Treating Cancer by Mimicking Infection

Author Manuscript Published OnlineFirst on May 23, 2019; DOI: 10.1158/1078-0432.CCR-18-1800 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Pathogen molecular pattern receptor agonists: treating cancer by mimicking infection

Running Title: Pattern recognition receptors in immuno-oncology

Mark Aleynick;1 Judit Svensson-Arvelund, PhD;1 Christopher R. Flowers, MD;2 Aurélien Marabelle, MD, PhD;3 Joshua D. Brody, MD.1,4,*

1Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA 2Winship Cancer Institute of Emory University, Emory University School of Medicine, Atlanta, GA 30307, USA 3Cancer Immunotherapy Program, Gustave Roussy, Villejuif 94800, France 4Hematology and Medical Oncology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA

*Correspondence: Joshua D. Brody [email protected]; 212-241-6756 Hess CSM Floor 5 Room 106 1470 Madison Avenue, New York, NY 10029

Disclosures:

Mark Aleynick has no conflicts of interest to disclose.

Judit Svensson-Arvelund has no conflicts of interest to disclose

Christopher Flowers has served as a consultant for: Abbvie, AstraZeneca, Bayer, BeiGene, Celgene (unpaid), Denovo Biopharma, Genentech/Roche (unpaid), Gilead, OptumRx, Karyopharm, Pharmacyclics/ Janssen, Spectrum. Dr. Flowers has received research funding from: Abbvie, Acerta, BeiGene, Celgene, Gilead, Genentech/Roche, Janssen Pharmaceutical, Millennium/Takeda, Pharmacyclics, TG Therapeutics, Burroughs Wellcome Fund, Eastern Cooperative Oncology Group, National Cancer Institute, ORIEN/M2Gen, and the V Foundation.

Aurélien Marabelle is the Principal Investigator of Clinical Trials from the following companies: Merck (MSD). Dr. Marabelle has served as a consultant for: Innate Pharma, Merck Serono, eTheRNA, Lytix pharma, Kyowa Kirin Pharma, Bayer, Novartis, BMS, Symphogen, Genmab, Amgen, Biothera, Nektar, GSK, Oncovir, Pfizer, Seattle Genetics, Flexus Bio, Roche/Genentech, OSE immunotherapeutics, Transgene, Gritstone, Merck (MSD), Cerenis, Protagen, Partner Therapeutics, Servier, Sanofi, Pierre Fabre, Molecular Partners, Roche, Pierre Fabre, Onxeo, EISAI, Bayer, Genticel, Rigontec, Daichii Sankyo, Imaxio, Sanofi, BioNTech, Corvus, GLG, Deerfield, Guidepoint Global, Edimark, System Analytics, imCheck, Sotio, Bioncotech, Pillar Partners, Boehringer Ingelheim. Dr. Marabelle has received research funding from Astrazeneca, BMS, Boehringer Ingelheim, Janssen Cilag, Merck, Novartis, Pfizer, Roche, Sanofi

Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 23, 2019; DOI: 10.1158/1078-0432.CCR-18-1800 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Joshua Brody has served as a consultant for: AstraZeneca, Bayer, Calithera, Celldex Therapeutics, Gilead, Merck, Pharmacyclics/ Janssen, Seattle Genetics, Teva. Dr. Brody has received research funding from: Acerta, Celgene, Gilead, Genentech/Roche, the Cancer Research Institute, and the Damon Runyon Cancer Research Foundation. Abstract

Immunotherapies such as checkpoint blockade have achieved durable benefits for patients with advanced stage cancer and have changed treatment paradigms. However, these therapies rely on a patient’s own a priori primed tumor-specific T cells, limiting their efficacy to a subset of patients. Because checkpoint blockade is most effective in patients with inflamed or ‘hot’ tumors, a priority in the field is learning how to ‘turn cold tumors hot’. Inflammation is generally initiated by innate immune cells which receive signals through pattern recognition receptors (PRRs) – a diverse family of receptors that sense conserved molecular patterns on pathogens, alarming the immune system of an invading microbe. Their immunostimulatory properties can reprogram the immune suppressive tumor microenvironment and activate antigen presenting cells (APC) to present tumors antigens, driving de-novo tumor-specific T cell responses. These features, among others, make PRR-targeting therapies an attractive strategy in immuno- oncology. Here, we discuss mechanisms of PRR activation, highlighting ongoing clinical trials and recent pre-clinical advances focused on therapeutically targeting PRRs to treat cancer.

Introduction

The interplay between cancer and the immune system is a double edged sword; the inflammation that recruits and activates intratumoral immune cells can either eliminate cancer cells or drive tumor progression in a context dependent manner (1). PRRs are a key family of proteins involved in the inflammatory response. They are expressed on a wide variety of innate and adaptive immune cells as well as tumor cells, and recognize both foreign pathogen associated molecular patterns (PAMPs) and self-derived damage associated molecular patterns (DAMPs) resulting from injury or cell death (1–3). There are 5 families of PRRs: toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), C-type lectin receptors (CLRs), RIG-I-like receptors (RLRs), and cytosolic DNA sensors (CDS) (2). Each PRR family possesses distinct immunomodulatory properties, making them attractive immunotherapeutic targets. Here, we discuss PRR mechanisms and clinical implications to provide a detailed overview of the role of PRRs in immuno-oncology.

TLRs in Cancer Immunotherapy

TLRs are the most widely studied PRR family, acknowledged in the 2011 Nobel Prize awarded to Drs. Steinman, Beutler, and Hoffman. There have been 10 TLRs identified in humans and 13 in mice (4); here we focus on the former. Structurally, TLRs are type I transmembrane proteins characterized by a ligand-binding N terminal ectodomain containing leucine-rich repeats, a single transmembrane domain, and a cytosolic Toll/IL-1R homology domain responsible for signal transduction (2). TLRs 1, 2, and 4-6 are located on the cell surface and recognize bacterial

membrane components such as lipids, proteins, lipoproteins (Fig. 1) (2,3), as well as several self- molecules, including extracellular matrix components, heat shock proteins (HSPs), and nuclear high-mobility group box 1 (HMGB1), often released as DAMPs from apoptotic or necrotic cells (1,3). Intracellular TLRs 3 and 7-9 are located within endosomes, and recognize viral and bacterial nucleic acids resulting from microbial replication or degradation upon entry into the cell (Fig. 1) (1–3). Their localization normally prevents intracellular TLRs from binding self nucleic acids. However, breakdown of this spatial separation may trigger autoimmune disease through recognition of self nucleic acids (3,5). Although TLR10 exists in humans, it has been difficult to study as it is non-functional in mice. Recent work suggests it may serve as a negative regulator of TLR signaling (6,7). TLRs dimerize upon binding their cognate ligand (Fig. 1), causing conformational changes that allow for the recruitment of adapter molecules (MyD88, TIRAP, TRIF, and TRAM), initiating signaling cascades that ultimately induce transcription of inflammatory mediators (2,3,8).

As TLRs are highly expressed on antigen presenting cells (APCs), targeting TLRs can activate APCs and trigger adaptive immune responses; intratumorally, this may shift a tolerogenic tumor microenvironment (TME) to become immunogenic. However, because TLR signaling triggers inflammatory and cell survival mechanisms, and certain tumors express TLRs, TLR activation could instead be tumorigenic in certain settings (1,9). Both TLR7 and TLR8 signaling have been implicated in driving lung cancer cell survival and chemotherapy resistance mechanisms (10,11). TLR4 signaling in breast cancer both enhances chemotherapeutic resistance and promotes angiogenesis and lymphatic metastasis (12,13). Tumor cells may also secrete heat shock proteins and extracellular matrix factors as DAMPs, stimulating an immunosuppressive program in tumor associated macrophages (TAMs) to promote angiogenesis and metastasis (14,15). One recent study demonstrated that mice lacking TLR3/7/9 cleared implanted tumors through spontaneous induction of an adaptive anti-tumor response (16). Similar effects are observed with other PRR families; galectin-9 signaling through the CLR dectin-1 on TAMs is pro-tumorigenic in mouse models of pancreatic ductal adenocarcinoma, although signaling through this receptor in other cancers may have the opposite effect (17). While several of these reports implicate DAMPs in tumor initiation and progression through chronic inflammation, other studies demonstrate that DAMPs released from dying tumor cells are the hallmark of immunogenic cell death, activating APCs in the TME to present tumor antigen (9,18). Despite the nuanced role of PRR signaling in cancer, in many contexts, therapies targeting PRR pathways have the ability to overcome immunosuppression or drive a de novo anti-tumor response by activating APCs to enhance tumor antigen presentation (Fig. 2). For the remainder of this review, we will focus on clinical trials and pre-clinical studies utilizing PRRs in this setting.

One of the few FDA approved TLR-targeting therapies in oncology is bacillus Calmette–Guérin (BCG), a strain of Mycobacterium bovis initially developed as a tuberculosis vaccine. Used as a urogenital cancer therapeutic for over 35 years, a large body of work has dissected its mechanism, demonstrating that BCG triggers an immune response by activating TLRs 2, 4, 9, and the NLR NOD2 (1,19,20). Several trials are combining BCG therapy with checkpoint blockade or have expanded BCG to other cancers, with varying degrees of success (21).

TLR3 is one of the more actively explored TLR targets, with 54 ongoing clinical trials using TLR3 agonists as single agents or in combination with other therapies to treat a broad list of malignancies (clinicaltrials.gov). TLR3 recognizes viral double stranded RNA (dsRNA), and can be targeted using synthetic dsRNA analogs such as polyinosinic-polycytidylic acid (poly- IC) (2,3,22). Poly-IC initially showed high toxicity and limited therapeutic benefit, but several poly-IC derivatives were subsequently created to improve efficacy (22). One such derivative is poly-ICLC, modified with poly-L-lysine and carboxymethylcellulose (Hiltonol, Oncovir) to increase stability in vivo, improving its interferon response to levels similar to those seen with attenuated viral infections (23,24). Another derivative, poly-IC12U (rintatolimod/Ampligen, Hemispherex Biopharma), adds unpaired bases that reduce stability, effectively reducing toxicity while generating robust DC/T cell responses (25). Pre-clinical data also suggests that rintatolimod recruits fewer Tregs to the TME versus unmodified poly-IC, possibly by losing ability to bind cytosolic RLRs (26). Recently completed trials using poly-ICLC demonstrate its potent ability to induce adaptive immune responses against a range of solid and hematopoietic cancers. A phase 1 study evaluating peptide pulsed DC vaccination in combination with poly-ICLC for pancreatic cancer showed promise with a 7.7-month median survival, an improvement over the 4.2-4.9-month survival seen with second line chemotherapy in metastatic pancreatic cancer (27). A phase 1/2a trial in smoldering multiple myeloma recently demonstrated that peptide + poly-ICLC vaccination increased numbers of antigen-specific CD8 T cells with an effector memory phenotype (28). Another study pinpointed TLR3+ DC as key mediators of tumor antigen cross-presentation, where an in situ vaccination combining poly- ICLC, Flt3 ligand, and local irradiation induced both partial and complete responses in non- Hodgkin’s lymphoma patients (29). A pilot study in transplant-ineligible hepatocellular carcinoma patients showed survival benefit compared to historical controls using local tumor irradiation followed by intratumoral poly-ICLC administration (30). Similarly, studies treating glioblastoma patients demonstrated impressive survival outcomes by combining poly-ICLC with irradiation and/or alkylating chemotherapy (31,32). In the pre-clinical setting, next generation DC vaccines are being explored, employing nanoparticles to selectively deliver tumor antigens + poly-IC to DCs in vivo, eliminating the need for ex vivo DC manipulation (33). Additionally, several groups have also demonstrated that TLR3 activation can help overcome resistance to checkpoint blockade, leading to ongoing academic and pharma trials planning to accrue > 400 patients studying combinations of poly-ICLC with PD-1, PD-L1, or CTLA-4 blockade to treat various cancers (e.g. NCT03121677, NCT03633110).

