US20080160015A1 - Anti-Idiotypic Antibodies Neutralizing the Inhibitory Activity of an Inhibitory Antibody Directed Against the C1 Domain of Factor VIII - Google Patents

Anti-Idiotypic Antibodies Neutralizing the Inhibitory Activity of an Inhibitory Antibody Directed Against the C1 Domain of Factor VIII Download PDF

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US20080160015A1
US20080160015A1 US11/817,018 US81701807A US2008160015A1 US 20080160015 A1 US20080160015 A1 US 20080160015A1 US 81701807 A US81701807 A US 81701807A US 2008160015 A1 US2008160015 A1 US 2008160015A1
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antibody
seq
factor viii
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Jean-Guy Gilles
Marc G. Jacquemin
Jean-Marie Saint-Remy
Christian Behrens
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LFB Biotechnologies SAS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the present invention is related to a monoclonal anti-idiotypic antibody directed against a Factor VIII inhibitory antibody which binds to the C1 domain of Factor VIII, as well as to a cell line producing this monoclonal anti-idiotypic antibody, to the use of this monoclonal anti-idiotypic antibody as medicament, and more particularly, to the use thereof for manufacturing a medicament for the treatment of haemophilia A.
  • Haemophilia A is a hereditary disease linked to an anomaly of chromosome X, which displays itself in the affected person by an inability to coagulate. This disease is the result of mutations on the gene of a protein involved in coagulation, the Factor VIII (FVIII) protein, which determine either a total absence of Factor VIII in the blood, or a partial deficit thereof.
  • FVIII Factor VIII
  • Haemophilia A is the most common of insufficiencies affecting blood coagulation: in France 1 male in 5000 is affected, which represents 80% of patients suffering from haemophilia.
  • the other type of haemophilia, haemophilia B affects 20% of patients suffering from haemophilia; it is caused by a deficiency in an other clotting factor, known as Factor IX.
  • heamophilia type A or B
  • heamophilia type A or B
  • Factor VIII for the treatment of haemophiliacs is available in form of blood derived medicaments provided by the Laboratoire Francais du Fractionêt et des Biotechnologies (LFB) or by international pharmaceutical laboratories, as well as in form of recombinant medicaments prepared by genetic engineering methods.
  • LLB Laboratoire Francais du Fractionêt et des Biotechnologies
  • the DNA coding Factor VIII has been isolated and expressed in mammalian cells (Wood et al., Nature (1984) 312: 330-337), and its amino acid sequence was deduced from cDNA.
  • Secreted Factor VIII is a glycoprotein with a molecular weight of 300 Kda (2332 amino acids), and plays a key role in the activation of intrinsic coagulation pathway.
  • Inactive FVIII consists of six regions: A1 (residues 1-372), A2 (residues 373-740), B (residues 741-1648), A3 (residues 1690-2019), C1 (residues 2020-2172) and C2 (residues 2173-2332), from the N-terminal extremity to the C-terminal extremity.
  • FVIII interacts with the von Willebrand Factor (vWF), which protects the FVIII against plasma proteases.
  • vWF von Willebrand Factor
  • FVIII dissociates from vWF upon cleavage by thrombin. This cleavage results in the elimination of the B domain and the formation of a heterodimer.
  • FVIII circulates in plasma in this form.
  • This heterodimer consists of a heavy chain (A1, A2) and of a light chain (A3, C1, C2).
  • FVIII When FVIII is infused to a haemophiliac patient, it binds to the von Willebrand Factor in the blood circulation of the patient.
  • Activated Factor VIII acts as a co-factor of activated Factor IX, accelerating the conversion of Factor X into activated Factor X.
  • Activated Factor X converts prothrombin into thrombin. Then the thrombin converts fibrinogen into fibrin, and clotting occurs.
  • the major problem encountered with Factor VIII administration is the appearance of antibodies directed against Factor VIII in the patient, referred to as ⁇ inhibiting antibodies >>. These antibodies neutralize the procoagulant activity of Factor VIII, which is inactivated as soon as infused. Thus, the administered clotting factor is destroyed before bleeding can be stopped, which leads to a serious complication thus causing the treatment to be ineffective. Further, some genetically non-haemophiliac patients may develop inhibitors against endogenous Factor VIII: this is called acquired haemophilia.
  • the anti-Factor VIII immune response is of the polyclonal IgG type belonging mostly to the IgG4 and IgG1 sub-class, and more rarely to IgG2.
  • the IgG3 subclass is never represented.
  • the light chain is often of Kappa type.
  • the overrepresentation of IgG4 is more pronounced with heamophiliacs having a long-term established inhibitor.
  • the C2 and A2 domains of the FVIII molecule are the favoured targets of the immune response although, in some cases, antibodies directed against the A3 domain are detected.
  • the recovered amounts are often higher than 100 ⁇ g per 10 mg of total IgGs (Gilles J G et al. (1993) Blood; 82: 2452-2461).
  • An animal model has been developed to study the formation of inhibitors of Factor VIII; rats immunized with human recombinant Factor VIII show a rapid immune response of the polyclonal type (Jarvis et al., Thromb Haemost. 1996 February; 75(2):318-25).
  • anti-Factor VIII antibodies interfere with function of Factor VIII are numerous, and include interference with the proteolytic cleavage of Factor VIII and with the interaction of Factor VIII with different partners, such as von Willebrand Factor (vWF), phospholipids (PL), Factor IX, activated Factor X (FXa) or APC (Activated Protein C).
  • vWF von Willebrand Factor
  • PL phospholipids
  • FXa activated Factor X
  • APC Active Protein C
  • BO2C11 a human monoclonal antibody directed against the C2 domain of Factor VIII, referred to as BO2C11 (IgG4kappa), produced from a library of memory B cells of a patient suffering from haemophilia A with inhibitors, has been isolated (Jacquemin et al., Blood 1998 Jul. 15; 92 (2):496-506).
