Comparison of two forms of catalytic antibody displayed on yeast-cell surface

Two forms of the Fab fragment of the catalytic antibody 6D9 were individually displayed on yeast-cell surface in fusion to the C-terminal half of -agglutinin: one was 6D9 Fab1, in which the light chain of the Fab (Lc) fragment is displayed on cell surface and the heavy chain of the Fab (Fd) fragment is secreted and linked to the Lc fragment with a disulfide bond; the other was 6D9 Fab2, in which the Fd fragment is displayed on cell surface and the Lc fragment is secreted and linked to the Fd fragment with a disulfide bond. Analysis by flow cytometry indicated that some 6D9 Fab2 fragments were unable to construct an appropriate conformation, and that most of the 6D9 Fab1 fragments displayed on yeast-cell surface exhibited higher binding affinity, stability, and catalytic activity. Conformation of the surface-displayed hetero-dimeric Fab fragment mainly depended on the intermolecular disulfide bond between the Lc and Fd fragments. The conformation of 6D9 Fab1 was more stable than that of Fab2. In the reducing environment of solution containing 25 nM DTT, the function of 6D9 Fab2 was almost completely lost. The successful display of 6D9 Fab1 on yeast-cell surface provides a novel approach to the engineering of catalytic antibodies.     

The disulfide bonds in antibody variable domains: effects on stability, folding in vitro, and functional expression in Escherichia coli.

The formation of the disulfide bonds in the variable domains VH and VL of the antibody McPC603 was found to be essential for the stability of all antigen binding fragments investigated. Exposure of the Fv fragment to reducing conditions in vitro resulted in irreversible denaturation of both VH and VL. In vitro refolding of the reduced Fv fragment was only possible when the disulfide bonds were allowed to form under oxidizing conditions. The analysis of a series of mutants of the Fv fragment, the Fab fragment and the single-chain Fv fragment, all secreted into the periplasm of Escherichia coli, in which each of the cysteine residues of the variable domains was replaced by a series of other amino acids, showed that functional antigen binding fragments required the presence of both the disulfide bond in VH and the one in VL. These results were also used to devise an alternative expression system based on the production of insoluble fusion proteins consisting of truncated beta-galactosidase and antibody domains, enzymatic cleavage, and refolding and assembly in vitro. This strategy should be useful for providing access to unstable antibody domains and fragments.     

Domain interactions in the Fab fragment: a comparative evaluation of the single-chain Fv and Fab format engineered with variable domains of different stability

Recombinant antibody fragments, most notably Fab and scFv, have become important tools in research, diagnostics and therapy. Since different recombinant antibody formats exist, it is crucial to understand the difference in their respective biophysical properties. We assessed the potential stability benefits of changing the scFv into the Fab format, the influence of the variable domains on the stability of the Fab fragment, and the influence of the interchain disulfide bond in the Fab fragment. To analyze domain interactions, the Fab fragment was broken down into its individual domains, several two-domain assemblies and one three-domain assembly. The equilibrium denaturation properties of these constructs were then compared to those of the Fab fragment. It was found that mutual stabilization occurred across the VH/VL and the CH1/CL interface, whereas the direct interaction between the V) and the CL domain had no influence on the stability of either domain. This observation can be explained by the different interfaces used for interaction. In contrast, the whole CH1CL and VHVL unit showed significant mutual stabilization, indicating a high degree of cooperation between the VH/VL and CH1/CL interface. The interchain disulfide bond in the Fab fragment plays an essential role in this stabilization. In addition to the effects of domain association on the thermodynamic (equilibrium) stability, Fab fragments differ from scFv fragments of similar equilibrium stability by having a very slow unfolding rate. This kinetic stabilization may increase significantly the resistance of Fab fragments against short time exposure to adverse conditions.     

Monoclonal antibodies against the lac carrier protein from Escherichia coli. 2. Binding studies with membrane vesicles and proteoliposomes reconstituted with purified lac carrier protein

Monoclonal antibodies 4B1 and 5F7 bind to distinct, nonoverlapping epitopes in the lac carrier protein. By use of immunofluorescence microscopy and radiolabeled monoclonal antibodies and Fab fragments, it is shown that both 4B1 and 5F7 bind to spheroplasts and to right-side-out vesicles, but only to a small extent to inside-out vesicles. Clearly, therefore, the lac carrier protein has an asymmetric orientation within the cytoplasmic membrane of Escherichia coli, and both epitopes are located on the periplasmic surface. In right-side-out vesicles, radiolabeled 4B1 binds with a stoichiometry of 1 mol of antibody per 2 mol of lac carrier protein, while radiolabeled 4B1 Fab fragments bind 1:1. Importantly, the intact antibody and its Fab fragments bind to proteoliposomes reconstituted with purified lac carrier protein with a stoichiometry very similar to that observed in right-side-out membrane vesicles. Thus, it seems highly likely that the orientation of the lac carrier protein in the reconstituted system is similar to that in the bacterial cytoplasmic membrane, at least with respect to 4B1 epitope.     

