Clinical Candidate Selection Research Articles

Optimization of an in vitro method for assessing pulmonary permeability of inhaled drugs using alveolar epithelial cells

IntroductionInhalation of drugs for the treatment of pulmonary diseases has been used since a long time. Due to lungs' larger absorptive surface area, delivery of drugs to the lungs is the method of choice for different disorders. Here we present the establishment of a comprehensive permeability model using Type II alveolar epithelial cells and Beclomethasone Dipropionate (BDP) as a model drug delivered by pressurized metered dose inhaler (pMDI). MethodsUsing Type II alveolar epithelial cells, the method was standardized for parameters viz., cell density, viability, incubation period and membrane integrity. The delivery and deposition of drug were using the pMDI device with a Twin Stage Impinger (TSI) modified to accommodate cell culture insert having monolayer of cells. The analytical method for simultaneous estimation of BDP and Beclomathasone-17-Monopropionate (17-BMP) was validated as per the bioanalytical guidelines. The extent and rate of absorption of BDP was determined by quantifying the amount of drug permeated and the data represented by calculating its apparent permeability. ResultsType II alveolar epithelial cells cultured at 0.55 × 105 cells/cm2 for 8–12 days under air-liquid interface were optimized for conducting permeability studies. The data obtained for absorptive transport showed a linear increase in the drug permeated against time for both BDP and 17-BMP along with proportional permeability profile. DiscussionWe have developed a robust in vitro model to study absorptive rate of drug transport across alveolar layer. Such models would create potential value during formulation development for comparative studies and selection of clinical candidates.

The dual GCGR/GLP-1R agonist survodutide: Biomarkers and pharmacological profiling for clinical candidate selection.

To describe the biomarker strategy that was applied to select survodutide (BI 456906), BI 456908 and BI 456897 from 19 dual glucagon receptor (GCGR)/ glucagon-like peptide-1 receptor (GLP-1R) agonists for in-depth pharmacological profiling, which led to the qualification of survodutide as the clinical development candidate. Potencies to increase cyclic adenosine monophosphate (cAMP) were determined in Chinese hamster ovary (CHO)-K1 cells stably expressing human GCGR and GLP-1R. Agonism for endogenously expressed receptors was investigated in insulinoma cells (MIN6) for mouse GLP-1R, and in rat primary hepatocytes for the GCGR. In vivo potencies to engage the GLP-1R or GCGR were determined, measuring improvement in oral glucose tolerance (30 nmol/kg) and increase in plasma fibroblast growth factor-21 (FGF21) and liver nicotinamide N-methyltransferase (NNMT) mRNA expression (100 nmol/kg), respectively. Body weight- and glucose-lowering efficacies were investigated in diet-induced obese (DIO) mice and diabetic db/db mice, respectively. Upon acute dosing in lean mice, target engagement biomarkers for the GCGR and GLP-1R demonstrated a significant correlation (Spearman correlation coefficient with p < 0.05) to the in vitro GCGR and GLP-1R potencies for the 19 dual agonists investigated. Survodutide, BI 456908 and BI 456897 were selected for in-depth pharmacological profiling based on the significant improvement in acute oral glucose tolerance achieved (area under the curve [AUC] of 54%, 57% and 60% vs. vehicle) that was comparable to semaglutide (AUC of 45% vs. vehicle), while showing different degrees of in vivo GCGR engagement, as determined by hepatic NNMT mRNA expression (increased by 15- to 17-fold vs. vehicle) and plasma FGF21 concentrations (increased by up to sevenfold vs. vehicle). In DIO mice, survodutide (30 nmol/kg/once daily), BI 456908 (30 nmol/kg/once daily) and BI 456897 (10 nmol/kg/once daily) achieved a body weight-lowering efficacy from baseline of 25%, 27% and 26%, respectively. In db/db mice, survodutide and BI 456908 (10 and 20 nmol/kg/once daily) significantly lowered glycated haemoglobin (0.4%-0.6%); no significant effect was observed for BI 456897 (3 and 7 nmol/kg/once daily). Survodutide was selected as the clinical candidate based on its balanced dual GCGR/GLP-1R pharmacology, engaging the GCGR for robust body weight-lowering efficacy exceeding that of selective GLP-1R agonists, while achieving antidiabetic efficacy that was comparable to selective GLP-1R agonism. Survodutide is currently being investigated in Phase 3 clinical trials in people living with obesity.

