta domains. SAgs may be endogenous,
e.g. MIs; bacterial, e.g. SEB, SEA; or viral, e.g. mouse mammary
tumour virus.
[0050] Alternatively, one may use immunostimulatory polynucleotide
sequences (ISS). The use of these sequences is known in the art,
for examples see Bauer et al. (1999) Immunology 97(4):699-705;
Klinman et al. (1999) Vaccine 17(1):19-25; Hasan et al. (1999) J
Immunol Methods 229(1-2):1-22; and others. For example, an
"immunostimulatory oligonucleotide" has been described as an
oligonucleotide that contains a cytosine/guanine dinucleotide
sequence and stimulates maturation and activation of DC. An
immunostimulatory oligonucleotide of interest may be between 2 to
100 base pairs in size and typically contain a consensus mitogenic
CpG motif represented by the formula: 5' X.sub.1 X.sub.2 CGX.sub.3
X.sub.4 3', where C and G are unmethylated, X.sub.1, X.sub.2,
X.sub.3 and X.sub.4 are nucleotides and a GCG trinucleotide
sequence is not present at or near the 5' and 3' termini (see U.S.
Pat. No. 6,008,200, Krieg et al., issued Dec. 28, 1999, herein
incorporated by reference).
[0051] Preferably the immunostimulatory oligonucleotides range
between 8 to 40 base pairs in size. In addition, the
immunostimulatory oligonucleotides are preferably stabilized
oligonucleotides, particularly preferred are phosphorothioate
stabilized oligonucleotides. In one embodiment, X.sub.1 X.sub.2 is
the dinucleotide GpA. In another embodiment X.sub.3 X.sub.4 is the
dinucleotide TpC or TpT.
[0052] The dose and protocol for delivery of the immunostimulant
will vary With the specific agent that is selected. Typically one
or more doses are administered. One particular advantage of the use
of ISS in the methods of the invention is that ISS exert
immunomodulatory activity even at relatively low dosages. Although
the dosage used will vary depending on the clinical goals to be
achieved, a suitable dosage range is one which provides from about
1 Fg to about 10,000 Fg, usually at least about 1,000 Fg of ISS in
a single dosage. Alternatively, a target dosage of ISS can be
considered to be about 1-10 femtomole in a sample of host blood
drawn within the first 24-48 hours after administration of ISS.
Based on current studies, ISS are believed to have little or no
toxicity at these dosage levels.
[0053] In an alternative embodiment, a non-replicating virus or
viral coat protein is used as the immunostimulant. Virions of
interest include herpes viruses, e.g. HSV, EBV, CMV, etc.;
adenoviruses, e.g. E1 deleted adenovirus; retroviruses; etc. The
virus may optionally comprise a marker gene, such as lacZ, in order
to track efficiency of infection. For examples, see Byrnes et al.
(1995) Neuroscience 66(4):1015-24; Wood et al. (1994) Gene Ther
1(5):283-91; and Kajiwara et al. (1997) Hum Gene Ther
8(3):253-65.
[0054] Injection of a non-replicating virus leads to an
inflammatory response, e.g. in brain or neural tissue. Much of this
inflammation is induced directly by the virion particles themselves
rather than through the expression of new proteins from the virus.
By two days there is a large increase in major histocompatibility
complex class I and P-selectin expression and a heavy infiltration
of leukocytes, mainly macrophages and T cells.
[0055] In an alternative embodiment, the CD25 depleted cells are
introduced into a pro-inflammatory environment either before or
during in vivo introduction to the host. A pro-inflammatory
environment can be induced by adding pro-inflammatory factors or
antibodies against anti-inflammatory (suppressor) factors, e.g.
IFN-.gamma., IL-12, TNF-alpha, anti-TGF-beta, anti-IL-10, in vivo
and/or in vitro prior to introduction into an animal.
