ICH M12 2024 vs FDA 2020 DDI Guidance. Exploring the Differences.

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The new ICH M12 guideline on drug interaction studies is welcomed by industry as it provides a harmonized approach which is expected to be implemented by the major regulatory authorities such as the US Food and Drug Administration (FDA), European Medicines Agency (EMA) and the Japanese Pharmaceuticals and Medical Devices Agency (PMDA) and presumably replace their respective existing guidance. In this blog, we explore the differences between the now finalized ICH M12 guideline adopted in May 2024 (draft for comment released 2022) and the previous US FDA 2020 guidance released in January 2020 with a focus on the in vitro assays.

Before we launch into the individual assays, one general observation in the new ICH M12 guideline is the replacement of the term ‘victim’ to ‘object’ to denote an investigational drug (usually a substrate) which is affected by a concomitant drug, and the replacement of the term ‘perpetrator’ to ‘precipitant’ to denote an investigational drug (typically an inhibitor or inducer) which affects a concomitant drug. This amendment only appeared in the final version of the ICH M12 guideline as it was one of the comments from review of the draft version.

Reaction Phenotyping

If we consider reaction phenotyping initially, the differences between the new ICH M12 and the FDA 2020 guidance are relatively minimal. Although the ICH M12 refers to all the enzymes mentioned in the FDA guidance, it also refers to some additional Phase II enzymes which may need to be evaluated including glutathione S-transferases (GSTs) and N-acetyl transferases. Both the ICH M12 and the FDA guidance have the same cut-off of ≥25% of total elimination identified using in vitro studies (reaction phenotyping and metabolite identification) and human mass balance data for determining if the enzyme needs further investigation in a clinical study. One key difference is that the FDA guidance recommends two methods should be used for reaction phenotyping – the first method using selective inhibitors in the presence of human liver microsome or hepatocytes and the second method using human recombinant enzymes. In the ICH M12 guidelines, only one of these methods is required.

Both the ICH M12 guideline and the FDA guidance have recommendations for evaluating metabolites in reaction phenotyping studies if the exposure levels of the metabolite results in clinically relevant changes in efficacy or safety.

Enzyme Inhibition

Moving onto enzyme inhibition, both the ICH M12 and FDA guidance recommend evaluating the main seven CYP isoforms for reversible and time dependent inhibition. For reversible inhibition, the cut-off for determining if a clinical study is required is the same for both the ICH M12 and FDA. However, for time dependent inhibition, 5 x C_max,u is used in the calculation in the ICH M12 whereas a higher safety factor of 50 x C_max,u was used in the 2020 FDA guidance, suggesting a less conservative approach is used in the new ICH M12 guideline.

One addition to the ICH M12 guideline, which is not specifically included in the previous FDA 2020 guidance, is the inclusion of reversible UGT inhibition. The ICH M12 recommends that UGT1A1, UGT1A4, UGT1A9, UGT2B7 and UGT2B15 inhibition should be evaluated if direct glucuronidation is one of the major elimination pathways of the investigational drug. Although it is considered an area of ongoing research, it is currently recommended in the ICH M12 that the same cut-off values should be used for UGT inhibition that is applied to CYP enzymes (i.e., C_max,u/K_i,u<0.02).

Enzyme Induction

For enzyme induction, the FDA guidance suggested either catalytic activity or mRNA could be used to assess induction. It also suggested that immortalized hepatic cell lines may be used to determine CYP induction potential. However, the new ICH M12 primarily recommends the analysis of mRNA (with the exception of CYP2C19 where catalytic activity should be measured) and that the CYP induction assessment should be performed in human hepatocytes. The ICH M12 guideline also addresses the issue of toxicity and recommends that cell viability assessment is performed before and at the end of the incubation.

For data analysis, both the ICH M12 and FDA cover the three basic methods (fold-change, relative induction score correlation method and basic kinetic model) and align on the cut-offs for indicating if an investigational drug has the potential to induce in vivo. However, for the basic mRNA fold method in the ICH M12 guideline describes assessing test drug concentration of 50 x C_max,u whereas the FDA only suggests testing up to 30 x C_max,u suggesting a more conservative approach now by the ICH M12. Furthermore, in the calculations for the correlation methods and the basic kinetic model, unbound EC₅₀ (EC_50,u) is specified in the ICH M12 whereas only EC₅₀ is referred to in the FDA guidance. Finally, the ICH M12 describes in more detail an indicative positive control response for CYP1A2, CYP2B6 and CYP3A4 of typically at least 6-fold to ensure sufficient sensitivity of system, whereas this level of detail is absent from FDA.

Transporter Substrate Identification

Both the ICH M12 guideline and the FDA DDI guidance (2020) recommend the same transporters are assessed. The method for testing and thresholds for clinical assessment are very similar between the FDA and ICH M12.

