TLR Agonists Research Articles

Vibrational spectroscopy coupled with machine learning sheds light on the cellular effects induced by rationally designed TLR4 agonists

In this work, we present the potential of Fourier transform infrared (FTIR) microspectroscopy to compare on whole cells, in an unbiased and untargeted way, the capacity of bacterial lipopolysaccharide (LPS) and two rationally designed molecules (FP20 and FP20Rha) to activate molecular circuits of innate immunity. These compounds are important drug hits in the development of vaccine adjuvants and tumor immunotherapeutics. The biological assays indicated that FP20Rha was more potent than FP20 in inducing cytokine production in cells and in stimulating IgG antibody production post-vaccination in mice. Accordingly, the overall significant IR spectral changes induced by the treatment with LPS and FP20Rha were similar, lipids and glycans signals being the most diagnostic, while the effect of the less potent molecule FP20 on cells resulted to be closer to control untreated cells. We propose here the use of FTIR spectroscopy supported by artificial intelligence (AI) to achieve a more holistic understanding of the cell response to new drug candidates while screening them in cells.

Differential susceptibility of cells infected with defective and intact HIV proviruses to killing by obatoclax and other small molecules.

Some drugs that augment cell-intrinsic defenses or modulate cell death/survival pathways have been reported to selectively kill cells infected with HIV or Simian Immunodeficiency Virus (SIV), but comparative studies are lacking. We hypothesized that these drugs may differ in their ability to kill cells infected with intact and defective proviruses. To investigate this hypothesis, drugs were tested ex vivo on peripheral blood mononuclear cells (PBMC) from nine antiretroviral therapy (ART)-suppressed individuals. We tested drugs currently in clinical use or human trials, including auranofin (p53 modulator), interferon alpha2A, interferon gamma, acitretin (RIG-I inducer), GS-9620/vesatolimod (TLR7 agonist), nivolumab (PD-1 blocker), obatoclax (Bcl-2 inhibitor), birinapant [inhibitor of apoptosis proteins (IAP) inhibitor], bortezomib (proteasome inhibitor), and INK128/sapanisertib [mammalian target of rapamycin mTOR] [c]1/2 inhibitor). After 6 days of treatment, we measured cell counts/viabilities and quantified levels of total, intact, and defective HIV DNA by droplet digital PCR (Intact Proviral DNA Assay). Obatoclax reduced intact HIV DNA [median = 27-30% of dimethyl sulfoxide control (DMSO)] but not defective or total HIV DNA. Other drugs showed no statistically significant effects. Obatoclax and other Bcl-2 inhibitors deserve further study in combination therapies aimed at reducing the intact HIV reservoir in order to achieve a functional cure and/or reduce HIV-associated immune activation.

The E3 Ubiquitin Protein Ligase LINCR Amplifies the TLR-Mediated Signals through Direct Degradation of MKP1.

Toll-like receptors (TLRs) induce innate immune responses through activation of intracellular signaling pathways, such as MAP kinase and NF-κB signaling pathways, and play an important role in host defense against bacterial or viral infections. Meanwhile, excessive activation of TLR signaling leads to a variety of inflammatory disorders, including autoimmune diseases. TLR signaling is therefore strictly controlled to balance optimal immune response and inflammation. However, its balancing mechanisms are not fully understood. In this study, we identified the E3 ubiquitin ligase LINCR/ NEURL3 as a critical regulator of TLR signaling. In LINCR-deficient cells, the sustained activation of JNK and p38 MAPKs induced by the agonists for TLR3, TLR4, and TLR5, was clearly attenuated. Consistent with these observations, TLR-induced production of a series of inflammatory cytokines was significantly attenuated, suggesting that LINCR positively regulates innate immune responses by promoting the activation of JNK and p38. Interestingly, our further mechanistic study identified MAPK phosphatase-1 (MKP1), a negative regulator of MAP kinases, as a ubiquitination target of LINCR. Thus, our results demonstrate that TLRs fine-tune the activation of MAP kinase pathways by balancing LINCR (the positive regulator) and MKP1 (the negative regulator), which may contribute to the induction of optimal immune responses.

Abstract LT08: Extracellular Vesicle Surface Display Enhances the Therapeutic Efficacy and Safety Profile of Cancer Immunotherapy

