Advances in phage display technology for drug discovery

Introduction: Over the past decade, several library-based methods have been developed to discover ligands with strong binding affinities for their targets. These methods mimic the natural evolution for screening and identifying ligand–target interactions with specific functional properties. Phage display technology is a well-established method that has been applied to many technological challenges including novel drug discovery.

Areas covered: This review describes the recent advances in the use of phage display technology for discovering novel bioactive compounds. Furthermore, it discusses the application of this technology to produce proteins and peptides as well as minimize the use of antibodies, such as antigen-binding fragment, single-chain fragment variable or single-domain antibody fragments like VHHs.

Expert opinion: Advances in screening, manufacturing and humanization technologies demonstrate that phage display derived products can play a significant role in the diagnosis and treatment of disease. The effects of this technology are inevitable in the development pipeline for bringing therapeutics into the market, and this number is expected to rise significantly in the future as new advances continue to take place in display methods. Furthermore, a widespread application of this methodology is predicted in different medical technological areas, including biosensing, monitoring, molecular imaging, gene therapy, vaccine development and nanotechnology.     

Rapid isolation of single-chain antibodies by phage display technology directed against one of the most potent marine toxins: Palytoxin

Several recombinant antibodies against one of the most potent marine toxins, Palytoxin (PlTX), were obtained using two naive human semi-synthetic phage display libraries (Tomlinson I and J) as an effective method for generating specific anti-toxin single-chain variable fragment (scFv) antibodies. After four rounds of panning and selection on free palytoxin adsorbed immunotubes, individual clones were isolated, sequenced and characterized by Enzyme-Linked Immunosorbent Assay (ELISA). Four phage-antibody clones specifically recognized the toxin. A competitive ELISA assay was optimized with one of these phage antibodies giving a very reproducible standard curve with a linear regression (R² = 0.9945), showing a working range of 0.0005–500 ng mL⁻¹. Several spiked shellfish samples were analysed by competitive ELISA to determine the accuracy of the assay, with a mean recovery rate of 90%. This study demonstrates that phage display libraries provide a valuable system for the easy and rapid generation of specific antibody fragments directed against difficult antigenic targets, such as free small molecules. Large-scale, low-cost production of anti-palytoxin scFv antibodies in Escherichia coli (E. coli) is an exciting prospect for the development of rapid and simple detection methods. Our results suggest that anti-palytoxin phage antibodies could be a valuable tool with competitive ELISA to detect palytoxin in natural shellfish samples.     

Development of an inhibitory antibody fragment to human tissue factor using phage display technology

Tissue factor is involved in the etiology of thrombotic diseases initiating the thrombosis associated with the inflammation that occurs during infection. The prevention of blood coagulation and inflammation is of primary importance in a number of pathological situations. A single‐chain variable antibody fragment of molecular weight of 26 kD that inhibits the action of human tissue factor was selected by phage display technology, purified and tested for its tissue factor inhibitory effect, purified on a protein A column, and its purity evaluated on SDS‐PAGE. The effects of the antibody fragment on prothrombin times, Factor Xa production, and thrombin generation were assessed with increasing fragment concentrations, using chromogenic and fluorometric substrates. The antibody fragment dose‐dependently prolonged the prothrombin time (IC₅₀=0.5 μM) and delayed the lag phase before the thrombin generation burst and the peak thrombin concentration in the thrombin generation assay. The effect on thrombin generation was more pronounced in thrombophilic plasma than in normal plasma. Antibody‐based tissue factor inhibitors therefore may provide an effective treatment for thrombotic disease without serious bleeding complications. Drug Dev Res 2009. © 2009 Wiley‐Liss, Inc.     

噬菌体展示技术体内筛选小鼠肾脏特异性多肽

目的利用噬菌体随机多肽文库对小鼠进行体内筛选,获得小鼠肾脏血管特异性多肽。方法将噬菌体多肽文库经尾静脉注射入小鼠体内,循环10 min后进行心脏灌注,收获小鼠肾脏,经过洗涤研磨后得到与肾脏血管特异性结合的噬菌体,该噬菌体体外扩增后被用于下一轮筛选,三轮筛选后随机挑取24个阳性噬菌体克隆送测序,并分析这些序列之间的共同序列。结果经过三轮筛选,特异性结合于小鼠肾脏血管的噬菌体得到富集,测序结果显示多肽序列VSASYHR出现的概率最大（62.5%）,其次为GQWGARG（25.0%）。结论利用噬菌体多肽文库对小鼠进行体内筛选可以得到小鼠肾脏血管特异性多肽。     

