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T-cell bispecific antibodies (TCBs) are a novel class of engineered immunoglobulins that unite monovalent binding to the T-cell receptor (TCR) CD3e chain and bivalent binding to tumor-associated antigens in order to recruit and activate T-cells for tumor cell killing. In vivo, T-cell activation is usually initiated via the interaction of the TCR with the peptide-HLA complex formed by the human leukocyte antigen (HLA) and peptides derived from intracellular proteins. TCR-like antibodies (TCRLs) that recognize pHLA-epitopes extend the target space of TCBs to peptides derived from intracellular proteins, such as those overexpressed during oncogenesis or created via mutations found in cancer. One challenge during lead identification of TCRL-TCBs is to identify TCRLs that specifically, and ideally exclusively, recognize the desired pHLA, but not unrelated pHLAs. In order to identify TCRLs suitable for TCRL-TCBs, large numbers of TCRLs have to be tested in the TCB format. Here, we propose a novel approach using chimeric antigen receptors (CARs) to facilitate the identification of highly selective TCRLs. In this new so-called TCRL-CAR-J approach, TCRL-candidates are transduced as CARs into Jurkat reporter-cells, and subsequently assessed for their specificity profile. This work demonstrates that the CAR-J reporter-cell assay can be applied to predict the profile of TCRL-TCBs without the need to produce each candidate in the final TCB format. It is therefore useful in streamlining the identification of TCRL-TCBs.

Directed molecular evolution has proven to be a very powerful concept for the generation of proteins with improved properties, such as increased activity, binding affinity, folding efficiency or enhanced chemical and/or thermodynamic stability. We review here advances in the selection of proteins carrying desired mutations from pools of proteins that mostly carry unfavourable alterations. A short overview of the concept of directed evolution with a discussion of randomisation strategies is given first. Two technologies for the selection of proteins, each with its own advantages, are then discussed: In Ribosome Display, all steps are carried out in a cell-free system, which allows one to create very large libraries (diversity > 1011), rapidly introduce mutations and thus obtain an iterative evolution. Examples with antibodies evolved for affinity or stability are discussed. In the Protein Fragment Complementation Assay, a library-versus-library selection is possible, that is, a simultaneous selection of binders against many targets. Examples with peptide and antibody libraries are discussed.