NZ619438B2 - Factor viii chimeric and hybrid polypeptides, and methods of use thereof - Google Patents

Abstract

Disclosed is a pharmaceutical composition comprising: (i) a chimeric polypeptide, which comprises a Factor VIII (FVIII) portion and a second portion; and (ii) at least one pharmaceutically acceptable excipient, wherein 15% to about 25% of the FVIII portion of the chimeric polypeptide comprises single chain FVIII and about 75% to about 85% of the FVIII portion of the chimeric polypeptide comprises processed FVIII, and wherein the composition is lyophilised. Also disclosed is use of the pharmaceutical composition of anyone of claims 1 to 24 in the manufacture of a medicament for preventing, decreasing or treating a bleeding episode in a human. e chain FVIII and about 75% to about 85% of the FVIII portion of the chimeric polypeptide comprises processed FVIII, and wherein the composition is lyophilised. Also disclosed is use of the pharmaceutical composition of anyone of claims 1 to 24 in the manufacture of a medicament for preventing, decreasing or treating a bleeding episode in a human.

Description

FACTOR VIII CHIMERIC AND HYBRID POLYPEPTIDES, AND S OF USE THEREOF REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY The content of the electronically submitted sequence listing in ASCII text file (Name: sequence1isting_ascii.txt; Size: bytes; and Date of Creation: ) filed with the application is incorporated herein by reference in its entirety.

Field of the Invention The present invention relates generally to the field of therapeutics for hemostatic disorders.

Background Art Hemophilia A is an X-linked bleeding disorder caused by mutations and/0r deletions in the factor VIII (FVIII) gene resulting in a ncy of FVIII activity (Peyvandi, F. et al.

Haemophilia 12:82-89 (2006). The disease is characterized by spontaneous hemorrhage and ive bleeding after trauma. Over time, the repeated bleeding into muscles and joints, which often begins in early childhood, results in hemophilic arthropathy and irreversible joint .

This damage is progressive and can lead to severely limited mobility ofj oints, muscle atrophy and chronic pain (Rodriguez-Merchan, E.C., Semin. Thromb. Hemost. 29:87-96 (2003), which is herein incorporated by reference in its ty).

The A2 domain is necessary for the gulant activity of the factor VIII molecule.

Studies show that porcine factor VIII has six-fold greater procoagulant ty than human factor VIII r, P., and E. T. Parker, J. Biol. Chem. 266:12481-12486 ), and that the difference in coagulant activity between human and porcine factor VIII appears to be based on a difference in amino acid sequence between one or more residues in the human and e A2 domains r, P., et al., J. Biol. Chem. 267:23652-23657 (1992)), incorporated herein by reference in its entirety.

Treatment of hemophilia A is by replacement therapy targeting restoration of FVIII ty to 1 to 5 % of normal levels to prevent spontaneous bleeding (Mannucci, P.M., et al., N.

Engl. J. Med. 344:1773-1779 (2001), which is herein incorporated by reference in its entirety).

There are plasma-derived and recombinant FVIII products available to treat bleeding episodes on- demand or to prevent bleeding episodes from occurring by treating prophylactically. Based on the short half-life of these products, r, e.g., 8-12 hours, treatment regimens require the 2012/045784 administration of frequent intravenous injections. Such frequent administration is painful and inconvenient.

Reduced mortality, prevention of joint damage and improved quality of life have been important achievements due to the development of plasma-derived and recombinant FVIII.

Prolonged protection from bleeding would represent another key ement in the treatment of hemophilia A patients. r, to date, no products that allow for prolonged hemostatic protection have been developed. Therefore, there remains a need for improved methods of treating hemophilia due to factor VIII deficiency that are more tolerable, longer lasting, and more ive than current therapies.

BRIEF SUMMARY OF THE ION The present invention provides methods of administering Factor VIII; methods of administering chimeric polypeptides comprising Factor VIII and hybrids of such chimeric polypeptides; chimeric polypeptides comprising Factor VIII and hybrids of such chimeric polypeptides; polynucleotides encoding such chimeric and hybrid polypeptides; cells comprising such polynucleotides; and methods of producing such chimeric and hybrid polypeptides using such cells.

The present ion provides a method of administering Factor VIII to a subject in need thereof, comprising administering to the subject a therapeutic dose of a ic Factor VIII polypeptide, e.g., a chimeric Factor VIII-Fc polypeptide, at a dosing interval at least about one and one-half times longer than the dosing interval required for an equivalent dose of said Factor VIII t the non-Factor VIII portion (a polypeptide consisting of said Factor VIII portion), e.g., without the Fc portion.

The dosing interval may be at least about one and one-half to six times longer, one and one-half to five times longer, one and one-half to four times longer, one and one-half to three times , or one and one-half to two times , than the dosing interval required for an equivalent dose of said Factor VIII t the non-Factor VIII portion (a polypeptide consisting of said Factor VIII portion), e.g., the Fc n. The dosing al may be at least about one and one-half, two, two and one-half, three, three and one-half, four, four and one-half, five, five and one-half or six times longer than the dosing al required for an equivalent dose of said Factor VIII without the non-Factor VIII portion (a polypeptide consisting of said Factor VIII portion), e. g., the Fc portion. The dosing interval may be about every five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen days or .

The dosing interval may be at least about one and one-half to 5, one and one-half, 2, 3, 4, or 5 days or longer.

WO 09627 The present invention also provides a method of stering Factor VIII to a subject in need thereof, comprising administering to the subject a therapeutic dose of a ic Factor VIII ptide, e.g., a chimeric Factor VIII-Fc polypeptide, to obtain an area under the plasma concentration versus time curve (AUC) at least about one and one-quarter times greater than the AUC ed by an equivalent dose of said Factor VIII Without the non-Factor VIII portion (a polypeptide consisting of said Factor VIII portion), e.g., Without the Fc portion.

The present invention also provides a method of administering Factor VIII to a subject in need thereof, comprising administering to the subject a therapeutic dose of a polypeptide comprising a Factor VIII and an Fc at a dosing interval of about every three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen days or longer.

The s of the invention may be practiced on a subject in need of prophylactic treatment or on-demand treatment.

On-demand treatment includes treatment for a ng e, hrosis, muscle bleed, oral bleed, hage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis (head trauma), gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central s system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, or bleeding in the illiopsoas sheath.

The subject may be in need of surgical prophylaxis, peri-operative management, or treatment for surgery. Such surgeries include, e. g., minor surgery, major surgery, tooth extraction, tonsillectomy, inguinal hemiotomy, synovectomy, total knee replacement, craniotomy, osteosynthesis, trauma surgery, intracranial surgery, intra-abdominal surgery, intrathoracic surgery, or joint replacement surgery.

For on-demand treatment, the dosing interval of said chimeric polypeptide is about once every 24-36, 24-48, 24-72, 24-96, 24-120, 24-144, 24-168, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours or longer.

The therapeutic doses that may be used in the methods of the invention are about 10 to about 100 IU/kg, more specifically, about 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 IU/kg, and more cally, about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 IU/kg.

The therapeutic doses that may be used in the methods of the invention are about 10 to about 150 IU/kg, more specifically, about 0, 110-120, 120-130, 130-140, 140-150 IU/kg, and more cally, about 110, 115, 120, 125, 130, 135, 140, 145, or 1501U/kg.

The subject in the methods of the invention is a human subject. The determination of dosing interval and AUC may be carried out in a single subject or in a population of subjects.

The Factor VIII (or Factor VIII portion of a chimeric polypeptide) is a human Factor VIII.

The Factor VIII (or Factor VIII portion of a ic polypeptide) may have a full or partial deletion of the B domain.

The Factor VIII (or Factor VIII portion of a chimeric polypeptide) may be at least 90% or 95% identical to a Factor VIII amino acid sequence shown in Table 2 without a signal sequence (amino acids 20 to 1457 of SEQ ID N02 or amino acids 4 to 2351 of SEQ ID NO:6). The Factor VIII (or Factor VIII portion of a chimeric polypeptide) may be identical to a Factor VIII amino acid sequence shown in Table 2 without a signal sequence (amino acids 20 to 1457 of SEQ ID N02 or amino acids 20 to 2351 of SEQ ID NO:6).

The Factor VIII (or Factor VIII portion of a ic polypeptide) may be at least 90% or 95% identical to a Factor VIII amino acid sequence shown in Table 2 with a signal ce (amino acids 1 to 1457 of SEQ ID N02 or amino acids 1 to 2351 of SEQ ID NO:6). The Factor VIII (or Factor VIII portion of a chimeric polypeptide) may be identical to a Factor VIII amino acid sequence shown in Table 2 with a signal ce (amino acids 1 to 1457 of SEQ ID N02 or amino acids 1 to 2351 of SEQ ID NO:6).

The Fc portion (or Fc portion of a chimeric polypeptide) may be at least 90% or 95% identical to the Fc amino acid sequence shown in Table 2 (amino acids 1458 to 1684 of SEQ ID N02 or amino acids 2352 to 2578 of SEQ ID NO:6). The Fc portion (or Fc portion of a chimeric polypeptide) may be identical to the Fc amino acid sequence shown in Table 2 (amino acids 1458 to 1684 of SEQ ID N02 or amino acids 2352 to 2578 of SEQ ID NO:6).

The chimeric polypeptide may comprise a sequence at least 90% or 95% identical to the Factor VIII and Fc amino acid sequence shown in Table 2A(i) t a signal sequence (amino acids 20 to 1684 of SEQ ID N02) or at least 90% or 95% identical to the Factor VIII and Fc amino acid sequence shown in Table 2A(i) with a signal sequence (amino acids 1 to 1684 of SEQ ID NO:2). The chimeric polypeptide may se a sequence identical to the Factor VIII and Fc amino acid sequence shown in Table 2A(i) without a signal sequence (amino acids 20 to 1684 of SEQ ID N02) or identical to the Factor VIII and Fc amino acid sequence shown in Table 2A(i) with a signal ce (amino acids 1 to 1684 of SEQ ID NO:2).

The chimeric polypeptide may be in the form of a hybrid comprising a second ptide in association with said chimeric polypeptide, wherein said second polypeptide comprises or consists essentially of an Fc.

The second polypeptide may comprise or consist essentially of a sequence at least 90% or 95% identical to the amino acid sequence shown in Table 2A(ii) without a signal sequence (amino acids 21 to 247 of SEQ ID NO:4) or at least 90% or 95% identical to the amino acid sequence shown in Table 2A(ii) with a signal sequence (amino acids 1 to 247 of SEQ ID NO:4). The second polypeptide may se or consist essentially of a sequence identical to the amino acid sequence shown in Table 2A(ii) without a signal sequence (amino acids 21 to 247 of SEQ ID NO:4) or cal to the amino acid sequence shown in Table 2A(ii) with a signal sequence (amino acids 1 to 247 of SEQ ID NO:4).

The chimeric polypeptide or hybrid may be stered as part of a pharmaceutical composition comprising at least one excipient.

The invention also provides the above-described chimeric and hybrid polypeptides lves, polynucleotides ng them, a cultured human embryonic cells comprising the polynucleotides, and methods of producing such chimeric and hybrid polypeptides, and the polypeptides produced by such methods.

The present invention also provide a chimeric polypeptide that has Factor VIII activity comprising a Factor VIII portion and a second portion, wherein the Factor VIII portion is processed Factor VIII comprising two chains, a first chain comprising a heavy chain and a second chain comprising a light chain, wherein said first chain and said second chain are associated by a metal bond. For example, at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% of the Factor VIII portion of the chimeric ptide is processed Factor VIII.

In addition, the present invention includes a chimeric polypeptide that has Factor VIII activity, n the Factor VIII portion is single chain Factor VIII. In one aspect, the single chain Factor VIII can contain an intact intracellular processing site. In one embodiment, at least about 1%, about 5%, about 10%, about 15%, about 20%, or about 25% of the Factor VIII portion of the chimeric polypeptide is single chain Factor VIII. In another embodiment, at least about %, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% of the Factor VIII portion of the chimeric polypeptide is single chain Factor VIII. In another aspect, the single chain FVIII does not contain an intracellular processing site. For e, the SCFVIII comprises a substitution or on at an amino acid position ponding to Arginine 1645, a substitution or mutation at an amino acid position corresponding to Arginine 1648, or a tution or mutation at amino acid positions ponding to Arginine 1645 and Arginine 1648 in full-length Factor VIII. The amino acid substituted at the amino acid position corresponding to Arginine 1645 is a different amino acid from the amino acid tuted at the amino acid on corresponding to Arginine 1648. In certain embodiments, the substitution or mutation is an amino acid other than arginine, e.g., alanine.

In some embodiments, the chimeric polypeptide comprising single chain Factor VIII has Factor VIII ty at a level comparable to a chimeric polypeptide consisting of two Fc portions and processed Factor VIII, which is fused to one of the two Fc portions, when the Factor VIII activity is measured in vitro by a chromogenic assay. In other embodiments, the chimeric polypeptide comprising single chain Factor VIII has Factor VIII activity in vivo comparable to a ic polypeptide consisting of two Fc portions and processed Factor VIII, which is fused to one of the two Fc portions. In still other embodiments, the chimeric polypeptide comprising single chain Factor VIII has a Factor Xa generation rate comparable to a ic polypeptide consisting of two Fc portions and processed Factor VIII, which is fused to one of the two Fc portions. In n embodiments, single chain Factor VIII in the chimeric polypeptide is vated by activated Protein C at a level comparable to processed Factor VIII in a chimeric polypeptide consisting of two Fc portions and processed Factor VIII. In yet other embodiments, the single chain Factor VIII in the chimeric polypeptide has a Factor IXa interaction rate comparable to processed Factor VIII in a chimeric polypeptide consisting of two Fc portions and sed Factor VIII. In further embodiments, the single chain Factor VIII in the chimeric polypeptide binds to von Willebrand Factor at a level comparable to processed Factor VIII in a chimeric polypeptide ting of two Fc portions and the processed Factor VIII.

The t invention further includes a composition sing a chimeric polypeptide having Factor VIII activity, wherein at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of said polypeptide comprises a Factor VIII portion, which is single chain Factor VIII, and a second portion, wherein said single chain Factor VIII is at least 90% or 95% identical to a Factor VIII amino acid ce shown in Table 2 t a signal sequence (amino acids 20 to 1457 of SEQ ID N02 or amino acids 20 to 2351 of SEQ ID NO:6). In one embodiment, the second portion can be an Fc. In another embodiment, the polypeptide is in the form of a hybrid comprising a second polypeptide, wherein said second polypeptide consists essentially of an Fc.

In other embodiments, the polypeptide has a half-life at least one and one-half to six times longer, one and one-half to five times , one and one-half to four times longer, one and one-half to three times , or one and one-half to two times longer to a polypeptide consisting of said Factor VIII.

Also provided is a method of treating a bleeding ion comprising administering a therapeutically effective amount of the composition. The treatment can be prophylactic treatment or on-demand treatment or perioperative. The bleeding coagulation disorder can be hemophilia.

In one embodiment, the subject that is treated is a pediatric subject.

The present invention is also directed to a method of preventing, sing, or treating a bleeding episode in a subject comprising administering to the subject an effective amount of a long-acting Factor VIII (FVIII) protein, wherein the subject expresses a high level of von Willebrand Factor (VWF) in plasma. In one embodiment, the subject has been identified as expressing a high level of VWF in plasma. The present invention is also directed to a method of preventing, decreasing, or treating a bleeding episode in a subject comprising: (a) identifying a t haVing high levels of VWF by measuring the level of VWF in the plasma of said subject, _ 6 _ wherein a VWF level of at least about 100 IU/dL identifies the subject as having a high level of VWF; and (b) administering to the subject an effective amount of a long-acting FVIII protein.

In one embodiment, the subject is a human. In another embodiment, the subject is a pediatric subject. In another embodiment, the subject has hemophilia A.

In one embodiment, the high level of VWF is at least about 100 IU/dL. In another embodiment, the high level ofVWF is between about 100 IU/dL and about 200 IU/dL. In r embodiment, the high level ofVWF is about 110 IU/dL, about 120 IU/dL, about 130 IU/dL, about 140 IU/dL, about 150 IU/dL, about 160 IU/dL, about 170 IU/dL, about 180 IU/dL, about 190 IU/dL, or about 200 IU/dL.

In one embodiment the subject has the blood serotype A, B, or AB.

In one embodiment, the long-acting FVIII protein has a half-life in said subject of between about 20 and about 40 hours. In another embodiment, the long-acting FVIII protein has a half-life of about 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, or 40 hours. In another ment, the long-acting FVIII protein has a half-life of n about 20 and 27 hours. In another embodiment, the long-acting FVIII n has a half-life that is at least about 1.2 times r than the half-life of said said long- acting FVIII protein when administered to an individual having average levels of VWF. In another embodiment, the long-acting FVIII protein has a half-life that is at least about about 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5-fold times greater than the half-life of said said long-acting FVIII protein when administered to an individual having e levels ofVWF.

In one embodiment, the effective amount of long-acting FVIII protein that is administered is at least about 2OIU/kg, at least about 25 IU/kg, at least about 3OIU/kg, at least about 35 IU/kg, at least about 4OIU/kg, at least about 45 IU/kg, at least about 50IU/kg, at least about 55 IU/kg, at least about 6OIU/kg, at least about 651U/kg, at least about 7OIU/kg, at least about 751U/kg, at least about 8OIU/kg, at least about g, or at least about 9OIU/kg. In another embodiment, the effective amount is at least about 65IU/kg to at least about 9OIU/kg. In another embodiment, the effective amount is 80 IU/kg.

In one embodiment,, the long-acting FVIII protein is stered every 72 hours or longer. In another embodiment, the long-acting FVIII protein is administered about once a week or . In another ment, the long-acting FVIII protein is administered about once every days, about once every two weeks, about once every 15 days, about once every 20 days, about once every three weeks, about once every 25 days, about once every four weeks, or about once every one month.

In one embodiment, the long-acting FVIII is administered at a dosage of 80 IU/kg once every 72 hours. In a further embodiment the cting FVIII is administered at a dosage of 80 IU/kg once every 72 hours to a pediatric subject.

In one embodiment, administration of the long-acting FVIII protein resolves greater than -20%, greater than 5-15%, greater than 5-lO%, greater than lO-20%, or greater than 10-15% of bleeding episodes. In one embodiment, the trough level of plasma Factor VIII:C in the subjects is maintained above l-3 or 3-5 IU/dl. In one embodiment, the administration prevents a bleeding episode in the subject. In another embodiment, the bleeding episode is spontaneous. In another embodiment, the administration resolves greater than 80-lOO%, greater than 80-90%, r than 85-90%, greater than 90-100%, greater than , or r than 95-100% of bleeding episodes.

In one embodiment, the stration maintains homeostatis in the population of the subjects in need of a surgery. In another embodiment, the long-acting FVIII protein is administered prior to, , or after the surgery. In another embodiment, the surgery is minor surgery, major surgery, tooth extraction, tonsillectomy, inguinal hemiotomy, synovectomy, total knee replacement, craniotomy, osteosynthesis, trauma surgery, intracranial surgery, intra- abdominal surgery, intrathoracic surgery, or joint ement surgery. In another embodiment, the surgery is an emergency surgery.

In one embodiment, the long-acting FVIII n has a ife longer than a polypeptide consisting of FVIII. In r ment, the long-acting FVIII protein is pegylated, hesylated, or polysialylated.

In one embodiment, the long-acting FVIII protein is a chimeric protein comprising a FVIII portion and a second portion. In another embodiment, the second portion is an Fc region, albumin, a PAS sequence, transferrin, CTP (28 amino acid C-terminal peptide (CTP) ofhCG with its 4 O-glycans), polyethylene glycol (PEG), hydroxyethyl starch (HES), albumin g ptide, albumin-binding small les, or two or more combinations f. In another embodiment, the second portion is fused to the amino-terminus or the carboxy-terminus of the FVIII portion. In another embodiment, the second portion is inserted between two amino acids in the FVIII portion. In another embodiment, the chimeric protein is a FVIIIFc monomer dimer . In another ment, the FVIII portion is a single chain. In another embodiment, the FVIII portion comprises a heavy chain and a light chain. In another embodiment, the FVIII portion comprises full-length factor VIII, mature factor VIII, or factor VIII with a full or partial deletion of the B domain. In another embodiment, the FVIII portion comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 1 to 1438 of SEQ ID NO: 2 or amino acids 1 to 2332 of SEQ ID NO: 6. In another embodiment, the FVIII portion comprises amino acids 1 to 1438 of SEQ ID NO: 2 or amino acids 1 to 2332 of SEQ ID NO: 6. In another embodiment, the chimeric polypeptide ses an Fc region Which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 1439 to 1665 of SEQ ID NO: 2 or amino acids 2333 to 2559 of SEQ ID NO: 6. In another embodiment, the second portion comprises amino acids 1439 to 1665 of SEQ ID NO: 2 or amino acids 2333 to 2559 of SEQ ID NO: 6. In another embodiment, the long-acting FVIII polypeptide is administered as part of a pharmaceutical composition comprising at least one excipient.

The invention also provides a method of treating a subject sed With bleeding disorder, sing measuring the half-life of FVIII-Fc in said subject, n a half-life that is at least about 1.2 times greater than the half-life of FVIII-Fc in a normal subject indicates the subject is a ate for long interval dosing, and administering a Fc polypeptide in an effective amount and at a dosing interval of at least 3 days.

The invention also provides a method of treating a subject diagnosed With ng disorder, comprising administering a FVIII-Fc polypeptide in an effective amount and at a dosing interval of at least 3 days to a subject, Wherein the half-life of FVIII-Fc in said subject is at least about 1.2 times greater than the half-life of FVIII-Fc When stered to a subject having average levels of VWF.

In one embodiment, the plasma half-life of FVIII-Fc in said subject is at least about 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5-fold times r than the plasma half-life of FVIII-Fc When administered to a subject having average levels of VWF. In another embodiment, the FVIII-Fc plasma half-life is between 20-40 hours. In another embodiment, the long-acting FVIII protein has a half-life of about 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, or 40 hours. In another embodiment, the long-acting FVIII protein has a ife of between about 20 and 27 hours.

The invention also es a method of treating a subject diagnosed With bleeding disorder, comprising measuring the half-life of a short-acting FVIII administered to said subject, Wherein a half-life that is at least about 1.2 times greater than the half-life of said short-acting FVIII in a subject having average VWF levels indicates that the subject is a candidate for long interval dosing, and administering a long-acting FVIII-Fc polypeptide in an effective amount and at a dosing interval of at least 3 days. In one embodiment, the half-life of the acting FVIII in said subject is at least about 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5-fold greater than the half-life of a acting FVIII When administered to a subject having average levels of VWF.

In one embodiment, the subject is a human. In r embodiment, the subject is a ric subject. In another embodiment, the subject has hemophilia A. In another embodiment, the subject has the blood serotype A, B, or AB.

In one ment, the long-acting FVIII-Fc is administered in an effective amount that is at least about 20IU/kg, at least about 25 IU/kg, at least about 30IU/kg, at least about 35 IU/kg, at least about g, at least about 45 IU/kg, at least about 501U/kg, at least about 55 IU/kg, at least about 601U/kg, at least about g, at least about 70IU/kg, at least about 75IU/kg, at least about 801U/kg, at least about 851U/kg, or at least about 90IU/kg. In another embodiment, the effective amount is at least about 651U/kg to at least about 901U/kg.

In one embodiment, the effective amount of the FVIII-Fc protein is stered about once every week, about once every 10 days, about once every two weeks, about once every 15 days, about once every 20 days, about once every three weeks, about once every 25 days, about once every four weeks, or about once every one month.

In one embodiment, the stration resolves greater than 5-20%, greater than 5-15%, greater than 5-10%, greater than , or greater than 10-15% of bleeding episodes. In one embodiment, the trough level of plasma Factor VIII:C in the subjects is maintained above 1-3 or 3-5 IU/dl.

In one embodiment, the administration prevents a bleeding episode in the subject. In one embodiment, the bleeding episode is spontaneous. In one embodiment, the administration resolves greater than 80-100%, greater than , greater than 85-90%, greater than 90-100%, greater than 90-95%, or greater than 95-100% of bleeding episodes. In one embodiment, the administration maintains tatis in the population of the subjects in need of a surgery. In one embodiment, the FVIII-Fc n is administered prior to, , or after the surgery. In one embodiment, the surgery is minor surgery, major surgery, tooth extraction, tonsillectomy, inguinal hemiotomy, synovectomy, total knee ement, craniotomy, osteosynthesis, trauma surgery, intracranial surgery, intra-abdominal surgery, horacic surgery, or joint replacement surgery.

In one embodiment the surgery is an emergency surgery.

In one embodiment, the FVIII-Fc protein has a half-life longer than a polypeptide consisting of FVIII. In one embodiment, the FVIII-Fc protein is pegylated, hesylated, or polysialylated. In one embodiment, the FVIII-Fc n is a FVIIIFc monomer dimer hybrid. In one embodiment, the FVIII portion is a single chain. In one embodiment, the FVIII portion comprises a heavy chain and a light chain. In one embodiment, the FVIII portion comprises full- length factor VIII, mature factor VIII, or factor VIII with a full or partial on of the B . In one embodiment, the FVIII portion comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 1 to 1438 of SEQ ID NO: 2 or amino acids 1 to 2332 of SEQ ID NO: 6. In one embodiment, the FVIII portion comprises amino acids 1 to 1438 of SEQ ID NO: 2 or amino acids 1 to 2332 of SEQ ID NO: 6. In one embodiment, the second portion of the chimeric polypeptide comprises an Fc region which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 1439 to 1665 of SEQ ID NO: 2 or amino acids 2333 to 2559 of SEQ ID NO: 6. In one embodiment, the second n comprises amino acids 1439 to 1665 of SEQ ID NO: 2 or amino acids 2333 to 2559 of SEQ ID NO: 6.

In one embodiment, the FVIII—Fc polypeptide is administered as part of a ceutical composition comprising at least one excipient.

The invention also provides a method for ining whether a subject diagnosed with bleeding disorder is a candidate for long interval dosing with a long- acting FVIII polypeptide, comprising measuring the expression levels of plasma VWF, wherein an VWF expression level of at least 100 IU/dL indicates that the subject is a candidate for long interval dosing using a cting FVIII polypeptide. In one ment, the VWF expression level is at least about 110 IU/dL, about 120 IU/dL, about 130 IU/dL, about 140 IU/dL, about 150 IU/dL, about 160 IU/dL, about 170 IU/dL, about 180 IU/dL, about 190 IU/dL, or about 200 IU/dL.

The invention also provides a method for determining whether a subject diagnosed with bleeding disorder is a candidate for long interval dosing of a long— acting FVIII polypeptide, comprising measuring the half—life of FVIII-Fe in said subject, wherein a half—life that is at least about 1.2-fold greater than the half—life of FVIII—Fe when administered to a t having average VWF levels indicates the subject is a candidate for long interval dosing. In one embodiment, the half-life of F’v’III—Fc is at least about 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5— fold greater than the half-life of FVIII-Fc when administered to a subject having average levels of VWF.

The invention also provides a method for ining r a subject diagnosed with bleeding disorder is a candidate for long interval dosing of a long- acting FVIII polypeptide, comprising measuring the half-life of short—acting FVIII in said subject, n a ife that is at least about 12—fold greater than the half—life of short—acting FVIII when administered to a subject having average VWF levels indicates the subject is a candidate for long interval dosing. In one embodiment, the half—life is at least about 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5— fold greater than the half—life of FVIII—Fe when administered to a subject having average levels of VWF. 1] Definitions of specific embodiments of the invention as claimed herein follow. [0058b] According to a first embodiment of the invention, there is provided a ceutical composition comprising: (i) a chimeric polypeptide, which comprises a Factor VIII (FVIII) portion and a second portion; and (ii) at least one pharmaceutically acceptable excipient, n 15% to about 25% of the FVIII portion of the chimeric polypeptide comprises single chain FVIII and about 75% to about 85% of the FVIII portion of the chimeric polypeptide comprises processed FVIII, and wherein the composition is lyophilized. [0058c] According to a second embodiment of the invention, there is provided use of the pharmaceutical composition of the first embodiment in the manufacture of a medicament for preventing, decreasing or treating a bleeding episode in a human. [0058d] According to a third embodiment of the invention, there is provided use of the ceutical composition of the first embodiment in the manufacture of a medicament for prophylactic treatment of a bleeding episode in a human. [0058c] According to a fourth embodiment of the invention, there is provided use of the pharmaceutical composition of the first embodiment in the manufacture of a medicament for on—demand treatment of a bleeding episode in a human.

] According to a fifth embodiment of the invention, there is provided use of the pharmaceutical composition of the first embodiment in the manufacture of a medicament for tailored lactic treatment of a bleeding episode in a human. [0058g] According to a sixth embodiment of the invention, there is provided use of the pharmaceutical composition of the first embodiment in the cture of a medicament for perioperative treatment of a subject before, after or during surgery.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES tic Representation of rFVIIIFc monomer.

FIGS. 2A—E. A—B. Non—reducing and reducing GE is of rFVIIIFc (processed or single chain). C. rFVIIIFc structure analyzed by LC/UV and LC/MS. D.

Total Ion Current (TIC) chromatogram (LC/MS map) of rFVIIIFc after thrombin cleavage. Major digestion ts are indicated. E. Deconvoluted Mass Spectrum of the A2 domain of rFVIIIFc and [Text continues on page 12] -lla- WO 09627 rBDDFVIII. Major products and their cognate masses are ted, corresponding to thrombin- cleaved A2 domain (S373 to R740) and two truncated products, S373 to Y729 and S373 to E720.

FIGS. 3A-C. Biochemical characterization of rFVIII-Fc: A. Activation of Factor X as a function of phospholipid vesicle concentration; B. Activation of Factor X as a function of FX concentration. C. Activation of Factor X as a function of Factor IXa concentration.

FIG 4. Activation of Factor X following ge by activated Protein C.

FIGS. 5A-D. Observed group mean FVIII activity (::SE) versus time profiles, sorted by dose level, grouped by compound (one stage assay, 25 IU/kg (A) or 65 IU/kg (B); and chromogenic assay, 25 IU/kg (C) or 65 IU/kg (D)) versus time.

FIGS. 6A-B. Observed group mean FVIII activity (::SE) versus time s, grouped by dose level and compound (one stage assay (A) or genic assay (B)) versus time.

FIGS. 7(A)-(C). In Vivo Efficacy of Single Chain FVIII:Fc in HemA Mouse Tail Vein Transection Model. (A) Single chain rFVIII:Fc doses are shown as squares, and processed rFVIII:Fc doses are shown as circles. (B) Percent survival following tail vein transection of 4.6 [Lg/kg, 1.38 [Lg/kg, and 0.46 [Lg/kg of rFVIIIFc or SC rFVIIIFc. (C) Percent of non-bleeders following tail vein transection of 4.6 [Lg/kg (black circle or inverted triangle), 1.38 [Lg/kg (triangle or d), and 0.46 [Lg/kg (square and gray ) of rFVIIIFc or SC rFVIIIFc, respectively.

Study Design. Fig. 8 depicts the study design of the phase l/2a study, which was a dose-escalation, sequential design to evaluate the safety and PK of rFVIIIFc compared with ADVATE® after a single intravenous dose of either 25 IU/kg (low dose Cohort A) or 65 IU/kg (high dose Cohort B).

Correlation of rFVIII Activity by One-Stage (aPTT) and Chromogenic Assays.

Correlation between one-stage ng (aPTT) and chromogenic assay results measuring FVIII activity (IU/mL) following injection ofADVATE® (Q) and rFVIIIFc ( ).

FIGS. lO(A)-(B). Group Mean Plasma FVIII Activity Pharmacokinetic Profiles for Low- Dose and High-Dose s. The plasma FVIII activity (one stage aPTT assay) versus time curve after a single intravenous injection of rFVIIIFc or ADVATE® are shown for (A) 25 IU/kg (low-dose cohort, n=6); and (B) 65 IU/kg (high dose cohort, n=10 E®]; n=9 [rFVIIIFc]).

Results presented are group mean :: standard error of mean (SEM).

FIGS. ll(A)-(B). Effect of VWF Antigen Levels on C1 and t1/2 of FVIII Activity after Injection of ADVATE® or rFVIIIFc. Correlation between VWF antigen levels and (A) the weight-adjusted c1 of ADVATE® .5415 and p=0.0012) and rFVIIIFc (122:0.5492 and 16); and (B) the tug of ® (122:0.7923 and p<o.0001) and rFVIIIFc (R2: 0.6403 and p=0.0003). Each dot represents an individual subject.

FIGS. l2(A)-(B). EX Vivo Whole Blood ROTEM® s for Individual Subjects After ion of ADVATE® or rFVIIIFc. Blood was sampled from subjects prior to and after treatment at doses of (A) 25 IU/kg ® and rFVIIIFc; and (B) 65 IU/kg ® and rFVIIIFc at specified time points. Clotting time was determined by NATEM initiated with Ca++ on a ROTEM® ment. Results presented are mean :: standard error of mean (SEM) from triplicate channel readings for each dual sample.

FIGS. 13(A)-(B). Activity comparison in thrombin generation assay (TGA). (A) SC rFVIIIFc showed a reduced endogenous in potential (ETP), and (B) a reduced peak thrombin compared to rFVIIIFc.

FIGS. 14(A)-(C): In Vitro ROTEM data. ROTEM (NATEM) results (Mean :: SD) for varying concentratios of XYNTHA, ADVATE< and rFVIIIFc spked in pooled whole blood obtained from naive HemA mice. (A). e clot time (CT) (Fig. 14A), (B). clot formation time (CFT) and (C). alpha angle.

