US 20150353911A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0353911A1 Salas et al. (43) Pub. Date: Dec. 10, 2015

(54) CHMERC CLOTTING FACTORS on Feb. 1, 2013, provisional application No. 61/801, 603, filed on Mar. 15, 2013, provisional application (71) Applicant: Biogen Idec MA Inc., Cambridge, MA No. 61/829,775, filed on May 31, 2013. (US) Publication Classification (72) Inventors: Joe Salas, Wayland, MA (US); Elena Kistanova, Brookline, MA (US); Vu (51) Int. Cl. Phong Hong, Cambridge, MA (US); CI2N 9/64 (2006.01) Adam R. Mezo, Carmel, IN (US); A638/48 (2006.01) Robert T. Peters, West Roxbury, MA A647/48 (2006.01) (US) (52) U.S. Cl. CPC ...... CI2N 9/6432 (2013.01); A61K 47/48238 (73) Assignee: Biogen Idec MA Inc., Cambridge, MA (2013.01); A61 K38/4846 (2013.01): CI2N (US) 9/6437 (2013.01); C12Y 304/21021 (2013.01); CI2Y 304/21006 (2013.01) Appl. No.: 14/406,160 (21) (57) ABSTRACT (22) PCT Fled: Jun. 7, 2013 The invention provides chimeric clotting factors comprising an activatable clotting factor and an enhancer moiety. The (86) PCT NO.: PCT/US13A44842 activatable clotting factor allows the chimeric clotting factor S371 (c)(1), to be activated at the site of coagulation. The enhancer moiety (2) Date: Dec. 5, 2014 can additionally improve procoagulation activities of the chi meric clotting factors. The chimeric clotting factors can fur ther be improved by fusion to a half-life extender, which Related U.S. Application Data improves a pharmacokinetics property of the chimeric clot (60) Provisional application No. 61/657,685, filed on Jun. ting factor. The invention also includes methods of making 8, 2012, provisional application No. 61/759,817, filed and methods of using these chimeric clotting factors. Patent Application Publication Dec. 10, 2015 Sheet 1 of 25 US 2015/0353911A1

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CHMERC CLOTTING FACTORS kinetic properties, have reduced clearance rates, have improved manufacturability, have reduced thrombogenicity, BACKGROUND OF THE INVENTION or have enhanced activity, or more than one of these charac 0001 Initiation of the extrinsic clotting pathway is medi teristics. ated by the formation of a complex between tissue factor, which is exposed as a result of injury to a vessel wall, and SUMMARY OF THE INVENTION Factor VIIa. This complex then converts Factors IX and X to 0006. The present invention provides a chimeric protein their active forms (Factor IXa and Xa). Factor Xa converts comprising (i) an activatable clotting factor (Ac), (ii) an limited amounts of prothrombin to thrombin on the tissue enhancer moiety (Em), and (iii) an optionally linker moiety factor-bearing cell. This resulting thrombin is then able to (L or L1) between the activatable clotting factor and the diffuse away from the tissue-factor bearing cell and activate enhancer moiety. The activatable clotting factor and the platelets, and Factors V and VIII, making Factors Va and enhancer moiety can be linked or associated with each other, VIIIa. Dung the propagation phase of coagulation, Factor Xa but may not be chemically crosslinked. The chimeric: protein is generated by Factor IXa (in complex with factor VIIIa) on can be represented by formula Ac-L-Em or Em-L-Ac, the surface of activated platelets. Factor Xa, in complex with wherein Ac comprises the activatable clotting factor; L com the cofactor Factor Va, activates prothrombin into thrombin, prises the optional linker moiety; and Em comprises the generating a thrombin burst. The cascade culminates in the enhancer moiety. conversion of fibrinogen to fibrin by thrombin, which results 0007. In one embodiment, the activatable clotting factor in the formation of a fibrin clot. Factor VII and tissue factor comprises a clotting factor Zymogen comprising a heavy are key players in the initiation of blood coagulation. chain (HC) and a light chain (LC) and a protease-cleavage site 0002 Factor VII is a plasma glycoprotein that circulates in inserted between the HC and the LC. The clotting factor blood as a single-chain Zymogen, which is catalytically inac Zymogen can be a FVII protein (e.g., FVII, functional frag tive. Although single-chain Factor VII may be converted to ments, derivatives, or variants thereof) or a FX protein (FX, two-chain Factor VIIa by a variety of factors in vitro, Factor functional fragments, derivatives, or variants thereof). Xa is an important physiological activator of Factor VII. The Embodiments of the present invention include a chimeric conversion of Zymogen Factor VII into the activated two protein further comprising a self-immolative moiety (e.g., chain occurs by cleavage of the link PABC) inserted between the protease-cleavage site and the ing the Arginine residue atamino acid position 152 and the Ile HC. The protease-cleavage site inserted between the HC and residue at amino acid position 153. In the presence of tissue the LC can be cleaved by a protease selected from thrombin factor, phospholipids and calcium , the two-chain Factor (factor IIa), factor XIa, factor XIIa, kallikrein, factor VIIa, VIIa activates Factor X or Factor IX. Factor VIIa is thought to factor IXa, factor Xa, or any combinations thereof, wherein be the physiologic initiator of the clotting cascade by acting at the protease-cleavage site is not naturally occurring in the the Surface of a TF-bearing cell and generating the initial clotting factor Zymogen. amount of thrombin that then diffuses to platelets to activate 0008. In another embodiment, the enhancer moiety com and prime them for the propagation phase of thrombingen prises a clotting cofactor, a procoagulant peptide, or an anti eration. Therapeutically, recombinant FVIIa acts by activat gen-binding moiety. Examples of the clotting cofactors ing Factor X on the Surface of activated platelets, bypassing include, but are not limited to, Tissue Factor, a fragment (e.g., the need for FIXa or FVIIIa to generate a thrombin burst soluble tissue factor), a variant, or a derivative thereofor FVa, during the propagation phase of coagulation. Since FVIIa has a fragment, a variant, or derivative thereof. Non-limiting relatively low affinity for platelets, recombinant FVIIa is examples of the antigen binding moiety include an antibody dosed at Supra-physiological levels. This process is thought to or an antigen-binding fragment thereof which is capable of be tissue factor-independent. binding to the FVII protein or the FX protein and enhances the 0003) Factor X is also synthesized as a single-chain activity of FVII or FX, respectively. polypeptide containing the light and heavy chains connected 0009. In some embodiments, the chimeric protein further by an Arg-Lys-Arg tripeptide. The single-chain molecule is comprises a heterologous moiety (Het) (e.g., a half-life then converted to the light and heavy chains by cleavage of extender) linked to the activatable clotting factor, the linker two (or more) internal peptide bonds. In plasma, these two moiety, or the enhancer moiety. Non-limiting examples of the chains are linked together by a disulfide bond, forming Factor half-life extender include an immunoglobulin constant region X. Activated Factor X, Factor Xa, participates in the final or a portion thereof (e.g., an Fc moiety or an FcRn binding common pathway whereby prothrombin is converted to partner), albumin, transferrin, an albumin binding moiety, a thrombin, which in turn converts fibrinogen to fibrin. PAS sequence, an XTEN sequence, a HES sequence, the B 0004 Clotting factors have been administered to patients subunit of the C-terminal peptide (CTP) of human chorionic to improve hemostasis for some time. The advent of recom gonadotropin, glycol (PEG), hydroxyethyl binant DNA technology has significantly improved treatment starch (HES), albumin-binding Small , or any com for patients with clotting disorders, allowing for the develop binations thereof. The immunoglobulin constant region or a ment of safe and consistent protein therapeutics. For example, portion thereof can comprise an Fc moiety oran FcRn binding recombinant activated factor VII has become widely used for partner. the treatment of major bleeding, such, as that which occurs in 0010. In other embodiments, the chimeric protein com patients having haemophilia A or B, deficiency of coagulation prises a first heterologous moiety (Het1) and a second heter Factors XI or VII, defective platelet function, thrombocytope ologous moiety (Het2). Either or both of Het1 and Het2 can nia, or von Willebrand’s disease. comprise a half-life extender, e.g., an immunoglobulin con 0005. Although such recombinant molecules are effective, stant region or a portion thereof (e.g., an Fc moiety oran FcRn there is a need for improved versions which localize the binding partner), albumin, transferrin, an albumin binding therapeutic to sites of coagulation, have improved pharmaco moiety, a PAS sequence, an XTEN sequence, a HES US 2015/03539 11 A1 Dec. 10, 2015 sequence, the B subunit of the C-terminal peptide (CTP) of 0027 (f) the first polypeptide comprises a structure rep human chorionic gonadotropin, polyethylene glycol (PEG), resented by the formula Em-L1-Het1, and the second hydroxyethyl starch (HES), albumin-binding small mol polypeptide comprises a structure represented by the ecules, or any combinations thereof. Optionally, in certain formula Ac-Het2: embodiments, the first heterologous moiety is linked to the 0028 (g) the first polypeptide comprises a structure activatable clotting factor via a first linker (L1), and the sec represented by the formula Em-Het1, and the second ond heterologous moiety is linked to an enhancer moiety via polypeptide comprises a structure represented by the a second linker (L2). The chimeric protein may comprise only formula Ac-Het2; and, one linker (either L1 or L2) or both linkers. 0029 (h) the first polypeptide comprises a structure 0011. In yet other embodiments, the chimeric protein represented by the formula Em-Het1, and the second comprises a first polypeptide chain and a second polypeptide polypeptide comprises a structure represented by the chain, wherein the first polypeptide chain comprises the acti formula Ac-L1-Het2 wherein Het1 and Het2 of the two Vatable clotting factor (Ac) and the second polypeptide chain polypeptide chains form a disulfide bond. comprises the enhancer moiety (Em), wherein the first 0030. In some embodiments, the chimeric protein is a polypeptide chain and the second polypeptide chain are asso single polypeptide chain. For example, the chimeric protein ciated with each other. For example, the first polypeptide can further comprise a sche linker (X) linked to the enhancer chain can comprise the activatable clotting factor (Ac), the moiety and the first heterologous moiety linked to the acti first heterologous moiety (Het1), and the first optional linker vatable clotting factor or the activatable clotting factor and the moiety (L1), and the second polypeptide chain can comprise second heterologous moiety linked to the enhancer moiety. the enhancer moiety (Em), the second heterologous moiety Examples of the single chain chimeric protein can comprise a (Het2), and the second optional linker moiety (L2), wherein formula selected from the first polypeptide chain and the second polypeptide chain 0031 (1) Ac-Het1-X-Em-Het2: are associated with each other. 0032 (2) Ac-Het1-X-Em-L2-Het2: 0012. In still other embodiments, the chimeric protein has 0033 (3) Ac-L1-Het1-X-Em-Het2: a structure selected from: 0034 (4) Ac-L1-Het1-X-Em-L2-Het2: 0013 (a) Ac linked to Het1 via the linker moiety, and 0035 (5) Het2-Em-X-Het1-Ac: Em linked to Het2: 0.036 (6) Het2-L2-Em-X-Het1-Ac: 0014 (b) Ac linked to Het1 via the first linker moiety, 0037 (7) Het2-Em-X-Het1-L1-Ac; or and Em linked to Het2 via the second linker moiety; 0038 (8) Het2-L2-Em-X-Het1-L1-At, 0.039 wherein (a) Ac is the activatable clotting factor, (0015 (c) Ac linked to Het1, and Em linked to Het2 via (b) L1 is the first optional linker moiety; (c) Het1 is the the linker moiety; first heterologous moiety; (d) X is the scFc linker; (e) Em (0016 (d) Ac linked to Het1, and Em linked to Het2: is the enhancer moiety, (f) L2 is the optional second 0017 (e) Em linked to Het1 via the linker moiety, and linker moiety; (g) Het2 is the second heterologous moi Ac linked to Het2: ety, and (h)(-) is, a peptide bond or one or more amino (0018 (f) Em linked to Het1 via the first linker moiety, acids. In one embodiment, the scle linker is a process and Ac linked to Het2 via the second linker moiety; able linker (cScP.c), which comprises at least one intra cellular processing site. The processable linker may be 0019 (g) Em linked to Het1, and Ac is linked to Het2via processed into two or more polypeptide chains by one or the linker moiety; or, more intracellular processing enzymes selected from a (0020 (h) Em linked to Het1, and Ac linked to Het2. yeast Kex2. PCSK1, PCSK2, PCSK3, PCSK4, PCSK5, 0021. In yet other embodiments, the chimeric protein PCSK6, PCSK7, or any combinations thereof. In one comprising two polypeptide chains can comprise. embodiment, the intracellular processing site is pro 0022 (a) the first polypeptide comprises a structure rep cessed by PCSK5. resented by the formula Ac-L1-Het1, and the second 0040. In some embodiments, the chimeric protein is poly polypeptide comprises a structure represented by the sialylated, pegylated, glycosylated, hesylated, gamma-car formula Em-Het2: boxylated, or any combinations thereof. 0023 (b) the first polypeptide comprises a structure 0041. Also included is a nucleic acid molecule encoding represented by the formula Ac-L1-Het1, and the second the chimeric protein or a complement thereof, a set of nucleic polypeptide comprises a structure represented by the acid molecules comprising a first nucleotide sequence (NA1) formula Em-L2-Het2: and a second nucleotide sequence (NA2), wherein NA1 encodes the first polypeptide of the two chain chimeric pro 0024 (c) the first polypeptide comprises a structure rep tein or a complement thereof, and NA2 encodes the second resented by the formula Ac-Het1, and the second polypeptide of the two chain chimeric protein or a comple polypeptide comprises a structure represented by the ment thereof, the vectors comprising the nucleic acid mol formula Em-Het2: ecule or the set of nucleic acid molecules, or the host cell 0025 (d) the first polypeptide comprises a structure comprising the vector or the set of vectors. represented by the formula Ac-Het1, and the second 0042. The invention is also directed to a pharmaceutical polypeptide comprises a structure represented by the composition comprising the chimeric protein, the nucleic formula Em-L1-Het2: acid molecule, the vector, and the host cell and to methods for 0026 (e) the first polypeptide comprises a structure rep treating, ameliorating, or preventing a bleeding disease or resented by the formula Em-L2-Het1, and the second disorder in a subject in need thereof comprising administer polypeptide comprises a structure represented by the ing to the Subject an effective amount of the chimeric protein, formula Ac-L1-Het2: the nucleic acid molecule or the set of nucleic acid molecules, US 2015/03539 11 A1 Dec. 10, 2015

the vector or the set of vectors, the host cell, or a pharmaceu ecule as an enhancer moiety and a heterologous moiety (Het), tical composition. The bleeding disease or disorder treatable shown as an Fc moiety for half-life extension. The construct or preventable by the composition is selected from hemar in FIG. 3A comprises a first polypeptide chain and a second throsis, muscle bleed, oral bleed, hemorrhage, hemorrhage polypeptide chain, wherein the first polypeptide chain com into muscles, oral hemorrhage, trauma, trauma capitis, prises a FVII light chain, a thrombin cleavage site, a FVII trointestinal bleeding, intracranial hemorrhage, intra-ab heavy chain, a first linker, and a first Fc moiety (Het1) in the dominal hemorrhage, intrathoracic hemorrhage, bone frac N terminus to C terminus order and the second polypeptide ture, central nervous system bleeding, bleeding in the chain comprises STF, a second linker, and a second Fc moiety retropharyngeal space, bleeding in the retroperitoneal space, (Het2) in the N-terminus to C terminus direction. The first and bleeding in the illiopsoas sheath. The composition of the linker and the second linker can be the same or different. The present invention can also be used to treat, ameliorate, or first Fc moiety and the second Fc moiety can be the same or prevent a coagulation factor deficiency in a mammalian Sub different. When the construct in FIG. 3A (i.e., a zymogen ject comprising administering to the Subject an effective (non-active form)) is dosed, the construct is resistant to pro amount of the chimeric protein, the nucleic acid molecule or tease inhibitors upon administration (prior to activation). the set of nucleic acid molecules, the vector or the set of When the clotting factors are activated by thrombinat the site vectors, the host cell, or the pharmaceutical composition, of an injury, as shown in FIG. 3B, the clotting factors can wherein the coagulation factor is selected from FVII, FVIIa, display high activity stimulated by the enhancer moiety. FVIII, FIX, or FXI. 0048 FIG. 4 shows a schematic diagram of generation of 0043. In a specific embodiment, the chimeric protein or the construct in FIG. 4B (similar to FIG. 3A). FIG. 4A (left the composition is used to treat, ameliorate, or prevent a construct) shows a single polypeptide sequence encoding a bleeding disease or disorderina bypass therapy. In the bypass FVII light chain, a thrombin cleavage site (ALRPR (SEQID therapy, the Subject may have developed or may be subject to NO: 1)), a FVII heavy chain, a first linker, a first Fc moiety develop an inhibitor against Factor VIII. (Het1), a first intracellular processing site (e.g., RRRR (SEQ 0044) The invention also includes methods of producing a ID NO: 2)), a second linker, a second intracellular processing chimeric protein comprising expressing a nucleotide site (e.g., RKRRKR (SEQID NO:3)), STF, a third linker, and sequence encoding the single chain chimeric protein, which a second Fc moiety (Het2) in the N-terminus to C-terminus comprises a sche linker, wherein the sche linker is cleaved order. A nucleotide sequence (FVIII-133) encoding the single intracellularly by an intracellular processing enzyme. The polypeptide sequence can be expressed in a host cell, and the chimeric protein produced by the nucleotide sequence would single polypeptide sequence undergoes intracellular process comprise two polypeptide chains due to the processing of the ing such that the first intracellular processing site and the intracellular processing site. second intracellular processing site are cleaved by a propep tide endopeptidase, e.g., PCSK5. The second linker inter BRIEF DESCRIPTION OF THE DRAWINGS posed between the first intracellular processing site and the 0045 FIG. 1 illustrates a schematic diagram of an exem second intracellular processing site can thus be removed by plary chimeric protein (e.g., a chimeric clotting factor) com PCSK5. FIG. 4B shows the final construct, after the linker is prising a thrombin-activatable or FXIa-activatable clotting removed by processing, which may contain a portion of the factor and an enhancer moiety (sometimes referred to as “an intracellular processing site which remains after cleavage. activity enhancing moiety'). FIG. 1A shows a FVII or FX This remaining linker portion may comprise a series of amino light chain with optional activation peptide for FX linked to a acids from about 1 to about 10, 1 to about 4. FIG. 4C shows an coagulation cascade protease cleavage site (e.g., thrombin SDS-PAGE of the thrombin-activatable FVII-Fc/STF-Fc chi cleavage site or FXIa cleavage site), which is further linked to meric protein under either non-reducing condition or reduc a FVII or FX heavy chain. The FVII or FX heavy chain is then ing condition as indicated. linked to an enhancer moiety via a linker. When the construct 0049 FIG. 5 shows an SDS-PAGE analysis of the throm in FIG. 1A is dosed as a Zymogen (non-active form), the bin-activatable FVII-Fc/sTF-Fc (FVII-133) under construct is resistant to protease inhibitors upon administra reducing condition. The second lane (i.e., eluate) shows the tion (prior to activation). When the clotting factors are acti purified thrombin-activatable FVII-Fc/sTF-Fc dimer. vated at the site of an injury as shown in FIG. 1B, the clotting, 0050 FIG. 6 shows data generated by thrombingeneration factors can display high activity stimulated by the enhancer assays to test the activity of FVII-133 and FVIIaFc. The moiety. activities of FVII-133 without or with Tissue Factor (TF) are 0046 FIG. 2 illustrates a schematic diagram of a chimeric shown as circle (O) and square (), respectively. The activi FVII protein comprising a thrombin-activatable molecule ties of FVIIaFc without or with TF are shown as triangle (A) and an enhancer moiety. FIG. 2A shows a FVII light chain or diamond (0), respectively. The y-axis shows nanomoles linked to a thrombin cleavage site, which is further linked to (nM) of thrombin, and the x-axis shows time. The construct a FVII heavy chain. The FVII heavy chain is then linked to an FVIIaFc consists of two polypeptide chains, a first chain enhancer moiety via a linker, resulting in a non-active and consisting of an activated FVII (FVIIa) linked to a first Fc Zymogen form. When dosed in animals, this Zymogen is region, and a second chain consisting of an Fc region. resistant to protease inhibitors in the circulation and can be 0051 FIG. 7 shows data generated by ROTEM assays converted to the activated form (FIG. 2B) at the site of an with mouse hemophilia B blood to test the activity of FVII injury. Activity of the clotting factors can be stimulated by an 133 and FVIIaFc. FIGS. 7A and 7B show clotting time and enhancer moiety. Examples for the enhancer moieties include alpha angle for FVII-133. FIGS. 7C and 7D show clotting soluble tissue factor (STF), procoagulant peptides and anti time and alpha angle of FVIIaFc and vehicle respectively. body fragments. 0052 FIG. 8A-C shows FVII activity measured by 0047 FIG.3 illustrates a schematic diagram of a molecule ROTEM assay in human hemophilia A blood. FVII-133, comprising a thrombinactivatable FVII molecule, a sTF mol FVII-184, and FVIIa were spiked into citrated human blood US 2015/03539 11 A1 Dec. 10, 2015

from hemophilia Adonors. Structure of FVII-133 is shown in 0060 FIG. 16A shows the general organization of a pro FIG. 4. FVII-184 is a mutant form of FVII-133 and is insen tease-activatable procoagulant compound of the invention. sitive to thrombin activation due to mutation of the Arg resi Het2, Pep2, Het1 and L are independently optional compo due, which is essential for thrombin cleavage, to Ala. FVIII nents. Pep 1 and Pep2 are polypeptides, at least one of which 184 is otherwise identical to FVII-133. Clotting Time (CT), is a clotting factor or a procoagulant peptide. Het1 and Het are Clotting Formation Time (CFT), and Alpha Angle for FVII heterologous moieties. L is a linker. Additional linkers can 133 (triangle), FVII-184 (square), and FVIIa (circle) were connect the different moieties; for example, a linker could be measured. The baseline clotting, time in the hemophilia A located between Pep2 and Het 1 (as shown in the diagram). donor is shown as diamond (()). FIGS. 8A, 8B, and 8C show Additional protease cleavable substrate and self-immolative a graph of the results of the clotting time, clotting formation spacer groups can be inserted at the N-terminus of other time, and alpha angle, respectively. FVIIa was used as a moieties Such as polypeptides or heterologous moieties. The control for FVIIa activity. diagram shows the optional insertion of Such a group at the 0053 FIG. 9 shows FVII-133 ex vivo efficacy in hemB N-terminus of Pep2. FIG.16B is a representation of an exem mice by ex vivo ROTEM assays. Clotting Time (CT) was plary procoagulant compound of the invention comprising a measured on blood collected from mice that were dosed via protease cleavable Substrate (Aa1 Aa2Aa3Aa4), a self-immo tail vein injection by vehicle, FVIIa, and FVII-133, respec lative spacer and a protein of interest (POI; e.g., a clotting tively. factor or procoagulant peptide); illustrating the fragmentation 0054 FIG. 10A-B shows the plasma level of FVII and of the compound and the release of the peptide or protein of FVII/ATIII complex as a function of time following admin interest after proteolytic cleavage of the cleavable substrate istration of the proteins. HemB mice were administered i.v. and 1.6 spontaneous fragmentation. with FVII-133, rFVIIaFc, or rFVIIa. The plasma samples at 0061 FIG. 17 is a representation of an alternative exem various time points were collected, and the FVII antigen level plary protease-activatable procoagulant compound of the (FIG. 10A) and the FVII-133/ATIII or rPVIIFc-ATIII com invention which comprises an exosite binding peptide (M). plex (FIG. 10B) were measured by ELISA. The PK proper The diagram illustrates the release of the peptide or protein of ties, including the Mean Residence Time (MRT), of the FVII interest (POI; e.g., a clotting factor or procoagulant peptide) 133 (dotted line, circle) and FVIIaFc (solid line, triangle) and the exosite binding peptide after proteolytic cleavage of a were generated by 2 compartmental analysis using Phoenix 6 cleavable Substrate (Aa1 Aa2Aa3Aa4) and 1.6 spontaneous program. fragmentation. 0055 FIG. 11 shows in vitro clotting time measured by 0062 FIG. 18 shows the release kinetics of the peptide ROTEM assays in human HemA blood. The clotting activity IVGGQE, which corresponds to the six N-terminal amino of FVII-212, which has the identical structure as FVII-133, acid residues of the heavy chain of the FXa clotting factor, was measured. The proteins were spiked in citrated human from different procoagulant compounds (Compounds 1, 2, Hema blood. The clotting was initiated by Calcium and the and 3) following treatment with 14 nM thrombin. clotting time was recorded by ROTEM machine under the 0063 FIG. 19 shows the release kinetics of the peptide NATEM program. X axis shows concentrations of either IVGGQE, which corresponds to the six N-terminal amino acid residues of the heavy chain of the FXa clotting factor, rFVIIa or FVII-212 in nM, and y axis shows the clotting time. from different procoagulant compounds (Compounds 1, 4, 5 0056 FIG. 12 shows in vitro clotting time measured by ROTEMassays in mouse HemA blood, which were collected and 6) following treatment with 1.4 nM thrombin. by vena cava bleeding. The proteins were spiked into citrated 0064 FIG. 20 shows the natural processing of factor VII to mouse HemA blood. The clotting was initiated by Calcium yield activated factor (FVIIa). and the clotting time was recorded in ROTEM machine under 0065 FIG. 21 is a representation of exemplary procoagul NATEM program. X axis shows the concentrations of spiked lant compounds of the invention comprising FVIIa clotting rFVIIa and FVII-212 in nM, and y axis shows the clotting factor. time. 0.066 FIG. 22A-B shows a flow diagram of a cleavable polypeptide, FVII-186 (FIG.22A) that can be processed by a 0057 FIG. 13 shows ex vivo efficacy in HemA mice. proprotein convertase (e.g., PACE) to a processed cleavable FVII-212 (triangle) was administered in HemA mice at 10 polypeptide (FIG. 22B). FIG. 22A shows a cleavable nmol/kg. Blood was collected via Vena cava with citrate and polypeptide comprising FVIILC (FVII light chain)—Propro corn trypsin inhibitor (CTI) as an anti-coagulant at various tein Convertase Processing Site by a proprotein convertase times after dosing, and the clotting activity was measured by (e.g., PACE processing site, e.g., 2x(SEQ ID NO: 3))— ROTEM analyzer under NATEM program. rFVIIa (circle) Linker1 SUMO Truncated FVIIHC (FVII heavy chain was used as a control. X axis shows the time (hr) following without IVGGKV (SEQ ID NO 60) at the N-terminus)– protein administration, and y axis shows the clotting time. Linker2 Fc Region2 Linker3—Fe Region2. FIG. 22B 0058 FIG. 14 shows pharmacokinetics of rFVIIa (circle), shows a schematic diagram of a cleavable polypeptide that FVII-212 (square), and rEVII/ATIII (triangle) in Hema mice. has been processed by PACE. The processed cleavable The concentration of the proteins (y axis) is plotted against polypeptide comprises two polypeptide chains, the first chain time (X axis). comprising FVIILC linked to the Proprotein Convertase pro 0059 FIG. 15. shows amildolytic activity of FVII-212 cessing site and the second chain comprising Linkerl— measured before and after thrombin activation using a chro SUMO Truncated FVIIHC (FVII heavy chain without mozyme t-PA substrate. FVII-212 with thrombin is repre IVGGKV (SEQID NO: 60) at the N-terminus) Linker2– sented as inverted triangle (the first line from top to bottom). Fc Region1–Linker3—Fe Region2. FIG.22C demonstrates rFVIIa is represented, as circle (the second line from top to non-reduced (lane 1) or reduced (lane 2) SDS-PAGE, show bottom). FVII-212 and thrombin are shown as the third and ing the above constructs and chains. (—) indicates a peptide fourth lines (the bottom lines). bond. US 2015/03539 11 A1 Dec. 10, 2015

0067 FIG. 23 A-C shows a flow diagram of (i) FVII-186 0070 Exemplary constructs of the invention are illustrated cleavage by a SUMO protease (FIG.23B) and (ii) its fusion to in the accompanying Figures and sequence listing. In one a thioester peptide (FIG. 23C). FIG. 23A is identical to the embodiment, the invention pertains to a polypeptide having construct in FIG.22B. FIG. 23B shows that, after FVII-186 is the structure as set forth in the Figures. In another embodi cleaved by a SUMO protease, the resulting cleaved polypep ment, the invention pertains to a polypeptide having the tide construct comprises two chains, the first chain compris sequence set forth in the accompanying sequence listing or ing FVIILC and Proprotein Convertase Site and the second the nucleic acid molecule encoding Such polypeptides. In one chain comprising Truncated FVIIHC(FVII heavy chain with embodiment, the invention pertains to a mature form of a out IVGGKV (SEQ ID NO: 60) at the N-terminus)– polypeptide having the sequence set forth in the accompany Linker2 Fc Region1—Linker3—Fe Region2. The first ing sequence listing. It will be understood that these con chain and the second chain are bound by a disulfide bond. structs and nucleic acid molecules encoding them can be used FIG. 23C shows that after the cleaved polypeptide construct to improve hemostasis in a subject. in FIG. 23B is ligated with a thioester peptide (D-Phe-Pip 0071. In order to provide a clear understanding of the Arg-PABC-IVGGKV-COSBn), the resulting construct com specification and claims, the following definitions are pro prises two polypeptide chains, the first chain comprising FVI vided below. ILC and Proprotein Convertase Processing Site and the second chain comprising Thrombin cleavage site FVIIHC I. Definitions (FVII heavy chain)—Linker2 Fc Region1—Linker3—Fe (0072. As used herein, the term “protein' or “polypeptide' Region2 (TA-FVII-186). FIG. 23D shows reducing SDS refers to a polymer of two or more of the natural amino acids PAGE indicating the constructs and chains: lane 1 shows or non-natural amino acids. marker; lane 2 shows FVII-186; lane 3 shows FVII-186 with 0073. The tern "amino acid includes alanine (Ala or A); SUMO protease reaction; lane 3 shows FVII-186 with SUMO arginine (Argor R); asparagine (ASnor N); aspartic acid (Asp protease reaction and conjugation with a positive control or D); cysteine (CyS or C); glutamine (Glin or Q); glutamic peptide; and lane 5 shows FVII-186 with SUMO protease acid (Glu or E); glycine (Gly or G); histidine (His or H): reaction and conjugation with PABC peptide. ( ) indicates a isoleucine (Ile or I): leucine (Leu or L); lysine (Lys or K); peptide bond. methionine (Met or M); phenylalanine (Phe or F); proline 0068 FIG. 24 shows FVIIa chromogenic assay after (Pro or P); serine (Ser or S); threonine (Thror T); tryptophan thrombin activation of TA-FVII-186. X axis indicates time (Trp or W); tyrosine (Tyr or Y); and valine (Val or V). Non (min), and Y axis indicates Absorbance (A405) measurement traditional amino acids are also within the scope of the inven for FVIIa activity. (x) shows FVIIa activity of a mixture of tion and include norleucine, omithine, norvaline, thrombin and hirudin. (D) indicates FVIIa activity of a mix homoserine, and other amino acid residue analogues such as ture of FVII-186, thrombin, and hirudin. (o) indicates FVIIa those described in Ellman et al. Meth. Enzym. 202:301-336 activity of a mixture of TA-FVII-186, thrombin, and hirudin. (1991). To generate such non-naturally occurring amino acid residues, the procedures of Noren et al. Science 244:182 DETAILED DESCRIPTION OF THE INVENTION (1989) and Ellman et al., Supra, can be used. Briefly, these procedures involve chemically activating a Suppressor tRNA 0069. The present invention relates to chimeric proteins with a non-naturally occurring amino acid residue followed comprising an activatable clotting factor and an enhancer by in vitro transcription and translation of the RNA. Intro moiety. The present invention is based, at least in part, on the duction of the non-traditional amino acid can also be achieved development of novel ways to enhance the efficacy, pharma using peptide chemistries known in the art. As used herein, cokinetic properties, and/or manufacturability of clotting fac the term “polar amino acid' includes amino acids that have tors. The activatable clotting factor is in a form that is acti net Zero charge, but have non-zero partial charges in different Vatable at the site of coagulation. For use in bypass therapy, portions of their side chains (e.g. M. F. W. S. Y. N, Q, C). exogenous clotting factors are only efficacious when given in These amino acids can participate in hydrophobic interac the activated form. However, such activated clotting factors tions and electrostatic interactions. As used herein, the term are rapidly inactivated by endogenous pathways (e.g. anti “charged amino acid' includes amino acids that can have thrombin III, TFPI), leading to their fast clearance and a short non-Zero net charge on their side chains (e.g. R. K. H. E. D). effective half-life in circulation. Giving higher doses does not These amino acids can participate in hydrophobic interac Solve this, problem as it can result in thrombogenic effects. tions and electrostatic interactions. Thus, in one embodiment, the invention pertains to an activ 0074 An "amino acid substitution” refers to the replace ity-enhanced chimeric protein constructs which comprise an ment of at least one existing amino acid residue in a prede activatable clotting factor fused to or associated with an termined amino acid sequence (an amino acid sequence of a enhancer moiety. The “activatable' clotting factor comprises starting polypeptide) with a second, different “replacement” a heavy chain and a light chain of a clotting factor Zymogen amino acid residue. An "amino acid insertion” refers to the and a heterologous protease cleavage site (i.e., not naturally incorporation of at least one additional amino acid into a occurring in the clotting factor Zymogen) inserted between predetermined amino acid sequence. While the insertion will the heavy chain and the light chain. These molecules circulate usually consist of the insertion of one or two amino acid as enhanced Zymogen fusion proteins and have a longer half residues, the present larger'peptide insertions', can be made, life than their activated counterparts due to the lack of inac e.g. insertion of about three to about five or even up to about tivation, but can readily be activated at the site of clotting due ten, fifteen, or twenty amino acid residues. The inserted resi to the cleavage of the heavy chain and the light chain by a due(s) may be naturally occurring or non-naturally occurring protease that is activated or localized at the site of clotting. as disclosed above. An "amino acid deletion” refers to the Incorprating an enhancer moiety can also improve their pro removal of at least one amino acid residue from a predeter coagulant activities. mined amino acid sequence. US 2015/03539 11 A1 Dec. 10, 2015

0075 Polypeptides may be either monomers or multim linkers at the N terminus of the cleavage site, but requires ers. For example, in one embodiment, a protein of the inven direct fusion at the C-terminus of the cleavage site to the tion is a dinner. A dimeric polypeptide of the invention may amino terminus of the heavy chain of the clotting factor. comprise two polypeptide chains or may consist of one 0078. As used herein, the term “gly-ser peptide linker' polypeptide chain (e.g., in the case of a ScFc molecule). In one refers to a peptide that consists of glycine and serine residues. embodiment, the dimers of the invention are homodimers, An exemplary gly/ser peptide linker comprises the amino comprising two identical monomeric Subunits or polypep acid sequence (Gly. Ser), (SEQ ID NO: 4) Another exem tides (e.g., two identical Fc moieties or two identical biologi plary gly/ser peptide linker comprises the amino acid cally active moieties). In another embodiment, the dimers of sequence S(Gly, Ser), (SEQID NO. 5) wherein n is an inte the invention are heterodimers, comprising two non-identical ger that is the same or higher than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. monomeric subunits or polypeptides (e.g., comprising two 15, 20, 25, 30, 35, 40, 46, 50, 55, 60, 70, 80,90, or 100. different clotting factors or portions thereof or one clotting 0079. In one embodiment, n=1. In one embodiment, n=2. factor only). See, e.g., U.S. Pat. No. 7,404.956, incorporated In another embodiment, n=3. In another embodiment, n=4. In herein by reference. another embodiment, n=5. In yet another embodiment, n=6. 0076. As used herein, the term "peptide linker(s).” “linker In another embodiment, n=7. nyet another embodiment, n=8. (s), or “linker moiety” refers to a peptide or polypeptide In another embodiment, n=9. In yet another embodiment, sequence (e.g., a synthetic peptide or polypeptide sequence) n=10. Another exemplary gly/ser peptide linker comprises which connects two domains in a linear amino acid sequence the amino acid sequence Ser(Gly Ser) (SEQ ID NO 5). In of a polypeptide chain. In one embodiment, the polypeptides one embodiment, n=1. In one embodiment, n=2. In a pre of invention are encoded by nucleic acid molecules that ferred embodiment, n=3. In another embodiment, n=4. In encode peptide linkers which either directly or indirectly another embodiment. In yet another embodiment, n=6. connect the two Fc moieties which make up the construct. 0080) “Derivatives” of polypeptides or proteins of the These linkers are referred to herein as “scFc linkers' and the invention are polypeptides or proteins which have been sch c linker is interposed between the two Fc moieties of a altered so as to exhibit additional features not found on the polypeptide which comprises it. If the scFc linker connects native polypeptide or protein. Also included as "derivatives’ two Fc moieties contiguously in the linear polypeptide are those peptides that contain one or more naturally occur sequence, it is a "direct linkage. In contrast, the Sclc linkers ring amino acid derivatives of the twenty standard amino may link the first Fc moiety to a binding moiety which is, in acids. A polypeptide or amino acid sequence "derived from turn, linked to the second Fc moiety, thereby forming an a designated polypeptide or protein refers to the origin of the indirect linkage. These scFc linkers permit the formation of a polypeptide. In one embodiment, the polypeptide or amino single chain genetic construct. In one embodiment, the acid sequence which is derived from aparticular sequence has polypeptides also comprise intracellular processing sites an amino acid sequence that is essentially identical to that which result in the sche linker being processed (a cscFc sequence or a portion thereof, wherein the portion consists of linker) and, in one embodiment, Substantially excised (e.g., at least about 10 to about 20 amino acids, at least about 20 to during processing by a cell). Thus, the resulting processed about 30 amino acids, or at least about 30 to about 50 amino polypeptide is a dimeric molecule comprising at least two acids, or which is otherwise identifiable to one of ordinary amino acid chains and Substantially lacking extraneous linker skill in the art as having its origin in the sequence. amino acid sequences. In some embodiments, all or Substan I0081 Polypeptides that are “variants' of another peptide tially all of the linker is excised, while in some embodiments, may have one or more mutations relative to the starting a portion of the cleavage site may remain, e.g., four arginines polypeptide, e.g., one or more amino acid residues which of the RRRR cleavage site. In another embodiment, the linker have been substituted with another amino acid residue or or peptide linker may not typically cleaved; however in cer which has one or more amino acid residue insertions or dele tain embodiments, such cleavage may be desirable. Exem tions. In one embodiment, the polypeptide comprises an plary positions of the linkers are shown in the accompanying amino acid sequence which is not naturally occurring. Such drawings. Linkers can be located between the activatable variants necessarily have less than 100% sequence identity or clotting factors, enhancing moieties, and/or heterologous similarity with the, starting polypeptide. In another embodi moieties, e.g., at the N or C terminus of these moieties. In one ment, the variant will have an amino acid sequence from embodiment, these linkers are not removed during process about 75% to less than 100% amino acid sequence identity or 1ng. similarity with the amino acid sequence of the starting 0077. A third type of linker which may be present in an polypeptide, for example, from about 80% to less than 100%, activatable clotting factor is herein referred to as a “cleavable from about 85% to less than 100%, from about 90% to less linker” which comprises a heterologous protease-cleavage than 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, site (e.g., a factor XIa or thrombin cleavage site) that are not 98%, 99%) and from about 95% to less than 100%, e.g., over naturally occurring in the clotting factor and which may the length of the variant molecule. In one embodiment, there include additional linkers on either the N terminal of Cter is one amino acid difference between a starting polypeptide minal or both sides of the cleavage site. Exemplary locations sequence and the sequence derived therefrom. Identity or for Such sites are shown in the accompanying drawings and similarity with respect to this sequence is defined herein as the include, e.g., placement between a heavy chain of a clotting percentage of amino acid residues in the candidate sequence factor Zymogen and a light chain of a clotting factor Zymogen. that are identical (i.e. same residue) with the starting amino In another embodiment, Such linkers can further comprises a acid residues, after aligning the sequences and introducing self-immolative moiety. For example, in one embodiment, a gaps, if necessary, to achieve the maximum percent sequence self-immolative moiety linked to a cleavable linker may be identity. fused to the N terminus of the heavy chain of a clotting factor. I0082. The term “fragment' when referring to polypeptides In Such cases, the cleavable linker may include additional and proteins of the present invention include any polypep US 2015/03539 11 A1 Dec. 10, 2015 tides or proteins which retain at least some of the properties of fusion), chemical conjugation or other means. For example, the reference polypeptide or protein. Fragments of polypep one way in which molecules or moieties can be linked tides include proteolytic fragments, as well as deletion frag employs peptide linkers which link the molecules or moieties mentS. via peptide bonds. The terms “genetically fused.” “geneti 0083. In one embodiments, polypeptides of the invention cally linked' or “genetic fusion” are used interchangeably comprise an amino acid sequence (e.g., at least one clotting and refer to the co-linear, covalent linkage or attachment of factor or Fc moiety or domain) derived from a human protein two or more proteins, polypeptides, or fragments thereof via sequence. However, polypeptides may comprise one or more their individual peptide backbones, through genetic expres amino acids from another mammalian species. For example, sion of a single polynucleotide molecule encoding those pro a clotting factor, Fc domain, or enhancing moiety may be teins, polypeptides, or fragments. Such genetic fusion results derived from a non-human species and included in the Subject in the expression of a single contiguous genetic sequence. polypeptides. Alternatively, one or more amino acids may be Preferred genetic fusions are in frame, i.e., two or more open present in a polypeptide which are derived from a non-human reading frames (ORFs) are fused to form a continuous longer species. In a particular embodiment, the polypeptides of the ORF, in a manner that maintains the correct reading frame of invention are not immunogenic. the original ORFs. Thus, the resulting recombinant fusion 0084. It will also be understood by one of ordinary skill in protein is a single polypeptide containing two or more protein the art that the polypeptides of the invention may be altered segments that correspond to polypeptides encoded by the Such that they vary in amino acid sequence from the naturally original ORFs (which segments are not normally so joined in occurring or native polypeptides from which they were nature). In this case, the single polypeptide is, cleaved during, derived, while retaining the desirable activity of the native processing to yield dimeric molecules comprising two polypeptides. For example, nucleotide or amino acid Substi polypeptide chains. tutions leading to conservative Substitutions or changes at “non-essential amino acid residues may be made. An iso 0088. As used herein the term “associated with refers to a lated nucleic acid molecule encoding a non-natural variant of covalent or non- formed between a first amino a polypeptide derived from an immunoglobulin (e.g., an Fc acid chain and a second amino acid chain. In one embodi domain, moiety, or antigen binding site) can be created by ment, the term “associated with means a covalent, non introducing one or more nucleotide Substitutions, additions or peptide bond or a non-covalent bond. In another embodiment, deletions into the nucleotide sequence of the immunoglobulin the term “associated with refers to a covalent, non-peptide Such that one or more amino acid Substitutions, additions or bond or a non-covalent bond that is not chemically deletions are introduced into the encoded protein. Mutations crosslinked. In some embodiments this association is indi may be introduced by standard techniques, such as site-di cated by a colon, i.e., (:). In another embodiment, it means a rected mutagenesis and PCR-mediated mutagenesis. covalent bond except a peptide bond. For example, the amino 0085. The polypeptides of the invention may comprise acid cysteine comprises a thiol group that can form a disulfide conservative amino acid substitutions at one or more amino bond or bridge with a thiol group on a second cysteine resi acid residues, e.g., at essential or non-essential amino acid due. In most naturally occurring IgG molecules, the CH1 and residues. A "conservative amino acid substitution' is one in CL regions are associated by a disulfide bond and the two which the amino acid residue is replaced with an amino acid heavy chains are associated by two disulfide bonds at posi residue having a similar side chain. Families of amino acid tions corresponding to 239 and 242 using the Kabat number residues having similar side chains have been defined in the ing system (position 226 or 229, EU numbering system). art, including basic side chains (e.g., lysine, arginine, histi Examples of covalent bonds include, but are not limited to, a dine), acidic side chains (e.g., aspartic acid, glutamic acid), peptide bond, a metal bond, a bond, a disulfide uncharged polar side chains (e.g., glycine, asparagine, bond, a sigma bond, a pi bond, a delta bond, a glycosidic glutamine, serine, threonine, tyrosine, cysteine), nonpolar bond, an agnostic bond, a , a dipolar bond, a Pi side chains (e.g., alanine, Valine, leucine, isoleucine, proline, backbond, a , a , a quadruple bond, a phenylalanine, methionine, tryptophan), beta-branched side quintuple bond, a sextuple bond, conjugation, hyperconjuga chains (e.g., threonine, Valine, isoleucine) and aromatic side tion, , , or antibonding. Non-limiting chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). examples of non-covalent bond include an ionic bond (e.g., Thus, a nonessential amino acid residue in a polypeptide may cation-pibond or salt bond), a metal bond, anhydrogen bond be replaced with another amino acid residue from the same (e.g., , , low-barrier side chain family. In another embodiment, a string of amino , or ), Van der Walls acids can be replaced with a structurally similar string that force, , a mechanical bond, a differs in order and/or composition of side chain family mem bond, aurophilicity, intercalation, stacking, entropic force, or bers. Alternatively, in another embodiment, mutations may be . introduced randomly along all or part of a coding sequence, I0089. As used herein, the term “chemically crosslinked' Such as by Saturation mutagenesis, and the resultant mutants refers to linking by covalent bonds between acid side, chains can be incorporated into polypeptides of the invention and of amino acids, either directly or via a linker, e.g., a peptide screened for their ability to bind to the desired target. linker. Chemical crosslinking does not include intramolecu I0086. In the context of polypeptides, a “linear sequence' lar or intermolecular disulfide bonds between Fc moieties of or a "sequence' is the order of amino acids in a polypeptide in a dimeric Fc region, or non-engineered disulfide bonds anamino to carboxyl terminal direction in which residues that between an amino acid of the activatable clotting factor and neighbor each other in the sequence are contiguous in the an amino acid of the enhancer moiety. Chemical crosslinking primary structure of the polypeptide. generally takes place by addition of a cross-linking agent, 0087. As used herein, the terms “linked,” “fused, or e.g., a heterobifunctional crosslinking agent. Examples of “fusion” refer to linkage via a peptide bonds (e.g., genetic chemical crosslinking includes one or more photo-reactive US 2015/03539 11 A1 Dec. 10, 2015

