Note: Descriptions are shown in the official language in which they were submitted.
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INHIBIT~RS OF FACTOR XII ACTIVATION AND - -
APPLICATIONS THEREOF
5This invention is in the area of irnmunology/biochemistry, and presents
inhibitors of Factor XIIa activation and medical applications of the inhibitor, alone or in
combination, for the prophylactic or therapeutic treatment of sepsis, and the prevention
of toxic side effects associated with cytokine therapy. The preferred inhibitors are
antibody which may include polyclonal, or monoclonal antibody, or fragments derived
10 therefrom, or recombinant constructs having the binding activity of such antibodies.
In the United States alone nosocomial bacterernia develops in about 191,000
patients, and of these about 75,000 die. Maki, D.G., 1981, Nosocomial Infeçt.,
(Dikson, R.E., Ed.), page 183, Yrke Medical Books, U.S.A.. Most of these deaths
are attributable to six major grarn-negative bacilli, and these are Pseudomonas
15 aeru~inosa, Escherichia coli~ Proteus, Klebsiella, Enterobacter and Serratia. The
cu~rent treatrnent for bacteremia is the adrninistration of antibiotics which,
unfortunately, have limited effectiveness.
Although the precise pathology of bacteremia is not completely elucidated, i~ isbelieved that bacterial endotoxins, lipopolysaccharides (LPS), are the primary causative
20 agents. LPS consist of at least three significant antigenic regions, the lipid A, core
polysaccharide, and O-specific polysaccharide. The latter is also referred to as O-
specific chain or simply O-antigen. The O-specif1c chain region is a long-chain
polysaccharide built up from repeating polysaccharide units. The number of
polysaccharide units differs among different bacterial species and may vary from one to
25 as many as six or seven monosaccharide units. While the O-specific chain varies
among different gram-negative bacteria, the lipid A and core polysaccharides are similar
if not identical. Since LPS plays a key role in sepsis, a variety of approaches have been ~ -
pursued to neutralize its activity. Presently, there is considerable work which suggest
that antibody to LPS will soon be a valuable clinical adjunct to the standard antibiotic
30 therapy.
LPS initiates a cascade of biochemical events that eventually causes the death of
the patient. One of the clinical symptoms of sepsis is intravascular coagulation which is
reflected in decreased plasma concentrations of various coagulation factors, such as
Factor XII. This aspect of the clinical course of the disease is consistent with in vitro
35 studies which have shown that LPS can activate both the contact system of intrinsic
coagulation, as well as the complement system. Morris, E.C., et al., 1974, LQf
Experimental Med., 140:797 and Morrison, D.C., et al., 1978, American Journal ofPatholo"v, ~:527.
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Factor XII, also known as Hageman Factor, is a plasma serine protease
zymogen. Mason, J.W., et al., 1979, Annals of Intemal Medicine, 73:545. Human
Factor XII is a single-chain Beta globulin of molecular weight 80,000. During
activation, it is cleaved to a two-chain enzyme, terrned Factor XlIa, consisting of a
heavy chain of 50,000 molecular weight and a light chain of 28,000 molecular weight.
Both chains are linked together by a single disulfide bridge. The light chain isenzymatically active, while the heavy chain binds to solid surfaces during contact
activation.
LPS leads to the activation of Factor XII, which in turn is involved in causing
coagulation, a major clinical manifestation of sepsis. In addition, activation of Factor
XII is involved in causing hypotensive reactions via activation of prekallikrein, which
in turn cleaves bradykinin from kininogen. Colman, 1984, J. Clin. Invest. 73:1249.
Thus, it will appreciated that agents that prevent the activation of Factor XII would be
valuable therapeutics or prophylactics that could be used to preven~ or treat disease.
Both monoclonal and polyclonal antibody to Fac~or XII have been described.
For example, monoclonal antibody that binds to the heavy chain of Factor XII is
described by Small et al., (1985) Blood, vol. 65: p. 202; Saito et al., (1985) Blood,
vol. 65: p. 1263; and Pixley et al., (1987) J. Biol. Chem., vol. 262: 10140.
Monoclonal antibody to the light chain of Factor XII are shown in PCT patent
application WO 89/11865.
Polyclonal antibody has been reported by Lammle et al., (1986) AnaL
Biochem. vol. 156: p.118 and Lamrnle et al., (1986) Thromb. Res. vol. 41: p. 747.
A first object of the invention is a description of inhibitors of Factor XII
activation that are useful medicaments for the prophylactic or therapeutic treatment of
sepsis.
A second object of the invention is a description of antibody inhibitors of Factor
XII activation that are useful medicarnents for the prophylactic or therapeutic treatment
of sepsis. Exemplary antibody would be polyclonal, monoclonal, or recombinant
constructs, or fragments derived therefTom having the binding activity of such antibody
or antibody fragments.
A third object of the invention is a description of antibody inhibitors of Factor
XII activation that are useful medicaments for the prophylactic or therapeutic treatment
of sepsis that are characterized in having some, but not necessarily solely the following
properties: substantially inhibits the amidolytic activity of Factor XIIa, indicating that it
affects the catalytic center of the molecule: reacts with native Factor XII: and is
substantially unreactive with Factor XIIa bound to Cl inhibitor.
WO 91/17258 PCr/US91/02990
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A fourth object of the invention is a description of methods for administrating
inhibitors of Factor XII activation that are useful medicaments for the prophylac~c or
therapeutic treatment of sepsis.
- These, and other objects of the invention, will be more fully understood after a
S consideration of the following description of the invention.
Figure 1 shows that the antibody, OT-2, binds to native Factor XII in plasma.
Figure 2 shows the effect of rnAb OT-2 that inhibits the amidolytic activity of
Factor XIIa.
Figure 3 shows that OT-2 inhibits activation of the contact system in plasma.
10 Inhibition occurs at concentrations equimolar to those of Factor XII. DXS stands for
dextran sulfate.
Figure 4 shows that mAb OT-2 inhibits activity of Factor XIIa bound to an
activator, glass, whereas a natural inhibitor of Factor XIIa, C1 esterase inhibitor (C1-
inh.) does not -:
Figure 5 shows that the antibody OT-2 does not bind to Factor XIIa-C1-
inhibitor complexes.
Several patents/patent applications and scientific references are referred to below
that discuss various aspeets of the materials and methods used to realize the invention.
Because the inventdon draws on these materials and methods, it is thus intended that all
20 of the references, in their entirety, be incorporated by reference.
The kernel of the instant invendon is the realizadon that Factor XII plays a keyrole in the onset and progression of sepsis, and that inhibition of its activation would
prevent or be useful for treating the disease. The preferred embodiment inhibitors ehat
would have this activity are antibody, or molecules with the binding activity of2~ antibody, but by no means should the invention be constructed so narrowly. Virtually
any composition that prevents the activation of Factor XII is intended to come within
the scope of the invention.
Exemplary antibody inhibitors of Factor ~I activation are preferably but not
exclusively polyclonal, monoclonal, recombinant antibody constructs, or fragments
30 derived therefrorn having the binding activity of such antibody or antibody fragments.
