Complement receptor 2 (CR2) targeting groups

Patent metadata

Field	Value
Publication number	US-9650447-B2
Application number	US-201113697693-A
Country	US
Kind code	B2
Filing date	May 13, 2011
Priority date	May 14, 2010
Publication date	May 16, 2017
Grant date	May 16, 2017

Abstract

Provided herein are compositions and methods directed to soluble proteins which can selectively deliver modulators of complement activity. Targeted delivery of these modulators is accomplished by selectively mutating particular amino acids in a targeting protein portion of the composition corresponding to at least the first two N-terminal SCR domains of CR2. Depending on the particular combination of mutations introduced into the targeting portion, a complement activity modulator can be selectively delivered to particular ligands of CR2 at sites where complement system activation or suppression is desired.

First claim

We claim:

1. A soluble composition capable of targeted delivery of a complement modulator to sites of complement system activation comprising a construct, wherein the construct comprises:
(a) a complement receptor 2 (CR2) portion comprising at least the first two N-terminal short consensus repeat (SCR) domains of CR2; and
(b) a complement modulator portion;
wherein the CR2 portion contains an alanine substitution at an amino acid position selected from the group consisting of N11 and Y64 of SEQ ID NO: 2 that decreases binding affinity of the CR2 portion for EBV gp350 relative to a construct in which the CR2 portion has the sequence of SEQ ID NO: 2, or the CR2 portion contains an alanine substitution at an amino acid position selected from the group consisting of S42 and K50 of SEQ ID NO: 2 that decreases binding of the CR2 portion for Interferon-alpha (IFNα) relative to a construct in which the CR2 portion has the sequence of SEQ ID NO: 2.

2. The soluble composition of claim 1 , wherein the alanine substitution of the CR2 portion is N11 or Y64 of SEQ ID NO: 2 and the construct exhibits decreased binding affinity for EBV gp350.

3. The soluble composition of claim 1 , wherein the construct is a fusion protein comprising a linker between the CR2 portion and the complement modulator portion.

4. The soluble composition of claim 1 , wherein the complement modulator portion comprises a complement inhibitor selected from the group consisting of human membrane complement protein (MCP)(SEQ ID NO:10), human decay accelerating factor (DAF)(SEQ ID NO:11), mouse DAF (SEQ ID NO:12), mouse complement receptor 1-related gene/protein y (Crry) (SEQ ID NO:4), human CD59 (SEQ ID NO:3), mouse CD59 isoform A (SEQ ID NO:6), mouse CD59 isoform 8 (SEQ ID NO:7), human complement receptor 1 (CR1) (SEQ ID NO:9), human factor H (SEQ ID NO:5), and mouse factor H (SEQ ID NO:8).

5. The soluble composition of claim 4 , wherein the complement inhibitor comprises human MCP (SEQ ID NO:10).

6. The soluble composition of claim 1 , wherein the complement modulator portion comprises a complement inhibitor selected from the group consisting of SCR1-4 of human MCP (amino acids 35-285 of SEQ ID NO: 10), SCR1-4 plus a serine/threonine-rich domain of human MCP (amino acids 35-326 of SEQ ID NO: 10), and an extracellular domain of MCP (amino acids 35-343 of SEQ ID NO:10).

7. The soluble composition of claim 4 , wherein the complement inhibitor comprises human DAF (SEQ ID NO: 11).

8. The soluble composition of claim 1 , wherein the complement modulator portion comprises a complement inhibitor selected from the group consisting of SCR1-4 of human DAF (amino acids 25-285 of SEQ ID NO:11) and SCR1-4 plus a 0-glycosylated serine/threonine-rich domain of human DAF (amino acids 25-353 of SEQ ID NO: 11).

9. The soluble composition of claim 4 , wherein the complement inhibitor comprises human CD59 (SEQ ID NO:3).

10. The soluble composition of claim 1 , wherein the complement modulator portion comprises a complement inhibitor comprising an extracellular domain of human CD59 lacking its GPI anchor (amino acids 26-101 of SEQ ID NO:3).

11. The soluble composition of claim 4 , wherein the complement inhibitor comprises human CR1 (SEQ ID NO:9).

12. The soluble composition of claim 1 , wherein the complement modulator portion comprises a complement inhibitor selected from the group consisting of SCR1-3 of human CR1 (amino acids of 42-234 of SEQ ID NO:9), SCR 1-4 of human CR1 (amino acids 42-295 of SEQ ID NO:9), SCR 1-10 of human CR1 (amino acids 42-684 of SEQ ID NO:9), SCR8-10 of human CR1 (amino acids of 491-684 of SEQ ID NO:9), SCR 8-11 of human CR1 (amino acids 491-745 of SEQ ID NO:9), SCR15-17 of human CR1 (amino acids of 941-1134 of SEQ ID NO:9), SCR15-18 of human CR1 (amino acids 941-1195 of SEQ ID NO:9), and SCR22-28 of human CR1 (amino acids 1394-1842 of SEQ ID NO:9).

