WO1991017258A1 - Inhibiteurs de l'activation du facteur xii et leurs applications - Google Patents

Inhibiteurs de l'activation du facteur xii et leurs applications Download PDF

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WO1991017258A1
WO1991017258A1 PCT/US1991/002990 US9102990W WO9117258A1 WO 1991017258 A1 WO1991017258 A1 WO 1991017258A1 US 9102990 W US9102990 W US 9102990W WO 9117258 A1 WO9117258 A1 WO 9117258A1
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antibody
factor xii
factor
inhibitor
leu
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PCT/US1991/002990
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English (en)
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Jan H. Nuijens
Cecile C. M. HUIJBREGTS
C. Erik HACK
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Cetus Corporation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention is in the area of immunology/biochemistry, and presents inhibitors of Factor Xlla 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 therefrom, or recombinant constructs having the binding activity of such antibodies.
  • nosocomial bacteremia develops in about 194,000 patients, and of these about 75,000 die. Maki, D.G., 1981, Nosocomial Infect..
  • LPS lipopolysaccharides
  • 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 studies which have shown that LPS can activate both the contact system of intrinsic coagulation, as well as the complement system. Morris, E.C., el al-, 1974, J. of Experimental Med.. 140:797 and Morrison, D.C., _i _!., 1978, American Journal of Pathology.22:527.
  • Factor XII also known as Hageman Factor, is a plasma serine protease zymogen.
  • Human Factor XII is a single-chain Beta globulin of molecular weight 80,000. During activation, it is cleaved to a two-chain enzyme, termed Factor Xlla, 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 is enzymatically active, while the heavy chain binds to solid surfaces during contact activation.
  • 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 medicaments for the prophylactic or therapeutic treatment of sepsis.
  • Exemplary antibody would be polyclonal, monoclonal, or recombinant constructs, or fragments derived therefrom 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 XUa, indicating that it affects the catalytic center of the molecule; reacts with native Factor XII; and is substantially unreactive with Factor XUa bound to Cl inhibitor.
  • a fourth object of the invention is a description of methods for administrating inhibitors of Factor XE activation that are useful medicaments for the prophylactic or therapeutic treatment of sepsis.
  • Figure 1 shows that the antibody, OT-2, binds to native Factor XII in plasma.
  • Figure 2 shows the effect of mAb OT-2 that inhibits the amidolytic activity of Factor Xlla.
  • Figure 3 shows that OT-2 inhibits activation of the contact system in plasma. 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 Xlla bound to an activator, glass, whereas a natural inhibitor of Factor XUa, Cl esterase inhibitor (Cl- inh.) does not.
  • Figure 5 shows that the antibody OT-2 does not bind to Factor Xlla-Cl- inhibitor complexes.
  • the kernel of the instant invention is the realization that Factor XII plays a key role 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 that would have this activity are antibody, or molecules with the binding activity of 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 XII activation are preferably but not exclusively polyclonal, monoclonal, recombinant antibody constructs, or fragments derived therefrom having the binding activity of such antibody or antibody fragments. Further, applications of bispecific antibody inhibitors are readily imagined.
  • “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 the six major gram-negative bacilli and these are Pseudomonas aeruginosa. 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 XUa” refers to activated Factor XII, without intending to denote whether or not the heavy chain is contained within this active species.
  • ⁇ -Factor XUa refers to activated Factor XH, 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 center.
  • ⁇ -Factor XUa refers to an active form of Factor XE 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.
  • ⁇ -Factor-XIIa is formed upon cleavage of the heavy chain of ⁇ -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.
  • substantially free means approximately 5% (w/w) or fewer contaminating antibodies in the composition.
  • modifications to antibody that increase its effectiveness include conjugation of a water soluble polymer.
  • the water soluble polymer is polyethylene glycol, or a functionally related molecule such as, for example, 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 that might occur when it is introduced in vivo.
  • an “antibody-producing cell line” is a clonal population or clone derived through mitosis of a single antibody-producing cell capable of stable growth in vitro for many generations.
  • “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.
  • variable chain alone can be expressed in bacteria with significant antigen binding activity.
  • Recombinant antibody monoclonal Fab antibody produced using the techniques described by Huse, W.D. ej_ al., 1989, Science 24 . 2:1275.
  • the term “incubation” means contacting antibodies and antigens under conditions that allow for the formation of antigen/antibody complexes (e.g., proper pH, temperature, time, medium, etc.).
