Nothing Special   »   [go: up one dir, main page]

WO2020115283A1 - Anticorps bispécifiques fixant le facteur ixa et le facteur x - Google Patents

Anticorps bispécifiques fixant le facteur ixa et le facteur x Download PDF

Info

Publication number
WO2020115283A1
WO2020115283A1 PCT/EP2019/084000 EP2019084000W WO2020115283A1 WO 2020115283 A1 WO2020115283 A1 WO 2020115283A1 EP 2019084000 W EP2019084000 W EP 2019084000W WO 2020115283 A1 WO2020115283 A1 WO 2020115283A1
Authority
WO
WIPO (PCT)
Prior art keywords
light chain
variable domain
bispecific antibody
heavy chain
factor
Prior art date
Application number
PCT/EP2019/084000
Other languages
English (en)
Inventor
Fritz Scheiflinger
Michael Dockal
Patrice DOUILLARD
Randolf Kerschbaumer
Sabine KNAPPE
Rudolf Hartmann
Nicolas Fischer
Franck Gueneau
Giovanni Magistrelli
Ulla Ravn
Original Assignee
Baxalta GmbH
Baxalta Incorporated
Novimmune Sa
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/EP2018/084038 external-priority patent/WO2020114615A1/fr
Application filed by Baxalta GmbH, Baxalta Incorporated, Novimmune Sa filed Critical Baxalta GmbH
Publication of WO2020115283A1 publication Critical patent/WO2020115283A1/fr

