U.S. patent application number 10/727383 was filed with the patent office on 2004-06-24 for method for treating multiple sclerosis.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Jardieu, Paula M., Montgomery, Bruce.
Application Number | 20040120960 10/727383 |
Document ID | / |
Family ID | 25463656 |
Filed Date | 2004-06-24 |
United States Patent
Application |
20040120960 |
Kind Code |
A1 |
Jardieu, Paula M. ; et
al. |
June 24, 2004 |
Method for treating multiple sclerosis
Abstract
A method is provided for administering to a mammal suffering
from, or at risk for, multiple sclerosis, an initial dosing of a
therapeutically effective amount of an anti-LFA-1 antibody,
followed by a subsequent intermittent dosing of a therapeutically
effective amount of the anti-LFA-1 antibody wherein the antibody is
administered to the mammal no more than once per week in the
intermittent dosing.
Inventors: |
Jardieu, Paula M.; (Uttica,
NY) ; Montgomery, Bruce; (Redwood City, CA) |
Correspondence
Address: |
GENENTECH, INC.
1 DNA WAY
SOUTH SAN FRANCISCO
CA
94080
US
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
25463656 |
Appl. No.: |
10/727383 |
Filed: |
December 2, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10727383 |
Dec 2, 2003 |
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10208112 |
Jul 29, 2002 |
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10208112 |
Jul 29, 2002 |
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09309749 |
May 11, 1999 |
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09309749 |
May 11, 1999 |
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08766008 |
Dec 13, 1996 |
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08766008 |
Dec 13, 1996 |
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08432543 |
May 2, 1995 |
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5622700 |
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08432543 |
May 2, 1995 |
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08287055 |
Aug 8, 1994 |
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08287055 |
Aug 8, 1994 |
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08128329 |
Sep 28, 1993 |
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08128329 |
Sep 28, 1993 |
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07933269 |
Aug 21, 1992 |
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Current U.S.
Class: |
424/164.1 |
Current CPC
Class: |
A61K 39/395 20130101;
A61P 37/00 20180101; C07K 16/2845 20130101; A61K 38/00 20130101;
A61P 37/06 20180101; A61K 39/395 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/164.1 |
International
Class: |
A61K 039/395; A61K
039/40 |
Claims
What is claimed is:
1. A method for treating a LFA-1-mediated disorder in a mammal
comprising administering to the mammal an initial dosing of a
therapeutically effective amount of an LFA-1 antagonist, followed
by a subsequent intermittent dosing of a therapeutically effective
amount of LFA-1 antagonist that is less than 100%, calculated on a
daily basis, of the initial dosing of the antagonist.
2. The method of claim 1 wherein the subsequent dosing is less than
about 50% of the initial dosing of the antagonist.
3. The method of claim 1 wherein the subsequent dosing is less than
about 25% of the initial dosing of the antagonist.
4. The method of claim 1 wherein the subsequent dosing is less than
about 10% of the initial dosing of the antagonist.
5. The method of claim 1 wherein the subsequent dosing is less than
about 2% of the initial dosing of the antagonist.
6. The method of claim 1 wherein the disorder is rejection of or by
a transplanted graft.
7. The method of claim 6 wherein the initial dosing takes place
before, during, and after transplantation has occurred.
8. The method of claim 1 further comprising administering an
effective amount of an immunosuppressive agent to the mammal.
9. The method of claim 6 further comprising administering an
effective amount of cyclosporin A to the mammal.
10. The method of claim 1 wherein the mammal is a human.
11. The method of claim 10 wherein the disorder is rejection of a
transplanted graft, and the donor of the graft and the recipient
are matched for HLA class II antigens.
12. The method of claim 1 wherein the subsequent dosing is carried
out for a longer time than the initial dosing.
13. The method of claim 6 wherein the initial dosing consists of
daily administration and the subsequent dosing is a dose
administered no more than about once a week.
14. The method of claim 13 wherein the initial dosing comprises
daily administration of antagonist for at least one week after the
graft implant and the subsequent dosing comprises administration of
antagonist no more than once biweekly for at least about 5 weeks
after the end of the initial dosing.
15. The method of claim 6 wherein the antagonist is an anti-CD11a
or anti-CD18 antibody and initial dosing terminates from about 1
day to 4 weeks after transplantation has occurred and commences
from about 1 week before transplantation occurs up to about
simultaneously with the transplantation.
16. The method of claim 6 wherein the dosing is given by
intravenous or subcutaneous injections.
17. The method of claim 1 wherein the antagonist is an anti-LFA-1
antibody or an anti-ICAM-1 antibody.
18. The method of claim 17 wherein the antibody is an anti-CD11a or
anti-CD18 antibody.
19. The method of claim 17 wherein the antibody is an anti-CD11a
antibody.
20. A method for increasing tolerance of a transplanted graft by a
mammalian host or of the host by a transplanted graft comprising
administering to the host an initial dosing of a therapeutically
effective amount of anti-LFA-1 antibody, followed by a subsequent
dosing of a therapeutically effective amount of anti-LFA-1 antibody
that is less than 100%, calculated on a daily basis, of the initial
dosing of anti-LFA-1 antibody.
Description
[0001] This is a continuation application filed under 37 CFR
.sctn.1.53(b) (1) claiming priority under 35 U.S.C. .sctn.120 to
application Ser. No. 10/208,112, filed on Jul. 29, 2002, which is a
continuation application of application Ser. No. 09/309,749, filed
on May 11, 1999 (now abandoned), which is a divisional application
of application Ser. No. 08/766,008, filed on Dec. 13, 1996 (now
abandoned), which is a continuation application of application Ser.
No. 08/432,543, filed on May 2, 1995 (U.S. Pat. No. 5,622,700),
which is a continuation application of application Ser. No.
08/287,055, filed on Aug. 8, 1994 (now abandoned), which is a
continuation application of application Ser. No. 08/128,329, filed
Sep. 28, 1993 (now abandoned), which is a continuation application
of application Ser. No. 07/933,269, filed on Aug. 21, 1992 (now
abandoned) which applications are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to a method for treating mammals,
preferably humans, who suffer from unwanted immune responses. In
particular, it relates to methods for ameliorating LFA-1-mediated
disorders, such as those caused by transplanted grafts and immune
diseases.
[0004] 2. Description of Background and Related Art
[0005] The treatment of disorders and diseases mediated by T
lymphocytes has been addressed through many routes. Rheumatoid
arthritis (RA) is one such disorder. Current therapy for RA
includes bed rest, application of heat, and drugs. Salicylate is
the currently preferred drug, particularly as other alternatives
such as immunosuppressive agents and adrenocorticosteroids can
cause greater morbidity than the underlying disease itself.
Nonsteroidal anti-inflammatory drugs are available, and many of
them have effective analgesic, anti-pyretic and anti-inflammatory
activity in RA patients. These include indomethacin,
phenylbutazone, phenylacetic acid derivatives such as ibuprofen and
fenoprofen, naphthalene acetic acids (naproxen), pyrrolealkanoic
acid (tometin), indoleacetic acids (sulindac), halogenated
anthranilic acid (meclofenamate sodium), piroxicam, and
diflunisal.
[0006] Other drugs for use in RA include anti-malarials such as
chloroquine, gold salts and penicillamine. These alternatives
frequently produce severe side effects, including retinal lesions
and kidney and bone marrow toxicity. Immunosuppressive agents such
as methotrexate have been used only in the treatment of severe and
unremitting RA because of their toxicity. Corticosteroids also are
responsible for undesirable side effects (e.g., cataracts,
osteoporosis, and Cushing's disease syndrome) and are not well
tolerated in many RA patients.
[0007] Another disorder mediated by T lymphocytes is rejection of
host or grafts after transplantation. Attempts to prolong the
survival of transplanted allografts and xenografts, or to prevent
graft versus host rejection, both in experimental models and in
medical practice, have centered mainly on the suppression of the
immune apparatus of the recipient. This treatment has as its aim
preventive immunosuppression and/or treatment of graft
rejection.
[0008] Examples of agents used for preventive immunosuppression
include cytotoxic drugs, anti-metabolites, corticosteroids, and
anti-lymphocytic serum. Nonspecific immunosuppressive agents found
particularly effective in preventive immunosuppression
(azathioprine, bromocryptine, methylprednisolone, prednisone, and
most recently, cyclosporin A) have significantly improved the
clinical success of transplantation. The nephrotoxicity of
cyclosporin A after renal transplantation has been reduced by
co-administration of steroids such as prednisolone, or prednisolone
in conjunction with azathioprine. In addition, kidneys have been
grafted successfully using anti-lymphocyte globulin followed by
cyclosporin A. Another protocol being evaluated is total lymphoid
irradiation of the recipient prior to transplantation followed by
minimal immunosuppression after transplantation.
[0009] Treatment of rejection has involved use of steroids,
2-amino-6-aryl-5-substituted pyrimidines, heterologous
anti-lymphocyte globulin, and monoclonal antibodies to various
leukocyte populations, including OKT-3. See generally J.
Pediatrics, 111: 1004-1007 (1987), and specifically U.S. Pat. No.
4,665,077.
[0010] The principal complication of immunosuppressive drugs is
infections. Additionally, systemic immunosuppression is accompanied
by undesirable toxic effects (e.g., nephrotoxicity when cyclosporin
A is used after renal transplantation) and reduction in the level
of the hemopoietic stem cells. Immunosuppressive drugs may also
lead to obesity, poor wound healing, steroid hyperglycemia, steroid
psychosis, leukopenia, gastrointestinal bleeding, lymphoma, and
hypertension.
[0011] In view of these complications, transplantation
immunologists have sought methods for suppressing immune
responsiveness in an antigen-specific manner (so that only the
response to the donor alloantigen would be lost). In addition,
physicians specializing in autoimmune disease strive for methods to
suppress autoimmune responsiveness so that only the response to the
self-antigen is lost. Such specific immunosuppression generally has
been achieved by modifying either the antigenicity of the tissue to
be grafted or the specific cells capable of mediating rejection. In
certain instances, whether immunity or tolerance will be induced
depends on the manner in which the antigen is presented to the
immune system.
[0012] Pretreating the allograft tissues by growth in tissue
culture before transplantation has been found in two murine model
systems to lead to permanent acceptance across MHC barriers.
Lafferty et al., Transplantation, 22: 138-149 (1976); Bowen et al.,
Lancet, 2:585-586 (1979). It has been hypothesized that such
treatment results in the depletion of passenger lymphoid cells and
thus the absence of a stimulator cell population necessary for
tissue immunogenicity. Lafferty et al., Annu. Rev. Immunol., 1: 143
(1983). See also Lafferty et al., Science, 188: 259-261 (1975)
(thyroid held in organ culture), and Gores et al., J. Immunol.,
137: 1482-1485 (1986) and Faustman et al., Proc. Natl. Acad. Sci.