Abstract As our understanding of the complex interactions between the immune system and cancer has progressed, so has the identification of novel targets and ligands that can modulate effective anti-cancer immune responses. While immunotherapy has emerged as a mainstay in cancer therapy, its efficacy has been limited by the potential for off-target toxicity against normal body cells. In this study, we present a nanoparticle-based delivery system that enhances the signaling efficacy of immunomodulatory ligands while concurrently limiting systemic toxicity. We demonstrate that extracellular vesicles (EVs), lipid bilayer enclosed nanoparticles released by cells, can be efficiently engineered to display multiple immunomodulatory ligands on their surface. Display of immunomodulatory ligands such as agonistic CD137 and CD40 antibodies on the EV surface significantly enhanced their therapeutic efficacy over equivalent doses of free ligands by inducing ligand multimerization, efficient receptor crosslinking and the formation of immune synapses. EVs conjugated with a complementary combination of immune stimulatory ligands and immune checkpoint inhibitors were able to shift the tumor immune milieu towards an anti-tumorigenic phenotype in a lung metastatic melanoma allograft and suppress tumor progression to a greater extent than an equivalent dose of free ligands. We also demonstrate the ability of EVs to co-deliver intraluminally loaded TLR agonists which synergize with surface displayed agonistic CD40 antibodies to induce effective anti-tumor responses and tumor regression in a metastatic autochthonous cell-derived model of PDAC. Remarkably, we also observed that EV-associated immunotherapeutics were limited in their biodistribution to the tumor microenvironment, resulting in significantly lower off-target toxicity than treatment with free ligands. In summary, we present an EV-based delivery approach that allows the use of potent cancer immunotherapy combinations to achieve superior anti-tumor responses at significantly lower doses with less side-effects than is possible with conventional administration methods for cancer immunotherapy. Citation Format: Migara Kavishka Jayasinghe, Dawn Liu Xiao Tian, Yock Sin Lay, Chang Yu Lee, Brendon Zhi Jie Yeo, Dong Van Hoang, Rebecca Carissa Prajogo, Boya Peng, Thach Tuan Pham, Minh Thi Nguyet Le. Extracellular Vesicle Surface Display Enhances the Therapeutic Efficacy and Safety Profile of Cancer Immunotherapy [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr LT08.

Single-B cell analysis correlates high-lactate secretion with stress and increased apoptosis

While cellular metabolism was proposed to be a driving factor of the activation and differentiation of B cells and the function of the resulting antibody-secreting cells (ASCs), the study of correlations between cellular metabolism and functionalities has been difficult due to the absence of technologies enabling the parallel measurement. Herein, we performed single-cell transcriptomics and introduced a direct concurrent functional and metabolic flux quantitation of individual murine B cells. Our transcriptomic data identified lactate metabolism as dynamic in ASCs, but antibody secretion did not correlate with lactate secretion rates (LSRs). Instead, our study of all splenic B cells during an immune response linked increased lactate metabolism with acidic intracellular pH and the upregulation of apoptosis. T cell-dependent responses increased LSRs, and added TLR4 agonists affected the magnitude and boosted LSRhigh B cells in vivo, while resulting in only a few immunoglobulin-G secreting cells (IgG-SCs). Therefore, our observations indicated that LSRhigh cells were not differentiating into IgG-SCs, and were rather removed due to apoptosis.

Modulating the mesenchymal stromal cell microenvironment alters exosome RNA content and ligament healing capacity.

Although mesenchymal stromal cell (MSC) based therapies hold promise in regenerative medicine, their clinical application remains challenging due to issues such as immunocompatibility. MSC-derived exosomes are a promising off-the-shelf therapy for promoting wound healing in a cell-free manner. However, the potential to customize the content of MSC-exosomes, and understanding how such modifications influence exosome effects on tissue regeneration remain underexplored. In this study, we used an in vitro system to compare the priming of human MSCs by 2 inflammatory inducers TNF-α and CRX-527 (a highly potent synthetic TLR4 agonist that can be used as a vaccine adjuvant or to induce anti-tumor immunity) on exosome molecular cargo, as well as on an in vivo rat ligament injury model to validate exosome potency. Different microenvironmental stimuli used to prime MSCs in vitro affected their exosomal microRNAs and mRNAs, influencing ligament healing. Exosomes derived from untreated MSCs significantly enhance the mechanical properties of healing ligaments, in contrast to those obtained from MSCs primed with inflammation-inducers, which not only fail to provide any improvement but also potentially deteriorate the mechanical properties. Additionally, a link was identified between altered exosomal microRNA levels and expression changes in microRNA targets in ligaments. These findings elucidate the nuanced interplay between MSCs, their exosomes, and tissue regeneration.

Size Tuning of Mesoporous Silica Adjuvant for One-Shot Vaccination with Long-Term Anti-Tumor Effect.

Despite recent clinical successes in cancer immunotherapy, it remains difficult to initiate a long-term anti-tumor effect. Therefore, repeated administrations of immune-activating agents are generally required in most cases. Herein, we propose an adjuvant particle size tuning strategy to initiate a long-term anti-tumor effect by one-shot vaccination. This strategy is based on the size-dependent immunostimulation mechanism of mesoporous silica particles. Hollow mesoporous silica (HMS) nanoparticles enhance the antigen uptake with dendritic cells around the immunization site in vivo. In contrast, hierarchically porous silica (HPS) microparticles prolong cancer antigen retention and release in vivo. The size tuning of the mesoporous silica adjuvant prepared by combining both nanoparticles and microparticles demonstrates the immunological properties of both components and has a long-term anti-tumor effect after one-shot vaccination. One-shot vaccination with HMS-HPS-ovalbumin (OVA)-Poly IC (PIC, a TLR3 agonist) increases CD4+ T cell, CD8+ T cell, and CD86+ cell populations in draining lymph nodes even 4 months after vaccination, as well as effector memory CD8+ T cell and tumor-specific tetramer+CD8+ T cell populations in splenocytes. The increases in the numbers of effector memory CD8+ T cells and tumor-specific tetramer+CD8+ T cells indicate that the one-shot vaccination with HMS-HPS-OVA-PIC achieved the longest survival time after a challenge with E.G7-OVA cells among all groups. The size tuning of the mesoporous silica adjuvant shows promise for one-shot vaccination that mimics multiple clinical vaccinations in future cancer immunoadjuvant development. This study may have important implications in the long-term vaccine design of one-shot vaccinations.

