Development of multivalent CAR T cells as dual immunotherapy and conditioning agents

Quenton Rashawn Bubb; Mohammad Balood; Gabe Eduardo Seir; Leah Swartzrock; Ethan Haslett; Katie Ho; Peng Xu; Saida G Wiltz; Elena Sotillo; Tanja A Gruber; Rebecca M Richards; Crystal L Mackall; Agnieszka Czechowicz

doi:10.1016/j.omton.2025.200944

Development of multivalent CAR T cells as dual immunotherapy and conditioning agents

Mol Ther Oncol. 2025 Jan 30;33(1):200944. doi: 10.1016/j.omton.2025.200944. eCollection 2025 Mar 20.

Authors

Quenton Rashawn Bubb^{1

2

3}, Mohammad Balood², Gabe Eduardo Seir^{2

4}, Leah Swartzrock^{2

3}, Ethan Haslett^{2

3}, Katie Ho^{2

3}, Peng Xu⁵, Saida G Wiltz^{2

3}, Elena Sotillo⁵, Tanja A Gruber², Rebecca M Richards⁶, Crystal L Mackall^{2

5

7}, Agnieszka Czechowicz^{2

3}

Affiliations

¹ Stem Cell Biology and Regenerative Medicine Graduate Program, Medical Scientist Training Program, Stanford University School of Medicine, Stanford, CA 94305, USA.
² Department of Pediatrics, Division of Hematology, Oncology, Stem Cell Transplantation and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
³ Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
⁴ Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.
⁵ Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA 94305, USA.
⁶ Department of Pediatrics, University of Wisconsin-Madison, Madison, WI 53792, USA.
⁷ Division of Blood and Stem Cell Transplantation and Cell Therapy, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.

Abstract

Hematopoietic stem cell transplantation (HSCT) is the only definitive cure for pediatric acute myeloid leukemia (AML). Despite adjustments in HSCT protocols and improvements in supportive care, 30% of high-risk patients who receive HSCT as part of their therapy still experience disease relapse with high transplant-related mortality. Relapsed AML has a dismal prognosis, and novel therapies are needed. To improve upon the status quo, HSCT would more effectively eliminate relapse-initiating leukemic cells and be delivered with safer, non-genotoxic conditioning. Here, we investigate hematopoietic cytokine receptors (HCRs) and identify that KIT, MPL, and FLT3 are collectively highly expressed in virtually all pediatric AML samples studied. Further, we establish proof-of-concept of a first-in-class chimeric antigen receptor (CAR) T cell that enables simultaneous targeting of KIT, MPL, and FLT3 through a single receptor, which we term the extracellularly linked concatemeric trivalent cytokine (ELECTRIC) CAR. ELECTRIC CARs exhibit potent cytotoxicity against normal and malignant hematopoietic cells in vitro and display anti-HCR activity in a murine xenograft model. We propose that the ELECTRIC system can be the foundation to developing a non-genotoxic, anti-leukemic conditioning regimen to enable safer, more durable efficacy with minimal toxicity.

Keywords: AML; CAR-T; HSCT; MT: Regular Issue; conditioning; pediatric AML; trispecific; trivalent.