title: Maleimide conjugation markedly enhances the immunogenicity of both human and murine idiotype-KLH vaccines.
abstract: The collection of epitopes present within the variable regions of the tumor-specific clonal immunoglobulin expressed by B cell lymphomas (idiotype, Id) can serve as a target for active immunotherapy. Traditionally, tumor-derived Id protein is chemically conjugated to the immunogenic foreign carrier protein keyhole limpet hemocyanin (KLH) using glutaraldehyde to serve as a therapeutic vaccine. While this approach offered promising results for some patients treated in early clinical trials, glutaraldehyde Id-KLH vaccines have failed to induce immune and clinical responses in many vaccinated subjects. We recently described an alternative conjugation method employing maleimide-sulfhydryl chemistry that significantly increased the therapeutic efficacy of Id-KLH vaccines in three different murine B cell lymphoma models, with protection mediated by either CD8(+) T cells or antibodies. We now define in detail the methods and parameters critical for enhancing the in vivo immunogenicity of human as well as murine Id-KLH conjugate vaccines. Optimal conditions for Id sulfhydryl pre-reduction were determined, and maleimide Id-KLH conjugates maintained stability and potency even after prolonged storage. Field flow fractionation analysis of Id-KLH particle size revealed that maleimide conjugates were far more uniform in size than glutaraldehyde conjugates. Under increasingly stringent conditions, maleimide Id-KLH vaccines maintained superior efficacy over glutaraldehyde Id-KLH in treating established, disseminated murine lymphoma. More importantly, human maleimide Id-KLH conjugates were consistently superior to glutaraldehyde Id-KLH conjugates in inducing Id-specific antibody and T cell responses. The described methods should be easily adaptable to the production of clinical grade vaccines for human trials in B cell malignancies.
authors.affiliation: Division of Hematology and Oncology, Department of Medicine, University of California, Los Angeles, Los Angeles, CA 90095-1678, USA.
authors.forename: Kamran
authors.initials: K
authors.lastname: Kafi
authors.forename: David J
authors.initials: DJ
authors.lastname: Betting
authors.forename: Reiko E
authors.initials: RE
authors.lastname: Yamada
authors.forename: Michael
authors.initials: M
authors.lastname: Bacica
authors.forename: Kristopher K
authors.initials: KK
authors.lastname: Steward
authors.forename: John M
authors.initials: JM
authors.lastname: Timmerman
mesh_terms: D000818:Animals; D019496:Cancer Vaccines / Q000276:immunology*; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D031521:Fractionation, Field Flow; D005615:Freezing; D005976:Glutaral / Q000276:immunology; D006433:Hemocyanins / Q000276:immunology*; D006801:Humans; D007130:Immunoglobulin Idiotypes / Q000276:immunology*; D008223:Lymphoma / Q000276:immunology; D008301:Maleimides / Q000276:immunology*; D051379:Mice; D010084:Oxidation-Reduction / Q000187:drug effects; D010316:Particle Size; D055550:Protein Stability / Q000187:drug effects; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology; D013997:Time Factors
chemical_list: D019496:Cancer Vaccines; D004220:Disulfides; D007130:Immunoglobulin Idiotypes; D008301:Maleimides; C043592:maleimide; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin; D005976:Glutaral; D004229:Dithiothreitol