纳米抗体研究进展

纳米抗体是由骆驼科动物缺失轻链的天然重链抗体的可变区(VHH)组成的单域抗体,其可变区的相对分子质量约为15×10~3。与传统抗体相比,纳米抗体具有相对分子质量小、亲和力高、稳定性高、溶解性好、免疫原性低、穿透力强、人源化简单等优势。基于纳米抗体的特性以及骆驼天然重链抗体的VHH单域抗体的特殊结构,使其在基础医学研究以及疾病诊断和治疗方面具有广阔的应用前景。     

具有高亲和力和稳定性的人源性抗PD-L1二硫键稳定Diabody的制备

目的：对天然噬菌体抗体库进行筛选并对抗体进行体外亲和力成熟,获得高亲和力人源性抗PD-L1抗体,然后对该抗体进行二硫键稳定改造,获得具有高亲和力和稳定性的人源性抗PD-L1的二硫键稳定Diabody。方法：首先以PD-L1重组蛋白为抗原在天然噬菌体Fab抗体库中筛选Fab抗体,其次分析结合能力较好的抗PD-L1的Fab抗体可变区基因中的热点,通过对轻链、重链CDR3区的7处热点随机突变构建噬菌体抗体突变库,从中筛选出亲和力得到提高的抗体。最后在抗体骨架区引入两个二硫键,构建二硫键稳定的抗PD-L1的ds-Diabody,并在毕赤酵母GS115中进行表达。结果：该方法筛选获得了6株特异性抗PD-L1噬菌体Fab抗体,对结合能力较好的其中一株抗体CDR3区的热点进行随机突变,成功构建库容为1.14×10⁸ CFU/mL的噬菌体抗体突变库,并从中筛选出亲和力提高约6倍的噬菌体抗体突变株。对该抗体骨架区进行二硫键引入,成功构建与表达二硫键稳定的ds-Diabody。结论：构建的ds-Diabody比Fab抗体与PD-L1结合亲和力高、稳定性好,为药物开发、肿瘤治疗等研究P...     

抗GM-CSF纳米抗体的筛选与鉴定

目的:制备抗粒细胞-巨噬细胞集落刺激因子(GM-CSF)纳米抗体,并测定其亲和力。方法:分离提取GM-CSF免疫后羊驼外周血淋巴细胞总RNA,PCR扩增得到纳米抗体基因片段,与载体pHEN1重组后克隆至大肠杆菌TG1以构建初始文库,经拯救构建得到噬菌体展示纳米抗体文库,并对其进行生物淘选;通过大肠杆菌BL21(DE3)原核表达阳性纳米抗体克隆,并测定其亲和力。结果:构建了多样性良好且库容量为1.37×10~9cfu的纳米抗体初始文库,3轮淘选后共筛选得到5株氨基酸序列差异性较大的纳米抗体,并对其中一株纳米抗体G1进行表达纯化,SDS-PAGE分析表明纳米抗体G1纯度较高,且有较高的亲和力(K_D=2.95×10~(-8)mol/L)。结论:制备了高亲和力的抗GM-CSF纳米抗体,可应用于相关炎症抗体药物研制和疾病监测等方面。     

纳米抗体的特性及其在免疫检测中的研究进展

纳米抗体(nanobody,Nb)作为抗体行业的新成员,近年来在免疫检测领域的发展突飞猛进.Nb具有分子量小、稳定性好、亲和力和特异性高、易于在原核表达系统中高质量生产等特性.应用Nb的检测试剂改善了传统依赖单克隆抗体检测试剂存在的运输和保存困难、试剂研发成本高、抗体试剂批间差异大的问题,并且提高了检测的灵敏度.Nb在...     