Links

Classifications

    • 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
    • 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
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention provides therapeutics for the treatment and/or prevention of bleeding disorders such as hemophilia A.
  • the present invention provides bispecific antibodies that bind to Factor IXa and Factor X.
  • Hemophilia A is a severe X-chromosome-linked recessive disorder caused by mutations in the factor VIII (FVIII) gene.
  • FVIII is involved in the intrinsic pathway of blood coagulation, and FVIII deficiency leads to blood either coagulating poorly, or barely at all.
  • FVIII deficiency alternatively known as hemophilia A, is one of the most common hemorrhagic disorders, and affects one in about 10,000 males (Stonebraker et al. (2012) Haemophilia 18(3):e91-4).
  • Hemophilia A has three grades of severity defined by factor FVIII plasma levels of 1% or less ("severe"), 2 to 5% ("moderate"), and 6 to 30% (“mild”) (White et al. (2001) Thromb. Haemost. 85:560); alternatively, a range of 5- ⁇ 40% is referred to in WFH“Guidelines for the Management of Hemohpilia” 2nd edition Haemophilia; Epub 6 JUL 2012. DOI: 10.1111/j.1365-2516.2012.02909.x. In severe forms of the disorder, the first bleeds typically appear at 5 to 6 months of age, whereas the first bleeds are delayed until about 1 to 2 years of age in the moderate form.
  • a bleed can appear spontaneously, or following minimum trauma. Approximately half of all patients with hemophilia A are classified as having the severe form of the disease. These patients experience severe bleeding starting in early childhood, and frequent episodes of spontaneous or excessive bleeding later in life. Bleeding commonly occurs into joints and muscles, and without appropriate treatment, recurrent bleeding can lead to irreversible hemoarthropathy (Manco-Johnson et al. (2007) N. Engl. J. Med. 357(6):535-44).
  • hemophilia A treatment An important goal of hemophilia A treatment is maintenance of FVIII plasma levels >1% (even more preferably at least 3% or even at least 5%), which reduces bleeding risk. To achieve this, intravenous recombinant or plasma-derived FVIII is administered frequently as prophylactic therapy.
  • this current standard of treatment of hemophilia A is difficult, has several drawbacks, and incurs a considerable physical and mental burden on patients and their families.
  • Figure 1 Octet analysis of binding of bispecific antibody W198V282 H-CK2 to either or both of Factor IXa and Factor X.
  • Figure 2 Thrombogram of bispecific antibody W198V282 H-CK2 at a concentration of 100 nM.
  • the invention provides bispecific antibodies that bind to Factor IXa and Factor X. These bispecific antibodies according to the invention have a procoagulant activity. These antibodies may also bind to Factor IX and/or Factor Xa. Bispecific antibodies according to the invention are defined in the claims and in the description of the invention below.
  • the bispecific antibodies of the invention are generated using the following steps:
  • Two antibodies having different specificities one antibody binding to Factor IXa and the other to Factor X), and sharing the same heavy chain variable domain but having different light chain variable domains, are isolated. This step is facilitated by the use of antibody libraries having a fixed heavy chain or transgenic animals containing a single VH gene.
  • the heavy chain variable domain is fused to the constant domain of a heavy chain, one light chain variable domain is fused to a Kappa constant domain and the other light chain variable domain is fused to a Lambda constant domain.
  • the light chain variable domain fused to the Kappa constant domain may be of the Kappa type and the light chain variable domain fused to the Lambda constant domain may be of the Lambda type.
  • the generation of hybrid light chains is possible, so that two light chain variable domains of the same type can be used to generate bispecific antibodies of the invention.
  • the three chains i.e.
  • one heavy chain and two different light chains are co-expressed in mammalian cells leading to the assembly and secretion in the supernatant of a mixture of three antibodies: two monospecific antibodies and one bispecific antibody carrying two different light chains.
  • the ratio of the different antibodies depends on the relative expression of the chains and their assembly into an IgG. Methods to tune these ratios and maximize the production of the bispecific antibodies of the present invention are also provided.
  • the antibody mixture is purified using standard chromatography techniques used for antibody purification.
  • the antibody mixture can be characterized and used as a multi targeting agent.
  • the bispecific antibody is purified using in a consecutive manner affinity chromatography media that bind specifically to human Kappa and human Lambda light chain constant domains. This purification process is independent of the sequence of the light chain variable domains and is thus generic for all bispecific antibodies of the invention generated by the steps described here.
  • the isolated bispecific antibody bearing a light chain containing a Kappa constant domain and a light chain containing a Lambda constant domain is characterized using different biochemical and immunological methods.
  • the bispecific antibody of the invention can be used for therapeutic intervention or as a research or diagnostic reagent.
  • the bispecific antibodies according to the invention may also be generated by any method for generating bispecific antibodies known in the art. Several such methods will be discussed further in the detailed description of the invention below.
  • the invention provides monoclonal antibodies carrying a different specificity in each antigen-binding site and including two copies of a single heavy chain polypeptide and a first light chain and a second light chain, wherein the first and second light chains are different.
  • the first light chain includes at least a Kappa constant domain. In some antibodies, the first light chain further includes a Kappa variable domain. In some antibodies, the first light chain further includes a Lambda variable domain. In some antibodies, the second light chain includes at least a Lambda constant domain. In some antibodies, the second light chain further includes a Lambda variable domain. In some antibodies, the second light chain further includes a Kappa variable domain. In some antibodies, the first light chain includes a Kappa constant domain and a Kappa variable domain, and the second light chain includes a Lambda constant domain and a Lambda variable domain.
  • the constant and variable framework region sequences are human.
  • Some methods also include the additional step of d) isolating the bispecific antibodies produced from the monospecific antibodies produced.
  • the isolation is accomplished by using one or more affinity chromatography purification steps.
  • the purification step or the purification steps is or are performed using Kappa constant domain specific, Lambda constant domain specific or both Kappa constant domain specific and Lambda constant domain specific affinity chromatography media.
  • a Kappa light chain variable domain is fused to a constant domain of the Kappa type. In some methods, a Kappa light chain variable domain is fused to a constant domain of the Lambda type. In some methods, a Lambda light chain variable domain is fused to a constant domain of the Kappa type. In some methods, a Lambda light chain variable domain is fused to a constant domain of the Lambda type.
  • step a) and b) are facilitated by the use of antibody libraries having a common heavy chain and diversity confined to the light chain variable domain.
  • the variable heavy chain domain that is fixed in one of such libraries can be based on different variable germline genes and have different sequences both in the CDR and framework regions.
  • such libraries are designed using different types of variable heavy chain domains and can be used to generate antibodies of the invention.
  • the antibody library is displayed on filamentous bacteriophage, at the surface of yeast, bacteria or mammalian cells or used for ribosome or other type of in vitro display.
  • Some methods also include the further step of e) recovering the bispecific antibody.
  • the bispecific antibody is recovered in step e) using an affinity chromatography purification step.
  • the purification step is performed using Kappa constant domain specific, Lambda constant domain or both Kappa constant domain specific and Lambda constant domain specific affinity chromatography media.
  • the second nucleic acid sequence encodes a Kappa-type light chain variable domain. In some methods, the second nucleic acid sequence encodes a Kappa-type constant domain. In some methods, the second nucleic acid sequence encodes a Lambda- type constant domain. In some methods, the second nucleic acid sequence encodes a Lambda-type light chain variable domain. In some methods, the second nucleic acid sequence encodes a Kappa-type constant domain. In some methods, the second nucleic acid sequence encodes a Lambda-type constant domain. In some methods, the third nucleic acid sequence encodes a Kappa-type light chain variable domain. In some methods, the third nucleic acid sequence encodes a Kappa-type constant domain.
  • the third nucleic acid sequence encodes a Lambda-type constant domain. In some methods, the third nucleic acid sequence encodes a Lambda-type light chain variable domain. In some methods, the third nucleic acid sequence encodes a Kappa-type constant domain. In some methods, the third nucleic acid sequence encodes a Lambda-type constant domain.
  • the invention also provides an antibody mixture that includes two monospecific antibodies and one bispecific antibody, all having a common heavy chain.
  • the bispecific antibody is any of the bispecific antibodies described herein or made using methods described herein.
  • the invention also provides methods of generating such an antibody mixture by a) isolating an antibody having a specificity determined by a heavy chain variable domain combined with a first light chain variable domain; b) isolating an antibody having a different specificity determined by the same heavy chain variable domain as the antibody of step a) combined with a second light chain variable domain; c) co-expressing in a cell: (i) the common heavy chain variable domain fused to an antibody heavy chain constant domain; (ii) the first light chain variable domain fused either to a light chain constant domain of the Kappa type or fused to a light chain constant domain of the Lambda type; and (iii) the second light chain variable domain fused to either to a light chain constant domain of the Kappa type or fused to a light chain constant domain of the Lambda type. Some methods also include the additional step of d) isolating the antibody mixture produced in step c) from cell culture supernatant.
  • Some methods also include the step of harvesting or otherwise purifying the two or more, for example, three or more, non-identical antibodies from the recombinant host cell or from a culture of the host cell.
  • the host cell is, for example, a mammalian cell.
  • non-identical antibodies include monospecific and bispecific antibodies.
  • the non-identical antibodies bind to different antigens, i.e. Factor IXa and X.
  • the two or more, for example, three or more, non-identical antibodies are independently selected from the group consisting of: IgGI, lgG2, lgG3, lgG4, IgAI, lgA2, IgD, IgE and IgM.
  • the two or more, for example, three or more, non-identical antibodies contain a modified Fc region that modifies the effector functions of the antibodies such as Antigen Dependent Cell mediated Cytotoxicity (ADCC), Complement Dependent Cytotoxicity (CDC), Antigen Dependent Cellular Phagocytosis (ADCP) or their pharmacokinetic properties by altering its binding the neonatal Fc Receptors (J Drug Target. 2014 May;22(4):269-78. doi: 10.3109/1061186X.2013.875030. Epub 2014 Jan 9.
  • Neonatal Fc receptor (FcRn) a novel target for therapeutic antibodies and antibody engineering. Wang Y1 , Tian Z, Thirumalai D, Zhang X).
  • the two or more, for example, three or more, non-identical antibodies contain a modification of the Fc region that extends the serum half-life of the antibodies.
  • the one or more nucleic acid sequences are stably expressed in the host cell.
  • the two or more, for example three or more, non-identical antibodies are produced by the host cell in vitro.
  • Some methods also include the additional steps of selecting at least one host cell by assaying the two or more, for example, three or more, non-identical antibodies produced by the recombinant host cell for their ability to bind a target antigen (i.e. Factor IXa or Factor X) and/or for a procoagulant activity; culturing the recombinant host cell; and isolating the three or more non-identical antibodies.
  • the antibodies can be isolated using any of the techniques described herein or any other suitable art-recognized method.
  • the different antibody light chains have identical constant domains. In some methods, the different antibody light chains have different constant domains.
  • animal refers to any multicellular eukaryotic heterotroph which is not a human.
  • the animal is selected from a group consisting of cats, dogs, pigs, ferrets, rabbits, gerbils, hamsters, guinea pigs, horses, rats, mice, cows, sheep, goats, alpacas, camels, donkeys, llamas, yaks, giraffes, elephants, meerkats, lemurs, lions, tigers, kangaroos, koalas, bats, monkeys, chimpanzees, gorillas, bears, dugongs, manatees, seals and rhinoceroses.
  • antibody refers to a molecule comprising at least one immunoglobulin domain that binds to, or is immunologically reactive with, a particular antigen.
  • the term includes whole antibodies and any antigen binding portion or single chains thereof and combinations thereof.
  • the term“antibody” in particular includes bispecific antibodies.
  • a typical type of antibody comprises at least two heavy chains (“HC") and two light chains (“LC”) interconnected by disulfide bonds.
  • Each “heavy chain” comprises a “heavy chain variable domain” (abbreviated herein as “VH”) and a “heavy chain constant domain” (abbreviated herein as “CH”).
  • the heavy chain constant domain typically comprises three constants domains, CH1 , CH2, and CH3.
  • Each “light chain” comprises a “light chain variable domain” (abbreviated herein as “VL”) and a “light chain constant domain” (“CL”).
  • the light chain constant domain (CL) can be of the kappa type or of the lambda type.
  • the VH and VL domains can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions ("CDR"), interspersed with regions that are more conserved, termed “framework regions” (“FW").
  • CDR Complementarity Determining Regions
  • Each VH and VL is composed of three CDRs and four FWs, arranged from amino-terminus to carboxy-terminus in the following order: FW1 , CDR1 , FW2, CDR2, FW3, CDR3, FW4.
  • the present disclosure inter alia presents VH and VL sequences as well as the subsequences corresponding to CDR1 , CDR2, and CDR3.
  • FW1 is the subsequence between the N-terminus of the VH and the N-terminus of H-CDR1
  • FW2 is the subsequence between the C-terminus of H-CDR1 and the N-terminus of H-CDR2
  • FW3 is the subsequence between the C-terminus of H-CDR2 and the N-terminus of H-CDR3
  • FW4 is the subsequence between the C-terminus of H-CDR3 and the C-terminus of the VH.
  • FW1 is the subsequence between the N-terminus of the VL and the N- terminus of L-CDR1
  • FW2 is the subsequence between the C-terminus of L-CDR1 and the N-terminus of L-CDR2.
  • FW3 is the subsequence between the C-terminus of L-CDR2 and the N-terminus of L-CDR3
  • FW4 is the subsequence between the C-terminus of L-CDR3 and the C-terminus of the VL.
  • variable domains of the heavy and light chains contain a region that interacts with an antigen, and this region interacting with an antigen is also referred to as an“antigen-binding site” or“antigen binding site” herein.
  • the constant domains of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • Exemplary antibodies of the present disclosure include typical antibodies, but also fragments and variations thereof such as scFvs, and combinations thereof where, for example, an scFv is covalently linked (for example, via peptidic bonds or via a chemical linker) to the N-terminus of either the heavy chain and/or the light chain of a typical antibody, or intercalated in the heavy chain and/or the light chain of a typical antibody.
  • exemplary antibodies of the present disclosure include bispecific antibodies.
  • antibody encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain variable fragment (scFv), disulfide stabilized scFvs, multispecific antibodies such as bispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen binding site.
  • antibody fragments such as Fab, Fab', F(ab')2, and Fv fragments
  • scFv single chain variable fragment
  • disulfide stabilized scFvs multispecific antibodies such as bispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen binding site.
  • An antibody can be of any the five major classes (isotypes) of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses thereof (e.g. lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively.
  • the different classes of immunoglobulins have different and well known subunit structures and three-dimensional configurations.
  • Antibodies can be naked or conjugated to other molecules such as therapeutic agents or diagnostic agents to form immunoconjugates.
  • bispecific antibody refers to an antibody, as defined above, that is able to bind to at least two different antigens through two different antigen binding sites.
  • the bispecific antibodies of the present invention are able to bind to Factor IXa and Factor X.
  • VH and VL domains i.e. the variable domains of the heavy chain and the light chain, respectively
  • CDR complementarity determining regions
  • FR framework regions
  • the extent of the framework region and CDRs has been precisely defined by a number of methods (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.91-3242; Chothia, C. et al. (1987) J. Mol.
  • an effective amount of an agent e.g., a therapeutic agent such as an antibody
  • an effective amount of an agent is, for example, an amount sufficient to reduce or decrease bleeding occurrences, as compared to the response obtained without administration of the agent.
  • effective amount can be used interchangeably with “effective dose,” “therapeutically effective amount,” or “therapeutically effective dose.”
  • втори ⁇ е IX refers to a protein that is synthesized by liver hepatocytes as a pre-prozymogen that requires extensive posttranslational modification.
  • the pre-prozymogen contains a pre-peptide (hydrophobic signal peptide) at its amino terminal that transports the growing polypeptide into the lumen of the Endoplasmic Reticulum (ER). Once inside the ER, this signal peptide is cleaved by a signal peptidase.
  • a pro-peptide functions as a recognition element for a vitamin K-dependent carboxylase (y-glutamyl carboxylase) which modifies 12 glutamic acid residues to gammacarboxyglutamyl (Gla) residues.
  • FIX is in a zymogen form.
  • FIX zymogen thus circulates as a 415 amino acid, single chain polypeptide.
  • the zymogen of FIX is activated by FXIa or by the tissue factor/FVIIa complex.
  • the first cleavage is at Arg 191 (Arg 145 in the mature FIX sequence), generating an inactive FIX- alpha.
  • the second cleavage at Arg226 removes 35 amino acids of the FIX activation peptide and results in a catalytically active molecule FlXa- beta.
  • This catalytically active FIXa not associated with FVIIIa is also referred to herein as free Factor IXa (free FIXa) or, briefly, as“Factor IXa” or“FIXa”.
  • This resulting heterodimer is held by a disulfide bridge at Cys178-Cys335.
  • the serine protease contains a catalytic triad of His267, Asp315, and Ser411.
  • Val227 can form a salt bridge with Asp410, which is a characteristic of active serine proteases.
  • Data concerning a non-limiting example of Factor IX has been deposited in UniProtKB under accession number P00740.
  • the bispecific antibody of the present invention preferentially binds to Factor IXa over the zymogen of Factor IX (i.e. over the protein referred to as“Factor IX” or “FIX” herein).
  • Fractor IXa or“FIXa” refers to the catalytically active Factor IXa molecule resulting from activation of the FIX zymogen by FXIa or by the tissue factor/FVIIa complex.
  • FIXa activity of FIXa is similar to the activity of FIX zymogen, i.e. relatively low.
  • the complex formation with cofactor FVIIIa represents a critical second phase of activation, after which the intrinsic Xase (tenase) complex (including FIXa) reaches an approximately 200,000-fold enhanced activity that is strictly specific toward the physiological substrate FX and restricted to the surface of activated platelets (van Dieijen et al., J Biol Chem. 1981 Apr 10;256(7):3433-42).
