Document resource
Background We have previously evaluated tumor-associated antigen-specific T lymphocyte therapy (TAA-T) targeting WT1, PRAME, and Survivin for the treatment of children with relapsed/refractory solid tumors. In this study we identified increased serum IL-8 in patients, often associated with progressive disease. IL-8 has been shown to contribute to an immunosuppressive tumor microenvironment (TME) and has been implicated in angiogenesis, tumor cell invasion, proliferation, and poor cell survival and response to immunotherapy. Attempts to abrogate IL-8 signaling to enhance anti-tumor efficacy of TAA-T have not previously been explored. We hypothesized that expression of a non-functional IL-8 receptor (IL-8 sink receptor, IL8sr) on the surface of TAA-T products targeting PRAME will neutralize IL-8 in the TME, enhance T cell trafficking, and improve the anti-tumor effects of TAA-T in vitro.Methods Three IL8sr constructs including the CD34 QBEND10 moieties were designed, and CD3/28 stimulated T cells were gene engineered to express these constructs using retroviral transduction. Surface expression of IL-8 sink receptor constructs was measured by flow cytometry for QBEND10. Transwell migration assays and ELISA assays for IL-8 were used to measure (i) IL-8 sinking and (ii) migration of T cells expressing IL-8 sink receptors in media containing IL-8, using non-transduced T cells as a control.Results We have successfully generated three IL8sr transgenes with stable expression of IL8sr (27-75%) on the surface of CD3/28-stimulated T cells. T cells expressing IL8sr constructs 1 and 2, mimicking the native CXCR1 and CXCR2, demonstrated enhanced migration into media supplemented with exogenous IL-8 compared to serum-free media. Additionally, T cells expressing constructs 1, 2, or 12 (based on an IL-8 antibody) demonstrated sinking of both exogenous IL-8 and tumor-conditioned media with high concentrations of IL-8 (up to 1000 pg/mL) with maximum sinking to undetectable levels.Conclusions We have demonstrated successful transduction of T cells with three IL8sr constructs with the ability to neutralize IL-8 and two constructs that support T cell migration to areas of increased IL-8 concentration. The expression of IL8sr constructs hold potential to improve TAA-Ts through enhanced migration to the tumor site, thereby supporting anti-tumor effects and persistence, and mitigating immune suppressive IL-8 effects in the TME. Future studies include: (i) transducing TAA-T products to express IL8sr and (ii) evaluating anti-tumor efficacy of IL8sr-transduced TAA-T in vitro and in murine models.