Design, engineering, and characterization of a bispecific killer cell engager with high affinity and specificity toward CD16a receptor on natural killer cells for cancer immunotherapy
Creators: name: Khoshtinat Nikkhoi, Shahryar
Publisher: name: No Publisher Supplied
Year: 2023

description: The efficacy of cancer antibody therapy heavily relies on the interaction between monoclonal antibodies (mAbs) and the CD16a receptor found on natural killer (NK) cells. This interaction plays a vital role in the destruction of cancer cells through a mechanism called antibody-dependent cell-mediated cytotoxicity (ADCC). However, the affinity and selectivity of the CD16a receptor for the Fc region of mAbs are often limited, leading to suboptimal anticancer activity and potential adverse effects. Recent clinical evidence has underscored the importance of enhancing the binding affinity between the Fc region and CD16a receptor, as it directly correlates with improved therapeutic outcomes in cancer patients. Consequently, the main objective of this study was to develop a bispecific killer cell engager (BiKE) that exhibits high affinity and specificity towards the CD16a receptor on NK cells, with the aim of enhancing cancer immunotherapy.The engineering of the BiKE involved a comprehensive approach, beginning with the immunization of a llama to generate specific single-domain antibodies, known as nanobodies or VHHs, against the CD16a receptor and HER2 (human epidermal growth factor receptor-2) antigens. The implementation of phage display technology allowed for the isolation of high-affinity anti-CD16a and anti-HER2 VHH clones, which were subsequently characterized using ELISA, flow cytometry, and biolayer interferometry (BLI) techniques. These analytical methods provided valuable insights into the physico-chemical properties of the VHH clones. The data obtained revealed a remarkable sub-nanomolar affinity between the anti-CD16a VHH and the CD16a antigen. Importantly, this interaction demonstrated specificity towards CD16a, as there was no cross-reactivity observed with CD16b-NA1 receptors on neutrophils or CD32b receptors on B cells. Similarly, the isolated anti-HER2 VHH displayed high affinity and specificity towards the HER2 antigen, without any cross-reactivity observed with HER1, HER3, and HER4. Building upon these findings, we formulated a hypothesis that fusing the anti-CD16a VHH with the anti-HER2 VHH could yield a BiKE capable of activating NK cells and effectively eliminating HER2+ cancer cells, surpassing the therapeutic efficacy of the current leading IgG-based mAb, trastuzumab. To test this hypothesis, the anti-HER2 VHH was recombinantly fused with the anti-CD16a VHH using a semi-flexible linker, resulting in the creation of the BiKE:HER2/CD16a molecule.The initial investigations focused on evaluating the capacity of BiKE:HER2/CD16a to stimulate NK cells, resulting in degranulation and the activation of various immune response factors including TNF-α and IFN-γ cytokines, as well as cytotoxic molecules such as granzyme B and perforin. The findings demonstrated that BiKE:HER2/CD16a exhibited superior NK cell activation compared to Trazimera® (trastuzumab).Subsequently, the functionality of BiKE:HER2/CD16a was assessed using both high-affinity haNK92 (CD16+, V176) and low-affinity laNK92 (CD16+, F176) effector cells against HER2+ ovarian and breast cancer cells. The ADCC assays revealed that the engineered BiKE:HER2/CD16a exhibited approximately 100-fold greater potency than Trazimera™ (trastuzumab), resulting in significantly enhanced ADCC activity.Another crucial aspect examined in this study was the epitope recognition of BiKE:HER2/CD16a on the CD16a antigen. Utilizing biolayer interferometry (BLI) analysis, it was discovered that BiKE:HER2/CD16a identified a distinct epitope on CD16a, which differed from the epitopes recognized by anti-CD16a IgG-based monoclonal antibodies (mAbs). This distinctive epitope recognition presents an opportunity not only for monotherapy but also for combination therapy with other antibody drugs, such as checkpoint inhibitors and antibody-drug conjugates. Consequently, BiKE:HER2/CD16a exhibits the potential to synergistically enhance therapeutic outcomes when utilized in conjunction with other immunotherapeutic approaches.In conclusion, the enhanced anticancer activity exhibited by BiKE:HER2/CD16a can be attributed to its elevated affinity and specificity towards the CD16a receptor on NK cells. This heightened interaction facilitates augmented activation of NK cells and subsequent release of key cytotoxic molecules, including granzyme B, perforin, TNF-α, and IFN-γ. These significant findings establish a solid groundwork for future investigations and clinical advancements in the realm of BiKE:HER2/CD16a, offering a promising pathway towards more precise and effective cancer immunotherapies.