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Supplementary Materials and Methods

Supplementary Figure S1. Epitope mapping of clones A-D by alanine scanning mutagenesis. Supplementary Figure S2. Analysis of the selectivity of ET1402L1 (clone B) towards AFP158. Supplementary Figure S3. Analysis of the potential risk for cross-presentation of AFP158 by normal cells. Supplementary Figure S4. Co-incubation of AFP-CAR T cells with antigen-positive cells induces production and release of cytokines. Supplementary Figure S5. AFP-CAR T cells can penetrate laminin-rich Matrigel and specifically kill AFP+/A2+ cells in vitro. Supplementary Figure S6. Representative quality control tests performed on each batch of AFP-CAR T cells manufactured for in vivo studies. Supplementary Figure S7. T cell subset analysis to determine distribution of TNaive, TCM TEM, and TEffector cell populations. Supplementary Figure S8. Analysis of human CD3+ T cells in Hep G2 s.c. tumors from i.v.- treated mice. Supplementary Figure S9. Determination of the number of AFP158/HLA-A*02:01 complexes expressed on the surface of liver cancer cells. Supplementary Table S1. Chimeric mouse IgG1 antibody binding affinity to AFP158/HLAA* 02:01.

Figure Legends for Sup. Figures 1-9 and Sup. Table 1

Purpose: The majority of tumor-specific antigens are intracellular and/or secreted and therefore inaccessible by conventional chimeric antigen receptor (CAR) T-cell therapy. Given that all intracellular/secreted proteins are processed into peptides and presented by class I MHC on the surface of tumor cells, we used alpha-fetoprotein (AFP), a specific liver cancer marker, as an example to determine whether peptide–MHC complexes can be targets for CAR T-cell therapy against solid tumors.Experimental Design: We generated a fully human chimeric antigen receptor, ET1402L1-CAR (AFP-CAR), with exquisite selectivity and specificity for the AFP158–166 peptide complexed with human leukocyte antigen (HLA)-A*02:01.Results: We report that T cells expressing AFP-CAR selectively degranulated, released cytokines, and lysed liver cancer cells that were HLA-A*02:01+/AFP+ while sparing cells from multiple tissue types that were negative for either expressed proteins. In vivo, intratumoral injection of AFP-CAR T cells significantly regressed both Hep G2 and AFP158-expressing SK-HEP-1 tumors in SCID-Beige mice (n = 8 for each). Moreover, intravenous administration of AFP-CAR T cells in Hep G2 tumor-bearing NSG mice lead to rapid and profound tumor growth inhibition (n = 6). Finally, in an established intraperitoneal liver cancer xenograft model, AFP-CAR T cells showed robust antitumor activity (n = 6).Conclusions: This study demonstrates that CAR T-cell immunotherapy targeting intracellular/secreted solid tumor antigens can elicit a potent antitumor response. Our approach expands the spectrum of antigens available for redirected T-cell therapy against solid malignancies and offers a promising new avenue for liver cancer immunotherapy. Clin Cancer Res; 23(2); 478–88. ©2016 AACR.