Toll-like Receptors 7 And 8 Research Articles

Abstract 7172: Pharmacokinetic/biomarker (PK/BMx) analysis of the toll-like receptor 7 and 8 (TLR7/8) agonist EIK1001 in phase 1 studies in participants (Pts) with solid tumors

Abstract Introduction: Immune checkpoint inhibitors (ICIs) relieve immunosuppression of tumor-reactive T cells and enhance antitumor immune response; however, not all patients benefit and some become refractory. EIK1001 is a TLR7/8 agonist that stimulates myeloid and plasmacytoid dendritic cells, activating immune and inflammatory responses. This dual activity provides another pathway, distinct from effects on checkpoint proteins, to enhance antitumor T-cell activity alone or in combination with ICIs. Methods: Adults with advanced solid tumors received a range of doses of EIK1001 (QW IV) alone and in combination with pembrolizumab (200 mg Q3W; NCT03486301) or atezolizumab (1200 mg Q3W; NCT04196530) in Phase 1 studies (n=128). PK samples were collected on Cycle 1 Day 1 (C1D1) and C1D8 predose and at 0.5, 1, 2, 4, 6, 8, 12, and 24 hrs postdose, with sparse sampling thereafter. Matched cytokine BMx samples were collected. The PK analysis population comprised pts with at least one measurable EIK1001 concentration (1563 observations from 94 pts). PK data were analyzed using non-linear mixed effects in Phoenix (v8.3). Covariate effects of the combination drugs were tested using a stepwise approach with a significance level of 0.05 for forward addition. All other covariates were evaluated using exploratory plots and formally tested using a full matrix approach. Empirical Bayes estimates were used to generate individual steady-state concentration-time profiles and calculate post-hoc PK parameters. Exploratory analyses of exposure-safety and exposure-efficacy were conducted. Results: EIK1001 PK was linear and dose-proportional over the doses studied. Tmax ranged from 4-12 hrs with no accumulation. Pop-PK analysis indicated that body size influenced clearance, but intrinsic factors of age, gender, tumor type, ECOG status, or hepatic/renal impairment (mild/moderate) did not. Combination drugs did not influence clearance. These attributes support BSA-based dosing and inform inclusion/exclusion criteria for future studies. On C1D1, time-dependent increases in IL-6, IL-8, IFNγ, and IP-10 returned to baseline within 24 hrs. An earlier IL-8 increase (Tmax 3-5 hr) is consistent with TLR8 activation on various cells including neutrophils, whereas the delayed increase of IFNγ/IP-10 (Tmax 5-8 hr) suggests a downstream effect. Dose-dependent increases in cytokines were observed, with median peak changes from baseline of 1.13- to 7.97-fold for IFNγ and 0.63- to 12.1-fold for IP-10. The 2 highest doses provided evidence of robust target engagement alone or in combination. Conclusion: The linear, dose-proportional PK of EIK1001 is well described by the Pop-PK model. Increases in IFNγ and IP-10 were consistent with the anticipated mechanism of action, with doses representing target engagement identified. Further development of EIK1001 is underway. Citation Format: Drew Rasco, Manish Patel, Melissa Johnson, Anthony Tolcher, David Sommerhalder, Omid Hamid, Angela Alistar, Jarema Kochan, Etah Kurland, Meihua Wang, Carolyn Cho, Sam Rebello. Pharmacokinetic/biomarker (PK/BMx) analysis of the toll-like receptor 7 and 8 (TLR7/8) agonist EIK1001 in phase 1 studies in participants (Pts) with solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7172.

Nucleotide modifications enable rational design of TLR7-selective ligands by blocking RNase cleavage.

Toll-like receptors 7 (TLR7) and 8 (TLR8) each sense single-stranded RNA (ssRNA), but their activation results in different immune activation profiles. Attempts to selectively target either TLR7 or TLR8 have been hindered by their high degree of homology. However, recent studies revealed that TLR7 and TLR8 bind different ligands resulting from the processing of ssRNA by endolysosomal RNases. We demonstrate that by introducing precise 2' sugar-modified bases into oligoribonucleotides (ORNs) containing known TLR7 and TLR8 binding motifs, we could prevent RNase-mediated degradation into the monomeric uridine required for TLR8 activation while preserving TLR7 activation. Furthermore, a novel, optimized protocol for CRISPR-Cas9 knockout in primary human plasmacytoid dendritic cells showed that TLR7 activation is dependent on RNase processing of ORNs and revealed a previously undescribed role for RNase 6 in degrading ORNs into TLR ligands. Finally, 2' sugar-modified ORNs demonstrated robust innate immune activation in mice. Altogether, we identified a strategy for creating tunable TLR7-selective agonists.

