Abstract
T cells that are genetically engineered to express chimeric antigen receptors (CAR T cells) have shown impressive clinical efficacy against B-cell malignancies. In contrast to these highly potent CD19-targeting CAR T cells, many of those directed against other tumor entities and antigens currently suffer from several limitations. For example, it has been demonstrated that many scFvs used as antigen-binding domains in CARs show some degree of oligomerization, which leads to tonic signaling, T cell exhaustion, and poor performance invivo. Therefore, in many cases alternatives to scFvs would be beneficial. Fortunately, due to the development of powerful protein engineering technologies, also non-immunoglobulin-based scaffolds can be engineered to specifically recognize antigens, thus eliminating the historical dependence on antibody-based binding domains. Here, we discuss the advantages and disadvantages of such engineered binding scaffolds, in particular with respect to their application in CARs. We review recent studies, collectively showing that there is no functional or biochemical aspect that necessitates the use of scFvs in CARs. Instead, antigen recognition can also be mediated efficiently by engineered binding scaffolds, as well as natural ligands or receptors fused to the CAR backbone. Finally, we critically discuss the risk of immunogenicity and show that the extent of nonhuman amino acid stretches in engineered scaffolds-even in those based on nonhuman proteins-is more similar to humanized scFvs than might be anticipated. Together, we expect that engineered binding scaffolds and natural ligands and receptors will be increasingly used for the design of CAR T cells.
Highlights
Immunotherapy for the treatment of cancer has experienced a breakthrough in the last decade
It has been demonstrated that many single-chain variable fragment (scFv) used as antigen-binding domains in Chimeric AnƟgen Receptor (CAR) show some degree of oligomerization, which leads to tonic signaling, T cell exhaustion, and poor performance in vivo
We expect that engineered binding scaffolds and natural ligands and receptors will be increasingly used for the design of CAR T cells
Summary
Immunotherapy for the treatment of cancer has experienced a breakthrough in the last decade. Target cells expressing high levels of EGFR were efficiently lysed by both types of CARs. In a recent study, we engineered two completely different binding scaffolds (rcSso7d and monobodies) to recognize hRBP4 only when loaded with an orally available small molecule drug called A1120. They harbor several advantages compared with engineered protein scaffolds: (i) They naturally occur in the human body, thereby minimizing the risk of potential immunogenicity; (ii) many of them can bind multiple targets, which broadens the range of applications and prevents escape due to antigen downregulation; and (iii) some of their ligands are upregulated upon stress conditions and can thereby often be found in the tumor microenvironment One example for such a natural receptor is natural killer group 2 member D (NKG2D), a type II transmembrane protein present on various immune cells, such as natural killer (NK) cells, CD8+ T cells, and cd T cells [94]. IL-13 muteins represent a further example of natural ligands, which have been engineered through introduction of a limited number of mutations to achieve a therapeutically optimal affinity
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