Abstract
BACKGROUND: 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. METHODS: 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. FINDINGS: 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. INTERPRETATION: 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. FUNDING: This study was mainly supported by Institutional Funds (K.S). A part of in vivo Nb characterisation was supported by NIH grant R01-CA285519 (K.S.) and the Nb dye conjugation and modelling was supported by NIH grant R01-AI165666 (M.R).