[0056] After administration of the T cells and co-factor, within
about 4 to 8 weeks the animals develop chronic inflammatory
disease. Scoring of the disease severity is based on physical
appearance, measurable ear thickness, cytokine expression, presence
of T cells at the lesion, etc. A more detailed analysis may utilize
histological section of various tissues, conveniently ear, eyelid,
tail, etc. Specific histological features include mononuclear cell
infiltration; high vascular density; etc.
[0057] To more fully characterize the disease, immunophenotypic
analysis may be performed to detect a variety of relevant antigenic
determinants. To characterize the types of immune cells present,
immunohistochemical stains for various leukocyte markers may be
performed. The expression of additional adhesion molecules that are
relevant to the pathophysiology of chronic inflammatory disease may
include mononuclear cell infiltrate; T cells at lesions; and the
expression in adjacent blood vessels of focal E-selectin,
P-selectin, ICAM-1 and diffuse vascular cell adhesion molecule-1
(VCAM-1) expression.
[0058] The subject animals are useful for screening candidate
therapeutic agents and treatment modalities. Through use of the
subject animals or cells derived therefrom, one can identify
ligands or substrates that affect the progression of chronic
inflammatory disease. Of particular interest are screening assays
for agents that have a low toxicity for human cells.
[0059] Drug screening protocols will generally include a panel of
animals, for example a test compound or combination of test
compounds, and negative and/or positive controls, where the
positive controls may be known immunosuppressive agents. Such
panels may be treated in parallel, or the results of a screening
assay may be compared to a reference database.
[0060] A wide variety of assays may be used for this purpose,
including histological analysis of effectiveness, determination of
the localization of drugs after administration, labeled in vitro
protein-protein binding assays, protein-DNA binding assays,
electrophoretic mobility shift assays, immunoassays for protein
binding, and the like. Depending on the particular assay, whole
animals may be used, or cells derived therefrom, particularly skin
cells, e.g. keratinocytes. Cells may be freshly isolated from an
animal, or may be immortalized in culture. Candidate therapies may
be novel, or modifications of existing treatment options.
[0061] For screening assays that use whole animals, a candidate
agent or treatment is applied to the subject animals. Typically, a
group of animals is used as a negative, untreated or
placebo-treated control, and a test group is treated with the
candidate therapy. Generally a plurality of assays are run in
parallel with different agent dose levels to obtain a differential
response to the various dosages. The dosages and routes of
administration are determined by the specific compound or treatment
to be tested, and will depend on the specific formulation,
stability of the candidate agent, response of the animal, etc.
[0062] The analysis may be directed towards determining
effectiveness in prevention of disease induction, where the
treatment is administered before induction of the disease, i.e.
prior to injection of the T cells and/or pro-inflammatory cytokine.
Alternatively, the analysis is directed toward regression of
existing lesions, and the treatment is administered after initial
onset of the disease, or establishment of moderate to severe
disease. Frequently, treatment effective for prevention is also
effective in regressing the disease.
[0063] In either case, after a period of time sufficient for the
development or regression of the disease, the animals are assessed
for impact of the treatment, by visual, histological,
immunohistological, and other assays suitable for determining
effectiveness of the treatment. The results may be expressed on a
semi-quantitative or quantitative scale in order to provide a basis
for statistical analysis of the results.
[0064] The term "agent" as used herein describes any molecule, e.g.
protein or pharmaceutical, with the capability of affecting the
severity of chronic inflammatory disease. An agent or treatment,
e.g. UV light, is administered to an animal of the invention, or to
cells derived therefrom. Antibodies specific for cytokines,
polyclonal activating agents, and T cell antigens are agents of
particular interest. Most preferably, according to another aspect
of the instant invention, the agents are monoclonal antibodies,
e.g. which neutralize lymphokines or block adhesion molecules.
[0065] Other candidate agents encompass numerous chemical classes,
typically organic molecules. Candidate agents comprise functional
groups necessary for structural interaction with proteins,
particularly hydrogen bonding, and typically include at least an
amine, carbonyl, hydroxyl or carboxyl group, preferably at least
two of the functional chemical groups. The candidate agents often
comprise cyclical carbon or heterocyclic structures and/or aromatic
or polyaromatic structures substituted with one or more of the
above functional groups. Candidate agents are also found among
biomolecules including, but not limited to: peptides, saccharides,
fatty acids, steroids, purines, pyrimidines, derivatives,
structural analogs or combinations thereof.
