Method of inhibiting apoptosis or promoting cell survival by providing a bi-specific protein comprising insulin-like growth factor IGF-1 and Annexin A5
Bi-specific fusion proteins with therapeutic uses are provided, as well as pharmaceutical compositions comprising such fusion proteins, and methods for using such fusion proteins to repair or regenerate damaged or diseased tissue.
1. A method of inhibiting apoptosis, the method comprising:
(a) providing a bi-specific protein having (1) an activator domain, wherein the activator domain comprises a variant of human insulin-like growth factor IGF-1 having at least 95% identity to wild type human IGF-1 and comprising one or more mutations, wherein the one or more mutations consist of a substitution at one or more positions corresponding to E3, Y24, Y31, Y60, and combinations thereof, and (2) a targeting domain, wherein the targeting domain comprises a variant of human Annexin A5 having at least 95% identity to wild type human Annexin A5 and comprising one or more mutations, wherein the one or more mutations consist of a substitution at the position corresponding to C316 and optionally at one or more positions corresponding to R63, K70, K101, E138, D139, N160 and combinations thereof; and
(b) administering to a patient in need thereof a therapeutically effective amount of the bi-specific protein whereby the Annexin A5 variant targets the bi-specific protein to a first cell of the tissue, wherein the cell expresses phosphatidylserine on the outer leaflet of the plasma membrane, and whereby upon exposure of the IGF-1 variant to an IGF-1 receptor at the surface of a second cell of the tissue, the IGF-1 variant specifically activates the IGF-1 receptor so as to inhibit apoptosis.
2. The method of claim 1 , wherein the IGF-1 variant induces the phosphorylation of serine/threonine protein kinase B (AKT).
3. The method of claim 1 , wherein the bi-specific protein further comprises a peptide linker.
4. The method of claim 1 , wherein the peptide linker is a human serum albumin, an Fc fragment or a variant thereof.
5. The method of claim 4 , wherein the human serum albumin or variant thereof has an amino acid sequence set forth in any one of SEQ ID NOs: 54-56, or 124.
6. The method of claim 1 , wherein the Annexin A5 variant is a non-internalizing variant of human Annexin A5 and wherein the bi-specific protein has a prolonged half-life as compared to a bi-specific protein comprising the amino acid sequence of wild-type human Annexin A5.
7. A method of promoting cell survival, the method comprising:
(a) providing a bi-specific protein having (1) an activator domain, wherein the activator domain comprises a variant of human insulin-like growth factor IGF-1 having at least 95% identity to wild type human IGF-1 comprising one or more mutations, wherein the one or more mutations consist of a substitution at one or more positions corresponding to E3, Y24, Y31, Y60, and combinations thereof, and (2) a targeting domain, wherein the targeting domain comprises a variant of human Annexin A5 having at least 95% identity to wild type human Annexin A5 comprising one or more mutations, wherein the one or more mutations consist of a substitution at the position corresponding to C316 and optionally at one or more positions corresponding to R63, K70, K101, E138, D139, N160 and combinations thereof; and
(b) administering to a patient in need thereof a therapeutically effective amount of the bi-specific protein whereby the Annexin A5 variant targets the bi-specific protein to a first cell of the tissue, wherein the cell expresses phosphatidylserine on the outer leaflet of the plasma membrane, and whereby upon exposure of the IGF-1 variant to an IGF-1 receptor at the surface of a second cell of the tissue, the IGF-1 variant specifically activates the IGF-1 receptor so as to promote cell survival.
8. The method of claim 7 , wherein the IGF-1 variant induces the phosphorylation of serine/threonine protein kinase B (AKT).
9. The method of claim 7 , wherein the bi-specific protein further comprises a peptide linker.
10. The method of claim 7 , wherein the peptide linker is a human serum albumin, an Fc fragment or a variant thereof.
11. The method of claim 10 , wherein the human serum albumin or variant thereof has an amino acid sequence set forth in any one of SEQ ID NOs: 54-56, or 124.
12. The method of claim 7 , wherein the Annexin A5 variant is a non-internalizing variant of human Annexin A5 and wherein the bi-specific protein has a prolonged half-life as compared to a bi-specific protein comprising the amino acid sequence of wild-type human Annexin A5.