Toll-like receptors (TLR-2 and TLR-4): putative role in the uterine inflammatory response in cows – A mini review

Metritis and endometritis are the most prevalent uterine inflammatory diseases in bovines and cause infertility and high economic loss to farmers. Many studies have been conducted to investigate the mechanism of pathology, immune response and therapeutics associated with these diseases. This review focuses mainly on the immune system present in the uterus and on the involvement of Toll-like receptors. This study aimed to provide a deep understanding of the structure, function and expression pathways of Toll-like receptors (TLR-2 and TLR-4) in the treatment of pathogenic microorganisms invading the uterus and their significance in inflammatory conditions such as metritis and clinical and subclinical endometritis. The genetic associations of TLRs with diseases and some therapeutic interventions are also briefly discussed. As such, TLRs-2 and 4 play a critical role in providing a uterine defense mechanism against foreign substances (embryos, sperm, and pathogenic bacteria). In the present era, the use of TLR agonists is being unfurled for treatment, and further exploration and study of this topic in the future are needed.

Development of Non-toxic Recombinant Ricin Vaccine and Evaluation of Vaccine Efficacy

Ricin is a highly toxic protein which is produced in the seeds of the castor oil plant. Ricin toxin A chain has ribosomal RNA N-glycosylase activity that irreversibly hydrolyses the N-glycosidic bond of the adenine residue at position 4324 within the 28S rRNA. In this study, we developed non-toxic recombinant ricin vaccine(R51) in <i>E. coli</i> expression system, and evaluated efficacy of the R51 according to adjuvants. When the R51 was administered using aluminum hydroxide as an adjuvant, the vaccine efficacy was higher than that of TLR agonists or aluminum phosphate. Because it is time-consuming to administer the vaccine three times at three-week intervals, we investigated the survival rate and antibody titer of mice according to the change of time interval of vaccination. Interestingly, there was no difference in survival rate and antibody titer when R51 was administered at 0, 1, and 3 weeks or 0, 2, and 4 weeks compared to when administered at 0, 3, and 6 weeks. Therefore, the developed R51 vaccine is promising to protect soldiers from Ricin attack.

Abstract LB040: MACO-355, a unique pan-LILR monoclonal antibody for cancer therapy, re-programs and stimulates immuno-suppressive macrophages in a novel ligand-binding blocking independent manner

Abstract Macrophages populate most solid tumors in large numbers and limit effective anti-tumoral immune responses. The leukocyte immunoglobulin-like receptors (LILR) such as e.g. LILRB1, LILRB2, and others are expressed on tumor associated macrophages. Despite sharing multiple ligands such as major histocompatibility complex class I G (HLA-G), these receptors regulate different macrophage functions such as phagocytosis or cytokine release. To date, therapeutic approaches to targeting LILRs have concentrated on blocking receptor-ligand interactions to relieve ligand-mediated immune suppression. MACO-355 is a fully human antibody binding multiple LILRA (activating) and LILRB (inhibiting) family members including LILRB1, LILRB2, LILRB3, LILRA4 and LILRA6, that does not neutralize ligand (HLA-G, HLA-A, HLA-E) binding to either LILRB1 or to LILRB2. Nevertheless, MACO-355 mediates macrophage reprogramming even under strong immune suppressive conditions, as measured e.g. by stimulation of the TLR agonist induced pro-inflammatory cytokine release in CSF1, TGFβ, IL-10, and IL-4 treated cells. Concordantly, stimulation of immuno-suppressive macrophages with MACO-355 also resulted in significantly higher levels of T cell expansion, proliferation, and INFγ release in a co-culture suppression assay compared to ligand blocking antibodies. Additionally, MACO-355 enhances macrophage phagocytosis irrespective of tumor cell HLA-G ligand status and NK tumor cell killing. All these in vitro activities translate into in vivo activity in A375 melanoma CDX tumor model comparable to that of anti-PD-1 and anti-PD-L1 Abs in humanized mice. Safety studies showed that despite its potent immune stimulatory activity, MACO-355 does not lead to spurious pro-inflammatory cytokine production in in vitro cytokine storm assays. Molecular studies showed MACO-355 binds a novel epitope distant from a ligand binding pocket on LILRs and requires both bi-valent binding and Fc effector function for full activity. This results in pronounced modulation of intracellular kinase signaling. MACO-355 is a unique receptor-ligand non-blocking pan-LILR antibody highly potent in pro-inflammatory reprogramming of immuno-suppressive macrophages, both in vitro and in vivo. This appears to occur via a novel mode of action in which MACO-355 clusters multiple LILRs with Fc-receptors into unconventional signaling hubs. Altogether the data support the future clinical evaluation of MACO-355 as a cancer therapeutic. Citation Format: Krzysztof B. Wicher, Moritz Haneklaus, Martha Lopez-Yrigoyen, Alicia Poindron, Carmen Rodriguez-Seoane, Maikel Fransen, Chantell Payton, Stephane M. Guillame, Luca Cassetta, Stephen Myatt, Carola Ries. MACO-355, a unique pan-LILR monoclonal antibody for cancer therapy, re-programs and stimulates immuno-suppressive macrophages in a novel ligand-binding blocking independent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB040.

