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Published on: July 12, 2018
Engineering of a novel subnanomolar affinity fibronectin III domain binder targeting human programmed death-ligand 1
Sindhuja Ramakrishnan1, Arutselvan Natarajan1, Carmel T Chan1
1Department of Radiology, Molecular Imaging Program at Stanford (MIPS), Stanford University, 318 Campus Drive, Stanford, CA 94305, USA.
Abstract:
The programmed death-ligand 1 (PD-L1) is a major checkpoint protein that helps cancer cells evade the immune system. A non-invasive imaging agent with rapid clearance rate would be an ideal tool to predict and monitor the efficacy of anti-PD-L1 therapy. The aim of this research was to engineer a subnanomolar, high-affinity fibronectin type 3 domain (FN3)-based small binder targeted against human PD-L1 (hPD-L1) present on tumor cells. A naive yeast G4 library containing the FN3 gene with three binding loop sequences was used to isolate high-affinity binders targeted to purified full-length hPD-L1. The selected binder clones displayed several mutations in the loop regions of the FN3 domain. One unique clone (FN3hPD-L1-01) with a 6x His-tag at the C-terminus had a protein yield of >5 mg/L and a protein mass of 12 kDa. In vitro binding assays on six different human cancer cell lines (MDA-MB-231, DLD1, U87, 293 T, Raji and Jurkat) and murine CT26 colon carcinoma cells stably expressing hPD-L1 showed that CT26/hPD-L1 cells had the highest expression of hPD-L1 in both basal and IFN-γ-induced states, with a binding affinity of 2.38 ± 0.26 nM for FN3hPD-L1-01. The binding ability of FN3hPD-L1-01 was further confirmed by immunofluorescence staining on ex vivo CT26/hPD-L1 tumors sections. The FN3hPD-L1-01 binder represents a novel, small, high-affinity binder for imaging hPD-L1 expression on tumor cells and would aid in earlier imaging of tumors. Future clinical validation studies of the labeled FN3hPD-L1 binder(s) have the potential to monitor immune checkpoint inhibitors therapy and predict responders.
Insights
Researchers engineered a novel, small, high-affinity binder targeting programmed death-ligand 1 (PD-L1) on tumor cells. This binder shows potential for non-invasive imaging to predict and monitor anti-PD-L1 cancer therapy efficacy.
Area of Science:
- Biotechnology
- Molecular Biology
- Immunology
Background:
- Programmed death-ligand 1 (PD-L1) is a key protein enabling cancer cells to evade immune responses.
- Non-invasive imaging agents with rapid clearance are needed to predict and monitor anti-PD-L1 therapy effectiveness.
Purpose of the Study:
- To engineer a high-affinity fibronectin type 3 domain (FN3)-based small binder targeting human PD-L1 (hPD-L1) on tumor cells.
- To develop a tool for non-invasive imaging of hPD-L1 expression for therapeutic monitoring.
Main Methods:
- A naive yeast G4 library expressing FN3 variants was used to isolate binders against purified hPD-L1.
- Binding affinity and specificity were assessed using in vitro assays on cancer cell lines and immunofluorescence staining on tumor sections.
- A unique clone, FN3hPD-L1-01, was selected and characterized for yield, mass, and binding properties.
Main Results:
- The engineered FN3hPD-L1-01 binder demonstrated subnanomolar affinity (2.38 ± 0.26 nM) for hPD-L1 expressed on CT26/hPD-L1 cells.
- High protein yield (>5 mg/L) and a small molecular mass (12 kDa) were achieved for the FN3hPD-L1-01 binder.
- Binding was confirmed on various human cancer cell lines and ex vivo tumor sections, indicating potential for in vivo imaging.
Conclusions:
- FN3hPD-L1-01 is a novel, small, high-affinity binder for imaging hPD-L1 expression on tumor cells.
- This binder could facilitate earlier tumor detection and aid in monitoring anti-PD-L1 immunotherapy.
- Further clinical validation of labeled binders may predict patient response to immune checkpoint inhibitors.
