Efficient bacterial production of functional antibody fragments using a phagemid vector

The so-called ‘in vitro evolutionary method’ using a phage display system has been applied for protein engineering of the antigen-binding fragment of antibodies (Fab) by conducting random mutagenesis at the antigen-binding site in combination with antigen-based biopanning. However, isolated phage clones displaying Fab cannot necessarily be used for efficient bacterial production of engineered Fab proteins, often due to deleterious defects in their proper folding abilities derived in compensation for the gain of high affinity for a particular antigen. We here report a new method of an efficient and direct bacterial expression system for the phagemid-coded Fab proteins without use of the helper phage. To overcome a low folding efficiency derived from somatic hypermutations, if any, we have established optimum conditions for bacterial cultivation and protein expression, utilizing unusually long cultivation time (>50 h) and very low temperature (25 °C) and thereby leading to the production and extracellular secretion of Fab proteins in a very high yield (3–15 mg/L of culture). The purified Fab folded correctly and could efficiently bind an antigen, as judged by circular dichroism and isothermal titration calorimetry, respectively.     

Development of phage biopanning strategies to identify affinity peptide ligands for kappa light chain Fab fragments

In this work, two phage biopanning strategies were developed to identify affinity peptides for a single Fab and multiple kappa Fabs. For the biopanning rounds, protein L beads were employed to bind Fab targets in a fixed orientation, and NHS functionalized magnetic beads were used to facilitate evaluation of low pH elution conditions. The resulting peptide sequences were synthesized and the binding to different Fabs was evaluated using fluorescence polarization. The first biopanning approach yielded a peptide with similar affinities for two forms of the Fab (recombinantly expressed and post papain-digestion) as well as the intact antibody. While moderate affinity was observed toward a murine variant of the Fab with the same complementarity determining regions (CDR) region but different framework, minimal binding occurred to a Fab with high sequence homology but containing different CDR loops. The second biopanning strategy yielded a peptide with affinity for all three kappa Fabs indicating that it may be a good lead for the development of more general affinity reagents for recombinant kappa Fabs. Finally, an affinity peptide column was developed, and its efficacy was demonstrated for Fab purification from a complex cell culture fluid mixture. The results presented in this article demonstrate that different peptide-based phage biopanning strategies can be effectively employed to identify affinity peptide leads for specific Fab and more general kappa Fab purifications.     

Selection of recombinant anti‐SH3 domain antibodies by high‐throughput phage display

Antibodies are indispensable tools in biochemical research and play an expanding role as therapeutics. While hybridoma technology is the dominant method for antibody production, phage display is an emerging technology. Here, we developed and employed a high‐throughput pipeline that enables selection of antibodies against hundreds of antigens in parallel. Binding selections using a phage‐displayed synthetic antigen‐binding fragment (Fab) library against 110 human SH3 domains yielded hundreds of Fabs targeting 58 antigens. Affinity assays demonstrated that representative Fabs bind tightly and specifically to their targets. Furthermore, we developed an efficient affinity maturation strategy adaptable to high‐throughput, which increased affinity dramatically but did not compromise specificity. Finally, we tested Fabs in common cell biology applications and confirmed recognition of the full‐length antigen in immunoprecipitation, immunoblotting and immunofluorescence assays. In summary, we have established a rapid and robust high‐throughput methodology that can be applied to generate highly functional and renewable antibodies targeting protein domains on a proteome‐wide scale.     

High-throughput affinity ranking of antibodies using surface plasmon resonance microarrays

A method was developed to rapidly identify high-affinity human antibodies from phage display library selection outputs. It combines high-throughput Fab fragment expression and purification with surface plasmon resonance (SPR) microarrays to determine kinetic constants (kon and koff) for 96 different Fab fragments in a single experiment. Fabs against human tissue kallikrein 1 (hK1, KLK1 gene product) were discovered by phage display, expressed in Escherichia coli in batches of 96, and purified using protein A PhyTip columns. Kinetic constants were obtained for 191 unique anti-hK1 Fabs using the Flexchip SPR microarray device. The highest affinity Fabs discovered had dissociation constants of less than 1 nM. The described SPR method was also used to categorize Fabs according to their ability to recognize an apparent active site epitope. The ability to rapidly determine the affinities of hundreds of antibodies significantly accelerates the discovery of high-affinity antibody leads.     