Immunogenicity and efficacy of VLA2001 vaccine against SARS-CoV-2 infection in male cynomolgus macaques

BackgroundThe fight against COVID-19 requires mass vaccination strategies, and vaccines inducing durable cross-protective responses are still needed. Inactivated vaccines have proven lasting efficacy against many pathogens and good safety records. They contain multiple protein antigens that may improve response breadth and can be easily adapted every year to maintain preparedness for future seasonally emerging variants.MethodsThe vaccine dose was determined using ELISA and pseudoviral particle-based neutralization assay in the mice. The immunogenicity was assessed in the non-human primates with multiplex ELISA, neutralization assays, ELISpot and intracellular staining. The efficacy was demonstrated by viral quantification in fluids using RT-qPCR and respiratory tissue lesions evaluation.ResultsHere we report the immunogenicity and efficacy of VLA2001 in animal models. VLA2001 formulated with alum and the TLR9 agonist CpG 1018™ adjuvant generate a Th1-biased immune response and serum neutralizing antibodies in female BALB/c mice. In male cynomolgus macaques, two injections of VLA2001 are sufficient to induce specific and polyfunctional CD4+ T cell responses, predominantly Th1-biased, and high levels of antibodies neutralizing SARS-CoV-2 infection in cell culture. These antibodies also inhibit the binding of the Spike protein to human ACE2 receptor of several variants of concern most resistant to neutralization. After exposure to a high dose of homologous SARS-CoV-2, vaccinated groups exhibit significant levels of protection from viral replication in the upper and lower respiratory tracts and from lung tissue inflammation.ConclusionsWe demonstrate that the VLA2001 adjuvanted vaccine is immunogenic both in mouse and NHP models and prevent cynomolgus macaques from the viruses responsible of COVID-19.

Strategic development of a self-adjuvanting SARS-CoV-2 RBD vaccine: From adjuvant screening to enhanced immunogenicity with a modified TLR7 agonist

Adjuvants enhance the body's immune response to a vaccine, often leading to better protection against diseases. Monophosphoryl lipid A analogues (MPLA, TLR4 agonists), α-galactosylceramide analogues (NKT cell agonists), and imidazoquinoline compounds (TLR7/8 agonists) are emerging novel adjuvants on market or under clinical trials. Despite significant interest in these adjuvants, a direct comparison of their adjuvant activities remains unexplored. We initially assessed the activities of various adjuvants from three distinct categories using the SARS-CoV-2 RBD trimer antigen. TLR4 and TLR7/8 agonists are discovered to elicit robust IgG2a/2b antibodies, which is crucial for eliciting antibody dependent cytotoxicity. While α-galactosylceramide analogs induced mainly IgG1 antibody. Then, because of the flexibility of the TLR7/8 agonist, we designed and synthesized a tri-component self-adjuvanting SARS-CoV-2 RBD vaccine, featuring a covalent TLR7 agonist and targeting mannoside. Animal studies indicated that this vaccine generated antigen-specific humoral immunity. Yet, its immunogenicity seems compromised, indicating the complexity of the vaccine.

Quantitative and Qualitative Distinctions between HIV-1 and SIV Reservoirs: Implications for HIV-1 Cure-Related Studies.

The persistence of the latent viral reservoir is the main hurdle to curing HIV-1 infection. SIV infection of non-human primates (NHPs), namely Indian-origin rhesus macaques, is the most relevant and widely used animal model to evaluate therapies that seek to eradicate HIV-1. The utility of a model ultimately rests on how accurately it can recapitulate human disease, and while reservoirs in the NHP model behave quantitatively very similar to those of long-term suppressed persons with HIV-1 (PWH) in the most salient aspects, recent studies have uncovered key nuances at the clonotypic level that differentiate the two in qualitative terms. In this review, we will highlight differences relating to proviral intactness, clonotypic structure, and decay rate during ART between HIV-1 and SIV reservoirs and discuss the relevance of these distinctions in the interpretation of HIV-1 cure strategies. While these, to some degree, may reflect a unique biology of the virus or host, distinctions among the proviral landscape in SIV are likely to be shaped significantly by the condensed timeframe of NHP studies. ART is generally initiated earlier in the disease course, and animals are virologically suppressed for shorter periods before receiving interventions. Because these are experimental variables dictated by the investigator, we offer guidance on study design for cure-related studies performed in the NHP model. Finally, we highlight the case of GS-9620 (Vesatolimod), an antiviral TLR7 agonist tested in multiple independent pre-clinical studies in which virological outcomes may have been influenced by study-related variables.

Abstract 2697: Simultaneous activation of innate immune suppressive components impairs antitumor efficacy of immunotherapy against the innate immune system