  • This macromolecular activation is assisted by low- molecular-weight agonists, including Ca2+ (Mathur et al., Biol. Chem., 272 (1997), pp.
  • procoagulant activity is used herein to define an activity that can promote coagulation, e.g. by promoting any process or reaction that contributes to coagulation (or by inhibiting any process or reaction that reduces coagulation). Accordingly, the term “procoagulant activity” encompasses (but is not limited to) one or more of the activities listed below:
  • a Factor-VI I la-like activity i.e. an activity that corresponds to an activity of activated
  • Factor VIII Enhancing Factor-IXa-mediated Factor X activation, as measured by a amidolytic (chromogenic or fluorogenic) assay based on FIXa-mediated FX activation.
  • the assay measures FXa through cleavage of an FXa specific peptide substrate.
  • the substrate is produced, giving a color that can be measured photometrically by absorbance.
  • clotting assays such as Activated partial thromboplastin time (APTT) measure the activity of the intrinsic and common pathways of coagulation.
  • Plasma is preincubated with an APTT reagent containing a contact activator, e.g. ellagic acid or kaolin, and phospholipid.
  • a contact activator e.g. ellagic acid or kaolin
  • Calcium chloride is added to promote fibrin clot formation.
  • Possible readouts are clotting time or clot wave form.
  • Enhancing thrombin generation as measured in a thrombin generation assay such as Calibrated Automated Thrombography (CAT).
  • CAT Calibrated Automated Thrombography
  • the thrombogram describes the concentration of thrombin in clotting plasma and is therefore a functional test of the hemostatic system.
  • the assay is based on the measurement of fluorescence that is generated by the cleavage of the fluorogenic substrate Z G G R AMC by thrombin over time.
  • thrombus formation under flow as measured by flow chamber systems such as the t-TAS (Total Thrombus-formation Analysis System).
  • This assay is used for the quantitative assessment of the thrombus formation process under variable flow. Blood flows through the analytical path of a microchip, platelets adhere and aggregate on the surface of collagen-coated capillaries, eventually resulting in an increase of flow pressure.
  • WBCT whole blood closure time
  • VWF Von Willebrand Factor
  • the aggregometer works on the basic principle of light transmission. As platelets aggregate, the light transmission of the sample increases. All methods described above can be applied to human blood-related specimen, as well as specimen of different animal species. Therefore, they are analytical tools that can be used to analyze ex vivo the procoagulant activity of samples for e.g. assessing the pharmacodynamic properties of antibodies in animal studies.
  • the present invention provides in-vivo methods and means for identifying a bispecific antibody or variants thereof with favorably characteristics typical of a therapeutic antibody as well as in-vivo methods for selecting the most qualified bispecific antibody and/or the preclinical testing of the therapeutic bispecific antibody.
  • an animal model is a living animal used during the research and development of human drugs, or for the purpose of better understanding the human disease mirrored by the animal model.
  • the animal model chosen will usually recapitulate the human pathophysiology, and the pharmacology, exaggerated pharmacology, safety, and/or toxicity of the administered drug candidate while the efficacy of the treatment is reflected in modifications of a prevalent disease state or when challenging healthy animals with an artificial disease inducing insult (trauma).
  • the drug candidate is administered either prophylactic or acutely through oral or parenteral routes including for example the intravenous and subcutaneous routes.
  • Animal models have been proven valuable and predictive in the selection and development of human treatments for hemophilia including factor replacement therapies or bispecific antibody mimicking factor activity.
  • Animal models refer to any animal classified including but not limited to rodents such as mouse, rat, guinea pig, hamster, rabbit, dog, cat, pig, cow, sheep, goat, horse, non-human primates. These include laboratory, domestic and farm animals, and also veterinary patients. Animal models make use of pharmacologic depletion or inhibition of procoagulant factors (anti-Factor VIII), and naturally occurring or genetically engineered modifications that are characterized by loss or gain of function of particular disease related genes and their encoded proteins. As an example animal models recapitulate the causative human loss of function of components in haemostatic regulation (Factor VIII, Factor IX, VWF, APC).
  • rodents such as mouse, rat, guinea pig, hamster, rabbit, dog, cat, pig, cow, sheep, goat, horse, non-human primates. These include laboratory, domestic and farm animals, and also veterinary patients. Animal models make use of pharmacologic depletion or inhibition of procoagulant factors (anti-Fa
  • the rodent is mouse or rat, guinea pig, or hamster.
  • the non-human model can e.g. be a rabbit, or more weight bearing animals like dog, sheep or a non human primate such as Cynomolgus macaque or Rhesus macaque.
  • the test-article shows pharmacokinetic behavior, specific target engagement and a pharmacologic profile that closely reflects those in human.
  • animal models in this invention can monitor for endpoints related to arterial or venous thrombosis, microvascular thrombosis, thrombolysis.
  • thrombosis/thrombolysis models apply ferric chloride, photochemicals, venous stasis, mechanical trauma, systemic epinephrine-collagen infusion, laser injury, spontaneous lysis of pulmonary embolism or microemboli and pharmacologic arterial thrombolysis.
  • the vascular site of investigation includes but is not limited to carotid or femoral arteries, jugular or femoral veins, mesenteric or cremasteric arterioles, small ear veins and arteries, muscle arterioles, tail veins, blood vessels of the nail.
  • animal models in this invention monitor for endpoints related to hemostasis and pharmacologic changes thereof after spontaneous or induced bleeding episodes.
  • Assessments can include those collected through spontaneous bleeds and when challenged by tail vein transection, tail-tip bleeding, vein puncture bleeding, cremaster injury model, ferric chloride carotid artery occlusion, nail-clipping, cutaneous injury (Surgicut), intramuscular injury, subutaneous exfoliation, or injury models inducing mechanical trauma to the joint or by spontaneous hemarthrosis.
  • Monitoring of disease modification includes but is not limited to endpoints assessing clinical signs, incidence and frequency of limping episodes, bleeding time, blood volume, measurements of bruised areas, joint swelling, blood hemoglobin levels, urinary hemoglobin; ex-vivo analyses of coagulation (ROTEM), APTT, factor-activity and thrombin generation; survival, re-bleeds, as well as pathological findings and changes in joint tissues, and internal bleeds in other tissues.
  • ROTEM ex-vivo analyses of coagulation
  • APTT APTT
  • factor-activity and thrombin generation survival, re-bleeds, as well as pathological findings and changes in joint tissues, and internal bleeds in other tissues.
  • Factor X refers to a vitamin-K dependent glycoprotein with a molecular weight of 58.5 kDa, which is secreted from liver cells into the plasma as a zymogen. Initially Factor X is produced as a prepropeptide with a signal peptide consisting in total of 488 amino acids.
  • the signal peptide is cleaved off by signal peptidase during export into the endoplasmic reticulum.
  • the propeptide sequence is cleaved off after gamma carboxylation took place at the first 11 glutamic acid residues at the N-terminus of the mature N-terminal chain.
  • a further processing step occurs by cleavage between Arg 182 and Ser 183. This processing step also leads concomitantly to the deletion of the tripeptide Arg180-Lys181-Arg182.
  • the resulting secreted factor X zymogen consists of an N-terminal light chain of 139 amino acids (M, 16,200) and a C-terminal heavy chain of 306 amino acids (M, 42,000) which are covalently linked via a disulfide bridge between Cys172 and Cys342.
  • the Factor X zymogen can be cleaved in its heavy chain by Factor IXa and consequently become activated after the release of an activation peptide resulting in a protein referred to as“Factor Xa” or“FXa”.
  • Factor X has been deposited in UniProtKB under accession number P00742.
  • the bispecific antibody of the present invention preferentially binds to the zymogen of Factor X (i.e. to the protein referred to as “Factor X” or“FX” herein) over Factor Xa.
  • the terms “individual”, “patient” or “subject” are used interchangeably in the present application to designate a human being and are not meant to be limiting in any way.
  • the “individual”,“patient” or“subject” can be of any age, sex and physical condition.
  • prevention refers to a set of hygienic, pharmacological, surgical and/or physical means used to prevent the onset and/or development of a disease and/or symptoms.
  • prevention encompasses prophylactic methods, since these are used to maintain the health of an animal or individual.
  • treatment and“therapy” refer to a set of hygienic, pharmacological, surgical and/or physical means used with the intent to cure and/or alleviate a disease and/or symptoms with the goal of remediating the health problem.
  • treatment and“therapy” include preventive and curative methods, since both are directed to the maintenance and/or reestablishment of the health of an individual or animal. Regardless of the origin of the symptoms, disease and disability, the administration of a suitable medicament to alleviate and/or cure a health problem should be interpreted as a form of treatment or therapy within the context of this application.
  • bispecific antibody formats In general, in order to overcome the limitations of monoclonal and monovalent antibody therapeutics that can only target a single antigen or to overcome the limitations of combinations of monovalent antibody therapeutics, intense efforts have aimed at multiple antigen targeting using bispecific antibody formats. Such antibodies carrying more than one specificity are of interest in biotechnology and have great potential as therapeutic agents enabling novel therapeutic approaches (Fischer and Leger, Pathobiology 2007; 74:3-14; Morrison SL Nature Biotechnol 2007; 25:1233-1234). Bispecific antibodies are advantageous as they allow for multiple targeting, they increase therapeutic potential, they address redundancy of biological systems, and they provide novel mechanisms of action. As validated single therapeutic targets become more and more exhausted, combinations allowed by bispecific antibodies provide a new and expansive universe of targets for therapeutic agents and applications.
  • Quadromas Quadromas and triomas can be generated by fusing either two hybridomas or one hybridoma with a B lymphocyte, respectively (Suresh MR et al., Methods Enzymol 1986; 121 : 210-228). In this case the simultaneous expression of two heavy and two light chains leads to the random assembly of 10 antibody combinations and the desired bispecific antibody (bsAb) represent only a small fraction of the secreted antibodies.
  • the bsAb has to be purified using a combination of chromatographic techniques.
  • a major limitation is that quadromas produce bsAb of rodent origin which limit their therapeutic potential due to immunogenicity issues.
  • bispecific antibody formats have been generated by genetic engineering techniques using antibody fragment such as scFv or Fab fragments as building blocks connected via polypeptide linkers. Formats based on linked antibody fragments include tandem scFv (BiTE), diabodies and tandem-diabodies (Kipriyanov SM. Methods Mol Biol 2003; 207:323-333; Korn T et al, Int J Cancer 2002; 100:690-697). These building blocks can further be linked to an antibody Fc region given rise to 'IgGlike' molecules.
  • These formats include diabody-Fc, tandem diabody-Fc, tandem diabody-CH3, (scFv)4-Fc and DVD-lg (Lu D et al, J Immunol Methods 2003; 279: 219-232 ; Lu D et al, J Biol Chem 2005; 280: 19665-19672 ; Lu D et al, J Biol Chem 2004; 279: 2856- 2865; Wu C et al., Nat Biotechnol 2007 25:1290-7).
  • Single domain based antibodies Single domain based antibodies.
  • Camelid, shark and even human V domains represent alternatives to antibodies but they also be used for bsAbs generation. They can be reformatted into a classical IgG in which each arm has the potential to bind two targets either via its VH or VL domain. This single domain-lgG would have biochemical properties similar to an IgG and potentially solve problems encountered with other bsAbs formats in terms of production and heterogeneity.
  • the bispecific antibodies compositions and methods provided herein overcome such development obstacles.
  • the bispecific antibodies provided herein have a common heavy chain, two light chains - one Kappa (K), one Lambda (L) - that each has a different specificity (i.e., two light chains, two specificities).
  • the bispecific antibodies do not contain any linkers or other modifications, including amino acid mutations.
  • the methods provided herein produce molecules having specific binding where diversity is restricted to the VL domain. These methods produce the bispecific antibodies through controlled co-expression of the three chains (one heavy chain, two different light chains), and purification of the bispecific antibody.
  • the bispecific antibodies described herein exhibit similar affinities for a given target as compared to the affinities of monospecific antibodies for that same target.
  • the bispecific antibodies described herein are virtually indistinguishable from standard IgG molecules.
  • the methods provided herein also provide the means of generating simple antibody mixtures of two monospecific antibodies and one bispecific antibody that are useful, for example, for multiple targeting without purification of the bispecific antibody from the mixture.
  • bispecific antibodies according to the invention that bind to Factor IXa and Factor X and are essentially identical in structure to a human antibody are provided.
  • This type of molecule is composed of two copies of the same heavy chain polypeptide, a first light chain variable domain fused to a constant Kappa domain and second light chain variable domain fused to a constant Lambda domain.
  • Each antigen-binding site displays a different antigen specificity to which both the heavy and light chain contribute.
  • the light chain variable domains can be of the Lambda or Kappa family and can be fused to a Lambda and Kappa constant domains, respectively.
  • bispecific antibodies of the invention by fusing a Kappa light chain variable domain to a constant Lambda domain for a first specificity and fusing a Lambda light chain variable domain to a constant Kappa domain for the second specificity (see Figure 3 of WO 2012/023053 A2).
  • the bispecific antibodies described herein, which are produced in accordance with these particular embodiments, are also referred to as IgG Kappa Lambda (“kl” in Greek letters, or“KL” as the abbreviation in English letters) antibodies or "KL bodies,” a fully human bispecific IgG format.
  • This KL body format allows the affinity purification of a bispecific antibody that is virtually indistinguishable from a standard IgG molecule with characteristics that are virtually indistinguishable from a standard monoclonal antibody and, therefore, favorable as compared to previous formats (see also WO 2012/023053 A2).
  • An essential step of the method in these particular embodiments is the identification of two antibody Fv regions (each composed by a variable light chain and variable heavy chain domain) having different antigen specificities that share the same heavy chain variable domain.
  • Two antibody Fv regions each composed by a variable light chain and variable heavy chain domain
  • Numerous methods have been described for the generation of monoclonal antibodies. (See, e.g., Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).
  • Fully human antibodies are antibody molecules in which there are no sequences derived from species other than human. Such antibodies are termed "human antibodies", or “fully human antibodies” herein; such human antibodies or fully human antibodies may contain artificially introduced changes in their amino acid sequence.
  • Human monoclonal antibodies can be prepared by using the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4 : 72); and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al, 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized and may be produced by using human hybridomas (see Cote, et al, 1983.
  • Monoclonal antibodies are generated, e.g., by immunizing an animal with a target antigen or an immunogenic fragment, derivative or variant thereof.
  • the animal is immunized with cells transfected with a vector containing a nucleic acid molecule encoding the target antigen, such that the target antigen is expressed and associated with the surface of the transfected cells.
  • a variety of techniques are well-known in the art for producing xenogenic non-human animals. For example, see U.S. Pat. No. 6,075,181 and No. 6,150,584, which is hereby incorporated by reference in its entirety.
  • the antibodies are obtained by screening a library that contains antibody or antigen binding domain sequences for binding to the target antigen.
  • This library is prepared, e.g., in bacteriophage as protein or peptide fusions to a bacteriophage coat protein that is expressed on the surface of assembled phage particles and the encoding DNA sequences contained within the phage particles (i.e., "phage displayed library").
  • Hybridomas resulting from myeloma/B cell fusions can be screened for reactivity to the target antigen.
  • Monoclonal antibodies are prepared, for example, using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes can be immunized in vitro.
  • the serendipitous identification of different antibodies having the same heavy chain variable domain but directed against different antigens is highly unlikely. Indeed, in most cases the heavy chain contributes largely to the antigen binding surface and is also the most variable in sequence. In particular the CDR3 on the heavy chain is the most diverse CDR in sequence, length and structure. Thus, two antibodies specific for different antigens will almost invariably carry different heavy chain variable domains.
  • the method described herein overcomes this limitation and greatly facilitates the isolation of antibodies having the same heavy chain variable domain by the use of antibody libraries in which the heavy chain variable domain is the same for all the library members and thus the diversity is confined to the light chain variable domain.
  • antibody libraries are described, for example, in application PCT/US2010/035619, filed May 20, 2010 and published on November 25, 2010 as PCT Publication No. WO 2010/135558 and application PCT/US2010/057780, filed November 23, 2010 each of which is hereby incorporated by reference in its entirety.
  • both domains can contribute to antigen binding.
  • antibody libraries containing the same heavy chain variable domain and either a diversity of Lambda variable light chains or Kappa variable light chains can be used in parallel for in vitro selection of antibodies against different antigens.
  • This approach enables the identification of two antibodies having a common heavy chain but one carrying a Lambda light chain variable domain and the other a Kappa light chain variable domain that can be used as building blocks for the generation of a bispecific antibody in the full antibody format of the invention (i.e. the KL body format).
  • the bispecific antibodies of the invention can be of different isotypes and their Fc portion can be modified in order to alter the binding properties to different Fc receptors and in this way modify the effector functions of the antibody as well as its pharmacokinetic properties (e.g.
  • another step optionally performed is the optimization of co expression of the common heavy chain and two different light chains into a single cell to allow for the assembly of a bispecific antibody of the invention. If all the polypeptides get expressed at the same level and get assembled equally well to form an antibody molecule then the ratio of monospecific (same light chains) and bispecific (two different light chains) should be 50%. However, it is likely that different light chains are expressed at different levels and/or do not assemble with the same efficiency. Therefore the methods of the invention also provide means to modulate the relative expression of the different polypeptides to compensate for their intrinsic expression characteristics or different propensities to assemble with the common heavy chain.
