Science Pool

Children diagnosed with TUBB4A leukodystrophy (Hypomyelination with Atrophy of Basal Ganglia and Cerebellum) experience a progressive and severe deterioration of recently acquired motor skills. Currently, there is no available cure for this condition. Evotec, in collaboration with SynaptixBio, is developing antisense oligonucleotides (ASOs) targeting TUBB4A mRNA to suppress gene expression, irrespective of specific mutations.

The research presented here represents a crucial segment of Evotec’s oligonucleotide-based drug discovery initiative. This study employs a combination of in vitro, in vivo, and bioinformatic methodologies to minimize the synthesis and testing of acutely toxic molecules in murine models.

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Heart failure is a global health challenge that demands innovative therapeutic approaches. Evotec’s pioneering iPSC-derived cardiomyocytes offer a promising solution, addressing the critical challenges of immune rejection, graft-induced arrhythmia, and scalable production.  

The growing global burden of heart failure

Heart failure (HF) remains a leading cause of mortality, affecting millions worldwide​¹​. Current estimations predict that there will be over 8 million people in the United States alone living with HF by 2030^​2​. The condition is exacerbated by the heart’s limited regenerative capacity, as cardiomyocytes — the cells responsible for heart muscle contraction — have minimal ability to regenerate after injury. Consequently, a single myocardial infarction can result in the irreversible loss of billions of cardiomyocytes, leading to heart failure with reduced ejection fraction (HFrEF)​³​. 

Traditional treatments focus on managing symptoms and slowing the progression of HF but fail to address the root cause — the loss of functional cardiac tissue. Over time, patients will reach the end stage of HF, experiencing a vastly diminished quality of life, with ventricular assist devices or rare heart transplantation being final treatment options. As the global population ages and cardiovascular risk factors rise, there is an urgent need for novel therapies that go beyond symptom management, to regenerate lost heart tissue and prevent HF from progressing. 

One promising avenue is regenerative cell therapy using induced pluripotent stem cells (iPSCs). In this article, we explore the current challenges in developing cell therapies for heart failure and Evotec’s innovative approach to overcoming them, with recent findings from their iPSC-derived cardiomyocyte (iCM) Heart Repair program. 

The promise of regenerative cell therapy for HF

Regenerative medicine, especially iPSC-based therapies, holds tremendous potential in addressing the unmet needs of HF patients. iPSCs can be reprogrammed from adult cells, and with unlimited proliferative capacity, they have the unique ability to differentiate into any cell type. 

Thus, iPSCs offer a renewable source of cardiomyocytes. This makes iPSC cell-derived cardiomyocytes (iCM) an ideal candidate for providing novel and curative HF therapies, with the potential to replenish lost heart cells in HF patients and restore cardiac function. However, the path to clinical application is fraught with challenges. 

Challenges in cardiac cell therapy

One of the primary challenges with allogeneic iPSC-based therapies is the prevention of immune rejection. Allogeneic iCM are recognized as foreign by the patient’s immune system. This leads to rejection unless immunosuppressive drugs are used​4​. However, long-term immunosuppression carries significant risks and is not a viable solution for all patients.  

Moreover, there’s the challenge of preventing graft-induced arrhythmias. These can occur when transplanted cardiomyocytes do not integrate properly with the host tissue, leading to irregular heartbeats.  

Another key challenge is successfully scaling up production to meet clinical needs. HF patients require over a billion iCM for effective treatment. However, producing large doses of iCM demands cost-effective, scalable processes. Additionally, ensuring consistent quality and purity of these cells at increased scale is critical for their therapeutic success. 

Evotec’s iCM Heart Repair program, in collaboration with the Medical Center Hamburg-Eppendorf “UKE”, has been developed to address these challenges. This collaborative program focuses on the development of first-class non-immunogenic iCM that can be produced at scale, offering a promising off-the-shelf treatment that can meet the global demand for HF therapies.  

Immune-shielded iCM are protected from T cells and NK cells

As part of Evotec’s iCM Heart Repair program, researchers at Evotec evaluated iCM immune-shielding strategies for tissue replacement in HF patients. In this study, the researchers first developed a robust production process of wildtype (wt) iCM cells, using the fully characterized GMP iPSC line in small-scale GMP-compliant bioreactors. Yield evaluation with flow cytometry demonstrated an exceptionally high purity of >97% cardiomyocyte marker, cardiac troponin T (cTNT).

 To investigate strategies to prevent allogeneic immune rejection, two genetically engineered iCM lines were chosen, human leukocyte antigen (HLA)- encoded class I and II knockout iCM, and iACT iCM​^5​​,6​ (panCELLa Induced Allogenic Cell Tolerance Stealth Cells™). These lines contain genetic modifications to inactivate human leukocyte antigen factors I and II (HLA-I/II KO), or overexpress immune-shielding factors (iACT), which were engineered to the iPSC prior to their differentiation. Thus, both iCM are designed to prevent immune cell activation and cytokine release.  

