Alu RNAs show Efficacy as Cancer Vaccine Adjuvants

Introduction: Cancer vaccines seek to mobilize existing CD8+ T cells and generate new antigen specific CD8+ T cells to combat tumors. Current cancer vaccines have had limited success due to low immunogenicity of cancer antigens, poor adjuvants, and inefficient carrier systems. One potential adjuvant strategy is to exploit the innate immune system's RNA sensing pathways through pattern recognition receptors (PRRs), which can activate pro-inflammatory signaling cascades. To avoid autoreactivity to self RNA, processing methods such as A-to-I editing keep RNA inert. When A to I editing is defunct, detection of RNA results in type I interferon and NF-κB pro-inflammatory responses. Lack of A to I editing is implicated in autoimmune diseases where PRRs recognize RNA derived from endogenous Alu elements. Alu elements are short interspersed nuclear elements commonly found in introns or 3’ and 5’ untranslated regions and makeup 10% of the human genome. There are multiple Alu RNAs, including AluJb which can activate type I interferon and NF-ĸB, and Alu5 that only activates type I interferon. Localized pharmacological activation of PRRs via unedited Alu RNA may be beneficial as adjuvants in new vaccine strategies to induce antigen specific CD8+ T cells. Unfortunately, RNA requires a carrier due to poor internalization, and when internalized, it is degraded in the endosome before it can reach its receptor in the cytosol. Herein, we show that Alu adjuvants delivered via a pH-responsive polymer-based nanoparticle (NP) delivery system can effectively deliver Alu RNA to induce an antigen-specific CD8+ T cell response.

Materials and

Methods: Polymer Synthesis and Characterization: Reversible addition-fragmentation chain transfer polymerization was used to formulate diblock copolymer poly [(polyethylene glycol) methacrylate)0.9-co-(pyridyl disulfide ethyl methacrylate)0.1]13.7kDa-block- [(dimethylamino ethyl methacrylate)0.5-co-(butyl methacrylate)0.5]25kDa (PEGMA-co-PDSMA-DB). 1H nuclear magnetic resonance (NMR) was used to calculate Polymer composition, degree of polymerization, and theoretical molecular weight. To form NPs polymer was dissolved in 200 proof ethanol (50 mg/mL), diluted to 10 mg/mL in PBS, then concentrated via centrifugal dialysis. OVA was thiolated using thiol-disulfide exchange reactions then conjugated to NPs. RNA was complexed to NPs in citrate buffer at pH=4 for 45 minutes at room temperature after which the pH was raised with 3x the volume of PBS. RNA NPs were quantified for size via dynamic light scattering (DLS) and complexation via gel electrophoresis.
In vitro experiments: The activity of RNA-NPs was assessed using THP1-Dual (human monocytes) and A549-Dual (human lung carcinoma) reporter cell lines. 50,000 cells were plated, and 24 hours later, cells were treated with Alu5, AluJb, or Poly(I:C) NPs. 24 hours after treatment, supernatants were collected for Quanti-luc and Quanti-blue assays, which are readouts of type I interferon and Nf-ĸb production, respectively.
In vivo experiments: Mice were treated subcutaneously in the center of the back on days 0, 7, and 14 with OVA-NPs complexed with 30 µg of Alu5, AluJb, or Poly(I:C). Mice were euthanized on day 21 for tetramer response and antibody titer. Alternatively, on day 38, mice were inoculated with 1,000,000 MC38-OVA cells on the right flank to determine if vaccination provides a protective effect.

Results, Conclusions, and Discussions: 1H NMR showed PEGMA-co-PDSMA block was approximately 13 kDa, while the DB block was approximately 25 kDa resulting in a 38 kDa polymer. Analysis via DLS revealed diameters of 59, 43, and 39 for Poly(I:C), AluJb, and Alu5, respectively. Gel electrophoresis displayed elevated levels of Alu complexation and OVA conjugation. In vitro analysis revealed that AluJb was the most potent activator of THP1-Dual and A549-Dual cells with the highest levels of both type I interferon and Nf-ĸb, while Alu5 performed similarly to Poly(I:C). For the duration of the vaccination regime, there was no significant weight loss with any of the adjuvants, suggesting administering our vaccine does not result in systemic toxicity. Evaluation of OVA tetramer response in the spleens of mice on day 21 post vaccine initiation, Alu5, AluJb, and Poly(I:C) NP treatments all elicited a stronger response than PBS treated control mice (Figure 1A). By increasing the frequency of antigen specific CD8+ T cells in the spleen, this may leave mice better equipped when challenged with cancer cells. Interestingly, there were no differences between the IgG2c antibody titer, but Alu5 NPs had a significantly higher IgG11 titer than Poly(I:C) NPs (Fig. 1B-C). Additionally, there were no significant differences between adjuvants when comparing tumor growth or survival following the MC38-OVA tumor challenge, but all adjuvants prolonged survival compared to PBS-treated control mice and unadjuvanted OVA-NPs (Fig. 1D-E). These data show that an adjuvant is necessary for efficacy and that Alu RNA’s can perform at a level like a widely used adjuvant. Overall, this work utilizes unique RNA elements as adjuvants to form a new vaccine platform which elicits CD8+ T cell responses and anti-tumor effects.

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