Influenza PR8 HA-specific Fab fragments produced by phage display methods

Anti-influenza hemagglutinin (HA) Fabs were isolated from a phage display library using purified HA of influenza virus A/Puerto Rico/8/34 (PR8; H1N1) as an antigen. Four Fab clones displaying a 25-50-fold higher binding signal to PR8 HA than the control were selected for further analysis and comparison with anti-PR8 monoclonal antibody (mAb). All four Fabs and mAb recognized the PR8 HA under non-reducing conditions but rarely bound to reduced PR8 HA. Inhibition of influenza virus infection on MDCK cells was observed with Fab1 and mAb in a dose-dependent manner while Fab3 and 4 exhibited only a partial inhibitory effect. Moreover, Fab1 clone and mAb exhibited cross-reactivity with the A/Peking/262/95 (A/Peking; H1N1) strain. The inhibitory effects of mAb on both influenza strains were more potent than Fab1, which is likely attributed to its higher affinity for the antigen. SPR analyses, in fact, revealed that Fab1 and mAb have K_D of 1.5 × 10⁻⁸ and 3.2 × 10⁻⁹ M, respectively. These results strongly suggest that phage library-derived Fabs can be readily prepared and that such HA-specific Fabs with inhibitory action on influenza infection may be used to treat influenza patients.     

Bacterial expression and purification of recombinant bovine Fab fragments.

We have previously described a recombinant phagemid expression vector, pComBov, designed for the production of native sequence bovine monoclonal antibodies (mAb) generated by antibody phage display. Bovine mAb Fab fragments isolated from libraries constructed using pComBov in Escherichia coli strain XL1-Blue, which is routinely used for antibodies expressed on the surface of phage, were expressed at very low yields. Therefore, a study was undertaken to determine optimal growth conditions for maximal expression of bovine Fab fragments in E. coli. By varying the E. coli strain, and the temperature and length of the culture growth, we were able to substantially increase the yield of soluble Fab fragments. A high yield of Fab fragments was found in the culture growth medium, which enabled us to devise a rapid and simple single-step method for the purification of native (nondenatured) Fabs based on immobilized metal affinity chromatography against a six-histidine amino acid carboxyl-terminal extension of the heavy-chain constant region. Using these methods we were able to express and purify antigen-specific bovine Fab fragments from E. coli.     

IgG Fab Fragments Forming Bivalent Complexes by a Conformational Mechanism That Is Reversible by Osmolytes

Generated by proteolytic cleavage of immunoglobulin, Fab fragments possess great promise as blocking reagents, able to bind receptors or other targets without inducing cross-linking. However, aggregation of Fab preparations is a common occurrence, which generates intrinsic stimulatory capacity and thwarts signal blockade strategies. Using a panel of biochemical approaches, including size exclusion chromatography, SDS-PAGE, mass spectrometry, and cell stimulation followed by flow cytometry, we have measured the oligomerization and acquisition of stimulatory capacity that occurs in four monoclonal IgG Fabs specific for TCR/CD3. Unexpectedly, we observed that all Fabs spontaneously formed complexes that were precisely bivalent, and these bivalent complexes possessed most of the stimulatory activity of each Fab preparation. Fabs composing bivalent complexes were more susceptible to proteolysis than monovalent Fabs, indicating a difference in conformation between the Fabs involved in these two different states of valency. Because osmolytes represent a class of compounds that stabilize protein folding and conformation, we sought to determine the extent to which the amino acid osmolyte l-proline might impact bivalent Fab complexation. We found that l-proline (i) inhibited the adoption of the conformation associated with bivalent complexation, (ii) preserved Fab monovalency, (iii) reversed the conformation of preformed bivalent Fabs to that of monovalent Fabs, and (iv) separated a significant percentage of preformed bivalent complexes into monovalent species. Thus, Fab fragments can adopt a conformation that is compatible with folding or packing of a bivalent complex in a process that can be inhibited by osmolytes.     

