U.S. patent application number 10/847212 was filed with the patent office on 2005-01-06 for alleviation of symptoms associated with inflammatory disease states.
Invention is credited to Rose, Lynn M..
Application Number | 20050002932 10/847212 |
Document ID | / |
Family ID | 26740253 |
Filed Date | 2005-01-06 |
United States Patent
Application |
20050002932 |
Kind Code |
A1 |
Rose, Lynn M. |
January 6, 2005 |
Alleviation of symptoms associated with inflammatory disease
states
Abstract
Disclosed are methods for the alleviation of symptoms associated
with inflammatory disease states, and more particularly to the
inhibition of amatory processes associated with the multiple
sclerosis disease, by administering a pharmaceutically effective
amount of antibody substance immunologically reactive with the
common .beta. chain (CD18) of human leukocyte integrins and/or
competes with mAb 60.3 for binding to human LPA-1.
Inventors: |
Rose, Lynn M.; (Seattle,
WA) |
Correspondence
Address: |
MARSHALL, GERSTEIN & BORUN LLP
6300 SEARS TOWER
233 S. WACKER DRIVE
CHICAGO
IL
60606
US
|
Family ID: |
26740253 |
Appl. No.: |
10/847212 |
Filed: |
May 17, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10847212 |
May 17, 2004 |
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08333947 |
Nov 3, 1994 |
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6737059 |
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08333947 |
Nov 3, 1994 |
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08060699 |
May 10, 1993 |
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08060699 |
May 10, 1993 |
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07915068 |
Jul 16, 1992 |
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Current U.S.
Class: |
424/144.1 ;
530/388.22 |
Current CPC
Class: |
A61P 29/00 20180101;
C07K 16/2845 20130101; C07K 2319/00 20130101; A61K 2039/505
20130101; A61K 38/00 20130101; C07K 2317/24 20130101 |
Class at
Publication: |
424/144.1 ;
530/388.22 |
International
Class: |
A61K 039/395; C07K
016/28; C12N 005/06 |
Claims
What is claimed is:
1. A method for inhibition of inflammatory processes and
alleviation of symptoms associated with the multiple sclerosis
disease state comprising administering a therapeutically effective
amount of an anti-CD18 integrin-antibody substance.
2. The method of claim 1 wherein said anti-CD18 antibody is mAb
60.3.
3. The method of claim 1 wherein said anti-CD18 antibody is mAb
23F2G.
4. The method of claim 1 wherein said anti-CD18 antibody is a
recombinant human antibody comprising a murine complimentarily
determining region of mAb 23F2G.
5. Hybridoma cell line 23F2G, A.T.C.C. HB11081.
6. Monoclonal antibody 23F2G produced by hybridoma cell line 23F2G,
A.T.C.C. HB 11081.
7. A method for inhibition of inflammatory processes and
alleviation of symptoms associated with the multiple sclerosis
disease state comprising administering a therapeutically effective
amount of monoclonal antibody which competes with mAb 60.3 for
binding to LFA-1.
Description
BACKGROUND OF THE INVENTION
[0001] The-present invention relates generally to methods for the
alleviation of symptoms associated with inflammatory disease
states, and more particularly to the inhibition of inflammatory
processes through administration of a pharmaceutically effective
amount of an antibody substance immunologically reactive with
molecules expressed on the surface of leukocytes.
[0002] Inflammation is a body process central to a number of
diseases and is the body's primary defense against infection. The
inflammatory process involves an orchestrated series of events
initiated in response to tissue damage. In all, cases, this
cellular damage ultimately leads to the influx of white blood cells
(leukocytes) to the site of injury. As leukocytes arrive at the
site of injury, they become metabolically activated and begin to
secrete specific proteins (mediators) that are generally of
defensive significance, for example, in the eradication of
bacteria.
[0003] If unregulated, the inflammatory state may persist as a
condition known as chronic inflammation. In this setting, the
mediators produced may amplify the inflammatory response and cause
damage to otherwise normal tissue. Depending upon the body site,
such tissue damage may result in chronic diseases such as
arthritis, multiple sclerosis, asthma, emphysema, ulcerative
colitis, and various autoimmune diseases.
[0004] Multiple sclerosis (MS) is a chronic disease characterized
by recurrent attacks of neurologic dysfunction due to lesions in
the central nervous system. These lesions, termed "plaques,"
represent areas of axonal demyelination which are the hallmark of
multiple sclerosis. The lesions contain inflammatory cells such as
lymphocytes, macrophages and neutrophils in areas where myelin is
being destroyed. The classic clinical features of multiple
sclerosis include impaired vision and weakness or paralysis of one
or more limbs. After a number of years, patients experience a slow,
steady deterioration of neurologic function. The disease course is
unpredictable and involves exacerbations and remissions in 75% of
patients. Although a few patients die within the first few years of
onset, the average duration of disease is greater than 30
years.
[0005] There are an estimated 250,000 cases of multiple sclerosis
in the United States, with approximately 10,000 new cases occurring
each year. The cause is unknown but epidemiology implicates
immunologic or infectious factors resulting in a chronic
inflammatory brain condition. Multiple sclerosis is a disease of
young adults, with 66% of cases occurring at ages 20-40; 60% of the
patients are women. Over one million physician visits occur
annually for multiple sclerosis in the United States alone;
however, there is currently no effective treatment-for multiple
sclerosis. Therapy is directed toward the reduction of the severity
of acute episodes, and prevention of relapses. In acute flare-ups,
steroids reduce severity and speed recovery. Experimental therapy
with, other immunosuppressive agents, such as cyclophosphamide, has
been tried, but with limited success.
[0006] A model for human multiple sclerosis is experimental
allergic encephalomyelitis (EAE), an acute inflammatory and
demyelinating disease of the central nervous system (CNS). [Rose,
et al., Clin. Immunol. Immunopathol., 59:1-15 (1991)]. For both EAE
and MS, there is considerable evidence that immunological and
inflammatory processes, contribute to the pathogenesis of the
disease. [Hauser, et al., Ann Neurol 13:418-425 (1983); Traugott,
et al., Cell. Immunol., 68:261-275 (1982); Rose, et al., Clin.
