Science Pool

Eloi Haudebourg¹, Yvan Eb-Levadoux¹, Catherine Pech¹, François Autelitano¹, Berenice Rotty¹, François Lantreibecq¹, Yannick Cogne¹, Navratan Bagwan¹, Alessia Cavaliere², Rossella Cardin², Alberto Vezzelli², Alessandro Greco²,
Philipp Ellinger³, Wiebke Afhueppe³, Karoline Droebner³, Winfried Wunderlich⁴

_{1 Evotec (France) SAS Toulouse, France
2 Aptuit (Verona) Srl, an Evotec Company, Verona, Italy)
3 Bayer AG Pharmaceuticals Research, Berlin, Germany
4 Evotec SE, Goettingen, Germany}

Introduction: While proteomics tools are increasingly used in the early steps of the drug development journey, its use remains limited to exploratory endpoints in clinical phase. Successful MS methods used in the exploratory and preclinical phases are adapted to antibody-based assays. For the present study, no antibody of the target engagement biomarker could be validated for animals in preclinical phase. An MS-based protein quantification method was therefore developed in Research Use Only (RUO) and ultimately transferred to a Good Clinical Practice (GCP) environment for validation and application to support preclinical development and clinical trial Phase 1 as a primary endpoint.

Methods: First, a high purity Stable Isotope Labelled (SIL) protein was produced in HEK293 cells cultured in SILAC medium and purified (anti-poly-histidine followed by IMAC and SEC). Second, a targeted MS assay was developed for the absolute quantification of biomarker of interest. Briefly 2 µL of plasma was processed on an iST kit (Preomics), peptides were injected using a microflow LC (M Class, Waters) on a C18-CSH column (Acquity, Waters) and quantified in MRM mode on a triple quadrupole (6500+, Sciex).

Preliminary data: Acceptable protein purity (> 80 % based on MS intensity) and excellent heavy label incorporation (>98 %) was archived. A targeted proteomics assay was first developed in RUO and transferred to a GCP facility where the method was validated according to FDA validation of bioanalytical methods for Industry and ICH-M10 guidelines. Specifically, the method validation included the assessment of precision and accuracy of the assay, surrogate matrix equivalence, lower limit of detection (100 ng/ml), concentration range, short- and long-term stability of the samples.
The validated, targeted MS method is routinely used to quantify a target engagement biomarker in a currently ongoing Phase 1 clinical trial.

Novel aspects: First time for the use of MS-based targeted proteomics to quantify proteins in clinical samples as primary endpoint.

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Oliver Kardell¹, Till Kindel¹, Frank Rolfs¹, Samira Vautrin¹, Barbara Kracher¹, Thomas Gronauer², Christine von Toerne² , Andreas Tebbe¹, Stefanie M. Hauck², Carleen Kluger¹

<sub>1. Evotec München GmbH, Anna-Sigmund-Str.5, D-82061 Neuried (Germany)
2. Helmholtz Zentrum München, Ingolstädter Landstraße 1,  D-85764 Neuherberg (Germany)</sub>

Liquid biopsies like plasma, cerebrospinal fluid (CSF), or urine are a less invasive and inexpensive alternative to tissue biopsies. Additionally, these body fluids are known to contain a multitude of known and yet to be revealed biomarkers that can be detected using mass spectrometry (MS) based proteomics. There are several distinct advantages of this technology: (I) it gives unbiased access to the biofluid proteome, (II) it is species independent, (III) it can reveal information about isoforms and post-translational modifications and (IV) it does not require availability of antibodies. Furthermore, MS based proteomics has been rapidly evolving over the last years due to the development of new generations of MS instruments such as timsTOF or Thermo Astral, advancements in sample preparation like nanoparticle-based protein enrichment, as well as novel AI based tools for data processing. The continuous progress results in increased depth and throughput of clinical proteomics.

However, this highly dynamic nature of the field can also be a drawback, as applications of mass spectrometry-based proteomics for larger clinical trials is often hindered by a lack of standardization across laboratories and studies. To address this problem and to enhance the integration of MS-based proteomics into medical research the CLINPSECT-M consortium and its MS laboratories conducted a round robin study on clinical specimens such as human plasma and CSF.

