Note: Descriptions are shown in the official language in which they were submitted.
CA 02226962 1998-02-16
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TITLE
Use of binding agents to CD47 and its ligands in the treatment or the
prophylaxis of various inflanvnatory, autoimmune and allergic diseases and in
the
treatment of graft rejection.
FIELD OF THE INVENTION
The present invention relates to new uses of binding agents to CD47 antigen
Zo or its ligands, and more particularly to monoclonal antibodies specific to
the CD47
or thrombospondin, in the treatment or prophylaxis of various inflammatory,
autoimmune and allergic diseases as well as in treatment of tumor metastasis,
cachexia and graft rejection.
15 BACKGROUND OF THE INVENTION
Integrin superfamily of adhesive receptors are transmembrane heterodimeric
molecules which function in cell-matrix and cell-cell adhesion (4, 5). The
CD47 Ag,
a surface glycoprotein of ~50 KD lvlW is physically and ftmctionally
associated
2 o wide X33 integrin mainly av(33 (the vitronectin receptor) on a variety of
cell types (6,
7). lntegrin av(33 is a highly promiscuous receptor recognizing Arg-Gly-Asp
(RGD)
in a wide variety of proteins as well as being expressed by many cell types
including
endothelial cells, osteoclasts, monocytes, activated lymphoid cells, platelet,
fibroblasts and malignant cells (8, 9). The av(33 integrin plays a role in
diverse
25 biologic processes such as cell migration and differentiation, himor cell
invasion,
angiogenesis, bone resorption and immune response (9-l2). Taken together, the
CA 02226962 1998-02-16
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role of av~33 in cell directed mobility underlies the importance of this
molecule in
development, wound repair, cancer and inflammation.
The cDNA sequence of CD47 Ag predicts a multispanning membrane protein
with 5 transmembrane domains; the large extracellular N-terminal domain is
homologous to members of LgV superfamily and harbours several potential N-
glycosylation sites ( 13, I4). It appears that CD47 Ag affects both ligand
affinity and
signal transduction of (33 integl-ins (6, 15). Indeed, mAb directed against
CD47 Ag
inhibited ligand binding to av(33 receptor and blocked activation of PMN
to phagocytes, respiratory burst, chemotaxis as well as stimulation of Ca+2
entry in
endothelial cells in response to RGD containing proteins. Recent studies have
shdwn that CD47 Ag is also involved in transendothelial and transepithelial
migration of neutrophils ( I G, 17).
Although CD47 does not bind to vitronectin, its natural ligand was recently
identified as being thrombospondin (TSI) ( 18), one of the several ligands of
av(33
to which it binds via RGD-containing sequence. TSI interacts with CD47 through
its cell binding domain (non RGD sequence). Both anti-TSI and anti-CD47 mAbs
partially inhibited TSI-stimulated Ca' 2 entrance in fibroblasts providing a
possible
2o mechanism for TSI directed cell mobility via CD47. Lt is also speculated
that TSI-
CD47 interactions would modulate the function of av(33 during angiogenesis (
18).
Interestingly, CD47 Ag is an ubiquitous molecule, present on a variety of cell
types
including lymphocytes and erythrocytes which express low levels or no av(33
integrins, respectively ( 19).
It is also known that the infiltrate at the extravascular site of inflammation
during acute (ex: following invasion by microorganisms) or chronic (ex:
CA 02226962 1998-02-16
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autoimmune) diseases consists of diverse accumulation of leukocytes (i.e., T,
B,
g~ranulocytes and macrophages). Despite the specific immune reaction that
triggers
the disease, the majority of cells found in the inflammatory infiltrate are
non-
specifically activated leukocytes. Recovery from the disease is intimately
related
to the regression of this infiltrate and this can be achieved by the
elimination of the
few Ag specific T cells strongly suggesting that rare cells may retnllate the
recruitment and most likely the function of the vast majority of non-specific
cells
( 1 ). Recently an "in vitro model" of non-specific T cell activation has been
developed whereby cocultures of human resting T cells with autologous
monocytes
1 o and LL-2 or IL-12 lead to large production of IFN-~y in the absence of Ag
(2).
Results obtained with this method indicates that CD40-CD40L interactions as
well
as 1 W I 2 are key relmlators of this bystander T cell activation and that
sCI)23 further
amplifies it by triggering monokine release by monocytes (ex: TNF-a, IL- l )
(3).
SUMMARY OF THE INVENTION
The present invention is directed to a plurality of new uses of binding agents
to CD47 antigen (Ag), and more particularly to new uses of monoclonal
antibodies
2 0 (mAbs) specific to the CD47. The invention i s based on the discovery that
mAbs
directed against CD47 Ag strongly abrogate both IFN-y production and monokine
release (i.e. LL-1, IL-12 and TNF-a), and dowrlregulate cytokine production by
anti-
CD3 or allogenic cell-stimulated T cells. These results were obtained with
both an
antibody named IOG2 produced by the applicant, and with different commercial
2 5 anti-CD47 antibodies, including an antibody named B6H 12 (ATCC HB-9771 ).
