Expression of Recombinant Antibodies

Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with “human-like” post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications

Analyses of transcriptional alterations during the mutualistic interaction between the model legume Medicago truncatula and the arbuscular mycorrhiza fungus Glomus intraradices, and characterisation of mycorrhiza-specific lectins

[no abstract

Application of M13 Phage Display for Identifying Immunogenic Proteins from Tick (\u3cem\u3eIxodes scapularis\u3c/em\u3e) Saliva

Background: Ticks act as vectors for a large number of different pathogens, perhaps most notably Borrelia burgdorferi, the causative agent of Lyme disease. The most prominent tick vector in the United States is the blacklegged tick, Ixodes scapularis. Tick bites are of special public health concern since there are no vaccines available against most tick-transmitted pathogens. Based on the observation that certain non-natural host animals such as guinea pigs or humans can develop adaptive immune responses to tick bites, anti-tick vaccination is a potential approach to tackle health risks associated with tick bites. Results: The aim of this study was to use an oligopeptide phage display strategy to identify immunogenic salivary gland proteins from I. scapularis that are recognized by human immune sera. Oligopeptide libraries were generated from salivary gland mRNA of 18 h fed nymphal I. scapularis. Eight immunogenic oligopeptides were selected using human immune sera. Three selected immunogenic oligopeptides were cloned and produced as recombinant proteins. The immunogenic character of an identified metalloprotease (MP1) was validated with human sera. This enzyme has been described previously and was hypothesized as immunogenic which was confirmed in this study. Interestingly, it also has close homologs in other Ixodes species. Conclusion: An immunogenic protein of I. scapularis was identified by oligopeptide phage display. MP1 is a potential candidate for vaccine development

Generation of Recombinant Antibodies against the beta-(1,6)-Branched beta-(1,3)-D-Glucan Schizophyllan from Immunized Mice via Phage Display

beta-(1,6)-Branched beta-(1,3)-D-glucans like schizophyllan from the basidiomycete Schizophyllum commune excite various immunostimulatory effects and have been clinically tested as adjuvants. Some of the glucans are also applicable in food or petrol industry due to their viscosity and temperature stability in aqueous solution. Antibodies against these glucans could be used as tool for analysis of glucan preparations or for further research of its bioactivity. Therefore, an immune phage display library was constructed from mice immunized with schizophyllan. Three recombinant monoclonal antibodies were isolated from this library by affinity selection (panning) on schizophyllan. The half-maximal effective concentration (EC50) values for those antibodies varied between 16.4 ng mL−1 and 21.3 ng mL−1. The clones showed binding specificity not only for schizophyllan but also for other beta-(1,6)-branched beta-(1,3)-D-glucans of similar macromolecular structure. Denaturation of the secondary structure led to a reduced antibody binding, indicating an epitope requiring the correct conformation of the triple helical structure of the glucans

A study on the desulfurization of sulfidic mine tailings for the production of a sulfur-poor residue

The mining industry generates large amounts of tailings every year. The most common destination for the tailings is deposition in tailings storage facilities (TSFs), which can have enormous dimensions. The management and storage of such large volumes of material pose many challenges in terms of dam stability and immobilization of hazardous contaminants that represent human-health and environmental risks, particularly for sulfide-containing materials. In addition, considerable amounts of precious and base metals can be lost in the tailings. Due to the economic value and growing industrial demand for precious and base metals, tailings may therefore be potential sources of secondary raw materials. This contribution investigates the flotation of pyrite-rich tailings, containing residual chalcopyrite, galena, and sphalerite, and high amounts of ultrafine particles. Flotation was used to recover the sulfide minerals and generate tailings with low sulfur content. The Cu-Pb-Zn-rich product could go to further treatment (e.g. (bio)hydrometallurgy) to recover the metals, while the low sulfur fraction could be used in the civil construction industry. Automated mineralogy (MLA) was used to provide quantitative mineralogical and textural data. Bench-scale experiments were performed by combining classic flotation and floc flotation (flotation of flocs of targeted minerals). Flotation of the material as received, as well as after classification into two fractions was performed. The samples as received and the coarser fraction (+37 µm) underwent classic flotation, while the finer fraction (−37 µm) was processed either by using the classic or the floc flotation approach. The flotation of the coarser particles provided higher sulfide recoveries, higher combined Cu-Pb-Zn grades in the concentrate (3.66 %), cleaner residues (1.6 % S), faster flotation rates, and reduced reagent consumption. Likewise, the results from the fine particle flotation allowed lower S content in the residues (3.4 % S) as compared to the flotation of the original material. The results of the use of floc flotation for the finer fraction show an increase in the mass pull with a slight increase in the recovery of sulfides. Overall, the development of a route to process the tailings proved to be promising and the use of a two-route approach indicates advantages as compared to a single route

