Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND PRODUCTS FOR REGULATING LECTIN COMPLEMENT
PATHWAY ASSOCIATED COMPLEMENT ACTIVATION
s Related Applications
This application claims priority under 35 U.S.C. ~119 to US Provisional Patent
Application No. 60/112,390, filed December 15, 1998, the entire contents of
which is
hereby incorporated by reference.
Government Support
~ o The present invention was supported in part by grants from the National
Institutes of Health
HI,56086, HL52886., and Ci1VI07592. The U.S. Government may retain certain
rights in the
invention.
Field of the Invention
The present invention relates to methods and products for regulating lectin
t s complement pathway (LCP,I associated complement <~ctivation. In
particular, the invention
relates to methods for inhibiting LCP associated complement activation by
contacting a
m~unmalian cell having a mannose binding lectin. (MBL) ligand with an MBL
inhibitor.
The invention also relates t~~ products which are MBL inhibitors, such as an
MBL binding
pe ptide.
Background Of The Invention
The immune system functions to defend the body against pathogenic bacteria,
viruses and parasites. Immunity against foreign pathogens usually involves the
complement
sy item. The complement ~;ystem is a cascade of 18 sequentially activated
serum proteins
2s which functions to recruit and activate other cells of the immune system,
effect cytolysis of
target cells and induce opsonization of foreign pathogens. Complement can be
activated by
the: presence of either antibody/antigen complexes, as in the classical
complement pathway,
or microbial surfaces, as iri the alternative complement pathway. Complement
activation
can also occur via the lectir, complement pathway (LCP). Lectins are
carbohydrate-binding
3o proteins that recognize oligosaccharide structures :present on cell
surfaces, the extracellular
m~~trix, and secreted glycoproteins. As shown i:n Figure 1, these distinct
activation
pathways ultimatel~~ converge at the common enzymatic step of serum protein C3
cleavage
to C3b and C3a. This in turn initiates the terminal steps of complement
function including
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the cleavage of CS to CSb and CSa and subsequent deposition of CSb-C9 onto the
target cell
membrane.
The LCP is an antil~~dy-independent cascade that is initiated by binding of
mannan
(or mannose) binding lectin (MBL) to cell surface carbohydrates on bacteria,
yeasts,
s parasitic protozoa, and viruses (Turner MW, "Marmose-binding lectin: The
pluripotent
molecule of the innate immune system", Immunol. Today, 1996;17:532-540). MBL
0600
kl;~a) is a member of the ce~llectin protein family and is structurally
related to the classical
complement C 1 subcomponent, C I q. Associated with MBL are two serine
proteases,
Mannose binding lectin associated serine protease, MASP-1 and MASP-2, which
show
~ o striking homology to the two C 1 q-associated serine proteases of the
classical complement
pa~:hway, C 1 r and C 1 s (Thief S, et aL, "A second serine protease
associated with mannan-
binding lectin that activate: complement", NaturES 1997;386:506-510 ). The
selectivity of
M~3L sugar binding is: N-acetyl-D-glucosannine (GIuNAc) > mannose > N-
acsaylmannosamine and fucose > maltose > glucose » galactose and N-
~s ac~;tylgalactosamine (Thief S, et al., "A second serine protease associated
with mannan-
binding lectin that activates complement", Nature 1997;386:506-510; Turner MW,
"Mannose-binding lectin: The pluripotent molecule of the innate immune
system",
Immunol. Today, 1996;17:532-540). Binding of the MBL/MASP complex to cell
surface
carbohydrates activates the LCP, which in turn activates the classical
complement pathway
2o independently of C 1 q, (: I r, C 1 s or antibodies (Fig. 1 ). Most if not
all the carbohydrate
moieties to which MBL birn~s are not normally expressed by unperturbed human
tissue.
Summary Of The Invention
The present invention relates to methods and products for regulating lectin
2s complement pathway (LCP) associated complement activation. Prior to the
instant
invention, it was known that LCP associated complement activation was a
mechanism used
by the body to recognize and destroy an invading microorganism. LCP activation
normally
occurs through the binding ~~f mannan-binding lecti.n (MBL) and its two
associated serine
pr~~teases, MASP-1 and M~,SP-2, to carbohydrates on the surface of
microorganisms. Once
3o MSL and MASP-l and MA.SP-2 are localized to th.e surface of the
microorganism,
complement begins to assemble, ultimately killing the microorganism. These
prior art
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teachings demonstrate that I~~IBI. is an important cellular component in the
process of the
eradication of infectious microorganisms. In fact, MBL deficiencies can result
in medical
disorders. A disease known as MBL deficiency, in which children are deficient
in MBL,
renders the children prone to the development of ini:ectious diseases.
s The present invention is based upon the surprising discovery that MBL
recognizes
specific carbohydrates or pc;ptides on the surface of mammalian endothelial
cells, causing
complement deposition through activation of the L,CP. According to U.S. Patent
No.
5,270,199 issued to Ecekowitz, MBL does not recognize the cell wall of human
and animal
cells. In contrast to these prior art teachings, it has been discovered,
according to the
~o invention, that MBL does rc;cognize specific sequences on the surface of
mammalian cells.
It has also been discovered that MBL deposition on the surface of mammalian
cells results
in ;activation of LCP., contributing to the development of diseased or damaged
tissue.
In one aspect, the invention is a method for inhibiting LCP-associated
complement
activation. The method includes the step of contacaing a mammalian cell having
a surface
~ s exposed MBL ligand with an effective amount of an MBL inhibitor to inhibit
cellular MBL
deposition and LCP-associated complement activation. In one illustrative
embodiment, the
method is an in vitro screening assay.
In another aspect, the invention is a method for inhibiting a cellular injury
mediated
by LCP-associated complerzent activation. The method includes the step of
administering
zo to a subject in need thereof an effective amount of an MBL inhibitor to
inhibit LCP
as<.~ociated complement activation.
In one embodiment of the methods of the invention, the MBL inhibitor is an
isolated
MJ3L binding peptide. In an illustrative embodiment, the isolated MBL binding
peptide has
an MBL binding CDR3 region or functional variant thereof. In some embodiments,
the
zs isolated MBL binding peptide is an antibody fragment. In other embodiments,
the isolated
M BL binding peptide is an ;antibody.
According to anotr~er embodiment of the methods of the invention, the MBL
inhibitor is an isolated MASP binding peptide. The: isolated MASP binding
peptide may
bind to either MASP-1 or MASP-2 or both, preventing MASP from participating in
the
3o L(:P.
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The cellular injury mediated by LCP-associated complement activation may
contribute to the development of injured tissue associated with a variety of
disorders. In
one embodiment, the cellular injury is associated with atherosclerosis. In
another
em'~odiment, the cellular injury is associated with arthritis, myocardial
infarction, ischemia
s and reperfusion, transplantation, CPB, stroke, ARDS, SLE, Lupus, or
dialysis.
The MBL inhibitor rnay be administered to the subject by any route known in
the
art. When the cellular injury is associated with the pulmonary system, the MBL
inhibitor
may be administered to the subject by an aerosol route of delivery.
According to another aspect of the invention, an MBL inhibitor is provided.
The
io MBL inhibitor is an isolated peptide that selectively binds to a human MBL
epitope and
inhibits LCP-associated complement activation.
In another aspect, l:he invention is a hybridoma cell line. In one
illustrative
embodiment, the hvbridoma cell line is the cell line deposited under ATCC
accession
nwnber HB-12621. In another embodiment, the hybridoma cell line is the cell
line
~s deposited under ATCC a~~cession number HB-12620. In another embodiment, the
hybridoma cell line is the cell line deposited under ,ATCC accession number HB-
12619.
According to vet another aspect, the invention is a composition of an MBL
inhibitor,
wherein the MBL inhibitor is an isolated binding peptide that selectively
binds to a human
M'.BL epitope and that inhibits LCP-associated complement activation. In an
illustrative
2o err~bodiment the composition is a pharmaceutical composition including an
effective
amount for treating an MBI~ mediated disorder of the isolated MBL binding
peptide and a
pharmaceutically acceptable: carrier. In one embodiment, the composition also
includes a
drag for the treatment of an MBL mediated disorder.
In one embodiment the isolated MBL binding peptide has an MBL binding CDR3 ~
2s re;~ion or a functional variant thereof of a monoclonal antibody produced
by hybridoma cell
line 3Fg deposited under A~CG(: accession number HB-12621. In another
embodiment the
isolated MBL binding peptide has an MBL binding CDR32 region or a functional
variant
thereof of a monoclonal antibody produced by lzybridoma cell line an9
deposited under
ATCC accession number 1-IB-12620. In another embodiment the isolated MBL
binding
3o pf;ptide has an MBI_ binding C:DR32 region or a functional variant thereof
of a monoclonal
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antibody produced by hybridoma cell line nMBm.2 deposited under ATCC accession
number
HB-12619.
The isolated peptide; may be an intact soluble monoclonal antibody. In one
embodiment the isolated peptide is monoclonal antibody ~3Fg~ produced by the
hybridoma
s cell line deposited under A'TCC Accession No. H:B-12621. In another
embodiment the
isolated peptide is monoclonal antibody~2Ay~ produced by the hybridoma cell
line deposited
under ATCC Accession No. HB-12620. In another embodiment the isolated peptide
is
monoclonal antibody hMBLl.2 produced by the hybridoma cell line deposited
under ATCC
Ac~~ession No. HB-12619. In an illustrative embodiment the isolated peptide is
a
~o humanized monoclonal anti~~ody.
According to some embodiments the isolated peptide is an antibody fragment.
The
isolated peptide, for instance, may be a monoclonal antibody fragment selected
from the
grcup consisting of an F(ab')z fragment, Fd fragment, and an Fab fragment. The
isolated
peptide may also be a peptide having a light chain C'DR2 region selected from
the group
~s consisting of a CDR2~3F8t of a monoclonal antibody produced by
hybridomat3F8~ deposited
under ATCC Accession No. HB-12621, a CDR2t2A~~t of a monoclonal antibody
produced by
hyhridoma Za,9 deposited under ATCC Accession No,. HB-12620, and a
CDR2~nMBLl.2) of a
monoclonal antibody produced by hybridoma~nMBLi.2~ deposited under ATCC
Accession No.
Hl=4-12619. In another em'oodiment the isolated peptide has a light chain CDR1
region
2o selected from the group consisting of a CDRI~3Fg~ of a monoclonal antibody
produced by
hybridomat3f~g? deposited under ATCC Accession No. HB-12621, a CDR1~2A9~ of a
monoclonal antibody produced by hybridomatZA9~ deposited under A'TCC Accession
No.
HB-12620, and a CDRI~nMBL~.z> of a monoclonal antibody produced by
hybridomatnMSr.A.2~
deposited under ATCC Accession No. HB-12619.
2s In another aspect, the invention is a composition, wherein the MBL
inhibitor is an
anti-MBL antibody that: (i;1 selectively binds to a human MBL epitope, and
(ii) prevents
L(:P activation.
In yet another aspect, the invention is a method for screening a subject for
susceptibiliy to treatment with an MBL inhibitor. The method includes the
steps of
so isolating a mammalian cell from a subject, and .detecting the presence of
an MBL on a
surface of the mammalian cell, wherein the presence of the MBL indicates that
the cell is
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susceptible to LCP-associated complement activation and that the subject is
susceptible to
treatment with an MBL inhibitor. In one embodiment, the method includes the
step of
co:ltacting the MBL with a detection reagent that selectively binds to the MBL
to detect the
prcaence of the MBL. The detection reagent in one embodiment is an isolated
MBL
s binding peptide.
A method for screening a subject for susceptibility to treatment with MBL
inhibitor
is provided in another aspect of the invention. The; method includes the steps
of contacting
a mammalian cell from a subject with a labeled isolated MBL binding peptide,
and
detecting the presence of .an MBL on the surface of the mammalian cell,
wherein the
Io pr~aence of the MBL indicates that the cell is susceptible to LCP-
associated complement
ac:ivation and that t:he subject is susceptible to treatment with an MBL
inhibitor. In one
embodiment, the mammalian cell is an endothelial hell.
Each of the limitations of the invention can encompass various embodiments of
the
invention. It is, therefore, anticipated that each of the limitations of the
invention involving
~ s any one element or combinations of elements can be included in each aspect
of the
invention.
Brief Description Of 'The Drawings
Figure 1 is a schematic depicting the antigen/antibody-dependent classical
complement pathway and the antibody-independent alternative and lectin
complement
2o pathways. All three path~Nays merge at C3 and lead to the formation of the
terminal
complement complex (CSb-9).
Figure 2 depicts a flow cytometry printout to demonstxate MBL deposition on
H1JVECs. MBL deposition on HUVECs subjecaed to zero (normoxia) or 24 hours of
hypoxia was studied by flow cytometry using a monoclonal anti-human MBL
antibody.
is MBL deposition (MFI =- 40 ~ 3) was significantly increased on hypoxic
HUVECs
re~~xygenated for 3 hours in 30% human serum compared to normoxic HUVECs (MFI
= 8 t
2). where MFI = mean fluorescent intensity.
Figure 3 is a graph depicting MBL deposition on HUVECs (ELISA). MBL
deposition on HUVECs subjected to zero (normoxia) or 24 hours of hypoxia
followed by 3
3o hours of reoxygenation was examined by ELISA using a monoclonal anti-human
MBL
artibody. MBL deposition on hypoxic I-iUVECs reoxygenated in the presence of
30%
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human serum (vehicle) was significantly greater than normoxic HUVECs or
hypoxic
HUVECs reoxygenated in 30% human serum treated with 30 mmol/L GIuNac.
Figure 4a is a graph depicting iC3b deposition following competitive
inhibition of
MEtL. iC3b deposition was studied by ELISA on HUVECs reoxygenated in the
presence of
s 30°,io human serum treate~~ with 30 mmol/L GIuNAc, D-mannose, or L-
mannose.
Deposition of iC3b on hypoxic HUVECs reoxygenated in 30% human serum (vehicle)
or
30~~o human serum treated. with L-mannose was significantly greater than
normoxic
HL~VECs. iC3b deposition, however, on HUVECs reoxygenated in 30% human serum
treated with GIuNAc or D-rr~annose did not significantly differ from normoxic
controls.
~o Figure 4b is a grapl-., depicting iC3b deposition following depletion of
MBL from
human serum. HUVECs were reoxygenated in the presence of MBL-depleted human
serum
to inhibit the lectin complement pathway. Deposition of iC3b (ELISA) on
hypoxic
HLJVECs reoxygenated in 30% human serum was significantly greater (p < 0.05)
than
noomoxic HUVECs. iC3b deposition, however, on hypoxic HUVECs reoxygenated in
30%
is Ml3L-depleted human cell was significantly less (p < 0.05) than hypoxic
HUVECs
rec~xygenated in 30% humem serum. When MBL, was added back to the MBL-depleted
human serum, iC3b deposition on the hypoxic/reoxygenated HUVECs was
significantly
greater than normoxic HUV ECs.