FIGS. 15(A)-(C). EX Vivo ROTEM data. ROTEM (NATEM) results (Mean :: SD) from HemA mice following a single intravenous administration of 50 IU/kg of XYNTHA, ADVATE, or rFVIIIFc at 5 min, 24, 48, 72, and 96 hours after dosing. (A). Average clot time (CT), (B).

Clot formation time (CFT), and (C). alpha angle.

FIGS. 16(A)-(E): Real-time evaluation of the interaction of rFVIIIFc and single chain (SC) rFVIIIFc with VWF, and real-time evaluation of thrombin mediated release of rFVIIIFc and SC rFVIIIFc from VWF. (A). Surface plasmon resonance (SPR) analysis of rFVIIIFc and SC rFVIIIFc affinity for VWF. Depicted are the binding curve and the 1:1 fit interaction model. The X-aXis shows time in seconds and the y-aXis shows response in response units (RU). (B).

Reference subtracted sensograms of thrombin-mediated release of activated rFVIIIFc, SC rFVIIIFc, and B-domain deleted rFVIII lacking Fc moieties (rBDD FVIII) at 25° C (top) and 37° C (bottom). The X-aXis shows time in seconds and the y-aXis shows response in se units (RU). Individual lines indicate the response at ent oc-thrombin concentrations. The uppermost line is the response at 0 U/mL oc-thrombin, and each uent line runs in order for oc-thrombin concentrations of 0.005, 0.01, 0.02, 0.04, 0.08, 0.16, 0.31, 0.63, 1.3, 2.5, 5, 10, and 20 U/mL. (C). Double reference cted sensograms of thrombin mediated release phase for rFVIIIFc, SC rFVIIIFc, and rBDD FVIII at 25° C (top) and 37° C m). The X-aXis shows time in seconds and the y—aXis shows response in response units (RU). Individual lines indicate response at different oc-thrombin concentrations. The ost line is the response at 0 U/mL oc- thrombin, and each subsequent line runs in order for oc-thrombin concentrations of 0.005, 0.01, 0.02, 0.04, 0.08, 0.16, 0.31, 0.63, 1.3, 2.5, 5.0, 10, and 20 U/mL. (D). Thrombin-mediated release rate as a function of time for Fc, SC rFVIIIFc, and rBDD FVIII at 25° C (top) and 37° C (bottom). The X-aXis shows time in seconds and the y-aXis shows response in response units (RU).

Individual lines indicate response at different oc-thrombin concentrations. The uppermost line is the response at 20 U/mL OL-tl’lI‘OInbil’l, and each subsequent line runs in order for oc-thrombin concentrations of 10, 5, 2.5, 1.3, 0.63, 0.31, 0.16, 0.08, 0.04, 0.02, 0.01, and 0.005 U/mL. (E).

Peak thrombin-mediated e rate as a function of thrombin concentration for rFVIIIFc, SC rFVIIIFc, and rBDD FVIII at 25° C (top) and 37° C m). EC50 is half maximal effective concentration. The x-axis is ombin concentration in U/mL and the y-axis is maximum release rate in RU/second.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method of treating Hemophilia A with Factor VIII (processed, single chain, or a combination thereof) using a longer dosing interval and/or greater AUC than is possible with currently known Factor VIII ts. The present invention also provides improved Factor VIII chimeric polypeptides and methods of tion.

Treatment of hemophilia A is by replacement therapy targeting restoration of FVIII activity to 1 to 5 % of normal levels to prevent spontaneous bleeding (Mannucci, P.M. et al., N.

Engl. J. Med. 344:1773-9 (2001), herein incorporated by reference in its entirety). There are plasma-derived and recombinant FVIII products available to treat bleeding episodes on-demand or to t bleeding es from occurring by treating prophylactically. Based on the short half-life of these products (8-12 hr) (White G.C., et al., Thromb. Haemost. 77:660-7 (1997); Morfmi, M., Haemophilia 9 (suppl 1):94-99; discussion 100 (2003)), treatment regimens require frequent intravenous administration, commonly two to three times weekly for prophylaxis and one to three times daily for and treatment (Manco-Johnson, M.J., et al., N. Engl. J. Med. 357:535-544 (2007)), each of which is incorporated herein by reference in its entirety. Such frequent administration is painful and inconvenient.

The present invention provides a method of stering Factor VIII to a human subject in need thereof (e.g., human patient), comprising administering to the subject a therapeutic dose of a chimeric Factor VIII polypeptide, e. g., a ic Factor c polypeptide, or a hybrid of such a polypeptide at a dosing interval at least about one and one-half times longer than the dosing interval required for an equivalent dose of said Factor VIII without the non-Factor VIII portion (a polypeptide ting of said Factor VIII portion), e.g., without the Fc portion. The t invention is also directed to a method of increasing dosing interval of Factor VIII administration in a human subject in need thereof comprising administering the chimeric Factor VIII polypeptide.

The dosing interval may be at least about one and one-half to six times longer, one and one-half to five times longer, one and one-half to four times longer, one and one-half to three times longer, or one and one-half to two times , than the dosing interval required for an equivalent dose of said Factor VIII without the non-Factor VIII portion (a polypeptide consisting of said Factor VIII portion), e.g., without the Fc portion. The dosing al may be at least about one and one-half, two, two and one-half, three, three and one-half, four, four and one-half, five, five and one-half or six times longer than the dosing interval ed for an lent dose of said Factor VIII without the non-Factor VIII portion (a polypeptide consisting of said Factor VIII portion), e. g., without the Fc portion. The dosing interval may be about every three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen days or longer.

The dosing interval may be at least about one and one-half to 5, one and one-half, 2, 3, 4, or 5 days or longer.

The present invention also provides a method of administering Factor VIII to a human subject in need thereof, comprising administering to the subject a therapeutic dose of a chimeric Factor VIII polypeptide, e.g., a chimeric Factor VIII-Fc polypeptide, or a hybrid of such a polypeptide to obtain an area under the plasma concentration versus time curve (AUC) at least about one and one-quarter times greater than the AUC obtained by an equivalent dose of said Factor VIII without non-Factor VIII n (a polypeptide consisting of said Factor VIII portion), e.g., without the Fc portion. The t invention thus includes a method of increasing or extending AUC of Factor VIII actiVity in a human patient in need thereof comprising administering the chimeric Factor VIII polypeptide.

The present invention also es a method of administering Factor VIII to a subject in need thereof, comprising administering to the t a therapeutic dose of a polypeptide comprising a Factor VIII and an Fc or a hybrid of such a polypeptide at a dosing interval of about every three, four, five, six, seven, eight, nine, ten, eleven, twelve, en, or fourteen days or longer.

The methods of the invention may be practiced on a subject in need of lactic treatment or on-demand treatment.

"Administering," as used herein, means to give a pharmaceutically able Factor VIII polypeptide of the invention to a subject Via a ceutically acceptable route. Routes of administration can be intravenous, e.g., intravenous injection and intravenous infusion.

Additional routes of administration include, e.g., aneous, intramuscular, oral, nasal, and pulmonary administration. ic polypeptides and hybrid proteins may be administered as part of a pharmaceutical composition sing at least one excipient.

”Area under the plasma concentration versus time curve (AUC),” as used , is the same as the term of art in pharmacology, and is based upon the rate and extent of absorption of Factor VIII following administration. AUC is determined over a specified time period, such as 12, 18, 24, 36, 48, or 72 hours, or for infinity using extrapolation based on the slope of the curve.

Unless otherwise specified herein, AUC is determined for infinity. The determination of AUC may be carried out in a single t, or in a population of ts for which the average is calculated.

A ”B domain” of Factor VIII, as used herein, is the same as the B domain known in the art that is defined by internal amino acid ce identity and sites of proteolytic cleavage by thrombin, e.g., residues Ser741-Arg1648 of full length human factor VIII. The other human factor VIII domains are defined by the following amino acid residues: A1, residues Ala1-Arg372; A2, residues Ser373-Arg740; A3, residues Ser1690-Ile2032; C1, es Arg2033-Asn2172; C2, residues Ser2173-Tyr2332. The A3-C1-C2 sequence includes residues Ser1690-Tyr2332. The remaining sequence, residues Glu1649-Arg1689, is usually referred to as the factor VIII light chain activation e. The ons of the boundaries for all of the domains, including the B domains, for porcine, mouse and canine factor VIII are also known in the art. In one embodiment, the B domain of Factor VIII is d (”B domain deleted factor VIII” or ”BDD FVIII”). An example of a BDD FVIII is REFACTO® binant BDD FVIII), which has the same sequence as the Factor VIII n of the sequence in Table 2A(i) (amino acids 1 to 1457 or 20 to 1457 of SEQ ID NO:2). In another embodiment, the B domain deleted Factor VIII contains an intact intracellular processing site, which corresponds to Arginine at residue 754 of B domain deleted Factor VIII, which corresponds to Arginine residue 773 of SEQ ID NO: 2, or e 1648 of full- length Factor VIII, which corresponds to Arginine residue 1667 of SEQ ID NO: 6. The sequence residue numbers used herein without referring to any SEQ ID Numbers correspond to the Factor VIII sequence without the signal e sequence (19 amino acids) unless otherwise ted.

For example, S743/Q1638 of full-length Factor VIII corresponds to S762/Q1657 of SEQ ID NO: 6 due to the 19 amino acid signal peptide sequence. In other embodiments, the B domain deleted FVIII comprises a substitution or mutation at an amino acid position corresponding to Arginine 1645, a substitution or mutation at an amino acid position ponding to Arginine 1648, or a substitution or mutation at amino acid positions corresponding to Arginine 1645 and Arginine 1648 in full-length Factor VIII. In some ments, the amino acid substituted at the amino acid position ponding to Arginine 1645 is a different amino acid from the amino acid substituted at the amino acid position corresponding to Arginine 1648. In certain embodiments, the substitution or mutation is an amino acid other than arginine, e.g., alanine.

A ”B domain deleted factor VIII” may have the full or partial deletions disclosed in US.

Patent Nos. 6,316,226, 6,346,513, 7,041,635, 5,789,203, 6,060,447, 886, 6,228,620, ,972,885, 6,048,720, 5,543,502, 5,610,278, 5,171,844, 5,112,950, 4,868,112, and 6,458,563, each of which is incorporated herein by reference in its entirety. In some embodiments, a B domain deleted factor VIII sequence of the present invention comprises any one of the deletions disclosed at col. 4, line 4 to col. 5, line 28 and examples 1-5 of US. Patent No. 6,316,226 (also in US 513). In some embodiments, a B domain deleted factor VIII of the present invention has a deletion disclosed at col. 2, lines 26-51 and examples 5-8 of US. Patent No. 5,789,203 (also US 447, US 5,595,886, and US 6,228,620). In some embodiments, a B domain deleted factor VIII has a deletion described in col. 1, lines 25 to col. 2, line 40 of US Patent No. ,972,885; col. 6, lines 1-22 and example 1 of US. Patent no. 720; col. 2, lines 17-46 of US. Patent No. 5,543,502; col. 4, line 22 to col. 5, line 36 of US. Patent no. 5,171,844; col. 2, lines 55-68, figure 2, and e 1 of US. Patent No. 5,112,950; col. 2, line 2 to col. 19, line 21 and table 2 of US. Patent No. 4,868,112; col. 2, line 1 to col. 3, line 19, col. 3, line 40 to col. 4, line 67, col. 7, line 43 to col. 8, line 26, and col. 11, line 5 to col. 13, line 39 of US. Patent no. 7,041,635; or col. 4, lines 25-53, of US. Patent No. 6,458,563. In some embodiments, a B domain deleted factor VIII has a deletion of most of the B domain, but still contains amino- terminal ces of the B domain that are essential for in vivo lytic processing of the primary translation t into two polypeptide chain (i.e., intracellular processing site), as disclosed in WO 91/09122, which is orated herein by reference in its entirety. In some embodiments, a B domain deleted factor VIII is constructed with a deletion of amino acids 747- 1638, i.e., Virtually a complete deletion of the B . Hoeben R.C., et al. J. Biol. Chem. 265 (13): 7318-7323 (1990), orated herein by reference in its ty. A B domain deleted factor VIII may also contain a deletion of amino acids 771—1666 or amino acids 868-1562 of factor VIII. Meulien P., et al. Protein Eng. 2(4): 301-6 (1988), incorporated herein by reference in its entirety. Additional B domain deletions that are part of the invention include, e. g.,: deletion of amino acids 982 through 1562 or 760 through 1639 (Toole et al., Proc. Natl. Acad. Sci. USA. 83:5939-5942 (1986)), 797 through 1562 (Eaton et al., Biochemistry 25:8343-8347 (1986)), 741 through 1646 (Kaufman (PCT published application No. WO 87/04187)), 747-1560 (Sarver et al., DNA 6:553-564 (1987)), 741 through 1648 (Pasek (PCT application No.88/OO831)), 816 through 1598 or 741 through 1689 (Lagner (Behring Inst. Mitt. (1988) No 82:16-25, EP 295597)), each of which is incorporated herein by reference in its entirety. Each of the ing deletions may be made in any Factor VIII sequence.

In one embodiment, the B domain deleted Factor VIII portion in the chimeric polypeptide is processed into two chains connected (or associated) by a metal bond, the first chain comprising a heavy chain (A1 -A2-partial B) and a second chain comprising a light chain (A3-C1-C2) . In another embodiment, the B domain deleted Factor VIII portion is a single chain Factor VIII. The single chain Factor VIII can comprise an intracellular processing site, which corresponds to ne at e 754 of B domain deleted Factor VIII (residue 773 of SEQ ID NO: 2) or at residue 1648 of full-length Factor VIII (residue 1657 of SEQ ID NO: 6).

The metal bond between the heavy chain and the light chain can be any metal known in the art. For example, the metals useful for the invention can be a divalent metal ion. The metals that can be used to associate the heavy chain and light chain include, but not limited to, Ca2+, Mn2+, or Cu2+. Fatouros et al., Intern. J. Pharm. 155(1): 121-131 (1997); Wakabayashi et al., JBC. 279(13): 12677-12684 (2004).

”Chimeric polypeptide,” as used herein, means a polypeptide that es within it at least two polypeptides (or subsequences or peptides) from different sources. Chimeric polypeptides may include, e.g., two, three, four, five, six, seven, or more ptides from different s, such as different genes, different cDNAs, or different animal or other species.

Chimeric polypeptides may include, e. g., one or more linkers joining the different subsequences.

Thus, the subsequences may be joined ly or they may be joined indirectly, via linkers, or both, within a single chimeric polypeptide. Chimeric polypeptides may include, e.g., additional peptides such as signal sequences and sequences such as 6His and FLAG that aid in protein purification or detection. In addition, chimeric polypeptides may have amino acid or peptide additions to the N— and/or C-termini.

In some ments, the ic polypeptide comprises a Factor VIII portion and a non-Factor VIII portion. Exemplary non-Factor VIII portions include, e. g., Fc, XTEN, albumin, a PAS sequence, transferrin, CTP (28 amino acid C-terminal peptide (CTP) of human chorionic gonadotropin (hCG) with its 4 O-glycans), hylene glycol (PEG), hydroxyethyl starch (HES), albumin binding polypeptide, and albumin-binding small molecules. Exemplary chimeric ptides of the ion include, e.g., ic Factor VIII-Fc polypeptides, chimeric Factor VIII-XTEN polypeptides, chimeric Factor VIII-albumin ptides, chimeric Factor VIII-PAS polypeptides, chimeric Factor VIII-transferrin polypeptides, chimeric Factor VIII-CTP polypeptides, chimeric Factor VIII-PEG polypeptides, chimeric Factor VIII-HES polypeptides, chimeric Facotr VIII-albumbin binding polypeptide ptides, and chimeric Factor VIII.- albumin-binding small molecule polypeptides.

Exemplary chimeric Factor VIII-Fc polypeptides include, e. g., SEQ ID N02 or 6 (Table 2), with or without their signal sequences and the ic Fc polypeptide of SEQ ID NO:4 (Table 2).

The chimeric polypeptide may comprise a ce at least 90% or 95% identical to the Factor VIII and Fc amino acid sequence shown in Table 2A(i) without a signal sequence (amino acids 20 to 1684 of SEQ ID N02) or at least 90% or 95% identical to the Factor VIII and Fc amino acid sequence shown in Table 2A(i) with a signal sequence (amino acids 1 to 1684 of SEQ ID NO:2), wherein the sequence has Factor VIII activity. The Factor VIII activity can be measured by activated Partial Thromboplastin Time (aPPT) assay, chromogenic assay, or other known s. The chimeric polypeptide may comprise a sequence identical to the Factor VIII and Fc amino acid sequence shown in Table 2A(i) without a signal sequence (amino acids 20 to 1684 of SEQ ID N02) or cal to the Factor VIII and Fc amino acid sequence shown in Table 2A(i) with a signal sequence (amino acids 1 to 1684 of SEQ ID NO:2).

As discussed above, exemplary chimeric ptides include Factor VIII fused to one or more XTEN polypeptides. Schellenburger et al., Nat. Biotech. 6-90 (2009), which is incorporated herein by reference in its entirety. The XTEN ptide can be fused to either the N—terminal end of FVHI or to the C-terminal end of FVHI. A protease site may be included between the XTEN portion and the Factor VIII portion to allow such processing. XTEN polypeptides include, e.g., those disclosed in WO 2009/023270, WO 2010/091122, WO 2007/103515, US 2010/0189682, and US 2009/0092582, each of which is incorporated herein by reference in its ty.

As discussed above, exemplary chimeric polypeptides also include Factor VIII fused to one or more albumin ptides, albumin binding polypeptides, or albumin-binding small molecules. In one embodiment, the albumin is human albumin. The albumin or n binding protein can be fused to either the N—terminal end of FVlll or to the C-terminal end of FVlll or inserted between two amino acids in FVlll. Examples of albumin, e.g., fragments thereof, that may be used in the present invention are known. e. g., U.S. Patent No. 7,592,010; U.S. Patent No. 6,686,179; and Schulte, Thrombosis Res. 124 Suppl. 2:S6-S8 (2009), each of which is orated herein by nce in its entirety.

The albumin binding polypeptides can compromise, without limitation, bacterial albumin- binding domains, albumin-binding peptides, or albumin-binding antibody fragments that can bind to albumin. Domain 3 from streptococcal protein G, as disclosed by Kraulis et al., FEBS Lett. 378:190-194 (1996) and Linhult et al., Protein Sci. 11:206-213 (2002) is an example ofa bacterial albumin-binding domain. es of albumin-binding peptides include a series of peptides having the core ce DICLPRWGCLW (SEQ ID NO: 7). See, e,g., Dennis et al., J. Biol.

Chem. 2002, 277: 35035-35043 (2002). Examples of albumin-binding antibody fragments are disclosed in Muller and Kontermann, Curr. Opin. Mo]. Ther. 9:319-326 (2007); Rooverset et al., Cancer Immunol. Immunother. 56:303-317 (2007), and Holt et al., Prot. Eng. Design Sci., 21 88 (2008), which are incorporated herein by nce in their entireties..

In certain aspects, a recombinant FVlll polypeptide of the invention ses at least one attachment site for a non-polypeptide small le, variant, or derivative that can bind to albumin thereof. An example of such albumin binding moieties is 2-(3 -maleimidopropanamido)- 6-(4-(4-iodophenyl)butanamido)hexanoate (“Albu” tag) as sed by Trusselet et al., Bioconjugate Chem. 20:2286-2292 (2009).

As discussed above, exemplary chimeric polypeptides also include Factor VIII fused to at least one B subunit of the C-terminal peptide (CTP) of human chorionic gonadotropin or fragment, variant, or derivative thereof. The CTP can be fused to Factor VIII either the N- terminal end of FVlll or to the C-terminal end of FVlll or inserted between two amino acids in FVlll. One or more CTP peptides fused to or ed into a recombinant protein is known to increase the in vivo half-life of that protein. See, e.g., U.S. Patent No. 5,712,122, incorporated by reference herein in its entirety. Exemplary CTP peptides include DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL (SEQ ID NO: 8) or PPPSLPSPSRLPGPSDTPILPQ. (SEQ ID NO: 9). See, e.g., U.S. Patent Application Publication No. US 2009/008741 1 A1, incorporated by reference.

As discussed above, ary ic polypeptides also include Factor VIII fused to at least one PAS sequence or fragment, variant, or derivative thereof. The PAS sequence can be fused to either the N—terminal end of FVIII or to the C-terminal end of FVIII or inserted between two amino acids in FVIII. A PAS peptide or PAS sequence, as used herein, means an amino acid sequence comprising mainly alanine and serine residues or comprising mainly alanine, serine, and proline residues, the amino acid sequence forming random coil conformation under physiological conditions. Accordingly, the PAS sequence is a building block, an amino acid polymer, or a ce cassette sing, consisting essentially of, or consisting of alanine, serine, and proline which can be used as a part of the heterologous moiety in the ic protein. An amino acid polymer also can form random coil conformation when residues other than alanine, , and proline are added as a minor tuent in the PAS sequence. By “minor constituent” is meant that that amino acids other than alanine, serine, and e can be added in the PAS sequence to a certain degree, e.g., up to about 12%, i.e., about 12 of 100 amino acids of the PAS sequence, up to about 10%, up to about 9%, up to about 8%, about 6%, about 5%, about 4%, about 3%, Le. about 2%, or about 1%, of the amino acids. The amino acids different from alanine, serine and e cab be selected from the group consisting of Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Val. Under physiological conditions, a PAS peptide forms a random coil conformation and thereby can mediate an increased in vivo and/or in vitro stability to a recombinant protein of the invention, and has procoagulant activity.

Non-limiting examples of the PAS peptides include ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 10), AAPASPAPAAPSAPAPAAPS (SEQ ID NO: 1 1), APSSPSPSAPSSPSPASPSS (SEQ ID NO: 12), APSSPSPSAPSSPSPASPS (SEQ ID NO: 13), SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 14), PSAPPAAASPAAPSAPPA (SEQ ID NO: 15), ASAAAPAAASAAASAPSAAA (SEQ ID NO: 16) or any variants, derivatives, fragments, or combinations f onal examples of PAS sequences are known from, e.g., US Pat. Publ. No. 2010/0292130 A1 and PCT Appl. Publ. No. A1.

European issued patent EP2173 890.

As discussed above, exemplary chimeric polypeptides also include Factor VIII fused to at least one transferrin peptide or fragment, variant, or derivative thereof At least one transferrin peptide can be fused to either the N—terminal end of FVIII or to the C-terminal end of FVIII or inserted between two amino acids in FVIII. Any transferrin can be fused to or inserted into a inant FVIII protein of the ion. As an example, wild-type human Tf (Tf) is a 679 amino acid protein, of approximately 75 KDa (not accounting for glycosylation), with two main domains, N (about 330 amino acids) and C (about 340 amino acids), which appear to originate from a gene duplication. See GenBank accession numbers NM001063, XM002793, M12530, XM039845, XM 039847 and S95936 cbi.nlm.nih.gov), all of which are herein incorporated by reference in their entirety.

Transferrin transports iron through transferrin receptor (TfR)-mediated endocytosis.

After the iron is released into an endosomal compartment and Tf-TfR complex is recycled to cell surface, the Tf is released back extracellular space for next cycle of iron transporting. Tf possesses a long half-life that is in excess of 14-17 days (Li et al., Trends Pharmacol. Sci. 23:206- 209 ).Transferrin fusion proteins have been studied for half-life extension, targeted deliver for cancer ies, oral delivery and sustained tion of proinsulin (Brandsma et al., Biotechnol. Adv., 29: 230-238 (2011); Bai et al., Proc. Natl. Acad. Sci. USA 92—7296 (2005); Kim et al., J. Pharmacol. Exp. Ther, 334:682-692 (2010); Wang et al., J. Controlled Release 155:386-392 (2011)).

As discussed above, exemplary chimeric polypeptides also e Factor VIII fused to at least one polyethylene glycol (PEG) moieties.

PEGylated FVIII can refer to a conjugate formed between FVIII and at least one polyethylene glycol (PEG) molecule. PEG is commercially available in a large variety of molecular weights and average molecular weight ranges. Typical examples of PEG average molecular weight ranges include, but are not limited to, about 200, about 300, about 400, about 600, about 1000, about 1300-1600, about 1450, about 2000, about 3000, about 3000-3750, about 3350, about 3000-7000, about 3500-4500, about 5000-7000, about 7000-9000, about 8000, about 10000, about 8500-11500, about 16000-24000, about 35000, about 40000, about 60000, and about 80000 daltons. These average molecular weights are ed merely as examples and are not meant to be limiting in any way.

A recombinant FVIII protein of the invention can be ted to e mono- or poly- (e.g., 2-4) PEG moieties. PEGylation can be carried out by any of the PEGylation reactions known in the art. Methods for preparing a PEGylated n product will generally include (i) reacting a ptide with polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions whereby the peptide of the invention becomes attached to one or more PEG groups; and (ii) obtaining the reaction product(s). In l, the optimal on conditions for the reactions will be determined case by case based on known parameters and the desired result.

There are a number of PEG attachment methods available to those d in the art, for example Malik F et al., Exp. Hematol. 20:1028-35 (1992); Francis, Focus on Growth Factors 3(2):4-10 (1992); European Pat. Pub. Nos. 384, EP0154316, and EP0401384; and International Pat. Appl. Pub. Nos. WO92/l 6221 and 4326. As a non-limiting example, FVIII variants can contain ne substitutions in one or more insertion sites in FVIII, and the cysteines can be further conjugated to PEG polymer. See Mei et al., Blood 116:270-279 (2010) and U.S. Patent No. 7,632,921, which are incorporated herein by reference in their entireties.

As discussed above, exemplary chimeric polypeptides also include Factor VIII fused to at least one hydroxyethyl starch (HES) polymer. HES is a derivative of naturally occurring amylopectin and is degraded by alpha-amylase in the body. HES exhibits advantageous biological properties and is used as a blood volume ement agent and in hemodilution therapy in the clinics. See, e.g., Sommermeyer et al., Krankenhauspharmazie 8:271-278 (1987); and Weidler et al., Arzneim.-Forschung/Drug Res. 41: 494-498 (1991).

HES is mainly characterized by the molecular weight bution and the degree of substitution. HES has a mean molecular weight (weight mean) of from 1 to 300 kD, from 2 to 200kD, from 3 to 100 kD, or from 4 to 70kD. yethyl starch can further exhibit a molar degree of tution of from 0.1 to 3, from 0.1 to 2,from 0.1 to 0.9, or from 0.1 to 0.8, and a ratio between C2:C6 substitution in the range of from 2 to 20 with t to the yethyl groups. HES with a mean molecular weight of about 130 kD is Voluven® from ius.

Voluven® is an artificial colloid, employed, e.g., for volume replacement used in the therapeutic indication for therapy and prophylaxis of hypovolaemia. There are a number of HES attachment methods available to those skilled in the art, e.g., the same PEG attachment methods described above.

] In some ments, a chimeric polypeptide comprising a Factor VIII portion has an increased half-life (t1/2) over a polypeptide consisting of the same Factor VIII portion without the non Factor VIII portion. A chimeric Factor VIII polypeptide with an increased tl/2 may be referred to herein as a long-acting Factor VIII. Long-acting chimeric Factor VIII polypeptides include, e.g., Factor VIII fused to Fc (including, e.g., chimeric Factor VIII polypeptides in the form of a hybrid such as a FVIIIFc monomer dimer hybrid; see Example 1, Fig. l, and Table 2A; and US Patent Nos. 7,404,956 and 7,348,004), Factor VIII fused to XTEN, and Factor VIII fused to albumin.

”Culture,” ”to culture” and ”culturing,” as used herein, means to incubate cells under in Vitro conditions that allow for cell grth or diVision or to maintain cells in a liVing state.

”Cultured cells,” as used , means cells that are propagated in Vitro.

] ”Factor VIII,” as used herein, means functional factor VIII polypeptide in its normal role in coagulation, unless otherwise specified. Thus, the term Factor VIII includes variant polypeptides that are onal. Factor VIII proteins can be the human, porcine, canine, and murine factor VIII proteins. As described in the Background Art section, the full length polypeptide and cleotide sequences are known, as are many functional fragments, s and modified versions. Examples of human factor VIII sequences are shown as uences in SEQ ID NOs:2 or 6 (Table 2). Factor VIII polypeptides include, e.g., full-length factor VIII, full- length factor VIII minus Met at the N—terminus, mature factor VIII (minus the signal sequence), mature factor VIII with an additional Met at the N—terminus, and/or factor VIII with a full or partial deletion of the B domain. Factor VIII variants include B domain deletions, whether partial or full deletions.

A great many functional factor VIII variants are known, as is discussed above and below.

In addition, hundreds of nonfunctional mutations in factor VIII have been identified in hemophilia patients, and it has been ined that the effect of these mutations on factor VIII function is due more to where they lie within the 3-dimensional structure of factor VIII than on the nature of the substitution (Cutler et al., Hum. Mutat. [9274-8 (2002)), incorporated herein by reference in its entirety. In addition, comparisons between factor VIII from humans and other species have identified ved residues that are likely to be required for function (Cameron et al., Thromb.

Haemost. 79:317-22 (1998); US 6,251,632), incorporated herein by reference in its entirety.

The human factor VIII gene was isolated and expressed in mammalian cells (Toole, J. J., et al., Nature 312:342-347 (1984); Gitschier, J., et al., Nature 312:326-330 (1984); Wood, W. I., et al., Nature 312:330-337 ; Vehar, G. A., et al., Nature 312:337-342 (1984); WO 87/04187; WO 88/08035; WO 88/03558; U.S. Pat. No. 4,757,006), each of which is incorporated herein by reference in its entirety, and the amino acid sequence was deduced from cDNA. Capon et al., U.S. Pat. No. 4,965,199, incorporated herein by reference in its entirety, discloses a recombinant DNA method for producing factor VIII in mammalian host cells and purification of human factor VIII. Human factor VIII expression in CHO (Chinese hamster ovary) cells and BHKC (baby r kidney cells) has been reported. Human factor VIII has been d to delete part or all of the B domain (U.S. Pat. Nos. 4,994,371 and 4,868,112, each of which is incorporated herein by reference in its ty), and replacement of the human factor VIII B domain with the human factor V B domain has been performed (U.S. Pat. No. 5,004,803, incorporated herein by reference in its entirety). The cDNA sequence encoding human factor VIII and ted amino acid sequence are shown in SEQ ID NOs:1 and 2, respectively, of US Application Publ. No. 2005/0100990, incorporated herein by reference in its entirety.

U.S. Pat. No. 5,859,204, Lollar, J. S., orated herein by nce in its entirety, reports functional mutants of factor VIII having reduced antigenicity and reduced immunoreactivity. U.S. Pat. No. 6,376,463, Lollar, J. S., incorporated herein by reference in its entirety, also reports mutants of factor VIII having reduced immunoreactivity. US Application Publ. No. 2005/0100990, Saenko et al., orated herein by nce in its entirety, reports functional mutations in the A2 domain of factor VIII.

A number of functional factor VIII les, including B-domain deletions, are disclosed in the following patents US 6,316,226 and US 6,346,513, both assigned to Baxter; US 7,041,635 assigned to In2Gen; US 5,789,203, US 6,060,447, US 5,595,886, and US 6,228,620 assigned to Chiron; US 5,972,885 and US 6,048,720 assigned to rum, US 502 and US ,610,278 assigned to Novo Nordisk; US 5,171,844 assigned to Immuno Ag; US 5,112,950 assigned to Transgene S.A.; US 4,868,112 assigned to Genetics Institute, each of which is incorporated herein by reference in its entirety.

The porcine factor VIII sequence is published, (Toole, J. J et al., Proc. Natl. Acad. Sci.

USA 83:5939-5942 (1986)), incorporated herein by reference in its entirety, and the complete porcine cDNA sequence ed from PCR amplification of factor VIII sequences from a pig spleen cDNA library has been reported (Healey, J. F. et al., Blood 88:4209-4214 (1996), incorporated herein by reference in its ty). Hybrid porcine factor VIII haVing substitutions of all domains, all subunits, and specific amino acid sequences were disclosed in US. Pat. No. 771 by Lollar and Runge, and in WO 93/20093, incorporated herein by reference in its entirety. More ly, the nucleotide and corresponding amino acid sequences of the A1 and A2 domains of porcine factor VIII and a chimeric factor VIII with porcine A1 and/or A2 domains tuted for the corresponding human domains were reported in W0 94/11503, incorporated herein by reference in its entirety. US. Pat. No. 5,859,204, Lollar, J. S., also discloses the porcine cDNA and deduced amino acid ces. US Pat. No. 6,458,563, incorporated herein by reference in its entirety assigned to Emory discloses a B-domain deleted porcine Factor VIII.

The Factor VIII (or Factor VIII portion of a ic polypeptide) may be at least 90% or 95% identical to a Factor VIII amino acid sequence shown in Table 2 without a signal sequence (amino acids 20 to 1457 of SEQ ID N02; and amino acids 20 to 2351 of SEQ ID NO:6), wherein said Factor VIII portion has Factor VIII actiVity. The Factor VIII (or Factor VIII portion of a chimeric polypeptide) may be identical to a Factor VIII amino acid sequence shown in Table 2 without a signal sequence (amino acids 20 to 1457 of SEQ ID N02; and amino acids 20 to 2351 of SEQ ID NO:6).

