Enhancing Anti-Cancer Immune Response by Acidosis-sensitive Nanobody Display

One of the main challenges with many cancer immuno-therapies is that they depend on biomarkers for targeting. These biomarkers are often associated with tumors but are not specific to a particular tumor, which can lead to damage in healthy tissues, resistance to treatment, and the need for customization for different types of cancer due to the variations in targets. A promising alternative approach is to target the acidic microenvironment found in most solid tumor types. This can be achieved using the pH (Low) Insertion Peptide (pHLIP), which inserts selectively into cell membranes in acidic conditions, sparing healthy tissues. pHLIP has shown potential for imaging, drug delivery, and surface display. For instance, we previously used pHLIP to display epitopes on the surfaces of cancer cells, enabling antibody-mediated immune cell recruitment and selective killing of cancer cells. In this study, we further this concept by directly fusing an anti-CD16 nanobody, which activates Natural Killer (NK) cells, to pHLIP, eliminating the need for antibody recruitment. Our results demonstrate pH-sensitive insertion into cancer cells, activation of the CD16 receptor on effector cells, and successful targeting and destruction of cancer cells by high-affinity CD16⁺ NK cells in two cancer cell lines.