  • 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. The selection of the appropriate incubation and separation techniques is within the skill of the art.
  • Cl inhibitor is meant a plasma glycoprotein 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 Cls, and the intrinsic coagulation system, Factor XIa, Factor XQa, and Kallikrein. Cl inhibitor also interacts with plasmin and tissue plasminogen activator. Cl inhibitor has the further property of itself being inactivated by proteases, notably elastase. It will, or course, be understood that intended to come within the scope of the definition of the Cl inhibitor are fragments of the molecule that maintain biologically activity.
  • Factor XII antibody producing immunologic cells are isolated from a mammal immunized with Factor XII, and 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.
  • 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 XH 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 Enzymology.4_5_: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 immune response to Factor XII.
  • An exemplary adjuvant is Freund's adjuvant.
  • the precise immunization schedule is generally not critical, and determinative of which procedure is employed, is the presence of Factor XH antibody in the host animal as measured by a suitable assay, described below.
  • a preferred immunization procedure consists of hyperimmunization with Factor XII by repeated intraperitoneal injections as described by Nuijens, J. H., _t_l., 1989, J. Biol. Chem.. 264:12941.
  • lymphocytes may be immunized in vitro.
  • immunization of peripheral blood cells may be achieved as described by Boss,
  • Enzvmologv. 121 Part One: 18, or Voss, B., 1986, Methods in Enzvmologv.121:27.
  • a number of j ⁇ vitro immunization systems have been shown to be effective for sensitizing human B-cells. Reading, C, 1982, J. of Immun. Methods.52:261.
  • Factor XII as immunogens
  • an alternative approach is to synthesize Factor Xlla peptides, and use these as immunogens.
  • 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 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 are well 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 known in the art, such as, for example, the Merrifield solid-phase method described in Science.222:341-347 (1985).
  • the procedure may use commercially available synthesizers such as a Biosearch 9500 automated 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 15-20 ⁇ m Vydac C4 PrepPAK column. Once clones are identified that secrete anti-peptide antibody, the antibody can be screened for binding and neutralizing activity to Factor XII.
  • synthesizers such as a Biosearch 9500 automated 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 15-20 ⁇ m Vydac C4 PrepPAK column.
  • Antibody to Factor Xlla may be either polyclonal, monoclonal, recombinant or fragments derived therefrom.
  • the antibody is preferably human or humanized, although non-human antibody will perform satisfactory.
  • the preparation of high-titer neutralizing polyclonal antibody can be realized by immunizing 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 XH) 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. 256:495, or modifications thereof that are known in the art. Using the screening assays described below, the specificity of antibody produced can be discerned.
  • a suitable plasmacytoma is Sp 2/0-Agl4 and is widely used by practitioners of this art.
  • 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
  • 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.
  • 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 partner in small amounts over a short period of time accompanied with gentle agitation.
  • 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.
  • hybrid cells After a period of several weeks, hybrid cells are apparent, and may be identified as to 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 lymphocytes sensitized with Factor XII either in vivo or in 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 above.
  • sensitized lymphocytes may be immortalized 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.
  • fusion partners are known in the art, and exemplary partners may be a mouse myeloma cell line, a heteromyeloma line, or a human myeloma line, or other immortalized cell line.
  • 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 April 18, 1985. The procedures for generating F3B6 are described in European Patent Application, Publication No. 174,204.
  • Epstein-Barr virus transformation Techniques applicable to the use of Epstein-Barr virus transformation and the production of immortal antibody secreting cell lines are presented by Roder, J. si al-, 1986, Methods in Enzymology. 121:140. Basically, the procedure consist of isolating 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. Subsequently, virally transformed 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 identified using standard methods known in the art.
  • the preferred embodiment of the instant invention is neutralizing Factor XII monoclonal antibody, such antibody(s) may be altered and still maintain biological activity.
  • antibody modified by reduction to various size fragments such as F(ab')2» Fab, Fv, or the like.
  • the hybrid cell lines that produce the antibody may be considered to be a source of the DNA that 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 al-; 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.
  • British patent apphcation, No. 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.
  • CDRs complementary determining regions
  • the preferred Factor XII antibody 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 supematants, 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.
  • the initial screening step is preferably done by RIA or ELISA assay.
  • Both assays are known in the art, and consists of binding Factor XII to a solid matrix, and assaying for antibody binding to Factor XII as 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., ⁇ ial-. 1987, Arthritis Rheum.2Q:607.
