Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy.

Lymphoid organs, in which antigen presenting cells (APCs) are in close proximity to T cells, are the ideal microenvironment for efficient priming and amplification of T-cell responses. However, the systemic delivery of vaccine antigens into dendritic cells (DCs) is hampered by various technical challenges. Here we show that DCs can be targeted precisely and effectively in vivo using intravenously administered RNA-lipoplexes (RNA-LPX) based on well-known lipid carriers by optimally adjusting net charge, without the need for functionalization of particles with molecular ligands. The LPX protects RNA from extracellular ribonucleases and mediates its efficient uptake and expression of the encoded antigen by DC populations and macrophages in various lymphoid compartments. RNA-LPX triggers interferon-α (IFNα) release by plasmacytoid DCs and macrophages. Consequently, DC maturation in situ and inflammatory immune mechanisms reminiscent of those in the early systemic phase of viral infection are activated. We show that RNA-LPX encoding viral or mutant neo-antigens or endogenous self-antigens induce strong effector and memory T-cell responses, and mediate potent IFNα-dependent rejection of progressive tumours. A phase I dose-escalation trial testing RNA-LPX that encode shared tumour antigens is ongoing. In the first three melanoma patients treated at a low-dose level, IFNα and strong antigen-specific T-cell responses were induced, supporting the identified mode of action and potency. As any polypeptide-based antigen can be encoded as RNA, RNA-LPX represent a universally applicable vaccine class for systemic DC targeting and synchronized induction of both highly potent adaptive as well as type-I-IFN-mediated innate immune mechanisms for cancer immunotherapy.

Uptake of synthetic naked RNA by skin-resident dendritic cells via macropinocytosis allows antigen expression and induction of T-cell responses in mice.

Intradermal administration of antigen-encoding RNA has entered clinical testing for cancer vaccination. However, insight into the underlying mechanism of RNA uptake, translation and antigen presentation is still limited. Utilizing pharmacologically optimized naked RNA, the dose-response kinetics revealed a rise in reporter signal with increasing RNA amounts and a prolonged RNA translation of reporter protein up to 30 days after intradermal injection. Dendritic cells (DCs) in the dermis were shown to engulf RNA, and the signal arising from the reporter RNA was significantly diminished after DC depletion. Macropinocytosis was relevant for intradermal RNA uptake and translation in vitro and in vivo. By combining intradermal RNA vaccination and inhibition of macropinocytosis, we show that effective priming of antigen-specific CD8(+) T-cells also relies on this uptake mechanism. This report demonstrates that direct antigen translation by dermal DCs after intradermal naked RNA vaccination is relevant for efficient priming of antigen-specific T-cells.

A non-functional neoepitope specific CD8+ T-cell response induced by tumor derived antigen exposure in vivo.

Cancer-associated mutations, mostly single nucleotide variations, can act as neoepitopes and prime targets for effective anti-cancer T-cell immunity. T cells recognizing cancer mutations are critical for the clinical activity of immune checkpoint blockade (ICB) and they are potent vaccine antigens. High frequencies of mutation-specific T cells are rarely spontaneously induced. Hence, therapies that broaden the tumor specific T-cell response are of interest. Here, we analyzed neoepitope-specific CD8+ T-cell responses mounted either spontaneously or after immunotherapy regimens, which induce local tumor inflammation and cell death, in mice bearing tumors of the widely used colon carcinoma cell line CT26. A comprehensive immune reactivity screening of 2474 peptides covering 628 transcribed CT26 point mutations was conducted. All tested treatment regimens were found to induce a single significant CD8+ T-cell response against a non-synonymous D733A point mutation in the Smc3 gene. Surprisingly, even though Smc3 D733A turned out to be the immune-dominant neoepitope in CT26 tumor bearing mice, neither T cells specific for this neoepitope nor their T cell receptors (TCRs) were able to recognize or lyse tumor cells. Moreover, vaccination with the D733A neoepitope did not result in anti-tumoral activity despite induction of specific T cells. This is to our knowledge the first report that neoepitope specific CD8+ T cells primed by tumor-released antigen exposure in vivo can be functionally irrelevant.

Improvement of In Vivo Expression of Genes Delivered by Self-Amplifying RNA Using Vaccinia Virus Immune Evasion Proteins.

