阅读说明：本技术 单链因子viii分子 (Single chain factor VIII molecules ) 是由 S·基斯特纳 C·昂格雷尔 J·道芬巴赫 P·赫伯纳 于 2020-05-08 设计创作，主要内容包括：本发明涉及重组因子VIII蛋白,其在单链中包括包含因子VIII的A1和A2结构域的重链部分和包含因子VIII的A3、C1和C2结构域的轻链部分,其中B-结构域在两个缺失中部分缺失,第一个导致存在可被凝血酶切割的确定的加工序列,并且第二个导致在位置R1664-R1667处不存在弗林蛋白酶切割识别位点。B-结构域的内部片段被保留。还提供了编码所述蛋白的核酸、宿主细胞和制备该蛋白的方法,以及包含所述蛋白、核酸或宿主细胞的药物组合物,其可用于治疗A型血友病。(The present invention relates to a recombinant factor VIII protein comprising in single chain a heavy chain portion comprising the a1 and a2 domains of factor VIII and a light chain portion comprising the A3, C1 and C2 domains of factor VIII, wherein the B-domain is partially deleted in two deletions, the first resulting in the presence of a defined processing sequence cleavable by thrombin and the second resulting in the absence of a furin cleavage recognition site at positions R1664-R1667. The internal fragment of the B-domain is retained. Also provided are nucleic acids encoding the proteins, host cells and methods of making the proteins, as well as pharmaceutical compositions comprising the proteins, nucleic acids or host cells, which are useful for treating hemophilia a.)

1. A recombinant factor VIII protein comprising, in a single chain, a heavy chain portion comprising the a1 and a2 domains of factor VIII and a light chain portion comprising the A3, C1 and C2 domains of factor VIII, wherein

a) In the recombinant factor VIII protein, 894 amino acids corresponding to the consecutive amino acids between F761 and P1659 of wild-type factor VIII as defined in SEQ ID NO 1 were deleted, resulting in a first deletion;

b) the recombinant factor VIII protein comprises a processing sequence spanning the first deletion site, the processing sequence comprising SEQ ID NO 2 or a sequence having at most one amino acid substitution in SEQ ID NO 2, wherein the processing sequence comprises a first thrombin cleavage site;

c) a deletion in the recombinant factor VIII protein of at least the amino acids R1664 to R1667 corresponding to wild-type factor VIII, resulting in a second deletion; and

d) the recombinant factor VIII protein comprises a second thrombin cleavage site, C-terminal to the second deletion and N-terminal to the A3 domain.

2. A recombinant factor VIII protein comprising, in a single chain, a heavy chain portion comprising the a1 and a2 domains of factor VIII and a light chain portion comprising the A3, C1 and C2 domains of factor VIII, wherein

a) The recombinant factor VIII protein comprises a processing sequence comprising SEQ ID NO 2 or a sequence having at most one amino acid substitution in SEQ ID NO 2, wherein the processing sequence comprises a first thrombin cleavage site;

b) optionally, directly on the C-terminal side of the processing sequence, the factor VIII protein comprises a heterologous sequence;

c) directly on the C-terminal side of the processing sequence or, if present, directly on the C-terminal side of the heterologous sequence, the factor VIII protein comprises a combined sequence having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11;

d) the recombinant factor VIII protein comprises a second thrombin cleavage site at the C-terminal side of SEQ ID NOS: 9-11,

wherein the factor VIII protein is optionally the factor VIII protein of claim 1.

3. The recombinant factor VIII protein of any one of the preceding claims, wherein the processing sequence is SEQ ID NO 4 or a sequence having at most one amino acid substitution in the sequence, wherein optionally S, Q or N of the C-terminal side of F, F is substituted.

4. The recombinant factor VIII protein of any one of the preceding claims, wherein the processing sequence is selected from the group consisting of SEQ ID NOs 4, 6, 7, or 8.

5. The recombinant factor VIII protein of any of the preceding claims, wherein the processing sequence is SEQ ID NO 4.

6. The recombinant factor VIII protein of any one of the preceding claims, wherein the amino acids corresponding to amino acids K1663 through L1674 of wild-type factor VIII are deleted, resulting in a second deletion.

7. The recombinant factor VIII protein according to any one of the preceding claims, which does not comprise a furin cleavage recognition site and does not comprise a sequence with more than 75% sequence identity to the furin cleavage recognition site RQR between the processing sequence and the combining sequence, preferably does not comprise a sequence with more than 40% sequence identity to SEQ ID NO. 15.

8. The recombinant factor VIII protein of any one of the preceding claims, comprising a processing sequence and a combined sequence at the C-terminal side of the processing sequence, wherein the sequence is selected from the group consisting of:

the combined sequence of SEQ ID NO. 4 and SEQ ID NO. 12,

the combined sequence of SEQ ID NO 5 and SEQ ID NO 12,

the combined sequence of SEQ ID NO 6 and SEQ ID NO 12,

the combined sequence of SEQ ID NO 7 and SEQ ID NO 12,

the combined sequence of SEQ ID NO 8 and SEQ ID NO 12,

the combined sequence of SEQ ID NO 3 and SEQ ID NO 13,

the combined sequence of SEQ ID NO.2 and SEQ ID NO. 13,

h.processing sequence of SEQ ID NO 3 and Combined sequence of SEQ ID NO 14,

wherein, optionally, the pooling sequence is directly at the C-terminal side of the processing sequence.

9. The recombinant factor VIII protein of any one of the preceding claims, further comprising a third thrombin cleavage site located between the A1 and A2 domains.

10. The recombinant factor VIII protein of any one of the preceding claims, which is a fusion protein having at least one heterologous fusion partner selected from the group consisting of an Fc region, an albumin binding sequence, albumin, a PAS polypeptide, a HAP polypeptide, a C-terminal peptide of the β subunit of chorionic gonadotropin, an albumin binding small molecule, polyethylene glycol, hydroxyethyl starch,

wherein, optionally, the heterologous fusion partner is inserted directly into the C-terminal side of the processing sequence.

11. A nucleic acid encoding the recombinant factor VIII protein of any one of the preceding claims, wherein the polynucleotide is optionally an expression vector suitable for expressing the recombinant factor VIII protein in a mammalian cell selected from the group comprising human cells.

12. A host cell comprising the nucleic acid of claim 11, wherein preferably the host cell is a mammalian cell comprising an expression vector suitable for expressing said recombinant factor VIII protein in said cell.

13. A method of producing a factor VIII protein, comprising culturing the host cell of claim 12 under conditions suitable for expression of the factor VIII protein and isolating the factor VIII protein, wherein the method optionally comprises preparing the factor VIII protein into a pharmaceutical composition.

14. A pharmaceutical composition comprising the recombinant factor VIII protein of any one of claims 1-10, the nucleic acid of claim 11, or the host cell of claim 12.

15. The pharmaceutical composition of claim 14, for use in the treatment of hemophilia a, wherein, optionally, the treatment is immune tolerance induction.

Summary of The Invention

In a first embodiment, the present invention provides a recombinant factor VIII protein comprising, in a single chain, a heavy chain portion comprising the a1 and a2 domains of factor VIII and a light chain portion comprising the A3, C1 and C2 domains of factor VIII, wherein

a) In the recombinant factor VIII protein, 894 amino acids corresponding to the consecutive amino acids between F761 and P1659 of wild-type factor VIII as defined in SEQ ID NO 1 were deleted, resulting in a first deletion;

b) the recombinant factor VIII protein comprises a processing sequence spanning the first deletion site, the processing sequence comprising SEQ ID NO 2 or a sequence having at most one amino acid substitution in SEQ ID NO 2, wherein the processing sequence comprises a first thrombin cleavage site;

c) a deletion in the recombinant factor VIII protein of at least the amino acids R1664 to R1667 corresponding to wild-type factor VIII, resulting in a second deletion; and

d) the recombinant factor VIII protein comprises a second thrombin cleavage site, C-terminal to the second deletion and N-terminal to the A3 domain.

In a second embodiment, the present invention provides a recombinant factor VIII protein comprising, in a single chain, a heavy chain portion comprising the a1 and a2 domains of factor VIII and a light chain portion comprising the A3, C1 and C2 domains of factor VIII, wherein

a) The recombinant factor VIII protein comprises a processing sequence comprising SEQ ID NO 2 or a sequence having at most one amino acid substitution in SEQ ID NO 2, wherein the processing sequence comprises a first thrombin cleavage site;

b) optionally, directly on the C-terminal side of the processing sequence, the factor VIII protein comprises a heterologous sequence;

c) directly on the C-terminal side of the processing sequence or, if present, directly on the C-terminal side of the heterologous sequence, the factor VIII protein comprises a combined sequence having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11;

d) the recombinant factor VIII protein comprises a second thrombin cleavage site at the C-terminal side of SEQ ID NO 9-11.

In a third embodiment, the factor VIII protein of the second embodiment (embodiment 2) is also the protein of the first embodiment (embodiment 1).

In a fourth embodiment, the recombinant factor VIII protein of any one of embodiments 1-3 comprises the sequence of SEQ ID NO 9.

In a fifth embodiment, in the recombinant factor VIII protein of any one of embodiments 1-4, corresponding to wild-type factor VIII as defined in SEQ ID NO 1

i) F761 and S1656;

ii) S762 and Q1657;

iii) Q763 and N1658; or

iv) N764 and P1659

Wherein the amino acids are part of the sequence defined in SEQ ID NO 2. This is due to the first deletion defined in the first embodiment.

