VEGFR2-specific FnCAR effectively redirects the cytotoxic activity of T cells and YT NK cells.

Sergey V Kulemzin,Andrey A Gorchakov,Andrey Y Zaritskey,Alexey V Petukhov,Olga Y Volkova,Anton N Chikaev,Daria A Matvienko,Valeriya V Kuznetsova,Alexandr V Taranin

doi:10.18632/oncotarget.24078

Abstract

T and NK cells armed with chimeric antigen receptors (CAR) are promising tools for the specific elimination of cancer cells. In most CAR designs implemented to date, the recognition of target cells is mediated by single-chain variable fragments (scFvs) derived from murine monoclonal antibodies. This format, however, has a number of limitations, including its relatively large size and potential immunogenicity in humans. In this study, we explored the feasibility of using human fibronectin type III domains (Fn3) as the antigen recognition domain in CARs. Human Fn3 domains have lower predicted immunogenicity compared to mouse-derived sequences, and a reduced molecular weight compared to scFvs. We created a functional CAR using a VEGFR2-specific Fn3 module replacing the conventional scFv. The resulting FnCAR specifically potentiates the cytotoxic activity of human T cells and YT NK cells in the presence of VEGFR2-positive targets. These findings demonstrate that Fn3 domains can be used in CARs for antigen recognition.

Highlights

T-cells expressing CD19-specific Chimeric Antigen Receptors (CARs) demonstrated remarkable anti-tumor activity in patients with advanced B-cell malignancies [1,2,3,4,5,6], and other chimeric antigen receptors (CAR) are currently being evaluated in the clinical and preclinical settings
Antigen-recognition module of the Fn3-based chimeric antigen receptor (FnCAR) obtained was based on the clone K10 described previously [13] whereas internal ribosome entry site (IRES)-linked copGFP reporter allowed convenient detection of the transduced cells
Several non-single-chain fragment variable (scFv) formats that partially fit this description have been tested in the CAR context, which include camelid single-domain antibodies (VHHs) [29,30,31,32], sea lamprey variable lymphocyte receptors (VLRs) [33], designed ankyrin repeat proteins (DARPins) [34], and affinity peptides [35,36,37]

Summary

Introduction

T-cells expressing CD19-specific Chimeric Antigen Receptors (CARs) demonstrated remarkable anti-tumor activity in patients with advanced B-cell malignancies [1,2,3,4,5,6], and other CARs are currently being evaluated in the clinical and preclinical settings. The antigen-recognition domains of most CARs contain scFv (single-chain fragment variable) sequences derived from well-characterized monoclonal antibodies specific to tumor-associated surface antigens. While the scFv-derived targeting domains inherit the specificity and versatility of monoclonal antibodies, they have a number of important limitations. The framework regions of scFvs have been shown to frequently produce CARs that are prone to tonic signaling—an unwanted effect that results in the premature exhaustion of CAR T-cells [7]

Methods

Results

Conclusion