Biopharma and biosimilar companies are increasingly considering final production costs during early-stage process development rather than blindly racing to the clinic with an inferior process that must undergo redesign during subsequent clinical stages. Furthermore, the most advanced companies have a laser-like focus on product quality and consider this early on in development. This ensures that they progress the best product candidate into the clinic and avoid costly failures.

Just-Evotec Biologics is helping partners with antibody product candidates achieve the highest product quality with Cost of Goods Manufactured (COGM) below $50/g by combining our new J.CHO™ High Expression System (J.CHO™) with our unique continuous manufacturing platform. This extraordinary productivity represents a 75% reduction in industry standard COGM and is driven by the exceptionally high titers exceeding 4g/L/day in perfusion achieved utilizing J.CHO™. This performance is equivalent to a titer of approximately 30 g/L in fed-batch mode.

The J.CHO™ High Expression System comprises:

1. Engineered GS knockout CHO-K1 host cell lines capable of delivering specific productivities more than 50 pg/cell/day and growing at target densities of 60-100 million cells/mL
2. Transposon-based expression vectors with strong promoter sequences allowing stable integration and high expression of genes-of-interest (GOI)
3. Proprietary chemically defined, protein-free and dual sourced perfusion cell culture media designed with cost-efficiency in mind

Just-Evotec Biologics developed product sales royalty-free cell lines to work perfectly with our upstream perfusion platform process and to scale seamlessly from 3L to 500L or 1000L bioreactors for clinical or commercial production.

Figure 1: Comparison of antibody yields in a 20-day continuous perfusion culture using an industrystandard CHO (Chinese Hamster Ovary) cell line

Seamlessly Integrating Product and Cell Line Development

Just-Evotec Biologics can leverage its unique J.MD™ Molecule Design suite of services to create multiple variants of an antibody candidate with improved developability characteristics. Our innovative high-throughput cell line development workflows allow us to produce up to 96 stable transfectant pools of variants and screen for productivity and product quality in parallel. Expression in stable pools produces more representative material for testing than from transients and by combining candidate development with cell line development we can save partners up to two months from your timelines. Most importantly, we identify antibody candidate variants with excellent manufacturability properties that have been shown to increase expression titer by 3-fold compared to parental sequences. We are delivering elevated levels of productivity for monospecific, bispecific- and multi-specific antibodies, Fc-fusion proteins, and single-chain Fv-antibody fusion proteins.

Perfusion Delivers Superior Product Quality

Just - Evotec Biologics has demonstrated that our perfusion platform delivers superior antibody product quality compared to fed-batch systems. Perfusion cultures give healthier cells with more complete glycosylation patterns while shorter product residence times in the bioreactor result in lower levels of oxidation and deamidation. We modulate media and bioreactor conditions to meet our partners’ product quality requirements.

Our J.CHO™ High Expression System provides partners with opportunities to refine the product quality attributes of their candidate. Partners may choose to do this for a variety of reasons, for example, we collaborate with companies developing biosimilar products that must match the product quality profile of innovator molecules and innovator companies developing novel biologics with unique features. Our additional capabilities within the J.CHO™ High Expression System service offering includes:

• FUT8 Knock Out Cell Line enabling afucosylated antibodies for enhanced ADCC and improved efficacy.
• Inducible Cell Lines for the controlled protein expression of cyto-toxic products used in next-generation therapies.
• Additional advanced glycoengineered cell lines capable of delivering a range of glycan modifications on biosimilar candidates that match those of innovator products.

Our cell line development process can take as little as 14-weeks and uses the latest high-throughput cell culture methods and analytics to maximize efficiency.

Figure 2: Typical cell line development program at Just-Evotec Biologics to select clones with optimum performance in continuous perfusion culture; DWP = deep-well plates, tfxn = transfection

Highest Titers and Best Product Quality

In conclusion, in an increasingly mature and competitive market, biopharma companies are finding ways to differentiate themselves based on product quality attributes and on cost. Just-Evotec Biologics is supporting partners with its new J.CHO™ High Expression System that integrates with its continuous manufacturing platform for antibodies and delivers the highest titers in the industry and the product quality our partners demand.

Learn more on our website

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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Addressing unmet challenges in CAR T cell therapeutics

CAR T cell therapies have revolutionized the treatment of hematological malignancies such as leukemia and lymphoma, however the manufacturing process is extremely costly and slow due to its bespoke nature. Allogeneic CAR-T cell therapy, using cells from healthy donors, provides an essential alternative which could lead to ‘off-the-shelf’ solutions instead. Induced pluripotent stem cells (iPSCs) provide a standardized and scalable approach. Evotec's recent study showcases iPSC-derived T cells targeting cancer cells with precision, hinting at a promising future toward accessible and standardized cancer immunotherapies.

