Purification of endogenous digitalis-like factor(s) from cord blood of neonate by immunoaffinity chromatography.

We have previously shown that antidigoxin antibodies may neutralize partially purified endogenous digitalis like factor(s) present in newborn (umbilical cord) plasma. We here report on the preparation of an immunoaffinity chromatographic system (high affinity digoxin-binding antibodies (Fab fragments) bound covalently to Sepharose) for the purification of endogenous digitalis like factor(s). Neonate plasma extract loses all its biological digitalis-like activity (erythrocyte 86Rb uptake inhibition) after absorption on Sepharose coupled to Fab fragments but not after absorption on uncoupled Sepharose. Endogenous digitalis like factor(s) absorbed to Sepharose coupled to Fab fragments can be eluted by methanol. Subsequent HPLC separation indicate that at least two molecular species with digitalis-like properties are retained by antibodies bound to Sepharose and can be recovered with methanol.     

Kinetics of anti-carcinoembryonic antigen antibody internalization: effects of affinity,bivalency, and stability

Theoretical analyses suggest that the cellular internalization and catabolism of bound antibodies contribute significantly to poor penetration into tumors. Here we quantitatively assess the internalization of antibodies and antibody fragments against the commonly targeted antigen carcinoembryonic antigen (CEA). Although CEA is often referred to as a non-internalizing or shed antigen, anti-CEA antibodies and antibody fragments are shown to be slowly endocytosed by LS174T cells with a half-time of 10–16 h, a time scale consistent with the metabolic turnover rate of CEA in the absence of antibody. Anti-CEA single chain variable fragments (scFvs) with significant differences in affinity, stability against protease digestion, and valency exhibit similar uptake rates of bound antibody. In contrast, one anti-CEA IgG exhibits unique binding and trafficking properties with twice as many molecules bound per cell at saturation and significantly faster cellular internalization after binding. The internalization rates measured herein can be used in simple computational models to predict the microdistribution of these antibodies in tumor spheroids. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Affinity maturation of Fab antibody fragments by fluorescent-activated cell sorting of yeast-displayed libraries

We report for the first time the affinity maturation of Fab antibody fragments using fluorescent-activated cell sorting (FACS) of yeast-displayed repertoires. A single yeast display vector which enables the inducible expression of an anchored heavy chain and a soluble light chain has been constructed. The assembly and functional display on the yeast cell surface of Fab antibodies specific for different protein targets has been demonstrated by flow cytometry and immunofluorescence microscopy. We have affinity matured a Fab antibody specific for the tetravalent antigen streptavidin using FACS of yeast-displayed repertoires diversified by error-prone polymerase chain reaction. A panel of variants with up to 10.7-fold improvement in affinity was obtained after selection. Two leading clones, R2H10 (3.2 nM) and R3B1 (5.5 nM), had mutations in light chain complementarity determining region 1 LC-CDR1 (H34R) and LC-CDR3 (Y96H or Y96F) and gave a 10.7-fold and 6.3-fold affinity improvement over the starting antibody, respectively. The ability to efficiently affinity mature Fab antibodies is an important component of the antibody development pipeline and we have shown that yeast display is an efficient method for this purpose.     

Purification and specificity of antibodies to inosine 5'-monophosphate

Antibodies to inosine 5'-monophosphate elicited in rabbits by immunization with a conjugate of IMP (oxidized with periodate) and bovine serum albumin have been purified by affinity chromatography. By the use of two affinity columns, Sepharose-IMP and Sepharose-oligo(I), the antibodies have been fractionated into three fractions. By gel diffusion, the three fractions were found to react with the conjugates of bovine serum albumin and IMP, GMP and AMP respectively. The association constants for the binding of the Fab fragments purified on the Sepharose-oligo(I) column and several haptens have been deduced from fluorescence experiments. It is shown that the base and the phosphate group play an important part in the binding of IMP to Fab fragments. No reaction has been found between the antibodies and poly(I).poly(C) by gel diffusion. However, the antibodies interact with poly(I).poly(C) since they decrease the thermal stability of poly(I).poly(C).     

Primary antibody-Fab fragment complexes: a flexible alternative to traditional direct and indirect immunolabeling techniques.

Immunolabeling with immune complexes of primary and secondary antibodies offers an attractive method for detecting and quantifying specific antigen. Primary antibodies maintain their affinity for specific antigen after labeling with Fab fragments in vitro. Incubation of these immune complexes with excess normal serum from the same species as the primary antibody prevents free Fab fragments from recognizing immunoglobulin. Effectively a hybrid between traditional direct and indirect immunolabeling techniques, this simple technique allows primary antibodies to be non-covalently labeled with a variety of reporter molecules as and when required. Using complexes containing Fab fragments that recognize both the Fc and F(ab')2 regions of IgG, we show that this approach prevents nonspecific labeling of endogenous immunoglobulin, can be used to simultaneously detect multiple antigens with primary antibodies derived from the same species, and allows the same polyclonal antibody to be used for both antigen capture and detection in ELISA.     

Inactivation and modification of superoxide dismutase by glyoxal: prevention by antibodies

Glyoxal is an endogenous compound, the levels of which are increased in various pathologies associated with hyperglycaemia and other related disorders. It has been reported to inactivate critical cellular enzymes by promoting their cross-linking and perpetuates advanced glycation end-product (AGE) formation. In this study, we used superoxide dismutase (SOD) as a model to investigate the ability of specific anti-enzyme antibodies and monomer Fab fragments to protect against glyoxal-induced deactivation and aggregate formation. We found that glyoxal deactivated SOD, in a concentration and time-dependent fashion. The enzymatic activity was monitored spectrophotometrically and it was found that enzyme lost approximately 95% of its original activity, when exposed to 10 mM glyoxal for 120 h. SDS-polyacrylamide gel electrophoresis demonstrated the formation of high molecular weight aggregates in SOD samples exposed to glyoxal. Surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) showed increase in relative molecular mass (M(r)), upon exposure to glyoxal. Specific anti-enzyme antibodies and monomer Fab fragments markedly inhibited SOD deactivation caused by glyoxal and decreased the extent of cross-linking or formation of aggregates. This protection by the antibodies or Fab fragments was specific since, other non-specific antibodies were not able to protect SOD. Previously, antibodies have been used to prevent aggregation of beta-amyloid peptides in Alzheimer and prion-protein disease. Our findings provide a new perspective, for use of antibodies to prevent the biomolecules against glycation-induced deactivation and alteration.     

抗人IgG Fc片段及Fab段单抗的鉴定及应用

本实验采用木瓜酶水解,SPA柱亲合层析等手段得到人IgGFc段及Fab段,以Sigma抗人IgGfFc段和抗人IgG Fab段单抗为标准品,鉴定了细胞库中抗人IgG系列的部分细胞株,得到特异性分泌抗人IgG Fc段和抗人IgG Fab段单抗的细胞各一株。 在上述实验基础上,用抗人IgG Fc及抗人IgG Fab单抗分别制备了Sepharose4B亲合层析柱,提纯了酶解人IgG Fc、Fab片段,经ELISA法鉴定,相互之间无交叉反应。同时用此方法制备了人抗HBe Fab片段,并将该片段进行了过氧化物酶标记,用来配制HBe ELISA诊断盒,证明其生物活性未受影响,而且消除了类风湿因子引起的HBe Ag假阳性现象。因抗HBe单抗来源困难,如采用HBe多抗制备ELISA试剂,本法将是提高质量的一个好方法。     

Serologic aspects of IgG4 antibodies. II. IgG4 antibodies form small, nonprecipitating immune complexes due to functional monovalency

