Understanding the metabolic burden of recombinant antibody production in Saccharomyces cerevisiae using a quantitative metabolomics approach

The cellular changes induced by heterologous protein expression in the yeast Saccharomyces cerevisiae have been analysed on many levels and found to be significant. However, even though high‐level protein production poses a metabolic burden, evaluation of the expression host at the level of the metabolome has often been neglected. We present a comparison of metabolite profiles of a wild‐type strain with those of three strains producing recombinant antibody variants of increasing size and complexity: an scFv fragment, an scFv–Fc fusion protein and a full‐length IgG molecule. Under producing conditions, all three recombinant strains showed a clear decrease in growth rate compared with the wild‐type strain and the severity of the growth phenotype increased with size of the protein. The levels of 76 intracellular metabolites were determined using a targeted (semi) quantitative mass spectrometry based approach. Based on unsupervised and supervised multivariate analysis of metabolite profiles, together with pathway activity profiling, the recombinant strains were found to be significantly different from each other and from the wild‐type strain. We observed the most prominent changes in metabolite levels for metabolites involved in amino acid and redox metabolism. Induction of the unfolded protein response was detected in all producing strains and is considered to be a contributing factor to the overall metabolic burden on the cells.     

Application of the Antibody-Inducing Activity of Glycosphingolipids to Human Diseases

Glycosphingolipids (GSLs) are composed of a mono-, di-, or oligosaccharide and a ceramide and function as constituents of cell membranes. Various molecular species of GSLs have been identified in mammalian cells due to differences in the structures of oligosaccharides. The oligosaccharide structure can vary depending on cell lineage, differentiation stage, and pathology; this property can be used as a cell identification marker. Furthermore, GSLs are involved in various aspects of the immune response, such as cytokine production, immune signaling, migration of immune cells, and antibody production. GSLs containing certain structures exhibit strong immunogenicity in immunized animals and promote the production of anti-GSL antibodies. By exploiting this property, it is possible to generate antibodies that recognize the fine oligosaccharide structure of specific GSLs or glycoproteins. In our study using artificially synthesized GSLs (artGSLs), we found that several structural features are correlated with the antibody-inducing activity of GSLs. Based on these findings, we designed artGSLs that efficiently induce the production of antibodies accompanied by class switching and developed several antibodies that recognize not only certain glycan structures of GSLs but also those of glycoproteins. This review comprehensively introduces the immune activities of GSLs and their application as pharmaceuticals.     

A longitudinal observational study of the dynamics of Mycoplasma bovis antibodies in naturally exposed and diseased dairy cows

Mycoplasma bovis is an important pathogen causing disease and substantial economic losses in cattle. However, knowledge of the dynamics of antibody responses in individual cows in the face of an outbreak is currently extremely limited. The use of commercial antibody tests to support clinical decision-making and for surveillance purposes is therefore challenging. Our objective was to describe the dynamics of M. bovis antibody responses in 4 Danish dairy herds experiencing an acute outbreak of M. bovis-associated disease, and to compare the antibody dynamics between dairy cows with different disease manifestations. A total of 120 cows were examined using a standardized clinical protocol and categorized into 4 disease groups: “mastitis,” “systemic,” “nonspecific,” and “none.” Paired blood and milk samples were collected and tested using a commercial M. bovis antibody–detecting ELISA. Plots of raw data and generalized additive mixed models with cow and herd as random effects were used to describe serum and milk antibody dynamics relative to the estimated time of onset of clinical disease. Cows with mastitis had high optical density measurement (ODC%) of antibodies in both milk and serum at disease onset. The estimated mean ODC% in milk was below the manufacturer's cut-off for the other groups for the entire study period. The estimated mean serum ODC% in the “systemic” group was high at onset of disease and stayed above the cut-off until 65 d after disease onset. However, the lower 95% confidence interval (CI) for the mean ODC% was only above the manufacturer's cut-off between 7 and 17 d after onset of disease. The CI of the “systemic” and “none” groups did not overlap at any time between the day of disease onset and 65 d after disease onset, and the estimated mean ODC% for both the “nonspecific” and “none” groups were generally below the cut-off for the majority of the study period. In conclusion, the serum antibody responses were highly dynamic and showed a high level of variation between individual cows. This strongly suggests that serology is unlikely to be useful for individual diagnosis of M. bovis-associated disease in dairy cows. However, it might still be useful for herd- or group-level diagnosis. Antibodies in milk were only increased in cows with M. bovis mastitis, indicating that milk antibody measurements only have diagnostic utility for cows with mastitis.     