  • BO2C11 recognizes the C2 domain of Factor VIII, and inhibits its binding to von Willebrand Factor and to phospholipids. It completely inhibits the procoagulation activity of native and activated Factor VIII.
  • a further example of monoclonal antibody is the BOIIB2 antibody directed against the A2 domain of Factor VIII.
  • the BOIIB2 antibody inhibits 99% of Factor VIII activity. By binding to the A2 domain, it can interfere with and inhibit the binding of FIXa, which contains a low affinity binding site within this region of FVIII, and thus inhibits the enzyme activity of FIXa.
  • the second conceivable way of action is its interference with the equilibrium between the heterodimeric form (A2:A1 and A3:C1:C2) of FVIII and the heterotrimeric form (A2 and A1 and A3:C1:C2) of FVIII by accelerating the dissociation of the A2 domain of these complexes, rendering them non-functional. (Ananyeva N M et al., (2004) Blood Coagul Fibrinolysis. Mar. 15(2):109-24. Review).
  • a mouse anti-idiotypic antibody known as 14C12, disclosed in the document WO 2004/014955, neutralizes in vivo, in a dose-dependent manner, the inhibitory properties of the anti-Factor VIII target antibody (monoclonal antibody BO2C11), which is directed against the C2 domain of Factor VIII.
  • the anti-Factor VIII immune response being polyclonal, mouse anti-idiotypic antibodies directed against the A2 domain of Factor VIII have also been developed (and described in the patent application FR 05 08320).
  • the A2 domain is a domain of 43 kD, the function of which is not well known but it has been demonstrated that inhibitory antibodies directed against the A2 domain of Factor VIII inhibit the function of Factor VIIIa by inhibiting the conversion of the complex FXase/FX in the transition state (Lollar et al., J Clin Invest. 1994 June; 93(6):2497-504, Fay et al., J Biol. Chem. 1996; 271(11) 6027-6032).
  • the immune response directed against Factor VIII is polyclonal, and, therefore, implies that inhibitory antibodies are directed against domains different from the A2 and C2 domains. Indeed, even if the study of epitopic specificities of anti-Factor VIII antibodies has shown that the majority of the inhibitors recognize limited zones of the Factor VIII molecule, located on the A2 domain of the heavy chain and/or on the C2 domain of the light chain, other epitopes are sometimes recognized. Indeed, some plasmas from patients contain antibodies capable to bind to the C1 domain of the light chain of Factor VIII (Moreau et al., 2000; 95(11):3435-441; Jacquemin et al,. 2000; 95(1):156-162).
  • the Applicant has attempted to develop a novel tool for treating haemophilia A enabling neutralization of inhibitory antibodies directed against the C1 domain of Factor VIII.
  • a first object of the invention relates to a monoclonal anti-idiotypic antibody directed against a Factor VIII human inhibitory antibody, the inhibitory antibody being directed against the C1 domain of Factor VIII, this anti-idiotypic antibody having at least one CDR region (Complementarity Determining Region) of each of the light chains of said antibody, in which the peptide sequence has at least 70% identity to a sequence selected from the sequences SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and at least one CDR region of each of the heavy chains of said antibody, in which the peptide sequence has at least 70% identity to a sequence selected from the sequences SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11.
  • the concerned CDR regions are the CDR1 and/or CDR2 and/or CDR3 regions.
  • sequences SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, are defined according to Kabat [Kabat et al., “Sequences of Proteins of Immunological Interest”, NIH Publication, 91-3242 (1991)].
  • the identity with each of the above-mentioned sequences is at least 80%, preferably at least 90%, 95%, 99%, and more preferably 100%.
  • the percentage of identity is calculated by aligning the two sequences to be compared and by counting the number of positions having an identical amino acid, this number being divided by the total number of amino acids of the sequence. In any case, these sequence differences do not affect at all either the affinity of the monoclonal antibody for its target, or its functionality.
  • ⁇ Inhibitory antibodies >> or ⁇ inhibitors >> of Factor VIII refers to antibodies which inhibit all or a part of the procoagulant activity of Factor VIII, namely by binding thereto, and particularly an anti-Factor VIII antibody the epitope of which is located on Factor VIII.
  • the antibody of the invention has the ability to neutralize at least 20%, advantageously at least 30%, advantageously at least 40%, advantageously at least 50%, advantageously at least 60%, and in an even more advantageous way, at least 70%, 80%, 90%, 99% or 100% of the coagulation inhibitory activity of inhibitory antibodies directed against the C1 domain of Factor VIII, which are the targets of the anti-idiotypic monoclonal antibodies of the invention.
  • This ability to neutralize the coagulation inhibitory activity of inhibitory antibodies can be determined by measuring the activity of Factor VIII in the presence of an inhibitory antibody and of an anti-idiotypic antibody in an assay such as the ⁇ Factor VIII chromogen test >> (Jacquemin et al. (1998) Blood 92. 494-506).
  • anti-idiotypic antibody refers to an antibody directed against the variable region of the target inhibitory antibodies.
  • the anti-idiotypic antibody of the invention is directed against inhibitory antibodies, of which the variable domain of the heavy chain of said antibody is related to the germ line DP-10.
  • inhibitory antibodies can be obtained from humans (for example from serum of patients containing inhibitory antibodies) or other animal species such as mouse, horse, goat, non-human primates, taken from a non-limiting list, by immunization with Factor VIII or fragments derived from Factor VIII, and more particularly with a fragment comprising all or a part of the C1 domain.
  • the target inhibitory antibody of the anti-idiotypic antibody of the invention recognizes the C1 domain in its native configuration.
  • the target inhibitory antibody of the anti-idiotypic antibody of the invention does not recognize the same domain being a R2150H mutation.
  • the monoclonal anti-idiotypic antibody of the invention can be of human or animal origin. In addition, it can be obtained using a variety of different methods. For example, cells producing anti-idiotypic antibodies can be obtained from peripheral blood lymphocytes of patients having anti-Factor VIII inhibitory antibodies or from healthy persons. These cells can be immortalized by use of techniques well known to those skilled in the art and selected with regard to the ability of the produced anti-idiotypic antibodies to neutralize inhibitory antibodies directed against Factor VIII.