  • ELISA assay as described by Smeenk, R.J.T., ⁇ ial-. 1987, Arthritis Rheum.2Q:607.
  • additional ELISA assay methods see Langone, J. and Van Vinakis, H., 1983, Methods of Enzymology. 92. Part E. and for a description of RIA assay see Miller si aL, 1983, Method in Enzym.. 121:433 Part I.
  • the initial screening step determines if the antibody binds to peptide conjugates bound to a solid matrix.
  • An additional assay for Factor XH antibody may be conducted which determines if antibody immunoprecipitates Factor XII from solution. For example, supematants being tested for the presence of antibody may be incubated with labelled Factor XH for an appropriate time to allow 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.
  • Factor XQ monoclonal antibody if Factor XQ monoclonal antibody is being screened, then labelled Factor XII will be indirectly bound to the bead and thereby immune-precipitated.
  • Factor XII antibody can then be quantitated using suitable detection methods known in the art dependent on the nature of the label used.
  • the material can be dissociated from the bead using standard techniques and identified by techniques known in the art, including gel electrophoresis.
  • the preferred electrophoresis procedure is Western Blot gel analysis as described by Bumette, 1981, Anal. Bio. Chem..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 0.1% ovalbumin (w/v).
  • 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 supernatant, or dilute ascites fluid containing Factor XII antibody, washed, and then antibody binding revealed with 1251-protein A for about 30-60 minutes.
  • the blots are washed, and subjected to autoradiography using X-ray film.
  • 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 (e.g. PVC or polystyrene) latex, or metal beads; plastic vessels; and the like. Many methods of immobilizing antibodies are also known in the art.
  • Such methods include covalent coupling, direct adsorption, physical entrapment, and attachment to a protein-coated surface.
  • the surface is first coated with a water-insoluble protein such as zein, collagen, fibrinogen, keratin, glutelin, etc.
  • the antibody is attached by simply contacting 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.
  • 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.
  • the label directly or indirectly results in a signal which is measurable and related to the amount of label present in the sample.
  • directly measurable labels can include radio-labels (e.g. 1251, 35S, 14C, etc.).
  • a preferred directly measurable label is an enzyme, conjugated to the antibody, which 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.
  • 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.
  • 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 enzyme reaction.
  • a nonradioactive signal such as optical density (or color intensity) produced by an 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 may be 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.
  • the amount of antibody administered per dose will be in the range of about 0.1 to 25 mg/kg of body weight, with the preferred dose being about 0.1 to 10 mg/kg of patient body weight.
  • the antibodies will be formulated in an injectable form combined with a pharmaceutically acceptable parenteral vehicle.
  • Such vehicles are well known in the art and examples include water, saline, Ringer's solution, dextrose solution, and solutions consisting of small amounts of the human serum albumin.
  • the vehicle may contain minor amounts of additives that maintain the isotonicity and stability of the antibody.
  • the preparation of such solutions is within the skill of the art.
  • 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 animal model system is baboon, and is described by Taylor, ej al-, 1987, J. of Clinical Inv..
  • MSAP mean systemic arterial pressure
  • Affinity purified Factor XII was used in some of the assays described below and was prepared as follows. About 350 ml of citrated plasma pulled from three human donors was made 0.1% (w/v) Tween 20, 10 mM EDTA, 10 mM benzamidine, 0.01% (w/v) STI, 0.05% (w/v) polybrene, and 0.01% NaN 3 . The plasma was filtered through a monoclonal antibody column 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.
  • the F3-Sepharose affinity column was prepared as described below. Subsequently, the column was washed with 100 ml of PBS containing 0.1% (w/v) Tween 20, 10 mM EDTA, and 0.5 M NaCl. This procedure removes most non- specifically absorbed protein.
  • 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 NaCl, 1 mM EDTA, and 0.02% NaN 3 , pH 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- activated Sepharose 4B.
  • Factor XII was determined, using SDS- PAGE, to have a molecular weight of approximately 80,000.
  • Example II Peptide Immunogens Based on the known amino acid sequence of Factor XII, peptides corresponding to neutralizing epitopes of the molecule are synthesized and used as immunogens to produce antibody. Peptides may be synthesized using the solid-phase method, described in detail in Merrifield R.B., 1985, Sci.. 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 ⁇ m Vydac C4 PrepPAK column.