A Study of the Process of Identifying New Drugs and Enabling Ways

The selection of clinical candidates, their synthesis, and characterization for therapeutic efficacy are all steps in the drug discovery process. This method underwent tremendous development over the past 50 years, beginning with a chemistry-centric approach and progressing through a more biological one to one that is more disease-specific. The development of molecular biology, along with improvements in screening and synthetic chemistry techniques, has made it possible to combine random screening with knowledge of the biological target. Although a strategic need drove this change, the astounding technological developments in chemistry and biology made it possible. The features of the new molecular entities will be refined during the course of numerous iterative screening runs required for this process, and the best balanced compounds will then move on to in vitro and in vivo testing in the selected disease model. 100 hits are thought to be found out of every 100,000 compounds tested. Only one of these 100 hits moves on to the lead compound stage. Most of these lead compounds—between 40% and 60%—fail ADMET testing. Despite the fact that the human genome project has revealed numerous unique and clinically verified targets in recent years, fewer new drugs are being launched, and the overall costs associated with developing a medicine are dramatically growing.

Bruton's Tyrosine Kinase Inhibitors with Distinct Binding Modes Reveal Differential Functional Impact on B-Cell Receptor Signaling.

Small molecule inhibitors of Bruton's tyrosine kinase (BTK) have been approved for the treatment of multiple B-cell malignancies and are being evaluated for autoimmune and inflammatory diseases. Various BTK inhibitors (BTKi) have distinct potencies, selectivity profiles, and binding modes within the ATP-binding site. On the basis of the latter feature, BTKis can be classified into those that occupy the back-pocket, H3 pocket, and the hinge region only. Hypothesizing that differing binding modes may have differential impact on the B-cell receptor (BCR) signaling pathway, we evaluated the activities of multiple BTKis in B-cell lymphoma models in vitro and in vivo. We demonstrated that, although all three types of BTKis potently inhibited BTK-Y223 autophosphorylation and phospholipase C gamma 2 (PLCγ2)-Y1217 transphosphorylation, hinge-only binders were defective in inhibiting BTK-mediated calcium mobilization upon BCR activation. In addition, PLCγ2 activation was effectively blocked by back-pocket and H3 pocket binders but not by hinge-only binders. Further investigation using TMD8 cells deficient in Rac family small GTPase 2 (RAC2) revealed that RAC2 functioned as a bypass mechanism, allowing for residual BCR signaling and PLCγ2 activation when BTK kinase activity was fully inhibited by the hinge-only binders. These data reveal a kinase activity-independent function of BTK, involving RAC2 in transducing BCR signaling events, and provide mechanistic rationale for the selection of clinical candidates for B-cell lymphoma indications.

Evaluation of bottom-up modeling of the blood-brain barrier to improve brain penetration prediction via physiologically based pharmacokinetic modeling.

Predicting the brain penetration of drugs has been notoriously difficult; however, recently, permeability-limited brain models have been constructed. Lead optimization for central nervous system compounds often focuses on compounds that have low transporter efflux, where passive permeability could be a main driver in determining cerebrospinal fluid (CSF)/brain concentrations. The main objective of this study was to evaluate the translatability of passive permeability data generated from different in vitro systems and its impact on the prediction of human CSF/brain concentrations using physiologically-based pharmacokinetic (PBPK) modeling. In vitro data were generated using gMDCK and parallel artificial membrane permeability assay-blood-brain barrier for comparison and predictions using a quantitative structure-activity relationship model were also evaluated. PBPK modeling was then performed for seven compounds with moderate-high permeability and a range of efflux in vitro, and the CSF/brain mass concentrations and Kpuu were reasonably predicted. This work provides the first step of a promising approach using bottom-up PBPK modeling for CSF/brain penetration prediction to support lead optimization and clinical candidate selection.

Validation of a Dendritic Cell and CD4+ T Cell Restimulation Assay Contributing to the Immunogenicity Risk Evaluation of Biotherapeutics.