Rationalization of gene regulation by a eukaryotic transcription factor: calculation of regulatory region occupancy from predicted binding affinities

The antigen-binding fragments (Fab) of antibodies are powerful tools in clinical therapy, molecular diagnostics and basic research. However, their principal applications require pure recombinant molecules in large amounts, which are challenging to obtain. Severe limitations in yield, folding and functionality are commonly encountered in bacterial production of Fab fragments. Secretion into the oxidizing periplasm generally results in low yield, whereas expression in the reducing cytoplasmic environment produces unfolded or non-functional protein. We hypothesized that an impaired reducing environment of the cytoplasm would permit correctly folded, functional cytoplasmic expression of Fabs with high yield. We used the Escherichia coli strain FA113, which has no activity of both thioredoxin and glutathione reductase, and thus has an oxidizing cytoplasmic environment. With the newly constructed vector pFAB1 we tested the cytoplasmic expression of two Fab fragments, which recognize the integral membrane protein NhaA, a bacterial Na(+)/H(+) antiporter. These antibodies differ in terms of DNA sequence and stability. Both antibody fragments were produced to very high yields (10-30 mg/l from bacterial cultures at an A(600 nm)=1.2-1.3). This is a factor 50-250 times higher than any other reported over-expression strategy for Fab fragments and currently represents the highest production rate ever been reported for antibody Fab fragments in bacteria grown to similar cell densities. The fragments are fully functional and can be efficiently purified by His-tag chromatography. Expression of active Fab fragments in the bacterial cytoplasm unlocks the possibility of using antibody specific targeting in an intracellular environment. Such a capacity opens new perspectives for investigating metabolic and regulatory pathways in vivo and also provides a powerful selection system for functional genomics.     

IgY‐binding peptide screened from a random peptide library as a ligand for IgY purification

Chicken egg yolk immunoglobulin (IgY) is a functional substitute for mammalian IgG for antigen detection. Traditional IgY purification methods involve multi‐step procedures resulting in low purity and recovery of IgY. In this study, we developed a simple IgY purification system using IgY‐specific peptides identified by T7 phage display technology. From disulfide‐constrained random peptide libraries constructed on a T7 phage, we identified three specific binding clones (Y4‐4, Y5‐14, and Y5‐55) through repeated biopanning. The synthetic peptides showed high binding specificity to IgY‐Fc and moderate affinity for IgY‐Fc (K_d: Y4‐4 = 7.3 ± 0.2 μM and Y5‐55 = 4.4 ± 0.1 μM) by surface plasmon resonance analysis. To evaluate the ability to purify IgY, we performed immunoprecipitation and affinity high‐performance liquid chromatography using IgY‐binding peptides; the result indicated that these peptides can be used as affinity ligands for IgY purification. We then used a peptide‐conjugated column to purify IgY from egg yolks pre‐treated using an optimized delipidation technique. Here, we report the construction of a cost‐effective, one‐step IgY purification system, with high purity and recovery. © 2017 The Authors. Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.     

Secretion of a functional Fab fragment in Escherichia coli and the influence of culture conditions

Summary Genes encoding a light chain and an Fd region (a variable region and a CH1 domain of a heavy chain) of a mouse-human chimeric antibody with specificity for human carcinoembryonic antigen (CEA) were fused to a DNA segment coding for the signal peptide of Escherichia coli ompF. E. coli cells harbouring an expression vector containing these genes downstream of a tac promoter were able to secrete a Fab fragment of the antibody efficiently. When the cells were cultured at 37° C and the inducer (isopropyl-\-d-thiogalactopyranoside, IPTG) concentration was 1 mm (standard conditions), production of functional Fab was very low (medium; 200 ng/l culture and periplasm; <90 ng/l culture). In order to optimize functional Fab production, we examined the influence of culture conditions (i.e. temperature and the inducer concentration) on secretion of the product. It was found that a 12.7-fold higher amount of Fab fragment could be produced at 30° C using 0.1 mm IPTG, as compared with standard conditions. Under these optimal conditions, functional Fab accumulated in the periplasm and culture medium for 10 h after induction and the total production level was found to reach approximately 4.5 mg/l culture. Correspondence to: T. Shibui     