Further, applieations of bispecific antibody inhibitors are readily imagined. U.S.
Patent No. 4,474,893.
To more clearly define the present invention, particular terms herein will be
employed according to the following definitions generally consistent with their usage in
35 the art.
"Sepsis" is herein defined to mean a disease resulting from bacterial infection
due to the bacterial endotoxin, lipopolysaccharide (LPS). It can be induced by at least
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the six major gram-negative bacilli and these are Pseudomonas aeru~inosa, Escherichia
coli, Proteus, Klebsiella, Enterobacter and Serratia. It is expected that sepsis induced
by gram-positive organisms may also be beneficially treated with the approaches
described herein.
"Factor XIIa" refers to activated Factor XII, without intending to denote
whether or no~ the heavy chain is contained within this active species.
"a-Factor XIIa" refers to activated Factor XII, that still contains the heavy
chain, this form consists of a disulfide linked dimer (Mr 80,000) of a heavy chain (Mr
50,000) which contains the binding site, and a light chain (Mr 28,000) which contains
the catalytic cenier.
"B-Factor XIla" refers to an active form of Factor XII with Mr 30,000, which
consists of the complete light chain (with the catalytic center) and a disulfide linked
fragment, Mr 2,000, of the original heavy chain. B-Factor-XIIa is formed upon
cleavage of the heavy chain of a-Factor-XIIa.
"Monoclonal antibody" refers to a composition of antibodies produced by a
clonal population (or clone~ derived through mitosis from a single antibody-producing
cell. A composition of monoclonal antibodies is "substantially free of other antibodies"
when it is substantially free of antibodies that are not produced by cells from the clonal
population. The term "substantially free" means approxirnately 5% (w/w) or fewercontaminating antibodies in the composition. Also intended to come within the scope of
the definition are modifications to antibody that increase its effectiveness. A preferred
modification includes conjugation of a water soluble polymer. Preferably the water
soluble polymer is polyethylene glycol, or a functionally related molecule such as, for
exarnple, polypropylene glycol homopolymers, polyoxyethylated polyols, and
polyvinyl alcohol. Derivatization of antibody with such water soluble polymers
increases its in vivo half-life, reduces its immunogenicity, and reduces or eliminates
aggregation of the protein and may reduce its immunogenicity and aggregation tha~
might occur when it is introduced in vivo. Derivatization of proteins generally, or
antibody specifically, with water soluble polymers such as those described above are
presented in U. S. Patent Nos. 4,179,337, issued December 18, 1979, to Davis _ ah,
entitled "Non-immunogenic polypeptides"; and 4,732,863, issued March 22, 1988, to
Tomasi, et al., entitled "PEG-modified antibody with reduced affinity for cell surface
Fc receptors", respectively.
An "antibody-producing cell line" is a clonal population or clone derived
through mitosis of a single antibody-producing cell capable of stable growth ]n v1tro
for many generations.
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"Recombinant antibody" refers to antibody wherein one portion of each of the
amino acid sequences of heavy and light chain is homologous to corresponding
sequences in antibody derived from a particular species or belonging to a particular
class, while the remaining segment of the chains is homologous to corresponding
sequences in another. Most commonly, in a recombinant antibody the variable region
of both light and heavy chain mirrors the variable regions of antibody derived from one
species of mammals, while the constant regions are homologous to the sequences in
antibody derived from another. However, this is not necessarily always the case; for
example, Ward, et al., 1989, Nat~lre, ~1:544, have shown that variable chain alone
can be expressed in bacteria with significant antigen binding activity. Also intended to
come within the scope of "Recombinant antibody" is monoclonal Fab antibody
produced using the techniques described by Huse, W.D. et al., 1989, Science
~:1275.
Two antibodies are "cross-blocking" or have a "shared epitope" when each
antibody effectively blocks the binding of the other antibody in a binding inhibition
assay. Thus, if antibodies A and B are cross-blocking, antibody A would not bind to
its antigen when the antigen had been preincubated with antibody B, and antibody B
would not bind to its antigen when the antigen had been preincubated with antibody A.
The term "binding affinity" or "Ka" of an antibody to its epitope, as used
herein, refers to a binding affinity calculated according to standard methods by the
formula Ka = 8/3(It-Tt), where It is the total molar concentration of inhibitor uptake a
50% tracer, and Tt is the total molar concentration of tracer. See Muller, 1980, L
Immunol. Methods, ~: 345-352.
As used herein, the term "incubation" means contacting antibodies and
25 antigens under conditions that allow for the forrnation of antigen/antibody complexes
(e.g., proper pH, temperature, time, medium, etc.). Also as used herçin, "separating"
refers to any method, usually washing, of separating a composition from a test support
or immobilized antibody, such that any unbound antigen or antibody in the composition
are removed and any antigen/antibody complexes on the support remain intact. Theselection of the appropriate incubation and separaùon techniques is within the skill of
the art.
By C1 inhibitor is meant a plasma glycopro~ein with a molecular weight of
about 105,000 that belongs to the super family of serine protease inhibitors. It inhibits
activated components of the classical pathway of complement, Clr and C1 s, and the
intrinsic coagulation system, Factor XIa, Factor XIIa, and Kallikrein. C1 inhibitor also
interacts with plasmin and tissue plasminogen activator. C1 inhibitor has the further
property of itself being inactivated by proteases, notably elastase. It will, or course, be
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understood that intended to come within the scope of the definition of the Cl inhibitor
are fragments of the molecule that maintain biologically activity.
In a preferred embodiment of the invention, Factor XII antibody producing
imrnunologic cells are isolated from a mammal immunized with Factor XII, and
S immortalized to yield antibody secreting cell lines e.g., hybridomas, triomas, quadroma
etc. Cell lines that secrete the desired antibody can be identified by assaying culture
supernatants for antibody activity. Thus, the invention can be broken down into three
- sections, and each section discussed separately. That is, the immunization procedure,
the cell immortalization procedure, and the identification of the desired Factor XII
antibody.
Factor XII produced using a variety of purification schemes may be used to
immunize an appropriate host animal. The preferred purification scheme is described by
Griffin and Cochrane, 1976, Methods Enzymologv, 45:56. The Factor XII so obtained
may be affinity purified as described below.
A variety of distinguishable immunization protocols may be employed, and
may consist of a primary intravenous, subcutaneous, or intraperitoneal immunization
followed by one or more boosts. A suitable adjuvant may be used to enhance the
irnmune response to Factor XII. An exemplary adjuvant is Freund's adjuvant. The
precise irnmunization schedule is generally not critical, and determinative of which
procedure is employed, is the presence of Factor XII antibody in the host animal as
measured by a suitable assay, described below. A preferred immunization procedure,
however, consists of hyperimmunization with Factor XII by repeated intraperitoneal
injections as described by Nuijens, J. H., et al., 1989, J. Biol. Chem.. 264:12941.
