Pathogen Molecular Pattern Receptor Agonists: Treating Cancer by Mimicking Infection Mark Aleynick1, Judit Svensson-Arvelund1, Christopher R

Published OnlineFirst May 23, 2019; DOI: 10.1158/1078-0432.CCR-18-1800

Review Clinical Cancer Research Pathogen Molecular Pattern Receptor Agonists: Treating Cancer by Mimicking Infection Mark Aleynick1, Judit Svensson-Arvelund1, Christopher R. Flowers2, Aurelien Marabelle3, and Joshua D. Brody1,4

Abstract

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

Introduction TLRs in Cancer Immunotherapy The interplay between cancer and the immune system is a TLRs are the most widely studied PRR family, acknowledged in double edged sword; the inflammation that recruits and acti- the 2011 Nobel Prize awarded to Drs. Steinman, Beutler, and vates intratumoral immune cells can either eliminate cancer Hoffman. There have been 10 TLRs identified in humans and 13 in cells or drive tumor progression in a context-dependent man- mice (4); here we focus on the former. Structurally, TLRs are type I ner (1). Pattern recognition receptors (PRR) are a key family of transmembrane proteins characterized by a ligand-binding N proteins involved in the inflammatory response. They are terminal ectodomain containing leucine-rich repeats, a single expressed on a wide variety of innate and adaptive immune transmembrane domain, and a cytosolic Toll/IL1R homology cells, as well as tumor cells, and recognize both foreign domain responsible for signal transduction (2). TLRs 1, 2, and pathogen-associated molecular patterns (PAMP) and self- 4–6 are located on the cell surface and recognize bacterial mem- derived damage-associated molecular patterns (DAMP) result- brane components such as lipids, proteins, lipoproteins (Fig. 1; ingfrominjuryorcelldeath(1–3). There are five families of refs. 2, 3), as well as several self-molecules, including extracellular PRRs: toll-like receptors (TLR), nucleotide-binding oligomeri- matrix components, HSPs, and nuclear high-mobility group zation domain (NOD)-like receptors (NLR), C-type lectin box 1 (HMGB1), often released as DAMPs from apoptotic or receptors (CLR), RIG-I–like receptors (RLR), and cytosolic DNA necrotic cells (1, 3). sensors (CDS; ref. 2). Each PRR family possesses distinct Intracellular TLRs 3 and 7–9 are located within endosomes, and immunomodulatory properties, making them attractive immu- recognize viral and bacterial nucleic acids resulting from micro- notherapeutic targets. Here, we discuss PRR mechanisms and bial replication or degradation upon entry into the cell (Fig. 1; clinical implications to provide a detailed overview of the role refs. 1–3). Their localization normally prevents intracellular TLRs of PRRs in immuno-oncology. 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 nonfunctional in 1Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, mice. Recent work suggests it may serve as a negative regulator of New York, New York. 2Winship Cancer Institute of Emory University, TLR signaling (6, 7). TLRs dimerize upon binding their cognate 3 Emory University School of Medicine, Atlanta, Georgia. Cancer Immuno- ligand (Fig. 1), causing conformational changes that allow for the 4 therapy Program, Gustave Roussy, Villejuif, France. Hematology and recruitment of adapter molecules (MyD88, TIRAP, TRIF, and Medical Oncology, Icahn School of Medicine at Mount Sinai, New York, New York. TRAM), initiating signaling cascades that ultimately induce tran- scription of inflammatory mediators (2, 3, 8). Corresponding Author: Joshua D. Brody, Icahn School of Medicine at As TLRs are highly expressed on antigen-presenting cells (APC), Mount Sinai, New York, NY 10029. Phone: 212-241-5426; Fax: 646-537- 9268; E-mail: [email protected] targeting TLRs can activate APCs and trigger adaptive immune responses; intratumorally, this may shift a tolerogenic tumor Clin Cancer Res 2019;XX:XX–XX microenvironment (TME) to become immunogenic. However, doi: 10.1158/1078-0432.CCR-18-1800 because TLR signaling triggers inflammatory and cell survival 2019 American Association for Cancer Research. mechanisms, and certain tumors express TLRs, TLR activation

www.aacrjournals.org OF1

Aleynick et al.

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 Cytosol Endosome MYD88 MYD88 MYD88 SYK 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 RX1 IKKα IKKβ dsDNA NL AIM2 ASC cGAS ProCaspase-1 IRF3 IRF7 Mitochondria NF-κBAP-1 cGAMP

STING

Endoplasm Cytosol reticulum

ISRE3 ISRE7 NF-κBAP-1 Nucleus Type 1 interferons Proinﬂammatory cytokines

Figure 1. Proinflammatory 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 proinflammatory cytokines and type 1 IFNs. Printed with permission from Mount Sinai Health System.

could instead be tumorigenic in certain settings (1, 9). Both of these reports implicate DAMPs in tumor initiation and pro- TLR7 and TLR8 signaling have been implicated in driving lung gression through chronic inﬂammation, other studies demon- cancer cell survival and chemotherapy resistance mechanisms strate that DAMPs released from dying tumor cells are the (10, 11). TLR4 signaling in breast cancer both enhances chemo- hallmark of immunogenic cell death, activating APCs in the therapeutic resistance and promotes angiogenesis and lymphatic TME to present tumor antigen (9, 18). Despite the nuanced metastasis (12, 13). Tumor cells may also secrete HSPs and role of PRR signaling in cancer, in many contexts, therapies extracellular matrix factors as DAMPs, stimulating an immuno- targeting PRR pathways have the ability to overcome immunosuppressive program in tumor-associated macrophages (TAM) suppression or drive a de novo antitumor response by activating to promote angiogenesis and metastasis (14, 15). One recent APCs to enhance tumor antigen presentation (Fig. 2). For the study demonstrated that mice lacking TLR3/7/9 cleared implant- remainder of this review, we will focus on clinical trials and ed tumors through spontaneous induction of an adaptive anti- preclinical studies utilizing PRRs in this setting. tumor response (16). Similar effects are observed with other One of the few FDA-approved TLR-targeting therapies in PRR families; galectin-9 signaling through the CLR dectin-1 on oncology is Bacillus Calmette-Guerin (BCG), a strain of TAMs is protumorigenic in mouse models of pancreatic ductal Mycobacterium bovis initially developed as a tuberculosis vaccine. adenocarcinoma, although signaling through this receptor in Used as a urogenital cancer therapeutic for over 35 years, a large other cancers may have the opposite effect (17). While several body of work has dissected its mechanism, demonstrating that

OF2 Clin Cancer Res; 2019 Clinical Cancer Research

Pattern Recognition Receptors in Immuno-oncology

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

Figure 2. PRR pathways in antitumor 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 antitumor immune response. Printed with permission from Mount Sinai Health System.

BCG triggers an immune response by activating TLRs 2, 4, and trials using poly-ICLC demonstrate its potent ability to induce 9, and the NLR NOD2 (1, 19, 20). Several trials are combining adaptive immune responses against a range of solid and hemato- BCG therapy with checkpoint blockade or have expanded BCG poietic cancers. A phase I study evaluating peptide-pulsed DC to other cancers, with varying degrees of success (21). vaccination in combination with poly-ICLC for pancreatic cancer TLR3 is one of the most actively explored TLR targets, with showed promise with a 7.7 month median survival, an improve- 54 ongoing clinical trials using TLR3 agonists as single agents or ment over the 4.2–4.9 month survival seen with second-line in combination with other therapies to treat a broad list of chemotherapy in metastatic pancreatic cancer (27). A phase I/IIa malignancies (clinicaltrials.gov). TLR3 recognizes viral double- trial in smoldering multiple myeloma recently demonstrated that stranded RNA (dsRNA), and can be targeted using synthetic peptide þ poly-ICLC vaccination increased numbers of antigen- dsRNA analogs such as polyinosinic-polycytidylic acid (poly-IC; specific CD8 T-cells with an effector memory phenotype (28). þ refs. 2, 3, 22). Poly-IC initially showed high toxicity and limited Another study pinpointed TLR3 DCs as key mediators of tumor therapeutic benefit, but several poly-IC derivatives were subse- antigen cross-presentation, where an in situ vaccination combin- quently created to improve efficacy (22). One such derivative ing poly-ICLC, Flt3 ligand, and local irradiation induced both is poly-ICLC, modified with poly-L-lysine and carboxymethylcel- partial and complete responses in patients with non-Hodgkin lulose (Hiltonol, Oncovir) to increase stability in vivo, improving lymphoma (29). A pilot study in patients with transplant- its interferon (IFN) response to levels similar to those seen with ineligible hepatocellular carcinoma showed survival benefit attenuated viral infections (23, 24). Another derivative, poly- compared with historical controls using local tumor irradiation IC12U (rintatolimod/Ampligen, Hemispherx Biopharma), adds followed by intratumoral poly-ICLC administration (30). Simi- unpaired bases that reduce stability, effectively reducing toxicity larly, studies treating patients with glioblastoma demonstrated while generating robust dendritic cell (DC)/T-cell responses (25). impressive survival outcomes by combining poly-ICLC with Preclinical data also suggests that rintatolimod recruits fewer irradiation and/or alkylating chemotherapy (31, 32). In the pre- regulatory T cells to the TME versus unmodified poly-IC, possibly clinical setting, next-generation DC vaccines are being explored, by losing ability to bind cytosolic RLRs (26). Recently completed employing nanoparticles to selectively deliver tumor antigens þ

www.aacrjournals.org Clin Cancer Res; 2019 OF3

Aleynick et al.

