CD73 is a cell surface 5'nucleotidase (NT5E) and key node in the catabolic process generating immunosuppressive adenosine in cancer. Using a murine monoclonal antibody surrogate of Oleclumab, we investigated the effect of CD73 inhibition in concert with cytotoxic therapies (chemotherapies as well as fractionated radiotherapy) and PD-L1 blockade. Our results highlight improved survival in syngeneic tumor models of colorectal cancer (CT26 and MC38) and sarcoma (MCA205). This therapeutic outcome was in part driven by cytotoxic CD8 T-cells, as evidenced by the detrimental effect of CD8 depleting antibody treatment of MCA205 tumor bearing mice treated with anti-CD73, anti-PD-L1 and 5-Fluorouracil+Oxaliplatin (5FU+OHP). We hypothesize that the improved responses are tumor microenvironment (TME)-driven, as suggested by the lack of anti-CD73 enhanced cytopathic effects mediated by 5FU+OHP on cell lines in vitro. Pharmacodynamic analysis, using imaging mass cytometry and RNA-sequencing, revealed noteworthy changes in specific cell populations like cytotoxic T cells, B cells and NK cells in the CT26 TME. Transcriptomic analysis highlighted treatment-related modulation of gene profiles associated with an immune response, NK and T-cell activation, T cell receptor signaling and interferon (types 1 & 2) pathways. Inclusion of comparator groups representing the various components of the combination allowed deconvolution of contribution of the individual therapeutic elements; highlighting specific effects mediated by the anti-CD73 antibody with respect to immune-cell representation, chemotaxis and myeloid biology. These pre-clinical data reflect complementarity of adenosine blockade with cytotoxic therapy, and T-cell checkpoint inhibition, and provides new mechanistic insights in support of combination therapy.

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.

A recombinant Newcastle Disease Virus (NDV), encoding either a human (NDVhuGM-CSF, MEDI5395) or murine (NDVmuGM-CSF) GM-CSF transgene, combined broad oncolytic activity with the ability to significantly modulate genes related to immune functionality in human tumor cells. Replication in murine tumor lines was significantly diminished relative to human tumor cells. Nonetheless, intratumoral injection of NDVmuGM-CSF conferred antitumor effects in three syngeneic models in vivo; with efficacy further augmented by concomitant treatment with anti–PD-1/PD-L1 or T-cell agonists. Ex vivo immune profiling, including T-cell receptor sequencing, revealed profound immune-contexture changes consistent with priming and potentiation of adaptive immunity and tumor microenvironment (TME) reprogramming toward an immune-permissive state. CRISPR modifications rendered CT26 tumors significantly more permissive to NDV replication, and in this setting, NDVmuGM-CSF confers immune-mediated effects in the noninjected tumor in vivo. Taken together, the data support the thesis that MEDI5395 primes and augments cell-mediated antitumor immunity and has significant utility as a combination partner with other immunomodulatory cancer treatments.

OBJECTIVES:Humanised mice have emerged as valuable models for pre-clinical testing of the safety and efficacy of immunotherapies. Given the variety of models available, selection of the most appropriate humanised mouse model is critical in study design. Here, we aimed to develop a model for predicting cytokine release syndrome (CRS) while minimising graft-versus-host disease (GvHD). METHODS:To overcome donor-induced variation, we directly compared the in vitro and in vivo immune phenotype of immunodeficient NSG mice reconstituted with human bone marrow (BM) CD34+ haematopoietic stem cells (HSCs), peripheral blood mononuclear cells (PBMCs) or spleen mononuclear cells (SPMCs) from the same human donors. SPMC engraftment in NSG-dKO mice, which lack MHC class I and II, was also evaluated as a strategy to limit GvHD. Another group of mice was engrafted with umbilical cord blood (UCB) CD34+ HSCs. Induction of CRS in vivo was investigated upon administration of the anti-CD3 monoclonal antibody OKT3. RESULTS:PBMC- and SPMC-reconstituted NSG mice showed short-term survival, with engrafted human T cells exhibiting mostly an effector memory phenotype. Survival in SPMC-reconstituted NSG-dKO mice was significantly longer. Conversely, both BM and UCB-HSC models showed longer survival, without demonstrable GvHD and a more naïve T-cell phenotype. PBMC- and SPMC-reconstituted mice, but not BM-HSC or UCB-HSC mice, experienced severe clinical signs of CRS upon administration of OKT3. CONCLUSION:PBMC- and SPMC-reconstituted NSG mice better predict OKT3-mediated CRS. The SPMC model allows generation of large experimental groups, and the use of NSG-dKO mice mitigates the limitation of early GvHD.

There are no approved drug therapies that can prevent or slow the progression of Parkinson's disease (PD). Accumulation and aggregation of α-synuclein protein is observed throughout the nervous system in PD. α-Synuclein is a core component of Lewy bodies and neurites that neuropathologically define PD, suggesting that α-synuclein may be a key causative agent in PD. Recent experimental data suggest that PD progression may arise due to spreading of pathological forms of extracellular α-synuclein throughout the brain via a cellular release, uptake and seeding mechanism. We have developed a high affinity α-synuclein antibody, MEDI1341, that can enter the brain, sequester extracellular α-synuclein and attenuate α-synuclein spreading in vivo. MEDI1341 binds both monomeric and aggregated forms of α-synuclein. In vitro, MEDI1341 blocks cell-to-cell transmission of pathologically relevant α-synuclein preformed fibrils (pffs). After intravenous injection into rats and cynomolgus monkeys, MEDI1341 rapidly enters the central nervous system and lowers free extracellular α-synuclein levels in the interstitial fluid (ISF) and cerebrospinal fluid (CSF) compartments. Using a novel lentiviral-based in vivo mouse model of α-synuclein spreading in the brain, we show that treatment with MEDI1341 significantly reduces α-synuclein accumulation and propagation along axons. In this same model, we demonstrate that an effector-null version of the antibody was equally as effective as one with effector function. MEDI1341 is now in Phase 1 human clinical trial testing as a novel treatment for α-synucleinopathies including PD with the aim to slow or halt disease progression.