Two routes for production and purification of Fab fragments in biopharmaceutical discovery research: Papain digestion of mAb and transient expression in mammalian cells

Fab (fragment that having the antigen binding site) of a monoclonal antibody (mAb) is widely required in biopharmaceutical research and development. At Centocor, two routes of Fab production and purification were used to enable a variety of research and development efforts, particularly, crystallographic studies of antibody–antigen interactions. One route utilizes papain digestion of an intact monoclonal antibody for Fab fragment production. After digestion, separation of the Fab fragment from the Fc (fragment that crystallizes) and residual intact antibody was achieved using protein A affinity chromatography. In another route, His-tagged Fab fragments were obtained by transient expression of an appropriate construct in mammalian cells, and typical yields are 1–20 mg of Fab fragment per liter of cell culture. The His-tagged Fab fragments were first captured using immobilized metal affinity chromatography (IMAC). To provide high quality protein sample for crystallization, Fabs from either proteolytic digestion or from direct expression were further purified using size-exclusion chromatography (SEC) and/or ion-exchange chromatography (IEC). The purified Fab fragments were characterized by mass spectrometry, SDS–PAGE, dynamic light scattering, and circular dichroism. Crystallization experiments demonstrated that the Fab fragments are of high quality to produce diffraction quality crystals suitable for X-ray crystallographic analysis.     

Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins

We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols.     

Neutralizing chimeric mouse-human antibodies against Burkholderia pseudomallei protease: expression, purification and characterization

A recombinant Fab monoclonal antibody (Fab) C37, previously obtained by phage display and biopanning of a random antibody fragment library against Burkholderia pseudomallei protease, was expressed in different strains of Escherichia coli. E. coli strain HB2151 was deemed a more suitable host for Fab expression than other E. coli strains when grown in media supplemented with 0.2 % glycerol. The expressed Fab fragment was purified by affinity chromatography on a Protein G-Sepharose column, and the specificity of the recombinant Fab C37 towards B. pseudomallei protease was proven by Western blotting, enzyme-linked immunosorbent assay (ELISA) and by proteolytic activity neutralization. In addition, polyclonal antibodies against B. pseudomallei protease were produced in rabbits immunized with the protease. These were isolated from high titer serum by affinity chromatography on recombinant-Protein A-Sepharose. Purified polyclonal antibody specificity towards B. pseudomallei protease was proven by Western blotting and ELISA.     

人源单克隆抗人免疫缺陷病毒1型抗体Fab段基因的获得

应用噬苏体抗体库技术有效地筛选出了多株抗ＨＩＶ－１人源单克隆抗体。以逆转录聚合酶链反应（ＲＴ－ＰＣＲ）从ＨＩＶ－１感染者外周血淋巴细胞中扩增抗体轻重链可变区基因,插入载体ｐＣＯＭＢ３,建立噬菌体抗体库。分别以ＨＩＶ－１ｇｐ１２０和ｇｐ１６０为固相抗原,经过多轮筛选,从中获得了多株抗ＨＩＶ－１ｇｐ４１、ｇｐ１２０和ｇｐ１６０的单克隆抗体Ｆａｂ段基因。抗ＨＩＶ特异性噬菌体抗体随抗体库的筛选高度富集,抗     

用硫氰酸盐洗脱法直接测定噬菌体抗体的相对亲和力

抗体与相应抗原的结合可以被硫氰酸盐洗脱而解离,抗体的亲和力越高则解离所需要的硫氰酸盐浓度就越大,这一原理在传统的免疫学实验中被用来测定单克隆抗体或多克隆抗体的相对亲和力。如果证明该原理同样适用于噬菌体抗体库技术,则可以建立一种直接测定噬菌体抗体相对亲和力的简便方法。首先将噬菌体抗体与工作浓度的硫氰酸盐共孵育,以证明这一过程并不影响其后的ELISA反应,然后参照硫氰酸盐洗脱法测定完整抗体分子和Fab段相对亲和力的方法,在ELISA实验中以酶标抗M13为二抗检测了5个单克隆噬菌体抗体的相对亲和力,并与相对应的可溶性Fab段的相对亲和力进行了比较。被测抗体中包括3个克隆的抗角蛋白抗体和2个克隆的抗乙型肝炎表面抗原抗体。结果发现,用硫氰酸盐洗脱法测定5个噬菌体抗体所得到的亲和力排序与测定其相应可溶性Fab段所得结果一致,表明硫氰酸盐洗脱法可作为一种简便快速的方法用来直接测定噬菌体抗体的相对亲和力。     