[0066] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides and oligopeptides.
Alternatively, libraries of natural compounds in the form of
bacterial, fungal, plant and animal extracts are available or
readily produced. Additionally, natural or synthetically produced
libraries and compounds are readily modified through conventional
chemical, physical and biochemical means, and may be used to
produce combinatorial libraries. Known pharmacological agents may
be subjected to directed or random chemical modifications, such as
acylation, alkylation, esterification, amidification, etc. to
produce structural analogs.
[0067] The therapeutic agents may be administered to patients in a
variety of ways, orally, topically, parenterally e.g.
subcutaneously, intramuscularly, intravascularly, etc. Depending
upon the manner of introduction, the compounds may be formulated in
a variety of ways. The concentration of therapeutically active
agent in the formulated pharmaceutical compositions may vary from
about 0.1-100 wt. %.
[0068] The pharmaceutical compositions can be prepared in various
forms, such as granules, tablets, pills, suppositories, capsules,
suspensions, salves, lotions and the like. Pharmaceutical grade
organic or inorganic carriers and/or diluents suitable for oral and
topical use can be used to make up compositions containing the
therapeutically-active compounds. Diluents known to the art include
aqueous media, vegetable and animal oils and fats. Stabilizing
agents, wetting and emulsifying agents, salts for varying the
osmotic pressure or buffers for securing an adequate pH value, and
skin penetration enhancers can be used as auxiliary agents.
[0069] It is to be understood that this invention is not limited to
the particular methodology, protocols, cell lines, animal species
or genera, constructs, and reagents described, as such may vary. It
is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments only, and is not
intended to limit the scope of the present invention which scope
will be determined by the language in the claims.
[0070] It must be noted that as used herein and in the appended
claims, the singular forms "a", "and", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a mouse" includes a plurality of such mice
and reference to "the cytokine" includes reference to one or more
cytokines and equivalents thereof known to those skilled in the
art, and so forth.
[0071] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this invention belongs. Although
any methods, devices and materials similar or equivalent to those
described herein can be used in the practice or testing of the
invention, the preferred methods, devices and materials are now
described.
[0072] All publications mentioned herein are incorporated herein by
reference for all relevant purposes, e.g., the purpose of
describing and disclosing, for example, the cell lines, constructs,
and methodologies that are described in the publications which
might be used in connection with the presently described invention.
The publications discussed above and throughout the text are
provided solely for their disclosure prior to the filing date of
the present application. Nothing herein is to be construed as an
admission that the inventors are not entitled to antedate such
disclosure by virtue of prior invention.
[0073] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the subject invention, and are
not intended to limit the scope of what is regarded as the
invention. Efforts have been made to ensure accuracy with respect
to the numbers used (e.g. amounts, temperature, concentrations,
etc.) but some experimental errors and deviations should be allowed
for. Unless otherwise indicated, parts are parts by weight,
molecular weight is average molecular weight, temperature is in
degrees centigrade; and pressure is at or near atmospheric.
EXPERIMENTAL
[0074] Animal Model for Chronic Inflammation
[0075] Mice. Female Balb/c mice (donor mice) were purchased from
Jackson Labs (Bar Harbor, Me.) or similar source, and C.B-17/lcr
scid/scid (recipient mice) were purchased from Taconic (Germantown,
N.Y.) or Charles River. All mice were housed in a specific pathogen
free environment and were used between 4-8 wk of age. Mice were
housed 2-5 per microisolator. All scid/scid mice were handled with
gloves under a class II hood, fed sterile food and water ad
libitum, and maintained in sterilized microisolators that are
changed twice weekly. Donor mice were housed in conventional cages
that were changed weekly.