Figure S is a graf~h depicting percent hemolysis as an indicator of classical
2o co::nplement pathway actimity. No significant differences in the serum
complement
hemolytic assay (CH;o) were observed between human serum or MBL-depleted human
semm, indicating that depletion of MBL did not inhibit or deplete classical
complement
pathway activity;
Figure 6 depicts a Western blot analysis of C3 activation following
2s hypoxia/reoxygenation using purified C2, C3, C4, and MBL. Western blot
analysis of the
C_~ and C3b a'-chain was performed under reduced conditions with a polyclonal
anti
human C3 antibody on thc: supernatants of norrr~oxic and hypoxic (12 hours)
HUVECs
reoxygenated for 3 hours ir, the presence of purified C2, C3, C4, and MBL.
Lanes 1 and 2
revresent normoxic HUVECs supernatant, lanes :3 and 4, hypoxic HUVECs
supernatant,
30 lane 5, purified C3 standard. and lane 6, purified C:3b standard. The
results demonstrate an
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increased band density of C3b a'-chain in the hypoxic/reoxygenated
supernatants compared
to the normoxic supernatants. The Figure is representative of five
experiments.
Figure 7 is a scan of a Western blot analysis of human MBL. Monoclonal
antibodies 3F8, hMBL 1.2, A9 or 1 C 10 were used for western blot analysis of
reduced
s M)=.L. Lanes l, 2, 3 and 4 represent staining of reduced human MBL with 10
p.g/ml of mAb
2A'~, hMBL 1.2, 1 C 10 or 3F8, respectively. A single band with an approximate
molecular
weight (MW) of 32 kDa (i.~~., consistent with MB:L) was observed with each
mAb. This
figure is representative of three separate experiment's.
Figure 8 is a graph depicting C3 deposition with inhibitors.
~o Figure 9 is a graph df;picting inhibition of VCAM-1 expression.
Reoxygenation of
hyhoxic HUVECs in 30% HS treated with PBS (Vehicle) induced a significant
increase in
VCAM-1 expression compared to normoxic cells incubated with 30% HS. Treatment
of the
30°,io HS with 3F8 (~ pg/ml) significantly inhibited'VC',AM-1
expression. The bars represent
the mean of 3 individual experiments. Brackets represent SEM. * represent
p<0.05
t s compared to the respective normoxia control. * * represent p<0.05 compared
to vehicle
tre~~ted hypoxia group.
Dfaailed Description Of The Invention
The invention relate, to methods and products for regulating and manipulating
lectin
2o complement pathv-av (LCP)-associated complement activation. As discussed
above, the
invention is based on the finding that LCP-associated complement activation
plays a role in
complement induced cellular injury of mammalian cells. It was discovered
according to the
invention that MBL interac~a with carbohydrates o~r peptides on the surface of
mammalian
cells in vitro and in vivo. The surface associated MBL leads to the
accumulation of
2s complement on the surface ~af the cell, ultimately leading to cell injury
or death. According
to the prior art, LCf-associated complement activation was predominantly
associated with
infectious microorganisms, suggesting that MBL deposition should be promoted
in order to
enhance the killing of infectious microorganism:>. It was discovered,
according to the
imrention, that in mammal~~ it is preferable to block MBL cellular
association, preventing
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LC'.P-associated complement activation rather than to promote it. The LCP is
not necessary
for eradication of ini:ectious microorganisms i;n adu.lt mammals, and in fact,
it contributes to
cellular injury associated with several types of disorders, such as
atherosclerosis, arthritis,
myocardial infarction, ischemia and reperfusion, transplantation, CPB, stroke,
ARDS, SLE,
s Lupus, or dialysis.
In one aspect, the invention is a method for inhibiting LCP-associated
complement
activation. The method includes the steps of contacting a mammalian cell
having surface
exposed MBL ligand with an effective amount of an MBL inhibitor to inhibit LCP-
associated complement activation.
~o The methods of the invention are useful for inhibiting LCP-associated
complement
ac:ivation on the surface of a mammalian cell having surface exposed MBL
ligand
(c~~rbohydrate or peptide groups) recognized by ME3L.. The mammalian cell may
be any cell
in which the cell surface c~;rbohydrates or peptides interact with MBI,. In
one illustrative
embodiment, the mammalian cell is an endothelial cell having a surface exposed
MBL
~s ligand. For instance, vascular endothelial cells have been shown in
subjects that have
sustained ischemic/reperfusion injury to express an MBL ligand. Mammalian
cells having
MBL ligands can easily be identified. For instance, an MBL binding assay
(e.g., such as
those described below) can be used to identify MBI:. ligands.
The method for inhibiting LCP-associated complement activatian may be used for
a
2o variety of in vitro and in vivo purposes. The method may be used, for
instance, as an in
vi~ro screening assay. The o vitro screening assay may be used to identify
compounds
which function as an MBL inhibitor, such as the assay described above, to
identify
m;~mmalian cells having surface exposed MBL ligands, or to detect
susceptibility of a
subject to treatment with MBL inhibitor. In order to screen a subject for
susceptibility to
2s treatment with an MBL inhibitor, a cell is isolated from the subject and
the presence of
MBL or the ability of MBL to bind to the surface is detected. If MBL is
present on the
surface of a cell or is able to bind to the surface of a cell, then the cell
is susceptible to LCP-
associated complement activation. If this is the case, then the subject is
susceptible to
trf~atment with an MBL inhibitor.
3o The methods of the invention are also useful in vivo when it is desirable
to inhibit
MBL deposition on a mammalian cell surface. For instance, the methods of the
invention
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are useful for treating an MBL mediated disorder. 'the MBL inhibitors can be
used alone as
a primary therapy or in combination with other therapeutics as an adjuvant
therapy to
enhance the therapeutic benefits of other medical treatments.
The mammalian cell is contacted with an MBL inhibitor. The step of
"contacting"
s as used herein refers to tle addition of the MBL inhibitor to a medium
containing a
mammalian cell. The medium may be an in vitro tissue culture or a biological
specimen, an
ex vivo sample, or in vivo. The step of contacting refers to the addition of
the MBL
inriibitor in such a manner that it will prevent L(JP-associated complement
activation
as:.ociated with the mammal ion cell.
~o An "MBL mediated disorder" as used herein is a disorder which involves
cellular
inj ury caused by LCP-associated complement activation. MBL disorders include,
for
instance, atherosclerosis, arthritis, myocardial infarction, ischemia and
reperfusion,
transplantation, CPB, stroke:, ARDS, SLE, Lupus, or dialysis. Each of these
disorders is
well-known in the art and is described, for instance., in Harrison's
principles of Internal
is ME~dicine (McGraw I-Iill, Inc;., New York).
Atherosclerosis and myocardial infarction can lead to ischemia-reperfusion
(I/R)
injury. One of the underlying mechanisms for I/R-induced injury is the hypoxic
and
reoxygenated environments created in affected tissues. Fluctuations in oxygen
content as
observed in these instances can create oxygen free radicals which have been
reported to,
2o among other things, modulate endothelial cell surface profile.
The invention also i;; useful for treating cellular injury arising from
ischemia/reperfusion associ;~ted with atherosclerosis and/or cardio-vascular
remodeling.
Injury to the vascular system can lead to a number of undesirable health
conditions,
including, for example, forms of atherosclerosis an<I arteriosclerosis that
are associated with
2s unwanted vascular smooth muscle cell proliferation. ,A common injury to the
vascular
sy,~tem occurs as a side effe~~t of a medical procedure for treating ischemic
heart disease.
Isc:hemia refers to a lack of oxygen due to inadequate perfusion of blood.
Ischemic heart
di:;ease is characterized by a disturbance in cardiac function due to an
inadequate supply of
oxygen to the heart. The m~~st common form of this disease involves a
reduction in the
30 lumen of coronary arteries, which limits coronary blood-flow. Under these
conditions the
carbohydrate or peptide residues of the cell surface become exposed or an MBL
ligand is
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synthesized, allowing MBL to associate with the cell surface and initiate the
LCP associated
complement activation.
When ischernic heart disease becomes very serious, then management must be
invasive. Until recently, ischemic heart disease was treated by coronary-
artery, bypass
s surgery. Less invasive procedures, however, now ihave been developed. These
procedures
involve the use of catheters introduced into the narrowed region of the blood
vessel ("the
stenosis") for mechanically disrupting, laser ablating or dilating the
stenosis.
The compositions rnay be administered in combination with other therapeutic
tre;~tments. The most widely used method to achieve revascularization of a
coronary artery
~ o is percutaneous transluminal coronary angioplasty. A flexible guide wire
is advanced into a
coronary artery and positioned across the stenosis. A balloon catheter then is
advanced over
the guide wire until the balloon is positioned across the stenosis. The
balloon then is
reF~eatedly inflated until the stenosis is substantially eliminated. This
procedure, as
compared to heart surgery, is relatively noninvasive and can result in
hospital stays of only
is three days. The procedure is an important tool :in the management of
serious heart
conditions.
An "MBL inhibitor" as used herein is a compound that prevents LCP-associated
complement activation. The MBL inhibitor may function by blocking MBL
deposition on
the surface of a mammalian cell or by blocking the association of MASP-1 or
MASP-2 or
2o C3b associated with MBL, deposition. The ability of an MBL inhibitor to
block MBL
deposition or prevent association of MASP-1, MA~SP-2, or C3b with MBL can be
detected
using routine in vitro binding assays, such as the following assay (also
described in the
Examples).
MBL deposition (or association with MASP-1, MASP-2, or C3b) can be measured
is by ELISA on normoxic H1JVECs and HUVECs subjected to 24 hr of hypoxia
followed by
3 r~r of reoxygenation in the presence of 30% human serum {HS) or 30% HS
treated with 3,
30, or 300 mmol/L of N-acetyl-D-glucosamine (GIuNAc) or with the putative
binding
peptide to inhibit competitively MBL deposition.
C3 and MBL specific cell surface ELISAs can be performed using peroxidase
3o conjugated polyclonal goat anti-human C3 antibody (Cappel, West Chester,
PA) and
monoclonal anti-human 1V1BL antibody (Biodesign, Kennebunk, ME, clone #131-1),
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respectively. HUVE;Cs are grown to confluence on 0.1% gelatinized 96-well
plastic plates
(C~~rning Costar, Cambridge, MA). The plates are then subjected to 0
(normoxia) or 24 hr
of hypoxia. Hypoxic strc;ss is maintained using a humidified sealed chamber
(Coy
Laboratory Products. Inc., Grass Lake, MI) at 37 "C gassed with 1% OZ, 5% C02,
balance
s NZ (Collard CD, et al., "Re~~xygenation of hypoxic; human umbilical vein
endothelial cells
ac~.ivates the classical cornp:lement pathway", Circadation, 1997;96:326-333).
Following the
specified period of normoxia or hypoxia, the cell media are aspirated and 100
~.1 of one the
following is added to each well: 1) 30% HS, 2) Hank's balanced salt solution,
3) 30% HS +
3, 30, or 300 mmol/L GIuNAc, 4) 30% HS + 3, 30., or 300 mmol/L D-mannose, 5)
30% HS
~o + :3, 30, 300 mmol/L L-mannose, 6) 30% MBL-depleted HS 7) 30% MBL-depleted
HS +
O.fi ~.g/ml MBL or 8) 30°/<. HS + 3, 30, or 300 :mmol/L putative MBL
binding peptide.
Additionally, 100 ~1 of purified MBL (3-300 ng,/ml) is added to select wells
to form a
standard curve for quantitative analysis of MBL deposition. 'the cells are
then
reoxygenated for 3 hr at 37°C in 95% air and 5% C'.O_Z. The cells are
washed and then fixed
~s with 1% paraformaldehyde (Sigma Chemical Co., St. Louis, MO) for 30 min.
The cells axe
thc;n washed and izicubated at 4 °C for 1.5 hr with 50 pl of peroxidase-
conjugated
polyclonal goat anti-human C3 antibody (1:1000 dilution) or monoclonal anti-
human MBL
antibody (1:1000 dilution). The MBL ELISA plates are then washed and incubated
for 1 hr
at 4 °C with 50 ~1 of pernxidase-conjugated polyclonal goat anti-mouse
IgG secondary
2o antibody (1:1000 dilution). After washing the cells, the plates are
developed with SO ~l of
ARTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfi~n:ic acid)), and read
(Molecular Devices,
SLnnyvale, CA) at 405 nrn. Background control~.s for the C3 ELISA consist of
cells to
which only the anti-human C3 antibody is added (i.e., no HS) or cells
incubated with 30%
heat-inactivated HS. Background controls for thc; MBL ELISA consist of cells
to which
2s only secondary antibody and an isotype control monoclonal antibody to
porcine CSa are
added. Background optical density is subtracted from all groups. ELISA
experiments are
generally performed 3 times using 6 wells per experimental group. Endothelial
C3 and
MBL deposition on normoxic vs. hypoxic HUVECs is analyzed by two-way analysis
of
variance (ANOVA).
3o The MBL inhibitor prevents LCP-associated complement activation. Whether a
particular compound can inhibit LCP-associated complement activation can also
be
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ass~~ssed using routine in vitro screening assays. For instance, the
Complement hemolytic
ass;~y (CHSO) described in t:he Examples below can be performed on MBL-
depleted HS in
order to demonstrate that MBL depletion inhibit :LC'.P-associated complement
activation.
Thf; assay may be performed, however, using MBL containing HS and adding an
MBL
s binding peptide and/or a control peptide.
In one illustrative embodiment, the MBL inhibitor is an isolated MBL binding
peytide. An "isolated MBL binding peptide " as used herein is a peptide which
binds to
MI=4L and inhibits LCP ass~~ciated complement activation. One method by which
MBL
binding peptides inhibit LC:I? associated complement activation is by binding
to MBL and
~o inhibiting MBL association with surface exposed MBL ligands. Additionally,
the MBL
binding peptide may bind to MBL and inhibit the association between MBL and
MASP-1 or
-2 ;and/or C3b. Several pepi;ides which bind to MBL or MASP have been
described in the
art. including Lanzrein, A.S. et al., "Mannan-binding lectin in human serum,
cerebrospinal
flu:.d and brain tissue and i~a role in Alzheimer's .disease", Department of
Pharmacology,
is University of Oxford, UK, May I1, 1998, Neuroreport, 9(7):1491-5; Jack,
D.L. et al.,
"A~tivation of complement by mannose-binding lectin on isogenic mutants of
Neisseria
meningitidis serogroup B", Immunobiology Unit, Institute of Child Health,
London, UK, J
Immunol, February 1, 1998,160(3):1346-53, Terai, I. et al., "Human serum
mannose-
binding lectin (MBL)-associated serine protease-1 (MASP-1): determination of
levels in
2o body fluids and identification of two forms in serum", Division of Clinical
Pathology,
Hokkaido Institute of Public Health, Sapporo, Japan, Clin. Exp. Immunol.,
Nov., 1997,
110(2):317-23; Endo, M. et al., "Glomerular depo;>ition of mannose-binding
lectin (MBL)
inc.icates a novel mechanism of complement activation in IgA nephropathy [In
Process
Citation]", Second Department of Internal Medicine, hlihon University School
of Medicine,
2s Tokyo, 3apan, Nephrol Dial Transplant, August 13, 1998, (8):1984-90;
Valdimarsson, H. et
al.. "Reconstitution of opsonizing activity by infusion of mannan-binding
lectin (MBL) to
Ml3L-deficient humans", Department of Immunology, University of Reykjavik,
Iceland,
Sc~nd. J. Immunol., August 1998, 48(2):116-23; T'hiel, S. et al., "The
concentration of the
C-type lectin, mannan-binding protein, in human plasma increases during an
acute phase
3o re~;ponse", Clin Exp. Immunol., Oct. 1992, 90(1):31-5. These peptides can
be tested for
their ability to inhibit the association between MBL, and MASP-1 or -2 and/or
C3b.