Immunogenicity, defined as the ability to provoke an immune response, can be either wanted (i.e., vaccines) or unwanted. The latter refers to an immune response to protein or peptide therapeutics, characterized by the production of anti-drug antibodies, which may affect the efficacy and/or the safety profiles of these drugs. Consequently, evaluation of the risk of immunogenicity early in the development of biotherapeutics is of critical importance for defining their efficacy and safety profiles. Here, we describe and validate a fit-for-purpose FluoroSpot-based in vitro assay for the evaluation of drug-specific T cell responses. A panel of 24 biotherapeutics with a wide range of clinical anti-drug antibody response rates were tested in this assay. We demonstrated that using suitable cutoffs and donor cohort sizes, this assay could identify most of the compounds with high clinical immunogenicity rates (71% and 78% for sensitivity and specificity, respectively) while we characterized the main sources of assay variability. Overall, these data indicate that the dendritic cell and CD4+ T cell restimulation assay published herein could be a valuable tool to assess the risk of drug-specific T cell responses and contribute to the selection of clinical candidates in early development.

Identification of a potential diagnostic signature for postmenopausal osteoporosis via transcriptome analysis.

Purpose: We aimed to establish the transcriptome diagnostic signature of postmenopausal osteoporosis (PMOP) to identify diagnostic biomarkers and score patient risk to prevent and treat PMOP. Methods: Peripheral blood mononuclear cell (PBMC) expression data from PMOP patients were retrieved from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were screened using the “limma” package. The “WGCNA” package was used for a weighted gene co-expression network analysis to identify the gene modules associated with bone mineral density (BMD). Least absolute shrinkage and selection operator (LASSO) regression was used to construct a diagnostic signature, and its predictive ability was verified in the discovery cohort. The diagnostic values of potential biomarkers were evaluated by receiver operating characteristic curve (ROC) and coefficient analysis. Network pharmacology was used to predict the candidate therapeutic molecules. PBMCs from 14 postmenopausal women with normal BMD and 14 with low BMD were collected, and RNA was extracted for RT-qPCR validation. Results: We screened 2420 differentially expressed genes (DEGs) from the pilot cohort, and WGCNA showed that the blue module was most closely related to BMD. Based on the genes in the blue module, we constructed a diagnostic signature with 15 genes, and its ability to predict the risk of osteoporosis was verified in the discovery cohort. RT-qPCR verified the expression of potential biomarkers and showed a strong correlation with BMD. The functional annotation results of the DEGs showed that the diagnostic signature might affect the occurrence and development of PMOP through multiple biological pathways. In addition, 5 candidate molecules related to diagnostic signatures were screened out. Conclusion: Our diagnostic signature can effectively predict the risk of PMOP, with potential application for clinical decisions and drug candidate selection.

Artemisinin Cocrystals for Bioavailability Enhancement. Part 2: In Vivo Bioavailability and Physiologically Based Pharmacokinetic Modeling.

We report the evaluation and prediction of the pharmacokinetic (PK) performance of artemisinin (ART) cocrystal formulations, that is, 1:1 artemisinin/orcinol (ART-ORC) and 2:1 artemisinin/resorcinol (ART2-RES), using in vivo murine animal and physiologically based pharmacokinetic (PBPK) models. The efficacy of the ART cocrystal formulations along with the parent drug ART was tested in mice infected with Plasmodium berghei. When given at the same dose, the ART cocrystal formulation showed a significant reduction in parasitaemia at day 4 after infection compared to ART alone. PK parameters including Cmax (maximum plasma concentration), Tmax (time to Cmax), and AUC (area under the curve) were obtained by determining drug concentrations in the plasma using liquid chromatography-high-resolution mass spectrometry (LC-HRMS), showing enhanced ART levels after dosage with the cocrystal formulations. The dose-response tests revealed that a significantly lower dose of the ART cocrystals in the formulation was required to achieve a similar therapeutic effect as ART alone. A PBPK model was developed using a PBPK mouse simulator to accurately predict the in vivo behavior of the cocrystal formulations by combining in vitro dissolution profiles with the properties of the parent drug ART. The study illustrated that information from classical in vitro and in vivo experimental investigations of the parent drug of ART formulations can be coupled with PBPK modeling to predict the PK parameters of an ART cocrystal formulation in an efficient manner. Therefore, the proposed modeling strategy could be used to establish in vitro and in vivo correlations for different cocrystals intended to improve dissolution properties and to support clinical candidate selection, contributing to the assessment of cocrystal developability and formulation development.

