545P - Antibody-fragment based immunotherapeutic approaches for medulloblastoma and rhabdomyosarcoma

Background

Medulloblastoma (MB) with SHH-MB as most frequent subtyp is the most common pediatric brain tumor. Conventional therapy can result in severe brain damage and high relapse rates. Thus, targeted less immunogenic immunotherapy is needed. Neo-antigens are rare in pediatric cancers due to their low mutational load. But, SHH-MB overexpresses the EGF-receptor, a well-characterized immunotherapeutic target. Even though it is also expressed at low levels in healthy tissue, it was our immunotherapeutic-target of choice. We established a platform in order to test the target specificity in SHH-MB using EGFR-specific antibody fragments (scFv) as part of fusion proteins with a SNAP-tag. Then, the scFvs were used as part of immunotoxins (ITs) and bispecific T-Cell engagers (BiTEs). Apart from EGFR, PIK3CA mutations occur in SHH-MB and may also serve as therapeutic target. Besides MB the pediatric tumor rhabdomyosarcoma (RMS) also shows EGFR overexpression and abnormal signaling in the PI3-kinase pathway. RMS, the most common pediatric soft tissue sarcoma, shows brain metastasis. The Pi3-kinase inhibitor BKM-120, can therefore tested in combination with EGFR-specific immunotherapy.

Methods

ScFv-SNAP fusions were coupled to Benzylguanin-modified fluorophores under nucleophilic substituent reaction. Specific binding of all constructs was proved with flow cytometry, internalization with confocal microscopy. Impact on cell viability was tested in cell viability assays, luminescent cytotoxicity assays and AnnexinV-apoptosis assays. In vitro- Blood Brain Barrier (BBB) models were generated from hiPSCs (human inducible pluripotent stem cells).

Results

The EGFR-specific scFv shows specific binding and internalization to target cells. The IT demonstrates IC₅₀-values in the low nanomolar range and specific apoptotic effects. In combination with BKM-120 the IC₅₀ decreases. BiTEs influence the cell viability with an effector to target cell ratio of 5:1 and show apoptotic effects. The IT and BKM-120 were used in the BBB-model showing preliminary results of permeability.

Conclusions

Targeted innovative scFv-based immunotherapy as highly specific and less immunogenic therapy for MB or RMS was established, also as combination therapy approach.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

Research Institute Childrens Cancer Center.

Funding

Mildred Scheel Cancer Career Centre HaTriCS4, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany.

Disclosure

All authors have declared no conflicts of interest.