Bi-specific therapeutic proteins and method of use thereof

A method for of promoting tissue regeneration or tissue survival, the method comprising:

• (a) providing a bi-specific protein having (i) a targeting domain having a binding specificity to a target molecule associated with the outer surface of a cell of a tissue, wherein the targeting domain comprises a variant of 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 (ii) an activator domain having a binding specificity to a receptor associated with the surface of a cell of the tissue, wherein the activator domain comprises a variant of human insulin-like growth factor IGF-1 having an N-terminal 13-residue extension and a substitution at the position corresponding to E3, and optionally a mutation at one or more positions corresponding to a deletion at position R37, a deletion of amino acids at the position corresponding to K68, S69 and A70, a substitution at one or more position corresponding to Y24, Y31, Y60, and combinations thereof, wherein the variant of IGF-1 decreases activation of the IGF-1 receptor relative to the wild-type IGF-1;
• 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, the IGF-1 variant specifically activates the IGF-1 receptor so as to promote tissue regeneration or tissue survival.

A method for of protecting tissue from damage, the method comprising:

• (a) providing a bi-specific protein having (i) a targeting domain having a binding specificity to a target molecule associated with the outer surface of a cell of a tissue, wherein the targeting domain comprises a variant of 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 (ii) an activator domain having a binding specificity to a receptor associated with the surface of a cell of the tissue, wherein the activator domain comprises a variant of human insulin-like growth factor IGF-1 having an N-terminal 13-residue extension and a substitution at the position corresponding to E3, and optionally a mutation at one or more positions corresponding to a deletion at position R37, a deletion of amino acids at the position corresponding to K68, S69 and A70, a substitution at one or more position corresponding to Y24, Y31, Y60, and combinations thereof, wherein the variant of IGF-1 decreases activation of the IGF-1 receptor relative to the wild-type IGF-1;
• 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, the IGF-1 variant specifically activates the IGF-1 receptor so as to protect tissue from damage.

A method of inhibiting apoptosis, the method comprising:

• (a) providing a bi-specific protein having (i) a targeting domain having a binding specificity to a target molecule associated with the outer surface of a cell of a tissue, wherein the targeting domain comprises a variant of 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 (ii) an activator domain having a binding specificity to a receptor associated with the surface of a cell of the tissue, wherein the activator domain comprises a variant of human insulin-like growth factor IGF-1 having an N-terminal 13-residue extension and a substitution at the position corresponding to E3, and optionally a mutation at one or more positions corresponding to a deletion at position R37, a deletion of amino acids at the position corresponding to K68, S69 and A70, a substitution at one or more position corresponding to Y24, Y31, Y60, and combinations thereof, wherein the variant of IGF-1 decreases activation of the IGF-1 receptor relative to the wild-type IGF-1;
• 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, the IGF-1 variant specifically activates the IGF-1 receptor so as inhibit apoptosis.