针对神经母细胞瘤（NB）和尤因肉瘤（EWS）的GFRA2靶向抗体药物偶联物的开发

Background: We recently defined the NB/EWS surfaceomes using integrative proteogenomics to prioritize proteins as candidate immunotherapeutic targets. GFRA2 was a top ranked candidate for both NB and EWS ( Clin Cancer Res 2023, Cancer Cell 2024). Aims: (1) Validate and assess mechanism of GFRA2 overexpression; (2) Identify selective antibody binders to GFRA2; (3) Engineer antibody drug conjugates (ADCs) and test for internalization and potency. Methods: ChIP- and RNA-sequencing of NB and RNA-sequencing following siRNA depletion of EWSR1::FLI1 were used to evaluate mechanisms of GFRA2 overexpression. GFRA2 abundance and cellular localization was evaluated by flow cytometry and immunofluorescence. Phage display was performed with recombinant GFRA2 extracellular domain protein as the bait and the highly conserved GFRA1 and GFRA3 recombinant proteins as counters. In parallel, we humanized the anti-GFRA2 murine HSAN antibody. While we plan on reporting on several linker-payload combinations, here we focus on the initial humanized HSAN antibody conjugated to pyrrolobenzodiazepine (PBD) via a cleavable linker, which was tested for internalization using live cell imaging and cytotoxicity across a panel of human NB and EWS preclinical models. Results: We identified a proximal super enhancer (Percentile: 97.4%-99.7%) or enhancer (94.4%-97.7%) in all 10 NB cell lines profiled. Depletion of EWSR1::FLI1 resulted in significantly decreased GFRA2 mRNA expression across 6 EWS cell lines (P<0.0001), and this was validated via immunoblotting. GFRA2 protein was expressed uniformly and to variable degrees on the cell surface of 10 NB cell lines, 8 NB patient-derived xenografts, and 6 EWS cell lines by flow cytometry and/or immunofluorescence. Patient tumor RNA-Seq showed 92.8% of NB (N=126; median=47.65) and 74% of EWS (N=85, median=11.47) had high expression defined as a GFRA2 TPM > 5. Phage display panning of fully human antibody fragments yielded three Fabs and one VH single domain antibody specific to GFRA2 with moderate binding affinity (50-500 nM). Humanized HSAN (co-hu-HSAN) showed high binding affinity specific to GFRA2 (1-10 nM). We then showed robust internalization of co-hu-HSAN across 6 NB cell lines. A co-hu-HSAN-PBD ADC showed potent and specific cytotoxicity across GFRA2+ cell lines (NB=2; median IC 50 =1.69;2.92pM, EWS=2; median IC 50 =9.81;12.43pM) with no cytotoxicity in GFRA2 null cell lines (N=2; median IC 50 =NA). Conclusions: GFRA2 is a lineage restricted oncoprotein abundantly expressed in both NB/EWS, as well as other human solid cancers. We show initial proof-of-concept for potent and specific cytotoxicity in both NB/EWS with the co-hu-HSAN-PBD ADC. We will report on efficacy testing of the co-hu-HSAN-PBD ADC across NB/EWS xenograft models. Work to affinity enhance the fully human binders, and progress with additional linker/payload combinations are ongoing and will be reported.