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Human complement c3 derivatives with cobra venom factor-like function

A cobra venom factor and complement technology, applied in the field of chimeric derivatives of human complement C3, can solve problems such as serum complement activity failure

Inactive Publication Date: 2007-08-08
UNIV OF HAWAII
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The combination of the long intrinsic half-life of CVF-containing enzymes and resistance regulation allows CVF to continuously activate C3 and C5 (and subsequently activate other complement components), ultimately leading to depletion of serum complement activity

Method used

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  • Human complement c3 derivatives with cobra venom factor-like function
  • Human complement c3 derivatives with cobra venom factor-like function
  • Human complement c3 derivatives with cobra venom factor-like function

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1: Production of human complement C3 / CVF hybrid protein

[0084] Replacing the human C3 sequence with a CVF sequence that represents important structural features of CVF-specific functions results in the generation of C3 derivatives with CVF-like functions. Accordingly, embodiments of the present invention relate to the generation of human C3 derivatives that exhibit complement-depleting CVF-specific functions by forming stable convertases for use as novel therapeutic agents that deplete complement in clinical diseases in which complement activation is part of the etiology. Since the structural changes of human C3 caused by the specific sequence of CVF are small, it is clear that the modified C3 molecules can show significantly reduced or even no immunogenicity.

[0085] The human C3 molecules in Table 1 were designed to contain specific CVF sequences in order to generate CVF-functional human C3 derivatives. Some embodiments of the invention provide for minor s...

Embodiment 2

[0098] Example 2: Expression of modified human C3 protein

[0099] The protein was produced in the Drosophila S2 cell system using the Drosophila Bip signal sequence to secrete the protein. Briefly, plasmid pMB / HC3-1550, pMB / HC3-1504, pMB / HC3-1496, pMB / HC3-1550 / 1617 and pMB / HC3-1348 were transfected into Drosophila S2 cells. S2 cells were transfected with a mixture of expression plasmids and pCoBlast in a 19:1 (w:w) ratio. After transfection, cells containing the two plasmids were selected with blasticidin (25 μg / ml). For expression, a 1 liter culture of transfected cells was grown in serum-free medium (Hi-Five plus L-glutamine) without blasticidin. When the cell density reaches 5×10 6 At 1 cell / ml, CuSO4 was added to a final concentration of 25 μM to induce recombinant protein production. Allow the cultures to express the recombinant protein for 4-5 days. The hybrid protein was then purified from the culture medium by combined ANX, Sephacryl H-300 and CM-FF chromatog...

Embodiment example 3

[0102] Embodiment 3: Activity test result of modified human complement C3 protein

[0103] The purified modified human C3 protein hybrids were subjected to various functional assays as follows.

[0104] Complement depletion

[0105] This test detects the ability of a protein to deplete complement in human (or other) serum. This test proceeds in two steps. In the first step, the protein of interest is usually diluted in buffer to expected concentration. A 10 microliter portion of the diluted protein was then mixed with undiluted serum. The mixture is incubated at 37°C for 3 hours, which allows the protein to activate complement by forming a C3 convertase. The subsequent formation of convertase activates C3 in serum. The amount of remaining complement activity is then determined by diluting the serum with antibody-sensitized sheep red blood cells that are readily lysed by complement when present in the serum and mixing with the serum. The reaction was allowed to proce...

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Abstract

A modified human complement C3 protein (C3) is disclosed comprising a substitution of a portion of a human C3 protein, with a corresponding portion of a Cobra Venom Factor protein (CVF) which results in a human C3 protein with CVF functions, but with substantially reduced immunogenicity. Advantageously, the C3 protein can be manipulated to contain at least one of the following CVF functions: increased stability of the C3 convertase and increased resistance to the actions of factors H and / or I. A large number of hybrid C3 proteins containing substitutions in the C-terminal portion of the alpha chain of C3 are presented and tested for the above functions. Methods of treatment of diseases such as reperfusion injury, autoimmune diseases, and other diseases of increased complement activation are presented as well as methods of increasing the effectiveness of gene therapeutics and other therapeutics.

Description

[0001] This international application claims priority to U.S. Provisional Applications 60 / 567,069 filed April 30, 2004, 60 / 653,247 filed February 14, 2005, and 60 / 667,352 filed March 30, 2005, each of which All are incorporated herein by reference in their entirety. field of invention [0002] The present invention generally relates to chimeric derivatives of human complement C3, wherein said chimeric derivatives of human complement C3 have a portion of human C3 protein replaced by a corresponding portion of cobra venom factor (CVF) protein. Preferably, the alpha chain portion of C3 is replaced with a corresponding portion of CVF. Background of the invention [0003] The third component of complement, C3, plays a key role in both the classical and alternative pathways of complement activation, and many physiological C3 activation products have important functions in immune responses and host defense responses. In the alternative pathway, the activated form of C3, C3b, is th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00C07K19/00
Inventor C－W·沃格尔D·C·弗里齐格尔
Owner UNIV OF HAWAII
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