Factor x binders promoting FX activation

Abstract

SUMMARY OF THE INVENTION The problem to be solved by the present invention is to provide a FX-binding agent, such as a FX antibody or a fragment thereof, capable of stimulating the activation of FX, i.e. the generation of FXa from FX.SOLUTION: The present invention relates to an antibody or an antigen-binding fragment thereof capable of binding to FX, wherein said antibody or antigen-binding fragment is capable of: a. stimulating FX activation; b. stimulating FX activation by FXI; c. stimulating FX activation; d. stimulating FX activation by FXI; e. rendering FX more susceptible to proteolysis; or f. rendering FX more susceptible to proteolysis by FXI.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to coagulation factor X binders and their use in the treatment of clotting disorders such as hemophilia.

[0002] Incorporation by reference of sequence listing This application is submitted with an electronic Sequence Listing, the entire contents of which are incorporated herein by reference. [Background technology]

[0003] In patients with coagulation disorders, such as people with hemophilia A and B, various steps in the coagulation cascade malfunction, for example, due to the absence or insufficient presence of clotting factors. Malfunction of any part of such a coagulation cascade results in insufficient blood clotting and potentially life-threatening bleeding or damage to internal organs, such as joints.

[0004] Coagulation factor VIII (FVIII) deficiency, commonly referred to as hemophilia A, is a congenital bleeding disorder that affects approximately 420,000 people worldwide, of which approximately 105,000 are currently diagnosed.

[0005] Patients with hemophilia A may receive clotting factor replacement therapy, such as exogenous FVIII. Conventional treatment consists of replacement therapy, provided as prophylaxis for bleeding episodes or as on-demand treatment. The current standard of care for patients with severe hemophilia A is prophylactic intravenous infusions of either plasma-derived or recombinant FVIII, or their long-acting variants, up to three times per week.

[0006] However, such patients are at risk of developing neutralizing antibodies, so-called "inhibitors," against such exogenous factors, rendering previously effective therapy ineffective. Hemophilia A patients with "inhibitors" (i.e., alloantibodies to FVIII) are a non-limiting example of a coagulation disorder that is partly congenital and partly acquired. Patients who have developed "inhibitors" to FVIII cannot receive conventional replacement therapy, either as a prophylactic or on-demand procedure.

[0007] Furthermore, exogenous clotting factors can only be administered intravenously, which causes considerable inconvenience and discomfort to the patient. For example, infants and young children may need to have an intravenous catheter surgically inserted into a chest vein to ensure venous access. This greatly increases the risk of developing a bacterial infection.

[0008] In bleeding individuals, coagulation is initiated by the tissue factor / factor VIIa (TF / FVIIa) complex when extravascular TF is exposed to activated FVII (FVIIa) in the blood. Formation of the TF / FVIIa complex leads to the activation of factor X (FX) to activated factor Xa (FXa), which, together with activated factor V (FVa), generates limited amounts of thrombin.

[0009] Small amounts of thrombin activate platelets. Activated platelets support the assembly and binding of the tenase complex, which consists of activated factor VIII (FVIIIa) and activated factor IX (FIXa). The tenase complex is a highly efficient catalyst for FX activation, and the FXa generated in this second step functions as the active protease in the FVa / FXa prothrombinase complex, responsible for the final thrombin burst. Thrombin cleaves fibrinogen to generate fibrin monomers, which polymerize to form a fibrin network that seals leaking blood vessels and stops bleeding. A rapid and extensive thrombin burst is a prerequisite for the formation of a robust and stable fibrin clot.

[0010] Inadequate FXa formation and reduced thrombin generation resulting from the absence of coagulation factor FVIII activity are the underlying predisposing factors for bleeding in hemophilia A patients.

[0011] As mentioned, the proteolytic conversion of FX to the enzymatically active form, FXa, can be achieved by a unique FX activation complex containing FIXa and its cofactor, FVIIIa. Cofactor binding increases the enzymatic activity of FIXa by approximately five orders of magnitude and is thought to occur through multiple mechanisms, as reviewed by Scheiflinger et al. (2008) J Thromb Haemost, 6:315-322. In particular, FVIIIa has been shown to stabilize the structure of FIXa, which increased its proteolytic activity toward FX (Kolkman JA, Mertens K (2000) Biochemistry, 39:7398-7405; Zogg T, Brandstetter H (2009) Biol Chem, 390:391-400). Based on this observation and recognizing that antibodies are versatile binding proteins that can mimic various protein-protein interactions, Scheiflinger et al. performed a screen for agonistic anti-FIXa antibodies characterized by their ability to enhance FX activation by FIXa in the presence of phospholipid surfaces and calcium, but in the absence of the natural cofactor FVIIIa. From a screen of 5280 hybridoma supernatants, 88 antibodies were found to display varying degrees of FIXa agonist activity.

[0012] Antibodies that bind to factor X / Xa have been previously characterized (US Pat. No. 8,062,635), and emicizumab (US Pat. No. 9,334,331), known as ACE910, has been approved as a bypassing agent that mimics the action of FVIIIa by binding to factor IX / IXa with one arm and factor X / Xa with the other. The anti-FX arm of emicizumab binds to the light chain of FX and is not specific for the zymogen, but rather binds to FX and FXa with comparable affinity. The anti-FX arm is thought to primarily recruit FX and bring it into proximity with FIXa.

[0013] Thus, while mimicking the overall activity of FVIIIa, the FVIII mimetic compound emicizumab appears to lack some of FVIIIa's unique specificity and function. Specifically, FVIIIa functions by binding to FIXa and the zymogen FX on phospholipid surfaces, stimulating FIXa activity by approximately 200,000-fold and subsequently promoting FX activation by releasing FXa.

[0014] Mutants of ACE910 that are stated to have improved function are disclosed in WO2018 / 021450.

[0015] Further bispecific anti-FIX / anti-FX FVIII mimetic compounds are disclosed in WO2018 / 098363.

[0016] The present invention relates to FX binders that stimulate the production of FXa. Such molecules are therefore potentially useful therapeutic agents, either as individual compounds or as components of FVIII mimetic compounds. Summary of the Invention [Means for solving the problem]

[0017] The present invention relates to a group of FX binding agents, such as FX antibodies or fragments thereof, that are capable of stimulating the activation of FX, i.e., the production of FXa from FX, as well as the identification of such FX binding agents.

[0018] The present invention further relates to a group of FX-binding agents, including antibodies and antigen-binding fragments thereof, containing immunoglobulin single variable domains, such as Nanobodies®, that are capable of binding to zymogens but not activated FXa.

[0019] Binding of FX by the FX binders disclosed herein stimulates subsequent activation of FX by FIXa. In one embodiment, binding occurs via a peptide extension in the activation peptide of FX. Thus, the FX binders disclosed herein have been observed to stimulate FX activation by making FX more susceptible to FIXa-mediated activation.

[0020] In one aspect, specific binding of the zymogen form of FX to the activated form (FXa) prevents capture of FX binders to nascent FXa, thereby increasing the amount of FX binder available for interaction with zymogen FX. Furthermore, specific binding to zymogen FX avoids potential inhibitory effects on FXa activity (such as interference with prothrombinase complex assembly).

[0021] The FX-binding agents disclosed herein exhibit unexpected functional properties by binding to FX in a manner that renders FX more susceptible to proteolysis by FIXa. It is the inventors' surprising finding that increased proteolysis of FX by FIXa can be achieved by binding FX in a manner that renders FX more susceptible to proteolysis by FIXa, and consequently more susceptible to activation. The art provides examples of anti-FIXa compounds that stimulate FIXa proteolytic activity, resulting in the conversion of FX to FXa. However, to the applicant's knowledge, no compounds capable of binding to FX and stimulating FX activation by rendering FX more susceptible to proteolysis by FIXa have been previously known.

[0022] One aspect of the present invention relates to FX binding agents, such as antibodies or antigen-binding fragments thereof, which are capable of stimulating FX activation. Another aspect of the present invention relates to the identification of such compounds.

[0023] A further aspect of the present invention relates to the use of such FX binders in methods of treatment, possibly, but not exclusively, as part of a FVIII mimetic compound. Stimulation of FX activation is useful for stimulating coagulation, and such molecules are therefore suitable for the treatment of coagulation disorders such as hemophilia, including hemophilia A and B.

[0024] Sequence Listing Overview SEQ ID NO:1 - Human Factor IX zymogen YNSGKLEEFV QGNLERECME EKCSFEEARE VFENTERTTE FWKQYVDGDQ CESNPCLNGG 60 SCKDDINSYE CWCPFGFEGK NCELDVTCNI KNGRCEQFCK NSADNKVVCS CTEGYRLAEN 120 QKSCEPAVPF PCGRVSVSQT SKLTRAEAVF PDVDYVNSTE AETILDNITQ STQSFNDFTR 180 VVGGEDAKPG QFPWQVVLNG KVDAFCGGSI VNEKWIVTAA HCVETGVKIT VVAGEHNIEE 240 TEHTEQKRNV IRIIPHHNYN AAINKYNHDI ALLELDEPLV LNSYVTPICI ADKEYTNIFL 300 KFGSGYVSGW GRVFHKGRSA LVLQYLRVPL VDRATCLRST KFTIYNNMFC AGFHEGGRDS 360 CQGDSGGPHV TEVEGTSFLT GIISWGEECA MKGKYGIYTK VSRYVNWIKE KTKLT SEQ ID NO:2 - Human Factor X zymogen ANSFLEEMKK GHLERECMEE TCSYEEAREV FEDSDKTNEF WNKYKDGDQC ETSPCQNQGK 60 CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 120 GKACIPTGPY PCGKQTLERR KR SVAQATSS SGEAPDSITW KPYDAADLDP TENPFDLLDF 180 NQTQPERGDN NLTR IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 240 AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 300 ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 360 NMFCAGYDTK QEDACQGDSG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 420 WIDRSMKTRG LPKAKSHAPE VITSSPLK underline : Activation peptide (aa 143-194). Bold: "C common minimal epitope" (aa 177-178) "Minimal common epitope" on SEQ ID NO:3-FX (shown in bold in SEQ ID NO:2) LL SEQ ID NO:4 - Heavy chain variable domain mAb 00916 QSVEESGGRL VTPGTPLTLT CTVSGFSLS S YAMS WVRQAP GKGLEWIG II STSGSTFYAT 60 WAKG RFTISK TSTTVDLKII SPTIEDTATY FCAR DMWAGS RYDFNI WGPG TLVTVSS 117 SEQ ID NO:5 - Light chain variable domain mAb 00916 ADIVMTQTPA SVEAAVGGTV TIKC QASQSI SSYLA WYQQK PGQRPKLLIY RASTLES GVP 60 SRFKGSGSGT QFTLTISDLE CADAATYYC Q TYYYSGGGSY ASA FGGGTEV VVK 113 SEQ ID NO:6—Heavy chain variable domain mAb 13F62 EVQLVESGGG QVKPGGSLRL SCAASGFTFS TSSIY WVRQA PGKGLEWVS S ISSGSSYIFY 60 ADSVKG RFTI SRDNAKNSLF LQMNSLRAED TAVYYCVS GY SRLLDY WGQG TLVTVSS 117 SEQ ID NO:7 - Light chain variable domain mAb 13F62 DVVMTQSPLS LPVTLGQPAS ISC RSSQSLV YSDGNTYLN W FQQRPGQSPR RLIY KVSNRD 60 S GVPDRFSGSGSGTDFTLKI SRVEAEDVGF YYC MQGTHWP LT FGGGTKVE IK 112 SEQ ID NO:8-Nb 701B09 EVQLVESGGGVVQPGGSLRLSCAAS GRTFSTYAMG WFRQAPGKEREFVA AISWSGSRTY YADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAA DATPANGELDY WGQGTLVTVSS SEQ ID NO:9-Nb 701C06 EVQLVESGGGVVQPGGSLRLSCAAS GRTFSTYAMG WFRQAPGKEREFVA AISRRGGRTY YADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAA DATARDGLLDY WGQGTLVTVSS SEQ ID NO: 10 - Nb 702C12 EVQLVESGGGVVQPGGSLRLSCVAS GRTFSRYAMG WFRQAPGKEREFVA AISRRGGSTNY ADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAA DYSSGDGYLDY WGQGTLVTVSS SEQ ID NO: 11-Nb 701D07 EVQLVESGGGVVQPGGSLRLSCAAS GGTLSRYAMG WFRQAPGKEREFVA AITRRGSRTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAA DLAPGDYALDY WGQGTLVTVSS SEQ ID NO: 12 - Nb 501A02 EVQLVESGGGLVQPGGSLRLSCAAS GFTFSNYEMS WVRQAPGKGLEWVS DIGSGGGVSTY YADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAMYYCAR GWTYGSDGMDY WGKGTLVTVSS SEQ ID NOs: 13-53 correspond to the sequences listed in Table 5 of Example 5. SEQ ID NO:54 - Nb 721E08 EVQLVESGGGVVQPGGSLRLSCAAS GRTFSTYSMG WFRQAPGKEREFVA AITRRGSRTY YADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAA DRRPADSYLDY WGQGTLVTVSS SEQ ID NO: 55 - Nb 729A04 EVQLVESGGGVVQPGGSLRLSCAAS GFTFSDYAMS WVRQAPGKGLEWVS GITSGGGRTY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCAA VRTLGLRGSYDY WGQGTLVTVSS SEQ ID NO:56 - Nb 729C08 EVQLVESGGGVVQPGGSLRLSCAAS GFTFSDYAMS WVRQAPGKGLEWVS GITGGGRTY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCAA AILTRTRRTYDY WGQGTLVTVSS SEQ ID NO:57 - Nb 730C03 EVQLVESGGGVVQPGGSLRLSCVAS GFTFSDYAMS WVRQAPGKGLEWVS GITSGSGRTY YADSVKGRFTISRDNSaKNTVYLQMNSLRPEDTALYYCAA AILRGYRKTYDY WGQGTLVTVSS