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The preferred compositions of the invention include an MBL inhibitor which is
an
isolated binding peptide that selectively binds to a human MBL epitope and
that inhibits
LC:P-associated complemem: activation. A ''human MBL epitope" as used herein
is a
portion of MBL which when contacted with an MBL-binding peptide inhibits LCP-
s associated complement activation by preventing the association between MBL
and the MBL
lig~.nd or MASP-1 or - 2 and/or C3b. Preferably the MBL epitope is a region of
the MBL
which interacts with any of t:ze three deposited monoclonal antibodies.
In another embodiment, the MBL inhibitor is an isolated MASP binding peptide.
An "isolated MASP binding peptide" as used herein refers to a peptide which
binds to
co M~.SP-I or MASP-2 and prevents LCP-associated complement activation by
preventing
M~~SP-1 or MASP-2 from forming a complex with MBL on the surface of a cell
thereby
preventing the resulting C3b deposition associated with the MBL-MASP complex.
In another embodimewt the MBL inhibitor is, a mannan binding peptide. A
"mannan
binding peptide" as used herein is a peptide which binds to the MBL ligand on
the surface
i s of a mammalian cell, preventing its interaction with the MBL-MASP complex.
The MBL
inhibitors may easily be prepared or identified by those of ordinary skill in
the art using
routine experiments since 1V1BL,, MASP, mannan and. C3b are all well known
compounds
which have been characterized and described extensively in the prior art.
The MBL, MASP, and mannan binding peptides of the invention can be identified
zo using routine assays, such a;~ the binding and LCP complement activation
assays described
above and elsewhere throughout this patent application.
The peptides of the invention are isolated peptides. As used herein, with
respect to
peytides, the term "isolated ;peptides" means that the peptides are
substantially pure and are
essentially free of other suostances with which they may be found in nature or
in vivo
2s systems to an extent practical and appropriate for their intended use. In
particular, the
peptides are sufficiently pure and are sufficiently free from other biological
constituents of
their hosts cells so as to be Laeful in, for example, producing pharmaceutical
preparations or
secuencing. Because an isolated peptide of the invention may be admixed with a
pharmaceutically acceptable carrier in a pharmaceutical preparation, the
peptide may
3o comprise only a small percentage by weight of the preparation. The peptide
is nonetheless
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substantially pure in that it has been substantially separated from the
substances with which
it rnay be associated in living systems.
MBL binding peptides also may easily be :>ynthesized or produced by
recombinant
means by those of skill in the art. Methods for preparing or identifying
peptides which bind
s to a particular target are well known in the art. Molecular imprinting, for
instance, may be
used for the de novo constn::ction of maeromoleeular structures such as
peptides which bind
to a particular molecule. See for example Kenneth J. Shea, Molecular
Imprinting of
Synthetic Network Polymers: The De Novo synthesis of Macromolecular Binding
and
Catalytic Sites, TRIP Vol. 2, No. 5, May 1994; Klaus Mosbach, Molecular
Imprinting,
~o Trends in Biochenz Sci., 1~~(9) January 1994; andl Wulff, G., in Polymeric
Reagents and
Catalysts (Ford, W. T., E~i.) AC.S Symposium Series No. 308, pp 186-230,
American
Chemical Society (1986). One method for prep~~ring mimics of MBL binding
peptides
involves the steps of: (i) polymerization of functional monomers araund a
known MBL
binding peptide or the bin~~ing region of an anti-MBL antibody (such as the
deposited
t s am:ibodies) (the template) that exhibits a desiredl activity; (ii) removal
of the template
molecule; and then I,iii) polymerization of a second class of monomers in the
void left by
the. template, to provide a new molecule which exhibits one or more desired
properties
wl-~ich are similar to that of the template. In addition to preparing peptides
in this manner
other MBL binding molecules which are MBL inhibitors such as polysaccharides,
2o nucleosides, drugs. nucleoproteins, lipoproteins, carbohydrates,
glycoproteins, steroids,
lipids, and other biologically active materials can ;also be prepared. This
method is useful
for designing a wide variety of biological mimics that are more stable than
their natural
co anterparts, because they are typically prepared by the free radical
polymerization of
functional monomers. resul:ing in a compound with a nonbiodegradable backbone.
Other
zs mf;thods for designing such molecules include for example drug design based
on structure
acrivity relationships which require the synthesis aaad evaluation of a number
of compounds
and molecular modeling.
Peptides which bind to the MBL may also be identified by conventional
screening
mcahods such as phage display procedures (e.g., met.hods described in Hart, et
al., J. Biol.
3o Chem. 269:12468 ( 1994);1. Hart et al. report a. filamentous phage display
library for
idE:ntifying novel peptide ligands for mammalian cell receptors. In general,
phage display
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libraries using, e.g., M13 or fd phage, are prepared using conventional
procedures such as
those described in the foregoing reference. The libracries display inserts
containing from 4
to 80 amino acid residues. The inserts optionally rc;present a completely
degenerate or a
biased array of peptides. I,igands that bind selectively to MBL are obtained
by selecting
s those phages which express on their surface a liga~ld that binds to the MBL.
These phages
thc;n are subjected to several cycles of reselection to identify the peptide
ligand-expressing
phages that have the most useful binding characteristics. Typically, phages
that exhibit the
best binding characteristics (e.g., highest affinity) arc: further
characterized by nucleic acid
analysis to identify the particular amino acid sequences of the peptides
expressed on the
~ o phage surface and the optirrmm length of the expressed peptide to achieve
optimum binding
to the MBL. Alternatively, such peptide ligands can be selected from
combinatorial
li>"~raries of peptides containing one or more amino acids. Such libraries can
further be
synthesized which contain non-peptide synthetic moieties which are less
subject to
enzymatic degradation com,aared to their naturally-occurring counterparts.
i s To determine whether a peptide binds to MBL any known binding assay may be
employed. For example, the peptide may be immobilized on a surface and then
contacted
with a labeled MBL. The ;mount of MBL which interacts with the peptide or the
amount
which does not bind to the peptide may then be quantitated to determine
whether the peptide
bi nds to MBL. A surface having the deposited monoclonal antibody immobilized
thereto
2o m ay serve as a positive com.rol.
Screening of peptides of the invention., also can be carried out utilizing a
ccmpetition assay. If the peptide being tested competes with the deposited
monoclonal
antibody, as shown by a decrease in binding of the deposited monoclonal
antibody, then it is
lil:ely that the peptide and the deposited monoclonal antibody bind to the
same, or a closely
2s related, epitope. Still another way to determine whether a peptide has the
specificity of the
deposited monoclonal antibody of the invention is 'to pre-incubate the
deposited monoclonal
antibody with MBL with which it is normally reactive, and then add the peptide
being tested
to determine if the peptide being tested is inhibited in its ability to bind
MBL. If the peptide
bc;ing tested is inhibited thc;n, in all likelihood, it has the same, or a
functionally equivalent,
3o epitope and specificity as the deposited monoclonail antibody.
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Using routine procedures known to those of ordinary skill in the art, one can
determine whether a peptide which binds to MBI, is useful according to the
invention by
determining whether the peptide is one which bIoc:ks MBL from binding to an
MBL ligand.
Such assays are described above and in the Examples section. Other assays will
be apparent
s to those of skill in the art, having read the present specification, which
are useful for
determining whether a peptide which binds to MBL also inhibitors LCP
associated
complement activation.
By using the deposi~.ed monoclonal antibodies of the invention, it is now
possible to
produce anti-idiotypic antibodies which can be used to screen other antibodies
to identify
~ o whether the antibody has the same binding specificity as the deposited
monoclonal
antibodies of the invention.. In addition, such a~zti~-idiotypic antibodies
can be used for
active immunization (Herlyn, et al., Science, 232:100., 1986). Such anti-
idiotypic antibodies
ca:n be produced using well-known hybridoma techniques (Kohler and Milstein,
Nature,
256:495, 1975). An anti-idiotypic antibody is an antibody which recognizes
unique
is determinants present on the deposited monoclonal antibodies. These
determinants are
located in the hypervariable region of the antibody. It is this region which
binds to a given
epitope and, thus, is respc.nsible for the specificity of the antibody. An
anti-idiotypic
antibody can be prepared by immunizing an animal with the deposited monoclonal
antibodies. The immunized animal will recognize and respond to the idiotypic
determinants
20 of the immunizing depos,.ted monoclonal antibodies and produce an antibody
to these
idiotypic determinants. By using the anti-idiotypic antibodies of the
immunized animal,
which are specific for the deposited monoclonal antibodies of the invention,
it is possible to
idc;ntify other clones with the same idiotype as the deposited monoclonal
antibody used for
immunization. Idiotypic identity between monoclonal antibodies of two cell
lines
2s de monstrates that the two monoclonal antibodies are the same with respect
to their
recognition of the same epitopic determinant. Thus, by using anti-idiotypic
antibodies, it is
possible to identify other h.ybridomas expressing monoclonal antibodies having
the same
ep itopic specificity.
It is also possible: to use the anti-idiotype technology to produce monoclonal
3o antibodies which mimic an epitope. For example:, an anti-idiotypic
monoclonal antibody
made to a first monoclonal ;antibody will have a binding domain in the
hypervariable region
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which is the image of the e~~itope bound by the first monoclonal antibody.
Thus, the anti-
idiotypic monoclonal antih~ody can be used for immunization, since the anti-
idiotype
monoclonal antibody binding domain effectively acts as an antigen.
Activation assays al~.o can be used to assess the relative inhibitory
concentrations of
s a peptide in an activation assay and to identify those peptides which
inhibit activation by at
least, e.g., 75%.
Other assays will be: apparent to those of skill in the art, having read the
present
specification, which are useful for determining whether a peptide which binds
to MBL also
inl:.ibits MBL activation.
t o In one embodiment the peptide that inhibits the activation of MBL is an
antibody or
a functionally active antibody fragment. Antibodies are well known to those of
ordinary
skill in the science of immunology. As used herein., the term "antibody" means
not only
int:~ct antibody molecules but also fragments of antibody molecules retaining
MBL binding
ability. Such fragments are also well known in the art and are regularly
employed both in
i s vit~~o and in vivo. In particular, as used herein, the term "antibody"
means not only intact
immunoglobulin molecules but also the well-known active fragments F(ab')2, and
Fab.
F(ab')2, and Fab fragments vrhich lack the Fc fragment of intact antibody,
clear more rapidly
from the circulation, and rr,ay have less non-specific; tissue binding of an
intact antibody
(V4'ahl et al., J. Nucl. Mecl. 24:316-325 (1983)). As is well-known in the
art, the
zo complementarity determini:zg regions (CDRs) of an antibody are the portions
of the
anl:ibody which are largely responsible for antibody specificity. The CDR's
directly interact
with the epitope of the antigen (see, in general, Clark, 1986; Roitt, 1991 ).
In both the heavy
ch~~in and the light chain variable regions of IgG imrnunoglobulins, there are
four
framework regions (FRI through FR4) separated respectively by three
complementarity
2s derermining regions (CDRI through CDR3). The: framework regions (FRs)
maintain the
tertiary structure of the paratope, which is the portion of the antibody which
is involved in
thc; interaction with the antigen. The CDRs, and in particular the CDR3
regions, and more
particularly the heavy chain CDR3 contribute to antibody specificity. Because
these CDR
regions and in particular th~~ CDR3 region confer .antigen specificity on the
antibody these
3o rel;ions may be incorporated into other antibodies or peptides to confer
the identical antigen
specificity onto that antibody or peptide.
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As discussed above ~:he MBL inhibitors of the present invention encompass in
some
embodiments of the invention MBL binding peptides which include a MBL binding
region
which specifically binds to human MBL and inhibits LCP assaciated complement
activation, e.g., by preventing MBL from interacting with MBL ligands. "MBL
ligands" as
s used herein are carbohydra~es or peptides with which MBL can interact.
Optionally the
MBL binding region is a MBL binding CDR3 region. A "MBL binding CDR3 region"
as
used herein is a CDR3 peptide sequence derived from the monoclonal antibodies
produced
by the hybridomas deposited with the ATCC under ATCC Accession No. (HB-12621
),
ATCC Accession No. (HB-12620), and ATCC Accession No. (HB-12619).
~o Three antibody producing hybridoma cell lines (3F8, 2A9, hMBLl.2) were
deposited by Applicants with the ATCC on December 15, 1998. Hybridoma 3F8
produces
monoclonal antibodoy ;FBA having binding specificity for MBL. Monoclonal
antibody 3Fa
includes the CDR3;FS regicm within its sequence. .As used herein "CDR3~3Fg~"
includes the
CDR3 region of monoclonal antibody~3Fx~. H;ybridomat2,a9> produces monoclonal
~s antibody~2A9> having binding specificity for MBL. Monoclonal antibody~2A9~
includes the
CLtR3~2A9~ region within its sequence. As used ',herein "CDR3~2A9~" includes
the CDR3
region of monoclonal <~ntibody 2p9. HybTl(IOma~hMBLl.2) produces monoclonal
antibody~nMem.z~ having binding specificity for MBL. Monoclonal
antibody~hMaL~.z>
includes the CDRJ~h~~BLl.2) region within its sequence. As used herein
"CDR3~,,MBL1.2~"
2o includes the CDR3 region of monoclonal antibody~hMSL~.2~. Each of
monoclonal antibody
3F8~ monoclonal antibody f'9, and monoclonal antlbady~hMBLl.2) specifically
bind to MBL
an~i prevent MBL from interacting with an MBL ligand.
The "MBL binding; C:DR3 region" refers to the CDR3(3Fg~, CDR3(zA9~ and
CI)R3~hMBt.~.2~ peptide sequesnces. In one embodiment the peptides of the
invention include
2s functional variants of the M:BL binding CDR3 region. A "functional variant"
as used herein
is ~t peptide having the sequence of the CDR3~3Fg~, CDR3(2A9), or
CDR3~hMaLl.z> regions with
conservative substitutions therein. As used herein., ".conservative
substitution" refers to an
arr;ino acid substitution whi~~h does not alter the relative charge or size
characteristics of the
pe;~tide in which the amino acid substitution is made. Conservative
substitutions of amino
3o acids include substitutians made amongst amino acids with the following
groups: (1)
M.I,L,V; (2) F,Y,Vv': (3) K,R,H; (4) A,G; (5) S,T; (6) Q,N; and, (7) E,D. Such
substitutions
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can be made by a variety of methods known to one of ordinary skill in the art.