Human IgG4 antibodies directed against phospholipase A, the P1 antigen from Dermatophago?des pteronyssinus extracts, and cat albumin were found unable to cross-link antigen. Previously, it was demonstrated that IgG4 antibodies, in contrast to IgG1 antibodies, did not cross-link Sepharose-bound antigen and antigen added in solution. To eliminate the possibility that this phenomenon was caused by preferential binding of both IgG4 Fab fragments to the solid-phase-bound antigen, cross-linking of antigen was studied in a fluid-phase system. In this test, incapability of IgG4 antibodies to bridge two antigens was also found. As a result of such a phenomenon, it is expected that immune complexes formed by IgG4 antibodies will be considerably smaller than complexes formed by IgG1. This was confirmed by analysis of the molecular size profiles of IgG1- and IgG4-containing immune complexes in sucrose-density gradients. Moreover, IgG1 was able to precipitate antigen in a radioimmunoprecipitation test, whereas precipitation was not demonstrable by the same amount of IgG4 antibodies. Even 3% polyethylene glycol 8,000 did not precipitate the small IgG4-containing immune complexes efficiently. The antibodies studied were of a high-affinity type, and there was no significant difference in association constants between IgG1 and IgG4 antibodies. Therefore, we were not able to confirm observations reported in the literature that the IgG4 subclass is associated with a low-affinity antibody response; probably, the affinity of the IgG4 antibodies was underestimated by other investigators because of the polyethylene glycol precipitation technique used to separate antibody-bound and free antigen. Our findings stress the point that IgG4 antibodies take a special place in the immune response upon chronic exposure to antigen.     

Oriented Immobilization of Anti-Pneumolysin Tagged Recombinant Antibody Fragments

Recombinant antibodies such as Fab and scFv are monovalent and small in size, although their functional affinity can be improved through tag-specific immobilization. In order to find the optimum candidate for oriented immobilization, we generated Fab and scFv fragments derived from an anti-pneumolysin monoclonal antibody PLY-7, with histidine and cysteine residues added in diverse arrangements. Tagged antibody fragments scFv-Cys7-His6, His6-scFv-Cys7, and Fab-Cys7 lost considerable affinity for the antigen; however, Fab-His6, Fab-Cys1, and scFv-His6-Cys1 were able to detect immobilized antigen, revealing that the position and number of histidine and cysteine residues are involved differently in the reactivity of antibody fragments. Random and orientated immobilizations were carried out using conventional polystyrene and commercial surface-pretreated ELISA plates. The best orientation performance was obtained with Fab-Cys1-biotin on streptavidin-coated plates with increased signal levels of 62%, while oriented immobilization of Fab-His6 and scFv-His6-Cys1 on nickel- and maleimide-coated plates failed to improve the ELISA sensitivity.     

Antibody-induced activation of p185HER2 in the human lung adenocarcinoma cell line Calu-3 requires bivalency

In the present study we utilized two previously described monoclonal antibodies (mAb), and their respective Fab portions, directed against the extracellular domain of p185^HER2, a transmembrane glycoprotein with intrinsic tyrosine kinase activity coded by theHER2/neu oncogene, to study the mechanism of mAb-induced receptor internalization and phosphorylation. Fluorescence scan analysis and direct binding of radiolabelled mAb and their Fab fragments showed that entire MGR2 and MGR3 mAb were reactive with similar binding affinity on two cell lines (Calu-3 and Sk-Br-3) overexpressing the p185^HER2 receptor, and unreactive on unrelated cells. The corresponding Fab fragments were positive on the related cells, but bound with diminished intensity and affinity. Entire MGR2 and MGR3 induced internalization in both Calu-3 and Sk-Br-3 cells, whereas their Fab portions were not internalized. When the bivalency of the MGR2 Fab fragment was artificially reconstituted by incubation with rabbit anti-(mouse IgG), internalization was obtained. Monovalent binding of the entire labelled antibodies, obtained in the presence of a saturating amount of unlabelled antibody, decreased both the rate and the final amount of internalized antibody. Metabolic labelling and immunoblotting experiments showed that incubation with entire MGR3 amplified the basal phosphorylation of the p185^HER2 receptor in Calu-3 and Sk-Br-3 cells, whereas MGR3 Fab decreased the signal. Taken together, our data indicate that antibody-mediated activation of p185^HER2 in Calu-3 and Sk-Br-3 cells occurs through the dimerization of receptor molecules and that bivalency of the activating antibody is mandatory for induction of internalization and phosphorylation of the receptor. Our data support an allosteric model of activation for the p185^HER2 receptor.     