Cell-based therapy, which involves the use of living cells to combat diseases, has recently seen remarkable growth, both in clinical applications and within the pharmaceutical industry. As a result, it is now considered one of the most promising therapeutic approaches for cancers.

In particular, chimeric antigen receptor (CAR) T cell therapy has demonstrated significant clinical success in recent years, particularly in the treatment of hematological malignancies. Several CAR-T therapies have received approval from regulatory bodies such as the Food and Drug Administration (FDA) and the European Medicines Agency (EMA), providing critical treatment for various hematological cancers [1].

However, autologous CAR-T cell therapy, which uses T cells isolated from the patient’s peripheral blood, is often slow, complex, and costly due to its bespoke nature [2]. Furthermore, manufacturing success is often dependent upon the availability and condition of the initial autologous T cells. Patients may have undergone prior treatments that compromise the quality and quantity of their immune cells, further complicating the production process and reducing the likelihood of success.

To overcome these challenges, researchers are exploring the use of allogeneic T cells sourced from healthy donors, aiming to create "off-the-shelf" therapies readily available for patients. This approach could streamline the production process and potentially allow for multiple modifications to target different tumor antigens, enhancing efficacy and accessibility.

While this approach represents a promising avenue for streamlining and standardizing T cell therapy, there are still inevitable drawbacks with the manufacturing process. Allogeneic T cells need to be extensively genetically modified to prevent alloreactivity and immunogenicity, as well as ensuring tumor-specific activity. However, engineering T cells presents significant challenges, including reduced production yield; genotoxicity due to off-target effects; and the development of an exhausted T cell phenotype and product owing to the need for prolonged ex vivo expansion [3].

Induced pluripotent stem cells (iPSCs) offer an alternative approach. iPSCs provide a standardized, scalable cell source that can be precisely engineered for therapeutic use [3]. These cells are easier to genetically engineer and have a much higher proliferative capacity, ensuring a stable and plentiful cell source. By establishing master cell banks of iPSCs, researchers can ensure consistent quality and quantity of starting materials, reducing variability across CAR-T or T-cell receptor (TCR)-T products and creating more accessible, standardized, and effective treatments for cancer patients. In this article, we will highlight a promising iPSC approach for targeting tumor cells, providing a pathway towards scalable and GMP-compliant off-the-shelf cancer therapies.

Developing off-the-shelf T cell therapies

iPSCs provide a crucial off-the-shelf source of therapeutic T cells, offering significant advantages in scalability and genetic engineering capabilities. By leveraging iPSC technology, researchers can generate T cells with the potential for infinite expansion and tailor them to possess specific therapeutic functions through straightforward genetic manipulation.

Importantly, genetic engineering of iPSCs enables the generation of fully modified clonal lines, facilitating rigorous safety assessments and ensuring consistent therapeutic outcomes. However, realizing the full potential of iPSC-derived T cell therapy is dependent on the development of a robust and scalable production process that meets Good Manufacturing Practice (GMP) standards. Moreover, it's essential that this process yields mature T cells expressing the TCRα and TCRβ isoforms, commonly known as αβ T cells, which constitute the majority of T cells.

However, current manufacturing methods often suffer from low differentiation efficiency and poor scalability, hindering widespread application [4]. This is partially due to the complex differentiation processes that are required to generate T cells from iPSCs. Standard T cell differentiation protocols rely on different types of murine feeder cells to support prolonged in vitro proliferation. These murine feeders are unable to divide and provide essential extracellular secretions for iPSC proliferation, hematopoietic progenitor induction, and T cell differentiation. However, each feeder requires different sets of serum and basal media for maintenance culture and co-culture with differentiating iPSCs, complicating safety, control, and reproducibility. As such, a significant part of developing “off the shelf” T cell therapies is the establishment of a feeder-free culture for all stages of iPSC differentiation.

Modified iPSC lines successfully target cancer cells

In a recent study, researchers from Evotec examined the production of CD8+ T cells using Evotec's fully scalable, GMP-compliant iPSC-derived αβT (iαβT) cell differentiation process. The researchers used a validated GMP iPSC line, which had been modified with a NY-ESO-1 specific TCR knock-in. This TCR targets NY-ESO-1, a cancer-germline antigen that is expressed in a wide range of tumor types.

