Anti-idiotype antibodies: an alternative approach to tumor immunotherapy

Studies presented in this paper examined the tumor-specific cellular and humoral immunity induced by anti-idiotype antibodies (Ab2s) 2F10 and 3A4. A panel of Ab2s was made against a monoclonal anti-L1210/GZL lymphoma, 11C1. The Ab2s were screened for their ability to block 11C1 binding to tumor, to induce tumor-specific DTH and CTL responses and to induce an anti-tumor humoral response. Two Ab2s, 2F10 and 3A4, were found to have similar fine specificity and to induce similar cellular and humoral responses. These were then examined for their ability to elicit tumor-protective immunity. Only preimmunization with 2F10 Ab2 protected animals from live tumor challenge, and in this paper the possible causes of this difference in otherwise similar Ab2s is discussed.     

Reorganization of arrays of prekeratin filaments during mitosis : Immunofluorescence microscopy with multiclonal and monoclonal prekeratin antibodies

Indirect immunofluorescent labelling of different epithelial cell lines for intermediate filaments of the prekeratin type revealed prominent changes in the organization of prekeratin during mitosis. In three out of four cell lines tested (Henle-407, A-431 and HeLa cells) the filamentous prekeratin networks disappeared at the initiation of mitosis and the immunofluorescent labelling was concentrated in small cytoplasmic bodies. This observation was obtained with both polyspecific rabbit anti-bovine prekeratin antibodies and with monospecific antibodies produced by mouse hybridomas. In a fourth cell line, PtK₂, prekeratin filaments were retained throughout mitosis, mainly in the mitotic poles, whereas the central areas of the cells were apparently devoid of filaments. The addition of colchicine to the different cultured cells induced alterations in the organization of prekeratin filaments which were usually manifested by the formation of thicker filament bundles. It did not induce the formation of the prekeratin-cytoplasmic bodies in interphase cells. However, upon prolonged incubation in the presence of colchicine, there was an increase in the number of mitotically arrested cells and a parallel increase in the number of cells containing prekeratin cytoplasmic bodies. It is thus proposed that the state of organization of prekeratin in these cells is cell-cycle-dependent and may be modulated to permit radical shape changes as those occurring during mitosis.     

Preparation of DNA and protein micro arrays on glass slides coated with an agarose film

A thin layered agarose film on microscope slides provides a versatile support for the preparation of arrayed molecular libraries. An activation step leading to the formation of aldehyde groups in the agarose creates reactive sites that allow covalent immobilization of molecules containing amino groups. Arrays of oligonucleotides and PCR products were prepared by tip printing. After hybridization with complementary fluorescence labeled nucleic acid probes strong fluorescence signals of sequence-specific binding to the immobilized probes were detected. The intensity of the fluorescence signals was proportional to the relative amount of immobilized oligonucleotides and to the concentration of the fluorescence labeled probe. We also used the agarose film-coated slides for the preparation of protein arrays. In combination with specific fluorescence labeled antibodies these protein arrays can be used for fluorescence linked immune assays. With this approach different protein tests can be performed in parallel in a single reaction with minimal amounts of the binding reagents.     

100-kD coated vesicle proteins: molecular heterogeneity and intracellular distribution studied with monoclonal antibodies

Proteins with molecular weights of around 100,000 (designated 100K) are found in all coated vesicles. Five monoclonal antibodies have been raised against the major 100K proteins of bovine brain coated vesicles, which migrate on SDS gels as three closely spaced bands. One antibody stains the middle band (band B), two stain both upper and lower bands (bands A and C), and two stain the lower band (band C) only. Thus, the polypeptides in bands A and C are related (but not identical), a result confirmed by NH2-terminal sequencing. Other tissues were found to express proteins corresponding to, and co-migrating with, bands B and C but not band A. Only the two antibodies that recognize both A and C stained fixed and permeabilized tissue culture cells; they both showed a punctate pattern in the plane of the plasma membrane. Double labeling with anti-clathrin antibodies confirmed that the dots correspond to coated pits and vesicles. However, perinuclear staining seen with anti-clathrin, corresponding to Golgi-derived coated vesicles, was conspicuously absent with the two monoclonal antibodies. Affinity-purified polyclonal antisera against the 100K proteins, reported earlier, gave perinuclear as well as punctate staining; these included one antiserum which gave mainly perinuclear staining (Robinson, M. S., and B. M. F. Pearse, 1986, J. Cell Biol., 102:48-54). Thus, different 100K proteins appear to be found in different membrane compartments. Since the 100K proteins are thought to lie between clathrin and the membrane proteins of the vesicle, these results may help to explain how different membrane proteins can be sorted into coated vesicles in different parts of the cell.     

