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110 IL-2 variant improves CAR-T functionality and efficacy against solid tumors
  1. Qi Dong,
  2. Wenjie Yin,
  3. Pengfei Jiang,
  4. Manli Yin,
  5. Tao Wang,
  6. Ping Wang,
  7. Xinxin Wang,
  8. William Wei Cao and
  9. Lianjun Shen
  1. Gracell Biotechnologies, Shanghai, China


Background Solid tumors remain a challenge for chimeric antigen receptor T (CAR-T) cell therapy due to lack of tumor-specific antigens, often-hard-to-penetrate tumor structure, and hostile tumor microenvironment (TME) for T-cell activation and survival. Interleukin (IL)-2 is an essential cytokine central to the initiation and maintenance of T-cell-mediated immune responses, which is usually downregulated in TME. IL-2 has been shown to improve the effectiveness of various T-cell-based therapies for solid tumors. However, systemic administration of IL-2 has been shown to elicit immunosuppression through regulatory T cells and cause capillary leak syndrome, both of which limit its use in T-cell immunotherapies, such as CAR-T. To improve the resistance of CAR-T to TME and to enhance its persistence, expansion and efficacy in vivo, we designed novel second generation mesothelin (MSLN)-specific CAR constructs (MSLN-CAR-T-IL-2tb) that incorporate secretory form of IL-2 variants (IL-2tb). IL-2tb produced by MSLN-CAR-T-IL-2tb improves cell viability, expansion, and potency, and reduces immunosuppression which could also potentially stimulate endogenous polyclonal tumor-infiltrating lymphocytes in solid tumors.

Methods MSLN-CAR-T-IL-2tb and MSLN-CAR-T cells were generated by lentiviral transduction. To assess in vitro proliferation, CAR-T cells were repeatedly cocultured with MSLN-expressing tumor cell lines and CAR+ T cells were enumerated. CAR-T cell apoptosis, memory phenotype and exhaustion were monitored by flow cytometry at various time points. MSLN-CAR-T-IL-2tb and MSLN-CAR-T cell resistance to TME was tested in vitro. Cytotoxicity was determined using RTCA- or luciferase-based assays. CAR-T tumoricidal activity in vivo was evaluated in human cell line-derived xenograft models using severe immunodeficient mice.

Results MSLN-CAR-T-IL-2tb and MSLN-CAR-T demonstrated comparable efficacies in short-term tumor killing assays in vitro against multiple tumor cell lines expressing varying levels of MSLN in the presence of exogenous IL-2. However, when cultured in the absence of IL-2, MSLN-CAR-T-IL-2tb was much longer-lasting than MSLN-CAR-T in terms of CAR-T cell viability, proliferation, and persistence. MSLN-CAR-T-IL-2tb was more cytotoxic against multiple MSLN-expressing tumor cells, including MDA-MB-231 and HCC70 (triple-negative breast cancers) and OVCAR-3 (ovarian cancer). Moreover, in multiple xenograft mouse models, MSLN-CAR-T-IL-2tb showed very potent and durable anti-tumor responses.

Conclusions MSLN-CAR-T cells expressing a secretory form of IL-2 variant were able to maintain long-term proliferation and cytotoxicity which could be partly due to the reduced immunosuppression in the TME. Autocrine and paracrine loops of IL-2 can further improve CAR-T functionality in solid tumor and could be a promising strategy for clinical application.

Ethics Approval All animal experiments were conducted in facilities accredited by the service providers’ Institutional Review Boards.

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