bonds by chemically connecting photo-Ile, photo-Met, and certain embodiments, the half-life extender can comprise an photo-Leu. See Suchanek et al., (2005) Nature methods, 2: attachment site for a non-polypeptide moiety Such as poly 261-267. ethylene glycol (PEG), hydroxyethyl starch (HES), polysialic 0090. As used herein, the term “Fc region' is defined as acid, or any derivatives, variants, or combinations of these the portion of a polypeptide which corresponds to the Fc elements. region of native immunoglobulin, i.e., as formed by the 0.095 As used herein the term "moiety” refers to a com dimeric association of the respective Fc domains of its two ponent part or constituent of a chimeric, polypeptide. heavy chains. A native Fc region is homodimeric and com 0096. As used herein, the term "enhancer moiety” refers to prises two polypeptide chains. In contrast, the term "geneti a molecule, fragment, derivative, or variant thereof or a com cally-fused Fc region' or “single-chain Fc region' (sclfc ponent of a polypeptide which is capable of enhancing the region), as used herein, refers to a synthetic dimeric Fc region procoagulantactivity of a clotting factor. In one embodiment, comprised of Fc domains, genetically linked within a single a chimeric clotting factor of the invention comprises an polypeptide chain (i.e., encoded in a single contiguous "enhancer moiety' which enhances the activity of the genetic sequence). polypeptide, e.g., by acting as a cofactor. Such a moiety may 0091. As used herein, the term “Fc domain refers to the be, e.g., a clotting cofactor, such as a soluble tissue factor portion of a single immunoglobulin heavy chain beginning in (STF), or a Factor Va protein, but does not include a targeting the hinge region just upstream of the papain cleavage site (i.e. moiety, e.g., a platelet targeting moiety. In another embodi residue 216 in IgG, taking the first residue of heavy chain ment, the enhancer moiety interacts with the activatable clot constant region to be 114) and ending at the C-terminus of the ting factor, thereby increasing procoagulant activity of the antibody. Accordingly, a complete Fc domain comprises at clotting factor. The enhancer moiety may be genetically fused least a hinge domain, a CH2 domain, and a CH3 domain. to the construct, chemically conjugated to the construct, or 0092. As used herein, the term “Fc domain portion” or “Fc linked to the construct via a linker. For example, enhancer moiety' includes an amino acid sequence of an Fc domain or moieties may be attached to a construct of the invention by derived from an Fc domain. In certain embodiments, an Fc formation of a bond between the enhancer moiety and an moiety comprises at least one of a hinge (e.g., upper, middle, activatable clotting factor of a construct, where the enhancer and/or lower hinge region) domain, a CH2 domain, a CH3 moiety comprises a first functional group and the activatable domain, a CH4 domain, or a variant, portion, or fragment clotting factor comprises a second functional group, and thereof. In other embodiments, an Fc moiety comprises a where the first and second functional groups are, capable of complete Fc domain (i.e., a hinge domain, a CH2 domain, and reacting with each other to form a . Exemplary a CH3 domain). In one embodiment, a Fc moiety comprises a enhancer moieties are described in more detail below. hinge domain (or portion thereof) fused to a CH3 domain (or (0097. As used herein, the term “self-immolative moiety” portion thereof). In another embodiment, an Fc moiety com refers to a molecule that can be included in a cleavable linker prises a CH2 domain (or portion thereof) fused to a CH3 to enhance its function. In one embodiment, a self-immola domain (or portion thereof). In another embodiment, an Fc tive moiety is interposed between a heavy chain of a clotting moiety consists of a CH3 domain or portion thereof. In factor Zymogen and a protease cleavage site. Such self-im another embodiment, an Fc moiety consists of a hinge domain molating moieties have the advantage that the cleavability of (or portion thereof) and a CH3 dormain (or portion thereof). the protease cleavage site is not negatively impacted by the In another embodiment, a Fc moiety consists of a CH2 terminal amino acid residue of the first moiety. Exemplary domain (or portion thereof) and a CH3 domain. In another self-immolative moieties are disclosed, e.g., in U.S. Pat. No. embodiment, arc moiety consists of a hinge domain (or por 7,375,078 and U.S. Pat. No. 7,754,681, which are incorpo tion thereof) and a CH2 domain (or portion thereof). In one rated herein by reference in their entirety. embodiment, an Fc moiety lacks at least a portion of a CH2 0098. As used herein, the term "heterologous moiety’ domain (e.g., all or part of a CH2 domain). refers to a moiety that does not naturally occur with the 0093. As used herein, the term “half-life” refers to a bio components of the chimeric protein, e.g., an activatable clot logical half-life of a particular polypeptide in vivo. Half-life ting factor, a linker moiety, or an enhancer moiety and/or is may be represented by the time required for half the quantity linked to or associated with the components of the chimeric administered to a subject to be cleared from the circulation protein. In one embodiment, the heterologous moiety is and/or other tissues in the animal. When a clearance curve of capable of extending the half-life of the activatable clotting a given polypeptide is constructed as a function of time, the factor. In another embodiment, the heterologous moiety curve is usually biphasic with a rapid C-phase and longer increases the hydrodynamic radius of the activatable or acti B-phase. The C-phase typically represents an equilibration of vated clotting factor. In other embodiments, a heterologous the administered chimeric polypeptide between the intra- and moiety improves one or more pharmacokinetic properties of extra-vascular space and is, in part, determined by the size of the clotting factor without significantly affecting its biologi the polypeptide. The B-phase typically represents the catabo cal activity or function (e.g., its procoagulantactivity). In still lism of the polypeptide in the intravascular space. Therefore, other embodiments, the heterologous moiety is a non in a particular embodiment, the term half-life as used herein polypeptide moiety, e.g., chemical modification or a combi refers to the half-life of the polypeptide in the B-phase. The nation of a peptide or polypeptide and a non-polypeptide typical B-phase half-life of a human antibody in humans is 21 moiety. In yet other embodiments, the heterologous moiety is days. a polypeptide. In some embodiments, the chimeric clotting 0094. As used herein, the term “half-life extender refers factor is linked or connected to the heterologous moiety by a to a heterologous moiety which increases the in vivo half-life linker. Non-limiting examples of heterologous polypeptide of a protein. In vivo half-life of a chimeric clotting factor of moieties comprise an immunoglobulin constant region or a the invention can be determined by any method known to portion thereof, albumin or a fragment thereof, an albumin those of skill in the art, e.g., FVII activity level assays. In binding moiety, a PAS sequence, a HAP sequence, transferrin US 2015/03539 11 A1 Dec. 10, 2015

or a fragment thereof, the B subunit of the C-terminal peptide binding conformation. In one embodiment, a ScPv linker (CTP) of human chorionic gonadotropin, albumin binding comprises or consists of an Schv linker peptide. In certain small molecule, an XTEN sequence, or two or more combi embodiments, an ScFv linker peptide comprises or consists of nations thereof. Non-limiting examples of the heterologous a gly-serpeptide linker. In other embodiments, an Schv linker non-polypeptide moiety include polyethylene glycol (PEG), comprises a disulfide bond. polysialic acid, hydroxyethyl starch (HES), a derivative 0103) The term “glycosylation” refers to the covalent link thereof, or any combinations thereof. Exemplary heterolo ing of one or more carbohydrates to a polypeptide. Typically, gous moieties include, e.g., FcRn binding moieties (e.g., glycosylation is a posttranslational event which can occur complete Fc regions or portions thereof which bind to FcRn), within the intracellular milieu of a cell or extract therefrom. single chain Fc regions (ScPc regions, e.g., as described in US The term glycosylation includes, for example, N-linked gly 2008/0260738, WO 2008/012543, or WO 2008/143.9545), cosylation (where one or more Sugars are linked to an aspar processable scle regions (comprising a cscFc regions as agine residue) and/or O-linked glycosylation (where one or described herein). more Sugars are linked to an amino acid residue having a 0099. In one embodiment an enhancer moiety for use in a hydroxyl group (e.g., serine or threonine). In one embodi construct of the invention comprises an antibody variant. The ment, a molecule of the invention is glycosylated. In another term “antibody variant' or “modified antibody' includes an embodiment, a molecule of the invention is aglycosylated. In antibody which does not occur in nature and which has an yet another embodiment, a molecule of the invention has amino acid sequence or amino acid side chain chemistry reduced glycosylation as compared to that in a wild type Fc which differs from that of a naturally-derived antibody by at region. least one amino acid or amino acid modification as described 0104. As used herein the term “disulfide bond' includes herein. As used herein, the term “antibody variant' includes the covalent bond formed between two sulfur . The synthetic forms of antibodies which are altered such that they amino acid cysteine comprises a thiol group that can form a are not naturally occurring, e.g., antibodies that comprise at disulfide bond or bridge with a second thiol group. In most least two heavy chain portions but not two complete heavy naturally occurring IgG molecules, the CH1 and CL regions chains (such as, domain deleted antibodies or minibodies); are linked by native disulfide bonds and the two heavy chains multispecific forms of antibodies (e.g., bispecific, trispecific, are linked by two native disulfide bonds at positions corre etc.) altered to bind to two or more different antigens or to sponding to 239 and 242 using the Kabat numbering system different epitopes on a single antigen); heavy chain molecules (position 226 or 229, EU numbering system). joined to scFv molecules; single-chain antibodies; diabodies; 01.05 The term “vector” or “expression vector” is used triabodies; and antibodies with altered effector function and herein to mean vectors used in accordance with the present the like. invention as a vehicle for introducing into and expressing a 0100. As used herein, the term "Gla domain refers to the desired polynucleotide in a cell. As known to those skilled in conserved membrane binding motif which is present in Vita the art, Such vectors may easily be selected from plasmids, min K-dependent proteins, such as as prothrombin, coagula phages, viruses or retroviruses. In general, vectors compat tion factors VII, IX and X, proteins C. S, and Z. These proteins ible with the instant invention will comprise a selection require vitamin K for the posttranslational synthesis of g-car marker, appropriate restriction sites to facilitate cloning of the boxyglutamic acid, an amino acid clustered in the N-terminal desired gene and the ability to enter and/or replicate in Gla domain of these proteins. All glutamic residues present in eukaryotic or prokaryotic cells. the domain are potential carboxylation sites and many of 0106 Numerous expression vector systems may be them are therefore modified by carboxylation. In the presence employed to produce the chimeric clotting factors of the of calcium ions, the Gla domain interacts with phospholipid invention. For example, one class of vector utilizes DNA membranes that include phosphatidylserine. The Gla domain elements which are derived from animal viruses such as also plays a role in binding to the FVIIa cofactor, tissue factor bovine papilloma virus, polyomavirus, adenovirus, vaccinia (TF). Complexed with TF, the Gla domain of FVIIa is loaded virus, baculovirus, retroviruses (RSV, MMTV or MOMLV) with seven Ca2+ ions, projects three hydrophobic side chains or SV40 virus. Additionally, cells which have integrated the in the direction of the cell membrane for interaction with DNA into their chromosomes may be selected by introducing phospholipids on the cell Surface, and has significant contact one or more markers which allow selection of transfected host with the C-terminal domain of TF. cells. The marker may provide for prototrophy to an aux 0101. As used herein the term “scEv molecule' includes otrophic host, biocide resistance (e.g., antibiotics) or resis binding molecules which consist of one light chain variable tance to heavy metals such as copper. The selectable marker domain (VL) or portion thereof, and one heavy chain variable gene can either be directly linked to the DNA sequences to be domain (VH) or portion thereof, wherein each variable expressed, or introduced into the same cell by cotransforma domain (or portion thereof) is derived from the same or dif tion. In one embodiment, an inducible expression system can ferent antibodies. Schv molecules preferably comprise an be employed. Additional elements may also be needed for schv linker interposed between the VH domain and the VL optimal synthesis of mRNA. These elements may include domain, ScFv molecules are known in the art and are signal sequences, splice signals, as well as transcriptional described, e.g., in U.S. Pat. No. 5,892,019, Ho et al. 1989. promoters, enhancers, and termination signals. In one Gene 77:51: Bird et al. 1988 Science 242:423; Pantoliano et embodiment, a secretion signal, e.g., any one of several well al. 1991. Biochemistry 30:101 17: Milenicetal. 1991. Cancer characterized bacterial leader peptides (e.g., pelB, phoA, or Research 51:6363; Takkinen et al. 1991. Protein Engineering ompA), can be fused in-frame to the N terminus of a polypep 4:837. tide of the invention to obtain optimal secretion of the 0102. A “scFv linker as used herein refers to a moiety polypeptide. (Lei et al. (1988), Nature, 331:543; Better et al. interposed between the VL and VH domains of the scFv. Schv (1988) Science. 240: 1041: Mullinax et al., (1990), PNAS, linkers preferably maintain the scFv molecule in an antigen 87:8095). US 2015/03539 11 A1 Dec. 10, 2015

01.07 The term “host cell refers to a cell that has been forms of genetic engineering or a heterologous amino acid transformed with a vector constructed using recombinant sequence may be present in a protein in which it is not natu DNA techniques and encoding at least one heterologous rally found. gene. In descriptions of processes for isolation of proteins 0110. As used herein, the term “cleavage site' or “pro from recombinant hosts, the terms “cell' and “cell culture' tease-cleavage site' refers to a site recognized by a protease. are used interchangeably to denote the Source of protein In one embodiment, a polypeptide has a protease-cleavage unless it is clearly specified otherwise. In other words, recov site cleaved by a protease that is activated during the clotting ery of protein from the “cells' may mean either from spun cascade, Such that cleavage of Such sites occurs at the site of down whole cells, or from the cell culture containing both the clot formation. Exemplary Such sites include e.g., those rec medium and the suspended cells. The host cell line used for ognized by thrombin, Factor XIa or Factor Xa. Exemplary protein expression is most preferably of mammalian origin; FXIa cleavage sites include, e.g., TQSFNDFTR (SEQ ID NO: 6) and SVSQTSKLTR (SEQ ID NO: 7). Exemplary those skilled in the art are credited with ability to preferen thrombin cleavage sites include, e.g., DFLAEGGGVR (SEQ tially determine particular host cell lines which are best suited IDNO:8), TTKIKPR(SEQID NO:9), LVPRG (SEQID NO: for the desired gene product to be expressed therein. Exem 10) and ALRPR (SEQ ID NO: 1). Other protease-cleavage plary host cell lines include but are not limited to, DG44 and sites are described in detail below. DUXB11 (Chinese Hamster Ovary lines, DHFR minus), 0111. As used herein, the term “processing site' or “intra HELA (human cervical carcinoma). CVI (monkey kidney cellular processing site' refers to a type of enzymatic cleav line), COS (a derivative of CVI with SV40 Tantigen), R1610 age site in a polypeptide which is the target for enzymes that (Chinese hamster fibroblast) BALBC/3T3 (mouse fibro function after translation of the polypeptide. In one embodi blast), PerC6 cells), HAK (hamster kidney line), SP2/O ment, such enzymes function during transport from the Golgi (mouse myeloma), P3x63-Ag3.653 (mouse myeloma), BFA lumen to the trans-Golgi compartment. Intracellular process 1c1 BPT (bovine endothelial cells), RATI (human lympho ing enzymes cleave polypeptides prior to secretion of the cyte) and 293 (human kidney). Host cell lines are typically protein from the cell. Examples of Such processing sites available from commercial services, the American Tissue include, e.g., those targeted by the PACE/furin (where PACE Culture Collection or from published literature. The polypep is an acronym for Paired basic Amino acid Cleaving Enzyme) tides of the invention can also be expressed in non-mamma family of endopeptidases. These enzymes are localized to the liancells Such as bacteria or yeast or plant cells. In this regard Golgi membrane and cleave proteins on the carboxyterminal it will be appreciated that various unicellular non-mammalian side of the sequence motif Arg-(any residue-(Lys or Arg)- microorganisms such as bacteria can also be transformed; i.e. Arg. As used herein the “furin family of enzymes includes, those capable of being grown in cultures or fermentation. e.g., furin, a yeast Kex2. PCSK1 (also known as PC1/Pc3), Bacteria, which are susceptible to transformation, include PCSK2 (also known as PC2), PCSK3 (also known as furin or members of the enterobacteriaceae. Such as strains of PACE), PCSK4 (also known as PC4), PCSK5 (also known as Escherichia coli or Salmonella, Bacillaceae, such as Bacillus PC5 or PC6), PCSK6 (also known as PACE4), or PCSK7 subtilis, Pneumococcus, Streptococcus, and Haemophilus (also known as PC7/LPC, PC8, or SPC7). Other processing influenzae. It will further be appreciated that, when expressed sites are known in the art. in bacteria, the polypeptides typically become part of inclu 0112 In constructs that include more than one processing sion bodies. The polypeptides must be isolated, purified and or cleavage site, it will be understood that Such sites may be then assembled into functional molecules. the same or different. 0113. In vitro production allows scale-up to give large 0108. In addition to prokaryotes, eukaryotic microbes amounts of the desired altered polypeptides of the invention. may also be used. Saccharomyces cerevisiae, or common Techniques for mammalian cell cultivation under tissue cul baker's yeast, is the most commonly used among eukaryotic ture conditions are known in the art and include homogeneous microorganisms although a number of other strains are com Suspension culture, e.g. in an airlift reactor or in a continuous monly available including Pichia pastoris. For expression in stirrer reactor, or immobilized or entrapped cell culture, e.g. Saccharomyces, the plasmid YRp7, for example, (Stinch in hollow fibers, microcapsules, on agarose microbeads or comb et al., (1979), Nature, 282:39: Kingsman et al., (1979), ceramic cartridges. If necessary and/or desired, the Solutions Gene, 7:141; Tschemperet al., (1980), Gene, 10:157) is com of polypeptides can be purified by the customary chromatog monly used. This plasmid already contains the TRP1 gene raphy methods, for example gel filtration, -exchange chro which provides a selection marker for a mutant strain of yeast matography, hydrophobic interaction chromatography (HIC, lacking the ability to grow in tryptophan, for example ATCC chromatography over DEAE-cellulose or affinity chromatog No. 44076 or PEP4-1 (Jones, (1977), Genetics, 85:12). The raphy. presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for 0114. As used herein, the phrase “subject that would ben detecting transformation by growth in the absence of tryp efit from administration of a polypeptide' or “subject in need tophan. thereof includes subjects, such as mammalian Subjects, that would benefit from administration of polypeptides of the 0109 As used herein the term “endogenous” refers to invention, e.g., to improve hemostasis. In one embodiment, molecules (e.g. nucleic acid and/or protein molecules) that the subjects include, but are not limited to, the individuals are naturally present in a cell. In contrast, the term “exog who have developed a FVIII inhibitor and thus are in need of enous” or "heterologous' refers to such molecules that are not a bypass therapy. In another embodiment, the Subjects also normally found in, a given context, e.g., in a cell or in a include the individuals who have not yet developed a FVIII polypeptide. For example, an exogenous or heterologous inhibitor, but have a tendency to develop a FVIII inhibitor. molecule may be introduced into a cell and are only present The Subject can be an adult or a minor (e.g., under 12 years after manipulation of the cell, e.g., by transfection or other old). US 2015/03539 11 A1 Dec. 10, 2015

0115. A “chimeric protein’ or “fusion protein', as used thrombasthenia). In liver failure (acute and chronic forms), herein, refers to any protein comprised of a first amino acid there is insufficient production of coagulation factors by the sequence derived from a first source, bonded, covalently or liver; this may increase bleeding risk. non-covalently, to a second amino acid sequence derived 0.121. The chimeric molecules of the invention can be used from a second source, wherein the first and second source are prophylactically. As used herein the term “prophylactic treat not the same. A first source and a second source that are not ment” refers to the administration of a molecule prior to a the same can include two different biological entities, or two bleeding episode. In one embodiment, the Subject in need of different proteins from the same biological entity, or a bio a general hemostatic agent is undergoing, or is about to logical entity and a non-biological entity. A chimeric protein undergo, Surgery. The chimeric protein of the invention can be can include for example, a protein derived from at least 2 administered prior to or after Surgery as a prophylactic. The different biological sources. A biological source can include chimeric protein of the invention can be administered during any non-synthetically produced nucleic acid or amino acid or after Surgery to control an acute bleeding episode. The sequence (e.g. a genomic or cDNA sequence, a plasmid or Surgery can include, but is not limited to, liver transplantation, viral vector, a native virion or a mutant or analog, as further liver resection, or stem cell transplantation described herein, of any of the above). A synthetic source can 0.122 On-demand treatment includes treatment for a include a protein or nucleic acid sequence produced chemi bleeding episode, hemarthrosis, muscle bleed, oral bleed, cally and not by a biological system (e.g. Solid phase synthe hemorrhage, hemorrhage into muscles, oral hemorrhage, sis of amino acid sequences). A chimeric protein can also trauma, trauma capitis (head trauma), gastrointestinal bleed include a protein derived from at least 2 different synthetic ing, intracranial hemorrhage, intra-abdominal hemorrhage, Sources or a protein derived from at least one biological intrathoracic hemorrhage, bone fracture, central nervous sys Source and at least one synthetic source. A chimeric protein tem bleeding, bleeding in the retropharyngeal space, bleeding may also comprise a first amino acid sequence derived from a in the retroperitoneal space, or bleeding in the illiopsoas first source, covalently or non-covalently linked to a nucleic sheath. The Subject may be in need of Surgical prophylaxis, acid, derived from any source or a small organic or inorganic peri-operative management, or treatment for Surgery. Such molecule derived from any source. The chimeric protein may Surgeries include, e.g., minor Surgery, major Surgery, tooth comprise a linker molecule between the first and second extraction, tonsillectomy, inguinal herniotomy, synovec amino acid sequence or between the first amino acid sequence tomy, total knee replacement, craniotomy, osteosynthesis, and the nucleic acid, or between the first amino acid sequence trauma Surgery, intracranial Surgery, intra-abdominal Surgery, and the Small organic or inorganic molecule. intrathoracic Surgery, or joint replacement surgery. 0116. As used herein, the term “clotting factor.” refers to I0123. As used herein the term “acute bleeding refers to a molecules, or analogs thereof, naturally occurring or recom bleeding episode regardless of the underlying cause. For binantly produced which prevent or decrease the duration of example, a Subject may have trauma, uremia, a hereditary a bleeding episode in a Subject. In other words, it means bleeding disorder (e.g., factor VII deficiency) a platelet dis molecules having pro-clotting activity, i.e., are responsible order, or resistance owing to the development of antibodies to for the conversion offibrinogen into a mesh of insoluble fibrin clotting factors. causing the blood to coagulate or clot. An “activatable clot 0.124 Treat, treatment, treating, as used herein refers to, ting factor” is a clotting factor in an inactive form (e.g., in its e.g., the reduction in severity of a disease or condition; the Zymogen form) that is capable of being converted to an active reduction in the duration of a disease course; the amelioration form. of one or more symptoms associated with a disease or con 0117 Clotting activity, as used herein, means the ability to dition; the provision of beneficial effects to a subject with a participate in a cascade of biochemical reactions that culmi disease or condition, without necessarily curing the disease or nates in the formation of a fibrin clot and/or reduces the condition, the prophylaxis of one or more symptoms associ severity, duration or frequency of hemorrhage or bleeding ated with a disease or condition. episode. 0.125. As used herein, the term "solid phase peptide syn 0118. Hemostasis, as used herein, means the stopping or thesis” refers to the in vitro synthesis of polypeptide mol slowing of bleeding or hemorrhage; or the stopping or slow ecules immobilized on a solid Surface. The general principle ing of blood flow through a blood vessel or body part. of SPPS is one of repeated cycles of coupling-wash-depro 0119 Hemostatic disorder, as used herein, means a geneti tection-wash. The free N-terminal amine of a solid-phase cally inherited or acquired condition characterized by a ten attached peptide is coupled to a single N-protected amino dency to hemorrhage, either spontaneously or as a result of acid unit. This unit is then deprotected, revealing a new N-ter trauma, due to an impaired ability or inability to form a fibrin minal amine to which a further amino acid may be attached. clot. Solid phase peptide synthesis was originally described in 0120 Examples of such disorders include the hemophil Merrifield et al., “Solid Phase Peptide Synthesis. I. The Syn ias. The three main forms are hemophilia A (factor VIII defi thesis of a Tetrapeptide'. J. Am. Chem. Soc. 85 (14): 2149 ciency), hemophilia B (factor IX deficiency or “Christmas 2154 (1963). For example, the compounds of the present disease') and hemophilia C (factor XI deficiency, mild bleed disclosure can be synthesised using Solid-phase peptide Syn ing tendency), Von Willebrand disease, factor Xi deficiency thesis as described in “Fmoc Solid Phase Peptide Synthe (PTA deficiency), Factor XII deficiency, deficiencies or struc sis—A Practical Approach, edited by W. C. Chan, P. D. tural abnormalities in fibrinogen, prothrombin, Factor V. Fac White, Oxford University Press, New York 2000 and refer tor VII, Factor X or factor XIII, Bernard-Soulier syndrome is ences therein. Solid phase peptide synthesis includes the Syn a defect or deficiency in GPIb. GPIb, the receptor for vWF, thesis of polypeptides comprising natural amino acids, can be defective and lead to lack of primary clot formation unnatural amino acids including D-amino acids, peptide?pro (primary hemostasis) and increased bleeding tendency), and tein backbone modification, and conjugation of peptidic and thrombasthenia of Glanzman and Naegeli (Glanzmann non-peptidic moieties. US 2015/03539 11 A1 Dec. 10, 2015

II. Chimeric Proteins moiety, and Em is an enhancer moiety. In one embodiment, 0126 The present invention is directed to a chimeric pro the linker moiety can be a peptide linker. Non-limiting tein comprising an activatable clotting factor and an enhancer examples of the peptide linkers are described in section (D) moiety. The activatable clotting factor in the chimeric protein below. In another embodiment, the linker moiety is a low is administered as an inactive form (i.e., Zymogen) and is complex polypeptide, e.g., an XTEN sequence. A linker moi activated by a protease after administration in Vivo, for ety useful for the chimeric protein comprises at least about example, at the site of an injury. Once the activatable clotting five, at least about 10, at least about 20, at least about 30, at factor is activated, the enhancer moiety associated with or least about 40, at least about 50, at least about 60, at least linked to the activated clotting factor can enhance the activity about 70, at least about 80, at least about 90, at least about 100, of the clotting factor by acting as a partner in the coagulation at least about 110, at least about 120 amino acids, at least 150 pathway. Therefore, the chimeric protein of the invention can amino acids, at least 200 amino acids, at least 500 amino also be described as an enhanced or improved Zymogen oran acids, at least 1000 amino acids, or at least 2000 amino acids. enhanced or improved Zymogen fusion protein (e.g., FVII I0131. In another aspect, the chimeric protein of the inven enhanced Zymogen fusion protein or FX enhanced Zymogen tion comprises an activatable clotting factor, an enhancer fusion protein). Examples of the activatable clotting factors moiety, and one or more heterologous moieties (sometimes useful for the chimeric protein include, but are not limited to, indicated herein as Het, Het1, or Het2). Heterologous moi Factor VII or Factor X as described in section (A) below. eties can comprise a heterologous polypeptide moiety, a non 0127. The activatable clotting factor is further improved polypeptide moiety, or both. The heterologous polypeptide by bringing an enhancer moiety (e.g., a clotting cofactor, e.g., moiety can be selected from an immunoglobulin constant Tissue Factor) in close proximity to the activatable clotting region or portion thereof, albumin or a fragment, derivative, factor. Thus, when the clotting factor is cleaved into a het or variant thereof, an albumin binding moiety, an albumin erodimer, the enhancer moiety can interact with the clotting biding Small molecule, a PAS sequence, an XTEN sequence, factor heterodimer and can induce conformational changes to a HAP sequence, transferrin or a fragment, derivative, or enhance the procoagulant activities. Examples of the variant thereof, or any combination thereof. In other embodi enhancer moiety useful for the invention include, but are not ments, the heterologous moiety is an immunoglobulin con limited to, a clotting cofactor, a procoagulant peptide, oran, stant region orportion thereof, e.g., an Fc moiety. In still other antigen binding moiety as described in section (B) below. In embodiments, the non-polypeptide moiety is selected from Some embodiments, the enhancer moiety interacts with the polyethylene glycol (PEG), polysialic acid, hydroxyethyl clotting factor without cleavage of the clotting factor into a starch (HES), a derivative thereof, or any combinations heterodimer. thereof. The heterologous moiety can be linked to the N-ter 0128. While cleavage of a light chain from a heavy chain minus or C-terminus of the activating clotting factor (either of a clotting factor makes two-chain activated form of the the light chain, the heavy chain, or both) or inserted between clotting factor, the clotting factor may still be present as a two amino acids within the

lent interaction, e.g., an ionic interaction, a hydrophobic In a particular embodiment, the association (:) is a disulfide interaction, a hydrophilic interaction, a Van der Waals inter bond between Het1 and Het2. In another embodiment, the action, a hydrogen bond. In other embodiments, (:) is a non association, i.e., (), is a non-covalent interaction, e.g., an peptide covalent bond. In still other embodiments, (:) is a ionic interaction, a hydrophobic interaction, a hydrophilic peptide bond. In yet other embodiments, (:) in formulas set interaction, a Van der Waals interaction, a hydrogen bond. In forth herein represents a physical or chemical association other embodiments, (:) is a non-peptide covalent bond. In still between two sequences, but not a chemical crosslinking, other embodiments, (:) is a peptide bond. In yet other embodi wherein a portion of the first sequence is in close proximity to ments, (:) in formulas set forth herein represents a physical or the second sequence such that the first sequence and the chemical association between two sequences, but not a second sequence interacts with each other upon activation of chemical crosslinking, wherein a portion of a first sequence is either or both the first sequence and the second sequence. in close proximity to a second sequence Such that the first 0134) Formulas set forth herein are merely non-limiting sequence and the second sequence interacts with each other examples of constructs of the present invention. The orienta upon activation of either or both the first sequence and the tion of the polypeptide formulas is shown from N-terminus Second sequence. (left) to C-terminus (right). For example, formula Ac-Het 1 0.136. In certain aspects, a chimeric protein, of the inven means formula NH2-Ac-Het1-COOH. In addition, (:) can be tion comprises a first polypeptide chain and a second an association or interaction between two polypeptide chains polypeptide chain, wherein the first polypeptide chain com by a covalent bond or a non-covalent bond between any part prises an activatable clotting factor and the second polypep of the first chain and any part of the second chain unless tide chain comprises an enhancer moiety, wherein the first otherwise noted. For example, formula Ac-Het1:Em-Het2 polypeptide chain and the second polypeptide chain are has two polypeptide chains, the first chain being Ac-Het1 and linked to or associated with each other. The chimeric protein the second chain being Em-Het2, wherein Ac in the first chain can further comprise a dimeric heterologous moiety region interacts or associates with Em in the second chain and/or comprising a first heterologous, moiety, Hetl (e.g., a first Fc Het1 in the first chain interacts or associates with Het2 in the moiety, e.g., Fl) and a second heterologous moiety, Het2 second chain. In some embodiments, (:) means a covalent, (e.g., a second Fc moiety, e.g., F2), wherein the first heter non-peptide bond or non-covalent bond. ologous moiety is in the first polypeptide chain and the second 0135) In a further aspect, a chimeric protein of the inven heterologous moiety is in the second polypeptide chain. For tion comprises an activatable clotting factor, an enhancer example, a chimeric protein can comprise a structure selected moiety, one or more linker moieties, and one or more heter from: ologous moieties. In one embodiment, the chimeric protein 0.137 (a) Ac linked to Het1 via the linker moiety, and comprises an activatable clotting factor (Ac), an enhancer Em linked to Het2: moiety (Em), one linker moiety (L), and one heterologous 0.138 (b) Ac linked to Het1 via the first linker moiety, moiety (Het), wherein the components arc linked to or asso and Em linked to Het2 via the second linker moiety; ciated with each other. The chimeric protein can be repre 0139 (c) Ac linked to Het1, and Emis linked to Het2via sented by a formula of Ac-L-Het: Het-L-Ac:Em, Em-L-Het: the linker moiety; Ac, Het-L-Em:Ac, Ac-L-Het-Em, or Em-Het-L-Ac. In 0140 (d) Ac linked to Het1, and Em linked to Het2: another embodiment, the chimeric protein comprises an acti Vatable clotting factor (Ac), an enhancer moiety (Em), two 0.141 (e) Em linked to Het1 via the linker moiety, and linker moieties (L1 and L2), and one heterologous moiety Ac linked to Het2: (Het). The chimeric protein can be represented by a formula 0.142 (f) Em linked to Het1 via the first linker moiety, of Ac-L1-Het-L2-Em and Em-L2-Het-L1-Ac. In other and Ac linked to Het2 via the second linker moiety; embodiments, the chimeric protein comprises an activatable 0.143 (g) Emlinked to Het1, and Ac is linked to Het2 via clotting factor (Ac), an enhancer moiety (Em), two linker the linker moiety; or, moieties (L1 and L2), and two heterologous moieties (Het 1 0.144 (h) Em linked to Het1, and Ac linked to Het2. and Het2), wherein the components are linked to or associ wherein Het1 and Het2 form a disulfide bond. ated with each other, The chimeric protein can be represented 0145 The chimeric protein comprising two polypeptides by a formula Ac-L1-Het1: Em-L2-Het2, Het1-L2-Ac:Em can also be represented as set forth below: L2-Het2, or Het1-L2-Ac: Het2-L2-Em, wherein Ac com 0146 (a) the first polypeptide comprises a structure rep prises, consisting essentially of or consisting of an activat resented by the formula Ac-L1-Het1, and the second able clotting factor, L1 comprises, consisting essentially of polypeptide comprises a structure represented by the or consisting of an first optional linker moiety, e.g., a first linker, Hetl comprises, consisting essentially of, or consist formula Em-Het2: ing of a first heterologous moiety (e.g., a first Fc moiety), Em 0147 (b) the first polypeptide comprises a structure comprises, consisting essentially of, or consisting of an represented by the formula Ac-L1-Het1, and the second enhancer moiety, L2 comprises, consisting essentially of, or polypeptide comprises a structure represented by the consisting of a second optional linker moiety, e.g., a second formula Em-L2-Het2: linker, Het2 comprises, consisting essentially of, or consist 0.148 (c) the first polypeptide comprises a structure rep ing of a second optional heterologous moiety (e.g., a second resented by the formula Ac-Het1, and the second Fc moiety), (—) comprises, consisting essentially of, or con polypeptide comprises a structure represented by the sisting of a peptide bond or one, or more amino acids, and (:) formula Em-L2-Het2: is an association between Ac-L1-Het1 and Em-L2-Het2. The 0.149 (d) the first polypeptide comprises a structure association (:) as set forth herein represents a covalent bond or represented by the formula Ac-Het1, and the second anon-covalent bond, e.g., at least one non-peptide bond. In polypeptide comprises a structure represented by the one embodiment, the association, i.e., (), is a covalent bond. formula Em-L1-Het2: US 2015/03539 11 A1 Dec. 10, 2015 14