The Factor VIII (or Factor VIII portion of a chimeric polypeptide) may be at least 90% or 95% identical to a Factor VIII amino acid sequence shown in Table 2 with a signal ce (amino acids 1 to 1457 of SEQ ID N02 and amino acids 1 to 2351 of SEQ ID NO:6), wherein said Factor VIII n has Factor VIII actiVity. The Factor VIII (or Factor VIII portion of a chimeric polypeptide) may be identical to a Factor VIII amino acid sequence shown in Table 2 with a signal sequence (amino acids 1 to 1457 of SEQ ID N02 and amino acids 1 to 2351 of SEQ ID NO:6).

”Equivalent dose,” as used herein, means the same dose of Factor VIII activity as expressed in International Units, which is independent of molecular weight of the polypeptide in question. One International Unit (IU) of factor VIII ty corresponds approximately to the quantity of factor VIII in one iter of normal human plasma. Several assays are available for measuring Factor VIII activity, including the European Pharmacopoeia chromogenic substrate assay and a one stage clotting assay.

”Fc,” as used herein, means functional neonatal Fc receptor (FcRn) binding partners, unless otherwise specified. An FcRn binding partner is any molecule that can be cally bound by the FcRn receptor with consequent active transport by the FcRn receptor of the FcRn binding partner. Thus, the term Fc includes any variants of IgG Fc that are onal. The region of the Fc portion of IgG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al. Nature 372:379 (1994), incorporated herein by reference in its entirety). The major contact area of the Fc with the FcRn is near the junction of the CH2 and CH3 domains. Fc-FcRn contacts are all within a single Ig heavy chain. The FcRn binding partners include, e. g., whole IgG, the Fc fragment of IgG, and other fragments of IgG that include the complete binding region of FcRn. The major contact sites e amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain and amino acid residues 385-387, 428, and 433-436 of the CH3 domain. References made to amino acid numbering of immunoglobulins or immunoglobulin fragments, or regions, are all based on Kabat et al. 1991, Sequences of Proteins of Immunological st, U. S. Department of Public Health, Bethesda; MD, incorporated herein by reference in its entirety. (The FcRn receptor has been isolated from several mammalian species including humans. The ces of the human FcRn, rat FcRn, and mouse FcRn are known (Story et al., J. Exp. Med. 180: 2377 (1994), incorporated herein by reference in its ty.) An Fc may comprise the CH2 and CH3 domains of an immunoglobulin with or without the hinge region of the globulin. Exemplary Fc variants are provided in and , incorporated herein by reference in its entirety.

] Fc (or Fc portion of a chimeric polypeptide) may contain one or more mutations, and combinations of mutations.

Fc (or Fc portion of a chimeric polypeptide) may contain mutations conferring increased ife such as M252Y, S254T, T256E, and combinations thereof, as sed in Oganesyan et al., Mol. Immunol. 46:1750 (2009), which is incorporated herein by reference in its entirety; H433K, N434F, and combinations thereof, as sed in o et al., Nat. Biotechnol. 23 :1283 (2005), which is incorporated herein by reference in its entirety; the mutants disclosed at pages 1-2, aph [0012], and Examples 9 and 10 of US 2009/0264627 A1, which is incorporated herein by reference in its entirety; and the mutants disclosed at page 2, paragraphs to [0021] of US 20090163699 A1, which is incorporated herein by reference in its entirety.

Fc (or Fc portion of a chimeric polypeptide) may also e, e.g., the following mutations: The Fc region of IgG can be modified according to well recognized ures such as site ed mutagenesis and the like to yield modified IgG or Fc fragments or portions thereof that will be bound by FcRn. Such ations include, e.g., modifications remote from the FcRn contact sites as well as ations within the t sites that preserve or even enhance binding to the FcRn. For example the following single amino acid residues in human IgG] Fc (Fcyl) can be substituted without significant loss of Fc binding affinity for FcRn: P23 8A, SZ39A, K246A, K248A, D249A, M252A, T256A, E258A, T26OA, D265A, SZ67A, H268A, E269A, D27OA, E272A, L274A, N276A, Y278A, D28OA, V282A, E283A, H285A, N286A, T289A, K29OA, R292A, E293A, E294A, Q295A, Y296F, N297A, SZ98A, Y3OOF, R301A, V303A, V305A, T307A, L309A, Q311A, D312A, N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A33OQ, A33OS, P331A, P3318, E333A, K334A, T335A, S337A, K338A, K34OA, Q342A, R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K36OA, N361A, Q362A, Y373A, S375A D376A, A378Q, E38OA, E382A, S383A, N384A, Q386A, E388A, N389A, N39OA, Y391F, K392A, L398A, S4OOA, D401A, D413A, K414A, R416A, Q418A, Q419A, N421A, V422A, S424A, E43OA, N434A, T437A, Q438A, K439A, S44OA, S444A, and K447A, where for example P23 8A represents wildtype proline substituted by alanine at on number 238. In addition to alanine other amino acids may be substituted for the wildtype amino acids at the positions ed above. ons may be introduced singly into Fc giVing rise to more than one hundred FcRn binding partners distinct from native Fc. Additionally, combinations of two, three, or more of these individual mutations may be introduced together, giVing rise to hundreds more FcRn binding partners. Certain of these mutations may confer new functionality upon the FcRn binding partner. For example, one embodiment incorporates N297A, ng a highly conserved N—glycosylation site. The effect of this mutation is to reduce immunogenicity, thereby enhancing circulating half-life of the FcRn binding partner, and to render the FcRn binding partner incapable of g to FcyRI, FcyRIIA, FcyRIIB, and FcyRIIIA, without compromising affinity for FcRn (Routledge et al. 1995, Transplantation 60:847, which is incorporated herein by reference in its entirety; Friend et al. 1999, lantation 68:1632, which is incorporated herein by reference in its entirety; Shields et al. 1995, J. Biol. Chem. 276:6591, which is orated herein by reference in its entirety).

Additionally, at least three human Fc gamma receptors appear to recognize a binding site on IgG within the lower hinge region, generally amino acids 234-237. Therefore, another example of new functionality and potential decreased immunogenicity may arise from mutations of this region, as for example by replacing amino acids 233-236 of human IgG] ”ELLG” to the corresponding sequence from IgG2 ”PVA” (with one amino acid deletion). It has been shown that FcyRI, FcyRII, and FcyRIII which mediate various effector functions will not bind to IgGl when such mutations have been introduced (Ward and Ghetie, Therapeutic Immunology 2:77 (1995), which is incorporated herein by reference in its ty; and Armour et al., Eur. J. Immunol. 29:2613 (1999), which is incorporated herein by reference in its entirety). As a further example of new functionality g from mutations described above affinity for FcRn may be increased beyond that of wild type in some instances. This increased affinity may reflect an sed ”on” rate, a decreased ”off‘ rate or both an increased ”on” rate and a decreased ”off‘ rate. Mutations ed to impart an increased affinity for FcRn include, e.g., T256A, T307A, E380A, and N434A (Shields et al. J. Biol. Chem. 276:6591 (2001), which is incorporated herein by reference in its entirety).

The Fc (or Fc portion of a chimeric polypeptide) may be at least 90% or 95% identical to the Fc amino acid sequence shown in Table 2 (amino acids 1458 to 1684 of SEQ ID N02 or amino acids 2352 to 2578 of SEQ ID NO:6). The Fc (or Fc n of a chimeric polypeptide) may be identical to the Fc amino acid sequence shown in Table 2 (amino acids 1458 to 1684 of SEQ ID N02 and amino acids 2352 to 2578 of SEQ ID NO:6).

"Hybrid” polypeptides and proteins, as used herein, means a combination of a chimeric ptide with a second ptide. The chimeric polypeptide and the second polypeptide in a hybrid may be associated with each other via protein-protein ctions, such as charge-charge or hydrophobic interactions. The chimeric polypeptide and the second polypeptide in a hybrid may be associated with each other via disulfide or other covalent bond(s). Hybrids are described in WC 2004/101740 and , each of which is incorporated herein by reference in its entirety. See also US. Patent Nos. 7,404,956 and 7,348,004, each of which is incorporated herein by reference in its entirety. The second polypeptide may be a second copy of the same ic polypeptide or it may be a non-identical chimeric ptide. See, e.g., Figure 1, Example 1, and Table 2. In one embodiment, the second ptide is a polypeptide comprising an Fc. In another ment, the chimeric polypeptide is a chimeric Factor VIII-Fc polypeptide and the second polypeptide consists essentially of Fc, e.g., the hybrid polypeptide of Example 1, which is a rFVIIIFc recombinant fusion protein consisting of a single molecule of inant B- domain d human FVIH (BDD-rFVIII) fused to the dimeric Fc domain of the human lgGl, with no intervening linker sequence. This hybrid polypeptide is referred to herein as FVIIIFc monomeric Fc fusion protein, FVIIIFc monomer hybrid, monomeric FVHHFc hybrid, and FVIIIFc monomer-dimer. See Example 1, Fig. 1, and Table 2A. The Examples provide preclinical and clinical data for this hybrid polypeptide.

The second polypeptide in a hybrid may comprise or consist essentially of a sequence at least 90% or 95% identical to the amino acid sequence shown in Table 2A(ii) without a signal sequence (amino acids 21 to 247 of SEQ ID NO:4) or at least 90% or 95% identical to the amino acid sequence shown in Table 2A(ii) with a signal sequence (amino acids 1 to 247 of SEQ ID NO-:4). The second polypeptide may comprise or consist essentially of a sequence cal to the amino acid sequence shown in Table 2A(ii) t a signal sequence (amino acids 21 to 247 of SEQ ID NO:4) or identical to the amino acid sequence shown in Table 2A(ii) with a signal sequence (amino acids 1 to 247 of SEQ ID NO:4).

Figure 1 is a schematic showing the structure of a B domain deleted factor VIII-Fc chimeric polypeptide, and its ation with a second polypeptide that is an Fc polypeptide. To obtain this hybrid, the coding sequence of human inant B-domain deleted FVIII was obtained by reverse transcription-polymerase chain reaction (RT-PCR) from human liver poly A RNA ech) using FVIII-specific primers. The FVIII sequence includes the native signal sequence for FVIII. The B-domain on was from serine 743 (S743; 2287 bp) to glutamine 1638 (Q1638; 4969 bp) for a total deletion of 2682 bp. Then, the coding sequence for human recombinant Fc was obtained by RT-PCR from a human leukocyte cDNA y ech) using Fc specific primers. Primers were designed such that the B-domain deleted FVIII sequence was fused ly to the N—terminus of the Fc sequence with no intervening linker. The FVIIIFc DNA sequence was cloned into the mammalian dual expression vector pBUDCE4.] rogen) under control of the CMV promoter. A second identical Fc sequence including the mouse ng signal sequence was obtained by RT-PCR and cloned downstream of the second promoter, EFla, in the expression vector pBUDCE4. 1.

The rFVIIIFc expression vector was transfected into human embryonic kidney 293 cells (HEK293H; InVitrogen) using Lipofectamine 2000 transfection reagent (InVitrogen). Stable clonal cell lines were generated by selection with Zeocin (InVitrogen). One clonal cell line, 3C4- 22 was used to generate FVIIIFc for characterization in Vivo. Recombinant FVIIIFc was produced and d (McCue et al. 2009) at Biogen Idec (Cambridge, MA). The transfection strategy described above was expected to yield three products, i.e., monomeric rFVIIIFc s, dimeric rFVIIIFc hybrids and dimeric Fc. However, there was essentially no dimeric rFVIIIFc detected in the conditioned medium from these cells. Rather, the conditioned medium contained Fc and monomeric rFVIIIFc. It is possible that the size of dimeric rFVIIIFc was too great and prevented efficient secretion from the cell. This result was beneficial since it rendered the purification of the monomer less complicated than if all three proteins had been present. The material used in these studies had a specific actiVity of approximately 9000 IU/mg.

] In one embodiment, the polypeptides of the invention are administered to a patient who expresses high level of von Willebrand factor (VWF). ”Subject" or ”patient” as used herein means a human indiVidual. A subject can be a patient who is currently suffering from a ng disorder or is expected to be in need of such a treatment. ct" can include an adult or a pediatric subject. The pediatric t can be a pediatric patient under the age of 12. The term ”pediatrics” as used herein is the branch of medicine that deals with the care of infants and en and the treatment of their diseases. In one ment, the subject is a pediatric patient who has a diagnosis of severe hemophilia A. In certain embodiments, pediatric subjects are treated with a long-acting Factor VIII polypeptide of the invention.

VWF is a plasma protein having a er ure in which the molecular weight of the various forms varies between approximately 230 kDa for each monomer subunit and up to more than 20 million Da in the multimer forms of greater molecular weight, thus forming the largest known soluble protein. Its plasma concentration is imately around 5-10 [Lg/ml (Siedlecki et al., Blood, vol 88: 2939-2950 (1996)) and the plasma form of smaller size is that corresponding to the dimer, with an approximate size of 500 kDa.

VWF has an essential role to play in primary haemostasis, being responsible for the adhesion of platelets to damaged vascular surfaces and therefore formation of the platelet plug on which the mechanisms for formation of the fibrin coagulate develop. It is suggested that the higher molecular weight multimers support platelet adhesion mechanisms to the sub-endothelium with greater efficiency and the clinical efficacy of VWF concentrates has been related to the concentration of these multimers of higher molecular weight (Metzner et al., Haemophilia 4:25- 32 (1998).

] Therefore, subjects expressing high levels of VWF would require less frequent dosing of FVIII compared to a subject who expresses lower or normal levels ofVWF. The average range of VWF in plasma is n about 50 IU/dL and about 200 IU/dL. In one embodiment, the average level of VWF in plasma is about 50 IU/dL. In r embodiment, a VWF level in plasma of at least about 100 IU/dL is considered a high VWF level. In another ment, a high level of VWF in plasma is between about 100 IU/dL and about 200 IU/dL. In another embodiment a high level of VWF in plasma is at least about 110 IU/dL, about 120 IU/dL, about 130 IU/dL, about 140 IU/dL, about 150 IU/dL, about 160 IU/dL, about 170 IU/dL, about 180 IU/dL, about 190 IU/dL, or about 200 IU/dL.

Therefore, in one embodiment, ts expressing at least about 100 IU/dL of plasma VWF are administered a long-acting FVIII polypeptide of the invention at a long interval dosing regimen. In one embodiment, the long-acting FVIII polypeptide is administered at a dosing al of at least about 3 days. In another embodiment, the long-acting FVIII polypeptide is administered at a dosing interval of at least about once every week, about once every two weeks, about once every 15 days, about once every 20 days, about once every three weeks, about once every 25 days, about once every four weeks, or about once every one month.

In one embodiment, the subjects were previously identified as having high levels of VWF. In certain embodiments, subjects having a blood serotype other than 0 (i.e., A, B, or AB) require less frequent dosing of long-acting FVIII because the long-acting FVIII has a longer half- life in these ts. In these subjects, the increased half-life is due to their ed VWF levels. er, pharmacokinetic data, defined as the study of the time course of drug absorption, bution, metabolism, and excretion, can be used as an fier of subjects eligible for longer or shorter dosing intervals using a long-acting FVIII polypeptide of the invention. Cinical pharmacokinetics is the application of pharmacokinetic principles to the safe and effective eutic management of drugs in an individual patient. The primary goals of clinical pharmacokinetics include enhancing efficacy and decreasing toxicity of a patient’s drug therapy. The pment of strong ations n drug concentrations and their pharmacologic responses has enabled clinicians to apply cokinetic principles to actual patient situations.

Thus, in one embodiment, the half-life of a FVIII-Fc ptide of the invention is used to fy patients who express high levels of VWF. The range of half-life of FVIII-Fc is between about 10 and about 40 hours, depending at least in part on the levels of VWF also present. On average however, the half-life of FVIII-Fc is about 18 hours. Generally, FVIII-Fc exhibits an increased half-life of at least about 12-fold in patients having high levels of VWF compared to the half-life of FVIII-Fc when administered to individuals having average levels of VWF. In one embodiment, FVIII-Fc exhibits an increased half-life of at least about 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5-fold compared to the half-life of FVIII-Fc when administered to individuals having average levels of VWF. In one embodiment, in subjects expressing high levels of VWF, the half-life of FVIII-Fc is between at least about 20 hours and about 40 hours. In another embodiment, the ife of FVIII-Fc is at least about 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, or 40 hours. In one embodiment the half-life of Fc is between about 20 and about 27 hours in subjects having high levels of VWF. Thus, in one embodiment, an increased half-life of FVIII-Fc compared to average values is indicative of a subject that is eligible for a longer dosing interval with a long-acting FVIII polypeptide of the invention.

In another embodiment, the half-life of a short-acting FVIII polypeptide is used to identify patients who express high levels of VWF. As used herein, the term ”short-acting FVIII" refers to a FVIII polypeptide in which no extenders of half-life have been added. In one embodiment, acting FVIII polypeptides consist of ength or B domain-deleted FVIII.

Examples of short-acting FVIII ptides are Advate® and ReFacto®.

Since the half-life of short-acting FVIII also varies depending at least in part on VWF levels, short-acting FVIII polypeptides can also be used to identify patients that are eligible for a longer dosing interval of a long-acting FVIII polypeptide of the ion. In one embodiment, the short-acting FVIII exhibits an increased half-life of at least about 12-fold in individuals expressing high levels of VWF compared to the half-life of the short-acting FVIII when administered to individuals having average levels of VWF. In another ment, the short- acting FVIII exhibits an increased half-life of at least about 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5-fold in individuals expressing high levels of VWF compared to the half- life of the acting FVIII when administered to individuals having average levels of VWF.

Thus, individuals that demonstrate an increased ife of at least about 1.2-fold when they are administered a short-acting FVIII are eligible for a longer dosing interval with a long-acting FVIII ptide of the invention.

”Dosing interval,” as used , means the dose of time that elapses between multiple doses being administered to a subject. The ison of dosing interval may be carried out in a single subject or in a population of subjects and then the average obtained in the population may be calculated.

The dosing interval when administering a chimeric Factor VIII polypeptide, e. g., a chimeric Factor VIII-Fc polypeptide (a polypeptide comprising a Factor VIII or a hybrid) of the invention may be at least about one and one-half times longer than the dosing interval required for an equivalent dose of said Factor VIII t the non-Factor VIII portion, e. g., without the Fc portion (a polypeptide consisting of said Factor VIII). The dosing interval may be at least about one and one-half to six times longer, one and one-half to five times , one and one-half to four times longer, one and one-half to three times longer, or one and one-half to two times longer, than the dosing interval required for an equivalent dose of said Factor VIII without the non-Factor VIII portion, e.g., without the Fc portion (a polypeptide consisting of said Factor VIII). The dosing interval may be at least about one and one-half, two, two and one-half, three, three and one-half, four, four and one-half, five, five and one-half or six times longer than the dosing al required for an equivalent dose of said Factor VIII without the non-Factor VIII portion, e. g., without the Fc portion (a polypeptide consisting of said Factor VIII).. The dosing al may be about every three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen days or longer. The dosing interval may be at least about one and one-half to 5, one and one-half, 2, 3, 4, or 5 days or longer. For on-demand ent, the dosing interval of said chimeric polypeptide or hybrid is about once every 24-36, 24-48, 24-72, 24-96, 24-120, 24-144, 24-168, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours or longer.

In one ment, the effective dose is 25-80 IU/kg (25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 62, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 IU/kg) and the dosing al is once every 3-5, 3-6, 3-7, 3, 4, 5, 6, 7, or 8 or more days, or three times per week, or no more than three times per week. In one embodiment, the effective dose is 80 IU/kg and the dosing interval is once every 3 days. In a further embodiment, the effective dose of 80 IU/kg given at a dosing interval of every 3 days is stered to a ric t. In another embodiment, the effective dose is 65 IU/kg and the dosing interval is once weekly, or once every 6-7 days. The doses can be administered repeatedly as long as they are necessary (e.g., at least , 20, 28, 30, 40, 50, 52, or 57 weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years).

In certain embodiments, the effective dose for on-demand treatment is 20-5OIU/Kg (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 IU/kg). The on-demand treatment can be one time dosing or repeated dosing.

For repeated dosing, the dosing interval can be every 12-24 hours, every 24-36 hours, every 24- 48 hours, every 36-48 hours, or every 48-72 hours. acting Factor VIII” is a Factor VIII having an increased half-life (also referred to herein as t1/2, t1/2 beta, elimination half-life and HL) over a reference Factor VIII. The increased half-life of a long-acting Factor VIII may be due to fusion to one or more non-Factor VIII polypeptides such as, e. g., Fc, XTEN, albumin, a PAS sequence, transferrin, CTP (28 amino acid C-terminal peptide (CTP) of hCG with its 4 ans), polyethylene glycol (PEG), yethyl starch (HES), albumin binding polypeptide, albumin-binding small molecules, or two or more combinations thereof. The increased half-life may be due to one or more modification, such as, e.g., pegylation. Exemplary long-acting Factor VIII polypeptides include, e.g., chimeric Factor VIII polypeptides comprising Fc, ic Factor VIII polypeptides comprising XTEN and chimeric Factor VIII polypeptides sing n. onal exemplary long-acting Factor VIII polypeptides include, e. g., pegylated Factor VIII.

The "reference" polypeptide, in the case of a long-acting chimeric Factor VIII polypeptide, is a polypeptide consisting essentially of the Factor VIII portion of the chimeric polypeptide, e.g., the same Factor VIII n without the Fc portion, without the XTEN portion, or without the albumin portion. Likewise, the reference polypeptide in the case of a modified Factor VIII is the same Factor VIII without the modification, e.g., a Factor VIII without the pegylation.

In some embodiments, the long-acting Factor VIII has one or more of the following properties when administered to a subject: a mean residence time (MRT) (activity) in said subject of about 14—41 .3 hours; a clearance (CL) (activity) in said subject of about .19 mL/hour/kg or less; a t1/2beta (activity) in said subject of about 11-264 hours; an incremental recovery (K value) (activity; observed) in said subject of about 1.3 8288 IU/dL per IU/kg; a Vss (activity) in said subject of about 37.7-79.4 mL/kg; and an AUC/dose in said subject of about 19.2-81.7 IU*h/dL per IU/kg.

In some embodiments, the long-acting Factor VIII has one or more of the following properties when administered to a patient population: a mean incremental recovery (K-Value) (activity; observed) greater that 1.38 IU/dL per IU/kg; a mean incremental recovery ue) (activity; observed) of at least about 1.5, at least about 1.85, or at least about 2.46 IU/dL per IU/kg. a mean clearance (CL) (activity) in said patient population of about 2.33 :: 1.08 mL/hour/kg or less; a mean clearance (CL) (activity) in said patient population of about 1.8-2.69 mL/hour/kg; a mean clearance (CL) (activity) in said patient population that is about 65% of the clearance of a polypeptide comprising said Factor VIII without modification; a mean mean residence time (MRT) (activity) in said patient population of at least about 26.3 : 8.33 hours; a mean MRT (activity) in said patient population of about 25.9 - 26.5 hours; a mean MRT (activity) in said patent population that is about 1.5 fold longer than the mean MRT of a polypeptide comprising said Factor VIII without modification; a mean t1/2beta (activity) in said t tion of about 18.3 :: 5.79 hours; a mean t1/2beta (activity)in said t population that is about 18 - 18.4 hours; a mean t1/2beta (activity) in said patient population that is about 1.5 fold longer than the mean t1/2beta of a polypeptide comprising said Factor VIII without modification; a mean incremental recovery (K value) (activity; ed) in said patient population of about 2.01 :: 0.44 IU/dL per IU/kg; a mean incremental recovery (K value) (activity; observed) in said patient population of about 1.85 - 2.46 IU/dL per IU/kg; a mean incremental recovery (K value) (activity; observed) in said patient population that is about 90 % of the mean incremental recovery of a polypeptide sing said Factor VIII without modification; a mean Vss ity) in said t population of about 55.1 :: 12.3 mL/kg; a mean Vss (activity) in said patient population of about 45.3 - 56.1 mL/kg; a mean AUC/dose (activity) in said patient population of about 49.9 :: 18.2 IU*h/dL per IU/kg a mean AUC/dose (activity) in said patient population of about 44.8 - 57.6 IU*h/dL per IU/kg.

In other embodiments, the long-acting Factor VIII has one or more of the following properties when administered to a patient population: a CmaX_OBS in said t administered with the chimeric ptide is comparable to the CmaX_OBS in a subject administered with the same amount of a ptide consisting of the full-length, mature Factor VIII when measured by a one stage (aPTT) assay or a two stage (chromogenic) assay; a Cmax_OBS in said t of about 60.5 IU/dL, about 60.5 :: 1 IU/dL, about 60.5 :: 2 IU/dL, about 60.5 :: 3 IU/dL, about 60.5 :: 4 IU/dL, about 60.5 :: 5 IU/dL, about 60.5 :: 6 IU/dL, about 60.5 ::7 IU/dL, about 60.5 :: 8 IU/dL, about 60.5 :: 9 IU/dL, or about 60.5 :: 10 IU/dL as measured by a one stage (aPTT) assay when about 25 IU/kg of the chimeric polypeptide is administered; a CmaX_OBS in said subject of about 53.1 — 69 lU/dL as measured by a one stage (aPTT) assay when about 25 IU/kg of the chimeric polypeptide is administered; a Cmax_OBS in said subject of about 119 lU/dL, about 119 :: 1 lU/dL, about 119 :: 2 lU/dL, about 119 :: 3 lU/dL, about 119 :: 4 IU/dL, about 119 :: 5 lU/dL, about 119 :: 6 IU/dL, about 119 :: 7 lU/dL, about 119 :: 8 lU/dL, about 119 :: 9 lU/dL, about 119 :: 10 lU/dL, about 119 :: 11 IU/dL, about 119 :: 12 IU/dL, about 119 ::131U/dL, about 119 :: 14 IU/dL, about 119 ::151U/dL, about 119 :: 16 lU/dL, about 119 :: 17 IU/dL, or about 119 :: 18 IU/dL, as ed by a one stage (aPTT) assay when about 65 IU/kg of the chimeric polypeptide is administered; a CmaLOBS in said subject of about 103 — 136 IU/dL as ed by a one stage (aPTT) assay when about 65 IU/kg of the chimeric polypeptide is administered; a CmaLOBS in said subject of about 76.5 lU/dL, about 76.5 :: 1 IU/dL, about 76.5 :: 2 lU/dL, about 76.5 :: 3 IU/dL, about 76.5 :: 4 IU/dL, about 76.5 :: 5 lU/dL, about 76.5 :: 6 lU/dL, about 76.5 :: 7 lU/dL, about 76.5 :: 8 IU/dL, about 76.5 :: 9 IU/dL, about 76.5 :: 10 lU/dL, about 76.5 :: 11 IU/dL, about 76.5 :: 12 lU/dL, about 76.5 :: 13 lU/dL, about 76.5 :: 14 lU/dL, or about 76.5 :: 15 lU/dL, as measured by a two stage (chromogenic) assay when about 25 lU/kg of the chimeric polypeptide is administered; a me_OBS in said subject of about 64.9 — 90.1 lU/dL as measured by a two stage ogenic) assay when about 25 lU/kg of the chimeric polypeptide is administered; a me_OBS in said subject of about 182 IU/dL, about 182 :: 2 IU/dL, about 182 :: 4 IU/dL, about 182 :: 6 lU/dL, about 182 :: 8 lU/dL, about 182 :: 10 IU/dL, about 182 :: 12 lU/dL, about 182 :: 14 lU/dL, about 182 :: 16 IU/dL, about 182 :: 18 lU/dL, or about 182 :: 20 IU/dL as measured by a two stage (chromogenic) assay when about 65 lU/kg of the chimeric polypeptide is administered; or a BS in said subject of about 146 — 227 lU/dL, about 146 :: 5 IU/dL, about 146 :: 10 lU/dL, about 227 :: 5 IU/dL, or about 146 :: 10 lU/dL as measured by a two stage (chromogenic) assay when about 65 IU/kg of the chimeric ptide is administered.

In certain embodiments, the long-acting Factor VIII has one or more of the following properties when stered to a patient population: a t1/2beta (activity) in said subject that is at least 1.48, 1.49, 1.50, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 157,158,159, 160,161, 1.62,1.63,1.64,1.65, 1.66, 167,168, 1.69,1.70, 1.71, 1.72,1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.80, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, or 1.90 times higher than the t1/2beta (activity) in a subject administered with the same amount of a polypeptide consisting of the full-length, mature Factor VIII when ed by a one stage (aPTT) assay or a two stage (chromogenic) assay; a t1/2beta (activity) in said subject of about 18.8 hours, 18.8 :: 1 hours, 18.8 :: 1 hours, 18.8 :: 2 hours, 18.8 :: 3 hours, 18.8 :: 4 hours, 18.8 :: 5 hours, 18.8 :: 6 hours, 18.8 :: 7 hours, 18.8 :: 8 hours, 18.8 :: 9 hours, 18.8 :: 10 hours, or 18.8 :: 11 hours as measured by a one stage (aPTT) assay; a t1/2beta (activity) in said subject of about 14.3 — 24.5 hours as measured by a one stage (aPTT) assay; a t1/2beta (activity) in said subject of about 16.7 hours, 16.7 :: 1 hours, 16.7 :: 2 hours, 16.7 :: 3 hours, 16.7 :: 4 hours, 16.7 :: 5 hours, 16.7 :: 6 hours, 16.7 :: 7 hours, 16.7 :: 8 hours, 16.7 :: 9 hours, 16.7 :: 10 hours, or 16.7 :: 11 hours as measured by a two stage (chromogenic) assay; a t1/2beta (activity) in said subject of about 13.8 — 20.1 hours as measured by a two stage (chromogenic) assay; a t1/2beta (activity) in said subject of about 19.8 hours, 19.8 :: 1 hours, 19.8 :: 2 hours, 19.8 :: 3 hours, 19.8 :: 4 hours, 19.8 :: 5 hours, 19.8 :: 6 hours, 19.8 :: 7 hours, 19.8 :: 8 hours, 19.8 :: 9 hours, 19.8 :: 10 hours, or 19.8 :: 11 hours as measured by a two stage (chromogenic) assay; or a t1/2beta (activity) in said subject of about 14.3 — 27.5 hours as measured by a two stage (chromogenic) assay.

] In certain embodiments, the long-acting Factor VIII has one or more of the following properties when administered to a patient population: a clearance (CL) (activity) in said t is 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, or 0.70 times lower than the clearance in a subject administered with the same amount of a ptide consisting of the full-length, mature Factor VIII when measured by a one stage (aPTT) assay or a two stage (chromogenic) assay; a nce (CL) (activity) in said subject of about 1.68 mL/hour/kg, 1.68 :: 0.1 mL/hour/kg, 1.68 :: 0.2 mL/hour/kg, 1.68 :: 0.3 r/kg, 1.68 :: 0.4 mL/hour/kg, 1.68 :: 0.5 mL/hour/kg, 1.68 :: 0.6 mL/hour/kg, or 1.68 :: 0.7 mL/hour/kg, as measured by a one stage (aPTT) assay when about 25 IU/kg of the chimeric polypeptide is administered; a clearance (CL) (activity) in said subject of about 1.31 — 2.15 mL/hour/kg as measured by a one stage (aPTT) assay when about 25 IU/kg of the chimeric polypeptide is administered; a clearance (CL) (activity) in said subject of about 2.32 mL/hour/kg, 2.32 :: 0.1 mL/hour/kg, 2.32 :: 0.2 r/kg, 2.32 :: 0.3 mL/hour/kg, 2.32 :: 0.4 mL/hour/kg, 2.32 :: 0.5 mL/hour/kg, 2.32 :: 0.6 mL/hour/kg, or 2.32 :: 0.7 r/kg as measured by a one stage (aPTT) assay when about 65 IU/kg of the chimeric polypeptide is administered; a clearance (CL) (activity) in said subject of about 1.64 — 3.29 mL/hour/kg as measured by oa ne stage (aPTT) assay when about 65 IU/kg of the ic ptide is administered; a clearance (CL) (activity) in said t of about 1.49 r/kg, 1.49 :: 0.1 mL/hour/kg, 1.49 :: 0.2 mL/hour/kg, 1.49 :: 0.3 mL/hour/kg, 1.49 :: 0.4 mL/hour/kg, 1.49 :: 0.5 mL/hour/kg, 1.49 :: 0.6 mL/hour/kg, or 1.49 :: 0.7 mL/hour/kg as measured by a two stage (chromogenic) assay when about 25 IU/kg of the chimeric polypeptide is administered; a clearance (CL) (activity) in said subject of about 1.16 — 1.92 mL/hour/kg as measured by a two stage (chromogenic) assay when about 25 IU/kg of the chimeric polypeptide is administered; WO 09627 a clearance (CL) (activity) in said subject of about 1.52 mL/hour/kg, 1.52 :: 0.1 mL/hour/kg, 1.52 :: 0.2 mL/hour/kg, 1.52 :: 0.3 mL/hour/kg, 1.52 :: 0.4 mL/hour/kg, 1.52 :: 0.5 mL/hour/kg, 1.52 :: 0.6 mL/hour/kg, or 1.52 :: 0.7 mL/hour/kg as measured by a two stage (chromogenic) assay when about 65 IU/kg of the chimeric polypeptide is administered; or a clearance (CL) (activity) in said subject of about 1.05-2.20 mL/hour/kg as measured by a two stage ogenic) assay when about 65 IU/kg of the chimeric polypeptide is administered.