Using this cell-line, the researchers established a feeder-free differentiation protocol to efficiently generate iαβT cells. Each stage of the process was rigorously monitored using flow cytometry and single-cell transcriptome analysis. From iPSCs enriched with the knock-in modification, hematopoietic progenitor cells (HPCs) were induced and differentiated into iαβT cells (Figure 1). Throughout differentiation, cells displayed T cell markers CD45, CD5, and CD7, and initiated NY-ESO-1-specific TCR expression.

Following activation of T cell differentiation by Notch signaling, the proportion of NY-ESO-1-TCR positive cells surged to over 95%. Transcriptome analysis confirmed the successful differentiation from pluripotent cells to those with a T cell-specific gene expression profile.

Figure 1: Morphology of cells during differentiation process. Evotec has developed a 3D scalable, feeder-free induction process of Hematopoietic Progenitor Cells (HPCs). After enrichment of CD34-positive cells, T cell differentiation is initiated by activation of Notch signaling in a feeder-free process that will be further developed based on Evotec’s know-how with other immune cell types.

Importantly, the iαβT cells were shown to express CD8α and CD8β, which are both crucial for cytotoxic T cell function. Co-culture experiments with NY-ESO-1 antigen presenting tumor cell lines confirmed the cytotoxic activity of iαβT cells and their ability to release cytokines such as TNF-α and IFN-γ (Figure 2).

Figure 2: Functional characterization of iαβT cell. iαβT cells were cocultured with a tumor cell line loaded with the NY-ESO-1 peptide or negative control peptides. Anti-CD3 antibodies were used as a positive control. Cytotoxic activity and the release of cytokines (TNF-α and IFN-γ) was analyzed.

These results demonstrate that the Evotec iαβT differentiation process can efficiently generate CD8+ T cells that secrete cytokines and show cytotoxic activity, indicating their potential as a promising cell source for TCR-T or CAR-T cancer immunotherapies.

Evotec’s in-house GMP production pipeline

Evotec has built an iPSC infrastructure that represents one of the largest and most sophisticated platforms in the industry. Its growing portfolio includes natural killer cells (iNK), macrophages (iMACs) and αβ and γδ T cells (iT) (Figure 3). Each type of immune cell can serve as a foundation for creating numerous differentiated allogeneic cell therapy products.

Figure 3: Evotec’s iPSC-based cell therapy pipeline for oncology

Evotec’s iPSC platform is closely connected to a variety of in-house key technologies, which - together with a strong focus on standardization, upscaling and quality control (QC) – enable the efficient generation, characterization, and differentiation of iPSCs. . Supported by Evotec’s world class GMP manufacturing facilities, novel allogeneic cell therapeutics can be developed without the complexities or production bottlenecks associated with autologous therapies.

Starting with genetically engineered iPSC GMP master cell banks, Evotec’s cell therapeutics manufacturing platform provides a fully integrated pipeline encompassing all stages from research to development and manufacturing of cell therapy products. From the initial project inception to clinical application, Evotec excels in efficiently producing a diverse array of "off-the-shelf" cell therapy products (Figure 4)

Figure 4: Schematic depiction of Evotec’s fully scalable GMP manufacturing process.

From tailor-made to off-the-shelf solutions

Allogeneic T cell platforms are driving the transition from customized to standardized T cell therapy, addressing the urgent need of patients both in cell quality, consistency, and delivery time. However, realizing the full potential of iPSC-derived T cell therapies requires the development of scalable and GMP-compliant production pipelines.

By producing a feeder-free culture for all stages of PSC differentiation, Evotec provides an efficient, reproducible, and scalable way to produce iPSC-derived αβT cells that can effectively target tumors. Thanks to Evotec’s expansive iPSC differentiation platform, iPSCs are one step closer to producing essential T cell-based cancer immunotherapies for the future.

