Design and Evaluation of Bispecific T cell Engaging Antibodies for the Treatment of Neuroblastoma

Abstract

Neuroblastoma, a cancer of the sympathetic nervous system, is the most common extracranial solid tumor in children, representing 10% of all pediatric malignancies. Despite aggressive treatment with intensive multi-modal therapies, most children with high-risk neuroblastoma will relapse, so novel therapeutic approaches are desperately needed. Immunotherapies have risen to the forefront of cancer treatments, exemplified by the ability of checkpoint inhibitors to induce anti-tumor responses in adult cancers refractory to conventional treatments. However, children with cancer, including those with neuroblastoma, have failed to benefit from these advances, attributed to intrinsic mechanisms of immune evasion. Thus, neuroblastoma presents a challenge that may serve as a model for the application of immunotherapies for other “immunologically cold” pediatric tumors. Bispecific T cell engaging antibodies are engineered proteins designed to simultaneously bind a tumor specific antigen and CD3 on T cells, thereby instructing endogenous lymphocytes to destroy the bridged tumor cell. Bispecific T cell Engagers (BTEs) are a rapidly evolving approach to treating cancer, with the number of clinically approved BTEs jumping from 1 to 8 since 2022. Although one BTE, blinatumomab, has improved outcomes in pediatric leukemia, the application of BTEs to pediatric solid tumors has proved challenging in part due to a scarcity of tumor-specific antigens. To identify potential neuroblastoma antigens suitable for BTE immunotherapy, we screened a diverse panel of neuroblastoma cell lines and identified B7H3, a surface protein with reported roles in immune evasion, as a candidate target. B7H3 overexpression was validated in high-risk neuroblastoma patient samples compared to adjacent peripheral tissues. Although there are over 100 distinct BTEs in preclinical development, few published works have directly compared different BTE designs and CD3 engagers. We designed, produced, and evaluated a suite of unique B7H3-CD3 BTEs to investigate the role of BTE architecture and CD3 affinity on the function of BTEs in vitro. We found that IgG-like BTEs with anti-CD3 scFvs linked to the carboxy-termini of the IgG light chains mediated potent anti-tumor activity in vitro, attributed to their bivalency for both B7H3 and CD3. This format, termed IgG-L-scFv, demonstrated an increase in potency with an increase in CD3 affinity, and yet demonstrated strictly target-dependent activity. From these studies, we identified a B7H3MGA271- CD3ADI24 IgG-L-scFv as the lead candidate BTE. Despite the promising potency of this molecule in functional assays, in neuroblastoma xenograft models, we found that efficacy was limited by insufficient recruitment of T cells in circulation. This finding highlights a crucial barrier to the application of BTEs and other T-cell targeting immunotherapies to neuroblastoma and by extension other pediatric solid tumors. A deeper understanding of the mechanisms regulating T cell trafficking may guide future development of immunotherapeutic strategies for children with high-risk neuroblastoma.