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The combination of native electrospray ionization with top-down fragmentation in mass spectrometry (MS) allows simultaneous determination of the stoichiometry of noncovalent complexes and identification of their component proteoforms and cofactors. Although this approach is powerful, both native MS and top-down MS are not yet well standardized, and only a limited number of laboratories regularly carry out this type of research. To address this challenge, the Consortium for Top-Down Proteomics initiated a study to develop and test protocols for native MS combined with top-down fragmentation of proteins and protein complexes across 11 instruments in nine laboratories. Here we report the summary of the outcomes to provide robust benchmarks and a valuable entry point for the scientific community., Competing Interests: Competing interests: N.L.K. is involved in entrepreneurial activities in top-down proteomics and consults for ThermoFisher Scientific. P.O.D. is the founder and principal of Eastwoods Consulting, providing business advisory services to life science companies. The remaining authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

CDK4/6 inhibition in combination with endocrine therapy is the standard of care for estrogen receptor (ER+) breast cancer, and although cytostasis is frequently observed, new treatment strategies that enhance efficacy are required. Here, we perform two independent genome-wide CRISPR screens to identify genetic determinants of CDK4/6 and endocrine therapy sensitivity. Genes involved in oxidative stress and ferroptosis modulate sensitivity, with GPX4 as the top sensitiser in both screens. Depletion or inhibition of GPX4 increases sensitivity to palbociclib and giredestrant, and their combination, in ER+ breast cancer models, with GPX4 null xenografts being highly sensitive to palbociclib. GPX4 perturbation additionally sensitises triple negative breast cancer (TNBC) models to palbociclib. Palbociclib and giredestrant induced oxidative stress and disordered lipid metabolism, leading to a ferroptosis-sensitive state. Lipid peroxidation is promoted by a peroxisome AGPAT3-dependent pathway in ER+ breast cancer models, rather than the classical ACSL4 pathway. Our data demonstrate that CDK4/6 and ER inhibition creates vulnerability to ferroptosis induction, that could be exploited through combination with GPX4 inhibitors, to enhance sensitivity to the current therapies in breast cancer., (© 2024. The Author(s).)

BamA is the central component of the essential β-barrel assembly machine (BAM), a conserved multi-subunit complex that dynamically inserts and folds β-barrel proteins into the outer membrane of Gram-negative bacteria. Despite recent advances in our mechanistic and structural understanding of BamA, there are few potent and selective tool molecules that can bind to and modulate BamA activity. Here, we explored in vitro selection methods and different BamA/BAM protein formulations to discover peptide macrocycles that kill Escherichia coli by targeting extreme conformational states of BamA. Our studies show that Peptide Targeting BamA-1 (PTB1) targets an extracellular divalent cation-dependent binding site and locks BamA into a closed lateral gate conformation. By contrast, PTB2 targets a luminal binding site and traps BamA into an open lateral gate conformation. Our results will inform future antibiotic discovery efforts targeting BamA and provide a template to prospectively discover modulators of other dynamic integral membrane proteins., (© 2024. The Author(s).)

Human genetics and preclinical studies have identified key contributions of TREM2 to several neurodegenerative conditions, inspiring efforts to modulate TREM2 therapeutically. Here, we characterize the activities of three TREM2 agonist antibodies in multiple mixed-sex mouse models of Alzheimer's disease (AD) pathology and remyelination. Receptor activation and downstream signaling are explored in vitro, and active dose ranges are determined in vivo based on pharmacodynamic responses from microglia. For mice bearing amyloid-β (Aβ) pathology (PS2APP) or combined Aβ and tau pathology (TauPS2APP), chronic TREM2 agonist antibody treatment had limited impact on microglia engagement with pathology, overall pathology burden, or downstream neuronal damage. For mice with demyelinating injuries triggered acutely with lysolecithin, TREM2 agonist antibodies unexpectedly disrupted injury resolution. Likewise, TREM2 agonist antibodies limited myelin recovery for mice experiencing chronic demyelination from cuprizone. We highlight the contributions of dose timing and frequency across models. These results introduce important considerations for future TREM2-targeting approaches., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

Introduction: Hearing health is a public health concern that affects the quality of life and can be disturbed by noise exposure, generating auditory and extra-auditory symptoms. Objective. To identify the hearing health status in adults living in Bogotá and its association with environmental noise exposure and individual and otological factors., Objective: To identify the hearing health status in adults living in Bogotá and its association with environmental noise exposure and individual and otological factors., Materials and Methods: We conducted a cross-sectional study using a database with 10,311 records from 2014 to 2018, consigned in a structured survey of noise perception and hearing screening. We performed a descriptive, bivariate, and binary logistic regression analysis., Results: Of the included participants, 35.4% presented hearing impairment. In the perception component, 13.0 % reported not hearing well; 28.8 % had extra-auditory symptoms, 53.3 % informed otological antecedents and 69.0 % presented discomfort due to extramural noise. In the logistic regression, the variables with the highest association for hearing impairment were living in noisy areas (OR = 1.50) (95% CI: 1.34-1.69), being male (OR = 1.85) (95% CI: 1.64-2.09), increasing age (for each year of life, the risk of hearing impairment increased 6%), and having history of extra-auditory symptoms (OR = 1.86) (95% CI: 1.66-2.08)., Conclusions: Hearing impairment is multi-causal in the studied population. The factors that promote its prevalence are increasing age, being male, smoking, ototoxic medications, living in areas with high noise exposure, and extra-auditory symptoms.

Ferritin is a multivalent, self-assembling protein scaffold found in most human cell types, in addition to being present in invertebrates, higher plants, fungi, and bacteria, that offers an attractive alternative to polymer-based drug delivery systems (DDS). In this study, the utility of the ferritin cage as a DDS was demonstrated within the context of T cell agonism for tumor killing. Members of the tumor necrosis factor receptor superfamily (TNFRSF) are attractive targets for the development of anticancer therapeutics. These receptors are endogenously activated by trimeric ligands that occur in transmembrane or soluble forms, and oligomerization and cell-surface anchoring have been shown to be essential aspects of the targeted agonism of this receptor class. Here, we demonstrated that the ferritin cage could be easily tailored for multivalent display of anti-OX40 antibody fragments on its surface and determined that these arrays are capable of pathway activation through cell-surface clustering. Together, these results confirm the utility, versatility, and developability of ferritin as a DDS.

The heterogeneity inherent in today's biotherapeutics, especially as a result of heavy glycosylation, can affect a molecule's safety and efficacy. Characterizing this heterogeneity is crucial for drug development and quality assessment, but existing methods are limited in their ability to analyze intact glycoproteins or other heterogeneous biotherapeutics. Here, we present an approach to the molecular assessment of biotherapeutics that uses proton-transfer charge-reduction with gas-phase fractionation to analyze intact heterogeneous and/or glycosylated proteins by mass spectrometry. The method provides a detailed landscape of the intact molecular weights present in biotherapeutic protein preparations in a single experiment. For glycoproteins in particular, the method may offer insights into glycan composition when coupled with a suitable bioinformatic strategy. We tested the approach on various biotherapeutic molecules, including Fc-fusion, VHH-fusion, and peptide-bound MHC class II complexes to demonstrate efficacy in measuring the proteoform-level diversity of biotherapeutics. Notably, we inferred the glycoform distribution for hundreds of molecular weights for the eight-times glycosylated fusion drug IL22-Fc, enabling correlations between glycoform sub-populations and the drug's pharmacological properties. Our method is broadly applicable and provides a powerful tool to assess the molecular heterogeneity of emerging biotherapeutics., (© 2024. The Author(s).)