poly-IC to DCs in vivo, eliminating the need for ex vivo DC enhances local immune response and directly induces apoptosis manipulation (33). In addition, several groups have also dem- in BCC cells (43). Imiquimod is being investigated in phase III onstrated that TLR3 activation can help overcome resistance to studies as a treatment for gynecologic cancers with promising checkpoint blockade, leading to ongoing academic and pharma early results (44), and in dozens of phase I and II trials in various trials planning to accrue >400 patients studying combinations of cancers, either alone or combination (clinicaltrials.gov). Other poly-ICLC with PD-1, PD-L1, or CTLA-4 blockade to treat various imidazoquinoline derivatives in the clinic include a topical gel cancers (e.g., NCT03121677 and NCT03633110). formulation of resiquimod, a more potent imidazoquinoline TLR4 is canonically involved in the recognition of bacterial investigated as an adjuvant to NY-ESO-1 vaccination for patients lipopolysaccharide (LPS), although it is also indirectly involv- with melanoma that has been shown to induce NY-ESO-specific ed in viral infection by recognizing DAMPs, such as HMGB1, CD8 T-cell responses (45). DSP-0509 (Boston Biomedical), a HSPs, and extracellular matrix components released from TLR7/8 agonist formulated for intravenous delivery, has shown infected or dying cells (1, 3, 8). Ongoing clinical efforts with preclinical efficacy in several tumor models and is now being TLR4 ligands in cancer immunotherapy include the FDA- investigated in a phase I trial (NCT03416335; ref. 46). MEDI9197 approved TLR4 agonist AS04 (GlaxoSmithKline), a monopho- (3M-052; Medimmune) is an imidazoquinoline formulated for sphoryl lipid A (MPLA) LPS derivative in alum. AS04 is used as intratumoral injection and optimal tumor retention to improve an adjuvant in the (human papillomavirus) HPV-16/18 vaccine safety. Preliminary phase I results (NCT02556463) demonstrate Cervarix, which in a landmark trial was shown to not only intratumoral immune cell infiltration and low serum MEDI9197 protect women from cervical cancer from the vaccine-inclusive levels, indicating effective retention in the TME (47). Similarly, strains, but was also cross-reactive against other oncogenic NKTR-262 (Nektar Therapeutics) is a TLR7/8 agonist formulated forms of HPV (34). Interestingly, two recent phase III trials for intratumoral retention to minimize systemic exposure (48) of a MAGE-A3 vaccine with AS15 (GlaxoSmithKline), an adju- and has shown potent efficacy, where treatment of one tumor site vant containing MPLA and a TLR9 agonist, both failed to led to clearance of untreated contralateral tumors in multiple improve patient survival, citing low CD8 T-cell responses in preclinical models, an abscopal effect often considered the holy patients (35, 36). Another TLR4 agonist, a synthetic analog of grail of intratumoral immunotherapy. These promising results glucopyranosyl lipid A engineered to decrease heterogeneity led to a recently opened phase I/II study of NKTR-262 in com- and minimize toxicity over natural lipid A formulated in a bination with a CD122 agonistic antibody and checkpoint stable emulsion (GLA-SE/G100; Immune Design), showed blockade (NCT03435640; ref. 49). A recent randomized study promise in a phase I study of Merkel Cell Lymphoma, where of platinum-based chemoimmunotherapy for head/neck can- 2 of 10 patients had durable sustained antitumor responses, cers demonstrated no overall survival benefit by adding the whereas 2 others had complete responses (37). Similarly, TLR8 agonist motolimod (Array Biopharma/Celgene), although encouraging clinical responses were seen in 26 patients with motolimod did improve survival in subsets of patients with þ follicular lymphoma receiving intratumoral G100 and radio- HPV tumors (50). therapy with or without PD-1 blockade, where >80% disease TLR9 recognizes unmethylated 20-deoxyribo(cytidine- control rates were seen in both groups, and addition of anti-PD- phosphate-guanosine) (CpG) motifs, which occur more fre- 1benefited relapsed and chemo-refractory patients (38). quently in prokaryotic DNA. Similar to TLRs 7/8, TLR9 is highly TLR5 is unique in that it does not recognize DAMPs as its expressed on pDCs, as well as on B cells, and is critical in the only ligand is bacterial flagellin, making it a potentially useful immune response to DNA viruses (3, 8). Synthetic CpG oligo- immunotherapeutic target (39). Two TLR5 agonists in clinical deoxynucleotides (ODN) potently activate TLR9-expressing development, entolimod and mobilan (Cleveland Bio Labs), immune cells and have been divided into four classes: Class A, have shown preclinical efficacy in several tumor models B, C, and P (51, 52). Class A ODNs contain palindromic phos- (39–41). Entolimod is a flagellin derivative engineered to reduce phodiester CpG central sequences with phosphorothioate G toxicity, currently being investigated in a phase II trial as a rich ends, allowing tetrad formation, enhanced stability, endo- neoadjuvant therapy for colorectal cancer (NCT02715882; somal uptake, and robust activation of pDC type 1 IFN responses. ref. 42). Mobilan is an adenovirus construct that upon infection Class B ODNs are short, linear phosphorothioate backbone induces co-expression of TLR5 and a secreted form of entolimod, ssDNA strands, and potent activators of B and natural killer creating an autocrine signaling loop and inflammatory sig- (NK) cells. Class C ODNs combine properties of class A nature in the TME (41). Mobilan has shown preclinical efficacy and B, activating both B and NK cells, as well as type 1 IFN for prostate cancer, which expresses high levels of the adenovirus pDC responses. Class P ODNs feature multiple palindromic receptor necessary for its entry, and is now in a phase I/II trial sequences and form multimeric structures, enhancing stability (NCT02844699; ref. 41). and immunostimulatory responses (52). The first ODN in Both TLR7 and TLR8 are functional in humans, whereas mice human trials was CpG7909 (agatolimod/PF-3512676/ProMune; only have functional TLR7. TLR7/8 recognizes single-stranded Pfizer), a class B ODN, which showed early promise both as RNA from RNA viruses, although RNA from certain bacterial an in situ vaccination and chemotherapy adjuvant (53–55). strains may also ligate these TLRs (8). TLR7/8 also recognizes However, a phase III lung cancer trial of chemotherapy with or purine analogs such as imidazoquinolines, as well as guanine without this ODN concluded that CpG7909 increased adverse derivatives and certain siRNA (3). Ligation of TLR7/8 triggers events without benefiting survival, curtailing its development. robust proinflammatory cytokine production, and is critical for Two other phase III trials that investigated CpG7909 as part of the activation of plasmacytoid DC (pDC), a key source of type 1 a MAGE-A3 vaccination also failed to demonstrate clinical IFNs (3, 8). The only FDA-approved TLR7/8 agonist is imiquimod benefit (35, 36). (Aldara; 3M Pharmaceuticals), an imidazoquinoline topical agent Despite failures with CpG7909, several CpG ODNs modified for the treatment of basal-cell carcinoma (BCC) that both to enhance efficacy and safety are in development. CMP-001