Generation and structure-activity relationships of novel imidazo-thienopyridine based TLR7 agonists: application as payloads for immunostimulatory antibody drug-conjugates

Pairing immunostimulatory small molecules with the targeting capability of an antibody has emerged as a novel therapeutic modality with the potential to treat a variety of solid tumors. A series of compounds based on an imidazo-thienopyridine scaffold were synthesized and tested for their ability to agonize the innate immune sensors toll-like receptor 7 and 8 (TLR7/8). Structure-activity relationship (SAR) studies revealed that certain simple amino-substituents could enable TLR7 agonism at low nanomolar concentrations. Drug-linkers containing either payload 1 or 20h were conjugated to the HER2-targeting antibody trastuzumab at the interchain disulfide cysteine residues using a cleavable valine-citrulline dipeptide linker and stochastic thiol-maleimide chemistry. In vitro, these immune-stimulating antibody drug-conjugates (ADCs) were found to induce cytokine release in a murine splenocyte assay when co-cultured with the HER2-high NCI-N87 cancer cell line. In vivo, tumor regression was observed with a single dose in an NCI-N87 gastric carcinoma xenograft model in BALB/c nude mice.

Abstract 1542: Generation of an antibody-drug conjugate-optimized TLR 7/8 agonist payload

Abstract Ligation of toll-like receptors 7 and 8 (TLR7/8) can potently activate innate immune cells, including tumor-associated macrophages, to prime downstream T cell activation and drive potent, lasting anti-tumor immunity. TLR7/8 agonists have long been pursued as an anti-cancer therapeutic because of their potential in re-programing the immune system but the clinical utility has been limited by systemic toxicity. Several means of decreasing this systemic toxicity have been investigated, including intra-tumoral administration, nanoparticle encapsulation and conjugation to a tumor-targeting antibody (antibody-drug conjugates, ADCs). ADCs are a clinically validated technology designed to target drugs to disease tissues to reduce the systemic toxicity of highly potent payloads and improve anti-tumor activity. We have developed an imidazoquinoline-based dual TLR7 and TLR8 small molecule agonist that has been specifically designed as an ADC payload. The initial compound was chosen from a set of imidazoquinoline-based small molecules using HEK293 reporter cells expressing either TLR7 or TLR8. This newly identified TLR7/8 agonist potently reactivated immunosuppressive macrophages to produce inflammatory cytokines, increased phagocytosis of tumor cells and enhanced T cell activation and proliferation. A lead payload candidate based on a modified version of the initial TLR7/8 agonist was designed to decrease drug permeability to minimize non-targeted systemic immune activation. The lead TLR7/8 agonist was shown to be significantly less potent than the first-generation compound as a small molecule but demonstrated enhanced immune-stimulating capability when conjugated to a direct immune-targeting antibody. The increased potency as an ADC is hypothesized to be driven by enhanced intracellular retention of the less cell permeable payload and/or a higher binding affinity to the TLR7 or 8 receptors, which was predicted via modeling. The greater in vitro potency of the lead payload was also observed in vivo in an MC38 syngeneic tumor model where 33% complete tumor cures were observed with the lead versus only tumor delay with the permeable version. Drug linkers employing the lead payload were evaluated by varying payload linkage chemistry, drug linker hydrophobicity, and drug release mechanisms using in vivo anti-tumor activity in different syngeneic mouse tumor models as a readout. The optimized drug linker design increased the in vitro and in vivo potency up to 4-fold when compared with the initial linker/payload. These data clearly demonstrate the potency of TLR7/8 agonists as immune stimulants in anti-cancer therapies and outline how their optimization through chemical modification can make them suitable payloads for ADCs. Citation Format: Kung-Pern Wang, Chris Neumann, Angela Epp, Weiping Zeng, Thomas Griffith, David Ferguson, Shyra Gardai, Alyson J. Smith. Generation of an antibody-drug conjugate-optimized TLR 7/8 agonist payload [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1542.