For example,
amino-acid substitutions may be made by PCR-directed mutation, site-directed
rnutagenesis
according to the method oKunkel (Kunkel, Prc~c. Nat. Acad. Sci. U.S.A. 82: 488-
492,
19$5), or by chemical synthesis of a gene encoding; the CDR3 region. These and
other
s methods for altering a CDR3 region peptide will b~e known to those of
ordinary skill in the
art and may be found in rc;ferences which compile such methods, e.g. Sambrook.
et al.,
Molecular Cloning.' A Lab~~ratory Manual, 2nd e;di ion, Cold Spring Harbor
Laboratory
Prcas, 1989. The activity of functionally equivalent variants of the MBL
binding CDR3
region can be tested by the Minding and activity assays discussed above.
~o For purposes of bre~rity the term "ATCC deposited hybridoma" is used
throughout
the' specification to refer to the three hybridomas deposited with the ATCC on
December
15, 1998. The term "deposited monoclonal antibody" is used to refer to each of
the
monoclonal antibodies (monoclonal antibodyt3Fgt, :monoclonal antibody~2A9~, or
monoclonal
an°.ibody~hMSL~.2> produced by the ATCC deposited hybridomas. For
purposes of
i s de Eniteness in the attached claims each of the hybridomas and monoclonal
antibodies is
specifically recited.
According to one embodiment, the peptide of the invention is an intact soluble
anti-
M:BL monoclonal antibody in an isolated form or in a pharmaceutical
preparation. An
intact soluble monoclonal antibody, as is well known in the art, is an
assembly of
2o polypeptide chains linked by disulfide bridges. Two principle polypeptide
chains, referred
to as the light chain and heavy chain, make up all major structural classes
(isotypes) of
antibody. Both heavy chair..s and light chains are further divided into
subregions referred to
as variable regions and constant regions. As used herein the term "monoclonal
antibody"
re~~ers to a homogenous population of immunog;lobulins which specifically bind
to an
2s epitope (i.e. antigenic determinant) of human MBL.
The peptide of the invention in one embodiment is, for example, the deposited
monoclonal antibody. The preparation and use of the deposited monoclonal
antibody is
described more fully in the attached Examples. In another embodiment the
peptide of the
invention is an intact antibody having the binding characteristics of the
deposited
3o m~~noclonal antibody. An antibody having the binding characteristics of the
deposited
monoclonal antibody is one: which binds to MBL and inhibits MBL from
interacting with
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M13L ligands. One of ordinary skill in the art can easily identify antibodies
having the
binding characteristics of the deposited monoclonal antibody using the
screening and
binding assays set forth in detail below.
In one set of embodiments, the peptide useful according to the methods of the
s present invention is an intact humanized anti-MBL monoclonal antibody in an
isolated form
or in a pharmaceutical preparation. The following examples of methods for
preparing
humanized monoclonal antibodies that interact~with MBL and inhibit LCP
associated
complement activation are exemplary and are provided for illustrative purposes
only.
A "humanized monoclonal antibody" as used herein is a human monoclonal
~ o antibody or functionally act:,ve fragment thereof having human constant
regions and a MBL
binding CDR3 region from a mammal of a species other than a human. Humanized
monoclonal antibodies rna;y be made by any method known in the art. Humanized
monoclonal antibodies, for c:xarnple, may be constructed by replacing the non-
CDR regions
of a non-human mammali~~n antibody with similar regions of human antibodies
while
~s retaining the epitopic specificity of the original antibody. For example,
non-human CDRs
and optionally some of thc; framework regions may be covalently joined to
human FR
and/or Fc/pFc' regions to produce a functional antibody. There are entities in
the United
States which will synthesize humanized antibodies; from specific murine
antibody regions
commercially, such as Protein Design Labs (Mountain View California).
2o European Patent Application 0239400, the entire contents of which is hereby
incorporated by reference, provides an exemplary teaching of the production
and use of
humanized monoclonal antibodies in which at least the CDR portion of a murine
(or other
non-human mammal) antibody is included in the humanized antibody. Briefly, the
following methods are useful for constructing a humanized CDR monoclonal
antibody
2s including at least a portion ~~f a mouse CDR. A first replicable expression
vector including
a suitable promoter operabhr linked to a DNA sequence encoding at least a
variable domain
of an Ig heavy or light chain and the variable domain comprising framework
regions from a
human antibody and a CDF: region of a murine antibody is prepared. Optionally
a second
replicable expression vector is prepared which includes a suitable promoter
operabiy linked
3o to a DNA sequence encoding at least the variable: domain of a complementary
human Ig
light or heavy chain respectively. A cell line is then transformed with the
vectors.
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Preferably the cell line is an immortalized mammalian cell line of lymphoid
origin, such as
a myeloma, hybridoma, trio ma, or quadroma cell line, or is a normal lymphoid
cell which
has been immortalized by transformation with a virus. The transformed cell
line is then
cultured under conditions I<:nown to those of skill in the art to produce the
humanized
s antibody.
As set forth in European Patent Application 0239400 several techniques are
well
known in the art for creating the particular antibody domains to be inserted
into the
replicable vector. (Preferre~~ vectors and recombinant techniques are
discussed in greater
detail below.) For example, the DNA sequence encoding the domain may be
prepared by
~o olit;onucleotide synthesis. ~.lternatively a synthetic gene lacking the CDR
regions in which
four framework regions are fused together with suitable restriction sites at
the junctions,
such that double stranded synthetic or restricted sulbcloned CDR cassettes
with sticky ends
could be ligated at the juncv:ions of the framework regions. Another method
involves the
preparation of the DNA sequence encoding the variable CDR containing domain by
~ s olil;onucleotide site-directed mutagenesis. Each of these methods is well
known in the art.
Th~:refore, those skilled in the art may construct humanized antibodies
containing a marine
CIJ~R region without destroying the specificity of they antibody for its
epitope.
In preferred embodiments, the humanized antibodies of the invention are human
monoclonal antibodies including at least the MBL binding CDR3 region of the
deposited
zo monoclonal antibody. As noted above, such humanized antibodies may be
produced in
which some or all of the FR regions of deposited monoclonal antibodies have
been replaced
by homologous human FR :°egions. In addition, the Fc portions may be
replaced so as to
produce IgA or IgM as wel:: as human IgG antibodies bearing some or all of the
CDRs of
the deposited monoclonal antibody. Of particular importance is the inclusion
of the
is deposited monoclonal antibody MBL binding CDR3 region and, to a lesser
extent, the other
CIr~Rs and portions of the framework regions of the deposited monoclonal
antibody. Such
humanized antibodies will have particular clinical utility in that they will
specifically
recognize human MBL buy: will not evoke an immune response in humans against
the
antibody itself. In a most preferred embodiment, a marine CDR is grafted into
the
3o framework region of a humor antibody to prepare thf: " humanized antibody."
See, e.g., L.
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Riechmann et al., Nature :3:32, 323 (1988); M. S. l~leuberger et al., Nature
314, 268 (1985)
and EPA 0 239 400 (published Sep. 30, 1987).
In addition to the deposited monoclonal antibodies, other antibodies (e.g.,
anti-MBL,
anti-MASP, anti-mannan-like antibodies) can be generated. The following is a
description
s of a method for de~-elopinl; a monoclonal antibody specific for MBL (MASP-1
or -2, or
m;mnan). The description i;~ exemplary and is provided for illustrative
purposes only.
Murine monoclonal antibodies may be made by any of the methods known in the
art
utilizing MBL as an immunogen. An example of a method for producing murine
monoclonals useful according to the invention is the following: Female Balb/C
mice were
to initially inoculated (i.p.) wish 250 ul of the following mixture: 250 pl
Titermax mixed with
100 p,g human MBL in 25G pl PBS. The following week and for three consecutive
weeks
thc; mice were injected with 5() p.g hMBL in 250 ~IPBS. On the 4th week the
mice were
injected with 25 ~g h-IBL in 250 p.l PBS and the mice were fused 4 days later.
The fusion protocol is adapted from Current Protocols in Immunology. The
is splenocytes were fused l:l with myelinoma fusion partner P301 from ATCC
using PEG
150 at 50% w/v. The fusec: cells were plated at a density of 1.25x 106/ m.
with 100 ~1/well
of a 96 well microtiter plate. 'The fusion media consisted of Deficient DME
high glucose,
Pen/Strep (50,000 LJ pen, 50,000 ~g strep per lite:r), 4 mM L-glutamine, 20%
fetal bovine
seoum, 10% thyroid enriched media, 1 % OPI, 1 °ro NEAA, 1 % HAT, and 50
p.M 2
2o me:rcaptoethanol. The cells were fed 100 pl/well on day one and 100/well
media were
exchanged on days '_'. 3, ~I, 7, 9, 11, and 13. The last media change before
primary
sc;.-eening consisted of HAT' substituted for the 1 % HT. All subsequent
feedings were done
with fusion media minus the minus HT or HAT. Screening was done with human MBL
pl;rted to plastic EL1S A plates (96 well plates). Purified hMBL was plated in
each well at
2s 50 p,l volume containing 'Z ~g/ml MBL in 2% sodium carbonate buffer. The
plates were
th~.n blocked with 3°,~o BSA. in PBS. Tissue culture media (50 pl) was
placed in the wells
and incubated for 1 hour at room temperature. The plates were washed and a
secondary
H1RP labeled goat anti-mouse IgG antibody was used for detection. Colorimetric
analysis
was done with ABTS and read at a405 nm. Positive controls consisted of a
polyclonal
3o antibody to human ~tBL. Cells are then grown in media consisting of the
following:
DiVIEM high glucose no-I-glut, sod, pyruvate 500m1 (Irvine Scientific #9024),
heat
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inactivated Hyclone 10%, 1 % Non-essential amino acids, 4mM L-gluamine, 100
U/ml
penicillin and 100 pg/ml streptomycin. All positive wells were then screened
for function
in a secondary screen.
Human monoclonal antibodies may be made: by any of the methods known in the
art,
s su~~h as those disclosed in L1S Patent No. 5,567,610, issued to Borrebaeck
et al.. US Patent
No. 565,354, issued to Ostt~erg, US Patent No. 5,571,893, issued to Baker et
al, Kozber, J.
Immunol. 133: 3001 (1984), Brodeur, et al., Monoclonal Antibody Production
Techniques
and Applications, p. 51-63 (Marcel Dekker, Inc, new York, 19$7), and Boerner
el al., J.
Immunol., 147: 86-95 (1991 ). In addition to the conventional methods for
preparing human
~ o monoclonal antibodies, such antibodies may also bc: prepared by immunizing
transgenic
animals that are capable of o~roducing human antibodies (e.g., Jakobovits et
al., PNAS USA,
90: 2551 (1993), Jakobovits et ai., Nature, 362: 255-258 (1993), Bruggermann
et al., Year
in Immuno., 7:33 (1993) anti US Patent No. 5,569,825 issued to Lonberg).
An example of one method for producing :human monoclonals useful according to
~s thc; invention is the following: Peripheral Bloocl Lymphocytes (PBL) are
isolated from
he;~lthy human donors using density centrifugation, and further separated into
B, T and
accessory (A) cells, described methods such as (Danie:lsson, L., Moller, S. A.
& Borrebaeck,
C..A.K. Immunology 61, 51-55 (1987)). PBL are fractionated into T and non-T
cells by
ro~~etting with 2-amino ethyl {isothiouronium brorrride) - treated sheep red
corpuscles, and
2o thc; latter cell population is incubated on Petri dishes coated with
fibronectin or autologous
plasma. Non-adherent cells (B-cells) are decanted" and adherent cells
(accessory cells) are
removed by IOmM EDTA. 'the B cells are stimulated with 50 p g Staphylococcus
aureus
Cowan I/ml and irradiated (2000R) T cells with 10 p g PWM/ml overnight. The
accessory
cells are stimulated with 5 1U gamma interferon/ml and 10 p m indomethacin.
The cell
2s populations are cultured in supplemented RPMI 1 fi40 which contains 10%
human AB
semm at a cell ratio of 2:1:0.4 (Ti:B:A) for a total of 6 days. The antigenic
dose of MBL is
1 la g/ml. The culture is supplemented with recombinant IL-2 (5 U/ml) and sPWM-
T (25%
by vol.), produced by described methods such. as (Danielsson, L., Moller, S.A.
&
Borrebaeck C.A.K. Immamology 61, 51-55 (198'1)). T cells (10 cells/ml)
suspended in
3o serum-free RPMI 1640 are incubated with 2.5 mM freshly prepared Leu-OMe for
40 min at
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room temperature. The cells are then washed 3 times in RPMI 1640 containing 2%
human
antibody serum.
In one embodiment of the invention the peptide containing a MBL binding region
is
a functionally active antibody fragment. Significantly, as is well-known in
the art, only a
s small portion of an antibody molecule, the parai:ope, is involved in the
binding of the
antibody to its epitope {see, in general, Clark, W.P;. (1986) The Experimental
Foundations
of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential
Immunology, 7th Ed., Black:well Scientific Publications, Oxford). The pFc' and
Fc regions
of the antibody, for examphs, are effectors of the complement cascade but are
not involved
~o in antigen binding. An antibody from which the pFe' region has been
enzymatically
cleaved, or which has been produced without the pFc' region, designated an
F(ab')2
fragment, retains both of the: antigen binding sites of an intact antibody. An
isolated F(ab')Z
fragment is referred to as a bivalent monoclonal fragment because of its two
antigen binding
sitc;s. Similarly, an antibody from which the Fc region has been enzymatically
cleaved, or
~ s which has been produced without the Fc region, designated an Fab fragment,
retains one of
the antigen binding sites of an intact antibody molecule. Proceeding further,
Fab fragments
consist of a covalently bound antibody light chain and a portion of the
antibody heavy chain
denoted Fd (heavy chain variable region). The Fd fragments are the major
determinant of
antibody specificity (a single Fd fragment may be associated with up to ten
different light
2o chs~ins without altering antibody specificity) and Fd fragments retain
epitope-binding ability
in isolation. The terms Fab, Fc, pFc', F(ab')2 and Fv are used consistently
with their
standard immunological me,~nings [Klein, Immunology (John Wiley, New York, NY,
1982);
Cl;~rk, W.R. (1986) The Experimental Foundations of Modern Immunology (Wiley &
Sons,
Inc., New York); Roitt, 1. ( 1991 ) Essential Immunology, 7th Ed., (Blackwell
Scientific
2s Publications, Oxford)].
As used herein the term "functionally active antibody fragment" means a
fragment
of an antibody molecule including a MBL binding peptide of the invention which
retains the
LC'.P associated complement inhibitory activity of an intact antibody having
the same
sp~.cificity such as the deposited monoclonal antibodies. Such fragments are
also well
3o known in the art and are regularly employed both ira vitro and in vivo. In
particular, well-
known functionally active antibody fragments include but are not limited to
F(ab')z, Fab, Fv
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ancl Fd fragments of antibodies. These fragments which lack the Fc fragment of
intact
antibody, clear more rapidly from the circulation, and may have less non-
specific tissue
binding than an intact antibody (Wahl et al., J. Nucf. Med. 24:316-325
(1983)). For
ex~unple, single-chain antibodies can be constructed in accordance with the
methods
s described in U.S. Patent No. 4,946,778 to Ladner et al. Such single-chain
antibodies
include the variable regions of the light and heavy chains joined by a
flexible linker moiety.
