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

• Published in: Journal for immunotherapy of cancer Vol. 12; no. 4; p. e008174
• Main Authors: 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
• Format: Journal Article
• Language: English
• Published: England BMJ Publishing Group Ltd 05.04.2024; BMJ Publishing Group LTD; BMJ Publishing Group
• Series: Original research
• Subjects: Antibodies; Antineoplastic Agents; Biotechnology; Blood cancer; CD8-Positive T-Lymphocytes; Cell Engineering; Cell Line, Tumor; Child; Cloning; CRISPR; CRISPR-Cas Systems - genetics; Cytomegalovirus; Fetal Blood; Flow cytometry; Genome editing; Hematologic Neoplasms; Humans; Immune Cell Therapies and Immune Cell Engineering; Leukemia; Leukemia, Myeloid, Acute - genetics; Leukemia, Myeloid, Acute - therapy; Lymphocytes; Pediatrics; Plasmids; R&D; Receptors, Antigen, T-Cell - genetics; Recurrence; Research & development; Stem cell transplantation; T cell Receptor - TCR; T cell receptors; CD8-Positive T-Lymphocytes; Pediatrics; Cell Engineering; Hematologic Neoplasms; T cell Receptor - TCR
• ISSN: 2051-1426; 2051-1426
• DOI: 10.1136/jitc-2023-008174

BackgroundHematopoietic 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.MethodsWe 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.ResultsThe 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.ConclusionsIn 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.