Engineering of Lipid Nanoparticles by the Multifunctionalization of the Surface with Amino Acid Derivatives for the Neutralization of a Target Toxic Peptide

Protein affinity reagents (e.g., antibodies) are often used for basic research, diagnostics, separations, and disease therapy. Although a lot of “synthetic” protein affinity reagents have been developed as a cost-effective alternative to antibodies, their low biocompatibility is a considerable problem for clinical application. Lipid nanoparticles (LNP) represent a highly biocompatible drug delivery agent. However, little has been reported that LNP itself works as a protein affinity reagent in living animals. Here, LNP is engineered for binding to and neutralizing a target toxic peptide in living animals by multifunctionalization with amino acid derivatives. Multifunctionalized LNP (MF-LNP) is prepared using amino acid derivative-conjugated lipids. Optimized MF-LNP exhibits nanomolar affinity to the target toxic peptide and inhibits toxic peptide-dependent hemolysis and cytotoxicity. In addition, MF-LNP captures and neutralizes the toxic peptide after intravenous injection in the bloodstream; in addition, MF-LNP does not release the toxic peptide in the accumulated organ. These results reveal the potential of using LNP as a highly biocompatible protein affinity reagent such as an antidote.     

Monoclonal antibody‐targeted polymeric nanoparticles for cancer therapy – future prospects

Although combination therapy for cancer utilising monoclonal antibodies in conjunction with chemotherapeutic drugs has resulted in increases in 5 year survivals, there nevertheless remains significant morbidity and mortality associated with systemic delivery of cytotoxic drugs. The advent of living radical polymerisation has resulted in complex and elegant nanoparticle structures that can be engineered to passively target a drug payload for cancer treatment. This presents a therapeutic modality whereby biodistribution and consequently systemic toxicity can be reduced, while focusing drug delivery to the tumour site. Nanoparticle delivery can be enhanced by attachment of a targeting monoclonal antibody fragment to facilitate tumour cell uptake through endocytosis, and so increase therapeutic efficacy. In this way, monoclonal antibodies can be supercharged by carrying a payload consisting of a cocktail of conventional chemotherapeutic drugs and siRNA. This review will focus on antibody‐targeted polymeric nanoparticles to cancer cells, and methods and technologies for synthesising such antibody‐targeted nanoparticles. The review is confined to polymeric‐based nanoparticles as these offer some advantages over liposomal nanoparticles and may circumvent some of the pitfalls in nanomedicine. Development of these antibody based polymeric nanoparticles and future directions for therapy are highlighted in this review. © 2014 Society of Chemical Industry     

Computer-based engineering of thermostabilized antibody fragments

We used the molecular modeling program Rosetta to identify clusters of amino acid substitutions in antibody fragments (scFvs and scAbs) that improve global protein stability and resistance to thermal deactivation. Using this methodology, we increased the melting temperature (T_m) and resistance to heat treatment of an antibody fragment that binds to the Clostridium botulinum hemagglutinin protein (anti-HA33). Two designed antibody fragment variants with two amino acid replacement clusters, designed to stabilize local regions, were shown to have both higher T_m compared to the parental scFv and importantly to retain full antigen binding activity after 2 hr of incubation at 70°C. The crystal structure of one thermostabilized scFv variants was solved at 1.6 Å and shown to be in close agreement with the RosettaAntibody model prediction.     