Alternatively, lymphocytes may be immunized in vitro. For example,
immunization of peripheral blood cells may be achieved as described by Boss,
Methods of Enzvmologv,121 (1), and in EPA 86106791.6. Note particularly m vitro
immunization techniques that can be used to produced either murine or human
monoclonal ~Procedures for Transforming Cells, pages 18-32, 140-174, Methods Qf
Enzvmolop,v, vol.l21, part 1). Such techniques are also described by Luben, R. and
Mohler, M., 1980, Molecular Immunologv,17:635, Reading, C. Methods in
Enzvmologv,121 (PartOne):18,orVoss,B., 1986,MethodsinEnzvmology, 1 1:27.
A number of in vitro immunization systems have been shown to be effective for
sensitizing human B-cells. Reading, C., 1982, J. of Tmmun. Methods, ~:261;
In lieu of using Factor XII as immuno_ens, an alternative approach is to
~ 35 synthesize Factor XIIa peptides, and use these as immunogens. For example,
; preferred peptides would be those that are at the catalytic enter of the molecule, and
preferably would encompass amino acid residues his at position 393, asp at position
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WO 91/17258 PCr/l'S9lJ02990
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442 or ser at position 544. The numbering follows the scheme put forth by Cool et al
1985, J. Biol. Chem. 260; 13666. The methods for making antibody to peptides arewell known in the art and generally require coupling the peptides to a suitable carrier
molecule, such as serum albumin. The peptides can be made by techniques well
S known in the art, such as, for example, the Merrifield solid-phase method described in
Science,232:341-347 (1985). The procedure may use cornmercially available
synthesizers such as a Biosearch 9500 au~omated peptide machine, with cleavage of the
blocked amino acids being achieved with hydrogen fluoride, and the peptides purified
by preparative HPLC using a Waters Delta Prep 3000 instrument, on a 15-2011m
Vydac C4 PrepPAK column. Once clones are identified Ihat secrete anti-peptide
antibody, the antibody can be screened for binding and neutralizing activity to Factor
XII.
Antibody to Factor XIla may be either polyclonal, monoclonal, recombinant or
fragments derived therefrom. The antibody is preferably h~man or humanized,
although non-human antibody will perforrn satisfactory.
The preparation of high-titer neutralizing polyclonal antibody can be realiæd byimrnunizing a variety of species and employing one of several different immunization
regimes. The preferred method of the instant invention is to immunize rabbits with
Factor XII prepared in complete Freund's adjuvant by injection into axial lymph nodes.
The animals are subsequently subjected to multiple boosts (containing about half the
original amount of Factor ~I~ in incomplete Freunds adjuvant at about 21-day
intervals. About 10 days following each 21-day interval, 20-30 ml of blood is
removed, the serum isolated and antibody isolated therefrom. This procedure may be
carried out for a period of several months.
Monoclonal antibody may be produced using Factor XII, or peptides/peptide
conjugates of these molecules as described above, and using the procedures described
by Kohler, G. and Milstein, C., 1975, Nature, ;~:495, or modifications thereof that
are known in the art. Using the screening assays described below, the specificity of
antibody produced can be discerned.
The initial work of Kohler and Milstein, above, involved fusing murine
lymphocytes and drug selectable plasmacytomas ~o produce hybridomas. A suitable
plasmacytoma is Sp 2/0-Agl4 and is widely used by practitioners of this art.
Subsequent to the work of Kohler and I~qilstein, the hybridoma technique has been
applied to produce hybrid cell lines that secrete human monoclonal antibodies. The
latter procedures are generally described in Abrams, P., 1986, Methods in
Enzvmologv,121: 107, but other modifications are known to those skilled in the art.
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Regardless of whether murine or human antibody is produced, the antibody secreting
cells are combined with the fusion partner and the cells fused with a suitable fusing
agent, preferably polyethylene glycol, and more preferably polyethylene glycol 1000.
The latter is added to a cell pellet containing the antibody secreting cells and the fusion
5 partner in small amounts over a short period of time accompanied with gentle agitation.
After the addition of the fusing agent, the cell mixture is washed to remove the fusing
agent and any cellular debris, and the cell mixture consisting of fused and unfused cells
seeded into appropriate cell culture chambers containing selective growth media. After
a period of several weeks, hybrid cells are apparent, and may be identified as to
10 antibody production and subcloned to ensure the availability of a stable hybrid cell line.
The preferred antibody is human monoclonal antibody which can be prepared
from Iymphocytes sensitized with Factor XII either in vivo or ~n vitro by
immortalization of antibody-producing hybrid cell lines, thereby making available a
permanent source of the desired antibody, using the cell fusion techniques described
15 above. Alternaively, sensitized lymphocytes may be imrnortalized by a combination of
two techniques, viral transformation and cell fusion. The preferred combination consist
of transforming antibody secreting cells with Epstein-barr virus, and subsequently
fusing the transformed cells to a suitable fusion partner. Such fusion partners are
known in the art, and exemplary partners may be a mouse myeloma cell line, a
20 heteromyeloma line, or a human myeloma line, or odher immortalized cell line. PCT
Patent Application No. 81/00957; Schlom et ~., 1980, PNAS USA, ~ Z:6841; Croce et
al., 1980, NatuTe7 ~:488. The preferred fusion partner is a mouse-human hetero-
hybrid, and more preferred is the cell line designated F3B6. This cell line is on deposit
with the American Type Culture Collection, Accession No. HB8785. It was deposited
~5 April 18, 1985. The procedures for generating F3B6 are described in European Patent
Application, Publication No. 174,204.
Techniques applicable to the use of Epstein-Barr virus transformation and dhe
production of immortal antibody secreting cell lines are presented by Roder, J. ~ al.,
1986, Metho~s in Enzvmo]o~y,121:140. Basically, the procedure consist of isolating
30 Epstein-Barr virus from a suitable source, generally an infected cell line, and exposing
the target antibody secreting cells to supernatants containing the virus. The cells are
washed, and cultured in an appropriate cell culture medium. Subsequendy, virallytransformed cells present in the cell culture can be identified by the presence of the
Epstein-Barr viral nuclear antigen, and transformed antibody secreting cells can be
35 identified using standard methods known in the art. ~ -
It will be apparent to dhose skilled in the art~, and as mentioned above, while the
preferred embodiment of the instant invention is neutralizing Factor XII monoclonal
. W O 91/17258 PC~r/US91/02990
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antibody, such antibody(s) may be altered and still maintain biological activity. Thus,
encompassed within the scope of the invention is antibody modified by reduction to
various size fragments, such as F(ab')2, Fab, Fv, or the like. Also, the hybrid cell
lines that produce the antibody may be considered to be a source of the DNA thatS encodes the desired antibody, which may be isolated and transferred to cells by known
genetic techniques to produce genetically engineered antibody. An example of the latter
would be the production of single chain antibody having the antibody combining site of
. the hybridomas described herein. Single chain antibody is described in U.S. Patent
No. 4,704,692.
A second example of genetically engineered antibody is recombinant, or
chimeric antibody. Methods for producing recombinant antibody are shown in U.S.
