Preclinical Assays Research Articles

Analyses of Off-Target Effects on Cardiac and Skeletal Muscles by Berberine, a Drug Used to Treat Cancers and Induce Weight Loss.

Previous reports from our laboratory describing the formation of myofibrils in cultured embryonic cardiac and skeletal muscle cells have proposed that myofibrillogenesis occurs in three steps of increasing protein organization: beginning with premyofibrils, followed by nascent myofibrils, and ending in mature myofibrils. Inhibitors of the ubiquitin proteasome system (UPS) prevented nascent myofibrils from progressing directly to mature myofibrils in cultured cardiac and skeletal muscle cells, supporting a three-step model of assembly in which some of the proteins in nascent myofibrils are proteolyzed to allow the assembly of mature myofibrils. Application of UPS inhibitors on cultured muscle cells suggests possible explanations for the off-target cardiac and skeletal muscle adverse effects of UPS drugs, which are used on cancer patients. Berberine, a plant derivative, has been used to treat various cancers, including multiple myelomas. In contrast to the use of UPS drugs, success was reported with Berberine in multiple myeloma patients with no off-target effects on their hearts. We have exposed cultured cardiac and skeletal muscle cells to Berberine, a ligase inhibitor of UHRF1 (ubiquitin-like with PHD and RING finger domains). Berberine inhibited myofibril assembly at the nascent myofibril stage in embryonic skeletal muscle cells but had no effect in the assembly of mature myofibrils in embryonic heart cells. RT-PCR experiments demonstrated Berberine inhibition of mRNA for muscle myosin II heavy chains but not for muscle actin mRNA in skeletal muscle cells. Berberine is also being used as a popular weight losing compound, because it is much cheaper and available without a prescription than the semaglutide containing weight losing drugs (Wegovy and Ozempic). In contrast to Berberine, semaglutide had no effects on myofibril assembly in culture assays for both cardiac and skeletal muscle cells. We postulate that analyses of cultured embryonic cardiac and skeletal muscle cells will provide a preclinical assay for the testing of novel cancer drugs with improved outcomes for patients, an important goal for cancer therapeutics.

Classification of psychedelics and psychoactive drugs based on brain-wide imaging of cellular c-Fos expression.

Psilocybin, ketamine, and MDMA are psychoactive compounds that exert behavioral effects with distinguishable but also overlapping features. The growing interest in using these compounds as therapeutics necessitates preclinical assays that can accurately screen psychedelics and related analogs. We posit that a promising approach may be to measure drug action on markers of neural plasticity in native brain tissues. We therefore developed a pipeline for drug classification using light sheet fluorescence microscopy of immediate early gene expression at cellular resolution followed by machine learning. We tested male and female mice with a panel of drugs, including psilocybin, ketamine, 5-MeO-DMT, 6-fluoro-DET, MDMA, acute fluoxetine, chronic fluoxetine, and vehicle. In one-versus-rest classification, the exact drug was identified with 67% accuracy, significantly above the chance level of 12.5%. In one-versus-one classifications, psilocybin was discriminated from 5-MeO-DMT, ketamine, MDMA, or acute fluoxetine with >95% accuracy. We used Shapley additive explanation to pinpoint the brain regions driving the machine learning predictions. Our results support a novel approach for characterizing and validating psychoactive drugs with psychedelic properties.