Mechanism of cholesteryl ester transfer protein inhibition by a neutralizing monoclonal antibody and mapping of the monoclonal antibody epitope

The plasma cholesteryl ester transfer protein (CETP, Mr 74,000) has a binding site for neutral lipid which can readily equilibrate with lipoprotein cholesteryl esters or triglycerides. Recently, a monoclonal antibody (TP2) was obtained which neutralizes the cholesteryl ester (CE) and triglyceride (TG) transfer activities of the CETP. In this report, the epitope of the inhibitory monoclonal antibody has been localized to a hydrophobic 26-amino acid sequence at the COOH terminus of CETP. The Fab fragments of TP2 caused partial (50%) inhibition of CE transfer and complete inhibition of TG transfer by the CETP. Similarly, the Fab fragments inhibited (37%) the binding of CE to the CETP and abolished the binding of TG to the CETP. Surprisingly, the TP2 Fab was also found to enhance the binding of CETP to plasma lipoproteins and to phospholipid vesicles. In conclusion, the TP2 monoclonal antibody inhibits lipid transfer by blocking the uptake of lipid by CETP. The COOH-terminal epitope may be in or near the neutral lipid binding site. Occupancy of this site by TP2 Fab fragments or by neutral lipid may result in a conformational change of CETP causing enhanced binding to lipoproteins or vesicles.     

Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system

We describe protein synthesis, folding and assembly of antibody fragments and full-length aglycosylated antibodies using an Escherichia coli-based open cell-free synthesis (OCFS) system. We use DNA template design and high throughput screening at microliter scale to rapidly optimize production of single-chain Fv (scFv) and Fab antibody fragments that bind to human IL-23 and IL-13α1R, respectively. In addition we demonstrate production of aglycosylated immunoglobulin G (IgG1) trastuzumab. These antibodies are produced rapidly over several hours in batch mode in standard bioreactors with linear scalable yields of hundreds of milligrams/L over a 1 million-fold change in scales up to pilot scale production. We demonstrate protein expression optimization of translation initiation region (TIR) libraries from gene synthesized linear DNA templates, optimization of the temporal assembly of a Fab from independent heavy chain and light chain plasmids and optimized expression of fully assembled trastuzumab that is equivalent to mammalian expressed material in biophysical and affinity based assays. These results illustrate how the open nature of the cell-free system can be used as a seamless antibody engineering platform from discovery to preclinical development of aglycosylated monoclonal antibodies and antibody fragments as potential therapeutics.Key words: cell-free protein synthesis, Fab antibody, aglycosylated antibodies, HER2, trastuzumab     

The human combinatorial antibody library HuCAL GOLD combines diversification of all six CDRs according to the natural immune system with a novel display method for efficient selection of high-affinity antibodies

This article describes the generation of the Human Combinatorial Antibody Library HuCAL GOLD. HuCAL GOLD is a synthetic human Fab library based on the HuCAL concept with all six complementarity-determining regions (CDRs) diversified according to the sequence and length variability of naturally rearranged human antibodies. The human antibody repertoire was analyzed in-depth, and individual CDR libraries were designed and generated for each CDR and each antibody family. Trinucleotide mixtures were used to synthesize the CDR libraries in order to ensure a high quality within HuCAL GOLD, and a β-lactamase selection system was employed to eliminate frame-shifted clones after successive cloning of the CDR libraries. With these methods, a large, high-quality library with more than 10 billion functional Fab fragments was achieved. By using CysDisplay, the antibody fragments are displayed on the tip of the phage via a disulfide bridge between the phage coat protein pIII and the heavy chain of the antibody fragment. Efficient elution of specific phages is possible by adding reducing agents. HuCAL GOLD was challenged with a variety of different antigens and proved to be a reliable source of high-affinity human antibodies with best affinities in the picomolar range, thus functioning as an excellent source of antibodies for research, diagnostic, and therapeutic applications. Furthermore, the data presented in this article demonstrate that CysDisplay is a robust and broadly applicable display technology even for high-throughput applications.     

Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system

We describe protein synthesis, folding and assembly of antibody fragments and full-length aglycosylated antibodies using an Escherichia coli-based open cell-free synthesis (OCFS) system. We use DNA template design and high throughput screening at microliter scale to rapidly optimize production of single-chain Fv (scFv) and Fab antibody fragments that bind to human IL-23 and IL-13α1R, respectively. In addition we demonstrate production of aglycosylated immunoglobulin G (IgG₁) trastuzumab. These antibodies are produced rapidly over several hours in batch mode in standard bioreactors with linear scalable yields of hundreds of milligrams/L over a 1 million-fold change in scales up to pilot scale production. We demonstrate protein expression optimization of translation initiation region (TIR) libraries from gene synthesized linear DNA templates, optimization of the temporal assembly of a Fab from independent heavy chain and light chain plasmids and optimized expression of fully assembled trastuzumab that is equivalent to mammalian expressed material in biophysical and affinity based assays. These results illustrate how the open nature of the cell-free system can be used as a seamless antibody engineering platform from discovery to preclinical development of aglycosylated monoclonal antibodies and antibody fragments as potential therapeutics.     

Renal metabolism of 3'-iodohippuryl N(epsilon)-maleoyl-L-lysine (HML)-conjugated Fab fragments

Renal localization of radiolabeled antibody fragments constitutes a problem in targeted imaging and radiotherapy. Recently, we reported use of a novel radioiodination reagent, 3'-[131I]iodohippuryl N(epsilon)-maleoyl-L-lysine (HML), that liberates m-iodohippuric acid before antibody fragments are incorporated into renal cells. In mice, HML-conjugated Fab demonstrated low renal radioactivity levels from early postinjection times. In this study, renal metabolism of HML-conjugated Fab fragments prepared by different thiolation chemistries and by direct radioiodination were investigated to determine the mechanisms responsible for the low renal radioactivity levels. Fab fragments were thiolated by 2-iminothiolane modification or by reduction of disulfide bonds in the Fab fragments, followed by conjugation with radioiodinated HML to prepare [131I]HML-IT-Fab and [125I]HML-Fab, respectively. In biodistribution studies in mice, both [131I]HML-IT-Fab and [125I]HML-Fab demonstrated significantly lower renal radioactivity levels than those of [125I]Fab. In subcellular distribution studies, [125I]Fab showed migration of radioactivity from the membrane to the lysosomal fraction of the renal cells from 10 to 30 min postinjection. On the other hand, the majority of the radioactivity was detected only in the membrane fraction at the same time points after injection of both [131I]HML-IT-Fab and [125I]HML-Fab. In metabolic studies, while [125I]Fab remained intact at 10 min postinjection, both HML-conjugated Fab fragments generated m-iodohippuric acid as a radiometabolite at the same postinjection time. [131I]HML-IT-Fab registered two radiometabolites (intact [131I]HML-IT-Fab and m-iodohippuric acid), whereas additional radiometabolites were observed with [125I]HML-Fab. This suggested that metabolism of both HML-conjugated Fab fragments would occur in the membrane fractions of the renal cells. The findings of this study reinforced our previous hypothesis that radiochemical design of antibody fragments that liberate radiometabolites that are excreted into the urine by the action of brush border enzymes would constitute a useful strategy to reduce renal radioactivity levels from early postinjection times.     

Influence of subunit-specific antibodies on the activity of the F0 complex of the ATP synthase of Escherichia coli. II. Effects of subunit c-specific polyclonal antibodies.

After incubation of F1-stripped everted membrane vesicles with antibodies against subunit c of the ATP synthase of Escherichia coli the proton translocation through the open F0 channel was blocked. Rebinding of F1 to those vesicles is affected by the antibody concentration used. In general, the use of F(ab')2 or Fab fragments prepared from anti-c antibodies gave similar results. However, using Fab fragments a higher amount of antigenic binding sites was necessary to block the F0 complex completely, whereas extremely low amounts of Fab fragments were necessary to inhibit the binding of F1. This can be explained by an antigenic determinant of subunit c, which is only accessible to the smaller Fab fragments with a molecular mass of approximately 50,000. Incubation of F1-containing everted membranes with anti-c antibodies showed that the binding of the antibodies resulted in a displacement of F1, while simultaneously the proton translocation through F0 has been blocked. Such a displacement can only be observed after incubation with IgG molecules or F(ab')2 fragments. Fab fragments were not able to displace the F1 part, indicating that the ability of antibodies and F(ab')2 fragments to produce cross-links is responsible for the loss of F1 from the membranes.     

