Modified factor vii polypeptides for subcutaneous administration and on-demand treatment

Abstract

Provided herein are modified FVII polypeptides, and modified FVIIa polypeptides, and methods of treatment of acute and episodic bleeding with modified FactorVIIa polypeptides. To effect treatment and use, in some embodiments, the modified polypeptides are subcutaneously administered to provide on-demand treatment. In some embodiments, the on-demand treatment is provided in a multiple dosing regimen over a twenty-four hour period. The subcutaneous administration of the modified polypeptides of the disclosure exhibit increased coagulant activity, potency, bioavailablilty and prolonged duration.

1 . A method of treating a bleeding event in a subject, comprising subcutaneously administering to the subject a dose of a modified Factor VIIa comprising modifications at least at a position corresponding to position 286 and at a position corresponding to position 298 in a FVII polypeptide comprising the sequence of amino acids set forth in SEQ ID NO: 3 or in a corresponding residue in a FVII polypeptide, wherein:
 the modification at position 286 is an amino acid replacement with Arg (R);   the modification at position 298 is an amino acid replacement with Gln (Q);   the subcutaneous administration of the modified FVIIa has increased activity or potency; and   a dose of the modified FVIIa is administered subcutaneously within about 5 or 4 or 3 or 2 or 1 or fewer hours or minutes before and/or after the bleeding event, whereby the amount of bleeding is reduced or stopped or the cause of the bleed is corrected or is healed.   
     
     
         2 . The method of  claim 1 , the modified FVIIa further comprising a modification at a position corresponding to position 128 in the FVII polypeptide having the sequence of amino acids set forth in SEQ ID NO: 3, wherein:
 the modification at position 128 is an amino acid replacement with Asn (N).   
     
     
         3 . The method of  claim 2 , the modified FVIIa further comprising a modification at a position corresponding to position 129 and in the FVII polypeptide having the sequence of amino acids set forth in SEQ ID NO: 3, wherein:
 the modification at position 129 is Ala (A).   
     
     
         4 . The method of  claim 1 , wherein the dose of the modified FVIIa is administered subcutaneously within about 5 or 4 or 3 or 2 or 1 or fewer hours or minutes before or after the bleeding event. 
     
     
         5 - 27 . (canceled) 
     
     
         28 . The method of  claim 1 , wherein:
 the bleeding is episodic or predictable by the subject; and   the subject is treated with a subcutaneous dose of the modified FVIIa before the bleeding starts.   
     
     
         29 . The method of  claim 1 , wherein:
 the bleeding event results from trauma or injury; and   the subject is treated with a subcutaneous dose of the modified FVIIa 15 minutes, 1, 2, 3, or 4 hours after the bleeding event.   
     
     
         30 . The method of  claim 1 , wherein:
 the subject has a hemophilia or other bleeding disorder or condition, and is undergoing surgery;
 a dose of the modified FVIIa is subcutaneously administered 5, 4, 3, 2, 1 hours or less before surgery; 
 the subject is treated with a FVIIa intravenously during surgery; and 
 a dose of the modified FVIIa is subcutaneously administered at least one time following surgery until there is no bleeding and/or risk of bleeding or until the subject is healed or the cause is corrected. 
   
     
     
         31 - 41 . (canceled) 
     
     
         42 . The method of  claim 1 , wherein:
 the bleed is episodic or predictable; and   the subject is pre-treated prior to the bleeding, and wherein the pre-treatment is effected about 4 hours or less, 3 hours or less, 2 hours or less, 1 hour or less, or at least 15 minutes before the bleed.   
     
     
         43 - 45 . (canceled) 
     
     
         46 . The method of  claim 1 , wherein the bleeding is the result of any one or more of surgery, trauma, injury, a wound, menstrual bleeding, a joint bleed, trauma, dental extraction, or bleeding gums. 
     
     
         47 - 50 . (canceled) 
     
     
         51 . The method of  claim 1 , wherein a dose or doses of the modified FVIIa is/are administered subcutaneously before the bleeding or after the bleeding starts. 
     
     
         52 - 53 . (canceled) 
     
     
         54 . The method of  claim 1 , wherein the modified FVIIa is administered subcutaneously a plurality of times until the bleeding stops or the wound heals or bleeding is corrected. 
     