Immunol. Immunopathol., 45:405-423 (1987). This is supported by the
presence of perivascular mononuclear cellular infiltrates in
lesions and macrophage-dependent phagocytosis of myelin in the CNS
white matter. [Prineas, et al., Lab. Invest., 38:409-421 (1978);
Alvord, J C Koetsier, editor; Handbook of Clinical Neurol,
3(47):467-502, Koetsier (ed.); Amsterdam; Elsevier Science
Publishers BV (1985)]. The mechanisms by which these blood cells
first recognize the brain as a target organ and then traverse the
blood brain barrier are not well understood.
[0007] The migration of blood cells into extravascular sites of
inflammation involves a complex series of events including: i)
recognition of an intravascular chemotactic stimulus, ii) adherence
to endothelium, iii) diapedesis across the endothelium, and iv)
migration through subendothelial connective tissue. [Snyderman, et
al. Science, 213:830-837 (1981)]. Endothelial cells (EC) found on
lumenal surfaces of blood vessels are the first cells that
leukocytes encounter during migration from the blood to the
extravascular space. Molecules expressed by both the leukocytes and
by the endothcelial cells are important in regulating the adhesive
interaction between these two cell types.
[0008] One family of leukocyte receptors, variously designated
"leukocyte integrins," "leukointegrins," and "CD11/CD18 integrins,"
are involved in cell-cell and cell-protein interactions of all
leukocytes. [Hynes, Cell, 48:549-554 (1987); Beatty, et al., J.
Immunol., 131:2913-2918(1983)]. The CD11/CD18 antigen family
consists of three heterodimers, each containing a unique
.alpha.-chain (CD11a, CD11b, or CD11c), and a common .beta.-chain
(CD18). [Sanchez-Madrid, et al., J. Exp. Med., 158:1785-1803
(1983)]. The CD11a/CD18 integrin is referred to as LFA-1; the
CD11b/CD18 integrin is referred to as Mac-1; the CD11c/CD18
integrin is referred to as p 150,95.
[0009] Numerous murine hybridomas have been generated which produce
monoclonal antibodies of varying isotype to the common .beta. chain
(CD18) of the leukocyte integrins. These include: mAb 1B4 [IgG2a;
Wright, et al., Proc. Nat'l. Acad. Sd. USA, 80:5699-5703 (1983)];
mAb 60.3 [IgG2a; Beatty, et al., J. Immunol., 131:2913-2918
(1983)]; mAb TS1/18 [IgG1; Sanchetz-Madrid, supra]; mAb H52
(Hildreth, et al., Science 244:1075-1803 (1989)]; and ATCC T1B 218
[IgG2a Kappa; Springer, et al., J. Exp. Med., 158:586-602 (1983)].
Production of chimeric and humanized monoclonal antibodies against
human CD18 is referred to in Law, et al., European Patent
Application 440 351 A2, published Aug. 7, 1992.
[0010] Monoclonal antibodies directed against the common
.beta.-chain of leukocyte integrins completely inhibit adherence of
PMNCs to un-activated endothelial cells and certain matrix proteins
in vitro [Harlan, et al., Blood, 66:167-178 (1985); Zimmerman, et
al., J. Clin. Invest., 81:531-537 (1988); Bohnsack, et al., J. Exp.
Med., 171:1221-1237 (1990); Luscinskas, et al., J. Immunol.,
142:2257-2263 (1989)]. Moreover, the systemic administration of
anti-CD11/CD18 antibodies inhibits, tissue accumulation of PMNCS.
[Springer, et al., Nature, 346:425-434 (1990); Jutila, et al.
Transplantation, 48:727-731 (1989); Arfors, et al., Blood,
60:338-340 (1987); Price, et al., J. Immunol., 139:4174-4177
(1987); and, Doerschuk, et al., J. Immunol., 144:2327-2333
(1990)].
[0011] Arfors, et al., supra, studied the effect of the anti-CD18
antibody, mAb 60.3, on induced PMN accumulation in vivo and found
that both PMN accumulation and PMN-dependent plasma leakage were
abolished in the inflammatory skin lesions of rabbits treated with
mAb 60.3 prior to intra dermal injection with the chemotactic
factors fMLP, leukotriene (LTB.sub.4) and C5a. These chemotactic
agents cause a significant increase in albumin extravasation;
extravasation of PMNs at chemotaxin-injective sites is followed by
plasma leakage. Curiously, histamine-induced PMN-independent plasma
leakage was unaffected by pre-treatment with mAb 60.3.
[0012] Hernandez, et al., Am. J. Physiol. 253:H699 (1987)
investigated whether PMNs mediate the increase in microvascular
permeability produced by ischemia-reperfusion (I/R) by treating
cats with either saline, anti-neutrophil serum (ANS), or mAb 60.3.
The results indicated that both PMN depletion and prevention of PMN
adherence significantly attenuated the increase in microvascular
permeability induced by I/R and that prevention of neutrophil
adherence with mAb 60.3 afforded protection against I/R-induced
microvascular injury. See also, U.S. Pat. No. 4,797,277. Similarly,
Vedder, et al., J. Clin. Invest., 81:939 (1988) concluded that
increased leukocyte adhesiveness plays an important role in the
development of multiple organ injury and death after general
ischemia-reperfusion and that this injury may be significantly
reduced by blocking leukocyte adherence functions with the mAb
60.3. Their results, however, did not rule out the possibility that
other leukocytes or other leukocyte adherence functions may also be
involved, as CD18 is present on all leukocytes.
[0013] Doerschuk, et al., supra, reported on the differential
effects of mAb 60.3 on neutrophil (PMN) migration to either
pulmonary or peritoneal sites of inflammation in rabbits.
Inflammation in the abdominal wall of rabbits was induced following
implantation of polyvinyl sponges containing one of the following
stimuli: Streptococcus pneumoniae, endotoxin (E. coli),
hydrochloric acid or phorbol myristate acetate (PMA). Peritoneal
inflammation was induced by instillation of S. pneumoniae and
pulmonary inflammation was induced by instilling intrabronchially
either S. pneumoniae, hydrochloric acid, E. coli endotoxin, or PMA.
Rabbits were pre-treated with either intra venous injection of mAb
60.3 or saline 20 minutes prior to initiating inflammation. The
results demonstrated that mAb 60.3 inhibited PMN emigration in
response to S. pneumoniae, hydrochloric acid, E. coli endotoxin, or
PMA into both the abdominal wall and the peritoneal cavity.
