Resurrection of a Clinical Antibody: Template ProteoGenomic de novo Proteomic Sequencing and Reverse Engineering of an Anti-Lymphotoxin Alpha Antibody
A mouse hybridoma antibody directed against a member of the TNF-superfamily, lymphotoxin alpha (LT-ɑ), was isolated from stored mouse ascites and purified to homogeneity. After more than a decade of storage the genetic material was not available for cloning, however biochemical assays with the ascites showed this antibody against LT-ɑ (LT-3F12) to be a pre-clinical candidate for the treatment of several inflammatory pathologies. We have successfully rescued the LT-3F12 antibody by performing mass spectrometric analysis, primary amino acid sequence determination by template proteogenomics, and synthesis of the corresponding recombinant DNA by reverse engineering. The resurrected antibody was expressed, purified and shown to demonstrate the desired specificity and binding properties in a panel of immuno-biochemical tests. The work described herein demonstrates the powerful combination of high throughput informatic proteomic de novo sequencing with reverse engineering to re-establish monoclonal antibody expressing cells from archived protein sample, exemplifying the development of novel therapeutics from cryptic protein sources.
The raw spectra used in the project can be downloaded from tranche using the hash key
The spectra after clustering using MSCluster can be downloaded here:
The matching of clustered spectra back to the raw spectra is here.
The database search results from InsPecT is here.
- The results of the template proteogenomic tool, GenoMS, is here.
Resurrection of a clinical antibody: template proteogenomic de novo
proteomic sequencing and reverse engineering of an anti-lymphotoxin
NE Castellana, K McCutcheon, V Pham, K Harden, A Nguyen, J Young, C Adams, K Schroeder, D Arnott, V Bafna, JL Grogan, JR Lill. (2010).
Proteomics, epub ahead of print.
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