Immunoglobulin Fab Fragments Research Articles

Editors' Selections from Relevant Scientific Publications

Editors' Selections from Relevant Scientific Publications

De novo discovery of peptide-based affinity ligands for the fab fragment of human immunoglobulin G

Antibody fragments and their engineered variants show true potential as next-generation therapeutics as they combine excellent targeting with superior biodistribution and blood clearance. Unlike full antibodies, however, antibody fragments do not yet have a standard platform purification process for large-scale production. Short peptide ligands are viable alternatives to protein ligands in affinity chromatography. In this work, an integrated computational and experimental scheme is described to de novo design 9-mer peptides that bind to Fab fragments. The first cohort of designed sequences was tested experimentally using human polyclonal Fab, and the top performing sequence was selected as a prototype for a subsequent round of ligand refinement in silico. The resulting peptides were conjugated to chromatographic resins and evaluated via equilibrium and dynamic binding studies using human Fab-κ and Fab-λ. The equilibrium studies returned values of binding capacities up to 32 mg of Fab per mL of resin with mild affinity (KD ∼ 10−5 M) that are conducive to high product capture and recovery. Dynamic studies returned values of product yield up to ∼90%. Preliminary purification studies provided purities of 83–93% and yields of 11–89%. These results lay the groundwork for future development of these ligands towards biomanufacturing translation.

Fab Fragment of Immunoglobulin Y Modulates NF-κB and MAPK Signaling through TLR4 and αVβ3 Integrin and Inhibits the Inflammatory Effect on R264.7 Macrophages.

High-purity Fab fragment and immunoglobulin Y (IgY) were prepared to evaluate their anti-inflammatory activity in the lipopolysaccharide (LPS)-induced Raw 264.7 macrophage system. Compared with IgY, the Fab fragment possessed a greater potency in inhibiting the inflammation by nitric oxide (NO)/inducible nitric oxide synthase (iNOS) and prostaglandin-E2 (PGE2)/cyclooxygenase-2 (COX-2) pathways. The Fab fragment attenuated the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10) to 38.07 ± 1.86-48.39 ± 11.33 pg/mL (63.1-71.0% inhibition), 31.59 ± 3.91-38.08 ± 4.44 pg/mL (72.4-77.1% inhibition), and 20.62 ± 0.46-21.91 ± 0.65 pg/mL (50-53% inhibition), respectively. Additionally, the Fab fragment significantly inhibited the translocation of nuclear transcription factor-κB (NF-κB) p65 and the phosphorylation of mitogen-activated protein kinase (MAPK) proteins, including ERK1/2 (41.5/33.2%), JNK1/2 (44.2/39.6%), and p38 (42.2%). The Fab fragment could be internalized into cells, and the pretreatment of RAW 264.7 macrophages with the Fab fragment reduced the mRNA expression of the Toll-like receptor (TLR4, 32.7-44.4% inhibition) and αVβ3 integrin (76.1% inhibition). In conclusion, Fab fragments regulated the TLR4 and αVβ3 integrin-mediated inflammatory processes by blocking the NF-κB and MAPKs pathways in the LPS-induced RAW 264.7 macrophage system.

Orphan designation: Ovine specific immunoglobulin (Fab) fragments raised against Vipera berus veno, Treatment of snakebite envenomation

Orphan designation: Ovine specific immunoglobulin (Fab) fragments raised against Vipera berus veno, Treatment of snakebite envenomation

Investigation of the interatomic interaction in immunoglobulin G fragments via X-ray photoelectron spectroscopy

The chemical structure of Fc and Fab fragments of immunoglobulin G (IgG) is studied by means of X-ray photoelectron spectroscopy. The purpose of studies is to apply the given technique in determining the chemical composition of fragments obtained via papain hydrolysis. Fragments with specified biological properties can be used as an effective agent in a vaccine developed for the treatment of human rheumatoid arthritis, which is prepared from the IgG Fc fragment.