OF4 Clin Cancer Res; 2019 Clinical Cancer Research

Pattern Recognition Receptors in Immuno-oncology

(Checkmate Pharmaceuticals), a class A ODN packaged within CLR in Cancer Immunotherapy a virus-like particle, potently activates intratumoral pDCs and CLRs are a large family of receptors that contain at least one overcomes resistance to checkpoint blockade; five trials with carbohydrate recognition domain, recognizing mannose, fructose, this ODN are ongoing for various solid tumors (clinicaltrials. and glucans presentonpathogens (reviewed inref.67; refs.2,8,69). gov; ref. 56). Tilsotolimod (IMO-2125; Idera Pharmaceuticals) Although classically associated with antifungal and mycobacterial is another TLR9 agonist that is being investigated in checkpoint immune responses, more recent evidence suggests CLRs are blockade refractory patients. In patients who failed anti-PD-1 involved in sensing numerous pathogens including bacteria, virus- therapy, tilsotolimod combined with ipilimumab CTLA-4 es, and helminths, as well as DAMPs (69–71). CLRs are mainly blockade improved objective tumor responses over ipilimu- expressed by DCs, although monocytes/macrophages, B cells, and mab alone, and this combination has entered a phase III trial neutrophils may also express CLRs. Most CLR family members are (NCT03445533; ref. 57). Lefitolimod (MGN1703, Mologen transmembrane receptors, although a few may be released as AG) is a novel class of ODN that lacks phosphorothionate soluble proteins, such as mannose-binding-lectin (4, 69). Upon backbone modifications and is instead "dumbbell shaped" to ligation, CLRs transduce signal by either associating with kinases prevent degradation (51). Demonstrating favorable safety and and phosphatases directly, or by recruiting ITAM-containing co- clinical efficacy, lefitolimod has initiated a phase III trial in receptors such as FcRg (Fig. 1; refs. 8, 69). Signaling ultimately metastatic colorectal cancer (NCT02077868; refs. 58, 59). converges on MAPK and NF-kB, allowing CLRs to influence Another class C agonist, SD-101 (Dynavax), is being investi- signaling cascades from other PRRs, tailoring the immune response gated in several trials, after showing both preclinical and against specific pathogens. In addition, many CLRs are internalized clinical efficacy in melanoma and as an in situ vaccination for after activation, bringing their ligand cargo within the cell for lymphoma, in combination with checkpoint blockade and degradation and subsequent antigen presentation, a critical process agonistic antibodies for T-cell costimulation (55, 60–62). in activating the adaptive immune response (69). DV281, a TLR9 agonist formulated for inhalation, is being Strategies targeting CLRs date back over two decades. Random- investigated as an adjuvant for PD-1 checkpoint blockade ized studies with a mannan-MUC1 fusion protein targeting man- therapy in lung cancer (NCT03326752), where intratumoral nose receptor (MR), vaccinating patients with breast cancer after injection of adjuvant is more challenging. Notably, another surgical resection, showed significant protection from recurrence, Dynavax TLR9 agonist tested in a large randomized trial did demonstrating the efficacy of CLR targeting and the importance demonstrate superior immunogenicity (seroconversion) when of adaptive immunity in preventing recurrence (72). Anti-CLR combined with HBsAg as comparedwithstandardHBVvacci- antibodies have also been used to target CLR-expressing DCs. nation, leading to its FDA approval. Potentially, this immu- CDX-1307 (Celldex Therapeutics) is an MR-specific antibody nostimulatory effect could portend success in cancer therapy as fused to human chorionic gonadotropin beta-chain (HCG-b), com- now shown with pathogen vaccines. monly overexpressed in various cancers (73). Vaccinating with CDX-1307, GM-CSF, poly-ICLC, and/or resiquimod to mature NLR in Cancer Immunotherapy DCs, most treated patients developed humoral response against HCG-b, and some developed T-cell responses. Combining NLRs are intracellular PRRs that recognize a diverse set of CDX-1401 (Celldex Therapeutics), an anti-DEC-205 antibody ligands including bacterial and viral PAMPs, as well as DAMPs fused to NY-ESO-1 antigen, with poly-ICLC and/or resiquimod, (reviewedinref.61;refs.2,3,8,63).OfseveralNLRfamilies, yielded humoral and T-cell responses in most patients that cor- the NLRC and NLRP families are the most well-studied (2, 63). related with stable disease (74). In addition, several patients NOD1 and NOD2 are prominent NLRC family members, who progressed saw dramatic benefit with subsequent check- which all contain N-terminal CARD domains. Similar to TLR2, point blockade, warranting studies of this combination therapy. NOD1 and NOD2 recognize components of the peptidoglycan CDX-1401 is currently being investigated in gynecologic bacterial cell wall, where NOD1 specifically recognizes gamma- (NCT02166905) and hematologic (NCT03358719) malignancies. D-glutamyl-meso-diaminopemelic acid and NOD2 recognizes CMB305 (Immune Design) is a Sindbis virus engineered to use muramyl dipeptide (Fig. 1; refs. 8, 63). NLRPs, NLRP3 being DC-SIGN as an attachment receptor, selectively infecting and the most well characterized, form part of the inflammasome, expressing NY-ESO-1 protein in DCs for antigen presentation which leads to production of proinflammatory IL-1b/IL-18 (75). CMB305 is being investigated in a phase III trial in syno- (Fig. 1). Besides several bacterial ligands, environmental pol- vial sarcoma (NCT03520959). Another therapy, Imprime PGG lutants such as asbestos and silica are known to initiate NLR (Biothera), uses IV yeast-derived soluble b-glucan, a dectin-1 inflammasomes (64). ligand, to sensitize patients and boost efficacy of targeted therapy Mifamurtide, a synthetic analog of muramyl tripeptide and and anti-PD-1 blockade, and is being investigated in several phase I NOD2 agonist, is approved in the European Union in combi- and II studies (clinicaltrials.gov) with promising early results. nation with chemotherapy to treat osteosarcoma (4, 65). The Interestingly, Imprime PGG shows a moderate toxicity profile, with TLR8 agonist, motolimod, is also a potent stimulator of the 22% of patients discontinuing treatment due to adverse events, NLRP3 inflammasome, likely because of the molecule's lipo- possibly because of the route of administration and the drug's philic structure; however the specific mechanism is still under potent activation of the complement cascade (76). investigation (50, 66). In addition, particulate adjuvants such as alum and saponins, often used in cancer vaccine formula- – tions including HPV and the previously mentioned MAGE-A3 Cytosolic Viral Sensors RLR and CDS vaccine studies (35, 36), are potent activators of the NLRP3 While TLR3 is responsible for detecting viral dsRNA within inflammasome, producing inflammatory cytokines to engender endosomal compartments, RLRs retinoic acid–inducible gene I adaptive immune responses (67, 68). (RIG-I), melanoma differentiation–associated gene 5 (MDA5),

www.aacrjournals.org Clin Cancer Res; 2019 OF5

Aleynick et al.

and laboratory of genetics and physiology 2 (LGP2) recognize function (8, 83, 84). In the context of infection, STING is a key cytosolic dsRNA (Fig. 1; reviewed in ref. 75; ref. 77). These sen- mediator of the immune response against intracellular bacteria, sors are critical in the host antiviral response and are expressed DNA viruses, and retroviruses; however, its ability to detect within most cell types, including cancer cells (4). Structurally, genomic DNA from dying tumor cells makes the STING path- RLR family members contain a C-terminal RNA binding domain, way potentially important for antitumor immune responses. a DExD/H central domain for ATP catalysis and activation, and The ability of the STING pathway to drive adaptive antitumor an N-terminal CARD domain that interacts with the downstream responses has generated significant interest, and recent studies effector molecule MAVS (also referred to as IPS-1) to convey suggest that efficacy of numerous DNA-damaging cancer ther- signaling. Short dsRNA with 50 triphosphate (50-PPP) ends are apies can in part be attributed to STING signaling. Chemother- preferentially recognized by RIG-I, whereas MDA5 recognizes apeutic agents cause DNA leakage into the cytosol, triggering a longer dsRNA fragments, including poly-IC. While LGP2 also STING-dependent type I IFN response (Fig. 2; ref. 85). STING recognizes dsRNA, this family member cannot convey down- signaling is also required for successful activation of adaptive stream signaling because it lacks a CARD domain, and is instead immunity and tumor clearance in response to both radiother- important in regulating RIG-I and MDA5 activation (8, 77). apy (86) and T-cell checkpoint blockade, as cGAS-STING sig- Stimulation of epithelial ovarian cancer cells with a RIG-I– naling enhances DC-mediated T-cell priming. Administration specific agonist triggers type I IFN release and immunogenic of adjuvant cGAMP synergized with checkpoint blockade in apoptosis, which effectively matures DCs upon phagocytosis vivo,presumablybyincreasingthetumorreactiveT-cell of these apoptotic cancer cells (78). In addition, activation of pool (87). Such studies highlight the importance of STING RIG-I using 50-PPP RNA or MDA5 using poly-IC causes apoptosis and type I IFNs in DC cross-presentation of tumor antigens for in human melanoma cells both in vitro and in vivo, whereas antitumor T-cell priming. adjacent nonmalignant cells are spared because of intact anti- Several STING-specific agonists recently entered clinical devel- apoptotic BCL-XL signaling (79). opment. Originally investigated as a vascular disrupting agent, The multimodal action of RLRs in immune cell activation STING agonist DMXAA (ASA404/vadimesan, Antisoma/Novar- while simultaneously triggering immunogenic cell death in tis) showed preclinical efficacy, but failed in a pivotal phase III cancer cells makes this pathway a particularly attractive immu- trial as a combination treatment with chemotherapy in non– notherapeutic target. The successes of poly-ICLC in clinical small cell lung cancer (NSCLC; ref. 88). It was later shown to be trials can in part be attributed to its dual agonistic activity on ineffective in patients due to STING polymorphisms that prevent TLR3 and MDA5. BO-112 (Bioncotech) is another poly-IC DMXAA binding (84, 89). MIW815 (ADU-S100, Aduro Biotech) derivative that potently activates RLR signaling in addition to is a cyclic dinucleotide human STING agonist currently in phase I TLR3, and is currently in phase I trials with promising early trials in combination with PD-1 (NCT03172936) or CTLA-4 results (NCT02828098; ref. 80). One synthetic RIG-I–specific blockade (NCT02675439). MK-1454 (Merck), a similar cyclic ligand, RGT100/MK-4621 (Merck) has initiated human trials, dinucleotide agonist currently in a phase I trial in combination after preclinical data demonstrated potent antitumor activity in with PD-1 blockade (NCT03010176) has shown favorable melanoma and colon carcinoma models (81). A phase I/II trial safety profiles and an objective response rate of 20% across in solid tumors began in 2017 (NCT03065023), yielding only several cancer types, with a median depth of response of approx- stable diseases as best response with intratumoral therapy (82). imately 80% (90). In addition, tumor clearance mediated by Pharmacokinetic studies show intratumorally administered antibody blockade of CD47, a classical "do not eat me" signal, MK-4621 is well retained in the TME, helping minimize adverse is STING dependent, where enhanced phagocytosis resulting events due to systemic toxicity. Numerous RLR agonists from CD47 blockade ultimately requires STING and type I IFN are currently in active preclinical development and will likely signaling to prime T cells and inhibit tumor growth (91). Blockade be seen in the clinic soon. of CD47 is currently the focus of several clinical trials in As cellular DNA is ordinarily restricted to the nucleus and both hematopoietic and solid tumors (clinicaltrials.gov). mitochondria, aberrant cytosolic DNA arising from viral infection or cell damage triggers immunogenic signaling by activating ubiquitously expressed CDS. To date, several pathways for Oncolytic Viruses sensing cytosolic DNA have been described. DNA can be tran- Among the most rapidly evolving therapeutic approaches scribed in the cytosol, generating a dsRNA molecule that can be in immuno-oncology is the use of oncolytic viruses (OV). recognized by RIG-I, triggering an inflammatory response to Either through the intrinsic permissiveness of tumor cells for cytosolic DNA in an RLR-dependent fashion (8). In addition, unchecked replication (including viral replication) or by direct- cytosolic DNA can be recognized by absent in melanoma ly engineering the viral genome, OVs can selectively infect and 2 (AIM2), prompting inflammasome assembly, resulting in kill tumor cells. Tumors are specifically susceptible to viral IL-1b/IL-18 production (Fig. 1; ref. 8). Perhaps the most impact- infection and replication, as many of the pathways required ful CDS is the stimulator of interferon genes (STING) pathway. for oncogenesis can be coopted by OVs. While loss of tumor Knockout studies indicate that STING is critical for host type I suppressors such as p53 and RB, activation of RAS and similar IFN and NF-kB responses to synthetic and viral DNA, whereas oncogenes, disruption of IFN signaling components, as well STING deletion had no impact on AIM2-mediated IL-1b pro- as a generally immunosuppressive TME all enable immune duction and the TLR9 CpG DNA response (83). STING is also escape and promote tumor growth, these pathways con- essential for a successful adaptive immune response to DNA currently promote OV infection, replication, and inhibit viral vaccination. Several other CDSs such as DNA-dependent acti- clearance, creating a permissive space for OV growth that is vator of IFN-regulatory factors (DAI) have been identified; preferential to nontransformed tissue (92). OV infection in however, deletion studies indicate they may serve redundant turn results in the immunogenic cell death of infected tumor

