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STRO-002 is a novel homogeneous folate receptor alpha (FolRα) targeting antibody–drug conjugate (ADC) currently being investigated in the clinic as a treatment for ovarian and endometrial cancers. Here, we describe the discovery, optimization, and antitumor properties of STRO-002. STRO-002 was generated by conjugation of a novel cleavable 3-aminophenyl hemiasterlin linker-warhead (SC239) to the nonnatural amino acid para-azidomethyl-L-phenylalanine incorporated at specific positions within a high affinity anti-FolRα antibody using Sutro's XpressCF+, which resulted in a homogeneous ADC with a drug–antibody ratio (DAR) of 4. STRO-002 binds to FolRα with high affinity, internalizes rapidly into target positive cells, and releases the tubulin-targeting cytotoxin 3-aminophenyl hemiasterlin (SC209). SC209 has reduced potential for drug efflux via P-glycoprotein 1 drug pump compared with other tubulin-targeting payloads. While STRO-002 lacks nonspecific cytotoxicity toward FolRα-negative cell lines, bystander killing of target negative cells was observed when cocultured with target positive cells. STRO-002 is stable in circulation with no change in DAR for up to 21 days and has a half-life of 6.4 days in mice. A single dose of STRO-002 induced significant tumor growth inhibition in FolRα-expressing xenograft models and patient-derived xenograft models. In addition, combination treatment with carboplatin or Avastin further increased STRO-002 efficacy in xenograft models. The potent and specific preclinical efficacy of STRO-002 supports clinical development of STRO-002 for treating patients with FolRα-expressing cancers, including ovarian, endometrial, and non–small cell lung cancer. Phase I dose escalation for STRO-002 is in progress in ovarian cancer and endometrial cancer patients (NCT03748186 and NCT05200364).

STRO-002 is an antibody-drug conjugate (ADC) composed of an anti-folate receptor alpha (FolRα) antibody conjugated to a tubulin-targeting hemiasterlin (SC209) warhead via a cleavable linker currently in clinical studies for ovarian and endometrial cancer. FolRα exhibits minimal expression in normal tissues but is overexpressed in several cancers such as ovarian, endometrial, and NSCLC. We previously showed that STRO-002 induces in vitro hallmarks of immunogenic cell death (ICD), such as increased calreticulin exposure and HMGB1 and ATP release in a FolRα dependent manner. Follow-up vaccination studies with murine MC38 tumor cells engineered to express human FolRα (MC38-hFolRa) were performed to further explore the significance of STRO-002-induced ICD and protective immunity in vivo. A single vaccination with STRO-002 or SC209 in vitro-treated MC38-hFolRa cells in immunocompetent mice resulted in 60% of animals rejecting a subsequent rechallenge with living MC38-hFolRa cells, while relapse was observed in most control mice. Including an additional booster vaccination further increased the proportion ( 90%) of animals remaining tumor free after rechallenge. These results demonstrate that tumor cells pre-treated with STRO-002 or SC209 undergo potent immunogenic cell damage which can, in turn, mount protective immunity in vivo. The ability of STRO-002 to induce ICD that elicits an effective and robust anti-tumor immune response may translate into several advantages in the clinic. Induction of ICD is likely to offer a complementary anti-tumor mechanism in combination therapy regimens such as with check point inhibitors or VEGF blockade. We have demonstrated that STRO-002 in combination with avelumab in MC38-hFolRα-bearing mice significantly enhanced efficacy and durable anti-tumor immunity. More recent rechallenge studies reveal that this combination treatment suppresses growth of not only MC38-hFolRα but also MC38 wild-type tumor cells, indicating formation of immunological memory with signs of epitope spreading. Additionally, co-administration of STRO-002 with anti-VEGF therapy demonstrates improved TGI compared to monotherapy (p < 0.0001) in human ovarian OV-90 tumors. The potent cytotoxic and immunostimulatory properties of STRO-002 may also contribute to enhancing efficacy in indications with low FolRα expression. Such tumor types include endometrial and NSCLC, in which STRO-002 has displayed potent monotherapy activity in preclinical studies. In a panel of endometrial PDX models, STRO-002 was significantly efficacious in 9/17 (53%) FolRα-positive tumors, including models with moderate and weak FolRα levels. In a NSCLC PDX model, a single dose of STRO-002 induced tumor regression and suppressed tumor growth for up to three months post-dose. In conclusion, the induction of ICD by STRO-002 has potential to increase anti-tumor activity in cancers with low FolRα expression. Citation Format: Cristina L. Abrahams, Andrew McGeehan, Jennifer Smith, Robert Yuan, Kshama A. Doshi, Sihong Zhou, Xiaofan Li, Cuong Tran, Gang Yin, Arturo Molina, Kristin Bedard, Trevor Hallam. Anti-FolRα ADC STRO-002 induces immunogenic cell death (ICD) to enhance anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5591.