0150 (e) the first polypeptide comprises a structure rep 0158 Each component of the chimeric proteins is resented by the formula Em-L2-Het1, and the second described below. polypeptide comprises a structure represented by the 0159 A. Activatable Clotting Factors formula Ac-L1-Het2: (0160) 1. Clotting Factors 0151 (f) the first polypeptide comprises a structure rep 0.161. In particular, the invention pertains to improved ver resented by the formula Em-L1-Het1, and the second sions of factors VII and X. These factors are all structurally polypeptide comprises a structure represented by the related in that in each the amino terminal end of the light chain formula Ac-Het2: is not amenable to the incorporation of additional moieties. 0152 (g) the first polypeptide comprises a structure Similarly, the amino terminal end of the heavy chain of these represented by the formula Em-Het1, and the second three clotting factors is not amenable to the incorporation of polypeptide comprises a structure represented by the additional moieties, with the exception of cleavable moieties, formula Ac-L1-Het2; and, i.e., moieties linked via a cleavage site or moieties which 0153 (h) the first polypeptide comprises a structure consist of a cleavage site. The chimeric clotting factor con represented by the formula Em-Het1, and the second structs of the invention were designed based on these shared polypeptide comprises a structure represented by the properties and it will be understood that although factor VII is often shown to illustrate exemplary embodiments of the formula Ac-Het2: invention, the Subject constructs may be made using factor 0154 wherein Het1 and Het2 of the two polypeptide VII or X. For example, one of skill in the art would understand chains form a disulfide bond. that the FVII portion of a construct of the invention could be 0155. In yet other aspects, the chimeric protein comprises substituted with a FX portion to make an enhanced version of an activatable clotting factor (Ac), an enhancer moiety (Em). one of these clotting factors. three linker moieties (L1, L2, and X), and two heterologous 0162 Clotting factors given for bypass therapy are effica moieties (Het1 and Het2), wherein the components are linked cious when given in the activated form, since exogenous to each other. The chimeric protein can comprise by a formula clotting factors are often not activated with sufficient kinetics selected from Ac-Het1-X-Em-Het2 or Het2-Em-X-Het1-Ac, to be effective. However, they are also rapidly inactivated by wherein Ac is an activatable clotting factor, Het1 is a first endogenous pathways (e.g., by antithrombin III or TFPI), heterologous moiety, X is a scFc linker, Em is an enhancer leading to clearance of the active form and a short effective moiety, and Het2 is a second heterologous moiety. The chi half-life. In order to prevent rapid inactivation by endogenous meric protein can also comprise one or more linker moieties. enzymes and clearance, the chimeric clotting factor of the For example, a chimeric protein can comprise a formula invention is constructed as an “activatable' form. Such acti selected from Ac-L1-Het1-X-Em-Het2. Ac-Het1-X-Em-L2 Vatable constructs circulate as an enhanced Zymogen with a Het2, Ac-L1-Het1-X-Em-L2-Het2, Het2-Em-X-Het1-L1 longer half-life, but can be readily cleaved at the site of Ac, Het2-L2-Em-X-Het1-Ac, or Het2-L2-Em-X-Het1-L1 clotting when necessary. Ac, wherein Ac is an activatable clotting factor, L1 is a first 0163 Exemplary chimeric clotting factor constructs of the optional linker moiety, Het1 is a first heterologous moiety, X invention are set forth in the accompanying Figures. The is a scFc linker, Em is an enhancer moiety, L2 is a second chimeric clotting factor useful for the invention is expressed optional linker moiety, and Het2 is a second heterologous in inactive form, is Subsequently administered as inactive moiety. form, and then is activated upon administration in vivo. Inac 0156. In one embodiment, either or both of the heterolo tive forms of Factors VII and X are single chain Zymogens. gous moieties (Het1 and Het2) are a heterologous polypep Active forms of Factors VII and X are comprised of dimeric tide moiety, which are the same or different. In another molecules in which the heavy chain and the light chain are embodiment, either or both of Het1 and Het2 are a non linked by a covalent bond, e.g., a disulfide bond. polypeptide moiety. In other embodiments, either or both of 0164. An activatable clotting factor comprises a light the heterologous, moieties (Het1 and Het2) can be a half-life chain of the clotting factor Zymogen linked to a protease extender. Examples of the half-life extender include, but are cleavage site, which is further linked to a heavy chain of the not limited to, an immunoglobulin constant region or a por clotting factor Zymogen. The light chain or the heavy chain of tion thereof, albumin, transferrin, an albumin binding moiety, the clotting factor Zymogen can include a fragment, a variant, a PAS sequence, a HES sequence, the B subunit of the C-ter a derivative, or an analog thereof that retains the function of minal peptide (CTP) of human chorionic gonadotropin, poly the light chain or the heavy chain of the clotting factor ethylene glycol (PEG), an XTEN sequence, hydroxyethyl Zymogen, respectively. starch (HES), albumin-binding small molecules, von Will 0.165. In one embodiment, a clotting factor of the invention ebrand Factor or a fragment, derivative, or variant thereof, or is a mature form of Factor VII or a variant thereof. Factor VII any combinations thereof Examples of the heterologous moi (FVII, F7; also referred to as Factor 7, coagulation factor VII, ety are shown in section (C) below. serum factor VII, serum prothrombin conversion accelerator, O157. In another embodiment, the first and second heter SPCA, proconvertin and eptacog alpha) is a serine protease ologous moieties (Het1 and Het2) are linked to each other by that is part of the coagulation cascade. FVII includes a Gla a peptide bond or a linker (e.g., ScPc linker (sometimes also domain, two EGF domains (EGF-1 and EGF-2), and a serine indicated as “X”)) or associated by a covalent or non-covalent protease domain (or peptidase S1 domain) that is highly con bond, e.g., a disulfide bond. For example, a Schc linker can served among all members of the peptidase S1 family of linka first Fc moiety and a second Fc moiety, thereby forming serine proteases, such as for example with chymotrypsin. a dimeric Fc region. The Sclc linker can further comprise an FVII occurs as a single chain zymogen (i.e., activatable FVII) intracellular processing site, which enables processing of the and a fully activated two-chain form. chimeric protein when expressed in a host cell. Examples of 0166 As used herein, the term “a FVII protein’ includes the sche linker are shown in section (C.3) below. wild-type FVII, mature FVII, full-length FVII, a functional US 2015/03539 11 A1 Dec. 10, 2015 15 fragment of FVII, a variant, or a derivative thereof. Exem acid and nucleotide sequences are disclosed in the Sequence plary FVII variants include those with increased specific Listing as a portion of SEQID NOs: 44 and 45, respectively. activity, e.g., mutations that increase the activity of FVII by 0.167 Factor VII or Factor X activation occurs when the increasing its enzymatic activity (Kcator Km). Such variants immediate upstream of a heavy chain of a FVII Zymogen or a FX Zymogen is cleaved. For example, FVII is activated when have been described in the art and include, e.g., mutant forms the immediate upsteam of the first residue of the FVII heavy of the molecule as described for example in Persson et al. chain, I.e., Ile-153, is cleaved. 2001. PNAS 98: 13583; Petrovan and Ruf. 2001. J. Biol. 0.168. In one embodiment, a clotting factor of the invention Chem. 276:6616; Persson et al. 2001 J. Biol. Chem. 276: is a mature form of Factor X. Factor X is a vitamin-K depen 29.195; Soejima et al. 2001. J. Biol. Chem. 276:17229; Soe dent glycoprotein of a molecular weight of 58.5kDa, which is jima et al. 2002. J. Biol. Chem. 247:49027. In one embodi secreted from liver cells into the plasma as a Zymogen. Ini ment, a variant form of FVII includes the mutations. tially factor X is produced as a prepropeptide with a signal Exemplary mutations include V158D-E296V-M298Q. In peptide consisting in total of 488 amino acids. The signal another embodiment, a variant form of FVII includes a peptide is cleaved off by signal peptidase during export into replacement of amino acids 608-619 (LQQSRKVGDSPN, the endoplasmatic reticulum, the propeptide sequence is corresponding to the 170-loop) from the FVII mature cleaved off after gamma carboxylation took place at the first sequence with amino acids EASYPGK from the 170-loop of 11 glutamic acid residues at the N-terminus of the mature trypsin. High specific activity variants of FIX are also known N-terminal chain. A further processing step occurs by cleav in the art. For example, Simioni et al. (2009 N.E. Journal of age between Arg182 and Ser183. This processing step also Medicine 361: 1671) describe an R338L mutation. Chang, et leads concomitantly to the deletion of the tripeptide Arg180 al. (1988JBC 273:12089) and Pierrietal. (2009 Human Gene Lys181-Arg182. The resulting secreted factor X Zymogen Therapy 20:479) describe an R338A mutation. Other muta consists of an N-terminal light chain of 139 amino acids (M. tions are known in the art and include those described, e.g., in 16,200) and a C-terminal heavy chain of 306 amino acids (M, Zogg and Brandstetter. 2009 Structure 17:1669; Sichler et al. 42,000) which are covalently linked via a disulfide bridge 2003. J. Biol. Chem. 278:4121; and Sturzebecher et al. 1997. between Cys 172 and Cys342. Further posttranslational pro FEBS Lett 412:295. The contents of these references are cessing steps include the beta.-hydroxylation of Asp103 as incorporated herein by reference. Exemplary FVII amino well as N- and O-type glycosylation. TABLE 1. Amino acid Sequence of Factor X zymogen (SEQ ID NO: 11) MGTPLHLWLL SASLAGLLL GESLFIRREO ANNILARVTR ANSFLEEMKK GHLERECMEE TCSYEEAREW FEDSDKTNEF WNKYKDGDOC ETSPCONOGK CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDOFC HEEONSVWCS CARGYTLADN GKACIPTGPY PCGKOTLERR KRSVAOATSS SGEAPDSITW KPYDAADLDP TENPFDLLDF NOTOPERGDN NLTRIWGGOE CKDGECPWOA LLINEENEGF CGGTILSEFY ILTAAHCLYO AKRFKWRWGD RNTEOEEGGE AWHEWEWWIK HNRFTKETYD FDIAWLRLKT PITFRMNWAP ACLPERDWAE STLMTOKTGI WSGFGRTHEK GROSTRLKML EVPYWDRNSC KLSSSFIITO NMFCAGYDTK QEDACQGDSG GPHWTRFKDT YWTGIWSWG EGCARKGKYG IYTKVTAFLK WIDRSMKTRG LPKAKSHAPE WITSSPLK

Nucleotide Sequence Encoding Factor X Zymogen (SEQ NO: 12) cactgcacct cgt.cctgctic agtgcct c cc tggctggc ct cctgctgctic ggggaaagtic tgttcatc.cg Cagggagcag gccaacaa.ca t cctggcgag ggit Cacgagg gccaattic ct ttcttgaaga gatgaagaaa gga cacct cq aaagagagtg Catggaagag acctgct cat acgalagaggc cc.gc.gaggto tittgaggaca gcgacaagac gaatgaattic tggaataaat acaaagatgg cgaccagtgt gagaccagtic cittgccagaa cCagggcaaa tgtaaagacg gcct C9ggga atacacctgc acctgtttag aaggatt.cga aggcaaaaac tgttgaattat t cacacggaa gctctgcagc Ctggacaacg gggactgttga c cagttctgc cacgaggaac agaactctgt ggtgtgct Co tgcgc.ccg.cg ggtacacc ct ggctgacaac ggcaaggc ct gcatt.cccac agggcc ctac CCCtgtggga aacagacc ct ggaacgcagg aagaggltdag tggccCaggc Caccagcagc agcggggagg cc cct gacag cat cacatgg aa.gc.catatg atgcagc.cga cctggacc cc accgagaacc c ctitcgacct gcttgactitc alaccagacgc agcctgagag gggcgacaac aac Ctcacca ggat.cgtggg aggcc aggaa tgcaaggacg gggagtgtcC Ctggcaggcc ctgct catca atgaggaaaa cgagggitttic tgttggtggala c cattctgag cgagttctac atcctaacgg cagcc cactg tot Ctaccala gccalagagat t caaggtgag gg taggggac cggalacacgg agcaggagga ggg.cggtgag gcggtgcacg aggtggaggt ggtcat Caag Cacaaccggit t cacaaagga gacctatgac tt cqacat cq cc.gtgctic.cg gct Caagacic CCCatcacct tcc.gcatgaa cgtggcgc.ct gcct gcct co cc.gagcgtga Ctgggcc.gag tccacgctga tgacgcagaa gacggggatt gtgagcggct tcgggcgcac ccacgagaag ggc.cggcagt ccaccaggct Caagatgctg gaggtgcc ct acgtggaccg Caac agctgc aagctgtc.ca gcagott cat cat cacc cag aacatgttct gtgc.cggcta cgacaccalag Caggaggatg CctgcCaggg ggacagoggg ggc.ccgcacg t caccc.gctt Caagga cacc tactitcgtga Caggcatcgt. Cagctgggga gagggctgttg cc.cgitaaggg galagtacggg atctacacca agg to accgc citt cotcaag tggat.cgaca ggtc catgaa alaccaggggg ttgcc.caagg ccalaga.gc.ca tgcc.ccggag qt catalacgt. cottctic catt aaagtga US 2015/03539 11 A1 Dec. 10, 2015

0169. It will be understood that in addition to wild type (0175 2. Protease-Cleavage Site (WT) versions of these clotting factors or biologically active 0176 A protease-cleavage site linking a light chain of a portions thereof, the present invention may also employ pre clotting factor Zymogen and a heavy chain of the clotting cursor truncated forms thereof that have activity, allelic vari factor Zymogen can be selected from any protease-cleavage ants and species variants, variants encoded by splice variants, site known in the art. In one embodiment, the protease-cleav and other variants, including polypeptides that have at least age site is cleaved by a protease selected from factor XIa, 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, factor XIIa, kallikrein, factor VIIa, factor IXa, factor Xa, 95%, 96%, 97%, 98%, 99% or more sequence identity to the factor IIa (thrombin), or any combinations thereof. The pro mature form of the clotting factor and which retain the ability tease-cleavage sites allow the light chain and the heavy chain to promote clot formation. For example, modified FVII of the clotting factor to be cleaved and dissociated from each polypeptides and variants thereof which retain at least one other at the site of injury. Exemplary FXIa cleavage sites activity of a FVII, such as TF binding, factor X binding, include, e.g., KLTR (SEQID NO: 13), DFTR (SEQID NO: phospholipid binding, and/or coagulant activity of a FVII 14), TOSFNDFTR (SEQ ID NO: 6) and SVSQTSKLTR may be employed. By retaining activity, the activity can be (SEQID NO: 7). Exemplary thrombin cleavage sites include, altered. Such as reduced or increased, as compared to a wild e.g., DFLAEGGGVR (SEQID NO: 8), TTKIKPR (SEQ ID type clotting factor so long as the level of activity retained is NO:9), LVPRG (SEQID NO: 10) and ALRPR (SEQID NO: sufficient to yield a detectable effect. Exemplary sequences of 1). clotting factors that can be used in the constructs of the 0177. In some embodiments, the protease-cleavage site invention are found in the accompanying sequence listing. can be combined with an intracellular processing site for 0170 Exemplary modified polypeptides include, but are efficient cleavage and activation. For example, an activatable not limited to, tissue-specific isoforms and allelic variants clotting factor in the chimeric protein may comprise a het thereof, synthetic molecules prepared by translation of erodimer, which comprises a light chain of a clotting factor nucleic acids, proteins generated by chemical synthesis, Such associated with a heavy chain of the clotting factor by a as syntheses that include ligation of shorter polypeptides, covalent bond, wherein the N-terminus of the heavy chain of through recombinant methods, proteins isolated from human the clotting factor is linked, to a protease-cleavage site The and non-human tissue and cells, chimeric polypeptides and protease-cleavage site can be cleaved off at the site of coagul modified forms thereof The instant clotting factors may also lation, thus activating the clotting factor. Such constructs can consist of fragments or portions of WT molecules that are of be designed by inserting an, intracellular processing site sufficient length or include appropriate regions to retain at between the light chain of the clotting factor Zymogen and the least one activity (upon activation if needed) of a full-length protease-cleavage site, which is linked to the heavy chain of mature polypeptide. Exemplary clotting factor variants are the clotting factor Zymogen. The intracellular processing site known in the art. inserted therein can be processed (cleaved) by an intracellular 0171 In one embodiment, activatable clotting factors are processing enzyme upon expression in a host cell, thereby modified to lack a Gla domain. In the case of Factor VII, the allowing formation of a Zymogen-like heterodimer. Gla domain is present at the amino terminus of the light chain Examples of the intracellular processing enzymes include and consists of amino acids 1-35. The GLA domain is respon furin, a yeast Kex2. PCSK1 (also known as PC1/Pc3), PCSK2 sible for the high-affinity binding of calcium ions. It starts at (also known as PC2), PCSK3 (also known as furin or PACE), the N-terminal extremity of the mature form of proteins and PCSK4 (also known as PC4), PCSK5 (also known as PC5 or ends with a conserved aromatic residue. A conserved Gla-X PC6), PCSK6 (also known as PACE4), or PCSK7 (also (3)-Gla-x-Cys motif is found in the middle of the domain known as PC7/LPC, PC8, or SPC7). Other processing sites which seems to be important for substrate recognition by the are known in the art. carboxylase. (0178. 3. Self-Immolative Moiety 0172. Using stopped-flow fluorescence kinetic measure 0179. In certain embodiments, the protease-cleavage site ments in combination with Surface plasmon resonance analy is linked to a heavy chain of a clotting factor Zymogen via a sis, the Gla domain has been found to be important in the self-immolative moiety. The term “self-immolative moiety’ sequence of events whereby the protease domain of FVIIa as used herein refers to a bifunctional chemical moiety which initiates contact with STF (Biochemical and Biophysical is capable of covalently linking together two spaced moieties Research Communications. 2005. 337: 1276). In addition, (e.g., a heavy chain of a clotting factor and a protein-cleavage clearance of clotting factors may be significantly mediated site) into a normally stable tripartate molecule. The self through Gla interactions, e.g., on liver cells and clearance immolative moiety will spontaneously separate from the sec receptors, e.g., EPCR. ond moiety (e.g., a heavy chain of a clotting factor) if it is 0173 Therefore, the Gla domain is responsible for medi bound to the first moiety (e.g., a protein-cleavage site) is ating clearance of clotting factors via multiple pathways. Such cleaved. as binding to liver cells, clearance receptors such as EPCR, 0180. In some aspects, the self-immolative moiety com etc. Thus, eliminating the Gla domain has beneficial effects prises an aminobenzyl carbamate group, an aminobenzyl on half-life of the clotting factors. The Gla domain of factor ether group, or an aminobenzyl carbonate group. In one VII comprises the uncommon amino acid-carboxyglutamic aspect, the self-immolative moiety is p-amino benzyl car acid (Gla), which plays a vital role in the binding of clotting, bamate (PABC). factors to negatively charged phospholipid surfaces. 0181 P-amino benzyl carbamate (PABC) is the most effi 0.174 Exemplary clotting, factors are those of mamma cient and most widespread connector linkage for self-immo lian, e.g., human, origin. The sequences of exemplary clotting lative site-specific prodrug activation (see, e.g., Carl et al. J. factors are presented in the accompanying sequence listing, Med. Chem. 24:479-480 (1981); WO 1981/001145; Rautio et e.g., alone or in the context of a chimeric clotting factor la, Nature Reviews Drug Discovery 7:255-270 (2008); Sim COnStruct. plicio et al., Molecules 13:519-547 (2008):). PABC allows US 2015/03539 11 A1 Dec. 10, 2015 the release of any amine drugs, peptides, and proteins upon aspects, the self-immolative moiety comprises a heterocyclic cleavage by a protease and 1.6 spontaneous fragmentation. ring (see., e.g., U.S. Pat. Nos. 7,375,078; 7.754,681). Numer 0182. The aromatic ring of the aminobenzyl group may ous homoaromatic (see, e.g., Carletal.J.Med. Chem. 24:479 optionally be substituted with one or more (e.g., R and/or R) (1981); Senter et al. J. Org. Chem.55:2975 (1990); Taylor et Substituents on the aromatic ring, which replace a hydrogen al. J. Org. Chem. 43:1197 (1978); Andrianomenjanahary et that is otherwise attached to one of the four non-substituted al. Bioorg. Med. Chem. Lett. 2: 1903 (1992)), and coumarin carbons that form the ring. As used herein, the symbol “R” (see, e.g., Weinstein et al. Chem. Commun. 46:553 (2010)), (e.g., R. R. R. Ra) is a general abbreviation that represents furan, thiophene, thiazole, oxazole, isoxazole, pyrrole, pyra a Substituent group as described herein. zole (see, e.g., Hay et al. J. Med. Chem. 46:5533 (2003)), 0183 Substituent groups can improve the self-immolative pyridine (see, e.g., Perry-Feigenbaum et al. Org. Biomol. ability of the p-aminobenzyl group (Hay et al., J. ChemSoc., Chem. 7:4825 (2009)), imidazone (see, e.g., Nailor et al. Perkin Trans. 1:2759-2770 (1999); see also, Sykes et al. J. Bioorg. Med. Chem. Lett. Z:1267 (1999); Hay and Denny, Chem. Soc., Perkin Trans. 1:1601-1608 (2000)). Tetrahedron Lett. 38:8425 (1997)), and triazole (see, e.g., 0184 The following formula shows the general topology Bertrand and Gesson, J. Org. Chem. 72:3596 (2007)) based of a p-amino benzyl immolative linker and the relative loca heteroaromatic groups that are self-immolative under both tions of an exemplary protease-cleavage site aqueous and physiological conditions are known in the art. (Aa Aa AasAa) and a heavy chain of a clotting factor (POI). See also, U.S. Pat Nos. 7,691.962; 7,091, 186: U.S. Pat. Publ. The formula indicates possible locations of R substituent Nos. US2006/0269480; US2010/0092496; US2010/ groups (R, R2, R). 0145036; US2003/0130189; US2005/0256030) 0188 Where substituent groups in the self-immolative linkers are specified by their conventional chemical formulae, (Formula I) written from left to right, they equally encompass the chemi cally identical substituents, which would result from writing the structure from right to left. For example, “ CHO is ls POI intended to also recite " OCH . Substituent groups in self-immolative, for example, R and/or R. Substituents in a p-aminobenzyl self-immolative linker as discuss above can include, e.g., alkyl, alkylene, alkenyl, alkyiyl, alkoxy, alky lamino, alkylthio, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, aryloxy, heteroaryl, etc. When a compound of the present disclosure includes more than one Substituent, 0185. The substituents, which may be a single , e.g., then each of the Substituents is independently chosen. a halogen, or a multi-atom group, e.g., methyl, are selected in (0189 B. Enhancer Moieties (Em) order to impact the stability of the aminobenzyl or the decom 0190. The present invention provides an improved or position product thereof. Electron withdrawal from the ring enhanced activatable clotting factor Such that by fusing the may be used to facilitate the spontaneous decomposition of activatable clotting factor to an "enhancer moiety, the prop the aminobenzyl group from the drug after cleavage of the erties of an activatable clotting factor of the invention are bond between the amino group of the aminobenzyl group and improved compared to the activatable clotting factor which is the adjacent peptide linkage. Exemplary aromatic group R. not fused to the enhancer moiety. The improved properties R, or R substituents include, for example, F, Cl, I, Br, OH, include a procoagulant activity of the clotting factor. The methyl, methoxy, NO, NH, NO, NHCOCH, N(CH), increase in the procoagulant activity is relative to the free, or NHCOCF, alkyl, haloalkyl, C-C alkylhalide, carboxylate, un-fused, activatable clotting factor. An enhancer moiety can Sulfate, Sulfamate, Sulfonate, etc. (see, e.g., U.S. Pat. Nos. be any molecule which has the ability to enhance the proco 7,091,186 and 7.659,241). The p-aminobenzyl linker can agulant activity of a clotting factor. The enhancer moiety comprise a heteroatom Z connected to the amino terminus of useful for the invention can have a physical interaction with the peptide or protein of interest protein. The term heteroa the activatable clotting factor, and the physical interaction can tom, as used herein, includes oxygen (O), nitrogen (N), Sulfur induce increase in procoagulant activity. (S), silicon (Si), boron (B) and phosphorus (P). In one 0191 The chimeric protein of the invention can comprise embodiment, the heteroatoms in Z are O, S or N. one or more than one enhancer moieties. Additionally, two or 0186. In some embodiments, only one of the four non more enhancer moieties may be linked to each other (e.g., via Substituted carbons in the p-aminobenzyl ring is Substituted. a linker) in series, and the tandem array operably linked to a In some other embodiments, two of the four non-substituted construct of the invention. When two or more enhancer moi carbons in the p-aminobenzyl ring are Substituted. In other eties are present in a chimeric clotting factor of the invention, embodiments, three of the four non-substituted carbons in the the moieties may be the same or different. p-aminobenzyl ring are substituted. In some embodiments, 0.192 In one embodiment, the enhancer moiety is located the four non-substituted carbons in the p-aminobenzyl ring on the C-terminus of the heavy chain of factor VII or factor X. are substituted. In another embodiment, the enhancer moiety is located on the 0187 Self-immolative elimination can take place, e.g., via N-terminus of the light chain of factor VII or factor X. In other 1.4 elimination, 1.6 elimination (e.g., PABC), 1.8 elimination embodiments, the enhancer moiety is located on the C-termi (e.g., p-amino-cinnamyl alcohol), cyclisation-elimination nus of the light chain of factor VII or factor X. In embodi (e.g., 4-aminobutanol ester and ethylenediamines), etc. In ments in which an Fc domain orportion thereof is employed, Some aspects, the self-immolative moiety can comprise, e.g., the enhancer moiety may be positioned at the Nor C terminus an cinnamyl, naphthyl, or biphenyl groups (see, e.g., Blen of the second Fc moiety, or the C-terminus of either or both Fc cowe et al. Polym. Chem. 2:773-790 (2011)). In some moieties. US 2015/03539 11 A1 Dec. 10, 2015

0193 In one embodiment, an enhancer moiety is not TABLE 2 genetically fused directly to a construct, but rather is linked via a linker or a chemical bond to the construct. For example, Tissue Factor Sequences enhancer moieties may be attached to a construct of the inven Tissue Factor Amino Acid Sequence- isoform 1 tion by formation of a bond between the enhancer moiety and (SEQ ID NO: 15) an Fc moiety of a construct, where the enhancer moiety METPAWPRWP RPETAVARTL LLGWVFAOVA GASGTTNTVA comprises a first functional group and the Fc moiety com AYNLTWKSTN FKTILEWEPK PVNOVYTVOI STKSGDWKSK prises a second functional group, and where the first and second functional groups are capable of reacting with each CFYTTDTECD LTDEIVKDWK OTYLARVFSY PAGNVESTGS other to form a chemical bond (see, e.g., U.S. Pat. No. 7,381, AGEPLYENSP EFTPYLETNL GOPTIOSFEO VGTKVNVTVE 408). DERTLWRRNN TFLSLRDWFG KDLIYTLYYW KSSSSGKKTA 0194 In certain embodiments, the enhancer moieties of the invention can be blood coagulation pathway proteins KTNTNEFLID VDKGENYCFS VOAVIPSRTV NRKSTDSPVE (e.g., cofactors), procoagulant peptides, or antigen binding CMGOEKGEFR EIFYIIGAVV FVVIILVIIL AISLHKCRKA molecules. Examples of enhancer moieties are found in the instant examples and Figures. Other molecules useful, as GVGOSWKENS PLNWS enhancer moieties can readily be selected by one of skill in the Tissue Factor Nucleic Acid Sequence art based upon the teaching herein. (SEQ ID NO: 16) ATGGAGACCCCTGCCTGGCCCCGGGTCCCGCGCCCCGAGACCG 0195 1. Clotting Cofactors 0196. An enhancer moiety useful for the chimeric protein CCGTCGCTCGGACGCTCCTGCTCGGCTGGGTCTTCGCCCAGGT can be a clotting cofactor. The "clotting cofactor as used GGCCGGCGCTTCAGGCACTACAAATACTGTGGCAGCATATAAT herein means a clotting factor that forms a complex with another clotting factor, e.g., Factor VII or Factor X, and TTAACTTGGAAATCAACTAATTTCAAGACAATTTTGGAGTGGG becomes an activated complex having procoagulant activity. AACCCAAACCCGTCAATCAAGTCTACACTGTTCAAATAAGCAC For example, a clotting cofactor for FVII is Tissue Factor, which forms the TF-FVIIa complex. A clotting cofactor for TAAGTCAGGAGATTGGAAAAGCAAATGCTTTTACACAACAGAC FX is FVa, which forms the prothrombinase complex and ACAGAGTGTGACCT CACCGACGAGATTGTGAAGGATGTGAAGC thereby activates prothrombin to thrombin. AGACGTACTTGGCACGGGTCTTCTCCTACCCGGCAGGGAATGT 0197) In one embodiment, the clotting factor zymogen is a FVII protein, and the clotting cofactor is a Tissue Factor (TF) GGAGAGCACCGGTTCTGCTGGGGAGCCTCTGTATGAGAACTCC polypeptide. Tissue Factor initiates blood coagulation by CCAGAGTTCACACCTTACCTGGAGACAAACCTCGGACAGCCAA forming a complex with circulating factor VII or VIIa. The ITF:VIIa complex activates factors IX or X by specific lim CAATTCAGAGTTTTGAACAGGTGGGAACAAAAGTGAATGTGAC ited proteolysis. TF plays a role in normal hemostasis by initiating the cell-surface assembly and propagation of the CGTAGAAGATGAACGGACTTTAGTCAGAAGGAACAACACTTTC coagulation protease cascade. TF is also known as coagula CTAAGCCTCCGGGATGTTTTTGGCAAGGACTTAATTTATACAC tion factor III, thiomboplastin, CD142, and F3. The full length tissue factor polypeptide has Accession Number TTTATTATTGGAAATCTTCAAGTTCAGGAAAGAAAACAGCCAA P13726-1 in UniProtKB entry and consists of the signal pep AACAAACACTAATGAGTTTTTGATTGATGTGGATAAAGGAGAA tide (amino acids 1 to 32), the extracellular domain (amino acids 33 to 251), the transmembrane domain (amino acids AACTACTGTTTCAGTGTTCAAGCAGTGATTCCCTCCCGAACAG 252 to 274) and the cytoplasmic domain (amino acids 275 to TTAACCGGAAGAGTACAGACAGCCCGGTAGAGTGTATGGGCCA 295), total of 295 amino acids. The nucleotide and amino acid sequences of Tissue Factor are represented herein as SEQID GGAGAAAGGGGAATTCAGAGAAATATTCTACATCATTGGAGCT NO: 16 and SEQ ID NO: 15, respectively. An isoform of Accession Number P13726-1 (No. P13726-2) contains a sub GTGGTATTTGTGGTCATCATCCTTGTCATCATCCTGGCTATAT Stitution of amino acids 199-238: TAKTNTNEFL . . . CTCTACACAAGTGTAGAAAGGCAGGAGTGGGGCAGAGCTGGAA TVNRKSTDSP-sySTSLELWYL. . . WGRAGRRTPH and a deletion of amino acids 239 to 295. Variants of human GGAGAACTCCCCACTGAATGTTTCATAA Tissue Factor include, but are not limited to, the polypeptides with the following mutations: T36A, I145V, R163W, or G281E. Also included is PCSK1 from a different species, 0198 The tissue factor polypeptide used for the present e.g., mouse, rat, monkey, dog, drosophila, orporcine. As used invention comprises an amino acid sequence, which is at least herein, a tissue factor polypeptide refers to a polypeptide 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, comprising the soluble ectodomain of Tissue Factor (STF) 99%, or 100% identical to amino acids 33-251 of SEQ ID (approximately amino acids 33-251), or functional variants, NO: 15, (STF), wherein the amino acid sequence is capable of fragments, analogues, or derivatives thereof. STF lacks the forming a complex with FVII or FVIIa. The term “TF pro transmembrane and cytoplasmic domains. The full length tein’ as used herein includes full-length TF, functional frag sequence of mature human Tissue Factor is disclosed in ments (e.g., an extracellular domain), variants, analogues, or Spicer et al. Proc. Natl. Acad. Sci., USA, 84, 5148-5152 derivatives thereof. The term "soluble TF as used herein (1987). includes any functional fragments, variants, analogues, or US 2015/03539 11 A1 Dec. 10, 2015 19 defivatives thereof that retain one or more activities of the heavy chain and a light chain. The full-length Factor V complete extracellular domain of TF. In one embodiment, polypeptide has Accession No. P12259 in UniProtKB entry soluble TF (and its functional fragments, variants, analogues, and consists of the signal peptide (amino acids 1 to 28), the or derivatives thereof) is capable of binding to FVII. In heavy chain (amino acids 29 to 737), the activation peptide (also called as connecting region, amino acids 734 to 1573), another embodiment, Soluble TF can act as a clotting cofactor and the light chain (amino acids 1574 to 2224). The nucle for FVII. otide and amino acid sequences of FV are represented herein 0199. In another embodiment, the clotting factor zymogen as SEQID NO: 18 and SEQID NO: 17, respectively. Variants is a FX protein, and the clotting cofactor is a FVa protein. A of human Factor V include, but are not limited to, the FVa protein serves as a critical cofactor for the prothrombi polypeptides with the following mutations: G15S, D107H, nase activity of factor Xa that results in the activation of R334G, R334T, I387T, M413T, R513K, R534Q, C613R, prothrombin to thrombin. Factor Va, the activated form of S775A, S781R, P809S, N817T, K858R, H865R, T915S, factor V, is composed of a heavy chain and a light chain, K925E, N969S, R980L, H1146Q, L1285I, H1327R, L1397F, non-covalently bound. The interaction between the two P1404S, E1530A, T1685S, Y1730C, L1749V, M1764V, chains is calcium-dependent. Factor V is also known as M1820I, R2102C, R2102H, M2148T, K2185R, or D2222G. coagulation factor V, activated protein C cofactor, proacce Also included is a Factor V protein from a different species, lerin, and labile factor and can be cleaved into two chains, a e.g., mouse, rat, monkey, dog, drosophila, or porcine. TABL 3 Factor V Sequences Factor V Amino Acid Sequence (SEO ID NO : 17) MFPGCPRLWW LWWLGTSWWG WGSOGTEAAQ LROFYVAAOG ISWSYRPEPT NSSLNLSVTS

FKKIWYREYE KEKPOS TISGLLGPTL YAEVGDIIKV HFKNKADKPL SIHPOGIRYS

KLSEGASYLD HTFPAEKMDD AWAPGREYTY EWSISEDSGP THDDPPCLTH IYYSHENLIE

DFNSGLIGPL ICK KGTLTE GGTOKTFDKO IVLLFAVFDE SKSWSOSSSL MYTVNGYWNG

TMPDITWCAH DHISWHLLGM SSGPELFSIH FNGOVLEONH HKVSAITLVS ATSTTANMTV

GPEGKWISS TPK HLOAGM QAYIDIKNCP KKTRNLKKIT REORRHMKRW EYFIAAEEVI

WDYAPWIPAN MDKKYRSOHL DNFSNOIGKHYKKWMYTOYE DESFTKHTVN PNMKEDGILG

PIIRAOVRDT KIW FKNMAS RPYSIYPHGV TFSPYEDEVN SSFTSGRNNT MIRAVOPGET

YTYKWNILEF DEPT ENDAQC LTRPYYSDVD IMRDIASGLI GLLLICKSRS LDRRGIORAA

DIEQQAVFAW FDEN KSWYLE DININKFCENP DEWKRDDPKF YESNIMSTIN GYWPESITTL

GFCFDDTWOW HFCSVGTONE ILTIHFTGHS FIYGKRHEDT LTLFPMRGES WTWTMDNWGT

WMLTSMNSSP RSKK LRLKFR DWKCIPDDDE DSYEIEEPPE STWMATRKMH DRLEPEDEES

DADYDYONR AAALIGIRSF R NSSLNOEEEE FNLTALALEN GTEFVSSNTD IIWGSNYSSP

SNISKFTWNN LAEPOKAPS H OOATTAGSPL RHILIGKNSVL NSSTAEHSSP YSEDPIEDPL

OPDWTGIRL SLGAGEFKSQ EHAKHKGPKW ERDOAAKHRF SWMKLLAHKW GRHLSODTGS

PSGMRPWED PSOD TGSPSR MRPWKDPPSD LLLLKOSNSS KILVGRWHLA SEKGSYEIIQ

DTDEDTAVNN WLISPONAS R AWGESTPLAN KPGKOSGHPK FPRVRHKSLO WRODGGKSRL

KKSOFLIKTR KKKK EKHTHH APLSPRTFHP LRSEAYNTFS ERRLKHSLWL HKSNETSLPT

DLNOTLPSM FGWIASLPD H NONSSNDTGO ASCPPGLYOT WPPEEHYOTF PIODPDOMHS

TSDPSHRSSS PELS EMLEYD RSHKSFPTDI SOMSPSSEHE WWOTVISPDL SOVTLSPELS

QTNLSPDLS TTLSPELIO R NLSPALGOMP ISPDLSHTTL SPDLSHTTLS LDLSOTNLSP

ELSOTNLSPA LGOM PLSPDL SHTTLSLDFS QTNLSPELSH MTLSPELSOT NLSPALGOMP

ISPDLSHTT SLDFSQTNLS PELSOTNLSP ALGOMPLSPD PSHTTLSLDL SOTNLSPELS

QTNLSPDLSE MPLFADLSOI PLTPDLDOMT LSPDLGETDL SPNFGOMSLS PDLSOVTLSP

DISDTTLLPD LSOISPPPDL DOIFYPSESS OSL.LLOEFNE SFPYPDLGOM PSPSSPTLND

TFLSKS KP WIWGLSKDGT DYIEIIPKEE WOSSEDDYAE IDYVPYDDPY KTDVRTNINS

US 2015/03539 11 A1 Dec. 10, 2015 21

TABLE 3 - continued Factor V Sequences

CAGAGCAGTT CAACCAGGGG AAACCTATAC TTATAAGTGG AACATCTTAG AGTTTGATGA

ACCCACAGAA AATGATGCCC AGTGCTTAAC AAGACCATAC TACAGTGACG, TGGACATCAT

GAGAGACATC GCCTCTGGGC TAATAGGACT ACTTCTAATC TGTAAGAGCA GATCCCTGGA

CAGGCGAGGA, ATACAGAGGG CAGCAGACAT CGAACAGCAG GCTGTGTTTG. CTGTGTTTGA

TGAGAACAAA AGCTGGTACC TTGAGGACAA CATCAACAAG TTTTGTGAAA ATCCTGATGA

GGTGAAACGT GATGACCCCA AGTTTTATGA, ATCAAACATC ATGAGCACTA TCAATGGCTA

TGTGCCTGAG. AGCATAACTA CTCTTGGATT CTGCTTTGAT GACACTGTCC AGTGGCACTT

CTGTAGTGTG. GGGACCCAGA ATGAAATTTT GACCATCCAC TTCACTGGGC ACTCATTCAT

CTATGGAAAG AGGCATGAGG ACACCTTGAC CCTCTTCCCC ATGCGTGGAG AATCTGTGAC

GGTCACAATG GATAATGTTG GAACTTGGAT GTTAACTTCC ATGAATTCTA GTCCAAGAAG

CAAAAAGCTG AGGCTGAAAT TCAGGGATGT TAAATGTATC CCAGATGATG ATGAAGACTC

ATATGAGATT TTTGAACCTC CAGAATCTAC AGTCATGGCT ACACGGAAAA TGCATGATCG

TTTAGAACCT GAAGATGAAG AGAGTGATGC TGACTATGAT TACCAAAGAC TGGCTGCGAC

AGCATTAGGA, ATCAGGTCAT TCCGAAACTC ATCATTGAAT CAGGAAGAAG AAGAGTTCAA

TCTTACTGCC CTAGCTCTGG AGAATGGCAC TGAATTCGTT TCTTCAAACA CAGATATAAT

TGTTGGTTCA AATTATTCTT CCCCAAGTAA TATTAGTAAG TTCACTGTCA, ATAACCTTGC

AGAACCTCAG AAAGCCCCTT CTCACCAACA AGCCACCACA GCTGGTTCCC CACTGAGACA

CCTCATTGGC AAGAACT CAG TTCTCAATTC TTCCACAGCA GAGCATTCCA GCCCATATTC

TGAAGACCCT ATAGAGGATC CTCTACAGCC AGATGTCACA. GGCATACGTC TACTTTCACT

TGGTGCTGGA GAATTCAAAA GTCAAGAACA TGCTAAGCAT AAGGGACCCA AGGTAGAAAG

AGATCAAGCA GCAAAGCACA. GGTTCTCCTG GATGAAATTA. CTAGCACATA AAGTTGGGAG

ACACCTAAGC CAAGACACTG GTTCTCCTTC CGGAATGAGG CCCTGGGAGG ACCTTCCTAG

CCAAGACACT GGTTCTCCTT CCAGAATGAG GCCCTGGAAG GACCCTCCTA GTGATCTGTT

ACTCTTAAAA CAAAGTAACT CATCTAAGAT TTTGGTTGGG AGATGGCATT TGGCTTCTGA

GAAAGGTAGC TATGAAATAA TCCAAGATAC TGATGAAGAC ACAGCTGTTA ACAATTGGCT

GATCAGCCCC CAGAATGCCT CACGTGCTTG. GGGAGAAAGC ACCOCTOTTG. CCAACAAGCC

TGGAAAGCAG. AGTGGCCACC CAAAGTTTCC TAGAGTTAGA CATAAATCTC TACAAGTAAG

ACAGGATGGA. GGAAAGAGTA. GACTGAAGAA. AAGCCAGTTT CTCATTAAGA CACGAAAAAA

GAAAAAAGAG. AAGCACACAC ACCATGCTCC TTTATCTCCC AGGACCTTTC ACCCTCTAAG

AAGTGAAGCC TACAACACAT TTTCAGAAAG AAGACTTAAG CATTCGTTGG. T.GCTTCATAA

ATCCAATGAA ACATCTCTTC CCACAGACCT CAATCAGACA TTGCCCTCTA TGGATTTTGG

CTGGATAGCC TCACTTCCTG ACCATAATCA GAATTCCTCA AATGACACTG GTCAGGCAAG

CTGTCCTCCA GGTCTTTATC AGACAGTGCC CCCAGAGGAA CACTATCAAA CATTCCCCAT

TCAAGACCCT GATCAAATGC ACTCTACTTC AGACCCCAGT CACAGATCCT. CTTCTCCAGA

GCTCAGTGAA. ATGCTTGAGT ATGACCGAAG TCACAAGTCC TTCCCCACAG ATATAAGTCA

AATGTCCCCT TCCTCAGAAC ATGAAGTCTG GCAGACAGTC ATCTCTCCAG ACCTCAGCCA

GGTGACCCTC. TCTCCAGAAC TCACCCAGAC AAACCTCTCT CCAGACCTCA GCCACACGAC

TCTCTCTCCA GAACTCATTC AGAGAAACCT TTCCCCAGCC CTCGGTCAGA TGCCCATTTC US 2015/03539 11 A1 Dec. 10, 2015 22

TABLE 3 - continued Factor V Sequences

TCCAGACCTC. AGCCATACAA CCCTTTCTCC AGACCTCAGC CATACAACCC TTTCTTTAGA

CCTCAGCCAG ACAAACCTCT CTCCAGAACT CAGTCAGACA AACCTTTCTC CAGCCCTCGG

TCAGATGCCC CTTTCTCCAG ACCTCAGCCA TACAACCCTT TCTCTAGACT TCAGCCAGAC

AAACCTCTCT CCAGAACTCA GCCATATGAC TCTCTCTCCA GAACTCAGTC AGACAAACCT

TTCCCCAGCC CTCGGTCAGA TGCCCATTTC TCCAGACCTC. ACCCATACAA CCCTTTCTCT

AGACTTCAGC CAGACAAACC TCTCTCCAGA ACTCAGTCAA ACAAACCTTT CCCCAGCCCT

CGGTCAGATG CCCCTTTCTC CAGACCCCAG CCATACAACC CTTTCTCTAG ACCTCAGCCA

GACAAACCTC. TCTCCAGAAC TCAGTCAGAC AAACCTTTCC CCAGACCTCA. GTGAGATGCC

CCTCTTTGCA GATCTCAGTC AAATTCCCCT TACCCCAGAC CTCGACCAGA TGACACTTTC

TCCAGACCTT GGTGAGACAG ATCTTTCCCC AAACTTTGGT CAGATGTCCC TTTCCCCAGA

CCTCAGCCAG GTGACTCTCT CTCCAGACAT CAGTGACACC ACCCTTCTCC CGGATCTCAG

CCAGATATCA CCTCCTCCAG ACCTTGATCA GATATTCTAC CCTTCTGAAT CTAGTCAGTC

ATTGCTTCTT CAAGAATTTA ATGAGTCTTT TCCTTATCCA GACCTTGGTC AGATGCCATC

TCCTTCATCT CCTACTCTCA ATGATACTTT TCTATCAAAG GAATTTAATC CACTGGTTAT

AGTGGGCCTC AGTAAAGATG GTACAGATTA. CATTGAGATC ATTCCAAAGG AAGAGGTCCA

GAGCAGTGAA GATGACTATG CTGAAATTGA TTATGTGCCC TATGATGACC CCTACAAAAC

TGATGTTAGG ACAAACATCA ACTCCTCCAG. AGATCCTGAC AACATTGCAG CATGGTACCT

CCGCAGCAAC AATGGAAACA GAAGAAATTA TTACATTGCT GCTGAAGAAA. TATCCTGGGA

TTATTCAGAA TTTGTACAAA. GGGAAACAGA TATTGAAGAC TCTGATGATA TTCCAGAAGA

TACCACATAT AAGAAAGTAG TTTTTCGAAA GTACCTCGAC AGCACTTTTA. CCAAACGTGA

TCCTCGAGGG GAGTATGAAG AGCATCTCGG AATTCTTGGT CCTATTATCA GAGCTGAAGT

GGATGATGTT ATCCAAGTTC GTTTTAAAAA TTTAGCATCC AGACCGTATT CTCTACATGC

CCATGGACTT TCCTATGAAA AATCATCAGA. GGGAAAGACT TATGAAGATG ACTCTCCTGA

ATGGTTTAAG GAAGATAATG CTGTTCAGCC AAATAGCAGT TATACCTACG TATGGCATGC

CACTGAGCGA TCAGGGCCAG PAAGTCCTGG CTCTGCCTGT CGGGCTTGGG CCTACTACTC

AGCTGTGAAC CCAGAAAAAG ATATTCACTC AGGCTTGATA GGTCCCCTCC TAATCTGCCA

AAAAGGAATA CTACATAAGG ACAGCAACAT GCCTATGGAC ATGAGAGAAT TTGTCTTACT

ATTTATGACC TTTGATGAAA AGAAGAGCTG GTACTATGAA. AAGAAGTCCC GAAGTTCTTG

GAGACT CACA TCCTCAGAAA TGAAAAAATC CCATGAGTTT CACGCCATTA ATGGGATGAT

CTACAGCTTG CCTGGCCTGA AAATGTATGA, GCAAGAGTGG CTCAGGTTAC ACCTGCTGAA

CATAGGCGGC. TCCCAAGACA TTCACGTGGT TCACTTTCAC GGCCAGACCT TGCTGGAAAA

TGGCAATAAA CAGCACCAGT TAGGGGTCTG GCCCCTTCTG CCTGGTTCAT, TTAAAACTCT

TGAAATGAAG CCATCAAAAC CTGGCTGGTG GCTCCTAAAC ACAGAGGTTG GAGAAAACCA.