[0025] The underlined sequences represent the CDRs of mAbs using Kabat numbering and definitions, whereas the CDRs of Nanobodies® (Nbs) are determined using Kabat numbering and AbM definitions as described in Kontermann and Dubel (2010, Eds., Antibody Engineering, vol. 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51). DETAILED DESCRIPTION OF THE INVENTION

[0026] FIX is a vitamin K-dependent coagulation factor structurally similar to factor VII, prothrombin, FX, and protein C. The circulating zymogen form consists of 415 amino acids divided into four distinct domains, including an N-terminal gamma-carboxyglutamic acid-rich (Gla) domain, two EGF domains, and a C-terminal trypsin-like serine protease domain. FIX circulates in plasma as a single-chain zymogen (SEQ ID NO: 1). Activation of FIX occurs by limited proteolysis at Arg145 and Arg180, releasing the activation peptide (residues 146-180 of SEQ ID NO: 1). Thus, activated FIX (FIXa) consists of residues 1-145 of SEQ ID NO: 1 (light chain) and residues 181-415 of SEQ ID NO: 1 (heavy chain).

[0027] FIXa is a trypsin-like serine protease that, as part of the tenase complex, plays a key role in hemostasis by generating most of the FXa required to support proper thrombin formation during coagulation.

[0028] FX is a vitamin K-dependent coagulation factor structurally similar to factor VII, prothrombin, FIX, and protein C. It is synthesized using a preprosequence containing a hydrophobic signal sequence that targets the protein for secretion. The propeptide is important for directing γ-carboxylation to the FX light chain. The human FX zymogen contains four distinct domains, including an N-terminal gamma-carboxyglutamic acid-rich (Gla) domain, two EGF domains, EGF1 (residues 46-82) and EGF2 (residues 85-125), and a C-terminal trypsin-like serine protease domain (residues 195-448). FX circulates in plasma as a two-chain zymogen, comprising residues 1-139 of SEQ ID NO:2 (light chain) and residues 143-448 of SEQ ID NO:2 (heavy chain). Activation of FX occurs by limited proteolysis at Arg194, resulting in the release of the activation peptide (residues 143-194). Thus, activated FX (FXa) is composed of residues 1 to 139 of SEQ ID NO:2 (light chain) and residues 195 to 448 of SEQ ID NO:2 (activated heavy chain).

[0029] As used herein, the term "binding agent" is understood in its broadest sense and includes, for example, lectins, proteins, polypeptides and peptides, as well as all FX-binding molecules or substances. FX-binding molecules include, for example, antibodies and antigen-binding fragments thereof (such as, but not limited to, single variable domain antibodies, Nanobodies®, Fab, Fab'2, Fv, and scFv), affibodies, adnectins, anticalins, DARPins, and avimers.

[0030] As used herein, the term "antibody" refers to a protein derived from an immunoglobulin sequence that can bind to an antigen or a portion thereof. The term antibody includes, but is not limited to, full-length antibodies of any class (or isotype), i.e., IgA, IgD, IgE, IgG, IgM, and / or IgY. An antibody that specifically binds to an antigen, or a fragment thereof, may bind exclusively to that antigen, or a portion or fragment thereof, or may bind to a limited number of homologous antigens. Typically, the simple terms "bind" or "bind to" are used, and it is generally understood that the binding of an antibody or its fragment is "specific." If an epitope is shared between homologous antigens, it is logical that the antibody can also bind to such highly homologous antigens. The term antibody includes antibodies that are multivalent, e.g., bivalent, such as bispecific antibodies.

[0031] A natural full-length antibody contains at least four polypeptide chains: two heavy chains (HC) and two light chains (LC) linked by disulfide bonds. In some cases, natural antibodies contain fewer than four chains, as in the case of IgNAR found in Chondrichthyes. One class of immunoglobulin with particular pharmaceutical interest is IgG. In humans, the IgG class can be divided into four subclasses, IgG1, IgG2, IgG3, and IgG4, based on the sequences of their heavy chain constant regions. Light chains can be divided into two types, kappa chains and lambda chains, based on differences in their sequence composition. IgG molecules are composed of two heavy chains linked by two or more disulfide bonds and two light chains, each linked to a heavy chain by a disulfide bond. IgG heavy chains contain a heavy chain variable domain (V H ) and up to three heavy chain constants (C H ) Domain: C H 1. C H 2, and C H 3. The light chain may comprise a light chain variable domain (V L ) and the light chain constant domain (C L ) may be included. V H and V LDomains can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs) or hypervariable regions (HvRs), interspersed with more conserved regions, called framework regions (FRs). H and V L A domain typically consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy and light chain variable domains, containing the hypervariable regions (CDRs), form a structure capable of interacting with an antigen, while the antibody constant region can mediate the binding of the immunoglobulin to host tissues or factors, including, but not limited to, various cells of the immune system (effector cells), Fc receptors, and the first component, C1q, of the C1 complex of the classical complement system.

[0032] The antibodies of the present invention may be monoclonal antibodies (mAbs), in the sense that they represent a set of unique heavy and light chain variable domain sequences expressed from a single B cell or by a clonal population of B cells. The antibodies of the present invention may be produced and purified using a variety of methods known to those skilled in the art. For example, the antibodies may be produced from hybridoma cells. The antibodies may be produced by B cell proliferation. The antibodies or fragments thereof may be recombinantly expressed in mammalian or microbial expression systems or by in vitro translation. The antibodies or fragments thereof may also be recombinantly expressed as cell surface binding molecules, for example, by phage display, bacterial display, yeast display, mammalian cell display, or ribosomal or mRNA display.

[0033] The antibodies of the present invention may be isolated. The term "isolated antibody" refers to an antibody that has been separated and / or recovered from other component(s) in the environment in which it was produced and / or purified from a mixture of components present in the environment in which it was produced.

[0034] Certain antigen-binding fragments of antibodies may be suitable in the context of the present invention, as it has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. The term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to bind to or recognize an antigen, such as FX or another target molecule, as described herein. Examples of antigen-binding fragments include Fab, Fab', Fab2, Fab'2, Fv (typically the V of a single arm of an antibody), and the like. L Domains and V H domain combinations), single-chain Fv (scFv) (see, e.g., Bird et al., Science 1988;242:423-426, and Huston et al., PNAS 1988;85:5879-5883), dSfv, Fd (typically a V H and C H 1 domain), a single V H and a single V L These include, but are not limited to, monovalent molecules comprising chains, minibodies, diabodies, triabodies, tetrabodies, and kappabodies (see, e.g., Ill et al. (1997) Protein Eng 10:949-57), as well as one or more isolated CDRs or functional paratopes, which isolated CDRs or antigen-binding residues or polypeptides can be associated or joined together to form functional antibody fragments. These antibody fragments may be obtained using conventional techniques known to those skilled in the art, and the fragments can be screened for utility in the same manner as intact antibodies.

[0035] "Fab fragments" of antibodies, including "Fab" and "Fab'2" fragments, can be derived from the antibody by cleavage of the heavy chain at the hinge region on the N-terminus or C-terminus of the hinge cysteine ​​residues connecting the heavy chains of the antibody. A "Fab" fragment contains the variable and constant domains of the light chain and the variable and constant domains of the heavy chain. H1 domain. An "Fab'2" fragment comprises a pair of "Fab" fragments generally covalently linked by their hinge cysteines. Fab' is formally derived from a Fab'2 fragment by cleavage of the hinge disulfide bond connecting the heavy chains in Fab'2. Other chemical bonds of antibody fragments besides disulfide bonds are also known in the art. Fab fragments retain the ability of the parent antibody to bind to its antigen, potentially with lower affinity. Fab and Fab' fragments are capable of monovalent binding, while Fab'2 fragments are capable of bivalent binding. Generally, Fab fragments contain a constant C H 2 domain and C H Fab fragments lack the Fc domain, i.e., the Fc portion where interaction with Fc receptors and C1q occurs. Thus, Fab fragments generally lack effector function. Fab fragments can be produced by methods known in the art, for example, by enzymatic cleavage of antibodies using papain to obtain Fab or pepsin to obtain Fab'2, and Fab fragments, including Fab, Fab', and Fab'2, can be produced recombinantly using techniques well known to those skilled in the art.

[0036] An "Fv" (fragment variable) fragment is an antibody fragment that contains a complete antigen recognition and binding site, and generally comprises a dimer of one heavy- and one light-chain variable domain in a naturally covalently linked association, e.g., in a single-chain variable domain fragment (scFv). In this configuration, the three hypervariable regions of each variable domain interact to form a V H -V L Collectively, the six hypervariable regions, or a subset thereof, define an antigen-binding site on the surface of the dimer and confer antigen-binding specificity to the antibody.

[0037] "Single-chain Fv" or "scFv" antibodies are antibodies that contain the V H Domains and V L Fv polypeptides generally comprise V domains, and these domains are present in a single polypeptide chain. H Domains and V LThe scFv may further comprise a polypeptide linker between the domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Pluckthun, 1994, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315.

[0038] "Single-chain Fab" or "scFab" antibodies are antibodies with V H , C H 1. V L and C L Fab polypeptides generally contain V domains, which enable the scFab to form the desired structure for antigen binding. H Domain and C L Between domains or V L Domain and C H It further includes any polypeptide linker between the 1 domain (Koerber et al., (2015) J Mol Biol. 427:576-86).

[0039] The term "diabody" refers to a small antibody fragment with two antigen-binding sites, wherein the fragments are bound to the same polypeptide chain (V H and V L ) in the light chain variable domain (V L ) connected to the heavy chain variable domain (V H By using a linker that is too short to allow pairing between two variable domains on the same chain, the variable domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0040] The term "linear antibody" refers to antibodies as described in Zapata et al. (1995) Protein Eng. 8:1057-1062. Briefly, these antibodies comprise a pair of tandem Fd segments (V) which, together with complementary light chain polypeptides, form a pair of antigen-binding regions. H -C H 1-V H -C H 1). Linear antibodies can be bispecific or monospecific.

[0041] Antibody fragments may be obtained using conventional recombinant or protein engineering techniques, and the fragments can be screened for binding to an antigen, such as FX, in the same manner as intact antibodies.

[0042] Antibody fragments of the invention can be produced by truncation, for example, by removal of one or more amino acids from the N-terminus and / or C-terminus of the polypeptide. Fragments can also be generated by one or more internal deletions.

[0043] The antibody of the present invention may be or comprise an antibody fragment or a variant of any one of the antibodies disclosed herein. The antibody of the present invention may be or comprise an antigen-binding portion of one of these antibodies or a variant thereof. For example, the antibody of the present invention may be an Fab fragment of one of these antibodies or a variant thereof, or a single-chain antibody derived from one of these antibodies or a variant thereof. The antibody of the present invention may also be a combination of a full-length antibody and a fragment thereof.

[0044] However, even a single variable domain containing only the three hypervariable regions specific for an antigen can retain the ability to recognize and bind antigen with high affinity, although usually at a lower affinity than the entire binding site (Cai and Garen (1996) PNAS 93:6280-6285). Naturally occurring camelid antibodies (V) containing only heavy chain variable domains HH) can bind to antigens (Desmyter et al., (2002) J. Biol. Chem. 277:23645-23650; Bond et al., (2003) J. Mol. Biol. 332:643-655). IgNAR (or V NAR Similar to the 10th FN3 domain, single domain antibody binding regions are available from cartilaginous fish. An alternative is offered by Creative Biolabs, which uses the human 10th fibronectin FN3 domain (10FN3 domain) as a scaffold. Such antibody molecules are called single variable domains, immunoglobulin single variable domains, or single variable domain fragments. The molecules are monomeric and typically range in size from 12 to 15 kDa.