  • a further method for producing the monoclonal anti-idiotypic antibody of the invention is through the immunization of animals, advantageously mice, by injection of Factor VIII inhibitory antibodies directed against the C1 domain of Factor VIII, then by fusion of spleen lymphocytes with a myeloma cell line, advantageously mouse myeloma, followed by the identification and the cloning of cell cultures producing the anti-idiotypic antibodies directed against the Factor VIII inhibitory antibodies.
  • each CDR region of the light chains of the anti-idiotypic antibody of the invention contains a peptide sequence having at least 70% identity with the sequences respectively identified as SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, and each CDR region of each of the heavy chains of said antibody contains a peptide sequence having at least 70% identity with the sequences respectively identified as SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11.
  • the CDR1 region of each of the light chains of the antibody of the invention contains a peptide sequence having at least 70% identity to the sequence SEQ ID NO: 12
  • the CDR2 region of each of the light chains of the antibody of the invention contains a peptide sequence having at least 70% identity to the sequence SEQ ID NO: 13
  • the CDR3 region of each of the light chains of the antibody of the invention contains a peptide sequence having at least 70% identity to the sequence SEQ ID NO: 14
  • the CDR1 region of each of the heavy chains of the antibody of the invention contains a peptide sequence having at least 70% identity to the sequence SEQ ID NO: 9
  • the CDR2 region of each of the heavy chains of the antibody of the invention contains a peptide sequence having at least 70% identity to the sequence SEQ ID NO: 10
  • the CDR3 region of each of the heavy chains of the antibody of the invention contains a peptide sequence having at least 70% identity to the sequence SEQ ID NO: 11.
  • the identity to each of the above-mentioned sequences
  • variable region of each of the light chains of the monoclonal anti-idiotypic antibody of the invention is coded by a nucleic acid sequence having at least 70% identity to the nucleic acid sequence SEQ ID NO: 16
  • variable region of each of the heavy chains of the monoclonal anti-idiotypic antibody is coded by a nucleic acid sequence having at least 70% identity to the nucleic acid sequence SEQ ID NO: 15.
  • a signal peptide can be added to sequences SEQ ID NO: 15 and SEQ ID NO: 16 to yield respectively, for example, the sequences SEQ ID NO: 1 and SEQ ID NO: 2, wherein neither the activity nor the specificity of the antibody of the invention are affected by such a signal peptide.
  • the sequence identity is at least 80%, and preferably from at least 95 to 99%.
  • the percentage of identity is calculated by alignment of 2 sequences to be compared and by counting the number of positions containing an identical nucleotide, this number is divided by the total number of nucleotides of the sequence.
  • Genetic code degeneration lea ds to the fact that the same amino acid can be coded by several triplets of different nucleotides. In any case, neither the affinity of the monoclonal antibody for its target nor its ability to neutralize the inhibitory activity of the target inhibitory antibodies are at all affected by these sequence differences.
  • variable region of each of the light chains of the monoclonal anti-idiotypic antibody is coded by the nucleic acid sequence SEQ ID NO: 16
  • variable region of each of the heavy chains of the monoclonal anti-idiotypic antibody is coded by the nucleic acid sequence SEQ ID NO: 15.
  • the peptide sequence of each of the variable regions of the light chains of the antibody of the invention is a sequence having at least 70% identity, and advantageously at least 80% or 90%, and yet more advantageously at least 99% identity to the sequence SEQ ID NO: 18.
  • a signal peptide can be added to the sequences SEQ ID NO: 17 and SEQ ID NO: 18 in order to yield, for example, respectively, the sequences SEQ ID NO: 3 and SEQ ID NO: 4, neither the activity nor the specificity of the antibody of the invention are affected at by such a signal peptide.
  • the peptide sequence of each of the variable regions of the heavy chains of the antibody of the invention is a sequence having at least 70% identity, and advantageously at least 80% or 90%, and yet more advantageously at least 99% identity to the sequence SEQ ID NO: 3.
  • the peptide sequence of each of the light chains of the antibody of the invention is a sequence having at least 70% identity, and advantageously at least 80% or 90%, and yet more advantageously at least 99% identity to the sequence SEQ ID NO: 4, and the peptide sequence of each of the heavy chains of the antibody of the invention is a sequence having at least 70% identity, and advantageously at least 80% or 90%, and yet more advantageously, at least 99% identity to the sequence SEQ ID NO: 3.
  • the peptide sequence of each of the light chains of the antibody of the invention is the sequence SEQ ID NO: 4.
  • the peptide sequence of each of the light chains of the antibody of the invention is the sequence SEQ ID NO: 3.
  • the peptide sequence deduced from the sequence SEQ ID NO: 16 is the sequence SEQ ID NO: 18, and the peptide sequence deduced from the sequence SEQ ID NO: 15 is the sequence SEQ ID NO: 17.
  • the variable region of each of the light chains of the monoclonal anti-idiotypic antibody of the invention has the peptide sequence SEQ ID NO: 18, and the variable region of each of the heavy chains of the monoclonal anti-idiotypic antibody of the invention has the peptide sequence SEQ ID NO: 17.
  • the target inhibitory antibody of the anti-idiotypic antibody of the invention is the antibody RHD5 deposited at the Belgian Co-ordinated Collections of Microorganisms/Plasmid Collection (BCCM/LMBP), Laboratorium voor Mole Les Biologie, University of Ghent, Technologiepark 297, B-9052 Zwijnaarede, Belgium, in August 2004, by the Collen Research Foundation, under the accession number LMBP 6165CB.
  • BCCM/LMBP Belgian Co-ordinated Collections of Microorganisms/Plasmid Collection
  • the antibody RHD5 is a human monoclonal IgG1 antibody directed against the C1 domain of Factor VIII produced initially from lymphocytes of a patient suffering from haemophilia A, namely an acquired severe haemophilia A with a high level of inhibitors.