OF6 Clin Cancer Res; 2019 Clinical Cancer Research

Pattern Recognition Receptors in Immuno-oncology

Table 1. Ongoing clinical trials using TLR agonists PRR Target Agent Combination Cancers investigated Phase Results Identifier TLR2/4/9 þ BCG aPD-L1 Non–muscle-invasive bladder III Ongoing NCT03528694 NOD2 cancer BCG aPD-1 Non–muscle-invasive bladder III Ongoing phase II NCT03711032 cancer results (104) BCG Mitomycin C High risk non–muscle-invasive III Ongoing NCT02948543 bladder cancer BCG Non–muscle-invasive bladder III Ongoing NCT03091660 cancer TLR3 Rintatolimod þ tumor cell Ovarian, fallopian tube, and primary I/II Ongoing NCT01312389 lysate vaccination peritoneal cancer Rintatolimod þ peptide GM-CSF Breast cancer I/II Ongoing NCT01355393 vaccination Poly-ICLC þ DC vaccine Metastatic pancreatic cancer I Results (27) NCT01410968 Poly-ICLC þ peptide Smoldering multiple myeloma I/IIa Results (28) NCT01718899 vaccination Poly-ICLC þ peptide Breast cancer I Results (105) NCT01532960 vaccination Poly-ICLC Cyclophosphamide þ Hepatocellular cancer I/II Results (30) NCT00553683 radiotherapy Poly-ICLC CDX-301 þ Low-grade B-cell lymphoma I/II Ongoing NCT01976585 Radiotherapy Poly-ICLC þ peptide aPD-1 þ Rituximab Follicular lymphoma I Ongoing NCT03121677 vaccination Poly-ICLC þ peptide aPD-1 Melanoma, NSCLC, head and neck I/II Ongoing NCT03633110 vaccination squamous cell carcinoma, urothelial, and renal cell carcinoma TLR4 þ TLR9 þ AS15 þ MAGE-A3 vaccine Stage III melanoma III Results (35) NCT00796445 NLRP3 AS15 þ MAGE-A3 vaccine NSCLC III Results (36) NCT00480025 TLR4 G100 Merkel cell carcinoma I Results (37) NCT02035657 G100 Cutaneous T-cell lymphoma II Ongoing NCT03742804 G100 aPD-1 þ Rituximab Follicular low-grade non-Hodgkin I/II Ongoing NCT02501473 lymphoma GSK1795091 aOX40, aICOS, or aPD-1 Advanced solid tumors I Ongoing NCT03447314 GLA-SE þ MART-1 Antigen Stage II–IV melanoma N/A Ongoing NCT02320305 vaccine TLR5 Entolimod Colorectal cancer II Ongoing NCT02715882 Entolimod Advanced or metastatic solid tumors I Results (42) NCT01527136 Mobilan Prostate cancer I/II Ongoing NCT02844699 TLR7/8 Imiquimod Cervical intraepithelial neoplasia III Results (44) NCT00941252 Resiquimod þ NY-ESO-1 Melanoma I Results (45) NCT00821652 vaccine DSP-0509 Neoplasms I Ongoing preclinical NCT03416335 results (46) MEDI9197 aPD-L1 Solid tumors I Results (47) NCT02556463 NKTR-262 aIL-2Rb þ aPD-1 Locally advanced or metastatic I Ongoing NCT03435640 solid tumors preclinical results (49) TLR8 þ NLRP3 Motolimod Cetuximab þ aPD-1 Head and neck squamous cell I Results (50) NCT02124850 carcinoma TLR9 CMP-001 aPD-L1 þ Radiotherapy NSCLC I Ongoing NCT03438318 CMP-001 aPD-1 þ aCTLA-4 þ Metastatic colorectal cancer I Ongoing NCT03507699 Radiotherapy CMP-001 aPD-1 Melanoma I Ongoing NCT03618641 CMP-001 aPD-1 Advanced melanoma Ib Ongoing early NCT03084640 results (56) CMP-001 aPD-1 Melanoma I Ongoing NCT02680184 Tilsotolimod aCTLA-4 Anti-PD-1 refractory melanoma III Ongoing phase II NCT03445533 results (57) Tilsotolimod aCTLA-4 or aPD-1 Metastatic melanoma I/II Ongoing NCT02644967 Lefitolimod Metastatic colorectal cancer III Ongoing phase II NCT02077868 results (58) Lefitolimod aCTLA-4 Advanced solid tumors 1 Ongoing NCT02668770 SD-101 Radiotherapy Low-grade B-cell lymphoma I/II Results (55) NCT02266147 SD-101 aPD-1 Metastatic melanoma/head and Ib/II Ongoing early NCT02521870 neck cancer results (60) SD-101 Anti-OX40 Antibody þ Low-grade B-cell non-Hodgkin I Ongoing preclinical NCT03410901 radiotherapy lymphomas results (61) DV281 aPD-1 NSCLC I Ongoing NCT03326752

www.aacrjournals.org Clin Cancer Res; 2019 OF7

Aleynick et al.

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 II Ongoing NCT03643133 DEC-205 þ TLR3 þ TLR7 CDX-1401 Poly-ICLC þ resiquimod Advanced cancers I/II Results (74) NCT00948961 DEC-205 þ TLR3 CDX-1401 Poly-ICLC þ epacadostat Ovarian, fallopian tube, and primary I/II Ongoing NCT02166905 peritoneal cancer in remission CDX-1401 Poly-ICLC þ aPD-1 þ decitabine Myelodysplastic syndrome or acute I Ongoing NCT03358719 myeloid leukemia DC-SIGN CMB305 Synovial sarcoma III Ongoing early NCT03520959 phase II results (106) Dectin-1 Imprime PGG Cetuximab þ paclitaxel þ NSCLC II Results (76) NCT00874848 carboplatin Imprime PGG aPD-1 NSCLC Ib/II Ongoing NCT03003468 Imprime PGG aPD-1 Advanced melanoma, triple- II Ongoing NCT02981303 negative breast cancer Imprime PGG Rituximab Relapsed indolent non-Hodgkin II Ongoing NCT02086175 lymphoma Imprime PGG aPD-L1 þ Bevacizumab Metastatic colorectal cancer I/II Ongoing NCT03555149