Comprehensive optimization of a single-chain variable domain antibody fragment as a targeting ligand for a cytotoxic nanoparticle

Antibody-targeted nanoparticles have the potential to significantly increase the therapeutic index of cytotoxic anti-cancer therapies by directing them to tumor cells. Using antibodies or their fragments requires careful engineering because multiple parameters, including affinity, internalization rate and stability, all need to be optimized. Here, we present a case study of the iterative engineering of a single chain variable fragment (scFv) for use as a targeting arm of a liposomal cytotoxic nanoparticle. We describe the effect of the orientation of variable domains, the length and composition of the interdomain protein linker that connects VH and VL, and stabilizing mutations in both the framework and complementarity-determining regions (CDRs) on the molecular properties of the scFv. We show that variable domain orientation can alter cross-reactivity to murine antigen while maintaining affinity to the human antigen. We demonstrate that tyrosine residues in the CDRs make diverse contributions to the binding affinity and biophysical properties, and that replacement of non-essential tyrosines can improve the stability and bioactivity of the scFv. Our studies demonstrate that a comprehensive engineering strategy may be required to identify a scFv with optimal characteristics for nanoparticle targeting.     

Purification and characterization of Fab fragments from anti-mouse NGF polyclonal antibodies

A functional role for Nerve Growth Factor (NGF) in the peripheral nervous system is well-documented, but a similar case for NGF in the central nervous system remains to be established. One approach to answering this question would be the availability of high-affinity monospecific Fab fragments obtained against NGF. In the present studies we describe the preparation and characterization of such Fab fragments from anti-mouse NGF polyclonal antibodies. Following their purification by the use of a NGF Sepharose-coupled affinity column, the Fab fragments were examined for biological competence in several ways. In vitro, the anti-Fab fragments blocked the neuronotrophic activity of NGF, as measured by the survival of chicken embryonic day 8 dorsal root ganglion neurons. In vivo, these Fab fragments, when administered systemically to neonatal rats, produced a decrease of noradrenaline levels in two sympathetically innervated organs, the heart and the spleen. These findings suggest that affinity purified Fab fragments of anti-NGF antibodies can be a useful tool for studying the physiological function of NGF in the nervous system.     

Comprehensive optimization of a single-chain variable domain antibody fragment as a targeting ligand for a cytotoxic nanoparticle

Antibody-targeted nanoparticles have the potential to significantly increase the therapeutic index of cytotoxic anti-cancer therapies by directing them to tumor cells. Using antibodies or their fragments requires careful engineering because multiple parameters, including affinity, internalization rate and stability, all need to be optimized. Here, we present a case study of the iterative engineering of a single chain variable fragment (scFv) for use as a targeting arm of a liposomal cytotoxic nanoparticle. We describe the effect of the orientation of variable domains, the length and composition of the interdomain protein linker that connects VH and VL, and stabilizing mutations in both the framework and complementarity-determining regions (CDRs) on the molecular properties of the scFv. We show that variable domain orientation can alter cross-reactivity to murine antigen while maintaining affinity to the human antigen. We demonstrate that tyrosine residues in the CDRs make diverse contributions to the binding affinity and biophysical properties, and that replacement of non-essential tyrosines can improve the stability and bioactivity of the scFv. Our studies demonstrate that a comprehensive engineering strategy may be required to identify a scFv with optimal characteristics for nanoparticle targeting.     

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     

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.     

Yeast mating for combinatorial Fab library generation and surface display

Yeast display of antibody fragments has proven to be an efficient and productive means for directed evolution of single chain Fv antibodies for increased affinity and thermal stability, and more recently for the display and screening of a non-immune library. In this paper, we describe an elegant and simple method for constructing large combinatorial Fab libraries for display on the surface of Saccharomyces cerevisiae, from modestly sized, and easily constructed, heavy and light chain libraries. To this end, we have constructed a set of yeast strains and a two vector system for heavy chain and light chain surface display of Fab fragments with free native amino termini. Through yeast mating of the haploid libraries, a very large heterodimeric immune Fab library was displayed on the diploids and high affinity antigen specific Fabs were isolated from the library.     

