Arthur Lewis

Antibody validation of immunohistochemistry for biomarker discovery: Recommendations of a consortium of academic and pharmaceutical based histopathology researchers

As biomarker discovery takes centre-stage, the role of immunohistochemistry within that process is increasing. At the same time, the number of antibodies being produced for “research use” continues to rise and it is important that antibodies to be used as biomarkers are validated for specificity and sensitivity before use. This guideline seeks to provide a stepwise approach for the validation of an antibody for immunohistochemical assays, reflecting the views of a consortium of academic and pharmaceutical based histopathology researchers. We propose that antibodies are placed into a tier system, level 1–3, based on evidence of their usage in immunohistochemistry, and that the degree of validation required is proportionate to their place on that tier.

Development of a mouse model mimicking key aspects of a viral asthma exacerbation.

Viral respiratory tract infections are known triggers of asthma exacerbations in both adults and children. The current standard of care, inhaled CS (corticosteroids) and LABAs (long-acting β2-adrenoceptor agonists), fails to prevent the loss of control that manifests as an exacerbation. In order to better understand the mechanisms underlying viral asthma exacerbations we established an in vivo model using the clinically relevant aeroallergen HDM (house dust mite) and the viral mimetic/TLR3 (Toll-like receptor 3) agonist poly(I:C). Poly(I:C) alone induced a similar neutrophilic inflammatory profile in the BAL (bronchoalveolar lavage) to that of HRV1b (human rhinovirus 1b) alone, accompanied by both elevated BAL KC (keratinocyte-derived chemokine) and IL-1β (interleukin-1β). When mice allergic to HDM were also challenged with poly(I:C) the neutrophilic inflammatory profile was exacerbated. Increased CD8(+) T-cell numbers, increased CD4(+) and CD8(+) cell activation and elevated KC and IL-1β were observed. No increases in Th2 cytokines or the eosinophil chemoattractant CCL11 [chemokine (C-C motif) ligand 11], above those induced by HDM alone, were observed. The poly(I:C)-exacerbated neutrophilia did not translate into changes in AHR (airways hyper-responsiveness), indicating that in this model inflammation and AHR are two mechanistically independent events. To test the clinical relevance of this model CS sensitivity was assessed using prednisone, a synthetic oral CS used to manage exacerbations in asthmatic patients already on maximal doses of inhaled CS. The increased neutrophils, and accompanying cytokines/chemokines KC and IL-1β induced by poly(I:C) challenge of HDM-sensitized and challenged mice were insensitive to oral prednisone therapy. In summary we have described a CS-resistant mouse model mimicking the key aspects of viral asthma exacerbation using the clinically relevant aeroallergen HDM and the viral mimic poly(I:C). This model may provide better understanding of disease mechanisms underlying viral exacerbations and could be used to build early confidence in novel therapeutic axes targeting viral asthma exacerbations in Th2 asthmatics.

Transformation of the tumour microenvironment by a CD40 agonist antibody correlates with improved responses to PD-L1 blockade in a mouse orthotopic pancreatic tumour model.

Despite the availability of recently developed chemotherapy regimens, survival times for pancreatic cancer patients remain poor. These patients also respond poorly to immune checkpoint blockade therapies (anti-CTLA-4, anti-PD-L1, anti-PD-1), which suggests the presence of additional immunosuppressive mechanisms in the pancreatic tumour microenvironment (TME). CD40 agonist antibodies (αCD40) promote antigen presenting cell (APC) maturation and enhance macrophage tumouricidal activity, and may therefore alter the pancreatic TME to increase sensitivity to immune checkpoint blockade. Here, we test whether αCD40 transforms the TME in a mouse syngeneic orthotopic model of pancreatic cancer, to increase sensitivity to PD-L1 blockade. We found that whilst mice bearing orthotopic Pan02 tumours responded poorly to PD-L1 blockade, αCD40 improved overall survival. αCD40 transformed the TME, upregulating Th1 chemokines, increasing cytotoxic T cell infiltration and promoting formation of an immune cell-rich capsule separating the tumour from the normal pancreas. Furthermore, αCD40 drove systemic APC maturation, memory T cell expansion, and upregulated tumour and systemic PD-L1 expression. Combining αCD40 with PD-L1 blockade enhanced anti-tumour immunity and improved overall survival versus either monotherapy. These data provide further support for the potential of combining αCD40 with immune checkpoint blockade to promote anti-tumour immunity in pancreatic cancer.

