靶向PSMA多价纳米抗体的制备及其生物学活性表征*

目的:构建原核表达系统,制备靶向前列腺特异性膜抗原(prostate-specific membrane antigen,PSMA)多价纳米抗体并初步评价其生物学活性。方法:Bglbrick法构建多价纳米抗体表达载体,转化至大肠杆菌表达并利用亲和层析法纯化。联合蛋白质电泳和Western blot验证纯化产物,BCA法检测表达量。通过免疫荧光和流式细胞术定性评估PSMA特异性亲和能力,细胞ELISA法定量检测PSMA亲和水平,流式细胞术检测内吞效率。结果:成功构建靶向PSMA单价、二价、三价和四价纳米抗体大肠杆菌表达菌株。发酵结果表明四种纳米抗体均能在摇瓶水平实现高效可溶表达,其中二价纳米抗体表达量最高[(259.14±23.56) mg/L],单价纳米抗体表达量最低[(100.58±6.27) mg/L]。亲和实验结果证实四种纳米抗体均能特异性识别并结合PSMA阳性肿瘤细胞,与单价纳米抗体相比,二价、三价和四价纳米抗体对PSMA亲和能力分别提高了3.32倍、2.29倍和2.03倍。最后的内吞实验显示四种纳米抗体均能被PSMA阳性肿瘤细胞高效摄取,30 min内的摄取率均在80%以上。结论:靶向PSMA的多价纳米抗体,尤其是二价纳米抗体,具有比单价纳米抗体更高的产量和亲和水平,且具备不亚于单价纳米抗体的内吞效率,是未来基于PSMA肿瘤诊疗试剂开发的重要候选。     

抗c-Myc标签纳米抗体的筛选与应用

目的: 以c-Myc-GST蛋白为靶分子,从纳米抗体噬菌体展示免疫文库中筛选能够特异性识别c-Myc标签(EQKLISEEDL)的纳米抗体。方法: 采用固相淘选技术,筛选出能与c-Myc标签特异性结合的噬菌体,phage-ELISA鉴定阳性克隆并测序,通过基因重组技术将阳性噬菌体编码的纳米抗体基因克隆至原核表达载体pET25b(+),再转化至大肠杆菌Rosetta(DE3),IPTG诱导表达,SDS-PAGE分析重组蛋白表达情况。采用间接ELISA和量子点免疫荧光法验证纳米抗体的结合活性和特异性。结果: 通过4轮固相淘选,具有结合活性的噬菌体克隆得到了有效富集,回收率提高了145倍,阳性率从20.83%提高至85.4%。将phage-ELISA鉴定显色值高的两个纳米抗体A25和A26分别进行了重组表达,SDS-PAGE结果显示均为可溶性表达,表达量为60 mg/L。间接ELISA结果表明重组蛋白A25和A26都能够识别c-Myc标签,量子点免疫荧光法验证得到纳米抗体A25能够对SP2/0细胞内的c-Myc蛋白进行检测。结论: 成功地筛选出与c-Myc标签结合的纳米抗体噬菌体克隆,构建了两个抗c-Myc标签纳米抗体的原核表达载体并实现了可溶性表达,为检测胞内c-Myc蛋白奠定了基础。     

High-level expression of <Emphasis Type="Italic">Camelid</Emphasis> nanobodies in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

Nanobodies (or VHHs) are single-domain antigen-binding fragments derived from Camelid heavy chain-only antibodies. Their small size, monomeric behaviour, high stability and solubility, and ability to bind epitopes not accessible to conventional antibodies make them especially suitable for many therapeutic and biotechnological applications. Here we describe high-level expression, in Nicotiana benthamiana, of three versions of an anti-hen egg white lysozyme (HEWL) nanobody which include the original VHH from an immunized library (cAbLys3), a codon-optimized derivative, and a codon-optimized hybrid nanobody comprising the CDRs of cAbLys3 grafted onto an alternative ‘universal’ nanobody framework. His6- and StrepII-tagged derivatives of each nanobody were targeted for accumulation in the cytoplasm, chloroplast and apoplast using different pre-sequences. When targeted to the apoplast, intact functional nanobodies accumulated at an exceptionally high level (up to 30% total leaf protein), demonstrating the great potential of plants as a nanobody production system.     

