Exploiting recombination in single bacteria to make large phage antibody libraries

The creation of large phage antibody libraries has become an important goal in selecting antibodies against any antigen. Here we describe a method for making libraries so large that the complete diversity cannot be accessed using traditional phage technology. This involves the creation of a primary phage scFv library in a phagemid vector containing two nonhomologous lox sites. Contrary to the current dogma, we found that infecting Cre recombinase-expressing bacteria by such a primary library at a high multiplicity of infection results in the entry of many different phagemid into the cell. Exchange of Vh and Vl genes between such phagemids creates many new V h/Vl combinations, all of which are functional. On the basis of the observed recombination, the library is calculated to have a diversity of 3x1011. A library created using this method was validated by the selection of high affinity antibodies against a large number of different protein antigens.     

人源噬菌体抗体库的构建及抗VEGF抗体的初步筛选分析

应用噬菌体表面呈递技术构建人抗体组合文库 .筛选获得了结合血管内皮细胞生长因子( VEGF)的人噬菌体 Fab抗体 ,并对所获抗体的多样性进行了进一步分析 .从不同人群外周血淋巴细胞提取总 RNA,经反转录后采用家族特异性免疫球蛋白可变区基因引物与免疫球蛋白信肽序列引物 ,通过改变 PCR条件或半套式扩增分别获得全部亚型的轻、重链抗体 Fab段 ,并重组到噬粒载体 p Comb3H中 ,经电转化大肠杆菌 XL- 1 Blue,构建了 1 .5× 1 0 8完整组合抗体库 .利用 VEGF12 1对该库经过 4轮固相筛选后 ,获得 1 2个可与 VEGF特异结合的阳性克隆 .酶谱分析表明了所获抗体克隆的多样性 .为通过基因工程改造 ,进一步获得可用于临床的人源 VEGF抗体奠定了基础 .     

人源天然ScFv噬菌体抗体库的构建及鉴定

噬菌体抗体库技术是获得治疗性抗体的一条重要途径。以20份健康人外周血为样本,通过提取淋巴细胞、逆转录－PCR（RT PCR）、抗体可变区基因的扩增、重叠PCR获得单链抗体（ScFv）基因,将ScFv克隆入噬粒载体,通过近300次的电转化获得了库容量为1.3×109的全人源天然ScFv噬菌体抗体库。通过随机挑克隆测序和用5种不同抗原筛选对抗体库进行了初步验证。随机测序表明抗体库具有较好的多样性,用5种不同抗原对其进行筛选,均获得了特异性噬菌体抗体的不同富集,表明成功构建了一个多样性良好的人源天然ScFv噬菌体抗体库。     

Polyclonal Fab phage display libraries with a high percentage of diverse clones to Cryptosporidium parvum glycoproteins

The protozoan parasite Cryptosporidium parvum is regarded as a major public health problem world-wide, especially for immunocompromised individuals. Although no effective therapy is presently available, specific immune responses prevent or terminate cryptosporidiosis and passively administered antibodies have been found to reduce the severity of infection. Therefore, as an immunotherapeutic approach against cryptosporidiosis, we set out to develop C. parvum-specific polyclonal antibody libraries, standardised, perpetual mixtures of polyclonal antibodies, for which the genes are available. A combinatorial Fab phage display library was generated from the antibody variable region gene repertoire of mice immunised with C. parvum surface and apical complex glycoproteins which are believed to be involved in mediating C. parvum attachment and invasion. The variable region genes used to construct this starting library were shown to be diverse by nucleotide sequencing. The library was subjected to one round of antigen selection on C. parvum glycoproteins or a C. parvum oocyst/sporozoite preparation. The two selected libraries showed specific reactivity to the glycoproteins as well as to the oocyst/sporozoite preparation, with 50-73% antigen-reactive members. Fingerprint analysis of individual clones from the two antigen-selected libraries showed high diversity, confirming the polyclonality of the selected libraries. Furthermore, immunoblot analysis on the oocyst/sporozoite and glycoprotein preparations with selected library phage showed reactivity to multiple bands, indicating diversity at the antigen level. These C. parvum-specific polyclonal Fab phage display libraries will be converted to libraries of polyclonal full-length antibodies by mass transfer of the selected heavy and light chain variable region gene pairs to a mammalian expression vector. Such polyclonal antibody libraries would be expected to mediate effector functions and provide optimal passive immunity against cryptosporidiosis.     

