Tandem CAR-T-Cell Therapy Targeting CD5 and CD7: Facilitating Condition-Free Engraftment of Epitope-Edited Allogeneic HSCs

Introduction:

Patients with refractory or relapsed T-cell acute lymphoblastic leukemia (R/R-T-ALL) face a dire prognosis. Even with salvage hematopoietic stem cell transplantation (HSCT), survival rates seldom exceed 30%, primarily due to disease relapse and transplant-related morbidity. This highlights the critical need for more effective and less toxic preconditioning regimens to improve HSCT outcomes. Recent multicenter studies have indicated that CD7 CAR-T-cell therapy can induce complete remission in approximately 90% of R/R T-ALL patients within one month of infusion. However, the long-term efficacy of CAR-T-cell therapy is compromised by antigen-negative relapse and severe infections, which often require additional bone marrow transplantation for consolidation. Despite these challenges, emerging clinical data support the integration of donor-derived CD7 CAR-T-cell therapy with HSCT, suggesting the viability of CAR-T-cell therapy as a preconditioning regimen. This study aimed to develop and evaluate a novel dual-target CD5-CD7 CAR-T-cell therapy as a preconditioning strategy for epitope-edited allogeneic HSCT, with the goal of establishing a novel treatment for R/R T-ALL.

Methods:

Leveraging established methodologies, we engineered Tan5-7 CAR-T cells that concurrently target the CD5 and CD7 antigens. We employed a base editor (BE) to modify the CD5- and CD7-binding epitopes in HSCs. In vivo experiments involved injecting T-ALL tumor cells intravenously via the tail vein, with subsequent in vivo imaging and flow cytometry performed to confirm tumor cell engraftment. The mice were randomly divided into groups: the experimental group underwent preconditioning with fludarabine and cyclophosphamide for three consecutive days before receiving Tan5-7 CAR-T-cell infusion; one control group received the same FC preconditioning followed by an infusion of control T cells (T_con+HSCT group); and the other control group underwent a standard HSCT preconditioning regimen followed by allogeneic bone marrow transplantation (standard HSCT group). We monitored the tumor load, CAR-T-cell proliferation, and spectrum of peripheral blood cells weekly after transplantation. The mice were also observed for indicators such as weight, temperature, hair loss, diarrhea, and kyphosis to assess graft-versus-host disease (GVHD), infections, and other adverse events, along with survival rates.

Results:

Tan5-7 CAR-T cells effectively eliminated T-ALL tumor cells both in vivo and in vitro, and depleted T, NK, and partial B cells from normal donor PBMCs. Unlike the T_con+HSCT group, where HSCs failed to engraft, the Tan5-7 CAR-T-cell infusion enabled successful allogeneic HSC engraftment without pretransplant conditioning, achieving engraftment efficiencies comparable to those of standard conditioning regimens, predominantly resulting in complete chimerism. Post-engraftment, there were no significant differences in the counts in myeloid cells, megakaryocytes, or platelets between the experimental and control groups. Compared with the standard HSCT group, the Tan5-7 CAR-T + HSCT group presented significantly lower counts of T, NK, and B cells post-transplant and a lower incidence of GVHD but a higher rate of infectious mortality.

We edited single amino acid mutations in CD5/CD7 antigen-binding epitopes in HSCT, enabling BE-T/NK/B cells to be resistant to Tan5-7 CAR recognition. Preliminary data indicated that the Tan5-7 CAR-T + BE-HSCT group presented significantly greater post-transplant T, NK, and B-cell counts compared with the Tan5-7 CAR-T + HSCT group, without a corresponding increase in short-term GVHD incidence. Additionally, the levels of engrafted T-cell counts and TCR clonal diversity appeared to be negatively correlated with GVHD occurrence, whereas T cells differentiated from engrafted HSCs did not significantly increase the risk of GVHD.

Conclusions:

Tan5-7 CAR-T-cell therapy effectively eliminates tumor cells and selectively depletes various immune effector cells, offering a viable alternative to traditional conditioning regimens for HSCT. Antigen epitope-edited HSCT restores peripheral blood immune cell counts without significantly increasing adverse reactions such as GVHD. This dual-target CAR-T-cell bridging strategy with antigen epitope-edited HSCT represents a promising therapeutic option for patients with R/R-T-ALL.