Novel Polystyrene-Binding Nanobody for Enhancing Immunoassays: Insights into Affinity, Immobilization, and Application Potential.

Nanobodies, which represent the next generation of antibodies due to their unique properties, face a significant limitation in their poor physical adsorption on solid supports. In this study, we successfully discovered polystyrene binding nanobodies from a synthetic nanobody library. Notably, bivalent nanobody B2 exhibited high affinity for polystyrene (0.7 nM for ELISA saturation binding analysis and 15.6 nM for isothermal titration calorimetry), displaying a pH-dependent behavior. Remarkably, hydrophobic and electrostatic interactions contribute minimally to the binding process. Molecular modeling provided insights into the interaction between B2 and polystyrene, revealing that the Trp51 residue within the CDR2 loop formed an aromatic H-bond with polystyrene at a distance of 2.74 Å, thus explaining the observed reduction in B2 affinity caused by Trp51 mutations. To explore B2's potential in protein immobilization, we constructed a bispecific nanobody by fusing B2 to an anticarcinoembryonic antigen nanobody 11C12, which cannot be immobilized on polystyrene through passive adsorption. Remarkably, the fusion construct achieved effective immobilization on polystyrene within 5 min by passing the need for periplasmic protein purification despite its low expression level. Moreover, the fusion construct demonstrated excellent linearity in the chemiluminescent enzyme immunoassay. For the first time, this study reports a simplified and seamless platform for the oriented immobilization of nanobody. Importantly, the entire process eliminated the need for protein purification, enabling efficient and rapid immobilization of fusion proteins directly from crude cell extracts, even when the expression level was low. Our developed process dramatically reduced the processing time from 2.5 days to just 5 min.

Isthmin-1 attenuates allergic Asthma by stimulating adiponectin expression and alveolar macrophage efferocytosis in mice.

BACKGROUND: Allergic asthma is a common respiratory disease that significantly impacts human health. Through in silico analysis of human lung RNASeq, we found that asthmatic lungs display lower levels of Isthmin-1 (ISM1) expression than healthy lungs. ISM1 is an endogenous anti-inflammatory protein that is highly expressed in mouse lungs and bronchial epithelial cells, playing a crucial role in maintaining lung homeostasis. However, how ISM1 influences asthma remains unclear. This study aims to investigate the potential involvement of ISM1 in allergic airway inflammation and uncover the underlying mechanisms. METHODS: We investigated the pivotal role of ISM1 in airway inflammation using an ISM1 knockout mouse line (ISM1-/-) and challenged them with house dust mite (HDM) extract to induce allergic-like airway/lung inflammation. To examine the impact of ISM1 deficiency, we analyzed the infiltration of immune cells into the lungs and cytokine levels in bronchoalveolar lavage fluid (BALF) using flow cytometry and multiplex ELISA, respectively. Furthermore, we examined the therapeutic potential of ISM1 by administering recombinant ISM1 (rISM1) via the intratracheal route to rescue the effects of ISM1 reduction in HDM-challenged mice. RNA-Seq, western blot, and fluorescence microscopy techniques were subsequently used to elucidate the underlying mechanisms. RESULTS: ISM1-/- mice showed a pronounced worsening of allergic airway inflammation and hyperresponsiveness upon HDM challenge. The heightened inflammation in ISM1-/- mice correlated with enhanced lung cell necroptosis, as indicated by higher pMLKL expression. Intratracheal delivery of rISM1 significantly reduced the number of eosinophils in BALF and goblet cell hyperplasia. Mechanistically, ISM1 stimulates adiponectin secretion by type 2 alveolar epithelial cells partially through the GRP78 receptor and enhances adiponectin-facilitated apoptotic cell clearance via alveolar macrophage efferocytosis. Reduced adiponectin expression under ISM1 deficiency also contributed to intensified necroptosis, prolonged inflammation, and heightened severity of airway hyperresponsiveness. CONCLUSIONS: This study revealed for the first time that ISM1 functions to restrain airway hyperresponsiveness to HDM-triggered allergic-like airway/lung inflammation in mice, consistent with its persistent downregulation in human asthma. Direct administration of rISM1 into the airway alleviates airway inflammation and promotes immune cell clearance, likely by stimulating airway adiponectin production. These findings suggest that ISM1 has therapeutic potential for allergic asthma.