In some embodiments, the cting Factor VIII has one or more of the following properties when administered to a patient population: a MRT in said t is at least 1.46, 1.47, 1.48, 1.49, 1.50, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 9,160, 161,162, .64,1.65,1.66, 1.67, 168,169, 1.70,1.71, 1.72, 1.73, 1.74,1.75, 1.76, 1.77, 1.78, 1.79, 1.80, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.90, 1.91, 1.92, or 1.93 times higher than the MRT in a subject administered with the same amount of a polypeptide consisting of the full-length, mature Factor VIII when meansured by a one stage (aPTT) assay or a two stage (chromogenic) assay; a MRT (activity) in said subject of about 27 hours, 27 :: 1 hours, 27 :: 2 hours, 27 :: 3 hours, 27 :: 4 hours, 27 :: 5 hours, 27 :: 6 hours, 27 :: 7 hours, 27 :: 8 hours, 27 :: 9 hours, or 27 :: 10 hours as meansured by a one stage (aPTT) assay; a MRT (activity) in said subject of about 20.6 — 35.3 hours as meansured by a one stage (aPTT) assay; a MRT (activity) in said subject of about 23.9 — 28.5 hours as measured by a two stage (chromogenic) assay; a MRT (activity) in said subject of about 19.8 — 28.9 hours as measured by a two stage (chromogenic) assay; or a MRT (activity) in said subject of about 20.5 — 39.6 hours as measured by a two stage (chromogenic) assay.

In other embodiments, the long-acting Factor VIII has one or more of the following properties when stered to a patient population: an incremental recovery in said t that is able to the Incremental Recovery in a subject administered with the same amount of a polypeptide consisting of the full-length, mature Factor VIII when measured by a one stage (aPTT) assay or a two stage (chromogenic) assay; an incremental recovery in said subject of about 2.44 IU/dL per IU/kg, 2.44 :: 0.1 IU/dL per IU/kg, 2.44 :: 0.2 IU/dL per IU/kg, 2.44 :: 0.3 IU/dL per IU/kg, 2.44 :: 0.4 IU/dL per IU/kg, 2.44 :: 0.5 IU/dL per IU/kg, 2.44 :: 0.6 IU/dL per IU/kg, 2.44 :: 0.7 IU/dL per IU/kg, 2.44 :: 0.8 IU/dL per IU/kg, 2.44 :: 0.9 IU/dL per IU/kg, 2.44 :: 1.0 IU/dL per IU/kg, 2.44 :: 1.1 IU/dL per IU/kg, or 2.44 :: 1.2 IU/dL per IU/kg as measured by a one stage (aPTT) assay when about 25 IU/kg of the chimeric polypeptide is administered; an incremental recovery in said subject of about 2.12 — 2.81 IU/dL per lU/kg as measured by a one stage (aPTT) assay when about 25 IU/kg of the chimeric polypeptide is administered; an incremental recovery in said subject of about 1.83 lU/dL per lU/kg, 1.83 :: 0.1 IU/dL per lU/kg, 1.83 :: 0.2 lU/dL per IU/kg, 1.83 :: 0.3 IU/dL per IU/kg, 1.83 :: 0.4 IU/dL per IU/kg, 1.83 :: 0.5 lU/dL per IU/kg, 1.83 :: 0.6 IU/dL per lU/kg, 1.83 :: 0.7 lU/dL per IU/kg, 1.83 :: 0.8 lU/dL per lU/kg, 1.83 :: 0.9 lU/dL per lU/kg, 1.83 :: 1.0 IU/dL per lU/kg, or 1.83 :: 1.1 lU/dL per lU/kg as measured by a one stage (aPTT) assay when about 65 IU/kg of the chimeric ptide is administered; an incremental recovery in said subject of about 1.59 — 2.10 IU/dL per lU/kg as measured by a one stage (aPTT) assay when about 65 IU/kg of the chimeric polypeptide is administered; an incremental recovery in said subject of about 3.09 IU/dL per lU/kg, 3.09 :: 0.1 IU/dL per lU/kg, 3.09 :: 0.2 IU/dL per IU/kg, 3.09 :: 0.3 lU/dL per IU/kg, 3.09 :: 0.4 lU/dL per lU/kg, 3.09 :: 0.5 lU/dL per IU/kg, 3.09 :: 0.6 IU/dL per lU/kg, 3.09 :: 0.7 lU/dL per IU/kg, 3.09 :: 0.8 lU/dL per lU/kg, 3.09 :: 0.9 lU/dL per lU/kg, 3.09 :: 1.0 IU/dL per IU/kg, 3.09 :: 1.1 IU/dL per IU/kg, 3.09 :: 1.2 IU/dL per lU/kg, or 3.09 :: 1.3 IU/dL per lU/kg, as measured by a two stage (chromogenic) assay when about 25 lU/kg of the chimeric polypeptide is administered; an incremental recovery in said subject of about 2.80 IU/dL per lU/kg, 2.80 :: 0.1 IU/dL per lU/kg, 2.80 :: 0.2 lU/dL per IU/kg, 2.80 :: 0.3 IU/dL per IU/kg, 2.80 :: 0.4 IU/dL per IU/kg, 2.80 :: 0.5 lU/dL per IU/kg, 2.80 :: 0.6 IU/dL per lU/kg, 2.80 :: 0.7 lU/dL per IU/kg, 2.80 :: 0.8 lU/dL per lU/kg, 2.80 :: /dL per IU/kg, 2.80 :: 1.01U/dL per IU/kg, 2.80 :: 1.1 IU/dL per lU/kg, or 2.80 :: 1.2 lU/dL per lU/kg, as measured by a two stage (chromogenic) assay when about 65 lU/kg of the chimeric ptide is administered; an incremental recovery in said subject of about 2.61-3.66 IU/dL per lU/kg as measured by a two stage (chromogenic) assay when about 25 lU/kg of the chimeric polypeptide is administered; or an incremental recovery in said t of about 2.24-3.50 IU/dL per lU/kg as measured by a two stage ogenic) assay when about 65 lU/kg of the chimeric polypeptide is administered.

In still other embodiments, the long-acting Factor VIII has one or more of the following properties when administered to a patient population: a Vss (activity) in said subject that is comparable to the Vss (activity) in a subject administered with the same amount of a polypeptide consisting of the full-length, mature Factor VIII when ed by a one stage (aPTT) assay or a two stage (chromogenic) assay; a Vss (activity) in said subject of about 45.5 mL/kg, 45.5 :: 1 mL/kg, 45.5 :: 2 mL/kg, 45.5 :: 3 mL/kg, 45.5 4 mL/kg, 45.5 5 mL/kg, 45.5 6 mL/kg, 45.5 7 mL/kg, 45.5 8 mL/kg, 45.5 9 mL/kg, 45.5 :: 10 mL/kg, or 45.5 :: 11 mL/kg, as measured by a one stage (aPTT) assay when about 25 lU/kg of the chimeric polypeptide is administered; a Vss (activity) in said t of about 39.3 — 52.5 mL/kg as measured by a one stage (aPTT) assay when about 25 lU/kg of the chimeric polypeptide is administered; a Vss (activity) in said subject of about 62.8 mL/kg, 62.8 :: 1 mL/kg, 62.8 :: 2 mL/kg, 62.8 :: 3 mL/kg, 62.8 :: 4 mL/kg, 62.8 :: 5 mL/kg, 62.8 :: 6 mL/kg, 62.8 :: 7 mL/kg, 62.8 :: 8 mL/kg, 62.8 :: 9 mL/kg, 62.8 :: 10 mL/kg, 62.8 :: 11 mL/kg, 62.8 :: 12 mL/kg, 62.8 :: 13 mL/kg, 62.8 :: 14 mL/kg, 62.8 :: 15 mL/kg, or 62.8 :: 16 mL/kg as measured by a one stage (aPTT) assay when about 65 lU/kg of the chimeric polypeptide is administered; a Vss (activity) in said subject of about 55.2 — 71.5 mL/kg as measured by a one stage (aPTT) assay when about 65 lU/kg of the chimeric polypeptide is administered; a Vss (activity) in said subject of about 35.9 mL/kg, 35.9 :: 1 mL/kg, 35.9 :: 2 mL/kg, 35.9 :: 3 mL/kg, 35.9 :: 4 mL/kg, 35.9 :: 5 mL/kg, 35.9 :: 6 mL/kg, 35.9 :: 7 mL/kg, 35.9 :: 8 mL/kg, 35.9 :: 9 mL/kg, 35.9 :: 10 mL/kg, 35.9 :: 11 mL/kg, 35.9 :: 12 mL/kg, or 35.9 :: 13 mL/kg, as measured by a two stage (chromogenic) assay when about 25 lU/kg of the chimeric polypeptide is stered; a Vss (activity) in said subject of about 30.4-42.3 mL/kg as measured by a two stage (chromogenic) assay when about 25 IU/kg of the chimeric polypeptide is administered; a Vss (activity) in said subject of about 43.4 mL/kg, 43.4 :: 1 mL/kg, 43.4 :: 2 mL/kg, 43.4 :: 3 mL/kg, 43.4 :: 4 mL/kg, 43.4 :: 5 mL/kg, 43.4 :: 6 mL/kg, 43.4 :: 7 mL/kg, 43.4 :: 8 mL/kg, 43.4 :: 9 mL/kg, 43.4 :: 10 mL/kg, 43.4 :: 11 mL/kg, 43.4 :: 12 mL/kg, 43.4 :: 13 mL/kg, 43.4 :: 14 mL/kg, 43.4 :: mL/kg, or 43.4 :: 16 mL/kg, as ed by a two stage (chromogenic) assay when about 65 lU/kg of the chimeric polypeptide is administered; or a Vss ity) in said subject of about 38.2-49.2 mL/kg as measured by a two stage (chromogenic) assay when about 65 IU/kg of the ic polypeptide is administered.

In yet other embodiments, the cting Factor VIII has one or more of the following properties when administered to a patient population: an AUCINF in said subject that is at least 1.45 1.46, 1.47, 1.48, 1.49, 1.50, 1.51, 1.52, 1.53, 1.54, 1.55, 156,157,158, 159,160, 1.61,1.62,1.63,1.64, 1.65, 7, 168,169, 1.70, 1.71,1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.80, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.90 times higher than the AUCINF in a subject stered with the same amount of a polypeptide consisting of the full-length, mature Factor VIII when meansured by a one stage (aPTT) assay or a two stage (chromogenic) assay; an AUCINF in said subject of about 1440 :: 316 dL per IU/kg as measured by a one stage (aPTT) assay when about 25 IU/kg of the chimeric polypeptide is administered; an AUCINF in said subject of about 1160 - 1880 hr*1U/dL per IU/kg as measured by a one stage (aPTT) assay when about 25 IU/kg of the chimeric polypeptide is administered; an AUCINF in said subject of about 1480 hr*IU/dL per lU/kg, 1480 :: 100 hr*1U/dL per lU/kg, 1480 :: 200 hr*1U/dL per lU/kg, 1480 :: 300 hr*1U/dL per IU/kg, 1480 :: 400 hr*IU/dL per IU/kg, 1480 :: 500 hr*1U/dL per lU/kg, 1480 :: 600 hr*1U/dL per lU/kg, 1480 :: 700 hr*IU/dL per IU/kg, 1480 :: 800 hr*lU/dL per lU/kg, 1480 :: 900 hr*IU/dL per lU/kg, or 1480 :: 1000 hr*1U/dL per IU/kg, as measured by a one stage (aPTT) assay when about 25 IU/kg of the chimeric polypeptide is administered; an AUCINF in said subject of about 2910 :: 1320 hr*IU/dL per lU/kg as ed by a one stage (aPTT) assay when about 65 IU/kg of the chimeric polypeptide is administered; an AUCINF in said t of about 1980 - 3970 hr*lU/dL per IU/kg as measured by a one stage (aPTT) assay when about 65 IU/kg of the chimeric polypeptide is stered; an AUCINF in said subject of about 2800 hr*IU/dL per lU/kg, 2800 :: 100 hr*lU/dL per lU/kg, 2800 :: 200 hr*IU/dL per lU/kg, 2800 :: 300 hr*IU/dL per lU/kg, 2800 :: 400 hr*IU/dL per lU/kg, 2800 :: 500 hr*IU/dL per lU/kg, 2800 :: 600 hr*IU/dL per lU/kg, 2800 :: 700 hr*IU/dL per lU/kg, 2800 :: 800 hr*IU/dL per lU/kg, 2800 :: 900 hr*IU/dL per IU/kg, or 2800 :: 1000 hr*lU/dL per IU/kg as measured by a one stage (aPTT) assay when about 65 IU/kg of the chimeric polypeptide is administered; an AUCINF in said subject of about 1660 hr*IU/dL per lU/kg, 1660 :: 100 hr*IU/dL per IU/kg, 1660 :: 200 hr*IU/dL per lU/kg, 1660 :: 300 hr*IU/dL per lU/kg, 1660 :: 400 hr*IU/dL per lU/kg, 1660 :: 500 hr*IU/dL per IU/kg, 1660 :: 600 hr*IU/dL per lU/kg, 1660 :: 700 hr*IU/dL per lU/kg, 1660 :: 800 hr*IU/dL per IU/kg, 1660 :: 900 hr*IU/dL per IU/kg, or 1660 :: 1000 hr*IU/dL per IU/kg as measured by a two stage ogenic) assay when about 25 lU/kg of the chimeric polypeptide is administered; an AUCINF in said subject of about 1300 - 2120 hr*IU/dL per IU/kg as measured by a two stage (chromogenic) assay when about 25 IU/kg of the ic polypeptide is administered; an AUCINF in said subject of about 4280 hr*IU/dL per lU/kg, 4280 :: 100 hr*IU/dL per IU/kg, 4280 :: 200 hr*IU/dL per IU/kg, 4280 :: 300 hr*IU/dL per IU/kg, 4280 :: 400 hr*IU/dL per IU/kg, 4280 :: 500 dL per IU/kg, 4280 :: 600 hr*IU/dL per lU/kg, 4280 :: 700 hr*IU/dL per lU/kg, 4280 :: 800 hr*IU/dL per lU/kg, 4280 :: 900 hr*IU/dL per lU/kg, 4280 :: 1000 hr*IU/dL per IU/kg, 4280 :: 1100 hr*IU/dL per lU/kg, 4280 :: 1200 hr*IU/dL per IU/kg, 4280 :: 1300 hr*IU/dL per IU/kg, 4280 :: 1400 hr*IU/dL per lU/kg, 4280 :: 1500 hr*lU/dL per IU/kg, or 4280 :: 1600 hr*IU/dL per lU/kg as measured by a two stage ogenic) assay when about 65 lU/kg of the chimeric polypeptide is administered; or an AUCINF in said subject of about 2960 - 6190 hr*IU/dL per IU/kg as measured by a two stage (chromogenic) assay when about 65 IU/kg of the chimeric polypeptide is administered.

] ”On-demand treatment,” as used , means treatment that is intended to take place over a short course of time and is in response to an ng condition, such as a bleeding e, or a perceived need such as planned surgery. Conditions that may require on-demand treatment include, e. g., a bleeding episode, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma s, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, or ng in the illiopsoas sheath. The subject may be in need of surgical prophylaxis, WO 09627 peri-operative management, or treatment for surgery. Such surgeries include, e. g., minor surgery, major surgery, tooth tion, tonsillectomy, inguinal herniotomy, ctomy, total knee replacement, craniotomy, osteosynthesis, trauma surgery, intracranial surgery, intra-abdominal surgery, intrathoracic surgery, or joint replacement surgery.

In one embodiment, on-demand treatment resolves greater than 80% (greater than 80%, greater than 81%, greater than 82%, greater than 83%, greater than 84%, greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, greater than 90%, greater than 91%, greater than 92%, r than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or 100%) or 80-100%, , 85-90%, 90-100%, 90-95%, or 95-100% of bleeds (e.g., spontaneous bleeds) in a single dose. In another embodiment, greater than 80% (greater than 81%, greater than 82%, greater than 83%, greater than 84%, greater than 85%, greater than 86%, r than 87%, greater than 88%, greater than 89%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, r than 95%, greater than 96%, greater than 97%, greater than 98%, or 100%) or 80-100%, 80-90%, 85-90%, 90-100%, 90-95%, or 95-100% of bleeding episodes are rated excellent or good by ians after on-demand treatment. In other embodiments, greater than 5%, (greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 11%, greater than 12%, greater than 13%, greater than 14%, greater than 15%, greater than 16%, greater than 17%, greater than 18%, greater than 19%, greater than 20%), or 5- %, 5-15%, 5-10%, 10-20%, or 10-15% of bleeding es are rated as fair by ians after on-demand treatment.

] ”Polypeptide,” ”peptide” and ”protein” are used interchangeably and refer to a polymeric compound comprised of covalently linked amino acid residues.

”Polynucleotide” and ”nucleic acid” are used interchangeably and refer to a polymeric compound comprised of covalently linked nucleotide residues. Polynucleotides may be DNA, cDNA, RNA, single stranded, or double ed, vectors, plasmids, phage, or Viruses.

Polynucleotides include, e.g., those in Table 1, Which encode the polypeptides of Table 2 (see Table 1). Polynucleotides also include, e.g., nts of the polynucleotides of Table 1, e.g., those that encode fragments of the polypeptides of Table 2, such as the Factor VIII, Fc, signal sequence, 6His and other fragments of the polypeptides of Table 2.

”Prophylactic treatment,” as used herein, means administering a Factor VIII polypeptide in le doses to a subject over a course of time to increase the level of Factor VIII activity in a subj ect's plasma. The increased level can be sufficient to decrease the incidence of spontaneous bleeding or to prevent bleeding, e.g., in the event of an unforeseen injury. During lactic treatment, the plasma protein level in the subject may not fall below the baseline level for that t, or below the level of Factor VIII that characterizes severe hemophilia (<1 IU/dl [1%]).

In one embodiment, the prophylaxis n is ”tailored” to the dual patient, for example, by determining PK data for each patient and administering Factor VIII of the invention at a dosing interval that maintains a trough level of 1-3% FVIII activity. Adjustments may be made when a subject experiences unacceptable ng episodes defined as 22 neous bleeding episodes over a rolling two-month period. In this case, adjustment will target trough levels of 3-5%. In another embodiment, prophylactic treatment results in prevention and control of ng, sustained control of bleeding, sustained protection from bleeding, and/or sustained benefit. Prophylaxis, e.g., sustained protection can be trated by an sed AUC to last measured time point (AUC-LAST) and reduced clearance, resulting in increased terminal t1/2 ed to short acting FVIII. Prophylaxis can be demonstrated by better Cmax, better Tmax, and/or r mean residence time versus short-acting FVIII. In some embodiments, prophylaxis results in no spontaneous bleeding es within about 24, 36, 48, 72, or 96 hours (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 96, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 , after injection (e.g., the last injection). In certain embodiments, prophylaxis results in r than 30% (e.g., greater than 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 96, 87, 88, 89, or 90%, for example, greater than 50%), mean reduction in annualized bleeding episodes with once weekly dosing (e. g., at 65 IU/kg). ”Therapeutic dose,” as used herein, means a dose that achieves a therapeutic goal, as described herein. The calculation of the required dosage of factor VIII is based upon the cal finding that, on average, 1 IU of factor VIII per kg body weight raises the plasma factor VIII actiVity by approximately 2 IU/dL.

The required dosage is determined using the following formula: Required units = body weight (kg) x desired factor VIII rise (IU/dL or % of normal) x 0.5 (IU/kg per IU/dL) The therapeutic doses that may be used in the methods of the invention are about 10-100 IU/kg, more specifically, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 IU/kg, and more specifically, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 IU/kg.

Additional therapeutic doses that may be used in the methods of the invention are about to about 150 IU/kg, more specifically, about 100-110, 110-120, 120-130, 130-140, 140-150 IU/kg, and more specifically, about 110, 115, 120, 125, 130, 135, 140, 145, or 150 IU/kg.

"Variant," as used herein, refers to a polynucleotide or polypeptide differing from the original polynucleotide or polypeptide, but retaining essential properties thereof, e.g., factor VIII coagulant actiVity or PC (FcRn binding) actiVity. Generally, variants are overall closely similar, W0 2013/009627 and, in many s, identical to the original polynucleotide or polypeptide. Variants include, e.g., polypeptide and polynucleotide nts, deletions, insertions, and modified versions of original polypeptides.

Variant polynucleotides may comprise, or alternatively consist of, a nucleotide sequence which is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% cal to, for example, the tide coding sequence in SEQ ID NO], 3, or 5 (the factor VIII portion, the Fc portion, individually or together) or the complementary strand thereto, the nucleotide coding sequence of known mutant and recombinant factor VIII or Fc such as those disclosed in the publications and patents cited herein or the complementary strand thereto, a nucleotide sequence encoding the polypeptide of SEQ ID N02, 4, or 6 (the factor VIII portion, the Fc portion, dually or together), and/or polynucleotide fragments of any of these nucleic acid les (e.g., those nts described herein). Polynucleotides which hybridize to these c acid molecules under stringent hybridization conditions or lower stringency conditions are also included as variants, as are polypeptides encoded by these polynucleotides as long as they are functional.

Variant polypeptides may comprise, or atively consist of, an amino acid sequence which is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to, for example, the polypeptide sequence shown in SEQ ID NOS:2, 4, or 6 (the factor VIII portion, the Fc portion, individually or together), and/or polypeptide fragments of any of these polypeptides (e.g., those fragments described herein).

By a nucleic acid having a nucleotide sequence at least, for example, 95% ”identical” to a reference nucleotide sequence, it is ed that the nucleotide sequence of the nucleic acid is identical to the nce sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference ce may be deleted or substituted with r nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be, for example, the entire sequence shown in SEQ ID NO:1 or 3, the ORF (open reading frame), or any fragment specified as described herein.

As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% cal to a nucleotide sequence or polypeptide of the present invention can be determined conventionally using known computer programs. In one embodiment, a method for determining the best overall match between a query sequence (reference or original ce) and a subject sequence, also referred to as a global ce ent, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al., Comp. App. Biosci. 6:237-245 (1990), which is herein orated by reference in W0 2013/009627 its entirety In a sequence ent the query and subject sequences are both DNA sequences.

An RNA sequence can be compared by converting U’s to T’s. The result of said global ce alignment is in percent ty. In another embodiment, parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch PenaltFl, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=l, Gap PenaltF5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter.

If the subject sequence is r than the query sequence because of 5’ or 3’ deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for 5’ and 3’ truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5’ or 3’ ends, relative to the query ce, the percent ty is corrected by calculating the number of bases of the query sequence that are 5’ and 3’ of the t sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. r a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, ated by the above FASTDB program using the ed parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of the present invention. Only bases outside the 5’ and 3’ bases of the subject sequence, as displayed by the FASTDB ent, which are not matched/aligned with the query sequence, are calculated for the es of manually adjusting the percent identity score.

For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5’ end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5’ end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5’ and 3’ ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score ated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5’ or 3’ of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5’ and 3’ of the subject sequence which are not matched/aligned with the query sequence are ly ted for. No other manual corrections are to made for the es of the present ion.

By a polypeptide having an amino acid sequence at least, for example, 95% ical” to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, (indels) or substituted with another amino acid. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the nce amino acid ce or anywhere between those terminal positions, interspersed either individually among residues in the reference ce or in one or more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequences of SEQ ID NO:2 (the factor VIII portion, the Fc portion, individually or together) or 4, or a known factor VIII or Fc polypeptide sequence, can be determined conventionally using known er programs. In one embodiment, a method for determining the best overall match between a query sequence (reference or original sequence) and a subject sequence, also referred to as a global sequence ent, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al., Comp. App. Biosci. 6:237-245(l990), incorporated herein by reference in its entirety. In a sequence ent the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global ce alignment is in percent identity. In another embodiment, parameters used in a FASTDB amino acid alignment are: Matrix=PAM O, k-tuple=2, Mismatch Penalty=l, Joining PenaltFZO, Randomization Group Length=0, Cutoff Score=l, Window Size=sequence length, Gap F5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N— or C-terminal deletions, not because of internal deletions, a manual tion must be made to the results. This is because the FASTDB program does not account for N— and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N— and C-termini, ve to the query sequence, the percent identity is corrected by calculating the number of residues of the query ce that are N— and C-terminal of the subject sequence, which are not matched/aligned with a corresponding t e, as a percent of the total bases of the query sequence. r a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified ters, to arrive at a final percent ty score. This final percent ty score is what is used for the purposes of the present invention. Only residues to the N— and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C- terminal residues of the subject sequence.

For example, a 90 amino acid residue t sequence is aligned with a 100 residue query sequence to determine t identity. The deletion occurs at the N-terminus of the subject sequence and ore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query ce) so 10% is subtracted from the percent identity score calculated by the FASTDB m. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject ce is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C- termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and inal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to made for the purposes of the present invention.

The cleotide variants may contain alterations in the coding regions, non-coding regions, or both. In one embodiment, the polynucleotide variants contain alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded ptide. In another embodiment, nucleotide ts are produced by silent tutions due to the degeneracy of the genetic code. In other embodiments, variants in which -10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination.

Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to others, e.g., a bacterial host such as E. coli).

Naturally occurring variants are called ”allelic variants,” and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an sm (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present ion.

Alternatively, turally occurring variants may be produced by mutagenesis techniques or by direct synthesis.

] Using known methods of protein engineering and inant DNA technology, variants may be generated to improve or alter the characteristics of the ptides. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the secreted protein without substantial loss of ical function. Ron et al., J. Biol. Chem. 268: 2984-2988 (1993), incorporated herein by reference in its entirety, reported variant KGF proteins having heparin binding ty even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216 (1988), incorporated herein by reference in its entirety.) Moreover, ample evidence trates that ts often retain a biological activity similar to that of the lly occurring protein. For example, Gayle and coworkers (J. Biol.

Chem 268:22105-22111 (1993), incorporated herein by reference in its entirety) conducted extensive onal analysis of human cytokine IL-la. They used random mutagenesis to generate over 3,500 individual IL-la mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple ons were examined at every possible amino acid position. The investigators found that ”[m]ost of the molecule could be altered With little effect on either [binding or ical activity].” (See Abstract.) In fact, only 23 unique amino acid sequences, out of more than 3,500 nucleotide sequences examined, produced a protein that significantly differed in actiVity from Wild-type.

As stated above, polypeptide variants include, e. g., modified polypeptides. Modifications include, e.g., acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flaVin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent ment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, ylation, ion of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor ion, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation (Mei et al., Blood [163270-79 (2010), Which is incorporated herein by reference in its entirety), proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, ion, transfer-RNA mediated on of amino acids to proteins such as arginylation, and tination. In some embodiments, Factor VIII is modified, e.g., pegylated, at any convenient location. In some embodiments, Factor VIII is pegylated at a surface d amino acid of Factor VIII, e.g., a surface exposed cysteine, Which may be an engineered cysteine. Id.. In some embodiments, modified Factor VIII, e.g., pegylated Factor VIII, is a long-acting Factor VIII. e of distribution at steady state ” as used herein, has the same meaning as the term used in pharmacology, Which is the apparent space (volume) into Which a drug distributes. Vss = the amount of drug in the body divided by the plasma concentration at steady state.

”About," as used herein for a range, modifies both ends of the range. Thus, ”about 10-20” means ”about 10 to about 20.” The chimeric polypeptide used herein can se processed Factor VIII or single chain Factor VIII or a combination thereof. ”Processed Factor VIII,” as used herein means Factor VIII that has been d at Arginine 1648 (for ength Factor VIII) or Arginine 754 (for B- domain deleted Factor VIII), i.e., intracellular sing site. Due to the cleavage at the intracellular processing site, processed Factor VIII comprises two polypeptide chains, the first chain being a heavy chain and the second chain being a light chain. For example, the processed Factor VIII-Fc fusion protein (i.e., Heavy chain and Light chain fused to Fc) run at approximately 90 kDa and 130 kDa on a non-reducing SDS-PAGE, respectively, and 90 kDa and 105 kDa on a reducing SDS-PAGE, tively. Therefore, in one embodiment, at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the Factor VIII portion in the chimeric polypeptide is processed Factor VIII. In another embodiment, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the Factor VIII portion in the chimeric polypeptide is processed Factor VIII. In a particular embodiment, the chimeric polypeptide comprising processed Factor VIII is purified (or ed) from the chimeric polypeptide comprising single chain Factor VIII, and at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the Factor VIII n in the chimeric polypeptide is processed Factor VIII.

”Single chain Factor VIII,” “SC Factor VIII,” or “SCFVIII” as used herein means Factor VIII that has not been cleaved at the Arginine site (residue 1648 for full-length Factor VIII (i.e., residue 1667 of SEQ ID NO: 6) or residue 754 for B-domain deleted Factor VIII (i.e., residue 773 of SEQ ID NO: 2). Therefore, single chain Factor VIII in the ic polypeptide used herein comprises a single chain. In one ment, the single chain Factor VIII contains an intact intracellular processing site. In another embodiment, the single chain Factor VIII of the ion comprises a substitution or mutation at an amino acid position corresponding to Arginine 1645, a substitution or mutation at an amino acid position corresponding to Arginine 1648, or a substitution or mutation at amino acid positions corresponding to Arginine 1645 and Arginine 1648 in full-length Factor VIII. In other embodments, the amino acid substituted at the amino acid position corresponding to Arginine 1645 is a different amino acid from the amino acid substituted at the amino acid position corresponding to Arginine 1648. In certain embodiments, the substitution or mutation is an amino acid other than arginine, e. g., isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, alanine, gine, aspartic acid, cysteine, glutamic acid, glutamine, e, proline, selenocysteine, serine, tyrosine, histidine, ornithine, pyrrolysine, or taurine. The single chain Factor VIII-Fc fusion n can run at approximately 220 kDa on a non reducing SDS-PAGE and at approximately 195 kDa on a reducing SDS-PAGE.

WO 09627 In one embodiment, the ic polypeptide comprising single chain Factor VIII is purified (or isolated) from the chimeric polypeptide comprising processed Factor VIII, and at least about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% of the Factor VIII portion of the chimeric polypeptide used herein is single chain Factor VIII. In another embodiment, at least about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35% of the Factor VIII portion of the chimeric polypeptide is single chain Factor VIII. In other embodiments, about 1%-about 10%, about 5%-about 15%, about 10%-about 20%, about 15%-about 25%, about 20%- about 30%, about 25%-about 35%, about 30%-about 40% of the Factor VIII portion of the ic polypeptide used herein is single chain Factor VIII. In a particular embodiment, about 1%, about 5%, about 10%, about 15%, about 20% about 25%, about 30%, about 35% of the Factor VIII portion of the chimeric polypeptide used herein is single chain Factor VIII. In other ments, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the Factor VIII portion of the chimeric polypeptide used herein is single chain Factor VIII. In some embodiments, the ratio of the single chain Factor VIII to the processed Factor VIII of the chimeric polypeptide is (a) about 25% of single chain Factor VIII and about 75% of processed Factor VIII; (b) about 20% of single chain Factor VIII and about 80% of sed Factor VIII; (c) about 15% of single chain Factor VIII and about 85% of processed Factor VIII; (d) about 10% of single chain Factor VIII and about 90% of processed Factor VIII; (e) about 5% of single chain Factor VIII and about 95% of processed Factor VIII; (I) about 1% of single chain Factor VIII and about 99% of processed Factor VIII; (g) about 100% of processed Factor VIII, (h) about 30% of single chain Factor VIII and about 70% of processed Factor VIII, (i) about 35% of single chain Factor VIII and about 65% of processed Factor VIII, or (j) about 40% of single chain Factor VIII and about 60% of processed Factor VIII. In other embodiments, the ratio of the single chain Factor VIII to the processed Factor VIII of the chimeric ptide is (a) about 30% of single chain Factor VIII and about 70% of processed Factor VIII; (b) about 40% of single chain Factor VIII and about 60% of processed Factor VIII; (c) about 50% of single chain Factor VIII and about 50% of processed Factor VIII; (d) about 60% of single chain Factor VIII and about 40% of processed Factor VIII; (e) about 70% of single chain Factor VIII and about 30% of processed Factor VIII; (f) about 80% of single chain Factor VIII and about 20% of processed Factor VIII; (g) about 90% of single chain Factor VIII and about 10% of processed Factor VIII; (h) about 95% of single chain Factor VIII and about 5% of processed Factor VIII; (i) about 99% of single chain Factor VIII and about 1% of processed Factor VIII; or (j) about 100% of single chain Factor VIII.

The Factor VIII portion in the ic polypeptide used herein has Factor VIII actiVity.

Factor VIII actiVity can be measured by any known methods in the art. For example, one of W0 2013/009627 those methods can be a chromogenic assay. The chromogenic assay mechanism is based on the principles of the blood coagulation cascade, where activated Factor VIII accelerates the conversion of Factor X into Factor Xa in the presence of activated Factor IX, phospholipids and calcium ions. The Factor Xa activity is ed by hydrolysis of a p-nitroanilide (pNA) substrate specific to Factor Xa. The initial rate of release of p-nitroaniline ed at 405 nM is directly proportional to the Factor Xa actiVity and thus to the Factor VIII activity in the sample. The chromogenic assay is recommended by the Factor VIII and Factor IX Subcommittee of the Scientific and Standardization Committee (SSC) of the ational y on Thrombosis and atsis (ISTH). Since 1994, the chromogenic assay has also been the reference method of the European Pharmacopoeia for the assignment of FVIII concentrate potency. Thus, in one embodiment, the chimeric polypeptide comprising single chain Factor VIII has Factor VIII activity comparable to a ic polypeptide sing processed Factor VIII (e.g., a ic polypeptide ting essentially of or consisting of two Fc portions and processed Factor VIII, wherein said processed Factor VIII is fused to one of the two Fc portions), when the Factor VIII activity is measured in Vitro by a chromogenic assay.

In r embodiment, the chimeric polypeptide comprising single chain Factor VIII of this invention has a Factor Xa generation rate comparable to a ic polypeptide comprising processed Factor VIII (e.g., a chimeric polypeptide consisting essentially of or consisting of two Fc portions and processed Factor VIII, wherein the processed Factor VIII is fused to one Fc of the two Fc portions).