8595 Brain Penetration Impedes SERD Efficacy in Breast Cancer Pre-clinical Models

Abstract Disclosure: S.E. Wardell: None. B. Chakraborty: None. S. Artham: None. M.A. Newlin: None. M. Kirkland: None. C. Chang: None. D.P. McDonnell: None. Most breast cancers express the estrogen receptor (ER, ESR1), and agents that target ER remain the cornerstone of interventions used to treat this disease at all stages. Aromatase inhibitors (AI), which block estrogen synthesis, and the selective ER downregulator (SERD) fulvestrant were developed with the goal of achieving absolute inhibition of ER signaling in tumors. However, it has been assumed that the clinical efficacy (and utility) of fulvestrant is somewhat limited by its poor pharmaceutical properties and the requirement that it be administered by intramuscular injection. This, together with the observation that the activating ESR1 mutations selected for during AI treatment are not effectively inhibited by fulvestrant, encouraged the development of new classes of SERDs that achieve sufficient exposure to saturate and degrade ER in tumors. The recent approval of the second-generation SERD elacestrant for the treatment of breast cancers harboring ESR1 mutations was a major advance in approaches to target ER in advanced breast cancer. Several additional oral SERDs have been developed and evaluated as potential treatments for advanced metastatic breast cancer. Despite showing substantial efficacy in pre-clinical assays that assess ER downregulation and gene transcription, and notably equivalent antitumor activities in cell line derived and PDX tumors in immunocompromised mice, the clinical development of the majority of these SERDs has been discontinued for lack of efficacy. In reviewing the available clinical data across many SERD programs, we noted that some drugs demonstrated a paradoxical, non-linear response curve with higher doses exhibiting less efficacy than lower doses. Probing this pharmacology in animal models we determined that the ability of SERDs to cross the blood brain barrier and attenuate ER signaling in the hypothalamus was a key differentiator of SERDs. In validated syngeneic models of breast cancer, we observed doses of fulvestrant having confirmed brain exposure stimulated tumor growth independent of tumor ER-status. We evaluated the ability of several next generation SERDs for their brain penetrance and demonstrated that SERD presence in the brain was associated with a stimulatory effect on tumor growth. Interestingly, administration of a low dose of 17b-estradiol (E2) together with a brain excluded SERD further repressed tumor growth, highlighting a potentially beneficial anti-tumor role for estrogen signaling in the brain. Furthermore, combination of a brain excluded SERM with E2 also resulted in increased tumor growth repression; thus, ER downregulation is dispensable for the cooperative activity observed for estrogens administered together with a peripheral ER antagonist. These data suggest that differences in tissue pharmacology, as opposed to differences in ability to degrade ER, is likely to be a major differentiator of ER modulators. Presentation: 6/3/2024

Quality by Design for Preclinical In Vitro Assay Development.

Quality by Design (QbD) is an approach to assay development to determine the design space, which is the range of assay variable settings that should result in satisfactory assay quality. Typically, QbD is applied in manufacturing, but it works just as well in the preclinical space. Through three examples, we illustrate the QbD approach with experimental design and associated data analysis to determine the design space for preclinical assays.

Mycobacterium abscessus strain variability in preclinical drug development: does it really matter?

Abstract Background New treatment options for Mycobacterium abscessus infections are urgently needed. Since a correlation between MICs and clinical outcomes is not clearly established, potency of novel drugs needs to be evaluated using additional in vitro drug activity assays. Preclinical drug activity assays generally use the M. abscessus type strain ATCC 19977. However, M. abscessus complex entails a genetically and morphologically diverse group, and it is questionable whether drug activity observed against ATCC 19977 is representative of drug activity against clinical M. abscessus isolates. Objectives To assess whether the relationship between MIC and the quantitative antimycobacterial activity of amikacin, imipenem and clofazimine differs between the ATCC 19977 strain and clinical M. abscessus isolates. Methods Experiments were performed with M. abscessus ATCC 19977 and a subset of six clinical isolates covering the three M. abscessus subspecies and the smooth and rough morphotypes. Cultures were exposed to the drugs at 4-fold increasing, MIC-standardized concentrations, and the mycobacterial load was assessed over time. Results Concentration- and time-dependent activity of amikacin, imipenem and clofazimine against the six clinical isolates was similar. Only slight variations in drug activity were observed between ATCC 19977 and clinical isolates. Conclusions Time- and concentration-dependent drug activity against the ATCC 19977 strain seems indicative for in vitro drug behaviour against M. abscessus complex clinical isolates. Including one clinical smooth morphotype isolate alongside ATCC 19977 seems appropriate for reliable interpretation of this particular in vitro drug activity assay as part of the M. abscessus preclinical drug development pipeline.

Developmental interplay between transcriptional alterations and a targetable cytokine signaling dependency in pediatric ETO2::GLIS2 leukemia