Introduction: Despite advances in cytotoxic and targeted therapies, recurrent disease remains the most significant obstacle to long-term survival in patients with childhood and adult leukemias. As part of our efforts to identify therapies that can be repurposed for immediate use in patients with leukemia, we interrogated a library of all available antibody-drug conjugates (ADCs) whose targets are expressed in leukemias (AML or ALL). A list of targets with available ADCs was merged with transcriptome data from over 3000 pediatric and adult leukemias (AML and ALL) to identify targets that are expressed in a large cohort of leukemias with immediate therapies for repurposing. The transcriptome data set included nearly 2000 pediatric AML cases sequenced as part of Target Pediatric AML (TpAML), 419 adult AML cases from TCGA LAML and the Beat AML program, as well as 853 ALL cases from COG and St. Jude trials. In this repurposing endeavor, CD74 emerged as the most expressed transcript in AML and ALL. CD74 encodes for a cell surface protein that associates with the class II major histocompatibility complex and is involved in the regulation of antigen presentation for immune response and B-cell differentiation. STRO-001 (Sutro Biopharma) is a CD74-directed, site-specific ADC developed for the treatment of multiple myeloma and lymphomas. Given broad expression of CD74 in leukemias, we evaluated the efficacy of STRO-001 in AML and ALL preclinical models. Methods: To characterize CD74 expression, RNA-seq data obtained from pediatric and adult AML and ALL patients was examined. Cell surface expression of CD74 was determined by flow cytometry using PE labeled anti-human CD74 antibody. The CD74-targeting ADC (STRO-001) was obtained from Sutro Biopharma.The preclinical efficacy of STRO-001 was evaluated against AML and ALL cell lines and patient samples expressing various levels of CD74 in vitro and in vivo. For in vivo studies, AML and ALL cell lines were transduced with GFP/Luciferase construct, and GFP+ cells were injected intravenously into NSG mice. Leukemia burden was measured by bioluminescence (IVIS) imaging weekly. Results: Transcriptomics analysis showed CD74 expression in a majority of adult and pediatric AML (>99% of cases) and at a much higher level compared CD33 and CD123 (targets currently developed for AML, Fig. 1A). CD74 is also broadly expressed in pediatric ALL, with a significant increase in expression observed compared to CD19 and CD22 (known targets in ALL, Fig. 1B). We confirmed that CD74 is expressed on the cell surface of AML blasts in primary patient samples (Fig. 1C) as well as AML and ALL cell lines (Fig. 1D). Given confirmation of cell surface expression of CD74, we investigated whether targeting CD74 can effectively eliminate leukemia cells. We evaluated the in vitro cytotoxicity of STRO-001 against K562 (a CML cell line that does not express CD74), AML cell lines (MV4;11 and NOMO-1), and ALL cell lines (REH1 and RS4;11) with varied CD74 expression. STRO-001 demonstrates target-specific cytotoxicity against CD74-expressing AML and ALL cell lines, but not K562 cells (Fig. 1E). STRO-001 exhibited high potency in CD74 expressing cells, with IC-50s of 41nM (MV4;11), 1.3nM (NOMO-1), 0.7nM (REH-1) and 3nM (RS4;11). In vivo studies in NSG mice transplanted with AML and ALL cell lines showed high potency. Treatment with STRO-001 at 3mg/kg once a week for 3 weeks effectively eradicated the leukemia in NOMO-1, REH-1, and RS4;11-bearing xenograft mice, while disease progression was observed in untreated control mice (Fig. 1F). We further evaluated the efficacy of STRO-001 in primary patient samples. Primary leukemia specimens from 3 patients with varied CD74 expression (Fig. 1G) were incubated with increasing concentrations of STRO-001 for 3 days. STRO-001 exhibited potent anti-leukemia activity against primary AML cells with IC-50s of 17.4nM, 12.8nM, and 4.07nM, respectively (Fig. 1H). Conclusion: Through transcriptomics profiling and validation of the cell surface expression by flow cytometry, we have identified CD74 as a viable therapeutic target for AML and ALL in children and adults. We further demonstrate that targeting CD74 with STRO-001 effectively eliminates leukemia cells both in vitro and in vivo, providing the preclinical data to compel evaluation of STRO-001 in clinical trials for childhood and adult leukemia. Figure 1 Figure 1. Disclosures Hylkema: Moderna: Current equity holder in publicly-traded company; Quest Diagnostics Inc: Current equity holder in publicly-traded company. Pardo: Hematologics, Inc.: Current Employment. Abrahams: Sutro Biopharma: Current Employment. Bedard: Sutro Biopharma: Current Employment. Molina: Sutro Biopharma: Current Employment. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: Equity Ownership. Loken: Hematologics, Inc.: Current Employment, Other: current equity holder in a privately owned company.