Cell-free production of functional antibody fragments

Botulinum neurotoxin serotype B (BoNT/B)-specific Fab was expressed in a cell-free protein synthesis system derived from an E. coli extract. The cell-free synthesized antibody fragment was found to be effective in neutralizing the toxicity of BoNT/B in animal studies. Expression of functional Fab required an appropriately controlled and stably maintained redox potential. Under an optimized redox condition, the cell extract, whose disulfide reducing activity had been exhausted, could generate bio-functional Fab molecules. Use of a cell extract enriched with molecular chaperones (GroEL/ES) and disulfide bond isomerases were effective in obtaining larger quantities of functional Fab. Under the optimized reaction conditions, approximately 30 μg of functional Fab was obtained after purification from 1 mL reaction mixture.     

Purification of antibody Fab and F(ab')2 fragments using Gradiflow technology

The Gradiflow, a preparative electrophoresis instrument designed to separate molecules on the basis of their size and charge, was used to purify antibody Fab and F(ab')2 fragments. The method described is charge based, utilizing the difference in the pI between the antibody Fab/F(ab')2 fragments and antibody Fc fragments that occur after enzyme digestion of whole antibody molecules. This method of purification was successful across a range of monoclonal and polyclonal antibodies. In particular, F(ab')2 fragments were purified from a number of mouse monoclonal antibodies (both IgG1 and IgG2a isotypes) and Fab fragments were purified from egg yolk IgY polyclonal antibodies. This is a rapid purification method which has advantages over alternative methods that usually comprise ion exchange and gel filtration chromatography. This method may be applicable to most antibody digest preparations.     

Immunological studies on bovine milk lipoprotein lipase. Effects of Fab fragments on enzyme activity

Rabbit antiserum was prepared against purified bovine mild lipoprotein lipase. Immunoelectrophoresis of lipoprotein lipase gave a single precipitin line against the antibody which was coincident with enzyme activity. The gamma-globulin fraction inhibited heparin-releasable lipoprotein lipase activity of bovine arterial intima, heart muscle and adipose tissue. The antibody also inhibited the lipoprotein lipase activity from adipose tissue of human and pig, but not that of rat and dog. Fab fragments were prepared by papain digestion of the gamma-globulin fraction. Fab fragments inhibited the lipoprotein lipase-catalyzed hydrolysis of dimyristoylphosphatidylcholine vesicles and trioleoylglycerol emulsions to the same extent. The Fab fragments also inhibited the lipolysis of human plasma very low density lipoproteins. The change of the kinetic parameters for the lipoprotein lipase-catalyzed hydrolysis of trioleoylglycerol by the Fab fragments was accompanied with a 3-fold increase in Km and a 10-fold decrease in Vmax. Preincubation of lipoprotein lipase with apolipoprotein C-II, the activator protein for lipoprotein lipase, did not prevent inhibition of enzyme activity by the Fab fragments. However, preincubation with dipalmitoylphosphatidylcholine-emulsified trioleoylglycerol or Triton X-100-emulsified trioleoylglycerol had a protective effect (remaining activity 7.0 or 25.8%, respectively, compared to 1.0 or 0.4% with no preincubation). The addition of both apolipoprotein C-II and substrate prior to the incubation with the Fab fragments was associated with an increased protective effect against inhibition of enzyme activity; remaining activity with dipalmitoylphosphatidylcholine-emulsified trioleoylglycerol was 40.6% and with Triton X-100-emulsified trioleoylglycerol, 45.4%. Human plasma very low density lipoproteins also protected against the inhibition of enzyme activity by the Fab fragments. These immunological studies suggest that the interaction of lipoprotein lipase with apolipoprotein C-II in the presence of lipids is associated with a conformational change in the structure of the enzyme such that the Fab fragments are less inhibitory. The consequence of a conformational change in lipoprotein lipase may be to facilitate the formation of an enzyme-triacylglycerol complex so as to enhance the rate of the lipoprotein lipase-catalyzed turnover of substrate to products.     

Electrostatic engineering of the interface between heavy and light chains promotes antibody Fab fragment production

Monoclonal antibodies and antibody fragments are used for diverse diagnostic and therapeutic applications. We have investigated the secretory production of Fab fragments from insect cells cotransfected with plasmid vectors carrying heavy- and light-chain genes. In the present study, to promote the formation of the disulfide bond between the heavy and light chains, some positively charged amino acid residues were introduced near the cysteine residue for the disulfide bond at the C-terminus of C_L, while some negatively charged amino acid residues were added near the cysteine residue for the disulfide bond at the C-terminus of C_H1. This electrostatic steering led to an increase in Fab secretions from insect cells.     