Immobilization of cells of Nicotiana tabacum L. on polyphenyleneoxide coated with poly-L-lysine

Cells of Nicotiana tabacum L. cv. Wisconsin 38 were immobilized on poly (2,6-dimethyl)-p-phenyleneoxide in powder form (Sorfix) coated with poly-L-lysine (molecular weight 40 000 daltons). The dependence of cell immobilization on the amount of bound polyL-lysine was estimated.Abbreviations MW molecular weight - dwt dry weight - fwt fresh weight     

Microalgae autoflocculation: an alternative to high-energy consuming harvesting methods

With increasing concerns regarding energy and environment, algae biofuel is generating considerable interest around the world. Nevertheless, the harvesting step required before downstream biomass processing is a major bottleneck. Commonly employed methods include addition of chemicals or use of mechanical equipment that increase dramatically the biofuel production cost. This review deals with naturally occurring processes that can help offset those costs by causing microalgae flocculation. Interaction theories are briefly reviewed. In addition, operational parameters such as pH, irradiance, nutrients, dissolved oxygen, and temperature effect on microalgae flocculation are evaluated. Finally, microalgae flocculation is also considered from an ecological point of view by taking advantage of their interaction with other microorganisms.     

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.     

Caprylic acid precipitation method for impurity reduction: An alternative to conventional chromatography for monoclonal antibody purification

We report the use of caprylic acid based impurity precipitation as (1) an alternative method to polishing chromatography techniques commonly used for monoclonal antibody purification and (2) an impurity reduction step prior to harvesting the bioreactor. This impurity reduction method was tested with protein A purified antibodies and with cell culture fluid. First, the operational parameters influencing precipitation of host cell proteins and high molecular weight aggregate in protein A pools were investigated. When used as a polishing step, the primary factor affecting purification and yield was determined to be pH. Caprylic acid precipitation was comparable to polishing IEX chromatography in reducing host cell protein and aggregate levels. A virus reduction study showed complete clearance of a model retrovirus during caprylic acid precipitation of protein A purified antibody. Caprylic acid mediated impurity precipitation in cell culture showed that the impurity clearance was generally insensitive to pH and caprylic acid concentration whereas yield was a function of caprylic acid concentration. Protein A purification of caprylic acid precipitated cell culture fluid generated less turbid product pool with reduced levels of host cell proteins and high molecular weight aggregate. The results of this study show caprylic acid precipitation to be an effective purification method that can be incorporated into a production facility with minimal cost as it utilizes existing tanks and process flow. Eliminating flow through chromatography polishing step can provide process intensification by avoiding the process tank volume constraints for high titer processes. Biotechnol. Bioeng. 2012; 109: 2589–2598. © 2012 Wiley Periodicals, Inc.     

Methodologies for the isolation of alternative binders with improved clinical potentiality over conventional antibodies

The availability of binders to different functional domains of the same protein or to physiologically co-operating proteins allows for the simultaneous inhibition of independent downstream signaling pathways. This multi-target approach represents a promising therapeutic strategy, as demonstrated in the case of the synergistic effect of anti-Her2 treatment based on the combined use of the trastuzumab and pertuzumab monoclonal antibodies that induce cellular cytotoxicity and impair the receptor dimerization, respectively. Therefore, a reliable selection method for the recovery of epitope-specific antibodies is highly needed. Animal immunization with short peptides resembling the epitope sequence for raising conventional antibodies represents an alternative. Panning phage displayed libraries of recombinant antibodies such as scFvs and nanobodies or of other peptide collections is another option. Although recombinant antibodies can provide the same specificity as conventional antibodies, they offer at least two further advantages: i) the protocols for the selection of epitope-specific antibodies can be rationally designed, and ii) their expression as multivalent, bispecific and biparatopic molecules is feasible. This review will analyze the recent literature concerning technical aspects related to the isolation, the expression as multivalent molecules, and the therapeutic applications of binders able to interfere with antigen functional domains. The term binder will be preferred when possible to include those molecules, such as peptides or affibodies, with at least some proven practical uses.     