Heterozygous mutations in the granulin ( GRN ) gene are a leading cause of frontotemporal lobar degeneration with TDP-43 aggregates (FTLD-TDP). Polymorphisms in TMEM106B have been associated with disease risk in GRN mutation carriers and protective TMEM106B variants associated with reduced levels of TMEM106B, suggesting that lowering TMEM106B might be therapeutic in the context of FTLD. Here, we tested the impact of full deletion and partial reduction of TMEM106B in mouse and iPSC-derived human cell models of GRN deficiency. TMEM106B deletion did not reverse transcriptomic or proteomic profiles in GRN-deficient microglia, with a few exceptions in immune signaling markers. Neither homozygous nor heterozygous Tmem106b deletion normalized disease-associated phenotypes in Grn -/- mice. Furthermore, Tmem106b reduction by antisense oligonucleotide (ASO) was poorly tolerated in Grn -/- mice. These data provide novel insight into TMEM106B and GRN function in microglia cells but do not support lowering TMEM106B levels as a viable therapeutic strategy for treating FTD- GRN ., Competing Interests: The authors have a published patent application WO 2019/154979 A1 related to this work., (© 2023 The Authors.)

During the course of biopharmaceutical production, heterologous protein expression in Chinese hamster ovary (CHO) cells imposes a high proteostatic burden that requires cellular adaptation. To mitigate such burden, cells utilize the unfolded protein response (UPR), which increases endoplasmic reticulum (ER) capacity to accommodate elevated rates of protein synthesis and folding. In this study, we show that during production the UPR regulates growth factor signaling to modulate growth and protein synthesis. Specifically, the protein kinase R-like ER kinase (PERK) branch of the UPR is responsible for transcriptional down-regulation of platelet-derived growth factor receptor alpha (PDGFRa) and attenuation of the IRE1-alpha (IRE1a) branch of the UPR. PERK knockout (KO) cell lines displayed reduced growth and viability due to higher rates of apoptosis despite having stabilized PDGFRa levels. Knocking out PERK in an apoptosis impaired (Bax/Bak double KO) antibody-expressing cell line prevented apoptotic cell death and revealed that apoptosis was likely triggered by increased ER stress and reactive oxygen species levels in the PERK KO hosts. Our findings suggest that attenuation of IRE1a and PDGFRa signaling by the PERK branch of the UPR reduces ER protein folding capacity and hence specific productivity of CHO cells in order to mitigate UPR and prevent apoptotic cell death. Last, Bax/Bak/PERK triple KO CHO cell lines displayed 2-3 folds higher specific productivity and titer (up to 8 g/L), suggesting that modulation of PERK signaling during production processes can greatly improve specific productivity in CHO cells., (© 2023 American Institute of Chemical Engineers.)

TGFβ signaling is associated with non-response to immune checkpoint blockade in patients with advanced cancers, particularly in the immune-excluded phenotype. While previous work demonstrates that converting tumors from excluded to inflamed phenotypes requires attenuation of PD-L1 and TGFβ signaling, the underlying cellular mechanisms remain unclear. Here, we show that TGFβ and PD-L1 restrain intratumoral stem cell-like CD8 T cell (T SCL ) expansion and replacement of progenitor-exhausted and dysfunctional CD8 T cells with non-exhausted T effector cells in the EMT6 tumor model in female mice. Upon combined TGFβ/PD-L1 blockade IFNγ hi CD8 T effector cells show enhanced motility and accumulate in the tumor. Ensuing IFNγ signaling transforms myeloid, stromal, and tumor niches to yield an immune-supportive ecosystem. Blocking IFNγ abolishes the anti-PD-L1/anti-TGFβ therapy efficacy. Our data suggest that TGFβ works with PD-L1 to prevent T SCL expansion and replacement of exhausted CD8 T cells, thereby maintaining the T cell compartment in a dysfunctional state., (© 2023. Springer Nature Limited.)

Recent advances in native mass spectrometry (MS) and denatured intact protein MS have made these techniques essential for biotherapeutic characterization. As MS analysis has increased in throughput and scale, new data analysis workflows are needed to provide rapid quantitation from large datasets. Here, we describe the UniDec processing pipeline (UPP) for the analysis of batched biotherapeutic intact MS data. UPP is built into the UniDec software package, which provides fast processing, deconvolution, and peak detection. The user and programming interfaces for UPP read a spreadsheet that contains the data file names, deconvolution parameters, and quantitation settings. After iterating through the spreadsheet and analyzing each file, it returns a spreadsheet of results and HTML reports. We demonstrate the use of UPP to measure the correct pairing percentage on a set of bispecific antibody data and to measure drug-to-antibody ratios from antibody-drug conjugates. Moreover, because the software is free and open-source, users can easily build on this platform to create customized workflows and calculations. Thus, UPP provides a flexible workflow that can be deployed in diverse settings and for a wide range of biotherapeutic applications.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with significant mortality. Prognostic biomarkers to identify rapid progressors are urgently needed to improve patient management. Since the lysophosphatidic acid (LPA) pathway has been implicated in lung fibrosis in preclinical models and identified as a potential therapeutic target, we aimed to investigate if bioactive lipid LPA species could be prognostic biomarkers that predict IPF disease progression. LPAs and lipidomics were measured in baseline placebo plasma of a randomized IPF-controlled trial. The association of lipids with disease progression indices were assessed using statistical models. Compared to healthy, IPF patients had significantly higher levels of five LPAs (LPA16:0, 16:1, 18:1, 18:2, 20:4) and reduced levels of two triglycerides species (TAG48:4-FA12:0, -FA18:2) (false discovery rate < 0.05, fold change > 2). Patients with higher levels of LPAs had greater declines in diffusion capacity of carbon monoxide over 52 weeks (P < 0.01); additionally, LPA20:4-high (≥median) patients had earlier time to exacerbation compared to LPA20:4-low (

Idiopathic pulmonary fibrosis is a progressive fibrotic disease characterized by excessive deposition of (myo)fibroblast produced collagen fibrils in alveolar areas of the lung. Lysyl oxidases (LOXs) have been proposed to be the central enzymes that catalyze the cross-linking of collagen fibers. Here, we report that, while its expression is increased in fibrotic lungs, genetic ablation of LOXL2 only leads to a modest reduction of pathological collagen cross-linking but not fibrosis in the lung. On the other hand, loss of another LOX family member, LOXL4, markedly disrupts pathological collagen cross-linking and fibrosis in the lung. Furthermore, knockout of both Loxl2 and Loxl4 does not offer any additive antifibrotic effects when compared to Loxl4 deletion only, as LOXL4 deficiency decreases the expression of other LOX family members including Loxl2 . On the basis of these results, we propose that LOXL4 is the main LOX activity underlying pathological collagen cross-linking and lung fibrosis.

