Search

The immune checkpoint protein B7-H4 plays an important role in the positive as well as the negative regulation of immune T-cell responses. When expressed on cancer cells, B7-H4 inhibits T-cell activity, and numerous types of cancer cells use upregulation of B7-H4 as a survival strategy. Thus, B7-H4 is a potential target for anticancer drug therapy. Unfortunately, the cell biology of this molecule has yet to be fully elucidated. Even basic properties, such as the nature of B7-H4 interactors, are controversial. In particular, the cis-inter-actors of B7-H4 on cancer cell plasma membranes have not been investigated to date. The present study used a proteomic proximity-labelling assay to investigate the molecular neighbours of B7-H4 on the surface of the human breast cancer cells SK-BR-3. By comparison to a comprehensive proteome analysis of SK-BR-3 cells, the proximity method detected a relatively small number of low abundance plasma membrane proteins highly enriched for proteins known to modulate cell adhesion and immune recognition. It may be inferred that these molecules contribute to the immunosuppressive behaviour that is characteristic of B7-H4 on cancer cells.

Infections of humans and livestock with African trypanosomes are treated with drugs introduced decades ago that are not always fully effective and often have severe side effects. Here, the trypanosome haptoglobin-haemoglobin receptor (HpHbR) has been exploited as a route of uptake for an antibody-drug conjugate (ADC) that is completely effective against Trypanosoma brucei in the standard mouse model of infection. Recombinant human anti-HpHbR monoclonal antibodies were isolated and shown to be internalised in a receptor-dependent manner. Antibodies were conjugated to a pyrrolobenzodiazepine (PBD) toxin and killed T. brucei in vitro at picomolar concentrations. A single therapeutic dose (0.25 mg/kg) of a HpHbR antibody-PBD conjugate completely cured a T. brucei mouse infection within 2 days with no re-emergence of infection over a subsequent time course of 77 days. These experiments provide a demonstration of how ADCs can be exploited to treat protozoal diseases that desperately require new therapeutics., Author summary Here we show that antibody-drug conjugates (ADCs) can be re-purposed from cancer immunotherapeutics to anti-protozoals by changing the specificity of the immunoglobulin to target a trypanosome cell surface receptor. Trypanosomes were used as a model system due to the availability of receptor null cell lines that allowed the unambiguous demonstration that ADCs targeted to a parasite surface receptor could be specifically internalised via receptor-mediated endocytosis. A single low dose of the resulting ADC was able to cure a stage 1 mouse model of trypanosome infection. We have used toxins and conjugation chemistry that are identical to anti-cancer ADCs demonstrating the ability to piggy-back onto the huge research efforts and resources that are being invested in the development of such ADCs. The potential for development of ADCs against a wide range of human pathogens is vast, where only epitope binding sites need vary in order to provide selectivity. This provides a far-reaching opportunity for the rapid development of novel anti-protozoals for the targeted killing of a wide range of pathogens that cause disease worldwide, especially in developing countries.

Cancer stem cells (CSCs) represent an important clinical entity given their role in tumor initiation, metastasis and patient relapse. Phenotypic selection allows for identification of novel antigens important in biological processes. Therefore, we undertook a phenotypic selection campaign using a designed ankyrin-repeat (DARPin) library to identify new cancer stem cell (CSC) targets. CSCs generated from late-stage pancreatic cancer patient-derived xenograft (PDX) models were used to pan a designed Ankyrin repeat (DARPin) library. The most prevalent target identified in this screen was Annexin A2 (ANXA2). ANXA2 is a member of the Annexin family that has diverse roles in cancer including proliferation and invasion and is widely expressed in cancer. Furthermore, expression of ANXA2 is linked to poor prognosis in pancreatic cancer. Targeting of Annexin A2 on the surface of pancreatic cancer cells using DARPins resulted in decreased frequency of CSCs both in vitro and in vivo in several pancreatic patient-derived xenografts. ANXA2 DARPins also reduced proliferation and tumor growth of HPAC cells both in vitro and in vivo. Mechanistically, we found that blocking ANXA2 resulted in decreased IL6 and IL8 secretion, two cytokines that have been linked to CSC expansion and survival. Additionally, we found that pharmacological blockade of ANXA2 resulted in decreased plasmin activation, a pathway that has been linked to dissemination of cancer cells. Together our data suggests that targeting of cell surface ANXA2 is effective at reducing CSCs and could be a novel target for the treatment of pancreatic cancer. Citation Format: Elaine M. Hurt, Matt Flynn, Paul Hynes, M. Carla Cabrera, Suneetha B. Thomas, Lilian van Vlerken-Ysla, Louise Slater, Alan Sandercock, Steven Rust, Ralph Minter, Ronald Herbst. Phenotypic selection screening reveals Annexin A2 as a pancreatic cancer stem cell target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 151.