T cell-independent eradication of experimental glioma by intravenous TLR7/8-agonist-loaded nanoparticles

Glioblastoma, the most common and aggressive primary brain tumor type, is considered an immunologically “cold” tumor with sparse infiltration by adaptive immune cells. Immunosuppressive tumor-associated myeloid cells are drivers of tumor progression. Therefore, targeting and reprogramming intratumoral myeloid cells is an appealing therapeutic strategy. Here, we investigate a β-cyclodextrin nanoparticle (CDNP) formulation encapsulating the Toll-like receptor 7 and 8 (TLR7/8) agonist R848 (CDNP-R848) to reprogram myeloid cells in the glioma microenvironment. We show that intravenous monotherapy with CDNP-R848 induces regression of established syngeneic experimental glioma, resulting in increased survival rates compared with unloaded CDNP controls. Mechanistically, CDNP-R848 treatment reshapes the immunosuppressive tumor microenvironment and orchestrates tumor clearing by pro-inflammatory tumor-associated myeloid cells, independently of T cells and NK cells. Using serial magnetic resonance imaging, we identify a radiomic signature in response to CDNP-R848 treatment and ultrasmall superparamagnetic iron oxide (USPIO) imaging reveals that immunosuppressive macrophage recruitment is reduced by CDNP-R848. In conclusion, CDNP-R848 induces tumor regression in experimental glioma by targeting blood-borne macrophages without requiring adaptive immunity.

Oral Cancer Cells Release Vesicles that Cause Pain.

Oral cancer pain is attributed to the release from cancers of mediators that sensitize and activate sensory neurons. Intraplantar injection of conditioned media (CM) from human tongue cancer cell line HSC-3 or OSC-20 evokes nociceptive behavior. By contrast, CM from noncancer cell lines, DOK, and HaCaT are non-nociceptive. Pain mediators are carried by extracellular vesicles (EVs) released from cancer cells. Depletion of EVs from cancer cell line CM reverses mechanical allodynia and thermal hyperalgesia. CM from non-nociceptive cell lines become nociceptive when reconstituted with HSC-3 EVs. Two miRNAs (hsa-miR-21-5p and hsa-miR-221-3p) are identified that are present in increased abundance in EVs from HSC-3 and OSC-20 CM compared to HaCaT CM. The miRNA target genes suggest potential involvement in oral cancer pain of the toll like receptor 7 (TLR7) and 8 (TLR8) pathways, as well as signaling through interleukin 6 cytokine family signal transducer receptor (gp130, encoded by IL6ST) and colony stimulating factor receptor (G-CSFR, encoded by CSF3R), Janus kinase and signal transducer and activator of transcription 3 (JAK/STAT3). These studies confirm the recent discovery of the role of cancer EVs in pain and add to the repertoire of algesic and analgesic cancer pain mediators and pathways that contribute to oral cancer pain.

MEDB-56. POx NPs mediated delivery of (TLR7/8) agonist resiquimod improves treatment outcomes in SHH medulloblastoma by targeting tumor associated macrophages

Cancer immunotherapy, the utilization of the patients’ own immune system to treat cancer, has emerged as a powerful new strategy in cancer treatment. Recent clinical data has demonstrated immunotherapy to be effective in a wide range of cancers, including lung, bladder, renal cell, colorectal, gastro-esophageal, and head and neck cancers5-11. However, clinical reports of immune-based treatments for medulloblastomas are scarce and preliminary. Therefore, there is a need in developing strategies to improve medulloblastoma immunotherapy. Our recent studies have confirmed that SHH medulloblastomas are enriched in Tumor Associated Macrophages (TAMs) and unlike other tumors, TAMs are associated with positive outcomes and play a positive role by impairing tumor growth. Overall, analysis of TME in medulloblastomas reveals TAMs as a potential therapeutic target. Resiquimod is a synthetic small molecule agonist of Toll-like receptors 7 and 8 (TLR7/8) that modulates innate immune cells. We have loaded resiquimod into ultra-high-capacity polyoxazoline (POx) block copolymers forming small, homogeneous nanoparticles (POx-resiquimod). Our recent study shows that loading into POx nanoparticles improves drug delivery to tumors and treatment with 3 injections of POx-res as the single-agent treatment results in a profound anti-tumor effect G-Smo mice while treatment with free drug shows no therapeutic benefit. Our data also shows that that the tumors of G-Smo mice are enriched with the mixed populations of anti-inflammatory and pro-inflammatory macrophages and the treatment with POx-resiquimod have enhanced infiltration of macrophages into the tumors, enhanced the repolarization of macrophages to M1 subtype and decreased tumor cells viability. These studies show for the first time that targeting the medulloblastoma TME with POx-resiquimod can produce a significant anti-tumor effect. Furthermore, combination of POx-resiquimod with radiation resulted in long term survivals, showing potential therapeutic combination. The expression of TLR7/8 on TAMs in patient-derived medulloblastoma samples suggests that resiquimod may produce similar anti-medulloblastoma effects in humans.

Identification of 2-Pyridinylindole-Based Dual Antagonists of Toll-like Receptors 7 and 8 (TLR7/8).