Find out more about Evotec’s industry leading cell therapy platform

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References

1. Chen, Y.J., Abila, B., & Mostafa Kamel, Y. (2023). CAR-T: What Is Next? Cancers, 15(3), 663. https://doi.org/10.3390/cancers15030663
2. Gajra, A., Zalenski, A., Sannareddy, A., Jeune-Smith, Y., Kapinos, K., & Kansagra, A. (2022). Barriers to Chimeric Antigen Receptor T-Cell (CAR-T) Therapies in Clinical Practice. Pharmaceutical Medicine, 36(3), 163–171. https://doi.org/10.1007/s40290-022-00428-w
3. Netsrithong, R., Garcia-Perez, L., & Themeli, M. (2024). Engineered T cells from induced pluripotent stem cells: From research towards clinical implementation. Frontiers in Immunology, 14. https://doi.org/10.3389/fimmu.2023.1325209
4. Iriguchi, S., Yasui, Y., Kawai, Y., Arima, S., Kunitomo, M., Sato, T., et al. (2021). A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy. Nature Communications, 12(1), 430. https://doi.org/10.1038/s41467-020-20658-3
   
   

Addressing unmet challenges in macrophage cell therapeutic

Macrophages are paving the way for exciting new opportunities in cancer therapy - overcoming barriers faced by T-cell therapeutics in targeting solid tumors. Discover the exciting world of macrophage cell therapeutics, the importance of manufacturing, and how Evotec’s proprietary cell therapy development pipeline is helping to shape tomorrow’s therapies.

Cell therapies have emerged as one of the most promising immunotherapeutic strategies in the fight against cancer. In particular, chimeric antigen receptor T-cell (CAR-T) therapy has become notable for its strong efficacy in generating targeted antitumor responses in a broad range of hematological malignancies. CAR-T cell therapies have been approved by the United States Food and Drug Administration (FDA) to treat a number of hematological cancers, having been shown to dramatically improve the outcomes of patients with B-cell malignancies and Multiple Myeloma.

Despite the clinical success of CAR-T cells against some hematological cancers, CAR-T cell therapy has shown limited efficacy against solid tumors, which account for approximately 90% of all cancer cases. Several factors contribute to their diminished efficacy when combatting solid tumors: The tumor microenvironment (TME) has clever immunosuppressive defense mechanisms, while T-cells exhibit poor infiltration into the tumor. Furthermore, there is a lack of solid tumor antigen targets that provide adequate specificity and safety [1].

To overcome the challenges faced in treating solid tumors, novel macrophage-based immunotherapies are gaining attention. Macrophages can infiltrate tumors more easily and bring favorable immunomodulatory characteristics. Furthermore, their phenotypic plasticity allows them to be easily re-engineered to prompt antitumor activity. Several macrophage reprograming approaches have been developed, including the use of gene editing tools to inhibit immunosuppressive genes [2].

While macrophage-based cell therapies have garnered promising results in preliminary trials, the majority are based on autologous macrophages. Implementing an autologous cell therapy approach brings complications to macrophage therapeutic production: Patient material is limited and tricky to work with, while the cell manufacturing phase often requires personalized genomic profiling and gene editing, which is both costly and time-consuming.

Induced pluripotent stem cell (iPSC)-derived macrophages (iMACs) offer the opportunity to overcome the production bottleneck associated with autologous cell therapies. By opting for a reliable, scalable GMP manufacturing process, it’s possible to create allogenic macrophage cell therapy products with consistent high quality. Furthermore, iPSCs enable straightforward introduction of genetic material for gene editing-based cellular engineering.

In this article, we will highlight a promising iMAC approach for targeting solid tumors, and discover how opting for an iMAC cell therapy product can eliminate the need for combination therapies.

Overcoming the CD47 defense mechanism

CD47 is a protein highly expressed on the surface of all solid tumor cells, and represents a key component of the TME’s defense – acting as a ‘don't eat me’ signal for phagocytic cells. CD47 is a natural ligand for SIRPα, a membrane protein expressed on macrophages. When a macrophage approaches a tumor cell, CD47-SIRPα interactions prevent the macrophage from phagocytosing the tumor cell [3].

Several agents that disrupt CD47-SIRPα signaling have entered clinical trials in recent years. Many of these therapies consist of monoclonal antibodies or antagonist drugs targeting either CD47 or SIRPα. While these have yielded varying degrees of success, combination treatments of anti-CD47 or anti-SIRPα inhibitors with additional tumor targeting antibodies have often been required to produce significant anticancer efficacy [4]

To improve upon the efficacy of therapeutics targeting the CD47-SIRPα axis, a novel iMAC cell therapy approach holds promise of blocking this critical checkpoint with greater reliability. By gene editing iPSCs to knockout (KO) the gene coding for SIRPα, it’s possible to generate a highly potent iMAC cell therapy product resistant to phagocytosis inhibition by CD47-expressing tumor cells.

