Enhancing Anti-Cancer Immune Response by Acidosis-Sensitive Nanobody Display.

One of the main challenges with many cancer immunotherapies is that biomarkers are needed for targeting. These biomarkers are often associated with tumors but are not specific to a particular tumor and can lead to damage in healthy tissues, resistance to treatment, or the need for customization for different types of cancer due to 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 under 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 explored 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 demonstrated the insertion of pH-sensitive agents 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.

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.

Selective Recruitment of Antibodies to Cancer Cells and Immune Cell-mediated Killing via In Situ Click Chemistry.

Many current cancer immunotherapies function by redirecting immune system components to recognize cancer biomarkers and initiate a cytotoxic attack. The lack of a universal tumor biomarker limits the therapeutic potential of these approaches. However, one feature characteristic of nearly all solid tumors is extracellular acidity. This inherent acidity provides the basis for targeted drug delivery via the pH-low insertion peptide (pHLIP), which selectively accumulates in tumors in vivo due to a pH-dependent membrane insertion propensity. Previously, we established that we could selectively decorate cancer cells with antigen-pHLIP conjugates to facilitate antibody recruitment and subsequent killing by engineered effector cells via antibody-depended cellular cytotoxicity (ADCC). Here, we present a novel strategy for opsonizing antibodies on target cell surfaces using click chemistry. We utilize pHLIP to facilitate selective tetrazine - trans-cyclooctene ligation of human IgGs to the cancer cell surface and induce ADCC. We demonstrate that our approach activates the primary ADCC signaling pathway via CD16a (FcγRIIIa) receptors on effector cells and induces the killing of cancer cell targets by engineered NK cells.

Targeted acidosis mediated delivery of antigenic MHC-binding peptides.

Cytotoxic T lymphocytes are the primary effector immune cells responsible for protection against cancer, as they target peptide neoantigens presented through the major histocompatibility complex (MHC) on cancer cells, leading to cell death. Targeting peptide-MHC (pMHC) complex offers a promising strategy for immunotherapy due to their specificity and effectiveness against cancer. In this work, we exploit the acidic tumor micro-environment to selectively deliver antigenic peptides to cancer using pH(low) insertion peptides (pHLIP). We demonstrated the delivery of MHC binding peptides directly to the cytoplasm of melanoma cells resulted in the presentation of antigenic peptides on MHC, and activation of T cells. This work highlights the potential of pHLIP as a vehicle for the targeted delivery of antigenic peptides and its presentation via MHC-bound complexes on cancer cell surface for activation of T cells with implications for enhancing anti-cancer immunotherapy.