O7 - Co-Localization of Lymph Node Therapeutics in Lymph Nodes Enhances T Cell Activation and Anti-Tumor Response

Introduction: : Lymph nodes (LNs) are essential tissues in generating and modulating immune response. Direct intra-lymph node (i.LN.) delivery in cancer vaccination and immunotherapy has become of interest owing to the ability to create locally-high doses of these components LNs without systemic or off-target tissue effects. For example, several recent immunotherapy trials involving soluble drugs have switched to direct LN delivery (e.g. Umoja, 2024), while a number of academic medical centers offer LN delivery of allergy shots to accelerate tolerance. Our lab has shown combining this route with diffusion-limited microparticle depots can be used to control retention of antigen and modulatory cues in LNs. These depots are too large to drain from LNs and instead slowly degrade releasing cues into the local LN environment. [1][2] However, the impact of loading and delivering immunotherapy cargos co-loaded in a single particle or in separate particle has not been investigated for LN therapeutics in which cargo are delivered directly to the LN microenvironment. This is important as this scheme offers platform flexibility and dramatically simpler chemistry and manufacturing control (CMC) considerations for regulatory approval. For example, MPs with a single component could be mixed during fill/finish prior to injection. Here we interrogate how the antigen and modulatory cues, when formulated into a single particle or individually into separate particles, alters immune cell response. We hypothesized that direct co-localization of antigen and adjuvant in LNs define therapeutic outcome of cancer vaccination.

Materials and

Methods: : Microparticle (MP) depots were synthesized to encapsulate model antigens (ovalbumin, OVA) and a molecular adjuvant (PolyIC) that activates immune cells. Poly(lactic-co-glycolic acid) (PLGA) MPs were synthesized by double emulsion. Several types of MP formulations were synthesized: (i) MPs containing both PolyIC and OVA, denoted (P+O)MP, (ii) particles containing only PolyIC (PMP), (iii) containing only OVA (OMP), and (iv) mixing PMP and OMP after synthesis (PMP+OMP) at a 1:1 ratio (w/w). (Fig. 1A) Bone-marrow derived dendritic cells (BMDCs) were treated with fluorescently labeled MP formulations to assess cellular uptake of different formulations on a single-cell level. For in vivo studies, OVA-specific CD8+ T cells were isolated from OT-I mice and adoptively transferred to MP treated C57BL/6J mice. Flow cytometry was used to quantify frequency of transferred cells in treated and untreated LNs. For tumor studies, mice were vaccinated with MP formulations then an OVA melanoma model was induced.

Results, Conclusions, and Discussions:: Confocal microscopy confirmed both (P+O)MP and PMP+OMP particle types were internalized and localized within the cells (Fig. 1B). To evaluate impact of T cell responses elicited by different MP formulations, we conducted an adoptive transfer study. In treated LNs, (P+O)MP and PMP+OMP recruited and expanded transferred OVA-specific CD8+ T cells at similar levels, while vehicle control did not (Fig. 1C), indicating the two formulations have similar abilities to induce CD8+ T cell response. In tumor study we further tested our hypothesis that co-localization of PolyIC and OVA in LN define therapeutic outcome of cancer vaccination. In addition to groups receiving MP treatments in one LN, two groups of mice receiving treatments in separate LNs were included in the study. In one group, PMP was delivered to the LN that later became tumor-draining (indicated as “Split LN (tdLN)”), while in the other group, PMP was delivered to the LN that remained non-tumor-draining (indicated as “Split LN (ndLN)”). We demonstrated that, compared with controls, (P+O)MP and PMP+OMP conferred significant tumor suppression in the B16-OVA tumor model. The two Split LN groups showed moderate levels of efficacy compared with vehicle, with no significant differences between each cohort (Fig. 1D). This result indicates that potent, antigen-specific T cell responses and anti-tumor immunity require co-localization of PolyIC and OVA within the same LN. However, as long as both particle types were present in the same LN, efficacy was independent of whether cargo was loaded in a single particle or separately in distinct particles mixed before injection.
Overall, our results indicate co-localization of MPs in LNs is a defining feature for efficacy, irrespective of whether the cargo are in a single particle or two particles in the lymph node. These data help inform design parameters that could simplify design of intra-LN immunotherapies by eliminating the need for complex CMC processes that require particles incorporating multiple immunotherapeutic components.

Acknowledgements and/or References (Optional):: [1] Kapnick, S. M. et al. PNAS. (2024). [2] Gammon, J. M. et al. Nat. Commun. 14, 681 (2023).