Human cytomegalovirus-specific T-cell receptor engineered for high affinity and soluble expression using mammalian cell display

Abstract

Edited by Peter Cresswell T-cell receptors (TCR) have considerable potential as therapeutics and antibody-like reagents to monitor disease progression and vaccine efficacy. Whereas antibodies recognize only secreted and surface-bound proteins, TCRs recognize otherwise inaccessible disease-associated intracellular proteins when they are presented as processed peptides bound to major histocompatibility complexes (pMHC). TCRs have been primarily explored for cancer therapy applications but could also target infectious diseases such as cytomegalovirus (CMV). However, TCRs are more difficult to express and engineer than antibodies, and advanced methods are needed to enable their widespread use. Here, we engineered the human CMV-specific TCR RA14 for high-affinity and robust soluble expression. To achieve this, we adapted our previously reported mammalian display system to present TCR extracellular domains and used this to screen CDR3 libraries for clones with increased pMHC affinity. After three rounds of selection, characterized clones retained peptide specificity and activation when expressed on the surface of human Jurkat T cells. We obtained high yields of soluble, monomeric protein by fusing the TCR extracellular domains to antibody hinge and Fc constant regions, adding a stabilizing disulfide bond between the constant domains and disrupting predicted glycosylation sites. One variant exhibited 50 nM affinity for its cognate pMHC, as measured by surface plasmon resonance, and specifically stained cells presenting this pMHC. Our work has identified a human TCR with high affinity for the immunodominant CMV peptide and offers a new strategy to rapidly engineer soluble TCRs for biomedical applications. Antibodies represent a large and growing class of successful therapeutics, by virtue of their abilities to tightly bind antigens that are secreted or expressed on a target cell surface. The

Massively parallel interrogation and mining of natively paired human TCRαβ repertoires

“…Furthermore, the primary T cells used in these TCR discovery approaches are difficult to culture and are a nonrenewable resource. Although TCRs can be engineered and affinity matured from artificial libraries 14 , 15 , these non-natural TCRs have not been subjected to endogenous thymic selection processes and can have off-target effects that are difficult to predict 16 . This has led to several tragic deaths caused by off-target effects 17 .…”

Affinity Maturation of a T-Cell Receptor-Like Antibody Specific for a Cytomegalovirus pp65-Derived Peptide Presented by HLA-A*02:01

“…For example, the CMVpp65 495-503 /HLA-A*02:01 complex was reported to be ~100 molecules/cell on the surface of CMV-infected fibroblasts [ 13 ]. Accordingly, TCRs or TCR-like Abs with high affinity and specificity are necessary for the sensitive detection or targeting of the low copy numbers of pMHC [ 10 , 11 , 22 ]. Though the H9 Ab detected the CMVpp65 495-503 /HLA-A*02:01 on the surface of CMV-infected fibroblasts [ 13 ], it has not been further developed.…”

“…Among the pp65-derived CTL epitope peptides, the 9-mer peptide 495 NLVPMVATV 503 (residues 495–503; hereafter referred to as CMVpp65 495-503 peptide) is the most immunogenic T cell epitope predominantly displayed on HLA-A*02:01, the most common MHC-I allele in the population [ 6 , 7 , 8 ]. Hence, detection and targeting of the highly prevalent CMVpp65 495-503 /HLA-A*02:01 complex on the surface of CMV-infected cells are crucial for the development of detection and/or therapeutic modalities [ 9 , 10 ]. T-cell receptors (TCRs) specifically recognize the peptide–MHC complex (pMHC), but their natural affinity is limited to ~1–100 μM [ 4 ].…”

Immune Literacy: Reading, Writing, and Editing Adaptive Immunity

“…Fast and inexpensive functional screening of transiently expressed TCRs in CD8+ and CD4+ Jurkats has been established, but the transient nature prevented further engineering approaches and selections. Current research in the field has been overwhelmingly focused on affinity-based selections of TCRs ( Kessels et al., 2001 ; Chervin et al., 2008 ; Malecek et al., 2013 ; Schmitt et al., 2017 ; Wagner et al., 2019 ; Spindler et al., 2020 ). As highlighted in previous sections, affinity maturation of scTCR-pMHC pairs has been done by phage and yeast display screening.…”

“…A number of reports demonstrate the use of mammalian cells for TCR screening and engineering, most of which involved viral transformations and affinity-based selections ( Kessels et al., 2001 ; Chervin et al., 2008 ; Malecek et al., 2013 ; Schmitt et al., 2017 ; Wagner et al., 2019 ; Spindler et al., 2020 ). In order to overcome the challenges related to TCR affinity and cross-reactivity, it is necessary to engineer TCR specificity not only on the basis of binding and function ( Rosskopf et al., 2018 ), but also while detecting cross-reactivity.…”