Bacteriophage Vehicles for Phage Display: Biology,Mechanism, and Application

The phage display technique is a powerful tool for selection of various biological agents. This technique allows construction of large libraries from the antibody repertoire of different hosts and provides a fast and high-throughput selection method. Specific antibodies can be isolated based on distinctive characteristics from a library consisting of millions of members. These features made phage display technology preferred method for antibody selection and engineering. There are several phage display methods available and each has its unique merits and application. Selection of appropriate display technique requires basic knowledge of available methods and their mechanism. In this review, we describe different phage display techniques, available bacteriophage vehicles, and their mechanism.     

Synthetic approach to the generation of antibody diversity

The in vitro antibody discovery technologies revolutionized the generation of target-specific antibodies that traditionally relied on the humoral response of immunized animals. An antibody library, a large collection of diverse, pre-constructed antibodies, can be rapidly screened using in vitro display technologies such as phage display. One of the keys to successful in vitro antibody discovery is the quality of the library diversity. Antibody diversity can be obtained either from natural B-cell sources or by the synthetic methods that combinatorially generate random nucleotide sequences. While the functionality of a natural antibody library depends largely upon the library size, various other factors can affect the quality of a synthetic antibody library, making the design and construction of synthetic antibody libraries complicated and challenging. In this review, we present various library designs and diversification methods for synthetic antibody library. From simple degenerate oligonucleotide synthesis to trinucleotide synthesis to physicochemically optimized library design, the synthetic approach is evolving beyond the simple emulation of natural antibodies, into a highly sophisticated method that is capable of producing high quality antibodies suitable for therapeutic, diagnostic, and other demanding applications. [BMB Reports 2015; 48(9): 489-494]     

Diversity of the Antibody Response to Tetanus Toxoid: Comparison of Hybridoma Library to Phage Display Library

Monoclonal antibodies are important tools in research and since the 1990s have been an important therapeutic class targeting a wide variety of diseases. Earlier methods of mAb production relied exclusively on the lengthy process of making hybridomas. The advent of phage display technology introduced an alternative approach for mAb production. A potential concern with this approach is its complete dependence on an in vitro selection process, which may result in selection of V_H-V_L pairs normally eliminated during the in vivo selection process. The diversity of V_H-V_L pairs selected from phage display libraries relative to an endogenous response is unknown. To address these questions, we constructed a panel of hybridomas and a phage display library using the spleen of a single tetanus toxoid-immunized mouse and compared the diversity of the immune response generated using each technique. Surprisingly, the tetanus toxoid-specific antibodies produced by the hybridoma library exhibited a higher degree of V_H-V_L genetic diversity than their phage display-derived counterparts. Furthermore, the overlap among the V-genes from each library was very limited. Consistent with the notion that accumulation of many small DNA changes lead to increased antigen specificity and affinity, the phage clones displayed substantial micro-heterogeneity. Contrary to previous reports, we found that antigen specificity against tetanus toxoid is encoded by both Vκ and V_H genes. Finally, the phage-derived tetanus-specific clones had a lower binding affinity than the hybridomas, a phenomenon thought to be the result of random pairing of the V-genes.     

A Novel Heavy Domain Antibody Library with Functionally Optimized Complementarity Determining Regions

