The cytokine, interleukin-33 (IL-33), has emerged as a promising therapeutic target in chronic respiratory diseases and viral exacerbations. Despite great progress being made with simple cell assays, aspects of IL-33 biology require further investigation in complex, translational systems. Consequently, the aim of this work was to develop a human precision-cut lung slice (huPCLS) platform suitable to explore IL-33 biology and test novel therapies. huPCLS were generated and cultured for up to 7 days. LDH levels and H&E staining were used to evaluate tissue viability and architecture with inflammatory cytokine release following LPS stimulation measured to assess huPCLS responsiveness. IL-33 stimulated huPCLS were analysed via bulk RNA-sequencing (RNA-seq) whilst immunohistochemistry was used to identify airway epithelial subsets. huPCLS were also exposed to human rhinovirus A (HRV-A) with cytokine release utilised as a readout of infection. LDH levels peaked 1 day post slicing and decreased with time in culture. Tissue architecture was maintained at day 7 versus day 0 whilst 24-hour LPS stimulation induced significant IL-6, IL-1β and TNFα release relative to unstimulated huPCLS. In pilot studies, IL-33 responsive genes were upregulated when IL-33-stimulated huPCLS were analysed by bulk RNA-seq. Furthermore, airway epithelial subsets known to be modulated by IL-33 could be identified in huPCLS. Finally, infection with HRV-A increased IFNγ and IL-5 release by huPCLS relative to control slices. In summary, a method to generate viable and responsive huPCLS has been developed with promising initial results demonstrating the potential of the platform to investigate IL-33 biology and screen novel therapies.

BACKGROUND:Epithelial damage, repair and remodelling are critical features of chronic airway diseases including chronic obstructive pulmonary disease (COPD). Interleukin (IL)-33 released from damaged airway epithelia causes inflammation via its receptor, serum stimulation-2 (ST2). Oxidation of IL-33 to a non-ST2-binding form (IL-33ox) is thought to limit its activity. We investigated whether IL-33ox has functional activities that are independent of ST2 in the airway epithelium.METHODS:In vitro epithelial damage assays and three-dimensional, air-liquid interface (ALI) cell culture models of healthy and COPD epithelia were used to elucidate the functional role of IL-33ox. Transcriptomic changes occurring in healthy ALI cultures treated with IL-33ox and COPD ALI cultures treated with an IL-33-neutralising antibody were assessed with bulk and single-cell RNA sequencing analysis.RESULTS:We demonstrate that IL-33ox forms a complex with receptor for advanced glycation end products (RAGE) and epidermal growth factor receptor (EGFR) expressed on airway epithelium. Activation of this alternative, ST2-independent pathway impaired epithelial wound closure and induced airway epithelial remodelling in vitro. IL-33ox increased the proportion of mucus-producing cells and reduced epithelial defence functions, mimicking pathogenic traits of COPD. Neutralisation of the IL-33ox pathway reversed these deleterious traits in COPD epithelia. Gene signatures defining the pathogenic effects of IL-33ox were enriched in airway epithelia from patients with severe COPD.CONCLUSIONS:Our study reveals for the first time that IL-33, RAGE and EGFR act together in an ST2-independent pathway in the airway epithelium and govern abnormal epithelial remodelling and muco-obstructive features in COPD.