Among nucleic acid-based delivery platforms, self-amplifying RNA (saRNA) vectors are of increasing interest for applications such as transient expression of recombinant proteins and vaccination. saRNA is safe and, due to its capability to amplify intracellularly, high protein levels can be produced from even minute amounts of transfected templates. However, it is an obstacle to full exploitation of this platform that saRNA induces a strong innate host immune response. In transfected cells, pattern recognition receptors sense double-stranded RNA intermediates and via activation of protein kinase R (PKR) and interferon signaling initiate host defense measures including a translational shutdown. To reduce pattern recognition receptor stimulation and unleash suppressed saRNA translation, this study co-delivered non-replicating mRNA encoding vaccinia virus immune evasion proteins E3, K3, and B18. It was shown that E3 is far superior to K3 or B18 as a highly potent blocker of PKR activation and of interferon (IFN)-ß upregulation. B18, in contrast, is superior in controlling OAS1, a key IFN-inducible gene involved in viral RNA degradation. By combining all three vaccinia proteins, the study achieved significant suppression of PKR and IFN pathway activation in vitro and enhanced expression of saRNA-encoded genes of interest both in vitro and in vivo. This approach promises to overcome key hurdles of saRNA gene delivery. Its application may improve the bioavailability of the encoded protein, and reduce the effective dose and correspondingly the cost of goods of manufacture in the various fields where saRNA utilization is envisioned.

FLT3 Ligand as a Molecular Adjuvant for Naked RNA Vaccines.

Intranodal immunization with antigen-encoding naked mRNA has proven to be an efficacious and safe approach to induce antitumor immunity. Thanks to its unique characteristics, mRNA can act not only as a source for antigen but also as an adjuvant for activation of the immune system. The search for additional adjuvants that can be combined with mRNA to further improve the potency of the immunization revealed Fms-like tyrosine kinase 3 (FLT3) ligand as a potent candidate. Systemic administration of the dendritic cell-activating FLT3 ligand prior to or along with mRNA immunization-enhanced priming and expansion of antigen-specific CD8(+) T cells in lymphoid organs, T-cell homing into melanoma tumors, and therapeutic activity of the intranodally administered mRNA. Both compounds demonstrate a successful combination in terms of boosting the immune response. This chapter describes methods for intranodal immunization with naked mRNA by co-administration of FLT3 ligand, which leads to strong synergistic effects.

Antitumor vaccination with synthetic mRNA: strategies for in vitro and in vivo preclinical studies.

Synthetic antigen-encoding mRNA is increasingly exploited as a tool for delivery of genetic information of complete antigens into professional antigen presenting dendritic cells for HLA haplotype-independent antigen-specific vaccination against cancer. Two strategies for mRNA-based antitumor vaccination have emerged into the clinical setting. One is transfection of autologous dendritic cells with synthetic mRNA for adoptive transfer into the patient. The other is direct injection of naked synthetic mRNA. Both methods have proven to be feasible and safe and to elicit antigen-specific immune responses. The design of novel synthetic vaccines employing synthetic mRNA requires further in-depth investigation of its bioavailability and immune pharmacology. This chapter summarizes the state-of-art in this field and describes methods elementary for preclinical studies of mRNA-based antitumor vaccine protocols.

mTOR inhibition improves antitumor effects of vaccination with antigen-encoding RNA.

Vaccination with in vitro transcribed RNA encoding tumor antigens is an emerging approach in cancer immunotherapy. Attempting to further improve RNA vaccine efficacy, we have explored combining RNA with immunomodulators such as rapamycin. Rapamycin, the inhibitor of mTOR, was used originally for immunosuppression. Recent reports in mouse systems, however, suggest that mTOR inhibition may enhance the formation and differentiation of the memory CD8(+) T-cell pool. Because memory T-cell formation is critical to the outcome of vaccination approaches, we studied the impact of rapamycin on the in vivo primed RNA vaccine-induced immune response using the chicken ovalbumin-expressing B16 melanoma model in C57BL/6 mice. Our data show that treatment with rapamycin at the effector-to-memory transition phase skews the vaccine-induced immune response toward the formation of a quantitatively and qualitatively superior memory pool and results in a better recall response. Tumor-infiltrating immune cells from these mice display a favorable ratio of effector versus suppressor cell populations. Survival of mice treated with the combined regimen of RNA vaccination with rapamycin is significantly longer (91.5 days) than that in the control groups receiving only one of these compounds (32 and 46 days, respectively). Our findings indicate that rapamycin enhances therapeutic efficacy of antigen-specific CD8(+) T cells induced by RNA vaccination, and we propose further clinical exploration of rapamycin as a component of immunotherapeutic regimens.

