Evidence for a role of endogenous opiates in postprandial somatostatin release

Previously, we have demonstrated the effects of exogenously administered opiates on somatostatin release in dogs and therefore the present study was designed to determine the effect of endogenous opiates via naloxone-induced opiate receptor blockade on somatostatin release. Additionally, plasma insulin and pancreatic polypeptide (PP) levels were determined in response to intragastrically instilled protein, carbohydrate and fat test meals in a group of eight conscious dogs. To all test meals either naloxone (4 mg) or saline was added. The rise of plasma somatostatin levels in response to liver extract, sucrose and fat was attenuated significantly by naloxone. Naloxone had no effect on the rise of postprandial plasma insulin and PP levels. The present data demonstrate that endogenous opiates have a stimulatory effect on postprandial somatostatin release in dogs which indicates a tight interaction that might be of relevance for nutrient homeostasis.     

A rapid procedure for the humanization of monoclonal antibodies

An efficient and rapid procedure for the humanization of murine monoclonal antibodies (MumAb) is described. It consists of site-directed mutagenesis (SDM) to transfer the murine complementarity-determining regions (MuCDR) onto human framework regions (HuFR), followed by polymerase chain reaction (PCR) of the SDM product. Using SDM/PCR, rapid and correct humanization of MumAb heavy chains is clearly demonstrated. Compared to current protocols this method considerably reduces the time and labour required to generate humanized mAb.     

Validation of new aromatase monoclonal antibodies for immunohistochemistry: progress report

Intratumoral aromatase is a potential therapeutic target for the treatment of postmenopausal estrogen-dependent breast cancers. Therefore, reliable methods should be developed for routine application for the detection of intratumoral aromatase. A multi-center collaborative group has been established to generate and validate new aromatase monoclonal antibodies (MAbs). A recombinant GST–aromatase fusion protein was expressed in baculovirus and the purified protein was used for immunization of mice either as a native or formalin-fixed antigen. Hybridomas were generated using standard techniques and screened biochemically prior to immunohistochemistry (IHC) evaluation in human placenta, ovary and breast cancer tissues. Twenty-three MAbs selected by biochemical assays were further evaluated by IHC of paraffin-embedded tissue sections including normal ovary, and placenta, and a small series of 10 breast carcinomas. Of the 23 MAbs, 2 (clones 677 and F2) were determined to specifically stain cell types known to express aromatase in normal tissues. In breast carcinomas staining of malignant epithelium, adipose tissue, normal/benign and stromal compartments was detected. IHC was performed and independently evaluated by three pathologists (HS, TJA and SGS), each using the same evaluation criteria for staining intensity and proportion of immunopositive cells. With these two MAbs, interpathologist and intralaboratory variations were minimal in comparison with differences which could be detected between tissue specimens and antibodies.     

An express morphine assay in aqueous samples by immunochromatography using monoclonal antibodies labeled with colloidal gold

A simple and accessible express method was developed for the detection of morphine in aqueous samples using competitive immunochromatography. A complex of colloidal gold with monoclonal antibodies to morphine was used as the detection agent. The detection limit for morphine in aqueous samples was 10 ng/ml, and the analysis time was 5 min.Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 1, 2005, pp. 108–112.Original Russian Text Copyright © 2005 by Lyubavina, Zinchenko, Lapenkov, Nikolaeva.     

Lipid binding properties of 4E10, 2F5, and WR304 monoclonal antibodies that neutralize HIV-1

Two human mAbs (2F5 and 4E10), originally derived from HIV-1-infected patients, are important, but rare, mAbs that exhibit broad cross-clade neutralizing activities against HIV-1. In addition to peptide sequences on the gp41 envelope protein, both antibodies reportedly also bound specifically to several phospholipid antigens. However, the phospholipid binding property of 2F5 has been disputed and, because of uncertainly regarding phospholipid binding, the modeling of neutralizing mechanisms has been difficult. To explore this issue, we examined the binding of 4E10 and 2F5 to a broad range of lipid antigens by ELISA. 4E10 and 2F5 both bound to a variety of purified phospholipids, and 4E10 bound, but 2F5 did not bind, to cardiolipin. Both mAbs also bound to a sulfated glycolipid, sulfogalactosyl ceramide (sulfatide), and to two neutral glycolipids, galactosyl ceramide and glucosyl ceramide, but not to other galactosyl glycolipids. 4E10, but not 2F5, also bound to cholesterol, although both mAbs bound to squalene. Interestingly, 4E10, but not 2F5, exhibited striking binding to lipid A, the lipid moiety of Gram-negative bacterial lipopolysaccharide. The binding properties of 4E10 to phospholipids, sulfatide, cholesterol, squalene, and lipid A were similar to those of a neutralizing murine mAb (WR304) induced by liposomes containing phosphatidylinositol phosphate and lipid A, although WR304 did not bind to neutral glycolipids. The discovery of a binding specificity of 4E10 for lipid A, a widely used vaccine adjuvant, suggests that innate immunity stimulated by lipid A could have played a role for induction of multispecific antibodies that simultaneously recognize both HIV-1 protein and lipid antigens.     

