Methods for treating factor x deficiency

Abstract

Compositions and methods for treating Factor X deficiency are disclosed. The compositions and methods include the use of a a FVIII mimetic bispecific antibody that binds Factor IXa and Factor X.

Description

[0001] METHODS FOR TREATING FACTOR X DEFICIENCY

[0002] By Benjamin J. Samelson-Jones Rodney Camire

[0003] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 703,322, filed October 4, 2024. The foregoing application is incorporated by reference herein.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to the fields of medicine and hematology. More specifically, the invention provides novel methods for treating Factor X deficiency.

[0006] BACKGROUND OF THE INVENTION

[0007] Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.

[0008] In response to vascular injury such as a cut, coagulation enzymes are activated in a stepwise manner, ultimately resulting in the formation of a blood clot at the site of injury. Thrombin is generated from its inactive precursor prothrombin in the final step of this cascade and subsequently produces the fibrous clot. Activated Factor IX (FIXa) is a key component of this system as it is the serine protease of the intrinsic Xase complex, which also comprises the co-factor activated Factor VIII (FVIIIa). This enzyme complex, assembled on cells with exposed anionic membranes, rapidly converts Factor X (FX) to activated Factor X (FXa). FXa and its co-factor, activated Factor V (FVa), form prothrombinase, the enzyme complex that activates thrombin.

[0009] Factor X deficiency is a rare genetic bleeding disorder that causes abnormally slow clotting process. Factor X deficiency is caused by a variant or mutation in the Factor X gene. Common symptoms of Factor X deficiency include nosebleeds, easy bruising, bleeding under the skin, bleeding of the gums, blood in the urine (hematuria), and prolonged or excessive bleeding following surgery or trauma. Severe FX deficiency can lead to life-threatening condition through the increased risk of intracranial hemorrhage, pulmonary hemorrhage, or excessive bleeding in the gastrointestinal tract. Current therapies, such as the administration of Factor X (e.g., Coagadex®), aim to restore circulating Factor X levels to at least 10-40% of normal. However, the administration of large amounts of Factor X can lead to the development of Factor X inhibitors. Therefore, there is a need for improved therapeutic methods.

[0010] SUMMARY OF THE INVENTION

[0011] In accordance with the present invention, methods for treating, inhibiting, and / or preventing treating Factor X deficiency in a subject are provided. In certain embodiments, the methods comprise administering a therapeutically effective amount of a FVIII mimetic bispecific antibody to the subject. In certain embodiments, the subject comprises a Factor X mutant or variant which causes Factor X deficiency. In certain embodiments, the subject comprises a Factor X mutant or variant which has a disrupted or reduced interaction or binding affinity between FX and FVIIIa and / or the Factor X mutant has reduced activation (e.g., compared to wild-type) by FIXa and FVIIIa (e.g., in the intrinsic Xase complex). In certain embodiments, the Factor X mutant comprises a mutation within the FVIIIa binding site of FX. In certain embodiments, the Factor X mutant comprises a mutation at R139, R247, T318 and / or R347. In certain embodiments, the Factor X mutant comprises a mutation selected from the group consisting of R139S, R139G, R247C, T318M, T318P, and / or R347C. In certain embodiments, the FVIII mimetic bispecific antibody binds Factor IXa and Factor X. In certain embodiments, the FVIII mimetic bispecific antibody is emicizumab or mim8. In certain embodiments, the method further comprises determining whether the subject comprises a Factor X deficiency mutant prior to treatment, optionally by obtaining a biological sample from the subject.

[0012] In accordance with the present invention, methods for increasing the coagulation of blood are provided. In certain embodiments, the methods comprise contacting the blood with a FVIII mimetic bispecific antibody. In certain embodiments, the blood comprises a Factor X mutant or variant which causes Factor X deficiency. In certain embodiments, the blood comprises a Factor X mutant which disrupts the interaction between FX and FVIIIa (e.g., compared to wild-type). In certain embodiments, the Factor X mutant has reduced activation (e.g., compared to wild-type) by FIXa and FVIIIa (e.g., in the intrinsic Xase complex). In certain embodiments, the Factor X mutant comprises a mutation within the FVIIIa binding site of FX. In certain embodiments, the Factor X mutant comprises a mutation at R139, R247, T318 and / or R347. In certain embodiments, the Factor X mutant comprises a mutation selected from the group consisting of R139S, R139G, R247C, T318M, T318P, and / or R347C. In certain embodiments, the FVIII mimetic bispecific antibody binds Factor IXa and Factor X. In certain embodiments, the FVIII mimetic bispecific antibody is emicizumab or mim8. In certain embodiments, the method further comprises determining the presence of the Factor X deficiency mutant prior to treatment or contacting with the FVIII mimetic bispecific antibody.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 A provides a schematic of a Factor Vlll-mimetic rescuing Factor X activity in a Factor X-deficiency causing variant. The deficient Factor X has dysfunctional assembly of the intrinsic Xase complex, optionally due to decreased affinity for Factor Villa caused by mutation(s) in Factor Villa-binding sites within Factor X. Figure IB provides examples of mutations causing Factor X deficiency. The (-) positions are present in the prepropeptide.

