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General control nonderepressible 2 (GCN2) protein kinase is a cellular stress sensor within the tumor microenvironment (TME), whose signaling cascade has been proposed to contribute to immune escape in tumors. Herein, we report the discovery of cell-potent GCN2 inhibitors with excellent selectivity against its closely related Integrated Stress Response (ISR) family members heme-regulated inhibitor kinase (HRI), protein kinase R (PKR), and (PKR)-like endoplasmic reticulum kinase (PERK), as well as good kinome-wide selectivity and favorable PK. In mice, compound

Agents that remodel the tumor microenvironment (TME), prime functional tumor-specific T cells, and block inhibitory signaling pathways are essential components of effective immunotherapy. We are evaluating live-attenuated, double-deleted Listeria monocytogenes expressing tumor antigens (LADD-Ag) in the clinic. Here we show in numerous mouse models that while treatment with nonrecombinant LADD induced some changes in the TME, no antitumor efficacy was observed, even when combined with immune checkpoint blockade. In contrast, LADD-Ag promoted tumor rejection by priming tumor-specific KLRG1+PD1loCD62L− CD8+ T cells. These IFNγ-producing effector CD8+ T cells infiltrated the tumor and converted the tumor from an immunosuppressive to an inflamed microenvironment that was characterized by a decrease in regulatory T cells (Treg) levels, a proinflammatory cytokine milieu, and the shift of M2 macrophages to an inducible nitric oxide synthase (iNOS)+CD206− M1 phenotype. Remarkably, these LADD-Ag–induced tumor-specific T cells persisted for more than 2 months after primary tumor challenge and rapidly controlled secondary tumor challenge. Our results indicate that the striking antitumor efficacy observed in mice with LADD-based immunotherapy stems from TME remodeling which is a direct consequence of eliciting potent, systemic tumor-specific CD8+ T cells.

Hematopoietic progenitor kinase 1 (HPK1) is an intracellular protein kinase that negatively regulates T cell signaling and proliferation. Upon T cell receptor (TCR) activation, active HPK1 phosphorylates the adaptor protein SLP76 in the TCR complex, recruiting the negative regulator 14-3-3 and targeting components of the TCR signaling complex for degradation. HPK1 thus limits the TCR signaling important for mounting an effective immune response against tumor cells. We are employing structure-guided drug design to develop potent small-molecule inhibitors of HPK1. Our compounds potently inhibit HPK1 in biochemical assays, reduce levels of phosphorylated SLP76 and concomitantly increase IL-2 production by Jurkat T cells. Importantly, our HPK1 inhibitors enhance cytokine production by human and mouse primary T cells above that observed with TCR activation alone. Treatment of mice with orally available HPK1 inhibitors results in increased activation of antigen-specific CD8+ T cells in vivo and decreased tumor growth as single agent and in combination with clinically relevant checkpoint inhibitor antibodies. Our work confirms the importance of HPK1 for T cell function and supports HPK1 as a promising next generation immuno-oncology target. Citation Format: George Katibah, Yamini Ohol, Cyril Bucher, Lavanya Adusumilli, Deepika Kaveri, Omar Robles, Michael Sun, Cynthia Cho, Heather Milestone, Rachel Ames, Scott Jacobson, Dan Nebalasca, Justy Gomez- Guagua, Jerick Sanchez, Molly Grandcolas, Steve Wong, Martin Brovarney, Chandru Ramana, Thant Zaw, Lan Nguyen, Parcharee Tivitmahaisoon, Andrew Ng, Anqi Ma, Blanca Gomez, Michelle Ko, Paul Leger, Jeffrey Jackson, Grant Shibuya, Anton Shakhmin, Delia Bradford, Mengshu Xu, Mikhail Zibinsky, Daniel Poon, David Wustrow, Paul Kassner, Dirk Brockstedt. Development of small-molecule HPK1 inhibitors to unleash tumor-specific T cell responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1646.