U.S.A., 78: 5156-5159 (1981) (islet cells treated with murine
anti-Ia antisera and complement before transplantation). Also,
thyroids taken from donor animals pretreated with lymphocytotoxic
drugs and gamma radiation and cultured for ten days in vitro were
not rejected by any normal allogeneic recipient. Gose and Bach,
J.Exp.Med., 149: 1254-1259 (1979). All of these techniques involve
depletion or removal of donor lymphocyte cells.
[0013] In some models such as vascular and kidney grafts, there
exists a correlation between Class II matching and prolonged
allograft survival, a correlation not present in skin grafts.
Pescovitz et al., J.Exp.Med., 160: 1495-1508 (1984); Conti et al.,
Transplant. Proc., 19: 652-654 (1987). Therefore, donor-recipient
HLA matching has been utilized. Additionally, blood transfusions
prior to transplantation have been found to be effective. Opelzet
al., Transplant. Proc., 4: 253 (1973); Persijn et al., Transplant.
Proc., 23: 396 (1979). The combination of blood transfusion before
transplantation, donor-recipient HLA matching, and
immunosuppression therapy (cyclosporin A) after transplantation was
found to improve significantly the rate of graft survival, and the
effects were found to be additive. Opelz et al., Transplant. Proc.,
17: 2179 (1985).
[0014] The transplantation response may also be modified by
antibodies directed at immune receptors for MHC antigens. Bluestone
et al., Immunol. Rev. 90: 5-27 (1986). Further, graft survival can
be prolonged in the presence of antigraft antibodies, which lead to
a host reaction that in turn produces specific immunosuppression.
Lancasteret al., Nature, 315: 336-337 (1985).
[0015] The immune response of the host to MHC antigens may be
modified specifically by using bone marrow transplantation as a
preparative procedure for organ grafting. Thus, anti-T-cell
monoclonal antibodies are used to deplete mature T cells from the
donor marrow inoculum to allow bone marrow transplantation without
incurring graft-versus-host disease. Mueller-Ruchholtz et al.,
Transplant Proc., 8: 537-541 (1976). In addition, elements of the
host's lymphoid cells that remain for bone marrow transplantation
solve the problem of immunoincompetence occurring when fully
allogeneic transplants are used.
[0016] Lymphocyte adherence to endothelium is a key event in the
process of inflammation. There are at least three known pathways of
lymphocyte adherence to endothelium, depending on the activation
state of the T cell and the endothelial cell. T cell immune
recognition requires the contribution of the T cell receptor as
well as adhesion receptors, which promote attachment of T cells to
antigen-presenting cells and transduce regulatory signals for T
cell activation. The lymphocyte function associated (LFA) antigen-1
(LFA-1, CD11a, .alpha.-chain/CD18, .beta.-chain) has been
identified as the major integrin receptor on lymphocytes involved
in these cell adherence interactions leading to several
pathological states. ICAM-1, the endothelial cell
immunoglobulin-like adhesion molecule, is a known ligand for LFA-1
and is implicated directly in graft rejection, psoriasis, and
arthritis.
[0017] LFA-1 is required for a range of leukocyte functions,
including lymphokine production of helper T cells in response to
antigen-presenting cells, killer T cell-mediated target cell lysis,
and immunoglobulin production through T cell-B cell interactions.
Activation of antigen receptors on T cells and B cells allows LFA-1
to bind its ligand with higher affinity.
[0018] Monoclonal antibodies (MAbs) directed against LFA-1 led to
the initial identification and investigation of the function of
LFA-1. Davignon et al., J. Immunol., 127: 590 (1981). LFA-1 is
present only on leukocytes [Krenskey et al., J. Immunol., 131: 611
(1983)], and ICAM-1 is distributed on activated leukocytes, dermal
fibroblasts, and endothelium. Dustinet al., J. Immunol., 137: 245
(1986).
[0019] Previous studies have investigated the effects of anti-CD11a
MAbs on many T-cell-dependent immune functions in vitro and a
limited number of immune responses in vivo. In vitro, anti-CD11a
MAbs inhibit T-cell activation [Kuypers et al., Res. Immunol., 140:
461 (1989)], T-cell-dependent B-cell proliferation and
differentiation [Davignon et al., supra; Fischer et al., J.
Immunol., 136: 3198 (1986)], target cell lysis by cytotoxic T
lymphocytes [Krensky et al., supra], formation of immune conjugates
[Sanders et al., J. Immunol., 137: 2395 (1986); Mentzer et al., J.
Immunol., 135: 9 (1985)], and the adhesion of T-cells to vascular
endothelium. Lo et al., J. Immunol., 143: 3325 (1989). Also, the
antibody 5C6 directed against CD11b/CD18 was found to prevent
intra-islet infiltration by both macrophages and T cells and to
inhibit development of insulin-dependent diabetes mellitis in mice.
Hutchings et al., Nature, 348: 639 (1990).
[0020] The observation that LFA-1-ICAM-1 interaction is necessary
to optimize T cell function in vitro, and that anti-CD11a MAbs
induce tolerance to protein antigens [Benjamin et al., Eur J.
Immunol., 18: 1079 (1988)] and prolongs tumor graft survival in
mice [Heagy et al., Transplantation, 37: 520-523 (1984)] was the
basis for testing the MAbs to these molecules for prevention of
graft rejection in humans.
[0021] Experiments have also been carried out in primates. For
example, based on experiments in monkeys it has been suggested that
a MAb directed against ICAM-1 can prevent or even reverse kidney
graft rejection. Cosimi et al., "Immunosuppression of Cynomolgus
Recipients of Renal Allografts by R6.5, a Monoclonal Antibody to
Intercellular Adhesion Molecule-1," in Springer et al. (eds.),
Leukocyte Adhesion Molecules (New York: Springer, 1988), p. 274;
Cosimi et al., J. Immunology, 144: 4604-4612 (1990). Furthermore,
the in vivo administration of anti-CD11a MAb to cynomolgus monkeys
prolonged skin allograft survival. Berlinet al., Transplantation,
53: 840-849 (1992).
[0022] The first successful use of a rat anti-murine CD11a antibody
(25-3; IgG1) in children with inherited disease to prevent the
rejection of bone-marrow-mismatched haploidentical grafts was
reported by Fischer et al., Lancet, 2: 1058 (1986). Minimal side
effects were observed. See also Fischer et al., Blood, 77: 249
(1991); van Dijken et al., Transplantation, 49: 882 (1990); and
Perez et al., Bone Marrow Transplantation, 4: 379 (1989).
Furthermore, the antibody 25-3 was effective in controlling
steroid-resistant acute graft-versus-host disease in humans. Stoppa
et al., Transplant. Int., 4: 3-7 (1991).
[0023] However, these results were not reproducible in leukemic
adult grafting with this MAb [Maraninchi et al., Bone Marrow
Transplant, 4: 147-150 (1989)], or with an anti-CD18 MAb, directed
against the invariant chain of LFA-1, in another pilot study. Baume
et al., Transplantation, 47: 472 (1989). Furthermore, a rat
anti-murine CD11a MAb, 25-3, was unable to control the course of
acute rejection in human kidney transplantation. LeMauff et al.,
Transplantation, 52: 291 (1991).
[0024] A review of the use of monoclonal antibodies in human
transplantation is provided by Dantal and Soulillou, Current
Opinion in Immunology, 3: 740-747 (1991).
[0025] A recent report showed that brief treatment with either
anti-LFA-1 or anti-ICAM-1 MAbs minimally prolonged the survival of
primarily vascularized heterotopic heart allografts in mice. Isobe
et al., Science, 255: 1125 (1992). However, combined treatment with
both MAbs was required to achieve long-term graft survival in this
model.
[0026] Independently, it was shown that treatment with anti-LFA-1
MAb alone potently and effectively prolongs the survival of
heterotopic (ear-pinnae) nonprimarily vascularized mouse heart
grafts using a maximum dose of 4 mg/kg/day and treatment once a
week after a daily dose. Nakakura et al., J. Heart Lung
Transplant., 11: 223 (1992). [See also The New York Times, p. B6
(Tuesday, Mar. 10, 1992) "New Technique in Lab Prevents Rejection
of Organ Transplants," by Sandra Blakeslee.] Nonprimarily
vascularized heart allografts are more immunogenic and more
resistant to prolongation of survival by MAbs than primarily
vascularized heart allografts. Warrenet al., Transplant. Proc., 5:
717 (1973); Trager et al., Transplantation, 47: 587 (1989). The
latter reference discusses treatment with antibodies against L3T4
using a high initial dose and a lower subsequent dose.
[0027] Another study on treating a sclerosis-type disease in
rodents using similar antibodies to those used by Nakakura et al.,
supra, is reported by Yednock et al., Nature, 356: 63-66
(1992).
[0028] Additional disclosures on the use of anti-LFA-1 antibodies
and ICAM-1, ICAM-2, and LFA-3 and their antibodies to treat
LFA-1-mediated disorders include WO 91/18011 published Nov. 28,
1991, WO 91/16928 published Nov. 14, 1991, WO 91/16927 published
Nov. 14, 1991, Can. Pat. Appln. 2,008,368 published Jun. 13, 1991,
WO 90/15076 published Dec. 13, 1990, WO 90/10652 published Sep. 20,
1990, EP 387,668 published Sep. 19, 1990, WO 90/08187 published
Jul. 26, 1990, EP 379,904 published Aug. 1, 1990, EP 346,078
published Dec. 13, 1989, U.S. Pat. Nos. 5,071,964, 5,002,869,
Australian Pat. Appln. 8815518 published Nov. 10, 1988, EP 289,949
published Nov. 9, 1988, and EP 303,692 published Feb. 22, 1989.
[0029] The above methods successfully utilizing anti-LFA-1 or
anti-ICAM-1 antibodies represent an improvement over traditional
immunosuppressive drug therapy; however, they advocate a higher
than minimum or fixed dosage of drug that we expect either to
unduly suppress the immune system (and create a signficant risk of
infection) or to be inadequate for long-term tolerance. There is a
need in the art to better treat disorders that are mediated by
LFA-1 such as autoimmune diseases, graft vs. host or host vs. graft
rejection, and T cell inflammatory responses, so as to minimize
side effects and sustain specific tolerance to self- or
xenoantigens.
[0030] Accordingly, it is an object of this invention to provide an
improved method for sustaining resistance to LFA-1-mediated
disorders with minimal side effects.
[0031] It is another object to prolong graft survival in
transplants.
[0032] It is a further object to minimize the toxicity and other
adverse effects arising from the use of large doses of
immunosuppressants in transplant patients.
