News – Page 6 – Drug Discovery Opinion

Once Bitten…

The concept of bispecific antibodies – monoclonal antibodies able to recognise and engage two different antigens – has been explored for over twenty years. Development of therapies based on the approach has, however, been hampered by difficulties in their construction, poor efficacy and undesirable side-effects.

One particular subset of bispecific antibodies, the so-called bispecific T-cell engager (BiTE®), has nevertheless begun to show promise. Blinatumomab, developed by Micromet, targets the CD19 receptor of B-cells and CD3 on T-cells and is designed to direct cytotoxic T-cells to B-cell tumours. Interim data from a phase I trial in Non-Hodgkin’s Lymphoma patients have shown signs of clinical efficacy and additional clinical trials in acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL) are ongoing.

More recently, the Micromet team have reported on preclinical data using BiTE® antibodies targeting the EGFR receptor and CD3, incorporating the binding domains of either panitumumab or cetuximab. Panitumumab and cetuximab, as well as EGFR kinase inhibitors, are marketed for treatment of colorectal cancer (CRC) and primarily inhibit CRC growth by interfering with EGFR signalling. CRC patients whose tumours have mutated KRAS or BRAF, however, are resistant to treatment. This latest study, published in Proceedings of the National Academy of Sciences, showed that both EGFR-specific BiTE® antibodies mediated potent redirected lysis of KRAS– and BRAF-mutated CRC lines by human T cells at subpicomolar concentrations. The cetuximab-based BiTE® antibody also inhibited growth of tumours from KRAS– and BRAF-mutated human CRC xenografts, whereas cetuximab was not effective. The researchers also report preliminary safety data in non-human primates and conclude that EGFR-specific BiTE® antibodies may have potential to treat CRC that does not respond to conventional antibodies.

Dial M3 for Memory

The cognitive improvement in Alzheimer’s disease patients brought about by treatment with acetyl cholinesterase inhibitors has been largely attributed to enhanced M1-muscarinic receptor signalling. Recently, however, studies with M1-receptor knockout mice and with more selective M1-receptor modulators have suggested that this receptor may not directly mediate learning and memory.

A team led by researchers at the University of Leicester has now suggested an alternative mechanism involving the M3-muscarinic receptor which is widely expressed in many brain regions, including the hippocampus. M3-receptor knockout mice were found to show a deficit in fear conditioning learning and memory. A knock-in mouse strain expressing a phosphorylation-deficient receptor also showed a deficit in fear conditioning learning, indicating that the learning process involves receptor phosphorylation. Agonist treatment and fear conditioning training led to phosphorylation of the M3-receptor in the hippocampus, confirming the importance of receptor phoshorylation on learning and memory. The phosphorylation-deficient receptor was expressed normally at the cell surface and was able to signal via the Gq/11 calcium pathway, but was uncoupled from phosphorylation-dependent processes such as receptor internalization and arrestin recruitment. The study, which is published in PNAS, suggests that an M3-receptor modulator that enhances phosphorylation/arrestin-dependent (non-G protein) signalling may be beneficial in treating cognitive disorders. ‘Biased’ ligands – those able to direct signalling of GPCRs selectively through the phosphorylation/arrestin-dependent pathway – have recently been described for a number of other GPCRs.

Breathe Easy

Researchers from the Wyss Institute for Biologically Inspired Engineering at Harvard University, Harvard Medical School and Children’s Hospital Boston have created a lung-on-a-chip which may help to speed pharmaceutical development by reducing reliance on animal models. The device consists of a layer of human alveolar cells separated from a layer of human endothelial cells by a porous membrane and mimics the boundary between the lung’s air sacs and capillaries. The cells on the chip can also be made to ‘breathe’ by cyclically applying a vacuum to increase the width of the membrane and stretch the cells before allowing them to contract again.

The, lung-on-a-chip has the potential to model the effects of environmental toxins, the inflammatory response to inhaled pathogens and the effectiveness of new drugs. Because the chip is transparent, responses can be captured in real time using high-resolution fluorescence microscopy. When E.Coli bacteria were introduced into the air on the ‘lung’ side of the chip, white blood cells on the ‘blood’ side of the chip migrated through the porous membrane into the air chamber to destroy the bacteria. ‘Breathing’ was found to enhance absorption of nanoparticles, some of which induced an inflammatory response and overproduction of free radicals by the lung cells.

The team are now exploring whether the system can mimic gas exchange between alveolar cells and the bloodstream and believe that the device provides proof-of-principle for the concept that organs-on-chips could replace many animal studies in the future.

The study is published in the journal Science.

Oestrogen – Better Out than In for Cardiovascular Health

Although oestrogen replacement lowers cardiovascular risk in post-menopausal women, treatment is associated with an increased risk of uterine and breast cancer.