However, in the same animals, mAb 60.3 did not alter PMN emigration
into the alveolar space, visceral pleura, or bronchial epithelium
in response to S. pneumoniae or hydrochloric acid. In contrast, mAb
60.3 markedly inhibited PMN emigration into the alveolar space in
response to E. coli endotoxin and abolished lung emigration in
response to PMA. The results demonstrated that there, are as yet
unidentified organ-specific and stimulus-specific differences in
PMN adherence mechanisms that could be due to differences in
response of pulmonary and systemic endothelium to identical stimuli
or, to the site-specific production of secondary mediators that
affect the PM1 or endothelial cell.
[0014] In addition to U.S. Pat. No. 4,797,277, which addresses
methods for reperfusion therapy with anti-leukocyte-integrin
antibody preparations, a number of quite generalized proposals for
therapeutic uses of such antibodies have recently been made. PCT WO
92/04034, published Mar. 19, 1992, generically proposes the use of
anti-CD18 antibodies in the treatment of endotoxin shock. PCT WO
92/03413, published Mar. 5, 1992, proposes use of CD18 peptide
fragments and-antibodies thereto in the treatment of a "disease".
European Patent Application No. 440 451, supra, illustrates
usefulness of recombinant humanized anti-CD18 antibodies in the
treatment of rabbit dermal inflammation but projects utility in
treatment of inflammation in "lung, central nervous system, kidney,
joints, endocardium pericardium, eyes, ears, skin, gastrointestinal
tract and urogenital systems." Although multiple sclerosisis
proposed as one of many disease states which "may be responsive to
recombinant human anti-CD18 antibody," no studies of the effect of
such antibodies on any chronic inflammatory disease states are
reported.
[0015] There thus continues to exist a need in the art for
therapeutic materials and regimens for the treatment of
inflammation associated with multiple sclerosis.
BRIEF SUMMARY OF THE INVENTION
[0016] The present invention provides novel and effective methods
for the treatment of inflammatory processes and the alleviation of
symptoms associated with the multiple sclerosis disease state
comprising administering a therapeutically effective amount of an
anti-CD18 and/or anti-LFA-1 antibody substance.
[0017] Antibody substances useful in practice of the present
invention include monoclonal and polyclonal antibodies, antibody
fragments, single chain antibodies, chimeric and/or CDR-grafted.
(including humanized) antibodies and the like which are
specifically immunoreactive with one or more epitopes presented by
the common .beta.-chain (CD18) of human leukocyte integrins.
Antibodies may be of any class or subclass including IgG, IgA, IgD,
IgE and/or IgM. Presently preferred is mouse monoclonal antibody
mAB 60.3 of the IgG2a isotype.
[0018] Also provided by the present invention are monoclonal
anti-human CD18 antibodies produced by hybridoma cell lines
designated 23F2G (A.T.C.C. HB 11081) 23I11B and 22F12C. These and
antibodies produced by hybridomas 22B3B and 22J4A are characterized
as competitive inhibitors of the binding of mAb 60.3 to human LFA-1
on Hut78 cells.
[0019] Other aspects and advantages of the present invention will
be apparent upon consideration of the enclosed detailed description
of an embodiment thereof, reference being had to the drawing
wherein:
[0020] FIGS. 1 and 2 present magnetic resonance images illustrating
results of practice of the invention in the Experimental Allergic
Encephalomyelitis (EAE) model system; and
[0021] FIGS. 3 and 4 represent histological results of practice of
the invention in the EAE model system.
DETAILED DESCRIPTION
[0022] The following detailed description illustrates practice of
the invention through use of the mouse monoclonal anti-CD18
antibody, mAb 60.3, to alleviate progression of Experimental
Allergic Encephalomyelitis (EAE) in macaque monkeys. Non-human
primates induced to develop EAE, an acute inflammatory disease of
the central nervous system and a model of the human disease MS,
were chosen because of the many immunological and
neurophysiological similarities EAE shares with MS (Rose, et al.,
supra.) and because the CD18 antigen recognized by mAb 60.3 is
present in high density on monkey peripheral blood leukocytes
(Rose, et al., Clin. Immunol. Immunopathol, 44:93-106 (1987). In
vivo administration of the antibody at the onset of clinical
disease significantly prolonged survival of five of six macaques
and in some cases completely reversed clinical appearance of
disease.
[0023] Example 1 relates to the induction of EAE in monkeys.
Example 2 describes treatment by mAb 60.3 antibody infusion and
Example 3 relates to the evaluation of disease progression as
monitored by a combination of clinical evaluation, blood analysis,
and magnetic resonance imaging, including immunocytochemical
staining of frozen tissue sections obtained post-mortem. Example 4
describes the preparation of hybridoma cell lines producing
anti-human CD18 antibodies and Example 5 relates use of monoclonal
antibodies from hybridoma 23F2G in treatment of EAE.
EXAMPLE 1
[0024] Induction of EAE
[0025] Eighteen male monkeys Macaca fasciculatis weighing 2-3 kg
were sensitized to myelin basic protein (BP) by the intra dermal
injection of 0.1 ml of an emulsion containing 5.0 mg of monkey BP
and 0.5 mg of heat-killed M. tuberculosis. Each animal was obtained
and housed by the Regional Primate Research Center at the
University of Washington, Seattle. The Primate Center conforms to
the National Institutes of Health Guide for the Care and Use of
Laboratory Animals. Ten days after sensitization to BP, each animal
was outfitted with a femoral catheter and tether to facilitate
blood sampling and the administration of treatments or anesthetics
for magnetic resonance imaging (MRI). The animals were maintained
on intravenous heparin (18 units/hour) to prevent blood clots from
forming in the intra venous lines.
[0026] The animals were randomly admitted to specific treatment
groups before they were sensitized to BP. This was done to schedule
MRI and to prevent the severity of clinical signs at the onset from
influencing the choice of therapy. Primate Center personnel,
responsible for deciding when animals should be treated or
sacrificed, were blinded regarding the treatment history of each
animal. Table 1 below provides a statement of criteria for
determination of clinical signs and grades of EAE in the
animals.
1TABLE 1 CLINICAL SIGNS AND GRADES OF EAE IN MACAQUES Score Signs ?