The toll-like receptors (TLRs) play key roles in activation of the innate immune system. Aberrant activation of TLR7 and TLR8 pathways can occur in the context of autoimmune disorders due to the elevated presence and recognition of self-RNA as activating ligands. Control of this unintended activation via inhibition of TLR7/8 signaling holds promise for the treatment of diseases such as psoriasis, arthritis, and lupus. Optimization of a 2-pyridinylindole series of compounds led to the identification of potent dual inhibitors of TLR7 and TLR8, which demonstrated good selectivity against TLR9 and other family members. The in vitro characterization and in vivo evaluation in rodent pharmacokinetic/pharmacodynamic and efficacy studies of BMS-905 is detailed, along with structural information obtained through X-ray cocrystallographic studies.

The efficacy of a 2,4-diaminoquinazoline compound as an intranasal vaccine adjuvant to protect against influenza A virus infection in vivo.

Adjuvants are substances added to vaccines to enhance antigen-specific immune responses or to protect antigens from rapid elimination. As pattern recognition receptors, Toll-like receptors 7 (TLR7) and 8 (TLR8) activate the innate immune system by sensing endosomal single-stranded RNA of RNA viruses. Here, we investigated if a 2,4-diaminoquinazoline-based TLR7/8 agonist, (S)-3-((2-amino-8-fluoroquinazolin-4-yl)amino)hexan-1-ol (named compound 31), could be used as an adjuvant to enhance the serological and mucosal immunity of an inactivated influenza A virus vaccine. The compound induced the production of proinflammatory cytokines in macrophages. In a dose-response analysis, intranasal administration of 1 µg compound 31 together with an inactivated vaccine (0.5 µg) to mice not only enhanced virus-specific IgG and IgA production but also neutralized influenza A virus with statistical significance. Notably, in a virus-challenge model, the combination of the vaccine and compound 31 alleviated viral infection-mediated loss of body weight and increased survival rates by 40% compared with vaccine only-treated mice. We suggest that compound 31 is a promising lead compound for developing mucosal vaccine adjuvants to protect against respiratory RNA viruses such as influenza viruses and potentially coronaviruses.

Abstract PO-105: Overcoming stromal barriers in PDA with a novel polymeric Toll-like receptor agonist

Abstract The complex tumor microenvironment (TME) of PDA creates a uniquely immune- and drug-privileged sanctuary that contributes to disease pathogenesis and treatment resistance. The profoundly immunosuppressive microenvironment of PDA includes abundant MDSC, Treg, M2-like TAM and a dearth of cytotoxic T-cells. Recent discoveries of highly expressed Toll-like receptors 7 and 8 (TLR7/8) in the desmoplastic stroma of PDA suggest that TLR7/8 agonists may be an effective immune-targeted therapy. TLR7/8 agonists may inhibit tumor growth by any of several mechanisms including myeloid cell reeducation, depletion of MDSC, and/or depletion of Treg, thereby activating cytotoxic CD8+ T cells. However, the circulatory half-life of most TLR7/8 agonists is relatively short, and elevated systemic levels and prolonged treatment can also cause severe adverse reactions that can limit their use and effectiveness. To address these concerns, we have developed a novel polymeric form of a TLR7/8 agonist that exploits a breakthrough synthetic technology to directly polymerize drugs into nanocarriers with defined architectures. These agents are designed to minimize adverse effects and increase efficacy through more favorable pharmacokinetics and enhanced delivery to desmoplastic tumors with release profiles tailorable over the range of days to weeks. This agent is in development in our murine preclinical trials program (MCTP) which is modeled on human clinical trials and involves randomized, blinded, placebo-controlled studies against the current standard-of-care using the KrasLSL-G12D/+;Trp53LSL-R172H/+;p48Cre/+ (KPC) model as our primary platform of the autochthonous disease. Exploratory experiments were performed to determine optimal dose and treatment schedule. KPC mice with ultrasound-imaging documented disease meeting enrollment criteria were subsequently randomized into one of two arms in 28-day pilot studies: control vehicle-treated and polymeric TLR7/8 (n=5-8 animals per arm). Daily health and behavior checks, serial body weight measurements, and complete blood count (CBC) profiles over the course of the study revealed no adverse health effects or overt toxicities. Mice were euthanized at study completion and all organs harvested for histological analyses; single cell suspensions from blood, spleen, peri-pancreatic lymph nodes and primary tumor were analyzed by FACS and/or CyTOF Helios cytometry (incorporating 30+ specific markers and with a particular focus on identifying specific states and subtypes of CD8+ T cells as well as markers of checkpoint activation to assess innate and adaptive immunity in treated versus untreated and tumor versus normal tissues). Preliminary analyses reveal response associated with increases in T cell activation markers and concomitant changes in inhibitory markers. M1 and M2 macrophage populations also appeared to be profoundly affected, suggesting targeting of specific immune cell types and distinct activation states. Citation Format: Christopher C. DuFort, Ciana L. Lopez, Martin C. Whittle, Vladimir Vlaskin, Aditi Vadodkar, Selvi Srinivasan, Patrick S. Stayton, Sunil R. Hingorani. Overcoming stromal barriers in PDA with a novel polymeric Toll-like receptor agonist [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-105.

