Lab Meeting: Maria Grazia Roncarolo
"Tr1 cells have the potential to not only provide symptom relief, but also reset the immune system to provide cures.”
The human immune system lends itself to militaristic description.
Within our bodies, microscopic “soldiers” fight a winner takes all battle between host and pathogen. When these “troops” are missing in action, chaos ensues: “During my rotation in pediatrics, I was exposed to children with primary immunodeficiencies. We didn't have any therapeutic options for these families,” remembers Dr. Maria Grazia Roncarolo, the George D. Smith Professor of Stem Cell and Regenerative Medicine at Stanford, and co-founder and President/Head of R&D at Tr1X.
Decades of careful research have shown that our immune soldiers are highly specialized. Some can attack and engulf pathogens directly, whereas others kill cells invaded by virus, or produce soluble factors that can protect against future assaults. Patients with rare genetic mutations can lack entire regiments of their immune army. For example, patients with ADA-severe combined immunodeficiency disease (SCID) are deficient in lymphocyte populations like B-, T- and NK cells. Over nearly two decades (working across France, Italy and the US), Roncarolo pioneered therapies for those with devastating rare diseases like ADA-SCID and Wiskott Aldrich Syndrome: “I helped create a relatively small institute in Italy, which then developed a huge pipeline for rare genetic diseases. This work led to two drugs now on the market [Strimvelis and Libmeldy] …this was a major milestone for gene therapy,” she says.
In addition to developing gene therapies for patients with immunodeficiencies, Roncarolo has made fundamental contributions to the field of immune tolerance. Intense immune battles can lead to collateral damage when healthy tissues are inadvertently exposed to toxic cytokines or destructive cells. In cases of transplant rejection or autoimmune disease, white blood cells attack healthy tissues directly. “[In autoimmunity] the immune system is totally dysregulated…multiple cell types are causing damage,” says Roncarolo. “What is needed is a system reset.”
Enter regulatory T cells (Tregs); peripheral emissaries whose job is to quiet the troops or kill unruly effector cells that refuse to stop fighting. Increased Treg activity is associated with immune tolerance, whereas loss of these cells can induce autoimmunity.
“We noticed that in some patients, despite mismatched stem cell transplants, there was tolerance [of the graft],” describes Roncarolo. In the 1980s, her group showed that in patients with mismatched grafts—transplants that the immune systems should attack—some individuals evaded rejection. Roncarolo isolated cells from these patients and hypothesized that they might drive tolerance. Working at DNAX in the 1990s, Roncarolo’s group showed that the cells from her patients were CD4+ T cells and produced extremely high levels of IL-10. Thus, they were the first to characterize type 1 regulatory T (Tr1) cells—a Treg subset that lacks expression of the classical FOXP3 marker.
Since their initial description, Tr1s and other Tregs have held therapeutic promise for conditions where the immune system targets healthy tissues. While Tregs have been effective in animal models of inflammatory bowel disease (IBD), type 1 diabetes (T1D) and graft versus host disease (GvHD), isolation and persistence of these cells after adoptive transfer in humans has been challenging in clinical trials.
Enter Tr1X (pr: “Trix”), a venture seeking to harness the power of Tr1 cells to combat GvHD, IBD and other autoimmune diseases. “Tr1 cells suppress immune activation through IL-10, which has pleiotropic functions…in theory these cells can reset the immune system in a way that other Tregs cannot,” describes Roncarolo. Co-founded by Roncarolo, David de Vries, Jan de Vries and Jonathan Perrin, Tr1x has developed a proprietary method to create high purity allogeneic Tr1 and CAR-Tr1 cells. These “off the shelf” solutions aim to provide enhanced durability and immune modulation, compared to classical Tregs. Importantly, their scalable production may (one day) expand patient reach: “current autologous Tregs, CD19 or BCMA effector cell therapies require a huge effort to collect and manufacture…we think we can modulate both B and T cells with our products and eventually provide access to more patients,” says Roncarolo. In April of this year, Tr1X announced IND clearance to test its lead Treg asset (TRX103) in Ph1 trials of patients with GvHD. The company also completed a $75M series A financing led by The Column Group in 2023, which will enable further development of their Treg pipeline. This includes an IND for TRX103 in Crohn’s (H2 2024) and a CAR-Tr1 asset (TRX319) for B-cell driven autoimmune disease.