Immune monitoring in cancer immunotherapy involves screening CD8+ T-cell responses against neoantigens, the tumor-specific peptides presented by Major histocompatibility complex Class I (MHCI) on the cell surface. High-throughput immune monitoring requires methods to produce and characterize small quantities of thousands of MHCI-peptide complexes that may be tested for a patient's T-cell response. MHCI synthesis has been achieved using a photocleavable peptide that is exchanged by the neoantigen; however, assays that measure peptide exchange currently disassemble the complex prior to analysis─precluding direct molecular characterization. Here, we use native mass spectrometry (MS) to profile intact recombinant MHCI complexes and directly measure peptide exchange. Coupled with size-exclusion chromatography or capillary-zone electrophoresis, the assay identified all tested human leukocyte antigen (HLA)/peptide combinations in the nanomole to picomole range with minimal run time, reconciling the synthetic and analytical requirements of MHCI-peptide screening with the downstream T-cell assays. We further show that the assay can be "multiplexed" by measuring exchange of multiple peptides simultaneously and also enables calculation of Vc 50 , a measure of gas-phase stability. Additionally, MHCI complexes were fragmented by top-down sequencing, demonstrating that the intact complex, peptide sequence, and their binding affinity can be determined in a single analysis. This screening tool for MHCI-neoantigen complexes represents a step toward the application of state-of-the-art MS technology in translational settings. Not only is this assay already informing on the viability of immunotherapy in practice, the platform also holds promise to inspire novel MS readouts for increasingly complex biomolecules used in the diagnosis and treatment of disease.

Acinetobacter baumannii is a clinically important, predominantly health care-associated gram-negative bacterium with high rates of emerging resistance worldwide. Given the urgent need for novel antibacterial therapies against A. baumannii , we focused on inhibiting lipoprotein biosynthesis, a pathway that is essential for envelope biogenesis in gram-negative bacteria. The natural product globomycin, which inhibits the essential type II signal peptidase prolipoprotein signal peptidase (LspA), is ineffective against wild-type A. baumannii clinical isolates due to its poor penetration through the outer membrane. Here, we describe a globomycin analog, G5132, that is more potent against wild-type and clinical A. baumannii isolates. Mutations leading to G5132 resistance in A. baumannii map to the signal peptide of a single hypothetical gene, which we confirm encodes an alanine-rich lipoprotein and have renamed lirL (prolipoprotein signal peptidase inhibitor resistance lipoprotein). LirL is a highly abundant lipoprotein primarily localized to the inner membrane. Deletion of lirL leads to G5132 resistance, inefficient cell division, increased sensitivity to serum, and attenuated virulence. Signal peptide mutations that confer resistance to G5132 lead to the accumulation of diacylglyceryl-modified LirL prolipoprotein in untreated cells without significant loss in cell viability, suggesting that these mutations overcome a block in lipoprotein biosynthetic flux by decreasing LirL prolipoprotein substrate sensitivity to processing by LspA. This study characterizes a lipoprotein that plays a critical role in resistance to LspA inhibitors and validates lipoprotein biosynthesis as a antibacterial target in A. baumannii .

Mitogen-activated protein kinases (MAPKs) drive key signaling cascades during neuronal survival and degeneration. The localization of kinases to specific subcellular compartments is a critical mechanism to locally control signaling activity and specificity upon stimulation. However, how MAPK signaling components tightly control their localization remains largely unknown. Here, we systematically analyzed the phosphorylation and membrane localization of all MAPKs expressed in dorsal root ganglia (DRG) neurons, under control and stress conditions. We found that MAP3K12/dual leucine zipper kinase (DLK) becomes phosphorylated and palmitoylated, and it is recruited to sphingomyelin-rich vesicles upon stress. Stress-induced DLK vesicle recruitment is essential for kinase activation; blocking DLK-membrane interaction inhibits downstream signaling, while DLK recruitment to ectopic subcellular structures is sufficient to induce kinase activation. We show that the localization of DLK to newly formed vesicles is essential for local signaling. Inhibition of membrane internalization blocks DLK activation and protects against neurodegeneration in DRG neurons. These data establish vesicular assemblies as dynamically regulated platforms for DLK signaling during neuronal stress responses., (© 2022 Genentech Inc. Published under the terms of the CC BY NC ND 4.0 license.)

Bone morphogenetic protein 1 (BMP1) belongs to the astacin/BMP1/tolloid-like family of zinc metalloproteinases, which play a fundamental role in the development and formation of extracellular matrix (ECM). BMP1 mediates the cleavage of carboxyl terminal (C-term) propeptides from procollagens, a crucial step in fibrillar collagen fiber formation. Blocking BMP1 by small molecule or antibody inhibitors has been linked to anti-fibrotic activity in the preclinical models of skin, kidney and liver fibrosis. Therefore, we reason that BMP1 may be important for the pathogenesis of lung fibrosis and BMP1 could be a potential therapeutic target for progressive fibrotic disease such as idiopathic pulmonary fibrosis (IPF). Here, we observed the increased expression of BMP1 in both human IPF lungs and mouse fibrotic lungs induced by bleomycin. Furthermore, we developed an inducible Bmp1 conditional knockout (cKO) mouse strain. We found that Bmp1 deletion does not protect mice from lung fibrosis triggered by bleomycin. Moreover, we found no significant impact of BMP1 deficiency upon C-term propeptide of type I procollagen (CICP) production in the fibrotic mouse lungs. Based on these results, we propose that BMP1 is not required for lung fibrosis in mice and BMP1 may not be considered a candidate therapeutic target for IPF., (© 2022. The Author(s).)

Partial reprogramming by expression of reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) for short periods of time restores a youthful epigenetic signature to aging cells and extends the life span of a premature aging mouse model. However, the effects of longer-term partial reprogramming in physiologically aging wild-type mice are unknown. Here, we performed various long-term partial reprogramming regimens, including different onset timings, during physiological aging. Long-term partial reprogramming lead to rejuvenating effects in different tissues, such as the kidney and skin, and at the organismal level; duration of the treatment determined the extent of the beneficial effects. The rejuvenating effects were associated with a reversion of the epigenetic clock and metabolic and transcriptomic changes, including reduced expression of genes involved in the inflammation, senescence and stress response pathways. Overall, our observations indicate that partial reprogramming protocols can be designed to be safe and effective in preventing age-related physiological changes. We further conclude that longer-term partial reprogramming regimens are more effective in delaying aging phenotypes than short-term reprogramming., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Detection of host cell protein (HCP) impurities is critical to ensuring that recombinant drug products, including monoclonal antibodies (mAbs), are safe. Mechanistic characterization as to how HCPs persist in drug products is important to refining downstream processing. It has been hypothesized that weak lipase-mAb interactions enable HCP lipases to evade drug purification processes. Here, we apply state-of-the-art methods to establish lipase-mAb binding mechanisms. First, the mass spectrometry (MS) approach of fast photochemical oxidation of proteins was used to elucidate putative binding regions. The CH1 domain was identified as a conserved interaction site for IgG1 and IgG4 mAbs against the HCPs phospholipase B-like protein (PLBL2) and lysosomal phospholipase A2 (LPLA2). Rationally designed mutations in the CH1 domain of the IgG4 mAb caused a 3- to 70-fold K D reduction against PLBL2 by surface plasmon resonance (SPR). LPLA2-IgG4 mutant complexes, undetected by SPR and studied using native MS collisional dissociation experiments, also showed significant complex disruption, from 16% to 100%. Native MS and ion mobility (IM) determined complex stoichiometries for four lipase-IgG4 complexes and directly interrogated the enrichment of specific lipase glycoforms. Confirmed with time-course and exoglycosidase experiments, deglycosylated lipases prevented binding, and low-molecular-weight glycoforms promoted binding, to mAbs. This work demonstrates the value of integrated biophysical approaches to characterize micromolar affinity complexes. It is the first in-depth structural report of lipase-mAb binding, finding roles for the CH1 domain and lipase glycosylation in mediating binding. The structural insights gained offer new approaches for the bioengineering of cells or mAbs to reduce HCP impurity levels. Abbreviations: CAN, Acetonitrile; AMAC, Ammonium acetate; BFGS, Broyden-Fletcher-Goldfarb-Shanno; CHO, Chinese Hamster Ovary; K D , Dissociation constant; DTT, Dithiothreitol; ELISA, Enzyme-linked immunosorbent assay; FPOP, Fast photochemical oxidation of proteins; FA, Formic acid; F(ab'), Fragment antibodies; HCP, Host cell protein; IgG, Immunoglobulin; IM, Ion mobility; LOD, Lower limit of detection; LPLA2, Lysosomal phospholipase A2; Man, Mannose; MS, Mass spectrometry; MeOH, Methanol; MST, Microscale thermophoresis; mAbs, Monoclonal antibodies; PPT1, Palmitoyl protein thioesterase; ppm, Parts per million; PLBL2, Phospholipase B-like protein; PLD3, Phospholipase D3; PS-20, Polysorbate-20; SP, Sphingomyelin phosphodiesterase; SPR, Surface plasmon resonance; TFA, Trifluoroacetic acid.