Abstract Cancer immunotherapy has shown noticeable clinical efficacy in many cancer types, while clinical benefits are only observed in 20-40% of treated patients. Therefore, combination cancer immunotherapy targeting multiple factors that limit antitumor immunity has been tested in the clinic. Accumulating evidence reveals that cancers that harbor limited immune cell infiltration (so-called immunologically cold tumors) respond poorly to current immunotherapy. Preclinical studies using animal models have shown that activation of innate immunity by TLR agonists or STING agonists can successfully remodel the TME to effectively induce antitumor immunity in those immunologically cold tumors. However, unexpectedly, all TLR agonists and STING agonists tested have not shown clinical efficacy in combination with immune checkpoint blockade therapy, including PD-1/PD-L1 blockade therapy. Hence, we addressed the mechanism(s) why stimulators of innate immunity failed to elicit effective antitumor immunity in cancer patients. We then employed OK-432, which reportedly stimulates TLR-2, TLR-4, and/or TLR-9, as OK-432 is widely used in Japan in the clinic. In vitro OK-432 treatment accelerated the maturation DCs, and efficiently induced CD8+ T cells in humans. However, the combination treatment with anti-PD-1 mAb and OK-432 failed to augment antitumor immunity in animal models using anti-PD-1 mAb monotherapy-resistant tumors; the data mirrors the failure of clinical trials of combination therapy with immune checkpoint blockade and TLR agonists or STING agonists in humans. Therefore, using the animal models, we explored the acquired resistance mechanism developed after combination treatment. While OK-432 treatment effectively stimulated DCs, innate immune suppressive cells, such as MDSCs, were concurrently accumulated in the TME. The accumulation of PMN-MDSCs after OK-432 treatment was induced in a CXCL1-CXCR2-dependent fashion. Accordingly, triple combination treatment with a PMN-MDSC inhibitor along with OK-432 and anti-PD-1 mAb attenuated the accumulation of PMN-MDSCs in the TME and improved the antitumor effect of the combination treatment. Taken together, we propose that successful combination treatment stimulating innate immunity against immunogenically cold cancers requires controlling the unwanted co-activation of innate immune suppressive cells. This study clearly demonstrates the mechanism of acquired resistance after administration of reagents that activate innate immunity, such as TLR agonists or STING agonists, and provides the basis for future successful combination therapy. Citation Format: Hitomi Nishinakamura, Sayoko Shinya, Takuma Irie, Shugo Sakihama, Tadaaki Ioroi, Takeo Naito, Keisuke Watanabe, Daisuke Sugiyama, Motohiro Tamiya, Tatsuya Yoshida, Tetsunari Hase, Takao Yoshida, Kennosuke Karube, Yuka Maeda, Shohei Koyama, Hiroyoshi Nishikawa. Simultaneous activation of innate immune suppressive components impairs antitumor efficacy of immunotherapy against the innate immune system [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 2697.

Abstract 715: Nanomedicines loaded with TLR agonists and inhibitors of immunosuppression to reprogram the tumor microenvironment

Abstract The stroma of solid tumors is populated by myeloid cells, which mostly represent macrophages. Tumor-associated macrophages (TAMs), strongly influenced by cancer cell-derived factors, are key drivers of immunosuppression and support tumor growth and spread to distant sites. Increasing evidence demonstrates their ability to hamper cancer patients' response to most treatments currently applied in the clinic, including immunotherapy. Therefore, strategies to counteract negative effects of TAMs are nowadays gaining momentum at preclinical, translational, and clinical levels. Here, we present the development and evaluation of nanoparticles (NPs) loaded with TLR agonists and/or inhibitors of immunosuppressive pathways to reprogram TAMs and the tumor microenvironment (TME) to unleash an effective immune response to fight against the tumor. Firstly, we have evaluated in vitro the ability of poly(I:C) and/or R848, agonists of TLR3 and TLR7/8 respectively, to reprogram TAMs into antitumor effector cells, and also the efficacy of Stattic and/or Galunisertib, inhibitors of STAT3 and TGF-β pathways respectively, to inhibit immunosuppression by cancer cells and/or TAMs. The best combinations of these drugs were encapsulated in nanoemulsions or polymeric nanocapsules for improved TAM-targeting and pharmacokinetics in vivo. These NPs were characterized for their physicochemical properties and also tested in vitro using primary human macrophages. For in vivo evaluation, subcutaneous and orthotopic murine models of lung cancer (CMT167) were used, showing antitumoral efficacy and TME reprogramming as evaluated by FACS, RNA analysis and multiplex immunofluorescence. Protamine-NCs-loaded with poly(I:C)+R848 and coated with an additional layer of hyaluronic acid functionalized with mannose were used to target the CD206 receptors, showing antitumoral efficacy mediated by TAM reprogramming, evaluated as higher CD86 while lower CD206 and Arg1 expression. Nanoemulsions with a PEGylated surfactant, encapsulating Stattic+Galunisertib+R848 showed faster antitumoral activity versus the free drugs. Experiments performed in IFN-γ KO mice and immune deficient mice (NSG and Balb/c nude) revealed that a fully functional immune system is crucial for the response to the treatment. In conclusion, our work demonstrates the antitumoral efficacy and reprogramming of the tumor microenvironment by combinations of TLR agonists and/or inhibitors of immunosuppression, which can be improved by nanotechnological approaches. Further investigations are ongoing to assess their antitumoral efficacy in other tumor models (i.e. breast and pancreatic cancer), with the final aim of clinical translation. Citation Format: Fernando Torres Andon, Alba Pensado-López, Clement Anfray, Aldo Ummarino, Iago Fernández-Mariño, Lucia Sanjurjo, Jose Crecente-Campo, Eduardo Fernández-Megía, Flavia Castro, Maria Jose Oliveira, Alfonso Calvo, Rosario García Campelo, Alberto Mantovani, Maria Jose Alonso, Paola Allavena. Nanomedicines loaded with TLR agonists and inhibitors of immunosuppression to reprogram the tumor microenvironment [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 715.

