Affinity-matured variants derived from nimotuzumab keep the original fine specificity and exhibit superior biological activity.

Nimotuzumab is a humanized monoclonal antibody against the Epidermal Growth Factor Receptor with a long history of therapeutic use, recognizing an epitope different from the ones targeted by other antibodies against the same antigen. It is also distinguished by much less toxicity resulting in a better safety profile, which has been attributed to its lower affinity compared to these other antibodies. Nevertheless, the ideal affinity window for optimizing the balance between anti-tumor activity and toxic effects has not been determined. In the current work, the paratope of the phage-displayed nimotuzumab Fab fragment was evolved in vitro to obtain affinity-matured variants. Soft-randomization of heavy chain variable region CDRs and phage selection resulted in mutated variants with improved binding ability. Two recombinant antibodies were constructed using these variable regions, which kept the original fine epitope specificity and showed moderate affinity increases against the target (3-4-fold). Such differences were translated into a greatly enhanced inhibitory capacity upon ligand-induced receptor phosphorylation on tumor cells. The new antibodies, named K4 and K5, are valuable tools to explore the role of affinity in nimotuzumab biological properties, and could be used for applications requiring a fine-tuning of the balance between binding to tumor cells and healthy tissues.

Nimotuzumab: beyond the EGFR signaling cascade inhibition.

One of the most known oncogenes is the epidermal growth factor receptor (EGFR) family. It activates multiple signaling cascades that promote carcinogenesis and immune evasion. Therefore, these molecules have been extensively targeted in cancer immunotherapy. Beyond EGFR signaling cascade inhibition, some of these agents are able to induce T-cell activation, transforming a passive therapy into a vaccine-like effect. Nimotuzumab is an IgG1 humanized monoclonal antibody directed against the extracellular domain of the EGFR blocking the binding to its ligands. It possesses unique pharmacodynamic properties, which allow treating patients for long-term periods and with very low toxicity. Based on its clinical effect, nimotuzumab has been approved in Cuba and abroad for the treatment of different epithelial tumors. Recently, new potential mechanisms of action of nimotuzumab involving the activation of the innate and adaptive immune response have been reported. This review summarizes the main properties of nimotuzumab in comparison with other EGFR-specific monoclonal antibodies, highlighting its capacity to activate an effective immune response. In addition, differential clinical effects of this antibody and ongoing clinical trials to deeply characterize the biomarkers of clinical benefit are shown.

Upregulation of HLA Class I Expression on Tumor Cells by the Anti-EGFR Antibody Nimotuzumab.

Defining how epidermal growth factor receptor (EGFR)-targeting therapies influence the immune response is essential to increase their clinical efficacy. A growing emphasis is being placed on immune regulator genes that govern tumor - T cell interactions. Previous studies showed an increase in HLA class I cell surface expression in tumor cell lines treated with anti-EGFR agents. In particular, earlier studies of the anti-EGFR blocking antibody cetuximab, have suggested that increased tumor expression of HLA class I is associated with positive clinical response. We investigated the effect of another commercially available anti-EGFR antibody nimotuzumab on HLA class I expression in tumor cell lines. We observed, for the first time, that nimotuzumab increases HLA class I expression and its effect is associated with a coordinated increase in mRNA levels of the principal antigen processing and presentation components. Moreover, using 7A7 (a specific surrogate antibody against murine EGFR), we obtained results suggesting the importance of the increased MHC-I expression induced by EGFR-targeted therapies display higher in antitumor immune response. 7A7 therapy induced upregulation of tumor MHC-I expression in vivo and tumors treated with this antibody display higher susceptibility to CD8+ T cells-mediated lysis. Our results represent the first evidence suggesting the importance of the adaptive immunity in nimotuzumab-mediated antitumor activity. More experiments should be conducted in order to elucidate the relevance of this mechanism in cancer patients. This novel immune-related antitumor mechanism mediated by nimotuzumab opens new perspectives for its combination with various immunotherapeutic agents and cancer vaccines.