BackgroundSeveral fusion oncogenes showing a higher incidence in pediatric acute myeloid leukemia (AML) are associated with heterogeneous megakaryoblastic and other myeloid features. Here we addressed how developmental mechanisms influence human leukemogenesis by ETO2::GLIS2, associated with dismal prognosis.MethodsWe created novel ETO2::GLIS2 models of leukemogenesis through lentiviral transduction and CRISPR-Cas9 gene editing of human fetal and post-natal hematopoietic stem/progenitor cells (HSPCs), performed in-depth characterization of ETO2::GLIS2 transformed cells through multiple omics and compared them to patient samples. This led to a preclinical assay using patient-derived-xenograft models to test a combination of two clinically-relevant molecules.ResultsWe showed that ETO2::GLIS2 expression in primary human fetal CD34+ hematopoietic cells led to more efficient in vivo leukemia development than expression in post-natal cells. Moreover, cord blood-derived leukemogenesis has a major dependency on the presence of human cytokines, including IL3 and SCF. Single cell transcriptomes revealed that this cytokine environment controlled two ETO2::GLIS2-transformed states that were also observed in primary patient cells. Importantly, this cytokine sensitivity may be therapeutically-exploited as combined MEK and BCL2 inhibition showed higher efficiency than individual molecules to reduce leukemia progression in vivo.ConclusionsOur study uncovers an interplay between the cytokine milieu and transcriptional programs that extends a developmental window of permissiveness to transformation by the ETO2::GLIS2 AML fusion oncogene, controls the intratumoral cellular heterogeneity, and offers a ground-breaking therapeutical opportunity by a targeted combination strategy.

Reevaluating safety pharmacology respiratory studies within the ICH S7A core battery: A multi-company evaluation of preclinical utility and clinical translation

Optimization of ICH safety guideline studies for inclusion into regulatory submissions is critical for resource conservation, animal use reduction, and efficient drug development. The ICH S7A guidance for Safety Pharmacology (SP) studies adopted in 2001 identified the core battery of studies to evaluate the acute safety of putative pharmaceutical molecules prior to First in Human (FIH) trials. To assess the utility of respiratory studies in predicting clinical AE's, seven pharmaceutical companies pooled preclinical and clinical respiratory findings. A large database of novel molecules included all relevant data from standard S7A respiratory (n = 459) and FIH studies (n = 309). The data were analyzed with respect to the progression of these molecules, clinical adverse event reporting of these same molecules, and achieved exposures. These S7A respiratory assay findings had no impact on compound progression, and only 12 of 309 drug candidates were ‘positive’ preclinically and reported a respiratory-related AE in clinical trials (i.e. cough, dyspnea, etc.), an overall incidence rate of 3.9%. Contingency tables/statistics support a lack of concordance of these preclinical assays. Overall, our extensive analysis clearly indicated that the preclinical respiratory assay fails to provide any prognostic value for detecting clinically relevant respiratory adverse events.

Rapid liver self-recovery: A challenge for rat models of tissue damage

Animal models, mainly murine, stay as a fundamental resource in diverse research pursuits, notably contributing to significant strides in discovering novel treatments for therapeutic applications. Preclinical assays must consider the existence of self-recovery mechanisms in the murine species to achieve a well-designed control group. This study focuses on unveiling the innate rapid regenerative capacity of rat liver by utilizing the thioacetamide-induced sub-chronic liver injury model. Employing histopathological, biochemical, and molecular liver function tests, we assessed the recovery of liver tissue functionality. Moreover, animals were housed with voluntary running wheels and locomotory activity was recorded and employed as an indirect index of overall animal recuperation. Remarkably, basal locomotory activity reestablished to normal levels only two weeks post-thioacetamide exposure. Our results raise vital considerations about the importance of temporal synchronicity in comparative assays to validate the real action of treatments, emphasizing the role of the rapid rat liver endogenous self-recovery.

Novel Penicillin Derivatives Against Selected Multiple-drug Resistant Bacterial Strains: Design, Synthesis, Structural Analysis, In Silico and In Vitro Studies.

The rising numbers of multiple drug-resistant (MDR) pathogens and the consequent antibacterial therapy failure that resulted in severe medical conditions push to illustrate new molecules with extended activity against the resistant strains. In this manner, chemical derivatization of known antibiotics is proposed to save efforts in drug discovery, and penicillins serve as an ideal in this regard. Seven synthesized 6-aminopenicillanic acid-imine derivatives (2a-g) were structure elucidated using FT-IR, 1H NMR, 13C NMR, and MS spectroscopy. In silico molecular docking and ADMET studies were made. The analyzed compounds obeyed Lipinski's rule of five and showed promising in vitro bactericidal potential when assayed against E. coli, E. cloacae, P. aeruginosa, S. aureus, and A. baumannii. MDR strains using disc diffusion and microplate dilution techniques. The MIC values were 8 to 32 μg/mL with more potency than ampicillin, explained by better membrane penetration and more ligand-protein binding capacity. The 2g entity was active against E. coli. This study was designed to find new active penicillin derivatives against MDR pathogens. The products showed antibacterial activity against selected MDR species and good PHK, PHD properties, and low predicted toxicity, offering them as future candidates that require further preclinical assays.