In a sixth embodiment, in the recombinant factor VIII protein of any one of embodiments 1-5, the processing sequence is SEQ ID No.2 or a sequence having at most one amino acid substitution in said sequence, wherein, optionally, S, Q or N of the C-terminal side of F, F is substituted.

In a seventh embodiment, in the recombinant factor VIII protein of any one of embodiments 1-5, the processing sequence is SEQ ID No. 3 or a sequence having at most one amino acid substitution in said sequence, wherein, optionally, S, Q or N of the C-terminal side of F, F is substituted.

In an eighth embodiment, in the recombinant factor VIII protein of any one of embodiments 1-5, the processing sequence is SEQ ID No. 4 or a sequence having at most one amino acid substitution in said sequence, wherein, optionally, S, Q or N of the C-terminal side of F, F is substituted.

In a ninth embodiment, in the recombinant factor VIII protein of any one of embodiments 1-5 or 8, the addition sequence is selected from the group consisting of SEQ ID NOs 4, 5, 6, 7 or 8.

In a tenth embodiment, in the recombinant factor VIII protein of embodiment 9, the processing sequence is SEQ ID NO 5. In an eleventh embodiment, in the recombinant factor VIII protein of embodiment 9, the processing sequence is SEQ ID NO 6. In a twelfth embodiment, in the recombinant factor VIII protein of embodiment 9, the processing sequence is SEQ ID NO 7. In a thirteenth embodiment, in the recombinant factor VIII protein of embodiment 9, the processing sequence is SEQ ID NO 8. In a fourteenth embodiment, in the recombinant factor VIII protein of embodiment 9, the processing sequence is SEQ ID NO 4.

In a fifteenth embodiment, in the recombinant factor VIII protein of any of the preceding embodiments, the processing sequence is located directly N-terminal to the amino acid corresponding to Q1675, I1681 or E1690 of wild-type factor VIII.

In a sixteenth embodiment, in the recombinant factor VIII protein of any of the preceding embodiments, the amino acids corresponding to amino acids K1663 through L1674 of wild-type factor VIII are deleted, resulting in a second deletion.

In a seventeenth embodiment, in the recombinant factor VIII protein of any one of embodiments 1 to 16, the amino acids corresponding to amino acids V1661 and L1674 of wild-type factor VIII as defined in SEQ ID No. 1, or the amino acids corresponding to amino acids L1662 and Q1675 of wild-type factor VIII, are adjacent to each other due to the second deletion. Both options may result in the same sequence.

In an eighteenth embodiment, in the recombinant factor VIII protein of any one of embodiments 1 to 16, the amino acids corresponding to amino acids V1661 and I1681 of wild-type factor VIII as defined in SEQ ID No. 1 are adjacent to each other due to the second deletion.

In a nineteenth embodiment, in the recombinant factor VIII protein of any one of embodiments 1 to 16, the amino acids corresponding to amino acids P1660 and I1681 of wild-type factor VIII as defined in SEQ ID No. 1 are adjacent to each other due to the second deletion.

In a twentieth embodiment, in the recombinant factor VIII protein of any one of embodiments 1-16, the amino acids corresponding to amino acids V1661 and E1690 of wild-type factor VIII as defined in SEQ ID No. 1 are adjacent to each other due to the second deletion.

In a twenty-first embodiment, the recombinant factor VIII protein of any of the preceding embodiments does not comprise a furin cleavage recognition site.

In a twenty-second embodiment, the recombinant factor VIII protein of any one of the preceding embodiments does not comprise a sequence having more than 75% (preferably, more than 50%) sequence identity to the furin cleavage recognition site RHQR between the processing sequence and the pooled sequence. The sequence corresponding to SEQ ID NO 15 containing the furin cleavage recognition site may be deleted. Optionally, the recombinant factor VIII protein of any one of the preceding embodiments does not comprise a sequence having more than 30% sequence identity to SEQ ID No. 15. Alternatively, it does not comprise a sequence having more than 40% sequence identity to SEQ ID NO. 15.

In a twenty-third embodiment, the recombinant factor VIII protein of any one of the preceding embodiments comprises a processing sequence and a combined sequence at the C-terminal side of said processing sequence, wherein the processing sequence is selected from the group comprising SEQ ID NOs 2, 3, 4, 5, 6, 7 or 8 and the combined sequence is selected from the group comprising SEQ ID NOs 12, 13 or 14.

In a twenty-fourth embodiment, in the recombinant factor VIII protein of the twenty-third embodiment, the processing sequence is SEQ ID NO. 4 and the consensus sequence is SEQ ID NO. 12. In a twenty-fifth embodiment, in the recombinant factor VIII protein of the twenty-third embodiment, the processing sequence is SEQ ID NO. 5 and the consensus sequence is SEQ ID NO. 12. In a twenty-sixth embodiment, in the recombinant factor VIII protein of the twenty-third embodiment, the processing sequence is SEQ ID NO 6 and the combined sequence is SEQ ID NO 12. In a twenty-seventh embodiment, in the recombinant factor VIII protein of the twenty-third embodiment, the processing sequence is SEQ ID NO 7 and the consensus sequence is SEQ ID NO 12. In a twenty-eighth embodiment, in the recombinant factor VIII protein of the twenty-third embodiment, the processing sequence is SEQ ID NO 8 and the consensus sequence is SEQ ID NO 12. In a twenty-ninth embodiment, in the recombinant factor VIII protein of the twenty-third embodiment, the processing sequence is SEQ ID NO 3 and the combined sequence is SEQ ID NO 13. In a thirtieth embodiment, in the recombinant factor VIII protein of the twenty-third embodiment, the processing sequence is SEQ ID NO.2 and the combined sequence is SEQ ID NO. 13. In a thirty-first embodiment, in the recombinant factor VIII protein of the twenty-third embodiment, the processing sequence is SEQ ID NO 3 and the consensus sequence is SEQ ID NO 14. In a thirty-second embodiment, in the recombinant factor VIII protein of any one of embodiments 23-31, the merging sequence is located directly C-terminal to the processing sequence.

In a thirty-third embodiment, the recombinant factor VIII protein of any one of the preceding embodiments is a fusion protein having at least one heterologous fusion partner selected from the group consisting of an Fc region, an albumin binding sequence, albumin, a PAS polypeptide, a HAP polypeptide, a C-terminal peptide of the β subunit of chorionic gonadotropin, an albumin binding small molecule, polyethylene glycol, hydroxyethyl starch.

In a thirty-fourth embodiment, in the recombinant factor VIII protein of embodiment 33, the heterologous fusion partner is inserted directly into the C-terminal side of the processing sequence. In a thirty-fifth embodiment, in the recombinant factor VIII protein of any one of embodiments 33 or 34, the heterologous fusion partner is inserted C-terminal to the C2 domain.

In a thirty-sixth embodiment, the recombinant factor VIII protein of any one of the preceding embodiments further comprises a third thrombin cleavage site located between the a1 and a2 domains.

As a thirty-seventh embodiment, the present invention further provides a nucleic acid encoding the recombinant factor VIII protein of any one of the preceding embodiments, wherein the polynucleotide is optionally an expression vector suitable for expressing the recombinant factor VIII protein in a mammalian cell, e.g., a human cell.

As a thirty-eighth embodiment, the present invention further provides a host cell comprising the nucleic acid of embodiment 37, wherein preferably the host cell is a mammalian cell comprising an expression vector suitable for expressing said recombinant factor VIII protein in said cell. The cell may be a human cell selected from the group comprising a Hek293 cell line or a CAP cell line. Preferably, the cell is a CAP cell line.

As a thirty-ninth embodiment, the present invention further provides a method of preparing a factor VIII protein comprising culturing the host cell of embodiment 38 under conditions suitable for expression of the factor VIII protein and isolating the factor VIII protein, wherein the method optionally comprises preparing the factor VIII protein as a pharmaceutical composition.

In a fortieth embodiment, the present invention provides a composition comprising the recombinant factor VIII protein of any one of embodiments 1-36, wherein the single chain protein factor VIII protein content of all factor VIII proteins is at least 90%.

As a forty-first embodiment, the present invention further provides a pharmaceutical composition comprising the recombinant factor VIII protein of any one of embodiments 1 to 36 or 40, the nucleic acid of embodiment 37 or the host cell of embodiment 38. The pharmaceutical composition may comprise a pharmaceutically acceptable solvent, such as water or a buffer, and/or a pharmaceutically acceptable excipient. In a forty-second embodiment, the pharmaceutical composition of embodiment 41 or the kit comprising the pharmaceutical composition of embodiment 41 further comprises an immunosuppressive agent, for example an immunosuppressive agent selected from the group comprising methylprednisolone, prednisolone, dexamethasone, cyclophosphamide, rituximab, and/or cyclosporine.

As a forty-third embodiment, the present invention further provides the pharmaceutical composition of any one of embodiments 41 or 42 for use in the treatment of hemophilia a, wherein optionally the treatment is Immune Tolerance Induction (ITI). In a forty-fourth embodiment, the pharmaceutical composition of any one of embodiments 41-43 is for use in treating a patient suffering from hemophilia a selected from the group comprising patients not previously treated with any factor VIII protein, patients previously treated with factor VIII protein, patients having an antibody response (including an inhibitory antibody response to factor VIII protein), and patients who have received ITI treatment, have an antibody response (including an inhibitory antibody response to factor VIII protein), or have not received ITI treatment.

In a forty-fifth embodiment, the present invention provides a vial, e.g., a pre-filled or ready-to-use syringe, containing the pharmaceutical composition of any one of embodiments 41-44.