Patent No. 4,816,567, to Cabilly, et ah; Japanese patent application, Serial No.84169370, filed August 15, 1984; British patent application 8422238, filed on
September 3,1984; and Japanese patent application, No. 85239543, filed October 28,
1985. Also, British patent application, l\lo. 867679, filed March 27, 1986, describes
methods for producing an altered antibody in which at least parts of the complementary
.- determining regions (CDRs) in the light or heavy chain variable domains have been
replaced by analogous parts of CDRs from an antibody of different specificity. Using
the procedures described therein it is feasible to construct recombinant antibody having
the CDR region of one species grafted onto antibody from a second species that has its
CDR region replaced.
Regardless of the type of antibody, polyclonal, monoclonal or recombinant., it
is desirable to purify the antibody by standard techniques as is known in the art, or as
described by Springer, 1980, Monoclonal Antibodies,: 194, (Eds. Kennett, T.
McKearn and K. Bechtol, Plenum Press, New York. Generally, this consists of at
least one amrnonium sulfate precipitation of the antibody using a 50% ammonium
sulfate solution. Antibody affinity colurnns may also be used.
The preferred Factor XII antibodv is denoted OT-2, and methods and
procedures for obtaining it are described below in the Example section.
Cell lines that secrete Factor XII antibody can be identified by assaying culture
supernatants, ascites fluid etc., for antibody. The preferred screening procedure
consists of two sequential steps. First, hybridomas are identified that secrete antibody;
and second, the antibody is assayed to determine if it exhibits neutralizing activity. The
latter consists of determining whether the antibody blocks the activation of Factor XII.
As applied to cell culture supernatants. the initial screening step is preferably
done by RIA or ELISA assay. Both assavs are known in the art~ and consists of
binding Factor XII to a solid matrix, and assaying for antibody binding to Factor XII as
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WO 91/17258 Pcr/US9l/02990
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revealed by a second, labelled antibody. If peptides are used as immunogen, the initial
screening step determines if the antibody binds to peptide conjugates bound to a solid
matrix.
The preferred assay is an ELISA assay as described by Smeenk, R.J.T., et al.,
1987, Arthn~is Rheum,30:607. For as description of additional ELISA assay
methods see Langone, J. and Van Vinakis, H., 1983, Methods of Enzvmolo~
Part E, and for a description of RIA assay see Miller et ah, 1983, Method in Enzvm
121 :433 Part I. If peptides are used as immunogen, the initial screening step
determines if the antibody binds to peptide conjugates bound to a solid matrix.
An additional assay for Factor XII antibody may be conducted which
determines if antibody immunoprecipitates Factor XII from solution. For example,supernatants being tested for the presence of antibody may be incubated with labelled
Factor XII for an appropriate time to allo~ antigen/antibody complexes to form. The
complex may be washed to removed any unreacted reagents, and next the antibody
complexes incubated with anti-xenotypic or anti-isotypic antibodies specific for the
monoclonal antibody being screened. These anti-xenotypic or anti-isotypic antibodies
may be immobilized, for example, on a plastic bead. Thus, if Factor XII monoclonal
antibody is being screened, then labelled Factor XII will be indirectly bound to the bead
and thereby imrnunoprecipitated. Factor XII antibody can then be quantitated using
suitable detection methods known in the art dependent on the nature of the label used.
Also, the material can be dissociated from the bead using standard techniques and
identified by techniques known in the art, including gel electrophoresis.
The preferre~ electrophoresis procedure is Western Blot gel analysis as
described by Burnette, 1981, Anal. Bio. Ch~ " 112:195. The Western blots are
blocked, washed, and probed preferably in 10 mM sodium phosphate buffer containing
150 mM sodium chloride (pH 7.4), with 0.1% bovine serum albumin (w/v), and 0.1C~c
ovalburnin (w/v). In addition, a detergent is preferably employed such as Tween 20 at
a concentration of about 0.1 %. Sodium azide may also be included in the solution at a
concentration of 0.02%. The blots are preferably first probed with either hybridoma
culture supernatan~, or dilute ascites fluid containing Factor XII antibody, washed, and ~ -
then antibody binding revealed with l2sI-protein A for about 30-60 minutes. The blots
are washed, and subjected to autoradiography using X-ray film.
To expedite the time it takes to assay for Factor XII antibody, several culture
supernatants may be combined and assayed simultaneously. If the mixture is positive,
then media from each well may subsequently be assayed independently to confirrn the
presence of antibody.
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WO 91/17258 PCI/I!S91/02990
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The antibodies employed in the present invention can be immobilized on any
appropriate solid test support by any appropriate technique. The solid test support can
be any suitable insoluble carrier material for the binding of antibodies and
immunoassays. Many such materials are known in the art, including, but not limited
to, nitrocellulose sheets or filters; agarose, resin, plastic te.g. PVC or polystyrene)
.~ latex, or metal beads; plastic vessels; and the like. Many methods of immobilizing
antibodies are also known in the art. See, e.g., Silman et al., 1966, Ann. Rev.
Biochem., ~: 873; Melrose, 1971, Rev. Pure & App. Chem., 21: 83; Cuatrecaas et
al., 1971, Meth. Enzvm., 22. Such methods include covalent coupling, direct
adsorption, physical entrapment, and attachment to a protein-coated surface. In the
latter method, the surface is first coated with a water-insoluble protein such as æin,
collagen, fibrinogen, keratin, glutelin, etc. The antibody is attached by simplycontacting the protein-coated surface with an aqueous solution of the antibody and
allowing it to dry.
Any combination of support and binding technique which leaves the antibody
immunoreactive, yet sufficiently immobilizes the antibody so that it can be retained with
any bound antigen during a washing, can be employed in the present invention. A
. preferred solid test support is a plastic bead.
As discussed above, the assay of the present invention employs a labelled
antibody. The label can be any type that allows for the detection of the antibody when -
bound to a support. Generally, the label directly or indirectly results in a signal which
is measurable and related to the amount of label present in the sample. For example,
directly measurable labels can include radio-labels (e.g. 125I, 35S, 14C, etc.). A
preferred directly measurable label is an enzyme, conjugated to the antibody, which
- 25 produces a color reaction in the presence of the appropriate substrate. (e.g. horseradish
peroxidase/o-phenylenediamine). An example of an indirectly measurable label is
antibody that has been biotinylated. The presence of this label is measured by
contacting it with a solution containing a labeled avidin complex, whereby the avidin
becomes bound to the biotinylated antibody. The label associated with the avidin is
then measured. A preferred example of an indirect label is the avidin/biotin system
employing an enzyme conjugated to avidin, the enzyme producing a color reaction as
described above.
Whatever label is selected, it results in a signal which can be measured and is
related to the amount of label in a sample. Common signals are radiation levels (when
radioisotopes are used), optical density (e.g. when enzyme color reactions are used)
and fluorescence (when fluorescent compounds are used). It is preferred to employ a
nonradioactive signal, such as optical density (or color intensity) produced by an
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- enzyme reaction. Numerous enzyme/substrated combinations are known in the
immunoassay art which can produce a suitable signal. See, e.g., U.S. Patent Nos.4,323,647 and 4,190,496, the disclosures of which are incorporated herein.
