High-Throughput Antibody Neutralization Screening in Massively Parallel Droplet Arrays.

Neutralizing antibodies provide rapid immune defense against infectious diseases, but are difficult to discover at scale because neutralization assays require live reporter cells and soluble monoclonal antibodies. Here we report Droplet Reporter Cell Testing for Neutralization (DrReCT-Neutralization) to screen antibody gene libraries for their ability to neutralize viral infections. We established the necessary engineered cell lines and validated the DrReCT screening platform using synthetic oligoclonal libraries, followed by an example discovery campaign that demonstrated scalable functional antibody data collection against viral diseases.

Application of the CRISPR/Cas9 Gene Editing Method for Modulating Antibody Fucosylation in CHO Cells.

Genetic engineering plays an essential role in the development of cell lines for biopharmaceutical manufacturing. Advanced gene editing tools can improve both the productivity of recombinant cell lines as well as the quality of therapeutic antibodies. Antibody glycosylation is a critical quality attribute for therapeutic biologics because the glycan patterns on the antibody fragment crystallizable (Fc) region can alter its clinical efficacy and safety as a therapeutic drug.

Recurrent hemichorea in an adolescent with systemic lupus erythematosus and previous ipsilateral cerebral infarction.

Systemic lupus erythematosus (SLE) can present with movement disorders, among which chorea is closely associated with antiphospholipid (aPL) antibodies. Brain imaging results obtained in patients with chorea are generally inconsistent with the clinical manifestation of chorea; moreover, medical tests for hemichorea, which are expected to reveal distinct localization, may show negative findings. Herein, we present a case of a 15-year-old girl with SLE who had a history of left cerebral infarction; tests revealed elevated aPL levels, and she developed recurrent left hemichorea 2 years later.

Cell activation-based screening of natively paired human T cell receptor repertoires.

Adoptive immune therapies based on the transfer of antigen-specific T cells have been used successfully to treat various cancers and viral infections, but improved techniques are needed to identify optimally protective human T cell receptors (TCRs). Here we present a high-throughput approach to the identification of natively paired human TCRα and TCRβ (TCRα:β) genes encoding heterodimeric TCRs that recognize specific peptide antigens bound to major histocompatibility complex molecules (pMHCs). We first captured and cloned TCRα:β genes from individual cells, ensuring fidelity using a suppression PCR.

Quality Control: Chain Pairing Precision and Monitoring of Cross-Sample Contamination: A Method by the AIRR Community.

New approaches in high-throughput analysis of immune receptor repertoires are enabling major advances in immunology and for the discovery of precision immunotherapeutics. Commensurate with growth of the field, there has been an increased need for the establishment of techniques for quality control of immune receptor data. Our laboratory has standardized the use of multiple quality control techniques in immunoglobulin (IG) and T-cell receptor (TR) sequencing experiments to ensure quality control throughout diverse experimental conditions.

Immortalization and functional screening of natively paired human T cell receptor repertoires.

Functional analyses of the T cell receptor (TCR) landscape can reveal critical information about protection from disease and molecular responses to vaccines. However, it has proven difficult to combine advanced next-generation sequencing technologies with methods to decode the peptide-major histocompatibility complex (pMHC) specificity of individual TCRs. We developed a new high-throughput approach to enable repertoire-scale functional evaluations of natively paired TCRs.

The interplay of protein engineering and glycoengineering to fine-tune antibody glycosylation and its impact on effector functions.

The N-glycan pattern of an IgG antibody, attached at a conserved site within the fragment crystallizable (Fc) region, is a critical antibody quality attribute whose structural variability can also impact antibody function. For tailoring the Fc glycoprofile, glycoengineering in cell lines as well as Fc amino acid mutations have been applied. Multiple glycoengineered Chinese hamster ovary cell lines were generated, including defucosylated (FUT8KO), α-2,6-sialylated (ST6KI), and defucosylated α-2,6-sialylated (FUT8KOST6KI), expressing either a wild-type anti-CD20 IgG (WT) or phenylalanine to alanine (F241A) mutant.