GAGAGCAGGG ATGCAAACGC CATTTCTTAT CATGGACAGA GACTGTAGGA TGCCAATGGG

ACTAAGCACT GGTATCATAT, CTGATTCACA GATCAAGGCT TCAGAGTTTC TGGGTTACTG

GGAGCCCAGA TTAGCAAGAT TAAACAATGG TGGATCTTAT AATGCTTGGA GTGTAGAAAA

ACTTGCAGCA GAATTTGCCT CTAAACCTTG GATCCAGGTG GACATGCAAA AGGAAGTCAT US 2015/03539 11 A1 Dec. 10, 2015

TABLE 3 - continued Factor V Sequences

AATCACAGGG ATCCAGACCC AAGGTGCCAA ACACTACCTG AAGTCCTGCT ATACCACAGA

GTTCTATGTA GCTTACAGTT CCAACCAGAT CAACTGGCAG ATCTTCAAAG GGAACAGCAC

AAGGAATGTG ATGTATTTTA ATGGCAATTC AGATGCCTCT ACAATAAAAG AGAATCAGTT

TGACCCACCT ATTGTGGCTA GATATATTAG GATCTCTCCA ACTCGAGCCT ATAACAGACC

TACCCTTCGA TTGGAACTGC AAGGTTGTGA. GGTAAATGGA TGTTCCACAC CCCTGGGTAT

GGAAAATGGA. AAGATAGAAA ACAAGCAAAT CACAGCTTCT TCGTTTAAGA AATCTTGGTG

GGGAGATTAC TGGGAACCCT TCCGTGCCCG TCTGAATGCC CAGGGACGTG TGAATGCCTG

GCAAGCCAAG GCAAACAACAATAAGCAGTG GCTAGAAATT GATCTACTCA AGATCAAGAA

GATAACGGCA ATTATAACAC AGGGCTGCAA GTCTCTGTCC TCTGAAATGT ATGTAAAGAG

CTATACCATC CACTACAGTG. AGCAGGGAGT GGAATGGAAA CCATACAGGC TGAAATCCTC

CATGGTGGAC AAGATTTTTG AAGGAAATAC TAATACCAAA. GGACATGTGA AGAACTTTTT

CAACCCCCCA ATCATTTCCA GGTTTATCCG TGTCATTCCT. AAAACATGGA ATCAAAGTAT

TGCACTTCGC CTGGAACTCT TTGGCTGTGA TATTTACTAG

0200. The FVaprotein used for the present invention com form a loop. In one example, the side chains of C and Care prises a heterodimer comprising a heavy chain and a light covalently linked (e.g., via a disulfide bond or an amide chain, wherein the heavy chain comprises a first amino acid bond). sequence, which is at least 60%, 70%, 75%, 80%, 85%, 90%, 0207. In Formula II, one, two or three additional amino 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids acids can be inserted anywhere between C and C. In one 29 to 737of SEQID NO: 17 and the light chain comprises a example according to any of the above embodiments, one or second amino acid sequence, which is at least 60%, 70%, two additional amino acids are optionally inserted into For 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% mula (I) anywhere between C and C. In another example, identical to amino acids 1574 to 2224 of SEQ ID NO: 18, one amino acid is optionally inserted into Formula II any wherein the first amino acid sequence and the second amino where between C and C. In another example, no amino acid acid sequence when formed the heterodimer is capable of is inserted between C and C. forming a complex with FX or FXa. The FVa protein as used 0208. In Formula II, L is L-leucine, A is L-alanine, S is herein includes full-length FVa, mature FVa, functional frag L-serine, and Y is L-tyrosine. In Formula II, one, two or three ments, variants, analogues, or derivatives thereof. of L, A, S, and Y are optionally replaced with an indepen 0201 2. Procoagulant Peptides dently selected replacementamino acid. In one example, one 0202 In other embodiments, the enhancer moiety is a or two of L, A, S, and Y are optionally replaced with an procoagulant peptide. A procoagulant peptide' is a low independently selected replacement amino acid. In another molecular weight compound (e.g., peptides or peptide deriva example, exactly one of L, A, S, and Y is optionally replaced tives) with pro-coagulant activity that can be used for the with an independently selected replacement amino acid. treatment of bleeding diathesis (e.g., blood coagulation dis 0209. In another embodiment, the enhancer moiety in a orders/coagulopathies, such as hemophilia A) or for the treat chimeric protein comprises a compound that contains a pep ment of deficiencies in at least one of FV, FVII, FVIII, FIX, tide of Formula III: FX, FXI, and VWF. In some embodiments, when a procoagul lant peptide is used as an enhancer moiety, it is capable of increasing the catalytic activity of the clotting factor to which (Formula III) it is fused. O RI O R3 O 0203. In one embodiment, the procoagulant peptide com H H H N N N prises a compound that includes: N N NH 0204 (a) an amino acid sequence including Formula II: O R2 O R4 CLASYC2 (Formula II) O 0205 or (b) a retro-, an inverso- or a retro-inverso variant N L2 of the amino acid sequence of (a). The present disclosure u-1 further provides pharmaceutically acceptable salts of the above compound. or a retro-, an inverso- or a retro-inverso variant thereof. 0206. In Formula II, C' and Care amino acids having a 0210. In Formula III, R', R, R and R are members side chain, wherein the side chains of C and C are linked to independently selected from amino acid side chains. In For US 2015/03539 11 A1 Dec. 10, 2015 24 mula III, L' and L are linker groups independently selected chemical methods that are well known in the art. Exemplary from Straight or branched alkylene, and straight or branched antigen-binding portions include Fv, Fab, Fab', and (Fab')2 as heteroalkylene. well as scFv molecules. 0211. In Formula III, Z is a linking moiety. In one 0217. In other embodiments, a chimeric clotting factor of example, Z is selected from an amino group, an amide group, the invention may comprise an enhancer moiety comprising a a disulfide group, a diselenide group, a —S Se—group, binding site from single chain binding molecule (e.g., a single alkylene, e.g., (C-C)alkylene, alkenyl, e.g., (C-C)alkenyl, chain variable region or schv). Techniques described for the alkynyl, e.g., (C-C)alkynyl, cycloalkyl (e.g., (C-Cs)cy production of single chain antibodies (U.S. Pat. No. 4,694, cloalkyl containing from 1 to 4 double bonds), heterocy 778; Bird, Science 242:423-442 (1988); Huston et al., Proc. cloalkyl (e.g., 3- to 8-membered heterocyclic ring comprising Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., from 1 to 6 heteroatoms selected from O, S and N), aryl (e.g., Nature 334:544-554 (1989)) can be adapted to produce single (C-C)aryl), and heteroaryl (e.g., 3- to 8-membered het chain binding molecules. Single chain antibodies are formed eroaryl comprising from 1 to 6 heteroatoms selected from O, by linking the heavy and light chain fragments of the FV region via an amino acid bridge, resulting in a single chain S and N). antibody. Techniques for the assembly of functional Fv frag 0212 Exemplary synthetic procoagulant peptides include, ments in E coli may also be used (Skerra et al., Science for example: 242:1038-1041 (1988)). 0218. In certain embodiments, a chimeric clotting factor (SEQ ID NO: 19) of the invention may comprise an enhancer moiety compris KLTCLASYCWLF; ing one or more binding sites or regions comprising or con sisting of a single chain variable region sequence (ScPV). (SEQ ID NO: 2O) Single chain variable region sequences comprise a single RRAPGKLTCLASYCWLFWTGIA; polypeptide having one or more antigen binding sites, e.g., a (SEQ ID NO: 21) VL domain linked by a flexible linker to a VH domain. Schv RRAPGKLQCLASYCWLFWTGIA; molecules can be constructed in a VH-linker-VL orientation or VL-linker-VHorientation. The flexible linker that links the (SEQ ID NO: 22) VL and VH domains that make up the antigen binding site PRIRTWGPGSRSASGKLTCLASYCWLFWTGIA; preferably comprises from about 10 to about 50 amino acid (SEQ ID NO; 23) residues. In one embodiment, the peptide linker is a gly-ser SKOGRPISPDRRAAGKLTCLASYCWLFWTGIA; peptide linker. An exemplary gly/ser peptide linker is of the (SEQ ID NO: 24) formula (Gly4Ser)n, whereinn is a positive integer (e.g., 1, 2, PRIRTWGPGSRSASGKST CLASYCWLFWTGIA; 3, 4, 5, or 6). Other peptide linkers are known in the art. Antibodies having single chain variable region sequences (SEQ ID NO: 25) (e.g. single chain Fv antibodies) and methods of making said SRIRTVSPGSRSASGKST CLASYCWLFWTGIA; single chain antibodies are well-known in the art (see e.g., Ho o et al. 1989. Gene 77:51; Bird et al. 1988 Science 242:423; (SEQ ID NO: 26) Pantoliano et al. 1991. Biochemistry 30:101 17: Milenicetal. PRSRTWGPGSRSASGKSTCLASYCWLFWTGIA. 1991. Cancer Research 51:6363; Takkinen et al. 1991. Pro tein Engineering 4:837). 0213 Exemplary procoagulant peptides are additionally 0219. In certain embodiments, a scFv molecule employed disclosed in U.S. 61/495,818, U.S. 61/600,237, U.S. 61/605, in a chimeric clotting factor of the invention is a stabilized 540, U.S. 61/496,540, U.S. 61/496,543, U.S. 61/496,544, scFv molecule. In one embodiment, the stabilized cFv mol U.S. 61/496,541, and U.S. 61/496,542, each of which is ecule may comprise a schv linker interposed between a VH incorporated herein by reference in its entirety. domain and a VL domain, wherein the VH and VL domains 0214) 3. Antibodies or Antigen Binding Sites are linked by a disulfide bond between an amino acid in the VH and an amino acid in the VL domain. In other embodi 0215. In other embodiments, the enhancer moiety com ments, the stabilized schv molecule may comprise a schv prises at least one antigen binding moiety (e.g., an antigen linker having an optimized length or composition. In yet other binding site of an antibody, antibody variant, or antibody embodiments, the Stabilized scEv molecule may comprise a fragment), a receptor binding portion of , or a ligand VH or VL domain having at least one stabilizing amino acid binding portion of a receptor. Exemplary antigen binding substitution(s). In yet another embodiment, a stabilized scFV molecules that can be used as enhancer moieties are disclosed molecule may have at least two of the above listed stabilizing in Andersen LMetal., J Biol Chem. 287: 8994-9001 (January features. 2012), incorporated herein by reference in its entirety, which 0220 Stabilized scFv molecules have improved protein discloses FVII activating antibodies and antibody derivatives stability or impart improved protein stability to the polypep used for increasing the procoagulantactivity of FVIIa and for tide to which it is operably linked. Preferred schv linkers of treating blood coagulation disorders such as hemophilia A the invention improve the thermal stability of a chimeric and hemorrhagic diathesis. clotting factor of the invention by at least about 2°C. or 3° C. 0216. The term “antigen-binding moiety” refers to a as compared to a conventional polypeptide. Comparisons can polypeptide fragment of an immunoglobulin, antibody, or be made, for example, between the schv molecules of the antibody variant which binds antigen or competes with intact invention. In certain embodiments, the stabilized scFv mol antibody (i.e., with the intact antibody from which they were ecule comprises a (Gly4Ser)4 schv linker and a disulfidebond derived) for antigen binding (i.e., specific binding). Antigen which links VHamino acid 44 and VL amino acid 100. Other binding portions can be produced by recombinant or bio exemplary stabilized scFv molecules which may be US 2015/03539 11 A1 Dec. 10, 2015

employed in the chimeric clotting factor of the invention are 1986)). It will further be appreciated that the monoclonal described in U.S. Provisional Patent Application No. 60/873, antibodies secreted by the subclones may be separated from 996, filed on Dec. 8, 2006 or U.S. patent application Ser. No. culture medium, ascites fluid or serum by conventional puri 11/725,970, filed on Mar. 19, 2007, each of which is incor fication procedures such as, for example, affinity chromatog porated herein by reference in its entirety. raphy (e.g., protein-A, protein-G, or protein-Laffinity chro 0221 Chimeric clotting factor of the invention may com matography), hydroxylapatite chromatography, gel prise a variable region orportion thereof (e.g. a VL and/or VH electrophoresis, or dialysis. domain) derived from an antibody using art recognized pro 0224 DNA encoding the desired monoclonal antibody or tocols. For example, the variable domain may be derived from binding site thereof may be readily isolated and sequenced antibody produced in a non-human mammal, e.g., murine, using any of the conventional procedures described Supra for guinea pig, primate, rabbit or rat, by immunizing the mammal the isolation of constant region domain sequences (e.g., by with the antigen or a fragment thereof. See Harlow & Lane, using oligonucleotide probes that are capable of binding spe Supra, incorporated by reference for all purposes. The immu cifically to genes encoding the heavy and light chains of noglobulin may be generated by multiple Subcutaneous or murine antibodies). The isolated and subcloned hybridoma infraperitoneal injections of the relevant antigen (e.g., puri cells serve as a preferred source of such DNA. More particu fied tumor associated antigens or cells or cellular extracts larly, the isolated DNA (which may be synthetic as described comprising Such antigens) and an adjuvant. This immuniza herein) may be used to clone the desired variable region tion typically elicits an immune response that comprises pro sequences for incorporation in the chimeric clotting factor of duction of antigen-reactive antibodies from activated spleno the invention. cytes or lymphocytes. 0225. In other embodiments, the binding site is derived 0222. While the variable region may be derived from poly from a fully human antibody. Human or Substantially human clonal antibodies harvested from the serum of an immunized antibodies may be generated in transgenic animals (e.g., mammal, it is often desirable to isolate individual lympho mice) that are incapable of endogenous immunoglobulin pro cytes from the spleen, lymph nodes or peripheral blood to duction (see e.g., U.S. Pat. Nos. 6,075,181, 5,939,598, 5,591, provide homogenous preparations of monoclonal antibodies 669 and 5,589,369, each of which is incorporated herein by (MAbs) from which the desired variable region is derived. reference). For example, it has been described that the Rabbits or guinea pigs are typically used for making poly homozygous deletion of the antibody heavy-chain joining clonal antibodies. Mice are typically used for making mono region in chimeric and germ-line mutant mice results in com clonal antibodies. Monoclonal antibodies can be prepared plete inhibition of endogenous antibody production. Transfer against a fragment by injecting an antigen fragment into a of a human immunoglobulin gene array to Such germ line mouse, preparing "hybridomas' and screening the hybrido mutant mice will result in the production of human antibodies mas for an antibody that specifically binds to the antigen. In upon antigen challenge. Another preferred means of generat this well-known process (Kohler et al., (1975), Nature, 256: ing human antibodies using SCID mice is disclosed in U.S. 495) the relatively short-lived, or mortal, lymphocytes from Pat. No 5,811,524 which is incorporated herein by reference. the mouse which has been injected with the antigen are fused It will be appreciated that the genetic material associated with with an immortal tumor cell line (e.g. a myeloma cell line), these human antibodies may also be isolated and manipulated thus, producing hybrid cells or “hybridomas’ which are both as described herein. immortal and capable of producing the antibody genetically 0226. In other aspects, the polypeptides of the invention encoded by the B cell. The resulting hybrids are segregated may comprise antigen binding sites, or portions thereof, into single genetic strains by selection, dilution, and regrowth derived from modified forms of antibodies. Exemplary such with each individual strain comprising specific genes for the forms include, e.g., minibodies, diabodies, triabodies, nano formation of a single antibody. They produce antibodies bodies, camelids, Dabs, tetravalent antibodies, intradiabodies which are homogeneous against a desired antigen and, in (e.g., Jendreyko et al. 2003. J. Biol. Chem. 278:47813), reference to their pure genetic parentage, are termed "mono fusion proteins (e.g., antibody cytokine fusion proteins, pro clonal'. teins fused to at least a portion of an Fc receptor), and bispe 0223 Hybridoma cells thus prepared are seeded and cific antibodies. Other modified antibodies are described, for grown in a Suitable culture medium that preferably contains example in U.S. Pat. No. 4,745,055; EP256,654; Faulkner et one or more substances that inhibit the growth or survival of al., Nature 298:286 (1982); EP 120,694; EP 125,023; Morri the unfused, parental myeloma cells. Those skilled in the art son, J. Immun. 123:793 (1979); Kohler et al., Proc. Natl. will appreciate that reagents, cell lines and media for the Acad. Sci. USA 77:2197 (1980); Raso et al., Cancer Res. formation, selection and growth of hybridomas are commer 41:2073 (1981); Morrison et al., Ann. Rev. Immunol. 2:239 cially available from a number of sources and standardized (1984); Morrison, Science 229:1202 (1985); Morrison et al., protocols are well established. Generally, culture medium in Proc. Natl. Acad. Sci. USA 81:6851 (1984); EP 255,694; EP which the hybridoma cells are growing is assayed for produc 266,663; and WO 88/03559. Reassorted immunoglobulin tion of monoclonal antibodies against the desired antigen. chains also are known. See, for example, U.S. Pat. No 4,444, Preferably, the binding specificity of the monoclonal antibod 878; WO 88/03565; and EP 68,763 and references cited ies produced by hybridoma cells is determined by immuno therein. precipitation or by an in vitro assay, Such as a radioimmu 0227. In another embodiment, a chimeric clotting factor of noassay (RIA) or enzyme-linked immunosorbent assay the invention comprises an antigen binding site or region (ELISA). After hybridoma cells are identified that produce which is a diabody or an antigen binding site derived there antibodies of the desired specificity, affinity and/or activity, from. Diabodies are dimeric, tetravalent molecules each hav the clones may be subcloned by limiting dilution procedures ing a polypeptide similar to Schv molecules, but usually hav and grown by standard methods (Goding, Monoclonal Anti ing a short (e.g., less than 10 and preferably 1-5) amino acid bodies: Principles and Practice, pp 59-103 (Academic Press, residue linker connecting both variable domains, such that the US 2015/03539 11 A1 Dec. 10, 2015 26

VL and VH domains on the same polypeptide chain cannot 0231. A heterologous moiety can comprise a heterologous interact. Instead, the VL and VH domain of one polypeptide polypeptide moiety, or a heterologous non-polypeptide moi chain interact with the VHand VL domain (respectively) on a ety, or both. In one specific embodiment, Het1 is a first het second polypeptide chain (see, for example, WO 02/02781). erologous moiety, e.g., a half-life extending molecule which In one embodiment, achimeric clotting factor of the invention is known in the art. In some embodiments, Het2 is a second comprises a diabody which is operably linked to the N-ter heterologous moiety that can also be a half-life extending minus and/or C-terminus of at least one genetically-fused Fc molecule which is known in the art. In some aspects, the region (i.e., ScFc region). heterologous moiety comprises a combination of a heterolo 0228. In certain embodiments, a chimeric clotting factor gous polypeptide and a non-polypeptide moiety. of the invention comprises a single domain binding molecule 0232. In certain embodiments, the first heterologous moi (e.g. a single domain antibody) as an enhancer moiety. Exem ety (e.g., a first Fc moiety) and the second heterologous plary single domain molecules include an isolated heavy moiety (e.g., a second Fc moiety) are associated with each chain variable domain (VH) of an antibody, i.e., a heavy chain other to form a dimer. In one embodiment, the second heter variable domain, without a light chain variable domain, and ologous moiety is a second Fc moiety, wherein the second Fc an isolated light chain variable domain (VL) of an antibody, moiety is linked to or associated with the first heterologous i.e., a light chain variable domain, without a heavy chain moiety, e.g., the first Fc moiety. For example, the second variable domain. Exemplary single-domain antibodies heterologous moiety (e.g., the second Fc moiety) can be employed in the binding molecules of the invention include, linked to the first heterologous moiety (e.g., the first Fc moi for example, the Camelid heavy chain variable domain (about ety) by a linker or associated with the first heterologous 118 to 136 amino acid residues) as described in Harpers moiety by a covalent or non-covalent bond Casterman, et al., Nature 363:446-448 (1993), and Dumou 0233. In some embodiments, the Het1 and Het2 heterolo lin, et al., Protein Science 11:500-515 (2002). Other exem gous moieties are peptides and polypeptides with either plary single domain antibodies include single VH or VL unstructured or structured characteristics that are associated domains, also known as Dabs(R (Domantis Ltd., Cambridge, with the prolongation of in vivo half-life when incorporated UK). Yet other single domain antibodies include shark anti in a chimeric protein of the invention. Non-limiting examples bodies (e.g., shark Ig-NARs). Shark Ig-NARs comprise a include albumin, albumin fragments, Fc fragments of immu homodimer of one variable domain (V-NAR)and five C-like noglobulins, the B subunit of the C-terminal peptide (CTP) of constant domains (C-NAR), wherein diversity is concen human chorionic gonadotropin, a HAP sequence, an XTEN trated in an elongated CDR3 region varying from 5 to 23 sequence, a transferrin or a fragment thereof, a PAS polypep residues in length. In camelid species (e.g., llamas), the heavy tide, polyglycine linkers, polyserine linkers, albumin-binding chain variable region, referred to as VHH, forms the entire moieties, or any fragments, derivatives, variants, or combina antigen-binding domain. The main differences between cam tions of these polypeptides. In other related aspects a heter elid VHH variable regions and those derived from conven ologous moiety can include an attachment site (e.g., a cys tional antibodies (VH) include (a) more hydrophobic amino teine amino acid) for a non-polypeptide moiety Such as acids in the light chain contact Surface of VHas compared to polyethylene glycol (PEG), hydroxyethyl starch (HES), poly the corresponding legion in VHH, (b) alonger CDR3 in VHH, sialic acid, or any derivatives, variants, or combinations of and (c) the frequent occurrence of a disulfide bond between these elements. In some aspects, a heterologous moiety con CDR1 and CDR3 in VHH. Methods for making single sisting of a cysteine amino acid that function as an attachment domain binding molecules are described in U.S. Pat. Nos. site for a non-polypeptide moiety Such as polyethylene glycol 6,005,079 and 6,765,087, both of which are incorporated (PEG), hydroxyethyl starch (HES), polysialic acid, or any herein by reference. Exemplary single domain antibodies derivatives, variants, or combinations of these elements. comprising VHH domains include Nanobodies(R (Ablynx 0234. In some embodiments, the heterologous moiety is a Nev., Ghent, Belgium). polypeptide comprising, consisting essentially of, or consist 0229 C. Heterologous Moieties (e.g., Het1, Het2, . . . . ing of at least about 10, 100, 200, 300, 400, 500, 600, 700, Het) 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 0230. Some embodiments of the invention comprise one 1800, 1900, 2000, 2500, 3000, or 4000 amino acids. In other or more heterologous moieties (indicated herein as “Het1 or embodiments, the heterologous moiety is a polypeptide com “Het2). In other embodiments, the chimeric protein of the prising, consisting essentially of, or consisting of about 100 to invention can comprise two heterologous moieties (“Het1' about 200 amino acids, about 200 to about 300 amino acids, and “Het2). In yet other embodiments, the chimeric protein about 300 to about 400 amino acids, about 400 to about 500 of the invention can comprise more than two heterologous amino acids, about 500 to about 600 amino acids, about 600 moieties, e.g., three, four, five, or more than five heterologous to about 700 amino acids, about 700 to about 800 amino acids, moieties. In some embodiments, all the heterologous moi about 800 to about 900 amino acids, or about 900 to about eties are identical. In some embodiments, at least one heter 1000 amino acids. ologous moiety is different from the other heterologous moi 0235. In certain embodiments, a heterologous moiety eties. In some embodiments, the chimeric protein of the improves one or more pharmacokinetic properties of the chi invention can comprise two, three or more than three heter meric protein without significantly affecting the biological ologous moieties in tandem. In other embodiments, the chi activity or function of the activatable clotting factor and/or the meric protein of the invention can comprise two, three, or enhancer moiety (e.g., procoagulant activity of a clotting more than heterologous moieties wherein at least an addi factor or a fragment thereof, or of activity enhancing property tional moiety (e.g., an activatable clotting factor, a linker of an enhancer moiety). moiety, a protease-cleavage site, a self-immolative moiety, an 0236. In certain embodiments, a heterologous moiety enhancer moiety, or combinations thereof) is interposed increases the in vivo and/or in vitro half-life of the clotting between two heterologous moieties. factor of the invention. In other embodiments, a heterologous US 2015/03539 11 A1 Dec. 10, 2015 27 moiety facilitates visualization or localization of the clotting another embodiment, the half-life of chimeric protein com factor of the invention or a fragment thereof (e.g., a fragment prising a half-life extender is extended about 2-fold to about comprising a heterologous moiety after proteolytic cleavage 10-fold, about 2-fold to about 9-fold, about 2-fold to about of the activatable clotting factor). Visualization and/or loca 8-fold, about 2-fold to about 7-fold, about 2-fold to about tion of the chimeric protein of the invention or a fragment 6-fold, about 2-fold to about 5-fold, about 2-fold to about thereof can be in vivo, in vitro, ex vivo, or combinations 4-fold, about 2-fold to about 3-fold, about 2.5-fold to about thereof. 10-fold, about 2.5-fold to about 9-fold, about 2.5-fold to 0237. In other embodiments, a heterologous moiety about 8-fold, about 2.5-fold to about 7-fold, about 2.5-fold to increases stability of the chimeric protein of the invention or about 6-fold, about 2.5-fold to about 5-fold, about 2.5-fold to a fragment thereof (e.g., a fragment comprising a heterolo about 4-fold, about 2.5-fold to about 3-fold, about 3-fold to gous moiety after proteolytic cleavage of the activatable clot about 10-fold, about 3-fold to about 9-fold, about 3-fold to ting factor). As used herein, the term “stability” refers to an about 8-fold, about 3-fold to about 7-fold, about 3-fold to art-recognized measure of the maintenance of one or more about 6-fold, about 3-fold to about 5-fold, about 3-fold to physical properties of the activatable clotting factor in about 4-fold, about 4-fold to about 6 fold, about 5-fold to response to an environmental condition (e.g., an elevated or about 7-fold, or about 6-fold to about 8 fold as compared to lowered temperature). In certain aspects, the physical prop the in vivo half-life of the corresponding protein lacking Such erty can be the maintenance of the covalent structure of the half-life extender. chimeric protein (e.g., the absence of proteolytic cleavage, 0241. In other embodiments, the half-life of the chimeric unwanted oxidation or deamidation). In other aspects, the protein comprising a half-life extender is at least about 17 physical property can also be the presence of the chimeric hours, at least about 18 hours, at least about 19 hours, at least protein in a properly folded State (e.g., the absence of soluble about 20 hours, at least about 21 hours, at least about 22 hours, or insoluble aggregates or precipitates). In one aspect, the at least about 23 hours, at least about 24 hours, at least about stability of the chimeric protein is measured by assaying a 25 hours, at least about 26 hours, at least about 27 hours, at biophysical property of the chimeric protein, for example least about 28 hours, at least about 29 hours, at least about 30 thermal stability, pH unfolding profile, stable removal of gly hours, at least about 31 hours, at least about 32 hours, at least cosylation, solubility, biochemical function (e.g., ability to about 33 hours, at least about 34 hours, at least about 35 hours, bind to a protein, receptor or ligand), etc., and/or combina at least about 36 hours, at least about 48 hours, at least about tions thereof. In another aspect, biochemical function is dem 60 hours, at least about 72 hours, at least about 84 hours, at onstrated by the binding affinity of the interaction. In one least about 96 hours, or at least about 108 hours. aspect, a measure of protein stability is thermal stability, i.e., 0242. In still other embodiments, the half-life of the chi resistance to thermal challenge. Stability can be measured meric protein comprising a half-life extender is about 15 using methods known in the art, Such as, HPLC (high perfor hours to about two weeks, about 16 hours to about one week, mance liquid chromatography), SEC (size exclusion chroma about 17 hours to about one week, about 18 hours to about one tography), DLS (dynamic light scatering), etc. Methods to week, about 19 hours to about one week, about 20 hours to measure thermal stability include, but are not limited to dif about one week about 21 hours to about one week, about 22 ferential scanning calorimetry (DSC), differential scanning hours to about one week, about 23 hours to about one week, fluorimetry (DSF), circular dichroism (CD), and thermal about 24 hours to about one week, about 36 hours to about one challenge assay. week, about 48 hours to about one week, about 60 hours to 0238. In certain aspects, a chimeric protein of the inven about one week, about 24 hours to about six days, about 24 tion comprises at least one half-like extender, i.e., a heterolo hours to about five days, about 24 hours to about four days, gous moiety which increases the in vivo half-life of the chi about 24 hours to about three days, or about 24 hours to about meric protein with respect to the in vivo half-life of the two days. corresponding chimeric protein lacking such heterologous 0243 In some embodiments, the average half-life per sub moiety. In vivo half-life of a chimeric protein can be deter ject of the chimeric protein comprising a half-life extender is mined by any method known to those of skill in the, art, e.g., about 15 hours, about 16 hours, about 17 hours, about 18 activity assays (chromogenic assay or one stage clotting hours, about 19 hours, about 20 hours, about 21 hours, about aPTT assay), ELISA, etc. 22 hours, about 23 hours, about 24 hours (1 day), about 25 0239. In some embodiments, the presence of one or more hours, about 26 hours, about 27 hours, about 28 hours, about half-life extenders results in the half-life of the chimeric 29 hours, about 30 hours, about 31 hours, about 32 hours, protein to be increased compared to the half-life of the cor about 33 hours, about 34 hours, about 35 hours, about 36 responding protein lacking Such one or more half-life extend hours, about 40 hours, about 44 hours, about 48 hours (2 ers. The half-life of the chimeric protein comprising a half days), about 54 hours, about 60 hours, about 72 hours (3 life extender is at least about 1.5 times, at least about 2 times, days), about 84 hours, about 96 hours (4 days), about 108 at least about 2.5 times, at least about 3 times, at least about 4 hours, about 120 hours (5 days), about six days, about seven times, at least about 5 times, at least about 6 times, at least days (one week), about eight days, about nine days, about 10 about 7 times, at least about 8 times, at least about 9 times, at days, about 11 days, about 12 days, about 13 days, or about 14 least about 10 times, at least about 11 times, or at least about days. 12 times longer than the in vivo half-life of the corresponding 0244 1. An immunoglobulin Constant Region or a Portion chimeric protein lacking such half-life extender. Thereof 0240. In one embodiment, the half-life of the chimeric 0245. In another aspect, a heterologous moiety comprises protein comprising a half-life extender is about 1.5-fold to one or more immunoglobulin constant region or a portion about 20-fold, about 1.5 fold to about 15 fold, or about 1.5 thereof (e.g., an Fc moiety). In one embodiment, a chimeric fold to about 10 fold longer than the in vivo half-life of the protein comprises an activatable clotting factor, an enhancer corresponding protein lacking such half-life extender. In moiety, and at least two heterologous moieties, a first heter US 2015/03539 11 A1 Dec. 10, 2015 28 ologous moiety comprises a first immunoglobulin constant specific primers based on the published heavy and light chain region or a portion thereof (e.g., a first Fc moiety), which is DNA and amino acid sequences. As discussed above, PCR linked to the activatable clotting factor and a second heter also may be used to isolate DNA clones encoding the anti ologous moiety comprises a second immunoglobulin con body light and heavy chains. In this case the libraries may be stant region or a portion thereof (e.g., a second Fc moiety), screened by consensus primers or larger homologous probes, which is linked to the enhancer moiety. The first immunoglo Such as mouse constant region probes. Numerous primer sets bulin constant region or a portion thereof and the second Suitable for amplification of antibody genes are known in the immunoglobulin constant region or a portion thereof can art (e.g., 5' primers based on the N-terminal sequence of form a covalent bond (e.g., a disulfide bond), thereby placing purified antibodies (Benhar and Pastan. 1994. Protein Engi the activatable clotting factor and the enhancer moiety close neering 7:1509); rapid amplification of cDNA ends (Ruberti, in proximity to allow interaction between the activated clot F. etal. 1994. J. Immunol. Methods 173:33); antibody leader ting factor and the enhancer moiety at the site of injury. sequences (Larricket al. 1989 Biochem. Biophys. Res. Com 0246. An immunoglobulin constant region is comprised of mun. 160:1250). The cloning of antibody sequences is further domains denoted CH (constant heavy) domains (CH1, CH2, described in Newman et al., U.S. Pat. No. 5,658,570, filed etc.). Depending on the isotype, (i.e. IgG, IgM, IgA IgD, or Jan. 25, 1995, which is incorporated by reference herein. IgE), the constant region can be comprised of three or four CH 0250) An immunoglobulin constant region used herein domains. Some isotypes (e.g. IgG) constant regions also con can include all domains and the hinge region or portions tain a hinge region. See Janeway et al. 2001, immunobiology, thereof. In one embodiment, the immunoglobulin constant Garland Publishing, N.Y., N.Y. region or a portion thereof comprises CH2 domain, CH3 0247 An immunoglobulin constant region or a portion domain, and a hinge region, i.e., an Fc domain or an FcRn thereof for producing the chimeric protein of the present binding partner. invention may be obtained from a number of different Sources. In one embodiment, an immunoglobulin constant 0251 An immunoglobulin constant region or a portion region or a portion thereof is derived from a human immuno thereof can bean FcRn binding partner. FcRn is active in adult globulin. It is understood, however, that the immunoglobulin epithelial tissues and expressed in the lumen of the intestines, constant region or a portion thereof may be derived from an pulmonary airways, nasal Surfaces, vaginal Surfaces, colon immunoglobulin of another mammalian species, including and rectal surfaces (U.S. Pat. No 6,485,726). An FcRn bind for example, a rodent (e.g. a mouse, rat, rabbit, guinea pig) or ing partner is a portion of an immunoglobulin that binds to non-human primate (e.g. chimpanzee, macaque) species. FcRn. Moreover, the immunoglobulin constant region or a portion 0252. The FcRn receptor has been isolated from several thereof may be derived from any immunoglobulin class, mammalian species including humans. The sequences of the including IgM, IgG, Ig|D, IgA and IgE, and any immunoglo human FcRn, monkey FcRn, rat FcRn, and mouse FcRn are bulin isotype, including IgG1, IgG2, IgG3 and IgG4. In one known (Story et al. 1994, J. Exp. Med. 180:2377). The FcRn embodiment, the human isotype IgG1 is used. receptor binds IgG (but not other immunoglobulin classes 0248. A variety of the immunoglobulin constant region Such as IgA, IgM, Ig), and IgE) at relatively low pH, actively gene sequences (e.g. human constant region gene sequences) transports the IgG transcellularly in aluminal to serosal direc are available in the form of publicly accessible deposits. tion, and then releases the IgG at relatively higher pH found in Constant region domains sequence can be selected having a the interstitial fluids. It is expressed in adult epithelial tissue particular effector function (or lacking a particular effector (U.S. Pat. Nos. 6,485,726, 6,030,613, 6,086,875; WO function) or with a particular modification to reduce immu 03/077834; US2003-0235536A1) including lung and intesti nogenicity. Many sequences of antibodies and antibody-en nal epithelium (Israel et al. 1997, Immunology 92:69) renal coding genes have been published and Suitable Ig constant proximal tubular epithelium (Kobayashi et al. 2002, Am. J. region, sequences (e.g. hinge, CH2, and/or CH3 sequences, Physiol. Renal Physiol. 282: F358) as well as nasal epithe or portions thereof) can be derived from these sequences lium, vaginal Surfaces, and biliary tree Surfaces. using art recognized techniques. The genetic material 0253 FcRn binding partners useful in the present inven obtained using any of the foregoing methods may then be tion encompass molecules that can be specifically bound by altered or synthesized to obtain polypeptides of the present the FcRn receptor including whole IgG, the Fc fragment of invention. It will further be appreciated that the scope of this IgG, and other fragments that include the complete binding invention encompasses alleles, variants and mutations of con region of the FcRn receptor. The region of the Fc portion of stant region DNA sequences. IgG that binds to the FcRn receptor has been described based 0249. The sequences of the immunoglobulin constant on X-ray crystallography (Burmeister et al. 1994, Nature region or a portion thereof can be cloned, e.g., using the 372:379). The major contact area of the Fc with the FcRn is polymerase chain reaction and primers which are selected to near the junction of the CH2 and CH3 domains. Fc-FcRn amplify the domain of interest. To clone a sequence of the contacts are all within a single Ig heavy chain. The FcRn immunoglobulin constant region or a portion thereof from an binding partners include whole IgG, the Fc fragment of IgG, antibody, mRNA can be isolated from hybridoma, spleen, or and other fragments of IgG that include the complete binding lymph cells, reverse transcribed into DNA, and antibody region of FcRn. The major contact sites include amino acid genes amplified by PCR. PCR amplification methods are residues 248,250-257 272,285,288,290-291, 308-311, and described in detail in U.S. Pat. Nos. 4,683, 195; 4,683,202; 314 of the CH2 domain and amino acid residues 385-387, 4,800,159: 4,965, 188; and in, e.g. “PCR Protocols: A Guide 428, and 433-436 of the CH3 domain. References made to to Methods and Applications' Innis et al. eds., Academic amino acid numbering of immunoglobulins or immunoglo Press, San Diego, Calif. (1990); Ho et al. 1989. Gene 77:51: bulin fragments, or regions, are all based on Kabat et al. 1991, Hortonetal. 1993. Methods Enzymol. 217:270). PCR may be Sequences of Proteins of Immunological Interest, U.S. initiated by consensus constant region primers or by more Department of Public Health, Bethesda, Md. US 2015/03539 11 A1 Dec. 10, 2015 29

