Multispecific Targeting with Synthetic Ankyrin Repeat Motif Chimeric Antigen Receptors

被引:43
作者
Balakrishnan, Ashwini [1 ]
Rajan, Anusha [1 ]
Salter, Alexander I. [1 ,2 ]
Kosasih, Paula L. [1 ]
Wu, Qian [1 ]
Voutsinas, Jenna [1 ]
Jensen, Michael C. [2 ,3 ]
Pluckthun, Andreas [4 ]
Riddell, Stanley R. [1 ,2 ]
机构
[1] Fred Hutchinson Canc Res Ctr, Div Clin Res, 1124 Columbia St, Seattle, WA 98104 USA
[2] Univ Washington, Seattle, WA 98195 USA
[3] Seattle Childrens Res Inst, Seattle, WA USA
[4] Univ Zurich, Dept Biochem, Zurich, Switzerland
关键词
CAR-T-CELLS; PROTEINS DARPINS; EXTRACELLULAR DOMAIN; B-ALL; CD19; SELECTION; BINDING; ESCAPE; CD8(+); HER2;
D O I
10.1158/1078-0432.CCR-19-1479
中图分类号
R73 [肿瘤学];
学科分类号
100214 ;
摘要
Purpose: The outgrowth of antigen-negative variants is a significant challenge for adoptive therapy with T cells that target a single specificity. Chimeric antigen receptors (CAR) are typically designed with one or two scFvs that impart antigen specificity fused to activation and costimulation domains of T-cell signaling molecules. We designed and evaluated the function of CARs with up to three specificities for overcoming tumor escape using Designed Ankyrin Repeat Proteins (DAR-Pins) rather than scFvs for tumor recognition. Experimental Design: A monospecific CAR was designed with a DARPin binder (E01) specific for EGFR and compared with a CAR designed using an anti-EGFR scFv. CAR constructs in which DARPins specific for EGFR, EpCAM, and HER2 were linked together in a single CAR were then designed and optimized to achieve multispecific tumor recognition. The efficacy of CAR-T cells bearing a multispecific DARPin CAR for treating tumors with heterogeneous antigen expression was evaluated in vivo. Results: The monospecific anti-EGFR E01 DARPin conferred potent tumor regression against EGFR(+) targets that was comparable with an anti-EGFR scFv CAR. Linking three separate DARPins in tandem was feasible and in an optimized format generated a single tumor recognition domain that targeted a mixture of heterogeneous tumor cells, each expressing a single antigen, and displayed synergistic activity when tumor cells expressed more than one target antigen. Conclusions: DARPins can serve as high-affinity recognition motifs for CAR design, and their robust architecture enables linking of multiple binders against different antigens to achieve functional synergy and reduce antigen escape.
引用
收藏
页码:7506 / 7516
页数:11
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