[0045] In particular, the immunoglobulin single variable domain or single variable domain may be a Nanobody® (as defined herein) or a suitable fragment thereof. For a general description of Nanobodies®, see e.g. WO2008 / 020079 (page 16).

[0046] The term "immunoglobulin single variable domain", which is used interchangeably with "single variable domain", defines a molecule in which an antigen-binding site is present and which is formed by a single immunoglobulin domain. This sets immunoglobulin single variable domains apart from "conventional" immunoglobulins or fragments thereof (e.g., Fab, scFv, etc.), but which, as mentioned above, are composed of two immunoglobulin domains, in particular two variable domains (heavy chain variable domains (V)). H ) and the light chain variable domain (V L ) interact to form the antigen-binding site. In contrast, the binding site of an immunoglobulin single variable domain consists of a single V H or V L The antigen-binding site of an immunoglobulin single variable domain is therefore formed by no more than three CDRs.

[0047] Thus, the terms "immunoglobulin single variable domain" and "single variable domain" do not include conventional immunoglobulins or fragments thereof, which require the interaction of at least two variable domains to form an antigen-binding site. However, these terms do include fragments of conventional immunoglobulins, in which the antigen-binding site is formed by a single variable domain.

[0048] Generally, a single variable domain is a polypeptide sequence consisting essentially of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), or a suitable fragment of such a polypeptide sequence (which usually contains at least a portion of the amino acid residues forming at least one of the CDRs). The amino acid sequence and structure of an immunoglobulin sequence, particularly an immunoglobulin single variable domain such as a Nanobody®, can be considered to consist of four framework regions or "FRs," referred to in the art and herein as, but not limited to, "framework region 1" or "FR1," "framework region 2" or "FR2," "framework region 3" or "FR3," and "framework region 4" or "FR4," respectively, with the framework regions being separated by three complementarity-determining regions or "CDRs," referred to in the art as "complementarity-determining region 1" or "CDR1," "complementarity-determining region 2" or "CDR2," and "complementarity-determining region 3" or "CDR3," respectively.

[0049] The total length of an immunoglobulin single variable domain is typically, for example, 112 to 115, most preferably 113 amino acid residues for Nanobodies® in the region of 110 to 120 amino acid residues.

[0050] Such single variable domains and fragments are most preferred because they comprise an immunoglobulin fold or are capable of forming an immunoglobulin fold under suitable conditions. As such, a single variable domain can form a single antigen-binding unit (i.e., bind to, for example, another variable domain (e.g., a V H / V L For example, a light chain variable domain sequence (e.g., V) can be used as long as it is capable of forming a functional antigen-binding unit (a functional antigen-binding unit consisting essentially of a single variable domain), such that a single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit, as is the case for variable domains present in conventional antibodies and scFv fragments, which need to interact (through interactions) to form a functional antigen-binding domain. L sequence) or a suitable fragment thereof, or a heavy chain variable domain sequence (e.g., V H Array or V H H sequence) or a suitable fragment thereof.

[0051] In one embodiment of the present invention, the immunoglobulin single variable domain comprises a light chain variable domain sequence (e.g., V L sequence), or heavy chain variable domain sequence (e.g., V H sequence), and more particularly, the immunoglobulin single variable domain may be a heavy chain variable domain sequence derived from a conventional four-chain antibody, or a heavy chain variable domain sequence derived from a heavy chain antibody.

[0052] For example, a single variable domain or immunoglobulin single variable domain (or an amino acid sequence suitable for use as an immunoglobulin single variable domain) may be used in the design of a (single) domain antibody, a "dAb" or an "sdAb" or a Nanobody® (V H H, humanized V H H, or camelized V H(Single) domain antibodies may be referred to as (single) domain antibodies, other single variable domains, or suitable fragments of any one of them. For a review of (single) domain antibodies, see, for example, EP 0368684. For the term "dAb", see, for example, Ward et al., 1989 (Nature 341:544), Holt et al., 2003 (Trends Biotechnol. 21:484), and published patent applications by Domantis Ltd., e.g., WO2004 / 068820, WO2006 / 030220, WO2006 / 003388, and others.

[0053] Thus, in the sense of the present invention, the term "immunoglobulin single variable domain" or "single variable domain" includes polypeptides derived from a non-human source, preferably from Camelidae, preferably Camelidae heavy chain antibodies (e.g., V H Hs). They may also be humanized (e.g., humanized V H Hs). Furthermore, the term includes polypeptides from non-camelid sources, such as mouse or human, that have been "camelized" (e.g., camelized V), as described, for example, in Davies and Riechmann, 1994 (FEBS 339:285), 1995 (Biotechonol. 13:475), and 1996 (Prot. Eng. 9:531), and Riechmann and Muyldermans, 1999 (J. Immunol. Methods 231:25). H domain). As used herein, the term "monospecific" antibody refers to an antibody that is capable of binding to one particular epitope (including, but not limited to, bivalent antibodies).

[0054] As used herein, the term "bispecific" antibody refers to an antibody that can bind to two different antigens or two different epitopes on the same antigen.

[0055] As used herein, the term "trispecific" antibody refers to an antibody that can bind to three different antigens, or three different epitopes on the same antigen, or three different epitopes present on two different antigens.

[0056] As used herein, the term "multispecific" antibody refers to an antibody that can bind to two or more different antigens or two or more different epitopes on the same antigen. Thus, multispecific antibodies include bispecific and trispecific antibodies.

[0057] Full-length IgG bispecific antibodies can be generated by fusing two separate hybridomas to form hybrid quadromas, generating a mixture of antibodies containing bispecific heterodimerized antibody fragments (Chelius D. et al., MAbs. 2010 May-Jun;2(3):309-319). Alternatively, bispecific heterodimerized antibodies can be generated using recombinant techniques. Heterodimerization can also be achieved by engineering the dimerization interface of the Fc region to promote heterodimerization. One example is the so-called knob-in-hole mutation, in which a sterically bulky side chain (knob) is introduced on one Fc that matches a sterically small side chain (hole) on the opposing Fc, thereby creating steric complementarity that promotes heterodimerization. Other methods for engineering heterodimerized Fc interfaces include electrostatic complementarity, fusion to non-IgG heterodimerization domains, or exploiting the natural Fab arm exchange phenomenon of human IgG4 to control heterodimerization. Examples of heterodimerized bispecific antibodies are described in detail in the literature, for example, (Klein C. et al., MAbs. 2012 Nov-Dec;4(6):653-663). Particular attention must be paid to the light chain of the heterodimeric antibody. Correct pairing of the LC and HC can be achieved by using a common light chain. Again, manipulation of the LC / HC interface can be used to facilitate heterodimerization or light chain crossover engineering, as in the case of CrossMab. Bispecifics can also be generated using in vitro antibody reconstitution under mild reducing conditions from two individual IgGs containing appropriate mutations (e.g., Labrijn et al., PNAS, 110, 5145-5150 (2013)). A native Fab arm exchange method has also been reported to ensure correct light chain pairing.

[0058] Multispecific antibody-based molecules can also be recombinantly expressed as fusion proteins combining natural modules of IgG to form multispecific and multivalent antibody derivatives as described in the literature. Examples of fusion antibodies are DVD-Ig, IgG-scFV, diabodies, DARTs, etc. Specific detection or purification tags, half-life extending moieties, or other components can be incorporated into the fusion protein. Additional non-IgG modalities can also be incorporated into the fusion protein. C Bispecific full-length antibodies based on heterodimerization are commonly referred to as asymmetric IgGs, regardless of the LC pairing method. In general, bispecific antibodies can be generated in a variety of molecular formats, as outlined by Brinkmann et al. (Brinkmann et al., The making of bispecific antibodies. Mabs 9, 182-212 (2017)).

[0059] Multispecific antibody-based molecules can also be generated by chemically binding or coupling individual full-length IgGs or coupling IgG fragments to form multispecific and multivalent antibody derivatives, as described in the literature. Examples of fusion antibodies include chemically conjugated Fab'2, IgG dimers, etc. Specific detection or purification tags, half-life extensions, or other components can be incorporated into the conjugated protein. Additional non-IgG polypeptides can also be incorporated into the fusion protein. Multispecific molecules can also be generated by combining recombinant and chemical methods, including those described above.

[0060] The anti-FX antibodies or antigen-binding fragments thereof disclosed herein can be used as part of a multispecific procoagulant antibody. Accordingly, one aspect of the present invention relates to individual component (intermediate) anti-FX antibodies or antigen-binding fragments thereof suitable for use in multispecific procoagulant antibodies, such as bispecific procoagulant antibodies. In one aspect, such bispecific (or multispecific) antibodies are capable of binding to FIX and / or its activated form, FIXa, and FX.

[0061] In one embodiment, the antibody of the present invention is a chimeric, human, or humanized antibody. Such antibodies can be generated, for example, by using a suitable antibody display or immunization platform, or any other suitable platform or method known in the art. As used herein, the term "human antibody" is intended to include antibodies having a variable domain in which at least a portion of the framework region and / or at least a portion of the CDRs are derived from human germline immunoglobulin sequences. (For example, a human antibody can have a variable domain in which both the framework and CDRs are derived from human germline immunoglobulin sequences.) Furthermore, if the antibody contains a constant region, the constant region, or a portion thereof, also is derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0062] In one embodiment, such human antibodies are human monoclonal antibodies. Such human monoclonal antibodies may be produced by hybridomas comprising B cells obtained from a transgenic non-human animal (e.g., a transgenic mouse having a genome comprising a repertoire of human immunoglobulin heavy and light chain gene segments fused to an immortalized cell).

[0063] Human antibodies may be isolated from sequence libraries constructed based on a selection of human germline sequences, and further diversified with natural and synthetic sequence diversity.

[0064] Human antibodies can be prepared by in vitro immunization of human lymphocytes followed by transformation of the lymphocytes with Epstein-Barr virus.

[0065] The term "human antibody derivatives" refers to any modified form of a human antibody, such as a conjugate of the antibody with another compound or antibody.

[0066] As used herein, the term "humanized antibody" refers to a human / non-human chimeric antibody containing sequences (CDRs or portions thereof) derived from a non-human immunoglobulin. Thus, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from at least the recipient's hypervariable region are replaced by residues from a hypervariable region of an antibody from a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, sequence composition, and functionality. In some cases, framework (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. An example of such a modification is the introduction of one or more so-called backmutations, which typically are amino acid residues derived from the donor antibody. Antibody humanization may be performed using recombinant techniques known to those skilled in the art (see, for example, Antibody Engineering, Methods in Molecular Biology, vol. 248, edited by Benny K. Lo). Suitable human recipient frameworks for both the light and heavy chain variable domains can be identified, for example, by sequence or structural homology. Alternatively, a fixed recipient framework may be used, for example, based on knowledge of the structure, biophysical properties, and biochemical properties. The recipient framework may be derived from a germline or mature antibody sequence. CDRs from a donor antibody can be grafted by CDR grafting. CDR-grafted humanized antibodies can be further optimized, for example, for affinity, functionality, and biophysical properties, by identifying key framework positions where reintroduction of amino acid residues from the donor antibody (backmutation) beneficially affects the properties of the humanized antibody. In addition to backmutation from the donor antibody, humanized antibodies can be engineered by introducing germline residues into the CDR or framework regions, removing immunogenic epitopes, site-directed mutagenesis, affinity maturation, etc.

[0067] In further embodiments, humanized antibodies comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, a humanized antibody will comprise at least one, and typically two, variable domains, in which all or substantially all CDRs correspond to those of a non-human immunoglobulin and in which all or substantially all FR residues are those of a human immunoglobulin sequence. The humanized antibody can also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0068] The term "humanized antibody derivative" refers to any modified form of a humanized antibody, such as a conjugate of the antibody with a chemical compound or a conjugate of the antibody with another antibody.

[0069] The term "chimeric antibody" as used herein refers to an antibody comprising antibody portions derived from two or more species. For example, the genes encoding such an antibody include genes encoding variable domains and genes encoding constant regions derived from two different species. For example, genes encoding the variable segments of a mouse monoclonal antibody may be combined with genes encoding the constant regions of an antibody of human origin.

