Combining CRISPR-Cas9 and TCR exchange to generate a safe and efficient cord blood-derived T cell product for pediatric relapsed AML.

Background: Hematopoietic cell transplantation (HCT) is an effective treatment for pediatric patients with high-risk, refractory, or relapsed acute myeloid leukemia (AML). However, a large proportion of transplanted patients eventually die due to relapse. To improve overall survival, we propose a combined strategy based on cord blood (CB)-HCT with the application of AML-specific T cell receptor (TCR)-engineered T cell therapy derived from the same CB graft.
Methods: We produced CB-CD8 ⁺ T cells expressing a recombinant TCR (rTCR) against Wilms tumor 1 (WT1) while lacking endogenous TCR (eTCR) expression to avoid mispairing and competition. CRISPR-Cas9 multiplexing was used to target the constant region of the endogenous TCRα ( TRAC ) and TCRβ ( TRBC ) chains. Next, an optimized method for lentiviral transduction was used to introduce recombinant WT1-TCR. The cytotoxic and migration capacity of the product was evaluated in coculture assays for both cell lines and primary pediatric AML blasts.
Results: The gene editing and transduction procedures achieved high efficiency, with up to 95% of cells lacking eTCR and over 70% of T cells expressing rWT1-TCR. WT1-TCR-engineered T cells lacking the expression of their eTCR (eTCR ^-/- WT1-TCR) showed increased cell surface expression of the rTCR and production of cytotoxic cytokines, such as granzyme A and B, perforin, interferon-γ (IFNγ), and tumor necrosis factor-α (TNFα), on antigen recognition when compared with WT1-TCR-engineered T cells still expressing their eTCR (eTCR ^+/+ WT1-TCR). CRISPR-Cas9 editing did not affect immunophenotypic characteristics or T cell activation and did not induce increased expression of inhibitory molecules. eTCR ^-/- WT1-TCR CD8 ⁺ CB-T cells showed effective migratory and killing capacity in cocultures with neoplastic cell lines and primary AML blasts, but did not show toxicity toward healthy cells.
Conclusions: In summary, we show the feasibility of developing a potent CB-derived CD8 ⁺ T cell product targeting WT1, providing an option for post-transplant allogeneic immune cell therapy or as an off-the-shelf product, to prevent relapse and improve the clinical outcome of children with AML.
Competing Interests: Competing interests: JK reports grants from Gadeta, Novartis, and Miltenyi Biotec and is the inventor of patents dealing with γδT cell-related aspects, as well as the cofounder and shareholder of Gadeta. ZS is an inventor of patents dealing with γδT cell-related aspects.
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