WO2020115281A1 - Proteinaceous molecules binding factor ixa and factor x - Google Patents

Proteinaceous molecules binding factor ixa and factor x Download PDF

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Publication number
WO2020115281A1
WO2020115281A1 PCT/EP2019/083998 EP2019083998W WO2020115281A1 WO 2020115281 A1 WO2020115281 A1 WO 2020115281A1 EP 2019083998 W EP2019083998 W EP 2019083998W WO 2020115281 A1 WO2020115281 A1 WO 2020115281A1
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lcdr1
lcdr2
lcdr3
binding
seq
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PCT/EP2019/083998
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English (en)
French (fr)
Inventor
Fritz Scheiflinger
Randolf Kerschbaumer
Michael Dockal
Patrice DOUILLARD
Sabine KNAPPE
Rudolf Hartmann
Andrea Iskenderian
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Baxalta GmbH
Baxalta Incorporated
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Priority claimed from PCT/EP2018/084037 external-priority patent/WO2020114614A1/en
Application filed by Baxalta GmbH, Baxalta Incorporated filed Critical Baxalta GmbH
Publication of WO2020115281A1 publication Critical patent/WO2020115281A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components

Definitions

  • the present invention provides therapeutics for the treatment and/or prevention of bleeding disorders such as hemophilia A.
  • the present invention provides proteinaceous molecules 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) or 5- ⁇ 40% according to WFH “Guidelines for the Management of Hemohpilia” 2nd edition Haemophilia; Epub 6 JUL 2012. DOI: 10.1111/j.1365- 2516.2012.02909.x.
  • 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.
  • hemophilia A treatment An important goal of hemophilia A treatment is maintenance of FVIII plasma levels >1%, 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 Diagrams of bispecific multivalent proteinaceous molecules.
  • The“3 by 1” topology is characterized by having three antigen-binding sites that bind to Factor IXa and one antigen-binding site that binds to Factor X.
  • The“1 by 3” topology is characterized by having three antigen-binding sites that bind to Factor X and one antigen-binding site that binds to Factor IXa.
  • The“2 by 2” topology is characterized by having two antigen-binding sites that bind to Factor X and two antigen-binding sites that binds to Factor IXa.
  • the antigen-binding sites that bind to Factor IXa and the antigen-binding sites that bind to Factor X may be switched around for the“2 by 2” constructs.
  • the binding modules of the“1 by 3” and “3 by 1” constructs are not limited to being attached to the C-terminus of the heavy chain, but may also be attached to the N-terminus of the heavy chain or the N- or C-terminus of the light chain. All binding modules depicted here are attached to the scaffold module through linkers.
  • FIG. 2 Diagram of trispecific multivalent proteinaceous molecule.
  • a trispecific multivalent proteinaceous molecule comprising a“IXa/X”-type scaffold.
  • the topology of this molecule can be characterized in that the molecule comprises an antigen-binding site that binds to Factor IXa, an antigen-binding site that binds to Factor X, and a third and fourth antigen-binding site that can bind to another target that enhances the procoagulant activity of the molecule.
  • the binding modules labeled“Z” may be any binding module that comprises an antigen-binding site that binds to anything other than Factor IXa or Factor X.
  • binding modules are not limited to being attached to the C-terminus of the heavy chain, but may also be attached to the N-terminus of the heavy chain or the N- or C-terminus of the light chain. All binding modules depicted here are attached to the scaffold module through linkers.
  • FIG. 3 Diagram of trispecific multivalent proteinaceous molecule.
  • a trispecific multivalent proteinaceous molecule comprising the features of a“2 by 2” topology with two additional binding modules comprising an antigen-binding site each.
  • the two additional antigen-binding sites bind to another target that enhances the procoagulant activity of the molecule.
  • the binding modules labeled“Z” may be any binding module that comprises an antigen-binding site that binds to anything other than Factor IXa or Factor X.
  • the antigen-binding sites that bind to Factor IXa and the antigen-binding sites that bind to Factor X may be switched around for any of the constructs depicted in Figure 3. All binding modules depicted here are attached to the scaffold module through linkers.
  • Figure 4 Diagrams of different topologies which are possible and encompassed by the present invention.
  • the binding modules labeled“Z” are optional and may be replaced by any binding module that comprises an antigen-binding site that binds to anything other than Factor IXa or Factor X. Further, the antigen-binding sites that bind to Factor IXa and the antigen-binding sites that bind to Factor X may be switched around for any of the constructs depicted in Figure 4.
  • two binding modules are attached to the N- terminus of the heavy chains through a linker, two binding modules are attached to the C- terminus of the light chains through a linker and two binding modules are attached to the C- terminus of the heavy chains through a linker.
  • Figure 5 Peak thrombin values of different proteinaceous molecules at a concentration of 100 nM. This Figure provides a comparison between a number of constructs that comprise similar antigen-binding site sequences but comprise different linkers.
  • Figure 6 Peak thrombin values of different proteinaceous molecules at a concentration of 100 nM. This Figure provides a comparison between a number of constructs that comprise similar antigen-binding site sequences but comprise different linkers.
  • Figure 7 Peak thrombin values of different proteinaceous molecules at a concentration of 100 nM. This Figure provides a comparison between a number of trispecific constructs that comprise bavituximab.
  • Figure 8 Peak thrombin values of different proteinaceous molecules at a concentration of 100 nM. This Figure provides a comparison between a number of trispecific constructs that comprise domain V of b2 ⁇ IgoorGq ⁇ b ⁇ h I.
  • Figure 9 Peak thrombin values of different proteinaceous molecules at a concentration of 100 nM. This Figure provides a comparison between a number of constructs that are bispecific and multivalent.
  • Figure 10 Peak thrombin values of different proteinaceous molecules at a concentration of 100 nM. This Figure provides a comparison between a number of different constructs with a 2 by 2 topology.
  • FIG 11 Results of the b2 ⁇ IgoorGq ⁇ b ⁇ h I ELISA described in Example 9. These results show that constructs comprising bavituximab are still able to bind domain II of b2- glycoprotein I.
  • Lex#4 is used as a negative control.
  • Lex#4 is a bispecific KL-body that comprises the VL domain of V217, the VL domain of W83 and a VH domain, wherein the VH domain is SEQ ID NO: 4.
  • 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).
  • Figure 12 Results of the Phospholipid ELISA described in Example 9. These results show that constructs comprising domain V of b2 ⁇ IgoorGq ⁇ b ⁇ h I are still able to bind to phosphatidylserine.
  • Lex#4 is used as a control.
  • Lex#4 is a bispecific KL-body that comprises the VL domain of V217, the VL domain of W83 and a VH domain, wherein the VH domain is SEQ ID NO: 4.
  • 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).
  • Figure 13 Results of the GPIIbllla ELISA described in Example 9. These results show that constructs comprising anti-LIBS are still able to bind to GPIIbllla.
  • Lex#1 is used as a negative control.
  • Lex#1 is identical to Lex#36 except that Lex#1 does not comprise a binding module.
  • the present invention provides a proteinaceous molecule comprising (i) a scaffold module comprising a first antigen-binding site and a second antigen-binding site, and (ii) at least a first binding module comprising a third antigen-binding site; wherein at least one of the antigen-binding sites binds to Factor IXa and at least one of the antigen-binding sites binds to Factor X.
  • the remaining antigen-binding site(s) i.e. the ones whose function has not been specified yet
  • a target e.g. phosphatidylserine, a phosphatidylserine-binding protein or a platelet surface marker
  • any of the proteinaceous molecules described herein has a procoagulant activity. Those proteinaceous molecules that have a high procoagulant activity are particularly preferred.
  • the present invention also provides a nucleic acid encoding the proteinaceous molecule of the present invention. Further, the present invention provides a cell (preferably a mammalian cell) comprising a nucleic acid of the present invention and/or a proteinaceous molecule of the present invention.
  • a cell preferably a mammalian cell
  • a method for producing the proteinaceous molecule of the present invention comprising expressing the proteinaceous molecule according to the present invention in a cell and purifying the proteinaceous molecule is also provided by the present invention.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the proteinaceous molecules of the present invention and a pharmaceutically acceptable carrier and/or diluent.
  • the present invention also provides the molecule or pharmaceutical composition of the present invention for use as a medicament. Further, the present invention provides the molecule 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 molecule. A method of treating and/or preventing a bleeding disorder wherein the patient or animal is administered a therapeutically effective amount of the molecule or pharmaceutical composition of the present invention is also provided. Further, the present invention provides the use of the molecule or pharmaceutical composition of the present invention for the manufacture of a medicament for the treatment and/or prevention of a bleeding disorder.
  • affibody refers to a protein that is derived from the Z domain of protein A and that been engineered to bind to a specific target (see Frejd & Kim, 2017. Exp Mol Med. 49(3): e306).
  • 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 target.
  • the term includes whole antibodies and any antigen binding portion or single chains thereof and combinations thereof; for instance, the term “antibody” in particular includes bivalent antibodies and bivalent 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
  • FW framework 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 a binding target, and this region interacting with a binding target 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 antibody 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 bivalent fragments and variations thereof such as a F(ab’)2.
  • antibody encompasses intact polyclonal antibodies, intact monoclonal antibodies, bivalent antibody fragments (such as F(ab')2), multispecific antibodies such as bispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, and any other modified immunoglobulin molecule comprising two antigen binding sites.
  • 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.
  • agent refers to a protein that is derived from the lipocalin and that been engineered to bind to a specific target (see Skerra, 2008. FEBS J. 275(11):2677-83).
  • antibody fragment refers to an antibody fragment comprising one constant and one variable domain of each of the heavy and light chain.
  • a Fab fragment may be obtained by digesting an intact monoclonal antibody with papain.
  • an“antigen-binding site” refers to a region of a scaffold module or binding module that allows for a non-covalent binding interaction with an intended target.
  • An“antigen-binding site” may be, for example, a binding site comprising three complementary determining regions such as in the case of an antibody, scFv or scFab or a binding interface such as the phospholipid-binding interface of 2-glycoprotein I.
  • binding module refers to any substance that binds to Factor IXa, Factor X, or any other target, which may enhance a pro-coagulating activity of the proteinaceous molecule of the present invention in comparison to the scaffold module per se.
  • binding modules include anticalin, repebody, monobody, scFv, Fab, scFab, affibody, fynomer, DARPin, nanobody, peptide aptamer, and nucleic acid aptamter.
  • bispecific molecule refers to a molecule that is able to bind to at least two different targets through two different antigen binding sites.
  • the bispecific molecules of the present invention are able to bind to Factor IXa and Factor X. It follows that a“trispecific molecule” is a molecule that is able to bind to at least three different targets through three different antigen binding sites.
  • the trispecific molecules of the present invention are able to bind to Factor IXa, Factor X and a further target in order to enhance a procoagulant activity.
  • multispecific molecule refers to a molecule that is able to bind more than two different targets through more than two different antigen binding sites.
  • DARPin designed ankyrin repeat proteins
  • 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 in 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. 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 called herein as free 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. Upon cleavage at Arg226, 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.
  • an antigen-binding site of the proteinaceous molecule of the present invention preferentially binds to free Factor IXa over the zymogen of Factor IX.
  • 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 83.
  • 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 is 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”.
  • an antigen-binding site of the proteinaceous molecule of the present invention preferentially binds to the zymogen of Factor X over Factor Xa.
  • the term“fynomer” refers to a protein that is derived from the SH3 domain of human Fyn kinase that has been engineered to bind to a specific target (see Bertschinger et al., 2007. Protein Eng Des Sel. 20(2):57-68).
  • 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.
  • linker refers to at least one atom that forms a covalent bond between two chemical entities.
  • the term“linker” may refer to at least one atom that forms a covalent bond between the scaffold module and another covalent bond to the binding module. If the scaffold module and binding module is linked solely through peptide bonds, the linker is referred to as a “peptide linker”. Otherwise, the linker is referred to as a“chemical linker”.
  • a “flexible peptide linker” comprises mostly small, non-polar or polar amino acids whereas a “rigid peptide linker” comprises alpha-helix forming sequences and/or are rich in proline residues (Chen et al., 2013. Adv Drug Deliv Rev. 65(10): 1357-1369).
  • the term“monobody” refers to a protein that is derived from a fibronectin type III domain that has been engineered to bind to a specific target (see Koide et al., 2013. J Mol Biol. 415(2): 393-405).
  • the term“nanobody” refers to a protein comprising the soluble single antigen-binding V- domain of a heavy chain antibody, preferably a camelid heavy chain antibody (see Bannas et al. , 2017. Front Immunol. 8:1603).
  • nucleic acid aptamer refers to a short synthetic single-stranded oligonucleotide that specifically binds to various molecular targets (see Ni et al., 2011. Curr Med Chem. 18(27) : 4206-4214) .
  • peptide aptamer refers to a short, 5-20 amino acid residue sequence that can bind to a specific target. Peptide aptamers are typically inserted within a loop region of a stable protein scaffold (see Reverdatto et al., 2015. Curr Top Med Chem. 15(12):1082-101 ).
  • phosphatidylserine may refer to both phosphatidyl-L-serine and phosphatidyl-D- serine. In some embodiments, the term“phosphatidylserine” refers to phosphatidyl-L-serine.
  • phosphatidylserine-binding protein refers to a protein that is capable of binding to phosphatidylserine.
  • Non-limiting examples include Protein Z, Tissue Factor, Factor VIII, LOX- 1 , lactadherin, an oxysterol binding protein, Protein C, Protein S, Factor II (prothrombin), Factor V, Factor VII, Factor IX, Factor IXa, Factor X, Factor Xa, Mer, 0 d b3 integrin, the CR3 complement receptor, the CR4 complement receptor, the phosphatidylserine receptor, an annexin, and b2 ⁇ oorGq ⁇ b ⁇ h I (Apo-H).
  • platelet surface marker refers to a molecule that can be found on the surface of a platelet which can be used to co-localize the proteinaceous molecule of the present invention to the surface of the platelet.
  • Non-limiting examples include CD61 , GPIb-IX, CD69, CD40, collagen chaperone HSP47, ephrin B1 , thiol isomerase protein ERP5, Hematopoietic progenitor kinase 1-interacting protein of 55 (HIP-55), glycoprotein VI, platelet glycoprotein 1 b, platelet-derived growth factor receptor, platelet endothelial aggregation receptor I, CD36, CD31 , MARKS, multimerin, integrin alpha llb/beta 3, triggering receptor expressed on myeloid cells (TREM) like transcript-1 (TLT-1), integrin-linked kinase (ILK), zyxin, collagen, P-selectin, Factor XIII, P-selectin glyco
  • 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.
  • 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 (Factor Villa).
  • 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.
  • the present invention provides in-vivo methods and means for identifying a proteinaceous molecule with favorably characteristics typical of a therapeutic drug as well as in-vivo methods for selecting the most qualified proteinaceous molecule and/or the preclinical testing of the therapeutic proteinaceous molecule.
  • 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 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 (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 (Factor VIII), and
  • the rodent is mouse or rat, guinea pig, or hamster.
  • the non-human model can 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.
  • the term“repebody” refers to a protein that is derived from a leucine-rich repeat module and that been engineered to bind to a specific target (see Lee et al., 2012. PNAS. 109(9): 3299- 3304).
  • scaffold module refers to a proteinaceous entity which comprises two antigen binding sites and may act as a support structure for one or more binding modules.
  • the binding modules may be attached to the scaffold module through a linker and/or the binding modules may be incorporated into any loop regions present in the scaffold module.
  • single-chain antigen-binding fragment refers to a fusion protein comprising one variable and one constant domain of the light chain of an antibody attached to one variable and one constant domain of the heavy chain of an antibody, wherein the heavy and light chains are linked together through a short peptide.
  • scFv single-chain variable fragment
  • scFv refers to a fusion protein comprising the variable domains of the heavy chain and light chain of an antibody linked to one another with a peptide linker.
  • the scFv is a disulfide stabilized Fv (dsFv).
  • 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 proteinaceous molecules disclosed herein are at least “trivalent” and may be, for example, “tetravalent” or “hexavalent”.
  • multivalent refers to a molecule that has at least three antigen-binding sites. Proteinaceous molecules of the present invention
  • the present invention provides a proteinaceous molecule comprising (i) a scaffold module comprising a first antigen-binding site and a second antigen-binding site, and (ii) at least a first binding module comprising a third antigen-binding site; wherein at least one of the antigen-binding sites binds to Factor IXa and at least one of the antigen-binding sites binds to Factor X.
  • the remaining antigen-binding site(s) can bind to a target (e.g. phosphatidylserine, a phosphatidylserine-binding protein or a platelet surface marker) in order to enhance procoagulant activity as measured e.g. in a thrombin generation assay and/or to a target associated with a site of injury (e.g. von Willebrand factor (VWF), fibrinogen or collagen) in order to localize the proteinaceous molecule to the site of injury.
  • VWF von Willebrand factor
  • the first binding module is attached to the scaffold module through a linker, or the first binding module is inserted within a loop region of the scaffold module. In some embodiments, the first binding module is attached to the scaffold module through a linker.
  • each individual binding module may be attached to the scaffold module through one or more linkers.
  • the binding modules may be in tandem such as in Figure 4B and 4E.