Here, the data from the round robin study is compared to internal benchmark studies on human biofluids that were performed at Evotec. In addition, while the focus of the round-robin study was to harmonize workflows for undepleted plasma and CSF and track interlaboratory reproducibility, the Evotec study also measured longitudinal platform stability and shows how nanoparticle-based proteomics using the Proteograph^TM can provide deeper coverage of the plasma proteome and at the same time overcome some of the challenges associated with undepleted workflows. 

Multicenter Collaborative Study to Optimize Mass Spectrometry Workflows of Clinical Specimens Oliver Kardell, Christine von Toerne, Juliane Merl-Pham, Ann-Christine König, Marcel Blindert, Teresa K. Barth, Julia Mergner, Christina Ludwig, Johanna Tüshaus, Stephan Eckert, Stephan A. Müller, Stephan Breimann, Pieter Giesbertz, Alexander M. Bernhardt, Lisa Schweizer, Vincent Albrecht, Daniel Teupser, Axel Imhof, Bernhard Kuster, Stefan F. Lichtenthaler, Matthias Mann, Jürgen Cox, and Stefanie M. Hauck, Journal of Proteome Research Article ASAP, DOI: 10.1021/acs.jproteome.3c00473

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Carleen Kluger, Evotec (München) GmbH

The advance of nanoparticle-based technologies (e.g. ProteographTM)  in combination with high-end mass spectrometry (MS) has increased the depth of proteome coverage for biofluids like plasma, serum or cerebrospinal fluid (CSF), making it possible to routinely analyze 2.000-3,000 proteins per individual biofluid sample. Over the last two years we have extensively used this technology at Evotec to screen multiple different patient cohorts, each containing up to a few hundred serum or plasma samples from patients with very different disease backgrounds, including patients with multiple co-morbidities and a dedicated cohort of healthy subjects that were hospitalized over-night to ensure maximum control of biofluid and data collection.

While in the past, most activities in the field of Clinical Proteomics were limited to a specific patient cohort in a clearly defined disease context, the next challenge is to integrate proteomics signatures from multiple cohorts into a unified framework. Here, we show how this can be achieved by minimizing technical variability during sample collection, sample preparation and MS measurement using highly automated nanoparticle-based proteomics approaches. 

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Data Integration: With Bioinformatics to Biological Knowledge

Tim Blokker, Christian Schiffmann, Micael Fernandes dos Reis, Christopher Bruhn, Christiane Honisch, Carleen Kluger, Barbara Kracher, Erik Schliep, Evotec International GmbH

PanHunter is a user-oriented and interactive web application allowing for in-depth data discovery across multi-omics datasets without the need for coding skills. The platform was originally built by Evotec’s bioinformatics team to handle large transcriptomics datasets generated by our industrialized high-throughput transcriptomics platform. In recent years, with the advancement of high-throughput mass spectrometry-based proteomics, major advances have been made to tailor PanHunter towards handling massive datasets from different proteomics data formats.

The strength and novelty of proteomics analysis in PanHunter is founded in the holistic approach to integrate multiple levels of data (e.g. gene, protein, and post-translational modification (PTM)) through the whole workflow starting from quality control (QC) over exploratory analysis, differential expression analysis down to functional analysis, and incorporation of biological knowledge from various sources. PanHunter can map between PTM IDs, UniProt IDs, and on transcriptomics level Ensemble IDs. This mapping allows the user to analyze features simultaneously in linked plots, with data stemming from transcriptomics, proteomics or PTM data.

Here, we provide examples to highlight how the expansion of PanHunter’s proteomics capabilities facilitates data integration and interpretation in the context of precision medicine, thus driving easier access to novel medicines across various therapeutic areas.

E.MPD, Evotec‘s molecular patient database, combining patient and omics data across multiple disease areas has been extended to cover autoimmune diseases in recent years. Integration of proteomics data from Evotecs’s high-throughput mass spectrometry-based proteomics platform with patient data in PanHunter enables novel insights into the regulation of autoimmune diseases on the level of the proteome.

Evotec’s ScreenPep platform allows for high-throughput screening of compounds of interest. To enable seamless data analysis after the screening, PanHunter’s signature database has been broadened with proteomics signatures from Mitchell et al. 2023. These signatures can be used to investigate toxicological effects and help decipher the mechanism of action of novel compounds.

