U.S. patent application number 10/108191 was filed with the patent office on 2002-12-19 for method of treating psoriasis using anti-interleukin 12 antibody.
Invention is credited to Ehrhardt, Rolf, Hong, Kenneth, Queen, Cary.
Application Number | 20020194631 10/108191 |
Document ID | / |
Family ID | 22339643 |
Filed Date | 2002-12-19 |
United States Patent
Application |
20020194631 |
Kind Code |
A1 |
Ehrhardt, Rolf ; et
al. |
December 19, 2002 |
Method of treating psoriasis using anti-interleukin 12 antibody
Abstract
Methods and compositions are provided for the creation and
screening of non-human animal models having many of the histologic
characteristics of human psoriasis. Immunocompromised host animals
are injected with a purified population of CD45Rb positive cells,
which are tolerant of the host major histocompatibility antigens,
but are mismatched at one or more minor antigens. The injected
cells are stimulated with a pro-inflammatory cytokine, e.g. IL-12,
and a polyclonal activating agent. The injected animals develop a
chronic skin disorder that includes histological features observed
in human psoriasis, e.g. rete pegs, severe acanthosis and
infiltration of Th1 cells into the dermis.
Inventors: |
Ehrhardt, Rolf; (San
Francisco, CA) ; Hong, Kenneth; (El Cerrito, CA)
; Queen, Cary; (Los Altos, CA) |
Correspondence
Address: |
HOWREY SIMON ARNOLD & WHITE, LLP
BOX 34
301 RAVENSWOOD AVE.
MENLO PARK
CA
94025
US
|
Family ID: |
22339643 |
Appl. No.: |
10/108191 |
Filed: |
March 26, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10108191 |
Mar 26, 2002 |
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09457912 |
Dec 8, 1999 |
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6410824 |
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60111642 |
Dec 9, 1998 |
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Current U.S.
Class: |
800/9 ; 424/85.2;
800/18 |
Current CPC
Class: |
C07K 2317/76 20130101;
C07K 16/24 20130101; A61K 39/00 20130101; C07K 14/5434 20130101;
C07K 16/244 20130101; A61K 38/208 20130101; A61K 35/12 20130101;
A61K 2039/505 20130101; A61P 17/06 20180101; A61K 39/3955 20130101;
A01K 67/0271 20130101 |
Class at
Publication: |
800/9 ; 800/18;
424/85.2 |
International
Class: |
A01K 067/027; A61K
038/20 |
Claims
What is claimed is:
1. A method for inducing a psoriasis-like syndrome in an animal,
the method comprising: transferring a purified CD45Rb positive T
cell population from a donor animal to an immunocompromised animal
host, wherein said T cell population is tolerant of the host major
histocompatibility antigens but is immunoreactive with one or more
of the host minor histocompatibility antigens; administering at
least one pro-inflammatory cytokine and at least one polyclonal
activating agent to said immunocompromised animal host; wherein
said host develops a disease having characteristics of human
psoriasis.
2. The method of claim 1, wherein said T cell population is
CD4.sup.+ CD45Rb.sup.hi.
3. The method of claim 1 wherein the donor and host animals are MHC
matched.
4. The method of claim 1, wherein said immunodeficient animal is an
immunodeficient rodent.
5. The method of claim 4, wherein said immunodeficient animal is a
scid-scid mouse.
6. The method of claim 1, wherein said pro-inflammatory cytokine is
interleukin-12.
7. The method of claim 6, wherein the dose of said IL-12 is at
least about 0.1 ng/gram weight of host, and not more than about 2
ng/gram weight of host.
8. The method of claim 7, wherein said IL-12 is administered at
about one day and at about three days after transferring said T
cell population.
9. The method of claim 1, wherein said polyclonal activating agent
is an endotoxin.
10. The method of claim 9, wherein the dose of said endotoxin is
from about 0.1 .mu.g/g weight of host to about 5 .mu.g/g weight of
host.
11. The method of claim 1, wherein said polyclonal activating agent
is a superantigen.
12. The method of claim 11, wherein said superantigen is a
bacterial superantigen.
13. The method of claim 12, wherein the dose of said superantigen
is from about 0.1 .mu.g/g weight of host to about 5 .mu.g/g weight
of host.
14. A method for screening a candidate therapy for efficacy in
treatment of psoriasis, the method comprising: transferring a
purified CD45Rb positive T cell population from a donor animal to
at least one immunocompromised animal host, wherein said T cell
population is tolerant of the host major histocompatibility
antigens but is immunoreactive with one or more of the host minor
histocompatibility antigens; administering at least one
pro-inflammatory cytokine and at least one polyclonal activating
agent to said immunocompromised animal host; wherein said host
develops a disease having characteristics of human psoriasis;
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.
15. The method of claim 14, wherein said T cell population is
CD4.sup.+ CD45Rb.sup.hi.
16. The method of claim 14 wherein said donor and host animals are
MHC matched.
17. The method of claim 14, wherein said therapy is treatment with
a candidate pharmaceutical agent.
18. The method of claim 17 wherein said candidate pharmaceutical
agent is a monoclonal antibody.
19. A method of claim 18 wherein said antibody binds to an antigen
selected from-the group of interferon gamma, interleukin 12,
E-selectin, P-selectin, CD3 or alphaE integrin subunit.
20. The method of claim 14, wherein said immunodeficient animal is
an immunodeficient mouse or rat.
21. The method of claim 20, wherein said immunodeficient animal is
a scid-scid mouse.
22. The method of claim 14, wherein said pro-inflammatory cytokine
is interleukin-12.
23. The method of claim 14, wherein said polyclonal activating
agent is an endotoxin.
24. The method of claim 14, wherein said polyclonal activating
agent is a superantigen.
25. A method of treating a patient suffering from psoriasis
comprising the step of administering to the patient an antibody
that binds to an antigen selected from the group of interferon
gamma, interleukin 12, E-selectin, P-selectin, CD3 or alphaE
integrin subunit.
26. A method of claim 25 wherein said antibody is a humanized
antibody.
27. A method of claim 26 wherein said antibody is the HuZAF,
HuEP5C7, or HuM291 antibody.
28. An immunodeficient mouse induced to exhibit a psoriasis-like
syndrome by transfer of minor histocompatability mismatched murine
CD4.sup.+ CD45RB.sup.hi T cells and administration of a
proinflammatory lymphokine and a polyclonal lymphocyte
activator.
29. A method of reducing the PASI of a patient suffering from
psoriasis by at least 50%, comprising treating the patient with a
neutralizing monoclonal antibody to interleukin 12.
30. The method of claim 29, wherein said antibody is humanized or
human.
31. A method of treating psoriasis patients comprising the steps of
(1) administering to the patients therapies that induce remission
of their psoriasis, and then (2) treating the patients with a
neutralizing monoclonal antibody to interleukin 12, wherein
treatment with said antibody prolongs the median time to relapse by
at least 50%.
32. The method of claim 31, wherein said antibody is humanized or
human.
Description
BACKGROUND
[0001] Psoriasis is a chronic skin disease, characterized by
scaling and inflammation. Psoriasis affects 1.5 to 2 percent of the
United States population, or almost 5 million people. It occurs in
all age groups and about equally in men and women. People with
psoriasis suffer discomfort, restricted motion of joints, and
emotional distress. When psoriasis develops, patches of skin
thicken, redden, and become covered with silvery scales, referred
to as plaques. Psoriasis most often occurs on the elbows, knees,
scalp, lower back, face, palms, and soles of the feet. The disease
also may affect the fingernails, toenails, and the soft tissues
inside the mouth and genitalia. About 10 percent of people with
psoriasis have joint inflammation that produces symptoms of
arthritis.
[0002] When skin is wounded, a wound healing program is triggered,
also known as regenerative maturation. Lesional psoriasis is
characterized by cell growth in this alternate growth program. In
many ways, psoriatic skin is similar to skin healing from a wound
or reacting to a stimulus such as infection, where the
keratinocytes switch o from the normal growth program to
regenerative maturation. Cells are created and pushed to the
surface in as little as 2-4 days, and the skin cannot shed the
cells fast enough. The excessive skin cells build up and form
elevated, scaly lesions. The white scale (called "plaque") that
usually covers the lesion is composed of dead skin cells, and the
redness of the lesion is caused by increased blood supply to the
area of rapidly dividing skin cells.
[0003] The exact cause of psoriasis in humans is not known,
although it is generally accepted that it has a genetic component,
and a recent study has established that it has an autoimmune
component. Whether a person actually develops psoriasis is
hypothesized to depend on something "triggering" its appearance.
Examples of potential "trigger factors" include systemic
infections, injury to the skin (the Koebner phenomenon),
vaccinations, certain medications, and intramuscular injections or
oral steroid medications.
[0004] The chronic skin inflammation of psoriasis is associated
with hyperplastic epidermal keratinocytes and infiltrating
mononuclear cells, including CD4+ memory T cells, neutrophils and
macrophages. Because of this highly mixed inflammatory picture and
the resulting complex interrelationships between these different
cells, it has been very difficult to dissect the mechanisms that
underlie the induction and progression of the disease.
[0005] Research into the pathogenesis and treatment of psoriasis
has long been hindered by the lack of suitable animal models.
Although several rodent models of skin inflammation have been
recently introduced, none of these models have the specific T cell
abnormalities that have been demonstrated as a primary cause for
the induction of disease. The development of improved animal models
having the clinical features associated with human psoriasis would
be of great benefit for screening potential therapies and
drugs.