  • each individual binding module is attached to the scaffold module separately through a linker such as in, for example, Figures 1-3 as well as Figure 4A, C, D and F.
  • the scaffold module is an antibody or a bivalent fragment thereof. In some embodiments, the scaffold module is an IgD, IgE or IgG. In some embodiments, the scaffold module is an IgG or a bivalent fragment thereof. In some embodiments, the scaffold module is an IgG. In some embodiments, IgG refers to lgG1.
  • the scaffold module is an IgG that comprises a mutation that reduces FcyR and C1q binding (see Wang et al., 2018. Protein Cell. 9(1): 63-73).
  • the mutation is a LALA mutation (see Xu et al., 2000. Cell Immunol. 200(1): 16-26).
  • the scaffold module is an IgG that comprises a mutation that improves FcRn-mediated transport in human systems.
  • the scaffold module is an IgG that comprises a mutation that increases its serum half-life in humans (see, for example, the mutations disclosed in WO 02/060919 A2).
  • the mutation is a YTE mutation (see Robbie et al., 2013. Antimicrob Agents Chemother. 57(12):6147-53).
  • the scaffold module is an IgG that comprises a further antigen binding site.
  • the further antigen-binding site binds to phosphatidylserine, a phosphatidylserine-binding protein or a platelet surface marker.
  • the further antigen-binding site may be introduced by modifying loop regions in the CH3 domain (see Wozniak-Knopp et al., 2010. Protein Eng Des Sel. 23(4):289-97).
  • the scaffold modules of the present invention can be divided into two groups:
  • the scaffold module is bispecific and binds to Factor IXa and Factor X;
  • the scaffold module is monospecific and binds to Factor IXa or Factor X.
  • the first antigen-binding site binds to Factor IXa
  • the second antigen-binding site binds to Factor X.
  • each of the first antigen-binding site and the second antigen-binding site binds to Factor IXa, or each of the first antigen-binding site and the second antigen-binding site binds to Factor X.
  • the bispecific scaffold module may be, for example, obtained through chemical crosslinking of two monospecific antibodies, the scaffold module may be a quadroma or any recombinant bispecific antibody disclosed herein.
  • the scaffold module is an antibody or a bivalent fragment thereof that comprises two heavy chains with identical HCDRs.
  • the scaffold module comprises two heavy chains comprising identical HCDRs, wherein HCDR1 is GFTFSSYA (SEQ ID NO: 1), HCDR2 is ISGSGGST (SEQ ID NO: 2) and HCDR3 is AKSYGAFDY (SEQ ID NO: 3).
  • the scaffold module is an antibody or a bivalent fragment thereof that comprises two heavy chains with identical VH domains. In some embodiments, the scaffold module comprises two heavy chains comprising identical VH domains, wherein the VH domain is SEQ ID NO: 4.
  • SEQ ID NO 4 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYY
  • the scaffold module is an antibody or a bivalent fragment thereof that comprises two identical heavy chains. In some embodiments, the scaffold module comprises two identical heavy chains comprising SEQ ID NO: 5.
  • the scaffold module comprises two identical heavy chains comprising SEQ ID NO: 405.
  • the scaffold module comprises two light chains.
  • one light chain variable domain is fused to a Kappa constant domain and the other variable light chain domain is fused to a Lambda constant domain. This allows for the purification of scaffold modules that comprise two antigen binding sites wherein one antigen binding site binds to one target and the other binds to another.
  • the Lambda constant domain comprises SEQ ID NO: 165 and the Kappa constant domain comprises SEQ ID NO: 166.
  • the scaffold module is an antibody or a bivalent fragment thereof that comprises two identical heavy chains as well as two light chains wherein one light chain variable domain is fused to a Kappa constant domain and the other variable light chain domain is fused to a Lambda constant domain (referred to as a “kl-body”).
  • the scaffold module comprises two antigen-binding sites, wherein one antigen binding site binds to Factor IXa and the other antigen-binding site binds to Factor X.
  • the scaffold module is an antibody or a bivalent fragment thereof that comprises:
  • HCDR1 is SEQ ID NO: 1
  • HCDR2 is SEQ ID NO: 2
  • HCDR3 is SEQ ID NO:3;
  • the LCDR1 , LCDR2 and LCDR3 of (ii) is the LCDR1 , LCDR2 and LCDR3 of W83, W88, W128, W127 or W162 disclosed in Table 1
  • the LCDR1 , LCDR2 and LCDR3 of (iii) is the LCDR1 , LCDR2 and LCDR3 of V43, V90, V141 , V149, V198, V202, V204, V212, or V217 disclosed in Table 2a.
  • the LCDR1 , LCDR2 and LCDR 3 of (ii) and (iii) are the LCDR1 , LCDR2 and LCDR3 of W88 and V198, LCDR1 , LCDR2 and LCDR3 of W127 and V202, LCDR1 , LCDR2 and LCDR3 of V149 and W128, LCDR1 , LCDR2 and LCDR3 of W128 and V198, LCDR1 , LCDR2 and LCDR3 of W128 and V141 , LCDR1 , LCDR2 and LCDR3 of W162 and V204, LCDR1 , LCDR2 and LCDR3 of W83 and V217, LCDR1 , LCDR2 and LCDR3 of W88 and V90, or LCDR1 , LCDR2 and LCDR3 of W83 and V43, respectively, as disclosed in Tables 1 and 2a.
  • the scaffold module is an antibody or a bivalent fragment thereof that comprises:
  • HCDR1 is SEQ ID NO: 1
  • HCDR2 is SEQ ID NO: 2
  • HCDR3 is SEQ ID NO:3;
  • the LCDR1 , LCDR2 and LCDR3 of (ii) is the LCDR1 , LCDR2 and LCDR3 of W206, W83, W88, W128, W127 or W162 disclosed in Table 1
  • the LCDR1 , LCDR2 and LCDR3 of (iii) is the LCDR1 , LCDR2 and LCDR3 of V245, V43, V90, V141 , V149, V198, V202, V204, V212, V305, V320 or V217 disclosed in Table 2b.
  • the LCDR1 , LCDR2 and LCDR 3 of (ii) and (iii) are the LCDR1 , LCDR2 and LCDR3 of W88 and V198, LCDR1 , LCDR2 and LCDR3 of W206 and V245, LCDR1 , LCDR2 and LCDR3 of W127 and V202, LCDR1 , LCDR2 and LCDR3 of V149 and W128, LCDR1 , LCDR2 and LCDR3 of W128 and V198, LCDR1 , LCDR2 and LCDR3 of W128 and V141 , LCDR1 , LCDR2 and LCDR3 of W162 and V204, LCDR1 , LCDR2 and LCDR3 of W83 and V217, LCDR1 , LCDR2 and LCDR3 of W88 and V90, or LCDR1 , LCDR2 and LCDR3 of W83 and V43, respectively, as disclosed in Tables 1 and 2b.
  • the scaffold module is an antibody or a bivalent fragment thereof that comprises:
  • HCDR1 is SEQ ID NO: 1
  • HCDR2 is SEQ ID NO: 2
  • HCDR3 is SEQ ID NO:3;
  • the LCDR1 , LCDR2 and LCDR3 of (ii) is the LCDR1 , LCDR2 and LCDR3 of W83 disclosed in Table 1
  • the LCDR1 , LCDR2 and LCDR3 of (iii) is the LCDR1 , LCDR2 and LCDR3 of V43, V90, V141 , V149, V155, V198, V202, V204, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289, V290, V296 or V196 disclosed in Table 2a.
  • the LCDR1 , LCDR2 and LCDR3 of (ii) is the LCDR1 , LCDR2 and LCDR3 of W88 disclosed in Table 1
  • the LCDR1 , LCDR2 and LCDR3 of (iii) is the LCDR1 , LCDR2 and LCDR3 of V43, V90, V141 , V149, V155, V198, V202, V204, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289, V290, V296 or V196 disclosed in Table 2a.
  • the LCDR1, LCDR2 and LCDR3 of (ii) is the LCDR1, LCDR2 and LCDR3 of W122 disclosed in Table 1
  • the LCDR1, LCDR2 and LCDR3 of (iii) is the LCDR1, LCDR2 and LCDR3 of V43, V90, V141, V149, V155, V198, V202, V204, V212, V217, V241, V242, V245, V249, V281, V282, V283, V284, V285, V286, V287, V288, V289, V290, V296 or V196 disclosed in Table 2a.
  • the LCDR1, LCDR2 and LCDR3 of (ii) is the LCDR1, LCDR2 and LCDR3 of W127 disclosed in Table 1
  • the LCDR1, LCDR2 and LCDR3 of (iii) is the LCDR1, LCDR2 and LCDR3 of V43, V90, V141, V149, V155, V198, V202, V204, V212, V217, V241, V242, V245, V249, V281, V282, V283, V284, V285, V286, V287, V288, V289, V290, V296 or V196 disclosed in Table 2a.
  • the LCDR1, LCDR2 and LCDR3 of (ii) is the LCDR1, LCDR2 and LCDR3 of W128 disclosed in Table 1
  • the LCDR1, LCDR2 and LCDR3 of (iii) is the LCDR1, LCDR2 and LCDR3 of V43, V90, V141 , V149, V155, V198, V202, V204, V212, V217, V241, V242, V245, V249, V281, V282, V283, V284, V285, V286, V287, V288, V289, V290, V296 or V196 disclosed in Table 2a.
  • the LCDR1, LCDR2 and LCDR3 of (ii) is the LCDR1, LCDR2 and LCDR3 of W133 disclosed in Table 1
  • the LCDR1, LCDR2 and LCDR3 of (iii) is the LCDR1, LCDR2 and LCDR3 of V43, V90, V141, V149, V155, V198, V202, V204,
  • the LCDR1, LCDR2 and LCDR3 of (ii) is the LCDR1, LCDR2 and LCDR3 of W159 disclosed in Table 1
  • the LCDR1, LCDR2 and LCDR3 of (iii) is the LCDR1, LCDR2 and LCDR3 of V43, V90, V141, V149, V155, V198, V202, V204, V212, V217, V241, V242, V245, V249, V281, V282,
  • the LCDR1, LCDR2 and LCDR3 of (ii) is the LCDR1, LCDR2 and LCDR3 of W162 disclosed in Table 1
  • the LCDR1, LCDR2 and LCDR3 of (iii) is the LCDR1, LCDR2 and LCDR3 of V43, V90, V141, V149, V155, V198, V202, V204, V212, V217, V241 , V242, V245, V249, V281, V282, V283, V284, V285, V286, V287, V288, V289, V290, V296 or V196 disclosed in Table 2a.
  • the LCDR1, LCDR2 and LCDR3 of (ii) is the LCDR1, LCDR2 and LCDR3 of W189 disclosed in Table 1
  • the LCDR1, LCDR2 and LCDR3 of (iii) is the LCDR1, LCDR2 and LCDR3 of V43, V90, V141, V149, V155, V198, V202, V204, V212, V217, V241, V242, V245, V249, V281, V282, V283, V284, V285, V286, V287, V288, V289, V290, V296 or V196 disclosed in Table 2a.
  • the LCDR1, LCDR2 and LCDR3 of (ii) is the LCDR1, LCDR2 and LCDR3 of W198 disclosed in Table 1
  • the LCDR1, LCDR2 and LCDR3 of (iii) is the LCDR1, LCDR2 and LCDR3 of V43, V90, V141, V149, V155, V198, V202, V204, V212, V217, V241, V242, V245, V249, V281, V282, V283, V284, V285, V286, V287, V288, V289, V290, V296 or V196 disclosed in Table 2a.
  • the LCDR1, LCDR2 and LCDR3 of (ii) is the LCDR1, LCDR2 and LCDR3 of W204 disclosed in Table 1
  • the LCDR1, LCDR2 and LCDR3 of (iii) is the LCDR1 , LCDR2 and LCDR3 of V43, V90, V141 , V149, V155, V198, V202, V204, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289, V290, V296 or V196 disclosed in Table 2a.
  • the LCDR1 , LCDR2 and LCDR3 of (ii) is the LCDR1 , LCDR2 and LCDR3 of W206 disclosed in Table 1
  • the LCDR1 , LCDR2 and LCDR3 of (iii) is the LCDR1 , LCDR2 and LCDR3 of V43, V90, V141 , V149, V155, V198, V202, V204, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289, V290, V296 or V196 disclosed in Table 2a.
  • the LCDR1 , LCDR2 and LCDR3 of (ii) is the LCDR1 , LCDR2 and LCDR3 of W207 disclosed in Table 1
  • the LCDR1 , LCDR2 and LCDR3 of (iii) is the LCDR1 , LCDR2 and LCDR3 of V43, V90, V141 , V149, V155, V198, V202, V204,
  • the LCDR1 , LCDR2 and LCDR3 of (ii) is the LCDR1 , LCDR2 and LCDR3 of W140 disclosed in Table 1
  • the LCDR1 , LCDR2 and LCDR3 of (iii) is the LCDR1 , LCDR2 and LCDR3 of V43, V90, V141 , V149, V155, V198, V202, V204, V212, V217, V241 , V242, V245, V249, V281 , V282,
  • the LCDR1 , LCDR2 and LCDR3 of (ii) is the LCDR1 , LCDR2 and LCDR3 of W196 disclosed in Table 1
  • the LCDR1 , LCDR2 and LCDR3 of (iii) is the LCDR1 , LCDR2 and LCDR3 of V43, V90, V141 , V149, V155, V198, V202, V204, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289, V290, V296 or V196 disclosed in Table 2a.
  • the scaffold module is an antibody or a bivalent fragment thereof that comprises:
  • HCDR1 is SEQ ID NO: 1
  • HCDR2 is SEQ ID NO: 2
  • HCDR3 is SEQ ID NO:3;
  • the LCDR1 , LCDR2 and LCDR3 of (ii) is the LCDR1 , LCDR2 and LCDR3 of W83 disclosed in Table 1
  • the LCDR1 , LCDR2 and LCDR3 of (iii) is the LCDR1 , LCDR2 and LCDR3 of V43, V245, V320, V305, V90, V141 , V149, V155, V198, V202, V204, V212, V217, V241 , V242, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289, V290, V296 or V196 disclosed in Table 2b.
  • the LCDR1 , LCDR2 and LCDR3 of (ii) is the LCDR1 , LCDR2 and LCDR3 of W88 disclosed in Table 1
  • the LCDR1 , LCDR2 and LCDR3 of (iii) is the LCDR1 , LCDR2 and LCDR3 of V43, V245, V320, V305, V90, V141, V149, V155, V198, V202, V204, V212, V217, V241, V242, V249, V281, V282, V283, V284, V285, V286, V287, V288, V289, V290, V296 or V196 disclosed in Table 2b.
  • the LCDR1, LCDR2 and LCDR3 of (ii) is the LCDR1, LCDR2 and LCDR3 of W122 disclosed in Table 1
  • the LCDR1, LCDR2 and LCDR3 of (iii) is the LCDR1, LCDR2 and LCDR3 of V43, V245, V320, V305, V90, V141, V149, V155, V198, V202, V204, V212, V217, V241, V242, V249, V281, V282, V283, V284, V285, V286, V287, V288, V289, V290, V296 or V196 disclosed in Table 2b.
  • the LCDR1, LCDR2 and LCDR3 of (ii) is the LCDR1, LCDR2 and LCDR3 of W127 disclosed in Table 1
  • the LCDR1, LCDR2 and LCDR3 of (iii) is the LCDR1, LCDR2 and LCDR3 of V43, V245, V320, V305, V90, V141, V149, V155, V198, V202, V204, V212, V217, V241, V242, V249, V281, V282, V283, V284, V285, V286, V287, V288, V289, V290, V296 or V196 disclosed in Table 2b.
  • the LCDR1, LCDR2 and LCDR3 of (ii) is the LCDR1, LCDR2 and LCDR3 of W128 disclosed in Table 1
  • the LCDR1, LCDR2 and LCDR3 of (iii) is the LCDR1, LCDR2 and LCDR3 of V43, V245, V320, V305, V90, V141, V149, V155, V198, V202, V204, V212, V217, V241, V242, V249, V281, V282, V283, V284, V285, V286, V287, V288, V289, V290, V296 or V196 disclosed in Table 2b.
  • the LCDR1, LCDR2 and LCDR3 of (ii) is the LCDR1, LCDR2 and LCDR3 of W133 disclosed in Table 1
  • the LCDR1, LCDR2 and LCDR3 of (iii) is the LCDR1, LCDR2 and LCDR3 of V43, V245, V320, V305, V90, V141, V149, V155, V198, V202, V204, V212, V217, V241 , V242, V249, V281, V282, V283, V284, V285, V286, V287, V288, V289, V290, V296 or V196 disclosed in Table 2b.
  • the LCDR1, LCDR2 and LCDR3 of (ii) is the LCDR1, LCDR2 and LCDR3 of W159 disclosed in Table 1
  • the LCDR1, LCDR2 and LCDR3 of (iii) is the LCDR1, LCDR2 and LCDR3 of V43, V245, V320, V305, V90, V141, V149, V155, V198, V202, V204, V212, V217, V241, V242, V249, V281, V282, V283, V284, V285, V286, V287, V288, V289, V290, V296 or V196 disclosed in Table 2b.