Tremelimumab combined with durvalumab in patients with mesothelioma (NIBIT-MESO-1): an open-label, non-randomised, phase 2 study

BACKGROUND Tremelimumab, an anti-CTLA4 monoclonal antibody, initially showed good activity when used alone in patients with mesothelioma, but did not improve the overall survival of patients who failed on first-line or second-line chemotherapy compared with placebo in the DETERMINE study. We aimed to investigate the efficacy and safety of first-line or second-line tremelimumab combined with durvalumab, an anti-PD-L1 monoclonal antibody, in patients with malignant mesothelioma. METHODS In this open-label, non-randomised, phase 2 trial, patients with unresectable pleural or peritoneal mesothelioma received intravenous tremelimumab (1 mg/kg bodyweight) and durvalumab (20 mg/kg bodyweight) every 4 weeks for four doses, followed by maintenance intravenous durvalumab at the same dose and schedule for nine doses. The primary endpoint was the proportion of patients with an immune-related objective response according to the immune-related modified Response Evaluation Criteria in Solid Tumors (RECIST; for pleural mesothelioma) or immune-related RECIST version 1.1 (for peritoneal mesothelioma). The primary analysis was done by intention to treat, whereas the safety analysis included patients who received at least one dose of study drug. This trial is registered with the European Clinical Trials Database, number 2015-001995-23, and ClinicalTrials.gov, number NCT02588131, and is ongoing but no longer recruiting patients. FINDINGS From Oct 30, 2015, to Oct 12, 2016, 40 patients with mesothelioma were enrolled and received at least one dose each of tremelimumab and durvalumab. Patients were followed-up for a median of 19·2 months (IQR 13·8-20·5). 11 (28%) of 40 patients had an immune-related objective response (all partial responses; confirmed in ten patients), with a median response duration of 16·1 months (IQR 11·5-20·5). 26 (65%) patients had immune-related disease control and 25 (63%) had disease control. Median immune-related progression-free survival was 8·0 months (95% CI 6·7-9·3), median progression-free survival was 5·7 months (1·7-9·7), and median overall survival was 16·6 months (13·1-20·1). Baseline tumour PD-L1 expression did not correlate with the proportion of patients who had an immune-related objective response or immune-related disease control, with immune-related progression-free survival, or with overall survival. 30 (75%) patients experienced treatment-related adverse events of any grade, of whom seven (18%) had grade 3-4 treatment-related adverse events. Treatment-related toxicity was generally manageable and reversible with protocol guidelines. INTERPRETATION The combination of tremelimumab and durvalumab appeared active, with a good safety profile in patients with mesothelioma, warranting further exploration. FUNDING Network Italiano per la Bioterapia dei Tumori Foundation, Associazione Italiana per la Ricerca sul Cancro, AstraZeneca, and Istituto Toscano Tumori.

Genetic methods of antibody generation and their use in immunohistochemistry.

Genetic methods of antibody generation offer a highly tuneable tool for the production of target specific reagents suitable for a wide range of applications, including immunohistochemistry. The direct linkage between binder phenotype and genotype enables the rapid identification and manipulation of specific binders into formats suitable for highly specific and sensitive detection of targets from soluble proteins to individual components of multi-protein structures within the context of living tissues. Here we review the types of genetic methods employed and binder formats available and demonstrate how mining huge combinatorial repertoires of binders can deliver diverse and exquisitely sensitive tools for the use in immunohistochemistry. Finally, we offer a perspective on how this approach might be further refined to routinely deliver binders for specific use in immunohistochemical studies.

