Landscape of immunogenic tumor antigens in successful immunotherapy of virally induced epithelial cancer

Abstract

Immunotherapy has clinical activity in certain virally associated cancers. However, the tumor antigens targeted in successful treatments remain poorly defined. We used a personalized immunogenomic approach to elucidate the global landscape of antitumor T cell responses in complete regression of human papillomavirus-associated metastatic cervical cancer after tumor-infiltrating adoptive T cell therapy. Remarkably, immunodominant T cell reactivities were directed against mutated neoantigens or a cancer germline antigen, rather than canonical viral antigens. T cells targeting viral tumor antigens did not display preferential in vivo expansion. Both viral and nonviral tumor antigen-specific T cells resided predominantly in the programmed cell death 1 (PD-1)-expressing T cell compartment, which suggests that PD-1 blockade may unleash diverse antitumor T cell reactivities. These findings suggest a new paradigm of targeting nonviral antigens in immunotherapy of virally associated cancers.

Fig. 1.. Therapeutic TIL used for successful treatment of patients with metastatic HPV⁺ cervical cancer targeted viral and non-viral tumor antigens.

(A and B) IFN-γ ELISPOT assay of (A) TIL-3775 and (B) TIL-3853, compared with pre-treatment PB T cells from these patients after co-culture with autologous DCs electroporated with RNA encoding HPV type-specific antigens or glycoprotein 100 (GP100, negative control). (C and D) Flow cytometric analysis (FCA) of CD137 expression on (C) TIL-3775 and (D) TIL-3853, and PB T cells, co-cultured with DCs electroporated with RNA encoding (C) HPV16-E6, HPV16-E7 or GP100, and (D) HPV18-E7 or GP100. (E) IFN-γ ELISPOT assay of TIL-3775 and PB T cells co-cultured with DCs electroporated with TMG-1 to TMG-19, or GP100 RNA. (F) FCA of CD137 expression on TIL-3775 and PB T cells upon co-culture with DCs electroporated with TMG-1, TMG-5, TMG-18 or GP100 RNA. (G) IFN-γ ELISPOT assay of TIL-3775 co-cultured with APCs pulsed with individual mutated 25-mer peptides encoded in the indicated TMG (table S2). Mutated 25-mers 3 and 10 encoded in TMG-1 and TMG-5, respectively, could not be synthesized. (H) Reactivity of TIL-3775 to the mutated (MUT) 25-mer peptides encoded by TMG-1 (SETDB1_E>D), TMG-5 (METTL17_E>K)_, and TMG-18 (ALDH1A1_N>I) or their wild-type (WT) counterparts pulsed on DCs. Error bars represent standard deviation of duplicate wells. (I) IFN-γ ELISPOT assay of TIL-3853 and PB T cells to DCs electroporated with the indicated cancer-germline antigens or GP100 RNA. (J) FCA of CD137 expression on TIL-3853 and PB T cells upon co-culture with DCs electroporated with KK-LC-1 or GP100 RNA. Phorbol 12-Myristate 13-Acetate and Ionomycin (P/I) stimulation was used as a positive control. T-cell reactivity was measured at 20–24 hours in all co-culture assays. “>” denotes off-scale values. All FCA’s are pre-gated on live⁺ CD3⁺ T cells. Experiments were performed as single determinations unless stated otherwise. All data, except E and G, are representative of ≥ two independent experiments.

Fig. 2.. Immunodominant anti-tumor T-cell responses in therapeutic TIL were directed against mutated neoantigens and the cancer-germline antigen KK-LC-1, rather than HPV antigens.