Transporter Inhibition

For transporter inhibition, the same transporters are recommended in the ICH M12 as the previous FDA guidance, however, the cut-off value for determining if a clinical study should be performed are different in a couple of instances. Firstly, for P-gp and BCRP inhibition where the investigative drug is administered by the parenteral route or if a metabolite is formed post absorption, the cut-off value in ICH M12 is an IC₅₀ of 50 x C_max,u, whereas in the FDA guidance it is 10x C_{max total}; important to note the additional difference for what is defined as [I]. This indicates a more conservative approach by the FDA when plasma protein binding is > 80%, or by ICH M12 when plasma protein binding is < 79%. The second instance applies to the cut-off value for MATE1 and MATE2-K, which has increased to an IC₅₀ of 50 x C_max,u rather than 10x C_max,u, indicating a more conservative approach by the ICH M12 for these renal transporters. The ICH M12 also specifically refers to unbound IC₅₀ (IC_50,u) in the calculations whereas the FDA guidance only references IC₅₀. In this regard, it is important to recognize that any correction of IC₅₀ for potential non-specific binding that might occur within a transporter inhibition assay would only be required if the assay did not incorporate a pre‑incubation step with investigational drug as standard methodology for all transporters prior to the co‑incubation with fresh investigational drug solution and probe substrate. With such standard methodologies, the inclusion of the pre‑incubation step would be anticipated to mask any non-specific binding sites therefore the co‑incubated concentrations of investigational drug would be nominal for IC₅₀ fitting purposes, i.e. IC₅₀ = IC₅₀,_u.

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The top challenges in commercial API manufacturing

Once an investigational medicinal product has demonstrated its safety and efficacy during clinical trials, the next challenge for its Sponsor is to secure a reliable supply chain for its commercial production. Understandably, commercial pharmaceutical manufacturing, including commercial API manufacturing, is a highly regulated area to ensure patients’ ongoing access to a consistently high quality of medicines that guarantees their safety and efficacy. Transitioning from a clinical pilot scale to eventually the larger plant scale required for commercial API production can be a long and arduous process.

The main challenges faced in the journey to commercialization include scaling up production, establishing and maintaining process consistency and reproducibility, and ensuring compliance with regulatory guidelines. In this blog, we explore these areas in detail, discussing how a strategic approach, robust planning, and the incorporation of innovative technologies, can help ensure your drug smoothly and successfully reaches the market.

Developing robust and scalable processes

Increasing the production of active pharmaceutical ingredients (APIs) and drug excipients from milligram to multi-kilogram quantities is arguably the biggest challenge faced in paving the way to reliable commercial pharmaceutical manufacturing. Without the adoption of strategic planning during the process development stage, unexpected issues may arise and lead to significant increases in your program timelines and costs.

Here are our top tips for effectively planning and developing robust and scalable API manufacturing processes, and achieving faster and more seamless scale-up that supports marketing approval and supply security requirements:

• Choice of starting materials – several considerations should be made when selecting the starting materials used in the manufacture of APIs and medicinal products. This includes quality, compatibility, long-term stability, affordability, safety, security of supply, and compliance with pharmaceutical regulatory guidelines. Ensuring your starting materials meet these criteria throughout the entire scale-up process, up to commercial scale, will pave the way for a safe and high-quality medicinal product.
• Choice of equipment and processing technology – where possible, scalable equipment and processing technology should be chosen in API and drug manufacturing. This will help to streamline the technology transfer process, reducing the time and costs required for process optimization and validation, and minimizing the risk of failure. Cost, availability, material compatibility, cleaning processes, and the ability to implement automation and control systems at commercial scale should be considered.
• Process development – the basis of every robust, scalable process is effective API process development. This requires the optimization of several components, including the selection of the synthetic route, and measures for process control and risk management. Scientific and risk-based approaches should be utilized to help gain an in-depth understanding of how any changes to the process during scale-up will affect the safety and quality of the product.
  
  

Read this blog for more detail on how to optimize API process development

Establishing and maintaining process consistency and reproducibility

Establishing and maintaining consistency and reproducibility during scale-up is a complex yet vital task in commercial pharmaceutical manufacturing. Increasing the vessel size and introducing any other necessary changes can drastically alter process performance and product quality. Failure to design and optimize the process to account for these differences could derail production, altering the safety, quality, and efficacy of the API.

The key to ensuring process consistency and reproducibility in commercial scale production lies in strategic planning during process development, with the use of systematic approaches that facilitate the implementation of a robust process control strategy

Implementation of systematic approaches

Several systematic approaches, such as design of experiments (DoE), quality by design (QbD), and process modeling and simulations performed with a software such as Dynochem® are used to improve knowledge of the product and the scale-up process. This includes the thorough impact assessment of any changes in processing parameters on process performance and final product quality.

DoE involves the use of factorial design to plan a series of experiments that test simultaneously variations in individual input factors. The aim of this is to gain an understanding of the individual and combined effects of parameters variability on the process performance and output. Following these experiments, mathematical models, such as response surface methodology (RSM), are used to identify the optimal set of process parameters as well as the acceptable operating envelope for the process.

DoE and process modeling and simulations can be integrated into a QbD approach, which incorporates statistical and analytical methodologies to enhance the process control strategy and build quality and risk management into the manufacturing process. This is achieved by identifying the critical process parameters (CPPs) and critical quality attributes (CQAs) that are associated with the quality, safety, and efficacy of the drug substance and excipients.

