B7-h3-specific engager T cells for the immunotherapy of pediatric solid tumors

Abstract

Background: B7-H3 positive tumors, including osteosarcoma, neuroblastoma, and high grade glioma, cause significant morbidity and mortality in pediatric patients despite aggressive management with multimodality therapy. Current B7-H3-targeted immune-therapies take advantage of the monoclonal antibody (MAb) 8H9, which is actively being evaluated in Phase I clinical trials. We now propose to develop a T-cell therapy approach targeting B7-H3 using Engager (ENG) T cells. ENG T cells, which secrete bispecific engager molecules consisting of single chain variable fragments (scFvs) specific for CD3 and a tumor antigen, are a new class of antigen-specific T cells, with the unique ability to redirect bystander T cells to tumor cells, amplifying antitumor effects. Objectives: To generate B7-H3-specific Engager (B7-H3-ENG) T cells, and preclinically evaluate their effector function in vitro and in vivo. Design/Method: B7-H3-ENG T cells were created by synthesizing a mini gene consisting of a leader sequence and a B7-H3-specific scFv derived from MAb 8H9. The mini gene was cloned into a SFG retroviral vector containing a CD3-specific scFv and mOrange separated by an internal ribosomal entry site. RD114-pseudotyped retroviral vectors were used to transduce CD3/CD28-activated human T cells. The effector function of B7-H3-ENG T cells was evaluated in vitro by performing coculture and cytotoxicity assays with B7-H3-positive osteosarcoma (LM7), neuroblastoma (CHLA255), and glioma (U373) cell lines, and a B7-H3-negative lung cancer (HTB-119) cell line. Results: Post transduction 30-60% of T cells were genetically modified as judged by mOrange expression. In coculture assay B7-H3-ENG T cells recognized B7-H3- positive target cells (LM7, CHLA255, U373) as judged by IFNg production and cytolytic activity, in contrast to B7-H3-negative cells (HTB-119). T cells secreting an engager molecule specific for an irrelevant antigen (CD19) did not recognize or kill any of the target cells. Conclusion: We have successfully generated B7-H3-ENG T cells and shown that these cells recognize and kill B7-H3-positive tumor cells in an antigendependent manner. In the future, we plan to extend our in vitro studies, and also evaluate antitumor activity in relevant preclinical animal models of pediatric solid tumors.