Thus, it is believed that anti-CD47 antibodies in general, and CD47 ligands
such as
thrombospondin could be usefiil in the treatment or prophylaxis of various
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inflammatory, autoimmune and allergic diseases as well as in treatment of
tumor
metastasis, cachexia and graft rejection. These human diseases include
rheumatoid
arthritis, lupus erythematosus, multiple sclerosis, diabetes, uveitis,
ulcerative colitis,
Crohn's disease, inflammatory bowel disease, thyroiditis, glomemlonephritis,
Sjogren disease, graft versus host disease (GVH), allergies, asthma, rhinitis
and
eczema.
Preferred binding agents include ligands, antibodies, fragments thereof or
artificial constmcts comprising antibodies or fragments thereof or artificial
i o constructs designed to mimic the binding of antibodies or fragments
thereof. Such
binding agents are discussed in Dougall et al in Tibtech ( 1994) 12:372-379.
They
include complete antibodies, F(ab')2 fragments, Fab fragments, Ev fragments,
ScFv
fragments, other fragments, CDR peptides and mimetics. These can be
obtained/prepared by those skilled in the a.rt. For example, enzyme digestion
can
be used to obtain F(ab')2 and Fab fragments (by subjecting an LgG to molecule
to
pepsin or papain cleavage respectively). References to "antibodies" in the
following
description should be taken to include all of the possibilities mentioned
above.
Recombinant antibodies may be used. The antibodies may be humanized or
chiinerised. The CDRs may be derived from a rat or mouse monoclonal antibody.
2 o The framework of the variable domains, and the constant domains, of the
altered
antibody may be derived from a human antibody. Such a humanized antibody
elicits
a negligible immune response when administered to a human compared to the
immune response mounted by a human against a rat or mouse antibody.
Alternatively, the antibody may be a chimeric antibody. A chimeric antibody
comprises an antigen binding region and a non-immunoglobuiin region. The
antigen
binding region is an antibody light chain variable domain or heavy chain
variable
CA 02226962 1998-02-16
domain. Typically, the chimeric antibody comprises both light and heavy chain
variable domains. The non-immunoglobulin region is fused as its C-terminus to
the
antigen binding region. The non-immunoglobulin region is typically a non-
immlmoglobulin protein and may be an enzyme region, a region derived ftom a
5 protein having known binding specificity, ftom a protein toxin or indeed
ftom any
protein expressed by a gene. The two regions of the chimeric antibody may be
connected via a cleavable linker sequence.
The antibody may be a human IgG such as IgG 1, IgG2, IgG3, IgG4, IgM,
io IgA, LgE or 1gD carrying rat or mouse variable regions (chimeric) or CDRs
(humanized). Primatizing techniques may also be used.
The binding agents of the present invention may be used alone or in
combination with immunosuppressive agents such as steroids, cyclosporin, or
antibodies such as an anti-lymphocyte antibody or more preferably with a
tolerance-
inducing, anti-autoimmune or anti-inflammatory agent such as a CD4 ' T cell
inhibiting agent, e.g. an anti-CD4 antibody (preferably a blocking or non-
depleting
antibody), an anti-CD8 antibody, a TNF antagonist e.g. an anti-TNF antibody or
TNF inhibitor e.g. soluble TNF receptor, or agents such as NSAIDs.
The binding agent will usually be supplied as part of a sterile,
pharmaceutically acceptable composition. This pharmaceutical composition may
be in any suitable form, depending upon the desired method of administering it
to
a patient. It may be provide in unit dosage form and may be provided as part
of a
kit. Such a kit would normally (although not necessarily) include instnlction
for use.
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Binding agent administrations are generally given parenterally, for example
intravenously, intramuscularly or sub-cutaneously. The binding agents are
generally
given by injection or by infitsion. For this purpose a binding agent is
formulated in
a pharmaceutical composition containing a pharmaceutically acceptable carrier
or
diluent. Any appropriate carrier or diluent may be used, for example isotonic
saline
solution. Stabilizers may be added such as a metal chelator to avoid copper-
induced
cleavage. A suitable chelator would be ED'1'A or sodium citrate. 'hhey may be
given orally or nasally by means of a spray, especially for treatment of
respiratory
disorders. They may be formulated as creams or ointments, especially for use
in
io treating skin disorders. They may be formulated as drops, or the like, for
administration to the eye, for use in treating disorders such as vernal
conjunctivitis.
For injectable solutions, excipients which may be used include, for example,
water,
alcohols, polyols, glycerine, and vegetable oils.
15 The pharmaceutical compositions may contain preserving agents, solubilizing
agents, stabilizing agents, wetting agents, emulsifiers, sweeteners,
colorants,
odorants, salts (substances of the present invention may themselves be
provided in
the form of a phannaceutically acceptable salt), buffers, coating agents or
antioxidants. They may also contain other therapeutically active agents.