Expression Cloning of Camelid Nanobodies Specific for Xenopus Embryonic Antigens

Developmental biology relies heavily on the use of conventional antibodies, but their production and maintenance involves significant effort. Here we use an expression cloning approach to identify variable regions of llama single domain antibodies (known as nanobodies), which recognize specific embryonic antigens. A nanobody cDNA library was prepared from lymphocytes of a llama immunized with Xenopus embryo lysates. Pools of bacterially expressed cDNAs were sib-selected for the ability to produce specific staining patterns in gastrula embryos. Three different nanobodies were isolated: NbP1 and NbP3 stained yolk granules, while the reactivity of NbP7 was predominantly restricted to the cytoplasm and the cortex. The isolated nanobodies recognized specific protein bands in immunoblot analysis. A reverse proteomic approach identified NbP1 target antigen as EP45/Seryp, a serine protease inhibitor. Given the unique stability of nanobodies and the ease of their expression in diverse systems, we propose that nanobody cDNA libraries represent a promising resource for molecular markers for developmental biology.     

Optimization of Recombinant Cytochrome P450 2C9 Protein Production in Escherichia coli DH5α by Statistically-Based Experimental Design

Cytochrome P450 (CYP) 2C9 is of major importance in drug metabolism. However, the low yield of recombinant CYP2C9 protein in E. coli strains prevents its extensive use in the study of in vitro drug metabolism. In the present study, Taguchi design and desirability function were first used to investigate the effect of medium components (glycerol, δ-ALA, IPTG, ampicillin, chloramphenicol, inoculum density, peptone, thiamine, trace elements, NH₄Cl, and MgSO₄) on recombinant human CYP2C9 production by E. coli DH5α. An L₁₂ (2¹¹) orthogonal array was used to design the experiments to screen out the most influential factors. The CYP concentration and the specific content of CYP were considered as two product quality variables. A desirability function was applied to combine these two qualities as a single objective function. Optimization via central composite design (CCD) was then undertaken to yield the best performance. The confirmation experiments indicated that the expression performance under the optimized conditions was better than those obtained under other conditions. A compromise between conflicting goals, such as achievement of good yield of recombinant CYP2C9 and facility of the following purification, was found by means of the desirability function D. This is the first report that combined Taguchi design and CCD, and performed experiments in a multiresponse framework to optimize the production of human CYP in a recombinant E. coli strain.     

Enhancement of extracellular bispecific anti‐MUC1 nanobody expression in E. coli BL21 (DE3) by optimization of temperature and carbon sources through an autoinduction condition

Escherichia coli is one of the most suitable hosts for production of antibodies and antibody fragments. Antibody fragment secretion to the culture medium improves product purity in cell culture and diminishes downstream costs. In this study, E. coli strain BL21 (DE3) harboring gene encoding bispecific anti‐MUC1 nanobody was selected, and the autoinduction methodology for expression of bispecific anti‐MUC1 nanobody was investigated. Due to the replacement of IPTG by lactose as inducer, less impurity and toxicity in the final product were observed. To increase both intracellular and extracellular nanobody production, initially, the experiments were performed for the key factors including temperature and duration of protein expression. The highest amount of nanobody was produced after 21 h at 33°C. The effect of different carbon sources, glycerol, glucose, lactose, and glycine as a medium additive at optimum temperature and time were also assessed by using response surface methodology. The optimized concentrations of carbon sources were obtained as 0.75% (w/v), 0.03% (w/v), 0.1% (w/v), and 0.75% (w/v) for glycerol, glucose, lactose, and glycine, respectively. Finally, the production of nanobody in 2 L fermenter under the optimized autoinduction conditions was evaluated. The results show that the total titer of 87.66 µg/mL anti‐MUC1 nanobody, which is approximately seven times more than the total titer of nanobody produced in LB culture medium, is 12.23 µg/L .     