cells, initiating de novo antitumor immune responses or boost- as the genetic payload, Swedish biotech Lokon recently opened ing existing responses through mechanisms discussed in the a trial of their lead candidate LOAd703, an adenovirus encod- above sections (Fig. 2). Cancer cells infected with the polio OV ing the costimulatory ligands CD40L and 4-1BBL PVSRIPO (Istari Oncology) release DAMPs (HMGB1, HSP60/ (NCT03225989). Upon infection, tumor and other cells in the 70/80) and PAMPs (viral dsRNA), activating DCs to drive a TME begin to express costimulatory ligands, helping to activate tumor-antigen–specific cytotoxic T-cell response (93). Similar NK effector cells and remodel the TME (97). OVs without a to other intratumorally delivered PRR agonists, the innate– therapeutic payload, including Pelareorep (Reolysin, Oncolytics adaptive immune axis is critical for OV therapy, as the ability to Biotech) and PVSRIPO, are in active clinical development as induce systemic antitumor immunity is antigen restricted to the well. Recently published phase I data shows PVSRIPO increased OV infected site. Using a Newcastle disease OV and contralat- 36 month overall survival to 21% in patients with recurrent eral B16 and MC38 tumors, Zamarin and colleagues demon- glioblastoma, a major increase from the 4% survival seen in strate that OV injection of one tumor results in immunity only historical controls (98). Taken together, all of these different against that same tumor type (94). Talimogene laherparepvec successful approaches with OVs substantiate the idea that induc- or T-VEC (Amgen), a modified herpes virus expressing GM-CSF, tion of immunogenic tumor cell death in combination with PRR was approved in 2015 for the treatment of late-stage metastatic agonism can drive effective adaptive immune responses. melanoma, earning a place for OV therapy in the clinic. In a landmark phase III study, intratumoral injection of T-vec caused complete resolution in 47% of injected lesions, as well Perspectives as 22% of noninjected visceral lesions, highlighting the power Pattern recognition receptors present potentially powerful of OV therapy to induce systemic antitumor immunity (95). T- weapons in the cancer immunotherapy armory. Their ability to VEC has already been effectively combined with CTLA-4 block- modulate numerous aspects of the tumor microenvironment, ade, where a randomized phase II study demonstrated an from APCs and their cross-talk with T cells, to directly modulating increase in objective response rates from 18% with CTLA-4 cancer cells themselves, allow these pathways to shape and monotherapy to 39% in the combination group (96), and is ultimately drive an antitumor immune response. As PRR agonists being investigated aggressively, including combinations with and oncolytic viruses trigger innate cells to activate adaptive PD-1 blockade (NCT02965716), with neoadjuvant chemother- immunity, combining these approaches with checkpoint block- apy (NCT02779855), and with preoperative radiotherapy ade therapy effectively presses the gas pedal while cutting the (NCT02453191). Other promising OV platforms in late devel- brakes, unleashing the full potential of immune effector cells. PRR opment include Pexa Vec (JX-594, SillaJen), a vaccinia virus agonism could additionally reverse resistance in checkpoint also engineered to express GM-CSF, currently in a phase III trial refractory tumors (99), and synergize with standard-of-care ther- for hepatocellular carcinoma in combination with the kinase apies including chemotherapy (100) and anti-CD20 targeting inhibitor sorafenib (NCT02562755). A modified Coxsackie against lymphoma (101); a variety of combinatorial approaches virus, CAVATAK (Viralytics), is currently in phase II trials for are being actively explored in clinical trials (Tables 1–4). Preclin- several indications (clinicaltrials.gov). Moving beyond GM-CSF ical approaches focused on developing next-generation agonists

Table 3. Ongoing clinical trials using RLR and CDS agonists PRR Target Agent Combination Cancers investigated Phase Results Identiﬁer RIG-I MK4621 Advanced solid tumors I/II Results (82) NCT03065023 MK4621 aPD-1 Advanced solid tumors I Ongoing NCT03739138 STING MIW815 aPD-1 Advanced solid tumors or lymphomas I Ongoing NCT03172936 MIW815 aCTLA-4 Advanced solid tumors or lymphomas I Ongoing NCT02675439 MK-1454 aPD-1 Advanced solid tumors or lymphomas I Ongoing NCT03010176 Early results (90)

OF8 Clin Cancer Res; 2019 Clinical Cancer Research

Pattern Recognition Receptors in Immuno-oncology

Table 4. Ongoing clinical trials using oncolytic viruses Virus Agent Combination Cancers investigated Phase Results Identiﬁer Herpes simplex T-Vec aCTLA-4 Melanoma Ib/II Results (96) NCT01740297 T-Vec aPD-1 Stage III/IV melanoma II Ongoing NCT02965716 T-Vec Paclitaxel Triple-negative breast cancer I/II Ongoing NCT02779855 T-Vec Radiotherapy Soft tissue sarcoma I/II Ongoing NCT02453191 Vaccinia poxvirus Pexa Vec Sorafenib Hepatocellular carcinoma III Ongoing NCT02562755 Pexa Vec aPD-1 Renal cell carcinoma Ib Ongoing NCT03294083 Pexa Vec aPD-1 Hepatocellular carcinoma I/IIa Ongoing NCT03071094 Coxsackievirus CAVATAK Stage IIIC–IV melanoma II Results (107) NCT01636882 CAVATAK aCTLA-4 Advanced melanoma I Ongoing NCT02307149 CAVATAK aPD-1 Advanced NSCLC I Ongoing NCT02824965 Adenovirus LOAd703 Pancreatic, ovarian, biliary, and colorectal cancer I/II Ongoing NCT03225989 Enterovirus PVSRIPO Recurrent glioblastoma I Results (98) NCT01491893 PVSRIPO Unresectable melanoma I Ongoing NCT03712358 Reovirus Pelareorep Paclitaxel Metastatic breast cancer II Results (108) NCT01656538 Pelareorep aPD-1 Advanced pancreatic cancer II Ongoing NCT03723915

are underway; one group recently developed an OV-packaged Disclosure of Potential Conflicts of Interest anti-CTLA4 antibody construct, effectively combining OV and C. R. Flowers reports receiving other commercial research support from checkpoint therapy into a single injection (102). Others have Abbvie, Acerta, BeiGene, Celgene, Gilead, Genentech/Roche, Janssen Pharma- fused Resiquimod nanoparticles to PD-1–targeting antibodies, ceuticals, Millennium/Takeda, Pharmacyclics, and TG Therapeutics, and is a þ consultant/advisory board member for Abbvie, AstraZeneca, Bayer, BeiGene, allowing PD-1 T cells to selectively deliver the TLR7 agonist to fi Celgene, Denovo Biopharma, Genentech/Roche, Gilead, Karyopharm, Phar- the tumor, reshaping the TME to improve T-cell in ltration and macyclics/Janssen, and Spectrum. A. Marabelle reports receiving commercial disease control (103). These approaches highlight the immuno- research grants from Transgene, speakers bureau honoraria from Amgen and modulatory potency of PRRs, where their ability to overcome MSD, and is a consultant/advisory board member for AstraZeneca, Lytix immunosuppression and drive adaptive immunity effectively Pharma, MSD, Bioncotech, and Oncovir. J.D. Brody reports receiving commer- enhances efficacy of concurrently administered therapies. With cial research grants from Merck, Genentech, and Bristol-Myers Squibb. No potential conflicts of interest were disclosed by the other authors. so many novel agonists being investigated preclinically and in the clinic, continued exploration and understanding of PRR pathways and targeting will help to shape treatment paradigms in immuno- Received January 29, 2019; revised April 8, 2019; accepted May 16, 2019; oncology. published first May 23, 2019.

References 1. Rakoff-Nahoum S, Medzhitov R. Toll-like receptors and cancer. Nat Rev 12. Yang H, Wang B, Wang T, Xu L, He C, Wen H, et al. Toll-like receptor 4 Cancer 2009;9:57–63. prompts human breast cancer cells invasiveness via lipopolysaccharide 2. Kawai T, Akira S. Toll-like receptors and their crosstalk with other innate stimulation and is overexpressed in patients with lymph node metastasis. receptors in infection and immunity. Immunity 2011;34:637–50. PLoS One 2014;9:e109980. 3. Kawai T, Akira S. The role of pattern-recognition receptors in innate 13. Volk-Draper L, Hall K, Griggs C, Rajput S, Kohio P, DeNardo D, et al. immunity: update on toll-like receptors. Nat Immunol 2010;11: Paclitaxel therapy promotes breast cancer metastasis in a TLR4-dependent 373–84. manner. Cancer Res 2014;74:5421–34. 4. Shekarian T, Valsesia-Wittmann S, Brody J, Michallet MC, Depil S, Caux C, 14. Li D, Wang X, Wu J-L, Quan W-Q, Ma L, Yang F, et al. Tumor-produced et al. Pattern recognition receptors: immune targets to enhance cancer versican V1 enhances hCAP18/LL-37 expression in macrophages through immunotherapy. Ann Oncol 2017;28:1756–66. activation of TLR2 and vitamin D3 signaling to promote ovarian cancer 5. Barton GM, Kagan JC. A cell biological view of toll-like receptor function: progression in vitro. PLoS One 2013;8:e56616. regulation through compartmentalization. Nat Rev Immunol 2009;9: 15. Kim S, Takahashi H, Lin WW, Descargues P, Grivennikov S, Kim Y, et al. 535–42. Carcinoma-produced factors activate myeloid cells through TLR2 to 6. Oosting M, Cheng S-C, Bolscher JM, Vestering-Stenger R, Plantinga TS, stimulate metastasis. Nature 2009;457:102–6. Verschueren IC, et al. Human TLR10 is an anti-inflammatory pattern- 16. Klein JC, Moses K, Zelinskyy G, Sody S, Buer J, Lang S, et al. Combined toll- recognition receptor. Proc Natl Acad Sci U S A 2014;111:E4478–84. like receptor 3/7/9 deficiency on host cells results in T-cell-dependent 7. Lee SM-Y, Yip T-F, Yan S, Jin D-Y, Wei H-L, Guo R-T, et al. Recognition of control of tumour growth. Nat Commun 2017;8:14600. double-stranded RNA and regulation of interferon pathway by toll-like 17. Daley D, Mani VR, Mohan N, Akkad N, Ochi A, Heindel DW, et al. Dectin receptor 10. Front Immunol 2018;9:516. 1 activation on macrophages by galectin 9 promotes pancreatic carcinoma 8. Takeuchi O, Akira S. Pattern recognition receptors and inflammation. Cell and peritumoral immune tolerance. Nat Med 2017;23:556–67. 2010;140:805–20. 18. Mellman I, Coukos G, Dranoff G. Cancer immunotherapy comes of age. 9. Pradere JP, Dapito DH, Schwabe RF. The Yin and Yang of toll-like Nature 2011;480:480–9. receptors in cancer. Oncogene 2014;33:3485–95. 19. Biot C, Rentsch C, Gsponer J, Birkh€auser FD, Jusforgues-Saklani H, 10. Chatterjee S, Crozet L, Damotte D, Iribarren K, Schramm C, Alifano M, Lema^tre F, et al. Preexisting BCG-specific T cells improve intravesical et al. TLR7 promotes tumor progression, chemotherapy resistance, and immunotherapy for bladder cancer. Sci Transl Med 2012;4:137ra72. poor clinical outcomes in non-small cell lung cancer. Cancer Res 2014;74: 20. Divangahi M, Mostowy S, Coulombe F, Kozak R, Guillot L, Veyrier F, et al. 5008–18. NOD2-deficient mice have impaired resistance to mycobacterium tuber- 11. Cherfils-Vicini J, Platonova S, Gillard M, Laurans L, Validire P, Caliandro culosis infection through defective innate and adaptive immunity. R, et al. Triggering of TLR7 and TLR8 expressed by human lung cancer cells J Immunol 2008;181:7157–65. induces cell survival and chemoresistance. J Clin Invest 2010;120: 21. Marabelle A, Kohrt H, Caux C, Levy R. Intratumoral immunization: a new 1285–97. paradigm for cancer therapy. Clin Cancer Res 2014;20:1747–56.

