Cloning of idiotype immunoglobulin genes in B cell lymphomas by anchored PCR and production of individual recombinant idiotype vaccines in Escherichia coli.
OBJECTIVE
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 kappa-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.
Bertinetti
Cristina
C
Department of Haematology/Oncology, Freiburg University Medical Center, Freiburg, Germany.
Simon
Frederic
F
Zirlik
Katja
K
Heining-Mikesch
Kristina
K
Pfeifer
Dietmar
D
Osterroth
Frank
F
Rosenthal
Felicia M
FM
Veelken
Hendrik
H
D019496:Cancer Vaccines / Q000235:genetics* / Q000302:isolation & purification / Q000627:therapeutic use; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000302:isolation & purification / Q000627:therapeutic use; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000302:isolation & purification / Q000627:therapeutic use; D007130:Immunoglobulin Idiotypes / Q000235:genetics* / Q000302:isolation & purification / Q000627:therapeutic use; D007147:Immunoglobulin Light Chains / Q000235:genetics* / Q000302:isolation & purification / Q000627:therapeutic use; D016393:Lymphoma, B-Cell / Q000235:genetics* / Q000628:therapy; D008224:Lymphoma, Follicular / Q000235:genetics* / Q000628:therapy; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000627:therapeutic use; D014611:Vaccination
D019496:Cancer Vaccines; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007130:Immunoglobulin Idiotypes; D007147:Immunoglobulin Light Chains; D011994:Recombinant Proteins