[0033] It is a still further object to provide the host with
selective tolerance to the antigen or agent causing the specific
immune disorder, so that the host has a reduced susceptibility to
infections and other assaults on the immune system that are
opportunistic when conventional immunosuppressive agents or dosages
are employed.
[0034] These and other objects will become apparent to one of
ordinary skill in the art.
SUMMARY OF THE INVENTION
[0035] These objects are accomplished by a method for treating a
LFA-1-mediated disorder in a mammal comprising administering to the
mammal an initial dosing of a therapeutically effective amount of
an LFA-1 antagonist, followed by a subsequent intermittent dosing
of a therapeutically effective amount of an LFA-1 antagonist that
is less than 100%, calculated on a daily basis, of the initial
dosing of LFA-1 antagonist, whereby the mammal has selective
tolerance of the disorder. Preferably, the LFA-1 antagonist is an
anti-LFA-l antibody, in particular anti-CD11a.
[0036] It was surprisingly found that specific tolerance is induced
by adjusting the dose regimen, and that it is not necessary to
maintain antagonist dosage at the same initial level over the
course of treatment. It was also surprising that the survival of
grafts upon transplantation was prolonged using a dosing regimen
where a high initial dose is given followed by a continuous
maintenance dose. Also unexpected was that this dosing scheme using
only one drug resulted in a selective tolerance of the host to the
agent causing the disorder, so that the host defense system was not
severely depressed.
[0037] The transplantation method herein is applicable to both
allografts and xenografts, and the use of xenografts overcomes the
difficulties encountered by the limited supply of tissue from
humans.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIGS. 1A and 1B illustrate the comparative efficacies and
potencies, respectively, of treatment with M17 (anti-CD11a MAb,
stars) and cyclosporin A (CsA, circles) days 1-13 for the
prolongation of heterotopic (ear-pinnae), nonprimarily vascularized
BALB/c heart graft survival in C3H mouse recipients.
[0039] FIG. 2 is an isobologram plot of data derived from the
quantal BALB/c to C3H mouse heart graft bioassay. This assay was
used to evaluate the effect of combined daily treatment with
different doses of M17 and CsA (n=5/dose group) on graft survival.
The percent viable grafts on post-transplant day 14 was used as the
endpoint of the assay.
[0040] FIG. 3 shows the effect of M17 dose (intraperitoneal, i.p.)
and schedule on the survival of heterotopic (ear-pinnae),
nonprimarily vascularized BALB/c heart grafts in C3H mouse
recipients (n=4-14/group). The Mann-Whitney U test was used to
determine levels of statistical significance (corrected for small
sample sizes) for the differences in survival times between groups.
p>0.05 was considered not significant (NS). The open circles are
2 mg/kg of rat IgG2a isotype control 0-13 days daily, the solid
squares are 2 mg/kg of M17 0-13 days daily, the solid circles are 2
mg/kg of M17 0-13 days every third day, the triangles are 2mg/kg of
M17 0-20 days daily, then weekly until day 98, and the stars are 8
mg/kg of M17 0-14 days daily, then 2 mg/kg biweekly until day
99.
[0041] FIG. 4 shows the effect of different M17 treatment schedules
on BALB/c ear-heart allograft survival in C3H recipient mice
presensitized to BALB/c alloantigens. The squares are rat IgG2a
isotype control, and the circles are M17, where solid is days -7 to
-3, shaded is days -7 to 13, and open is days 0 to 13.
[0042] FIG. 5 shows the effect of treatment with M17 on relative
proportions of spleen lymphocyte populations determined by flow
cytometry. The open bars are no treatment, the shaded bars are
treatment with rat IgG2a isotype control, and the solid bars are
treatment with M17, where the treatment is 4 mg/kg/day from day 0
to day 13, i.p.
[0043] FIG. 6 shows the effect of in vivo treatment with M17 on the
in vitro proliferative response of C3H spleen cells to ConA. The
shaded portion of the graph of mean percent change in .sup.3H-TdR
incorporation versus ConA concentration represents inhibition,
while the unshaded portion represents activation. The p values are
versus the control.
[0044] FIG. 7 shows the effect of treatment with M17 on the host
versus graft popliteal lymph node (PLN) hyperplasia assay. The p
values are versus the control.
[0045] FIG. 8 shows the inhibitory effects of anti-CD18 (open
circles), anti-CD11a (solid circles), anti-ICAM (open squares), and
the isotype control (solid squares) on the human mixed lymphocyte
response.
[0046] FIG. 9 shows the effect of control antibodies (solid bars),
H52 (solid slashed bars), anti-CD11b (medium shaded bars),
anti-CD11a (open slashed bars), anti-CD18 (open bars), and
anti-gp120(dark shaded bars) on cytotoxic T lymphocyte target cell
killing.
[0047] FIG. 10 shows the effect of M17 on contact sensitivity of
mice to an immunogen, dinitrofluorobenzene.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0048] I. Definitions
[0049] The term "LFA-1-mediated disorders" refers to pathological
states caused by cell adherence interactions involving the LFA-1
receptor on lymphocytes. Examples of such disorders include T cell
inflammatory responses such as inflammatory skin diseases including
psoriasis; responses associated with inflammatory bowel disease
(such as Crohn's disease and ulcerative colitis); adult respiratory
distress syndrome; dermatitis; meningitis; encephalitis; uveitic;
allergic conditions such as eczema and asthma and other conditions
involving infiltration of T cells and chronic inflammatory
responses; skin hypersensitivity reactions (including poison ivy
and poison oak); atherosclerosis; leukocyte adhesion deficiency;
autoimmune diseases such as rheumatoid arthritis, systemic lupus
erythematosus (SLE), diabetes mellitus, multiple sclerosis,
Reynaud's syndrome, autoimmune thyroiditis, experimental autoimmune
encephalomyelitis, Sjorgen's syndrome, juvenile onset diabetes, and
immune responses associated with delayed hypersensitivity mediated
by cytokines and T-lymphocytes typically found in tuberculosis,
sarcoidosis, polymyositis, granulomatosis and vasculitis;
pernicious anemia; diseases involving leukocyte diapedesis; CNS
inflammatory disorder, multiple organ injury syndrome secondary to
septicaemia or trauma; autoimmune haemolytic anemia; myethemia
gravis; antigen-antibody complex mediated diseases; all types of
transplantations, including graft vs. host or host vs. graft
disease; etc.
[0050] "Treating" such diseases includes therapy, prophylactic
treatment, prevention of rejection of grafts, and induction of
tolerance of grafts on a long-term basis.
[0051] "Initial" dosing means dosing that is not the last dosing
administered in the treatment and means dosing administered before
and/or at the time that the disorder is first incurred (or first
apparent or first diagnosed), e.g., the day when transplantation of
a graft occurs, preferably at least at the time when the disorder
is first incurred, apparent, or diagnosed. The initial dosing need
not be a single dose, but it is not the last dose. "Subsequent"
dosing is dosing that follows the initial dosing and includes the
last dose administered for the treatment. This latter dosing is a
maintenance dose ordinarily not sufficient alone to tolerate a
second graft.
[0052] The term "graft" as used herein refers to biological
material derived from a donor for transplantation into a recipient.
Grafts include such diverse material as, for example, isolated
cells such as islet cells, tissue such as the amniotic membrane of
a newborn, bone marrow, hematopoietic precursor cells, and organs
such as skin, heart, liver, spleen, pancreas, thyroid lobe, lung,
kidney, tubular organs (e.g., intestine, blood vessels, or
esophagus), etc. The tubular organs can be used to replace damaged
portions of esophagus, blood vessels, or bile duct. The skin grafts
can be used not only for burns, but also as a dressing to damaged
intestine or to close certain defects such as diaphragmatic hernia.
The graft is derived from any mammalian source, including human,
whether from cadavers or living donors. Preferably the graft is
bone marrow or an organ such as heart and the donor of the graft
and the host are matched for HLA class II antigens.
[0053] The term "mammal" refers to any animal classified as a
mammal, including humans, domestic and farm animals, and zoo,
sports, or pet animals, such as dogs, horses, cats, cows, etc.
Preferably, the mammal herein is human.
[0054] The term "mammalian host" as used herein refers to any
compatible transplant recipient. By "compatible" is meant a
mammalian host that will accept the donated graft. Preferably, the
host is human. If both the donor of the graft and the host are
human, they are preferably matched for HLA class II antigens so as
to improve histocompatibility.
[0055] The term "donor" as used herein refers to the mammalian
species, dead or alive, from which the graft is derived.
Preferably, the donor is human. Human donors are preferably
volunteer blood-related donors that are normal on physical
examination and of the same major ABO blood group, because crossing
major blood group barriers possibly prejudices survival of the
allograft. It is, however, possible to transplant, for example, a
kidney of a type O donor into an A, B or AB recipient.
[0056] The term "transplant" and variations thereof refers to the
insertion of a graft into a host, whether the transplantation is
syngeneic (where the donor and recipient are genetically
identical), allogeneic (where the donor and recipient are of
different genetic origins but of the same species), or xenogeneic
(where the donor and recipient are from different species). Thus,
in a typical scenario, the host is human and the graft is an
isograft, derived from a human of the same or different genetic
origins. In another scenario, the graft is derived from a species
different from that into which it is transplanted, such as a baboon
heart transplanted into a human recipient host, and including
animals from phylogenically widely separated species, for example,
a pig heart valve, or animal beta islet cells or neuronal cells
transplanted into a human host.
[0057] The term "LFA-1 antagonist" generally refers to an antibody
directed against either CD11a or CD18 or both, but also includes
ICAM-1, soluble forms of ICAM-1 (e.g., the ICAM-1 extracellular
domain, alone or fused to an immunoglobulin sequence), antibodies
to ICAM-1, and fragments thereof, or other molecules capable of
inhibiting the interaction of LFA-1 and ICAM-1.
[0058] The term "anti-LFA-1 antibody" or "anti-LFA-1 MAb" refers to
an antibody directed against either CD11a or CD18 or both. The
anti-CD11a antibodies include, e.g., MHM24 [Hildreth et al., Eur.
J. Immunol., 13: 202-208 (1983)], R3.1 (IgG1) [R. Rothlein,
Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Conn.],
25-3 (or 25.3), an IgG1 available from Immunotech, France [Olive et
al., in Feldmann, ed., Human T cell Clones. A new Approach to
Immune Regulation, Clifton, N.J., Humana, 1986 p. 173], KBA (IgG2a)
[Nishimura et al., Cell. Immunol., 107: 32 (1987); Nishimura et
al., ibid., 94: 122 (1985)], M7/15 (IgG2b) [Springer et al.,
Immunol. Rev., 68: 171 (1982)], IOT16 [Vermot Desroches et al.,
Scand. J. Immunol., 33: 277-286 (1991)], SPVL7 [Vermot Desroches et
al., supra], and M17 (IgG2a), available from ATCC, which are rat
anti-murine CD11a antibodies.