Background: Chronic obstructive pulmonary disease (COPD) exacerbations are heterogenous and profoundly impact the disease trajectory. Bioactive lipid lysophosphatidic acid (LPA) has been implicated in airway inflammation but the significance of LPA in COPD exacerbation is not known. The aim of the study was to investigate the utility of serum LPA species (LPA16:0, 18:0, 18:1, 18:2, 20:4) as biomarkers of COPD exacerbation., Patients and Methods: LPA species were measured in the baseline placebo sera of a COPD randomized controlled trial. Tertile levels of each LPA were used to assign patients into biomarker high, medium, and low subgroups. Exacerbation rate and risk were compared among the LPA subgroups., Results: The levels of LPA species were intercorrelated (rho 0.29-0.91). Patients with low and medium levels of LPA (LPA16:0, 20:4) had significantly higher exacerbation rate compared to the respective LPA-high patients [estimated rate per patient per year (95% CI)]: LPA16:0-low = 1.2 (0.8-1.9) (p = 0.019), LPA16:0-medium = 1.3 (0.8-2.0) (p = 0.013), LPA16:0-high = 0.5 (0.2-0.9); LPA20:4-low = 1.4 (0.9-2.1) (p = 0.0033), LPA20:4-medium = 1.2 (0.8-1.8) (p = 0.0089), LPA20:4-high = 0.4 (0.2-0.8). These patients also had earlier time to first exacerbation (hazard ratio (95% CI): LPA16:0-low = 2.6 (1.1-6.0) (p = 0.028), LPA16:0-medium = 2.7 (1.2-6.3) (p = 0.020); LPA20.4-low = 2.8 (1.2-6.6) (p = 0.017), LPA20:4-medium = 2.7 (1.2-6.4) (p = 0.021). Accordingly, these patients had a significant increased exacerbation risk compared to the respective LPA-high subgroups [odd ratio (95% CI)]: LPA16:0-low = 3.1 (1.1-8.8) (p = 0.030), LPA16:0-medium = 3.0 (1.1-8.3) (p = 0.031); LPA20:4-low = 3.8 (1.3-10.9) (p = 0.012), LPA20:4-medium = 3.3 (1.2-9.5) (p = 0.025). For the other LPA species (LPA18:0, 18:1, 18:2), the results were mixed; patients with low and medium levels of LPA18:0 and 18:2 had increased exacerbation rate, but only LPA18:0-low patients had significant increase in exacerbation risk and earlier time to first exacerbation compared to the LPA18:0-high subgroup., Conclusions: The study provided evidence of association between systemic LPA levels and exacerbation in COPD. Patients with low and medium levels of specific LPA species (LPA16:0, 20:4) had increased exacerbation rate, risk, and earlier time to first exacerbation. These non-invasive biomarkers may aid in identifying high risk patients with dysregulated LPA pathway to inform risk management and drug development., (© 2021. The Author(s).)

Deletion of the pyruvate kinase muscle (PKM) gene, which is involved in conversion of phosphoenolpyruvate to pyruvate, has been shown to curb lactogenic behavior in Chinese hamster ovary (CHO) cells. This study describes the generation of pyruvate kinase muscle isoforms 1 and 2 knockout (PKM-KO) and pyruvate kinase muscle isoform-1 knockout (PKM1-KO) CHO host cells to understand metabolic shifts that reduce lactate secretion in these cells. Glucose and amino acids uptake levels in wild-type (WT), PKM-KO, and PKM1-KO stable cell lines, expressing two different antibodies, were analyzed in 14-day fed-batch production assays using different vessels. PKM-KO and PKM1-KO cells consumed more glucose per cell, altered amino acids metabolism, had higher flux of pyruvate into the tricarboxylic acid (TCA) cycle, and as previously shown reduced lactate secretion levels compared with the WT cells. Additionally, both PKM-KO and PKM1-KO cells had higher specific productivity and lower cell growth rates compared with the WT cells. Our findings suggest that rewiring the flux of pyruvate to the TCA cycle by deletion of PKM or PKM1 reduced cell growth and increased specific productivity in CHO cells. Overall, PKM1-KO cells had similar product quality and comparable or better titers relative to the WT cells, hence, targeted deletion of this isoform for curbing lactogenic behavior in CHO cells is suggested., (© 2021 American Institute of Chemical Engineers.)

Mass spectrometry is gaining momentum as a method of choice to de novo sequence antibodies (Abs). Adequate sequence coverage of the hypervariable regions remains one of the toughest identification challenges by either bottom-up or top-down workflows. Methods that efficiently generate mid-size Ab fragments would further facilitate top-down MS and decrease data complexity. Here, we explore the proteases Cathepsins L and D for forming protein fragments from three IgG1s, one IgG2, and one bispecific, knob-and-hole IgG1. We demonstrate that high-resolution native MS provides a sensitive method for the detection of clipping sites. Both Cathepsins produced multiple, albeit specific cleavages. The Abs were cleaved immediately after the CDR3 region, yielding ~ 12 kDa fragments, that is, ideal sequencing-sized. Cathepsin D, but not Cathepsin L, also cleaved directly below the Ab hinge, releasing the F(ab')2. When constrained by the different disulfide bonds found in the IgG2 subtype or by the tertiary structure of the hole-containing bispecific IgG1, the hinge region digest product was not produced. The Cathepsin L and Cathepsin D clipping motifs were related to sequences of neutral amino acids and the tertiary structure of the Ab. A single pot (L + D) digestion protocol was optimized to achieve 100% efficiency. Nine protein fragments, corresponding to the VL, VH, CL, CH1, CH2, CH3, CL + CH1, and F(ab')2, constituted ~ 70% of the summed intensities of all deconvolved proteolytic products. Cleavage sites were confirmed by the Edman degradation and validated with top-down sequencing. The described work offers a complementary method for middle-down analysis that may be applied to top-down Ab sequencing. ENZYMES: Cathepsin L-EC 3.4.22.15, Cathepsin D-EC 3.4.23.5., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Mass spectrometry (MS) plays a key role throughout all stages of drug development and is now as ubiquitous as other analytical techniques such as surface plasmon resonance, nuclear magnetic resonance, and supercritical fluid chromatography, among others. Herein, we aim to discuss the history of MS, both electrospray and matrix-assisted laser desorption ionization, specifically for the analysis of antibodies, evolving through to denaturing and native-MS analysis of newer biologic moieties such as antibody-drug conjugates, multispecific antibodies, and interfering nucleic acid-based therapies. We discuss challenging therapeutic target characterization such as membrane protein receptors. Importantly, we compare and contrast the MS and hyphenated analytical chromatographic methods used to characterize these therapeutic modalities and targets within biopharmaceutical research and highlight the importance of appropriate MS deconvolution software and its essential contribution to project progression. Finally, we describe emerging applications and MS technologies that are still predominantly within either a development or academic stage of use but are poised to have significant impact on future drug development within the biopharmaceutic industry once matured. The views reflected herein are personal and are not meant to be an exhaustive list of all relevant MS performed within biopharmaceutical research but are what we feel have been historically, are currently, and will be in the future the most impactful for the drug development process.