[0015] Figures 2A-2C show FX activity in a one-stage activated partial thromboplastin time (aPTT) assay with conditioned media in the absence (left bar) or presence (right bar) of 300 nM emicizumab. Bars represent mean of multiple transfections. UC: untransfected control.

[0016] Figure 3 A shows FX activity in a one-stage activated partial thromboplastin time (aPTT) assay without a Factor VIII mimetic (left), with 300 nM emicizumab (center), or with 34 nM Mim8 (right; except R247C). Error bars represent the mean ± standard deviation of the mean of > 3 independent transfections.

[0017] Figure 3B provides thrombograms showing thrombin generation. Wild-type (WT) FX, unconditioned media (UC), and FX deficiency-causing FX variants were studied with 0 nM (black) and 300 nM emicizumab (grey). Thrombin generation was triggered in the presence of 4 pM PCPS with 1 pM FXIa (top row) and 0.07 pM tissue factor (bottom row). The results are representative of 2 thrombograms from 2 independent transfections.

[0018] DETAILED DESCRIPTION OF THE INVENTION

[0019] The recent advent of bispecific antibody Factor VIII (FVIII)-mimetics has revolutionized the clinical care of hemophilia A (HA, FVIII deficiency). Activated factor IX (FIXa) and F Villa are responsible for the proteolytic activation of Factor X (FX), the rate limiting step of clot formation. Herein, it is shown that patients with FX deficiency, such as those caused by mutations that result in dysfunctional Xase complex formation, have improvement in their hemostatic function using bispecific antibody FVIII-mimetics. The Xase complex comprises Factor IXa (FIXa) and its cofactor coagulation Factor Villa (FVIIIa) and catalyzes the activation of Factor X (FX) to Factor Xa (FXa), stimulating thrombin production in the blood coagulation cascade.

[0020] Specifically, FVIII-mimetics can have efficacy in patients with Factor X deficiency with loss-of-function FX variants due to restricted FX activation by the FVIIIa / FIXa complex. The effect of emicizumab, a bispecific antibody FVIII-mimetic, on recombinant FX variants identified as causing FX-deficiency is shown herein. Certain FX variants show a dramatic improvement in the FX levels with the addition of emicizumab, which is sufficient for therapeutic efficacy against Factor X deficiency.

[0021] Factor X is a serine protease zymogen and is a substrate for both the extrinsic

[0022] (tissue factor / FVIIa) and intrinsic (FVIIIa / FIXa) tenase enzyme complexes which cleave the Arg-Ile scissile bond in FX thereby releasing a 52-amino acid activation peptide generating FXa. Gene ID: 2159 and GenBank Accession Nos. NM_000504.4 and

[0023] NP 000495.1 provide examples of the amino acid and nucleotide sequences of wild-type human Factor X preproprotein. An example of the amino acid sequence of the human Factor X protein is:

[0024] 1 ANSFLEEMKK GHLERECMEE TCSYEEAREV FEDSDKTNEF WNKYKDGDQC

[0025] 51 ETSPCQNQGK CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC 101 HEEQNSWCS CARGYTLADN GKACIPTGPY PCGKQTLERR KRSVAQATSS 151 SGEAPDS ITW KPYDAADLDP TENPFDLLDF NQTQPERGDN NLTRIVGGQE 201 CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ AKRFKVRVGD 251 RNTEQEEGGE AVHEVEWIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301 ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC 351 KLSSSFI ITQ NMFCAGYDTK QEDACQGDSG GPHVTRFKDT YFVTGIVSWG 401 EGCARKGKYG IYTKVTAFLK WIDRSMKTRG LPKAKSHAPE VITSSPLK ( SEQ ID NO : 1 )

[0026] The underlined amino acids are certain of the amino acids discussed herein. Bolded and italicized amino acids are certain of the amino acids discussed herein with improved activity in the presence of a FVIII mimetic.

[0027] Factor X can be synthesized as a prepropetide with a signal peptide and a propeptide. The cleavage of the propeptide yields a protein with a new terminus sequence of Ala-Asn-Ser, depicted in SEQ ID NO: 1. Factor X is cleaved into a mature two-chain form (light and heavy) at the excision of the tripeptide RKR to generate the Factor X zymogen. The two chains are linked via at least one disulfide bond. An example of the amino acid sequence of the human Factor X light chain is: ANSFLEEMKK GHLERECMEE TCSYEEAREV FEDSDKTNEF WNKYKDGDQC ETSPCQNQGK CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSWCS CARGYTLADN GKACIPTGPY PCGKQTLER ( SEQ ID NO : 2 )

[0028] An example of the amino acid sequence of the human Factor X heavy chain is:

[0029] SVAQATSS SGEAPDSITW KPYDAADLDP TENPFDLLDF NQTQPERGDN NLTRIVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ AKRFKVRVGD RNTEQEEGGE AVHEVEWIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFI ITQ NMFCAGYDTK QEDACQGDSG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK WIDRSMKTRG LPKAKSHAPE VITSSPLK ( SEQ ID NO : 3 )

[0030] Factor X is activated by the cleavage of the activation peptide to yield a new aminoterminal sequence of the heavy chain. An example of the amino acid sequence of the human Factor Xa light chain is:

[0031] ANSFLEEMKK GHLERECMEE TCSYEEAREV FEDSDKTNEF WNKYKDGDQC ETSPCQNQGK CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC

[0032] HEEQNSWCS CARGYTLADN GKACIPTGPY PCGKQTLER ( SEQ ID NO : 2 )

[0033] An example of the amino acid sequence of the human Factor Xa heavy chain is:

[0034] IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ

[0035] AKRFKVRVGD RNTEQEEGGE AVHEVEWIK HNRFTKETYD FDIAVLRLKT

[0036] PITFRMNVAP ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFI ITQ NMFCAGYDTK QEDACQGDSG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK WIDRSMKTRG LPKAKSHAPE VITSSPLK ( SEQ ID NO : 4 )

[0037] Nucleic acid molecules which encode Factor X and FXa can be readily determined from the provided amino acid sequences.

[0038] The instant invention encompasses methods of inhibiting, treating, and / or preventing Factor X deficiency. In certain embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a FVIII mimetic (mimicking) bispecific antibody. The FVIII mimetic bispecific antibody may mimic the activity of F Villa in the intrinsic Xase complex (e.g., by bringing FIXa and FX into proximity). In certain embodiments, the FVIII mimetic bispecific antibody comprises a first binding domain (e.g., an antigen binding antibody fragment, such as a Fab arm) which binds FIXa and / or FIX and a second binding domain (e.g., an antigen binding antibody fragment, such as a Fab arm) which binds FX and / or FXa. The FVIII mimetic bispecific antibody, by binding both FIXa and FX brings the two proteins into proximity such that FIXa activates FX to FXa. In certain embodiments, the FVIII mimetic bispecific antibody comprises an Fc region. In certain embodiments, the FVIII mimetic bispecific antibody comprises an anti-FIXa heavy chain, an anti-FX heavy chain, and two light chains (optionally identical or different). In certain embodiments, the FVIII mimetic bispecific antibody is emicizumab (ACE910; HEMLIBRA®; Genentech;

[0039] DrugBank Accession Number DB 13923; Lenting, et al. Blood (2017) 130(23):2463- 2468; Walsh, et al., J. Managed Care Med., 22(2):65-69). In certain embodiments, the FVIII mimetic bispecific antibody is mim8 (denecimig; UNII: EUV85RR8DJ (drugs. neats. io / drug / EUV85RR8DJ); Novo Nordisk; Kjellev et al., Blood (2019) 134 (Supplement l): 96; Ostergaard et al., Blood (2021) 138(14): 1258-1268). In certain embodiments, the FVIII mimetic bispecific antibody is NXT007 (Teranishi-Ikawa, et al. (2024) J. Thromb. Haemost., 22:430-40). In certain embodiments, the FVIII mimetic bispecific antibody is described in WO2018 / 047813, U.S. Patent Application Publication No. 2020 / 0148787 or U.S. Patent No. 10,759,870 or 11,150,254 (each incorporated herein by reference).

[0040] In certain embodiments, the subject being treated has a mutation in FX (e.g., not wild-type FX). In certain embodiments, the subject comprises a FX mutant or variant which causes Factor X deficiency. In certain embodiments, the FX mutation disrupts the interaction between FX and FVIIIa. In certain embodiments, the FX mutation lowers the binding affinity of FX for FVIIIa (e.g., compared to wild-type FX). In certain embodiments, the FX mutation decreases the FIXa and FVIIIa induced activation of FXa (e.g., compared to wild-type FX). In certain embodiments, the FX mutation is located within the FVIIIa binding site of FX.

[0041] In certain embodiments, the FX mutation is a mutation set forth in Figure IB. In certain embodiments, the FX mutation is at a position presented in Figure IB. In certain embodiments, the FX mutation is at R139, R247, T318 and / or R347. In certain embodiments, the FX mutation is not Cl 11R, E269K, Y279H, R287W, or M362T. In certain embodiments, the FX mutation is not Cl 11R, A234S, A235T, E269K, Y279H, R287W, M362T, W421R, or S425P.

[0042] In certain embodiments, the mutation at R139 is not R139C. In certain embodiments, the mutation at R139 is R139H, R139L, R139P, R139S, or R139G. In certain embodiments, the mutation at R139 is R139S or R139G. In certain embodiments, the mutation at R247 is R247C. In certain embodiments, the mutation at T318 is not T318S. In certain embodiments, the mutation at T318 is T318A, T318K, T318M, or T318P. In certain embodiments, the mutation at T318 is T318M or T318P. In certain embodiments, the mutation at R347 is R347C or R347H, particularly R347C.

[0043] The methods of the instant invention may further comprise determining if the subject to be treated (e.g., a subject with Factor X deficiency) has a mutant FX. In certain embodiments, the FX mutation lowers the binding affinity of FX for F Villa (e.g., compared to wild-type FX). In certain embodiments, the FX mutation decreases the FIXa and F Villa activation of FX (e.g., compared to wild-type FX). In certain embodiments, the FX mutation is located within the FVIIIa binding site of FX. In certain embodiments, the method further comprises obtaining a biological sample (e.g., whole blood, cells (e.g., blood cells), plasma, purified or isolated FX, etc.) from the subject.