Differential Inhibition of Human Atherosclerotic Plaque–Induced Platelet Activation by Dimeric GPVI-Fc and Anti-GPVI Antibodies: Functional and Imaging Studies

BackgroundGlycoprotein VI (GPVI) is the essential platelet collagen receptor in atherothrombosis, but its inhibition causes only a mild bleeding tendency. Thus, targeting this receptor has selective antithrombotic potential. ObjectivesThis study sought to compare compounds interfering with platelet GPVI–atherosclerotic plaque interaction to improve current antiatherothrombotic therapy. MethodsHuman atherosclerotic plaque–induced platelet aggregation was measured in anticoagulated blood under static and arterial flow conditions (550/s, 1,100/s, and 1,500/s). Inhibition by dimeric GPVI fragment crystallizable region of IgG (Fc) masking GPVI binding sites on collagen was compared with that of 3 anti-GPVI antibodies: BLO8-1, a human domain antibody; 5C4, a fragment antigen-binding (Fab fragment) of monoclonal rat immunoglobulin G; and m-Fab-F, a human recombinant sFab against GPVI dimers. ResultsGPVI-Fc reduced plaque-triggered platelet aggregation in static blood by 51%, BLO8-1 by 88%, and 5C4 by 93%. Under arterial flow conditions, BLO8-1 and 5C4 almost completely inhibited platelet aggregation while preserving platelet adhesion on plaque. Inhibition by GPVI-Fc, even at high concentrations, was less marked but increased with shear rate. Advanced optical imaging revealed rapid persistent GPVI-Fc binding to collagen under low and high shear flow, upstream and downstream of plaque fragments. At low shear particularly, platelets adhered in plaque flow niches to GPVI-Fc–free segments of collagen fibers and recruited other platelets onto aggregates via ADP and TxA2 release. ConclusionsAnti-GPVI antibodies inhibit atherosclerotic plaque-induced platelet aggregation under static and flow conditions more effectively than GPVI-Fc. However, potent platelet inhibition by GPVI-Fc at a higher shear rate (1,500/s) suggests localized antithrombotic efficacy at denuded or fissured stenotic high-risk lesions without systemic bleeding. The compound-specific differences have relevance for clinical trials targeting GPVI-collagen interaction combined with established antiplatelet therapies in patients with spontaneous plaque rupture or intervention-associated plaque injury.

Evaluation of coagulation in vitro with platelet mapping and rapid TEGTM on thrombelastography

Objective To evaluate performance of PlateletMapping(R) and RapidTEGTM based on thrombelastography by blocking platelet function with Reopro.Methods PlateletMapping was carried out with whole blood from healthy volunteers mixed with Reopro in vitro in a serial of titration.TEG(R) ACT of heparinized blood was tested with Rapid TEG kits.Linearity, repeatability and validity were calculated for two methods.Results MA activated by Kaolin, AA and ADP decreased with the increase of the concentration of Reopro. Inhibition rates (%)for AA and ADP induced aggregation were repeatable in channels and systems.At the Reopro levels of (1-4) × 10-2 mg, inhibition rate increased statistically( AA:27.99% ± 2.8% vs 63.37% ± 0.0% ,t = 21.9, P ＜ 0.01;ADP: 35.9% ± 0.56% vs 91.42% ± 1.14%,t=58.9,P ＜ 0.01 ) after addition of Reopro.Dose-dependent effect relationship could be seen between TEG(R) ACT and heparin;In Rapid TEG assay, measurement repeatability of K, α angle, MA and TEG(R)ACT were all good ( CV ＜ 5% ) except for R.Conclusions PlateletMapping(R) is sensitive to the inhibition of platelet function with good precision with dose-dependent effect.Moreover, Rapid TEG provides analysis of the overall coagulation function besides monitoring heparin therapy. Key words: Thrombelastograghy; Platelet function tests; Platelet aggregation; Antibodies,monoclonal; Immunoglobulin fab fragments; Heparin

Polymeric Osteopontin Employs Integrin α9β1 as a Receptor and Attracts Neutrophils by Presenting a de Novo Binding Site

