Enhancing Focused Ultrasound-Inducible CAR-T Cell Therapy in the Inhibitory Tumor Microenvironment with TGFβ Signaling Redirectors

Introduction: : Chimeric antigen receptor (CAR)-T cells can treat several blood cancers by targeting overexpressed or cancerous antigen markers but struggle to repeat this efficacy with solid tumors. The immunosuppressive tumor microenvironment of solid tumors presents multiple inhibitory factors to immune cells, one is through the secretion of TGFβ, a cytokine that promotes T cell quiescence. Focused ultrasound/heat controllable CAR expression allows for the restriction of CAR T cell killing to the tumor site. However, its therapeutic window is reduced due to low sustained expression. To combat the immunosuppressive environment and sustain CAR expression after heat-induced expression, a synthetic receptor was engineered onto CAR-T cells to strengthen their inflammatory activity against tumors and sustain CAR expression through an NFkB-responsive positive feedback mechanism. This synthetic receptor, TG18, uses the Transforming Growth Factor β (TGFβ) receptor's extracellular domain and the transmembrane and intracellular domain of the IL18 receptor to reverse an immunosuppressive signal into an inflammatory signal. While proven to facilitate tumor death in vitro and in vivo, the original unoptimized TG18 sequence suffers from a low expression efficiency and potential for unwanted T cell activation in normal tissues due to its constitutive expression. In this study, we first optimized the expression level of TG18 receptor by codon optimization. To reduce the potential side effects of constitutively expressed TG18 receptor, we developed the heat-inducible TG18 and evaluated its expression dynamics in vitro. Combining TG18 with heat-inducible CAR can improve CAR-T cell safety and efficacy in the tumor microenvironment.

Materials and

Methods: : To increase the expression efficiency of TG18, the version based on the consensus human sequence for each component was submitted for codon optimization. Molecular cloning with the optimized TG18 segment was performed using the pHR backbone, with plasmid construction done by Gibson assembly, and lentivirus was produced by a calcium phosphate precipitation-based technique. To reduce the size of the gene insert for improved lentiviral packaging, GFP in the original design was removed in favor of the FLAG peptide tag, placed after the signal recognition particle sequence of TG18. The separated vector design is depicted in Figure 1A prior to optimization and FLAG tagging, and the updated heat-inducible design is shown in Figure 1B. FLAG tagging was verified using an anti-FLAG antibody surface staining on Jurkat and primary T cells. FLAG-tagged TG18 function was verified in flow cytometry with Jurkat cells using an NFkB reporter. The CAR expression dynamics of constitutively expressed and heat-inducible TG18 designs were evaluated by flow cytometry at 6, 12, 24, and 48 hours after heat shock with CAR antibody staining. To assess TG18+CAR killing capability, donor CAR T cells were cocultured with tumor target Fluc+ U-87 MG cells at 1:1 E:T ratio. CAR T cell killing capacity was measured by luciferase assay.

Results, Conclusions, and Discussions:: Codon optimization improved the expression level compared to the original human consensus-derived TG18 in Jurkat and primary T cells. FLAG has been successfully added to the TG18 receptor, with no impact on the function of the TG18 as indicated by the NFkB reporter assay. Successful peptide tagging using the FLAG sequence was a key finding for future viral vector design, as it allows for independence from transduced fluorescent reporters to verify the transduction of a receptor gene, as was done for the construct in Figure 1B, while maintaining low immunogenicity. Initial CAR T cell in vitro killing with constitutively expressed TG18 showed significant killing of U-87 glioblastoma cells, supporting the feedback design as shown in Figure 1C. Dynamics comparison of the CAR expression in Jurkat, depicted in Figure 1D, with either heat-inducible or constitutive TG18 showed higher sustained expression levels compared to the group with no feedback designs over 48 hours. In summary, we have improved the original TG18 designs and developed the heat-inducible TG18 receptor, which can utilize environmental TGFβ, an immune suppressive cytokine, to trigger immune-stimulating signaling pathways for improved CAR expression on transformed T cells. Unlike the original TG18 design, the heat-inducible receptor alleviates concerns of unwanted stimulatory signaling in normal tissues expressing high levels of TGFβ. Coupling this design with heat-inducible CAR expression will enable specific cytotoxicity at the tumor site for unwanted CAR expression, which can potentially lead to on-target off-tumor cell death in the constitutive design. Furthermore, this TGFβ redirector and feedback concept can be applied to signaling domains of other cytokine receptors, such as IL-2, IL-12, IL-15, and IL-23, to trigger various signaling pathways in different tumor contexts.

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