Using the QbD methodology, the determined parameters and quality attributes are used to establish a design space. For this, statistical tools are used to explore how combinations of CPPs might interact and impact on CQAs. This helps manufacturers optimize the production of APIs and drug excipients, ensuring the design of reproducible processes that minimize batch failure risks and significantly reduce batch-to-batch variability.

Click here to learn more on how to apply QbD principles to drug development and manufacturing

Adoption of advanced process control systems

The process knowledge gained from experimental modeling approaches, such as QbD and DoE, can be used to develop advanced process control systems. These commonly utilize process analytical technology (PAT), which monitors process parameters on-line in real-time.

When PAT is integrated into advanced process control systems, the analytical data may be used to make automatic system adjustments and maintain process parameters within their predefined limits. This reduces manual intervention and creates a closed-loop control system, allowing for the immediate detection of deviations in process conditions and subsequent feedback control.

By immediately correcting any deviations in the process, advanced process control systems enable real-time release testing, assuring manufacturers that the processes have remained consistent, and that the product meets quality and safety standards. By increasing efficiency and consistency, these systems also help to reduce waste, costs, and product cycle times.

Regulatory compliance

Compliance with current good manufacturing practice (cGMP) regulations is critical to ensuring the production of consistent, high-quality, pharmaceutical products. API manufacturing plants are subject to strict regulations, with complex and ever-evolving requirements, stringent quality standards, and severe consequences in case of non-compliance. To comply with cGMP guidelines, several robust management systems must be in place, including those for data integrity, process control, risk management, and supply chain management:

Data integrity

Consistent, accurate, timely, and complete records are required to provide regulators and stakeholders with the confidence that your medicinal product meets all safety and quality standards. Clarity, consistency, and conciseness of the documents must be maintained across the entire product lifecycle. The development of a robust documentation system can help manufacturers with this, establishing effective procedures for naming, authoring, reviewing, approving, updating, storing, and distributing documents.

Control strategy

There are increasing requirements for a clear, well-defined, and scientifically justified process control strategy in cGMP applications. This should include the selection and evaluation of starting materials, followed by approaches including QbD and DoE to link materials attributes and process parameters to product CQAs. These approaches are used to establish a design space and plan control measures to ensure that CQAs are met and a sound process validation methodology is implemented. Furthermore, the process control and validation strategy should be adapted to the increased production scale throughout all stages of the product’s lifecycle.

Risk management strategy

cGMP guidelines require a systematic approach to risk assessment in pharmaceutical manufacturing. This involves the process of identifying, assessing, controlling and reviewing risks based on their potential for impacting the performance of the process and the quality of the product. Risk management plays a central role in scaling up to commercial production in order to mitigate significant quality risks such as cross-contamination and minimize health and safety risks to operators, especially when handling highly potent APIs. Several tools are available for risk assessment, including failure mode and effects analysis (FMEA).

Supply chain management systems

In the pharmaceutical industry, securing patients’ access to drug supplies post-marketing authorization is a regulator’s number one priority. Regulatory guidelines are designed to cover the entire supply chain, from the supply of raw materials to be introduced in cGMP manufacturing operations through to the manufacturing, packaging, labeling, and distribution of the final product.

Digitalizing the management of manufacturing activities can enhance the visibility and efficiency of inventory management, product monitoring, and data exchange throughout the supply chain. Additionally, supply chain management systems should be designed to incorporate risk management strategies for the prediction, prioritization, and mitigation of risks of product stock-outs. This is not only essential for regulatory compliance and patient access, but it will also increase supply chain efficiency to help overcome issues such as inadequate forecasting, long lead times and build-up of working capital.

How to master commercial pharmaceutical manufacturing

The journey to commercialization can be challenging. Scaling up production while maintaining process consistency, product quality, and regulatory compliance, requires expert process development capabilities, and the adoption of innovative science and risk management methodologies. A common pitfall for the Sponsor of an innovative therapy is to under-estimate the complexity and intricacy of this enterprise, which involves the coordinated optimization of strategies for process control, risk management, data management, and supply chain management.

With ever-evolving regulatory requirements and the increasing urge to shorten drug development timelines, getting your drug to market can seem like a daunting undertaking. That’s why taking some of the pressure off your organization by outsourcing your drug development and manufacturing activities to an expert partner can be the smartest decision. This will ensure your drug is commercialized in the fastest and most cost-efficient way possible, utilizing expertise, facilities, equipment, and processes to anticipate and overcome any challenges thrown at your program with ease.

Evotec offers an integrated end-to-end solution for innovative drug R&D, with the capabilities to support all phases of your drug development program. Your projects are in safe hands with our team of expert scientists who are pioneers in QbD, process design, scale-up, and validation, operating to full cGMP within FDA, MHRA, AIFA and BfArM approved facilities.