[0044] In certain embodiments, the FX mutation is at one of the locations set forth herein or is one of the FX mutations set forth herein. In certain embodiments, the FX mutation is a mutation set forth in Figure IB. In certain embodiments, the FX mutation is at a position presented in Figure IB. In certain embodiments, the FX mutation is at R139, R247, T318 and / or R347. In certain embodiments, the FX mutation is not Cl 11R, E269K, Y279H, R287W, or M362T. In certain embodiments, the FX mutation is not Cl HR, A234S, A235T, E269K, Y279H, R287W, M362T, W421R, or S425P.

[0045] In certain embodiments, the mutation at R139 is not R139C. In certain embodiments, the mutation at R139 is R139H, R139L, R139P, R139S, or R139G. In certain embodiments, the mutation at R139 is R139S or R139G. In certain embodiments, the mutation at R247 is R247C. In certain embodiments, the mutation at T318 is not T318S. In certain embodiments, the mutation at T318 is T318A, T318K, T318M, or T318P. In certain embodiments, the mutation at T318 is T318M or T318P. In certain embodiments, the mutation at R347 is R347C or R347H, particularly R347C.

[0046] In certain embodiments, the method further comprises administering an additional therapeutic agent (e.g., human FX (e.g., Coagedex®), prothrombin complex (e.g., Balfaxar®), vitamin K therapy, and / or immunosuppressive therapy (e.g., corticosteroid (e.g., prednisolone))) for Factor X deficiency to the subject.

[0047] Whether a subject has a mutant FX can be determined by any method known in the art. In certain embodiments, a nucleic acid encoding FX from a subject may be analyzed. For example, all or part of a nucleic acid sequence encoding FX (e.g., gene, mRNA (optionally converted to cDNA), etc.) may be determined or sequenced from a biological sample from the subject and then it can be determined whether the encoded for FX comprises any mutations compared to wild-type FX. In certain embodiments, the FX protein from a subject may be analyzed. For example, all or part of the amino acid sequence from the FX from the subject may be determined and compared to the wild-type FX sequence. Binding assays or activation assays may also be performed with the FX from the subject (e.g., isolated from the subject or recombinantly produced) may also be performed to determine if the FX from the subject has a lower binding affinity for F Villa (e.g., compared to wild-type FX) or has decreased FIXa and FVIIIa activation (e.g., compared to wild-type FX).

[0048] The instant invention also encompasses methods of increasing the coagulation of blood (e.g., compared to the blood without treatment and / or to wild-type blood). For example, the method may decrease the clot time of the blood (e.g., compared to the blood clot time without treatment and / or to wild-type blood). In certain embodiments, the method decreases the clot time of the blood to less than a minute, particularly less than about 40 seconds. In certain embodiments, the method decreases the clot time of the blood to about normal clot time for blood. The method may be performed in vitro or in vivo. The methods may further comprise determining if the blood, cell, and / or subject has a mutant FX (e.g., as described hereinabove, such as by sequencing).

[0049] In certain embodiments, the method comprises contacting the blood with a FVIII mimetic bispecific antibody. In certain embodiments, the FVIII mimetic bispecific antibody comprises a first binding domain (e.g., an antigen binding antibody fragment, such as a Fab arm) which binds FIXa / FIX and a second binding domain (e.g., an antigen binding antibody fragment, such as a Fab arm) which binds FX / FXa. The FVIII mimetic bispecific antibody, by binding both FIXa and FX brings the two proteins into proximity such that FIXa activates FX to FXa. In certain embodiments, the FVIII mimetic bispecific antibody comprises an Fc region. In certain embodiments, the FVIII mimetic bispecific antibody comprises an anti-FIX heavy chain, an anti-FX heavy chain, and two light chains (optionally identical or different). In certain embodiments, the FVIII mimetic bispecific antibody is emicizumab (ACE910; HEMLIBRA®; Genentech; DrugBank Accession Number DB 13923; Lenting, et al. Blood (2017) 130(23):2463-2468; Walsh, et al., J. Managed Care Med., 22(2):65-69). In certain embodiments, the FVIII mimetic bispecific antibody is mim8 (Novo Nordisk; Kjellev et al., Blood (2019) 134 (Supplement l): 96; Ostergaard et al., Blood (2021) 138(14): 1258-1268). In certain embodiments, the FVIII mimetic bispecific antibody is NXT007 (Teranishi-Ikawa, et al. (2024) J. Thromb. Haemost., 22:430-40). In certain embodiments, the FVIII mimetic bispecific antibody is described in WO2018 / 047813, U.S. Patent Application Publication No. 2020 / 0148787, or U.S. Patent No. 10,759,870 or 11,150,254 (each incorporated herein by reference).

