Today at NecstGen, we had the pleasure of meeting with Assistant Professor Françoise Carlotti from Leiden University Medical Center to discuss our collaboration within the broader RegMed XB U(iP)Scale project, which recently received funding. A central goal of the RegMed XB Diabetes Moonshot is to leverage induced pluripotent stem cells (iPSCs) to drive forward regenerative medicine solutions for Type 1 diabetes.
Type 1 diabetes is an autoimmune disorder caused by the destruction of insulin-producing beta cells, which impairs the body’s ability to control blood sugar levels. Without proper intervention, this condition can result in severe health complications and severely impact the quality of life for patients. At present, disease management relies on lifelong administration of external insulin via injections or pumps, alongside continuous monitoring of glucose levels. An iPSC-derived therapy has the potential to restore the body’s ability to produce insulin to control blood sugar levels, ultimately rendering external insulin administration obsolete.
The U(iP)Scale project has two main objectives: scaling up the manufacturing of insulin-producing islets derived from iPSCs and improving the design of a first-generation open-delivery device. Building on insights from previous large-scale preclinical and surgical studies, these efforts aim to support the successful translation of this technology into clinical applications.
At NecstGen, our role is to demonstrate the reliable scale-up of stem cell-derived islets, building upon the pioneering research led by Françoise Carlotti’s team. By using bioreactors, we aim to facilitate the scalable manufacturing of regenerative therapies. First, we will undertake technology transfer into the process development laboratories of NecstGen, where our team will collaborate with LUMC to create a cost-efficient manufacturing process resulting in a clinically relevant scale for regenerative therapy.
This project represents a significant step toward creating a sustainable and scalable source of insulin-producing cells, providing renewed hope for people living with Type 1 diabetes.
Partners: Leiden University Medical Center, Maastricht University, NecstGen, DON, Diabetesfonds
Pan Cancer T, a preclinical biotech focussing on the development of TCR-T cells, has extended its collaboration with Leiden ground NecstGen to include the clinical development and manufacturing of retroviral vectors. The parties have agreed to collaborate on the development of a retroviral vector manufacturing and testing process. The process will enable Pan Cancer T to utilise its licenced, stable producer technology to supply clinical vectors. NecstGen will develop a suitable, scalable manufacturing and testing procedure, establish a Master Cell Bank of the producer cell line, and be responsible for the clinical supply of viral vectors expressing the proprietary PCT-1 COSTIM TCR construct. The vector is to be used for TRC-T cell production in trials targeting Triple Negative Breast Cancer and Multiple Myeloma. This agreement follows an earlier agreement between Pan Cancer T and NecstGen, in which a commercially viable TCR-T cell production process was arranged.
Reinout Hesselink, VP of Process Development and CMC of Pan Cancer T says: We are very glad we have been able to extend our relationship with NecstGen to include the viral vector process. As both our vector- and our cell process are developed at the same CDMO, this agreement gives us the opportunity to develop a lean supply chain. This should speed up our clinical manufacturing capabilities.
The fact that we will be using a stable producer cell line, which allows for serum-free suspension culture, means that we will have a saleable, cost-efficient manufacturing process for one of our most critical materials.
Paul Bilars, CEO of NecstGen BV, adds that this agreement confirms our position as a go-to CDMO for cell and gene therapies. After assisting Pan Cancer T with their cell manufacturing process, we will now assist them with establishing their viral vector process.
This will be an interesting vector manufacturing process for us, and our technical capabilities as a CDMO, not only for cellular drug products but also for vector products, will be useful. We can run the process in our vector unit at different scales supporting preclinical, clinical and possibly commercial supply.
On Pan Cancer T
Pan Cancer T is an immunotherapy company developing next-generation TCR-T cell therapies for solid cancer.
The Company´s products are based on a form of T cell therapy called TCR-T therapy. It exploits the abilities of T cells to recognise and kill tumour cells. Pan Cancer T has developed a highly specific TCR targeted against Rophorin-1, which is highly expressed in tumour tissue of patients suffering from Triple Negative Breast Cancer and Multiple Myeloma. It has also developed a proprietary costimulatory molecule to enhance the activity and longevity of the TCR-expressing T cells.
Pan Cancer T´s differentiated Next Generation approach thus addresses two major obstacles in the field. Firstly, we work on a unique set of 30 untapped targets, exclusively and robustly expressed in multiple solid cancers. Secondly, our augmented T cells are engineered for enhanced durability in order to drive deeper and more durable clinical responses in multiple solid tumours.
On NecstGen
The Netherlands Center for the Clinical Advancement of Stem Cell and Gene Therapies (NecstGen) comprises a state-of-the-art facility for development and GMP manufacturing. NecstGen additionally offers cleanroom rental to allow organisations to maintain control of production while de-risking the capital investment required.
