Efficient construction of a highly useful phage-displayed human antibody repertoire

We have constructed a highly useful phage-displayed human antibody repertoire with limited cloning efforts. Our strategy was to maximize diversity during the first steps of library construction through the use of various lymphoid sources from several donors, inclusion of different immunoglobulin isotypes, and performance of multiple separate amplification reactions with all possible combinations within a complex primer set. The resulting variable region collections were cloned to form a moderate size library, composed by 4.25x10(8) single chain antibody fragments. This repertoire was successfully used to retrieve binders to seven model antigens: six proteins and one 12 aa peptide. Binding affinities reached nanomolar and even subnanomolar range. Sequence diversity and V-gene usage variability among binders were proven. Our approach was not focused on absolute library size, but on a high quality sampling of variable regions from the human antibody repertoire.     

Effects of signal sequence on phage-displayed disulfide-stabilized single chain antibody variable fragment (sc-dsFv) libraries

Phage-displayed single chain variable fragment (scFv) libraries are powerful tools in antibody engineering. Disulfide-stabilized scFv (sc-dsFv) with an interface disulfide bond is structure-wise more stable than the corresponding scFv. A set of recently discovered signal sequences replacing the wild type (pelB) signal peptidase cleavage site in the c-region has been shown to be effective in rescuing the expression of sc-dsFv libraries on the phage surface. However, the effects of the other regions of the signal sequence on the expression of the sc-dsFv libraries and on the formation of the interface disulfide bond in the phage-displayed sc-dsFv have not been clear. In this work, selected novel signal sequence variants in the h-region were shown to be equally effective in promoting sc-dsFv library expression on the phage surface; the expression level and complexity of the sc-dsFv libraries were comparable to the corresponding scFv libraries produced with the wild-type (pelB) signal sequence. The interface disulfide bond in the phage-displayed sc-dsFv was proven to form to a large extent in the library variant ensemble generated with signal sequence variants in both the h-region and the c-region. The sc-dsFv engineering platform established in this work can be applied to many of the known scFv molecules which are in need of a more stable version for the applications under harsh conditions or for longer shelf-life.     

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]     

Selection and characterisation of binders based on homodimerisation of immunoglobulin V(H) domains

The antigen-binding surface of antibodies is formed by the heterodimerisation of the two variable domains of the light (V(L)) and heavy (V(H)) chains. We have previously described the spontaneous formation of V(H) dimers (VHD) in both bacteria and mammalian cells. The self-association of a single domain produces a homo-VHD, in which the two identical V(H) domains generate a unique symmetric surface for antigen binding that is never found in the normal V(L)/V(H) antibody binding site. We developed a phagemid vector for the construction of phage display libraries in which a cysteine residue, introduced at the C-terminus of the only V(H) cloned, allowed display of homo-VHDs. Panning of the library on different proteins yielded antigen specific binders against lysozyme, glutathione S-transferase and streptavidin. A lysozyme specific homo-VHD was further characterised with an apparent affinity determined to be 216+/-6.6 nM. Importantly, the results showed that its binding activity was fully dependent on the dimerisation of both identical V(H) domains.     

An antibody library for stabilizing and crystallizing membrane proteins - selecting binders to the citrate carrier CitS

Co-crystallization of membrane proteins with antibody fragments may emerge as a general tool to facilitate crystal growth and improve crystal quality. The bound antibody fragment enlarges the hydrophilic part of the mostly hydrophobic membrane protein, thereby increasing the interaction area for possible protein-protein contacts in the crystal. Additionally, it may restrain flexible parts or lock the membrane protein in a defined conformational state. For successful co-crystallization trials, the antibody fragments must be stable in detergents during the extended period of crystal growth and must be easily produced in amounts necessary for crystallography. Therefore, we constructed a library of antibody Fab fragments from a framework subset of the HuCAL GOLD library (Morphosys, Munich, Germany). By combining the most stable and well expressed frameworks, V(H)3 and V(kappa)3, with the further stabilizing constant domains, a Fab library with the desired properties was obtained in a standard phage display format. As a proof of principle, we selected binders with phage display against the detergent-solubilized citrate transporter CitS of Klebsiella pneumoniae. We describe efficient methods for the immobilization of the membrane protein during selection, for ELISA screening, and for BIAcore evaluation. We demonstrate that the selected Fab fragments form stable complexes with native CitS and recognize conformational epitopes with affinities in the low nanomolar range.     

