Department of Oncology of the First Affiliated Hospital, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, 230027, Anhui, China.
Hefei National Laboratory for Physical Sciences at Microscale, The CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences, University of Science and Technology of China, Hefei, 230027, Anhui, China.
Microb Cell Fact. 2023 Apr 11;22(1):67. doi: 10.1186/s12934-023-02082-6.
Natural killer (NK) cell-based immunotherapies have demonstrated substantial potential for the treatment of hematologic malignancies. However, its application is limited due to the difficulty in the production of a large number of NK cells in vitro and the insufficient therapeutic efficacy against solid tumors in vivo. Engineered antibodies or fusion proteins targeting activating receptors and costimulatory molecules of NK cells have been developed to encounter these problems. They are mostly produced in mammalian cells with high cost and long processing times. Yeast systems, such as Komagataella phaffii, present a convenient manipulation of microbial systems with the key advantages of improved folding machinery and low cost.
In this study, we designed an antibody fusion protein scFvCD16A-sc4-1BBL, composed of the single chain variant fragment (scFv) of anti-CD16A antibody and the three extracellular domains (ECDs) of human 4-1BBL in a single-chain format (sc) with the GS linker, aiming to boost NK cell proliferation and activation. This protein complex was produced in the K. phaffii X33 system and purified by affinity chromatography and size exclusion chromatography. The scFvCD16A-sc4-1BBL complex showed comparable binding abilities to its two targets human CD16A and 4-1BB as its two parental moieties (scFvCD16A and monomer ECD (mn)4-1BBL). scFvCD16A-sc4-1BBL specifically stimulated the expansion of peripheral blood mononuclear cell (PBMC)-derived NK cells in vitro. Furthermore, in the ovarian cancer xenograft mouse model, adoptive NK cell infusion combined with intraperitoneal (i.p) injection of scFvCD16A-sc4-1BBL further reduced the tumor burden and prolonged the survival time of mice.
Our studies demonstrate the feasibility of the expression of the antibody fusion protein scFvCD16A-sc4-1BBL in K. phaffii with favourable properties. scFvCD16A-sc4-1BBL stimulates PBMC-derived NK cell expansion in vitro and improves the antitumor activity of adoptively transferred NK cells in a murine model of ovarian cancer and may serve as a synergistic drug for NK immunotherapy in future research and applications.
自然杀伤 (NK) 细胞为基础的免疫疗法已显示出治疗血液恶性肿瘤的巨大潜力。然而，由于在体外大量生产 NK 细胞的困难以及在体内对实体瘤的治疗效果不足，其应用受到限制。靶向 NK 细胞的激活受体和共刺激分子的工程抗体或融合蛋白已被开发出来以解决这些问题。它们主要在哺乳动物细胞中产生，成本高且处理时间长。毕赤酵母属（Komagataella phaffii）等酵母系统具有便于操作微生物系统的特点，具有改善的折叠机制和低成本的关键优势。
在这项研究中，我们设计了一种抗体融合蛋白 scFvCD16A-sc4-1BBL，由抗 CD16A 抗体的单链变异片段 (scFv) 和人 4-1BBL 的三个细胞外结构域 (ECDs) 组成，以 sc 形式（sc）与 GS 接头连接，旨在促进 NK 细胞的增殖和激活。该蛋白复合物在毕赤酵母 X33 系统中产生，并通过亲和层析和分子筛层析进行纯化。scFvCD16A-sc4-1BBL 复合物与其两个亲本分子（scFvCD16A 和单体 ECD(mn)4-1BBL）对其两个靶标人类 CD16A 和 4-1BB 的结合能力相当。scFvCD16A-sc4-1BBL 特异性刺激外周血单核细胞 (PBMC) 衍生的 NK 细胞在体外扩增。此外，在卵巢癌异种移植小鼠模型中，过继性 NK 细胞输注联合腹腔（i.p）注射 scFvCD16A-sc4-1BBL 进一步减少了肿瘤负担并延长了小鼠的生存时间。
我们的研究表明，在毕赤酵母属中表达抗体融合蛋白 scFvCD16A-sc4-1BBL 是可行的，具有良好的性质。scFvCD16A-sc4-1BBL 刺激 PBMC 衍生的 NK 细胞在体外扩增，并提高了卵巢癌小鼠模型中过继转移 NK 细胞的抗肿瘤活性，可能成为未来 NK 免疫治疗的协同药物在研究和应用中。