Exploiting the mutanome for tumor vaccination.

Multiple genetic events and subsequent clonal evolution drive carcinogenesis, making disease elimination with single-targeted drugs difficult. The multiplicity of gene mutations derived from clonal heterogeneity therefore represents an ideal setting for multiepitope tumor vaccination. Here, we used next generation sequencing exome resequencing to identify 962 nonsynonymous somatic point mutations in B16F10 murine melanoma cells, with 563 of those mutations in expressed genes. Potential driver mutations occurred in classical tumor suppressor genes and genes involved in proto-oncogenic signaling pathways that control cell proliferation, adhesion, migration, and apoptosis. Aim1 and Trrap mutations known to be altered in human melanoma were included among those found. The immunogenicity and specificity of 50 validated mutations was determined by immunizing mice with long peptides encoding the mutated epitopes. One-third of these peptides were found to be immunogenic, with 60% in this group eliciting immune responses directed preferentially against the mutated sequence as compared with the wild-type sequence. In tumor transplant models, peptide immunization conferred in vivo tumor control in protective and therapeutic settings, thereby qualifying mutated epitopes that include single amino acid substitutions as effective vaccines. Together, our findings provide a comprehensive picture of the mutanome of B16F10 melanoma which is used widely in immunotherapy studies. In addition, they offer insight into the extent of the immunogenicity of nonsynonymous base substitution mutations. Lastly, they argue that the use of deep sequencing to systematically analyze immunogenicity mutations may pave the way for individualized immunotherapy of cancer patients.

Tumor vaccination using messenger RNA: prospects of a future therapy.

While the endeavor to vaccinate against cancer has been pursued for over 20 years, only recently was the first tumor vaccine approved. Among the different antigen formats assessed for vaccination, coding messenger RNA (mRNA) is emerging as a particularly attractive option. It can code for all types of transcript based proteins, is easy and cost efficient to produce, has a favorable safety profile and enables induction of combined immune responses. Within the last few years major developments have been achieved in this field. Clinical approaches use mRNA either for direct administration or for engineering of adoptively transferred dendritic cells. However, there are still challenges to be overcome for successful clinical application of mRNA-based immunotherapies.

FLT3 ligand enhances the cancer therapeutic potency of naked RNA vaccines.

Intranodal immunization with antigen-encoding naked RNA may offer a simple and safe approach to induce antitumor immunity. RNA taken up by nodal dendritic cells (DC) coactivates toll-like receptor (TLR) signaling that will prime and expand antigen-specific T cells. In this study, we show that RNA vaccination can be optimized by coadministration of the DC-activating Fms-like tyrosine kinase 3 (FLT3) ligand as an effective adjuvant. Systemic administration of FLT3 ligand prior to immunization enhanced priming and expansion of antigen-specific CD8(+) T cells in lymphoid organs, T-cell homing into melanoma tumors, and therapeutic activity of the intranodal RNA. Unexpectedly, plasmacytoid DCs (pDC) were found to be essential for the adjuvant effect of FLT3 ligand and they were systemically expanded together with conventional DCs after treatment. In response to FLT3 ligand, pDCs maintained an immature phenotype, internalized RNA, and presented the RNA-encoded antigen for efficient induction of antigen-specific CD8(+) T-cell responses. Coadministration of FLT3 ligand with RNA vaccination achieved remarkable cure rates and survival of mice with advanced melanoma. Our findings show how to improve the simple and safe strategy offered by RNA vaccines for cancer immunotherapy.

Highly specific auto-antibodies against claudin-18 isoform 2 induced by a chimeric HBcAg virus-like particle vaccine kill tumor cells and inhibit the growth of lung metastases.