Recent advances in cancer metabolism suggest that targeting amino acid metabolism represents a promising strategy for the development of novel therapeutic agents. Tumor, stromal and myeloid-derived suppressor cells (MDSC) within the tumor microenvironment (TME) create a nutrient-poor environment that inhibit immune function and support tumor growth. GCN2 (general control nonderepressible 2), a stress response kinase, plays a key role in maintaining cellular homeostasis under a wide range of stressors. Phosphorylation of GCN2 (pGCN2) in response to stress leads to inhibition of global protein synthesis and subsequently leads to 1) T cell anergy and apoptosis, 2) enhanced MDSC-dependent immune suppression and 3) tumor cell survival. Collectively, these roles suggest that GCN2 inhibition could have both a direct anticancer and an immune-activating effect. Treating nutrient-deprived T cells in vitro with a RAPT GCN2 inhibitor (RPT-GCN2i) rescued CD4+ and CD8+ T cell proliferation and effector functions. The RPT-GCN2i also reversed T cell suppression mediated by MDSCs derived from healthy donors or cancer patients. Using syngeneic mouse tumor models, we demonstrated that translational induction of activating transcription factor 4 (ATF4) is a strong marker of GCN2 pathway activation in vivo. Oral administration of an RPT-GCN2i exhibited notable drug-target occupancy and potently inhibited GCN2 kinase and ATF4 in the TME. RPT-GCN2i as a single agent and in combination with checkpoint blockade or angiogenesis inhibitor (anti-VEGFR) led to delays in tumor growth rate in various syngeneic tumor models. In addition, GCN2 inhibition redirected MDSC within the TME from a suppressive to inflammatory phenotype through downregulation of Arg1 and iNOS. Our results show that inhibition of GCN2 is an attractive approach for enhancing antitumor immune response and therefore GCN2 is a promising therapeutic target for the treatment of cancer. Citation Format: Lisa Marshall, Buvana Ravishankar, Urvi Kolhatkar, Mengshu Xu, Lavanya Adusumilli, Deepa Pookot, Thant Zaw, Chandru Ramana, Raashi Sreenivasan, Mikhail Zibinsky, Jeffrey Jackson, Grant Shibuya, Paul Leger, Omar Robles, Anqi Ma, Andrew Ng, Anton Shakhmin, Justy Guagua, Scott Jacobson, Steve Wong, Delia Bradford, Tracy L. McGaha, M Teresa Ciudad, James E. Talmadge, Holly C. Britton, George Katibah, Gene Cutler, David Wustrow, Paul D. Kassner, Dirk G. Brockstedt. Targeting the stress response kinase GCN2 to restore immunity in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3153.

The accumulation of DNA in the cytosol serves as a key immunostimulatory signal associated with infections, cancer and genomic damage1,2. Cytosolic DNA triggers immune responses by activating the cGAS/STING pathway3. The binding of DNA to the cytosolic enzyme cGAMP synthase (cGAS), activates its enzymatic activity, leading to the synthesis of a second messenger, cyclic[G(2’,5’)pA(3’,5’)] (2’3’-cGAMP)4–8. 2’3’-cGAMP, a cyclic dinucleotide (CDN), activates the protein ‘stimulator of interferon genes’ (STING)9, which in turn activates the transcription factors IRF3 and NF-κB promoting the transcription of genes encoding type I interferons and other cytokines and mediators that stimulate a broader immune response. Exogenous 2’3’-cGAMP and other CDNs, including CDNs produced by bacteria and synthetic CDNs used in cancer immunotherapy, must traverse the cell membrane to activate STING in target cells. How these charged CDNs pass through the lipid bilayer is unknown. Here we used a genome-wide CRISPR interference screen to identify the reduced folate carrier SLC19A1 as the major CDN transporter for uptake of synthetic and naturally occurring CDNs. CDN uptake and functional responses are inhibited by depleting SLC19A1 from cells and enhanced by overexpressing SLC19A1. In both cell lines and primary cells ex vivo, CDN uptake is inhibited competitively by folate and blocked by the SLC19A1 inhibitor sulfasalazine, a medication approved for the treatment of inflammatory diseases. The identification of SLC19A1 as the major transporter of CDNs into cells has far reaching implications for the immunotherapeutic treatment of cancer10, transport of 2’3’-cGAMP from tumor cells to other immune cells to trigger the anti-tumor immune response11, host responsiveness to CDN-producing pathogenic microorganisms12, and potentially in certain inflammatory diseases.

