Cancer immunotherapy has emerged as a revolutionary approach in oncology, complementing the classical standard of care chemo- or radiotherapy strategies. As the field advances, there is an increasing focus on intracellular antigens, presented as peptides on major histocompatibility complex (pMHC), which offers a vast repertoire of potential targets, including those specific for individual patients. Their targeting thus opens doors to novel personalized treatments, potentially improving therapeutic outcomes. Natural cell-surface peptide major histocompatibility complexes (pMHCs) are recognized by T cell receptors (TCRs) which have been intensively studied for their therapeutic anti-tumor potential. However, the intrinsic capability of TCRs to cross-react with many pMHCs presents a major barrier for their practical application. In consequence, TCRs and current TCR mimetics (TCRms) based on antibody fragments, must undergo extensive and laborious specificity profiling to ensure safety. Thus, the development of safe TCRs/TCRms does not currently align well with the timeframes required for personalized clinical medicine. To respond to this unmet need, the current work explores two complementary strategies that may allow the rapid selection of highly peptide-specific pMHC engagers and the assessment of their safety profiles in real clinical time.
For the first arm of the project, we considered the structural basis for TCR cross-reactivity and developed a novel human single-domain antibody (sdAb) library suitable for the selection of peptide-specific pMHC binders. Selected candidates proved to be functional in a CAR therapeutic format when assessed by Jurkat cell NFAT reporter assays, and at least one clone could direct primary T cells to kill a relevant tumor cell line. However, a major constraint on the functionality of these sdAbs is their generally poor affinity. Structural studies conducted on several solved sdAb:pMHC complexes provided us with valuable insights into their observed properties, which has allowed us to define improvements for future library designs. The presented findings highlight a significant potential of such sdAbs for peptide-specific targeting of pMHCs.
The second arm of the project concerned the construction and validation of a robust peptide-diversified pMHC phage display library for TCRs/TCRms profiling. Initially, we explored several designs and approaches to identify suitable expression formats for the production of functional, soluble TCRs/TCRms as panning targets. In parallel, we developed and evaluated the performance of novel pMHC libraries for phage display centered around a controlled, “human-like”, peptide diversity. Panning against clinically interesting TCRs revealed clear enrichment motifs, with subsequent reporter assays confirming that such phage-enriched peptides were indeed functionally recognized. Further, an interrogation of the human proteome using a consensus cross-reactivity search motif led to the identification of a self, non-cognate, peptide capable of triggering reporter activity.
In summary, this work presents tools and strategies to assist in the development of therapeutics targeting pMHC. In particular, the marriage of novel, platform compatible, sdAb-based TCRm discovery, together with ‘plug-and-play’ pMHC library technology for cross-reactivity safety profiling of TCRs/TCRms offers significant potential for real-time personalized therapy.