RELEVANT LITERATURE
[0006] Schon et al. (1997) Nat Med. 3:183-8 induced a murine
psoriasis-like disorder by reconstituting scid/scid mice with naive
CD4.sup.+ T cells. This model, however, lacked certain distinctive
histological hallmarks of the human disease and included some
characteristics that are absent from human patients with psoriasis,
Nickoloff et al. (1997) Nat Med. 3:475-6. Other mouse models for
psoriasis have also utilized immunodeficient animals. Sugai et al.
(1998) J Dermatol Sci 17:85-92 transplanted human psoriatic lesions
onto SCID mice. The human skin grafts were generally well
maintained during this period, but the histological and
immunohistochemical findings characteristic of psoriasis, except
for acanthosis and hyperkeratosis, gradually disappeared as
lymphocytic infiltration of the psoriatic lesions declined.
Yamamoto et al. (1998) J Dermatol Sci 17:8-14 injected
staphylococcal enterotoxin B-stimulated lymphocytes subcutaneously
under full-thickness psoriatic skin grafted onto severe combined
immunodeficient (SCID) mice.
[0007] Gottlieb et al. (1995) Nat Med. 1:442-7 treated patients
with fragments of diphtheria toxin linked to human interleukin-2
(DAB3891L-2), which selectively targets activated T cells but not
keratinocytes. They showed significant clinical improvement,
indicating that T cells and not keratinocytes are the primary
pathogenic component in the disease.
[0008] Sundberg et al. (1997) Pathobiology 65(5):271-86 describe
the development and progression of psoriasiform dermatitis and
systemic lesions in the flaky skin (fsn) mouse mutant. Flaky skin
(fsn) mutant mice were originally described as a mouse model for
psoriasis accompanied by hematological abnormalities. However,
homozygous (fsn/fsn) mice develop a number of other pathological
changes.
[0009] Hong et al. (1999) J. Immunol. 162:7480-7491 (which is
herein incorporated by reference for all purposes) describe
improved animal models of psoriasis.
SUMMARY OF THE INVENTION
[0010] Non-human animal models are provided for psoriasis. The
animals develop a disease having many of the characteristics of
human psoriasis. The animals are useful for testing and screening
for biologically active -agents for the treatment of psoriasis.
Naive, immunocompetent T lymphocytes are transferred into an
immunodeficient animal host, along with at least one
pro-inflammatory cytokine and a polyclonal activating agent. The
engrafted T cells are tolerant to the major histocompatibility
antigens of the host animal, but are mismatched at one or more
minor histocompatibility loci. The engrafted animals develop a
chronic skin disorder that includes histological features observed
in human psoriasis, e.g. rete pegs, severe acanthosis and
infiltration of Th1 cells into the dermis. These animals provide a
useful model for the specific pathogenic requirements of Th1
promoting cytokines and cells for the development of psoriasiform
lesions, and into the prevention and treatment of psoriasis in
humans.
[0011] In another embodiment, the animal model has been used to
discover a treatment for psoriasis comprising administration to
patients of monoclonal antibodies that bind to and neutralize
interleukin 12 (IL-12) or gamma-interferon (.gamma. -IFN).
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a graph depicting the results of treating
psoriatic mice with antibody to IL-12. Each cluster of bars
corresponds to one experiment. The average histology score
represented by each bar is printed above it. .alpha.-IL-12 means
anti-IL-12.
[0013] FIG. 2 is a graph depicting the disease severity from weeks
14 to 18, as measured by average ear thickness, of psoriatic mice
treated with anti-IL-12 antibody compared to mice treated with an
isotype-matched control antibody (Isotype).
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0014] Non-human animal models having many of the histologic
characteristics of human psoriasis are provided. An
immunocompromised host animal is injected with a purified
population of CD45Rb positive cells from a donor animal of the same
or related species. Preferably, the injected cells will be
CD4.sup.+ CD45Rb.sup.hi T cells, where CD45Rb.sup.hi indicates that
the cells are in the upper half of T cells for CD45Rb expression.
The host and donor animals are tolerant of the host major
histocompatibility antigens, e.g., are of the same MHC haplotype
(MHC matched) but are mismatched at one or more minor antigens. The
injected cells are stimulated with a pro-inflammatory cytokine,
e.g. IL-12, and/or a polyclonal activating agent, before and/or
after CD45Rb positive cells are transferred to the host. The
animals develop a chronic skin disorder that includes histological
features observed in human psoriasis, e.g. rete pegs, severe
acanthosis and infiltration of Th1 cells into the dermis.
[0015] These animals provide a useful model for the specific
pathogenic requirements of Th1 promoting cytokines and cells for
the development of psoriasiform lesions, and into the prevention
and treatment of psoriasis in humans. 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 the disease, the role of
specific T cell subsets and cytokines, and the role of specific
antigens in activation of the disease associated T cells.
[0016] 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 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
which results in an inability to undergo germlne DNA rearrangement
at the loci encoding immunoglobulins and T-cell antigen receptors
or to a genetic defect in thymus development (nu/nu).
[0017] 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 TIMTM 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/lnu. Transgenic mice, rats and pigs are
available which lack functional B cells and T cells due to a
homozygous disruption in the CD3.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/+.
[0018] Additional loss of immune function in the host animal may be
achieved by decreasing the number of endogenous macrophages before,
during, or after transfer of T cells, e.g. the reduction of
macrophages by administration of dichloromethylene diphosphonate
(Cl.sub.2MDP) encapsulated in liposomes.
[0019] 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.
[0020] 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, Bl, 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.
[0021] The immunocompromised host animals are injected with a
purified population of CD45Rb positive T cells isolated from an
immunocompetent donor. 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 one or more
minor histocompatibility antigens of the recipient. 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.
[0022] 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. It has been suggested that some
cutaneous antigens may serve as a trigger for psoriasis (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
psoriasis. 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.
[0023] 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.
[0024] 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.
[0025] The injected cell population comprises naive,
immunocompetent T cells. A convenient marker for this population is
CD45Rb, which is highly expressed in naive T cells and B cells
(Serra-Pages et al. (1993) Tissue Antigens 42:441). A further
separation may be obtained by sorting for CD4 positive cells, which
enriches for T helper cells. Preferably, the injected cells will be
CD4.sup.+ CD45Rb.sup.hi T cells, where CD45Rb.sup.hi indicates that
the cells are in the upper half of T cells for CD45Rb
expression.
[0026] T cells are conveniently isolated from secondary immune
organs, e.g. spleen, lymph node, thymus, etc. Cells may also be
isolated from peripheral blood, cord blood, aphoresis product, etc.
For isolation of cells from tissue, an appropriate solution may be
used to disperse the spleen, lymph nodes, etc. Such solution will
generally be a balanced salt solution, conveniently supplemented
with fetal calf serum or other naturally occurring factors, in
conjunction with an acceptable buffer at low concentration,
generally from 5-25 mM. Convenient buffers include HEPES, phosphate
buffers, lactate buffers, etc. Otherwise lymphocytes may be
released from the tissue in accordance with conventional
methods.
[0027] Separation of the desired cells for engraftment will then
use affinity separation to provide a substantially pure population.
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.
[0028] 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.
[0029] 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.
[0030] 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. 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.
[0031] The labeled cells are then separated-as to the expression of
CD4SRb and 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.
[0032] Compositions highly enriched for the desired T cells are
achieved in this manner. The subject population will be preferably
at or about 90% or more of the cell composition, and most
preferably be at or about 95% or more of the cell composition. 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 cells may optionally be expanded by use of
growth factors or stromal cells associated with T cell
proliferation and differentiation.
[0033] 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
1.times.10.sup.5 and not more than about 1.times.10.sup.6, more
usually at least about 2.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.
[0034] Prior to injection, concurrently with injection of cells,
and/or following injection, the T cells are stimulated with a
polyclonal activating agent and/or at least one pro-inflammatory
cytokine. In a preferred embodiment of the invention, the
stimulation comprises injection into the T cell recipient animal of
both a polyclonal activating agent and a pro-inflammatory cytokine.
Administration of either or both agents is by any convenient
method, e.g. systemic injection, which may be intravascular,
subcutaneous, intraperitoneal, etc.; patches or implants; by
inhalation, etc. Usually such stimulus is administered within about
1 day after the cells are injected, and an optional follow-up dose
of the cytokine may be given within about 3 days. While some
aspects of psoriasis are induced by cells alone, the severity of
disease is greatly enhanced by administration of this immune
stimulus.
[0035] Suitable pro-inflammatory cytokines include IL-12, IL-2,
IL-15, IL-18, TNF.alpha., IL-1.alpha., IL-6, VEGF, and GM-CSF.
Preferably the cytokine will have the same species of origin as the
T cells, although some cytokines have sufficient cross-reactivity
that the protein from one species will broadly stimulate cells from
other species.
[0036] Of particular interest is IL-12. A preferred dose of IL-12
is at least about 0.5 ng/gram weight of recipient, and not more
than about 2 ng/gram weight of recipient. IL-12 and its biological
activity is described in Trinchieri (1998) Int Rev Immunol.
16(3-4):365-96; Gately et al. (1998) Annu Rev Immunol. 16:495-521.
The cDNA cloning of IL-12 is described in Wolf et al. (1991) J.
Immun. 146:3074-3081. Also of interest is the use of IL-18, which
is described by Okamura et al. (1995) Nature 378:88-91. The
cytokines can readily be produced in recombinant form by expressing
the appropriate nucleic acid sequences, which are available in
public databases, e.g. mouse IL-18 sequence may be found at Genbank
accession number D49949. Dosage ranges for cytokines are readily
determined using standard practice, e.g. dosing in 10-fold
increments to determine effective ranges.
[0037] In a preferred embodiment of the invention, the
pro-inflammatory cytokines are administered in conjunction with a
polyclonal activating agent. Polyclonal activating agents of
interest 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. Mls;
bacterial, e.g. SEB, SEA; or viral, e.g. mouse mammary tumor virus.