Mc;thods for obtaining a sinl;le domain antibody ("Fd") which comprises an
isolated
variable heavy chain single domain, also have been reported (see, for example,
Ward et al.,
Nature 341:644-646 (1989), disclosing a method of'sc:reening to identify an
antibody heavy
~o chstin variable region (VH single domain antibody) with sufficient affinity
for its target
epitope to bind thereto in isolated form). Methods for making recombinant Fv
fragments
ba >ed on known antibody heavy chain and light chain variable region sequences
are known
in the art and have been described, e.g., Moore et al., I;JS Patent No.
4,462,334. Other
references describing the use and generation of antibody fragments include
e.g., Fab
~ s fragments (Tijssen. Practice and Theory of Enzyme Immunoassays (Elsevieer,
Amsterdam,
1985)), Fv fragments (Hochman et al., Biochemistrw 12: 1130 (1973); Sharon et
al.,
Bi~xhemistry I5: 1591 (1976); Ehrilch et al., U.S. Patent No. 4,355,023) and
portions of
am.ibody molecules ('Audilore-Hargreaves, U.S. patent No. 4,470,925). Those
skilled in the
art rnay construct amibody fragments from various portions of intact
antibodies without
Zo de;~troying the specificity of the antibodies for the MBL epitope.
Functionally active antibody fragments also encompass "humanized antibody
fra.gments." As one skilled in the art will recognize, such fragments can be
prepared by
traditional enzymatic cleavage of intact humanized antibodies. If, however,
intact
antibodies are not susceptible to such cleavage, because of the nature of the
construction
2s involved, the noted constru~~tions can be prepared with immunoglobulin
fragments used as
thc: starting materials: or, if recombinant techniques are used, the DNA
sequences,
th~;mselves, can be tailored to encode the desired "i:ragment" which, when
expressed, can be
combined in vivo or in vitr~~. by chemical or biological means, to prepare the
final desired
intact immunoglobulin frag:_nent.
3o In addition to the identification of peptides from libraries etc. the
peptides of the
invention including those containing the MBL binding CDR3 region may easily be
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synthesized or produced by recombinant means. Such methods are well known to
those of
orJinary skill in the art. Peptides can be synthesi2;ed for example, using
automated peptide
synthesizers which are commercially available. The peptides can be produced by
recombinant techniques by incorporating the DNA expressing the peptide into an
expression
s vector and transforming cells with the expression vector to produce the
peptide.
The sequence of the CDR regions, for instance, for use in synthesizing
peptides of
thc.~ invention, may be determined by methods known in the art. The heavy
chain variable
region is a peptide which generally ranges from lU0 to 150 amino acids in
length. The light
chain variable region is a peptide which generally ranges from 80 to 130 amino
acids in
~o length. The CDR sequences within the heavy .and light chain variable
regions which
include only approximately 3-25 amino acid sequences may easily be sequenced
by one of
ordinary skill in the art. The: peptides may even be synthesized by commercial
sources such
as by the Scripps Protein ann Nucleic Acids Core Sequencing Facility (La Jolla
California).
The sequences responsible for the specificity of the deposited monoclonal
antibody
is can easily be determined by one of ordinary skill in the art so that
peptides according to the
invention can be prepared using recombinant DN,~1 'technology. There are
entities in the
United States which will perform this function commercially, such as Thomas
Jefferson
University and the Scripps Protein and Nucleic Acids Core Sequencing Facility
(La Jolla
California). For example, the variable region cDNA can be prepared by
polymerase chain
2o reaction from the deposited hybridoma RNA using degenerate or non-
degenerate primers
(derived from the amino acid sequence). The cDNA can be subcloned to produce
sufficient
quantities of double stranded DNA for sequencing by conventional sequencing
reactions or
equipment.
Once the nucleic acid sequences of the heavy chain Fd and light chain variable
2s domains of the deposited MBL monoclonal antibody are determined, one of
ordinary skill
in the art is now enabled to produce nucleic acids which encode this antibody
or which
encode the various antibody fragments, humanized antibodies, or peptides
described above.
It is contemplated that such nucleic acids will be operably joined to other
nucleic acids
forming a recombinant vector for cloning or for expression of the peptides of
the invention.
3o The. present invention includes any recombinant vector containing the
coding sequences, or
part thereof, whether for prokaryotic or eukaryotic transformation,
transfection er gene
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therapy. Such vectors may be prepared using conventional molecular biology
techniques,
known to those with skill in the ari, and would comprise DNA coding sequences
for the
CC~R3 region and additional variable sequences contributing to the specificity
of the
antibodies or parts thereof, as well as other non-specific peptide sequences
and a suitable
s promoter either with (Whittle et al., Protein Eng. 1:499, 1987 and Burton et
al., Science
26fi:1024-1027, 1994) or w~.thout (Marasco et al., Proc. Natl. Acad. Sci.
(USA) 90:7889,
1993 and Duan et al., Proc. Natl. Acad. Sci. (USA) 91:5075-5079,1994) a signal
sequence
for export or secretion. Such vectors may be transformed or transfected into
prokaryotic
(Hose et al., .Science 246:12'75, 1989, Ward et al., Nature 341: 644-646,
1989; Marks et al.,
~o J. l~ol. Biol_ 222:581, 1991 and Barbas et al., Proc, Ncxtl. Acad. Sci.
(U~fA) 88:7978, 991) or
eul<:aryotic (Whittle et al., 1987 and Burton et al., 1994) cells or used for
gene therapy
(Marasco et al., 1993 and Cnan et al., 1994) by conventional techniques, known
to those
with skill in the art.
As used herein, a "vector" may be any of a number of nucleic acids into which
a
is desired sequence may be inserted by restriction and Iigation for transport
between different
genetic environments or for expression in a host cell. Vectors are typically
composed of
DT A although RNA vectors are also available. Vectors include, but are not
limited to,
pla~mids and phagemids. A cloning vector is one which is able to replicate in
a host cell,
anci which is further characterized by one or more endonuclease restriction
sites at which
zo the vector may be cut in a determinable fashion and into which a desired
DNA sequence
may be ligated such that the: new recombinant vector retains its ability to
replicate in the
ho:.t cell. In the case of plasmids, replication of the desired sequence may
occur many times
as the plasmid increases in copy number within the host bacterium or just a
single time per
ho~,t before the host reproduces by mitosis. In the ease of phage, replication
may occur
2s act:.vely during a lytic phase or passively during a lysogenic phase. An
expression vector is
one: into which a desired I):VA sequence may be inserted by restriction and
ligation such
that it is opexably joined v.o regulatory sequence, and may be expressed as an
RNA
transcript. Vectors may further contain one or more marker sequences suitable
for use in
the identification of cells which have or have not been transformed or
transfected with the
3o vector. Markers include, fo;- example, genes encoding proteins which
increase or decrease
either resistance or sensitivity to antibiotics or other compounds, genes
which encode
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en:ymes whose activities are detectable by standard assays known in the art
(e.g.,
13-~;alactosiidase or alkaline phosphatase), and genes which visibly affect
the phenotype of
tra:lsformed or transfected cells, hosts, colonies or :plaques. Preferred
vectors are those
capable of autonomous replication and expression of the structural gene
products present in
s the DNA segments to which they are operably joined.
The expression vectors of the present invention include regulatory sequences
operably
joined to a nucleotide sequence encoding one of the; peptides of the
invention. As used
herein, the term "regulatory sequences" means nucleotide sequences which are
necessary
for or conducive to the transcription of a nucleotide sequence which encodes a
desired
~ o peptide and/or which are necessary for or conducive to the translation of
the resulting
transcript into the desired peptide. Regulatory sequences include, but are not
limited to, S'
sequences such as operator, promoters and riboso~mc: binding sequences, and 3'
sequences
such as polyadenylation signals. The vectors of the invention may optionally
include 5'
leader or signal sequences, S' or 3' sequences encoding fusion products to aid
in protein
is purification, and various markers which aid in the identification or
selection of
transformants. The choice and design of an appropriate vector is within the
ability and
discretion of one of ordinary skill in the art. The subsequent purification of
the peptides
ma.y be accomplished by am/ of a variety of standard means known in the art.
A preferred vector for screening peptides, but not necessarily preferred for
the mass
2o production of the peptides of the invention, is a recombinant DNA molecule
containing a
nu~~leotide sequence that codes for and is capable of expressing a fusion
peptide containing,
in the direction of amino- to carboxy-terminus, (1) a prokaryotic secretion
signal domain,
(2) a peptide of the invention, and, optionally, (3) a fusion protein domain.
The vector
includes DNA regulatory sequences for expressing the fusion peptide,
preferably
2s prokaryotic regulatory sequences. Such vectors can be constructed by those
with skill in the
art and have been described by Smith et al. (Science 228:1315-1317, 1985),
Clackson et al.
(:~'~ture 352:624-628, 1991 ); Kang et al. (in "Methods: A Companion to
Methods in
Enzymology: Vol. 2", R.A. Lerner and D.R. Burton, ed. Academic Press, NY, pp
111-
118,1991 ); Barbas et al. (Pnoc. Natl. Acad. S'ci. (L'S~l) 88:7978-7982, 1991
), Roberts et al.
30 (P.roc. Natl. Acad. Sci. (USfI) 89:2429-2433, 1992)
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A fusion peptide ma:r be useful for purification of the peptides of the
invention. The
fusion domain may, for example, include a poly-His tail which allows for
purification on
Ni-~- columns or the maltose: binding protein of the commercially available
vector pMAL
(New England BioLabs, Be~rerly, MA). A currently preferred, but by no means
necessary,
s fusion domain is a filamentous phage membrane anchor. This domain is
particularly useful
for screening phage display libraries of monoclonal antibodies but may be of
less utility for
the mass production of antibodies. The filamentouso phage membrane anchor is
preferably a
domain of the cpIII or cp'1III coat protein capable of associating with the
matrix of a
filamentous phage particle, i:hereby incorporating the fusion peptide onto the
phage surface,
io to enable solid phase binding to specific antigens or epitopes and thereby
allow enrichment
and selection of the specific antibodies or fragments. encoded by the phagemid
vector.
The secretion signal is a leader peptide domain of a protein that targets the
protein
membrane of the host cell, such as the periplasmic membrane of gram negative
bacteria. A
preferred secretion signal fc~r E. coli is a pelB secretion signal. The
predicted amino acid
is residue sequences of the secretion signal domain :from two pelB gene
producing variants
from Erwirria carotova are described in Lei, et al. (Nature 381:543-546,
1988). The leader
secuence of the pelB protein has previously been used as a secretion signal
for fusion
proteins (Better, et al., Science 240:1041-1043, 1988; Sastry, et al., Proc.
Natl. Acad. Sci
(USA) 86:5728-5732, 1989; and Mullinax, et al., Proc. Natl. Acad S'ci. (USA)
87:8095-
20 80!9, 1990). Amino acid re;~idue sequences for other secretion signal
peptide domains from
E. coli useful in this invention can be found in Olives, In Neidhard, F.C.
(ed.), Escherichia
coli and Salmonella Typhimurium, American Society for Microbiology,
Washington, D.C.,
1:56-69 (1987).
To achieve high levels of gene expression in E. coli, it is necessary to use
not only
2s strong promoters to generate large quantities of mRNA, but also ribosome
binding sites to
ensure that the mRNA is effciently translated. In E. coli, the ribosome
binding site
includes an initiation codon (AUG) and a sequence 3-9 nucleotides long located
3-11
nucleotides upstream from the initiation codon (Shine, et al., Nature 254:34,
1975). The
sequence, AGGAGGU. which is called the Shine-Dalgarno (SD) sequence, is
3o complementary to the 3' end of E. coli 16S rRNA. Binding of the ribosome to
mRNA and
the sequence at the 3' end of the mRNA can be affected by several factors:
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(I) The degree oi' complementarity between the SD sequence and 3' end of the
16S rRNA.
(ii) The spacing and possibly the DNA sequence lying between the SD sequence
and the AUG (Roberts, et al., Proc. l1~'atl. Acad. Sci. (LISA) 76:760.,1979a:
s Roberts, et al., Proc. Natl. Acad. Sci. (USA) 76:5596, 1979b; Guarente, et
al., Science :09:1428, 1980; and Guarente, et al., C.'ell 20:543, 1980).
Optimization is achieved by measuring the level of expression of genes in
plasmids in which this spacing is systematically altered. Comparison of
different mRrJAs shows that there a:re statistically preferred sequences from
positions -20 to +13 (where the A of the AUG is position 0) (Gold, et al.,
Arrnu. Rev. Microbiol. 35:365, 1981). L eader sequences have been shown to
influence translation dramatically (Roberts, et al., 1979a, b supra).
(iii) The nucleotide sequence following the AUG, which affects ribosome
binding (Taniguchi, et al., J. Mol. Binl_, 118:533, 1978).
i s The. 3' regulatory sequences define at least one termination (stop) codon
in frame with and
ope;rably joined to the heterologous fusion peptide.
In preferred embodiments with a prokaryotic: expression host, the vector
utilized
includes a prokayotic origin of replication or replicon, i.e., a DNA sequence
having the
ability to direct autonomous replication and maintenance of the recombinant
DNA molecule
2o extra-chromosomally in a prokaryotic host cell, such as a bacterial host
cell, transformed
therewith. Such origins of replication are well I<;nown in the art. Preferred
origins of
replication are those that are efficient in the host organism. A preferred
host cell is E. toll.
Fo:- use of a vector in E. toll, a preferred origin of replication is ColE1
found in pBR322
and a variety of other common plasmids. Also preferred is the plSA origin of
replication
2s found on pACYC and its derivatives. The ColEl and plSA replicons have been
extensively
utilized in molecular biology, are available on a variety of plasmids and are
described by
Sa~nbrook. et al.. :Molecular Cloning: A Laboratory Manual, 2nd edition, Cold
Spring
Harbor Laboratory Press, 1989).
In addition. those embodiments that include a prokaryotic replicon preferably
also
3o include a gene whose expret~sion confers a selective advantage, such as
drug resistance, to a
banterial host transformed therewith. Typical bacterial drug resistance genes
are those that
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confer resistance to ampicillin, tetracycline, neo:mycin/kanamycin ar
chloramphenicol.
Vectors typically also contain convenient restriction sites for insertion of
translatable DNA
seq uences. Exemplary vectors are the plasmids pUC 18 and pUC 19 and derived
vectors
such as pcDNAII available fiom Invitrogen, (San Diego, CA).
s When the peptide of the invention is an antibody including both heavy chain
and
light chain sequences, these sequences may be encoded on separate vectors or,
more
conveniently, may be expressed by a single vector.. 'the heavy and light chain
may, after
translation or after secretion, form the heterodirneric structure of natural
antibody
molecules. Such a heterodirneric antibody may or rnay not be stabilized by
disulfide bonds
~o between the heavy and light chains.
