Modified truncated complement system regulators

a complement system and regulator technology, applied in the field of modified and/or shortened forms of complement system regulators, can solve the problem that the complete inhibition of the complement system on a long-term basis is not likely to be desirable in most individuals, and achieve the effect of reducing tissue damage and suppressing transplant rejection

Inactive Publication Date: 2008-05-22
WASHINGTON UNIV IN SAINT LOUIS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0030]These analogs are useful in controlling the complement system, and thus may be useful in the treatment of autoimmune diseases, the suppression of rejection of transplants, in diagnosis and the reduction in tissue damage associated with myocardial infarctions and cerebral vascular accidents. They may also play a role in the diagnosis of conditions associated with complement activation and immune complex formation.

Problems solved by technology

Complete inhibition of the complement system on a long-term basis is not likely to be desirable in most individuals.

Method used

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  • Modified truncated complement system regulators
  • Modified truncated complement system regulators
  • Modified truncated complement system regulators

Examples

Experimental program
Comparison scheme
Effect test

example 1

Binding Specificity of Truncated CR1

[0108]The cDNA which encodes the first 543 amino acids of mature CR1 (SCR 1-8 and ½ of SCR-9) was transfected into COS cells to obtain a secreted protein designated CR1-4.

[0109]Two mouse monoclonal anti-CR1 antibodies were used to determine the immunoreactivity of CR1-4, and as a method to assay for this protein. Antibody E11 (Hogg, N., et al., Eur. J. Immunol. (1984) 14:236-243), and 3D9 (O'Shea, J. J., et al., J. Immunol. (1985) 134:2580-2587), recognized CR1 and bound to the recombinantly produced CR1-4.

[0110]Binding to C4b or to C3b was tested using either C4b or iC3 (C3 containing a broken thioester bond analogous in reactivity and function to C3b) bound to a Sepharose™ support as described by Dykman, T., et al., Proc. Natl. Acad. Sci. USA (1983) 80:1698-1702, and Cole, J. L., et al., Proc. Natl. Acad. Sci. USA (1985) 82:859-863. Binding to these solid-supported substrates was verified by testing with an ELISA for CR1.

[0111]The C4b derivatize...

example 2

Construction of CR1-4 Analogs

[0113]Various mutated forms of CR1-4 were constructed and tested for binding activity to C4b and iC3 as described above in Example 1. Table 2 shows the analogs constructed and the binding and cofactor activities of the analogs. In Table 2, the modifications are shown by the number of the amino acid and the conversion effected. The numbers correspond to those set forth in FIGS. 2A and 2B and FIGS. 3A and 3B which set forth the amino sequences of SCR-1 (SEQ ID NO: 1), SCR-2, (SEQ ID NO: 3) SCR-8 (SEQ ID NO: 2) and SCR-9 (SEQ ID NO: 4) in CR1.

[0114]The combined changes shown in truncated forms should be predictive of the activity of a modified full length protein. There is strong evidence available that establishes three primary binding and cofactor sites in the major polymorphic form (30 SCRs) of CR1. One site interacts almost exclusively with C4b (SCRs 1-4) while the other two sites, nearly identical in amino acid sequence, interact with both C4b and C3b ...

example 5

DAF Analogs

[0133]The membrane-bound complement regulator DAF facilitates the dissociation of C3b and C4b-containing convertases but does not bind C3b or C4b, nor does it serve as cofactor for factor I-mediated proteolytic inactivation of C3b or C4b. It would be desirable, under certain situations, to increase the complement regulatory activity of DAF or of truncated, recombined or hybrid forms of DAF.

[0134]Based on amino sequence homology, DAF SCRs 2-3 corresponds to the CR1 active regions. Homology between DAF SCRs 2-3 and CR1 SCRs 1-2 is 40% while homology between DAF SCRs 2-3 and CR1 SCRs 8-9 is 39%. Since there are several unmatched amino acids in these alignments, the position numbers of DAF do not precisely match those of CR1.

[0135]One or more substitutions are introduced into the DAF SCRs 2-3. Specific substitutions designed to confer C3b and C4b cofactor activity and binding activity are selected from the following:

[0136]DAF position(s) enumerated as in Lublin and Atkinson, ...

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Abstract

Analogs of regulators of complement activation (RCA) proteins which have altered specificities and affinities for the targets C3b and / or C4b are described. These analogs are obtained by substituting amino acids which affect the complement inhibitory activities of these proteins, substituting, rearranging or adding SCRs (short consensus repeats) or SCR regions to the proteins, deleting amino acid sequences, and combinations thereof.

Description

RELATED APPLICATIONS[0001]This application is a continuation of U.S. application Ser. No. 08 / 126,505, filed Sep. 24, 1993 (Now U.S. Pat. No. 6,897,290 B1), which is a continuation-in-part of U.S. application Ser. No. 07 / 695,514, filed May 3, 1991 (Now abandoned). The entire teachings of U.S. application Ser. No. 08 / 126,505 (Now U.S. Pat. No. 6,897,290 B1) are incorporated herein by reference.BACKGROUND OF THE INVENTION[0002]The invention relates to modified and / or shortened forms of complement regulators derived from regulatory proteins of complement activation (RCA), especially CR1.[0003]The complement system serves to aid in the removal of foreign substances and of immune complexes from animal hosts. This system and its regulation is reviewed by Hourcade, D., et al., Advances in Immunol (1989) 45: 381-416. Briefly, the complement system generates, either by a “classical pathway” or an “alternative pathway,” C3b which binds to target immune complexes or foreign substances and marks...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/00C07K14/00C12N15/11C12N15/00A61P43/00C12N5/06A01K67/00C12P21/04A61P9/00A61P9/10A61P37/00C07K14/435C07K14/47C07K14/705C12N5/10C12N15/09C12N15/12C12P21/02C12R1/91G01N33/53
CPCA01K67/0275A01K2217/05A01K2267/03C07K2319/00C07K14/472C07K14/70596A61K38/00A61P9/00A61P9/10A61P37/00A61P43/00
Inventor ATKINSON, JOHN P.HOURCADE, DENNISKRYCH, MALGORZATA
Owner WASHINGTON UNIV IN SAINT LOUIS
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