The validation of new aromatase monoclonal antibodies for immunohistochemistry--a correlation with biochemical activities in 46 cases of breast cancer

Intratumoral aromatase is a therapeutic target for the treatment of post-menopausal estrogen-dependent breast cancers. Therefore, reliable methods should be developed for routine application for the detection of intratumoral aromatase. Immunohistochemistry (IHC) is considered one of the most suitable methods in this regard. A multi-centre collaborative group has been established to generate and validate new aromatase monoclonal antibodies. We have selected two monoclonal antibodies, #677 against native aromatase protein and F2 against formalin-fixed protein for this purpose. With these two monoclonal antibodies 43 cases of invasive ductal carcinoma, which had been previously assayed for aromatase activity by product isolation methodology, were immunostained in three laboratories in UK, USA and Japan and independently evaluated by three pathologists (H.S., T.A. and S.G.S.). Staining of malignant epithelium, adipose tissue, normal/benign and stromal compartments of the tumors were assessed by estimating the proportion of positive staining cells and the relative intensity of staining in this fashion. Immunoreactivity could be detected in each component of the tissue specimens but a significant positive correlation with biochemical activity was detected only in malignant epithelium stained with 677 not in other components with #677 and not in any of the components. Staining using F2 as a primary antibody did not produce a positive correlation in any components with aromatase activity. These results suggest that we now have a monoclonal antibody against aromatase (#677) which may be used to stain archival materials. A methodology and scoring system is recommended whereby staining significantly correlates with aromatase activity of the resected tissue specimens of breast cancer.     

Combining phage display with de novo protein sequencing for reverse engineering of monoclonal antibodies

The enormous diversity created by gene recombination and somatic hypermutation makes de novo protein sequencing of monoclonal antibodies a uniquely challenging problem. Modern mass spectrometry-based sequencing will rarely, if ever, provide a single unambiguous sequence for the variable domains. A more likely outcome is computation of an ensemble of highly similar sequences that can satisfy the experimental data. This outcome can result in the need for empirical testing of many candidate sequences, sometimes iteratively, to identity one which can replicate the activity of the parental antibody. Here we describe an improved approach to antibody protein sequencing by using phage display technology to generate a combinatorial library of sequences that satisfy the mass spectrometry data, and selecting for functional candidates that bind antigen. This approach was used to reverse engineer 2 commercially-obtained monoclonal antibodies against murine CD137. Proteomic data enabled us to assign the majority of the variable domain sequences, with the exception of 3–5% of the sequence located within or adjacent to complementarity-determining regions. To efficiently resolve the sequence in these regions, small phage-displayed libraries were generated and subjected to antigen binding selection. Following enrichment of antigen-binding clones, 2 clones were selected for each antibody and recombinantly expressed as antigen-binding fragments (Fabs). In both cases, the reverse-engineered Fabs exhibited identical antigen binding affinity, within error, as Fabs produced from the commercial IgGs. This combination of proteomic and protein engineering techniques provides a useful approach to simplifying the technically challenging process of reverse engineering monoclonal antibodies from protein material.     

Anti-tumor effects of depleting and non-depleting anti-CD27 monoclonal antibodies in immune-competent mice

CD27 plays an important role in T-cell co-stimulation and is also expressed on lymphomas. In the present study, we generated novel depleting and non-depleting monoclonal antibodies (mAbs) against mouse CD27 and characterized their co-stimulatory activity in vitro and anti-tumor effects in immune-competent mice bearing syngeneic T-cell lymphoma (EG7) expressing or lacking CD27. A profound anti-tumor effect was observed with a non-depleting mAb (RM27-3E5), but not with a depleting mAb (RM27-3C1), against either EG7/CD27(+) or EG7/CD27(−) tumors, which was associated with the induction of EG7-specific cytotoxic T lymphocytes (CTLs). Consistently, the anti-tumor effect of RM27-3E5 was abolished in T cell-deficient nude mice. These results indicate that a non-depleting agonistic mAb against CD27 is promising for cancer therapy by co-stimulating tumor-specific CTL induction.     

