Resurrection of a clinical antibody: template proteogenomic de novo proteomic sequencing and reverse engineering of an anti-lymphotoxin-? antibody.

ABSTRACT: A mouse hybridoma antibody directed against a member of the tumour necrosis factor (TNF)-superfamily, lymphotoxin-alpha (LT-?), 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-? (LT-3F12) to be a preclinical candidate for the treatment of several inflammatory pathologies. We have successfully rescued the LT-3F12 antibody by performing MS 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 reestablish monoclonal antibody-expressing cells from archived protein sample, exemplifying the development of novel therapeutics from cryptic protein sources.

SUBMITTER: Castellana NE 

PROVIDER: S-EPMC3253889 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature