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CHIPS Résumé de rapport

Project ID: 517128
Financé au titre de: FP6-MOBILITY
Pays: Sweden

Final Activity Report Summary - CHIPS (Chemotaxis inhibitory protein of staphylococci)

The immediate objective of the project is to develop novel drug candidates for the treatment and basic disease modification of inflammatory disorders. A first approach involves de-immunisation of an already identified molecule, (CHIPS) derived from Staphylococci. The CHIPS molecule was developed by UMCU, and the group has shown the therapeutic potential of the substance. 2007 our lead candidate, ADC-1004 was chosen.

De-immunisation of the parent CHIPS molecule
In the first round of de-immunisation random mutagenesis was used to create mutations in the CHIPS wt protein. Libraries of mutants were phage selected and HT screening of mutants for decreased binding to anti-CHIPS antibodies were performed. Nine clones with improved characteristics were used as starting material for FIND recombinantion.

Repeated library design (3 rounds of FIND in total), HT screening of CHIPS mutants and characterisation of selected mutants was performed. The final round of FIND libraries and screening was finished in October 2006. Top ten mutants were selected for further characterisation in functional assays. To be able to evaluate the mutants in functional assays we needed purified proteins.

During the exchange of Erika we realised that the protocol for purification of mutants could not be the same as for purification of wt CHIPS. The mutants were mostly expressed as insoluble proteins. Therefore, the second exchange (Ingrid) was focused only on establishment of a new protocol for purification of CHIPS mutants. She continued her work initiated in Lund when she came back to Utrecht and finally by the end of the year she managed to establish a protocol for purification of inclusion bodies.

During 2007 27 new CHIPS variants were created from the top ten mutants selected during 2006. The amino acid changes found in our CHIPS mutants were changed into other amino acids (suggested by our collaborator; Saromics).

Secondly, new combination of mutations was created in some of our site-directed mutagenesis mutants. The different CHIPS mutants were expressed and analysed for anti-CHIPS antibody binding and for binding to the C5a receptor. All mutants had low binding to anti-CHIPS antibodies, however some of the mutants showed decreased binding to the receptorThe different mutants were analysed in different assay for diminished binding to serum antibodies. They were further characterised in functional activity. Our candidate drug has been selected and it is called, ADC-1004. ADC-1004 is a full antagonist for the C5aR, inhibiting the activation and chemotaxis of neutrophils at comparable level as wildtype CHIPS. ADC-1004 has lost almost all binding to serum antibodies, 0,4 % binding compared to wildtype CHIPS binding.

A spin-off project was initiated in milestone 1, this approach used peptide phage display libraries to select peptides that bind anti-CHIPS abs. 4 different unique peptides were identified corresponding to 14 aminoacids in the CHIPS protein. Soluble peptides corresponding to the four sequences identified in the phage selections were synthesised and were analysed in ELISA and column based assays. The epitopes were mapped on the surface of the CHIPS molecule and individual amino acid changes in CHIPS were introduced, the mutants were analysed in an ELISA for decreased binding to the anti-CHIPS antibodies. Unique positions important for binding the antibodies were identified. This work has been summarised in an article.

Small antibody fragments that mimic CHIPS action
The objective for the project is to identify an antibody that mimics CHIPS actions, that is inhibition of C5a induced activation of neutrophils. In the selections of scFv fragment with affinity to C5aR the phage library, n-CoDeR from Bioinvent was used. The C5aR binding phages were enriched by using a biotinylated peptide corresponding to the N-terminal part of the C5aR. After 3-4 rounds of positive selection 120 clones were analysed in a peptide ELISA and most of the clones were positive. However, no antibodies binding the receptor could be selected. The project was therefore stopped.


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