     
         55 . The method of  claim 1 , wherein a single dose of the modified FVIIa is from about 10 μg/kg to 30 μg/kg, 10 μg/kg to 60 μg/kg, 10 μg/kg to 90 μg/kg, 10 μg/kg to 120 μg/kg, 30 μg/kg to 60 μg/kg, 30 μg/kg to 90 μg/kg, 30 μg/kg to 120 μg/kg, 10 μg/kg to 500 μg/kg, or 15 μg/kg to 400 μg/kg, or 15 μg/kg to 350 μg/kg, or 20 μg/kg to 400 μg/kg, or 20 μg/kg to 350 μg/kg, or 30 μg/kg to 350 μg/kg, or 25 μg/kg to 350 μg/kg, based on the weight of the treated subject. 
     
     
         56 - 60 . (canceled) 
     
     
         61 . The method of  claim 1 , wherein a single subcutaneous dose of the modified FVIIa is in a volume of about 1 mL to 2 mL, or 1.25 mL to 1.5 mL, or 1 mL to 10 mL. 
     
     
         62 . The method of  claim 1 , further comprising administering any one or more of an additional coagulant treatment or factor, an anti-tissue factor pathway inhibitor (TFPI) antibody, and an RNA interference (RNAi) therapeutic targeting antithrombin (AT). 
     
     
         63 - 68 . (canceled) 
     
     
         69 . The method of  claim 1 , wherein the subject has any one or more of blood coagulation disorders, hematologic disorders, hemorrhagic disorders, hemophilia A, hemophilia B, hemophilia A with inhibitors, hemophilia B with inhibitors, Factor VII deficiency, Glanzmann thrombasthenia, and acquired hemophilia, and/or wherein the subject is taking anti-coagulant therapy, and/or wherein the subject has autoantibodies to factor VIII or factor IX, and other bleeding disorders. 
     
     
         70 - 71 . (canceled) 
     
     
         72 . The method of  claim 69 , wherein the subject has hemophilia, and the hemophilia is congenital or acquired. 
     
     
         73 - 74 . (canceled) 
     
     
         75 . The method of  claim 1 , wherein a single subcutaneous dose of the modified FVIIa is about 60 μg/kg to about 120 μg/kg, or about 60 μg/kg, or about 10 to about 20 μg/kg, or about 10 μg/kg, or about 20 μg/kg based on the weight of the treated subject. 
     
     
         76 - 83 . (canceled) 
     
     
         84 . The method of  claim 1 , wherein the modified FVIIa has a potency greater than the FVIIa of SEQ ID NO: 3. 
     
     
         85 . The method of  claim 1 , wherein the modified FVIIa has increased coagulant activity, compared to wild-type FVIIa of SEQ ID NO: 3, in the absence of tissue factor and/or in the presence of tissue factor. 
     
     
         86 . (canceled) 
     
     
         87 . The method of claim  186 , wherein the modified FVIIa has k cat /k m  in a tissue-factor dependent assay that is greater than 100%, 150%, 200%, or 250% or more than unmodified FVIIa (SEQ ID NO: 3) in the same assay and/or wherein the modified FVIIa has coagulation activity that is at least 1.5, or 3, or 4, or 5 times the activity of unmodified FVIIa of SEQ ID NO: 3 in the same assay. 
     
     
         88 - 90 . (canceled) 
     
     
         91 . The method of  claim 1 , wherein coagulation activity of the modified FVIIa polypeptide is at least 110%, 150%, 200%, 250%, 300%, 400%, 500% or more of the coagulation activity of an unmodified FVIIa polypeptide that has the primary amino acid sequence set forth in SEQ ID NO: 3. 
     
     
         92 . The method of  claim 1 , wherein the modified FVIIa has any one or more of increased serum half-life, an increased terminal elimination half-life, greater coagulation activity, and greater potency compared to an unmodified FVIIa that has the primary amino acid sequence set forth in SEQ ID NO: 3. 
     
     
         93 . (canceled) 
     
     
         94 . The method of  claim 1 , wherein:
 the modified FVIIa polypeptide, when in an activated form, exhibits procoagulant activity, wherein:   the procoagulant activity is greater than procoagulant activity of a FVIIa polypeptide having the primary amino acid sequence set forth in SEQ ID NO: 3.   
     
