Abstract NG11: Orthogonal IL-9 receptor signaling reprograms T cells to obviate conditioning chemotherapy before adoptive cell therapy
Authors: Kalbasi Anusha , Tariveranmoshabad Mito , Escuin-Ordenas Helena , Kremer Sarah , Su Leon L. , Picton Lora , Parisi AnushaGiulia , Garcia Christopher , Ribas Antoni 
Published: July 2021

Abstract
Background: Adoptive cell therapy using genetically engineered T cells - such as chimeric antigen receptor (CAR) or T cell receptor (TCR) modified T cells - is an effective therapy for patients with cancer. However, successful adoptive cell therapies (ACT) require conditioning chemotherapy in order to deplete a patient's endogenous T cells. Patients may also be treated with interleukin-2 (IL2) to promote expansion of adoptively transferred cells, causing significant toxicity and resulting in counterproductive expansion of regulatory T cells. A synthetic orthogonal IL2 (oIL2) can be used to selectively expand adoptively transferred T cells engineered with a synthetic orthogonal interleukin-2 receptor (oIL2R) in vivo, without activating the wildtype IL2 receptor of endogenous T cells. In a lymphodepleted host treated with oIL2, the oIL2R-engineered tumor-specific T cells were equally as effective as tumor-specific T cells treated with wildtype IL2. We hypothesized that selective expansion of adoptively transferred cells using orthogonal interleukin/interleukin-receptor pairs may circumvent the need for lymphodepletion prior to ACT. Methods: To evaluate the impact of orthogonal interleukin signaling on efficacy of adoptively transferred tumor-specific T cells, we used gp100-specific pmel-1 T cells and the gp100+ B16-F10 mouse melanoma model. Activated pmel-1 T cells were engineered with retroviral vectors encoding one of two orthogonal interleukin receptors designed by our collaborator (Dr. Christopher Garcia): orthogonal interleukin-2 receptor (oIL2R) or orthogonal interleukin-9 receptor (oIL9R). The oIL2R consists of a mutant IL2Rb chain that selectively binds orthogonal IL2 (oIL2), but does not bind wildtype IL2. In turn, oIL2 does not bind the wildtype IL2Rb. The oIL9R consists of the extracellular and transmembrane domains of the oIL2R, but the intracellular domain of IL9Ra. Thus, both orthogonal interleukin receptors are activated upon stimulation with oIL2. T cells engineered with oIL9R signal through STAT1, STAT3 and STAT5, compared to oIL2R T cells which predominantly signal through STAT5. We hypothesized that orthogonal interleukin signaling through the IL-9 receptor pathway may promote superior anti-tumor efficacy because this signaling pathway results in activation of STAT1, STAT3, and STAT5 signaling, in contrast to primarily STAT5 signaling activation observed with IL-2 signaling. Sorted oIL2 or oIL9R pmel-1 T cells were used for downstream experiments. For in vivo experiments, tumor-bearing mice were lymphodepleted (or not) with total body irradiation one day prior to adoptive cell transfer (ACT) of 4.0-6.0 × 106 gp100-activated pmel-1 T cells. Mice were also treated with mIL2 or oIL2 for 5 consecutive days starting on the day of adoptive transfer. Tumor volume and survival were assessed. Peripheral blood was examined for adoptively transferred pmel-1 T cells (Thy1.1+CD8+) at various timepoints. Tumors and spleens from mice were harvested for analysis by multiplex immunohistochemistry (IHC) and mass cytometry. In vitro, oIL2 and/or oIL9R pmel-1 T cells were cultured with either wildtype murine IL-2 (mIL2) or orthogonal IL-2 (oIL2). Proliferation was quantified and T cell phenotype was evaluated by flow cytometry. Activated C3H splenocytes transduced with oIL2R and oIL9R were used as a secondary model. After coculture with mIL2 or oIL2, oIL2R and oIL9R pmel-1 T cells were cocultured with nuclear RFP+ B16-F10 tumor cells at various effector:target ratios to assess cell