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. 2021 Dec 27;14(1):106.
doi: 10.3390/cancers14010106.

Identification of Neoantigens in Two Murine Gastric Cancer Cell Lines Leading to the Neoantigen-Based Immunotherapy

Koji Nagaoka  1 , Changbo Sun  1 , Yukari Kobayashi  1 , Takayuki Kanaseki  2 , Serina Tokita  2   3 , Toshihiro Komatsu  4 , Kazuhiro Maejima  5 , Junichiro Futami  6 , Sachiyo Nomura  7 , Keiko Udaka  4 , Hidewaki Nakagawa  5 , Toshihiko Torigoe  2 , Kazuhiro Kakimi  1
Affiliations

Identification of Neoantigens in Two Murine Gastric Cancer Cell Lines Leading to the Neoantigen-Based Immunotherapy

Koji Nagaoka et al. Cancers (Basel). .

Abstract

To develop combination immunotherapies for gastric cancers, immunologically well-characterized preclinical models are crucial. Here, we leveraged two transplantable murine gastric cancer cell lines, YTN2 and YTN16, derived from the same parental line but differing in their susceptibility to immune rejection. We established their differential sensitivity to immune checkpoint inhibitors (ICI) and identified neoantigens. Although anti-CTLA-4 mAbs eradicated YTN16 tumors in 4 of 5 mice, anti-PD-1 and anti-PD-L1 mAbs failed to eradicate YTN16 tumors. Using whole-exome and RNA sequencing, we identified two and three neoantigens in YTN2 and YTN16, respectively. MHC class I ligandome analysis detected the expression of only one of these neoantigens, mutated Cdt1, but the exact length of MHC binding peptide was determined. Dendritic cell vaccine loaded with neoepitope peptides and adoptive transfer of neoantigen-specific CD8+ T cells successfully inhibited the YTN16 tumor growth. Targeting mutated Cdt1 had better efficacy for controlling the tumor. Therefore, mutated Cdt1 was the dominant neoantigen in these tumor cells. More mCdt1 peptides were bound to MHC class I and presented on YTN2 surface than YTN16. This might be one of the reasons why YTN2 was rejected while YTN16 grew in immune-competent mice.

Keywords: DC vaccine; adoptive cell therapy (ACT); checkpoint inhibitor; gastric cancer; neoantigen.

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Conflict of interest statement

Kazuhiro Kakimi reports grants from TAKARA BIO Inc. outside the submitted work. The Department of Immunotherapeutics, The University of Tokyo Hospital, is an endowed department by TAKARA BIO Inc. The other authors have no competing interests to disclose.