Learn more about our API and drug product manufacturing capabilities

Active pharmaceutical ingredient (API) manufacturing for new chemical entities (NCEs) is a lengthy undertaking involving many complex chemical processes, including the production of intermediates and purification and isolation steps. Optimizing the manufacturing process, through rational API process design and development, is the key to enabling fast, safe, reproducible, and cost-effective API production. And with the increasing pressure on accelerating drug development while staying within budget and meeting increasingly complex and stringent regulatory requirements, optimizing the API process development workflow has never been more crucial.

However, designing and executing an effective API process development strategy is no small or easy feat. From the early phases of drug development through to commercial manufacturing, the fine-tuning of many individual steps is required to optimize and de-risk API production. This blog outlines some of the key considerations involved in successfully designing and developing an optimal commercial API manufacturing process, including the selection of the synthetic route and regulatory starting materials, the development of a highly effective process control strategy, as well as risk management methodologies, and process safety considerations.

Synthetic route selection

When designing or selecting a synthetic route, it’s important that you get it right, as the use of suboptimal, inefficient routes can drain resources and substantially increase your time to market. Indeed, changing the synthetic route at the late development stage can prove very costly and time-consuming, not least due to the regulatory constraints being faced; in the worst-case scenario, such a change can even trigger the requirement for additional in vivo studies of the drug.

A key consideration for selecting the synthetic route for any type of chemical API is efficiency. Ideally, the chosen synthetic route requires the least number of steps and results in the shortest time to market.

In this regard, scalability is a critical aspect. All chosen materials, unit operations, processing equipment, and conditions should be suitable for scale-up to enable the manufacture of both clinical and commercial scale API batches. Building robustness into the synthetic route by selecting chemical transformations for ease of scalability will help maintain high levels of reproducibility, quality, and cost-effectiveness at increasing scales. This will ultimately ensure continuity in API supply by minimizing the time and costs involved in technology transfer activities.

Additional essential considerations in API synthetic route selection and optimization include those outlined by the SELECT principle: safety, environmental impact, legal requirements, economics, control, and throughput. These principles were first proposed in a 2006 consortium of pharmaceutical manufacturers, including AstraZeneca, GSK, and Pfizer, providing a good basis for selecting or designing a robust, commercially viable synthetic route.

Starting material selection

The selection and introduction of all processing materials, including raw materials, intermediates, solvents, and reagents, will depend on the chosen synthetic route and the API. However, all materials should have well-defined chemical properties, structures, and impurity profiles.

Additionally, a thorough understanding of the selection criteria for starting materials is essential, as outlined in global regulatory guidelines, including ICH Q7 ‘Good manufacturing practice for active pharmaceutical ingredients’. This requires a risk-based approach, which entails gaining a detailed understanding of how changes in the proposed API starting materials can influence the critical quality attributes (CQAs) of the drug substance, including its impurity profile, and the consequences this may have on the medicinal product’s quality.

All starting materials should also be evaluated for their security of supply, in addition to their quality, safety, and environmental impact. It is of particular importance to develop a robust supply chain strategy for custom-made starting materials, including the evaluation and qualification of reliable suppliers.

Developing a process control strategy (PCS)

Each process involved in API manufacturing requires the definition of a unique set of process parameters, such as mixing, temperature, pressure, and time. These parameters must be adequately monitored and tightly controlled to avoid the formation of impurities and prevent inconsistencies in output quality and yield. To develop an effective PCS, several considerations should be made, including the control of input materials, and the use of quality-by-design (QbD) principles to characterize the process and develop appropriate analytical methods to be implemented for quality control.

Quality-by-design (QbD)

QbD is a systematic, rigorous, data-driven approach that should be adopted to improve process and quality control in API manufacturing. QbD relies on predefined objectives to gain a thorough understanding of process control. This starts with determining the drug’s quality target product profile (QTPP), which consists of several design specifications that ensure the product is safe to use and has the desired therapeutic effect.

Based on the definition of the QTPP for the medicinal product, the API CQAs, i.e. the measurable properties of the compound that characterize its quality, including purity, potency, particle size, and stability, are identified. Once API CQAs are defined, the manufacturing process must be thoroughly studied and characterized to evaluate the critical process parameters (CPPs), which are the variables, such as temperature, pH, agitation, and processing time, that impact process performance and consequently product quality.

Essentially, QbD involves the consideration of all materials and processing parameters that could influence product quality. By gaining a sound scientific understanding of the processes through the execution of a rational experimental design, e.g. using a statistical design of experiments (DoE) methodology, this approach enables the elaboration of a more effective control strategy. As documented in the international pharmaceutical guidelines, ICH Q8, Q9, and Q10, the QbD methodology enhances drug development by helping to reduce the risk of batch failure and improving final product quality, safety, and consistency.

Learn more about the role of QbD in drug development

Analytical method development

QbD principles can also be extended to analytical method development. The API’s CQAs provide a good basis for selecting the most appropriate analytical methods and determining their parameters. Well-designed test methods are paramount to supporting the accurate and reliable monitoring of intermediates and product quality, in line with international quality standards and regulatory guidelines.