Suitable dosages of the substance of the present invention will vary,
depending upon factors such as the disease or disorder to be treated, the
route of
administration and the age and weight of the individual to be treated. Without
being
bound by any particular dosages, it is believed that for instance for
parenteral
administration, a daily dosage of from 0.01 to 50 mg/kg of a binding agent of
the
present invention (usually present as part of a pharmaceutical composition as
indicated above) may be suitable for treating a typical adult. More suitably
the dose
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might be 0.05 to 10 mg/kg, such as 0.1 to 2 mg/kg. This dosage may be repeated
as often as appropriate. Typically administration may be 1 to 7 times a week.
If
side effects develop the amount and/or frequency of the dosage can be reduced.
A
typical unit dose for incorporation into a pharmaceutical composition would
thus be
at least 1 mg of binding agent, suitably 1 to 1000 mg.
BRIEF DESCRIPTION Oh' TIDE DRAW1NGS
io The invention will be better understood from the following detailed
description given with reference to the accompanying drawings.
Fig. 1 and Fig. 2 are graphical representations which show 1 OG2 and B61-i 12
mAbs recognizing CD47 antigen. Untransfected COS and COS cell lines
transiently
i5 expressing CD47 Ag (Fig. 1) or stable CHO-transfected cell line with CD47
cDNA
(Fig. 2) were stained with either 1 OG2 (Fig. l and 2) or B6H 12 (Fig. 2) mAbs
or
isotype-matched cont mAbs as described in materials and methods. One
representative experiment out of 3.
2 o Figures 3 to 5 are graphical representations which show the measurements
of IFN-y production by autologous monocytes. T cells ( 1 X l 0''/ml) were
cultured in
the presence of autologous monocytes (2.Sx105/ml) and stimulated by LL-2 (50
U/ml) (Fig. 3), LL-2 (2 U/ml) plus sCD23 (25 ng/ml) (Fig. 4) or IL-:15 (100
ng/ml)
plus sCD23 (25 ng/ml) (Fig. 5) in the presence or absence of anti-CD47 mAbs
used
2 5 at 5 ~.g/ml final concentration. IFN-y production was measured in the CSN
by RIA
after 3 days culture. Data represent mean ~ SE1VI of 5 experiments (p<0.001 ).
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Figures 6 is a graphical representation which shows that anti-CD47 mAbs
suppressed in a dose-dependent manner IL-2 plus sCD23 induced IFN-y
production.
Cultures of T cells and monocytes were stimulated with IL-2 ( l 0 U/ml) and
sCD23
(25 ng/ml) in the presence of various concentration of anti-CD47 tnAb (clone 1
OG2)
(Panel A), F(ab')2 fragments of clone B6H 12 (Panel B) or different anti-CD47
mAbs
(Panel C). IFN-'y production was measured after 3 days cultures. Data is one
representative experiment out of 3.
Figures 7 to 9 are graphical representations which show Anti-CD47 mAbs
i o suppressed IL- I 2 producti on. T cells ( 1 X 10~/ml) were cocultured with
autologous
monocytes (2. S X 105/ml) as described in Fi gures 3-5 . After 3 days
cultures, I L- l 2
p40 release was measured in the culture supernatant by ELISA. Data represent
mean ~ SEM of 5 (Fig. 7), 4 (Fig. 8) and 1 out of 3 experiments (Fig. 9)
(p<0.01 ).
Figures IO is a graphical representation which shows that Anti-CD47 mAbs
suppressed IL-l 2 plus sCD23-induced IFN-y production. T cells (l X 10''/ml)
were
cultured with autologous monocytes (2.SX105/ml) and stimulated by IL-l 2 (40
pM)
and sCD23 (25 ng/ml) in the presence or absence of anti-CD47 mAbs (2.5
~.g/ml).
IFN-~y production was measured in the CSN after 3 days culture. Data represent
a o mean ~ SD of 4 experiments (p<0.0 I ).
Figures I 1 is a graphical representation which shows that F(ab')2 and Fab
fragments of anti-CD47 mAb suppressed IFN-y production. T cells ( 1X 106/ml)
were
cocttltt~red with monocytes (2.SX 105/ml) and stimulated by IL-2 (20 U/ml)
with or
without F(ab')2 or Fab fragments of anti-CD47 mAb (clone B6H 12). IFN-y was
measured after 3 days of culhtre. Shown is one representative experiment out
of 2.
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Figures 12 is a graphical representation which shows that clones 1 OG2 and
B6H.12 recognize different CD47 epitopes. .Iurkat T cell line (Panel A) and Tl-
IP-1
monocyte cell line (Panel B) were stained with various concentrations of clone
l OG2, B6H l2 tnAbs and isotype-control matched mAbs as described in materials
and methods.
Figures 13 is a graphical representation which shows the cellular distribution
of l OG2 and B6H12 antigens on dendritic cells and erythrocytes. Erythrocytes
(Fig.
13a) and dendritic cells (Fig. 13b) were stained with either 1 OG2 or B6H 12
mAbs
i o as described in materials and methods. One representative experiment out
of 3 .