Roncarolo has already changed the game for patients suffering from rare genetic disorders. With the team at Tr1X, she is building upon decades of work in immune tolerance to expand the reach of cell therapy to patients suffering from autoimmune attack. Yet battling a dysregulated immune system isn’t easy. Roncarolo knows from experience that only human testing will reveal whether they are winning the war: “The goal is to show that our cells can truly work from within: that they can reset the patient's immune system, activate endogenous regulatory cells and secure long-term remission or cure.”
Below is an interview with Dr. Maria Grazia Roncarolo from May of 2024:
What got you interested in science and medicine initially?
Despite coming from a family of businesspeople, I always knew I wanted to be a doctor. I can't explain why, but it was a dream I carried with me from a young age. When I was in junior high school, I started volunteering on an ambulance crew, which deepened my desire to pursue a career in medicine. I never shared this dream with my father because he had already charted out clear paths for each of his three daughters. My destiny, according to him, was to become a chemical engineer. He envisioned me working in our family-owned business, which produced industrial paints. For the longest time, I respected his wishes and kept my medical aspirations to myself. During my last year of high school, my father passed away unexpectedly. After his passing, amidst the grief and turmoil within the family, I was also torn between honoring my father's plan for me and following my own dream. In the end, it was my mother and sisters who gave me the courage to pursue medicine. They told me to follow my calling and enroll in medical school, reassuring me that they would manage the family business. In Europe, you can go straight to medical school after high school, which means six years of study plus one year of internship. Thus, while my sisters took on roles in the company, I embarked on my medical journey at the age of 18.
What was medical school training like in Europe?
The initial years of medical school in Europe were quite basic: physics, chemistry, and mathematics—foundational topics that are comparable to what you would study in [a US] college. By the end of the second year, we started to learn physiology and biochemistry. By the fourth year, we started studying pathology. I remember at the time, pathology came as a shock to me. The approach was highly empirical, lacking a lot of the precision and clarity we have today because of tools such as genomics, precision medicine, or biomarkers. Diagnoses were based on patterns and correlations that could fit multiple diseases, making the process feel more like guesswork than science. I found this deeply disappointing. I realized I wanted evidence-based medicine, not medicine based on empirical observations and correlations. The experience inspired me to pursue research. I began working in a lab at the start of my fifth year of medical school. It was during my pediatrics rotation that I found my true calling and research focus. I encountered children with primary immunodeficiency, often referred to as "bubble boys." These children suffered from severe combined immunodeficiency (SCID), a condition we now know encompasses over 37 different diseases under a single clinical phenotype. At that time, treatment options were extremely limited. Bone marrow transplants were just beginning to be explored, and my medical school had not yet adopted this practice. Seeing these children, and the lack of therapeutic options available to them, was heart-wrenching. We had to tell parents that we did not have any curative solutions. This experience solidified my decision to focus on pediatric immunology. I wanted to find solutions, not just manage symptoms. Thus, at this point in my training, I decided to pursue research in pediatric immunology and complete my residency in pediatrics, followed by a fellowship in clinical immunology. This path into pediatric immunology was serendipitous, yet it felt like the perfect fit. The desire to help those children and find solutions to their conditions guided me through my career, shaping my work in cell and gene therapy.
[During your medical training did you get exposure to research?]
During my first year of residency in pediatrics, I was fortunate enough to win a prestigious grant that allowed me to spend a year doing research abroad. Initially, I considered going to New York's Memorial Sloan Kettering Cancer Center, but ultimately decided to go to France. This grant not only supported my salary but also covered some of my research expenses. With special permission, I went to Lyon, France, for what was supposed to be a 1-year research stint. That one year turned into 15 months, during which I immersed myself in groundbreaking research. It was a period of intense learning and growth, both professionally and personally. Upon completing my residency, I faced an exciting yet challenging decision. I had enrolled in a fellowship program in Clinical Immunology at the University of Milan, but at the same time, my French mentor offered me an assistant professor position in Lyon. Determined to take advantage of both opportunities, I received special permission to pursue both simultaneously. This arrangement meant that I would spend most of my time in Lyon, working as what was then called a "Maître de conférences" or assistant professor at the university, and travel to Milan one week each month to fulfill my fellowship requirements. It was a demanding schedule, but the experience was incredibly enriching. Balancing these dual roles was not easy, but it allowed me to broaden my expertise and strengthen my connections in the medical and research communities across two countries. This period laid the foundation for my future career, blending clinical practice with cutting-edge research, and reinforced my commitment to advancing the field of pediatric immunology.
A lot of your early work was in the field of cell and gene therapy – leading the first stem cell-based gene therapy for ADA-SCID What was it like to be part of these clinical trials, and how did it inform your future research?