     
         95 . (canceled) 
     
     
         96 . The method of  claim 1 , wherein the modified FVIIa polypeptide is two-chain activated Factor VII (FVIIa) polypeptide comprising the amino acid sequence of SEQ ID NO: 280 or comprising the amino acid sequence of SEQ ID NO: 138 cleaved between the arginine at position 152 and the isoleucine at position 153. 
     
     
         97 . (canceled) 
     
     
         98 . The method of  claim 1 , wherein the modified FVIIa polypeptide has at least 90% amino acid sequence identity to SEQ ID NO: 280, wherein the amino acids corresponding to positions 128, 129, 286 and 298 of SEQ ID NO: 280 are invariant. 
     
     
         99 . The method of  claim 96 , wherein the first and second chains of the two-chain polypeptide consist respectively of amino acids 1-152 and 153-406 of SEQ ID NO: 280. 
     
     
         100 . The method of  claim 1 , wherein the modified FVIIa polypeptide comprises one or more amino acid modification(s) that increases resistance to antithrombin-III, increases binding and/or affinity to phospholipids, increases affinity for tissue factor, increases intrinsic activity, increases TF-dependent activity, increases coagulant activity, alters the conformation of the polypeptide to alter zymogenicity, increases catalytic or coagulant activity by shifting the equilibrium between highly active and less active FVIIa conformations in favor of the highly active conformations, increases resistance to proteases, decreases glycosylation, increases glycosylation, reduces immunogenicity, increases stability, and/or facilitates chemical group linkage. 
     
     
         101 . The method of  claim 1 , wherein the primary sequence of the unmodified FVIIa polypeptide consists of the sequence of amino acids set forth in SEQ ID NO: 3. 
     
     
         102 . The method of  claim 1 , wherein the modified FVIIa polypeptide is post-translationally modified, wherein a post-translational modification is any one or more of O-linked glycosylation, N-linked glycosylation, carboxylation of glutamic acid to γ-carboxyglutamic acid, and hydroxylation of aspartic acid to β-hydroxyaspartic acid. 
     
     
         103 - 108 . (canceled) 
     
     
         109 . The method of  claim 1 , wherein the subcutaneous administration of the modified FVIIa has increased terminal elimination half-life compared to an intravenous administration of the modified FVIIa. 
     
     
         110 . The method of  claim 1 , wherein a dose of the modified FVIIa is administered in a multiple dosing regimen, and wherein at least one dose of the multiple dosing regimen comprises about 30 μg/kg, about 45 μg/kg, about 60 μg/kg, about 90 μg/kg, or about 120 μg/kg of body weight of the subject. 
     
     
         111 - 127 . (canceled) 
     
     
         128 . The method of  claim 1 , wherein the subcutaneous administration of the modified FVIIa has an activity or potency greater than an intravenous administration of the modified FVIIa polypeptide and/or greater than an intravenous administration of an FVIIa polypeptide that is unmodified and/or greater than a subcutaneous administration of an FVIIa polypeptide that is unmodified. 
     
     
         129 - 131 . (canceled) 
     
     
         132 . The method of  claim 1 , wherein the modified FVIIa comprises the amino acid sequence set forth in SEQ ID NO: 280 or SEQ ID NO: 138. 
     
     
         133 . A method of providing an on-demand treatment to a subject experiencing a bleed or to a subject likely to experience a bleed, comprising administering to the subject a subcutaneous dose of a modified FVIIa comprising modifications at least at a position corresponding to position 286 and at a position corresponding to position 298 in a FVII polypeptide comprising the sequence of amino acids set forth in SEQ ID NO: 3 or in a corresponding residue in a FVII polypeptide, wherein:
 the modification at position 286 is an amino acid replacement with Arg (R);   the modification at position 298 is an amino acid replacement with Gln (Q); and   the dose is about 10 to about 120 μg/kg of body weight of the subject.   
     
     
         134 . The method of  claim 133 , the modified FVIIa further comprising a modification at a position corresponding to position 128 in the FVII polypeptide having the sequence of amino acids set forth in SEQ ID NO: 3, wherein
 the modification at position 128 is an amino acid replacement with Asn (N).   
     
     
         135 . The method of  claim 134 , the modified FVIIa further comprising a modification at a position corresponding to position 129 in the FVII polypeptide having the sequence of amino acids set forth in SEQ ID NO: 3, wherein:
 the modification at position 129 is Ala (A).   
     
     
         136 . The method of  claim 133 , wherein the subcutaneous dose of a modified FVIIa is administered in a multiple dosing regimen, and wherein each dose of the multiple dosing regimen occurs about 2 to about 6 hours apart for a predetermined time period. 
     
     
         137 - 143 . (canceled) 
     
     
         144 . The method of  claim 1 , wherein the subject has any one or more of hemophilia A, hemophilia B, hemophilia A with inhibitors, hemophilia B with inhibitors, hemophilia C, Factor VII deficiency, Glanzmann thrombasthenia, acquired hemophilia, or is taking anti-coagulant therapy. 
     
     
         145 . (canceled) 
     
     
         146 . The method of  claim 144 , wherein the subject has a hemophilia, and the hemophilia is congenital or acquired. 
     
     
         147 - 151 . (canceled) 
     
     
         152 . The method of  claim 133 , wherein the on-demand treatment comprises administering the treatment to a subject experiencing a bleed or a subject likely to experience a bleed. 
     
     
         153 . The method of  claim 152 , wherein the subject is experiencing a bleed and the subcutaneous dose is administered about 1 minute to about 1 hour, or about 2, or about 3, or about 4 hours after onset of the bleed, or wherein the subject is likely to experience a bleed and the subcutaneous dose is administered about 1 minute to about 1 hour, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7 hours before the likelihood of a bleed. 
     
     
         154 - 162 . (canceled) 
     
     
         163 . The method of  claim 133 , wherein the subcutaneous administration of the modified FVIIa has an activity or potency greater than an intravenous administration of the modified FVIIa polypeptide, and/or greater than an intravenous administration of an FVIIa polypeptide that is unmodified, and/or greater than a subcutaneous administration of an FVIIa polypeptide that is unmodified. 
     
     
         164 - 166 . (canceled) 
     
     
         167 . The method of  claim 133 , wherein the modified FVIIa polypeptide is two-chain activated Factor VII (FVIIa) polypeptide comprising the amino acid sequence of SEQ ID NO: 280 cleaved between the arginine at position 152 and the isoleucine at position 153. 
     
     
         168 . A pharmaceutical composition for a single dosage subcutaneous administration, comprising a single therapeutically effective dose of a modified FVIIa in a pharmaceutically acceptable carrier for subcutaneous administration for an on-demand treatment of a bleed; wherein the modified FVIIa comprises modifications at least at a position corresponding to position 286 and at a position corresponding to position 298 in a FVII polypeptide comprising the sequence of amino acids set forth in SEQ ID NO: 3 or in a corresponding residue in a FVII polypeptide, wherein:
 the modification at position 286 is an amino acid replacement with Arg (R);   the modification at position 298 is an amino acid replacement with Gln (Q); and   the modified FVIIa has increased activity or potency.   
     
     
         169 . The pharmaceutical composition of  claim 168 , wherein the modified FVIIa has an activity or potency greater than an intravenous administration of the modified FVIIa polypeptide, and/or greater than an intravenous administration of an FVIIa polypeptide that is unmodified, and/or greater than a subcutaneous administration of an FVIIa polypeptide that is unmodified. 
     
     
         170 - 171 . (canceled) 
     
     
         172 . The pharmaceutical composition of  claim 168 , the modified FVIIa further comprising modifications at a position corresponding to position 128 in the FVII polypeptide having the sequence of amino acids set forth in SEQ ID NO: 3, wherein:
 the modification at position 128 is an amino acid replacement with Asn (N).   
     
     
         173 . The pharmaceutical composition of  claim 172 , the modified FVIIa further comprising modifications at a position corresponding to position 128 and at a position corresponding to position 129 in the FVII polypeptide having the sequence of amino acids set forth in SEQ ID NO: 3, wherein:
 the modification at position 128 is an amino acid replacement with Asn (N); and   the modification at position 129 is Ala (A).   
     
     
         174 . The pharmaceutical composition of  claim 168 , wherein the amount of modified FVIIa is from 100 μg to 35 mg in a volume of 1 ml to 10 ml. 
     
     
         175 - 178 . (canceled) 
     
     
         179 . A container, comprising the pharmaceutical composition of  claim 168 , wherein the container is a syringe or injector pen and wherein the pharmaceutical composition is lyophilized. 
     
     
         180 - 182 . (canceled) 
     
     
         183 . An on-demand method of treating a bleed in a subject, comprising subcutaneously administering to the subject the pharmaceutical composition of  claim 168 . 
     
     
         184 . (canceled)