Preclinical Strategies to Identify Off-Target Toxicity of High-Affinity TCRs

• Published in: Molecular therapy Vol. 26; no. 5; pp. 1206 - 1214
• Main Authors: Bijen, Helena M., van der Steen, Dirk M., Hagedoorn, Renate S., Wouters, Anne K., Wooldridge, Linda, Falkenburg, J.H. Frederik, Heemskerk, Mirjam H.M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 02-05-2018; Elsevier Limited; American Society of Gene & Cell Therapy
• Subjects: Acute myeloid leukemia; Adoptive transfer; adoptive T cell therapy; Alanine; Amino acids; Antigens; Cancer; Cloning; Cross-reactivity; Epithelial cells; Epstein-Barr virus; Experiments; Fibroblasts; Gene expression; Histocompatibility antigen HLA; Immunotherapy; Keratinocytes; Leukemia; Lymphocytes; Lymphocytes B; Lymphocytes T; Minor histocompatibility antigens; Mutagenesis; Myeloid leukemia; off-target toxicity; Original; Patients; Peptides; preclinical screening; Proteins; SCT; T cell receptors; Target recognition; TCR; Toxicity; adoptive T cell therapy; TCR; off-target toxicity; preclinical screening; SCT
• ISSN: 1525-0016; 1525-0024
• DOI: 10.1016/j.ymthe.2018.02.017

Adoptive transfer of T cells engineered with a cancer-specific T cell receptor (TCR) has demonstrated clinical benefit. However, the risk for off-target toxicity of TCRs remains a concern. Here, we examined the cross-reactive profile of T cell clone (7B5) with a high functional sensitivity for the hematopoietic-restricted minor histocompatibility antigen HA-2 in the context of HLA-A*02:01. HA-2pos Epstein-Barr virus-transformed B lymphoblastic cell lines (EBV-LCLs) and primary acute myeloid leukemia samples, but not hematopoietic HA-2neg samples, are effectively recognized. However, we found unexpected off-target recognition of human fibroblasts and keratinocytes not expressing the HA-2 antigen. To uncover the origin of this off-target recognition, we performed an alanine scanning approach, identifying six out of nine positions to be important for peptide recognition. This indicates a low risk for broad cross-reactivity. However, using a combinatorial peptide library scanning approach, we identified a CDH13-derived peptide activating the 7B5 T cell clone. This was confirmed by recognition of CDH13-transduced EBV-LCLs and cell subsets endogenously expressing CDH13, such as proximal tubular epithelial cells. As such, we recommend the use of a combinatorial peptide library scan followed by screening against additional cell subsets to validate TCR specificity and detect off-target toxicity due to cross-reactivity directed against unrelated peptides before selecting candidate TCRs for clinical testing.
Adoptive transfer of T cells engineered with a cancer-specific T cell receptor (TCR) has demonstrated clinical benefit. To validate TCR specificity and detect off-target toxicity before selecting candidate TCRs for clinical testing, Bijen et al. recommend the use of a combinatorial peptide library scan followed by screening against additional cell subsets.