[0050] In certain embodiments, the blood comprises a mutant FX. In certain embodiments, the FX mutant or variant causes Factor X deficiency. In certain embodiments, the FX mutation disrupts the interaction between FX and FVIIIa. In certain embodiments, the FX mutation lowers the binding affinity of FX for FVIIIa (e.g., compared to wild-type FX). In certain embodiments, the FX mutation decreases the FIXa and FVIIIa activation of FX (e.g., compared to wild-type FX). In certain embodiments, the FX mutation is located within the FVIIIa binding site of FX. In certain embodiments, the FX mutation is at one of locations set forth herein or is one of the FX mutations set forth herein. In certain embodiments, the FX mutation is a mutation set forth in Figure IB. In certain embodiments, the FX mutation is at a position presented in Figure IB. In certain embodiments, the FX mutation is at R139, R247, T318 and / or R347. In certain embodiments, the FX mutation is not Cl 11R, E269K, Y279H, R287W, or M362T. In certain embodiments, the FX mutation is not Cl 11R, A234S, A235T, E269K, Y279H, R287W, M362T, W421R, or S425P.

[0051] In certain embodiments, the mutation at R139 is not R139C. In certain embodiments, the mutation at R139 is R139H, R139L, R139P, R139S, or R139G. In certain embodiments, the mutation at R139 is R139S or R139G. In certain embodiments, the mutation at R247 is R247C. In certain embodiments, the mutation at T318 is not T318S. In certain embodiments, the mutation at T318 is T318A, T318K, T318M, or T318P. In certain embodiments, the mutation at T318 is T318M or T318P. In certain embodiments, the mutation at R347 is R347C or R347H, particularly R347C.

[0052] In accordance with the instant invention, methods of determining whether a subject (e.g., a subject with Factor X deficiency) can be treated with a FVIII mimetic bispecific antibody are provided. In certain embodiments, the method comprises obtaining blood or a fraction thereof (e.g., whole blood, cells, plasma, purified or isolated FX, etc.) from the subject. In certain embodiments, the method comprises determining if the subject has a mutant FX (e.g., as described hereinabove, such as by sequencing). In certain embodiments, the method comprises contacting blood or a fraction thereof (e.g., plasma, purified or isolated FX, etc.) with a FVIII mimetic bispecific antibody and determining if FX activity is increased (e.g., compared to the absence of the FVIII mimetic bispecific antibody), wherein an increase in FX activity indicates the subject can be treated with a FVIII mimetic bispecific antibody. In certain embodiments, FX activity is measured by a clotting assay (e.g., an aPTT assay). In certain embodiments, FX activity is measured by rotational thromboelastometry. In certain embodiments, FIX activity is measured by thrombin generation.

[0053] The FVIII mimetic bispecific antibody may be administered as part of a composition with a pharmaceutically acceptable carrier. The compositions of the instant invention may be conveniently formulated for administration with any carrier, particularly any pharmaceutically acceptable carrier(s). Except insofar as any conventional carrier is incompatible with the agents to be administered, its use in the pharmaceutical composition is contemplated. For example, the active agents may be formulated with an acceptable medium such as sterile liquid, water, aqueous solutions, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents or suitable mixtures thereof. The concentration of the active agents in the chosen medium may be varied and the medium may be chosen based on the desired route of administration of the pharmaceutical preparation. Except insofar as any conventional media or agent is incompatible with the active agents to be administered, its use in the pharmaceutical preparation is contemplated.

[0054] The compositions of the present invention can be administered by any suitable route, for example, by infusion, injection or other modes of administration such as controlled release devices. In certain embodiments, the composition is delivered by injection (e.g., to the bloodstream). In certain embodiments, the composition is delivered by intravenous injection. In certain embodiments, the composition is delivered subcutaneously. The compositions of the instant invention may be directly administered or applied to the site of bleeding (e.g., by injection). In general, pharmaceutical compositions and carriers of the present invention comprise, among other things, pharmaceutically acceptable buffers, diluents, liquids (such as water, saline, glycerol, sugars and ethanol), preservatives, stabilizing agents, solubilizers, emulsifiers, wetting agents, pH buffering substances adjuvants and / or carriers. Such compositions can include diluents of various buffer content (e.g., saline, Tris HC1, acetate, phosphate), pH and ionic strength; and additives such as detergents and solubilizing agents (e.g., polysorbate 80), anti oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., benzyl alcohol) and bulking substances (e.g., lactose, mannitol). For example, the preparation can be formulated with a buffer containing salts, such as NaCl, CaCh, and amino acids, such as glycine and / or lysine, and in a pH range from 6 to 8. The pharmaceutical compositions may be formulated in aqueous solutions (e.g., physiologically compatible buffers). Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. The compositions of the invention may also be incorporated into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc., or into liposomes or micelles, or mixed with phospholipids or micelles to increase stability. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of components of a pharmaceutical composition of the present invention. Exemplary pharmaceutical compositions and carriers are provided, e.g., in “Remington’s Pharmaceutical Sciences” by E.W. Martin (Mack Pub. Co., Easton, Pa.) and “Remington: The Science and Practice of Pharmacy” by Alfonso R. Gennaro (Lippincott Williams & Wilkins) which are herein incorporated by reference. The pharmaceutical composition of the present invention can be prepared, for example, in liquid form, deep-frozen, or can be in dried powder form (e.g., lyophilized). In a particular embodiment, when the preparation is stored in lyophilized form, it may be dissolved into a visually clear solution using an appropriate reconstitution solution prior to administration.