TLR4 is canonically involved in the recognition of bacterial lipopolysaccharide (LPS), although it is also indirectly involved in viral infection by recognizing DAMPs – such as HMGB1, HSPs and extracellular matrix components, - released from infected or dying cells (1,3,8). Ongoing clinical efforts with TLR4 ligands in cancer immunotherapy include the FDA approved TLR4 agonist AS04 (GlaxoSmithKline), a monophosphoryl lipid A (MPLA) LPS derivative in alum. AS04 is used as an adjuvant in the HPV-16/18 vaccine Cervarix, which in a landmark trial was shown to not only protect women from cervical cancer from the vaccine-inclusive strains, but was also cross-reactive against other oncogenic forms of HPV (34). Interestingly, two recent phase 3 trials of a MAGE-A3 vaccine with AS15 (GlaxoSmithKline), an adjuvant containing MPLA and a TLR9 agonist, both failed to improve patient survival, citing low CD8 T cell responses in patients (35,36). Another TLR4 agonist, a synthetic analog of glucopyranosyl lipid A engineered to decrease heterogeneity and minimize toxicity over natural lipid A formulated in

a stable emulsion (GLA-SE/G100; Immune Design), showed promise in a phase 1 study of Merkel Cell Lymphoma , where 2 of 10 patients had durable sustained anti-tumor responses while 2 others had complete responses (37). Similarly encouraging clinical responses were seen in 26 patients with follicular lymphoma receiving intratumoral G100 and radiotherapy with or without PD-1 blockade, where >80% disease control rates were seen in both groups, and addition of anti-PD-1 benefited relapsed and chemo-refractory patients (38).

TLR5 is unique in that it does not recognize DAMPs as its only ligand is bacterial flagellin; making it a potentially useful immunotherapeutic target (39). Two TLR5 agonists in clinical development, entolimod and mobilan (Cleveland Bio Labs), have shown pre-clinical efficacy in several tumor models (39–41). Entolimod is a flagellin derivative engineered to reduce toxicity, currently being investigated in a phase 2 trial as a neo-adjuvant therapy for colorectal cancer (NCT02715882) (42). Mobilan is an adenovirus construct that upon infection induces co- expression of TLR5 and a secreted form of entolimod, creating an autocrine signaling loop and inflammatory signature in the TME (41). Mobilan has shown pre-clinical efficacy for prostate cancer, which expresses high levels of the adenovirus receptor necessary for its entry, and is now in a phase 1/2 trial (NCT02844699) (41).

Both TLR7 and TLR8 are functional in humans, while mice only have functional TLR7. TLR7/8 recognize single stranded RNA from RNA viruses, although RNA from certain bacterial strains may also ligate these TLRs (8). TLR7/8 also recognize purine analogs such as imidazoquinolines, as well as guanine derivatives and certain siRNA (3). Ligation of TLR7/8 triggers robust proinflammatory cytokine production, and is critical for the activation of plasmacytoid DC (pDC), a key source of type 1 interferons (3,8). The only FDA approved TLR7/8 agonist is imiquimod (Aldara; 3M Pharmaceuticals), an imidazoquinoline topical agent for the treatment of basal cell carcinoma (BCC) that both enhances local immune response and directly induces apoptosis in BCC cells (43). Imiquimod is being investigated in phase 3 studies as a treatment for gynecologic cancers with promising early results (44), and in dozens of phase 1 and 2 trials in various cancers, either alone or combination (clinicaltrials.gov). Other imidazoquinoline derivatives in the clinic include a topical gel formulation of resiquimod, a more potent imidazoquinoline investigated as an adjuvant to NYESO-1 vaccination for melanoma patients that has been shown to induce NYESO specific CD8 T cell responses (45). DSP-0509 (Boston Biomedical), a TLR7/8 agonist formulated for intravenous (IV) delivery, has shown pre- clinical efficacy in several tumor models and is now being investigated in a phase 1 trial (NCT03416335) (46). MEDI9197 (3M-052; Medimmune) is an imidazoquinoline formulated for intratumoral injection and optimal tumor retention to improve safety. Preliminary phase 1 results (NCT02556463) demonstrate intratumoral immune cell infiltration and low serum MEDI9197 levels, indicating effective retention in the TME (47). Similarly, NKTR-262 (Nektar Therapeutics) is a TLR7/8 agonist formulated for intratumoral retention to minimize systemic exposure (48) and has shown potent efficacy, where treatment of one tumor site led to clearance of untreated contralateral tumors in multiple pre-clinical models, an abscopal effect often considered the holy grail of intratumoral immunotherapy. These promising results led to a recently opened phase 1/2 study of NKTR-262 in combination with a CD122 agonistic antibody and checkpoint blockade (NCT03435640) (49). A recent randomized study of platinum-based chemoimmunotherapy for head/neck cancers demonstrated no overall survival benefit by adding

the TLR8 agonist motolimod (Array Biopharma/Celgene), although motolimod did improve survival in subsets of patients with HPV+ tumors (50).

TLR9 recognizes unmethylated 2′-deoxyribo(cytidine-phosphate-guanosine) (CpG) motifs which occur more frequently in prokaryotic DNA. Similarly to TLRs 7/8, TLR9 is highly expressed on pDCs, as well as on B cells, and is critical in the immune response to DNA viruses (3,8). Synthetic CpG oligodeoxynucleotides (ODN) potently activate TLR9 expressing immune cells and have been divided into four classes: Class A, B, C and P (51,52). Class A ODNs contain palindromic phosphodiester CpG central sequences with phosphotionate G rich ends, allowing tetrad formation, enhanced stability, endosomal uptake, and robust activation of pDC type 1 interferon responses. Class B ODNs are short, linear phosphorothionate backbone ssDNA strands, and potent activators of B and NK cells. Class C ODNs combine properties of class A and B, activating both B and NK cells as well as type 1 interferon pDC responses. Class P ODNs feature multiple palindromic sequences and form multimeric structures, enhancing stability and immunostimulatory responses (52). The first ODN in human trials was CpG7909 (agatolimod/PF-3512676/ProMune; Pfizer), a class B ODN, which showed early promise both as an in situ vaccination and chemotherapy adjuvant (53–55). However, a phase 3 lung cancer trial of chemotherapy with or without this ODN concluded that CpG7909 increased adverse events without benefiting survival, curtailing its development. Two other phase 3 trials that investigated CpG7909 as part of a MAGE-A3 vaccination also failed to demonstrate clinical benefit (35,36).

Despite failures with CpG7909, several CpG ODNs modified to enhance efficacy and safety are in development. CMP-001 (Checkmate Pharmaceuticals), a class A ODN packaged within a virus-like particle, potently activates intratumoral pDCs and overcomes resistance to checkpoint blockade; 5 trials with this ODN are ongoing for various solid tumors (clinicaltrials.gov) (56). Tilsotolimod (IMO-2125; Idera Pharmaceuticals) is another TLR9 agonist that is being investigated in checkpoint blockade refractory patients. In patients who failed anti-PD-1 therapy, tilsotolimod combined with ipilimumab CTLA-4 blockade improved objective tumor responses over ipilimumab alone, and this combination has entered a phase 3 trial (NCT03445533) (57). Lefitolimod (MGN1703, Mologen AG) is a novel class of ODN that lacks phosphorothionate backbone modifications and is instead ‘dumbbell shaped’ to prevent degradation (51). Demonstrating favorable safety and clinical efficacy, lefitolimod has initiated a phase 3 trial in metastatic colorectal cancer (NCT02077868) (58,59). Another class C agonist, SD-101 (Dynavax) is being investigated in several trials, after showing both pre- clinical and clinical efficacy in melanoma and as an in situ vaccination for lymphoma, in combination with checkpoint blockade and agonistic antibodies for T cell co-stimulation (55,60– 62). DV281, a TLR9 agonist formulated for inhalation, is being investigated as an adjuvant for PD-1 checkpoint blockade therapy in lung cancer (NCT03326752), where intratumoral injection of adjuvant is more challenging. Notably, another Dynavax TLR9 agonist tested in a large randomized trial did demonstrate superior immunogenicity (seroconversion) when combined with HBsAg as compared to standard HBV vaccination, leading to its FDA approval. Potentially, this immunostimulatory effect could portend success in cancer therapy as now shown with pathogen vaccines.

NLR in Cancer Immunotherapy

NLRs are intracellular PRRs that recognize a diverse set of ligands including bacterial and viral PAMPs as well as DAMPs [Reviewed in Ref. 61] (2,3,8,63). Of several NLR families, the NLRC and NLRP families are the most well studied (2,63). NOD1 and NOD2 are prominent NLRC family members, which all contain N-terminal CARD domains. Similar to TLR2, NOD1 and NOD2 recognize components of the peptidoglycan bacterial cell wall, where NOD1 specifically recognizes gamma-D-glutamyl-meso-diaminopemelic acid (iE-DAP) and NOD2 recognizes muramyl dipeptide (MDP) (Fig. 1) (8,63). NLRPs, NLRP3 being the most well characterized, form part of the inflammasome, which leads to production of the proinflammatory IL-1β/IL-18 (Fig. 1). Besides several bacterial ligands, environmental pollutants such as asbestos and silica are known to initiate NLR inflammasomes (64).

Mifamurtide, a synthetic analog of muramyl tripeptide and NOD2 agonist, is approved in the EU in combination with chemotherapy to treat osteosarcoma (4,65). The TLR8 agonist motolimod is also a potent stimulator of the NLRP3 inflammasome, likely because of the molecule’s lipophilic structure, however the specific mechanism is still under investigation (50,66). Additionally, particulate adjuvants such as alum and saponins, often used in cancer vaccine formulations including HPV and the previously mentioned MAGE-A3 vaccine studies (35,36), are potent activators of the NLRP3 inflammasome, producing inflammatory cytokines to engender adaptive immune responses (67,68).

CLR in Cancer Immunotherapy

CLRs are a large family of receptors that contain at least one carbohydrate recognition domain, recognizing mannose, fructose, and glucans present on pathogens [Reviewed in Ref. 67] (2,8,69). Although classically associated with anti-fungal and mycobacterial immune responses, more recent evidence suggests CLRs are involved in sensing numerous pathogens including bacteria, viruses, and helminths, as well as DAMPs (69–71). CLRs are mainly expressed by DCs, although monocytes/macrophages, B cells, and neutrophils may also express CLRs. Most CLR family members are transmembrane receptors, although a few may be released as soluble proteins, such as mannose-binding-lectin (4,69). Upon ligation, CLRs transduce signal by either associating with kinases and phosphatases directly, or by recruiting ITAM-containing adaptor proteins such as FcR (Fig. 1) (8,69). Signaling ultimately converges on MAPK and NF-B signaling, allowing CLRs to influence signaling cascades from other PRRs, tailoring the immune response against specific pathogens. Additionally, many CLRs are internalized after activation, bringing their ligand cargo within the cell for degradation and subsequent antigen presentation, a critical process in activating the adaptive immune response (69).

