Linking the brain, gut and bacteria in neurological disease.

The lifetime risk of developing neurological disease is influenced by variety of factors: genetics, cardiovascular health, and of course, age and neuroscience research continues to uncover more subtle links. 

Recent work elaborates on a long-suspected connection between that occasionally troublesome leftover, the appendix, and Parkinson’s disease risk, while other researchers have raised the possibility of the brain having its own microbiome, with implications for a bacterial influence on the risk and course of neurological disease.

Alzheimer’s disease (AD) and Parkinson’s disease (PD) are characterised by the accumulation of mis-folded proteins in the brain: amyloid β and tau proteins in the case of AD, and α-synuclein in PD, where it is the main constituent of “Lewy bodies”- clumps of aggregated protein found within neurons and a hallmark of PD and other dementias. Mutations within the α-synuclein gene are found in familial PD and efforts are ongoing to determine the normal function of α-synuclein and whether preventing its aggregation or accumulation in the brain might be of benefit.

PD causes both motor and non-motor symptoms and gut-associated problems such as constipation and impaired emptying of the stomach are common years before the onset of motor symptoms. That the aberrant form of α-synuclein can be found throughout the gastrointestinal tract in individuals with PD has been known for several years, although this also the case in those who don’t develop PD.

   

The highest levels of aberrant α-synuclein are found the appendix, raising the possibility that it serves as reservoir for dysfunctional protein which makes its way to the brain via the vagus nerve and potentiates the transformation of normal α-synuclein into the aggregated form. Circumstantial evidence for a link between the appendix and PD risk has been found in a large epidemiological study, with individuals who had undergone surgical severing of the vagus nerve (usually to manage hard to treat chronic duodenal ulcers) being at  lower risk of developing PD.

  

Defining the role of the appendix in PD has proved elusive, and three recent epidemiological studies failed to find any obvious link. By analysing medical records from over 1.6m Swedes from 1964, a research group at the Van Andel Institute has established that appendectomy reduces the risk of developing PD by around 20%, although this protective effect was only apparent in individuals living in rural areas. Pesticide and herbicide exposure are linked with a higher risk of PD and appendectomy may in some way mitigate environment-related PD risk.  Further analysis indicated that appendectomy delayed the onset of PD by an average of more than three and a half years in those who had undergone appendix removal 30 years or more before.

Biochemical analysis of appendix samples from healthy individuals and those with PD identified aberrant forms of α-synuclein. These were present in 46 of 48 normal individuals.  Mixing normal appendix tissue with normal α-synuclein resulted in the protein being broken down into forms resembling those found in PD brain samples.

 

Although far from being a recommendation for elective appendectomy, the finding that aberrant α-synuclein is common in healthy people suggests that PD risk requires its migration to the brain. Finding ways of confining, or even eliminating, the protein from the appendix could conceivably reduce PD risk.

It’s postulated that appendix might play a role in monitoring and restoring the gut microbiome and that inflammation results in changes which favour bacteria which generate “pro-PD” metabolites. That the gut flora might directly influence the neurological environment is not as far-fetched as it once would have seemed although a poster presentation given at the Society for Neuroscience annual meeting suggests the possibility that a local, rather than distant, microbiome might potentially influence conditions in the brain.

University of Alabama researchers have found that rod-shaped structures first observed on electron microscopic examination of brain samples from schizophrenics are, in fact, bacteria. These were most abundant in the substantia nigra, hippocampus and prefrontal cortex but rarer in the striatum. Bacteria were also found within brain cells, particularly in the ends of astrocytes closest to the blood-brain barrier locations, in dendrites, glial cells and in and around myelinated axons.

To rule out sample contamination, the group compared fresh brain samples from mice raised in a normal environment and those born and maintained in a germ-free environment: bacteria were only found in the former. Nucleic acid sequencing indicated that most of the bacteria belonged to groups commonly found in the gut, although their means of passage to the brain- whether from blood, the nose or through the nervous system.

Since bacteria were found in the brains of both normal individuals and those with schizophrenia, there’s no obvious causal relationship, but, as the study of gut, oral and skin microbiomes has shown, bacterial nutrients and metabolites can cause subtle but important changes in cell and organ function. Whether the presence of bacteria in the brain truly indicates a permanent ecosystem and not merely a post-mortem artefact remains to be established. But, as with the appendix and PD, confirmation that the brain is indeed influenced by local (or distant) bacteria may help better define neurological disease risk and uncover new means of treatment and prevention.

Melanoma immunotherapy: Can vaccines and cell therapies expand on immune checkpoint inhibitor successes?

Mestastatic melanoma cells

Tracking the major cancer meetings has kept me occupied throughout October and into November but left me with plenty of material for this, and future, blog articles. A presentation at the UK’s NCRI conference on the increasing mortality rate from melanoma in men (but not women, where mortality rates are generally declining or stabilising), while alarming, did remind me of just how far melanoma treatment has advanced in a few short years.  

Prior to the availability of anti-CLTA-4 and anti-PD-1 immune checkpoint inhibitors, overall survival from metastatic melanoma in developed countries was around 25% after three years: combination immune checkpoint inhibitor treatment has stretched this to over 60%, and use following surgery (“adjuvant” use) significantly improves recurrence free survival.

Despite these successes, a significant need remains for alternative treatments for those who fail, or are intolerant of, current immune inhibitor checkpoint regimens and a gamut of investigational immunotherapies including “personalized” or “individualised” peptide and mRNA therapeutic vaccines, cell therapies and oncolytic virus therapies are in active clinical development.

That the immune system recognises melanoma as being “not self” has been known for decades and means of usefully exploiting this distinction long precede the discovery of immune checkpoints. Attempts to effectively boost the anti-melanoma immune response through injection of the Bacille Calmette–Guérin (BCG) tuberculosis vaccine were made in the 1970s, with mixed success. The potent immunomodulators, interferon alpha (IFNα) and interleukin 2 (IL-2), were approved for use in melanoma in the 1990s and still have a role in the treatment of metastatic disease and adjuvant therapy.

Melanoma has long been an attractive target for cancer vaccine development. A variety of melanoma antigens common to a majority of tumours - “tumour-associated antigens” (TAAs), including gp100, GM2; tryosinase, MART-1 and MAGE-A3, have been exploited, either alone or in combination, in cell-based and peptide therapeutic vaccines.

Cell-based vaccines (as either intact or processed tumour cells or as cell-free lysates) offer the advantage of presenting a spectrum of TAAs, although neither patient-derived (autologous) nor cultured tumour cell-derived (allogeneic) cell-based melanoma vaccines, such as Melacine® (GSK/Schering) and Canvaxin® (CancerVax/Serono), have made it through pivotal studies. M-VAX (AVAX), a chemically-modified autologous cell vaccine, has been in late-stage development limbo for over a decade.

Historically, peptide vaccines have fared no better, with a pivotal study of Oncophage® (Antigenics), the manufacture of which involved isolation of heat shock protein-peptide complexes from autologous tumour cells, being abandoned, and a Phase III study of a MAGE-A3 peptide vaccine (GSK) being terminated due to lack of obvious efficacy over placebo.

Adoptive cell transfer (ACT) involves the collection, isolation, ex vivo expansion and (re)-infusion of autologous tumour-associated cytotoxic T-cells. ACT using tumour-infiltrating lymphocytes (TIL) has occasionally attained response rates of 40%-50% and complete remission in 10% to 25% of patients with extensive metastatic disease: however, the complexity of ACT has essentially confined it to clinical studies and compassionate use.

As is the case with other cancer indications, decades of disappointment and inconsistency have not curbed the academic and commercial pursuit of effective melanoma immunotherapies. Applying recent advances in technology- next generation sequencing; gene transfer and editing; nucleic acid delivery- to melanoma vaccine and cell therapy development might just make these old dogs capable of new tricks.

Cancer vaccine efficacy is blunted by the immunosuppressive tumour microenvironment: combination with immune checkpoint inhibitors is an obvious means of increasing the odds of success and a number of studies combining therapeutic vaccines with anti-PD-1 or anti-CTLA-4 immune checkpoint inhibitors are underway. These agents may eventually be joined or replaced, by one or more of the “next wave” of immune-oncology drugs directed at LAG3, CSF1-R, GITR or at targets in the innate immune systems which can fire up the immune response.

Imlygic® (T-VEC: Amgen), an oncolytic virus therapy is“vaccine-like” in effect, activating both the innate immune system and revealing hidden tumour antigens (“neoantigens”) to the adaptive immune system through tumour lysis. Combination with ipilimumab has shown improvement in response rates over Imlygic® alone, and a Phase III combination study with pembrolizumab (KEYNOTE-034) is ongoing. Early-stage studies of CAVATAK®, an investigational virotherapy acquired by Merck & Co from Viralytics earlier this year, has shown promise when combined with either pembrolizumab or ipilimumab.

The application of next generation sequencing technology and bioinformatics could offer a practical route to bespoke melanoma vaccines, with antigen selection and vaccine composition being determined by tumour and patient genetic makeup. Neon Therapeutics is currently trialling a synthetic peptide vaccine (NEO-PV-01) using sequencing of tumour biopsy material to formulate a selection of up to 20 peptide-mimicking patient-specific neoantigens. NantBioScience is pursuing a similar personalization strategy, with expression of patient-specific neoantigens in yeast cells (YE-NEO-001).

The in vivo expression of melanoma (and other cancer antigens) through the introduction of the corresponding mRNA sequence is receiving increasing attention. Lipid complex mRNA vaccines, encoding multiple melanoma TAAs or a personalised selection of antigens, are now in early clinical studies (Lipo-MERIT and RO7198457: BioNTech).

mRNA has brought a new twist to dendritic cell (DC) vaccination, where DCs isolated from the patient are loaded with melanoma antigens to optimise their processing and efficiency of presentation to the immune system. eTheRNA’s TriMix technology combines mRNA encoding melanoma antigens with mRNA encoding proteins known to enhance DC activation and maturation and to promote both helper and cytotoxic T cell production. Durable clinical responses have been achieved in melanoma patients who had failed previous treatments when the TriMix-DC-MEL vaccine was administered in combination with ipilimumab.

Gene transfer may open up additional ACT strategies for melanoma. T-cell receptor (TCR) gene transfer allows the generation of antigen-specific lymphocytes from patient T-cells Early studies with melanoma antigen-specific TCRs have shown modest response rates, although several have been marred by severe adverse events due to the “off-target” destruction of normal melanocytes.

The utility of ACT may be significantly improved through chimeric antigen receptor (CAR-T) technology, where T-cell antigen receptors are engineered to combine binding, signalling and co-stimulatory domains. Pilot CAR-T studies are underway. Improvements in TIL ACT may be possible by using CRISPR-CAS9 gene editing to increase the ability of T-cells to home in on tumours.

Next generation cell therapies, including DC vaccination, are likely to benefit from the broader expanding commercial interest in CAR-T and TCR therapies which will likely lead to further improvements in manufacture and assist in establishing the logistics necessary to delivery patient-specific treatments. Growing use of semi- or wholly-automated cell product processing will ultimately reduce costs and make the treatment of larger number of patients viable.

Effective melanoma immunotherapy had been a long time in coming, but as immune checkpoint inhibitor therapy has shown, revolution is possible. Experimental melanoma immunotherapies still have a lot to prove, but with the aid of across the board advances in immuno-oncology and other disciplines, we may finally see vaccine and cell-based approaches becoming practical and valuable treatment options.

Photo credit: Valencia, JC. NCI Center for Cancer Research

Zika virus: a whistle-stop tour

Zika virus (false colour transmission
electron microscope image)

In a recent piece on mRNA vaccines[mRNA vaccine technology: industry is getting the message] , I mentioned Zika virus development as an indication for this emerging technology. only to realise my ignorance of this high profile pathogen (shameful, since I spent my post-doc years working in a school of tropical medicine)

First stop in filling the knowledge gap was a visit to the ever-useful World Health Organisation website which features a comprehensive “Zika timeline”. First discovered in 1947 in monkeys living in a Ugandan forest (which gave its name to the virus), and shortly after in species of Aedes mosquito, epidemiological studies conducted in the 60s and 80s indicated widespread human exposure to the Zika virus in Africa and Asia, with infection largely associated with no, or only mild, symptoms.

The more sinister nature of Zika infection emerged in 2007 and 2008, with the first confirmed large scale outbreak on the Micronesian island of Yap, and evidence that infection could be sexually transmitted. Further outbreaks occurred on various Pacific Islands during 2013 and 2014 and pointed to a link between Zika infection and birth defects and with Guillain–Barré  syndrome, a rare autoimmune disease which affects the nervous system. A year later, an epidemic characterized by a skin rash bit otherwise mild symptoms was reported in north-eastern Brazil, but was not recognised as being due to Zika virus.

By October 2015, an increasing numbers of microcephaly cases (newborns with small heads, indicative of abnormal brain development) were being reported. Further Zika outbreaks occurred in several South American and Caribbean countries over the following year, with the first cases (via sexual transmission) being identified in the continental United States in 2016.

Being generally asymptomatic, the prevalence of Zika infection is not easy to quantify, but mosquito-transmitted infection has been reported in over 80 countries (with transmission ongoing in over 60 of these). Over 1.3 million people are thought to have been infected in Brazil alone during the 2015 outbreak. In common with other vector-borne diseases, the spread of Zika owes something to human mobility, although social factors, principally the inability to afford protection against mosquitoes and high population density have been identified as key drivers.

The Zika virus has several properties that contribute to its ready transmission and to its devastating effect on foetal development. The virus is highly persistent in whole blood (up to 100 days) and in the male reproductive tract, allowing sexual transmission. Zika has a preference for certain cell types that facilitate the passage of infection through the placenta; animal studies suggest that viral preference extends to neural progenitor cells essential for normal cortical development. Whether Zika infection is a direct cause of Guillain–Barré  syndrome  has not been established.

As with malaria, Zika’s vulnerability lies in its dependence on mosquito vectors: and targeted insecticide use, management of standing water and the conscientious use of bed nets and repellents can significantly reduce transmission. Like malaria, control initiatives are vulnerable to political and economic factors, including climate change-related changes in mosquito distribution and abundance. Unlike malaria, Zika has a simple lifecycle; as with other flaviviruses (including the causative agents of yellow fever and Japanese encephalitis), infection should, in theory, be preventable through vaccination.

Vaccine development efforts are almost contemporary with the Zika outbreak itself, beginning in the second half of 2015 with the genetic analysis of Brazilian Zika isolates, with the first clinical study of a Zika vaccine being reported in late 2017. Around 45 candidate vaccines have been developed through academic, governmental and industrial efforts, with nine of these reaching the clinic, representing both established (inactivated virus) and experimental approaches (DNA and mRNA vaccines). The WHO’s initial requirement is for a vaccine that can be deployed in response to outbreaks with the primary goal of preventing congenital Zika syndrome through minimizing virus carriage in the immediate population.

Early clinical studies have established that vaccination can elicit aneutralizing antibody responses, although, while an accepted hallmark of flavivirus vaccine efficacy, the importance of neutralising antibodies, and the minimum levels needed to establish protection have still to be established in the context of Zika infection. Other important unknowns include the duration of effective vaccine-induced immunity and whether the reproductive tract can be protected from infection.

              

Perversely, given the untold misery arising from Zika outbreaks around the globe, the virus’s propensity for neural progenitor cells may offer a new means of treating the most aggressive and intractable form of brain tumour, glioblastoma.

Chinese researchers have found that an experimental live attenuated Zika vaccine functions as an oncolytic virotherapy [Going viral] in an animal model of glioblastoma, specifically infecting and destroying glioma stem cells thought to be responsible for the inevitable recurrence of the tumour. Elimination was also observed using glioma stem cells isolated from individual patients.

Photo credit: Credit: NIH/NIAID

Gene editing: off the bench and into the clinic

As the first sponsored CRISPR-Cas9 clinical study gets underway,
industry has high hopes that genome editing will eventually make gene
therapy a mainstream treatment for both inherited and non-inherited
diseases. 

Recent progress in gene therapy, with successful proof of concept being reported in a range of otherwise untreatable conditions and the grant of marketing authorisations for commercially-developed treatments makes it easy to forget that these successes were built on almost three decades of endeavour and regular disappointment.

Gene therapy restores aberrant or missing biological functions through the introduction of functional genes carried into the cell using engineered viruses or by zapping with electrical pulses. A more involved process is the alteration of specific nucleotide sequences within a faulty gene to restore or 
modify function- so-called “gene editing”. 

Around the turn of the millennium, certain bacterial genomes were observed to have multiple copies of repeated DNA sequences which read the same in both directions -“clustered regularly interspaced palindromic repeats” (CRISPR), which were subsequently found to be responsible for the exquisitely specific recognition of infecting viruses. In 2012, it was shown that CRISPR could be exploited to edit genes in mammalian cells, prompting an explosion in translational science, patent wrangles and an influx of venture funding. 

Reduced to its bare bones, CRISPR gene editing utilises a short piece of “guide” RNA, designed to bind to a specific sequence of DNA, complexed with an enzyme able to cut double-stranded DNA, with the bacterial enzyme “Cas9” being widely used. Once within the nucleus, the guide RNA latches onto its complementary DNA sequence, directing the enzyme to cut in the right place: the cell’s DNA repair mechanism completes the editing process. 

CRISPR-Cas9 (and other CRISPR based systems) have brought relative simplicity and low cost to gene editing, providing a powerful research tool with application across biological disciplines and a route to significant advances in agriculture and the treatment of both inherited and non-inherited disease.

American and European regulators have been understandably cautious with respect to CRISPR clinical studies. As with other forms of gene therapy, CRISPR-Cas9 editing is not without potential risk, such as unwanted alteration of DNA sequences other than the target gene sequence, or the triggering of immune responses to the Cas9 enzyme (obtained from one or other of two common skin bacteria) or through introduced RNA being recognised as being  “foreign”. 

Chinese investigators have been less troubled by the unknowns surrounding CRISPR clinical development:  first in man studies began in 2010, with around 80 subjects being treated in cancer and HIV gene editing studies by February this year. Several Chinese research groups have explored  gene editing in non-viable, and most recently, viable human embryos.  

CRISPR Therapeutics, in partnership with Vertex,  successfully addressed undisclosed FDA concerns raised in May this year and are now recruiting beta thalassemia patients for a Phase I/II gene editing study. Like sickle cell disease, beta thalassemia arises from an inherited defect in the gene encoding the oxygen-carrying protein, haemoglobin. 

Rather than directly editing the haemoglobin gene, the CRISPR-Cas9 complex (designated CTX001) is designed to alter a DNA sequence responsible for the shutdown of foetal haemoglobin production, in the hope that turning the switch back on will produce sufficient levels of functional haemoglobin. The editing process is performed ex vivo, with blood-forming stem cells being extracted from the patient, transfected with the CRISPR-Cas9 complex, expanded and then returned by infusion. 

CRISPR Therapeutics and its peers, Editas Medicine and Intellia Therapeutics have ambitions to develop gene editing therapies for a range of genetic conditions that result in metabolic dysfunction (Hurler syndrome, glycogen storage disease, alpha-1 antitrypsin deficiency, transthyretin amyloidosis) and visual impairment (Leber congenital amaurosis, Usher syndrome), as well as cystic fibrosis and Duchenne muscular dystrophy (DMD). Exonics Therapeutics recently published data indicating that their “SingleCut” CRISPR technology was able to  restore near normal levels of dystrophin expression in an animal model of DMD. 

CRISPR may prove to be more efficient and flexible means of engineering “off the shelf” (allogeneic) cancer-fighting T cell therapies, reducing the cost and complexity of current patient-specific CAR-T therapy  [ASCO Clinical Cancer Advances 2018. And the winner is...].

Pre-CRISPR era gene editing methodologies may also prove to have therapeutic application. Sangamo Therapeutics has used a zinc finger nuclease-based approach to achieve “in body” editing of a faulty gene in four individuals with Hunter syndrome, an enzyme disorder that prevents the breakdown of complex sugars, leading to harmful accumulation. A decrease in urinary complex sugar level was observed in two individuals, although not an increase in normal enzyme levels in the blood. Full interpretation of the significance of these results will take time but importantly, “in body” editing raised no safety issues. 

Clinical development of  gene editing-based therapies will continue to advance cautiously (at least in the West), but is likely to can to benefit from experience gained in viral and non-viral vector design now being exploited in other forms of gene therapy and growing regulatory familiarity with gene therapy risk-benefit assessment. 

Image credit: Credit: Jill George, NIH.

mRNA vaccine technology: industry is getting the message

Can mRNA technology take vaccine
design and development into a
post-Jennerian era?

Vaccination is second only to the provision of drinking water with respect to global public health benefit, and while  still some way short of universal  vaccine coverage, hundreds of millions of children and adults are protected each year  from formerly lethal  infections.

Vaccine development has never lacked ingenuity, but progress has arguably been evolutionary rather than revolutionary. The concept of injecting selected components of infectious agents rather than whole microorganisms would not greatly surprise Edward Jenner.

Twenty-first century vaccinology faces major challenges. Conventional manufacturing and deployment capabilities are taxed by the annual challenge of shifting influenza strains [A single shot] or in mounting a large-scale response to  “swine ‘flu”-like global pandemics; shorter development cycles are needed to tackle outbreaks of Zika and Ebola viruses, or whatever emerging pathogens tomorrow might bring; no effective vaccines exist for infections common in both developed and emerging economies, including HIV, Chlamydia, cytomegalovirus and tuberculosis.

Revolutions in vaccinology have proved elusive. The prospect of replacing expensive and complex vaccine manufacture with the injection of chemically-synthesised strands of DNA encoding one or more vaccine components has been pursued since the 1990s. Although simple in concept, and despite efforts to optimise DNA delivery, low potency and unresolved safety issues have confined DNA vaccination to a few animal health products. Synthetic peptide vaccines designed to mimic and present a desirable selection of antigenic sequences, struggle to elicit robust immune responses and confer effective protection.

High profile buy-ins by vaccine industry majors suggest that a true technological revolution might, finally, be on the horizon. Protein synthesis requires DNA code to be rewritten in the form  of another nucleic acid, messenger RNA (“mRNA”), which is then translated by ribosomes, the cell’s protein factories. That the transcription of DNA can be circumvented through direct injection of mRNA to produce the  corresponding protein has been known for decades, but the instability of mRNA,  and the complication that unmodified mRNA is itself highly immunogenic,  led to the exploration of mRNA vaccination  being sidelined by less technically demanding DNA and recombinant protein approaches.

Across the board advances in ease of delivery, chemical modification to improve stability and increased duration of protein production in vivo are rapidly making mRNA vaccination a viable proposition.  Clinical trials are underway in both infectious disease indications including influenza, Zika virus and HIV infection (the latter exploring therapeutic rather than prophylactic potential), and in solid and haematological cancers. The majority of cancer studies exploit the properties of specialised antigen-presenting cells (dendritic cells) which can be readily isolated from patients, loaded with tumour antigen encoding mRNA and then returned by infusion [Dendritic cell vaccines: back to the future].

Recent licensing agreements between mRNA vaccine developers and leading vaccine companies add to a growing list of industrial, governmental and non-for-profit collaborations aimed at leveraging  the benefits that mRNA technology might bring to infectious disease vaccination: higher immunogenicity; inherent safety and rapid, low-cost, scalable manufacture.

Pfizer’s $425 million headline collaboration with mRNA vaccine developer BioNTech is focused on building better flu vaccines which can be manufactured quickly and cheaply. Another mRNA vaccine pioneer, Translate Bio, entered in an $805 million headline agreement with Sanofi Pasteur in June covering five undisclosed infectious disease agents with the option to expand the collaboration to other pathogens. CureVac AG has infectious disease mRNA vaccine partnerships with both Sanofi Pasteur and Johnson & Johnson, while GSK and Novartis are collaborating on mRNA vaccine development.

Early clinical data obtained using directly injected flu and rabies mRNA vaccines can best be described as “modestly encouraging” and it will be several years before which, if any, of the various flavours of mRNA technology  can claim to be a viable route to cost-effective, large scale vaccination,  and/or  serve as a solution to  problem pathogens. The picture may become clearer with a second Phase I study of Moderna Therapeutics’s flu vaccine candidate and a Zika virus Phase I study due to complete before year end.

Image credit: Wikipedia (public domain image)

Tumour-treating fields: moving beyond glioblastoma treatment

Tumour treating fields: more than just
fun with physics

Glioblastoma (GBM), that most aggressive of brain cancers, is notoriously resistant to treatment and a variety of leading edge approaches, including immunotherapy, virotherapy and gene therapy  are under investigation as means of extending survival by a meaningful amount.

One of the few treatments to have gained regulatory approval in the last decade is a medical device, Optune™, a custom-made, shower cap-like array of electrodes worn constantly on a shaven scalp, which is claimed to disrupt and slow tumour growth through the application of low intensity, alternating electrical current- so-called “tumour treating fields (TTFs).

I’m probably not alone in being reminded of 19^th century quackery and the various galvanic treatments advocated by the beauty industry, but Optunes’s developer, NovoCure, a publicly traded US-based company, has managed to navigate the rocky path of clinical development to satisfy both American and European regulators, although approval was not without controversy.

Optune™ (then designated NovoTTF-100A) received FDA approval for the treatment of recurrent GBM in April 2011, on the back of clinical data that hinted at, rather than conclusively established, a degree of benefit comparable to chemotherapy, a comparison in itself made complex by differences in investigator choices of chemotherapy regimen. The FDA panel was accused of being overly influenced by pleas from GBM patients and their families during an open public hearing. Approval did come with the rider that NovoCure conduct a post-approval study to establish non-inferiority of TTF treatment to chemotherapy.

FDA approval for the treatment of newly-diagnosed GBM in conjunction with chemotherapy was granted in 2015 on the back of interim clinical data gathered from almost 700 GBM patients receiving radiotherapy and chemotherapy following surgery. The addition of TTF treatment increased the median time to progression, and overall survival, by almost three months over chemotherapy alone.

Neuro-oncologists remain divided on Optune™, being, with reason, critical of a pivotal clinical study that did not include sham treatment as a control, and the vagueness of the TTF mechanism of action, stated as involving “misalignment” of charged proteins critical to cell division. On the other hand, TTF in combination with chemotherapy is recommended in the authoritative National Comprehensive Cancer Center treatment guidelines. Despite the requirement of near constant wear (a minimum of 18 hours a day) and high treatment cost (around $20,000 per month with as yet limited reimbursement, Optune™ is currently in use by over 2,000 GBM patients in the US, Europe and Japan.