SIRPα knockout in iMACs improves phagocytosis of tumor cells

A recent study conducted by Evotec researchers investigated the activity of SIRPα KO iMACs manufactured via Evotec’s proprietary 3D iMAC differentiation process. Preliminary studies demonstrated that the SIRPα KO iMACs retain typical phenotype comparable to wild-type (WT) iMACs. Next, the researchers tested the phagocytic activity of SIRPα KO iMACs and WT iMACs against cultured Raji cells, a cell line commonly used as a preclinical tumor model that expresses CD47 and the tumor target CD20.

Raji cells were co-cultured for 20 hours with WT or SIRPα KO iMACs in the presence of different treatments (isotype controls, anti-CD20 antibody, anti-CD47 antibody or combined anti-CD20 + anti-CD47 (combo)). The tumor cell uptake by macrophages known as antibody-dependent cellular phagocytosis (ADCP) was then monitored by live-cell imaging via Incucyte (Figure 1).

Figure 1: Antibody-dependent cellular phagocytosis (ADCP) of RAJI cells by WT and SIRPα KO iMACs. Data expressed as mean ± SEM.

In the presence of anti-CD20 antibody for tumor targeting, SIRPα KO iMACs (represented by the dark blue graph line) showed augmented phagocytosis that was equivalent to WT iMACs treated with a combination of CD20- and CD47-targeting antibodies (black line). In addition to increased phagocytosis, the tumor-killing capacity of SIRPα KO iMACs loaded with anti-CD20 antibody was found to be comparable to WT iMACs in the presence of anti-CD47 antibody.

This finding has significant implications for iMAC-based anti-cancer cell therapy development. It demonstrates that the novel allogenic SIRPα KO iMAC cell product developed by Evotec overcomes the need for a treatment combination with anti-CD47 or anti-SIRPα checkpoint inhibitors, and has great potential to serve as the basis to develop innovative treatments for solid tumors.

Evotec’s scalable cell therapeutics platform

Good Manufacturing Practice (GMP) manufacturing ensures that cell therapies are produced in a consistent, controlled environment to meet stringent quality standards. This is crucial for cell therapies as it ensures product safety, efficacy, and reproducibility, laying the foundation for successful clinical outcomes and regulatory approval. Although the allogenic SIRPα KO iMAC cells used in the discussed phagocytosis study were of Research Use Only (RUO) grade, Evotec possesses in-house GMP manufacturing capabilities to generate GMP-grade iMAC cell therapy products.

The iMAC platform optimized for solid tumors is one of several iPSC-based cancer cell therapies developed by Evotec. Their growing portfolio includes natural killer cells (iNK), macrophages (iMACs) and αβ and γδ T cells (iT) (Figure 2). Each immune cell type can be leveraged to create multiple differentiated allogenic cell therapy products.

Figure 2: Evotec’s iPSC-based cell therapy pipeline for oncology

Evotec’s growing iPSC cancer cell therapy platform can be used as the basis to develop novel allogenic cell therapeutics without the complexities or production bottleneck associated with autologous therapies. Supported by Evotec’s world class GMP manufacturing facilities, the cell therapy platform is fully scalable, and empowers developers with reliable, highly pure, ready-edited cell therapy products.

The iPSC-based oncology cell therapy platforms represent a wider iPSC pipeline for cancer cell therapy and beyond. Evotec’s industry leading iPSC platform has been developed with the aim to industrialize the use of iPSC technology in terms of throughput, reproducibility and robustness for the development off-the-shelf allogeneic cell therapies. Starting with a GMP iPSC master cell bank, Evotec’s cell therapy manufacturing platform provides full scalability and wide versatility in the numerous cell types that can be generated. An integrated iPSC gene editing platform enables functional optimization of the individual cell therapy products, ensuring that they are optimally tailored for their intended therapeutic use. (Figure 3).

Figure 3: Schematic depiction of Evotec’s fully scalable GMP-compliant cell therapeutics manufacturing platform

A bright future ahead for cell therapeutics

Macrophage cell therapies hold much promise in combatting solid tumors with greater efficacy versus CAR-T cell therapies. Overcoming TME defense mechanisms like the CD47-SIRPα axis is key to optimizing macrophages to exhibit maximum antitumor behavior. iMACs derived from iPSCs offer distinct advantages over autologous macrophage therapies, enabling a consistent, scalable platform for clinical development.

Editing iMACs by knocking out SIRPα enhanced their phagocytosis activity against tumor cells. Evotec’s 3D iMAC differentiation platform facilitates the genetic engineering of iPSCs to create an innovative allogenic SIRPα KO iMAC cell therapy product against solid tumors. This is one of many exciting projects happening at Evotec, who are supporting the development of novel cell therapies based on various cell types.

