Abstract 3605: Human chimeric orthogonal IL9 receptor signaling promotes stemness and polyfunctionality for adoptive T cell therapy of cancer
Authors: Tariveranmoshabad Mito , Su Leon L. , Sun Amy L. , Picton Lora K. , Ribas Antoni , Garcia K. Christopher , Kalbasi Anusha 
Published: June 2022

Background: Orthogonal cytokine-receptor pairs allow selective in vivo manipulation of T cells after adoptive cell therapy (ACT) of cancer. Using mouse models, we previously described a chimeric orthogonal receptor (o9R) comprised of an orthogonal IL-2Rβ extracellular domain (ECD) and an IL-9R intracellular domain (ICD). Adoptive transfer of tumor-specific T cells engineered with o9R showed superior anti-tumor activity in mice treated with orthogonal IL-2 (oIL2). To evaluate the translational potential of o9R signaling, we generated and evaluated a human chimeric orthogonal IL-2Rβ ECD/IL-9R ICD (ho9R).

Methods: We hypothesized that ho9R in tumor-specific T cells would result in distinct signaling, phenotypic and functional properties compared to the human orthogonal IL-2Rβ (ho2R), mirroring our observations in the mouse system. Activated T cells were retrovirally transduced with two vectors: one encoding either ho9R or ho2R and another encoding a T cell receptor (TCR) specific for NY-ESO-1 in the context of HLA*0201. Transduced and sorted T cells were exposed to mouse serum albumin-bound human orthogonal IL2 (MSA-hoIL2) or wildtype IL-2 (MSA-hIL2) for 20 mins to evaluate STAT-1, -3 and -5 phosphorylation. We assessed proliferation and memory differentiation of transduced T cells cultured in MSA-hoIL2 or MSA-hIL2 for one week or after repetitive tumor challenge using an HLA*0201+ NY-ESO-1+ melanoma tumor cell line (M407-nRFP) in the presence of MSA-hoIL2 or MSA-hIL2. After four tumor challenges, T cells were restimulated to quantify intracellular cytokine production. Comparisons between ho2R and ho9R T cells were made using unpaired t-tests or 2-way ANOVA.

Results: Consistent with the mouse system, human T cells signaling through ho9R activated pSTAT-1, -3 and -5, whereas ho2R signaling primarily activated pSTAT5. Similar to the mouse system, ho9R signaling resulted in weaker proliferation than ho2R or wildtype IL-2 signaling (p&lt;0.001). Despite weaker proliferation, ho9R signaling expanded a population of TSCM cells (CD45RA+CD27+CCR7+CD95+; p&lt;0.0001). The difference in T cell phenotype between ho9R/NYESO1-TCR and ho2R/NYESO1-TCR T cells persisted even after four challenges with M407-nRFP in the presence of MSA-hoIL2: ho9R/NYESO1-TCR T cells retained more CD45RA+CD27+ and TSCM cells (p&lt;0.01), and expressed higher levels of CD62L (p&lt;0.05) and CXCR3 (p&lt;0.001). Upon re-exposure to antigen after four tumor challenges, ho9R/NYESO1-TCR T cells expressed more IFNγ, TNFα, and IL-2 (p&lt;0.0001) and exhibited greater polyfunctionality than ho2R/NYESO1-TCR T cells.

Conclusion: In tumor-specific T cells ho9R signaling activates STAT-1, -3, and -5, promotes stemness, and maintains effector polyfunctionality despite repetitive antigen stimulation, providing human evidence for its potential in adoptive T cell therapy of cancer.

Citation Format: Mito Tariveranmoshabad, Leon L. Su, Amy L. Sun, Lora K. Picton, Antoni Ribas, K. Christopher Garcia, Anusha Kalbasi. Human chimeric orthogonal IL9 receptor signaling promotes stemness and polyfunctionality for adoptive T cell therapy of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3605.