  • the LCDR1, LCDR2 and LCDR3 of (ii) is the LCDR1, LCDR2 and LCDR3 of W162 disclosed in Table 1
  • the LCDR1, LCDR2 and LCDR3 of (iii) is the LCDR1, LCDR2 and LCDR3 of V43, V245, V320, V305, V90, V141, V149, V155, V198, V202, V204, V212, V217, V241, V242, V249, V281, V282, V283, V284, V285, V286, V287, V288, V289, V290, V296 or V196 disclosed in Table 2b.
  • the LCDR1, LCDR2 and LCDR3 of (ii) is the LCDR1, LCDR2 and LCDR3 of W189 disclosed in Table 1
  • the LCDR1, LCDR2 and LCDR3 of (iii) is the LCDR1, LCDR2 and LCDR3 of V43, V245, V320, V305, V90, V141, V149, V155, V198, V202, V204, V212, V217, V241, V242, V249, V281, V282, V283, V284, V285, V286, V287, V288, V289, V290, V296 or V196 disclosed in Table 2b.
  • the LCDR1, LCDR2 and LCDR3 of (ii) is the LCDR1, LCDR2 and LCDR3 of W198 disclosed in Table 1
  • the LCDR1, LCDR2 and LCDR3 of (iii) is the LCDR1, LCDR2 and LCDR3 of V43, V245, V320, V305, V90, V141, V149, V155, V198, V202, V204, V212, V217, V241 , V242, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289, V290, V296 or V196 disclosed in Table 2b.
  • the LCDR1 , LCDR2 and LCDR3 of (ii) is the LCDR1 , LCDR2 and LCDR3 of W204 disclosed in Table 1
  • the LCDR1 , LCDR2 and LCDR3 of (iii) is the LCDR1 , LCDR2 and LCDR3 of V43, V245, V320, V305, V90, V141 , V149, V155, V198, V202, V204, V212, V217, V241 , V242, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289, V290, V296 or V196 disclosed in Table 2b.
  • the LCDR1 , LCDR2 and LCDR3 of (ii) is the LCDR1 , LCDR2 and LCDR3 of W206 disclosed in Table 1
  • the LCDR1 , LCDR2 and LCDR3 of (iii) is the LCDR1 , LCDR2 and LCDR3 of V43, V245, V320, V305, V90, V141 , V149, V155, V198, V202, V204, V212, V217, V241 , V242, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289, V290, V296 or V196 disclosed in Table 2b.
  • the LCDR1 , LCDR2 and LCDR3 of (ii) is the LCDR1 , LCDR2 and LCDR3 of W207 disclosed in Table 1
  • the LCDR1 , LCDR2 and LCDR3 of (iii) is the LCDR1 , LCDR2 and LCDR3 of V43, V245, V320, V305, V90, V141 , V149, V155, V198, V202, V204, V212, V217, V241 , V242, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289, V290, V296 or V196 disclosed in Table 2b.
  • the LCDR1 , LCDR2 and LCDR3 of (ii) is the LCDR1 , LCDR2 and LCDR3 of W140 disclosed in Table 1
  • the LCDR1 , LCDR2 and LCDR3 of (iii) is the LCDR1 , LCDR2 and LCDR3 of V43, V245, V320, V305, V90, V141 , V149, V155, V198, V202, V204, V212, V217, V241 , V242, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289, V290, V296 or V196 disclosed in Table 2b.
  • the LCDR1 , LCDR2 and LCDR3 of (ii) is the LCDR1 , LCDR2 and LCDR3 of W196 disclosed in Table 1
  • the LCDR1 , LCDR2 and LCDR3 of (iii) is the LCDR1 , LCDR2 and LCDR3 of V43, V320, V305, V90, V141 , V149, V155, V198, V202, V204, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289, V290, V296 or V196 disclosed in Table 2b.
  • the LCDR1 , LCDR2 and LCDR3 of (ii) is the LCDR1 , LCDR2 and LCDR3 of W206 disclosed in Table 1
  • the LCDR1 , LCDR2 and LCDR3 of (iii) is the LCDR1 , LCDR2 and LCDR3 of V43, V320, V305, V90, V141 , V149, V155, V198, V202, V204, V212, V217, V241 , V242, V245, V249, V281 , V282, V283, V284, V285, V286, V287, V288, V289, V290, V296 or V196 disclosed in Table 2b.
  • the scaffold module is an antibody or a bivalent fragment thereof that comprises:
  • VH domain is SEQ ID NO:
  • the VL of (ii) is the VL of W83, VL of W88, VL of W128, VL of W127, or VL of W162 disclosed in Table 3
  • the VL of (iii) is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V198, VL of V202, VL of V204, VL of V212, or VL of V217 disclosed in Table 4a.
  • the VL of (ii) and (iii) are the VL of W88 and V198, VL of W127 and V202, VL of V149 and W128, VL of W128 and V198, VL of W128 and V141 , VL of W162 and V204, VL of W83 and V217, VL of W88 and V90, or VL of W83 and V43, respectively, disclosed in Tables 3 and 4a.
  • the scaffold module is an antibody or a bivalent fragment thereof that comprises:
  • VH domain is SEQ ID NO: 4;
  • the VL of (ii) is the VL of W206, VL of W83, VL of W88, VL of W128, VL of W127, or VL of W162 disclosed in Table 3, and the VL of (iii) is the VL of V43, VL of V245, VL of V305, VL of V320, VL of V90, VL of V141 , VL of V149, VL of V198, VL of V202, VL of V204, VL of V212, or VL of V217 disclosed in Table 4b.
  • the VL of (ii) and (iii) are the VL of W206 and V245, VL of W88 and V198, VL of W127 and V202, VL of V149 and W128, VL of W128 and V198, VL of W128 and V141 , VL of W162 and V204, VL of W83 and V217, VL of W88 and V90, or VL of W83 and V43, respectively, disclosed in Tables 3 and 4b.
  • the scaffold module is an antibody or a bivalent fragment thereof that comprises:
  • VH domain is SEQ ID NO: 4;
  • the VL of (ii) is the VL of W83 disclosed in Table 3, and the VL of (iii) is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4a.
  • the VL of (ii) is the VL of W88 disclosed in Table 3, and the VL of (iii) is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4a.
  • the VL of (ii) is the VL of W122 disclosed in Table 3, and the VL of (iii) is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4a.
  • the VL of (ii) is the VL of W127 disclosed in Table 3, and the VL of (iii) is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4a.
  • the VL of (ii) is the VL of W128 disclosed in Table 3, and the VL of (iii) is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4a.
  • the VL of (ii) is the VL of W133 disclosed in Table 3, and the VL of (iii) is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4a.
  • the VL of (ii) is the VL of W159 disclosed in Table 3, and the VL of (iii) is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4a.
  • the VL of (ii) is the VL of W162 disclosed in Table 3, and the VL of (iii) is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4a.
  • the VL of (ii) is the VL of W189 disclosed in Table 3, and the VL of (iii) is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4a.
  • the VL of (ii) is the VL of W198 disclosed in Table 3, and the VL of (iii) is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4a.
  • the VL of (ii) is the VL of W204 disclosed in Table 3, and the VL of (iii) is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4a.
  • the VL of (ii) is the VL of W206 disclosed in Table 3, and the VL of (iii) is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4a.
  • the VL of (ii) is the VL of W207 disclosed in Table 3, and the VL of (iii) is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4a.
  • the VL of (ii) is the VL of W140 disclosed in Table 3, and the VL of (iii) is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4a.
  • the VL of (ii) is the VL of W196 disclosed in Table 3, and the VL of (iii) is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4a.
  • the scaffold module is an antibody or a bivalent fragment thereof that comprises:
  • VH domain is SEQ ID NO:
  • the VL of (ii) is the VL of W83 disclosed in Table 3, and the VL of (iii) is the VL of V320, VL of V305, VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4b.
  • the VL of (ii) is the VL of W88 disclosed in Table 3, and the VL of (iii) is the VL of V320, VL of V305, VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4b.
  • the VL of (ii) is the VL of W122 disclosed in Table 3, and the VL of (iii) is the VL of V320, VL of V305, VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4b.
  • the VL of (ii) is the VL of W127 disclosed in Table 3, and the VL of (iii) is the VL of V320, VL of V305, VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4b.
  • the VL of (ii) is the VL of W128 disclosed in Table 3, and the VL of (iii) is the VL of V320, VL of V305, VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4b.
  • the VL of (ii) is the VL of W133 disclosed in Table 3, and the VL of (iii) is the VL of V320, VL of V305, VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4b.
  • the VL of (ii) is the VL of W159 disclosed in Table 3, and the VL of (iii) is the VL of V320, VL of V305, VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4b.
  • the VL of (ii) is the VL of W162 disclosed in Table 3, and the VL of (iii) is the VL of V320, VL of V305, VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4b.
  • the VL of (ii) is the VL of W189 disclosed in Table 3, and the VL of (iii) is the VL of V320, VL of V305, VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4b.
  • the VL of (ii) is the VL of W198 disclosed in Table 3, and the VL of (iii) is the VL of V320, VL of V305, VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4b.
  • the VL of (ii) is the VL of W204 disclosed in Table 3, and the VL of (iii) is the VL of V320, VL of V305, VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4b.
  • the VL of (ii) is the VL of W206 disclosed in Table 3, and the VL of (iii) is the VL of V320, VL of V305, VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4b.
  • the VL of (ii) is the VL of W207 disclosed in Table 3, and the VL of (iii) is the VL of V320, VL of V305, VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4b.
  • the VL of (ii) is the VL of WHO disclosed in Table 3, and the VL of (iii) is the VL of V320, VL of V305, VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4b.
  • the VL of (ii) is the VL of W196 disclosed in Table 3, and the VL of (iii) is the VL of V320, VL of V305, VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4b.
  • the VL of (ii) is the VL of W206 disclosed in Table 3, and the VL of (iii) is the VL of V320, VL of V305, VL of V43, VL of V90, VL of V141 , VL of V149, VL of V155, VL of V198, VL of V202, VL of V204, VL of V212, VL of V217, VL of V241 , VL of V242, VL of V245, VL of V249, VL of V281 , VL of V282, VL of V283, VL of V284, VL of V285, VL of V286, VL of V287, VL of V288, VL of V289, VL of V290, VL of V296 or VL of V196 disclosed in Table 4b.
  • the monospecific scaffold module may comprise two identical light chains and two identical heavy chains. This allows for the expression of a single symmetrical molecular entity to be affinity purified from a culture media as one would with a monoclonal antibody.
  • the scaffold module is an antibody or bivalent fragment thereof that comprises:
  • HCDR1 is SEQ ID NO: 1
  • HCDR2 is SEQ ID NO: 2
  • HCDR3 is SEQ ID NO:3;
  • the LCDR1 , LCDR2 and LCDR3 of each of the two light chains is the LCDR1 , LCDR2 and LCDR3 of W83, LCDR1 , LCDR2 and LCDR3 of W88, LCDR1 , LCDR2 and LCDR3 of W128, LCDR1 , LCDR2 and LCDR3 of W127, or LCDR1 , LCDR2 and LCDR3 of W162 disclosed in Table 1 , or the LCDR1 , LCDR2 and LCDR3 of each of the two light chains is the LCDR1 , LCDR2 and LCDR3 of V43, LCDR1 , LCDR2 and LCDR3 of V90, LCDR1 , LCDR2 and LCDR3 of V141 , LCDR1 , LCDR2 and LCDR3
  • the scaffold module is an antibody or bivalent fragment thereof that comprises:
  • HCDR1 is SEQ ID NO: 1
  • HCDR2 is SEQ ID NO: 2
  • HCDR3 is SEQ ID NO:3
  • two light chains each comprising the LCDR1 , LCDR2 and LCDR3 of any one of the Factor X binding light chains disclosed in Table 1 , or the Factor IXa binding light chains disclosed in Table 2b.
  • the LCDR1 , LCDR2 and LCDR3 of each of the two light chains is the LCDR1 , LCDR2 and LCDR3 of W206, LCDR1 , LCDR2 and LCDR3 of W83, LCDR1 , LCDR2 and LCDR3 of W88, LCDR1 , LCDR2 and LCDR3 of W128, LCDR1 , LCDR2 and LCDR3 of W127, or LCDR1 , LCDR2 and LCDR3 of W162 disclosed in Table 1 , or the LCDR1 , LCDR2 and LCDR3 of each of the two light chains is the LCDR1 , LCDR2 and LCDR3 of V43, LCDR1 , LCDR2 and LCDR3 of V90, LCDR1 , LCDR2 and LCDR3 of V141 , LCDR1 , LCDR2 and LCDR3 of V149, LCDR1 , LCDR2 and LCDR3 of V198, LCDR1 , LCDR2 and LCDR3 of V320, LCDR1 ,
  • the scaffold module is an antibody or a bivalent fragment thereof that comprises:
  • VH domain is SEQ ID NO: 4;
  • the VL of each of the two light chains is the VL of W83, VL of W88, VL of W128, VL of W127, or VL of W162 disclosed in Table 3, or the VL of each of the two light chains is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V198, VL of V202, VL of V204, VL of V212, or VL of V217 disclosed in Table 4a.
  • the scaffold module is an antibody or a bivalent fragment thereof that comprises:
  • VH domain is SEQ ID NO: 4;
  • the VL of each of the two light chains is the VL of W206, VL of W83, VL of W88, VL of W128, VL of W127, or VL of W162 disclosed in Table 3, or the VL of each of the two light chains is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V320, VL of V305, VL of V155, VL of V245, VL of V198, VL of V202, VL of V204, VL of V212, or VL of V217 disclosed in Table 4b.
  • Table 1 LCDRs of Factor X binding light chains
  • the scaffold module is an antibody or a bivalent fragment thereof that comprises a modified hinge domain wherein a linker is inserted between the CH1 and CH2 domain of the antibody.
  • the scaffold module comprises two identical heavy chains comprising SEQ ID NO: 5 wherein a linker is inserted between residue C219 and D220 of SEQ ID NO: 5.
  • the scaffold module comprises two identical heavy chains comprising SEQ ID NO: 5 wherein a flexible linker is inserted between residue C219 and D220 of SEQ ID NO: 5.
  • the scaffold module comprises two identical heavy chains comprising SEQ ID NO: 5 wherein 1 , 2, 3 or 4 repeats of GGGGS (SEQ ID NO: 6) or GSAGSAAGSGEF (SEQ ID NO: 7) is inserted between residue C219 and D220 of SEQ ID NO: 5.
  • the scaffold module comprises two identical heavy chains comprising SEQ ID NO: 5 wherein GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 8) or GSAGSAAGSGEF (SEQ ID NO: 7) is inserted between residue C219 and D220 of SEQ ID NO: 5.
  • the first and, optionally, any further binding module is attached to the scaffold module.
  • Any linker can be employed to attach the one or more binding module(s) to the scaffold module. The skilled person is aware of a number of different linkers which could be used.
  • the linker is a chemical linker or a peptide linker some embodiments, the linker is a peptide linker.
  • peptide linkers can be classified as flexible or rigid depending on the sequence and structure of the linker.
  • at least one or all of the linker(s) is or are a flexible peptide linker.
  • flexible linkers provide an advantage in a thrombin generation assay in comparison to more rigid linkers.
  • the linker is cleavable.
  • the linker may comprise a disulfide bond or a protease cleavage site.
  • the linker comprises a thrombin or Factor Xa cleavage site.
  • the linker is selected from Table 5. In some embodiments, the linker is a flexible linker selected from Table 5.
  • the linker comprises SEQ ID NO: 8.
  • the linker comprises or consists of SEQ ID NO: 168 or SEQ ID NO: 169. This may be preferable because a short linker which cannot be post-translationally modified during protein expression would result in a reduced heterogeneity and improve the developability of the proteinaceous molecule as a drug.
  • the proteinaceous molecules of the present invention may comprise one or more binding modules.
  • the binding modules may increase the number of antigen-binding sites on the proteinaceous molecule that bind to Factor IXa/Factor X, or they may add a further specificity making the proteinaceous molecule multispecific, e.g. trispecific.
  • the antigen-binding site of the binding module binds to Protein Z, Tissue Factor, Factor VIII, LOX-1 , lactadherin, an oxysterol binding protein, Protein C, Protein S, Factor II (prothrombin), Factor V, Factor VII, Factor IX, Factor IXa, Factor X, Factor Xa, Mer, a5b3 integrin, the CR3 complement receptor, the CR4 complement receptor, the phosphatidylserine receptor, an annexin, b2-9 ⁇ goorGq ⁇ b ⁇ h I, CD61 , GPIb-IX, CD69, CD40, collagen chaperone HSP47, ephrin B1 , thiol isomerase protein ERP5, Hematopoietic progenitor kinase 1-interacting protein of 55 (HIP-55), glycoprotein VI, platelet glycoprotein 1 b, platelet-derived growth factor receptor, platelet endothelial
  • the antigen-binding site of the binding module binds to phosphatidylserine, a phosphatidylserine-binding protein or a platelet surface marker.