Optimizing effector functions of monoclonal antibodies via tailored N-glycan engineering using a dual landing pad CHO targeted integration platform.

Monoclonal antibodies (mAbs) eliminate cancer cells via various effector mechanisms including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which are influenced by the N-glycan structures on the Fc region of mAbs. Manipulating these glycan structures on mAbs allows for optimization of therapeutic benefits associated with effector functions. Traditional approaches such as gene deletion or overexpression often lead to only all-or-nothing changes in gene expression and fail to modulate the expression of multiple genes at defined ratios and levels. In this work, we have developed a CHO cell engineering platform enabling modulation of multiple gene expression to tailor the N-glycan profiles of mAbs for enhanced effector functions. Our platform involves a CHO targeted integration platform with two independent landing pads, allowing expression of multiple genes at two pre-determined genomic sites. By combining with internal ribosome entry site (IRES)-based polycistronic vectors, we simultaneously modulated the expression of α-mannosidase II (MANII) and chimeric ß-1,4-N-acetylglucosaminyl-transferase III (cGNTIII) genes in CHO cells. This strategy enabled the production of mAbs carrying N-glycans with various levels of bisecting and non-fucosylated structures. Importantly, these engineered mAbs exhibited different degrees of effector cell activation and CDC, facilitating the identification of mAbs with optimal effector functions. This platform was demonstrated as a powerful tool for producing antibody therapeutics with tailored effector functions via precise engineering of N-glycan profiles. It holds promise for advancing the field of metabolic engineering in mammalian cells.

Manufacturability and functionality assessment of different formats of T-cell engaging bispecific antibodies.

T-cell-engaging bispecific antibodies (T-bsAbs) are promising immunotherapies for cancer treatment due to their capability of redirecting T-cells toward destroying tumor cells. Numerous T-bsAb formats have been developed, each with advantages and disadvantages in terms of developability, immunogenicity, effector functions, and pharmacokinetics. Here, we systematically compared T-bsAbs produced using eight different formats, evaluating the effect of molecular design of T-bsAbs on their manufacturability and functionality. These eight T-bsAb formats were constructed using antigen-binding fragments (Fabs) and single-chain variable fragments (scFvs) of antibodies linked to the crystallizable fragment (Fc) domain of immunoglobulin G. To ensure a fair comparison of growth and production data, we used recombinase-mediated cassette exchange technology to generate the T-bsAb-producing CHO cell lines. The produced T-bsAbs were assessed for their purification profile and recovery, binding capability, and biological activities. Our findings indicated that the manufacturability of bsAbs was adversely affected with increased number of scFv building blocks, while the functionality was affected by the combination of multiple factors, including the binding affinity and avidity of targeting moieties and the flexibility and geometry of formats. These results provide valuable insights into the impact of the format design on the optimal production and function of T-bsAbs.

Enhancing pharmacokinetic and pharmacodynamic properties of recombinant therapeutic proteins by manipulation of sialic acid content.

The circulatory half-life of recombinant therapeutic proteins is an important pharmacokinetic attribute because it determines the dosing frequency of these drugs, translating directly to treatment cost. Thus, recombinant therapeutic glycoproteins such as monoclonal antibodies have been chemically modified by various means to enhance their circulatory half-life. One approach is to manipulate the N-glycan composition of these agents. Among the many glycan constituents, sialic acid (specifically, N-acetylneuraminic acid) plays a critical role in extending circulatory half-life by masking the terminal galactose that would otherwise be recognised by the hepatic asialoglycoprotein receptor (ASGPR), resulting in clearance of the biotherapeutic from the circulation. This review aims to provide an illustrative overview of various strategies to enhance the pharmacokinetic/pharmacodynamic properties of recombinant therapeutic proteins through manipulation of their sialic acid content.