Osteopontin (OPN) is a cytokine and ligand for multiple members of the integrin family. OPN undergoes the in vivo polymerization catalyzed by cross-linking enzyme transglutaminase 2, which consequently increases the bioactivity through enhanced interaction with integrins. The integrin alpha9beta1, highly expressed on neutrophils, binds to the sequence SVVYGLR only after intact OPN is cleaved by thrombin. The SVVYGLR sequence appears to be cryptic in intact OPN because alpha9beta1 does not recognize intact OPN. Because transglutaminase 2-catalyzed polymers change their physical and chemical properties, we hypothesized that the SVVYGLR site might also be exposed on polymeric OPN. As expected, alpha9beta1 turned into a receptor for polymeric OPN, a result obtained by cell adhesion and migration assays with alpha9-transfected cells and by detection of direct binding of recombinant soluble alpha9beta1 with colorimetry and surface plasmon resonance analysis. Because the N-terminal fragment of thrombin-cleaved OPN, a ligand for alpha9beta1, has been reported to attract neutrophils, we next examined migration of neutrophils to polymeric OPN using time-lapse microscopy. Polymeric OPN showed potent neutrophil chemotactic activity, which was clearly inhibited by anti-alpha9beta1 antibody. Unexpectedly, mutagenesis studies showed that alpha9beta1 bound to polymeric OPN independently of the SVVYGLR sequence, and further, SVVYGLR sequence of polymeric OPN was cryptic because SVVYGLR-specific antibody did not recognize polymeric OPN. These results demonstrate that polymerization of OPN generates a novel alpha9beta1-binding site and that the interaction of this site with the alpha9beta1 integrin is critical to the neutrophil chemotaxis induced by polymeric OPN.

Novel System to Achieve One-Pot Modification of Cargo Molecules with Oligoarginine Vectors for Intracellular Delivery

There is a growing number of reports showing the usefulness of cell-penetrating peptides (CPPs) including oligoarginines for intracellular delivery of macromolecules. Although the covalent attachment of the CPP segments to the cargo molecules is usually required to ensure effective delivery, conventional methods of conjugation need several manipulation steps that are often time-consuming and laborious. Here, we report a novel approach to allow easy modification of cargo molecules with oligoarginine CPPs. The key feature is the employment of oligoarginines (R8 and R12) equipped with a sulfosuccinimidylsuberyl moiety (BS(3)-R8 and -R12). One-pot modification is achieved simply by mixing BS(3)-R8 and -R12 with cargos in an aqueous buffer. The usefulness of this approach was exemplified through the conjugate formation with Fab fragments of immunoglobulin G, amino-functionalized poly(ethylene glycol)s (amino-PEGs), and amino quantum dots (amino-QDs), yielding an efficient cellular uptake.

An actin–myosin functional assay for analysis of smooth muscle (anti-microfilament) autoantibodies in human plasma

The detection of serum autoantibodies to smooth muscle (SMA) on rodent gastric mucosa by indirect immunofluorescence (IIF) has long been an immunodiagnostic marker for autoimmune hepatitis type 1 (AIH-1). The reactive antigenic moieties are cytoskeletal proteins which include polymeric F-actin as judged by the staining of microfilaments of tissue by IIF. However, their specificity for actin in AIH-1 can be and usually is uncertain. Using an in vitro functional assay, we compared the effects of Fab fragments of immunoglobulin (IgG) prepared from SMA-positive plasma from two patients with the effects of Fabs from 10 healthy subjects. Fabs are incorporated into an assay where actin (the putative antigen) activates skeletal muscle heavy meromyosin (HMM) ATPase activity. The data from these functional assays provide new insights into the significance of anti-microfilament assays in the diagnosis, and perhaps also pathogenesis, of AIH-1.

Acid wash in determining cellular uptake of Fab/cell‐permeating peptide conjugates

Successful intracellular delivery of various bioactive molecules has been reported using cell-permeating peptides (CPPs) as delivery vectors. To determine the effects of CPPs on the cellular uptake of immunoglobulin Fab fragment, conjugates of a radio-iodinated Fab fragment with CPPs (CPP-(125)I-Fab) derived from HIV-1 TAT, HIV-1 REV, and Antennapedia (ANP) were prepared. These vectors are rich in basic amino acids, and their strong adsorption on cell surfaces often results in overestimation of internalized peptides. Cell wash with an acidic buffer (0.2M glycine-0.15M NaCl, pH 3.0) was thus employed in this study to remove cell-surface adsorbed CPP-(125)I-Fab conjugates. This procedure enabled clearer understanding of the methods of internalization of CPP-(125)I-Fab conjugates. The kinetics of internalization of REV-(125)I-Fab conjugate was rapid, and a considerable fraction of REV-(125)I-Fab was taken up by HeLa cells as early as 5 min after administration. It was also shown that cellular uptake of these conjugates was significantly inhibited in the presence of endocytosis/ macropinocytosis inhibitors, in the order REV-(125)I-Fab > or = TAT-(125)I-Fab > or = ANP-(125)I-Fab; this order was the same as for effectiveness of intracellular delivery. Simultaneous cell washing with phosphate-buffered saline (PBS) and this acidic buffer effectively separated the internalized conjugates from the cell-surface-adsorbed ones, and considerable differences were observed in these amounts dependent on the employed CPPs.

