纳米抗体应用于感染疾病的研究现状

纳米抗体独特的分子特性使其适用于疾病诊断治疗的许多不同领域。抗病毒、细菌、寄生虫感染以及毒素中和等应用领域的纳米抗体正处于不同的研发阶段。研发中的纳米抗体不仅以病原体表面抗原为靶点,调动宿主免疫系统的补体固定和调理吞噬杀伤来清除病原体,而且中和病原体产生的毒素,显著降低对宿主细胞结构和功能的损害作用,降低感染的严重程度。抗体工程技术突飞猛进的发展,推动了抗感染纳米抗体的探索及尝试。临床试验中的2个抗病毒纳米抗体均处于试验后期阶段,相关结果尚需进一步观察。不断的研究和推进或将使纳米抗体在未来的感染疾病应用中占据一席之地。     

基于表面电荷对抗体分子状态调控的研究

抗体功能化纳米粒子(Ab-NPs)由于高亲和力和特异性的优势而得到广泛应用,但抗体分子在纳米粒子表面的分子状态及应用过程中的非特异性吸附均会影响其抗原结合能力。研究制备了一系列不同表面电性和电荷密度的二氧化硅纳米粒子,并分别通过物理吸附和共价偶联法对抗α-HCG(人绒毛促性腺激素)单克隆抗体进行了固定。实验表明抗体在正电荷纳米粒子表面倾向于Fab朝外,随电荷密度升高抗体固定量减少,抗原结合强度降低;在负电荷纳米粒子表面抗体取向随电荷密度的增高逐渐趋向于Fc片段朝外,抗原结合强度降低。相对于稳定的共价偶联法,物理吸附抗体的纳米粒子在非特异性吸附血清蛋白后会引起抗体取向的重排,使得更多的抗体呈现出Fab片段朝外的取向,从而导致抗原结合能力增加。通过研究电性和电荷密度对表面抗体取向的调控将有助于开发具有高灵敏度、高特异性的生物检测体系。     

应用流式细胞仪检测聚合物纳米胶束表面抗体偶联效率

目的探索应用流式细胞仪(flow cytometer,FCM)检测纳米级聚合物胶束(polymeric micelles,PMs)表面偶联抗体效率的方法。方法将DMPE-PEG-Maleimide、DPPE-PEG-Maleimide、DSPE-PEG-Maleimide、DOPE-PEG-Malei-mide、DLPE-PEG-Maleimide聚合物纳米胶束分别与Anti-GOLPH2抗体共同孵育,使其彼此偶联;再与SNU-423细胞共同孵育,采用FCM检测SNU-423细胞膜表面GOLPH2抗原的阳性表达率,并比较各类聚合物纳米胶束与AntiGOLPH2抗体的相对偶联效率。结果 SNU-423细胞表面GOLPH2抗原的阳性表达率为1.23%;在各类聚合物纳米胶束中,DLPE-PEG-Maleimide与Anti-GOLPH2抗体的相对偶联效率最高,且与抗体浓度呈正相关。结论应用FCM检测聚合物纳米胶束表面抗体偶联效率的方法,能在一定程度上反映抗体与聚合物纳米胶束的抗体偶联情况,其检测结果具有一定的参考价值。     

抗幽门螺杆菌细胞空泡毒素纳米抗体的制备及鉴定

目的:获取抗幽门螺杆菌(Helicobacter pylori,Hp)细胞空泡毒素纳米抗体并鉴定其结合和抑制幽门螺杆菌的能力.方法:用灭活的幽门螺杆菌免疫羊驼,以重组蛋白VacA为靶点,从噬菌体展示文库中筛选与幽门螺杆菌特异性结合的纳米抗体,间接phage-ELISA鉴定结合重组蛋白VacA能力并诱导其进行原核表达,鉴...     

