Citation
- Authors: Raman S. et al.
- Year: 2021
- Journal: Cell Rep Med 2 100344
- Applications: in vitro / DNA / FectoPRO
- Cell type: FreeStyle 293-F
Method
Isolation and preparation of D3-GPC2-IgG1
- A naive human Fab phage display library constructed from peripheral blood B cells of 50 healthy donors was used for selection of Fabs against purified recombinant GPC2 ectodomain (R&D Systems, Inc., #2304) as previously described.
- Briefly, the isolated Fabs were expressed, purified, and tested for binding to the GPC2 ectodomain through ELISA and the best binder, designated as D3-GPC2-Fab, was converted to a full-length human IgG1.
- The full length IgG1 DNA construct was transiently transfected into FreeStyle™ 293-F cells for antibody production and the D3-GPC2-IgG1 was purified on a protein A column.
Abstract
Glypican 2 (GPC2) is a MYCN-regulated, differentially expressed cell-surface oncoprotein and target for immune-based therapies in neuroblastoma. Here, we build on GPC2's immunotherapeutic attributes by finding that it is also a highly expressed, MYCN-driven oncoprotein on small-cell lung cancers (SCLCs), with significantly enriched expression in both the SCLC and neuroblastoma stem cell compartment.By solving the crystal structure of the D3-GPC2-Fab/GPC2 complex at 3.3 Å resolution, we further illustrate that the GPC2-directed antibody-drug conjugate (ADC; D3-GPC2-PBD), that links a human GPC2 antibody (D3) to DNA-damaging pyrrolobenzodiazepine (PBD) dimers, binds a tumor-specific, conformation-dependent epitope of the core GPC2 extracellular domain. We then show that this ADC induces durable neuroblastoma and SCLC tumor regression via induction of DNA damage, apoptosis, and bystander cell killing, notably with no signs of ADC-induced in vivo toxicity. These studies provide preclinical data to support the clinical translation of ADCs targeting GPC2.