Strategies for antibody-mediated cancer immunotherapy, such as active immunization with virus-like particle (VLP)-based vaccines, are gaining increasing attention. We developed chimeric hepatitis B virus core antigen (HBcAg)-VLPs that display a surface epitope of the highly selective tumor-associated cell lineage marker claudin-18 isoform 2 (CLDN18.2) flanked by a mobility-increasing linker. Auto-antibodies elicited by immunization with these chimeric HBcAg-VLPs in 2 relevant species (mouse and rabbit) bind with high precision to native CLDN18.2 at physiologic densities on the surface of living cells but not to the corresponding epitope of the CLDN18.1 splice variant that differs by merely one amino acid. The induced auto-antibodies are capable of efficiently killing CLDN18.2 expressing cells in vitro by complement-dependent and antibody-dependent cell-mediated cytotoxicity. Moreover, they provide partial protective immunity against the challenge of mice with syngeneic tumor cells stably expressing CLDN18.2. Our study provides a first proof-of-concept that immunization combining VLPs as antigen carriers with specific conformational epitopes of a highly selective differentiation antigen may elicit auto-antibodies with high cytocidal and tumoricidal potential.

Intranodal vaccination with naked antigen-encoding RNA elicits potent prophylactic and therapeutic antitumoral immunity.

Although naked antigen-encoding RNA has entered clinical testing, basic knowledge on how to apply this promising novel vaccine format is still pending. By comparing different administration routes, we observed surprisingly potent antigen-specific T-cell immunity upon intranodal injection of naked antigen-encoding RNA. RNA was selectively uptaken by resident dendritic cells, propagated a T-cell attracting and stimulatory intralymphatic milieu, and led to efficient expansion of antigen-specific CD8+ as well as CD4+ T cells. By intranodal treatment of mice with repeated cycles of RNA, we achieved de novo priming of naïve T cells, which became potent cytolytic effectors capable of homing to primary and secondary lymphatic tissues as well as memory T cells. In tumor-bearing mice intralymphatic RNA vaccination elicited protective and therapeutic antitumor immune responses, resulting in a remarkable survival benefit as compared with other treatment regimens. This is the first report of strong systemic antigen-specific Th1-type immunity and cancer cure achieved with naked antigen-encoding RNA in preclinical animal models.

Increased antigen presentation efficiency by coupling antigens to MHC class I trafficking signals.

Genetic modification of vaccines by linking the Ag to lysosomal or endosomal targeting signals has been used to route Ags into MHC class II processing compartments for improvement of CD4+ T cell responses. We report in this study that combining an N-terminal leader peptide with an MHC class I trafficking signal (MITD) attached to the C terminus of the Ag strongly improves the presentation of MHC class I and class II epitopes in human and murine dendritic cells (DCs). Such chimeric fusion proteins display a maturation state-dependent subcellular distribution pattern in immature and mature DCs, mimicking the dynamic trafficking properties of MHC molecules. T cell response analysis in vitro and in mice immunized with DCs transfected with Ag-encoding RNA showed that MITD fusion proteins have a profoundly higher stimulatory capacity than wild-type controls. This results in efficient expansion of Ag-specific CD8+ and CD4+ T cells and improved effector functions. We used CMVpp65 and NY-ESO-1 Ags to study preformed immune responses in CMV-seropositive individuals and cancer patients. We show that linking these Ags to the MITD trafficking signal allows simultaneous, polyepitopic expansion of CD8+ and CD4+ T cells, resulting in distinct CD8+ T cell specificities and a surprisingly broad and variable Ag-specific CD4+ repertoire in different individuals.

Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells.

Adoptive transfer of dendritic cells (DCs) transfected with in vitro-transcribed, RNA-encoding, tumor-associated antigens has recently entered clinical testing as a promising approach for cancer immunotherapy. However, pharmacokinetic exploration of RNA as a potential drug compound and a key aspect of clinical development is still pending. While investigating the impact of different structural modifications of RNA molecules on the kinetics of the encoded protein in DCs, we identified components located 3' of the coding region that contributed to a higher transcript stability and translational efficiency. With the use of quantitative reverse transcription-polymerase chain reaction (RT-PCR) and eGFP variants to measure transcript amounts and protein yield, we showed that a poly(A) tail measuring 120 nucleotides compared with a shorter one, an unmasked poly(A) tail with a free 3' end rather than one extended with unrelated nucleotides, and 2 sequential beta-globin 3' untranslated regions cloned head to tail between the coding region and the poly(A) tail each independently enhanced RNA stability and translational efficiency. Consecutively, the density of antigen-specific peptide/MHC complexes on the transfected cells and their potency to stimulate and expand antigen-specific CD4+ and CD8+ T cells were also increased. In summary, our data provide a strategy for optimizing RNA-transfected DC vaccines and a basis for defining release criteria for such vaccine preparations.