Small-molecule Immunomodulators Research Articles

CD40 agonist engineered immunosomes modulated tumor microenvironment and showed pro-immunogenic response, reduced toxicity, and tumor free survival in mice bearing glioblastoma

CD40 agonist antibodies (αCD40) have shown promising anti-tumor response in both preclinical and early clinical studies. However, its systemic administration is associated with immune- and hepato-toxicities which hampers its clinical usage. In addition, αCD40 showed low tumor retention and induced PD-L1 expression which makes tumor microenvironment (TME) immunosuppressive. To overcome these issues, in this study, we have developed a multifunctional Immunosome where αCD40 is conjugated on the surface and RRX-001, a small molecule immunomodulator was encapsulated inside it. Immunosomes showed higher tumor accumulation till 96 h of administration and displayed sustained release of αCD40 in vivo. Immunosomes significantly delayed tumor growth and showed tumor free survival in mice bearing GL-261 glioblastoma by increasing the population of CD45+CD8+ T cells, CD45+CD20+ B cells, CD45+CD11c+ DCs and F4/80+CD86+ cells in TME. Immunosome significantly reduced the population of T-regulatory cells, M2 macrophage, and MDSCs and lowered the PD-L1 expression. Moreover, Immunosomes significantly enhanced the levels of Th1 cytokines (IFN-γ, IL-6, IL-2) over Th2 cytokines (IL-4 and IL-10) which supported anti-tumor response. Most interestingly, Immunosomes averted the in vivo toxicities associated with free αCD40 by lowering the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), IL-6, IL-1α and reduced the degree of liver damage. In addition, Immunosomes treated long-term surviving mice showed tumor specific immune memory response which prevented tumor growth upon rechallenge. Our results suggested that this novel formulation can be further explored in clinics to improve in vivo anti-tumor efficacy of αCD40 with long-lasting tumor specific immunity while reducing the associated toxicities.

Small Molecule Immunomodulators as Next-Generation Therapeutics for Glioblastoma.

Glioblastoma (GBM), the most aggressive astrocytic glioma, remains a therapeutic challenge despite multimodal approaches. Immunotherapy holds promise, but its efficacy is hindered by the highly immunosuppressive GBM microenvironment. This review underscores the urgent need to comprehend the intricate interactions between glioma and immune cells, shaping the immunosuppressive tumor microenvironment (TME) in GBM. Immunotherapeutic advancements have shown limited success, prompting exploration of immunomodulatory approaches targeting tumor-associated macrophages (TAMs) and microglia, constituting a substantial portion of the GBM TME. Converting protumor M2-like TAMs to antitumor M1-like phenotypes emerges as a potential therapeutic strategy for GBM. The blood-brain barrier (BBB) poses an additional challenge to successful immunotherapy, restricting drug delivery to GBM TME. Research efforts to enhance BBB permeability have mainly focused on small molecules, which can traverse the BBB more effectively than biologics. Despite over 200 clinical trials for GBM, studies on small molecule immunomodulators within the GBM TME are scarce. Developing small molecules with optimal brain penetration and selectivity against immunomodulatory pathways presents a promising avenue for combination therapies in GBM. This comprehensive review discusses various immunomodulatory pathways in GBM progression with a focus on immune checkpoints and TAM-related targets. The exploration of such molecules, with the capacity to selectively target key immunomodulatory pathways and penetrate the BBB, holds the key to unlocking new combination therapy approaches for GBM.

Small Molecule Targeting Immune Cells: A Novel Approach for Cancer Treatment.