Sex differences in the social motivation of rats: Insights from social operant conditioning, behavioural economics, and video tracking

BackgroundSocial behaviour plays a key role in mental health and wellbeing, and developing greater understanding of mechanisms underlying social interaction—particularly social motivation—holds substantial transdiagnostic impact. Common rodent behavioural assays used to assess social behaviour are limited in their assessment of social motivation, whereas the social operant conditioning model can provide unique and valuable insights into social motivation. Further characterisation of common experimental parameters that may influence social motivation within the social operant model, as well as complementary methodological and analytical approaches, are warranted.MethodsThis study investigated the effects of biological sex, housing condition, and time-of-day, on social motivation using the social operant model. This involved training rats to lever press (FR1) for 60-s access to a social reward (same-sex conspecific stimulus). Subjects were male and female Wistar rats, housed under individual or paired conditions, and sessions were conducted either in the mid-late light phase (ZT6-10) or early-mid dark phase (ZT13-17). A behavioural economics approach was implemented to measure social demand and the influence of stimulus partner sex (same- vs. opposite-sex stimulus) on social operant responding. Additionally, video tracking analyses were conducted to assess the degree of convergence between social appetitive and consummatory behaviours.ResultsBiological sex, housing conditions, the interaction between sex and housing, and stimulus partner sex potently influenced social motivation, whereas time-of-day did not. Behavioural economics demonstrated that sex, housing, and their interaction influence both the hedonic set-point and elasticity of social demand. Video analysis of social interaction during social operant sessions revealed that social appetitive and consummatory behaviours are not necessarily convergent, and indicate potential social satiety. Lastly, oestrus phase of female experimental and stimulus rats did not impact social motivation within the model.ConclusionsSocial isolation-dependent sex differences exist in social motivation for rats, as assessed by social operant conditioning. The social operant model represents an optimal preclinical assay that comprehensively evaluates social motivation and offers a platform for future investigations of neurobiological mechanisms underlying sex differences in social motivation. These findings highlight the importance of continued consideration and inclusion of sex as a biological variable in future social operant conditioning studies.Plain English summaryHumans are social creatures—our everyday interactions with others and the support this provides play a key role in our wellbeing. For those experiencing mental health conditions, people’s motivation to engage with others can wane, which can lead them to withdraw from those who support them. Therefore, to develop better treatment strategies for these conditions, we need to gain a deeper understanding of social motivation. Studying social behaviour in animals can facilitate this investigation of social motivation as it allows for a causal understanding of underlying neurobiology that is not possible in human experiments. An optimal way to study social motivation in animals is using the social operant conditioning model, where rats learn to press a lever that opens a door and allows them to interact with another rat for a short time. This study characterised the social operant model by testing whether sex, housing conditions, time-of-day, and the sex of the stimulus partner influence rats’ motivation to seek interaction with another rat. We found that female rats were more socially motivated than males, and that rats living alone were more motivated than those living with another rat; interestingly, this effect of housing affected females more than males. Regardless of sex, rats were more motivated to interact with a rat of the opposite sex. These findings provide insights into sex differences in social motivation in rats and new insights into the social operant model which will help guide future research into social motivation and other mental health conditions.

Delocalized quinolinium-macrocyclic peptides, an atypical chemotype for CNS penetration.

Macrocyclic drugs can address an increasing range of molecular targets but enabling central nervous system (CNS) access to these drugs has been viewed as an intractable problem. We designed and synthesized a series of quinolinium-modified cyclosporine derivatives targeted to the mitochondrial cyclophilin D protein. Modification of the cation to enable greater delocalization was confirmed by x-ray crystallography of the cations. Critically, greater delocalization improved brain concentrations. Assessment of the compounds in preclinical assays and for pharmacokinetics identified a molecule JP1-138 with at least 20 times the brain levels of a non-delocalized compound or those reported for cyclosporine. Levels were maintained over 24 hours together with low hERG potential. The paradigm outlined here could have widespread utility in the treatment of CNS diseases.

Synthesis, Anticancer Activity, and Docking Studies of Novel Hydroquinone-Chalcone-Pyrazoline Hybrid Derivatives.