The increased cancer risk is linked to oestrogen’s action at nuclear receptors but researchers at UT Southwestern Medical Center have now found that a subpopulation of oestrogen receptors outside the cell nucleus mediate the beneficial cardiovascular effects. The extra-nuclear receptors in endothelial cells are important for blood vessel maintenance and repair and also regulate production of nitric oxide which has a number of beneficial cardiovascular effects. The team have found that an oestrogen-macromolecule complex which is excluded from the nucleus is highly effective in stimulating the extra-nuclear receptors. Similar dendrimer conjugates have been successfully used as drug delivery device in animal models and the oestrogen complex was shown to provide cardiovascular protection in high cholesterol ovariectomized female mice without stimulating growth of breast or uterine cancer. The team believe that such oestrogen-macromolecule complexes could provide cardiovascular protection for both men and women and are creating molecules that may be suitable for use in humans.

The study is published in the Journal of Clinical Investigation.

For cells, too, 2-D is not the same as 3-D

Cell culture experiments to screen for compounds that can inhibit cell migration – and potentially metastasis of cancer cells – are typically carried out in a 2-D environment, but researchers at Johns Hopkins University and the University of Washington suggest that results from such experiments may be, at best, misleading. The team has shown that the way in which cells move in a 3-D environment, such as the human body, is different both qualitatively and quantitatively from the way they move in a 2-D environment such as a culture dish. When cells are grown in 2-D, they develop broad fan-shaped protrusions called lamella along their leading edge which help them to move forward. Macromolecular assemblies known as focal adhesions which can last for up to several minutes are also formed. These focal adhesions mediate cell signalling, force transduction and adhesion. In 3-D, the cells take on a more spindle-like appearance, with two pointed protrusions at opposite ends and focal adhesions – if they form at all – are so small and short-lived that they cannot be resolved by microscopy. The authors suggest that the shape and movement of cells in 2-D culture experiments are artifacts of the environment and could produce misleading results in studies to test the effects of drugs on cell motility. This may explain why positive results from cell culture experiments do not always translate into efficacy in animal models.

Even in cell culture systems designed to more closely mimic a 3-D environment, the cells may be only partially embedded in a matrix and produce misleading results. Using live-cell microscopy, the team showed that, when cells are fully embedded in a 3-D matrix, focal adhesion proteins do not form aggregates, but are distributed throughout the cytoplasm. The focal adhesion proteins still modulate cell motility, but not in the same way as in a 2-D environment. Because loss of adhesion and increased motility are hallmarks of cancer cells, it is important to understand cell motility under physiological conditions and to use culture techniques that most closely mimic this.

The study is published in Nature Cell Biology.

Why Bacteria Turn Bad

bad bacteria — Image: Flickr - beneneuman

The human body is host to a plethora of microorganisms and, for the most part, their presence has no ill effects. Some, particularly intestinal bacteria, even provide benefit. From a microbial perspective, harming the host does not have any obvious survival benefit (unless it enables infection to spread, such as the sneezing induced by the cold virus). So why is it that inoffensive organisms occasionally turn nasty, evolving properties that are damaging or even deadly to us? A study funded by the US Public Health Service and the Wellcome Trust provides one answer to the question.

Since many pathogens interact with their host at mucosal surfaces and have to compete with other microflora, scientists at the University of Pennsylvania School of Medicine and Oxford University used a mouse model of nasal infection to investigate whether competition between microbes promoted virulence. They found that Haemophilus influenzae was able to out-compete Streptococcus pneumoniae by recruiting the host’s immune system. S. pneumoniae is normally harmless and ignored by the immune system, but the immune response stimulated by H. influenzae has unintended consequences. S. pneumoniae strains with polysaccharide capsules that confer resistance to the immune attack are able to survive at the expense of non-resistant strains, resulting in a S. pneumoniae population dominated by the resistant phenotype. Unfortunately, the resistant strains are also more dangerous – if they are able to enter the bloodstream they can multiply unchecked and go on to cause pneumonia, septicaemia and meningitis. So in this battle between S. pneumoniae and H. influenzae, with weapons provided by the host, S. pneumoniae prevails at the expense of the host.

The study is published in Current Biology.

Saying it with Flowers

The process of cell competition is believed to provide a mechanism to optimise tissue ‘fitness’ during development by eliminating weaker cells from the overall cell population. First described in Drosophila, a number of genes have been linked to cell competition but the precise details of the process are poorly understood. A new study conducted by scientists at the Spanish National Cancer Centre (CNIO), however, has furthered our understanding.

Using a combination of genomic analysis and functional assays, the team investigated how cells of Drosophila wing imaginal discs distinguished ‘winner’ and ‘loser’ cells. They found that six genes were upregulated early in loser cells and five of these encoded cell membrane proteins, suggesting that cell-cell communication is critical in the initial stages of cell competition. One of these membrane proteins, Flower (Fwe), was examined in detail.

Fwe is conserved in multicellular organisms and in the Drosophila study was found to be required and sufficient to label cells as winners or losers. The win/lose decision is mediated by three differentially expressed forms of fwe (fwe^ubi, fwe^LoseA and fwe^LoseB) and cells are identified as losers when relative differences in fwe^ubi and fwe^Lose levels are detected – stress conditions that uniformly affect the entire population result in cell survival. Although further work is necessary to elucidate the detail, the team proposes that, in outcompeted cells, the fwe transcript is alternatively spliced and fwe^Lose isoforms are induced at the expense of fwe^ubi. It is likely that downregulation of fwe^ubi and upregulation of fwe^Lose both contribute to establish the lose/win decision.