In order to activate Factor X to Factor Xa, activated Factor IX r IXa) hydrolyses one arginine-isoleucine bond in Factor X to form Factor Xa in the presence of Ca2+, membrane phospholipids, and a Factor VIII cofactor. Therefore, the interaction of Factor VIII with Factor IX is critical in coagulation pathway. In certain embodiments, the chimeric ptide comprising single chain factor VIII can ct with Factor IXa at a rate comparable to a chimeric polypeptide comprising processed Factor VIII (e.g., a chimeric polypeptide consisting essentially of or consisting of two Fc portions and processed Factor VIII, wherein the processed Factor VIII is fused to one Fc of the two Fc portions).

In addition, Factor VIII is bound to von Willebrand Factor while inactive in circulation.

Factor VIII degrades rapidly when not bound to VWF and is released from VWF by the action of thrombin. In some embodiments, the chimeric polypeptide comprising single chain Factor VIII binds to von rand Factor at a level comparable to a chimeric polypeptide comprising processed Factor VIII (e.g., a chimeric polypeptide consisting ially of or consisting of two Fc portions and processed Factor VIII, wherein the processed Factor VIII is fused to one Fc of the two Fc portions).

Factor VIII can be inactivated by activated protein C in the ce of calcium and phospholipids. ted protein C cleaves Factor VIII heavy chain after Arginine 336 in the A1 domain, which disrupts a Factor X substrate interaction site, and cleaves after Arginine 562 in the A2 domain, which enhances the iation of the A2 domain as well as ts an interaction site with the Factor IXa. This cleavage also bisects the A2 domain (43 kDa) and tes A2-N (18 kDa) and A2-C (25 kDa) domains. Thus, ted protein C can catalyze multiple cleavage sites in the heavy chain. In one embodiment, the chimeric polypeptide comprising single chain Factor VIII is vated by activated Protein C at a level comparable to a chimeric polypeptide comprising processed Factor VIII (e.g., a ic polypeptide consisting essentially of or consisting of two Fc portions and processed Factor VIII, wherein the processed Factor VIII is fused to one Fc of the two Fc portions).

In other embodiments, the chimeric polypeptide comprising single chain Factor VIII has Factor VIII activity in Vivo comparable to a chimeric polypeptide comprising processed Factor VIII (e. g., a chimeric ptide consisting essentially of or consisting of two Fc portions and processed Factor VIII, wherein the processed Factor VIII is fused to one Fc of the two Fc portions). In a particular embodiment, the chimeric ptide comprising single chain Factor VIII is capable of protecting a HemA mouse at a level comparable to a chimeric polypeptide comprising processed Factor VIII (e.g., a chimeric polypeptide ting essentially of or consisting of two Fc portions and processed Factor VIII, wherein said processed Factor VIII is fused to one Fc of the two Fc portions) in a HemA mouse tail vein transection model.

The term ”comparable” as used herein means a compared rate or level ed from using the chimeric polypeptide is equal to, substantially equal to, or similar to the reference rate or level.

The term "similar" as used herein means a compared rate or level has a difference of no more than % or no more than 15% from the reference rate or level (e.g., FXa generation rate by a chimeric polypeptide consisting essentially of or consisting of two Fc portions and processed Factor VIII, wherein said processed Factor VIII is fused to one Fc of the two Fc portions). The term ”substantially equal” means a compared rate or level has a ence of no more than 0.01%, 0.5% or 1% from the reference rate or level.

The present invention further includes a composition comprising a ic polypeptide having Factor VIII activity, wherein at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 99% of the chimeric polypeptide comprises a Factor VIII portion, which is single chain Factor VIII and a second portion. In r embodiment, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the chimeric polypeptide in the composition is single chain Factor VIII. In other embodiments, the second portion is an Fc, XTEN, albumin, a PAS sequence, transferrin, CTP (28 amino acid C- terminal peptide (CTP) of hCG with its 4 O-glycans), polyethylene glycol (PEG), hydroxyethyl starch (HES), albumin binding polypeptide, albumin-binding small molecules, or two or more ations thereof. In still other embodiments, the composition of the present invention comprises a combination of a chimeric ptide comprising processed Factor VIII and a chimeric polypeptide comprising single chain Factor VIII, (a) wherein about 30% of the Factor VIII portion of the chimeric polypeptide is single chain Factor VIII, and about 70% of the Factor VIII n of the chimeric polypeptide is processed Factor VIII; (b) wherein about 40% of the Factor VIII portion of the chimeric polypeptide is single chain Factor VIII, and about 60% of the Factor VIII portion of the chimeric polypeptide is processed Factor VIII; (c) wherein about 50% of the Factor VIII portion of the chimeric polypeptide is single chain Factor VIII, and about 50% of the Factor VIII portion of the ic polypeptide is processed Factor VIII; (d) wherein about 60% of the Factor VIII portion of the chimeric ptide is single chain Factor VIII and about 40% of the Factor VIII portion of the chimeric polypeptide being processed Factor VIII; (e) wherein about 70% of the Factor VIII portion of the chimeric polypeptide is single chain Factor VIII and about 30% of the Factor VIII portion of the chimeric polypeptide is processed Factor VIII; (f) wherein about 80% of the Factor VIII n of the chimeric polypeptide is single chain Factor VIII and about 20% of the Factor VIII portion of the chimeric polypeptide is processed Factor VIII; (g) wherein about 90% of the Factor VIII portion of the ic polypeptide is single chain Factor VIII and about 10% of the Factor VIII portion of the ic ptide is processed Factor VIII; (h) wherein about 95% of the Factor VIII portion of the chimeric polypeptide is single chain Factor VIII and about 5% of the Factor VIII portion of the chimeric polypeptide is processed Factor VIII; (i) wherein about 99% of the Factor VIII portion of the ic polypeptide is single chain Factor VIII and about 1% of the Factor VIII portion of the chimeric polypeptide is processed Factor VIII; or (j) n about 100% of the Factor VIII portion of the chimeric polypeptide is single chain Factor VIII.

In certain embodiments, the ition of the present ion has Factor VIII actiVity comparable to the composition comprising processed Factor VIII (e. g., a composition comprising a chimeric polypeptide, which consists essentially of or consists of two Fc portions and processed Factor VIII, wherein said processed Factor VIII is fused to one of the two Fc portions), when the Factor VIII activity is measured in Vitro by a chromogenic assay.

In other embodiments, the composition of the invention has a Factor Xa generation rate comparable to a composition comprising processed Factor VIII (e. g., a composition comprising a chimeric polypeptide, which consists essentially of or ts of two Fc portions and processed Factor VIII, wherein the processed Factor VIII is fused to one Fc of the two Fc portions). In still other ments, the composition comprising single chain factor VIII can interact with Factor IXa at a rate comparable to a composition comprising processed Factor VIII (e. g., a composition comprising a chimeric polypeptide, which consists essentially of or ts of two Fc portions and processed Factor VIII, wherein the processed Factor VIII is fused to one Fc). In further embodiments, the single chain Factor VIII in the chimeric polypeptide of the present ition is inactivated by activated Protein C at a level comparable to processed Factor VIII in a ic polypeptide of a ition (e.g., a composition comprising a chimeric polypeptide, which consists essentially of or consists of two Fc portions and processed Factor VIII, wherein the processed Factor VIII is fused to one Fc of the two Fc portions). In a ular embodiment, the composition comprising single chain Factor VIII has Factor VIII activity in Vivo comparable to the composition comprising processed Factor VIII (e.g., a ition comprising a chimeric polypeptide, which consists essentially of or consists of two Fc portions and processed Factor VIII, wherein the processed Factor VIII is fused to one Fc of the two Fc portions). In some embodiments, the composition comprising single chain Factor VIII of the invention is capable of protecting HemA mouse at a level comparable to the composition comprising processed Factor VIII (e.g., a composition comprising a chimeric polypeptide, which consists essentially of or consists of two Fc portions and processed Factor VIII, wherein said processed Factor VIII is fused to one Fc of the two Fc portions) in HemA mouse tail vein transection model.

] The present invention further provides a method for treating a bleeding condition in a human subject using the composition of the invention. An exemplary method comprises administering to the subject in need thereof a therapeutically effective amount of a pharmaceutical composition/formulation comprising a chimeric polypeptide haVing Factor VIII actiVity, wherein at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 99% of the chimeric polypeptide comprises a Factor VIII portion, which is single chain Factor VIII, and a second portion.

The bleeding condition can be caused by a blood coagulation disorder. A blood ation disorder can also be referred to as a coagulopathy. In one example, the blood coagulation disorder, which can be treated with a pharmaceutical composition of the current disclosure, is hemophilia or von Willebrand disease (vWD). In another e, the blood coagulation disorder, which can be treated with a ceutical composition of the t disclosure is hemophilia A.

In some embodiments, the type of bleeding associated with the bleeding ion is selected from hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra- abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system ng, bleeding in the retropharyngeal space, ng in the retroperitoneal space, and bleeding in the illiopsoas sheath.

In other embodiments, the t suffering from bleeding condition is in need of treatment for surgery, including, e.g., surgical prophylaxis or peri-operative ment. In one example, the surgery is selected from minor surgery and major y. Exemplary surgical procedures include tooth extraction, tonsillectomy, inguinal omy, synovectomy, craniotomy, ynthesis, trauma surgery, intracranial surgery, intra-abdominal surgery, intrathoracic surgery, joint replacement surgery (e.g., total knee replacement, hip replacement, and the like), heart surgery, and caesarean section.

In another example, the subject is concomitantly treated with FIX. Because the nds of the invention are capable of activating FIXa, they could be used to pre-activate the FIXa ptide before administration of the FIXa to the subject.

The methods of the invention may be practiced on a subject in need of prophylactic ent or on-demand treatment.

The ceutical itions comprising at least 30% of single chain Factor VIII may be formulated for any appropriate manner of administration, ing, for example, topical (e. g., transdermal or ocular), oral, buccal, nasal, vaginal, rectal or parenteral administration.

The term parenteral as used herein includes subcutaneous, intradermal, intravascular (e. g., intravenous), intramuscular, spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intrasynovial and intraperitoneal ion, as well as any similar injection or infusion techniqueThe composition can be also for example a suspension, emulsion, sustained release formulation, cream, gel or powder. The composition can be formulated as a itory, with traditional binders and carriers such as triglycerides.

In one example, the pharmaceutical formulation is a liquid formulation, e. g., a buffered, isotonic, aqueous solution. In another example, the pharmaceutical composition has a pH that is physiologic, or close to physiologic. In other examples, the aqueous formulation has a physiologic or close to physiologic osmolarity and salinity. It can contain sodium chloride and/or sodium acetate. In some examples, the composition of the present invention is lized.

HaVing now described the present ion in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention. All patents and ations referred to herein are expressly incorporated by reference.

Exanqfles Example 1 Cloning, expression andpurification 0frFVYIIFc All molecular biology procedures were med following standard techniques. The coding sequence of human FVIII nk accession number NM_000132), including its native signal sequence, was obtained by reverse transcription-polymerase chain ons (RT-PCR) from human liver polyA RNA. Due to the large size of FVIII, the coding sequence was obtained in several sections from separate RT-PCR reactions, and assembled through a series of PCR ons, restriction digests and ligations into an ediate cloning vector containing a B domain deleted (BDD) FVIII coding region with a fusion of serine 743 (S743) to glutamine 1638 (Q1638), eliminating 2682 bp from the B domain of full length FVIII. The human IgG1 Fc sequence (e.g., k accession number Y14735) was obtained by PCR from a leukocyte cDNA library, and the final expression cassette was made in such a way that the BDD FVIII sequence was fused ly to the N—terminus of the Fc sequence (hinge, CH2 and CH3 domains, beginning at D221 of the IgGl ce, EU numbering) with no intervening linker. For expression of the Fc chain alone, the mouse ng (kappa) light chain signal sequence was created with synthetic ucleotides and added to the Fc coding sequence using PCR to enable secretion of this protein product. The FVHIFc and Fc chain coding sequences were cloned into a dual expression vector, pBudCE4.1 (Invitrogen, Carlsbad, CA).

HEK 293H cells (Invitrogen, Carlsbad, CA) were transfected with the pSYN—FV111-013 plasmid using Lipofectamine transfection reagent (Invitrogen, Carlsbad, CA)), and a stable cell line was selected with zeocin. Cells were grown in serum free suspension culture, and rFVHIFc protein d from clarified harvest media using a four column purification process, including a FVIH-specific affinity purification step (McCue J. et al., J. Chromatogr. A., [216(45): 7824-30 (2009)), followed by a combination of anion ge columns and a hydrophobic ction column.

Example 2 Biochemical characterization Processed recombinant FVIII-Fc (rFVIHFc) is synthesized as two polypeptide chains, one chain consisting of HI (S743-Q1638 fusion, 1438 amino acids) fused to the Fc domain (hinge, CH2 and CH3 domains) of IgG1 (226 amino acids, extending from D221 to G456, EU numbering), for a total chain length of 1664 amino acids, the other chain consisting of the same Fc region alone (226 amino acids). Though cells transfected with the FVIIIFc/Fc dual expression plasmid were expected to secrete three products (FVHIFc dimer, FVHIFc monomer, and Fc dimer), only the FVHIFc monomer and Fc dimer were detected in conditioned media. Purified FVIIIFc was analyzed by non-reducing and reducing SDS-PAGE is (Figure 2A and, B).

For the nonreduced SDS-PAGE, bands were found migrating at approximately 90 kDa and 130 kDa, consistent with the predicted molecular weights of the FVIIIFc heavy chain (HC) and light chain-dimeric Fc fusion (LCFc2) (Figure 2A, lane 3). A third band was also detected at approximately 220 kDa, consistent with the predicted molecular weight for single chain FVIIIFc (SC FVIIIFc; HC+LCFc2), in which the arginine residue at position 754 (1648 with respect to the full length sequence) is not cleaved during secretion. For the d SDS-PAGE analysis, major bands were seen migrating at approximately 25 kDa, 90 kDa, 105 kDa, and 195 kDa, consistent with the predicted molecular weights for the single chain Fc, HC, LCFc, and SC FVIIIFc (Figure 2B, lane 3). Cotransfection with human PC5, a member of the proprotein convertase subtlisin/kexin (PCSK) type proteases, resulted in full processing of the rFVIIIFc product (Figure 2A, B, lane 2).

] Densitometry analysis of several batches of rFVIIIFc after SDS-PAGE indicated greater than 98% purity of the expected bands. Size exclusion tography (SEC) was also used to assess the degree of aggregation present, and all batches were found to have ate levels at 0.5% or less. ] rFVIIIFc ure was further analyzed by thrombin cleavage, reduction, and analysis by LC/UV and LC/MS. The four Factor VIII fragments generated by thrombin (by cleavages at three arginine residues, at positions 372, 740 and 795 (795 corresponds to 1689 with respect to the full length FVIII ce), can be detected by UV absorbance (Figure 2C), ponding to the following segments of the protein: Fc (peak 1), light-chain-Fc (peak 2); the A1 domain from the heavy chain (peak 3) and the A2 domain from the heavy chain (peak 4). The 14 amino acid B domain linker and N 6 kDa a3-related peptides are not detected by UV absorbance due to their small size.

Analysis of the thrombin digestion of rFVIIIFc by HPLC/MS ed further detailed information of the four main domains as well as the N6 kDa a3 related peptides, and was compared to REFACTO®, a CHO-derived recombinant III protein (rBDD FVIII), using the same s. FVHI samples were passed through ent-OUTDTG-100X columns (GBioscience, Maryland Heights, M0) for the removal of Tween, fully digested with thrombin, d and analyzed either by RP-HPLC-UV (POROS R1/10, Applied Biosystems) or RP- HPLC-MS (Agilent 1200 d to an Agilent 6210TOF mass spectrometer) using gradients of acetonitrile in water + 0.1% formic acid . Peptide sequence was also confirmed with LysC peptide mapping, analyzed by RP-HPLC/MS (Thermo an LTQ-XL-ETD).

As expected, the total ion current (TIC) chromatogram of rFVIIIFc (Figure 2D) appears similar to the UV chromatogram (Figure 2C). Five of the expected six products can be detected by LC/MS, including two forms of the a3 acidic region generated from the processed and single chain isoforms, as well as the thrombin used for the digestion. An additional truncated form of the a3 acidic region was also observed, and is described more fully below. rBDD FVIII yielded a similar TIC chromatogram, but without the free Fc chain and having a different mass for the LC compared to the rFVIIIFc LC-Fc, consistent with the lack of an Fc region (data not shown).

Due to the heterogeneity of glycosylation over much of the protein, the deconvoluted mass spectra for the Al, LC-Fc, and Fc regions are complex and therefore the identity of all of the lar ions have not been established. However, the observed mass for the three major peaks from the Fc region were found to match the G0, G1, and G2 isoforms found on IgG molecules, corresponding to biantennary oligosaccharides terminating in O, l or 2 galactose residues. The deconvoluted mass a of the a3-related es and the A2 domain e the most definitive data, as there are no heterogeneity in the posttranslational modifications in these regions, allowing the expected masses to be identified guously.

The 6 kDa N—terminal peptide released from the LC after ge of R1689 is predicted to se the a3 acidic region (amino acids E1649 to R1689) if derived from the processed isoform, and the 14 amino acid truncated B domain fused to the a3 acidic region if derived from the single chain isoform. Both rFVIIIFc and rBDD FVIII were found to contain both forms of the a3 region, proportional to the expected levels based on SDS-PAGE analysis. In on, both proteins contained a truncated form of the a3 region corresponding to amino acids Dl658-Rl689, as has been reported for other FVIII ts, though this was found in greater abundance in rBDD FVIII than in rFVIIIFc.

The A2 domain contains three ial tyrosine sulfation sites, but no glycosylation sites that could result in complex heterogeneity, and therefore the exact masses of this region can be calculated. In on to the primary expected peak in the deconvoluted mass spectrum of rFVIIIFc correlating to the mass of the S373 to R740 sequence (Figure 2E), two additional forms were identified corresponding to known truncations of the FVIII HC, correlating with an A2 domain truncated at E720 and Y729. These reported truncated forms were also observed directly in the oluted um of rBDD FVIII A2 (Figure 2E). Both rFVIIIFc and rBDD FVIII A2 contained r relative amounts of the form truncated at Y729 while the rBDD FVIII A2 domain contained a notably greater level of the form truncated at E720 as compared to the same form in Fc e 2E).

The primary sequence of rFVIIIFc was confirmed by peptide mapping with lysyl endopeptidase (Lys-C) digests followed by UV and mass spectrometric detection. Of the 99 theoretical peptides produced from rFVIIIFc, 81 were detected, corresponding to 98% of the total sequence. The posttranslational modifications of rFVIIIFc were also characterized by this method. FVHI contains 6 potential tyrosine sulfation sites, corresponding to positions 346, 718, 719, 723, 1664, and 1680. Fully ed peptides corresponding to these siX sites were found, with trace amounts of non-sulfated peptide ponding to position 1680 as assessed by integration of the total ion chromatogram in the mass spectra, and no detectable non-sulfated peptides corresponding to the other positions. BDD FVHI also contains 6 potential N- glycosylation sites, four of which have been reported to be glycosylated in inant FVHI products. tent with this, rFVIIIFc was found to have the same 4 sites glycosylated; N239 and N2118 were found to contain high e structures, while N41 and N1810 were found to contain more complex carbohydrates, similar to those found on rBDD FVIII. The Fc region N- linked glycosylation was found to match the G0, G1, and G2 isoforms found with the thrombin map by LC/MS. FVHI has been reported to have osylation sites at Ser 741 and 743 that are partially occupied, and this was found to be the case with rFVIIIFc as well.

The rFVIIIFc polypeptide produced without cotransfected processing s exhibited -25% single chain FVIIIFc (SC FVIIIFc), which differs from processed rFVIIIFc by a single e bond between R754 and E755 (R1648/E1649 with respect to the full length FVHI). This m was purified and characterized in all of the biochemical assays described above, and found to be comparable to rFVIIIFc as shown below. The activity of d single chain FVIIIFc was found to be similar to rFVIIIFc in a chromogenic assay as well as by the various functional assays described below.

Measurement ofFVIIIActivity by Chromogenic and One-Stage says FVIII activity was measured by a FVIII chromogenic assay. The average specific ty from four separate batches of rFVIIIFc was found to be 9762 :: 449 IU/mg by the chromogenic assay, corresponding to 2148::99 IU/nmol. The average specific activity from fourteen separate batches of rFVIIIFc was found to be 8460 :: 699 IU/mg by the aPTT assay, and 9348 :: 1353 IU/mg by the genic assay, corresponding to 1861::154 and 2057::298 IU/nmol, respectively. FVIII activity of single chain FVIII:Fc was also measured by the chromogenic assay and compared to the completely processed rFVHI:Fc or rFVHI:Fc DS ining about % single chain rFVIII:Fc). As Table 3A shows, single chain rFVIIIFc showed no significant difference in FVIII activity compared to the Factor VIII activity of completely processed FVIIIFc or rFVIIIFc DS by the chromogenic assay, both in the presence and the absence of von Willebrand Factor (VWF). Table 3B shows that full activity of SCrFVHIFc, as measured by one- stage activated partial thromboplastin time (aPTT) assay, was observed in the absence ofVWF.

WO 09627 TABLE _3A_FVHI Activity by Chromogenic Assay Chromogenic ActiVity(1U/mg) FVIIIFcDS (25% NP) (RECDO9-013) 9066 2.49 FVIII depleted Single chain rFVIIIFc (purified from RECD 8194 2.72 plasma 19189013) Completely Processed rFVIIIFc 9577 8.34 (purified from an engineered cell line) FVIIIFcDS (25% NP) (RECD09-013) 10801 8.92 FVIII and VWF depleted Single chain rFVIIIFc 9498 4.70 plasma (purified from RECD 19189013) Completely Processed rFVIIIFc 9569 4.54 (purified from an engineered cell line) depleted supplemented with human Completely-processed rFVIIIFc *CV=coefficient of variation TABLE _3B_FV111 ty by aPTT assay Matrix Sample Coagulation (aPTT) Specific (IU/mg) rFVIIIFcDS (25% NP) 8210 5.88 (RECD09-013) epleted . . (purified from RECD 19189013) Completely sed rFVIIIFc 8683 3.57 (purified from an engineered cell line) rFVIIIFcDS (25% NP) 15621 6.47 FVIII and (RECD09-013) VWF depleted _ _ 13572 2.41 plasma S1ngle cha1n rFVIIIFc (purified from RECD 19189013) Completely Processed rFVIIIFc 15170 10.42 (purified from an engineered cell line) FVIII/VWF- rFVIIIFc 7.4 depleted supplemented with human Completely-processed rFVIIIFc 8495 4 .0 In one-stage clotting assay (APTT), SC rFVIIIFc trated a 60% decrease in activity when the plasma has normal VWF level, suggesting the potential role ofVWF in the activation of SC rFVIIIFc. This ation was further confirmed by addition of human VWF back to the VWF-depleted plasma (Table 3), where the coagulant ty of SC rFVIIIFc was reduced to the same level as in congenital FVIII-deficient plasma.

Activity in Xase Complex FVIII activity was also measured in the context of the Xase complex, by incubating activated FIX and thrombin-activated O® or rFVIIIFc protein on a olipid surface in the presence of calcium, and monitoring the conversion of FX to FXa as measured by cleavage of a chromogenic or fluorogenic substrate, from which FXa generation rates were determined.

This assay was then modified by varying one component of the assay while keeping the others constant in order to examine the interactions with each individual component.

The FXa generation rate was determined as a function of g phospholipid concentrations for Fc DS, rBDD FVIII, and single chain rFVIIIFc (Figure 3A), using synthetic phospholipid vesicles (25% phosphotadyl serine/75% phosphotadyl choline). Both proteins were found to have a r ty profile, with peak actiVity at approximately 156 “M phospholipids.

The FXa tion rate was then determined as a function of varying FX concentrations, and Km and Vmax values calculated (Figure 3B). The activity profiles for rFVIIIFc DS, rBDD FVIII, and single chain rFVIIIFc were found to be similar, with similar Km and Vmax (Table 4).

Finally, the FXa generation rate was determined as a function of varying FIX trations (Figure 3C). The activity profiles appeared similar, with similar Kd and Vmax (Table 4). Similar results were obtained using platelets as a phospholipid source (unpublished data, June 2009).

TABLE 4. FXa Generation Parameters for FVIII Proteins on Phospholipids Lipid Source Km (nM) Vmax n) I'FVIIIFC DS 55.0 :: 5.9 65.6 :: 8.6 %PS-75%PC I‘BDD FVIII 51.0 :: 8.7 73.5 :: 10.1 NP I‘FVIIIFC 53.2 :: 7.5 56.0 :: 13.8 TABLE 5. FIXa Interactions with FVIII Proteins Lipid Source Km (nM) Vmax (nM/min) Fc DS 28-__ 0.4 45-__ 0.3 %PS—75%PC rBDD FVlll 2.5 :: 0.3 4.0 :: 1.0 NP rFVllch Inactivation by APC Once active, FVIII is inactivated by cleavage by activated protein C (APC), as well as by dissociation of the A2 domain. c and rBDD FVIII were both activated by thrombin, then incubated with APC for different times and ty determined in a FXa generation assay e 4). In the absence of in activation, little FXa generation was detected, and this was increased cantly with in digestion. Treatment with APC for 90 min led to a significant decrease in FXa generation rates, similar to non-activated samples, and these results were similar for rFVIHFc DS, rBDD FVIII, and single chain Fc.

Affinityfor vWF FVIII interactions with von Willebrand factor (vWF) were measured by real-time biomolecular interaction analysis (BLAcore), based on surface Plasmon resonance (SPR) technology, to determine the kinetics of g of rFVIIIFc and rBDD FVIII towards vWF (Table 6). Kinetic rate parameters of Ka (on-rate) and Kd (off-rate), and the affinity KD (Kd/Ka), were determined for each FVIII interaction under identical conditions. Both rFVIHFc and rBDD FVIII were found to have a low nM binding affinity (KD) for vWF, of 1642037 and 084620.181 nM, respectively. The proteins had similar off-rates, with a two fold difference in e resulting in a two fold difference in the affinity.

TABLE 6. Biocore g Analysis of FVIII Proteins to vWF _--Kinetic rate parameters Off-rate/On-rate On-rate (M- 1 s-1) Off-rate (s-1) KD(M) ro111Fch thf 5 7.92:1.51x105 1.25:1.12x10'3 0.37X10'9 NP rFVHIFc thf 5 8.66 1.10x105 1.09 0.09x10'3 1.28 0.22x10'9 rBDD FVIII thf 5 13.7 1.50x105 1.14 0.12x10'3 0.846 0.181X10'9 As shown in Table 6, the affinity ofrFV111:Fc DS or single chain rFVIIIFc with vWF was found to be in the low nM range, approximately two fold greater than that of BDD FVIII alone.

At physiological concentrations, this would result in a slight decrease in the percentage of rFVIIIFc (processed or single chain) complexed with vWF as compared to free FVIII, however in vivo studies have indicated that the half life of rFVIHFc is cantly prolonged over full length or BDD FVIII despite this slightly lower affinity, and ore this does not appear to compromise the half life of the molecule. It may be possible that the free rFVIIIFc is more efficiently recycled through the FcRn pathway and therefore this may bute to a greater prolongation of half life.

Aflim'tyfor vWF and Thrombin-mediated Releasefrom vWF Recombinant B-domain d Factor VIIIFc (rFVIHFc) was expressed in HEK293 cells. During biosynthesis in HEK293 cells, most of the rFVIHFc is processed by limited proteolysis to generate a FVIII heavy chain (HC) and a FVIII light chain (LC) to which the Fc moiety is attached. Spontaneous disassociation of the HC and LC in plasma, and during storage of FVIII drug products, is thought to contribute to a loss of FVIII activity. The ing portion of the biosynthesized rFVIIIFc, which is not processed, forms a single chain isoform of rFVIIIFc (SC rFVIIIFc), which may provide enhanced manufacturability and stability compared to the processed rFVHIFc.

This e describes an assay comparing the interaction of SC Fc with von Willebrand factor (vWF) in relation to the interaction of rFVIIIFc with vWF. Interactions with VWF were measured by real-time biomolecular ction analysis (BLAcore), based on surface Plasmon resonance (SPR) technology, to determine the kinetics of binding of rFVIIIFc and SC rFVIHFc towards vWF (Table 11 and Figure 16A). FVIII-free human plasma derived VWF was immobilized by amine coupling on the surface of a biosensor at levels low enough to prevent mass transport limitation. rFVIIIFc and SC rFVIHFc were sequentially injected in single-cycle kinetics mode at trations ranging from 0.13 to 5.0 nM. Sensorgram data was fit to a 1:1 interaction model.

Kinetic rate parameters of Ka (on-rate) and Kd (off-rate), and the affinity KD (Kd/Ka), were determined for each FVIII interaction under identical conditions. Both rFVIIIFc and SC c were found to have a low nM binding affinity (KD) for vWF, of 0.34:0.1 and O.31::O.1 nM, respectively. Both isoforms also had similar on-rates and off-rates.

TABLE 11. Biocore Binding Analysis of FVIII Proteins to vWF _--Kinetic rate parameters Off-rate/On-rate Analyte Ligand N e (M-ls-l) Off-rate (s-1) KD(M) rFVIIIFc thf 6 2.6:0.4x105 .3X10'4 3.4:0.1x10'10 seroch thf 6 2.7:0.1x105 .4X10'4 3.1:0.1x10'1° WO 09627 Next, thrombin-mediated release of rFVIIIFc, SC rFVIIIFc, and B-domain deleted FVIII lacking Fc es (rBDD FVHI) was measured at both 25° C and 37° C. Human vWF was immobilized by amine coupling at similar levels on three flow cells on the biosensor surface. The remaining flow cell served as a blank for reference purposes. The Fc and SC rFVIIIFc proteins were captured by vWF and allowed to slowly disassociate, and the concentrations of rFVIIIFc and SC rFVIIIFc were adjusted to obtain equimolar capture levels by the end of the dissociation phase. Human oc-thrombin solutions were prepared by 2-fold serial dilution and applied at concentrations that ranged from 0.005 to 20 U/mL, resulting in proteolytic release of FVHIa species from vWF. Thrombin ed release of activated rFVIIIFc, SC Fc, and rBDD FVIII from vWF was red in real-time using an SPR-based optical biosensor (Figure 16B). Following blank reference subtraction (Figure 16C), the release rate as a function of oc- thrombin concentration was determined (Figure 16D). The thrombin half l effective concentration (EC50) for SC rFVIIIFc was 12:] U/mL compared to 39:03 U/mL for rFVIIIFc at ° C and was 152] U/mL for SC rFVIIIFc compared to 4.8202 U/mL for rFVIIIFc at 37° C (Figure 16E). rFVIIIFc had a similar thrombin EC50 value compared to rBDD FVIII, having values of 39:03 U/mL and 33:03 U/mL, respectively at 25° C and values of 4.8::0.2 U/mL and 40:02 U/mL, respectively at 37° C. SC rFVIIIFc had a thrombin EC50 value that was approximately 3-fold higher than rFVIIIFc. This impairment of thrombin mediated release from vWF may underlie the specific reduction in specific activity for SC rFVIIIFc observed in the aPTT assay in which vWF was t (Table 3B).

Example 3 A Phase I/Ila, open-label, crossover, dose-escalation, multi-center, and first-in-human study was designed to evaluate the safety, tolerability, and pharmacokinetics of a single dose of Fc in subjects with severe (defined as <1 IU/dL [1%] endogenous factor VIII [FVHI]) hemophilia A. A total of approximately 12 previously treated patients were enrolled and dosed with rFVIIIFc at 25 or 65 IU/kg. After the screening (scheduled within 28 days prior to the first dose of the ® [rFVIII], the nce comparator agent) and a minimum of 4-days (96 hours) elapsing with no FVIII treatment prior to the first injection, approximately 6 subjects received a single 25 IU/kg dose of ADVATE® followed by a 3-day (72 hours) pharmacokinetic (PK) profile then crossover and receive a 25 IU/kg single, open-label dose of Fc for a 7-day (168 hours) PK profiling. The first 3 ts were dosed tially. For the first three (3) subjects dosed with 25 IU/kg of rFVIIIFc, each subject underwent an inhibitor assessment at 14— days (336 hours) post-injection of rFVIIIFc. Dosing of the next subject (for the first three subjects only) occurred once the inhibitor testing is completed. After the 3rd subject completed the 14 day inhibitor assessment, the remaining three subjects at 25 lU/kg and the six subjects at 65 IU/kg began enrollment tially at least 1 day apart within each dose group.

One week after the last subject received the 25 IU/kg dose of the rFVHIFc, approximately 6 unique subjects were recruited for the 65 IU/kg . Each subject in the 65 IU/kg cohort received a single 65 IU/kg dose of ADVATE® followed by a 4-day (96 hours) PK profiling then crossover and receive a 65 IU/kg single, open-label dose of c for a 10-day (240 hours) profiling. If a bleeding episode ed before the first injection of rFVIHFc in any cohort, subject’s pre-study FVIII product was used for treatment and an interval of at least 4 days had to pass before receiving the first injection of rFVHIFc for the PK profile.

All subjects were followed for a 14-day (336 hours) and 28 day safety evaluation period after administration of c 25 IU/kg or 65 IU/kg for safety. All subjects underwent pharmacokinetic sampling pre- and post-dosing along with blood samples for analysis of FVIII activity at designated time points.