Antibodies to morphine in workers exposed to opiates at a narcotics manufacturing facility and evidence for similar antibodies in heroin abusers

According to the International Narcotics Control Board, over 45,000 kg of morphine and 54,000 kg of codeine were ethically manufactured in 1986 at three facilities in the United States. Little information exists about possible adverse health effects associated with workplace exposure to opiate compounds in this industry. Because there are no specific federal standards for workplace exposure to narcotic dusts, exposure-control defaults to the nuisance dust standard (10 mg/m3, as an 8 hr time-weighted average). Narcotics manufacturing workers were evaluated for anti-morphine IgG before and 10 mo. after the implementation of an improved respiratory protection program (RPP). Significantly elevated IgG levels were measured before the improved RPP (P less than 0.005). After the improved RPP, a significant reduction was observed (P less than 0.001), suggesting that specific antibody levels could be used as biomarkers of exposure. Inhibition studies showed that the antibodies were specifically directed against morphine with some cross reactivity with morphine derivatives. Preliminary results are also shown which indicate that similar anti-morphine antibodies are present in the sera of intravenous heroin abusers. Elevated levels (P less than 0.05) of anti-morphine antibodies were detected in sera from heroin abusers, providing evidence that similar antibodies may be produced from non-occupational exposure to opiates. These finding have potentially far-reaching implications for addiction research and drug testing.     

Spectroscopic evidence for a porphobilinogen deaminase-tetrapyrrole complex that is an intermediate in the biosynthesis of uroporphyrinogen III

Incubation of porphobilinogen (PBG) with PBG deaminase from Rhodopseudomonas sphaeroides in carbonate buffer (pH 9.2) to total PBG consumption resulted in low yields of uroporphyrinogen I (uro'gen I). In the reaction mixture a pyrrylmethane accumulated, which at longer incubation periods was transformed into uro'gen I. The accumulated pyrrylmethane gave an Ehrlich reaction which was different from that of a 2-(aminomethyl)dipyrrylmethane or 2-(aminomethyl)tripyrrane. It resembled that of a bilane (tetrapyrrylmethane) but was different from that of a 2-(hydroxymethyl)bilane. The 13C NMR spectra of incubations carried out with [11-13C]PBG indicated that the pyrrylmethane was a tetrapyrrole with methylene resonances at 22.35-22.50 ppm. It was loosely bound to the deaminase, and when separated from the enzyme by gel filtration or gel electrophoresis, it immediately cyclized to uro'gen I. No enzyme-bound methylene could be detected by its chemical shift, suggesting that its line width must be very broad. When uro'gen III-cosynthase was added to the deaminase-tetrapyrrole complex, uro'gen III was formed at the expense of the latter in about 75% yield. The tetrapyrrole could only be partially displaced from the enzyme by ammonium ions, although a small amount of 2-(aminomethyl)bilane was always formed together with the tetrapyrrole intermediate. A protonated uro'gen I structure for this intermediate was ruled out by incubations using [2,11-13C]PBG. Uro'gen III formation from 2-(hydroxymethyl)bilane (HMB) and from the deaminase-tetrapyrrole intermediate was compared by using deaminase-cosynthase and cosynthase from several sources. It was found that while the HMB inhibited uro'gen III formation at higher concentrations and longer incubation times, uro'gen III formation from the complex did not decrease with time.     

Looking for therapeutic antibodies in next-generation sequencing repositories

ABSTRACT

Recently it has become possible to query the great diversity of natural antibody repertoires using next-generation sequencing (NGS). These methods are capable of producing millions of sequences in a single experiment. Here we compare clinical-stage therapeutic antibodies to the ~1b sequences from 60 independent sequencing studies in the Observed Antibody Space database, which includes antibody sequences from NGS analysis of immunoglobulin gene repertoires. Of 242 post-Phase 1 antibodies, we found 16 with sequence identity matches of 95% or better for both heavy and light chains. There are also 54 perfect matches to therapeutic CDR-H3 regions in the NGS outputs, suggesting a nontrivial amount of convergence between naturally observed sequences and those developed artificially. This has potential implications for both the legal protection of commercial antibodies and the discovery of antibody therapeutics.     

Preparation and partial characterization of monoclonal antibodies specific for the nascent non-triple helical form of the type IV collagen alpha 1 chain

This report describes the preparation and partial characterization of monoclonal antibodies that are reactive specifically with the nascently produced non-triple helical form of the type IV collagen α1 chain, designated as NTH α1(IV). These antibodies were nonreactive with the α1 chain of the type IV collagen in the triple-helical conformation. Three antibodies, #141, #179 and #370, with different epitopes in NTH α1(IV) were found to be reactive with the nascent polypeptide secreted from human normal cells and a human carcinoma cell line. The antibodies with different epitopes may provide a key method for elucidating the physiological function and tissue distribution of NTH α1(IV), which is distinct from the chain derived from triple-helical type IV collagen.     