Lysophospholipids are bioactive signaling molecules derived from cell membrane glycerophospholipids or sphingolipids and are highly regulated under normal physiological conditions. Lysophosphatidic acids (LPAs) are a class of lysophospholipids that act on G-protein-coupled receptors to exert a variety of cellular functions. Dysregulation of phospholipase activity and consequently LPA synthesis in serum have been linked to inflammation, such as seen in chronic obstructive pulmonary disease (COPD). The accurate measurement of phospholipids is critical for evaluating their dysregulation in disease. In this study, we optimized experimental parameters for the sensitive measurement of LPAs. We validated the method based on matrix, linearity, accuracy, precision, and stability. An investigation into sample extraction processes emphasized that the common practice of including low concentration of hydrochloric acid in the extraction buffer causes an overestimation of lipid recovery. The liquid chromatography gradient was optimized to separate various lysophospholipid classes. After optimization, detection limits of LPA were sufficiently sensitive for subsequent analysis, ranging from 2 to 8 nM. The validated workflow was applied to a cohort of healthy donor and COPD patient sera. Eight LPA species were identified, and five unique species of LPA were quantified. Most LPA species increased significantly in COPD patients compared to healthy donors. The correlation between LPAs and other demographic parameters was further investigated in a sample set of over 200 baseline patient sera from a COPD clinical trial. For the first time, LPAs other than the two most abundant and readily detectable moieties are quantified in COPD patients using validated methods, opening the door to downstream biomarker evaluation in respiratory disease.

Lipoprotein diacylglyceryl transferase (Lgt) catalyzes the first step in the biogenesis of Gram-negative bacterial lipoproteins which play crucial roles in bacterial growth and pathogenesis. We demonstrate that Lgt depletion in a clinical uropathogenic Escherichia coli strain leads to permeabilization of the outer membrane and increased sensitivity to serum killing and antibiotics. Importantly, we identify G2824 as the first-described Lgt inhibitor that potently inhibits Lgt biochemical activity in vitro and is bactericidal against wild-type Acinetobacter baumannii and E. coli strains. While deletion of a gene encoding a major outer membrane lipoprotein, lpp , leads to rescue of bacterial growth after genetic depletion or pharmacologic inhibition of the downstream type II signal peptidase, LspA, no such rescue of growth is detected after Lgt depletion or treatment with G2824. Inhibition of Lgt does not lead to significant accumulation of peptidoglycan-linked Lpp in the inner membrane. Our data validate Lgt as a novel antibacterial target and suggest that, unlike downstream steps in lipoprotein biosynthesis and transport, inhibition of Lgt may not be sensitive to one of the most common resistance mechanisms that invalidate inhibitors of bacterial lipoprotein biosynthesis and transport. IMPORTANCE As the emerging threat of multidrug-resistant (MDR) bacteria continues to increase, no new classes of antibiotics have been discovered in the last 50 years. While previous attempts to inhibit the lipoprotein biosynthetic (LspA) or transport (LolCDE) pathways have been made, most efforts have been hindered by the emergence of a common mechanism leading to resistance, namely, the deletion of the gene encoding a major Gram-negative outer membrane lipoprotein lpp . Our unexpected finding that inhibition of Lgt is not susceptible to lpp deletion-mediated resistance uncovers the complexity of bacterial lipoprotein biogenesis and the corresponding enzymes involved in this essential outer membrane biogenesis pathway and potentially points to new antibacterial targets in this pathway.

Fibroblastic reticular cells (FRCs) are specialized stromal cells that define tissue architecture and regulate lymphocyte compartmentalization, homeostasis, and innate and adaptive immunity in secondary lymphoid organs (SLOs). In the present study, we used single-cell RNA sequencing (scRNA-seq) of human and mouse lymph nodes (LNs) to identify a subset of T cell-zone FRCs defined by the expression of Gremlin1 (Grem1) in both species. Grem1-CreER T2 knock-in mice enabled localization, multi-omics characterization and genetic depletion of Grem1 + FRCs. Grem1 + FRCs primarily localize at T-B cell junctions of SLOs, neighboring pre-dendritic cells and conventional dendritic cells (cDCs). As such, their depletion resulted in preferential loss and decreased homeostatic proliferation and survival of resident cDCs and compromised T cell immunity. Trajectory analysis of human LN scRNA-seq data revealed expression similarities to murine FRCs, with GREM1 + cells marking the endpoint of both trajectories. These findings illuminate a new Grem1 + fibroblastic niche in LNs that functions to maintain the homeostasis of lymphoid tissue-resident cDCs.

Mutations in the GBA1 gene encoding glucocerebrosidase (GCase) are linked to Gaucher (GD) and Parkinson's Disease (PD). Since some GD and PD patients develop ocular phenotypes, we determined whether ocular phenotypes might result from impaired GCase activity and the corresponding accumulation of glucosylceramide (GluCer) and glucosylsphingosine (GluSph) in the Gba1 D409V/D409V knock-in (Gba KI/KI; "KI") mouse. Gba KI mice developed age-dependent pupil dilation deficits to an anti-muscarinic agent; histologically, the iris covered the anterior part of the lens with adhesions between the iris and the anterior surface of the lens (posterior synechia). This may prevent pupil dilation in general, beyond an un-responsiveness of the iris to anti-muscarinics. Gba KI mice displayed atrophy and pigment dispersion of the iris, and occlusion of the iridocorneal angle by pigment-laden cells, reminiscent of secondary open angle glaucoma. Gba KI mice showed progressive thinning of the retina consistent with retinal degeneration. GluSph levels were increased in the anterior and posterior segments of the eye, suggesting that accumulation of lipids in the eye may contribute to degeneration in this compartment. We conclude that the Gba KI model provides robust and reproducible eye phenotypes which may be used to test for efficacy and establish biomarkers for GBA1-related therapies.

Plasma membrane rupture (PMR) is the final cataclysmic event in lytic cell death. PMR releases intracellular molecules known as damage-associated molecular patterns (DAMPs) that propagate the inflammatory response 1-3 . The underlying mechanism of PMR, however, is unknown. Here we show that the cell-surface NINJ1 protein 4-8 , which contains two transmembrane regions, has an essential role in the induction of PMR. A forward-genetic screen of randomly mutagenized mice linked NINJ1 to PMR. Ninj1 -/- macrophages exhibited impaired PMR in response to diverse inducers of pyroptotic, necrotic and apoptotic cell death, and were unable to release numerous intracellular proteins including HMGB1 (a known DAMP) and LDH (a standard measure of PMR). Ninj1 -/- macrophages died, but with a distinctive and persistent ballooned morphology, attributable to defective disintegration of bubble-like herniations. Ninj1 -/- mice were more susceptible than wild-type mice to infection with Citrobacter rodentium, which suggests a role for PMR in anti-bacterial host defence. Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. The discovery of NINJ1 as a mediator of PMR overturns the long-held idea that cell death-related PMR is a passive event.