Abstract 6352: Preclinical discovery of ARX622: A site-specific, TLR7 agonist, HER2-targeted immune-stimulatory antibody drug conjugate for treatment of multiple solid tumor types

Abstract Traditional antibody-drug conjugates leverage the specificity of antibodies to deliver cytotoxic payloads to tumor microenvironments (TMEs), thereby reducing systemic toxicities associated with non-targeted chemotherapeutics. Analogously, a novel class of immune-stimulating antibody conjugates (ISACs) has recently emerged to selectively deliver synthetic immune agonists to the TME without systemic immune activation. The first generation of ISACs and the majority of approved and investigational ADCs contain payloads linked to native lysine or cysteine residues, which can lead to heterogenous drug-antibody ratios (DARs), reduced antibody stability, and labile conjugation, all of which limit therapeutic index. Here we describe the generation and preclinical characterization of ARX622, a site-specific TLR7-selective agonist ISAC targeting HER2. To avoid the instability issues of previous generation ADCs and ISACs, ARX622 has a homogenous and stable DAR via oxime bond-mediated payload conjugation to a synthetic amino acid, para-acetyl phenylalanine (pAF), which is recombinantly incorporated at defined heavy chain positions. Similar to cytotoxic ADCs built on this site-specific conjugation platform, ARX622 ISAC and total mAb pharmacokinetic (PK) profiles in non-human primates (NHPs) are indistinguishable, demonstrating in vivo stability and minimal payload deconjugation. Accordingly, ARX622 displays an extended PK profile in mice compared to a DAR-matched random cysteine ISAC containing the same antibody and payload. In the presence of HER2-expressing cells, ARX622 selectively induces proinflammatory cytokine production, type I and III IFN secretion, and APC maturation. In a syngeneic tumor model, ARX622 promotes complete tumor regressions, immunologic memory, and epitope spreading. In xenograft systems, single doses of ARX622 induce complete regression of large, established tumors (>1000mm3), and robustly inhibit tumor growth in mice that have progressed through prior treatment with HER2-targeted mAbs or ADCs. In HER2-low xenografts, ARX622 monotherapy inhibits tumor growth, and promotes complete regression of late-stage (800mm3) tumors when sequentially combined with a HER2-ADC. Based on PK data at efficacious doses in mice and tolerated exposures in NHP toxicity studies, ARX622 has an estimated therapeutic index of ~60x. Taken together, ARX622’s mAb-like properties, high stability, broad immune mechanism induction, strong anti-tumor activity alone or in combination with ADCs, and wide therapeutic index make it a strong candidate for further development toward treating patients with a variety of HER2-expressing solid tumors. Citation Format: David Mills, Ji Young Kim, Erica Wood, Keith Tatsukawa, Nick Knudsen, Jay Nelson, Lillian Skidmore, Kedar GC, Hon Tran, Manoj Pal, Hamidreza Hashemi, Ying Buechler, Shawn Zhang, Dan O'Connor. Preclinical discovery of ARX622: A site-specific, TLR7 agonist, HER2-targeted immune-stimulatory antibody drug conjugate for treatment of multiple solid tumor types [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 6352.

Abstract 6746: A B-cell targeted TLR9 agonist antibody conjugate potentiates cancer vaccine efficacy and rejuvenates vaccine responses in the elderly

Abstract TLR9 agonists (unmethylated CpG oligodeoxynucleotides) are clinically validated as vaccine adjuvants and cancer therapeutics1,2. We developed a Toll-like Receptor Agonist Antibody Conjugate (TRAAC) comprised of a potent TLR9 agonist (T-CpG) conjugated to an antibody against CD22, a receptor restricted to B cells (TAC-001). Systemically administered TAC-001 delivers T-CpG to B cells, resulting in TLR9 activation, and innate and adaptive anti-tumor immunity3. TAC-001 is being evaluated as an immunotherapy in a Phase 1 clinical trial in patients with advanced solid tumors (NCT05399654)4. In this study, we investigated whether TAC-001 mouse surrogate, mCD22 TRAAC, can potentiate cancer vaccine efficacy using preclinical vaccination models. Human Nectin-4 ectodomain protein (N4, 94% homology to its mouse ortholog) was used as a neoantigen-like vaccine, alone and in combination with mCD22 TRAAC, MPLA (a TLR4 agonist), or unconjugated T-CpG. Mice were immunized, then challenged 2 months later with syngeneic CT26 tumors expressing N4. Mice in treatment combination cohorts that resisted tumor development were split into two groups and rechallenged 3.5 months later with a more aggressive tumor, syngeneic 4T1 or 4T1 expressing N4. The combination of vaccine with mCD22 TRAAC was the only treatment able to inhibit growth of the 4T1-N4 tumor. No efficacy was observed in mice inoculated with parental 4T1, thereby demonstrating neoantigen vaccine specific anti-tumor immunity. An MHCI restricted peptide of HER2/neu, GP2, fused to a B cell epitope peptide, P4, and conjugated to KLH, was used as another vaccine model5. GP2-P4-KLH was administered to mice alone or in combination with mCD22 TRAAC or unconjugated T-CpG. Ex vivo GP2/GP2-P4 stimulation of lymphoid organ immune cells from mice treated with vaccine plus mCD22 TRAAC resulted in elevated Granzyme B, IFNγ and IL17A levels, indicating that mCD22 TRAAC enhances T cell activation and cytotoxic activity. Lastly, to investigate the effect of mCD22 TRAAC in elderly mice, SARS-CoV-2 spike protein was used as a vaccine. Spike was administered alone or in combination with mCD22 TRAAC, MPLA or T-CpG. Combining mCD22 TRAAC with vaccine resulted in high levels of spike neutralizing IgG in both youth and elderly cohorts, showing that mCD22 TRAAC rejuvenates vaccine responses in elderly. In all studies, mCD22 TRAAC led to the most potent vaccine recall response, with high antigen specific IgG titers skewing towards a Th1 phenotype, as shown by elevated levels of effector function antibodies of IgG2a subclass and increased IFNγ production. These findings provide a strong rationale for combining TAC-001 with cancer vaccines. References1Hyer et al., Vaccine 2018; 36(19) 2Long et al., Annals of Oncology 2018; 29(8) 3Kuo et al., Cancer Research 2021; 81(13)4Perez et al., JITC 2023; 11(1) 5Nordin et al., Cancers 2021; 13(19) Citation Format: Maja Z. Bonacorsi, Amy Chen, Ons Harrabi, Min Li, Emma R. Sangalang, Danielle Fontaine, Janet Sim, Pavel Strop, Hong I. Wan, Maria Jose Costa. A B-cell targeted TLR9 agonist antibody conjugate potentiates cancer vaccine efficacy and rejuvenates vaccine responses in the elderly [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 6746.

