Combining CRISPR-Cas9 and TCR exchange to generate a safe and efficient cord blood-derived T cell product for pediatric relapsed AML
Lo Presti, Vania; Meringa, Angelo; Dunnebach, Ester; van Velzen, Alice; Moreira, Aida Valera; Stam, Ronald W; Kotecha, Rishi S; Krippner-Heidenreich, Anja; Heidenreich, Olaf T; Plantinga, Maud; Cornel, Annelisa; Sebestyen, Zsolt; Kuball, Jurgen; van Til, Niek P; Nierkens, S
(2024) Journal for ImmunoTherapy of Cancer, volume 12, issue 4
(Article)
Abstract
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
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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.
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Keywords: Antineoplastic Agents, CD8-Positive T-Lymphocytes, CRISPR-Cas Systems/genetics, Cell Line, Tumor, Child, Fetal Blood, Humans, Leukemia, Myeloid, Acute/genetics, Receptors, Antigen, T-Cell/genetics, Recurrence, Journal Article
ISSN: 2051-1426
Publisher: BioMed Central Ltd.
Note: Publisher Copyright: © Author(s) (or their employer(s)) 2024.
(Peer reviewed)