Inhibitory Monoclonal Antibodies and Their Recombinant Derivatives Targeting Surface-Exposed Carbonic Anhydrase XII on Cancer Cells

Monoclonal and recombinant antibodies are widely used for the diagnostics and therapy of cancer. They are generated to interact with cell surface proteins which are usually involved in the development and progression of cancer. Carbonic anhydrase XII (CA XII) contributes to the survival of tumors under hypoxic conditions thus is considered a candidate target for antibody-based therapy. In this study, we have generated a novel collection of monoclonal antibodies (MAbs) against the recombinant extracellular domain of CA XII produced in HEK-293 cells. Eighteen out of 24 MAbs were reactive with cellular CA XII on the surface of live kidney and lung cancer cells as determined by flow cytometry. One MAb 14D6 also inhibited the enzymatic activity of recombinant CA XII as measured by the stopped-flow assay. MAb 14D6 showed the migrastatic effect on human lung carcinoma A549 and renal carcinoma A498 cell lines in a ‘wound healing’ assay. It did not reduce the growth of multicellular lung and renal cancer spheroids but reduced the cell viability by the ATP Bioluminescence assay. Epitope mapping revealed the surface-exposed amino acid sequence (35-FGPDGENS-42) close to the catalytic center of CA XII recognized by the MAb 14D6. The variable regions of the heavy and light chains of MAb 14D6 were sequenced and their complementarity-determining regions were defined. The obtained variable sequences were used to generate recombinant antibodies in two formats: single-chain fragment variable (scFv) expressed in E. coli and scFv fused to human IgG1 Fc fragment (scFv-Fc) expressed in Chinese Hamster Ovary (CHO) cells. Both recombinant antibodies maintained the same specificity for CA XII as the parental MAb 14D6. The novel antibodies may represent promising tools for CA XII-related cancer research and immunotherapy.     

VWF,Platelets and the Antiphospholipid Syndrome

The antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity with the persistent presence of antiphospholipid antibodies (aPLs). Laboratory criteria for the classification of APS include the detection of lupus anticoagulant (LAC), anti-cardiolipin (aCL) antibodies and anti-β2glycoprotein I (aβ2GPI) antibodies. Clinical criteria for the classification of thrombotic APS include venous and arterial thrombosis, along with microvascular thrombosis. Several aPLs, including LAC, aβ2GPI and anti-phosphatidylserine/prothrombin antibodies (aPS/PT) have been associated with arterial thrombosis. The Von Willebrand Factor (VWF) plays an important role in arterial thrombosis by mediating platelet adhesion and aggregation. Studies have shown that aPLs antibodies present in APS patients are able to increase the risk of arterial thrombosis by upregulating the plasma levels of active VWF and by promoting platelet activation. Inflammatory reactions induced by APS may also provide a suitable condition for arterial thrombosis, mostly ischemic stroke and myocardial infarction. The presence of other cardiovascular risk factors can enhance the effect of aPLs and increase the risk for thrombosis even more. These factors should therefore be taken into account when investigating APS-related arterial thrombosis. Nevertheless, the exact mechanism by which aPLs can cause thrombosis remains to be elucidated.     

龙血素A和龙血素B的酶联免疫检测方法的建立

建立了龙血素A和龙血素B间接竞争酶联免疫吸附分析方法,并用于测定龙血素A和龙血素B的含量。分别合成龙血素A和龙血素B人工抗原制备其多克隆抗体,建立二者间接竞争酶联免疫吸附分析方法并评估其分析性能。结果表明:抗龙血素A血清和抗龙血素B血清的效价分别达到8 000和30 000;龙血素A和龙血素B的最低检测限分别为3.9ng·mL-1和26.1ng·mL-1,批内精密度分别为CV≤10.7%和CV≤9.9%,批间精密度分别为CV≤11.5%和CV≤12.3%,回收率分别为87.4%~110.8%和109.8%~113.5%,与各自的6种类似物的交叉反应率最大分别为9.20%和3.64%,在4种龙血竭药物中的最大含量和最小含量分别相差4.05倍和3.57倍。龙血素A和龙血素B间接竞争酶联免疫吸附分析方法的检测精密度、准确度和特异性均较好,可快捷有效地用于药物检测和分析。