Cloning of idiotype immunoglobulin genes in B cell lymphomas by anchored PCR and production of individual recombinant idiotype vaccines in Escherichia coli.

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.

Transplanted bone marrow cells preferentially home to the vessels of in situ generated murine tumors rather than of normal organs.

Transplanted bone marrow-derived (BM) cells have been shown to home into the tumor vessels of s.c. implanted tumor models and to functionally contribute to tumor neoangiogenesis and tumor growth. However, whether BM cells contribute to the vessels of in situ developing tumors remains unknown. We have taken advantage of the in situ generation of mammary tumors in transgenic mice carrying the polyoma virus middle T oncogene (MMTV-PyVT) to determine whether transplanted BM cells home to and incorporate into the intratumoral vessels. Unfractionated BM from lacZ+ROSA 26 mice was used to rescue irradiated MMTV-PyVT transgenic mice or their wild-type congenics. All transgenic mice were sacrificed when they developed easily palpable mammary tumors. BM cells recruited and incorporated into the vasculature were identified by coexpression of lacZ and CD31, evidence that these cells had a distinctive, elongated appearance and that they lined the vessel structures. We found that BM cells home to and incorporate into 1.3% of the vessels of all in situ generated mammary adenocarcinomas examined (n=8). In contrast, BM cells did not recruit into the vessels of colon or liver of the tumor-bearing mice. Whether these cells contribute to new vessel formation via vasculogenesis or angiogenesis or simply attach to, and integrate into, the growing tips or shafts of pre-existing vessels has to be determined. BM could be used as a vehicle for the specific transport of antiangiogenic signals into the tumor vascular bed.

Lack of effector cell function and altered tetramer binding of tumor-infiltrating lymphocytes.

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.