It is of interest that bacterial infection may provide a trigger
for induction of psoriasis in patients. The choice of polyclonal
activating agent provides a means of assessing the potency of
different superantigens and mitogens on development of the disease
in the subject animal model.
[0038] The polyclonal activating agent is administered at a dose
that is sufficient to activate the injected T cells, but which is
below the dose at which there is observed to be systemic toxicity.
The appropriate dose for different agents is readily determined by
one of skill in the art. For example, with LPS the dose is usually
at least about 0.1 .mu.g/g weight of recipient and not more than
about 5 .mu.g/g weight of recipient, preferably about 1 .mu.g/g
weight. A similar range of doses is used for bacterial
superantigens, such as SEA and SEB.
[0039] After administration of the T cells, cytokine(s) and
polyclonal activating agent, within about 4 weeks the animals
develop a disease that closely mimics human psoriasis. Scoring of
the disease severity is based on physical appearance and ear
thickness. Symptoms include erythema on one or more locations,
generally first appearing on the ears and face; and scaling over
the body surface. Severe scaling is defined as covering more than
about 20% of the surface of the animal. Measurement of ear
thickness is conventional, using a micrometer, etc.
[0040] A more detailed analysis may utilize histological section of
various tissues, conveniently ear, eyelid, tail, etc. Specific
histological features include acanthosis; mononuclear cell
infiltration, thickening of epidermis; high vascular density; rete
pegs; hyperplasia of epidermis and keratinocytes; microabscesses;
thinning of the granular cell layer; pustule formation; and
destruction of granular cell layers. Such features are well
described in the literature, for examples see Carroll et al. (1995)
Cell 83:957-968; Sundberg et al. (1997) Pathobiology 65:271-286;
and Wrone-Smith and Nickoloff (1996) J. Clin. Invest.
98:1878-1887.
[0041] To more fully characterize the lesions, immunophenotypic
analysis may be performed to detect a variety of relevant antigenic
determinants. The extent of keratinocyte proliferation may be
assessed by immunostaining to detect proliferating cell nuclear
antigen (PCNA). Other indicators of keratinocyte activation include
HLA-DR, integrin, keratin 16, and involucrin expression. 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 psoriasis may include T cells within the
epidermis and dermis; and the expression in adjacent blood vessels
of focal E-selectin, and diffuse vascular cell adhesion molecule-1
(VCAM-1) expression.
[0042] A distinctive feature of the human disease, which may also
be found in the animals of the invention, is the abnormal presence
of rete pegs. In the epidermis, the interface with the dermis is
uneven. The epidermal papillae or rete pegs are ridges and grooves
that are matched by a corresponding connective tissue pattern of
the dermis, called dermal papillae (this makes up the papillary
layer of the dermis). These projections of epidermis created by
dermal papillae appear as columns of cells in section.
Drug Screening Assays
[0043] In addition to assessment of the role in disease induction
of various components, e.g. minor histocompatibility antigens; T
cell sub-populations; pro-inflammatory cytokines; and polyclonal
triggers; 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 psoriasis. Of
particular interest are screening assays for agents that have a low
toxicity for human cells.
[0044] 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. Currently
available treatment for psoriasis includes:
1TABLE I THERAPY NOTES Antibiotics, Infections may worsen (flare)
psoriasis antimicrobials Cyclosporine Suppresses the body's immune
system; use for more than one year not recommended Methotrexate
Effective in psoriasis and psoriatic arthritis; Lifetime cumulative
dose of 4.5 grams has been associated with up to 25% risk of
liver/cirrhosis/fibrosis Hydroxyurea, NSAIDS, Sulfasalazine,
6-thioguanine Retinoids--acitretin, Etretinate, labeled for
psoriasis, etretinate, has been replaced by acitretin isotretinoin
ULTRAVIOLET LIGHT Hospitalization, Outpatient day For severe
psoriasis: Intensive nurse treatment administered therapy of
day-long UV and prescription topicals for 30 days or more
Phototherapy (UVB) Minimum 20-40 treatments to clear psoriasis;
additional treatments may prolong clearance Photochemotherapy
(PUVA) Combines ingestion, soaking, or painting with psoralen
medication before exposure to UVA light. Minimum 20 treatments to
produce substantial clearing; additional to prolong clearance Home
phototherapy (UVB) Durable medical equipment for home use to
prolong clearance in physician-selected patients. TOPICAL AND
INTRALESIONAL THERAPY Anthralin Compounded in various strengths,
can be combined with UV exposure Calcipotriene Topical vitamin D is
first in this class to be approved for psoriasis Coal Tar
Compounded in various strengths, can be combined with UV exposure
Corticosteroids-- Applied to skin low strength Corticosteroids--
Various potencies, applied to skin, moderate to potent injected
into lesions, or taken orally Emollients Preserves skin flexibility
Keratolytics-- Compounded in various strengths, salicylic acid used
with tar or emollients Tazarotene First topical vitamin A
derivative approved for psoriasis
[0045] 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.
[0046] 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. Frequently, treatment effective for
prevention is also effective in regressing the disease.
[0047] 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.
[0048] The term "agent" as used herein describes any molecule, e.g.
protein or pharmaceutical, with the capability of affecting the
severity of psoriasis. 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.
[0049] By way of example but not limitation, the antibodies may be
specific for IL-12, or .gamma.-IFN. Exemplary antibodies include
HuZAF, a humanized anti-IFN-.gamma. antibody (described in commonly
assigned U.S. provisional patent application No. 60/110,523, filed
Dec. 1, 1998); HuEP5C7, a humanized anti-E/P selectin antibody
(described in U.S. Pat. No. 5,622,701); and HuM291, a humanized
anti-CD3 antibody (described in PCT publication WO 96/26964).
[0050] 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.
[0051] 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.
[0052] 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. %.
[0053] 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.
[0054] Therapy with Antibodies that Neutralize IL-12 or Gamma
Interferon
[0055] In other embodiments of the present invention, agents that
block the effects of the lymphokines interleukin 12 (IL-12) or
gamma interferon (IFN-.gamma.) are used to treat a patient with
psoriasis. These agents may be small molecules as described above,
but are preferably proteins. In preferred embodiments, the agent is
an antibody, especially a monoclonal antibody, that binds IL-12 or
IFN-.gamma.. The monoclonal antibody may be derived from any
convenient species, e.g., mouse, rat, hamster, etc. by methods well
known in the art (see, generally, Harlow & Lane, Antibodies, A
Laboratory Manual, Cold Spring Harbor Press, NY, 1988). Preferably,
the antibody will be chimeric (see, e.g., Cabilly et al., U.S. Pat.
No. 4,816,567 which is herein incorporated by reference for all
purposes) or humanized, i.e., formed by linking the complementary
determining regions (CDRs) of a non-human antibody to an
essentially human framework and constant region by recombinant DNA
techniques (see, e.g., Queen et al., U.S. Pat. No. 5,585,089, which
is herein incorporated by reference for all purposes).
Alternatively, the antibody may be human, as made, e.g., by trioma
technology (see, e.g., U.S. Pat. No. 4,634,664) or from transgenic
animals (see, e.g., Lonberg et al., WO 93/12227 and Kucherlapati,
WO 91/10741, each of which is herein incorporated by reference for
all purposes) or by phage display methods (see, e.g., Dower et al.,
WO 91/17271 and McCafferty et al., WO 92/01047, and Winter WO
92/20791, each of which is herein incorporated by reference for all
purposes).
[0056] Preferably, the monoclonal antibody will have binding
affinity (association constant) for its antigen of at least
10.sup.8 M.sup.-1 or more preferably 10.sup.9 M.sup.-1 or higher.
Most preferably, the antibody will neutralize IL-12 or IFN-.gamma.,
that is block one or more of its biological functions, for example
the ability to bind to its cellular receptor. Other functions that
may be blocked by a neutralizing antibody are exemplified for
IFN-.gamma. in commonly assigned U.S. provisional patent
application No. 60/110,523 and for IL-12 in Gately et aL, U.S. Pat.
No. 5,780,597 and Gately et al. WO 99/37682 (each of which is
herein incorporated by reference for all purposes), e.g., blocking
the IFN-.gamma.-inducing function of IL-12. Preferably, a
concentration of 0.001, 0.005, 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2, 5,
or 10 .mu.g/ml of the antibody will block 25%, 50%, 90%, 95%, 99%
or essentially 100% of the function of the respective lymphokine,
especially when the lymphokine is also used at one of these
concentrations or at a molar concentration that is 0.005, 0.01,
0.05, 0.1, 0.25, 0.5 or 1.0 of the concentration of the antibody.
Exemplary IFN-.gamma. neutralizing antibodies are HuZAF or other
humanized forms of AF2 (as described in U.S. provisional patent
application No. 60/110,523). Exemplary IL-12 neutralizing
antibodies are 5F2, 16F2, 16G2, and 20E11 (as described in Gately
et al. WO 99/37682) and their chimeric and humanized forms. Other
preferred antibodies have the same or overlapping epitopes as the
aforementioned antibodies, that is they compete (cross-block) with
them for binding to antigen, or in which some of the same amino
acids of the antigen are shown by in vitro mutagenesis to
contribute to binding. Particularly preferred anti-IL-12 antibodies
will bind to the p75 IL-12 heterodimer but not to the individual
subunits such as p40.
[0057] Alternatively, instead of an antibody, an extracellular
portion of a cellular receptor (or its binding subunit) for IL-12
or IFN-.gamma. can be recombinantly linked to the Fc region of a
human immunoglobulin (e.g., IgG1) to improve its half-life or other
properties. See, e.g., Ashkenazi et al. (1991), Proc. Natl. Acad.