[0070] The fragment crystallizable region of an antibody ("Fc region" / "Fc domain") is the C-terminal region of an antibody, which comprises the hinge and constant C domains. H 2 and C HThe Fc region comprises three domains. The Fc domain may interact with cell surface receptors called Fc receptors, as well as several proteins of the complement system. The Fc region allows the antibody to interact with the immune system. In one embodiment of the invention, an antibody can be engineered to contain modifications within the Fc region, typically to alter one or more of its functional properties, such as serum half-life, complement fixation, Fc receptor binding, protein stability, and / or antigen-dependent cellular cytotoxicity, or lack thereof, among others. Additionally, the antibodies of the invention can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter its glycosylation, again to alter one or more of the antibody's functional properties. An IgG1 antibody may carry a modified Fc domain containing one or more, and possibly all, of the following mutations, which would result in reduced affinity for certain Fc gamma receptors (L234A, L235E, and G237A) and reduced C1q-mediated complement fixation (A330S and P331S) (residue numbering according to the EU index), respectively:

[0071] The isotype of the antibody of the present invention can be IgG, e.g., IgG1, e.g., IgG2, e.g., IgG4. If desired, the class of the antibody can be "switched" by known techniques. For example, an antibody originally generated as an IgM molecule can be class-switched to an IgG antibody. Class-switching techniques can also be used to convert one IgG subclass to another, e.g., from IgG1 to IgG2 or IgG4, from IgG2 to IgG1 or IgG4, or from IgG4 to IgG1 or IgG2. Antibody engineering can also be performed by combining regions from different IgG subclasses to generate constant region chimeric molecules.

[0072] In one embodiment, C H The hinge region of 1 is modified to change, e.g., increase or decrease, the number of cysteine ​​residues in the hinge region. This approach is further described, for example, in U.S. Patent No. 5,677,425 by Bodmer et al.

[0073] The constant region may be modified to stabilize the antibody, for example, to reduce the risk of a bivalent antibody separating into two monovalent VH-VL fragments. For example, in the IgG4 constant region, residue S228 (according to the EU numbering index and S241 according to Kabat) may be mutated to a proline (P) residue to stabilize inter-heavy chain disulfide bridge formation in the hinge (see, e.g., Angal et al., Mol Immunol. 1993;30:105-8).

[0074] Antibodies or fragments thereof can be defined in terms of their complementarity-determining regions (CDRs). The terms "complementarity-determining region" or "hypervariable region," as used herein, refer to the regions of an antibody in which the amino acid residues involved in antigen binding are located. Hypervariable regions or CDRs can be identified as the regions with the highest variability in the amino acid alignment of antibody variable domains. Databases such as the Kabat database can be used to identify CDRs, which are defined, for example, as including amino acid residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) of the light chain variable domain and residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) of the heavy chain variable domain (Kabat et al., 1991; Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242). Alternatively, the CDRs can be defined as those residues from the "hypervariable loops" (residues 26-33 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, J. Mol. Biol. 1987;196:901-917). Typically, the numbering of amino acid residues in this region is performed according to the method described in Kabat et al., supra. As used herein, phrases such as "Kabat position," "Kabat residue," and "according to Kabat" refer to this numbering system for heavy chain variable domains or light chain variable domains. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to shortening of, or insertion into, the framework (FR) or CDR of the variable domain. For example, the heavy chain variable domain may include an amino acid insertion after residue 52 of CDR H2 (residues 52a, 52b, and 52c according to Kabat) and residues inserted after heavy chain FR residue 82 (such as residues 82a, 82b, and 82c according to Kabat).The Kabat numbering of residues can be determined for a given antibody by alignment of the antibody's sequence with a "standard" Kabat numbered sequence at the regions of homology.

[0075] V H The amino acid residues of the H domain are similar to those of the V H domain from Camelidae, as shown, for example, in Figure 2 of Riechmann and Muyldermans (1999, J. Immunol. Methods 231:25-38). H As applied to the H domain, V given by Kabat et al. H They may be numbered according to the general numbering of domains. H Alternative methods for numbering the amino acid residues of VH domains, which can also be applied to H domains, are known in the art. The CDRs can also be determined by different methods. In the Kabat CDR determination, H FR1 of H includes amino acid residues at positions 1 to 30, and V H CDR1 of H comprises amino acid residues at positions 31 to 35, and V H FR2 of H includes amino acids 36-49, and V H CDR2 of H comprises amino acid residues at positions 50 to 65, and V H FR3 of H includes amino acid residues 66-94, and V H CDR3 of H comprises amino acid residues 95 to 102, and V H FR4 of H includes amino acid residues at positions 103-113.

[0076] In this document, the CDR sequences of single variable domain antibodies (Nanobodies®) are determined using the AbM definition by Kontermann and Dubel (2010, Eds., Antibody Engineering, vol. 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51). According to this method, FR1 comprises the amino acid residues at positions 1 to 25, CDR1 comprises the amino acid residues at positions 26 to 35, FR2 comprises the amino acid residues at positions 36 to 49, CDR2 comprises the amino acid residues at positions 50 to 58, FR3 comprises the amino acid residues at positions 59 to 94, CDR3 comprises the amino acid residues at positions 95 to 102, and FR4 comprises the amino acid residues at positions 103 to 113 (according to Kabat numbering).

[0077] In this document, the CDR sequences of IgG antibodies (including full-length IgG antibodies and their Fab fragments) are determined using the Kabat definition by Kontermann and Dubel (2010, Eds., Antibody Engineering, vol. 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51). According to this method, the CDRs of the light chain variable domain are defined as positions 24-34 (CDR1), positions 50-56 (CDR2), and positions 89-97 (CDR3), while the CDRs of the heavy chain variable domain are defined as positions 31-35 (CDR1), positions 50-65 (CDR2), and positions 95-102 (CDR3).

[0078] The term "framework region" or "FR" residues, as defined herein, refer to those V residues that are not within the CDRs. H or V L Refers to amino acid residues.

[0079] In one embodiment, an antibody or antigen-binding fragment thereof of the present invention may comprise the CDRs from one or more of the specific antibodies disclosed herein.

[0080] The term "procoagulant antibody" refers to an antibody that enhances blood clotting, for example, by accelerating the process of blood clotting and / or by increasing the enzymatic activity of one or more clotting factors.

[0081] The term "procoagulant activity" refers to the ability of a compound, such as an antibody, to enhance blood clotting, for example, by accelerating the process of blood clotting and / or by increasing the enzymatic activity of one or more clotting factors.

[0082] V H Domains and V H For H domains, as is well known in the art, it should be noted that the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e., one or more positions according to Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat numbering). This generally means that the Kabat numbering may or may not correspond to the actual numbering of amino acid residues in the actual sequence. H Domains and V H The total number of amino acid residues in the H domain is usually in the range of 110-120, often in the range of 112-115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein. The antibodies of the invention may comprise CDRs from one or more of the specific antibodies disclosed herein.

[0083] The term "antigen" (Ag) refers to a molecular entity used for immunization of an immunocompetent vertebrate to generate antibodies (Abs) that recognize the Ag. As used herein, Ag is referred to more broadly and is generally intended to include the target molecule specifically recognized by an Ab, and thus includes fragments or mimics of the molecule used in the immunization process or other processes, such as phage display, used to generate Abs.

[0084] As used herein, the term "epitope" is defined in the context of molecular interactions between an "antigen-binding polypeptide" such as an antibody (Ab) and its corresponding antigen (Ag). Generally, "epitope" refers to the area or region on an Ag to which an Ab specifically binds, i.e., the area or region that is in physical contact with the Ab. Physical contact can be defined using various criteria (e.g., distance cutoffs of 2-6 Å, such as 3 Å, 3.5 Å, 4 Å, 4.5 Å, 5 Å, or solvent accessibility) for atoms within the Ab and Ag molecules.

[0085] FX / FXa may contain a number of different epitopes, which may include, but are not limited to, (1) linear peptide epitopes, (2) structural epitopes consisting of one or more non-contiguous amino acids located close to each other in the mature FX / FXa structure, and (3) epitopes consisting of either all or part of a molecular structure covalently attached to FX / FXa, such as a carbohydrate group.

[0086] The epitope of a given antibody (Ab) / antigen (Ag) pair can be described and characterized to different levels of detail using various experimental and computational epitope mapping methods. Experimental methods include mutagenesis, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, hydrogen-deuterium exchange mass spectrometry (HDX-MS), and various competitive binding methods, all of which are known in the art. Because each method relies on unique principles, the description of an epitope is closely related to the method by which it was determined. Therefore, depending on the epitope mapping method used, the epitope of a given Ab / Ag pair can be described differently.

[0087] The epitope and paratope of a given antibody (Ab) / antigen (Ag) pair can be identified by routine methods. For example, the general location of the epitope may be determined by assessing the ability of the antibody to bind to different fragments or variants of FX. Also, the specific amino acids in FX that contact the antibody (epitope) and the specific amino acids in the antibody that contact FX (paratope) may be determined using routine methods. An antibody according to the invention may be able to compete with another molecule, such as a naturally occurring ligand or receptor or another antibody, for binding to FX.

[0088] Competitive binding assays can be performed in which binding of an antibody to a target is compared to binding of the target by another ligand of that target, such as another antibody.

[0089] Antibodies that bind to the same antigen can be characterized for their ability to simultaneously bind to their common antigen and may be subject to "competitive binding" / "binning." In this context, the term "binning" refers to a method of grouping antibodies that bind to the same antigen. Antibody "binning" may be based on competitive binding of two antibodies to their common antigen in assays based on standard techniques.

[0090] The "bin" of an antibody is defined using a reference antibody. If the second antibody cannot bind to the antigen at the same time as the reference antibody, the second antibody is said to belong to the same "bin" as the reference antibody. In this case, the reference antibody and the second antibody competitively bind to the same part of the antigen and are considered "competing antibodies." If the second antibody can bind to the antigen at the same time as the reference antibody, the second antibody is said to belong to a separate "bin." In this case, the reference antibody and the second antibody do not competitively bind to the same part of the antigen and are considered "non-competing antibodies."

[0091] Antibody "binning" does not provide direct information about the epitope.

[0092] Competing antibodies, i.e., antibodies belonging to the same "bin," may have the same epitope, overlapping epitopes, or even distinct epitopes. The latter is the case when a reference antibody bound to that epitope on the antigen occupies the space required for a second antibody to contact that epitope on the antigen ("steric hindrance"). Non-competing antibodies generally have distinct epitopes. Thus, in some embodiments, the antibodies of the present invention will bind to the same epitope as at least one of the antibodies specifically disclosed herein.

[0093] Competitive assays for determining whether an antibody binds to the same epitope as the anti-FX antibody disclosed herein or competes with it for binding are known in the art. Exemplary competitive assays include immunoassays (e.g., ELISA assays, RIA assays), surface plasmon resonance analysis (e.g., using a BIAcore™ instrument), biolayer interferometry, and flow cytometry. Typically, competitive assays involve the use of an antigen bound to a solid surface or expressed on a cell surface, a test FX-binding antibody, and a reference antibody. The reference antibody is labeled, and the test antibody is unlabeled. Competitive inhibition is measured by determining the amount of labeled reference antibody bound to the solid surface or cells in the presence of the test antibody. Typically, the test antibody is present in excess of the reference antibody (e.g., 1x, 5x, 10x, 20x, 100x, 1000x, 10000x, or 100000x). Antibodies identified as competitive in competition assays (i.e., competing antibodies) include antibodies that bind to the same epitope as the reference antibody, or to an overlapping epitope, as well as antibodies that bind to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody to create steric hindrance.

[0094] In an exemplary competitive assay, a reference anti-FX or anti-FXa antibody is biotinylated using commercially available reagents. The biotinylated reference antibody is mixed with serial dilutions of the test antibody or unlabeled reference antibody (self-competition control) to obtain mixtures with various molar ratios (e.g., 1, 5, 10, 20, 100, 1,000, 10,000, or 100,000 times) of the test antibody (or unlabeled reference antibody) to the labeled reference antibody. The antibody mixture is then added to an FX or FXa polypeptide-coated ELISA plate. The plate is then washed, and horseradish peroxidase (HRP)-strepavidin is added to the plate as a detection reagent. The amount of labeled reference antibody bound to the target antigen is detected after addition of a chromogenic substrate (e.g., TMB (3,3',5,5'-tetramethylbenzidine) or ABTS (2,2''-azino-di-(3-ethylbenzthiazoline-6-sulfonate)), as known in the art. Optical density readings (OD units) are measured using a spectrometer (e.g., a SpectraMax® M2 spectrometer (Molecular Devices)). The response (OD units) corresponding to zero percent inhibition is determined from wells that do not contain any competing antibody. The response (OD units) corresponding to 100% inhibition, i.e., assay background, is determined from wells that do not contain any labeled reference or test antibody. The percent inhibition of labeled reference antibody against FX or FXa by test antibody (or unlabeled reference antibody) at each concentration is calculated as follows: % inhibition = (1 - (OD units - 100% inhibition) / (0% inhibition - 100% inhibition)) * 100.

