Microvesicles derived from recombinant yeast having haemostatic activities and uses thereof

1. A topical composition comprising a pharmaceutically acceptable vehicle and tissue factor (TF)-bearing yeast derived microvesicles, said TF-bearing yeast derived microvesicles comprising (i) a yeast membrane, and (ii) a tissue factor (TF) protein or a variant thereof having pro-coagulant activity, wherein a portion of said tissue factor (TF) protein or variant thereof having pro-coagulant activity is integrated in said yeast membrane, and wherein said TF variant thereof having pro-coagulant activity is a mature tissue factor protein having at least one modification selected from the group consisting of: the inclusion of a tag bound to the N-terminal or C-terminal domain; the elimination of the signal peptide; the modification of a N-glycosylation site to make it non-functional; the elimination of all or part of the domain responsible for binding to FVIIa; and the introduction of a mutation in the domain responsible for binding to FVIIa which results in a substantially reduced affinity for FVIIa.

2. The topical composition according to claim 1 , wherein said TF protein, or a variant thereof having pro-coagulant activity, is glycosylated.

3. The topical composition according to claim 1 that is a pharmaceutical composition comprising pharmaceutically acceptable vehicles, carriers or excipients.

4. The topical composition according to claim 1 , wherein said TF protein, or a variant thereof having pro-coagulant activity, is human TF.

5. The topical composition according to claim 1 , wherein said TF protein, or a variant thereof having pro-coagulant activity comprises a tag and said tag is a histidine tag (His-tag).

6. The topical composition according to claim 1 , wherein said TF variant having pro-coagulant activity is the mature TF protein without the signal peptide.

7. The topical composition according to claim 1 , wherein said TF variant having pro-coagulant activity comprises the interaction domain to Factor X (aa 174-251, SEQ ID NO: 13), the transmembrane region (aa 252-274, SEQ ID NO: 14), and the cytoplasmic tail (aa 275-295, SEQ ID NO: 15) of human TF protein, and lacks, partially or completely, the domain responsible for binding to FVIIa.

8. The topical composition according to claim 1 , wherein said TF variant having pro-coagulant activity comprises the interaction domain to Factor X (aa 174-251, SEQ ID NO: 13), the transmembrane region (aa 252-274, SEQ ID NO: 14), and the cytoplasmic tail (aa 275-295, SEQ ID NO: 15) of the human TF protein and a histidine tag.

9. The topical composition according to claim 1 , wherein said TF variant having pro-coagulant activity is human mature rTF protein containing at least one non-functional N-glycosylation site or sites corresponding to the N-glycosylation sites NLT at positions 11-13, NVT at positions 124-126, or NNT at positions 137-139 in the mature human rTF (SEQ ID NO: 17).

10. The topical composition according to claim 9 wherein said TF variant having pro-coagulant activity has one or more Asn-to-Ala mutations at the Asn residues in positions corresponding to positions 11, 124 or 137 in the mature human rTF (SEQ ID NO: 17).

11. The topical composition according to claim 1 , wherein the N-terminal domain of said TF protein or variant thereof having pro-coagulant activity faces the exoplasmic side of said yeast membrane.

12. The topical composition according to claim 1 , wherein the N-terminal domain of said TF protein or variant thereof having pro-coagulant activity faces the endoplasmic side of said yeast membrane.

13. The topical composition according to claim 1 , wherein said TF-bearing yeast derived microvesicles have a size between 0.2 and 0.1 μm.

14. The topical composition according to claim 1 , wherein said TF variant having pro-coagulant activity is human mature TF protein containing a histidine tag and a non-functional N-glycosylation site corresponding to Asn124Ala mutation.

15. A method for the treatment of haemorrhages comprising topically administering a composition according to claim 1 to a subject in need thereof.

16. A method for treating a disease which requires promoting cellular migration and/or angiogenesis, said method comprising topically administering a pharmaceutical composition according to claim 1 to a subject in need thereof.

17. A process for the manufacture of a topical composition according to claim 1 which comprises:

a) subjecting a culture of recombinant yeast cells which express TF protein or a variant thereof having pro-coagulant activity as defined in claim 1 to fermentation under conditions which allow the expression of said TF protein or variant thereof;

b) pelleting the product resulting from the fermentation of step a), to render a fermentation product;

c) subjecting said fermentation product from step b) to homogenization in the absence of detergents, to render a fermentation homogenate; and

d) subjecting said fermentation homogenate from step c) to separation, to render a pellet and a clarified yeast extract (CYE) containing said TF-bearing yeast derived microvesicle;

e) collecting said clarified yeast extract (CYE) containing said TF-bearing yeast derived microvesicle; and

f) if desired, isolating or purifying said TF-bearing yeast derived microvesicles having pro-coagulant activity.

18. The process according to claim 17 , wherein said purifying is carried out by using one or more purification methods selected from the group consisting of size partitioning, tag purification, and immunoaffinity chromatography.

19. The process according to claim 17 , wherein said purifying is size partitioning performed by tangential flow filtration and/or using membrane filters that retain microvesicles having a diameter of 0.1 to 0.2 μm and/or size exclusion chromatography.