# A nanobody-stem cell platform targeting innate and adaptive immune axis in the tumour microenvironment

## TL;DR

A new cell-based platform using nanobodies and stem cells targets immune suppression in tumors, potentially improving cancer immunotherapy.

## Contribution

A novel cell-based platform using nanobodies and stem cells to target both innate and adaptive immune suppression in the tumor microenvironment.

## Key findings

- Biparatopic nanobodies against CSF-1R and PD-1 showed superior efficacy in reprogramming tumor-associated macrophages and T cells.
- Stem cells releasing these nanobodies reduced tumor growth and enhanced immune activation in mouse models.
- The platform demonstrated therapeutic efficacy in a glioblastoma model post-surgery.

## Abstract

Tumour associated macrophages (TAMs) and exhausted T cells are dominant in the immuno-suppressive tumour microenvironment (TME) and pose a challenge to effective cancer immunotherapy in solid tumours.

In this study, we immunised llamas and generated monovalent and biparatopic nanobodies (Nbs) against colony stimulating factor 1 receptor (CSF-1R) and programmed death receptor 1 (PD-1) to re-educate TAMs and overcome T cell exhaustion, respectively, in the TME. To circumvent short systemic half-life and low peak concentrations of Nbs in the TME, we developed a platform of allogenic off-the-shelf stem cells (SC) releasing biparatopic PD-1 and CSF-1R Nbs and tested its efficacy in different mouse models.

Nbs targeting CSF-1R and PD-1 inhibited the CSF1/CSF-1R and PD-1/PD-L1 pathways, respectively, with biparatopic Nbs demonstrating superior efficacy and functionality compared with their monovalent counterparts. Locoregional SC mediated release of biparatopic CSF-1R and PD-1 Nbs, reduced tumour growth by increasing T cell numbers, enhancing T cell activation, and by shifting the macrophage polarisation towards a pro-inflammatory phenotype. Moreover, the presence of both Nbs improved dendritic cells (DCs) activation within the TME. Finally, we show that biocompatible gel encapsulated SC releasing Nbs against PD-1 and CSF-1R have therapeutic efficacy in a highly immunosuppressive glioblastoma model post-tumour resection.

Taken together, our findings establish a cell-based Nb platform targeting both innate and adaptive immune axis within the TME, which has the potential to facilitate treatment of solid cancers that are otherwise refractory to conventional immunotherapies.

This study was mainly supported by Institutional Funds (K.S). A part of in vivo Nb characterisation was supported by 10.13039/100000002NIH grant R01-CA285519 (K.S.) and the Nb dye conjugation and modelling was supported by 10.13039/100000002NIH grant R01-AI165666 (M.R).