Hong Haofei, Zhou Zhifang, Zhou Kun, Liu Shaozhong, Guo Zhongwu, Wu Zhimeng
Key Laboratory of Carbohydrate Chemistry & Biotechnology , Ministry of Education , School of Biotechnology , Jiangnan University , Wuxi , 214122 , China . Email:
Department of Chemistry , University of Florida , 214 Leigh Hall , Gainesville , Florida 32611 , USA . Email:
Chem Sci. 2019 Aug 28;10(40):9331-9338. doi: 10.1039/c9sc03840j. eCollection 2019 Oct 28.
Nanobodies are a class of camelid-derived single-domain antibodies that have many potential advantages over conventional antibodies and have been utilized to develop new therapeutic strategies for cancer and other diseases. However, nanobodies lack the Fc region of a conventional antibody, which possesses many functions, , eliciting antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), essential for effective immunotherapy. The small molecular size of nanobodies also leads to poor pharmacokinetics, such as short half-life. To address these deficiencies, an endogenous antibody-based strategy to reconstitute the Fc functions for nanobodies was developed. As a proof-of-principle, an anti-human EGFR nanobody, 7D12, was selected to conduct C-terminal modification with the dinitrophenyl (DNP) hapten through Sortase A-mediated site-specific ligation. It was expected that the DNP motif would recruit endogenous human -DNP antibodies to indirectly reinstate the Fc functions. The resultant nanobody-DNP conjugates were shown to exhibit specific and high affinity binding to human EGFR expressed on target cancer cells. It was further proved that in the presence of -DNP antibody, these conjugates could mediate potent ADCC and CDC and exhibit significantly elongated half-life . Ultimately, it was proven in severe combined immunodeficiency (SCID) mice that treatment with the nanobody 7D12-DNP conjugate and -DNP mouse serum could inhibit xenograft tumor growth efficiently. In view of the abundance of -DNP and other endogenous antibodies in the human blood system, this could be a generally applicable approach employed to reconstitute the Fc functions for nanobodies and develop nanobody-based cancer immunotherapy and other therapies.
纳米抗体是一类源自骆驼科动物的单域抗体，与传统抗体相比具有许多潜在优势，已被用于开发针对癌症和其他疾病的新治疗策略。然而，纳米抗体缺乏传统抗体的Fc区域，而Fc区域具有许多功能，如引发抗体依赖性细胞毒性（ADCC）和补体依赖性细胞毒性（CDC），这些对于有效的免疫治疗至关重要。纳米抗体的小分子尺寸还导致药代动力学较差，例如半衰期短。为了解决这些缺陷，开发了一种基于内源性抗体的策略来重建纳米抗体的Fc功能。作为原理验证，选择了一种抗人表皮生长因子受体（EGFR）纳米抗体7D12，通过分选酶A介导的位点特异性连接，用二硝基苯基（DNP）半抗原进行C端修饰。预计DNP基序将募集内源性人抗DNP抗体，以间接恢复Fc功能。结果表明，所得的纳米抗体-DNP缀合物对靶癌细胞上表达的人EGFR具有特异性和高亲和力结合。进一步证明，在存在抗DNP抗体的情况下，这些缀合物可以介导有效的ADCC和CDC，并表现出显著延长的半衰期。最终，在严重联合免疫缺陷（SCID）小鼠中证明，用纳米抗体7D12-DNP缀合物和抗DNP小鼠血清治疗可以有效抑制异种移植肿瘤的生长。鉴于人血液系统中存在丰富的抗DNP和其他内源性抗体，这可能是一种普遍适用的方法，用于重建纳米抗体的Fc功能，并开发基于纳米抗体的癌症免疫疗法和其他疗法。