A vector for expression of heterodimeric antibodies, such as the intact
antibodies of
the invention or the F(ab')2, Fab or Fv fragment antibodies of the invention,
is a
recombinant DNA molecule adapted for receiving and expressing translatable
first and
second DNA sequences. That is, a DNA expression vector for expressing a
heterodimeric
is antibody provides a system :for independently cloning (inserting) the two
translatable DNA
sequences into two separate cassettes present in the vector, to form two
separate cistrons for
exyressing the first and second peptides of a heterodirneric antibody. T'he
DNA expression
vector for expressing two cis,trons is referred to as a dicistronic expression
vector.
Preferably, the vecv:or comprises a first cassette that includes upstream and
zo downstream DNA regulatory sequences operably joined via a sequence of
nucleotides
adapted for directional ligation to an insert DNA.. The upstream translatable
sequence
preferably encodes the secretion signal as described above. The cassette
includes DNA
regulatory sequences for expressing the first antic>ody peptide that is
produced when an
insert translatable DNA sequence (insert DNA) is directionally inserted into
the cassette via
zs the sequence of nucleotides ;adapted for directional ligation.
The dicistronic exprcasion vector also contains a second cassette for
expressing the
second antibody peptide. The second cassette includes a second translatable
DNA sequence
that preferably encodes a secretion signal, as described above, operably
joined at its 3'
terminus via a sequence of nucleotides adapted far directional ligation to a
downstream
3o DrdA sequence of the vector that typically defines at least one stop codon
in the reading
frame of the cassette. The second translatable DNA sequence is operably joined
at its 5'
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tern..inus to DNA regulatory sequences forming the 5' elements. The second
cassette is
cap2~ble, upon insertion of a translatable DNA sequence (insert DNA), of
expressing the
second fusion peptide comprising a secretion signal with a peptide coded by
the insert
DN~~.
s The peptides of the present invention rnay also, of course, be produced by
eukaryotic
cell; such as CHO cells, human hybridomas, immortalized B-lymphoblastoid
cells, and the
like. In this case, a vector is constructed in which eukaryotic regulatory
sequences are
operably joined to the nucleotide sequences encoding the peptide. The design
and selection
of an appropriate eukaryotic vector is within the ability and discretion of
one of ordinary
o skill in the art. The subsequent purification of the peptides may be
accomplished by any of
a variety of standard means known in the art.
In another embodiment, the present invention provides host cells, both
prokaryotic
and eukaryotic, transformed ~~r transfected with, and therefore including, the
vectors of the
present invention.
t s As used herein, a coding sequence and regulatory sequences are said to be
"operably
joined" when they are covulently linked in such a way as to place the
expression or
transcription of the coding sequence under the influence or control of the
regulatory
sequences. If it is desired that the coding sequences be translated into a
functional peptide,
two DNA sequences are said to be operably joined if induction of a promoter in
the 5'
2o regulatory sequences results in the transcription of tlhe coding sequence
and if the nature of
the linkage between the two DNA sequences does not ( 1 ) result in the
introduction of a
frame-shift mutation, (2) i:m:erfere with the ability of the promoter region
to direct the
transcription of the coding sc;quences, or (3) interfere with the ability of
the corresponding
RN.~ transcript to be translated into a protein. Thus, a promoter region would
be operably
zs joined to a coding sequence if the promoter region were capable of
effecting transcription of
that DNA sequence such tlmt the resulting transcript might be translated into
the desired
peptide.
The precise nature of the regulatory sequences needed for gene expression may
vary
between species or cell type., but shall in general include, as necessary, 5'
non-transcribing
3o and 5' non-translating sequences involved with initiation of transcription
and translation
respectively, such as a TATA box, capping sequence, CAAT sequence, and the
like.
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Especially, such 5' non-transcribing regulatory sequences will include a
promoter region
which includes a promoter sequence for transcriptional control of the operably
joined gene.
Regulatory sequences may also include enhanc;er sequences or upstream
activator
sequences, as desired.
s According to the methods of the invention, the compositions may be
administered in
a ~~harmaceutically acceptable composition. In general, pharmaceutically-
acceptable
carriers for monoclonal antibodies, antibody fragments, and peptides are well-
known to
tho;~e of ordinary skill in the art. As used herein, a pharmaceutically-
acceptable carrier
means a non-toxic material that does not interfere 'with the effectiveness of
the biological
~o activity of the active ingredients, i.e., the ability of the MBL inhibitor
to inhibit LCP
associated complement activation. Pharmaceutically acceptable carriers include
diluents,
fillers, salts, buffers. stabilizers, solubilizers and other materials which
are well-known in
the art. Exemplary pharmaceutically acceptable carriers for peptides in
particular are
des~,ribed in U.S. Patent No. 5,211,657. The peptides of the invention may be
formulated
~s rote preparations in solid, semi-solid, liquid or gaseous forms such as
tablets, capsules,
powders, granules, ointments, solutions, depositories, inhalants (e.g.,
aerosols) and
injections, and usual ways for- oral, parenteral or surgical administration.
The invention also
embraces locally administering the compositions of the invention, including as
implants.
According to the methods of the invention the compositions can be administered
by
2o injection by gradual infusion over time or by any other medically
acceptable mode. The
administration may, for example, be intravenous, intraperitoneal,
intramuscular, intracavity,
subcutaneous or transdermal. Preparations for pa.renteral administration
includes sterile
aqueous or nonaqueous solutions, suspensions andl emulsions. Examples of
nonaqueous
sohrents are propylene glycol, polyethylene glycol, vegetable oil such as
olive oil, an
2s injeetable organic esters such as ethyloliate. Aqueous carriers include
water,
alcoholic/aqueous solutions, emulsions or suspensions., including saline and
buffered media.
Par~nterai vehicles include sodium chloride solution, Ringer's dextrose,
dextrose and
sodium chloride, lactated Kinger's or fixed oils. Intravenous vehicles include
fluid and
nutrient replenishers, electrolyte replenishers, (such as those based on
Ringer's dextrose),
3o and the like. Presen~atives ;end other additives may also be present such
as, for example,
ant:.microbials, antioxidants, chelating agents, and inert gases and the like.
Those of skill in
CA 02347734 2001-04-18
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the art can readily determine the various pararneters for preparing these
alternative
pharmaceutical compositions without resort to undue experimentation. When the
compositions of the inventio n are administered for the treatment of pulmonary
disorders the
compositions may be delivered for example by aerosol.
s The compositions o:f the invention are administered in therapeutically
effective
am~~unts. As used herein, an "effective amount" of the inhibitor of the
invention is a dosage
which is sufficient to inhibit the increase in, m<~intain or even reduce the
amount of
undesireable LCP associated complement activation. The effective amount is
sufficient to
produce the desired effect of inhibiting associated cellular injury until the
symptoms
~o ass~~ciated with the MBL mediated disorder are ameliorated or decreased.
Preferably an
effc;ctive amount of the peptide is an effective .amount for preventing
cellular injury.
Generally, a therapeutically effective amount may vary with the subject's age,
condition,
and. sex, as well as the extent of the disease in the subject and can be
determined by one of
skill in the art. The dosage may be adjusted by the: individual physician or
veterinarian in
i s the event of any complication. A therapeutically effective amount
typically will vary from
about 0.01 mg/kg to about _'~00 mg/kg, were typically from about 0.1 mg/kg to
about 200
mg.~kg, and often from about 0.2 mg/kg to about 20 mg/kg, in one or more dose
administrations daily, for one or several days (.depending of course of the
mode of
administration and the factors discussed above). A preferred concentration of
the inhibitor
2o is a concentration which is equimolar to the concentration of MBL in the
plasma of a
subject. The normal plasma concentration of MBI_, c;an be assessed clinically.
A normal
range of MBL is 1-2yg/ml MBL,/plasma.
One of skill in the art can determine what an effective amount of an inhibitor
is by
scr~:ening the ability of the inhibitor to inhibit the LCP associated
complement activation in
2s an in vitro assay. The activity of the inhibitor canr be defined in terms
of the ability of the
inhibitor to inhibit LCP associated complement activation. An exemplary assay
for
measuring the ability of a putative inhibitor of the invention to inhibit LCP
associated
complement activation is provided in the Examplc;s and has been discussed
above. The
exemplary assay is predictv.ve of the ability of an inhibitor to inhibit LCP
associated
3o complement activation in vivo and, hence, can be used to select inhibitors
for therapeutic
applications.
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The MBL inhibitors may be administered in a physiologically acceptable
carrier.
The: term "physiologically-a~~ceptable" refers to a non-toxic material that is
compatible with
the biological systems such of a tissue or organism, The physiologically
acceptable carrier
must be sterile for in viva administration. The characteristics of the carrier
will depend on
s the route of administration. The characteristics of the carrier will depend
on the route of
adrninistration.
The invention further provides detestably labeled, immobilized and toxin
conjugated
forms of the peptides, antibodies and fragments thereof. The antibodies may be
labeled
using radiolabels, fluorescent labels, enzyme labels, fivee radical labels,
avidin-biotin labels,
~o or bacteriophage labels, using techniques known to the art (Chard,
Laboratory Technigues
in Biology, "An lntroduct.on to Radioimmunoassay and Related Techniques,"
North
Holland Publishing Company (1978).
Typical fluorescern: labels include fluorescein isothiocyanate, rhodamine,
phycoerythrin, phycocyanin. allophycocyanin, and fluorescamine.
~ s Typical chemiluminescent compounds include luminol, isoluminol, aromatic
acridinium esters, imidazole~, and the oxalate esters.
Typical biolumines<;ent compounds include luciferin, and luciferase. Typical
en.,ymes include alkaline phosphatase, 13-galactosidase, glucose-6-phosphate
dehydrogenase, maleate dehydrogenase, glucose oxidase, and peroxidase.
2o The invention also includes methods for screening a subject for
susceptibility to
tre;~tment with an MBL inhibitor. In one aspect, the method is accomplished by
isolating a
mammalian cell from a subject and detecting the presence of an MBL or an MBL
ligand on
a ~~urface of the mammalian cell. The presence o:f the MBL indicates that the
cell is
su~;ceptible to LCP-associated complement activiation, and that the subject is
susceptible to
2s treatment with an MBL inhibitor. The mammalian cell may be isolated by any
method
known in the art, for instance by a biopsy. Another method for accomplishing
the screening
as~,ay involves the steps of contacting a mammalian cell from the subject with
a labeled
isolated MBL binding peptide and detecting the presence of an MBL on the
surface of the
mammalian cell. This assay may be performed in vitro, ex vivo, or in vivo.
Many labels
3o v~~l~iich can be used to observe the MBL binding peptide interacting with
the mammalian cell
area known in the art under each of these conditions. For instance,
radioactive compounds
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can be used in vitro, and other biocompatible labels can be used ex vivo or in
vivo. Once the
subjects are identified which are susceptible to treatment with an MBL
inhibitor, the
subjects can then be treated ;according to the methods of the invention.
The following examples are provided to ilhustrate specific instances of the
practice
s of the present invention and are not to be construed as limiting the present
invention to these
ex~unples. As will be apparent to one of ordinary skill in the art, the
present invention will
fine application in a variety ~f compositions and methods.
Examples
~o Ex,~mple 1:MBL and Complement Deposition on Human Coronary Arteries.
Isolation and Purification ofMBL. MBL and associated MASPs were purified from
human plasma. MBL was isolated from human plasma as previously described{Tan,
Chung, et al. 1996 Biochem. J. 319, 329-332}. Briefly, human plasma was mixed
with 7%
PEG3500 (w:v). The pellet was collected by centrifugation and resuspended in
TBS-Ca2+
is [50 mM Tris, 150 mM N;~CI, 0.05% Tween 20 and 20 mM CaCl2 at pH 7.8]. The
supernatant was applied to a mannan-Sepharose column (25 ml, Sigma). The
column was
washed with TBS-C'.a2+ with 109mM EDTA]. "I'he protein containing supernatant
was
calcified to 40 mM calcium and then applied to a maltose-Sepharose column (5
ml). The
column was washed with T'I3S-Ca2+ and then eluted. with TBS-Ca2+ containing
100 mM N-
2o ace~tylglucosamine. Western analysis and SDS-PAGE established purity for
MBL, and the
ab:;ence of IgG and IgM. Purified MBL and associated MASPs were analyzed by
SD S/PAGE. Western blotting was performed to rule out IgG andlor IgM
contamination.
Production of Anti-.~luman MBL Antibodies. Purified human MBL was used to
immunize rabbits to produ~~e polyclonal anti-human. MBI. antibodies (Harlow E,
et al.,
2s An!ibodies: A laboratoy manual. Cold Spring Harbor, NY, Cold Spring Harbor
La~oratory, 1988). Adult rabbits were injected with 100 p,g of MBL emulsified
in complete
Fre;und's adjuvant. Booster immunizations ( 100 ~g of MBL in incomplete
Freund's
adjuvant) were started 4 wk after the priming immunir..ation and continued at
4 wk intervals.
Polyclonal IgG anti-human MBL antibody (R2.2) was purified from sera by
protein G
3o affinity chromatography.
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Human Coronary Ar,!ery Immunohistochemistry. Immunohistochemical analysis of
MH~L, C3d, IgG, IgW, transferrin, and haptoglobin deposition was performed on
tissue
spe~~imens from normal (rn-=14) and atherosclerotic human epicardial coronary
arteries
(n=18) obtained at autopsy (Department of Pathology, University of Helsinki,
Finland) from
s patients who expired secor,,dary to an acute myocardial infarction (MI). The
control
spe~eimens were histologically normal coronary arteries obtained from patients
who died
from non-cardiovascular causes. The mean (~ SD) MI age (time difference
between the
beginning of the clinical episode and death) was :5 =~ 5 days. The mean (~ SD)
age of
patients suffering from acute MI was 65 t 15 years compared to 66 ~ 24 years
for the
to control patients. Infarcted myocardium was identified macroscopically at
autopsy by
dis<;olor, pallor, and hyperemia. To improve macroscopic diagnosis, a slice of
non-fixed
myocardium was incubated in nitroblue tetrazolium solution that leaves the
damaged
my~~cardium unstained. Histopathological first signs of infarction were wavy
myocardial
fibers and myocytolysis followed by signs of coagulation necrosis (i.e.,
edema, hemorrhage,
is neutrophil infiltration and pyknosis of nuclei). Infarcts older than 24 hr
showed signs of
total coagulative necrosis with loss of nuclei and striations together with
heavy interstitial
neutrophil infiltration. Coronary blood vessel samples for indirect
imrnunofluorescence (IFI~) microscopy were snap frozen in liquid nitrogen and
stored at -
80 "C until analyzed. Frozen sections (4 pm) were air dried and fixed in -20
°C acetone for
20 10 ~nin. The tissue samples were then incubated for 30 min at 22 °C
with either polyclonal
rab'oit anti-human C3d (Dakopatts, Glostrup, Denmt~rk), MBL (polyclonal R2.2),
IgG, IgM,
trar~sferrin, or haptoglobin antibody (all from Behringwerke AG, Germany).
