Identification of the Targets of T-cell Receptor Therapeutic Agents and Cells by Use of a High-Throughput Genetic Platform

Abstract

◥T-cell receptor (TCR)-based therapeutic cells and agents have emerged as a new class of effective cancer therapies. These therapies work on cells that express intracellular cancer-associated proteins by targeting peptides displayed on MHC receptors. However, crossreactivities of these agents to off-target cells and tissues have resulted in serious, sometimes fatal, adverse events. We have developed a high-throughput genetic platform (termed "PresentER") that encodes MHC-I peptide minigenes for functional immunologic assays and determines the reactivities of TCR-like therapeutic agents against large libraries of MHC-I ligands. In this article, we demonstrated that PresentER could be used to identify the on-andoff targets of T cells and TCR-mimic (TCRm) antibodies using in vitro coculture assays or binding assays. We found dozens of MHC-I ligands that were cross-reactive with two TCRm antibodies and two native TCRs and that were not easily predictable by other methods.

Rejection of immunogenic tumor clones is limited by clonal fraction

“…The PresentER system we have employed does have some potential biochemical caveats that may impact the interpretation of the data. While we have not observed alternative cleavage patterns in individual peptides we have studied ( Gejman, 2018b ), in a library setting we cannot test if every minigene is properly yielding its encoded MHC-I ligand. For instance, some encoded peptides may not bind well to MHC-I or may only bind in the presence of an MHC loading chaperone.…”

“…Expression of the peptide and its display on MHC-I does not require proteasomal degradation or peptide processing, thus enabling precise definition of the exact epitope displayed to the immune system. As previously described ( Gejman, 2018a ), Transporter associated with antigen presentation (Tap) deficient cell lines expressing PresentER antigen minigenes lead to surface presentation of the encoded MHC-I peptide, detectable by multiple modalities, including fluorescently labeled antibodies directed to specific MHC-I ligands, mass spectrometry based immunopeptidomics and antigen-specific T cell reactivity. To demonstrate the applicability of PresentER antigen minigenes to study MHC-I ligand immunogenicity, we first asked if cancer cells encoding known immunogenic (mouse Tyrp1/gp75 TAYRYHLL W223A,H224Y ( Dyall et al, 1998 ); mouse Ddx5/p68 SNFVFAGI S551F ( Dubey et al, 1997 ); synthetic SIYRYYGL ( Udaka et al, 1996 ); synthetic VTFVFAGL ( Dubey et al, 1997 ); chicken ovalbumin SIINFEKL) or non-immunogenic (mouse Serpinf1/Pedf MSIIFFLPL ( Wang et al, 2006 ); scrambled chicken ovalbumin FEKIILSN; mouse Ndufa4/dEV8 EQYKFYSV ( Holler et al, 2003 ); mouse Ddx5/p68 SNFVSAGI ( Dubey et al, 1997 ); mouse Tyrp1/gp75 TWHRYHLL ( Dyall et al, 1998 ); Mouse Trp2 SVYDFFVWL) MHC-I ligands would be rejected by wild-type (WT) animals.…”

Development of a T‐cell receptor mimic antibody targeting a novel Wilms tumor 1‐derived peptide and analysis of its specificity

“…A detailed biochemical evaluation of a well‐characterized TCRm‐Ab, ESK1, by crystallographic studies explained the mechanism of its recognition of other peptides that share sequence similarity with the target peptide 24 . Gejman et al 25,26 developed the PresentER antigen presentation platform to identify the precise peptide epitope recognized by a TCRm‐Ab and TCR. These results demonstrate the nature of TCRm‐Abs, as well as TCR, having cross–reactivity with nontarget peptides.…”

An Effective COVID-19 Vaccine Needs to Engage T Cells

“…Decoding matched T cell clonotypes, phenotypes and antigenic epitopes in COVID-19 patients will also answer the important questions as to which characteristics of the T cell repertoire explain the higher risk of the elderly, and how HLA genetic diversity contributes to the SARS-CoV-2 specific T cell repertoire and immune response (53). Such studies are now possible thanks to recent advances in single cell sequencing technologies combined with largescale HLA tetramer or HLA reporter gene-based epitope library screening technologies (38,(58)(59)(60)(61)(62)(63)(64)(65)(66).…”