HIGH THROUGHPUT CALI FOR TARGET VALIDATION IN METASTASIS

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Metastasis is one of the most devastating aspects of cancer. Thus, discovering therapies that inhibit metastasis is an important goal in cancer treatment. In order to do this, proteins involved in cancer cell invasiveness that could be targets for therapeutic drugs must be validated and this is often a rate-limiting step in drug discovery. The overall objective is to develop an enhanced high throughput screen (HTS) using chromophore assisted laser inactivation (CALI) for target validation of surface proteins that act in cancer cell invasiveness. CALI targets laser energy using a dye-labeled antibody to inactivate the function of the bound antigen. In the R21 phase, an HTS Transwell assay will be developed and used to show that CALI will increased markedly the number of phage display antibodies that disrupt invasiveness. Single chain Fv fusion phage libraries that bind specifically to surface proteins of HT-1080 human fibrosarcoma cells will be generated and used for a pilot screen (n=96 antibodies) using CALI. The achievement of these aims will establish the feasibility of an HTS using CALI for the validation of targets involved in cancer cell invasiveness. In the R33 phase, a full scale automated screen will be conducted (n= 10,000 antibodies) and protein targets validated by this screen will be identified using high resolution mass spectrometry. This proposed technology will provide a low cost and rapid means of target validation and will contribute a number of targets for anti-metastasis drug discovery. The methods developed are general; they will have an application to cell processes in cancer and other diseases. As such, they will be of great utility for pharmaceutical companies and academic labs.