纳米抗体筛选技术研究进展

纳米抗体(nanobody, Nb)是在骆驼科血清中发现的一种新型抗体,具有体积小、特异性强、稳定性高、易于表达和能识别隐藏的抗原表位等优势,在各个领域具有广泛的应用价值。本文介绍了纳米抗体筛选与优化过程,包括纳米抗体文库构建、体外展示和亲和力成熟3个重要技术阶段的分类与特点。其中,简要描述了天然、免疫及半合成/合成文库的制备方法与重要参数,并系统介绍了应用噬菌体、酵母、细菌、核糖体/mRNA和真核细胞等表面展示系统,以及酵母双杂交、高通量测序和质谱鉴定方法,共8种不同体外展示技术进行快速筛选的方法及其优缺点,汇总用于提升纳米抗体功能可靠性的体外及计算机辅助亲和力成熟技术平台,为综合运用各种技术手段快速获得稳定、可靠、特异的纳米抗体类药物或诊断制剂提供了参考。     

单域抗体的研究和应用进展

传统IgG抗体分子一般由轻链和重链组成,轻链包含1个可变区(VL)和1个恒定区(CL),重链包含1个可变区(VH)和3个恒定区(CH1,CH2,CH3)。单域抗体(Single domain antibody,sdAb),是指缺失抗体轻链而只有重链可变区的一类抗体,因其分子量小,也被称为纳米抗体(Nanobody)。20世纪90年代,单域抗体最早在骆驼科动物中被发现,之后在护士鲨、大星鲨和鳐鱼等软骨鱼纲动物中也发现了类似的抗体。单域抗体虽然结构简单,但仍然可以达到与传统抗体相当甚至更高的与特异抗原结合的亲和力。相比于传统抗体,单域抗体具有分子量小、稳定性强、易于重组表达等优点。近年来在生物学基础研究和医学临床应用方面均备受关注并被广泛应用。文中将从单域抗体的结构特征、理化性质、筛选方法及其在生物医学领域的重要应用进展进行综述。     

纳米抗体在传染病的预防、诊断和治疗中的应用 *

传染病是一种由致病性微生物引起,能够影响人类身体健康甚至引发严重社会危机的传播性疾病。近年来,新冠、埃博拉等传染病的恶性暴发促使人们寻找更为高效便捷的防治手段以遏制疾病的进程。抗体在传染病防治中的应用引起了广泛关注,palivizumab是目前唯一被批准应用于呼吸道合胞病毒在免疫力低下人群的预防的单克隆抗体。纳米抗体(nano-antibody, Nb)是目前已知的能与抗原稳定结合的最小功能性单域抗体,具有稳定性高、亲水性强、易于表达和改造等优势。独特的分子特性使其在病毒、细菌、寄生虫等引发的传染病的预防、诊断和治疗中展现出良好的应用前景,相关研究显示纳米抗体对艾滋、流感、新型冠状病毒等都有很好的治疗效果。重点叙述纳米抗体的结构特点及其在传染性疾病中的研究进展。     

基于核酸适配体的DNA纳米结构在肿瘤研究中的应用进展

DNA纳米结构具有强大的分子载带量、良好的稳定性、可编辑性和生物相容性等特点,是纳米材料领域的研究热点.核酸适配体是一段短的寡核苷酸序列(RNA或ssDNA),能够折叠成特定的三维结构与靶标高特异性、高亲和力的结合.将核酸适配体的分子识别特性和DNA纳米结构相结合,可将靶向识别、生物成像及药物递送等特点集于一体,在生命...     

二硫键Cys139-Cys206影响人胰蛋白酶的稳定性

胰蛋白酶是一种丝氨酸蛋白酶,可特异切割精氨酸及赖氨酸C端肽键。重组人源阳离子胰蛋白酶(recombinant human cationic trypsin:rht1)的稳定性明显高于重组人源阴离子胰蛋白酶(recombinant human anionic trypsin:rht2)。比较ht1和ht2的氨基酸序列和三维结构,二者的氨基酸序列同源性为95%,rht1比rht2多1对二硫键Cys139-Cys206。为解释该二硫键对其稳定性的作用,构建rht1的Cys139-Cys206二硫键缺失突变体rht1-dC139S-C206S和rht2的增加该对二硫键的突变体rht2-S139C-S206C。进行了重组表达和纯化,并测定rht1、rht2及两个突变体的酶学性质,对比其稳定性。结果发现,与野生型rht1、rht2相比,突变体的酶学动力学参数km和kcat值与最适pH均未有较大差异。但在pH3～12条件下的稳定性,rht1-dC139S-C206S比rht1低46.6%;rht2-S139C-S206C比rht2高30.3%。对比其热稳定性,40℃保温4 h,rht1残余活性为92.4%,而rht1-dC139S-C206S降为60%。60℃保温4 h,rht2-S139C-S206C残余活性为83.3%,而rht2完全失活。表明了该二硫键Cys139-Cys206对人胰蛋白酶稳定性的重要性。进一步进行结构模拟和分析,解释了该对二硫键对稳定性影响的机制。     

