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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

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Publications

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

Castellana Natalie E NE McCutcheon Krista K Pham Victoria C VC Harden Kristin K Nguyen Allen A Young Judy J Adams Camellia C Schroeder Kurt K Arnott David D Bafna Vineet V Grogan Jane L JL Lill Jennie R JR

Proteomics 20110105 3

A mouse hybridoma antibody directed against a member of the tumour necrosis factor (TNF)-superfamily, lymphotoxin-alpha (LT-α), 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-α (LT-3F12) to be a preclinical candidate for the treatment of several inflammatory pathologies. We have successfully rescued the LT-3F12 a ...[more]

PMID: 21268269

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Project description:We have carried out proteogenomic analysis of the breast cancer transcriptomic and proteomic data, available at The Clinical Proteomic Tumor Analysis Consortium resource, to identify novel peptides arising from alternatively spliced events as well as other noncanonical expressions. We used a pipeline that consisted of de novo transcript assembly, six frame-translated custom database, and a combination of search engines to identify novel peptides. A portfolio of 4,387 novel peptide sequences initially identified was further screened through PepQuery validation tool (Clinical Proteomic Tumor Analysis Consortium), which yielded 1,558 novel peptides. We considered the dataset of 1,558 validated through PepQuery to understand their functional and clinical significance, leaving the rest to be further verified using other validation tools and approaches. The novel peptides mapped to the known gene sequences as well as to genomic regions yet undefined for translation, 580 novel peptides mapped to known protein-coding genes, 147 to non-protein-coding genes, and 831 belonged to novel translational sequences. The novel peptides belonging to protein-coding genes represented alternatively spliced events or 5' or 3' extensions, whereas others represented translation from pseudogenes, long noncoding RNAs, or novel peptides originating from uncharacterized protein-coding sequences-mostly from the intronic regions of known genes. Seventy-six of the 580 protein-coding genes were associated with cancer hallmark genes, which included key oncogenes, transcription factors, kinases, and cell surface receptors. Survival association analysis of the 76 novel peptide sequences revealed 10 of them to be significant, and we present a panel of six novel peptides, whose high expression was found to be strongly associated with poor survival of patients with human epidermal growth factor receptor 2-enriched subtype. Our analysis represents a landscape of novel peptides of different types that may be expressed in breast cancer tissues, whereas their presence in full-length functional proteins needs further investigations.

Dataset Information

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

Publications

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

Similar Datasets

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