Recombinant factor VIII proteins
First Claim
Patent Images
1. A recombinant FVIII protein comprising:
- a first polypeptide comprising Formula I;
(A1)-a1-(A2)-a2-[B]; and
a second polypeptide comprising Formula II;
a3-(A3)-(C1);
wherein the first polypeptide and the second polypeptide are fused or exist as a heterodimer;
wherein, a) A1 is an A1 domain of FVIII;
b) A2 is an A2 domain of FVIII;
c) [B] is a B domain of FVIII, a fragment thereof, or is deleted;
d) A3 is an A3 domain of FVIII;
e) C1 is a C1 domain of FVIII;
f) a1, a2, and a3 are acidic spacer regions;
wherein the A1 domain comprises a permissive loop-1 (A1-1) region and a permissive loop-2 (A1-2) region;
wherein the A2 domain comprises a permissive loop-1 (A2-1) region and a permissive loop-2 (A2-2) region;
wherein the A3 domain comprises a permissive loop-1 (A3-1) region and a permissive loop-2 (A3-2) region;
wherein a first half-life extending and a second half-life extending moiety are inserted within the FVIII protein immediately downstream of amino acids which correspond to amino acids in mature native human FVIII selected from the group consisting of;
a amino acids 18 and 1656 of SEQ ID NO;
1;
b amino acids 40 and 1656 of SEQ ID NO;
1;
c amino acids 399 and 1656 of SEQ ID NO;
1;
d amino acids 403 and 1656 of SEQ ID NO;
1;
e amino acids 1656 and 1720 of SEQ ID NO;
1;
f amino acids 1656 and 1900 of SEQ ID NO;
1;
g amino acids 18 and 399 of SEQ ID NO;
1;
h amino acids 26 and 399 of SEQ ID NO;
1; and
i amino acids 40 and 399 of SEQ ID NO;
1;
wherein the first half-life extending moiety or the second half-life extending moiety is selected from;
(a) albumin;
(b) albumin-binding polypeptide;
(c) Fc;
(d) PAS;
(e) the C-terminal peptide (CTP) of the β
subunit of human chorionic gonadotropin;
(f) polyethylene glycol (PEG);
(g) hydroxyethyl starch (HES);
(h) albumin-binding small molecules;
(i) a clearance receptor, or fragment thereof, wherein the clearance receptor blocks binding of the recombinant FVIII protein to FVIII clearance receptors;
(j) a low-density lipoprotein receptor-related protein 1 (LRP1) or a FVIII-binding fragment thereof;
(k) an XTEN; and
(l) any combination of (a)-(j); and
wherein the recombinant FVIII protein exhibits procoagulant activity.
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Abstract
Provided are recombinant Factor VIII proteins, e.g., human Factor VIII proteins with heterologous moieties inserted into flexible permissive loops located in the Factor VIII A domains, while retaining the procoagulant activity of Factor VIII.
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Citations
20 Claims
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1. A recombinant FVIII protein comprising:
- a first polypeptide comprising Formula I;
(A1)-a1-(A2)-a2-[B]; and
a second polypeptide comprising Formula II;
a3-(A3)-(C1);wherein the first polypeptide and the second polypeptide are fused or exist as a heterodimer; wherein, a) A1 is an A1 domain of FVIII;
b) A2 is an A2 domain of FVIII;
c) [B] is a B domain of FVIII, a fragment thereof, or is deleted;
d) A3 is an A3 domain of FVIII;
e) C1 is a C1 domain of FVIII;
f) a1, a2, and a3 are acidic spacer regions;wherein the A1 domain comprises a permissive loop-1 (A1-1) region and a permissive loop-2 (A1-2) region; wherein the A2 domain comprises a permissive loop-1 (A2-1) region and a permissive loop-2 (A2-2) region; wherein the A3 domain comprises a permissive loop-1 (A3-1) region and a permissive loop-2 (A3-2) region; wherein a first half-life extending and a second half-life extending moiety are inserted within the FVIII protein immediately downstream of amino acids which correspond to amino acids in mature native human FVIII selected from the group consisting of; a amino acids 18 and 1656 of SEQ ID NO;
1;b amino acids 40 and 1656 of SEQ ID NO;
1;c amino acids 399 and 1656 of SEQ ID NO;
1;d amino acids 403 and 1656 of SEQ ID NO;
1;e amino acids 1656 and 1720 of SEQ ID NO;
1;f amino acids 1656 and 1900 of SEQ ID NO;
1;g amino acids 18 and 399 of SEQ ID NO;
1;h amino acids 26 and 399 of SEQ ID NO;
1; andi amino acids 40 and 399 of SEQ ID NO;
1;wherein the first half-life extending moiety or the second half-life extending moiety is selected from; (a) albumin; (b) albumin-binding polypeptide; (c) Fc; (d) PAS; (e) the C-terminal peptide (CTP) of the β
subunit of human chorionic gonadotropin;(f) polyethylene glycol (PEG); (g) hydroxyethyl starch (HES); (h) albumin-binding small molecules; (i) a clearance receptor, or fragment thereof, wherein the clearance receptor blocks binding of the recombinant FVIII protein to FVIII clearance receptors; (j) a low-density lipoprotein receptor-related protein 1 (LRP1) or a FVIII-binding fragment thereof; (k) an XTEN; and (l) any combination of (a)-(j); and wherein the recombinant FVIII protein exhibits procoagulant activity. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 14, 15, 16)