Why All the Fury over Furin?

Analysis of the SARS-CoV-2 sequence revealed a multibasic furin cleavage site at the S1/S2 boundary of the spike protein distinguishing this virus from SARS-CoV. Furin, the best-characterized member of the mammalian proprotein convertases, is an ubiquitously expressed single pass type 1 transmembrane protein. Cleavage of SARS-CoV-2 spike protein by furin promotes viral entry into lung cells. While furin knockout is embryonically lethal, its knockout in differentiated somatic cells is not, thus furin provides an exciting therapeutic target for viral pathogens including SARS-CoV-2 and bacterial infections. Several peptide-based and small-molecule inhibitors of furin have been recently reported, and select cocrystal structures have been solved, paving the way for further optimization and selection of clinical candidates. This perspective highlights furin structure, substrates, recent inhibitors, and crystal structures with emphasis on furin’s role in SARS-CoV-2 infection, where the current data strongly suggest its inhibition as a promising therapeutic intervention for SARS-CoV-2.

Combining Laser Resurfacing and Facial Rejuvenation Surgery.

Facial aging is a multifactorial process involving skin laxity, volume loss, facial rhytids and solar elastosis. Surgical interventions can address the laxity of the skin and restore volume loss but do not fully correct the texture of the skin. Historically, there were concerns over the safety of combining laser resurfacing with facelifting procedures, but now there is much evidence to support the safe use of this combination treatment. When done appropriately, the combination of laser resurfacing with facial rejuvenation surgery can provide a synergistic effect for optimal clinical results. Here, we review the many laser options available and how they may be combined safely with surgical facial rejuvenation. We also review clinical contexts and candidate selection to provide safe, reliable results and minimize complications.

Managing the Impact of Immunogenicity in an Era of Immunotherapy: From Bench to Bedside.

Biotherapeutics have revolutionized our ability to treat life-threatening diseases. Despite clinical success, the use of biotherapeutics has sometimes been limited by the immune response mounted against them in the form of anti-drug antibodies (ADAs). The multifactorial nature of immunogenicity has prevented a standardized approach for assessing this and each of the assessment methods developed so far does not exhibit high enough reliability to be used alone, due to limited predictiveness. This prompted the Roche Pharma Research and Early Development (pRED) Immunogenicity Working Group to establish an internal preclinical immunogenicity toolbox of in vitro/in vivo approaches and accompanying guidelines for a harmonized assessment and management of immunogenicity in early development. In this article, the complex factors influencing immunogenicity and their associated clinical ramifications are discussed to highlight the importance of an end-to-end approach conducted from lead optimization to clinical candidate selection. We then examine the impact of the resulting lead candidate categorization on the design and implementation of a multi-tiered ADA/immunogenicity assay strategy prior to phase I (entry into human) through early clinical development. Ultimately, the Immunogenicity Toolbox ensures that Roche pRED teams are equipped to address immunogenicity in a standardized manner, paving the way for lifesaving products with improved safety and efficacy.

CD28 Co-Stimulus Achieves Superior CAR T Cell Effector Function against Solid Tumors Than 4-1BB Co-Stimulus.

Simple SummaryEfficient trafficking and survival of CAR T cells within the hostile tumor microenvironment are important prerequisites for potent solid tumor attack that have not yet been achieved. We deployed monospecific murine instead of polyclonal human T cells for CAR T cell generation to evaluate second generation L1CAM- and HER2-specific CARs with different spacer length and either the CD28 or 4-1BB co-stimulatory domain in mouse models of neuroblastoma and ovarian carcinoma. This mouse-in-mouse approach ensured CAR T cell trafficking unhindered by species-specific discrepancies and demonstrated superior solid tumor attack by CAR T cells harboring the CD28 compared to 4-1BB co-stimulatory domain. Our approach has the potential to improve prediction and selection of promising clinical CAR candidates against solid tumors in the future.Spacer or co-stimulatory components in chimeric antigen receptor (CAR) design influence CAR T cell effector function. Few preclinical mouse models optimally support CAR candidate pre-selection for clinical development. Here we use a model in which murine CAR T cells can be exploited with human tumor xenografts. This mouse-in-mouse approach avoids limitations caused by species-specific factors crucial for CAR T cell survival, trafficking and function. We compared trafficking, expansion and tumor control for T cells expressing different CAR construct designs targeting two antigens (L1CAM or HER2), structurally identical except for spacer (long or short) or co-stimulatory (4-1BB or CD28) domains to be evaluated. Using monoclonal, murine-derived L1CAM-specific CAR T cells in Rag-/- mice harboring established xenografted tumors from a human neuroblastoma cell line revealed a clear superiority in CAR T cell trafficking using CD28 co-stimulation. L1CAM-targeting short spacer-CD28/ζ CAR T cells expanded the most at the tumor site and induced initial tumor regression. Treating patient-derived neuroblastoma xenografts with human L1CAM-targeting CAR T cells confirmed the superiority of CD28 co-stimulus. CD28 superiority was also demonstrated with HER2-specific CAR T cells (targeting ovarian carcinoma xenografts). Our findings encourage incorporating CD28 signaling into CAR design for adoptive T cell treatment of solid tumors.