Using in vitro assays, the two iCM lines were co-cultured with primed T cells or expanded natural killer (NK) cells. The subsequent release of the cytokine IFNγ and killing of the iCM was measured to investigate the immune-suppressing potential of HLA-I/II KO and iACT immune-shielded iCM. 

Figure 1: Wild type iCM (wt), HLA-I/II KO, and iACT immune-shielded iCM were co-cultured with primed T cells or expanded NK cells of human donors. iCM survival and IFNγ release from immune cells was measured after 6-24 hours.

Results of the in vitro co-culture assays (Figure 1) showed iACT iCM provide efficient protection against NK cells, but only moderate protection against T cells. Contrastingly, HLA-I/II KO iCM were shown to be vulnerable to NK cells due to the “missing-self-response”, but highly efficient against T cells. 

Engineering an improved iCM cell line  

To improve the therapeutic potential of immune-shielded iCM, Evotec has developed its proprietary EvoCloaking iPSC line. The novel cell line is based on HLA-I/II KO, with an additional innovative anti-NK cell strategy to increase its immune-shielding properties. 

In vitro data supports that EvoCloaking iPSC-derived iCM can reduce cytokine release and are protected against both T cells and NK cells. This means the proprietary cell line offers an innovative strategy to ensure the acceptance and persistence of engrafted iCM, improving the safety and long-term function of the therapy, while avoiding the need for immunosuppressants. 

In addition to its immune-shielding properties, EvoCloaking iPSC-derived iCM are genetically engineered to safeguard against tumorgenicity, with a drug inducible kill-switch allowing for the elimination of undesired proliferating cells. Importantly, the proprietary cell line will also contain a genetic modification to avoid graft-induced arrhythmia in derived iCM. This is supported by in vitro data, and is currently tested on arrhythmia-prone pigs.

EvoCloaking iCM are highly pure cells optimized for off-the-shelf product use as a single cell suspension. In heart injury guinea pig models, efficient iCM engraftment has been demonstrated when injected as single cell suspensions. Consequently, catheter-based interventional administration should be compatible with this product format.

Evotec's scalable therapeutics platform

The iCM Heart Repair Program aims to overcome challenges associated with large-scale iCM production. Using Evotec’s GMP-compatible manufacturing infrastructure, scalable and cost-effective bioreactor processes have been established for iPSC expansion and differentiation in 3D. Furthermore, predictive in-process controls and advanced in silico modeling have been implemented into the differentiation process, to optimize cell yield and purity. 

Evotec’s cell therapy pipeline expansion to include immune-shielded iCM is testament to its commitment to allogeneic cell therapeutics. Our unique end-to-end platform and expertise support the development and manufacturing of off-the-shelf iPSC therapies for a range of diseases and conditions, from cancer and autoimmune disease to diabetes and cardiovascular disease. The integrated platform covers development stages from early exploration to GMP compliant scale-up for further research, pre-clinical, and clinical studies. 

Figure 2: An overview of Evotec’s end-to-end platform for iPSC-based therapeutics

The future of heart repair

The development of immune-shielded iPSC-derived cardiomyocytes represents a significant advancement in the field of regenerative medicine. This therapy could revolutionize the treatment of heart failure, offering a solution that addresses the underlying cause of the disease, rather than just managing symptoms. Integrated iCM development and manufacturing processes are also ensuring the scalable, cost-effective production of this therapy, holding great potential for meeting the increasing global demand for curative HF treatment.

Evotec’s commitment to innovation and excellence in the field of regenerative medicine positions it as a leader in the development of next-generation therapies. By addressing the key challenges in iCM therapy, including scalable production, immune rejection, and graft-induced arrhythmias, Evotec is paving the way for a new era in heart failure treatment.

Moreover, the broader impact of this research by Evotec extends beyond heart failure, as the principles of immune-shielding and scalable bioprocessing can be applied to other cell therapies, opening new avenues for treating a range of diseases.

Discover more about Evotec’s innovative iPSC-based therapies

Download the iCM research poster

References

(1)    Savarese, G.; Becher, P. M.; Lund, L. H.; Seferovic, P.; Rosano, G. M. C.; Coats, A. J. S. Global Burden of Heart Failure: A Comprehensive and Updated Review of Epidemiology. Cardiovasc Res 2023, 118 (17), 3272–3287. https://doi.org/10.1093/CVR/CVAC013.
(2)    Golla, M. S. G.; Hajouli, S.; Ludhwani, D. Heart Failure and Ejection Fraction. StatPearls 2024.
(3)    Laflamme, M. A.; Murry, C. E. Regenerating the Heart. Nature Biotechnology 2005 23:7 2005, 23 (7), 845–856. https://doi.org/10.1038/nbt1117.
(4)    Lanza, R.; Russell, D. W.; Nagy, A. Engineering Universal Cells That Evade Immune Detection. Nature Reviews Immunology 2019 19:12 2019, 19 (12), 723–733. https://doi.org/10.1038/s41577-019-0200-1.
(5)    Harding, J.; Vintersten-Nagy, K.; Yang, H.; Tang, J. K.; Shutova, M.; Jong, E. D.; Lee, J. H.; Massumi, M.; Oussenko, T.; Izadifar, Z.; Zhang, P.; Rogers, I. M.; Wheeler, M. B.; Lye, S. J.; Sung, H. K.; Li, C. J.; Izadifar, M.; Nagy, A. Immune-Privileged Tissues Formed from Immunologically Cloaked Mouse Embryonic Stem Cells Survive Long Term in Allogeneic Hosts. Nature Biomedical Engineering 2023 8:4 2023, 8 (4), 427–442. https://doi.org/10.1038/s41551-023-01133-y.
(6)    Harding, J.; Vintersten-Nagy, K.; Shutova, M.; Yang, H.; Tang, J. K.; Massumi, M.; Izaidfar, M.; Izadifar, Z.; Zhang, P.; Li, C.; Nagy, A. Induction of Long-Term Allogeneic Cell Acceptance and Formation of Immune Privileged Tissue in Immunocompetent Hosts. bioRxiv 2019, 716571. https://doi.org/10.1101/716571.