A novel series of antitumor hybrids was synthesized using 1,4-benzohydroquinone and chalcone, furane, or pyrazoline scaffolds. This were achieved through isosteric substitution of the aryl group of the chalcone β-carbon with the furanyl moiety and structural modification of the α,β-unsaturated carbonyl system. The potential antitumor activity of these hybrids was evaluated in vivo on MCF-7 breast adenocarcinoma and HT-29 colorectal carcinoma cells, demonstrating cytotoxic activity with IC50 values ranging from 28.8 to 124.6 µM. The incorporation of furan and pyrazoline groups significantly enhanced antiproliferative properties compared to their analogues and precursors (VII-X), which were inactive against both neoplastic cell lines. Compounds 4, 5, and 6 exhibited enhanced cytotoxicity against both cell lines, whereas compound 8 showed higher cytotoxic activity against HT-29 cells. Molecular docking studies revealed superior free-energy values (ΔGbin) for carcinogenic pathway-involved kinase proteins, with our in silico data suggesting that these derivatives could be promising chemotherapeutic agents targeting kinase pathways. Among all the synthesized PIBHQ compounds, derivatives 7 and 8 exhibited the best drug-likeness properties, with values of 0.53 and 0.83, respectively. ADME results collectively suggest that most of these compounds hold promise as potential candidates for preclinical assays.

Immune-related adverse events of antibody-based biological medicines in cancer therapy.

Recombinant antibodies (Abs) are an integral modality for the treatment of multiple tumour malignancies. Since the Food and Drug Administration (FDA) approval of rituximab as the first monoclonal antibody (mAb) for cancer treatment, several mAbs and antibody (Ab)-based therapies have been approved for the treatment of solid tumour malignancies and other cancers. These Abs function by either blocking oncogenic pathways or angiogenesis, modulating immune response, or by delivering a conjugated drug. The use of Ab-based therapy in cancer patients who could benefit from the treatment, however, is still limited by associated toxicity profiles which may stem from biological features and processes related to target binding, alongside biochemical and/or biophysical characteristics of the therapeutic Ab. A significant immune-related adverse event (irAE) associated with Ab-based therapies is cytokine release syndrome (CRS), characterized by the development of fever, rash and even marked, life-threatening hypotension, and acute inflammation with secondary to systemic uncontrolled increase in a range of pro-inflammatory cytokines. Here, we review irAEs associated with specific classes of approved, Ab-based novel cancer immunotherapeutics, namely immune checkpoint (IC)-targeting Abs, bispecific Abs (BsAbs) and Ab-drug-conjugates (ADCs), highlighting the significance of harmonization in preclinical assay development for safety assessment of Ab-based biotherapeutics as an approach to support and refine clinical translation.

Review on Pre-clinical Antimicrobial Assay

Review on Pre-clinical Antimicrobial Assay

Epidermal Growth Factor Receptor Targeting in Colorectal Carcinoma: Antibodies and Patient-Derived Organoids as a Smart Model to Study Therapy Resistance.

Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide. Therefore, the need for new therapeutic strategies is still a challenge. Surgery and chemotherapy represent the first-line interventions; nevertheless, the prognosis for metastatic CRC (mCRC) patients remains unacceptable. An important step towards targeted therapy came from the inhibition of the epidermal growth factor receptor (EGFR) pathway, by the anti-EGFR antibody, Cetuximab, or by specific tyrosine kinase inhibitors (TKI). Cetuximab, a mouse-human chimeric monoclonal antibody (mAb), binds to the extracellular domain of EGFR thus impairing EGFR-mediated signaling and reducing cell proliferation. TKI can affect the EGFR biochemical pathway at different steps along the signaling cascade. Apart from Cetuximab, other anti-EGFR mAbs have been developed, such as Panitumumab. Both antibodies have been approved for the treatment of KRAS-NRAS wild type mCRC, alone or in combination with chemotherapy. These antibodies display strong differences in activating the host immune system against CRC, due to their different immunoglobulin isotypes. Although anti-EGFR antibodies are efficient, drug resistance occurs with high frequency. Resistant tumor cell populations can either already be present before therapy or develop later by biochemical adaptations or new genomic mutations in the EGFR pathway. Numerous efforts have been made to improve the efficacy of the anti-EGFR mAbs or to find new agents that are able to block downstream EGFR signaling cascade molecules. Indeed, we examined the importance of analyzing the anti-EGFR antibody-drug conjugates (ADC) developed to overcome resistance and/or stimulate the tumor host's immunity against CRC growth. Also, patient-derived CRC organoid cultures represent a useful and feasible in vitro model to study tumor behavior and therapy response. Organoids can reflect tumor genetic heterogeneity found in the tissue of origin, representing a unique tool for personalized medicine. Thus, CRC-derived organoid cultures are a smart model for studying the tumor microenvironment and for the preclinical assay of anti-EGFR drugs.