An alternative assay to hydrophobic interaction chromatography for high-throughput characterization of monoclonal antibodies

The effectiveness of therapeutic monoclonal antibodies (mAbs) is governed not only by their bioactivity, but also by their biophysical properties. Assays for rapidly evaluating the biophysical properties of mAbs are valuable for identifying those most likely to exhibit superior properties such as high solubility, low viscosity and slow serum clearance. Analytical hydrophobic interaction chromatography (HIC), which is performed at high salt concentrations to enhance hydrophobic interactions, is an attractive assay for identifying mAbs with low hydrophobicity. However, this assay is low throughput and thus not amenable to processing the large numbers of mAbs that are commonly generated during antibody discovery. Therefore, we investigated whether an alternative, higher throughput, assay could be developed that is based on evaluating antibody self-association at high salt concentrations using affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS). Our approach is to coat gold nanoparticles with polyclonal anti-human antibodies, use these conjugates to immobilize human mAbs, and evaluate mAb self-interactions by measuring the plasmon wavelengths of the antibody conjugates as a function of ammonium sulfate concentration. We find that hydrophobic mAbs, as identified by HIC, generally show significant self-association at low to moderate ammonium sulfate concentrations, while hydrophilic mAbs typically show self-association only at high ammonium sulfate concentrations. The correlation between AC-SINS and HIC measurements suggests that our assay, which can evaluate tens to hundreds of mAbs in a parallel manner and requires only small (microgram) amounts of antibody, will enable early identification of mAb candidates with low hydrophobicity and improved biophysical properties.     

An alternative assay to hydrophobic interaction chromatography for high-throughput characterization of monoclonal antibodies

The effectiveness of therapeutic monoclonal antibodies (mAbs) is governed not only by their bioactivity, but also by their biophysical properties. Assays for rapidly evaluating the biophysical properties of mAbs are valuable for identifying those most likely to exhibit superior properties such as high solubility, low viscosity and slow serum clearance. Analytical hydrophobic interaction chromatography (HIC), which is performed at high salt concentrations to enhance hydrophobic interactions, is an attractive assay for identifying mAbs with low hydrophobicity. However, this assay is low throughput and thus not amenable to processing the large numbers of mAbs that are commonly generated during antibody discovery. Therefore, we investigated whether an alternative, higher throughput, assay could be developed that is based on evaluating antibody self-association at high salt concentrations using affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS). Our approach is to coat gold nanoparticles with polyclonal anti-human antibodies, use these conjugates to immobilize human mAbs, and evaluate mAb self-interactions by measuring the plasmon wavelengths of the antibody conjugates as a function of ammonium sulfate concentration. We find that hydrophobic mAbs, as identified by HIC, generally show significant self-association at low to moderate ammonium sulfate concentrations, while hydrophilic mAbs typically show self-association only at high ammonium sulfate concentrations. The correlation between AC-SINS and HIC measurements suggests that our assay, which can evaluate tens to hundreds of mAbs in a parallel manner and requires only small (microgram) amounts of antibody, will enable early identification of mAb candidates with low hydrophobicity and improved biophysical properties.     

Spectroscopic diagnosis of anti-phospholipid antibodies by visible and near-infrared spectroscopy in SLE patients' plasma samples

The purpose of this study was to investigate whether visible and near-infrared (Vis-NIR) spectroscopy can be used for diagnoses of anti-phospholipid syndrome (APS). Vis-NIR spectra from 90 plasma samples [anti-phospholipid antibodies (aPLs)-positive group, n=48; aPLs-negative group, n=42] were subjected to principal component analysis (PCA) and soft independent modeling of class analogy (SIMCA) to develop multivariate models to discriminate between aPLs-positive and aPLs-negative. Both PCA and SIMCA models were further assessed by the prediction of 84 masked other determinations. The PCA model predicted successful discrimination of the masked samples with respect to aPLs-positive and aPLs-negative. The SIMCA model predicted 42 of 48 (87.5%) aPLs-positive patients and 33 of 36 (91.7%) aPLs-negative patients of Vis-NIR spectra from masked samples correctly. These results suggest that Vis-NIR spectroscopy combined with multivariate analysis could provide a promising tool to objectively diagnose APS.     