Prodromal signs, including weight loss, anorexia, yawning, slow
response to stimuli, irritability .+-. Mild neurologic signs,
including "headache" (acute distress), "apathy" (indifference),
hypokinesia, drooling, clumsiness in using limbs, nystagmus +
Moderate neurologic signs, including akinesia, blindness, ataxia,
tremor, ptosis, seizures, paresis, incontinence ++ Severe
neurologic signs including somnolence, paraplegia, hemiplegia, or
quadriplegia +++ Moribund state with semicoma, coma, decerebration
or decortication D Death
EXAMPLE 2
[0027] Treatment By Antibody Infusion
[0028] At the onset of definite clinical signs (.+-. to +) six EAE
animals received a bolus injection of mAb 60.3 (2 mg/kg) and
dexamethasone (4 mg/kg). Dexamethasone was administered to reduce
the incidence of severe acute EAE which is rapidly fatal within 48
hours unless treated immediately. Sever acute EAE occurs in
approximately 25% of animals induced to develop EAE and is caused
by edema in areas involving or impinging upon the brain stem or
cerebral cortex. Following injection of the bolus, these six
animals were treated by continuous infusion of mAb 60.3 (2
mg/kg/day) for seven days. Over the seven day treatment period, the
dose of dexamethasone, which started at 4 mg/kg, was halved every
two days until only 1 mg had been in effect for three days. Six
control EAE animals were treated only with dexamethasone, following
the same protocol outlined above, and six additional controls were
treated with continuous infusion of saline.
[0029] Animals that improved and/or stabilized as a result of
therapy were sacrificed six weeks after the onset of clinical
signs. Animals that experienced a clinical relapse (clinical signs
.gtoreq.++) some time after the cessation of treatment and before
six weeks had elapsed, were sacrificed shortly after the onset of
the relapse. Animals that did not respond well to therapy and
continued to deteriorate clinically (clinical signs .gtoreq.++)
were sacrificed without completing the treatment program. The
statistical significance of differences in survival times in each
treatment group was determined by two-tailed Student t
analysis.
EXAMPLE 3
[0030] Evaluation of Disease Progression
[0031] Disease progression was monitored by a combination of
clinical evaluation, blood analysis and magnetic resonance imaging.
Clinical evaluations were performed twice daily.
[0032] As indicated in Table 2 below, all of the animals in this
study developed clinical signs of EAE 13-26 days after
sensitization to myelin basic protein.
[0033] In Table 2, listing of animal numbers in boldface identified
animals scanned by MRI; the Onset/Grade column reflects the day of
onset after sensitization and the severity of clinical signs (see
Table 1) at onset; the Survival/Grade column reflects days of
survival post-onset and severity of clinical signs at time of
sacrifice.
2TABLE 2 THERAPEUTIC RESULTS Animals Onset/Grade Treatment
Survival/Grade 89070 17 days/+ 60.3 + dex 2 days/D 89075 21 days/+
60.3 + dex 42 days/.+-. 89080 21 days/+ 60.3 + dex 42 days/.+-.
89069 18 days/.+-. 60.3 + dex 42 days/.+-. 89071 18 days/.+-. 60.3
+ dex 21 days/++ 89074 22 days/+ 60.3 + dex 13 days/+ 87143 21
days/.+-. dex alone 17 days/++ 87125 24 days/.+-. dex alone 1
day/++ 87209 21 days/+ dex alone 2 days/++ 89077 16 days/.+-. dex
alone 1 day/D 87200 22 days/+ dex alone 13 days/++ 87069 26
days/.+-. dex alone 9 days/++ 84228 13 days/+ saline 5 days/D 84218
19 days/+ saline 3 days/D 84291 25 days/+ saline 1 day/D 86140 26
days/+ saline 3 days/D 86187 19 days/+ saline 1 day/++ 86209 15
days/+ saline 1 day/++
[0034] As indicated in Table 2, all eighteen animals developed
clinical signs of EAE which ranged in severity from .+-. to + at
the onset of disease. Six of the eighteen EAE animals (33%) died of
severe acute EAE 1-5 days after onset. Four of the six had been
treated with saline, one with dexamethasone, and one with a
combination of dexamethasone and mAb 60.3. The remaining twelve
animals were, sacrificed at various times after, onset either
because they had deteriorated clinically or because they had
completed the six-week post-treatment protocol.
[0035] Out of six mAb-treated animals, five either improved or
stabilized with continuing, but not worsening, clinical signs.
Animal 89071 improved, but suffered an acute relapse two weeks
after the cessation of treatment and was sacrificed at 21 days.
Animal 89074 was sacrificed at 13 days, one week after the
cessation of treatment, to investigate an abnormal lipid signal
detected by magnetic resonance spectroscopy (MRS), a technique
being developed for studies of demyelination in the nonhuman
primate [Richards et al., New York, Soc. Mag. Reson. Med. 2: p.1043
(1990)]. This animal had improved as a result of therapy and was
clinically stable at the time of sacrifice. Animals 89075, 89080,
and 89069 were all clinically stable six weeks after onset. The
mean survival time of mAb 60.3-treated animals was 30 days. Control
animals treated with saline (excluding animal 87220) survived 1-5
days after onset, with a mean survival time of 3 days. Animals
treated with dexamethasone survived 1-17 days afteronset, with a
mean survival time of 7 days, 3 of 6 dying of their disease 1-2
days after onset, and 3 of 6 surviving 9,13 and 17 days,
respectively, until they were sacrificed due to deteriorating
clinical signs (.gtoreq.++).
[0036] Magnetic Resonance Imaging
[0037] MRI was used to map the anatomic distribution of the lesions
in all six of the mAb-treated EAE animals, two
dexamethasone-treated animals, and two saline-treated animals.
Conventional spin-warp imaging was performed using a General
Electric CSI-II. NMR imager/spectrometer (2 Tesla magnet) as
described in Rose et al., Biomed. and Pharmacother., 43:347-353
(1989). Images were acquired once a week before the onset of
clinical signs and twice a week after the onset of clinical signs.
The animals were anesthetized with ketamine and positioned in the
magnet so that the center of the brain was at magnet iso-center.
Coronal T2-weighted images (TE=80 msec, TR=3000 msec) were used to
find abnormal and normal brain regions. Lesions were detected in
60% of the scanned animals on or before the onset of clinical
signs. Most lesions were localized in the cerebral white, matter,
optic tracts, visual radiations or brainstem. The lesions were
identified as areas of increased brightness (white) in a
T2-weighted MR image. Of the four control animals that were
scanned, only one survived long enough to obtain post-treatment
scans. In this dexamethasone-treated animal (87069) a brainstem
lesion, detected one day before the onset of clinical signs,
progressed in size and intensity until the animal was sacrificed
nine days later as shown in FIG. 1. Plate A represents a "normal"
brain scan, 21 days after sensitization; Plate B reveals detection
of a brainstem lesion two days after onset of clinical signs i.e.,
28 days post sensitization: Plates C and D illustrate gradual
progression of the brainstem lesion 35 and 38 days after
sensitization, respectively.