Abstract 2461: Discovery of an orally available and liver-targeted ALPK1 agonist for cancer immunotherapy

Abstract Boosting anti-tumor immune responses can be achieved by activating the innate immune system. Despite the ongoing development of numerous STING and TLR agonists for this purpose, none of them have progressed to late-stage clinical evaluation. Alpha protein kinase-1 (ALPK1) is an intracellular innate immune receptor newly identified for its ability to detect ADP-heptose, a metabolite released during the biosynthesis and degradation of lipopolysaccharides (LPS). Since heptose is not naturally present in mammalian cells, the recognition of heptose metabolites by ALPK1 serves as a reliable mechanism to identify bacterial infections. When ADP-heptose binds to ALPK1, it initiates the phosphorylation of TIFA, thereby triggering a cascade of immune responses. Systemic administration with ADP-heptose induces robust ALPK1-dependent cytokine responses in mice. While high doses of ADP-heptose demonstrate anti-tumor effects, its instability and low in vivo potency hinder its clinical development. Through a SBDD medicinal chemistry effort, a novel, stable, and potent ADP-heptose analog, designated PTT-936, has been discovered and is progressing into clinical evaluation in cancer patients. PTT-936 exhibits remarkable nanomolar potencies for ALPK1 activation in various cellular assays. Notably, in human PBMCs, PTT-936 induces both concentration-dependent release of cytokines and activation of NK and T cells. Consistent with in vitro studies, oral administration of PTT-936 triggered significant cytokine responses in mice at microgram dose levels. However, overt toxicities were observed only at milligram dose levels, indicating a wide therapeutic window for this compound. In syngeneic tumor models, oral administration of PTT-936 induced dose-dependent tumor growth inhibition, which correlated with elevations in several cytokine biomarkers. Mechanistic studies show that PTT-936, working exclusively through the ALPK1 pathway, elicited strong innate and adaptive immune responses, as evidenced by robust cytokine induction and immune cell activation and recruitment. In syngeneic mouse models, the anti-tumor effects of PTT-936 were significantly hindered by either CD8+ T cell depletion or neutralization of select cytokines. Besides monotherapy studies, compelling evidence demonstrates strong synergistic anti-tumor effects when combining PTT-936 with immune checkpoint inhibitors. Intriguingly, PTT-936 exhibits favorable tolerability and a wider therapeutic index compared to certain clinical-stage STING and TLR agonists. Furthermore, PTT-936 does not enhance cytokine elevations induced by checkpoint inhibitors, which have been linked to adverse events (including CRS) observed in clinical combination studies. The dosing frequency of PTT-936 in the upcoming first-in-human clinical trial is carefully designed to prioritize both tolerability and patient compliance. Citation Format: Yupeng Wang, Nana Du, Xiaona Dong, Xuebing Sun, Yuan Hong, Xiaolong Yang, Zhijiang Ji, Qianyu Wang, Yun Lv, Zhichen Shi, Yajuan Gao, Xiaofu Hou, Yaxin Zhang, Juan Wang, Rui Mao, Yinxin Liu, Liang Li, Jingjin Ding, Hexiang Wang, Nan Hu, Counde O’Yang, Zhihong Li, Keith Bley, Feng Shao. Discovery of an orally available and liver-targeted ALPK1 agonist for cancer immunotherapy [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 2461.