Conventional and cancer immunotherapies encompass diverse strategies to address various cancer types and stages. However, combining these approaches often encounters limitations such as non-specific targeting, resistance development, and high toxicity, leading to suboptimal outcomes in many cancers. The tumor microenvironment (TME) is orchestrated by intricate interactions between immune and non-immune cells dictating tumor progression. An innovative avenue in cancer therapy involves leveraging small molecules to influence a spectrum of resistant cell populations within the TME. Recent discoveries have unveiled a phenotypically diverse cohort of innate-like T (ILT) cells and tumor hybrid cells (HCs) exhibiting novel characteristics, including augmented proliferation, migration, resistance to exhaustion, evasion of immunosurveillance, reduced apoptosis, drug resistance, and heightened metastasis frequency. Leveraging small-molecule immunomodulators to target these immune players presents an exciting frontier in developing novel tumor immunotherapies. Moreover, combining small molecule modulators with immunotherapy can synergistically enhance the inhibitory impact on tumor progression by empowering the immune system to meticulously fine-tune responses within the TME, bolstering its capacity to recognize and eliminate cancer cells. This review outlines strategies involving small molecules that modify immune cells within the TME, potentially revolutionizing therapeutic interventions and enhancing the anti-tumor response.

Dissecting regulatory T cell expansion using polymer microparticles presenting defined ratios of self-antigen and regulatory cues.

Biomaterials allow for the precision control over the combination and release of cargo needed to engineer cell outcomes. These capabilities are particularly attractive as new candidate therapies to treat autoimmune diseases, conditions where dysfunctional immune cells create pathogenic tissue environments during attack of self-molecules termed self-antigens. Here we extend past studies showing combinations of a small molecule immunomodulator co-delivered with self-antigen induces antigen-specific regulatory T cells. In particular, we sought to elucidate how different ratios of these components loaded in degradable polymer particles shape the antigen presenting cell (APC) -T cell interactions that drive differentiation of T cells toward either inflammatory or regulatory phenotypes. Using rapamycin (rapa) as a modulatory cue and myelin self-peptide (myelin oligodendrocyte glycoprotein- MOG) - self-antigen attacked during multiple sclerosis (MS), we integrate these components into polymer particles over a range of ratios and concentrations without altering the physicochemical properties of the particles. Using primary cell co-cultures, we show that while all ratios of rapa:MOG significantly decreased expression of co-stimulation molecules on dendritic cells (DCs), these levels were insensitive to the specific ratio. During co-culture with primary T cell receptor transgenic T cells, we demonstrate that the ratio of rapa:MOG controls the expansion and differentiation of these cells. In particular, at shorter time points, higher ratios induce regulatory T cells most efficiently, while at longer time points the processes are not sensitive to the specific ratio. We also found corresponding changes in gene expression and inflammatory cytokine secretion during these times. The in vitro results in this study contribute to in vitro regulatory T cell expansion techniques, as well as provide insight into future studies to explore other modulatory effects of rapa such as induction of maintenance or survival cues.

Photothermal enhanced polyphenol-based nanofibers ameliorate catalytic efficiency of ferroptosis for synergistic tumor therapy

Ferroptosis is an emerging antitumor treatment modality with the superiority for evading apoptotic cell death pathway. However, how to elevate catalytic efficacy of Fe2+-mediated Fenton reaction and efficiently elicit ferroptosis remain enormously challenging. Herein, inspired by hyperthermia-enhanced Fenton reaction kinetics, we firstly designed iron-polyphenol-aspirin-coordinated nanochelates for photothermal-enhanced ferroptosis antitumor immunotherapy. Specifically, we modulated optimal mass feeding ratio of gallic acid (GA), aspirin (ASA), Fe (II) and polyvinylpyrrolidone (PVP) to construct novel nanofibrous GA-ASA-Fe (II) metalchelates named GAFs. The variations in size and structure allowed the nanomedicines to avoid the risk of premature renal clearance in vivo, compared with the reported ultrasmall GA-Fe (II) nanocomplexes (GFs). Under NIR laser irradiation, GAFs could constantly amplify toxic hydroxyl radicals (OH) generation and deplete excessive GSH to induce more accumulation of lethal lipid peroxidation (LPO), thereby triggering ferroptosis pathway in vitro and in vivo. Besides, the introduction of ASA could inhibit cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2), in combination with photothermal-enhanced ferroptosis tumor therapy to induce immunogenic cell death (ICD), promote maturation of dendritic cells (DCs) and activate cytotoxic T cells for synergistic antitumor immunotherapy. GAFs with laser irradiation exhibited the capacity of inhibition of pulmonary metastasis. This work presented a strategy for incorporating small molecule immunomodulator into the metal-polyphenolic coordination to ameliorate its deficiencies, thereby inspiring a new design concept for tumor treatment.

