ES2575629T3 - Factor VIII polypeptide - Google Patents

Description

image 1

image2

image3

image4

image5

image6

image7

image8

image9

5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

55

derivatives of FVIII and, for example, the protein dihydrofolate reductase (DHFR), it is appropriate to select the host according to the type of DHFR protein used. If wild-type DHFR protein is employed, it is preferable to select a host cell lacking DHFR, thus allowing the use of the DHFR coding sequence as a marker of successful transfection in selective medium lacking hypoxanthine, glycine and thymidine.

On the other hand, if the DHFR protein with low binding affinity with methotrexate (MTX) is used as the regulatory sequence, it is not necessary to use DHFR-resistant cells. The mutant DHFR is resistant to MTX, therefore, the MTX-containing media can be used as selection means with the proviso that the host cells themselves are sensitive to MTX. Alternatively, a wild-type DHFR gene can be used as a marker of amplification in a host cell that does not lack DHFR, provided that a second selectable marker of the drug, such as hygromycin resistance, is employed. The examples that are set forth describe the use of CHO cells (CHO-DBX11 cells) resistant to MTX as host cells and expression vectors that employ the CMV and SV40 promoter as regulatory sequences to direct the expression of the FVIII and DHFR derivatives , respectively.

Variant and mutant polynucleotides

Nucleic acid variants of this type include those produced by substitutions, deletions or additions of nucleotides. Substitutions, deletions or additions may involve one or more nucleotides. Alterations in the amino acid sequence can produce substitutions, deletions or additions of amino acids, conservative or non-conservative. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the polypeptides of the present invention or portions thereof. In this respect, conservative substitutions are also preferred.

The invention allows the use of sequences in expression vectors, as well as to transfect host cells and cell lines, these cells being prokaryotic or eukaryotic. The invention also allows the purification of the polypeptides expressed from the expression vector. The expression vector may contain various molecular markers to facilitate purification. Subsequently, the artificial expression structure obtained can be transformed into any chosen host cell. Cell lysates from the host cell are isolated by established methods well known in the art.

Variant and mutant polypeptides

To improve or alter the characteristics of the FVIII polypeptide of the present invention, genetically modified amino acids can be employed. Recombinant DNA technology known to those skilled in the art can be used to create new mutant polypeptides including substitutions, deletions, single or multiple amino acid additions, or fusion proteins. Such modified polypeptides can show, for example, an increase / decrease in activity or an increase / decrease in stability. In addition, they can be purified with higher yield and show a better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions.

Antibodies

In one embodiment, the present invention is directed to the detection of the presence of a FVIII polypeptide using a variety of detection methods. One way to detect a Factor VIII polypeptide is to label a ligand that specifically binds to the FVIII polypeptide. A ligand of this type can be an antibody. The purified FVIII polypeptide can be used to produce a monoclonal or polyclonal antibody. Factor VIII polypeptide fragments can also be used to produce a monoclonal or polyclonal antibody. A monoclonal or polyclonal antibody obtained subsequently can be used to determine the presence of a FVIII polypeptide in various samples including, but not limited to cells, tissues and body fluids such as blood, serum, plasma and urine. The FVIII polypeptide can be tested using a variety of molecular biological methods, including in situ hybridization, immunoprecipitation, immunofluorescent staining, Western blot analysis and so forth, but are not limited thereto. An ELISA can be carried out using a monoclonal antibody against an FVIII polypeptide to determine the amount of FVIII polypeptide in the biological sample, including body fluids from humans believed to be suffering from a blood coagulation disorder, such like hemophilia.

Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F (ab ') fragments, fragments produced by a Fab expression library. , anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies for antibodies of the invention), and fragments that bind to epitopes of any of the foregoing. The term "antibody", as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, ie, molecules that contain an antigen-binding site that binds immunospecifically to an antigen. The immunoglobulin molecules of the invention can be of any type (for example, IgG, IgE, IgM, IgD, IgA and IgY), class (for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of molecule of immunoglobulin.

eleven

image10

image11

5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

55

mosomics of the nucleic acids encoding the antibody (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86: 89328935 (1989); Zijlstra et al., Nature 342: 435-438 (1989).

