Rapid production of specific vaccines for lymphoma by expression of the tumor-derived single-chain Fv epitopes in tobacco plants

Abstract

Rapid production of protein-based tumorspecific vaccines for the treatment of malignancies is possible with the plant-based transient expression system described here. We created a modified tobamoviral vector that encodes the idiotype-specific single-chain Fv fragment (scFv) of the immunoglobulin from the 38C13 mouse B cell lymphoma. Infected Nicotiana benthamiana plants contain high levels of secreted scFv protein in the extracellular compartment. This material reacts with an anti-idiotype antibody by Western blotting, ELISA, and affinity chromatography, suggesting that the plant-produced 38C13 scFv protein is properly folded in solution. Mice vaccinated with the affinity-purified 38C13 scFv generate >10 g͞ml anti-idiotype immunoglobulins. These mice were protected from challenge by a lethal dose of the syngeneic 38C13 tumor, similar to mice immunized with the native 38C13 IgM-keyhole limpet hemocyanin conjugate vaccine. This rapid production system for generating tumorspecific protein vaccines may provide a viable strategy for the treatment of non-Hodgkin's lymphoma.Most B cell malignancies are incurable and are increasing in frequency in the populations of industrial nations (1, 2). Although B cell tumors are characterized by extreme variability in treatment and prognosis (3), they share a common feature that makes them ideal for the development of patientspecific cancer vaccines. Each clone of malignant B cells expresses a unique cell surface immunoglobulin (Ig)-a tumorspecific marker. The tumor's surface Ig, when made immunogenic by conjugation to keyhole limpet hemocyanin (KLH) and administered with an adjuvant, has been used to vaccinate patients in chemotherapy-induced remission (4, 5). When an immune response is triggered by such vaccination, patients have a superior clinical outcome (6, 7).Unfortunately, Igs are difficult proteins to produce. Currently, Igs for patient therapies are created by fusion of tumor cells to a transformed human͞mouse heteromyeloma cell (8, 9). Hybridomas are screened for secreted patient tumorspecific Ig and expanded for large-scale production of the protein. Besides the labor and expense involved, a drawback of hybridoma production systems is the unpredictable loss of chromosomes and of tumor-specific Ig protein expression over time. This phenomenon currently limits the application of the therapy, in terms of both the quantity of vaccine per patient and the total number of patients that can be treated. To expand the scope of individualized non-Hodgkin's lymphoma (NHL) therapies, a source of abundant, safe, easily purified vaccine needs to be generated in a time frame of weeks rather than months or years.An appealing alternative to multichain whole Ig vaccines is singe-chain variable region (scFv) vaccines. Consisting of just the hypervariable domains from the tumor-specific Ig, these proteins recreate the antigen-binding site of the native Ig (10-12), are a fraction of the size, and can be expressed in several expression systems (13-17), including transgenic plants (...

Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants

“…A plant virus vector has been used to produce a tumorspecific vaccine transiently in tobacco for the treatment of lymphoma 9 . The antibody genes for expression of an scFv were derived from a mouse B-cell lymphoma.…”

Variations in “rescuability” of immunoglobulin molecules from different forms of human lymphoma: implications for anti-idiotype vaccine development

“…This phenomenon might imply that the most crucial step in this strategy for producing Id vaccine is the biological stabilization of an Id-secreting hybridoma. This is a problem that, per se, is not easily surmountable when dealing with rescue fusions and might even accelerate the transition towards recombinant technologies [23,24] Finally, at the time of writing it is not clear whether the recent identification of acquired, potential glycosylation sites within the amino acid sequences of tumor-specific immunoglobulins [25][26][27] may have an impact on either idiotypic vaccination as a whole or the choice of the methods by which the Id-containing immunoglobulin is rescued.…”

Transient Virus Expression Systems for Recombinant Protein Expression in Dicot- and Monocotyledonous Plants

“…In order to provide higher antigen content to vaccines, single chain antibodies (scFvs) were constructed from tumor-derived idiotypic antibodies to provide simpler and more sequence-focused vaccines for clinical testing. This vaccine strategy was shown to be effective in murine models of NHL [49] and a GENEWARE ® -based production methodology was developed that could produce >80% of scFvs from human tumor samples [50]. Sixteen patients were enrolled in a Phase I clinical trial under the regulatory oversight of the US Food and Drug Administration [31,51].…”