Identification of Cross-Reactive Targets of TCR-Based Therapeutic Agents within the Human Proteome

TCR-based therapeutic cells and agents (antigen-adapted T cell infusions, TCR-engineered T cells, ImmTACs, and TCR mimic antibodies) have emerged as a new class of effective cancer therapeutics. Cross-reactivities of these agents to off-target cells and tissues are poorly understood, difficult to predict, and have resulted in serious, sometimes fatal adverse reactions. There are no reliable techniques for determining the on-target and off-target reactivity of these agents. We sought to develop a method for predicting reactivities of TCR-based agents, starting with three well-defined tools and systems. Wilms Tumor 1 (WT1) and Preferentially expressed antigen in melanoma (PRAME) are overexpressed protein-coding genes in many hematologic malignancies with restricted expression in normal tissue. The ability to target cells expressing these genes is limited because their protein products are intracellular and no small molecules to these drugs exist. T cell receptor (TCR) mimic antibodies (TCRm) can bind to 8-11aa peptide antigens in the context of major histocompatibility complexes (MHC) on the surface of cells. The peptide MHC (pMHC) targets of TCR and TCRm can be derived from wild-type or mutated, intracellular or extracellular proteins, thereby allowing TCRm therapeutics to access a universe of proteins that traditional antibodies cannot reach. We have generated two TCRm antibodies, one directed to a HLA-A*02:01 peptide from the Wilms Tumor 1 protein (WT1 126-134: RMFPNAPYL) and one to the Preferentially Expressed Antigen In Melanoma (PRAME 300-309: ALYVDSLFFL) protein. Like the TCR on T cells, TCRm can cross-react with off-target peptides that share sequence homology. In order to identify endogenous cross-reactive peptides for these therapeutic drugs, we have created a minigene-based method (termed "PresentER") to genetically encode pMHC on model target cells. PresentER is an easily clonable, Illumina-sequencing compatible genetic system capable of expressing a single MHC-I peptide ligand (8-12 amino acid long). This system is highly specific to the encoded linear amino acid sequence and does not require proteasome cleavage, peptide transport or peptide processing for presentation on MHC-I. PresentER expressing cells can stimulate T cells, bind to TCR and TCRm and be used in pooled screens to discover ligands of TCR and TCRm. We have cloned a library of ~12,000 exomic HLA-A*02:01 ligands and performed a pooled screen of pMHC that can be recognized by the TCRm. The utility of PresentER was validated in its first application to find dozens of MHC-I ligands encoded in the human proteome that were cross-reactive with the ESK1 TCRm. The PresentER screen revealed MHC-I ligands of ESK1 and Pr20 that were not predictable by other methods. PresentER may be used to discover the ligandome of T cell receptors and T cell receptor mimic antibodies in a native, cell-based context.