Related Experiment Video
Updated: Oct 4, 2025
Non-Viral Engineering of Primary Human T Cells via Homology-Mediated End-Joining Targeted Integration of Large DNA Templates
Published on: May 9, 2025
CRISPR-based gene disruption and integration of high-avidity, WT1-specific T cell receptors improve antitumor T cell
Eliana Ruggiero1, Erica Carnevale1, Aaron Prodeus2
1Experimental Hematology Unit, Division of Immunology, Transplantation and Infectious Diseases, Ospedale San Raffaele Scientific Institute, 20132 Milan, Italy.
Abstract:
T cell receptor (TCR)-based therapy has the potential to induce durable clinical responses in patients with cancer by targeting intracellular tumor antigens with high sensitivity and by promoting T cell survival. However, the need for TCRs specific for shared oncogenic antigens and the need for manufacturing protocols able to redirect T cell specificity while preserving T cell fitness remain limiting factors. By longitudinal monitoring of T cell functionality and dynamics in 15 healthy donors, we isolated 19 TCRs specific for Wilms' tumor antigen 1 (WT1), which is overexpressed by several tumor types. TCRs recognized several peptides restricted by common human leukocyte antigen (HLA) alleles and displayed a wide range of functional avidities. We selected five high-avidity HLA-A*02:01-restricted TCRs, three that were specific to the less explored immunodominant WT137-45 and two that were specific to the noncanonical WT1-78-64 epitopes, both naturally processed by primary acute myeloid leukemia (AML) blasts. With CRISPR-Cas9 genome editing tools, we combined TCR-targeted integration into the TCR α constant (TRAC) locus with TCR β constant (TRBC) knockout, thus avoiding TCRαβ mispairing and maximizing TCR expression and function. The engineered lymphocytes were enriched in memory stem T cells. A unique WT137-45-specific TCR showed antigen-specific responses and efficiently killed AML blasts, acute lymphoblastic leukemia blasts, and glioblastoma cells in vitro and in vivo in the absence of off-tumor toxicity. T cells engineered to express this receptor are being advanced into clinical development for AML immunotherapy and represent a candidate therapy for other WT1-expressing tumors.
Insights
Researchers identified novel T cell receptors (TCRs) targeting Wilms' tumor antigen 1 (WT1) for cancer immunotherapy. Engineered T cells demonstrated potent anti-tumor activity against acute myeloid leukemia and other cancers, with potential for clinical development.
Area of Science:
- Immunology
- Oncology
- Gene Therapy
Background:
- T cell receptor (TCR)-based therapies offer potential for durable cancer responses by targeting intracellular antigens.
- Limitations include identifying shared oncogenic antigen-specific TCRs and manufacturing methods that preserve T cell function.
- Wilms' tumor antigen 1 (WT1) is overexpressed in multiple cancer types, making it a promising therapeutic target.
Purpose of the Study:
- To isolate and characterize novel TCRs specific for Wilms' tumor antigen 1 (WT1).
- To engineer T cells with enhanced anti-cancer activity using CRISPR-Cas9 gene editing for potential immunotherapy.
- To evaluate the efficacy and safety of engineered T cells against WT1-expressing tumors.
Main Methods:
- Longitudinal monitoring of T cell functionality in healthy donors to isolate WT1-specific TCRs.
- Selection of high-avidity TCRs, including those targeting WT137-45 and WT1-78-64 epitopes.
- CRISPR-Cas9 genome editing for TCR-targeted integration and TCR constant gene knockout to optimize T cell function.
- In vitro and in vivo evaluation of engineered T cells against acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and glioblastoma cells.
Main Results:
- Isolated 19 TCRs specific for WT1, recognizing various HLA-restricted peptides with diverse avidities.
- Selected five high-avidity HLA-A*02:01-restricted TCRs, including novel specificities for WT137-45 and WT1-78-64 epitopes.
- Engineered T cells exhibited enhanced function, enriched memory stem T cell populations, and demonstrated potent, antigen-specific killing of AML, ALL, and glioblastoma cells in vitro and in vivo.
- No off-tumor toxicity was observed with the lead WT137-45-specific TCR.
Conclusions:
- Novel WT1-specific TCRs have been identified, offering a promising avenue for cancer immunotherapy.
- Engineered T cells with a unique WT137-45-specific TCR demonstrate significant anti-tumor efficacy and safety.
- These engineered T cells are advancing to clinical development for AML and hold potential for treating other WT1-expressing malignancies.