Mitchell, D.C., Kuljanin, M., Li, J. et al. A proteome-wide atlas of drug mechanism of action. Nat Biotechnol 41, 845–857 (2023). https://doi.org/10.1038/s41587-022-01539-0

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At Evotec, our expert immunologists drive your immuno-oncology projects from target identification to clinical trials. We offer comprehensive support for integrated projects and specialized expertise at critical stages. Our services include bespoke in vitro immunological assays, genetic engineering of primary immune cells, and advanced preclinical models. By integrating cutting-edge technologies and leveraging our extensive clinician network, we ensure the successful translation of innovative cancer immunotherapies from the lab to the clinic. Partner with Evotec to advance your cancer immunotherapy research and achieve breakthrough results.

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In the ever-evolving landscape of biomedical research, proteomics has emerged as a crucial field for advancing drug discovery and development and biomarker discovery. Evotec stands at the forefront of this scientific frontier, offering cutting-edge mass spectrometry-based proteomics solutions. Here’s how Evotec’s innovative technologies and comprehensive services deliver exceptional value to customers, driving breakthroughs in clinical research, such as discovery of stratification biomarkers and diagnostic markers for the discovery of novel targets.

Revolutionizing Proteomics with Advanced Platforms

ScreenPep™ Platform: Evotec’s ScreenPep™ platform is a high-throughput, automated system designed for deep-coverage proteomics of cell lines, tissues or biofluids. When applied to plasma or serum it can identify up to 1,500 proteins , making it incredibly efficient and cost-effective. This platform is ideal for large-scale studies, in which sample quantity may be limited but extensive protein identification is required.

Proteograph™ Technology: As the only Center of Excellence in Europe offering Proteograph™ technology, Evotec provides an unparalleled capability to identify up to 5,000 proteins from human biofluids such as serum, plasma, and cerebrospinal fluid (CSF). This technology leverages nanoparticle-based methods to achieve deep proteome coverage, significantly enhancing the scope of biomarker discovery and translational research.

Integrated MultiOmics Solutions: Evotec excels in integrating proteomics data with genomics, transcriptomics, and metabolomics, offering a holistic approach to biomarker discovery. This integrated solution, facilitated by Evotec’s PanHunter platform, allows for comprehensive data analysis and interpretation, enabling researchers to uncover complex biological insights and accelerate drug development processes.

Customized and Scalable Services: Understanding that each research project has unique requirements, Evotec offers highly customized services tailored to meet specific client needs. From optimizing study designs to adapting sample preparation workflows for difficult and rare samples, Evotec ensures that every project receives the precise attention and expertise it demands.

Exceptional Expertise and Infrastructure: With over 20 years of experience and more than 50 mass spectrometers across its sites, Evotec is one of the largest providers of proteomics services worldwide. Their dedicated team of bioinformaticians and proteomics experts continuously develops and refines in-house pipelines, ensuring the highest standards in data quality and analysis.

High-End Mass Spectrometry: Utilizing cutting-edge mass spectrometry instruments, Evotec guarantees high-sensitivity and high-precision proteomics. These advanced technologies are crucial for detecting low-abundance proteins and analyzing post-translational modifications, providing deeper insights into protein functions and interactions.

Advantages of Mass Spectrometry-Based Proteomics

Unbiased Protein Measurement: Unlike antibody or aptamer-based techniques, mass spectrometry provides an unbiased measurement of all proteins containing tryptic peptides. This comprehensive approach allows for a more accurate and complete proteome analysis, essential for discovering new biomarkers and therapeutic targets. The quantification is also not impacted by any conformational changes.

Versatility Across Species: Evotec’s proteomics solutions are not limited to human samples; they are also applicable to various species, including animals used in health studies. This versatility broadens the research applications and facilitates translational research from preclinical models to clinical settings.

Detection of Protein Isoforms and Modifications: Mass spectrometry is uniquely capable of detecting protein isoforms and post-translational modifications, such as phosphorylation and ubiquitination. This capability is vital for understanding protein functions and regulatory mechanisms, paving the way for novel therapeutic strategies.

Cutting-Edge Technology and Continuous Innovation: Evotec’s commitment to innovation is exemplified by their continuous development of nanoparticle-based proteomics in collaboration with Seer Inc. This ongoing enhancement ensures that Evotec remains at the forefront of proteomics research, providing customers with the most advanced and reliable technologies available.