The Factor XII antibody described herein, alone or in combination, may be
5 used to passively immunize a host organism suffering from bacteremia or sepsis, or at
risk with respect to bacterial infection. Treatment will generally consist of
administering the antibodies parenterally, and preferably intravenously. The dose and
administration regime will be a function of whether the antibody is being administered
therapeutically or prophylactically, and the patient's medical history. Typically, the
10 amount of antibody administered per dose will be in the range of about 0. I to 25 mg/kg
of body weight, with the preferred dose being about 0.1 to 10 mg/kg of patient body
weight. For parenteral administration, the antibodies will be formulated in an injectable
form combined with a phatmaceutically acceptable parenteral vehicle. Such vehicles are
well known in the art and examples include water, saline, Ringer s solution, dextrose
15 solution, and solutions consisting of small amounts of the human serum albumin. The
vehicle may contain minor amounts of additives that maintain the isotonicity andstability of thç antibody. The preparation of such solutions is within the skill of the art.
Typically, the antibodies will be formulated in such vehicles at a concentration of about
2-8.0 mg/ml to about 100 mg/ml.
The effectiveness of the subject Factor XII antibody in the treatment of sepsis
can be demonstrated in one of several animal model systems. The preferred animalmodel system is baboon, and is described by Taylor, et al., 1987, J. of Clinical~,
79:918, and by Taylor, ç~ al., 1988, Circulatorv Shock, 26:227. Briefly, this consist
of infusing a lethal dose of E. coli, about 4 x 1010 organisms per kilogram of body
weight administered over a 2-hour period. This is sufficient to kill 100% of the test
animals in a period ranging from 16-32 hours. The animals are anesthetized with
sodium pentobarbital in the cephalic vein through a percutaneous catheter. They are
also orally intubated and positioned on their right side on a heating pad. Blood samples
are removed from the femoral vein which is aseptically cannulated in the hind limb.
The percutaneous catheter is used to infuse the E. ~ organisms. Blood samples are
taken at desired time intervals and assayed for white blood cells hematocrit, platelet
Ievels, and fibrinogen. Additionally, mean systemic arterial pressure (MSAP) may be
monitored with a transducer (Stratham P2306, Porter ) pressure gauge. Changes inthese parameters may be prognostic of a patients ability to withstand exposure to a
lethal dose of bacteria.
Having described what the applicants believe their invention to be, the
following examples are presented to illustrate the invention. and are not to be construed -
: . . - - - - , :. . ., . ~, .............................. :
., , ., . . . . . . . ~. . .
, - . ~ : ~ . ':
WO 91/17258 PCr/US9t/OZ990
13 2~2~4~ -
as limiting the scope of the invention. For example, variation in the source, type, or
method of prodl~cing antibodies; different labels and/or signals; test supports of
different materials and configurations; different irnmobilization methods may beemployed without departing from the scope of the present invention.
Example I
P~eparation of Factor XII
Factor XII was prepared by the method described by Griffin and Cochrane,
1976, Methods Enzvmolo(Jv, 4~:56, which utilizes ion-exchange chromatography on
10 DEAE-Sephadex and SP-Sephadex.
Affinity purified Factor XII was used in some of the assays described below
and was prepared as follows. About 350 ml of citraled plasma pulled from three
human donors was made 0.1% (w/v) Tween 20, 10 tnM EDTA, 10 rnM benzamidine,
0.01% (w/v) STI, 0.05% (w/v) polybrene, and 0.01% NaN3. The plasma was filtered
15 through a monoclonal antibody colurnn consisting of the monoclonal antibody F3
coupled to Sepharose. The F3 antibody binds to both native and activated Factor XII in
the light chain region. The antibody is described by Nuijens et al., 1989, J. Biol.
Chem., vol. 264, 12941. The F3-Sepharose affinity column was prepared as described
below. Subsequently, the colurnn was washed with 100 rnl of PBS containing 0.1%
20 (w/v) Tween 20, 10 mM EDTA, and û.S M NaCl. This procedure removes most non-
specifically absorbed protein.
Next, Factor XII was eluted from the affinity column with 3 M KSCN in PBS,
and the resulting eluate (50 ml) was dialyzed against buffer that consisted of 4 mM
sodium acetate, 2 mM acetic acid, 015 M NaCI, 1 mM EDTA, and 0.02% NaN3, pH
25 5.2. This procedure has a yield of about 45% of Factor XII. The Factor XII
preparation had a specific coagulant activity of 33 U/mg of protein, and displayed no
amidolytic towards the chromogenic substrate S2302.
The affinity column was prepared using the antibody F3. The column was
prepared using a 50% ammonia sulphate fraction of the antibody coupled to CNBr-
30 activated Sepharose 4B.
By the above purification method Factor XII was determined, using SDS-
PAGE, to have a molecular weight of approximately 80,000.
.
; , ' ` ~ :'
WO 91/17258 PCI/US9l/02ss0
2~ i4;f~3 14
Example rl
Pepnde Imrnuno~ens
Based on the known amino acid sequence of Factor XII, peptides
corresponding to neutralizing epitopes of the molecule are synthesized and used as
5 immunogens to produce antibody. Peptides may be synthesiæd using the solid-phase
method, described in detail in Merrifield R.B., 1985, ~,232:341-347, on a
Biosearch 9500 automated peptide machine, cleaved with hydrogen fluoride, and
purified by preparative HPLC using a Waters Delta Prep 3000 instrument, on a 15-20
~Lm Vydac C4 PrepPAK column. The preferred peptides for producing antibody to
10 Factor XII have the following amino acid sequence:
1. N-cys-trp-val-leu-thr-ala-ala-his-cys-leu-gln-asp-C (his = residue 393).
2. N-phe-ser-pro-val-ser-tyr-gln-his-asp-leu-ala-leu-C (asp = residue 442).
3. N-gly-thr-asp-ala-cys-gln-gly-asp-ser-gly-gly-pro-leu-C (ser=residue
544).
Before using the peptides to make antibody they are conjugated to a suitable
carrier molecule to enhance eliciting an antibody response. These procedures aredescribed in U.S. Patent No. 4,762,706, inventors McCormick, et al.. Suitable
carriers are keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA). The
conjugation is achieved via a sulfhydryl group of a cysteine residue that, if necessary,
20 is added to the amino or carboxyl terminal end of the peptides. A heterobifunctional
crosslinking reagent, N-maleimido-6-atnino caproyl ester of 1-hydroxy-2-nitro-benzene-
4-sulfonic acid sodium salt, is prepared by the following procedure.
One molar equivalent (2.24 g) of 4-hydroxy-3-nitro-benzene sulfonic acid
sodium salt (HNTSA) is mixed together with one molar equivalent (2.06 g) of
25 dicyclohexylcarbodiimide and one molar equivalent (2.10 g) of N-maleimido-6-
aminocaproic acid in 25 ml of dimethylforrnamide (DMF) at room temperature
overnight. A white precipitate of dicyclohexyl urea is formed. The precipitate is
filtered and 300 ml diethyl ether is added to the mother liquor. After about 10 minutes
to 4 hours a gummy solid precipitated from the mother liquor is formed. This solid will
30 contain 58% of active HNSA ester and 42% of free HNSA.
