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.
Tags: Neuroscience, Respiratory, Oncology, Kidney diseases, Women's health, Presentations, Blog, Formulation & CMC, Biologics, Age-Related Diseases, IND Enabling Studies/Preclinical Development, Anti-Infectives, Immunology & Inflammation, Metabolic Disease & Complications, Rare Diseases, Clinical Development
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:
Tags: Blog, ADME/DMPK, IND Enabling Studies/Preclinical Development
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.
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.
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.
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.
Tags: Induced pluripotent stem cells, Blog, In vitro Biology, In vivo Pharmacology, Immunology & Inflammation
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:
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:
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.
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
Tags: Articles & Whitepapers, Blog, In vitro Biology, Proteomics, Metabolomics & Biomarkers, Age-Related Diseases, Clinical Development
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.
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.
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 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.
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
Tags: Oncology, Induced pluripotent stem cells, Blog, Biologics, In vitro Biology
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.
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.
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.
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
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
Tags: Oncology, Induced pluripotent stem cells, Blog, Biologics, In vitro Biology
In the realm of drug discovery, unveiling the intricate interactions between bioactive compounds and cellular targets is paramount. Evotec leads the charge with its pioneering chemical proteomic applications, aimed at target deconvolution and selectivity profiling.
At the heart of Evotec's approach lies Cellular Target Profiling™, an unbiased and proteome-wide methodology that meticulously identifies and quantifies compound interactions with both on- and off-targets within the cellular milieu. Leveraging high-end quantitative mass spectrometry, this platform offers unparalleled insights into specific cellular targets, enabling precise target identification and determination of target-specific dissociation constants.
Central to this chemical proteomics approach is photoaffinity labelling coupled with mass spectrometry, allowing for the covalent capture of target proteins within live cells. This technique not only identifies target proteins but also visualizes compound-target interactions, shedding light on binding site locations within protein targets and complexes.
Evotec's chemical proteomic arsenal extends beyond target deconvolution to encompass diverse small molecule compounds. Activity-Based Protein Profiling (ABPP) offers a comprehensive view of enzyme classes, while KinAffinity® provides rapid target profiling of kinase inhibitors in cell and tissue samples. Unlike traditional biochemical kinase panel screenings, KinAffinity® evaluates inhibitors' target affinities across a spectrum of native kinases within their physiological cellular environment, facilitating hit-to-lead optimization with unprecedented precision.
With a track record of success in profiling various compounds, Evotec's expertise in quantitative proteomics stands as a beacon innovation in drug discovery. For those seeking advanced insights into target deconvolution, drug selectivity and activity profiling, Evotec's experts are posed to offer tailored solutions.
Cardiotoxicity is one of the leading causes of drug attrition. To address this, there is a need for improved predictive screens which can be applied at an early stage in drug development to ensure only safe compounds progress to the clinic.
Drug-induced cardiotoxicity can manifest itself via various different mechanisms which makes detection challenging. Either functional or structural changes can occur, and these effects can be direct or indirect. Functional cardiotoxicity results from acute alteration in the heart function often as a consequence of electrophysiological effects. Structural cardiotoxicity is associated with alterations in cell and tissue morphology and can manifest itself as cell death, inflammatory changes and fibrosis. These morphological changes can take time to present themselves clinically and, therefore, sensitive techniques which can detect early molecular changes are valuable from a predictive perspective.
Combining transcriptomics with artificial intelligence is showing great potential in providing this transformative improvement in predictive safety assessment. In fact, the value of this approach in drug-induced liver injury (DILI) has been demonstrated in a recent poster presented by Cyprotex. At SOT in Salt Lake City from 10-14 March 2024, Cyprotex showcased how this technique can also be applied to cardiotoxicity. The research evaluated 42 compounds (33 cardiotoxicants and 9 non-cardiotoxicants) used in a variety of therapeutic indications. The compounds were assessed at 2 time points and 8 concentrations in a beating cardiac organ model using human iPSC-derived cardiomyocytes. At the end of the incubation period, three different analytical methods were compared; high content screening (HCS) (cell count, cellular ATP, mitochondrial mass, mitochondrial membrane potential, cellular calcium levels, DNA structure and nuclear size), calcium transience (wave amplitude, frequency, full peak width and full decay time) and transcriptomics (high-throughput RNA sequencing).
The HCS and calcium transience assays were valuable in detecting structural and functional cardiotoxicants, respectively. However, the synergism of combining these assays with transcriptomics was demonstrated by an overall improved cardiotoxicity risk prediction. Additionally, transcriptomics analysis provided detailed mechanistic information and identified specific pathway responses. Combined, the three approaches (HCS, calcium transience and transcriptomics analysis) gave excellent cardiotoxicity prediction metrics of 100% specificity, 82% sensitivity and 86% accuracy at 10x Cmax and 89% specificity, 91% sensitivity and 90% accuracy at 25x Cmax. The transcriptomics analysis improved the overall sensitivity by identifying various molecular mechanisms of structural toxicity such as alterations in cardiac pathways, genotoxicity, ER stress and mitochondrial toxicity.
It is not just cardiomyocytes which can be affected by drug induced toxicity, non-cardiomyocytes such as fibroblasts and endothelial cells may also be impacted. Organotypic models developed using different cell types, therefore, are likely to be more representative both structurally and functionally. In the future, Cyprotex is extending its research to evaluate a number of different cell-based models and different organ-specific toxicities using the transcriptomics approach. Our cardiac safety database is also growing and we now have identified approximately 140 compounds for testing in our models. The use of transcriptomics and artificial intelligence is accelerating the development of new cell-based models in the field of drug-induced toxicity leading to a new paradigm in in vitro safety testing.
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The endocrine system comprises of a network of glands and organs that produce and release hormones to control various bodily functions. Endocrine disruptors, either natural or man-made, can affect the endocrine system by interfering with the normal actions of hormones. This can lead to serious health problems such as cancer, birth defects and developmental disorders. Many of the traditional endocrine disruption testing approaches use radiolabelled material or involve animal models and so have high cost and low efficiency, in addition to ethical concerns. Accurate low-cost screening models are required to flag up chemicals which are potential endocrine disruptors at an early stage.
At the Society of Toxicology (SOT) conference on March 10-14, 2024, Cyprotex presented a poster titled, ‘Establishment of a High Throughput Endocrine Disruptor Screening Panel of Assays for Rapid Screening of Chemicals’. The research developed a panel of 384 well high throughput cell-based hormone activation assays and in vitro hormone receptor binding assays with a 24-48 hr turnaround time. A range of chemicals with different potencies for the endocrine receptors were assessed.
Cell-based endocrine receptor activation assays expressing the human androgen receptor (AR), the human estrogen receptor (ERα or ERβ) or the the human thyroid hormone receptor (TR) were used to assess the chemicals at a range of concentrations at 37°C over 24 hr. Upon ligand activation, the hormone receptor binds to the promoter sequence linked to a luciferase reporter gene and the activation is quantified by luminescence. Cell viability was also assessed in parallel.
Hormone receptor competitor binding assays were also used to assess potential AR or ER ligands by measuring fluorescence polarization. A fluorescently-tagged ligand is added to the AR or ER in the presence of the competitor test chemical. If the test chemical binds then it prevents the formation of the fluorescent ligand/receptor complex and a decrease in polarisation is observed. The extent of the shift in polarization was used to determine the relative affinity of the test chemical for the hormone receptor.
The results observed for the known set of chemicals were consistent with literature values and data corresponded well between the two assays. Additional steroidogenesis assays are being developed to detect hormone levels of AR and ER in H295R cell culture supernatants following exposure to endocrine disruptors.
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