In a significant advancement for cancer immunotherapy, researchers have developed a novel approach using mRNA-encoded nanobodies that effectively combat colorectal cancer in preclinical models.
An original study published in eGastroenterology demonstrates how lipid nanoparticle-delivered mRNA producing anti-PD-L1 nanobodies can suppress tumour growth in both sporadic and colitis-associated colorectal cancer, offering new hope for patients with cancers resistant to conventional immunotherapies.
Colorectal cancer remains the third most common cancer in the United States and the second leading cause of cancer-related deaths. While immunotherapies targeting immune checkpoint molecules like PD-1 and PD-L1 have revolutionised cancer treatment, their effectiveness against colorectal cancer has been limited. Most patients with colorectal cancer have microsatellite stable tumours that respond poorly to these treatments, creating an urgent need for more effective therapeutic strategies.
Conventional monoclonal antibodies used in immunotherapy face several limitations: their large molecular size restricts tumour penetration, they can trigger immune-related adverse effects, and they're costly to produce. Additionally, anti-PD-L1 antibodies have shown disappointing results in treating colitis-associated colorectal cancer, a particularly aggressive form of the disease linked to chronic intestinal inflammation.
This study turned to nanobodies – unique single-domain antibodies originally discovered in camelids and sharks. These miniature antibodies offer significant advantages over conventional antibodies: their small size (approximately 15 kDa versus 150 kDa for conventional antibodies) allows for better tumour penetration, they have lower immunogenicity, and they demonstrate high stability and binding affinity.
However, nanobodies have their own limitation: their small size leads to rapid clearance by the kidneys, shortening their therapeutic window. To address this, the researchers developed a novel quadruple nanobody format, connecting four individual anti-PD-L1 nanobodies with flexible linkers to create a larger molecule that remains in circulation longer while maintaining excellent tissue penetration.
Leveraging technology made famous by COVID-19 vaccines, the team used lipid nanoparticles to deliver nucleoside-modified mRNA encoding these anti-PD-L1 nanobodies. This approach bypasses several limitations of conventional nanobody production, which typically requires complex expression systems in bacteria, yeast, or mammalian cells with risks of toxin contamination and misfolding.
The mRNA-LNP platform enables the body to continuously produce therapeutic nanobodies, maintaining effective levels throughout treatment. The researchers engineered both monomeric and quadruple forms of anti-PD-L1 nanobody mRNA and tested their efficacy in multiple mouse models of colorectal cancer.
The results revealed striking differences between the two formats. While monomeric nanobody mRNA showed some effect against tumour growth, the quadruple nanobody mRNA demonstrated significantly superior efficacy. The larger quadruple nanobody remained in circulation approximately twice as long as the monomeric version, providing sustained therapeutic activity.
In mice with sporadic colorectal cancer tumours, quadruple nanobody mRNA-LNP treatment significantly inhibited tumour progression, with effects becoming apparent after the third injection. Serum analysis confirmed that the quadruple format achieved higher blood levels and slower clearance, explaining its enhanced therapeutic performance.
Perhaps most impressively, the quadruple nanobody mRNA-LNP treatment successfully reduced tumour incidence in colitis-associated colorectal cancer – a context where conventional PD-L1 antibodies have failed. The treatment significantly diminished tumour numbers in both wild-type mice and genetically susceptible models that typically develop more aggressive disease.
The researchers uncovered the mechanism behind this success: the treatment fundamentally reshaped the tumour immune microenvironment. It markedly reduced infiltration of myeloid-derived suppressor cells and tumour-associated macrophages – two key cell types that create an immunosuppressive environment allowing tumours to evade immune detection. Simultaneously, it increased infiltration of CD8+ T cells, the immune system's primary cancer-fighting cells.
The study went further to demonstrate that the nanobody mRNA approach directly affects immune cell development. When researchers treated bone marrow hematopoietic stem cells with nanobody mRNA-LNPs in vitro, the treatment significantly inhibited their differentiation into macrophages and reduced expression of immunosuppressive markers including PD-L1, CD80, CD86, and CD206. This finding suggests the therapy may work not only by activating existing immune cells but also by preventing the development of new immunosuppressive cells that support tumour growth.
The research team proposes that human versions of these quadruple anti-PD-L1 nanobody mRNA-LNPs could represent a promising new immunotherapy approach for colorectal cancer patients. The technology combines the advantages of nanobodies – superior tissue penetration and low immunogenicity – with the proven safety and efficacy of the mRNA-LNP delivery platform. While the current study focused on single-agent therapy, the researchers note that combination approaches using multiple nanobodies targeting different immune checkpoints in a single mRNA construct, or combining nanobody mRNA with chemotherapy or radiotherapy, might yield even better results.
- Chu W-M, Ma L, Hew B, et al. Immunotherapy against colorectal cancer via delivery of anti-PD-L1 nanobody mRNA. eGastroenterology 2025;3:e100106. doi:10.1136/egastro-2024-100106