Abstract 5051: RenMab Mouse: A leading platform for fully human antibody generation

Abstract With the development of immune-oncology, therapeutic antibodies have been proven to be extraordinarily effective for cancer treatment. Conventional human antibody discovery process can be divided into steps including target selection, target validation, screening preparation, hits generation, leads selection, lead optimization, and clinical candidate selection. To accelerate antibody development process, Biocytogen has developed RenMab Mouse, a fully human antibody mouse whose entire variable regions were replaced by human Ig heavy chain and κ light chain through Biocytogen's unique Mb-scale chromosome engineering technology. RenMab Mouse provides a remarkably efficient therapeutic antibody discovery platform for fully human antibody generation, characterization, and rapid in vivo efficacy screening. We have characterized RenMab Mouse with the following features. 1. The entire mouse variable regions were replaced with complete human genome DNA in situ for complete human &amp; mouse regulatory elements. The gene regulation of RenMab mouse is highly consistent with that of human. 2. Mouse constant region remains to ensure normal immune cell population, development and maturation. Immune system of RenMab Mouse has been proven to be almost identical to that of wild type mice. RenMab Mouse showed normal antibody immune responses to antigens. 3. RenMab Mouse generates a highly diverse repertoire of fully human antibody variable regions through V(D)J recombination. This capability can lead to improved hit rates in antibody drug discovery. In conclusion, RenMab Mouse, a fully human antibody mouse developed by Biocytogen's chromosome engineering technology, has been proven to exhibit normal immune cell profile and rapid immune response. Combined with existing inventory of single, double or triple target humanized mouse models and a world-leading proprietary gene editing platform, Biocytogen is dedicated to providing a one-stop solution for antibody discovery and development from target validation to IND application. Citation Format: Gary Ng, Huizhen Zhao, Yabo Zhang, Lili Liu, Hui Lu, Shuwen Huang, Yuelei Shen. RenMab Mouse: A leading platform for fully human antibody generation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5051.

Medicines discovery for auditory disorders: Challenges for industry.

Currently, no approved medicines are available for the prevention or treatment of hearing loss. Pharmaceutical industry productivity across all therapeutic indications has historically been disappointing, with a 90% chance of failure in delivering a marketed drug after entering clinical evaluation. To address these failings, initiatives have been applied in the three cornerstones of medicine discovery: target selection, clinical candidate selection, and clinical studies. These changes aimed to enable data-informed decisions on the translation of preclinical observations into a safe, clinically effective medicine by ensuring the best biological target is selected, the most appropriate chemical entity is advanced, and that the clinical studies enroll the correct patients. The specific underlying pathologies need to be known to allow appropriate patient selection, so improved diagnostics are required, as are methodologies for measuring in the inner ear target engagement, drug delivery and pharmacokinetics. The different therapeutic strategies of protecting hearing or preventing hearing loss versus restoring hearing are reviewed along with potential treatments for tinnitus. Examples of current investigational drugs are discussed to highlight key challenges in drug discovery and the learnings being applied to improve the probability of success of launching a marketed medicine.