In a forty-sixth embodiment, the present invention provides a method of treatment comprising administering an effective amount of the pharmaceutical composition of any one of embodiments 41-44 to a patient in need thereof, e.g., a patient suffering from hemophilia a, which may be selected from the group of patients defined herein.

Drawings

Figure 1 shows comparative single chain FVIII protein constructs in which the furin cleavage recognition site was deleted. The variants AC _ SC-V1(V1) and V3(V3) have native thrombin cleavage sites (PR/SV or SC/SV, respectively) and have only a single deletion of aa (amino acids) 760-. The variants AC _ SC-V2(V2) and-V4 (V4) comprise different thrombin cleavage sites (PR/VA or IR/SV respectively) compared to V1 and V2, wherein V2 comprises a VA sequence that can be considered to be derived from the B-domain based on V1. Based on V3, V4 contained an insertion of DPR-IRSV-VAQ at the deletion site.

FIG. 2 shows wild-type FVIII (A) and single chain FVIII protein constructs AC _ SC-V0(B), -V5(C), -V6(D), and-V7 (E) (alternatively, abbreviated as V0, V5, V6, V7) that are based on RectorAmino acid sequence AC-6rs-Ref SC, two deletions are introduced. The arrow indicates the thrombin cleavage site. Sig is the signal peptide (19 aa). Bold and italicized letters indicate the amino acids of the thrombin cleavage recognition site that are cleaved after aa 759 (i.e., in the processing sequence of the construct of the invention). The furin cleavage recognition site is underlined in the wild-type protein. Italicized numbers in parentheses relate to amino acid numbering in the wild-type sequence.

Figure 3 comparison of in vitro functionality of unpurified single chain FVIII variants. Cell culture supernatants of CAP-T cells expressing the double-chain FVIII molecule AC-6rs-REF and the single-chain FVIII variants AC _ SC-V0, -V1, -V2, -V5, -V6, -V7 were analyzed for chromogenic FVIII activity (A), actin FSL-induced FVIII coagulation activity (B), total FVIII protein amount (C) as indicated by FVIII antigen levels. The specific chromogenic activity was calculated as the ratio of chromogenic FVIII activity to FVIII antigen, shown as% (D). The specific coagulation activity was calculated as the ratio of coagulation FVIII activity to FVIII antigen, shown as% (E). n is 2.

FIG. 4 shows V0 and ReFactorComparison of the stability (determined by the chromogenic activity) in vitro for 24 hours (A) and 14 days (B). In A, stability was assayed in buffer, ReFactoIndicated by diamonds and V0(AC-SC) by squares. In B, REFACT is shownIn FVIII preparationsIn the wash (diamonds) and in FVIII-depleted plasma (FVIII-dp) (squares), and V0 in the buffer (triangles) and in FVIII-dp (crosses).

FIG. 5 shows V0 (solid line) and ReFacto(dotted line) regression curve of normalized activity (determined by chromogenic activity) as a result of non-compartmental analysis of in vivo pharmacokinetic studies in hemophilia a mice.

Sequence listing

Wild type factor VII of SEQ ID NO 1

2 PRSFSQNPP minimal processing sequence of SEQ ID NO

3 PRSFSQNPPV processing sequence of SEQ ID NO

4 PRSFSQNPPVL processing sequence

5 PRSXSQNPPVL processing sequence of SEQ ID NO

6 PRSFXQNPPVL processing sequence of SEQ ID NO

7 PRSFSXNPPVL processing sequence of SEQ ID NO

8 PRSFSQXPPVL processing sequence of SEQ ID NO

9 QSDQEEIDYD merge sequences, e.g., in V0

10 IDYDDTI pooled sequences, e.g. in V5+ V6

11 EMKKEDFD merge sequences, e.g. in V7, SEQ ID NO

12Q 1675 to R1708 von V0, SEQ ID NOs, pooled sequences, e.g., in V0

13I 1681 to R1708 von V6, SEQ ID NO, the sequences being combined, for example, in V6

14E 1690 to R1708 von V7, incorporated sequences, e.g., in V7

SEQ ID NO. 15 KRHQREITRTT sequence comprising a furin cleavage recognition site deleted in the protein of the invention

16V 0 aa sequence

17V 1 aa sequence of SEQ ID NO

18V 2aa sequence of SEQ ID NO

19V 3aa sequence of SEQ ID NO

20V 4aa sequence of SEQ ID NO

21V 5aa sequence of SEQ ID NO

22V 6aa sequence of SEQ ID NO

23V 7aa sequence of SEQ ID NO

24V 0 na sequence of SEQ ID NO

25V 5 na sequence of SEQ ID NO

26V 6 na sequence of SEQ ID NO

27V 7 na sequence of SEQ ID NO

28 AC-6rs-REFaa sequence of SEQ ID NO

29 AC-6rs aa sequence of SEQ ID NO

30 AC-6rs-REF na sequence of SEQ ID NO

31 SVEMKKEDF incorporation of the sequence in V1

32 DSYEDISAYLLSKNNAIEPR sequence on the N-terminal side of the processing sequence and including 2aa of the processing sequence, the sequence N-terminal to the thrombin cleavage site

Part of the FVIII sequence SEQ ID NO 33-38.

Detailed Description

In a first embodiment, the present invention provides a recombinant factor VIII protein comprising, in a single chain, a heavy chain portion comprising the a1 and a2 domains of factor VIII and a light chain portion comprising the A3, C1 and C2 domains of factor VIII, wherein

a) In the recombinant factor VIII protein, 894 amino acids corresponding to the consecutive amino acids between F761 and P1659 of wild-type factor VIII as defined in SEQ ID NO 1 were deleted, resulting in a first deletion;

b) the recombinant factor VIII protein comprises a processing sequence spanning the first deletion site, the processing sequence comprising SEQ ID NO 2 or a sequence having at most one amino acid substitution in SEQ ID NO 2, wherein the processing sequence comprises a first thrombin cleavage site;

c) a deletion in the recombinant factor VIII protein of at least the amino acids R1664 to R1667 corresponding to wild-type factor VIII, resulting in a second deletion; and

d) the recombinant factor VIII protein comprises a second thrombin cleavage site, C-terminal to the second deletion and N-terminal to the A3 domain.

The present inventors have explained novel FVIII single chain variants in which the region comprising the B-domain deletion is shorter than in other single chain FVIII variants. In particular, by retaining internal fragments of the B-domain, the inventors have sought to form a processing sequence that is closer to the wild-type processing sequence (see SEQ ID NO. 2). Preferably, the part of the processing sequence corresponds to a truncated B-domain, and thus the processing sequence is embedded in a sequence derived from wild-type FVIII. As a result of these findings, the resulting FVIII protein exhibits high levels of expression, low characteristics of fragments and by-products, and in particular a high specific activity as demonstrated by different biological activity assays. Furthermore, the present inventors have found that certain amniotic fluid cell lines are particularly suitable for high level expression of functional molecules.

Further advantages and preferred embodiments are explained elsewhere in this specification.

Side-by-side comparison with either prior art double-stranded FVIII (using morocog Alfa sequences, see examples) or single-stranded variants (variants V1 and V2, see examples) showed binding of high expression levels with improved specific activity. FVIII proteins according to the invention also gave much better results than other single chain variants (V3, V4) designed and tested.

The skilled person understands the term FVIII (or factor VIII) and knows the structure and biological function of wild type FVIII and typical variants thereof. In addition to the sequences specified above, the FVIII protein of the invention may be designed as deemed suitable and advantageous by the skilled person. In particular, the factor VIII protein of the invention should generally comprise all essential parts and domains known to be important for biological function. For example, preferably, the FVIII protein further comprises domains corresponding, substantially corresponding and/or functionally corresponding to the a and C domains of wild type FVIII. It may further comprise additional portions and domains. For example, preferably, the FVIII protein further comprises a1 domain between the a1 and a2 domains and a2 domain C-terminal to the a2 domain, wherein the processing sequence is C-terminal to the a2 domain. The part of the processing sequence corresponds to the truncated B-domain. On the C-terminal side of the processing sequence, the FVIII protein comprises at least one truncated a3 domain, which may comprise a merging sequence as defined herein. Prior to processing, the factor VIII proteins of the invention may also comprise a signal sequence. Any or all of the domains may be wild-type (wt) FVIII domains, or they may be different from wild-type domains, for example, as known in the art or as deemed appropriate by the skilled person. The domains are preferably included in the protein in this order, i.e., from the N-terminus to the C-terminus of the protein.

The FVIII protein according to the invention should have at least one biological activity or function of the wt FVIII protein, in particular in terms of coagulation function. FVIII protein should be cleaved by thrombin, leading to activation. Preferably, the thrombin recognition and/or thrombin cleavage sites correspond to or substantially correspond to those of wild-type FVIII. It is then able to form a complex with activated coagulation factor IXa and coagulation factor X, and the light chain is able to bind to a phospholipid bilayer, e.g. the cell membrane of (activated) platelets.

The biological activity of FVIII can be determined by assaying the chromogenic or clotting activity of the protein, as described herein. Generally, chromogenic activity is taken as a measure of biological activity.

