RESUMEN
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 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.
Asunto(s)
Vacunas contra el Cáncer/genética , Fragmentos Fab de Inmunoglobulinas/genética , Cadenas Pesadas de Inmunoglobulina/genética , Idiotipos de Inmunoglobulinas/genética , Cadenas Ligeras de Inmunoglobulina/genética , Linfoma de Células B/genética , Linfoma Folicular/genética , Vacunas contra el Cáncer/aislamiento & purificación , Vacunas contra el Cáncer/uso terapéutico , Escherichia coli/genética , Humanos , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Fragmentos Fab de Inmunoglobulinas/uso terapéutico , Cadenas Pesadas de Inmunoglobulina/aislamiento & purificación , Cadenas Pesadas de Inmunoglobulina/uso terapéutico , Idiotipos de Inmunoglobulinas/aislamiento & purificación , Idiotipos de Inmunoglobulinas/uso terapéutico , Cadenas Ligeras de Inmunoglobulina/aislamiento & purificación , Cadenas Ligeras de Inmunoglobulina/uso terapéutico , Linfoma de Células B/terapia , Linfoma Folicular/terapia , Reacción en Cadena de la Polimerasa/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/uso terapéutico , VacunaciónRESUMEN
Tumor-specific CD8 T cell responses to MCA102 fibrosarcoma cells expressing the cytotoxic T cell epitope gp33 from lymphocytic choriomeningitis virus were studied. MCA102(gp33) tumors grew progressively in C57BL/6 mice, despite induction of peripheral gp33-tetramer(+) T cells that were capable of mediating antiviral protection, specific cell rejection, and concomitant tumor immunity. MCA102(gp33) tumors were infiltrated with a high number ( approximately 20%) of CD11b(+)CD11c(-) macrophage-phenotype cells that were able to cross-present the gp33 epitope to T cells. Tumor-infiltrating CD8 T cells exhibited a highly activated phenotype but lacked effector cell function. Strikingly, a significant portion of tumor-infiltrating lymphocytes expressed TCRs specific for gp33 but bound MHC tetramers only after cell purification and a 24-h resting period in vitro. The phenomenon of "tetramer-negative T cells" was not restricted to tumor-infiltrating lymphocytes from MCA102(gp33) tumors, but was also observed when Ag-specific T cells derived from an environment with high Ag load were analyzed ex vivo. Thus, using a novel tumor model, allowing us to trace tumor-specific T cells at the single cell level in vivo, we demonstrate that the tumor microenvironment is able to alter the functional activity of T cells infiltrating the tumor mass.