[0038] Following treatment with mAb 60.3, the MRI lesions detected
in 5 of 6 animals diminished in both size and intensity. Three
animals (89075, 89071, and 89074) had evidence of brainstem lesions
at the onset of clinical signs. Although such lesions are
frequently fatal if untreated [Shaw et al., Ann. Neurol. 24:738-748
(1988)], these lesions disappeared in all three animals following
treatment with mAb 60.3. FIG. 2 demonstrates the progression of the
brainstem lesion in animal 89071. In the Figure, Plate A
illustrates a normal brain scan 15 days post sensitization; Plate B
shows a brainstem lesion at the onset of clinical signs, i.e., 18
days after sensitization; Plates C through F illustrate gradual
resolution of the brainstem lesion through antibody treatment as
monitored at 22, 25, 29 and 32 days post sensitization; Plate G
illustrates reappearance of a lesion in the same region which was
accompanied by clinical relapse 39 days post sensitization and 14
days after cessation of treatment. Enlarging lesions are identified
as prominent areas of increased intensity (white) on the scans. As
noted above, two weeks after the treatment was discontinued, 89071
clinically relapsed and MR images demonstrated a larger, more
intense lesion in the brainstem. The relapse was not treated and
the animal was sacrificed.
[0039] Animal 89080 developed a major hemorrhagic lesion in the
lateral geniculate nucleus and striatum. Hemorrhagic lesions are
usually fatal and usually do not respond to treatment (Shaw et al.,
supra) but this lesion almost completely disappeared following
administration of mAb 60.3. Animal 89069 had the mildest disease of
any of the animals, as determined by MRI, with several small, but
intense, sub-cortical white matter lesions that disappeared
following treatment.
[0040] Two of the longest surviving animals (89080 and 89069)
developed a chronic disease characterized by the appearance of new,
smaller, MRI-detectable lesions 25 and 22 days, respectively, after
the cessation of treatment. These secondary lesions were not as
severe as the first lesions and were accompanied by milder clinical
signs, which were not treated. Animal 89074 was sacrificed 13 days
after the onset of disease, to investigate an abnormal lipid signal
detected by MRS. At the time of sacrifice, six days after stopping
treatment, his initial lesions were gone, but a new smaller
subclinical lesion had appeared on the opposite side of the brain.
No new lesions were ever detected in animal 89075 after cessation
of treatment.
[0041] Cryostat sections of cerebellum or cerebrum were stained
with hematoxylin and eosin and by the immunoperoxidase technique in
conjunction with mAbs to human leukocyte membrane antigens in
order: to analyze the composition of the cellular infiltrates.
Immunocytochemical staining with mAb 60.3, which recognizes a CD18
determinant common to all leukocytes, was used to confirm the
presence of leukocytes in EAE lesions. A tissue section obtained
from the brain of animal 89070 and stained with mAb 60.3 is shown
in FIG. 3. In Panel A, at 390.times. magnification and stained with
rabbit anti-mouse IgG, murine IgG coated cells are revealed in the
lumen of blood vessels but not in brain tissue; and, in Panel B, at
390.times. magnification and stained with mAb 60.3, massive
infiltration of PMBCs is seen in the exudate and extending out into
adjacent white matter.
[0042] Animal 89070 was the only mAb-treated animal to die before
completion of treatment. Brain tissue taken from this animal was
stained with a goat anti-mouse Ig antibody in order to determine
whether the treatment mAb had entered the CNS before the animal
died. MAb 60.3 reacts with both leukocytes and brain microglia. If
this mAb had crossed the blood-brain-barrier during the treatment
period, it was expected that a staining pattern similar to that
shown in FIG. 3, Panel B might be obtained. Alternatively, if the
treatment mAb entered the CNS, bound only to blood leukocytes, the
microglia would not have been stained. Instead, murine Ig-coated
cells were only detectable in the vessel lumen, and not at all in
the perivascular spaces. The absence of any positively stained
leukocytes in the lesion stained with goat anti-mouse Ig, suggests
that the animal died of an inflammatory condition existing before
treatment was initiated and that the treatment antibody was given
too late to be effective.
[0043] Effect of Treatments on Leukocyte Counts
[0044] The hematologic hallmarks of untreated EAE are a progressive
leukocytosis and lymphopenia prior to the onset of clinical signs.
The leukocytosis can represent as much as a four-fold increase in
the number of circulating PMNC and no significant changes in the
frequency or absolute numbers of monocytes, eosinophils, or
basophils. Following successful treatments of EAE, the absolute
numbers of lymphocytes and PMNC return, fairly soon after the
initiation of treatment, to pre-sensitization levels. Thus,
continuous monitoring of blood leukocytes provides a useful measure
of disease progression which complements the clinical evaluation
and magnetic resonance imaging.
[0045] Blood was drawn on a weekly basis until the onset of
clinical disease and then more frequently. One-half ml was sent to
the hematology laboratory for WBC and differential counts. The
results are set out in Table 3 below providing white blood cell
number (.times.10.sup.3) per mm.sup.3 whole blood. "NA" indicates
that data was not available. A dashed line indicates that the
animal was sacrificed at this time.