The cell-surface glycoprotein B7-H4 is overexpressed in a range of solid tumors including breast cancer, ovarian serous carcinoma, endometrial carcinoma, and cholangiocarcinoma, yet has limited expression in normal tissue, making it an attractive target for an antibody-drug conjugate (ADC). This presentation describes for the first time the development of AZD8205, a B7-H4 targeted ADC incorporating a novel topoisomerase 1 inhibitor (TOP1i) linker-warhead, AZ’0133 which was designed to exploit the full potential of B7-H4 as an ADC target. Initially, we investigated a series of more than 35 TOP1i compounds as warheads and achieved activity in a clinically relevant nM range. We further optimized the conjugation site and chemistry to reduce the potential for aggregation while maintaining potency, overcoming major synthetic challenges to deliver a robust synthetic route amenable to scale-up. Finally, with a series of optimized linker-warheads, we explored the impact of linker-warhead design on ADC hydrophobicity, stability, efficacy, pharmacokinetics and tolerability culminating in the development of AZD8205. The primary mechanism of action of AZD8205 is intracellular delivery of the TOP1i warhead to B7-H4 positive cells, leading to DNA damage and apoptotic cell death. AZD8205 drove bystander killing of target negative cells in mixed cultures in vitro, which is further supported by robust antitumor activity observed in in vivo studies with patient-derived xenograft (PDX) tumors with heterogeneous target expression, representing multiple tumor indications. In a study of 26 human TNBC PDX tumors, a single IV administration of 3.5 mg/kg AZD8205 provided an overall response rate of 69% (tumor regression of 30% or greater from baseline) and complete responses observed in 9/26 (36%) of models. To understand the biology underlying antitumor response, we conducted a multiparametric analysis including genomics, proteomics and computational pathology and found that deeper antitumor activity was observed in models with elevated B7-H4 expression as well as in models with defects in DNA damage repair (DDR). To further exploit the DNA damage elicited by the TOP1i warhead, we examined combinations of AZD8205 with small molecules, including a novel PARP1 selective inhibitor, in a BRCA wild type MDA-MB-468 model. These data suggest that AZD8205 is a promising therapeutic candidate for the treatment of B7-H4 positive solid tumors. A first in human phase 1 study in patients with advanced solid tumors is currently ongoing (NCT05123482). Citation Format: Krista Kinneer, Niall J. Dickinson, Luke Masterson, Thais Cailleau, Ian Hutchinson, Balakumar Vijayakrishnan, Nazzareno Dimasi, R. James Christie, Mary McFarlane, Kathryn Ball, Arthur Lewis, Sofia Koch, Lee Brown, Yue Huang, Anton I. Rosenbaum, Jiaqi Yuan, Si Mou, Noel R. Monks, Jon Chesebrough, Ravinder Tammali, Judith Anderton, Darrin Sabol, Frances Anne Tosto, Philipp Wortmann, Zachary A. Cooper, Pauline Ryan, John Hood, Carlos Fernandez Teruel, Carlos Serra Traynor, Andy Pike, Michael Davies, Elisabetta Leo, Kimberly Cook, Nadia Luheshi, Philip W. Howard, Puja Sapra. Discovery and first disclosure of AZD8205, a B7-H4-targeted antibody-drug conjugate utilizing a novel topoisomerase I linker-warhead [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1765.

Antibody-Drug-Conjugates (ADCs) are biopharmaceutical drugs designed for targeted tumor therapy, meant to improve therapeutic index by restricting drug delivery to tumor cells that express the target antigen. ADCs bind to the target molecule on the cell membrane, which triggers internalization, linker cleavage, and ultimately drug release inside the target cell. Prospective patient selection can be done by quantifying the level of target expression in the tumor using immuno-histochemistry (IHC). However, this process typically involves pathologists and is time consuming, expensive, and prone to human bias. We have developed a supervised deep learning algorithm that segments IHC images of invasive tumor epithelium into individual epithelial cells and their membrane, cytoplasm and nucleus with high accuracy. On unseen test data its performance for epithelial cell detection and segmentation is comparable to the inter-pathologist consensus. With our algorithm, we can describe the target molecule distribution of individual cells in a fully quantitative fashion after using standard IHC methods: we call our approach Quantitative Continuous Score (QCS). We applied QCS to interrogate the mechanism of action of AZD8205, a B7-H4 directed ADC incorporating a novel topoisomerase I linker-warhead. Pharmacodynamic effects were evaluated in vivo, using a human tumor xenograft mouse model and the cell line HT29-huB7-H4 Clone 26, engineered to express human B7-H4. After tumors grew in volume to approximately 250 to 300 mm3, animals were randomized and each mouse received an IV injection of either AZD8205 (1.25, 3.5, or 7 mg/kg) or control articles. Tumors were collected at designated timepoints, fixed in 10% neutral buffered formalin and subsequently embedded into paraffin blocks. IHC and QCS were then used to examine human IgG, γH2AX foci, cleaved caspase-3, and epithelial cell density in tumor samples over time. Using our novel approach we could quantitatively measure the level of AZD8205 bound to tumor cells, with the highest level of ADC on the cell membrane detected at 24-48 hrs. Increased dose levels accelerated the binding kinetics of the drug and led to to a 4- and 3-fold excess of γH2AX and CC-3 respectively, as well as more cells being killed, with up to 2/3 of all epithelial cells dead at the highest dose studied. In summary, we here set the basis for future mechanistic investigation of model systems using computational pathology to improve our understanding of ADC effects. Computational pathology has the potential to determine molecule abundance quantitatively, increase throughput and avoid human bias. Our data implies QCS has the potential to identify patients who may respond to AZD8205, which we will interrogate further and integrate into future clinical studies. Citation Format: Philipp Wortmann, Tze Heng Tan, Susanne Haneder, Andrea Ennio Storti, Ansh Kapil, Jon Chesebrough, Daniel Sutton, Michal Sulikowski, Arthur Lewis, Sofia Koch, Steve Sweet, Zifeng Song, David Chain, Yeoun Jin Kim, Nadia Luheshi, Krista Kinneer, Zachary A. Cooper, Marlon Rebelatto, Günter Schmidt, Hadassah Sade, J. Carl Barrett. Development and implementation of image analysis-based Quantitative Continuous Score (QCS) for B7-H4 IHC to understand AZD8205 pharmacodynamics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 452.