  • the preferred peptides for producing antibody to Factor XII have the following amino acid sequence:
  • a suitable carrier molecule to enhance eliciting an antibody response.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • the conjugation is achieved via a sulfhydryl group of a cysteine residue that, if necessary, is added to the amino or carboxyl terminal end of the peptides.
  • a heterobifunctional crosslinking reagent, N-maleimido-6-amino caproyl ester of l-hydroxy-2-nitro-benzene- 4-sulf ⁇ nic acid sodium salt is prepared by the following procedure.
  • HNSA 4-hydroxy-3-nitro-benzene sulfonic acid sodium salt
  • DMF dimethylformamide
  • 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 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 unreacted free HNSA which is the contaminating material in the HNSA ester preparation.
  • Addition of very small amounts of concentrated strong base (such as 5N 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 Sephadex 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 ammonia 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 purified mal-sac HNSA ester is dissolved in 1 ml of distilled water. A 10 ⁇ l aliquot of this solution is diluted into 1 ml of 10 mM phosphate buffer at pH 7.0. The absorbance at 406 nm is used to calculate the concentration of free HNSA as described above.
  • 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.
  • a total of 22 mg (20 ⁇ moles) 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 7.0.
  • each diluted aliquot is recorded using a Hewlett-Packard spectrophotometer and the absorbance at 406 nm measured.
  • a total of 50 ⁇ l 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 reaction mixtures. The results show that the reaction rate is essentially linear over a 15- minute period.
  • reaction is stopped by applying the reaction mixture to aPDIO desalting Sephadex G-25 column (Pharmacia, Inc.) equilibrated with 0.1 M phosphate buffer at pH 6.0. It is found that 2.6 x lO- 3 moles/1 of the ester reacts, and thus 25.9% of the 59 epsilon-amino groups of BSA are presumably substituted. Thus, the product contains 16 mal-sac groups per molecule.
  • mal-sac-BS A (or mal-sac-KLH)
  • the product of the first reaction is isolated by applying the reaction mixture to a PD10 desalting Sephadex G-25 column equilibrated with 0.1 M phosphate buffer at pH 6.0.
  • the column is eluted with 0.1 M phosphate buffer 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 analyzed for changes in amino acid composition. These peptide conjugates may be used to immunize animals, or lymphocytes in vitro to produce the desired antibody.
  • Example III Immunization with Factor XII or Peptide Immunogens and the Production of Hybridomas The following describes the immunization of mice with Factor XII with the aim of isolating immunized lymphocytes 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.
  • mice were hyperimmunized with repeated intraperitoneal injections of 25 ⁇ g of Factor XH 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.
  • spleens were removed from the immunized animals and spleens immunized with non- affinity purified Factor XII prepared as described above.
  • spleens from immunized mice were removed and the splenocytes fused to the murine myeloma cell line, SP 2/0Agl4.
  • mice were sacrificed and splenocytes teased from immunized spleens, and washed in seram free Dulbecco's Modified Eagles medium. Similarly, SP 2/0Agl4 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 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 10 5 cells/well in Dulbecco's Modified Eagles medium supplemented with 20% fetal calf serum. After 24 hours, the cells were fed a 2x solution of hypoxanthine and 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 this method the hybridoma OT-2 was identified and isolated.
  • Antibody may be produced in vitro 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 10 6 /ml, and a week later the supematants are collected, and concentrated using a hollow fiber device. 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 M glycine, pH 8.9.
  • the protein A column is equilibrated with the latter buffer, and the concentrate added to the column, the column washed, and antibody eluted off the column with 100 mM sodium citrate buffer, pH 6.0. Those peaks containing antibody are pooled, dialyzed against phosphate buffered saline, and stored until used.
  • Peripheral blood lymphocytes are isolated from septic patients, and then infected with Epstein-Barr viras 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. Standard laboratory techniques are utilized to effect these 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 population 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 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.
  • the appropriate antigen in this instance Factor XII or peptide immunogens derived from Factor XII
  • 6- well plates (Costar) are coated with 1-20 ⁇ g of Factor XII or peptide immunogens per well in phosphate buffered saline at 4°C 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 4 ⁇ C, and subsequently washed with phosphate buffered saline BSA.