Today a number of synthetic antibody libraries of different formats have been created and used for the selection of a large number of recombinant antibodies. One of the determining factors for successful isolation of recombinant antibodies from libraries lies in the quality of the libraries i.e. the number of correctly folded, functional antibodies contained in the library. Here, we describe the construction of a novel, high quality, synthetic single domain antibody library dubbed Predator. The library is based on the HEL4 domain antibody with the addition of recently reported mutations concerning the amino acid composition at positions critical for the folding characteristics and aggregation propensities of domain antibodies. As a unique feature, the CDR3 of the library was designed to mimic the natural human immune response by designating amino acids known to be prevalent in functional antibodies to the diversity in CDR3. CDR randomizations were performed using trinucleotide synthesis to avoid the presence of stop codons. Furthermore a novel cycle free elongation method was used for the conversion of the synthesized single stranded DNA containing the randomized CDRs into double stranded DNA of the library. In addition a modular approach has been adopted for the scaffold in which each CDR region is flanked by unique restrictions sites, allowing easy affinity maturation of selected clones by CDR shuffling. To validate the quality of the library, one round phage display selections were performed on purified antigens and highly complex antigen mixtures such as cultured eukaryotic cells resulting in several specific binders. The further characterization of some of the selected clones, however, indicates a reduction in thermodynamic stability caused by the inclusion the additional mutations to the HEL4 scaffold.     

Phage antibody fragments library combining a single human light chain variable region with immune mouse heavy chain variable regions

We describe the construction of a phage antibody fragments library which combines, in a single cloning step, a synthetic human light chain variable region (V(L)) with a diverse set of heavy chain variable regions, from a mouse immunized with the prostate specific antigen (PSA). Despite V(L) restriction, selection from this library rendered two different single chain Fv antibody fragments, specifically recognizing PSA. The human V(L), used as a general partner for mouse heavy chains, was constructed by linking the germline A27 gene and the J(K)1 minigene segment, both of which are prominently involved in human antibody responses. Our approach offers a fast and simple way to produce half-human molecules, while keeping the advantage of immunizing animals for high affinity antibodies.     

基于细胞的抗单增李斯特菌单域重链抗体的筛选及鉴定

【目的】获得针对单增李斯特菌的特异性单域重链抗体,并对筛选过程中特异性克隆的富集规律进行分析,为筛选具有种属特异性的噬菌体展示抗体提供参考。【方法】采用固相筛选技术,以热灭活的单增李斯特菌菌体为抗原,通过四轮常规筛选和一轮消减筛选,从驼源天然噬菌体展示文库中筛选针对单增李斯特菌的单域重链抗体。采用Phage-ELISA法,对后四轮筛选洗脱物中随机挑选的噬菌体进行鉴定,阳性克隆进行基因测序及结合特异性分析。通过多序列比对分析将获得的基因序列进行分组和统计。【结果】成功筛选到2株单增李斯特菌特异性的单域重链抗体。【结论】在优化的筛选条件下,基于全细胞的筛选方法能够获得特异性识别单增李斯特菌的单域重链抗体,消减筛选对于去除非特异性克隆是有效的和必要的。     

Phage versus phagemid libraries for generation of human monoclonal antibodies

Non-immune (na?ve) phage antibody libraries have become an important source of antibodies for reagent, diagnostic, and therapeutic use. To date, reported na?ve libraries have been constructed in phagemid vectors as fusions to pIII, yielding primarily single copy (monovalent) display of antibody fragments. For this work, we subcloned the single chain Fv (scFv) gene repertoire from a na?ve phagemid antibody library into a true phage vector to create a multivalently displayed scFv phage library. Compared to monovalently displayed scFv, multivalent phage display resulted in improved efficiency of display as well as antibody selection. A greater number of antibodies were obtained and at earlier rounds of selection. Such increased efficiency allows the screening for binding antibodies after a single round of selection, greatly facilitating automation. Expression levels of antigen-binding scFv were also higher than from the phagemid library. In contrast, the affinities of scFv from the phage library were lower than from the phagemid library. This could be overcome by utilizing the scFv in a multivalent format, by affinity maturation, or by converting the library to monovalent display after the first round of selection.     