MHC class I chain-related protein A and B (MICA and MICB) are predominantly expressed intracellularly in tumour and normal tissue

Background:Major histocompatibility complex (MHC) class I chain-related protein A (MICA) and MHC class I chain-related protein B (MICB) are polymorphic proteins that are induced upon stress, damage or transformation of cells which act as a ‘kill me’ signal through the natural-killer group 2, member D receptor expressed on cytotoxic lymphocytes. MICA/B are not thought to be constitutively expressed by healthy normal cells but expression has been reported for most tumour types. However, it is not clear how much of this protein is expressed on the cell surface.Methods:Using a novel, well-characterised antibody and both standard and confocal microscopy, we systematically profiled MICA/B expression in multiple human tumour and normal tissue.Results:High expression of MICA/B was detected in the majority of tumour tissues from multiple indications. Importantly, MICA/B proteins were predominantly localised intracellularly with only occasional evidence of cell membrane localisation. MICA/B expression was also demonstrated in most normal tissue epithelia and predominantly localised intracellularly. Crucially, we did not observe qualitative differences in cell surface expression between tumour and MICA/B expressing normal epithelia.Conclusions:This demonstrates for the first time that MICA/B is more broadly expressed in normal tissue and that expression is mainly intracellular with only a small fraction appearing on the cell surface of some epithelia and tumour cells.

Divergent roles for Clusterin in Lung Injury and Repair

Lung fibrosis is an unabated wound healing response characterized by the loss and aberrant function of lung epithelial cells. Herein, we report that extracellular Clusterin promoted epithelial cell apoptosis whereas intracellular Clusterin maintained epithelium viability during lung repair. Unlike normal and COPD lungs, IPF lungs were characterized by significantly increased extracellular Clusterin whereas the inverse was evident for intracellular Clusterin. In vitro and in vivo studies demonstrated that extracellular Clusterin promoted epithelial cell apoptosis while intercellular Clusterin modulated the expression of the DNA repair proteins, MSH2, MSH6, OGG1 and BRCA1. The fibrotic response in Clusterin deficient (CLU−/−) mice persisted after bleomycin and it was associated with increased DNA damage, reduced DNA repair responses, and elevated cellular senescence. Remarkably, this pattern mirrored that observed in IPF lung tissues. Together, our results show that cellular localization of Clusterin leads to divergent effects on epithelial cell regeneration and lung repair during fibrosis.

Abstract 1451: Analysis of expression MHC class I chain-related gene A and B (MICA/B) in normal and tumor tissue

MHC class I chain-related gene A and B (MICA and MICB) are highly polymorphic proteins that are induced upon stress, damage or transformation of cells which act as a “kill me” signal through the NKG2D receptor expressed on Natural Killer, CD8+ and γδ T cells. Experimentally, the MIC/NKG2D axis has been shown to be important for the recognition of tumour cells by cytotoxic cells of the immune system and many tumours have evolved strategies to evade the detection by NKG2D expressing cells, e.g. by shedding MIC from the cell surface. Expression of MIC has been reported for most tumour types and in normal gastrointestinal tract epithelium but the published data is often difficult to interpret. Additionally, it is not clear how much of the protein is expressed on the cell surface as MIC cell surface expression is known to be regulated tightly on multiple levels. A validated MICA/B IHC assay was developed using an in-house tool antibody to profile multiple frozen human normal and tumour tissue microarrays (TMA’s) by both standard and confocal microscopy techniques. Using a stringently characterised novel antibody that detects MICA as well as MICB this study describes the expression patterns in a wide range of tumours and normal tissues. With this method we generated data with unprecedented resolution, which enabled us to analyse the expression of MICA and MICB not only on the cellular but also on the sub-cellular level. Citation Format: Hormas Ghadially, Lee Brown, Arthur Lewis, Meggan Czapiga, Viia Valge-Archer, Robert W. Wilkinson. Analysis of expression MHC class I chain-related gene A and B (MICA/B) in normal and tumor tissue. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1451.