  • 10 7 lymphocytes in 1 ml of PBS/BSA are added to each well of the six well plates. The lymphocytes are allowed to incubate on the plates for 70 minutes, after which any nonadherent cells are removed by aspiration. The adherent cells are incubated with cell culture medium (IMDM, Sigma Chemical Co., St. Louis, Missouri) containing 10% fetal calf seram.
  • IMDM cell culture medium
  • the adherent cells are subjected to Epstein-Barr viras transformation by adding an equal amount of culture media obtained from growing the Epstein-Barr viras infected marmoset cell line, B95-8, or similar cell line, and thus containing the viras, to media bathing the adherent cells.
  • the cells are cultured in this environment at 37°C for 3 hours, and in this way the lymphocytes in the adherent cell population are subjected to Epstein-Barr infection. Following the infection period, the cells are washed and plated onto 96 well microtitre plates at a density of about 10 - 10 5 cells/well in IMDM medium, plus 10% fetal calf seram, and 30% conditioned medium.
  • the latter is derived from a lymphoblastoid cell line, preferably JW5.
  • the medium also contains 5 x lO- 5 M 2-mercaptoethanol, 50 ⁇ g ml gentamycin sulfate (Sigma), and 600 ng ml cyclosporine A (Sandimmune, Sandoz, Basel, Switzerland).
  • cell culture supematants are combined and screened for the desired antibody binding activity as described below.
  • Positive 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 Hybridomas 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 line F3B6 is a heteromyeloma that is sensitive to growth in media containing 100 ⁇ M hypoxanthine, 5 ⁇ g ml azaserine and 5 ⁇ M 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.
  • binding of native Factor XII to OT-2 may be determined as follows. Microtiter plates were coated overnight at 4°C with native Factor XII purified from plasma by affinity chromatography (Nuijens, J.H. si al., 1989, J. Biol. Chem.. 264:12941). The concentration of Factor XII used to coat the plates was 2 ⁇ g/ml in phosphate buffered saline (PBS), pH 7.4. The plates were washed and incubated 60 minutes at 37°C with dilutions of mAb OT-2 in PBS containing 0.1% (w/v) Tween 20.
  • PBS phosphate buffered saline
  • Inhibition of contact activation in plasma by mAb OT-2 was shown by measuring a decrease in kallikrein-Cl -inhibitor and Factor XII-Cl -inhibitor complexes. Activation was initiated by dextran sulfate.
  • 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 ⁇ g/ml in PBS, were added, and the mixture was incubated for 20 minutes at 37° C.
  • mAb 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 concentration of 100 ⁇ g ml for 20 minutes at 37°C.
  • the beads were washed with saline, and Cl -inhibitor complexes bound to the beads were detected by a subsequent incubation with polyclonal i- ⁇ I-anti-Cl-inhibitor antibodies.
  • the antibody F3 was similarly coupled to Sepharose, and also assayed for Factor Xlla- Cl-inhibitor complex binding activity.
  • the effectiveness of the Factor XII antibody, OT-2, in a baboon sepsis model system would be tested essentially as described by Taylor, si al., 1987, J. of Clinical Inv.. 22:918, and by Taylor, si al-, 1988, Circulator y Shock. 2.2:227. 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 XTJ antibody is effective in treating sepsis by preventing the death, or prolonging the lives of septic animals.
  • a lethal dose of E. eoli consists of approximately 4 x 10 10 organisms.

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Abstract

Compositions et procédés de traitement prophylactique ou thérapeutique de la scepticémie comprenant un inhibiteur de l'activation du facteur XII, l'inhibiteur étant de préférence un anticorps et présentant les propriétés suivantes: il se lie au facteur XII qu'il inactive dans du plasma, il inhibe l'activité enzymatique du facteur XIIa, et il ne réagit pas avec des complexes de facteur XII-C1.