Figures 14 is a graphical representation which shows that anti-CD47 mAbs
suppressed TNF-a production by purified monocytes. Enriched monocytes
(2X 105/ml) were stimulated by sCD23 (25 ng/ml) or LPS ( 10 ~.g/ml) in the
presence
i5 or absence of 2.5 pg/mI anti-CD47 mAbs (clone 1 OG2 or B6HI2). After
overnight
culture, TNF-a, IL-1 Vii, IL-8 and PGE2 were measured in the CSN by ELISA.
Data
represent mean, ~ SD of 8 (Panel A) and 3 experiments (Panel B) (p<0.001 ).
Figures 15 is a graphical representation which shows that sCD23
ao costimulates IL-2 or IL,-15-induced IL-12 p40 release. T cells (106/ml)
were cultured
with autologous monocytes (2XIOShnl) and stimulated with IL-2 (50 U/ml) or IL-
15
(100 ng/ml) in the presence or absence of sCD23 (25 ng/ml). Anti-CD47 or
isotype-control matched mAb (5 ~ g/ml) were added to the cultures. After 3
days
culhtre, IL-12 p40 release was measured in the CSN. Data represent mean ~ SD
of
25 4 experiments.
CA 02226962 1998-02-16
Figures 16 is a graphical representation which shows that anti-CD47 mAb
suppresses IL-l2 p75 production induced by T-cell dependent or independent
costimulatory signals. Monocytes (106/ml) were stimulated with SAC alone, SAC
plus IFN~y or sCD40L plus IFN~y and GM-CSF in the presence of anti-CD47 or
5 control mAb. After overnight culhire, IL-12 p75 release was measured in the
CSN.
Data represent mean ~ SD of 7 experiments.
Figm-es 17 is a graphical representation which shows the effect of anti-CD47
mAb on SAC or SAC plus IFNy-induced monokine release. Monocytes (10~/ml)
s o were stimulated with SAC plus 1 FN~y in the presence of anti-CD47 or
isotype
control-matched mAb. Monokine release (i.e. 1L-1 (3, 1L-6, '1'NFa and 1L-10)
were
measured in the CSN after overnight culture. Data represent mean ~ SD of 5
experiments.
Figures 18 is a g~rapllical representation which slows that anti-CD47 mAb
suppresses Ag-dependent T cell LFN-y response. Purified T cells ( l X 1
U~'/ml) were
stimulated by soluble anti-CD3 mAb (clone 64.1 ) plus I L; 2 (25 U/ml) or I L-
12 (60
PM) with or without anti-CD47 mAb (5 tlg/ml). 3H thymidine uptake was measured
during the last 16 hrs of 5 days culhare and CSN were collected for the
measurement
of IFNy production. Data represent mean ~ SD of 3 experiments (p<0.05).
Figures 19 is a graphical representation which shows that anti-CD47 mAbs
suppresses allogeneic mixed lymphocyte reaction. T cells (0. S X 106/ml) were
coculhired with allogeneic mitomycin C-treated dendritic cells (0.3XI05/ml) in
96-
a 5 well U-bottom plate in the presence or absence of anti-CD47 mAb (5 ~
g/ml). 'H-
thymidine uptake was measured after 5 days culture. Data are one
representative
experiment out of 3.
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Fig~ires 20 is a graphical representation which shows that anti-CD47 mAbs
specifically suppresses IgE synthesis with no effect on B cell proliferation.
Tonsillar
B cells ( 1 X 106/ml) were stimulated by soluble CD40-ligand (sCD40L) ( 1
tlg/ml)
and IL-4 (10 ng/ml) in the presence or absence of anti-CD47 mAbs (5 ~g/ml)
(clone
I OG2 or B6H12). 3H-thymidine uptake (B-cell proliferation) was measured after
5
days culture and IgE production after 14 days. Data represent mean ~ SD of 8
experiments (p<0.001 ).
1o DETAILED DESCRIPTION OF THE INVENTION
Production of mAbs
Clone l OG2 secreting anti-CD47 antibodies (IgM class) has been produced
according to conventional procedures such as described by Kohler and Milstein
(Nature ( 1975) 256:495-497). Accordingly, a non-IgG secreting mouse myeloma
cell line (NSI) rendered azag~.ianine resistant are ftised to spleen cells
from
immunized mice with Jurkat T cell line to obtain hybrid cells that produce
large
amounts of monoclonal antibody. This method employed polyethylene-glycol
(PEG) as the fusing agent followed by selection in HAT medium (hypoxanthine,
2 o aminopterin and thymidine). Screening of mAbs was performed according to
their
"anti-inflammatory biological activity: i.e. inhibition of IFN-y response" in
T
cells/monocytes coculture system in the absence of TCR engagement.
