Funding from the WPP has been critical not only to support this project, but to provide the springboard for me to pursue new and exciting research directions for my growing lab. As someone new to Wisconsin, I have felt grateful to the WPP community during this time of transition and look forward to integrating into the community for many years to come.
At a Glance
This project, Engineering CAR T Cells to Overcome Variable Antigen Density in Acute Myeloid Leukemia, led by Rebecca Richards, MD, PhD, will evaluate precision methods for measuring key indicators of success for a targeted cancer immunotherapy to expand treatment options for patients with acute myeloid leukemia (AML), which typically has low treatment response rates and poses a challenge for inducing remission after relapse. The results of this project will support efforts to improve other targeted immunotherapeutic strategies for AML through broader application of the study methods.
The Challenge
Over 350 Wisconsinites will be diagnosed with acute myeloid leukemia (AML) each year, equating to a 1 in 200 lifetime risk. AML is the most common leukemia in the United States and has a dismal prognosis with only a 31 percent overall survival rate five years after diagnosis. Immunotherapies harness the potency and specificity of the immune system to target cancer with the goal of minimizing short- and long-term toxicities. Chimeric antigen receptor (CAR) T-cell therapy is a type of cancer treatment in which immune cells are engineered to better recognize and destroy cancer cells. It has been particularly effective in treating certain blood cancers, but work is needed to develop it for other types of cancer like AML.
Project Goals
The overarching goal of this study is to design CAR T cells that could lead to new clinical trials in the hopes of increasing the number of patients who benefit from CAR T cell therapy. This goal will be addressed through three specific aims:
Develop a multiparameter quantitative flow cytometry panel for AML genotype-phenotype correlation.
Identify an antigen density threshold for AML CAR T cell efficacy and for stable synapse formation.
Engineer CAR T cells to enhance cytotoxicity at lower antigen densities.
Progress Update
During the first year of funding, this project made progress toward understanding how antigen density influences the effectiveness of CAR T cell therapy for acute myeloid leukemia (AML). The research successfully established the foundational tools, datasets and collaborations necessary to advance this work, while generating early findings that inform future therapeutic strategies.
Under Aim 1, the team developed and validated a multiparameter quantitative flow cytometry panel capable of measuring antigen density across multiple AML targets simultaneously. This panel, which includes CD33, CD123, CD38, and CD93, revealed significant variability in antigen expression both across and within AML cell lines. Notably, CD33 was consistently expressed above the threshold required for CAR T cell activation, while CD123 expression was closer to this threshold, highlighting its potential role as a limiting factor in treatment effectiveness. In parallel, the project expanded access to primary AML patient samples through newly secured biobanking resources and institutional approvals, positioning the team to conduct future genotype-phenotype analyses using patient-derived data.
Under Aim 2, the research demonstrated that antigen density directly impacts CAR T cell cytotoxicity. Experiments showed that CAR T cells more effectively eliminated AML cells with higher antigen densities, and identified differences in performance between CAR constructs with distinct co-stimulatory domains (CD28 vs. 4-1BB). These findings are particularly relevant for optimizing CAR T cell design in clinical settings. Additionally, innovative collaboration with a bioengineering lab enabled the development of a synthetic system to model varying antigen densities. This approach demonstrated that higher antigen densities lead to stronger T cell activation, while additional co-stimulatory signaling may help overcome low antigen density conditions, offering a potential strategy to improve therapeutic response.
Progress toward Aim 3 has begun, with early work focused on engineering CAR T cell variants to enhance activity against low antigen density targets. Initial findings from Aims 1 and 2 support the feasibility of this approach and guide the design of these next-phase experiments.
Despite some implementation challenges, including technical optimization of assays, limited availability of viable biobank samples and unexpected toxicity in animal models, the project successfully adapted by refining methodologies, securing alternative sample sources and adjusting experimental approaches. These adaptations ensured continued progress without compromising research goals. Importantly, this work has already extended beyond its initial scope by catalyzing new collaborations and securing additional funding through a pilot grant. The project has also strengthened research infrastructure and positioned the team to disseminate findings through future publications and broader scientific engagement.
Looking to the Future
This work has laid the foundation for development of novel CAR T cells that prevent AML from escaping detection. Dr. Richards and her team will continue testing these CAR T cells in pre-clinical models, with the goal of initiating a clinical trial to test their safety and efficacy in patients with AML.
Lasting Impact
Even though CAR T cells have worked well in some types of leukemias, translation to AML has been slow. Dr. Richards and her team have demonstrated that antigen density is a major determinant of CAR T cell success or failure in AML. The tools developed through this funding can be adapted to a variety of AML CAR T cell clinical trials, both to identify the patients who are most likely to respond, and to monitor response to therapy. Together, this work will expand the number of patients who benefit from this powerful immunotherapy.