The impact of sialylation linkage-type on the pharmacokinetics of recombinant butyrylcholinesterases.

Chinese hamster ovary (CHO) cells typically produce glycoproteins with N-glycans terminating in α-2,3 sialylation. Human cells produce glycoproteins that include α-2,3 and α-2,6 sialic acids. To examine the impact of altering protein sialylation on pharmacokinetic properties, recombinant human butyrylcholinesterase (BChE) was produced in CHO cells by knocking out the α-2,3 sialyltransferase genes followed by overexpression of the α-2,6 sialyltransferase (26BChE) enzyme.

Distributions of discrete spherical particles with a constant susceptibility can lead to echo time dependent phase shifts which deviate from theories.

Purpose: When an object contains a distribution of discrete magnetic inclusions with a constant susceptibility, the MRI signal inside the object may no longer be determined analytically by assuming that the object is uniform or magnetic inclusions are completely random. Through simulations and experiments with spherical particles inside cylinders, this work is to study the signal behavior in the static dephasing regime.

Methods: MRI complex images of long cylinders containing spherical particles with different arrangements were simulated and compared to similar experimental phantom data.

Combining Butyrated ManNAc with Glycoengineered CHO Cells Improves EPO Glycan Quality and Production.

Sodium butyrate (NaBu) is not only well-known for enhancing protein production, but also degrades glycan quality. In this study, butyrate supplied by the precursor molecule 1,3,4-O-Bu ManNAc is applied to overcome the negative effects of NaBu on glycan quality while simultaneously increasing the productivity of the model recombinant erythropoietin (EPO). The beneficial impact of 1,3,4-O-Bu ManNAc on EPO glycan quality, while evident in wild-type CHO cells, is particularly pronounced in glycoengineered CHO cells with stable overexpression of β-1,4- and β-1,6-N-acetylglucosaminyltransferases (GnTIV and GnTV) and α-2,6-sialyltransferase (ST6) enzymes responsible for N-glycan antennarity and sialylation.

Removing unwanted background phase with a reference phantom for applications in susceptibility quantification.

Purpose: A method of removing the background phase with a reference phantom but without overcorrecting the induced phase from objects of interest is proposed. Several factors during the imaging procedure and post-processing are investigated for their accuracies.

Methods: A method using a reference phantom to remove eddy currents as well as using the least squares fit to quantify susceptibility and to remove the background phase is proposed.

Antibody glycoengineering strategies in mammalian cells.

As a key parameter impacting functional and structural heterogeneity, protein glycosylation is a critical quality attribute for antibody biotherapeutic manufacturing. The glycan patterns on recombinant antibodies, particularly on the conserved fragment crystallizable (Fc) region, can have significant effects on an antibody's functional activities including clearance rate, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and anti-inflammatory activity. In this review, we examined specific glycan attachments (fucosylation, sialylation, galactosylation, high-mannose, and bisecting glycans) and their importance to antibody properties.

Butyrated ManNAc analog improves protein expression in Chinese hamster ovary cells.

The chemical additive sodium butyrate (NaBu) has been applied in cell culture media as a direct and convenient method to increase the protein expression in Chinese hamster ovary (CHO) and other mammalian cells. In this study, we examined an alternative chemical additive, 1,3,4-O-Bu ManNAc, for its effect on recombinant protein production in CHO. Supplementation with 1,3,4-O-Bu ManNAc for two stable CHO cell lines, expressing human erythropoietin or IgG, enhanced protein expression for both products with negligible impact on cell growth, viability, glucose utilization, and lactate accumulation.

A study of MRI gradient echo signals from discrete magnetic particles with considerations of several parameters in simulations.

Modeling MRI signal behaviors in the presence of discrete magnetic particles is important, as magnetic particles appear in nanoparticle labeled cells, contrast agents, and other biological forms of iron. Currently, many models that take into account the discrete particle nature in a system have been used to predict magnitude signal decays in the form of R2* or R2' from one single voxel. Little work has been done for predicting phase signals.