The cellular tagging by Flower isoforms may have biomedical implications beyond cell competition since imbalances in cell fitness also occur during ageing, cancer formation and metastasis.

The study is published in Developmental Cell.

How ApoE4 May Increase Alzheimer’s Risk

ApoE is a lipid transport protein with roles in transport of dietary lipids, regulation of plasma cholesterol, and protection from atherosclerosis. In humans, there are three variants of ApoE (ApoE2, ApoE3 and ApoE4) and one of these, ApoE4, has been linked to earlier onset of Alzheimer’s disease. The mechanisms underlying the increased risk of Alzheimer’s disease remain unclear but researchers at UT Southwestern Medical Center have now shown that the ApoE4 variant reduces surface expression of receptors involved in synaptic plasticity by sequestering the receptors inside the cell.

ApoE interacts with members of the LDL receptor family and one of the receptors for ApoE, Apoer2, also acts as a signalling receptor for reelin, a protein that is important in the developing brain but also enhances NMDA receptor activity and increases long-term potentiation (LTP) in the adult brain. ApoE4 was found to reduce surface expression of NMDA and AMPA receptors as well as Apoer2 receptors, thereby impairing glutamatergic neurotransmission. β-Amyloid peptide, a hallmark of Alzheimer’s disease, suppresses LTP and the ability of reelin to counter the effects of β-amyloid peptide was almost completely abolished in mice expressing human ApoE4. The team are now trying to understand whether it is possible to build on their findings to develop new treatments for Alzheimer’s disease.

The study is published in the Proceedings of the National Academy of Sciences.

NSAIDs and Cancer – Another Piece of the Puzzle

Regular use of NSAIDS has been linked to reduced incidence of certain types of cancer but the underlying protective mechanisms are unclear. Some of the anticancer effects are believed to be mediated through inhibition of COX-2, but a study led by investigators at Sanford-Burnham Medical Research Institute has now identified another mechanism by which the sulindac sulfide (the NSAID metabolite of sulindac) inhibits tumour growth. The team found that sulindac sulfide induces apoptosis by binding to retinoid X receptor-α (RXRα), a member of the nuclear hormone receptor family which had been already been identified as a potential target for cancer therapy. In cancer cells, levels of RXRα are often reduced, at least in part because of proteolytic processing to a truncated form, tRXRα. As with other nuclear receptors, RXRα regulates transcription of target genes by binding to DNA response elements but accumulating evidence suggests that RXRα may also have extranuclear activity. Both RXRα and tRXRα can exist in the cytoplasm and the study showed that cytoplasmic tRXRα can activate the PI3K/AKT survival pathway by interaction with the p85a subunit of PI3K, leading to anchorage-independent cell growth in vitro, and tumour growth in animals. Sulindac sulfide was found to inhibit the tRXRα-mediated PI3K/AKT activation, suggesting that the compound could provide a useful lead for anti-cancer drugs targeting this pathway.

The use of NSAIDs to reduce the incidence of cancer has been limited by the risk of major cardiovascular events and the Sanford-Burnham have identified an analogue of sulindac sulfide, K-80003 which has improved affinity for RXRα but lacks significant COX-2 inhibitory activity. K-80003 inhibited the growth of cancer cells in vitro and in animals and would be expected to have reduced COX-2-associated side effects.

The study is published in the journal Cancer Cell.

Dual-receptor T-cells may Trigger MS

Multiple sclerosis (MS) is an autoimmune disorder in which T-cells attack and damage the fatty myelin sheaths around the axons of the brain and spinal cord, disrupting the conduction of electrical signals along the nerve fibres. Although both genetic factors and viral infections have been suggested to contribute to the development of MS, no single virus has been conclusively linked to the disease and other mechanisms could also play a role. Animal models induced by CD8⁺ T-cells show similarities to human MS, and researchers at the University of Washington investigating the causes of MS have engineered mice that over-express CD8⁺ T-cells that recognise myelin basic protein (MBP), a candidate autoantigen in MS.

When infected with vaccinia virus engineered to produce MBP, the infection should activate the CD8⁺ T-cells to attack virally infected cells and also other cells that produce MBP. As expected, mice infected with the engineered virus developed MS-like disease but, surprisingly, symptoms were also triggered by infection with wild-type virus. This suggested that the engineered CD8⁺ T cells expressed a second receptor that recognised wild-type virus and subsequent cross-breeding experiments confirmed that some of the CD8⁺ T cells did indeed have receptors for both MBP and wild-type virus. Once activated by the virus, the dual-receptor CD8⁺ T cells were than able to attack cells producing MBP.

The study suggests a role for dual-receptor cells in autoimmune diseases and could explain how infection with a common virus triggers MS in genetically predisposed people, whilst having no lasting effects in most of the population. In the ‘dual-receptor model’, autoimmune activation could be triggered by a chance event leading to T-cells that recognise both MBP and a viral antigen. The prevalence of dual-receptor T cells is presently unclear and the team plan to assess whether they are more common in MS patients.

The study is published in Nature Immunology.