人源抗狂犬病毒单克隆抗体Fab段基因的获得和表达

运用噬菌体表面呈现（phage display）技术获得了人源抗狂犬病毒糖蛋白基因工程单克隆抗体Fab段基因及其表达。从狂犬病毒PM株Vero细胞疫苗免疫的人抗凝血中分离获得外周淋巴细胞,提取细胞总RNA,通过RTPCR方法,用一组人IgG Fab基因4特异性引物,从合成的cDNA中扩增了一组轻链和重链Fab段基因,将轻链和重链Fab段基因,将轻链和重链先后克隆入噬菌体载体pComb3,成功地建立了抗狂犬病毒抗原的方法,对此抗体库进行富积筛选表达,成功地获得了抗狂犬病毒的人源单抗Fab段基因及其在大肠杆菌中的有效表达,对其中一株单抗G10进行了较为系统的分析,发现它与一株鼠源中和性狂犬病毒糖蛋白特异性单抗存在竞争,证实该单抗能识别狂犬病毒糖蛋白,其序列资料分析表明,该单抗为一株新的抗狂犬病毒人源基因工程抗体。     

噬菌体抗体库技术在肿瘤抗体药物研究中的进展及应用

目的:综述噬菌体抗体库技术的研究进展,介绍该技术的原理,构建,筛选和应用,为抗肿瘤抗体药物研发提供参考。方法:采用文献综述的方法,筛选近5年来噬菌体抗体库技术试验论文,对噬菌体抗体库技术的原理,构建,筛选和应用进行总结。结果:噬菌体抗体库主要分为免疫抗体库和非免疫抗体库两大类;噬菌体抗体库筛选技术包括亲和筛选、细胞筛选和生物体内筛选三种;噬菌体抗体库技术主要应用于肿瘤标志物的识别和肿瘤诊断,抗肿瘤抗体药物的筛选和制备。结论:噬菌体抗体库技术方便、快速、高效,可以在体外环境下培养,这些特点决定了其在肿瘤标志物的发现和肿瘤抗体药物研发中的广泛应用。目前噬菌体抗体库技术还存在一定缺陷,但技术的不断发展和革新必然使噬菌体抗体库技术成为研制抗体药物的新思路,极大促进了肿瘤抗体药物的研发。     

驼源天然单域重链抗体库的构建与鉴定

从未经主动免疫的健康羊驼(Lama pacos)外周血淋巴细胞中提取总RNA,反转录后作为第一轮PCR的模板。根据重链抗体保守区域设计引物,经巢式PCR法扩增获得了全套重链抗体可变区基因,将其克隆至噬菌粒pHEN1,电转化大肠杆菌TG1得到初级抗体库NAL,含有2×10⁷个独立克隆,菌落PCR和Hinf I酶切分析结果显示,克隆效率大于97%,文库的多样性良好。辅助噬菌体救援后,得到噬菌体展示文库命名为NA-PDL,滴度达10¹³CFU/ml。以真菌毒素人工抗原DON-MBSA为目标抗原,对NA-PDL进行了淘选,第二轮洗脱物中,阳性克隆率达36.4%,提示针对目标抗原的噬菌体颗粒得到了有效富集,文库NA-PDL多样性较好,为后续淘选针对特定抗原的单域重链抗体奠定了基础。     

天然兔源噬菌体单链抗体库的构建与鉴定

目的:构建天然兔源噬菌体单链抗体库。方法:采用RT-PCR法从未免疫的兔子脾脏中克隆得到抗体重链可变区(VH)与轻链可变区(VL)基因,重叠PCR将VH和VL拼接成scFv片段,将scFv连接到噬菌粒pComb3XSS上,电转入XL1-Blue菌中,得到单链抗体库,并用此抗体库筛选抗肌酸激酶抗体。结果:构建了容量为4×108,基因重组率95%的单链抗体库,DNA指纹图谱显示抗体库多样性良好。以肌酸激酶为抗原,从该库中筛到3株抗肌酸激酶的抗体。结论:分析表明构建的天然兔源单链抗体库质量良好,可用于快速筛选、制备多种单链抗体。     

Effective use of nitrocellulose-blotted antigens for phage display monoclonal antibody selection

The combinatorial phage display library approach to antibody repertoire cloning offers a powerful tool for the isolation of specific antibodies to defined target antigens. Panning strategy is often a very critical point for selecting antibody displayed on the surface of bacteriophages. Most selection strategies described to date have relied on the availability of purified and often recombinant antigen, providing the possibility to perform selections on a well defined antigen source. However, when the antigen is difficult to purify by means of laborious and time-consuming chromatography procedures, panning of phage antibody libraries has to be performed on complex antigen sources such as cell surfaces or tissue sections, or even by in vivo selection methods. This provides a series of technical and experimental complications. In the present work, we successfully generated a mouse monoclonal antibody fragment from a phage display library directed against protein E7 of HPV18 avoiding antigen purification as for immunizing mice as for antibody library selection. Our work demonstrates the feasibility of phage antibody selections on antigens transferred to a nitrocellulose membrane as solid support, using one-dimensional polyacrylamide gel electrophoresis system as the only practice to separate a given antigen present in bacterial crude cell lysate.     