For in-process analysis, chromatographic methods, like high-performance liquid chromatography (HPLC) and gas chromatography (GC), are commonly preferred to monitor process execution due to their accuracy and reliability. More recently, the development of process analytical technology (PAT) based on the use of in-line spectroscopic methods, such as Fourier transform infrared (FTIR) spectroscopy, Raman spectroscopy, and focused beam reflectance measurement (FBRM), has helped to improve process control by enabling real-time process monitoring.

When choosing the best analytical method for each process, molecule type and stability arekey considerations. These attributes drive requirements for achieving the appropriate sensitivity and robustness of each assay, including the choice of detection method, range of detection, sample preparation procedure, and analysis time, in addition to cost and ease of routine implementation.

Implementing quality risk management

Risk of failures associated with the facilities, equipment, materials, personnel, processing, product testing, and storage are inherent to API manufacturing. Effective risk management strategies are required to prevent these risks from impacting the quality, efficacy and safety of the API, and from causing costly delays. Recognizing this, ICH guideline Q9 outlines a framework for quality risk management (QRM) applied to pharmaceutical products. This is a strategic approach that involves assessing, controlling, and continuously reviewing potential risks that could occur in API manufacturing, using the following steps:

• Risk assessment – this step considers the identification of hazards, the probability of them occurring, and the evaluation of the severity of their consequences.
• Risk control – this next step consists of the use of a decision-making tool to identify and implement control strategies designed to either eliminate or reduce the probability of occurrence, and lessen the severity of the impact down to a level that assures the quality, efficacy, and safety of the product.
• Risk review – this step is essential to maintaining the effectiveness of the quality risk management framework since it is designed to incorporate the risk assessment and control activities described above while reviewing the new data and knowledge generated throughout the product lifecycle activities.

Several risk management tools are available to aid quality risk management, including failure mode effects analysis (FMEA) and its extension, failure mode, effects, and criticality analysis (FMECA). FMECA is a critical risk analysis tool derived from FMEA and used to determine the potential sources of failure during the execution of the manufacturing process, evaluating the severity and likelihood of such failure, in addition to how it can be detected before it occurs.

Process safety

The development of API manufacturing processes requires particular attention to health and safety considerations to avoid serious and potentially fatal hazards. In addition to ensuring that operators are effectively protected from direct exposure to chemicals, particularly those with high potency, the process chemist also needs to ensure that the safety of the API manufacturing process is carefully assessed and established.

The first step to developing an effective process safety strategy consists of performing hazard assessments for each processing step. These require a detailed understanding of the chemical processes involved, in addition to several other components, including the use of material safety data sheets (MSDS) to take into account specific chemical risks associated with the starting materials and API, and the consideration of any requirement for specific operator training, worker exposure control measures, and compliance with regulatory standards. Although conducting thorough hazard assessments and using these to develop safe processes is time-consuming, it is essential to manage the operational risks associated with small molecule drug development.

Key considerations and future directions for API process development

In summary, a clear API process development strategy is essential to streamline and de-risk drug development, helping your drug reach the market in the most cost and time-effective way possible. Additionally, utilizing science- and risk-based methodologies in your strategy is essential for successful API production and for demonstrating a thorough understanding of the product and the process, including how any changes in the manufacturing process will affect the safety, efficacy, and quality of the final product.

Looking to the future, the adoption of innovative technologies, such as high-throughput experimentation (HTE) and predictive process modeling, will undoubtedly change the landscape of API process development. These methods can effortlessly generate large amounts of data, aiding the prediction of how processes will respond to varied reaction conditions and scale-up configurations. These data-driven techniques will further help to improve decision-making and design more efficient, robust, and scalable API manufacturing processes.

Outsourcing your API process development With these many complex considerations, outsourcing your API process development can maximize your chances of success while taking the pressure off your drug development teams. Leveraging the scientific expertise and tried-and-tested tools and methodologies offered by a specialist partner to develop your processes will pave the way for streamlined and de-risked API manufacturing. When looking to outsource your API process development, you’re in safe hands with Evotec. The combination of our 25 years of experience in the development and manufacturing of small molecule APIs and our end-to-end shared R&D platform offers a fully integrated approach to process research and analytical development, supplying APIs for use in pre-clinical development, non-clinical use, and clinical trials, all the way through to commercial supplies.

Head to our API capabilities webpage to find out more about how we can optimize your API process development strategy, and de-risk and accelerate your drug development program.

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Autoimmune diseases (ADs) represent a significant challenge in healthcare, affecting millions worldwide. These conditions arise when the body's immune system mistakenly attacks healthy tissues, leading to chronic inflammation and tissue damage. While current treatments for ADs focus on immunosuppression, they often come with significant side effects and provide only symptomatic relief. In recent years, there has been growing interest in cell-based therapies, particularly CAR (chimeric antigen receptor) cell therapy, as a potential solution to address the underlying causes of ADs.  

Autoimmune diseases encompass a wide range of conditions, including rheumatoid arthritis, lupus, multiple sclerosis and inflammatory bowel disease, among others. Despite their diverse manifestations, these diseases share a common underlying pathology of immune dysregulation. In healthy individuals, the immune system is finely tuned to distinguish between self and non-self antigens, but in autoimmune disorders, this distinction becomes blurred, leading to an attack on the body's own tissues. 