The delivery of the nucleic acids to a patient can be direct, in which case the patient is exposed directly to the nucleic acid or to vectors carrying the nucleic acid, or indirect, in which case, the cells are first transformed with the nucleic acids in vitro and , then, they are transplanted to the patient. These two methodologies are known, respectively, as in vivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are administered directly in vivo, where they are expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, for example, by building them as part of an appropriate nucleic acid expression vector and administering them so that they become intracellular, for example, by infection using defective or attenuated retroviral vectors or other viral vectors, or by direct injection of naked DNA, or by coating with lipids or receptors the cell surface or transfection agents, encapsulation in liposomes, microparticles or microcapsules, or by administering them bound to a peptide that is known to enter the nucleus, administering them bound to a ligand subject to receptor-mediated endocytosis (see, for example, Wu and Wu, J. Biol. Chem. 262: 4429-4432 (1987)) (which can be used to target cell types that specifically express the receivers), and so on. In another embodiment, nucleic acid-ligand complexes can be formed wherein the ligand comprises a fusogenic viral peptide to destroy endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be directed in vivo for uptake and specific expression in cells, directing it to a specific receptor. Alternatively, the nucleic acid can be introduced intracellularly and incorporated into the DNA of the host cell for expression, by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86: 8932-8935 (1989); Zijlstra et al. ., Nature 342: 435-438 (1989)).

In a specific embodiment, viral vectors containing nucleic acid sequences encoding the polypeptide are used. The nucleic acid sequences encoding the polypeptide to be used in gene therapy are cloned into one or several vectors, which facilitates delivery of the gene to a patient. Retroviral vectors, adenoviral vectors and adeno-associated viruses are examples of viral vectors that can be used. The retroviral vectors contain the necessary components for a correct packaging of the viral genome and integration into the DNA of the host cell.

Another methodology for gene therapy involves transferring a gene to cells in tissue culture by methods such as electroporation, lipofection, calcium phosphate mediated transfection or viral infection. In general, the transfer method includes the transfer to the cells of a selectable marker. The cells are then selected to isolate those cells that have captured and are expressing the transferred gene. These cells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior to in vivo administration of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, transfer gene mediated by microcells, fusion of spheroplasts and so on. Numerous methods are known in the art for introducing foreign genes into cells and can be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not affected. The technique should provide a stable transfer of the nucleic acid to the cell, so that the cell can express the nucleic acid and can be inherited and expressed preferably in its cell line.

Cells into which a nucleic acid can be introduced for the purposes of gene therapy, comprise any desired cellular type, available, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, for example, as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, and so on.

In a preferred embodiment, the cell used for gene therapy is autologous to the patient.

In an embodiment in which recombinant cells are used in gene therapy, the nucleic acid sequences encoding the polypeptide are introduced into the cells in such a manner that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo. for the therapeutic effect. In a specific embodiment, stem or progenitor cells are used. Any stem and / or progenitor cell that can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention.

In a specific embodiment, the nucleic acid to be introduced for the purposes of gene therapy comprises an inducible promoter functionally linked to the coding region, such that the expression of the nucleic acid can be monitored, controlling the presence or absence of the inducer. appropriate transcription.

Therapeutic composition

14 5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

55

60

In one embodiment, the present invention relates to the treatment of blood coagulation diseases. In this way, the therapeutic compound of the invention can be administered to human patients suffering from

or they are prone to suffer from the disease, providing compounds that stimulate the coagulation of the blood. In particular, the disease can be hemophilia, in particular, hemophilia A.