[0076] Induction of chronic skin inflammation. Briefly, splenocytes
were collected from 5-10 week old donor mice (Balb/c) and donor
population was either enriched for CD4+ and depleted of CD25+
cells, or depleted of CD25+ cells only. The collected cell
population was injected subcutaneously (s.c.) into C.B-17/lcr
scid/scid mice, aged 4-8 weeks (usually 3.times.10.sup.5 to
5.times.10.sup.5 cells per mouse in 200-400 .mu.L). A systemic
(s.c., i.p. or i.v.) injection of an immuno-modulating agent was
given 24 hours following cell transfer. Alternatively, these
co-injections were given at the same time to one week after. The
co-injection was, alternatively, repeated every other day or once a
week for the entire course of the experiment.
[0077] Day 0:
[0078] Cell Selection: Balb/c mouse spleens were collected and
homogenized by pressing through 100.mu. cell strainer (Falcon) and
suspended in cold PBS supplemented with 10% FBS. Cell suspension
was then centrifuged (400G) and the cell pellet was retained,
resuspended in 2 ml warm RBC lysing buffer (37 C.) per spleen, and
incubated 3 minutes at 37.degree. C. The cells were then washed
with cold PBS+10% FBS and again centrifuged. The cell pellet was
then resuspended in 5 ml cold PBS+10% FBS and we added CD4+
selection beads (Dynal) 25 .mu.l per spleen to the cell suspension.
This mixture was then incubated for 30 minutes at 4.degree. C. on a
rotator and using a magnetic particle collector (MPC) the bead-cell
complexes were collected and rinsed three times with PBS+10% FCS.
The cell-bead complexes were then resuspended in 5 ml warm media
(DME, RPMI)+10% FBS, and 20 .mu.l/spleen CD4 DetachaBead (Dynal)
was added and incubated for 45 minutes RT on rotator to remove
beads from the cell surface. We removed beads using a MPC, rinsed
them twice with PBS+10% FBS and retained supernatants. The
resultant cell quantities were 5-8 million cells per spleen. We
then resuspend these cells in 1 ml PBS+10% FBS with 8 .mu.l/spleen
anti-CD25-Biotin conjugated mAb and incubate 20 minutes 4.degree.
C. Again, cells were washed and resuspended in 1 mL PBS+10% FBS
with 25-30 .mu.l steptavidin beads and incubate for 20 minutes at
4.degree. C. Bead-CD25+ cell complexes were removed using an MPC.
The cells were once again, collected, washed, and the pellet was
checked for stray beads which were removed if necessary using
MPC.
[0079] Scid/scid mice were then injected with
1.times.10.sup.5-5.times.10.- sup.6 cells in 200-400 .mu.l PBS sc.
(all SC injections were done with the mice under general
anesthesia).
[0080] After 24 hours all recipient mice were given a co-injection
of 20 .mu.g LPS (or 10 .mu.g SEB) s.c. Disease causing T cells then
proliferated and began to induce inflammation over a period of 4
weeks.
[0081] Week 4-6: Disease expression period. Beginning on week 4,
measurements of skin thickness were taken from both ears to monitor
level of disease expression and incidence.
[0082] The thickness of the skin on the ear was measured using a
Dyer micrometer. The micrometer was first modified to better
perform in the measurement of soft tissue. The contact pads are
reduced to 4 mm, and the spring tension is reduced to <3
lbs.
[0083] Psoriatic mice were selected for use in compound screening
based on ear thickness and clinical phenotype. Mice were then
randomly assigned to experimental groups.
[0084] Week 6-10: Treatment Period (2-4 Weeks)
[0085] Administration of experimental compounds (which included
antibodies, small molecules, chemicals, viral vectors, drugs) was
conducted regularly (once, twice or 3 times per week or daily) for
2-4 weeks at a dose relative to appropriate mg/kg dosages. In
general, administration of all compounds were given systemically
(SC, IP or IV). Along with the experimental compounds, control
groups were run simultaneously with injections of PBS, a negative
control (isotype control), and a positive control (anti-IL-12,
anti-TNF.alpha., corticosteroids, or other known compounds that
result in the resolution of psoriatic lesions).