Strategies targeting CLRs date back over two decades. Randomized studies with a mannan- MUC1 fusion protein targeting mannose receptor (MR), vaccinating breast cancer patients after surgical resection, showed significant protection from recurrence, demonstrating the efficacy of CLR targeting and the importance of adaptive immunity in preventing recurrence (72). Anti- CLR antibodies have also been used to target CLR expressing DCs. CDX-1307 (Celldex Therapeutics) is an MR specific antibody fused to human chorionic gonadotropin beta-chain

(hCG-β), commonly overexpressed in various cancers (73). Vaccinating with CDX-1307, GM- CSF, poly-ICLC and/or resiquimod to mature DCs, most treated patients developed humoral response against hCG-β, and some developed T cell responses. Combining CDX-1401 (Celldex Therapeutics) - an anti-DEC-205 antibody fused to NY-ESO-1 antigen - with poly-ICLC and/or resiquimod, yielded humoral and T cell responses in most patients that correlated with stable disease (74). Additionally, several patients who progressed saw dramatic benefit with subsequent checkpoint blockade, warranting studies of this combination therapy. CDX-1401 is currently being investigated in gynecologic (NCT02166905) and hematologic (NCT03358719) malignancies. CMB305 (Immune Design) is a Sindbis virus engineered to use DC-SIGN as an attachment receptor, selectively infecting and expressing NY-ESO-1 protein in DCs for antigen presentation (75). CMB305 is being investigated in a phase 3 trial in synovial sarcoma (NCT03520959). Another therapy, Imprime PGG (Biothera), uses IV yeast-derived soluble B- glucan, a dectin-1 ligand, to sensitize patients and boost efficacy of targeted therapy and anti-PD- 1 blockade, and is being investigated in several phase 1 and 2 studies (clinicaltrials.gov) with promising early results. Interestingly, Imprime PGG shows a moderate toxicity profile, with 22% of patients discontinuing treatment due to adverse events, possibly because of the route of administration and the drug’s potent activation of the complement cascade (76).

Cytosolic Viral Sensors – RLR & CDS

While TLR3 is responsible for detecting viral dsRNA within endosomal compartments, RLRs retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2) recognize cytosolic dsRNA (Fig. 1) [Reviewed in Ref 75] (77). These sensors are critical in the host antiviral response and are expressed within most cell types, including cancer cells (4). Structurally, RLR family members contain a C- terminal RNA binding domain, a DExD/H central domain for ATP catalysis and activation, and an N-terminal CARD domain that interacts with the downstream effector molecule MAVS (also referred to as IPS-1) to convey signaling. Short dsRNA with 5’ triphosphate (5’-PPP) ends are preferentially recognized by RIG-I, while MDA5 recognizes longer dsRNA fragments, including poly-IC. While LGP2 also recognizes dsRNA, this family member cannot convey downstream signaling because it lacks a CARD domain, and is instead important in regulating RIG-I and MDA5 activation (8,77). Stimulation of epithelial ovarian cancer cells with a RIG-I specific agonist triggers type I interferon release and immunogenic apoptosis, which effectively matures DCs upon phagocytosis of these apoptotic cancer cells (78). Additionally, activation of RIG-I using 5’-PPP RNA or MDA5 using poly-IC causes apoptosis in human melanoma cells both in vitro and in vivo, while adjacent non-malignant cells are spared because of intact anti-apoptotic BCL-XL signaling (79).

The multimodal action of RLRs in immune cell activation while simultaneously triggering immunogenic cell death in cancer cells makes this pathway a particularly attractive immunotherapeutic target. The successes of poly-ICLC in clinical trials can in part be attributed to its dual agonistic activity on TLR3 and MDA5. BO-112 (Bioncotech) is another poly-IC derivative that potently activates RLR signaling in addition to TLR3, and is currently in phase 1 trials with promising early results (NCT02828098) (80). One synthetic RIG-I-specific ligand, RGT100/MK-4621 (Merck) has initiated human trials, after pre-clinical data demonstrated

potent anti-tumor activity in melanoma and colon carcinoma models (81). A phase 1/2 trial in solid tumors began in 2017 (NCT03065023), yielding only stable diseases as best response with intratumoral therapy (82). Pharmacokinetic studies show intratumorally-administered MK-4621 is well retained in the TME, helping minimize adverse events due to systemic toxicity. Numerous RLR agonists are currently in active pre-clinical development and will likely be seen in the clinic soon.

As cellular DNA is ordinarily restricted to the nucleus and mitochondria, aberrant cytosolic DNA arising from viral infection or cell damage triggers immunogenic signaling by activating ubiquitously expressed CDS. To date, several pathways for sensing cytosolic DNA have been described. DNA can be transcribed in the cytosol, generating a dsRNA molecule that can be recognized by RIG-I, triggering an inflammatory response to cytosolic DNA in an RLR dependent fashion (8). Additionally, cytosolic DNA can be recognized by absent in melanoma 2 (AIM2), prompting inflammasome assembly, resulting in IL-1β/IL-18 production (Fig. 1) (8). Perhaps the most impactful CDS is the stimulator of interferon genes (STING) pathway. Knockout studies indicate that STING is critical for host type I interferon and NF-B responses to synthetic and viral DNA, whereas STING deletion had no impact on AIM2 mediated IL-1β production and the TLR9 CpG DNA response (83). STING is also essential for a successful adaptive immune response to DNA vaccination. Several other CDSs such as DNA-dependent Activator of IFN-regulatory factors have been identified, however deletion studies indicate they may serve redundant function (8,83,84). In the context of infection, STING is a key mediator of the immune response against intracellular bacteria, DNA viruses, and retroviruses, however its ability to detect genomic DNA from dying tumor cells makes the STING pathway potentially important for anti-tumor immune responses.

The ability of the STING pathway to drive adaptive anti-tumor responses has generated significant interest, and recent studies suggest that efficacy of numerous DNA-damaging cancer therapies can in part be attributed to STING signaling. Chemotherapeutic agents cause DNA leakage into the cytosol, triggering a STING dependent type I interferon response (Fig. 2) (85). STING signaling is also required for successful activation of adaptive immunity and tumor clearance in response to both radiotherapy (86) and T cell checkpoint blockade, as cGAS-STING signaling enhances DC mediated T cell priming. Administration of adjuvant cGAMP synergized with checkpoint blockade in vivo, presumably by increasing the tumor-reactive T cell pool (87). Such studies highlight the importance of STING and type I interferons in DC cross-presentation of tumor antigens for anti-tumor T cell priming.

Several STING-specific agonists recently entered clinical development. Originally investigated as a vascular disrupting agent, STING agonist DMXAA (ASA404/vadimesan, Antisoma/Novartis) showed pre-clinical efficacy, but failed in a pivotal phase 3 trial as a combination treatment with chemotherapy in NSCLC (88). It was later shown to be ineffective in patients due to STING polymorphisms that prevent DMXAA binding (84,89). MIW815 (ADU- S100, Aduro Biotech) is a cyclic dinucleotide human STING agonist currently in phase 1 trials in combination with PD-1 (NCT03172936) or CTLA-4 blockade (NCT02675439). MK-1454 (Merck), a similar cyclic dinucleotide agonist currently in a phase 1 trial in combination with PD-1 blockade (NCT03010176) has shown favorable safety profiles and an objective response rate of 20% across several cancer types, with a median depth of response of ~80% (90).

Additionally, tumor clearance mediated by antibody blockade of CD47, a classical ‘don’t eat me’ signal, is STING-dependent, where enhanced phagocytosis resulting from CD47 blockade ultimately requires STING and type I interferon signaling to prime T cells and inhibit tumor growth (91). Blockade of CD47 is currently the focus of several clinical trials in both hematopoietic and solid tumors (clinicaltrials.gov).

Oncolytic Viruses

Amongst the most rapidly evolving therapeutic approaches in immuno-oncology is the use of oncolytic viruses (OVs). Either through the intrinsic permissiveness of tumor cells for unchecked replication (including viral replication) or by directly engineering the viral genome, OVs can selectively infect and kill tumor cells. Tumors are specifically susceptible to viral infection and replication, as many of the pathways required for oncogenesis can be coopted by OVs. While loss of tumor suppressors such as p53 and RB, activation of RAS and similar oncogenes, disruption of interferon signaling components, as well as a generally immunosuppressive TME all enable immune escape and promote tumor growth, these pathways concurrently promote OV infection, replication, and inhibit viral clearance, creating a permissive space for OV growth that is preferential to non-transformed tissue (92). OV infection in turn results in the immunogenic cell death of infected tumor cells, initiating de novo anti-tumor immune responses or boosting existing responses through mechanisms discussed in the above sections (Fig 2). Cancer cells infected with the polio OV PVSRIPO (Istari Oncology) release DAMPs (HMGB1, HSP60/70/80) and PAMPs (viral dsRNA), activating DCs to drive a tumor-antigen-specific cytotoxic T cell response (93). Similar to other intra-tumoraly delivered PRR agonists, the innate-adaptive immune axis is critical for OV therapy, as the ability to induce systemic anti- tumor immunity is antigen-restricted to the OV infected site. Using a Newcastle disease OV and contralateral B16 and MC38 tumors, Zamarin et al. demonstrate that OV injection of one tumor results in immunity only against that same tumor type (94). Talimogene laherparepvec or T- VEC (Amgen), a modified herpes virus expressing GM-CSF, was approved in 2015 for the treatment of late stage metastatic melanoma, earning a place for OV therapy in the clinic. In a landmark phase 3 study, intratumoral injection of T-vec caused complete resolution in 47% of injected lesions, as well as 22% of non-injected visceral lesions, highlighting the power of OV therapy to induce systemic anti-tumor immunity (95). T-VEC has already been effectively combined with CTLA-4 blockade, where a randomized phase 2 study demonstrated an increase in objective response rates from 18% with CTLA-4 monotherapy to 39% in the combination group (96), and is being investigated aggressively, including combinations with PD-1 blockade (NCT02965716), with neoadjuvant chemotherapy (NCT02779855), and with preoperative radiotherapy (NCT02453191). Other promising OV platforms in late development include Pexa Vec (JX-594, SillaJen), a vaccinia virus also engineered to express GM-CSF, currently in a phase 3 trial for hepatocellular carcinoma in combination with the kinase inhibitor sorafenib (NCT02562755). A modified Coxsackie virus, CAVATAK (Viralytics), is currently in phase 2 trials for several indications (clinicaltrials.gov). Moving beyond GM-CSF as the genetic payload, Swedish biotech Lokon recently opened a trial of their lead candidate LOAd703, an adenovirus encoding the costimulatory ligands CD40L and 4-1BBL (NCT03225989). Upon infection, tumor and other cells in the TME begin to express costimulatory ligands, helping to activate NK effector cells and remodel the TME (97). OVs without a therapeutic payload, including

Pelareorep (Reolysin, Oncolytics Biotech) and PVSRIPO, are in active clinical development as well. Recently published phase 1 data shows PVSRIPO increased 36 month overall survival to 21% in recurrent glioblastoma patients, a major increase from the 4% survival seen in historical controls (98). Taken together, all of these different successful approaches with OVs substantiate the idea that induction of immunogenic tumor cell death in combination with PRR agonism can drive effective adaptive immune responses.