  • This antibody belongs to the sub-class IgG1, and originates from the germ line DP-10.
  • the epitope recognized by said antibody on Factor VIII is the C1 domain in its native configuration, but not the same domain with a R2150H mutation.
  • the antibody RHD5 can inhibit up to 98% of Factor VIII activity.
  • the antibody of the invention refers also to any modified antibody having the features of the invention, in which one or more amino acid(s) have been substituted or deleted. Such a substitution or deletion can be located on any position in the molecule. In the case where several amino acids have been substituted or deleted, any combination of substitution or deletion can be considered. Such sequence modifications of the variable regions of the antibody of the invention can be carried out in order to increase the number of residues likely to come into contact with the anti-idiotypic antibody of the invention and with the target inhibitor antibody.
  • the anti-idiotypic antibody is a mouse antibody.
  • this mouse monoclonal anti-idiotypic antibody is a IgG1kappa.
  • the monoclonal antibody of the invention is a chimeric antibody.
  • ⁇ Chimeric antibody it is to be understood that it refers to an antibody in which the variable regions of the light chains and of the heavy chains belong to a different species than the constant regions of the light chains and of the heavy chains.
  • the antibody of the invention also contains the constant regions of light and heavy chains belonging to a non-murine species.
  • all non-murine mammalian families and species are capable of being used, and in particular, for example, man, monkey, muridae (except the mouse), suidae, bovidae, equidae, felidae, canidae, as well as birds.
  • the chimeric antibodies of the invention can be constructed using standard techniques for recombinant DNA, well known by those skilled in the art, and more particularly through the use of the ⁇ chimeric >> antibody construction techniques described, for example by Morrison et al., Proc. Natl. Acad. Sci. U.S.A., 81. pp. 6851-55 (1984), where use is made of recombinant DNA technology to replace the constant region of a heavy chain and/or the constant region of a light chain of an antibody originating from a non-human mammal with the corresponding regions of a human immunoglobulin.
  • the antibody of the invention is a human hybrid antibody, that is to say a chimeric antibody, the constant part of which is human.
  • This embodiment of the invention enables a reduction in the immunogenicity of the antibody in humans, and thereby improves its efficacy upon therapeutic administration to man.
  • the antibody of the invention is a humanized antibody.
  • Such an antibody can be obtained by association of one or more CDR region(s) (Complementarity Determining Region) of a monoclonal antibody of a non-human species with human framework regions (highly conserved regions of variable regions, known as frameworks), such a manufacturing process being taught in the state of the art (Jones et al., Nature (1986) 321:522; Riechmann et al., Nature (1988) 332:323).
  • CDR region(s) Complementarity Determining Region
  • Such a humanized antibody directed against the variable domain of inhibitory antibodies recognizing the C1 domain of FVIII can contain human framework regions and one or more CDR regions of the sequences SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14.
  • a particular humanized antibody of the invention is a humanized antibody directed against the variable domain of inhibitory antibodies recognizing the C1 domain of FVIII, the CDR regions of which are regions of sequence SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14.
  • the monoclonal anti-idiotypic antibody of the invention is the antibody 18B6 produced by the hybridoma 18B6 deposited under the registration number CNCM I-3559, on Jan. 24, 2006, at the Collection Nationale de Cultures de Microorganismes (CNCM, 25 rue du Dondel Roux, 75724 Paris Cedex 15).
  • the variable region of each of the light chains of the monoclonal anti-idiotypic antibody 18B6 is coded by the nucleic acid sequence SEQ ID NO: 16
  • the variable region of each of the heavy chains of the monoclonal anti-idiotypic antibody 18B6 is coded by the nucleic acid sequence SEQ ID NO: 15.
  • the method for obtaining the hybridoma 18B6 is described in the ⁇ Examples >> section of the present document.
  • the monoclonal anti-idiotypic antibody of the invention refers also to any antibody comprising fragments of the antibody 18B6, and more particularly any antibody comprising the variable region of the light chain and/or the variable region of the heavy chain of the antibody 18B6, or any fragment of the variable region of the light chain and/or the variable region of the heavy chain of the antibody 18B6.
  • ⁇ Fragments it is meant a F(ab′)2 fragment or a Fab′ fragment or a Fab fragment or a CDR region or any modified version of any of these fragments or region.
  • the monoclonal anti-idiotypic antibody of the invention is a F(ab′)2 fragment or a Fab′ fragment or a Fab fragment or a CDR region or any modified version of any of these fragments or region.
  • the enzymatic digestion of immunoglobulins with papain generates 2 identical fragments called ⁇ Fab fragment >> (Fragment Antigen Binding), and a Fc fragment (crystallizable fraction).
  • the Fc fragment is the support for the effector functions of immunoglobulins.
  • F(ab′)2 fragment is generated where both Fab fragments remain linked by two disulfide bonds, and the Fc fragment is split into several peptides.
  • the F(ab′)2 fragment is formed by two Fab′ fragments (one Fab′ fragment consisting of a Fab and a hinge region), linked by intercatenary disulfide bonds in order to form a F(ab′)2.
  • Such fragments which contain the binding site of the antibody, may have lost some of the properties of a whole antibody from which they are derived, such as the ability to activate the complement or to bind the Fcgamma receptors. However, these fragments have not lost the ability of the whole antibody to neutralize the inhibitor antibody.
  • the invention refers also to the F(ab′)2, Fab′, Fab fragments, or to the CDR region or any modified version of any of these fragments or region of the antibody 18B6. Particularly, these fragments have preserved the ability of the whole antibody to neutralize RHD5 antibodies.
  • a further object of the invention is a stable cell line producing an antibody such as described above.
  • the stable cell line of the invention can be of human or animal origin.
  • the stable cell line of the invention can originate from human immortalized cells.