Leveraging an advanced simulated moving bed approach to achieve 3-component separation for enhanced impurity removal in a non-affinity cation exchange capture step.

One of the key challenges in downstream bioprocessing is to obtain products of high purity in a productive fashion through the effective removal of process and product related impurities. While a classical simulated moving bed (SMB) system operation can typically achieve a 2-component separation between the weakly bound impurities and target species, here we present an advanced SMB approach that can achieve a 3-component separation, including the removal of the strongly bound impurities from the target species. As a proof-of-concept, we demonstrate the enhanced removal of strongly bound host cell proteins (HCP) from the target monoclonal antibody (mAb) through the utilisation of the advanced SMB approach in a non-affinity cation exchange (CEX) capture step. In this way, 1 less polishing step was required to achieve the therapeutic requirements of < 100 ppm HCP and the overall process recovery was increased by ~ 6% compared to the corresponding process that utilised a batch CEX operation. The non-affinity CEX capture platform technology established through the utilisation of the advanced SMB approach presented here can potentially be further applied to address the downstream processing challenges presented by other challenging biotherapeutic modalities to yield a final target product with improved purity and recovery.

Harnessing ceramic hydroxyapatite as an effective polishing strategy to remove product- and process-related impurities in bispecific antibody purification.

Bispecific antibody (bsAb), a novel therapeutic modality, provides excellent treatment efficacy, yet poses numerous challenges to downstream process development, which are mainly due to the intricate diversity of bsAb structures and impurity profiles. Ceramic hydroxyapatite (CHT), a mixed-mode medium, allows proteins to interact with its calcium sites (C-sites) through metal affinity and/or its phosphate sites (P-sites) through cation exchange interactions. This dual-binding capability potentially offers unique bind and elute behaviours for different proteins of interest, resulting in optimal product purity when suitable elution conditions are employed. In this study, the effectiveness of CHT as a polishing step for bsAb purification was investigated across three model molecules and benchmarked against the traditional cation exchange chromatography (CEX). For both asymmetric and symmetric IgG-like bsAb post Protein A eluates, at least 97% product purity was achieved after CHT polishing. CHT delivered a superior aggregate clearance to CEX, resulting in low high molecular weight (HMW) impurities (0.5%) and low process-related impurities in the product pools. Moreover, CHT significantly mitigated "chromatography-induced aggregation" whereas eightfold more HMW was generated by CEX. This study illustrated the developability of CHT in effectively eliminating low molecular weight (LMW) impurities through post-load-wash (PLW) optimization, resulting in an additional reduction of up to 48% in LMW impurities. A mechanistic explanation regarding the performance of impurity removal and mitigation of the chromatography-induced aggregation by CHT was proposed, illustrating unique CHT capability is potentially driven by C-site cooperation, of which effectiveness could depend on the bsAb composition and size.

Vector design for enhancing expression level and assembly of knob-into-hole based FabscFv-Fc bispecific antibodies in CHO cells.

Background: Two-armed FabscFv-Fc is a favoured bispecific antibody (BsAb) format due to its advantages of the conventional IgG structure. Production of FabscFv-Fc requires expression of three polypeptide chains, one light chain (LC), one heavy chain (HC) and a scFv fused to the Fc (scFvFc) at optimal ratios. Methods: We designed a set of internal ribosome entry site (IRES)-mediated multi-cistronic vectors tailoring to various expression ratios of the three polypeptides to study how the chain ratios affect the FabscFv-Fc production. Results: Expression of HC and scFvFc chains at 1:1 ratio and excess LC gave the highest yield of correctly assembled product. Compared to the use of IRES and multiple promoters, using 2A peptides for co-expression of the three polypeptides gave the highest titre and correctly assembled product. Conclusion: The results obtained in this work provide insights to the impacts of hetero-chain ratios on the BsAb production.