NIMG-48. TLR7/8-AGONIST-LOADED NANOPARTICLES REPROGRAM TUMOR-ASSOCIATED MYELOID CELLS FOR EFFECTIVE IMMUNOTHERAPY OF EXPERIMENTAL GLIOMA AND MRI-BASED TREATMENT MONITORING

Abstract Drivers of glioblastoma progression include the immunosuppressive tumor microenvironment (TME), dominated by tumor-associated myeloid cells. Therefore, we investigated a new approach targeting the myeloid compartment to reprogram myeloid cells in the TME using a β-cyclodextrin nanoparticle (CDNP) formulation encapsulating the toll-like receptor 7 and 8 (TLR7/8) agonist R848. Biodistribution confirmed specific targeting of CDNP-R848 to tumor-associated macrophages (TAMs) (labeling efficiency: 34.0% ± 22.2%), whereas tumor microglia (5.4% ± 4.4%) and splenic macrophages (13.2% ± 0.7%) revealed less uptake. Interestingly, intravenous application of CDNP-R848 induced strong tumor regression with an overall response rate of 80% (2.5% complete response, 52.5% partial response and 25% stable disease, n=40 mice) in Gl261 syngeneic experimental gliomas, while CDNP vehicle treated animals showed exponential tumor growth (100% progressive disease, n=12 mice). As advanced imaging is essential to monitor intracranial disease and possibly predict response and resistance, we performed high resolution magnetic resonance imaging using ultrasmall iron oxide nanoparticles (USPIO) for macrophage tracking. Increased levels of USPIO uptake in vehicle treated animals compared to CDNP-R848 treated animals were found as an early marker of responding mice (ΔT2*: -11.7 ± 4.2 vs -4.0 ± 2.8 ms, p=0.01). This correlated with an increased influx of myeloid cells into the TME of vehicle treated animals and showed a strong correlation of macrophage recruitment and USPIO uptake (R2: 0.78, p=0.004). Mechanistically, phenotyping of macrophages (CD45high/CD11b+) indicated a pro-inflammatory shift of TAMs with an increased infiltration of pro-inflammatory F4/80+/MHCII+ macrophages during CDNP-R848 treatment. Surprisingly, the anti-tumor effect of CDNP-R848 was independent of CD8+ T cells, CD4+ T cells or NK cells during selective depletion experiments. In summary, this work demonstrates the ability of myeloid-targeted therapies to re-shape the tumor microenvironment for an effective immunotherapy of glioma.

A Redox-Responsive Nanovaccine Combined with A2A Receptor Antagonist for Cancer Immunotherapy.

In situ vaccination can trigger an antitumor immune response. However, the therapeutic effect is still limited since the high expression of adenosine binding to G protein-coupled receptor A2AR induces an immunosuppressive effect. In this work, a new formulation is presented with the combination of a nanovaccine based on redox-responsive polymer micelles and A2AR antagonist SCH58261. The micelles simultaneously encapsulate immunogenic cell death (ICD) inducer doxorubicin (DOX) and adjuvant toll-like receptor 7 and 8 (TLR7/8) agonist R848, acting as the potent in situ vaccines. A high concentration of glutathione in tumor cells leads to the disintegration of these micelles, releasing DOX and R848 to mediate ICD, inducing the activation of dendritic cells and initiating an immune response. Meanwhile, A2AR antagonist SCH58261, a generation immune checkpoint blocker, inhibits the immunosuppressive adenosinergic pathway in the tumor microenvironment, activating natural killer (NK) cells and CD8+ T cells, and inhibiting the proliferation of regulatory T cells. Therefore, this formulation can trigger a robust systemic antitumor immune response.

Evolution of Toll-like receptor 7/8 agonist therapeutics and their delivery approaches: From antiviral formulations to vaccine adjuvants.