  • this cell line can originate from immortalized cells of animal origin, for example mice.
  • a preferred example of a cell line obtained in this embodiment of the invention is the line 18B6, deposited at CNCM under the number I-3559.
  • the stable cell line of the invention is a line which has integrated a genetic construction allowing the expression of the antibody of the invention at the desired point of the genome. The step consisting of obtaining such a cell is a stable transfection.
  • This step can be applied to any type of cells as long as they can be maintained in in vitro culture.
  • Stable transfection requires integration of the genetic construction, which can be carried out by homologous recombination or randomly.
  • the presence of a positive selection cassette in the genetic construction comprising the gene of interest which confers antibiotic resistance to the cell, for example, attests to the insertion of the transgene into the cell genome.
  • a long term producer cell line is obtained from the antibody of the invention, for example 18B6, which can be maintained in in vitro culture.
  • the stable cell line expressing an antibody of the invention can be selected from the group consisting of a human cell line, a rodent cell line, for example a mouse cell line, SP2/0, YB2/0, IR983F, a human myeloma such as Namalwa, or any other cell of human origin such as PERC6, CHO cell lines, namely CHO-K-1, CHO-Lec10, CHO-Lec1, CHO-Lec13, CHO Pro-5, CHO dhfr- (CHO DX B11, CHO DG44), or further cell lines selected from Wil-2, Jurkat, Vero, Molt-4, COS-7, 293-HEK, BHK, K6H6, NSO, SP2/0-Ag 14 and P3X63Ag8.653.
  • a human cell line for example a mouse cell line, SP2/0, YB2/0, IR983F, a human myeloma such as Namalwa
  • PERC6
  • a further particular subject matter of the invention is the hybridoma 18B6 deposited under the registration number CNCM I-3559 at the Collection Nationale de Cultures de Microorganismes (CNCM).
  • the variable region of each of the light chains of the monoclonal anti-idiotypic antibody produced by the hybridoma 18B6 is coded by the nucleic acid sequence SEQ ID NO: 16
  • the variable region of each of the heavy chains of the monoclonal anti-idiotypic antibody produced by the hybridoma 18B6 is coded by the nucleic acid sequence SEQ ID NO: 15.
  • the antibody produced by the hybridoma 18B6 is the antibody 18B6, and a method for obtaining the hybridoma 18B6 is described in the “Examples” Section of the present document.
  • a further subject matter of the invention is a DNA fragment of the sequence SEQ ID NO: 15 encoding the variable region of the heavy chain of the antibody of the invention such as previously described.
  • This DNA fragment can be inserted into a vector enabling the expression of a polypeptide, preferably of an antibody, the variable region of the heavy chain of said antibody is coded by the nucleic acid sequence SED ID NO: 15, the derived peptide sequence of which is the sequence SEQ ID NO: 17, in order to be introduced and maintained in a host cell.
  • This vector enables the expression of this foreign nucleic acid fragment in the host cell because it contains the sequences (promoter, polyadenylation sequence, selection gene) essential for this expression.
  • any mammalian cell can be used as the host cell, that is as the cell expressing the polypeptide or the antibody of the invention, for example SP2/0, YB2/0, IR983F, a human myeloma such as Namalwa, or any other cell of human origin such as PERC6, CHO cell lines, namely CHO-K-1, CHO-Lec10, CHO-Lec1, CHO-Lec13, CHO Pro-5, CHO dhfr-(CHO DX B11, CHO DG44), or other lines selected from Wil-2, Jurkat, Vero, Molt-4, COS-7, 293-HEK, BHK, K6H6, NSO, SP2/0-Ag 14 and P3X63Ag8.653.
  • any mammalian cell can be used as the host cell, that is as the cell expressing the polypeptide or the antibody of the invention, for example SP2/0, YB2/0, IR983F, a human myeloma
  • a further object of the invention is a DNA fragment of the sequence SEQ ID NO: 16 coding the variable region of the light chain of an antibody of the invention such as previously described.
  • This DNA fragment can be inserted into a vector enabling the expression of a polypeptide, preferably of an antibody, the variable region of the light chain of said antibody is coded by the nucleic acid sequence SED ID NO: 16, the deduced peptide sequence thereof is the sequence SEQ ID NO: 18, in order to be introduced into and maintained in a host cell.
  • This vector enables the expression of this foreign nucleic acid fragment in the host cell because it contains the sequences (promoter, polyadenylation sequence, selection gene) essential for this expression.
  • any mammalian cell can be used as host cell, that is as the cell expressing the polypeptide or the antibody of the invention, for example SP2/0, YB2/0, IR983F, a human myeloma as Namalwa, or any other cell of human origin as PERC6, CHO cell lines, especially CHO-K-1, CHO-Lec10, CHO-Lec1, CHO-Lec13, CHO Pro-5, CHO dhfr-(CHO DX B11, CHO DG44), or other lines selected from Wil-2, Jurkat, Vero, Molt-4, COS-7, 293-HEK, BHK, K6H6, NSO, SP2/0-Ag 14 and P3X63Ag8.653.
  • a further object of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody of the invention and at least an excipient and/or at least one pharmaceutically acceptable carrier.
  • the monoclonal anti-idiotypic antibody contained in the pharmaceutical composition of the invention is the antibody 18B6, a fragment or a region derived from 18B6, or even a chimeric or humanized antibody comprising the variable regions or the CDRs of 18B6, and such as previously described in the present document.
  • the pharmaceutical composition of the invention can be formulated into any excipient which can be tolerated by a patient to be treated. Examples of such excipients include water, saline solutions, Ringer's solution, dextrose solutions, and any other suitable aqueous physiological solution.
  • the excipient can also contain low amounts of additives, such as substances increasing the isotonicity and the stability of the composition.
  • excipients include phosphate buffer, bicarbonate buffer, and Tris buffer.
  • Standard formulations can be in the form of liquids for injection or solid formulations which can be resuspended in a suitable liquid prior to administration.