Influence of Grain Orientation and Grain Boundary Features on Local Stress State of Cu-8Al-11Mn Alloy Investigated Using Crystal Plasticity Finite Element Method.

Reducing the local stress in the vicinity of the grain boundaries is a favorable way to improve the super-elastic properties of super-elastic alloys. The crystal plasticity finite element method (CPFEM) was applied in this study to simulate the deformation behavior and local stress of a super-elastic Cu-8Al-11Mn (wt.%) alloy containing single grains with various orientations, columnar grains with different misorientation angles, and tri-crystals with distinct grain boundary morphologies. The results indicated that the stress distribution of single grains presented obvious orientation dependence during deformation. Uniformly distributed stress was observed in grains with orientations of 0° and 90° when more slip systems were activated during deformation. With the increase in the misorientation angles of columnar grains, the stresses in the vicinity of the grain boundaries increased, which was related to the difference in the shear stress of the slip systems in adjacent grains. When the difference in the shear stress of the slip systems in two adjacent grains was large, a local stress concentration formed in the vicinity of the grain boundary. Compared with the triple-junction grain boundaries, the local stresses of the straight and vertical grain boundaries were smaller, which was closely related to the number of activated slip systems on both sides of the grain boundary. The above results were obtained experimentally and could be used to design super-elastic alloys with high performance.

Development of a T cell-redirecting bispecific antibody targeting B-cell maturation antigen for the suppression of multiple myeloma cell growth.

Background: Multiple myeloma (MM) is the second most common hematological malignancy. It has emerged as one of the next possible hematological diseases amenable to immunotherapy. B-cell maturation antigen (BCMA), a member of the tumor necrosis factor receptor superfamily, is highly expressed in MM cells and is one target with the most potential for developing MM-targeting immunotherapy. Other than the FDA-approved BCMA-targeting CAR T-cell therapy, such as Abecma and CARVYKTI, T cell-engaging multi-specific antibody is another promising therapeutic modality for BCMA-targeting MM treatment. We develop a T-cell redirecting BCMA-targeting bispecific antibody (bsAb) and evaluate its anti-MM activity. Methods: We first generated several clones of mouse anti-human BCMA monoclonal antibodies using DNA immunization. One of the anti-BCMA antibodies was then used to design and produce a T cell-redirecting BCMA × CD3 bsAb in CHO cells. Finally, we examined the effect of the bsAb on MM cell growth both in vitro and in vivo. Results: The BCMA × CD3 bsAb was designed in a FabscFv format and produced in CHO cells with good yield and purity. Moreover, the bsAb can trigger robust T cell proliferation and activation and induce efficient T cell-mediated MM cell killing in vitro. Using a MM xenograft mouse model, we demonstrate that the bsAb can effectively suppress MM cell growth in vivo. Conclusions: Our results suggest that the BCMA × CD3 bsAb in the FabscFv format can efficiently inhibit MM cell growth and have promising potential to be developed into a therapeutic antibody drug for the treatment of MM.

Excellent removal of knob-into-hole bispecific antibody byproducts and impurities in a single-capture chromatography.