The pharmacokinetic data for the Phase I/Ila clinical trial demonstrated the following results for FVIIIFc. FVIIIFc had about a 50% increase in systemic exposure F), about 50% reduction in clearance (Cl), and about 50-70% increase in elimination half-life and MRT compared to ADVATE® (full length rFVHI). In on, FVIHFc showed increased C168, TBLPl, TBLP3, and TBLP5 values ed to ADVATE®.

AUCINF Area under the concentration-time curve from zero to infinity Beta HL Elimination phase half-life; also referred to as t1 /2[3 C168 ted FVIIIFc activity above baseline at approximately 168 h after dose Cl Clearance MRT Mean residence time TBLPl Model-predicted time after dose when FVIIIFc activity has declined to approximately 1 IU/dL above baseline TBLP3 Model-predicted time after dose when FVIIIFc activity has declined to approximately 3 IU/dL above baseline TBLP5 Model-predicted time after dose when FVIIIFc activity has declined to approximately 5 IU/dL above baseline A recombinant B-domain-deleted factor VIII-Fc (rFVIHFc) fusion protein has been created as an approach to extend the half-life of FVIII. The cokinetics (PK) of rFVIHFc were compared to rFVIII in hemophilia A mice. We found that the terminal half-life was twice as long for rFVIHFc ed to rFVIII. In order to confirm that the underlying mechanism for the ion of half-life was due to the protection of rFVIHFc by FcRn, the PK were evaluated in FcRn knockout and human FcRn transgenic mice. A single intravenous dose (125 lU/kg) was administered and the plasma concentration measured using a chromogenic ty assay. The Cmax was similar between rFVllch and rFVlll (XYNTHA®) in both mouse strains. However, while the half-life for rFVllch was comparable to that of rFVlll in the FcRn knockout mice, the half-life for rFVllch was ed to approximately twice longer than that for rFVlll in the hFcRn transgenic mice. These results confirm that FcRn mediates or is responsible for the prolonged ife of rFVHIFc compared to rFVIH. Since hemostasis in whole blood measured by rotation thromboelastometry (ROTEM®) has been shown to ate with the efficacy of coagulation s in bleeding models of hemophilia mice as well as in clinical applications, we sought to evaluate the ex vivo efficacy of rFVllch in the hemophilia A mice using ROTEM®.

Hemophilia A mice were administered a single intravenous dose of 50 IU/kg rFVHIFc, XYNTHA® (FVHI) or ADVATE® (FVIH). At 5 minutes post dose, clot formation was similar with respect to clotting time (CT), clot formation time (CFT) and (x-angle. However, rFVllch showed cantly improved CT at 72 and 96 hr post dose, and CFT and (x-angle were also improved at 96 hrs compared to both XYNTHA® (FVHI) and ADVATE® (FVIII), consistent with prolonged PK of rFVHIFc. Therefore construction of an Fc fusion of FVIII produces a molecule with a defined ism of action that has an increased half-life and the potential to provide ged protection from bleeding.

Example 5 This Example presents final is results for FVIII activity from 16 patients treated with 25 and 65 lU/kg FVIII products. See Example 3.

In this Example, rFVHIFc is a recombinant fusion protein comprised of a single molecule of recombinant B-domain deleted human FVIII (BDD-rFVlll) fused to the dimeric Fc domain of the human lgGl, with no intervening linker ce. This protein construct is also ed to herein as rFVllch heterodimeric hybrid protein, FVllch monomeric Fc fusion protein, FVllch r hybrid, monomeric FVlllch hybrid, and FVllch monomer-dimer. See Example 1, Fig. l, and Table 2A.

Preclinical studies with rFVllch have shown an approximately 2-fold prolongation of the half-life of rFVlll activity compared to commercially available rFVlll products. The rationale for this study was to evaluate the safety and tolerability of a single dose of rFVllch in frozen liquid formulation and e data on the PK in severe hemophilia A ts. For this study, 16 evaluable subjects were available for PK evaluation. Single administration of two doses of both rFVHIFc and ADVATE® at a l dose of 25 (n=6) and 65 lU/kg of body weight (n=10) were infused intravenously over approximately 10 minutes. Blood samples for plasma PK assessments were obtained before infusion, as well as up to 10 days after dosing. The PK of FVIII activity for both ADVATE® and rFVIHFc were characterized in this study using a model-dependent method.

OBJECTIVES ] The primary objective of this study was to assess the safety and tolerability of single administration of two doses of rFVllch (25 and 65 lU/kg) in usly treated ts (PTPs) aged 12 and above with severe hemophilia A.

The secondary objectives were to determine the pharmacokinetics (PK) parameters determined by pharmacodynamic (PD) activity of FVIII over time after a single administration of or 65 lU/kg of rFVHIFc compared to ADVATE® in one-stage clotting and chromogenic assays.

Study Design (See Example 3) Blood samples were collected for FVIII activity PK evaluations at the screening visit (within 28 days prior to dosing ®); on Day 0 (injection of ADVATE®) pre-inj ection and at 10 and 30 minutes and l, 3, 6, and 9 hours post-injection; on Day 1 at 24 hours post-injection of ADVATE®; on Day 2 at 48 hours njection of ADVATE®; on Day 3 at 72 hours post- injection of ADVATE®; and on Day 4 at 96 hours post-injection of high dose of ADVATE® (Cohort B only).

Blood samples were ted for FVIII activity PK evaluations on the day of rFVHIFc injection just prior to the administration of rFVllch, at 10 and 30 minutes and l, 3, 6, and 9 hours post-injection of h; on Day 1 at 24 hours post-inj ection of rFVllch; on Days 2 through 5 at 48, 72, 96, and 120 hours njection of rFVllch; on Day 7 at 168 hours njection of rFVllch; on Days 8, 9, and 10 at 192, 216, and 240 hours post-injection of high dose of h (Cohort B only). FVIII activity was also measured at the final study visit (28 days post-injection of rFVllch) at 672 hours post-inj ection of rFVllch.

Pharmacokinetic Modeling and Calculations Abbreviations TBLPl = Model-predicted time after dose when FVIII activity has declined to approximately 1 lU/dL above baseline.

TBLP3 = Model-predicted time after dose when FVIII activity has declined to approximately 3 lU/dL above baseline KV_M = CmaX_M/Actual Dose (lU/kg) KV_OB = CmaX_OB/Actual Dose (lU/kg) IVR_M = 100 X CmaX_M >< Plasma Volume (dL) / Total Dose in 1U; where plasma volume in mL = (23.7 X Ht in cm) -- (9.0 X Wt in kg) — 1709.

IVR_OB = 100 X CmaX_OB >< Plasma Volume (dL) / Total Dose in 1U; where plasma volume in mL = (23.7 X Ht in cm) -- (9.0 X Wt in kg) — 1709.

RESULTS -Dose cokinetics tage Assay) Observed FVIII ty increased sharply after the short IV infusion of either ADVATE® or rFVIIIFc, with mean (::SD) model-predicted CmaX values of 56.6 :: 4.74 and 121 :: 28.2 IU/dL for ADVATE® and 55.6 8.18 and 108 :: 16.9 IU/dL for rFVIIIFc for the 25 and 65 IU/kg dose groups, respectively. All ADVATE®- and rFVHIFc-treated patients had dose-related ses in FVIII activity. The observed increase in both CmaX and AUCINF was slightly less than proportional to dose over the dose range ted.

] After the end of the infusion, the decline of the observed FVIII activity exhibited monoeXponential decay characteristics until the baseline level was reached. The rate of decline in FVIII activity was slower for rFVIIIFc than for ADVATE® with mean (::SD) model-predicted elimination half-life values of 11.9 :: 2.98 and 10.4 = 3.03 hr for ADVATE® and 18.0 3.88 and 18.4 :: 6.99 hr for rFVIHFc for the 25 and 65 IU/kg dose groups, respectively. Elimination half- life values appeared to be dose-independent over the dose range evaluated for both FVIII products.

Total systemic FVIII exposure (assessed by AL'CINF) was N 48% and 61% greater following rFVHIFc administration than ADVATE® at 25 and 65 IU/kg dose levels, respectively.

Mean (::SD) model-predicted AUCINF values were 974 :: 259 and 1810 :: 606 hr*IU/dL for ADVATE® and 1440 316 and 2910 :: 1320 hr*IU/dL for rFVHIFc for the 25 and 65 IU/kg dose groups, respectively.

Similar to ation half-life, the MRT was prolonged for rFVIIIFc relative to ADVATE®. Mean (::SD) model-predicted MRT values were 17.1 :: 4.29 and 14.9 :: 4.38 hr for ADVATE® and 25.9 :: 5.60 and 26.5 :: 10.1 hr for rFVHIFc for the 25 and 65 IU/kg dose groups, tively. MRT values ed to be dose-independent over the dose range evaluated for both FVHI products.

In addition, primary PK parameter values for CL and V were determined. CL values for rFVHIFc only accounted for N 66% of those observed for ADVATE® at equivalent doses. Mean (::SD) model-predicted CL values were 2.70 :: 0.729 and 4.08 :: 1.69 mL/hr/kg for ADVATE® and 1.80 :: 0.409 and 2.69 :: 1.25 mL/hr/kg for rFVIIIFc for the 25 and 65 IU/kg dose groups, respectively. V values were comparable between ADVATE® and c with mean (::SD) model-predicted V values of 43.9 :: 4.27 and 56.1 :: 13.4 mL/kg for ADVATE® and 45.3 7.23 W0 2013/009627 and 61.6 :: 10.6 mL/kg for rFVHIFc for the 25 and 65 lU/kg dose groups, respectively. Slight increases in mean CL and V values were noted with increasing dose of ADVATE® and rFVHIFc; however, the increase in rd deviations at the 65 lU/kg dose coupled with limited dose levels confounded an assessment of the dose-dependency of these parameters. For example, the CV% geometric mean CL value for the rFVllch treatment group increased from 23.0% (25 IU/kg) to 48.6% (65 IU/kg).

] In addition to the primary PK parameters, secondary PK parameters (e.g. K-values, IVR, etc.) were determined to evaluate FVIII on of effect. Evidence of PK difference was also observed with rFVllch demonstrating increased TBLPland TBLP3 values compared to ADVATE® at equivalent doses. IVR and K-values for ADVATE® and rFVllch appeared to be comparable. A slight se in TBLPl and TBLP3 values were observed with increasing dose of ADVATE® and rFVHIFc. In contrast, slight decreases in mean IVR and K-values were noted with increasing dose of ® and rFVllch. As previously indicated, an ment of the dose dependency of these parameters is confounded by limited dose levels.

Mean (:SD) observed TBLPl were 2.88 :: 0.733 and 2.93 :: 0.848 lU/dL per lU/kg for ADVATE® and 4.28 :: 0.873 and 5.16 :: 2.02 IU/dL per lU/kg for rFVllch for the 25 and 65 lU/kg dose groups, tively. Mean (:SD) observed TBLP3 were 2.06 :: 0.527 and 2.26 :: 0.666 IU/dL per lU/kg for ADVATE® and 3.09 :: 0.623 and 3.93 :: 1.59 IU/dL per lU/kg for c for the 25 and 65 lU/kg dose , respectively.

Mean IVR and K-values calculated using ed CmaX values (subtracted with baseline and residual drug within the model) were generally greater than values determined using model- predicted CmaX values; consistent with slight underestimation of the observed peak activity using the one-compartment model. Mean (:SD) observed K—values were 2.57 :: 0.198 and 2.13 :: 0.598 lU/dL per lU/kg for ADVATE® and 2.46 :: 0.330 and 1.85 :: 0.332 IU/dL per lU/kg for rFVHIFc for the 25 and 65 lU/kg dose groups, respectively. Mean (:SD) observed IVR values were 94.1 :: .6 and 85.8 :: 16.5 % for ADVATE® and 89.5 :: 11.9 and 74.8 :: 6.72 % for rFVHIFc for the 25 and 65 lU/kg dose groups, respectively.

-Dose Pharmacokinetics (Chromogenic Assay) Observed FVIII activity increased sharply after the short IV infusion of either ADVATE® or rFVHIFc, with mean (:SD) model-predicted CmaX values of 70.2 :: 9.60 and 157 :: 38.6 IU/dL for ADVATE® and 70.3 :: 10.0 and 158 :: 34.7 IU/dL for rFVllch for the 25 and 65 lU/kg dose groups, respectively.

All ADVATE®- and rFVllch-treated patients had dose-related increases in FVIII activity. The observed se in both CmaX and AUCINF was ly less than proportional to dose over the dose range evaluated.

] After the end of the infusion, the decline of the observed FVIII activity exhibited monoeXponential decay characteristics until the baseline level was reached. The rate of decline in FVIII activity was slower for rFVIIIFc than for ADVATE® with mean (:SD) model-predicted elimination half-life values of 10.7 :: 1.98 and 10.3 :: 3.27 hr for ADVATE® and 16.2 :: 2.92 and 19.0 :: 7.94 hr for Fc for the 25 and 65 IU/kg dose groups, tively. Elimination half- life values appeared to be dose-independent over the dose range evaluated for both FVIII products.

Total systemic FVIII exposure (assessed by ) was N 53% and 84% greater following rFVIIIFc administration than ADVATE® at 25 and 65 IU/kg dose levels, respectively.

Mean (:SD) model-predicted AUCINF values were 1080 :: 236 and 2320 :: 784 hr*IU/dL for ADVATE® and 1650 :: 408 and 4280 :: 1860 hr*IU/dL for rFVIIIFc for the 25 and 65 IU/kg dose groups, respectively.

Similar to elimination half-life, the MRT was prolonged for rFVIIIFc relative to ADVATE®. Mean (:SD) predicted MRT values were 15.3 :: 2.86 and 14.8 :: 4.72 hr for ADVATE® and 23.4 :: 4.22 and 27.3 :: 11.4 hr for rFVIIIFc for the 25 and 65 IU/kg dose groups, respectively. MRT values appeared to be dose-independent over the dose range ted for both FVIII products.

In addition, primary PK parameter values for CL and V were determined. CL values for rFVIIIFc only accounted for N 58-66% of those observed for ADVATE® at equivalent doses.

Mean (:SD) model-predicted CL values were 2.39 :: 0.527 and 3.21 :: 1.40 mL/hr/kg for ADVATE® and 1.57 :: 0.349 and 1.86 :: 0.970 mL/hr/kg for rFVIIIFc for the 25 and 65 IU/kg dose groups, respectively. V values were able between ADVATE® and rFVIIIFc with mean (:SD) model-predicted V values of 35.8 :: 5.52 and 43.6 :: 11.2 mL/kg for ADVATE® and .9 :: 6.65 and 42.7 :: 8.91 mL/kg for Fc for the 25 and 65 IU/kg dose groups, respectively. Increases in mean CL and V values were noted with increasing dose of ADVATE® and rFVIIIFc; however, the increase in standard deviations at 65 IU/kg coupled with limited dose levels nded an assessment of the dose-dependency of these ters.

In addition to the primary PK parameters, secondary PK parameters (e.g. K-Values, IVR, etc.) were determined to evaluate FVIII duration of effect. Evidence of PK difference was also ed with rFVIIIFc demonstrating sed nd TBLP3 values compared to ADVATE® at equivalent doses. IVR and K-Values for ADVATE® and rFVIIIFc appeared to be comparable.

A slight increase in TBLPl and TBLP3 values were observed with increasing dose of ADVATE® and rFVIIIFc. In contrast, slight decreases in mean IVR and K-Values were noted with increasing dose of ADVATE® and rFVIIIFc. As preViously indicated, an assessment of the dose dependency of these parameters is confounded by limited dose levels.

Mean (:SD) observed TBLPl were 2.70 :: 0.511 and 3.09 :: 0.978 IU/dL per lU/kg for ADVATE® and 4.06 :: 0.798 and 5.66 :: 2.38 lU/dL per lU/kg for rFVHIFc for the 25 and 65 lU/kg dose groups, tively. Mean (:SD) observed TBLP3 were 1.98 :: 0.377 and 2.39 :: 0.718 IU/dL per IU/kg for ADVATE® and 3.04 :: 0.598 and 4.44 :: 1.84 IU/dL per IU/kg for rFVHIFc for the 25 and 65 lU/kg dose groups, respectively.

Mean IVR and K-values calculated using observed Cmax values (subtracted with baseline and residual drug within the model) were generally greater than values determined using model- predicted Cmax values; tent with slight underestimation of the observed peak activity using the one-compartment model. Mean (:SD) observed K—values were 3.08 :: 0.429 and 2.85 :: 0.721 lU/dL per lU/kg for ADVATE® and 3.12 :: 0.451 and 2.92 :: 0.985 lU/dL per lU/kg for rFVllch for the 25 and 65 lU/kg dose , tively. Mean (:SD) observed IVR values were 112 :: 14.5 and 116 :: 26.9 % for ADVATE® and 113 :: 16.3 and 117 :: 33.6 % for rFVllch for the 25 and 65 lU/kg dose groups, respectively.

CONCLUSIONS All ADVATE®- and rFVIHFc-treated patients had comparable elated increases in Cmax and AUCINF over the dose range evaluated. Peak plasma levels of ADVATE® and rFVllch activity were generally observed within the first hour after the end of the infusion and remained detectable for several days after dosing. After the end of infusion, the decline in baseline corrected FVIII activity exhibited monoexponential decay until the baseline was reached for both products. Parameter values for elimination half-life and MRT appeared to be dose- independent over the dose range evaluated for both FVIII products. Slight increases in mean CL and V values were noted with increasing dose of ADVATE® and rFVHIFc; however, increased intersubj ect ility at the 65 lU/kg coupled with limited dose levels confounded an assessment of the dose-dependency of these parameters.

Comparison of rFVllch and ADVATE® activity PK ed an approximate 48-61% (One-Stage Assay) or 53-84% (Chromogenic Assay) increase in systemic exposure, approximate -40% reduction in clearance, and an approximate 50-80% increase in both elimination half-life and MRT for rFVHIFc relative to ADVATE® at comparable doses. Evidence of PK difference was also observed with rFVllch trating increased TBLPl and TBLP3 values ed to ADVATE® at equivalent doses. IVR and es for ADVATE® and rFVllch appeared to be comparable.

] The PK parameters obtained from the Chromogenic Assay results generally agreed with those from the One-Stage Assay, except that the Chomogenic Assay yielded a higher estimation of exposure ters (e. g., Cmax, AUCINF, etc.).

With the observed ements in PK, rFVllch may provide a prolonged protection from bleeding, allowing less frequent injections for individuals with Hemophilia A.

Example 6 On the basis of the interim PK analysis from the first in-human study of rFVlll:Fc (Example 3), the A-LONG study was designed. A-LONG is an open label, multi-center evaluation of the safety, pharmacokinetics, and efficacy of recombinant Factor VIII Fc fusion (FVIII:Fc) in the prevention and treatment of bleeding in previously treated subjects with severe hemophilia A (defined as <1 lU/dL [<l%] endogenous FVIII).

Approximately 106 ts will be enrolled into one of three ns: a tailored prophylaxis regimen (arm 1), a weekly dosing regimen (arm 2), and an on-demand regimen (arm Arm 1: Tailored laxis Regimen Arm 1 will e an overall group and a PK subgroup. Approximately 66 subjects will be enrolled. The initial regimen will be twice weekly at 25 lU/kg on the first day, followed by 50 IU/kg on the fourth day of the week. Subjects will ster rFVllch on this weekly prophylaxis regimen until PK results for rFVllch are available. Based on these results, a tailored prophylaxis regimen will be established for each individual, in which the dose and interval will be determined to maintain a trough level of 1-3% FVIII activity. Each subject will then administer his dually tailored prophylaxis regimen throughout the study. ts will be monitored throughout the study and ongoing dose and interval adjustments will be made. Adjustments will only be made when a subject experiences ptable bleeding episodes defined as 22 spontaneous bleeding episodes over a g two- month period. In this case, adjustment will target trough levels of 3-5%.

Arm 2: Weekly Dosing Regimen Approximately 20 subjects will be ed/randomized and undergo abbreviated rFVllch PK profiling as follows: Washout of at least 96 hours; a single dose of rFVllch 65 lU/kg; Abbreviated sampling beginning on rFVllch Day 0, including pre-inj ection and 10 (::2) s, 3 hours (:: 15 minutes), 72 (:: 2) hours [Day 3], and 96 (:: 2) hours [Day 4] from the start of injection. Following the abbreviated PK profiling, subjects will then administer a fixed dose of 65 lU/kg rFVllch every 7 days at least for 28 weeks and up to 52 weeks. 2012/045784 Arm 3: On-demand Regimen A minimum of 10 major surgeries in at least 5 subjects will be evaluated in the study.

Maj or surgery is defined as any al procedure (elective or emergent) that involves general anesthesia and/or respiratory assistance in which a major body cavity is penetrated and exposed, or for which a substantial impairment of physical or physiological functions is produced (e.g., laparotomy, thoracotomy, craniotomy, joint replacement, and limb amputation).

For prophylaxis during surgery, subjects will be treated with 20 to 50 IU/kg rFVllch every 12 to 24 hours. Prior to surgery, the physician will review the subj ect's rFVllch PK profile and assess the dose regimen of Factor VIII ement generally required for the type of planned y and the clinical status of the subject. Recommendation for the appropriate dosing of rFVllch in the al treatment period, including any rehabilitation time, will take these factors into consideration.

] The primary objectives of this study are (a) to evaluate the safety and tolerability of rFVllch administered as prophylaxis, on-demand, and surgical treatment regimens; and (b) to evaluate the efficacy of rFVllch administered as prophylaxis, on-demand, and surgical treatment regimens. The secondary objectives of this study are (a) to characterize the PK profile of rFVllch and compare the PK of FVllch with the currently ed product, ADVATE®; (b) to evaluate individual responses with FVllch; and (c) to evaluate FVllch consumption.

Primary Objectives 0 To te safety and tolerability of rFVllch administered as prophylaxis, weekly, on- demand, and surgical treatment regimens 0 To evaluate the efficacy of rFVllch administered as tailored prophylaxis, and, and surgical ent regimens Secondary Objectives 0 To characterize the PK profile of rFVllch and compare the PK of rFVllch with the currently marketed product, ADVATE® 0 To evaluate individual responses with rFVHIFc 0 To characterize the range of dose and schedules required to tely t bleeding in a prophylaxis regimen; maintain homeostasis in a surgical setting; or to treat bleeding episodes in an on-demand, weekly treatment, or prophylaxis g 0 To evaluate rFVllch consumption (e.g., total ized rFVllch consumption per subject) WO 09627 Example 7 Clinical ROTEM® Assessment In the study in Example 7, in addition to the measurement of plasma FVIII activity by one-stage activated partial thromboplastin time (aPTT) assay, whole blood rotational thromboelastometry (ROTEM®) has also been explored to assess the improvement in global hemostasis by rFVIIIFc and ADVATE® in 2 subjects, cally, I in the low dose cohort and 1 in the high dose cohort. ] rFVIIIFc and ADVATE® appear to be ably active in clot formation when spiked into subj ects' blood prior to Fc treatment. The clotting time (CT) was linear with t to the dose of rFVIIIFc and ADVATE® in the range of approximately 1% of 100% of normal, and the dose response was comparable between rFVIIIFc and ADVATE® in the same subject. ing dosing with ADVATE® and subsequently Fc, citrated whole blood was sampled at various time points and the clot formation ing recalcification was monitored by ROTEM®. Despite the variable baseline CT due to residue FVIII levels prior to ADVATE® or rFVIIIFc dosing, both products effectively corrected the CT to comparable levels 30 minutes post-inj ection. In addition, the improvement in CT was better sustained at and after 3 hours post- injection of 25 IU/kg of rFVIIIFc relative to ADVATE® in the subject dosed at this low dose.

However, the differential improvement of rFVIIIFc versus ADVATE® was much less appreciable at the 65 IU/kg dose.

Example 8 HemA mice were used for tail clip s. The mice were first anesthetized and then injected with 4.6 [Lg/kg, 1.38 [Lg/kg, or 0.46 [Lg/kg of either processed rFVIIIFc (Drug Substance, which contain about 75%-85% processed Fc) and purified single chain rFVIIIFc. After the injection, the tail was cut from the tip and immediately placed into a tube to collect blood. tage of protection on survival was measured for rFVIIIFc processed (drug substance) and single chain FVIIIFc as shown in Table 7 and Figure 7.

TABLE 7. In Vivo Efficacy of rFVIII:Fc DS and Single chain rFVIIIFc Dose (pg/kg) 4.6 1.38 0.46 % of Protection FVIIIFc DS 93 52 19 on Survival Single chain 93 64 14 rFVIIIFc As shown in Table 7 and Figure 7, the protection on survival by single chain rFVIIIFc is comparable to processed rFVIIIFc (DS).

Clotting activity by in vitro ROTEM ] The clotting potency of rFVIIIFc was further explored in whole blood Rotational Thromboelastometry (ROTEM) over a range of concentrations, and compared to both rBDD FVIII (Xyntha) and recombinant full length FVIII II; Advate). For in Vitro ROTEM, rFVIII proteins were spiked in triplicate into citrated pooled blood collected from the vena cava of -6 male HemA mice to the final concentration of 0, 0.1, 1, 10, and 100% of normal plasma FVIII level. The clot was initiated by the addition of CaC12 (NATEM) and clotting time (CT), clot formation time (CFT), angle and maximum clot firmness (MCF) were recorded on the ROTEM system (Pentapharm GmbH, Munich, Germany). The clotting time (CT), clot formation time (CFT), and alpha angle for the three proteins spiked in HemA mouse blood at escalating doses from 0.1 — 100 % of normal FVIII levels are shown in Figure 14. In the wide range of 0.1 to 100% of normal, the CT and CFT are comparable among rFVIIIFc, rBDD FVIII and rflFVIII.

The alpha angle is only significantly different (p<0.05) between rFVIIIFc and rBDD FVIII at %. ng activity by ex vivo ROTEM The pharmacodynamics of rFVIIIFc, as measured by ROTEM, was ed to rBDD FVIII and rflFVIII after a single intravenous injection into Hemophilia A mice. For eX Vivo ROTEM, male HemA mice were injected intravenously with a single dose of 50 IU/kg Fc, ADVATE®, or XYNTHA®, and 5 mice sacrificed at each time point (5 minutes, 24, 48, 72 and 96 hours post ). Individual citrated whole blood collected from the vena cava was immediately analyzed by NATEM on the ROTEM system and parameters measured as above.

The CT, CFT, and alpha angle were ined for samples taken from 5 min to 96 hours after dosing, and shown in Figure 15. At 5 min, all are comparably effective resulting in similar CT, CFT and alpha angle (Figure 15A — C). However, rFVIIIFc demonstrated a significantly improved (p<0.05) CT at 72 and 96 hrs, CFT and alpha angle at 96 hrs (Figures 15A — C) relative to rBDD FVIII and rfiFVIII .

Example 9 ] Recombinant factor VIIIFc (rFVIIIFc) is comprised of a B domain deleted (BDD) rFVIII protein genetically fused to the Fc domain of human immunoglobulin G1 (IgGl). Prior to secretion from HEK 293 cells, most of the rFVIIIFc is sed into a FVIII heavy chain (HC) and light chain (LC+Fc). In circulation, rFVIIIFc is complexed with von Willebrand factor (VWF) and ed upon activation in a manner that is indistinguishable from native FVIII.

Spontaneous dissociation of the HC and LC is thought to contribute to the loss of FVIII activity in plasma and during storage of FVHI drug products. Here we describe a single chain non-processed isoform of rFVllch (SC rFVllch), which may provide superior manufacturability and enhanced stability compared to native FVlll.

] SC rFVllch was purified from rFVllch, which contains a fraction of the non-processed isoform. Compared to rFVllch, SC rFVllch showed equivalent chromogenic activity but approximately 60% reduced actiVity by the one stage (aPTT) assay, (Table 3A-B). Thrombin tion assay (TGA) was performed using calibrated ted thrombogram (from Thrombinoscope®). In a thrombin generation assay (TGA), SC rFVIHFc also showed a d thrombin potential (Figure 13A), and peak thrombin (Figure 13B) compared to rFVllch.

However, as shown in Table 3B, full actiVity of SC rFVllch by aPTT was observed in the absence of VWF, suggesting release from VWF may be delayed due to covalent linkage of the a3 acidic region to the HC after Arg 1680 cleavage in SC rFVllch, in contrast to a3 release and dissociation from fully processed FVHI. Delayed dissociation from vWF may explain the reduced activity ed in the aPTT assay and TGA, while full activity was observed in the two-stage chromogenic assay. A reduced rate of activation in the ce of VWF was ed in a modified chromogenic substrate assay with limiting thrombin as FVlll activator.

In vivo function of SC rFVllch was assessed in the HemA mouse tail vein transection (TVT) model. HemA male mice were treated with indicated doses of either rFVllch drug product or SC h 48 hours prior to TVT. Tail re-bleeding and survival were monitored hourly up to 12 hours post TVT with final observation performed at 24-hour post TVT. SC rFVllch and the rFVllch demonstrated equivalent in Vivo y in this model, with an ED50 of 1.17 [Lg/kg and 1.23 ug /kg respectively when TVT was performed at 48 hours post infusion (Figure 7(A)).

Comparable 24 hour post TVT survival curves (p Z 0.65) (Figure 7(B)) and ed rates (Figure 7(C)) in HemA mice were ed for the SC rFVllch and rFVllch at each tested dose level, indicating that SC rFVllch was equally effective as rFVllch despite its lower apparent aPTT ty. The delayed in Vitro activation of SC rFVllch in the presence of VWF therefore appears to have no significant impact on its in Vivo efficacy. Thus, SC h represents a novel and efficacious isoform of h with potential clinical applications. Further studies will be required to demonstrate enhanced t ity in the context of this Fc fusion protein.

Example 10 ] Current factor VIII (FVlll) products display a half-life (tug) of approximately 8-12 hours, requiring frequent intravenous injections for prophylaxis and treatment of hemophilia A patients. rFVllch is a recombinant fusion protein composed of a single molecule of FVlll covalently linked to the Fc domain of human IgG1 to extend circulating rFVlll half-life. This first-in-human _ 74 _ WO 09627 study in previously-treated male ts with severe hemophilia A investigated safety and pharmacokinetics of rFVllch. n subjects received a single dose of ADVATE® at 25 or 65 IU/kg followed by an equal dose of rFVllch. Most adverse events were unrelated to study drug. None of the study subjects developed anti-FVllch antibodies or inhibitors. Across dose levels, as compared with ADVATE®, rFVllch showed 1.54 to 1.71-fold longer elimination tm and mean nce time, 1.49 to 1.56-fold lower clearance, and 1.48 to 1.56-fold higher total systemic exposure. ADVATE® and rFVllch had comparable dose-dependent peak plasma concentrations and recoveries. Time to 1% FVIII activity above baseline was imately 1.53 to old longer than ADVATE® across dose levels. Thus, rFVllch may offer a viable therapeutic approach to achieve ged hemostatic protection and less frequent dosing in patients with hemophilia A.

Hemophilia A is an inherited bleeding disorder that results in frequent spontaneous and traumatic bleeding into the joints and soft tissues. Mannucci PM, Tuddenham EGD, N Engl J Med, 344:1773-1779 (2001). When inadequately treated, this bleeding leads to chronic arthropathy, disability, and increased risk of death. Soucie JM et al., Blood. 96(2):437-442 (2000).

Plasma-derived FVIII (deVIII) and recombinant human FVIII (rFVlll) products are utilized for treatment (on-demand therapy) and prevention (prophylaxis therapy) of ng episodes. rFVlll was developed to reduce the risk of blood-bome pathogen transmission following the widespread contamination of plasma products with HIV and hepatitis viruses, and to secure an adequate supply of FVIII product. However, hemostatic protection with current FVIII ts is temporally limited due to a short half-life (t1/2) of approximately 8-12 hours, requiring prophylactic injections three times per week or every other day for most patients in order to maintain FVIII levels above 1%, a level that has been established as protective against most spontaneous ng episodes. Manco-Johnson et al., New Engl JMed. 357(6):535-44 (2007).

Many s have shown that, even at high doses, on-demand therapy is not ive in preventing arthropathy. Aledort L. et (11., JIntern Med. 236:391-399 (1994); Petrini P. et (11., Am JPediatr Hematal 011601.132280-287 (1991). The benefits of prophylactic therapy have been demonstrated in numerous al studies 4’ 6'15 and Manco-Johnson et al., supra, established that children started on primary prophylaxis after their first joint bleed had significantly fewer bleeds and less joint damage than children d on-demand.

Compared to on-demand ent, prophylactic therapy also decreases disability, hospitalization rate, and time lost from school or work;6’16 and improves quality of life for patients and their families.17 However, prophylactic therapy often requires use of l venous access devices in children, and their attendant risks of infection, sepsis, and thrombosis. In addition, e the benefits, ance of and compliance with prophylaxis decreases with age, in part 2012/045784 because of inconvenience and invasiveness.18’19 Thus, a rFVIII product with a prolonged plasma t1/2 would potentially be of benefit. Lillicrap D., Current Opinion in Hematology 17:393-397 (2010). rFVIIIFc is a recombinant fusion protein composed of a single molecule of B-domain deleted rFVIII covalently linked to the human IgG1 Fc domain. Potential advantages of Fc-fusion proteins include better tolerability and prolonged hemostatic protection, and the Fc domain represents a natural le with no known inherent ty.21’22 ment to the IgG1 Fc domain permits g to the neonatal Fc receptor (FcRn), which is expressed in many cell types, including elial cells. FcRn expression remains stable throughout life and is responsible for protecting IgG1 and Fc-fusion proteins from lysosomal degradation, thus ging the t1/2 of the protein.21’23 Numerous proteins within the circulation are internalized into the cells lining the vasculature via nonspecific pinocytosis and are trafficked to mal and lysosomal degradation pathways.