Identification of single-chain antibody fragments specific against SARS-associated coronavirus from phage-displayed antibody library

To develop early diagnostic reagents, effective vaccines, and even drugs against SARS-associated coronavirus (SARS-CoV), the human single fold single-chain antibody fragments, (scFv) libraries I+J (Tomlinson I+J) were used to identify novel scFvs, which can specifically bind to SARS-CoV. Interestingly, two scFvs (B5 and B9) exhibited higher binding specificity to SARS-CoV with the OD(450) value 0.608 and 0.545, respectively, and their coding sequences shared the identical sequence composed of V(H) gene (351bp) and V(L) gene (327bp), so the two scFvs were uniformly named as SA59B and chosen for further analysis. SA59B scFv was expressed in soluble form in Escherichia coli HB2151 and purified by immobilized metal affinity chromatography. The soluble 30kDa SA59B scFv-antibody was verified in SDS-PAGE and Western-blot. The purified SA59B scFv-antibody was labeled with HRP by the glutaraldehyde method, and the concentration of HRP and SA59B scFv-antibody in the SA59B-HRP solution reached 2.4 and 2.28mg/ml, respectively. Then, the binding ability of SA59B-HRP to SARS-CoV was evaluated by ELISA with S/N of 11.6, indicating higher binding specificity between them. Finally, both the SA59B sequence specificity and its application for diagnosis, prophylaxis or therapy of SARS were discussed.     

Optimal construction of non-immune scFv phage display libraries from mouse bone marrow and spleen established to select specific scFvs efficiently binding to antigen

Monoclonal antibodies (MAbs) are widely applied in basic research, medicine, and the pharmaceutical industry. Recently, applications and generations of MAbs have been increasingly attracting attention in many research areas since MAbs could be produced in large quantities with the development of genetic technology and antibody engineering. On the other hand, in recent years, phage display system has been developed for high-throughput isolation and generation of novel MAbs that have high affinity with various antigens. This technology is capable of constructing "Library" containing billions of phage repertoires displaying various antibody fragments, and rapid selection of a specific MAb from this phage library. Additionally, this technology has a great advantage that MAbs can be generated without immunization to animals. However, there are still relatively few reports confirming that useful MAbs can be derived from non-immune antibody libraries. The latter, as undertaken by current methods, seem unable to achieve the high quality required to produce useful MAbs for any desired antigen because cloning of antibody gene from non-immune donors is inefficient. This problem is caused by the fact that their RT-PCR primer sets, PCR conditions, and efficiency of subcloning through construction of antibody gene library cannot encompass all the antibody diversity. In an attempt to overcome some of these earlier problems, here we describe an optimized method to establish a high quality, non-immune library from mouse bone-marrow and spleen, and assess its diversity in terms of content of multiple antibodies for a wide antigenic repertoire. As an example of the application of the methodology, we describe the selection of specific MAbs binding to Luciferase and identify at least 18 different clones. Using this non-immune mouse antibody library, we also obtained MAbs for VEGF, VEGF receptor 2, TNF-alpha, and Pseudomonas Exotoxin, confirming the high quality of the library and its suitability for this application.     

噬菌体抗体库筛选技术研究进展

噬菌体抗体库技术是一项新兴的基因工程抗体技术,应用这项技术获得高特异性抗体的关键之一就是筛选环节。根据抗原性质以及筛选目的的不同,筛选方法的选择也不相同,各种筛选策略的优化对中和抗体的获得有至关重要的作用。     

B3HM细胞免疫小鼠脾细胞scFv噬菌体展示文库的构建及表达

目的: 利用噬菌体展示技术构建B3HM细胞免疫小鼠的脾细胞表达scFv文库。方法: 用人骨髓细胞系B3HM细胞免疫小鼠,取其脾细胞采用RT-PCR方法扩增VH 和Vk基因并克隆入噬菌体展示表达载体,构建scFv文库,测定文库的库容量,BstNI酶切单克隆分析文库的多样性,对文库进行富集检测,鉴定单克隆噬菌体与B3HM细胞结合反应。结果:文库的库容为5×106cfu,单克隆的BstNI酶切图谱显示多样性,单克隆噬菌体抗体与B3HM细胞呈阳性反应。结论:噬菌体展示文库的成功构建为寻找新的致白血病相关基因,阐明白血病发病机理奠定了基础。     

A neutralizable epitope is induced on HGF upon its interaction with its receptor cMet

A new conformational neutralizable epitope is created on heptocyte growth factor (HGF), when it interacts with its receptor, cMet. By immunizing rabbits with HGF-cMet complex, we successfully generated a monoclonal antibody (SFN68) that inhibits HGF-cMet interaction, and blocks the biological function mediated by HGF. To define the epitope, we screened out an epitope-mimicking peptide, KSLSRHDHIHHH, from a phage display of combinatorial peptide library. In molecular mimicry this peptide bound to cMet and inhibited HGF-cMet interaction. No humoral response was induced to this epitope-mimicking peptide when immunization was done with HGF alone.     