The analysis consists of dissolving a small amount of the precipitate in
phosphate buffer at pH 7.0 and measuring the absorbance at 406 nm; this reading
provides the amount of unreac~ed free HNSA which is the contaminating material in the
HNSA ester preparation. Addition of very small amounts of concentrated strong base
35 (such as SN NaOH) instantly hydrolyses the ester formed and a second reading is
taken. Subtraction of the first reading from the second yields the amount of ester in the
original material. The solid is then dissolved in DMF and placed on a LH20 Sephade:;
. . ~ . , .. ~
WO 91/17258 PCI-/I IS91/02990
column and eluted with DMF so that the ester is separated from the contaminating free
HNSA. The progress of purification is monitored by thin layer chromatography using
eluting solvents of chloroform, acetone and acetic acid (6:3:1 vol/vol). The product is
positively identified as mal-sac HNSA ester by its reactivity with amine. The yield of
the pure ester is estimated to be approximately 30% of theoretical; the purified material
consists of 99% ester.
The ester thus obtained is found to dissolve fully in water and is stable in water
for several hours, provided no nucleophiles are added. When placed in 1 N arnrnonia
the ester produces the corresponding amide with a portion hydrolyzed to free acid. The
purified ester is found to be stable for extended periods when stored dessicated.
About 0.5 mg of the purifled mal-sac HNSA ester is dissolved in 1 ml of
distilled water. A 10 111 aliquot of this solution is diluted into 1 ml of 10 rn,M phosphate
buffer at pH 7Ø The absorbance at 406 nm is used to calculate the concentrarion of
free HNSA as described above. When 50 111 of 4.8N sodium hydroxide solution is
added to the diluted aliquot of ester and mixed, the absorbance of the solution at 406
nm increases significantly, indicating that the hydroxide nucleophile rapidly hydrolyses
the ester to component acid and free HNSA anion.
The difference between the post-base and initial free HNSA concentration
represents the concentration of ester. From the actual concentration of ester and protein
amino groups the amount of ester to be added to the protein solution to achieve the
desired degree of substitution can be calculated.
Thé punfied HNSA ester is then reacted with BSA as follows (the reaction with
KLH is similar to this procedure):
A total of 22 mg (20 llmoles) of BSA (of molecular weight 66,296) is dissolved
in 2.0 ml of 0.1 M phosphate buffer at pH 7.5 to yield a total amine concentration of
1.0 x 10-2 moles per liter (assuming 59 lysines/BSA molecule). A calculated amount
(11 mg, 2.35 x 10-5 moles) of the above-prepared mal-sac HNSA ester (97.7% pure) in
powder form is dissolved in 2.0 ml of BSA solution. The reaction is carried out at
room temperature. Ten ~l aliquots are removed from the solution at timed intervals and
are each diluted into 1.0 ml of 0.01 M phosphate buffer at pH 7Ø The spectrum of
each diluted aliquot is recorded using a Hewlett-Packard spectrophotometer and the
absorbance at 406 nm measured. A total of 50 111 of 4.8N NaOH is then added to each
aliquot, each aliquot is mixed and its spectrum retaken, and the absorbance at 406 nm
measured.
From the absorbance at 406 nm before and after addition of base the
concentration of ester remaining and the percent ester that reacts are determined for the
WO ~1/17258 PCI /US91/02990
2~2~43 16
reaction mixtures. The results show that the reaction rate is essentially linear over a 15- -
rninute period.
After 15 minutes of reaction time, the reaction is stopped by applying the
reaction mixture to a PD10 desalting Sephadex G-25 column (Pharmacia, Inc.)
equilibrated with 0.1 M phosphate buffer at pH 6Ø It is found that 2.6 x 10-3 moles/1 ~ ~ -
of the ester reacts, and thus 25.9% of the 59 epsilcn-arnino groups of BSA are
presumably substituted. Thus, the product contains 16 mal-sac groups per molecule.
The product of the first reaction, mal-sac-BSA (or mal-sac-KLH), is isolated b~ -
applying the reaction mixture to a PD10 desalting Sephadex G-25 column equilibrated
with 0.1 M phosphate buffer at pH 6Ø The column is eluted with 0.1 M phosphatebuffer in 1.0 ml fractions. The column elution is followed by monitoring the
absorbance spectrum, and peak fractions containing the mal-sac BSA are pooled.
The peptides synthesized as described above are added and the pooled mixture
is stirred at room temperature overnight. The conjugates are subjected to extensive
dialysis against distilled water and lyophilization, and in some cases are anal~vzed for
changes in amino acid composition. These peptide conjugates may be used to
immunize animals, or Iymphocytes in vitro to produce the desired antibody.
Example m
Immunization with Factor Xll or Peppde Immunogens
and the Production of Hvbridomas
The following describes the immunization of mice with Factor XII with the aim
of isolating immunized lyrnphocytes and producing murine hybridomas. It will be
further appreciated that the procedure can be employed to produce antibody against
Factor XII peptides, synthesized and conjugated as described above.
Generally, the procedures described in the following references are followed forgenerating hybridomas. Shulman, et al., 1978, ~a~, ~:269; Oi, ç~ ~1., in Selected .-.
Methods in Cellular Immunolo~v, p 351 (Mischell & Schiigi eds. 1980). Foung, et
al., 1983, Proc. Nat'l Acad. Sci. USA, ~2:7484. Further references include, Gerhard,
et .~1., 1978, Proc. Nat'l Acad. Sci.USA, ~: 1510; Monoclonal Antibodies (R.
Kennett, T. McKearn, & K. Bechtol eds. 1980); Schreier et al., 1980, Hvbridoma
~ç~; Monoclonal Antibodies and T-Cell Hvbridomas (G. Harnrnerling, U.
Hammerling, & J. Kearney eds. 1981); Kozbor et al., 1982, Proc. Nat'l Açad. Sci.~, ~: 6651; Jonak et al., 1983, Hvbridoma, _: 124; Monoclonal Antibodies and
Functional Cell Lines (R. Kennett, K. Bechtol, ~ T. McKearn eds.1983); Kozbor etal., 1983, Immunolo~Jv Todav, _:72-79; Shulman et al., 1982, Nature,276: 269-270;
" ~ . . .
, - . : . .
..
WO 91/172S8 PCI/US91/02990
17 2a~2~
Oi et al., 1980, Selected Methods and Cellular Immunolo ,v, pg.351-371 (B. Mischell
: & S. Schiigi Eds.); Foung e~t al., 1983, Proc. Nat'l Acad. Sci. USA,12:7484-7488.
The preferred procedure is described by Nuijens J. H., et ah, 1989, The
Journal of Biolo~ical Chemistrv,264:12941. Briefly, Balb/c mice were
5 hyperimmunized with repeated intraperitoneal injections of 25 ~Lg of Factor XII which
consisted of a first injection in complete Freund's adjuvant, followed by subsequent
boosts at 3, 6, and 9 weeks after the initial injection. Four days after the final boost,
spleens were removed from the immunized animals and spleens immunized with non-
affmity purified Factor XII prepared as described above. Three days later, spleens
10 from immunized mice were removed and the splenocytes fused to the murine myeloma
cell line, SP 2/0Agl4.