[0095] Those skilled in the art will understand that similar assays can be performed to determine whether two or more anti-FX / FXa antibodies share binding regions, bins, and / or competitively bind to an antigen. Those skilled in the art will also understand that competitive assays can be performed using a variety of detection systems known in the art.

[0096] A test antibody competes with a reference antibody for binding to an antigen if an excess of one antibody (e.g., 1, 5, 10, 20, 100, 1000, 10000, or 100,000 fold) inhibits binding of the other antibody by, for example, at least 50%, 75%, 90%, 95%, or 99%, as measured in a competitive binding assay. An example of a competitive assay is provided in Methods section 5.

[0097] The term "binding affinity" is used herein as a measure of the strength of a non-covalent interaction between two molecules, for example, an antibody or fragment thereof and an antigen. The term "binding affinity" is used to describe a monovalent interaction.

[0098] The binding affinity between two molecules, for example, an antibody or fragment thereof and an antigen, is determined by a monovalent interaction and is expressed as an equilibrium dissociation constant (K D ) can be quantified by determining the K D can be determined by measuring the kinetics of complex formation and dissociation, for example, by SPR. The rate constants corresponding to the association and dissociation of the monovalent complex are the association rate constant k a (or k on ), and the dissociation rate constant k d (or k off ) is called K D is the formula K D =k d / k a via k a and k d and related.

[0099] According to the above definition, the binding affinities associated with different molecular interactions, such as comparing the binding affinities of different antibodies to a given antigen, are calculated by the K of the individual antibody / antigen complexes. D The comparison may be by value comparison. The value of dissociation constant can be determined directly by well-known method.Standard assays for evaluating the binding ability of ligands such as antibodies directed to targets are known in the art, and include, for example, ELISA, Western blot, RIA and flow cytometry analysis.The binding kinetics and binding affinity of antibodies can also be evaluated by standard assays known in the art, such as SPR.An example of SPR binding assay is provided in Example 2 herein.

[0100] An FX binder, such as an antibody or fragment thereof according to the present invention, binds 1×10 of its target -4 M or less, 1×10 -5 M or less, 1×10 -6 M or less, 1×10 -7 M or less, 1×10 -8 M or less, or 1 x 10 -9 M or less, or 1 x 10 -10 M or less, 1×10 -11 M or less, 1×10 -12 M or less, 1×10 -13 M or less, or 1 x 10 -14 K below M D In one embodiment, the K of the antibody according to the invention D may be less than 100 μM, e.g., less than 50 μM, e.g., less than 10 μM. D is less than 9 μm, such as less than 8 μm, for example less than 7 μm, such as less than 6 μm, for example less than 5 μm. D is less than 4 μm, such as less than 3 μm, for example less than 2 μm, such as less than 1 μm.

[0101] identity The term "identity" as known in the art refers to the relationship between the sequences of two or more polypeptides, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptides, as determined by the number of matches between strings of two or more amino acid residues. "Identity" measures the percentage of exact matches between the smaller of two or more sequences, with gap alignment (if any) accommodated by a particular mathematical model or computer program (i.e., "algorithm"). The identity of related polypeptides can be readily calculated by known methods.

[0102] In the present invention, similarity and identity were determined using Needleman from EMBOSS-6.6.0 (Needleman et al., J. Mol. Biol. 1970;48:443-453) using parameters of 10 and 0.5 for gap opening and gap extension, respectively (gapopen=10, gapextend=0.5).

[0103] Pharmaceutical preparations In another aspect, the invention provides compositions and formulations comprising the compounds of the invention, such as the antibodies described herein. For example, the invention provides pharmaceutical compositions comprising an antibody of the invention, or an antigen-binding fragment thereof, formulated together with a pharmaceutically acceptable carrier.

[0104] Therefore, one object of the present invention is to provide a pharmaceutical formulation comprising such an antibody or antigen-binding fragment thereof present at a concentration of 0.1 mg / mL to 500 mg / mL, the formulation having a pH of 2.0 to 10.0. The formulation may further comprise one or more of a buffer system, a preservative, a tonicity agent, a chelating agent, a stabilizer, or a surfactant, and various combinations thereof. The use of preservatives, tonicity agents, chelating agents, stabilizers, and surfactants in pharmaceutical compositions is well known to those skilled in the art. Reference may be made to Remington: The Science and Practice of Pharmacy, 19th Edition, 1995.

[0105] In one embodiment, the pharmaceutical formulation is an aqueous formulation. Such formulations are typically solutions or suspensions, but may also include colloids, dispersions, emulsions, and multi-phase materials. The term "aqueous formulation" is defined as a formulation containing at least 50% w / w water. Similarly, the term "aqueous solution" is defined as a solution containing at least 50% w / w water, and the term "aqueous suspension" is defined as a suspension containing at least 50% w / w water.

[0106] In another embodiment, the pharmaceutical formulation is a lyophilized formulation to which solvents and / or diluents are added prior to use.

[0107] In a further embodiment, a pharmaceutical formulation comprises an aqueous solution of such an antibody and a buffer in which the antibody is present at a concentration of 0.1 mg / mL or greater, the formulation having a pH of about 2.0 to about 10.0.

[0108] In one embodiment, the invention relates to an injection device having the contents of the composition. In some embodiments, the pharmaceutical compositions of the invention are intended for use in and / or contained within an injection device. In some embodiments, the injection device is a disposable, pre-filled, multi-dose FlexTouch®-style pen (supplier: Novo Nordisk A / S, Denmark). In some embodiments, the injection device is a single-shot device.

[0109] In some embodiments, the injection device is a fixed dose device, such as one configured to deliver multiple predetermined doses of medication, sometimes referred to as a multiple fixed dose device or a fixed dose, multi-shot device.

[0110] Administration The compounds of the present invention, such as antibodies or antigen-binding fragments thereof, or compositions containing such compounds, may be administered parenterally, such as intravenously, intramuscularly, or subcutaneously. Alternatively, the compounds of the present invention, such as antibodies or antigen-binding fragments thereof, or compositions containing such compounds may be administered parenterally, such as orally or topically. The compounds or compositions may be administered prophylactically. Alternatively, the compounds or compositions may be administered therapeutically (as needed).

[0111] Dosage The dose of the compound delivered may be about 0.01 mg to 500 mg of compound per day, preferably about 0.1 mg to 250 mg per day, more preferably about 0.5 mg to about 250 mg per day, and may be administered daily, weekly, biweekly, or monthly as an initial and maintenance dose depending on the severity of the condition. The appropriate dose may also be adjusted for a particular compound based on its characteristics, including its in vivo half-life or mean residence time and its biological activity. For example, the compound may be administered once weekly in one embodiment, once every other week in another embodiment, or once monthly in another embodiment, and in any of these embodiments, may be administered at a dose of, for example, 0.1, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mg per kg of body weight.

[0112] The compositions containing the compounds disclosed herein can be administered for preventive treatment and / or in some embodiments for therapeutic treatment.In therapeutic use, the compositions are administered to a subject who already suffers from a disease, such as any bleeding disorder as described above, in an amount sufficient to cure, alleviate, or partially prevent the disease and its complications.An amount sufficient to achieve this is defined as a "therapeutically effective amount".As understood by those skilled in the art, the amount effective for this purpose depends on the severity of the disease or injury, and the subject's weight and general condition.

[0113] In one aspect, the present invention relates to FX-binding molecules that can stimulate the activation of FX to generate FXa. As noted above, such functionality is desirable in the search for new and improved therapies to improve coagulation function, particularly in hemophilia patients.

[0114] In one embodiment, the present invention relates to an FX-binding agent, such as an antibody or antigen-binding fragment thereof, that binds to human FX and stimulates FX activation.

[0115] In one embodiment, the present invention relates to an FX-binding agent, such as an antibody or antigen-binding fragment thereof, which is capable of binding to human FX and stimulating FX activation. Of particular interest is that the observed activation occurs in the absence of a FIX-binding moiety, such as FVIII or a FVIII-mimetic compound, including a FIXa-binding agent that brings FX and FIXa together. In one embodiment, the FX-binding agent, such as an antibody or antigen-binding fragment thereof, binds to human FX, thereby stimulating its proteolysis by FIXa to generate FXa.

[0116] In one embodiment, the antibody or antigen-binding fragment thereof stimulates FXa production independent of the presence of a FIXa-binding moiety. In one embodiment, the antibody or antigen-binding fragment thereof is capable of stimulating FXa production independent of the presence of a FIXa-binding moiety.

[0117] In one embodiment, an FX-binding agent, such as an antibody or antigen-binding fragment thereof, capable of binding to FX, can make FX more susceptible to proteolysis, independent of the presence of a FIXa-binding moiety. In one embodiment, an FX-binding agent, such as an antibody or antigen-binding fragment thereof, capable of binding to FX, can make FX more susceptible to proteolysis by FIXa. In one embodiment, an FX-binding agent, such as an antibody or antigen-binding fragment thereof, capable of binding to FX, can make FX more susceptible to proteolysis, independent of the presence of a FIXa-binding moiety.

[0118] The ability of an FX binder to stimulate FXa production can be tested by a suitable assay known to those skilled in the art. See Example 3 herein, which provides a suitable in vitro assay for testing the activation function of a given FX binder. In one embodiment, an FX binder, such as an antibody or antigen-binding fragment thereof capable of binding to FX, stimulates FX activation as determined by Example 3. An FX binder is stimulatory, or capable of stimulating FX activation, when an increase in FXa is measured in the presence of the FX binder compared to an equivalent assay performed without the FX binder or a non-activated FX binder.

[0119] The ability of an FX binder to promote FIXa-mediated FX activation is considered to be present when an increase in the rate of FX activation is observed as described in the assay according to Example 3 herein for a given FX binder compared to the absence of the FX binder, i.e., when a fold increase in FX activation of greater than 1 is observed, such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9. In preferred embodiments, the observed fold increase in FX activation is greater than 2-fold, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100-fold or greater. In one embodiment, the fold increase in FX activation is between 2-100,000-fold. In one such embodiment, the fold increase in FX activation is between 2-500-fold. In one such embodiment, the fold increase in FX activation is between 3-500-fold. In another such embodiment, the fold increase in FX activation is between 4-500-fold. In another such embodiment, the increase in FX activation is between 5 and 100 fold. In another embodiment, the increase in FX activation is between 10 and 100 fold, for example between 10 and 83 fold.

[0120] To avoid the binding activity of the FX binder affecting the assay measurements as described herein in Example 3, the assay is preferably performed using a monovalent FX binder. If the FX binder is a bivalent or multivalent FX binder, the activation assay should be performed using a monovalent antigen-binding fragment thereof.

[0121] One aspect of the present invention relates to an FX binder, such as an antibody or antigen-binding fragment thereof, that binds to human FX more strongly than FXa. In one embodiment, the FX binder does not bind to FXa. In another embodiment, the FX binder binds to FX preferentially over FXa. In one such embodiment, the FX binder binds to FXa with low affinity. Where FX is distinguished from FXa by the presence of an activation peptide, the present invention, in one aspect, relates to an FX binder, such as an antibody or antigen-binding fragment thereof, that binds to the activation peptide of human FX. The activation peptide of human FX is defined by amino acid residues 143-194 of SEQ ID NO:2.

[0122] In one embodiment, the antibody or antigen-binding fragment thereof binds to the activation peptide of FX defined by amino acid residues 143-194 of SEQ ID NO: 2. In a further embodiment, the antibody or antigen-binding fragment thereof binds to amino acid residues 170-185 within the activation peptide of FX defined by SEQ ID NO: 2. The covalent binding site of the identified FX-activated FX binders was identified based on peptide sequencing performed as described in Example 5. The common minimal peptide epitope was determined and found to contain the peptide "LL" within the activation peptide of FX, corresponding to amino acid residues 177-178 of human FX, as identified by SEQ ID NO:2.

[0123] In one embodiment, the present invention relates to an FX binding agent, such as an antibody or antigen-binding fragment thereof, capable of binding to "LL" in the activation peptide of FX, which corresponds to amino acid residues 177-178 of human FX as identified by SEQ ID NO:2.