纳米抗体应用于肿瘤治疗的研究现状

纳米抗体(nanobodies,Nbs)具有较小的相对分子质量,其独特的分子结构使其适用于疾病诊断治疗等诸多领域,由于其缺少Fc段,不能引发普通单抗药物在肿瘤治疗中重要的细胞毒作用,在肿瘤治疗领域的前景一度不被看好,但随着药物研究的不断深入及更合理的设计,Nbs在该领域的应用正在不断扩展。目前研发中的Nbs是以直接拮抗作用、与效应分子相连、与药物递送系统相连等多种形式参与肿瘤治疗,并可联合应用于靶向性放射核素疗法及光动力疗法中。本文就Nbs的成药特性、Nbs在肿瘤治疗中的应用及目前处于临床试验阶段Nbs药物的研究现状作一综述。     

麻疹活疫苗免疫后血凝抑制抗体与中和抗体的比较

本文对无麻疹干扰的人工封闭地区麻疹活疫苗免疫后的 HI 及 Nt 抗体进行了比较。结果显示,免疫成功后,HI 抗体阴转持续≤5年和6～10年者,分别有82%和43.8%的人均可测出不同水平的 Nt 抗体,证明 HI 抗体降至<1∶2仍具有免疫力。初免与再免者,在 HI 抗体阴转持续时间与 Nt 抗体之间关系没有差别,说明只要初免成功,其 Nt 抗体比较牢固。免疫成功者再显性或隐性感染后3年,仍有较高的 HI 和 Nt 抗体,这与人工再免后1～2年内,大部分人 HI 抗体还原到再免前水平有明显的不同。     

抗体药物免疫原性的研究进展

抗体药物是生物技术药物研发领域中公认的研究热点。在抗体药物研制过程中,免疫原性一直是限制其临床应用的重要因素,不但严重影响其安全性和有效性,有时还会导致严重的临床后果。本文主要分析了抗体药物本身引起免疫原性的多种因素以及针对这些因素的改进方法,即通过抗体的人源化、去免疫化、糖基化修饰、抗体分子小型化和多聚体问题的改善来降低抗体药物的免疫原性。     

红细胞自身抗体的检测及结果分析

目的分析红细胞自身抗体阳性患者的临床诊断,探讨红细胞自身抗体的临床意义。方法采用微柱凝胶抗球蛋白试验,筛检和鉴定患者红细胞血型不规则抗体,根据抗体是否凝集其自身红细胞,鉴别同种抗体或自身抗体;对鉴定存在红细胞自身抗体的患者,进行红细胞直接抗球蛋白试验,并分析其临床诊断。结果在46000名检测红细胞血型不规则抗体的患者中,检出23名红细胞自身抗体阳性,其中红细胞直接抗球蛋白试验阳性者13名。在23名红细胞自身抗体阳性者中,临床诊断为自身免疫溶血性疾病6名,系统性红斑狼疮5名,Evans综合征2名,自身免疫性肝炎2名,药物性急性自身免疫溶血性贫血1名,过敏性紫癜性肾炎1名,Ⅰ型糖尿病酮症酸中毒1名,溃疡性结肠炎1名,4名未检出与自身免疫相关。结论红细胞自身抗体可出现于多种自身免疫性疾病或与自身免疫相关疾病患者的血浆中,检测红细胞自身抗体对自身免疫性疾病的诊断及治疗有参考意义。     

丙型肝炎病毒分片段抗体检测及优势抗原表位分析

目的检测丙型肝炎病毒(HCV)分片段抗体,并分析HCV优势抗原表位,为HCV抗原、抗体检测及疫苗研究提供依据。方法收集经ELISA法检测抗HCV阳性血清样品90份,使用总抗体检测试剂及抗HCV分片段试剂进行检测,并用荧光定量PCR对抗体弱阳性及阴性样品进行复核,分析不同片段出现的阳性率,从而分析HCV的优势抗原表位。结果 90份抗HCV阳性的样品经总抗体检测试剂检测,其中阳性78份(86.67%);90份抗HCV阳性的样品中,分片段试剂检测1个片段以上阳性样品59份(65.56%);其中C、NS3、NS4及NS5片段抗体阳性分别为56份(94.92%)、57份(96.61%)、42份(71.19%)及47份(79.66%),NS3及C片段抗体是HCV的优势抗体。结论 HCV-C及NS3表位是HCV的优势抗原表位;HCV分片段抗体与总抗体检测试剂之间阳性检出率存在一定差异。     