Cryptic inv(16)(p13.3q24.3) leading to the CBFA2T3-GLIS2 (CBF/GLIS) oncogenic fusion is exclusively seen in infants with AML and is associated with adverse outcome. Infants with this fusion are uniformly refractory to conventional therapies and despite intensive and myeloablative therapies, virtually all patients relapse with survival less than 15% (Smith et. al. 2020). In the effort to discover actionable targets for this highly refractory leukemia, we interrogated the genome and transcriptome of nearly 3,000 AML cases, including 45 cases of CBF/GLIS AML, and contrasted this to the transcriptome in normal hematopoietic tissues. Initial computational effort to identify CBF/GLIS-specific targets focused on genes that are overexpressed in CBF/GLIS AML and silent in normal hematopoietic tissues providing us with 194 candidate genes. Filtering this library of genes based on cell surface expression and prevalence in target population narrowed the target gene list to six cell surface encoding genes (FOLR1, FRAS1, HPSE2, KLRF2, MEGF10 and PCDH19). FOLR1 encodes folate receptor alpha and given available therapeutic options with FOLR1-targeting agents, this gene was selected for further studies and therapeutic assessment of available targeted therapies. Based on RNA-seq data, FOLR1 is uniquely expressed in CBF/GLIS AML as it is absent in other AML and in normal hematopoietic cells (Figure 1A) providing an opportunity to specifically target leukemia cells while sparing normal hematopoiesis. Cell surface expression of FOLR1 on AML blasts was confirmed by flow cytometry, which further shows AML-restricted expression of FOLR1 on AML cells but not in normal hematopoietic subsets in individual CBF/GLIS patient samples (Figure 1B, C). We previously showed that forced expression of CBF/GLIS fusion transcript in cord blood hematopoietic stem/ progenitor cells (CB HSPCs) induces malignant transformation that fully recapitulates primary CBF/GLIS AML (Hylkema et. al. 2019 ASH). We also demonstrated that CBF/GLIS-transduced CB HSPCs upregulate FOLR1 expression indicating that FOLR1 expression is causally associated with CBF/GLIS expression (Figure 1D). Given cell surface expression of FOLR1 on CBF/GLIS AML cells, we next investigated the preclinical efficacy of targeting FOLR1 using FOLR1-directed site-specific antibody-drug conjugate (STRO-002, Sutro Biopharma). In vitro efficacy of STRO-002 was tested against MV4;11 AML cell line engineered to express FOLR1 (MV4;11 FOLR1+, Figure 1D) and CBF/GLIS-transduced CB HPSCs. STRO-002 exhibited high cytotoxicity against MV4;11 FOLR1+ and CBF/GLIS-transduced CB HPSCs with IC50s of 0.1nM and 4.2nM, respectively (Figure 1E). In vivo efficacy of STRO-002 was evaluated in MV4;11 FOLR1+ NSG xenograft models, which showed potent activity that led to complete leukemia clearance after 3 weekly doses of STRO-002 at 2.5 and 5 mg/kg (Figure 1F). The anti-leukemia effects of STRO-002 resulted in significant increase in survival (p=0.002, Figure 1G). STRO-002 showed similar in vivo efficacy in the xenograft mice bearing CBF/GLIS-transduced CB HSPCs (Figure 1H) suggesting highly potent activity of STRO-002 against FOLR1+ AML cells. In this study, we utilize a computational approach to discover AML-restricted targets (high expression in AML, silent in normal) that are previously elusive to identify actionable targets for high-risk CBF/GLIS AML. From this discovery pipeline, we demonstrate FOLR1 to be highly and uniquely expressed in CBF/GLIS AML but not in normal counterparts, providing a strategy to target leukemia cells without impacting normal hematopoiesis. Finally, we demonstrate that targeting FOLR1 with STRO-002 antibody drug conjugate effectively eliminates CBF/GLIS-positive AML in vitro and in vivo, providing a promising approach to eradicate leukemia and improve outcomes in this high-risk AML subtype. Figure 1 Figure 1. Disclosures Hylkema: Quest Diagnostics Inc: Current equity holder in publicly-traded company; Moderna: Current equity holder in publicly-traded company. Pardo: Hematologics, Inc.: Current Employment. Abrahams: Sutro Biopharma: Current Employment. Bedard: Sutro Biopharma: Current Employment. Molina: Sutro Biopharma: Current Employment. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: equity ownership. Loken: Hematologics, Inc.: Current Employment, Other: current equity holder in a privately owned company.