  • This modulation can be achieved via promoter strength, the use of internal ribosome entry sites (IRES) featuring different efficiencies or other types of regulatory elements that can act at transcriptional or translational levels as well as acting on mRNA stability.
  • IRES internal ribosome entry sites
  • Different promoters of different strength could include CMV (Immediate-early Cytomegalovirus virus promoter); EFI- la (Human elongation factor la-subunit promoter); Ubc (Human ubiquitin C promoter); SV40 (Simian virus 40 promoter).
  • CMV immediate-early Cytomegalovirus virus promoter
  • EFI- la Human elongation factor la-subunit promoter
  • Ubc Human ubiquitin C promoter
  • SV40 Synimian virus 40 promoter
  • IRES have also been described from mammalian and viral origin. (See e.g., Hellen CU and Sarnow P. Genes Dev 2001 15: 1593-612). These IRES can greatly differ
  • the modulation of the expression can also be achieved by multiple sequential transfections of cells to increase the copy number of individual genes expressing one or the other light chain and thus modify their relative expressions (i.e. altering the ratio of the two different light chains introduced into the cell).
  • the co-expression of the heavy chain and two light chains generates a mixture of three different antibodies into the cell culture supernatant: two monospecific bivalent antibodies and one bispecific bivalent antibody.
  • the latter has to be purified from the mixture to obtain the molecule of interest.
  • the method described herein greatly facilitates this purification procedure by the use of affinity chromatography media that specifically interact with the Kappa or Lambda light chain constant domains such as the CaptureSelect Fab Kappa and CaptureSelect Fab Lambda affinity matrices (BAC BV, Holland).
  • This multi-step affinity chromatography purification approach is efficient and generally applicable to antibodies of the invention (see also Figure 8A of WO 2012/023053 A2).
  • the invention also provides a means of producing simple antibody mixtures of two or more monospecific antibodies and one or more bispecific antibody according to the invention that share the same heavy chain and can be purified using standard chromatography techniques used for monoclonal antibody purification. (See e.g., Lowy, I et al. N Engl J Med 2010; 362: 197-205; Goudsmit, J . et al. J Infect Dis. 2006. 193, 796-801). Such simple mixtures can be used as multi-targeting agents for therapeutic usage.
  • the bispecific antibodies were purified using a three-step affinity chromatography procedure: (1) capture of IgG (mono- and bi-), (2) Kappa select: capture IgG containing a Kappa light chain(s), and (3) Lambda select: capture IgG containing a Lambda light chain.
  • Kappaselect and Lambdaselect are affinity chromatography media developed by BAC, BV and GE Healthcare.
  • the purified bispecific antibodies can be characterized as described in WO 2012/023053 A2.
  • the methods of generating bispecific antibodies use a complete serum-free chemically defined process.
  • These methods incorporate the most widely used mammalian cell line in pharmaceutical industry, the Chinese Hamster Ovary (CHO) cell line, but also other cell lines such as PEAK cells (ATCC CRL-2828).
  • CHO Chinese Hamster Ovary
  • PEAK cells ATCC CRL-2828
  • any mammalian cell line suitable for protein expression may be used, including e.g. also HEK293 cells.
  • the methods described are used to generate both semi stable and stable cell lines. However, also transiently transfected cell lines could be used.
  • the methods can be used to manufacture the bispecific antibodies of the invention at small scale (e.g., in an Erlenmeyer flask) and at mid-scale (e.g., in 25L Wave bag).
  • the methods are also readily adaptable for larger scale production of the bispecific antibodies, as well as antibody mixtures of the invention.
  • bispecific antibodies are produced in mammalian cells.
  • mammalian cells that may be used in accordance with the present invention include BALB/c mouse myeloma line (NSO/1 , ECACC No: 85110503); human retinoblasts (PER.C6, CruCell, Leiden, The Netherlands); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (HEK293 or 293 cells subcloned for growth in suspension culture, Graham et al., J.
  • human fibrosarcoma cell line e.g., HT1080
  • baby hamster kidney cells BHK21 , ATCC CCL 10
  • Chinese hamster ovary cells +/-DHFR CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216, 1980
  • CHO EBNA Daramola O. et al., Biotechnol. Prog., 2014, 30(1):132-41
  • CHO GS Fean L. et al., Biotechnol. Bioeng. 2012, 109(4):1007-15
  • mouse sertoli cells TM4, Mather, Biol.
  • monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.
  • bispecific antibodies are produced from human cells.
  • bispecific antibodies are produced from CHO cells or HEK cells or HT1080 cells.
  • a host cell is selected for generating a cell line based on certain preferable attributes or growth under particular conditions chosen for culturing cells. It will be appreciated by one skilled in the art, such attributes may be ascertained based on known characteristic and/or traits of an established line (i.e. a characterized commercially available cell line) or though empirical evaluation.
  • a cell line may be selected for its ability to grow on a feeder layer of cells.
  • a cell line may be selected for its ability to grow in suspension.
  • a cell line may be selected for its ability to grow as an adherent monolayer of cells.
  • a cell line may be chosen for preferential post translational modifications (e.g., glycosylation).
  • such cells can be used with any tissue culture vessel or any vessel treated with a suitable adhesion substrate.
  • a suitable adhesion substrate is selected from the group consisting of collagen (e.g. collagen I, II, II, or IV), gelatin, fibronectin, laminin, vitronectin, fibrinogen, BD MatrigelTM, basement membrane matrix, dermatan sulfate proteoglycan, Poly-D-Lysine and/or combinations thereof.
  • an adherent host cell may be selected and modified under specific growth conditions to grow in suspension. Such methods of modifying an adherent cell to grown in suspension are known in the art. For example, a cell may be conditioned to grow in suspension culture, by gradually removing animal serum from the growth media over time.
  • cells that are engineered to express a bispecific antibody may comprise a transgene that encodes a bispecific antibody described herein.
  • Cells can be engineered to express the bispecific antibody in a transient or a stable expression system.
  • the nucleic acids encoding bispecific antibodies may contain regulatory sequences, gene control sequences, promoters, non-coding sequences and/or other appropriate sequences for expressing the bispecific antibody.
  • the coding region is operably linked with one or more of these nucleic acid components.
  • bispecific antibodies are expressed using a batch culture method.
  • batch culture duration may be for 7-14 days.
  • the batch culture may be for 14-21 days.
  • bispecific antibodies are expressed using a perfusion culture method (collection of culture medium over time each day).
  • bispecific antibodies are expressed using a pseudoperfusion culture method (daily collection of culture medium at a single time point with replacement with fresh medium).
  • specific feeding regimens/media may be used to promote optimal bispecific antibody production (improved glycan, reduce aggregate, improved kappa/lambda body ratio).
  • the cell density may be controlled/maintained to promote optimal bispecific antibody production (reduced aggregate, improved heavy/light chain, improved kappa/lambda body ratio).
  • FIG. 16 of WO 2012/023053 A2 demonstrates purification and product integrity testing of exemplary bispecific antibodies purified from a semi-stable cell line.
  • the bispecific antibodies were purified using the following three-step affinity chromatography procedure: (i) Protein A purification to capture IgG molecules, including both monospecific and bispecific; (ii) KappaSelect purification to capture IgG containing Kappa light chain(s); (iii) LambdaSelect purification to capture IgG containing Lambda light chain.
  • the present invention takes a different approach that is based on the production of a simple mixture of antibodies that have the standard characteristics of productivity and scalability of monoclonal antibodies and provides efficient and generic means to purify the bispecific antibody in KL body format from the mixture or to purify the antibody mixture, wherein the bispecific antibody binds to Factor IXa and Factor X.
  • the KL-bodies produced according to these particular embodiments conserve the structure of a classical human IgG. Therefore, they have the advantage of a reduced risk of immunogenicity, as compared to certain other formats of bispecific antibodies, and thus the advantage of being particularly well suited for long-term administration to a subject, e.g. to treat a chronic disease (such as hemophilia A).
  • the bispecific antibody of the present invention is a bispecific antibody that binds to Factor IXa and Factor X and that has a procoagulant activity, the bispecific antibody comprising a first heavy chain and a first light chain and a second heavy chain and a second light chain.
  • the bispecific antibody of the present invention may comprise two heavy chains (i.e. the first heavy chain and the second heavy chain) with identical CDRs, whereas the CDRs of the first light chain and the CDRs of the second light chain differ from each other.
  • Preferred embodiments of the present invention are those bispecific antibodies of the present invention that have a high procoagulant activity.
  • the antibody comprises a first heavy chain and a second heavy chain each comprising in its variable domain the CDRs H-CDR1 , H-CDR2 and H- CDR3, wherein H-CDR1 is GFTFSSYA (SEQ ID NO: 1), H-CDR2 is ISGSGGST (SEQ ID NO: 2) and H-CDR3 is AKSYGAFDY (SEQ ID NO:3), and the antibody further comprises a first light chain and a second light chain comprising in its variable domain the CDRs of one of the Identifiers set out in Table 1A and Table 2A below, respectively.
  • the antibody comprises a first heavy chain and a second heavy chain each comprising in its variable domain the CDRs H-CDR1 , H-CDR2 and H- CDR3, wherein H-CDR1 is GFTFSSYA (SEQ ID NO: 1), H-CDR2 is ISGSGGST (SEQ ID NO: 2) and H-CDR3 is AKSYGAFDY (SEQ ID NO:3), and the antibody further comprises a first light chain and a second light chain comprising in its variable domain the CDRs of one of the Identifiers set out in Table 1 B and Table 2B below, respectively.
  • Table 1 B CDRs of Factor X binding light chains
  • the bispecific antibody comprises L1-CDR1 , L1-CDR2 and L1- CDR3 of W83 according to Table 1A in the variable domain of the first light chain, and L2- CDR1 , L2-CDR2 and L2-CDR3 of V141 , V149, V155, V198, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289, V290 or V296 according to
  • the bispecific antibody comprises L1-CDR1 , L1-CDR2 and L1-CDR3 of W122 according to Table 1A in the variable domain of the first light chain, and L2-CDR1 , L2-CDR2 and L2-CDR3 of V141 , V149, V155, V198, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289, V290 or V296 according to Table 2A in the variable domain of the second light chain, and two heavy chains (i.e. the first heavy chain and the second heavy chain) each comprising identical CDRs in its variable domain, namely H-CDR1 (SEQ ID NO: 1), H-CDR2 (SEQ ID NO: 2) and H-CDR3
  • the bispecific antibody comprises L1-CDR1 , L1- CDR2 and L1-CDR3 of W128 according to Table 1A in the variable domain of the first light chain, and L2-CDR1 , L2-CDR2 and L2-CDR3 of V141 , V149, V155, V198, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289, V290 or V296 according to Table 2A in the variable domain of the second light chain, and two heavy chains (i.e.
  • the bispecific antibody comprises L1- CDR1 , L1-CDR2 and L1-CDR3 of W133 according to Table 1A in the variable domain of the first light chain, and L2-CDR1 , L2-CDR2 and L2-CDR3 of V141 , V149, V155, V198, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289, V290 or V296 according to Table 2A in the variable domain of the second light chain, and two heavy chains (i.e.
  • the bispecific antibody comprises L1-CDR1 , L1-CDR2 and L1-CDR3 of W159 according to Table 1A in the variable domain of the first light chain, and L2-CDR1 , L2-CDR2 and L2-CDR3 of V141 , V149, V155, V198, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289, V290 or V296 according to Table 2A in the variable domain of the second light chain, and two heavy chains (i.e.
  • the bispecific antibody comprises L1-CDR1 , L1-CDR2 and L1-CDR3 of W189 according to Table 1A in the variable domain of the first light chain, and L2-CDR1 , L2-CDR2 and L2-CDR3 of V141 , V149, V155, V198, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289, V290 or V296 according to Table 2A in the variable domain of the second light chain, and two heavy chains (i.e.
  • the bispecific antibody comprises L1-CDR1 , L1-CDR2 and L1-CDR3 of W198 according to Table 1A in the variable domain of the first light chain, and L2-CDR1 , L2-CDR2 and L2-CDR3 of V141 , V149, V155, V198, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289, V290 or V296 according to Table 2A in the variable domain of the second light chain, and two heavy chains (i.e.
  • the bispecific antibody comprises L1-CDR1 , L1-CDR2 and L1-CDR3 of W204 according to Table 1A in the variable domain of the first light chain, and L2-CDR1 , L2-CDR2 and L2-CDR3 of V141 , V149, V155, V198, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289, V290 or V296 according to Table 2A in the variable domain of the second light chain, and two heavy chains (i.e.
  • the bispecific antibody comprises L1-CDR1 , L1-CDR2 and L1-CDR3 of W206 according to Table 1A in the variable domain of the first light chain, and L2-CDR1 , L2-CDR2 and L2-CDR3 of V141 , V149, V155, V198, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289, V290 or V296 according to Table 2A in the variable domain of the second light chain, and two heavy chains (i.e.
  • the bispecific antibody comprises L1-CDR1 , L1-CDR2 and L1- CDR3 of W207 according to Table 1A in the variable domain of the first light chain, and L2- CDR1 , L2-CDR2 and L2-CDR3 of V141 , V149, V155, V198, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289, V290 or V296 according to Table 2A in the variable domain of the second light chain, and two heavy chains (i.e.
  • the bispecific antibody comprises L1-CDR1 , L1-CDR2 and L1-CDR3 of W127 according to Table 1 B in the variable domain of the first light chain, and L2-CDR1 , L2-CDR2 and L2-CDR3 of V141 , V149, V155, V198, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289, V290 or V296 according to Table 2A in the variable domain of the second light chain, and two heavy chains (i.e. the first heavy chain and the second heavy chain) each comprising identical CDRs in its variable domain, namely H-
  • the bispecific antibody comprises L1-CDR1 , L1-CDR2 and L1- CDR3 of W83 according to Table 1 B in the variable domain of the first light chain, and L2- CDR1 , L2-CDR2 and L2-CDR3 of V141 , V149, V155, V198, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289, V290, V296, V380, V390,
  • the bispecific antibody comprises L1-CDR1 , L1-CDR2 and L1-CDR3 of W122 according to Table 1 B in the variable domain of the first light chain, and L2-CDR1 , L2-CDR2 and L2-CDR3 of V141 , V149, V155, V198, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284,
  • H-CDR1 SEQ ID NO: 1
  • H-CDR2 SEQ ID NO: 2
  • H-CDR3 SEQ ID NO:3
  • the bispecific antibody comprises L1- CDR1 , L1-CDR2 and L1-CDR3 of W128 according to Table 1 B in the variable domain of the first light chain, and L2-CDR1 , L2-CDR2 and L2-CDR3 of V141 , V149, V155, V198, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289,
  • H-CDR1 SEQ ID NO: 1
  • H-CDR2 SEQ ID NO: 2
  • H-CDR3 SEQ ID NO:3
  • the bispecific antibody comprises L1-CDR1 , L1-CDR2 and L1-CDR3 of W133 according to Table 1 B in the variable domain of the first light chain, and L2-CDR1 , L2-CDR2 and L2-CDR3 of V141 , V149, V155, V198, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289, V290, V296, V380, V390, V386,
  • the bispecific antibody comprises L1-CDR1 , L1-CDR2 and L1-CDR3 of W159 according to Table 1 B in the variable domain of the first light chain, and L2-CDR1 , L2-CDR2 and L2-CDR3 of V141 , V149, V155, V198, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284,
  • H-CDR1 SEQ ID NO: 1
  • H-CDR2 SEQ ID NO: 2
  • H-CDR3 SEQ ID NO:3
  • the bispecific antibody comprises L1- CDR1 , L1-CDR2 and L1-CDR3 of W189 according to Table 1 B in the variable domain of the first light chain, and L2-CDR1 , L2-CDR2 and L2-CDR3 of V141 , V149, V155, V198, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289,
  • H-CDR1 SEQ ID NO: 1
  • H-CDR2 SEQ ID NO: 2
  • H-CDR3 SEQ ID NO:3
  • the bispecific antibody comprises L1-CDR1 , L1-CDR2 and L1-CDR3 of W198 according to Table 1 B in the variable domain of the first light chain, and L2-CDR1 , L2-CDR2 and L2-CDR3 of V141 , V149, V155, V198, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289, V290, V296, V380, V390, V386, V297, V299, V387, V243, V410, V298, V426, V205, V429, V305, V204, V202, V424, V246, V425, V320, V244, V401 or V402 according to Table 2B in the variable domain of the second light chain, and two heavy chains (i.e.
  • the bispecific antibody comprises L1-CDR1 , L1-CDR2 and L1-CDR3 of W204 according to Table 1 B in the variable domain of the first light chain, and L2-CDR1 , L2-CDR2 and L2-CDR3 of V141 , V149, V155, V198, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284,
  • H-CDR1 SEQ ID NO: 1
  • H-CDR2 SEQ ID NO: 2
  • H-CDR3 SEQ ID NO:3
  • the bispecific antibody comprises L1- CDR1 , L1-CDR2 and L1-CDR3 of W206 according to Table 1 B in the variable domain of the first light chain, and L2-CDR1 , L2-CDR2 and L2-CDR3 of V141 , V149, V155, V198, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289,
  • H-CDR1 SEQ ID NO: 1
  • H-CDR2 SEQ ID NO: 2
  • H-CDR3 SEQ ID NO:3
  • the bispecific antibody comprises L1-CDR1 , L1-CDR2 and L1-CDR3 of W207 according to Table 1 B in the variable domain of the first light chain, and L2-CDR1 , L2-CDR2 and L2-CDR3 of V141 , V149, V155, V198, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289, V290, V296, V380, V390, V386,
  • the bispecific antibody comprises L1-CDR1 , L1-CDR2 and L1-CDR3 of W127 according to Table 1 B in the variable domain of the first light chain, and L2-CDR1 , L2-CDR2 and L2-CDR3 of V141 , V149, V155, V198, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284,
  • H-CDR1 SEQ ID NO: 1
  • H-CDR2 SEQ ID NO: 2
  • H-CDR3 SEQ ID NO:3
  • the bispecific antibody comprises the three CDRs (L1-CDR1 , L1- CDR2, L1-CDR3 and L2-CDR1 , L2-CDR2, L2-CDR3) of W128 and V241 , W128 and V242, W128 and V245, W206 and V245, W198 and V245, W159 and V245, W133 and V245,
  • the bispecific antibody further comprises two heavy chains (i.e. the first heavy chain and the second heavy chain) each comprising identical CDRs in its variable domain, namely H-CDR1 (SEQ ID NO: 1), H-CDR2 (SEQ ID NO: 2) and H-CDR3 (SEQ ID NO:3).
  • the bispecific antibody comprises the three CDRs (L1-CDR1 , L1- CDR2, L1-CDR3 and L2-CDR1 , L2-CDR2, L2-CDR3) of W128 and V241 , W128 and V242, W128 and V245, W206 and V245, W198 and V245, W159 and V245, W133 and V245,
  • H-CDR1 SEQ ID NO: 1
  • H-CDR2 SEQ ID NO: 2
  • H-CDR3 SEQ ID NO:3
  • the bispecific antibody comprises the three CDRs (L1-CDR1 , L1- CDR2, L1-CDR3 and L2-CDR1 , L2-CDR2, L2-CDR3) of W198 and V282, W198 and V284, W198 and V285, W198 and V286, or W198 and V288, respectively, according to Tables 1A and 2A, respectively, in its first and second light chain, respectively, and the bispecific antibody further comprises two heavy chains (i.e. the first heavy chain and the second heavy chain) each comprising identical CDRs in its variable domain, namely H-CDR1 (SEQ ID NO: 1), H-CDR2 (SEQ ID NO: 2) and H-CDR3 (SEQ ID NO:3).
  • H-CDR1 SEQ ID NO: 1
  • H-CDR2 SEQ ID NO: 2
  • H-CDR3 SEQ ID NO:3
  • the bispecific antibody comprises the three CDRs (L1-CDR1 , L1- CDR2, L1-CDR3 and L2-CDR1 , L2-CDR2, L2-CDR3) of W198 and V282, W198 and V284, W198 and V285, W198 and V286, W198 and V288, W206 and V380, W198 and V390, W198 and V386, W198 and V297, W198 and V299, W198 and V387, W198 and V380, W128 and V244, W198 and V401 , W198 and V402, W198 and V424, or W198 and V425, respectively, according to Tables 1 B and 2B, respectively, in its first and second light chain, respectively, and the bispecific antibody further comprises two heavy chains (i.e. the first heavy chain and the second heavy chain) each comprising identical CDRs in its variable domain, namely H-CDR1 (SEQ ID NO: 1), H-CDR2 (SEQ ID NO: 2) and H