Fc proteins interact with FcRn, resident within endosomes. Endosomes containing FcRn direct the Fc fusion proteins back to the plasma membrane, releasing them into circulation in a pH-dependent manner,24 thereby avoiding lysosomal degradation. This recycling approach has been used successfully to extend the tug of therapeutic biologics; a number of PC fusion-based drugs have been approved for clinical use (eg etanercept, romiplostim) and others are in development.25’26 Preclinical data for rFVIIIFc indicate that FVIII can be rescued from degradation by a natural tive pathway mediated by FcRn, thus ing t1/2. In Hemophilia A mice and dogs, terminal plasma t1/2 for rFVIIIFc was approximately 2 times longer than with rFVIII.27’28 Based on these data, we conducted a first-in-human clinical study to investigate the safety and PK of a long-lasting rFVIIIFc fusion n in subjects with hemophilia A.

Study Design: This open-label, dose-escalation, multicenter Phase l/2a study in previously treated patients with severe hemophilia A investigated the safety of rFVIIIFc and its pharmacokinetics (PK) compared with ADVATE® (antihemophilic factor [recombinant], plasma/albumin-free method, octocog alfa, Baxter care). This study was med in accordance with the US CFR and ICH Guidelines on Good Clinical Practices. Prior to any testing, approval from ipating Institutional Review Boards and written informed consents from all subjects were obtained. The study design was sequential; a single dose of ADVATE® was stered at 25 or 65 IU/kg followed by an equal dose of rFVIIIFc (. Both drugs were injected intravenously over approximately 10 minutes. The two dose levels were expected to bracket the typical therapeutic dose . Subjects were followed for 28 days after receiving rFVIIIFc for safety analyses, including testing for anti-FVIII antibodies and tors at 14 and 28 days post-inj ection. Plasma FVIII activity was ed in subjects before injection, 10 and 30 s, 1, 3, 6, 9, 24, 48, 72, 96, 120, and 168 hours (7 days) after rFVllch injection, with additional samples at 192, 216, and 240 hours (10 days) for subjects dosed at 65 IU/kg of rFVllch. Plasma FVIII ty was measured at the same time points after ADVATE® ent, through 72 hours for the 25 lU/kg group and 96 hours for the 65 lU/kg group.

Subjects: Male subjects were at least 12 years of age with severe hemophilia A (defined as FVIII activity level < 1%) and had at least 100 documented prior exposure days to FVIII concentrates (deVlll or ). Subjects with known hypersensitivity to mouse or hamster n, history of tor or detectable inhibitor titer at screening, or who were taking any medications that could affect hemostasis or ic immunosuppressive drugs, or who experienced an active bacterial or Viral infection (other than hepatitis or HIV) within 30 days of screening were excluded. Subj ect’s genotype was recorded at study entry, when known.

Treatment Product: The human rFVllch and Fc transgenes were stably transfected into HEK293 cells and the cell line was extensively tested for stability, sterility, and Viral contamination to ensure safety. The purified drug t is composed of a monomeric B- domain-deleted FVIII covalently linked through its carboxy-terminus to the N—terminus of an Fc monomer, which forms a disulfide bond with a second Fc monomer during synthesis and secretion from the cells. rFVllch was purified by tography and nanoflltration, and was fully active in one-stage and chromogenic ng assays relative to commercially available rFVlll preparations. It was supplied as a frozen liquid ning 1000 IU per 2 mL of solution and formulated with L-histidine (pH 7), sodium chloride, calcium chloride, sucrose, mannitol, and Polysorbate 20. For injection, the product was d with saline solution (0.9% NaCl).

Outcome Measures: The y objective of the study was safety, ted through physical examination, ing of treatment-emergent adverse events (AEs), development of antibodies, and laboratory monitoring over time. The secondary objectives included parameters derived from PK analyses. Laboratory assessments ed prothrombin time, activated partial oplastin time (aPTT), international normalized ratio, levels of D-dimer, von Willebrand factor (VWF) antigen, standard hematology and blood chemistry tests, and urinalysis.

FVIII actiVity was measured by the one-stage clotting (aPTT) assay on a Siemens BCS-XP analyzer using commercial reagents (Dade Actin FSL) with calibration against a normal reference plasma (Precision Biologics CRYOcheckT'V') traceable to the World Health Organization (WHO) 5th lntemational Standard (1S) for human plasma. In addition to the aPTT assay, FVIII activity was measured by a chromogenic substrate assay29 using a commercially available kit (Aniara BIOPHEN FVIII:C) that complies with European Pharmacopoeia recommendations. The chromogenic assay was calibrated t normal human reference plasma (Instrumentation Laboratories ORKE45), which also had a potency assigned against the WHO 5th 1S human plasma standard.

] The lower limit of fication (LLOQ) for the one-stage and chromogenic assays was 0.5 IU/dL and 0.4 IU/dL, respectively. FVIII inhibitors were measured by the - gen-modified Bethesda assay and less than 0.6 BU/mL was considered negative. Anti- rFVIIIFc antibodies were assessed using a specific bridging electrochemiluminescent immunoassay which uses biotin and sulfo-tagged rFVIIIFc. Assay sensitivity was determined to be 89 ng/mL using an anti-human FVIII monoclonal antibody as a surrogate control. Exploratory whole blood rotation thromboelastometry (ROTEM®) was performed in two ts, one from each dose level, at various time points to assess the improvement in global hemostasis following injection with ADVATE® and rFVIIIFc.

Pharmacokinetic es: A user-defined one-compartment disposition model, which automatically estimates the endogenous FVIII level and subsequent residual decay, was ed in WinNonLin for analysis of the dual subject plasma FVIII activity-versus-time data following a single administration of ® or rFVIIIFc. Actual sampling times, doses, and duration of ion were used for calculations of parameters including maximum activity (Cmax), t1/2, clearance (CL), volume of distribution at steady-state (VSS), area under the curve (time zero extrapolated to infinity [AUCINF]), mean residence time (MRT), and incremental recovery.

Monte Carlo Simulation of rFVIIIFc Activity-Versus-Time Profile - To construct FVIII activity-time profiles following dosing ns of 25 IU/kg or 65 IU/kg, a Monte Carlo tion was ted using the population PK model of ADVATE® and rFVIIIFc. The mean estimates of model parameters (CL, volume of distribution) in the tested population, the inter- individual variance, and the residual variability were estimated based on the one-stage (aPTT) clotting assay activity data of ADVATE® and rFVIIIFc from 16 subjects in this Phasel/2a study.

Five hundred ts were ted with 15 sampling points for each subject for each dosing regimen. The percentage of the population with FVIII activity above or equal to 1% and 3% at ent times following different dosing regimens ofADVATE® or rFVIIIFc was estimated.

Statistical Analyses - Selected PK parameters for rFVIIIFc and ADVATE® were compared using an analysis of variance model. PK parameters were log-transformed for these analyses and estimated means, mean differences, and confidence intervals on the log-scale were transformed to obtain tes for geometric means, geometric mean ratios (GMR), and confidence intervals, respectively, on the original scale. The GMR is the geometric mean of the intra-subj ect ratio of the rFVIHFc PK parameter value to the ADVATE® PK parameter value.

Results Subject Disposition- Nineteen subjects were enrolled in the study; 16 underwent PK evaluation for both ADVATE® and rFVHIFc. One subject dministered his previous product prior to completing the wash-out period following the dose with ADVATE® and was thus excluded from the PK analysis, but was included in the safety analysis. Three subjects were discontinued from the study before receiving either study drug: one voluntarily withdrew; a second was withdrawn by the Investigator for non-compliance; and one was awn at the Sponsor’s request due to completion of study enrollment. Of the ts dosed, six subjects ed 25 IU/kg and 10 ts received 65 IU/kg of both ADVATE® and rFVIIIFc. Mean age was 40.3 years (23 to 61 years). Genotypic identification was collected for seven subjects; inversion of intron 22 was reported in six subjects; and a frame-shift defect was reported in one subject. The genotype was unknown for nine subjects. Thirteen subjects had hepatitis C dies, four of whom were also positive for HIV.

Safety - Forty-four treatment-emergent AEs were reported by 11 (69%) subjects during the treatment and follow-up periods. This included the day of dosing with Advate or rFVIIIFc through a 28-day post-dosing observation period. The majority of events were considered mild and none led to withdrawal from the study. One event, dysgeusia, occurred ently in one t while receiving a 65 IU/kg dose of rFVIIIFc and was considered related to rFVIIIFc.

One subject experienced an anxiety attack after receiving 65 IU/kg of rFVIIIFc which resulted in 21 AEs, 19 of which were graded as mild, and two of which (headache and photophobia) were rated as moderate. Neither was deemed related to rFVIIIFc by the Investigator.

] No serious ng episodes were reported. No evidence of allergic reactions to injection was detected. All plasma samples tested negative for FVIII inhibitors and anti-rFVIIIFc antibodies. No signs of injection site reactions were observed. No clinically meaningful changes in al laboratory values were reported.

Pharmacokinetics: Correlation Between aPTT and Chromogenic Activity for rFVIIIFc in Plasma - ADVATE® and rFVIIIFc activities were determined in the same assays using commercially available reagents and calibration against normal human plasma standards. There was a strong correlation between the results obtained by the one-stage ng assay and the chromogenic assay in samples that had an activity above the LLOQ. Correlation coefficients (Pearson R2) of 0.94 and 0.95 were observed between the two assay results for 151 samples following ADVATE® dosing and 185 samples following rFVIIIFc dosing, respectively.

Compared to the aPTT results, the chromogenic FVIII activities were, on average, 21% higher for ADVATE® and 32% higher for rFVIIIFc, not statistically cant (Figure 9). This observation led to a slightly higher estimation of exposure parameters by the genic ment for both drugs. The apparent higher FVIII recoveries by the genic assay are typical for recombinant FVIII ts tested in clinical assays, and are in agreement with most other marketed FVIII products.30'32 Improved cokinetics for rFVIIIFc - The primary PK estimates were derived from one-stage (aPTT) clotting assay activity data. In subjects who received 25 or 65 IU/kg of ADVATE® followed by an equal dose of rFVIIIFc, the plasma FVIII activity rose sharply and reached Cmax within the first hour following . The subsequent decline of the observed FVIII activity ted monoexponential decay characteristics until the baseline FVIII actiVity was d (Figure 10). The Cmax increased proportionally to the dose, but was comparable between equal doses of ADVATE® and rFVHIFc (Table 8) The total exposure (AUCINF) also increased proportionally to the dose. However, the AUCINF of rFVHIFc was 1.48 and 1.56-fold greater than that ofADVATE® at 25 IU/kg (p=0.002) and 65 IU/kg (p<0.001), respectively (Table 8).

Table 8. PK Parameters by One-Stage (aPTT) Assay for rFVIIIFc and ADVATE® Per Dose Group Parameter Dose: 25 IU/kg (N=6) Dose: 65 IU/kg (N=9) ADVATE® rFVIIIFc Geom. Mean ADVATE® Fc Geom. Mean Geom. Geom. Ratio Geom. Mean Geom. Mean Ratio hkm1 hkm1 95%Cm(6 95%CH 95%CH 95%Cm(6 [95% C1] [95% C1] value) value) C;ML}DB (1952 (1895 S 636 605 [0819,r11] 133 119 [0795,r01] [591,683] [53.1,691H m=0mm [105,168] [103,136] m=00m) AUCINF 1.48 r56 (hr*IIJ/d14 994 [r26,r76] 1800 2800 83] ) [723,1370] 09= [1350,2400] [1980,3970] 0002) t1/2 (hr) 122 188 188 [r40,r69] [r54,r89] [914,163] [148,238] [876,139] [143,245] @<ODM) @<ODM) r54 r71 175 270 158 270 MRT (hr) [r40,r69] [r54,r89] [131,234] [213,342] [126,199] [206,353] @<ODM) @<ODM) 0673 0642 249 r68 [0569 361 232 [0541 (mL/hour/ [180,345] [131,215] 0796] [271,483] [r64,329] 0753] (p==(1002) @<ODM) 43.9 1.04 r09 Vss 454 574 628 [393, [0.947,].13] Ul976,L22] (mL/kg) [393,525] [483,683] [552,7r5] 490] o = 0.357 .=(1107 Increment Recovery 0952 0894 256 244 204 r83 GlUdL [236,278] [212,281] [r61,259] [159,210] (p==(x444) (p=00a» ILng) C1= Confidence Interval; Geom. Mean = ric Mean; OBS = observed. Estimated means, 95% CI for means, and mean ences were ormed to obtain estimated geometric means, 95% CI for geometric means, and ric mean ratios, respectively.

The tl/Z, MRT, CL, and VSS ed to be independent of dose (Table 8). The geometric mean t1/2 of rFVIIIFc was 18.8 hours for both the 25 IU/kg and 65 IU/kg dose groups. This represents a 1.54 and 1.70-fold improvement over that of ADVATE® (12.2 hours and 11.0 hours) at equivalent doses (p<0.001), respectively (Table 8). The same intra-subject improvement was observed in the MRT of rFVIIIFc (27.0 hours for both dose groups) ed with ADVATE® (17.5 hours for the 25 IU/kg and 15.8 hours for the 65 IU/kg) (p<0.001). Consistent with improvement in the tm and MRT was a corresponding 1.49 and 1.56-fold reduction in intra- subject CL at doses of 25 IU/kg (p=0.002) and 65 IU/kg (p<0.001), respectively. There were no _ 81 _ 2012/045784 significant differences in VSS and incremental recovery between ADVATE® and rFVIIIFc.

Therefore, within each subject, Fc trated an improved PK profile compared with ADVATE®. It was also observed that the patients with shorter half-life on ADVATE® had shorter half-life on rFVIIIFc, and patients with longer ife on ADVATE® had longer half-life on rFVIIIFc.

The improved PK profile of rFVIIIFc resulted in increased time post-dosing to 1% FVIII activity which was 1.53 and 1.68-fold longer respectively, than with ADVATE® at 25 IU/kg (p<0.001) and 65 IU/kg (p<0.001) (data not shown), suggesting a potentially longer therapeutic duration for rFVIIIFc.

The favorable PK profile of rFVIIIFc relative to ADVATE® was also demonstrated by FVIII activity measured in the chromogenic assay (Table 9), which was comparable to data derived from aPTT assays. The estimation of re, ie, Cmax and , was slightly higher, however, based on the chromogenic assay than on the one-stage (aPTT) clotting assay for both ADVATE® and rFVIIIFc.

WO 09627 Table 9. PK Parameters by Two-Stage (Chromogenic) Assay for rFVIIIFc and ® Per Dose Group Parameter Dose: 25 IU/kg (N=6) Dose: 65 IU/kg (N=9) ADVATE® rFVIIIFc Geom. Mean ADVATE® rFVIIIFc Geom. Mean Geom. Geom. Mean Ratio Geom. Geom. Ratio Mean [95% CI] [95% CI] (p- Mean Mean [95% CI] (p- [95% CI] value) [95% CI] [95% CI] value) cm_OBs 1.01 1.04 (IU/dL) 75.5 76.5 [0.940, 1.09] 175 182 [0.900, 1.20] [65.5, 87.1] [64.9, 90.1] (p = 0.686) [143,215] [146, 227] (p = 0.571) AUCINF 1.57 2270 4280 1.89 1060 1660 (hr*IU/dL [1.38, 1.80] [1670, [2960, [1.61, 2.21] ) [822, 1360] [1300, 2120] (p < 0.001) 3070] 6190] (p < 0.001) 1.59 1.84 (hr) 10.5 16.7 10.8 19.8 t1/2 [1.35, 1.87] [1.60, 2.12] [8.49, 12.9] [13.8, 20.1] [8.16, 14.2] [14.3, 27.5] (p < 0.001) (p < 0.001) 1.59 1.85 .0 23.9 15.4 28.5 MRT (hr) [1.35, 1.87] [1.61, 2.12] [12.2, 18.6] [19.8, 28.9] [11.7, 20.4] [20.5, 39.6] (p < 0.001) (p< 0.001) 0.636 0.530 2.35 1.49 [0.557, 2.87 1.52 , (mL/hour/ [1.80, 3.06] [1.16,1.92] 0.727] [2.12, 3.89] [1.05, 2.20] 0.620] . < 0.001 . < 0.001 1.01 0.975 .5 35.9 44.5 43.4 [0.898, 1.14] [0.863,1.10] [30.5, 41.3] [30.4, 42.3] [36.7, 54.1] [38.2, 49.4] . = 0.822 . = 0.653 Increment al 1.01 1.04 Recovery 3.05 3.09 2.70 2.80 [0.940, 1.09] [09001.20] [2.62, 3.54] [2.61, 3.66] [220,331] [2.24, 3.50] (IU/dL per (p = 0.679) (p = 0.571) IU/kg) CI = Confidence Interval; Geom. Mean = Geometric Mean; OBS = observed. Estimated means, 95% CI for means, and mean differences were transformed to obtain estimated geometric means, 95% CI for geometric means, and geometric mean , respectively.

Correlation n von Willebrand Factor and Disposition of rFVIIIFc - Because the majority of FVIII in circulation is in complex with VWF33 and because the genome-wide association study has identified that the genetic determinants of FVIII levels are primarily dependent on VWF levels,34 we examined the association n VWF and rFVIIIFc. A strong correlation was observed between VWF levels and CL and t1/2 for both Fc and ADVATE®.

As shown in Figure 10, as the level of VWF increased, the CL of rFVIIIFc (p=0.0016) and of ADVATE® (p=0.0012) decreased.

The opposite relationship was observed between the level ofVWF and t1/2. As the level of VWF increased, the t1/2 of rFVIIIFc (p=0.0003) and of ADVATE® (p<0.0001) increased. This W0 09627 correlation suggests that the Fc moiety of rFVIIIFc does not alter the role of VWF in protecting FVHI from clearance.

Effects of Prolonged PK of rFVIIIFc on Whole Blood ROTEM® - Prior to administration of study drug, blood from one subject in each dose group was spiked with an equal dose of rFVIIIFc or ADVATE® and analyzed by whole blood ROTEM®. Clotting time (CT) was linear with respect to the dose in the range of approximately 1% to 100% of normal, and the dose response was comparable between rFVIIIFc and ® in the same subject (data not shown), indicating comparable potency of rFVIIIFc and ADVATE® in clot formation.

Despite the variable baseline CT due to residual FVIII levels prior to the administration of ADVATE® or rFVIIIFc, both products effectively corrected the CT to comparable levels 30 minutes osing (Figure 12). The improvement in CT was better sustained by rFVIIIFc than ADVATE® after 3 hours following a dose of 25 IU/kg (Figure 12A), and after 24 hours following a dose of 65 IU/kg (Figure 12B). rFVIIIFc was well tolerated by subjects at both doses. There were no clinically significant changes observed in hematology, blood chemistry, or urinalysis parameters. The majority of AEs were mild, unrelated to rFVIIIFc, and resolved without sequelae. No serious AEs or deaths ed during the study, and no subjects at either dose developed neutralizing or binding antibodies to rFVIIIFc. rFVIIIFc demonstrated a cantly improved FVIH actiVity PK profile ve to ADVATE®, with tm and MRT across dose levels being 1.54 to 1.71-fold , as measured by the one-stage (aPTT) clotting assay and 1.59 to 1.84-fold longer by the two-stage chromogenic assay. The prolonged actiVity of rFVIIIFc predicts possible prolonged efficacy, allowing for a less frequent dosing regimen in the lactic treatment of patients with Hemophilia A.

Adopting the PK parameters d from this study, the Monte Carlo simulation predicts that a higher percentage of patients receiVing rFVIIIFc will n FVHI levels above 1% or 3% as compared with patients receiVing equal doses ofADVATE® (Table 10). For e, at a dose of 25 IU/kg, 12.2% of ADVATE® patients versus 71.2% of rFVIIIFc patients are predicted to have FVHI trough levels above 1% on Day 4; at a dose of 65 IU/kg, 11.0% TE® patients versus 66.4% of rFVIIIFc patients are predicted to have FVHI levels above 3% on Day 4. Clinical trials in larger s of patients are planned to confirm results from this Phase 1/2a study and from the Monte Carlo simulation predictions.

Table 10. Predicted Percentage of Subjects Achieving FVIII Trough Levels Above 1% and 3% ofNormal at a Specified Dose Regimen ofADVATE® or rFVllch Timepoint following ADVATE® h dosing(DaY) IU/k_ 65 IU/k_ 25 IU/k_ 65 lU/k_ t of Sub'ects with FVIII Trouh Levels above 1% Percent of Subjects with FV .6 34.6 11.0 ] Despite the success of PC fusion technology in prolonging circulating t1/2 for a variety of protein therapeutics, rFVIH was considered too large to successfully produce dimeric Fc fusions.

We thus created a monomeric Fc fusion protein whereby a single effector molecule was covalent linked to a dimeric Fc, ng binding to intracellular FcRn and subsequent recycling.2122’ In vitro coagulation assays demonstrate no loss of specific activity for rFVHIFc, compared to B- domain deleted or native FVIII, by either clotting or chromogenic assays, using commercially available reagents and commonly used FVIII reference rds (JAD, TL, SCL, et al., manuscript ted , 2011). In addition, these results indicate that rFVllch can be reliably assayed in a clinic setting by either the one-stage assay or the chromogenic method.

In summary, this Phase 1/2a clinical trial is the first trial to demonstrate the safety and prolonged t1/2 of rFVllch in patients with severe hemophilia A. A pivotal Phase 3 study is ongoing with h to establish effective prophylaxis dosing regimens for individuals with hemophilia A. e 11 A novel -chain (SC) isoform of factor VIII (FVIH), resulting from incomplete proteolysis at residue R1648 during biosynthesis, may provide superior manufacturability and stability ve to native FVlll. A single recombinant B domain deleted factor VIII molecule fused to an immunoglobulin Fc domain (rFVllch) and its purified SC counterpart (SC-rFVllch) exhibited similar specific activity in one stage ng assays using plasma depleted of von Willebrand factor (VWF), but SC-rFVIHFc exhibited lower specific activity in the presence of VWF. This study was undertaken to determine if VWF-bound rFVHIFc, SC-rFVIHFc and rBDD- FVIII (XYNTHA®, O AF®) differ with respect to thrombin-mediated proteolytic release from VWF.

Equimolar amounts of rFVIIIFc, SC-rFVIIIFc, and rBDD-FVHI were captured on an optical biosensor chip on which human VWF had been immobilized by amine coupling. Human (x-thrombin at a range of trations was d over the chip surface, and the rates of FVIII release from immobilized VWF were monitored in real time. The half maximal ive concentration (ECSO) of (x-thrombin was determined for each FVIII species. (x-thrombin ECSO values for rFVHIFc and rBDD-FVIH were comparable (3.7 :: 0.2 U/mL and 3.2 :: 0.3 U/mL, respectively), whereas the ECSO value for SC-rFVIHFc was greater than 3- fold higher (11.7 :: 0.9 U/mL). This finding that SC-rFVIIIFc is released more slowly from VWF than are either rFVHIFc or rBDD-FVIH is consistent with a previously observed finding regarding the actiVities of rFVIIIFc and SC-rFVIIIFc in a age clotting assay (aPTT) in which SC-FVIIIFc had a lower apparent actiVity only when VWF was present in the assay plasma sample. However, all samples possessed equivalent activities in a mouse ng model, indicating that responsiveness of FVIII preparations to in in the release of FVIII from VWF does not correlate with efficacy in vivo.

