MYCN-targeting vaccines and immunotherapeutics

ABSTRACT

Amplification and concomitant overexpression of the MYCN oncogene is a frequent event in many malignancies including the childhood tumors, neuroblastoma and medulloblastoma. MYCN is only expressed in a defined time frame during early developmental processes,¹ which is beneficial for approaches combatting tumor-specific MYCN. However, MYCN is a transcription factors that was considered a poor drug target, until recent approaches suggested that down-regulation of MYCN could be possible by indirect targeting using Aurora kinase inhibitors or BET inhibitors. These concepts were proven using preclinical models^2–6 and are now entering clinical trials.

KEYWORDS:

• DNA vaccination
• minigene
• MYCN
• neuroblastoma

In neuroblastoma (NB), a common solid tumor of childhood, the MYCN oncogene is amplified in _˜20 % of cases.⁷ Following induction chemotherapy and consolidation with high dose chemotherapy and stem cell rescue, maintenance treatment regimens focus on passive immunotherapy by targeting NB-specific expression of the disialoganglioside GD2 with GD2-specific monoclonal antibodies (MAbs). As GD2 is devoid of any intracellular signal transduction component, the mechanism of action of this approach is the induction of complement-dependent (CDC) and antibody-dependent cellular cytotoxicity (ADCC). In this context, treatment with mouse-human chimeric Mab ch14.18 showed superior survival rates when used as a single agent approach⁸ or in combination with IL-2 and GM-CSF.⁹ These two studies from independent cooperative groups demonstrated the potential of targeted immunotherapies in neuroblastoma. Ongoing clinical trials in the context of passive immunotherapy address the role of cytokines as well as the potential of novel delivery methods by long term continuous infusion. The specific components of ch14.18 were also used to design GD2-specific chimeric antigen receptor-engineered (CAR) T cells. These were generated and administered to NB patients in the context of a phase I study. Complete remission was achieved in 3/11 patients with active disease and persistence of CARs in vivo > 6 weeks was found to correlate with clinical outcome.¹⁰ One disadvantage of passive immunotherapy is the absence of long lasting and persistent immunity against the malignancy. Based on the high relapse rate in NB combined with limited strategies for therapeutic intervention new approaches are urgently needed.¹¹

Globally, neuroblastomas escape from destruction by the immune system using a combinatorial strategy involving MYCN-dependent downregulation of MHC molecules¹² and inhibition of NKT cells, which in turn causes up-regulation of tumor-associated macrophages (TAMs,^13,14 Earlier reports also indicated that MYCN-specific cytotoxic T cells (CTLs) are present in neuroblastoma patients harboring tumor-specific MYCN amplification.¹⁵ Surprisingly, little is known on the usefulness of MYCN as a target for cancer immunotherapy.¹⁶ Peptide vaccination using a HLA-A2 restricted peptide derived from MYCN has been shown to effectively induce a cytotoxic T cell response.¹⁷ In principle, vaccination against MYCN as a tumor antigen could be an interesting strategy, especially for those patients with MYCN amplification and thus high MYCN expression.

In a recent paper, DNA vaccination has been investigated as a means to exploit high MYCN expression on tumor cells and thus to overcome MYCN immune-suppressive activities in neuroblastoma.¹⁸ Here, the following improvements over previous attempts were incorporated in the study design: first, use of a “minigene” avoided transfer of a potentially harmful gene sequence to a mammalian host; second, an attenuated S. typhimurium strain (SL7207) was used as DNA delivery vehicle.¹⁹ Immunocompetent mice were immunized with a MYCN minigene displaying high binding affinity to MHC class I H2-K^k by three subsequent oral applications of S. typhimurium (SL7207) carrying the MYCN constructs. Mice were then randomly assigned to one of two groups receiving either mouse NB cells with low and high MYCN expression, respectively, as syngeneic grafts. Tumor volume was significantly reduced by vaccination with a MYCN minigene displaying high affinity to MHC class I H2-K^k in comparison to vaccination with a minigene epitope displaying low MHC class I affinity. Additionally, vaccination with full-length MYCN-cDNA was also less effective in reducing tumor volume. MYCN-DNA vaccination induced a cytotoxic MYCN-specific anti-NB immune response involving IFN- and increased target cell lysis. Interestingly, absence of MYCN in tumor cells abrogated IFN- release. Importantly, no signs of autoimmunity were noted. Ex vivo, splenocytes from MYCN-vaccinated mice receiving MYCN-expressing tumor cells presented with significant higher specific cytotoxicity toward MYCN-high expressing tumor cells or SCK mammary carcinoma cells pulsed with MYCN-peptides.¹⁸

Taken together, strategies to exploit MYCN as a tumor-associated antigen for immune therapy deserve further functional validation. In tumors with high MYCN expression, targeting MYCN could be useful to overcome MYCN-mediated immune suppression. While passive immune therapies in a MYCN-driven disease such as high-risk, MYCN amplified neuroblastoma, are already in clinical use, vaccination strategies have the potential to evoke long lasting effects by inducing a memory immune response. Here, strategies involving multi-peptide cancer vaccines^20–22 might be an attractive route also for MYCN-based therapies. Alternatively, DNA vaccination should be further evaluated as a cost-effective and easy-to-handle option.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

A.S. acknowledges the support of the Deutsche Forschungsgemeinschaft (DFG) within the Collaborative Research Center SFB 876 (http://sfb876.tu-dortmund.de), “Providing Information by Resource-Constrained Analysis”, subproject C1. The funder had no role in decision to publish or preparation of this manuscript.