Abstract 4613: Identification of tumor necrosis factor receptor II as a regulatory T cell target for cancer immunotherapy using designed ankyrin repeat protein phenotypic selections

Regulatory T cells (Treg cells) are observed within multiple tumor types and contribute to immune evasion. Effective and specific targeting of Treg cells by antibodies is hampered by the lack of a unique surface marker. With the aim of identifying such a marker, human Treg cells were isolated from peripheral blood, expanded in vitro and used as a source of antigen in phage display selections with a diverse library of designed ankyrin repeat proteins (DARPins). The resulting DARPins were screened for binding to multiple cell-types, and approximately thirty with preferential binding to Treg cells were identified. All of these DARPins were determined to bind to the third and/or fourth cysteine-rich extracellular domains of tumor necrosis factor receptor II (TNFRII, also known as TNFRSF1B or CD120b). Expression of TNFRII has previously been demonstrated on Treg cells, and TNFRII is shown here also to be expressed on CD8+ T cells following exposure to cognate antigen and on the surface of tumor-infiltrating T cells in the 4T1 mouse tumor model. Furthermore, TNFRII mRNA was detected at increased levels in tumor-infiltrating leukocytes isolated from cancer patients. Functional characterisation of the anti-TNFRII DARPins and mAbs revealed an ability to enhance T-cell activation in response to polyclonal and antigen specific stimuli, and in Treg cell suppression assays. These studies demonstrate the utility of DARPins for phenotypic selections to identify drug targets using primary human lymphocytes and highlight TNFRII as a potential therapeutic target enriched on Treg cells. Citation Format: Geoff Williams, Judith Anderton, Vahe Bedian, Jane Coates Ulrichsen, Andrea Gonzalez-Munoz, Sandrine Guillard, Olivia Harris, James Hair, Andrew Leishman, Arthur Lewis, Jacques Moissan, Ralph Minter, Bina Mistry, Julie Parmentier, Edmund Poon, Amy Popple, Steve Rust, Alan Sandercock, Ross Stewart, Viia Valge-Archer, Robert W. Wilkinson. Identification of tumor necrosis factor receptor II as a regulatory T cell target for cancer immunotherapy using designed ankyrin repeat protein phenotypic selections. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4613. doi:10.1158/1538-7445.AM2014-4613

Abstract 1562: Choosing the right preclinical model for cancer immunotherapy: The “SyngenOmic” toolbox..

Recent FDA approvals of ipilimumab and sipuleucel-T for the treatment of metastatic melanoma and castrate-resistant prostate cancer respectively have validated the approach to modulate the immune system for the clinical treatment of cancer. MedImmune Oncology, has an extensive pipeline and expertise in investigational Immune Mediated Therapy in Cancer (IMT-C) drugs (e.g. tremelimumab and MEDI4736), and is heavily invested in this novel era of cancer therapeutics. Pre-clinical assessment of the validity of potential IMT-C drugs can be enabled by the use of syngeneic tumours established in immuno-competent animals. MedImmune9s pharmacology team has lead efforts to develop such models, to a point that a panel of 15 different validated syngeneic tumour types is available in house for studies to progress projects. The intention of selecting relevant models and of minimizing animal experimentation, reduces the number of models tested for each project, and allows for detailed genetic and proteomic characterization of treatment dependent effects within our models. Utilising both in vitro and in vivo approaches, we have generated transcriptomic and genomic data for our cell lines, the resulting implanted tumours and for the relevant lymphatic organs (draining lymph node and spleen). Using key proteomic/cell markers through IHC and FACS analysis, we are also evaluating the immune profile/status of our tumour bearing animals in parallel. Finally, in order to establish the disease relevance and prognostic value of our models we are analyzing our cell panel for the presence of somatic mutations in 50 genes previously implicated in human cancers. Once completed, the resulting dataset will hopefully help pre-clinical scientists to refine their in vivo plans and deliver more clinically-relevant pharmacology packages for the progression of cancer immunotherapy drug candidates. Citation Format: James A. Harper, John Prime, Athula Herath, Jane Howell, Marianna Papaspyridonos, Amy Popple, Judith Anderton, Michelle Morrow, Arthur Lewis, Liz Henley, Matthew McCourt, Richard Sainson. Choosing the right preclinical model for cancer immunotherapy: The “SyngenOmic” toolbox. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1562. doi:10.1158/1538-7445.AM2013-1562