13. The soluble composition of claim 4 , wherein the complement inhibitor comprises human factor H (SEQ ID NO:5).

14. The soluble composition of claim 1 , wherein the complement modulator portion comprises a complement inhibitor selected from the group consisting of SCR1-4 of human factor H (amino acids 21-262 of SEQ ID NO:5), SCR1-5 of human factor H (amino acids 21-320 of SEQ ID NO:5), SCR1-8 of human factor H (amino acids 21-507 of SEQ ID NO:5), and SCR1-18 of human factor H (amino acids 21-1104 of SEQ ID NO:5).

15. The soluble composition of claim 4 , wherein the complement inhibitor comprises mouse factor H (SEQ ID NO:8).

16. The soluble composition of claim 1 , wherein the complement modulator portion comprises a complement inhibitor selected from the group consisting of SCR1-4 of mouse factor H (amino acids 19-264 of SEQ ID NO:8), SCR1-5 of mouse factor H (amino acids 19-322 of SEQ ID NO:8), SCR1-8 of mouse factor H (amino acids 19-507 of SEQ ID NO:8), and SCR1-18 of mouse factor H (amino acids 19-1109 of SEQ ID NO:8).

17. The soluble composition of claim 1 , wherein the complement modulator portion comprises a complement activator is selected from the group consisting of human IgG 1 , human IgG 1 Fc domain, human IgM, human IgM Fc domain, mouse IgG 3 , mouse IgG 3 Fc domain, mouse IgM, mouse IgM Fc domain, and cobra venom factor (CVF).

18. A method for making a construct that selectively binds to one or more complement component 3 (C3) proteolytic fragments but does not selectively bind to EBV gp350 or IFNα, wherein the method comprises:
(a) mutating a CR2 portion of the construct to an alanine at a position selected from the group consisting of: N11 and Y64 of SEQ ID NO:2; or
(b) mutating one or more amino acids in a CR2 portion of the construct to an alanine at a position selected from the group consisting of: S42 and K50 of SEQ ID NO:2, wherein the construct comprises:
(i) a CR2 portion comprising at least the first two N-terminal SCR domains of the CR2 protein; and
(ii) a complement modulator portion.

19. The method of claim 18 , wherein the method further comprises mutating one or more amino acids in the CR2 portion of the construct at a position selected from the group consisting of: R13, Y16, A22, R28, S32, R36, K41, K48, K57, K67, Y68, R83, G84, and R89 of SEQ ID NO:2.

20. A method of reducing the binding affinity of a construct for EBV-gp350, wherein the construct comprises:
(a) a CR2 portion comprising at least the first two N-terminal SCR domains of CR2; and
(b) a complement modulator portion,
the method comprising mutating an amino acid residue of the CR2 portion selected from the group consisting of N11 and Y64 of SEQ ID NO:2 to alanine.

21. A method of reducing the binding affinity of a construct for IFNα, wherein the construct comprises:
(a) a CR2 portion comprising at least the first two N-terminal short consensus repeat (SCR) domains of CR2; and
(b) a complement modulator portion,
the method comprising mutating an amino acid residue of the CR2 portion selected from the group consisting of S42 and K50 of SEQ ID NO:2 to alanine.

22. The soluble composition of claim 1 , wherein the alanine substitution of the CR2 portion is at position S42 or K50 of SEQ ID NO: 2 and the construct exhibits decreased binding affinity for IFNα.

23. The soluble composition of claim 1 , wherein the construct exhibits comparable binding affinity for at least one C3 proteolytic fragment selected from the group consisting of C3d, iC3dg, C3dg, and a cell-bound fragment of C3b that binds to CR2 compared to a construct in which the CR2 portion does n

Assignees

• Kovacs James M
• Hannan Jonathan P
• Holers V Michael
• Univ Colorado Regents

Inventors

• Kovacs James M
• Hannan Jonathan P
• Holers V Michael

Classifications

• A61P37/06

  Immunosuppressants, e.g. drugs for graft rejection · CPC title

• A61P7/06

  Antianaemics · CPC title

• A61P9/00

  Drugs for disorders of the cardiovascular system · CPC title

• A61P5/14

  of the thyroid hormones, e.g. T3, T4 · CPC title

• A61P37/02

  Immunomodulators · CPC title