从大容量噬菌体抗体库获取人源性抗角蛋白抗体ScFv及Diabody的构建

目的从大容量噬菌体抗体库筛选人源性抗角蛋白抗体,并构建双体抗体(Diabody)。方法以固相化的角蛋白对构建的大容量噬菌体抗体库进行筛选,经3～4轮筛选后,挑取克隆,ELISA法鉴定其特异性,并对部分抗角蛋白阳性抗体克隆基因进行DNA序列分析。选取活性好的克隆基因进行改造,构建Diabody表达载体。结果在抗体库的筛选过程中可见明显的富集现象,获得了29株可与角蛋白特异性结合的人源单链抗体,选取4个克隆基因进行序列分析,结果表明,4个克隆的轻链基因均属于λ轻链第1亚群,1号和8号克隆的重链基因属于人IgG第2亚群,6号和29号克隆分别属于第1和第3亚群。从表达抗角蛋白抗体的集落中挑选一个进行基因改造,构建的Diabody表达载体所表达的Diabody活性较高。结论利用噬菌体抗体技术获得了人源性抗角蛋白抗体,并改造成应用前景较好的Diabody,为开发银屑病治疗性抗体奠定了基础。     

Easily Established and Multifunctional Synthetic Nanobody Libraries as Research Tools

Nanobodies, or VHHs, refer to the antigen-binding domain of heavy-chain antibodies (HCAbs) from camelids. They have been widely used as research tools for protein purification and structure determination due to their small size, high specificity, and high stability, overcoming limitations with conventional antibody fragments. However, animal immunization and subsequent retrieval of antigen-specific nanobodies are expensive and complicated. Construction of synthetic nanobody libraries using DNA oligonucleotides is a cost-effective alternative for immunization libraries and shows great potential in identifying antigen-specific or even conformation-specific nanobodies. This review summarizes and analyses synthetic nanobody libraries in the current literature, including library design and biopanning methods, and further discusses applications of antigen-specific nanobodies obtained from synthetic libraries to research.     

Enthalpy- versus Entropy-Driven Molecular Recognition in the Era of Biologics

Our laboratory has recently identified two nanobodies (small antibodies produced by camelids)—Nb1 and Nb6—that bind efficiently to epithelial growth factor (EGF) and inhibit its ability to activate its receptor (EGFR). Because of the relevance of the EGF/EGFR axis as a target in oncology, these new nanobodies have promising therapeutic potential. This article, however, is focused on another feature of these nanobodies: their distinct thermodynamic signatures. Nb1 binds to EGF through an entropy-driven mechanism whereas Nb6 binds to this factor under enthalpic control. We discuss the advantages and disadvantages of each mechanism in the contexts of traditional medical chemistry (small-molecule drugs) and also of biological drugs. In this latter case, the implications in terms of selectivity are far from being clearly established and further experimental data are required. Their monomeric natures, high stability, and ease of recombinant production make nanobodies ideally suited for thermodynamic studies. Moreover, nanobodies, thanks to their simpler structures in comparison with conventional antibodies, might provide better understanding of the structural basis of the thermodynamic parameters of antigen recognition.     

A New in Silico Antibody Similarity Measure Both Identifies Large Sets of Epitope Binders with Distinct CDRs and Accurately Predicts Off-Target Reactivity

Developing a therapeutic antibody is a long, tedious, and expensive process. Many obstacles need to be overcome, such as biophysical properties (issues of solubility, stability, weak production yields, etc.), as well as cross-reactivity and subsequent toxicity, which are major issues. No in silico method exists today to solve such issues. We hypothesized that if we were able to properly measure the similarity between the CDRs of antibodies (Ab) by considering not only their evolutionary proximity (sequence identity) but also their structural features, we would be able to identify families of Ab recognizing similar epitopes. As a consequence, Ab within the family would share the property to recognize their targets, which would allow (i) to identify off-targets and forecast the cross-reactions, and (ii) to identify new Ab specific for a given target. Testing our method on 238D2, an antagonistic anti-CXCR4 nanobody, we were able to find new nanobodies against CXCR4 and to identify influenza hemagglutinin as an off-target of 238D2.     