Perspectives

Pattern recognition receptors present potentially powerful weapons in the cancer immunotherapy armory. Their ability to modulate numerous aspects of the tumor microenvironment, from APCs and their crosstalk with T cells, to directly modulating cancer cells themselves, allow these pathways to shape and ultimately drive an anti-tumor immune response. As PRR agonists and oncolytic viruses trigger innate cells to activate adaptive immunity, combining these approaches with checkpoint blockade therapy effectively presses the gas pedal while cutting the brakes, unleashing the full potential of immune effector cells. PRR agonism could additionally reverse resistance in checkpoint refractory tumors (99), and synergize with standard of care therapies including chemotherapy (100) and anti-CD20 targeting against lymphoma (101); a variety of combinatorial approaches are being actively explored in clinical trials (Tables 1-4). Pre-clinical approaches focused on developing next-generation agonists are underway – one group recently developed an OV-packaged anti-CTLA4 antibody construct, effectively combining OV and checkpoint therapy into a single injection (102). Others have fused Resiquimod nanoparticles to PD-1 targeting antibodies, allowing PD-1+ T cells to selectively deliver the TLR7 agonist to the tumor, reshaping the TME to improve T cell infiltration and disease control (103). These approaches highlight the immunomodulatory potency of PRRs, where their ability to overcome immunosuppression and drive adaptive immunity effectively enhances efficacy of concurrently administered therapies. With so many novel agonists being investigated pre-clinically and in the clinic, continued exploration and understanding of PRR pathways and targeting will help to shape treatment paradigms in immuno-oncology.

References:

1. Rakoff-Nahoum S, Medzhitov R. Toll-like receptors and cancer [Internet]. Nat. Rev. Cancer. Nature Publishing Group; 2009 [cited 2018 Oct 7]. page 57–63. Available from: http://www.nature.com/articles/nrc2541 2. Kawai T, Akira S. Toll-like Receptors and Their Crosstalk with Other Innate Receptors in Infection and Immunity [Internet]. Immunity. Cell Press; 2011 [cited 2018 Oct 7]. page 637–50. Available from: https://www.sciencedirect.com/science/article/pii/S1074761311001907?via%3Dihub 3. Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: Update on toll-like receptors [Internet]. Nat. Immunol. Nature Publishing Group; 2010 [cited 2018 Oct 7]. page 373–84. Available from: http://www.nature.com/articles/ni.1863 4. Shekarian T, Valsesia-Wittmann S, Brody J, Michallet MC, Depil S, Caux C, et al. Pattern recognition receptors: Immune targets to enhance cancer immunotherapy. Ann Oncol

[Internet]. 2017 [cited 2018 Sep 13];28:1756–66. Available from: https://watermark.silverchair.com/mdx179.pdf?token=AQECAHi208BE49Ooan9kkhW_Er cy7Dm3ZL_9Cf3qfKAc485ysgAAA7owggO2BgkqhkiG9w0BBwagggOnMIIDowIBADCCA5w GCSqGSIb3DQEHATAeBglghkgBZQMEAS4wEQQMvwyW36e0VaohRvFmAgEQgIIDbfC9FtD iRLENyK7O6aVLj904B4L94o3xzKQeBHQPHC3l8fY3 5. Barton GM, Kagan JC. A cell biological view of toll-like receptor function: Regulation through compartmentalization [Internet]. Nat. Rev. Immunol. Nature Publishing Group; 2009 [cited 2018 Oct 7]. page 535–41. Available from: http://www.nature.com/articles/nri2587 6. Oosting M, Cheng S-C, Bolscher JM, Vestering-Stenger R, Plantinga TS, Verschueren IC, et al. Human TLR10 is an anti-inflammatory pattern-recognition receptor. Proc Natl Acad Sci [Internet]. 2014 [cited 2018 Oct 7];111:E4478–84. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25288745 7. Lee SM-Y, Yip T-F, Yan S, Jin D-Y, Wei H-L, Guo R-T, et al. Recognition of Double-Stranded RNA and Regulation of Interferon Pathway by Toll-Like Receptor 10. Front Immunol [Internet]. 2018 [cited 2018 Oct 7];9:516. Available from: http://www.ncbi.nlm.nih.gov/pubmed/29616030 8. Takeuchi O, Akira S. Pattern Recognition Receptors and Inflammation [Internet]. Cell. 2010 [cited 2018 Sep 13]. page 805–20. Available from: http://www.ncbi.nlm.nih.gov/pubmed/20303872 9. Pradere JP, Dapito DH, Schwabe RF. The Yin and Yang of Toll-like receptors in cancer [Internet]. Oncogene. 2014 [cited 2018 Sep 13]. page 3485–95. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23934186 10. Chatterjee S, Crozet L, Damotte D, Iribarren K, Schramm C, Alifano M, et al. TLR7 promotes tumor progression, chemotherapy resistance, and poor clinical outcomes in non-small cell lung cancer. Cancer Res [Internet]. 2014 [cited 2018 Sep 16];74:5008–18. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25074614 11. Cherfils-Vicini J, Platonova S, Gillard M, Laurans L, Validire P, Caliandro R, et al. Triggering of TLR7 and TLR8 expressed by human lung cancer cells induces cell survival and chemoresistance. J Clin Invest [Internet]. American Society for Clinical Investigation; 2010 [cited 2018 Oct 10];120:1285–97. Available from: http://www.ncbi.nlm.nih.gov/pubmed/20237413 12. Yang H, Wang B, Wang T, Xu L, He C, Wen H, et al. Toll-like receptor 4 prompts human breast cancer cells invasiveness via lipopolysaccharide stimulation and is overexpressed in patients with lymph node metastasis. Dileepan KN, editor. PLoS One [Internet]. 2014 [cited 2018 Sep 13];9:e109980. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25299052 13. Volk-Draper L, Hall K, Griggs C, Rajput S, Kohio P, DeNardo D, et al. Paclitaxel therapy promotes breast cancer metastasis in a TLR4-dependent manner. Cancer Res [Internet]. 2014 [cited 2018 Sep 16];74:5421–34. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25274031 14. Li D, Wang X, Wu J-L, Quan W-Q, Ma L, Yang F, et al. Tumor-Produced Versican V1 Enhances hCAP18/LL-37 Expression in Macrophages through Activation of TLR2 and Vitamin D3 Signaling to Promote Ovarian Cancer Progression In Vitro. Wang Q, editor.

PLoS One [Internet]. 2013 [cited 2018 Oct 9];8:e56616. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23424670 15. Kim S, Takahashi H, Lin WW, Descargues P, Grivennikov S, Kim Y, et al. Carcinoma- produced factors activate myeloid cells through TLR2 to stimulate metastasis. Nature [Internet]. 2009 [cited 2018 Sep 16];457:102–6. Available from: http://www.ncbi.nlm.nih.gov/pubmed/19122641 16. Klein JC, Moses K, Zelinskyy G, Sody S, Buer J, Lang S, et al. Combined toll-like receptor 3/7/9 deficiency on host cells results in T-cell-dependent control of tumour growth. Nat Commun [Internet]. Nature Publishing Group; 2017 [cited 2018 Oct 16];8:14600. Available from: http://www.nature.com/doifinder/10.1038/ncomms14600 17. Daley D, Mani VR, Mohan N, Akkad N, Ochi A, Heindel DW, et al. Dectin 1 activation on macrophages by galectin 9 promotes pancreatic carcinoma and peritumoral immune tolerance. Nat Med [Internet]. Nature Publishing Group; 2017 [cited 2018 Nov 14];23:556–67. Available from: http://www.nature.com/doifinder/10.1038/nm.4314 18. Mellman I, Coukos G, Dranoff G. Cancer immunotherapy comes of age. Nature [Internet]. Nature Publishing Group; 2011 [cited 2018 Oct 6];480:480–9. Available from: http://www.nature.com/articles/nature10673 19. Biot C, Rentsch C, Gsponer J, Birkh"auser F, Hélène J-S, Lema\^itre F, et al. Preexisting {BCG-specific} T cells improve intravesical immunotherapy for bladder cancer. Sci Transl Med [Internet]. American Association for the Advancement of Science; 2012 [cited 2018 Oct 15];4:137ra72. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22674550 20. Divangahi M, Mostowy S, Coulombe F, Kozak R, Guillot L, Veyrier F, et al. NOD2-Deficient Mice Have Impaired Resistance to Mycobacterium tuberculosis Infection through Defective Innate and Adaptive Immunity. J Immunol [Internet]. American Association of Immunologists; 2008 [cited 2018 Nov 12];181:7157–65. Available from: http://www.ncbi.nlm.nih.gov/pubmed/18981137 21. Marabelle A, Kohrt H, Caux C, Levy R. Intratumoral immunization: a new paradigm for cancer therapy. Clin Cancer Res [Internet]. American Association for Cancer Research; 2014 [cited 2018 Oct 15];20:1747–56. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24691639 22. Martins KA, Bavari S, Salazar AM. Expert Review of Vaccines Vaccine adjuvant uses of poly-IC and derivatives. 2014 [cited 2018 Oct 16]; Available from: http://www.tandfonline.com/action/journalInformation?journalCode=ierv20 23. Caskey M, Lefebvre F, Filali-Mouhim A, Cameron MJ, Goulet J-P, Haddad EK, et al. Synthetic double-stranded RNA induces innate immune responses similar to a live viral vaccine in humans. J Exp Med [Internet]. Rockefeller University Press; 2011 [cited 2018 Oct 16];208:2357–66. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22065672 24. Stahl-Hennig C, Eisenblätter M, Jasny E, Rzehak T, Tenner-Racz K, Trumpfheller C, et al. Synthetic Double-Stranded RNAs Are Adjuvants for the Induction of T Helper 1 and Humoral Immune Responses to Human Papillomavirus in Rhesus Macaques. Schiller JT, editor. PLoS Pathog [Internet]. Public Library of Science; 2009 [cited 2018 Oct 16];5:e1000373. Available from: http://dx.plos.org/10.1371/journal.ppat.1000373 25. Navabi H, Jasani B, Reece A, Clayton A, Tabi Z, Donninger C, et al. A clinical grade poly I:C- analogue (Ampligen®) promotes optimal DC maturation and Th1-type T cell responses of