Other parts of the FVIII protein of the invention may be designed according to the needs of the skilled person, but preferably retain high FVIII bioactivity. As shown in the examples, the present invention allows for the production of FVIII proteins with high biological activity as measured, for example, by chromogenic activity. Thus, preferably, the FVIII protein according to the invention has a chromogenic activity at least comparable to the activity of the wt protein, i.e. it has at least 50% of the chromogenic activity of the wt protein (SEQ ID NO: 1). Preferably, the FVIII protein according to the invention has at least 80%, at least 100% or more than 100% of the chromogenic activity of wt protein. Preferably, the chromogenic activity is also ReFacto(International non-patent name: Morocog Alfa) (a commercially available B-Domain deleted FVIII (pfeira)) at least 80%, at least 90%, at least 100% or more than 100% of the chromogenic activity.

As defined in a), 894 amino acids in the FVIII according to the invention, corresponding to the consecutive amino acids between F761 and P1659 of the wild type factor VIII as defined in SEQ ID No. 1, were deleted in the factor VIII protein according to the invention, resulting in a first deletion. In certain embodiments, in particular, the term "corresponding to" is understood to mean "identical" starting from the amino acid numbering in FVIII without deletion or insertion.

For a particular amino acid which may be mutated as compared to wt, the amino acid corresponding to aa of the wild type is determined by alignment, for example using EMBOSS Needle (based on the Needleman-Wunsch algorithm; settings: MATRIX: "BLOSUM 62", GAP OPEN: "20", GAP EXTEND: "0.5", END GAP PENALTY: "false", END GAP OPEN: "10", END GAP EXTEND: "0.5").

To assess the sequence identity of two polypeptides, this alignment can be performed in two steps: I. global protein alignments were performed using EMBOSS Needle (settings: MATRIX: "BLOSUM 62", GAP OPEN: "20", GAP EXTEND: "0.5", END GAP PENALTY: "false", END GAP OPEN: "10", END GAP EXTEND: "0.5") to identify specific regions of highest similarity. Exact sequence identity was defined by comparing the fully overlapping polypeptide sequences identified in (I) using an EMBOSS Needle (settings: MATRIX: "BLOSUM 62", GAP OPEN: "20", GAP EXTEND: "0.5", END GAP PENALTY: "false", END GAP OPEN: "10", END GAP EXTEND: "0.5") while excluding a second alignment of unpaired amino acids.

The "between" does not include the recited amino acids, for example, it means that the recited amino acids are retained. "deletion" or "deleted" does not necessarily mean that the protein is actually prepared by deleting an amino acid previously present in a precursor molecule, but is defined only as the absence of the amino acid, regardless of the preparation of the molecule. For example, proteins can be produced based on nucleic acids prepared by de novo synthesis or by genetic engineering techniques.

As defined in b), the recombinant factor VIII protein comprises a processing sequence spanning the first deletion site, said processing sequence comprising SEQ ID No. 2(PRSFSQNPP) or a sequence having at most one amino acid substitution in SEQ ID No.2, wherein said processing sequence comprises a first thrombin cleavage site. Thus, at least one amino acid of the processing sequence corresponds to the amino acid on the C-terminal side of the deletion, and at least one amino acid of the processing sequence corresponds to the amino acid on the N-terminal side of the deletion. The processing sequence comprises SEQ ID NO 2 or a sequence having at most one amino acid substitution in SEQ ID NO 2, i.e. the processing sequence may be longer. In particular, the processing sequence is selected from the group comprising SEQ ID NO 2, 3, 4, 5, 6, 7 or 8. The inventors have found that the processing sequences of the invention are capable of particularly good cleavage by thrombin.

Generally, the processing sequence is NO longer than SEQ ID NO 4. The processing sequence may be located directly on the C-terminal side of the sequence from the a2 domain, for example the wt a2 domain sequence. The first two N-terminal amino acids of the processing sequence may already belong to the a2 domain. Preferably, the amino acid on the direct N-terminal side of the processing sequence is E.

2 can be substituted, for example, to reduce immunogenicity. Optionally, S, Q or N of the C-terminal side of F, F is substituted.

The processing sequence may be SEQ ID No.2 or a sequence having at most one amino acid substitution in said sequence, wherein, optionally, S, Q or N of the C-terminal side of F, F is substituted. For example, the FVIII proteins of V0, V5, V6 and V7 of the present invention comprise SEQ ID NO 2. The processing sequence of V6 consists of SEQ ID NO 2.

Alternatively, the processing sequence may be SEQ ID NO 3(PRSFSQNPPV) or a sequence having at most one amino acid substitution in said sequence, wherein, optionally, S, Q or N on the C-terminal side of F, F is substituted. For example, the FVIII proteins of V0, V5, and V7 of the present invention comprise SEQ ID NO 3. The processing sequences of V5 and V7 consist of SEQ ID NO 3.

Alternatively, the processing sequence may be SEQ ID NO 4(PRSFSQNPPVL) or a sequence having at most one amino acid substitution in said sequence, wherein, optionally, S, Q or N on the C-terminal side of F, F is substituted. For example, the inventors have shown that L at the C-terminus of the processing sequence (as in SEQ ID NO:4, 5, 6, 7 or 8) confers particularly good activity on FVIII. It has been found that the processing sequence of FVIII protein V0 of the present invention consists of SEQ ID NO. 4, which is a specific embodiment of SEQ ID NO. 5-8.

The substitution processing sequences SEQ ID NO 5(PRSXSQNPPVL), SEQ ID NO 6(PRSFXQNPPVL), SEQ ID NO 7(PRSFSXNPPVL) and SEQ ID NO 8(PRSFSQXPPVL) are variants, wherein X can be any naturally occurring amino acid. Optionally, X is a conservative substitution, i.e., a hydrophobic amino acid is substituted with a hydrophobic amino acid, a hydrophilic amino acid is substituted with a hydrophilic amino acid, an aromatic amino acid is substituted with an aromatic amino acid, an acidic amino acid is substituted with an acidic amino acid and a basic amino acid is substituted with a basic amino acid, as compared to the corresponding amino acid in SEQ ID NO 4.

As defined in c), in the FVIII protein of the invention the amino acids corresponding to amino acids R1664 to R1667 of wild type factor VIII are deleted, resulting in a second deletion. These amino acids correspond to the furin cleavage recognition site of wt FVIII. Thus, the protein is not substantially cleaved by furin. In the composition, at least 80%, optionally at least 90% or at least 95% of the FVIII protein of the invention is present in single chain form.

The recombinant factor VIII protein of the invention comprises a second thrombin cleavage site, as defined in d), at the C-terminal side of the second deletion and N-terminal of the A3 domain. Thus, upon activation, the portion of the FVIII protein between the thrombin cleavage site and the second thrombin cleavage site in the processing sequence is cleaved from the activated FVIII protein.

As a second embodiment, the invention also provides a recombinant factor VIII protein comprising, in single chain, a heavy chain portion comprising the A1 and A2 domains of factor VIII and a light chain portion comprising the A3, C1 and C2 domains of factor VIII, wherein,

a) the recombinant factor VIII protein comprises a processing sequence comprising SEQ ID NO 2 or a sequence having at most one amino acid substitution in SEQ ID NO 2, wherein the processing sequence comprises a first thrombin cleavage site;

b) optionally, directly on the C-terminal side of the processing sequence, the factor VIII protein comprises a heterologous sequence;

c) directly on the C-terminal side of the processing sequence or, if present, directly on the C-terminal side of the heterologous sequence, the factor VIII protein comprises a combined sequence having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID No. 9(QSDQEEIDYD), SEQ ID No. 10 (idydti) and SEQ ID No. 11 (EMKKEDFD);

d) the recombinant factor VIII protein comprises a second thrombin cleavage site at the C-terminal side of SEQ ID NO 9-11.

The factor VIII protein is optionally a factor VIII protein as described above in the first embodiment. In any case, the definitions provided herein apply to both embodiments.

As described in b), optionally, directly on the C-terminal side of the processing sequence, the factor VIII protein comprises a heterologous sequence. The heterologous sequence is not present in the same relative position in the wild-type protein, and it optionally does not consist of the same number of amino acids as the sequence deleted from the protein of the invention. Preferably, the heterologous sequence comprises a non-FVIII sequence of at least 10, optionally at least 20, at least 30 or at least 40 amino acids not present in wild type FVIII and preferably has less than 30% sequence identity to any wild type FVIII fragment, optionally less than 25% sequence identity to any wild type FVIII fragment. In particular, it may have less than 20% sequence identity to any wt FVIII sequence from the B-domain or the a3 domain. However, the heterologous sequence may comprise a subsequence found in FVIII. Optionally, at least 60% of the heterologous sequence is non-FVIII sequence.

As described in C), directly on the C-terminal side of the processing sequence or, if present, directly on the C-terminal side of the heterologous sequence, the factor VIII protein comprises a consensus sequence having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID No. 9(QSDQEEIDYD), SEQ ID No. 10 (idydti) and SEQ ID No. 11 (EMKKEDFD). The term "pooled sequence" illustrates that this sequence is pooled together with the processing sequence into the final sequence, and optionally directly on the C-terminal side of the sequence, which can be achieved by a deletion in wt FVIII. The pooled sequence may also be referred to as an "a 3-derived" sequence, which exemplifies the origin of the sequence. It does not necessarily mean that the sequence corresponds to all of the a3 sequences contained in the recombinant factor VIII protein of the invention. Preferably, the N-terminal side of the defined pooled sequences is free of a 3-derived amino acids, but, especially for SEQ ID NOS: 9-11, the C-terminal side of the defined sequences may be present with a 3-derived amino acids, e.g., as defined in SEQ ID NOS: 12, 13 or 14.

