Cloning of Idiotype Immunoglobulin Genes in B Cell Lymphomas by Anchored PCR and Production of Individual Recombinant Idiotype Vaccines in E. coli.

The hypervariable regions (idiotypes) of the immunoglobulins (Ig) expressed by lymphoma cells represent individual and specific tumor antigens. Purified or recombinant lymphoma-derived idiotype (Id) may be formulated into an individual vaccine and administered to lymphoma patients to elicit a therapeutic tumor-specific host immune response. We have developed an expression strategy based on anchored PCR amplification of lymphoma-derived cDNA. The Id variable (V) gene segments are inserted into a prokaryotic expression vector, and recombinant Fab fragment corresponding to the native Id protein is expressed in E.coli. This approach was applied to 106 biopsies with confirmed lymphoma involvement (47 follicular, 14 mantle cell, 6 diffuse large cell NHL, 22 myelomas, 6 CLL, and 6 other NHL types) and 8 non-lymphomatous control samples with the aim to investigate the feasibility of this production strategy in a large cohort of NHL patients, and to assess the yield and purity of the recombinant Id vaccines under large-scale production. According to clonality criteria defined to minimize the likelihood of mistakenly interpreting occasional repeat sequences (observed with low frequency in the control biopsies) as lymphoma-derived Id, cloning of Id VH and VL segments was successful in 95% of lymphoma biopsies. Expression of recombinant Fab fragment was successful (> 1 mg) in 93% of lymphomas. Fab yield was variable and ranged from 1.2 to 250 mg, whereas Fab purity was >95% in 95% of the cases after affinity chromatography purification. One Fab preparation only was contaminated with E.coli protein by western blot. MM had a trend towards superior Fab yields but this finding may merely reflect a selection for cases with secreted paraprotein, i.e. Ids with favorable folding properties and solubility. No other NHL type was associated with inferior expression levels. Fabs with lambda-type light chain had a trend towards lesser yield (p=0.0016) and purity (p=0.029) than kappa-Fabs. As expected, 25 of 38 follicular lymphoma cases had acquired novel potential glycosylation sites within their Id V segments (preferentially in CDR regions), whereas 34 of 40 other lymphomas lacked such sites (p<0.0001). Nevertheless, such sites did not influence the Fab yield (p=0.97). All other structural Id features (isotype, V gene usage, hypermutation status, CDRIII charge) also had no adverse influence on Fab expression. In summary, A-PCR cloning of Ig transcripts has a high success rate for the identification and analysis of Id genes in all major types of B-cell lymphoma. A-PCR with expression of idiotype protein in E. coli as a recombinant Fab fragment provides a solid basis for production of individual Id vaccines of all major IgH and IgL classes. The short culture time required for Id expression in bacteria in comparison to eukaryotic cell cultures may facilitate clinical trials and reduce production costs.