Figures

Figure 1
Figure 1
Immunological characterization of YTN2 and YTN16 in terms of tumor growth and sensitivity to checkpoint inhibitors: (a) Mice (n = 5) were inoculated with 5 × 106 YTN2 or YTN16 cells on day 0. Anti-PD-1 (clone RMP1-14, 200 µg/mouse), anti-PD-L1 (clone 10F9G2, 200 µg/mouse) or anti-CTLA-4 (clone 9H10, 100 µg/mouse) mAbs were administered on days 0, 4, and 8. Tumor volumes were monitored every 2 or 3 days. Arrows indicate the days of antibody injection. (b) Tumor volumes on day 21 were shown. * p < 0.05, ** p < 0.01, one-way ANOVA with Dunnett’s test for multiple comparisons. (c) Mice (n = 5) were inoculated with 5 × 106 YTN16 cells on day 0. Anti-CTLA-4 and anti-CD8α (clone 53-6.7, 200 µg/mouse) mAbs were injected on days 0, 4 and 8, and 4, 8, and 12, respectively. Tumor volumes were monitored every 2 or 3 days. Arrows and arrowheads indicate the days of anti-CTLA-4 and anti-CD8α injection, respectively. (d) Tumor volumes on day 26 were shown. ** p < 0.01, one-way ANOVA with Dunnett’s test for multiple comparisons.
Figure 2
Figure 2
Identification of neoantigens: (a) YTN2-reactive CD8+ T cell lines were established from the spleens of mice that had rejected YTN2 tumors. They responded to both YTN2 and YTN16 tumor cells and produced IFN-γ. (b) YTN16-reactive CD8+ T cell lines were established from splenocytes of mice that had rejected YTN16 following treatment with anti-PD-1 and/or anti-CTLA-4 mAbs. IFN-γ production by YTN16-reactive CD8+ T cells stimulated with YTN16 or YTN2 was quantified. Venn diagrams indicate (c) missense mutations identified by WES, (d) expressed mutations filtered by RNA-Seq data (FPKM ≥ 30 and RNA VAF ≥ 0.04), and (e) candidate neoepitopes of YTN2 and YTN16 cells. (f) YTN16-reactive CD8+ T cell lines were stimulated with 11 candidate peptides. IFN-γ production in the culture supernatant was evaluated by ELISA. Data are based on 3 independently established YTN16-reactive CD8+ T cell lines. A dotted line indicates mean + 2 SD of IFN-γ production by the unstimulated cells.
Figure 3
Figure 3
Three neoantigen-reactive CD8+ T cell lines. The established neoantigen-reactive CD8+ T cell lines were stimulated with the indicated peptides or cells, and their reactivity was evaluated by IFN-γ production: (a) mCdt1-reactive CD8+ T cells responded to YTN2 and YTN16; they did not respond to B16F10, LLC1 or MC38 cell lines. (b) mScarb2-reactive CD8+ T cells responded to YTN16; they did not respond to YTN2, B16F10, LLC1, or MC38 cell lines. (c) mZfp106-reactive CD8+ T cells responded to YTN2 and YTN16; they did not respond to B16F10, LLC1, or MC38 cell lines.
Figure 4
Figure 4
Identification of neoantigen-specific CD8+ T cells in the tumor. Mice (n = 3) were inoculated with 5 × 106 YTN2 or YTN16 cells on day 0. Tumors were harvested on day 17 and tumor-infiltrating cells were analyzed by flow cytometry. Dot plots show frequencies of mCdt1- and mZfp106-reactive CD8+ T cells in YTN2 (a) and mCdt1, mScarb2 and mZfp106-reactive CD8+ T cells in YTN16 (b).
Figure 5
Figure 5
Identification of 10-mer mCdt1 peptides by MHC class I ligandome analysis: (a) Venn diagrams indicate the numbers of H-2Db- and H-2Kb-binding peptides identified in YTN2 and YTN16. (b) The numbers of H-2Db- and H-2Kb-mutated binding peptides identified in YTN2 and YTN16. (c) The amino acid sequence and predicted IC50 value of the mCdt1 peptide identified in YTN2.
Figure 6
Figure 6
Functional affinities of three neoantigens: (a) MHC class I binding prediction values of 9-mer and 10-mer mCdt1 epitopes. (b) mCdt1-reactive CD8+ T cells were stimulated with 9-mer or 10-mer mCdt1 peptides at the indicated concentrations. IFN-γ production was evaluated by intracellular cytokine staining. (c) MHC class I binding prediction values of 8-mer, 9-mer, and 10-mer mScarb2 peptides. (d) mScarb2-reactive CD8+ T cells were stimulated with 8-mer, 9-mer, or 10-mer mScarb2 peptides at the indicated concentrations. IFN-γ production was evaluated by intracellular cytokine staining. (e) An IGV screenshot of the WES reads of the mZfp106 gene. (f) mZfp106-reactive CD8+ T cells were stimulated at the indicated concentrations with mZfp106 A656T&C659R, wild-type, A656T or C659R peptides. IFN-γ production was evaluated by intracellular cytokine staining.
Figure 7
Figure 7
Anti-tumor effects of neoantigen-based immunotherapy: (a) Mice were vaccinated with neoepitope peptide-pulsed DCs twice biweekly. Two weeks after the second vaccine, the mice were sacrificed, splenocytes were stimulated with corresponding short peptides for 4 h, and IFN-γ production was evaluated by intracellular cytokine staining. (b) Mice (n = 5) were inoculated with 5 × 106 YTN16. DCs pulsed with neoepitope peptides were injected subcutaneously 5 days after tumor inoculation. Tumor volumes were monitored every 2 or 3 days. (c) Tumor volumes on day 10 were shown. ** p < 0.01, *** p < 0.0001, one-way ANOVA with Dunnett’s test for multiple comparisons. (d) Mice (n = 5) were inoculated with 5 × 106 YTN16. Six days later, 1 × 107 neoantigen-reactive CD8+ T cells were injected intravenously. Tumor volumes were monitored every 2 or 3 days. (e) Tumor volumes on day 22 were shown. * p < 0.05, *** p < 0.0001, one-way ANOVA with Dunnett’s multiple comparison test.
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