The formulation of therapeutic compounds is generally known in the art and can be conveniently referred to Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., USA. For example, it can be administered from about 0.05 μg to about 20 mg per kilogram of body weight per day. The dosage regimen can be adjusted to provide an optimal therapeutic response. For example, several divided doses may be administered daily or the dose may be reduced proportionally as indicated by the exigencies of the therapeutic situation. The active compound can be administered in a convenient manner such as orally, intravenously (when water-soluble), intramuscular, subcutaneous, intranasal, intradermal or suppository or by an implant (eg, using slow-release molecules by the intraperitoneal route or using cells, for example, monocytes or dendritic cells sensitized in vitro and passively transferred to the receptor). Depending on the route of administration, the peptide may be required to be coated with a material to protect it from the action of enzymes, acids and other natural conditions that may inactivate said ingredients.

For example, the low lipophilicity of the peptides will allow them to be destroyed in the gastrointestinal tract by enzymes capable of cleaving peptide bonds and in the stomach by acid hydrolysis. In order to administer the peptides by another route than parenteral administration, they can be coated or administered with a material that prevents their inactivation. For example, the peptides can be administered in an adjuvant, co-administered with enzyme inhibitors or in liposomes. The adjuvants contemplated herein include resorcinols, nonionic surfactants such as polyoxyethylene oleyl ether and polyethylene n-hexadecyl ether. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropyl fluorophosphate (DEP) and Trasylol. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes.

The active compounds can also be administered parenterally or intraperitoneally. The dispersions can also be prepared in liquid glycerol, polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (when they are water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that it has a capacity to be injected comfortably. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or a dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol and the like), suitable mixtures thereof and vegetable oils. Proper fluidity can be maintained, for example, with the use of a coating such as lecithin, by the maintenance of the required particle size, in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, chlorobutanol, phenol, sorbic acid, thiomersal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be caused by the use in the composition of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent, with various other ingredients mentioned above, as required, followed by sterilization by filtration. Generally, dispersions are prepared by incorporating the various sterile active ingredients into a sterile vehicle containing the basic dispersion medium and the other required ingredients from those mentioned above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization technique which produces a powder of the active ingredient plus any additional desired ingredient, from a solution thereof. previously sterile filtered.

When the peptides are suitably protected as described above, the active compound can be administered orally, for example, with an inert diluent or with an edible assimilable carrier, or it can be included in a hard shell gelatin capsule or soft, or it can be pressed in the form of tablets, or it can be incorporated directly into the diet food. For oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, ingestible wafers and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may vary, of course, and may conveniently be between about 5 to about 80% of the unit's weight. The amount of active compound in such therapeutically useful compositions is such that an adequate dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that a unit dosage form orally contains between about 0.1 μg and

fifteen

image12

image13

pleta. The cohesive ends of the vector digested with EcoNI were blunted by the Klenow fragment of DNA polymerase I for ligation. This ligated vector was designated pCR2.1-deltaEcoNI_FVIII and used as a template for further, accurate deletion mutagenesis.

The oligonucleotide primers were designed to perform a series of precise deletions in frame. Each primer is paired with the sequences flanking both sides of the segments to be deleted. The delta-747, delta-754, delta-761, delta-768, delta-775 and delta-782 primers generate the fusion sites of Arg747-Gln1659, Lys754-Gln1659, Ile761-Gln1659, Lys768-Gln1659, His775-Gln1659 and Ile782-Gln1659, which are respectively:

(delta-747: 5'-CTTCTCCCAGAATTCAAGACAAGAGGAAATTGACTATG-3 '(SEQ ID NO: 8));

(delta-754: 5'-CCTAGCACTAGGCAAAAGCAAGAGGAAATTGACTATG-3 '(SEQ ID NO: 9));

(delta-761: 5'-CAATTTAATGCCACCACAATTCAAGAGGAAATTGACTATG-3 '(SEQ ID NO: 10));

(delta-768: 5'-CAGAAAATGACATAGAGAAGCAAGAGGAAATTGACTATG-3 '(SEQ ID NO: 11));

(delta-775: 5'-GACCCTTGGTTTGCACACCAAGAGGAAATTGACTATG-3 '(SEQ ID NO: 12));

(delta-782: 5'-GCACACAGAACACCTATGCCTAAAATACAAGAGGAAATTGACTATGATGATACC-3 '(SEQ ID NO: 13)).

