SUMMARY Despite its success in several clinical trials, cancer immunotherapy remains limited by the rarity of targetable tumor-specific antigens, tumor-mediated immune suppression, and toxicity triggered by systemic delivery of potent immunomodulators. Here, we present a proof-of-concept immunomodulatory gene circuit platform that enables tumor-specific expression of immunostimulators, which could potentially overcome these limitations. Our design comprised de novo synthetic cancer-specific promoters and, to enhance specificity, an RNA-based AND gate that generates combinatorial immunomodulatory outputs only when both promoters are mutually active. These outputs included an immunogenic cell-surface protein, a cytokine, a chemokine, and a checkpoint inhibitor antibody. The circuits triggered selective T cell-mediated killing of cancer cells, but not of normal cells, in vitro. In in vivo efficacy assays, lentiviral circuit delivery mediated significant tumor reduction and prolonged mouse survival. Our design could be adapted to drive additional immunomodulators, sense other cancers, and potentially treat other diseases that require precise immunological programming.

Forkhead-associated (FHA) domains recognize phosphothreonines, and SQ/TQ cluster domains (SCDs) contain concentrated phosphorylation sites for ATM/ATR-like DNA-damage-response kinases. The Rad53-SCD1 has dual functions in regulating the activation of the Rad53-Dun1 checkpoint kinase cascade but with unknown molecular mechanisms. Here we present structural, biochemical, and genetic evidence that Dun1-FHA possesses an unprecedented diphosphothreonine-binding specificity. The Dun1-FHA has >100-fold increased affinity for diphosphorylated relative to monophosphorylated Rad53-SCD1 due to the presence of two separate phosphothreonine-binding pockets. In vivo, any single threonine of Rad53-SCD1 is sufficient for Rad53 activation and RAD53-dependent survival of DNA damage, but two adjacent phosphothreonines in the Rad53-SCD1 and two phosphothreonine-binding sites in the Dun1-FHA are necessary for Dun1 activation and DUN1-dependent transcriptional responses to DNA damage. The results uncover a phospho-counting mechanism that regulates the specificity of SCD, and provide mechanistic insight into a role of multisite phosphorylation in DNA-damage signaling.

NKp30 is a natural cytotoxicity receptor (NCR) that is expressed on natural killer (NK) cells and recognizes B7-H6, which is expressed on several types of tumors but few normal cells. To target effector T cells against B7-H6-positive tumors, we have developed several chimeric antigen receptors (CAR) based on NKp30, which contain the CD28 and/or CD3ζ signaling domains with the transmembrane domains from CD3ζ, CD28 or CD8α, respectively. The data show that chimeric NKp30-expressing T cells responded to B7-H6-positive tumor cells. The NKp30 CAR expressing T cells produced IFN-γ and killed B7-H6 ligand-expressing tumor cells, and this response was dependent upon ligand expression on target cells but not on MHC expression. PBMC-derived dendritic cells (DCs) also express NKp30 ligands, including immature DCs (iDCs), and they can stimulate NKp30 CAR-bearing T cells to produce IFN-γ, but to a lesser extent. The addition of a CD28 signaling domain significantly enhanced the activity of the NKp30 CAR in a PI3-kinase-dependent manner. Adoptive transfer of T cells expressing a chimeric NKp30 receptor containing a CD28 signaling domain inhibited the growth of a B7-H6-expressing murine lymphoma (RMA/B7-H6) in vivo. Moreover, mice that remained tumor-free were resistant to a subsequent challenge with the wildtype RMA tumor cells, suggesting the generation of immunity against other tumor antigens. Overall, this study demonstrates the specificity and therapeutic potential of adoptive immunotherapy with NKp30 CAR-expressing T cells against B7-H6+ tumor cells in vivo.