Selection of SARS-Coronavirus-specific B cell epitopes by phage peptide library screening and evaluation of the immunological effect of epitope-based peptides on mice

Antibodies to SARS-Coronavirus ( SARS-CoV)-specific B cell epitopes might recognize the pathogen and interrupt its adherence to and penetration of host cells. Hence, these epitopes could be useful for diagnosis and as vaccine constituents. Using the phage-displayed peptide library screening method and purified Fab fragments of immunoglobulin G (IgG Fab) from normal human sera and convalescent sera from SARS-CoV-infected patients as targets, 11 B cell epitopes of SARS-CoV spike glycoprotein (S protein) and membrane protein (M protein) were screened. After a bioinformatics tool was used to analyze these epitopes, four epitope-based S protein dodecapeptides corresponding to the predominant epitopes were chosen for synthesis. Their antigenic specificities and immunogenicities were studied in vitro and in vivo. Flow cytometry and ELISPOT analysis of lymphocytes as well as a serologic analysis of antibody showed that these peptides could trigger a rapid, highly effective, and relatively safe immune response in BALB/c mice. These findings might aid development of SARS diagnostics and vaccines. Moreover, the role of S and M proteins as important surface antigens is confirmed.

Distribution of Immunoglobulin Fab Fragment Conjugated with HIV-1 REV Peptide following Intravenous Administration in Rats

HIV-1 REV peptide (positions 34-50) is well-known as a cell-permeating peptide. In this study, we investigated the distribution of Fab fragment of immunoglobulin conjugated with REV peptide (REV-Fab) following intravenous administration in rats, and compared with those of the native Fab fragment (nFab). Radioiodinated REV-Fab or nFab ((125)I-REV-Fab or (125)I-nFab, respectively) was given in a single intravenous dose of 2 mg/kg (3 MBq/kg). Total radioactive and TCA-insoluble radioactive concentrations in blood, whole-body autoradiography (ARG), and urinary excretion rates were assayed following administration. Regarding blood and plasma, total radioactive and TCA-insoluble radioactive concentrations for (125)I-REV-Fab were remarkably lower than those for (125)I-nFab. In the whole-body ARG at 4 h after administration, (125)I-REV-Fab produced remarkably higher radioactivity in the adrenal gland, spleen, and liver, compared to (125)I-nFab. Regarding urinary excretion rates, approximately 70% of the radioactive dose was excreted in the form of a low-molecular-weight component by 24 h after administration for both samples. (125)I-REV-Fab may penetrate quickly from blood to adrenal gland, spleen, liver, and other tissues after intravenous administration to rats, and then did not stay in situ and was digested and excreted mostly via the renal route by 24 h. With these features, cell-permeating peptides are expected to help the development of new antibody pharmaceuticals.

Isolation and some properties of fimbriae of oral Streptococcus intermedius.

Streptococcus intermedius 1208-1 carried linear fiber-like fimbriae that extended radially from the cell surface. The fimbriae were isolated by pipetting and sonication and were purified by ammonium sulfate precipitation followed by a column chromatography series. Heat treatment in the presence of sodium dodecyl sulfate resulted in the dissociation into smaller molecules. Rabbit antiserum raised against the purified protein reacted with fimbriae on the surface of bacteria under immunogold staining. Serotype g or g-related strains produced the fimbriae and aggregated in human saliva. The aggregation was inhibited by the anti-fimbriae immunoglobulin Fab fragment or the purified fimbriae.

Improving the tolerance of a protein a analogue to repeated alkaline exposures using a bypass mutagenesis approach.