[0055] The compositions described herein will generally be administered to a patient as a pharmaceutical preparation. The term “patient” or “subject”, as used herein, refers to human or animal subjects. The compositions of the instant invention may be employed therapeutically, under the guidance of a physician.

[0056] The dose and dosage regimen of the compositions according to the invention that are suitable for administration to a particular patient may be determined by a physician considering the patient’s age, sex, weight, general medical condition, and the specific condition for which the active agent is being administered and the severity thereof (e.g., the severity of the bleeding). The physician may also take into account the route of administration, the pharmaceutical carrier, and the particular agent’s biological activity.

[0057] Selection of a suitable pharmaceutical preparation will also depend upon the mode of administration chosen. For example, the compositions of the invention may be administered by direct injection to a desired site. In this instance, a pharmaceutical preparation comprises the active agents of the instant invention dispersed in a medium that is compatible with the site of injection. The compositions of the instant invention may be administered by any method. For example, the compositions can be administered, without limitation, intravenously. Pharmaceutical preparations for injection are known in the art. If injection is selected as a method for administering the compositions, steps must be taken to ensure that sufficient amounts of the molecules reach their target cells to exert a biological effect.

[0058] A pharmaceutical preparation of the invention may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to a physically discrete unit of the pharmaceutical preparation appropriate for the patient undergoing treatment. Each dosage should contain a quantity of active ingredient calculated to produce the desired effect in association with the selected pharmaceutical carrier. Procedures for determining the appropriate dosage unit are well known to those skilled in the art. Dosage units may be proportionately increased or decreased based on the weight of the patient. Appropriate concentrations for alleviation of a particular pathological condition may be determined by dosage concentration curve calculations, as known in the art.

[0059] In accordance with the present invention, the appropriate dosage unit for the administration of the composition may be determined by evaluating the toxicity of the molecules in animal models. Various concentrations of active agents in pharmaceutical preparations may be administered to mice or other animal models, and the minimal and maximal dosages may be determined based on the beneficial results and side effects observed as a result of the treatment. Appropriate dosage unit may also be determined by assessing the efficacy of the treatment in combination with other standard drugs. The dosage units of the compositions of the instant invention may be determined individually or in combination with each treatment according to the effect detected.

[0060] The invention includes, but is not limited to, the embodiments of the following numbered paragraphs:

[0061] 1. A method of treating or inhibiting Factor X deficiency in a subject in need thereof comprising administering a therapeutically effective amount of a Factor VIII (FVIII) mimetic bispecific antibody to said subject, wherein said subject comprises a Factor X (FX) mutant. 2. The method of paragraph 1, wherein activated FVIII (FVIIIa) has decreased binding affinity for the FX mutant compared to wild-type FX and / or the FX mutant has decreased activation by FIXa and FVIIIa compared to wild-type FX.

[0062] 3. The method of paragraph 1 or 2, wherein said FX mutant comprises a mutation within the FVIIIa binding site of FX.

[0063] 4. The method of any one of paragraphs 1-3, wherein said FX mutant comprises a mutation at R139, R247, T318 and / or R347.

[0064] 5. The method of any one of paragraphs 1-4, wherein said FX mutant comprises a mutation selected from the group consisting of R139S, R139G, R247C, T318M, T318P, and / or R347C.

[0065] 6. The method of any one of paragraphs 1-5, wherein said FVIII mimetic bispecific antibody binds Factor IXa and Factor X.

[0066] 7. The method of any one of paragraphs 1-6, wherein said FVIII mimetic bispecific antibody is emicizumab.

[0067] 8. The method of any one of paragraphs 1-6, wherein said FVIII mimetic bispecific antibody is mim8.

[0068] 9. The method of any one of paragraphs 1-8, further comprising determining said subject comprises said Factor X mutant prior to administering said FVIII mimetic bispecific antibody.

[0069] 10. The method of paragraph 9, wherein determining said subject comprises said Factor X mutant comprises at least partially sequencing a nucleic acid encoding Factor X from said subject.

[0070] 11. The method of any one of paragraphs 1-10, further comprising obtaining a biological sample from said subject. 12. A method for increasing blood coagulation comprising contacting blood with a Factor VIII (FVIII) mimetic bispecific antibody, wherein said blood comprises a Factor X (FX) mutant.

[0071] 13. The method of paragraph 12, wherein said FX mutant causes Factor X deficiency.

[0072] 14. The method of paragraph 12 or 13, wherein activated FVIII (F Villa) has decreased binding affinity for the FX mutant compared to wild-type FX.

[0073] 15. The method of any one of paragraphs 12-14, wherein the FX mutant has decreased activation by FIXa and FVIIIa compared to wild-type FX.

[0074] 16. The method of any one of paragraphs 12-15, which is an in vitro method.

[0075] 17. The method of any one of paragraphs 12-16, wherein said Factor IX mutant comprises a mutation within the FVIIIa binding site of FX.

[0076] 18. The method of any one of paragraphs 12-17, wherein said FX mutant comprises a mutation at R139, R247, T318 and / or R347.

[0077] 19. The method of any one of paragraphs 12-18, wherein said FX mutant comprises a mutation selected from the group consisting of R139S, R139G, R247C, T318M, T318P, and / or R347C.