STING is a signaling protein encoded by the human TMEM173 gene that is central to the host response to infection. Upon binding cyclic dinucleotides (CDNs), STING activates transcriptional programs that orchestrate innate and adaptive immune responses ([1][1], [2][2]). For this reason, STING is a

Significance The development of therapeutic cancer vaccines using recombinant microorganisms has been pursued for many decades. However, the underlying mechanisms of therapeutic cancer vaccines remain unclear. Here we compare recombinant Listeria-based cancer vaccines to synthetic long peptide and adenovirus delivery systems for tumor antigens, and describe immunologic correlates of antitumor efficacy of Listeria-based cancer vaccines. Our results show that the profound antitumor efficacy requires tumor microenvironment (TME) remodeling that depends on tumor-specific CD8+ T cells induced by live-attenuated double-deleted Listeria monocytogenes expressing cognate tumor antigens. Together, this work highlights the importance of cognate tumor antigen expression by cancer vaccines and pinpoints the relationship between induced tumor antigen-specific immunity and the TME., Agents that remodel the tumor microenvironment (TME), prime functional tumor-specific T cells, and block inhibitory signaling pathways are essential components of effective immunotherapy. We are evaluating live-attenuated, double-deleted Listeria monocytogenes expressing tumor antigens (LADD-Ag) in the clinic. Here we show in numerous mouse models that while treatment with nonrecombinant LADD induced some changes in the TME, no antitumor efficacy was observed, even when combined with immune checkpoint blockade. In contrast, LADD-Ag promoted tumor rejection by priming tumor-specific KLRG1+PD1loCD62L− CD8+ T cells. These IFNγ-producing effector CD8+ T cells infiltrated the tumor and converted the tumor from an immunosuppressive to an inflamed microenvironment that was characterized by a decrease in regulatory T cells (Treg) levels, a proinflammatory cytokine milieu, and the shift of M2 macrophages to an inducible nitric oxide synthase (iNOS)+CD206− M1 phenotype. Remarkably, these LADD-Ag–induced tumor-specific T cells persisted for more than 2 months after primary tumor challenge and rapidly controlled secondary tumor challenge. Our results indicate that the striking antitumor efficacy observed in mice with LADD-based immunotherapy stems from TME remodeling which is a direct consequence of eliciting potent, systemic tumor-specific CD8+ T cells.

SummarySpontaneous tumor-initiated T cell priming is dependent on IFN-β production by tumor-resident dendritic cells. On the basis of recent observations indicating that IFN-β expression was dependent upon activation of the host STING pathway, we hypothesized that direct engagement of STING through intratumoral (IT) administration of specific agonists would result in effective anti-tumor therapy. After proof-of-principle studies using the mouse STING agonist DMXAA showed a potent therapeutic effect, we generated synthetic cyclic dinucleotide (CDN) derivatives that activated all human STING alleles as well as murine STING. IT injection of STING agonists induced profound regression of established tumors in mice and generated substantial systemic immune responses capable of rejecting distant metastases and providing long-lived immunologic memory. Synthetic CDNs have high translational potential as a cancer therapeutic.

Interferon (IFN) responses play key roles in cellular defense against pathogens. Highly expressed IFN-induced proteins with tetratricopeptide repeats (IFITs) are proposed to function as RNA binding proteins, but the RNA binding and discrimination specificities of IFIT proteins remain unclear. Here we show that human IFIT5 has comparable affinity for RNAs with diverse phosphate-containing 5′-ends, excluding the higher eukaryotic mRNA cap. Systematic mutagenesis revealed that sequence substitutions in IFIT5 can alternatively expand or introduce bias in protein binding to RNAs with 5′ monophosphate, triphosphate, cap0 (triphosphate-bridged N7-methylguanosine), or cap1 (cap0 with RNA 2′- O -methylation). We defined the breadth of cellular ligands for IFIT5 by using a thermostable group II intron reverse transcriptase for RNA sequencing. We show that IFIT5 binds precursor and processed tRNAs, as well as other RNA polymerase III transcripts. Our findings establish the RNA recognition specificity of the human innate immune response protein IFIT5.

