2021 White Paper on Recent Issues in Bioanalysis: ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry (Part 2 - Recommendations on Biomarkers/CDx Assays Development & Validation, Cytometry Validation & Innovation, Biotherapeutics PK LBA Regulated Bioanalysis, Critical Reagents & Positive Controls Generation).

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included three Main Workshops and seven Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "context of use" [COU]); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations on ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 9 and 11 (2022), respectively.

Discovery of Orally Bioavailable Purine-Based Inhibitors of the Low-Molecular-Weight Protein Tyrosine Phosphatase.

Obesity-associated insulin resistance plays a central role in the pathogenesis of type 2 diabetes. A promising approach to decrease insulin resistance in obesity is to inhibit the protein tyrosine phosphatases that negatively regulate insulin receptor signaling. The low-molecular-weight protein tyrosine phosphatase (LMPTP) acts as a critical promoter of insulin resistance in obesity by inhibiting phosphorylation of the liver insulin receptor activation motif. Here, we report development of a novel purine-based chemical series of LMPTP inhibitors. These compounds inhibit LMPTP with an uncompetitive mechanism and are highly selective for LMPTP over other protein tyrosine phosphatases. We also report the generation of a highly orally bioavailable purine-based analogue that reverses obesity-induced diabetes in mice.

A flow cytometric assay for HLA-DR expression on monocytes validated as a biomarker for enrollment in sepsis clinical trials.

PURPOSE: Decreased expression of HLA-DR on monocytes (mHLA-DR) is a reliable indicator of immunosuppression in patients with sepsis and is correlated with increased risk of secondary infection and mortality. A flow cytometry-based laboratory developed test for the measurement of mHLA-DR in whole blood was validated for clinical trial enrollment, which is considered medical decision-making, for patients with severe sepsis or septic shock. METHODS: The BD Quantibrite™ anti-HLA-DR/anti-monocyte reagent measures antibodies bound per cell of HLA-DR on CD14+ monocytes. The mHLA-DR assay was planned to support inclusion/exclusion of patients for a clinical trial and was validated according to New York State Department of Health (NYSDOH) requirements for a new non-malignant leukocyte immunophenotyping assay. RESULTS: Normal, healthy donor and sepsis patient samples were stable up to 72 h post-collection in Cyto-Chex BCT phlebotomy tubes. Pre-determined acceptance criteria were met for precision parameters (average %CV ≤ 20%) and global laboratory-to-laboratory comparisons (average %Δ ≤ 20%). The approaches taken to evaluate and report accuracy, analytical specificity and sensitivity, reportable range, reference interval, and the proposed multi-level quality control were accepted by NYSDOH. CONCLUSIONS: In this study, the validation strategy necessary when the intended use of assay results changes from exploratory to medical decision making (patient enrollment), which successfully resulted in regulatory approval, is described.

Best practices for the development, analytical validation and clinical implementation of flow cytometric methods for chimeric antigen receptor T cell analyses.

Chimeric Antigen Receptor (CAR) T cells are recognized as efficacious therapies with demonstrated ability to produce durable responses in blood cancer patients. Regulatory approvals and acceptance of these unique therapies by patients and reimbursement agencies have led to a significant increase in the number of next generation CAR T clinical trials. Flow cytometry is a powerful tool for comprehensive profiling of individual CAR T cells at multiple stages of clinical development, from product characterization during manufacturing to longitudinal evaluation of the infused product in patients. There are unique challenges with regard to the development and validation of flow cytometric methods for CAR T cells; moreover, the assay requirements for manufacturing and clinical monitoring differ. Based on the collective experience of the authors, this recommendation paper aims to review these challenges and present approaches to address them. The discussion focuses on describing key considerations for the design, optimization, validation and implementation of flow cytometric methods during the clinical development of CAR T cell therapies.

Combination Treatment of the Oral CHK1 Inhibitor, SRA737, and Low-Dose Gemcitabine Enhances the Effect of Programmed Death Ligand 1 Blockade by Modulating the Immune Microenvironment in SCLC.