不同给药途径的治疗性纳米抗体研究进展

骆驼科及鲨鱼科动物血清中天然存在的纳米抗体具有不同于传统单克隆抗体的独特结构和分子量,这为抗体药物开发提供了全新的思路。纳米抗体较小的分子量和优异的稳定性使其在给药方面具有更大的灵活性,可以在一定程度上克服传统单克隆抗体在给药途径方面存在的局限性。同时,较小的分子量使纳米抗体具有双重药代动力学特征,既有优异的组织渗透性,又表现出快速的血液清除。重点介绍纳米抗体的药物代谢动力学特征和进一步改善药代动力学的方法,综述不同给药途径的纳米抗体药物研究进展,对其治疗特定疾病的可行性、安全性以及治疗效果进行分析,以期为纳米抗体药物研发中给药途径的选择提供参考。     

What can disulfide bonds tell us about protein energetics, function and folding: simulations and bioninformatics analysis

We study the impact of disulfide bonds on protein stability and folding. Using lattice model simulations, we show that formation of a disulfide bond stabilizes a protein to an extent that depends on the distance along the chain between linked cysteine residues. However, the impact of disulfide bonds on folding kinetics varies broadly, from acceleration when disulfides are introduced in or close to the folding nucleus, to slowing when disulfides are introduced outside the nucleus. Having established the effect of disulfide bonds on stability, we study the correlation between the number of disulfide bonds and the composition of certain amino acid classes with the goal to use it as a statistical probe into factors that contribute to stability of proteins. We find that the number of disulfides is negatively correlated with aliphatic hydrophobic but not aromatic content. It is surprising that we observe a strong correlation of disulfide content with polar (Q,S,T,N) amino acid content and a strong negative correlation with charged (E,D,K,R) content. These findings provide insights into factors that determine protein stability and principles of protein design as well as possible relations of disulfide bonds and protein function.     

Cellular quality control screening to identify amino acid pairs for substituting the disulfide bonds in immunoglobulin fold domains

We are interested in determining which amino acid pairs can be substituted for the disulfide (S-S) bonds in proteins without disrupting their native structures under physiological conditions. In this study, we focused on the intradomain S-S bonds in Ig fold domains and aimed to determine a simple rule for replacement of their S-S bonds. The cysteines of four different Ig fold domains were mutated randomly, and the amino acid pairs substituted for the S-S bonds were screened by the method utilizing a cellular quality control system. Among the 36 selected mutants, 31 were natively folded without S-S bonds, as judged from the cooperativity of thermal unfolding. In addition, the selected mutant llama heavy chain antibodies retained antigen-binding affinity. At least two of the pairs Ala:Ala, Ala:Val, Val: Ala, and Val:Val were found in the selected mutants for all four different Ig fold domains, and they were stably folded at 30 degrees C. This suggests that examination of these four pairs could be enough to obtain natively folded Ig fold domains without S-S bonds.     

Engineering disulfide bonds within an antibody

Antibodies have evolved to function in oxidative, extracellular environments. A pair of cysteines in close proximity will oxidatively react to form a disulfide bond that fixes and stabilizes the tertiary structure of a protein. Immunoglobulin G (IgG) includes several disulfide bonds, and the patterns of inter-chain disulfide bonds characterize different IgG sub-classes. Moreover, the Ig-fold domains are characterized by a buried intra-domain disulfide bond, which is important for its structural stability. However, the intra-domain disulfide bond can be replaced without crucial effects on the structure and function, if the domain structure is intrinsically stable or has been stabilized by protein engineering. In previous studies, disulfide bonds were removed by amino-acid substitution indicating that Val and/or Ala (i.e. Ala–Ala, Ala–Val, Val–Ala, and Val–Ala) pairs were preferred for cysteine replacement in the Ig-fold domain. As such, these mutations may be useful for the intracellular use of antibodies. Recently, additional intra-domain disulfide bonds have been shown to stabilize Ig-fold domains and whole IgGs. In heavy chain variable or light chain variable domains, the introduction of additional disulfide bonds into the framework region did not reduce antigen-binding affinity, suggesting that generating disulfide bonds may be a method for stabilizing IgG and antibody fragments, such as the antigen-binding fragment, and single-chain and single-domain antibodies. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

