Development of next generation antibody-based immunotherapy for childhood cancer neuroblastoma

Abstract

BackgroundHigh risk neuroblastoma poses a significant clinical problem in paediatric oncology and new treatment strategies are needed. Antibody therapy specific for neuroblastoma surface molecule GD2 has been shown to improve outcome. Here we develop a new generation antibody-based therapeutic agent named BiTE (Bi-specific T cell Engager) that combines antibody specificity with potent T cell functions. MethodsTo identify the BiTE format with maximum activity against neuroblastoma, BiTEs were constructed with single chain variable fragments (scFv) derived from three different CD3-specific antibodies, scFv from two different GD2-specific antibodies, and three different flexible linkers between these CD3 (ie, T cell) and GD2 (ie, neuroblastoma) binders. Specific binding to CD3-positive and GD2-positive cell lines and cytolytic activity at a range of BiTE concentrations was assessed. FindingsSeven different BiTE formats have been successfully produced and purified. Whereas all seven BiTEs constructed showed binding to GD2-positive and CD3-positive cell lines and absence of binding to target antigen-negative cells, mean fluorescence intensity of CD3 binding (14·6 arbitrary units, 24·6, 255) and of GD2 binding (1366, 4258) indicated a range of affinities for CD3 and GD2. For each BiTE, lysis of GD2-target cells was only observed in the presence of T cells. Similarly, secretion of interferon γ by activated T cells only occurred in a CD3-specific and GD2-specific manner. All seven BiTEs induced GD2-specific cytolytic activity. Cytolytic activity was achieved at the lowest EC50 (ie, <1 ng/mL BiTE) for the BiTE format with highest GD2 affinity and moderate CD3 affinity. InterpretationOur findings demonstrate that the optimum BiTE design for tumour targeting can be identified by functional testing of BiTE formats incorporating CD3-specific scFvs and tumour antigen-specific scFvs with a range of binding affinities. Our optimum BiTE construct shows remarkable in-vitro activity approaching that of blinatumomab, which has proven clinical activity against leukaemia; we aim to develop this BiTE into a new therapeutic agent for neuroblastoma. FundingGreat Ormond Street Hospital Institute of Child Health Biomedical Research Centre, Wellcome Trust.