Define Critical Parameters of Trastuzumab-Mediated ADCC Assays via Assay Optimization Processes, Focusing on the Impact of Cryopreserved Effector Cells on Assay Performance.

The mechanisms of mAb-induced ADCC have been well established. However, the ADCC bioassays used to quantify mAb-induced ADCC require continued development/refinement to properly assess and compare the potency of newly developed therapeutic mAbs and biosimilars to meet regulatory requirements. We used trastuzumab and a lactate dehydrogenase (LDH)-based ADCC bioassay as a model to define critical parameters of the ADCC bioassay, describing how several bioassay parameters, including preparation of effector cells, E/T ratio, target cell selection, bioassay media components, and treatment time can influence the data quality of the ADCC activity.

A unified view on enzyme catalysis by cryo-EM study of a DNA topoisomerase.

The theories for substrate recognition in enzyme catalysis have evolved from lock-key to induced fit, then conformational selection, and conformational selection followed by induced fit. However, the prevalence and consensus of these theories require further examination. Here we use cryogenic electron microscopy and African swine fever virus type 2 topoisomerase (AsfvTop2) to demonstrate substrate binding theories in a joint and ordered manner: catalytic selection by the enzyme, conformational selection by the substrates, then induced fit.

Visualizing the DNA repair process by a photolyase at atomic resolution.

Photolyases, a ubiquitous class of flavoproteins, use blue light to repair DNA photolesions. In this work, we determined the structural mechanism of the photolyase-catalyzed repair of a cyclobutane pyrimidine dimer (CPD) lesion using time-resolved serial femtosecond crystallography (TR-SFX). We obtained 18 snapshots that show time-dependent changes in four reaction loci.

Atezolizumab Induces Necroptosis and Contributes to Hepatotoxicity of Human Hepatocytes.

Atezolizumab is an immune checkpoint inhibitor (ICI) targeting PD-L1 for treatment of solid malignancies. Immune checkpoints control the immune tolerance, and the adverse events such as hepatotoxicity induced by ICIs are often considered as an immune-related adverse event (irAE). However, PD-L1 is also highly expressed in normal tissues, e.

Mutational and biophysical analyses reveal a TFIIIC binding region in the TFIIF-related Rpc53 subunit of RNA polymerase III.

The TFIIF-like Rpc53/Rpc37 heterodimer of RNA polymerase (pol) III is involved in various stages of transcription. The C-terminal region of Rpc53 dimerizes with Rpc37 to anchor on the lobe domain of the pol III cleft. However, structural and functional features of the Rpc53 N-terminal region had not been characterized previously.

Comparative Characterization of Different Molecular Formats of Bispecific Antibodies Targeting EGFR and PD-L1.

We generated two IgG1-like bispecific antibodies (BsAbs) with different molecular formats, symmetrical DVD-Ig and asymmetrical knob-in-hole (KIH), targeting the same antigens, EGFR and PD-L1 (designated as anti-EGFR/PD-L1). We performed the physiochemical and biological characterization of these two formats of anti-EGFR/PD-L1 BsAbs and compared some key quality attributes and biological activities of these two formats of BsAbs. Physiochemical binding characterization data demonstrated that both formats bound EGFR and PD-L1.

Serial crystallography captures dynamic control of sequential electron and proton transfer events in a flavoenzyme.

Flavin coenzymes are universally found in biological redox reactions. DNA photolyases, with their flavin chromophore (FAD), utilize blue light for DNA repair and photoreduction. The latter process involves two single-electron transfers to FAD with an intermittent protonation step to prime the enzyme active for DNA repair.

Structural and biological insights into Klebsiella pneumoniae surface polysaccharide degradation by a bacteriophage K1 lyase: implications for clinical use.

Background: K1 capsular polysaccharide (CPS)-associated Klebsiella pneumoniae is the primary cause of pyogenic liver abscesses (PLA) in Asia. Patients with PLA often have serious complications, ultimately leading to a mortality of ~ 5%. This K1 CPS has been reported as a promising target for development of glycoconjugate vaccines against K.

Enzymology and Dynamics by Cryogenic Electron Microscopy.

Cryogenic electron microscopy (cryo-EM) has revolutionized the field of structural biology, particularly in solving the structures of large protein complexes or cellular machineries that play important biological functions. This review focuses on the contribution and future potential of cryo-EM in related emerging applications-enzymatic mechanisms and dynamic processes. Work on these subjects can benefit greatly from the capability of cryo-EM to solve the structures of specific protein complexes in multiple conditions, including variations in the buffer condition, ligands, and temperature, and to capture multiple conformational states, conformational change intermediates, and reaction intermediates.

Tumor Extrinsic Factors Mediate Primary T-DM1 Resistance in HER2-Positive Breast Cancer Cells.

To explore if the tumor microenvironment contributes to the primary resistance of HER2-positive breast cancer cells to T-DM1, we examined whether Matrigel, a basement membrane matrix that provides a three-dimensional (3D) cell culture condition, caused the primary resistance of HER2-positive, T-DM1-sensitive breast cancer cells (JIMT1 and SKBR-3 cells) to T-DM1. This is different from the conventional approach such that the cells are exposed with escalated doses of drug to establish a drug-resistant cell line. We found that these cells were able to grow and form spheroids on the Matrigel in the presence of T-DM1.

Mechanisms contributing to ado-trastuzumab emtansine-induced toxicities: a gateway to better understanding of ADC-associated toxicities.