Exploring single-domain antibody thermostability by molecular dynamics simulation

Abstract

Single-domain antibodies also known as nanobodies are recombinant antigen-binding domains that correspond to the heavy-chain variable region of camelid antibodies. Previous experimental studies showed that the nanobodies have stable and active structures at high temperatures. In this study, the thermal stability and dynamics of nanobodies have been studied by employing molecular dynamics simulation at different temperatures. Variations in root mean square deviation, native contacts, and solvent-accessible surface area of the nanobodies during the simulation were calculated to analyze the effect of different temperatures on the overall conformation of the nanobody. Then, the thermostability mechanism of this protein was studied through calculation of dynamic cross-correlation matrix, principal component analyses, native contact analyses, and root mean square fluctuation. Our results manifest that the side chain conformation of some residues in the complementarity-determining region 3 (CDR3) and also the interaction between α-helix region of CDR3 and framework2 play a critical role to stabilize the protein at a high temperature.

Communicated by Ramaswamy H. Sarma     

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.     

Neutralization of SARS‐CoV‐2 by highly potent,hyperthermostable, and mutation‐tolerant nanobodies

Monoclonal anti‐SARS‐CoV‐2 immunoglobulins represent a treatment option for COVID‐19. However, their production in mammalian cells is not scalable to meet the global demand. Single‐domain (VHH) antibodies (also called nanobodies) provide an alternative suitable for microbial production. Using alpaca immune libraries against the receptor‐binding domain (RBD) of the SARS‐CoV‐2 Spike protein, we isolated 45 infection‐blocking VHH antibodies. These include nanobodies that can withstand 95°C. The most effective VHH antibody neutralizes SARS‐CoV‐2 at 17–50 pM concentration (0.2–0.7 µg per liter), binds the open and closed states of the Spike, and shows a tight RBD interaction in the X‐ray and cryo‐EM structures. The best VHH trimers neutralize even at 40 ng per liter. We constructed nanobody tandems and identified nanobody monomers that tolerate the K417N/T, E484K, N501Y, and L452R immune‐escape mutations found in the Alpha, Beta, Gamma, Epsilon, Iota, and Delta/Kappa lineages. We also demonstrate neutralization of the Beta strain at low‐picomolar VHH concentrations. We further discovered VHH antibodies that enforce native folding of the RBD in the E. coli cytosol, where its folding normally fails. Such “fold‐promoting” nanobodies may allow for simplified production of vaccines and their adaptation to viral escape‐mutations.     

Targeting high affinity and epitope-distinct oligoclonal nanobodies to HER2 over-expressing tumor cells

Modern anti-HER2 antibody therapy tends to exploit a panel of different antibodies against different epitopes on the antigen. For this aim, nanobodies are very striking targeting agents and can be easily produced against any cell-specific membrane antigen. The oligoclonal nanobodies can be used to block more than one functional epitope on a target antigen and inhibit the generation of escape variants associated with cancer therapy. In this study, 12 nanobody clones selected from an immune camel library were examined for their ability to differ between tumor markers. These oligoclonal nanobodies targeted breast cancer cells better than each individual nanobody. In epitope mapping, several nanobodies overlapped in the epitope recognized by trastuzumab and some of the non-overlapping nanobodies could affect the binding of trastuzumab to HER2. This study demonstrates that the oligoclonal nanobodies are potential therapeutic tools that can be used instead of, or in combination with trastuzumab to assess tumor viability during treatment.     