Proline-Rich Chaperones Are Compared Computationally and Experimentally for Their Abilities to Facilitate Recombinant Butyrylcholinesterase Tetramerization in CHO Cells.

Human butyrylcholinesterase (BChE), predominantly tetramers with a residence time of days, offers the potential to scavenge organophosphorus pesticides and chemical warfare agents. Efficient assembly of human BChE into tetramers requires an association with proline-rich peptide chaperones. In this study, the incorporation of different proline-rich peptide chaperones into BChE is investigated computationally and experimentally.

SnapShot: N-Glycosylation Processing Pathways across Kingdoms.

Post-translational modification of proteins with carbohydrates shapes their localization and function. This SnapShot presents the core pathways from different organisms that install these complex and highly variable structures.

Combinatorial genome and protein engineering yields monoclonal antibodies with hypergalactosylation from CHO cells.

One of the key quality attributes of monoclonal antibodies is the glycan pattern and distribution. Two terminal galactose residues typically represent a small fraction of the total glycans from antibodies. However, antibodies with defined glycosylation properties including enhanced galactosylation have been shown to exhibit altered properties for these important biomedical modalities.

Glycoengineering of CHO Cells to Improve Product Quality.

Chinese hamster ovary (CHO) cells represent the predominant platform in biopharmaceutical industry for the production of recombinant biotherapeutic proteins, especially glycoproteins. These glycoproteins include oligosaccharide or glycan attachments that represent one of the principal components dictating product quality. Especially important are the N-glycan attachments present on many recombinant glycoproteins of commercial interest.

A novel sugar analog enhances sialic acid production and biotherapeutic sialylation in CHO cells.

A desirable feature of many therapeutic glycoprotein production processes is to maximize the final sialic acid content. In this study, the effect of applying a novel chemical analog of the sialic acid precursor N-acetylmannosamine (ManNAc) on the sialic acid content of cellular proteins and a model recombinant glycoprotein, erythropoietin (EPO), was investigated in CHO-K1 cells. By introducing the 1,3,4-O-Bu ManNAc analog at 200-300 µM into cell culture media, the intracellular sialic acid content of EPO-expressing cells increased ∼8-fold over untreated controls while the level of cellular sialylated glycoconjugates increased significantly as well.

Integrated Genome and Protein Editing Swaps α-2,6 Sialylation for α-2,3 Sialic Acid on Recombinant Antibodies from CHO.

Immunoglobin G with α-2,6 sialylation has been reported to have an impact on antibody-dependent cellular cytotoxicity and anti-inflammatory efficacy. However, production of antibodies with α-2,6 sialylation from Chinese hamster ovary cells is challenging due to the inaccessibility of sialyltransferases for the heavy chain N-glycan site and the presence of exclusively α-2,3 sialyltransferases. In this study, combining mutations on the Fc regions to allow sialyltransferase accessibility with overexpression of α-2,6 sialyltransferase produced IgG with significant levels of both α-2,6 and α-2,3 sialylation.

Transparent deoxyribonucleic acid substrate with high mechanical strength for flexible and biocompatible organic resistive memory devices.

Biocompatible deoxyribonucleic acid (DNA), with high mechanical strength, was employed as the substrate for a Ag nanowire (Ag NW) pattern and then used to fabricate flexible resistor-type memory devices. The memory exhibited typical write-once-read-many (WORM)-type memory features with a high ON/OFF ratio (10), long-term retention ability (10 s) and excellent mechanical endurance.

Quantifications of in vivo labeled stem cells based on measurements of magnetic moments.

Cells labeled by super paramagnetic iron-oxide (SPIO) nanoparticles are more easily seen in gradient echo MR images, but it has not been shown that the amount of nanoparticles or the number of cells can be directly quantified from MR images. This work utilizes a previously developed and improved Complex Image Summation around a Spherical or Cylindrical Object (CISSCO) method to quantify the magnetic moments of several clusters of SPIO nanoparticle labeled cells from archived rat brain images. With the knowledge of mass magnetization of the cell labeling agent and cell iron uptake, the number of cells in each nanoparticle cluster can be determined.