血源性人免疫球蛋白-NK细胞重定向分子偶联物的构建与效应检测

doi:10.3969/j.issn.1671-2587.2026.04.007

临床输血与检验 ›› 2026, Vol. 28 ›› Issue (4): 503-511.DOI: 10.3969/j.issn.1671-2587.2026.04.007

血源性人免疫球蛋白-NK细胞重定向分子偶联物的构建与效应检测

李辰昊^1,2, 贾俊婷², 袁方², 章冰艳², 付振朋², 王夏璐¹, 张嵘¹, 马玉媛²

¹沈阳药科大学,辽宁本溪 117004;
²军事医学研究院国家药品监督管理局血液制品质量控制重点实验室,北京 100850

收稿日期:2026-07-08 出版日期:2026-08-20 发布日期:2026-08-26
通讯作者: 马玉媛,主要从事血液制品研究,（E-mail）mayuyuan07@hotmail.com。共同通信作者：张嵘,主要从事先天性免疫分子作用机制研究,（E-mail）zhangrong7110@163.com。
作者简介:李辰昊,主要从事生物化学与分子生物学研究,（E-mail）13081371283@163.com。并列第一作者：贾俊婷,主要从事血液制品研究,（E-mail）junting1006@163.com。

Construction and Efficacy Evaluation of Blood-derived Human Immunoglobulin-NK Cell Retargeting Molecule Conjugates

LI Chenhao^1,2, JIA Junting², YUAN Fang², ZHANG Bingyan², FU Zhenpeng², WANG Xialu¹, ZHANG Rong¹, MA Yuyuan²

¹Shenyang Pharmaceutical University, Benxi 117004;
²Academy of Military Medical Sciences, NMPA Key Laboratory for Quality Control of Blood Products, Beijing 100850

Received:2026-07-08 Online:2026-08-20 Published:2026-08-26

摘要/Abstract

摘要： 目的基于化学偶联法构建血源性人免疫球蛋白-NK细胞重定向分子偶联物,并评估其抗病毒感染效力。方法以前期筛选的含较高效价抗水痘-带状疱疹病毒（varicella-zoster virus, VZV）抗体人免疫球蛋白制品为原料,利用Traut's试剂和Sulfo-SMCC制备不同偶联程度的人免疫球蛋白与NK细胞重定向分子（抗CD16a scFv）偶联物（以下简称IgG×αCD16a）,通过非还原SDS-PAGE对偶联产物进行表征,使用流式细胞术鉴定IgG×αCD16a与效应细胞（NK细胞）的结合能力。通过乳酸脱氢酶释放法检测ADCC效应、ELISA检测细胞杀伤介质（颗粒酶B、穿孔素）和细胞因子（TNF-α、IFN-γ）释放,系统评估偶联产物IgG×αCD16a与未偶联人免疫球蛋白对VZV感染细胞的杀伤能力。结果 IgG×αCD16a偶联率可达30%以上;IgG×αCD16a可与NK细胞结合,结合比例高于未偶联人免疫球蛋白;与未偶联人免疫球蛋白相比,IgG×αCD16a可显著提高对VZV感染细胞的ADCC效应以及细胞因子（TNF-α、IFN-γ）的释放水平。结论基于化学偶联构建的血源性人免疫球蛋白-NK细胞重定向分子化学偶联物可有效提升血源性人免疫球蛋白的抗病毒感染效果,为新一代高效抗病毒感染抗体药物的研发提供新的思路。

关键词: 血源性人免疫球蛋白, NK细胞, 重定向分子, 化学偶联, 抗病毒

Abstract: Objective Based on the chemical coupling method, the blood-derived human immunoglobulin-NK cell retargeting molecular conjugate was constructed and its antiviral effect was evaluated. Methods Using human immunoglobulin products with high anti-VZV antibody titers as raw materials, the conjugates of human immunoglobulin and NK cell redirection molecule (anti-CD16a scFv) with different coupling degrees, IgG×αCD16a, were prepared by Traut's reagent and Sulfo-SMCC. The conjugated products were characterized by non-reducing SDS-PAGE, and the binding ability of IgG×αCD16a to effector cells (NK cells) was determined by flow cytometry. ADCC activity was detected by LDH release assay, the release of cell killing mediators (granzyme B, perforin) and cytokines (TNF-α, IFN-γ) was detected by ELISA. The killing ability of coupled product IgG×αCD16a and unconjugated human immunoglobulin on VZV infected cells was systematically evaluated. Results The coupling rate of IgG×αCD16a can reach more than 30%. IgG×αCD16a can bind to NK cells, and the binding ratio is higher than that of unconjugated human immunoglobulin. Compared with unconjugated human immunoglobulin, IgG×αCD16a significantly enhanced the ADCC activity on VZV infected cells and the release level of cytokines (TNF-α, IFN-γ). Conclusion The molecular chemical conjugate of blood-derived human immunoglobulin -NK cell redirection based on chemical coupling can effectively improve the antiviral effect of blood-derived human immunoglobulin, and provide a new idea for the development of a new generation of highly effective anti-viral antibody drugs.