In addition to the mutagenic primers described above, a selection primer (5'-CGTGATCCATGTCGACGCCTGCTTGCC-3 '(SEQ ID NO: 14)) which changes the unique original NcoI site by SalI, was used for site-directed mutagenesis with the plasmid pCR2 .1-deltaEcoNI_FVIII as a mold. Restriction digestion was carried out to select the positive clones, which were further verified by sequencing. The finally verified clones were named dB747, dB754, dB761, dB768, dB775 and dB782, respectively.

Example 2B - Generation of plasmids containing a new sequence of N-glycan at the fusion sites

To avoid exposure of a new epitope with a non-natural amino acid sequence, in the binding region of the heavy chain and the light chain, we created an N-glycosylation recognition sequence (Asn-X-Ser / Thr in which X can be any amino acid) at the fusion sites, joining Asn at positions 745, 757 and 764 of domain B, to amino acids at positions 1650, 1653 and 1656 located near amino acids Ser or Thr at positions 1651, 1654 and 1657, which generate a glycosylation site linked to N at the fusion sites. The oligonucleotide primers were designed to perform a series of precise deletions in frame. pCR2.1-deltaEcoNI_FVIII was used as a template for an accurate mutagenesis by additional deletion. Each primer is paired with the sequences flanking both sides of the segments to be deleted. The primers N-745-1650, N-7451653, N-745-1656, N-757-1650, N-757-1653, N-757-1656, N-764-1650, N-764-1653 and N- 764-1656 generate the fusion sites Asn745-Ile1650, Asn745-Thr1653, Asn745-Gln1656, Asn757-Ile1650, Asn757-Thr1653, Asn757-Gln1656, Asn764-Ile1650, Asn764-Thr1653 and Asn764-Gln1656. The nucleotide sequences of the oligonucleotide primers are the following:

(N-745-1650: 5'-CAAGAAGCTTCTCCCAGAAAATAACTCGTACTACTCTTC-3 '(SEQ ID NO: 15));

(N-745-1653: 5'-CAAGAAGCTTCTCCCAGAAAACTACTCTTCAGTCAGTC-3 '(SEQ ID NO: 16));

(N-745-1656: 5'-CAAGAAGCTTCTCCCAGAAACAGTCAGATCAAGAGGAAATTG-3 '(SEQ ID NO: 17));

(N-757-1650: 5'-CTAGGCAAAAGCAATTTAATATAACTCGTACTACTCTTC-3 '(SEQ ID NO: 18));

(N-757-1563: 5'-CTAGGCAAAAGCAATTTAATACTACTCTTCAGTCAGTC-3 '(SEQ ID NO: 19));

(N-757-1656: 5'-CTAGGCAAAAGCAATTTAATCAGTCAGATCAAGAGGAAATTG-3 '(SEQ ID NO: 20));

(N-764-1650: 5'-CACCACAATTCCAGAAAATATAACTCGTACTACTCTTC-3 '(SEQ ID NO: 21));

(N-764-1653: 5'-CACCACAATTCCAGAAAATACTACTCTTCAGTCAGTC-3 '(SEQ ID NO: 22));

(N-764-1656: 5'-CACCACAATTCCAGAAAATCAGTCAGATCAAGAGGAAATTG-3 '(SEQ ID NO: 23)).

In addition to the mutagenic primers described above, a selection primer (5'-CGTGATCCATGTCGACGCCTGCTTGCC-3 '(SEQ ID NO: 24)), which changes the unique original site of NcoI by SalI, was used for site-directed mutagenesis using the Plasmid pCR2.1-deltaEcoNI_FVIII as a template. Restriction digestion was carried out to select the positive clones, which were further verified by sequencing. The verified clones were finally named dBN (45-50), dBN (45-53), dBN (45-56), dBN (57-50), dBN (57-53), dBN (57-56), dBN ( 64-50), dBN (64-53) and dBN (64-56), respectively.