CAR cell therapy offers a promising approach to treating ADs by depleting disease-driving immune cells and rebalancing immune homeostasis. CAR T cells, which are engineered to express synthetic receptors targeting antigens expressed on the surface of pathogenic cells, have shown remarkable success in treating certain cancers. Now, researchers are exploring the potential of CAR T cells and their counterparts, CAR iNK (natural killer) cells, in targeting the aberrant autoreactive cells implicated in autoimmune diseases to reset the immune sytsem¹. We believe that cell therapy approaches could provide long-lasting drug-free remission and potentially a curative treatment for AD patients.

Understanding CAR Therapeutics

CAR T cell therapy first emerged as a groundbreaking treatment modality for hematological malignancies, such as leukemia and lymphoma. Building on this clinical success, researchers have recently turned their attention to applying CAR cell therapy to treat autoimmune diseases. 

In the context of ADs, CAR T cells have been investigated for their ability to target autoreactive T cells or B cells. B cells, in particular, play a central role in many autoimmune disorders by producing autoantibodies and driving inflammation. One promising target for CAR cell therapy in ADs is the cell surface protein CD19. High-level expression of CD19 is maintained throughout the majority of B-cell differentiation stages. By targeting CD19-positive B cells, CAR T cells or CAR NK cells can selectively eliminate the autoreactive B cell populations responsible for driving autoimmune responses². 

A recent early clinical study showed promising results for using CD19 CAR T cell therapy to treat ADs. Patients with systemic sclerosis, severe Systemic Lupus Erythematosus (SLE), and idiopathic inflammatory myositis showed remission following therapy³. However, CAR NK cell therapy may be beneficial over CAR T cell therapy, particularly in ADs that have dysfunctional T cells. CAR T cell therapy may also result in side-effects such as GvHD (Graft-versus-host disease, worsening the AD symptoms), neurotoxicity, and cytokine release syndrome; CAR NK cell therapy may offer a safer alternative⁴. 

Despite the potential of CAR cell therapy in ADs, several challenges remain, including scalability, persistence, and off-target effects. Evotec's innovative approach to addressing these challenges involves the use of induced pluripotent stem cells (iPSCs) to generate allogeneic off-the-shelf CAR iNK cells with enhanced scalability and precision. iPSCs, reprogrammed from adult somatic cells, offer a potentially inexhaustible source of immune cells that can be genetically engineered and differentiated into various cell types to tackle a range of diseases.

iPSC-Derived CD19 CAR iNK Cells for Targeted B Cell Depletion

A study by researchers at Evotec investigated the performance of iPSC-derived CD19 CAR iNK cells as a novel therapeutic approach for ADs. Using Evotec’s validated GMP iPSC line, researchers produced genetically modified cells with a knock-in of CD19-CAR. The modified cells successfully differentiated into CD19 CAR iNK cells using a feeder-free 3D differentiation process, which could be confirmed by flow cytometry. The established protocol can ensure high purity and functionality of the resulting cells. Results showed that iNKs generated from the GMP iPSC line were homogenous and phenotypically comparable to blood-derived (BD) NK cells form healthy donors.  

Figure 1: Cytotoxicity against SLE patient B cells. NK killing assays of effector cells - iNK without CAR (WT), CD19 CAR iNK or healthy donor BD NK cells (BD-NK) co-cultured 1:1 (E:T) with SLE patient B cells, + 10μg/ml anti-CD20 antibody (Obinutuzumab (Obi)) or isotype control (Iso) (n=2). 

In vitro experiments demonstrated the cytotoxic effector function of CD19 CAR iNK cells in selectively targeting and eliminating CD19-positive B cells. Co-culture assays using patient-derived primary B cells from patients with SLE autoimmune disease, showed robust cytotoxicity of CD19 CAR iNK cells. The CD19 CAR iNK cells were more efficient than iNK cells without a CAR or BD NK cells in depleting the patient-derived primary B cells. These findings highlight the therapeutic potential of CD19 CAR iNK cells in treating ADs, offering a targeted and scalable alternative to conventional immunosuppressive therapies.

Evotec's Scalable Therapeutics Approach

Evotec's commitment to allogeneic cell therapy innovation is exemplified by its scalable therapeutics approach, which leverages cutting-edge technologies and infrastructure to develop next-generation therapies for ADs. Central to this approach is the use of iPSCs as a platform for generating CAR iNK cells with enhanced scalability and precision. By introducing CARs targeting CD19 into iPSCs and differentiating them into iNK cells, Evotec aims to create scalable and precise off-the-shelf therapies for ADs. 

Figure 2: Evotec’s pipeline to co-create iPSC-based cell therapeutics with partners in Inflammation & Immunology.

Evotec's end-to-end process for iPSC-based therapeutics encompasses differentiation, gene editing, preclinical and clinical development, ensuring the efficient generation and characterization of CAR iPSC-derived cells. By utilizing validated GMP iPSC lines and GMP-compatible differentiation protocols, Evotec ensures the safety and quality of its allogeneic cell therapy products, paving the way for clinical translation.