High precision immunoscanning electron microscopy using Fab fragments coupled to ultra-small colloidal gold.

Ultra-small colloidal gold (less than 1 nm), bound to Fab fragments provides the shortest practical specific marker system to date and can be used in concert with field emission scanning electron microscopes to precisely locate antigenic sites. An "in-lens" FE-SEM equipped with a highly sensitive single crystal YAG-detector for backscattered electrons, as well as the use of advanced specimen preparation techniques based on cryofixation, are among the indispensible prerequisites. A T-even type Escherichia coli bacteriophage, Tu II*-46, was chosen to study properties of the immunogold labeling system. Distinct regions on the tail fibers of this phage were labeled with Fab fragments derived from antibodies against the related phage Tu II*-6. The tail fibers are composed of pairs of homologous proteins, thus offering two identical antigenic sites at the same locus on the tail fibers. Fab fragments can be visualized in the SEM at high accelerating voltage (30 kV) without any additional marker. This permits comparison of the labeling characteristics of unmarked and colloidal gold-marked Fab fragments. Unmarked Fab fragments often bind by pairs (two singlet Fab fragments bound opposed to each other along the axis of the tail fiber). The labeling efficiency of unmarked Fab fragments was greater than that of ultra-small gold-labeled Fab fragments. Binding by pairs was not seen after labeling with ultra-small gold-Fab fragments. The conjugates used in this study exhibited one colloidal gold per Fab fragment.     

Engineering an improved IgG4 molecule with reduced disulfide bond heterogeneity and increased Fab domain thermal stability

The integrity of antibody structure, stability, and biophysical characterization are becoming increasingly important as antibodies receive increasing scrutiny from regulatory authorities. We altered the disulfide bond arrangement of an IgG4 molecule by mutation of the Cys at the N terminus of the heavy chain constant domain 1 (C(H)1) (Kabat position 127) to a Ser and introduction of a Cys at a variety of positions (positions 227-230) at the C terminus of C(H)1. An inter-LC-C(H)1 disulfide bond is thus formed, which mimics the disulfide bond arrangement found in an IgG1 molecule. The antibody species present in the supernatant following transient expression in Chinese hamster ovary cells were analyzed by immunoblot to investigate product homogeneity, and purified product was analyzed by a thermofluor assay to determine thermal stability. We show that the light chain can form an inter-LC-C(H)1 disulfide bond with a Cys when present at several positions on the upper hinge (positions 227-230) and that such engineered disulfide bonds can consequently increase the Fab domain thermal stability between 3 and 6.8 °C. The IgG4 disulfide mutants displaying the greatest increase in Fab thermal stability were also the most homogeneous in terms of disulfide bond arrangement and antibody species present. Importantly, mutations did not affect the affinity for antigen of the resultant molecules. In combination with the previously described S241P mutation, we present an IgG4 molecule with increased Fab thermal stability and reduced product heterogeneity that potentially offers advantages for the production of IgG4 molecules.     

Direct selection of antibodies from complex libraries with the protein fragment complementation assay

The aim of the present study was to develop the protein fragment complementation assay (PCA) for the intracellular selection of specific binding molecules from the fully synthetic HuCAL antibody library. Here, we describe the first successful selections of specific antibodies by PCA, and we discuss the opportunities and limitations of this approach. First, we enriched an antibody specific for the capsid protein D of bacteriophage lambda (gpD) by ten successive rounds of competitive liquid culture selection. In an independent approach, we selected a specific antibody for the c-Jun N-terminal kinase 2 (JNK2) in a single-step selection setup. In order to obtain specific antibodies in only a single PCA selection round, the selection system was thoroughly investigated and several strategies to reduce the amount of false positives were evaluated. When expressed in the cytoplasm of Escherichia coli, the PCA-selected scFv antibody fragments could be purified as soluble and monomeric proteins. Denaturant-induced unfolding experiments showed that both antibody fragments are stable molecules, even when the disulfide bonds are reduced. Furthermore, antigen-specificity of the PCA-selected antibody fragments is demonstrated by in vivo and in vitro experiments. As antigen binding is retained regardless of the antibody redox state, both PCA-selected antibody fragments can tolerate the loss of disulfide bridge formation. Our results illustrate that it is possible to select well-expressed, stable, antigen-specific, and intracellular functional antibodies by PCA directly.     

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.