Cell separation and culture conditions
Morrocyte.s: PBMC were isolated by density gradient centrifugation of
heparinized
blood from normal healthy volunteers using Lymphoprep (Nycomed, Oslo,
Norway). Monocytes were prepared as described (2). Briefly, 1'BMC were
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resuspended at 50 X 106 cells/ml in RPMI 1 G40 containing 10% FCS
(BioWittaker,
Inc., Walkersville, MD) and incubated 40 min at 4°C under rotation
(to allow
aggregation of monocytes) followed by 10 min incubation on ice. Pellets of
aggregated enriched monocytes were further separated from non-aggregated PBMC
by a gradient of FCS and another 10 min incubation on ice. Enriched monocyte
preparations were further depleted in T and/or NK cells by rosetting with S-(2-
aminoethyl) isothiouronium bromide (Aldrich Chemical Co., Milwaukee, W I)
treated sheep red blood cells (AET-SRBC). Monocyte purity was shown to be
>95% by flow cytometry (FACScan, Becton Dickinson) using phycoerythrin-
1 o conjugated anti-CD 14 mAb (Becton Dickinson). For some experiments,
monocytes
were positively selected according to CD14 expression by means of a FACSort
(Becton Dickinson), and monocyte purity was >99% CD 14' cells. Cellular
viability
was >90% using trypan blue exclusion.
15 T cells: Enriched T cell populations were obtained ftom the monocyte-
depleted
PBMC by rosetting with AET-SRBC and treatment with ammonium chloride. To
obtain highly pm-ified T cells, rosette forming cells were washed and
incubated for
20 min at 37°C in Lympho-Kwik T (One Lambda, Los Angeles, CA). Cell
purity
was assessed by flow cytometry (FACScan, Becton Dickinson) using phycoerythrin-
2o conjugated anti-CD3 mAb (Becton Dickinson) and shown to be >98% in all the
cases.
All cultures were performed in complete seem-free HB 1 O1 medium (lrvine
Scientific, Santa Ana, CA) supplemented with 2 mM glutamine, 1 W M sodium
25 pymvate, 10 mM I~epes, l00 LU penicillin and I00 yg/ml streptomycin. When
cultured alone, monocytes were incubated in Sml sterile Falcon tubes (Becton
Dickinson, Lincoln Park, NJ) at 2 X 105 cells/ml for cytokine measurement in
the
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presence of polytnyxin B ( 10 Itg/ml) (Sigma Chem., St. Louis, MO). For
coculture
experiments, T cells ( 106 cells/ml) were incubated with monocytes or B cells
(2 X
105 cells/ml) in 24-well Falcon plates.
Reagents
Human recombinant IL-2 was kindly provided by Dr. D. Bron (Instihtt
Bordet, Brussels, Belgium). IL-4 and soluble CD40L, was a gift from Immunex
(Seattle, WA), IL-10 was received from Dr. K. Moore (DNAX, Palo Alto, CA), 1L-
12 was a generous gift from Dr. M. Gately (HofFmann-La Roche, Nutley, NJ) and
1 o used at 40 pM. Endotoxin-free (< 15 pg/ml as determined by the chromogenic
Limulus amebocyte lysate, QCL-1000, BioWhittaker Inc., Walkersville, MD)
affinity-purified sCD23 was prepared in our laboratory from CSN of CHO cell
line
transfected with human cDNA encoding for as 148 to 321 of the CD23 molecule.
The concentration of 25 ng/ml sCD23 used throughout this shtdy was selected on
the basis of previously reported dose-response curves. Recombinant TNFa was
kindly provided by Dr. W. Fiers (State University, Ghent, Belgimn). B6H I 2
mAb
was purchased, at the ATCC (clone HB-9771: U.S. Pa.t. 5,057,604).
Cytotluorimetric analysis
2 o Immlunofluorescence was performed on various cells and cell lines
according
to standard techniques using both anti-CD47 mAbs in the presence of normal
human
Igs (150 gg/ml). PE-conjugated streptavidin were obtained from Ancell and
biotinylated goat anti-human IgG + IgM was purchased from Tago. After
staining,
cells were analyzed with a FACScan (Becton Dickinson & Co.).
CA 02226962 1998-02-16
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Lymphokine determinations
IFN-'y and IL-10 were measured by a sandwich solid-phase RIA. Anti-LFN-
y mAb (clone 42.25) was used to coat the solid phase and'25I-labeled anti-IFN-
y
mAb (clone KM48, purchased from Dimension Labs. lllC., Mississauga, Ont.,
Canada) as detecting probe; anti-I I~ 10 clone 9D7 for coating and anti-I L-10
clone
1268 for labelling. TNF-a was assessed using a sandwich ELISA employing
mouse mAb to human TNF-a (clone T 144. B, kindly provided by Dr. T. Nakajima,
St. Marianna University School of Medicine, Kawasaki, Japan) and a polyclonal
rabbit anti-TNF-a received from Dr. J. Tavernier (Roche Research Institute,
Ghent,
1o Belgium). IFN-'y, 1L-10 and TNF-a assays were calibrated against
international
standards obtained from the National Institute of Biological Standards and
Control
(Hertfordshire, England). The detection limit for the IFN-y and IL-10 RLA is
30
pg/ml and is 45 pg/ml for the TNF-aELLSA. LL-1 (3 was measured by ELISA kits
purchased from R & D Systems. IL-12 p40 and IL-12 p75 were measured by a
15 two-site sandwich ELISA employing clone 2.4 A 1 or clone 20C2 as capture
mAbs
and clone 4D6 as second mAb. Samples were analyzed in serial 5 fold dilutions
in
duplicate; the sensitivity of the assay is 10 pg/ml.