A fluorescent imaging method for analyzing the biodistribution of therapeutic monoclonal antibodies that can distinguish intact antibodies from their breakdown products

Many monoclonal antibodies have been developed for therapy over the last 2 decades. In the development of therapeutic antibodies, the preclinical assessment of an antibody's biodistribution is important for the prediction of the antibody's efficacy and safety. For imaging analyses of such biodistributions, radioisotope (RI) labeling and fluorescence labeling methods are typically used, but the resulting data are limited because these methods cannot distinguish breakdown products from intact antibodies. To resolve this problem, we developed a novel method using fluorescent resonance energy transfer (FRET)-type labeling and a spectral unmixing tool. With FRET-type labeling (labeling with 2 species of fluorophore), different fluorescence properties of labeled intact antibodies and their breakdown products (the hydrolyzed/digested type of breakdown products) are made visible. With the spectral unmixing tool, the fluorescence of a solution containing the intact antibody and its breakdown products could be unmixed in proportion to their contents. Moreover, when labeled antibodies that targeted either human epidermal growth factor receptor-2 or epidermal growth factor receptor were injected into nude mice implanted subcutaneously with tumor cells, the accumulation of the injected labeled antibodies and their breakdown products in the tumor could be separately analyzed by both whole-mouse imaging and a tumor homogenate analysis. These results suggest that our method using FRET-type labeling and a spectral unmixing tool could be useful in distinguishing breakdown products from intact antibodies.     

Anti-carcinoembryonic antigen single-chain variable fragment antibody variants bind mouse and human neonatal Fc receptor with different affinities that reveal distinct cross-species differences in serum half-life

Serum half-life of IgG is controlled by the neonatal Fc receptor (FcRn) that interacts with the IgG Fc region and may be increased or decreased as a function of altered FcRn binding. Preclinical evaluations of modified IgGs are frequently carried out in mice, but such IgGs may bind differently to mouse and human FcRn (mFcRn and hFcRn). Here, we report a detailed characterization of a matched set of mouse-human chimeric T84.66 scFv-Fc variants with specificity for the tumor carcinoembryonic antigen and mutations in the FcRn-binding site. Binding to soluble mFcRn and hFcRn was measured using in vitro assays, and the results were compared with blood clearance in vivo in normal (mFcRn bearing) and hFcRn transgenic mice. All variants bound better to mFcRn than to hFcRn. The loss of affinity varied among the mutants, however, and also the hierarchy of binding differed depending on the receptor. The mutations had no major impact on binding to the classical Fcγ receptors. Importantly, the trend of blood clearance in both strains of mice correlated with the hierarchy of binding obtained using soluble FcRn. Consequently, in vitro interaction analysis of engineered IgGs regarding their cross-species FcRn binding ability provides information for prediction of in vivo pharmacokinetics.     

Evaluation of three serological tests for detection of anti-candidal antibodies in diagnosis of invasive candidiasis

Immunoblot detection of antibody against 47 KD cytoplasmic antigen ofCandida albicans was evaluated in diagnosis of invasive candidiasis and compared to whole cell agglutination and gel diffusion tests for detection of anticandidal antibody in 64 patients. The patients included 17 with culture proved candidemia, 34 with significant candiduria (more than 10,000 colony forming units per ml of urine) and 13 with nonsignificant candiduria. Antibody against 47 KD antigen was found to be the best indicator for diagnosis of invasive candidiasis even in patients with malignancy. The sensitivity of this procedure was 82.4%, specificity 86.7%, positive predictive value 77.8%, negative predictive value 89.7% and efficacy 85.1%. The gel diffusion procedure lacked in sensitivity whereas whole cell agglutination lacked in specificity. Detection of antibody against 47 KD antigen proved to be a valuable adjunct in the diagnosis of invasive candidiasis.     

Spectroscopic evidence revealing polyproline II structure in hydrophobic, putatively elastomeric sequences encoded by specific exons of human tropoelastin

Elastin, the protein responsible for tissue elasticity, is contained in arterial walls, lungs, and skin. Given the cassette like organization of the human tropoelastin gene, giving rise to alternating exons encoding for crosslink domains and elastomeric domains, it is tempting to suggest that polypeptides encoded by different exons could adopt structures independent of the other exons. The results obtained with the polypeptide sequences encoded by exons 3, 7, and 30 of human tropoelastin are described. It is shown that these hydrophobic exons may partly assume the polyproline II (PPII) structure, as found by circular dichroism studies in aqueous solution. Classical Raman spectroscopy evidences a specific sharp band at 1314 cm(-1), which is assigned to the PPII structure adopted by these exons in the solid state. As these sequences are among those putatively responsible for elastomeric properties, these findings are of particular interest in relation to the current models of the elasticity of elastin.