Surface-Bioengineered Extracellular Vesicles Seeking Molecular Biotargets in Lung Cancer Cells.

Personalized medicine is a new approach to modern oncology. Here, to facilitate the application of extracellular vesicles (EVs) derived from lung cancer cells as potent advanced therapy medicinal products in lung cancer, the EV membrane was functionalized with a specific ligand for targeting purposes. In this role, the most effective heptapeptide in binding to lung cancer cells (PTHTRWA) was used. The functionalization process of EV surface was performed through the C- or N-terminal end of the heptapeptide. To prove the activity of the EVs functionalized with PTHTRWA, both a model of lipid membrane mimicking normal and cancerous cell membranes as well as human adenocarcinomic alveolar basal epithelial cells (A549) and human normal bronchial epithelial cells (BEAS-2B) have been exposed to these bioconstructs. Magnetic resonance imaging (MRI) showed that the as-bioengineered PTHTRWA-EVs loaded with superparamagnetic iron oxide nanoparticle (SPIO) cargos reach the growing tumor when dosed intravenously in NUDE Balb/c mice bearing A549 cancer. Molecular dynamics (MD) in silico studies elucidated a high affinity of the synthesized peptide to the α5β1 integrin. Preclinical safety assays did not evidence any cytotoxic or genotoxic effects of the PTHTRWA-bioengineered EVs.

Response to targeted and immune therapy in patients with NSCLC and BRAF non-V600E mutations: A retrospective, multicenter analysis within the national Network Genomic Medicine Lung Cancer (nNGM) in Germany.

8591 Background: Non-small-cell lung cancer (NSCLC) poses a significant global health challenge, with multiple treatment strategies targeting oncogenic drivers. The BRAF V600E mutation, found in 1-3% of NSCLC patients, is effectively targeted by dabrafenib and trametinib, achieving considerable clinical benefits. However, BRAF non-V600E mutations, constituting half of all BRAF mutations, are challenging to treat due to their heterogeneity and lack of information concerning outcome upon targeted and immune therapies. Methods: In order to investigate outcome of patients with BRAF non-V600E mutations upon targeted and (chemo)immune therapy we conducted a retrospective analysis within the national Network Genomic Medicine (nNGM) lung cancer in Germany and. In addition, we evaluated the MEK/ERK activation potential of the BRAF non-V600E mutations from our cohort and their sensitivity to dabrafenib and/or trametinib in vitro. Results: Twenty patients with 11 different BRAF non-V600E mutations were identified. From these patients, 15% achieved partial remission, 40% stable disease, and 40% progressive disease under TKI-therapy. Progression-free survival (PFS) upon targeted therapy ranged from 0.9 to 53.5 months. Notably, patients with MEK/ERK-activating BRAF non-V600E mutations exhibited persistent responses (mPFS 9 months). Specifically, patients with the high-activity mutants p.V600D, p.V600K and p.K601E showed responses to TKI therapy (mPFS 9.2 months). Similar outcome was achieved in patients with activating p.G466V (PFS 4.9 months), p.G469R (PFS 9 months), p.G469V (mPFS 28.2 months) and p.V600_K601delinsE (mPFS 5.6 months) mutations. Interestingly, outcome upon treatment with (chemo)immune therapy for these patients with activating mutations was similar compared to targeted therapy (mPFS 15.0 and 11.7 months). Expectedly, patients with kinase-dead mutants (p.G466R, p.D594E, p.D594G) were not responding to TKIs (mPFS 2.5 months) and had considerably better outcomes under (chemo)immune therapy (mPFS 11.0 and 11 months). Conclusions: Patients with activating BRAF non-V600E mutations can be treated efficiently with BRAF/MEK TKI or (chemo)immune therapy while patients with non-activating mutations do not benefit from targeted therapy but from (chemo)immune therapy. Hence, it is of key importance to apply preclinical activation assays showing good correlations to clinical outcomes in our study prior to applying targeted therapy in patients with unknown BRAF non-V600E mutations. Altogether, our study helps to guide personalized treatment decisions in this patient population.