Abstract 6639: Mechanisms of action of a killed, bacteria-based, multiple immune receptor agonist in development for pulsed anti-tumor immunotherapy

Abstract Activation of immune receptors, such as Toll-like (TLR), NOD-like (NLR) and Stimulator of Interferon Genes (STING) is required for efficient innate and adaptive immunity. Gram-negative bacteria (G-NB) contain multiple TLR, NOD and STING agonists. Potential utility of G-NB for cancer immunotherapy is supported by observations of tumor regression in the setting of infection and Coley’s Toxins. Coley reported that intravenous (i.v.) administration was likely most effective but produced toxicity. The discovery of TLRs and their agonists, particularly the potent/multi-functional TLR4 agonist lipopolysaccharide (LPS)-endotoxin, comprising ~75% of the outer membrane of G-NB, suggests that it may be both a critical active ingredient and responsible for dose-limiting toxicity of i.v. G-NB. We have produced killed, intact bacteria products from non-pathogenic G-NB with ~90% reduction of LPS activity. One product, Decoy10, contained TLR2,4,8,9, NOD2 and STING agonist activity and exhibited reduced i.v. toxicity in mice and rabbits relative to unprocessed cells. Decoy10 and closely related Decoy20 produced single agent activity or combination-mediated durable regressions, with immunological memory, in mice with syngeneic or human breast, colorectal, hepatocellular, pancreatic carcinomas or non-Hodgkin’s lymphoma. Regressions were observed in combination with chemotherapy, a non-steroidal anti-inflammatory drug, anti-PD-1, or rituximab, were associated with induction of 18-26 plasma cytokines/chemokines, activation of innate and adaptive immune pathways in tumors, and were dependent on NK, CD4+ and CD8+ T cells (Newman, Cancer Res 2023;83(7 Suppl):Abstract nr 4165). These and preliminary clinical results demonstrating rapid clearance of Decoy20 with transient induction of >50 plasma cytokines/chemokines (Newman et al., J Immunother Cancer 2023;11(Suppl 2)782-E:A1770) are supportive of a “pulse-prime” mechanism, whereby Decoy bacteria produce transient, but broad innate and adaptive immune activation. We have now analyzed immune activation by Decoy bacteria in vitro using human peripheral blood mononuclear cells (PBMCs). Decoy10 enhanced polarization of CD14+ monocytes to M1 macrophages and inhibited Treg polarization. Decoy10 enhanced monocyte-derived dendritic cell maturation and enhanced Th1, Th2 and Th17 CD4+ polarization, with Th1 at 10-100-fold lower Decoy10 concentrations than Th2 or Th17. Decoy10 also activated NKT and T cells, and enhanced PBMC-mediated tumor cell killing. The results demonstrate that Decoy bacteria stimulate polarization, maturation, or activation of cellular mediators of innate and adaptive anti-tumor immune responses, inhibit an immune-suppressive mechanism, and enhance immune cell killing of tumor cells. The results are consistent with and significantly extend our previously reported in vivo data. Citation Format: Michael J. Newman, Deepak Singh. Mechanisms of action of a killed, bacteria-based, multiple immune receptor agonist in development for pulsed anti-tumor immunotherapy [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 6639.

Abstract 2465: A novel agonist of CD137 as a single agent shows cancer immunoprevention efficacy against various tumor types that is not dependent on the trained immunity

Abstract Introduction: We recently demonstrated that pretreatment of mice with SA-4-1BBL, a novel oligomeric form of CD137 agonist, as a single agent prevents the development of various tumor types in mice. The tumor immunoprevention efficacy is long-lasting (>14 weeks) and contingent upon CD4+ immune cells, NK cells, and IFN-γ, but not CD8+ T and B cells. Trained immunity mediated by previously activated myeloid cells has been shown to have efficacy against cancer. The objective of this study was twofold: i) assess the role of trained immunity and ii) elucidate the mechanistic underlying of cancer immunoprevention. Study Design: Wild-type C57BL/6 mice and various transgenic (CD4+ OT-II.Rag+/+, CD4+ OT-II.Rag−/−, CD8+ OT-I/Rag2−/- KO, and Rag1−/−) mice on C57BL/6 background were pretreated subcutaneously with SA-4-1BBL or an agonistic Ab that does not have immunoprevention efficacy (3H3) twice two weeks apart. Mice were challenged two weeks later with a cervical cancer cell line (TC-1) and monitored for tumor development. A group of mice was also treated with monophosphoryl lipid A (MPLA), an agonist of TLR4 involved in trained immunity, using the above scheme. Mice treated with saline served as the control throughout all studies. Deep immunophenotyping was conducted to assess immune correlates of cancer immunoprevention. Results: Pretreatment with SA-4-1BBL prevented tumor growth in wild-type mice (> 60%), whereas all MPLA treated mice developed tumor. SA-4-1BBL did not show tumor immunoprevention efficacy in mice lacking adaptive immune cells (Rag1−/- KO), OT-II cells on Rag−/- or Rag+/+ background, and OT-I on Rag2−/− background. Deep immunophenotyping revealed a significant increase in the absolute number of CD4+ T central memory and CD4+ T stem cell-like memory cells, NK cells, NKT cells, and various myeloid cells in mice treated with SA-4-1BBL as compared with those treated with the 3H3 Ab or saline treated controls. Conclusions: These results demonstrate that cancer immunoprevention efficacy of SA-4-1BBL requires a diverse T cell receptor repertoire as OT-II mice on Rag+/+ background, restricted to a TCRβ chain, but have all other myeloid and NK cells, do not show efficacy. Furthermore, treatment with MPLA involved in trained immunity has no efficacy. Altogether, these observations do not implicate trained immunity as a mechanism of cancer immunoprevention. Elucidation of the mechanistic basis of SA-4-1BBL immunoprevention may have significant clinical implications. Citation Format: Feyza Nur Arguc, Mohammad Tarique, Vahap Ulker, Esma S. Yolcu, Haval Shirwan. A novel agonist of CD137 as a single agent shows cancer immunoprevention efficacy against various tumor types that is not dependent on the trained immunity [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 2465.