Preferably, the factor VIII protein may comprise a pooled sequence selected from the group consisting of SEQ ID NO 9, 10 or 11. The inventors could prove advantageous if the FVIII protein comprises a longer sequence corresponding to part of the a3 domain of the C-terminus of SEQ ID NO 9, preferably resulting in the sequence of SEQ ID NO 12. The same applies to SEQ ID NO 10, preferably the sequence giving rise to SEQ ID NO 13, and to SEQ ID NO 11, preferably the sequence giving rise to SEQ ID NO 14. Thus, preferably, the recombinant factor VIII protein comprises SEQ ID NO 9. For example, the V0 protein comprises SEQ ID NO 9. It also comprises SEQ ID NO 10 and SEQ ID NO 11, which are derived (at least in part) from the a3 region on the C-terminal side of SEQ ID NO 9. V5 and V6 comprise SEQ ID NO 10 and V7 comprises SEQ ID NO 11.

The factor VIII protein of the present invention may also be defined as corresponding to wild-type factor VIII as defined in SEQ ID NO 1

i) F761 and S1656;

ii) S762 and Q1657;

iii) Q763 and N1658; or

iv) N764 and P1659

Wherein the amino acids are part of the sequence defined in SEQ ID NO 2. This is due to the first deletion defined in the first embodiment. This is due to the first deletion defined in the first embodiment. The skilled person will note that this particular construction results in maintaining the amino acids identical without and with deletions. Thus, the structure of the protein is less affected by the deletion than other deletions of the B-domain known in the art. "adjacent" in the context of the present invention is synonymous with "directly adjacent" and relates to the secondary structure of the protein.

In an embodiment of the invention wherein no heterologous sequence is inserted directly into the C-terminal side of the processing sequence, it is further preferred that the processing sequence is located directly N-terminal to the amino acids Q1675, I1681 or E1690 corresponding to wild-type factor VIII. This occurs, for example, in Q1675 in V0, I1681 in V5 and V6, and E1690 in V7.

In the factor VIII proteins of the present invention, the 7N-terminal amino acids of the alpha 3-domain are preferably absent (i.e., deleted), i.e., the alpha 3-domain is partially deleted or truncated.

In the factor VIII protein of the invention, the amino acids corresponding to amino acids K1663 to L1674 of wild-type factor VIII may be deleted, resulting in a second deletion. This is the case, for example, in V0 and V5-V7.

In the FVIII protein according to the invention, the amino acids corresponding to amino acids V1661 and L1674 of wild type factor VIII as defined in SEQ ID No. 1 may be adjacent to each other or the amino acids corresponding to amino acids L1662 and Q1675 of wild type factor VIII may be adjacent to each other due to the second deletion. V0 is an example of a protein suitable for use herein. In the case where the heterologous sequence is inserted directly into the C-terminal side of the processing sequence, the amino acids corresponding to amino acids L1662 and Q1675 of wild-type factor VIII are not adjacent to each other.

In other FVIII proteins of the invention, e.g. V5, the amino acids corresponding to amino acids V1661 and I1681 of wild type factor VIII as defined in SEQ ID NO:1 are adjacent to each other due to the second deletion.

In other FVIII proteins of the invention, e.g. V6, the amino acids corresponding to amino acids P1660 and I1681 of wild type factor VIII as defined in SEQ ID NO:1 are adjacent to each other due to the second deletion.

In other FVIII proteins according to the invention, e.g. V7, the amino acids corresponding to amino acids V1661 and E1690 of wild type factor VIII as defined in SEQ ID No. 1 are adjacent to each other due to the second deletion.

The recombinant factor VIII proteins of the present invention do not comprise a furin cleavage recognition site. Preferably, it also does not comprise a sequence having more than 75% sequence identity to the furin cleavage recognition site RHQR between the processing sequence and the merging sequence. Deletion of the furin cleavage recognition site has been found to be superior to other mutations, e.g., substitutions. Optionally, the recombinant factor VIII protein of the invention does not comprise a sequence with more than 50% sequence identity to the furin cleavage recognition site RHQR between the processing sequence and the pooled sequence.

Preferably, other amino acids near the furin cleavage recognition site are also deleted. Thus, preferably, the FVIII proteins of the present invention do not comprise a sequence having more than 30% sequence identity to SEQ ID NO:15(KRHQREITRTT, amino acids K1663-T1673 of SEQ ID NO:1) comprising a furin cleavage recognition site. Optionally, they do not comprise a sequence having more than 40% sequence identity to SEQ ID NO. 15.

Since the furin cleavage recognition site is absent, the protein is substantially not cleaved by furin, and the single chain protein factor VIII protein content in all factor VIII proteins is at least 90%.

Particularly good results in terms of expression and activity have been found for the recombinant factor VIII protein of the present invention comprising a processing sequence and a combined sequence at the C-terminal side of said processing sequence, wherein the processing sequence is selected from the group comprising SEQ ID NO 2, 3, 4, 5, 6, 7 or 8 and the combined sequence is selected from the group comprising SEQ ID NO 12, 13 or 14.

Thus, the present invention also provides a recombinant factor VIII protein comprising a processing sequence and a combined sequence at the C-terminal side of said processing sequence, wherein the processing sequence is selected from the group comprising SEQ ID NO 2, 3, 4, 5, 6, 7 or 8 and the combined sequence is selected from the group comprising SEQ ID NO 12, 13 or 14.

For example, the processing sequence may be SEQ ID NO. 4 and the sequence SEQ ID NO. 12 is combined, for example in construct V0. The processing sequence may also be SEQ ID NO 5 and the combined sequences SEQ ID NO 12. The processing sequence may be SEQ ID NO 6 and the combined sequences SEQ ID NO 12. The processing sequence may be SEQ ID NO 7 and the combined sequences SEQ ID NO 12. The processing sequence may be SEQ ID NO 8 and the combined sequences SEQ ID NO 12. The processing sequence may be SEQ ID NO 3 and the combined sequence SEQ ID NO 13, for example in V5. The processing sequence may be SEQ ID NO 2 and the combined sequence SEQ ID NO 13, for example in V6. The processing sequence may be SEQ ID NO 3 and the combined sequence SEQ ID NO 14, for example as in V7. Optionally, the pooling sequence is located directly at the C-terminal side of the processing sequence. Alternatively, a heterologous sequence may be inserted between the processing sequence and the combining sequence, e.g., as defined below for the fusion partner.

The recombinant factor VIII proteins of the invention typically further comprise a third thrombin cleavage site between the a1 and a2 domains. It may also contain further thrombin cleavage sites as long as biological function is maintained, but this is not essential.

Preferred FVIII proteins of the invention comprise the amino acid sequence of any one of SEQ ID NO 16(V0), 21(V5), 22(V6) or 23(V7), preferably the mature protein of SEQ ID NO 16 (i.e., NO signal sequence), or a fusion protein of any of these proteins.

Thus, the present invention provides a recombinant factor VIII molecule comprising an amino acid sequence according to SEQ ID NO 16, 21, 22 or 23 (each without a signal sequence), or a fusion protein comprising at least one of these sequences. Proteins comprising the amino acid sequence according to SEQ ID NO 16 (without signal sequence) have proven to have particularly good properties. The signal sequence corresponds to amino acids 1-19 of the respective protein. In mature proteins commonly used, in particular for pharmaceutical purposes, the signal sequence is often absent. However, signal sequences may also be comprised in the proteins of the invention.

Fusion partners may be used to extend the in vivo plasma half-life of FVIII proteins of the invention. In one embodiment, the recombinant factor VIII protein of the invention is a fusion protein with at least one heterologous fusion partner, preferably a fusion partner that extends the in vivo plasma half-life of the FVIII protein. The fusion partner may, for example, be selected from the group comprising an Fc region, albumin, an albumin binding sequence, a PAS polypeptide, a HAP polypeptide, a C-terminal peptide of the β subunit of chorionic gonadotropin, an albumin binding small molecule, and combinations thereof. FVIII protein may alternatively or additionally be covalently linked to non-protein fusion partners such as PEG (polyethylene glycol) and/or HES (hydroxyethyl starch). PAS polypeptides or PAS sequences are polypeptides comprising an amino acid sequence comprising predominantly alanine and serine residues or predominantly alanine, proline and serine residues, which PAS sequences form a random coil conformation under physiological conditions, as defined in WO 2015/023894. The HAP polypeptide or sequence is a homo-type amino acid polymer (HAP) comprising, for example, glycine as defined in WO 2015/023894 or a repeated sequence of glycine and serine. Potential fusions, fusion partners and combinations thereof are described in more detail in, for example, WO 2015/023894.

Optionally, for certain therapeutic applications, the recombinant FVIII protein may be fused to an Fc region. Fusions to the Fc region can be used to extend half-life and reduce immunogenicity.

The inventors have found that the heterologous fusion partner may advantageously be inserted directly into the C-terminal side of the processing sequence and/or the C-terminal side of the C2 domain. The inventors have found that these positions facilitate fusion while retaining good biological activity of the FVIII protein. Optionally, the fusion protein further comprises at least one linker.

