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

Download the Poster

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

“The intracellular exchange assay design enables studying the cumulative effect of modulators from the intracellular face of ion channels.” 

Background:

Intracellular binding sites of ion channels are targets for many drugs (example: anesthetics targeting voltage gated sodium channels). Pharmacological manipulation of the intracellular (IC) environment is as critical as the extracellular (EC) environment.  

In manual patch clamp, once the gigaseal is established and the patch is in whole cell mode, manipulating the IC environment by pipette solution exchange is a technical feat few can achieve (reference).  

This is where the intracellular exchange (ICE) assay developed by Evotec on Sophion Qube changes the game. 

The assay design: 

As is illustrated below (fig. 1), an EC salt solution is added to each well in a 384-well QChip via an inlet well and the same can be removed via a waste well, both from the top (fig. 1). On the other hand, the IC salt solution has only one inlet/outlet channel i.e. for both perfusing in and out. 

In the ICE assay, cells are patched on this QChip in the whole-cell mode using a negative pressure via the patch hole. The cells are thus surrounded by the EC solution while the cytosol is accessed via the IC solution.  

Figure 1: Architecture of each well in a 384-well Qchip (left). A single hole Qchip with the indicated intracellular (IC) solution inlet.

Hereafter, the magic begins: after one or more stabilization stages to obtain baseline activity, the IC solution is completely replaced with a new IC solution in sequential liquid periods. Our ICE assay has been developed to execute such IC exchanges up to four times (fig. 2), replacing the previous solution completely with a new solution each time.  

Figure 2: Liquid periods in the ICE assay with the stabilization periods to obtain baseline activity followed by four rounds of IC solution exchange. 

Thus, modulators can be delivered to the cytosolic face of the ion channel cumulatively.  

All the while, the cells are maintained in whole cell configuration in the QChip and the seal resistance does not drop below the QC threshold (40 MΩ, fig. 3). The ion channel target is observed to respond to each increasing concentration of the modulator post-exchange (fig. 3). 

Figure 3: (a) Seal resistance during sequential liquid periods interspersed with removal of the Qchip from the bio-chip interface, (b) Current vs time (IT) plot. Signal from the target ion channel responds to cumulatively increasing concentration of the modulator in cytosolic side. 

Achieve more with less reagents and resources: 

• With four rounds of exchange, the volume of the IC solution required is only 4 ml for 3-4 experiments.  
• The assay has a throughput of up to 26 compounds per Qchip for a 3-point dose response profiling.  
• The assay is very resource-friendly also in terms of chemistry, since only minute amounts of compound are required. 
• An experiment with four exchanges lasts only about 90 minutes.  

In a proprietary assay developed by Evotec, it was possible to profile about 700 compounds in 70 runs performed over 64 days.  

Key learnings: 

• The IC exchange assay can cumulatively test three increasing concentrations of the same compound, delivered to the cytosolic face, providing pre- and post-compound data.  
• A complete solution exchange is possible between each concentration, thus preventing compound cross-contamination.  
• The assay can also be adapted to different intracellular ion concentrations, pH values, or signaling molecules.  
• Druggability of ion channels as a family of proteins is greatly enhanced. 

No matter whether your drug discovery program is in the hit ID, hit-to-lead, lead optimization or early safety assessment, the ICE assay is useful for high-throughput hit profiling at any point.  

Companies interested in using Evotec’s technology are welcome to contact Evotec at info@evotec.com. 

In many diseases, genes are not altered, but their regulation is disturbed, and only recently have approaches emerged to target gene regulation as a therapeutic tool.

How are genes and their expression regulated? While the central dogma of the 1960s DNA->RNA->protein still holds true, recent advances in molecular biology have provided much insight into how cells regulate which genes are transcribed into mRNA and how translation of RNA into proteins is regulated. It is now known that mRNA can undergo many chemical modifications, induced by a variety of enzymes. These modifications affect mRNA maturation, stability, and lifespan, as well as the rate and duration of translation and mRNA degradation. Likewise, some small modifications can either prematurely terminate protein synthesis, reduce peptide yield, or alter the amino acid sequence of the translated protein.

While the existence of post-transcriptional RNA modifications has been known for more than 30 years, their mechanisms, functional consequences and connection with human diseases including cancers have only recently been elucidated, making RNA epitranscriptomics an unexplored and exciting field for drug discovery. Meanwhile, more than 150 RNA modifications have been reported and approximately 300 RNA-modifying enzymes have a potential as novel therapeutic targets.

Evotec's partner Storm Therapeutics is amongst the first companies to pursue RNA-modifying enzymes as drug targets. The company identified RNA methyltransferases, a class of approximately 75 enzymes, as the most promising target class. Some have been identified as important regulators of cancer development and progression and thus represent promising novel antitumoral targets. Storm focused on METTL3, an enzyme involved in the co-transcriptional methylation of internal adenosine residues in eukaryotic mRNAs. This enzyme regulates fundamental aspects of mRNA life cycle, such as splicing, transport to the cytoplasm, stability, and translation into protein. It was known that in AML cell lines, knocking out METTL3 leads to a pronounced antiproliferative effect associated with a reduction of the BCL2 (anti-apoptotic factor). In other models, a marked upregulation of genes associated with innate immunity, such as those in the interferon (IFN) signaling pathway was demonstrated following METTL3 depletion.