[0086] Mice were observed and data recorded for ear thickness and
total body weight on a weekly basis. Body weight was monitored to
help monitor the overall health of the animal, e.g. exclude viral
infection and colitis.
[0087] Week 8-12: Evaluation Period (1-2 Weeks after Last Injection
of Experimental Compound)
[0088] After the completion of treatment period with the
experimental compounds, including positive and negative controls a
period of at least one or two weeks was allowed to pass to confirm
that the drug did or did not have an effect on the severity of
disease. Thus the time from the first injection to the end of this
waiting period was generally 3-4 weeks in all experiments. At the
end of this period, mice were sacrificed, biopsies from both ears
taken, and 6 cross sections were made, stained (H and E) and
evaluated in blind fashion by at least 2 investigators (given
histology score ranged 0-4). Biopsies from all other skin areas
were occasionally taken as well.
[0089] Skin biopsies were taken from the ear by removing the ear
entirely by making the cut below the base of the ear. This method
was required to make an adequate observation of the organ as
possible From the base of the ear to the tip the tissue tends to
become thinner. Often mild disease is easier to detect at the base
of the ear. The histology score was determined by evaluating 6
sections (2 cross sections made from the tip, middle and base
sections of the ear). Other biopsies are useful to support the data
collected from the ear. Such cases included extremely severe
clinical cases where hair loss occurs indicating involvement of
other regions of skin.
[0090] Scid/scid mice engrafted with T cells have been shown in
previous studies to come down with some incidence of colitis. It
was found in our experiments that this procedure could be used with
immunomodulatory co-factors to create organ specific inflammation,
e.g. psoriasis, colitis, etc. Because of the immunostimulating
properties of bacterial mitogens or bacterial superantigens it was
initially tested whether the co-administration of such agents would
have a positive effect on the induction of disease with this novel
scid/scid transfer model.
[0091] Disease induction, severity, and chronicity. Initially the
animals were tested to determine the percentage of mice that came
down with disease. The experimental data represented in FIGS. 1 and
2 comes from a group of 40 scid/scid mice that received a transfer
of CD4+/CD25- T cells as described above. In, brief, naive
scid/scid mice were injected sc. with 3-6.times.10.sup.5
CD4.sup.+CD25.sup.- cells on day 0, followed by a sc. injection of
20 .mu.g LPS 24 hours later. The mice were then handled with normal
husbandry for 4 at which time clinical signs of psoriasis begin
appear on the ears in the form of reddened, thickened skin. On week
five 50% of the animals were considered diseased (>=25 .mu.m
skin thickness). The incidence of disease improved by week 6 to
76%, and reached 96% on week 7. This data is from one experiment
and is representative of 6 experiments.
[0092] The psoriasis in this model is significant it does reach
high severity. The normal scid/scid mouse skin thickness in the ear
is 18-22 .mu.m. As seen in FIG. 1 the distribution of severity
attainable in this model has a majority of the mice expressing
severe levels of disease (Each mark represents a single ear
measurement. n=80. Normal ear thickness: 19-22, Mild disease 25-30
.mu.m. Moderate disease 31-39 .mu.m Severe disease >40 .mu.m).
As seen in FIG. 2, the progress of disease can be monitored and the
severity of disease is scaleable. The mild to moderately diseased
animals are shown to come down with disease (ear thickness becomes
>25 .mu.m) between week 7 and 8. In mice that have more severe
disease by week 8 will show a more aggressive development of
psoriasis and will become disease (develop skin thickness >25
.mu.m) starting on week 5.
[0093] In order to determine that this disease was a chronic
inflammation, several mice from various experiments were observed
for greater than 14 weeks after cell transfer. In FIG. 3 one group
of 10 animals which were induced with psoriasis by the standard
protocol were observed for 17 weeks after the transfer of T cells.