(A and B) IFN-γ ELISPOT assay of autologous pre-treatment PB T cells retrovirally transduced with TCRs identified from infused TIL of patients. (A) Reactivity of PB T cells from patient 3775 transduced with the 13 identified TCRs to peptide pools of HPV16-E6, HPV16-E7 and GP100 (negative control), and wild-type (WT) and mutated (MUT) SETDB1_E>D, METTL17_E>K, and ALDH1A1_N>I 25-mer peptides pulsed on autologous DCs. (B) Reactivity of PB T cells from patient 3853 transduced with the 2 identified TCRs stimulated with autologous DCs pulsed with peptide pools of HPV18-E7, KK-LC-1 and GP100 (negative control). T-cell reactivity was measured at 20–24 hours in co-culture assays. The TCR transduction efficiencies were >40%, as determined by anti-mouse TCRB constant region flow cytometry (data not shown). P/I stimulation was used as a positive control in all co-culture assays. “>” denotes off-scale values. Experiments were performed as single determinations. Data are representative of two independent experiments. (C and D) Frequency and rank of the individual tumor antigen-specific TCRB clonotypes identified in infused TIL as determined by TCRB deep sequencing analysis. (C) In TIL-3775, CD8⁺ clonotypes were all mutation-specific (rank 1, 2, 3, 4, 8, 10, 61 and 65), the HPV16-E6-specific (rank 6) and two HPV16-E7-specific (rank 11 and 24) clonotypes. CD4⁺ clonotypes were two HPV16-E7-specific (rank 9 and 33) clonotypes. (D) In TIL-3853, the KK-LC-1-specific clonotype (rank 1) was CD8⁺ and the HPV18-E7-specific clonotype (rank 2) was CD4⁺. Pie charts display the sum of the frequencies of individual TCRB clonotypes with indicated tumor antigen specificity in the infused TIL. White color indicates the frequency of TCRB clonotypes with undefined specificity. (E and F) Functional avidity assay testing by IFN-γ enzyme-linked immunospot assay (ELISA) of autologous PB T cells transduced with the indicated tumor-antigen specific TCRs co-cultured with autologous antigen-presenting cells pulsed with titrated concentrations of cognate peptides (fig. S4). (E and F) Functional avidity of (E) 13 identified TCRs from patient 3775 and (F) two TCRs from patient 3853. Reactivity of TCR transduced T cells (TCR Td) and untransduced T cells (UT) (control T cells) are shown for HPV-specific TCRs (E and F) and KK-LC-1-specific TCR (F). Reactivity of TCR transduced T cells against mutated (MUT) and wild-type (WT) cognate epitopes are shown for mutation-specific TCRs (E). HLA-class I restricted CD8⁺-T-cell derived TCRs (rank 1, 2, 3, 4, 6, 8, 10, 11, 24, 61 and 65 in TIL-3775 (Figure 2C); rank 1 in TIL-3853 (Figure 2D)) were transduced into PB CD8⁺ T cells, and CD4⁺-T-cell derived TCRs (rank 9 and 33 in TIL-3775 (Figure 2C); rank 2 in TIL-3853 (Figure 2D)) were transduced into PB CD4⁺ T cells. TCR transduced T cells were enriched by anti-mouse TCRB constant region to yield >90% CD8⁺ or CD4⁺ TCR transduced T cells in each case for use in co-culture assays (data not shown). Error bars represent standard deviation of duplicate wells. Data are representative of two independent experiments.

Fig. 3.. Repopulation of peripheral blood (PB) by infused T cells targeting viral and non-viral tumor antigens throughout cancer regression and remission.

(A and B) Reactivity of PB T cells from before (PRE) and after (POST) treatment was assessed by IFN-γ ELISPOT assay against peptides from tumor antigens identified in patient’s infused TIL. (A) Reactivity of PB T cells from patient 3775 from before and 1 month (M) after treatment to peptide pools of HPV16-E6, HPV16-E7 and GP100 (negative control), and mutated (MUT) and wild-type (WT) SETDB1_E>D, METTL17_E>K, and ALDH1A1_N>I 25-mer peptides pulsed on autologous DCs. (B) Reactivity of PB T cells from patient 3853 from before, 0.3 and 5.6 M after treatment to peptide pools of HPV18-E7, KK-LC-1 and GP100 (negative control) pulsed on autologous DCs. T-cell reactivity was measured at 20–24 hours in co-culture assays. Error bars represent standard deviation of duplicate wells. “>” denotes off-scale values. P/I stimulation was used as a positive control in all co-culture experiment. (C and D) Frequency (f) and rank of individual tumor antigen-specific TCRB clonotypes as identified within (C) TIL-3775 and (D) TIL-3853 among PB mononuclear cells (PBMC) pre- and post-treatment (at the indicated months (M)), as determined by TCRB deep sequencing. Pie charts display the sum of the frequencies of individual TCRB clonotypes with indicated tumor antigen specificity in the infused TIL.

Fig. 4.. Programmed cell death 1 (PD-1) expression identifies both viral and non-viral tumor antigen-specific T cells in the circulation of patients with metastatic cervical carcinoma before treatment.

(A) Expression of PD-1 on CD4⁺ and CD8⁺ T cells from PB before treatment for (left) patient 3775 and (right) patient 3853. (B and C) Frequency of individual tumor antigen-specific TCRB clonotypes as identified for (B) patient 3775 in TIL-3775 and for (C) patient 3853 in TIL-3853 (Fig. 2D) within sorted PD-1⁻ and PD-1⁺ PB CD4⁺ and CD8⁺ T-cell subsets was determined by TCRB deep sequencing analysis. Graphs display CD4⁺ and CD8⁺ clonotypes. The specificity of each TCRB clonotype and its rank in the infused TIL is indicated next to the symbol. ND in (B) indicates clonotypes not detected (<0.001%) in sorted PD-1⁻ population (symbols shown on the x-axis).