  • the bispecific antibody comprises the three CDRs (L1-CDR1 , L1- CDR2, L1-CDR3 and L2-CDR1 , L2-CDR2, L2-CDR3) of W198 and V282, W206 and V380, or W198 and V380, respectively, according to Tables 1 B and 2B, respectively, in its first and second light chain, respectively, and the bispecific antibody further comprises two heavy chains (i.e. the first heavy chain and the second heavy chain) each comprising identical CDRs in its variable domain, namely H-CDR1 (SEQ ID NO: 1), H-CDR2 (SEQ ID NO: 2) and H-CDR3 (SEQ ID NO:3).
  • H-CDR1 SEQ ID NO: 1
  • H-CDR2 SEQ ID NO: 2
  • H-CDR3 SEQ ID NO:3
  • the bispecific antibody of the present invention may comprise two heavy chains with identical variable domains.
  • the antibody comprises two heavy chains comprising identical variable domains wherein the variable domain comprises SEQ ID NO: 97.
  • the bispecific antibody comprises the V L of the Factor X binding arm of any one of antibodies 1 to 37 according to Table 3, and the bispecific antibody further comprises the V L of the Factor IXa binding arm of any one (i.e. the same one, or a different one) of antibodies 1 to 37 according to Table 3, and the bispecific antibody further comprises two heavy chains comprising identical variable domains wherein the heavy chain variable domain comprises SEQ ID NO: 97.
  • the bispecific antibody comprises the V L of the Factor X binding arm of any one of antibodies 1 to 63 according to Table 3, and the bispecific antibody further comprises the V L of the Factor IXa binding arm of any one (i.e. the same one, or a different one) of antibodies 1 to 63 according to Table 3, and the bispecific antibody further comprises two heavy chains comprising identical variable domains wherein the heavy chain variable domain comprises SEQ ID NO: 97.
  • the bispecific antibody comprises both the V L of the Factor X binding arm and the V L of the Factor IXa binding arm of any one of antibodies 1 to 37 according to Table 3, and the bispecific antibody further comprises two heavy chains comprising identical variable domains wherein the heavy chain variable domain comprises SEQ ID NO: 97.
  • the bispecific antibody comprises both the V L of the Factor X binding arm and the V L of the Factor IXa binding arm of any one of antibodies 1 to 63 according to Table 3, and the bispecific antibody further comprises two heavy chains comprising identical variable domains wherein the heavy chain variable domain comprises SEQ ID NO: 97.
  • the bispecific antibody comprises both the V L of the Factor X binding arm and the V L of the Factor IXa binding arm of any one of antibodies 12, 14 to 16 and 18 according to Table 3, and the bispecific antibody further comprises two heavy chains comprising identical variable domains wherein the heavy chain variable domain comprises SEQ ID NO: 97.
  • the bispecific antibody comprises both the V L of the Factor X binding arm and the V L of the Factor IXa binding arm of any one of antibodies 12, 14 to 16, 18, 38 to 43, 45 and 59 to 63 according to Table 3, and the bispecific antibody further comprises two heavy chains comprising identical variable domains wherein the heavy chain variable domain comprises SEQ ID NO: 97.
  • the bispecific antibody comprises both the V L of the Factor X binding arm and the V L of the Factor IXa binding arm of any one of antibodies 12, 38 and 45 according to Table 3, and the bispecific antibody further comprises two heavy chains comprising identical variable domains wherein the heavy chain variable domain comprises SEQ ID NO: 97.
  • the bispecific antibody of the present invention may comprise two identical heavy chains.
  • the bispecific antibody comprises two identical heavy chains comprising SEQ ID NO: 172.
  • the bispecific antibody comprises two identical heavy chains comprising SEQ ID NO: 462.
  • the heavy chain comprising SEQ ID NO: 462 can be used instead of the heavy chain comprising SEQ ID NO: 172 in any bispecific antibody disclosed herein.
  • the bispecific antibody comprises two identical heavy chains comprising one or more mutations that give rise to an extended half-life of the 0 bispecific antibody (see e.g. WO 02/060919 A2).
  • the bispecific antibody comprises two identical heavy chains comprising SEQ ID NO: 463.
  • the heavy chain comprising SEQ ID NO: 463 contains the mutations M252Y, S254T and T256E, and may give rise to an extended half-life of a bispecific antibody containing said heavy chain.
  • the heavy chain comprising SEQ ID NO: 463 can be used instead of the heavy chain comprising SEQ ID NO: 172 in any bispecific antibody disclosed herein.
  • the bispecific antibody comprises both the light chain of the Factor X binding arm and the light chain of the Factor IXa binding arm of any one of antibodies 1 to 37 according to Table 4, and the bispecific antibody further comprises two identical heavy chains each comprising SEQ ID NO: 172.
  • the bispecific antibody comprises both the light chain of the Factor X binding arm and the light chain of the Factor IXa binding arm of any one of antibodies 1 to 63 according to Table 4, and the bispecific antibody further comprises two identical heavy chains each comprising SEQ ID NO: 172.
  • the bispecific antibody comprises both the light chain of the Factor X binding arm and the light chain of the Factor IXa binding arm of any one of antibodies 1 to 37 according to Table 4, and the bispecific antibody further comprises two identical heavy chains each comprising SEQ ID NO: 462.
  • the bispecific antibody comprises both the light chain of the Factor X binding arm and the light chain of the Factor IXa binding arm of any one of antibodies 1 to 63 according to Table 4, and the bispecific antibody further comprises two identical heavy chains each comprising SEQ ID NO: 462.
  • the bispecific antibody comprises both the light chain of the Factor X binding arm and the light chain of the Factor IXa binding arm of any one of antibodies 1 to 37 according to Table 4, and the bispecific antibody further comprises two identical heavy chains each comprising SEQ ID NO: 463.
  • the bispecific antibody comprises both the light chain of the Factor X binding arm and the light chain of the Factor IXa binding arm of any one of antibodies 1 to 63 according to Table 4, and the bispecific antibody further comprises two identical heavy chains each comprising SEQ ID NO: 463.
  • the bispecific antibody comprises both the light chain of the Factor X binding arm and the light chain of the Factor IXa binding arm of any one of antibodies 12, 14 to 16 and 18 according to Table 4, and the bispecific antibody further comprises two identical heavy chains each comprising SEQ ID NO: 172.
  • the bispecific antibody comprises both the light chain of the Factor X binding arm and the light chain of the Factor IXa binding arm of any one of antibodies 12, 14 to 16, 18, 38 to 43, 45 and 59 to 63 according to Table 4, and the bispecific antibody further comprises two identical heavy chains each comprising SEQ ID NO: 172.
  • the bispecific antibody comprises both the light chain of the Factor X binding arm and the light chain of the Factor IXa binding arm of any one of antibodies 12, 38 and 45 according to Table 4, and the bispecific antibody further comprises two identical heavy chains each comprising SEQ ID NO: 172.
  • the bispecific antibody comprises both the light chain of the Factor X binding arm and the light chain of the Factor IXa binding arm of any one of antibodies 12, 14 to 16 and 18 according to Table 4, and the bispecific antibody further comprises two identical heavy chains each comprising SEQ ID NO: 462.
  • the bispecific antibody comprises both the light chain of the Factor X binding arm and the light chain of the Factor IXa binding arm of any one of antibodies 12, 14 to 16, 18, 38 to 43, 45 and 59 to 63 according to Table 4, and the bispecific antibody further comprises two identical heavy chains each comprising SEQ ID NO: 462.
  • the bispecific antibody comprises both the light chain of the Factor X binding arm and the light chain of the Factor IXa binding arm of any one of antibodies 12, 38 and 45 according to Table 4, and the bispecific antibody further comprises two identical heavy chains each comprising SEQ ID NO: 462.
  • the bispecific antibody comprises both the light chain of the Factor X binding arm and the light chain of the Factor IXa binding arm of any one of antibodies 12, 14 to 16 and 18 according to Table 4, and the bispecific antibody further comprises two identical heavy chains each comprising SEQ ID NO: 463.
  • the bispecific antibody comprises both the light chain of the Factor X binding arm and the light chain of the Factor IXa binding arm of any one of antibodies 12, 14 to 16, 18, 38 to 43, 45 and 59 to 63 according to Table 4, and the bispecific antibody further comprises two identical heavy chains each comprising SEQ ID NO: 463.
  • the bispecific antibody comprises both the light chain of the Factor X binding arm and the light chain of the Factor IXa binding arm of any one of antibodies 12, 38 and 45 according to Table 4, and the bispecific antibody further comprises two identical heavy chains each comprising SEQ ID NO: 463.
  • the light chain CDRs and light chain variable domains (VL) of the following Tables 5-8 can be combined with the above-described heavy chain CDRs (SEQ ID NO: 1-3) and heavy chain variable domain (SEQ ID NO: 97), respectively, to form the Factor X binding arm or the Factor IXa binding arm of an antibody (e.g. a bispecific antibody), as applicable:
  • the antigen bound i.e. Factor X or Factor IXa
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibodies of the present invention and a pharmaceutically acceptable carrier and/or diluent.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody of the present invention and a pharmaceutically acceptable carrier or diluent.
  • pharmaceutically acceptable carrier or “pharmaceutically acceptable diluent” means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and, without limiting the scope of the present invention, include: additional buffering agents; preservatives; co-solvents; antioxidants, including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (e.g., Zn-protein complexes); biodegradable polymers, such as polyesters; salt-forming counterions, such as sodium, polyhydric sugar alcohols; amino acids, such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactitol, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinis
  • a pharmaceutical composition as described herein may also contain other substances. These substances include, but are not limited to, cryoprotectants, lyoprotectants, surfactants, bulking agents, anti-oxidants, and stabilizing agents. In some embodiments, the pharmaceutical composition may be lyophilized.
  • cryoprotectant includes agents which provide stability to the antibody against freezing-induced stresses, by being preferentially excluded from the antibody’s surface. Cryoprotectants may also offer protection during primary and secondary drying and long-term product storage.
  • cryoprotectants include sugars, such as sucrose, glucose, trehalose, mannitol, mannose, and lactose; polymers, such as dextran, hydroxyethyl starch and polyethylene glycol; surfactants, such as polysorbates (e.g., PS-20 or PS-80); and amino acids, such as glycine, arginine, leucine, and serine.
  • a cryoprotectant exhibiting low toxicity in biological systems is generally used.
  • a lyoprotectant is added to a pharmaceutical composition described herein.
  • the term "lyoprotectant” as used herein includes agents that provide stability to the antibody during the freeze-drying or dehydration process (primary and secondary freeze drying cycles), by providing an amorphous glassy matrix and by binding with the antibody’s surface through hydrogen bonding, replacing the water molecules that are removed during the drying process. This helps to minimize product degradation during the lyophilization cycle, and improve the long-term product stability.
  • Non-limiting examples of lyoprotectants include sugars, such as sucrose or trehalose; an amino acid, such as monosodium glutamate, non-crystalline glycine or histidine; a methylamine, such as betaine; a lyotropic salt, such as magnesium sulfate; a polyol, such as trihydric or higher sugar alcohols, e.g., glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol; propylene glycol; polyethylene glycol; pluronics; and combinations thereof.
  • the amount of lyoprotectant added to a pharmaceutical composition is generally an amount that does not lead to an unacceptable amount of degradation of the strain when the pharmaceutical composition is lyophilized.
  • a bulking agent is included in the pharmaceutical composition.
  • bulking agents may also impart useful qualities in regard to modifying the collapse temperature, providing freeze-thaw protection, and enhancing the strain stability over long-term storage.
  • Non-limiting examples of bulking agents include mannitol, glycine, lactose, and sucrose.
  • Bulking agents may be crystalline (such as glycine, mannitol, or sodium chloride) or amorphous (such as dextran, hydroxyethyl starch) and are generally used in formulations in an amount from 0.5% to 10%.
  • pharmaceutically acceptable carriers such as those described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) may also be included in a pharmaceutical composition described herein, provided that they do not adversely affect the desired characteristics of the pharmaceutical composition.
  • pharmaceutically acceptable carrier means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include: additional buffering agents; preservatives; co-solvents; antioxidants, including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (e.g., Zn- protein complexes); biodegradable polymers, such as polyesters; salt-forming counterions, such as sodium, polyhydric sugar alcohols; amino acids, such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactitol, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, gal
  • the pharmaceutical composition may be a solution which is suitable for intravenous, intramuscular, conjunctival, transdermal, intraperitoneal and/or subcutaneous administration (e.g. subcutaneous administration using a device).
  • the pharmaceutical composition may be in a form suitable for nasal administration or oral administration.
  • the bispecific antibody according to the present invention in one embodiment is formulated into a physiologically-acceptable pharmaceutical composition
  • a carrier i.e. , vehicle, adjuvant, buffer, or diluent.
  • the particular carrier employed is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the bispecific antibody, and by the route of administration.
  • Physiologically- acceptable carriers are well known in the art.
  • Illustrative pharmaceutical forms suitable for injectable use include without limitation sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (for example, see U.S. Patent No. 5,466,468).
  • a pharmaceutical composition comprising a bispecific antibody provided herein is optionally placed within containers, along with packaging material that provides instructions regarding the use of such pharmaceutical compositions.
  • such instructions include a tangible expression describing the reagent concentration, as well as, in certain embodiments, relative amounts of excipient ingredients or diluents that may be necessary to reconstitute the pharmaceutical composition.
  • a physiologically-acceptable composition such as a pharmaceutical composition comprising a bispecific antibody described herein
  • a pharmaceutical composition is applied or instilled into body cavities, absorbed through the skin or mucous membranes, ingested, inhaled, and/or introduced into circulation.
  • a composition comprising a bispecific antibody is administered intravenously, intraarterially, or intraperitoneally to introduce the bispecific antibody of the invention into circulation.
  • Non-intravenous administration also is appropriate.
  • a pharmaceutical composition comprising the bispecific antibody orally, topically, sublingually, vaginally, rectally, pulmonary; through injection by intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intra-ocular, intraportal, intralesional, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intranasal, urethral, or enteral means; by sustained release systems; or by implantation devices.
  • the bispecific antibody is administered regionally via intraarterial or intravenous administration feeding a region of interest, e.g., via the femoral artery for delivery to the leg.
  • the bispecific antibody is incorporated into a microparticle as described in, for example, U.S. Patents 5,439,686 and 5,498,421 , and U.S. Patent Publications 2003/0059474, 2003/0064033, 2004/0043077, 2005/0048127, 2005/0170005, 2005/0142205, 2005/142201 , 2005/0233945, 2005/0147689. 2005/0142206, 2006/0024379, 2006/0260777,
  • the composition is administered via implantation of a membrane, sponge, or another appropriate material on to which the desired bispecific antibody has been absorbed or encapsulated.
  • the device in one aspect is implanted into any suitable tissue, and delivery of the desired bispecific antibody is in various aspects via diffusion, timed-release bolus, or continuous administration.
  • the bispecific antibody is administered directly to exposed tissue during surgical procedures or treatment of injury, or is administered via transfusion of blood procedures.
  • Therapeutic delivery approaches are well known to the skilled artisan, some of which are further described, for example, in U.S. Patent No. 5,399,363.
  • the pharmaceutical composition may further comprise common excipients and carriers which are known in the state of the art.
  • the pharmaceutical composition may further comprise cryoprotectants, lyoprotectants, surfactants, bulking agents, anti-oxidants, stabilizing agents and pharmaceutically acceptable carriers.
  • the present invention also provides the bispecific antibody or pharmaceutical composition of the present invention for use as a medicament. Further, the present invention provides the bispecific antibody or pharmaceutical composition of the present invention for use in a method of treating and/or preventing a bleeding disorder, wherein a patient or animal is administered a therapeutically effective amount of the bispecific antibody.
  • the present invention provides the use of the bispecific antibody or pharmaceutical composition of the present invention for the manufacture of a medicament for the treatment and/or prevention of a bleeding disorder.
  • the coagulation or bleeding disorder is caused by the absence of a coagulation factor.
  • the coagulation or bleeding disorder may be hemophilia or von Willebrand disease.
  • the coagulation or bleeding disorder is hemophilia A or acquired hemophilia.
  • the coagulation or bleeding disorder is hemophilia A.
  • the coagulation or bleeding disorder is acquired hemophilia where the subject no longer produces FVIII.
  • the bispecific antibodies or pharmaceutical compositions of the present invention may be administered to a subject with mild hemophilia A, moderate hemophilia A, or severe hemophilia A.
  • the bispecific antibodies or pharmaceutical compositions disclosed herein may be administered to a subject with Factor VIII plasma levels of 6% to 40%, 2% to 5%, or 1% or less.
  • the bispecific antibodies or pharmaceutical compositions of the present invention may be administered to a subject with hemophilia A or suspected of having hemophilia A when there is an external wound on the subject.
  • the bispecific antibodies or pharmaceutical compositions of the present invention may be administered to a subject with hemophilia A or suspected of having hemophilia A with an existing external wound on the subject.
  • bispecific antibodies or pharmaceutical compositions of the present invention may be administered to a subject with an external wound until the wound has healed.
  • the wound may include, but is not limited to, an abrasion, a laceration, a puncture, or an avulsion.
  • the bispecific antibodies or pharmaceutical compositions of the present invention may be administered to a subject with hemophilia, A or suspected of having hemophilia A, prior to, during, or after surgery, a serious injury, or dental work.
  • the bispecific antibodies or pharmaceutical compositions of the present invention may be administered to a subject with hemophilia A, or suspected of having hemophilia A, and has experienced spontaneous bleeding.
  • the bispecific antibodies or pharmaceutical compositions of the present invention may be administered to a subject with hemophilia A, or suspected of having hemophilia A, and has experienced bleeding once, twice, or more times in a week.
  • the bispecific antibodies or pharmaceutical compositions of the present invention may be administered to a subject of any age group suffering from, or suspected of having hemophilia A.
  • the bispecific antibodies or pharmaceutical compositions of the present invention may be administered to a child of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 ,15, 16, or 17 years of age suffering from, or suspected of having hemophilia A.