[0059] Examples of anti-CD18 antibodies include MHM23 [Hildreth et
al., supra], M18/2 (IgG2a) [Sanches-Madrid et al., J. Exp. Med.,
158: 586 (1983)], H52 [Fekete et al., J. Clin. Lab Immunol., 31:
145-149 (1990)], Mas191c [Vermot Desroches et al., supra], IOT18
[Vermot Desroches et al., supra], 60.3 [Taylor et al., Clin. ExP.
Immunol., 71: 324-328 (1988)], and 60.1 [Campana et al., Eur. J.
Immunol., 16: 537-542 (1986)].
[0060] Other examples of suitable LFA-1 antagonists, including
antibodies, are described in Hutchings et al., supra, WO 91/18011
published Nov. 28, 1991, WO 91/16928 published Nov. 14, 1991, WO
91/16927 published Nov. 14, 1991, Can. Pat. Appln. 2,008,368
published Jun. 13, 1991, WO 90/15076 published Dec. 13, 1990, WO
90/10652 published Sep. 20, 1990, EP 387,668 published Sep. 19,
1990, EP 379,904 published Aug. 1, 1990, EP 346,078 published Dec.
13, 1989, U.S. Pat. No. 5,071,964, U.S. Pat. No. 5,002,869,
Australian Pat. Appln. 8815518 published Nov. 10, 1988, EP 289,949
published Nov. 9, 1988, and EP 303,692 published Feb. 22, 1989.
[0061] The antibody is appropriately from any source, including
chicken and mammalian such as rodent, goat, primate, and human.
Preferably, the antibody is from the same species as the species to
be treated, and more preferably the antibody is humanized (i.e.,
has all human components) and the host is human. While the antibody
can be a polyclonal or monoclonal antibody, preferably it is a
monoclonal antibody, which can be prepared by conventional
technology. The antibody is an IgG-1, -2, -3, or -4, IgE, IgA, IgM,
IgD, or an intraclass chimera in which Fv or a CDR from one class
is substituted into another class. The antibody may have an Fc
domain capable of an effector function or may not be capable of
binding complement or participating in ADCC.
[0062] The term "immunosuppressive agent" as used herein for
adjunct therapy refers to substances that act to suppress or mask
the immune system of the host into which the graft is being
transplanted. This would include substances that suppress cytokine
production, downregulate or suppress self-antigen expression, or
mask the MHC antigens. Examples of such agents include
2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat. No.
4,665,077,supra, the disclosure of which is incorporated herein by
reference), azathioprine (or cyclophosphamide, if there is an
adverse reaction to azathioprine); bromocryptine; glutaraldehyde
(which masks the MHC antigens, as described in U.S. Pat. No.
4,120,649, supra); anti-idiotypic antibodies for MHC antigens and
MHC fragments; cyclosporin A; steroids such as
glucocorticosteroids, e.g., prednisone, methylprednisolone, and
dexamethasone; cytokine or cytokine receptor antagonists including
anti-interferon-.gamma., -.beta., or -.alpha. antibodies;
anti-tumor necrosis factor-.alpha. antibodies; anti-tumor necrosis
factor-.beta. antibodies; anti-interleukin-2 antibodies and
anti-IL-2 receptor antibodies; anti-L3T4 antibodies; heterologous
anti-lymphocyte globulin; pan-T antibodies, preferably anti-CD3 or
anti-CD4/CD4a antibodies; soluble peptide containing a LFA-3
binding domain (WO 90/08187 published Jul. 26, 1990),
streptokinase; TGF-.beta.; streptodornase; RNA or DNA from the
host; FK506; RS-61443; deoxyspergualin; rapamycin; T-cell receptor
(Cohen et al., U.S. Pat. No. 5,114,721); T-cell receptor fragments
[Offner et al., Science, 251: 430-432 (1991)]; copending U.S. Ser.
No. 07/853,362 filed Mar. 18, 1992, the disclosure of which is
incorporated herein by reference; Howell, WO 90/11294; Ianeway,
Nature, 341: 482 (1989); and Vandenbark, WO 91/01133]; and T cell
receptor antibodies (EP 340,109) such as T10B9. These agents are
administered at the same time or at separate times from the CD11a
or CD18 antagonists as used in this invention, and are used at the
same or lesser dosages than as set forth in the art.
[0063] The preferred adjunct immunosuppressive agent will depend on
many factors, including the type of disorder being treated
including the type of transplantation being performed, as well as
the patient's history, but a general overall preference is that the
agent be selected from cyclosporin A, a glucocorticosteroid (most
preferably prednisone or methylprednisolone), OKT-3 monoclonal
antibody, azathioprine, bromocryptine, heterologous anti-lymphocyte
globulin, or a mixture thereof.
[0064] "Increasing tolerance of a transplanted graft" by a host
refers to prolonging the survival of a graft in a host in which it
is transplanted, i.e., suppressing the immune system of the host so
that it will better tolerate a foreign transplant.
[0065] "Intermittent" or "periodic" dosing is a dosing that is
continuous for a certain period of time and is at regular intervals
that are preferably separated by more than one day.
[0066] "Selective tolerance" of the disorder refers to a tolerance
by the host's immune system for the specific agent causing the
disorder, but retaining the ability of the host to reject a second
allogeneic or xenogeneic graft. Preferably, the tolerance is such
that the immune system is left otherwise intact.
[0067] II. Modes for Carrying out the Invention
[0068] Superior immunosuppressive efficacy is seen with a treatment
regimen that uses early induction with a high dose of LFA-1
antagonist followed by extended treatment with a lower dose of
antagonist.
[0069] If antibodies are employed as the antagonist, they are
prepared by any suitable technique. LFA-1 or either of itsa or
.beta.chains or any other appropriate immunogen may be used to
induce the formation of anti-LFA-1 or anti-ICAM antibodies, which
are identified by routine screening. Such antibodies may either be
polyclonal or monoclonal antibodies, or antigen binding fragments
of such antibodies (such as, for example, F(ab) or F(ab).sub.2
fragments). The antibodies are monovalent or polyvalent for LFA-1
or ICAM-1, and are monospecific for LFA-1 or ICAM-1 or are
polyspecific for LFA-1 or ICAM-1 and a predetermined antigen. An
LFA-1 antagonist is used in a single course of therapy, different
antagonists are used at different stages in therapy (e.g., the
initial or sustaining dose), or mixtures thereof are employed
(e.g., antibodies to ICAM-1 and to LFA-1).
[0070] Polyclonal antibodies to LFA-1 or ICAM-1 generally are
raised in animals by multiple subcutaneous (s.c.) or
intraperitoneal (i.p.) injections of the CD11a or CD18 polypeptide
or dimer thereof or ICAM-1, together with an adjuvant. It may
be-useful to conjugate the LFA-1 or ICAM-1 antigen polypeptide
(including its chains and fragments containing the target amino
acid sequence) to a protein that is immunogenic in the species to
be immunized, e.g., keyhole limpet hemocyanin, serum albumin,
bovine thyroglobulin, or soybean trypsin inhibitor using a
bifunctional or derivatizing agent, for example, maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde,
succinic anhydride, SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where R
and R.sup.1 are different alkyl groups.
[0071] The route and schedule for antibody stimulation of the host
animal or cultured antibody-producing cells therefrom are generally
in keeping with established and conventional techniques for
antibody stimulation and production. While mice are frequently
employed as the test model, it is contemplated that any mammalian
subject including human subjects or antibody-producing cells
obtained therefrom can be manipulated according to the processes of
this invention to serve as the basis for production of mammalian,
including human, hybrid cell lines.
[0072] Animals are typically immunized against the immunogenic
conjugates or derivatives by combining 1 mg or 1 .mu.g of conjugate
(for rabbits or mice, respectively) with 3 volumes of Freund's
complete adjuvant and injecting the solution intradermally at
multiple sites. One month later the animals are boosted with 1/5 to
{fraction (1/10)} the original amount of conjugate in Freund's
incomplete adjuvant (or other suitable adjuvant) by subcutaneous
injection at multiple sites. Seven to 14 days later animals are
bled and the serum is assayed for antibody titer. Animals are
boosted until the titer plateaus. Preferably, the animal is boosted
with the conjugate of the same LFA-1 or ICAM polypeptide, but
conjugated to a different protein and/or through a different
cross-linking agent. Conjugates also can be made in recombinant
cell culture as protein fusions. Also, aggregating agents such as
alum are used to enhance the immune response.
[0073] Monoclonal antibodies are prepared by recovering immune
cells--typically spleen cells or lymphocytes from lymph node
tissue--from immunized animals and immortalizing the cells in
conventional fashion, e.g., by fusion with myeloma cells or by
Epstein-Barr (EB)-virus transformation and screening for clones
expressing the desired antibody. The hybridoma technique described
originally by Kohler and Milstein, Eur J. Immunol., 6: 511 (1976)
and also described by Hammerling et al., In: Monoclonal Antibodies
and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981) has been
widely applied to produce hybrid cell lines that secrete high
levels of monoclonal antibodies against many specific antigens.
[0074] It is possible to fuse cells of one species with another.
However, it is preferable that the source of the immunized
antibody-producing cells and the myeloma be from the same
species.
[0075] The hybrid cell lines can be maintained in culture in vitro
in cell culture media. The cell lines producing the antibodies can
be selected and/or maintained in a composition comprising the
continuous cell line in hypoxanthine-aminopterin thymidine (HAT)
medium. In fact, once the hybridoma cell line is established, it
can be maintained on a variety of nutritionally adequate media.
Moreover, the hybrid cell lines can be stored and preserved in any
number of conventional ways, including freezing and storage under
liquid nitrogen. Frozen cell lines can be revived and cultured
indefinitely with resumed synthesis and secretion of monoclonal
antibody.
[0076] The secreted antibody is recovered from tissue culture
supernatant by conventional methods such as precipitation, ion
exchange chromatography, affinity chromatography, or the like. The
antibodies described herein are also recovered from hybridoma cell
cultures by conventional methods for purification of IgG or IgM, as
the case may be, that heretofore have been used to purify these
immunoglobulins from pooled plasma, e.g., ethanol or polyethylene
glycol precipitation procedures. The purified antibodies are
sterile filtered.