Sci. USA 88:10535-9 and Moosmayer et aL (1995), J. Interferon
Cytokine Res. 15:1111-5 (each of which is herein incorporated by
reference for all purposes) for methods of constructing such fusion
proteins ("immunoadhesins"). Receptors for IL-12 and IFN-.gamma.
are respectively described in Chua et al. (1994), J. Immunol.
153:128-36 and Presky et al. (1996), Proc. Natl. Acad. Sci. USA
93:14002-7; and Aguet et al. (1988), Cell 55:273-80 (each of which
is herein incorporated by reference for all purposes). The fusion
protein will bind to and neutralize IL-12 or IFN-.gamma.
respectively and will have properties similar to an antibody and
may therefore be used for the treatment of psoriasis. In what
follows, the term "antibody" will be understood to also encompass
fusion proteins of this type.
[0058] For administration to patients, the antibody (or fusion
protein) drug will typically be formulated in a pharmaceutically
acceptable carrier. A variety of aqueous carriers can be used,
e.g., water for injection (WFI), or water buffered with phosphate,
citrate, acetate, etc. to a pH typically of 5.0 to 8.0, most often
6.0 to 7.0, and/or containing salts such as sodium chloride,
potassium chloride, etc. to make isotonic. The carrier may also
contain excipients such as human serum albumin, polysorbate 80,
sugars or amino acids such as arginine, histidine or glycine to
protect the active protein. The concentration of antibody in these
formulations may vary widely from about 0.01 to 100 mg/ml but will
most often be in the range 1 to 10 mg/ml. The formulated antibody
is particularly suitable for parenteral administration, and may be
administered as an intravenous infusion or by subcutaneous,
intramuscular or intravenous injection, and may also be
administered by injection at the site of disease, e.g., into the
psoriatic lesions.
[0059] Doses of the drug will typically contain from 0.01 to 100 mg
antibody (or fusion protein) but most often from 0.1 to 1, or 1, 2
or 5 to 10 mg per kilogram body weight or as a unit dose, in an
amount sufficient to alleviate the disease without causing
unacceptable side effects ("therapeutically effective dose"). The
antibody drug may be administered once or multiple times, e.g., 1,
2 or 3 times per day, week or month for one to several days, weeks,
months or years, or chronically, depending upon the nature and
severity of the disease. The antibody will often be administered
after or in combination with one or more other immunosuppressive
drugs or other therapies, for example, corticosteroids,
cyclosporine, methotrexate, phototherapy (with or without PUVA) or
others listed in Table 1 above. The anti-IL-12 or anti-IFN-.gamma.
antibody may also be used together or in combination with other
antibodies, for example to adhesion molecules or lymphokines such
as CD11a, CD40 ligand, IL-8 or to their receptors (e.g., IL-2
receptor).
[0060] The severity of psoriasis is measured by the Psoriasis Area
Severity Index (PASI) (see e.g., Fleischer et al. (1999), J.
Dermatol. 26:210-215 and Tanew et al. (1999), Arch Dermatol.
135:519-524) or various psoriasis global assessment scores such as
Physician's Global Assessment (PGA) which are well-known to those
skilled in the art of clinical trials for psoriasis. Treatment with
the (e.g., humanized or human) antibody to IL-12 or IFN-.gamma.
will reduce the PASI (or global assessment score) by at least 25%
or 40% but preferably 50% or even 60 or 70% or more in at least 50%
but preferably 60-70% or 75% or more of the patients treated.
Alternatively, such treatment will cause a greater reduction in a
smaller group or patients, i.e., at least 75% but preferably 80-90%
or more or even essentially complete clearance, in at least 20% to
25% but preferably 30% to 40% or even 50% or more of the patients.
This reduction will typically last at least 2 but preferably 3 or
more months, or even 4 to 6 or more months, but most preferably a
year or longer, either while treatment with the antibody is
continued or after it is stopped. Typically, in a clinical trial
(e.g., a phase II or phase III trial), the improvement in PASI or
score in the patients treated with the anti-IL-12 or
anti-IFN-.gamma. antibody, relative to the control group of
patients receiving no treatment or placebo or another agent, will
be statistically significant, for example at the p=0.05 or 0.01 or
even 0.001 level.
[0061] Alternatively, the antibody to IL-12 or IFN-.gamma. can be
administered after remission has already been induced by another
drug, for example corticosteroids, cyclosporine, methotrexate,
phototherapy (with or without PUVA) or others listed in Table 1
above. In this case, treatment with the antibody will increase the
median time to relapse (e.g., 50% worsening in PASI) by at least
40% but preferably 50% and most preferably 60-70% or more or even
100% (doubling) or more. Typically, in a clinical trial (e.g., a
phase 11 or phase III trial), this increase of time to relapse in
the patients treated with the anti-IL-12 or anti-IFN-.gamma.
antibody, relative to the control group of patients receiving no
treatment or placebo or another agent, will be statistically
significant, for example at the p=0.05 or 0.01 or even 0.001
level.
[0062] 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.
[0063] 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.
[0064] 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.
[0065] 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.
[0066] 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
EXAMPLE 1
[0067] Materials and Methods
[0068] Mice.
[0069] Female BALB/cj mice and BALB/cj-IFN.gamma..sup.-/- mice
(donor mice) were purchased from Jackson Labs (Bar Harbor, Me.).
C.B-17/lcr scid/scid mice and C.B-17 scid-beige double mutant mice
(recipient mice) were purchased from Taconic (Germantown, N.Y.).
All mice were housed in a specific pathogen free environment at the
Protein Design Labs animal facility and were used between 4-12
weeks of age. Sentinel mice were used to screen for the following
pathogens: MHV, sendai, PVM, REO3, TMEV GDVIII, M. pulmonis and
parvovirus. Random screens of mice for pinworms were also
conducted. None of the pathogens listed above were detected at
anytime. Mice were housed 2-5 per microisolator. All scid/scid or
scid/beige mice were handled with gloves under a class II hood, fed
sterile food and water ad libitum, and maintained inside a laminar
flow tent (Bioclean Maywood, N.J.) in sterile microisolators that
were changed weekly. Donor mice were housed in conventional cages
that were changed weekly.
[0070] Cell Purification, and Injection into scid/scid Mice.
[0071] Spleens were collected from 6-12 week old donor mice
(BALB/cj or IFN.gamma..sup.-/--BALB/cj) and splenocytes were
isolated by mechanical homogenization of whole spleens. CD4.sup.+ T
cells were selected by positive selection. In brief, a cell
suspension of pooled splenocytes from 4-5 donor mice was incubated
with anti-CD4 (L3T4) antibody coated magnetic beads (DYNABEADS:
Catalog # 114.05, Dynal, Lake Success, N.Y.) for 20-30 minutes at
4.degree. C. and separated by magnetic cell sorting with a Dynal
Magnetic Particle Concentrator (MPC). Cells were removed from the
cell-bead complex with Dynal DETACHaBEAD, and isolated from beads
using a Dynal MPC. The resulting CD4.sup.+ enriched population was
>90% pure. The cell suspension (10.times.10.sup.6 cells/ml) was
then incubated with Fc block (anti-CD32, PharMingen, 01241A) (10
.mu.g/ml) and labeled with anti-CD4-FITC (PharMingen, 9004D) and
anti-CD45RB-PE (PharMingen, 01145A) (both at 10 .mu.g/ml), for 30
minutes at 4.degree. C., washed, and sorted using a FACSTAR (Becton
Dickinson, San Jose, Calif.) cell sorter. Double positive cells
(CD4.sup.+/CD45Rb.sup.+) were collected, selecting the cells that
expressed high levels of CD45Rb (brightest 45%). The collected cell
population was >90% pure and viable. Cells were then washed in
cold potassium buffered saline (PBS Sigma D8662) and resuspended in
PBS at 1.5.times.10.sup.6 cells/mL. C.B-1 7/lcr scid/scid mice,
aged 4-6 weeks, were injected intravenously with 3.times.10.sup.5
cells each, 200 .mu.L total volume into 10 the tail vein.
[0072] Induction and Treatment of Psoriasiform Lesions in scid
Mice.
[0073] To study the effect of microbial products and IL-12,
recipient mice were treated as follows: A control group received
CD4.sup.+CD45Rb.sup.hi sorted cells with no additional treatment. A
second group was given 20 .mu.g lipopolysaccharide (LPS) from
Salmonella enteritidis (Sigma L-2012) i.p. per mouse on day 1 after
cell transfer. A third group received 10 ng IL-12 (Pharmingen, CA)
alone per mouse delivered i.p. on days 1 and 3. The final group was
injected i.p. with a combination of LPS and IL-12. Dosage studies
were conducted using 2, 10, and 100 ng doses of IL-12 in
conjunction with 20 .mu.g LPS. The LPS and IL-12 injection was
given on day 1 following T cell transfer and an additional dose of
IL-12 was administered on day 3. In additional studies, LPS (20
.mu.g) and IL-12 (10 ng) were administered once weekly for 3 weeks.
Some experimental groups received 10 .mu.g Staphylococcal
enterotoxin B protein from Staphylococcus aureus (Sigma catalog
S4881) i.p. per mouse once on day 1 following T cell transfer.
[0074] To study the role of IFN.gamma., T cells from
BALB/c-IFN.gamma..sup.-/- mice (Jackson labs) T cells were isolated
by the same methods described above. Recipient scid/scid mice were
also co-injected with 20 .mu.g LPS and 10 ng IL-12 on day 1 and 10
ng of IL-12 on day 3. In addition, scid-beige mice (Taconic), that
are T-, B- and NK-cell deficient, were used as recipient mice for
IFN.gamma..sup.-/- T cell transfer in some experiments. For
interventional studies, 0.5 mg anti-IL-12 (clone C17.8, PharMingen,
San Diego, Calif.) was given i.p. to mice on day 7 and 35. Control
mice received either PBS or rat IgG (Sigma) on the same day.