INTRODUCTION: Despite the enthusiasm surrounding cancer immunotherapy, most SCLC patients show very modest response to immune checkpoint inhibitor monotherapy treatment. Therefore, there is growing interest in combining immune checkpoint blockade with chemotherapy and other treatments to enhance immune checkpoint blockade efficacy. Based on favorable clinical trial results, chemotherapy and immunotherapy combinations have been recently approved by the U.S. Food and Drug Administration for frontline treatment for SCLC. METHODS AND RESULTS: Here, we show that combined treatment of SRA737, an oral CHK1 inhibitor, and anti-programmed death ligand 1 (PD-L1) leads to an antitumor response in multiple cancer models, including SCLC. We further show that combining low, non-cytotoxic doses of gemcitabine with SRA737 + anti-PD-L1/anti-PD-1 significantly increased antitumorigenic CD8+ cytotoxic T cells, dendritic cells, and M1 macrophage populations in an SCLC model. This regimen also led to a significant decrease in immunosuppressive M2 macrophage and myeloid-derived suppressor cell populations, as well as an increase in the expression of the type I interferon beta 1 gene, IFNß, and chemokines, CCL5 and CXCL10. CONCLUSIONS: Given that anti-PD-L1/anti-PD-1 drugs have recently been approved as monotherapy and in combination with chemotherapy for the treatment of SCLC, and that the SRA737 + low dose gemcitabine regimen is currently in clinical trials for SCLC and other malignancies, our preclinical data provide a strong rational for combining this regimen with inhibitors of the PD-L1/PD-1 pathway.

Metabolism at the Max: How Vertebrate Organisms Respond to Physical Activity.

Activity metabolism is supported by phosphorylated reserves (adenosine triphosphate, creatine phosphate), glycolytic, and aerobic metabolism. Because there is no apparent variation between vertebrate groups in phosphorylated reserves or glycolytic potential of skeletal muscle, variation in maximal metabolic rate between major vertebrate groups represents selection operating on aerobic mechanisms. Maximal rates of oxygen consumption in vertebrates are supported by increased conductive and diffusive fluxes of oxygen from the environment to the mitochondria. Maximal CO2 efflux from the mitochondria to the environment must be matched to oxygen flux, or imbalances in pH will occur. Among vertebrates, there are a variety of modes of locomotion and vastly different rates of metabolism supported by a variety of cardiorespiratory architectures. However, interclass comparisons strongly implicate systemic oxygen transport as the rate-limiting step to maximal oxygen consumption for all vertebrate groups. The key evolutionary step that accounts for the approximately 10-fold increase in maximal oxygen flux in endotherms versus ectotherms appears to be maximal heart rate. Other variables such as ventilation, pulmonary/gill, and tissue diffusing capacity, have excess capacity and thus are not limiting to maximal oxygen consumption. During maximal activity, the ratio of ventilation to respiratory system blood flow is remarkably similar among vertebrates, and CO2 extraction efficiency increases while oxygen extraction efficiency decreases, suggesting that the respiratory system provides the largest resistance to maximal CO2 flux. Despite the large variation in modes of activity and rates of metabolism, maximal rates of oxygen and CO2 flux appear to be limited by the cardiovascular and respiratory systems, respectively.

Discovery of Sulfonamidebenzamides as Selective Apoptotic CHOP Pathway Activators of the Unfolded Protein Response.

Cellular proteins that fail to fold properly result in inactive or disfunctional proteins that can have toxic functions. The unfolded protein response (UPR) is a two-tiered cellular mechanism initiated by eukaryotic cells that have accumulated misfolded proteins within the endoplasmic reticulum (ER). An adaptive pathway facilitates the clearance of the undesired proteins; however, if overwhelmed, cells trigger apoptosis by upregulating transcription factors such as C/EBP-homologous protein (CHOP). A high throughput screen was performed directed at identifying compounds that selectively upregulate the apoptotic CHOP pathway while avoiding adaptive signaling cascades, resulting in a sulfonamidebenzamide chemotype that was optimized. These efforts produced a potent and selective CHOP inducer (AC50 = 0.8 µM; XBP1 > 80 µM), which was efficacious in both mouse embryonic fibroblast cells and a human oral squamous cell cancer cell line, and demonstrated antiproliferative effects for multiple cancer cell lines in the NCI-60 panel.

Identification and characterization of novel human glucose-6-phosphate dehydrogenase inhibitors.

Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme of the pentose phosphate pathway, converting glucose-6-phosphate to 6-phosphoglucono-δ-lactone with parallel reduction of NADP(+). Several human diseases, including cancer, are associated with increased G6PD activity. To date, only a few G6PD inhibitors have been available. However, adverse side effects and high IC(50) values hamper their use as therapeutics and basic research probes. In this study, we developed a high-throughput screening assay to identify novel human G6PD (hG6PD) inhibitors. Screening the LOPAC (Sigma-Aldrich; 1280 compounds), Spectrum (Microsource Discovery System; 1969 compounds), and DIVERSet (ChemBridge; 49 971 compounds) small-molecule compound collections revealed 139 compounds that presented ≥50% hG6PD inhibition. Hit compounds were further included in a secondary and orthogonal assay in order to identify false-positives and to determine IC(50) values. The most potent hG6PD inhibitors presented IC(50) values of <4 µM. Compared with the known hG6PD inhibitors dehydroepiandrosterone and 6-aminonicotinamide, the inhibitors identified in this study were 100- to 1000-fold more potent and showed different mechanisms of enzyme inhibition. One of the newly identified hG6PD inhibitors reduced viability of the mammary carcinoma cell line MCF10-AT1 (IC(50) ~25 µM) more strongly than that of normal MCF10-A cells (IC(50) >50 µM).

A comparative meta-analysis of maximal aerobic metabolism of vertebrates: implications for respiratory and cardiovascular limits to gas exchange.

Maximal aerobic metabolic rates (MMR) in vertebrates are supported by increased conductive and diffusive fluxes of O(2) from the environment to the mitochondria necessitating concomitant increases in CO(2) efflux. A question that has received much attention has been which step, respiratory or cardiovascular, provides the principal rate limitation to gas flux at MMR? Limitation analyses have principally focused on O(2) fluxes, though the excess capacity of the lung for O(2) ventilation and diffusion remains unexplained except as a safety factor. Analyses of MMR normally rely upon allometry and temperature to define these factors, but cannot account for much of the variation and often have narrow phylogenetic breadth. The unique aspect of our comparative approach was to use an interclass meta-analysis to examine cardio-respiratory variables during the increase from resting metabolic rate to MMR among vertebrates from fish to mammals, independent of allometry and phylogeny. Common patterns at MMR indicate universal principles governing O(2) and CO(2) transport in vertebrate cardiovascular and respiratory systems, despite the varied modes of activities (swimming, running, flying), different cardio-respiratory architecture, and vastly different rates of metabolism (endothermy vs. ectothermy). Our meta-analysis supports previous studies indicating a cardiovascular limit to maximal O(2) transport and also implicates a respiratory system limit to maximal CO(2) efflux, especially in ectotherms. Thus, natural selection would operate on the respiratory system to enhance maximal CO(2) excretion and the cardiovascular system to enhance maximal O(2) uptake. This provides a possible evolutionary explanation for the conundrum of why the respiratory system appears functionally over-designed from an O(2) perspective, a unique insight from previous work focused solely on O(2) fluxes. The results suggest a common gas transport blueprint, or Bauplan, in the vertebrate clade.

Macrophage p38 mitogen-activated protein kinase activity regulates invariant natural killer T-cell responses during Borrelia burgdorferi infection.

The interaction of macrophages with infectious agents leads to the activation of several signaling cascades, including mitogen-activated protein (MAP) kinases, such as p38. We now demonstrate that p38 MAP kinase-mediated responses are critical components to the immune response to Borrelia burgdorferi. The pharmacological and genetic inhibition of p38 MAP kinase activity during infection with the spirochete results in increased carditis. In transgenic mice that express a dominant negative form of p38 MAP kinase specifically in macrophages, production of the invariant natural killer T (iNKT) cell-attracting chemokine MCP-1 and of the antigen-presenting molecule CD1d are significantly reduced. The expression of the transgene therefore results in the deficient infiltration of iNKT cells, their decreased activation, and a diminished production of interferon γ (IFN-γ), leading to increased bacterial burdens and inflammation. These results show that p38 MAP kinase provides critical checkpoints for the protective immune response to the spirochete during infection of the heart.

Pathway-selective suppression of chemokine receptor signaling in B cells by LPS through downregulation of PLC-ß2.