Structural and Computational Studies of the SARS-CoV-2 Spike Protein Binding Mechanisms with Nanobodies: From Structure and Dynamics to Avidity-Driven Nanobody Engineering

Nanobodies provide important advantages over traditional antibodies, including their smaller size and robust biochemical properties such as high thermal stability, high solubility, and the ability to be bioengineered into novel multivalent, multi-specific, and high-affinity molecules, making them a class of emerging powerful therapies against SARS-CoV-2. Recent research efforts on the design, protein engineering, and structure-functional characterization of nanobodies and their binding with SARS-CoV-2 S proteins reflected a growing realization that nanobody combinations can exploit distinct binding epitopes and leverage the intrinsic plasticity of the conformational landscape for the SARS-CoV-2 S protein to produce efficient neutralizing and mutation resistant characteristics. Structural and computational studies have also been instrumental in quantifying the structure, dynamics, and energetics of the SARS-CoV-2 spike protein binding with nanobodies. In this review, a comprehensive analysis of the current structural, biophysical, and computational biology investigations of SARS-CoV-2 S proteins and their complexes with distinct classes of nanobodies targeting different binding sites is presented. The analysis of computational studies is supplemented by an in-depth examination of mutational scanning simulations and identification of binding energy hotspots for distinct nanobody classes. The review is focused on the analysis of mechanisms underlying synergistic binding of multivalent nanobodies that can be superior to single nanobodies and conventional nanobody cocktails in combating escape mutations by effectively leveraging binding avidity and allosteric cooperativity. We discuss how structural insights and protein engineering approaches together with computational biology tools can aid in the rational design of synergistic combinations that exhibit superior binding and neutralization characteristics owing to avidity-mediated mechanisms.     

Nanobody Paratope Ensembles in Solution Characterized by MD Simulations and NMR

Variable domains of camelid antibodies (so-called nanobodies or V_HH) are the smallest antibody fragments that retain complete functionality and therapeutic potential. Understanding of the nanobody-binding interface has become a pre-requisite for rational antibody design and engineering. The nanobody-binding interface consists of up to three hypervariable loops, known as the CDR loops. Here, we structurally and dynamically characterize the conformational diversity of an anti-GFP-binding nanobody by using molecular dynamics simulations in combination with experimentally derived data from nuclear magnetic resonance (NMR) spectroscopy. The NMR data contain both structural and dynamic information resolved at various timescales, which allows an assessment of the quality of protein MD simulations. Thus, in this study, we compared the ensembles for the anti-GFP-binding nanobody obtained from MD simulations with results from NMR. We find excellent agreement of the NOE-derived distance maps obtained from NMR and MD simulations and observe similar conformational spaces for the simulations with and without NOE time-averaged restraints. We also compare the measured and calculated order parameters and find generally good agreement for the motions observed in the ps–ns timescale, in particular for the CDR3 loop. Understanding of the CDR3 loop dynamics is especially critical for nanobodies, as this loop is typically critical for antigen recognition.     

Camelid Single-Domain Antibodies: Promises and Challenges as Lifesaving Treatments

Since the discovery of camelid heavy-chain antibodies in 1993, there has been tremendous excitement for these antibody domains (VHHs/sdAbs/nanobodies) as research tools, diagnostics, and therapeutics. Commercially, several patents were granted to pioneering research groups in Belgium and the Netherlands between 1996–2001. Ablynx was established in 2001 with the aim of exploring the therapeutic applications and development of nanobody drugs. Extensive efforts over two decades at Ablynx led to the first approved nanobody drug, caplacizumab (Cablivi) by the EMA and FDA (2018–2019) for the treatment of rare blood clotting disorders in adults with acquired thrombotic thrombocytopenic purpura (TPP). The relatively long development time between camelid sdAb discovery and their entry into the market reflects the novelty of the approach, together with intellectual property restrictions and freedom-to-operate issues. The approval of the first sdAb drug, together with the expiration of key patents, may open a new horizon for the emergence of camelid sdAbs as mainstream biotherapeutics in the years to come. It remains to be seen if nanobody-based drugs will be cheaper than traditional antibodies. In this review, I provide critical perspectives on camelid sdAbs and present the promises and challenges to their widespread adoption as diagnostic and therapeutic agents.     