One of the main reasons I chose to go to Lyon instead of MSKCC was because of their innovative fetal stem cell transplant program for patients with SCID. In cases where patients didn't have an HLA-matched sibling donor, Lyon was experimenting with fetal liver stem cell transplants. I was very interested to explore this as a curative option for SCID and other monogenic diseases like metachromatic leukodystrophy and thalassemia. During my investigations, I made two key observations. First, I found that some patients succeeded with transplants despite a complete donor-host mismatch--indicating an active process of tolerance. This sparked my interest in understanding this tolerance mechanism. Secondly, we attempted fetal stem cell transplants in utero to avoid the high GVHD risk associated with post-birth transplants. However, we soon realized that even in utero transplants could result in GVHD and rejection. These insights led me to shift from allogeneic fetal transplants to an autologous stem cell gene therapy approach: using the patient's own engineered cells to reduce GVHD risk and harness the immune system more effectively.
It was in one of these patients that exhibited active tolerance where the first observation of Tr1 regulatory cells was made. Despite the limited tools to detect these regulatory cells, we published evidence of this mechanism in the late 1980s. My journey toward uncovering the function of these Tr1 cells continued at DNAX, a Bay area-based biotech company founded by three Stanford professors Arthur Kornberg, Paul Berg and Charles Yanofsky. The timing of my arrival at DNAX was somewhat fortuitous. DNAX had just cloned the mouse IL-10 gene, and wanted to clone the human IL-10 gene. By this time, I had found these regulatory cells in my patients but had not yet figured out how they function. After joining DNAX, I gave some of these patient-derived cells to Kevin Moore, one of my colleagues at DNAX. His postdoc came back to me after five days saying that the cells I had given him produced copious amounts of IL-10 and that he had cloned the human IL-10 gene! Thus, if I had to look back, being part of those early clinical trials in monogenic inherited immunological diseases is what led to the discovery of the Tr1 cells and took me down the research path of trying to understand how these cells function.
Can you walk us through the tools available at the time in immunology that helped in your research?
When I reflect on the tools available during my early research in immunology, I can identify three significant steps that marked the evolution of the field and profoundly impacted my work. The first step occurred during my time in medical school with the advent of monoclonal antibodies which enabled the advancement of immunofluorescence techniques to investigate the immune system. Of course, the process was still quite labor intensive and time-consuming since we examined slides under a microscope and had to manually count the positive cells. The second pivotal moment came when I returned to Lyon in 1986. I had joined an immunology lab supported by Schering-Plough, which had incredible resources, including advanced technology such as the fluorescence-activated cell sorter (FACS). This lab attracted top talent, including a researcher who would later become my husband. They introduced me to single-cell cloning, allowing us to characterize biological cells at the single-cell level. This technology enabled us to investigate different cells from our patients more precisely, providing deeper insights into their immune functions. The third major advancement happened when I moved to the DNAX in the Bay Area. DNAX was at the forefront of DNA recombination technology, enabling us to clone cytokines and their receptors, which is what allowed us to clone human IL-10 and obtain the necessary reagents to study cytokine production and expression in cells. These advancements were crucial as they also coincided with the sequencing of the human genome in the late 1990s. At Stanford and DNAX, we focused on interpreting the newly sequenced genes and generating biomarkers to identify them, which was transformative for our understanding of immunology.
Can you walk us through some of your early discoveries about Tr1 cells and how you balanced this with your work in gene therapy?
Reflecting on my early discoveries about Tr1 cells, I consider it one of my major scientific achievements even though (to date) my clinical impact has been more pronounced in treating monogenic diseases through gene therapy. In the early days after discovering the Tr1 cells, we determined that these cells were distinct from Th1 and Th2 cells based on their cytokine production profile and receptor expression. We demonstrated their suppressive function and found that these cells exist not only in transplant patients but also in the general population. We submitted our findings to Nature, but the paper's publication was delayed by almost a year because we had to show that Tr1 cells also existed in mice and were relevant in a mouse model of disease. We eventually demonstrated this using a model of IBD/Crohn’s disease.