  • the bispecific antibodies or pharmaceutical compositions of the present invention thereof may be administered to an infant suffering from or suspected of having hemophilia A.
  • the bispecific antibodies or pharmaceutical compositions of the present invention may be administered to a subject who is an infant of 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 months of age suffering from, or suspected of having hemophilia A.
  • the bispecific antibodies or pharmaceutical compositions of the present invention are administered to a subject at an early age before the first episode of bleeding.
  • administering the bispecific antibodies or pharmaceutical compositions of the present invention before the first episode of bleeding protects against further bleeding and development of joint damage in the future.
  • administering a bispecific antibodies or pharmaceutical compositions of the present invention to subjects may have the following effects, but is not limited to, hemostasis, reduced pain, and improved mobility.
  • the subject has developed, has a tendency to develop, or is at risk to develop an inhibitor against Factor VIII ("FVIII").
  • the inhibitor against FVIII is a neutralizing antibody against FVIII.
  • the subject is undergoing treatment with FVIII or is a candidate for treatment with FVIII, e.g., FVIII replacement therapy.
  • the bleeding episode is the result of hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the illiopsoas sheath, or any combinations thereof.
  • the present invention also provides a method of treating a blood coagulation disorder in a subject in need thereof, comprising administering to the subject an effective amount of the bispecific antibody or pharmaceutical composition of the present invention.
  • the blood coagulation disorder is hemophilia A or hemophilia B, preferably hemophilia A.
  • the subject is a human subject.
  • the subject is undergoing or has undergone FVIII replacement therapy.
  • the bispecific antibody or pharmaceutical composition of the present invention is administered in combination with a hemophilia therapy.
  • the hemophilia therapy is a FVIII replacement therapy.
  • the bispecific antibody or pharmaceutical composition of the present invention is administered before, during or after administration of the hemophilia therapy.
  • the bispecific antibody is administered prophylactically.
  • the bispecific antibody or pharmaceutical composition of the present invention is administered intravenously or subcutaneously.
  • administration of the bispecific antibodies or pharmaceutical compositions of the present invention reduces the frequency of break-through bleeding episodes, spontaneous bleeding episodes, or acute bleeding. In some aspects, administration of bispecific antibodies or pharmaceutical compositions of the present invention reduces the annualized bleed rate by 5%, 10%, 20%, 30%, or 50%.
  • the bispecific antibodies or pharmaceutical compositions of the present invention may be administered by any route appropriate to the condition to be treated.
  • the bispecific antibodies or pharmaceutical compositions of the present invention will typically be administered parenterally, i.e. , infusion, subcutaneous, intramuscular, intravenous, or intradermal.
  • the bispecific antibodies or pharmaceutical compositions of the present invention are administered subcutaneously.
  • the bispecific antibodies or pharmaceutical compositions of the present invention are administered intermittently or discontinuously.
  • dose levels of the bispecific antibodies of the present invention for example, administered via injection, such as subcutaneous injection, range from about 0.0001 mg/kg to about 100 mg/kg bodyweight.
  • the bispecific antibodies or pharmaceutical compositions of the present invention are administered until disease progression or unacceptable toxicity.
  • Phage were eluted with 100 nM TEA for 30 minutes at room temperature on a rotary mixer. Eluted phage and beads were neutralized with Tris-HCI 1M pH 7.4 and directly added to 10 ml of exponentially growing TG1 cells and incubated for one hour at 37 °C with slow shaking (90 rpm). An aliquot of the infected TG1 was serial diluted to titer the selection output. The remaining infected TG1 were spun at 3800 rpm for 10 minutes and resuspended in 2 ml 2xTY and spread on 2xTYAG (2xTY medium containing 100 pg/ml ampicillin and 2% glucose) agar Bioassay plates.
  • Phage rescue 50 pi of cell suspension obtained from previous selection rounds were added to 50 ml of 2xTYAG and grown at 37 °C with agitation (240 rpm) until an ODeoo of 0.3 to 0.5 is reached. The culture was then super-infected with 1.2x10 11 M13K07 helper phage and incubated for one hour at 37°C (90 rpm). The medium was changed by centrifuging the cells at 3800 rpm for 10 minutes, removing the medium and resuspending the pellet in 50 ml of 2xTYAK (100 pg/ml ampicillin; 50 pg/ml kanamycin). The culture is then grown overnight at 30°C (240 rpm). The next day, 10 mI of the phage containing supernatant was used for the next round of selection.
  • EXAMPLE 2 Screening for scFv binding to hFIXa or hFX
  • scFv periplasmic preparation for binding assays Individual TG1 clones were inoculated into a 96-well deep well plate containing 0.9 ml per well of 2xTYAG medium (0.1 % glucose) and grown at 37 °C for 5-6 hours (240 rpm). 100 mI per well of 0.2 mM IPTG in 2xTY medium were then added to give a final concentration of 0.02 mM IPTG. The plate was incubated overnight at 30 °C with shaking at 240 rpm. The deep well plate was centrifuged at 3200 rpm for 10 minutes at 4°C and the supernatant carefully removed.
  • 2xTYAG medium 0.1 % glucose
  • the pellets were resuspended in 150 mI TES buffer (50 mM Tris-HCI (pH 8), 1 mM EDTA (pH 8), 20% sucrose, complemented with Complete protease inhibitor, Roche).
  • a hypotonic shock was produced by adding 150 mI of diluted TES buffer (1 :5 TES:water dilution) and incubation on ice for 30 minutes. The plate was centrifuged at 4000 rpm for 10 minutes at 4 °C to pellet cells and debris. The supernatants were carefully transferred into a 96-well microtiter plate and kept on ice for immediate testing in functional assays or binding assays.
  • Binding Screening of scFv for binding to hFIXa or hFX was tested in a homogenous assay using Celllnsight technology. The following reagents were mixed in each well of a 384-well clear bottom plate (Corning): 30 mI of a streptavidin polystyrene bead suspension (Polysciences; 3000 beads/well) coated with biotinylated hFIXa or hFX or a control protein (hFII); 60 mI of blocked scFv periplasmic preparation; 10 mI of detection buffer (PBS containing mouse anti-c-myc antibody at 5 pg/ml; anti-mouse Fc AlexaFluor® 647 diluted 1 :200).
  • detection buffer PBS containing mouse anti-c-myc antibody at 5 pg/ml
  • DNA was extracted using the Zyppy-96 Plamisd Miniprep kit (Zymo Research). 5 pi of the eluted DNA was sequenced using the fdtseqlong primer, 5’-GTCGTCTTT CCAGACGTT AGT AAAT G-3’ (SEQ ID NO: 288).
  • EXAMPLE 3 Fixed VH candidates reformatting into (monospecific) IgG and transient expression in mammalian cells
  • scFv candidates against hFIXa or hFX were reformatted into IgG and expressed by transient transfection into PEAK cells.
  • the VH and VL sequences of selected scFv were amplified with specific oligonucleotides and cloned into an expression vector containing the heavy and light chain constant domains and the constructions were verified by sequencing.
  • the expression vectors were transfected into mammalian cells using the Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific, Waltham, MA).
  • Transient transfections were performed using a mix containing 30 pg of DNA and 42 pL of Lipofectamine 2000 transfection reagent (Invitrogen) in 2 mL of DMEM for 107 cells per T175 flask in 50 mL of complete DMEM.
  • IgG expression was measured using the Octet RED96 with protein A-coated biosensors (Pall ForteBio). According to antibody concentration, supernatants were harvested 7 to 10 days after transfection and clarified by centrifugation 10 min at 2000 rpm. Total IgGs were purified by one affinity chromatography step using the FcXL resin (Thermo Fischer Scientific).
  • KL bodies One way to assemble KL bodies is to co-express a complete variable light chain of the kappa type (i.e. variable and constant kappa domains) with a complete variable light chain of the lambda type (i.e. variable and constant lambda domains) and a common heavy chain (i.e. variable and constant heavy domains).
  • a complete variable light chain of the kappa type i.e. variable and constant lambda domains
  • lambda type i.e. variable and constant lambda domains
  • a common heavy chain i.e. variable and constant heavy domains
  • two variable domain of the same type i.e. two kappa variable domains or two lambda variable domains
  • variable domain of a lambda light chain can be fused to a constant domain of the kappa type or conversely the variable domain of kappa a light chain can be fused to a constant domain of the lambda type as described in US 2012/0184716 and WO 2012/023053.
  • hybrid chains were generated for several anti- FlXa and anti-FX arms.
  • Downstream purification of bsAb containing hybrid chain follows the same strategy and affinity purification resins, as one light chain contains a kappa constant domain and the other contains a lambda constant domain.
  • EXAMPLE 5 Expression and purification of bispecific antibodies carrying a Lambda and a Kappa constant light chain domain.
  • simultaneous expression of one heavy chain and two lights chain in the same cell can lead to the assembly of three different antibody forms, two monospecific antibodies and one bsAb.
  • Simultaneous expression can be achieved in different ways such as that the transfection of multiple vectors expressing some of the chains to be co-expressed or by using vectors that drive multiple gene expression.
  • a vector pNovi kHl (i.e. pNovi Kappa H Lambda) was previously generated to allow for the co-expression of one heavy chain, one Kappa light chain and one Lambda light chain as described in US 2012/0184716 and WO 2012/023053, each of which is hereby incorporated by reference in its entirety.
  • the expression of the three genes is driven by human cytomegalovirus promoters (hCMV) and the vector also contains a glutamine synthetase gene (GS) that enables the selection and establishment of stable cell lines.
  • hCMV human cytomegalovirus promoters
  • GS glutamine synthetase gene
  • the VH and VL gene of the anti-FIXa or the anti-FX were cloned in the above- mentioned vector pNovi kHl, for transient expression in mammalian cells.
  • IgG expression was measured using the Octet RED96 with protein A-coated biosensors (Pall ForteBio). According to antibody concentration, supernatants were harvested 7 to 10 days after transfection.
  • IgGs were purified by one affinity chromatography step using the FcXL resin (Thermo Fischer Scientific). Then, two additional affinity chromatography steps were required to isolate the KL body and eliminate the two monospecific mAbs: one purification on the KappaSelect resin (GE Healthcare) to eliminate the (i.e. IgG comprising two light chains with constant domain of Lambda type) and one purification on the LambdaFabSelect resin (GE Healthcare) to get rid of IgGio (i.e. IgG comprising two light chains with constant domain of Kappa type).
  • KappaSelect resin GE Healthcare
  • LambdaFabSelect resin GE Healthcare
  • the final product was quantified using the Nanodrop.
  • Purified bispecific antibodies were analyzed by electrophoresis in denaturing and reducing conditions.
  • the Agilent 2100 Bioanalyzer was used with the Protein 80 kit as described by the manufacturer (Agilent Technologies, Santa Clara, CA, USA). 4 pl_ of purified samples were mixed with sample buffer supplemented with dithiothreitol (DTT; Sigma Aldrich, St. Louis, MO). Samples were heated at 95°C for 5 min and then loaded on the chip.
  • DTT dithiothreitol
  • Antibodies 1 to 63 according to the present invention were analyzed for their binding to the targets FIXa and FX.
  • Bio-Layer Interferometry is a label-free technology for measuring biomolecular interactions. It is an optical analytical technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized protein on the biosensor tip, and an internal reference layer. The binding between the ligand-protein immobilized on the biosensor tip surface and an analyte in solution produces an increase in optical thickness at the biosensor tip, which results in a wavelength shift (nm shift), which is a direct measure of the change in thickness of the biological layer.
  • nm shift wavelength shift
  • Protein A biosensors were rehydrated in running buffer (HNaCT buffer) for 10 min, then conditioned by 3 cycles of: regeneration buffer (5secs, 10mM glycine pH 1.7) followed by neutralization buffer (5secs, HNaCT). Baseline signal was recorded for 120 secs before dipping the biosensors for 10min in the solution containing bsAb at 10ug/ml_.
  • Protein A biosensors loaded with bsAb were dipped into a solution containing human FIXa (hFIXa) or human FX (hFX) or a mixture containing hFIXa + hFX, each at 10ug/ml_. The association was recorded for 10min followed by a dissociation of 10min in running buffer. Finally, biosensors were regenerated by 3 cycles of regeneration buffer (5secs, 10mM glycine pH 1.7) followed by neutralization (5 secs, HNaCT).
  • KL bodies incorporating either one dummy kappa light chain or one dummy lambda light chain that - when combined with the common heavy chain - does not confer binding to either of FIXa or FX were used as negative controls.
  • these dummy KL bodies incorporate only one specificity, either anti-FIXa or anti-FX, on one arm of the bispecific antibody - whereas the other (dummy) arm does not bind to FIXa or FX.
  • Example 8 Calibrated automated thrombography assay in FVIII deficient human plasma
  • Antibodies 1 to 63 according to the present invention were analyzed for a procoagulant activity using a thrombin generation assay (wherein prothrombin is activated to thrombin by FXa, which is an essential reaction in the coagulation pathway).
  • thrombin generation (TG) was evaluated for FVIII deficient patient plasma pool via calibrated automated thrombography (CAT), a method described by Hemker et al (Pathophysiology of Haemostasis and Thrombosis, 2003; 33:4-15). The data generated in this assay, i.e.
  • thrombograms and values derived therefrom such as peak thrombin (see below), describe the concentration of thrombin in clotting plasma and is therefore a functional test of a procoagulant activity.
  • the assay is based on the measurement of fluorescence that is generated by the cleavage of the fluorogenic substrate Z-G-G-R-AMC by thrombin over time.
  • a thrombin calibrator is included to correct for inner filter effects, different coloration of plasma, substrate depletion and instrumental differences.
  • the FXIa-triggered CAT in FVIII deficient human plasma was used for functional characterization of purified bispecific antibodies.
  • 80 pl_ of pre-warmed (37°C) hemophilia A patient platelet-poor plasma pool (George King Bio-Medical Inc., Overland Park, KS, USA) or normal pooled plasma treated with anti-human FVII l-inhibitor plasma from goat (polyclonal, at 150 BU/mL, heat-inactivated; also referred to as “FVIII-inhibited plasma” below) was added to each well of a 96-well micro-plate (Immulon 2HB, U-bottom; Thermo Electron).
  • the plasma was pretreated with 62.5 pg/mL corn trypsin inhibitor (Hematologic Technologies Inc., Essex Junction, VT, USA or Enzyme Research Laboratories, South Bend, IN, USA) to prevent pre-activation of the plasma.
  • 62.5 pg/mL corn trypsin inhibitor Hematologic Technologies Inc., Essex Junction, VT, USA or Enzyme Research Laboratories, South Bend, IN, USA
  • samples were prediluted in sample buffer (25mM Histidine/125 mM NaCI pH6.0), which also served as formulation buffer for all antibodies.
  • the prediluted antibody samples (10 pL) were added, resulting in antibody concentrations in plasma of 10 to 300 nM.
  • a pooled normal human plasma and, as a negative control, hemophilia A plasma pool was used (George King Bio-Medical Inc., Overland Park, KS, USA); FVIII-inhibited plasma (see above) was used as a further negative control.
  • sample buffer was added instead of diluted bispecific antibody.
  • Emicizumab i.e. either Hemlibra or a sequence identical analogue (SIA) thereof was measured at 10-300 nM.
  • SIA is a purified, bispecific, bivalent anti-FIX(a), anti-FX(a) antibody produced in HEK293 cells with identical sequence as Hemlibra, based on the published amino acid sequence (WHO Drug Information Vol. 29, No. 2, 2015 - Proposed International Nonproprietary Names, List 113. World Health Organization). Head-to-head comparison of SIA and Hemlibra showed equivalent activity in in the CAT assay described here.
  • Thrombin generation was triggered via the intrinsic pathway by 5 pL purified plasma-derived human FXIa (Enzyme Research Laboratories, South Bend, IN, USA) and 5 pL of MP reagent (Thrombinoscope BV, Maastricht, The Netherlands) containing a phospholipid mixture composed of phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine (48 pM).
  • a plasma concentration of 500 pM FXIa was used to provide a high sensitivity to FVIII in the assay system.
  • Thrombin generation was started by dispensing 20 pL of FluCa reagent (Thrombinoscope BV, Maastricht, The Netherlands) containing fluorogenic substrate and Hepes buffered CaCL (100 mM) into each well. Fluorescence measurements were performed in a Fluoroskan Ascent® reader (Thermo Labsystems, Helsinki, Finland; filters 390 nm excitation and 460 nm emission) at 37°C for 90 minutes with 20 seconds measurement intervals. The parameters of the resulting TG curves were calculated using the ThrombinoscopeTM software (Thrombinoscope BV, Maastricht, The Netherlands).
  • thrombin calibrator With the thrombin calibrator as a reference, the molar concentration of thrombin in the test wells was derived.
  • bispecific antibodies of the invention have a particularly high procoagulant activity.
  • the bispecific antibodies with antibody numbers 12, 14 to 16, 18, 38 to 43, 45 and 59 to 63 show very high procoagulant activity already at antibody concentrations such as 100 nM, 30 nM and even 10 nM, as reflected in Table 10 above.
  • these bispecific antibodies of the invention outperform emicizumab at these concentrations. This superior property may allow administration of the above-mentioned bispecific antibodies of the invention to a patient at a lower dose, as compared to known bispecific antibodies such as emicizumab.
  • the superior procoagulant activity which these antibodies of the invention display already at relatively low antibody concentrations, as shown above, may allow for longer treatment intervals, as it may be possible to wait for a longer time before the next dose must be administered in order to maintain the therapeutic effect.
  • Example 9 Bispecific antibodies with light chain specificity for factor IXa and X improve thrombin generation in hemophilia A plasma
  • Bispecific antibodies partially mimic the function of activated factor (F)VIII by bridging FIXa and FX. They facilitate FX activation and improve hemostatic potential under hemophilic conditions.
  • the kl body platform enables the development of bsAbs in which the light chains drive specific target binding.
  • the native human IgG architecture of kl bodies is suitable for long-term therapy.
  • Aim To identify bsAbs targeting FIXa and FX that significantly increase thrombin generation in hemophilia A patient plasma.
  • Methods Antibodies against FIX(a) and FX(a) were selected using phage display. To produce kl bodies, one common heavy and two light chains were co-expressed. Functional screening of cell culture supernatants identified numerous hits which were further optimized.
  • Promising bsAbs were purified and subjected to activity testing by a FXIa-triggered thrombin generation (TG) assay in hemophilia A plasma and Octet measurements to assess binding properties.
  • TG FXIa-triggered thrombin generation
  • the bsAbs varied in their specificity, binding capacity, target binding region, and/or their procoagulant effect in the TG assay. For example, TG increased gradually in a bsAb concentration range of 10-300 nM. Some bsAbs reached a maximum of >350 nM peak thrombin. In comparison, a normal plasma control showed peak thrombin of 525 nM.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Diabetes (AREA)
  • Genetics & Genomics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des anticorps bispécifiques qui se fixent au facteur IXa et au facteur X et qui ont une activité procoagulante.
PCT/EP2019/084000 2018-12-07 2019-12-06 Anticorps bispécifiques fixant le facteur ixa et le facteur x WO2020115283A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
PCT/EP2018/084038 WO2020114615A1 (fr) 2018-12-07 2018-12-07 Anticorps bispécifiques se liant au facteur ixa et au facteur x
EPPCT/EP2018/084038 2018-12-07
US201962796233P 2019-01-24 2019-01-24
US62/796,233 2019-01-24

