Engineering and characterizing human T cell receptors for cancer immunotherapies
The T cell receptor (TCR) is an heterodimer that binds to a short peptide bound to a product of the major histocompatibility complex (MHC). The chains each contain variable (V) and constant (C) region domains, followed by a transmembrane region. Each V domain contains three loops, called complementarity-determining regions (CDR1, CDR2, and CDR3), which interact with the peptide (pep)MHC antigen. The conserved docking angle of the six CDRs over the pepMHC antigens is thought to confer maximal signaling capabilities and it places the two most hypervariable loops (CDR3s) over the most diverse portion of the antigen (the peptide). Recent adoptive T cell trials have shown promising results using TCRs directed towards pepMHC complexes expressed in various types of malignancies. The process of immunological tolerance leads to the deletion of T cells with TCRs against possible cancer peptide self- antigens. Those TCRs that remain typically do not have the required affinity to mediate a sufficient T cell response. To overcome this, TCRs can be engineered for higher- affinity ex vivo and reintroduced into patient’s peripheral T cells. In chapter two, a TCR with a switch in peptide specificity was characterized. In previous studies, a TCR called RD1-MART1HIGH was engineered to bind to the cancer peptide MART1/HLA-A2, using libraries of mutants form the parental TCR, which is specific for a viral peptide, Tax/HLA-A2. The switch in peptide specificity was achieved by a limited number of CDR substitutions. To understand which residues contributed to the new peptide specificity and TCR binding, single codon libraries were created at every CDR residue. Libraries were pooled and sorted for binding to MART1/HLA-A2 to produce a sequence fitness landscape, which measured the effect that each individual substitution had on pepMHC binding. The sequence fitness landscape of RD1- MART1HIGH was compared to a sequence fitness landscape of its parent TCR A6 to identify the key residues involved with peptide specificity. Our collaborators (Professor Brian Baker at Notre Dame) solved the structure of the RD1-MART1HIGH TCR/MART1/HLA-A2 complex and this structure showed that the switch in specificity was accomplished by the reorientation of the TCR over the pep/HLA-A2 and the distributed action of many CDR residues. Despite the reorientation of the TCR, RD1- MART1HIGH TCR was able to mediate MART1/HLA-A2-specific activity when introduced into T cells. In chapter three, I analyzed the enriched mutants from the sequence fitness landscape. Mutants were characterized as individual mutants and combined as double and triple mutants. When combined, the enriched mutations from the sequence fitness landscape were able to increase the affinity of RD1-MART1HIGH by nearly 100-fold. Additionally, the surface expression level on yeast was substantially increased. The enriched mutants were compared to a high-affinity clone of RD1-MART1HIGH isolated from a directed evolution approach. Chapter four is an analysis of the activity and specificity of two different high- affinity TCRs in T cells. A high-affinity TCR for the cancer antigen WT1/HLA-A2 and another TCR called T1 that is specific for the melanoma antigen MART1/HLA-A2 were characterized. These TCRs were analyzed both as traditional full-length TCRs consisting of variable and constant domains as well as single chain signaling (SCS) constructs, which consist of TCR variable domains linked to intracellular signaling domains. These constructs were analogous to the chimeric antigen receptors (CARs) currently in adoptive T cell clinical trials. Despite being expressed at lower surface levels than their SCS counter parts, the full-length TCRs exhibited greater peptide sensitivity to pepMHC. The results of these studies provided insight into the differences in sensitivities of the CAR compared to the TCR formats for adoptive T cell therapies. In addition, they provided information about the specificity of the two high-affinity TCRs in an adoptive T cell form. Chapter five, as a prelude to studying a different human TCR, describes the engineering of a TCR for high-affinity against the survivin/HLA-A2 complex. Survivin is an anti-apoptotic protein overexpressed in a variety of cancers. A TCR isolated from peripheral lymphocytes was stabilized as a single-chain TCR (consisting of variable domains linked by a flexible linker) on the surface of yeast. Libraries were created in the CDR3α and CDR3β domains of the TCR and sorted for binding to survivin/HLA-A2. Two high-affinity clones were isolated that bind to survivin/HLA-A2 with nanomolar affinity.
University of Illinois - Urbana-Champaign
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