Selection of a Clinical Lead TCR Targeting Alpha-Fetoprotein-Positive Liver Cancer Based on a Balance of Risk and Benefit

Abstract

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer with a poor prognosis and limited therapeutic options. Alpha-fetoprotein (AFP), an established clinical biomarker of HCC, has been employed as an attractive target for T cell-based immunotherapy against this disease given its high expression in the tumor and restricted expression in normal tissues. We have identified a number of T cell receptors (TCRs) recognizing the HLA-A*02:01 restricted AFP158-166 peptide FMNKFIYEI, providing a TCR candidate pool for identifying TCRs with optimal clinical benefit. To select the ideal AFP TCR for clinical use, we evaluated the efficacy and safety profile of 7 TCRs by testing their potency toward AFP-expressing HCC cells and their specificity based upon reactivity to normal and transformed cells covering a wide variety of primary cell types and HLA serotypes. Furthermore, we assessed their cross-reactivity to potential protein candidates in the human genome by an extensive alanine scan (X-scan). We first selected three TCR candidates based on the in vitro anti-tumor activity. Next we eliminated two potential cross-reactive TCRs based on their reactivity against normal and transformed cells covering a variety of primary cell types and HLA serotypes, respectively. We then excluded the potential cross-reactivity of the selected TCR with a protein candidate identified by X-scan. At present we have selected an AFP TCR with the optimal affinity, function, and safety profile, bearing properties that are expected to allow AFP TCR redirected T cells to specifically differentiate between AFP levels on tumor and normal tissues. An early phase clinical trial using T cells transduced with this TCR to treat HCC patients (NCT03971747) has been initiated.

Keywords: T cell receptor (TCR); X-scan; alloreactivity; alpha-fetoprotein (AFP); cross-reactivity; hepatocellular carcinoma (HCC); immunotherapy.

Copyright © 2020 Luo, Cui, Cai, Zhu, Yang, Patrick, Zhu, Huang, Yao, Yao, He and Ji.

Figures

Figure 1

AFP TCR1, 2, and 3 demonstrate comparable potent reactivity toward the target cells in vitro. (A) Alignment of amino acid sequence of CDR3 regions of the α (left) and β (right) chains of the 7 AFP TCRs. (B) FACS analysis of the surface expression of TCR β chain and AFP tetramer staining of T cells 8 days after transducing of indicated AFP TCRs. The percentage of TCR β (top) or AFP tetramer (bottom) positive cells as indicated. (C) Cytokine production and degranulation of AFP TCR T cells upon encountering HepG2 target cells as revealed by intracellular staining of IL-2, IFN-γ (top), TNF-α and CD107a (bottom). (D) IFN-γ concentration in the supernatant of overnight, 1:1 ratio co-culture of AFP TCR T cells with HepG2 target cells or control Huh7 cells measured by ELISA. Data represents the mean + s.d. of quadruplicate co-culture samples. (E) Cytolytic capacity of AFP TCR T cells toward HepG2 target cells over a 4-day co-culture. Data represents the mean of triplicate samples derived from RTCA instrument, as compared to target only wells. (E:T): effect to target ratio. Colors are represented as in (D). (F) Time to eradicate 50% of target cells (KT50) of different AFP TCR T cells at the indicated effect to target (E:T) ratio. Colors are represented as in (D). T cells transduced with TCR 8 and TCR 11 did not eradicate 50% of the target by the end of the analysis at E:T = 0.25:1, thus were not plotted. Data shown in b-f is representative of at least 3 independent experiments on T cells isolated from 3 healthy donors. UT, untransduced T cells.

Figure 2

AFP TCR 2 shows lower than background activity toward primary neurons and no alloreactivity compared to TCR 1 and 3. (A) IFN-γ concentration in the supernatant of overnight, 1:1 ratio co-culture of the indicated AFP TCR T cells with iCell derived neurons measured by ELISA. Data represents the mean + s.d. of the triplicate co-cultures and is representative of T cells prepared from 2 healthy donors. (B) IFN-γ concentration in the supernatant of overnight, 1:1 ratio co-culture of the indicated AFP TCR T cells with a panel of Epstein-Barr virus transformed B cell lines expressing various HLA alleles measured by ELISA. Data represents the mean + s.d. of quadruplicate co-cultures and is representative of T cells prepared from 2 healthy donors. HepG2 and Huh7 cells were included as positive and negative controls, respectively, in both (A,B). *P < 0.05 (unpaired two-tailed Student's t-test).

Figure 3

AFP TCR 2 displays no reactivity toward a variety of HLA-A*02:01+ primary cells. (A) IFN-γ concentration in the supernatant of overnight, 1:1 ratio co-culture of UT or T cells expressing AFP TCR 2 with primary hepatocytes measured by ELISA. T cells were prepared from 2 healthy donors and human primary hepatocytes were obtained from 2 adult HLA-A*02:01+ donors. Data represents the mean + s.d. of quadruplicate co-cultures. (B) 4-1BB upregulation by T cells after co-culture as described in a. Cells were further analyzed by FACS for surface expression of 4-1BB on total CD3+ untransduced T cells or AFP TCR 2 transduced T cells. HepG2 and Huh7 cells were included as positive and negative controls, respectively, in both (A,B). (C) Summary of reactivity from co-culture experiment of T cells expressing AFP TCR 2 with primary hepatocytes, primary lung and kidney epithelial cells, and iCell derived primary cells including neurons, astrocytes, cardiomyocyte, and endothelial cells.

Figure 4

AFP TCR 2 shows no alloreactivity. (A) IFN-γ concentration in the supernatant of overnight, 1:1 ratio co-culture of UT or AFP TCR 2 transduced T cells with a panel of 14 Epstein-Barr virus transformed B cell lines expressing various HLA alleles measured by ELISA. Data represents the mean + s.d. of quadruplicate co-cultures and is representative of 3 healthy donors. A total of 38 transformed B cell lines were tested. (B) Frequencies of class I HLA serotypes in Chinese Han population. Serotypes were sorted by frequency for each subclass from high to low; those covered by the 38 transformed B cell lines in our test are shown in blue while those not covered are shown in orange. For HLA-B, only the top 20 serotypes are displayed. (C) Summary of the number of class I HLA serotypes covered in this study and corresponding cumulative percentage in Chinese Han population.

Figure 5

AFP TCR 2 is unlikely to cross-react with other proteins in the human genome. (A) Graphic presentation of X-scan results showing the replaceability of each amino acid of AFP158−166 epitope. Peptides with replacement of each amino acid residue with every other 19 amino acids were used to activate the TCR 2 transduced T cells. The amount of IFN-γ produced by AFP TCR 2 transduced T cells after stimulation with mutated epitope (mut) was compared to that from wild type AFP158−166 peptide (wt) and the ratios are presented. Dashed line indicates the cut-off for tolerance. Data represents the mean + s.d. of duplicate co-cultures and is representative of 3 healthy donors. (B) ENPP1 and AFP expression in the indicated cell lines tested by Western blot. GAPDH was used as loading control. (C) IFN-γ concentrations in the supernatant of overnight, 1:1 ratio co-culture of UT or AFP TCR 2 transduced T cells with the indicated cell lines measured by ELISA. Data represents the mean + s.d. of quadruplicate co-cultures and is representative of 3 healthy donors.