  • the phosphatidylserine-binding protein is Protein C, Protein Z, Protein S, Tissue Factor, Factor II (prothrombin), Factor V, Factor VII, Factor VIII, Factor IX, Factor IXa, Factor X, Factor Xa, Mer, LOX-1 , 0 d b3 integrin, lactadherin, the CR3 complement receptor, the CR4 complement receptor, the phosphatidylserine receptor, an oxysterol binding protein, an annexin (preferably annexin V) or b2-9 ⁇ goorGq ⁇ b ⁇ h I.
  • annexin preferably annexin V
  • the binding module binds to a platelet surface marker, wherein the platelet surface marker is CD61 , GPIb-IX, CD69, CD40, collagen chaperone HSP47, ephrin B1 , thiol isomerase protein ERP5, Hematopoietic progenitor kinase 1-interacting protein of 55 (HIP-55), glycoprotein VI, platelet glycoprotein 1b, platelet-derived growth factor receptor, platelet endothelial aggregation receptor I, CD36, CD31 , MARKS, multimerin, integrin alpha llb/beta 3, triggering receptor expressed on myeloid cells (TREM) like transcript-1 (TLT-1), integrin-linked kinase (ILK), zyxin, collagen, P-selectin, Factor XIII, P-selectin glycoprotein ligand-1 , integrin alpha 6 beta 1 , thrombospondin, von Willebrand factor, G6B
  • the binding module may be linked to the scaffold module through a linker or may be inserted into a loop region present in the scaffold module.
  • the scaffold module is an IgG or bivalent fragment thereof and the binding module is attached to the C- or N- terminus of a light or heavy chain.
  • the binding module is inserted between the CH1 and CH2 domain, i.e. around the hinge region.
  • the scaffold module is an antibody or bivalent fragment thereof comprising a heavy and a light chain and the binding module is attached to the C- or N-terminus of the light or heavy chain.
  • the scaffold module is an antibody or bivalent fragment thereof comprising two heavy and two light chains and the binding module is attached to the C- or N- terminus of a light or heavy chain.
  • the antigen-binding site of the binding module binds to Factor IXa or Factor X.
  • the binding module is an anticalin, repebody, monobody, scFv, Fab, scFab, affibody, fynomer, DARPin, nanobody, peptide aptamer, or nucleic acid aptamter.
  • the binding module is a scFv or scFab.
  • the binding module is a scFv.
  • the binding module is a scFv or scFab that comprises (i) an LCDR1 , LCDR2, and LCDR3 of any one of the Factor X binding light chains disclosed in Table 1 , or the Factor IXa binding light chains disclosed in Table 2a, and (ii) an HCDR1 , HCDR2, and HCDR3, wherein HCDR1 is SEQ ID NO: 1 , HCDR2 is SEQ ID NO: 2 and HCDR3 is SEQ ID NO: 3.
  • the LCDR1 , LCDR2 and LCDR3 are that of V198, V202, W128, W88, W127, V212, or W162 disclosed in Tables 1 and 2a.
  • the binding module is a scFv or scFab that comprises (i) an LCDR1 , LCDR2, and LCDR3 of any one of the Factor X binding light chains disclosed in Table 1 , or the Factor IXa binding light chains disclosed in Table 2b, and (ii) an HCDR1 , HCDR2, and HCDR3, wherein HCDR1 is SEQ ID NO: 1 , HCDR2 is SEQ ID NO: 2 and HCDR3 is SEQ ID NO: 3.
  • the LCDR1 , LCDR2 and LCDR3 are that of V198, W206, W198, V202, W128, W88, W127, V212, or W162 disclosed in Tables 1 and 2b.
  • the binding module is a scFv or scFab that comprises (i) the VL of any one of the Factor X binding light chains disclosed in Table 3 or the Factor IXa binding light chains disclosed in Table 4a, and (ii) SEQ ID NO: 4 (VH domain).
  • (i) is the VL of V198, V202, W128, W88, W127, V212 or W162 of Tables 3 and 4a.
  • the binding module is a scFv or scFab that comprises (i) the VL of any one of the Factor X binding light chains disclosed in Table 3 or the Factor IXa binding light chains disclosed in Table 4b, and (ii) SEQ ID NO: 4 (VH domain).
  • (i) is the VL of V198, W206, W198, V202, W128, W88, W127, V212, or W162 of Tables 3 and 4b.
  • the binding module is a scFv that comprises (i) a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of any one of the Factor X binding light chains disclosed in Table 1 , or the Factor IXa binding light chains disclosed in Table 2a, and wherein FW1 is SYVLTQPPSVSVAPGKTARITCGGD (SEQ ID NO: 179), FW2 is VHWYQQKPGQAPVLVIY (SEQ ID NO: 180), FW3 is
  • the LCDR1 , LCDR2 and LCDR3 are that of V198, V202, W128, W88, W127, V212, or W162 disclosed in Tables 1 and 2a.
  • the binding module is a scFv that comprises (i) a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of any one of the Factor X binding light chains disclosed in Table 1 , or the Factor IXa binding light chains disclosed in Table 2b, and wherein FW1 is SYVLTQPPSVSVAPGKTARITCGGD (SEQ ID NO: 179), FW2 is VHWYQQKPGQAPVLVIY (SEQ ID NO: 180), FW3 is
  • the LCDR1 , LCDR2 and LCDR3 are that of V198, W206, W198, V202, W128, W88, W127, V212, or W162 disclosed in Tables 1 and 2b.
  • the binding module binds to phosphatidylserine, a platelet surface marker, or a phosphatidylserine-binding protein, wherein the phosphatidylserine-binding protein is Protein Z, Tissue Factor, Factor VIII, LOX-1 , lactadherin, an oxysterol binding protein, Protein C, Protein S, Factor II (prothrombin), Factor V, Factor VII, Mer, 0 d b3 integrin, the CR3 complement receptor, the CR4 complement receptor, the phosphatidylserine receptor, an annexin (preferably annexin V) or b2-9 ⁇ oorGq ⁇ b ⁇ h I.
  • the phosphatidylserine-binding protein is Protein Z, Tissue Factor, Factor VIII, LOX-1 , lactadherin, an oxysterol binding protein, Protein C, Protein S, Factor II (prothrombin), Factor V, Factor VII
  • the binding module binds to a target associated with a site of injury (e.g. von Willebrand factor (VWF), fibrinogen or collagen) in order to localize the proteinaceous molecule to the site of injury, instead of or in addition to binding to phosphatidylserine, a platelet surface marker or a phosphatidylserine-binding protein as mentioned above.
  • a site of injury e.g. von Willebrand factor (VWF), fibrinogen or collagen
  • the binding module is an anticalin, repebody, monobody, scFv, Fab, scFab, affibody, fynomer, DARPin, nanobody, peptide aptamer, or nucleic acid aptamter.
  • the binding module is a scFv or scFab.
  • the binding module is a scFv.
  • the binding module is a scFv or scFab that comprises a VL domain and a VH domain, wherein the VL domain comprises LCDR1 , LCDR2 and LCDR3 and the VH domain comprises HCDR1 , HCDR2 and HCDR3, wherein:
  • LCDR1 is QDIGSS (SEQ ID NO: 293)
  • LCDR2 is ATS (SEQ ID NO: 294)
  • LCDR3 is
  • HCDR1 is GYSFTGYN (SEQ ID NO: 296)
  • HCDR2 is
  • IDPYYGDT (SEQ ID NO: 297) and HCDR3 is VKGGYYGHWYFDV (SEQ ID NO: 298);
  • LCDR1 is SLRSYY (SEQ ID NO: 299)
  • LCDR2 is GKN (SEQ ID NO: 300)
  • LCDR3 is
  • HCDR1 is GFTFSSYA (SEQ ID NO: 302)
  • HCDR2 is
  • INGSGGST SEQ ID NO: 303
  • HCDR3 is AKTRRKVFDY (SEQ ID NO: 304); or
  • LCDR1 is GNIHNY (SEQ ID NO: 305)
  • LCDR2 is NAK (SEQ ID NO: 306)
  • LCDR3 is QHFWSTPYT (SEQ ID NO: 307)
  • HCDR1 is GFTFSSYI (SEQ ID NO: 308)
  • HCDR2 is IRSGGDNT (SEQ ID NO: 309)
  • HCDR3 is AIYYGNYGGLAY (SEQ ID NO: 310).
  • the binding module is a scFv or scFab that comprises a VL domain and a VH domain, wherein the VL domain is selected from SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186 and the VH domain is selected from SEQ ID NO: 187, SEQ ID NO: 188 or SEQ ID NO: 189.
  • the VL domain is SEQ ID NO: 184 and the VH domain is SEQ ID NO: 187
  • the VL domain is SEQ ID NO: 185 and the VH domain is SEQ ID NO: 188
  • the VL domain is SEQ ID NO: 186 and the VH domain is SEQ ID NO: 189.
  • the binding module is SEQ ID NO: 190, SEQ ID NO: 191 or SEQ ID NO: 192.
  • the binding module is a scFv, scFab or Fab comprising the VH and VL domain of any one of 1-13 according to Table 6.
  • the binding module is a scFv, scFab or Fab comprising the VH and VL domain of any one of 1-13 according to Table 7.
  • Table 7 VL and VH sequences of humanized anti-LIBS constructs
  • the binding module comprises the phosphatidylserine-binding domain of a phosphatidylserine-binding protein.
  • Such domains are known in the art and can be easily identified by the skilled persons (see, for example, WO 2006/079120 A2, the contents of which are herein incorporated in their entirety by reference).
  • the binding module comprises domain V of b2 ⁇ oorGq ⁇ b ⁇ h I (see WO 2006/079120 A2).
  • the binding module comprises KASCKVPVKKATVVYQGERVKIQEKFKNGMLHGDKVSFFCKNKEKKCSYTEDAQCIDGTIE VPKCFKEHSSLAFWKTDASDVKPC (SEQ ID NO: 178).
  • the proteinaceous molecule is bispecific and multivalent.
  • the scaffold module comprises two antigen-binding sites that bind to Factor IXa or Factor X, or the scaffold module comprises an antigen-binding site that binds to Factor IXa and an antigen-binding site that binds to Factor X.
  • FIGS 1 and 4 disclose examples of bispecific multivalent molecules encompassed by the present invention.
  • the bispecific multivalent molecules of the present invention can be subcategorized into two categories depending on the scaffold module used: 1) scaffold modules which comprise a first antigen-binding site that binds to Factor IXa and a second antigen-binding site that binds to Factor X (referred to as“IXa/X scaffold”) and 2) scaffold modules which comprise two antigen-binding sites wherein both antigen-binding sites bind to Factor IXa or Factor X (referred to as“monospecific scaffold).
  • the IXa/X scaffold is an antibody.
  • the monospecific scaffold is an antibody.
  • a single binding module can be attached to an IXa/X scaffold through any of the linkers and positions previously disclosed or the binding module may be inserted into a loop region.
  • the binding module comprises an antigen-binding site that binds to any of the other targets, i.e. not Factor IXa or Factor X, disclosed herein.
  • An IXa/X scaffold can also be made bispecific multivalent.
  • a single binding module can be attached to an IXa/X scaffold through any of the linkers and positions previously disclosed or the binding module may be inserted into a loop region.
  • the binding module comprises an antigen-binding site that binds to Factor IXa or Factor X.
  • the monospecific scaffold can only be made bispecific multivalent if only one binding module is attached/inserted.
  • a single binding module can be attached to a monospecific scaffold through any of the linkers and positions previously disclosed or the binding module may be inserted into a loop region.
  • the binding module comprises an antigen-binding site that binds to Factor IXa or Factor X.
  • the proteinaceous molecule in order to make a trispecific multivalent proteinaceous molecule comprising a monospecific scaffold, it is necessary for the proteinaceous molecule to comprise two binding modules.
  • the first binding module is attached to the monospecific scaffold through a linker, or the first binding module is inserted into a loop region.
  • the second binding module may be attached to the scaffold module in a similar or different way as the first binding module.
  • the antigen-binding site of the first binding module should bind to Factor IXa or Factor X and the antigen-binding site of the second binding module should bind to another target (i.e. not Factor IXa or Factor X) to make the proteinaceous molecule trispecific.
  • Proteinaceous molecules which have a “3 by 1” or “1 by 3” topology are depicted in Figure 1. These proteinaceous molecules are bispecific multivalent and comprise a larger number of antigen-binding sites directed to one of the targets than the other.
  • the present invention provides a proteinaceous molecule comprising:
  • a scaffold module comprising a first antigen-binding site that binds to Factor IXa and a second antigen-binding site that binds to Factor X;
  • the present invention provides a proteinaceous molecule comprising:
  • HCDR1 is SEQ ID NO: 1
  • HCDR2 is SEQ ID NO: 2
  • HCDR3 is SEQ ID NO:3;
  • binding modules that are scFvs or scFabs comprising:
  • HCDR1 is SEQ ID NO: 1
  • HCDR2 is SEQ ID NO: 2
  • HCDR3 is SEQ ID NO: 3.
  • the LCDR1 , LCDR2 and LCDR3 of (a)(ii) is the LCDR1 , LCDR2 and LCDR3 of W83, LCDR1 , LCDR2 and LCDR3 of W88, LCDR1 , LCDR2 and LCDR3 of W128, LCDR1 , LCDR2 and LCDR3 of W127, or LCDR1 , LCDR2 and LCDR3 of W162 disclosed in Table 1
  • the LCDR1 , LCDR2 and LCDR3 of (a)(iii) is the LCDR1 , LCDR2 and LCDR3 of V43, LCDR1 , LCDR2 and LCDR3 of V90, LCDR1 , LCDR2 and LCDR3 of V141 , LCDR1 , LCDR2 and LCDR3 of V149, LCDR1 , LCDR2 and LCDR3 of V198, LCDR1 , LCDR2 and LCDR3 of V202, LCDR1 , LCDR2 and LCDR3 of V204, LCDR
  • the LCDR1 , LCDR2 and LCDR3 of (a)(ii) and (a)(iii) are the LCDR1 , LCDR2 and LCDR3 of W88 and V198, LCDR1 , LCDR2 and LCDR3 of W127 and V202, LCDR1 , LCDR2 and LCDR3 of V149 and W128, LCDR1 , LCDR2 and LCDR3 of W128 and V198, LCDR1 , LCDR2 and LCDR3 of W128 and V141 , LCDR1 , LCDR2 and LCDR3 of W162 and V204, LCDR1 , LCDR2 and LCDR3 of W83 and V217, LCDR1 , LCDR2 and LCDR3 of W88 and V90, or LCDR1 , LCDR2 and LCDR3 of W83 and V43, respectively, as disclosed in Tables 1 and 2a, and the LCDR1 , LCDR2 and LCDR3 of (b)(i) are that of V198, V202
  • the present invention provides a proteinaceous molecule comprising:
  • HCDR1 is SEQ ID NO: 1
  • HCDR2 is SEQ ID NO: 2
  • HCDR3 is SEQ ID NO:3;
  • binding modules that are scFvs or scFabs comprising:
  • HCDR1 is SEQ ID NO: 1
  • HCDR2 is SEQ ID NO: 2
  • HCDR3 is SEQ ID NO: 3.
  • the LCDR1 , LCDR2 and LCDR3 of (a)(ii) is the LCDR1 , LCDR2 and LCDR3 of W206, LCDR1 , LCDR2 and LCDR3 of W83, LCDR1 , LCDR2 and LCDR3 of W88, LCDR1 , LCDR2 and LCDR3 of W128, LCDR1 , LCDR2 and LCDR3 of W127, or LCDR1 , LCDR2 and LCDR3 of W162 disclosed in Table 1
  • the LCDR1 , LCDR2 and LCDR3 of (a)(iii) is the LCDR1 , LCDR2 and LCDR3 of V43, LCDR1 , LCDR2 and LCDR3 of V90, LCDR1 , LCDR2 and LCDR3 of V245, LCDR1 , LCDR2 and LCDR3 of V141 , LCDR1 , LCDR2 and LCDR3 of V149, LCDR1 , LCDR2 and LCDR3 of V198, LCDR
  • the LCDR1 , LCDR2 and LCDR3 of (a)(ii) and (a)(iii) are the LCDR1 , LCDR2 and LCDR3 of W206 and V245, LCDR1 , LCDR2 and LCDR3 of W88 and V198, LCDR1 , LCDR2 and LCDR3 of W127 and V202, LCDR1 , LCDR2 and LCDR3 of V149 and W128, LCDR1 , LCDR2 and LCDR3 of W128 and V198, LCDR1 , LCDR2 and LCDR3 of W128 and V141 , LCDR1 , LCDR2 and LCDR3 of W162 and V204, LCDR1 , LCDR2 and LCDR3 of W83 and V217, LCDR1 , LCDR2 and LCDR3 of W88 and V90, or LCDR1 , LCDR2 and LCDR3 of W83 and V43, respectively, as disclosed in Tables 1 and 2b, and the LCDR1 , LCDR2 and LCDR3
  • the present invention provides a proteinaceous molecule comprising: (a) an antibody or a bivalent fragment thereof that comprises:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of any one of the Factor X binding light chains disclosed in Table 1 , or the Factor IXa binding light chains disclosed in Table 2a, and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183.