Find out more about Evotec’s industry leading cell therapy platform

Download the Poster

References

1. Chen, K., Liu, M.L., Wang, J.C., Fang, S. CAR-macrophage versus CAR-T for solid tumors: The race between a rising star and a superstar. Biomol Biomed. Advanced online release. 2023. https://doi.org/10.17305/bb.2023.9675
2. Mishra, A. K., Banday, S., Bharadwaj, R., Ali, A., Rashid, R., et al. Macrophages as a Potential Immunotherapeutic Target in Solid Cancers. Vaccines. 2022;11(1), 55. https://doi.org/10.3390/vaccines11010055
3. Willingham, S. B., Volkmer, J. P., Gentles, A. J., Sahoo, D., Dalerba, P., et al. The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors. PNAS USA, 2012;109(17), 6662–6667. https://doi.org/10.1073/pnas.1121623109
4. Willingham, S. B., Volkmer, J. P., Gentles, A. J., Sahoo, D., Dalerba, P., et al. The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors. PNAS USA, 2012;109(17), 6662–6667. https://doi.org/10.1073/pnas.1121623109

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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Cardiotoxicity is one of the leading causes of drug attrition. To address this, there is a need for improved predictive screens which can be applied at an early stage in drug development to ensure only safe compounds progress to the clinic.

Drug-induced cardiotoxicity can manifest itself via various different mechanisms which makes detection challenging. Either functional or structural changes can occur, and these effects can be direct or indirect. Functional cardiotoxicity results from acute alteration in the heart function often as a consequence of electrophysiological effects. Structural cardiotoxicity is associated with alterations in cell and tissue morphology and can manifest itself as cell death, inflammatory changes and fibrosis. These morphological changes can take time to present themselves clinically and, therefore, sensitive techniques which can detect early molecular changes are valuable from a predictive perspective.

Combining transcriptomics with artificial intelligence is showing great potential in providing this transformative improvement in predictive safety assessment. In fact, the value of this approach in drug-induced liver injury (DILI) has been demonstrated in a recent poster presented by Cyprotex. At SOT in Salt Lake City from 10-14 March 2024, Cyprotex showcased how this technique can also be applied to cardiotoxicity. The research evaluated 42 compounds (33 cardiotoxicants and 9 non-cardiotoxicants) used in a variety of therapeutic indications. The compounds were assessed at 2 time points and 8 concentrations in a beating cardiac organ model using human iPSC-derived cardiomyocytes. At the end of the incubation period, three different analytical methods were compared; high content screening (HCS) (cell count, cellular ATP, mitochondrial mass, mitochondrial membrane potential, cellular calcium levels, DNA structure and nuclear size), calcium transience (wave amplitude, frequency, full peak width and full decay time) and transcriptomics (high-throughput RNA sequencing).

The HCS and calcium transience assays were valuable in detecting structural and functional cardiotoxicants, respectively. However, the synergism of combining these assays with transcriptomics was demonstrated by an overall improved cardiotoxicity risk prediction. Additionally, transcriptomics analysis provided detailed mechanistic information and identified specific pathway responses. Combined, the three approaches (HCS, calcium transience and transcriptomics analysis) gave excellent cardiotoxicity prediction metrics of 100% specificity, 82% sensitivity and 86% accuracy at 10x C_max and 89% specificity, 91% sensitivity and 90% accuracy at 25x C_max. The transcriptomics analysis improved the overall sensitivity by identifying various molecular mechanisms of structural toxicity such as alterations in cardiac pathways, genotoxicity, ER stress and mitochondrial toxicity.

It is not just cardiomyocytes which can be affected by drug induced toxicity, non-cardiomyocytes such as fibroblasts and endothelial cells may also be impacted. Organotypic models developed using different cell types, therefore, are likely to be more representative both structurally and functionally. In the future, Cyprotex is extending its research to evaluate a number of different cell-based models and different organ-specific toxicities using the transcriptomics approach. Our cardiac safety database is also growing and we now have identified approximately 140 compounds for testing in our models. The use of transcriptomics and artificial intelligence is accelerating the development of new cell-based models in the field of drug-induced toxicity leading to a new paradigm in in vitro safety testing.