Key words: Blood-derived human immunoglobulin, NK cells, Redirecting molecules, Chemical conjugation, Antiviral

中图分类号:

R392.11

李辰昊, 贾俊婷, 袁方, 章冰艳, 付振朋, 王夏璐, 张嵘, 马玉媛. 血源性人免疫球蛋白-NK细胞重定向分子偶联物的构建与效应检测[J]. 临床输血与检验, 2026, 28(4): 503-511.

LI Chenhao, JIA Junting, YUAN Fang, ZHANG Bingyan, FU Zhenpeng, WANG Xialu, ZHANG Rong, MA Yuyuan. Construction and Efficacy Evaluation of Blood-derived Human Immunoglobulin-NK Cell Retargeting Molecule Conjugates[J]. JOURNAL OF CLINICAL TRANSFUSION AND LABORATORY MEDICINE, 2026, 28(4): 503-511.

参考文献

[1] 马玉媛,向思龙,王卓,等. 特异性人免疫球蛋白与传染性疾病的防治[J]. 军事医学,2015,39(3):220-224.
[2] Wightman P,Kelton W.Recent developments and future clinical prospects of polyclonal antibody therapies[J]. Expert Opin Biol Ther,2025,25(12):1285-1298.
[3] Davey R T Jr,Fernández-Cruz E,Markowitz N,et al. Anti-influenza hyperimmune intravenous immunoglobulin for adults with influenza A or B infection (FLU-IVIG): a double-blind,randomised,placebo-controlled trial[J]. Lancet Respir Med,2019,7(11):951-963.
[4] van Griensven J,Edwards T,de Lamballerie X,et al. Evaluation of convalescent plasma for Ebola virus disease in Guinea[J]. N Engl J Med,2016,374(1):33-42.
[5] Pinto A K,Hassert M,Han X B,et al.The ability of zika virus intravenous immunoglobulin to protect from or enhance zika virus disease[J]. Front Immunol,2021,12: 717425.
[6] Tharmalingam T,Han X B,Wozniak A,et al.Polyclonal hyper immunoglobulin: A proven treatment and prophylaxis platform for passive immunization to address existing and emerging diseases[J]. Hum Vaccin Immunother,2022,18(2):1886560.
[7] ITAC INSIGHT 013 Study Group. Hyperimmune immunoglobulin for hospitalised patients with COVID-19 (ITAC): a double-blind,placebo-controlled, phase 3,randomised trial INSIGHT 013 Study Group. Hyperimmune immunoglobulin for hospitalised patients with COVID-19 (ITAC): a double-blind,placebo-controlled, phase 3,randomised trial[J]. Lancet,2022,399(10324): 530-540.
[8] Fox J M,Roy V,Gunn B M,et al. Optimal therapeutic activity of monoclonal antibodies against chikungunya virus requires Fc-FcγR interaction on monocytes[J]. Sci Immunol,2019,4(32):eaav5062.
[9] Bournazos S,DiLillo D J,Goff A J,et al. Differential requirements for FcγR engagement by protective antibodies against Ebola virus[J]. Proc Natl Acad Sci U S A,2019,116(40):20054-20062.
[10] DiLillo D J,Palese P,Wilson P C,et al. Broadly neutralizing anti-influenza antibodies require Fc receptor engagement for in vivo protection[J]. J Clin Invest,2016,126(2):605-610.
[11] Yamin R,Jones A T,Hoffmann H H,et al.Fc-engineered antibody therapeutics with improved anti-SARS-CoV-2 efficacy[J]. Nature,2021,599(7885):465-470.
[12] Mackin S R,Desai P,Whitener B M,et al.Fc-γR-dependent antibody effector functions are required for vaccine-mediated protection against antigen-shifted variants of SARS-CoV-2[J]. Nat Microbiol,2023,8(4): 569-580.
[13] Adams L E,Leist S R,Dinnon K H 3rd,et al. Fc-mediated pan-sarbecovirus protection after alphavirus vector vaccination[J]. Cell Rep,2023,42(4):112326.
[14] Gorman M J,Patel N,Guebre-Xabier M,et al.Fab and Fc contribute to maximal protection against SARS-CoV-2 following NVX-CoV2373 subunit vaccine with Matrix-M vaccination[J]. Cell Rep Med,2021,2(9):100405.