[0078] 20. The method of any one of paragraphs 12-19, wherein said FVIII mimetic bispecific antibody binds Factor IXa and Factor X.

[0079] 21. The method of any one of paragraphs 12-20, wherein said FVIII mimetic bispecific antibody is emicizumab.

[0080] 22. The method of any one of paragraphs 12-20, wherein said FVIII mimetic bispecific antibody is mim8. 23. The method of any one of paragraphs 12-22, further comprising determining said blood comprises said Factor X mutant prior to administering said FVIII mimetic bispecific antibody.

[0081] 24. The method of paragraph 23, wherein determining said blood comprises said Factor X mutant comprises at least partially sequencing a nucleic acid encoding Factor X from said blood.

[0082] 25. A method of identifying a subject with Factor X deficiency as being responsive or treatable with a Factor VIII (FVIII) mimetic bispecific antibody, said method comprises determining the mutation in the Factor X (FX) mutant.

[0083] 26. The method of paragraph 25, wherein activated FVIII (F Villa) has decreased binding affinity for the FX mutant compared to wild-type FX and / or the FX mutant has decreased activation by FIXa and FVIIIa compared to wild-type FX.

[0084] 27. The method of paragraph 25 or 26, wherein said FX mutant comprises a mutation within the FVIIIa binding site of FX.

[0085] 28. The method of any one of paragraphs 25-27, wherein said FX mutant comprises a mutation at R139, R247, T318 and / or R347.

[0086] 29. The method of any one of paragraphs 25-28, wherein said FX mutant comprises a mutation selected from the group consisting of R139S, R139G, R247C, T318M, T318P, and / or R347C.

[0087] 30. The method of any one of paragraphs 25-29, wherein said FVIII mimetic bispecific antibody binds Factor IXa and Factor X.

[0088] 31. The method of any one of paragraphs 25-30, wherein said FVIII mimetic bispecific antibody is emicizumab.

[0089] 32. The method of any one of paragraphs 25-30, wherein said FVIII mimetic bispecific antibody is mim8. 33. The method of any one of paragraphs 25-32, comprising at least partially sequencing a nucleic acid encoding Factor X from said subject.

[0090] 34. The method of any one of paragraphs 25-33, further comprising obtaining a biological sample from said subject.

[0091] Definitions

[0092] Various terms relating to the biological molecules of the present invention are used hereinabove and also throughout the specification and claims.

[0093] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0094] The term “substantially pure” refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.), particularly at least 75% by weight, or at least 90-99% or more by weight of the compound of interest. Purity may be measured by methods appropriate for the compound of interest (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).

[0095] “Pharmaceutically acceptable” indicates approval by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0096] A “carrier” refers to, for example, a diluent, adjuvant, preservative (e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., polysorbate 80), emulsifier, buffer (e.g., Tris HC1, acetate, phosphate), antimicrobial, bulking substance (e.g., lactose, mannitol), excipient, auxiliary agent or vehicle with which an active agent of the present invention is administered. Pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E.W. Martin (Mack Publishing Co., Easton, PA); Gennaro, A. R., Remington: The Science and Practice of Pharmacy, (Lippincott, Williams and Wilkins); Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y.; and Kibbe, et al., Eds., Handbook of Pharmaceutical Excipients, American Pharmaceutical Association, Washington.

[0097] An “antibody” or “antibody molecule” is any immunoglobulin, including antibodies and fragments thereof, that binds to a specific antigen. As used herein, antibody or antibody molecule contemplates intact immunoglobulin molecules, immunologically active portions of an immunoglobulin molecule (e.g., antigen-binding fragment), and fusions of immunologically active portions of an immunoglobulin molecule.

[0098] The term “bispecific” as used herein means the polypeptide (e.g., antibody or antigen binding fragment) is capable of specifically binding at least two target entities.

[0099] As used herein, the term “immunologically specific” refers to proteins / polypeptides, particularly antibodies, that bind to one or more epitopes of a protein or compound of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.

[0100] As used herein, the term “subject” refers to an animal, particularly a mammal, particularly a human.

[0101] A “therapeutically effective amount” of a compound or a pharmaceutical composition refers to an amount effective to prevent, inhibit, treat, or lessen the symptoms of a particular disorder or disease. The treatment of a disease or disorder herein may refer to curing, relieving, and / or preventing the disease or disorder, the symptom(s) of it, or the predisposition towards it.

[0102] As used herein, the term “therapeutic agent” refers to a chemical compound or biological molecule including, without limitation, nucleic acids, peptides, proteins, and antibodies that can be used to treat a condition, disease, or disorder or reduce the symptoms of the condition, disease, or disorder.

[0103] The following example is provided to illustrate various embodiments of the present invention. The example is illustrative and are not intended to limit the invention in any way.

