title: Cloning of idiotype immunoglobulin genes in B cell lymphomas by anchored PCR and production of individual recombinant idiotype vaccines in Escherichia coli.
abstract: 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.
authors.affiliation: Department of Haematology/Oncology, Freiburg University Medical Center, Freiburg, Germany.
authors.forename: Cristina
authors.initials: C
authors.lastname: Bertinetti
authors.forename: Frederic
authors.initials: F
authors.lastname: Simon
authors.forename: Katja
authors.initials: K
authors.lastname: Zirlik
authors.forename: Kristina
authors.initials: K
authors.lastname: Heining-Mikesch
authors.forename: Dietmar
authors.initials: D
authors.lastname: Pfeifer
authors.forename: Frank
authors.initials: F
authors.lastname: Osterroth
authors.forename: Felicia M
authors.initials: FM
authors.lastname: Rosenthal
authors.forename: Hendrik
authors.initials: H
authors.lastname: Veelken
mesh_terms: 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
chemical_list: D019496:Cancer Vaccines; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007130:Immunoglobulin Idiotypes; D007147:Immunoglobulin Light Chains; D011994:Recombinant Proteins