0254 Fc regions or FcRn binding partners bound to FcRn another mammalian species, including for example, a rodent can be effectively shuttled across epithelial barriers by FcRn, (e.g. a mouse, rat, rabbit, guinea pig) or non-human primate thus providing a non-invasive means to systemically admin (e.g. chimpanzee, macaque) species. Moreover, the polypep ister a desired therapeutic molecule. Additionally, fusion pro tide of the Fc domains or portions thereof may be derived teins comprising an Fc region oran FcRn binding partner are from any immunoglobulin class, including IgM, IgG, Ig|D. endocytosed by cells expressing the FcRn. But instead of IgA and IgE, and any immunoglobulin isotype, including being marked for degradation, these fusion proteins are IgG1, IgG2, IgG3 and IgG4. In another embodiment, the recycled out into circulation again, thus increasing the in vivo human isotype IgG1 is used. half-life of these proteins. In certain embodiments, the por 0259. In certain embodiments, the Fc variant confers a tions of immunoglobulin constant regions are an Fc region or change in at least one effector function imparted by an Fc an FcRn binding partner that typically associates, via disul moiety comprising said wild-type Fc domain (e.g., an fide bonds and other non-specific interactions, with another improvement or reduction in the ability of the Fc region to Fc region or another FcRn binding partner to form dimers and bind to Fc receptors (e.g. FcyRJ, FcyRII, or FcyRIII) or higher order multimers. complement proteins (e.g. Cld), or to triggerantibody-depen 0255. Two FcRn receptors can bind a single Fc molecule. dent cytotoxicity (ADCC), phagocytosis, or complement-de Crystallographic data Suggest that each FcRn molecule binds pendent cytotoxicity (CDCC)). In other embodiments, the Fc a single polypeptide of the Fc homodimer. In one embodi variant provides an engineered cysteine residue. ment, linking the FcRn binding partner, e.g., an Fc fragment 0260 The Fc moiety of the invention may employ art of an IgG, to a biologically active molecule provides a means recognized Fc variants which are known to impart a change of delivering the biologically active molecule orally, buccally, (e.g., an enhancement or reduction) in effector function and/ Sublingually, rectally, vaginally, as an aerosol administered or FcR or FcRn binding. Specifically, a binding molecule of nasally or via a pulmonary route, or via an ocular route. In the invention may include, for example, a change (e.g., a another embodiment, the chimeric protein can be adminis Substitution) at one or more of the amino acid positions dis tered invasively, e.g., Subcutaneously, intravenously. closed in International PCT Publications WO88/07089A1, 0256 An FcRn binding partner region is a molecule or WO96/14339A1, WO98/05787A1, WO98/23289A1, portion thereof that can be specifically bound by the FcRn WO99/51642A1, WO99/58572A1, WO00/09560A2, receptor with consequent active transport by the FcRn recep WO00/32767A1, WO00/42072A2, WO02/44215A2, tor of the Fc region. Specifically bound refers to two mol WO02/060919A2, WO03/074569A2, WOO4/016750A2, ecules forming a complex that is relatively stable under physi WOO4/029207A2, WOO4/035752A2, WOO4/063351A2, ologic conditions. Specific binding is characterized by a high WOO4/074455A2, WOO4/099249A2, WO05/040217A2, affinity and a low to moderate capacity as distinguished from WOO4/044859, WO05/070963A1, WO05/077981A2, nonspecific binding which usually has a low affinity with a WO05/092925A2, WO05/123780A2, WO06/019447A1, moderate to high capacity. Typically, binding is considered WO06/047350A2, and WO06/085967A2; US Patent Publi specific when the affinity constant KA is higher than 10'M', cation Nos. US2007/0231329, US2007/0231329, US2007/ or higher than 10 M'. If necessary, non-specific binding can 0237765, US2007/0237766, US2007/0237767, US2007/ be reduced without substantially affecting specific binding by 0243188, US20070248603, US20070286859, varying the binding conditions. The appropriate binding con US20080057056; or U.S. Pat. Nos. 5,648,260; 5,739,277; ditions such as concentration of the molecules, ionic strength 5,834,250; 5,869,046; 6,096,871; 6,121,022: 6,194.551; of the solution, temperature, time allowed for binding, con 6.242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; centration of a blocking agent (e.g. serum albumin, milk 6,821,505; 6,998,253; 7,083,784; 7,404,956, and 7,317,091, casein), etc., may be optimized by a skilled artisan using each of which is incorporated by reference herein. In one routine techniques. embodiment, the specific change (e.g., the specific Substitu 0257. In certain embodiments, a chimeric protein of the tion of one or more amino acids disclosed in the, art) may be invention comprises one or more truncated Fc regions that are made at one or more of the disclosed amino acid positions. In nonetheless sufficient to confer Fc receptor (FcR) binding another embodiment, a different change at one or more of the properties to the Fc region. For example, the portion of an Fc disclosed amino acid positions (e.g., the different Substitution region that binds to FcRn (i.e., the FcRn binding portion) of one or more amino acid position disclosed in the art) may comprises from about amino acids 282-438 of IgG1, EU be made. numbering (with the primary contact sites being amino acids 0261 The Fc moiety or FcRn binding partner of IgG can 248, 250-257, 272, 285,288, 290-291, 308-311, and 314 of be modified according to well recognized procedures such as the CH2 domain and amino acid residues 385-387, 428, and site directed mutagenesis and the like to yield modified IgG or 433-436 of the CH3 domain. Thus, an Fc region of the inven Fc fragments or portions thereofthat will be bound by FcRn. tion may comprise or consist of an FcRn binding portion. Such modifications include modifications remote from the FcRn binding portions may be derived from heavy chains of FcRn contact sites as well as modifications within the contact any isotype, including IgG1, IgG2, IgG3 and IgG4. In one sites that preserve or even enhance binding to the FcRn. For embodiment, an FcRn binding portion from an antibody of example, the following single amino acid residues in human the human isotype IgGI is used In another embodiment, an IgG1 Fc (Fc Y1) can, be substituted without significant loss of FcRn binding portion from an antibody of the human isotype Fc binding affinity for FcRn: P238A, S239A, K246A, IgG4 is used. K248A, D249A, M252A, T256A, E258A, T260A, D265A, 0258. The Fc moieties denoted as F, F1, or F2 herein may S267A, H268A, E269A, D270A, E272A, L274A, N276A, be obtained from a number of different sources. In one Y278A, D280A, V282A, E283A, H285A, N286A, T289A, embodiment, an Fc moiety of the polypeptide is derived from K290A, R292A, E293A, E294A, Q295A, Y296F, N297A, a human immunoglobulin. It is understood, however, that an S298A, Y300F, R301A, V303A, V305A, T307A, L309A, Fc moiety may be derived from an immunoglobulin of Q311A, D312A, N315A, K317A, E318A, K320A, K322A, US 2015/03539 11 A1 Dec. 10, 2015 30

S324A, K326A, A327Q, P329A, A330O, P331A, E333A, one or more disulfide bonds with another immunoglobulin K334A, T335A, S337A, K338A, K340A, Q342A, R344A, constant region or a portion thereof. The disulfide bond by the E345A, Q347A, R355A, E356A, M358A, T359A, K360A, immunoglobulin constant region or a portion thereof places N361A, Q362A, Y373A, S375A, D376A, A378Q, E380A, the first polypeptide comprising an activatable clotting factor E382A, S383A, N384A, Q386A, E388A, N389A, N390A, and the second polypeptide comprising the enhancer moiety Y391F, K392A, L398A, S400A, D401A, D413A, K414A, together so that upon activation of the clotting factor, the R416A, Q418A, Q419A, N421A, V422A, S424A, E430A, enhancer moiety is available to enhance activity of the clot N434A, T437A, Q438A, K439A, S440A, S444A, and ting factor. The hinge region or a portion thereof can further K447A, where for example P238A represents wild type pro be linked to one or more domains of CH1, CH2, CH3, a line substituted by alanine at position number 238. As an fragment thereof, or any combinations thereof. example, a specific embodiment incorporates the N297A 0266. In certain embodiments, the immunoglobulin con mutation, removing a highly conserved N-glycosylation site. stant region or a portion thereof is hemi-glycosylated. For In addition to alanine other amino acids may be substituted example, the chimeric protein comprising two Fc moieties or for the wild type amino acids at the positions specified above. FcRn binding partners may contain a first, glycosylated, Fc Mutations may be introduced singly into Fc giving rise to moiety (e.g., a glycosylated CH2 region) or FcRn binding more than one hundred Fc regions distinct from the native Fc. partner and a second, aglycosylated, Fc moiety (e.g., an agly Additionally, combinations of two, three, or more of these cosylated CH2 region) or FcRn binding partner. In one individual mutations may be introduced together, giving rise embodiment, a linker may be interposed between the glyco to hundreds more Fc moieties. Moreover, one of the Fc, Sylated and aglycosylated Fc moieties. In another embodi moiety of a construct of the invention may be mutated and the ment, the Fc moiety or FcRn binding partner is fully glyco other Fc moiety of the construct not mutated at all, or they Sylated, i.e., all of the Fc moieties are glycosylated. In other both may be mutated but with different mutations. embodiments, the Fc moiety may be aglycosylated, i.e., none 0262 Certain of the above mutations may confer new of the Fc moieties are glycosylated. functionality upon the Fc moiety or FcRn binding partner. For 0267 In certain embodiments, a chimeric protein of the example, one embodiment incorporates N297A, removing a invention comprises an amino acid substitution to an immu highly conserved N-glycosylation site. The effect of this noglobulin constant region or a portion thereof (e.g., Fc vari mutation is to reduce immunogenicity, thereby enhancing ants), which alters the antigen-independent effector functions circulating half-life of the Fc region, and to render the Fc. of the Ig constant region, in particular the circulating, half-life region incapable of binding to FcyRI. FcyRIIA, FcyRIIB, and of the protein. FcyRIIIA, without compromising affinity for FcRn (Rout 0268 Such proteins exhibit either increased or decreased ledge et al. 1995, Transplantation 60:847; Friend et al. 1999, binding to FcRn when compared to proteins lacking these Transplantation 68:1632; Shields et al. 1995, J. Biol. Chem. Substitutions and, therefore, have an increased or decreased 276:6591). As a further example of new functionality arising half-life in serum, respectively. Fc variants with improved from mutations described above affinity for FcRn may be affinity for FcRn are anticipated to have longer serum half increased beyond that of wild type in Some instances. This lives, and Such molecules have useful applications in methods increased affinity may reflect an increased “on” rate, a of treating mammals where long half-life of the administered decreased “off” rate or both an increased “on” rate and a polypeptide is desired, e.g., to treat a chronic disease or dis decreased “off” rate. Examples of mutations believed to order (see e.g., U.S. Pat. Nos. 7,348,004, 7,404.956, and impart an increased affinity for FcRn include, but not limited 7,862,820). In contrast, Fc variants with decreased FcRn to, T256A, T307A, E380A, and N434A (Shields et al. 2001, binding affinity are expected to have shorter half-lives, and J. Biol. Chem. 276:6591). Such molecules are also useful, for example, for administra 0263. Additionally, at least three human Fc gamma recep tion to a mammal where a shortened circulation time may be tors appear to recognize a binding site on IgG within the lower advantageous, e.g. for in vivo diagnostic imaging or in situ hinge region, generally amino acids 234-237. Therefore, ations where the starting polypeptide has toxic side effects another example of new functionality and potential decreased when present in the circulation for prolonged periods. Fc immunogenicity may arise from mutations of this region, as variants with decreased FcRn binding affinity are also less for example by replacing amino acids 233-236 of human likely to cross the placenta and, thus, are also useful in the IgG1 “ELLG' to the corresponding sequence from IgG2 treatment of diseases or disorders in pregnant women. In “PVA' (with one amino acid deletion). It, has been shown that addition, other applications in which reduced FcRn binding FcyRI. FcyRII, and FcyRIII, which mediate various effector affinity may be desired, include those applications in which functions will not bind to IgG1 when such mutations have localization the brain, kidney, and/or liver is desired. In one been introduced. Ward and Ghetie 1995, Therapeutic Immu exemplary embodiment, the chimeric protein of the invention nology 2:77 and Armour et al. 1999, Eur. J. Immunol. exhibits reduced transport across the epithelium of kidney 29:2613. glomeruli from the vasculature. In another embodiment, the 0264. In one embodiment, the immunoglobulin constant chimeric protein of the invention exhibits reduced transport region or a portion thereof, e.g., an Fc moiety, is a polypeptide across the blood brain barrier (BBB) from the brain, into the including the sequence PKNSSMISNTP (SEQ ID NO 27) vascular space. In one embodiment, a protein with altered and optionally further including a sequence selected from FcRn binding, comprises at least one Fc moiety or FcRn HQSLGTQ (SEQ ID NO: 28), HQNLSDGK (SEQ ID NO binding partner (e.g., one or two Fc regions or FcRn binding 29), HQNISDGK (SEQID NO 30), or VISSHLGQ (SEQID partners) having one or more amino acid Substitutions within NO:31) (U.S. Pat. No. 5,739,277). the “FcRn binding loop' of an Ig constant region. 0265. In another embodiment, the immunoglobulin con 0269. The FcRn binding loop is comprised of amino acid stant region or a portion thereof comprises an amino acid residues 280–299 (according to EU numbering) of a wild sequence in the hinge region or a portion thereof that forms type, full-length, Fc region. In other embodiments, an Ig US 2015/03539 11 A1 Dec. 10, 2015 constant region or a portion thereof in a chimeric protein of example, in one embodiment, a polypeptide of the invention the invention having altered FcRn binding affinity comprises is capable of binding, via its scFc region, to at least one Fc at least one Fc region or FcRn binding partner having one or receptor (e.g. an FcRn, an FcyR receptor (e.g., FcyRIII), or a more amino acid substitutions within the 15 AFcRn "contact complement protein (e.g. C1q)) in order to improve half-life Zone.” As used herein, the term 15 A FcRn "contact zone” or trigger an immune effector function (e.g., antibody-depen includes residues at the following positions of a wild-type, dent cytotoxicity (ADCC), phagocytosis, or complement-de full-length Fc moiety: 243-261,275-280, 282-293,302-319, pendent cytotoxicity (CDCC) and/or to improve manufactur 336-348, 367, 369, 372-389, 391, 393, 408, 424, 425-440 ability). (EU numbering). In other embodiments, a Ig constant region 0274) A variety of polypeptides of alternative designs are or a portion thereof of the invention having altered FcRn within the scope of the invention. For example, in one binding affinity comprises at least one Fc moiety or FcRn embodiment, a polypeptide comprises the moieties: binding partner having one or more amino acid substitutions at an amino acid position corresponding to any one of the following EU positions: 256, 277-281, 283-288, 303-309, in linear sequence from the amino to carboxy terminus 313, 338,342,376, 381,384, 385, 387,434 (e.g., N434A or wherein A, if present, is an activatable clotting factor or N434K), and 438. Exemplary amino acid substitutions which portion thereof, F1 is a first immunoglobulin constant region altered FcRn binding activity are disclosed in International or a portion thereof. P1 is a first intracellular processing site, PCT Publication No WO05/047327 which is incorporated by L is a Sclc linker, P2 is a second intracellular processing site; reference herein. B is an enhancer moiety, F2 is a second immunoglobulin 0270. An Fc moiety or FcRn binding partner used in the constant region or a portion thereof, and ' ' represents a invention may also comprise an art recognized amino acid peptide bond. Formula ( ) comprises at least a P1 or a substitution which alters the glycosylation of the chimeric P2 and optionally both. P1 and P2, if both present, can be the protein. For example, the Fc moiety or FcRn binding partner same or different. Formula ( ) comprises at least a F1, of the chimeric protein linked to an activatable clotting factor a F2, or both. F1 and F2, if both present, can be the same or or an enhancer moiety may comprise an Fc moiety having, a different. mutation leading to reduced glycosylation (e.g., N- or 0275 3. CTP O-linked glycosylation) or may comprise an altered glyco 0276. In certain aspects, a chimeric protein of the inven form of the wild-type Fc moiety (e.g., a low fucose or fucose tion comprises at least one heterologous moiety comprising free glycan). one B subunit of the C-terminal peptide (CTP) of human 0271 In one embodiment, a chimeric protein of the inven chorionic gonadotropin or fragment, variant, or derivative tion may comprise a genetically fused Fc region (i.e., ScPc thereof. One or more CTP peptides inserted into a recombi region) having two or more of its constituent Ig constant nant protein is known to increase the in vivo half-life of that region or portion thereof independently selected from the Ig protein. See, e.g., U.S. Pat. No. 5,712,122, incorporated by constant region or portion thereof described herein. In one reference herein in its entirety. embodiment, the Fc domains of a dimeric Fc region are the (0277 Exemplary CTP peptides include same. In another embodiment, at least, two of the Fc domains are different. For example, the Fc moieties or FcRn binding DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL (SEQ ID partners of the proteins of the invention comprise the same NO:32) or SSSSKAPPPSLPSPSRLPGPSDTPILPQ. (SEQ number of amino acid residues or they may differ in length by ID NO: 33). See, e.g., U.S. Patent Application Publication one or more amino acid residues (e.g., by about 5 amino acid No. US 2009/0087411 A1, incorporated by reference. residues (e.g., 1, 2, 3, 4, or 5 amino acid residues), about 10 (0278 4. XTEN Sequence residues, about 15 residues, about 20 residues, about 30 resi 0279. In some embodiments, a heterologous moiety in the dues, about 40 residues, or about 50 residues). In yet other chimeric protein comprises one or more XTEN sequences, embodiments, the Fc moieties or FcRn binding partners of the fragments, variants, or derivatives thereof. As used here protein of the invention may differin sequence at one or more “XTEN sequence” refers to extended length polypeptides amino acid positions. For example, at least two of the Fc with non-naturally occurring, Substantially non-repetitive moieties or FcRn binding partners may differ at about 5 sequences that are composed mainly of Small hydrophilic amino acid positions (e.g., 1, 2, 3, 4, or 5 amino acid posi amino acids, with the sequence having a low degree or no tions), about 10 positions, about 15 positions, about 20 posi secondary or tertiary structure under physiologic conditions. tions, about 30 positions, about 40 positions, or about 50 As a heterologous moiety, XTENs can serve as a half-life positions). extension moiety. In addition, XTEN can provide desirable 0272. 2. Sclfc Regions properties including but are not limited to enhanced pharma 0273. In one embodiment, the invention provides for cokinetic parameters and solubility characteristics. unprocessed chimeric polypeptides comprising an activat 0280. The incorporation of a heterologous moiety com able clotting factor, an enhancer moiety, and at least one prising an XTEN sequence into a chimeric protein of the genetically fused Fc region or portion thereof within a single invention can confer to the chimeric protein one or more of polypeptide chain (i.e., polypeptides comprising a single the following advantageous properties: conformational flex chain Fc (ScFc) region). The unprocessed polypeptides com ibility, enhanced aqueous solubility, high degree of protease prise at least two immunoglobulin constant regions or por resistance, low immunogenicity, low binding to mammalian tions thereof (e.g., Fc moieties or domains (e.g., 2, 3, 4, 5, 6, receptors, or increased hydrodynamic (or Stokes) radii. or more Fc moieties or domains)) within the same linear 0281. In certain aspects, an XTEN sequence can improve polypeptide chain that are capable of folding (e.g., intramo pharmacokinetic properties such as extending in vivo half lecularly or intermolecularly folding) to form one functional life or increasing total exporSure (area under the curve sch c region which is linked by an Fc peptide linker. For (AUC)), so that a chimeric protein of the invention displays US 2015/03539 11 A1 Dec. 10, 2015 32 prolonged efficacy for controlling bleeds compared to a chi Dennis et al., J. Biol. Chem. 2002, 277:35035-35043 (2002). meric protein with the same but without the XTEN heterolo Examples of albumin-binding antibody fragments are dis gous moiety. closed in Muller and Kontermann, Curr. Opin. Mol. Ther. 0282. Examples of XTEN sequences that can be used as 9:319-326 (2007); Rooverset al., Cancer Immunol. Immu heterologous moieties in chimeric proteins of the invention nother. 56:303-317 (2007), and Holt et al., Prot. Eng. Design are disclosed, e.g., in U.S. Pat. Nos. 7,855,279 and 7,846,445, Sci., 21:283-288 (2008), which are incorporated herein by U.S. Patent Publication Nos. 2009/0092582 A1, 2010/ reference in their entireties. An example of such albumin 0239554 A1, 2010/0323956 A1, 2011/0046060 A1, 2011/ binding moiety is 2-(3-maleimidopropanamido)-6-(4-(4-io 0046061 A1, 2011/0077199 A1, 2013/0017997 A1, or 2012/ dophenyl)butanamido) hexanoate (Albu’ tag) as disclosed 0263701 A1, or 2011/0172146 A1, or International Patent by Trusselet al., Bioconjugate Chem. 20:2286-2292 (2009). Publication Nos. WO 2010091122 A1, WO 2010144502 A2, 0290 Fatty acids, in particular long chain fatty acids WO 2010144.508 A1, WO 2011028228A1, WO 2011028229 (LCFA) and long chain fatty acid-like albumin-binding com A1, or WO 2011028344 A2, or International Application No. pounds can be used to extend the in vivo halt-life of chimeric PCT/US2011/48517, filed Aug. 19, 2011, each of which is proteins of the invention. An example of a LCFA-like albu incorporated by reference herein in its entirety. min-binding compound is 16-(1-(3-(9-(((2,5-dioxopyrroli 0283) 5. Albumin or Fragment, Derivative, or Variant din-1-yloxy)carbonyloxy)-methyl)-7-sulfo-9H-fluoren-2- Thereof ylamino)-3-oxopropyl)-2,5-dioxopyrrolidin-3-ylthio) 0284. In certain embodiments, the chimeric protein of the hexadecanoic acid (see, e.g., WO 2010/140148). invention comprises a heterologous moiety comprising albu 0291 7. PAS Sequence minor a functional fragment thereof Human serum albumin 0292. In other embodiments, at least one heterologous (HSA, or HA), a protein of 609 amino acids in its fall-length moiety is a PAS sequence. A PAS sequence, as used herein, form, is responsible for a significant proportion of the osmotic means an amino acid sequence comprising mainly alanine pressure of serum and also functions as a carrier of endog and serine residues or comprising, mainly alanine, serine, and enous and exogenous . The term "albumin' as used proline residues, the, amino acid sequence forming random herein includes full-length albuminor a functional fragment, coil conformation under physiological conditions. Accord variant, derivative, or analog thereof. Examples of albuminor ingly, the PAS sequence is a building block, an amino acid the fragments or variants thereof are disclosed in US Pat. polymer, or a sequence cassette comprising, consisting essen Publ. Nos. 2008/0194481A1, 2008/0004206 A1, 2008/ tially of, or consisting of alanine, serine, and proline which 0161243 A1, 2008/0261877 A1, or 2008/0153751A1 or PCT can be used as a part of the heterologous moiety in the chi Appl. Publ. Nos. 2008/0334.13 A2, 2009/058322A1, or 2007/ meric protein. Yet, the skilled person is aware that an amino 021494 A2, which are incorporated herein by reference in acid polymer also may form random coil conformation when their entireties. residues other than alanine, serine, and proline are added as a 0285. In one embodiment, the chimeric protein of the minor constituent in the PAS sequence. The term “minor invention comprises albumin, a fragment, or a variant thereof constituent as used herein means that amino acids other than which is further linked to a heterologous moiety selected from alanine, serine, and proline may be added in the PAS an immunoglobulin constant legion or portion thereof (e.g., sequence to a certain degree, e.g., up to about 12%, i.e., about an Fc region), a PAS sequence, HES, PEG, or any combina 12 of 100 amino acids of the PAS sequence, up to about 10%, tions thereof. i.e. about 10 of 100 amino acids of the PAS sequence, up to 0286 6. Albumin Binding Moiety about 9%, i.e., about 9 of 100 amino acids, up to about 8%, 0287. In certain embodiments, the heterologous moiety is i.e., about 8 of 100 amino acids, about 6%, i.e., about 6 of 100 an albumin binding moiety, which comprises an albumin amino acids, about 5%, i.e., about 5 of 100 amino acids, about binding peptide, a bacterial albuminbinding domain, an albu 4%, i.e., about 4 of 100 amino acids, about 3%, i.e., about 3 of min-binding antibody fragment, or any combinations thereof. 100 amino acids, about 2%, i.e., about 2 of 100 amino acids, 0288 For example, the albumin binding protein can be a about 1%, i.e., about 1 of 100 of the amino acids. The amino bacterial albumin binding protein, an antibody oran antibody acids different from alanine, serine and proline may be fragment including domain antibodies (see U.S. Pat. No. selected from Arg, ASn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, 6,696.245). An albumin binding protein, for example, can be Lys, Met, Phe, Thr, Trp, Tyr, or Val. a bacterial albumin binding domain, Such as the one of strep 0293 Under physiological conditions, the PAS sequence tococcal protein G (Konig, T. and Skerra, A. (1998).J. Immu stretch forms a random coil conformation and thereby can mol. Methods 218, 73-83). Other examples of albumin bind mediate an increased in vivo and/or in vitro stability to the ing peptides that can be used as conjugation partner are, for chimeric protein. Since the random coil domain does not instance, those having a Cys-Xaa-Xaa-Xaa-Xaa-Cys adopt a stable structure or function by itself, the biological consensus sequence, wherein Xaa is Asp, ASn, Ser, Thr, or activity mediated by the activatable clotting factor in the Trp, Xaa is ASn, Gln, His, Ile, Leu, or Lys; Xaa- is Ala, Asp, chimeric protein is essentially preserved. In other embodi Phe, Trp, or Tyr; and Xaa, is Asp, Gly, Leu, Phe, Ser, or Thr ments, the PAS sequences that form random coil domain are (SEQID NO:34) as described in US patent application 2003/ biologically inert, especially with respect to proteolysis in 0069395 or Dennis et al. (Dennis et al. (2002).J. Biol. Chem. blood plasma, immunogenicity, isoelectric point/electrostatic 277, 35035-35043). behaviour, binding to cell Surface receptors or internalisation, 0289 Domain 3 from streptococcal protein G, as dis but are still biodegradable, which provides clear advantages closed by Kraulis et al., FEBS Lett. 378:190-194 (1996) and over synthetic polymers such as PEG. Linhultet al., Protein Sci. 11:206-213 (2002) is an example of 0294 Non-limiting examples of the PAS sequences form a bacterial albumin-binding domain. Examples of albumin ing random coil conformation comprise an amino acid binding peptides include a series of peptides having the core sequence selected from ASPAAPAPASPAAPAPSAPA (SEQ sequence DICLPRWGCLW (SEQ ID NO: 35). See, e.g., ID NO:36), AAPASPAPAAPSAPAPAAPS (SEQ ID NO: US 2015/03539 11 A1 Dec. 10, 2015

37), APSSPSPSAPSSPSPASPSS (SEQ ID NO 38), selected from an immunoglobulin constant region or portion APSSPSPSAPSSPSPASPS (SEQ ID NO 39), SSP thereof (e.g., an Fc moiety), a PAS sequence, HES, albumin, SAPSPSSPASPSPSSPA (SEQ ID NO 40), AASPAAPSAP fragment, or variant thereof, or any combinations thereof. In PAAASPAAPSAPPA (SEQ ID NO: 41), ASAAA yet other embodiments, the chimeric protein comprises a PAAASAAASAPSAAA (SEQ ID NO: 42) or any clotting factor or fragment thereof, a second clotting factor or combinations thereof. Additional examples of PAS sequences fragment thereof, and a PEG heterologous moiety, wherein are known from, e.g., US Pat. Publ. No. 2010/0292130 A1 the chimeric protein further comprises a heterologous moiety and PCT Appl. Publ. No. WO 2008/155134A1. selected from an immunoglobulin constant region or portion 0295 8. HAP Sequence thereof (e.g., an Fc moiety), a PAS sequence, HES, albumin, 0296. In certain embodiments, at least one heterologous fragment, or variant thereof, or any combinations thereof. In moiety is a glycine-rich homo-amino-acid polymer (HAP). other embodiments, the chimeric protein comprises a clotting The HAP sequence can comprise a repetitive sequence of factor or fragment thereof, a synthetic procoagulant polypep glycine, which has at least 50 amino acids, at least 100 amino tide, and a PEG heterologous moiety, wherein the chimeric acids, 120 amino acids, 140 amino acids, 160 amino acids, protein further comprises a heterologous moiety selected 180 amino acids, 200 amino acids, 250 amino acids, 300 from an immunoglobulin constant region or portion thereof amino acids, 350 amino acids, 400 amino acids, 450 amino (e.g., an Fc region), a PAS sequence, HES, albumin, frag acids, or 500 amino acids in length. In one embodiment, the ment, or variant thereof, or any combinations thereof. In other HAP sequence is capable of extending half-life of a moiety embodiments, the chimeric protein comprises two synthetic fused to or linked to the HAP sequence. Non-limiting procoagulant peptides and a PEG heterologous moiety, examples of the HAP sequence includes, but are not limited to wherein the chimeric protein further comprises a heterolo (Gly), (Gly Ser), or S(Gly-Ser), whereinn is 1,2,3,4, 5, 6, gous moiety selected from an immunoglobulin constant 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In one region orportion thereof (e.g., an Fc region), a PAS sequence, embodiment, n is 20, 21, 22, 23, 24, 25, 26, 26, 28, 29, 30, 31, HES, albumin, fragment, or variant thereof, or any combina 32, 33, 34, 35, 36, 37, 38, 39, or 40. In another embodiment, tions thereof. In yet another embodiment, the chimeric pro n is 50, 60, 70, 80,90, 100, 110, 120, 130, 140, 150, 160, 170, tein comprises a clotting factor or fragment thereof, a clotting 180, 190, or 200. factor cofactor (e.g., Factor Va if the clotting factor in Factor 0297 9. Transferrin or Fragment Thereof X; or Tissue Factor if the clotting factor is Factor VII), and a 0298. In certain embodiments, at least one heterologous PEG heterologous moiety, wherein the chimeric protein fur moiety is transferrin or a fragment thereof. Any transferrin ther comprises a heterologous moiety selected from an immu may be used to make the chimeric proteins of the invention. noglobulin constant region or portion thereof (e.g., an Fc As an example, wild-type human TF (TF) is a 679 amino acid region), a PAS sequence, HES, albumin, fragment, or variant protein, of approximately 75 KDa (not accounting for glyco thereof, or any combinations thereof. sylation), with two main domains, N (about 330 amino acids) 0302 Also provided by the invention are chimeric pro and C (about 340 amino acids), which appear to originate teins of the invention comprising heterologous moieties from a gene duplication. See GenBank accession numbers which may provide additional advantages such as increased NM001063, XM002793, M12530, XM039845, XM 039847 solubility, stability and circulating time of the polypeptide, or and S95936 (www.ncbi.nlm.nih.gov/), all of which are herein decreased immunogenicity (see U.S. Pat. No. 4,179,337). incorporated by reference in their entirety. Transferrin com Such heterologous moieties for modification can be selected prises two domains, N domain and C domain. N domain from water soluble polymers including, but not limited to, comprises two Subdomains, N1 domain and N2 domain, and polyethylene glycol, ethylene glycol/propylene glycol C domain comprises two Subdomains, C1 domain and C2 copolymers, carboxymethylcellulose, dextran, polyvinyl domain. alcohol, or any combinations thereof. 0299. In one embodiment, the transferrin heterologous 0303. The polymer can be of any molecular weight, and moiety includes a transferrin splice variant. In one example, a can be branched or unbranched. For polyethylene glycol, in transferrin splice variant can be a splice variant of human one embodiment, the molecular weight is between about 1 transferrin, e.g., Genbank Accession AAA61140. In another kDa and about 100 kDa for ease in handling and manufactur embodiment, the transferrin portion of the chimeric protein ing. Other sizes may be used, depending on the desired profile includes one or more domains of the transferrin sequence, (e.g., the duration of Sustained release desired, the effects, if e.g., N domain, C domain, N1 domain, N2 domain, C1 any on biological activity, the ease in handling, the degree or domain, C2 domain or any combinations thereof. lack of antigenicity and other known effects of the polyeth 0300 10. Polymer, e.g., Polyethylene Glycol (PEG) ylene glycol to a protein or analog). For example, the poly 0301 In other embodiments, at least one heterologous ethylene glycol may have an average molecular weight of moiety is a soluble polymer known in the art, including, but about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, not limited to, polyethylene glycol, ethylene glycol/propy 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500,9000, lene glycol copolymers, carboxymethylcellulose, dextran, or 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, polyvinyl alcohol. In some embodiments, the chimeric pro 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, tein comprising a PEG heterologous moiety further com 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, prises a heterologous moiety selected from an immunoglo 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, bulin constant region orportion thereof (e.g., an Fc region), a 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, PAS sequence, HES, albumin, fragment, or variant thereof, or 90,000, 95,000, or 100,000 kDa. any combinations thereof. In still other embodiments, the 0304. In some embodiments, the polyethylene glycol may chimeric protein comprises an activatable clotting factor or have a branched structure. Branched polyethylene glycols are fragment thereofanda PEG heterologous moiety, wherein the described, for example, in U.S. Pat. No 5,643,575; Morpurgo chimeric protein further comprises a heterologous moiety et al., Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev US 2015/03539 11 A1 Dec. 10, 2015 34 et al., Nucleosides Nucleotides 18:2745-2750 (1999); and preferably 0.1 to 0.8, and a ratio between C2:C6 substitution Caliceti of al., Bioconjug. Chem. 10:638-646 (1999), each of in the range of from 2 to 20 with respect to the hydroxyethyl which is incorporated herein by reference in its entirety. groups. A non-limiting example of HES having a mean 0305 The number of polyethylene glycol moieties molecular weight of about 130 kD is a HES with a degree of attached to each chimeric protein of the invention (i.e., the substitution of 0.2 to 0.8 such as 0.2,0.3, 0.4,0.5,0.6,0.7, or degree of Substitution) may also vary. For example, the 0.8, preferably of 0.4 to 0.7 such as 0.4,0.5,0.6, or 0.7. In a PEGylated chimeric protein may be linked, on average, to 1, specific embodiment, HES with a mean molecular weight of 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene about 130 kD is VOLUVENOR) from Fresenius. VOLUVENR) glycol molecules. Similarly, the average degree of Substitu is an artificial colloid, employed, e.g., for Volume replace tion within ranges such as 1-3, 2-4, 3-5, 4-6, 5–7, 6-8, 7-9, ment used in the therapeutic indication for therapy and pro 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, phylaxis of hypovolemia. The characteristics of VOLU 17-19, or 18-20 polyethylene glycol moieties per protein VENR) are a mean molecular weight of 130,000+/-20,000 D, molecule. Methods for determining the degree of substitution a molar substitution of 0.4 and a C2:C6 ratio of about 9:1. In are discussed, for example, in Delgado et al., Crit. Rev. Thera. other embodiments, ranges of the mean molecular weight of Drug Carrier Sys. 9:249-304 (1992). hydroxyethyl starch are, e.g., 4 to 70 kD or 10 to 70 kD or 12 0306 In some embodiments, the chimeric protein can be to 70 kD or 18 to 70 kD or 50 to 70 kD or 4 to 50 kD or 10 to PEGylated. A PEGylated chimeric protein comprises at least 50 kD or 12 to 50 kD or 18 to 50 kD or 4 to 18 kD or 10 to 18 one polyethylene glycol (PEG) molecule. In other embodi kD or 12 to 18 kD or 4 to 12 kD or 10 to 12 kD or 4 to 10 kD. ments, the polymer can be water-soluble. Non-limiting In still other embodiments, the mean molecular weight of examples of the polymer can be poly(alkylene oxide), poly hydroxyethyl starch employed is in the range of from more (vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline, or than 4 kD and below 70 kD such as about 10 kD, or in the poly(acryloylmorpholine). Additional types of polymer-con range of from 9 to 10 kD or from 10 to 11 kD or from 9 to 11 jugation to clotting factors are disclosed in U.S. Pat. No. kD, or about 12 kD, or in the range of from 11 to 12 kD) or 7,199,223. See also, Singh et al. Curr. Med. Chem. 15:1802 from 12 to 13 kD or from 11 to 13 kD, or about 18 kD, or in 1826 (2008). the range of from 17 to 18 kD or from 18 to 19 kD or from 17 0307 11. Hydroxyethyl Starch (HES) to 19 kD. or about 30 kD, or in the range of from 29 to 30, or 0308. In certain embodiments, at least one heterologous from 30 to 31 kD, or about 50 kD, or in the range of from 49 moiety is a polymer, e.g., hydroxyethyl starch (HES) or a to 50 kD or from 50 to 51 kD or from 49 to 51 kD. derivative thereof Hydroxyethyl starch (HES) is a derivative 0312. In certain embodiments, the heterologous moiety of naturally occurring amylopectin and is degraded by alpha can be a mixture of hydroxyethyl starches having different amylase in the body. HES is a substituted derivative of the mean molecular weights and/or different degrees of substitu carbohydrate polymer amylopectin, which is present in corn tion and/or different ratios of C2:C6 substitution. Therefore, starch at a concentration of up to 95% by weight. HES exhib mixtures of hydroxyethyl starches may be employed having its advantageous biological properties and is used as a blood different mean molecular weights and different degrees of Volume replacement agent and in hemodilution therapy in the substitution and different ratios of C2:C6 substitution, or clinics (Sommermeyer et al., Krankenhaus.pharmazie, 8(8), having different mean molecular weights and different 271-278 (1987); and Weidler et al., Arzneim.-Forschung/ degrees of substitution and the same or about the same ratio of Drug Res., 41, 494-498 (1991)). C2:C6 substitution, or having different mean molecular 0309 Amylopectin contains glucose moieties, wherein in weights and the same or about the same degree of substitution the main chain alpha-1,4-glycosidic bonds are present and at and different ratios of C2:C6 substitution, or having the same the branching sites alpha-1,6-glycosidic bonds are found. The or about the same mean molecular weight and different physical-chemical properties of this molecule are mainly degrees of substitution and different ratios of C2:C6 substi determined by the type of glycosidic bonds. Due to the nicked tution, or having different mean molecular weights and the alpha-1,4-glycosidic bond, helical structures with about six same or about the same degree of substitution and the same or glucose-monomers per tarn are produced. The physico about the same ratio of C2:C6 substitution, or having the chemical as well as the biochemical properties of the polymer same or about the same mean molecular weights and different can be modified via substitution. The introduction of a degrees of substitution and the same or about the same ratio of hydroxyethyl group can be achieved via alkaline hydroxy C2:C6 substitution, or having the same or about the same ethylation. By adapting the reaction conditions it is possible mean molecular weight and the same or about the same to exploit the different reactivity of the respective hydroxy degree of substitution and different ratios of C2:C6 substitu group in the unsubstitated glucose monomer with respect to a tion, or having about the same mean molecular weight and hydroxyethylation. Owing to this fact, the skilled person is about the same degree of substitution and about the same ratio able to influence the substitution pattern to a limited extent. of C2:C6 substitution. 0310 HES is mainly characterized by the molecular 0313 12. Polysialic Acids (PSA) weight distribution and the degree of substitution. The degree 0314. In certain embodiments, at least one heterologous of substitution, denoted as DS, relates to the molar substitu moiety is a polymer, e.g., polysialic acids (PSAs) or a deriva tion, is known to the skilled people. See Sommermeyer et al., tive thereof. Polysialic acids (PSAs) are naturally occurring Krankenhaus.pharmazie, 8(8), 271-278 (1987), as cited unbranched polymers of Sialic acid produced by certain bac above, in particular p. 273. terial strains and in mammals in certain cells Roth J., et al. 0311. In one embodiment, hydroxyethyl starch has a mean (1993) in Polysialic Acid. From Microbes to Man, eds Roth.J., molecular weight (weight mean) of from 1 to 300 kD. from 2 Rutishauser U., Troy F. A. (Birkhäuser Verlag, Basel, Swit to 200kD, from 3 to 100kD, or from 4 to 70 kD. hydroxyethyl zerland), pp. 335-348. They can be produced in various starch can further exhibit a molar degree of substitution of degrees of polymerisation from n=about 80 or more sialic from 0.1 to 3, preferably 0.1 to 2, more preferred, 0.1 to 0.9, acid residues down to n=2 by limited acid hydrolysis or by US 2015/03539 11 A1 Dec. 10, 2015