  • the useful carriers for preparing the pharmaceutical composition of the invention advantageously have the function of increasing the half-life of the therapeutic composition in the animal or patient, or enabling the controlled release of the active ingredient.
  • Such carriers can be organic and synthetic polymers and further chemical compounds capable of disseminating the medicaments at a normal rate or disseminating them only in certain environments, and can also be liposomes, this list being not limitative.
  • the pharmaceutical composition of the invention moreover, comprises at least an anti-idiotypic antibody directed against the inhibitory antibody binding to a domain different from the C1 domain of Factor VIII.
  • This other antibody can be an anti-idiotypic antibody directed against an inhibitor antibody binding to the A1, or A3, or B, A2 or C2 domains of Factor VIII.
  • a patient suffering from haemophilia A having developed inhibitory antibodies, exhibits most frequently several types of inhibitory antibodies.
  • the amounts and the nature of the different types of inhibitory antibodies are not fixed but may change during the patient's life.
  • the different inhibitory antibodies of a same patient are thus directed against the different domains of Factor VIII, and it is particularly advantageous to treat the patient not with one but with several types of anti-idiotypic antibody, directed against the different inhibitory antibodies.
  • the pharmaceutical composition comprises a monoclonal anti-idiotypic antibody directed against an inhibitory antibody binding to the C2 domain of Factor VIII and/or an inhibitory antibody binding to the A2 domain of Factor VIII, and the monoclonal antibody of the invention.
  • the A2 and C2 domains are the main targets of the anti-Factor VIII immune reaction.
  • a pharmaceutical composition comprising a mixture of anti-idiotypic antibodies directed against inhibitory antibodies binding to the C1 domain of Factor VIII and of anti-idiotypic antibodies directed against inhibitory antibodies binding to the C2 domain, enables neutralization of at least 70%, and advantageously at least 80% or 90% of all inhibitory antibodies present in a patient.
  • the pharmaceutical composition of the invention comprises the antibody 14C12 (deposited under the number LMBP 5878CB at the Belgian Coordinated Collections of Microorganisms) and/or the antibody 30D1 (deposited at CNCM under the number I-3450).
  • the pharmaceutical composition comprises the chimeric antibody 14C12 deposited at the CNCM under the number I-3510 and/or a chimeric or humanized antibody derived from the antibody 30D1, that is an antibody comprising the variable regions of the antibody 30D1.
  • a further object of the invention is the use of the antibody of the invention as a medicament.
  • a further object of the invention is the use of the antibody of the invention for manufacturing a medicament.
  • a medicament is used for reducing and/or preventing and/or treating bleeding in a patient suffering from haemophilia comprising inhibitory antibodies directed against the C1 domain of Factor VIII.
  • a further object of the invention is the use of the antibody of the invention for manufacturing a medicament intended for the treatment of type A haemophilia.
  • the thus treated type A haemophilia is a haemophilia with inhibitors.
  • This type of haemophilia treated with the antibody of the invention can be inborn or acquired.
  • the antibody of the invention makes treatment by injection of Factor VIII to a patient effective, since the activity of Factor VIII is no longer inhibited by inhibitory antibodies.
  • a further object of the invention is the use of the antibody of the invention for neutralisation of the in vitro or in vivo inhibitory activity of an inhibitory antibody directed against the C1 domain of Factor VIII. This process can be carried out in order to deplete the inhibitory antibodies directed against the C1 domain of Factor VIII from the blood of a patient, and afterwards to re-inject the treated blood to said patient.
  • a further object of the invention is related to a medicament comprising an antibody of the invention, preferentially the antibody 18B6.
  • a further object of the invention is the use of the antibody for adsorption of the inhibitory antibodies, by way of example in order to purify Factor VIII inhibitory antibodies.
  • a further object of the invention is the use of the antibody of the invention for detection and/or purification of Factor VIII inhibitory antibodies.
  • the general processes carrying out such methods of detection and purification are well known to those skilled in the art.
  • the use of an immuno-purification column containing beads with the antibody of the invention grafted on their surface can be mentioned. Only the molecules recognized by the antibody will affix themselves to the beads. The others will pass through the column. In order to recover the molecule, an increase of the ionic strength of the solvent is sufficient.
  • FIG. 1 increase (mean value) for the 4 mice in the binding of anti-idiotypic antibodies to RHD5.
  • FIG. 2 direct binding of the anti-idiotypic antibody 18B6 to the insolubilized antibody RHD5.
  • FIG. 3 inhibition of the binding of the antibody RHD5 to insolubilized recombinant FVIII (recFVIII).
  • FIG. 4 neutralisation of RHD5 by 18B6.
  • the human lymphoblastoid cell line RHD5 described here below was obtained by immortalization of B lymphocytes of a patient suffering from acquired haemophilia A having developed an immune response to Factor VIII, according to the procedure described in the document Jacquemin et al. (1998), Blood 92, 496-506 and in the patent application WO 2005/016455.
  • the cell line producing the monoclonal anti-C1 RHD5 antibody was deposited at the Belgian Co-ordinated Collections of Microorganisms/Plasmid Collection (BCCM/LMBP), Laboratorium voor Mole Les Biologie, University of Ghent, Technologiepark 297, B-9052 Zwijnaarede, Belgium, in August 2004, by the Collen Research Foundation, under the accession number LMBP 6165CB.
  • the nucleotide sequence of the variable region of the heavy chain of the RHD5 antibody is sequence SEQ ID NO: 5
  • the nucleotide sequence of the variable region of the light chain of the RHD5 antibody is sequence SEQ ID NO: 6.
  • the peptide sequence corresponding to the sequence SEQ ID NO: 5 is sequence SEQ ID NO: 7
  • the peptide sequence corresponding to the sequence SEQ ID NO: 6 is sequence SEQ ID NO: 8.
  • antibodies exhibiting the required properties can be produced by immunization of animals.
  • human Factor VIII is injected into mice with an adjuvant.