Fc galactosylation is a critical quality attribute for anti-tumor recombinant immunoglobulin G (IgG)-based monoclonal antibody (mAb) therapeutics with complement-dependent cytotoxicity (CDC) as the mechanism of action. Although the correlation between galactosylation and CDC has been known, the underlying structure-function relationship is unclear. Heterogeneity of the Fc N-glycosylation produced by Chinese hamster ovary (CHO) cell culture biomanufacturing process leads to variable CDC potency. Here, we derived a kinetic model of galactose transfer reaction in the Golgi apparatus and used this model to determine the correlation between differently galactosylated species from CHO cell culture process. The model was validated by a retrospective data analysis of more than 800 historical samples from small-scale and large-scale CHO cell cultures. Furthermore, using various analytical technologies, we discovered the molecular basis for Fc glycan terminal galactosylation changing the three-dimensional conformation of the Fc, which facilitates the IgG1 hexamerization, thus enhancing C1q avidity and subsequent complement activation. Our study offers insight into the formation of galactosylated species, as well as a novel three-dimensional understanding of the structure-function relationship of terminal galactose to complement activation in mAb therapeutics.

The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product's primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information on the common TD/MD MS methods and practices in the field. A panel of three mAbs was selected and centrally provided to 20 laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). Various MS instrument platforms and ion dissociation techniques were employed. The present study confirms that TD/MD MS tools are available in laboratories worldwide and provide complementary information to the BU approach that can be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. A primary limitation revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS.

Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function 1,2 . LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics 3,4 . Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains 5 , our understanding of the bacterial factors that regulate LPS biogenesis is incomplete. Here we characterize the inner membrane protein PbgA and report that its depletion attenuates the virulence of Escherichia coli by reducing levels of LPS and outer membrane integrity. In contrast to previous claims that PbgA functions as a cardiolipin transporter 6-9 , our structural analyses and physiological studies identify a lipid A-binding motif along the periplasmic leaflet of the inner membrane. Synthetic PbgA-derived peptides selectively bind to LPS in vitro and inhibit the growth of diverse Gram-negative bacteria, including polymyxin-resistant strains. Proteomic, genetic and pharmacological experiments uncover a model in which direct periplasmic sensing of LPS by PbgA coordinates the biosynthesis of lipid A by regulating the stability of LpxC, a key cytoplasmic biosynthetic enzyme 10-12 . In summary, we find that PbgA has an unexpected but essential role in the regulation of LPS biogenesis, presents a new structural basis for the selective recognition of lipids, and provides opportunities for future antibiotic discovery.

Accumulation of unfolded antibody chains in the ER triggers ER stress that may lead to reduced productivity in therapeutic antibody manufacturing processes. We identified UBR4 and UBR5 as ubiquitin E3 ligases involved in HC ER-associated degradation. Knockdown of UBR4 and UBR5 resulted in intracellular accumulation, enhanced secretion, and reduced ubiquitination of HC. In concert with these E3 ligases, PDIA3 was shown to cleave ubiquitinated HC molecules to accelerate HC dislocation. Interestingly, UBR5, and to a lesser degree UBR4, were down-regulated as cellular demand for antibody expression increased in CHO cells during the production phase, or in plasma B cells. Reducing UBR4/UBR5 expression before the production phase increased antibody productivity in CHO cells, possibly by redirecting antibody molecules from degradation to secretion. Altogether we have characterized a novel proteolysis/proteasome-dependent pathway involved in degradation of unfolded antibody HC. Proteins characterized in this pathway may be novel targets for CHO cell engineering., (© 2020 Genentech, Inc.)

The data supplied in this work are related to the research article entitled "Characterization of Bispecific and Mispaired IgGs by Native Charge-Variant Mass Spectrometry" (Phung et al., 2019). This data article describes a powerful analytical platform using native weak cation exchange chromatography coupled to a high-resolution mass spectrometer, charge variant mass spectrometry (CV-MS), to characterize bispecific and mispaired antibody species. Elution order is investigated through analytical methods and molecular modeling in an effort to understand the intrinsic charge, size and shape differences of these molecules., Competing Interests: WP, SP, MD, WS, PL, BW, DF, CS, PJC, JRL and WS are current employees of Genentech, Inc., which develops and markets drugs for profit. GH and PS were employees for Genentech at the time of these experiments. AOB is a former employee of Thermo Fisher Scientific, the company who manufactures and markets the chromatography and mass spectrometry instrumentation used., (© 2020 The Author(s).)

Conventionally, hydrophobic interaction chromatography (HIC) uses mobile phases with high salt concentration that are not compatible with mass spectrometry (MS). Here we describe development of an HIC method coupled with MS detection (HIC-MS) utilizing an aqueous mobile phase with a low concentration of a volatile salt for characterizing recombinant monoclonal antibody (mAb) post-translational modifications (PTMs). The ability of HIC to separate the oxidation and free thiol variants of the mAbs enables their isolation and rapid characterization of these attributes under native conditions, an important step toward understanding the role they play.

Emerging research suggests that multiple tumor compartments can influence treatment responsiveness and relapse, yet the search for therapeutic resistance mechanisms remains largely focused on acquired genomic alterations in cancer cells. Here we show how treatment-induced changes occur in multiple tumor compartments during tumor relapse and can reduce benefit of follow-on therapies. By using serial biopsies, next-generation sequencing, and single-cell transcriptomics, we tracked the evolution of multiple cellular compartments within individual lesions during first-line treatment response, relapse, and second-line therapeutic interventions in an autochthonous model of melanoma. We discovered that although treatment-relapsed tumors remained genetically stable, they converged on a shared resistance phenotype characterized by dramatic changes in tumor cell differentiation state, immune infiltration, and extracellular matrix (ECM) composition. Similar alterations in tumor cell differentiation were also observed in more than half of our treatment-relapsed patient tumors. Tumor cell-state changes were coincident with ECM remodeling and increased tumor stiffness, which alone was sufficient to alter tumor cell fate and reduce treatment responses in melanoma cell lines in vitro. Despite the absence of acquired mutations in the targeted pathway, resistant tumors showed significantly decreased responsiveness to second-line therapy intervention within the same pathway. The ability to preclinically model relapse and refractory settings-while capturing dynamics within and crosstalk between all relevant tumor compartments-provides a unique opportunity to better design and sequence appropriate clinical interventions.

