RESUMO
We report on the design of a phase I, non-randomized, open-label study of idiotypic DNA vaccination in patients with B-cell non-Hodgkin's lymphoma (ISRCTN31090206). The study uses DNA fusion gene vaccination encoding patient-specific single chain variable fragment, or idiotype, linked to an immunostimulatory sequence. Two types of immunostimulatory sequence are being explored: potato virus X coat protein and human chemokine MIP3α. Linear polyethylenimine with low molecular weight (8 kDa) is used as a synthetic vehicle for vaccine delivery. Humoral and T-cellular immune responses to vaccination will be measured by ELISA and ELISPOT, respectively. The primary study endpoints are safety, tolerability and immunogenicity of DNA-PEI vaccination.
Assuntos
Adjuvantes Imunológicos/efeitos adversos , Ensaios Clínicos Fase I como Assunto , Linfoma de Células B/terapia , Linfoma não Hodgkin/terapia , Polietilenoimina/efeitos adversos , Vacinas de DNA/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/genética , Adolescente , Adulto , Idoso , Autoanticorpos/sangue , Proteínas do Capsídeo/administração & dosagem , Proteínas do Capsídeo/efeitos adversos , Proteínas do Capsídeo/genética , Quimiocina CCL20/administração & dosagem , Quimiocina CCL20/efeitos adversos , Quimiocina CCL20/genética , Ensaio de Imunoadsorção Enzimática , ELISPOT , Feminino , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Masculino , Pessoa de Meia-Idade , Polietilenoimina/administração & dosagem , Potexvirus/genética , Linfócitos T/imunologia , Vacinas de DNA/administração & dosagem , Adulto JovemRESUMO
AIM: Idiotype, the unique part of immunoglobulin molecule expressed on the surface of B-cells, represents a specific antigen for vaccination against lymphoma. We have developed a rapid method for immunoglobulin variable fragments cloning, assembling and expression of recombinant idiotype protein in Escherichia coli. METHODS: PCR with specially designed panel of primers was used for direct amplification of variable regions of tumor immunoglobulin. Overlapping extension PCR, restriction and ligation was applied for assembling and cloning of vaccine construction. Idiotype protein was purified by metal-chelate chromatography. RESULTS: Methods of idiotype cloning from lymphoma cells and production of recombinant protein were developed and optimized. Several samples of idiotypic proteins originating from B-cell lines and lymphoma patients were produced. CONCLUSION: The proposed method of vaccine production is relatively cheap, not very laborious and requires as long as 6-7 week to perform. The expressed protein was soluble, did not accumulate in inclusion bodies and harvested at sufficient for vaccination quantity and concentration.