  • the VL of (a)(ii) is the VL of W83, VL of W88, VL of W128, VL of W127, or VL of W162 disclosed in Table 3, and the VL of (a)(iii) is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V198, VL of V202, VL of V204, VL of V212, or VL of V217 disclosed in Table 4a.
  • the VL of (a)(ii) and (a)(iii) are the VL of W88 and V198, VL of W127 and V202, VL of V149 and W128, VL of W128 and V198, VL of W128 and V141 , VL of W162 and V204, VL of W83 and V217, VL of W88 and V90, or VL of W83 and V43, respectively, disclosed in Tables 3 and 4a
  • the LCDR1 , LCDR2 and LCDR3 of (b)(i) are that of V198, V202, W128, W88, W127, V212, or W162 disclosed in Tables 1 and 2a.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of any one of the Factor X binding light chains disclosed in Table 1 , or the Factor IXa binding light chains disclosed in Table 2b, and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183.
  • the VL of (a)(ii) is the VL of W206, VL of W83, VL of W88, VL of W128, VL of W127, or VL of W162 disclosed in Table 3, and the VL of (a)(iii) is the VL of V245, VL of V43, VL of V90, VL of V141 , VL of V149, VL of V198, VL of V202, VL of V204, VL of V212, or VL of V217 disclosed in Table 4b.
  • the VL of (a)(ii) and (a)(iii) are the VL of W206 and V245, VL of W88 and V198, VL of W127 and V202, VL of V149 and W128, VL of W128 and V198, VL of W128 and V141 , VL of W162 and V204, VL of W83 and V217, VL of W88 and V90, or VL of W83 and V43, respectively, disclosed in Tables 3 and 4b, and the LCDR1 , LCDR2 and LCDR3 of (b)(i) are that of W206, V198, V202, W128, W88, W127, V212, or W162 disclosed in Tables 1 and 2b.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of V198 disclosed in Table 2a, and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of V198 disclosed in Table 2a, and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of V202 disclosed in Table 2a, and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of V128 disclosed in Table 1 , and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of W128 disclosed in Table 1 , and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of W88 disclosed in Table 1 , and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of W128 disclosed in Table 1 , and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183.
  • the present invention provides a proteinaceous molecule comprising: (a) an antibody or a bivalent fragment thereof that comprises:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of W127 disclosed in Table 1 , and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of W162 disclosed in Table 1 , and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of W206 disclosed in Table 1 , and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183.
  • the binding modules are attached to the N- or -C terminus of the heavy or light chain through a linker. In some embodiments, the binding modules are attached to the C-terminus of the heavy or light chain. In some embodiments, the first binding module is attached to the C-terminus of the first heavy chain through a linker, and the second binding module is attached to C-terminus of the second heavy chain through the linker.
  • the proteinaceous molecule comprises both the heavy chain sequence and the two LC sequences of any one of Lex#3, Bax#137, Bax#148, Bax#146, Lex#2, Lex#12, Bax#138 or Bax#145 disclosed in Table 8.
  • the proteinaceous molecule comprises both the heavy chain sequence and the two LC sequences of any one of Lex#3, Lex#131 , Bax#137, Bax#148, Bax# 146, Lex#2, Lex#12, Bax#138 or Bax#145 disclosed in Table 8.
  • the proteinaceous molecule consists of two copies of the heavy chain sequence, one copy of the LC with lambda constant domain sequence, and one copy of the LC with kappa constant domain sequence of any one of Lex#3, Bax#137, Bax#148, Bax# 146, Lex#2, Lex#12, Bax#138 or Bax#145 disclosed in Table 8.
  • the proteinaceous molecule consists of two copies of the heavy chain sequence, one copy of the LC with lambda constant domain sequence, and one copy of the LC with kappa constant domain sequence of any one of Lex#3, Lex#131 , Bax#137, Bax#148, Bax#146, Lex#2, Lex#12, Bax#138 or Bax#145 disclosed in Table 8.
  • Proteinaceous molecules which have a“2 by 2” topology are depicted in Figure 1. These proteinaceous molecules are bispecific multivalent and comprise an equal number of antigen-binding sites that bind to Factor IXa or Factor X. This sort of topology uses a monospecific scaffold.
  • the present invention provides a proteinaceous molecule comprising:
  • a scaffold module wherein the scaffold module comprises a first and second antigen binding site that bind to Factor IXa or Factor X;
  • the third and fourth antigen-binding sites bind to Factor IXa or Factor X and the proteinaceous molecule comprises two antigen-binding sites that bind to Factor IXa and two antigen-binding sites that bind to Factor X.
  • the present invention provides a proteinaceous molecule comprising:
  • HCDR1 is SEQ ID NO: 1
  • HCDR2 is SEQ ID NO: 2
  • HCDR3 is SEQ ID NO:3;
  • binding modules that are scFvs or scFabs comprising:
  • the LCDR1 , LCDR2 and LCDR3 of (a)(ii) is the LCDR1 , LCDR2 and LCDR3 of W83, LCDR1 , LCDR2 and LCDR3 of W88, LCDR1 , LCDR2 and LCDR3 of W128, LCDR1 , LCDR2 and LCDR3 of W127, or LCDR1 , LCDR2 and LCDR3 of W162 disclosed in Table 1 , or the LCDR1 , LCDR2 and LCDR3 of (a)(ii) is the LCDR1 , LCDR2 and LCDR3 of V43, LCDR1 , LCDR2 and LCDR3 of V90, LCDR1 , LCDR2 and LCDR3 of V141 , LCDR1 , LCDR2 and LCDR3 of V149, LCDR1 , LCDR
  • the present invention provides a proteinaceous molecule comprising:
  • HCDR1 is SEQ ID NO: 1
  • HCDR2 is SEQ ID NO: 2
  • HCDR3 is SEQ ID NO:3;
  • binding modules that are scFvs or scFabs comprising:
  • HCDR1 is SEQ ID NO: 1
  • HCDR2 is SEQ ID NO: 2
  • HCDR3 is SEQ ID NO: 3
  • the LCDR1 , LCDR2 and LCDR3 of (a)(ii) is the LCDR1 , LCDR2 and LCDR3 of W206, LCDR1 , LCDR2 and LCDR3 of W83, LCDR1 , LCDR2 and LCDR3 of W88, LCDR1 , LCDR2 and LCDR3 of W128, LCDR1 , LCDR2 and LCDR3 of W127, or LCDR1 , LCDR2 and LCDR3 of W162 disclosed in Table 1 , or the LCDR1 , LCDR2 and LCDR3 of (a)(ii) is the LCDR1 , LCDR2 and LCDR3 of V155, LCDR1 , LCDR2 and LCDR3 of V245, LCDR1 , LCDR2 and LCDR3 of V43, LCDR1 , LCDR2
  • the present invention provides a proteinaceous molecule comprising:
  • HCDR1 is SEQ ID NO: 1
  • HCDR2 is SEQ ID NO: 2
  • HCDR3 is SEQ ID NO:3;
  • binding modules that are scFvs or scFabs comprising:
  • HCDR1 is SEQ ID NO: 1
  • HCDR2 is SEQ ID NO: 2
  • HCDR3 is SEQ ID NO: 3
  • the proteinaceous molecule comprises at least two antigen-binding sites that bind to Factor IXa and at least two antigen binding sites that bind to Factor X.
  • the LCDR1 , LCDR2 and LCDR3 of each of the two light chains are identical, and the LCDR1 , LCDR2 and LCDR3 of each of the two binding modules are identical.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of any one of the Factor X binding light chains disclosed in Table 1 , or the Factor IXa binding light chains disclosed in Table 2a, and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183,
  • the proteinaceous molecule comprises at least two antigen-binding sites that bind to Factor IXa and at least two antigen binding sites that bind to Factor X.
  • the VL of (a)(ii) is the VL of W83, VL of W88, VL of W128, VL of W127, or VL of W162 disclosed in Table 3, or the VL of (a)(ii) is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V198, VL of V202, VL of V204, VL of V212, or VL of V217 disclosed in Table 4a
  • the LCDR1 , LCDR2 and LCDR3 of (b)(i) are that of V198, V202, W128, W88, W127, V212, or W162 disclosed in Tables 1 and 2a.
  • the VL of each of the two light chains are identical, and the LCDR1 , LCDR
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of any one of the Factor X binding light chains disclosed in Table 1 , or the Factor IXa binding light chains disclosed in Table 2b, and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183,
  • the proteinaceous molecule comprises at least two antigen-binding sites that bind to Factor IXa and at least two antigen binding sites that bind to Factor X.
  • the VL of (a)(ii) is the VL of W206, VL of W83, VL of W88, VL of W128, VL of W127, or VL of W162 disclosed in Table 3, or the VL of (a)(ii) is the VL of V245, VL of V155, VL of V320, VL of V305, VL of V43, VL of V90, VL of V141 , VL of V149, VL of V198, VL of V202, VL of V204, VL of V212, or VL of V217 disclosed in Table 4b, and the LCDR1 , LCDR2 and LCDR3 of (b)(i) are that of W206, W198, V198, V202, W128, W88, W127, V212, or W162
  • the present invention provides a proteinaceous molecule comprising: (a) an antibody or bivalent fragment thereof that comprises:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of W198 disclosed in Table 1 , and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183,
  • the proteinaceous molecule comprises at least two antigen-binding sites that bind to Factor IXa and at least two antigen binding sites that bind to Factor X.
  • the VL of each of the two light chains are identical, and the LCDR1 , LCDR2 and LCDR3 of each of the two binding modules are identical.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of W198 disclosed in Table 1 , and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183,
  • the proteinaceous molecule comprises at least two antigen-binding sites that bind to Factor IXa and at least two antigen binding sites that bind to Factor X.
  • the VL of each of the two light chains are identical, and the LCDR1 , LCDR2 and LCDR3 of each of the two binding modules are identical.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • binding modules that are scFvs comprising: (i) a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of W128 disclosed in Table 1 , and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183,
  • the proteinaceous molecule comprises at least two antigen-binding sites that bind to Factor IXa and at least two antigen binding sites that bind to Factor X.
  • the VL of each of the two light chains are identical, and the LCDR1 , LCDR2 and LCDR3 of each of the two binding modules are identical.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of W128 disclosed in Table 1 , and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183,
  • the proteinaceous molecule comprises at least two antigen-binding sites that bind to Factor IXa and at least two antigen binding sites that bind to Factor X.
  • the VL of each of the two light chains are identical, and the LCDR1 , LCDR2 and LCDR3 of each of the two binding modules are identical.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of W198 disclosed in Table 1 , and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183, wherein the proteinaceous molecule comprises at least two antigen-binding sites that bind to Factor IXa and at least two antigen binding sites that bind to Factor X.
  • the VL of each of the two light chains are identical, and the LCDR1 , LCDR2 and LCDR3 of each of the two binding modules are identical.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of W206 disclosed in Table 1 , and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183,
  • the proteinaceous molecule comprises at least two antigen-binding sites that bind to Factor IXa and at least two antigen binding sites that bind to Factor X.
  • the VL of each of the two light chains are identical, and the LCDR1 , LCDR2 and LCDR3 of each of the two binding modules are identical.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of W127 disclosed in Table 1 , and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183,
  • the proteinaceous molecule comprises at least two antigen-binding sites that bind to Factor IXa and at least two antigen binding sites that bind to Factor X.
  • the VL of each of the two light chains are identical, and the LCDR1 , LCDR2 and LCDR3 of each of the two binding modules are identical.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of W198 disclosed in Table 1 , and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183,
  • the proteinaceous molecule comprises at least two antigen-binding sites that bind to Factor IXa and at least two antigen binding sites that bind to Factor X.
  • the VL of each of the two light chains are identical, and the LCDR1 , LCDR2 and LCDR3 of each of the two binding modules are identical.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of W128 disclosed in Table 1 , and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183,
  • the proteinaceous molecule comprises at least two antigen-binding sites that bind to Factor IXa and at least two antigen binding sites that bind to Factor X.
  • the VL of each of the two light chains are identical, and the LCDR1 , LCDR2 and LCDR3 of each of the two binding modules are identical.
  • the present invention provides a proteinaceous molecule comprising: (a) an antibody or bivalent fragment thereof that comprises:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of W198 disclosed in Table 1 , and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183,
  • the proteinaceous molecule comprises at least two antigen-binding sites that bind to Factor IXa and at least two antigen binding sites that bind to Factor X.
  • the VL of each of the two light chains are identical, and the LCDR1 , LCDR2 and LCDR3 of each of the two binding modules are identical.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of W127 disclosed in Table 1 , and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183,
  • the proteinaceous molecule comprises at least two antigen-binding sites that bind to Factor IXa and at least two antigen binding sites that bind to Factor X.
  • the VL of each of the two light chains are identical, and the LCDR1 , LCDR2 and LCDR3 of each of the two binding modules are identical.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of V212 disclosed in Table 2a, and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183,
  • the proteinaceous molecule comprises at least two antigen-binding sites that bind to Factor IXa and at least two antigen binding sites that bind to Factor X.
  • the VL of each of the two light chains are identical, and the LCDR1 , LCDR2 and LCDR3 of each of the two binding modules are identical.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of W127 disclosed in Table 1 , and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183,
  • the proteinaceous molecule comprises at least two antigen-binding sites that bind to Factor IXa and at least two antigen binding sites that bind to Factor X.
  • the VL of each of the two light chains are identical, and the LCDR1 , LCDR2 and LCDR3 of each of the two binding modules are identical.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of V212 disclosed in Table 2a, and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183,
  • the proteinaceous molecule comprises at least two antigen-binding sites that bind to Factor IXa and at least two antigen binding sites that bind to Factor X.
  • the VL of each of the two light chains are identical, and the LCDR1 , LCDR2 and LCDR3 of each of the two binding modules are identical.
  • the binding modules are attached to the N- or -C terminus of the heavy or light chain through a linker.
  • the binding modules are attached to the C-terminus of the heavy or light chain.
  • the first binding module is attached to the C-terminus of the first heavy chain through a linker
  • the second binding module is attached to C-terminus of the second heavy chain through the linker.
  • the proteinaceous molecule comprises both the heavy chain sequence and the light chain sequence of any one of Lex#84, Lex#85, Lex#86, Lex#87, Lex#88, Lex#96, Lex#97, Lex#60, Lex#63, Lex#71 , Lex#72, Lex#73, Lex#74, Lex#75, Bax# 144, Lex#59, Lex#53, Lex#99, Lex#102, Lex#103, or Lex#105 disclosed in Table 9.
  • the proteinaceous molecule comprises both the heavy chain sequence and the light chain sequence of any one of Lex#95, Lex#121 , Lex#129, Lex#143, Lex# 153, Lex# 157, Lex#161 , Lex#167, Lex#168, Lex#169, Lex#84, Lex#85, Lex#86, Lex#87, Lex#88, Lex#96, Lex#97, Lex#60, Lex#63, Lex#71 , Lex#72, Lex#73, Lex#74, Lex#75, Bax# 144, Lex#59, Lex#53, Lex#99, Lex#102, Lex#103, or Lex#105 disclosed in Table 9.
  • the proteinaceous molecule comprises both the heavy chain sequence and the light chain sequence of Lex#153 disclosed in Table 9.
  • the proteinaceous molecule consists of two copies of the heavy chain sequence and two copies of the light chain sequence of any one of Lex#84, Lex#85, Lex#86, Lex#87, Lex#88, Lex#96, Lex#97, Lex#60, Lex#63, Lex#71 , Lex#72, Lex#73, Lex#74, Lex#75, Bax# 144, Lex#59, Lex#53, Lex#99, Lex#102, Lex#103, or Lex#105 disclosed in Table 9.
  • the proteinaceous molecule consists of two copies of the heavy chain sequence and two copies of the light chain sequence of any one of Lex#95, Lex#121 , Lex#129, Lex#143, Lex#153, Lex#157, Lex#161 , Lex#167, Lex#168, Lex#169, Lex#84, Lex#85, Lex#86, Lex#87, Lex#88, Lex#96, Lex#97, Lex#60, Lex#63, Lex#71 , Lex#72, Lex#73, Lex#74, Lex#75, Bax#144, Lex#59, Lex#53, Lex#99, Lex#102, Lex#103, or Lex#105 disclosed in Table 9.
  • the proteinaceous molecule consists of two copies of the heavy chain sequence and two copies of the light chain sequence of Lex#153 disclosed in Table 9.
  • the proteinaceous molecule is trispecific and multivalent.
  • FIGS 2 and 3 disclose examples of trispecific multivalent molecules encompassed by the present invention.
  • the trispecific multivalent molecules of the present invention can be subcategorized into two categories depending on the scaffold module used: 1) scaffold modules which comprise a first antigen-binding site that binds to Factor IXa and a second antigen-binding site that binds to Factor X (referred to as“IXa/X scaffold”) and 2) scaffold modules which comprise two antigen-binding sites wherein both antigen-binding sites bind to Factor IXa or Factor X (referred to as“monospecific scaffold).
  • Proteinaceous molecules which have a trispecific“IXa/X scaffold” topology are depicted in Figure 2. These proteinaceous molecules are trispecific multivalent and comprise at least three antigen-binding sites that bind to different targets.