Around the same time, I returned to Milan to establish a research institute for gene therapy. Although I continued my work on Tr1 cells, my primary focus shifted towards building the San Raffaele-Telethon Institute for Gene therapy (TIGET) and developing a pipeline of stem cell gene therapy products. This effort culminated in the approval of Strimvelis for ADA-SCID and Libmeldy for metachromatic leukodystrophy: both significant milestones for the field and for the institute, which went on to grow substantially in size and capability. It was also during my time in Milan that I developed some initial technology to generate Tr1 cells in vitro for patients undergoing allogeneic stem cell transplantation. We even managed to treat patients and demonstrate safety and efficacy, but the process was not yet optimized for broader clinical trials. When I joined Stanford in 2014, my primary goal was to build the Center for Definitive and Curative Medicine (CDCM), which included setting up a GMP facility and establishing a robust clinical trial office. Despite these major responsibilities in setting up these new institutions, I balanced my time and continued to work on Tr1 cells within my lab. We optimized a Tr1-based cellular product and then went on to conduct two promising clinical trials. These trials involved using a product derived from the donor of the allogeneic stem cell transplant, which contained approximately 10% Tr1 cells. These trials showed promising data, demonstrating both safety and initial signs of efficacy but also highlighted several challenges. The challenges we uncovered is what ultimately led to the creation of Tr1X later on.
What were some of the initial obstacles in thinking about translating these findings about Tr1 cells to the clinic?
Broadly speaking, there are overarching challenges in the cell therapy space. For instance, some of the advanced therapies like CAR-T cells that have become commercial products and gone on to save lives have a very high price tag. While the high cost of CAR-T therapy is justified by its life-saving potential, it does limit accessibility as not all patients can benefit from it. In addition, the need to use patient’s derived cells to engineer the CAR-T limit the feasibility. For instance, patients who are immunosuppressed or have undergone extensive chemotherapy might not have viable cells to extract for CAR-T production. Even when cells can be extracted, manufacturing them successfully can be a challenge. Another major obstacle is the logistics involved. The process of leukapheresis (extracting the patient’s white blood cells), manufacturing the CAR-T cells, and then shipping them to the clinical site on time, is complex and fraught with delays and complications. These logistical hurdles can prevent timely treatment and reduce the therapy’s overall effectiveness. Despite the promise of cell therapy, the number of patients treated with CAR-T is still a fraction of those treated with biologics like anti-PD-1 and anti-CTLA-4 antibodies or small molecule therapies. Biologics are more scalable and accessible, partly because they don't face the same manufacturing and logistical challenges. Thus, as we were thinking about translating our research findings surrounding Tr1 cells, we understood that for cell therapy to truly compete with biologics [cost and patient accessibility] these practical issues need to be addressed. Developing more efficient manufacturing processes, improving logistical frameworks, and finding ways to make these therapies more broadly available to patients in need was on the top of our mind.
Our experience with the initial Tr1-based clinical trials had revealed several challenges in generating autologous Tr1 cells. The feasibility of producing these cells at a clinical scale was difficult, and the cost of goods was prohibitively high. Given these challenges, it became evident that the most viable path forward was to develop an off-the-shelf product that did not rely on the limitations inherent to natural regulatory T cells. Unlike effector cells, natural regulatory T cells, including Tr1 and FOXP3+ Tregs, have intrinsic limitations when it comes to commercial viability, such as poor growth and expansion capabilities. These limitations made it clear to us that a new strategy was needed. This ultimately led to the creation of Tr1X, which engineers CD4+ T cells to become Tr1-like cells through genetic modifications, allowing them to mimic the natural Tr1 cells' regulatory functions without their limitations. By starting with cells from allogeneic sources rather than the patient’s own cells and using lentiviral vectors to introduce specific genes, we can reprogram these CD4+ T cells into Tr1-like cells, which have better growth and expansion properties, offering a more feasible and scalable solution for therapeutic use.
At what point did you start to consider translating the years of research on Tr1 cells that you had done until that point into a private venture? What is the founding story of Tr1X?
I'm a strong believer in academia's ability to translate research into real-world applications. The freedom to operate and make clinical trial decisions independently of business or financial pressures is invaluable in my opinion. This belief is what drove me to Stanford, where I went on to build the CDCM. The aim was to create a research center that brings together high-impact research investigators across therapeutic areas with best-in-class translational capabilities and proven leadership to accelerate the development process from the bench to the bedside, particularly in cell and gene therapy. The CDCM's success to date really demonstrates the value of such infrastructure with over 30 Phase I clinical trials in the pipeline currently, all representing potentially transformative treatments.
Having said that, there are obvious limitations within academia as well. Developing off-the-shelf technologies, for example, is nearly impossible within an academic setting due to resource constraints. Significant funding is often required to bring a product to the clinic—far beyond typical academic grants. I have always believed that there are two main factors that should drive the formation of a company: 1. The need for substantial capital and 2. The speed of development. In a biotech environment, with adequate capital and the right team, progress can be rapid. In academia, you are part of a larger system with broader priorities, which can slow things down. In our case, it became clear that while Tr1 cells could be developed within an academic setting, advancing our engineered Tr1 cells and making “off the shelf” products, required forming a company. This transition allowed us to leverage the capital, speed, and focused resources necessary to advance our work on Tr1 cells and bring these life-saving therapies to the clinic.