  • Monoclonal anti-human antibodies are obtained by fusion of spleen lymphocytes with a mouse myeloma cell line.
  • Cell supernatants producing the anti-Factor VIII antibodies are identified and cloned by limiting dilution.
  • a general description of such methods can be found in ⁇ Current Protocols in Immunology, Chapter 2, John Wiley & Sons, Inc, 1994>>. Further selections of inhibitors exhibiting the desired properties are described hereafter.
  • mice Four 6 week old Balb/c female mice were sub-cutaneously injected (SC) thrice in the footpad, with 10 ⁇ g of the human anti-C1 domain of FVIII RHD5 antibody suspended in a complete Freund's adjuvant (ACF) (1st immunization) then in an incomplete Freund's adjuvant (AIF).
  • ACF complete Freund's adjuvant
  • AIF incomplete Freund's adjuvant
  • the first bloodletting (bloodletting 0) was performed prior to immunization (bleeding Day 0 (D0)), then the injections and bloodletting proceeded as follows:
  • an ELISA assay with direct binding is performed.
  • either the RHD5 antibody, or a control IgG1 at 3 ⁇ g/ml were insolubilized, 50 ⁇ l/well, un Glycine buffer, over night at 4° C.
  • Glycine buffer 0.1M Glycine, 0.17M NaCl, pH 9.2
  • Magic Buffer 50 mM Tris, 0.17M NaCl, 1% BSA, pH 7.2
  • the system is incubated with a 1 ⁇ g/ml solution of goat polyclonal mouse anti-IgG antibodies labelled with HRP (horseradish peroxidase) (Bio-Rad) for 2 hours at room temperature (50 ⁇ l/well) (dilution in Magic Buffer). Then, the system is washed 3 times with PBS/Tween, and revelation is carried out with a chromogen (Ortho-phenyl diamine) and the intensity of the obtained coloration is read using a reader with filters corresponding to wavelengths 490/650 nm (reader Emax Molecular Devithese, Sunnyvale, Calif.).
  • the cells were successively expanded in a DMEM medium (Dulbecco's Modified Eagle Medium) containing hypoxanthine and thymidine according to the principle of limit dilutions and the clones tested positive detected in direct binding ELISA assay, such as previously described in point II.
  • DMEM medium Dulbecco's Modified Eagle Medium
  • the specificity of the binding was confirmed by insolubilizing a human IgG1 antibody having an irrelevant specificity, and produced in the Laboratory.
  • tests 1 to 3 were repeated during clones expansion, in different volumes of medium from 200 ⁇ l to 5 ml.
  • Test 1 measurement in well of 200 ⁇ l
  • Test 2 measurement in well of 1 ml
  • Test 3 measurement in bottle of 5 ml Results obtained during different epitope screenings are resumed in the following Table
  • Recombinant Factor VIII (recFVIII) (Baxter) at 2 ⁇ g/ml in a glycine buffer, 50 ⁇ l/well, was insolubilized, then left for 2 hours at room temperature.
  • the RHD5 antibody (or an irrelevant IgG1) at 0.6 ⁇ g/ml final concentration was pre-incubated over 2 hours with the culture supernatants in a dilution 1/1, 1 ⁇ 2 and 1 ⁇ 4 in Magic Buffer.
  • the wells were washed 3 times with a PBS/Tween buffer, then saturated with 100 ⁇ l/well of Magic Buffer (30 min at room temperature).
  • the culture supernatant was incubated with 50 ⁇ l of RHD5 (or irrelevant IgG1) (2 hours at room temperature, Magic Buffer), then, 3 washings were performed.
  • RHD5 or irrelevant IgG1
  • the RHD5 antibodies bound to insolubilized recFVIII were detected by addition of 50 ⁇ l/well of a mouse polyclonal human anti-IgG HRP-labelled (Southern Biotechnology) antibodies solution of 1 ⁇ g/ml in Magic Buffer.
  • the RHD5 antibody is incubated at a concentration of 1 ⁇ g/ml with supernatants of different clones selected during the test of inhibition (diluted 3 times, 6 times, 12 times and 24 times) in Magic Buffer at 37° C. After 30 min, the FVIII Kogenate (Bayer) at 0.5 U/ml final was added, then a complementary incubation of 30 min at 37° C. was carried out. The samples were diluted 30 ⁇ in Magic Buffer, then the reagents of the chromogenic DADE test (Factor VIII chromogenic, Dade Behring Gmbh, Marburg, Germany) were added following the manufacturer's instructions.
  • Antibody 18B6 was selected to be used in the following experiences, as a function of the results and neutralisation curves.
  • the anti-idiotypic antibody 18B6 was produced in a DMEM culture medium. This production was followed by purification on a Protein G affinity column (which enables purification, then concentration of the antibodies, and thus to ascertain further the obtained anti-idiotypic antibody specificity). Purification: 18B6: production of 8 ml at 8.48 mg/ml
  • ELISA assay direct binding of the anti-idiotypic antibody 18B6 to insolubilized antibody RHD5
  • the direct binding of the anti-idiotypic antibody 18B6 to the insolubilized antibody RHD5 is illustrated in FIG. 2 .
  • the curve shows that the binding of antibody 18B6 to RHD5 is dose-dependent.
  • ELISA assay inhibition of antibody RHD5 binding to insolubilized recombinant FVIII.
  • the inhibition of RHD5 antibody binding to insolubilized recombinant FVIII was measured according to the protocol described in point IV.
  • the concentration of used RHD5 is equal to 2 ⁇ g/ml.
  • the protocol is the same as described in point V with a final RHD5 concentration of 0.4 ⁇ g/ml and a curve of purified anti-idiotypic antibody from 4 to 0.002 ⁇ g/ml final concentration.
  • the binding kinetics of the anti-idiotypic antibody 18B6 to inhibitor RHD5 antibody was evaluated by use of the ⁇ Surface plasmon resonance Biacore >> method using the Pharmacia Biosensor BIAcore (Pharmacia Biosensor AB, Uppsala, Sweden).