Applications of nanobodies in plant science and biotechnology

Key message

Present review summarizes the current applications of nanobodies in plant science and biotechnology, including plant expression of nanobodies, plant biotechnological applications, nanobody-based immunodetection, and nanobody-mediated resistance against plant pathogens.

Abstract

Nanobodies (Nbs) are variable domains of heavy chain-only antibodies (HCAbs) isolated from camelids. In spite of their single domain structure, nanobodies display many unique features, such as small size, high stability, and cryptic epitopes accessibility, which make them ideal for sophisticated applications in plants and animals. In this review, we summarize the current applications of nanobodies in plant science and biotechnology, focusing on nanobody expression in plants, plant biotechnological applications, determination of plant toxins and pathogens, and nanobody-mediated resistance against plant pathogens. Prospects and challenges of nanobody applications in plants are also discussed.

     

Structural Basis of Epitope Recognition by Heavy-Chain Camelid Antibodies

Truncated versions of heavy-chain antibodies (HCAbs) from camelids, also termed nanobodies, comprise only one-tenth the mass of conventional antibodies, yet retain similar, high binding affinities for the antigens. Here we analyze a large data set of nanobody–antigen crystal structures and investigate how nanobody–antigen recognition compares to the one by conventional antibodies. We find that nanobody paratopes are enriched in aromatic residues just like conventional antibodies, but additionally, they also bear a more hydrophobic character. Most striking differences were observed in the characteristics of the antigen's epitope. Unlike conventional antibodies, nanobodies bind to more rigid, concave, conserved and structured epitopes enriched with aromatic residues. Nanobodies establish fewer interactions with the antigens compared to conventional antibodies, and we speculate that high binding affinities are achieved due to less unfavorable conformational and more favorable solvation entropy contributions. We observed that interactions with antigen are mediated not only by three CDR loops but also by numerous residues from the nanobody framework. These residues are not distributed uniformly; rather, they are concentrated into four structurally distinct regions and mediate mostly charged interactions. Our findings suggest that in some respects nanobody–antigen interactions are more similar to the general protein–protein interactions rather than antibody–antigen interactions.     

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。     

纳米抗体在癌症治疗中的应用

骆驼科动物的体内会产生一种缺失轻链的抗体,被称为重链抗体,又叫做nanobody。这种抗体只包含一个可变区,具有高亲和力、高稳定性、强组织穿透性、高效表达等优点,同时具有低毒性和低免疫原性等特性,适用于诊断、治疗和充当多种领域的实验研究工具。文中将主要讨论nanobody在癌症治疗中的应用,为nanobody的进一步研发提供思路。     

Selection of non-competitive leptin antagonists using a random nanobody-based approach

The adipocyte-derived cytokine leptin acts as a metabolic switch, connecting the body's metabolism to high-energy consuming processes such as reproduction and immune responses. Accumulating evidence suggests that leptin plays a role in human pathologies, such as autoimmune diseases and cancer, thus providing a rationale for the development of leptin antagonists. In the present study, we generated and evaluated a panel of neutralizing nanobodies targeting the LR (leptin receptor). A nanobody comprises the variable domain of the naturally occurring single-chain antibodies found in members of the Camelidae family. We identified three classes of neutralizing nanobodies targeting different LR subdomains: i.e. the CRH2 (cytokine receptor homology 2), Ig-like and FNIII (fibronectin type?III) domains. Only nanobodies directed against the CRH2 domain inhibited leptin binding. We could show that a nanobody that targets the Ig-like domain potently interfered with leptin-dependent regulation of hypothalamic NPY (neuropeptide Y) expression. As a consequence, daily intraperitoneal injection increased body weight, body fat content, food intake, liver size and serum insulin levels. All of these characteristics resemble the phenotype of leptin and LR-deficient animals. The results of the present study support proposed models of the activated LR complex, and demonstrate that it is possible to block LR signalling without affecting ligand binding. These nanobodies form new tools to study the mechanisms of BBB (blood-brain barrier) leptin transport and the effect of LR inhibition in disease models.     