[0075] Clinical Evaluation.
[0076] Mice were evaluated by three different investigators at
weekly intervals commencing on week 4 and ending on week 10. To
record disease progression semi-quantitative clinical scores from 0
to 4 were given based on physical appearance and ear thickness.
0=no skin or ear symptoms; 1=mild, moderate erythema on ears or
eyelids with mild thickening of the ear; (<2% of the body
surface) 2=moderate to severe erythema on one location (mostly ear
and face) (2-10% of the body surface), mild scaling; 3=severe
erythema at two or more sites (ear, face, trunk) (>10% of the
body surface), severe scaling; 4=very severe, extensive erythema
throughout the body (>20% of the body surface) with severe
scaling. Specific observations were noted based on fur condition,
ear manifestations, eyelid appearance, presence of inflammation on
limbs and tail. Ear thickness was determined using a modified
spring micrometer (Oditest; Dyer, Inc). Measurements were taken
from the same part of the ear for all data time points from both
the right and left ear. The micrometer was allowed to settle while
on the ear to prevent tissue edema from affecting final
measurement.
[0077] Histopathologic Analysis and Immunohistology of Skin Tissue
Samples.
[0078] Necropsies were performed on mice at week 10-12 after cell
transfer. Tissue samples from ear, eyelid and tail were collected
and fixed in paraformaldehyde solution and submitted to Comparative
Bioscience (Sunnyvale, Calif.) for section preparation and
analysis. To record disease severity, 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. Mice which had
ear thickness of 25 .mu.m or less with no additional clinical signs
were automatically given a histology score of zero without section
analysis. 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.
[0079] Tissue samples were collected and embedded in Tissue Tek OCT
compound and frozen with dry ice for cryostat-cut sections. Tissue
sections (5 .mu.m) were fixed in 100% acetone and stained with
PE-conjugated IL-12 mAb (p40170) (Pharmingen, clone C17.8). Tissues
were evaluated as positive or negative based on visual fluorescent
microscopy detection.
[0080] Skin Infiltrating Lymphocyte Cell Isolation.
[0081] Skin infiltrating lymphocytes (SIL) were isolated via enzyme
digestion. In short, skin, ears, and eyelids were minced with
sterile scissors, and the pieces washed with HBSS over a 100 mm
nylon cell strainer (Falcon) to remove surface debris. Infiltrating
cells were liberated by incubating the cut pieces in 25 mL of warm
(37.degree. C.) HBSS media without Ca/Mg (BioWhiftaker,
Walkersville, Md. 10-543F) supplemented with 25 mM HEPES buffer
(BioWhittaker 17-737E) and 10% FCS (HyClone Labs Inc., Logan, UT
SH30071.03) for 20 min at 37.degree. C. The remaining pieces were
washed over nylon mesh, resuspended in RPMI 1640 media
(BioWhittaker 12-702F) augmented with 25 mM HEPES buffer, 10% FCS,
400 U/mL DNAse (Boehringer Mannheim Biochemicals, Indianapolis,
Ind. 104159), 400 U/mL collagenase (Boehringer Mannheim 1088874),
and incubated 90 minutes at 37.degree. C. on a rocker. The
resulting cell suspension was filtered sequentially through a 100
.mu.m and 40 .mu.m nylon mesh filter and then washed twice in RPMI
1640 supplemented with 25 mM HEPES and 10% FCS.
[0082] In vitro Stimulation of SIL and Detection of Cytokines.
[0083] SIL were resuspended at 10.sup.6/mL in complete media RPMI
1640 supplemented with 10% FBS (HyClone), 5.times.10.sup.-5 M
2-mercaptoethanol (Sigma), 2 mM glutamine (Life Technologies), 10
U/mL penicillin/100 .mu.g streptomycin (Life Technologies) and 15
mM HEPES. CD4.sup.+ sorted T cells were resuspended at
2.5.times.10.sup.5/mL. 200 .mu.l per well of this suspension was
then placed in a 96 well tissue culture plate (Falcon 3072) and
incubated for 48 hours with .alpha.CD3 (PDL, clone 145-2C11) and
.alpha.CD28 (PharMingen), each at 1 .mu.g/ml. Supernatants from
three different culture wells were collected and tested by ELISA
for IFN.gamma., TNF.alpha., and IL-4. The ELISA procedure involved
coating a 96 well flat bottom Immulon 4 plate (Dynatech Labs, Inc
011-010-3850) overnight at 4.degree. C. with 50 .mu.l of a 2
.mu.g/mL solution of anti-IFN.gamma., anti-TNF.alpha. or anti-IL-4
antibody (all from Pharmingen) in carbonate buffer. Plates were
then washed with PBS/Tween (0.05% Tween 20 in PBS) and blocked with
200 .mu.l sterile solution of PBS with 3% BSA (Sigma bovine albumin
A7030) for 1 hour at 37.degree. C. In between all of the following
steps, plates were washed with PBS/Tween. IFN.gamma., IL-4 and
TNF.alpha. standards as well as sample supernatants were then added
to wells and incubated for 2 hours at 37.degree. C. Biotin
conjugated secondary antibodies for anti-IFN.gamma.,
anti-TNF.alpha., and anti-IL-4 (all antibodies from Pharmingen)
were then added to the respective plates at 2 .mu.g/mL in 3%
BSA/PBS solution and incubated for 1 hour at 37.degree. C. Horse
radish peroxidase (HRP) labeled streptavidin (Jackson
ImmunoResearch Labs 016-030-084) was then added at a concentration
of 1 .mu.g/mL. O-Phenylenediamine (Sigma 4664) was then used as
substrate buffer per manufacturer's protocol. Assay was then read
on a Molecular Devices (Sunnyvale, Calif.) plate reader and data
analyzed using SOFTmax.TM. software.
[0084] Results
[0085] Treatment of scid/scid Mice Restored with
CD4.sup.+/CD45Rb.sup.hi T Cells with LPS Plus Low and Medium Doses
of IL-12 Results in Increased Incidence and Severity of Psoriasis;
High Doses of IL-12 Prevent Disease Induction.
[0086] Previous studies have demonstrated that scid/scid mice
reconstituted with minor haplotype mismatched
CD4.sup.+/CD45Rb.sup.hi Balb/c T cells sometimes develop chronic
skin inflammation that resembles human psoriasis. In initial
experiments, it was found that when BALB/c CD4.sup.+/CD45Rb.sup.hi
T cells alone were transferred to C.B-17 scid/scid mice, only a few
animals exhibited psoriasiform lesions and the expression of
disease was rather mild. This finding was consistent with previous
observations made by Schon et al. (1997), supra. Because bacterial
mitogens or bacterial superantigens have been shown to be potent
modulators of cell-mediated immune responses, and IL-12 has been
demonstrated to play an important role in the induction of various
autoimmune conditions, it was initially tested whether the
co-administration of such agents would have an effect on the
induction of psoriasis in the scid/scid transfer model. As shown in
Table 2, when C.B-17 scid/scid mice were reconstituted with BALB/c
CD4.sup.+/CD45Rb.sup.hi T cells alone, only 38% of the mice
developed psoriasiform skin lesions and only 27% of the mice
developed severe forms of disease. When LPS was co-administered
alone, we observed a slight increase in disease incidence (50%),
but the severity of the lesions remained similar to lesions in mice
that had received cells alone. Similarly, co-administration of a
medium dose (10 ng/mouse) of IL-12 alone on day 1 and 3 following T
cell transfer led to an apparent increase in disease incidence
(67%) without affecting disease severity.
2TABLE 2 In vivo administration of IL-12 in combination with LPS
after CD4.sup.+/CD45Rb.sup.hi T cell transfer leads to a
significant increase in disease expression. Average Post Cell
Transfer Disease Histology Severe Treatment.sup.1 Incidence.sup.2
Score.sup.3 Disease.sup.4 PBS 10/26 (38%) 1.1 .+-. 1.4 7/26 (27%)
IL-12 medium 2/3 (67%) 0.75 .+-. 1 0/3 (0%) LPS 2/4 (50%) 0.6 .+-.
0.7 0/4 (0%) LPS + IL-12 low 6/11 (55%) 1.9 .+-. 1.0 4/11 (36%) LPS
+ IL-12 medium 24/33 (73%) 2.25 .+-. 1.1 14/33 (42%) LPS + IL-12
high 0/4 (0%) 0.0 0/4 (0%) LPS + IL-12 medium.sup.5 8/10 (80%) 2.5
.+-. 1 5/10 (50%) LPS + IL-12 medium.sup.6 0/8 (0%) ND 0/8 (0%) SEB
3/4 (75%) ND 2/4 (50%) .sup.1After CD4+/CD45Rb.sup.hi cells
isolated from BALB/cj spleens were transferred to scid/scid mice,
the recipient mice were given the following treatments. The PBS
group received sorted cells with no additional treatment. The IL-12
group received long IL-12 alone per mouse delivered i.p. on days 1
and 3 in addition to cells on day 0. The LPS group was given 20
.mu.g Lipopolysaccharide (LPS) from Salmonella enteritidis (Sigma
L-2012) per mouse on day 1 # after cell transfer. # Dosage studies
were conducted using the low (2 ng), medium (10 ng), or high (100
ng) doses of IL-12 in conjunction with 20 .mu.g LPS. The LPS and
IL-12 injections were given on day 1 and an additional dose of
IL-12 was administered on day 3. In a different series of
experiments LPS (20 .mu.g) and IL-12 (10 ng) were injected once a
week for three weeks. Other mice received 10 .mu.g Staphylococcal
enterotoxin B protein from Staphylococcus aureus (Sigma catalog
S4881) # per mouse once on day one. .sup.2Disease incidence is
reported as number of mice with disease over 12 weeks of time:
criteria being ear thickness .gtoreq. 26 .mu.m or clinical score of
.gtoreq. 1. Ear thickness of normal scid/scid mice: 21 .+-. 1 .mu.m
(n = 10). .sup.3Histological scores of diseased mice from 1 to 4
were given based on the severity of inflammation of ear, skin or
eyelid. Mice which had ear thickness of 25 .mu.m or less were
categorized as undiseased (average ear thickness of undiseased mice
22 .mu.m .+-. 1) and automatically excluded from section analysis.
