Abstract
Background: Chimeric antigen receptor (CAR) T-cell therapy is an effective treatment for relapsed and refractory acute lymphoblastic leukemia (R/R ALL). However, autologous CAR-T cells derived from tumor patients often exhibit poor proliferation capacity, exhaustion, and inefficacy. The preparation cycle for autologous CAR-T is lengthy and costly. To overcome these limitations, we investigated the anti-leukemic activity of allogeneic CAR-T cells derived from umbilical cord blood (UCB).
Methods: Cryopreserved UCB units from healthy donors were processed with a Pan-T isolation kit. CRISPR/Cas9 was used to disrupt TRBC, HLA-A and HLA-B, followed by lentiviral transduction encoding a CD19-directed CAR and cytomegalovirus IL-10 (cmvIL-10), generating allogeneic CD19 CAR-T cells (UCAR-T). To assess immunogenicity, irradiated UCAR-T were co-cultured with allogeneic PBMC and proliferation was quantified. For graft-versus-host disease (GvHD) risk, UCAR-T were either stimulated with anti-CD3 (OKT3) or co-cultured with irradiated allogeneic PBMC, and CD25 expression or proliferation was monitored. Cytotoxic specificity was evaluated in vitro against CD19⁺ targets and in vivo using NOD/Shi-scid/IL-2Rγnull (NOG) mice engrafted with NALM6-ZsGreen-Luc cells. Finally, the clinical efficacy and safety of UCAR-T were explored in a patient with relapsed ALL harboring a Tp53 gene mutation (ChiCTR1900025676). The patient’s tumor burden before CAR-T therapy was 9.33%. After chemotherapy with fludarabine combined with cyclophosphamide, 1×10⁶/kg of UCAR-T cells were infused.
Results: Post-editing flow cytometry revealed 89.7 % CD19-CAR⁺ cells, 99.8 % CD3⁻ cells, 93.5 % HLA-A⁻ cells, 74.1 % HLA-B⁻ cells, and 71.5 % double HLA-A⁻/HLA-B⁻ T cells. In immunogenicity assays, PBMC proliferation upon exposure to irradiated UCAR-T was markedly lower than with unedited CAR-T, indicating substantially reduced allo-immunogenicity. OKT3 stimulation failed to up-regulate CD25 expression level on UCAR-T, and co-culture with irradiated allogeneic PBMC elicited no significant UCAR-T proliferation, confirming minimal GvHD potential. In vitro, UCAR-T mediated 98 % and 75 % lysis of Nalm6 cells at effector-to-target ratios of 1:16 and 1:32, respectively, within 72 h. In the NALM6-NOG model, UCAR-T achieved complete tumor eradication by day 60 with 100 % survival, whereas vehicle-treated mice required euthanasia by day 15 due to tumor burden >3,500 mm³. Collectively, these pre-clinical data demonstrate that UCAR-T cells exhibit low immunogenicity, negligible allo-reactivity, and potent anti-tumor efficacy, supporting their translation into early-phase clinical testing.
In the clinical study, the patient developed persistent fever on day 6 (D6) after UCAR-T cell infusion, and UCAR-T cells were detected in the peripheral blood by flow cytometry. On day 9 (D9), the expansion of UCAR-T cells in the peripheral blood peaked at 1982 cells/μL. During treatment, the patient experienced grade 2 cytokine release syndrome (CRS), which was alleviated after treatment with toccb and dexamethasone. Bone marrow evaluations were performed at 2, 4, and 8 weeks post-treatment, with minimal residual disease (MRD) testing consistently negative. The patient remains under ongoing follow-up.
Conclusions: Umbilical cord blood-derived UCAR-T cells exhibit low immunogenicity and minimal GvHD-inducing potential, while demonstrating in vivo expansion capability and tumor-killing efficacy. Preliminary clinical studies indicate that UCAR-T therapy shows promising effectiveness and safety in treating relapsed ALL, though further validation through large-scale clinical trials remains necessary.