Lymphocyte activation leads to changes in chemokine receptor expression. There are limited data, however, on how lymphocyte activators can alter chemokine signaling by affecting downstream pathways. We hypothesized that B cell-activating agents might alter chemokine responses by affecting downstream signal transducers, and that such effects might differ depending on the activator. We found that activating mouse B cells using either anti-IgM or lipopolysaccharide (LPS) increased the surface expression of CCR6 and CCR7 with large increases in chemotaxis to their cognate ligands. By contrast, while anti-IgM also led to enhanced calcium responses, LPS-treated cells showed only small changes in calcium signaling as compared with cells that were freshly isolated. Of particular interest, we found that LPS caused a reduction in the level of B-cell phospholipase C (PLC)-ß2 mRNA and protein. Data obtained using PLC-ß2(-/-) mice showed that the ß2 isoform mediates close to one-half the chemokine-induced calcium signal in resting and anti-IgM-activated B cells, and we found that calcium signals in the LPS-treated cells were boosted by increasing the level of PLC-ß2 using transfection, consistent with a functional effect of downregulating PLC-ß2. Together, our results show activator-specific effects on responses through B-cell chemokine receptors that are mediated by quantitative changes in a downstream signal-transducing protein, revealing an activity for LPS as a downregulator of PLC-ß2, and a novel mechanism for controlling chemokine-induced signals in lymphocytes.

Brain-penetrating 2-aminobenzimidazole H(1)-antihistamines for the treatment of insomnia.

The benzimidazole core of the selective non-brain-penetrating H(1)-antihistamine mizolastine was used to identify a series of brain-penetrating H(1)-antihistamines for the potential treatment of insomnia. Using cassette PK studies, brain-penetrating H(1)-antihistamines were identified and in vivo efficacy was demonstrated in a rat EEG/EMG model. Further optimization focused on strategies to attenuate an identified hERG liability, leading to the discovery of 4i with a promising in vitro profile.

p38 mitogen-activated protein kinase controls NF-kappaB transcriptional activation and tumor necrosis factor alpha production through RelA phosphorylation mediated by mitogen- and stress-activated protein kinase 1 in response to Borrelia burgdorferi antigens.

The interaction of Borrelia burgdorferi, the causative agent of Lyme borreliosis, with phagocytic cells induces the activation of NF-kappaB and the expression of proinflammatory cytokines including tumor necrosis factor alpha (TNF-alpha). B. burgdorferi-induced TNF-alpha production is also dependent on the activation of p38 mitogen-activated protein (MAP) kinase. The specific contribution of these signaling pathways to the response of phagocytic cells to the spirochete and the molecular mechanisms underlying this response remain unresolved. We now show that p38 MAP kinase activity regulates the transcriptional activation of NF-kappaB in response to spirochetal lysate stimulation of phagocytic cells. The regulation occurs at the nuclear level and is independent of the translocation of the transcription factor to the nucleus or its capacity to bind to specific DNA target sequences. In RAW264.7 cells, p38alpha MAP kinase regulates the phosphorylation of NF-kappaB RelA. p38 MAP kinase phosphorylates the nuclear kinase mitogen- and stress-activated protein kinase 1 (MSK1). MSK1 in turn phosphorylates the transcriptionally active subunit of NF-kappaB, RelA. The repression of MSK1 expression with small interfering RNA results in reduced RelA phosphorylation and a significant decrease in the production of TNF-alpha in response to B. burgdorferi lysates. Overall, these results clarify the contribution of the signaling pathways that are activated in response to the interaction of spirochetes with phagocytic cells to TNF-alpha production. Our results situate p38 MAP kinase activity as a central regulator of the phagocytic proinflammatory response through MSK1-mediated transcriptional activation of the transcription factor NF-kappaB.

Design, synthesis, and biological evaluation of simplified alpha-keto heterocycle, trifluoromethyl ketone, and formyl substituted folate analogues as potential inhibitors of GAR transformylase and AICAR transformylase.

A series of simplified alpha-keto heterocycle, trifluoromethyl ketone, and formyl substituted folate analogues lacking the benzoylglutamate subunit were prepared and examined as potential inhibitors of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide transformylase (AICAR Tfase).

Multidrug resistance reversal activity of key ningalin analogues.

