In the field of hematologic oncology, treating acute myeloid leukemia (AML) has always been challenging, especially for relapsed or refractory patients. The limitations of traditional chemotherapy underscore the urgent need for innovative treatments. With advancements in immunotherapy, CAR-T cell therapy has introduced new breakthroughs in AML treatment. The 2024 American Society of Clinical Oncology (ASCO) Annual Meeting was held from May 31 to June 4 in Chicago, USA. The "Phase I Study of Performance-Enhanced CD33 CAR-T Cells in Relapsed or Refractory AML Patients" was presented as a poster, aiming to explore the safety and preliminary efficacy of a novel CAR-T cell therapy in this patient group. "Oncology Frontier - Hematology Frontier " invited the study’s first author, Director Jing Pan from Beijing GoBroad Boren Hospital, to provide an in-depth interpretation of the research and share insights into advancements in AML diagnosis and treatment.

Oncology Frontier – Hematology Frontier: At this ASCO meeting, you and your team had a study selected for poster presentation, investigating the application of performance-enhanced CD33 CAR-T cells in relapsed/refractory AML. Could you introduce the background and main findings of this study?

Professor Jing Pan: I am honored to introduce our team’s research selected for presentation at the ASCO meeting. Our study focuses on the application of performance-enhanced CD33 CAR-T cells in treating relapsed/refractory acute myeloid leukemia (AML). Although CAR-T cell therapy has been studied clinically for over a decade, and some related drugs have been marketed, its application in myeloid leukemia is still in the early stages. The main reasons are: first, the targets for CAR-T therapy are not ideal, often causing significant damage to hematopoietic stem cells and normal myeloid cells. Second, CAR-T therapy has considerable toxicity. Third, the overall efficacy of CAR-T therapy has not exceeded 50%.

In early clinical explorations, we studied some conventional CAR-T therapy targets such as CD33, CD123, and CLEC12A, but the results were not satisfactory. I participated in the EVOLVE project, led by Professor Nirali N. Shah from the Pediatric Oncology Branch of the National Institutes of Health (NIH), which reported at the 2023 American Society of Hematology (ASH) Annual Meeting. Professor Shah’s team found that CD33 CAR-T therapy, despite its widespread expression and frequent use in myeloid leukemia, showed extreme efficacy variability: for some patients, it was almost ineffective. After infusion, CAR-T cells exhibited poor expansion in vivo, failing to achieve expected therapeutic effects. For others, despite showing some efficacy, the therapy caused severe toxic reactions, placing a significant burden on patients. Through discussions with Professor Shah, we recognized the common understanding that CAR-T therapy targeting CD33 in myeloid leukemia showed unsatisfactory results in clearing CD33-positive cells from peripheral blood. The effectiveness of CAR-T therapy relies on the expansion and persistence of CAR-T cells in the body. Unlike CD19 CAR-T therapy, which typically results in sustained CAR-T cells and reduced target cells, this was not observed with myeloid CAR-T therapy.

Based on this discovery, our team began adjusting the structure of conventional CD33 CAR-T cells in 2019 to enhance their performance and efficacy. We hypothesized that insufficient in vivo expansion of CAR-T cells was due to their weak performance. Our early clinical exploration revealed another phenomenon: severe activation of macrophages during myeloid CAR-T cell response. Macrophages also express CD33, and if CAR-T cells are weak, they are quickly phagocytosed by macrophages. Our preclinical research, soon to be published, supports this finding. To address this, we adopted an innovative structural design, combining performance-enhanced CD33 CAR-T with second-generation lentiviral CAR-T. Early preclinical and human data showed that these CAR-T cells could rapidly expand in vivo and effectively inhibit macrophage activation. A significant highlight of our research is that almost all patients’ CD33-positive cells were cleared. This marks an important advance in the in vivo clearance of targets by CD33 CAR-T therapy. In terms of safety, while all four enrolled patients experienced cytokine release syndrome (CRS), only one patient with a high tumor burden experienced grade 4 CRS; other toxic reactions were effectively controlled. Long-term follow-up showed that, except for one patient with CD33-positive relapse, others remained in disease-free survival during the two-year follow-up. We plan to further adjust the CAR-T structure in this preliminary study to achieve better efficacy and safety, ultimately aiming for clinical application.