Allosteric Determinants of the SARS-CoV-2 Spike Protein Binding with Nanobodies: Examining Mechanisms of Mutational Escape and Sensitivity of the Omicron Variant

Structural and biochemical studies have recently revealed a range of rationally engineered nanobodies with efficient neutralizing capacity against the SARS-CoV-2 virus and resilience against mutational escape. In this study, we performed a comprehensive computational analysis of the SARS-CoV-2 spike trimer complexes with single nanobodies Nb6, VHH E, and complex with VHH E/VHH V nanobody combination. We combined coarse-grained and all-atom molecular simulations and collective dynamics analysis with binding free energy scanning, perturbation-response scanning, and network centrality analysis to examine mechanisms of nanobody-induced allosteric modulation and cooperativity in the SARS-CoV-2 spike trimer complexes with these nanobodies. By quantifying energetic and allosteric determinants of the SARS-CoV-2 spike protein binding with nanobodies, we also examined nanobody-induced modulation of escaping mutations and the effect of the Omicron variant on nanobody binding. The mutational scanning analysis supported the notion that E484A mutation can have a significant detrimental effect on nanobody binding and result in Omicron-induced escape from nanobody neutralization. Our findings showed that SARS-CoV-2 spike protein might exploit the plasticity of specific allosteric hotspots to generate escape mutants that alter response to binding without compromising activity. The network analysis supported these findings showing that VHH E/VHH V nanobody binding can induce long-range couplings between the cryptic binding epitope and ACE2-binding site through a broader ensemble of communication paths that is less dependent on specific mediating centers and therefore may be less sensitive to mutational perturbations of functional residues. The results suggest that binding affinity and long-range communications of the SARS-CoV-2 complexes with nanobodies can be determined by structurally stable regulatory centers and conformationally adaptable hotspots that are allosterically coupled and collectively control resilience to mutational escape.     

Combining magnetic nanoparticle with biotinylated nanobodies for rapid and sensitive detection of influenza H3N2

Our objective is to develop a rapid and sensitive assay based on magnetic beads to detect the concentration of influenza H3N2. The possibility of using variable domain heavy-chain antibodies (nanobody) as diagnostic tools for influenza H3N2 was investigated. A healthy camel was immunized with inactivated influenza H3N2. A nanobody library of 8 × 10⁸ clones was constructed and phage displayed. After three successive biopanning steps, H3N2-specific nanobodies were successfully isolated, expressed in Escherichia coli, and purified. Sequence analysis of the nanobodies revealed that we possessed four classes of nanobodies against H3N2. Two nanobodies were further used to prepare our rapid diagnostic kit. Biotinylated nanobody was effectively immobilized onto the surface of streptavidin magnetic beads. The modified magnetic beads with nanobody capture specifically influenza H3N2 and can still be recognized by nanobodies conjugated to horseradish peroxidase (HRP) conjugates. Under optimized conditions, the present immunoassay exhibited a relatively high sensitive detection with a limit of 50 ng/mL. In conclusion, by combining magnetic beads with specific nanobodies, this assay provides a promising influenza detection assay to develop a potential rapid, sensitive, and low-cost diagnostic tool to screen for influenza infections.     

Ferritin as a Platform for Creating Antiviral Mosaic Nanocages: Prospects for Treating COVID-19

Infectious diseases are a continues threat to human health and the economy worldwide. The latest example is the global pandemic of COVID-19 caused by SARS-CoV-2. Antibody therapy and vaccines are promising approaches to treat the disease; however, they have bottlenecks: they might have low efficacy or narrow breadth due to the continuous emergence of new strains of the virus or antibodies could cause antibody-dependent enhancement (ADE) of infection. To address these bottlenecks, I propose the use of 24-meric ferritin for the synthesis of mosaic nanocages to deliver a cocktail of antibodies or nanobodies alone or in combination with another therapeutic, like a nucleotide analogue, to mimic the viral entry process and deceive the virus, or to develop mosaic vaccines. I argue that available data showing the effectiveness of ferritin-antibody conjugates in targeting specific cells and ferritin-haemagglutinin nanocages in developing influenza vaccines strongly support my proposals.     

毒死蜱人工抗原的合成及多克隆抗体制备

由毒死蜱与3-巯基丙酸在碱性条件下反应,合成了毒死蜱半抗原。然后通过碳二亚胺法将毒死蜱半抗原分别与牛血清白蛋白(BSA)和鸡卵清白蛋白(OVA)共价偶联,得到了免疫抗原和包被抗原。用免疫抗原免疫兔子获得了高效价的多克隆抗体,抗血清效价达到了1:160000。通过试验确立了毒死蜱标准曲线,检测限为0.5μg.L-1,抑制中浓度I50为18.2μg.L-1,检测线性范围为1.8～1000μg.L-1。