Isolation of a novel neutralizing antibody fragment against human vascular endothelial growth factor from a phage-displayed human antibody repertoire using an epitope disturbing strategy

Following the clinical success of Bevacizumab, a humanized monoclonal antibody that blocks the interaction between vascular endothelial growth factor (VEGF) and its receptors, the search for new neutralizing antibodies targeting this molecule has continued until now. We used a human VEGF variant containing three mutations in the region recognized by Bevacizumab to direct antibody selection towards recognition of other epitopes. A total of seven phage-displayed antibody fragments with diverse binding properties in terms of inter-species cross-reactivity and sensitivity to chemical modifications of the antigen were obtained from a human phage display library. All of them were able to recognize not only the selector mutated antigen, but also native VEGF. One of these phage-displayed antibody fragments, denominated 2H1, was shown to compete with the VEGF receptor 2 for VEGF binding. Purified soluble 2H1 inhibited in a dose dependent manner the ligand-receptor interaction and abolished VEGF-dependent proliferation of human umbilical vein endothelial cells. Our epitope disturbing strategy based on a triple mutant target antigen was successful to focus selection on epitopes different from a known one. Similar approaches could be used to direct phage isolation towards the desired specificity in other antigenic systems.     

鼠源性抗雄性特异性抗原噬菌体Fab抗体的制备及分析

利用噬菌体抗体库筛选技术获得抗雄性特异性抗原的噬菌体Fab抗体,首次采用雄鼠脾细胞对鼠源性抗雄性特异性抗原噬菌体Fab抗体库进行3轮亲和富集和2轮雌鼠脾细胞吸附,对筛选后特异性噬菌体Fab抗体进行ELISA分析,重组率鉴定及基因测序分析。结果显示,5次筛选后的15个菌落中有9个能产生抗雄性特异性抗原特异性噬菌体抗体,噬菌体Fab抗体的基因重组率为60%,E5克隆的重链、轻链可变区序列分别属于VH1和VκⅣ基因家族,这为挑选出高亲和力的抗雄性特异性抗原噬菌体Fab抗体奠定了实验基础,将推进雄性特异性抗原及其抗体的研究进程,并为性别控制研究开创新途径。     

Antibody VH and VL recombination using phage and ribosome display technologies reveals distinct structural routes to affinity improvements with VH-VL interface residues providing important structural diversity

In vitro selection technologies are an important means of affinity maturing antibodies to generate the optimal therapeutic profile for a particular disease target. Here, we describe the isolation of a parent antibody, KENB061 using phage display and solution phase selections with soluble biotinylated human IL-1R1. KENB061 was affinity matured using phage display and targeted mutagenesis of VH and VL CDR3 using NNS randomization. Affinity matured VHCDR3 and VLCDR3 library blocks were recombined and selected using phage and ribosome display protocol. A direct comparison of the phage and ribosome display antibodies generated was made to determine their functional characteristics.     

Antibody VH and VL recombination using phage and ribosome display technologies reveals distinct structural routes to affinity improvements with VH-VL interface residues providing important structural diversity

In vitro selection technologies are an important means of affinity maturing antibodies to generate the optimal therapeutic profile for a particular disease target. Here, we describe the isolation of a parent antibody, KENB061 using phage display and solution phase selections with soluble biotinylated human IL-1R1. KENB061 was affinity matured using phage display and targeted mutagenesis of VH and VL CDR3 using NNS randomization. Affinity matured VHCDR3 and VLCDR3 library blocks were recombined and selected using phage and ribosome display protocol. A direct comparison of the phage and ribosome display antibodies generated was made to determine their functional characteristics.