3TABLE 3 WBC ANALYSES OF MAB-TREATED EAE ANIMALS ANIMALS: 89070
89075 89080 89069 89071 89074 Day Presens 8.1 8.7 14.0 8.0 7.8 13.3
Onset 14.6 16.2 16.4 9.3 12.3 18.1 Day 2 -- 27.1 33.2 22.2 22.6
49.9 Day 4 49.6 53.1 31.6 41.4 72.0 Day 7 61.2 61.7 16.4 90.7 88.6
Day 10 20.7 18.2 20.0 12.8 14.6 Day 14 33.3 9.6 8.2 18.4 13.4 Day
21 8.7 12.9 8.9 37.1 -- Day 28 8.7 19.8 6.7 -- Day 35 7.9 16.4 6.9
Day 42 NA NA 8.6 WBC ANALYSIS OF CONTROL EAE ANIMALS DEXAMETHASONE
CONTROL Animal: 87143 87125 87209 89077 87200 87069 Presens 4.7 6.1
6.7 13.5 8.9 14.1 Onset 9.6 9.0 14.6 32.2 12.4 16.3 Day 2 3.1 -- --
-- 5.4 NA Day 4 3.7 3.6 NA Day 7 3.8 7.0 NA Day 10 8.7 10.7 -- Day
14 7.9 -- Day 21 -- SALINE CONTROL Animal: 84228 84218 84291 86140
86187 86209 Presens 11.0 7.0 13.3 9.7 8.3 9.7 Onset 14.3 9.0 21.9
23.6 14.2 13.5 Day 2 20.2 8.4 22.5 29.0 -- -- Day 4 30.1 8.0 -- --
Day 7 -- --
[0046] In animals treated with mAb 60.3 and dexamethasone, a
delayed resolution of the leukocytosis was observed as compared to
animals treated with dexamethasone alone as revealed in Table 3. In
two of the dexamethasone-treated animals that survived 9 days or
longer, the white blood cell counts dropped to pre-sensitization
levels two days after the initiation of treatment. In mAb
60.3-treated animals, a pronounced leukocytosis (4-12 fold above
presensitization values) was observed which lasted for the entire
treatment period. In these animals, the WBC returned to
presensitization levels 3-7 days only after the treatment period
had ended. Transient episodes of lymphopenia and leukocytosis
continued to be observed in the surviving animals, and could be
correlated in some cases with episodes of clinical relapse.
[0047] Reaction of the treatment mAb with blood leukocytes was
confirmed by staining cells with anti-murine Kappa chain mAb
(Becton-Dickinson, San Jose, Calif.). Flow cytometric analysis was
performed with a FACSCAN Analyzer (Becton Dickinson, San Jose,
Calif.). Forward and right-angle scatter gates were set on
lymphocytes or large polymorphonuclear granulocytes for analysis of
staining patterns. The results for lymphocytes are set out in Table
4 below.
4TABLE 4 % MURINE Ig-COATED CELLS ANIMALS: Day 89070 89075 89080
89069 89071 89074 Presens 0 0 0 0 0 0 Onset 0 0 0 0 0 0 Day 2 --
96.6 97.7 94.2 96.7 98.2 Day 4 93.8 95.2 91.2 97.0 49.8 Day 7 95.2
99.1 94.9 95.4 27.2 Day 10 0 2.6 11.2 19.6 1.4
[0048] The dose of mAb given: to the animals was in all instances
sufficient to coat >95% of circulating white blood cells and, as
indicated in Table 5, to maintain detectable levels of free
antibody in circulation. Data in the Table represent micrograms of
circulating, murine IgG per mm.sup.3 whole blood.
5TABLE 5 CIRCULATING MURINE IgG ANIMALS: Day 89070 89075 89080
89069 89071 89074 Presens 0 0 0 0 0 0 Onset 0 0 0 0 0 0 Day 2 -- 65
70 15 28 34 Day 4 80 70 65 25 15 Day 7 90 58 4 7 28 Day 10 0 0 0 0
0
[0049] After 7 days, the injected mAb was no longer detectable in
the circulation. The disappearance of the murine Ig coincided with
the appearance of monkey anti-murine Ig antibodies in the
circulation of each animal.
[0050] Neuropathology
[0051] To confirm the diagnosis of EAE, the-brains and spinal cords
of each BP-sensitized animal were examined postmortem. The animals
were euthanized by Nembutal overdose and brains and spinal cords
were removed and dissected so that fresh-frozen samples could be
obtained before the larger blocks were fixed in 10% neutral
formalin. Representative blocks of tissue were embedded in paraffin
and sections were stained with hematoxylin and eosin,
gallocyanin-Darrow red, luxol fast blue (LFB) combined with
periodic acid Schiff (PAS) and hematoxylin and with Holmes' axon
stain alone or combined with LFB to differentiate demyelination
from necrosis. Frozen sections of mirror-image blocks were stained
with Oil Red O (ORO) [Kil, Stain Technology, 47:271, (1972)] to
identify lipid-filled macrophages.
[0052] All eighteen EAE animals were evaluated histologically
postmortem for evidence of EAE. All had microscopic evidence of
either mild to severe acute EAE or hyperacute EAE that generally
correlated with the severity and duration of the disease. All but 2
of the 12 control animals showed either predominantly hyperacute
lesions characterized by hemorrhages, subtotal necrosis,
demyelination, and diffuse massive infiltrations of neutrophils
into the CNS parenchyma; or severe acute perivascular lesions which
were more compact, more confined to the perivascular spaces than
the hyperacute lesions and composed predominantly of lymphocytes
and macrophages, but some neutrophils, and with abundant
demyelination as well as some axonal debris. The two exceptional
control monkeys, with EAE of 13-17 days duration, had a mixture of
lesions ranging from mild to severe demyelinating lesions, composed
predominantly of lymphocytes and macrophages with abundant myelin
debris and little or no axonal reaction. All but one of the
mAb-treated monkeys had a combination of: (a) older well developed
demyelinated plaques with abundant myelin debris, sudanophilic
lipid and little or no axonal reaction or lymphocytic infiltrate;
and (b) more recent lesions corresponding to the terminal untreated
relapses, which were compact, confined to perivascular spaces, with
varying proportions of lymphocytes, monocytes, or neutrophils
(consistent with diagnoses of mild to severe acute EAE) and some
myelin debris. These two types of lesions, found in the same animal
(#89069), are shown in FIG. 4.
[0053] In FIG. 4, Panel A, at two-fold magnification, numerous
inflammatory (area 1) and/or demyelinating (area 2) lesions can be
ascertained in the white matter; in Panel B, at 325.times.
magnification, area 2 of Panel A is revealed as being composed
predominantly of macrophages scattered diffusely through the tissue
and about blood vessels, with the small black granules constituting
PAS positive glyco-lipoproteins; and in Panels C and D, at
130.times. and 325.times. magnification, respectively, area 1 of
Panel A is revealed as being composed predominantly of PMNCs and no
PAS positive insoluble glycoproteins.