Cancer stem cells (CSCs) represent an important subpopulation of tumor cells that are untouched and perhaps even expanded after chemotherapy and radiation. This population has been shown in murine models of cancer to be solely responsible for the regrowth of tumors following chemotherapy, underscoring the importance of therapeutically targeting this population. Therefore, we sought to find novel targets against CSCs using a phenotypic selection screen. Using a designed ankyrin repeat (DARpin) library, we panned cells disaggregated from tumor spheres from three patient-derived xenograft models of late stage pancreatic cancer to enrich for DARPins that bind CSCs. Once released from the cells, the DARPins were further analyzed for binding to both normal, pancreatic cancer total tumor cells and pancreatic CSCs in order to choose targets that showed either pancreatic cancer-specific or pancreatic CSC-specific binding. These DARPins were then tested in a sphere formation assay, an EZH2-based high content imaging screen, and a standard proliferation assay. Those DARPins showing good cancer-specific binding profiles were also tested for internalization and killing using a saporin-conjugated secondary antibody. Using this approach, we identified 30 DARPins of interest for either direct inhibitory antibody approaches or for potential antibody drug conjugate or nanoparticle delivery approaches. Identification of the proteins bound by each of the DARPins was done using either immunopreciptation followed by liquid chromatography tandem-mass spectrometry or through Retrogenix's cell expression microarray technology. Identified targets included known CSC antigens such as integrin alpha6 (CD49f) as well as other potentially novel targets. These results highlight the possibilities of finding therapeutic targets for CSCs and opens up an exciting possibility for novel therapies. Citation Format: Elaine M. Hurt, Matt Flynn, Suneetha Thomas, Lilian van Vlerken-Ysla, Alan Sandercock, Steven Rust, Minter Ralph, Robert Hollingsworth. Phenotypic selection screening reveals cancer stem cell therapeutic targets. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4218. doi:10.1158/1538-7445.AM2015-4218

Lung cancer is the most common type of cancer, with approximately 222,000 new cases expected each year in the United States. Approximately 72% of the individuals diagnosed with lung cancer will fall victim to the disease. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, but current treatments will not cure the disease for most patients. In an effort to identify novel therapeutics to address this unmet need, a phenotypic screening campaign was initiated. This type of target identification approach allows the discovery of novel drug targets by selection of antibodies based on their ability to elicit a specific desired phenotype. Once antibodies with the desired function are isolated, the targets to which the “hit” antibodies bind can be identified using a variety of approaches including IP-mass spec. Although phenotypic screens have been performed previously, the current effort has important advantages over those described previously. Specifically, this included the use of several unique screening assays run in parallel to allow the evaluation of multiple phenotypes simultaneously. Also unique was the use of primary material from NSCLC patients for both selection and functional screening. Specifically, we demonstrate the use of primary tumor cells in high throughput screening assays. We show the utility of incorporating novel screening assays including anoikis and spheroids with multiple endpoint readouts. Finally we provide data describing a series of “hit” antibodies isolated in the screen which had a variety of activities across the screening assays. These studies show the utility of function-first screening in the identification of unique potential therapeutic targets for oncology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 974. doi:1538-7445.AM2012-974