Synthesis and immunomodulatory activity of novel amino acid analogues of fluoxetine

Immunomodulators are assigned as a robust tool for the treatment of various medical conditions like AIDS, cancer and autoimmune diseases. In this work, we have synthesized a series of fluoxetine-based small-molecule immunomodulators. Molecules were synthesized by a convergent strategy, where mitsunobu reaction was conducted to synthesize the common starting material. The novel analogues displayed immunomodulatory potential as indicated by the anti-inflammatory effect derived through the compound’s T cell proliferation inhibitory property. The synthesized novel analogues manifested encouraging biological responses, assure their application as immunomodulators.

Data-driven discovery of innate immunomodulators via machine learning-guided high throughput screening.

The innate immune response is vital for the success of prophylactic vaccines and immunotherapies. Control of signaling in innate immune pathways can improve prophylactic vaccines by inhibiting unfavorable systemic inflammation and immunotherapies by enhancing immune stimulation. In this work, we developed a machine learning-enabled active learning pipeline to guide in vitro experimental screening and discovery of small molecule immunomodulators that improve immune responses by altering the signaling activity of innate immune responses stimulated by traditional pattern recognition receptor agonists. Molecules were tested by in vitro high throughput screening (HTS) where we measured modulation of the nuclear factor κ-light-chain-enhancer of activated B-cells (NF-κB) and the interferon regulatory factors (IRF) pathways. These data were used to train data-driven predictive models linking molecular structure to modulation of the NF-κB and IRF responses using deep representational learning, Gaussian process regression, and Bayesian optimization. By interleaving successive rounds of model training and in vitro HTS, we performed an active learning-guided traversal of a 139 998 molecule library. After sampling only ∼2% of the library, we discovered viable molecules with unprecedented immunomodulatory capacity, including those capable of suppressing NF-κB activity by up to 15-fold, elevating NF-κB activity by up to 5-fold, and elevating IRF activity by up to 6-fold. We extracted chemical design rules identifying particular chemical fragments as principal drivers of specific immunomodulation behaviors. We validated the immunomodulatory effect of a subset of our top candidates by measuring cytokine release profiles. Of these, one molecule induced a 3-fold enhancement in IFN-β production when delivered with a cyclic di-nucleotide stimulator of interferon genes (STING) agonist. In sum, our machine learning-enabled screening approach presents an efficient immunomodulator discovery pipeline that has furnished a library of novel small molecules with a strong capacity to enhance or suppress innate immune signaling pathways to shape and improve prophylactic vaccination and immunotherapies.

Photothermal immunotherapy of melanoma using TLR-7 agonist laden tobacco mosaic virus with polydopamine coat

Photothermal therapy (PTT) is a promising cancer treatment that debulks tumors locally while priming immune responses. However, PTT as a standalone treatment approach often has limited systemic efficacy, motivating the development of synergistic combination approaches. Toward this goal, herein, the tobacco mosaic virus (TMV) was loaded with a small molecule immunomodulator, toll-like receptor 7 agonist (1V209), and its surface was coated with photothermal biopolymer polydopamine (PDA). The resulting 1V209-laden and PDA-coated TMV was used to treat B16F10 dermal melanoma in C57BL/6 mice. 1V209-TMV-PDA was intratumorally injected and irradiated using an 808-nm near infrared laser. 60 % of the mice receiving PTT with intratumoral 1V209-TMV-PDA + laser remained alive at the end point – in contrast to only 20 % survivors were observed in the control group. Immunological analysis indicates systemic anti-tumor immunity being induced by the combination therapy with a greater number of tumor-specific T cells (as determined by a splenocyte assay). This study highlights the potential of TMV versatility as a multifunctional nano-platform for combined PTT-immunotherapy.