[0104] EXAMPLE

[0105] The armamentarium of hemophilia therapy is rapidly expanding (Arruda, et al., Blood 130(21):2251-2256). HA is now frequently treated with the subcutaneously administered FVIII mimetic bispecific antibody emicizumab (Young, G., Thromb. Hemostasis (2021) Blood 138(26)2750-2751). Emicizumab has an excellent safety record as a monotherapy and is approved for people with hemophilia A of all ages, including infants. While the subcutaneous administration of a FVIII mimetic bispecific antibody has been revolutionary for HA, there remains a significant need to develop new therapies for other blood disorders such as Factor X deficiency. Herein, specific Factor X deficiency variants are identified that can be treated with Factor VIII mimetics such as emicizumab.

[0106] FX variants were transiently expressed from HEK293 cells and FX activity was measured using a one-stage activated partial thromboplastin time (aPTT) based clotting assay in FX-deficient plasma or FX-deficient plasma with 300 nM emicizumab. As seen in Figure 2A, FX variants R139S, R347C, R139G, T318M, and T318P showed significant and dramatic improvement in activity with emicizumab. Similarly, Figure 2B shows enhanced FX activity with therapeutic concentration of emicizumab and FX variants R139G, R139S, R247C, and T318M. Notably, certain FX variants presented in Figure 2C did not show significant recovery with emicizumab while others demonstrated a less dramatic recovery in activity with emicizumab.

[0107] Further studies were performed with certain of the FX variants. Again, FX variants were transiently expressed from HEK293 cells. FX activity was determined using an activated partial thromboplastin time (aPTT)-based assay from a standard curve of plasma derived FX (Prolytix, Essex Junction, VT) with 0 nM FVIII mimetics. The activity of the FX deficiency-causing FX variants was measured in 0 nM mimetics (left), 300 nM emicizumab (middle), and 34 nM Mim8 (right) (R247C not tested with mim8). As seen in Fig. 3A, mim8 also led to increased FX activity as with emicizumab.

[0108] The ability of the FX variants to increase thrombin generation in the presence of emicizumab was also studied. Thrombograms are provided in Fig. 3B of wild-type (WT) FX, unconditioned media (UC), and FX deficiency-causing FX variants with 0 nM (black) and 300 nM emicizumab (grey). Thrombin generation was triggered in the presence of 4 pM PCPS (phosphatidylcholine-phosphatidylserine) with 1 pM FXIa (top) and 0.07 pM tissue factor (bottom). As seen in Fig. 3B, the tested FX variants increased thrombin generation. While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.

Claims

WHAT IS CLAIMED IS1. A method of treating or inhibiting Factor X deficiency in a subject in need thereof comprising administering a therapeutically effective amount of a Factor VIII (FVIII) mimetic bispecific antibody to said subject, wherein said subject comprises a Factor X (FX) mutant.

2. The method of claim 1, wherein activated FVIII (F Villa) has decreased binding affinity for the FX mutant compared to wild-type FX and / or the FX mutant has decreased activation by FIXa and FVIIIa compared to wild-type FX.

3. The method of claim 1, wherein said FX mutant comprises a mutation within the FVIIIa binding site of FX.

4. The method of claim 1, wherein said FX mutant comprises a mutation at R139, R247, T318 and / or R347.

5. The method of claim 1, wherein said FX mutant comprises a mutation selected from the group consisting of R139S, R139G, R247C, T318M, T318P, and / or R347C.

6. The method of claim 1, wherein said FVIII mimetic bispecific antibody binds Factor IXa and Factor X.

7. The method of claim 1, wherein said FVIII mimetic bispecific antibody is emicizumab.

8. The method of claim 1, wherein said FVIII mimetic bispecific antibody is mim8.

9. The method of any one of claims 1-8, further comprising determining said subject comprises said Factor X mutant prior to administering said FVIII mimetic bispecific antibody.

10. The method of claim 9, wherein determining said subject comprises said Factor X mutant comprises at least partially sequencing a nucleic acid encoding Factor X from said subject.

11. The method of claim 9, further comprising obtaining a biological sample from said subject.

12. A method for increasing blood coagulation comprising contacting blood with a Factor VIII (FVIII) mimetic bispecific antibody, wherein said blood comprises a Factor X (FX) mutant.

13. The method of claim 12, wherein said FX mutant causes Factor X deficiency.

14. The method of claim 12, wherein activated FVIII (FVIIIa) has decreased binding affinity for the FX mutant compared to wild-type FX.

15. The method of claim 12, wherein the FX mutant has decreased activation by FIXa and FVIIIa compared to wild-type FX.

16. The method of claim 12 which is an in vitro method.

17. The method of claim 12, wherein said Factor IX mutant comprises a mutation within the FVIIIa binding site of FX.

18. The method of claim 12, wherein said FX mutant comprises a mutation at R139, R247, T318 and / or R347.

19. The method of claim 12, wherein said FX mutant comprises a mutation selected from the group consisting ofR139S, R139G, R247C, T318M, T318P, and / or R347C.

20. The method of claim 12, wherein said FVIII mimetic bispecific antibody binds Factor IXa and Factor X.

21. The method of claim 12, wherein said FVIII mimetic bispecific antibody is emicizumab.

22. The method of claim 12, wherein said FVIII mimetic bispecific antibody is mim8.

23. The method of any one of claims 12-22, further comprising determining said blood comprises said Factor X mutant prior to administering said FVIII mimetic bispecific antibody.

Images