Imidazoquinoline derivatives (IMDs) and related compounds function as synthetic agonists of Toll-like receptors 7 and 8 (TLR7/8) and one is FDA approved for topical antiviral and skin cancer treatments. Nevertheless, these innate immune system-activating drugs have potentially much broader therapeutic utility; they have been pursued as antitumor immunomodulatory agents and more recently as candidate vaccine adjuvants for cancer and infectious disease. The broad expression profiles of TLR7/8, poor pharmacokinetic properties of IMDs, and toxicities associated with systemic administration, however, are formidable barriers to successful clinical translation. Herein, we review IMD formulations that have advanced to the clinic and discuss issues related to biodistribution and toxicity that have hampered the further development of these compounds. Recent strategies aimed at enhancing safety and efficacy, particularly through the use of bioconjugates and nanoparticle formulations that alter pharmacokinetics, biodistribution, and cellular targeting, are described. Finally, key aspects of the biology of TLR7 signaling, such as TLR7 tolerance, that may need to be considered in the development of new IMD therapeutics are discussed.

BDB001, an intravenously administered toll-like receptor 7 and 8 (TLR7/8) agonist, in combination with pembrolizumab in advanced solid tumors: Phase 1 safety and efficacy results.

2512 Background: BDB001 is an intravenously administered TLR 7/8 dual agonist immune modulator capable of reprogramming dendritic cells to produce antitumor activities. BDB001 monotherapy has demonstrated favorable tolerability and robust systemic immune activation leading to durable clinical responses in a phase I dose escalation trial. Here, we report on the safety and efficacy of BDB001 in combination with pembrolizumab in a phase I dose escalation trial in advanced solid tumors (NCT03486301). Methods: BDB001-101 is a phase 1, open label, dose escalation/expansion trial of BDB001 (IV, Q1W) in combination with pembrolizumab (IV, Q3W) in patients with advanced solid tumors. The primary endpoint was safety and tolerability. Secondary endpoints included efficacy, pharmacokinetics and pharmacodynamic profiling of immune activation. Results: Twenty-three subjects with 13 different tumor types were enrolled across 4 dose levels. Sixty one percent were female, median age was 63 years (range, 33-86), median number of prior therapies was 3 (range, 1-8), and 48% of tumors had progressed on prior anti-PD-(L)1 therapy. Overall, BDB001 in combination with pembrolizumab was well tolerated and dose-limiting toxicities were not observed. The most common treatment related adverse events (TRAEs) were fever (39.1%), fatigue (39.1%), chills/rigor (34.8%), pruritus/rash (21.7%), and nausea (13.0%). Most of these TRAEs were grade 1 or 2 and transient. Only 3 (13.0%) subjects experienced grade 3 TRAEs of fatigue, rash, stomatitis, and alkaline phosphatase elevation. There were no grade 4 or 5 TRAEs. Pharmacodynamic evaluation of plasma cytokine levels showed robust increases in interferon gamma and interferon inducible protein-10 (IP-10) at BDB001 Dose Level 3 and 4. Preliminary efficacy evaluation of the 14 subjects treated at Dose Level 3 and 4 showed durable and deep clinical responses in 4 (29%) subjects with anti-PD-(L)1 mAb refractory melanoma, hepatocellular carcinoma, cholangiocarcinoma, and platinum-resistant ovarian carcinoma. The responses were observed by the initial efficacy assessment at 9-weeks, with some seen as early as 4-weeks. In addition, 4 (29%) subjects had stable disease for a disease control rate of 57%. To date, median time on treatment is 14.4 weeks (range, 6.0 – 42.1+) with 3 subjects still active on treatment. Conclusions: Intravenously administered BDB001 in combination with pembrolizumab is well tolerated. Rapid and deep clinical responses were observed, supported by robust systemic immune activation. BDB001 in combination with pembrolizumab is a promising novel therapeutic option for patients with advanced solid tumors and is being evaluated in an ongoing dose expansion trial. Clinical trial information: NCT03486301.

A polyamidoamine (PAMAM) derivative dendrimer with high loading capacity of TLR7/8 agonist for improved cancer immunotherapy

Tumor associated macrophages (TAMs) tend to exhibit tumor-promoting M2 phenotype and contribute to the development of immunosuppressive microenvironment of solid tumors. Reprograming TAMs from M2 into tumoricidal M1 phenotype is robust for stimulating tumor immunosuppressive microenvironment (TIME). In this study, we developed a poly(amidoamine) (PAMAM) derivative dendrimer (denoted as fourth generation-N,N-diethylaminoethyl (G4-DEEA)) for efficient loading of Toll-like receptor 7 and 8 (TLR7/8) agonist (R848) to remodel the TIME for potent cancer immunotherapy. G4-DEEA exhibited a high loading capacity of R848 up to 35.9 wt% by taking advantage of its dendritic structure. The resulting formulation (designated as G4-DEEA@R848) effectively polarized M2 macrophages into M1 phenotype in vitro, and improved the maturation and activation of antigen-presenting cells. In the 4T1 orthotopic breast cancer model, G4-DEEA@R848 showed a stronger tumor inhibitory effect than free drug. The mechanistic studies suggested that G4-DEEA@R848 could significantly stimulate the TIME by repolarizing TAMs into M1 phenotype, reducing the presence of immunosuppressive myeloid cells and increasing the infiltration of tumor cytotoxic T cells. This study provides a simple but effective dendrimer-based strategy to improve the formulation of R848 for improved cancer immunotherapy.