  • the present invention provides a proteinaceous molecule comprising:
  • a scaffold module comprising a first antigen-binding site that binds to Factor IXa and a second antigen-binding site that binds to Factor X;
  • the third and fourth antigen-binding sites bind to phosphatidylserine, a phosphatidylserine-binding protein or a platelet surface marker;
  • the phosphatidylserine-binding protein is Protein Z, Tissue Factor, Factor VIII, LOX- 1 , lactadherin, an oxysterol binding protein, Protein C, Protein S, Factor II (prothrombin), Factor V, Factor VII, Mer, 0 d b3 integrin, the CR3 complement receptor, the CR4 complement receptor, the phosphatidylserine receptor, an annexin (preferably annexin V) or b2- glycoprotein I.
  • the present invention provides a proteinaceous molecule comprising: (a) an antibody or a bivalent fragment thereof that comprises: (i) two heavy chains comprising identical HCDRs, wherein HCDR1 is SEQ ID NO: 1 , HCDR2 is SEQ ID NO: 2 and HCDR3 is SEQ ID NO:3;
  • binding modules comprising an antigen-binding site each, wherein both of the antigen-binding sites bind to phosphatidylserine, a phosphatidylserine-binding protein or a platelet surface marker;
  • the phosphatidylserine-binding protein is Protein Z, Tissue Factor, Factor VIII, LOX- 1 , lactadherin, an oxysterol binding protein, Protein C, Protein S, Factor II (prothrombin), Factor V, Factor VII, Mer, 0 d b3 integrin, the CR3 complement receptor, the CR4 complement receptor, the phosphatidylserine receptor, an annexin (preferably annexin V) or b2- glycoprotein I.
  • the LCDR1 , LCDR2 and LCDR3 of (a)(ii) is the LCDR1 , LCDR2 and LCDR3 of W83, LCDR1 , LCDR2 and LCDR3 of W88, LCDR1 , LCDR2 and LCDR3 of W128, LCDR1 , LCDR2 and LCDR3 of W127, or LCDR1 , LCDR2 and LCDR3 of W162 disclosed in Table 1
  • the LCDR1 , LCDR2 and LCDR3 of (a)(iii) is the LCDR1 , LCDR2 and LCDR3 of V43, LCDR1 , LCDR2 and LCDR3 of V90, LCDR1 , LCDR2 and LCDR3 of V141 , LCDR1 , LCDR2 and LCDR3 of V149, LCDR1 , LCDR2 and LCDR3 of V198, LCDR1 , LCDR2 and LCDR3 of V202, LCDR1 , LCDR2 and LCDR3 of V204, LCDR
  • the LCDR1 , LCDR2 and LCDR3 of (a)(ii) and (a)(iii) are the LCDR1 , LCDR2 and LCDR3 of W88 and V198, LCDR1 , LCDR2 and LCDR3 of W127 and V202, LCDR1 , LCDR2 and LCDR3 of V149 and W128, LCDR1 , LCDR2 and LCDR3 of W128 and V198, LCDR1 , LCDR2 and LCDR3 of W128 and V141 , LCDR1 , LCDR2 and LCDR3 of W162 and V204, LCDR1 , LCDR2 and LCDR3 of W83 and V217, LCDR1 , LCDR2 and LCDR3 of W88 and V90, or LCDR1 , LCDR2 and LCDR3 of W83 and V43, respectively, as disclosed in Tables 1 and 2a.
  • the present invention provides a proteinaceous molecule comprising: (a) an antibody or a bivalent fragment thereof that comprises:
  • HCDR1 is SEQ ID NO: 1
  • HCDR2 is SEQ ID NO: 2
  • HCDR3 is SEQ ID NO:3;
  • one light chain comprising the LCDR1 , LCDR2 and LCDR3 of any one of the Factor IXa binding light chains disclosed in Table 2b; and (b) at least two binding modules comprising an antigen-binding site each, wherein both of the antigen-binding sites bind to phosphatidylserine, a phosphatidylserine-binding protein or a platelet surface marker;
  • the phosphatidylserine-binding protein is Protein Z, Tissue Factor, Factor VIII, LOX- 1 , lactadherin, an oxysterol binding protein, Protein C, Protein S, Factor II (prothrombin), Factor V, Factor VII, Mer, 0 d b3 integrin, the CR3 complement receptor, the CR4 complement receptor, the phosphatidylserine receptor, an annexin (preferably annexin V) or b2- glycoprotein I.
  • the LCDR1 , LCDR2 and LCDR3 of (a)(ii) is the LCDR1 , LCDR2 and LCDR3 of W83, LCDR1 , LCDR2 and LCDR3 of W206, LCDR1 , LCDR2 and LCDR3 of W88, LCDR1 , LCDR2 and LCDR3 of W128, LCDR1 , LCDR2 and LCDR3 of W127, or LCDR1 , LCDR2 and LCDR3 of W162 disclosed in Table 1
  • the LCDR1 , LCDR2 and LCDR3 of (a)(iii) is the LCDR1 , LCDR2 and LCDR3 of V43, LCDR1 , LCDR2 and LCDR3 of V320, LCDR1 , LCDR2 and LCDR3 of V305, LCDR1 , LCDR2 and LCDR3 of V90, LCDR1 , LCDR2 and LCDR3 of V141 , LCDR1 , LCDR2 and LCDR3 of V149, LCDR
  • the LCDR1 , LCDR2 and LCDR3 of (a)(ii) and (a)(iii) are the LCDR1 , LCDR2 and LCDR3 of W88 and V198, LCDR1 , LCDR2 and LCDR3 of W206 and V245, LCDR1 , LCDR2 and LCDR3 of W127 and V202, LCDR1 , LCDR2 and LCDR3 of V149 and W128, LCDR1 , LCDR2 and LCDR3 of W128 and V198, LCDR1 , LCDR2 and LCDR3 of W128 and V141 , LCDR1 , LCDR2 and LCDR3 of W162 and V204, LCDR1 , LCDR2 and LCDR3 of W83 and V217, LCDR1 , LCDR2 and LCDR3 of W88 and V90, or LCDR1 , LCDR2 and LCDR3 of W83 and V43, respectively, as disclosed in Tables 1 and 2b.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • the phosphatidylserine-binding protein is Protein Z, Tissue Factor, Factor VIII, LOX- 1 , lactadherin, an oxysterol binding protein, Protein C, Protein
  • the VL of (a)(ii) is the VL of W83, VL of W88, VL of W128, VL of W127, or VL of W162 disclosed in Table 3, and the VL of (a)(iii) is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V198, VL of V202, VL of V204, VL of V212, or VL of V217 disclosed in Table 4a.
  • the VL of (a)(ii) and (a)(iii) are the VL of W88 and V198, VL of W127 and V202, VL of V149 and W128, VL of W128 and V198, VL of W128 and V141 , VL of W162 and V204, VL of W83 and V217, VL of W88 and V90, or VL of W83 and V43, respectively, disclosed in Tables 3 and 4a.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • the phosphatidylserine-binding protein is Protein Z, Tissue Factor, Factor VIII, LOX- 1 , lactadherin, an oxysterol binding protein, Protein C, Protein S, Factor II (prothrombin), Factor V, Factor VII, Mer, 0 d b3 integrin, the CR3 complement receptor, the CR4 complement receptor, the phosphatidylserine receptor, an annexin (preferably annexin V) or b2- glycoprotein I.
  • the VL of (a)(ii) is the VL of W206, VL of W83, VL of W88, VL of W128, VL of W127, or VL of W162 disclosed in Table 3, and the VL of (a)(iii) is the VL of V320, VL of V305, VL of V43, VL of V90, VL of V141 , VL of V149, VL of V245, VL of V198, VL of V202, VL of V204, VL of V212, or VL of V217 disclosed in Table 4b.
  • the VL of (a)(ii) and (a)(iii) are the VL of W88 and V198, VL of W206 and V245, VL of W127 and V202, VL of V149 and W128, VL of W128 and V198, VL of W128 and V141 , VL of W162 and V204, VL of W83 and V217, VL of W88 and V90, or VL of W83 and V43, respectively, disclosed in Tables 3 and 4b.
  • the present invention provides a proteinaceous molecule comprising: (a) an antibody or a bivalent fragment thereof that comprises:
  • VH domain is SEQ ID NO: 4;
  • VL domain comprises SEQ ID NO: 184 and the VH domain comprises SEQ ID NO: 187
  • VL domain comprises SEQ ID NO: 185 and the VH domain comprises SEQ ID NO: 188
  • VL domain comprises SEQ ID NO: 186 and the VH domain comprises SEQ ID NO: 189.
  • the VL of (a)(ii) is the VL of W83, VL of W88, VL of W128, VL of W127, or VL of W162 disclosed in Table 3, and the VL of (a)(iii) is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V198, VL of V202, VL of V204, VL of V212, or VL of V217 disclosed in Table 4a.
  • the VL of (a)(ii) and (a)(iii) are the VL of W88 and V198, VL of W127 and V202, VL of V149 and W128, VL of W128 and V198, VL of W128 and V141 , VL of W162 and V204, VL of W83 and V217, VL of W88 and V90, or VL of W83 and V43, respectively, disclosed in Tables 3 and 4a.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • VL domain comprises SEQ ID NO: 184 and the VH domain comprises SEQ ID NO: 187
  • VL domain comprises SEQ ID NO: 185 and the VH domain comprises SEQ ID NO: 188
  • VL domain comprises SEQ ID NO: 186 and the VH domain comprises SEQ ID NO: 189.
  • the VL of (a)(ii) is the VL of W83, VL of W206, VL of W88, VL of W128, VL of W127, or VL of W162 disclosed in Table 3, and the VL of (a)(iii) is the VL of V320, VL of V305, VL of V245, VL of V43, VL of V90, VL of V141 , VL of V149, VL of V198, VL of V202, VL of V204, VL of V212, or VL of V217 disclosed in Table 4b.
  • the VL of (a)(ii) and (a)(iii) are the VL of W206 and V245, W88 and V198, VL of W127 and V202, VL of V149 and W128, VL of W128 and V198, VL of W128 and V141 , VL of W162 and V204, VL of W83 and V217, VL of W88 and V90, or VL of W83 and V43, respectively, disclosed in Tables 3 and 4b.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain comprises SEQ ID NO: 4;
  • the VL of (a)(ii) is the VL of W83, VL of W88, VL of W128, VL of W127, or VL of W162 disclosed in Table 3, and the VL of (a)(iii) is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V198, VL of V202, VL of V204, VL of V212, or VL of V217 disclosed in Table 4a.
  • the VL of (a)(ii) and (a)(iii) are the VL of W88 and V198, VL of W127 and V202, VL of V149 and W128, VL of W128 and V198, VL of W128 and V141 , VL of W162 and V204, VL of W83 and V217, VL of W88 and V90, or VL of W83 and V43, respectively, disclosed in Tables 3 and 4a.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain comprises SEQ ID NO: 4;
  • the VL of (a)(ii) is the VL of W83, VL of W206, VL of W88, VL of W128, VL of W127, or VL of W162 disclosed in Table 3, and the VL of (a)(iii) is the VL of V43, VL of V320, VL of V305, VL of V245, VL of V90, VL of V141 , VL of V149, VL of V198, VL of V202, VL of V204, VL of V212, or VL of V217 disclosed in Table 4b.
  • the VL of (a)(ii) and (a)(iii) are the VL of W88 and V198, VL of W206 and V245, VL of W127 and V202, VL of V149 and W128, VL of W128 and V198, VL of W128 and V141 , VL of W162 and V204, VL of W83 and V217, VL of W88 and V90, or VL of W83 and V43, respectively, disclosed in Tables 3 and 4b.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain comprises SEQ ID NO: 4;
  • the VL of (a)(ii) is the VL of W83, VL of W88, VL of W128, VL of W127, or VL of W162 disclosed in Table 3, and the VL of (a)(iii) is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V198, VL of V202, VL of V204, VL of V212, or VL of V217 disclosed in Table 4a.
  • the VL of (a)(ii) and (a)(iii) are the VL of W88 and V198, VL of W127 and V202, VL of V149 and W128, VL of W128 and V198, VL of W128 and V141 , VL of W162 and V204, VL of W83 and V217, VL of W88 and V90, or VL of W83 and V43, respectively, disclosed in Tables 3 and 4a, and the binding module comprises SEQ ID NO: 178.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain comprises SEQ ID NO: 4;
  • the VL of (a)(ii) is the VL of W83, VL of W88, VL of W206, VL of W128, VL of W127, or VL of W162 disclosed in Table 3, and the VL of (a)(iii) is the VL of V43, VL of V320, VL of V305, VL of V90, VL of V141 , VL of V245, VL of V149, VL of V198, VL of V202, VL of V204, VL of V212, or VL of V217 disclosed in Table 4b.
  • the VL of (a)(ii) and (a)(iii) are the VL of W206 and V245, VL of W88 and V198, VL of W127 and V202, VL of V149 and W128, VL of W128 and V198, VL of W128 and V141 , VL of W162 and V204, VL of W83 and V217, VL of W88 and V90, or VL of W83 and V43, respectively, disclosed in Tables 3 and 4b, and the binding module comprises SEQ ID NO: 178.
  • the binding modules are attached to the N- or -C terminus of the heavy or light chain through a linker. In some embodiments, the binding modules are attached to the C-terminus of the heavy or light chain. In some embodiments, the first binding module is attached to the C-terminus of the first heavy chain through a linker, and the second binding module is attached to C-terminus of the second heavy chain through the linker.
  • the proteinaceous molecule comprises both the heavy chain sequence and the two light chain (LC) sequences of any one of Bax#142, Lex#38, Lex#39, Bax#87, Bax#89, Lex#36, Lex#40 or Lex#41 disclosed in Table 10.
  • the proteinaceous molecule consists of two copies of the heavy chain sequence, one copy of the LC with lambda constant domain sequence, and one copy of the LC with kappa constant domain sequence of any one of Bax#142, Lex#38, Lex#39, Bax#87, Bax#89, Lex#36, Lex#40 or Lex#41 disclosed in Table 10.
  • Proteinaceous molecules which have a“2 by 2 trispecific” topology are depicted in Figure 3. These proteinaceous molecules are trispecific multivalent and comprise an equal number of antigen-binding sites that bind to Factor IXa, Factor X or another target. This sort of topology uses a monospecific scaffold.
  • the present invention provides a proteinaceous molecule comprising: (i) a scaffold module, wherein the scaffold module comprises a first and second antigen binding site that bind to Factor IXa or Factor X;
  • the third and fourth antigen-binding sites bind to Factor IXa or Factor X
  • the fifth and sixth antigen-binding site bind to phosphatidylserine, a phosphatidylserine-binding protein or a platelet surface marker
  • the proteinaceous molecule comprises two antigen-binding sites that bind to Factor IXa, two antigen-binding sites that bind to Factor X and two antigen binding sites that bind to phosphatidylserine, a phosphatidylserine-binding protein or a platelet surface marker
  • the phosphatidylserine-binding protein is Protein Z, Tissue Factor, Factor VIII, LOX- 1 , lactadherin, an oxysterol binding protein, Protein C, Protein S, Factor II (prothrombin), Factor V, Factor VII, Mer, 0 d b3 integrin, the CR3 complement receptor, the CR4 complement receptor, the phosphatidylserine receptor, annexins (preferably annexin V) or b2 ⁇ oorGq ⁇ b ⁇ h I .
  • the present invention provides a proteinaceous molecule comprising:
  • HCDR1 is SEQ ID NO: 1
  • HCDR2 is SEQ ID NO: 2
  • HCDR3 is SEQ ID NO:3;
  • binding modules that are scFvs or scFabs comprising:
  • HCDR1 is SEQ ID NO: 1
  • HCDR2 is SEQ ID NO: 2
  • HCDR3 is SEQ ID NO: 3
  • binding modules comprising an antigen-binding site each, wherein both of the antigen-binding sites bind to phosphatidylserine, a phosphatidylserine-binding protein or a platelet surface marker;
  • the proteinaceous molecule comprises two antigen-binding sites that bind to Factor IXa, two antigen-binding sites that bind to Factor X and two antigen-binding sites that bind to phosphatidylserine, a phosphatidylserine-binding protein or a platelet surface marker; and the phosphatidylserine-binding protein is Protein Z, Tissue Factor, Factor VIII, LOX-1 , lactadherin, an oxysterol binding protein, Protein C, Protein S, Factor II (prothrombin), Factor V, Factor VII, Mer, 0 d b3 integrin, the CR3 complement receptor, the CR4 complement receptor, the phosphatidylserine receptor, an annexin (preferably annexin V) or b2- glycoprotein I.
  • the phosphatidylserine-binding protein is Protein Z, Tissue Factor, Factor VIII, LOX-1 , lacta
  • the LCDR1 , LCDR2 and LCDR3 of (a)(ii) is the LCDR1 , LCDR2 and LCDR3 of W83, LCDR1 , LCDR2 and LCDR3 of W88, LCDR1 , LCDR2 and LCDR3 of W128, LCDR1 , LCDR2 and LCDR3 of W127, or LCDR1 , LCDR2 and LCDR3 of W162 disclosed in Table 1 , or the LCDR1 , LCDR2 and LCDR3 of (a)(ii) is the LCDR1 , LCDR2 and LCDR3 of V43, LCDR1 , LCDR2 and LCDR3 of V90, LCDR1 , LCDR2 and LCDR3 of V141 , LCDR1 , LCDR2 and LCDR3 of V149, LCDR1 , LCDR2 and LCDR3 of V198, LCDR1 , LCDR2 and LCDR3 of V202, LCDR1 , LCDR2 and LCDR3 of V204, LCDR1
  • the LCDR1 , LCDR2 and LCDR3 of each of the two light chains are identical, the LCDR1 , LCDR2 and LCDR3 of each of the two binding modules of (b) are identical and the two binding modules of (c) bind to the same target.
