CD19-Specific T Cells Developed to Persist When Antigen Load Is Low: Implications for Treating Minimal Residual Disease After Hematopoietic Stem-Cell Transplantation

Clinical responses after adoptive immunotherapy are associated with in vivo expansion and persistence of the transferred antigen-specific T cells. Chimeric antigen receptor (CAR)-modified T cells receive activation, survival, and proliferative signaling via the introduced immunoreceptor when docked with a tumor associated antigen (TAA). However, patients who are conditioned with myeloablative chemotherapy followed by hematopoietic stem-cell transplantation (HSCT) have minimum residual disease and low levels of TAA. Under such conditions, adoptively transferred CD19-specific CAR⁺ T cells may fail to numerically expand and persist leading to compromised therapeutic potential. Therefore, we hypothesized that signaling outside of the CAR may improve the engraftment of genetically modified T cells. Interleukin (IL)-15 is a pro-survival cytokine that promotes T-cell memory and in vivo anti-tumor activity. However, systemic delivery of soluble IL-15 can lead to toxicity. To deliver physiologic and localized IL-15-mediated signaling, we generated a mutein of IL-15 that is expressed as a membrane-bound molecule (mbIL15) on CAR⁺ T cells. The mbIL15 construct was co-electro-transferred with a CD19-specific CAR (on Day 0) into primary human T cells as two Sleeping Beauty DNA transposon plasmids.  Clinically relevant numbers of mbIL15⁺CAR⁺ T cells was generated by co-culture on CD19⁺ artificial antigen presenting cells. Signaling through the IL­-15 receptor complex in genetically modified T cells was validated by phosphorylation of STAT5 and these T cells demonstrated redirected specific lysis of CD19⁺ tumor targets equivalent to CAR⁺ T cells. Furthermore, we observed that signaling generated by mbIL15 supported the emergence of mbIL15⁺CAR⁺ T cells with a memory-like phenotype including the capacity to secrete IL-2 and maintained telomere length. Adoptive transfer of mbIL15⁺CAR⁺ T cells into immunodeficient mice exhibited sustained persistence in the absence of exogenous cytokine support and CD19 TAA. In mice bearing a CD19⁺ leukemia, the mbIL15⁺CAR⁺ T cells demonstrated both persistence and anti-tumor effects. These data demonstrate that mbIL15 can be co-expressed on CAR⁺ T cells resulting in enhanced in vivo persistence without the need for TAA or exogenous cytokine support. In summary, this cytokine fusion molecule: (i) provides stimulatory signals via STAT5 leading to augmented in vivo T-cell persistence while maintaining tumor-specific functionality, (ii) maintains T-cell subsets that preserves memory potential in the pre-infusion product, (iii) eliminates the need and cost for IL-2 for T-cell expansion and persistence, and (iv) mitigates the need for clinical-grade soluble IL-15. These results have implications for the design of adoptive immunotherapy clinical trials to evaluate whether mbIL15⁺CAR⁺ T cells can improve the graft-versus-tumor effect after HSCT.