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 tumor-specific 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 micrograms/ml 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 tumor-specific protein vaccines may provide a viable strategy for the treatment of non-Hodgkin's lymphoma.

Figure 1

Schematic diagram of the viral expression vector used to produce murine 38C13 scFv NHL antigen in Nicotiana benthamiana host plants. Shown are the sequences encoding rice α-amylase signal peptide fused in-frame to the SphI site (underlined), introduced by PCR, 5′ of the 38C13 heavy chain. Also indicated in the linear map are the positions of the SP6 transcription start site, the 126-kDa, 183-kDa, and 30-kDa proteins from TMV, and the tomato mosaic virus coat protein, as well as pBR322 sequences for bacterial propagation.

Figure 2

Protein analysis of N. benthamiana plants 2 weeks after inoculation. Lanes 1 and 2, Coomassie-stained SDS/PAGE of IF extract from viral constructs expressing rice α-amylase protein (lane 1) or of 38C13 scFv IF extract (lane 2) run under nonreducing conditions. Lanes 3 and 4, Western blot of a duplicate gel probed with anti-38C13-specific monoclonal antibody S1C5 containing the control extract (lane 3) or 38C13 scFv (lane 4). Lanes 5 and 6 show S1C5 Western blots of 38C13 scFv-containing IF prepared under reducing conditions (but separated by PAGE under nonreducing conditions) or IF containing an 38C13 scFv protein that has no cysteine at amino acid 3. Lanes 7 and 8, Coomassie stain of the starting IF extract (lane 7) and of single-pass, S1C5 affinity-purified 38C13 scFv protein (lane 8). Lane 9, silver stain of affinity-purified protein.

Figure 3

Anti-38C13 titers in vaccinated animals. Ten days after the third vaccination, serum from each group of 10 C3H mice vaccinated with 38C13 scFv in the absence or presence of the adjuvant QS-21, or with 38C13-IgM coupled to KLH and injected with QS-21, was pooled and analyzed with the native 38C13 IgM as the target. Anti-38C13 IgG1 and IgG2a serum levels were quantitated by comparison with purified isotype-specific mouse anti-38C13 standards. Concentrations of IgG1 (hatched bars) and IgG2a (solid bars) are shown for all three 38C13 vaccine groups. No detectable anti-38C13 was measured in the control QS-21 vaccine group.

Figure 4

Mice immunized with plant-derived scFv protein are protected from tumor challenge. Tumor protection was measured from the time of tumor implantation (day 0) and is plotted as percent survival. These results are representative of two experiments. While all vaccinated groups statistically differed from the susceptible control (P < 0.00001), there was no statistical segregation among vaccinated groups (see tabulation in the graph).

Figure 5

Plant-produced 38C13 scFv does not contain carbohydrates. PAS stain was used to reveal the total carbohydrate content of 10 μg of 38C13 IgM (lane 1) or 5 and 25 μg of HRP (lanes 2 and 3) but not in 5 or 25 μg of 38C13 scFv made in plants (lanes 4, 5, and 6a). Coomassie staining was done in parallel on lane 6b, which contains 25 μg of 38C13 scFv, as well as on size standards (lane 7, in kDa) as indicated.