Comparative study on optical performance and visual outcomes between two diffractive multifocal lenses: AMO Tecnis® ZMB00 and AcrySof® IQ ReSTOR® Multifocal IOL SN6AD1

ABSTRACT Purpose: To compare the optical performance and visual outcomes between two diffractive multifocal lenses: AMO Tecnis® ZMB00 and AcrySof® ReSTOR® SN6AD1. Methods: This prospective, non-randomized comparative study included the assessment of 74 eyes in 37 patients referred for cataract surgery and candidates for multifocal intraocular lens implants. Exclusion criteria included existence of any other eye disease, previous eye surgery, high axial myopia, preoperative corneal astigmatism of >1.00 cylindrical diopter (D), and intraoperative or postoperative complications. Ophthalmological evaluation included the measurement of uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), distance-corrected near visual acuity (DCNVA), and distance-corrected intermediate visual acuity (DCIVA), with analysis of contrast sensitivity (CS), wavefront, and visual defocus curve. Results: Postoperative UDVA was 0.09 and 0.08 logMAR in the SN6AD1 and ZMB00 groups, respectively (p=0.868); postoperative CDVA was 0.04 and 0.02 logMAR in the SN6AD1 and ZMB00 groups, respectively (p=0.68); DCIVA was 0.17 and 0.54 logMAR in the SN6AD1 and ZMB00 groups, respectively (p=0.000); and DCNVA was 0.04 and 0.09 logMAR in the SN6AD1 and ZMB00 groups, respectively (p=0.001). In both cases, there was an improvement in the spherical equivalent and UDVA (p<0.05). Under photopic conditions, the SN6AD1 group had better CS at low frequencies without glare (p=0.04); however, the ZMB00 group achieved better sensitivity at high frequencies with glare (p=0.003). The SN6AD1 and ZMB00 lenses exhibited similar behavior for intermediate vision, according to the defocus curve; however, the ZMB00 group showed a shorter reading distance than the SN6AD1 group. There were no significant differences regarding aberrometry between the two groups. Conclusion: Both lenses promoted better quality of vision for both long and short distances and exhibited a similar behavior for intermediate vision. The SN6AD1 and ZMB00 groups showed better results for CS under photopic conditions at low and high spatial frequencies, respectively

Developing Recombinant Antibodies by Phage Display Against Infectious Diseases and Toxins for Diagnostics and Therapy

Antibodies are essential molecules for diagnosis and treatment of diseases caused by pathogens and their toxins. Antibodies were integrated in our medical repertoire against infectious diseases more than hundred years ago by using animal sera to treat tetanus and diphtheria. In these days, most developed therapeutic antibodies target cancer or autoimmune diseases. The COVID-19 pandemic was a reminder about the importance of antibodies for therapy against infectious diseases. While monoclonal antibodies could be generated by hybridoma technology since the 70ies of the former century, nowadays antibody phage display, among other display technologies, is robustly established to discover new human monoclonal antibodies. Phage display is an in vitro technology which confers the potential for generating antibodies from universal libraries against any conceivable molecule of sufficient size and omits the limitations of the immune systems. If convalescent patients or immunized/infected animals are available, it is possible to construct immune phage display libraries to select in vivo affinity-matured antibodies. A further advantage is the availability of the DNA sequence encoding the phage displayed antibody fragment, which is packaged in the phage particles. Therefore, the selected antibody fragments can be rapidly further engineered in any needed antibody format according to the requirements of the final application. In this review, we present an overview of phage display derived recombinant antibodies against bacterial, viral and eukaryotic pathogens, as well as microbial toxins, intended for diagnostic and therapeutic applications