www.aacrjournals.org Clin Cancer Res; 2019 OF9

Aleynick et al.

22. Martins KA, Bavari S, Salazar AM. Vaccine adjuvant uses of poly-IC and induces immunity by stimulating an NK-dendritic-CD8þ T-cell axis. derivatives. Expert Rev Vaccines 2015;14:447–59. Proc Natl Acad Sci U S A 2016;113:E874–83. 23. Caskey M, Lefebvre F, Filali-Mouhim A, Cameron MJ, Goulet J-P, Haddad 40. Leigh ND, Bian G, Ding X, Liu H, Aygun-Sunar S, Burdelya LG, et al. EK, et al. Synthetic double-stranded RNA induces innate immune A flagellin-derived toll-like receptor 5 agonist stimulates cytotoxic responses similar to a live viral vaccine in humans. J Exp Med 2011; lymphocyte-mediated tumor immunity. PLoS One 2014;9:e85587. 208:2357–66. 41. Mett V, Komarova EA, Greene K, Bespalov I, Brackett C, Gillard B, et al. 24. Stahl-Hennig C, Eisenbl€atter M, Jasny E, Rzehak T, Tenner-Racz K, Mobilan: a recombinant adenovirus carrying Toll-like receptor 5 self- Trumpfheller C, et al. Synthetic double-stranded RNAs are adjuvants activating cassette for cancer immunotherapy. Oncogene 2018;37: for the induction of T helper 1 and humoral immune responses to 439–49. human papillomavirus in rhesus macaques. PLoS Pathog 2009;5: 42. Bakhribah H, Dy GK, Ma WW, Zhao Y, Opyrchal M, Purmal A, et al. A e1000373. phase I study of the toll-like receptor 5 (TLR5) agonist, entolimod in 25. Navabi H, Jasani B, Reece A, Clayton A, Tabi Z, Donninger C, et al. A patients (pts) with advanced cancers. J Clin Oncol 2015;33:3063. clinical grade poly I:C-analogue (Ampligen) promotes optimal DC mat- 43. Vidal D, Matias-Guiu X, Alomar A. Open study of the efficacy and uration and Th1-type T cell responses of healthy donors and cancer mechanism of action of topical imiquimod in basal cell carcinoma. patients in vitro. Vaccine 2009;27:107–15. Clin Exp Dermatol 2004;29:518–25. 26. Theodoraki M-N, Yerneni S, Sarkar SN, Orr B, Muthuswamy R, Voyten J, 44. Grimm C, Polterauer S, Natter C, Rahhal J, Hefler L, Tempfer CB, et al. et al. Helicase-driven activation of NFkB-COX2 pathway mediates the Treatment of cervical intraepithelial neoplasia with topical imiquimod. immunosuppressive component of dsRNA-driven inflammation in the Obstet Gynecol 2012;120:152–9. human tumor microenvironment. Cancer Res 2018;78:4292–302. 45. Sabado RL, Pavlick A, Gnjatic S, Cruz CM, Vengco I, Hasan F, et al. 27. Mehrotra S, Britten CD, Chin S, Garrett-Mayer E, Cloud CA, Li M, et al. Resiquimod as an immunologic adjuvant for NY-ESO-1 protein vacci- Vaccination with poly(IC:LC) and peptide-pulsed autologous dendritic nation in patients with high-risk melanoma. Cancer Immunol Res 2015; cells in patients with pancreatic cancer. J Hematol Oncol 2017;10:82. 3:278–87. 28. Nooka AK, Wang ML, Yee AJ, Kaufman JL, Bae J, Peterkin D, et al. 46. Ota Y, Otsubo T, Koroki J, Hirose Y, Koga-Yamakawa E, Murata M, et al. Assessment of safety and immunogenicity of PVX-410 vaccine with or Novel intravenous injectable TLR7 agonist, DSP-0509, synergistically without lenalidomide in patients with smoldering multiple myeloma. enhanced antitumor immune responses in combination with anti-PD-1 JAMA Oncol 2018;4:e183267. antibody [abstract]. In: Proceedings of the AACR Annual Meeting; 2018 29. Hammerich L, Marron TU, Upadhyay R, Svensson-Arvelund J, Dhainaut Apr 14–18; Chicago, IL. Philadelphia (PA): AACR; 2018. Abstract nr. M, Hussein S, et al. Systemic clinical tumor regressions and potentiation 4726. of PD1 blockade with in situ vaccination. Nat Med 2019;25:814–24. 47. Gupta S, Grilley-Olson J, Hong D, Marabelle A, Munster P, Aggarwal R, 30. De La Torre AN, Contractor S, Castaneda I, Cathcart CS, Razdan D, Klyde et al. Safety and pharmacodynamic activity of MEDI9197, a TLR 7/8 D, et al. A Phase I trial using local regional treatment, nonlethal irradi- agonist, administered intratumorally in subjects with solid tumors ation, intratumoral and systemic polyinosinic-polycytidylic acid polyly- [abstract]. In: Proceedings of the AACR Annual Meeting; 2017 Apr 1– sine carboxymethylcellulose to treat liver cancer: in search of the abscopal 5; Washington, DC. Philadelphia (PA): AACR; 2017. Abstract nr. CT091. effect. J Hepatocell Carcinoma 2017;2017:4–111. 48. Lee M. NKTR-262: prodrug pharmacokinetics in mice, rats, and dogs 31. Butowski N, Chang SM, Junck L, DeAngelis LM, Abrey L, Fink K, et al. A [abstract]. In: Proceedings of the AACR Annual Meeting; 2018 Apr 14–18; phase II clinical trial of poly-ICLC with radiation for adult patients with Chicago, IL. Philadelphia (PA): AACR; 2018. Abstract nr. 2755. newly diagnosed supratentorial glioblastoma: a North American Brain 49. Kivim€ae S, Rubas W, Pena R, Mclaughlin J, Hennessy M, Kirksey Y, et al. Tumor Consortium (NABTC01-05). J Neurooncol 2009;91:175–82. Harnessing the innate and adaptive immune system to eradicate treated 32. Rosenfeld MR, Chamberlain MC, Grossman SA, Peereboom DM, Lesser and distant untreated solid tumors. J Immunother Cancer 2017;5:P275. GJ, Batchelor TT, et al. A multi-institution phase II study of poly-ICLC and 50. Ferris RL, Saba NF, Gitlitz BJ, Haddad R, Sukari A, Neupane P, et al. Effect radiotherapy with concurrent and adjuvant temozolomide in adults with of adding motolimod to standard combination chemotherapy and cetux- newly diagnosed glioblastoma. Neuro Oncol 2010;12:1071–7. imab treatment of patients with squamous cell carcinoma of the head and 33. Han HD, Byeon Y, Jang J-H, Jeon HN, Kim GH, Kim MG, et al. In vivo neck. JAMA Oncol 2018;4:1583–8. stepwise immunomodulation using chitosan nanoparticles as a platform 51. Kapp K, Schneider J, Schneider L, Gollinge N, J€ansch S, Schroff M, et al. nanotechnology for cancer immunotherapy. Sci Rep 2016;6:38348. Distinct immunological activation profiles of dSLIM and ProMune 34. Paavonen J, Naud P, Salmeron J, Wheeler C, Chow S-N, Apter D, et al. depend on their different structural context. Immunity Inflamm Dis 2016; Efficacy of human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine 4:446–62. against cervical infection and precancer caused by oncogenic HPV types 52. Samulowitz U, Weber M, Weeratna R, Uhlmann E, Noll B, Krieg AM, et al. (PATRICIA): final analysis of a double-blind, randomised study in young A novel class of immune-stimulatory CpG oligodeoxynucleotides unifies women. Lancet 2009;374:301–14. high potency in type I interferon induction with preferred structural 35. Dreno B, Thompson JF, Smithers BM, Santinami M, Jouary T, Gutzmer R, properties. Oligonucleotides 2010;20:93–101. et al. MAGE-A3 immunotherapeutic as adjuvant therapy for patients with 53. Brody JD, Ai WZ, Czerwinski DK, Torchia JA, Levy M, Advani RH, et al. resected, MAGE-A3-positive, stage III melanoma (DERMA): a double- In situ vaccination with a TLR9 agonist induces systemic lymphoma blind, randomised, placebo-controlled, phase 3 trial. Lancet Oncol 2018; regression: a phase I/II study. J Clin Oncol 2010;28:4324–32. 19:916–29. 54. Manegold C, van Zandwijk N, Szczesna A, Zatloukal P, Au JSK, Blasinska- 36. Vansteenkiste JF, Cho BC, Vanakesa T, De Pas T, Zielinski M, Kim MS, et al. Morawiec M, et al. A phase III randomized study of gemcitabine and Efficacy of the MAGE-A3 cancer immunotherapeutic as adjuvant therapy cisplatin with or without PF-3512676 (TLR9 agonist) as first-line treat- in patients with resected MAGE-A3-positive non-small-cell lung cancer ment of advanced non-small-cell lung cancer. Ann Oncol 2012;23:72–7. (MAGRIT): a randomised, double-blind, placebo-controlled, phase 3 55. Frank MJ, Reagan PM, Bartlett NL, Gordon LI, Friedberg JW, Czerwinski trial. Lancet Oncol 2016;17:822–35. DK, et al. In situ vaccination with a TLR9 agonist and local low-dose 37. Bhatia S, Miller NJ, Lu H, Vandeven NV, Ibrani D, Shinohara M, et al. radiation induces systemic responses in untreated indolent lymphoma. Intratumoral G100, a TLR4 agonist, induces anti-tumor immune Cancer Discov 2018;8:1258–69. responses and tumor regression in patients with Merkel cell carcinoma. 56. Milhem M, Gonzales R, Medina T, Kirkwood JM, Buchbinder E, Mehmi I, Clin Cancer Res 2019;25:1185–95. et al. Intratumoral toll-like receptor 9 (TLR9) agonist, CMP-001, in 38. Flowers C, Panizo C, Isufi I, Herrera AF, Okada C, Cull EH, et al. Long term combination with pembrolizumab can reverse resistance to PD-1 inhi- follow-up of a phase 2 study examining intratumoral G100 alone and in bition in a phase Ib trial in subjects with advanced melanoma [abstract]. combination with pembrolizumab in patients with follicular lymphoma In: Proceedings of the AACR Annual Meeting; 2018 Apr 14–18; Chicago, [abstract]. In: Proceedings of the 60th Annual Meeting and Exposition; IL. Philadelphia (PA): AACR; 2018. Abstract nr. CT144. 2018 Dec 1–4; Washington, DC. 57. Diab A, Rahimian S, Haymaker CL, Bernatchez C, Andtbacka RHI, James 39. Brackett CM, Kojouharov B, Veith J, Greene KF, Burdelya LG, Gollnick SO, M, et al. A phase 2 study to evaluate the safety and efficacy of Intratumoral et al. Toll-like receptor-5 agonist, entolimod, suppresses metastasis and (IT) injection of the TLR9 agonist IMO-2125 (IMO) in combination with

