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The adjuvant effect of melanin is superior to incomplete Freund’s adjuvant in subunit/peptide vaccines in mice

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Abstract

Peptide vaccines represent an attractive alternative to conventional anti-tumor therapies, but have not yet achieved significant clinical efficacy with commonly used formulations. Combination of short antigenic peptides, synthetic melanin and TLR9 agonist (Toll-like receptor 9, CpG-28) was reported as highly efficient to trigger strong CD8 + T-cell responses. We compared this vaccine approach to the standard adjuvant formulation that combines the incomplete Freund’s adjuvant (IFA) and CpG-28, using either an ovalbumin epitope (pOVA30) or a spontaneously occurring tumor neoepitope (mAdpgk).

Melanin-based vaccine induced significantly higher cytotoxic T lymphocytes (CTL) responses than IFA-based vaccine in both pOVA30- and mAdpgk-targeted vaccines. The anti-tumor efficacy of melanin-based vaccine was further assessed in mice, grafted either with E.G7-OVA cells (E.G7 cells transfected with ovalbumin) or with MC38 cells that spontaneously express the mAdpgk neoepitope. Melanin-based vaccine induced a major inhibition of E.G7-OVA tumor growth when compared to IFA-based vaccine (p < 0.001), but tumors eventually relapsed from day 24. In the MC38 tumor model, no significant inhibition of tumor growth was observed. In both cases, tumor escape appeared related to the loss of antigen presentation by tumor cells (loss of ovalbumin expression in E.G7-OVA model; poor presentation of mAdpgk in MC38 model), although the CTL responses displayed an effector memory phenotype, a high cytolytic potential and low programmed cell death-1 (PD1) expression.

In conclusion, synthetic melanin can be efficiently used as an adjuvant to enhance T-cells response against subunit vaccine antigens and compared favorably to the classic combination of IFA and TLR9 agonist in mice.

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Abbreviations

ACK:

Ammonium–chloride–potassium

APC:

Antigen-presenting cell

CCR7:

C–C chemokine receptor type 7

CTL:

Cytotoxic T lymphocyte

CTLA4:

Cytotoxic T-lymphocyte-associated protein 4

DMEM:

Dulbecco modified Eagle medium

EDTA:

Ethylenediaminetetraacetic acid

ICI:

Immune check-point inhibitor

IFA:

Incomplete Freund’s adjuvant

IFNγ:

Interferon gamma

LAG3:

Lymphocyte-activation gene 3

MHC:

Major histocompatibility complex

PBS:

Phosphate-buffered saline

PD1:

Programmed cell death 1

qRT-PCR:

Quantitative real-time reverse transcriptase–polymerase chain reaction

RNA:

Ribonucleic acid

SEM:

Standard error of the mean

SFC:

Spot-forming cell

TAA:

Tumor-associated antigens

TIL:

Tumor-infiltrating lymphocyte

TIM3:

T-cell immunoglobulin mucin-3

TLR9:

Toll-like receptor 9

Treg:

Regulatory T cells

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Acknowledgments

The authors thank the Association Oligocyte and the Association pour le development des neurosciences a Avicenne (ADNA) for supporting our research.

Funding

No funding sources were used for this report.

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Authors and Affiliations

  1. PARCC, INSERM, UMR-970, Université de Paris, 56 rue Leblanc, 75015, Paris, France

    Stefania Cuzzubbo, Thi Tran, Corinne Tanchot, Eric Tartour & Antoine F. Carpentier

  2. Laboratoire de Recherches Biochirurgicales (Fondation Carpentier), Assistance Publique-Hôpitaux de Paris (AP-HP), Hôpital Européen Georges Pompidou, 75015, Paris, France

    Claire Banissi, Marie Sophie Rouchon & Antoine F. Carpentier

  3. Service d’Immunologie Biologique, Assistance Publique-Hôpitaux de Paris (AP-HP), Hôpital Européen Georges Pompidou, 75015, Paris, France

    Eric Tartour

  4. Service de Neurologie, Assistance Publique-Hôpitaux de Paris (AP-HP), Hôpital Saint-Louis, 75010, Paris, France

    Antoine F. Carpentier

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  1. Stefania Cuzzubbo
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Contributions

SC, CT, ET and AFC contributed to the study concept and design of experiments. SC, CB, MSR and TT performed the experiments. CB and TT helped in methodology and software analysis. SC and CB were involved in data analysis and interpretation of results. SC and AFC wrote the preliminary version of the paper. All authors participated in critical review and revision of the final manuscript.

Corresponding author

Correspondence to Stefania Cuzzubbo.

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Conflict of interest

The AP/HP (Assistance Publique de Hopitaux de Paris) filed a provisional patent application on this method. AF Carpentier & C Banissi are listed as inventors. AF Carpentier holds shares in Altevax inc. and is consultant for BMS. The authors declare that there are no other conflicts of interest.

Ethical approval and ethical standards.

Experiments were conducted on female C57BL/6 mice. All animal experiments were approved by the ethics committee of Paris Descartes University (Project APAFIS #5337 N° 2016021517305775) and performed in accordance with European Union guidelines for animal experiments.

Human and animal rights

Animal source: Female C57BL/6 aged 5 to 6 week old were purchased from Janvier Labs (Le Genest-Saint-Isle, France) and kept under specific-pathogen-free conditions.

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Published as a poster abstract at the 5th Immunotherapy of Cancer Conference: [1]

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Cuzzubbo, S., Banissi, C., Rouchon, M.S. et al. The adjuvant effect of melanin is superior to incomplete Freund’s adjuvant in subunit/peptide vaccines in mice. Cancer Immunol Immunother 69, 2501–2512 (2020). https://doi.org/10.1007/s00262-020-02631-7

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