Stabilization of antibody VH-domains by proteolytic selection

Single variable domains of antibodies represent the smallest antigen binding fragments but are less stable than when associated with their cognate variable domains. Here we have attempted to improve the thermodynamic stability of a model heavy chain variable domain (V_H) by “proteolytic selection” a method whereby the protease-resistance of the displayed protein is coupled to the infectivity of a filamentous bacteriophage. The gene encoding the heavy chain variable domain was taken from the anti-lysozyme antibody HyHEL-10, mutated at random by error-prone PCR, and displayed on filamentous bacteriophage by fusion between the domains of the phage p3 protein. As the entire p3 protein is required for phage infectivity, treatment of the phage library with trypsin at an elevated temperature (which leads to cleavage of p3 fusions with unfolded variable domains) selects for infectious phages bearing the more stable variable domains. After several rounds of selection, a mutant (S65G/T70S/D99N) was obtained with improved stability (T_m=58.5 °C and ΔG_25°C=6.3 kcal/mol compared to 51.6 °C and 4.2 kcal/mol for the parent domain). These mutations are conservative and the mutant domain retains the ability to pair with its cognate light chain variable domain in an Fv fragment and to mediate binding to lysozyme. Our results show that the thermodynamic stability of antibody single domains can be improved by “proteolytic selection” and this may represent a step towards making useful antibody single domains for biotechnological application.     

从噬菌体表面展示肽库中筛选衣原体单克隆抗体识别的抗原表位

利用抗体捕获法,经三轮淘洗,从表面展示随机肽序列的噬菌体文库中筛选到与衣原体单克隆抗体C17特异结合的噬菌体克隆,其一致序列为：（L／I）PGGS（P／W）,竞争抑制实验表明含特异序列的克隆能与天然抗原竞争。据此,我们认为此序列为衣原体的B细胞抗原表位。     

噬菌体抗体库筛选技术

在过去的20年中,噬菌体抗体库筛选技术被广泛的应用于抗体筛选、疾病治疗,临床诊断以及基础研究之中。在该技术的应用过程中,快速有效的筛选出最适合的单克隆抗体并进行可靠和高效的数据管理和分析是十分重要的。文中总结了近年来噬菌体抗体库的高通量筛选方法并且对数据管理做了简要介绍。     

Antibodies for proteomic research: comparison of traditional immunization with recombinant antibody technology

Antibodies play a pivotal role in studying the expression and function of proteins. Proteomics studies require the generation of specific and high‐affinity antibodies against large numbers of proteins. While traditional animal‐based antibody generation is laborious, difficult to automate, and therefore less suited to keep up with the requirements of proteomics research, the use of recombinant in vitro antibody technology might offer a solution to this problem. However, it has not been demonstrated yet that such antibodies are at least as useful as conventional antibodies for typical proteomics applications. Here we generated novel recombinant Fab antibody fragments from the naïve HuCAL® GOLD library against a number of targets derived from a mouse cDNA library. We compared these antibodies with polyclonal antisera produced against the same targets and show that these recombinant antibodies are useful reagents for typical applications like Western blotting or immunohistochemistry.     

A large library based on a novel (CH2) scaffold: Identification of HIV-1 inhibitors

Isolated immunoglobulin CH2 domains were proposed as scaffolds for selection of binders with potential effector functions. We tested the feasibility of this approach by constructing a large (size 5 × 10¹⁰) library where all amino acids in two loops (BC and FG) were mutated to four residues (Y, A, D, or S). Three binders were selected from this library by panning against a gp120-CD4 complex. The strongest binder, m1a1, recognized specifically a highly conserved CD4i epitope and inhibited to various extents seven out of nine HIV-1 isolates from different clades. The loop BC and the conformational state of the scaffold are critical for its binding. These results provide a proof of concept for the potential of CH2 as a scaffold for construction of libraries containing potentially useful binders. The newly identified HIV-1 inhibitors could be further improved to candidate therapeutics and/or used as research reagents for exploration of conserved gp120 structures.     