[0124] In one embodiment, the present invention relates to an FX binding agent, such as an antibody or antigen-binding fragment thereof, capable of binding to an activation peptide of FX, wherein the minimal consensus epitope determined by the peptide sequence is comprised of "LL" (amino acid residues 177-178 of SEQ ID NO: 2).

[0125] In one embodiment, an FX binding agent, such as an antibody or antigen-binding fragment thereof, binds to the activation peptide of FX identified by amino acid residues 143-194 of SEQ ID NO:2, and the minimal consensus epitope determined by the peptide sequence is comprised of "LL" (amino acid residues 177-178 of SEQ ID NO:2).

[0126] It is well known in the art that antigen-binding molecules can take various forms. Although a series of well-known molecular forms are described herein, it should be noted that this should not be considered an exhaustive list, and that those skilled in the art can easily apply the teachings of the present application to their preferred molecular forms.

[0127] In one embodiment, the FX-binding agent is monovalent. In one such embodiment, the FX-binding agent is, for example, a (single) domain antibody or antibody fragment thereof, or a Nanobody®, V H H, humanized V H H, or camelized V H In one embodiment, the FX binder is an immunoglobulin single variable domain, such as a domain. In one embodiment, the FX binder is a Fab. In one embodiment, the FX binder is bivalent. In one embodiment, the FX binder is a whole monoclonal antibody (mAb) or an antigen-binding fragment of a mAb.

[0128] Aspects of the present invention relate to the identification of FX-binding agents capable of stimulating FX activation. In one such embodiment, a method is provided comprising the steps of: a) providing an FX-binding agent, b) testing the FX-binding agent in an in vitro assay suitable for testing the FX-activating function of a given FX-binding agent, and c) selecting an FX-binding agent capable of stimulating FX activation.

[0129] As noted above, an FX-binding agent capable of stimulating FX activation may be a desirable component of a bispecific, or trispecific, or even multispecific molecule.

[0130] Thus, one aspect of the present invention relates to bi-, tri-, and multispecific molecules, such as antibody molecules (or antigen-binding fragments thereof) that comprise at least one FX-binding portion that provides FX stimulation.

[0131] Aspects of the invention relate to the use of a molecule, such as an antibody or antigen-binding fragment thereof, described herein for the manufacture of a medicament.

[0132] Aspects of the invention relate to molecules, such as antibodies or antigen-binding fragments thereof, described herein, for use in methods of treatment.

[0133] Aspects of the invention relate to the use of molecules such as antibodies or antigen-binding fragments thereof described herein for the treatment of clotting disorders such as hemophilia.

[0134] An aspect of the invention relates to a method of treatment comprising administering to an individual in need thereof a therapeutically effective dose of a molecule, such as an antibody or antigen-binding fragment thereof, described herein. In one embodiment, the method is for treating a patient with a clotting disorder such as hemophilia, particularly hemophilia A, with or without inhibitors. In one embodiment, the method is for treating a patient with hemophilia B, with or without inhibitors.

[0135] Embodiment 1. An antibody or antigen-binding fragment thereof capable of binding to the activation peptide of FX specified by amino acid residues 143 to 194 of SEQ ID NO:2.

[0136] 2. An antibody or antigen-binding fragment thereof capable of binding to one or more of amino acid residues 170 to 185 within the activation peptide of FX identified by SEQ ID NO:2.

[0137] 3. An antibody or antigen-binding fragment thereof capable of binding to "LL" in the activation peptide of FX corresponding to amino acid residues at positions 177-178 of human FX as identified by SEQ ID NO:2.

[0138] 4. An antibody or antigen-binding fragment thereof capable of binding to the activation peptide of FX, wherein the minimal common epitope determined by peptide array comprises "LL" (AA 177-178 of SEQ ID NO: 2).

[0139] 5. An antibody or antigen-binding fragment thereof capable of binding to FX and stimulating FX activation.

[0140] 6. An antibody or antigen-binding fragment thereof capable of binding to FX, which antibody or antigen-binding fragment thereof is capable of stimulating FX activation by FIXa.

[0141] 7. An antibody or antigen-binding fragment thereof capable of binding to FX, which antibody or antigen-binding fragment thereof is capable of stimulating FX activation by FIXa independent of the presence of a FIXa binder.

[0142] 8. An antibody or antigen-binding fragment thereof capable of binding to FX according to any one of embodiments 1 to 4, wherein the antibody or antigen-binding fragment thereof is capable of stimulating FX activation.

[0143] 9. An antibody or antigen-binding fragment thereof capable of binding to FX according to any one of embodiments 5 to 8, wherein the ability of the antibody or antigen-binding fragment thereof to stimulate FX activation is determined as described in Example 3 herein.

[0144] 10. An antibody or antigen-binding fragment thereof capable of binding to FX, which antibody or antigen-binding fragment thereof stimulates FX activation by FIXa independent of the presence of a FIXa binding moiety.

[0145] 11. An antibody or antigen-binding fragment thereof capable of binding to FX according to any one of embodiments 5 to 10, wherein stimulation of FX activation is determined as described in Example 3 herein.

[0146] 12. An antibody or antigen-binding fragment thereof capable of binding to FX, which can make FX more susceptible to activation.

[0147] 13. An antibody or antigen-binding fragment thereof capable of binding to FX, which can make FX more susceptible to proteolysis by FIXa.

[0148] 14. An antibody or antigen-binding fragment thereof capable of binding to FX, which antibody or antigen-binding fragment can render FX more susceptible to proteolysis by FIXa, independent of the presence of a FIXa-binding moiety.

[0149] 15. An antibody or antigen-binding fragment thereof capable of binding to FX according to any one of embodiments 12 to 14, wherein the ability of the antibody or antigen-binding fragment thereof to make FX more susceptible to proteolysis is determined as described in Example 3 herein.

[0150] 16. An antibody or antigen-binding fragment thereof capable of binding to FX according to any one of embodiments 1 to 4, wherein the antibody or antigen-binding fragment thereof is capable of stimulating FX activation according to any one of embodiments 5 to 11.

[0151] 17. The antibody or antigen-binding fragment thereof of any one of embodiments 1 to 4, wherein the antibody or antigen-binding fragment thereof is a single variable domain antibody that stimulates FX activation of any one of embodiments 5 to 11.

[0152] 18. An antibody or antigen-binding fragment thereof capable of binding to FX described in any one of embodiments 1 to 4, wherein the antibody or antigen-binding fragment thereof is a single variable domain antibody that can make FX more susceptible to proteolysis described in any one of embodiments 12 to 15.

[0153] 19. The monovalent antigen-binding fragment thereof is a. Stimulate FX activation or b. Stimulates FX activation by FIXa, c.Can stimulate FX activation? d.Can it stimulate FX activation by FIXa? e.FX can be made more susceptible to proteolysis, or f. An antibody or antigen-binding fragment thereof capable of binding to FX according to any one of embodiments 5 to 18, which can make FX more susceptible to proteolysis by FIXa.

[0154] 20. The antibody or antigen-binding fragment has an equilibrium dissociation constant (K D 20. An antibody or antigen-binding fragment thereof capable of binding to FX according to any one of embodiments 1 to 19, having a binding affinity determined as follows:

[0155] 21. The antibody or antigen-binding fragment thereof of any one of embodiments 1 to 20, wherein the antibody or antigen-binding fragment is monovalent.

[0156] 22. The antibody or antigen-binding fragment thereof is an immunoglobulin single variable domain or antibody fragment thereof, such as a domain antibody, Nanobody®, V H H, humanized V H H, or camelized V H 22. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 21, which is a domain.

[0157] 23. The antibody or antigen-binding fragment thereof of any one of embodiments 1 to 22, wherein the antibody or antigen-binding fragment thereof is a Fab, scFab, Fv, or scFv.

[0158] 24. The antibody or antigen-binding fragment thereof of any one of embodiments 1 to 15, wherein the antibody or antigen-binding fragment thereof is bivalent.

[0159] 25. The antibody or antigen-binding fragment thereof of embodiment 24, wherein the antibody or antigen-binding fragment is a mAb of Fab'2.

[0160] 26. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 25, wherein the heavy chain variable domain is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the sequence defined by SEQ ID NO: 8, 9, 10, 11, 54, 55, 56, or 57, respectively.

[0161] 27. The antibody or antigen-binding fragment thereof a. three heavy chain CDR sequences with up to 10 amino acid residue changes compared to the three CDR sequences of the heavy chain variable domain identified by SEQ ID NO: 8; b. three heavy chain CDR sequences having up to 10 amino acid residue changes compared to the three CDR sequences of the heavy chain variable domain identified by SEQ ID NO: 9; c. three heavy chain CDR sequences having up to 10 amino acid residue changes compared to the three CDR sequences of the heavy chain variable domain identified by SEQ ID NO: 10; d. three heavy chain CDR sequences having up to 10 amino acid residue changes compared to the three CDR sequences of the heavy chain variable domain identified by SEQ ID NO: 11; e. three heavy chain CDR sequences having up to 10 amino acid residue changes compared to the three CDR sequences of the heavy chain variable domain identified by SEQ ID NO: 54; f. three heavy chain CDR sequences having up to 10 amino acid residue changes compared to the three CDR sequences of the heavy chain variable domain identified by SEQ ID NO: 55; g. three heavy chain CDR sequences having up to 10 amino acid residue changes compared to the three CDR sequences of the heavy chain variable domain identified by SEQ ID NO: 56; or 26. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 25, comprising three heavy chain CDR sequences having up to 10 amino acid residue changes compared to the three CDR sequences of the heavy chain variable domain identified by SEQ ID NO: 57.

[0162] 28. The antibody or antigen-binding fragment thereof according to embodiment 27, wherein the three heavy chain CDR sequences have up to 9, such as 8, such as 7, or such as 6 amino acid changes compared to the three CDRs of the specified SEQ ID NO.

[0163] 29. The antibody or antigen-binding fragment thereof of embodiment 28, wherein the three heavy chain CDR sequences have up to 5, such as 4, such as 3, such as 2, or up to 1 amino acid changes compared to the three CDRs of the specified SEQ ID NO.

[0164] 30. An antibody or antigen-binding fragment thereof comprising or consisting of SEQ ID NO: 8, 9, 10, 11, 54, 55, 56, or 57.

[0165] 31. An antibody or antigen-binding fragment thereof comprising three CDRs of SEQ ID NO: 8, 9, 10, 11, 54, 55, 56, or 57.

[0166] 32. An antibody or antigen-binding fragment thereof that competes with a reference antibody or antigen-binding fragment comprising or consisting of SEQ ID NO: 8, 9, 10, 11, 54, 55, 56, or 57.

[0167] 33. An antibody or antigen-binding fragment thereof comprising the heavy chain variable domain (SEQ ID NO: 4) and the light chain variable domain (SEQ ID NO: 5) of mAb 00916.

[0168] 34. An antibody or antigen-binding fragment thereof comprising the heavy chain variable domain (SEQ ID NO: 6) and the light chain variable domain (SEQ ID NO: 7) of mAb 13F62.

[0169] 35. An antibody or antigen-binding fragment thereof comprising the three CDRs of the heavy chain variable domain (SEQ ID NO: 4) and the three CDRs of the light chain variable domain (SEQ ID NO: 5) of mAb 00916.

[0170] 36. An antibody or antigen-binding fragment thereof comprising the three CDRs of the heavy chain variable domain (SEQ ID NO: 6) and the three CDRs of the light chain variable domain (SEQ ID NO: 7) of mAb 13F62.

[0171] 37. An antibody or antigen-binding fragment thereof that competes with a reference antibody or antigen-binding fragment comprising the heavy chain variable domain (SEQ ID NO: 4) and light chain variable domain (SEQ ID NO: 5) of mAb 00916.

[0172] 38. An antibody or antigen-binding fragment thereof that competes with a reference antibody or antigen-binding fragment comprising the heavy chain variable domain (SEQ ID NO: 6) and light chain variable domain (SEQ ID NO: 7) of mAb 13F62.

[0173] 39. The antibody or antigen-binding fragment thereof according to any one of embodiments 26 to 38, wherein the antibody or antigen-binding fragment is capable of stimulating FX activation according to any one of embodiments 5 to 9.

[0174] 40. An antibody or antigen-binding fragment thereof according to any one of embodiments 26 to 38, wherein the antibody or antigen-binding fragment stimulates FX activation according to any one of embodiments 9 to 11.

[0175] 41. An antibody or antigen-binding fragment thereof according to any one of embodiments 26 to 38, wherein the antibody or antigen-binding fragment is capable of making the FX according to any one of embodiments 12 to 15 more susceptible to proteolysis.