Summary: Intratumoral (IT) STING activation results in tumor regression in preclinical models, yet factors dictating the balance between innate and adaptive anti-tumor immunity are unclear. Here, clinical candidate STING agonist ADU-S100 (S100) is used in an IT dosing regimen optimized for adaptive immunity to uncover requirements for a T cell-driven response compatible with checkpoint inhibitors (CPIs). In contrast to high-dose tumor ablative regimens that result in systemic S100 distribution, low-dose immunogenic regimens induce local activation of tumor-specific CD8+ effector T cells that are responsible for durable anti-tumor immunity and can be enhanced with CPIs. Both hematopoietic cell STING expression and signaling through IFNAR are required for tumor-specific T cell activation, and in the context of optimized T cell responses, TNFα is dispensable for tumor control. In a poorly immunogenic model, S100 combined with CPIs generates a survival benefit and durable protection. These results provide fundamental mechanistic insights into STING-induced anti-tumor immunity. : Intratumoral STING pathway activation is a promising therapeutic approach to treat cancer. While high doses of STING agonist are effective at clearing injected tumors, Sivick et al. find that lower doses of STING agonist are optimal for generating robust systemic tumor-specific T cell responses and durable anti-tumor immunity. Keywords: STING, cyclic dinucleotide, intratumoral, ImmunoOncology, anti-tumor immunity, CD8+ T cell, checkpoint inhibitor, ADU-S100, type I interferon, abscopal immunity

SummaryAtopic dermatitis (AD) is a chronic itch and inflammatory disorder of the skin that affects one in ten people. Patients suffering from severe AD eventually progress to develop asthma and allergic rhinitis, in a process known as the “atopic march.” Signaling between epithelial cells and innate immune cells via the cytokine thymic stromal lymphopoietin (TSLP) is thought to drive AD and the atopic march. Here, we report that epithelial cells directly communicate to cutaneous sensory neurons via TSLP to promote itch. We identify the ORAI1/NFAT calcium signaling pathway as an essential regulator of TSLP release from keratinocytes, the primary epithelial cells of the skin. TSLP then acts directly on a subset of TRPA1-positive sensory neurons to trigger robust itch behaviors. Our results support a model whereby calcium-dependent TSLP release by keratinocytes activates both primary afferent neurons and immune cells to promote inflammatory responses in the skin and airways.

Interferon-induced proteins, including the largely uncharacterized interferon-induced tetratricopeptide repeat (IFIT) protein family, provide defenses against pathogens. Differing from expectations for tetratricopeptide repeat (TPR) proteins and from human IFIT1, IFIT2, and IFIT3, we show that human IFIT5 recognizes cellular RNA instead of protein partners. In vivo and in vitro, IFIT5 bound to endogenous 5'-phosphate-capped RNAs, including transfer RNAs. The crystal structure of IFIT5 revealed a convoluted intramolecular packing of eight TPRs as a fold that we name the TPR eddy. Additional, non-TPR structural elements contribute to an RNA binding cleft. Instead of general cytoplasmic distribution, IFIT5 concentrated in actin-rich protrusions from the apical cell surface colocalized with the RNA-binding retinoic acid-inducible gene-I (RIG-I). These findings establish compartmentalized cellular RNA binding activity as a mechanism for IFIT5 function and reveal the TPR eddy as a scaffold for RNA recognition.

Activation of the STING pathway by intratumoral (IT) injection of synthetic cyclic dinucleotides (CDNs) induces stable tumor regression in preclinical models, yet the underlying immune correlates are not fully understood. ADU-S100, a CDN under clinical evaluation, was administered IT with an optimized dosing regimen to explore the immune requirements for antitumor efficacy in mouse syngeneic tumor models. We show that CD8+ T cells are necessary and sufficient for durable antitumor immunity elicited by ADU-S100 and that activation of STING in hematopoietic cells mediates CD8+ T cell induction. Both type I IFN and TNFα, which are induced by STING pathway activation, influence the antitumor immune response. The combination of ADU-S100 and anti-PD1 treatment enhances CD8+ T cell-dependent, noninjected tumor control that correlates with an enhanced effector profile of CD8+ T cells in the tumor. Combination of ADU-S100 with checkpoint inhibition also enhances durable immunity in a poorly immunogenic tumor model. Together, these results elucidate the immune correlates to STING-mediated antitumor efficacy and highlight the potential of combining STING agonists with checkpoint inhibition in the clinic. Citation Format: Anthony L. Desbien, Kelsey Sivick Gauthier, Leticia Corrales, Gabrielle Reiner, Laura Hix Glickman, George Katibah, Thomas E. Hudson, Uyen Vu, Natalie H. Surh, Brian Francica, Weiwen Deng, David B. Kanne, Justin J. Leong, Chudi Ndubaku, Ken Metchette, Jeffery M. McKenna, Steven L. Bender, Meredith L. Leong, Thomas W. Dubensky Jr., Andrea van Elsas, Sarah M. McWhirter. Intratumoral activation of STING with a synthetic cyclic dinucleotide elicits antitumor CD8 T-cell immunity that effectively combines with checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 631.