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease which presents a grave prognosis for diagnosed patients. Nintedanib (a triple tyrosine kinase inhibitor) and pirfenidone (unclear mechanism of action) are the only approved therapies for IPF, but have limited efficacy. The pathogenic mechanisms of this disease are not fully elucidated; however, a role for mast cells (MCs) has been postulated.ObjectivesThe aim of this work was to investigate a role for MCs in IPF and to understand whether nintedanib or pirfenidone could impact MC function.Methods and resultsMCs were significantly elevated in human IPF lung and negatively correlated with baseline lung function (FVC). Importantly, MCs were positively associated with the number of fibroblast foci, which has been linked to increased mortality. Furthermore, MCs were increased in the region immediately surrounding the fibroblast foci, and co-culture studies confirmed a role for MC–fibroblast crosstalk in fibrosis. Nintedanib but not pirfenidone inhibited recombinant stem cell factor (SCF)–induced MC survival. Further evaluation of nintedanib determined that it also inhibited human fibroblast-mediated MC survival. This was likely via a direct effect on ckit (SCF receptor) since nintedanib blocked SCF-stimulated ckit phosphorylation, as well as downstream effects on MC proliferation and cytokine release. In addition, nintedanib ablated the increase in lung MCs and impacted high tissue density frequency (HDFm) in a rat bleomycin model of lung fibrosis.ConclusionNintedanib inhibits MC survival and activation and thus provides a novel additional mechanism by which this drug may exert anti-fibrotic effects in patients with IPF.

Abstract Antibodies are the most common tools in basic science and clinical research. Over the past several years there has been an ongoing discussion around antibody validation in light of poor experimental reproducibility between laboratories. This is in part due to the cross-reactivity between antibodies and off-target proteins and the variability between different antibody batches. The resulting experimental irreproducibility leads to wasted resources and compromises the advancement of science. Abcam partners with various research institutes and biotech companies to in-license hybridoma cell lines producing monoclonal antibodies. Through this in-licensing program, Abcam has taken steps to help ensure target-specificity of valuable clones. Our in-licensing program utilizes our ISO9001-accredited production method to ensure each batch performs in-line with previous batches. Where possible, all in-licensed antibodies are also tested in knockout (KO) material to confirm the specificity of the in-licensed clone and subsequent batches. Through our partnership with Horizon Discovery to test antibodies with KO cells, target genes are mutated via CRISPR-Cas9 within a haploid cell line, which results in a frameshift and a complete loss of gene expression. KO cell lines provide a true negative control for antibody validation that allows us to confirm the specificity of clones we in-license and produce. Alongside KO, we are making use of our expertise in recombinant antibodies to explore the conversion of selected in-licensed hybridomas to recombinant antibodies. A key issue with monoclonals is degradation of the clone over time, leading to inconsistent batches and irreproducible results. By sequencing and cloning the antibody, we expect to guarantee continued specificity and reproducibility; therefore, ensuring our licensors see long term impact and value generation for their innovations. Here we present KO data for several in-licensed published clones, validated in both western blot and immunocytochemistry. The antibodies have been tested in KO and wild-type cells to confirm their specificity. By providing researchers with reliable and specific antibodies that work as intended, we hope to minimize wasted resources and improve reproducibility. In addition to our KO data, we present data from the recombinant conversion of clone NAT105 to PD1 and show comparable, if not superior, performance when compared with the hybridoma. Citation Format: Sam Heaton, Sofia Koch, Jiangyan Ge, Joshua Rizzo, Michael Prater, Hanna Dreja, Bruce Hamilton, Alejandra Solache. In-licensing and knockout validation of monoclonal hybridomas to improve antibody reproducibility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2164.

At Abcam we are constantly improving our manufacturing processes and adopting more robust antibody validation technologies as they are developed. Recently we have implemented the use of human knockout (KO) cell lines to interrogate the specificity of our antibodies on a large scale. This has been possible through a partnership with Horizon Discovery and the use of their KO cell lines generated using CRISPR/Cas9 technology. PTEN, is a well-known tumour suppressor that plays a critical role in the host immune response. Recent research has highlighted the importance of this protein with regards to a) lineage stability and homeostasis in Treg cells (1) and b) negative regulation of the NK cell immune response (2, 3). In both cases PTEN acts by regulation of the PI3-kinase signalling pathway. We have identified several monoclonal antibodies to both PTEN and PI3K and confirmed their specificity by KO validation. We performed this analysis in either denatured (western blot) or native (immunocytochemistry) conditions. Our KO models represent true negative controls and provide confidence that these antibodies are binding to their intended target. This technology has allowed us to develop a growing range of over 650 KO-validated antibodies, including a number that are applicable to immunology/oncology research.

