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. 2024 Feb 1:15:1345037.
doi: 10.3389/fimmu.2024.1345037. eCollection 2024.

Tumor epitope spreading by a novel multivalent therapeutic cellular vaccine targeting cancer antigens to invariant NKT-triggered dendritic cells in situ

Satoru Yamasaki  1 , Kanako Shimizu  1   2 , Shin-Ichiro Fujii  1   2   3
Affiliations

Tumor epitope spreading by a novel multivalent therapeutic cellular vaccine targeting cancer antigens to invariant NKT-triggered dendritic cells in situ

Satoru Yamasaki et al. Front Immunol. .

Abstract

Introduction: Cancer is categorized into two types based on the microenvironment: cold and hot tumors. The former is challenging to stimulate through immunity. The immunogenicity of cancer relies on the quality and quantity of cancer antigens, whether recognized by T cells or not. Successful cancer immunotherapy hinges on the cancer cell type, antigenicity and subsequent immune reactions. The T cell response is particularly crucial for secondary epitope spreading, although the factors affecting these mechanisms remain unknown. Prostate cancer often becomes resistant to standard therapy despite identifying several antigens, placing it among immunologically cold tumors. We aim to leverage prostate cancer antigens to investigate the potential induction of epitope spreading in cold tumors. This study specifically focuses on identifying factors involved in secondary epitope spreading based on artificial adjuvant vector cell (aAVC) therapy, a method established as invariant natural killer T (iNKT) -licensed DC therapy.

Methods: We concentrated on three prostate cancer antigens (prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), and prostatic acid phosphatase (PAP)). By introducing allogeneic cells with the antigen and murine CD1d mRNA, followed by α-galactosylceramide (α-GalCer) loading, we generated five types of aAVCs, i.e, monovalent, divalent and trivalent antigen-expressing aAVCs and four types of prostate antigen-expressing cold tumors. We evaluated iNKT activation and antigen-specific CD8+ T cell responses against tumor cells prompted by the aAVCs.

Results: Our study revealed that monovalent aAVCs, expressing a single prostate antigen, primed T cells for primary tumor antigens and also induced T cells targeting additional tumor antigens by triggering a tumor antigen-spreading response. When we investigated the immune response by trivalent aAVC (aAVC-PROS), aAVC-PROS therapy elicited multiple antigen-specific CD8+ T cells simultaneously. These CD8+ T cells exhibited both preventive and therapeutic effects against tumor progression.

Conclusions: The findings from this study highlight the promising role of tumor antigen-expressing aAVCs, in inducing efficient epitope spreading and generating robust immune responses against cancer. Our results also propose that multivalent antigen-expressing aAVCs present a promising therapeutic option and could be a more comprehensive therapy for treating cold tumors like prostate cancer.

Keywords: cancer; cytotoxic T cell; dendritic cell; iNKT cell; immunotherapy.

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Conflict of interest statement