Human embryonic stem cells and induced pluripotent stem cells (hESCs and hiPSCs) are powerful tools for biomedical research. Realizing the full potential of these cells requires efficient genetic modification. However, techniques to generate cell type specific lineage reporters as well as reliable tools to disrupt, repair or overexpress genes by gene targeting are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc finger nuclease (ZFN) mediated genome editing. First, using ZFNs specific for the OCT4 locus we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Secondly, we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally, we targeted the PITX3 gene, demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs.

Agricultural biotechnology is limited by the inefficiencies of conventional random mutagenesis and transgenesis. Because targeted genome modification in plants has been intractable, plant trait engineering remains a laborious, time-consuming and unpredictable undertaking. Here we report a broadly applicable, versatile solution to this problem: the use of designed zinc-finger nucleases (ZFNs) that induce a double-stranded break at their target locus. We describe the use of ZFNs to modify endogenous loci in plants of the crop species Zea mays. We show that simultaneous expression of ZFNs and delivery of a simple heterologous donor molecule leads to precise targeted addition of an herbicide-tolerance gene at the intended locus in a significant number of isolated events. ZFN-modified maize plants faithfully transmit these genetic changes to the next generation. Insertional disruption of one target locus, IPK1, results in both herbicide tolerance and the expected alteration of the inositol phosphate profile in developing seeds. ZFNs can be used in any plant species amenable to DNA delivery; our results therefore establish a new strategy for plant genetic manipulation in basic science and agricultural applications.

We describe here the use of zinc finger nucleases (ZFNs) for somatic and germline disruption of genes in zebrafish (Danio rerio), where targeted mutagenesis was previously intractable. ZFNs induce a targeted double-strand break in the genome that is repaired to generate small insertions and deletions. We designed ZFNs targeting the zebrafish golden and no tail/Brachyury genes. In both cases, injection of ZFN-encoding mRNA into 1-cell embryos yielded a high percentage of animals carrying distinct mutations at the ZFN-specified position and exhibiting expected loss-of-function phenotypes. Disrupted ntl alleles were transmitted from ZFN mRNA-injected founder animals in over half the adults tested at frequencies averaging 20%. The frequency and precision of gene disruption events observed, in combination with the ability to design ZFNs against any locus, open fundamentally novel avenues of experimentation, and suggest that ZFN technology may be widely applied to many organisms that allow mRNA delivery into the fertilized egg.

Chromosomal translocations are signatures of numerous cancers and lead to expression of fusion genes that act as oncogenes. The wealth of genomic aberrations found in cancer, however, makes it challenging to assign a specific phenotypic change to a specific aberration. In this study, we set out to use genome editing with zinc finger (ZFN) and transcription activator-like effector (TALEN) nucleases to engineer, de novo, translocation-associated oncogenes at cognate endogenous loci in human cells. Using ZFNs and TALENs designed to cut precisely at relevant translocation breakpoints, we induced cancer-relevant t(11;22)(q24;q12) and t(2;5)(p23;q35) translocations found in Ewing sarcoma and anaplastic large cell lymphoma (ALCL), respectively. We recovered both translocations with high efficiency, resulting in the expression of the EWSR1–FLI1 and NPM1–ALK fusions. Breakpoint junctions recovered after ZFN cleavage in human embryonic stem (ES) cell–derived mesenchymal precursor cells fully recapitulated the genomic characteristics found in tumor cells from Ewing sarcoma patients. This approach with tailored nucleases demonstrates that expression of fusion genes found in cancer cells can be induced from the native promoter, allowing interrogation of both the underlying mechanisms and oncogenic consequences of tumor-related translocations in human cells. With an analogous strategy, the ALCL translocation was reverted in a patient cell line to restore the integrity of the two participating chromosomes, further expanding the repertoire of genomic rearrangements that can be engineered by tailored nucleases.