Development of thermosensitive resiquimod-loaded liposomes for enhanced cancer immunotherapy

Resiquimod (R848) is a toll-like receptor 7 and 8 (TLR7/8) agonist with potent antitumor and immunostimulatory activity. However, systemic delivery of R848 is poorly tolerated because of its poor solubility in water and systemic immune activation. In order to address these limitations, we developed an intravenously-injectable formulation with R848 using thermosensitive liposomes (TSLs) as a delivery vehicle. R848 was remotely loaded into TSLs composed of DPPC: DSPC: DSPE-PEG2K (85:10:5, mol%) with 100 mM FeSO4 as the trapping agent inside. The final R848 to lipid ratio of the optimized R848-loaded TSLs (R848-TSLs) was 0.09 (w/w), 10-fold higher than the previously-reported values. R848-TSLs released 80% of R848 within 5 min at 42 °C. These TSLs were then combined with αPD-1, an immune checkpoint inhibitor, and ultrasound-mediated hyperthermia in a neu deletion (NDL) mouse mammary carcinoma model (Her2+, ER/PR negative). Combined with αPD-1, local injection of R848-TSLs showed superior efficacy with complete NDL tumor regression in both treated and abscopal sites achieved in 8 of 11 tumor bearing mice over 100 days. Immunohistochemistry confirmed enhanced CD8+ T cell infiltration and accumulation by R848-TSLs. Systemic delivery of R848-TSLs, combined with local hyperthermia and αPD-1, inhibited tumor growth and extended median survival from 28 days (non-treatment control) to 94 days. Upon re-challenge with reinjection of tumor cells, none of the previously cured mice developed tumors, as compared with 100% of age-matched control mice. The dose of R848 (10 μg for intra-tumoral injection or 6 mg/kg for intravenous injection delivered up to 4 times) was well-tolerated without weight loss or organ hypertrophy. In summary, we developed R848-TSLs that can be administered locally or systematically, resulting in tumor regression and enhanced survival when combined with αPD-1 in mouse models of breast cancer.

Abstract 2199: Improvement of ADCP response by re-education of tumor-associated macrophages with Resiquimod-encapsulated liposomes

Abstract Background: Tumor-associated macrophages (TAMs) are the major part of the tumor microenvironment 1, which are classified as two groups - the tumor-suppressing M1 type and the tumor-supporting M2 type 2. Thus, TAMs could act as a double-edged sword. Reprogramming TAMs from M2 to M1 type would improve the efficacy of anti-tumor therapies 3. TAMs may influence the efficacy of therapeutic anti-tumor antibodies. One of the modes of action of therapeutic antibodies is an antibody-dependent cellular phagocytosis (ADCP) 1. However, it is still controversial whether the status of TAMs influences ADCP response 4,5. Resiquimod (R848) is an agonist for Toll-like receptor 7 and 8 (TLR7/8) that can reprogram TAMs from M2 to M1 6. In this study, we evaluated whether R848-induced M1 type TAMs could enhance ADCP response. Therefore, we encapsulated R848 into liposomes, intending to achieve TAM-specific R848 delivery, TAMs re-education, ADCP improvement, and enhancement of therapeutic efficacy of antibodies. Methods: For ADCP assay, mouse bone marrow derived macrophages (BMDM) were differentiated into M1 and M2 as TAM models. Phagocytosis of EGFP-expressing cancer cells (Raji, A431, and SKBR3) by TAMs was evaluated by FACS. For TAM re-education, M2 TAMs were incubated with R848. Expression level of macrophage markers (CD80 and CD206) was measured by FACS. Biodistribution of liposomal R848 (R848-LPs) in WiDr cell-bearing nude mice was evaluated by in vivo imaging and FACS. In vivo TAM re-education ability, ADCP enhancement ability, and tumor growth inhibition ability of R848-LPs combined with therapeutic antibody were investigated in WiDr cell-bearing nude mice. Results: In vitro ADCP assay showed that M1 macrophages elicited the more efficient ADCP response than M2 macrophages towards Raji, A431, and SKBR3 using Rituximab (α-CD20 antibody), α-EGFR antibody (528), and Trastuzumab (α-HER2 antibody), respectively. Upon treatment with R848-LPs, macrophages showed an M1 polarization and an enhanced ADCP response in vitro. In vivo imaging and FACS analysis showed that R848-LPs rapidly accumulated in TAMs at tumor site. R848-LP treatment efficiently enhanced the ADCP response towards WiDr using α-EGFR antibody (528). By the treatment of R848-LPs, a significant increased ratio of CD80/CD206 was observed. WiDr-bearing nude mice treated with R848-LP combined with α-EGFR antibody (528) suppressed tumor growth compared with control groups (non-treatment and α-EGFR antibody without R848-LPs). Conclusions: Liposomal R848 is a potential TAM re-education agent, which could significantly enhance the therapeutic effect of antibodies by improving ADCP. Keywords: tumor-associated macrophage; therapeutic antibodies; antibody-dependent cellular phagocytosis; Resiquimod; liposomes

