Epitope Editing Protects Hematopoietic Cells from CD123 Immunotherapy for Acute Myeloid Leukemia

Genetics Bioengineering Hematology Life Sciences Immunology Oncology Medicine
epitope editing CD123 immunotherapy acute myeloid leukemia hematopoietic stem cells CAR-T

Epitope Prime Editing Shields Hematopoietic Cells from CD123 Immunotherapy: A Novel Therapeutic Strategy for Acute Myeloid Leukemia

Research Background and Problem Statement

Acute Myeloid Leukemia (AML) is a malignant blood disease characterized by the abnormal differentiation of myeloid hematopoietic stem and progenitor cells (HSPCs). Its incidence is increasing globally, making it extremely difficult to treat. Current standard treatments include chemotherapy and allogeneic hematopoietic stem cell transplantation, but they have high relapse rates, with post-relapse survival generally less than 18 months, necessitating new treatment strategies. In recent years, targeted therapies, such as chimeric antigen receptor T-cell therapy (CAR-T), have shown promise, aiming to precisely kill cancer cells by targeting specific antigens on the surface of AML cells. However, several clinical studies have shown that this treatment often causes “on-target, off-tumor” toxicity in AML patients, meaning that CAR-T cells attack healthy hematopoietic cells expressing the same antigen while targeting AML cells. CD123 is an important molecular marker in AML, expressed in most AML patients. However, because normal hematopoietic precursor cells also express CD123, anti-CD123 CAR-T therapy in AML can lead to severe side effects. Therefore, to improve the safety and efficacy of CAR-T cell therapy, the research team designed a novel gene-editing strategy to protect healthy cells from CAR-T cell toxicity by precisely editing the CD123 epitope.

Research Methods and Process

This study, led by Zhang Ying’s team at the Frontier Science Center for Immunology and Metabolism, Zhongnan Hospital of Wuhan University, utilized base editors (BE) and prime editors (PE) as gene-editing tools to precisely modify the CD123 antigen epitope. Through three rounds of epitope screening and validation in vitro and in vivo models, the research team explored the optimal editing sites and strategies to effectively prevent CAR-T cells from killing healthy cells without affecting the normal function of CD123. The specific steps are as follows:

  1. Epitope Screening: The research team initially used single-chain variable fragment (scFv) affinity analysis with the CD123 protein to screen 21 amino acid sites in the N-terminal domain of the CD123 protein for mutation screening. Seven candidate sites were eventually identified where the scFv of CAR-T cells no longer had high-affinity binding.
  2. Base Editing Optimization: To increase the editing efficiency, the research team further screened base editors, increasing editing efficiency from 5.9% to 78.9% while avoiding complex byproduct generation. In vitro model tests of R84 and L30 site editing showed that only the R84 site could maintain CD123 protein expression and normal function.
  3. In Vivo Experimental Validation: HSPCs edited at the R84 site were injected into humanized mouse models. Results showed these edited cells had selective resistance to CAR-T cells and could normally differentiate into myeloid cells while expressing CD123, effectively reducing the non-tumor toxicity associated with CAR-T therapy.

Experimental Results and Data Analysis

  1. Epitope Screening and Editing Effects: The study showed that the R84 site at the N-terminal of CD123 retained selective resistance against anti-CD123 CAR-T cells after base editing, with a pronounced inhibition of CAR-T cell cytotoxicity. Upon editing the R84 site of the CD123-expressing AML cell line MOLM-13, these cells exhibited a significant survival rate in co-culture experiments with CAR-T cells.
  2. Byproduct Analysis: To further verify editing specificity, the research team used guide-seq technology to confirm that base editing at the R84 site did not produce significant off-target effects, demonstrating high precision.
  3. Application of Prime Editors: Compared to base editors, prime editors can generate fewer byproducts at specific sites. The team further optimized prime editors to improve editing purity and efficiency, successfully achieving precise double mutation editing of R84 and V85M sites.
  4. In Vivo Function Validation: By transplanting HSPCs edited at the R84 site into humanized immunodeficient mice, results showed that these cells could effectively differentiate even 16 weeks post-transplantation, maintaining protection against CAR-T cell attacks on normal hematopoietic function.

Conclusion and Research Value

Through precise epitope editing of HSPCs from AML patients, this study proposes a novel strategy that combines hematopoietic stem cell transplantation with gene editing, protecting patients’ healthy cells from non-tumor toxicity during CAR-T treatment. This strategy not only offers a potential safe solution for AML CAR-T cell treatment but also provides hope for treating other CD123-expressing hematological diseases.

Research Highlights and Innovation

  1. Innovative Editing Strategy: Unlike traditional CAR-T therapies, this study utilized base and prime editors for precise protection of healthy cells, significantly reducing the risk of off-target toxicity.
  2. Efficient Editing Tool Optimization: The research team performed multiple optimizations on base and prime editors, achieving a high editing efficiency of 78.9% with low byproduct rates, ensuring high specificity in editing.
  3. Broad Application Prospects: This technology is not only applicable to AML but can also be used for other CD123-related diseases through precise modification of specific epitopes, achieving a more personalized and safer form of immunotherapy.

Outlook and Potential Limitations

Although the study validates the effectiveness of CD123 epitope editing in vitro and in vivo models, further verification of its long-term safety and efficacy is required for actual clinical application. Moreover, as an emerging gene-editing therapy, potential immune reactions need to be thoroughly evaluated. Future research should further improve the safety of editing tools and develop more convenient clinical translation technologies to enable personalized treatment of AML patients.

This study offers new ideas for AML treatment by editing epitopes of healthy cells to reduce the non-tumor toxicity associated with CAR-T therapy, demonstrating broad application prospects.