[0077] While routinely mouse monoclonal antibodies are used, the
invention is not so limited; in fact, human antibodies may be used
and may prove to be preferable. Such antibodies can be obtained by
using human hybridomas (Cote et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p. 77 [1985]). In fact, according to
the invention, techniques developed for the production of chimeric
antibodies (Morrison et al., Proc. Natl. Acad. Sci., 81: 6851
[1984]; Neuberger et al., Nature, 312: 604 [1984]; Takeda et al.,
Nature, 314: 452 [1985]; EP 184,187; EP 171,496; EP 173,494; PCT WO
86/01533; Shaw et al., J. Nat. Canc. Inst., 80: 1553-1559 [1988];
Morrison, Science, 229: 1202-1207 [1985]; and Oi et al.
BioTechniques, 4: 214 [1986]) by splicing the genes from a mouse
antibody molecule of appropriate antigen specificity together with
genes from a human antibody molecule of appropriate biological
activity (such as ability to bind ICAM-1) can be used; such
antibodies are within the scope of this invention.
[0078] Techniques for creating recombinant DNA versions of the
antigen-binding regions of antibody molecules (known as Fab
fragments), which bypass the generation of monoclonal antibodies,
are encompassed within the practice of this invention. One extracts
antibody-specific messenger RNA molecules from immune system cells
taken from an immunized animal, transcribes these into
complementary DNA (cDNA), and clones the cDNA into a bacterial
expression system. One example of such a technique suitable for the
practice of this invention was developed by researchers at
Scripps/Stratagene, and incorporates a proprietary bacteriophage
lambda vector system that contains a leader sequence that causes
the expressed Fab protein to migrate to the periplasmic space
(between the bacterial cell membrane and the cell wall) or to be
secreted. One can rapidly generate and screen great numbers of
functional Fab fragments for those that bind the antigen. Such
LFA-1- or ICAM-binding molecules (Fab fragments with specificity
for the LFA-1 or ICAM polypeptide) are specifically encompassed
within the term "antibody" as it is defined, discussed, and claimed
herein.
[0079] Typically, the LFA-1 antagonist used in the method of this
invention is formulated by mixing it at ambient temperature at the
appropriate pH, and at the desired degree of purity, with
physiologically acceptable carriers, i.e., carriers that are
non-toxic to recipients at the dosages and concentrations employed.
The pH of the formulation depends mainly on the particular use and
the concentration of antagonist, but preferably ranges anywhere
from about 3 to about 8. Formulation in an acetate buffer at pH 5
is a suitable embodiment.
[0080] The LFA-1 antagonist for use herein is preferably sterile.
Sterility is readily accomplished by sterile filtration through
(0.2 micron) membranes. LFA-1 antagonist ordinarily will be stored
as an aqueous solution, although lyophilized formulations for
reconstitution are acceptable.
[0081] The antagonist composition will be formulated, dosed, and
administered in a fashion consistent with good medical practice.
Factors for consideration in this context include the particular
disorder being treated, the particular mammal being treated, the
clinical condition of the individual patient, the cause of the
disorder, the site of delivery of the agent, the method of
administration, the scheduling of administration, and other factors
known to medical practitioners. The "therapeutically effective
amount" of LFA-1 antagonist to be administered will be governed by
such considerations, and is the minimum amount necessary to
prevent, ameliorate, or treat the LFA-1-mediated disorder,
including treating rheumatoid arthritis, reducing inflamatory
responses, inducing tolerance of immunostimulants, preventing an
immune response that would result in rejection of a graft by a host
or vice-versa, or prolonging survival of a transplanted graft. Such
amount is preferably below the amount that is toxic to the host or
renders the host significantly more susceptible to infections.
[0082] As a general proposition, the initial pharmaceutically
effective amount of the LFA-1 antagonist administered parenterally
per dose will be in the range of about 0.1 to 20 mg/kg of patient
body weight per day, with the typical initial range of LFA-1
antagonist used being 0.3 to 15 mg/kg/day.
[0083] As noted above, however, these suggested amounts of antibody
are subject to a great deal of therapeutic discretion. The key
factor in selecting an appropriate dose and scheduling is the
result obtained, as indicated above. For example, relatively higher
doses may be needed initially for the treatment of ongoing and
acute graft rejection, or at a later stage for the treatment of
acute rejection, which is characterized by a sudden decline in
graft function.
[0084] Where the subsequent dosing is less than 100% of initial
dosing, it is calculated on the basis of daily dosing. Thus, for
example, if the dosing regimen consists of daily injections of 2
mg/kg/day for 2 weeks followed by a biweekly dose of 0.5 mg/kg/day
for 99 days, this would amount to a subsequent dose of about 1.8%
of the initial dose, calculated on a daily basis (i.e.,
2/day/100%=0.5/14 days/x%, x=.about.1.8%). Preferably, the
subsequent dosing is less than about 50%, more preferably, less
than about 25%, more preferably, less than about 10%, still more
preferably, less than about 5%, and most preferably, less than
about 2% of the initial dosing of LFA-1 antagonist.
[0085] To obtain the most efficacious results, depending on the
disorder, the initial dosing is given as close to the first sign,
diagnosis, appearance, or occurrence of the disorder as possible or
during remissions of autoimmune disorders. Preferably the initial
dosing begins before exposure to antigen, as in the case with
transplanted grafts. Furthermore, when the initial dosing is prior
to or substantially contemporaneous with exposure to antigen, it is
preferred that the subsequent dosing is carried out for a longer
period of time than the initial dosing, particularly for
transplants, and that it be a continuous intermittent maintenance
dose that need not be continuous for the life of the patient.
[0086] The preferred scheduling is that the initial dosing (i.e.,
administered before or at the time of the undesired immune response
at a dose administered no less frequently than daily up to and
including continuously by infusion) and the subsequent dosing is a
dose administered periodically no more than about once a week. More
preferably, depending on the specific disorder, and particularly
for transplantation, the initial daily dosing is administered for
at least about one week, preferably at least about 2 weeks, after
the exposure to antigen, e.g., graft, or initiation of an acute
immune response (as in autoimmune disorders), and the subsequent
dosing is administered no more than once biweekly (preferably once
biweekly) for at least about 5 weeks, preferably for at least about
10 weeks, after the initial dosing is terminated.
[0087] In another preferred embodiment, particularly if the
antagonist is anti-CD11a or anti-CD18 antibodies, initial dosing
terminates from about 1 day to 4 weeks after transplantation has
occurred, more preferably from about 1 week to 3 weeks, more
preferably from about 2 weeks to 3 weeks, and commences from about
1 week before transplantation occurs up to about simultaneously
with the transplantation.
[0088] The LFA-1 antagonist is administered by any suitable means,
including parenteral, subcutaneous, intraperitoneal,
intrapulmonary, and intranasal, and, if desired for local
immunosuppressive treatment, intralesional administration
(including perfusing or otherwise contacting the graft with the
antagonist before transplantation). Parenteral infusions include
intramuscular, intravenous, intraarterial, intraperitoneal, or
subcutaneous administration. In addition, the LFA-1 antagonist is
suitably administered by pulse infusion, particularly with
declining doses of the LFA-1 antagonist. Preferably the dosing is
given by injections, most preferably intravenous or subcutaneous
injections, depending in part on whether the administration is
brief or chronic.
[0089] The LFA-1 antagonist need not be, but is optionally
formulated with one or more agents currently used to prevent or
treat the disorder in question. For example, in rheumatoid
arthritis, the antibody may be given in conjunction with a
glucocorticosteroid. In addition, T cell receptor peptide therapy
is suitably an adjunct therapy to prevent clinical signs of
autoimmune encephalomyelitis. Offner et al., supra. For
transplants, the antibody may be administered concurrently with or
separate from an immunosuppressive agent as defined above, e.g.,
cyclosporin A, to modulate the immunosuppressant effect. The
effective amount of such other agents depends on the amount of
LFA-1 antagonist present in the formulation, the type of disorder
or treatment, and other factors discussed above. These are
generally used in the same dosages and with administration routes
as used hereinbefore or about from 1 to 99% of the heretofore
employed dosages.
[0090] The various autoimmune disorders described above are treated
with LFA-1 antagonists in such a fashion as to induce immune
tolerance to the self antigen under attack as a result of the
disorder. In this regard, autoimmune disorders resemble host versus
graft rejection and are treated with LFA-1 antagonists in analogous
fashion. However, in these disorders the patient is already
mounting an immune response to the target antigen, unlike the case
with transplants prior to grafting. Thus, it is desirable to first
induce and maintain a transient state of immunosuppression by
conventional methods in such patients, e.g. by the conventional use
of cyclosporin A or other conventional immunosuppressive agents
(alone or together with LFA-1 antagonist), or to monitor the
patient until the occurrence of a period of remission (an absence
or substantial lessening of pathological or functional indicia of
the autoimmune response).
[0091] Preferably, transient immunosuppression is induced by T cell
depletion using conventional therapy, e.g. as described further in
Example 1. This is then followed by the administration of the LFA-1
antagonist in order to prevent rebound when the immunosuppressive
inducing agent is withdrawn or when remission otherwise would
abrogate. Alternatively, the remission patient's condition is
closely monitored for signs of flare, and immediately upon the
initial functional or biochemical appearance of flare the initial
dosing regimen is started and continued until the flare subsides.
The LFA-1 administration during this period constitutes the initial
dose described elsewhere herein.
[0092] In the case of autoimmune disorders the initial dose will
extend about from 1 week to 16 weeks. Thereafter, the lower dose
maintenance regimen of LFA-1 antagonist is administered in
substantially the same fashion as set forth herein for the
amelioration of graft or host rejection, although in some instances
it is desirable to extend the subsequent or sustaining dose for
lengthier periods than with grafts. In an embodiment of this
invention, if an antigen or a composition containing the antigen is
known to be responsible for the autoimmune response then the
antigen is administered to the patient (optionally with IL-1 and/or
gamma interferon) after the initial LFA-1 antagonist dose and the
antagonist dose maintained thereafter in order to suppress the
regeneration of an autoimmune response against the antigen while
minimally immunosuppressing the patient's response to other
antigens.
[0093] The patient optimally will be isolated, preferably in an
aseptic environment such as is currently used in transplant
practice, at the time of initial treatment with LFA-1 antagonist.
The patient should be free of any infection. It is not necessary to
sustain these conditions during the maintenance dose, and in fact
this is one of the advantages of this invention, i.e., that the
patient is able to mount a substantially normal immune response to
ambient antigens (other than the graft or self antigen) while being
treated with the maintenance dosing.
[0094] The invention herein is particularly amenable to prolonging
survival and increasing tolerance of transplanted grafts. The
transplants are optionally functionally monitored systematically
during the critical postoperative period (the first three months)
using any suitable procedure. One such procedure is radionuclide
intravenous angiography using 99Tcm-pertechnetate, as described by
Thomsen et al., Acta Radiol., 29: 138-140 (1988). In addition, the
method herein is amenable to simultaneous, multiple organ perfusion
and transplantation (Toledo-Pereyra and MacKenzie, Am. Surg., 46:
161-164 (1980).