Application of the Tissue Composition–Based Model to Minipig for Predicting the Volume of Distribution at Steady State and Dermis-to-Plasma Partition Coefficients of Drugs Used in the Physiologically Based Pharmacokinetics Model in Dermatology

The minipig continues to build a reputation as a viable alternative large animal model to predict humans in dermatology and toxicology studies. Therefore, it is essential to describe and predict the pharmacokinetics in that species to speed up the clinical candidate selection. Essential input parameters in whole-body physiologically based pharmacokinetic models are the tissue-to-plasma partition coefficients and the resulting volume of distribution at steady-state (Vss). Mechanistic in vitro– and in silico–based models used for predicting these parameters of tissue distribution of drugs refer to the tissue composition–based model (TCM). Robust TCMs were initially developed for some preclinical species (e.g., rat and dog) and human; however, there is currently no model available for the minipig. Therefore, the objective of this present study was to develop a TCM for the minipig and to estimate the corresponding tissue composition data. Drug partitioning into the tissues was predominantly governed by lipid and protein binding effects in addition to drug solubilization and pH gradient effects in the aqueous phase on both sides of the biological membranes; however, some more complex tissue distribution processes such as drug binding to the collagen-laminin material in dermis and a restricted drug partitioning into membranes of tissues for compounds that are amphiphilic and contain sulfur atom(s) were also challenged. The model was validated by predicting Vss and the dermis-to-plasma partition coefficients (Kp-dermis) of 68 drugs. The prediction of Kp-dermis was extended to humans for comparison with the minipig. The results indicate that the extended TCM provided generally good agreements with observations in the minipig showing that it is also applicable to this preclinical species. In general, up to 86% and 100% of the predicted Vss values are respectively within 2-fold and 3-fold errors compared to the experimentally determined values, whereas these numbers are 78% and 94% for Kp-dermis when the anticipated outlier compounds are not included. Binding data to dermis are comparable between minipigs and humans. Overall, this study is a first step toward developing a mechanistic TCM for the minipig, with the aim of increasing the use of physiologically based pharmacokinetic models of drugs for that species in addition to rats, dogs, and humans because such models are used in preclinical and clinical transdermal studies.

The Role of Drug Metabolites in the Inhibition of Cytochrome P450 Enzymes.

Following the drug administration, patients are exposed not only to the parent drug itself, but also to the metabolites generated by drug-metabolizing enzymes. The role of drug metabolites in cytochrome P450 (CYP) inhibition and subsequent drug-drug interactions (DDIs) have recently become a topic of considerable interest and scientific debate. The list of metabolites that were found to significantly contribute to clinically relevant DDIs is constantly being expanded and reported in the literature. New strategies have been developed for better understanding how different metabolites of a drug candidate contribute to its pharmacokinetic properties and pharmacological as well as its toxicological effects. However, the testing of the role of metabolites in CYP inhibition is still not routinely performed during the process of drug development, although the evaluation of time-dependent CYP inhibition during the clinical candidate selection process may provide information on possible effects of metabolites in CYP inhibition. Due to large number of compounds to be tested in the early stages of drug discovery, the experimental approaches for assessment of CYP-mediated metabolic profiles are particularly resource demanding. Consequently, a large number of in silico or computational tools have been developed as useful complement to experimental approaches. In summary, circulating metabolites may be recognized as significant CYP inhibitors. Current data may suggest the need for an optimized effort to characterize the inhibitory potential of parent drugs metabolites on CYP, as well as the necessity to develop the advanced in vitro models that would allow a better quantitative predictive value of in vivo studies.

Pfizer, Wave team on metabolic therapy

Pfizer and Wave Life Sciences are joining to develop nucleic acid therapies targeting metabolic diseases. The partners will combine Wave’s stereopure drug development platform with Pfizer’s hepatic targeting technology for enhancing delivery to the liver. Pfizer will advance up to five programs from discovery through selection of clinical candidates, at which point it can license programs. Two targets have been declared. Wave will get $40 million up front and could take in $187 million if all five programs are successful.

Toward an Evidence-Based Nonclinical Road Map for Evaluating the Efficacy of New Tuberculosis (TB) Drug Regimens: Proceedings of a Critical Path to TB Drug Regimens-National Institute of Allergy and Infectious Diseases In Vivo Pharmacology Workshop for TB Drug Development.

