Co-Stimulation of CD28/CD40 Signaling Molecule Potentiates CD19CAR-T Cell Functions and Stemness

Introduction: Adoptive T-cell therapy of gene-modified to express CD19 chimeric antigen receptor (CD19CAR) has achieved promising clinical outcomes in B-cell malignancies. However, the recurrences after therapies still occur due to the poor CAR-T persistence. Various approaches have been studied including signaling domain modification to improve T-cell efficacy. The nuclear factor-κB (NF-κB) which interestingly exists in both T- and B-cells is a crucial signaling pathway for T-cell persistence. Furthermore, CAR-T memory signatures drive robust T-cell proliferation. Consequently, we developed the third generation CD19CAR incorporated dual T- and B-cell costimulatory molecules; CD28 and CD40 (CD19.28.40z), to enhance CAR-T cell proliferation and tumoricidal activity.

Methods: The anti-CD19CAR incorporated CD28/CD40 co-stimulatory domain; CD19scFv-CD28TM-CD28/CD40-CD3z-tEGFR, was generated. The CAR gene was subsequently cloned into retroviral packaging cells and transduced into primary CD3 cells. The CAR⁺-T cells were enriched using biotinylated anti-EGFR mAb and further expanded with anti-CD3/CD28 beads or gamma-irradiated EBV-LCL cells. These expanded cells were then used for downstream experiments compared with control CD19CARs including CD19.28z, CD19.BBz, and CD19.40z CAR-T cells.

Results: All CD19CAR genes were successfully transduced into primary CD3cells. We first determined the downstream T-cell signaling and found the higher baseline pNF-kB and pRelB of CD19.28.40z CAR-T cells compared to others. We then investigated the ability of CD19 CAR T-cell proliferation by coculturing with gamma-irradiated CD19-expressing K562 cells. Surprisingly, the novel CD19.28.40z exhibited robust T-cell proliferation and persistence throughout 2 weeks of the culture regardless of IL-2 supplementation. The long-term co-culture assay unveiled that CD19.28.40z CAR-T cells could effectively eradicate tumor cell growth until the end of the experiment at an effector to target cell ratio of 1:16, compared to control CAR T-cells. In term of cytokine secretion assays, the similar level of IL-2, TNF-a, and IFN-g concentrations were observed among CAR constructs after being stimulated with target cells. To mimic persistent antigen stimulation in vivo, we performed three consecutive weekly antigen stimulation that exhibited the greater CD19.28.40z CAR-T cell expansion without influencing T-cell fate and exhaustion. We interrogated the intrinsic genes underlying CD19.28.40z CAR-T cell responses compared to conventional CAR-T cells following CD19 antigen exposures using RNA-sequencing. The results confirmed the significant up-regulated genes associated with T-cell proliferation and differentiation, NF-κB pathway, WNT pathway, and CD4+ phenotypes as well as down-regulated genes related to apoptosis, exhaustion, and glycolysis pathway (Figure 1). Regarding T-cell memory phenotypes, we observed the enrichment of genes toward T-cell stemness particularly SELL, IL-7r, TCF7, and KLF2 compared to conventional CAR-T cells. To evaluate the potential anti-tumor efficiency in vivo, we studied NALM-6- and Raji-bearing NSG mice treated with a low CD19CAR-T cell dosage to assess the functional limits of different CAR structures. The CD19.28.40z CAR-T cells demonstrated potent tumoricidal activity as well as prolonged overall survival in both NSG mice models compared to control CAR-T cells (Figure 2).

Conclusions: The CD28/CD40 costimulatory molecule potentiates NF-κB signaling and memory signatures enhancing CD19CAR-T cell proliferation and persistence which translates into greater anti-tumor efficacy in both in vitro and xenograft models. The simplified structural modification of dual T-/B-cell signaling molecule could be advantageous to maximize the CAR-T cell functions and stemness.

No relevant conflicts of interest to declare.