Measurements of the ears of 10 mice were averaged (20 ears in
total). In this experiment the average measurement (n=20) was
>25 .mu.m on week 5. It was found that the diseased condition
not only developed from moderate to severe levels (>30 .mu.m)
but also lasted for 12 weeks. This is an adequate time period to
demonstrate that the disease does not resolve itself. The
chronicity of disease may be attributed to the autoantigens that
drive the disease. It is also observed that the severity of the
disease is narrow ranged (between 32-36 .mu.m) for a period of 6
weeks (week 9-15). This data was found to be representative of 4
separate experiments.
[0094] Co-factor injections are important for their mitogenic
properties. In one experiment five mice were compared with the
standard induction protocol which included an injection of 20 .mu.g
LPS on day one to 5 mice that received the same cells but were not
given the injection of LPS. Starting on week 6 the mice that
received the LPS displayed a higher severity of psoriasis like
disease. By week 7 the mice that received the LPS had an average
ear thickness of 36.4 .mu.m.+-.0.7 compared to 30 .mu.m.+-.1.2 in
the mice that did not receive LPS. Both groups of mice had an
incidence of disease of 100% (5/5), shown in FIG. 4. Data
represents the average of 10 measurements of skin thickness (1 per
ear, 2 per mouse n=5 mice), Error bars=SEM. p=3.6.times.10.sup.-3
Therefore, the data shows that the use of bacterial antigens
results in an increased severity of disease.
[0095] The effect of different co-factors on disease induction was
assessed. All mice were induced with the same cell population
described in the standard protocol. Each group received a single
injection of co-factor after cell transfer as follows. No
Co-injection: 200 .mu.l PBS s.c on day 1. LPS: 20 .mu.g LPS s.c.
diluted in PBS on day 1. LPS+Whole Cells: LPS and 10.sup.6
CD25.sup.- cells s.c. on day 14. Viral Vector: 1.times.10.sup.6
viral particles injected s.c. in 200 .mu.l PBS. SEB: 10 .mu.g SEB
diluted in 200 .mu.l PBS injected s.c.
[0096] The viral vector is the Adenovirus serotype 5 with deletion
of E1 and E3 genes. The transgene is the LacZ gene under the
control of the cytomegalovirus-IE promoter. The dose (10.sup.6 VP)
is the virus particle count, not the infections dose (our virus
particle dose is equivalent to 10.sup.5 TCID 50). The CD25.sup.-
cells were selected from Balb/C spleen without other enrichment,
and so included all cells including CD8, B, NK cell. The results
are shown in FIG. 10. It can be seen that the viral vector
co-factor produced a very strong response.
[0097] Phenotype of disease inducing cells. The sorted
CD4.sup.+/CD25.sup.- cell population was tested for purity, by
staining with florescein conjugated antibodies against CD4 and CD25
and analysis using a FacsCalibur (Becton Dickenson) and Cell Quest
Software. The population was found to stain positively for CD4 and
negatively for CD25 on greater than 97% of the cells.
[0098] The sorted CD4.sup.+/CD25.sup.- cell population was also
tested by staining with fluorescein conjugated antibodies against
CD45RB. It was found that the disease inducing cells have a
heterogeneous phenotype of CD45RB hi and low. Thus, the induction
of disease is CD45 independent.
[0099] Experiments were also performed demonstrating that secondary
transfer of whole spleen cell suspensions from a primary host with
disease induced as described above, results in the transfer of
disease. Whole spleens from diseased mice (induced by standard
protocol) were treated with red blood cell lysing buffer, and
reinjected into naive scid/scid mice. Each mouse received
2.5.times.10.sup.5 cells s.c. (n=3).
[0100] It was further shown that a suspension of unfractionated
spleen cells depleted of CD25 positive cells can be used to induce
disease The host animals were injected with 500,000 splenocytes
from normal Balb/C mice, that were depleted of CD25 cells by the
same magnetic bead-antibody method described above, in combination
with LPS. Cells included in this population, in addition to CD4 T
cells, are CD8 T cells, B cells, NK cells, macrophages, dendritic
cells etc. The result was an induction of disease.