  • the present invention provides a proteinaceous molecule comprising:
  • HCDR1 is SEQ ID NO: 1
  • HCDR2 is SEQ ID NO: 2
  • HCDR3 is SEQ ID NO:3;
  • binding modules that are scFvs or scFabs comprising:
  • HCDR1 is SEQ ID NO: 1
  • HCDR2 is SEQ ID NO: 2
  • HCDR3 is SEQ ID NO: 3
  • binding modules comprising an antigen-binding site each, wherein both of the antigen-binding sites bind to phosphatidylserine, a phosphatidylserine-binding protein or a platelet surface marker;
  • the proteinaceous molecule comprises two antigen-binding sites that bind to Factor IXa, two antigen-binding sites that bind to Factor X and two antigen-binding sites that bind to phosphatidylserine, a phosphatidylserine-binding protein or a platelet surface marker; and the phosphatidylserine-binding protein is Protein Z, Tissue Factor, Factor VIII, LOX-1 , lactadherin, an oxysterol binding protein, Protein C, Protein S, Factor II (prothrombin), Factor V, Factor VII, Mer, 0 d b3 integrin, the CR3 complement receptor, the CR4 complement receptor, the phosphatidylserine receptor, an annexin (preferably annexin V) or b2- glycoprotein I.
  • the phosphatidylserine-binding protein is Protein Z, Tissue Factor, Factor VIII, LOX-1 , lacta
  • the LCDR1 , LCDR2 and LCDR3 of (a)(ii) is the LCDR1 , LCDR2 and LCDR3 of W83, LCDR1 , LCDR2 and LCDR3 of W88, LCDR1 , LCDR2 and LCDR3 of W128, LCDR1 , LCDR2 and LCDR3 of W127, or LCDR1 , LCDR2 and LCDR3 of W162 disclosed in Table 1 , or the LCDR1 , LCDR2 and LCDR3 of (a)(ii) is the LCDR1 , LCDR2 and LCDR3 of V43, LCDR1 , LCDR2 and LCDR3 of V245, LCDR1 , LCDR2 and LCDR3 of V90, LCDR1 , LCDR2 and LCDR3 of V141 , LCDR1 , LCDR2 and LCDR3 of V149, LCDR1 , LCDR2 and LCDR3 of V198, LCDR1 , LCDR2 and LCDR3 of V202, LCDR1
  • the LCDR1 , LCDR2 and LCDR3 of each of the two light chains are identical, the LCDR1 , LCDR2 and LCDR3 of each of the two binding modules of (b) are identical and the two binding modules of (c) bind to the same target.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of any one of the Factor X binding light chains disclosed in Table 1 , or the Factor IXa binding light chains disclosed in Table 2a, and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183;
  • binding modules comprising an antigen-binding site each, wherein both of the antigen-binding sites bind to phosphatidylserine, a phosphatidylserine-binding protein or a platelet surface marker;
  • the proteinaceous molecule comprises two antigen-binding sites that bind to Factor IXa, two antigen-binding sites that bind to Factor X and two antigen-binding sites that bind to phosphatidylserine, a phosphatidylserine-binding protein or a platelet surface marker; and the phosphatidylserine-binding protein is Protein Z, Tissue Factor, Factor VIII, LOX-1 , lactadherin, an oxysterol binding protein, Protein C, Protein S, Factor II (prothrombin), Factor V, Factor VII, Mer, 0 d b3 integrin, the CR3 complement receptor, the CR4 complement receptor, the phosphatidylserine receptor, an annexin (preferably annexin V) or b2- glycoprotein I.
  • the phosphatidylserine-binding protein is Protein Z, Tissue Factor, Factor VIII, LOX-1 , lacta
  • the VL of (a)(ii) is the VL of W83, VL of W88, VL of W128, VL of W127, or VL of W162 disclosed in Table 3, or the VL of (a)(ii) is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V198, VL of V202, VL of V204, VL of V212, or VL of V217 disclosed in Table 4a, and the LCDR1 , LCDR2 and LCDR3 of (b)(i) are that of V198, V202, W128, W88, W127, V212, or W162 disclosed in Tables 1 and 2a.
  • the VL of each of the two light chains are identical
  • the LCDR1 , LCDR2 and LCDR3 of each of the two binding modules of (b) are identical and the two binding modules of (c) bind to the same target.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of any one of the Factor X binding light chains disclosed in Table 1 , or the Factor IXa binding light chains disclosed in Table 2b, and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183;
  • binding modules comprising an antigen-binding site each, wherein both of the antigen-binding sites bind to phosphatidylserine, a phosphatidylserine-binding protein or a platelet surface marker;
  • the proteinaceous molecule comprises two antigen-binding sites that bind to Factor IXa, two antigen-binding sites that bind to Factor X and two antigen-binding sites that bind to phosphatidylserine, a phosphatidylserine-binding protein or a platelet surface marker; and the phosphatidylserine-binding protein is Protein Z, Tissue Factor, Factor VIII, LOX-1 , lactadherin, an oxysterol binding protein, Protein C, Protein S, Factor II (prothrombin), Factor V, Factor VII, Mer, 0 d b3 integrin, the CR3 complement receptor, the CR4 complement receptor, the phosphatidylserine receptor, an annexin (preferably annexin V) or b2- glycoprotein I.
  • the phosphatidylserine-binding protein is Protein Z, Tissue Factor, Factor VIII, LOX-1 , lacta
  • the VL of (a)(ii) is the VL of W83, VL of W88, VL of W128, VL of W127, or VL of W162 disclosed in Table 3, or the VL of (a)(ii) is the VL of V43, VL of V245, VL of V90, VL of V141 , VL of V149, VL of V198, VL of V202, VL of V204, VL of V212, or VL of V217 disclosed in Table 4b, and the LCDR1 , LCDR2 and LCDR3 of (b)(i) are that of W198, V198, V202, W128, W88, W127, V212, or W162 disclosed in Tables 1 and 2b.
  • the VL of each of the two light chains are identical
  • the LCDR1 , LCDR2 and LCDR3 of each of the two binding modules of (b) are identical and the two binding modules of (c) bind to the same target.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of any one of the Factor X binding light chains disclosed in Table 1 , or the Factor IXa binding light chains disclosed in Table 2a, and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183;
  • the proteinaceous molecule comprises two antigen-binding sites that bind to Factor IXa, two antigen-binding sites that bind to Factor X and two antigen-binding sites that bind to phosphatidylserine, a phosphatidylserine-binding protein or a platelet surface marker; and the phosphatidylserine-binding protein is Protein Z, Tissue Factor, Factor VIII, LOX-1 , lactadherin, an oxysterol binding protein, Protein C, Protein S, Factor II (prothrombin), Factor V, Factor VII, Mer, 0 d b3 integrin, the CR3 complement receptor, the CR4 complement receptor, the phosphatidylserine receptor, an annexin (preferably annexin V) or b2- glycoprotein I.
  • the phosphatidylserine-binding protein is Protein Z, Tissue Factor, Factor VIII, LOX-1 , lacta
  • the VL of (a)(ii) is the VL of W83, VL of W88, VL of W128, VL of W127, or VL of W162 disclosed in Table 3, or the VL of (a)(ii) is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V198, VL of V202, VL of V204, VL of V212, or VL of V217 disclosed in Table 4a, and the LCDR1 , LCDR2 and LCDR3 of (b)(i) are that of V198, V202, W128, W88, W127, V212, or W162 disclosed in Tables 1 and 2a.
  • the VL of each of the two light chains are identical
  • the LCDR1 , LCDR2 and LCDR3 of each of the two binding modules of (b) are identical and the two binding modules of (c) are identical.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of any one of the Factor X binding light chains disclosed in Table 1 , or the Factor IXa binding light chains disclosed in Table 2b, and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183;
  • the proteinaceous molecule comprises two antigen-binding sites that bind to Factor IXa, two antigen-binding sites that bind to Factor X and two antigen-binding sites that bind to phosphatidylserine, a phosphatidylserine-binding protein or a platelet surface marker; and the phosphatidylserine-binding protein is Protein Z, Tissue Factor, Factor VIII, LOX-1 , lactadherin, an oxysterol binding protein, Protein C, Protein S, Factor II (prothrombin), Factor V, Factor VII, Mer, 0 d b3 integrin, the CR3 complement receptor, the CR4 complement receptor, the phosphatidylserine receptor, an annexin (preferably annexin V) or b2- glycoprotein I.
  • the phosphatidylserine-binding protein is Protein Z, Tissue Factor, Factor VIII, LOX-1 , lacta
  • the VL of (a)(ii) is the VL of W83, VL of W88, VL of W128, VL of W127, or VL of W162 disclosed in Table 3, or the VL of (a)(ii) is the VL of V43, VL of V245, VL of V90, VL of V141 , VL of V149, VL of V198, VL of V202, VL of V204, VL of V212, or VL of V217 disclosed in Table 4b, and the LCDR1 , LCDR2 and LCDR3 of (b)(i) are that of W198, V198, V202, W128, W88, W127, V212, or W162 disclosed in Tables 1 and 2b.
  • the VL of each of the two light chains are identical
  • the LCDR1 , LCDR2 and LCDR3 of each of the two binding modules of (b) are identical and the two binding modules of (c) are identical.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of any one of the Factor X binding light chains disclosed in Table 1 , or the Factor IXa binding light chains disclosed in Table 2a, and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183, and
  • the proteinaceous molecule comprises two antigen-binding sites that bind to Factor IXa, two antigen-binding sites that bind to Factor X and two antigen-binding sites that bind to phosphatidylserine, a phosphatidylserine-binding protein or a platelet surface marker; and the phosphatidylserine-binding protein is Protein Z, Tissue Factor, Factor VIII, LOX-1 , lactadherin, an oxysterol binding protein, Protein C, Protein S, Factor II (prothrombin), Factor V, Factor VII, Mer, 0 d b3 integrin, the CR3 complement receptor, the CR4 complement receptor, the phosphatidylserine receptor, an annexin (preferably annexin V) or b2- glycoprotein I.
  • the phosphatidylserine-binding protein is Protein Z, Tissue Factor, Factor VIII, LOX-1 , lacta
  • the VL of (a)(ii) is the VL of W83, VL of W88, VL of W128, VL of W127, or VL of W162 disclosed in Table 3, or the VL of (a)(ii) is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V198, VL of V202, VL of V204, VL of V212, or VL of V217 disclosed in Table 4a, and the LCDR1 , LCDR2 and LCDR3 of (b)(i) are that of V198, V202, W128, W88, W127, V212, or W162 disclosed in Tables 1 and 2a.
  • the VL of each of the two light chains are identical
  • the LCDR1 , LCDR2 and LCDR3 of each of the two binding modules of (b) are identical and the two binding modules of (c) are identical.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of any one of the Factor X binding light chains disclosed in Table 1 , or the Factor IXa binding light chains disclosed in Table 2b, and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183, and
  • the proteinaceous molecule comprises two antigen-binding sites that bind to Factor IXa, two antigen-binding sites that bind to Factor X and two antigen-binding sites that bind to phosphatidylserine, a phosphatidylserine-binding protein or a platelet surface marker; and the phosphatidylserine-binding protein is Protein Z, Tissue Factor, Factor VIII, LOX-1 , lactadherin, an oxysterol binding protein, Protein C, Protein S, Factor II (prothrombin), Factor V, Factor VII, Mer, 0 d b3 integrin, the CR3 complement receptor, the CR4 complement receptor, the phosphatidylserine receptor, an annexin (preferably annexin V) or b2- glycoprotein I.
  • the phosphatidylserine-binding protein is Protein Z, Tissue Factor, Factor VIII, LOX-1 , lacta
  • the VL of (a)(ii) is the VL of W83, VL of W88, VL of W128, VL of W127, or VL of W162 disclosed in Table 3, or the VL of (a)(ii) is the VL of V43, VL of V245, VL of V90, VL of V141 , VL of V149, VL of V198, VL of V202, VL of V204, VL of V212, or VL of V217 disclosed in Table 4b, and the LCDR1 , LCDR2 and LCDR3 of (b)(i) are that of W198, V198, V202, W128, W88, W127, V212, or W162 disclosed in Tables 1 and 2b.
  • the VL of each of the two light chains are identical
  • the LCDR1 , LCDR2 and LCDR3 of each of the two binding modules of (b) are identical and the two binding modules of (c) are identical.
  • the present invention provides a proteinaceous molecule comprising: (a) an antibody or bivalent fragment thereof that comprises:
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of any one of the Factor X binding light chains disclosed in Table 1 , or the Factor IXa binding light chains disclosed in Table 2a, and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183;
  • the proteinaceous molecule comprises two antigen-binding sites that bind to Factor IXa, two antigen-binding sites that bind to Factor X and two antigen-binding sites that bind to phosphatidylserine, a phosphatidylserine-binding protein or a platelet surface marker; and the phosphatidylserine-binding protein is Protein Z, Tissue Factor, Factor VIII, LOX-1 , lactadherin, an oxysterol binding protein, Protein C, Protein S, Factor II (prothrombin), Factor V, Factor VII, Mer, 0 d b3 integrin, the CR3 complement receptor, the CR4 complement receptor, the phosphatidylserine receptor, an annexin (preferably annexin V) or b2- glycoprotein I.
  • the phosphatidylserine-binding protein is Protein Z, Tissue Factor, Factor VIII, LOX-1 , lacta
  • the VL of (a)(ii) is the VL of W83, VL of W88, VL of W128, VL of W127, or VL of W162 disclosed in Table 3, or the VL of (a)(ii) is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V198, VL of V202, VL of V204, VL of V212, or VL of V217 disclosed in Table 4a, and the LCDR1 , LCDR2 and LCDR3 of (b)(i) are that of V198, V202, W128, W88, W127, V212, or W162 disclosed in Tables 1 and 2a.
  • the VL of each of the two light chains are identical
  • the LCDR1 , LCDR2 and LCDR3 of each of the two binding modules of (b) are identical and the two binding modules of (c) are identical.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of any one of the Factor X binding light chains disclosed in Table 1 , or the Factor IXa binding light chains disclosed in Table 2b, and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183;
  • the proteinaceous molecule comprises two antigen-binding sites that bind to Factor IXa, two antigen-binding sites that bind to Factor X and two antigen-binding sites that bind to phosphatidylserine, a phosphatidylserine-binding protein or a platelet surface marker; and the phosphatidylserine-binding protein is Protein Z, Tissue Factor, Factor VIII, LOX-1 , lactadherin, an oxysterol binding protein, Protein C, Protein S, Factor II (prothrombin), Factor V, Factor VII, Mer, 0 d b3 integrin, the CR3 complement receptor, the CR4 complement receptor, the phosphatidylserine receptor, an annexin (preferably annexin V) or b2- glycoprotein I.
  • the phosphatidylserine-binding protein is Protein Z, Tissue Factor, Factor VIII, LOX-1 , lacta
  • the VL of (a)(ii) is the VL of W83, VL of W88, VL of W128, VL of W127, or VL of W162 disclosed in Table 3, or the VL of (a)(ii) is the VL of V43, VL of V245, VL of V90, VL of V141 , VL of V149, VL of V198, VL of V202, VL of V204, VL of V212, or VL of V217 disclosed in Table 4b, and the LCDR1 , LCDR2 and LCDR3 of (b)(i) are that of W198, V198, V202, W128, W88, W127, V212, or W162 disclosed in Tables 1 and 2b.
  • the VL of each of the two light chains are identical
  • the LCDR1 , LCDR2 and LCDR3 of each of the two binding modules of (b) are identical and the two binding modules of (c) are identical.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of W198 disclosed in Table 1 , and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183;
  • the proteinaceous molecule comprises two antigen-binding sites that bind to Factor IXa, two antigen-binding sites that bind to Factor X and two antigen-binding sites that bind to phosphatidylserine.
  • the VL of each of the two light chains are identical
  • the LCDR1 , LCDR2 and LCDR3 of each of the two binding modules of (b) are identical and the two binding modules of (c) are identical.
  • the present invention provides a proteinaceous molecule comprising:
  • VH domain is SEQ ID NO: 4;
  • a VL domain consisting of LCDR1 , LCDR2, LCDR3 and four framework regions, wherein the LCDR1 , LCDR2 and LCDR3 are the LCDR1 , LCDR2 and LCDR3 of W127 disclosed in Table 1 , and wherein FW1 is SEQ ID NO: 179, FW2 is SEQ ID NO: 180, FW3 is SEQ ID NO: 181 and FW4 is SEQ ID NO: 182, and
  • VH domain (ii) a VH domain, wherein the VH domain is SEQ ID NO: 183;
  • the proteinaceous molecule comprises two antigen-binding sites that bind to Factor IXa, two antigen-binding sites that bind to Factor X and two antigen-binding sites that bind to phosphatidylserine.
