The molecular rules driving TCR cross-reactivity are poorly understood and, consequently, it is unclear the extent to which TCRs targeting the same Ag recognize the same off-target peptides. We determined TCR-peptide-HLA crystal structures and, using a single-chain peptide-HLA phage library, we generated peptide specificity profiles for three newly identified human TCRs specific for the cancer testis Ag NY-ESO-1 157-165-HLA-A2. Two TCRs engaged the same central peptide feature, although were more permissive at peripheral peptide positions and, accordingly, possessed partially overlapping peptide specificity profiles. The third TCR engaged a flipped peptide conformation, leading to the recognition of off-target peptides sharing little similarity with the cognate peptide. These data show that TCRs specific for a cognate peptide recognize discrete peptide repertoires and reconciles how an individual's limited TCR repertoire following negative selection in the thymus is able to recognize a vastly larger antigenic pool.

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Immunotherapeutic strategies that drive activation of cytotoxic T cells possess significant potential to eradicate tumours. Whereas monoclonal antibodies are restricted to targeting secreted or cell surface proteins, T cell receptors (TCRs) are able to recognise a wider range of targets. This is achieved through binding to short peptide fragments derived from proteins that are degraded intracellularly and presented at the cell surface by human leukocyte antigens (HLAs). Natural cancer specific TCRs however, have weak affinities and cancer cells often develop escape mechanisms to avoid destruction by T cells. To overcome this, we have developed Immune mobilising monoclonal TCRs Against Cancer (ImmTACs); a new class of soluble bi-specific molecules comprising affinity-enhanced, monoclonal T cell receptors (mTCRs) fused to an anti-CD3 scFv. ImmTACs target peptides presented by HLA, and through the anti-CD3 effector, re-direct cytotoxic T cells to achieve highly specific and potent tumour cell killing. At Immunocore, we have developed an integrated in-house process for the generation of ImmTACs and here describe the critical engineering steps involved. T-cell clones that specifically recognise validated cancer antigens are isolated from peripheral blood lymphocytes and the TCR-encoding sequences are identified by RACE. To confirm antigen binding, TCR á and â chains are expressed as inclusion bodies in bacteria, co-refolded in vitro, and their binding to the target peptide:HLA tested by Surface Plasmon Resonance (SPR). The affinity of the TCR is then enhanced up to a million-fold through directed evolution, utilising phage display. Individual mutants are screened by SPR and combined to generate ImmTACs with pM affinities (KD) and binding half-lives of many hours. A range of biochemical and cellular assays are then performed to assess the potency and specificity of each ImmTAC generated. This process has been successfully applied to produce ImmTACs for a wide range of targets, demonstrating the robustness of the platform. Our lead candidate, IMCgp100, is undergoing Phase IIa clinical trials in patients with advanced malignant melanoma. This reagent, which specifically targets the gp100 (280-288) peptide presented by HLA-A2 on melanoma cells, is well tolerated and shows very promising therapeutic potential. Citation Format: Andrew Knox, Fiona Chester, Frayne Bianchi, Sarah Bailey, Lucie Bouard, Nathaniel Liddy, Giovanna Bossi, Jane Harper, Joseph Dukes, Samantha Paston, Tara Mahon, Jessie Gavarret, Peter Molloy, Malkit Sami, Emma Baston, Brian Cameron, Alex Powlesland, Penio Todorov, Andrew Johnson, Martin Ebner, Yvonne McGrath, Namir Hassan, Annelise Vuidepot, Bent Jakobsen. Developing high affinity, soluble T cell receptors for the treatment of cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4872.