Vascular endothelial cell growth factor II

Claim 2

The vascular endothelial growth factor II of claim 1 wherein the growth factor is isolated and purified from human cells, cell culture media or tissues.

Claim 3

The vascular endothelial growth factor II of claim 1 wherein the factor is mitogenic for mammalian vascular endothelial cells.

Claim 4

Mammalian vascular endothelial growth factor II comprising a heterodimer with an apparent non-reduced molecular mass of about 58,000 daltons, with said homoodimer made up of two A subunits having an apparent reduced molecular mass of about 23,000 daltons each with said vascular endothelial growth factor substantially free of impurities and mitogenic for mammalian vascular endothelial cells.

Claim 5

Mammalian vascular endothelial growth factor comprising a homodimer comprising two B subunits with said endothelial growth factor substantially free of impurities and mitogenic for mammalian vascular endothelial cells.

Claim 6

A protein comprising an amino acid sequence substantially corresponding to all or a portion of the amino acid sequence of rat vascular endothelial growth factor II with said vascular endothelial gowth factor having a B subunit selected from the group consisting of the 135 amino acid form and the 115 amino acid form and having an A subunit selected from the g roup consisting of the 188 amino acid form, the 164 amino acid form and the 120 amino acid form and other microheterogeneous forms thereof with said growth factor being free of other proteins.

Claim 7

Vascular endothelial growth factor comprising an A subunit of at least the amino acid sequence as shown in Figure 4 and a B subunit of at least the amino acid sequence as shown in Figure 5 and any microheterogeneous or truncated forms thereof which are biologically active and are substantially free of impurities.

Claim 8

Vascular endothelial growth factor comprising an A subunit of at least the nucleotide sequence as shown in Figure 4 and an B subunit of at least the nucleotide sequence as shown in Figure 6 and any microheterogeneous or truncated forms thereof which are biologically active and are substantially free of impurities.

Claim 9

Vascular endothelial growth factor comprising two B subunits of at least the nucleotide sequence as shown in figure 6 or figure 7 and any microheterogeneous or truncated forms thereof which are biologically active and are substantially free of impurities.

Claim 10

A process for the isolation of vascular endothelial growth factor II in substantially pure form which comprises the sequence of steps of: a. isolation of conditioned growth media; b. cation exchange chromatography; c. lectin affinity chromatography; d. cation exchange high performance liquid chromatography; e. metal-chelate affinity chromatography; f. reverse-phase high performance liquid chromatography; and g. collecting the substantially pure vascular endothelial growth factor II.

Claim 11

The process of claim 10 wherein: a. isolation of conditioned growth media by filtration; b. cation exchange chromatography with adsorption on carboxymethyl Sephadex C-50 cation exchange resin at pH 6.0 followed by a rinse with 0.05 M sodium phosphate, pH 6.0, 0.15 M NaCl and elution with 0.05 sodium phosphate, pH 6.0, containing 0.6 M NaCl; c. lectin affinity chromatography with adsorption on concanavalin A agarose resin at pH 6.0 followed by elution with 0.05 M sodium acetate, pH 6.0, containing 1 mM CaCl₂, 1 mM MnCl₂, 0.1 M NaCl and 0.32 M alpha-methyl mannoside and 0.28 M alpha-methyl glucoside; d. cation exchange high performance liquid chromatography with adsorption on Polyaspartic Acid WCX, equilibrated in 0.05 M sodium phosphate, pH 6.0 followed by elution with about 0 to about 0.75 M NaCl linear gradient; e. metal-chelate chromatography with adsorption on Pharmacia Chelating Sepharose 6B charged with excess Cu⁺⁺ followed by elution with a gradient of 0.5 mM to 100 mM imidazole in buffer containing about 0.05 M sodium phosphate, about pH 7.0, about 2 M NaCl; f. reverse phase high performance liquid chromatography with adsorption on a C₄ column equilibrated in 0.1 % trifluoroacetic acid followed by elution with a gradient of 0 to 67 % (v/v) of acetonitrile in 0.1 % trifluoroacetic; and g. collecting the substantially pure vascular endothelial growth factor II.

Claim 12

The process of Claim 10 for the isolation of vascular endothelial growth factor II from GS-9L cell culture fluid.

Claim 13

A tissue repairing pharmaceutical composition comprising a pharmaceutical carrier and an effective tissue repairing amount of the purified vascular endothelial growth factor II of any one of Claims 1 or 4 to 9.

Claim 14

The use of the vascular endothelial growth factor II of any one of Claims 1 or 9 to 9 for the manufacture of a medicament capable of promoting tissue repair.

Claim 17

The use of a synthetic vessel prepared by the method of Claim 16 for the manufacture of a medicament suitable for use as a vessel implant whereby, after implantation, endothelial cells migrate into and grow on the artificial surface.

Claim 18

The use of vascular endothelial growth factor II for the manufacture of a medicament for the stimulation of the growth of vascular endothelial cells in vivo whereby either vascular repair, or neovascularization, or both, is promoted.

Claim 19

A purified and isolated DNA sequence encoding the 188 amino acid A subunit of vascular endothelial growth factor II.

Claim 20

A purified and isolated DNA sequence encoding the 164 amino acid A subunit of vascular endothelial growth factor II.

Claim 21

A purified and isolated DNA sequence encoding the 120 amino acid A subunit of vascular endothelial growth factor II.

Claim 22

A purified and isolated DNA sequence encoding the 135 amino acid B subunit of vascular endothelial growth factor II.

Claim 23

A purified and isolated DNA sequence encoding the 115 amino acid B subunit of vascular endothelial growth factor II.

Claim 24

Vascular endothelial growth factor II DNA comprising an A subunit DNA sequence selected from the group consisting of: a DNA sequence encoding an 188 amino acid form, a DNA sequence encoding an 164 amino acid form and a DNA sequence encoding a 120 amino acid form, with said A subunit DNA operably attached to a B subunit DNA sequence selected from the group consisting of: a DNA sequence encoding a 135 amino acid form; and a DNA sequence encoding a 115 amino acid form.

Claim 25

Heterodimeric vascular endothelial growth factor DNA comprising an A subunit DNA sequence encoding a 164 amino acid form operably attached to a B subunit DNA sequence encoding a 135 amino acid form.

Claim 26

Heterodimeric vascular endothelial growth factor DNA comprising an A subunit DNA sequence encoding a 164 amino acid form operably attached to a B subunit DNA sequence encoding a 115 amino acid form.

Claim 27

Homodimeric vascular endothelial growth factor DNA comprising a B subunit DNA sequence selected form the group consisting of: a B subunit DNA sequence encoding a 135 amino acid form and a B subunit DNA sequence encoding a 115 amino acid form.

Claim 28

A vector containing the DNA sequence of any one of claims 24 to 27.

Claim 29

A host cell transformed by the vector of claim 2 8 containing the DNA sequence encoding vascular endothelial growth factor II.

Claim 30

A process for the preparation of vascular endothelial growth factor II comprising culturing the transformed host cell of claim 29 under conditions suitable for the expression of vascular endothelial growth factor II and recovering vascular endothelial growth factor II.

Claim 31

Vascular endothelial growth factor II made by the process of claim 30.