Exploiting sequence and stability information for directing nanobody stability engineering

BackgroundVariable domains of camelid heavy-chain antibodies, commonly named nanobodies, have high biotechnological potential. In view of their broad range of applications in research, diagnostics and therapy, engineering their stability is of particular interest. One important aspect is the improvement of thermostability, because it can have immediate effects on conformational stability, protease resistance and aggregation propensity of the protein.MethodsWe analyzed the sequences and thermostabilities of 78 purified nanobody binders. From this data, potentially stabilizing amino acid variations were identified and studied experimentally.ResultsSome mutations improved the stability of nanobodies by up to 6.1 °C, with an average of 2.3 °C across eight modified nanobodies. The stabilizing mechanism involves an improvement of both conformational stability and aggregation behavior, explaining the variable degree of stabilization in individual molecules. In some instances, variations predicted to be stabilizing actually led to thermal destabilization of the proteins. The reasons for this contradiction between prediction and experiment were investigated.ConclusionsThe results reveal a mutational strategy to improve the biophysical behavior of nanobody binders and indicate a species-specificity of nanobody architecture.General significanceThis study illustrates the potential and limitations of engineering nanobody thermostability by merging sequence information with stability data, an aspect that is becoming increasingly important with the recent development of high-throughput biophysical methods.     

Increase in Silk Production by the Silkworm,Bombyx morì L., due to Oral Administration of a Juvenile Hormone Analog

(R)-1,3-butanediol ((R)-1,3-BD) is an important substrate for the synthesis of industrial chemicals. Despite its large demand, a bioprocess for the efficient production of 1,3-BD from renewable resources has not been developed. We previously reported the construction of recombinant Escherichia coli that could efficiently produce (R)-1,3-BD from glucose. In this study, the fermentation conditions were optimized to further improve 1,3-BD production by the recombinant strain. A batch fermentation was performed with an optimized overall oxygen transfer coefficient (82.3?h^?1) and pH (5.5); the 1,3-BD concentration reached 98.5?mM after 36?h with high-yield (0.444?mol (mol glucose)^?1) and a high maximum production rate (3.63?mM?h^?1). In addition, a fed-batch fermentation enabled the recombinant strain to produce 174.8?mM 1,3-BD after 96?h cultivation with a yield of 0.372?mol (mol glucose)^?1, a maximum production rate of 3.90?mM?h^?1, and a 98.6% enantiomeric excess (% ee) of (R)-1,3-BD.     

Nanobodies, a promising tool for species-specific diagnosis of Taenia solium cysticercosis

Taenia solium cysticercosis is a major helminth zoonosis in developing countries. Pigs are the intermediate hosts mediating transmission of infection. Specific assays to diagnose living cysts in pigs are lacking. The monoclonal-based antigen detection ELISA is genus-specific and cross-reactions with Taenia hydatigena hamper the use of this test to screen pigs. We, therefore, aimed to introduce nanobodies, camelid-derived single-domain antibodies specific for T. solium cysticercosis, to develop unambiguous tests. Nanobodies were cloned following immunization of two dromedaries with T. solium antigen and eight T. solium-specific nanobodies were selected after phage display. Their binding characteristics and potential for the diagnosis of porcine cysticercosis were investigated. The nanobodies do not cross-react with T. hydatigena, Taenia saginata, Taenia crassiceps or Trichinella spiralis and were categorized into four epitope-binding groups. The target protein was identified as 14 kDa diagnostic glycoprotein (Ts14), but the nanobodies also reacted with other proteins of the same family. Nanobodies were tested in a sandwich ELISA with cyst fluid, and one particular nanobody detected its cognate serum antigens in a species-specific inhibition ELISA. Considering their beneficial production and stability properties, these highly specific nanobodies constitute a promising tool to diagnose cysticercosis after further improvement of the sensitivity and future assay validation.