Abstract
BACKGROUND: Monoclonal antibodies (mAbs) targeting immune checkpoint molecules such as programmed death ligand 1 (PD-L1), which is expressed in both immune and tumour cells, are conventional immunotherapy approaches. Although approved as monotherapy for the first-line treatment of several cancers, mAbs targeting PD-L1 have shown limited efficacy in colorectal cancer (CRC). Here, we investigated if nucleic acids translated into anti-PD-L1 nanobodies (PDL1Nbs) effectively suppress CRC tumourigenesis in mouse models. METHODS: Mice were transplanted with MC-38 mouse sporadic CRC (sCRC) cells or challenged with azoxymethane and dextran sodium sulfate, a combination treatment that induces colitis-associated CRC (CAC). The tumour-bearing mice were treated with a PDL1Nb-encoding plasmid DNA (pDNA) delivered via polymers, or treated with PDL1Nb-encoding nucleoside-modified messenger RNA (PDL1Nb mRNA) delivered via lipid nanoparticles (LNP). Moreover, bone marrow haematopoietic stem cells (BMHSCs) were differentiated and maturated by treating growth factors in the presence of PDL1Nb mRNA-LNP or control luciferase mRNA-LNP with/without lipopolysaccharide. We examined sCRC tumour proliferation and growth, CAC tumour incidences and numbers, tumour infiltration of immune cells and bone marrow-derived macrophages (BMDMs). RESULTS: Polymer delivery of PDL1Nb pDNA efficiently repressed sCRC progression in tumour-bearing mice. Intriguingly, LNP delivery of the quadruple PDL1Nb (qPDL1Nb) mRNA showed a greater efficacy than the delivery of the monomeric PDL1Nb (mPDL1Nb) mRNA in suppressing sCRC tumour progression. Moreover, qPDL1Nb mRNA-LNP treatment significantly reduced CAC incidence. Mechanistically, PD-L1 blockade by qPDL1Nb resulted in marked decreases in tumour-infiltrating myeloid-derived suppressor cells and tumour-associated macrophages, as well as expression of PD-L1, but increases in tumour-infiltrating CD3(+)CD8(+) cells during CAC tumourigenesis. Notably, in vitro LNP delivery of PDL1Nb mRNA into BMHSCs significantly inhibited their differentiation and maturation into BMDMs and strikingly reduced the expression of PD-L1, CD80, CD86 and CD206 in BMDMs. CONCLUSION: These results suggest that the PDL1Nb therapy is effective for both CAC and sCRC and using qPDL1Nb mRNA-LNP is a promising alternative strategy for CRC immunotherapy.