18

image14

Determination of the FVIII antigen level (FVIII: Ag) and the procoagulant activity of FVIII (FVIII: C) in the supernatant of cultures of BHK21 transfected with the expression vector derived from FVIII, 24 h after transfection (results expressed in mU / ml or% / ml)

Derivative

FVIII: Ag (mU / ml) FVIII: C (% / ml)

dBN (45-56)

88.6 ± 6.37 7.2 ± 0.75

dBN (57-50)

87.9 ± 11.1 8.1 ± 0.37

dBN (57-53)

82.4 ± 3.24 7.3 ± 0.54

dBN (57-56)

86: 5 ± 8.15 7.6 ± 0.69

dBN (64-50)

87.4 ± 7.38 8.2 ± 0.53

dBN (64-53)

80.7 ± 5.56 7.5 ± 0.64

dBN (64-56)

84.9 ± 3.42 7.9 ± 0.41

FVIII full length

5.5 ± 0.53 0.3 ± 0.06

The results show that the FVIII derivatives that have undergone deletion are biologically active in a blood coagulation assay, and that the highest levels of protein were obtained with the artificial structures of FVIII derivatives, rather than with the FVIII in length complete In addition, the FVIII: C and FVIII: Ag ratio

5 for the derivatives of this invention is greater than that of full-length FVIII, which indicates that the FVIII derivatives may be more stable after secretion in culture media. As shown in Table 3, the increases in FVIII (FVIII: C) activity of the recombinant FVIII derivatives over time, after incubation, were higher than compared to FVIII's full length recombinant.

10 Example 4 - Substituting Pro for Phe at position 739

Pro739 in the FVIII variants with the deleted B domain described above, was modified using the site-directed mutagenesis method. An oligonucleotide primer (5'-AACAATGCCATTGAATTCAGAAGCTTCTCCCAG-3 '(SEQ ID NO: 25)) was designed to introduce the substitution of Pro for Phe at position 739. The entire mutagenesis procedures were identical to Example 2 described above. The vectors that possessed a Pro739Phe substitution in each FVIII derivative with the deleted B domain, were named dB747-739F, dB754-739F, dB761-739F, dB768-739F, dB775-739F, dB782739F, dBN (45-50) -739F , dBN (45-53) -739F, dBN (45-56) -739F, dBN (57-50) -739F, dBN (57-53) -739F, dBN (57-56) 739F, dBN (64-50 ) -739F, dBN (64-53) -739F and dBN (64-56) -739F, respectively. The resulting DNAs were cloned into the mammalian expression vector, prepared, transfected and the resulting samples were subjected to

20 test as described above. As shown in Table 4, it was observed that dB761-739F and dB782-739F generated more activity than dB761 and dB782, respectively, increased activity after mutation of proline to a phenylalanine at position 739.

Table 3

Determination of the procoagulant activity of FVIII (FVIII: C) in the supernatant of cultures of BHK21 transfected with the expression vector derived from FVIII, 24 h and 48 h after transfection (results expressed in% / ml)

Derivative

24 h 48 h

dB-747

7.8 ± 0.09 15.9 ± 0.85

dB-754

9.0 ± 0.28 17.9 ± 0.43

dB-761

8.0 ± 0.22 16.0 ± 0.69

dB-768

7.7 ± 0.31 18.0 ± 0.19

dB-775

7.3 ± 0.44 14.9 ± 0.34

dB-782

6.2 ± 0.12 14.2 ± 0.91

 image15

image16

FVIII full length

0.3 ± 0.09 0.2 ± 0.06

twenty

image17

image18

5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

Example 7 - Activation of full-length recombinant FVIII and FVIII derivatives with thrombin

Full length recombinant FVIII and FVIII derivatives were compared in a study of kinetics of activation with thrombin. Activation was measured in a classical coagulation test (APTT) after incubation in the presence of a catalytic amount of thrombin.

