Development of DNA vaccines against B-cell neoplasms

Abstract

For the development of DNA vaccines for B-cell malignancies, three aspects were studied. First, tumour Ig genes of B-cell malignancies were analysed to gain insight into the nature of the tumour antigen and the cell of origin in B-cell tumours. Transcripts of V_HDJ_H-constant region (V_H-C) sequences of the rare B-cell malignancy hairy cell leukemia (HLC) were studied. They were found mutated, with a low level of intraclonal heterogeneity, and multiple Ig isotype transcripts with identical V_H sequence were identified in single cells. These findings indicated that clonally-related multiple isotypes co-existed in single hairy cells, which were arrested at the point of isotype switch, where RNA processing precedes deletional recombination. Second, DNA vaccines encoding the scFv-FrC fusion gene were prepared from patients with B-cell malignancies selected for clinical trials. Each vaccine was characterised in vitro and in mice prior to human immunization. As a prelude to clinical use for lymphoma, the conformational status of the scFv encoded protein was evaluated in mice by monitoring the ability of the human DNA scFv-FrC vaccines to induce antibodies (Abs) directed against the private Id determinants of the tumour Ig. Non cross-reacting Abs specific to the individual patient's Ig were detected. In each case, there was clear evidence that Abs against private determinants were produced. Ab responses were predominantly driven by Th2 cells. These data demonstrated that DNA scFv-FrC fusion vaccines produced folded protein displaying Id determinants able to induce Ab against natural tumor Ig and set the scene for the application of DNA scFv-FrC vaccines to patients with B-cell malignancies. Third, a DNA based approach was developed with a second tumor antigen for myeloma, MUC1 glycoprotein. Initially, using a panel of anti-MUC1 monoclonal Abs and an RT-PCR technique, MUC1 was confirmed to be expressed by neoplastic myeloma cells. Then, using the same strategy applied to Ig determinants, DNA vaccines encoding human MUC1 protein alone or fused to the FrC were tested in mice for their ability to induce anti-tumor immunity. Fusion of MUC1 to FrC amplified anti-MUC1 Ab responses, but did not appear to increase protection against challenge with MUC1 expressing tumour cells. CD8 T cells seemed to be more important since their depletion in vivo abolished tumour protection. Two peptides that were able to induce cytotoxicity against MUC1 in vitro were identified and will be included in a new generation of DNA vaccines.

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