healthy donors and cancer patients in vitro. Vaccine [Internet]. Elsevier; 2009 [cited 2018 Oct 16];27:107–15. Available from: https://www.sciencedirect.com/science/article/pii/S0264410X08013911?via%3Dihub 26. Theodoraki M-N, Yerneni S, Sarkar SN, Orr B, Muthuswamy R, Voyten J, et al. Helicase- Driven Activation of NFκB-COX2 Pathway Mediates the Immunosuppressive Component of dsRNA-Driven Inflammation in the Human Tumor Microenvironment. Cancer Res [Internet]. American Association for Cancer Research; 2018 [cited 2018 Oct 16];78:4292– 302. Available from: http://www.ncbi.nlm.nih.gov/pubmed/29853604 27. Mehrotra S, Britten CD, Chin S, Garrett-Mayer E, Cloud CA, Li M, et al. Vaccination with poly(IC:LC) and peptide-pulsed autologous dendritic cells in patients with pancreatic cancer. J Hematol Oncol [Internet]. BioMed Central; 2017 [cited 2018 Oct 16];10:82. Available from: http://jhoonline.biomedcentral.com/articles/10.1186/s13045-017-0459- 2 28. Nooka AK, Wang M (Luhua), Yee AJ, Kaufman JL, Bae J, Peterkin D, et al. Assessment of Safety and Immunogenicity of PVX-410 Vaccine With or Without Lenalidomide in Patients With Smoldering Multiple Myeloma. JAMA Oncol [Internet]. 2018 [cited 2018 Oct 16];e183267. Available from: http://oncology.jamanetwork.com/article.aspx?doi=10.1001/jamaoncol.2018.3267 29. Hammerich L, Marron TU, Upadhyay R, Svensson-Arvelund J, Dhainaut M, Hussein S, et al. Systemic clinical tumor regressions and potentiation of PD1 blockade with in situ vaccination. Nat Med [Internet]. Nature Publishing Group; 2019;1. Available from: https://doi.org/10.1038/s41591-019-0410-x 30. De La Torre AN, Contractor S, Castaneda I, Cathcart CS, Razdan D, Klyde D, et al. A Phase I trial using local regional treatment, nonlethal irradiation, intratumoral and systemic polyinosinic-polycytidylic acid polylysine carboxymethylcellulose to treat liver cancer: in search of the abscopal effect. J Hepatocell Carcinoma [Internet]. 2017 [cited 2018 Oct 16];2017:4–111. Available from: http://dx.doi.org/10.2147/JHC.S136652 31. Butowski N, Chang SM, Junck L, DeAngelis LM, Abrey L, Fink K, et al. A phase II clinical trial of poly-ICLC with radiation for adult patients with newly diagnosed supratentorial glioblastoma: a North American Brain Tumor Consortium (NABTC01-05). J Neurooncol [Internet]. Springer US; 2009 [cited 2018 Oct 16];91:175–82. Available from: http://link.springer.com/10.1007/s11060-008-9693-3 32. Rosenfeld MR, Chamberlain MC, Grossman SA, Peereboom DM, Lesser GJ, Batchelor TT, et al. A multi-institution phase II study of poly-ICLC and radiotherapy with concurrent and adjuvant temozolomide in adults with newly diagnosed glioblastoma. Neuro Oncol [Internet]. Oxford University Press; 2010 [cited 2016 Sep 20];12:1071–7. Available from: http://www.ncbi.nlm.nih.gov/pubmed/20615924 33. Han HD, Byeon Y, Jang J-H, Jeon HN, Kim GH, Kim MG, et al. In vivo stepwise immunomodulation using chitosan nanoparticles as a platform nanotechnology for cancer immunotherapy. Sci Rep [Internet]. Nature Publishing Group; 2016 [cited 2018 Oct 17];6:38348. Available from: http://www.nature.com/articles/srep38348 34. Paavonen J, Naud P, Salmerón J, Wheeler C, Chow S-N, Apter D, et al. Efficacy of human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine against cervical infection and precancer caused by oncogenic HPV types (PATRICIA): final analysis of a double-blind,

randomised study in young women. Lancet [Internet]. Elsevier; 2009 [cited 2018 Oct 17];374:301–14. Available from: https://www.sciencedirect.com/science/article/pii/S0140673609612484?via%3Dihub 35. Dreno B, Thompson JF, Smithers BM, Santinami M, Jouary T, Gutzmer R, et al. MAGE-A3 immunotherapeutic as adjuvant therapy for patients with resected, MAGE-A3-positive, stage III melanoma (DERMA): a double-blind, randomised, placebo-controlled, phase 3 trial. Lancet Oncol [Internet]. Elsevier; 2018 [cited 2018 Oct 24];19:916–29. Available from: https://www.sciencedirect.com/science/article/pii/S1470204518302547?via%3Dihub 36. Vansteenkiste JF, Cho BC, Vanakesa T, De Pas T, Zielinski M, Kim MS, et al. Efficacy of the MAGE-A3 cancer immunotherapeutic as adjuvant therapy in patients with resected MAGE-A3-positive non-small-cell lung cancer (MAGRIT): a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet Oncol [Internet]. Elsevier; 2016 [cited 2018 Oct 24];17:822–35. Available from: https://www.sciencedirect.com/science/article/pii/S1470204516000991#bib9 37. Bhatia S, Miller NJ, Lu H, Vandeven NV, Ibrani D, Shinohara M, et al. Intratumoral G100, a TLR4 agonist, induces anti-tumor immune responses and tumor regression in patients with Merkel cell carcinoma. Clin Cancer Res [Internet]. American Association for Cancer Research; 2018 [cited 2018 Oct 17];clincanres.0469.2018. Available from: http://www.ncbi.nlm.nih.gov/pubmed/30093453 38. Flowers C, Panizo C, Isufi I, Herrera AF, Okada C, Cull EH, et al. Long Term Follow-up of a Phase 2 Study Examining Intratumoral G100 Alone and in Combination with Pembrolizumab in Patients with Follicular Lymphoma. ASH; 2018 [cited 2019 Jan 6]. Available from: https://ash.confex.com/ash/2018/webprogram/Paper117932.html 39. Brackett CM, Kojouharov B, Veith J, Greene KF, Burdelya LG, Gollnick SO, et al. Toll-like receptor-5 agonist, entolimod, suppresses metastasis and induces immunity by stimulating an NK-dendritic-CD8+ T-cell axis. Proc Natl Acad Sci U S A [Internet]. National Academy of Sciences; 2016 [cited 2018 Oct 27];113:E874-83. Available from: http://www.ncbi.nlm.nih.gov/pubmed/26831100 40. Leigh ND, Bian G, Ding X, Liu H, Aygun-Sunar S, Burdelya LG, et al. A Flagellin-Derived Toll-Like Receptor 5 Agonist Stimulates Cytotoxic Lymphocyte-Mediated Tumor Immunity. Piccirillo CA, editor. PLoS One [Internet]. Public Library of Science; 2014 [cited 2018 Oct 25];9:e85587. Available from: http://dx.plos.org/10.1371/journal.pone.0085587 41. Mett V, Komarova EA, Greene K, Bespalov I, Brackett C, Gillard B, et al. Mobilan: a recombinant adenovirus carrying Toll-like receptor 5 self-activating cassette for cancer immunotherapy. Oncogene [Internet]. Nature Publishing Group; 2018 [cited 2018 Oct 24];37:439–49. Available from: http://www.nature.com/doifinder/10.1038/onc.2017.346 42. Bakhribah H, Dy GK, Ma WW, Zhao Y, Opyrchal M, Purmal A, et al. A phase I study of the toll-like receptor 5 (TLR5) agonist, entolimod in patients (pts) with advanced cancers. J Clin Oncol [Internet]. 2015;33:3063. Available from: http://ascopubs.org/doi/abs/10.1200/jco.2015.33.15_suppl.3063 43. Vidal D, Matias-Guiu X, Alomar A. Open study of the efficacy and mechanism of action of

topical imiquimod in basal cell carcinoma. Clin Exp Dermatol [Internet]. Wiley/Blackwell (10.1111); 2004 [cited 2018 Oct 28];29:518–25. Available from: http://doi.wiley.com/10.1111/j.1365-2230.2004.01601.x 44. Grimm C, Polterauer S, Natter C, Rahhal J, Hefler L, Tempfer CB, et al. Treatment of Cervical Intraepithelial Neoplasia With Topical Imiquimod. Obstet Gynecol [Internet]. 2012 [cited 2018 Oct 28];120:152–9. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22914404 45. Sabado RL, Pavlick A, Gnjatic S, Cruz CM, Vengco I, Hasan F, et al. Resiquimod as an immunologic adjuvant for NY-ESO-1 protein vaccination in patients with high-risk melanoma. Cancer Immunol Res [Internet]. American Association for Cancer Research; 2015 [cited 2018 Oct 28];3:278–87. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25633712 46. Ota Y, Otsubo T, Koroki J, Hirose Y, Koga-Yamakawa E, Murata M, et al. Abstract 4726: Novel intravenous injectable TLR7 agonist, DSP-0509, synergistically enhanced antitumor immune responses in combination with anti-PD-1 antibody. Cancer Res [Internet]. 2018;78:4726 LP-4726. Available from: http://cancerres.aacrjournals.org/content/78/13_Supplement/4726.abstract 47. Gupta S, Grilley-Olson J, Hong D, Marabelle A, Munster P, Aggarwal R, et al. Abstract CT091: Safety and pharmacodynamic activity of MEDI9197, a TLR 7/8 agonist, administered intratumorally in subjects with solid tumors. Cancer Res [Internet]. 2017;77:CT091 LP-CT091. Available from: http://cancerres.aacrjournals.org/content/77/13_Supplement/CT091.abstract 48. Lee M. Abstract 2755: NKTR-262: Prodrug pharmacokinetics in mice, rats, and dogs. Cancer Res [Internet]. 2018;78:2755 LP-2755. Available from: http://cancerres.aacrjournals.org/content/78/13_Supplement/2755.abstract 49. Kivimäe S, Rubas W, Pena R, Mclaughlin J, Hennessy M, Kirksey Y, et al. Harnessing the innate and adaptive immune system to eradicate treated and distant untreated solid tumors. J Immunother Cancer [Internet]. 2017 [cited 2018 Oct 28];5:P275. Available from: https://www.nektar.com/application/files/8015/1033/6150/NKTR-262-NKTR- 214__2017SITC_Poster-P275.pdf.pdf 50. Ferris RL, Saba NF, Gitlitz BJ, Haddad R, Sukari A, Neupane P, et al. Effect of Adding Motolimod to Standard Combination Chemotherapy and Cetuximab Treatment of Patients With Squamous Cell Carcinoma of the Head and Neck. JAMA Oncol [Internet]. 2018 [cited 2018 Oct 28]; Available from: http://oncology.jamanetwork.com/article.aspx?doi=10.1001/jamaoncol.2018.1888 51. Kapp K, Schneider J, Schneider L, Gollinge N, Jänsch S, Schroff M, et al. Distinct immunological activation profiles of dSLIM® and ProMune® depend on their different structural context. Immunity, Inflamm Dis [Internet]. Wiley-Blackwell; 2016 [cited 2018 Nov 1];4:446–62. Available from: http://doi.wiley.com/10.1002/iid3.126 52. Samulowitz U, Weber M, Weeratna R, Uhlmann E, Noll B, Krieg AM, et al. A Novel Class of Immune-Stimulatory CpG Oligodeoxynucleotides Unifies High Potency in Type I Interferon Induction with Preferred Structural Properties. Oligonucleotides [Internet]. 2010 [cited 2018 Nov 3];20:93–101. Available from: www.liebertpub.com 53. Brody JD, Ai WZ, Czerwinski DK, Torchia JA, Levy M, Advani RH, et al. In situ vaccination