Stimulator of Interferon Genes (STING) is a critical component of the cytosolic DNA sensing pathway of the innate immune system. STING is activated by cyclic dinucleotides (CDNs), a product of the intracellular enzyme, cyclic GMP-AMP synthase (cGAS), in response to presence of cytosolic DNA, including tumor-derived DNA. Production of type I interferon within the tumor microenvironment (TME), mediated by the STING pathway, leads to the priming and activation of systemic tumor antigen-specific CD8+ T-cell immunity and tumor regression. Therapeutic activation of STING through intratumoral (IT) administration of CDNs results in anti-tumor efficacy and long-lived survival in several mouse syngeneic tumor models. Rational design of synthetic CDN derivatives has shown that certain modifications alter STING binding, increase cellular potency, enhance maturation of human dendritic cells to promote in vitro T cell expansion, and are able to broadly activate all human STING haplotypes. Mechanistic studies in mouse tumor models demonstrate that CDNs mediate anti-tumor immunity by inducing an acute innate immune response, leading to collapse of the injected tumor, and promoting a tumor-specific CD8+ T cell response that protects against tumor re-challenge. Anti-tumor efficacy is enhanced by combination with immune checkpoint inhibitors, informing future clinical development. The ability to elicit innate and adaptive anti-tumor immunity via activation of STING in the TME demonstrates that CDNs have high translational potential for the treatment of patients with advanced/metastatic solid tumors. The design of an ongoing Phase 1 first-in-human clinical study to evaluate the safety, tolerability and possible antitumor activity of ADU-S100 in subjects with cutaneously accessible tumors and lymphomas will also be presented. Citation Format: Sarah M. McWhirter, Laura Hix Glickman, Tony Desbien, Kelsey Sivick Gauthier, David Kanne, Shailaja Kasibhatla, Jie Li, AnneMarie Culazzo Pferdekamper, George Katibah, Ed Lemmens, Leticia Corrales, Meredith Leong, Chudi Ndubaku, Justin Leong, Leonard Sung, Lianxing Zheng, Charles Cho, Yan Feng, Jeffery M. McKenna, John A. Tallarico, Steven L. Bender, Thomas W. Dubensky, Jr.. STING activation in the tumor microenvironment using a synthetic human STING-activating cyclic dinucleotide induces potent antitumor immunity [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B020.