抗人B细胞淋巴瘤噬菌体单链抗体库的构建

目的构建抗人B细胞淋巴瘤单链抗体(ScFv)库。方法从人B细胞淋巴瘤细胞系Raji细胞免疫的BALB/c小鼠脾细胞中提取mRNA,RT-PCR扩增重、轻链可变区基因片断,连接成ScFv基因并扩增,将其克隆到噬菌体载体pHEN1中,构建成单链噬菌体抗体库。结果抗体库容量为3.4×106,约100%的噬菌体基因中有ScFv基因的插入。结论成功构建了抗人B细胞淋巴瘤噬菌体单链抗体库,方便进一步筛选抗人B细胞淋巴瘤抗体。     

Monoclonal antibody fragment from combinatorial phage display library neutralizes alpha-latrotoxin activity and abolishes black widow spider venom lethality, in mice.

Alpha-latrotoxin (alpha-ltx), a component of the venom of black widow spiders (BWSV), binds to higher vertebrates presynaptic nerve terminals, stimulating massive neurotransmitter release. This neurotoxic protein is responsible for most of the symptoms elicited in men by the bite of black widow spider (BWS), i.e. a neurological syndrome named latrodectism. By reasoning that targeting this single component would abrogate most of the effect of BWS envenomation, we took advantage of the antibody phage display technology to generate monoclonal Fab fragments able to bind and neutralize the alpha-ltx. To this aim, we immunized Balb/c mice with purified toxin and cloned their antibody repertoire in the pCombIII phage display vector. By combining a high-stringency affinity selection with a sensitive 45Ca(2+) uptake assay, we isolated a Fab fragment (FM1) able to bind the alpha-ltx in the low nM range and neutralize its ionophore activity, in vitro and in vivo. After the onset of overt symptomatology, administration of FM1 to experimentally envenomed mice induced remission of symptoms and prevented lethality. Since alpha-ltx is the only molecule responsible for the great toxicity of BWS bites in mammals, the FM1 Fab, highly effective in neutralizing the toxin in vivo, represents a promising immunotherapy reagent for treating latrodectic patients.     

Evolution of phage display technology: from discovery to application

Phage display technology as a selection-based system is an attractive method for evolution of new biological drugs. Unique ability of phage libraries for displaying proteins on bacteriophage surfaces enable them to make a major contribution in diverse fields of researches related to the diagnosis and therapy of diseases. One of the great challenges facing researchers is the modification of phage display technology and the development of new applications. This article reviews the molecular basis of phage display library, and summarizes the novel and specific applications of this technique in the field of biological drugs development including therapeutic antibodies, peptides, vaccines, and catalytic antibodies.     

抗体药物的设计及其研究进展

先进的展示技术为重组抗体的筛选和改造提供了有效而灵活的方法.通过展示技术可以获得更适用于诊断和治疗的优化分子,而后者不能直接通过动物免疫接种的方式获得.展示技术可通过自动化或与新一代测序技术和阵列技术联合应用得到进一步提高,形成高通量筛选技术平台,用于筛选多种对应大量基因组编码靶标的抗体.从头设计抗体已开始成为制造新抗体的一种策略,但仍具有技术挑战性.然而,抗体-抗原结构数据库的不断增加和计算工具的不断开发,都表明这些挑战是可以克服的.预计未来以物理分子模拟知识为基础的计算方法,将成为提高抗体设计准确性和成功性的主要驱动力.     

Analysis of the substrate recognition domain determinants of botulinum type B toxin using phage display.

The botulinum neurotoxin endopeptidases appear to recognise their intracellular protein substrates via two distinct sites: the cleavage site sequence and a 'recognition site' motif. In the present study phage display has been employed to generate a library of vesicle-associated membrane protein (VAMP2) variants in which the toxin recognition motif (part of the SNARE motif ELDDRADA) has been modified. VAMP (1-94) was displayed on the surface of M13 bacteriophage and this fragment was recognised and cleaved by botulinum neurotoxin type B (BoNT/B). A phage-displayed library was constructed in which six residues of the recognition domain (VAMP residues 63-68; wild-type sequence LDDRAD) were randomised, and a selection method established for identifying cleaved VAMP variants. Sequence analysis of 24 clones revealed that 5 contained two acidic residues although none corresponded to the native sequence. Cleavage was reduced compared to wild-type VAMP, and cleavage of mutants containing no acidic residues was also observed. The data are discussed in relation to the substrate recognition mechanism of BoNT/B.     