Figure 3: Evotec’s end-to-end process for iPSC-based therapeutics. 

The scalability of Evotec's approach enables the production of large quantities of CAR iNK cells, making them suitable for widespread use in treating ADs. Additionally, the precision of iPSC-derived CAR iNK cells allows for targeted and personalized therapies tailored to individual patients' needs, reducing the risk of off-target effects and enhancing treatment efficacy.

Future Potential with Evotec

Evotec's iPSC-derived CD19 CAR iNK cells represent a promising new approach to treating autoimmune diseases. By harnessing the power of iPSC and CAR technology, allogeneic cell therapy can help revolutionize the treatment landscape for ADs, offering patients a targeted, scalable and potentially curative alternative to conventional therapies. 

As research in this field continues to advance, Evotec remains at the forefront of allogeneic cell therapy innovation, driving the development of next-generation treatments for ADs (Figure 2). Evotec’s GMP-compliant production pipelines provides an efficient, reproducible, and scalable way to produce CAR iNK cells derived from iPSC for clinical development. With its commitment to precision medicine and scalable therapeutics, Evotec is well-positioned to meet the growing demand for effective and accessible off-the-shelf therapies for autoimmune diseases.

See more iPSC-based Cell Therapies for I&I Diseases

To discover more about this research, download our scientific poster

References: 

(1) Blache, U.; Tretbar, S.; Koehl, U.; Mougiakakos, D.; Fricke, S. CAR T Cells for Treating Autoimmune Diseases. RMD Open 2023, 9 (4), e002907. https://doi.org/10.1136/rmdopen-2022-002907.

(2) Jin, X.; Xu, Q.; Pu, C.; Zhu, K.; Lu, C.; Jiang, Y.; Xiao, L.; Han, Y.; Lu, L. Therapeutic Efficacy of Anti-CD19 CAR-T Cells in a Mouse Model of Systemic Lupus Erythematosus. Cell. Mol. Immunol. 2021, 18 (8), 1896–1903. https://doi.org/10.1038/s41423-020-0472-1.

(3) Müller Fabian; Taubmann Jule; Bucci Laura; Wilhelm Artur; Bergmann Christina; Völkl Simon; Aigner Michael; Rothe Tobias; Minopoulou Ioanna; Tur Carlo; Knitza Johannes; Kharboutli Soraya; Kretschmann Sascha; Vasova Ingrid; Spoerl Silvia; Reimann Hannah; Munoz Luis; Gerlach Roman G.; Schäfer Simon; Grieshaber-Bouyer Ricardo; Korganow Anne-Sophie; Farge-Bancel Dominique; Mougiakakos Dimitrios; Bozec Aline; Winkler Thomas; Krönke Gerhard; Mackensen Andreas; Schett Georg. CD19 CAR T-Cell Therapy in Autoimmune Disease — A Case Series with Follow-Up. N. Engl. J. Med. 2024, 390 (8), 687–700. https://doi.org/10.1056/NEJMoa2308917.

(4) Műzes, G.; Sipos, F. CAR-Based Therapy for Autoimmune Diseases: A Novel Powerful Option. Cells 2023, 12 (11), 1534. https://doi.org/10.3390/cells12111534.

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

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

Download the presentation

There is a sigh of relief as the new ICH M12 guideline for drug interaction studies is finally released. The new harmonised guideline, adopted in May 2024, is now likely to be implemented by all the major regulatory agencies who have been actively involved in its creation including the US FDA, the EMA and the Japanese PMDA. Previously, each authority had their individual DDI guidance all with distinct differences in protocols and interpretation, leading to practical challenges in designing and interpreting DDI studies to meet all the recommendations. The new ICH M12 aims to simplify the process by providing a single set of guidelines for the designing, conducting and interpreting metabolic enzyme- or transporter-mediated drug-drug interaction (DDI) studies. The guideline covers both in vitro and clinical DDI studies. It provides a consistent approach to replace existing recommendations from the main regulatory authorities.

The ICH M12 harmonised guideline is concentrated predominantly on small molecules. The DDI of biologics is only briefly covered with a focus on monoclonal antibodies and antibody-drug conjugates. Recommendations on how to address metabolite-mediated interactions and the use of model-based evaluations and DDI predictions are also included.

Watch out for our series of blogs on the key differences between the previous US FDA, EMA and Japanese PMDA guidance and the new ICH M12 and how you might be impacted by the changes. We are also busy updating our popular Everything you need to know about ADME and our DDI guides – we will let you know as soon as these are available!

Read the new ICH M12 guideline on drug interaction studies

We are on hand to assist you with designing, conducting and interpreting your DDI study according to the new ICH M12 guideline:

Get in touch

Inflammatory Bowel Disease (IBD) is the umbrella term for a group of diseases characterized by chronic, idiopathic and remitting inflammation of the gastrointestinal tract. Common symptoms include diarrhea, abdominal pain, and fatigue. However, extraintestinal manifestations such as inflammatory arthralgias/arthritides, primary sclerosing cholangitis (PSC), ocular or cutaneous involvement are often present and add to the complexity of the clinical picture.