The immune system is complex and traditional preclinical toxicity models often fail to predict the human immune response. Cyprotex has developed a panel of immune-specific in vitro toxicity assays using human cell-based models to understand the immunogenic and cytotoxic potential of various drug modalities such as small molecules, biologics and oligonucleotides. Both inter-individual and population wide responses can be assessed using our on-site biobank of HLA-types immunocompetant cells.

Read our fact sheet to learn more about our immunotoxicity services including the PBMC cytokine storm panel, the T-cell immunogenicity risk panel, the PBMC proliferation assay, the PBMC cytotoxicity assay and the dendritic cell activation assay. Cyprotex also offers a range of skin sensitization assays such as the DPRA assay, the KeratinoSens^TM assay and the U-SENS^TM assay.

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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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To gain insights into IgAN pathophysiology and unravel changes in the composition of immune cell subsets, we performed a comprehensive analysis of humoral and cell immunity of IgAN patients and HC. Our results converge to indicate that IgAN is characterized by immune abnormalities in gut mucosa and that unconventional T cells may play a critical role in disease pathophysiology.


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Learn about innovative strategies to succeed in hit ID.

Watch the webinar series!

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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In 2022, Evotec embarked on a transformative journey by committing to the Science Based Targets initiative (SBTi) Net Zero strategy. 

This ambitious plan aligns with a 1.5°C trajectory and includes the following key targets:

• 50% Reduction in Scope 1 and 2 GHG Emissions by 2032: From a 2021 baseline, Evotec aims to halve its greenhouse gas emissions.
  
  
• 100% Renewable Electricity by 2026: Transitioning to fully renewable electricity sources.
  
  
• Switching to Renewable Heating: Moving from natural gas to renewable electricity for heating.
  
  
• Energy Efficiency Improvements: Reducing electricity intensity per square meter and transitioning to low Global Warming Potential (GWP) refrigerants.

These initiatives are crucial steps towards Evotec’s goal of achieving Net Zero by 2045.

Figure 1 - Evotec's Scope 1 and 2 sustainability targets

Thermal Assessment and Efficiency Projects

In the same year, Armstrong International conducted a Thermal Assessment study at Evotec’s Verona plant. The study aimed to optimize primary energy consumption and reduce emissions. Armstrong’s engineering team, in collaboration with Evotec’s Facility and Sustainability department, analyzed thermal load data to identify potential efficiency projects.

The focus was on the utilities area, specifically the production of hot water using a steam exchanger. By analyzing thermal load profiles and the energy availability from evaporative cooling towers, the team identified the optimal size for a new heat pump. This innovative approach allows the use of waste heat as a source for the heat pump, significantly reducing natural gas consumption.

Figure 2 - Right sizing through heat load analysis [kWt]

Cutting-Edge Heat Pump Technology

The Armstrong+Combitherm industrial heat pump, equipped with a twin-screw compressor and using R1234ze refrigerant (GWP = 7), was chosen for its low environmental impact. The heat pump operates at a high efficiency with a COP (coefficient of performance) of 4.6 at full capacity and features noise reduction technology to comply with local regulations.

Figure 3 - New heat pump

Figure 4 - Estimated project energy and emissions savings

Project Outcomes and Sustainability Impact

The project, completed on a turnkey basis by Armstrong, is expected to save 1,604 tons of CO2 and approximately 760,000 cubic meters of natural gas annually. Additionally, through CO.EN’s consultancy as an Energy Saving Company (ESCo), Evotec secured “white certificates” for five years, amounting to around 357 certificates per year, equivalent to €89,000 annually.

Figure 5 - Natural gas consumption data after heat pump start-up

A Holistic Approach to Decarbonization

Armstrong’s Circular Thermal® and Pinch analysis methodologies provide a comprehensive decarbonization roadmap. This approach ensures the best integration of available technologies, guaranteeing significant reductions in primary energy consumption and emissions.

Evotec and Armstrong International’s partnership exemplifies a shared vision for sustainability, with a clear focus on achieving decarbonization targets through innovative engineering and strategic planning.