The fusion procedure that was followed is described by Kohler & Milstein,
1975, Nature,2S6:495, as modified by Fendly et ah, in Hvbridoma, 6:359 (1987).
Briefly, mice were sacrificed and splenocytes teased from immunized spleens, and15 washed in serum free Dulbecco's Modified Eagles medium. Similarly, SP V0Agl4
myeloma cells were washed, and combined with the splenocytes in a 5: 1 ratio of spleen
cells to myeloma cells. The cell mixture was pelleted, media removed and fusion
- . affected by the addition of 1.0 ml of 40% (v/v) solution of polyethylene glycol 1500 by
dropwise addition over 60 seconds at room temperature, followed by a 60-second
20 incubation at 37 C. To the cell suspension with gentle agitation was added 9 ml of
Dulbecco's Modified Eagles medium over 5 minutes. Cell clumps in the mixture were
gently resuspended, the cells washed to remove any residual PEG and plated at about 2
. x 105 cells/well in Dulbecco's Modifled Eagles medium supplemented with 20% fetal
calf serum. After 24 hours, the cells were fed a 2x solution of hypoxanthine and25 azaserine selection medium. The cells were plated in a total of 20 microtiter plates,
which corresponded to 1960 wells. About 9-10 days later, 80% of the wells exhibited
good cell growth, and these were screened for antibody to Factor XII. Using thismethod the hybridoma OT-2 was identified and isolated.
.. Antibody may be produced in ~ritro from the hybridoma cell line OT-2 by
culturing the cell line in 1 liter roller-bottles in IMDM media supplemented with 2%
fetal calf serum,50 uM 2-mercaptoethanol, and penicillin and streptomycin. The cells
are grown to a density of about 106/ml, and a week later the supernatants are collected,
and concentrated using a hollow fiber de~ice. To purify the antibody on a protein A
column (Pharmacia) solid NaCl is added to the concentrate to a final concentration of 3
M. This solution is diluted 1:1 with a solution consisting of 3 M NaCl and 1.5 Mglycine, pH 8.9. The protein A column is equilibrated with the latter buffer. and the
- ~ . . ,:
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WO 91/17258 PCI/US91/02990
2~3'~ ) 18
concentrate added to the column, the column washed, and antibody eluted off the
column with 100 mM sodium citra~e buffer, pH 6Ø Those peaks containing antibody
are pooled, dialyzed against phosphate buffered saline, and stored until used.
Peripheral blood Iymphocytes are isolated from septic patients, and then
5 infected with Epstein-BaIr virus and the infected lymphocytes immortalized by fusion
to a selectable myeloma cell line, and the hybrid cell lines so generated isolated and
characterized as to antibody production. More specifically, mononuclear cells are
separated on Ficoll-hypaque (Pharmacia), and monocytes depleted from the mixture by
adherence to plastic. S~andard laboratory techniques are utilized to effect these
10 procedures. Next, nonadherent cells are enriched for antibody producers by antigen-
specific panning. Panning is a technique generally known in the art, and involves
incubation of a popula~ion of antibody secreting cells on a plastic surface coated with
the appropriate antigen, in this instance Factor XII or peptide immunogens derived
from Factor XII, and produced as described in Example I. Those cells that express
15 antibody on their surface bind antigen, and consequently adhere to the plastic surface,
whereas cells that do not express cell surface antibody, do not adhere and can be
removed by washing. Thus, specific antibody secreting cells are enriched for by this
technique.
More specifically, 6-well plates (Costar) are coated with 1-20 llg of Factor XII20 or peptide immunogens per well in phosphate buffered saline at 4C overnight. The
wells are blocked after the overnight incubation period with phosphate buffered saline
containing 1% bovine serum albumin for at least 1 hour at 4C, and subsequently .
washed with phosphate buffered salinelBSA. Next, 107 lymphocytes in 1 ml of
PBS/BSA are added to each well of the six well plates. The Iymphocytes are allowed
25 to incubate on the plates for 70 minutes, after which any nonadherent cells are rernoved
by aspiration. The adherent cells are incubated with cell culture medium (IMDM,
Sigma Chemical Co., Sl. Louis, Missouri) containing 10% fetal calf serum.
The adherent cells are subjected to Epstein-Barr virus transformation by adding
an equal amount of culture media obtained from growing the Epstein-Barr virus
30 infected marmoset cell line, B95-8, or similar cell line, and thus containing the virus, to
media bathing the adherent cells. The cells are cultured in this environment at 37C for
3 hours, and in this way the Iymphocytes in the adherent cell population are subjected
to Eps~ein-Barr infection. Following the infection period, the cells are washed and
plated onto 96 well microtitre plates at a density of about 10~ - 105 cells/well in IMDM
35 medium, plus 10% fetal calf serum, and 30~70 conditioned medium. The latter is derived
from a lymphoblastoid cell line, preferably ~W5. The medium also contains S x 10-5 M
.. . . .. .', .: ' :
wO 91/17258 Pcr/US9l/02990
19 2~2~3
2-mercaptoethanol, 50 !lg/ml gentamycin sulfate (Sigma), and 600 ng/ml cyclosporine
A (Sandimmune, Sandoz, Basel, Switzerland).
After about 14 to 21 days of incubation, cell culture supernatants are combined
and screened for the desired antibody binding activity as described below. Positive
5 hybridomas are subcultured at low density, retested for activity, and grown up and
fused to the cell line F3B6 using polyethylene glycol and the plate fusion technique
known in the art. The latter technique is described by Larrick, J.W., 1985, Human
Hvbndomas and Monoclonal Antibodies, E.G. Engleman, S.K.H. Foung, J.W.,
Larrick, and A.A. Raubitschek, (Eds.), Plenum Press, New York, pg 446. The cell
10 line F3B6 is a heteromyeloma that is sensitive to growth in media containing 100 ~M
hypoxanthine, 5 ~g/ml azaserine and 5 IlM ouabain. It is on deposit with the American
Type Culture Collection with Accession No. HB8785. Finally, the resulting hybrids
are again screened to insure that they produce the desired antibody.
Example lV
Proper~ies of QT-2
Binding of native Factor XII to OT-2 was detected by incubating fresh plasma.
with OT-2 coupled to Sepharose. Bound Factor XII was assessed by a subsequent
incubation with polyclonal l2sI-anti-Factor XII antibodies. For comparative purposes,
20 binding of the mAb-F3 to Factor XII was also determined. Details of the procedure are
described by Nuijens, J.H. et al., 1989, Blood, 72:1704, for the radioimmunoassay
procedure performed with the anti-Factor XII mAb-F3. The results are shown in figure
1. It is apparent from the figure that both antibodies bind to Factor Xll.
Additionally, binding of native Factor XII to OT-2 may be deterrnined as
25 follows. Microtiter plates were coated overnight at 4C with native Factor XII purified
from plasma by affinity chromatography (Nuijens, J.H. et ah, 1989, J. Biol. Chem.,
264:12941). The concentration of Factor XII used to coat the plates was 2 llg/ml in
phosphate buffered saline (PBS), pH 7.4. The plates were washed and incubated 60~inutes at 37~C with dilutions of mAb OT-2 in PBS containing 0.1 % (w/v) Tween 20.