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The endocrine system comprises of a network of glands and organs that produce and release hormones to control various bodily functions. Endocrine disruptors, either natural or man-made, can affect the endocrine system by interfering with the normal actions of hormones. This can lead to serious health problems such as cancer, birth defects and developmental disorders. Many of the traditional endocrine disruption testing approaches use radiolabelled material or involve animal models and so have high cost and low efficiency, in addition to ethical concerns. Accurate low-cost screening models are required to flag up chemicals which are potential endocrine disruptors at an early stage.

At the Society of Toxicology (SOT) conference on March 10-14, 2024, Cyprotex presented a poster titled, ‘Establishment of a High Throughput Endocrine Disruptor Screening Panel of Assays for Rapid Screening of Chemicals’. The research developed a panel of 384 well high throughput cell-based hormone activation assays and in vitro hormone receptor binding assays with a 24-48 hr turnaround time. A range of chemicals with different potencies for the endocrine receptors were assessed.

Cell-based endocrine receptor activation assays expressing the human androgen receptor (AR), the human estrogen receptor (ERα or ERβ) or the the human thyroid hormone receptor (TR) were used to assess the chemicals at a range of concentrations at 37°C over 24 hr. Upon ligand activation, the hormone receptor binds to the promoter sequence linked to a luciferase reporter gene and the activation is quantified by luminescence. Cell viability was also assessed in parallel.

Hormone receptor competitor binding assays were also used to assess potential AR or ER ligands by measuring fluorescence polarization. A fluorescently-tagged ligand is added to the AR or ER in the presence of the competitor test chemical. If the test chemical binds then it prevents the formation of the fluorescent ligand/receptor complex and a decrease in polarisation is observed. The extent of the shift in polarization was used to determine the relative affinity of the test chemical for the hormone receptor.

The results observed for the known set of chemicals were consistent with literature values and data corresponded well between the two assays. Additional steroidogenesis assays are being developed to detect hormone levels of AR and ER in H295R cell culture supernatants following exposure to endocrine disruptors.

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Identifying and developing safer and more effective food additives are essential for a healthy growing population. Regulators such as the FDA and EFSA are responsible for monitoring the safety of these food additives. Current in vitro approaches for assessing genotoxicity of these additives present a lack of consistency in the literature regarding incubation time and analysis.

At the Society of Toxicology (SOT) conference on March 10-14, 2024, Cyprotex presented a poster titled, ‘Validation of a new genotoxicity pre-screening package for food additives’. The research evaluates a high content screening approach with robust data analysis which would be suitable as an early stage genotoxicity screening package for de-risking food additives.

Genotoxins are chemicals that cause DNA or chromosomal damage. This can be assessed using in vitro assays such as the phosphorylation of histone H2AX (pH2AX) and histone H3 (pH3), and the micronucleus test (MNT; OECD guideline 487). By assessing both pH2AX and pH3, it allows for assessment of clastogens (pH2AX) and aneugens (pH3). Clastogens are substances that result in structural damage to the chromosome through DNA double strand breaks. Aneugens are substances which result in the daughter cell having an abnormal number of chromosomes due to deletion or insertion of a whole chromosome. The in vitro MNT detects micronuclei which are formed from the misincorporation of chromosomal material that might be structurally and/or genetically damaged, due to interactions with clastogens and/or aneugens interactions. It is an approach recommended by the regulatory authorities.

For the pH2AX and pH3 assays, HepG2 cells were dosed with the food additives over 24hr. For the in vitro MNT, CHO-K1 cells were dosed with the food additives over 24hr. All assays used automated high content screening with robust data analysis to identify potential genotoxicity. From the 12 food additives assessed, 83% were correctly identified in at least one of the methods. Both of the false negatives (benzoic acid and tartrazine) have been reported to induce DNA damage under certain conditions but not others. This disparity in the literature may explain our results.

In summary, Cyprotex have developed an early stage high throughput screening approach to assess the genotoxic potential of food additives using a panel of assays to determine clastogenic and aneugenic potential in addition to micronucleus formation. As well as genotoxicity markers, the assays provide valuable additional information on cell survival, membrane integrity and cell cycle information.

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Predicting DILI (drug-induced liver injury) is challenging compared to other organ-specific toxicities. Translation from animals to humans is poor and, mechanistically, DILI can be complex. As a consequence, DILI continues to be one of the leading causes of attrition during drug development. Better human relevant models are required to improve early stage DILI prediction. Cyprotex is committed to researching and developing approaches to improve the prediction of DILI using human cell-based models in combination with novel techniques such as toxicogenomics and artificial intelligence (AI).