Conclusion

Evotec’s clinical proteomics capabilities offer a robust, scalable, and precise solution for drug development and biomarker discovery. By leveraging advanced technologies, integrated MultiOmics approaches, and customized services, Evotec empowers researchers to make significant scientific advancements. Partner with Evotec to unlock new possibilities in your research and drive the future of healthcare innovation.

For more information, please contact us at info@evotec.com or visit our website at PanOmics - Technology Platforms - Evotec Website (English)

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In the quest to revolutionize immunotherapy, the identification of naturally presented peptides bound to HLA class I and II molecules from cancerous cells holds paramount importance. These peptides, crucial for developing immunotherapy-based treatments, also offer promising avenues for combating infectious diseases. Evotec's pioneering immunopeptidomics platform stands at the forefront, enabling the unbiased discovery of novel immunotherapeutic targets.

This comprehensive approach extends beyond cancer research, facilitating the identification of diagnostic and monitoring biomarker signatures across normal and altered cells in cohort studies. It sheds light on the intricate interplay between T cells and MHC-presenting cells, deepening our understanding of immunobiology.

Evotec's meticulously crafted experimental strategy, coupled with its state-of-the-art capabilities in high-end quantitative mass spectrometry, achieves unparalleled sensitivity. This precision is essential for distinguishing disease-specific neoantigens from their normally presented counterparts. By integrating whole exome sequencing and transcriptomics data, the platform empowers the discovery of neoepitopes, while advanced statistics and bioinformatics tools enable comprehensive data analysis and interpretation, facilitating peptide prioritization.

The platform's capabilities are exemplified by its ability to identify up to a thousand peptides per sample, providing direct detection of presented peptides, surpassing computation-intensive in silico predictions. Moreover, validation and accurate quantification of individual peptides are ensured through targeted mass spectrometry (PRM-MS).

In essence, Evotec's immunopeptidomics platform represents a transformative leap in the field, offering unparalleled insights into immunobiology and neoantigen identification, with profound implications for the development of immunotherapy-based treatments and the fight against various diseases.

For further inquiries, contact Evotec's experts at info@evotec.com or learn more here 

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Evotec offers state-of-the-art mass spectrometry-based proteomics solutions to support drug development and biomarker discovery. Our ScreenPep™ platform enables high-throughput, automated plasma and serum proteomics with deep coverage, identifying up to 1,000 proteins from minimal sample volumes. As Europe's exclusive provider of Proteograph™ technology, we can identify up to 5,000 proteins from human biofluids, enhancing biomarker discovery. Integrated with genomics, transcriptomics, and metabolomics data, Evotec's proteomics services offer a holistic approach to translational research.

With over 20 years of experience and advanced technologies like timsTOF and Orbitrap, Evotec delivers precise, comprehensive, and scalable proteomics solutions tailored to customer needs.
read out factsheet to learn more about our clinical proteomics and how we can support your drug development

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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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In the realm of drug discovery, unveiling the intricate interactions between bioactive compounds and cellular targets is paramount. Evotec leads the charge with its pioneering chemical proteomic applications, aimed at target deconvolution and selectivity profiling.

At the heart of Evotec's approach lies Cellular Target Profiling™, an unbiased and proteome-wide methodology that meticulously identifies and quantifies compound interactions with both on- and off-targets within the cellular milieu. Leveraging high-end quantitative mass spectrometry, this platform offers unparalleled insights into specific cellular targets, enabling precise target identification and determination of target-specific dissociation constants.

Central to this chemical proteomics approach is photoaffinity labelling coupled with mass spectrometry, allowing for the covalent capture of target proteins within live cells. This technique not only identifies target proteins but also visualizes compound-target interactions, shedding light on binding site locations within protein targets and complexes.

Evotec's chemical proteomic arsenal extends beyond target deconvolution to encompass diverse small molecule compounds. Activity-Based Protein Profiling (ABPP) offers a comprehensive view of enzyme classes, while KinAffinity® provides rapid target profiling of kinase inhibitors in cell and tissue samples. Unlike traditional biochemical kinase panel screenings, KinAffinity® evaluates inhibitors' target affinities across a spectrum of native kinases within their physiological cellular environment, facilitating hit-to-lead optimization with unprecedented precision.

With a track record of success in profiling various compounds, Evotec's expertise in quantitative proteomics stands as a beacon innovation in drug discovery. For those seeking advanced insights into target deconvolution, drug selectivity and activity profiling, Evotec's experts are posed to offer tailored solutions.

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