  • the VL of each of the two light chains are identical
  • the LCDR1 , LCDR2 and LCDR3 of each of the two binding modules of (b) are identical and the two binding modules of (c) are identical.
  • the binding modules are attached to the N- or -C terminus of the heavy or light chain through a linker. In some embodiments, the binding modules are attached to the C-terminus of the heavy or light chain. In some embodiments, the binding modules are attached to the C-terminus of the heavy chain and the C-terminus of the light chain.
  • the proteinaceous molecule comprises both the heavy chain sequence and light chain sequence of either Lex#68 or Lex#69 disclosed in Table 11.
  • the proteinaceous molecule comprises both the heavy chain sequence and light chain sequence of any one of Lex#134, Lex#135, Lex#68 or Lex#69 disclosed in Table 11.
  • the proteinaceous molecule consists of two copies of the heavy chain sequence and two copies of the light chain sequence of either Lex#68 or Lex#69 disclosed in Table 11. In some embodiments, the proteinaceous molecule consists of two copies of the heavy chain sequence and two copies of the light chain sequence of any one of Lex#134, Lex#135, Lex#68 or Lex#69 disclosed in Table 11.
  • 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 or antigen-binding fragment thereof 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 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
  • 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 through abilities such as retargeting and/or increased specificity. 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.
  • 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).
  • a first approach coined 'knob into hole' aims at forcing the pairing of two different IgG heavy chains by introducing mutations into the CH3 domains to modify the contact interface (Ridgway JB et al., Protein Eng 1996; 9 : 617-621). On one chain amino acids with large side chains were introduced, to create a 'knob'. Conversely, bulky amino acids were replaced by amino acids with short side chains to create a 'hole' into the other CH3 domain. By coexpressing these two heavy chains, more than 90% heterodimer formation was observed ('knobhole') versus homodimers formation ('hole -hole' or 'knob-knob').
  • camelids lamas and camels
  • cartilaginous fish nourse sharks
  • V-domains fused to a Fc demonstrating that a single domain can confer high affinity binding to an antigen.
  • 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 IXa/X scaffold 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 (see Delves et al., 2017. Roitt’s Essential Immunology 13 th edition. ISBN: 978-1-118-41577-1 for a description of Kappa and Lambda light chains).
  • Each antigen-binding site displays a different 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 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 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”.
  • An essential step of the method of producing KL-bodies 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. Numerous methods have been described for the generation of monoclonal antibodies and fragments thereof. (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 site 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 are then 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.
  • 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.
  • the bispecific antibodies 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.
  • Another step which may be 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. 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 disclosed herein 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.
  • the modulation of the expression can also be applied to the expression of proteinaceous molecules of the present invention.
  • Purified bispecific antibodies can be characterized as described in WO 2012/023053 A2.
  • the polynucleotide may be in an expression plasmid.
  • the expression plasmid may have any number of origins of replication known to those of ordinary skill in the art.
  • the polynucleotide or expression plasmid may be introduced into the host cell by any number of ways known to those of ordinary skill in the art. For example, a flow electroporation system, such as the MaxCyte GT®, MaxCyte VLX®, or MaxCyte STX® transfection systems, can be used to introduce the polynucleotide or expression plasmid into the host cell.
  • the polynucleotide sequence may be contained on one plasmid, or on more than one plasmid.
  • the heavy chain and light chain sequences would be on the same plasmid.
  • the heavy chains are on one plasmid and the light chains are on a separate plasmid.
  • the ratio of the plasmids is 1 :1.
  • the ratio of the plasmids is altered such as 1 :1 , 1 :5, 1 :10.
  • the antibody variant plasmid contains a selection marker for selecting stable cell lines.
  • different selection makers are on different plasmids. For example, one selection marker could be on the heavy chain plasmid while an alternate selection marker is on the light chain plasmid.
  • the host cell expresses the nucleic acid.
  • the host cell may express antibody variants at a level sufficient for fed-batch cell culture scale or other large scale.
  • Alternative methods to produce antibody variants at a large scale include roller bottle cultures, bioreactor batch cultures, perfusion and pseudoperfusion methods.
  • an antibody variant protein is produced by cells cultured in suspense. In some embodiments, an antibody variant protein is produced by adherent cells.
  • an antibody variant may be produced by any available means.
  • an antibody variant may be recombinantly produced by utilizing a host cell system engineered to express an antibody variant-encoding nucleic acid.
  • an antibody variant can be produced in vivo by mRNA therapeutics or AAV/lentiviral gene therapy.
  • antibody variants 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. Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
  • antibody variants are produced from human cells.
  • recombinant antibody variants 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. In some embodiments, 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. In some embodiments a cell line may be chosen for preferential post translational modifications (e.g., glycosylation). In some embodiments, such cells can be used with any tissue culture vessel or any vessel treated with a suitable adhesion substrate. In some embodiments, 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.
  • collagen e.g. collagen I, II, II, or IV
  • gelatin fibronectin
  • laminin laminin
  • vitronectin fibrinogen
  • fibrinogen BD MatrigelTM
  • basement membrane matrix e.g., dermatan sulfate proteoglycan, Poly
  • 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.
  • 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 recombinant antibody variant may comprise a transgene that encodes a recombinant antibody variant described herein. Cells can be engineered to express the antibody variant in a transient or a stable expression system.
  • nucleic acids encoding recombinant antibody variants may contain regulatory sequences, gene control sequences, promoters, non-coding sequences and/or other appropriate sequences for expressing the recombinant antibody variant.
  • the coding region is operably linked with one or more of these nucleic acid components.
  • antibody variants are expressed using a batch culture method. In some embodiments batch culture duration may be for 7-14 days. In some embodiments the batch culture may be for 14-21 days. In some embodiments antibody variants are expressed using a perfusion culture method (collection of culture medium over time each day). In some embodiments, antibody variants are expressed using a pseudoperfusion culture method (daily collection of culture medium at a single time point with replacement with fresh medium). In some embodiments specific feeding regimens/media may be used to promote optimal antibody variant production (improved glycan, reduce aggregate, improved kappa/lambda body ratio). In some embodiments the cell density may be controlled/maintained to promote optimal antibody variant production (reduced aggregate, improved heavy/light chain, improved kappa/lambda body ratio).
  • Total IgGs from all multivalent and trispecific antibodies were first purified by one affinity chromatography step using a Protein A resin (eg. Mab Select Sure resin from GE Healthcare). Elution was performed with 100mM Glycine pH3.0 and neutralized with 1 M Tris- HCI pH 9.
  • a Protein A resin eg. Mab Select Sure resin from GE Healthcare
  • IgGs were purified using a LambdaFabSelect resin (eg. from GE Healthcare) to get rid of IgGio (i.e. IgG comprising two light chains of Kappa type) followed by a Kappa resin (eg. Kappa XL from Thermo or KappaSelect from GE Healthcare) to eliminate the (i.e. IgG comprising two light chains of Lambda type).
  • a LambdaFabSelect resin eg. from GE Healthcare
  • Kappa resin eg. Kappa XL from Thermo or KappaSelect from GE Healthcare
  • the second purification step could either be a SEC column (eg. Hi Prep 16/60 S-300 Hr SEC Column (17-1167-01)) or a Ceramic Hydroxyapatite (type I) Column (HA).
  • SEC column eg. Hi Prep 16/60 S-300 Hr SEC Column (17-1167-01)
  • HA Ceramic Hydroxyapatite
  • additional traditional chromatography could also be used to achieve the desired purity and homogeneity of the antibody (eg. Ion exchange, multimodal or hydrophobic resins).
  • the Protein A eluted and neutralized antibodies were concentrated with a 100kd VivaSpin (P/N 28-9323-63) and 2.5mL of the concentrated antibody at ⁇ 15mg/mL was loaded (Running Buffer was 20mM HEPES, 150mM NaCI pH 6.5).
  • Final purified antibody was pooled based on SDS-PAGE and SEC-HPLC analysis demonstrating purity and homogeneity of the purified antibody. Final pool dialyzed into Storage Buffer 20mM HEPES, 150mM NaCI pH 6.5.
  • other methods of buffer exchange could be used, such as desalting columns, or untrafiltration to achieve final buffer storage conditions.
  • ProteinA purified antibody was dialyzed overnight at 4°C in dPBS.
  • the antibody was loaded at 30mg per mL onto the column (loading and wash buffer was 10mM Na2HPC>4 pH6.5).
  • a surface neutralization step using 25mM Tris 25mM NaCI, 5mM Na2HPC>4 pH 7.7 was performed for 5 column volumes (CV) and the antibody was then eluted with elutionl (10CV Step at 100% Elution Buffer 2M NaCI 10mM Na 2 HP0 4 100mM MES pH 6.5) followed by elution2 (10CV gradient at 100% Elution Buffer 500mM Na2HPC>4 pH 6.5).
  • Final purified antibody was pooled based on SDS-PAGE and SEC-HPLC analysis demonstrating purity and homogeneity of the purified antibody. Final pool dialyzed into Storage Buffer 20mM HEPES, 150mM NaCI pH 6.5.
  • Other methods of buffer exchange could be used, such as desalting columns, or ultrafiltration to achieve final buffer storage conditions.
  • the present invention comprises the items listed below. These items may be combined with any of the above aspects or embodiments.
  • a proteinaceous molecule having procoagulant activity comprising:
  • a scaffold module comprising a first antigen-binding site and a second antigen-binding site
  • Item 1 refers to a proteinaceous molecule which has three antigen-binding sites.
  • the target of one of the antigen-binding sites is not particularly limited as long as the binding to the target allows for procoagulant activity.
  • Item 2 defines that the first binding module and any one or more other binding modules are attached to the scaffold module through a linker. Possible linkers which could be used in this embodiment and any subsequent embodiment have been disclosed above.
  • Item 4 defines that the scaffold module if a“IXa/X” scaffold as discussed previously. These scaffold modules per se are bispecific.
  • the possible targets of the third antigen-binding site of the first binding module are defined.
  • the binding module may make the proteinaceous molecule trivalent bispecific, or trivalent trispecific.
  • the phosphatidylserine- binding protein is Protein Z, Tissue Factor, Factor VIII, LOX-1 , lactadherin, an oxysterol binding protein, Protein C, Protein S, Factor II (prothrombin), Factor V, Factor VII, Mer, a5b3 integrin, the CR3 complement receptor, the CR4 complement receptor, the phosphatidylserine receptor, an annexin, or b2 ⁇ oorGq ⁇ b ⁇ h I, preferably wherein the annexin is annexin V.
  • Item 6 refers to proteinaceous molecules which are trivalent trispecific.
  • the binding module provides the third valency and specificity.
  • This proteinaceous molecule will be similar to that of Figure 2 except that proteinaceous molecules only comprising one binding module are also encompassed.
  • Item 7 refers to proteinaceous molecules which are trivalent bispecific.
  • the binding module provides the third valency.
  • This proteinaceous molecule will be similar to the“3 by 1” or“1 by 3” molecules in Figure 1 except that proteinaceous molecules only comprising one binding module are also encompassed.
  • the first binding module of the trivalent bispecific embodiments comprising a IXa/X scaffold are further defined in item 8.
  • HCDR1 is SEQ ID NO: 1
  • HCDR2 is SEQ ID NO: 2
  • HCDR3 is SEQ ID NO: 3;
  • the first binding module is defined to comprise CDRs of binding arms that have been demonstrated to bind to Factor IXa or Factor X.
  • VH domain (i) a VH domain, wherein the VH domain is SEQ ID NO: 183;
  • FW1 is SEQ ID NO: 179
  • FW2 is SEQ ID NO: 180
  • FW3 is SEQ ID NO: 181
  • FW4 is SEQ ID NO: 182.
  • the first binding module is defined to comprise variable domain sequences that have been demonstrated to bind to Factor IXa or Factor X.
  • the first binding module of the trivalent trispecific embodiments comprising a IXa/X scaffold are further defined in item 12.
  • VL domain is SEQ ID NO: 184 and the VH domain is SEQ ID NO: 187,
  • VL domain is SEQ ID NO: 185 and the VH domain is SEQ ID NO: 188, or
  • VL domain is SEQ ID NO: 186 and the VH domain is SEQ ID NO: 189.
  • the first binding module is further defined through sequence information.
  • SEQ ID NO: 184 and SEQ ID NO: 187 are comprised in bavituximab which is known to bind domain II of b2 ⁇ IgoorGq ⁇ b ⁇ h I.
  • SEQ ID NO: 185 and SEQ ID NO: 188 are comprised in PS72 which is known to bind phosphatidylserine (Bujak et al., 2015. Invest New Drugs. 33(4):791-800).
  • SEQ ID NO: 186 and SEQ ID NO: 189 are comprised in anti-LIBS which is known to bind GPIIbllla (Stoll et al., 2007. Arterioscler Thromb Vase Biol. 27(5):1206-12).
  • the first binding module is defined by a specific sequence, wherein SEQ ID NO:190 is bavituximab, SEQ ID NO: 191 is PS72, and SEQ ID NO: 192 is anti-LIBS.
  • the first binding module is domain V of b2 ⁇ IgoorGq ⁇ b ⁇ h I which is known to bind phosphatidylserine.
  • the “IXa/X” scaffold is defined by a number of structural features.
  • item 19 restricts the“IXa/X” scaffold to a kl-body.
  • HCDR1 is SEQ ID NO: 1
  • HCDR2 is SEQ ID NO: 2
  • HCDR3 is SEQ ID NO:3;
  • the scaffold module is defined to comprise CDR sequences of binding arms that have been demonstrated to bind to Factor IXa or Factor X.
  • V sequences specific Factor IXa-binding arm sequences
  • W specific Factor X-binding arm sequences
  • VH domain is SEQ ID NO:
  • the scaffold module is defined to comprise variable domain sequences of binding arms that have been demonstrated to bind to Factor IXa or Factor X. 25.
  • V sequences specific Factor IXa-binding arm sequences
  • W specific Factor X- binding arm sequences
  • V sequences specific Factor IXa-binding arm sequences
  • W specific Factor X-binding arm sequences
  • the proteinaceous molecules comprise a monospecific scaffold wherein both antigen-binding sites of the scaffold module bind to Factor IXa or Factor X.
  • Embodiments with monospecific scaffolds comprising only one binding module comprising only one antigen-binding site can only be bispecific trivalent because the proteinaceous molecule must comprise at least one antigen-binding site that binds to Factor IXa and one antigen binding site that binds to Factor X.
  • 28. The proteinaceous molecule according to item 27, wherein the third antigen-binding site binds to Factor IXa or Factor X.
  • the antigen-binding site of the first binding module binds to Factor IXa or Factor X.
  • This proteinaceous molecule will be similar in structure as the“2 by 2” topology depicted in Figure 1 except that this embodiment includes proteinaceous molecules comprising only a single binding module.
  • This sort of topology could be achieved by, for example, fusing a Factor X-binding scFv to the C-terminus of a Factor IXa-binding light chain comprising a Kappa constant domain and using a second light chain that binds to Factor IXa that comprises a Lambda constant domain.
  • Items 29-32 then define the first binding module that binds to Factor IXa or Factor X in a similar manner as in items 8-11.
  • HCDR1 is SEQ ID NO: 1
  • HCDR2 is SEQ ID NO: 2
  • HCDR3 is SEQ ID NO: 3;
  • VH domain (i) a VH domain, wherein the VH domain is SEQ ID NO: 183;
  • FW1 is SEQ ID NO: 179
  • FW2 is SEQ ID NO: 180
  • FW3 is SEQ ID NO: 181
  • FW4 is SEQ ID NO: 182.
  • the monospecific scaffold is further defined by incorporating more and more structural and sequence information.
  • the Factor IXa- and Factor X-binding arms recited previously are also recited in these items.
  • HCDR1 is SEQ ID NO: 1
  • HCDR2 is SEQ ID NO: 2
  • HCDR3 is SEQ ID NO:3;
  • an embodiment of any one of items 36-38 is one where the two light chains comprise identical LCDR1 , LCDR2 and LCDR3 sequences.
  • the scaffold module comprises:
  • VH domain is SEQ ID NO: 4;
  • VL of each of the two light chains is the VL of W83, VL of W88, VL of W128, VL of W127, or VL of W162 disclosed in Table 3.
  • VL of each of the two light chains is the VL of V43, VL of V90, VL of V141 , VL of V149, VL of V198, VL of V202, VL of V204, VL of V212, or VL of V217 disclosed in Table 4a.
  • an embodiment of any one of items 39-41 is one where the two light chains comprise identical VL sequences.
  • Item 42 describes an embodiment wherein the proteinaceous molecule comprises a first and a second binding module.
  • Proteinaceous molecules encompassed by this embodiment include the tetravalent proteinaceous molecules depicted in Figures 1 and 2.
  • the second binding module contributes to the tetravalency of the proteinaceous molecule and may also contribute by making the proteinaceous molecule tri or tetra-specific.
  • Item 42 describes an embodiment wherein the first and second binding modules are the same and items 43-53 allows for the second binding module to be different from the first binding module (allows for tetraspecificity).

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