Vaccine adjuvants are essential to drive a protective immune response in cases where vaccine antigens are weakly immunogenic, where vaccine antigen is limited, or where an increase in potency is needed for a specific population, such as the elderly. To discover novel vaccine adjuvants, we used a high-throughput screen (HTS) designed to identify small-molecule agonists of the RIG-I-like receptor (RLR) pathway leading to interferon regulatory factor 3 (IRF3) activation. RLRs are a group of cytosolic pattern-recognition receptors that are essential for the recognition of viral nucleic acids during infection. Upon binding of viral nucleic acid ligands, the RLRs become activated and signal to transcription factors, including IRF3, to initiate an innate immune transcriptional program to control virus infection. Among our HTS hits were a series of benzothiazole compounds from which we designed the lead analog, KIN1148. KIN1148 induced dose-dependent IRF3 nuclear translocation and specific activation of IRF3-responsive promoters. Prime-boost immunization of mice with a suboptimal dose of a monovalent pandemic influenza split virus H1N1 A/California/07/2009 vaccine plus KIN1148 protected against a lethal challenge with mouse-adapted influenza virus (A/California/04/2009) and induced an influenza virus-specific IL-10 and Th2 response by T cells derived from lung and lung-draining lymph nodes. Prime-boost immunization with vaccine plus KIN1148, but not prime immunization alone, induced antibodies capable of inhibiting influenza virus hemagglutinin and neutralizing viral infectivity. Nevertheless, a single immunization with vaccine plus KIN1148 provided increased protection over vaccine alone and reduced viral load in the lungs after challenge. These findings suggest that protection was at least partially mediated by a cellular immune component and that the induction of Th2 and immunoregulatory cytokines by a KIN1148-adjuvanted vaccine may be particularly beneficial for ameliorating the immunopathogenesis that is associated with influenza viruses.

Arenaviruses are a significant cause of hemorrhagic fever, an often-fatal disease for which there is no approved antiviral therapy. Lassa fever in particular generates high morbidity and mortality in West Africa, where the disease is endemic, and a recent outbreak in Nigeria was larger and more geographically diverse than usual. We are developing LHF-535, a small-molecule viral entry inhibitor that targets the arenavirus envelope glycoprotein, as a therapeutic candidate for Lassa fever and other hemorrhagic fevers of arenavirus origin. Using a lentiviral pseudotype infectivity assay, we determined that LHF-535 had sub-nanomolar potency against the viral envelope glycoproteins from all Lassa virus lineages, with the exception of the glycoprotein from the LP strain from lineage I, which was 100-fold less sensitive than that of other strains. This reduced sensitivity was mediated by a unique amino acid substitution, V434I, in the transmembrane domain of the envelope glycoprotein GP2 subunit. This position corresponds to the attenuation determinant of Candid#1, a live-attenuated Junín virus vaccine strain used to prevent Argentine hemorrhagic fever. Using a virus-yield reduction assay, we determined that LHF-535 potently inhibited Junín virus, but not Candid#1, and the Candid#1 attenuation determinant, F427I, regulated this difference in sensitivity. We also demonstrated that a daily oral dose of LHF-535 at 10 mg/kg protected mice from a lethal dose of Tacaribe virus. Serial passage of Tacaribe virus in LHF-535-treated Vero cells yielded viruses that were resistant to LHF-535, and the majority of drug-resistant viruses exhibited attenuated pathogenesis. These findings provide a framework for the clinical development of LHF-535 as a broad-spectrum inhibitor of arenavirus entry and provide an important context for monitoring the emergence of drug-resistant viruses., Author summary Lassa fever is a viral hemorrhagic fever disease that is transmitted to humans primarily through contact with the urine or feces of infected rodents. The disease is endemic in West Africa, and an unusually large outbreak occurred in Nigeria in early 2018. The case fatality rate was 25% among confirmed cases, underscoring the need for an effective antiviral therapy. Here, we evaluated the small-molecule drug LHF-535, which targets the arenavirus envelope glycoprotein, for broad-spectrum activity against Lassa viruses of different lineages and related arenaviruses that cause hemorrhagic fever diseases in South America. We also selected for LHF-535-resistant viruses and characterized their genotype and phenotype. Using a combination of surrogate systems and wild-type viruses, we determined that all tested Lassa virus strains and New World hemorrhagic fever arenaviruses were sensitive to LHF-535. Sensitivity to the drug was modulated by specific amino acid changes in the viral envelope glycoprotein, and the majority of emerging drug-resistant viruses were attenuated for virulence. Similarly, the live-attenuated vaccine strain for Argentine hemorrhagic fever was also resistant to LHF-535. These findings indicate that LHF-535 targets a viral virulence determinant, the mutation of which may result in the emergence of drug-resistant viruses, but with reduced capacity for virulence.

