Subtle Affinity-Enhancing Mutations in a Myelin Oligodendrocyte Glycoprotein-Specific TCR Alter Specificity and Generate New Self-Reactivity

Abstract

We describe a simple iterative approach to augment TCR affinity, which we studied using a myelin oligodendrocyte glycoproteinspecific TCR. We hypothesized that single amino acid modifications in TCR CDR3 could enhance TCR sensitivity through focal interactions with antigenic peptide while minimizing the risk of cross-reactivity observed previously in TCR more broadly mutagenized using in vitro evolution techniques. We show that this iterative method can indeed generate TCR with Ag sensitivity 100-fold greater than the parental receptor and can endow TCR with coreceptor independence. However, we also find that single amino acid mutations in the CDR3 can alter TCR fine specificity, affecting recognition requirements for Ag residues over most of the length of the MHC binding groove. Furthermore, minimal changes in surface-exposed CDR3 amino acids, even the addition of a single hydroxyl group or conversion of a methyl or sulfhydryl moiety to a hydroxyl, can confer modified Ag-specific TCR with new self-reactivity. In vivo modeling of modified TCR through retroviral TCR gene transfer into Rag ؊/؊ mice confirmed the biological significance of these altered reactivities, although it also demonstrated the feasibility of producing Ag-specific, positively selecting, coreceptor-independent receptors with markedly increased Ag sensitivity. These results affirm the possibility of readily generating affinity-enhanced TCR for therapeutic purposes but demonstrate that minimal changes in TCR CDR3 L imits on the TCR repertoire imposed by developmental selection and peripheral tolerance restrict the availability of T cells that can effectively target tumors or some pathogens. To overcome this, there has been growing interest in therapeutically skewing the T cell compartment through the enforced expression on T cells of cloned Ag-specific TCR, thereby promoting desirable immune responses (1, 2). One recent clinical trial, for example, showed that tumors in a subset of metastatic melanoma patients remitted after the infusion of CTL redirected with retrovirally transduced tumor Ag-specific TCR (3). Redirection of regulatory T lymphocytes with introduced autoantigen-specific TCR has similarly been proposed for use in the treatment of autoimmune conditions (4) and has been a subject of our interest. However, TCR gene therapy is currently limited by the availability within the natural TCR repertoire of Ag-specific receptors with desirable therapeutic properties. Both tumor-specific and autoantigen-specific TCR are often of low affinity. T cells transduced with these frequently express low levels of receptor that must compete with endogenous TCR for expression (5). Target ligand may be weakly expressed or expressed in the context of insufficient costimulation. Simple approaches to engineer TCR response characteristics, particularly affinity, are therefore desirable and may aid in the creation of receptors with optimized therapeutic potential.Essentially two approaches have been used to manipulate TCR affinity, and studies of these have ...

CDR3β sequence motifs regulate autoreactivity of human invariant NKT cell receptors

“…We comprehensively investigated the autoreactive strength of this unprecedentedly large set of human iNKT TCRs and identified three CDR3β amino acid sequence motifs that were associated with strong autoreactivity: a VD region with 2 or more acidic amino acids, usage of the Jβ2-5 allele, and a CDR3β region of 13 amino acids in length. Our findings are underpinned by the previous reports that an acidic amino acid composition, J usage, and the CDR3β region amino acid length individually affected the affinities of conventional TCRs [35-37]. Importantly, human iNKT TCRs highly reactive for self-ligands reported to date indeed harbor 2 out of the 3 sequence motifs (Supplementary Table 1) [2, 10, 34, 38].…”

Preferential Use of Public TCR during Autoimmune Encephalomyelitis

“…An overall enhanced compatibility with MHC may thereby lead to the increased utilization of public TCR in diverse responses. An element of enhanced self-reactivity would be expected to accompany any generically increased binding fitness of TCR for MHC, and indeed this has been observed in mutational analyses increasing TCR affinity (39, 40). …”

NFM Cross-Reactivity to MOG Does Not Expand a Critical Threshold Level of High-Affinity T Cells Necessary for Onset of Demyelinating Disease

“…4). For MOG 35-55 , evidence suggested that amino acid residues P-1 and P-2 outside of the core nonamer contribute to MHC anchoring and to TCR binding in individual clones (44, 45, 54, 55) with implications for T cell recognition of NFM 15-35 (28). We focused on modulating NFM at P1 (T20Y) to mirror MOG at that position because Petersen et al .…”