Oncology Frontier – Hematology Frontier: In this study, you used P2A to link an enhancement molecule to human CD33 scFv to improve CAR-T cell performance. Could you explain the principle and effects of this improvement?

Professor Jing Pan: As mentioned, we adopted an innovative method to improve CAR-T cell performance, specifically targeting CD33. The main challenges we faced were the unsatisfactory in vivo efficacy of CAR-T cells and the potential severe clinical side effects caused by macrophage activation.

To address these issues, our team employed two main strategies. First, we used a P2A sequence to link an enhancement molecule to the human CD33 single-chain variable fragment (scFv). This enhancement molecule aims to significantly boost CAR-T cell expansion in a short period, quickly producing therapeutic effects. This molecule is currently patent-pending, so details cannot be fully disclosed. Second, we took measures to inhibit excessive macrophage activation. Overactivation of macrophages can lead to macrophage activation syndrome (MAS), presenting severe safety issues in clinical settings. By improving the CAR-T cell design, we aimed to reduce these side effects and enhance overall treatment safety.

Our research showed that these improvements enabled CAR-T cells to rapidly expand in vivo and avoid severe side effects even with macrophage activation. CD33-positive cells were quickly cleared, and macrophage activation decreased, providing a relatively safe treatment option for patients.

Oncology Frontier – Hematology Frontier: In this study, the issue of CD33-positive normal cell depletion was observed. What potential risks could this pose, and what research plans and improvement measures do you and your team have to address this issue?

Professor Jing Pan: Depletion of CD33-positive normal cells is indeed a complex and challenging issue. As mentioned, CD33 is widely expressed in myeloid leukemia, making it an ideal target. However, since CD33 is also expressed on macrophages, for patients with high tumor burdens, CAR-T cell expansion prolonged the clearance of macrophages, increasing treatment-related toxicity. This not only raises clinical risks for patients but also limits the safety and effectiveness of CAR-T therapy.

To address this, our team has formulated research plans and improvement measures. First, we aim to rapidly clear CD33-positive tumor cells to shorten treatment time and reduce the impact on normal cells, thereby minimizing overall patient damage. Second, we plan to further enhance CAR-T cell performance to inhibit excessive macrophage activation, improving treatment safety. We are actively developing a second-generation product incorporating these improvements, anticipating validation in future studies. Additionally, our entire CAR-T structure, from antibody screening to overall design, is independently developed with our own patented technology.

We believe that through continuous research and innovation, we can overcome these challenges and provide safer, more effective treatment options for patients. We look forward to showcasing these improvements in future clinical studies and ultimately applying them in clinical practice.

Oncology Frontier – Hematology Frontier: Lastly, could you share your impressions of attending the ASCO meeting and highlight any significant research in CAR-T treatment for AML?

Professor Jing Pan: Attending the ASCO meeting has been an honor and an exciting experience. It is a grand event that brings together the most cutting-edge research from around the world, providing valuable opportunities to exchange the latest findings and treatment strategies with peers globally.

In terms of CAR-T treatment for AML, there were no groundbreaking studies presented at this meeting. Unfortunately, treatment strategies for refractory/relapsed myeloid leukemia and myelodysplastic syndromes (MDS) still rely mainly on traditional chemotherapy regimens, such as the “3+7” induction chemotherapy (anthracycline and cytarabine), and the combination of targeted drugs and hypomethylating agents. Research progress in antibody drugs is relatively limited. Specifically, CD33 monoclonal antibody gemtuzumab ozogamicin (GO), though approved by the FDA and EMA, is mainly used in AML patients with favorable genetic backgrounds and core factor positivity, not in resistant patients, limiting its potential application in a broader patient population.

In hematologic research, I am particularly interested in antibody-drug conjugates (ADCs) targeting CD123, which I believe have promising potential to improve treatment outcomes for refractory/relapsed AML patients. Additionally, I am keenly following developments in new logic-gated CAR-T therapies and bispecific CAR-Ts. These innovative strategies aim to further enhance the efficacy of CAR-T therapy in myeloid leukemia. Overall, although CAR-T therapy in AML has not yet achieved a breakthrough, I remain optimistic about the future of this field. I believe that with continued research and technological innovation, we will bring more hope to AML patients.