To target the Th2 cytokine-rich breast cancer TME, Bajgain et al. developed mucin 1 TCR, CD3ζ 41BB (signals 1 and 2) CAR T cells that incorporated an inverted chimeric cytokine receptor with an IL4 receptor exodomain and an IL7 receptor endodomain for signal 3. These CAR T cells thus possessed all 3 signals required for T cell activation and persistent function, and were less exhausted and more cytolytic compared to CAR T cells lacking at least one of the signals, resulting in durable tumor control and rejection in vitro and in vivo in mice with engrafted breast cancer.
BACKGROUND: The adoptive transfer of T cells redirected to tumor via chimeric antigen receptors (CARs) has produced clinical benefits for the treatment of hematologic diseases. To extend this approach to breast cancer, we generated CAR T cells directed against mucin1 (MUC1), an aberrantly glycosylated neoantigen that is overexpressed by malignant cells and whose expression has been correlated with poor prognosis. Furthermore, to protect our tumor-targeted cells from the elevated levels of immune-inhibitory cytokines present in the tumor milieu, we co-expressed an inverted cytokine receptor linking the IL4 receptor exodomain with the IL7 receptor endodomain (4/7ICR) in order to transform the suppressive IL4 signal into one that would enhance the anti-tumor effects of our CAR T cells at the tumor site. METHODS: First (1G - CD3zeta) and second generation (2G - 41BB.CD3zeta) MUC1-specific CARs were constructed using the HMFG2 scFv. Following retroviral transduction transgenic expression of the CAR+/-ICR was assessed by flow cytometry. In vitro CAR/ICR T cell function was measured by assessing cell proliferation and short- and long-term cytotoxic activity using MUC1+ MDA MB 468 cells as targets. In vivo anti-tumor activity was assessed using IL4-producing MDA MB 468 tumor-bearing mice using calipers to assess tumor volume and bioluminescence imaging to track T cells. RESULTS: In the IL4-rich tumor milieu, 1G CAR.MUC1 T cells failed to expand or kill MUC1+ tumors and while co-expression of the 4/7ICR promoted T cell expansion, in the absence of co-stimulatory signals the outgrowing cells exhibited an exhausted phenotype characterized by PD-1 and TIM3 upregulation and failed to control tumor growth. However, by co-expressing 2G CAR.MUC1 (signal 1 - activation + signal 2 - co-stimulation) and 4/7ICR (signal 3 - cytokine), transgenic T cells selectively expanded at the tumor site and produced potent and durable tumor control in vitro and in vivo. CONCLUSIONS: Our findings demonstrate the feasibility of targeting breast cancer using transgenic T cells equipped to thrive in the suppressive tumor milieu and highlight the importance of providing transgenic T cells with signals that recapitulate physiologic TCR signaling - [activation (signal 1), co-stimulation (signal 2) and cytokine support (signal 3)] - to promote in vivo persistence and memory formation.
Author Info: (1) Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children's Hospital and Houston Methodist Hospital, Houston, TX, 77030, USA. Interdepartmental Program in Tr
Author Info: (1) Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children's Hospital and Houston Methodist Hospital, Houston, TX, 77030, USA. Interdepartmental Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, TX, 77030, USA. (2) Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children's Hospital and Houston Methodist Hospital, Houston, TX, 77030, USA. Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, 10330, Thailand. (3) Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children's Hospital and Houston Methodist Hospital, Houston, TX, 77030, USA. (4) Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children's Hospital and Houston Methodist Hospital, Houston, TX, 77030, USA. Interdepartmental Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, TX, 77030, USA. (5) Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children's Hospital and Houston Methodist Hospital, Houston, TX, 77030, USA. (6) Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children's Hospital and Houston Methodist Hospital, Houston, TX, 77030, USA. (7) Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children's Hospital and Houston Methodist Hospital, Houston, TX, 77030, USA. (8) Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children's Hospital and Houston Methodist Hospital, Houston, TX, 77030, USA. (9) Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children's Hospital and Houston Methodist Hospital, Houston, TX, 77030, USA. (10) Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children's Hospital and Houston Methodist Hospital, Houston, TX, 77030, USA. jfvera@txch.org.