with a TLR9 agonist induces systemic lymphoma regression: a phase I/II study. J Clin Oncol [Internet]. 2010 [cited 2016 Sep 18];28:4324–32. Available from: http://www.ncbi.nlm.nih.gov/pubmed/20697067 54. Manegold C, van Zandwijk N, Szczesna A, Zatloukal P, Au JSK, Blasinska-Morawiec M, et al. A phase III randomized study of gemcitabine and cisplatin with or without PF-3512676 (TLR9 agonist) as first-line treatment of advanced non-small-cell lung cancer. Ann Oncol [Internet]. Oxford University Press; 2012 [cited 2018 Nov 1];23:72–7. Available from: https://academic.oup.com/annonc/article-lookup/doi/10.1093/annonc/mdr030 55. Frank MJ, Reagan PM, Bartlett NL, Gordon LI, Friedberg JW, Czerwinski DK, et al. In Situ Vaccination with a TLR9 Agonist and Local Low-Dose Radiation Induces Systemic Responses in Untreated Indolent Lymphoma. Cancer Discov [Internet]. 2018 [cited 2018 Nov 1];8:1258–69. Available from: www.aacrjournals.org 56. Milhem M, Gonzales R, Medina T, Kirkwood JM, Buchbinder E, Mehmi I, et al. Abstract CT144: Intratumoral toll-like receptor 9 (TLR9) agonist, CMP-001, in combination with pembrolizumab can reverse resistance to PD-1 inhibition in a phase Ib trial in subjects with advanced melanoma. Cancer Res [Internet]. Philadelphia: AACR; 2018 [cited 2018 Nov 4]. page CT144 LP-CT144. Available from: http://www.abstractsonline.com/pp8/#!/4562/presentation/11133 57. Dia A, Xx, Xx, Xx, Xx, xx. A phase 2 study to evaluate the safety and efficacy of Intratumoral (IT) injection of the TLR9 agonist IMO-2125 (IMO) in combination with ipilimumab (ipi) in PD-1 inhibitor refractory melanoma. J Clin Oncol [Internet]. 2018 [cited 2018 Nov 4];36:suppl; abstr 9515. Available from: https://meetinglibrary.asco.org/record/159086/abstract 58. Thomas M, Ponce-Aix S, Navarro A, Riera-Knorrenschild J, Schmidt M, Wiegert E, et al. Immunotherapeutic maintenance treatment with toll-like receptor 9 agonist lefitolimod in patients with extensive-stage small-cell lung cancer: results from the exploratory, controlled, randomized, international phase II IMPULSE study. Ann Oncol [Internet]. 2018 [cited 2018 Nov 1]; Available from: https://academic.oup.com/annonc/advance- article/doi/10.1093/annonc/mdy326/5076437 59. Krarup AR, Abdel-Mohsen M, Schleimann MH, Vibholm L, Engen PA, Dige A, et al. The TLR9 agonist MGN1703 triggers a potent type I interferon response in the sigmoid colon. Mucosal Immunol [Internet]. Nature Publishing Group; 2018 [cited 2018 Nov 4];11:449– 61. Available from: http://www.nature.com/doifinder/10.1038/mi.2017.59 60. Ribas A, Medina T, Kummar S, Amin A, Kalbasi A, Drabick JJ, et al. SD-101 in Combination with Pembrolizumab in Advanced Melanoma: Results of a Phase 1b, Multicenter Study. Cancer Discov [Internet]. 2018;CD-18-0280. Available from: http://cancerdiscovery.aacrjournals.org/lookup/doi/10.1158/2159-8290.CD-18-0280 61. Sagiv-Barfi I, Czerwinski DK, Levy S, Alam IS, Mayer AT, Gambhir SS, et al. Eradication of spontaneous malignancy by local immunotherapy [Internet]. Sci. Transl. Med. 2018. Available from: http://stm.sciencemag.org/ 62. Levy R, Reagan PM, Friedberg JW, Bartlett NL, Gordon LI, Leung A, et al. SD-101, a Novel Class C CpG-Oligodeoxynucleotide (ODN) Toll-like Receptor 9 (TLR9) Agonist, Given with Low Dose Radiation for Untreated Low Grade B-Cell Lymphoma: Interim Results of a Phase 1/2 Trial. Blood [Internet]. 2016 [cited 2019 Mar 16];128. Available from:

http://www.bloodjournal.org/content/128/22/2974?sso-checked=true 63. Saxena M, Yeretssian G. NOD-Like Receptors: Master Regulators of Inflammation and Cancer. Front Immunol [Internet]. Frontiers; 2014 [cited 2018 Nov 5];5:327. Available from: http://journal.frontiersin.org/article/10.3389/fimmu.2014.00327/abstract 64. Dostert C, Petrilli V, Van Bruggen R, Steele C, Mossman BT, Tschopp J. Innate Immune Activation Through Nalp3 Inflammasome Sensing of Asbestos and Silica. Science (80- ) [Internet]. 2008 [cited 2019 Jan 6];320:674–7. Available from: http://www.ncbi.nlm.nih.gov/pubmed/18403674 65. Chou AJ, Kleinerman ES, Krailo MD, Chen Z, Betcher DL, Healey JH, et al. Addition of muramyl tripeptide to chemotherapy for patients with newly diagnosed metastatic osteosarcoma: A report from the Children’s Oncology Group. Cancer [Internet]. Wiley- Blackwell; 2009 [cited 2018 Nov 8];115:5339–48. Available from: http://doi.wiley.com/10.1002/cncr.24566 66. Dietsch GN, Lu H, Yang Y, Morishima C, Chow LQ, Disis ML, et al. Coordinated activation of toll-like receptor8 (TLR8) and NLRP3 by the TLR8 agonist, VTX-2337, ignites tumoricidal natural killer cell activity. Nishikawa H, editor. PLoS One [Internet]. Public Library of Science; 2016 [cited 2018 Nov 12];11:e0148764. Available from: https://dx.plos.org/10.1371/journal.pone.0148764 67. Li H, Willingham SB, Ting JP-Y, Re F. Cutting Edge: Inflammasome Activation by Alum and Alum’s Adjuvant Effect Are Mediated by NLRP3. J Immunol [Internet]. American Association of Immunologists; 2008 [cited 2018 Nov 12];181:17–21. Available from: http://www.ncbi.nlm.nih.gov/pubmed/18566365 68. Cibulski SP, Rivera-Patron M, Mourglia-Ettlin G, Casaravilla C, Yendo ACA, Fett-Neto AG, et al. Quillaja brasiliensis saponin-based nanoparticulate adjuvants are capable of triggering early immune responses. Sci Rep [Internet]. Nature Publishing Group; 2018 [cited 2018 Nov 12];8:13582. Available from: http://www.nature.com/articles/s41598- 018-31995-1 69. Geijtenbeek TBH, Gringhuis SI. Signalling through C-type lectin receptors: Shaping immune responses [Internet]. Nat. Rev. Immunol. Nature Publishing Group; 2009 [cited 2018 Nov 8]. page 465–79. Available from: http://www.nature.com/articles/nri2569 70. Yamasaki S, Ishikawa E, Sakuma M, Hara H, Ogata K, Saito T. Mincle is an ITAM-coupled activating receptor that senses damaged cells. Nat Immunol [Internet]. Nature Publishing Group; 2008 [cited 2018 Nov 8];9:1179–88. Available from: http://www.nature.com/articles/ni.1651 71. Zhang JG, Czabotar PE, Policheni AN, Caminschi I, San Wan S, Kitsoulis S, et al. The Dendritic Cell Receptor Clec9A Binds Damaged Cells via Exposed Actin Filaments. Immunity [Internet]. Elsevier; 2012 [cited 2018 Nov 8];36:646–57. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22483802 72. Vassilaros S, Tsibanis A, Tsikkinis A, Pietersz GA, McKenzie IFC, Apostolopoulos V. Up to 15-year clinical follow-up of a pilot Phase III immunotherapy study in stage II breast cancer patients using oxidized mannan-MUC1. Immunotherapy [Internet]. 2013 [cited 2018 Nov 13];5:1177–82. Available from: https://www.futuremedicine.com/doi/10.2217/imt.13.126 73. Morse MA, Chapman R, Powderly J, Blackwell K, Keler T, Green J, et al. Phase I study

utilizing a novel antigen-presenting cell-targeted vaccine with toll-like receptor stimulation to induce immunity to self-antigens in cancer patients. Clin Cancer Res [Internet]. American Association for Cancer Research; 2011 [cited 2018 Nov 13];17:4844– 53. Available from: http://www.ncbi.nlm.nih.gov/pubmed/12114402 74. Dhodapkar M V, Sznol M, Zhao B, Wang D, Carvajal RD, Keohan ML, et al. Induction of antigen-specific immunity with a vaccine targeting NY-ESO-1 to the dendritic cell receptor DEC-205. Sci Transl Med [Internet]. American Association for the Advancement of Science; 2014 [cited 2018 Nov 13];6:232ra51. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24739759 75. Pollack SM. The potential of the CMB305 vaccine regimen to target NY-ESO-1 and improve outcomes for synovial sarcoma and myxoid/round cell liposarcoma patients [Internet]. Expert Rev. Vaccines. Taylor & Francis; 2018 [cited 2018 Nov 13]. page 107– 14. Available from: https://www.tandfonline.com/doi/full/10.1080/14760584.2018.1419068 76. Thomas M, Sadjadian P, Kollmeier J, Lowe J, Mattson P, Trout JR, et al. A randomized, open-label, multicenter, phase II study evaluating the efficacy and safety of BTH1677 (1,3–1,6 beta glucan; Imprime PGG) in combination with cetuximab and chemotherapy in patients with advanced non-small cell lung cancer. Invest New Drugs [Internet]. 2017 [cited 2018 Nov 14];35:345–58. Available from: http://link.springer.com/10.1007/s10637-017-0450-3 77. Loo YM, Gale M. Immune Signaling by RIG-I-like Receptors. Immunity [Internet]. 2011 [cited 2018 Sep 13];34:680–92. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21616437 78. Kübler K, Gehrke N, Riemann S, Böhnert V, Zillinger T, Hartmann E, et al. Targeted activation of RNA helicase retinoic acid - Inducible gene-I induces proimmunogenic apoptosis of human ovarian cancer cells. Cancer Res [Internet]. American Association for Cancer Research; 2010 [cited 2018 Nov 16];70:5293–304. Available from: http://www.ncbi.nlm.nih.gov/pubmed/20551064 79. Besch R, Poeck H, Hohenauer T, Senft D, Häcker G, Berking C, et al. Proapoptotic signaling induced by RIG-I and MDA-5 results in type I interferon–independent apoptosis in human melanoma cells. J Clin Invest [Internet]. 2009 [cited 2018 Sep 16];119:2399–411. Available from: http://www.ncbi.nlm.nih.gov/pubmed/19620789 80. Calles A, Rodriguez-Ruiz M, Soria A, Marquez Rodas I, Ponz-Sarvisé M, Martín M, et al. Intratumoral BO-112, a double-stranded RNA (dsRNA), alone and in combination with systemic anti-PD-1 in solid tumors. Ann Oncol [Internet]. 2018 [cited 2019 Mar 16];29. Available from: http://www.bioncotech.com/wp-content/uploads/POSTER-Marquez- Rodas-et-al-Poster-Discussion-abstract-LBA39_BO_02Oct.pdf 81. Barsoum J, Renn M, Schuberth C, Jakobs C, Schwickart A, Schlee M, et al. Abstract B44: Selective stimulation of RIG-I with a novel synthetic RNA induces strong anti-tumor immunity in mouse tumor models. Cancer Immunol Res [Internet]. 2017;5:B44 LP-B44. Available from: http://cancerimmunolres.aacrjournals.org/content/5/3_Supplement/B44.abstract 82. Middleton MR, Wermke M, Calvo E, Chartash E, Zhou H, Zhao X, et al. Phase I/II, multicenter, open-label study of intratumoral/intralesional administration of the retinoic

