Designed ankyrin repeat proteins are effective targeting elements for chimeric antigen receptors

Abstract

BackgroundAdoptive cell transfer of tumor-specific T lymphocytes (T cells) is proving to be an effective strategy for treating established tumors in cancer patients. One method of generating these cells is accomplished through engineering bulk T cell populations to express chimeric antigen receptors (CARs), which are specific for tumor antigens. Traditionally, these CARs are targeted against tumor antigens using single-chain antibodies (scFv). Here we describe the use of a designed ankyrin repeat protein (DARPin) as the tumor-antigen targeting domain.MethodsWe prepared second generation anti-HER2 CARs that were targeted to the tumor antigen by either a DARPin or scFv. The CARs were engineered into human and murine T cells. We then compared the ability of CARs to trigger cytokine production, degranulation and cytotoxicity.ResultsThe DARPin CARs displayed reduced surface expression relative to scFv CARs in murine cells but both CARs were expressed equally well on human T cells, suggesting that there may be a processing issue with the murine variants. In both the murine and human systems, the DARPin CARs were found to be highly functional, triggering cytokine and cytotoxic responses that were similar to those triggered by the scFv CARs.ConclusionsThese findings demonstrate the utility of DARPins as CAR-targeting agents and open up an avenue for the generation of CARs with novel antigen binding attributes.

T-cells “à la CAR-T(e)” – Genetically engineering T-cell response against cancer

“…Indeed, they contain ankyrin repeats (ARs) which are one of the most common protein binding motifs found in nature (usually stretches of 33 aminoacid forming a β-turn and two anti-parallel α-helices). These can be engineered to bind efficiently Her2 providing an alternative to the 4D5 murine scFv-based CAR [176,177]. Similarly, adnectins are affinity molecules derived from a fibronectin domain and can serve as an alternative to scFv in CAR configuration [178] though the complex structure of the two last examples may trigger immunogenicity.…”

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“…Conversely, a disadvantage of using scFv-binding domains is their tendency for oligomerization, which can lead to tonic signalling and T-cell exhaustion 17 . Alternative binding domains have been used in preclinical studies, including receptors 18 , ligands 19 , cytokines 20,21 , DARPins (designed ankyrin repeat proteins) 22 , adnectins 23 , Fc receptor fragments 24 , nanobodies 25 , peptides 26 and variable lymphocyte receptors 27 . The relative efficacy, safety and immunogenicity of these alternative binders have not yet been fully investigated and are the focus of substantial ongoing work.…”

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“…DARPins are more compact than scFv and are thermodynamically stable. They do not need VH‐VL pairing to bind targets specifically . They do not contain cysteine; hence, they are resistant to protease and can be expressed in bacteria.…”

“…They do not need VH-VL pairing to bind targets specifically. [95] They do not contain cysteine; hence, they are resistant to protease and can be expressed in bacteria. Because of their small size, penetrate better in tumour cells.…”