Multiple strategies have been developed to facilitate the efficient production of bispecific IgG (BsIgG) in single host cells. For example, we previously demonstrated near quantitative (≥90%) formation of BsIgG of different species and isotypes by combining 'knob-into-hole' mutations for heavy chain heterodimerization with engineered antigen-binding fragments (Fabs) for preferential cognate heavy/light chain pairing. Surprisingly, in this study we found high yield (>65%) of BsIgG 1 without Fab engineering to be a common occurrence, i.e., observed for 33 of the 99 different antibody pairs evaluated. Installing charge mutations at both C H 1/C L interfaces was sufficient for near quantitative yield (>90%) of BsIgG 1 for most (9 of 11) antibody pairs tested with this inherent cognate chain pairing preference. Mechanistically, we demonstrate that a strong cognate pairing preference in one Fab arm can be sufficient for high BsIgG 1 yield. These observed chain pairing preferences are apparently driven by variable domain sequences and can result from a few specific residues in the complementarity-determining region (CDR) L3 and H3. Transfer of these CDR residues into other antibodies increased BsIgG 1 yield in most cases. Mutational analysis revealed that the disulfide bond between heavy and light chains did not affect the yield of BsIgG 1 . This study provides some mechanistic understanding of factors contributing to antibody heavy/light chain pairing preference and subsequently contributes to the efficient production of BsIgG in single host cells.

Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.

The integrity of the mammalian epidermis depends on a balance of proliferation and differentiation in the resident population of stem cells 1 . The kinase RIPK4 and the transcription factor IRF6 are mutated in severe developmental syndromes in humans, and mice lacking these genes display epidermal hyperproliferation and soft-tissue fusions that result in neonatal lethality 2-5 . Our understanding of how these genes control epidermal differentiation is incomplete. Here we show that the role of RIPK4 in mouse development requires its kinase activity; that RIPK4 and IRF6 expressed in the epidermis regulate the same biological processes; and that the phosphorylation of IRF6 at Ser413 and Ser424 primes IRF6 for activation. Using RNA sequencing (RNA-seq), histone chromatin immunoprecipitation followed by sequencing (ChIP-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) of skin in wild-type and IRF6-deficient mouse embryos, we define the transcriptional programs that are regulated by IRF6 during epidermal differentiation. IRF6 was enriched at bivalent promoters, and IRF6 deficiency caused defective expression of genes that are involved in the metabolism of lipids and the formation of tight junctions. Accordingly, the lipid composition of the stratum corneum of Irf6 -/- skin was abnormal, culminating in a severe defect in the function of the epidermal barrier. Collectively, our results explain how RIPK4 and IRF6 function to ensure the integrity of the epidermis and provide mechanistic insights into why developmental syndromes that are characterized by orofacial, skin and genital abnormalities result when this axis goes awry.

Multiple myeloma (MM) arises from malignant immunoglobulin (Ig)-secreting plasma cells and remains an incurable, often lethal disease despite therapeutic advances. The unfolded-protein response sensor IRE1α supports protein secretion by deploying a kinase-endoribonuclease module to activate the transcription factor XBP1s. MM cells may co-opt the IRE1α-XBP1s pathway; however, the validity of IRE1α as a potential MM therapeutic target is controversial. Genetic disruption of IRE1α or XBP1s, or pharmacologic IRE1α kinase inhibition, attenuated subcutaneous or orthometastatic growth of MM tumors in mice and augmented efficacy of two established frontline antimyeloma agents, bortezomib and lenalidomide. Mechanistically, IRE1α perturbation inhibited expression of key components of the endoplasmic reticulum-associated degradation machinery, as well as secretion of Ig light chains and of cytokines and chemokines known to promote MM growth. Selective IRE1α kinase inhibition reduced viability of CD138 + plasma cells while sparing CD138 - cells derived from bone marrows of newly diagnosed or posttreatment-relapsed MM patients, in both US- and European Union-based cohorts. Effective IRE1α inhibition preserved glucose-induced insulin secretion by pancreatic microislets and viability of primary hepatocytes in vitro, as well as normal tissue homeostasis in mice. These results establish a strong rationale for developing kinase-directed inhibitors of IRE1α for MM therapy., Competing Interests: Conflict of interest statement: J.M.H., A.L.T., A.S., S.A.M., D.A.L., M. Lu, Y.-C.A.C., J.Q., K.T., D.K., E.S., M.M., M.R., H.A.W., W.W., K.C., S.K., M.H.B., S.T.L., W.S., M. Lorenzo, J.W., J.L., T.D.B., A.H., B.H., A.G., R.M.W., D.L., M.-G.B., J.R., and A.A. were employees of Genentech, Inc. during performance of this work., (Copyright © 2019 the Author(s). Published by PNAS.)

IgA antibodies have broad potential as a novel therapeutic platform based on their superior receptor-mediated cytotoxic activity, potent neutralization of pathogens, and ability to transcytose across mucosal barriers via polymeric immunoglobulin receptor (pIgR)-mediated transport, compared to traditional IgG-based drugs. However, the transition of IgA into clinical development has been challenged by complex expression and characterization, as well as rapid serum clearance that is thought to be mediated by glycan receptor scavenging of recombinantly produced IgA monomer bearing incompletely sialylated N-linked glycans. Here, we present a comprehensive biochemical, biophysical, and structural characterization of recombinantly produced monomeric, dimeric and polymeric human IgA. We further explore two strategies to overcome the rapid serum clearance of polymeric IgA: removal of all N-linked glycosylation sites creating an aglycosylated polymeric IgA and engineering in FcRn binding with the generation of a polymeric IgG-IgA Fc fusion. While previous reports and the results presented in this study indicate that glycan-mediated clearance plays a major role for monomeric IgA, systemic clearance of polymeric IgA in mice is predominantly controlled by mechanisms other than glycan receptor clearance, such as pIgR-mediated transcytosis. The developed IgA platform now provides the potential to specifically target pIgR expressing tissues, while maintaining low systemic exposure.

Tissue-resident macrophages require specific milieus for the maintenance of defining gene-expression programs. Expression of the transcription factor GATA6 is required for the homeostasis, function and localization of peritoneal cavity-resident macrophages. Gata6 expression is maintained in a non-cell autonomous manner and is elicited by the vitamin A metabolite, retinoic acid. Here, we found that the GATA6 transcriptional program is a common feature of macrophages residing in all visceral body cavities. Retinoic acid-dependent and -independent hallmark genes of GATA6 + macrophages were induced by mesothelial and fibroblastic stromal cells that express the transcription factor Wilms' Tumor 1 (WT1), which drives the expression of two rate-limiting enzymes in retinol metabolism. Depletion of Wt1 + stromal cells reduced the frequency of GATA6 + macrophages in the peritoneal, pleural and pericardial cavities. Thus, Wt1 +  mesothelial and fibroblastic stromal cells constitute essential niche components supporting the tissue-specifying transcriptional landscape and homeostasis of cavity-resident macrophages., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Innovative protein engineering and chemical conjugation technologies have yielded an impressive number of drug candidates in clinical development including >80 antibody drug conjugates, >60 bispecific antibodies, >35 Fc-fusion proteins and >10 immuno-cytokines. Despite these innovations, technological advances are needed to address unmet medical needs with new pharmacological mechanisms. Age-related eye diseases are among the most common causes of blindness and poor vision in the world. Many such diseases affect the back of the eye, where the inaccessibility of the site of action necessitates therapeutic delivery via intravitreal (IVT) injection. Treatments administered via this route typically have vitreal half-lives <10 days in humans, requiring frequent administration. Since IVT injection is burdensome to patients, there exists a strong need to develop therapeutics with prolonged residence time in the eye. We report here a strategy to increase retention of a therapeutic fragment antibody (Fab) in the eye, using an anti-complement factor D Fab previously optimized for ocular delivery. Polyethylene glycol structures, varying in length, geometry and degree of branching, were coupled to the Fab via maleimide-activated termini. A screening strategy was developed to allow for key determinants of ocular half-life to be measured in vitro. After compound selection, a scalable process was established to enable tolerability and pharmacokinetic studies in cynomolgus monkeys, demonstrating an increase in vitreal half-life with no associated adverse events. Further, we show that the technique for compound selection, analytical characterization, and scalable production is general for a range of antibody fragments. The application of the technology has broad impact in across many therapeutic areas with the first major advancement in the treatment of an important ocular disease., Competing Interests: The authors have read the journal's policy and have the following competing interests: W.S., P.E.H., N.P., M.T.P., C.B., W.S., A.M., K.M.L., V.B., H.B., S.C., A.K., and R.F.K. are employees of Genentech. G.H. is an employee of BGI Americas, a genome sequencing company. J.M.S. is an employee of Boeringer Ingleheim, a pharmaceutical company. However, G.H. and J.M.S. were employees of Genentech through the completion of the study. There are no patents, products in development or marketed products associated with this research to declare. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