30 After a second wash, plates were incubated for another 60 minutes at 37C with 1 to
1000 goat-anti-mouse immunoglobulins antibodies conjugated with horseradish
peroxidase and diluted in PBS-Tween. After a wash, peroxidase activity was detecled
as described by Smeenk et ah, 1987, Arthr. Rheum., 30:607. Using this assay it was
shown that OT-2 binds to Factor XII.
Inhibition of amidolytic activity of ~-Faclor XIIa by rnAb OT-2. Twenty-five
111 of 0.5 IlM of ~-Factor XlIa was incubated with 25 111 of 0.5 IlM OT-2 or 25 ~1 of
0.5 ~lM mAb F3 for 60 minutes at 37C. Then 100 111 of the chromogenic substrate
.
:
.
. '
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wO 91/17258 , Pcr/ussl/02990
2~2~
S2302 at a concentralion of 2 mM was added and the release of pNA was followed by
measuring A405 nm at indicated time-points. The results are shown in Figure 2. It is
apparent from the data that OT-2 nearly completely inhibits the arnidolytic activity of ~- -
Factor XIIa. In contrast F3, had little or no effect.
Inhibition of contact activation in plasma by mAb OT-2 was shown by
. measuring a decrease in kallikrein-C1-inhibitor and Factor XII-Cl-inhibitor complexes.
Activation was initiated by dextran sulfate. For comparative purposes, the effect of
mAb-F3 was also determined. Briefly, one volume of fresh plasma, containing 10 mM
. EDTA, was incubated with one volume of PBS, with or without various concentrations
of the monoclonal antibodies for 60 minutes at 37~C. Then, 2 volumes of dextran
sulphate (MW 500,000), 100 llg/ml in PBS, were added~ and the rnixture was
incubated for 20 minutes at 37C. Next, the ex~ent of contact activation in the mixture
was assessed by measuring kallikrein-Cl-inhibitor and Factor XIIa-Cl inhibitor . . .
complexes using the assay described by ~uijens, J.H., et al., 1989, Blood, 1~:14W
The results are shown in Figure 3, along with the appropriate controls. It is apparent
that contact activation is inhibited by OT-2 as judged by a decrease in both kallikrein-C1-
inhibitor and Factor XIIa-C1 complexes. In contrast, little or no inhibition was caused
; byF3.
A second experiment was conducted which showed that the antibody OT-2
inhibits contact activated Factor XII. The procedure whereby Factor XII is bound to
glass is described by Nuijens et al (1989) J. Biol. Chem., vol. 264: p. 12941. The
effect of the antibody at different concentrations was determined by measuring adecrease in pRkallikrein activation which was demonstrated by a decreased generation
of kall~crein-C1-inhibitor complexes in plasma, expressed as mU per rnl. For
comparative purposes, the effect of the C1 inhibitor was also tested. Figure 4 shows,
surprisingly, that OT-2, but not C1 inhibitor inhibits the activity of bound Factor XII.
rnAb OT-2 coupled to Sepharose was incubated for 4 hours at room temperature
with fresh plasma in which the contact system was, or was not activated by incubation
with dextran sulphate at a concentra~ion of 100 ~g/ml for 20 minutes at 37C. The
beads were washed with saline, and C1-inhibitor complexes bound to the beads were -
detected by a subsequent incubation with polyclonal l2sI-anti-C1-inhibitor antibodies.
The antibody F3 was similarly coupled to Sepharose, and also assayed for Factor XIIa- .
C1-inhibitor complex binding activity. Details of the procedures have been described
for a similar radioin~nunoassay with polyclonal antibodies (~uijens, J.H., et ah,
1987, Thromb. Haemost., 58:778). As shown on the right side of Figure 5, OT-2
does not bind to Factor XIIa-Cl-inhibitor complexes. In contrast to OT-2, it was
, - , , . , . . - ~ ., .~ :,............ .
;
,, . . . ~ ,
:. ~ ~ ' :: , :
WO 91/17258 PCI/US9t/02990
21 . 2Q~2~3
additionally shown that the mAb F3 does bind to Factor XIIa-C1-inhibitor complexes
(left side of Figure 5).
Exarnple V
Factor XTI Antibodv for the Treatment of Sepsis
The effectiveness of the Factor XII antibody, OT-2, in a baboon sepsis model
system would be tested essentially as described by Taylor, et al., 1987, J. Qf~Clinical
918, and by Taylor, et al., 1988, Circul3torv Shock, 26:227. Briefly, this
consists of first measuring Factor XII levels in baboon plasma in response to a lethal
dose of E. coli., and secondly, determining if Factor XII antibody is effective in
treating sepsis by preventing the death, or prolonging the lives of septic animals. A
lethal dose of E. coli consists of approximately 4 x 101 organisms.
After administration of a lethal dose of E. coli, Factor XII levels start to
decrease and Baboons invariably die within 16-32 hours. Taylor, et ah, 1987, J. of
Clinical Inv.,79:918, and by Taylor, et ah,1988, Circulatorv Shock, ~6:227.
The effectiveness of the OT-2 monoclonal in preventing the death or
prolonging the life of baboons is tested using an administration routine wherein the
antibody is delivered in physiological saline at 5.0 mg of antibody per kg of body
weight simultaneously with the bacterial challenge. The monoclonal antibody would
considerably extend the lifetime of the baboons that receive the dose of antibody and
survive for at least 40-60 hours, respectively. Recall that baboons that receive a lethal
dose of E. coli invariably die within 16-32 hours.
Deposition of Cultures
The hybridomas used in the above exarnples, to illustrate the method of the
present invention were deposited in and accepted by the American Type Culture
Collection (ATCC),12301 Parklawn Drive, Roclcville, Miaryland, USA, under the
terrns of the Budapest Treaty. The deposit dates and the accession numbers are given
below:
Culture Deposit Date Accession No.
Hybridoma OT-2
Hybridoma F3
These deposits were made under the Budapest Treaty and will be maintained
and made accessible according to the pro- isions thereof.
Availability of the deposited cell lines are not to be construed as a license topractice the invention in contravention of ~he rights granted under the authority of any
government in accordance with its patent laws.
.
WO ~1/17258 P~/l lS91/02990
2~
Also, the present invention is not to be considered limited in scope by the
deposited hybridomas, since they are in~ended only to be illustrative of particular
aspects of the invention. Any animal cell line (including any hybridoma) which can be
used for production of protein according to the methods described in this patentS application is considered within the scope of this invention. Further, various.. ~ modifications of the invention in addition to those shown and described herein apparent .
-
: ~ to those skilled in the art from the preceding description are considered to fall within the
scope of the appended claims.
The present invention has been described with reference to specific
10 embodiments. However, this application is intended to cover those chan~es andsubstitutions which may be made by those skilled in the art without departing from the
spirit and the scope of the appended claims
,
.
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