Viral sensing by RIG-I and downstream activation of antiviral defenses along with the induction of innate immune cytokines is essential from protection against influenza A virus (IAV) infection. We have identified a novel, small-molecule RIG-I agonist, KIN1148, which binds and activates RIG-I to signal the activation of IRF3 and the innate immune response. We are developing this molecule as an adjuvant to enhance vaccination against pandemic H1N1 (pH1N1) IAV. Ex vivo treatment of dendritic cells with KIN1148 leads to their activation and maturation. We determined the ability of KIN1148 to enhance suboptimal IAV vaccine responses in vivo. Administration of KIN1148 leads enhanced protection during high dose pH1N1 infection following a single, intramuscular administration of KIN1148 with IAV vaccine. This increase in protection is accompanied by a significant reduction in virus titers, as well as lung pathology. Analysis of the immune response induced following vaccination with KIN1148 as well as challenge demonstrates an increase in chemoattractant cytokines, germinal center B cells, IAV-specific antibodies, and IAV-specific CD4 and CD8 T cells compared to vaccination alone, indicating the induction of a broad anti-IAV immune response. Together these results demonstrate that prophylactic drug targeting of the RIG-I pathway with a small molecule enhances vaccine protection and highlight the potential of KIN1148 to enhancing vaccines against RNA virus infection.

There is a growing need for novel antiviral therapies that are broad spectrum, effective, and not subject to resistance due to viral mutations. Using high-throughput screening methods, including computational docking studies and an interferon-stimulated gene 54 (ISG54)-luciferase reporter assay, we identified a class of isoflavone compounds that act as specific agonists of innate immune signaling pathways and cause activation of the interferon regulatory factor (IRF-3) transcription factor. The isoflavone compounds activated the ISG54 promoter, mediated nuclear translocation of IRF-3, and displayed highly potent activity against hepatitis C virus (HCV) and influenza virus. Additionally, these agonists efficiently activated IRF-3 in the presence of the HCV protease NS3-4A, which is known to blunt the host immune response. Furthermore, genomic studies showed that discrete innate immune pathways centered on IRF signaling were regulated following agonist treatment without causing global changes in host gene expression. Following treatment, the expression of only 64 cellular genes was significantly induced. This report provides the first evidence that innate immune pathways dependent on IRF-3 can be successfully targeted by small-molecule drugs for the development of novel broad-spectrum antiviral compounds.