acid–inducible gene I (RIG-I) activator MK-4621 in patients with advanced or recurrent tumors. Ann Oncol [Internet]. Oxford University Press; 2018 [cited 2019 Jan 8];29. Available from: https://academic.oup.com/annonc/article/doi/10.1093/annonc/mdy424.016/5141574 83. Ishikawa H, Ma Z, Barber GN. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity. Nature [Internet]. Nature Publishing Group; 2009 [cited 2018 Nov 20];461:788–92. Available from: http://www.nature.com/doifinder/10.1038/nature08476 84. Chen Q, Sun L, Chen ZJ. Regulation and function of the cGAS–STING pathway of cytosolic DNA sensing. Nat Immunol [Internet]. Nature Publishing Group; 2016 [cited 2018 Nov 20];17:1142–9. Available from: http://www.nature.com/articles/ni.3558 85. Härtlova A, Erttmann SF, Raffi FA, Schmalz AM, Resch U, Anugula S, et al. DNA Damage Primes the Type I Interferon System via the Cytosolic DNA Sensor STING to Promote Anti- Microbial Innate Immunity. Immunity [Internet]. Cell Press; 2015 [cited 2018 Nov 21];42:332–43. Available from: https://www.sciencedirect.com/science/article/pii/S1074761315000382?via%3Dihub 86. Deng L, Liang H, Xu M, Yang X, Burnette B, Arina A, et al. STING-Dependent Cytosolic DNA Sensing Promotes Radiation-Induced Type I Interferon-Dependent Antitumor Immunity in Immunogenic Tumors. Immunity [Internet]. Cell Press; 2014 [cited 2018 Nov 23];41:843–52. Available from: https://www.sciencedirect.com/science/article/pii/S1074761314003951?via%3Dihub 87. Wang H, Hu S, Chen X, Shi H, Chen C, Sun L, et al. cGAS is essential for the antitumor effect of immune checkpoint blockade. Proc Natl Acad Sci [Internet]. National Academy of Sciences; 2017 [cited 2018 Nov 21];114:1637–42. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28137885 88. Lara Jr PN, Douillard J-Y, Nakagawa K, von Pawel J, McKeage MJ, Albert I, et al. Randomized Phase III Placebo-Controlled Trial of Carboplatin and Paclitaxel With or Without the Vascular Disrupting Agent Vadimezan (ASA404) in Advanced Non-Small-Cell Lung Cancer. J Clin Oncol [Internet]. 2011 [cited 2018 Nov 21];29:2965–71. Available from: www.jco.org 89. Corrales L, Glickman LH, McWhirter SM, Kanne DB, Sivick KE, Katibah GE, et al. Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity. Cell Rep [Internet]. Cell Press; 2015 [cited 2018 Nov 19];11:1018–30. Available from: https://www.sciencedirect.com/science/article/pii/S2211124715004325?via%3Dihub 90. Harrington KJ, Brody J, Ingham M, Strauss J, Cemerski S, Wang M, et al. Preliminary results of the first-in-human (FIH) study of MK-1454, an agonist of stimulator of interferon genes (STING), as monotherapy or in combination with pembrolizumab (pembro) in patients with advanced solid tumors or lymphomas. Ann Oncol [Internet]. Oxford University Press; 2018 [cited 2019 Jan 7];29:mdy424.015-mdy424.015. Available from: https://academic.oup.com/annonc/article/doi/10.1093/annonc/mdy424.015/5141570 91. Liu X, Pu Y, Cron K, Deng L, Kline J, Frazier WA, et al. CD47 blockade triggers T cell- mediated destruction of immunogenic tumors. Nat Med [Internet]. Nature Publishing

Group; 2015 [cited 2018 Nov 21];21:1209–15. Available from: http://www.nature.com/articles/nm.3931 92. Pikor LA, Bell JC, Diallo J-S. Oncolytic Viruses: Exploiting Cancer’s Deal with the Devil. Trends in Cancer [Internet]. Cell Press; 2015 [cited 2018 Dec 1];1:266–77. Available from: https://www.sciencedirect.com/science/article/pii/S2405803315000515?via%3Dihub#bi b0465 93. Brown MC, Holl EK, Boczkowski D, Dobrikova E, Mosaheb M, Chandramohan V, et al. Cancer immunotherapy with recombinant poliovirus induces IFN-dominant activation of dendritic cells and tumor antigen-specific CTLs. Sci Transl Med [Internet]. American Association for the Advancement of Science; 2017 [cited 2018 Dec 1];9:eaan4220. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28931654 94. Zamarin D, Holmgaard RB, Subudhi SK, Park JS, Mansour M, Palese P, et al. Localized oncolytic virotherapy overcomes systemic tumor resistance to immune checkpoint blockade immunotherapy. Sci Transl Med [Internet]. American Association for the Advancement of Science; 2014 [cited 2018 Dec 1];6:226ra32. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24598590 95. Andtbacka RHI, Ross M, Puzanov I, Milhem M, Collichio F, Delman KA, et al. Patterns of Clinical Response with Talimogene Laherparepvec (T-VEC) in Patients with Melanoma Treated in the OPTiM Phase III Clinical Trial. Ann Surg Oncol [Internet]. Springer International Publishing; 2016 [cited 2018 Dec 2];23:4169–77. Available from: http://link.springer.com/10.1245/s10434-016-5286-0 96. Chesney J, Puzanov I, Collichio F, Singh P, Milhem MM, Glaspy J, et al. Randomized, Open-Label Phase II Study Evaluating the Efficacy and Safety of Talimogene Laherparepvec in Combination With Ipilimumab Versus Ipilimumab Alone in Patients With Advanced, Unresectable Melanoma. J Clin Oncol [Internet]. American Society of Clinical Oncology; 2018 [cited 2019 Jan 7];36:1658–67. Available from: http://ascopubs.org/doi/10.1200/JCO.2017.73.7379 97. Eriksson E, Milenova I, Wenthe J, Hle MS, Leja-Jarblad J, Ullenhag G, et al. Shaping the tumor stroma and sparking immune activation by CD40 and 4-1BB signaling induced by an armed oncolytic virus. Clin Cancer Res [Internet]. American Association for Cancer Research; 2017 [cited 2018 Dec 4];23:5846–57. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28536305 98. Desjardins A, Gromeier M, Herndon JE, Beaubier N, Bolognesi DP, Friedman AH, et al. Recurrent Glioblastoma Treated with Recombinant Poliovirus. N Engl J Med [Internet]. Massachusetts Medical Society; 2018 [cited 2019 Jan 7];379:150–61. Available from: http://www.nejm.org/doi/10.1056/NEJMoa1716435 99. Fu J, Kanne DB, Leong M, Glickman LH, McWhirter SM, Lemmens E, et al. STING agonist formulated cancer vaccines can cure established tumors resistant to PD-1 blockade. Sci Transl Med [Internet]. 2015 [cited 2018 Sep 13];7:283ra52-283ra52. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25877890 100. Li J, Song W, Czerwinski DK, Varghese B, Uematsu S, Akira S, et al. Lymphoma immunotherapy with CpG oligodeoxynucleotides requires TLR9 either in the host or in the tumor itself. J Immunol [Internet]. 2007 [cited 2016 Sep 26];179:2493–500. Available from: http://www.ncbi.nlm.nih.gov/pubmed/17675511

101. Cheadle EJ, Lipowska-Bhalla G, Dovedi SJ, Fagnano E, Klein C, Honeychurch J, et al. A TLR7 agonist enhances the antitumor efficacy of obinutuzumab in murine lymphoma models via NK cells and CD4 T cells. Leukemia [Internet]. Nature Publishing Group; 2017 [cited 2019 Jan 10];31:1611–21. Available from: http://www.nature.com/doifinder/10.1038/leu.2016.352 102. Hamilton JR, Vijayakumar G, Palese P. A Recombinant Antibody-Expressing Influenza Virus Delays Tumor Growth in a Mouse Model. Cell Rep [Internet]. Cell Press; 2018 [cited 2018 Dec 4];22:1–7. Available from: https://www.sciencedirect.com/science/article/pii/S221112471731834X?via%3Dihub 103. Schmid D, Park CG, Hartl CA, Subedi N, Cartwright AN, Puerto RB, et al. T cell-targeting nanoparticles focus delivery of immunotherapy to improve antitumor immunity. Nat Commun [Internet]. Nature Publishing Group; 2017 [cited 2018 Oct 28];8:1747. Available from: http://www.nature.com/articles/s41467-017-01830-8 104. de Wit R, Kulkarni GS, Uchio E, Singer EA, Krieger L, Grivas P, et al. 864O Pembrolizumab for high-risk (HR) non–muscle invasive bladder cancer (NMIBC) unresponsive to bacillus Calmette-Guérin (BCG): Phase II KEYNOTE-057 trial. Ann Oncol [Internet]. 2018;29:mdy283.073-mdy283.073. Available from: http://dx.doi.org/10.1093/annonc/mdy283.073 105. Dillon PM, Petroni GR, Smolkin ME, Brenin DR, Chianese-Bullock KA, Smith KT, et al. A pilot study of the immunogenicity of a 9-peptide breast cancer vaccine plus poly-ICLC in early stage breast cancer. J Immunother Cancer [Internet]. BioMed Central; 2017 [cited 2018 Oct 16];5:92. Available from: https://jitc.biomedcentral.com/articles/10.1186/s40425-017-0295-5 106. Chawla S, Van Tine BA, Pollack S, Ganjoo K, Elias A, Riedel RF, et al. A phase 2 study of CMB305 and atezolizumab in NY-ESO-1+ soft tissue sarcoma: Interim analysis of immunogenicity, tumor control and survival. Ann Oncol [Internet]. 2017;28:mdx387.007- mdx387.007. Available from: http://dx.doi.org/10.1093/annonc/mdx387.007 107. Andtbacka RH, Curti BD, Hallmeyer S, Feng Z, Paustian C, Bifulco C, et al. Phase II calm extension study: Coxsackievirus A21 delivered intratumorally to patients with advanced melanoma induces immune-cell infiltration in the tumor microenvironment. J Immunother Cancer [Internet]. BioMed Central; 2015 [cited 2019 Jan 8];3:P343. Available from: http://jitc.biomedcentral.com/articles/10.1186/2051-1426-3-S2-P343 108. Bernstein V, Ellard SL, Dent SF, Tu D, Mates M, Dhesy-Thind SK, et al. A randomized phase II study of weekly paclitaxel with or without pelareorep in patients with metastatic breast cancer: final analysis of Canadian Cancer Trials Group IND.213. Breast Cancer Res Treat [Internet]. 2018 [cited 2019 Jan 9];167:485–93. Available from: http://www.ncbi.nlm.nih.gov/pubmed/29027598

Table 1. Ongoing Clinical Trials using TLR Agonists PRR Target Agent Combination Cancers Investigated Phase Results Identifier Non-muscle-invasive Bladder TLR2/4/9 + BCG aPD-L1 3 Ongoing NCT03528694 Cancer NOD2 BCG aPD-1 Non-muscle-invasive Bladder 3 Ongoing NCT03711032