Preclinical immunogenicity risk assessment of biotherapeutics using CD4 T cell assays.

T-cell dependent antibody responses to biotherapeutics remain a challenge to the optimal clinical application of biotherapeutics because of their capacity to impair drug efficacy and their potential to cause safety issues. To minimize this clinical immunogenicity risk, preclinical assays measuring the capacity of biotherapeutics to elicit CD4 T cell response in vitro are commonly used. However, there is considerable variability in assay formats and a general poor understanding of their respective predictive value. In this study, we evaluated the performance of three different CD4 T cell proliferation assays in their capacity to predict clinical immunogenicity: a CD8 T cell depleted peripheral blood mononuclear cells (PBMC) assay and two co-culture-based assays between dendritic cells (DCs) and autologous CD4 T cells with or without restimulation with monocytes. A panel of 10 antibodies with a wide range of clinical immunogenicity was selected. The CD8 T cell depleted PBMC assay predicted the clinical immunogenicity in four of the eight highly immunogenic antibodies included in the panel. Similarly, five antibodies with high clinical immunogenicity triggered a response in the DC: CD4 T cell assay but the responses were of lower magnitude than the ones observed in the PBMC assay. Remarkably, three antibodies with high clinical immunogenicity did not trigger any response in either platform. The addition of a monocyte restimulation step to the DC: CD4 T cell assay did not further improve its predictive value. Overall, these results indicate that there are no CD4 T cell assay formats that can predict the clinical immunogenicity of all biotherapeutics and reinforce the need to combine results from various preclinical assays assessing antigen uptake and presentation to fully mitigate the immunogenicity risk of biotherapeutics.

Changing the Landscape of Solid Tumor Therapy from Apoptosis-Promoting to Apoptosis-Inhibiting Strategies.

The many limitations of implementing anticancer strategies under the term "precision oncology" have been extensively discussed. While some authors propose promising future directions, others are less optimistic and use phrases such as illusion, hype, and false hypotheses. The reality is revealed by practicing clinicians and cancer patients in various online publications, one of which has stated that "in the quest for the next cancer cure, few researchers bother to look back at the graveyard of failed medicines to figure out what went wrong". The message is clear: Novel therapeutic strategies with catchy names (e.g., synthetic "lethality") have not fulfilled their promises despite decades of extensive research and clinical trials. The main purpose of this review is to discuss key challenges in solid tumor therapy that surprisingly continue to be overlooked by the Nomenclature Committee on Cell Death (NCCD) and numerous other authors. These challenges include: The impact of chemotherapy-induced genome chaos (e.g., multinucleation) on resistance and relapse, oncogenic function of caspase 3, cancer cell anastasis (recovery from late stages of apoptosis), and pitfalls of ubiquitously used preclinical chemosensitivity assays (e.g., cell "viability" and tumor growth delay studies in live animals) that score such pro-survival responses as "lethal" events. The studies outlined herein underscore the need for new directions in the management of solid tumors.

Harnessing the Cross-Neutralisation Potential of Existing Antivenoms for Mitigating the Outcomes of Snakebite in Sub-Saharan Africa.

Over 32,000 individuals succumb to snake envenoming in sub-Saharan Africa (sSA) annually. This results from several factors, including a lack of antivenom products capable of neutralising the venoms of diverse snake species in this region. Most manufacturers produce polyvalent antivenoms targeting 3 to 16 clinically important snake species in sSA. However, specific products are unavailable for many others, especially those with a restricted geographic distribution. While next-generation antivenoms, comprising a cocktail of broadly neutralising antibodies, may offer an effective solution to this problem, given the need for their clinical validation, recombinant antivenoms are far from being available to snakebite victims. One of the strategies that could immediately address this issue involves harnessing the cross-neutralisation potential of existing products. Therefore, we assessed the neutralisation potency of PANAF-Premium antivenom towards the venoms of 14 medically important snakes from 13 countries across sSA for which specific antivenom products are unavailable. Preclinical assays in a murine model of snake envenoming revealed that the venoms of most snake species under investigation were effectively neutralised by this antivenom. Thus, this finding highlights the potential use of PANAF-Premium antivenom in treating bites from diverse snakes across sSA and the utility of harnessing the cross-neutralisation potential of antivenoms.