Human papillomavirus (HPV) types from the beta genus (beta-HPVs) have been implicated in the development of skin cancer. A potentially important aspect of their carcinogenic role is the ability of the E6 protein to degrade the proapoptotic family member Bak, which gives cells the ability to survive UV damage. However, it is unknown if the ability to degrade Bak is limited to certain beta-HPV types or whether E6 expression in keratinocytes affects other proteins important for apoptosis signaling. We tested the abilities of E6 proteins from several representative members of the beta-HPVs to degrade Bak and protect UV-treated keratinocytes from apoptosis. The E6 proteins of the beta-HPV type 5 (HPV5), -8, -20, -22, -38, -76, -92, and -96, as well as the alpha genus HPV HPV16, all degraded Bak or prevented its accumulation following UV treatment but did not degrade Bak constitutively. In addition, when tested using HPV16 E6 (16E6) and 8E6 as representative E6 proteins from the alpha and beta genera, respectively, Bak degradation was dependent on the E3 ubiquitin ligase, E6AP. Other important regulators of apoptotic signaling were examined and found to be unperturbed by the expression of the beta-HPV E6 proteins. Importantly, the expression of beta-HPV E6 proteins protected keratinocytes from apoptosis to the same extent as 16E6-expressing cells. In conclusion, several of the beta-HPV types possess the ability to protect UV-treated keratinocytes from apoptosis by reducing levels of Bak in those cells, thus blocking the intrinsic apoptotic pathway.

RIG-I is a pathogen recognition receptor that upon RNA virus infections serves to trigger innate immunity to program the adaptive immune response and is essential for the control of infection. RIG-I signaling initiates antiviral programs that restrict virus infection and activate many cell types including dendritic cells and macrophages for antigen presentation and cytokine production. To develop novel small molecule immune adjuvants, we conducted a high-throughput screen to identify RIG-I agonist molecules from which we derived a lead compound, KIN1148. KIN1148 associates with and activates recombinant RIG-I protein in cell-free assays. Binding to endogenous RIG-I in whole cell extracts promoted KIN1148 association with known RIG-I signaling partners including TRIM25, 14-3-3e, DHX15, TBK1 and NEMO and their assembly on the MAVS adaptor protein. Genomics analyses revealed that KIN1148 activation of RIG-I promotes an IRF3- and NF-kB-dependent transcriptional signature that features the expression of innate immune genes, chemokines and cytokines including CCL2, CCL3, CCL4, CCL7, CXCL10, IL-1b and IL-8 from macrophage-like THP-1 cells and in vivo in mouse tissues. KIN1148 treatment promoted the maturation of primary human dendritic cells to enhance antigen-driven activation of CD8 + T cells in cell culture models. Importantly, when administered to mice in combination with either an experimental influenza A virus or West Nile virus vaccine, KIN1148 enhanced the humoral immune response and protection of mice over vaccine alone in subsequent virus challenge. Thus, we have identified a novel small molecule RIG-I agonist that activates the innate immune response to promote enhanced vaccine immunity against RNA viruses. In parallel work, we have identified a distinct family of compounds that exhibit potent antiviral activity against a broad range of RNA viruses including West Nile, hepatitis C and dengue viruses. Collectively, our work demonstrates the strong potential for developing small molecule agonists that activate RIG-like receptor signaling for prophylactic and therapeutic control of pathogenic RNA viruses. This work is supported by funding from NIH/NIAID ( HHSN272200900035C , HHSN272201300023C and 1R01AI098943 ).

RIG-I-like receptors (RLR) are cytoplasmic pathogen recognition receptors that detect pathogen-associated molecular patterns (PAMP) within viral RNA and initiate the innate antiviral immune response that is essential for controlling many RNA virus infections. Viral PAMP engagement of RLR initiates a signaling cascade and transcriptional program that culminates in the expression of antiviral genes that restrict virus replication and spread and induces the secretion of cytokines and chemokines that support the development of adaptive immunity. We hypothesize that agonists that activate RLR signaling pathways will induce innate and adaptive immune responses that can be used to enhance vaccine protection to RNA virus infections. Using a high throughput cell-based screening approach, we have now identified drug-like compounds that target the RLR pathway and drive IRF-3 activation. We found that these compounds differentially induce proinflammatory cytokine and chemokine production, and the expression of antiviral and antigen presentation genes in treated cells. On their own, the compounds exhibit varying efficacy in limiting RNA virus infections. Moreover, the compounds promote dendritic cell maturation and enhance antigen-dependent activation of effector T cell function. Importantly, when paired with a vaccine at suboptimal dose, the compounds elicited an immune response that protected mice from lethal influenza virus challenge. The compounds also elicited an immune response to suboptimal doses of an experimental West Nile virus vaccine that protected mice from virus infection. Our study thus identifies novel immune-modulating molecules that function as innate immune agonists of RLR signaling to enhance vaccine-induced immunity. Our analyses reveal the strong potential of developing these RLR agonists as a new class of adjuvants for RNA virus vaccines. Our study demonstrates that targeting RLR signaling for innate immune induction is a viable approach for new immune therapies.