[15] McMahan K,Yu J Y,Mercado N B,et al. Correlates of protection against SARS-CoV-2 in Rhesus macaques[J]. Nature,2021,590(7847):630-634.
[16] Fox J M,Roy V,Gunn B M,et al.Enhancing the therapeutic activity of hyperimmune IgG against chikungunya virus using FcγRIIIa affinity chromatography[J]. Front Immunol,2023,14:1153108.
[17] Wang X Y,Gomozkova M,Li S Q,et al.Selective fluorination of Fc glycans enhances antibody-mediated effector functions[J]. Proc Natl Acad Sci U S A,2026, 123(12):e2530653123.
[18] Fan Q R,Chen H,Wei G G,et al.A review of conjugation technologies for antibody drug conjugates[J]. Antib Ther,2025,8(2):157-170.
[19] Wu G Y,Yuan Z H,Chen M,et al.Antibody-drug conjugates (ADCs): a review of structural design, technological evolution,and future perspectives[J]. Molecules,2026,31(7):1180.
[20] Beck A,Goetsch L,Dumontet C,et al.Strategies and challenges for the next generation of antibody-drug conjugates[J]. Nat Rev Drug Discov,2017,16(5):315-337.
[21] Zappala F,Higbee-Dempsey E,Jang B,et al. Rapid,site-specific labeling of "off-the-shelf” and native serum autoantibodies with T cell-redirecting domains[J]. Sci Adv,2022,8(18):eabn4613.
[22] Lum L G,Ramesh M,Thakur A,et al.Targeting cytomegalovirus-infected cells using T cells armed with anti-CD3×anti-CMV bispecific antibody[J]. Biol Blood Marrow Transplant,2012,18(7):1012-1022.
[23] Giavridis T,van der Stegen S J C,Eyquem J,et al. CAR T cell-induced cytokine release syndrome is mediated by macrophages and abated by IL-1 blockade[J]. Nat Med, 2018,24(6):731-738.
[24] Morris E C,Neelapu S S,Giavridis T,et al.Cytokine release syndrome and associated neurotoxicity in cancer immunotherapy[J]. Nat Rev Immunol,2022,22(2):85-96.
[25] Santurio D S,Barros L R C,Glauche I,et al. Mathematical modeling unveils the timeline of CAR-T cell therapy and macrophage-mediated cytokine release syndrome[J]. PLoS Comput Biol,2025,21(4):e1012908.
[26] Schlub T E,Sun J C,Walton S M,et al.Comparing the kinetics of NK cells,CD4,and CD8 T cells in murine cytomegalovirus infection[J]. J Immunol,2011,187(3): 1385-1392.
[27] Welsh R M,Waggoner S N.NK cells controlling virus-specific T cells: Rheostats for acute vs. persistent infections[J]. Virology,2013,435(1):37-45.
[28] Hernández-Blanco C,Al-Akioui-Sanz K,Herrera L,et al. The phase I RELEASE clinical trial to evaluate the safety of NK cells in COVID-19[J]. iScience,2025,28(2): 111698.
[29] Liu E L,Marin D,Banerjee P,et al.Use of CAR-transduced natural killer cells in CD19-positive lymphoid tumors[J]. N Engl J Med,2020,382(6):545-553.

血源性人免疫球蛋白-NK细胞重定向分子偶联物的构建与效应检测

Construction and Efficacy Evaluation of Blood-derived Human Immunoglobulin-NK Cell Retargeting Molecule Conjugates

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 3

编辑推荐

Metrics

本文评价

关于本刊

作者中心

在线期刊

访问统计

联系我们

[1]	李然, 孙丽艳, 陈婷婷, 曾劲峰. 白细胞滤器中的NK及T细胞回收数量及其相关因素研究^*[J]. 临床输血与检验, 2024, 26(4): 451-456.
[2]	曹腾宇, 孙莉萍, 于洋. NK细胞免疫治疗恶性肿瘤的研究进展[J]. 临床输血与检验, 2024, 26(2): 267-273.
[3]	王颖颖, 郭笑寒, 臧亮, 邓雪莲. 1例核酸检测混检无反应性的HIV感染献血者的检出^*[J]. 临床输血与检验, 2021, 23(3): 389-391.