0 = no signs of inflammation; 1 = very low level and 2 = low level
of mononuclear cell infiltration, mild thickening of epidermis; # 3
= high level of mononuclear cell infiltration, high vascular
density, thickening of the epidermis # (acanthosis, rete pegs and
hyperplasia of epidermis and keratinocytes); 4 = very extensive
mononuclear cell infiltration in epidermis and dermis, very high
vascular density, extreme thickening of epidermis, pustule
formation and loss of granular cell layers. p versus PBS control:
LPS + IL-12 low, p < 0.1, LPS + IL-12 medium, p < 0.008.
Statistical analysis was performed using the # two-tailed student t
test. .sup.4Severe disease induction calculated as the number of
animals that received an average histology score of .gtoreq.2.5
and/or a clinical score of .gtoreq.3. .sup.5LPS (20 .mu.g) and
IL-12 (10 ng) was given once a week for 3 weeks. .sup.63 .times.
10.sup.5 unsorted CD4.sup.+ T cells (CD45Rb.sup.hi and
CD45Rb.sup.lo) were transferred into scid/scid mice. LPS (20 .mu.g)
and IL-12 (10 ng) was given once a week for 3 weeks.
[0087] In contrast, recipient mice that received either 1 or 10 ng
IL-12 on day 1 and day 3 along with 20 .mu.g of LPS on day 1
following T cell transfer showed an increase in disease severity
and incidence. In particular, the administration of a medium doses
of IL-12 (10 ng/mouse) along with LPS showed a 73% incidence of
disease with an average histology score of the diseased mice of
2.25.+-.1.1, which was significantly higher than when PBS was
co-administered (p<0.008). In addition, the percentage of
animals with severe disease in the IL-12 medium dose group was also
higher (42%) when compared to the IL-12 low dose group (36%) and
significantly higher when compared to the PBS control (27%).
Interestingly, co-administration of LPS (20 .mu.g/mouse) and a high
dose of IL-12 (100 ng/mouse) completely inhibited disease
development (incidence 0%). When LPS and medium doses of IL-12 (10
ng) were co-administered once a week for three weeks the incidence
of disease was 80% (8/10) with an average clinical score of
2.5.+-.1 (Table 2). This particular induction protocol was also
associated with an accelerated onset of disease as the animals in
this group came down with disease as soon as 4 weeks after T cell
transfer. In contrast, animals that had received only one dose of
LPS and IL-12 (10 ng) developed disease at an average of 6-8 weeks
after T cell transfer, and animals that received T cells only
developed signs of disease at an average of week 8 to 10 after T
cell transfer.
[0088] Animals that had received unsorted CD4.sup.+ T cells never
came down with disease even if they were treated with three
administrations of LPS (20 .mu.g) and IL-12 (10 ng) (Table 2),
indicating that LPS and IL-12 administration can only act on naive
T cells and the regulatory effects of CD45Rb.sup.lo cells can not
be overcome by the administration of microbial factors and IL-12.
Of note, mice that received no T cells or T cells alone were housed
together with mice that received T cells plus LPS and IL-12, to
ensure that other exogenous factors did not play a role in the
induction of disease.
[0089] In a different set of experiments, we tested whether other
microbial products such as SEB exert an influence on disease
expression as well. As shown in Table 2, SEB was also able to
induce disease at a higher incidence and severity (average clinical
score 1.5.+-.0.9) than cells alone; thus demonstrating that the
ability of bacterial constituents to modulate the expression of
psoriasiform lesions is not unique to LPS.
[0090] Additionally, in separate cell transfer studies, it was
found that scid/scid mice that received cells isolated from the
skin lesions of diseased mice did not develop psoriasis unless LPS
and IL-12 was co-administered, indicating that the transfer of
inflammatory psoriatic T cells alone is not sufficient to induce a
chronic inflammatory response in the skin.
[0091] Psoriatic Skin Lesions of scid/scid Mice Treated with LPS
and IL-12 Resemble Closely Human Pathology.
[0092] Animals that received CD4.sup.+/CD45Rb.sup.hi cells in
conjunction with LPS and IL-12 developed disease symptoms as soon
as 4-8 weeks after cell transfer. Mice that showed no clinical
signs of disease at week 10 post T cell transfer remained disease
free for an additional 4-6 weeks of observation. Thus, mice were
monitored beginning on week 4 and necropsies were performed on
subject animals between week 10-12. Clinical signs of disease
consistently included increased ear redness, and thickened skin on
ears and eyelids. Some animals also showed signs of significant
skin inflammation on the tail. In more severe cases, skin
inflammation was observed throughout the body with increased
scaling and hair loss (clinical score 4). Ear thickness typically
varied from a base line of 21.+-.1.1 .mu.m in undiseased animals to
a pathological range of 26 to 50 .mu.m. Skin that became severely
affected consistently became scaly, ulcerated, and typically showed
plaque-type elevation. Skin inflammation in psoriatic mice ranged
from mild, around the base of the ears and around the eyelids
(clinical score 1-2), to severe hair loss that extended to over 75%
of their body (clinical score 3-4). Since ear thickness correlated
very well with the severity of disease and clinical scores, we used
the measurement of ear thickness as an indicator for overall skin
inflammation in most experiments.
[0093] Other psoriasis-like models in mice have been criticized for
not possessing the histological characteristics found in human
forms of disease. Differences in the mouse skin structures were
deemed responsible for these discrepancies. An absence of rete pegs
or ridge elongation in mice, a major hallmark in human disease, was
attributed to the relatively flat dermoepidermal junction in mice.
Histological analysis of the lesions induced by our protocol was
performed by taking biopsies of skin samples from several areas of
diseased mice and examining 3 .mu.m sections stained with
hematoxylin and eosin. Samples taken from the ear, eyelid and tail
of diseased mice that had received T cells plus LPS (20
.mu.g/mouse) and IL-12 (10 ng/mouse) were prepared for histologic
evaluation and microscopically examined by an independent
pathologist. Most lesions were found to have typical signs of
parakeratosis and hyperkeratosis with some ulcer or erosion and
pustule formation. Also noted was a thickening of the epidermis
(acanthosis) with proliferation of the keratinocytes and moderately
deep rete pegs in the subcutis; the inflammatory cell infiltration
consisted of primarily mononuclear cells composed of lymphocytes
with fewer monocytes, macrophages and plasma cells. Variable
numbers of neutrophils with a few eosinophils were also seen.
Capillaries and other vessels were numerous, contained large
numbers of marginated neutrophils and were typically surrounded by
lymphocytes. The combination of these characteristics indicate that
the psoriasiform lesions in this model are very comparable to those
found in humans. Sections from normal scid/scid mice that received
no T cells were taken at the same age as diseased mice. The
epidermis is 1-2 cells thick and the dermis contains almost no
lymphocytes. In-addition, the density of vessels is sparse. The
junction of the dermis to the epidermis is straight and contains no
abscesses.
[0094] CD4.sup.+ T Cells from the Skin of Mice with Psoriasis are
CD45Rb.sup.lo and Produce High Levels of IFN.gamma. and Low Levels
of IL-4.
[0095] To compare the activation/cytokine profile of skin
infiltrating lymphocytes (SIL), we purified this population from
the skin lesions of diseased and undiseased mice. Isolated SIL were
stimulated in vitro with anti-CD3 and anti-CD28 for 48 hrs and
supernatants were tested for the production of IFN.gamma.,
TNF.alpha. and IL-4. Lymphocytes isolated from the skin of mice
that received T cells but showed no clinical signs of disease did
not secrete any detectable levels of IFN.gamma. or IL-4. In
contrast, cells from diseased mice expressed very high levels of
IFN.gamma. and TNF.alpha. and low levels of IL-4 (Table 3). Nave
CD4.sup.+/CD45Rb.sup.hi donor cells from spleens of BALB/c were
stimulated in a similar fashion and showed no detectable levels of
any cytokine tested. Furthermore, the majority of CD4+ cells
isolated from the inflamed tissue of diseased were CD45Rb.sup.lo.
The data suggests that the majority of naive T cells transferred
into scid/scid mice differentiate in the micro-environment of the
skin into Th1-like memory/effector T cells.
3TABLE 3 CD4.sup.+ T cells isolated from psoriatic lesions of
scid/scid mice produce high levels of interferon-gamma and low
levels of IL-4 T cell Source IFN.gamma. (pg) IL-4 (pg) TNF.alpha.
(pg) Skin from scid/scid-diseased.sup.1 22987 .+-. 648 468 .+-. 79
4654 .+-. 946 Skin from scid/scid-undiseased.sup.2 .ltoreq.20
.ltoreq.15 .ltoreq.35 CD4+/CD45RB.sup.hi cells from .ltoreq.20
.ltoreq.15 .ltoreq.35 BALB/c spleens.sup.3 IFN.sub..gamma., IL-4
and TNF.alpha. production by SIL or CD4+ SIT was measured as
described in Material and Methods. Data represents the mean and SD.