Key analogue derivatives of the ningalins, potent multidrug resistance (MDR) reversal compounds, were examined resulting in the discovery of a potent MDR reversal agent that hypersensitizes P-gp resistant tumor cell lines to front-line conventional therapeutic agents.

Rational design, synthesis, evaluation, and crystal structure of a potent inhibitor of human GAR Tfase: 10-(trifluoroacetyl)-5,10-dideazaacyclic-5,6,7,8-tetrahydrofolic acid.

Glycinamide ribonucleotide transformylase (GAR Tfase) has been the target of anti-neoplastic intervention for almost two decades. Here, we use a structure-based approach to design a novel folate analogue, 10-(trifluoroacetyl)-5,10-dideazaacyclic-5,6,7,8-tetrahydrofolic acid (10-CF(3)CO-DDACTHF, 1), which specifically inhibits recombinant human GAR Tfase (K(i) = 15 nM), but is inactive (K(i) > 100 microM) against other folate-dependent enzymes that have been examined. Moreover, compound 1 is a potent inhibitor of tumor cell proliferation (IC(50) = 16 nM, CCRF-CEM), which represents a 10-fold improvement over Lometrexol, a GAR Tfase inhibitor that has been in clinical trials. Thus, this folate analogue 1 is among the most potent and selective inhibitors known toward GAR Tfase. Contributing to its efficacious activity, compound 1 is effectively transported into the cell by the reduced folate carrier and intracellularly sequestered by polyglutamation. The crystal structure of human GAR Tfase with folate analogue 1 at 1.98 A resolution represents the first structure of any GAR Tfase to be determined with a cofactor or cofactor analogue without the presence of substrate. The folate-binding loop of residues 141-146, which is highly flexible in both Escherichia coli and unliganded human GAR Tfase structures, becomes highly ordered upon binding 1 in the folate-binding site. Computational docking of the natural cofactor into this and other apo or complexed structures provides a rational basis for modeling how the natural cofactor 10-formyltetrahydrofolic acid interacts with GAR Tfase, and suggests that this folate analogue-bound conformation represents the best template to date for inhibitor design.

10-Formyl-5,10-dideaza-acyclic-5,6,7,8-tetrahydrofolic acid (10-formyl-DDACTHF): a potent cytotoxic agent acting by selective inhibition of human GAR Tfase and the de novo purine biosynthetic pathway.

The synthesis of 10-formyl-DDACTHF (3) as a potential inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) is reported. Aldehyde 3, the corresponding gamma- and alpha-pentaglutamates 21 and 25 and related agents were evaluated for inhibition of folate-dependent enzymes including GAR Tfase and AICAR Tfase. The inhibitors were found to exhibit potent cytotoxic activity (CCRF-CEM IC(50) for 3=60nM) that exceeded their enzyme inhibition potency [K(i) (3)=6 and 1 microM for Escherichia coli GAR and human AICAR Tfase, respectively]. Cytotoxicity rescue by medium purines, but not pyrimidines, indicated that the potent cytotoxic activity is derived from selective purine biosynthesis inhibition and rescue by AICAR monophosphate established that the activity is derived preferentially from GAR versus AICAR Tfase inhibition. The potent cytotoxic compounds including aldehyde 3 lost activity against CCRF-CEM cell lines deficient in the reduced folate carrier (CCRF-CEM/MTX) or folylpolyglutamate synthase (CCRF-CEM/FPGS(-)) establishing that their potent activity requires both reduced folate carrier transport and polyglutamation. Unexpectedly, the pentaglutamates displayed surprisingly similar K(i)'s versus E. coli GAR Tfase and only modestly enhanced K(i)'s versus human AICAR Tfase. On the surface this initially suggested that the potent cytotoxic activity of 3 and related compounds might be due simply to preferential intracellular accumulation of the inhibitors derived from effective transport and polyglutamation (i.e., ca. 100-fold higher intracellular concentrations). However, a subsequent examination of the inhibitors against recombinant human GAR Tfase revealed they and the corresponding gamma-pentaglutamates were unexpectedly much more potent against the human versus E. coli enzyme (K(i) for 3, 14nM against rhGAR Tfase versus 6 microM against E. coli GAR Tfase) which also accounts for their exceptional cytotoxic potency.