Nanobodies in cell-mediated immunotherapy: On the road to fight cancer.

The immune system is essential in recognizing and eliminating tumor cells. The unique characteristics of the tumor microenvironment (TME), such as heterogeneity, reduced blood flow, hypoxia, and acidity, can reduce the efficacy of cell-mediated immunity. The primary goal of cancer immunotherapy is to modify the immune cells or the TME to enable the immune system to eliminate malignancies successfully. Nanobodies, known as single-domain antibodies, are light chain-free antibody fragments produced from Camelidae antibodies. The unique properties of nanobodies, including high stability, reduced immunogenicity, enhanced infiltration into the TME of solid tumors and facile genetic engineering have led to their promising application in cell-mediated immunotherapy. They can promote the cancer therapy either directly by bridging between tumor cells and immune cells and by targeting cancer cells using immune cell-bound nanobodies or indirectly by blocking the inhibitory ligands/receptors. The T-cell activation can be engaged through anti-CD3 and anti-4-1BB nanobodies in the bispecific (bispecific T-cell engagers (BiTEs)) and trispecific (trispecific T-cell engager (TriTEs)) manners. Also, nanobodies can be used as natural killer (NK) cell engagers (BiKEs, TriKEs, and TetraKEs) to create an immune synapse between the tumor and NK cells. Nanobodies can redirect immune cells to attack tumor cells through a chimeric antigen receptor (CAR) incorporating a nanobody against the target antigen. Various cancer antigens have been targeted by nanobody-based CAR-T and CAR-NK cells for treating both hematological and solid malignancies. They can also cause the continuation of immune surveillance against tumor cells by stopping inappropriate inhibition of immune checkpoints. Other roles of nanobodies in cell-mediated cancer immunotherapy include reprogramming macrophages to reduce metastasis and angiogenesis, as well as preventing the severe side effects occurring in cell-mediated immunotherapy. Here, we highlight the critical functions of various immune cells, including T cells, NK cells, and macrophages in the TME, and discuss newly developed immunotherapy methods based on the targeted manipulation of immune cells and TME with nanobodies.

The Enhanced Cytotoxic Effects of the p28-Apoptin Chimeric Protein As A Novel Anti-Cancer Agent on Breast Cancer Cell Lines.

BACKGROUNDS: Peptide-based drugs have shown promising results in overcoming the limitations of chemotherapeutic drugs by providing a targeted therapy approach to cancer. However, the response rate of targeted therapies is limited, in large part due to the intra- and inter-heterogeneity of tumors. METHODS: In this study, we engineered a novel chimeric protein composed of the p28 peptide as a tumor-homing killer peptide and apoptin as a killer peptide. We evaluated its cytotoxicity against MCF7 and MDA-MB-231 breast cancer cells and HEK-293 normal cells by the MTT assay. Different linkers were evaluated when designing the chimeric protein. Three-dimensional structure predictions of chimeric proteins with different linkers were carried out by Modeller 9.19, and their validation and analysis were performed by RAMPAGE. RESULTS: Results showed that a cleavable linker, including furin cleavage sites, is preferred over other linkers. The chimeric protein was then successfully expressed in E. coli and purified by affinity chromatography under native conditions, then confirmed by SDS-PAGE and Western blot analysis. Compared with apoptin alone, the chimeric protein showed significantly higher toxicity against breast cancer cell lines in a dose-dependent manner. The IC50 values of the chimeric protein for MCF7 and MDA-MB-231 cells were 38.55 µg/mL and 43.11 µg/mL, respectively. There was no significant cytotoxic effect on the normal HEK293 cell line. CONCLUSION: This study demonstrates that fusion of p28 peptide to a potent protein could provide an effective method for tumor targeting. Further, in vitro and in vivo studies of this novel chimeric protein are underway.