Abstract

Treatment of lymphoma patients with tumor-specific immunoglobulin (idiotype, Id) coupled to the immunogenic carrier protein keyhole limpet hemocyanin (Id-KLH) has shown promising results in phase 2 clinical trials. However, vaccines fail to elicit anti-Id immune responses in some patients, thus prompting the search for ways to improve the immunogenicity of Id-KLH vaccines. Current Id vaccine trials utilize tumor-specific Id proteins secreted by tumor-myeloma hybridomas, or recombinant Id proteins produced in mammalian lymphoid cells, bacteria, or insect cells. We now provide evidence that terminal mannose carbohydrate structures, characteristic of recombinant proteins produced in insect cells, lead to Id proteins with significantly enhanced immunostimulatory properties compared to Id proteins derived from mammalian sources. Monocyte-derived human dendritic cells (DCs) were incubated with fluorescently labeled Id proteins produced in insect or mammalian cell cultures. Insect cell-derived Id demonstrated substantially higher binding to DCs compared to the Id from a mammalian source by flow cytometry, and only the insect cell Id showed reduced binding when the DCs were preincubated with mannose receptor inhibitors. These results demonstrated that the insect cell-derived Id resulted in better targeting to DCs compared to the mammalian Id. When insect cell-derived Id proteins were coupled to KLH using glutaraldehyde and co-cultured with immature human DCs, increased expression of CD80 and CCR7 was observed by flow cytometry, indicating DC maturation. Upregulation of these markers was blocked by pre-treatment with anti-mannose receptor antibody. In tumor therapy studies, mice with 4-day established A20 murine B cell lymphoma were treated with 3 weekly injections of Id-KLH plus GM-CSF and followed for survival. A20 bearing mice treated with Id-KLH containing insect cell-derived A20 Id displayed improved survival compared with mice treated with hybridoma-derived A20 Id-KLH (61% vs. 46%, respectively). Anti-Id antibodies against A20 murine B cell lymphoma were assessed by ELISA following Id-KLH immunization. Both insect and mammalian sources of Id generated similar levels of anti-Id antibodies, showing no impairment in antibody responses due to the differences in glycoslylation. Anti-A20 cytotoxic T lymphocyte (CTL) activity was measured in splenic T cells from Id-KLH-immunized mice. Here, induction of CD8+ CTLs by insect cell-derived A20 Id-KLH was significantly greater than CTL induction following immunization with hybridoma-derived A20 Id-KLH (P=0.0061). To determine the importance of the T cell response in A20 tumor killing in vivo, mice were depleted of CD4+ and CD8+ T cell subsets, challenged with A20 tumor, and then vaccinated 4 days later as in the experiment above. Mice depleted of CD8+ T cells all succumbed to tumor demonstrating a critical role for CD8+ T cells in A20 tumor eradication. In conclusion, our data comparing sources of recombinant Id protein tumor antigens used in therapeutic cancer vaccines suggest that post-translational modifications, namely terminal mannose residues, can significantly influence the immunological properties and eventual therapeutic efficacy of the product.

Author notes

Disclosure:Employment: D.P. Gold, F. Rosas, X. Mu, and D. McDonnel are all employees of Favrille, Inc. Consultancy: J. M. Timmerman has served as a consultant to Favrille. Research Funding: J.M. Timmerman has received research funding from Favrille, Inc. Honoraria Information: J.M. Timmerman has received honoraria from Favrille, Inc. for consulting work.