TABLE 1: Polynucleotide Sequences A. B-Domain Deleted FVIIIFc 1i) B-Domain Deleted FVllch Chain DNA Sequence (FVIII signal peptide underlined, Fc region in bold) (SEQ ID NO:1, which encodes SEQ ID NO:2) 661 A TGCAAATAGA GCTCTCCACC TGCTTCTTTC 72“ TGTGCCTTTT GCGATTCTGC TTTAGTGCCA CCAGAAGATA CTACCTGGGT GCAGTGGAAC 78“ TGTCATGGGA CTATATGCAA AGTGATCTCG TGCC TGTGGACGCA AGATTTCCTC 84“ CTAGAGTGCC TTTT AACA CCTCAGTCGT GTACAAAAAG ACTCTGTTTG 90“ TAGAATTCAC GGATCACCTT TTCAACATCG CTAAGCCAAG GCCACCCTGG ATGGGTCTGC 96“ TAGGTCCTAC CATCCAGGCT GAGGTTTATG ATACAGTGGT CATTACACTT ATGG “02“ CTTCCCATCC TGTCAGTCTT CATGCTGTTG GTGTATCCTA CTGGAAAGCT GGAG “O8“ ATGA TGATCAGACC AGTCAAAGGG AAGA TGATAAAGTC TTCCCTGGTG “l4“ GAAGCCATAC ATATGTCTGG CAGGTCCTGA AAGAGAATGG TCCAATGGCC TCTGACCCAC “20“ TGTGCCTTAC CTACTCATAT CTTTCTCATG TGGACCTGGT AAAAGACTTG AATTCAGGCC “26“ TCATTGGAGC CCTACTAGTA TGTAGAGAAG GGAGTCTGGC CAAGGAAAAG ACACAGACCT “32“ TGCACAAATT TATACTACTT TTTGCTGTAT AAGG GAAAAGTTGG CACTCAGAAA “38“ CAAAGAACTC CTTGATGCAG GATAGGGATG CTGCATCTGC TCGGGCCTGG CCTAAAATGC “44“ ACACAGTCAA TGGTTATGTA AACAGGTCTC TGCCAGGTCT GATTGGATGC CACAGGAAAT “50“ CAGTCTATTG GCATGTGATT GGAATGGGCA CCACTCCTGA AGTGCACTCA CTCG “56“ AAGGTCACAC ATTTCTTGTG AGGAACCATC GCCAGGCGTC CTTGGAAATC TCGCCAATAA “62“ CTTTCCTTAC TGCTCAAACA CTCTTGATGG ACCTTGGACA ACTG TTTTGTCATA “68“ TCTCTTCCCA TGAT GGCATGGAAG CTTATGTCAA AGTAGACAGC TGTCCAGAGG “74“ AACCCCAACT GAAA AATAATGAAG AAGCGGAAGA CTATGATGAT ACTG “80“ ATTCTGAAAT GGATGTGGTC AGGTTTGATG ATGACAACTC TCCTTCCTTT ATTC “86“ GCTCAGTTGC CAAGAAGCAT CCTAAAACTT GGGTACATTA CATTGCTGCT GAAGAGGAGG “92“ ACTGGGACTA TGCTCCCTTA GTCCTCGCCC CCGATGACAG TAAA AGTCAATATT 98. TGAACAATGG GCGG ATTGGTAGGA AGTACAAAAA AGTCCGATTT ATGGCATACA 204.. CAGATGAAAC CTTTAAGACT CGTGAAGCTA ATGA ATCAGGAATC CCTT 210“ TACTTTATGG GGAAGTTGGA GACACACTGT TGATTATATT TAAGAATCAA GCAAGCAGAC 216“ CATATAACAT CTACCCTCAC GGAATCACTG ATGTCCGTCC TTTGTATTCA AGGAGATTAC 222“ CAAAAGGTGT AAAACATTTG AAGGATTTTC CAATTCTGCC AATA TTCAAATATA 228“ AATGGACAGT GACTGTAGAA GATGGGCCAA CTAAATCAGA TCCTCGGTGC CTGACCCGCT 234“ ATTACTCTAG TTTCGTTAAT ATGGAGAGAG ATCTAGCTTC AGGACTCATT GGCCCTCTCC 240“ TCATCTGCTA CAAAGAATCT GTAGATCAAA GAGGAAACCA GATAATGTCA GACAAGAGGA 246“ TCCT TGTA TTTGATGAGA ACCGAAGCTG GTACCTCACA GAGAATATAC 252“ AACGCTTTCT CCCCAATCCA GCTGGAGTGC AGCTTGAGGA TCCAGAGTTC CAAGCCTCCA 258“ ACATCATGCA CAGCATCAAT GGCTATGTTT TTGATAGTTT GCAGTTGTCA GTTTGTTTGC 264“ ATGAGGTGGC ATACTGGTAC ATTCTAAGCA TTGGAGCACA GACTGACTTC CTTTCTGTCT 270“ TCTTCTCTGG ATATACCTTC AAACACAAAA TGGTCTATGA AGACACACTC ACCCTATTCC 276“ CATTCTCAGG TGTC TTCATGTCGA TGGAAAACCC AGGTCTATGG ATTCTGGGGT 282“ GCCACAACTC AGACTTTCGG AACAGAGGCA TGACCGCCTT GGTT TCTAGTTGTG 288“ ACAAGAACAC TGGTGATTAT TACGAGGACA GTTATGAAGA TATTTCAGCA TACTTGCTGA 294“ GTAAAAACAA TGCCATTGAA CCAAGAAGCT TCTCTCAAAA CCCACCAGTC TTGAAACGCC 300“ ATCAACGGGA AATAACTCGT ACTACTCTTC AGTCAGATCA AGAGGAAATT GACTATGATG 306“ ATACCATATC AGTTGAAATG AAGAAGGAAG ATTTTGACAT TTATGATGAG GATGAAAATC 312“ AGAGCCCCCG CAGCTTTCAA AAGAAAACAC GACACTATTT TATTGCTGCA GTGGAGAGGC 318“ ATTA TGGGATGAGT CCAC ATGTTCTAAG AAACAGGGCT CAGAGTGGCA 324“ CTCA GTTCAAGAAA GTTGTTTTCC AGGAATTTAC TGATGGCTCC TTTACTCAGC 330“ CCTTATACCG TGGAGAACTA AATGAACATT TGGGACTCCT GGGGCCATAT GCAG 336“ AAGTTGAAGA TAATATCATG GTAACTTTCA GAAATCAGGC CTCTCGTCCC TATTCCTTCT 342“ ATTCTAGCCT TATTTCTTAT GAGGAAGATC AGAGGCAAGG ACCT AACT 348“ TTGTCAAGCC TAATGAAACC AAAACTTACT TTTGGAAAGT GCAACATCAT ATGGCACCCA 354“ CTAAAGATGA GTTTGACTGC AAAGCCTGGG CTTATTTCTC TGATGTTGAC CTGGAAAAAG 360“ ATGTGCACTC AGGCCTGATT GGACCCCTTC TGGTCTGCCA CACA CTGAACCCTG 366“ CTCATGGGAG ACAAGTGACA GTACAGGAAT TTGCTCTGTT TTTCACCATC r? T F? GA“?GAGA 372“ CCAAAAGCTG GTACTTCACT GAAAATATGG AAAGAAACTG CAGGGCTCCC TGCAATATCC 378“ AGATGGAAGA TCCCACTTTT AAAGAGAATT ATCGCTTCCA TGCAATCAAT GGCTACATAA 384“ rTGGAFTACAC’? ACCTGGCTTA GTAATGGCTC AGGATCAAAG GATTCGATGG TATCTGCTCA 390“ GCATGGGCAG CAATGAAAAC ATCCATTCTA TTCATTTCAG TGGACATGTG rTFTCACFTGFTAC 396“ GAAAAAAAGA AFTAAA CTGT ACAATCTCTA TCCAGGTGTT ACAG l\ 02“ TGGAAATGTT ACCATCCAAA GCTGGAATTT GGCGGGTGGA ATGCCTTATT GGCGAGCATC l\l\l\l\l\l\l\l\l\l\l\l\l\l\l\ 08“ TACATGCTGG GATGAGCACA CTTTTTCTGG TGTACAGCAA TCAG ACTCCCCTGG 14“ GAATGGCTTC TGGACACATT AGAGATTTTC AGATTACAGC TTCAGGACAA TATGGACAGT “ GGGCCCCAAA GCTGGCCAGA CTTCATTATT CCGGATCAAT CTGG AAGG 26“ TTTC TTGGATCAAG GTGGATCTGT TGGCACCAAT GAFTFTAFTFTCAC GGCATCAAGA 32“ CCCAGGGTGC CCGTCAGAAG TTCTCCAGCC TCTACATCTC rTCAGTFTFTAFTC AFTCAFTGTAFTA 38“ GTCTTGATGG GAAGAAGTGG TFTAFTC GAGGAAATTC CACTGGAACC TTAATGGTCT 44“ TCTTTGGCAA TGTGGATTCA GGAFTAA ATAT TCTTAACCCT CCAAFTFTAFTFTG 50“ CTCGATACAT CCGTTTGCAC CCAAC’?CA’?’T ATAGCATTCG CAGCACTCTT CGCATGGAGT 56“ GCTG ’? GA“? ’? rTAAA’? AGTTGCAGCA TGCCATTGGG AATGGAGAGT AAAGCAATAT 62“ CAGATGCACA TGCT TCATCCTACT rTFTACCAAFTA’? CACC TGGTCTCCTT 68“ CAAAAGCTCG ACTTCACCTC CAAGGGAGGA GTAATGCCTG GAGACCTCAG GFTGAAFTAAFTC 74“ CAAAAGAGTG GCTGCAAGTG GACTTCCAGA AGACAATGAA AGTCACAGGA GFTAACFTACFTC 80“ AGGGAGTAAA ATCTCTGCTT ACCAGCATGT ATGTGAAGGA CATC TCCAGCAGTC 86“ GCCA TCAGTGGACT CTCTTTTTTC AGAATGGCAA AGFTAAAGGFTT TTTCAGGGAA 92“ GACFTC CTTCACACCT AACT CTCTAGACCC ACCGTTACTG ACTCGCTACC l\ 98“ TTCGAATTCA CCCCCAGAGT TGGGTGCACC AGATTGCCCT GAGGATGGAG GTTCTGGGCT 504“ GCGAGGCACA GGACCTCTAC GACAAAACTC ACACATGCCC ACCGTGCCCA GCTCCAGAAC 5101 TCCTGGGCGG AGTC TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT 5161 CCCGGACCCC TGAGGTCACA TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA 5221 AGTTCAACTG GTACGTGGAC GGCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG 5281 AGCAGTACAA CAGCACGTAC CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC 5341 TGAATGGCAA GGAGTACAAG TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA 5401 AAACCATCTC CAAAGCCAAA GGGCAGCCCC CACA GGTGTACACC CTGCCCCCAT 5461 CCCGGGATGA GCTGACCAAG AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC 5521 CCAGCGACAT CGCCGTGGAG TGGGAGAGCA AGCC CAAC TACAAGACCA 5581 CGCCTCCCGT GTTGGACTCC GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTGGACA 5641 AGAGCAGGTG GCAGCAGGGG AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA 5701 ACCACTACAC GCAGAAGAGC CTCTCCCTGT CTCCGGGTAA A (ii) Fc DNA sequence (mouse IgK signal peptide underlined) (SEQ ID NO:3, Which encodes SEQ ID NO:4[ 798" A'1GGA GACAGACACA 804" C CCiGCiA GGGLAC GC GC C GGG i CCAGG CTGGTGACAA AACTCACACA 810" TGCCCACCG" GCCCAGCACC TGAACTCC"G CCGL CAGiC CC Ci CCCCCCA 816" AAGG ACACCC CA GA C CCCGG ACCCC CACA GCG GG GG GGAC 822" G"GAGCCACG CiGA GG CAAGi C AAC GG GGACGGCG GGAGG GCAL 828" AATGCCAAGA CAAAGCCGCG GGAGGAGCAG TACAACAGCA CGiACCGiG GG CAGCG C 834" C CACCG CC GCACCAGGA C"GGCTGAAT GAGT GCAA GG"CTCCAAC 840" AAAGCCC"CC CAGCCCCCA" CGAGAAAACC ATC"CCAAAG CCAAAGGGCA GCCCCGAGAA 846" CCACAGG"GT ACACCCTGCC CCCATCCCGC GATGAGC"GA CCAAGAACCA GG CAGCC G 852" ACC GCC GG CAAAGGCL C AiCCCAGC GACA CGCCG LGGAG GGGA GAGCAA"GGG 858" CAGCCGGAGA ACAACTACAA GACCACGCCL CCCG Gi GG ACLCCGACGG C CC iC C 864" CTC"ACAGCA AGCTCACCG" GGACAAGAGC AGGTGGCAGC AGGGGAACG i C C CA GC 870" iCCGiGA GC ALGAGGCLC GCACAACCAC TACACGCAGA AGAGCCiCiC CC G C CCG 876" GGTAAA B. Full Length FVIIIFc 1i) Full Leng’gh FVIIIFC DNA ce (FVIII signal peptide underlined, Fc region in bold) (SEQ ID I\O:5, Which encodes SEQ ID NO:6) 66" AiG CAAA AGAGC CiG 72" C C C G GCC iiiGC GA C GC AG GCCACC AGAAGATAC" ACCTGGGTGC 78" AG GGAAC G CA GGGACi A A GCAAAG GA C CGG GAGC GCCiG GGACGCAAG 84" A CC CC AGAG GCCAA AA C CC Al CAACACC iCAG CG G ACAAAAAGAC 90" C G G A GAA CACGG A CACC CAACA CGC AAGCCAAGGC CACCCTGGA" 96" GGG C GC A GGiCC ACCA CCAGGC GA GG iA GA ACAG GG CA ACAC AA "02" GAACA GGC CCCA CC G CAG C CA GC G GG GiAiCCiAC GGAAAGC C "08" r"GAGGGAGC GAA A GA G A CAGACCAG r"CAAAGGGAG AAAGAAGA G A AAAG C "14" CCC"GGTGGA AGCCA ACA A G C GGCA GG CC GAAA GAGAAiGG C CC C "20" r"GACCCAC G GCCi ACC ACiCA A C iC CA G G GACC GGiAA AAGAC GAA "26" CAGGCC C AiiGGAGCCC AC AG A G AGAGAAGGG AG C GGCCA AGGAAAAGAC "32" ACAGACC G CACAAA i A AC AC GC G Al GA GAAGGGA AAAGT"GGCA "38" C"CAGAAACA AAGAAC CC GA GCAGGA AGGGA GC GCAiC GCiC GGGCC"GGCC "44" AAAA GCAC ACAG CAA G G LA G AAA CAGGiC C G CCAGG CiGA GGA GCCA "50" CAGGAAA CA G C A GGC A G GA GG AA GGGCACC AC CC GAAG GCAC CAA "56" A iCC CGAA GG CACACA C G GAG GAACCA"CGC CAGGCGiCC GGAAA C C- "62" GCCAA AAC CC AC G C CAAACACi C GAiGGAC Ci GGACAG CiAC G "68" G CA AiC C CCCACC AACA GA GG CA GGAAGC iA GiCAAAG AGACAGC"G "74" AGGAA CCCCAAC AC GAA GAAAAA AA GAAGAA GCGGAAGAC A GA GA GA "80" Cl AC GA C GAAA GG A G GG CAG G GA GA GACAAC CiC C CC A "86" CCAAAi CGC CAGi GCCA AGAAGCA CC AAAAC iGG G ACA ACA GC GC GA "92" AGAGGAGGAC GGGACiAiG C CCCi AG CC CGCCCCC GA"GACAGAA G A AAAAG "98" CAA A G AACAA GGCC C CAGCGGA GG AGGAAG TACAAAAAAG CCGA A 204" CACA GA GAAACC i AAGAC CG GAAGC A CAGCA GAA CAGGAA C 210" GGGACC A Ci iA GGGG AAG GGAGA CACAC Gi G A A All A AGAA CAAGC 216" AAGCAGACCA A AACA C ACCC CACGG AAiCAC GA G CCG CC GiA CAAG 222" GAGA ACCA AAAGG G AA AACA LGAA GGA 1 CCA A C GCCAG GAGAAA A 228" CAAA A AAA GGACAG GA C G AGAAGA r"GGGCCAAC AAAiCAGA C CiCGG GCC 234" GACCCGCiA AC C AGi CG AA A GGAGAGAGA C 240" CCC C CCiC A GCiACA AAGAA C G AGA CAAAGA G 246" CAAGAGGAA G A CC G C GiAi GA GAGAAC C 252" GAA A ACAA CGC C CC CCAA CCAGC GGAG GCAG C GAGGAiC CAGAG CCA 258" AGCC"CCAAC A CA GCACA GCAiCAA GG CiA G ii GA AG LGC AGL G CAG 264" G GCA GAGG GGCA AC GG ACA C AAGCA GGAGCACAGA C GAC CC 270" C G C iC C C GGA A ACC CAA ACACAAAA G G CiA GAAG ACACAC"CAC 276" CC A CCCA C CAGGAG AAAC G C CA GiCGA G GAAAACCCAG G C A GGA 282" C GGGG GC iCAG AC CGGAA CAGAGGCA"G ACCGCCiiAC GAAGG i C 288" AG G GAC AAGAACAC G G GA A A CGAGGACAGi ALGAAGA A CAGCA A 294" C GC GAGi AA G CCA GAACC Ci C CCCAGAA CAAGACACCC 300" AGCAC AGG CAAAAGCAA AA GCCAC CACAAT"CCA GAAAA GACA AGAGAAGAC 306" GACCC GG GCACACA GAACACC Al GCCiAAAA A CAAAA G C CC C AG GA 312" G1 GAiG C Ci GCGAC AGAG CC AC CCACA GGG C AiCCi A C GA C CCA 318" AGAAGCCAAA A GAGAC C GA GA CCAiCACC GGAGCAA"AG ACAG AA AA 324" CAGCC G Ci GAAA GACAC AC CAGGCC ACAGCiCCA CACAG GGGG ACAiGG Al 330" ACCCC GAG CAGGCCiCC AA AAGA 1 AAA GAGAAA C"GGGGACAA C"GCAGCAAC 336" AGAG GAAG AAAC GA CAAAG C AG ACA CA AA AA C GA J. CAACAA 342" CCA CAGAC AA 1 GGCAG CAGGiAC GA AA ACAAG iCCi AGGAC GTA" 348" GCCAG CA A GA AG C AA AGA AC CAC CiA i GGCAAAAAG CA C CCCC 354" AC GAG C GG GGACC C GAGC GAG GAAGAAAA AA GA CAA AG iGiiAGA 360" A CAGG A A GAA AGCC AAGAAAG C A GGGGAAAA AA G A CG CAACAGAGAG 366" GG AGG A AAAGGGA AAAGAGC CA GGACCiGC iiG GAC W 372" C AL CAAA G AGCA Ci iiG AAA GACAAACAAA AC CCAA W 378" AA AGAAAG AC CACA G GGCCCA C A iAi AA GAGAA AG O 384" GCAAAA A A AGAAAG G CAC GAG AAAAAAG"G ACACC C)W l— OW C)WOWC) 390" AA GC A G GACAAAAA G ACAGCi GAGGC AAA CA AiG CE A AAAAC AC 396" iCA CAAAA AACA GGAAA GG CCAACA GAAAAAAGAG GGCCCCA O CACCAGA"GC 402" ACAAAA CCA GA A GiCGi C AAGA GCiAi Ci G CCAGAA"CAG CAAGG GGA 408" ACAAAGGACT CA"GGAAAGA AC C C GAA CiC GGGCAA GGCCCCAG"C CAAAGCAAT" 414" AG A CCi A GGACCAGAAA AA C G GGA AGG CAGAAi i CiiG C G ACAA 420" AG GGiAG A GGAAAGGGiG AA ACAAA GGACG AGGA C CAAAGAGA TGGTT"TTCC l\l\l\l\l\l\l\l\l\l\l\l\ 26" AAGCAGCAGA AACC All C iACiAAC i GGA AAiiiA CALGAAAA A AiACACACAA 32" TCAAGAAAAA AAAA"TCAGG AAGAAATAGA AAAGAAGGAA ACA AAiCC AAGAGAA G 38" AG GCC CAGA ACA A CAG GAC GG CAC AAGAA iiCA GAAGA ACC C 44" AC AGGCAAAA G AGAAGG C A A GACGGG GCA AiGC C CAG AC CA 50" AGA CA1 AAA G AL CAACAAA "AGAACAAAG ACAG C C 56" AAAAAAAGGG GAGGAAGAAA AC GGAAGG C GGGAAA AAGC G AGA 62" GAAA GCACCACAA GGA A C CC AA ACAAGC CAGCAGAA i CACGCA 68" ACG AG AAG AGAGC 1 GA AACAA iCAG AC CCCACLA ACAG GAAAA 74" AAGGALAA G GGA CC"CAACCCA G"GGTCCAAA AACA"GAAAC GACCCC 80" GAGCACCC"C AC ACAA GA GAAGGAGAAA GGGGCCAL A CiCC 86" Ci AiCAGA GGAGiCA AG CA CCC CAA GCAAA AGA ACC 92" AAAG CCALC A AGACC A A iA C GACCA A i 198" CCAAGACAAC CCAGCAGC AiCi A AGA GA CCA 504" AGAAAGCAG AAGGAGCCAA AAAAAA AAC Ci LC AG 510" Ci GGAGA G AAAGAGAGG GGC CCC G AG"G CCACAAAT"C 516" AG"CACATAC C G C CCCGAAA CCAGAC GC CCAAAACA"C 522" TGGCAAAG CAAAAG CA CA 1 A AAGGACC A CCC ACGGA 528" AACiAGCAA GGG GCCA C GGA C CG GGGAGCC C CAGGGAAC 534" AGAGGGAGCG A GAAGCAAA CAGACC"GGA AAAGi CCC CiGAGAGi 540" AGCAACAGAA AGAC CCC C CAAGC GA CC C G iGGGA AA 546" CCACiA GG AC CAAAAGAAGA G GGAAA CAAGAGAAG" 552" AACAGC ii A ACCAi i G CCC GAAC GC G GAAA 558" AA"AGCAGCA ATAAA"GAGG GACAAAA"AA GCCCGAAA ACC 564" AGG AGGAC GAAAGGC GC C CAAAA CCCACCAG GAAACGCC W1"CAACGGGA 570" AA AAC CG ACiAC C 1C AG CAGA CA AGAGGAAA GA W ACCALA C 576" GAAAiG AAGAAGGAAG A GACA 1A GA GAG WGAGCCCCCG 582" AAGAAAACAC GACAC A i H O GGGA A 588" AGCiCCCCAC A G C AAG O G CCC CA 594" G Gi CC AGGAA AC AC CAGC OO ALACCG 600" AA GAACA GGGAC CC WWG GAAGA 606" G AAC CA GAAA"CAGGC W C AGCCi 612" GAGGAAGA"C AGAGGCAAGG H G CAAGCC 618" AAAAC AC GGAAAG GGCACCCA O 1"AAAGATGA 624" AAAGCC GGG C A GGAAAAAG W G GCAC C 630" GGACCCC C GGiC GAACCCiG O CA GGGAG 636" O ACAGGAA GC C GA GAGA CCAAAAGC"G 642" OEE A GG AAAGAAAC"G CC AGA 648" WAAGAGAA A CGC CCA AA GGA 654" GGC O AAiGGC CA GCA GGGCAG 660" CAA"GAAAAC W CCA LC CAC AC GAAAAAAAGA 666" GGAG A AAA W GGCAC GAGACAG G 672" ACCA"CCAAA OO GGAA GGCGAGCALC ACAiGC GG 678" GA"GAGCACA O AC CCCC GG GAAiGGC C 684" "GGACACATT WOWOW A GGACAGL GGGCCCCAAA 690" GC"GGCCAGA O AAGG AGCCCii C 696" iGGAiCAAG O GGCACCAA GGCA"CAAGA CCCAGGG"GC 702" CCG"CAGAAG CiACA C A CA GiA A G C GA GG 708" GAAGAAG"GG GAGGAAA 1 AA GG C Ci GGCAA 714" G CCAA C CGAiACAi 720" CCG CA CGCA GGAG GA G 726" GA GCAGCA AAAGCAA A CAGA GCACA 732" GA CA CCiAC GGiC CC CAAAAGCTCG 738" AC CAAGGGAGGA CAG G GAA AA C AG'"G 744" GC GAC CCAGA CACAGGA G AC C AGGGAG"AAA 750" A ACCAGCA G CC CA C CCAGCAG"C AAGATGGCCA 756" iCAGiGGAC C C 11 1C AAAGG i CAGGGAA A"CAAGACTC 762" C CACACC G GG GAAC ACCGi AC G AC CGCiACC i CGAA LCA 768" CCCCCAGAG iGGG GCACC GCCCL GAGGA GGAG G CiGGGCi GCGAGGCACA 774" GGACCTCTAC GACAAAACTC ACACATGCCC CCCA GCTCCAGAAC TCCTGGGCGG 7801 ACCGTCAGTC TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT CCCGGACCCC 7861 TGAGGTCACA TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA AGTTCAACTG 7921 GGAC GGCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG AGCAGTACAA 7981 CAGCACGTAC CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC TGAATGGCAA 8041 GGAGTACAAG TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA AAACCATCTC 8101 CAAAGCCAAA GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT CCCGGGATGA 8161 CAAG AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC CCAGCGACAT 8221 CGCCGTGGAG TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA CGCCTCCCGT 8281 GTTGGACTCC GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTGGACA AGAGCAGGTG 8341 GCAGCAGGGG AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA ACCACTACAC 8401 GCAGAAGAGC CTGT CTCCGGGTAA A (ii) Fc (same sequence as A (ii) (SEQ ID NO:3))] Table 2: Polypeptide Sequences A. B-Domain d FVIII-Fc Monomer Hybrid (BDD FVIIIFc monomer dimer): created by coexpressing BDD FVIIIFc and Fe chains.

Construct = HC-LC-Fc fusion. An Fc expression cassette is cotransfected with BDDFVlll-Fc to generate the BDD FVIIIFc monomer-. For the BDD c chain, the Fc sequence is shown in bold; HC sequence is shown in double underline; remaining B domain sequence is shown in italics. Signal peptides are underlined. i B domain deleted Fc chain 19 amino acid si al se uence underlined SE ID NO:2 Q hCLLRFCFS ATRRYYHGAV?HSWDYMQSDHG?HPVDARFPPRVP<SFPFNTSVVY<KTHhVflhTDHHEN W GL HGPT QAflVYDTVV"THK MASiPVSLHAVGVSYW<ASflGAflYDDQTSQRfl<fl33<VhPGGSiTYVWQVLK ENGPMASDPHCHTYSYHSiVDHV<DL SGL"GAH'VCR?GSHAK?KTQTHi<h HHhAVhDLG<SWiSETK S; QDRDAASARAWP<M1TV GYVNRSHPGL"GC1RKSVYW1VIG GTTPflViS hHflGiThHVR HRQA m Up SP ThHTAQTHL DHGQFHHFCi"SSHQHDG DSCPflflPQHR (N flflAflDY DDHTDS? UVVRFDDDNSPSE Q RSVA<<1P<TWViY AAflflflDWDYAPHVHAPDDRSY<SQYL GPQ IGR<Y<<V Nh AYTDLTEKTRflA QHflSG HGPHHYG?VGDTHH hK QASRPY YPiG TDVRPHYS'RHP<GV<H DhP HPGfl VTVEDGPT<SDPRCLTRYYSSFV M?RDLASGU"GPUU CY<flSV QRG Q: S '% V HhSVhDfl RSWYHTfl QREHP PAGVQHflDPflhQAS HS GYVhDSHQHSVCHi?VAYWYI US"GAQTDFLSVFFSGYTF<1< VY?DTHTHhPhSGflTVh S fl PGHW"HGCi SDF? RG SS CD< TGDYYflDSYflD SAYHHS<N A flPRSfSQNPPVLKRHQRfl TRTTHQSDQflfl DYDDT SVfl K<fl 3h) YDflDfl QSPRSFQK<TRiYh AAVflRHWDYG SSSPiVL? RAQSGSVPQF<<VVFQEFTDGSFTQP KYRGflH fliHGHLGPY RAflVflDN IVTERNQASRPYSEYSSH SYflflDQRQGALPR< hV<P LT<TYEW T<DEFDCKAWAYFSDVDH? A DViSGL"GPHHVCHT T; PAHGRQVTVQflhAHhhT hDflT<S . .R CRAPCN Q fl KRhiA NGY DTHPGHV AQDQR"RWYHHS GS fl {S HhS GHVFTVR<<flflYK ALYNLYPGVhflTV 3 HPSKAG WRVflCL GfliHiAG STHFHVYSN<CQTPLG ASG ASGQYGQWAP<'IAR'IHYSGS: AWSTKflPJ: SW KVD' ' Shu HG KTQGARQ <FSSHY"SQF MYSHDG<<WQTYRGNSTGTLMVFFG VDSSG KiN h PP ARY RLiPTiYS"RSTHR ?LMGCDLN ' flS<A SDAQ TASSYFTNMFATWSPSKARHHHQGRS AWRPQVNNPK?WHQVDFQ(TMKVTGV TTQGV<SHHTSMYV<flhL SSSQDGHQWTLFFQNG<V<VFQGNQDSFTPVVNSHDPPHLTRYL?"{PQSWV HQ AL? flVHGCflAQ3LYDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS VFSCSVMHEALHNHYTQKSLSLSPGK ii) Fc chain 120 amino acid heterologous signal peptide from mouse ng chain underlined) (SEQ ID NO:4) ?TDTHLHWVLLHWVPGSTG D{THTCPPCPAPVHHGGPSVFLFPP{PKDTLM SRTPnVTCVVVDVSHflDPflV<hNWYVDGVEVH A<TKP KnflQYNSTYQVVSVHTVHHQDWLNG(VYKCKVSNKAUPAP nKT SKA(GQPREPQVYTHPPSRDTLTKNQ.|.

V<GEYPSD AVanSNGQPfl NYKTTPPVLDSDGSFFLYSKLTVD{SQWQQGNVFSCSV HEALH -lYTQ (S' .S' .SPG< B. Full length c monomer hybrid (Full length FVIIIFc monomer dimer): created by coexpressing FVIIIFc and Fc chains.

Construct = HC-B-LC-Fc fusion. An Fc expression cassette is cotransfected with full length FVlll-Fc to generate the full length FVIIIFc monomer. For the FVIIIFc chain, the Fc sequence is shown in bold; HC sequence is shown in double underline; B domain sequence is shown in italics. Signal peptides are underlined. i) Full length FVIIIFc chain (FVIII signal peptide ined (SEQ ID NO:6) Q flLSTChthLLQFCFS ATQQYYUGAVTHSWDYMQSDIGTHPVDARFPPRVP<SFPFNTSVVY<KTHhVflhTDHHEN A<PRPPW GL UGPT QAflVYDTVV_TIK MASiPVSLHAVGVSYW<AS*IGAfiYDDQTSQQfi(flDD<VhPGGSiTYVWQVLK ENGPMASDPHCUTYSYUSiVDIV<DL SGL GAI VCRTGSHAKTKTQTIi<h UHhAVhDLG<SWHS_ETK S; QDQDAASARAWP(MiTVNGYVNQSUPGL'GCiQKSVYWiVI_G GTTPfiViS hUfiGiThIVQNHQQA WIT SP ThHTAQTHL DHGQFHHFCi"SSHQH DG EAYVKVDSCPflflPQHR {N flflAflDY DDITDSTIM iN- '11 U U DNSPSh Q RSVA<<1P<TWV1Y AA. flflDWDYAPHVIAPDDRSY<SQYL GPQU/U/UU :GRKY<<V 'hN AYTDLTEKTRflA QHflSG HGPHHYGTVGDTHH hKNQASQPY YPiG TDVRPHYS' NHPKGV<H H<DhP HPGfl VTVEDGPT(SDPRCLTRYYSSFVNMTQDLASGU"GPUU CY<flSV RGNQ: S D<R V HhSVhDfl RSWYHTfl QRhHP PAGVQHflDPflhQAS MHS GYVhDSHQHSVCHadoTVAYWY.Z US"GAQTDFLSVFFSGYTFKi{MVYTDTLTLhPhSGflTVhMS fl GCi SDFR RG TALLKVSS CD< TGDYYflDSYflD SAYLHSKN A flPRSfSQNSRHPSTRQKQfNATT1PbND1bK1DPW1AHR1PMPK IQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPE SGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFG TESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVS 1SLLKYNKTSNNSATNRKTH1DGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMS NKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEK SVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHLNNTHNQLKKlbe1bKKL1L1Qb NVVLPQ1H1V1G1KN£MKNL£LLSTRQNVLGSYDGAYAPVLQDFRSLNDSTNRTKKHYAHESKKGLLLNLL GLGNQTKQ1VbKYAC11R1SPNTSQQNfVTQRSKRALKQPRLPLLLTbLbKR11VDDTSTQWSKNMKHLTP STLTQTDYNFKFKGATTQSPLSDCWTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAAS YRKKDSGVQESSHFLQGAKKNNLSLA1LTLLMTGDQRLVGSLGISAINSVTYKKVLNTVLPKPDLPKTSGK VELLPKV DPLAWDNHYG1Q1PKLLWKSQLKSPLK1A£KKKD1/TSTNACFSNHATAATNFGQNKPFTFVTWAKQGRTE RLCSQNPPVLKRHQRfl TQTT IQSDQflfl DYDDT SVfl KKflDhD YDflDfl QSPRSFQKKTRiYh AAVfl RLWDYGMSSSPHV_1% RAQSGSVPQF<<VVFQEFTDGSFTQPIYRGflH fliHGILGPY N VTh RNQASRPYSEYSSI RQGALPR<NhV<P W<VQHiMAPT<33F3CKAWAYFSDVDHT<D VHSGL"GPIIVCHT T; PAHGRQVTVQflhAHhhT hDflTKSWYhTflN fl? CRAPCN h<L Y? hHA NGY DTLPGHV AQDQR"RWYHHSMGS fl {S HhSGHVhTVR<<flTIYK ATIYNLYPGVETITVn LPSKAG WRVflCL GfliHiAG STHFHVYSN<CQTPLG ASGi RDhQ TASGQYGQWAPKIAQHHYSGS: AWSTKflPhSW KVD'HAP HG KTQGARQ<hSSIY SQh MYSHDG<<WQTYRGNSTGTLMVFFG V DSSG KiN h PP ARY RLiPTiYS"RSTHR TLMGCDTINSCSMPHG flS<A SDAQ TNMFA TWSPSKARHH'IQGRS AWRPQVNNPKT HQVDFQ<TMKVTGVTTQGV<SHHTSMYV<*IhL SSSQDGHQWT LFFQNG<V<VFQGNQDSFTPVVNSIDPPITTQYL%"1PQSWVHQ ALQ *VIGCTAQDTYDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS ii) Fc chain 120 amino acid heterologous signal peptide from mouse ng chain underlined) (SEQ ID NO:4[ 'ETDT' .T.' .WVT.T.' .WVPGSTG D (THTCPPCPAP'? .' .GGPSVFLFPP {PKDTTIM SRTP fiVTCVVVDVSH fiDP *.V (J: NWYV DGVEVH A<TKP Rfl fiQYNSTYRVVSV' .TV' .HQDWT.NG (”TYKCKVSNKA' IPAP flKT SKA {GQPREPQVYT' 'TTEKNQ.|.

VS'.TCHV<GJ:YPSD AV*-W*-SNGQP*- NYKTTPPVLDSDGSFFLYSKLTVD(SRWQQGNVFSCSV HEALH {YTQ (S' .S' .SPG< Cited References FOWSQSAB Aledort L. et al., n Med. .‘236:391-399 (1994) Petrini P. et al., Am JPediatr Hematol Oncol. 13:280-287 (1991).

Aznar J. et al., Haemophilia 6(3):170-176 (2000).

Feldrnan B. et al., J Thromb Haemost. 4:1228-1236 (2006).

Kreuz W. et al., Haemophilia 4:413-417 (1998).

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Claims (35)

What we claim is:
1. l. A pharmaceutical composition sing: (i) a chimeric polypeptide, which ses a Factor VIII (FVHI) portion and a second portion; and (ii) at least one pharmaceutically acceptable excipient, wherein 15% to about 25% of the FVIII portion of the chimeric ptide comprises single chain FVIII and about 75% to about 85% of the FVHI portion of the ic polypeptide comprises processed FVHI, and wherein the composition is lyophilized.
2. 2. The pharmaceutical composition of claim 1, n: (i) about 25% of the FVIII portion of the chimeric polypeptide ses single chain FVlIl and about 75% of the FVHI portion of the chimeric polypeptide comprises processed FVIII; (ii) about 20% of the FVHI portion of the ic polypeptide comprises single chain FVIH and about 80% of the FVIH portion of the chimeric polypeptide comprises processed FVIII; or (iii) about 15% of the FVHI portion of the chimeric polypeptide comprises single chain FVIII and about 85% of the FVHI portion of the chimeric polypeptide comprises processed FVHI.
3. 3. The pharmaceutical composition of claim 1 or claim 2, wherein the single chain FVIII comprises a heavy chain and a light chain connected by a peptide bond.
4. 4. The pharmaceutical composition of any one of claims 1 to 3, wherein the single chain FVIH. FVIII has FVIH activity comparable to a ptide consisting of processed
5. 5. The pharmaceutical composition of any one of claims 1 to 4, wherein the processed of FVIII and a FVIIl comprises two chains including a first chain comprising a heavy chain second chain second chain comprising a light chain of FVIH and wherein the first chain and the are associated by a metal bond.
6. 6. The pharmaceutical composition of any one of claims 1 to 5, wherein the chimeric polypeptide is a cting FVIII polypeptide.
7. 7. The pharmaceutical composition of any one of claims 1 to 6, wherein the second portion C—terminal comprises an Fc region, an albumin, a PAS sequence, a transferrin, a 28 amino acid starch peptide of hCG with its 4 O—glycans (CTP), a polyethylene glycol (PEG), a hydroxyethyl —96— (HES), an albumin binding polypeptide, an albumin—binding small molecule or two or more combinations thereof.
8. 8. The pharmaceutical composition of any one of claims 1 to 7, wherein the second n ses an Fc region.
9. 9. The pharmaceutical ition of any one of claims 1 to 8, wherein the chimeric polypeptide is a FVIIch monomer dimer hybrid.
10. 10. The pharmaceutical composition of any one of claims 1 to 9, wherein the FVIH portion comprises B—domain deleted FVIH or full—length mature FVHI.
11. 11. The pharmaceutical composition of claim 10, wherein the single chain FVIII or the processed FVIII comprises B-domain deleted FVIII. 1 to 11, wherein residue 1645
12. 12. The pharmaceutical composition of any one of claims in the single chain and/or residue 1648 corresponding to ength mature FVIII is/are arginine FVIH.
13. 13. The pharmaceutical composition of any one of claims 1 to 11, wherein residue 1645 tuted or mutated in and/or residue 1648 corresponding to full—length mature FVIH is/are the single chain FVHI compared to wild type FVIII. pharmaceutical composition of any one of claims 4 to 13, wherein the FVHI
14. 14. The activity is measured in vitro by a chromogenic assay.
15. 15. The pharmaceutical composition of any one of claims 1 to 14, wherein the chimeric polypeptide has a half-life longer than a polypeptide consisting of the FVIII n. is at least about 1.5
16. 16. The pharmaceutical composition of claim 15, wherein the half—life fold longer than the half—life of the polypeptide consisting of the FVIH portion.
17. 17. The pharmaceutical composition of any one of claims 1 to 16, wherein the composition chain FVHI. is more stable than a composition sing processed FVIII t single
18. 18. The pharmaceutical composition of any one of claims 1 to 17, wherein said composition is formulated for administration to a human.
19. 19. The pharmaceutical composition of any one of claims 1 to 18, wherein said composition is formulated for parenteral administration to a human.
20. 20. The pharmaceutical composition of any one of claims 1 to 19, wherein said composition is formulated for subcutaneous, intradermal, ascular, intravenous, uscular, spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intrasynovial or eritoneal injection to a human.
21. 21. Use of the pharmaceutical composition of any one of claims 1 to 20 in the manufacture of a medicament for preventing, decreasing or treating a ng episode in a human.
22. 22. Use of the pharmaceutical composition of any one of claims 1 to 20 in the manufacture of a medicament for prophylactic treatment of a bleeding episode in a human.
23. 23. Use of the pharmaceutical composition of any one of claims 1 to 20 in the manufacture of a medicament for on—demand treatment of a bleeding episode in a human.
24. 24. Use of the pharmaceutical composition of any one of claims 1 to 20 in the manufacture of a medicament for tailored prophylactic treatment of a ng episode in a human.
25. 25. The use any one of claims 21 to 24, wherein the bleeding episode is associated with a disease oral or condition comprising hemarthrosis, muscle bleed, bleed, hage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis (head trauma), gastrointestinal bleeding, intracranial hemorrhage, intra—abdominal hemorrhage, intrathoracic hemorrhage, bone re, l nervous system ng, bleeding in the retropharyngeal in the illiopsoas sheath. space, bleeding in the eritoneal space or bleeding
26. 26. Use of the pharmaceutical composition of any one of claims 1 to 20 in the manufacture of a medicament for perioperative treatment of a subject before, after or during surgery.
27. 27. The use of claim 26, wherein the tooth surgery is minor surgery, major surgery, extraction, tonsillectomy, al herniotomy, synovectomy, total knee ement, craniotomy, osteosynthesis, trauma surgery, intracranial surgery, intra-abdominal surgery, intrathoracic surgery or joint replacement surgery.
28. 28. The use of any one of claims 21 to 27, n the medicament is formulated to provide 30 IU/kg—40 IU/kg, a therapeutically effective amount of 10 IU/kg-20 IU/kg, 20 IU/kg—30 IU/kg, -98— 40 lU/kg—SO IU/kg, 50 IU/kg—60 lU/kg, 60 lU/kg-70 IU/kg, 70 SO IU/kg, 80 IU/kg—90 lU/kg, 9O IU/kg-lOO lU/kg, 100 IU/kg-l 10 IU/kg, 110 IU/kg-120 IU/kg, 120 IU/kg—13O lU/kg, 130 IU/kg—l40 IU/kg or 140 lU/kg—lSO IU/kg of the ceutical composition.
29. 29. The use of any one of claims 21 to 28, wherein said medicament is formulated to be administered at a dosing interval of once every 24-36, 24—48, 24—72, 24-96, 24—120, 24—144 or 24—168 hours or .
30. 30. A pharmaceutical composition as defined in claim 1 and as substantially described herein with reference to one or more of the accompanying examples.
31. 3 1. Use of the pharmaceutical composition of claim 30 in the manufacture of a medicament for ting, decreasing or treating a bleeding episode in a human.
32. 32. Use of the pharmaceutical composition of claim 30 in the manufacture of a medicament for prophylactic treatment of a bleeding episode in a human.
33. 33. Use of the ceutical composition of claim 30 in the manufacture of a medicament for on—demand ent of a bleeding episode in a human.
34. 34. Use of the pharmaceutical composition of claim 30 in the manufacture of a medicament for tailored prophylactic treatment of a bleeding episode in a human.
35. 35. Use of the pharmaceutical composition of claim 30 in the manufacture of a medicament for perioperative treatment of a subject before, after or during surgery. Biogen Hemophilia Inc. By the patent attorneys for the applicant CULLENS