# In vitro characterization of cellular responses elicited by endosomal TLR agonists encapsulated in Qβ virus-like particles

## TL;DR

This study compares how different TLR agonists in virus-like particles activate immune cells, finding that TLR7/8a VLPs are most effective at stimulating T cells and NK cells.

## Contribution

The study is the first to compare the immune-activating properties of TLR7a, TLR7/8a, and TLR9a agonists encapsulated in Qβ VLPs.

## Key findings

- TLR7/8a VLPs were most effective at activating NK and T cells compared to TLR7a and TLR9a VLPs.
- TLR7/8a VLP-mediated T cell activation was dependent on direct contact with pDCs.
- Neutralizing TNFα suppressed TLR7/8a VLP-induced T cell activation, but not IFNα.

## Abstract

Despite promising clinical data for Toll-like receptor-9 agonists encapsulated in virus-like particles (TLR9a VLPs), the relative potency and mechanisms of TLR7a and dual TLR7/8a VLPs remain undefined. TLR9a VLPs, also known as Vidutolimod or CMP-001 is a novel TLR9a encapsulated in Qβ VLPs, which can activate plasmacytoid DCs (pDCs) and promote T cell activation. Other endosomal TLRs such as TLR7 and TLR8 expressed in DCs have been studied in several preclinical and clinical studies; however, their immune-activating properties when encapsulated in VLPs have not been tested before. Here, we utilized a series of in vitro experiments to test and compare immune cell activation stimulated by agonists to TLR7 (TLR7a), TLR7/8 (TLR7/8a), and TLR9 (TLR9a) when encapsulated in Qβ VLPs.

Activation of immune cells (monocytes, natural killer (NK) cells, T cells, pDCs, and monocytic DCs (mDCs)) in response to TLR7a, TLR7/8a and TLR9a VLPs, was evaluated using flow cytometry, intracellular cytokine staining (ICS) and ELISA. Neutralizing cytokine antibodies, immune cell depletion kits and transwell models were used to determine the contribution of select cytokines and antigen presenting cells (APCs) in VLP-mediated immune cell activation.

Results showed that all three VLPs activated pDCs and monocytes. However, TLR7/8a VLPs were most effective at NK and T cell activation compared to the other VLPs. NK cells were a major source of IFNγ, whereas pDCs were the main source of IFNα and TNFα production in response to the VLPs. Neutralizing antibodies against TNFα (but not IFNα) showed significant suppressive effects on TLR7/8a VLP-mediated activation of CD4 + and CD8 + T cells. Depletion of APCs completely abrogated TLR7/8 VLP-mediated activation of CD4 + and CD8 + T cells. Lastly, TLR7/8a VLP-mediated activation of T cells was highly dependent on direct contact with pDCs (and not DC1 and DC2 subsets).

In summary, endosomal TLRa VLPs all have the ability to activate pDCs, however, combined TLR7/8 activation using TLR7/8a VLPs was significantly more effective than the other VLPs at activating T cells and was dependent on direct contact with pDCs. Therefore, TLR7/8a VLPs may potentially induce a robust anti-tumor immune response and warrant further investigation for cancer therapy.