Immunoglobulin determinations
a o IgE was measured by sandwich radioimmlu~oassays (RIA). Clone 89 (mAb
anti-IgE) was used as coating mAb and ' 25I clone 4.15 (anti-IgE) mAb as
detecting
probe; sensitivity of the assay was < 150 pg/ml.
Expression cloning of molecule recognized by lOG2 mAb
25 A cDNA library was prepared from Jurkat T cell line, that expressed high
level of lOG2 epitope, according to the method described by Seed et al
(Proc.Natl.Acad.Sci. USA ( 1987) 84:3365-3369). Briefly, the cDNA library was
CA 02226962 1998-02-16
used to transfect COS cells by DEAF-dextran method. After 3 days transfection,
COS cells were harvested and incubated with 1 OG2 mAb at 4°C for 1 h.
Unbound
mAb was removed by washing and the COS cells were incubated in petri dishes
coated with goat anti-mouse IgM antibodies. After 2 hrs, the unbound cells
were
5 extensively washed with PBS; the cells adhering to the plates were lysed and
the
episomal DNA was prepared. The cDNA was used to transform bacteria. The
antibiotics resistant colonies were amplified a.nd pooled. Plasmids were
prepared
from pools of 50 colonies and used to transfect COS cells. Single colonies
from the
positive pools were amplified and their plasmids were tested for their ability
to
1o transfer lOG2 epitope into COS cells. The positive clone was subsequently
cloned
and cDNA was sequenced.
Statistical analysis
Paired Shident's t test have been used to assess level of significance
(*<0.05;
15 **p<0.01, ***p<0.001 )
Explanation and significance of the foregoing results are as follows:
ao Clone lOG2 mAb is directed against CD47 antigen.
1t has been previously reported that soluble CD23 activated monocytes
to contribute to the antigen-independent stimulation of T cells (2). During
the
screening for mAbs that might regaate this bystander T cell response, the
applicant
found clone lOG2, this clone secreting antibodies having anti-inflammatory
properties. Using mammalian vector expression cloning method and 1 OG2 mAb,
the
cDNA encoding CD47 Ag was cloned from Jurkat T cell line cDNA library. The
CD47 cDNA was transiently expressed in COS 7 cell line. As shown in Fig. l ,
CA 02226962 1998-02-16
16
lOG2 mAb strongly reacted with CD47-transfectants with no staining of
untransfected cell lines. Next, the applicant prepared stable CD47
transfectants in
CHO cell line and found similar pattern of staining with clone l OG2 and B6H
12
mAb (a commercially available CD47 mAb binding to CD47 Ag) mAbs establishing
that lOG2 recognized CD47 Ag (Fig. 2).
Anti-CD47 mAbs suppressed I L.-2 and I L-1 S-induced 1 F'N-y production in the
presence or absence of sCD23.
As shown in Fig. 3-S, anti-CD47 mAbs (clone l OG2 and B6H 12), inhibited
1o IL-2 and IL-15 stimulated 1FN-y response not only in the presence but also
in the
absence of sCD23. The inhibitory effect of anti-CD47 mAbs is dose-dependent
(Fig.
6). Interestingly, thrombospondin, the naW ral ligand of CD47, similarly
suppressed
IL-2-induced IFN~y production in T/monocytes cocultures (Table Il). The
applicant
has previously demonstrated that the IL-2 or IL-15 induced LFN-y production
was
strictly dependent on CD40-CD40L interactions and on endogenous IL- I 2
production as shown by the inhibitory effects of both anti-IL-12 and anti-
CD40L
Abs on IFN-'y, production and of anti-CD40L mAb on IL-12 release (3). The
applicant therefore examined the efFect of these anti-CD47 mAbs on 1L-12
secretion. The data in Fig. 7-9 indicated that, like anti-CD40L mAb, both anti-
CD47
2o mAbs strongly suppressed IL-2 or IL-15-induced IL-12 production; however,
by
contrast to anti-CD40L mAb, addition of exogenous II~ 12 failed to restore IFN-
y
production (Fig. 10). Taken together, these data indicated that anti-CD47 mAbs
inhibited IFN-y production not only by reducing 1L-12 release belt also by
diminishing T cell response to IL-12.
To further analyze the mechanisms of inhibitory activity of anti-CD47 mAbs
in T cells/monocytes coculW res, the applicant prepared F(ab')2 and monovalent
Fab
CA 02226962 1998-02-16
17
fragments of CD47 mAb. As shown in Fig. 11, divalent or monovalent ftagments
of CD47 mAb significantly suppressed IFNy response suggesting that the anti-
CD47 mAb mediated its activity by either delivering a negative signal to the
cell
through the cross-linking of CD47 Ag via its divalent Fab (not via its Fc
fragment
bound to FcyR) or by inhibiting the interactions between CD47 and its natural
ligand, the thrombospondin-derived macrophages.