A phage‐displayed single‐chain Fab library optimized for rapid production of single‐chain IgGs

Phage‐displayed synthetic antibody (Ab) repertoires have become a major source of affinity reagents for basic and clinical research. Specific Abs identified from such libraries are often screened as fragments antigen binding (Fabs) produced in bacteria, and those with desired biochemical characteristics are reformatted for production as full‐length immunoglobulin G (IgG) in mammalian cells. The conversion of Fabs to IgGs is a cumbersome and often rate‐limiting step in the development of Abs. Moreover, biochemical properties required for lead IgG development are not always shared by the Fabs, and these issues are not uncovered until a significant effort has been spent on Abs that ultimately will not be useful. Thus, there is a need for simple and rapid techniques to convert phage‐displayed Fabs to IgGs at an early stage of the Ab screening process. We report the generation of a highly diverse phage‐displayed synthetic single‐chain Fab (scFab) library, in which the light and heavy chains were tethered with an optimized linker. Following selection, pools of scFabs were converted to single‐chain IgGs (scIgGs) en masse, enabling facile screening of hundreds of phage‐derived scIgGs. We show that this approach can be used to rapidly screen for and select scIgGs that target cell‐surface receptors, and scIgGs behave the same as conventional IgGs.     

通过构建轻链二级库对人源抗TNF-α抗体进行亲和力成熟

通过构建轻链二级库的方法,对人源抗ＴＮＦ－α单抗Ｆａｂ进行轻链置换,并筛选出具有更高亲和力的人源抗ＴＮＦ－α单抗的Ｆａｂ。首先用ＲＴ－ＰＣＲ技术扩增正常人全套抗体轻链基因,并与已获得的人源抗ＴＮＦ－α单抗的重链基因配对,构建人源抗ＴＮＦ－α噬菌体抗体轻链二级库,然后筛选与ＴＮＦ－α具有更高亲和力的克隆,经过三轮的生物淘筛（ｂｉｏｐａｎｎｉｎｇ）,获得了比原来的人源抗ＴＮＦ－α单抗Ｆａｂ具有更高亲和     

Locking the Elbow: Improved Antibody Fab Fragments as Chaperones for Structure Determination

Antibody Fab fragments have been exploited with significant success to facilitate the structure determination of challenging macromolecules as crystallization chaperones and as molecular fiducial marks for single particle cryo-electron microscopy approaches. However, the inherent flexibility of the “elbow” regions, which link the constant and variable domains of the Fab, can introduce disorder and thus diminish their effectiveness. We have developed a phage display engineering strategy to generate synthetic Fab variants that significantly reduces elbow flexibility, while maintaining their high affinity and stability. This strategy was validated using previously recalcitrant Fab–antigen complexes where introduction of an engineered elbow region enhanced crystallization and diffraction resolution. Furthermore, incorporation of the mutations appears to be generally portable to other synthetic antibodies and may serve as a universal strategy to enhance the success rates of Fabs as structure determination chaperones.     

Neutralization of Burkholderia pseudomallei protease by Fabs generated through phage display

The isolation of therapeutic and functional protease inhibitors in vitro via combinatorial chemistry and phage display technology has been described previously. Here we report the construction of a combinatorial mouse-human chimeric antibody fragment (Fab) antibody library targeted against the protease of the tropical pathogen, Burkholderia pseudomallei. The resulting library was biopanned against the protease, and selected clones were analyzed for their ability to function as protease inhibitors. Three families of Fabs were identified by restriction fingerprinting, all of which demonstrated high specificity towards the protease of B. pseudomallei. Purified Fabs also demonstrated the capacity to inhibit B. pseudomallei protease activity in vitro, and this inhibitory property was exclusive to the pathogenic protease. Thus these recombinant antibodies are candidates for immunotherapy and tools to aid in further elucidation of the mechanism of action of the B. pseudomallei protease.