[0101] The general health of the animals with inflammatory disease
was monitored not only by daily observations but also by measuring
their body weight. In previous studies, the induction of
inflammation often involved colitis, which results in a general
decrease in health of the animal. We observed three groups of 5
psoriatic mice in each group and found that even after the
induction of the inflammatory disease the average weight of the
mice stayed very consistent in all groups. In another study 5
psoriatic animals with an average ear thickness starting at 35
.mu.m and increasing to 45 .mu.m (moderate to severe severity) were
found to have an average body mass holding between 20-22 mg (normal
healthy body mass 19-23 mg) for the entire course of disease
progression, and up to 11 weeks post cell transfer. This
demonstrates that even severely affected psoriatic mice remain
healthy for an extended period of time.
[0102] To demonstrate that the disease was not an artifact of the
mechanical manipulation of the mice, a study was conducted to show
that the transferred cells were the cause of disease. Naive
scid/scid mice were given sc. Injections containing
2.5.times.10.sup.5 whole (un-enriched) spleen cells from psoriatic
mice that had been induced to develop psoriasis with
CD4.sup.+/CD25.sup.- cells. All of the test subjects came down with
disease (3 out of 3 at week 8). It may be noted that the spleens
from diseased scid/scid mice are small. This study shows that the
cells transferred in this invention are the cause of disease and
that the cells maintain their disease causing properties even after
multiple animal transfers.
[0103] Administration of anti IL-12 mAb. There have been many
examples of the effective anti-inflammatory effect of anti IL-12
mAb treatment. In animal model of the present invention, it was
also found to cause a reduction in psoriasis lesions. Animals were
tested for the effect of treatment during ongoing psoriatic
disease. The disease was induced by the standard protocol, and
treatment began on week 9. The treatment with the anti-IL-12 mAb
was 1 mg/mouse/week. In the initial experiments (n=5 untreated, n=5
treated) mice received injections of anti-IL-12 mAb at weeks 9 and
10, and were observed for 2 weeks after the final injection.
Control animals were left untreated, isotype treated animals
received an isotype matched monoclonal antibody against a non mouse
antigen. Anti-IL-12 treatment resulted in a skin thickness
improvement of -7 .mu.m compared to an increase in skin thickness
of +2 .mu.m in control isotype treated animals and untreated
animals, as shown in FIG. 7.
[0104] In a separate study, anti IL-12 mAb was used in a positive
control group, to determine if an experimental anti-mouse antigen
antibody had anti-inflammatory properties. In this experiment there
were 5 mice per experimental group: positive control (anti IL-12),
negative control (isotype matched anti Human antigen mAb), and the
experimental mAb labeled BSK Ab001. At week 12 the animals were
sacrificed for histology of the skin tissue. Where the isotype
control mice had an average histology score of 2.7 on a scale of
0-4, the anti IL-12 treated mice had a histology score of only 0.5
indicating a nearly complete resolution of psoriatic lesions FIG.
8. Therefore 2 injections of anti-IL-12 were shown to be an
effective treatment by clinical observations and by histology,
shown in FIG. 5. Treatment began 9 weeks after T cell transfer,
injections given on week 1 and 2, all animals received 1 mg/dose.
Control animals were left untreated, isotype control animals
received an isotype matched monoclonal antibody against a human
antigen. p=0.02.
[0105] An experimental antibody against a mouse antigen (BSK001)
was tested, and found to have no effect on psoriasis lesions. The
skin thickness on week 5 of disease did not decrease, as shown in
FIG. 6. Each group consisted of 5 mice and all were induced with
psoriasis by standard protocol. The treatment for each group was 1
mg of antibody per dose: positive control (anti IL-12 mAb),
negative control (isotype matched anti-human antigen mAb), and the
experimental (mAb labeled BSK Ab001), began on the third week,
after disease had begun with moderate to severe psoriasis. Where as
the mice in the positive control group demonstrated reduced skin
thickness (7 .mu.m improvement), the experimental mAb BSK Ab001
group of mice had an increase in ear thickness of 19 .mu.m 2 weeks
after the final injection, p=5.0.times.10.sup.-9. The isotype
negative control mice completed the treatment and observation
period (total of 4 weeks) with no change in ear thickness.