Cellular distribution of Ag binding to AIM mAbs.
The applicant next examined a panel of cell lines for their reactivity to
clone
1 OG2. As shown in Table I, clone IOG2 reacted to most of the cell lines (T,
B,
monocytic and erythroleukemia cell lines) with tle exception of THP 1
monocytic
cell line exclusively stained by BH612 mAb and not by 1 OG2 mAb (Fig. 12).
Both
anti-CD47 mAbs stained all leukocytes (T, B and macrophages) (Table I);
erythrocytes (Fig. 13a) and dendritic cells (Fig. 13b) are reacting with BI-
i612 but
s 5 not with 1 OGZ mAb. All together, these results strongly suggested that
both mAbs
reacted with different epitopes or different molecular forms of a common
antigen.
Anti-CD47 mAbs inhibit inflammatory mediators release by monocytes.
IFN-'y and TNF-a are directly implicated in the pathogenesis of chronic
a o inflammatory disease as shown by in vivo studies using neutralizing mAbs.
Monocyte-dependent T cell IFN-'y production involved TNF-a and IL-12
production as well as interactions between costimulatory surface molecules
(CD40-
CD40L; LFA3-CD2, B7-B7 ligands). The applicant therefore examined the
reg~~latory activity of anti-CD47 mAbs on monokine release by pm-ified
monocytes.
25 Bacterial stimuli (i.e. lipolysaccharides (LPS) or staphylococcus aureus
Cowan 1
(SAC) or sCD23 were used to trigger TN F-a, IL-1 ~3 or IL- I 2 production. The
data
in Fig. I4 and table IV indicated that anti-CD47 mAbs strongly inhibited sCD23
CA 02226962 1998-02-16
18
induced TNF-a release, without affecting LPS induced TNF-a production.
Similarly, sCD23-induced 1L-I ~3 and PGE2 production were suppressed by anti-
CD47 mAbs. Although sCD23 did not trigger IL- I 2 release by purified
monocytes,
it costimulated IL-12 production in T cells/monocytes cocultures system. As
shown
in Fig. 15, CD47 mAb strongly decreased sCD23 costimulatory activity on IL-12
secretion. Most sfikingly, CD47 mAb also suppressed IL-l 2 production by
purified
monocytes stimulated by T-cell independent (i.e. SAC) or dependent signals
(i.e.
sCD40L) (Fig. 16). Of interest, thrombospondin significantly reduced SAC and
IFN~y-activated IL-12 p70 release by monocytes (Table III). As reported for
LPS-
1o induced TNFa, CD47 mAb failed to inhibit SAC plus LFNy induced TNFa release
(Fig. 17). The SAC-induced secretion of other monocyte products (i.e. IL1~3,
IL-6,
1L-10) remained largely unaffected.
Taken together, these results indicated that anti-CD47 mAbs displayed potent
suppressing activity on inflammatory mediators release underlying their
inhibitory
effect on IFN-y production.
Anti-CD47 mAbs suppress IL-12 and anti-CD3-induced IFN-y production and
allogeneic mixed lymphocyte reaction.
a o The applicant next examined the biological activity of anti-CD47 mAbs on
Ag-dependent T cell stimulation. As depicted in Fig. 18, anti-CD47 mAbs
inhibited
anti-CD3-induced T cell proliferation and IFN-y production by purified T
cells.
Similar selective suppression of anti-CD3-induced T cell response to LL-12
were
obtained using purified CD4 or CD8 subpopulations (not shown). Of interest,
pokweed mitogen-induced IFN-y is also suppressed by anti-CD47 mAbs (data not
shown). The inhibitory activity by anti-CD47 mAbs of Ag-dependent T cell
activation was also observed in mixed lymphocyte reaction (MLR). Irradiated
CA 02226962 1998-02-16
19
allogeneic non-T cells enriched populations or purified dendritic cells were
used as
allostiinulators of adult peripheral purified CD4+ T cells. As shown in Fig.
19, anti-
CD47 mAbs inhibited primary mixed lymphocyte reaction as measured by 3H
thyrnidine uptake; the applicant also tested the effect of mAbs on secondary
stimulation, and found that triggering of CD47 Ag in primary cultures lead to
a state
of hyporesponsiveness of T cells in secondary cultures (not detailed).
Anti-CD47 mAbs specifically suppress IgE synthesis without significantly
affecting B cell proliferation.
1o Since resting B cells strongly express CD47 Ag, the applicant examined the
effect of CD47 ligation on B cell proliferation and differentiation. Purified
tonsillar
B cells were stimulated by trimeric soluble CD40L (sCD40L) in the presence of
IL-
4. As shown in Fig. 20 and table V, anti-CD47 mAbs did not interfere with B
cell
proliferation as measured by 3H thymidine uptake at day 5, while they strongly
1 s inhibited in a dose-dependent manner (not shown) II~ 4-induced IgE
synthesis.