Staphylococcal protein A (SPA) is a cell surface protein expressed by Staphylococcus aureus. It consists of five repetitive domains. The five SPA-domains show individual interaction to the Fc-fragment as well as certain Fab-fragments of immunoglobulin G (IgG) from most mammalian species. Due to the high affinity and selectivity of SPA, it has a widespread use as an affinity ligand for capture and purification of antibodies. One of the problems with proteinaceous affinity ligands in large-scale purification is their sensitivity to alkaline conditions. SPA however, is considered relatively stable to alkaline treatment. Nevertheless, it is desirable to further improve the stability in order to enable an SPA-based affinity medium to withstand even longer exposure to the harsh conditions associated with cleaning-in-place (CIP) procedures. For this purpose, a protein engineering strategy, which was used earlier for stabilization and consists of replacing the asparagine residues, is employed. Since Z in its "nonengineered" form already has a significant tolerance to alkaline treatment, small changes in stability due to the mutations are difficult to assess. Hence, in order to enable detection of improvements regarding the alkaline resistance of the Z domain, we chose to use a bypass mutagenesis strategy using a mutated variant Z(F30A) as a surrogate framework. Z(F30A) has earlier been shown to possess an affinity to IgG that is similar to the wild-type but also demonstrates decreased structural stability. Since the contribution of the different asparagine residues to the deactivation rate of a ligand is dependent on the environment and also the structural flexibility of the particular region, it is important to consider all sensitive amino acids one by one. The parental Z-domain contains eight asparagine residues, each with a different impact on the alkaline stability of the domain. By exchanging asparagine 23 for a threonine, we were able to increase the stability of the Z(F30A) domain in alkaline conditions. Also, when grafting the N23T mutation to the Z scaffold, we were able to detect an increased tolerance to alkaline treatment compared to the native Z molecule.

Lipid Cubic Phases as Stable Nanochannel Network Structures for Protein Biochip Development: X-ray Diffraction Study

Biocompatible nanochannel and mesoporous structures of a three-dimensional (3D) periodicity, facilitating protein entrapment at native conformations, constitute organized protective media of interest for the fast developing fields of protein biochips and affinity biosensors. Three-dimensional periodic nanochannel structures possessing reactive chemical groups, surface-exposed to the aqueous phase, are formed here in self-assembled mixtures of a nonlamellar lipid glycerylmonooleate (MO) and a maleimide(triethylene glycol)ether lipid (MTEG) under physiological hydration conditions and are investigated by high-resolution time-resolved synchrotron X-ray diffraction in the interval from 3 to 96 °C. The mechanism of formation and the structural parameters of two nonlamellar cubic phases Q224 and Q229 (of space groups Pn3m and Im3m) are monitored. The structural phase behavior of the MO/MTEG-based immunocubosome and proteocubosome mixtures, obtained upon incubation with immunoglobulin Fab fragments, whole IgG, albumin, transferrin, and fibrinogen at high concentrations, indicates that the cubic lattices exist in excess water as nanostructured channel networks. With the particular lipid composition of interest, the protein type only finely tunes these channel networks and they appear to be stable over a broad temperature interval. A new mechanism of protein entrapment within the 3D cubosome lipid networks is proposed.

Scaffolds for protein crystallisation.

In the absence of a method to ensure that crystals can be obtained for any given protein, the possibility of developing scaffolds for protein crystallisation becomes attractive. Among several approaches that could yield scaffolds, two are particularly promising: the first is based on immunoglobulin Fab fragments and immunoglobulin binding proteins while the second is based on fusion proteins. In the Fab based scaffold, the protein of interest is the antigen recognised by the antibody. In the second case, it is a protein fused to one of the scaffold components. The operational difference between the two methods is the existence of a flexible covalent tether compared to a highly specific interaction. The relative merits and disadvantages of each approach are discussed here. We also describe a lattice obtained through a combinatorial approach which appears to have the required properties to be considered a scaffold. The system makes use of an Fab derived from a rheumatoid factor and an Fc-fusion protein. The Fc-fusion system is ideal for enhanced expression of glycoproteins in mammalian cells and provides a useful tag for their purification. The molecular replacement shows a mode of binding for this rheumatoid factor that is not competitive with bacterial Fc-binding proteins. Hence it may be possible to generalize the method to include bacterial expression of fusion proteins with either protein A or protein G as the fusion partner.

