CAR T cell therapy for breast cancer: harnessing the tumor milieu to drive T cell activation

Abstract

BackgroundThe 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.MethodsFirst (1G - CD3ζ) and second generation (2G - 41BB.CD3ζ) 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.ResultsIn 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (10.1186/s40425-018-0347-5) contains supplementary material, which is available to authorized users.

Single residue in CD28-costimulated CAR-T cells limits long-term persistence and antitumor durability

“…ligands for costimulatory molecules (21), providing cytokine signaling (45)(46)(47)(48)(49), and the prevention of inhibitory signals (50,51).…”

Adoptive cell therapy of triple negative breast cancer with redirected cytokine-induced killer cells

“…17 Other ACT therapies, including the novel genetic redirection of T cells following the engineering with TCR or CAR receptors, are being explored to assess the potential activity and safety in TNBC patients. 18,19 Recently, indeed, a number of clinical trials are recruiting TNBC patients to assess the potential activity and safety of anti-mesothelin or anti-MUC1 18 CAR-T cell infusions. Despite the striking outcomes achieved with the infusion of CD19 CAR-T cells in hematological malignances, 3 the experience in solid tumors has not yet produced the same encouraging results, due to the difficulties of how to select the appropriate target, to address T cells to the tumor and/or metastases, and to reduce the risk of toxicities.…”

Adoptive Cell Therapy—Harnessing Antigen-Specific T Cells to Target Solid Tumours

“…The use of combinatorial antigen sensing circuits, i.e., synthetic Notch, where engagement of a tissue-specific antigen by a surface receptor induces transcription of a CAR recognising a tumour-associated antigen [42][43][44] Reduction of CAR T cell affinity [45] Designing CAR T cells targeting antigens that contain tumour-specific modifications/mutations (i.e., mutated variant III of epidermal growth factor receptor; EGFRvIII) [46][47][48] Overcoming immunosuppressive tumour microenvironment (TME) CAR T cell transduction with dominant-negative transforming growth factor β receptor II (dnTGF-βRII)-a decoy receptor for immunosuppressive TGFβ produced by tumour [49] Introducing switch receptors transforming inhibitory cytokine signals into a stimulus (i.e., fusing immunosuppressive IL-4 receptor exodomain to the immunostimulatory IL-7 receptor endodomain) [50,51] Introduction of hypoxia-inducible factor 1-alpha (HIF-1α), a transcription factor stabilised in response to hypoxia, to CAR T cells resulting in increased CAR expression specifically in hypoxic TME [52] Co-expression of catalase to protect CAR T cells, as well as bystander T cells, from reactive oxygen species (ROS) in TME [53] Inhibition of adenosine receptors with their antagonists or shRNA to prevent immunosuppressive effects exerted by tumour derived adenosine [54] The remaining issue of CAR T cell therapy is the loss of expression of targeted antigen by the tumour during the treatment [39,47]. It has been suggested that only patients with homogenous antigen expression on all cancer cells should be treated with this approach to avoid recurrence.…”