NecstGen is open to the world and supports academic, small, and large industrial therapy developers to translate research and early-stage clinical programs into patient treatments. One of NecstGen’s primary aims is to support the development of therapeutics for orphan diseases that may be underserved but are nevertheless catastrophic to people who suffer from them.
NecstGen is a mission-driven CDMO owned by Leiden University Medical Center (LUMC). This large-scale investment to support academia and industry was undertaken to address the barriers to entry for the creation of next-generation therapeutics, including:
Investment: The high cost of capital equipment, facilities, research, and development.
Limited capacity: Market availability of development and manufacturing slots.
Know-how: Knowledge, expertise, and talent acquisition.
Intellectual property: Complex ownership and control of the technologies required to realise new therapies successfully.
If you missed our recent webinar on Reducing Lentivirus Costs: A DOE Case Study, you can now watch the replay and gain valuable insights into cost-efficiency strategies for Cell & Gene Therapy manufacturing.
Lentiviral vectors (LV) play a critical role in CAR T therapies, but the high cost of plasmid DNA used in transfection-based production remains a significant challenge. During the webinar, Diederik Lokhorst, Viral Vector Specialist at NecstGen, shared how our team leverages Design of Experiments (DOE) to address this challenge, reduce the Cost of Goods (COG), and make lentivirus-based therapies more affordable.
What You’ll Learn in the Replay: – An inside look at transfection-based lentivirus production
– DOE as a tool to understand and improve your manufacturing process
– A Case study applying DOE to reduce LV Manufacturing Costs for CAR T
NecstGenis hosting a CGT Circle event on October 16th at our site in Leiden supporting The CGT Circle‘s mission to harness the collective power and experience of women in the field of Cell and Gene Therapy.
Join for an afternoon with brilliant speakers sharing insights on their career, lessons learned, and valuable advice and connect over networking drinks.
Don’t miss out on this event and use the registration link below to sign up.
Chimeric Antigen Receptor (CAR) T cell therapy represents a transformative approach in medicine, particularly in oncology. This method offers personalised treatment by harnessing a patient’s immune system to target and eliminate (cancer) cells. These novel therapies undergo a meticulous journey from target discovery to clinical application, starting with three fundamental steps: target identification, CAR design, and preclinical testing. Here, we want to focus on these initial steps, emphasising their crucial role in developing efficacious and safe CAR T cell therapies.
Target Identification
The basis of efficacious CAR T cell therapy is in selecting an appropriate target antigen for respective target cells. The target antigen must meet several criteria to ensure the therapy’s efficacy and safety.
Target Antigens in CAR T Cell Therapy
An antigen is any substance that causes the immune system to raise a specific immune response against it. In CAR T cell therapy, CARs are used to redirect T cells to recognise and eliminate cells expressing a specific target antigen. The target antigen must be highly specific to the target cells to avoid unwanted side effects such as the killing of healthy cells and tissues, which can be a side effect of the treatment. One significant target antigen that has been approved by the EMA (European Medicines Agency) is BCMA (B-cell Maturation Antigen). CAR T cell therapies like Abecma (Idecabtagene Vicleucel) and Carvykti (Ciltacabtagene Autoleucel) have been approved for treating relapsed or refractory multiple myeloma. These therapies modify T cells to target BCMA, which is commonly expressed on multiple myeloma cells. (Source)
Antigen Selection Criteria for CAR T Cell Therapy
The ideal target antigen should be:
Highly Expressed on Cancer Cells: Ensuring that the therapy targets most cancer cells.
Minimally Present on Healthy Cells: Reducing the risk of off-target effects and associated toxicities.
Uniformly Expressed Across Cancer Cells: Providing consistent targeting of all malignant cells. However, marker expression by (solid) tumours may change over time leading to tumour escape from the therapy.
Understanding the characteristics of a potential therapy, including safety, efficacy, and toxicity profile of a CAR T cell product as early as possible is essential for guiding strategic decisions and helps de-risking investment in further clinical development.
Assessing whether a specific antigen is (or is not) expressed on a specific tissue can be done using these methods:
In Silico Analysis: Bioinformatics tools analyse gene expression data to predict potential antigens highly specific to cancer cells.
In Vitro Studies: Laboratory experiments using cancer cell lines and patient-derived cells help confirming antigen.
In Vivo Models: Animal studies, typically in mice, help assessing whether the antigen is expressed on a specific tissue.
Chimeric Antigen Receptor (CAR) Design
The next critical step is generating the CAR construct.