Bispecific antibodies (bsAbs) are therapeutically promising due to their ability to bind to two different antigens. However, the bsAb byproducts and impurities, including mispaired homodimers, half-antibodies, light chain mispairings, antibody fragments and high levels of high molecular weight (HMW) species, all pose unique challenges to their downstream processing. Here, using two knob-into-hole (KiH) constructs of bsAbs as model molecules, we demonstrate the excellent removal of bsAb byproducts and impurities in a single Protein A chromatography under optimized conditions, including hole-hole homodimer mispaired products which are physicochemically very similar to the target bsAbs and still present even with the use of the KiH format, though at reduced levels. The removal occurs through the incorporation of an intermediate low-pH wash step and optimal elution conditions, achieving ~ 60% monomeric purity increase in a single Protein A step, without the introduction of sequence-specific bsAb modifications to specifically induce differential Protein A binding. Our results also suggest that the higher aggregation propensity of bsAbs may cause aggregation during the column process, hence an optimization of the appropriate loading amount, which may be lower than that of monoclonal antibodies (mAbs), is required. With the use of loading at 50% of 10% breakthrough (QB10) at 6-min residence time, we show that an overall high monomer purity of 92.1-93.2% can be achieved with good recovery of 78.4-90.6% within one capture step, which is a significant improvement from a monomer purity of ~ 30% in the cell culture supernatant (CCS). The results presented here would be an insightful guidance to all researchers working on the purification process development to produce bispecific antibodies, especially for knob-into-hole bispecific antibodies.

Effective flow-through polishing strategies for knob-into-hole bispecific antibodies.

Bispecific antibodies (bsAbs), though possessing great therapeutic potential, are extremely challenging to obtain at high purity within a limited number of scalable downstream processing steps. Complementary to Protein A chromatography, polishing strategies play a critical role at removing the remaining high molecular weight (HMW) and low molecular weight (LMW) species, as well as host cell proteins (HCP) in order to achieve a final product of high purity. Here, we demonstrate using two knob-into-hole (KiH) bsAb constructs that two flow-through polishing steps utilising Capto Butyl ImpRes and Capto adhere resins, performed after an optimal Protein A affinity chromatography step can further reduce the HCP by 17- to 35-fold as well as HMW and LMW species with respect to monomer by ~ 4-6% and ~ 1%, respectively, to meet therapeutical requirement at 30-60 mg/mL-resin (R) load. This complete flow-through polishing strategy, guided by Design of Experiments (DoE), eliminates undesirable aggregation problems associated with the higher aggregation propensity of scFv containing bsAbs that may occur in the bind and elute mode, offering an improved ease of overall process operation without additional elution buffer preparation and consumption, thus aligning well with process intensification efforts. Overall, we demonstrate that through the employment of (1) Protein A chromatography step and (2) flow-through polishing steps, a final product containing < 1% HMW species, < 1% LMW species and < 100 ppm HCP can be obtained with an overall process recovery of 56-87%.

Gold Nanoparticle-based "Mix and Measure" Fluorimetric Assays to Quantify Antibody Titer.

Monoclonal antibodies (mAbs) for treatment of human diseases are typically human or humanized Immunoglobulin G (IgG) produced in mammalian cell lines. A rapid, less tedious, and high throughput method to quantify mAbs is in demand to accelerate mAb production efficiency. To quantify mAb titer, we developed gold nanoparticle (AuNPs)-based "mix and measure" fluorimetric assays by exploiting AuNPs' fluorescence quenching ability. The AuNPs are functionalized by an Fc binding protein, i. e. protein G, which binds human IgG and fluorescently labeled rat IgG (Alexa Fluor 488-rat IgG) with differential affinity. The assays can be in competition or displacement format. The competitive binding of human IgG drug and the labelled rat IgG to protein G-coated AuNP lead to varied fluorescent intensity that is proportional to the amount of human IgG analte; or the displacement of the labelled rat IgG from protein G-coated AuNP by human IgG can lead to fluorescent recovery that is also proportionally related to human IgG concentration. The assays can quantify therapeutic mAbs in the range of 10-1,000 mg/L, demonstrated for Herceptin, Avastin, and Humira in cell culture media. The assays have fast turn over time (within 15 min). They can be performed in microplates and are suitable for high throughput "on-line" or "at-line" measurement in mAbs production lines.

Multiplexed engineering glycosyltransferase genes in CHO cells via targeted integration for producing antibodies with diverse complex-type N-glycans.