The extent of afucosylation, which refers to the absence of core fucose on Fc glycans, can correlate positively with the antibody-dependent cellular cytotoxicity (ADCC) activity of a monoclonal antibody (mAb). Therefore, it is important to maintain consistent afucosylation during cell culture process scale-up in bioreactors for a mAb with ADCC activity. However, there is currently a lack of understanding about the impact of partial pressure of carbon dioxide (pCO 2 )-a parameter that can vary with bioreactor scale-on afucosylation. Using a small-scale (3 L) bioreactor model that can modulate pCO 2 levels through modified configurations and gassing strategies, we identified three cell culture process parameters that influence afucosylation of a mAb produced by a recombinant Chinese Hamster Ovary (CHO) cell line: pCO 2 , media hold duration (at 37°C), and manganese. These three-independent parameters demonstrated a synergistic effect on mAb afucosylation; increase in pCO 2 , media hold duration, and manganese consistently increased afucosylation. Our investigations into the underlying mechanisms through proteomic analysis indicated that the synergistic interactions downregulated pathways related to guanosine diphosphate-fucose synthesis and fucosylation, and upregulated manganese transport into the CHO cells. These new findings highlight the importance of considering potential differences in culture environment and operations across bioreactor scales, and understanding the impact of their interactions on product quality., (© 2019 Wiley Periodicals, Inc.)

Chinese hamster ovary (CHO) cell lines are used to express a variety of therapeutic proteins. However, lactogenic behavior displayed by some CHO cell lines during manufacturing processes may result in a decline in viability, productivity, and possible alterations in product quality. In cultured cells, lactate is produced during glycolysis through irreversible conversion of phosphoenolpyruvate to pyruvate and then lactate via sequential function of pyruvate kinase and lactate dehydrogenase (LDH) enzymes. In the process of cell line development (CLD), two lactogenic cell lines expressing different antibody molecules are identified. The lactogenic behaviors of these cell lines can be differentially mitigated through optimization of either nutrient feeds or culture pH, depending on the cell line. Analysis of various proteins involved in the glycolysis pathway reveal a direct correlation between the pyruvate kinase muscle-1 (PKM-1) isoform levels and lactogenic behavior. CRISPR mediated knockout of the PKM-1 isoform abolishes lactate accumulation even under lactogenic conditions. Furthermore, a cell line lacking expression of both PKM-1 and PKM-2 enzymes capable of maintaining productivity, viability, and growth without displaying lactogenic behavior is identified. Targeted deletion of PKM in CHO cells may be tolerated due to expression of PKL (liver) and PKR (red blood cell) isoforms of pyruvate kinase. All together, these findings suggest that PKM-1 up-regulation during antibody production could trigger lactogenic behavior and that this enzyme is dispensable for CHO cell survival., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Post-translational modification of chemokines mediated by the dipeptidyl peptidase DPP4 (CD26) has been shown to negatively regulate lymphocyte trafficking, and its inhibition enhances T cell migration and tumor immunity by preserving functional chemokine CXCL10. By extending those initial findings to pre-clinical models of hepatocellular carcinoma and breast cancer, we discovered a distinct mechanism by which inhibition of DPP4 improves anti-tumor responses. Administration of the DPP4 inhibitor sitagliptin resulted in higher concentrations of the chemokine CCL11 and increased migration of eosinophils into solid tumors. Enhanced tumor control was preserved in mice lacking lymphocytes and was ablated after depletion of eosinophils or treatment with degranulation inhibitors. We further demonstrated that tumor-cell expression of the alarmin IL-33 was necessary and sufficient for eosinophil-mediated anti-tumor responses and that this mechanism contributed to the efficacy of checkpoint-inhibitor therapy. These findings provide insight into IL-33- and eosinophil-mediated tumor control, revealed when endogenous mechanisms of DPP4 immunoregulation are inhibited.

Lupus nephritis (LN) is a severe clinical manifestation of systemic lupus erythematosus (SLE) associated with significant morbidity and mortality. Assessment of severity and activity of renal involvement in SLE requires a kidney biopsy, an invasive procedure with limited prognostic value. Noninvasive biomarkers are needed to inform treatment decisions and to monitor disease activity. Proteinuria is associated with disease progression in LN; however, the composition of the LN urinary proteome remains incompletely characterized. To address this, we profiled LN urine samples using complementary mass spectrometry-based methods:  protein gel fractionation, chemical labeling using tandem mass tags, and data-independent acquisition. Combining results from these approaches yielded quantitative information on 2573 unique proteins in urine from LN patients. A multiple-reaction monitoring (MRM) method was established to confirm eight proteins in an independent cohort of LN patients, and seven proteins (transferrin, α-2-macroglobulin, haptoglobin, afamin, α-1-antitrypsin, vimentin, and ceruloplasmin) were confirmed to be elevated in LN urine compared to healthy controls. In this study, we demonstrate that deep mass spectrometry profiling of a small number of patient samples can identify high-quality biomarkers that replicate in an independent LN disease cohort. These biomarkers are being used to inform clinical biomarker strategies to support longitudinal and interventional studies focused on evaluating disease progression and treatment efficacy of novel LN therapeutics.

Site-specific conjugation of small molecules to antibodies represents an attractive goal for the development of more homogeneous targeted therapies and diagnostics. Most site-specific conjugation strategies require modification or removal of antibody glycans or interchain disulfide bonds or engineering of an antibody mutant that bears a reactive handle. While such methods are effective, they complicate the process of preparing antibody conjugates and can negatively impact biological activity. Herein we report the development and detailed characterization of a robust photoaffinity cross-linking method for site-specific conjugation to fully glycosylated wild-type antibodies. The method employs a benzoylphenylalanine (Bpa) mutant of a previously described 13-residue peptide derived from phage display to bind tightly to the Fc domain; upon UV irradiation, the Bpa residue forms a diradical that reacts with the bound antibody. After the initial discovery of an effective Bpa mutant peptide and optimization of the reaction conditions to enable efficient conjugation without concomitant UV-induced photodamage of the antibody, we assessed the scope of the photoconjugation reaction across different human and nonhuman antibodies and antibody mutants. Next, the specific site of conjugation on a human antibody was characterized in detail by mass spectrometry experiments and at atomic resolution by X-ray crystallography. Finally, we adapted the photoconjugation method to attach a cytotoxic payload site-specifically to a wild-type antibody and showed that the resulting conjugate is both stable in plasma and as potent as a conventional antibody-drug conjugate in cells, portending well for future biological applications.