Early mobilisation in critically ill COVID-19 patients: a subanalysis of the ESICM-initiated UNITE-COVID observational study

Background Early mobilisation (EM) is an intervention that may improve the outcome of critically ill patients. There is limited data on EM in COVID-19 patients and its use during the first pandemic wave. Methods This is a pre-planned subanalysis of the ESICM UNITE-COVID, an international multicenter observational study involving critically ill COVID-19 patients in the ICU between February 15th and May 15th, 2020. We analysed variables associated with the initiation of EM (within 72 h of ICU admission) and explored the impact of EM on mortality, ICU and hospital length of stay, as well as discharge location. Statistical analyses were done using (generalised) linear mixed-effect models and ANOVAs. Results Mobilisation data from 4190 patients from 280 ICUs in 45 countries were analysed. 1114 (26.6%) of these patients received mobilisation within 72 h after ICU admission; 3076 (73.4%) did not. In our analysis of factors associated with EM, mechanical ventilation at admission (OR 0.29; 95% CI 0.25, 0.35; p = 0.001), higher age (OR 0.99; 95% CI 0.98, 1.00; p ≤ 0.001), pre-existing asthma (OR 0.84; 95% CI 0.73, 0.98; p = 0.028), and pre-existing kidney disease (OR 0.84; 95% CI 0.71, 0.99; p = 0.036) were negatively associated with the initiation of EM. EM was associated with a higher chance of being discharged home (OR 1.31; 95% CI 1.08, 1.58; p = 0.007) but was not associated with length of stay in ICU (adj. difference 0.91 days; 95% CI − 0.47, 1.37, p = 0.34) and hospital (adj. difference 1.4 days; 95% CI − 0.62, 2.35, p = 0.24) or mortality (OR 0.88; 95% CI 0.7, 1.09, p = 0.24) when adjusted for covariates. Conclusions Our findings demonstrate that a quarter of COVID-19 patients received EM. There was no association found between EM in COVID-19 patients' ICU and hospital length of stay or mortality. However, EM in COVID-19 patients was associated with increased odds of being discharged home rather than to a care facility. Trial registration ClinicalTrials.gov: NCT04836065 (retrospectively registered April 8th 2021)

A Cassette Vector System for the Rapid Cloning and Production of Bispecific Tetravalent Antibodies

Bivalent single chain (sc)Fv-Fc antibodies have been used for years as recombinant alternatives of natural immunoglobulins. We have extended this approach to the scFv-Fc-scFv antibody format to obtain tetravalent antigen binding and the possibility to generate bispecific antibodies. We developed a mammalian expression vector system to construct tetravalent scFv-Fc-scFv antibodies with two NcoI+NotI compatible cloning sites flanking the Fc gene fragment. We demonstrated direct cloning from single chain antibody gene libraries and tested various scFv combinations. Transient production of scFv-Fc-scFv antibodies in human embryonic kidney (HEK) 293T cells achieved volumetric yields of up to 10 mg/L. However, expression levels were strongly dependent on the carboxyterminal scFv and the scFv combination. All scFv-Fc-scFv antibodies exclusively formed disulfide-linked homodimers. Antigen binding studies revealed dual specificity for all scFv-Fc-scFv employing different scFv fragments. Comparison of C-reactive protein (CRP) specific monovalent scFv LA13-IIE3, bivalent scFv-Fc and Fc-scFv LA13-IIE3, and tetravalent scFv-Fc-scFv (scFv LA13-IIE3 in combination with scFvs LA13-IIE3, TOB4-B11, or TOB5-D4) revealed an up to 500-fold increased antigen binding. This novel scFv-Fc-scFv antibody expression system allows simple and fast testing of various scFv combinations