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Cloning of idiotype immunoglobulin genes in B cell lymphomas by anchored PCR and production of individual recombinant idiotype vaccines in Escherichia coli
Author(s) -
Bertinetti Cristina,
Simon Frederic,
Zirlik Katja,
HeiningMikesch Kristina,
Pfeifer Dietmar,
Osterroth Frank,
Rosenthal Felicia M.,
Veelken Hendrik
Publication year - 2006
Publication title -
european journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.904
H-Index - 84
eISSN - 1600-0609
pISSN - 0902-4441
DOI - 10.1111/j.1600-0609.2006.00740.x
Subject(s) - idiotype , immunoglobulin idiotypes , biology , recombinant dna , antibody , escherichia coli , microbiology and biotechnology , virology , immunogenicity , antigen , immunology , gene , monoclonal antibody , genetics
Objectives: Individual immunoglobulins expressed by B‐cell lymphomas represent tumor‐specific antigens (‘idiotypes’). Immunization with idiotype in follicular lymphoma patients may induce specific immune responses, sustained progression‐free survival, and disappearance of minimal residual disease. Manufacturing of idiotype vaccines has mostly relied on heterohybridomas established from viable lymphoma cells. This paper describes the feasibility of production of GMP‐grade idiotype vaccines as recombinant Fab fragments in Escherichia coli . Methods: IgH and IgL transcripts were analyzed by anchored PCR from 106 lymphoma and nine control biopsies. Lymphoma‐derived V segments were inserted into prokaryotic expression plasmids. Recombinant idiotype Fab fragments were expressed in E. coli in a fermentation system. Results: Idiotype IgH and IgL transcripts were identified in 95% of 106 lymphoma biopsies according to stringent clonality criteria. Large‐scale idiotype expression was successful in 69 of 78 cases (89%) and yielded a median of 17 mg (range: 1.2–250 mg) recombinant Fab protein. After affinity chromatography, median vaccine purity was 99% heterodimeric Fab protein (range: 72–100%). Bacterial protein contamination was detectable in one vaccine only. Fab proteins with IgL lambda chains had a tendency for inferior yield and lesser purity than κ ‐type Fabs. Among other structural idiotype features (isotype, V family usage, somatic hypermutation pattern, novel glycosylation sites, CDR III net charge), no consistent influences on Fab yield or purity were detected. Conclusions: Anchored PCR cloning and subsequent protein expression in E. coli provides a reliable technological basis for clinical idiotype vaccination trials.