After washing
wit:1 PBS, the specimens were then stained with an appropriate fluorescein
isothiocyanate
(FI'rC)-conjugated secondary antibody. Controls consisted of specimens
incubated with
2s nor.immune sera or the secondary antibody alone. The slides were then
mounted with
Mowiol and examined with au~ Olympus Standard microscope equipped with a
filter specific
for FITC-fluorescence.
Results. Atherosclerotic coronary arteries obtained from patients suffering
from
acute MI demonstrated specific MBL and C3d deposits on the endothelium,
intima, and
3o media Immunohistochemical. analysis of human coronary arteries, and in
particular, the IFL
mic;roscopical demonstration of MBL and C3d deposition in an atherosclerotic
human
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coronary artery was performed. MBL and C3d were observed co-localized within
the
atherosclerotic lesion. M>3L staining of a normal coronary artery was also
performed.
An.tisera against human transferrin, haptoglobin, IgG, and IgM did not stain
normal or
atherosclerotic human coronary arteries.
s Additionally, MBL was observed to co-localize with C3d, with staining
intensity
being greatest in ruptured atherosclerotic plaques. Specifically, MBL and C3d
deposition
appeared to be greatest in the lipid core and surrounding areas of this core
in atherosclerotic
lesions. No MBL deposits were seen on normal coronary arteries, although the
basement
membrane sometimes appeared to stain lightly for MBL. Further, antisera
against human
~o transferrin, haptoglobin, Ig~3, and IgM did not stain normal human coronary
arteries or
ath.erosclerotic lesions in weasels obtained from acute MI patients.
Similarly, no staining
was observed in control experiments in which human coronaries were stained
with non
immune rabbit serum or with the secondary antibody only. These data
demonstrated that
M13L co-localized with complement in human coronary atherosclerotic lesions in
patients
t s wl-.o have died of acute MI.
Example 2: Endothelial Hy~~oxia/Reoxygenation Effects MBL Deposition.
Cell Culture. Hum;~n umbilical vein endothelial cells (HUVECs) were harvested
with 0.1% collagenase (Worthington Biochemical Corp., Freehold, NJ) and
suspended in
2o Mc;dia 199 containing 20% heat-inactivated bovine calf serum (Gibco Life
Technologies
Inc., Grand Island, NY). The cells were initially seeded in either 75 cmz
flasks or 100 mm
Petri dishes (Corning Costar, Cambridge, MA), and incubated at 37°C in
95% air and 5%
CO2. When confluent, the endothelial cells were passaged with 0.5% trypsin-
EDTA.
Endothelial cell purity was assessed by phase microscopic "cobblestone
appearance",
2s uptake of fluorescent acetylated low-density lipoprotein and the presence
of won Willebrand
facaor. All experiments were conducted on HUVE(~s during passages 1-3.
MBL-depleted Human Serum (HS). HS was depleted of MBL by affinity
chromatography using manr~an cross-linked to 4% beaded agarose (Sigma Chemical
Co., St.
Lcuis, MO). All operations were performed at 4 '°C. HS was treated with
2 mmol/L
3o ethylenediamine tetraacetai:e (EDTA) and phen,ylrnethanesulfonyl fluoride
(PMSF) to
inhibit complement activation and was applied to a mannan column equilibrated
with
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loading buffer (1.25 mmol/I, NaCI, 10 mmol/L imidazole, 20 mmol/L CaCl2, pH
7.8). The
resultant eluent was dialyzed overnight in Hank's buffered salt solution
containing Mg2+
anti Ca2+.
Flow Cytometry. HUVECs were grown to confluence in 100-mm Petri dishes
s coated with gelatin. MBI, deposition was measured by flow cytometry in
normoxic
HL~VECs and HUVECs subjected to 24 hr of hypoxia followed by 3 hr of
reoxygenation in
the presence of 30% HS. After washing the cells in Ca2+ free or sufficient
buffer, the cells
we re fixed, scraped, and then incubated with 20 ~,g,~ml of monoclonal anti-
human MBL
antibody (Biodesign, Kenr~ebunk, ME, clone # l!. 31-1 ) or isotype control
monoclonal
~o antibody to porcine CSa for 1.5 hr at 4 °C. The cells were then
washed and incubated with
a FITC-conjugated goat anti-mouse IgG secondary antibody for 1 hr at 4
°C. MBL
deposition on HUVECs was measured by florescence activated cell sarting (FACS)
using
the FACSort flow cytometer (Becton Dickinson, San Jose, CA). All flow
cytometry
experiments were performed in triplicate.
~s Enryme-LinlcEd Imrr~unoabsorbent Assay (ELISA) Experiments. C3 and MBL
specific cell surface ELISA;~ were developed using; peroxidase-conjugated
polyclonal goat
anti-human C3 antibody (C'.appel, West Chester, lPA) and monoclonal anti-human
MBL
antibody (Biodesign, Kennebunk, ME, clone #131-l ), respectively. HUVECs were
grown to
confluence on 0.1 % gelatinized 96-well plastic plates (Corning Costar,
Cambridge, MA).
2o Th~~ plates were then subjecaed to 0 (normoxia) or 24 hr of hypoxia.
Hypoxic stress was
maintained using a humidified sealed chamber (Cov Laboratory Products, Inc.,
Grass Lake,
MI) at 37 °C gassed with 1°,'° 02, 5% C02, balance
N(Collard CD, et al., "Reoxygenation
of hypoxic human umbilical vein endothelial cells activates the classical
complement
pathway", Circulation 199'x;96:326-333). Following the specified period of
normoxia or
Zs hypoxia, the cell media were aspirated and 100 ~.1 o:f one of the following
was added to each
well: 1) 30% HS, 2,1 Hank's balanced salt solution, 3) 30% HS + 3, 30, or 300
mmol/L
GIoNAc, 4) 30% HS + 3, 30, or 300 mrnol/L D-mannose, 5) 30% HS + 3, 30, 300
mmol/L
L-mannose, 6) 30% MBL-depleted HS + 3F8 (0, 20, 50 ~g/ml)or 7) 30% MBL-
depleted HS
+ (f.6 ~g/ml MBL. Additionally, 100 pl of purified MBL (3-300 ng/ml) was added
to select
3o wells to form a standard curve for quantitative analysis of MBL deposition.
The cells were
then reoxygenated for 3 hr at 37°C in 95% air and _'>% COZ. The cells
were washed and
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then fixed with 1% paraforrnaldehyde (Sigma Chemical Co., St. Louis, MO) for
30 min.
The cells were then washed and incubated at 4 °C l:or 1.5 hr with 50
p.l of peroxidase-
conjugated polyclonal goat anti-human C3 antibody (1:1000 dilution) or
monoclonal anti-
human MBL antibody ( 1:1000 dilution). The MB:L ELISA plates were then washed
and
s incubated for 1 hr at 4 °C', with 50 pl of peroxidase-conjugated
polyclonal goat anti-mouse
IgG secondary antibody (1:1000 dilution). After washing the cells, the plates
were
developed with 50 p.1 of ABTS (2,2'-azino-bis(3-ethy:lbenzthiazoline-6-
sulfonic acid)), and
react (Molecular Devices, S unnyvale, CA) at 405 nm. Background controls for
the C3
EL1SA consisted of cells to which only the anti-hutrian C3 antibody was added
(i.e., no HS)
~o or cells incubated with 30% .'teat-inactivated HS. Background controls for
the MBL ELISA
consisted of cells to which only secondary antibody and an isotype control
monoclonal
antibody to porcine C~a were added. Background optical density was subtracted
from all
groups. All ELISA experiments were performed _'3 times using 6 wells per
experimental
group. Endothelial C3 and MBL deposition on n~ormoxic vs. hypoxic HUVECs was
~s analyzed by two-way analysis of variance (ANOVA).
Results. Flow cytc~metric analysis (Figure 2) of endothelial MBL deposition
rev~:aled that the mean fluorescent intensity (MFI) of hypoxic HUVECs (24 hr)
reo:~cygenated (3 hr) in 30°,~o HS was significantly greater than
normoxic HUVECs or
hyf~oxic HUVECs reoxygenated in buffer alone.. Further, MBL deposition was not
20 observed following hypoxia/reoxygenation if the cells were washed in Caz+-
free buffer.
Thus, MBL deposition on hypoxic/reoxygenated HLJVECs was Ca2+-dependent.
In order to further confirm these findings, MBI~ deposition was measured by
ELISA
on normoxic HUVECs and HUVECs subjected to 24 hr of hypoxia followed by 3 hr
of
reoxygenation in the presence of 30% HS or 30% I-1S treated with 3, 30, or 300
mmol/L of
2s N-acetyl-D-glucosarnine (GIuNAc) to competitively inhibit MBL deposition.
MBL
def~osition on hypoxic HL~VECs reoxygenated i.n 30% HS was significantly
greater
(approximately 3-fold increase; p<0.05) than on normoxic HUVECs or HUVECs
reoxygenated in HS treated with GIuNAc (Figure 3). Addition of GIuNAc to the
HS
significantly inhibited MBI. deposition on hypoxic/reoxygenated HUVECs in a
dose-
3o deyendent manner with 3, 30 and 300 mmol/L of GIuNAc attenuating MBL
deposition
40==4%, 715% and 9613°/~, respectively. Finally, quantitative analysis
of the standard
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curve formed by the addi~.ion of purified human MBL (3-300 ng/ml) revealed
that
approximately 3 ng or 8.3x10~s fmol of MBL. maximally deposits per well (e.g.,
48"?001000 moleculeslcell) of hypoxic/reoxygenated HUVECs assuming 2x10s
HUVECs/well and a MBL MW of 600 kDa. 'Chas, hypoxia/reoxygenation increased
s endothelial MBL deposition.
Exaunple 3: Deposition of iC'3b Following Competitive Inhibition of MBL.
HUVEC cell culture ;end quantitation of iC3b deposition by ELISA were
performed
as outlined in Example 2.
~o Results. HUVECs vrere subjected to 0 or 24 hr of hypoxia followed by 3 hr
of
reo:~cygenation in the presen<;e of 30% HS or 30% HS treated with 3, 30, or
300 mmol/L
GIuNAc, D-mannose or L-mannose in order to inhibit MBL deposition, LCP
activation and
iC3b deposition. Deposition of iC3b on hypoxic H1:IVECs reoxygenated in 30% HS
or
30°~o HS treated with L-:nannose was signific;amtly greater
(approximately 3-fold;
~ s OD4os=0.140.01; p<0.05) than normoxic HUVECs (OD4os=O.OSt0.01 ) or hypoxic
HUVECs reoxygenated in H~~ treated with GIuNAc or D-mannose (Figure 4a).
Further, D-
matrrrose, but not I,-mannose, inhibited iC3b deposition on
hypoxic/reoxygenated HUVECs
in dose-dependent manner with 3, 30 and 300 m~mol/L of D-maumose attenuating
iC3b
deposition 192%, ~:?t3% a,nd 962%, respectively. Thus, these data demonstrated
that
2o inhibition of MBL deposition using GIuNAc or D-mannose during reoxygenation
significantly attenuated complement activation and iC3b deposition following
reo:cygenation of hypoxic endothelial cells. Further., inhibition of iC3b
deposition with
maumose was stereospecifrc as L-mannose in concentrations up to 300 mmol/L did
not
inhibit iC3b deposition (Figure 4a).
Example 4: Deposition of iC3b Following MBL Depletion and Reconstitution.
HUVEC cell culture and quantitation of iC3b deposition by ELISA were carried
out as in Example 2.
Results. HU~'ECs vrere subjected to 0 or 24 hr of hypoxia followed by 3 hr of
3o reo:~ygenation in the presenr.e of 30% HS, 30% MBL,-depleted HS or 30% MBL-
depleted
HS to which MBL was added back (Figure 4b). Deposition of iC3b on hypoxic
HUVECs
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reoxygenated in HS was significantly greater (p<0.05) than on normoxic HUVECs.
However, iC3b deposition cm hypoxic HUVECs reoxygenated in MBL-depleted HS was
significantly less (p<0.05) than on hypoxic HUVECs reoxygenated in HS. When
MBL was
adc.ed back to the MBL-depleted HS, iC3b deposition on HUVECs following 24 hr
of
s hypoxia and 3 hr of reoxygenation was restored. These data demonstrated that
reoxygenation of hypoxic human endothelial cells activated the LCP leading to
increased
deposition of iC3b.
Ex~unple 5: Complement hemolytic assay (CHSOLof"MBL-depleted HS.
~o Methods. Hemolytic assays were completed as previously described by us
{A::nsterdam, Stahl, et al., Limitation of reperfusion injury by a monoclonal
antibody to CSa
during myocardial infarction. in pigs, Am. J. Physiol. Heart Circ. Physiol.
1995; 268:H448-
H457} {Lennon, Collard, et al., Complement-induced endothelial dysfunction in
rabbits:
me~~hanisms, recovery, and ;sender differences, Am. J. Physiol. Heart Circ.
Physiol., 1996;
is 27(I:H1924-H1932}{Vakeva, Agah, et al. Myocardial infarction and apoptosis
after
myocardial ischemia and re perfusion. Role of the terminal complement
components and
inhibition by anti-CS therapy., Circulation 1998; 97:2259-2267}. Briefly,
chicken red blood
cel;,s were sensitized with sl:.eep anti-chicken antibodies. Serial dilutions
of sera were then
used to lyse the cells. Hemolytic activity was calculated by using 0.1% Triton
X100 and
Zo PB S as positive and negative controls, respectively. C>ptical density was
read at 550 nm on
a plate reader. Percent hemolytic activity was calculated as follows:
(Sample OD - PBS) / (Triton. OD - PBS) X 100 = % hemolytic OD
Samples were run in triplicate and at three determinations per group were
performed.
Results. Complement hemolytic assay (CH<;o) was performed on the MBL-depleted
2s HS in order to demonstrate; that MBL depletion did not alter the classical
complement
pathway. CHso of the MBI,-depleted HS revealed classical complement pathway
activity
similar to that of complete 1-1S (Figure 5). Similar :findings were observed
when antibodies
3F3, and 2A9 were used. 'thus, the decrease in iC',3b deposition on hypoxic
HUVECs
reoxygenated in MBL-depleted serum was not a result of altered classical
pathway
3o complement components (i.e., C: l q, C 1 r, and C 1 s).
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Ex~unple 6: Western blot analysis of C3 activation following
hypoxia/reoxygenation using
purified C2, C3, C4, and ME~L.
Western Blot. HU~~ECs were grown to confluence in 96 well plates and then
subjected to normoxia or h~rpoxia (24 hr). The cells were then washed with
GVB+ and
s reoxygenated for 3 hr in the presence of 50 Pl of the following complement
cocktail: MBL
(1.~! p,g/ml), C2 (8 p.g/ml), C3 (400 ~g/ml), and C~4 (200 p,g/ml) (C2, C3,
and C4 were
purchased from Advanced Research Technologies:; San Diego, CA). These
complement
concentrations were represc;ntative of the concentrations normally present in
30% HS.
Following reoxygenation, the supernatants were collected and the protein
concentration
io determined (BioRad, Hercules, CA). Five p.g of total protein was then
resolved by 9%
SDS-PAGE under reduced conditions. The gel was then transferred to
nitrocellulose,
blocked, and probed for the C3 and C3b a'-chain by western blot (Collard CD,
"Rc:oxygenation of hypoxic human umbilical vein endothelial cells activates
the classical
complement pathway", Circ:ulation 1997;96:326-333). Purified C3 and C3b
{Advanced
~s Re:;earch Technologies; San Diego, CA) served as internal standards for MW
comparisons
of the cleaved C3 a'-chain. This experiment was performed 5 times (n=5).
Results. Western blot analysis of the C3 and C3b a'-chain was performed under
reduced conditions on the supernatants of normo:Kic and hypoxic (12 hr) HUVECs
reoxygenated (3 hr) in the presence of purified C2, C3, C4, and MBL (Figure
6). A
2o sig:zificant increase in the C3b a'-chain band density was observed in the
hypoxic/reoxygenated supernatants (Lanes 2 and 4) compared to the normoxic
supernatants
(Lz.nes 1 and 3). These results demonstrated LC'.P-mediated activation of C3
following
endothelial hypoxic/reoxygenation independent of natural antibody or Cl. Thus,
complement activation following endothelial h;ypoxia/reoxygenation appeared to
be
2s mediated by the LCP and not the classical complement pathway.
Ex;~mple 7: Microphysiometer evaluation of HUVF;C receptor-ligand activation.
Microphysiometry. C'.hanges in 1-IUVEC ext:racellular acidification rate (EAR)
were
evaluated by use of a Cytosensor micraphysiometer (Molecular Devices,
Sunnyvale, CA).
3o HtIVECs were grown to 75% confluence on gelatin-coated (I%) transwell
capsules and
subjected to 24 hr of hypo:Kia followed by 3 hr o~f rreoxygenation. Following
30 min of
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equilibration in modified RF'MI containing 1 mmol/L phosphate buffer
(Molecular Devices,
Sunnyvale, CA), the EARS were determined (Gronert K, et al., "Characterization
of human
neutrophil and endothelial cell ligand-operated extacellular acidification
rate by
mi~.rophysiometry: Impact of reoxygenation", J.Pharmacol.Exp.Ther.
1998;285:252-261).
s HLfVECs were perfizsed with 300-1500 ng/ml of purified MBL (dialyzed in the
modified
RPMI) for 30 sec before the first rate measurement and perfusion was
maintained for 40
min. As a positive control, the HUVECs were perfused with media alone for 15
min
following MBL exposure ar,.d then stimulated with histamine (1 pmol/L, 15 min
perfusion)
to evoke extracellular acidvfication. Each concentration of MBL was analyzed
in two
~o independent chambers containing normoxic or hypoxic/reoxygenated HUVECs.
The
HLfVEC response to each MBL concentration was evaluated in 3 separate
experiments
(n==3).
Results. Microphysiometry was performed on normoxic and hypoxic/reoxygenated
HLfVECs in order to determine if MBL evoked receptor-mediated changes in the
~s endothelial EAR. Neither perfusion (40 min) of normoxic or hypoxic {12 hr)
/
reoxygenated (3 hr) I~IJVEC'.s with purified MBL (30(I -1500 ng/ml) evoked a
change in the
EAR, whereas all cells rem~~ined responsive to the agonist histamine. Thus,
MBL did not
evoke receptor-mediated changes in the EAR in normoxic or hypoxic/reoxygenated
HL~VECs. These data indicated that MBL binding t:o reoxygenated HUVECs
occurred via a
2o MI3L ligand and not a classi~~al receptor.
Ex;~mple 8: Preparation and Characterization of Monoclonal Antibodies to Human
MBL.
Female Balb/C mice were initially inoculated (i.p.) with 250 ul of the
following
mi;tture: 250 pl Titermax mixed with 100 pg human MBL in 250 pl PBS. The
following
2s week and for three consecutive weeks the mice were injected with 50 pg hMBL
in 250
pIPBS. On the 4th r~-eek the mice were injected with 25 pg MBL in 250 pl PBS
and the
mi~~e were fused 4 days later. 'The fusion protocol was adapted from Current
Protocols in
Immunology. The splenocytes were fused 1:1 with myelinoma fusion partner P301
from
ATCC using PEG 150 at 50'% w/v. The fused cells were plated at a density of
1.25x106/ m.
3o with 100 p,l/well of a 96 well microtiter plate. The: fusion media
consisted of Deficient
DME high glucose, Pen/Strep (50,000 U pen, 50,000 ~g strep per liter), 4 mM L-
glutamine,
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20~'i° fetal bovine serum, 10'% thyroid enriched media, 1 % OPI, 1 %
NEAA, 1 % HAT, and
50 p.M mercaptoethanol. 7'he cells were fed 100 ul/ well on day one and
100/well media
were exchanged on days 2, 3, 4, 7, 9, 11, and 13. T'he last media change
before primary
screening consisted of HAT substituted for the 1 % HT . All subsequent
feedings were done
s with fusion media minus the minus HT or HAT. S<;reening was done with human
MBL
plated to plastic ELISA plates (96 well plates). Purified hMBL was plated in
each well at
50 pl volume containing 2 ug/ml MBL in 2% sodium carbonate buffer. The plates
were
then blocked with 3% BSA in PBS. Tissue culture media (50 pl) was placed in
the wells
and incubated for 1 hour at room temperature. Tlle plates were washed and a
secondary
to HR.P labeled goat anti-mouse IgG antibody was used for detection.
Colorimetric analysis
was done with ABTS and read at a405 nm. Positive controls consisted of a
polyclonal
antibody to human MBL. Cells are then grown in media consisting of the
following:
DWEM high glucose no-I-glut, sod, pyruvate :500m1 (Irvine Scientific #9024),
heat
inactivated Hyclone 10%, 1 % Non-essential amino acids, 4mM L-gluamine, 100
U/ml
~ s penicillin and 100 ~tg/ml streptomycin. All positive wells were then
screened for function
in ~i secondary screen.
Fu.netional Screen fon Anti-II~IBL antibodies.
Methods. The functional screen for inhibition ~of MBL function by anti-human
MBL
2o antibodies was adapted from the literature{Super, Lc;vinsky, et al., The
level of mannan-
binding protein regulates the binding of complc;ment-derived opsonins to
mannanand
zymosan at low serum concentrations, Clin. Exp. Immunol. 1990; 79:144-150}.
Briefly, 100
pl of meannan (0.5 mg/ml in sodium carbonate/bicarbonate buffer, pH 9.6) was
added to
R:LA/EIA plates at 4(: overnight. The plates were then washed 3 times in
PBS/0.5% Tween
2s pH 7.3, once in PBS and finally in veronal-buffered saline. Human serum is
diluted to 4%
in VBS containing 5 mM (".a2~ and Mgz+. Diluted sera and tissue culture
supernatant or
purified antibody (~~arious concentrations) are then 1:1 to a mannan-coated
well to yield a
final volume or 100 ~l at a concentration of 2% human sera. The plate is then
incubated at
37~~ for 30 min. Positive ar,~d negative controls consist of human sera
without and with 100
3o mr~I N-acetlglucosamine (GluNac). The plates are then washed four times in
PBS/Tween.
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The plates are then incubated with an anti-human C3 polyclonal antibody
coupled with HRP
( 1 hours at RT), washed and developed with ABTS and read at 405 nm.
Results. Antibody production and characl:erization. Following a primary screen
using a solid phase antibody-capture ELISA, we identified 1 I clones that
recognized human
s M13L. After limiting dilution and isotyping, we identified eight mAbs that
recognized
human MBL in an antibody~~capture ELISA. Clones 3F8, 2A9, and hMBLl.2 were
isotyped
as mouse IgG~k, where as clone 1C10 was a mouse IgG2b. The other hybridomas
produced
Iglvl antibodies and were not included in this study.
Western blot analysis was used to determine that the mAbs recognized MBL. As
~o shown in Figure 7, antibodies 2A9 (Lane 1), hM:BI,1.2 (Lane 2), 1C10 (Lane
3) or 3F8
(L;~ne 4) recognized purified and reduced human )VIBL [i.e., molecular weight
(MW) ~32
kl;~]. Thus, these antibodie~~ are specific for human MBL. Clones hMBLl.2, 2A9
and 3F8
have been deposited at the International Desposito~y Authority with A'TCC
designations of
HI3-12619, HB-12620 and I-IB-12621, respectively.
~ s The most potent inhibitor of MBL induced complement activiation, N-
acc;tylglucosamine (GIuNAc) inhibited C3 deposition to plastic in mannan
coated plates in a
dope-dependent manner with an EC50 of approxirr~ately 1 nM. Similarly, 2A9 and
hMBL
l.a; inhibited C3 deposition with and EC50 of approximately 30 and 50 nM,
respectively.
Are isotype control antibody that recognizes MBh by solid phase ELISA did not
inhibit
2o M13L dependent C3 deposition. Thus, these antibodies are approximately 105 -
106 times
more potent than GIuNAc. The data represent 3 separate experiments with at
least 4
observations per experiment. HUBECs were hypoxic for 24 hours and then
reoxygenated in
30% human sera. iC3b deposition was then normalized to normoxic cells. An
approximate
190% increase in iC3b deposition on hypoxic cells was observed following
reoxygenation
zs (Fi gore 8). 3F8 attenuated iC3b deposition on hypoxic/reoxygenated HUVECs
in a dose-
de pendent manner. These: data demonstrate thz~t specific inhibition of MBL
with an
antibody attenuates complement activation and iC3b deposition following
hypoxia/reoxygenation of human endothelial calls. *p<0.05 compared to all
groups; n+2.
3o En.ample 9:
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Complement activation and deposition following HUVEC oxidative stress. To
chz~racterize further the functional properties of these novel mAbs and to
demonstrate
specifically the role of MBI, in complement activation following oxidative
stress of human
endothelial cells, we assessed MBL and C3 deposition on hypoxic human
endothelial cells
s fol owing reoxygenation in human sera.
Western blot analysis. To demonstrate the complement inhibitory action of
these
anti-human MBL mAbs, hypoxic HUVECs were reoxygenated in human sera treated
with
PBS (vehicle), 3F8, hMB:Ll.2, 2A9, or 1C10 (50 pg/ml final concentration).
Cell
membrane bound proteins were resolved by SDS-PAGE under reduced conditions,
~o transferred to membranes, and analyzed for human C3dg (i.e., part of the a-
chain of iC3b).
Th~~ a- and [3-chain of iC;sb were the only C3 stainable bands present on the
cellular
membranes. A representative C3dg band for vehicle, 3F8-, hMBL 1.2-, 2A9- and 1
C 10-
tre~~ted cells was observed. We observed a significant decrease in C3dg band
intensity on
cells reoxygenated in human sera treated with either 3F8, 2A9 or hMBLl.2.
However, the
is non-functional clone, 1C10, did not decrease iC3b deposition (i.e., C3dg
band intensity) on
the endothelial membranes. These data further support the role of MBL-
dependent
complement activation following reoxygenation of hypoxic HUVECs. Further,
these data
confirm that clone 1 C10 is an isotype control mAb that does not functionally
inhibit MBL.
Confocal microscopy studies. Dual labeling for MBL and C3 deposition on
2o normoxic and hypoxic HUVECs was performed to demonstrate co-Localization of
these
complement components and MBL-dependent complement pathway activation.
Normoxic
and hypoxic HUVE(a were reoxygenated in 30% HS treated with and without mAb
3F8 (5
pg.~ml) or 1 C 10 (50 p,g/ml). MBL (blue), C3 (green) and nuclei (red) were
then stained on
the same slide and anzlyzed by immunofluorescent confocal microscopy. Small
amounts of
2s C3 and MBL staining were observed under normoxic conditions, confirming our
finding of
low level C3 deposition under normoxic conditions, confirming our fording of
low level C3
deposition under normoxic conditions. C3 and IvIBL staining on
hypoxic/reoxygenated
HtJVECs was significantly greater than normoxic H1:IVECs. Clone 1C10 failed to
inhibit
C3 or MBL deposition following oxidative stress. C3 and MBL staining was
significantly
3o de~~reased on hypoxic/reox:/genated HUVECs treated with mAb 3F8 (5 p.g/ml)
to levels
below those observed under normoxic conditions (similar results were observed
with mAbs
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hMBL 1.2 or 2A9). It was observed that MBL and C3 co-localize on human
endothelial
ce:.ls under the conditions outlined above. These data demonstrate that
functional inhibition
of MBL with a mAb attenuates C3 deposition following oxidative stress of human
endothelial cells.
s
Example 10:
Methods: VCAM-1 ELISA. Briefly, HUVECs were grown to confluence on 0.1%
gelatinized 96-well plastic plates and then subjected to 0 or 12 hr of
hypoxia. The cell media was
them aspirated and HBSS, 30°/~ HS or 30% HS treated with 3F8 (5 p.g/ml)
was added to each well.
t 0 The cells were then reoxygenaied for 3 hr at 37°C in 95'% air and
5% C02. The cells were washed,
fixed, washed again, and incubated at 4°C for 1.5 hr with the anti-
human VCAM-1 mAb (clone
6G 10 obtained from the Developmental Studies Hybridorria Bank, University of
Iowa, Iowa City,
IA). A peroxidase-conjugated goat anti-mouse secondary antibody (Cappel, West
Chester, PA) was
then used. An inappropriate isotype control antibody (nnA.b GSI to porcine
CSa) was used to assess
t 5 bankground optical density an~~ fluorescence was subtracted from the data.
These experiments (6
wells per experimental group) were performed 3 times (n=~3).
Results: Inhibition of VCAM-I expression following oxidative stress. We have
demonstrated that oxidative stress of HUVECs activate, complement and results
in CSb-9 dependent
VCAM-1 induction. Thus, we: examined VCAM-1 expression by ELISA to demonstrate
further the
20 functional significance of MBL inhibition. As shown in Figure 9 and
confirming our own findings,
reoxygenation of hypoxic 1-II1'VECs in 30% HS treated 'with PBS (vehicle)
resulted in a significant
increase in VCAM-I protein expression. Treatment of 30'% HS with 3F8 (S pg/ml)
significantly
attenuated VCAM-1 expression. Since VCAM-1 expression in this model is
mediated by CSb-9,
these data demonstrated that C:Sb-9 formation is dependent on MBL depositian
and lectin pathway
25 activation.
The foregoing writven specification is considered to be sufficient to enable
one
skilled in the art to practice the invention. The present invention is not to
be limited in
sc~~pe by examples provided. since the examples are intended as a single
illustration of one
asy~ect of the invention and other functionally equivalent embodiments are
within the scope
30 of the invention. Various modifications of the invention in addition to
those shown and
described herein will become apparent to those skilled in the art from the
foregoing
description and fall within the scope of the appended claims. The advantages
and objects of
th~~ invention are not necessarily encompassed by each embodiment of the
invention.
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All references, patents and patent publications that are recited in this
application are
incorporated in their entiret~~ herein by reference.
W~~ claim:
