Science Pool

Continuous biomanufacturing is reducing the cost of goods of biopharmaceuticals. Achieving continuous manufacturing requires expertise in equipment design.

Download the highlights of Andrea Isby's presentation at Repligen's DSP Workshop in Estonia from May 23rd, 2024 to learn more. 

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High-throughput screening methodologies have accelerated downstream development for monoclonal antibodies by enabling parallelized evaluation of chromatographic resins across a range of conditions. However, scientists must now interpret results in a meaningful and consistent way.

Learn how Just - Evotec Biologics' Downstream Data Browser automates visualization of high-throughput datasets, fits response surface statistical models, standardizes report results from a high-throughput screening method and facilitates comparison across molecules allowing the accelerated development of continuous biomanufacturing processes.

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Biomarkers are very useful tools for drug developers as well as for clinicians. In drug research and development, they add value as they improve the success rate of clinical trials. In the clinic, they validate the eligibility of patients as well as the efficacy of an approved treatment. In the recent Evotec webinar on aging, Elizabeth van der Kam, SVP, Translational Biomarkers and Human Sample Management, gave an overview on biomarkers in general and the role of biomarkers in aging.

In fact, the success rate of clinical studies can by doubled by introducing biomarkers early on, that can predict efficacy and potential safety issues. Biomarkers also may be important to reduce costs by running smarter trails in smaller groups of patients and if translated to companion diagnostics, biomarkers enhance the readiness of payers to reimburse a novel drug, but they also enable higher profits as the drug can be sold together with a diagnostic test. Therefore, Evotec´s strategy is to develop a biomarker as early as possible during the R&D process.

Types of biomarkers

There are several types of biomarkers. Useful for early studies are biomarkers that demonstrate target engagement, meaning they show that a drug candidate hits the target in the relevant organ and triggers a response. However, target engagement not necessarily means that this is relevant for the disease.

Another classification consists of surrogate biomarkers, which exhibit correlations with the disease or its progression and could hold relevance in the context of the disease More useful are efficacy markers which are not just correlated but causative for the disease. Another important class of biomarkers are safety biomarkers which, as an example, alert a clinical trial leader or a physician that the drug also hits another target and could potentially cause an issue. Then there are stratification markers indicating the likelihood of a patient to respond to treatment. This is important as non-responders should not be included in trials or prescribed an ineffective treatment. Last but not least, there are diagnostic and prognostic biomarkers that help to better understand the disease and its progression, to establish the right dosage, assess efficacy and predict disease progression and monitor the patients.

In any case, a biomarker needs to be translatable and relevant, and its measurement should be feasible, robust, reliable, and durable.

Biomarkers in aging

The situation is complex in aging. Chronological age is not the best inclusion criterium for clinical trials of medicines trying to improve the health span of elderly patients as chronological age can be very different from biological age.

But how to define biological age? What markers are out there? Of course, there are a lot of markers of biological age, e.g., body composition, body fat, physical appearance and function, muscle mass, grip strength, walking speed, balance, wrinkles, grey hair, but also blood-based changes in terms of hormone and vitamin levels and progressing diseases such as poor eyesight, osteoporosis, declining kidney function, and many more.

However, none of these markers is sufficient as a stand-alone data point. Some of the changes observed in elderly people can also be found in younger people or in patients with non-age-related diseases. The best biomarkers are the ones that can be established without subjective assessments.

The situation is further complicated by the fact that aging is not a disease, and that any intervention should be made early before the onset of typical signs of aging. Ideally, one would have biomarkers that can tell which category of older people will develop certain diseases. At present however, there are biomarkers indicating changes in many pathways and targets, but these often only indicate a certain chance of getting a disease.

The challenge

At present, biomedicine does not have access to markers that can predict certain biological deteriorations, let alone predict potential success of a treatment. And how to define a subpopulation and forecast treatment success without waiting for years to see an effect?

Currently one of the best overall indicators of biological aging is inflammaging. It demonstrates changes in the immune system, inflammation, and an imbalance in the innate or the adaptive immune system, thereby predicting a high risk of unhealthy aging. However, inflammaging can also be caused by lifestyle and gender, so it is not an ideal biomarker. Recently, under review of the U.S. National Institute for Aging, the TAME BIO (Targeted Ageing with MEtformin) project tried to establish a basis for future biomarker discovery and validation and accelerate the pace of ageing-research. 

The project started out with more than 200 potential biomarker candidates that were screened for feasibility, dependency on gender, and environmental factors, etc., bringing down the list of candidates to less than 90. Then they were assessed for disease-relation, robustness, their association to multi-morbidity and the usefulness to clinical trials, leaving a final set of eight candidates. This was, however, a purely theoretical exercise and whether these candidates are useful in real life needs to be proven. At present, the jury is still out on useful biomarkers for trials and therapies to prolong health span and quality and duration of life.

Learn more in the webinar "A Spotlight on Ageing" by Elizabeth van der Kam, SVP, Translational Biomarkers and Human Sample Management at Evotec

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Watch on demand now!

Our 3 part "Spotlight on Aging" webinar series is now available to stream on demand.

In the last fifty years, life expectancy has increased dramatically thanks to advances in science, medicine, and public awareness. The population is living longer, but with it comes the challenge of improving Healthy Life Expectancy.

Join our speakers in our innovative 3-part webinar series where they will openly discuss 3 key topics:

• Therapeutic approaches for aging and age-related disease
• Aging and precision medicine
• Why older adults should not be underrepresented in clinical trials. 

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Can we age healthier?

Due to better hygiene and medicines, the aging population has been growing steadily over the last 30 years. By the year 2031 1.4 billion people will be aged 60 or over, comprising one in six of the worlds population.. This figure will reach 2.1 billion by 2050, with 426 million being aged 80 or more. Unsurprisingly, the UN declared 2021 -2030 the Decade of Healthy Aging. The aging global population brings considerable societal challenges. In developed nations, old age increases the financial pressure on healthcare systems because healthcare spending rises sharply with age. This is in part due to the increased use of medications with advanced years, but also the associated support and care costs.

Increasing healthspan

However, the underlying problem is not life span but healthspan. Being of advanced age does not necessarily mean being frail, sick and in need of care. Already today, there are many healthy seniors living an active life. To promote this notion, the WHO set the goal to provide every person in every country in the world the opportunity to live a long and healthy life. This is encapsulated perfectly with the definition of healthy aging being ‘the process of developing and maintaining the functional ability that enables well-being in older age’. Functional ability means having the capabilities to be and to do what people have reason to value, i.e. meeting not only basic needs but also allowing them to learn, grow, and make decisions, to be mobile, to build and maintain relationships, and to contribute to society. This is very different from life extension calculated as accumulation of human years.

What is biological aging?

Aging can be defined as a time-dependent decline in body function and is observed in virtually all living organisms. The accumulation of cellular damage due to dysfunction in multiple biochemical systems increases the susceptibility to disease and ultimately results in death. This is most likely a result of evolution, once an organism has reached sexual maturity to enable reproduction and raising offspring, it makes no biological sense to invest more energy in maintaining the organism. However, the case is more complex for long-lived mammals, with offspring that need to be protected for a protracted time to enable them to reach sexual maturity. Hence mammals have evolved sophisticated and very efficient repair and maintenance mechanisms in order to correct any cellular dysfunction. With this perspective in mind, aging can be defined as the gradual deterioration of this biological maintenance. We can then view improving healthspan through the lens of slowing this decline or improving restorative or regenerative capacity within tissues and organs.

Already, science has identified a number of factors that can contribute or accelerate the aging process and these include genetic predisposition, obesity, smoking and the status of the gut microbiome. Downstream of these drivers is frequently low-level chronic inflammation – ‘inflammaging’ that contributes to the gradual deterioration in cellular and tissue function. The immune system itself is also subject to decline over time, meaning that many of the protective and repair mechanisms of the adaptive and innate immune system become less effective over time. A combination of these factors are thought to contribute to the common diseases associated with advanced age, such as cancer, cardiovascular disease, chronic liver and kidney diseases, type-2 diabetes and dementia. We are all familiar with how these diseases lead to a reduced life expectancy, but also significant impairment in the individual’s quality of life.

At the cellular and molecular level, there are key biochemical mechanisms that promote gradual deterioration of function across all cell types, that we believe underpins loss of physiological performance - consistent with the process of aging. These are well recognized as the classical ‘hallmarks of aging’. These include the emergence of cellular senescence and the senescence-associated secretory phenotype- which drives much of the chronic inflammation in tissues. Telomere shortening, genomic instability and the accumulation of genetic damage which can lead to tumour formation, epigenetic changes, exhaustion of stem cells, altered cell-cell communication, loss of control of proteostasis, aberrant nutrient sensing and mitochondrial dysfunction. In addition there are more generalised drivers of the aging process such as those linked to dysbiosis.

There is therefore no single driver of biochemical and physiological aging, but the concerted influence of an array of many contribuing factors at play. These complex systems can seem daunting to address, but within each of these pathways there are potential points for pharmacological intervention that constitute targets for drug discovery programs. The goal of pharmacotherapy can therefore be viewed as intervening in key node points to slow the accumulation of dysfunctional processes and maintain cellular integrity for longer. Targeting these fundamental pathological mechanisms will be key to providing a systems-wide (i.e. holistic) benefit to the individual.

How to prolong health span?

It is known that medical interventions, good health practice, refraining from smoking, eating appropriately, and exercising, can all help protect our health. But as mentioned above, there are opportunities to improve healthspan, through targeting key points within the pathways that are recognized as the classical hallmarks of aging.

Developing novel senolytics - challenges and opportunities

One of the hallmarks of aging that has been very well characterized is cellular senescence. This occurs when cells reach the end of their ability to divide resulting in stasis, a state associated with the ‘senescence associated secretory phenotype’ (SASP). Under these conditions, the senescent cells release a cocktail of proinflammatory mediators which in the young targets them for removal by the immune system. However, in the aged, where the immune system itself is subject to gradual decline, senescent cells continue to release the cytokines, chemokines, growth factors and other bioactive components which contribute to inflammaging. As such, the use of senolytic agents which are aimed at killing off senescent cells by suppressing the pathways that keep them alive, is receiving significant attention in the aging field. According to a recent report in Nature Medicine, around 20 clinical trials of senolytic compounds are ongoing. Clearly, blocking the ‘keep me alive’ signals in the cell and triggering cell death offers a compelling approach in the treatment of cancer, but such a powerful pharmacological mechanism carries risk. In keeping with this, at present the majority of the ongoing clinical studies are for very severe indications and not aging. However, it seems likely that if the risk-benefit of such a pharmacological approach in man is understood and favourable, senolytics could find utility in aging.

An additional or maybe complimentary approach would also involve the immune system. A healthy lifestyle suppresses pro-inflammatory mediators and at a very simplistic level, agents which inhibit inflammation may prove beneficial. However, inflammation also plays a very important protective function in the body and so such an approach may not prove advantageous for chronic treatment. Conversely, boosting the performance of the immune cells seems like a more viable approach since it would enable the body to fight off infectious agents and correct and repair cellular and tissue dysfunction in a more effective way. The stem cells giving rise to immune cells reside in the bone marrow and their accessibility means their biology is very well understood, especially with respect to stem cell maturation and differentiation. Manipulation of these precursor cells in a positive way could offer the potential to regenerate the immune system and hence slow many of the downstream effects of inflammaging and the damaging consequences of infectious disease, which we know is more prevalent in the aged population.

Before we go in search of completely novel agents to address aging, there may already be therapeutic agents available which may be beneficial. Metformin and rapamycin are generic and widely used in the treatment of Type 2 diabetes and as an immunosuppressant for organ rejection, respectively. However, several clinical studies have suggested that there are health benefits to these agents which seem to be driven by pharmacology that lies outside of that recognized in their primary indications. In the preclinical setting, both compounds have been reported as extending lifespan in mice, but these findings have proven controversial. Metformin improves insulin sensitivity, so it seems reasonable to assume that metformin reduces the risk of cellular damage and oxidative stress by improving cellular homeostasis. On a more global level, improved glycaemic control will reduce the emergence of some cardiovascular disease and peripheral nerve damage. The immunosuppressive effects of rapamycin can be theoretically linked to a dampening of inflammaging, however it seems that the agent may have a more cryptic pharmacological effect associated with improving energy homeostasis in cells.

The bisphosphonates are a group of compounds used clinically in the treatment of osteoporosis, but there are observational studies emerging from the clinic suggesting that they could have beneficial effects on human health beyond that associated with bone homeostasis.

Collectively, these widely used agents may have uncovered key pathways in which to focus efforts to identify more potent or selective agents to address cellular aging. What is required is a deeper mechanistic understanding of the cellular pharmacology of these drugs to determine where best to intervene. A key approach to address this is the possibility of using phenotypic screens in cellular models which capture one or more of the hallmarks of aging, to determine modes of action of known agents. In addition, such models can also be used in a blind fashion to screen libraries of compounds to uncover completely novel pathways and identify agents that may be beneficial.

One of the big challenges in the search for treatments to improve healthspan is having robust endpoints by which to measure efficacy. Clearly extension in chronological time to death provides a very clear endpoint, but it seems likely that clinical trials aimed purely at increasing longevity are a long way off. As such, there is a growing need to accurately measure biological age, as chronological age fails to capture the heterogeneity of signs and symptoms with which people age. For example, we probably all have family members who we think look and behave much younger than we know their chronological age to be. How do we measure healthspan in the context of a clinical setting? How do we define quality of life? These are big challenges but we do have the ability to measure directly improvements in, for example, heart or liver function, muscle strength, mobility, cognition and the performance of the immune system. It seems probable that the identification of pharmacological agents that improve healthspan will be found via exploration in multiple surrogate indications where hard endpoints can be measured and beneficial or detrimental effects become clear.

We need to continue to develop biomarkers and translational strategies which are able to inform us of whole-body cellular ‘health’ and with the gathering interest in this area, we will likely have an increasing array of tools to more accurately assess biological age over time.

However, we should always remember that patients don’t care about biomarkers. They are interested in whether they ‘feel’ better, i.e. can meet their basic needs, whether they can learn and grow and make decisions, can be mobile, build and maintain relationships and contribute to society. That is the patient’s perspective which is encapsulated perfectly in the goals as stated by the WHO.

So, while there is a growing understanding of potential ways we can measure and improve healthspan, there are some challenges in clinical development of novel anti-aging compounds. Study subjects may be aged but otherwise healthy, leading to ethical considerations associated with treating healthy patients in a preventative manner. There is no regulatory path at present, and there is the fundamental question of who is going to pay for agents that improve healthspan. Currently there is an argument over whether old age can be regarded as a disease or not. This is irrelevant as approval of any novel agents or use of an existing therapeutic in an age-related condition will require properly controlled, randomized clinical trials. Given the likely heterogeneity in such a trial population and the differing rates at which individuals age (i.e. manifest the hallmarks of aging), the trials will need to be large and long in duration. There would be parallels to the many trials in Alzheimer's disease, where the cost is enormous and efficacy hard to find. Moreover, it seems probable that like the thinking around AD, treatments should start early before symptoms appear. An additional confounder is the likely variations in ADME in individuals. We know elderly subjects often have reduced hepatic and renal function which could introduce significant variability in the exposure to novel agents.

We can meet these challenges as we believe there is significant will within society to succeed. We all share the common goal of living long and healthy lives.

If you’d like to hear how Evotec has developed capabilities to measure the hallmarks of aging which can support efforts to identify novel agents to treat age-related disease then reach out to us. You can also learn more from our webinar "Therapeutic approaches for aging and age-related diseases" by Steve England, SVP, Head of in vitro Biology and Disease Area Lead for Aging and Senescence at Evotec.

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iPSC and age-related diseases: Case study AMD
Age-related macular degeneration (AMD) is a leading cause of vision loss in people over the age of 50, accounting for 90% of blindness in this population. There are dry and wet forms of the disease and to date, there are limited treatment options for the wet form and none for dry AMD. The exact cause of the disease is unknown, but it is suspected that it results from a combination of hereditary and environmental factors, with smoking and diet being implicated. Globally, more than 190 million people are affected by AMD and this figure is expected to increase to more than 285 million by the year 2040. The estimated global cost of the disease currently stands at $343bn, with $255bn in direct healthcare costs.

Significant research effort has been targeted towards identification of the genes involved in permanent vision loss through photoreceptor and/or retinal pigment epithelium (RPE) cell dysfunction or cell death. So far, mutations in over 250 genes are known to be involved. Despite a good understanding of the genotype-phenotype relationship in AMD, this has not translated into predictive in vitro and in vivo models to understand disease mechanisms.