digestion with neuraminidases, or by fractionation of the embodiment, Substantially excised (e.g., during processing natural, bacterially derived forms of the polymer. The com by a cell). Thus, the processed molecule is a dimeric molecule position of different polysialic acids also varies such that comprising at least two amino acid chains and Substantially there are homopolymeric forms i.e. the alpha-2.8-linked lacking extraneous linker amino acid sequences. In some polysialic acid comprising the capsular polysaccharide of E. embodiments, all or substantially all of the linker is excised, coli strain K1 and the group-B meningococci, which is also while in some embodiments, a portion of the intracellular found on the embryonic form of the neuronal cell processing site may remain, e.g., four arginines of the RRRR molecule (N-CAM). Heteropolymeric forms also exist— cleavage site. Such as the alternating alpha-2.8 alpha-2.9 polysialic acid of 0321. In another embodiment, another type of peptide E. coli strain K92 and group C polysaccharides of N. menin linker, herein referred to as a “linker moiety” may be used to gitidis. Sialic acid may also be found in alternating copoly connect different moieties, e.g., an activatable clotting factor mers with monomers other than sialic acid such as group to an enhancer moiety, an activatable clotting factor to a W135 or group Y of N. meningitidis. Polysialic acids have heterologous moiety, and/or an enhancer moiety to a heter important biological functions including the evasion of the ologous moiety. This type of peptide linkers may provide immune and complement systems by pathogenic bacteria and flexibility to the polypeptide molecule. Linkers are not typi the regulation of glial adhesiveness of immature neurons cally cleaved, however Such cleavage may be desirable. during foetal development (wherein the polymer has an anti Exemplary positions of linkers are shown in the accompany adhesive function) Cho and Troy, P.N.A.S., USA, 91 (1994) ing drawings. Linkers can be located between the activatable 11427-11431, although there >are no known receptors for clotting factor and the enhancer moiety, the activatable clot polysialic acids in mammals. The alpha-2.8-linked polysialic ting factor and the heterologous moiety linked thereto, or the acid of E. coli Strain K1 is also known as colominic acid and enhancer moiety and the heterologous moiety linked thereto, is used (in various lengths) to exemplify the present invention. e.g., at the Nor C terminus of these moieties. In one embodi Various methods of attaching or conjugating polysialic acids ment, these linkers are not removed during processing. to a polypeptide have been described (for example, see U.S. 0322. A third type of linker which may be present in a Pat. No. 5,846,951: WO-A-0187922, and US 2007/0191597 chimeric protein of the invention is a protease cleavable linker A1, which are incorporated herein by reference in their entire which comprises a cleavage site (i.e., a protease cleavage site ties. Substrate, e.g., a factor XIa, Xa, or thrombin cleavage site) 0315 13. Clearance Receptors and which may include additional linkers on either the N-ter 0316. In certain aspects, the in vivo half-life of an activat minal of C-terminal or both sides of the cleavage site. These able clotting factor in a chimeric protein of the invention can cleavable linkers when incorporated into a clotting factor be extended where the chimeric protein comprises at least one Zymogen result in a chimeric molecule having a heterologous heterologous molecule comprising a clearance receptor, frag cleavage site. Exemplary locations for Such sites are shown in ment, variant, or derivative thereof. In specific aspects the accompanying drawings and include, e.g., between the wherein the therapeutic peptide is Factor X, soluble forms of light chain and heavy chain of the clotting factor Zymogen, clearance receptors, such as the low density lipoprotein-re between the heavy chain of the clotting factor Zymogen and a lated protein receptor LRP 1, or fragments thereof, can block first heterologous moiety, between the enhancer moiety and a binding of Factor X to clearance receptors and thereby extend second heterologous moiety. its in vivo half-life. 0323. In one embodiment, an unprocessed polypeptide of 0317 LRP1 is a 600 kDa integral membrane protein that is the instant invention comprises two or more Fc domains or implicated in the receptor-mediate clearance of a variety of moieties linked via a cscFc linker to form an Fc region com proteins, such as Factor X. See, e.g., Narita et al., Blood prised in a single polypeptide chain. The cscFc linker is 91:555-560 (1998). flanked by at least one intracellular processing site, i.e., a site 0318 D. Linker Moieties (L, L1, or L2) cleaved by an intracellular enzyme. Cleavage of the polypep 03.19 Linker moieties useful for the present invention can tide at the at least one intracellular processing site results in a be either a peptide linker or a non-peptide linker. In one polypeptide which comprises at least two polypeptide chains. embodiment, the peptide linker can be synthetic. In one embodiment, an cscFc linker links F1 or F2 to, e.g., an 0320. As used herein, the term "peptide linkers’ refers to activatable clotting factor, optionally via an intracellular pro a peptide or polypeptide sequence (e.g., a synthetic peptide or cessing site or an enhancer moiety via an intracellular pro polypeptide sequence) which connects two domains in a lin cessing site. earamino acid sequence of a polypeptide chain. The polypep 0324. As is set forth above, other peptide linkers may tides of invention are encoded by nucleic acid molecules that optionally be used in a construct of the invention, e.g., to encode peptide linkers which either directly or indirectly connectan activatable clotting factor oran enhancer moiety to connect the two immunoglobulin constant regions orportions an Fc moiety. Some exemplary locations of linkers that can be thereof (e.g., Fc moieties) which make up the construct. used in connection with the invention include, e.g., polypep These linkers are referred to herein as “scFc linkers’. If the tides comprising GlySeramino acids Such as those set forth in ScFc linker connects two Fc moieties contiguously in the the accompanying figures and described in more detail below. linear polypeptide sequence, it is a “direct linkage. In con In one embodiment, a linker may be adjacent to one or more tract, the sche linkers may link the first Fc moiety to a binding moieties each independently selected from activatable clot moiety which is, in turn, linked to the second Fc moiety, ting factor, heterologous moiety, e.g., Fc, cleavage site, and an thereby forming an indirect linkage. These scFc linkers (X) enhancer moiety. result in the formation of a single chain genetic construct. 0325 In one embodiment, the peptide linker is synthetic, However, in one embodiment, the scFc polypeptides also i.e., non-naturally occurring. In one embodiment, a peptide comprise intracellular processing sites which result in the linker includes peptides (or polypeptides) (which may or may ScFc linker being cleavable (an cscFc linker) and, in one not be naturally occurring) which comprise an amino acid US 2015/03539 11 A1 Dec. 10, 2015 36 sequence that links or genetically fuses a first linear sequence amino acids in length. In one embodiment, a peptide linker of of amino acids to a second linear sequence of amino acids to the invention is 20 or 30 amino acids in length. which it is not naturally linked or genetically fused in nature. 0329. In some embodiments, the peptide linker can com For example, in one embodiment the peptide linker may prise at least two amino, at least three, at least four, at least comprise non-naturally occurring polypeptides which are five, at least 10, at, least 20, at least 30, at least 40, at least 50, modified forms of naturally occurring polypeptides (e.g., at least 60, at least 70, at least 80, at least 90, or at least 100 comprising a mutation Such as an addition, Substitution or amino acids. In other embodiments, the peptide linker can deletion). In another embodiment, the peptide linker may comprise at least 200, at least 300, at least 400, at least 500, at comprise non-naturally occurring amino acids. In another least 600, at least 700, at least 800, at least 900, or at least embodiment, the peptide linker may comprise naturally 1,000 amino acids. In some embodiments, the peptide linker occurring amino acids occurring in a linear sequence that can comprise at least about 10, 20, 30, 40, 50, 60, 70, 80,90, does not occur in nature. In still another embodiment, the 100, 150, 200,300,400,500, 600, 700, 800,900, 1000, 1100, peptide linker may comprise a naturally occurring polypep 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 tide sequence. amino acids. The peptide linker can comprise 1-5 amino 0326 For example, in certain embodiments, a peptide acids, 1-10 amino acids, 1-20 amino acids, 10-50 amino linker can be used to fuse identical Fc moieties, thereby acids, 50-100 amino acids, 100-200 amino acids, 200-300 forming a homodimeric Schc region. In other embodiments, a amino acids, 300-400 amino acids, 400-500 amino acids, peptide linker can be used to fuse different Fc moieties (e.g. a 500-600 amino acids, 600-700 amino acids, 700-800 amino wild-type Fc moiety and an Fc moiety variant), thereby form acids, 800-900 amino acids, or 900-1000 amino acids. ing a heterodimeric Schc region. 0330 Peptide linkers can be introduced into polypeptide 0327. In another embodiment, a peptide linker comprises sequences using techniques known in the art. Modifications or consists of a gly-ser linker. In one embodiment, a sce or can be confirmed by DNA sequence analysis. Plasmid DNA cScFc linker comprises at least a portion of an immunoglo can be used to transform host cells for stable production of the bulin hinge and a gly-ser linker. As used herein, the term polypeptides produced. 'gly-ser linker” refers to a peptide that consists of glycine and serine residues. An exemplary gly/ser linker comprises an III. Preparation of Polypeptides amino acid sequence of the formula (Gly Ser)n (SEQID NO: 0331 A variety of methods are available for recombi 4), wherein is a positive integer (e.g., 1, 2, 3, 4, 5, 6,7,8,9, or nantly producing a chimeric protein of the invention. In one 10). An example of gly/ser linker is (Gly-Ser), (SEQID NO: embodiment, the invention relates to a nucleic acid construct 4), (Gly-Ser) (SEQID NO: 4), or (Gly-Ser). (SEQID NO: comprising a nucleic acid sequence encoding the chimeric 4) Another exemplary gly-ser linker is GGGSSGGGSG proteins of the invention. It will be understood that because of (SEQID NO: 43). In certain embodiments, said gly-ser linker the degeneracy of the code, a variety of nucleic acid may be inserted between two other sequences of the peptide sequences will encode the amino acid sequence of the linker (e.g., any of the peptide linker sequences described polypeptide. The desired polynucleotide can be produced by herein). In other embodiments, a gly-ser linker is attached at de novo solid-phase DNA synthesis or by PCR mutagenesis one or both ends of another sequence of the peptide linker of an earlier prepared polynucleotide. (e.g., any of the peptide linker sequences described herein). In 0332 Oligonucleotide-mediated mutagenesis is one yet other embodiments, two or more gly-ser linker are incor method for preparing a Substitution, in-frame insertion, or porated in series in a peptide linker. In one embodiment, a alteration (e.g., altered codon) to introduce a codon encoding peptide linker of the invention comprises at least a portion of an amino acid Substitution (e.g., into an Fc variant moiety). an upper hinge region (e.g., derived from an IgG1, IgG2. For example, the starting polypeptide DNA is altered by IgG3, or IgG4 molecule), at least a portion of a middle hinge hybridizing an oligonucleotide encoding the desired mutation region (e.g., derived from an IgG1, IgG2, IgG3, or IgG4 to a single-stranded DNA template. After hybridization, a molecule) and a series of gly/ser amino acid residues (e.g., a DNA polymerase is used to synthesize an entire second gly/ser linker such as (Gly-Ser)n) (SEQ ID NO 4)). complementary strand of the template that incorporates the 0328 Peptide linkers of the invention are at least one oligonucleotide primer. In one embodiment, genetic engi amino acid in length and can be of varying lengths. In one neering, e.g., primer-based PCR mutagenesis, is sufficient to embodiment, a peptide linker of the invention is from about 1 incorporate an alteration, as defined herein, for producing a to about 50 amino acids in length. As used in this context, the polynucleotide encoding a polypeptide of the invention. term “about indicates +/- two amino acid residues. Since 0333 For recombinant production, a polynucleotide linker length must be a positive interger, the length of from sequence encoding the chimeric protein is inserted into an about 1 to about 50 amino acids in length, means a length of appropriate expression vehicle, i.e., a vector which contains from 1-3 to 48-52 amino acids in length. In another embodi the necessary elements for the, transcription and translation ment, a peptide linker of the invention is from about 10 to of the inserted coding sequence, or in the case of an RNA viral about 20 amino acids in length. In another embodiment, a vector, the necessary elements for replication and translation. peptide linker of the invention is from about 15 to about 50 0334. The nucleic acid encoding the chimeric protein is amino acids in length. In another embodiment, a peptide inserted into the vector in proper reading frame. The expres linker of the invention is from about 20 to about 45 amino sion vector is then transfected into a suitable target cell which acids in length. In another embodiment, a peptide linker of the will express the polypeptide. Transfection techniques known invention is from about 15 to about 35 or about 20 to about 30 in the art include, but are not limited to, calcium phosphate amino acids in length. In another embodiment, a peptide precipitation (Wigler et al. 1978, Cell 14: 725) and electropo linker of the invention is from about 1,2,3,4,5,6,7,8,9, 10, ration (Neumann et al. 1982, EMBO, J. 1: 841). A variety of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, host-expression vector systems may be utilized to express the 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000, or 2000 chimeric proteins described herein in eukaryotic cells. In one US 2015/03539 11 A1 Dec. 10, 2015 37 embodiment, the eukaryotic cell is an animal cell, including Cells which have integrated the DNA into their chromosomes mammalian cells (e.g. 293 cells, PerC6, CHO, BHK, Cos. may be selected by introducing one or more, markers which HeLa cells). When the chimeric protein is expressed in a allow selection of transfected host cells. The marker may eukaryotic cell the DNA encoding the chimeric protein may provide for prototrophy to an auxotrophic host, biocide resis also code for a signal sequence that will permit the chimeric tance (e.g., antibiotics) or resistance to heavy metals such as protein to be secreted. One skilled in the art will understand copper. The selectable marker gene can either be directly that while the protein is translated the signal sequence is linked to the DNA sequences to be expressed, or introduced cleaved by the cell to form the mature chimeric protein. into the same cell by cotransformation. Various signal sequences are known in the art e. g., native 0339. A preferred expression vector is NEOSPLA (U.S. factor VII signal sequence, native factor IX signal sequence Pat. No. 6,159,730). This vector contains the cytomegalovi and the mouse IgK light chain signal sequence. Alternatively, rus promoter/enhancer, the mouse beta globin major pro where a signal sequence is not included the chimeric protein moter, the SV40 origin of replication, the bovine growth can be recovered by lysing the cells. hormone polyadenylation sequence, neomycin phospho 0335 The chimeric protein of the invention can be synthe transferase exon 1 and exon 2, the dihydrofolate reductase sized in a transgenic animal. Such as a rodent, goat, sheep, pig, gene and leader sequence. This vector has been found to result or cow. The term “transgenic animals' refers to non-human in very high level expression of antibodies upon incorpora animals that have incorporated a foreign gene into their tion of variable and constant region genes, transfection in genome. Because this gene is present in germline tissues, it is cells, followed by selection in G418 containing medium and passed from parent to offspring. Exogenous genes are intro methotrexate amplification. Vector systems are also taught in duced into single-celled embryos (Brinster et al. 1985, Proc. U.S. Pat. Nos. 5,736,137 and 5,658,570, each of which is Natl. Acad.Sci. USA 82: 4438). Methods of producing trans incorporated by reference in its entirety herein. This system genic animals are known in the art including transgenics that provides for high expression levels, e.g., >30 pg/cell/day. produce immunoglobulin molecules (Wagner et al. 1981, Other exemplary vector Systems are disclosed e.g., in U.S. Proc. Natl. Acad. Sci. USA 78: 6376; McKnight et al. 1983, Pat. No. 6,413,777. Cell 34:335; Brinsteretal. 1983, Nature 306:332: Ritchie et 0340. In other embodiments the polypeptides of the inven al. 1984, Nature 312: 517: Baldassarre et al. 2003, Therio tion of the instant invention may be expressed using polycis genology 59: 831; Roblet al. 2003, Theriogenology 59: 107: tronic constructs. In these expression systems, multiple gene Malassagne et al. 2003, Xenotransplantation 10 (3): 267). products of interest Such as multiple polypeptides of multi 0336. The expression vectors can encode for tags that per mer binding protein may be produced from a single polycis mit for easy purification or identification of the recombi tronic construct. These systems advantageously use an inter nantly produced protein. Examples include, but are not lim nal ribosome entry site (IRES) to provide relatively high ited to, vectorpUR278 (Ruttier et al. 1983, EMBO.J. 2: 1791) levels of polypeptides of the invention in eukaryotic host in which the chimeric protein described herein coding cells. Compatible IRES sequences are disclosed in U.S. Pat. sequence may be ligated into the vector in frame with the lac No. 6,193.980 which is also incorporated herein. Those Z coding region so that a hybrid protein is produced; pGEX skilled in the art will appreciate that such expression systems vectors may be used to express proteins with a glutathione may be used to effectively produce the full range of polypep S-transferase (GST) tag. These proteins are usually soluble tides disclosed in the instant application. and can easily be purified from cells by adsorption to glu 0341 More generally, once the vector or DNA sequence tathione-agarose beads followed by elution in the presence of encoding a polypeptide has been prepared, the expression free glutathione. The vectors include cleavage sites (e. g. vector may be introduced into an appropriate host cell. That PreCission Protease (Pharmacia, Peapack, N.J.)) for easy is, the host cells may be transformed. Introduction of the removal of the tag after purification. plasmid into the host cell can be accomplished by various 0337 For the purposes of this invention, numerous expres techniques well known to those of skill in the art. These sion vector systems may be employed. These expression vec include, but are not limited to, transfection (including elec tors are typically replicable in the host organisms either as trophoresis and electroporation), protoplast fusion, calcium episomes or as an integral part of the host chromosomal DNA. phosphate precipitation, cell fusion with enveloped DNA, Expression vectors may include expression control microinjection, and infection with intact virus. See, Ridgway, sequences including, but not limited to, promoters (e.g., natu A. A. G. "Mammalian Expression Vectors' Chapter 24.2, pp. rally-associated or heterologous promoters), enhancers, sig 470-472 Vectors, Rodriguez and Denhardt, Eds. (Butter nal sequences, splice signals, enhancer elements, and tran worths, Boston, Mass. 1988). Most preferably, plasmid intro Scription termination sequences. Preferably, the expression duction into the host is via electroporation. The transformed control sequences are eukaryotic promoter systems in vectors cells are grown under conditions appropriate to the produc capable of transforming or transfecting eukaryotic host cells. tion of the light chains and heavy chains, and assayed for Expression vectors may also utilize DNA elements which are heavy and/or light chain protein synthesis. Exemplary assay derived from animal viruses such as bovine papilloma virus, techniques include enzyme-linked immunosorbent assay polyoma virus, adenovirus, vaccinia virus, baculovirus, ret (ELISA), radioimmunoassay (RIA), or flourescence-acti roviruses (RSV. MMTV or MOMLV), cytomegalovirus vated cell sorter analysis (FACS), immunohistochemistry and (CMV), or SV40 virus. Others involve the use of polycis the like. tronic systems with internal ribosome binding sites. 0342. As used herein, the term “transformation' shall be 0338 Commonly, expression vectors contain selection used in abroad sense to refer to the introduction of DNA into markers (e.g., amplicillin-resistance, hygromycin-resistance, a recipient host cell that changes the genotype and conse tetracycline resistance or neomycin resistance) to permit, quently results in a change in the recipient cell. detection of those cells transformed with the desired DNA 0343 Along those same lines, “host cells' refers to cells sequences (see, e.g., Itakura et al., U.S. Pat. No. 4,704,362). that have been transformed with vectors constructed using US 2015/03539 11 A1 Dec. 10, 2015 recombinant DNA techniques and encoding at least one het (that may be autocatalytically cleaved), a Subtilisin-like cata erologous gene. In descriptions of processes for isolation of lytic domain characterized by Asp, His, Ser, and ASn/Asp polypeptides from recombinant hosts, the terms “cell' and residues, and a Homo B domain that is, also essential for “cell culture' are used interchangeably to denote the source catalytic activity and characterized by an Arg-Gly-Asp of polypeptide unless it is clearly specified otherwise. In other (RGD) sequence. PACE, PACE4, and PC5 also include a words, recovery of polypeptide from the “cells' may mean Cys-rich domain, the function of which is unknown. In addi either from spun down whole cells, or from the cell culture tion, PC5 has isoforms with and without a transmembrane containing both the medium and the Suspended cells. domain; these different isoforms are known as PC5B and 0344) The host cell line used for protein expression is most PC5A, respectively. Comparison between the amino acid preferably of mammalian origin; those skilled in the art are sequence of the catalytic domain of PACE and the amino acid credited with ability to preferentially determine particular sequences of the catalytic domains of other, members of this host cell lines which are best suited for the desired gene family of proprotein convertases reveals the following product to be expressed therein. Exemplary host cell lines degrees of identity: 70 percent for PC4; 65 percent for PACE4 include, but are not limited to, DG44 and DUXB11 (Chinese and PC5; 61 percent for PC1/PC3:54 percent for PC2; and 51 Hamster Ovary lines, DHFR minus), HELA (human cervical percent for LPC/PC7/PC8/SPC7. Nakayama K (1997) Bio carcinoma). CVI (monkey kidney line), COS (a derivative of chem J. 327:625-35. CVI with SV40 T antigen), R1610 (Chinese hamster fibro (0349 PACE and PACE4 have been reported to have par blast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney tially overlapping but distinct Substrates. In particular, line), SP2/O (mouse myeloma), P3.times.63-Ag3.653 PACE4, in striking contrast to PACE, has been reported to be (mouse myeloma), BFA-1c1 BPT (bovine endothelial cells), incapable of processing the precursor polypeptide of FIX. RAJI (human lymphocyte), PerC6, and 293 (human kidney). Wasley L. C et al. (1993) J Biol Chem. 268:8458-65; Host cell lines are typically available from commercial ser Rehemtulla A et al. (1993) Biochemistry. 32:11586-90. vices, the American Tissue Culture Collection or from pub 0350 U.S. Pat. No. 5,840,529, issued to Seidah et al., lished literature. discloses nucleotide and amino acid sequences for human 0345. In one embodiment, a host cell endogenously PC7 and the notable ability of PC7, as compared to other PC expresses an enzyme (or the enzymes) necessary to cleave a family members, to cleave HIV gp160 to gp120 and gp41. ScFc linker (e.g., if such a linker is present and contains 0351 Nucleotide and amino acid sequences of rodent PC5 intracellular processing site(s)) during processing to form the were first described as PC5 by Lusson Jetal. (1993) Proc Natl mature polypeptide. During this processing, the sclc linker AcadSci USA 90:6691-5 and as PC6 by Nakagawa Tet al. may be substantially removed to reduce the presence of extra (1993) J Biochem (Tokyo) 113:132-5. U.S. Pat. No. 6,380, neous amino acids. In another embodiment of the invention, a 171, issued to Day et al., discloses nucleotide and amino acid host cell is transformed to express one or more enzymes sequences for human PC5A, the isoform without the trans which are exogenous to the cell Such that processing of a ScPc membrane domain. The sequences of these enzymes and linker occurs or is improved. method of cloning them are known in the art. 0346. In one embodiment an enzyme which may be 0352 Genes encoding the polypeptides of the invention endogenously or exogenously expressed by a cell is a member can also be expressed in non-mammalian cells Such as bac of the furin family of enzymes. Complete cDNA and amino teria or yeast or plant cells. In this regard it will be appreciated acid sequences of human furin (i.e., PACE) were published in that various unicellular non-mammalian microorganisms 1990. Van den Ouweland A M et al. (1990) Nucleic Acids Such as bacteria can also be transformed; i.e., those capable of Res. 18:664; Erratum in: Nucleic Acids Res. 18:1332 (1990). being grown in cultures or fermentation. Bacteria, which are 0347 U.S. Pat. No. 5,460.950, issued to Barr et al., susceptible to transformation, include members of the entero describes recombinant PACE and the coexpression of PACE bacteriaceae, Such as stains of Escherichia coli or Salmo with a substrate precursor polypeptide of a heterologous pro nella, Bacillaceae, such as Bacillus subtilis, Pneumococcus, tein to improve expression of active, mature heterologous Streptococcus, and Haemophilus influenzae. It will further be protein. appreciated that, when expressed in bacteria, the polypep 0348 U.S. Pat. No. 5,935,815, issued to van de Venet al., tides typically become part of inclusion bodies. The polypep likewise describes recombinant human furin (i.e., PACE) and tides must be isolated, purified and then assembled into func the coexpression of furin with a substrate precursor polypep tional molecules. tide of a heterologous protein to improve expression of active, 0353. In addition to prokaryates, eukaryotic microbes may mature heterologous protein. Possible Substrate precursors also be used Saccharomyces cerevisiae, or common baker's disclosed in this patent include a precursor of Factor IX. Other yeast, is the most commonly used among eukaryotic micro family members in the mammalian furin/subtilisin/Kex2p organisms although a number of other strains are commonly like proprotein convertase (PC) family in addition to PACE available. are reported to include PCSK1 (also known as PC1/Pc3), 0354 For expression in Saccharomyces, the plasmid PCSK2 (also known as PC2), PCSK3 (also known as furin or YRp7, for example, (Stinchcomb et al., Nature, 282:39 PACE), PCSK4 (also known as PC4), PCSK5 (also known as (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et PC5 or PC6), PCSK6 (also known as PACE4), or PCSK7 al., Gene, 10:157 (1980)) is commonly used This plasmid (also known as PC7/LPC, PC8, or SPC7). While these various already contains the TRP1 gene which provides a selection members share certain conserved overall structural features, marker for a mutant strain of yeast lacking the ability to grow they differin their tissue distribution, subcellular localization, in tryptophan, for example ATCC No. 44076 or PEP4-1 cleavage specificities, and preferred Substrates. For a review, (Jones, Genetics, 85:12 (1977)). The presence of the trpl see Nakayama K (1997) Biochem J. 327:625-35. Similar to lesion as a characteristic of the yeast host cell genome then PACE, these proprotein convertases generally include, begin provides an effective environment for detecting transforma ning from the amino terminus, a signal peptide, a propeptide tion by growth in the absence of tryptophan. US 2015/03539 11 A1 Dec. 10, 2015 39

0355. Other yeast hosts such Pichia may also be raphy, HPLC purification, gel electrophoresis and the like employed. Yeast expression vectors having expression con (see generally Scopes, Protein Purification (Springer-Verlag, trol sequences (e.g., promoters), an, origin of replication, N.Y., (1982)) and see specifically the methods used in the termination sequences and the like as desired. Typical pro instant Examples. Substantially pure proteins of at least about moters include 3-phosphoglycerate kinase and other glyco 90 to 95% homogeneity are preferred, and 98 to 99% or more lytic enzymes. Inducible yeast promoters include, among homogeneity most preferred, for pharmaceutical uses. others, promoters from alcohol dehydrogenase, isocyto 0362. In other embodiments, the chimeric clotting factor chrome C, and enzymes responsible for methanol, maltose, can be produced by combining recombinant DNA technology and galactose utilization. with chemical synthesis. For example, the present invention 0356 Alternatively, polypeptide-coding nucleotide includes a method of transfecting a host cell with a polynucle sequences can be incorporated in transgenes for introduction otide encoding a chimeric clotting factor comprising a light into the genome of a transgenic animal and Subsequent chain of a clotting factor, a protease cleavable site (e.g., expression in the milk of the transgenic animal (see, e.g., SUMO), a truncated heavy chain of the clotting factor, an Deboer et al., U.S. Pat. No. 5,741,957, Rosen, U.S. Pat. No. optional linker, and an enhancer moiety. Small Uoicquitin-like 5,304,489, and Meade et al., U.S. Pat. No. 5,849,992). Suit Modifier (or SUMO) is a member of the ubiquitin (Ub) and able transgenes include coding sequences for polypeptides in ubiquitin-like (Ubl) family. Post-translational attachment of operable linkage with a promoter and enhancer from a mam SUMO to target proteins occurs through an enzymatic cas mary gland specific gene. Such as casein or beta lactoglobulin. cade analogous to the ubiquitin conjugation cascade (E1-E2 0357. In vitro production allows scale-up to give large E3 enzymes), ultimately resulting in formation of an isopep amounts of the desired polypeptides. Techniques for mam tide bond between the Ub/Ubl C-terminal residue and malian cell cultivation under tissue culture conditions are Substrate lysine residue. known in the art and include homogeneous Suspension cul ture, e.g. in an airlift reactor or in a continuous stirrer reactor, 0363 SUMO Protease, a highly active cysteinyl protease or immobilized or entrapped cell culture, e.g. in hollow fibers, also known as Ulp, is a recombinant fragment of Ulp1 (Ubl microcapsules, on agarose microbeads or ceramic cartridges. specific protease 1) from Saccharomyces cerevisiae. SUMO If necessary and/or desired, the Solutions of polypeptides can Protease cleaves in a highly specific manner, recognizing the be purified by the customary chromatography methods, for tertiary structure of the ubiquitin-like (UBL) protein, SUMO, example gel filtration, ion-exchange chromatography, chro rather than an amino acid sequence. The protease can be used matography over DEAE-cellulose or (immuno-)affinity chro to cleave SUMO from recombinant fusion proteins. The matography, e.g., after preferential biosynthesis of a synthetic sequence of the SUMO protein comprises: hinge region polypeptide or prior to or Subsequent to the HIC chromatography step described herein. An affinity tag (SEO ID NO : 65) sequence (e.g. a His(6) tag) may optionally be attached or SLODSEVNOEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLM included within the polypeptide sequence to facilitate down EAFAKROGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREOIGG stream purification. 0358. In one embodiment, a host cell of the invention 0364. In some embodiments, the present invention comprises a genetic construct encoding a polypeptide com includes a method of transfecting a host cell with a polynucle prising a sce linker and one or more enzymes that can otide encoding a chimeric clotting factor comprising a light process a cscFc linker. The construct and the enzyme(s) can chain of a clotting factor, an optional intracellular processing be expressed using a single vector or two vectors. The chi site, a protease cleavable site (e.g., SUMO), a truncated heavy meric protein produced by the genetic construct encoding, a chain of the clotting factor, an optional linker, and an ScFc linker canthus have an additional polypeptide chain due enhancer moiety, wherein the chimeric clotting factor is to the intracellular processing. In some embodiments, the expressed. In certain embodiments, the truncated heavy chain chimeric protein may contain the cleaved protease cleavage does not comprise one or more amino acids from the N-ter site (e.g., RRRR). minus corresponding to the wildtype heavy chain. The heavy 0359. In one embodiment, the invention pertains to chain is missing one or more amino acids to expose a natu nucleic acid molecules which encode a polypeptide of the rally occurring cysteine residue on FVII or FX for chemical invention. In one embodiment, the nucleic acid molecule ligation to a thioester peptide. In one embodiment, the amino encodes a chimeric protein comprising an enhancer moiety acids missing from the truncated heavy chain are six amino and an activatable clotting factor selected from activatable acids, e.g., IVGGKV (SEQID NO: 60) for FVII or IVGGQE FVII or activatable FX, wherein the enhancer moiety (SEQID NO: 61) for FX. In another embodiment, the amino enhances activities of FVII and FX. In another embodiment, acids missing from the truncated heavy cahin are 11 amino the nucleic acid molecule encodes a chimeric protein com acids, e.g., IVGGKVCPKGE (SEQID NO: 62) for FVII or prising an enhancer moiety, an activatable clotting factor, and IVGGQECKDGE (SEQ ID NO: 63) for FX). In other optionally a linker moiety between the activatable clotting embodiments, the host cell further comprises a polynucle factor and the enhancer moiety. otide sequence encoding an intracellular processing enzyme, 0360. In another embodiment, the invention pertains to a thereby processing the light chain of the clotting factor from nucleic acid molecule encoding a polypeptide comprising the chimeric clotting factor. The light chain of the clotting FVII, which FVII which comprises a heterologous enzymatic factor can form a disulphide bond with the heavy chain of the cleavage site activatable by a component of the clotting cas clotting factor. cade. 0365. In certain embodiments, the method further com 0361. Once expressed, the chimeric clotting factor can be prises combining (or adding) a SUMO protease to the recom purified according to standard procedures of the art, including binantly expressed chimeric clotting factor, wherein the ammonium sulfate precipitation, affinity column chromatog SUMO protease cleaves SUMO from the chimeric clotting US 2015/03539 11 A1 Dec. 10, 2015 40 factor. The cleavage of SUMO can expose the N-terminus of any mucosal Surface, e.g., orally, Sublingually, buccally, Sub the truncated heavy chain of the clotting factor (e.g., CyS) for lingually, nasally, rectally, vaginally or via pulmonary route. further reaction. The chimeric protein can be implanted within or linked to a 0366. In other embodiments, the method further com biopolymer solid support that allows for the slow release of prises adding a thioester peptide to be linked to the N-termi the chimeric protein to the desired site. nus of the truncated heavy chain of the clotting factor, e.g., at 0372 For oral administration, the pharmaceutical compo Cys. In one embodiment, the thioester peptide can comprise a sition can take the form of tablets or capsules prepared by thrombin cleavage site (e.g., D-Phe-Pip-Arg). In another conventional means. The composition can also be prepared as embodiment, the thioester peptide comprises a thrombin a liquid for example a syrup or a Suspension. The liquid can cleavage site (e.g., D-Phe-Pip-Arg) and a self-immolative include Suspending agents (e.g. Sorbitol syrup, cellulose linker (e.g., PABC). In other embodiments, the thioester pep derivatives or hydrogenated edible fats), emulsifying agents tide comprises a thrombin cleavage site (e.g., D-Phe-Pip (lecithin or acacia), non-aqueous vehicles (e.g. almond oil, Arg), a self-immolative linker (e.g., PABC), and the one or oily esters, ethyl alcohol, or fractionated vegetable oils), and more amino acids identical to the amino acids missing from preservatives (e.g. methyl or propyl-p-hydroxybenzoates or the N-terminus of the truncated heavy chain of the clotting Sorbic acid). The preparations can also include flavoring, factor. In one embodiment, the one or more amino acids in the coloring and Sweetening agents. Alternatively, the composi thioester peptide comprises six amino acids missing from the tion can be presented as a dry product for constitution with truncated heavy chain (e.g., IVGGKV (SEQ ID NO: 60) for water or another suitable vehicle. FVII or IVGGQE (SEQ ID NO: 61) for FX). In another 0373 For buccal and sublingual administration the com embodiment, the one or more amino acids in the thioester position may take the form of tablets, lozenges or fast dis peptide comprises 11 amino acids missing from the truncated Solving films according to conventional protocols. heavy chain (e.g., IVGGKVCPKGE (SEQ ID NO: 62) for 0374 For administration by inhalation, the chimeric pro FVII or IVGGQECKDGE (SEQID NO: 63) for FX). There teins for use according to the present invention are conve fore, when the thioester peptide is fused to the truncated niently delivered in the form of an aerosol spray from a heavy chain of the clotting factor, the chimeric clotting factor pressurized pack or nebulizer (e.g. in PBS), with a suitable can comprise an activatable clotting factor, an optional linker, propellant, e.g., dichlorodifluoromethane, trichlorofluo and an enhancing moiety, wherein the activatable clotting romethane, dichlorotetrafluoromethane, carbon dioxide or factor comprises the thrombin cleavage site (e.g., D-Phe-Pip other suitable gas. In the case of a pressurized aerosol the Arg), the self-immolative linker (e.g., PABC), and the full dosage unit can be determined by providing a valve to deliver length heavy chain of the clotting factor. a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator can beformulated containing a IV. Methods of Administering Polypeptides of the Invention powder mix of the compound and a suitable powder base Such 0367 The invention also relates to a method of treating, as lactose or starch. ameliorating, or preventing a hemostatic disorder to a subject 0375. In one embodiment, the route of administration of comprising administering a therapeutically effective amount the polypeptides of the invention is parenteral. The term of a chimeric protein of the Invention. The treatment, ame parenteral as used herein includes intravenous, intraarterial, lioration, and prevention by the chimeric protein can be a intraperitoneal, intramuscular, Subcutaneous, rectal or vagi bypass therapy. The Subject in the bypass therapy may have nal administration. The intravenous form of parenteral already developed an inhibitor to a clotting factor, e.g., Factor administration is preferred. While all these forms of admin VIII, or is subject to developing a clotting factor inhibitor. istration are clearly contemplated as being within the scope of 0368 Compositions for administration to a subject the invention, a form for administration would be a solution include nucleic acid molecules which comprise a nucleotide for injection, in particular for intravenous or intraarterial sequence encoding a chimeric clotting factor of the invention injection or drip. Usually, a suitable pharmaceutical a Surfac (for gene therapy applications) as well as polypeptide mol tant (e.g. polysorbate), optionally a stabilizer agent (e.g. ecules. human albumin), etc. However, in other methods compatible 0369. In one embodiment, a chimeric protein composition with the teachings herein, the polypeptides can be delivered of the invention is administered in combination with at least directly to the site of the adverse cellular population thereby one other agent that promotes hemostasis. Said other agent increasing the exposure of the diseased tissue to the therapeu that promotes hemostasis in a therapeutic with demonstrated tic agent. clotting activity. As an example, but not as a limitation, hemo 0376 Preparations for parenteral administration include static agent can include Factor V. Factor VII, Factor VIII, sterile aqueous or non-aqueous solutions, Suspensions, and Factor IX, Factor X, Factor XI, Factor XII, Factor XIII, pro emulsions. Examples of non-aqueous are propylene thrombin, or fibrinogen or activated forms of any of the pre glycol, polyethylene glycol. Vegetable oils such as olive oil, ceding. The clotting factor of hemostatic agent can also and injectable organic esters such as ethyl oleate. Aqueous include anti-fibrinolytic drugs, e.g., epsilon-amino-caproic carriers include water, alcoholic/aqueous Solutions, emul acid, tranexamic acid. sions or Suspensions, including saline and buffered media. In 0370. In one embodiment of the invention, the composi the Subject invention, pharmaceutically acceptable carriers tion (e.g., the polypeptide or nucleic acid molecule encoding include, but are not limited to, 0.01-0.1M and preferably the polypeptide) is one in which the clotting factor is present 0.05M phosphate buffer or 0.8% saline. Other common in activatable form when administered to a subject. Such an parenteral vehicles include Sodium phosphate Solutions, activatable molecule can be activated in vivo at the site of Ringer's dextrose, dextrose and sodium chloride, lactated clotting after administration to a Subject. Ringer's, or fixed oils. Intravenous vehicles include fluid and 0371. The chimeric protein of the invention can be admin nutrient replenishers, electrolyte replenishers, such as those istered intravenously, Subcutaneously, intramuscularly, or via based on Ringer's dextrose, and the like. Preservatives and US 2015/03539 11 A1 Dec. 10, 2015

other additives may also be present such as for example, dosages may be titrated using routine methods knownto those antimicrobials, antioxidants, chelating agents, and inert of skill in the art to optimize safety and efficacy. and the like. 0382. In one embodiment, the dose of a biologically active 0377 More particularly, pharmaceutical compositions moiety (e.g., comprising FVII), can range from about 90 to Suitable for injectable use include sterile aqueous solutions 270 ug/kg or 0.090 to 0.270 mg/kg. In another embodiment, (where water soluble) or dispersions and sterile powders for the dose of a biologically active moiety (e.g., comprising FX), the extemporaneous preparation of sterile injectable solutions can range from about 1 lug/kg to 400 mg/kg. or dispersions. In Such cases, the composition must be sterile 0383 Dosages can range from 1000 ug/kg to 0.1 ng/kg and should be fluid to the extent that easy syringability exists. body weight. In one embodiment, the dosing range is lug/kg It should be stable under the conditions of manufacture and to 100 ug/kg. The protein can be administered continuously storage and will preferably be preserved against the contami or at specific timed intervals. In vitro assays may be employed nating action of microorganisms, such as bacteria and fungi. to determine optimal dose ranges and/or schedules for admin The carrier can be a or dispersion medium containing, istration. In vitro assays that measure clotting factor activity for example water, ethanol, polyol (e.g., glycerol, propylene are known in the art, e.g., STA-CLOTVIIa-rTF clotting assay. glycol and liquid polyethylene glycol, and the like), and Suit Additionally, effective doses may be extrapolated from dose able mixtures thereof. The proper fluidity can be maintained, response curves obtained from animal models, e.g., a hemo for example, by the use of a coating Such as lecithin, by the philiac dog (Mount et al. 2002, Blood 99 (8): 2670). maintenance of the required particle size in the case of dis 0384 Doses intermediate in the above ranges are also persion and by the use of Surfactants. intended to be within the scope of the invention. Subjects can 0378 Prevention of the action of microorganisms can be be administered Such doses daily, on alternative days, weekly achieved by various antibacterial and antifungal agents, for or according to any other schedule determined by empirical example, parabens, chlorobutanol, phenol, ascorbic acid, analysis. An exemplary treatment entails administration in thimerosal and the like. In many cases, it will be preferable to multiple dosages over a prolonged period, for example, of at include isotonic agents, for example, Sugars, polyalcohols, least six months. In some methods, two or more polypeptides Such as manntitol, Sorbitol, or Sodium chloride in the compo may be administered simultaneously, in which case the dos sition. Prolonged absorption of the injectable compositions age of each polypeptide administered falls within the ranges can be brought about by including in the composition an agent indicated. which delays absorption, for example, aluminum monostear 0385 Polypeptides of the invention can be administered ate and gelatin. on multiple occasions. Intervals between single dosages can be daily, weekly, monthly or yearly. Intervals can also be 0379. In any case, sterile injectable solutions can be pre irregular as indicated by measuring blood levels of modified pared by incorporating an active compound (e.g., a polypep polypeptide or antigen in the patient. Alternatively, polypep tide by itself or in combination with other active agents) in the tides can be administered as a Sustained release formulation, required amount in an appropriate solvent with one or a in which case less frequent administration is required. Dosage combination of ingredients enumerated herein, as required, and frequency vary depending on the half-life of the polypep followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile tide in the patient. vehicle, which contains a basic dispersion medium and the 0386 The dosage and frequency of administration can required other ingredients from those enumerated above. In vary depending on whether the treatment is prophylactic or the case of sterile powders for the preparation of sterile inject therapeutic. In prophylactic applications, compositions con able solutions, the preferred methods of preparation are taining the polypeptides of the invention or a cocktail thereof drying and freeze-drying, which yields a powder of are administered to a patient not already in the disease state to an active ingredient plus any additional desired ingredient enhance the patient's resistance or minimize effects of dis from a previously sterile-filtered solution thereof. The prepa ease. Such an amount is defined to be a “prophylactic effec rations for injections are processed, filled into containers such tive dose.” A relatively low dosage is administered at rela as ampoules, bags, bottles, Syringes or vials, and sealed under tively infrequent intervals over a long period of time. Some aseptic conditions according to methods known in the art. patients continue to receive treatment for the rest of their Further, the preparations may be packaged and sold in, the lives. form of a kit. Such articles of manufacture will preferably 0387 Polypeptides of the invention can optionally be have labels or package inserts indicating that the associated administered in combination with other agents that are effec compositions are useful for treating a Subject Suffering from, tive in treating the disorder or condition in need of treatment or predisposed to clotting disorders. (e.g., prophylactic or therapeutic). 0388. As used herein, the administration of polypeptides 0380. The pharmaceutical composition can also beformu of the invention in conjunction or combination with, an lated for rectal administration as a Suppository or retention adjunct therapy means the sequential, simultaneous, coexten enema, e.g., containing conventional Suppository bases Such sive, concurrent, concomitant or contemporaneous adminis as cocoa butter or other glycerides. tration or application of the therapy and the disclosed 0381 Effective doses of the compositions of the present polypeptides. Those skilled in the art will appreciate that the invention, for the treatment of conditions vary depending administration or application of the various components of upon many different factors, including means of administra the combined therapeutic regimen may be timed to enhance tion, target site, physiological state of the patient, whether the the overall effectiveness of the treatment. A skilled artisan patient is human or an animal, other medications adminis (e.g. a physician) would be readily be able to discern effective tered, and whether treatment is prophylactic or therapeutic. combined therapeutic regimens without undue experimenta Usually, the patient is a human but non-human mammals tion based on the selected adjunct therapy and the teachings of including transgenic mammals can also be treated. Treatment the instant specification. US 2015/03539 11 A1 Dec. 10, 2015 42

