Targeting leukaemias with a therapeutic human antibody


Currently marketed anticancer monoclonal antibodies (mAbs) recognise extracellular proteins or those expressed on the cell surface. Generally these are not tumour-specific, as oncogenic proteins tend to be nuclear or cytoplasmic. These intracellular proteins can however, be presented on the cell surface as T cell epitopes by the major histocompatibility complex (MHC). These epitopes are recognised by T cell receptors (TCRs). Therefore, the generation of ‘TCR-like’ mAbs that can recognise cell surface epitopes that are derived from tumours are an exciting potential cancer therapy.

Dao et al., recently published a paper in Science Translational Medicine, in which they have exploited phage display technology to produce a specific mAb (ESK1) against Wilms tumor 1 (WT1), an oncoprotein that is overexpressed in both leukaemia and a range of solid tumours such as ovarian cancer and mesothelioma, but is rare in normal tissues. It was also recently ranked as a top cancer target for immunotherapy by the NIH. ESK1 targets a 9mer WT1 derived peptide (RMF) that is processed and presented by HLA-A0201. RMF induces cytotoxic T cells that are able to kill WT1+ tumour cells in vitro.

ESK1 was shown to bind to acute myeloid leukaemia (AML) CD34+/CD33+ cells expressing HLA-A02 and WT1 but not to normal peripheral blood mononuclear cells. The specificity of the binding was confirmed with other cell types with no general cross-reactivity to healthy or leukemic cells that do not express WT1. Radioimmunoassay experiments then demonstrated that there was adequate RMF expressed on the surface of many cancer and leukaemia cells, whilst the levels of epitope on WT1 negative healthy cells are low.

mAbs can cause cytotoxicity in four ways: antibody-dependent cell-mediated cytotoxicity (ADCC), complement-mediated cytotoxicity (CMC), antibody-dependent cellular phagocytosis (ADCP) and inducing apoptosis. ESK1 was shown to be active in ADCC assays against the following cell types: JMN mesothelioma, BV173 leukaemia, ovarian carcinoma, colon carcinoma cell lines and AML cells. No other form of mAb-mediated cytotoxicity was observed.

The efficacy of ESK1 was then tested in vivo in mice that had been xenografted with BV173 acute lymphoblastic leukaemia (ALL) cells or BA25 acute lymphocytic leukaemia. Two intravenous doses of 100ug of ESK1, when administered in conjunction with human effector cells, suppressed the growth of  leukemic cells in both animal models. Prolonged or, in some cases, leukaemia-free survival were observed. Two control studies confirmed that the RMF/A2 epitope was required for the therapeutic effect observed. Further, no evidence of toxicity was observed in transgenic mice when given therapeutic doses of ESK1.

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Survival of NSG mice with BV173 leukaemia. **P<0.01 for all treatment groups compared to untreated control animals or animals treated with isotype control hIgG (log-rank Mantel-Cox test).

The ESK1 antibody could therefore be promising as a new cancer drug, with a large clinical impact for those patients with WT1+ tumours or leukaemia with HLA-A02 expression.

 

Aggregation false positives in cell based assays?


An article dealing with the common problem of compounds that are false positives in screening assays was published recently (http://www.ncbi.nlm.nih.gov/pubmed/23437772).  One cause of compounds acting as false positives in screening assays is that they can self-aggregate, forming colloidal particles. This aggregation effectively sequesters the protein from its target and prevents activity. This has been a common problem in drug screening assays, particularly with soluble protein methods. In this publication the Shoichet lab at University of California, and others, have been investigating a further scope of the problem by examining  GPCR assays using a cell based format.

They took four compounds that were known to form aggregates and measured the activity against a variety of receptors using the Beta- Arrestin assay. The results show that these compounds were acting as antagonists against the receptors when they were stimulated with their agonist ligand, and this activity could be reversed with the addition of detergent or the use of centrifugation.

They also observed inverse agonism when the compounds were tested against the receptor in the absence of the activating ligand of the receptor, maybe via membrane perturbation.

It all highlights a type of assay artefact, which was thought to be more prevalent in soluble protein assays, can also have a bearing in cell based formats.The steps show by the authors (centrifugation and detergent usage) should be included to reduce the chance of false positives even if you are using a cell based method.

gw1Figure extracted from: Sassano, M. F., Doak, A. K., Roth, B. L., & Shoichet, B. K. (2013). Colloidal aggregation causes inhibition of g protein-coupled receptors. Journal of medicinal chemistry, 56(6), 2406–14. doi:10.1021/jm301749y

 

Assessment of Cancer Genes for Drug Discovery


The Cancer Gene Census documents a list of genes which when genetically altered are known to contribute directly to cancer.

A recent paper by Patel et al describes a systematic, computational protocol, that they have used to identify which of these genes code for proteins that would be possible candidate targets, suitable for therapeutic modulation in the treatment of cancer.  A suite of analyses were undertaken to explore the biological and chemical space of these proteins (shown below).

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Following the computational analysis, the authors prioritize these proteins for drug development.  First they identified twenty-five proteins already known to be drug targets, with compounds with full FDA approval.  They suggest that some of the compounds may be useful for repurposing in different types of cancer.  For instance Smoothened SMO is the target of Vismodegib was recently approved for the treatment of basal cell carcinoma.  By mining multi-omic data from The Cancer Genome Atlas the authors suggest that Vismodegib might also be of use in treating Multiforme Glioblastoma (GB), as SMO was over-expressed in 95% of the GB samples analysed.

A  further eight-six proteins had active chemical compounds with submicromolar activity in biochemical or binding assays reported in the Chembl database.

They also explored which proteins had a known structure and predicted potential druggable pockets. Figure 2 illustrates the three-dimensional structure of GNAS with the druggable cavity displayed as a surface.  GNAS has an activating dominant mutation in pituitary adenoma, and further activating mutations have also been identified in kidney, thyroid, adenocortical, colorectal and Leydig tumours.  The authors suggest that small-molecule inhibitors of this enzyme regulator may have potential therapeutic applications.

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Of the 488 cancer gene census proteins, the authors identify 103 with good evidence of chemical tractability and group them by  “drug development” risk. They identify 46 proteins, whose genes are known to be genetically altered in cancer, whose structures are predicted to be druggable, with few or no know active small molecule modulators, that may be potential therapeutic targets. They suggest that these targets indicate new biological areas for chemical exploration in the treatment of cancer, but they also represent a high potential drug development risk.

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