Publications (1)

Publication Number Publication Date
WO2020115283A1 true WO2020115283A1 (fr) 2020-06-11

Family

ID=68848252

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2019/084000 WO2020115283A1 (fr) 2018-12-07 2019-12-06 Anticorps bispécifiques fixant le facteur ixa et le facteur x

Country Status (1)

Country Link
WO (1) WO2020115283A1 (fr)

Citations (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5399363A (en) 1991-01-25 1995-03-21 Eastman Kodak Company Surface modified anticancer nanoparticles
US5439686A (en) 1993-02-22 1995-08-08 Vivorx Pharmaceuticals, Inc. Methods for in vivo delivery of substantially water insoluble pharmacologically active agents and compositions useful therefor
US5466468A (en) 1990-04-03 1995-11-14 Ciba-Geigy Corporation Parenterally administrable liposome formulation comprising synthetic lipids
US5498421A (en) 1993-02-22 1996-03-12 Vivorx Pharmaceuticals, Inc. Composition useful for in vivo delivery of biologics and methods employing same
US6075181A (en) 1990-01-12 2000-06-13 Abgenix, Inc. Human antibodies derived from immunized xenomice
US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
WO2002060919A2 (fr) 2000-12-12 2002-08-08 Medimmune, Inc. Molecules a demi-vies longues, compositions et utilisations de celles-ci
US6528624B1 (en) 1998-04-02 2003-03-04 Genentech, Inc. Polypeptide variants
US20030059474A1 (en) 1999-10-18 2003-03-27 Scott Terrence L. Sustained release microspheres
US20030064033A1 (en) 2001-08-16 2003-04-03 Brown Larry R. Propellant-based microparticle formulations
WO2004003019A2 (fr) * 2002-06-28 2004-01-08 Domantis Limited Ligand
US20040043077A1 (en) 2000-10-27 2004-03-04 Brown Larry R. Production of microspheres
US20050048127A1 (en) 2003-07-22 2005-03-03 Larry Brown Small spherical particles of low molecular weight organic molecules and methods of preparation and use thereof
US20050142206A1 (en) 2003-07-18 2005-06-30 Larry Brown Methods for fabrication, uses and compositions of small spherical particles prepared by controlled phase separation
US20050142205A1 (en) 2003-07-18 2005-06-30 Julia Rashba-Step Methods for encapsulating small spherical particles prepared by controlled phase separation
US20050147689A1 (en) 2003-12-30 2005-07-07 Egilmez Nejat K. Method for inhibiting the growth of gastrointestinal tract tumors
US20060024379A1 (en) 2004-05-12 2006-02-02 Larry Brown Protein microspheres having injectable properties at high concentrations
US20060260777A1 (en) 2005-04-27 2006-11-23 Julia Rashba-Step Surface-modified microparticles and methods of forming and using the same
US20070092452A1 (en) 2003-07-18 2007-04-26 Julia Rashba-Step Methods for fabrication, uses, compositions of inhalable spherical particles
US20070207210A1 (en) 2004-05-12 2007-09-06 Brown Larry R Protein Microspheres Retaining Pharmacokinetic and Pharmacodynamic Properties
US20070281031A1 (en) 2006-06-01 2007-12-06 Guohan Yang Microparticles and methods for production thereof
US20080026068A1 (en) 2001-08-16 2008-01-31 Baxter Healthcare S.A. Pulmonary delivery of spherical insulin microparticles
WO2009080253A1 (fr) 2007-12-21 2009-07-02 F. Hoffmann-La Roche Ag Anticorps bivalents bispécifiques
WO2010135558A1 (fr) 2009-05-20 2010-11-25 Novimmune S.A. Banques de polypeptides synthétiques et procédés de création de variants polypeptidiques naturellement diversifiés
WO2012023053A2 (fr) 2010-08-16 2012-02-23 Novimmune S.A. Procédé de production d'anticorps multispécifiques et multivalents
WO2013088259A2 (fr) * 2011-10-19 2013-06-20 Novimmune S.A. Procédés de purification d'anticorps
WO2016166014A1 (fr) * 2015-04-17 2016-10-20 F. Hoffmann-La Roche Ag Polythérapie avec des facteurs de coagulation et des anticorps polyspécifiques
WO2018098363A2 (fr) * 2016-11-23 2018-05-31 Bioverativ Therapeutics Inc. Anticorps bispécifiques se liant au facteur ix de coagulation et au facteur x de coagulation
WO2018141863A1 (fr) * 2017-02-01 2018-08-09 Novo Nordisk A/S Anticorps procoagulants
WO2018215835A1 (fr) * 2017-05-26 2018-11-29 Novimmune Sa Anticorps anti-cd47 x anti-mésothéline et procédés d'utilisation correspondants

