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。