OF10 Clin Cancer Res; 2019 Clinical Cancer Research

Pattern Recognition Receptors in Immuno-oncology

ipilimumab (ipi) in PD-1 inhibitor refractory melanoma. J Clin Oncol advanced non-small cell lung cancer. Invest New Drugs 2017;35: 36,2018 (suppl; abstr 9515). Available from: https://meetinglibrary.asco. 345–58. org/record/159086/abstract. 77. Loo YM, Gale M. Immune signaling by RIG-I-like receptors. Immunity 58. Thomas M, Ponce-Aix S, Navarro A, Riera-Knorrenschild J, Schmidt M, 2011;34:680–92. Wiegert E, et al. Immunotherapeutic maintenance treatment with 78. Kubler€ K, Gehrke N, Riemann S, Bohnert€ V, Zillinger T, Hartmann E, et al. toll-like receptor 9 agonist lefitolimod in patients with extensive- Targeted activation of RNA helicase retinoic acid - Inducible gene-I stage small-cell lung cancer: results from the exploratory, controlled, induces proimmunogenic apoptosis of human ovarian cancer cells. randomized, international phase II IMPULSE study. Ann Oncol 2018; Cancer Res 2010;70:5293–304. 29:2076–84. 79. Besch R, Poeck H, Hohenauer T, Senft D, H€acker G, Berking C, et al. 59. Krarup AR, Abdel-Mohsen M, Schleimann MH, Vibholm L, Engen PA, Proapoptotic signaling induced by RIG-I and MDA-5 results in type I Dige A, et al. The TLR9 agonist MGN1703 triggers a potent type I interferon–independent apoptosis in human melanoma cells. J Clin interferon response in the sigmoid colon. Mucosal Immunol 2018;11: Invest 2009;119:2399–411. 449–61. 80. Calles A, Rodriguez-Ruiz M, Soria A, Marquez Rodas I, Ponz-SarviseM, 60. Ribas A, Medina T, Kummar S, Amin A, Kalbasi A, Drabick JJ, et al. SD-101 Martín M, et al. Intratumoral BO-112, a double-stranded RNA (dsRNA), in combination with pembrolizumab in advanced melanoma: results of a alone and in combination with systemic anti-PD-1 in solid tumors. phase 1b, multicenter study. Cancer Discov 2018;8:1250–7. Ann Oncol 2018;29:732. 61. Sagiv-Barfi I, Czerwinski DK, Levy S, Alam IS, Mayer AT, Gambhir SS, et al. 81. Barsoum J, Renn M, Schuberth C, Jakobs C, Schwickart A, Schlee M, et al. Eradication of spontaneous malignancy by local immunotherapy. Abstract B44: Selective stimulation of RIG-I with a novel synthetic RNA Sci Transl Med 2018;10:eaan4488. induces strong anti-tumor immunity in mouse tumor models [abstract]. 62. Levy R, Reagan PM, Friedberg JW, Bartlett NL, Gordon LI, Leung A, et al. In: Proceedings of the AACR Special Conference on Tumor Immunology SD-101, a novel class C CpG-oligodeoxynucleotide (ODN) toll-like and Immunotherapy; 2016 Oct 20–23; Boston, MA. Philadelphia (PA): receptor 9 (TLR9) agonist, given with low dose radiation for untreated AACR; Cancer Immunol Res 2017. Abstract nr B44. low grade B-cell lymphoma: interim results of a phase 1/2 trial. Blood 82. Middleton MR, Wermke M, Calvo E, Chartash E, Zhou H, Zhao X, et al. 2016;128:2974. Phase I/II, multicenter, open-label study of intratumoral/intralesional 63. Saxena M, Yeretssian G. NOD-like receptors: master regulators of inflam- administration of the retinoic acid–inducible gene I (RIG-I) activator mation and cancer. Front Immunol 2014;5:327. MK-4621 in patients with advanced or recurrent tumors. Ann Oncol 2018; 64. Dostert C, Petrilli V, Van Bruggen R, Steele C, Mossman BT, Tschopp J. 29:mdy424–016. Innate immune activation through Nalp3 inflammasome sensing of 83. Ishikawa H, Ma Z, Barber GN. STING regulates intracellular DNA- asbestos and silica. Science 2008;320:674–7. mediated, type I interferon-dependent innate immunity. Nature 2009; 65. Chou AJ, Kleinerman ES, Krailo MD, Chen Z, Betcher DL, Healey JH, et al. 461:788–92. Addition of muramyl tripeptide to chemotherapy for patients with 84. Chen Q, Sun L, Chen ZJ. Regulation and function of the cGAS–STING newly diagnosed metastatic osteosarcoma: a report from the Children's pathway of cytosolic DNA sensing. Nat Immunol 2016;17:1142–9. Oncology Group. Cancer 2009;115:5339–48. 85. H€artlova A, Erttmann SF, Raffi FA, Schmalz AM, Resch U, Anugula S, et al. 66. Dietsch GN, Lu H, Yang Y, Morishima C, Chow LQ, Disis ML, et al. DNA damage primes the type I interferon system via the cytosolic DNA Coordinated activation of toll-like receptor8 (TLR8) and NLRP3 by the sensor STING to promote anti-microbial innate immunity. Immunity TLR8 agonist, VTX-2337, ignites tumoricidal natural killer cell activity. 2015;42:332–43. PLoS One 2016;11:e0148764. 86. Deng L, Liang H, Xu M, Yang X, Burnette B, Arina A, et al. STING- 67. Li H, Willingham SB, Ting JP-Y, Re F. Cutting edge: inflammasome dependent cytosolic DNA sensing promotes radiation-induced type I activation by alum and alum's adjuvant effect are mediated by NLRP3. interferon-dependent antitumor immunity in immunogenic tumors. J Immunol 2008;181:17–21. Immunity 2014;41:843–52. 68. Cibulski SP, Rivera-Patron M, Mourglia-Ettlin G, Casaravilla C, Yendo 87. Wang H, Hu S, Chen X, Shi H, Chen C, Sun L, et al. cGAS is essential for ACA, Fett-Neto AG, et al. Quillaja brasiliensis saponin-based nanoparti- the antitumor effect of immune checkpoint blockade. Proc Natl Acad Sci culate adjuvants are capable of triggering early immune responses. Sci Rep U S A 2017;114:1637–42. 2018;8:13582. 88. Lara PN Jr, Douillard J-Y, Nakagawa K, von Pawel J, McKeage MJ, Albert I, 69. Geijtenbeek TBH, Gringhuis SI. Signalling through C-type lectin recep- et al. Randomized phase III placebo-controlled trial of carboplatin and tors: shaping immune responses. Nat Rev Immunol 2009;9:465–79. paclitaxel with or without the vascular disrupting agent vadimezan 70. Yamasaki S, Ishikawa E, Sakuma M, Hara H, Ogata K, Saito T. Mincle is an (ASA404) in advanced non-small-cell lung cancer. J Clin Oncol 2011; ITAM-coupled activating receptor that senses damaged cells. 29:2965–71. Nat Immunol 2008;9:1179–88. 89. Corrales L, Glickman LH, McWhirter SM, Kanne DB, Sivick KE, Katibah 71. Zhang JG, Czabotar PE, Policheni AN, Caminschi I, San Wan S, Kitsoulis S, GE, et al. Direct activation of STING in the tumor microenvironment leads et al. The dendritic cell receptor Clec9A binds damaged cells via exposed to potent and systemic tumor regression and immunity. Cell Rep 2015;11: actin filaments. Immunity 2012;36:646–57. 1018–30. 72. Vassilaros S, Tsibanis A, Tsikkinis A, Pietersz GA, McKenzie IFC, 90. Harrington KJ, Brody J, Ingham M, Strauss J, Cemerski S, Wang M, et al. Apostolopoulos V. Up to 15-year clinical follow-up of a pilot Phase III Preliminary results of the first-in-human (FIH) study of MK-1454, immunotherapy study in stage II breast cancer patients using oxidized an agonist of stimulator of interferon genes (STING), as monotherapy mannan-MUC1. Immunotherapy 2013;5:1177–82. or in combination with pembrolizumab (pembro) in patients 73. Morse MA, Chapman R, Powderly J, Blackwell K, Keler T, Green J, et al. with advanced solid tumors or lymphomas. Ann Oncol 2018;29: Phase I study utilizing a novel antigen-presenting cell-targeted vaccine mdy424.015. with toll-like receptor stimulation to induce immunity to self-antigens in 91. Liu X, Pu Y, Cron K, Deng L, Kline J, Frazier WA, et al. CD47 blockade cancer patients. Clin Cancer Res 2011;17:4844–53. triggers T cell-mediated destruction of immunogenic tumors. Nat Med 74. Dhodapkar MV, Sznol M, Zhao B, Wang D, Carvajal RD, Keohan ML, 2015;21:1209–15. et al. Induction of antigen-specific immunity with a vaccine targeting 92. Pikor LA, Bell JC, Diallo J-S. Oncolytic viruses: exploiting cancer's deal NY-ESO-1 to the dendritic cell receptor DEC-205. Sci Transl Med 2014; with the devil. Trends Cancer 2015;1:266–77. 6:232ra51. 93. Brown MC, Holl EK, Boczkowski D, Dobrikova E, Mosaheb M, 75. Pollack SM. The potential of the CMB305 vaccine regimen to target NY- Chandramohan V, et al. Cancer immunotherapy with recombinant ESO-1 and improve outcomes for synovial sarcoma and myxoid/round poliovirus induces IFN-dominant activation of dendritic cells and tumor cell liposarcoma patients. Expert Rev Vaccines 2018;17:107–14. antigen-specific CTLs. Sci Transl Med 2017;9:eaan4220. 76. Thomas M, Sadjadian P, Kollmeier J, Lowe J, Mattson P, Trout JR, et al. 94. Zamarin D, Holmgaard RB, Subudhi SK, Park JS, Mansour M, Palese P, A randomized, open-label, multicenter, phase II study evaluating the et al. Localized oncolytic virotherapy overcomes systemic tumor resis- efficacy and safety of BTH1677 (1,3-1,6 beta glucan; Imprime PGG) in tance to immune checkpoint blockade immunotherapy. Sci Transl Med combination with cetuximab and chemotherapy in patients with 2014;6:226ra32.

