Incorporation of a Novel CD16-Specific Single-Domain Antibody into Multispecific Natural Killer Cell Engagers With Potent ADCC

• Published in: Molecular pharmaceutics Vol. 18; no. 6; pp. 2375 - 2384
• Main Authors: van Faassen, Henk, Jo, Dong-Hyeon, Ryan, Shannon, Lowden, Michael J, Raphael, Shalini, MacKenzie, C. Roger, Lee, Seung-Hwan, Hussack, Greg, Henry, Kevin A
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 07-06-2021
• Subjects: Animals; Antibodies, Bispecific - genetics; Antibodies, Bispecific - metabolism; Antibody-Dependent Cell Cytotoxicity; Antigens, Neoplasm - metabolism; Biological Assay; Camelids, New World; GPI-Linked Proteins - antagonists & inhibitors; GPI-Linked Proteins - genetics; Humans; Immunotherapy - methods; Jurkat Cells; Killer Cells, Natural - metabolism; Killer Cells, Natural - transplantation; Macaca fascicularis; Mice; Neoplasms - immunology; Neoplasms - therapy; Primary Cell Culture; Protein Domains - genetics; Receptors, IgG - antagonists & inhibitors; Receptors, IgG - genetics; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Single-Domain Antibodies - genetics; Single-Domain Antibodies - metabolism; bispecific NK cell engager; VHH; cancer immunotherapy; CD16; nanobody; natural killer cell; single-domain antibody
• ISSN: 1543-8384; 1543-8392
• DOI: 10.1021/acs.molpharmaceut.1c00208

Multispecific antibodies that bridge immune effector and tumor cells have shown promising preclinical and clinical efficacies. Here, we isolated and characterized novel llama single-domain antibodies (sdAbs) against CD16. One sdAb, NRC-sdAb048, bound recombinant human and cynomolgus monkey CD16 ectodomains with equivalent affinity (K D: 1 nM) but did not recognize murine CD16. Binding was similar for human CD16a expressed on NK cells and CD16b (NA2) expressed on neutrophils but dramatically weaker (K D: ∼6 μM) for the CD16b (NA1) allotype. The sdAb stained primary human peripheral blood NK cells. Irrespective of fusion orientation and linker length, bispecific sdAb–sdAb and sdAb–scFv dimers (anti-CD16/EGFR, anti-CD16/HER2, and anti-CD16/CD19) retained full binding affinity for each target, coengaged both antigens simultaneously, elicited ADCC against target antigen-expressing tumor cells in a reporter bioassay, and triggered target-specific activation and degranulation of primary NK cells as measured via interferon-γ and CD107a expression. These molecules may have applications in cancer immunotherapy.