Cancer Phase 2 Results(104) High Risk Non-muscle- BCG Mitomycin C 3 Ongoing NCT02948543 invasive Bladder Cancer Non-muscle-invasive Bladder BCG 3 Ongoing NCT03091660 Cancer Rintatolimod + tumor cell Ovarian, Fallopian Tube, and 1/2 Ongoing NCT01312389 lysate vaccination Primary Peritoneal Cancer Rintatolimod + Peptide GM-CSF Breast Cancer 1/2 Ongoing NCT01355393 Vaccination Poly-ICLC + DC Vaccine Metastatic Pancreatic Cancer 1 Results(27) NCT01410968 Poly-ICLC + Peptide Smoldering Multiple 1/2a Results(28) NCT01718899 Vaccination Myeloma Poly-ICLC + Peptide Breast Cancer 1 Results(105) NCT01532960 Vaccination Cyclophosphamide + TLR3 Poly-ICLC Hepatocellular Cancer 1/2 Results(30) NCT00553683 Radiotherapy CDX-301 + Low-Grade B-cell Poly-ICLC 1/2 Ongoing NCT01976585 Radiotherapy Lymphoma Poly-ICLC + Peptide aPD-1 + Rituximab Follicular Lymphoma 1 Ongoing NCT03121677 Vaccination Melanoma, Non-Small Cell Lung Cancer, Head and Neck Poly-ICLC + Peptide aPD-1 Squamous Cell Carcinoma, 1/2 Ongoing NCT03633110 Vaccination Urothelial, and Renal Cell Carcinoma AS15 + MAGE-A3 Stage III Melanoma 3 Results(35) NCT00796445 TLR4 + TLR9 vaccine + NLRP3 AS15 + MAGE-A3 Non-Small Cell Lung Cancer 3 Results(36) NCT00480025 vaccine G100 Merkel Cell Carcinoma 1 Results(37) NCT02035657 G100 Cutaneous T-Cell Lymphoma 2 Ongoing NCT03742804 Follicular Low-Grade Non- G100 aPD-1+ Rituxumab 1/2 Ongoing NCT02501473 Hodgkin’s Lymphoma TLR4 aOX40, aICOS, or aPD- GSK1795091 Advanced Solid Tumors 1 Ongoing NCT03447314 1 GLA-SE + MART-1 Stage II-IV Melanoma N/A Ongoing NCT02320305 antigen vaccine Entolimod Colorectal Cancer 2 Ongoing NCT02715882 Advanced or Metastatic Solid TLR5 Entolimod 1 Results(42) NCT01527136 Tumors Mobilan Prostate Cancer 1/2 Ongoing NCT02844699 Cervical Intraepithelial NCT00941252 Imiquimod 3 Results(44) Neoplasia Resiquimod + NY-ESO-1 NCT00821652 Melanoma 1 Results(45) Vaccine Ongoing NCT03416335 DSP-0509 Neoplasms 1 Pre-clinical TLR7/8 Results(46) NCT02556463 MEDI9197 aPD-L1 Solid Tumors 1 Results(47)

Ongoing NKTR-262 Locally Advanced or aIL-2Rβ + aPD-1 1 Pre-clinical NCT03435640 Metastatic Solid Tumors Results(49) TLR8 + Head and Neck Squamous NCT02124850 Motolimod Cetuximab + aPD-1 1 Results(50) NLRP3 Cell Carcinoma CMP-001 aPD-L1 + Radiotherapy Non-Small Cell Lung Cancer 1 Ongoing NCT03438318 TLR9 aPD-1+ aCTLA-4 + NCT03507699 CMP-001 Metastatic Colorectal Cancer 1 Ongoing Radiotherapy

CMP-001 aPD-1 Melanoma 1 Ongoing NCT03618641 Ongoing CMP-001 aPD-1 Advanced Melanoma 1b NCT03084640 Early Results(56) CMP-001 aPD-1 Melanoma 1 Ongoing NCT02680184 Anti-PD-1 Refractory Ongoing Tilsotolimod aCTLA-4 3 NCT03445533 Melanoma Phase 2 Results(57) Tilsotolimod aCTLA-4 or aPD-1 Metastatic Melanoma 1/2 Ongoing NCT02644967 Ongoing Lefitolimod Metastatic Colorectal Cancer 3 NCT02077868 Phase 2 Results(58) Lefitolimod aCTLA-4 Advanced Solid Tumors 1 Ongoing NCT02668770 SD-101 Radiotherapy Low-grade B-cell Lymphoma 1/2 Results(55) NCT02266147 Metastatic Melanoma / Head Ongoing SD-101 aPD-1 1b/2 NCT02521870 and Neck Cancer Early Results(60) Ongoing anti-OX40 antibody + Low-grade B-cell Non- SD-101 1 Pre-clinical NCT03410901 Radiotherapy Hodgkin Lymphomas Results(61) DV281 aPD-1 Non-small Cell Lung Cancer 1 Ongoing NCT03326752

Table 2. Ongoing Clinical Trials Using NLR and CLR Agonists PRR Target Agent Combination Cancers Investigated Phase Results Identifier NOD2 Mifamurtide Chemotherapy High Risk Osteosarcoma 2 Ongoing NCT03643133 DEC-205 + Poly-ICLC + CDX-1401 Advanced Cancers 1/2 Results(74) NCT00948961 TLR3 + TLR7 Resiquimod Ovarian, Fallopian Tube, and CDX-1401 Poly-ICLC + Primary Peritoneal Cancer in 1/2 Ongoing NCT02166905 Epacadostat DEC-205 + Remission TLR3 Myelodysplastic Syndrome or CDX-1401 Poly-ICLC + aPD-1 + Acute Myeloid Leukemia 1 Ongoing NCT03358719 Decitabine

Ongoing DC-SIGN CMB305 Synovial Sarcoma 3 Early Phase 2 NCT03520959 Results(106) Cetuximab + Paclitaxel Imprime PGG + Carboplatin Non-Small Cell Lung Cancer 2 Results(76) NCT00874848

Imprime PGG aPD-1 Non-Small Cell Lung Cancer 1b/2 Ongoing NCT03003468 Dectin-1 Advanced Melanoma, Triple Imprime PGG aPD-1 2 Ongoing NCT02981303 Negative Breast Cancer Rituximab Relapsed Indolent Non- Imprime PGG 2 Ongoing NCT02086175 Hodgkin Lymphoma Imprime PGG aPD-L1 + Bevacizumab Metastatic Colorectal Cancer 1/2 Ongoing NCT03555149

Table 3. Ongoing Clinical Trials Using RLR and CDS Agonists PRR Target Agent Combination Cancers Investigated Phase Results Identifier MK4621 Advanced Solid Tumors 1/2 Results(82) NCT03065023 RIG-I MK4621 aPD-1 Advanced Solid Tumors 1 Ongoing NCT03739138 Advanced Solid Tumors or MIW815 aPD-1 1 Ongoing NCT03172936 Lymphomas Advanced Solid Tumors or STING MIW815 aCTLA-4 1 Ongoing NCT02675439 Lymphomas MK-1454 Advanced Solid Tumors or Ongoing aPD-1 1 NCT03010176 Lymphomas Early Results(90)

Table 4. Ongoing Clinical Trials Using Oncolytic Viruses PRR Target Agent Combination Cancers Investigated Phase Results Identifier T-Vec aCTLA-4 Melanoma 1b/2 Results(96) NCT01740297 T-Vec aPD-1 Stage III/IV Melanoma 2 Ongoing NCT02965716 Herpes simplex T-Vec Paclitaxel Triple Negative Breast Cancer 1/2 Ongoing NCT02779855 T-Vec Radiotherapy Soft Tissue Sarcoma 1/2 Ongoing NCT02453191 Pexa Vec Sorafenib Hepatocellular Carcinoma 3 Ongoing NCT02562755 Vaccinia poxvirus Pexa Vec aPD-1 Renal Cell Carcinoma 1b Ongoing NCT03294083 Pexa Vec aPD-1 Hepatocellular Carcinoma 1/2a Ongoing NCT03071094 CAVATAK Stage IIIC-IV Melanoma 2 Results(107) NCT01636882 Coxsackievirus CAVATAK aCTLA-4 Advanced Melanoma 1 Ongoing NCT02307149 Advanced Non-Small Cell CAVATAK aPD-1 1 Ongoing NCT02824965 Lung Cancer Pancreatic, Ovarian, Biliary, Adenovirus LOAd703 1/2 Ongoing NCT03225989 and Colorectal Cancer PVSRIPO Recurrent Glioblastoma 1 Results(98) NCT01491893 Enterovirus PVSRIPO Unrescectable Melanoma 1 Ongoing NCT03712358 Pelareorep Paclitaxel Metastatic Breast Cancer 2 Results(108) NCT01656538 Reovirus Pelareorep aPD-1 Advanced Pancreatic Cancer 2 Ongoing NCT03723915

Figure 1. Pro-inflammatory signaling pathways downstream of PRRs. Upon binding their respective ligands, each PRR conveys signal through specific adaptor molecules and signaling pathways, ultimately converging on production of pro-inflammatory cytokines and type 1 interferons. Printed with permission from ©Mount Sinai Health System.

Figure 2. PRR pathways in anti-tumor immunity. Therapeutic activation of PRR pathways can induce immunogenic cell death in cancer cells, releasing DAMPs and tumor associated antigens. PRR ligands can reprogram immunosuppressive TAMs, and activate DCs to cross-present tumor antigens, stimulating a cytotoxic anti-tumor immune response. Printed with permission from ©Mount Sinai Health System. Abbreviations: TLR: Toll-Like Receptor; RLR: RIG-I-Like Receptor; NLR: NOD-Like Receptor; CLR: C-Type Lectin Receptor; CDS: Cytosolic DNA Sensor; TAM: Tumor Associated Macrophage

Figure 1:

TriAcyl DiAcyl lipo- lipo- LPS protein protein Flagellin β-glucan Mannan

TLR4 TLR1/2 TLR2/6 TLR5 Dectin-1 Dectin-2 LPB MD2

Plasma membrane CD14

TIRAP TIRAP TIRAP MYD88 FCRγ SYK Cytosol Endosome MYD88 MYD88 MYD88 dsRNA CpG DNA IL-1β TLR4 ssRNA CARD9 BCL10 Pro-IL-1β 5’-PPP TLR3 IL-18 short dsRNA Long TLR7/8 TLR9 IRAK4 MALT1 Pro-IL-18 dsRNA IRAK1 and/or iE-DAP MDP IRAK2 Caspase-1 Flagellin RIG-I TRAM NOD1 NOD2 MDA5 TRIF MYD88 TRAF6 TRIF MYD88 NAIP RIP2 Alum NLRC4 ASC LGP2 TAB2 TAB3 NLRP3 ASC ProCaspase-1 TRAF3 TAK1 MAPK ProCaspase-1

MAVS signaling TBK1 IKK-i NEMO Cytoslic IKKα IKKβ dsDNA NLRX1 AIM2 ASC cGAS ProCaspase-1 IRF3 IRF7 Mitochondria NF-κB AP-1 cGAMP

STING

Endoplasm Cytosol reticulum

ISRE3 ISRE7 NF-κB AP-1 Nucleus Type 1 interferons Proinﬂammatory cytokines

Figure 2:

TAM Poly-ICLC TLR BCG Oncolytic RLR Dendritic virus cell

Tumor cells CLR

NLR TLR

RLR RLR Tumor antigen Immunogenic CDS Radiotherapy cross- cell death Chemotherapy presentation CDS

Viral genome Tumor DNA NK cell Tumor-associated antigen CD8+ Cytokines, chemokines, T cell type 1 interferons Cytotoxic antitumor immune response

Pathogen molecular pattern receptor agonists: treating cancer by mimicking infection

Mark Aleynick, Judit Svensson-Arvelund, Christopher R. Flowers, et al.

Clin Cancer Res Published OnlineFirst May 23, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-18-1800

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/early/2019/05/23/1078-0432.CCR-18-1800. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.