[0054] Summary of Results
[0055] Monitoring disease progression by a combination of clinical
evaluation, blood cell fluctuations, and magnetic resonance imaging
(MRI) revealed the following. Antibody treated animals survived
significantly longer than either dexamethasone- or saline-treated
animals (p<0.02; p<0.001; respectively). The effectiveness of
this therapy is further confirmed by visualization by magnetic
resonance imaging (MRI). The almost complete disappearance of major
lesions in the brainstems of three different animals was
unprecedented as brainstem lesions are rapidly fatal in untreated
animals. The disappearance of MRI-detectable lesions following
administration of mAb 60.3 demonstrates a potent effect on the
inflammatory reactions producing edema in the brain lesions. MAb
60.3 is known to inhibit plasma leakage in vivo [Arfors et al,
Blood, 60: 338-340, (1987)], probably the most important factor in
improvement in MRI-detectable lesions, as changes in the tissue
water characteristics can be visualized by changes in image
intensity on the MRI scans.
[0056] The severity of clinical signs at the onset of EAE was not
uniform because individual monkeys responded differently to
sensitization with myelin BP. Nevertheless, there was no
correlation between the severity of clinical signs at the onset of
EAE and the ability of animals to respond to treatment. Of the six
mAB-treated animals, four were + at the onset of clinical signs and
2 were .+-.. Of the twelve control animals (six
dexamethasone-treated and six saline-treated), 8 were + and 4 were
.+-., essentially the same distribution as the mAb-treated group.
No correlation was found between the severity of clinical signs at
the onset and the potentially stressful procedure of MRI
examination under general anesthesia. Of the ten animals scanned by
MRI, six were + at the onset of clinical disease and four were
.+-.. One might have expected the additional handling involved in
scanning the animals, as well as the frequent administration of
anesthetics, to result in a generalized suppression of the immune
response. [Wall et al., Lab Animals, 19:138-144 (1985), Thomas et
al., Clin. Exp. Immunol., 47:457-466 (1982)]. However, the results
do not support this hypothesis.
[0057] The dexamethasone-treated animals did have longer mean
survival time (7 days) than the saline-treated controls (3 days),
but the difference was not statistically significant (p<0.1).
Thus, although dexamethasone slowed down the disease process, it
had little apparent effect on the final outcome of the disease.
Co-administration of mAb 60.3, on the other hand, significantly
prolonged survival and in some cases completely reversed the
clinical and MRI appearances of EAE.
EXAMPLE 4
[0058] Six to twelve week old Balb/c mice (Charles River
Biotechnical Services, Inc., Wilmington, Mass., IACUC #901103) were
immunized with human T cell line Hut 78 to generate anti-CD18
antibodies. For each of two hybridoma-generating fusions
(designated fusions 22 and 23) two Balb/c mice were bled
retro-orbitally for the collection of pre-immune serum on day 0. On
day 2,each animal received a total of 5.times.10.sup.6 Hut 78 cells
in 0.2 ml sterile PBS intravenously. The mice were then immunized
at two week intervals for six weeks and then at monthly intervals
for three months. The final monthly boost was of glutaraldehyde
fixed Hut 78 cells. Cells were fixed by mixing equal volumes of
cells (2.times.10.sup.7) and glutaraldehyde (0.1% so that the final
concentration of the fixative was equal to 0.05%. The mixture was
incubated with swirling for thirty seconds and an equal volume of
0.2M glycine (in PBS) was added to block the reaction. The reaction
mixture was washed once with 0.2M glycine (in PBS) and twice with
PBS. Cells were resuspended in: PBS at a concentration of
5.times.10.sup.6 cells per 0.2 ml PBS. Immune serum was collected
via retro-orbital bleeding on day 56 and tested by FACS to
determine its reactivity with the immunogen (Hut 78 cells). Three
days prior to the projected fusion date, the animals were immunized
by intravenous injection with a final dose of 5.times.10.sup.6
glutaraldehyde fixed cells.
[0059] For each fusion, the spleen from the mouse displaying the
highest immune serum titer against the immunogen was removed
sterilely. A single-cell suspension was formed by grinding the
spleen between the frosted ends of two glass microscope slides
submerged in serum free RPMI 1640, supplemented with 2 mM
L-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100
.mu.g/ml streptomycin (RPMI)(Gibco, Canada). The cell suspension
was filtered through sterile 70-mesh Nitex cell strainer (Becton
Dickinson, Parsippany, N.J.), and washed twice by centrifuging at
200 g for 5 minutes and resuspending the pellet in 20 ml serum free
RPMI. Thymocytes taken from 3 naive Balb/c mice were prepared in a
similar manner.
[0060] NS-1 myeloma cells, kept in log phase in RPMI with 11% fetal
bovine serum (FBS)(Hyclone Laboratories, Inc., Logan, Utah.) for
three days prior to fusion, were centrifuged at 200 g for 5
minutes, and the pellet was washed twice as described in the
foregoing paragraph. After washing, each cell suspension brought to
a final volume of 10 ml in serum free RPMI, and 10 .mu.l was
diluted 1:100. 20 .mu.l of each dilution was removed, mixed with 20
.mu.l 0.4% trypan blue stain in 0.85% saline (Gibco), loaded onto a
hemacytometer (Baxter Healthcare Corp., Deerfield, Ill.) and
counted.
[0061] For each fusion, 2.times.10.sup.8 spleen cells were combined
with 4.times.10.sup.7 NS-1 cells, centrifuged and the supernatant
was aspirated the cell pellet was dislodged by tapping the tube and
2 ml of 37.degree. C. PEG 1500 (50% in 75 mM Hepes, pH
8.0)(Boehringer Mannheim) was added with stirring over the course
of 1 minute, followed by adding 14 ml of serum free RPMI over 7
minutes. An additional 16 ml RPMI was added and the cells were
centrifuged at 200 g for 10 minutes. After discarding the
supernatant, the pellet was resuspended in 200 ml RPMI containing
15% FBS, 100 .mu.M sodium hypoxanthine, 0.4 .mu.M aminopterin, 16
.mu.M thymidine (HAT) (Gibco), 25 units/ml IL-6 (Boehringer
Mannheim) and 1.5.times.10.sup.6 thymocytes/ml. The suspension was
dispensed into ten 96-well flat bottom tissue culture plates
(Corning, United Kingdom) at 200 .mu.l/well. Cells in plates were
fed on days 2, 4 and 6 days post fusion by aspirating approximately
100 .mu.l/well plating medium described above except containing 10
units/ml IL-6 and lacking thymocytes.