Moreover, anti-CD47 mAbs were likely to block IgE-class switching since they
also
suppressed 1gE synthesis by 1L-4-stimulated naive ( sIgM' slgD' ) B cel Is and
inhibited the expression of germ line E transcripts (not shown). The
inhibitory effect
was not reversed by addition of neutralizing mAbs to TGF-(3, a potent
inhibitor of
a o Ig synthesis nor by IL-6 (not shown). Taken together, anti-CD47 mAbs
appeared
as strong anti-inflammatory agents since they also interfered with the humoral
response of the allergic reaction.
IL-I2 is a potent proinflammatory and immunoregulatory cytokine which
25 plays a crucial role in innate and adaptive Th 1 response. IL-12 is
released during
the early stage of infection caused by a large variety of bacteria,
intracellular
pathogens, fungi and certain viruses. IL-12 is also produced in the absence of
CA 02226962 1998-02-16
infection, following interaction of CD4+ T and dendritic cells/monocytes. It
has
been reported that Thl response associated with chronic or autoimmune disease
remains IL-12-dependent. Indeed, IL-12 neutralization (by anti-IL-12 Ab)
reveals
to be an effective treatment of experimental bowel disease, auto-immure
5 encephalitis and insulin-dependent diabetes.
Therefore, the inhibition by CD47 ligation of proinflammatory cytokine
(including IL-12) production by monocytes and IL-12 responsiveness by T cells
could permit to downregulate inflammatory response on which new therapeutic
io strategies to chronic disorders could be based.
CA 02226962 1998-02-16
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~a
6) Brown E. , L. Hooper, T. Ho, and H. Grresham. 1990. Integrin-associated
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~3
12. Brooks P. C. , A. M. P. Montgomery ) M. Rosenfeld, R. A. Reisfeld, T. Hu,
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D. A. Cheresh. 1994. Integrin a~1~3 antagonists promote tumor regression by
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L/
17. Parkos C.A., S.P. Colgan) T.W. Liang, A. Nusrat, A.E. Bacarra, D.K.
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CA 02226962 1998-02-16
Table 1 Cellular distribution
: of lOG2 antigen on
human cell lines
Freshly Human cell lines
isolated
human
leucocytes
sCD23 lOG2 sCD23 lOG2
T cells +++ ++++ T cell lines ++ ++++
(Jurkat, CEM, HUT 78)
B cells ++ ++ B cell lines ++ ++
(RPMI 822f, Raji, WIL-2,
Daudi)
Monocytes + + Monocyte cell lines ++ ++
( 11937)
THP-I - _
Erythrocytes- - Erythroleukemia cell lines + +
(K5621 KS62-CR2)
CA 02226962 1998-02-16
a~
U
O
U
O ~ M
M V1 0~0
U
O
z
0
U w
E~
N
1~ I~ O v0
b G ~ M ~ c o
E~
H
U
N
z + d' N O ~ M
p
00 ~ N O M
+
H
O
O
o z
U
A'' ~ N M wt V'1
W v~
. CA 02226962 1998-02-16
w
O
O O O
...
4 .
F,
O
G
_
b
o E
~ o g
N ~ 0~0
.-r
aw ~ v
p', 0 0
w
~ N
H
O ~ ~3
z
N ~ O O
cd 4,
_
w ~ pp O O
o ..-) 3
'-' CV 1b4
O
~o .-i
O
G
~! ..,
'G
~ G
N M
CA 02226962 1998-02-16
TABLE IV (Appendix to I~g. 14a)
Effect of lOG2 and B6H12 mAbs on sCD23-induced TNFa production by purified
monocytes.
TNFa (pg/ml)
Cont mAb lOG2
Exp. 1 993 347
Exp. 2 1392 483
Exp. 3 396 110
Cont mAb B6H12
Exp. 4 845 347
Exp. 5 1177 166
Exp. 6 1152 252
Monocytes were stimulated overnight with sCD23 (25 ng/ml) in the presence of
anti-CD47 mAbs
(clone l OG2 or B6H 12) or isotype-matched cont mAbs. TNFa was measured in the
CSN by
specific ELISA.
CA 02226962 1998-02-16
TABLE V (Appendix to Fig. 20)
Effect of lOG2 and B6H12 mAb on IIr4-induced IgE synthesis by CD40-activated B
cells.
3H-Thymidine Uptake (X103 CP1V1) IgE (ng/ml)
A Cont mAb lOG2 Cont mAb 1062'
Exp. 1 2.8 3.9 51 26
Exp. 2 33 42 31 15
Exp. 3 47 48 79 28
B Cont mAb B6H12 Cont mAb B6H12'
Exp. 4 4.8 4.5 23 4.5
Exp. 5 87.1 60.4 97 18
Exp. 6 77.1 63.1 80 27.5
lOG2 mAb and B6H12 mAbs are used at 20 ~g/ml and 10 ~g/ml respectively.