[0095] In some instances, it is desirable to modify the surface of
the graft so as to provide positively or negatively charged groups,
as by using a suitable amino acid or polymer or by attaching a
physiologically acceptable source of charged functional groups. For
example, a negatively charged surface is appropriate for blood
vessels to diminish blood clotting. It also is desirable in certain
circumstances to render the surface hydrophobic or hydrophilic by
coupling, e.g., phenylalanine, serine or lysine to the surface. An
immunosuppressive agent particularly effective for these surface
modifications is glutaraldehyde.
[0096] As mentioned above, before transplantation an effective
amount of the antibody is optionally administered to induce
tolerance of the graft. The same dose and schedule as used for
initial post-transplantation may be employed. Furthermore, prior to
transplantation the graft is optionally contacted with a
TGF-.beta.composition as described in U.S. Pat. No. 5,135,915, the
disclosure of which is incorporated by reference. Briefly, the
contact suitably involves incubating or perfusing the graft with
the composition or applying the composition to one or more surfaces
of the graft. The treatment generally takes place for at least one
minute, and preferably from 1 minute to 72 hours, and more
preferably from 2 minutes to 24 hours, depending on such factors as
the concentration of TGF-.beta. in the formulation, the graft to be
treated, and the particular type of formulation. Also as noted, the
graft is simultaneously or separately perfused with LFA-1
antagonist. Perfusion is accomplished by any suitable procedure.
For example, an organ can be perfused via a device that provides a
constant pressure of perfusion having a pressure regulator and
overflow situated between a pump and the organ, as described by DD
213,134 published Sept. 5, 1984. Alternatively, the organ is placed
in a hyperbaric chamber via a sealing door and perfusate is
delivered to the chamber by a pump that draws the fluid from the
reservoir while spent perfusate is returned to the reservoir by a
valve, as described in EP 125,847 published Nov. 21, 1984.
[0097] After the graft is treated, it is suitably stored for
prolonged periods of time or is used immediately in the transplant
procedure. Storage life can be enhanced as described above by using
a blood substitute in the formulation (e.g., perfluorochemical
emulsion), or by perfusing the graft with a formulation of a
TGF-.beta. containing chilled isotonic agent and anticoagulant
followed by glycerol to allow for freezing of removed organs with
no destruction of the cells, as described in JP 60061501 published
Apr. 9, 1985. In addition, the organs can be preserved with known
perfusion fluids (containing TGF-.beta.and/or LFA-1 antagonist as
noted) while the organs are cooled to freezing temperatures, to
preserve the organ semi-permanently without cell necrocytosis, as
described by U.S. Pat. Nos. 4,462,215 and 4,494,385.
[0098] Respecting cardiac transplants specifically, Parent et al.,
Cryobiology, 18: 571-576 (1981) reports that cold coronary
perfusion prior to transplantation at 5.degree. C. increases
protection of the homograft during the initial period of
implantation. Any of these procedures, or others, are within the
scope of this invention if deemed necessary for graft
preservation.
[0099] Before transplantation, the graft is preferably washed free
of the TGF-.beta. composition, as by soaking it in a physiological
saline solution or by other means appropriate for this purpose. It
is not desirable to remove the LFA-1 antagonist prior to
transplantation.
[0100] Also, prior to transplantation, the host is optionally given
one or more donor-specific blood transfusions to aid in graft
survival. An alternative procedure is to subject the host to total
lymphoid irradiation prior to or after the transplantation
operation. Any other pre-transplant procedures that would be
beneficial to the particular transplant recipient can be performed
as part of the method of this invention.
[0101] The invention will be more fully understood by reference to
the following examples. They should not, however, be construed as
limiting the scope of the invention. All literature citations are
incorporated by reference.
EXAMPLE 1
Murine Heart Graft Model Using Anti-CD11a Antibody
[0102] Neonatal BALB/c (H-2.sup.d) hearts were transplanted into
the dorsal ear pinnae of male adult (8 to 10 weeks old) C3H
(H-2.sup.k) mice as described in Babany et al., J. Pharmacol. Exp.
Ther., 244: 259 (1988). All mice were raised under specific
pathogen-free conditions and were obtained from the Department of
Comparative Medicine, Stanford University Medical Center. The
reagents employed were M17 (clone M17/4.411.9, rat IgG2a
anti-murine CD11a MAb purified from ascites, obtainable from the
American Type Culture Collection, Rockville, Md. (ATCC Accession
No. TIB 217), cyclosporin A (CsA; i.v. formulation, Sandoz, East
Hanover, N.J.), or IgG2a (rat isotype control, Zymed. S. San
Francisco, Calif.). M17 (n=3-9/dose group) or CsA (n=5-20/dose
group) was administered to the mice daily i.p. for two weeks
starting on the day of transplantation (M17) or on the first
post-transplant day (CsA).
[0103] The results are shown in FIGS. 1A and 1B, representing
efficacy and potency, respectively. Grafts in untreated (n=105) or
in rat IgG2a isotype control-treated (n=9) mice were rejected in
10.63.+-.0.1 days (median graft survival time [MST].+-.S.E.; median
survival=10.0 days) and 10.0.+-.0.3 days (median survival=10.0
days), respectively. Compared to CsA, M17 prolonged graft survival
much more effectively and potently. The MST of mice treated with 4
mg/kg/day M17 was extended to 58 days, whereas the MST was only 24
days in mice treated with a maximally tolerated dose (25 mg/kg/day)
of CsA (FIG. 1A). In fact, 2 mg/kg/day M17 prolonged graft survival
significantly more than 25 mg/kg/day CsA (p<0.05). The highest
M17 dose (4 mg/kg/day) administered produced no observable
toxicity.
[0104] To compare quantitatively the relative immunosuppressive
potencies of M17 and CsA to obtain dose-response curves for
immunosuppression, a quantal in vitro murine heart allograft
bioassay was used. Morris, Transplant Rev., 6: 39 (1992). For each
dose group, the mean percent beating heart allografts on day 14 as
a function of log.sub.10 dose was fit by logistic regression. When
the ED.sub.50s of M17 and CsA were compared (the doses are
expressed as nmol/kg to control for the difference in molecular
weight between these two drugs), M17 was approximately 5000 times
more potent than CsA. (The ED.sub.50s for M17 and CsA were 1.48
nmol/kg and 8.10 pmole/kg, respectively.) See FIG. 1B. There is a
dramatic disparity between the potency of M17 and CsA.
[0105] Since it is likely that any new immunosuppressive agent
initially will be used clinically in combination with CsA,
different doses of M17 and CsA were administered in combination for
two weeks to C3H recipients of BALB/c heart grafts. To determine
whether M17 and CsA interact to produce immunosuppression that is
antagonistic, additive, or synergistic, isobologram analysis was
used to evaluate graft survival data. According to the geometric
isobologram method described by Berenbaum, Pharmacol. Rev., 41: 93
(1989), the additivism isobole is defined as the line joining the
minimum M17 and CsA doses required for 100% graft survival. FIG. 2
shows that M17 and CsA do not interact antagonistically; rather,
combined treatment with M17 and CsA produced 100% graft survival at
doses that are predicted if these drugs interact additively.
[0106] To determine whether treatment with M17 for more than two
weeks increases graft survival, 2 mg/kg/day of M17 was administered
daily for three weeks followed by weekly treatment until day 98
post-transplant. Despite cessation of treatment on day 98, some
heart grafts continued to contract for more than 90 additional days
(FIG. 3). In another group, recipient mice were treated with 8
mg/kg/day of M17 daily for two weeks followed by biweekly doses of
2 mg/kg until day 99. This treatment regimen substantially
increased the number of heart grafts surviving indefinitely
compared to mice treated with the lower dose of M17 (FIG. 3). Since
treatment in the early post-operative period with 8 mg/kg/day of
M17 induced long-term graft survival more effectively than
treatment with 2 mg/kg/day, high peri-operative blood levels of M17
may be critical for long-term graft survival.
[0107] Because extended treatment with 2 mg/kg/day of M17 produced
indefinite graft survival in some recipients, the specificity of
this immunosuppression was investigated in these animals by
transplanting C57BL/6 (H-2.sup.b) heart grafts into their
contralateral ear pinnae 154 days after the primary grafts. The
primary BALB/c grafts were not rejected even though the C57 grafts
were rejected promptly; the MST for C57 grafts was not
significantly (p>0.05) different from the MST of C57 grafts
transplanted into non-immunosuppressed mice (Table 1). Therefore,
limited treatment with M17 did not cause long-term non-specific
immunosuppression, since the immune systems in treated mice were
fully capable of responding to third-party alloantigens.
1TABLE 1 Survival of C57 heart grafts compared to BALB/c primary
grafts in C3H mice that accepted primary graft for more than 154
days M17 Treatment with 2 mg/kg/day, i.p., day 0-20, then weekly
until day 98 BALB/c graft C57 No treatment survival (days) graft
survival (days) C57 graft survival (days) p value.sup.a >190 7 8
NS >190 14 11 >190 14 11 >200 41 14 .sup.aThe Mann-Whitney
U test (corrected for small sample sizes) was used to determine the
level of statistical difference between C57 graft survival in
M17-treated and untreated mice. p > 0.05 was considered not
significant (NS).
[0108] Normally, prolonged treatment with MAbs elicits a xenogeneic
antibody response that limits the therapeutic efficacy of prolonged
MAb treatment. Norman, Sem. Nephrology, 12: 315 (1992). It was
found here, however, that extended treatment with M17 was more
effective than brief treatment (FIG. 3). The results herein
suggested that the xenogeneic response of mice to M17 differed from
the responses to other MAbs. Mouse anti-rat antibodies in the sera
of M17-treated mice were determined by an ELISA that used M17 as
the capture antibody and was developed with a horseradish
peroxidase-conjugated rat anti-mouse IgG antibody. Results of these
studies showed that the xenogeneic antibody response was inversely
related to the M17 treatment dose. For example, mice treated with
0.25 mg/kg/day of M17 produced an anti-rat immunoglobulin response
by day 15, but mice treated for the same time with 4 mg/kg/day of
M17 did not respond to rat immunoglobulin.
[0109] To characterize further the ability of M17 to suppress graft
rejection, a model of accelerated rejection was used. Primary
BALB/c hearts were transplanted into C3H recipients to sensitize
these mice to BALB/c alloantigens and were removed four days later.