Therapeutic antibodies are decorated with complex-type N-glycans that significantly affect their biodistribution and bioactivity. The N-glycan structures on antibodies are incompletely processed in wild-type CHO cells due to their limited glycosylation capacity. To improve N-glycan processing, glycosyltransferase genes have been traditionally overexpressed in CHO cells to engineer the cellular N-glycosylation pathway by using random integration, which is often associated with large clonal variations in gene expression levels. In order to minimize the clonal variations, we used recombinase-mediated-cassette-exchange (RMCE) technology to overexpress a panel of 42 human glycosyltransferase genes to screen their impact on antibody N-linked glycosylation. The bottlenecks in the N-glycosylation pathway were identified and then released by overexpressing single or multiple critical genes. Overexpressing B4GalT1 gene alone in the CHO cells produced antibodies with more than 80% galactosylated bi-antennary N-glycans. Combinatorial overexpression of B4GalT1 and ST6Gal1 produced antibodies containing more than 70% sialylated bi-antennary N-glycans. In addition, antibodies with various tri-antennary N-glycans were obtained for the first time by overexpressing MGAT5 alone or in combination with B4GalT1 and ST6Gal1. The various N-glycan structures and the method for producing them in this work provide opportunities to study the glycan structure-and-function and develop novel recombinant antibodies for addressing different therapeutic applications.

Nonsymmetrical Segregation of Solutes in Periodic Misfit Dislocations Separated Tilt Grain Boundaries.

Interfacial segregation is ubiquitous in mulit-component polycrystalline materials and plays a decisive role in material properties. So far, the discovered solute segregation patterns at special high-symmetry interfaces are usually located at the boundary lines or are distributed symmetrically at the boundaries. Here, in a model Mg-Nd-Mn alloy, we confirm that elastic strain minimization facilitated nonsymmetrical segregation of solutes in four types of linear tilt grain boundaries (TGBs) to generate ordered interfacial superstructures. Aberration-corrected high-angle annular dark-field scanning transmission electron microscopy observations indicate that the solutes selectively segregate at substitutional sites at the linear TGBs separated by periodic misfit dislocations to form such two-dimensional planar structures. These findings are totally different from the classical McLean-type segregation which has assumed the monolayer or submonolayer coverage of a grain boundary and refresh understanding on strain-driven interface segregation behaviors.

IL-10 Enhances Human Natural Killer Cell Effector Functions via Metabolic Reprogramming Regulated by mTORC1 Signaling.

Cell metabolism plays a pivotal role in regulating the effector functions of immune cells. Stimulatory cytokines, such as interleukin (IL)-2 or IL-12 and IL-15, activate glycolysis and oxidative phosphorylation in natural killer (NK) cells to support their enhanced effector functions. IL-10, a pleiotropic cytokine, is known to suppress macrophage activation but stimulate NK cells. However, it remains unclear if IL-10 has an effect on the metabolism of human NK cells and if so, what metabolic mechanisms are affected, and how these metabolic changes are regulated and contribute to the effector functions of NK cells. In this study, we demonstrate that IL-10 upregulates both glycolysis and oxidative phosphorylation in human NK cells, and these metabolic changes are crucial for the enhanced effector functions of NK cells. Mechanistically, we unravel that IL-10 activates the mammalian target of rapamycin complex 1 (mTORC1) to regulate metabolic reprogramming in human NK cells.

A comprehensive CHO SWATH-MS spectral library for robust quantitative profiling of 10,000 proteins.