Patent Citations (34)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6075181A (en) 1990-01-12 2000-06-13 Abgenix, Inc. Human antibodies derived from immunized xenomice
US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
US5466468A (en) 1990-04-03 1995-11-14 Ciba-Geigy Corporation Parenterally administrable liposome formulation comprising synthetic lipids
US5399363A (en) 1991-01-25 1995-03-21 Eastman Kodak Company Surface modified anticancer nanoparticles
US5439686A (en) 1993-02-22 1995-08-08 Vivorx Pharmaceuticals, Inc. Methods for in vivo delivery of substantially water insoluble pharmacologically active agents and compositions useful therefor
US5498421A (en) 1993-02-22 1996-03-12 Vivorx Pharmaceuticals, Inc. Composition useful for in vivo delivery of biologics and methods employing same
US6528624B1 (en) 1998-04-02 2003-03-04 Genentech, Inc. Polypeptide variants
US20030059474A1 (en) 1999-10-18 2003-03-27 Scott Terrence L. Sustained release microspheres
US20040043077A1 (en) 2000-10-27 2004-03-04 Brown Larry R. Production of microspheres
WO2002060919A2 (fr) 2000-12-12 2002-08-08 Medimmune, Inc. Molecules a demi-vies longues, compositions et utilisations de celles-ci
US20080026068A1 (en) 2001-08-16 2008-01-31 Baxter Healthcare S.A. Pulmonary delivery of spherical insulin microparticles
US20030064033A1 (en) 2001-08-16 2003-04-03 Brown Larry R. Propellant-based microparticle formulations
WO2004003019A2 (fr) * 2002-06-28 2004-01-08 Domantis Limited Ligand
US20070092452A1 (en) 2003-07-18 2007-04-26 Julia Rashba-Step Methods for fabrication, uses, compositions of inhalable spherical particles
US20050142206A1 (en) 2003-07-18 2005-06-30 Larry Brown Methods for fabrication, uses and compositions of small spherical particles prepared by controlled phase separation
US20050142201A1 (en) 2003-07-18 2005-06-30 Julia Rashba-Step Methods for fabrication, uses and compositions of small spherical particles of hGH prepared by controlled phase separation
US20050142205A1 (en) 2003-07-18 2005-06-30 Julia Rashba-Step Methods for encapsulating small spherical particles prepared by controlled phase separation
US20050170005A1 (en) 2003-07-18 2005-08-04 Julia Rashba-Step Methods for encapsulating small spherical particles prepared by controlled phase separation
US20050233945A1 (en) 2003-07-18 2005-10-20 Larry Brown Methods for fabrication, uses and compositions of small spherical particles of insulin prepared by controlled phase separation
US20050048127A1 (en) 2003-07-22 2005-03-03 Larry Brown Small spherical particles of low molecular weight organic molecules and methods of preparation and use thereof
US20050147689A1 (en) 2003-12-30 2005-07-07 Egilmez Nejat K. Method for inhibiting the growth of gastrointestinal tract tumors
US20070207210A1 (en) 2004-05-12 2007-09-06 Brown Larry R Protein Microspheres Retaining Pharmacokinetic and Pharmacodynamic Properties
US20060024379A1 (en) 2004-05-12 2006-02-02 Larry Brown Protein microspheres having injectable properties at high concentrations
US20060260777A1 (en) 2005-04-27 2006-11-23 Julia Rashba-Step Surface-modified microparticles and methods of forming and using the same
US20070281031A1 (en) 2006-06-01 2007-12-06 Guohan Yang Microparticles and methods for production thereof
WO2009080253A1 (fr) 2007-12-21 2009-07-02 F. Hoffmann-La Roche Ag Anticorps bivalents bispécifiques
WO2010135558A1 (fr) 2009-05-20 2010-11-25 Novimmune S.A. Banques de polypeptides synthétiques et procédés de création de variants polypeptidiques naturellement diversifiés
WO2012023053A2 (fr) 2010-08-16 2012-02-23 Novimmune S.A. Procédé de production d'anticorps multispécifiques et multivalents
US20120184716A1 (en) 2010-08-16 2012-07-19 Novlmmune S.A. Methods for the Generation of Multispecific and Multivalent Antibodies
WO2013088259A2 (fr) * 2011-10-19 2013-06-20 Novimmune S.A. Procédés de purification d'anticorps
WO2016166014A1 (fr) * 2015-04-17 2016-10-20 F. Hoffmann-La Roche Ag Polythérapie avec des facteurs de coagulation et des anticorps polyspécifiques
WO2018098363A2 (fr) * 2016-11-23 2018-05-31 Bioverativ Therapeutics Inc. Anticorps bispécifiques se liant au facteur ix de coagulation et au facteur x de coagulation
WO2018141863A1 (fr) * 2017-02-01 2018-08-09 Novo Nordisk A/S Anticorps procoagulants
WO2018215835A1 (fr) * 2017-05-26 2018-11-29 Novimmune Sa Anticorps anti-cd47 x anti-mésothéline et procédés d'utilisation correspondants

Non-Patent Citations (58)

* Cited by examiner, † Cited by third party
Title
"Antibody Engineering Lab Manual", SPRINGER-VERLAG, article "Protein Sequence and Structure Analysis of Antibody Variable Domains"
"ASHP Handbook on Injectable Drugs", 1986, TOISSEL, pages: 622 - 630
"Guidelines for the Management of Hemohpilia", 6 July 2012, HAEMOPHILIA
"Pharmaceutics and Pharmacy Practice", 1982, J. B. LIPPINCOTT CO., pages: 238 - 250
"Proposed International Nonproprietary Names, List 113", vol. 29, 2015, WORLD HEALTH ORGANIZATION, article "WHO Drug Information"
"Remington's Pharmaceutical Sciences", 1980
"UniProtKB", Database accession no. P00742
AL-LAZIKANI ET AL., JMB, vol. 273, 1997, pages 927 - 948
BAJAJ ET AL., PROC. NATL. ACAD. SEI. USA, vol. 89, 1992, pages 152 - 156
CHOTHIA, C. ET AL., J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
COLE ET AL.: "MONOCLONAL ANTIBODIES AND CANCER THERAPY", 1985, ALAN R. LISS, INC., pages: 77 - 96
COTE ET AL., PROC NATL ACAD SCI USA, vol. 80, 1983, pages 2026 - 2030
DARAMOLA O. ET AL., BIOTECHNOL. PROG., vol. 30, no. 1, 2014, pages 132 - 41
DAVIS JH ET AL., PEDS, vol. 23, 2010, pages 195 - 202
DAY SMREEVE JLMYERS DDFAY WP: "Murine thrombosis models", THROMB HAEMOST., vol. 92, no. 3, 2004, pages 486 - 94
DENISE E. SABATINOTIMOTHY C. NICHOLSELIZABETH MERRICKSDWIGHT A. BELLINGERROLAND W. HERZOGPAUL E. MONAHAN: "Animal Models of Hemophilia", PROG MOL BIOL TRANSL SCI., vol. 105, 2012, pages 151 - 209
ENFIELDTHOMPSON, BLOOD, vol. 64, 1984, pages 821 - 831
FAN L. ET AL., BIOTECHNOL. BIOENG., vol. 109, no. 4, 2012, pages 1007 - 15
FISCHERLEGER, PATHOBIOLOGY, vol. 74, 2007, pages 3 - 14
GLENNIE MJ ET AL., J EXP MED, vol. 175, 1992, pages 217 - 225
GOUDSMIT, J ET AL., J INFECT DIS., vol. 193, 2006, pages 796 - 801
GRAHAM ET AL., J. GEN VIROL., vol. 36, 1977, pages 59
HARLOW ELANE D: "Antibodies: A Laboratory Manual", 1988, COLD SPRING HARBOR LABORATORY PRESS
HELLEN CUSARNOW P, GENES DEV, vol. 15, 2001, pages 1593 - 612
HEMKER ET AL., PATHOPHYSIOLOGY OF HAEMOSTASIS AND THROMBOSIS, vol. 33, 2003, pages 4 - 15
J DRUG TARGET., vol. 22, no. 4, May 2014 (2014-05-01), pages 269 - 78
KABAT, E. A. ET AL.: "Sequences of Proteins of Immunological Interest", 1991, U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES, NIH PUBLICATION
KEMPTONWHITE, BLOOD, vol. 113, no. 1, 2009, pages 11 - 7
KIPRIYANOV SM, METHODS MOL BIOL, vol. 207, 2003, pages 323 - 333
KOHLERMILSTEIN, NATURE, vol. 256, 1975, pages 495
KORN T ET AL., INT J CANCER, vol. 100, 2002, pages 690 - 697
KOZBOR ET AL., IMMUNOL TODAY, vol. 4, 1983, pages 72
LEFRANC ET AL., DEV. COMP. IMMUNOL., vol. 27, 2003, pages 55 - 77
LEFRANC ET AL., DEV. COMP. IMMUNOL., vol. 29, 2005, pages 185 - 203
LEFRANC ET AL., NUCLEIC ACIDS RESEARCH, vol. 43, 2015, pages D413 - D422
LEFRANC, COLD SPRING HARB PROTOC., 1 June 2011 (2011-06-01)
LEFRANC, IMMUNOLOGY TODAY, vol. 18, 1997, pages 509
LEFRANC, THE IMMUNOLOGIST, vol. 7, 1999, pages 132 - 136
LOWY, I ET AL., N ENGL J MED, vol. 362, 2010, pages 197 - 205
LU D ET AL., J BIOL CHEM, vol. 279, 2004, pages 2856 - 2865
LU D ET AL., J BIOL CHEM, vol. 280, 2005, pages 19665 - 19672
LU D ET AL., J IMMUNOL METHODS, vol. 279, 2003, pages 219 - 232
MANCO-JOHNSON ET AL., N. ENGL. J. MED., vol. 357, no. 6, 2007, pages 535 - 44
MATHER ET AL., ANNALS N.Y. ACAD. SCI., vol. 383, 1982, pages 44 - 68
MATHER, BIOL. REPROD., vol. 23, 1980, pages 243 - 251
MATHUR ET AL., BIOL. CHEM., vol. 272, 1997, pages 23418 - 23426
MORRISON SL, NATURE BIOTECHNOL, vol. 25, 2007, pages 1233 - 1234
NAOKI UCHIDA ET AL: "A first-in-human phase 1 study of ACE910, a novel factor VIII-mimetic bispecific antibody, in healthy subjects", BLOOD, vol. 127, 1 December 2015 (2015-12-01), pages 1633 - 1641, XP055442821, DOI: 10.1182/blood-2015-06- *
RIDGWAY JB ET AL., PROTEIN ENG, vol. 9, 1996, pages 617 - 621
STEPHEN ET AL., PROC NATL ACAD SCI USA, vol. 97, 2000, pages 1536 - 1541
STONEBRAKER ET AL., HAEMOPHILIA, vol. 18, no. 3, 2012, pages e91 - 4
STROHL, WR CURR OPIN BIOTECHNOL, vol. 6, 2009, pages 685 - 91
SURESH MR ET AL., METHODS ENZYMOL, vol. 121, 1986, pages 210 - 228
URLAUBCHASIN, PROC. NATL. ACAD. SCI. USA, vol. 77, 1980, pages 4216
VAN DIEIJEN ET AL., J BIOL CHEM., vol. 256, no. 7, 10 April 1981 (1981-04-10), pages 3433 - 42
VAUGHAN ET AL., NAT. BIOTECH., vol. 14, 1996, pages 309 - 314
WHITE ET AL., THROMB. HAEMOST., vol. 85, 2001, pages 560
WU C ET AL., NAT BIOTECHNOL, vol. 25, 2007, pages 1290 - 7

Similar Documents

Publication Publication Date Title
CN110248964B (zh) 促凝血抗体
JP2024170616A (ja) Ｍａｓｐ－２依存性の補体活性化を阻害するための組成物
JP2024026169A (ja) 第ｘｉ因子抗体および使用方法
JP6783754B2 (ja) 組織因子経路インヒビター（１〜１６１）上の２つのエピトープに結合する能力がある抗体
TWI716059B (zh) 經改良的促凝血抗體
CZ2002935A3 (cs) Protilátky a deriváty protilátek k faktoru IX nebo faktoru IXa
CN105209497B (zh) 能够特异性结合组织因子途径抑制物上的两个表位的抗体
TW201605904A (zh) 治療凝血病變之新穎方法及抗體
JP2021502984A (ja) Ｆｘ活性化を促進する第ｘ因子結合剤
CN110023339A (zh) 凝血因子结合蛋白及其应用
EP3109320B1 (fr) Nouvel anticorps anti-pai-1 humain
CN104812402A (zh) 针对活化蛋白C (aPC)的单克隆抗体
WO2020114615A1 (fr) Anticorps bispécifiques se liant au facteur ixa et au facteur x
WO2020115281A1 (fr) Molécules protéiques se liant au facteur ixa et au facteur x
WO2020114614A1 (fr) Molécules protéiques se liant au facteur ixa et au facteur x
CA3118178A1 (fr) Anticorps activateurs du plasminogene tissulaire et procede d'utilisation associe
WO2020115283A1 (fr) Anticorps bispécifiques fixant le facteur ixa et le facteur x
JP6445440B2 (ja) 抗プロテインｃ抗体による出血性疾患の治療
WO2021210667A1 (fr) Molécule de liaison à un antigène bispécifique, composition associée à celle-ci et utilisation, kit et procédé de production de composition
RU2810748C2 (ru) Усовершенствованные прокоагулянтные антитела
RU2810094C2 (ru) Прокоагулянтные антитела
KR20240152428A (ko) 활성화된 단백질 c에 대한 인간 항체 및 이의 용도
BR112021000823B1 (pt) ANTICORPOS PRÓ-COAGULANTES CAPAZES DE ESTIMULAR A ATIVIDADE ENZIMÁTICA DE FIXa EM DIREÇÃO A FX, COMPOSIÇÃO FARMACÊUTICA COMPREENDENDO OS DITOS ANTICORPOS E SEU USO NO TRATAMENTO DA HEMOFILIA A

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19818009

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19818009

Country of ref document: EP

Kind code of ref document: A1