The First Stages of CAR T Cell Therapy Development

The First Stages of CAR T Cell Therapy Development

From Discovery to Preclinical Development: What are the critical stages to look out for?

Introduction

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.

https://www.ema.europa.eu/en/news/meeting-highlights-pharmacovigilance-risk-assessment-committee-prac-8-11-january-2024#ema-inpage-item-64904

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.

        A schematic overview of a Chimeric Antigen Receptor (CAR)

        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, 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 Testing

             

            Following 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.

              Cell and Gene Therapy companies in Europe

              Cell and Gene Therapy companies in Europe

              European Cell & Gene Therapy Companies

              European Landscape For Cell & Gene Therapy companies

              At NecstGen, we aim to support industry partnerships and growth. And to enable the next generation of therapies, it is pivotal to have oversight of the gene and therapy landscape to start from, which is why we created this helpful tool. Feel free to use and share it, so together, we challenge today’s possibilities and enable the unthinkable.

               

              Did we miss your organisation? 

              The biotechnology business is booming around the globe. Increased private equity investments in biotech, global cross continent acquisition deals, and IPOs have risen to record levels. This boom in development will enable the global biotech industry to surpass its older sibling, the pharmaceutical industry, in interest. One area of biotech that holds a particularly great promise to meet patients’ unmet needs is Cell and Gene Therapy. Such therapies defined as ATMPs (Advanced Therapy Medicinal Products) have a substantial therapeutic potential to treat the patients that current treatments may fail. Although their development can be complex, this is not holding back drug developers, innovation continues in leaps and bounds. But where are most Cell and Gene Therapy developers located? This article and map share insights into the ATMP landscape in Europe and the existence of several hubs

              Cell & Gene Therapy Companies in the United Kingdom

              With three hotspots in the UK, the Britons lead as Europe’s biotech hub for breakthrough life-science start-ups. McKinsey wrote a fascinating report covering how this hub has matured relative to its peers and what lies ahead on its road to playing a leading role in the Cell and Gene Therapy sector globally.

              The report based its results on a Biotech Innovation Index, which assessed the biotech sector on discovery, translation, growth capital, and various impact indicators. Although they do not directly target ATMPs in the study, the large hubs you can see on the map here definitely confirm their findings.

              In the UK, most Cell and Gene Therapy companies are located in one of these three clusters:

              Cell & Gene Therapy Companies in the Netherlands

              The Netherlands has several biotech hubs, all connected through a strong infrastructure network despite its size. Its central location in Europe makes it no surprise that EMA relocated its headquarters to Amsterdam following Brexit in 2020. The Netherlands is also home to several knowledge centres with extensive capacity in Cell and Gene Therapies

              Investments into the Leiden University Medical Center (LUMC) have accelerated patient access to innovative Cell & Gene Therapies, including stem-cell therapies. NecstGen is a fruit of those investments and supports organisations worldwide to develop novel therapies for patients. In addition, Utrecht is home to the Utrecht Cell Therapy Facility: a hub for ATMP development specialised in Cell and Tissue based therapies.

              In the Netherland, most Cell and Gene Therapy companies are located in these clusters:

              Switzerland

              Switzerland is known for being the home country of pharmaceutical giants Roche and Novartis and many other leading biotech companies. Together, the combined sector contributes over 40% of Swiss exports. Over 300 biotech start-ups are located in the country and specialise in diverse fields such as oncology, antibodies, and orthopaedics. It’s not a surprise that multiple Swiss companies have already worked on advancing their presence in Cell & Gene Therapy.

              Novartis opened its Cell and Gene Therapy facility back in 2019, for manufacturing Kymriah. Last month, Cytiva opened its new manufacturing facility in Grens (Link) and served as the base of operations regarding their Cell and Gene Therapy-related operations and a training centre for European customers.

              Swiss Biotech companies are located around the following three clusters:

              Italy

              The Italian biotechnological sector is concentrated around the northern parts of the country. The North-Western part of Italy is where companies specialising in drug development and new therapeutic approaches appear to be located, including for Cell & Gene Therapies.

              Italy’s ATMP-specific service provider market ranks third  in Europe. Therefore, it may not also surprise that some of the earliest advanced therapies and four out of seventeen authorised for the European market originate from Italian academic research. Nature published an article on the challenging yet promising future of ATMP development in Italy.

              In Italy, most biotech companies specialised in Cell and Gene Therapy are located in Milan and surroundings.

              Germany

              Even though there is no clear hotspot in which Cell and Gene Therapy-focussed companies are settled in Germany, the sector has seen immense growth. This builds on the underlying strength of Germany in R&D and manufacturing  in Pharma. The country is home to 660 biotech companies with a total of 50,000 employees. Of those 660 companies, many are focused on Cell & Gene Therapy.

              In 2021, more than 29 active clinical trials evaluating CAR-modified immune cells took place, most of which involved CAR-T cells. In Germany, over 50 clinical studies have been conducted in Gene Therapy, making Germany a country heavily involved in the development of Cell & Gene Therapies. Nature published an interesting report on the past and future of Gene Therapy in Germany.

              The German Cell and Gene Therapy companies are not clustered, but are located all throughout the country, demonstrating the broad strength of innovation locally.

              France

              Named as one of the best three biotech centres in Europe by McKinsey, France is home to 720 biotech companies with a combined workforce of over 50,000 employees.France has a thriving academic environment, which can be seen from the number of biotech firms backed by academic research, which was forty-six percent iin 2017.

              It may come as no surprise that France is one of the leading countries in Cell and Gene Therapy research. The French government and biotech companies invested in Gene Therapy development early on, and it paid off. More than 10-world leading biotech companies and research groups operate from France, such as Genethon. While most companies and institutes involved in Cell and Gene Therapy development, such as Institut Imagine or Institut Vision are located in Paris and its surroundings, there are several other biotech hubs in France.

              Sweden

              Sweden definitely has the ambition to rank globally as a Cell and Gene Therapies player. They share the aspiration of the countries mentioned above to provide patients with innovative treatments and have even started a program specifically tailored to ATMPs, Vinnova. By following this programme, Sweden aims to be a leader in advanced therapies by 2030!

              The complete program involves partners, including research companies, universities, the Swedish Medical Products Agency, patient representatives, and the pharmaceutical industry’s trade association. Therefore, we can consider their combined perspectives and goals the driving force behind the accelerated, developing landscape ATMPs.

              We can find a hotspot of ATMP companies located around the Ideon Science Park in Lund

              Belgium

              Finally, we mention Belgium on our list of hotspots for Cell and Gene Therapy developers. The Belgium biotech industry is thriving, with more than 140 operating biotechs despite its small size. Known for its vivid biotech landscape, Belgium is also developing their share in ATMP development.

              Compared to surrounding countries, large companies always favoured Belgium as a country for their manufacturing operations. Shortly, UCB plans to open up a new gene therapy facility in Braine-l’Alleud, and we can expect it to be operational in 2024.

               

              Related Questions

              Which Cell Therapies are approved?

              In these figures, we gathered and visualised overviews of approved ATMPs over the past years for you.

              What does the Cell Therapy Development process look like?

              From idea to treatment, you’ll face changing requirement and development challenges. View the figure to see how knowledge of the process inversely relates to freedom to make changes to your process.  

              Our experts are only a message away to help you understand the impact of any of these aspects and make informed decisions on outsourcing.

              We’d be happy to discuss and help you bring cell therapies to patients.

              Cell and Gene Therapy Companies in Europe

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