FVIII fusion protein and use thereof
Disclosed are a coagulation factor VIII fusion protein which is conjugated with polyalkylene glycol and which has an extended cyclic half-life, a preparation method therefor and a use thereof, wherein the active moiety of the coagulation factor VIII is directly linked or indirectly linked by a peptide linker to a fusion partner, and the fusion protein is further conjugated to a polyalkylene glycol. The half-life is significantly increased compared to an FVIII fusion protein modified without polyalkylene glycol.
1. A polyalkylene glycol-conjugated coagulation factor VIII fusion protein, wherein the coagulation factor VIII active moiety (FVIII) is directly or indirectly linked to a fusion partner for prolonging half-life via a peptide linker to form the fusion protein, and the fusion protein is further conjugated to the polyalkylene glycol;
wherein the fusion partner is an immunoglobulin Fc fragment and the polyalkylene glycol is 40 kD branched polyethylene glycol;
the coagulation factor VIII active moiety is linked to the fusion partner via a peptide linker and the peptide linker includes a flexible peptide segment and a rigid peptide segment;
the rigid peptide segment is the carboxy terminal peptide of human chorionic gonadotropin β subunit and the rigid peptide segment comprises an amino acid sequence selected from:
(i)
(SEQ ID NO: 11)
PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ;
(ii)
(SEQ ID NO: 12)
SSSSKAPPPSLPSPSRLPGPSDTPILPQ;
(iii)
(SEQ ID NO: 13)
SSSSKAPPPS;
and
(iv)
(SEQ ID NO: 14)
SRLPGPSDTPILPQ;
and
the flexible peptide segment has a general formula (GS)a(GGS)b(GGGS)c(GGGGS)d, where a, b, c and d are integers greater than or equal to 0, and a+b+c+d≥1, and the flexible peptide segment has a sequence selected from the group consisting of:
(i)
(SEQ ID NO: 6)
GSGGGSGGGGSGGGGS;
(ii)
(SEQ ID NO: 7)
GSGGGGSGGGGSGGGGSGGGGSGGGGS;
(iii)
(SEQ ID NO: 8)
GGGGSGGGGSGGGGSGGGGS;
(iv)
(SEQ ID NO: 9)
GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS;
and
(v)
(SEQ ID NO: 10)
GGGSGGGSGGGSGGGSGGGS.
2. The polyalkylene glycol-conjugated coagulation factor VIII fusion protein according to claim 1 , wherein the coagulation factor VIII active moiety is a full-length or truncated human coagulation factor VIII derived from human, and wherein the full-length or truncated human coagulation factor VIII may contain one or more amino acid mutations, provided that it still retains the FVIII activity.
3. The polyalkylene glycol-conjugated coagulation factor VIII fusion protein according to claim 1 , wherein the conjugation of the fusion protein and the branched polyethylene glycol is at a random or specific position, and the conjugation position is selected from the group consisting of a free amino group, sulfhydryl group, sugar group and carboxyl group.
4. The polyalkylene glycol-conjugated coagulation factor VIII fusion protein according to claim 3 , wherein a modifier is used for the conjugation, and the modifier is represented by:
wherein 0≤m2≤6, 0≤m3≤6, and mPEG represents a monomethoxy terminated polyethylene glycol group.
5. A pharmaceutical composition comprising an effective amount of the polyalkylene glycol-conjugated coagulation factor VIII fusion protein according to claim 1 , and a pharmaceutically acceptable carrier.
6. A method for preventing and/or treating hemorrhagic diseases, comprising administering to an subject in need thereof the polyalkylene glycol-conjugated coagulation factor VIII fusion protein according to claim 1 , wherein the hemorrhagic disease is selected from hemorrhagic diseases in patients with congenital or acquired deficiency of FVIII, and spontaneous or surgical bleeding in patients with hemophilia A.
7. The polyalkylene glycol-conjugated coagulation factor VIII fusion protein according to claim 2 , wherein the coagulation factor VIII active moiety comprises the amino acid sequence shown in SEQ ID NO: 1 or 2.
8. The polyalkylene glycol-conjugated coagulation factor VIII fusion protein according to claim 1 , wherein the fusion partner is an IgG Fc fragment with reduced ADCC effect and/or CDC effect and/or enhanced binding affinity to the FcRn receptor, and wherein the IgG Fc fragment having an amino acid sequence selected from the group consisting of:
(i) the amino acid sequence shown in SEQ ID NO: 3,
(ii) the amino acid sequence shown in SEQ ID NO: 4, and
(iii) the amino acid sequence shown in SEQ ID NO: 5.
9. The polyalkylene glycol-conjugated coagulation factor VIII fusion protein according to claim 1 , wherein the branched polyethylene glycol is end-capped with methoxy.
10. The polyalkylene glycol-conjugated coagulation factor VIII fusion protein according to claim 1 , wherein the peptide linker comprises the amino acid sequence shown in SEQ ID NO: 15.