PCT/US1991/002990 1990-05-10 1991-05-01 Inhibiteurs de l'activation du facteur xii et leurs applications WO1991017258A1 (fr)

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Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0577699A1 (fr) * 1991-03-11 1994-01-12 The General Hospital Corporation Compositions et procedes inhibant l'activation du facteur actif xiii
EP0637632A2 (fr) * 1993-08-06 1995-02-08 Nippon Zoki Pharmaceutical Co. Ltd. Méthode pour la mesure de l'activité d'une substance d'essai
EP0637633A2 (fr) * 1993-08-06 1995-02-08 Nippon Zoki Pharmaceutical Co. Ltd. Méthode pour la détermination de l'activité d'une substance d'essai
US6903184B1 (en) * 1998-03-02 2005-06-07 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Multiple antigenic peptides immunogenic against Streptococcus pneumonia
WO2007122371A1 (fr) * 2006-04-13 2007-11-01 Axis-Shield Diagnostics Limited TRAITEMENT DIRIGÉ CONTRE LE FACTEUR XIIa
EP2371857A1 (fr) * 2010-04-01 2011-10-05 CSL Behring GmbH Inhibiteurs de facteur XII pour traiter la maladie de poumon interstitiel
WO2012158003A1 (fr) 2011-05-17 2012-11-22 Tetra, Sia Nouvel inhibiteur du facteur xii
EP2548892A1 (fr) 2011-07-22 2013-01-23 CSL Behring GmbH Anticorps inhibiteurs monoclonal contre Factor XII/XIIa et leurs utilisations
WO2013014092A1 (fr) 2011-07-22 2013-01-31 Csl Behring Gmbh Anticorps monoclonaux inhibiteurs anti-facteur xii/xiia et leurs utilisations
WO2013113774A1 (fr) 2012-01-31 2013-08-08 Csl Behring Gmbh Inhibiteurs du facteur xii destinés au traitement de troubles inflammatoires neurologiques
WO2014135694A1 (fr) 2013-03-08 2014-09-12 Csl Behring Ag Traitement et prévention d'une lésion d'ischémie reperfusion distante
WO2014207199A1 (fr) 2013-06-28 2014-12-31 Csl Behring Gmbh Polythérapie utilisant un inhibiteur du facteur xii et un inhibiteur de c1
US20150315292A1 (en) * 2012-12-07 2015-11-05 Vanderbilt University Antibodies against factor xii and uses thereof
WO2015193457A1 (fr) 2014-06-18 2015-12-23 Csl Behring Gmbh Thérapie à l'aide d'un inhibiteur du facteur xii dans un trouble neurotraumatique
US9518128B2 (en) 2011-12-14 2016-12-13 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules
US9518129B2 (en) 2011-12-14 2016-12-13 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
WO2019030706A1 (fr) 2017-08-10 2019-02-14 Janssen Pharmaceutica Nv Molécules d'anticorps anti-thrombine et méthodes d'utilisation en chirurgie orthopédique
WO2019035055A1 (fr) 2017-08-16 2019-02-21 Janssen Pharmaceutica Nv Molécules d'anticorps anti-thrombine et procédés d'utilisation avec des agents antiagrégants plaquettaires
WO2019246176A1 (fr) * 2018-06-19 2019-12-26 Regeneron Pharmaceuticals, Inc. Anticorps anti-facteur xii/xiia et leurs utilisations
US10913802B2 (en) 2015-07-21 2021-02-09 Dyax Corp. Monoclonal antibody inhibitor of factor XIIA
US11198870B2 (en) 2002-11-14 2021-12-14 Thermo Fisher Scientific Inc. Methods and compositions for selecting siRNA of improved functionality
US11390687B2 (en) 2015-01-02 2022-07-19 Takeda Pharmaceutical Company Limited Bispecific antibodies against plasma kallikrein and Factor XII

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WO1989011865A1 (fr) * 1988-06-09 1989-12-14 Temple University Of The Commonwealth System Of Hi Anticorps monoclonaux contre la region a chaine legere du facteur humain xii et procedes de preparation et d'utilisation de ces anticorps
WO1990008835A1 (fr) * 1989-01-27 1990-08-09 Coagen Limited ESSAI IMMUNITAIRE ET ANTICORPS MONOCLONAL DU FACTEUR XIIa β DE COAGULATION DU SANG

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WO1989011865A1 (fr) * 1988-06-09 1989-12-14 Temple University Of The Commonwealth System Of Hi Anticorps monoclonaux contre la region a chaine legere du facteur humain xii et procedes de preparation et d'utilisation de ces anticorps
WO1990008835A1 (fr) * 1989-01-27 1990-08-09 Coagen Limited ESSAI IMMUNITAIRE ET ANTICORPS MONOCLONAL DU FACTEUR XIIa β DE COAGULATION DU SANG

Non-Patent Citations (1)

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Title
THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 264, no. 22, 5 August 1989, The American Society for Biochemistry and Molecular Biology, Inc. (US), J.H. NUIJENS et al.: "Activation of the contact system of coagulation by a monoclonal antibody directed against a neodeterminant in the heavy chain region of human coagulation factor XII (Hageman factor)", pages 12941-12949, see the whole article (cited in the application) *

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EP0577699A4 (fr) * 1991-03-11 1995-03-15 Gen Hospital Corp Compositions et procedes inhibant l'activation du facteur actif xiii.