[0176] 42. A bi-, tri-, or multispecific molecule comprising an antibody or antigen-binding fragment according to any one of embodiments 1 to 41.

[0177] 43. A bi-, tri-, or multispecific molecule comprising an antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 42, wherein said antibody or antigen-binding fragment thereof is a single variable domain antibody.

[0178] 44. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 43, and optionally one or more pharmaceutically acceptable carrier(s).

[0179] 45. Use of the antibody or antigen-binding fragment thereof or composition according to any one of embodiments 1 to 44 for the manufacture of a medicament for use in the treatment of a clotting disorder, such as hemophilia A, with or without inhibitors, or such as hemophilia B, with or without inhibitors.

[0180] 46. ​​The antibody or antigen-binding fragment thereof or composition according to any one of embodiments 1 to 44 for use in the treatment of a coagulation disorder, such as hemophilia A, with or without inhibitors, or such as hemophilia B, with or without inhibitors.

[0181] 47. A method for treating a subject suffering from a blood clotting disorder, such as hemophilia, comprising administering to the subject an antibody or antigen-binding fragment thereof or composition described in any one of embodiments 1 to 46.

[0182] 48. The method according to embodiment 47, wherein the coagulation disorder or blood clotting disorder is hemophilia A or B, with or without inhibitors.

[0183] 49. A method for identifying an antibody according to any one of embodiments 1 to 25, comprising the steps of: a) providing an FX binder; b) testing the FX binder in an in vitro assay suitable for testing the FX activation function of the FX binder provided in step a; and c) selecting an FX binder capable of stimulating FX activation.

[0184] 50. A method for identifying an antibody according to any one of embodiments 5 to 23, comprising the steps of: a) providing an FX binder; b) testing the FX binder in an in vitro assay suitable for testing the FX-activating function of the FX binder provided in step a; and c) selecting an FX binder that can make FX more susceptible to proteolysis, for example by FIXa.

[0185] 51. A eukaryotic cell expressing an antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 43.

[0186] 52. A kit comprising an antibody or antigen-binding fragment thereof or composition according to any one of embodiments 1 to 44, and instructions for use.

[0187] Examples and Methods General method Preparation of FX-conjugated Nanobodies General molecular biology For general molecular biology techniques, see Molecular Cloning: A Laboratory Manual (3rd ed., 2001, Sambrook, Fritsch and Maniatis, eds., CSHL Press, Cold Spring Harbor, NY).

[0188] 1. Immunization and Libraries After approval by the Ethical Committee of the Ablynx Camelid Facility (LA1400575), one llama and one alpaca were immunized with FX (Haemotologic Technologies, VT USA).

[0189] Cloning of heavy chain-only antibody fragment repertoires and preparation of phage immune libraries were carried out as follows.

[0190] After the final immunogen injection, blood samples were collected. Peripheral blood mononuclear cells (PBMCs) were prepared from these blood samples using Ficoll-Hypaque according to the manufacturer's instructions (Amersham Biosciences, Piscataway, NJ, US). Total RNA was extracted from PBMCs and used as starting material for RT-PCR, essentially as described in WO2005 / 044858. H H / Nanobody®-encoding DNA segments were amplified. Briefly, Nanobody®-encoding DNA segments were cloned into the phagemid vector pAX212, allowing the generation of phage particles displaying Nanobodies® fused with His6 and FLAG3 tags. Phages were then prepared and stored according to standard protocols.

[0191] Synthesis Library The synthetic library was generated by cloning synthetic Nanobody® gene fragments into the phagemid vector pAX190, which has the same characteristics as pAX212 described above, but with differences in multiple cloning sites.

[0192] Library screening of nanobodies that bind to FX Nanobody® phage display selection was performed on the generated immune and synthetic libraries, which underwent one to four successive enrichment rounds against different combinations of immobilized human FX (Haemotologic Technologies, VT, USA) and cynomolgus FX (Novo Nordisk in-house production).

[0193] To specifically enrich for FX-selective Nanobodies relative to FXa, certain experiments used excess soluble FXa for competition during incubation of the library with immobilized FX.

[0194] To specifically enrich for Nanobodies selective for FX over other structurally related coagulation factors, certain experiments used excess soluble FIX for competition during incubation of the library with immobilized FX.

[0195] Approximately 4500 individual clones from the selection output were screened for binding by ELISA against human and cynomolgus FX, FXa, or FIX (using periplasmic extracts from E. coli cells expressing the Nanobodies). Approximately 1500 clones were identified that showed specific binding to human FX / FXa, with the majority showing cross-binding to cynomolgus FX. Some clones showed preferential binding to FX over FXa. Sequence analysis of ELISA-positive clones identified approximately 700 unique sequences of Nanobodies that bound to FX / FXa.

[0196] 2. Generation of Nanobody® Expression Constructs Sequence analysis of Nanobodies from the phage display selection output was performed according to commonly published procedures (Pardon et al. (2014) Nat Protoc 9:674).

[0197] Nanobody®-containing DNA fragments obtained by PCR using specific combinations of forward FR1 and reverse FR4 primers, each carrying a unique restriction site, were digested with the appropriate restriction enzymes and ligated into the corresponding cloning cassette of a Nanobody® expression vector (described below). The ligation mixture was then transformed into electrocompetent Escherichia coli TG1 (60502, Lucigen, Middleton, WI) or TOP10 (C404052, ThermoFisher Scientific, Waltham, MA) cells, which were then grown under the appropriate antibiotic selection pressure (kanamycin or zeocin). Resistant clones were verified by Sanger sequencing of plasmid DNA (LGC Genomics, Berlin, Germany).

[0198] Monovalent Nanobodies were expressed in E. coli TG1 from a plasmid expression vector containing the lac promoter, a kanamycin resistance gene, an E. coli origin of replication, and a Nanobody® cloning site preceded by the coding sequence for the OmpA signal peptide. In frame with the Nanobody® coding sequence, the vector encodes C-terminal FLAG3 and HIS6 tags. The signal peptide directs expressed Nanobodies to the periplasmic compartment of the bacterial host.

[0199] Expression and purification of Nanobodies General expression of Nanobodies in E. coli E. coli TG-1 cells containing the Nanobody® construct of interest were grown in baffled shaker flasks containing "5052" auto-induction medium (0.5% glycerol, 0.05% glucose, 0.2% lactose + 3 mM MgSO4) for 2 hours at 37°C, followed by 29 hours at 30°C. Cell pellets from overnight frozen E. coli expression cultures are then dissolved in PBS (1 / 12.5 of the original culture volume) and incubated for 1 hour at 4°C with gentle agitation. Finally, the cells are pelleted once more, and the supernatant containing the secreted protein into the periplasmic space is retained.

[0200] Nanobodies® in P. pastoris Common manifestations of P. pastoris cells containing the Nanobody® construct of interest were grown in BGCM medium for 2 days (30°C, 200 rpm). On day 3, the medium was switched to BMCM and the construct was further grown (30°C, 200 rpm) and induced after 8 hours with 0.5% v / v methanol. The following day, the construct was induced with 0.5% v / v methanol in the morning, midday, and evening. On day 5, the cells were centrifuged and the supernatant (containing the secreted Nanobody®) was collected.

[0201] General purification of Nanobodies® HIS6-tagged Nanobodies® were purified by immobilized metal affinity chromatography (IMAC) on either Ni-Excel (GE Healthcare) or Ni-IDA / NTA (Genscript) resin with imidazole (for the former) or acidic elution (for the latter), followed by a desalting step in PBS (PD column with Sephadex G25 resin, GE Healthcare) and, if necessary, gel filtration chromatography (Superdex column, GE Healthcare).

[0202] 3. Thermal Shift Assay The thermal stability of the anti-FX Nanobodies® was determined using a thermal shift assay (TSA) performed in a 96-well plate on a LightCycler 480II instrument (Roche). One Nanobody® per row was analyzed according to the following pH range: 3.5 / 4 / 4.5 / 5 / 5.5 / 6 / 6.5 / 7 / 7.5 / 8 / 8.5 / 9. Per well, 5 μL of Nanobody® sample (0.8 mg / mL in PBS) was added to 5 μL of Sypro Orange (40x in MilliQ water, Invitrogen catalog number S6551) and 10 μL of buffer (100 mM phosphate, 100 mM borate, 100 mM citrate, and 115 mM NaCl, pH range 3.5-9). An applied temperature gradient (37-99 °C at a rate of 0.03 °C / s) induces unfolding of the Nanobodies, thereby exposing their hydrophobic patches. Sypro Orange binds to these hydrophobic patches, resulting in an increase in fluorescence intensity (Ex / Em = 465 / 580 nm). The inflection point of the first derivative of the fluorescence intensity curve at pH 7 serves as a measure of the melting temperature (Tm).

[0203] 4. Analytical Size Exclusion Chromatography The potential occurrence of aggregation and oligomerization of Nanobodies® was investigated by analytical size-exclusion chromatography (SEC). For this purpose, 8 μg of Nanobody® sample (0.5 mg / mL in PBS) and 10 μL of BioRad standard (catalog no. 151-1901, diluted 1 / 10 in PBS) were injected onto a Waters Xbridge column (particle size: 3.5 μm, pore size 200 Å, internal diameter 7.8 mm) via a Dionex Ultimate 3000. Arginine buffer [10 mM phosphate + 150 mM arginine + 10% 1-propanol + 0.02% NaN3 (pH 7.0)] was used as the mobile phase, and a flow rate of 0.5 mL / min was applied. Overall SEC performance was determined by considering three parameters relative to the 1.35 kDa BioRad standard peak: peak area of ​​the main peak (>90%=pass), recovery (>80%=pass), and retention time.

[0204] 5. Competition Assay Antibodies (such as Nanobodies®) that bind to the same or similar epitopes can be characterized using a competition assay, which can be performed as follows.

[0205] 25 μL (1 μg / mL) of human plasma-derived FX (Haematologic Technologies Inc, USA) is immobilized in a microtiter plate (Nunc, Wiesbaden, Germany). After washing and blocking the wells, the "binning" mAb (mAb 00916) is added at a concentration of 1 μM in a buffer containing 50 mM HEPES pH 7.4, 2.5 mM CaCl, 1% BSA, and 0.05% Tween 20 (Sigma), or a buffer-only control, and pre-incubated with the coated human FX for 30 minutes. Test antibodies (here, different Nanobodies®) are then added and allowed to bind for 1 hour. Unbound test antibody (Nanobody®) and bound mAb are washed away, and bound Nanobody® is detected using horseradish peroxidase (HRP)-conjugated anti-FLAG mAb (Sigma, Cat. No. A8592) followed by an enzymatic reaction in the presence of the substrate esTMB (3,3',5,5'-tetramentylbenzidine) (SDT, Brussels, Belgium).

[0206] For "binned" mAbs, the percent inhibition observed in the presence of each of the test antibodies (Nanobodies®) is calculated according to the following formula:

number

[0207] An antibody molecule is generally considered to compete if greater than 50% inhibition is observed.

[0208] [Examples 1 to 5] Example 1: Anti-FX Nanobodies® From the pool of anti-FX Nanobodies, a group of Nanobodies (FX and non-FXa) that selectively bind FX zymogen were selected for further study. The sequences of the Nanobodies are defined by SEQ ID NOs: 8-11 and 54-57. Competition experiments with mAb 00916 (IgG1 isotype, variable sequences defined by SEQ ID NOs: 4 and 5) as a "binning" antibody were performed, and Nanobodies Nb 701B09, Nb 701C06, Nb 702C12, Nb 701D07, Nb 721E08, Nb 729A04, Nb 729C08, and Nb 730C03 were all shown to compete with mAb 00916. A control Nanobody®, Nb 501A02 (SEQ ID NO: 12), which binds to a different region of FX, was also included; no competition with mAb 00916 was observed for this Nanobody®. The results are shown in Table 1 below and show that all compounds except Nb 501A02 compete with mAb 00916 for binding to FX, i.e., for binding to epitopes on FX that are the same as or overlap with mAb 00916. [Table 1]

[0209] Example 2: SPR analysis of anti-FX Nanobody® binding to FX The binding of purified anti-FX Nanobodies (Nb) to human plasma-derived FX (Haematologic Technologies Inc, USA) was probed by Surface Plasmon Resonance (SPR) (Biacore T200).