抗核抗体对不同抗原的免疫应答

目的　探讨体内产生抗核抗体的抗原。方法　分别以羊红细胞、伤寒沙门菌及鸡血清免疫小鼠 ,同时设立生理盐水对照组 ,每组 10只 ,采用商品化酶联免疫法检测抗核抗体试剂盒 ,将酶结合物 (酶标抗人IgG)换成酶标金黄色葡萄球菌A蛋白 (SPA) ,建立检测抗核抗体的SPA ELISA法 ,并使其性能保持不变 ,用其检测小鼠的抗核抗体。结果　新建立的检测抗核抗体的SPA ELISA法与原法检测同一批标本 ,相关系数为 0 931,P <0 0 1。小鼠血清抗核抗体检测结果 (阳性率 ) :羊红细胞组为 90 % (9 10 ) ,伤寒沙门菌为 70 % (7 10 ) ,鸡血清组为 10 0 % (10 10 ) ,生理盐水对照组未检出抗核抗体。各实验组与对照组比较差异有显著性 (P <0 0 1)。结论　体内抗核抗体可能是一类由不同抗原刺激非特异产生的抗体。     

An approach towards development of monoclonal IgY antibodies against SARS CoV-2 spike protein (S) using phage display method: A review

The present state of diagnostic and therapeutic developmental race for vaccines against the SARS CoV-2 (nCOVID-19) focuses on prevention and control of this global pandemic which also represents a critical challenge to the global health community. Although development of novel vaccines can prevent the SARS CoV-2 infections, it is still impeded by several other factors and therefore novel approaches towards treatment and management of this disease is the urgent need. Passive immunotherapy plays a vital role as a possible alternative to meet this challenge and among various antibody sources, chicken egg yolk antibodies (IgY) can be used as an alternative to mammalian antibodies which have been previously studied against SARS CoV outbreak in China. In this review, we discuss the strategies for the use of chicken egg yolk (IgY) antibodies in the development of rapid diagnosis and immunotherapy against SARS CoV-2. Also, IgY antibodies have previously been used against various respiratory bacterial and viral infections in humans and animals. Compared to mammalian antibodies (IgG), chicken egg yolk antibodies (IgY) have greater binding affinity to specific antigens, ease of extraction and lower production costs, hence possessing remarkable pathogen-neutralizing activity of pathogens in respiratory and lungs. We provide an overall importance for the use of monoclonal chicken egg yolk antibodies (IgY) using phage display method describing their potential passive immunotherapeutic application for the treatment and prevention of SARS CoV-2 infection which is simple, fast and safe way of approach for treating patients effectively.     

In vitro optimization of liposomal nanocarriers prepared from breast tumor cell specific phage fusion protein

Fusion proteins created by phage display peptides with tumor cell specificity and the pVIII major coat protein of filamentous phages have been explored recently as a simple and cost-effective means for preparing tumor-targeted liposomes that improve the cytotoxicity of anticancer drugs in vitro. The next step in the development of this approach is the optimization of the liposome composition for the maximum targeting activity and subsequent testing in vivo. This study aimed to investigate the impact of preparation protocols, lipid composition and phage protein content on the targeting efficiency of phage protein-modified liposomes. Analysis of size, zeta potential and morphology was used to investigate the effect of preparation protocols on the stability and homogeneity of the phage liposomes. A previously developed coculture targeting assay and a factorial design approach were used to determine the role of lipid composition of the liposomal membrane on the target cell specificity of the phage liposomes. Western blot combined with proteinase K treatment detected the orientation of targeted phage protein in liposomal membrane. Phage protein, DPPG and PEG_2k-PE showed positive effects on target specificity of phage liposomes. The results served to identify optimal formulation that offer an improved liposomal affinity for target tumor cells over the non-optimized formulation.     

单克隆抗体制备技术研究进展

 抗体药物的开发离不开抗体制备技术。随着分子生物学和二代测序技术的发展以及流式细胞分选技术的广泛应用,抗体制备技术也得以不断发展。此文概述了鼠杂交瘤技术、噬菌体展示技术、转基因小鼠技术等经典的单克隆抗体制备技术,重点介绍了新兴的单细胞逆转录PCR技术。     