Crystallization of macromolecular complexes:: stoichiometric variation screening

Theoretically a crystal may contain both complexed and uncomplexed molecules simultaneously in the same lattice. Since we seldom screen for such possibilities, such occurrences are only rarely reported. Here we propose that stoichiometry should be one of the parameters to be screened in the crystallization of macromolecular complexes. By allowing for non-biologically significant stoichiometries, we may increase the chances of crystallizing a macromolecular complex and of selecting arrangements which crystallize better or yield more ordered crystals. Although biological forces tend to be stronger than lattice-building interactions, in the crystal the latter will dominate numerically. By allowing for a varied stoichiometry we permit a wider selection of lattice-building contacts and increase the probability of crystallization. From these theoretical considerations we have developed methodology compatible with classical solubility screening and other well-established crystallization principles. We discuss this technique, stoichiometric variation screening (SVS), as part of a multicomponent system for the enhancement of crystallization of macromolecular complexes. We present this technique as an extension of reverse screening and illustrate the complementarity in the methodology. We present two examples of the use of SVS: the complexes between an immunoglobulin Fab fragment and two bacterial proteins, namely the D domain of protein A from Staphylococcus aureus (SpA) and a single domain of protein L from Peptostreptococcus magnus (PpL). In the first example there are 3 Fab molecules and only 2 SpA D domains (domD) (2 complexed and 1 unliganded Fab), in the second 2 Fabs and only 1 PpL domain (1 complexed and 1 unliganded Fab). SVS has the added and unique advantage that in the same crystal we have information on both the unliganded and complexed states under precisely identical conditions: one structure, two answers. Together with a combinatorial method for complex crystallization based on immunoglobulin-binding domains, it may enhance the probability of crystallization by well over a factor of ten.

The animal research kit (ARK) can be used in a multistep double staining method for human tissue specimens.

The newly developed Animal Research Kit (ARK) offers a simple and economic way of biotinylating mouse primary antibodies for background-free immunostaining of mouse and rat tissue specimens. Biotinylation involves the use of a biotinylated goat anti-mouse immunoglobulin Fab fragment mixed with a mouse primary antibody and subsequent blocking with normal mouse immunoglobulin. Because a reliable immunoenzyme double staining procedure on human tissue specimens with two unlabeled mouse primary antibodies of identical subclass is almost impossible, we have tested the performance of ARK biotinylation of one primary antibody in a multistep indirect/direct staining protocol. The multistep double staining procedure involved the subsequent application of an unlabeled mouse monoclonal antibody (MAb) 1 detected with an enzyme-labeled EnVision reagent, normal mouse serum for blocking, followed by a biotinylated mouse MAb 2 and enzyme-labeled streptavidin. Alkaline phosphatase and peroxidase enzymatic activities were developed last. Double staining results obtained with an ARK biotinylated reagent were compared with a truly biotinylated reagent using N-hydroxy succinimide-biotin for conjugation. It appeared that both biotinylation procedures revealed identical double staining results. Although a limited number of antibody combinations have been tested, it is clear that this double staining procedure will be successful for many antibody pairs.

Rapid expression cloning of human immunoglobulin Fab fragments for the analysis of antigen specificity of B cell lymphomas and anti-idiotype lymphoma vaccination

An expression system for rapid and standardized production of human recombinant immunoglobulin Fab fragments in E. coli was developed. Functional folding of the Fab fragments was accomplished by the dicistronic expression vector pFab.γκ containing specialized leader sequences to direct the immunoglobulin heavy and light chains to the periplasmic bacterial space. A C-terminal hexahistidine tag of the Fd chain facilitated metal affinity chromatography and purification to homogeneity as assessed by SDS PAGE and silver staining. Specific antigen recognition by a hybridoma-derived Fab fragment was indistinguishable from that of the corresponding monoclonal antibody. This protocol may be useful for analysis of the antigen specificity of human B cells and for convenient production of lymphoma-derived idiotype protein for vaccination strategies. To obtain unmodified immunoglobulin cDNA sequences from small human biopsies for insertion into pFab.γκ, oligo(dG)-tailed cDNA was amplified with an oligo(dC)- and nested μ or κ constant region-specific primers. Using single sets of primers for each class of immunoglobulin transcripts, the products of this anchored PCR reflected the relative abundance of the starting cell population and permitted reliable identification of clonal, lymphoma-derived sequences for subsequent expression cloning.