Despite recent advances in treatment, breast cancer remains the second-most common cause of cancer death among American women. A greater understanding of the molecular characteristics of breast tumors could ultimately lead to improved tumor-targeted treatment options, particularly for subsets of breast cancer patients with unmet needs. Using an unbiased genomics approach to uncover membrane-localized tumor-associated antigens (TAAs), we have identified glial cell line derived neurotrophic factor (GDNF) family receptor α 1 (GFRA1) as a breast cancer TAA. Immunohistochemistry (IHC) revealed that GFRA1 displays a limited normal tissue expression profile coupled with overexpression in specific breast cancer subsets. The cell surface localization as determined by fluorescence-activated cell sorting (FACS) and the rapid internalization kinetics of GFRA1 makes it an ideal target for therapeutic exploitation as an antibody-drug conjugate (ADC). Here, we describe the development of a pyrrolobenzodiazepine (PBD)-armed, GFRA1-targeted ADC that demonstrates cytotoxicity in GFRA1-positive cell lines and patient-derived xenograft (PDX) models. The safety profile of the rat cross-reactive GFRA1-PBD was assessed in a rat toxicology study to find transient cellularity reductions in the bone marrow and peripheral blood, consistent with known off-target effects of PBD ADC’s. These studies reveal no evidence of on-target toxicity and support further evaluation of GFRA1-PBD in GFRA1-positive tumors.

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.

Abstract 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.

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.

Receptor mediated transcytosis harnessing the cellular uptake and transport of natural ligands across the blood-brain barrier (BBB) has been identified as a means for antibody delivery to the CNS. In this study, we characterized bispecific antibodies in which a BBB-crossing antibody fragment FC5 was used as a BBB carrier. Cargo antibodies were either a high-affinity, selective antibody antagonist of the metabotropic glutamate receptor-1 (BBB-mGluR1), a widely abundant CNS target, or an IgG that does not bind the CNS target (BBB-NiP). Both BBB-NiP and BBB-mGluR1 demonstrated a similar 20-fold enhanced rate of transcytosis across an in vitro BBB model compared with mGluR1 IgG fused to a control antibody fragment. All 3 bispecific antibodies exhibited identical pharmacokinetics in vivo Comparative assessment of BBB-NiP and BBB-mGluR1 revealed that, whereas their serum pharmacokinetics and BBB penetration were identical, their central disposition (brain levels) and elimination (cerebrospinal fluid levels) were widely different, due to central target-mediated removal of the mGluR1-engaging antibody. Central mGluR1 target engagement after systemic administration was demonstrated by a dose-dependent inhibition of mGluR-1-mediated thermal hyperalgesia and by colocalization of the antibody with thalamic neurons involved in mGluR1-mediated pain processing. We demonstrate the feasibility of targeting central G-protein-coupled receptors using a BBB-crossing bispecific antibody approach and emerging principles that govern brain distribution and disposition of these antibodies. These data will be important for designing safe and selective CNS antibody therapeutics.-Webster, C. I., Caram-Salas, N., Haqqani, A. S., Thom, G., Brown, L., Rennie, K., Yogi, A., Costain, W., Brunette, E., Stanimirovic, D. B. Brain penetration, target engagement, and disposition of the blood-brain barrier-crossing bispecific antibody antagonist of metabotropic glutamate receptor type 1.

Abstract The TGF-ß type-1 receptor activin receptor-like kinase 1 (Alk1) is predominantly expressed on endothelial cells, regulates vascular morphogenesis, and has been implicated in pathological angiogenesis. Alk1 expression is increased in the vasculature of a broad range of tumours and agents that disrupt of Alk1 signalling in tumour endothelial cells may be potential therapeutics for the treatment of a variety of solid tumours. Alk1 has been described as a receptor for the bone morphogenic proteins, BMP9 and BMP10. In order to study the effects of Alk1 signalling in endothelial cells in vitro and in the vasculature in vivo we used a soluble antagonist Alk1-Fc protein consisting of the extracellular domain of the Alk1 protein fused to the Fc domain of IgG1. In vitro, Alk1-Fc inhibited BMP9-dependent Alk1 signalling via phosphorylation and nuclear translocation of SMAD1/5 in endothelial cells and modulated endothelial cell functions such as an ability to form tube- like structures. In mice, physiological levels of BMP9 could be detected in serum by ELISA, consistent with detectable levels of phosphorylated SMAD1/5 in the lung, a site of high Alk1 vascular expression. In mice, the administration of Alk1-Fc effectively blocked SMAD1/5 phosphorylation in the lung, suggesting that in this tissue Alk1 ligands are the predominant driver of lung SMAD1/5 activation and that signalling is efficiently antagonised by Alk1-Fc. Despite suppression of Alk1 signalling in the lung and expression of Alk1 on the vasculature associated with tumours, Alk1-Fc did not have profound effects on tumour size or microvessel density in mice bearing tumours, however administration of Alk1-Fc in neonatal mice did cause defects in angiogenesis of the retinal vasculature demonstrating that in some contexts Alk1 ligands play an important role in regulating the angiogenic process. Citation Format: Gareth C. Davies, James A. Harper, Richard C. Sainson, Lee Brown, Grace Opoku-Ansah, Alison Smith, Matthew McCourt, James W. Legg. Activin receptor-like kinase 1 (Alk1) ligands modulate endothelial cell function in vitro and in vivo. [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 363. doi:10.1158/1538-7445.AM2013-363