.sup.12.0 .times. 10.sup.5 cells from psoriasiform lesions of 5-10
recipient scid/scid mice that had developed disease following
reconstitution with CD4.sup.+ /CD45Rb.sup.hi cells from normal
BALB/c mice along with LPS and IL-12 were cultured for 48 hrs. Data
is one value representative of 3 experiments showing similar
values. .sup.22.0 .times. 10.sup.5 cells from the skin of 8-10
recipient scid/scid mice that did not show disease symptoms were
cultured for 48 hrs. Data is from one experiment representative of
2 independent experiments showing similar values. .sup.32.0 .times.
10.sup.5 CD4.sup.+ /CD45RB.sup.hi cells sorted by flow cytometry
from spleen cells isolated from BALB/cj mice were cultured for 48
hrs. Cell purity .gtoreq. 90%.
[0096] IFN.gamma.-deficient CD4.sup.+/CD45Rb.sup.hi T cells are
able to induce psoriasiform lesions in scid mice. To examine
whether IFN.gamma. has a primary role in the induction of
psoriasiform lesions, we first transferred naive T cells from
IFN.gamma.-deficient (IFN.gamma..sup.-/-) mice into C.B-17
scid/scid mice. Interestingly, we found that despite the lack of
IFN.gamma., CD4.sup.+/CD45Rb.sup.hi T cells from IFN.gamma..sup.-/-
donors were able to induce psoriasiform lesions in scid/scid mice.
Disease induction occurred with similar frequency, but ear
thickness in diseased IFN.gamma..sup.-/- T cell scid/scid mice was,
on average, less than in control mice, and skin lesions on eyes and
face were present, but were less pronounced. The average clinical
score of the diseased mice was 0.9.+-.1.0, and only one case of
severe psoriasis (clinical score .gtoreq.3) was noted. In addition,
disease onset was delayed (10-12 weeks after cell transfer), when
compared to control mice (average of 6-8 weeks after cell
transfer). Consistent with these observations, it appeared that in
particular the hyperkeratosis in the skin of IFN.gamma..sup.-/- T
cell scid/scid mice was less pronounced.
[0097] The absence of donor derived IFN.gamma. production was
verified by testing the supernatants of isolated lymphocytes from
the skin of diseased CD4.sup.+/CD45Rb.sup.hi IFN.gamma..sup.-/-
reconstituted scid/scid mice after 48 hrs. of stimulation with
anti-CD3 and anti-CD28. No detectable levels of IFN.gamma.
(.ltoreq.30 pg) were found in any of the samples when tested by
ELISA (Table 3). The expression of TNF.alpha. was also measured and
found to be elevated, but significantly less than the TNF.alpha.
levels observed in mice reconstituted with CD4.sup.+/CD45Rb.sup.hi
T cells from wild-type animals (Table 3 or 4).
4TABLE 4 Anti-IL-12 treatment reduces T cell inflammatory responses
significantly, IFN-.sub..gamma..sup.-/- inflammatory T cells
produce TNF-.alpha.. T cell Source IFN.sub..gamma.(pg) IL-4 (pg)
TNF.alpha.(pg) Control mice.sup.1 56380 .+-. 7940 76 .+-. 5 698
.+-. 195 Anti-IL-12 treated mice.sup.2 300 .+-. 7 46 .+-. 4.1
.ltoreq.35 Mice reconstituted with .ltoreq.20 69 .+-. 7.3 122 .+-.
31 CD4.sup.+ /CD45RB.sup.hi IFN.sub..gamma. BALB/c T-cells.sup.3
IFN.sub..gamma., IL-4 and TNF.alpha. production by 2.0 .times.
10.sup.5 SIL was measured as described in Material and Methods.
Data represents the mean and SD. All mice were sacrificed at week
10 after T cell transfer. Control and treatment groups consisted of
4-5 mice. Lymphocytes from each group were pooled for in vitro
stimulation regardless of disease status. .sup.1Cells were isolated
from psoriasiform lesions of 5-10 diseased scid/scid mice following
reconstitution with CD4.sup.+ /CD45Rb.sup.hi and LPS plus IL-12
administration. Mice did not receive any treatment. Data is from
one experiment representative of 3 independent experiments showing
similar values. .sup.2Mice were treated twice i.p. with 0.5 mg of
anti-IL-12 mAb (Clone C17.8 rat IgG.sub.1) on day 7 and 35. Mice
were sacrificed at week 10 after T cell transfer. Data is from one
experiment representative of 2 independent experiments showing
similar values. .sup.3Cells isolated from psoriatic skin from the
ears and eyelids of recipient scid/scid mice that were
reconstituted with CD4.sup.+/CD45RB.sup.hi cell from BALB/cj
IFN.sub..gamma..sup.-/- mice.
[0098] To further rule out that minute levels of IFN.gamma.
secreted by host NK cells are sufficient to induce disease, we
injected IFN.gamma..sup.-/- T cells into scid-beige mice. These
mice carry in addition to the scid mutations, the beige mutation
which causes a deficiency in NK cells in addition to the T, B cell
deficiency already present in the scid mutation. Scid/beige mice
that received IFN.gamma..sup.-/- CD4.sup.+/CD45Rb.sup.hi T cells
also developed a very significant increase in ear thickness,
however again, the onset of disease was significantly delayed and
the incidence of disease was reduced (Table 5) when compared to
mice that had received IFN.gamma..sup.+/+ CD4.sup.+/CD45Rb.sup.hi T
cells. In addition, the severity of disease as measured by ear
thickness and clinical score (Table 5) was attenuated.
Interestingly, despite the presence of severe mononuclear cell
infiltration, the acanthosis, consistent with above results, was
less pronounced in these animals. These results indicate that
IFN.gamma. regulates keratinocyte proliferation, but not
mononuclear cell infiltration/activation in psoriasis.
5TABLE 5 Incidence and clinical score of CD4.sup.+/CD45Rb.sup.hi
scid mice Average Clini- Severe T Cell/Recipient.sup.1
Incidence.sup.5 cal Score.sup.6 Disease.sup.7
IFN.sub.65.sup.-/-/scid/scid.sup.2 9/10 (90%) 2.4 .+-. 0.7 5/10
(50%) IFN.sub.65.sup.-/-/ 1/10 (10%) 0.1 .+-. 0.3 0/10 (0%)
scid/scid .alpha.-IL-12.sup.3 IFN.sub.65.sup.+/+/scid/scid.sup.4
5/9 (56%) 0.9 .+-. 1.0 1/9 (11%) IFN.sub.65.sup.+/+/ 3/7 (43%) 1.0
.+-. 1.3 2/7 (29%) scid/scid beige.sup.4 .sup.1All mice were
reconstituted with either IFN.sub.65 .sup.+/+ or IFN.sub.65
.sup.-/- CD4+ CD45Rb.sup.hi T cells and LPS plus IL-12. .sup.2Mice
received either PBS or control Ab (rat IgG1). .sup.3Antibody
treatment is described in material and method and Table 3.
.sup.4Scid/scid and scid/beige mice that were reconstituted with
CD4.sup.+/CD45 RB.sup.hi cell from BALB/cj IFN.sub.65.sup.-/- mice.
.sup.5Disease incidence is reported as number of mice with disease
over 10 weeks of time: criteria being ear thickness .gtoreq. 26
.mu.m or clinical score of .gtoreq. 1. .sup.6Average clinical score
.+-. SD was assessed of all mice in the group as described in
material and methods. p versus IFN.sub.65.sup.+/+scid/scid:
anti-IL-12: p < 0.0000001, IFN.sub.65.sup.-/-scid/scid: p <
0.003, IFN.sub.65.sup.-/-scid/beige: p < 0.01. Statistical
analysis was performed using the two-tailed student t test.
.sup.7Severe disease induction calculated as the number of animals
that received an average histology score of 2.5 and/or a clinical
score of .gtoreq. 3.
[0099] IL-12 is highly expressed in psoriasiform lesions and in
vivo neutralization of IL-12 downregulates TNF.alpha. and
IFN.gamma. and inhibits disease development We next focused on
IL-12, since this proinflammatory cytokine plays a key role in the
induction of IFN.gamma.. We performed immunohistochemical studies
to detect the presence of heterodimeric IL-12 in inflamed tissue.
There is a very significant amount of IL-12 (p35/p40, (p70))
heterodimer expressed in the tissue of diseased
CD4.sup.+/CD45Rb.sup.hi treated mice. In contrast, significantly
less staining could be observed in CD4.sup.+/CD45Rb.sup.hi treated
animals that were injected with anti-IL-12 mAb (0.5 mg/mouse) on
day 7 and 35.
[0100] To further evaluate the role of IL-12 in the induction of
psoriasiform lesions, we administered anti-IL-12 mAb (0.5 mg) at
day 7 and 35 after T cell transfer to scid/scid mice that had
received wild type CD4.sup.+/CD45Rb.sup.hi T cells. Two doses of
anti-IL-12 mAb were given to maintain a high enough Ab titer over
the entire period of disease induction after the transfer of
CD4.sup.+/CD45Rb.sup.hi T cells. In -two independent experiments,
mice (group of 5) that were treated with anti-IL-12 mAb were
completely protected from developing disease. Only one mouse, out
of 10, developed visible mild psoriasis in the form of slight hair
loss and erythema around the eyelids (clinical score 1), however,
this mouse and all other mice treated with anti-IL-12 mAb did not
develop any increase in ear thickness. In contrast, control mice
that were treated with rat IgG control antibodies or with PBS
showed an incidence of 90% (9 out of 10 mice developed disease)
with an average clinical score of 2.4.+-.0.7. The higher incidence
and severity of disease in this group was also associated with a
significantly higher increase in ear thickness over time.
[0101] The ears and skin of mice that received anti-IL-12 were
examined for the cytokine production of infiltrating lymphocytes.