Briefly, how does the Tr1X platform work? How is this platform differentiated from peers isolating FOXP3+ Tregs for therapeutic intent (e.g. in RA, MS, transplants)? [Can speak to unmet need/efficacy, scalability/cost, feasibility etc.]
In parallel with the biological characterization of Tr1 cells (1997), our team worked extensively with FOXP3+ Tregs (2001). In fact, we were among the first to describe human FOXP3+ Tregs. However, over the years, we found that Tr1 cells offer broader and more flexible modes of action compared to FOXP3+ Tregs. Tr1 cells suppress effector T cells mainly through IL-10, a pleiotropic cytokine that not only drives their differentiation but also inhibits inflammation, downregulates antigen-presenting cells, and creates a tolerogenic microenvironment. This broad action suggests that Tr1 cells could potentially reset the immune system, offering a curative approach rather than just suppression. Moreover, Tr1 cells are induced in the periphery, making them easier to generate through genetic engineering.
At its core, the Tr1X platform is quite simple. It leverages the natural properties of CD4+ T cells, reprogramming them into Tr1-like cells (TRX cells) through genetic engineering. This process begins with the isolation of normal CD4+ T cells from peripheral blood, which are then transduced with a lentiviral vector encoding two critical components: human IL-10 and a truncated NGF receptor (tNGFR). The IL-10 cytokine is essential for the differentiation and function of Tr1 cells, while the tNGFR serves a dual purpose, allowing for the selection and purification of successfully engineered cells during the manufacturing process. It also enables the tracking of these cells in patients’ blood post-infusion using monoclonal antibodies specific to the NGF receptor (CD271). Through a proprietary reprogramming process, the CD4+ T cells are then transformed into Tr1-like cells, mimicking the functions of natural Tr1 cells by producing IL-10 and exerting regulatory functions. The initial product from this platform, TRX103, targets disease prevention but also active disease treatment, particularly conditions related to allogeneic stem cell transplantation and Inflammatory Bowel Disease. These TRX103 cells are very pure regulatory T cells designed to modulate immune responses and prevent graft-versus-host disease (GVHD) and other immune-mediated complications. We also have a second, more advanced platform where the TRX cells are further engineered to express a chimeric antigen receptor (CAR), enabling them to target specific antigens associated with B- and T-cell mediated autoimmune diseases. For example, our second product, TRX319, is designed to target B cells in autoimmune diseases where B cell activity is a significant component of the pathology, such as certain forms of lupus and other B cell-mediated autoimmune conditions.
[On additional indications for CARTr1 platform]
As you know, there's incredible excitement surrounding the use of CAR-T therapies, particularly targeting CD19, for conditions like myasthenia gravis and lupus. I believe our product TRX319 has a distinct advantage because it not only targets B cells but also modulates T cell responses and reduces inflammation. This dual action could give us an edge in terms of mode of action and safety. One of the significant challenges with current CAR-T therapies is the need for extensive lymphodepletion, typically achieved with high doses of cyclophosphamide and fludarabine. For those of us in the field of stem cell transplantation, we know these drugs are far from benign and can be tough on patients. If our product can achieve effective results with less or no lymphodepletion, it would be a major advantage. Having said that, the field of CD19-targeted CAR-T therapies has become quite crowded, particularly for an indication like lupus. Thus, even though we believe our product might be superior in lupus, we are also actively considering other autoimmune diseases for our initial indications. Our goal is to identify the conditions where our therapy can make the most significant impact while navigating the competitive landscape effectively. The final indication selection will be made soon as we continue to scale up the process and produce the clinical-grade vector.
[On the biggest challenges Tr1X needs to solve going forward]
Our overarching goal is to show that Tr1 cells can offer a comprehensive solution for autoimmune diseases and transplant-related conditions, paving the way for broader applications and ultimately transforming patient outcomes. The primary challenge will be to prove the efficacy of engineered Tr1 cells. While early trials with FOXP3+ Tregs have shown safety, their efficacy to date has been limited. We need to demonstrate that Tr1X’s engineered Tr1 cells can not only provide symptomatic relief but also potentially cure underlying conditions by resetting the immune system and thus achieve long term remissions.
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