(J-386) Efficacy of Sublingually Delivered IL-1β3 and TNF Dual Cytokine Immunotherapy

Currently, many diseases are treated with monoclonal antibodies or small molecule drugs that are intended to neutralize pathogenic cytokines, such as those involved in chronic inflammatory pathways [1]. However, these therapies come with notable disadvantages such as the high required frequency of dosage and the potential for development of anti-drug antibodies, which diminish treatment efficacy over time [2, 3]. These drawbacks have driven the emergence of research into active immunotherapies with cytokines, which have the potential to limit production of anti-drug antibodies and neutralize multiple sources that contribute to pathology [4, 5, 6].

Moreover, traditional therapies are primarily delivered via parenteral routes of administration such as subcutaneous or intravenous and require the use of needles, which is associated with challenges including poor patient compliance and undesirable effects or responses at the injection site. The development of alternative needle-free delivery approaches, such as sublingual delivery is therefore highly motivated. Sublingual delivery with supramolecular nanofibers has been leveraged by the Collier lab for painless and straightforward administration of vaccines to raise mucosal and systemic responses [7, 8].

The objective of this study was therefore twofold: to design multi-epitope peptide vaccines capable of targeting both TNF and IL-1β, and secondly, to investigate the efficacy of these dual cytokine formulations via sublingual delivery as compared to subcutaneous delivery. As key inflammatory regulators, IL-1β and TNF cytokines were selected for such development and validation of peptide-based dual cytokine immunotherapies that produce anti-IL-1β and anti-TNF immune responses when sublingually delivered. Overall, these results will not only contribute to furthering our understanding of IL-1β and TNF blockade therapy for treatment of chronic inflammation but will also enable the design of needle-free strategies for active immunotherapy in other inflammatory conditions.

Peptides were synthesized on a Liberty Blue peptide synthesizer using standard Fmoc chemistry, then purified using high performance liquid chromatography. Peptides were characterized using matrix-assisted laser desorption ionization mass spectroscopy and Transmission Electron Microscopy. Sublingual immunizations for groups 1-3 were prepared by dissolving lyophilized peptides (along with c-di-AMP adjuvant) in sterile water to a concentration of 5 mM. Immunizations for group 4 were first dry mixed for 30 minutes, then dissolved in sterile water to 8 mM and stored at 4°C overnight. Three hours before administration, subcutaneous immunizations were brought to a concentration of 2 mM in 1X PBS by adding sterile water and 10X PBS, then incubated at room temperature until delivery.

Four experimental groups, each containing five C57BL/6 female mice, were immunized with IL-1β3 and/or TNF, conjugated to the Coil-29 nanofiber platform. Group 1 received TNF-C29, group 2 received IL-1β3-C29, and groups 3 and 4 received co-assembled IL-1β3-C29 and TNF-C29. Groups 1-3 received sublingual delivery of 8 µL of immunization, while group 4 mice were given two 50 µL injections subcutaneously above the shoulders. All groups received initial immunizations at week 0, with boosts of the same formulation at weeks 3, 6 and 9. Blood was collected at weeks 1, 4, 7, and 10 to evaluate serum IgG and IgA responses against IL-1β3 and TNF using ELISA. All mice were euthanized at week 10, and spleens were collected for ELISpot.

Initial responses to subcutaneously delivered immunizations were greater than or equal to responses elicited by sublingual groups (Fig. 2). However, after the third boost, responses to all sublingual groups were higher than responses to the subcutaneous group, demonstrating possible advantages in treatment efficacy may be achieved by sublingual delivery. Additionally, while the single epitope and dual epitope sublingual immunizations elicited similar responses after the third boost, responses to TNF in the combination group were visible after only the second boost and closely mimicked responses from the monoclonal TNF group across all points. Contrarily, responses to IL-1β3, despite still mimicking the trends of the monoclonal IL-1β3 immunization, were consistently lower across all points. These observations suggest that while dual cytokine treatments may be as effective as monoclonal therapies, responses to each constituent epitope take effect at different points in time.

Evaluating IgG subclasses, it was shown that single epitope formulations elicited higher IgG2b responses relative to other IgG subclasses, while combination immunizations elicited comparatively balanced responses across IgG1 and IgG2b (Fig. 3). Additionally, the subcutaneous immunizations showed the greatest responses to IgG1, yet elicited relatively low responses for the remaining subclasses, indicating a general systemic rather than a specific response. 

Finally, ELISpot indicated that sublingual groups containing TNF showed relatively balanced Th1 and Th2 biasing responses (Fig. 5), which is concurrent with findings from previous studies involving the use of c-di-AMP adjuvants [9]. Unexpectedly, however, the group that received IL-1β3-C29 showed a Th2-slanted response. Despite these unexpected results, it is important to note that all sublingually delivered groups elicited higher responses than the subcutaneously delivered immunizations.

These results are important to the verification of sublingually delivered dual cytokine therapies as effective treatments against disease, such as chronic inflammatory diseases which are the number one threat to human health according to the World Health Organization [10]. Future studies may investigate the ability to use the sublingual pathway, as well as other mucosal delivery routes such as intranasal, to ultimately develop clinical treatments that leverage increased bioavailability of therapies, lower required dosages, and non-invasive delivery to improve patient outcomes [11].

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