SF received research funding and honoraria from Astellas Pharma, Inc. KS received honoraria from Astellas Pharma Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Establishment of monovalent aAVCs co-expressing CD1d and prostate tumor antigens for cancer therapy. (A) aAVCs expressing PSMA were created by electroporating NIH3T3 cells with PSMA and murine CD1d mRNA, followed by loading with α-galactosylceramide (α-GalCer). PSMA protein expression level was assessed using western blotting. (B) CD1d surface expression on aAVC-PSMA was assessed by flow cytometry (open, aAVC-PSMA; shaded, isotype). (C) aAVC-PSA was prepared as described in (A), with PSA being transfected instead. PSA protein expression in aAVC-PSA was measured using western blot analysis. (D) CD1d surface expression on aAVC-PSA was assessed using flow cytometry (open, aAVC-PSA; shaded, isotype). (E) aAVC-PAP was prepared as described in (A), with PAP transfected instead. The PAP protein in aAVC-PAP was measured using western blot analysis. (F) as similar to (A), but CD1d surface expression on aAVC-PAP was assessed by flow cytometry (open, aAVC-PAP; shaded, isotype).
Figure 2
Figure 2
iNKT cell activation by aAVCs. (A, C, E) α-GalCer presentation on aAVC-PSMA (A), aAVC-PSA (B), and aAVC-PAP (C). aAVCs were co-cultured with the Vα14 iNKT cell hybridoma 1.B2 for 24 h (upper), and IL-2 production in the culture supernatant was evaluated using IL-2 ELISA (n=4, mean ± SEM) (lower); ***P<0.001 (Mann-Whiteny). (B, D, F) C57BL/6 mice were injected intravenously with 5 × 105 aAVC-PSMA (B), aAVC-PSA (D), and aAVC-PAP (F). Spleens were removed after 3 days, and splenic iNKT was analyzed after staining with CD19-FITC, CD1d-dimer+ APC, and 7-AAD (upper). Representative dot plots showing the frequency of splenic iNKT cells (lower left) and summary(n=3, mean ± SEM) (lower right) were shown. **P<0.01, ***P<0.001(Mann-Whiteny).
Figure 3
Figure 3
Antitumor response by aAVCs in a prophylactic model. (A) Establishment of B16-PSMA (A), B16-PSA (C), and B16-PAP (E) cells. After introducing the PSMA, PSA, or PAP gene into B16, stable PSMA, PSA, or PAP protein expression was verified using western blot analysis. (B, D, F) Mice were immunized with or without 5 × 105 aAVC-PSMA, aAVC-PSA, or aAVC-PAP on day 0. Following this, mice were challenged with 5 × 105 B16-PSMA (B), B16-PSA (D), or B16-PAP (F), respectively on day 14 (n=5 per group, mean ± SEM); *P<0.05. ***P<0.001.
Figure 4
Figure 4
Relation of iNKT cells in host to antigen-specific T cell immunity by aAVC therapy. (A) Preparation of MNCs and OT-1 cells. Frequency of iNKT cells in B cell-depleted MNCs from WT and CD1d-/- mice (right) and purity of OT-1 CD8+ T cells (left) were analyzed by flow cytometry. OT-1 CD8+ T cells were isolated from spleen and lymph nodes of Ly5.1 OT-1Tg mice using CD8 MACS beads. The purity of OT -1 cells was analyzed using CD8a-FITC and Va2-PE. (B) Experimental protocol. The MNCs from WT mice and CD1d-/- mice (40x106/mouse) at the ratio of 50:50 (%) or 100:0(%) were transferred to Rag1-/- mice. OT-1 cells (1x105/mouse) were transferred 6 h later. aAVC-OVA cells (5x105/mouse) were administered the following day. (C, D) Frequency and cytokine production of OVA-specific CD8+ T cells. A week later, the Kb/SIINFEKL-tetramer+ CD8+T cells (C) and OVA257–264 peptide specific cytokine production (IFN-γ and TNF-α) (D) were analyzed. The data are representative data from two experiments independently and each data are also provided.
Figure 5
Figure 5
Antitumor response by aAVCs in a therapeutic model. The antitumor response was evaluated in a therapeutic model in which the therapy was administered at a tumor volume of around 50 mm3. (A) Mice were inoculated with 5 × 105 B16-PSMA cells and treated with or without 5 × 105 aAVC-PSMA on day 12 (n=8 per group, mean ± SEM); *P<0.05. (B) Mice were inoculated with 5 × 105 B16-PSA cells and treated with or without 5 × 105 aAVC-PSA on day 7 (n=6 per group, mean ± SEM); ***P<0.001. (C) Mice were inoculated with 5 × 105 B16-PAP cells and treated with or without 5 × 105 aAVC-PAP on day 10 (n=7–8 per group, mean ± SEM); ***P<0.001.
Figure 6
Figure 6
Induction of tumor epitope spreading by aAVC vaccination. (A, B) B16-PSMA/PSA/PAP cells were established using a retroviral vector expressing PSMA, PSA, or PAP, respectively. Tumor expression was verified using RT-PCR and western blotting. (C, E, G) Antitumor effect of various prostate antigen-expressing aAVC therapies. Mice were inoculated with 5 × 105 B16-PSMA/PSA/PAP cells and treated with or without 5 × 105 aAVC-PSMA (C), aAVC-PSA (E), or aAVC-PAP (G) on day 7 (n=5 per group, mean ± SEM); *P<0.05. (D, F, H) Epitope spread of prostate antigens in CD8+ T cells after aAVC therapy. Ten days after vaccination with aAVC-PSMA (B), aAVC-PSA (D), or aAVC-PAP (F), PSMA-, PSA-, or PAP-specific CD8+ T cells were analyzed using an IFN-γ ELISPOT assay. For this, splenic CD8+ T cells isolated from the immunized mice were cultured with splenic CD11c+ dendritic cells (DCs) from naïve mice that had been cultured in the presence or absence of PSMA, PSA, or PAP-PepTivator for 24 h. (n=4 per group, mean ± SEM); *P<0.05; ***P<0.001; ns: not significant, according to Tukey’s test.
Figure 7
Figure 7
Establishment of a multivalent aAVC and prostate cancer antigen-specific CD8+ T cell response. (A) Establishment of multivalent artificial adjuvant vector cells for the prostate (aAVC-PROS). Three prostate cancer antigen-expressing artificial adjuvant cells (aAVC-PSMA/PSA/PAP and aAVC-PROS) were established by co-transfecting NIH3T3 cells with PSMA, PSA, PAP, and murine CD1d mRNA and then loaded with α-GalCer. (B) Tumor protein antigen in aAVC-PROS. The amount of PSMA, PSA, and PAP was determined using western blot analysis. (C) The prostate antigen-specific CD8+ T cell response induced by aAVC. Mice were intravenously immunized with 5 × 105 aAVC-PROS. After one week, PSMA-, PSA-, or PAP-specific CD8+ T cells were analyzed using an IFN-γ ELISPOT assay. Splenic CD8+ T cells isolated from the immunized mice were cultured with splenic CD11c+ DCs from naïve mice that had been cultured in the presence or absence of PSMA-, PSA-, or PAP-PepTivator for 24 h. (n=4 per group, mean ± SEM) *P<0.05; ***P<0.001; ns: not significant, according to Tukey’s test.
Figure 8
Figure 8
Comparison of the antitumor effect of divalent and trivalent prostate antigen-expressing aAVCs. Divalent artificial adjuvant cells (aAVC-PSMA/PSA) were established by co-transfecting NIH3T3 cells with PSMA, PSA, and murine CD1d mRNA. (A) Mice were inoculated with 5 × 105 B16-PSMA/PSA/PAP cells and treated with or without 5 × 105 divalent aAVC-PSMA/PSA or trivalent aAVC-PROS on day 7. (B) Comparison of the antitumor effects of monovalent (aAVC-PSMA, aAVC-PSA, or aAVC-PAP), divalent (aAVC-PSMA/PSA), or trivalent aAVCs (aAVC-PROS). The tumor size was evaluated by comparing untreated mice with treated mice on days 10 (Early phase) and 19 (Late phase). *Plt;0.05, **P<0.01, ***P<0.001; ns: Tukey’s test.
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References