[0210] Briefly, anti-His mAb (MAB050 from R&D Systems) was immobilized onto a CM4 sensor chip using standard amine coupling chemistry. Anti-FX Nanobodies (10 nM) were injected for 1 minute at a flow rate of 10 μL / min according to Table 3. Subsequently, 4096, 1024, 256, 64, 16, 4, and 0 nM FX were injected for 4 minutes at a flow rate of 30 μL / min to allow binding to the anti-FX Nb, followed by a 5-minute injection of running buffer (10 mM HEPES, 150 mM NaCl, 5 mM CaCl, 0.05% (v / v) Surfactant P20, 1 mg / mL bovine serum albumin, pH 7.4) to allow dissociation from the anti-FX Nanobody®. The running buffer was also used to dilute the anti-FX Nb and FX samples. Chip regeneration was achieved using a regeneration buffer consisting of 3 M MgCl, a contact time of 30 seconds, and a flow rate of 30 μL / min. Binding data were collected at 25° C. and analyzed with a 1:1 model using BiaEvaluation 4.1, supplied by the manufacturer (Biacore AB, Uppsala, Sweden).

[0211] In all cases, the combined sensograms were analyzed using K D showed a rapid on and rapid off coupled kinetic profile that precludes decision making. D The values ​​are determined based on steady-state analysis. The analysis yielded the binding constants reported in Table 2. The strongest binder identified was Nb702C12, while the other Nanobodies® tested had slightly higher K D and low R max Combine with FX. [Table 2]

[0212] Example 3: Activity of anti-FX Nanobodies in FXa generation assays The ability of monovalent anti-FX Nanobodies® (Nbs) to promote FIXa-mediated FX activation is thought to occur through multiple mechanisms, as reviewed in Scheiflinger et al. (2008) J Thromb Haemost, 6:315-322. Also included for comparison was the Fab of mAb 13F62 (IgG1 isotype, variable sequences identified by SEQ ID NOs: 6 and 7), which was also found to bind to the activation peptide. Additionally, anti-FX antibodies such as those disclosed in US 9,334,331 (J327 and L404-k therein) and more recently WO2018 / 021450 (J327 D31H and JYL280 therein) and WO2018 / 098363 (BIIB-12-917 represented by SEQ ID NO: 427 and 615 therein), which correspond to the anti-FX arm of ACE910, have been specified as suitable for use as part of anti-FIX / anti-FX bispecific FVIII mimetic compounds and were further included for comparison.

[0213] Each compound was tested individually at concentrations ranging from 0-1000 nM, 0-2000 nM, 0-4000 nM, or 0-8000 nM by preincubating for 10 min with 1-1.5 nM human plasma-derived FIXa (Haematologic Technologies Inc, USA) and 500 μM 25:75 ratio phosphatidylserine:phosphatidylcholine phospholipid vesicles (Haematologic Technologies Inc, USA) in assay buffer (50 mM HEPES, 100 mM NaCl, 5 mM CaCl, 0.1% (w / v) PEG 8000, 1 mg / mL bovine serum albumin, pH 7.3). The reaction was initiated by the addition of human plasma-derived FIXa (Haematologic Technologies Inc, USA) to a final concentration of 100 nM and a reaction volume of 50 μL. After 20 minutes of activation at room temperature with agitation (1000 RPM), the reaction was quenched by the addition of 25 μL of quench buffer (50 mM HEPES, 100 mM NaCl, 60 mM EDTA, 0.1% (w / v) PEG 8000, 1 mg / mL bovine serum albumin, pH 7.3). The amount of FXa produced was determined by adding 25 μL of 2 mM S-2765 chromogenic substrate (Chromogenix, Sweden), followed by measuring absorbance at 405 nm for 10 minutes in a microplate reader to determine chromogenic substrate conversion. The observed change in absorbance at 405 nm (ΔAbs405 / min) was fitted to a quadratic coupling equation (Equation 1) to obtain peak stimulatory activity and EC50.

[0214] Table 3 shows the fitted maximum increase in FXa production rate (fold increase in FX activation) and fitted K D The EC50 values ​​are listed. Most of the tested nanobodies stimulated FIXa-mediated FX activation (approximately 4- to 83-fold over FIXa alone), whereas the Fab of mAb 13F62 showed 1.5-fold activation and Nb 501A02 showed no activation. Fitted EC50 values ​​are not provided for the Fab of 13F62 and FIXa alone.

number

[0215] In the formula, Y i is the substrate conversion (ΔAbs405 / min) observed at a given FX binder concentration (i), [FX] and [binder] are the total concentrations FX and FX binder, respectively, and K D is the apparent dissociation constant of the FX binder, and Y max and Y0 are the maximum and initial signals (ΔAbs405 / min), respectively. [Table 3]

[0216] Example 4: Effect of anti-FX Nanobodies on FXa amidolytic activity To exclude the possibility that the fold increase in activation observed in Example 3 was due to a positive effect of the Nanobodies on FXa amidolytic activity, the ability of monovalent anti-FX Nanobodies to stimulate FXa amidolytic activity was investigated as follows: 5 nM human plasma-derived FIXa (Haematologic Technologies Inc, USA) was preincubated for 10 minutes with 50 μM of a 25:75 ratio of phosphatidylserine:phosphatidylcholine phospholipid vesicles (Haematologic Technologies Inc, USA) in assay buffer (50 mM HEPES, 100 mM NaCl, 5 mM CaCl, 0.1% (w / v) PEG 8000, 1 mg / mL bovine serum albumin, pH 7.3). Human plasma-derived FX (Haematologic Technologies Inc, USA) was added to the reaction at a final concentration of 100 nM and a final activation volume of 40 μL. After 20 minutes of incubation at room temperature with agitation (1000 RPM), the FX activation reaction was terminated by the addition of 25 μL of quench buffer (50 mM HEPES, 100 mM NaCl, 60 mM EDTA, 0.1% (w / v) PEG 8000, 1 mg / mL bovine serum albumin, pH 7.3). Subsequently, 10 μL of 25 μM of each FX binder was added to the generated FXa, and 25 μL of 2 mM S-2765 chromogenic substrate (Chromogenix, Sweden) was added. Chromogenic substrate conversion was determined by measuring absorbance at 405 nm (ΔAbs405 / min) in a microplate reader. Relative FXa amidolytic activity (a rel ) is calculated using Equation 2 below:

number

[0217] Binder is the amidolytic activity of the FXa / FX binder complex, and aFXa is the amidolytic activity of FXa alone.

[0218] Table 4 shows the relative FXa amidolytic activity for individual FX binders and it was concluded that the increased FX activation was not due to Nanobodies® having increased FXa amidolytic activity. [Table 4]

[0219] Example 5: Binding epitopes of anti-FX Nanobodies® To determine whether the binding epitopes of anti-FX Nanobodies® and mAb 13F62 antibody are located within the activation peptide (AP) of human FX, an ELISA assay was set up.

[0220] Forty-one unique 12-mer peptide fragments spanning 52 residues of the activation peptide with one amino acid spacing were immobilized in microtiter plate wells and tested by subsequent incubation with Nanobody® / antibody, and detection of bound ligand was performed by addition of a secondary HRP-labeled antibody.

[0221] The peptides were C-terminally linked to biotin and immobilized in individual wells of a microtiter plate pre-coated with 1 μg / mL streptavidin using 50 μL of a 1 μg / mL peptide solution. Each well was washed with wash buffer (10 mM Tris, 150 mM NaCl, 2.5 mM CaCl, 0.05% Tween 20, pH 8.60) followed by the addition of 50 μL of 2 μg / mL FLAG-tagged anti-FX Nanobody®. After 1 hour, unbound Nanobody® / antibody was washed away using wash buffer.

[0222] Bound anti-FX activating peptide ligand was detected by first binding an HRP-conjugated secondary antibody (Anti-FLAG mAb M2-Peroxidase (HRP) (Sigma-Aldrich, USA) for FLAG-tagged nanobodies, or Fcγ Fragment Specific Peroxidase AffiniPure F(ab')2 Fragment Goat Anti-Human IgG (Jackson ImmunoResearch Laboratories, Inc., US) or Peroxidase AffiniPure Goat Anti-Mouse IgG (Jackson ImmunoResearch Laboratories, Inc., US) for antibodies) for 1 hour, followed by addition of 100 μL of TMB-1 ELISA substrate (Kem-En-Tec Diagnostics, Denmark).

[0223] A minimal epitope was then deduced from the set of peptides that yielded a signal above baseline by identifying the consensus sequence contained in the set of peptides (Table 5) bound by each FX binder. The first residue of the epitope was defined by the last amino acid in the first consecutive ELISA-positive peptide, while the last residue of the epitope was defined as the first residue of the last consecutive ELISA-positive peptide.

[0224] Thus, Table 5 shows the ELISA signals from consecutive peptides spanning the FX AP for several different anti-FX Nanobodies® (Nb) and anti-FX mAb 13F62. ELISA signals are normalized to background signal (bbg). The gray area indicates the positive binding signal used to determine the minimal epitope. [Table 5-1] [Table 5-2]

[0225] For each FX binder, the minimal epitope was defined as the amino acid sequence common among the ELISA-positive peptides. The determined minimal epitopes within the AP are summarized in Table 6. [Table 6]

[0226] By correlating the data in Tables 5 and 6 with the data in Table 3, it is observed that for FX binders whose minimal epitope on FX is, for example, "PFDLLDF," the ability to stimulate FX activation is observed.

[0227] Subsequently, from Table 6, the common minimal epitope can be identified as "LL".

[0228] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will occur to those skilled in the art. It is therefore to be understood that the appended claims are intended to cover all such modifications and changes that fall within the true spirit of the invention.

Claims

1. An antibody or antigen-binding fragment thereof capable of binding to FX, said antibody or antigen-binding fragment comprising: a. Can stimulate FX activation? b. Can stimulate FX activation by FIXa? c. stimulates FX activation; d. stimulates FX activation by FIXa; e. FX can be made more susceptible to proteolysis, or f. An antibody or antigen-binding fragment thereof that can make FX more susceptible to proteolysis by FIXa.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the activation / proteolysis of FX is determined as described in Example 3 of the present specification.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is capable of binding to the activation peptide of FX (amino acid residues 143 to 194 of SEQ ID NO: 2).

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof is capable of binding to the activation peptide of FX, and the common minimal epitope determined by peptide array comprises amino acid residues "LL" (amino acid residues 177-178 of SEQ ID NO: 2).

5. An antibody or antigen-binding fragment thereof capable of binding to FX, a. comprising the heavy chain variable domain (SEQ ID NO: 4) and light chain variable domain (SEQ ID NO: 5) of mAb 00916; or b. comprising the three CDRs of the heavy chain variable domain (SEQ ID NO: 4) and the three CDRs of the light chain variable domain (SEQ ID NO: 5) of mAb 00916; or c. comprising the heavy chain variable domain (SEQ ID NO: 6) and light chain variable domain (SEQ ID NO: 7) of mAb 13F62; d. comprising the three CDRs of the heavy chain variable domain (SEQ ID NO: 6) and the three CDRs of the light chain variable domain (SEQ ID NO: 7) of mAb 13F62; or e. comprising SEQ ID NO: 8, 9, 10, 11, 54, 55, 56 or 57; or f. An antibody or antigen-binding fragment thereof comprising three CDRs of SEQ ID NO: 8, 9, 10, 11, 54, 55, 56, or 57.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the sequence identified by SEQ ID NO: 8, 9, 10, 11, 54, 55, 56, or 57, respectively.

7. An antibody or antigen-binding fragment thereof capable of binding to FX, which competes with a reference antibody or antigen-binding fragment thereof according to claim 5.

8. the antibody or antigen-binding fragment a. Can stimulate FX activation? b. Can stimulate FX activation by FIXa? c. stimulates FX activation; d. stimulates FX activation by FIXa; e. FX can be made more susceptible to proteolysis, or f) The antibody or antigen-binding fragment thereof according to claim 5 or 7, which can make FX more susceptible to proteolysis by FIXa.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment thereof stimulates FX activation by FIXa, independently of the presence of a FIXa-binding agent.

10. the antibody or antigen-binding fragment has an equilibrium dissociation constant (K Ｄ The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, having a binding affinity determined as follows:

11. The antibody or antigen-binding fragment is monovalent, e.g., an immunoglobulin single variable domain or antigen-binding fragment thereof, Nanobody®, V Ｈ H, humanized V Ｈ H, camelization V Ｈ The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, which is a domain, for example, a Fab, scFab, Fv or scFv.

12. The antibody or antigen-binding fragment thereof is bivalent, e.g., a full-length mAb or a Fab' ２ The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10,

13. A bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12.

14. 14. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 for the manufacture of a medicament for use in the treatment of hemophilia, such as hemophilia A or B, with or without an inhibitor.

15. 15. The antibody or antigen-binding fragment thereof of any one of claims 1 to 14 for use in the treatment of hemophilia, such as hemophilia A or B, with or without inhibitors.

Images