However, tackling new classes of enzymes such as RNA methyltransferases requires breaking new ground in assay development, screening, and downstream hits to guide progression. To address these challenges, Storm and Evotec began collaborating in 2016, which later evolved into an integrated drug discovery and development alliance focused on novel small molecule RNA epigenetic drugs for oncology and other diseases. Using Evotec's fully integrated small molecule drug discovery and development platform, including biomarker support (development of a m6A-mRNA level evaluation technic), STC-15 was identified. STC-15 is a potent, selective small molecule inhibitor of the mRNA modifying enzyme, METTL3. STC-15 was developed from high throughput screening to candidate nomination in less than three years.

In preclinical cancer models, treatment with STC-15 significantly inhibits tumor growth. In addition, a profound cell-intrinsic interferon response was observed, following an accumulation of double-stranded RNA. In mouse models, the induction of innate immunity mechanisms, such as the interferon pathway, enhanced T-cell mediated cancer cells killing. This work has been recently published in Cancer Discovery, a leading cancer journal (Guirguis, Ofir-Rosenfeld et al., 2023). Notably, it activated innate immune pathways and inhibited tumor growth as effectively as anti-PD1 therapy in some models. In addition, the data showed that the combination of the two agents resulted in significantly greater activity, leading to tumor regression and durable anti-cancer immunity. Detailed investigation of the mechanism of action of the two treatments revealed that they act independently, providing a strong rationale for their combination. This added significantly to previous studies where STC-15 demonstrated efficacy in leukemia models through mechanisms such as inhibition of leukemia stem cell function (Yankova et al., Nature, 2021). Furthermore, additional combination studies revealed a high degree of synergy between STC-15 and Venetoclax, a BLC2 inhibitor and standard of care therapy for acute myeloid leukemia (AML) patients.

This data provided the rationale for the development of STC-15 both as a monotherapy and as a combination partner for immune checkpoint inhibitors or with BCL2 inhibitors for the treatment of solid tumors and leukemias, respectively. Following the selection of STC-15 as a first-in-class development candidate in 2020, the seamless integration from project initiation to IND using Evotec's INDiGO platform led to the entry of STC-15 into Phase I clinical trials in 2022. The orally bioavailable, highly selective METTL3 inhibitor is being developed for the treatment of solid tumors and may also have potential in AML.

A phase 1, multi-center, open-label, first-in-human study is evaluating multiple ascending daily oral doses of STC-15 in a 3+3 cohort design. The study is designed to systematically evaluate the safety and tolerability, pharmacokinetics, pharmacodynamics, and clinical activity of STC-15 in adult patients with advanced malignancies. Dose levels for further evaluation in expansion cohorts will be selected based on all available PK, PD, target engagement (including m6A-mRNA level evaluation), efficacy, safety, and tolerability data, including long-term safety data beyond dose-limiting toxicities (DLTs).

Patient enrolment started in November 2022, and the company anticipates top-line results in 2024.

The development demonstrates the benefits of Evotec's integrated, accelerated IND-enabling platform to support the exploration of novel and exciting biology to maximize innovation, to execute efficiently with rapid and seamless integration from target to IND.

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Antimicrobial resistance (AMR) is one of the biggest health threats worldwide. Key to countering AMR is the development of novel anti-infective drugs. The limitations of animal models and clinical trial design have emphasised the importance of nonclinical pharmacokinetics/pharmacodynamics (PK/PD) platforms which provide a detailed understanding of the relationship between the fate of the antimicrobial compound in the body (PK) and the impact of exposure to the compound on the target microbes (PD). This allows us to optimise dosing regimens to maximise the efficacy of antimicrobial compounds (microbial killing) while minimising toxicity and the risk of the emergence of AMR.

What is the HFIM?

The HFIM is a system of pumps, tubing and microfibers that mimics the body, allowing in vitro assessment of anti-infective compounds under more relevant conditions. It consists of a central reservoir and tubing used as a circulating system, and a hollow fibre cartridge with thousands of permeable capillaries. The extra capillary space (ECS) outside the fibres within the cartridge contains the target organism. During operation, the drug-infused growth medium in the central reservoir is continuously pumped to the hollow fibre cartridge, rapidly passing through the capillaries into the ECS. This continuous flow ensures that nutrients, oxygen, and test compounds are continuously refreshed while waste products are removed. To simulate drug clearance, fresh medium is added to the central reservoir effectively diluting the drug from the system. Accordingly, this balance of drug supply/clearance can effectively simulate the drug’s PK profile.

Why choose the HFIM as PK/PD model?

It is the most capable in vitro system for PK/PD determination for anti-infective compounds, against bacteria and fungi. It is a dynamic model capable of simulating almost any given concentration-time profile for one or more compounds, even if they have very different half-lives.. The Hollow Fibre Infection Model is not limited by in vivo model availability, compatibility of PK profiles, dosing or sampling frequency, or study duration, which is extremely important for understanding PK/PD relationships and the risk of AMR over clinically relevant treatment times. Various cartridges with fibres manufactured from different materials are available to optimise the HFIM for microbial growth and compound performance.

In conclusion, the HFIM is a versatile in vitro PK/PD platform which can accelerate the development of antibacterial and antifungal compounds, contributing to the fight against AMR. 

If you’d like to learn more about the uses of the Hollow Fibre Infection Model you can download our white paper here

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

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

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

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

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Can we age healthier?

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

Increasing healthspan

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

What is biological aging?

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

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

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

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

How to prolong health span?

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

Developing novel senolytics - challenges and opportunities

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

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

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

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

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

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

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

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

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

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

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

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