Engineered high-affinity nanobodies recognizing staphylococcal Protein A and suitable for native isolation of protein complexes

In addition to its high affinity for antibody Fc domains, staphylococcal Protein A has been shown to bind certain Fab domains. We investigated this in order to develop a small, recombinant Protein A-binding alternative to immunoglobulin G (IgG) from nanobodies, single-domain antibodies derived from a camelid variant IgG’s variable region. We engineered a nanobody with affinity solely for Protein A as well as a dimerized version of higher affinity for typical multidomain Protein A constructs. Because this recombinant nanobody can be immobilized using a cleavable crosslinker, it has proven to be suitable for the isolation and mild elution of protein complexes in native conditions.     

Characterization of disulfide-linked heterodimers containing apolipoprotein D in human plasma lipoproteins.

Human plasma apolipoprotein (apo) D is a glycoprotein with an apparent molecular weight of 29,000 M(r). It is present, mainly, in high density lipoproteins (HDL) and very high density lipoproteins (VHDL). Western blot analysis of HDL and VHDL using rabbit antibodies to human apoD revealed major immunoreactive bands at 29,000 and 38,000 M(r), with minor bands ranging from 50,000 to and 80,000 M(r). Only the 29,000 M(r) band corresponding to apoD remained when the electrophoresis was conducted under reducing conditions, demonstrating that apoD is cross-linked to other proteins via disulfide bonds. The broad pattern of immunoreactivity was also observed under nonreducing conditions when the blood was collected into a solution of sulfhydryl-trapping reagents, or when these reagents were added to the isolated lipoproteins. These results indicated that the disulfide bonds were not the result of disulfide exchange during the experimental procedures. On the basis of amino acid sequencing and reactions to antibodies, the 38,000 M(r) band was identified as an apoD-apoA-II heterodimer. The apoD-apoA-II was also demonstrated in plasma. In both HDL and plasma, the apoD-apoA-II heterodimer constituted the major form of apoD. Disulfide-linked heterodimers of apoD and apoB-100 were also found in low and very low density lipoproteins, and in whole plasma. It is concluded that a fraction of human apoD, like other cysteine-containing apolipoproteins, exists as a disulfide-linked heterodimer with other apolipoproteins in all major human lipoprotein fractions.     

The disulfide bonds in antibody variable domains: effects on stability, folding in vitro, and functional expression in Escherichia coli.

The formation of the disulfide bonds in the variable domains VH and VL of the antibody McPC603 was found to be essential for the stability of all antigen binding fragments investigated. Exposure of the Fv fragment to reducing conditions in vitro resulted in irreversible denaturation of both VH and VL. In vitro refolding of the reduced Fv fragment was only possible when the disulfide bonds were allowed to form under oxidizing conditions. The analysis of a series of mutants of the Fv fragment, the Fab fragment and the single-chain Fv fragment, all secreted into the periplasm of Escherichia coli, in which each of the cysteine residues of the variable domains was replaced by a series of other amino acids, showed that functional antigen binding fragments required the presence of both the disulfide bond in VH and the one in VL. These results were also used to devise an alternative expression system based on the production of insoluble fusion proteins consisting of truncated beta-galactosidase and antibody domains, enzymatic cleavage, and refolding and assembly in vitro. This strategy should be useful for providing access to unstable antibody domains and fragments.     

OX133, a monoclonal antibody recognizing protein-bound N-ethylmaleimide for the identification of reduced disulfide bonds in proteins

In vivo, enzymatic reduction of some protein disulfide bonds, allosteric disulfide bonds, provides an important level of structural and functional regulation. The free cysteine residues generated can be labeled by maleimide reagents, including biotin derivatives, allowing the reduced protein to be detected or purified. During the screening of monoclonal antibodies for those specific for the reduced forms of proteins, we isolated OX133, a unique antibody that recognizes polypeptide resident, N-ethylmaleimide (NEM)-modified cysteine residues in a sequence-independent manner. OX133 offers an alternative to biotin-maleimide reagents for labeling reduced/alkylated antigens and capturing reduced/alkylated proteins with the advantage that NEM-modified proteins are more easily detected in mass spectrometry, and may be more easily recovered than is the case following capture with biotin based reagents.     