The protein may be further glycosylated and/or sulfated. Preferably, post-translational modifications of the protein, such as glycosylation and/or sulfation, occur in human cells. Particularly suitable posttranslational modification profiles can be achieved using CAP cells, in particular CAP-T cells or CAP-Go cells ((WO 2001/36615; WO 2007/056994; WO 2010/094280; WO 2016/110302)). CAP cells available from Cevec Pharmaceuticals GmbH (Colon, Germany) are derived from human amniotic fluid cells because they are isolated abdominally during conventional amniocentesis. The obtained amniotic cells were transformed with adenovirus functions (E1A, E1B and pIX functions) and subsequently adapted to growth in suspension in serum-free medium.

wt FVIII is typically bound by vWF. vWF prevents degradation and may positively influence immune tolerance. Thus, in certain embodiments of the inventionThe protein should be capable of binding to vWF. For example, the binding potency of a FVIII protein of the invention to vWF is ReFacto10% -100%, 10% -90%, 20-80%, 30-70%, 40-60% or 50-60% of the binding potency to vWF, as determined by ELISA-based methods. vWF binding is mediated in particular by amino acid positions Y1683 and Y1699. If vWF binding is desired, these should not be mutated.

In certain cases, it may be the case or even desirable that the FVIII protein is not able to bind to vWF. This may be the case, for example, if stabilization is mediated by a different means than vWF binding. To avoid vWF binding, e.g. amino acids Y1683 and/or Y1699 may be mutated, or vWF binding may be sterically hindered by different binding partners.

Advantageously, the FVIII protein according to the invention can be sufficiently stable for pharmaceutical use. The inventors can demonstrate the stability and ReFactor of the FVIII proteins of the invention in vitro and in vivoSee the examples for comparable stability. Thus, the proteins of the invention are preferably sufficiently stable in human plasma in vitro and/or in vivo, in particular in vivo. Preferably, the FVIII protein of the invention has a half-life in human plasma (in patients without inhibitor) of about at least 6 hours, preferably at least 12 hours, at least 18 hours, at least 24 hours, or at least 30 hours in vivo. The FVIII protein may be a FVIII protein without a fusion partner, as defined herein, or it may be a fusion protein as defined herein. However, as shown in the examples, the indicated half-life can already be obtained without a fusion partner. In the presence of one or more fusion partners, the half-life of the FVIII protein may be the same, or even longer.

The invention also provides nucleic acids encoding the recombinant factor VIII proteins of the invention. The polynucleotide may be an expression vector, for example suitable for expressing the recombinant factor VIII protein in mammalian cells, such as human cells.

The nucleic acid preferably encodes a FVIII with an N-terminal signal sequence, e.g., the 19aa signal sequence of SEQ ID NO 1. Preferred nucleic acids of the invention encode SEQ ID NO 16 and 21-23(V0, V5, V6, V7), or optionally, fusion proteins thereof. They may be SEQ ID NOS: 24-27. The nucleic acid of the present invention may be a DNA molecule or an RNA molecule. The nucleic acid may be optimized for expression in a host cell, for example, in a human cell.

The expression vector comprises a sequence encoding the FVIII protein, preferably in codon-optimized form, under the functional control of a suitable promoter, which may be a constitutive or inducible promoter. The promoter may be one which is not associated with expression of FVIII in nature, such as EF-1. alpha. or a heterologous promoter, such as CMV or SV 40. It may further comprise prokaryotic and/or eukaryotic selection markers, such as ampicillin resistance and dihydrofolate reductase (dhfr), and an origin of replication, for example the SV40 origin and/or the pBR322 origin. By "codon-optimized" is meant optimized for expression in a host cell, preferably for expression in a human host cell.

Alternatively, the nucleic acid may be a vector suitable for gene therapy, e.g., suitable for gene therapy of a human patient. Vectors suitable for gene therapy are known in the art, e.g., viral-based vectors, such as adenovirus-or adeno-associated virus (AAV) -based or retrovirus-based (e.g., lentiviral vectors, etc.), or non-viral-based vectors, such as, but not limited to, miniplasmids and minicircle-or transposon-based vectors. The AAV-based vectors of the invention can be, for example, packaged in AAV particles for gene therapy in patients with hemophilia a.

The invention also provides host cells comprising a nucleic acid of the invention. The host cell may be a bacterial cell, a plant cell, a fungal cell, a yeast cell or an animal cell. Preferably, the host cell is an animal cell, in particular a mammalian cell comprising an expression vector suitable for expressing said recombinant factor VIII protein in said cell. The host cell is preferably a human cell comprising an expression vector suitable for expressing said recombinant factor VIII protein in said human cell. Cells can be transfected transiently or stably with the nucleic acids of the invention. The cell may be a cell line, a primary cell or a stem cell. For protein production, the cell is typically a cell line, e.g., a HEK cell, such as a HEK-293 cell, a CHO cell, a BHK cell, a human embryonic retina cell, such as Crucell' sper.c. 6, or a human amniotic fluid cell, such as CAP. For the treatment of a human patient with a protein, the host cell is preferably a human cell, such as a HEK293 cell line or a CAP cell line (e.g., a CAP-T cell or a CAP-Go cell). The inventors have found that in CAP cell lines, particularly high single chain content of FVIII protein of the invention is produced. In CAP cells, CAP-T cells are preferably used for transient expression, while CAP-Go cells can be used to create stable cell lines, imparting advantageous glycosylation characteristics to FVIII molecules.

The cells may be autologous cells of a hemophilia a patient, suitable for producing FVIII in the patient after transfection and reintroduction into the patient. The cells may be stem cells, such as hematopoietic stem cells, but are preferably not embryonic stem cells, particularly when the patient is a human. The cells may also be hepatocytes, sinusoidal liver endothelial cells, or platelets.

Cell lines expressing a protein of the invention can also be used in methods of making a protein of the invention, including culturing the cells under conditions suitable for expression of a FVIII protein and purifying the protein, e.g., using a variety of methods known to the skilled artisan, e.g., as described herein. Such purification methods may include standard harvesting procedures for cell removal, such as centrifugation, followed by chromatographic steps, such as affinity chromatography, and methods of exchanging FVIII protein into a suitable buffer. The present invention therefore also provides a method for preparing a factor VIII protein, comprising culturing a host cell of the invention under conditions suitable for expression of the factor VIII protein and isolating the factor VIII protein, wherein the method optionally comprises preparing the factor VIII protein as a pharmaceutical composition.

The present invention therefore provides a pharmaceutical composition comprising a recombinant factor VIII protein of the invention, a nucleic acid of the invention or a host cell of the invention. Such pharmaceutical combinationsThe substance may comprise suitable excipients, such as a buffer, a stabilizer, a bulking agent, a preservative, another (e.g. recombinant) protein, or a combination thereof. In the context of the present invention, "a" and "an" are understood to mean one or more, if not explicitly stated otherwise. Suitable buffers for formulation may, for example, contain 205mM NaCl, 5.3mM CaCl in distilled water₂6.7mM L-histidine, 1.3% sucrose and 0.013% Tween 20 and has a pH of 7.0(FVIII formulation buffer). The buffer was used in the experiments described herein if not otherwise stated. The preparation of FVIII may be sterile, e.g. sterile filtered, especially for in vivo use.

The skilled person may suitably prepare the pharmaceutical composition as desired, e.g. for intravenous (i.v.) or subcutaneous application, intraperitoneal or intramuscular application. Generally, it is used for slow i.v. bolus administration. Continuous infusion is suitable, for example, for patients requiring admission to hospital for severe bleeding or surgery. Oral applications which may contribute to tolerance induction are also possible, for example after expression in plants. The pharmaceutical composition may be for sustained release.

The pharmaceutical composition comprising FVIII may be lyophilized. The dosage and treatment regimen may be selected as appropriate, for example for the prevention of bleeding or for intermittent on-demand treatment of bleeding episodes. The decision to administer the drug can be made by a physician. Administration depends on the patient, e.g., body weight, FVIII status, etc. For example, FVIII according to the invention may be administered intravenously at a dose of 0.5 to 250IU/kg body weight, typically 0.5 to 200IU/kg body weight every 0.5 to 6 days, depending on the severity of the disease. The present invention also provides pharmaceutical compositions comprising a FVIII protein of the invention in combination with an immunosuppressant (e.g. methylprednisolone, prednisolone, dexamethasone, cyclophosphamide, rituximab and/or cyclosporine) and/or it may be administered substantially simultaneously (e.g. within minutes to 12 hours) with such an agent.

Pharmaceutical compositions, e.g. comprising a protein of the invention, may be used for the treatment of patients in need thereof, in particular hemophilia a patients, e.g. patients suffering from acquired hemophilia or congenital hemophilia a involving an autoimmune response to FVIII. Mammals such as mice or dogs can be treated with the pharmaceutical compositions of the present invention, but the patient is typically a human patient.

The pharmaceutical compositions of the present invention may also be used to treat patients previously treated with recombinant and/or plasma factor VIII proteins. The pharmaceutical composition may, for example, be used for Immune Tolerance Induction (ITI) therapy in patients with antibodies comprising an inhibitory antibody response to recombinant and/or plasma factor VIII proteins. The compositions of the invention are therefore also useful in rescuing ITI. The pharmaceutical compositions may also be advantageously used in patients already having an antibody response, including an inhibitory antibody response to recombinant and/or plasma factor VIII proteins, e.g., patients already receiving ITI treatment. The pharmaceutical composition may also be advantageously used in patients who already have an antibody response, including an inhibitory antibody response to recombinant and/or plasma factor VIII proteins, and who have not been treated with ITI.

The invention also provides vials, e.g., syringes, comprising the pharmaceutical compositions of the invention. The syringe may be a pre-filled syringe, for example a ready-to-use syringe.

All publications cited herein are incorporated herein in their entirety. The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.

