Giang Kim Anh, Boxaspen Thorstein, Diao Yumei, Nilvebrant Johan, Kosugi-Kanaya Mizuha, Kanaya Minoru, Krokeide Silje Zandstra, Lehmann Fredrik, Svensson Gelius Stefan, Malmberg Karl-Johan, Nygren Per-Åke
Department of Protein Science, Div. Protein Engineering, AlbaNova University Center, KTH Royal Institute of Technology, S-114 21 Stockholm, Sweden.
Department of Cancer Immunology, Institute for Cancer Research, Oslo University Hospital, N-0424 Oslo, Norway; Precision Immunotherapy Alliance, Institute of Clinical Medicine, University of Oslo, N-0313 Oslo, Norway.
N Biotechnol. 2023 Nov 25;77:139-148. doi: 10.1016/j.nbt.2023.09.002. Epub 2023 Sep 4.
We describe the development and characterization of the (to date) smallest Natural Killer (NK) cell re-directing human B Cell Maturation Antigen (hBCMA) x CD16 dual engagers for potential treatment of multiple myeloma, based on combinations of small 58 amino acid, non-immunoglobulin, affibody affinity proteins. Affibody molecules to human CD16a were selected from a combinatorial library by phage display resulting in the identification of three unique binders with affinities (K) for CD16a in the range of 100 nM-3 µM. The affibody exhibiting the highest affinity demonstrated insensitivity towards the CD16a allotype (158F/V) and did not interfere with IgG (Fc) binding to CD16a. For the construction of hBCMA x CD16 dual engagers, different CD16a binding arms, including bi-paratopic affibody combinations, were genetically fused to a high-affinity hBCMA-specific affibody. Such 15-23 kDa dual engager constructs showed simultaneous hBCMA and CD16a binding ability and could efficiently activate resting primary NK cells and trigger specific lysis of a panel of hBCMA-positive multiple myeloma cell lines. Hence, we report a novel class of uniquely small NK cell engagers with specific binding properties and potent functional profiles.
我们描述了（迄今为止）最小的重定向人自然杀伤（NK）细胞的人B细胞成熟抗原（hBCMA）x CD16双特异性衔接子的开发与特性，该衔接子基于由58个氨基酸组成的小型非免疫球蛋白亲和体亲和蛋白组合，用于多发性骨髓瘤的潜在治疗。通过噬菌体展示从组合文库中筛选出与人CD16a结合的亲和体分子，鉴定出三种对CD16a具有亲和力（K）的独特结合物，亲和力范围为100 nM至3 μM。表现出最高亲和力的亲和体对CD16a同种异型（158F/V）不敏感，且不干扰IgG（Fc）与CD16a的结合。为构建hBCMA x CD16双特异性衔接子，将不同的CD16a结合臂，包括双对位亲和体组合，基因融合至高亲和力的hBCMA特异性亲和体上。此类15 - 23 kDa的双特异性衔接子构建体显示出同时结合hBCMA和CD16a的能力，并且能够有效激活静息的原代NK细胞，并触发一组hBCMA阳性多发性骨髓瘤细胞系的特异性裂解。因此，我们报道了一类新型的独特小型NK细胞衔接子，其具有特异性结合特性和强大的功能特性。