killing and cytokine production. Results: In the absence of lymphodepletion, pmel-1 T cells administered with mIL2 do not expand in vivo and do not have anti-tumor efficacy against B16-F10 tumor-bearing mice. Consistent with our hypothesis, oIL2R pmel-1 T cells expand and persist in vivo even in the absence of lymphodepletion when mice are treated with oIL2 but not mIL2 (53.7 versus 10.8 Thy1.1+CD8+ cells per 10mL blood; P=0.01). However, this improved expansion and persistence did not result in improved anti-tumor efficacy or survival for mice treated with oIL2R pmel-1 T cells and oIL2 in the absence of lymphodepletion across multiple experiments. We next evaluated the expansion and persistence of oIL9R pmel-1 T cells in vivo in the absence of lymphodepletion. Similar to oIL2R pmel-1 T cells, oIL9R pmel-1 T cells expand and persist in vivo even in the absence of lymphodepletion when mice are treated with oIL2 but not mIL2 (108.8 versus 5.7 Thy1.1+CD8+ cells per 10mL blood; P=0.004). But unlike the oIL2R pmel-1 T cells, adoptive transfer of oIL9R pmel-1 T cells in mice treated with oIL2 (versus mIL2) did result in delayed tumor growth and prolonged survival in the absence of lymphodepletion (mean tumor volume 155mm3 versus 1736mm3, P=0.02; Log-rank test, P=0.002). Thus, while both oIL2R and oIL9R pmel-1 T cells can expand in vivo after ACT in the absence of lymphodepletion, only oIL9R pmel-1 T cells result in anti-tumor efficacy and prolonged survival. We hypothesized that other functional differences between oIL9R and oIL2R T cells are responsible for the discrepancy in anti-tumor activity. We found that oIL9R pmel-1 T cells have improved tumor infiltration. Tumors from mice treated with oIL2R and oIL9R pmel-1 T cells were examined by mass cytometry. Of 18 phenotypic opt-SNE clusters, the only cluster with differential abundance was the cluster assigned to adoptively transferred pmel-1 T cells, which was present at greater abundance in tumors from mice treated with oIL9R pmel-1 T cells (log2(fold change) = 2.45, P=1.86 × 10−5). This data was corroborated by multiplex IHC, which demonstrated a higher quantity of CD8+ and CD8+PD1+ T cells in tumors from mice treated with oIL9R pmel-1 T cells, compared to oIL2R pmel-1 T cells. In vitro, oIL9R pmel-1 T cells, but not oIL2R pmel-1 T cells, upregulate lymphocyte homing marker CD62L upon exposure to oIL2 (P&lt;0.0001). In addition, oIL9R pmel-1 T cells lose expression of CD44, indicating a shift toward a naïve/T-stem cell memory phenotype, which is associated with improved anti-tumor efficacy. These phenotypic changes were corroborated in activated C3H T cells engineered with oIL9R T cells and exposed to oIL2. oIL9R pmel-1 T cells exposed to oIL2 for 48 hours prior to coculture with B16-F10 tumor cells had superior in vitro tumor killing than oIL2R pmel-1 T cells exposed to oIL2 (16.6% versus 58.2% tumor confluence at 70 hours, P=0.001). Likewise, oIL9R pmel-1 T cells exposed to oIL2 for 48 hours prior to coculture produced higher amounts of IFNg (15,318 versus 11,530 pg/mL, P=0.03). Conclusions: ACT with oIL9R reprogrammed pmel-1 T cells induce antitumor activity in the absence of conditioning chemotherapy. The oIL9R genetically programmed T cells have superior effector function upon encountering antigen and have improved in vivo activity through diverse effects on T cell phenotype, trafficking and function in non-lymphodepleted hosts. This opens a path toward obviating conditioning chemotherapy prior to ACT in patients with cancer.
Citation Format: Anusha Kalbasi, Mito Tariveranmoshabad, Helena Escuin-Ordenas, Sarah Kremer, Leon L. Su, Lora Picton, AnushaGiulia Parisi, Christopher Garcia, Antoni Ribas. Orthogonal IL-9 receptor signaling reprograms T cells to obviate conditioning chemotherapy before adoptive cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr NG11.