US5620688A (en) * 1991-03-11 1997-04-15 Bristol-Myers Squibb Company Methods of inhibiting the activation of Factor XIII
EP0577699A1 (fr) * 1991-03-11 1994-01-12 The General Hospital Corporation Compositions et procedes inhibant l'activation du facteur actif xiii
EP0637632A2 (fr) * 1993-08-06 1995-02-08 Nippon Zoki Pharmaceutical Co. Ltd. Méthode pour la mesure de l'activité d'une substance d'essai
EP0637633A2 (fr) * 1993-08-06 1995-02-08 Nippon Zoki Pharmaceutical Co. Ltd. Méthode pour la détermination de l'activité d'une substance d'essai
EP0637633A3 (fr) * 1993-08-06 1998-01-07 Nippon Zoki Pharmaceutical Co. Ltd. Méthode pour la détermination de l'activité d'une substance d'essai
EP0637632A3 (fr) * 1993-08-06 1998-01-07 Nippon Zoki Pharmaceutical Co. Ltd. Méthode pour la mesure de l'activité d'une substance d'essai
US8642048B2 (en) 1998-03-02 2014-02-04 The United States of America, as represented by the Secretary of the Department of Health and Human Services, Centers for Disease Control and Prevention Multiple antigenic peptides immunogenic against Streptococcus pneumonia
US6903184B1 (en) * 1998-03-02 2005-06-07 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Multiple antigenic peptides immunogenic against Streptococcus pneumonia
US7501132B2 (en) 1998-03-02 2009-03-10 The United States Of America As Represented By The Department Of Health And Human Services Multiple antigenic peptides immunogenic against Streptococcus pneumonia
US11198870B2 (en) 2002-11-14 2021-12-14 Thermo Fisher Scientific Inc. Methods and compositions for selecting siRNA of improved functionality
WO2007122371A1 (fr) * 2006-04-13 2007-11-01 Axis-Shield Diagnostics Limited TRAITEMENT DIRIGÉ CONTRE LE FACTEUR XIIa
US9150638B2 (en) 2010-04-01 2015-10-06 Justus-Liebig-Universität Giessen Factor XII inhibitors for treating interstitial lung disease
EP2371857A1 (fr) * 2010-04-01 2011-10-05 CSL Behring GmbH Inhibiteurs de facteur XII pour traiter la maladie de poumon interstitiel
WO2012158003A1 (fr) 2011-05-17 2012-11-22 Tetra, Sia Nouvel inhibiteur du facteur xii
KR20140054112A (ko) * 2011-07-22 2014-05-08 체에스엘 베링 게엠베하 억제성 항-ｘｉｉ/ｘｉｉａ 인자 모노클로날 항체 및 이들의 사용
KR102042982B1 (ko) * 2011-07-22 2019-11-11 체에스엘 베링 게엠베하 억제성 항-ｘｉｉ/ｘｉｉａ 인자 모노클로날 항체 및 이들의 사용
WO2013014092A1 (fr) 2011-07-22 2013-01-31 Csl Behring Gmbh Anticorps monoclonaux inhibiteurs anti-facteur xii/xiia et leurs utilisations
JP2014523253A (ja) * 2011-07-22 2014-09-11 ツェー・エス・エル・ベーリング・ゲー・エム・ベー・ハー 阻害性抗第ｘｉｉ／ｘｉｉａ因子モノクローナル抗体及びそれらの使用
EP2548892A1 (fr) 2011-07-22 2013-01-23 CSL Behring GmbH Anticorps inhibiteurs monoclonal contre Factor XII/XIIa et leurs utilisations
US10513560B2 (en) 2011-07-22 2019-12-24 Csl Behring Gmbh Methods of administering inhibitory anti-factor XII/XIIA monoclonal antibodies
US20170121423A1 (en) * 2011-07-22 2017-05-04 Csl Behring Gmbh Inhibitory anti-factor xii/xiia monoclonal antibodies and their uses
CN103687878A (zh) * 2011-07-22 2014-03-26 德国杰特贝林生物制品有限公司 抑制性的抗因子xii/xiia单克隆抗体及其用途
CN103687878B (zh) * 2011-07-22 2018-01-09 德国杰特贝林生物制品有限公司 抑制性的抗因子xii/xiia单克隆抗体及其用途
AU2012289001B2 (en) * 2011-07-22 2016-03-03 Csl Behring Gmbh Inhibitory anti -factor XII/XIIa monoclonal antibodies and their uses
US11345759B2 (en) 2011-07-22 2022-05-31 Csl Behring Gmbh Methods of administering inhibitory anti-factor XII/XIIa monoclonal antibodies