0389. It will further be appreciated that the polypeptides of include, but are not limited to, hemophilia A, hemophilia B, the instant invention may be used in conjunction or combi von Willebrand's disease, Factor XI deficiency (PTA defi nation with an agent or agents (e.g. to provide a combined ciency), Factor XII deficiency, as well as deficiencies or struc therapeutic regimen). Exemplary agents with which a tural abnormalities in fibrinogen, prothrombin, Factor V. Fac polypeptide of the invention may be combined include agents tor VII, Factor X, or Factor XIII. that represent the current standard of care for a particular 0398. In one embodiment, the hemostatic disorder is an disorder being, treated. Such agents may be chemical or bio inherited disorder. In one embodiment, the subject has hemo logic in nature. The term “biologic’’ or “biologic agent” refers philia A, and the chimeric protein comprises protease-acti to any pharmaceutically active agent made from living organ vatable Factor VII linked to or associated with an enhancer isms and/or their products which is intended for use as a moiety. In another embodiment, the Subject has hemophilia A therapeutic. and the chimeric clotting factor comprises protease-activat 0390 The amount of agent to be used in combination with able Factor VII linked to or associated with an enhancer the polypeptides of the instant invention may vary by Subject moiety. In another embodiment, the subject has hemophilia B or may be administered according to what is known in the art. and the chimeric protein comprises protease-activatable Fac See for example, Bruce A Chabner et al., Antineoplastic tor VII or Factor X linked to or associated with an enhancer Agents, in GOODMAN & GILMAN'S THE PHARMACOLOGICAL BASIS OF moiety. In another embodiment, the subject has inhibitory THERAPEUTICS 1233-1287 (Joel G. Hardman et al., eds.,9" ed antibodies to Factor VIII or Factor VIIIa and the chimeric 1996). In another embodiment, an amount of Such an agent clotting factor comprises protease-activatable Factor VII consistent with the standard of care is administered. linked to or associated with an enhancer moiety. In yet 0391 As previously discussed, the polypeptides of the another embodiment, the subject has inhibitory antibodies present invention, may be administered in a pharmaceutically against Factor IX or Factor IXa and the chimeric protein effective amount for the in vivo treatment of clotting disor comprises protease-activatable Factor VII linked to or asso ders. In this regard, it will be appreciated that the polypeptides ciated with an enhancer moiety. In other embodiments, the of the invention can beformulated to facilitate administration subject has inhibitory antibodies to Factor VIII or Factor and promote stability of the active agent. Preferably, pharma VIIIa and the chimeric clotting factor comprises protease ceutical compositions in accordance with the present inven activatable Factor X linked to or associated with an enhancer tion comprise a pharmaceutically acceptable, non-toxic, ster moiety. In yet another embodiment, the subject has inhibitory ile carrier Such as physiological saline, non-toxic buffers, antibodies against Factor IX or Factor IXa and the chimeric preservatives and the like. Of course, the pharmaceutical protein comprises protease-activatable Factor X linked to or compositions of the present invention may be administered in associated with an enhancer moiety. single or multiple doses to provide for a pharmaceutically 0399. The chimeric clotting factor of the invention can be effective amount of the polypeptide. used to prophylactically treat a Subject with a hemostatic 0392. In one embodiment, a chimeric clotting factor of the disorder. The chimeric clotting factor of the, invention can be invention can be administered as a nucleic acid molecule. used to treat an acute bleeding episode in a subject with a Nucleic acid molecules can be administered using techniques hemostatic disorder. known in the art, including via vector, plasmid, liposome, 0400. In one embodiment, the hemostatic disorder is the DNA injection, electroporation, gene gun, intravenously result of a deficiency in a clotting factor, e.g., Factor VII, injection or hepatic artery infusion. Vectors for use in gene Factor IX, or Factor VIII. In another embodiment, the hemo therapy embodiments are known in the art. static disorder can be the result of a defective clotting factor. 0393. In keeping with the scope of the present disclosure, 04.01. In another embodiment, the hemostatic disorder can the chimeric clotting factors of the invention may be admin bean acquired disorder. The acquired disorder can result from istered to a human or other animal in accordance with the an underlying secondary disease or condition. The unrelated aforementioned methods of treatment in an amount Sufficient condition can be, as an example, but not as a limitation, to produce a therapeutic or prophylactic effect. cancer, an autoimmune disease, or pregnancy. The acquired 0394 The chimeric proteins of the invention have many disorder can result from old age or from medication to treat an uses as will be recognized by one skilled in the art, including, underlying secondary disorder (e.g. cancer chemotherapy). but not limited to methods of treating, a Subject with a disease 0402. The invention also relates to methods of treating a or condition. The disease or condition can include, but is not Subject that does not have a hemostatic disorder or a second limited to, hemostatic disorders. ary disease or condition resulting in acquisition of a hemo 0395. In one embodiment, the invention relates to a static disorder. The invention thus relates to a method of method of treating a subject having a hemostatic disorder treating a Subject in need of a general hemostatic agent com comprising administering a therapeutically effective amount prising administering, atherapeutically effective amount of at of at least one chimeric protein of the invention. least, one chimeric protein of the invention. For example, in 0396 The chimeric proteins of the invention treat or pre one embodiment, the Subject in need of a general hemostatic vent a hemostatic disorder by promoting the formation of a agent is undergoing, or is about to undergo, Surgery. The fibrin clot. The chimeric protein of the invention can activate chimeric protein of the invention can be administered prior to any member of a coagulation cascade. The clotting factor can or after Surgery as a prophylactic. The chimeric protein of the be aparticipant in the extrinsic pathway, the intrinsic pathway invention can be administered during or after Surgery to con or both. trol an acute bleeding episode. The Surgery can include, but is 0397. A chimeric protein of the invention can be used to not limited to, liver transplantation, liver resection, or stem treat hemostatic disorders, e.g., those known to be treatable cell transplantation. with the particular clotting factor present in the chimeric 0403. In another embodiment, the chimeric protein of the protein. The hemostatic disorders that may be treated by invention can be used to treat a Subject having an acute bleed administration of the chimeric protein of the invention ing episode who does not have a hemostatic disorder. The US 2015/03539 11 A1 Dec. 10, 2015 acute bleeding episode can result from severe trauma, e.g., PC5 was subjected to protein A immunoprecipitation. Briefly, Surgery, an automobile accident, wound, laceration gun shot, cell culture Supernatant was mixed with approximately 50 ul or any other traumatic event resulting in uncontrolled bleed of protein A-Sepharose 50% slurry and incubated at 4° C. 1ng. with rocking for 1 hour, then centrifuged to pellet the protein 04.04 This invention is further illustrated by the following A beads. Beads were washed twice by resuspending in 1 ml of examples which should not be construed as limiting. The PBS, spinning and aspirating. The beads were resuspended contents of all references, patents and published patent appli with sodium dodecylsulfate-polyacrylamide gel electro cations cited throughout this application are incorporated phoresis (SDS-PAGE) buffer under reducing or nonreducing herein by reference. conditions, heated for 5 minutes at 100° C., spun down and loaded on SDS-PAGE gels and run according to standard EXAMPLES protocols. Under nonreducing conditions, 1 band with the 04.05 Throughout the examples, the following materials expected molecular weight for the thrombin-activatable and methods were used unless otherwise stated. FVII-Fc/sTF-Fc dimer was observed (FIG. 4C). Under reduc ing conditions 2 bands were observed representing the throm Example 1 bin-activatable FVII-Fc subunit and the sTF-Fc subunit. Cloning of FVII-133 Example 4 0406. The DNA sequence comprising nucleotides from Small Scale Purification of FVII-133 the HindIII site to the first EcoRI site of FVII-133 was syn thesized and subcloned into the HindIII/EcoRI sites of 0410 FcRn Load Adjustment Buffer (0.5 ml) were added pBUD-CE4.1 (Invitrogen), generating an intermediate con to 5 ml of filtered conditioned media from cells transiently struct. Next, the DNA region comprising nucleotides from the transfected with FVII-133. The pH-adjusted media (-5.5 ml) first EcoRI site to the Second EcoRI site of FVII-133 was was concentrated using a 30,000 MWCO, 15 ml centrifugal synthesized and subcloned into the EcoRI sites of the inter filter units (catalog #UFC 903008). The media was centri mediate construct to generate FVII-133 (FIG. 4A). fuged for 10' at 4000 rpm to ~200 ul volume, transferred to a tube and the volume adjusted to 400 ul with equilibration Example 2 buffer. 10 ul of FcRn resin were added and the mixture was rotated overnight, at 4°C. The conditioned media with resin Transient Expression of FVII-133 was loaded into mini-column and centrifuged for 30" at 2000 rpm. The column was washed extensively with equilibration 04.07 For expression of FVII-133, HEK-293-F cells were buffer. The protein was eluted with 30 ul of elution buffer. grown in Freestyle media (Invitrogen) Supplemented with Elution Buffer contains 50 mM Tris, 250 mM. NaCl, and vitamin K3 (Sigma Aldrich, St. Louis, Mo.) to 2 ug/liter 0.02% Tween-80 (a pH 7.5. FcRn Load Adjustment Buffer (growth media) as suspension cells at 37°C./10% CO2. Cells contains 0.5M MES and 0.2% Tween-80 (a) pH 6.0. FcRn were subcultured every three to four days by seeding at cell resin contains soluble FcRn conjugated to Sepharose 4 Fast density of 5x10 cells/ml. Flow. Equilibration buffer contains 10 mM MES, 250 mM 0408 Twenty-four hours prior to transfection, cells were NaCl, 0.02% Tween-80 (a) pH 6.2 seeded at a density of 7x10 cells/ml in growth media. On the 0411. The eluted material was analyzed by SDS PAGE day of transfection, a transfection solution was made with a under reducing conditions. Two bands with the, expected volume equal to 5% of the total volume of the cell culture to be transfected. In the transfection solution DNA was added molecular weight for the thrombin-activatable FVII-Fc and (final concentration 20 mg/L) to a freshly made solution of sTF-Fc subunits were observed (FIG. 5). PEI (60 mg/L) in growth media. The solution was swirled for Example 5 30 seconds and incubated for five minutes, at room tempera ture before adding directly to the cell culture. Four hours later Activity of FVII-133 by Prothrombin Time Assay a volume equal to the cell culture volume of OptiCHO (Invit rogen) supplemented with vitamin K3 and 200 mM 0412. The activity of the FVIIaFc and purified FVII-133 L-glutamine was added to the cells. The cell culture was was determined by the prothrombin time using Dade Innovin allowed to grow as shown above and daily media samples reagents (Siemens catalog number 539196), which measure were taken to assess protein expression. On the day of har the activity of total FVII, including FVIIa. Manufacturer rec vest, the cells were spun down and the media filtered, in ommendations were followed. We observed an activity of preparation for protein purification or protein analysis by approximately 10,000 IU/mg for FVIIaFc but the activity of protein A pulldown. For expression of FVII-133, a plasmid FVII-133 was below the level of quantitation. Therefore, in encoding FVII-133 was contransfected with a plasmid encod the absence of thrombin, FVII-133 remained as a zymogen ing the propeptide endopeptidase PC5 to ensure cleavage of and inactive form. the propeptide endopeptidase sites in the linker connecting the Fc to sTF (FIG. 4A). Example 6 Example 3 Activity FVII-133 and FVI FVIIaFc in Thrombin Generation Assays Analysis of Protein Generated from Transient Transfections 0413. Thrombin generation assays were performed in a Fluoroskan Ascent fluorometer (Thermo Scientific) and 04.09 For analysis of protein from transient transfections, reagents and analysis Software from Thrombinoscope, the conditioned media from cotransfections of FVII-133 with according to manufacturer's recommendations. Briefly, US 2015/03539 11 A1 Dec. 10, 2015 44 human platelets were washed in 5.4 mM trisodium citrate, Example 9 146 mM NaCl, pH 6.8 and resuspended in FVIII-deficient human plasma (Siemens) to generate platelet-rich plasma In Vitro Activity of FVII-133 in Human Hemophilia (PRP) at a platelet concentration of 2x10 platelets/ml. Each A Blood by Rotational Thromboelastometry Assays reaction contained FVIII-deficient PRP calibrator (Thrombi 0416) To determine the clotting activity, FVII-133 protein noscope) or Tyrode's buffer (15 mM Hepes pH 7.4, 138 mM was spiked into citrated human whole blood from hemophilia NaCl, 2.7 mMKC1, 1 mMMgCl, 5.5 mM dextrose, 1 mg/ml A donors. Clot was initiated by adding CaCl2, the clotting BSA), FVII-133 or FVIIaFc (50 nM final concentration), and time, clotting formation time, and a-angle were measured on lipidated tissue factor (PRP reagent at a 1/8 dilution, Throm the ROTEM analyzer (Pentapharm) following the manufac binoscope) where indicated. Tissue factor, as a complex with turer's recommendations. FVIIa and FVII-184 were tested in endogenous FVIIa, is expected to activate the reaction by parallel; whereas FVIIa was used as a comparator for activity, inducing the generation of Small amounts of thrombin. As FVII-184 was employed as a control for determining the shown in FIG. 6, FVII-133 displayed much greater activity contribution of endogenous FVIIa that could be activated by than FVIIaFc in the presence or in the absence of tissue factor the fusion protein itself, because FVII-184 was designed to be (TF). Interestingly FVII-133 showed similar activity in the insensitive to thrombinactivation by mutating the Arg essen presence or in the absence of TF, but prothrombin time assays tial for the thrombin cleavage site to Ala, but otherwise the show that FVII-133 has no activity in the absence of throm same as FVII-133. As shown in FIG. 8, the clotting time in bin. This suggests that trace amounts of thrombin or TF (that this hemophilia A donor recorded to be 2500 sec. Spiking can in turn generate thrombin with endogenous FVIIa) in the FVII-133 in the whole blood resulted in a much shorter clot PRP are sufficient to activate FVII-133. Furthermore, these ting time in a dose dependent manner. The clotting time data show that FVII-133 has the potential for high activity recorded on 10 and 2 nM of FVII-133 was shorter than that of once activated. 50, 10 nM of rFVIIa respectively. Based on its clotting time Example 7 profile, the activity of FVII-133 was estimated to be at least 10-fold higher than that of FVIIa. The faster clotting time of Activity of FVII-433 and FYIIaFc in Rotational FVII-133 correlated with its shorter clotting formation time Thromboelastometry Assays and higher C.-angle, which agree with the higher hemostatic activity of FVII-133. Inactivating the thrombin cleavage site 0414 Citrated blood from FIX-deficient mice was used of FVII133 considerably reduced the activity; as the activity for these experiments. Thromboelastography was carried out of FVII-184, which only became detectable at 50 nM, was in a ROTEM analyzer (Pentapharm) according to the manu much lower than that of FVIIa, indicating the activity of facturer's recommendations. Briefly, 280 ul of blood were FVII-133 was indeed contributed by its own thrombin acti transferred to a prewarmed ROTEM plastic cup and spiked vated FVIIa, but not by the endogenous FVII/FVIIa. with FVIIaFc or FVII-133 to a final concentration of 50 nM. EXTEM reagent (TF and Calcium) was added to initiate the Example 10 reaction. Clotting time (CT) and alpha angle were measured. As shown in FIG. 7, both FVII-133 and FVIIaFc displayed Ex Vivo Efficacy of FVII-133 in hemB Mice by clotting times and alpha angles that were significantly lower ROTEM Analysis or higher, respectively, than vehicle. Lower clotting times and higher alpha angles indicate increased hemostatic activity. 0417. To assess the ex vivo efficacy, hemophilia B (hemB) These data show that FVII-133 can display high activity when mice were dosed at 20 nmol/kg of FVII-133 or control protein activated by thrombin. rFVIIa via tail vein injection. At 2.5 hour after dosing, blood was collected in CTI at 9:1 ratio from mice via vena cava Example 8 bleeding. Blood was re-calcified, and immediately measured on the ROTEM analyzer. As shown in FIG. 9, whereas the Generation of Alternative Chimeric Clotting Factors average clotting time in hemB blood measured was about Comprising an Activatable FVII and an Enhancer 1800 sec, the clotting time of whole blood from mice injected Moiety with FVII-133 was much shorter, approaching 500 sec, indi 0415 Constructs will be generated for the expression of cating that FVII-133 was active in vivo. thrombin-activatable FVII followed by a linker and an Example 11 enhancer moiety (also referred to as “an enhancer moiety') as illustrated in FIG. 2. In one embodiment, the enhancer moiety Improved PK Profile of FVII-133 Over rFVIIaFc will be soluble tissue factor (residues 1-219 from mature sequence). In another embodiment, the enhancer moiety will 0418 FVII-133 was designed to circulate as FVII be procoagulant peptides SYN3731 or SYN3524 (see Intl Zymogen, thus it is predicted to be less Susceptible to anti Appl. No. PCT/US2012/041777, filed Jun. 9, 2012 and pub thrombin III (ATIII) mediated clearance. To evaluate the lished as WO 2012/170969, which is incorporated herein by pharmacokinetic properties of FVII-133, the purified FVII reference in its entirety). In another embodiment the enhancer 133, rFVIIaFc and rFVIIa were administrated intravenously moiety will be an antibody fragment derived from antibodies into hemB mice (n=4) at 10 nmol/kg, and plasma samples that increase the activity of FVIIa. For example, antibodies were collected via Vena cava bleeding at various times and that enhance the activity of FVIIa were described in Andersen analyzed for FVII antigen, and FVII-ATIII complex by L Metal.J. Biol Chem. 287: 8994-9001 (Jan. 24, 2012). These ELISA assays. Pharmacokinetic parameters were assessed by constructs will be transiently expressed in mammalian cells, PK modeling using Phoenix program (Pharsight). Since purified and tested for activity (prothrombin time, thrombin rFVIIa was cleared much more rapidly than either FVII-133 generation and ROTEM assays) as previously described. or rFVIIaFc (FIG. 10A), we used rFVIIaFc as a comparator US 2015/03539 11 A1 Dec. 10, 2015

for the pharmacokinetic analysis of FVII-133 (FIG. 10B). PK properties; and 3) resistance to ATIII inhibition contrib Plasma concentration of the protein Versus time curve was utes, at least partly, to the PK improvement in FVII-212. found to best fit in a two compartmental model; all the PK parameters indicated a marked improvement of FVII-133 Example 13 over rRVIIaFc, with longer terminal half-life (Beta halflife of 16.56 hour vs 7.78 hour, respectively) and longer mean resi Amidolytic Activity of FVII-212 dence time (MRT 19.29 hour vs. 7.45 hour) as shown in Table 0424 Amidolytic activity of FVII-212 was measured 4. before and after thrombin activation using a chromozyme t-PA substrate. For thrombin activation, FVII-212 (100 nM) TABLE 4 was treated with thrombin (50 nM) for 20 minutes at 37° C. PK Parameters: FWII-133 wSFVIIaFc. Thrombin was subsequently inhibited with hirudin (250 nM). 0425. As FIG. 15 shows, FVII-212 displayed no activity Alpha CL AUC prior to thrombin activation. Following thrombin activation, t12 Beta t 2 MRT (mL/ Vss (hour * the amidolytic activity associated with FVII-212 was greater Treatment (hour) (hour) (hour) hour?kg) (mL/kg) pmol/mL) than that observed for equal molar levels of rEVIIa. These FVII-133 O43 16.56 1929 17.59 339.31 738.96 data demonstrate that the activity of FVII-212 is dependent on FWIIaFC 1.41 7.78 7.45 73.15 S44.79 273.42 thrombin activation. 0419. In addition, whereas a significant amount of rFVI Example 14 IaFc/ATIII complex was detected, the FVII-133/ATIII com plex in plasma was almost undetectable. Together, these Thrombin-Activatable Procoagulant Compounds results indicated that FVII-133 successfully escaped the with PABC Self-Immolative Linker ATIII-mediated clearance, leading to a marked improvement 0426 Six different peptides, designated Compound 1 to 6, in PK profile over rFVIIaFc. were used in the experiments disclosed herein (TABLE 5). The sequence in Compounds 1 to 6 reproduces the coupling Example 12 of a thrombin cleavable substrate and a self-immolative spacer to the N-terminus of a clotting factor or a fragment Prolonged Ex Vivo Efficacy of FVII-212 Over thereof, in this specific example, FX. These compounds com rFVIIa in HemA Mice prise the six N-terminal amino acid residues of the heavy 0420 FVII-212 is identical to FVII-133, but encoded by chain of the FXa clotting factor, Ile-Val-Gly-Gly-Gln-Glu, an improved expression vector. Similar to FVII-133, FVII and serve as a model to show the applicability of the proco 212 displayed higher in vitro clotting activity than rFVIIa in agulant compound design disclosed hereinto clotting factors. human HemA blood per ROTEMassays (FIG. 11). In mouse Hema blood, however, the activity of FVII-212 was found to TABLE 5 be similar to rRVIIa (FIG. 12), indicating FVII-212 is less Compound Structure active in mouse than human. 1 (D-Phe)-Pip-Arg-PABC-Ile-Val-Gly-Gly-Gln-Glu-NH2 0421. To assess the ex vivo efficacy of FVII-212 and cor (D-Phe)-Pip-Arg-Ile-Val-Gly-Gly-Gln-Glu-NH2 roborate the finding by example 10 (ex vivo efficacy of FVII Ala-Leu-Arg-Pro-Arg-Ile-Val-Gly-Gly-Gln-Glu-NH2 133 in HemB mice), FVII-212 was administrated in Hema (D-Phe)-Pro-ArgPABC-Ile-Val-Gly-Gly-Gln-Glu-NH2 mice at 10 nmol/kg, blood was sampled at various times, and Ala-Leu-Val-Pro-ArgPABC-Ile-Val-Gly-Gly-Gln-Glu-NH2 the clotting activity was measured by ROTEM analyzerunder Ala-Leu-Val-Pro-Arg-Ile-Val-Gly-Gly-Gln-Glu-NH2 NATEM program. rEVIIa was included in parallel as a com parator. As shown in FIG. 13, although the clotting time from Pip is pipecolic acid, the blood collected at 5 min following dosing was similar (D-Phe) is a-Phenylalanine. between rFVII-212 and rRVIIa treated groups, the blood col The sequences of the thrombin substrate are underlined. lected from the later time points of FVII-212 group clotted The location PABC self-immolative linker is indicated by a box. faster than that from the corresponding time point of rEVIIa 0427 FIG. 18 depicts the cleavage of Compounds 1, 2 and group. The results indicate the prolonged ex-Vivo efficacy of 3 by 14 nM thrombin. In this specific example, 50 L of FVII-2121 compared to rFVIIa. peptide (1 mM) in water was added to 900 uL PBS, followed 0422. As indicated in Example 11, FVII-133 displayed by 50 uL of thrombin (278 nM, 10 g/mL), giving the fol improved PK properties over rRVIIaFc in HemB mice, which lowing approximate initial concentrations: thrombin 14 nM. were thought to contribute to the prolonged efficacy of this peptide=50LM. The mixture was incubated at room tempera protein. Similar to FVII-133 in HemB mice, the clearance of ture. Aliquots (95 uL) at various time points were quenched FVII-212 in HemA mice was considerably slower than that of with 5uL of hirudin (2M) and injected into the HPLC (C-18 rFVIIa (FIG. 14), represented by a longer terminal half-life of column, CHCN/HO, 0 to 70% over 12 minutes, 60° C. 0.5 about 18 hours (compared to the half-life about 1 hour in mL/min, v280 nm). The decreases of peptide peak areas Hema plasma of rEVIIa when measured by rFVIIas activity) were used to calculate yield. as well as the absence of rFVII-212-ATIII complex in treated 0428 Compared to Compounds 2 and 3, the construct HemA mice. incorporating the thrombin-cleavable synthetic Substrate 0423 Taken together, this example extended the previous D-Phe-Pip-Arg and the self-immolative spacer PABC (Com finding and demonstrated that 1) FVII-212 displays pro pound 1) was a better substrate for thrombin. The incorpora longed ex vivo efficacy over rRVIIa in Hema mice; 2) the tion of PABC to Compound 1 led to at least 10-fold increase prolonged efficacy of FVII-212 is resulted from its improved in cleavage rate compared to that of Compound 2. US 2015/03539 11 A1 Dec. 10, 2015 46

0429 FIG. 19 depicts the cleavage of Compounds 1, 4, 5 MES 100 mM. NaCl, pH 6.2, the bound material was eluted and 6 by 1.4 nM thrombin. Compounds 1,4 and 5 incorporate with 10 mM Tris, 250 mM. NaCl, pH 8.0 and analyzed with PABC and different thrombin-cleavable substrates. 50 uL of SDS-PAGE. peptide (1 mM) in water was added to 900 uL PBS. The 0434 FVII-186 was cleaved by a SUMO protease as fol mixture was incubated at 37°C. for 30 min, followed by 50LL lows. FVII-186 (0.83 mg/mL, 10 uI) was incubated with 10 of thrombin (27.8 nM, 1 g/mL), giving the following uL of 100 mM HEPES, 20 mM CaCl, 0.004% Tween 80 approximate initial concentrations: thrombin -1.4 nM, pep containing 0.4 mM oxidized Glutathione (GSSG), 20 nM tide-50 uM. The mixture was incubated at 37° C. Aliquots Glutathione (GSH), 0.2 uL SUMO protease (Invitrogen Cat. (95 uL) at various time points were quenched with 5 uL of No. 12588-018) for 48 hours at room temperature. Reducing hirudin (2 uM) and injected into the HPLC (C-18 column, 51)5-PAGE (FIG. 23, lane 3) showed almost complete con CHCN/HO, 0 to 70% over 12 minutes, 60° C. 0.5 mL/min, version of FVII-186 to the desired FVIIHC. v=280 nm) The decreases of peptide peak areas were used to 0435 For SUMO protease cleavage of FVII-186 and calculate yield. native chemical ligation with a thioester peptide, FVII-186 0430 Compound 1 was a better substrate for thrombin (0.83 mg/mL, 10 uL) was incubated with 10 uI of 100 mM than Compounds 4 and 5. At 1.4 nM, a physiological relevant HEPES, 20 mM.CaCl, 0.004% Tween 80 containing 0.4 mM concentration of thrombin, 30% of Compound 1 was quickly SYN470 as a positive control peptide, 0.4 mM GSSG, 20 mM cleaved and released. In contrast, thrombin-mediated release GSH, 0.2 U/uL SUMO protease (Invitrogen Cat. No. 12588 of peptide IVGGQE from Compound 6 without PABC linker 018) for 48 hours at room temperature. Reducing SDS-PAGE was not observed. (Figure it, lane 4) showed complete disappearance of the FVIIHC band and a single new band as the conjugate of the Example 15 positive peptide control and the FVIIHC. 0436. In order to synthesize Thrombin Activatable FVII Thrombin Activatable FVII-186 with SUMO 186 (TA-FVII-186), FVII-186 (0.83 mg/mL, 200 uL) was Cleavage Site incubated with 200 uL of 100 mM HEPES, 20 mM CaCl, 0.004% Tween 80 containing 0.4 mM FVII-PABC peptide 0431. For cloning of FVII-186, the DNA sequence com (i.e., D-Phe-Pip-Arg-PABC-IVGGKV-COSBn) (SEQ ID prising nucleotides from the HindIII site to the EcoRI site of NO: 66), 0.4 mM GSSG, 20 mM GSH, 0.2 U/uL SUMO FVII-186 (Table it) was synthesized. The DNA was sub protease (Invitrogen Cat. No. 12588-018) for 48 hours at cloned into the HindIII/EcoRI sites of pcDNA. room temperature and analyzed by reducing SDS-PAGE 0432) To transiently express FVII-186, HEK-293-F cells (FIG. 23, lane 5). Reaction mixture was placed in a 0.5 mL were grown in suspension in FREESTYLE(R) media (Invitro dialysis cassette with 10k MWCO and dialyzed against 1 L of gen) supplemented with vitamin K3 (Sigma Aldrich, St. 10 mM Tris, 250 mM. NaCl, pH 8.0 containing 0.4 mM Louis, Mo.) to 2 ug/liter (growth media) as Suspension cells at GSSG, 2 mM GSH for 24 hours at 4°C. The conjugate was 37° C./10% CO. Cells were subcultured every three to four further purified by rhFcRn-sepharose column as described. days by seeding at cell density of 5x10 cells/ml. Twenty-four 0437 FVIIa Chromogenic assay was performed after hours prior to transfection, cells were seeded at a density of Thrombin cleavage and activation of TA-FVII-186 (FIG. 24). 7x10 cells/ml in growth media. On the day of transfection, a This assay measures the FX activation activity by measuring transfection solution was made with a volume equal to 5% of the ability of FVIIa to activate FX, as determined by measur the total volume of the cell culture to be transfected. In the ing levels of a chromogenic Substrate that is cleaved by acti transection solution, DNA was added (final concentration 20 vated FX (FXa). TA-FVII-186 (200 nM) was activated with mg/L) to a freshly made solution of PEI (60 mg/L) in growth Thrombin (140 nM) for 20 minutes at 37° C. Hirudin was media. The solution was swirled for 30 seconds and incubated added to quench Thrombin. STF-PL mixture (ASTACLOTR for five minutes at room temperature before adding directly to FVII-rTF kit), FX, and PEFACHROME(R) FXa substrate were the cell culture. Four hours later a volume equal to the cell added and reaction was monitored by measuring absorbance culture volume of OPTICHOTM (Invitrogen) supplemented at 405 nm. FVII-186 missing the six N-terminal amino acids with vitamin K3 and 200 mM L-glutamine was added to the was not active in the presence of thrombin. Only TA-FVII cells. The cell culture was allowed to grow as shown above 186 with a thrombin cleavage site connected to the complete and daily media samples were taken to assess protein expres heavy chain FVII showed activity after thrombin cleavage. sion. On the day of harvest, the cells were spun down, and the The resulted activity demonstrated that the FVII PABC pep media filtered in preparation for protein purification or pro tide was successfully conjugated to the N-terminal cysteine of tein analysis by protein A pulldown. For expression of FVII the truncated heavy chain of FVII, the crucial N-terminal 186, a plasmid encoding FVII-186 was contransfected with a isoleucine residue was generated upon cleavage by thrombin, plasmid encoding the proprotein convertase PACE to ensure and the formed protein had the essential structure for activity. intracellular processing and cleavage of the proprotein con 0438. The present invention has been described above vertase cleavage sites (2x(RKR) SEQID NO3) in the linker with the aid of functional building blocks illustrating the connecting the FVII light chain to SUMO (FIG.22). implementation of specified functions and relationships 0433) To purify FVII-186, conditioned medium was thereof. The boundaries of these functional building blocks loaded onto a 25-mL column of Q SEPHAROSE(R) Fast Flow have been arbitrarily defined herein for the convenience of the (GE HealthCare Life Sciences) after adjustment of pH to 7.4 description. Alternative boundaries can be, defined so long as with 2.0 M Tris, pH 8.0. Column was washed with 10 mM the specified functions and relationships thereof are appro MES, 50mM NaCl, pH 6.5. The protein was eluted with 10 priately performed. mM MES, 100 mM. NaCl, 20 mM CaCl pH 6.5. The frac 0439. The foregoing description of the specific embodi tions containing FVII-186 were pooled and loaded onto a ments will so fully reveal the general nature of the invention 25-mL column of rhFcRn-sepharose after adjustment of pH that others can, by applying knowledge within the skill of the to 6.2 with 0.5 M MES, pH 5.5. After washing with 50 mM art, readily modify and/or adapt for various applications such

US 2015/03539 11 A1 Dec. 10, 2015 54

- Continued

SEQUENCES

SEO ID NO: 4 >HAPylation motif., n = 1 to 400 (Gly4Ser) in

SEO ID NO. 59 CTP DSSSSKAPPPSLPSPSRLPGPSDTPILPQ

SEO ID NO: 65 SUMO SLODSEVNOEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKRTTPLERLMEAFAKR QGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGG

SEO ID NO: 66 >FVII-PABC Peptide D-Phe-Pip-Arg-PABC-IVGGKV-COSBn

SEQUENCE LISTING

<16O is NUMBER OF SEO ID NOS: 64

<210s, SEQ ID NO 1 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: cleavage site <4 OOs, SEQUENCE: 1 Ala Lieu. Arg Pro Arg 1. 5

<210s, SEQ ID NO 2 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: intracellular processing site <4 OOs, SEQUENCE: 2 Arg Arg Arg Arg 1.

<210s, SEQ ID NO 3 &211s LENGTH: 6 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: intracellular processing site <4 OOs, SEQUENCE: 3 Arg Lys Arg Arg Lys Arg 1. 5

<210s, SEQ ID NO 4 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: peptide linker 22 Os. FEATURE: <221s NAME/KEY: REPEAT <222s. LOCATION: (1) . . (5) US 2015/03539 11 A1 Dec. 10, 2015 55

- Continued <223> OTHER INFORMATION: may be repeated 1-400 times <4 OOs, SEQUENCE: 4 Gly Gly Gly Gly Ser 1. 5

<210s, SEQ ID NO 5 &211s LENGTH: 6 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: peptide linker 22 Os. FEATURE: <221s NAME/KEY: REPEAT <222s. LOCATION: (2) . . (6) <223> OTHER INFORMATION: n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 46, 50, 55, 60, 70, 80, 90 or 100

<4 OOs, SEQUENCE: 5 Ser Gly Gly Gly Gly Ser 1. 5

<210s, SEQ ID NO 6 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: cleavage site < 4 OO SEQUENCE: 6 Thr Glin Ser Phe Asn Asp Phe Thr Arg 1. 5

<210s, SEQ ID NO 7 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: cleavage site <4 OO > SEQUENCE: 7 Ser Val Ser Glin Thr Ser Lys Lieu. Thr Arg 1. 5 1O

<210s, SEQ ID NO 8 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: cleavage site <4 OOs, SEQUENCE: 8 Asp Phe Lieu Ala Glu Gly Gly Gly Val Arg 1. 5 1O

<210s, SEQ ID NO 9 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: cleavage site

<4 OOs, SEQUENCE: 9 Thir Thr Lys Ile Llys Pro Arg 1. 5 US 2015/03539 11 A1 Dec. 10, 2015 56

- Continued

<210s, SEQ ID NO 10 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: cleavage site <4 OOs, SEQUENCE: 10 Lieu Val Pro Arg Gly 1. 5

<210s, SEQ ID NO 11 &211s LENGTH: 488 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 11 Met Gly Arg Pro Lieu. His Lieu Val Lieu. Lieu. Ser Ala Ser Lieu Ala Gly 1. 5 1O 15 Lieu. Lieu. Lieu. Lieu. Gly Glu Ser Lieu. Phe Ile Arg Arg Glu Glin Ala Asn 2O 25 3O Asn. Ile Lieu Ala Arg Val Thr Arg Ala Asn. Ser Phe Lieu. Glu Glu Met 35 4 O 45 Llys Lys Gly. His Lieu. Glu Arg Glu. Cys Met Glu Glu Thir Cys Ser Tyr SO 55 6 O Glu Glu Ala Arg Glu Val Phe Glu Asp Ser Asp Llys Thr Asn. Glu Phe 65 70 7s 8O Trp Asn Llys Tyr Lys Asp Gly Asp Glin Cys Glu Thir Ser Pro Cys Glin 85 90 95 Asn Glin Gly Lys Cys Lys Asp Gly Lieu. Gly Glu Tyr Thr Cys Thr Cys 1OO 105 11 O Lieu. Glu Gly Phe Glu Gly Lys Asn. Cys Glu Lieu. Phe Thr Arg Llys Lieu. 115 12 O 125 Cys Ser Lieu. Asp Asn Gly Asp Cys Asp Glin Phe Cys His Glu Glu Glin 13 O 135 14 O Asn Ser Val Val Cys Ser Cys Ala Arg Gly Tyr Thr Lieu Ala Asp Asn 145 150 155 160 Gly Lys Ala Cys Ile Pro Thr Gly Pro Tyr Pro Cys Gly Lys Glin Thr 1.65 17O 17s Lieu. Glu Arg Arg Lys Arg Ser Val Ala Glin Ala Thir Ser Ser Ser Gly 18O 185 19 O Glu Ala Pro Asp Ser Ile Thir Trp Llys Pro Tyr Asp Ala Ala Asp Lieu. 195 2OO 2O5 Asp Pro Thr Glu ASn Pro Phe Asp Lieu. Lieu. Asp Phe Asin Glin Thr Glin 21 O 215 22O

Pro Glu Arg Gly Asp Asn. Asn Lieu. Thir Arg Ile Val Gly Gly Glin Glu 225 23 O 235 24 O

Cys Lys Asp Gly Glu. Cys Pro Trp Glin Ala Lieu. Lieu. Ile Asin Glu Glu 245 250 255

Asn Glu Gly Phe Cys Gly Gly. Thir Ile Leu Ser Glu Phe Tyr Ile Leu 26 O 265 27 O Thir Ala Ala His Cys Lieu. Tyr Glin Ala Lys Arg Phe Llys Val Arg Val 27s 28O 285

US 2015/03539 11 A1 Dec. 10, 2015 58

- Continued gcggtgcacg aggtggaggit ggt catcaag cacaa.ccggit to acaaagga gacct atgac 96.O titcgacat cq ccgtgctic cq got caaga cc cc.cat cacct tcc.gcatgaa cqtggcgc.ct O2O gcctgcct Co. ccgagcgtga Ctgggc.cgag tocacgctga tigacgcagaa gacggggatt O8O gtgagcggct tcgggcgcac ccacgagaag ggc.cggcagt ccaccaggct Caagatgctg 14 O gaggtgcc ct acgtggaccg caa.ca.gctgc aagctgtc.ca gcagctt cat cat caccCag 2OO alacatgttct gtgc.cggcta cacaccalag caggaggatg cct gccaggg gga cagcggg 26 O ggc.ccgcacg tcaccc.gctt Caaggacacic tactt.cgtga Caggcatcgt cagctgggga 32O gagggctgtg ccc.gta aggg galagtacggg atctacacca agg to accgc Ctt cotcaag 38O tggat.cgaca ggtc.catgaa alaccaggggc titgcc calagg cca agagcca tocc C9gag 44 O gtcatalacgt cct ct c catt aaagtga 467

<210s, SEQ ID NO 13 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: cleavage site <4 OOs, SEQUENCE: 13 Llys Lieu. Thir Arg 1.