Figures 7A and 7B show a comparison of activation kinetics with thrombin, human recombinant FVIII (rH FVIII) and FVIII derivatives. Some FVIII derivatives are activated more strongly with 7-fold increments after 5 minutes of incubation with thrombin. Two derivatives of FVIII, dBN (64-53) and dBN (64-56), are activated in the same manner as rh FVIII. Although some FVIII derivatives have higher increases in activation, they are not activated more rapidly than the other derivatives or than full length FVIII, thereby showing that the FVIII derivatives are not preactivated. This is important for the purposes of its therapeutic use.

The greatest increases in thrombin activation of some derivatives can be explained by their lower activation threshold. Lower amounts of thrombin generated at a site of vascular injury can cause an increased activation of FVIII derivatives, which allows these derivatives to act as procoagulant molecules with an increase in efficacy, compared to other derivatives with FVIII activity. In other words, FVIII derivatives, except dBN (64-53) and dBN (64-56), can be activated in an event well before the events of blood coagulation. Consequently, FVIII derivatives with a greater increase in activation can be administered to patients with hemophilia A, at a much lower dose and a lower frequency than other molecules with FVIII activity. This significantly reduces the risk of a production of inhibitory antibodies in patients. In addition, this also further reduces the production and medication costs.

All references cited in this specification are incorporated by reference in their entirety.

Those skilled in the art will recognize, or be able to determine using only routine experimentation, many equivalents of the specific embodiments of the invention, specifically described herein. It is intended that such equivalents be included in the scope of the claims.

The preferred embodiments of the present invention are:

Item 1: A Factor VIII polypeptide comprising an internal deletion of one or more amino acids between 1649 and 1688 fused to any amino acid sequence in domain B from about 741 to 782, relative to an amino acid sequence of human Factor VIII of full length (SEQ ID NO: 1).

Point 2: The Factor VIII polypeptide according to item 1, wherein the internal deletion is from amino acids 746 to 1649, 746 to 1652, 746 to 1655, 758 to 1649, 758 to 1652, 758 to 1655, 765 to 1649 , 765 to 1652, 765 to 1655, 748 to 1658, 755 to 1658, 762 to 1658, 769 to 1658, 776 to 1658 or 783 to 1658.

Point 3: Factor VIII polypeptide according to item 1, which is a single chain.

Point 4: The Factor VIII polypeptide according to item 1, wherein the proline at position 739 is replaced by another amino acid.

Item 5: Factor VIII polypeptide according to item 1, which comprises the tripeptide sequence (Asn-X-Thr

or Asn-X-Ser) encompassing the fusion sites between the amino acid Asn at positions 745, 757 or 764, and the amino acid Thr or Ser at positions 1651, 1654 or 1657, relative to an amino acid sequence of the Factor VIII full length human (SEQ ID NO: 1).

Item 6: The Factor VIII polypeptide according to item 1, represented by the following formula with the following domains linked domains:

H-S-L

where

(i)

 domain H represents a polypeptide sequence comprising substantially Ala-1 up to Arg740 of human Factor VIII according to SEQ ID NO: 1;

(ii)

the S domain represents a polypeptide spacer-type linker comprising up to about 60 amino acids, wherein the N-terminal end of the S domain is approximately residue 740, and the C-terminal end of the S domain ends at approximately residue 1688 of the Factor VIII human according to SEQ ID NO: 1; Y

(iii) the L domain represents a polypeptide sequence comprising substantially Arg-1689 up to Tyr-2332 of human Factor VIII according to SEQ ID NO: 1.

Item 7: The Factor VIII polypeptide according to item 6, wherein the S domain comprises a sequence of

2. 3

Claims (1)

image 1