At the Society of Toxicology (SOT) conference on March 10-14, 2024, Cyprotex presented a poster titled, ‘An AI Approach to Drug-Induced Liver Injury Risk: Prediction of Safe Maximum Doses from Toxicogenomics Profiles’. The research evaluated 128 compounds from the FDA Liver Toxicity Knowledge Base – 68 of these compounds were associated with DILI and 60 of these compounds were not associated with DILI. Transcriptomics profiles were generated after dosing primary human hepatocytes in triplicate at 8 concentrations over 24 hr.

Machine learning is a subset of artificial intelligence which is used to find patterns, make decisions and optimise outcomes. In this study, the high throughput transcriptomics profiles of a set of known DILI-positive and DILI-negative compounds were used to train a supervised machine learning model to predict a safe maximum C_max for novel compounds. When interpreting the results, a compound was predicted as DILI-positive if the true C_max was above the predicted safe C_max, and a compound was predicted as DILI-negative if the true C_max was below the predicted safe Cmax. The model achieved the following metrics on the test set (assuming 40x C_max level and 90% DILI score threshold):

The poster provides a detailed insight into two DILI-positive (TAK-875 and bosentan) and two DILI-negative compounds (dopamine and caffeine) to demonstrate the power of the transcriptomics and AI in predicting DILI as well as identifying specific mechanisms of toxicity. The model was able to capture the importance of cholestasis-associated genes in DILI.

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Just - Evotec Biologics is constructing a new biomanufacturing facility in Toulouse, France. The facility applies the company’s successful J.POD design featuring a single-use continuous cell culture manufacturing platform set inside production-on-demand modules within a ballroom manufacturing space. The investment of approximately €150 million was announced in April 2021 and the company broke ground on the project in September 2022. In October last year the building shell was completed and the autonomous cleanroom POD installation occurred at the beginning of this year. Now the equipment will be installed into the cleanroom PODs ready for the facility to be operational in the second half of 2024.

At the core of this endeavour lies the innovative J.POD biomanufacturing facility developed by Just – Evotec Biologics. J.POD facilities contain the company’s continuous manufacturing platform for antibodies and other therapeutic proteins with an Fc-region. The continuous process is so highly intensified that it can be contained within production-on-demand modules that sit within a ballroom cleanroom space. The design is central to the company’s mission: to design and apply groundbreaking technologies that dramatically expand global access to biotherapeutics.

The J.POD design is commercial biologic manufacturing-ready but can also easily deliver batches for clinical trials. The ability to modulate capacity easily depending on the lifecycle stage of the molecules is one of the advantages of continuous manufacturing.

Transitioning to Continuous Manufacturing

Traditional fed-batch manufacturing methods have long been the standard for producing biologics. However, switching to continuous manufacturing with a high degree of intensification reduces the cost of goods manufactured (COGM) of biologics to less than $50 per gram by reducing the cost of building and running biomanufacturing facilities.

The use of pod modules in the design of J.POD Toulouse allow for greater agility, readily expandable facilities, and lower risk. Unlike traditional methods that require scaling up to larger production trains, the J.POD approach ensures flexibility in meeting demand fluctuations. This is extremely valuable to companies that launch new products and find it difficult to predict the ramp up in market demand.

By expanding into Europe, J.POD Toulouse enhances the company’s ability to support customers based in Europe, effectively. With facilities on both sides of the Atlantic, the company is providing supply chain security by having duplicated capacity in two geopolitically stable regions.

Just - Evotec Biologics' announcement last year of a multi-year, long-term tech partnership with Sandoz to develop and manufacture multiple biosimilars in J.POD facilities demonstrate the industry’s readiness to embrace continuous production methods.

Process Development Capabilities

To support manufacturing operations J.POD Toulouse facility houses robust process development capabilities, including:

1. Cell Line Development: Streamlining the creation of high-yield cell lines for antibody production.
2. Upstream & Downstream Process Development: Optimizing the entire continuous production process, from cell culture to purification.
3. Formulation Development: Crafting stable and effective formulations for therapeutic molecules.

Conclusion

J.POD Toulouse prepares to open its doors with its commitment to cost-effectiveness, scalability, and supply chain security. This facility stands poised to transform the way we produce lifesaving biotherapeutics. Watch this space—J.POD Toulouse is about to make waves in Europe and beyond.

This project benefits from French government funding as part of the Investments for the future Programme (programme d’investissements d’avenir in French) and is also supported economically by the Occitanie Region.