Rational design of antisense oligonucleotides modulating the activity of TLR7/8 agonists

Oligonucleotide-based therapeutics have become a reality, and are set to transform management of many diseases. Nevertheless, the modulatory activities of these molecules on immune responses remain incompletely defined. Here, we show that gene targeting 2′-O-methyl (2′OMe) gapmer antisense oligonucleotides (ASOs) can have opposing activities on Toll-Like Receptors 7 and 8 (TLR7/8), leading to divergent suppression of TLR7 and activation of TLR8, in a sequence-dependent manner. Surprisingly, TLR8 potentiation by the gapmer ASOs was blunted by locked nucleic acid (LNA) and 2′-methoxyethyl (2′MOE) modifications. Through a screen of 192 2′OMe ASOs and sequence mutants, we characterized the structural and sequence determinants of these activities. Importantly, we identified core motifs preventing the immunosuppressive activities of 2′OMe ASOs on TLR7. Based on these observations, we designed oligonucleotides strongly potentiating TLR8 sensing of Resiquimod, which preserve TLR7 function, and promote strong activation of phagocytes and immune cells. We also provide proof-of-principle data that gene-targeting ASOs can be selected to synergize with TLR8 agonists currently under investigation as immunotherapies, and show that rational ASO selection can be used to prevent unintended immune suppression of TLR7. Taken together, our work characterizes the immumodulatory effects of ASOs to advance their therapeutic development.

Optimization of 8-oxoadenines with toll-like-receptor 7 and 8 activity

Toll-like receptors 7 and 8 (TLR7/8) agonists are potent immunostimulants that are attracting considerable interest as vaccine adjuvants. We recently reported the synthesis of a new series of 2-O-butyl-8-oxoadenines substituted at the 9-position with various linkers and N-heterocycles, and showed that TLR7/8 selectivity, potency and cytokine induction could be modulated by varying the alkyl linker length and the N-heterocyclic ring. In the present study, we further optimized the oxoadenine scaffold by investigating the effect of different substituents at the 2-position of the oxoadenine on TLR7/8 potency/selectivity, cytokine induction and DC maturation in human PBMCs. The results show that introducing a 1-(S)-methylbutoxy group at the 2-position of the oxoadenine significantly increased potency for TLR7/8 activity, cytokine induction and DC maturation.

Synthetic Toll-like Receptors 7 and 8 Agonists: Structure-Activity Relationship in the Oxoadenine Series.

Toll-like receptors 7 and 8 (TLR7/8) are broadly expressed on antigen-presenting cells, making TLR7/8 agonists likely candidates for the development of new vaccine adjuvants. We previously reported the synthesis of a new series of 8-oxoadenines substituted at the 9-position with a 4-piperidinylalkyl moiety and demonstrated that TLR7/8 selectivity and potency could be modulated by varying the length of the alkyl linker. In the present study, we broadened our initial structure–activity relationship study to further evaluate the effects of N-heterocycle ring size, chirality, and substitution on TLR7/8 potency, receptor selectivity, and cytokine (IFNα and TNFα) induction from human peripheral blood mononuclear cells (PBMCs). TLR7/8 activity correlated primarily to linker length and to a lesser extent to ring size, while ring chirality had little effect on TLR7/8 potency or selectivity. Substitution of the heterocyclic ring with an aminoalkyl or hydroxyalkyl group for subsequent conjugation to phospholipids or antigens was well tolerated with the retention of both TLR7/8 activity and cytokine induction from human PBMCs.