US9518127B2 (en) 2011-07-22 2016-12-13 Csl Behring Gmbh Inhibitory anti-factor XII/XIIA monoclonal antibodies and their uses
US9856326B2 (en) 2011-07-22 2018-01-02 Csl Behring Gmbh Methods of administering inhibitory anti-factor XII/XIIA monoclonal antibodies
US9856325B2 (en) 2011-07-22 2018-01-02 Csl Behring Gmbh Nucleic acids encoding inhibitory anti-factor XII/XIIa monoclonal antibodies
US20170137536A1 (en) * 2011-07-22 2017-05-18 Csl Behring Gmbh Inhibitory anti-factor xii/xiia monoclonal antibodies and their uses
US9518128B2 (en) 2011-12-14 2016-12-13 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules
US10370454B2 (en) 2011-12-14 2019-08-06 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
US9518129B2 (en) 2011-12-14 2016-12-13 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
US9988463B2 (en) 2011-12-14 2018-06-05 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
US9988461B2 (en) 2011-12-14 2018-06-05 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
US11155637B2 (en) 2011-12-14 2021-10-26 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
US10287363B2 (en) 2011-12-14 2019-05-14 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
US9605082B2 (en) 2011-12-14 2017-03-28 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
WO2013113774A1 (fr) 2012-01-31 2013-08-08 Csl Behring Gmbh Inhibiteurs du facteur xii destinés au traitement de troubles inflammatoires neurologiques
US9957329B2 (en) 2012-01-31 2018-05-01 Csl Behring Gmbh Factor XII inhibitors for the treatment of neurological inflammatory disorders
US9574013B2 (en) * 2012-12-07 2017-02-21 Vanderbilt University Antibodies against factor XII and uses thereof
US20150315292A1 (en) * 2012-12-07 2015-11-05 Vanderbilt University Antibodies against factor xii and uses thereof
US10286047B2 (en) 2013-03-08 2019-05-14 Csl Behring Gmbh Treatment and prevention of remote ischemia-reperfusion injury
US10973891B2 (en) 2013-03-08 2021-04-13 Csl Behring Gmbh Treatment and prevention of remote ischemia-reperfusion injury
WO2014135694A1 (fr) 2013-03-08 2014-09-12 Csl Behring Ag Traitement et prévention d'une lésion d'ischémie reperfusion distante
WO2014207199A1 (fr) 2013-06-28 2014-12-31 Csl Behring Gmbh Polythérapie utilisant un inhibiteur du facteur xii et un inhibiteur de c1
WO2015193457A1 (fr) 2014-06-18 2015-12-23 Csl Behring Gmbh Thérapie à l'aide d'un inhibiteur du facteur xii dans un trouble neurotraumatique
US11390687B2 (en) 2015-01-02 2022-07-19 Takeda Pharmaceutical Company Limited Bispecific antibodies against plasma kallikrein and Factor XII
US10913802B2 (en) 2015-07-21 2021-02-09 Dyax Corp. Monoclonal antibody inhibitor of factor XIIA
WO2019030706A1 (fr) 2017-08-10 2019-02-14 Janssen Pharmaceutica Nv Molécules d'anticorps anti-thrombine et méthodes d'utilisation en chirurgie orthopédique
WO2019035055A1 (fr) 2017-08-16 2019-02-21 Janssen Pharmaceutica Nv Molécules d'anticorps anti-thrombine et procédés d'utilisation avec des agents antiagrégants plaquettaires
WO2019246176A1 (fr) * 2018-06-19 2019-12-26 Regeneron Pharmaceuticals, Inc. Anticorps anti-facteur xii/xiia et leurs utilisations
CN112437682A (zh) * 2018-06-19 2021-03-02 里珍纳龙药品有限公司 抗因子XII/XIIa抗体和其用途

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