Recent reports have provided the mechanistic insight of how innate immune activation promotes priming of anti-tumor immunity and inform the development of clinical approaches to facilitate this process. Spontaneous T cell infiltration of melanoma lesions in humans is correlated with a type I interferon (IFN) transcriptional profile in the tumor microenvironment (TME) and infiltration of lymphocytes, indicative of ongoing innate immune recognition within the tumor. Substantial evidence indicates that tumor infiltrating lymphocytes (TILs), including activated CD8+ T cells, is predictive of a positive clinical outcome in response to several immunotherapy strategies. Similarly, in mice bearing melanoma, there is a correlation between expression of IFN-β by tumor-resident dendritic cells (DCs), and spontaneous priming of tumor-specific immunity. Induction of IFN-β expression and co-regulated IFN-responsive genes and pro-inflammatory chemokines is dependent upon activation of the STING (Stimulator of Interferon Genes) pathway, mediated through sensing of tumor dsDNA in TME-resident CD8α+ DCs by cyclic GMP-AMP (cGAMP) synthase (cGAS), which in turn synthesizes cGAMP. The cyclic dinucleotide (CDN) cGAMP produced by cGAS is the natural STING agonist ligand. Thus, the cGAS-STING signaling axis has emerged as a central node for sensing damage in the host. We hypothesized that direct activation of the STING pathway in the TME by intratumoral (IT) injection of specific CDNs would be an effective therapeutic strategy to promote broad tumor-initiated T cell priming against an individual's tumor antigen repertoire. There are five variant human STING alleles that exist at varying frequencies. While the natural STING ligand cGAMP activates signaling in all variants, other natural CDNs, including those produced by bacteria, have structural differences and are unable to activate particular STING variants, such as the REF (R232H) allele, informing the development of compounds that activate all human STING alleles. We sought to develop synthetic CDN compounds with increased activity in human cells as well as the ability to engage all known polymorphic human STING molecules. Using human 293T cell lines engineered to express the various STING proteins, we screened a large panel of CDN derivatives that varied in purine nucleotide base, structure of the phosphate bridge linkage, and substitution of the non-bridging oxygen atoms at the phosphate bridge with sulfur atoms. ADU-S100 is composed of two adenosine monophosphate (AMP) analogues cyclized via a 2’-5’ (non-canonical) and a 3’-5’ (canonical) phosphodiester bond, and was selected for clinical translation based on properties of enhanced cellular uptake, human STING activation, stability and anti-tumor efficacy, as compared to bacterial and mammalian derived CDNs. Induced cytokine expression from a panel of donor human peripheral blood mononuclear cells (PBMCs) expressing a variety of STING haplotypes, including donors with a homozygous haplotype for the refractory human REF allele, indicates that ADU-S100 activates STING across a diverse human population. Direct engagement of STING through IT administration of ADU-S100 results in effective anti-tumor therapy and long-term survival in various mouse syngeneic tumor models. IT injection of ADU-S100 also generates substantial systemic immune responses capable of rejecting distant metastases and provided long-lived immunologic memory. A bell-shaped ADU-S100 dose response curve (which varied based on tumor model) delineated regression of injected tumor, induction of tumor-specific CD8+ T cell immunity, and regression of distal non-injected tumors (abscopal effect), and/or protection against autologous tumor challenge. At low dose levels, regression of the treated tumor was suboptimal. At optimal doses, regression of the treated and distal untreated tumors, or protection against tumor re-challenge was observed, and correlated with induction of a robust tumor Ag-specific CD8+ T cell response. At higher dose levels there was a loss of protection against tumor re-challenge which correlated with increases in acute systemic cytokines and reduction in CD8+ T cell responses even though growth of the treated tumor was inhibited. These results suggest that the mechanism of ADU-S100-induced tumor regression is due to both an acute pro-inflammatory cytokine response and also tumor-specific CD8+ T cell immunity. The local anti-tumor effect without systemic immunity is consistent with well-established data in which excessive innate immune stimulation and induction of pro-inflammatory cytokines such as TNF-α are known to inhibit both priming of CD8+ T cell immunity and establishment of a stable and self-renewing memory CD8+ T cell population. In addition, anti-tumor efficacy was enhanced by combination with immune checkpoint inhibitors, for example α-PD1, informing future clinical development. By virtue of the ability to elicit innate and T cell-mediated anti-tumor immunity in the TME, these results demonstrate that CDNs have high translational potential for the treatment of patients with advanced/metastatic solid tumors. A Phase 1 clinical study to evaluate the safety and tolerability and possible anti-tumor effects in subjects with cutaneously accessible non UV-induced and UV-induced malignancies or lymphomas given repeated IT doses of ADU-S100 is planned. Citation Format: Laura Hix Glickman, Leticia Corrales, David B. Kanne, Shailaja Kasibhatla, Jie Li, Anne Marie Culazzo Pferdekamper, Kelsey Sivick Gauthier, George E. Katibah, Justin J. Leong, Leonard Sung, Ken Metchette, Weiwen Deng, Anthony L. Desbien, Chudi Ndubaku, Lianxing Zheng, Charles Cho, Yan Feng, Jeffery M. McKenna, John A. Tallarico, Steven L. Bender, Sarah M. McWhirter, Thomas F. Gajewski, Thomas W. Dubensky. Direct activation of STING in the tumor microenvironment leads to potent and systemic tumor regression and immunity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr SY39-02.