While there were very few lymphocytes present in the skin of
anti-IL-12 treated animals, these were isolated and tested for
IFN.sub.7, IL-4 and TNF.alpha. production. Only low levels of
IFN.gamma., IL-4 and TNF.alpha. were detected in the supernatants
of cells isolated from treated animals when compared to the
cytokine production of supernatants obtained from control animals
(Table 4).
[0102] The results above are corroborated by the analysis of the
histopathological sections obtained from animals that were treated
with anti-IL-12 mAb. Mice that had been treated with 0.5 mg of
anti-IL-12 mAb on day 7 and 35 lacked any signs of significant
inflammation, acanthosis or hyperkeratosis. Thus, anti-IL-12
administration seems to prevent the development of psoriasiform
lesions by inhibiting keratinocyte hyperproliferation and
mononuclear cell infiltration most likely by downregulating both
IFN.gamma. and TNF.alpha. production.
[0103] Discussion
[0104] The above data demonstrates how immunomodulatory stimuli, in
this case microbial antigens and the proinflammatory lymphokine
IL-12, affect the ability of T cells to induce psoriasis in this
newly developed CD4.sup.+/CD45Rb.sup.hi T cells transfer model. It
is shown that co-administration of LPS and IL-12 (1 and 10 ng) led
to a more rapid onset and to an increased incidence of psoriasis in
C.B-17 scid mice. In addition, the observed lesions in treated mice
were also more severe. In additional experiments, it was
demonstrated that co-administration of SEB also led to a
significant increase in disease incidence and expression. These
findings suggest the importance of bacterial or viral infections
before the appearance of psoriasiform lesions. Most notably, the
skin lesions that developed by this method of induction were
characteristic and remarkably similar to human psoriatic lesions,
exhibiting most clinical and histological hallmarks. The scaling
and thickening of skin evident macroscopically was due to marked
hyper-, parakeratosis and acanthosis. It is further documented in
the microscopic appearance of rete pegs, ulcer and pustule
formation and the often severe epidermal hyperplasia. The
inflammatory cell infiltration was primarily mononuclear and
composed of lymphocytes with fewer monocytes, macrophages and
plasma cells. Moreover, the majority of CD4.sup.+ T cells isolated
from psoriasiform lesions express low levels of CD45Rb, which is
consistent with the fact that recent studies in humans found that T
cells isolated psoriatic plaques exhibit a memory phenotype. Beside
the similarity to the human histology observed in this study, there
are also some differences. Most notable is the absence of CD8+ T
cells in the epidermis of psoriatic plaques, which can be found in
human psoriasis. So far, however, a primary role for CD8+ T cells
in the pathophysiology of psoriasis has not been identified and
successful initial therapies targeting CD4+ T cells rather than
CD8+ T cells point toward the CD4+ T cells as the primary culprit
of the disease.
[0105] Several mechanisms could be responsible for the
disease-promoting effects of LPS, IL-12 and SEB. First, these
immunomodulators may assist in the proliferation and
differentiation of nave Th0 cells to Th1 cells. Recent studies have
shown that microbial products such as LPS can directly stimulate
TNF.alpha., IL-6 and to a lesser extent IL-12 production by murine
skin-derived dendritic cells; and thus may be responsible for
setting the proinflammatory condition for autoreactive T cells.
CD45Rb.sup.hi T cells after the transfer into SCID mice require
additional IL-12 dependent signals in order to develop into
autoimmune effector cells.
[0106] In addition to their immunostimulatory effects on
macrophages and their effects on the development of Th1 effector
cells, LPS, IL-12 and SEB may also modulate leukocyte trafficking
in recipient mice to result in the cutaneous localization of Th1
effector cells. LPS promotes leukocyte recruitment by stimulating
endothelial cell expression of E-selectin, ICAM-1, and VCAM-1
adhesion molecules both directly and through its ability to induce
the production of IL-1, TNF-.alpha. and IFN-.gamma.. The
proinflammatory effects of these cytokines on leukocyte adhesion
and migration is also well-known.
[0107] IFN.gamma. and IL-12 are two very important immunoregulatory
cytokines, that have been shown to play an important role in the
development of autoimmune disorders. IL-12 primarily activates NK
and T cells, while IFN.gamma. primarily activates macrophages and
induces the upregulation of class II molecules on tissue cells. One
might have expected IFNy to be crucial for the induction of
autoimmune effector cells in psoriasis. The above data, however,
indicate that IFN.gamma. may only participate in the disease
process by enhancing disease severity, most likely by promoting
keratinocyte proliferation, but clearly not by inducing and
maintaining pathogenic, inflammatory T cells in psoriatic skin.
This is supported by the fact that the histology observed in
lesions of mice that received IFN.gamma..sup.-/- donor T cells
showed slightly lesser or equal signs of inflammation but
hyperkeratosis or acanthosis was clearly diminished in scid/scid or
scid/beige mice. Moreover, although clinical severity of disease,
as measured by ear thickness and macroscopic observation, was
attenuated when compared to control animals, the penetrance of
disease was very similar. This was also found to be true when
IFN.gamma..sup.-/- T cells were transferred into T, B- and NK cell
deficient scid/beige mice.
[0108] These results indicate that IFN.gamma. is an important
co-factor in the induction of aberrant keratinocyte
proliferation.
[0109] The absence of host-derived IFN.gamma. was verified by the
measurement of IFN.gamma. from anti-CD3 and anti-CD28 stimulated
whole cells isolated from the inflammatory lesions of animals that
received IFN.gamma..sup.-/- T cells. As expected, we were unable to
detect any measurable level of IFN.gamma., thus further ruling out
the possibility that IFN.gamma. might be secreted by non-T cells in
the skin, including NK-cells.
[0110] In contrast to IFN.gamma., an absolute requirement for IL-12
in the development of chronic psoriasiform lesions in scid/scid
mice was demonstrated by several observations made in these
studies. First, medium and low doses of IL-12 (1 and 10 ng/mouse)
administered following donor T cell transfer resulted in a higher
incidence and severity of disease. Moreover, in situ staining of
inflamed tissue revealed a significant present of heterodimeric
IL-12 (p70), while IL-12 staining was not present at all in
non-inflamed control tissue. Most importantly, in vivo
neutralization of IL-12 with a mAb reacting against IL-12 p70
heterodimer 7 days following T cell transfer was able to completely
abrogate disease induction. An interesting aspect of these studies
was that high doses of IL-12 (100 ng/mouse) inhibited disease
induction instead of promoting disease development.
[0111] In summary, the chronic skin disorder described in this
study included features that are normally only observed in human
psoriasis, such as rete pegs, severe acanthosis and infiltration of
Th1 cells into the dermis. The clinical and histopathological
abnormalities were greatly enhanced by the in vivo administration
of LPS and IL-12, demonstrating an important role of infectious
agent(s) in the pathogenesis of the disease. Moreover, it is
demonstrated for the first time that the induction of psoriasiform
lesions was dependent on IL-12, but independent of IFN-.gamma..
These results provide insight into the specific pathogenic
requirements of Th1 promoting cytokines and cells for the
development of psoriasiform lesions, and into the prevention and
treatment of psoriasis in humans.
EXAMPLE 2
[0112] Animals were tested for the effect of treatment with
antibodies to IL-12 on on-going and late-stage psoriatic disease.
The disease was induced by the methods described in Example 1,
i.e., using 3.times.10.sup.5 CD4.sup.+CD45Rb.sup.hi cells
transferred into scid/scid mice followed by injection of LPS and
IL-12. Treatment with the anti-IL-12 antibody was always at 1
mg/mouse/dose. The results of four separate experiments are shown
in FIG. 1. In the initial experiment, (n=5 untreated; n=2 treated),
the mice received injections of anti-IL-12 at weeks 9 and 10 (i.e.,
about 5 weeks after the appearance of psoriatic symptoms) and were
sacrificed for histology at week 14. Whereas the untreated mice had
an average histology score of 2.5, indicating moderate to high
symptoms, the treated mice had a histology score of only 0.25,
indicating almost no symptoms. Hence, two injections of the
anti-IL-12 antibody were highly effective in alleviating
established symptoms of psoriasis.
[0113] In a second experiment, 3 groups of mice were either left
untreated, treated with an isotype-matched control antibody not
binding to IL-12, or treated with the anti-IL-12 antibody (n=4 for
each group). Treatment was at weeks 9 and 10, and histology
specimens were taken at week 12. -Again, the untreated -or
control-treated mice had substantial levels of psoriatic symptoms,
and the anti-IL-12 treated mice had much lower levels. In a third
experiment, mice were untreated (n=5) or treated with anti-IL-12 at
weeks-9 and 10 (n=-6), but half the treated mice were sacrificed
for histology at week 12 as before, and half later at week 17. The
treated mice again showed a dramatic reduction in histology score,
which was largely maintained at week 17, seven weeks after
treatment was stopped.
[0114] Finally, in a fourth experiment (n=4 for groups treated with
anti-IL-12 and isotype-matched control antibody), treatment was not
initiated until week 14, about 10 weeks after psoriatic symptoms
appeared, so the symptoms were very well-established (ear thickness
>40 .mu.m). Treatment was weekly from weeks 14 through 17.
Despite the well-established disease, anti-IL-12 treatment was
dramatically effective in reducing the symptoms (to average
histology score 0.25). This result was confirmed by weekly
measurement of ear thickness, an independent measure of disease
severity, as shown in FIG. 2.
[0115] By combining all these experiments, the average histology
score of the untreated or control-treated mice at sacrifice was
2.53, whereas the average histology score of the mice treated with
anti-IL-12 antibody was only 0.65, a highly statistically
significant result (p=1.7.times.10-9 by Student's two-tailed
t-test).
* * * * *