Enhanced catabolism of low density lipoproteins in rat by lactosaminated Fab fragment. A new carrier of macromolecules to the liver

Proteins conjugated with lactose residues exhibit enhanced hepatic uptake mediated by the galactose receptor. In this study, we demonstrate that lactosaminated Fab fragments (lac-Fab) of IgG can induce hepatic catabolism of specific antigens, especially low density lipoproteins (LDL). lac-Fab and human LDL-lac-Fab complex exhibited specific uptake in isolated rat hepatocytes. In vivo in the rat, lactosamination enhanced plasma clearance of Fab fragments 2-fold and hepatic localization 20-fold. Fab fragments retained their affinity after lactosamination. Hepatic uptake of rat 125I-IgG complexed in vitro with anti-rat lac-Fab was increased almost 5-fold, compared to rat 125I-IgG alone. Injection of rats with anti-LDL lac-Fab induced plasma clearance and hepatic uptake of tracer amounts of previously injected human 125I-LDL, which decreased 50% 10 min after injection of lac-Fab, with 30% present in the liver. Asialofetuin completely inhibited these processes. After a bolus of 6 mg of human LDL, administration of anti-LDL lac-Fab reduced the serum cholesterol of rats to basal values within 2.5 h. These findings suggest that lactosaminated Fab fragments of specific IgGs are effective reagents for inducing hepatic uptake of macromolecules through the galactose receptor. lac-Fab specific for LDL may be an effective hypocholesterolemic agent in vivo.     

Fab MOR03268 triggers absorption shift of a diagnostic dye via packaging in a solvent-shielded Fab dimer interface

Molecular interactions between near-IR fluorescent probes and specific antibodies may be exploited to generate novel smart probes for diagnostic imaging. Using a new phage display technology, we developed such antibody Fab fragments with subnanomolar binding affinity for tetrasulfocyanine, a near-IR in vivo imaging agent. Unexpectedly, some Fabs induced redshifts of the dye absorption peak of up to 44 nm. This is the largest shift reported for a biological system so far. Crystal structure determination and absorption spectroscopy in the crystal in combination with microcalorimetry and small-angle X-ray scattering in solution revealed that the redshift is triggered by formation of a Fab dimer, with tetrasulfocyanine being buried in a fully closed protein cavity within the dimer interface. The derived principle of shifting the absorption peak of a symmetric dye via packaging within a Fab dimer interface may be transferred to other diagnostic fluorophores, opening the way towards smart imaging probes that change their wavelength upon interaction with an antibody.     

Internalization and cycling of the T cell antigen receptor. Role of protein kinase C

The dynamics of the T cell antigen receptor on a murine antigen specific T cell hybridoma have been analyzed using a monoclonal anti-receptor antibody. When this antibody, A2B4-2, is bound to surface receptors, no internalization is seen at 4 degrees C. Upon warming to 37 degrees C, between 20 and 30% of the antibody molecules are internalized over 20-30 min as measured by sensitivity to external acid. This level of internalization is identical if monovalent Fab fragments are used. In contrast, cross-linking of the anti-receptor antibody with a second antibody leads to rapid internalization of 100% of prebound surface A2B4-2. Phorbol 12-myristate 13-acetate (PMA) leads to the rapid internalization of up to 65% of the surface A2B4-2 or A2B4-2 Fab fragments. This effect requires protein kinase C and can be completely inhibited by depleting this kinase from the cells by long term treatment with high doses of PMA. Pretreatment of the T cells with PMA leads to a 40-50% drop in surface T cell antigen receptor expression. Despite the loss of surface receptors, the uptake of A2B4-2 in PMA-treated cells at 37 degrees C is identical to that seen in control cells. The total uptake of A2B4-2 at 37 degrees C is 25-30% greater than the number of surface receptors in control cells and about 100-150% greater than the number of surface receptors in PMA-treated cells. At steady state the percentage of total A2B4-2 on the cell surface is 75% for control cells and 38% for PMA-treated cells. The good agreement of these numbers with the percent internalization of a cohort of surface receptors suggests that all receptors are constantly cycling. The effect of PMA is to alter the kinetic parameters of this cycling, thus changing the steady state distribution of receptors between the plasma membrane and internal, presumably endosomal compartments. Measurement of initial rates of internalization suggests that the PMA effect can be largely explained by an increase in the internalization rate constant.