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Chimeric antigen receptor (CAR) T cell therapies have demonstrated durable and potentially curative therapeutic efficacy against B cell leukemia in clinical trials. A CAR strategy can target any tumor surface antigens as long as an antigen-binding receptor can be generated. New CARs which target solid tumors and have the potential to target multiple tumor types are needed. In this study, B7H6, a ligand for the NK cell activating receptor NKp30, was targeted to create a CAR which targets multiple tumor types. B7H6 is expressed on various primary human tumors, including leukemia, lymphoma, and gastrointestinal stromal tumors (GISTs), but it is not constitutively expressed on normal tissues. B7H6-specific CAR T cells have robust cellular cytotoxicity and IFN-γ secretion when co-cultured with B7H6+ tumor cells, and they exhibit little self-reactivity to immature dendritic cells (iDCs) or pro-inflammatory monocytes. In vivo, B7H6-specific CAR T cells greatly enhanced the survival of RMA/B7H6 lymphoma bearing mice. The long-term survivor mice were protected against a B7H6-deficient tumor re-challenge. This CAR therapy also decreased tumor burden in a murine ovarian cancer model. In conclusion, B7H6-specific CARs have the potential to treat B7H6+ hematologic and solid tumors.

Targeting cancer through the use of effector T cells bearing chimeric antigen receptors (CARs) leads to elimination of tumors in animals and patients, but recognition of normal cells or excessive activation can result in significant toxicity and even death. CAR T cells based on modified NKG2D receptors are effective against many types of tumors, and their efficacy is mediated through direct cytotoxicity and cytokine production. Under certain conditions, their ligands can be expressed on non-tumor cells, so a better understanding of the potential off tumor activity of these NKG2D CAR T cells is needed. Injection of very high numbers of activated T cells expressing CARs based on murine NKG2D or DNAM1 resulted in increased serum cytokines (IFNγ, IL-6, MCP-1) and acute toxicity similar to cytokine release syndrome. Acute toxicity required two key effector molecules in CAR T cells – perforin and GM-CSF. Host immune cells also contributed to this toxicity, and mice with severe immune cell defects remained healthy at the highest CAR T cell dose. These data demonstrate that specific CAR T cell effector mechanisms and the host immune system are required for this cytokine release-like syndrome in murine models.

Substantial evidence showed that T cells are the key effectors in immune-mediated tumor eradication. However, most T cells do not exhibit anti-tumor specificity. In this study, a bispecific T cell engager (BiTE) approach was utilized to direct T cells to recognize B7H6+ tumor cells. B7H6 is a specific ligand for the NK cell activating receptor, NKp30. B7H6 is expressed on various types of primary human tumors, including leukemia, lymphoma, and gastrointestinal stromal tumors (GISTs), but it is not constitutively expressed on normal tissues. In this study, data show that B7H6-specific BiTEs direct T cells to mediate cellular cytotoxicity and IFN-γ secretion upon co-culturing with B7H6+ tumors. Furthermore, B7H6-specific BiTE exhibited no self-reactivity to pro-inflammatory monocytes. In vivo, B7H6-specific BiTE greatly enhanced the survival benefit of RMA/B7H6 lymphoma bearing mice through perforin and IFN-γ effector mechanisms. In addition, long term survivor mice were protected against a RMA lymphoma tumor re-challenge. The B7H6-specific BiTE therapy also decreased tumor burden in murine melanoma and ovarian cancer models. In conclusion, B7H6-specific BiTE activates host T cells and has the potential to treat various B7H6+ hematological and solid tumors.

Cell state-specific promoters constitute essential tools for basic research and biotechnology because they activate gene expression only under certain biological conditions. Synthetic Promoters with Enhanced Cell-State Specificity (SPECS) can be superior to native ones, but the design of such promoters is challenging and frequently requires gene regulation or transcriptome knowledge that is not readily available. Here, to overcome this challenge, we use a next-generation sequencing approach combined with machine learning to screen a synthetic promoter library with 6107 designs for high-performance SPECS for potentially any cell state. We demonstrate the identification of multiple SPECS that exhibit distinct spatiotemporal activity during the programmed differentiation of induced pluripotent stem cells (iPSCs), as well as SPECS for breast cancer and glioblastoma stem-like cells. We anticipate that this approach could be used to create SPECS for gene therapies that are activated in specific cell states, as well as to study natural transcriptional regulatory networks.