In order to improve the safety of novel therapeutic drugs, better understanding of the mechanisms of action is important. Ado-trastuzumab emtansine (also known as T-DM1) is an antibody-drug conjugate (ADC) approved for the treatment of HER2-positive breast cancer. While the treatment with T-DM1 results in significant efficacy in the selected patient population, nonetheless, there are concerns with side effects such as thrombocytopenia and hepatotoxicity.

A Novel Bispecific Antibody Targeting EGFR and VEGFR2 Is Effective against Triple Negative Breast Cancer via Multiple Mechanisms of Action.

Both EGFR and VEGFR2 frequently overexpress in TNBC and cooperate with each other in autocrine and paracrine manner to enhance tumor growth and angiogenesis. Therapeutic mAbs targeting EGFR (cetuximab) and VEGFR2 (ramucirumab) are approved by FDA for numerous cancer indications, but none of them are approved to treat breast cancers. TNBC cells secrete VEGF-A, which mediates angiogenesis on endothelial cells in a paracrine fashion, as well as promotes cancer cell growth in autocrine manner.

Trastuzumab Blocks the Receiver Function of HER2 Leading to the Population Shifts of HER2-Containing Homodimers and Heterodimers.

HER2, a member of the Erythroblastosis Protein B/Human Epidermal Growth Factor Receptor (ErbB/HER) family of receptor tyrosine kinase, is overexpressed in 20~30% of human breast cancers. Trastuzumab, a HER2-targeted therapeutic monoclonal antibody, was developed to interfere with the homodimerization of HER2 in HER2-overexpressing breast cancer cells, which attenuates HER2-mediated signaling. Trastuzumab binds to the domain IV of the HER2 extracellular domain and does not directly block the dimerization interface of HER2-HER2 molecules.

Identification of fidelity-governing factors in human recombinases DMC1 and RAD51 from cryo-EM structures.

Both high-fidelity and mismatch-tolerant recombination, catalyzed by RAD51 and DMC1 recombinases, respectively, are indispensable for genomic integrity. Here, we use cryo-EM, MD simulation and functional analysis to elucidate the structural basis for the mismatch tolerance of DMC1. Structural analysis of DMC1 presynaptic and postsynaptic complexes suggested that the lineage-specific Loop 1 Gln244 (Met243 in RAD51) may help stabilize DNA backbone, whereas Loop 2 Pro274 and Gly275 (Val273/Asp274 in RAD51) may provide an open "triplet gate" for mismatch tolerance.

Vibrio cholerae biofilm scaffolding protein RbmA shows an intrinsic, phosphate-dependent autoproteolysis activity.

Vibrio cholerae is the causative agent of the diarrheal disease cholera, for which biofilm communities are considered to be environmental reservoirs. In endemic regions, and after algal blooms, which may result from phosphate enrichment following agricultural runoff, the bacterium is released from biofilms resulting in seasonal disease outbreaks. However, the molecular mechanism by which V.

Cell proliferation and invasion are regulated differently by EGFR and MRP1 in T-DM1-resistant breast cancer cells.

We recently reported that T-DM1-resistant JIMT1 (T-DM1R-JIMT1) cells exhibited high invasive activity via EGFR and integrin cooperated pathways and gained cross-resistance to doxorubicin. Here, we show that EGFR positively coordinates with MRP1 in T-DM1R-JIMT1 cells to contribute to cross-resistance to doxorubicin. Downregulating EGFR and MRP1 inhibits T-DM1R-JIMT1 cell growth and re-sensitizes T-DM1R cells to doxorubicin, suggesting that dual targeting EGFR and MRP1 could serve as a therapeutic approach to overcome T-DM1 resistance.

Development of Novel Irreversible Pyruvate Kinase M2 Inhibitors.

As cancer cells undergo metabolic reprogramming in the course of tumorigenesis, targeting energy metabolism represents a promising strategy in cancer therapy. Among various metabolic enzymes examined, pyruvate kinase M2 type (PKM2) has received much attention in light of its multifaceted function in promoting tumor growth and progression. In this study, we reported the development of a novel irreversible inhibitor of PKM2, compound , that exhibits a differential tumor-suppressive effect among an array of cancer cell lines.

Atezolizumab potentiates Tcell-mediated cytotoxicity and coordinates with FAK to suppress cell invasion and motility in PD-L1 triple negative breast cancer cells.

Immune check point inhibitors targeting programmed cell death protein-1 (PD-1) and its ligand (PD-L1) have shown clinical success in treatment of human malignancies. Triple negative breast cancer (TNBC), which is primarily characterized by high heterogeneity and presence of tumor infiltrating lymphocytes, remains therapeutic challenge due to unavailability of approved targeted therapy. Therapeutic potential of immune check point inhibitors for TNBC patients is under active clinical investigation.

. 9°N DNA polymerase exhibits 3'-esterase activity that can be harnessed for DNA sequencing.

It was reported in 1995 that T7 and Taq DNA polymerases possess 3'-esterase activity, but without follow-up studies. Here we report that the 3'-esterase activity is intrinsic to the . 9°N DNA polymerase, and that it can be developed into a continuous method for DNA sequencing with dNTP analogs carrying a 3'-ester with a fluorophore.

Human DNA Polymerase μ Can Use a Noncanonical Mechanism for Multiple Mn-Mediated Functions.

Recent research on the structure and mechanism of DNA polymerases has continued to generate fundamentally important features, including a noncanonical pathway involving "prebinding" of metal-bound dNTP (MdNTP) in the absence of DNA. While this noncanonical mechanism was shown to be a possible subset for African swine fever DNA polymerase X (Pol X) and human Pol λ, it remains unknown whether it could be the primary pathway for a DNA polymerase. Pol μ is a unique member of the X-family with multiple functions and with unusual Mn preference.