Tuning T‐Cell Receptor Affinity to Optimize Clinical Risk‐Benefit When Targeting Alpha‐Fetoprotein–Positive Liver Cancer

Abstract

Patients with hepatocellular carcinoma (HCC) have a poor prognosis and limited therapeutic options. Alpha‐fetoprotein (AFP) is often expressed at high levels in HCC and is an established clinical biomarker of the disease. Expression of AFP in nonmalignant liver can occur, particularly in a subset of progenitor cells and during chronic inflammation, at levels typically lower than in HCC. This cancer‐specific overexpression indicates that AFP may be a promising target for immunotherapy. We verified expression of AFP in normal and diseased tissue and generated an affinity‐optimized T‐cell receptor (TCR) with specificity to AFP/HLA‐A*02+ tumors. Expression of AFP was investigated using database searches, by qPCR, and by immunohistochemistry (IHC) analysis of a panel of human tissue samples, including normal, diseased, and malignant liver. Using in vitro mutagenesis and screening, we generated a TCR that recognizes the HLA‐A*02‐restricted AFP158‐166 peptide, FMNKFIYEI, with an optimum balance of potency and specificity. These properties were confirmed by an extension of the alanine scan (X‐scan) and testing TCR‐transduced T cells against normal and tumor cells covering a variety of tissues, cell types, and human leukocyte antigen (HLA) alleles. Conclusion: We have used a combination of physicochemical, in silico, and cell biology methods for optimizing a TCR for improved affinity and function, with properties that are expected to allow TCR‐transduced T cells to differentiate between antigen levels on nonmalignant and cancer cells. T cells transduced with this TCR constitute the basis for a trial of HCC adoptive T‐cell immunotherapy.

Identification of an HLA‐A*24:02‐restricted α‐fetoprotein signal peptide‐derived antigen and its specific T‐cell receptor for T‐cell immunotherapy

“…In general, TCRs obtained from the peripheral T‐cell repertoire exhibit low affinity for tumor‐associated antigen‐derived peptides because of negative selection in the thymus. The functional avidity of a TCR, within a certain range, is directly correlated with its ligand binding affinity and therefore T cells expressing low‐affinity TCRs have limited clinical efficacy. Optimizing TCR affinity, while avoiding non‐specificity, becomes essential for the development of TCR‐T therapy .…”

“…The functional avidity of a TCR, within a certain range, is directly correlated with its ligand binding affinity and therefore T cells expressing low‐affinity TCRs have limited clinical efficacy. Optimizing TCR affinity, while avoiding non‐specificity, becomes essential for the development of TCR‐T therapy . KWV3.1 reported here has a relatively low affinity of ~54·8 μ m .…”

“…Another concern is toxicity associated with TCR‐T therapy. AFP is not strictly restricted in tumor cells: a low level of AFP expression can be detected in non‐malignant liver cells and other normal tissues . Careful adjustment of the antigen sensitivity of AFP‐specific TCRs might be required to avoid autoreactivity.…”

“…α ‐Fetoprotein (AFP) is expressed during embryonic development and transcriptionally repressed immediately after birth . It was considered that AFP was not expressed, or was expressed at almost undetectable levels in healthy tissues . However, AFP becomes transcriptionally reactivated and is highly expressed in approximately 50%–80% of HCC patients .…”

“…[14][15][16] It was considered that AFP was not expressed, or was expressed at almost undetectable levels in healthy tissues. 17 However, AFP becomes transcriptionally reactivated and is highly expressed in approximately 50%-80% of HCC patients. [18][19][20] As a result, AFP is an attractive target for TCR-T therapy.…”