Thus, mice (n=5-10/group) were presensitized with temporary BALB/c
heterotopic (ear-pinnae), nonvascularized heart grafts from days -7
to -3 at which time the ears bearing these primary grafts were
removed. After transplantation of secondary BALB/c heart grafts on
day 0, the individual survival times and the MSTs (horizontal lines
in FIG. 4) were determined and are shown in FIG. 4 for each
treatment group. Levels of statistical significance between graft
survival times in the different treatment groups and the control
group were computed using the Mann-Whitney U test (corrected for
small sample sizes). p>0.05 was considered not significant
(NS).
[0110] Secondary grafts transplanted into the contralateral ear
pinnae of mice treated i.p. with 4 mg/kg of isotype control rat
IgG2a from days -7 to 13 all underwent accelerated rejection and
failed to beat. See FIG. 4.
[0111] Different schedules (relative to the day of primary
grafting) were used to administer 4 mg/kg/day of M17 i.p. to
recipients. One group of mice was treated with 4 mg/kg/day of M17
from the day on which the primary hearts were grafted until two
weeks after transplantation of secondary grafts. The MST of grafts
in these mice was 42 days. This schedule and dose of M17 prevented
sensitization since the MST of grafts in these mice did not differ
significantly (p>0.05) from the MST of grafts in non-sensitized
recipients treated with 4 mg/kg/day of M17 for the first two weeks
after transplantation. Results from other experiments showed that
the timing of M17 treatment was more important than the duration of
treatment. The MST of secondary grafts in mice in which treatment
with M17 was delayed until the day of secondary heart
transplantation was significantly less (p<0.05) than the MST of
grafts in non-sensitized mice treated identically with M17.
Therefore, M17 treatment did not eliminate alloreactivity once
sensitization had occurred.
[0112] In other experiments, it was shown that the MST of secondary
grafts in mice treated with M17 for only the four days during which
the primary grafts were in place was not significantly different
(p>0.05) from the MST of secondary grafts in mice treated from
the day of primary heart transplantation until two weeks after
transplantation of the secondary graft. Thus, M17 needed to be
administered only during the period of sensitization to prevent
accelerated rejection. The MST of secondary grafts in mice treated
with M17 for the four days preceding implantation of the secondary
graft was also not significantly (p>0.05) different from the MST
of grafts in non-sensitized recipients treated with M17 from day
0-13. Thus, brief pre-treatment with M17 was no less effective than
a longer M17 treatment course post-transplant.
[0113] Delaying M17 treatment (2 mg/kg/day) until post-transplant
day 4 or 6, when severe rejection is evident in untreated mice,
prolonged graft survival only slightly over the control (MSTs of 16
and 14 days, respectively). Similarly, others have found that an
anti-CD11a MAb does not reverse acute renal allograft rejection in
humans. LeMauff et al., supra. These results, along with the in
vitro findings of Davignon et al., supra, and the conclusion of
Springer et al., Annu. Rev. Immunol., 5: 223 (1987), suggest that
treatment early in the immune response is critical for the
immunosuppressive efficacy of M17.
[0114] The histology of spleens, thymuses, and lymph nodes from
mice (n=5) treated with 4 mg/kg/day of M17 for two weeks was
determined using flow cytometric studies. Percent total T cells
(anti-Thy 1.2-FITC), CD4 (anti-L3T4-PE), and CD8 (anti-Lyt 2-FITC)
T cell subsets, B cells (anti-B220-PE; all MAbs from Caltag, S. San
Francisco, Calif.), and LFA-1+ (anti-CD11a-FITC, which recognize a
different epitope than M17; clone 2D7, Pharmingen, San Diego,
Calif.) spleen cells from mice that were untreated or from mice 14
days after daily i.p. treatment with 4 mg/kg of isotype control rat
IgG2a or M17 (n=5/group) were determined using a Profile II
(Coulter Ejpics, Hialeah, Fla.).
[0115] The flow cytometric studies (FIG. 5) showed an increase in
percent splenic T cells expressing pan T, CD4, and CD8 markers.
Furthermore, the percent of LFP-1+ spleen cells did not differ
substantially among M17-, IgG2a-, or non-treated mice. Moreover,
there was no evidence of a decrease in yield of leukocytes per
spleen in M17-treated mice compared to controls. Therefore, M17
does not appear to cause immunosuppression by central or peripheral
lymphoid depletion. Furthermore, complete blood counts in these
mice showed that M17 treatment did not suppress the number of
lymphocytes compared to IgG2a-treated (n=3) control mice.
[0116] Since treatment with M17 does not cause T cell depletion and
since the results herein and those of others [Isobe et al., supra)
show that LFA-1 is expressed after treatment with anti-LFA-1 MAbs,
M17 may cause immunosuppression by functional inactivation of T
cells. Thus, the function of immune cells from mice treated daily
for two weeks with 4 mg/kg/day of M17 was assessed by determining
the proliferative response of spleen cells to ConA in vitro. After
14 days of daily i.p. treatment with 4 mg/kg/day of either isotype
control IgG2a (n=5) or M17 (n=5), spleens were removed and cells
cultured in KC2000 (Hazelton Biologics, Lenexa, Kans.) in 96-well
plates for 3 days with different concentrations of ConA (Vector,
Burlingame, Calif.). Cell proliferation was assessed by pulsing
with .sup.3H-TdR (ICN Radiochemicals, Irvine, Calif., specific
activity 6.7 Ci/mM) for 16-18 hours and the .sup.3H-TdR
incorporation was determined by scintillation spectroscopy
(Packard, Downers Grove, Ill.). For each ConA concentration, the
mean disintegrations/min (dpm) was computed after subtracting
background dpm for cells from control mice and M17-treated mice.
The data were expressed as the percent change in dpm in spleen
cells from M17-treated mice relative to dpm in spleen cells from
control mice. p>0.05 determined using the Student's t test was
considered not significant (NS).
[0117] FIG. 6 shows that at low ConA concentrations, the
proliferative response of spleen cells from mice treated with M17
was activated 150-200% more than the response of spleen cells from
IgG2a-treated control mice. These data show that treatment with M17
does not impair T cell activation.
[0118] The host versus graft popliteal lymph node (PLN) hyperplasia
assay was used to investigate the mechanisms by which M17
suppresses the response to alloantigens in vivo. On day 0,
2.5.times.10.sup.6 irradiated BALB/c spleen cells were injected
into each left hind footpad of C3H mice (n=5/group) and the mice
were treated immediately with either isotype control IgG2a or with
M17 or beginning one or two days after cell injection. On day 4,
the right and left PLNs were removed and the mean difference in
weight between the left PLNs and right PLNs was determined for each
treatment group. Levels of statistical significance of PLN weight
differences between M17 treatment groups and the control group were
computed using the Mann-Whitney U test (corrected for small sample
sizes).
[0119] The increase in left PLN weights after alloantigenic
stimulation in IgG2a-treated control mice as shown in FIG. 7 is
caused by cell proliferation and recruitment of lymphoid cells into
the PLN due to altered cell migration. Treatment with M17
significantly suppressed increases in PLN weights regardless of
whether treatment was begun on day 0 or on day 2. This inhibition
could be caused by effects of M17 on the proliferative response to
alloantigens or by its effects on lymphocyte trafficking or both.
Since others have shown that treatment with anti-CD11a MAb inhibits
lymphocyte homing to peripheral lymph nodes in mice [Hamann et al.,
J. Immunol., 140: 693 (1988)], this effect may be one of the
mechanisms by which heart allograft survival is prolonged in
M17-treated mice, although this is only one theory and the
invention is not limited thereto.
[0120] In summary, administration of anti-CD11a antibody at the
time of alloantigenic stimulation using a high initial dosing of
antibody followed by a lower subsequent dosing produces long-term
allograft survival and prevents sensitization to alloantigens in
the difficult mouse heterotopic ear-heart model without lymphoid
cell depletion. A state of selective unresponsiveness develops:
mice that failed to reject their initial grafts reject third party
grafts. Since M17 produced long-term graft survival even without
the co-administration of anti-ICAM-1 MAb, suppression of the immune
system by anti-adhesion molecule MAbs may depend more on blocking
the interaction between LFA-1 and ICAM-1 than on preventing the
interaction of ICAM-1 with its other receptors, Mac-l and CD43.
[0121] Treatment with anti-CD4 used alone or in combination with
total lymphoid irradiation [Trager et al., supra] or with anti-CD3
MAb used alone or in combination with anti-CD2 MAb prolongs
allografts far less effectively in this model than M17 treatment
despite the substantial T cell depletion or near complete decrease
in CD3 cell surface expression these treatments produce. In
addition, treatment with M17 is much more effective, far more
potent, and has a greater therapeutic index than CsA. The ear-heart
model has been used extensively to evaluate other new xenobiotic
immunosuppressants and analysis of the data indicates that
treatment with M17 for prevention of rejection is more effective
and has a higher therapeutic index than mycophenolate mofetil
(RS-61443) [Morris et al., Transplant. Proc., 22: 1659 (1990)],
brequinar [Murphy and Morris, Med. Sci. Res., 19: 835 (1991)], or
FK506 [Morris et al., Transplant. Proc., 22: 1638 (1990)].
EXAMPLE 2
Encephalomyelitis Model Using Anti-CD11a Antibody
[0122] Experimental autoimmune encephalomyelitis (EAE) is an
inflammatory condition of the central nervous system with
similarities to multiple sclerosis. In both diseases, circulating
leukocytes penetrate the blood-brain barrier and damage myelin,
resulting in impaired nerve conduction and paralysis.
[0123] EAE is induced in Lewis rats by subcutaneous injection of 50
.mu.g guinea-pig basic protein (GPBP) [Vandenbark et al., Nature,
341: 541 (1989)]+400 .mu.g Mycobacteria in complete Freund's
adjuvant (CFA). One group of rats is untreated and one group of
rats is injected subcutaneously with 1-10 mg/kg/day daily of M17
mixed with CFA and 100 .mu.g Mycobacteria at either day -4, day -3,
day -2, day -1, day 0, day 1, day 2, day 3, day 4, day 5, day 6,
day 7, day 8, day 9, day 10, day 11, day 12, or day 13, with day 0
being the day when the GPBP is given. This administration is
continued up to day 21. Then, on days 28 and 35, each rat is
injected subcutaneously with 0.25-2.5 mg/kg/day of M17 mixed with
CFA and 100 .mu.g Mycobacteria. The average onset of EAE in this
model is 14 days and average length of paralysis is 6 days. The
severity of EAE in the experimental rat model is reduced by
administration of the anti-LFA-1 antibody, M17.