Partnership on iPSC-derived RPE cells
To drive the search for treatments for this devastating disease, it is vital to identify and develop new models which enable elucidation of retinal disease mechanisms and reliably predict the efficacy of therapeutic compounds. To address these challenges, Evotec has teamed up with the Centre for Regenerative Therapies (CRTD) in Dresden, Germany. The CRTD has a longstanding interest in degenerative processes and has contributed significantly to our understanding of retinal diseases in recent years. The joint TargetRD project is based on Evotec’s know-how in induced pluripotent stem cell (iPSC) technology, generating iPSC-derived RPE cells from AMD patients.

RPE cells are essential for visual function and a key component for light detection by photoreceptor neurons. They also are crucial for maintaining the blood-retina-barrier, for the transport of diverse biomolecules, ions, and fluids in and out of the retinal tissue, and recycling of the visual chromophore retinal molecule.

Even more important is their ability for phagocytosis: A single RPE cell is in contact with about 30 photoreceptor cells and responsible for the phagocytosis and removal of the distal portions of the photoreceptor outer segments that are phagocytozed in the course of a day. This is an important process with up to 10% eliminated daily, meaning the entire population of photoreceptor outer segments is turned over every 2 weeks. Maintaining, repairing, or replacing RPE cells therefore is crucial for the management of AMD, but also for other retinal degenerative diseases.

So far, research has been hampered by limited access to RPE cells. There are immortalized retinal cell lines available, but they lack the typical cell morphology or function of their in vivo counterparts, e.g. pigmentation, polarization, and expression of certain proteins. Likewise, artificial organoids, so-called 3D-cups, are not ideal since they are difficult to grow and differentiate and not suited for high-throughput profiling of compounds.

This situation is now improving as Evotec and CRTD succeeded in developing a protocol to efficiently and robustly produce high quality human iPSC-derived RPE cells at industrial scale from patient cells. This means that partners can not only investigate disease pathology directly in this highly relevant retinal cell type but for the first time also study disease phenotypes and mechanisms within the context of a patient’s genome. Moreover, the TargetRD platform enables the study of individual, overlapping functional and morphological changes from iPSC-RPE cells derived from patients with different genetic backgrounds, providing an opportunity to unravel complex AMD disease phenotypes.

Promising first results
Already, the partners were able to show the utility of the TargetRD platform by analyzing iPSC-RPE cells carrying a patient mutation resulting a lysosomal storage disorder. Amongst other symptoms, patients with this type of mutation develop severe retinal degeneration, leading to complete blindness early in life. Using the various phenotypic and functional assays established, it was possible to demonstrate impaired trans-epithelial resistance in patient cells. This indicates that in this case, patient RPE cells are unable to form the tight monolayer required for normal RPE cell function. Furthermore, patient RPE cells have, as expected, impaired lysosomal activity and are unable to phagocytose photo- receptor outer segments (POS) at the same rate as control RPE cells. Since all assays are able to support high-throughput screening, the partners can use the TargetRD platform to identify phenotypic and functional disease phenotypes from patient cells, enabling novel drug discovery approaches.

Furthermore, the project combines Evotec’s drug discovery expertise and the academic excellence of the CRTD to achieve significant progress towards developing therapies much needed by the patients.

Both partners believe this is a very promising approach that enables successful drug discovery programs for retinal degenerations coupled to a high likelihood of successful translation into the clinic.

N-Methyl-D-aspartate receptors (NMDARs) are members of the ionotropic glutamate receptor family and play a crucial role in learning and memory by regulating synaptic plasticity. Activation of NMDARs containing GluN2A, one of the NMDAR subunits, has been recently identified as a promising therapeutic approach for neuropsychiatric diseases such as schizophrenia, depression, and epilepsy.

Identification of a new hit for GluN2A PAMs is however difficult due to the similarity of PAM binding sites between GluN2A and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPARs), another member of the ionotropic glutamate receptor family.

In this collaborative publication with Takeda, we focus on:

• Identification of an hit compound with moderate AMPAR-binding activity, though a Ca²⁺ influx-based high throughput screening campaign with a compound set including an internal AMPAR-focused compound library
• The strategy using a structure-based drug design (SBDD) approach to minimize the AMPAR-binding activity while improving GluN2A activity
• The use of the potent and brain-penetrable GluN2A-selective positive allosteric modulators GluN2A PAM discovered as in vivo tool exhibiting significant neuroplastic enhancement in the rat hippocampus 24h after oral administration, having potential application for cognitive enhancement in neuropsychiatric diseases

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