肺癌特异性结合多肽的体外筛选和鉴定

目的应用噬菌体随机肽库技术筛选出与肺癌细胞特异性结合的多肽。方法以人肺癌细胞NCI-H1299为靶细胞,人胚肺细胞MRC-5为吸附细胞,对噬菌体随机12肽库进行3轮全细胞减性筛选后,随机挑取噬菌体克隆进行ELISA鉴定;对亲和力较高的阳性克隆进行DNA测序并翻译为氨基酸序列;化学合成异硫氰酸荧光素标记的多肽(FITC-ZS-5),采用细胞和组织免疫荧光法鉴定FITC-ZS-5与肺癌细胞的亲和力及特异性。结果通过3轮减性筛选后,与NCI-H1299细胞结合的噬菌体克隆得到有效的富集;ELISA结果显示5号克隆对NCI-H1299细胞亲和力最高,将其命名为Phage ZS-5;测序结果显示Phage ZS-5所表达的多肽序列在国内外均未见报道,细胞及组织免疫荧光实验结果显示FITC-ZS-5对肺癌细胞及组织具有较高的亲和力和特异性。结论应用噬菌体随机肽库技术筛选到肺癌靶向性多肽ZS-5,为肺癌的靶向治疗和诊断奠定基础。     

Identification of a novel peptide ligand targeting visceral adipose tissue via transdermal route by in vivo phage display

To find novel peptide ligands targeting visceral adipose tissue (visceral fat) via transdermal route, in vivo phage display screening was conducted by dermal administration of a phage-peptide library to rats and a peptide sequence, CGLHPAFQC (designated as TDA1), was identified as a targeting ligand to visceral adipose tissue through the consecutive transdermal biopannings. Adipocyte-specific affinity and transdermal activity of the TDA1 were validated in vitro and targeting ability of the dermally administered TDA1 to visceral adipose tissue was also confirmed in vivo. TDA1 was effectively translocated into systemic circulation after dermal administration and selectively targeted visceral adipose tissue without any preference to other organs tested. Fluorescent microscopic analysis revealed that the TDA1 could be specifically localized in the hair follicles of the skin, as well as in the visceral adipose tissue. Thus, we inferred that dermally administered TDA1 would first access systemic circulation via hair follicles as its transdermal route and then could target visceral fat effectively. The overall results suggest that the TDA1 peptide could be potentially applied as a homing moiety for delivery of anti-obesity therapeutics to visceral fat through the convenient transdermal pathway.     

深度学习辅助药物发现的研究进展

深度学习技术近年来取得了重大突破,被应用于医学、药学等多个领域。聚焦深度学习在创新药物发现中的发展和应用,对深度学习被用于蛋白结构预测、药物靶标预测、药物-靶标相互作用预测、药物合成路线设计、从头药物分子设计以及药物吸收、分布、代谢、排泄和毒性（ADMET）预测等代表性案例进行详细综述,同时总结了现有方法面临的问题和可能的解决思路,以期为深度学习辅助药物发现相关方法的发展和应用提供借鉴与思考。     

大鼠皮肤和肌肉挫伤mRNA差异显示技术中总RNA的提取和检测

目的研究大鼠皮肤和肌肉挫伤总RNA提取对差异显示技术的影响。方法使用Invitrogen TRIzol Reagent提取总RNA,由于皮肤与肌肉所含的蛋白比较多,所以改进了传统的提取步骤,可以减少杂质的残留。结果改进的Invitrogen TRIzol Reagent提取的RNA呈现28SrRNA,18S rRNA和5S rRNA3条较清晰的条带,其A260/A280值可达1.913,具有很高的纯度。结论大鼠皮肤和肌肉mRNA差异显示技术中,皮肤与肌肉总RNA的质量是十分关键的因素,通过对传统方法进行改进,建立起较完善的大鼠皮肤和肌肉mRNA差异显示技术体系,为开展损伤时间的分子鉴定与功能基因的分离、克隆奠定基础。     

Advances in nasal drug delivery through tight junction technology

New approaches for enhancing intranasal drug delivery based on recent discoveries on the molecular biology of tight junctions (TJ) are significantly improving the bioavailability of ‘non-Lipinsky’ small molecules, and peptide, protein and oligonucleotide drugs. As knowledge of the structure and function of the TJ has developed, so has the ability to identify mechanism-based TJ modulators using high-throughput molecular biology-based screening methods. The present review focuses on recent developments on the TJ protein complex as a lipid raft-like membrane microdomain, the emerging role of unique endocytic pathways in regulating TJ dynamics, and the utility of techniques such as RNA interference and phage display to study TJ components and identify novel peptides and related molecules that can modulate their function. Experimental and statistical methodologies used for the identification of new classes of TJ modulators are described, which are capable of reversibly opening TJ barriers with broad potential to significantly improve intranasal and, eventually, oral drug delivery. The development of an advanced intranasal formulation for the obesity therapeutic PYY_3-36, the endogenous Y2 receptor agonist is also reviewed.