Stimulator of Interferon Genes (STING) is a critical signaling sensor of the innate immune system. STING binds cyclic dinucleotides (CDN) produced by an intracellular enzyme in response to presence of intracellular DNA, including tumor-derived DNA. STING-mediated production of host type I interferon within the tumor microenvironment (TME) leads to the priming and activation of systemic tumor antigen-specific CD8+ T-cell immunity and tumor regression. A novel synthetic CDN derivative (ADU-S100), with superior STING-activating and anti-tumor properties, was developed for clinical translation. ADU-S100 has enhanced cellular uptake properties and stability, as compared to bacterial- and mammalian-derived CDNs. Induced cytokine expression from a panel of donor human peripheral blood mononuclear cells (PBMCs) expressing a variety of STING alleles, including a homozygous haplotype for the most refractory human allele (R232H), indicate that ADU-S100 activates STING across a diverse human population. Direct engagement of STING through intratumoral (IT) administration of ADU-S100 results in effective anti-tumor therapy and long-term survival in various mouse syngeneic tumor models. IT injection of ADU-S100 also generates substantial systemic immune responses capable of rejecting distant metastases and provides long-lived immunologic memory. Mechanistic studies demonstrate that STING-mediated anti-tumor immunity is due in part to an acute pro-inflammatory cytokine response as well as a tumor-specific CD8+ T cell response. Anti-tumor efficacy is enhanced by combination with immune checkpoint inhibitors, for example anti-PD1, informing future clinical development. By virtue of the ability to elicit innate and T cell-mediated anti-tumor immunity in the TME, these results demonstrate that CDNs have high translational potential for the treatment of patients with advanced/metastatic solid tumors. Citation Format: Laura Hix Glickman, David B. Kanne, Shailaja Kasibhatla, Jie Li, AnneMarie Culazzo Pferdekamper, Kelsey Sivick Gauthier, Weiwen Deng, Anthony L. Desbien, George E. Katibah, Justin J. Leong, Leonard Sung, Ken Metchette, Chudi Ndubaku, Lianxing Zheng, Charles Cho, Yan Feng, Jeffrey M. McKenna, John A. Tallarico, Steven L. Bender, Thomas W. Dubensky, Sarah M. McWhirter. STING activation in the tumor microenvironment with a synthetic human STING-activating cyclic dinucleotide leads to potent anti-tumor immunity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1445.

The development of zinc finger nucleases for targeted gene modification can benefit from rapid functional assays that directly quantify activity at the endogenous target. Here we describe a simple procedure for quantifying mutations that result from DNA double-strand break repair via non-homologous end joining. The assay is based on the ability of the Surveyor nuclease to selectively cleave distorted duplex DNA formed via cross-annealing of mutated and wild-type sequence.

The development of zinc finger nucleases for targeted gene modification can benefit from rapid functional assays that directly quantify activity at the endogenous target. Here we describe a simple procedure for quantifying mutations that result from DNA double-strand break repair via non-homologous end joining. The assay is based on the ability of the Surveyor nuclease to selectively cleave distorted duplex DNA formed via cross-annealing of mutated and wild-type sequence.

Spontaneous tumor-initiated T cell priming is dependent on IFN-β production by tumor-resident dendritic cells. Based on recent observations indicating that IFN-β expression was dependent upon activation of the host STING (Stimulator of Interferon Genes) pathway, we hypothesized that direct engagement of STING through intratumoral administration of specific agonists would result in effective anti-tumor therapy. To this end, we generated novel synthetic cyclic dinucleotide (CDN) derivatives with superior STING-activating and anti-tumor properties. The most potent molecule contains a 2′-5′ and 3′-5′ phosphate bridge that increases its affinity for STING at least 10-fold. Additionally, sulfur atoms at the non-bridging oxygens of the internucleotide phosphate bridge enable both superior STING binding affinity, as well as resistance to host cell phosphodiesterases. Importantly, the lead molecule activates PBMCs from a panel of human donors representing all known STING alleles, including homozygotes for the most refractory allele. Intratumoral injection of the lead CDN molecule induced profound regression of established B16 melanoma tumors, generated substantial systemic immune responses capable of rejecting distant metastases and provided long-lived immunologic memory. Cures in entire cohorts of mice bearing well-established 4T1 mammary and CT26 colon carcinoma models correlated with induction of cytokine-mediated innate and adaptive antigen-specific T cell immunity, both systemically and in the tumor microenvironment. Immune activation and anti-tumor efficacy was lost in mice encoding non-functional STING. In vivo activation of STING with the lead CDN administered by multiple routes demonstrated a high safety and tolerability margin in preclinical toxicology studies. In addition, pharmacokinetic analysis indicates that the lead CDN compound was rapidly cleared from systemic circulation, consistent with pulsatile STING activation and transient systemic cytokine and hematological changes. These results demonstrate that CDNs have high translation potential for treatment strategies to induce activation of the tumor microenvironment, leading to effective tumor-initiated T cell priming and anti-tumor immunity. Citation Format: Laura Hix Glickman, David B. Kanne, Kelsey E. Gauthier, George E. Katibah, Justin J. Leong, Ken Metchette, Thomas W. Dubensky, Sarah M. McWhirter. Potent in situ cancer immunotherapy with synthetic human STING-activating cyclic dinucleotides. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4272. doi:10.1158/1538-7445.AM2015-4272