[0062] On day 10 culture supernatants were taken from each well,
pooled by column or row and analyzed for the presence of an
antibody capable of competing with an FITC-60.3 conjugate (prepared
according to Goding, in "Monoclonal Antibodies: Principles and
Practice," 2d Ed., p. 241-280, Academic Press, New York, 1986) for
binding to Hut78 cells. The competition assay was performed as
follows. Twenty-five .mu.l of a cell suspension containing
10.times.10.sup.6 Hut78 cells/ml was combined with 25 .mu.l of a
substantial dose of FITC-60.3 (.times.2560.congruent.0.8 .mu.g/ml)
and 50 ml .mu.l of fusion supernatant and incubated for 30 min at
4.degree. C., washed by adding culture media to the cell mixture
and centrifuging at 18000 rpm for three minutes. The supernatant
was removed by aspiration and the pellet was washed twice with flow
cytometric analysis (FACS) medium containing RPMI 2% PBS and 0.1%
NaN.sub.3. The reaction mixture was fixed by addition of 1%
paraformaldehyde (pH 7.2 in PBS) and transferred to polystyrene
tubes for FACS using a Becton Dickenson FACscan analyzer. Of the
960 hybridomas generated in fusion 22, the supernatant of hybridoma
No. 22F12C inhibited binding of FITC-60.3 by about 90%;
supernatants from hybridoma Nos. 22J4A and 22B3B inhibited binding
by about 40% and 10% respectively. Two hybridomas from fusion 23,
23F2G and 23I11B, produced antibodies which inhibited FITC-60.3
binding by .gtoreq.95%. These five hybridomas were cloned.
Hybridoma cell line 23F2G was deposited with the American Type
Culture Collection on Jun. 30, 1992 and accorded accession number
A.T.C.C. HB 11081. The isotype of the antibody produced by
hybridoma cell line 23F2G (referred to herein as mAb 23F2G) was
determined to be IgG2a. Hybridomas 22F12C and 23I11B also produced
antibodies of the IgG2a isotype, while hybridomas 22B3B and 22J4A
produced IgG1 antibodies.
[0063] Antibodies from hybridomas 22F12C, 23F2G and 23I11B were all
found, in varying degrees, to: (1) block adhesion of human T cells
to activated HUVEC monolayers; (2) block aggregation of 13-acetate
(PMA) activated Con A blast cells phorbol 12-myristate; and (3)
induce demargination of white blood cells in rabbits. The ability
of the IgG1 antibodies produced by hybridomas 22B3B and 22J4A to
block adhesion is as yet undetermined; mAb 22J4A was able to block
aggregation, but mAb 22B3B was not; both antibodies induced
demargination, but to a lesser degree than the others or mAb 60.3.
Based on the screening results noted above, it appears likely that,
notwithstanding the ability to compete for binding of mAb 60.3 to
LPA-1 on Hut 78 cells, mAb 22B3B and 22J4A are specific for an
epitope present on the CD11a component of LFA-1 or an epitope
associated with the CD11a/CD18 heterodimer rather than with an
epitope of CD18.
[0064] A comparison was made between the amino acid sequence of the
variable region of mAb 60.3 presented by L. Harris at San
Francisco, Calif. on May 26-27, 1992 in a poster presented at a
conference entitled "Cell Adhesion Molecules In Inflammation"
organized by International Business Communications (South Natick,
Mass.) and the amino acid sequence of the variable region of mAb
23F2G. Gross differences were found, despite the fact that 23F2G
competed with 60.3 for binding to Hut78 cells. While these
differences are suggestive of the possibility that the antibodies
bind adjacent, different epitopes on CD18, confirmation of this
possibility will have to await definitive epitope-mapping
studies.
EXAMPLE 5
[0065] Hybridoma 23F2G was amplified by the ascites method and
purified by affinity chromatography on Protein A under pyrogen-free
conditions. From 25ml of ascites, 120 mg of antibody preparation
was isolated. This preparation contained only about 4 endotoxin
units per mg.
[0066] Two Macaca fascicularis monkeys were then involved in a
study to preliminarily determine the ability of mAb ?23F2G to
alleviate EAB symptoms. The EAE induction procedures were
essentially as in Example 1. Two animals, Nos. 89161 and 89186,
sensitized to 1.0 mg of purified monkey basic protein and developed
hyperacute hemorrhagic EAE 18 and 20 days post sensitization,
respectively. Animal 89161 died within 12 hours of onset and before
treatment with dexamethasone could be initiated. Animal 89186 was
treated for a week with both dexamethasone (3 days at 4 mg/kg, 2
days at 2 mg/kg and 2. days at 1 mg/kg) and mAb 23F2G (7 days at 2
mg/kg, i.v.). The animal improved dramatically with complete
resolution of paralysis three days after initiation of treatment.
Lesions detected by MRI resolved four days after cessation of
treatment and the animal improved dramatically with complete
resolution of paralysis. Eight days following cessation of
treatment, the animal relapsed and was sacrificed. Hematologic
analysis revealed that mAb 23F2G caused steady demargination of
leukocytes which peaked on the last day of treatment. The white
blood cell count rose from 22.7.times.10.sup.3 on the day of
clinical onset to 82.times.10.sup.3/mm.sup.3 on the last day of
treatment. The antibody showed saturation of blood lymphocytes at
the dose administered.
[0067] The foregoing examples describe a method for treatment for
inhibiting inflammatory processes and alleviating symptoms
associated with inflammatory disease states by administering a
reagent, such as an antibody, that blocks leukocyte adhesion and
migration to inflammatory sites. While the present invention has
been described in terms of specific methods and compositions, it is
understood that variations and modifications wit occur to those
skilled in the art upon consideration of the description of the
present invention. As one example, while two anti-CD18 mAbs, 60.3
and 23F2G, have been demonstrated to be useful in practice of
methods of the invention, it is expected that any antibody which
competes with mAb 60.3 and/or with 23F2G for binding to LFA-1 will
also be useful, whether or not such antibody recognizes on epitope
present on CD18. As another example, while mAb 23F2G is a mouse
monoclonal antibody, recombinant human antibody preparations
comprising the murine complimentarily determining regions of the
antibody are being prepared and are expected to be equally suitable
for practice of methods of the invention. Accordingly, it is
intended in the appended claims to cover all such equivalent
variations which come within the scope of the invention as
claimed.
* * * * *