Examples

1. Production of Single-chain variants and determination of biological Activity

In the course of developing new recombinant type a haemophilia therapeutics, the possibility of using single chain FVIII molecules lacking furin cleavage recognition sites to inhibit enzymatic cleavage has been tested. The single chain FVIII molecules may be advantageous in terms of stability during purification and storage, but may also have in vivo benefits in terms of stability, e.g. for subcutaneous administration and plasma half-life. Thus, DNA plasmids encoding different B-domain truncated FVIII single-chain variants were designed, each with a separate deletion including the furin cleavage recognition site, and their function was assessed using an in vitro assay.

Materials and methods

Preparation of constructs

As basic FVIII sequence for cloning, Refacto was usedWherein 6 restriction sites are added by silent mutation in order to simplify cloning. Some of these restriction sites were again excluded due to codon optimization. The basic sequence is AC-6rs-REF (SEQ ID NO: 28).

For constructs encoding FVIII of the invention and comparative constructs also analyzed in this context, the entire FVIII sequence or the DNA region encoding 550-600bp from the FVIII a2 domain to the A3 domain was synthesized. The completely synthesized DNA is codon optimized. The DNA fragment is flanked at the 5 'end by EcoRV restriction sites and at the 3' end by EcoRI restriction sites, and these restriction sites are also present in the basic FVIII sequence used. The restriction of the DNA insert and FVIII backbone plasmid allows for targeted ligation and generation of FVIII single stranded plasmids. Fully synthetic FVIII DNA is flanked at the 5 'end by a HindIII restriction site and at the 3' end by a NotI restriction site.

By transforming Escherichia coli K12 with the plasmid, and amplifying the transformed bacteria under ampicillin selection and plasmid extraction, a large number of plasmids can be prepared. Genetic engineering work was performed by Thermo Fisher Scientific after design using VectorNTI software (Thermo Fisher Scientific, Massachusetts, USA).

Culture of CAP-T cells

CAP-T cells (Cevec Pharmaceuticals,germany) was prepared in a laboratory medium supplemented with 4mM GlutaMAX (Thermo Fisher Scientific, 35050038) and 5 μ g/ml blisticin (Thermo Fisher Scientific, R21001; complete PEM medium). To thaw the cells, the required amount of frozen vials was transferred to a 37 ℃ water bath. After thawing, each vial was transferred to 10ml of frozen complete PEM medium. The cell suspension was centrifuged at 150x g for 5 minutes.In this washing step, dimethyl sulfoxide (DMSO) used for cryopreservation was removed. The pellet was resuspended in 15ml of warm complete PEM medium and transferred to a 125ml shake flask. Cells were incubated at 37 ℃ in the presence of 5% CO₂The culture is carried out in a humidified incubator under the atmosphere of (2). The shake flask was set on a shaking platform and rotated at 185rpm with an orbit of 50 mm.

Subculture of cells was performed every 3 to 4 days. Fresh cultures were set to 0.5x10 by transferring the required amount of cultured cell suspension to a new flask and adding complete PEM medium⁶Cells/ml. In the case where the transferred cell suspension exceeded 20% of the total volume, the suspension was centrifuged at 150x g for 5 minutes and the pellet was resuspended in fresh complete PEM medium. The cell suspension volume of each flask was 20% of the total flask volume.

At least 3 subcultures were performed after thawing before transfection experiments were performed.

Expression of proteins in CAP-T cells by transient transfection

Using a 4D-Nucleofector^TM(Lonza, Basel, Switzerland) transfected CAP-T cells. For each transfection, 10 × 10⁶Individual CAP-T cells were centrifuged at 150x g for 5 minutes in a 15ml conical tube. Considering the volume of the pellet and the volume of the plasmid solution, the cells were resuspended in 95. mu.l of supplemented SE buffer. Then, 5. mu.g of each plasmid was added to the cell suspension, followed by gentle mixing. The solution was transferred to 100. mu.l of Nucleocuvettes. The transfection procedure used was ED-100. After transfection, cells from one Nucleocuvette were transferred to 125ml shake flasks containing 12.5ml complete PEM medium. Cells were cultured for 4 days as described above. On day 4, cells were harvested by centrifugation at 150x g for 5 minutes. Larger amounts of protein can be produced by combining the 12.5ml method described above.

Chromogenic FVIII Activity

FVIII activity was determined by chromogenic assay. In this two-step assay, FIXa and FVIIIa activate FX in the first step. In the second step, activated FX hydrolyzes the chromogenic substrate, resulting in a color change, which can be measured at 405 nm. Since calcium and phospholipid are present in optimal amounts and there is an excess of FIXa and FX, the activation rate of FX depends only on the amount of active FVIII in the sample.

The reagent for chromogenic FVIII activity assay is obtained fromSPFVIII kit. The kit comprises phospholipid and calcium chloride (CaCl)₂) Trace thrombin, substrate S-2765, a mixture of FIXa and FX and thrombin inhibitor I-2581. The inhibitor is added to prevent hydrolysis of the substrate by the thrombin constructed during the reaction. All dilutions were performed in distilled water or Tris-BSA (TBSA) buffer containing 25mM Tris, 150mM sodium chloride (NaCl) and 1% Bovine Serum Albumin (BSA), pH set at 7.4. Each sample was diluted at least 1:2 with FVIII-depleted plasma. Further dilution was performed using TBSA buffer.

The assay was performed using BCS XP (siemens healthcare, erilangen, germany), a fully automatic hemostasis analyzer. All reagents, including water, TBSA buffer and sample, were inserted into the analyzer. For each sample, the analyzer mixed 34. mu.l calcium chloride, 20. mu.l TBSA buffer, 10. mu.l sample, 40. mu.l water, 11. mu.l phospholipid, and 56. mu.l FIXa-FX mixture. The mixture was incubated for 300 seconds. Then, 50. mu.l of S-2765+ I-2581 was added to the reaction. After addition of the substrate, the absorption at 405nm was measured in 200 seconds.

To calculate the amount of active FVIII, the software of the analyzer evaluated the slope of the kinetics measured between 30 and 190 seconds after the start of the reaction. The results correlate with a calibration curve generated using Biological Reference Preparations (BRPs) of FVIII. BRP activity is expressed in IU/ml. However, IU/ml can be assumed to be equivalent to U/ml. Results are expressed as "percent normal". These results are converted to U/ml, since 100% of normal FVIII activity corresponds to 1U FVIII activity per ml.

Blood coagulation activity FSL

In addition to the two-stage chromogenic assay (see above), one-stage coagulation was also performedAssay to determine the amount of active FVIII. FVIII-depleted plasma, CaCl during this assay₂The activator actin FSL and the sample containing FVIII are mixed in one step. Activators cause the production of FXIa, which activates FIX. FVIIIa, FIXa and FX construct the tenase complex and FX is activated. Further activation of prothrombin and fibrinogen ultimately leads to the formation of a fibrin clot. The time required to form a clot, i.e., the activated partial thromboplastin time (aPTT), was measured. The aPTT varies with the amount of FVIII.

Coagulation assays were performed using BCSXP. TBSA buffer, FVIII-depleted plasma, actin FSL, CaCl₂And a sample insertion analyzer. Samples were diluted at least 1:2 with FVIII-depleted plasma. Further dilution was performed using TBSA buffer. For each sample, the analyzer mixes 45 μ l of TBSA buffer, 5 μ l of sample, 50 μ l of FVIII-depleted plasma, and 50 μ l of actin FSL. By adding 50. mu.l CaCl₂The reaction was started. The analyzer measures the time required for clot formation.

To calculate the amount of active FVIII, the software of the analyzer evaluated the baseline extinction at 405nm at the beginning of the reaction. Over a period of 200 seconds, all of the following extinction degrees were analyzed for differences from the baseline extinction degrees. The first time point at which the prescribed threshold value is exceeded is determined as the clotting time. The results correlate with a calibration curve generated using BRP of FVIII.

FVIII antigen ELISA

Use ofAgELISA (diagnostic Stago, Asniemer SeineCeedex, France) determines the amount of FVIII antigen. In this sandwich ELISA, FVIII was used in combination with the mouse monoclonal antibody human FVIIIF (ab')₂Fragments were combined and the fragments were coated onto plates by the manufacturer. Bound FVIII was detected by a mouse monoclonal anti-human FVIII antibody coupled to peroxidase. Peroxidase-conjugated antibodies bind to FVIII in the presence of FVIII, and can be detected by addition of Tetramethylbenzidine (TMB) solution. Upon reaction with peroxidase, the TMB changed from a clear solution to a blue-green solution. After a short period of time, by adding sulfuric acid (H)₂SO₄) The reaction was terminated and the solution was made yellow. The amount of bound FVIII is related to the intensity of yellow, which can be measured at 450 nm. The final amount of FVIII is calculated using a calibration curve generated by measuring at least five serial dilutions of a calibrator with known antigen concentration.