[0106] These results were confirmed by histology scores composed of
the average of histology scores given to each ear of the
experimental groups histology. Semiquantitive histological scores
from 0 to 4 were given based on the severity of inflammation.
Initial histological evaluation was performed by an independent
outside pathologist. In later studies evaluation was blindly
conducted by three different investigators. 0=no signs of
inflammation; 1=very low focal areas of infiltration, mild
acanthosis; 2=low level of mononuclear cell infiltration, mild
thickening of epidermis, mild to moderate acanthosis 3=high level
of mononuclear cell infiltration, high vascular density, thickening
of the epidermis (acanthosis, rete pegs and hyperplasia of
epidermis and keratinocytes, microabscesses, thinning of the
granular cell layer 4=very extensive infiltration in epidermis and
dermis, very high vascular density, extreme thickening of
epidermis, pustule formation and destruction of granular cell
layers.
[0107] In another study, it was found that treatment with anti CD43
mAb showed no effect on the progression of disease (see FIG. 7). In
this experiment all mice were induced with disease by standard
protocol. Five mice per group were utilized to compare the affects
of anti-CD43 to untreated mice. The treated group received a
treatment regimen of 2 injections on week 7 and 8, of 1 mg per
mouse mAb given sc. The mice were observed for 2 weeks after the
final injection and no significant differences between the groups
were found as observed by ear thickness. Anti CD43 was selected as
a possible therapeutic mAb due to previous studies that showed this
antibody has the ability to abrogate semi-chronic diseases and to
prevent the induction of disease. Due to the lack of improvement in
disease severity in our model we determined that anti CD43 mAb is
not an effective treatment in truly chronic disease models. From
this we show that the model is selective and does not react to all
antibodies against mouse antigens.
[0108] Animals were tested to compare the effect of an experimental
oral compound (BSK 002) on the induction and severity of psoriatic
disease. Twenty animals were induced with psoriasis by the standard
protocol and were divided into 2 groups: autoclaved water treated
with BSK 002, and normal autoclaved water. The effect of compound
BSK002 was compared to a negative control at week 4, and it was
found that 33% (3/9) of animals without BSK 002 compared to 0%
(0/10) mice with BSK002 showed a disease state (ear thickness
>=25 .mu.m). At week nine, while both groups had diseased
animals, the group without BSK002 administration developed a higher
severity (30 .mu.m vs 24 .mu.m) as well as higher penetrance 100%
(9/9) compared to 50% (5/10). Hence the presence of the
experimental compound BSK002 slows down the onset of psoriasis and
reduces both penetrance and severity (shown in FIG. 8). On week 11
all animals had disease (100%; 10/10) in the normal water group
while the treated water group had 80% (8/10). At week 9:
p=1.7.times.10.sup.-5, week 11: p=3.4.times.10.sup.-2, week 17:
p=4.3.times.10.sup.-2.
[0109] Animals were tested to determine the effect of cyclosporin A
on the development of disease (shown in FIG. 9), with injections of
the compound at 17, 18 and 19 weeks post-T cell transfer. It was
found that the presence of the cyclosporin A reduced the severity
of the disease, p=3.6.times.10.sup.-2.
[0110] The known immunosuppressant methylprednisone was also found
to control the disease. After injection with methylprednisone, at a
dose of 40 mg/kg twice/day for 8 days, the ear thickness improved
immediately compared to the control animals which received
injections of PBS. After 8 days of treatment the skin thickness was
reduced from 33.7 to 22.4 .mu.m p=2.01.times.10.sup.-6
[0111] To demonstrate that the disease could be induced at a site
other than skin, a study was conducted in which CD25 negative
effector cells were transferred into scid/scid mice without
co-injection into the skin. Food and gut flora are acting as
co-factor antigen(s) in this model set-up. After, 6-8 weeks animals
developed severe colitis as measured by weight (average weight
15.8.+-.0.6 (n=4), Normal weight is 19-23).
* * * * *