www.aacrjournals.org Clin Cancer Res; 2019 OF11

Aleynick et al.

95. Andtbacka RHI, Ross M, Puzanov I, Milhem M, Collichio F, Delman KA, 102. Hamilton JR, Vijayakumar G, Palese P. A recombinant antibody- et al. Patterns of Clinical Response with Talimogene Laherparepvec expressing influenza virus delays tumor growth in a mouse model. (T-VEC) in Patients with Melanoma Treated in the OPTiM Phase III Cell Rep 2018;22:1–7. Clinical Trial. Ann Surg Oncol 2016;23:4169–77. 103. Schmid D, Park CG, Hartl CA, Subedi N, Cartwright AN, Puerto RB, et al. T 96. Chesney J, Puzanov I, Collichio F, Singh P, Milhem MM, Glaspy J, et al. cell-targeting nanoparticles focus delivery of immunotherapy to improve Randomized, open-label phase II study evaluating the efficacy and safety antitumor immunity. Nat Commun 2017;8:1747. of talimogene laherparepvec in combination with ipilimumab versus 104. de Wit R, Kulkarni GS, Uchio E, Singer EA, Krieger L, Grivas P, et al. 864O ipilimumab alone in patients with advanced, unresectable melanoma. Pembrolizumab for high-risk (HR) non–muscle invasive bladder cancer J Clin Oncol 2018;36:1658–67. (NMIBC) unresponsive to bacillus Calmette-Guerin (BCG): phase II 97. Eriksson E, Milenova I, Wenthe J, Hle MS, Leja-Jarblad J, Ullenhag G, et al. KEYNOTE-057 trial. Ann Oncol 2018;29:mdy283.073. Shaping the tumor stroma and sparking immune activation by CD40 and 105. Dillon PM, Petroni GR, Smolkin ME, Brenin DR, Chianese-Bullock KA, 4-1BB signaling induced by an armed oncolytic virus. Clin Cancer Res Smith KT, et al. A pilot study of the immunogenicity of a 9-peptide breast 2017;23:5846–57. cancer vaccine plus poly-ICLC in early stage breast cancer. J Immunother 98. Desjardins A, Gromeier M, Herndon JE, Beaubier N, Bolognesi DP, Cancer 2017;5:92. Friedman AH, et al. Recurrent glioblastoma treated with recombinant 106. Chawla S, Van Tine BA, Pollack S, Ganjoo K, Elias A, Riedel RF, et al. A poliovirus. N Engl J Med 2018;379:150–61. phase 2 study of CMB305 and atezolizumab in NY-ESO-1þ soft tissue 99. Fu J, Kanne DB, Leong M, Glickman LH, McWhirter SM, Lemmens E, et al. sarcoma: Interim analysis of immunogenicity, tumor control and surviv- STING agonist formulated cancer vaccines can cure established tumors al. Ann Oncol 2017;28:mdx387.007. resistant to PD-1 blockade. Sci Transl Med 2015;7:283ra52. 107. Andtbacka RH, Curti BD, Hallmeyer S, Feng Z, Paustian C, Bifulco C, 100. Li J, Song W, Czerwinski DK, Varghese B, Uematsu S, Akira S, et al. et al. Phase II calm extension study: Coxsackievirus A21 delivered Lymphoma immunotherapy with CpG oligodeoxynucleotides requires intratumorally to patients with advanced melanoma induces immune- TLR9 either in the host or in the tumor itself. J Immunol 2007;179: cell infiltration in the tumor microenvironment. J Immunother Cancer 2493–500. 2015;3:P343. 101. Cheadle EJ, Lipowska-Bhalla G, Dovedi SJ, Fagnano E, Klein C, Honey- 108. Bernstein V, Ellard SL, Dent SF, Tu D, Mates M, Dhesy-Thind SK, et al. A church J, et al. A TLR7 agonist enhances the antitumor efficacy of randomized phase II study of weekly paclitaxel with or without pelar- obinutuzumab in murine lymphoma models via NK cells and CD4 T eorep in patients with metastatic breast cancer: final analysis of Canadian cells. Leukemia 2017;31:1611–21. Cancer Trials Group IND.213. Breast Cancer Res Treat 2018;167:485–93.

OF12 Clin Cancer Res; 2019 Clinical Cancer Research

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

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/2019/07/16/1078-0432.CCR-18-1800. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.