<210s, SEQ ID NO 14 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: cleavage site <4 OOs, SEQUENCE: 14 Asp Phe Thir Arg 1.

<210s, SEQ ID NO 15 &211s LENGTH: 295 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 15 Met Glu Thr Pro Ala Trp Pro Arg Val Pro Arg Pro Glu Thir Ala Val 1. 5 1O 15 Ala Arg Thir Lieu Lleu Lieu. Gly Trp Val Phe Ala Glin Val Ala Gly Ala 2O 25 3O Ser Gly. Thir Thr Asn Thr Val Ala Ala Tyr Asn Lieu. Thir Trp Llys Ser 35 4 O 45

Thr Asn Phe Llys Thr Ile Lieu. Glu Trp Glu Pro Llys Pro Val Asn Glin SO 55 6 O

Val Tyr Thr Val Glin Ile Ser Thr Lys Ser Gly Asp Trp Llys Ser Lys 65 70 7s 8O

Cys Phe Tyr Thr Thr Asp Thr Glu. Cys Asp Lieu. Thr Asp Glu Ile Val 85 90 95

Lys Asp Wall Lys Glin Thr Tyr Lieu Ala Arg Val Phe Ser Tyr Pro Ala 1OO 105 11 O US 2015/03539 11 A1 Dec. 10, 2015 59

- Continued Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn 115 12 O 125 Ser Pro Glu Phe Thr Pro Tyr Lieu. Glu Thir Asn Lieu. Gly Glin Pro Thr 13 O 135 14 O Ile Glin Ser Phe Glu Glin Val Gly Thr Llys Val Asn Val Thr Val Glu 145 150 155 160 Asp Glu Arg Thr Lieu Val Arg Arg Asn. Asn. Thir Phe Lieu. Ser Lieu. Arg 1.65 17O 17s Asp Val Phe Gly Lys Asp Lieu. Ile Tyr Thr Lieu. Tyr Tyr Trp Llys Ser 18O 185 19 O Ser Ser Ser Gly Lys Llys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu 195 2OO 2O5 Ile Asp Val Asp Llys Gly Glu Asn Tyr Cys Phe Ser Val Glin Ala Val 21 O 215 22O Ile Pro Ser Arg Thr Val Asn Arg Llys Ser Thr Asp Ser Pro Val Glu 225 23 O 235 24 O Cys Met Gly Glin Glu Lys Gly Glu Phe Arg Glu Ile Phe Tyr Ile Ile 245 250 255 Gly Ala Val Val Phe Val Val Ile Ile Lieu Val Ile Ile Lieu Ala Ile 26 O 265 27 O Ser Lieu. His Lys Cys Arg Lys Ala Gly Val Gly Glin Ser Trp Llys Glu 27s 28O 285

ASn Ser Pro Lieu. ASn Wal Ser 29 O 295

<210s, SEQ ID NO 16 &211s LENGTH: 888 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 16 atggaga.ccc ctgcctggcc ccgggtc.ccg cgc.ccc.gaga cc.gc.cgt.cgc ticggacgctic 6 O

Ctgctcggct gggtct tcgc C caggtggcc ggcgctt cag gCact acaaa tactgtggca 12 O gcatataatt taacttggaa atcaactaat ttcaagacaa ttittggagtg ggaac ccaaa 18O cc.cgtcaatc aagttctacac togttcaaata agcactaagt caggagattg gaaaa.gcaaa 24 O tgcttt taca Caacagacac agagtgtgac ct caccgacg agattgttgaa ggatgtgaag 3OO

Cagacgtact tccacgggt Cttct Cotac cc.ggcaggga atgtggagag caccggttct 360 gctggggagc Ctctgt atga gaact cocca gagttcacac Cttacctgga gacaaacct C 42O ggacagccaa caattcagag titttgaacag gtgggaacaa aagtgaatgt gaccgtagaa 48O gatgaacgga Ctttagt cag aaggaacaac actitt CCtaa goctocggga tigtttittggc 54 O aaggacittaa tittata cact ttatt attgg aaatc.ttcaa gttcaggaaa gaaaa.ca.gc.c 6OO aaaacaaaca ctaatgagtt tttgattgat gtggataaag gagaaaact a citgttt cagt 660 gttcaa.gcag tatt ccctic ccgaacagtt aaccggaaga gtacaga cag ccc.gg tagag 72 O tgtatgggcc aggagaaagg gga attcaga gaaat attct acat cattgg agctgtggta 78O tttgtggit catcatcc ttgt catcatcc tig gctatat citc tacacaagtg tagaaaggca 84 O ggagtggggc agagctggala ggaga actico C Cactgaatg titt catala 888

<210s, SEQ ID NO 17 US 2015/03539 11 A1 Dec. 10, 2015 60

- Continued

&211s LENGTH: 2224 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 17 Met Phe Pro Gly Cys Pro Arg Lieu. Trp Val Lieu Val Val Lieu. Gly Thr 1. 5 1O 15 Ser Trp Val Gly Trp Gly Ser Glin Gly Thr Glu Ala Ala Glin Lieu. Arg 2O 25 3O Glin Phe Tyr Val Ala Ala Glin Gly Ile Ser Trp Ser Tyr Arg Pro Glu 35 4 O 45 Pro Thr Asn Ser Ser Lieu. Asn Lieu Ser Val Thr Ser Phe Llys Lys Ile SO 55 6 O Val Tyr Arg Glu Tyr Glu Pro Tyr Phe Llys Lys Glu Lys Pro Glin Ser 65 70 7s 8O Thir Ile Ser Gly Lieu. Lieu. Gly Pro Thir Lieu. Tyr Ala Glu Val Gly Asp 85 90 95 Ile Ile Llys Val His Phe Lys Asn Lys Ala Asp Llys Pro Lieu. Ser Ile 1OO 105 11 O His Pro Glin Gly Ile Arg Tyr Ser Lys Lieu. Ser Glu Gly Ala Ser Tyr 115 12 O 125 Lieu. Asp His Thir Phe Pro Ala Glu Lys Met Asp Asp Ala Val Ala Pro 13 O 135 14 O Gly Arg Glu Tyr Thr Tyr Glu Trp Ser Ile Ser Glu Asp Ser Gly Pro 145 150 155 160 Thr His Asp Asp Pro Pro Cys Lieu. Thr His Ile Tyr Tyr Ser His Glu 1.65 17O 17s Asn Lieu. Ile Glu Asp Phe Asn. Ser Gly Lieu. Ile Gly Pro Lieu. Lieu. Ile 18O 185 19 O Cys Llys Lys Gly Thr Lieu. Thr Glu Gly Gly Thr Gln Lys Thr Phe Asp 195 2OO 2O5 Lys Glin Ile Val Lieu Lleu Phe Ala Val Phe Asp Glu Ser Lys Ser Trp 21 O 215 22O Ser Glin Ser Ser Ser Leu Met Tyr Thr Val Asn Gly Tyr Val Asn Gly 225 23 O 235 24 O Thr Met Pro Asp Ile Thr Val Cys Ala His Asp His Ile Ser Trp His 245 250 255 Lieu. Leu Gly Met Ser Ser Gly Pro Glu Lieu Phe Ser Ile His Phe Asn 26 O 265 27 O Gly Glin Val Lieu. Glu Glin Asn His His Llys Val Ser Ala Ile Thr Lieu. 27s 28O 285 Val Ser Ala Thr Ser Thr Thr Ala Asn Met Thr Val Gly Pro Glu Gly 29 O 295 3 OO

Lys Trp Ile Ile Ser Ser Lieu. Thr Pro Llys His Leu Glin Ala Gly Met 3. OS 310 315 32O

Glin Ala Tyr Ile Asp Ile Lys Asn. Cys Pro Llys Llys Thr Arg Asn Lieu. 3.25 330 335 Llys Lys Ile Thir Arg Glu Glin Arg Arg His Met Lys Arg Trp Glu Tyr 34 O 345 35. O

Phe Ile Ala Ala Glu Glu Val Ile Trp Asp Tyr Ala Pro Val Ile Pro 355 360 365 Ala Asn Met Asp Llys Llys Tyr Arg Ser Glin His Lieu. Asp Asn. Phe Ser US 2015/03539 11 A1 Dec. 10, 2015 61

- Continued

37 O 375 38O Asn Glin Ile Gly Lys His Tyr Lys Llys Val Met Tyr Thr Glin Tyr Glu 385 390 395 4 OO Asp Glu Ser Phe Thr Lys His Thr Val Asn Pro Asn Met Lys Glu Asp 4 OS 41O 415 Gly Ile Lieu. Gly Pro Ile Ile Arg Ala Glin Val Arg Asp Thir Lieu Lys 42O 425 43 O Ile Val Phe Lys Asn Met Ala Ser Arg Pro Tyr Ser Ile Tyr Pro His 435 44 O 445 Gly Val Thr Phe Ser Pro Tyr Glu Asp Glu Val Asn Ser Ser Phe Thr 450 45.5 460 Ser Gly Arg Asn Asn Thr Met Ile Arg Ala Val Glin Pro Gly Glu Thr 465 470 47s 48O Tyr Thr Tyr Lys Trp Asn Ile Leu Glu Phe Asp Glu Pro Thr Glu Asn 485 490 495 Asp Ala Glin Cys Lieu. Thir Arg Pro Tyr Tyr Ser Asp Wall Asp Ile Met SOO 505 51O Arg Asp Ile Ala Ser Gly Lieu. Ile Gly Lieu. Lieu. Lieu. Ile Cys Llys Ser 515 52O 525 Arg Ser Lieu. Asp Arg Arg Gly Ile Glin Arg Ala Ala Asp Ile Glu Glin 53 O 535 54 O Glin Ala Val Phe Ala Val Phe Asp Glu Asn Llys Ser Trp Tyr Lieu. Glu 545 550 555 560 Asp Asn. Ile Asn Llys Phe Cys Glu Asn Pro Asp Glu Wall Lys Arg Asp 565 st O sts Asp Pro Llys Phe Tyr Glu Ser Asn Ile Met Ser Thr Ile Asin Gly Tyr 58O 585 59 O Val Pro Glu Ser Ile Thr Thr Lieu. Gly Phe Cys Phe Asp Asp Thr Val 595 6OO 605 Gln Trp His Phe Cys Ser Val Gly Thr Glin Asn Glu Ile Lieu. Thir Ile 610 615 62O His Phe Thr Gly His Ser Phe Ile Tyr Gly Lys Arg His Glu Asp Thr 625 630 635 64 O Lieu. Thir Lieu Phe Pro Met Arg Gly Glu Ser Val Thr Val Thr Met Asp 645 650 655 Asn Val Gly. Thir Trp Met Lieu. Thir Ser Met Asn Ser Ser Pro Arg Ser 660 665 67 O Llys Llys Lieu. Arg Lieu Lys Phe Arg Asp Wall Lys Cys Ile Pro Asp Asp 675 68O 685 Asp Glu Asp Ser Tyr Glu Ile Phe Glu Pro Pro Glu Ser Thr Val Met 69 O. 695 7 OO Ala Thr Arg Llys Met His Asp Arg Lieu. Glu Pro Glu Asp Glu Glu Ser 7 Os 71O 71s 72O

Asp Ala Asp Tyr Asp Tyr Glin Asn Arg Lieu Ala Ala Ala Lieu. Gly Ile 72 73 O 73

Arg Ser Phe Arg Asn. Ser Ser Lieu. Asn Glin Glu Glu Glu Glu Phe Asn 740 74. 7 O

Lieu. Thir Ala Lieu Ala Lieu. Glu Asin Gly Thr Glu Phe Val Ser Ser Asn 7ss 760 765

Thr Asp Ile Ile Val Gly Ser Asn Tyr Ser Ser Pro Ser Asn Ile Ser 770 775 78O US 2015/03539 11 A1 Dec. 10, 2015 62

- Continued

Llys Phe Thr Val Asn. Asn Lieu Ala Glu Pro Gln Lys Ala Pro Ser His 78s 79 O 79. 8OO Glin Glin Ala Thir Thr Ala Gly Ser Pro Lieu. Arg His Lieu. Ile Gly Lys 805 810 815 Asn Ser Val Lieu. Asn Ser Ser Thr Ala Glu. His Ser Ser Pro Tyr Ser 82O 825 83 O Glu Asp Pro Ile Glu Asp Pro Lieu. Glin Pro Asp Val Thr Gly Ile Arg 835 84 O 845 Lieu. Lieu. Ser Lieu. Gly Ala Gly Glu Phe Llys Ser Glin Glu. His Ala Lys 850 855 860 His Lys Gly Pro Llys Val Glu Arg Asp Glin Ala Ala Lys His Arg Phe 865 87O 87s 88O Ser Trp Met Lys Lieu. Lieu Ala His Llys Val Gly Arg His Lieu. Ser Glin 885 890 895 Asp Thr Gly Ser Pro Ser Gly Met Arg Pro Trp Glu Asp Leu Pro Ser 9 OO 905 91 O Gln Asp Thr Gly Ser Pro Ser Arg Met Arg Pro Trp Lys Asp Pro Pro 915 92 O 925 Ser Asp Lieu. Lieu Lleu Lleu Lys Glin Ser Asn. Ser Ser Lys Ile Lieu Val 93 O 935 94 O Gly Arg Trp His Lieu Ala Ser Glu Lys Gly Ser Tyr Glu Ile Ile Glin 945 950 955 96.O Asp Thr Asp Glu Asp Thr Ala Val Asn. Asn Trp Lieu. Ile Ser Pro Glin 965 97O 97. Asn Ala Ser Arg Ala Trp Gly Glu Ser Thr Pro Lieu Ala Asn Llys Pro 98O 985 99 O Gly Lys Glin Ser Gly His Pro Llys Phe Pro Arg Val Arg His Llys Ser 995 1OOO 1005 Lieu. Glin Val Arg Glin Asp Gly Gly Lys Ser Arg Lieu Lys Llys Ser O1O O15 O2O Glin Phe Lieu. Ile Llys Thir Arg Llys Llys Llys Lys Glu Lys His Thr O25 O3 O O35 His His Ala Pro Leu Ser Pro Arg Thr Phe His Pro Leu Arg Ser O4 O O45 OSO Glu Ala Tyr Asn. Thir Phe Ser Glu Arg Arg Lieu Lys His Ser Lieu. O55 O6 O O65 Val Lieu. His Llys Ser Asn. Glu Thir Ser Lieu Pro Thr Asp Lieu. Asn Of O O7 O8O Gln Thr Lieu Pro Ser Met Asp Phe Gly Trp Ile Ala Ser Leu Pro O85 O9 O O95 Asp His Asn Glin Asn. Ser Ser Asn Asp Thr Gly Glin Ala Ser Cys 1 OO 105 10 Pro Pro Gly Lieu. Tyr Glin Thr Val Pro Pro Glu Glu. His Tyr Glin 115 12 O 25 Thr Phe Pro Ile Glin Asp Pro Asp Glin Met His Ser Thr Ser Asp 13 O 135 4 O

Pro Ser His Arg Ser Ser Ser Pro Glu Lieu Ser Glu Met Lieu. Glu 145 15 O 55

Tyr Asp Arg Ser His Llys Ser Phe Pro Thr Asp e Ser Glin Met 16 O 1.65 70 US 2015/03539 11 A1 Dec. 10, 2015 63

- Continued Ser Pro Ser Ser Glu. His Glu Val Trp Gln Thr Val Ile Ser Pro 17s 18O 185 Asp Leu Ser Glin Val Thr Lieu Ser Pro Glu Lieu Ser Glin Thr Asn 190 195 2OO Lieu. Ser Pro Asp Leu Ser His Thr Thr Lieu Ser Pro Glu Lieu. Ile 2O5 21 O 215 Glin Arg Asn Lieu Ser Pro Ala Leu Gly Gln Met Pro Ile Ser Pro 22O 225 23 O Asp Leu Ser His Thr Thr Lieu Ser Pro Asp Leu Ser His Thr Thr 235 24 O 245 Lieu. Ser Lieu. Asp Lieu. Ser Glin Thr Asn Lieu. Ser Pro Glu Lieu. Ser 250 255 26 O Gln Thr Asn Lieu Ser Pro Ala Leu Gly Gln Met Pro Leu Ser Pro 265 27 O 27s Asp Leu Ser His Thr Thr Lieu Ser Lieu. Asp Phe Ser Glin Thr Asn 28O 285 29 O

Lieu. Ser Pro Glu Lieu. Ser His Met Thir Lieu. Ser Pro Glu Lieu. Ser 295 3OO 305 Gln Thr Asn Lieu Ser Pro Ala Leu Gly Gln Met Pro Ile Ser Pro 310 315 32O Asp Leu Ser His Thr Thr Lieu Ser Lieu. Asp Phe Ser Glin Thr Asn 3.25 33 O 335 Lieu Ser Pro Glu Lieu Ser Glin Thr Asn Lieu Ser Pro Ala Lieu. Gly 34 O 345 350 Gln Met Pro Leu Ser Pro Asp Pro Ser His Thr Thr Lieu. Ser Lieu. 355 360 365 Asp Lieu. Ser Glin Thir Asn Lieu. Ser Pro Glu Lieu. Ser Glin Thr Asn 37O 375 38O Lieu. Ser Pro Asp Lieu. Ser Glu Met Pro Lieu. Phe Ala Asp Lieu. Ser 385 390 395 Glin Ile Pro Leu. Thr Pro Asp Lieu. Asp Gln Met Thr Lieu. Ser Pro 4 OO 405 41 O Asp Leu Gly Glu Thir Asp Leu Ser Pro Asn Phe Gly Gln Met Ser 415 42O 425 Lieu. Ser Pro Asp Leu Ser Glin Val Thr Lieu Ser Pro Asp Ile Ser 43 O 435 44 O Asp Thir Thr Lieu. Leu Pro Asp Leu Ser Glin Ile Ser Pro Pro Pro 445 450 45.5 Asp Lieu. Asp Glin Ile Phe Tyr Pro Ser Glu Ser Ser Glin Ser Lieu. 460 465 47 O Lieu. Leu Gln Glu Phe Asin Glu Ser Phe Pro Tyr Pro Asp Leu Gly 47s 48O 485

Gln Met Pro Ser Pro Ser Ser Pro Thr Lieu. Asn Asp Thr Phe Lieu. 490 495 SOO

Ser Lys Glu Phe Asn Pro Lieu Val Ile Val Gly Lieu. Ser Lys Asp 5 OS 510 515

Gly Thr Asp Tyr Ile Glu Ile Ile Pro Lys Glu Glu Val Glin Ser 52O 525 53 O

Ser Glu Asp Asp Tyr Ala Glu Ile Asp Tyr Val Pro Tyr Asp Asp 535 54 O 545

Pro Tyr Llys Thr Asp Val Arg Thr Asn. Ile Asn. Ser Ser Arg Asp US 2015/03539 11 A1 Dec. 10, 2015 64

- Continued

550 555 560 Pro Asp Asn. Ile Ala Ala Trp Tyr Lieu. Arg Ser Asn. Asn Gly Asn 565 st O sts Arg Arg Asn Tyr Tyr Ile Ala Ala Glu Glu Ile Ser Trp Asp Tyr 58O 585 590 Ser Glu Phe Val Glin Arg Glu Thir Asp Ile Glu Asp Ser Asp Asp 595 6OO 605 Ile Pro Glu Asp Thir Thr Tyr Lys Llys Val Val Phe Arg Llys Tyr

Lieu. Asp Ser Thr Phe Thir Lys Arg Asp Pro Arg Gly Glu Tyr Glu

Glu. His Lieu. Gly Ile Lieu. Gly Pro Ile Ile Arg Ala Glu Val Asp

Asp Val Ile Glin Val Arg Phe Lys Asn Lieu Ala Ser Arg Pro Tyr 655 660 665 Ser Lieu. His Ala His Gly Lieu. Ser Tyr Glu Lys Ser Ser Glu Gly 670 675 68O Llys Thr Tyr Glu Asp Asp Ser Pro Glu Trp Phe Lys Glu Asp Asn 685 69 O. 695 Ala Val Glin Pro Asn Ser Ser Tyr Thr Tyr Val Trp His Ala Thr 7 OO 7Os 71O Glu Arg Ser Gly Pro Glu Ser Pro Gly Ser Ala Cys Arg Ala Trp 715 72O 725 Ala Tyr Tyr Ser Ala Val Asn Pro Glu Lys Asp Ile His Ser Gly 73 O 73 74 O Lieu. Ile Gly Pro Lieu. Lieu. Ile Cys Glin Lys Gly Ile Lieu. His Llys 74. 7 O 7ss Asp Ser Asn Met Pro Met Asp Met Arg Glu Phe Val Lieu Lleu Phe 760 765 770 Met Thr Phe Asp Glu Lys Llys Ser Trp Tyr Tyr Glu Lys Lys Ser 775 78O 78s Arg Ser Ser Trp Arg Lieu. Thir Ser Ser Glu Met Lys Llys Ser His 79 O 79. 8OO Glu Phe His Ala Ile Asin Gly Met Ile Tyr Ser Leu Pro Gly Lieu. 805 810 815 Llys Met Tyr Glu Glin Glu Trp Val Arg Lieu. His Lieu. Lieu. Asn. Ile 82O 825 83 O Gly Gly Ser Glin Asp Ile His Val Val His Phe His Gly Glin Thr 835 84 O 845 Lieu. Lieu. Glu Asn Gly Asn Lys Gln His Glin Lieu. Gly Val Trp Pro 850 855 86 O

Lieu. Lieu Pro Gly Ser Phe Llys Thir Lieu. Glu Met Lys Ala Ser Lys 865 87 O 87s

Pro Gly Trp Trp Lieu. Lieu. Asn Thr Glu Val Gly Glu Asn Glin Arg 88O 885 890

Ala Gly Met Glin Thr Pro Phe Lieu. Ile Met Asp Arg Asp Cys Arg 895 9 OO 905

Met Pro Met Gly Lieu Ser Thr Gly Ile Ile Ser Asp Ser Glin Ile 910 915 92 O

Lys Ala Ser Glu Phe Lieu. Gly Tyr Trp Glu Pro Arg Lieu Ala Arg 925 93 O 935 US 2015/03539 11 A1 Dec. 10, 2015 65

- Continued

Lieu. Asn. Asn Gly Gly Ser Tyr Asn Ala Trp Ser Val Glu Lys Lieu. 1940 1945 1950 Ala Ala Glu Phe Ala Ser Llys Pro Trp Ile Glin Val Asp Met Glin 1955 1960 1965 Lys Glu Val Ile Ile Thr Gly Ile Glin Thr Glin Gly Ala Lys His 1970 1975 198O Tyr Lieu Lys Ser Cys Tyr Thr Thr Glu Phe Tyr Val Ala Tyr Ser 1985 1990 1995 Ser Asn Glin Ile Asn Trp Glin Ile Phe Lys Gly Asn. Ser Thr Arg 2OOO 2005 2010 Asn Val Met Tyr Phe Asin Gly Asn Ser Asp Ala Ser Thr Ile Lys 2015 2O2O 2O25 Glu Asn Glin Phe Asp Pro Pro Ile Val Ala Arg Tyr Ile Arg Ile 2O3O 2O35 2O4. O Ser Pro Thr Arg Ala Tyr Asn Arg Pro Thr Lieu. Arg Lieu. Glu Lieu. 2O45 2OSO 2O55 Gln Gly Cys Glu Val Asin Gly Cys Ser Thr Pro Leu Gly Met Glu 2O60 2O65 2. Of O Asn Gly Lys Ile Glu Asn Lys Glin Ile Thr Ala Ser Ser Phe Llys 2O75 2O8 O 2O85 Llys Ser Trp Trp Gly Asp Tyr Trp Glu Pro Phe Arg Ala Arg Lieu. 2O90 2095 21OO Asn Ala Glin Gly Arg Val Asn Ala Trp Glin Ala Lys Ala Asn. Asn

Asn Lys Glin Trp Lieu. Glu Ile Asp Lieu. Lieu Lys Ile Llys Lys Ile

Thr Ala Ile Ile Thr Glin Gly Cys Llys Ser Leu Ser Ser Glu Met

Tyr Val Lys Ser Tyr Thir Ile His Tyr Ser Glu Gln Gly Val Glu

Trp Llys Pro Tyr Arg Lieu Lys Ser Ser Met Val Asp Llys Ile Phe 2 6 5 2 70 21.75 Glu Gly Asn Thr Asn Thr Lys Gly His Val Lys Asn Phe Phe Asn 2 8 O 2 85 219 O Pro Pro Ile Ile Ser Arg Phe Ile Arg Val Ile Pro Llys Thir Trp 21.95 22 OO 22O5 Asn Glin Ser Ile Ala Lieu. Arg Lieu. Glu Lieu. Phe Gly Cys Asp Ile 221 O 2215 222 O Tyr

<210s, SEQ ID NO 18 &211s LENGTH: 66.75 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 18 atgttcc.cag gctgcc cacg cct ctgggtc. Ctggtggtct tdgcaccag Ctggg taggc 6 O tgggggagcc aagggacaga agcggcacag cta aggcagt t ctacgtggc tigcticagggc 12 O atcagttgga gct accqacc tdagcc.caca aact caagtt tdaatctitt c td taact tcc 18O tittaagaaaa ttgttctacag agagtatgaa ccatattitta agaaagaaaa accacaatct 24 O

US 2015/03539 11 A1 Dec. 10, 2015 67

- Continued

Cagccagatg tca cagggat acgt.ctact t t cacttggtg Ctggagaatt Caaaagttcaa 2580 gaacatgcta agcatalaggg accca aggta gaaagagatc alagcagcaaa gCacaggttc 264 O t cctggatga aattactago a cataaagtt gggagacacic taa.gc.caaga cactggttct 27 OO

CCtt Coggaa taggc cctg ggaggacctt cct agccaag acactggttc. tcc titccaga 276 O atgaggcc ct ggaagg acco toctagtgat citgttactict taaaacaaag taact catct 282O aagattittgg ttgggagatg gcatttggct tctgagaaag gtagctatga aataatccala 288O gatact gatg aagacacagc tigittaacaat tictgatca gcc.cccagaa to ct cacgt 294 O gcttggggag aaa.gcaccCC tottgccaac aagcctggaa agcagagtgg ccacccaaag 3 OOO titt.cct agag ttagacataa at Ctctacaa gta agaCagg atggaggaala gagtag actg 3 O 6 O aagaaaagcc agtttct cat taaga cacga aaaaagaaaa aagagaag.ca cacacac cat 312 O gctic ctittat citcc.gaggac ctitt caccct ctaagaagtg aagcc tacaa cacatttitca 318O gaaagaagaci ttaa.gcattc gttggtgctt cataaatcca atgaaacat c tict tcc.caca 324 O gacct caatc agacattgcc ctic tatggat tittggctgga tag cct cact tcc tdac cat 33 OO aatcagaatt cotcaaatga cactggtcag goaa.gctgtc. citccaggtot titat cagaca 3360 gtgcc.cccag aggaacacta t caaacattc cc cattcaag accct gatca aatgcactict 342O actt cagacc ccagt cacag atcct cittct c cagagctica gtgaaatgct tdag tatgac 3480 cgaagt caca agt cct tccc cacagatata agt caaatgt cccct tcctic agaacatgaa 354 O gtctggcaga cagt catc to tccagacctic agc.caggtga ccct ct citcc agaacticago 36OO cagacaaacc tict citccaga cct cagocac acgactic tot citccagaact cattcagaga 366 O aacctitt.ccc cagccct cqg toagatgc cc atttctic cag acct cagcca tacaa.ccctt 372 O tctic cagacc ticago catac aaccotttct ttagacctica gccagacaaa cct ct ct coa 378 O gaacticagtic agacaaacct ttctic cagcc ct cqgtcaga tigc cc ctitt c ticcagacctic 384 O agccatacaa ccctitt ct ct agactitcago cagacaaacc tict ct coaga acticago cat 3900 atgact ct ct citccagaact cagt cagaca aacct titccc cagcc ct cqg toagatgcc c 396 O atttct coag acct cago ca tacaa.ccct t t ct ctagact t cago cagac aaacct citct 4 O2O ccagaactica gtcaaacaaa cctitt cocca gcc ct cqgtc agatgcc cct ttct c cagac 4 O8O cc.ca.gc.cata caa.ccctitt c tictag acctic agc.ca.gacaa acct ct citcc agaacticagt 414 O cagacaaacc titt coccaga cct cagtgag atgcc cct ct ttgcagatct cagtcaaatt 42OO cc ccttaccc cagacct cqa coagatgaca ctittctic cag accttggtga gacagat citt 426 O t ccc caaact ttggtcagat gtc. cctitt co coagacct ca gcc aggtgac tot ct ct coa 432O gacatcagtg acaccaccct tct cocqqat ct cagccaga tat caccitcc ticcagacctt 438 O gatcagatat t ct acc ctitc tdaatctagt cagt cattgc titcttcaaga atttaatgag 4 44 O tcttitt cott atccagacct togg to agatg ccatcto citt catct cotac tot caatgat 4500 acttitt citat caaaggaatt taatccactg gttatagtgg gcc to agitaa agatggtaca 456 O gattacattg agat catt CC aaaggaagag gtc.ca.gagca gtgaagatga citatgctgaa 462O attgattatg togc cctatga tigacic cctac aaaactgatgttaggacaaa catcaacticc 468O tccagagatc Ctgacaac at tcagcatgg tacct cogca gcaacaatgg aaa.ca.galaga 474. O aatt attaca ttgctgctga agaaatat co tdggattatt cagaatttgt acaaagggaa 48OO US 2015/03539 11 A1 Dec. 10, 2015 68

- Continued acagatattg aagacitctga tgatatt cca gaagatacca catataagaa agtagtttitt 486 O cgaaagtacc ticgacagcac ttt taccalaa. cgtgatcctic gaggggagta tgaagagcat 492 O citcggaattic ttggtccitat tat cagagct galagtggatg atgttatcca agttcgttitt 498O aaaaatttag catccagacc gtatt citcta catgcc catg gactitt.ccta tgaaaaatca 5040 t cagagggaa agacittatga agatgact ct Cctgaatggit ttalaggalaga taatgctgtt cago caaata gcagttatac Ctacgitatgg catgccactg agcgatcagg gcc agaaagt 516 O

Cctggctctg. cctgtcgggc ttgggcctac tacticagotg tgaac Coaga aaaagatatt 522 O

Cact caggct tataggit co CCt CCtaatc. tgccaaaaag gaatact aca taaggacagc 528 O alacatgccta tigacatgag agaatttgtc ttact attta tgacctittga tgaaaagaag 534 O agctgg tact atgaaaagaa gtc.ccgaagt tcttggagac to acatcCt C agaaatgaaa 54 OO aaatcc catg agttt cacgc Cattaatggg atgat ctaca gcttgcctgg Cctgaaaatg 546 O tatgagcaag agtgggtgag gttacacctg Ctgaacatag gcggctic cca aga catt cac 552O gtggttcact ttcacggc.ca gaccttgctg gaaaatggca ataaa.ca.gca Ccagttaggg 558 O gtctggcc cc ttctgcctgg ttcatttaala act cittgaaa tgaaggcatc aaaac Ctggc 564 O tggtggct Co taalacacaga ggttggagaa alaccagagag Cagggatgca aacgc.cattt st OO

Cttat catgg acagagactg taggatgc.ca atgggactaa gCactggitat catat ctdat 576. O t cacagat.ca aggctt caga gtttctgggit tactgggagc ccagattagc aagattaaac 582O aatggtggat Cttataatgc ttggagtgta gaaaaacttg Cagcagaatt tgcct ctaaa 588 O

CCttggat.cc aggtggacat gcaaaaggaa gtcataatca Cagggat.cca gacccalaggt 594 O gccaaacact acctgaagtic ctgctatacc acagagttct atgtagctta cagttccaac 6 OOO cagat caact ggcagat citt Calaagggaac agcacaagga atgtgatgta ttittaatggc aatt cagatg cct ctacaat aaaagagaat cagtttgacc cacct attgt ggctagatat 612 O attaggat ct citccaact cq agcctataac agacct accc titcgattgga actgcaaggt 618O tgtgaggtaa atggatgttc cacaccc.ctg gg tatggaaa atggaaagat agaaaacaag 624 O caaatcacag citt citt cott taagaaatct tggtggggag attactggga accct tcc.gt 63 OO gcc.cgtctga atgccCaggg acgtgttgaat gcctgcaag cCaaggcaaa caacaataag 636 O

Cagtggctag aaattgat ct act caagatc aagaagataa cggcaattat alacac agggc 642O tgcaagttctic tdtcct ctda aatgitatgta aagagctata CCatcCacta Cagtgagcag 648 O ggagtggaat ggaalaccata Caggctgaaa t cct c catgg tggacaagat ttittgaagga 654 O aatactaata ccaaaggaca tgttgaagaac titt tt calacc CCC caat Cat titccaggttt 66OO atcc.gtgtca ttcctaaaac atggaatcaa agtattgcac titcgc.ctgga act ctittggc 666 O tgttgat attt actag 6675

SEQ ID NO 19 LENGTH: 12 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: procoagulant peptide

SEQUENCE: 19 Llys Lieu. Thir Cys Lieu Ala Ser Tyr Cys Trp Lieu. Phe 1. 5 US 2015/03539 11 A1 Dec. 10, 2015 69

- Continued

<210s, SEQ ID NO 2 O &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: procoagulant peptide

<4 OOs, SEQUENCE: 2O Arg Arg Ala Pro Gly Llys Lieu. Thir Cys Lieu Ala Ser Tyr Cys Trp Lieu 1. 5 1O 15 Phe Trp Thr Gly Ile Ala 2O

<210s, SEQ ID NO 21 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: procoagulant peptide

<4 OOs, SEQUENCE: 21 Arg Arg Ala Pro Gly Llys Lieu. Glin Cys Lieu Ala Ser Tyr Cys Trp Lieu 1. 5 1O 15 Phe Trp Thr Gly Ile Ala 2O

<210s, SEQ ID NO 22 &211s LENGTH: 32 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: procoagulant peptide

<4 OOs, SEQUENCE: 22 Pro Arg Ile Arg Thr Val Gly Pro Gly Ser Arg Ser Ala Ser Gly Lys 1. 5 1O 15 Lieu. Thir Cys Lieu Ala Ser Tyr Cys Trp Leu Phe Trp Thr Gly Ile Ala 2O 25 3O

<210s, SEQ ID NO 23 &211s LENGTH: 32 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: procoagulant peptide

<4 OOs, SEQUENCE: 23 Ser Lys Glin Gly Arg Pro Ile Ser Pro Asp Arg Arg Ala Ala Gly Lys 1. 5 1O 15

Lieu. Thir Cys Lieu Ala Ser Tyr Cys Trp Leu Phe Trp Thr Gly Ile Ala 2O 25 3O

<210s, SEQ ID NO 24 &211s LENGTH: 32 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: procoagulant peptide

<4 OOs, SEQUENCE: 24 Pro Arg Ile Arg Thr Val Gly Pro Gly Ser Arg Ser Ala Ser Gly Lys US 2015/03539 11 A1 Dec. 10, 2015 70

- Continued

1. 5 1O 15 Ser Thr Cys Lieu Ala Ser Tyr Cys Trp Leu Phe Trp Thr Gly Ile Ala 2O 25 3O

<210s, SEQ ID NO 25 &211s LENGTH: 32 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: procoagulant peptide

<4 OOs, SEQUENCE: 25 Ser Arg Ile Arg Thr Val Ser Pro Gly Ser Arg Ser Ala Ser Gly Lys 1. 5 1O 15 Ser Thr Cys Lieu Ala Ser Tyr Cys Trp Leu Phe Trp Thr Gly Ile Ala 2O 25 3O

<210s, SEQ ID NO 26 &211s LENGTH: 32 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: procoagulant peptide

<4 OOs, SEQUENCE: 26 Pro Arg Ser Arg Thr Val Gly Pro Gly Ser Arg Ser Ala Ser Gly Lys 1. 5 1O 15 Ser Thr Cys Lieu Ala Ser Tyr Cys Trp Leu Phe Trp Thr Gly Ile Ala 2O 25 3O

<210s, SEQ ID NO 27 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: immunoglobulin constant region <4 OOs, SEQUENCE: 27 Pro Lys Asn Ser Ser Met Ile Ser Asn Thr Pro 1. 5 1O

<210s, SEQ ID NO 28 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: immunoglobulin constant region

<4 OOs, SEQUENCE: 28 His Glin Ser Leu Gly Thr Glin 1. 5

<210s, SEQ ID NO 29 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: immunoglobulin constant region

<4 OOs, SEQUENCE: 29 His Glin Asn Lieu. Ser Asp Gly Lys 1. 5 US 2015/03539 11 A1 Dec. 10, 2015 71

- Continued

<210s, SEQ ID NO 3 O &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: immunoglobulin constant region

<4 OOs, SEQUENCE: 30 His Glin Asn. Ile Ser Asp Gly Lys 1. 5

<210s, SEQ ID NO 31 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: immunoglobulin constant region

<4 OOs, SEQUENCE: 31 Val Ile Ser Ser His Lieu. Gly Glin 1. 5

<210s, SEQ ID NO 32 &211s LENGTH: 32 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: CTP peptide <4 OOs, SEQUENCE: 32 Asp Pro Arg Phe Glin Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser 1. 5 1O 15 Lieu Pro Ser Pro Ser Arg Lieu Pro Gly Pro Ser Asp Thr Pro Ile Leu 2O 25 3O

<210s, SEQ ID NO 33 &211s LENGTH: 28 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: CTP peptide <4 OOs, SEQUENCE: 33 Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg 1. 5 1O 15 Lieu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln 2O 25

<210s, SEQ ID NO 34 &211s LENGTH: 6 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: albumin binging protein 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Xaa is Asp, Asn., Ser, Thr, or Trp 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Xaa is Asn., Gln, H is, Ile, Leu, or Lys 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (4) ... (4) US 2015/03539 11 A1 Dec. 10, 2015 72

- Continued <223> OTHER INFORMATION: Xaa is Ala, Asp, Phe, Trp, or Tyr 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Xaa is Asp, Gly, Leu, Phe, Ser, or Thr <4 OOs, SEQUENCE: 34 Cys Xaa Xala Xala Xaa Cys 1. 5

<210s, SEQ ID NO 35 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: albumin binding peptide <4 OOs, SEQUENCE: 35 Asp Ile Cys Lieu Pro Arg Trp Gly Cys Lieu. Trp 1. 5 1O

<210s, SEQ ID NO 36 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PAS sequence

<4 OOs, SEQUENCE: 36

Ala Ser Pro Ala Ala Pro Ala Pro Ala Ser Pro Ala Ala Pro Ala Pro 1. 5 1O 15

Ser Ala Pro Ala 2O

<210s, SEQ ID NO 37 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PAS sequence

<4 OO > SEQUENCE: 37

Ala Ala Pro Ala Ser Pro Ala Pro Ala Ala Pro Ser Ala Pro Ala Pro 1. 5 1O 15

Ala Ala Pro Ser 2O

<210s, SEQ ID NO 38 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PAS sequence

<4 OOs, SEQUENCE: 38

Ala Pro Ser Ser Pro Ser Pro Ser Ala Pro Ser Ser Pro Ser Pro Ala 1. 5 1O 15

Ser Pro Ser Ser 2O

<210s, SEQ ID NO 39 &211s LENGTH: 19 212. TYPE: PRT US 2015/03539 11 A1 Dec. 10, 2015 73

- Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PAS sequence

<4 OOs, SEQUENCE: 39

Ala Pro Ser Ser Pro Ser Pro Ser Ala Pro Ser Ser Pro Ser Pro Ala 1. 5 1O 15

Ser Pro Ser

<210s, SEQ ID NO 4 O &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PAS sequence <4 OOs, SEQUENCE: 4 O

Ser Ser Pro Ser Ala Pro Ser Pro Ser Ser Pro Ala Ser Pro Ser Pro 1. 5 1O 15

Ser Ser Pro Ala 2O

<210s, SEQ ID NO 41 &211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PAS sequence

<4 OOs, SEQUENCE: 41

Ala Ala Ser Pro Ala Ala Pro Ser Ala Pro Pro Ala Ala Ala Ser Pro 1. 5 1O 15

Ala Ala Pro Ser Ala Pro Pro Ala 2O

<210s, SEQ ID NO 42 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PAS sequence

<4 OOs, SEQUENCE: 42

Ala Ser Ala Ala Ala Pro Ala Ala Ala Ser Ala Ala Ala Ser Ala Pro 1. 5 1O 15

Ser Ala Ala Ala 2O

<210s, SEQ ID NO 43 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: gly-ser linker

<4 OOs, SEQUENCE: 43 Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly 1. 5 1O

<210s, SEQ ID NO 44 &211s LENGTH: 366 O &212s. TYPE: DNA