Components of a CAR
A CAR consists of four primary components:
Extracellular Domain: Derived from a monoclonal antibody, this domain binds to the target antigen on cancer cells.
Hinge Region: Provides flexibility to the CAR, facilitating effective binding to the antigen.
Transmembrane Domain: Anchors the CAR to the T cell’s surface.
Intracellular Signalling Domain: This part of the CAR includes co-stimulatory domains (CD28 or 4-1BB) and an activation domain (CD3ζ), which trigger T cell activation and proliferation upon antigen binding.
A schematic overview of a CAR can be found in the figure below.
Figure 1. Schematic overview of a CAR. The schematic overview shows key components. The extracellular antigen-binding domain binds to the target antigen on cancer cells. The hinge region, a flexible segment, links this domain to the transmembrane domain, aiding positioning and motility. Within the antigen-binding domain, the extracellular domain identifies the specific antigen. The transmembrane domain anchors the CAR in the T cell membrane, while the intracellular signalling domain transmits activation signals upon antigen binding, triggering the T cell’s response.
Engineering and Optimisation of CAR T Cells
The process of engineering and optimisation of a CAR begins with synthesising the CAR gene. This gene is then incorporated into for example a viral vector, often a lentivirus or retrovirus. Introducing the CAR gene into T cells by using a viral vector is called viral transduction. The engineered CAR is then tested in cell lines to confirm its functionality. The next step is the optimisation of the CAR T cells, which includes but is not restricted to:
Affinity Tuning: Adjusting the binding strength of the CAR to the antigen to achieve a balance between efficacy and safety.
Signal Modulation: Enhancing the T cell’s response to ensure effective cancer cell destruction without causing excessive activation that could lead to toxicity.
Figure 2. Overview of CAR T cell therapy development. The process starts with harvesting peripheral blood cells from the patient. Then, mononuclear cells are isolated from the blood. If needed, the T cells are isolated from the mononuclear cells. These (isolated) T cells are then genetically modified to express chimeric antigen receptor (CAR) that target specific (cancer) cells. Following activation of the T cells, [Mv1] the insertion of the CAR gene is performed using a viral vector (viral transduction) or non-viral transfection methods. The modified T cells are expanded to clinically relevant numbers. Only after completing quality control testing and certification by a Qualified Person (QP), the CAR T cells can be administered to the patient, providing for a targeted and personalised treatment.
Preclinical Testing
Preclinical testing is used for the evaluation of CAR T cells in controlled environments to ensure their efficacy and safety before proceeding to a first in human clinical trial. The efficacy and safety of CAR T cells are tested using in vitro and in vivo methods.
In Vitro Testing
During in vitro testing, various standard assays are performed to understand more about the characteristics of the engineered CAR T cell, such as its cytotoxicity, proliferation, and cytokine secretion levels.
Cytotoxicity Assays: Assessing the CAR T cells’ ability to kill target cells and to leave non-target cells untouched.
Proliferation Assays: Measuring the expansion capacity and survival of CAR T cells upon activation by the target antigen.
Cytokine Secretion Assays: Evaluating the secretion of cytokines as an indication for T cell activation and potential toxicity.
In vivo TestingFollowing successful in vitro testing, CAR T cells are evaluated in vivo using clinically relevant animal models:
Toxicity Studies: These studies assess potential side effects and determine the maximum tolerated dose, which is a challenge as the weight of a mouse is only about 25 grams.
Efficacy Studies: Animal models are used to test the CAR T cells’ ability to effectively reduce the tumor burden and improve survival. The safety profile and mechanism of action of the CAR T cell therapy are being assessed.
Regulatory Compliance
Preclinical studies must comply with Good Laboratory Practice (GLP) standards to ensure reliability and reproducibility of the experiments. Comprehensive documentation of findings is crucial for future regulatory submissions, such as an Investigational Medicinal Product Dossier (IMPD) application.
As an academic institution or company aiming to develop a CAR T cell therapy, collaboration with a Contract Development and Manufacturing Organization (CDMO) can be invaluable. CDMOs offer specialised expertise, resources, and capacity, that contributes to streamline the development process, enhance scalability, and ensure regulatory compliance.
Conclusion
The first stage of CAR T cell therapy development, encompassing target identification, CAR design, and preclinical testing, is fundamental in contributing to the therapy’s safety and efficacy. By meticulously planning and executing each step, therapy developers aim to bring safe and effective CAR T therapies from the laboratory to the patient.
NecstGen is a non-profit CDMO and Centre of Excellence for Cell and Gene Therapy located in a purpose-built GMP facility in Leiden, The Netherlands. Dedicated to the field of Cell and Gene Therapies, we provide expertise and capacity.