Industrial bioconversion of renewable resources as an alternative to conventional chemistry

There are numerous possibilities for replacing chemical techniques with biotechnological methods based on renewable resources. The potential of biotechnology (products, technologies, metabolic pathways) is for the most part well known. Often the costs are still the problem. Biotechnological advances have the best chances for replacing some fine chemicals. While the raw material costs are less of a consideration here, the environmental benefit is huge, as chemical-technical processes often produce a wide range of undesirable/harmful by-products or waste. In the case of bulk chemicals (<US $1/kg) the product price is affected mainly by raw material costs. As long as fossil raw materials are still relatively inexpensive, alternatives based on renewable resources cannot establish themselves. Residues and waste, which are available even at no cost in some cases, are an exception. The introduction of new technologies for the efficient use of such raw materials is currently being promoted. The utilisation of residual wood, plant parts, waste fat, and crude glycerol, for example, provides great potential. For industrial chemicals (US $2–4/kg), process and recovery costs play a greater role. Here, innovative production technologies and product recovery techniques (e.g. on-line product separation) can increase competitiveness.     

Specificity of monoclonal antibodies to an EBV transformed B-cell line

Monoclonal antibodies were produced against an Epstein Barr virus (EBV) transformed human B-cell line with the following HLA specificities: HLA A2, B27, Cw2, Dr3,2. Antibodies from three clones, Mab B1, Mab B2 and B3 reacted with human Ia-like molecules on peripheral blood B cells, some monocytes, CLL cells, lymphoblastoid B-cell lines and some mixed leukocyte culture (MLC)-activated T cells, but were unreactive with leukoagglutinin (LA) and Concanavalin A (Con A)-activated T blasts and T-cell lines. Antibodies obtained from three other clones (Mab 4, 5 and 6) reacted with a mw 80,000 protein present on peripheral lymphocytes and on most of the lymphoblastoid T-and B-cell lines tested.     

Preparation and characterization of monoclonal antibodies to an N-linked oligosaccharide

Two monoclonal antibodies to an N-linked oligosaccharide, MT-5 and MT-9, have been prepared by immunization with a pyridylaminated, asialylated, galactosylated, fucosylated, bisected biantennary sugar. The reactivity of these antibodies was monitored by their reaction with human asialoglycophorin in a solid-phase enzyme-linked immunosorbent assay. Both antibodies reacted with the sugar chains of various human glycoproteins such as immunoglobulin G, transferrin, gamma-glutamyl transpeptidase, alpha 1-acid glycoprotein, and alpha-fetoprotein. Treatment of asialoglycophorin with beta-N-acetylhexosaminidase or alpha-mannosidase resulted in reduction of the binding to these antibodies. The reactivity of MT-5 to asialoglycophorin was slightly inhibited by D-mannose and N-acetylglucosamine, whereas that of MT-9 was inhibited by D-mannose, N-acetyl-D-glucosamine, chitobiose, and L-fucose. The epitope specificity of MT-5 appears to be a sugar chain containing biantennary N-acetyl-D-glucosamine residues, the bisected N-acetyl-D-glucosamine residue, and a trimannosyl core. The epitope to which MT-9 is directed may be a complex made up of beta-mannose, chitobiose, and L-fucose. These studies indicate that immunization with pyridylaminated sugars can produce antibodies that recognize N-linked oligosaccharides. Monoclonal/polyclonal antibodies to the N-linked sugar chains of glycopeptides would be useful in such studies of proteins.     

An immunocytochemical investigation with monoclonal antibodies to somatostatin

Summary Four monoclonal antibodies specific for somatostatin have been produced and characterized. These antibodies were used to assess the anatomical relationship of somatostatin-containing cells in the pancreas and gastrointestinal tract of man, baboon and rat with ten other peptide-containing endocrine cells. The peptides investigated were gastrin, cholecystokinin, motilin, secretin, neurotensin, gastric inhibitory polypeptide, gut-glucagon, pancreatic glucagon, pancreatic polypeptide and insulin.The only regions in which somatostatin cells were seen in close contact with another endocrine cell were in the pancreas and the gastric antrum. In the pancreas somatostatin cells were commonly seen in close contact with insulin, glucagon and pancreatic polypeptide cells and infrequent contact was demonstrable with the gastrin-immunoreactive cells in the antrum of both rat and man. In all other cases no evidence was obtained for a close anatomical relationship between somatostatin cells and the other enteroendocrine cells.     