[0124] In another experiment, pooled groups of B10.PL mice are
challenged subcutaneously at the base of the tail with 120 .mu.g of
the encephalitogenic MBP 1-9NAc peptide [Urban et al., Cell, 54:
577-592 (1988)] in CFA. At 24 and 74 hours after injection, the
mice are injected with 6.times.10.sup.9 heat-killed Bordetella
pertussis intravenously according to Zamvil et al., Nature, 324:
258-260 (1986). One group of mice is untreated and one group of
mice is injected subcutaneously with 1.5-13 mg/kg/day daily of M17
in normal saline at either day -4, day -3, day -2, day -1, day 0,
day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day
10, day 11, day 12, or day 13, with day 0 being the day when MBP
peptide is given. This administration is continued up to day 21.
Then, on days 28 and 35, each mouse is injected subcutaneously with
0.3-4 mg/kg/day of M17. The mice are observed daily for clinical
signs of EAE. The average day of onset for the untreated mice
developing EAE in this model is 8-12 days. The severity of EAE in
the experimental mouse model is reduced by administration of the
anti-LFA-l antibody, M17.
EXAMPLE 3
In vitro Mixed Lymphocyte Culture Model Using Anti-CD18
Antibody
[0125] This mixed lymphocyte culture model, which is an in vitro
model of transplantation [A.J. Cunningham, "Understanding
Immunology," Transplantation Immunoloqy, p. 157-159 (1978)],
examines the effects of various .alpha.-ICAM, .alpha.-LFA-1
antibodies, and soluble ICAM in both the proliferative and effector
arms of the human mixed lymphocyte response.
[0126] I. Protocol:
[0127] A. Mixed Lymphocyte Response
[0128] Part 1: Isolation of Cells: Mononuclear cells from
peripheral blood (PBMC) were separated from heparinized whole blood
drawn from healthy donors. Blood was diluted 1:1 with saline,
layered, and centrifuged at 2500.times.g for 30 minutes on LSM (6.2
g Ficoll and 9.4 g sodium diatrizoate per 100 ml) (Organon
Technica, N.J.). Cells were resuspended in RPMI 1640 medium (GIBCO,
Grand Island, N.Y.) supplemented with 5% heat-inactivated pooled
human AB serum (Peninsula Memorial Blood Bank, Burlingame, Calif.),
1 mM sodium pyruvate, 3 mM L-glutamine, 1 mM nonessential amino
acids, 500 .mu.g/ml penicillin, 50 .mu.g/ml streptomycin, 50
.mu.g/ml Gentamycin (GIBCO), and 5.times.10.sup.-5M
2-mercaptoethanol (Sigma, St. Louis, Mo.).
[0129] Part 2: Mixed Lymphocyte Response (MLR): One way human mixed
lymphocyte cultures were established in 96-well flat-bottomed
microtiter plates. Briefly, 1.5.times.10.sup.5 responder PBMC in
200 .mu.l of complete medium were co-cultured with an equal number
of allogeneic irradiated (3000 rads) stimulator PBMC. Soluble
ICAM-1 or anti-integrin antibodies [MHM24 (anti-CD11a) and H52
(anti-CD18), described in the references set forth above] were
added at the initiation of cultures. Cultures were incubated at
37.degree. C. in 5% Cp for 5 days, then pulsed with 1 .mu.Ci/well
of .sup.3H-thymidine (6.7 Ci/mmol, NEN, Boston, Mass.) for 16
hours. Cultures were harvested on a PHD cell harvester (Cambridge
Technology, Inc., Watertown, Mass.). [.sup.3H]TdR incorporation was
measured with a Beckman scintillation counter (LS6800) and
triplicate determinations were averaged. Data are expressed as net
cpm. The mean [.sup.3H]-TdR incorporated by control cultures was
<1000 cpm.
[0130] B. Cytotoxic T Lymphocyte (CTL) Assay:
[0131] Part 1: Generation of CTL: (CTLs were generated in a 7-day
mixed lymphocyte culture, except cultures were scaled up to
generate large numbers of cells.) Peripheral blood lymphocytes from
two unrelated donors were isolated with LSM in conventional
fashion. Cells were adjusted to 3.times.10.sup.6 cells/ml with
human MLR media described above. Lymphocytes from one donor were
irradiated with 3000 rads from a cesium source and were designated
"stimulator" cells. The second donor's lymphocytes were named
"responder" cells. Five ml of each of the responder and stimulator
cells were combined in a Corning T-25 cm.sup.2 tissue culture flask
and incubated for seven days at 37.degree. C., 5% CO.sub.2 in
air.
[0132] Part 2: Generation of target cells: Three days prior to
harvest of the CTLs, lymphocytes were isolated from the donor whose
lymphocytes were used as the stimulator cells in Part 1. These
cells were adjusted to 1.times.10.sup.6 cells/ml in human MLR
media. Next, ten ml of cells and a 1:500 dilution of Difco PHA-P
were combined in a Corning T-25 cm.sup.2 tissue culture flask and
incubated for 3 days at 37.degree. C., 5% CO.sub.2 in air.
[0133] Part 3: CTL Killing Assay (A 4-hour.sup.51 CR-release
assay):
[0134] After 7 days of culture, CTLs (effector cells) were
collected, washed three times, then adjusted to 1.times.10.sup.7
cells/ml. Target cells were collected and washed two times. Target
cells were labeled with 150 .mu.Ci Na.sup.51CrO.sub.4 (5 mCi/ml:
Amersham Corp., Arlington Heights, Ill.) for approximately 1 hour
at 37.degree. C., 5% CO.sub.2 in air. Cells were washed four times,
counted, and adjusted to 2.times.10.sup.5 cells/ml. The CTL killing
assay was set up in a Corning 96-well round-bottom plate. A total
of 200 .mu.l cells was added per well. 50 .mu.l of target cells and
100 .mu.l of effector cells at various concentrations, and 50 .mu.l
of antibodies [H52, anti-CD11b, anti-CD11a, anti-CD18, and
anti-gp120 (7F11), which are all publicly available] at 500 ng/ml
were added in triplicate to the plate.
[0135] After four hours of incubation at 37.degree. C., 5% CO.sub.2
in air the supernatants were harvested (Skatron, Rockville, Md.)
and their radioactivity was determined in an automatic gamma
counter (Micromedic Systems, Horsham, Pa.). Percent specific
cytotoxicity was calculated at 100.times.[cpm of test supernatants
of effector cells and target cells incubated together (experimental
release)]-[cpm of supernatants of target cells incubated alone
(spontaneous release)]/{[cpm after lysis of target cells with 2%
NP-40 (maximum release)]-[spontaneous release]}. Results determined
were the mean of triplicate cultures +/-SD. Spontaneous release of
target cells alone was <10% of maximum for all experiments.
[0136] II. Results:
[0137] A. Mixed Lymphocyte Response
[0138] The results of the mixed lymphocyte response are shown in
FIG. 8. It is clear that the anti-CD18 antibody has an inhibitory
effect on the human mixed lymphocyte response, similar to that of
the anti-CD11a response.
[0139] B. CTL Assay
[0140] The results of the effect of various antibodies on CTL
target cell killing are shown in FIG. 9. They indicate that only
H52, anti-CD11a, and anti-CD18 inhibit lysis of the cells.
[0141] It would be reasonably expected from the in vitro data above
that the LFA-1 antagonist would function in an in vivo setting,
i.e., in a transplantation.
EXAMPLE 4
Contact Sensitivity Model Using Anti-CD11a Antibody
[0142] The contact sensitivity model described below is a model for
treating psoriasis.
[0143] Protocol:
[0144] Day 0: Sensitization
[0145] BALB/c mice (4-6 weeks old) obtained from Charles River were
divided into four treatment gruops containing 6-8 mice per group.
The mice were anesthetized i.p. with
Ketamine/Xylazene/Acepromazine. A patch approximately 3.times.3
cm.sup.2 was shaved on the abdomen of all the mice. A total of 50
.mu.l of 10 mg/ml dinitrofluorobenzene (DNFB) was applied topically
to the hair-free abdomen of the mice in groups 2-14. A pipetman was
used to deliver the dose, enabling the wide end of the tip to be
used to spread the DNFB over the skin.
[0146] Days 0-5: Administration of the Antibodies
[0147] A total of 20 .mu.g of the M17 antibody was injected i.p. on
the days designated as follows: Group 1-days 0 and 1; Group 2-days
4 and 5; Group 3-days 0-5; and Group 4-rat IgG2a isotype
control.
[0148] Days 0-5: Challenge
[0149] The mice were anesthetized with Metaphane. With a pipetmen,
5 .mu.l of of DNFB was applied topically to each side of the left
pinnae of the mice in groups 2-14 (5 .mu.l/side). The wide end of
the pipet tip was used to spread the DNFB over the ear. To each
side of the right pinnae of the mice in groups 2-14 was applied
topically 5 .mu.l of the diluent of DNFB.
[0150] 8 to 10 Hours Later after Challenge
[0151] A total of 0.1 ml of 2 .mu.Ci .sup.125I-UdR was injected
i.v. into the tail vein of all mice.
[0152] 16 to 18 Hours after .sup.125I-UdR Injection
[0153] The mice were sacrificed with CO.sub.2. Both pinnae were cut
off at the hairline of all mice. Left and right pinnae were put in
separate tubes. Each pinnae was placed into appropriately labeled
12.times.75 polystyrene snap cap tubes. The pinnae were stored at
-20.degree. C.
[0154] Results:
[0155] The results are shown in FIG. 10. Those mice treated with
M17 all exhibited a decreased sensitivity to DNFB as compared to
the control. Those mice treated at days 0 and 1 and at days 0-5
exhibited the greatest decrease in sensitivity.
[0156] It would be expected that a decreased maintenance dose less
than the 20 .mu.g used in this experiment given intermittently
after the initial dose would further decrease the sensitivity of
the mice to the immunogen. Further, it would be reasonably expected
that the in vivo mice data described above may be extrapolated to
horses, cows, and other mammals, correcting for the body weight of
the mammal in accordance with recognized veterinary and clinical
procedures. Humans are believed to respond in this manner as well.
Thus, it would be reasonably expected that in man the dosing
regimen herein would have a beneficial restorative effect on immune
function mediated by LFA-1 in all patients.
[0157] The treatment herein is expected to provide a higher
therapeutic index than conventional and current therapy by
minimizing toxicity to various parts of the body, including the
kidneys (especially in the first few weeks after transplantation
when the kidneys are most susceptible), the liver, the pancreas,
the bones, the bone marrow, and the central nervous and immune
systems. It is also expected to lessen the occurrence of both acute
and chronic rejection. Further, the drug is expected to be useful
in patients at high risk who are receiving regrafts and have a low
one-year graft survival rate. Finally, the drug is expected to
decrease morbidity in the patients, thus reducing the overall cost
of transplantation.
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