Novel tuberculosis (TB) drug regimens are urgently needed, and their development will be enabled by improved preclinical approaches that more effectively inform and ensure safe selection of clinical candidates and drug combination/regimens. An evidence-based approach for the assessment of nonclinical models supporting TB drug development has been proposed by a joint partnership between the National Institute of Allergy and Infectious Diseases (NIAID) and the Critical Path to TB Drug Regimens (CPTR) Consortium.

Dose-response curve slope helps predict therapeutic potency and breadth of HIV broadly neutralizing antibodies.

A new generation of HIV broadly neutralizing antibodies (bnAbs) with remarkable potency, breadth and epitope diversity has rejuvenated interest in immunotherapeutic strategies. Potencies defined by in vitro IC50 and IC80 values (50 and 80% inhibitory concentrations) figure prominently into the selection of clinical candidates; however, much higher therapeutic levels will be required to reduce multiple logs of virus and impede escape. Here we predict bnAb potency at therapeutic levels by analysing dose–response curve slopes, and show that slope is independent of IC50/IC80 and specifically relates to bnAb epitope class. With few exceptions, CD4-binding site and V3-glycan bnAbs exhibit slopes >1, indicative of higher expected therapeutic effectiveness, whereas V2-glycan, gp41 membrane-proximal external region (MPER) and gp120–gp41 bnAbs exhibit less favourable slopes <1. Our results indicate that slope is one major predictor of both potency and breadth for bnAbs at clinically relevant concentrations, and may better coordinate the relationship between bnAb epitope structure and therapeutic expectations.

Abstract 2583: Refinement of inhibitors of the KRAS-signaling naocluster protein, CNKSR1, that block oncogenic KRAS signaling and growth

Abstract Introduction: KRAS, the predominant form of mutated RAS (mut-KRAS), is found in ∼25% of patient tumors across many cancer types and plays a critical role in driving tumor growth and resistance to therapy. CNKSR1 (connector enhancer of kinase suppressor of Ras 1) a protein associated with KRAS in the RAS membrane signaling nanocluster and we have shown it is critical for mut-KRAS but not wild type (wt)-KRAS signaling and cell proliferation. CNKSR1 is a multi-domain protein with a pleckstrin homology (PH) domain, which we have exploited to discover inhibitors that selectively inhibit mut-KRAS tumor growth. The initial leads identified were found to inhibit mut-KRAS signaling and growth of mut-KRAS isogenic NSCLC cell lines (IC50 ∼ 30 μM) and compound refinement provided PHT-7390 and 7391 (IC50 5 to 10 μM). Importantly, they did not block the growth of wt-KRAS cell lines at concentrations up to 100 μM. In mice the agents had antitumor activity againsta mut-KRAS xenograft model and improved the activity of EGFR inhibitors without observable toxicity, but had poor pharmacokinetic properties.Methods: PHusis has used its in silico computational modeling of the PH-domain of CNK1 to optimize its lead inhibitors to improve potency and pharmacokinetic properties. In silico compound refinement has been paired with surface plasmon resonance spectroscopy (SPR) and in vitro cell line screening to provide the latest panel of highly potent CNK1 inhibitors that are moving toward late preclinical development. Results: A panel of analogues of PHT-7390 and 7391 were synthesized with the aim of improving potency and pharmacokinetic properties. This structural optimization yielded several analogues with up to 100 fold greater potency than PHT-7390 to mut-KRAS NSCLC lines (IC50 10-100 nM) while retaining the differential in effect against wt-KRAS lines without activity up to 100 μM. Target engagement studies have shown that the agents inhibit mut-KRAS signaling in similar fashion as KRAS knockdown with siRNA, confirming on target effects in cells. Pharmacokinetic properties were also improved and agents have moved to clinical candidate selection. Conclusion: Potent and specific inhibitors of CNK1 PH-domain with improved pharmacokinetics have been identified. These novel PH-domain inhibitors now provide a therapeutic potential for patients with oncogenic KRAS for which there is currently no effective therapy. Citation Format: D. Lynn Kirkpatrick, Martin Indarte, Mike Scott, Assael Madrigal, Geoffrey Grandjean, Garth Powis. Refinement of inhibitors of the KRAS-signaling naocluster protein, CNKSR1, that block oncogenic KRAS signaling and growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2583. doi:10.1158/1538-7445.AM2015-2583