An immunocytochemical investigation with monoclonal antibodies to somatostatin

Four monoclonal antibodies specific for somatostatin have been produced and characterized. These antibodies were used to assess the anatomical relationship of somatostatin-containing cells in the pancreas and gastrointestinal tract of man, baboon and rat with ten other peptide-containing endocrine cells. The peptides investigated were gastrin, cholecystokinin, motilin, secretin, neurotensin, gastric inhibitory polypeptide, gut-glucagon, pancreatic glucagon, pancreatic polypeptide and insulin. The only regions in which somatostatin cells were seen in close contact with another endocrine cell were in the pancreas and the gastric antrum. In the pancreas somatostatin cells were commonly seen in close contact with insulin, glucagon and pancreatic polypeptide cells and infrequent contact was demonstrable with the gastrin-immunoreactive cells in the antrum of both rat and man. In all other cases no evidence was obtained for a close anatomical relationship between somatostatin cells and the other enteroendocrine cells.     

Peptibodies: A flexible alternative format to antibodies

Peptibodies or peptide-Fc fusions are an attractive alternative therapeutic format to monoclonal antibodies. They consist of biologically active peptides grafted onto an Fc domain. This approach retains certain desirable features of antibodies, notably an increased apparent affinity through the avidity conferred by the dimerization of two Fcs and a long plasma residency time. Peptibodies can be made in E. coli using recombinant technology. The manufacturing process involves fermentation and downstream processing, including refolding and multiple column chromatographic steps, that result in overall yields and quality suitable for commercial development. Romiplostim, marketed under the brand name Nplate®, is the first peptibody to be approved by the United States Food and Drug Administration and the European Medicines Agency and is indicated for the treatment of immune thrombocytopenic purpura. AMG 386, a peptibody antagonist to angiopoietins 1 and 2, is being evaluated in Phase 3 clinical testing in combination with chemotherapy in women with ovarian cancer. AMG 819, a peptibody targeting nerve growth factor for pain has also progressed to clinical trials. These peptibodies illustrate the versatility of the modality.     

Cup plant,an alternative to conventional silage from a LCA perspective

Purpose

The growing awareness of the importance of biodiversity in agroecosystems in increasing and ensuring the supply of biomass has led to heightened interest from governments and farmers in alternative crops. This article assesses one such alternative crop, cup plant (Silphium perfoliatum L.), in terms of the environmental aspects of cultivation for forage production. Many studies have previously focused on cup plant, but so far, this plant has not been assessed using the life cycle assessment (LCA) method.

Materials and methods

This study compares the environmental load of cup plant with the most commonly grown silage crops in Central European conditions—maize—and with another common forage crop—lucerne using LCA. The system boundaries include all the processes from cradle to farm gate and both mass-based (1 ton of dry matter) and area-based (1 ha of monoculture) functional units were chosen for the purposes of this study. The results cover the impact categories related to the agricultural LCAs, and the ReCiPe Midpoint (H) characterization model was used for the data expression, by using SimaPro 9.0.0.40 software.

Results

This study compares the cultivation of cup plant with the most commonly grown silage crop in Central European conditions—maize—and with another common forage crop—lucerne. The paper shows the potential of cup plant to replace conventional silage (maize and lucerne silage mix) with certain environmental savings in selected impact categories, and importantly, while still maintaining the same performance levels in dairy farming as with conventional silage, as already reported in previous publications. For the Czech Republic alone, this would, in practice, mean replacing up to 50,000 ha of silage maize and reducing the environmental load by about tens of percent or more within the various impact categories and years of cultivation.

Conclusion

Cup plant can replace the yield and quality of silage maize, represents a lower environmental load per unit of production and unit of area and generally carries many other benefits. Thus, cup plant is a recommendable option for dairy farming. Given the recent experience and knowledge of the issue, the cup plant can be considered an effective alternative to conventional silage.

     

Peptibodies: A flexible alternative format to antibodies

Peptibodies or peptide-Fc fusions are an attractive alternative therapeutic format to monoclonal antibodies. They consist of biologically active peptides grafted onto an Fc domain. This approach retains certain desirable features of antibodies, notably an increased apparent affinity through the avidity conferred by the dimerization of two Fcs and a long plasma residency time. Peptibodies can be made in E. coli using recombinant technology. The manufacturing process involves fermentation and downstream processing, including refolding and multiple column chromatographic steps, that result in overall yields and quality suitable for commercial development. Romiplostim, marketed under the brand name Nplate®, is the first peptibody to be approved by the United States Food and Drug Administration and the European Medicines Agency and is indicated for the treatment of immune thrombocytopenic purpura. AMG 386, a peptibody antagonist to angiopoietins 1 and 2, is being evaluated in Phase 3 clinical testing in combination with chemotherapy in women with ovarian cancer. AMG 819, a peptibody targeting nerve growth factor for pain has also progressed to clinical trials. These peptibodies illustrate the versatility of the modality.