- a first polypeptide comprising Formula I;
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12. A recombinant FVIII protein comprising:
- a first polypeptide comprising Formula I;
(A1)-a1-(A2)-a2-[B]; and
a second polypeptide comprising Formula II;
a3-(A3)-(C1);wherein the first polypeptide and the second polypeptide are fused or exist as a heterodimer; wherein, a) A1 is an A1 domain of FVIII;
b) A2 is an A2 domain of FVIII;
c) [B] is a B domain of FVIII, a fragment thereof, or is deleted;
d) A3 is an A3 domain of FVIII;
e) C1 is a C1 domain of FVIII;
f) a1, a2, and a3 are acidic spacer regions;wherein the A1 domain comprises a permissive loop-1 (A1-1) region and a permissive loop-2 (A1-2) region; wherein the A2 domain comprises a permissive loop-1 (A2-1) region and a permissive loop-2 (A2-2) region; wherein the A3 domain comprises a permissive loop-1 (A3-1) region and a permissive loop-2 (A3-2) region; wherein a first half-life extending moiety is inserted within the FVIII protein immediately downstream of amino acid 26 of SEQ ID NO;
1, corresponding to mature native human FVIII;wherein a second half-life extending moiety is inserted within the FVIII protein immediately downstream of amino acid 403 of SEQ ID NO;
1, corresponding to mature native human FVIII;wherein a third half-life extending moiety is inserted within the FVIII protein immediately downstream of amino acid 1656 of SEQ ID NO;
1, corresponding to mature native human FVIII;wherein the first half-life extending moiety, the second half-life extending moiety, or the third half-life extending moiety is selected from; (a) albumin; (b) albumin-binding polypeptide; (c) Fc; (d) PAS; (e) the C-terminal peptide (CTP) of the β
subunit of human chorionic gonadotropin;(f) polyethylene glycol (PEG); (g) hydroxyethyl starch (HES); (h) albumin-binding small molecules; (i) a clearance receptor, or fragment thereof, wherein the clearance receptor blocks binding of the recombinant FVIII protein to FVIII clearance receptors; (j) a low-density lipoprotein receptor-related protein 1 (LRP1) or a FVIII-binding fragment thereof; (k) an XTEN; and (l) any combination of (a)-(j); and wherein the recombinant FVIII protein exhibits procoagulant activity. - View Dependent Claims (17, 18, 19)
- a first polypeptide comprising Formula I;
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13. A recombinant FVIII protein comprising:
- a first polypeptide comprising Formula I;
(A1)-a1-(A2)-a2-[B]; and
a second polypeptide comprising Formula II;
a3-(A3)-(C1);wherein the first polypeptide and the second polypeptide are fused or exist as a heterodimer; wherein, a) A1 is an A1 domain of FVIII;
b) A2 is an A2 domain of FVIII;
c) [B] is a B domain of FVIII, a fragment thereof, or is deleted;
d) A3 is an A3 domain of FVIII;
e) C1 is a C1 domain of FVIII;
f) a1, a2, and a3 are acidic spacer regions;wherein the A1 domain comprises a permissive loop-1 (A1-1) region and a permissive loop-2 (A1-2) region; wherein the A2 domain comprises a permissive loop-1 (A2-1) region and a permissive loop-2 (A2-2) region; wherein the A3 domain comprises a permissive loop-1 (A3-1) region and a permissive loop-2 (A3-2) region; wherein a first half-life extending moiety is inserted within the FVIII protein immediately downstream of amino acid 26 of SEQ ID NO;
1, corresponding to mature native human FVIII;wherein a second half-life extending moiety is inserted within the FVIII protein immediately downstream of amino acid 1656 of SEQ ID NO;
1, corresponding to mature native human FVIII;wherein a third half-life extending moiety is inserted within the FVIII protein immediately downstream of amino acid 1720 of SEQ ID NO;
1, corresponding to mature native human FVIII;wherein the first half-life extending moiety, the second half-life extending moiety, or the third half-life extending moiety is selected from; (a) albumin; (b) albumin-binding polypeptide; (c) Fc; (d) PAS; (e) the C-terminal peptide (CTP) of the β
subunit of human chorionic gonadotropin;(f) polyethylene glycol (PEG); (g) hydroxyethyl starch (HES); (h) albumin-binding small molecules; (i) a clearance receptor, or fragment thereof, wherein the clearance receptor blocks binding of the recombinant FVIII protein to FVIII clearance receptors; (j) a low-density lipoprotein receptor-related protein 1 (LRP1) or a FVIII-binding fragment thereof; (k) an XTEN; and (l) any combination of (a)-(j); and wherein the recombinant FVIII protein exhibits procoagulant activity. - View Dependent Claims (20)
- a first polypeptide comprising Formula I;
Specification