The two most common forms of IBD are Crohn's disease and ulcerative colitis. Crohn's disease can cause inflammation anywhere in the gastrointestinal tract from the mouth to the anus, while ulcerative colitis is usually confined to the colon, where it can cause inflammation and ulceration. In about one in ten people, the two diseases cannot be clearly distinguished, and the condition is called indeterminate colitis. With approximately 6-8 million IBD patients worldwide (2 million in Europe and 1.5 million in North America), there is a huge unmet medical need.

IBD is an immune-mediated disease, but the exact causes are unknown. Crohn's disease, for example, involves a complex interplay of genetic predisposition, immune dysregulation, environmental influences, and microbial factors.

This complexity poses many challenges for drug development, as exemplified by the recent failure of a drug approved for ulcerative colitis that failed in late-stage clinical trials for Crohn's disease. The important lesson here is the need for proper patient stratification.

With its multimodal approach and patient stratification tools, Evotec is well positioned to develop innovative medicines. The Company is focused on development of IBD medicines through in-house research and through collaborations. Evotec’s strategy is modality agnostic, utilizing Evotec’s entire scope of technology platforms - from small molecules to biologics as well as iPSC-based cell therapies.

The Evotec approach

Drug discovery at Evotec always starts with patient data. Evotec's drug discovery efforts are based on its proprietary panOmics approach and a proprietary portfolio of molecular patient databases (E.MPD). panOmics combines both data generation and analysis platforms to industrialize OMICs data generation and AI/ML-based omics data analysis. Based on proprietary molecular patient data, panOmics fundamentally improves the understanding of disease processes, in vitro and in vivo disease modeling, identification of novel high value targets, biomarker discovery and patient selection.

Another integral aspect of drug development at Evotec is precision medicine. For this approach, Evotec has developed a comprehensive patient stratification toolbox via its panOmics-driven diagnostic approach – EVOgnostic.  

Therefore, IBD patients are an integral part of a pilot study in which Evotec is performing plasma and metabolomic analyses on samples from autoimmune disease patients to combine these clinical data with experimental data and data science approaches to identify potentially novel biomarker signatures.

Evotec aims to develop medicines that allow an early intervention and or, ideally, a cure for patients suffering from IBD. Evotec is engaged in several drug discovery programs tackling various aspects of IBD such as restoration of epithelial barrier function, modulation of inflammation, or resolution of intestinal fibrosis. Depending on the different aspects of the disease. Evotec’s experts are engaged in the IBD community and you can see our recent poster summarizing our IBD activities here. 

Selected collaborations

Evotec also is constantly seeking to enhance its capabilities through strategic investments and collaborations. For example, in 2022 it invested in IMIDomics Inc., a company focused on immune-mediated inflammatory diseases (IMIDs). IBD constitutes a large part of IMIDs. The aim is to jointly develop and use IMIDomics' Precision Discovery™ Engine. This technology enables a deep understanding of how inflammatory diseases work in patients. It applies a combination of clinical and computational expertise to clinical data and biological samples from more than 17,000 patients in a biobank, generating proprietary biomolecular signatures. 

With the Crohn's & Colitis Foundation, Evotec is advancing drug discovery for two novel IBD drug targets originating from academic research. The targets address fibrosis, the excessive accumulation of scar tissue in the intestinal wall, and the restoration of intestinal barrier function in IBD to reduce the increased intestinal permeability and chronic intestinal inflammation often seen in IBD patients.

Efficacy models in IBD

Another challenge in IBD is the lack of adequate animal models. While more than two dozen mouse and rat models of colitis have been developed and implemented, the multifactorial etiology and highly heterogeneous manifestations of the disease have prevented the development of a model that fully represents the pathophysiology of human IBD and related complications. Each mouse model has its strengths in elucidating the pathogenesis of colonic inflammation, fibrosis, or CAC, but each has a self-limiting nature and shows marked variability in drug development. While these IBD models cannot fully recapitulate the disease features commonly seen in humans (genetic and environmental influences, gut microbiota interactions, etc.), they have led to the identification of three key elements for disease etiology: T lymphocytes (T cells) mediate chronic intestinal inflammation; intestinal inflammation is initiated and maintained by certain commensal intestinal bacteria; the onset and severity of the disease is largely dependent on the genetic background.

Therefore, Evotec has carefully selected several preclinical models that recapitulate key aspects of IBD: inflammation, leukocyte trafficking, breakdown of epithelial barrier integrity, T cell-mediated injury. These models are routinely used and complemented by the current gold standard: the T cell transfer model of chronic colitis. This mouse model best reflects the clinical pathology observed in IBD and dissects the initiation, induction, and regulation of T cell-mediated immunopathology. 

In summary, Evotec is advancing breakthrough solutions for IBD using the most advanced technologies and platforms available. The Company's primary focus is on precision medicine and leading-edge approaches with the goal of providing tailored, effective, and minimally invasive treatments by taking into account the unique characteristics of each patient.

Download our poster from the IBD Innovate Conference

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

WATCH ON DEMAND

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