Pegilodecakin, a Pegylated Human IL-10 (AM0010), Enhances the Cytotoxicity of CAR-T Cells in Vitro and In Vivo

Introduction

Clinical outcomes with immune cell therapies, including chimeric antigen receptor (CAR) T cells, rely on expansion of the anti-tumor T cells for successful therapeutic outcomes. Recent studies on Pegilodecakin, a pegylated interleukin-10 (IL-10), have demonstrated that doses greatly exceeding typical endogenous levels can drive a productive tumor specific T cell expansion and response. In a large Phase I/Ib study, Pegilodecakin achieved objective responses across multiple tumor types, alone and in combination with chemotherapies and immune checkpoint inhibitors, including Programmed Cell Death-1 (PD-1). Agents that improve the functional expansion of CAR-T cells, post adoptive transfer, hold promise to improve the therapeutic efficacy of the CAR-T therapies in patients. Here we report on early studies which demonstrate that Pegilodecakin significantly enhances the anti-tumor cytotoxic T lymphocyte (CTL) activity of CAR-T cells.

Methods

In Vitro RTCA Assays: The engineered Primary CD19 CAR-T cells were generated by isolating human T cells from whole blood and transducing these T cells with a CD19-targeted CAR. The CD19 CAR-T cells were then activated, expanded, and tested in a Real-time Cytotoxicity Assay (RTCA) against HeLa cells stably expressing human CD19 (CD19-HeLa), at multiple effector-to-target (E:T) ratios. The CTL activity or cytotoxicity was measured through cell-sensor impedance in an electronic microtiter plate. Cytotoxicity was measured for CD19 CAR-T alone or in combination with varied concentrations of Pegilodecakin, and was directly compared with controls, including Pegilodecakin alone or Pegilodecakin with non-transduced T cells. To functionally validate the CTL activity, we measured the levels of Granzyme-B and Interferon-gamma (IFNg) in the culture supernatants at the end of the RTCA by ELISA.

In Vivo Raji Leukemia Model: 6-8 week old female NSG Mice NOD.Cg-Prkdc^scid IL2rg^tm1Wjl/SzJ (NOD scid gamma), 5 mice per group, were injected with 0.5E6 Raji cells engineered to express luciferase (Raji-luc) on Day 0, IV. Tumor progression was imaged on Day 0 and weekly thereafter for 6 weeks using the Spectrum In Vivo Imaging System (IVIS). Mice were treated with varying numbers of non-transduced/CD19 CAR-T cells alone or in combination with 0.5mg/kg Pegilodecakin. Specifically, non-transduced T/CD19 CAR-T cells were administered on Days 2 and 9, with daily Pegilodecakin where indicated. Blood was collected weekly for immunophenotypic analyses. Serum was analyzed for IFNg and Granzyme B via ELISA.

Results

The RTCA assay revealed that Pegilodecakin in combination with the CD19 CAR-T showed a significant increase (p<0.001) in CTL activity against CD19-HeLa cells as compared to the CD19-CAR-T alone. Functionally, when combined with Pegilodecakin, CD19-CAR-T cells produced significantly higher levels of Granzyme-B (p<0.0005) and IFNg (p<0.02) in the RTCA Cytotoxicity Assay. Controls, including Pegilodecakin alone (without CAR-T or non-transduced T cells) and non-transduced T cells in combination with Pegilodecakin, had significantly lower CTL activity by RTCA or ELISA (Granzyme-B or IFNg).

In the Raji-leukemia model, the Pegilodecakin combination with 5E6 CD19 CAR-T had a significant tumor control benefit by IVIS imaging, as directly compared with CD19 CAR-T monotherapy. A modest dose dependent benefit with 2.5E6 CD19 CAR-T was also observed.

Conclusions

We demonstrate that when combined with Pegilodecakin, the cytotoxic activity of the CD19-CAR-T is significantly improved in in vitro and in vivo models. Also, this combination demonstrates significantly improved functional activity of these CD19-CAR T cells via enhanced Granzyme-B and IFNg activity. The in vivo synergistic activity of Pegilodecakin with CD19 CAR-T demonstrates superior tumor control with potentially better depth of response. These results suggest Pegilodecakin/CAR-T therapy combination may provide longer in vivo persistence of the CAR T products offering better clinical outcomes.