Impact of Polymer-TLR-7/8 Agonist (Adjuvant) Morphology on the Potency and Mechanism of CD8 T Cell Induction

Abstract

Small molecule Toll-like receptor-7 and -8 agonists (TLR-7/8a) can be used as vaccine adjuvants to induce CD8 T cell immunity but require formulations that prevent systemic toxicity and focus adjuvant activity in lymphoid tissues. Here, we covalently attached TLR-7/8a to polymers of varying composition, chain architecture and hydrodynamic behavior (∼300 nm submicrometer particles, ∼10 nm micelles and ∼4 nm flexible random coils) and evaluated how these parameters of polymer-TLR-7/8a conjugates impact adjuvant activity in vivo. Attachment of TLR-7/8a to any of the polymer compositions resulted in a nearly 10-fold reduction in systemic cytokines (toxicity). Moreover, both lymph node cytokine production and the magnitude of CD8 T cells induced against protein antigen increased with increasing polymer-TLR-7/8a hydrodynamic radius, with the submicrometer particle inducing the highest magnitude responses. Notably, CD8 T cell responses induced by polymer-TLR-7/8a were dependent on CCR2+ monocytes and IL-12, whereas responses by a small molecule TLR-7/8a that unexpectedly persisted in vaccine-site draining lymph nodes (T1/2 = 15 h) had less dependence on monocytes and IL-12 but required Type I IFNs. This study shows how modular properties of synthetic adjuvants can be chemically programmed to alter immunity in vivo through distinct immunological mechanisms.

Nanoparticles Presenting Potent TLR7/8 Agonists Enhance Anti-PD-L1 Immunotherapy in Cancer Treatment

“… 28 − 32 Moreover, recent studies of TLR7/8a-functional macromolecular constructs indicate that mannose functionalization can increase recognition and internalization by mannose-binding C-type lectins. 14 , 33 We therefore hypothesized that introduction of mannose to the NP surface alongside TLR7/8a may improve NP uptake and therefore receptor activation. 14 , 33 We used RAW-Blue transgenic mouse macrophage reporter cells (Invivogen) to evaluate the potency of interferon regulatory factor (IRF) activation by TLR7/8a-tethered NPs.…”

“… 14 , 33 We therefore hypothesized that introduction of mannose to the NP surface alongside TLR7/8a may improve NP uptake and therefore receptor activation. 14 , 33 We used RAW-Blue transgenic mouse macrophage reporter cells (Invivogen) to evaluate the potency of interferon regulatory factor (IRF) activation by TLR7/8a-tethered NPs. In these assays, the cells were incubated with TLR7/8a at a range of concentrations (0.08–10 μg/mL) either in free form or tethered to PEG–PLA NPs (TLR7/8a NPs) at different densities to generate concentration-dependent activation curves.…”

“…For example, Lynn et al found that the morphology of NPs formed from TLR7/8a-conjugated polymers had a significant impact on both induction of cytotoxic T cells and antibody production against a co-presented antigen. 13 , 14 Nuhn et al have likewise shown that pH-responsive polymeric micelles with conjugated TLR7/8a ligands are effective in eliciting a local immune response with low toxicity that synergizes with PD-L1 antibody antagonists. 15 , 16 …”

Peptide–TLR-7/8a conjugate vaccines chemically programmed for nanoparticle self-assembly enhance CD8 T-cell immunity to tumor antigens

“…1a). The LPs were then administered to mice alone or in combination with an imidazoquinoline-based TLR-7/8a as a source of adjuvant, which was either covalently attached to the LPs or provided as a particle (PP-7/8a) 33,34 admixed with the LPs ( Fig. 1a and Supplementary Fig.…”

Determining Whether Agonist Density or Agonist Number Is More Important for Immune Activation via Micoparticle Based Assay

“…While this evidence strongly supports agonist number, it does not rule out that particle density can influence immune responses in other ways. TLR agonist density alters downstream responses including Th1/Th2 bias, CD8 T-cell responses, and Fc receptor density (10,(22)(23)(24). Taking into account these finding, we propose a potential two-step process in which a cell is activated and then uses density and concentration to inform the type of response that develops.…”