Recent data suggests that TTF may be of benefit in other forms of solid tumour. Two completed pilot studies of TTF in combination with standard chemotherapy in malignant mesothelioma and pancreatic cancer patients indicated improvements over historical progression-free survival and one year survival rates obtained with chemotherapy alone.

An apparent improvement in progression-free survival was observed in a small study conducted patients with recurrent ovarian cancer is more difficult to interpret due to difference in the treatment histories of individual patients and historical chemotherapy comparators.

Nonetheless, NovoCure is sponsoring three pivotal TTF studies in pancreatic cancer, non-small cell lung cancer (NSCLC) and in brain metastases arising from NSCLC, with final data from the latter study anticipated in 2020. TTF faces tougher competition in these indications from advances in precision chemotherapy and immuno-oncology, but should TTF even prove to be non-inferior to conventional therapies, it could find use in individuals intolerant of, or unresponsive to, other forms of cancer treatment.  Success may also serve to disarm TTF’s critics, as might elucidation of the putative mechanism(s) of action, along with the acid test of undertaking a  TTF study that includes a sham treatment control arm.

Image credit: Wikipedia(creative commons licence)

Holding back the years. Drug development takes on the ageing process.

Can the selective removal of old cells

 modify age-related conditions?

An oft-quoted Irish proverb advises “Do not resent growing old. Many are denied the privilege”.  Compared with the alternative, we should perhaps be more accepting of the physical (and cognitive) decline that time brings to all of us. But rising life expectancy has consequences, not least in healthcare costs: postponing or even reversing age-related decline has a societal importance far beyond personal vanity.

The impact of age is readily apparent at the cellular level, and a variety of alterations in the mechanisms which regulate normal cell division and function, including genetic stability; energy production, and cell-to-cell communication – collectively “cellular senescence” – have been identified. Ageing cells secrete a variety of pro-inflammatory proteins and are more abundant in failing hearts, joints and eyes. The relationship between senescence and organ dysfunction is misty, but animal studies in which an artificially-aged type of stem cell was transplanted into young mice resulted in a decrease in their physical capabilities and lifespan when compared to control animals.

Moreover, treatment with agents hypothesised to be capable of triggering programmed cell death (apoptosis) in senescent cells had a positive effect on physical function and lifespan in the stem cell-transplanted mice and on levels of secreted proteins associated with cellular senescence.

Drugs designed to selectively remove ageing cells- “senolytics”- are just beginning to make their way into clinical development. Unity Biotechnology’s strategy is the elimination of ageing cells in specific disease states. UBX0101 targets the interaction of the proteins MDM and p53, the latter being involved in cell-cycle arrest and apoptosis. A Phase I study is recruiting subjects with osteoarthritis, who will each receive a single intra-articular injection into the knee. A preclinical candidate, UBX1967 targets Bcl-2, another pro-apoptotic protein, and is intended for the treatment of age-related ophthalmological conditions.

Several other candidate senolytics are in preclinical evaluation. Two target the interaction between p53 and a DNA-binding protein: FOX04, a pro-apoptopic peptide (FOX04-DRI: Cleara Biotech), and an undisclosed small molecule (Antoxerene). Oisin Biotechnologies is developing a plasmid-based gene therapy to selectively ablate senescent cells.

Recently published results suggest that pharmaceutical intervention might be capable of bolstering ageing immune systems. RTB101, being developed by ResTORbio in partnership with Novartis has completed a Phase II study conducted in elderly volunteers. RTB101 is a combination of two drugs, both of which act on targets within a long-studied, multi-protein complex, “the mechanistic target of rapamycin” (TORC1), which is critical for the activation of protein translation.

The 264 study volunteers (65 years or over) received either the drug combination, one or other of the drugs or placebo tablets for six weeks and were then given a flu vaccination two weeks later. At 10-month follow-up, those receiving both drugs had the lowest rate of self-reported respiratory infection; blood tests indicated that the combination (but not single drug treatment) resulted in more complete TORC1 inhibition and that genes which control virus-fighting Type 1 interferon production were upregulated.  The authors postulated that other mechanisms might also contribute to this apparent reduction in “immunosenescence”.

Time to start planning for that 100^th birthday celebration? Probably not (unless centenarians run in the family), but there’s a reasonable expectation that one or more of the current development strategies in, or close to, clinical study, will eventually lead to useful medications, although perhaps in specific age-related disease states rather than universal elixirs of youth.

Photo credit: Linnaea Mallette https://www.publicdomainpictures.net

Gut instinct

It's what's inside that counts- microbiome research is 

starting to pin down how gut bacteria contribute

to a variety of disease states.

It's estimated that the number of Intestinal bacteria is roughly equal to the number of cells in our bodies and, intuitively, one would expect such a substantial and active biomass (around 200g in a 70kg adult) to have some influence on health and disease.

Increasingly sophisticated means of profiling the multitude of bacterial species found in the gut microbiome and the metabolites they produce are beginning to identify factors that contribute to inflammatory bowel disease, cardiovascular disease, metabolic diseases and how individuals respond to cancer immunotherapy. 

The role of the microbiome in gut conditions has, as might be expected, received particular attention. Inflammatory bowel disease (IBD) falls largely into two distinct types: ulcerative colitis (UC), which is confined to the large intestine; and Crohn's disease (CD), which may affect any part of the digestive tract. Both are chronic conditions characterised by periods of remission and flare, and are treatable but not curable. IBD is immune-mediated, although the trigger(s) remain largely undefined: genetics; the integrity of the gut surface, and environmental factors (diet, stress, antibiotic use) which may change the gut flora all play a part. 

Analysis of the gut microbiome in IBD sufferers suggests that both UC and CD are associated with different "microbial signatures", with certain bacterial species being present in abundance, and other species being absent or present only in low numbers. Bacterial degradation of mucin, a protective glycoprotein which lines and protects the gut is one possible contributory mechanism, as are changes in microbial fatty acid and amino acid biosynthesis. Association is by no means proof of causation- changes in the microbiome- “dysbiosis”- might result in chronic inflammation, but the reverse may also be the case.

Alteration of the microbiome through antibiotic treatment is associated with better clinical outcomes in IBD, but also appears to be a risk factor for the development of CD. There is no compelling evidence that probiotics- the "good bacteria" of marketing campaigns- are of benefit in IBD. While understandably unappealing, re-balancing the gut flora through "faecal microbial transplantation" (FMT) has been shown to be of benefit in small studies involving IBD patients. 

A recent study throws light on the relationship between one particular bacterial metabolite, ascorbate (vitamin C) and IBD. Microbially produced vitamin C does not contribute to our nutritional requirement but its presence in the gut may be significant. University of California researchers investigated 139 different microbial metabolites associated with CD for their effects on human T cells which mediate inflammatory processes. Fifteen metabolites were identified as having an effect on T cells, of which only ascorbate served as an inhibitor of T cell activation, possibly through altering glycolysis, a key energy-producing pathway.

That certain bacterial species associated with CD are capable of ascorbate production adds a little more circumstantial weight to the hypothesis, but further elucidation of the relationship between disease and gut ascorabate levels and the presence or absence of ascorbate-producing species is required before practical use can be made of this observation. 

The enzyme indoleamine-2,3-dioxygenase (IDO) has featured in a couple of my blog pieces as a target of great interest in cancer immunotherapy. IDO is an important regulator of tryptophan levels and increased IDO activity contributes to unwanted immunosuppression within the tumour microenvironment. The clinical performance of IDO inhibiting drugs in combination with established immuno-oncology agents has been less than stellar, leading to the large scale abandonment of further development [IDO a no-go: what’s up next in immuno-oncology combination therapy?]

Intriguingly and unexpectedly, IDO, or more accurately, the lack of it, has been found to have a beneficial effect on gut microbes in an animal model of obesity. IDO activity is increased in obesity and French researchers have shown that IDO action in the gut results in a "rewiring" of tryptophan metabolism which in turn impacts on insulin sensitivity, lipid metabolism in the liver, the integrity of the gut surface and levels of chronic inflammation. 

These effects are mediated through production of IL-22, a signalling protein involved in immune regulation and liver and gut epithelial cell survival. Knocking out IDO activity allows gut bacteria to direct tryptophan metabolism in a benign manner. The composition of the gut microbiome was found to be different between mice with active and inactive IDO. 

It's a leap to suppose that IDO inhibitors might eventually be repurposed as treatments for obesity, but better understanding of the interplay between "immunometabolic" mediators such as IDO and the microbiome could open up paths to better therapies or means of either preventing metabolic disease or identifying individuals at higher risk of obesity.

Photo credit: Darryl Leja, National Human Genome Research Institute, National Institutes of Health.

Targeting tooth decay

For now, brushing beats biopharma

in caries prevention

Back in the late 90s, I was involved in the licensing of an early-stage peptide drug aimed at preventing tooth decay by blocking the anchoring of the acid-producing (and enamel destroying) bacterium, Streptococcus mutans. As is not unusual in small biopharma, development was never progressed, but I’ve remained interested in the concept of caries prevention through pharmaceutical intervention. 

Tooth decay is an infectious disease, the principal villains being S.mutans, S.sobrinus and other acid-producing/acid-loving bad actors which outstrip other bacteria present on the tooth surface and modify the local environment to their advantage. S.mutans breaks down the sugars found in our diet and the resulting acid initiates and continues the destructon of enamel and dentine, an essential tooth mineral. Caries risk is influenced by a variety of factors, the most obvious being sugar consumption and oral hygiene habits, although the recent identification of highly virulent S.mutans strains by Swedish researchers associated with mutations in saliva proteins indicates that genetics may play a part.

As a bacterial disease, it’s not surprising that vaccination has been pursued as a possible means of caries prevention. Saliva contains secretory IgA (sIgA), a class of antibody evolved for the protection of mucosal surfaces and oral protection is theoretically possible providing effective levels of specific sIgA can be generated. Vaccination is also a low-cost and practical means of protecting large populations from an early age. 

Since the 70s, a multitude of potential vaccine targets expressed by both S.mutans and S.sobrinus have been identified and evaluated in a variety of formulations and different delivery routes (systemic, oral, intranasal), largely in animal models and a small number of volunteers studies. Despite more recently applied technical sophistication (including DNA vaccines and synthetic proteins combining several bacterial targets), development has never moved out of the transitional phase and the challenge of eliciting an effective, long-lasting antibody response remains. 