Sequential window acquisition of all theoretical fragment-ion spectra (SWATH) is a data-independent acquisition (DIA) strategy that requires a specific spectral library to generate unbiased and consistent quantitative data matrices of all peptides. SWATH-MS is a promising approach for in-depth proteomic profiling of Chinese hamster Ovary (CHO) cell lines, improving mechanistic understanding of process optimization, and real-time monitoring of process parameters in biologics R&D and manufacturing. However, no spectral library for CHO cells is publicly available. Here we present a comprehensive CHO global spectral library to measure the abundance of more than 10,000 proteins consisting of 199,102 identified peptides from a CHO-K1 cell proteome. The robustness, accuracy and consistency of the spectral library were validated for high confidence in protein identification and reproducible quantification in different CHO-derived cell lines, instrumental setups and downstream processing samples. The availability of a comprehensive SWATH CHO global spectral library will facilitate detailed characterization of upstream and downstream processes, as well as quality by design (QbD) in biomanufacturing. The data have been deposited to ProteomeXchange (PXD016047).

Effect of grain morphology on the degradation behavior of Mg-4 wt% Zn alloy in Hank's solution.

The degradation behavior of Mg-4 wt% Zn alloy with three different microstructures was examined in Hank's solution at 37 °C by electrochemical measurements and immersion tests in this study. The results show that the sample with cellular structure exhibits a more positive corrosion potential, lower corrosion current density, larger impedance and more protective film than samples with columnar dendritic and equiaxed dendritic structure. The higher corrosion resistance is attributed to the preferred orientation, eliminating susceptible grain boundaries and reduced secondary phases.

A Wrapper Feature Subset Selection Method Based on Randomized Search and Multilayer Structure.

The identification of discriminative features from information-rich data with the goal of clinical diagnosis is crucial in the field of biomedical science. In this context, many machine-learning techniques have been widely applied and achieved remarkable results. However, disease, especially cancer, is often caused by a group of features with complex interactions. Unlike traditional feature selection methods, which only focused on finding single discriminative features, a multilayer feature subset selection method (MLFSSM), which employs randomized search and multilayer structure to select a discriminative subset, is proposed herein. In each level of this method, many feature subsets are generated to assure the diversity of the combinations, and the weights of features are evaluated on the performances of the subsets. The weight of a feature would increase if the feature is selected into more subsets with better performances compared with other features on the current layer. In this manner, the values of feature weights are revised layer-by-layer; the precision of feature weights is constantly improved; and better subsets are repeatedly constructed by the features with higher weights. Finally, the topmost feature subset of the last layer is returned. The experimental results based on five public gene datasets showed that the subsets selected by MLFSSM were more discriminative than the results by traditional feature methods including LVW (a feature subset method used the Las Vegas method for randomized search strategy), GAANN (a feature subset selection method based genetic algorithm (GA)), and support vector machine recursive feature elimination (SVM-RFE). Furthermore, MLFSSM showed higher classification performance than some state-of-the-art methods which selected feature pairs or groups, including top scoring pair (TSP), k-top scoring pairs (K-TSP), and relative simplicity-based direct classifier (RS-DC).

An IRES-Mediated Tricistronic Vector for Efficient Generation of Stable, High-Level Monoclonal Antibody Producing CHO DG44 Cell Lines.

The generation of stable, high-level monoclonal antibody (mAb) producing cell lines remains a major challenge in biopharmaceutical industry. The commonly used plasmid vectors for mAb expression, which express light chain (LC), heavy chain (HC), and selection marker genes on separate vectors or via multiple promoters on a single vector, are not able to accurately control the ratio of LC over HC expression and tend to result in non-expressing clones. To overcome these issues, we have developed a tricistronic vector using two internal ribosome entry sites (IRES) to express the LC, HC, and dihydrofolate reductase (DHFR) selection marker genes in one transcript. In this tricistronic vector, the three genes are under the control of a hapten-modified human cytomegalovirus (hCMV) promoter containing a core CpG island element (IE) to enhance the production stability. The LC gene is arranged as the first cistron followed by a wild-type IRES to control the HC expression. Such design expresses excess LC polypeptides which enhance mAb expression level and reduce aggregate. A mutated IRES with attenuated strength is applied on DHFR to reduce its expression for enhancing the stringency of selection for high producers. This vector allows easy generation of stable, high mAb producing CHO DG44 pools and clones for antibody development and manufacturing.