The calibrator and control were reconstituted with 500 μ l of distilled water 30 minutes before the start of the ELISA. After this incubation time, the calibrant was diluted 1:10 in the phosphate buffer provided. This represents the starting concentration. The calibrator was further serially diluted 1:2 to 1: 64. Since the concentration of the calibrant contained about 1U/ml FVIII, the starting concentration corresponded to 0.1U/ml FVIII, depending on the batch, while the final dilution contained about 0.0016U/ml FVIII. The control was diluted 1:10 and 1:20 with phosphate buffer. All samples were diluted with phosphate buffer, depending on their previously determined activity (see above), in order to be in the middle of the calibration curve. After dilution of FVIII sample, control and calibrator, 200 μ l of each solution was applied per well in duplicate. In addition, two wells were filled with 200 μ l phosphate buffer as a blank. Plates were incubated with film cover at room temperature for 2 hours. During this period, peroxidase-conjugated anti-human FVIII antibody was reconstituted with 8ml of phosphate buffer and incubated at room temperature for 30 minutes. After antigen immobilization, the wells were washed five times with the supplied washing solution, which was diluted with distilled water 1:20 in advance. Immediately after washing, 200 μ l of peroxidase-conjugated anti-human FVIII antibody was added to each well and incubated for 2 hours at room temperature with a membrane cover. After that, the plate was washed five times as before. To reveal the amount of bound FVIII, 200 μ l of TMB solution was added to each well and incubated accurately for 5 minutes at room temperature. By adding 50. mu.l of 1MH to each well₂SO₄To terminate the reaction. After incubation for 15 min at room temperature, the absorbance of each well was measured at 450nm using a POLA Rstar omega microplate reader (BMGLAZTECH, Ortenberg, Germany).

The ELISA results were calculated using MARS software (BMGLAbtech). In the first step, blank correction was performed for all wells and the mean of the replicates was calculated. Then, a 4-parameter fit was applied to calculate the concentration from the calibration curve. From this calibration curve, the amount of FVIII antigen in each well was determined. In the final step, these values are corrected by a dilution factor to give the amount of FVIII antigen per sample.

Results and discussion

Different variants of single chain FVIII molecules were generated and analysed (figures 1, 2). In all variants, the furin cleavage recognition site was deleted. The variants AC _ SC-V1(V1) and V3(V3) have a native thrombin cleavage site (PR/SV or SC/SV, respectively) and only one deletion of aa760-1687(V1) and aa 731-1687 (V3). The variants AC _ SC-V2(V2) and-V4 (V4) comprise different thrombin cleavage sites (PR/VA or IR/SV respectively) compared to V1 and V3, wherein V2 further comprises a VA sequence that can be considered to be derived from the B-domain based on V1. Based on V3, V4 contained an insertion of DPR-IRSV-VAQ at the deletion site (FIG. 1). None of the constructs V1-V4 comprise the processing sequence claimed in the present invention, e.g., the sequence NPP is included in the context of the thrombin cleavage site of interest.

The inventors carried out further experiments by generating additional constructs, designated AC _ SC-V0, -V5, -V6 and-V7 (or simply V0, V5, V6 and V7), as shown in fig. 2. In a less straightforward manner, they are transferred from a single deletion of the B-domain to create two deletions, which are bridged by retaining a short segment of the B-domain. Thus, a processing sequence is generated which appears to be closer to the wild-type sequence. Constructs based on RefactoThe amino acid sequence AC-6rs-Ref, by introducing two deletions, i.e. a protein of the invention comprising a processing sequence as defined herein. Thus, V0, V5, V6 and V7 represent FVIII proteins according to the invention.

Western blots of V0-V7 showed that expression of all constructs analyzed was predominantly as single chain molecules (not shown), represented by a double band at about 180kD, and no or little heavy chain band at about 80 kD.

Expression levels and in vitro function of different single-chain FVIII variants were assessed compared to the double-chain FVIII variant AC-6rs-REF, which has the same properties as factorThe same amino acid sequence and used as a baseline molecule during the development of treatments for hemophilia a. Naturally, this double-stranded FVIII is expected to provide the best functional result.

As described above, CAP-T cells were transiently transfected with the corresponding plasmid DNA encoding AC-6rs-REF or a different single-stranded molecule, repeated twice. After four days in culture, the cells were centrifuged and the cell culture supernatant was used directly to determine (I) chromogenic FVIII activity, (II) FVIII antigen corresponding to the amount of total FVIII protein, and (III) FVIII clotting activity induced by actin FSL.

In comparison of construct V1-V4 with FVIII double-stranded molecule AC-6rs (SEQ ID NO:29), the chromogenic activity and the specific chromogenic activity of the expressed single-stranded construct indicate that the ratio of chromogenic activity to FVIII antigen is lower than for the double-stranded molecule. In more detail, the color developing activity and specific color developing activity of V3 and V4 are lower than those of V1 and V2. In turn, the chromogenic and specific chromogenic activities of the expressed constructs V1 and V2 were still lower than that of the double-stranded molecules (data not shown).

In a comparison of V1, V2, V0, V5, V6, V7 and double-stranded FVIII (AC-6rs-REF), as shown in fig. 3A, the double-stranded FVIII control reached the highest chromogenic FVIII activity level of about 1U/ml (all measured in the supernatant of transfected cells). The single-chain variants AC _ SC-V0, -V5 and-V6 showed a chromogenic activity of about 0.7U/ml while AC _ SC-V1, -V2 and-V7 showed about 0.4 to 0.5U/ml.

FVIII protein amounts were also found to be highest in the double-stranded AC-6rs-REF control, about 2.4U/ml (FIG. 3C). Single chain variants are expressed in the range of 1.4U/ml for AC _ SC-V2 up to 2.0U/ml for AC _ SC-V5. The one-stage coagulation assay generally resulted in lower activity values compared to the two-stage chromogenic FVIII assay (fig. 3B). AC _ SC-V0, AC-6rs-REF, AC _ SC-V5 and AC _ SC-V6 showed about 0.3U/ml, while AC _ SC-V7 showed about 0.2U/ml of blood coagulation activity, and AC _ SC-V1 and-V2 showed very low blood coagulation activity.

The specific chromogenic activity represents the ratio of chromogenic FVIII activity to FVIII antigen level, i.e. they represent a decisive measure of activity. From a patient point of view, a high specific activity is desirable, since this means that better treatment can be achieved with less material. In this case, the specific activity was calculated as the ratio of chromogenic FVIII activity to FVIII antigen, expressed in%. As shown in FIG. 3D, the specific activity of AC _ SC-V0 was the highest (44%). The double-stranded AC-6rs-REF control reached a specific activity of 40%, followed by the activities of AC _ SC-V5, -V6 and V7. Specific clotting activity was calculated as the ratio of FVIII clotting activity to FVIII antigen expressed as%. As shown in FIG. 3E, the specific activity of AC _ SC-V0 (22.8%) was the highest, followed by-V6 (16.9%), V5 (15.5%), V7 (14.8%). The double-stranded AC-6rs-REF control reached a lower specific clotting activity of 13.9%, whereas the single-stranded constructs AC _ SC-V1 and-V2 (prior art) reached only 6.7% and 9.6%, respectively.

In summary, the in vitro function of different single-chain FVIII molecules AC _ SC-V0, -V1, -V2, -V5, -V6 and-V7 was evaluated compared to double-chain FVIII, AC-6 rs-REF. Thus, all FVIII molecules were analyzed using a two-stage chromogenic activity assay, a one-stage coagulation assay and FVIII antigen ELISA to measure total FVIII protein amounts. Since FVIII antigen levels can be determined, it was observed that all FVIII variants were expressed. Furthermore, all molecules are generally functional, as activity values can be determined in both chromogenic and thrombogenic assays. However, the lowest activity was determined for the single chain variants AC _ SC-V1 and AC _ SC-V2. Although AC _ SC-V7 performed moderately in these activity assays, AC _ SC-V0, -V5, -V6 and-V7 performed best as single-chain variants. The double-stranded AC-6rs-REF control overall exhibited the highest protein amount and chromogenic FVIII activity. The specific activities of AC _ SC-V0, AC _ SC-V5, AC _ SC-V6 and AC _ SC-V7, which represent the ratio of chromogenic activity to FVIII antigen or the ratio of coagulation activity to FVIII antigen, are optimal as indicators of protein function.

2. Stability and pharmacokinetic data

Prior to in vitro stability experiments and in vivo experiments, FVIII single chain proteins were purified by standard purification procedures, including cell removal and supernatant concentration, followed by purification of FVIII protein by affinity chromatography and rebuffering into FVIII formulation buffer.

V0 and ReFactor were analyzed in both FVIII-deficient buffer and plasma over 24 hours and 14 daysThe chromogenic and clotting activities of (1). V0 and ReFactoComparable in stability (fig. 4).

V0 and commercial double-stranded FVIII Refactor were analyzed in micePharmacokinetic data of (d). Coagulation factors (200IU (chromogenic activity)/kg body weight) were injected into female hemophilia A mice (FVIII-deficient mice, Jax No B6; 129S-F8) at 6ml/kg body weight via tail vein in a single intravenous injection^tm1Kaz/J, Charles River Laboratories, Sulzfeld, Germany). Blood was collected at 0.5, 4, 8, 12 and 20 hours after treatment, followed by extraction of citrate plasma by centrifugation. Plasma samples were analyzed for chromogenic FVIII activity and FVIII protein amount (FVIII antigen). Pharmacokinetic evaluation was performed by analyzing the original values and performing non-compartmental analysis (NCA) using phoenixwinnonlin8.1(Certara, USA).

In this study, ReFactoA half-life better than the average was obtained, with an antigen half-life of about 7.4 hours and FVIII chromogenic activity half-life of 6.9 hours. In contrast, the half-lives of the antigenic and chromogenic activities of V0 were 6.1 and 6.8 hours, respectively. The maximum concentration after intravenous administration was observed in the samples collected 0.5 hours after injection, 2.16IU/ml and 2.02IU/ml, respectively. Thus, both variants showed comparable half-lives (fig. 5) and comparable maximum concentrations after injection in terms of antigen and chromogenic activity.

Thus, transforming in vitro findings into animal models, the inventors were able to ascertain the advantageous properties of FVIII variants according to the invention also in vivo situations.