Almost as old a concept is “passive” immunisation- the use of bacteria-specific antibodies delivered in a mouth rinse or gel to sequester unwanted acid-producers. While the concept has been established in animal models and a few clinical studies, formulations based on mouse, bovine, chicken and even plant-grown antibodies have remained as curiosities. 

A recent approach combines advances in antibody generation to probe for targets actually present on living S.mutans  and S.sobrinus  and evolve corresponding antibody fragments which retain their specific binding properties. Fragments can be produced cheaply at large scale, and while successful in reducing caries in an animal model, formulation to give effective protection without frequent reapplication poses a formidable problem.

A variety of both natural and synthetic peptides with antimicrobial action have been evaluated as a means of ablating S.mutans and other acid-producers, although with mixed results. Clinical trials of a “specifically targeted antimicrobial peptide”- STAMP, designated C1652 and sponsored by a small US biotech company, C3J Therapeutics, are underway. C1652 was designed to act only on S.mutans to avoid unwanted effects on the oral microbiome. 

Interestingly, C1652 treatment appears to prevent S.mutans recolonization after treatment, although it’s too early to tell whether this useful property will be observed in human studies. A collaboration between Johnson & Johnson, a pharmaceutical and consumer healthcare giant,  and the University of Pennsylvania School of Dental Medicine is looking at the feasibility of low-cost antimicrobial peptide production in plants. 

Effective immunological or antimicrobial-based protection against decay probably remains decades away, but drug-based means of repairing the damage might be on the horizon. Researchers at the Dental Institute, Kings College London have shown that, tideglusib, a drug originally investigated as a potential Alzheimer’s disease treatment, acts upon stem cells present in dental pulp to stimulate dentine production. This process does occur naturally, but at a level too low to result in a robust repair. 

Tideglusib inhibits an enzyme, glycogen synthase kinase 3 (GSK-3), yhat normally serves as a brake on dentine production. When drug-loaded protein sponges were place in cavities formed in mouse teeth, sufficient dentine was produced to repair the tooth within six weeks. Optimised GSK-3 inhibitors could proof to be even more effective restoratives. 

In the meantime, the toothbrush, dentist and the fluoridation of drinking water remain our best defence against the ravages of S.mutans and its companions. 

Photo credit: Thegreenj, Wikipedia Creative Commons

Bugs as drugs?

Lactococcus lactis: Potential
beyond cheesemaking? 

More microbiological nostalgia, this time prompted by an article in Nature concerning another decades-old concept, the therapeutic use of genetically engineered bacteria.

The early 90s saw a spate of clinical studies involving bacteria (Clostridium and Salmonella species) capable of growing and multiplying within the oxygen-poor environment found within solid tumours, their natural anti-cancer properties enhanced through engineered expression of enzymes capable of activating cancer drugs. Response rate were far from compelling, although interest remains in their use as oncolytic “vaccines".

Listeria monocytogenes is under evaluation as a live vector for the delivery of tumour-specific antigens across a variety of cancer indications, but the treatment-related death of a cervical cancer patient (and subsequent clinical hold) in a Phase I/II combination study involving Advaxis’s L.monocytogenes HPV vaccine has raised questions as to whether the vector or AstraZeneca’s immune checkpoint, Imfinzi® (durvalumab) is the culprit.

More recent efforts are directed towards conditions other than cancer and are exploiting advances made in synthetic biology. A US company, Synlogic, is applying “therapeutic programming” to repurpose commensal bacteria by stitching in DNA encoding for environmental sensors and metabolic switches. Clinical studies have recently commenced with an E.coli strain engineered to break down phenylalanine, an amino acid that accumulates to harmful effect in those with the hereditary condition phenylketonuria, and with another engineered strain capable of breaking down ammonia in those with life-threatening blood levels arising from genetic disease or through liver failure.

Intrexon (through a subsidiary, ActoBio Therapeutics and various collaborators) is looking to repurpose the cheese makers’ friend, Lactobacillus lactis. A clinical study is underway with a strain that produces trefoil factor I, a human protein involved in the maintenance and repair of mucosal epithelium, in subjects with oral mucositis, a painful inflammation of the lining of the mouth and a common side effect of radio- and chemotherapy.

Another strain in the clinic produces an antibody fragment against tumour necrosis factor, the target of several established and effective therapies for inflammatory bowel disease and a study is planned with a strain which produces a form of insulin thought to trigger the autoimmune destruction of insulin-secreting cells in Type 1 diabetes, along with a tolerance-inducing cytokine.

While the genetic manipulation of well-characterised bacteria is relatively straightforward, the design and development of effective therapeutic strains is not without problems. Mutation and the loss of inserted plasmids can result in reversion of engineered bacteria to their wild state. Predicting the potency of modified bacteria and the likelihood of successful colonisation remains complex and uncertain. As demonstrated by antibiotic resistance, bacteria are notoriously promiscuous when it comes to sharing DNA and there is a risk that the ability to express therapeutic proteins might be passed to other bacterial species. The gut and oral microbiomes play subtle and important roles in human health and might be detrimentally altered through colonisation by engineered strains.

While none of these challenges are insurmountable, and with admiration at the ingenuity exercised in designing and developing bacterial therapeutics, I suspect that the same treatment objectives can be more readily achieved by established, less problematic, technologies, although the convergence of synthetic biology and growing understanding of the human microbiome opens some intriguing, if not near-horizon, possibilities for short or long term beneficial manipulation.

Photo credit: Kenneth Toda, University of Wisconsin