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.
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