    1. Boettcher AN, Usman A, Morgans A, VanderWeele DJ, Sosman J, Wu JD. Past, current, and future of immunotherapies for prostate cancer. Front Oncol (2019) 9:884. doi: 10.3389/fonc.2019.00884 - DOI - PMC - PubMed
    1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. . Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin (2021) 71(3):209–49. doi: 10.3322/caac.21660 - DOI - PubMed
    1. Jones CU, Hunt D, McGowan DG, Amin MB, Chetner MP, Bruner DW, et al. . Radiotherapy and short-term androgen deprivation for localized prostate cancer. N Engl J Med (2011) 365(2):107–18. doi: 10.1056/NEJMoa1012348 - DOI - PubMed
    1. Arndt C, Feldmann A, Koristka S, Cartellieri M, Dimmel M, Ehninger A, et al. . Simultaneous targeting of prostate stem cell antigen and prostate-specific membrane antigen improves the killing of prostate cancer cells using a novel modular T cell-retargeting system. Prostate (2014) 74(13):1335–46. doi: 10.1002/pros.22850 - DOI - PubMed
    1. Wang J, Cheng X, Jin Y, Xia B, Qin R, Zhang W, et al. . Safety and clinical response to combined immunotherapy with autologous iNKT cells and PD-1(+)CD8(+) T cells in patients failing first-line chemotherapy in stage IV pancreatic cancer. Cancer Res Commun (2023) 3(6):991–1003. doi: 10.1158/2767-9764.CRC-23-0137 - DOI - PMC - PubMed

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The author(s) declare financial support was received for the research, authorship, and/or publication of this article. The authors declare that this study received funding from Astellas Pharma, Inc. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.
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