  • the RHD5 antibodies were immobilized on the activated surface of a CM5 probe.
  • the anti-idiotypic antibodies 18B6 were infused in different RHD5 concentrations immobilized on the surface of the probe.
  • the association and dissociation constants were determined:
  • the IsoStrip system by Roche was used (colorimetric strip).
  • the antibody 18B6 was identified as a IgG1 Kappa.
  • mRNA of hybridoma producing the anti-idiotypic antibody 18B6 was isolated, using a Quick Prep Micro mRNA Purification Kit (Amersham Pharmacia Biotech, Uppsala, Sweden).
  • the cDNA was synthesized by use of First-strand cDNA Synthesis Kit (Amersham Pharmacia Biotech).
  • the cDNA encoding the heavy chain (VH) and the light chain (VL) was amplified by PCR (Polymerase Chain Reaction) using specific primers corresponding to different families of genes potentially found in the mouse.
  • PCR products were isolated from an agarose gel 1.5% by means of QIA quick Gel Extraction Kit (Qiagen, Hilden, Germany) and cloned with pGEM-T Easy Vector system (Promega, Madison, Wis.). Plasmidic DNA of positive colonies was isolated by means of High Pure Plasmid Isolation Kit (Roche Diagnostics, Mannheim, Germany) and sequenced in both directions with Seqenase (US Biochemical, Cleveland, Ohio).

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US20070065425A1 (en) * 2005-08-04 2007-03-22 Laboratoire Francais Du Fractionnement Et Des Biotechnologies Anti-idiotypic antibody neutralizing the inhibitor activity of a factor viii inhibitor antibody
US20110097754A1 (en) * 2008-07-02 2011-04-28 Lfb Biotechnologies Method for measuring activated factor vii level in a sample
WO2018071676A1 (en) * 2016-10-12 2018-04-19 Bioverativ Usa Inc. Anti-c1s antibodies and methods of use thereof
US10450382B2 (en) 2012-11-02 2019-10-22 Bioverativ Usa Inc. Anti-complement C1s antibodies
US10457745B2 (en) 2012-10-25 2019-10-29 Bioverativ Usa Inc. Anti-complement C1s antibodies
US10729767B2 (en) 2015-04-06 2020-08-04 Bioverativ Usa Inc. Humanized anti-C1s antibodies and methods of inhibiting C1s cleavage

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WO2009024653A1 (fr) * 2007-08-23 2009-02-26 Lfb Biotechnologies Anticorps anti-idiotypigues neutralisant l'activite inhibitrice d'un anticorps inhibiteur dirige contre le domaine c1 du facteur viii
AU2010290131C1 (en) 2009-08-24 2015-12-03 Amunix Operating Inc. Coagulation factor VII compositions and methods of making and using same
FR2969761A1 (fr) * 2010-12-22 2012-06-29 Lfb Biotechnologies Procede de dosage d'anticorps diriges contre le facteur viii
CA2864126A1 (en) 2012-02-15 2013-08-22 Biogen Idec Ma Inc. Recombinant factor viii proteins
SI3564260T1 (sl) 2012-02-15 2023-02-28 Bioverativ Therapeutics Inc. Sestavki faktorja VIII in postopki njegove izdelave in uporabe
EP3033097B1 (de) 2013-08-14 2021-03-10 Bioverativ Therapeutics Inc. Faktor viii-xten fusionen sowie ihre verwendungen.
CN108472337B (zh) 2015-08-03 2022-11-25 比奥贝拉蒂治疗公司 因子ix融合蛋白以及其制备和使用方法

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FR508320A (fr) 1920-01-12 1920-10-07 Deynoux Freres Fermoir pour sacs et objets analogues
EP1194528B1 (de) * 1999-07-14 2007-03-07 D. Collen Research Foundation vzw Monoklonaler Antikörper der Faktor VIII selbst in molarem Überschuß nur teilweise inaktiviert und eine Methode zur Herstellung solch eines Antikörpers
EP1388544A1 (de) 2002-07-31 2004-02-11 D. Collen Research Foundation vzw Anti-Idiotypische Antikörper gegen Factor VIII-Inhibitor und Verwendungen davon
EP1706079B1 (de) * 2003-08-14 2013-02-20 ThromboGenics N.V. Antikörper gegen faktor viii mit modifizierter glykosilierung in der variablen region
FR2889534B1 (fr) 2005-08-04 2012-11-02 Lab Francais Du Fractionnement Anticorps anti-idiotypique neutralisant l'activite inhibitrice d'un anticorps inhibiteur du facteur viii

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US20070065425A1 (en) * 2005-08-04 2007-03-22 Laboratoire Francais Du Fractionnement Et Des Biotechnologies Anti-idiotypic antibody neutralizing the inhibitor activity of a factor viii inhibitor antibody
US8071094B2 (en) * 2005-08-04 2011-12-06 Laboratoire Francais du Fractionement Et des Biotechnologies Anti-idiotypic antibody neutralizing the inhibitor activity of a factor VIII inhibitor antibody
US20110097754A1 (en) * 2008-07-02 2011-04-28 Lfb Biotechnologies Method for measuring activated factor vii level in a sample
US10457745B2 (en) 2012-10-25 2019-10-29 Bioverativ Usa Inc. Anti-complement C1s antibodies
US10450382B2 (en) 2012-11-02 2019-10-22 Bioverativ Usa Inc. Anti-complement C1s antibodies
US10729767B2 (en) 2015-04-06 2020-08-04 Bioverativ Usa Inc. Humanized anti-C1s antibodies and methods of inhibiting C1s cleavage
US11246926B2 (en) 2015-04-06 2022-02-15 Bioverativ Usa Inc. Polynucleotides encoding anti-C1s antibodies
WO2018071676A1 (en) * 2016-10-12 2018-04-19 Bioverativ Usa Inc. Anti-c1s antibodies and methods of use thereof

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