Domain-level stability of an antibody monitored by reduction, differential alkylation, and mass spectrometry analysis

Human immunoglobulin G1 (IgG1) contains 12 domains, and each has an intrachain disulfide bond that connects the two layers of antiparallel β-sheets. These intrachain disulfide bonds are shielded from solvents under native conditions. Therefore, accessibility of the disulfide bonds to reduction under conditions that unfold antibody has the potential to be a good indicator of the thermodynamic stability of each domain. The stability of a recombinant monoclonal antibody at the domain level was investigated using a novel method involving reduction of the disulfide bonds in the presence of increasing amounts of guanidine hydrochloride and alkylation with [¹²C]iodoacetic acid, which was followed by reduction of the remaining disulfide bonds and alkylation with [¹³C]iodoacetic acid. The percentage of modification by [¹²C]iodoacetic acid of each cysteine residue was calculated using mass spectra of the cysteine-containing tryptic peptides and used to follow the unfolding of each domain. It demonstrated that the CH2 domain was the least stable domain of the antibody, whereas the CH3 domain was the most stable domain of the antibody. Other domains showed intermediate resistance to the denaturant concentration, similar to the overall unfolding transition monitored by the intrinsic tryptophan fluorescence wavelength shift.     

Structural consequences of cysteine substitutions C1977Y and C1977R in calcium-binding epidermal growth factor-like domain 30 of human fibrillin-1

The largest group of disease-causing mutations affecting calcium-binding epidermal growth factor-like (cbEGF) domain function in a wide variety of extracellular and transmembrane proteins is that which results in cysteine substitutions. Although known to introduce proteolytic susceptibility, the detailed structural consequences of cysteine substitutions in cbEGF domains are unknown. Here, we studied pathogenic mutations C1977Y and C1977R, which affect cbEGF30 of human fibrillin-1, in a recombinant three cbEGF domain fragment (cbEGF29-31). Limited proteolysis, 1H NMR, and calcium chelation studies have been used to probe the effect of each substitution on cbEGF30 and its flanking domains. Analysis of the wild-type fragment identified two high affinity and one low affinity calcium-binding sites. Each substitution caused the loss of high affinity calcium binding to cbEGF30, consistent with intradomain misfolding, but the calcium binding properties of cbEGF29 and cbEGF31 were surprisingly unaffected. Further analysis of mutant fragments showed that domain packing of cbEGF29-30, but not cbEGF30-31, was disrupted. These data demonstrate that C1977Y and C1977R have localized structural effects, confined to the N-terminal end of the mutant domain, which disrupt domain packing. Cysteine substitutions affecting other cbEGF disulfide bonds are likely to have different effects. This proposed structural heterogeneity may underlie the observed differences in stability and cellular trafficking of proteins containing such changes.     

Formation of native-like mammalian sperm cell chromatin with folded bull protamine

The DNA of most vertebrate sperm cells is packaged by protamines. The primary structure of mammalian protamine I can be divided into three domains, a central DNA binding domain that is arginine-rich and amino- and carboxyl-terminal domains that are rich in cysteine residues. In native bull sperm chromatin, intramolecular disulfide bonds hold the terminal domains of bull protamine folded back onto the central DNA binding domain, whereas intermolecular disulfide bonds between DNA-bound protamines help stabilize the chromatin of mature mammalian sperm cells. Folded bull protamine was used to condense DNA in vitro under various solution conditions. Using transmission electron microscopy and light scattering, we show that bull protamine forms particles with DNA that are morphologically similar to the subunits of native bull sperm chromatin. In addition, the stability provided by intermolecular disulfide bonds formed between bull protamine molecules within in vitro DNA condensates is comparable with that observed for native bull sperm chromatin. The importance of the bull protamine terminal domains in controlling the bull sperm chromatin morphology is indicated by our observation that DNA condensates formed under identical conditions with a fish protamine, which lacks cysteine-rich terminal domains, do not produce as uniform structures as bull protamine. A model is also presented for the bull protamine.DNA complex in native sperm cell chromatin that provides an explanation for the positions of the cysteine residues in bull protamine that form intermolecular disulfide bonds.