Functional improvement of antibody fragments using a novel phage coat protein III fusion system

Functional expressions of proteins often depend on the presence of host specific factors. Frequently recombinant expression strategies of proteins in foreign hosts, such as bacteria, have been associated with poor yields or significant loss of functionality. Improvements in the performance of heterologous expression systems will benefit present-day quests in structural and functional genomics where high amounts of active protein are required. One example, which has been the subject of considerable interest, is recombinant antibodies or fragments thereof as expressions of these in bacteria constitute an easy and inexpensive method compared to hybridoma cultures. Such approaches have, however, often suffered from low yields and poor functionality. A general method is described here which enables expressions of functional antibody fragments when fused to the amino-terminal domain(s) of the filamentous phage coat protein III. Furthermore, it will be shown that the observed effect is neither due to improved stability nor increased avidity.     

Enhanced antibody affinity to Japanese encephalitis virus E protein by phage display

Obtaining antibodies with high affinity and specificity against antigens are required for the development of therapeutic and diagnostic antibodies. In this study, the contributions to binding affinity in the CDR2 and CDR3 regions of two monoclonal antibodies E3.3 and 2H2 were investigated by random mutagenesis in a phage-display synthetic oligonucleotide library. One high-affinity clone (CDR3-30) was obtained with a 3-fold increase of the dissociation constant, resulting from the changes in amino acids at residues 95, 97, and 98 in the CDRH3 region. Analysis of the predicted structure by modeling suggested that the contributions of mutated residues in the CDR3 region to the binding affinity involved not only complementarity between antigen and CDR3, but also interaction between heavy and light chains. The information gained from this study may benefit the design of vaccines and therapeutic antibodies against Japanese encephalitis virus infection.     

Domain libraries: Synthetic diversity for de novo design of antibody V-regions

A completely synthetic gene library encoding the variable light (VL) immunoglobulin domains has been constructed in vitro. The library was constructed by assembling a set of six oligodeoxyribonucleotides (oligos) using the polymerase chain reaction (PCR). Three out of the six overlapping oligonucleotides were synthesized with randomized complementarity determining regions (CDR) with the codon pattern, (NNS)_n, where N is any of the four nucleotides (nt) and n is the number of codons with variation in the CDR. The framework regions, taken from the D1.3 anti-lysozyme antibody (Ab), were kept intact. Overlapping regions of approx. 20 nt, together with two additional flanking primers carrying the desired restriction sites, allowed the construction of a library in one single PCR reaction. The VL library was cloned into the phage display vector pEXmide3, and ten randomly picked clones were sequenced. These sequences exhibited complete diversity in all the three CDR and the codons for five canonical amino acid (aa) residues were kept intact and identified. Seven clones contained the full-length gene for the VL domain while deletions were observed in three clones. The restricted use of nt at the third position successfully avoided the stop codons TGA and TAA, whereas the stop codon TAG is read as Gln in an amber suppressor strain. We call this synthetic Ab diversity Domain Library, and it represents an example of syntheticlibraries with extensive, multiple randomized sequences. The use of Domain Libraries opens up the possibility for design in Ab engineering, e.g., additional CDR regions can be added or their length varied. Furthermore, the use of synthetic gene libraries, constructed with the Domain Library strategy, is not limited to the construction of synthetic Ab fragments, but can be used in the design of other types of proteins.     

Identification of preferred substrate sequences of microbial transglutaminase from Streptomyces mobaraensis using a phage-displayed peptide library

Microbial transglutaminase (TGase) from Streptomyces mobaraensis (MTG) has been used in many industrial applications because it effectively catalyzes the formation of covalent cross-linking between glutamine residues in various substrate proteins and lysine residues or primary amines. To better understand the sequence preference around the reactive glutamine residue by this enzymatic reaction, we screened preferred peptide sequences using a phage-displayed random peptide library. Most of the peptides identified contained a consensus sequence, which was different from those previously found for mammalian TGases. Of these, most sequences had a specific reactivity toward MTG when produced as a fusion protein with glutathione-S-transferase. Furthermore, the representative sequence was found to be reactive even in the peptide form. The amino acid residues in the sequence critical for the reactivity were further analyzed, and the possible interaction with the enzyme has been discussed in this paper.