The Hippo pathway, by tightly controlling the phosphorylation state and activity of the transcription cofactors YAP and TAZ is essential during development and tissue homeostasis whereas its deregulation may lead to cancer. Recent studies have linked the apicobasal polarity machinery in epithelial cells to components of the Hippo pathway and YAP and TAZ themselves. However the molecular mechanism by which the junctional pool of YAP proteins is released and activated in epithelial cells remains unknown. Here we report that the tumour suppressor ASPP2 forms an apical-lateral polarity complex at the level of tight junctions in polarised epithelial cells, acting as a scaffold for protein phosphatase 1 (PP1) and junctional YAP via dedicated binding domains. ASPP2 thereby directly induces the dephosphorylation and activation of junctional YAP. Collectively, this study unearths a novel mechanistic paradigm revealing the critical role of the apical-lateral polarity complex in activating this localised pool of YAP in vitro, in epithelial cells, and in vivo, in the murine colonic epithelium. We propose that this mechanism may commonly control YAP functions in epithelial tissues.

Epithelial to mesenchymal transition (EMT), and the reverse mesenchymal to epithelial transition (MET), are known examples of epithelial plasticity that are important in kidney development and cancer metastasis. Here we identify ASPP2, a haploinsufficient tumour suppressor, p53 activator and PAR3 binding partner, as a molecular switch of MET and EMT. ASPP2 contributes to MET in mouse kidney in vivo . Mechanistically, ASPP2 induces MET through its PAR3-binding amino-terminus, independently of p53 binding. ASPP2 prevents β-catenin from transactivating ZEB1, directly by forming an ASPP2–β-catenin–E-cadherin ternary complex and indirectly by inhibiting β-catenin’s N-terminal phosphorylation to stabilize the β-catenin–E-cadherin complex. ASPP2 limits the pro-invasive property of oncogenic RAS and inhibits tumour metastasis in vivo . Reduced ASPP2 expression results in EMT, and is associated with poor survival in hepatocellular carcinoma and breast cancer patients. Hence, ASPP2 is a key regulator of epithelial plasticity that connects cell polarity to the suppression of WNT signalling, EMT and tumour metastasis.

Significance Squamous cell carcinoma (SCC) is one of the most common human tumor types with high malignancy. Most SCC mouse models involve multiple genetic mutations. Here we present the ASPP2 Δexon3/+ Balb/c mouse as a haploinsufficient SCC mouse model and identify ASPP2 as a suppressor of SCC and a potent repressor of p63 expression. The finding that ASPP2 represses p63 expression via NF-κB provides a molecular insight into how inflammatory signaling may affect SCC development. All these demonstrate that ASPP2 may serve as a molecular signature of SCC and the characterized ASPP2 Δexon3/+ Balb/c mouse may provide a much-needed experimental model to extend our understanding of SCC as well as enable us to develop better strategies to treat SCC.

Inhibitor of apoptosis-stimulating protein of p53 (iASPP) is the most ancient member of the ASPP family of proteins and an evolutionarily conserved inhibitor of p53. iASPP is also a binding partner and negative regulator of p65RelA. Because p65RelA and the p53 family members often have opposite effects in controlling cell fate, it is important to understand the cellular context in which iASPP can regulate their activities. To address this question and to study the biological importance of iASPP in vivo, we generated a transgenic mouse in which iASPP expression is controlled by the Cre/loxP recombination system. We observed that iASPP is able to prevent premature cellular senescence in mouse embryonic fibroblasts. iASPP loss resulted in increased differentiation of primary keratinocytes both in vitro and in vivo. In stratified epithelia, nuclear iASPP often colocalized with p63 in the nuclei of basal keratinocytes. Consistent with this, iASPP bound p63 and inhibited the transcriptional activity of both TAp63α and ΔNp63α in vitro and influenced the expression level of p63-regulated genes such as loricrin and involucrin in vivo. In contrast, under the same conditions, p65RelA was frequently expressed as a cytoplasmic protein in the suprabasal layers of stratified epithelia and rarely colocalized with nuclear iASPP. Thus, iASPP is likely to control epithelial stratification by regulating p63's transcriptional activity, rather than p65RelA's. This study identifies iASPP as an inhibitor of senescence and a key player in controlling epithelial stratification.