Biomimetic Nanocarriers Guide Extracellular ATP Homeostasis to Remodel Energy Metabolism for Activating Innate and Adaptive Immunity System.

Metabolic interventions via targeting intratumoral dysregulated metabolism pathways have shown promise in reinvigorating antitumor immunity. However, approved small molecule immunomodulators often suffer from ineffective response rates and severe off‐target toxicity. ATP occupies a crucial role in energy metabolism of components that form the tumor microenvironment (TME) and influences cancer immunosurveillance. Here, a nanocarrier‐assisted immunometabolic therapy strategy that targets the ATP‐adenosine axis for metabolic reprogramming of TME is reported. An ecto‐enzyme (CD39) antagonist POM1 and AMP‐activated protein kinase (AMPK) agonist metformin are both encapsulated into cancer cell‐derived exosomes and used as nanocarriers for tumor targeting delivery. This method increases the level of pro‐inflammatory extracellular ATP (eATP) while preventing the accumulation of immunosuppressive adenosine and alleviating hypoxia. Elevated eATP triggers the activation of P2X7‐NLRP3‐inflammasome to drive macrophage pyroptosis, potentiates the maturation and antigen capacity of dendritic cells (DCs) to enhance the cytotoxic function of T cells and natural killer (NK) cells. As a result, synergistic antitumor immune responses are initiated to suppress tumor progress, inhibit tumor distant metastases, provide long‐term immune memory that offers protection against tumor recurrence and overcome anti‐PD1 resistance. Overall, this study provides an innovative strategy to advance eATP‐driven antitumor immunity in cancer therapy.

Targeted delivery of a STING agonist to brain tumors using bioengineered protein nanoparticles for enhanced immunotherapy

Immunotherapy is emerging as a powerful tool for combating many human diseases. However, the application of this life-saving treatment in serious brain diseases, including glioma, is greatly restricted. The major obstacle is the lack of effective technologies for transporting therapeutic agents across the blood-brain barrier (BBB) and achieving targeted delivery to specific cells once across the BBB. Ferritin, an iron storage protein, traverses the BBB via receptor-mediated transcytosis by binding to transferrin receptor 1 (TfR1) overexpressed on BBB endothelial cells. Here, we developed bioengineered ferritin nanoparticles as drug delivery carriers that enable the targeted delivery of a small-molecule immunomodulator to achieve enhanced immunotherapeutic efficacy in an orthotopic glioma-bearing mouse model. We fused different glioma-targeting moieties on self-assembled ferritin nanoparticles via genetic engineering, and RGE fusion protein nanoparticles (RGE-HFn NPs) were identified as the best candidate. Furthermore, RGE-HFn NPs encapsulating a stimulator of interferon genes (STING) agonist (SR717@RGE-HFn NPs) maintained stable self-assembled structure and targeting properties even after traversing the BBB. In the glioma-bearing mouse model, SR717@RGE-HFn NPs elicited a potent local innate immune response in the tumor microenvironment, resulting in significant tumor growth inhibition and prolonged survival. Overall, this biomimetic brain delivery platform offers new opportunities to overcome the BBB and provides a promising approach for brain drug delivery and immunotherapy in patients with glioma.

The Landscape of COVID-19 Research in the United States: a Cross-sectional Study of Randomized Trials Registered on ClinicalTrials.Gov.

ImportanceSARS-CoV-2 has infected over 200 million people worldwide, resulting in more than 4 million deaths. Randomized controlled trials are the single best tool to identify effective treatments against this novel pathogen.ObjectiveTo describe the characteristics of randomized controlled trials of treatments for COVID-19 in the United States launched in the first 9 months of the pandemic.Design, Setting, and ParticipantsWe conducted a cross-sectional study of all completed or actively enrolling randomized, interventional, clinical trials for the treatment of COVID-19 in the United States registered on www.clinicaltrials.gov as of August 10, 2020. We excluded trials of vaccines and other interventions intended to prevent COVID-19.Main Outcomes and MeasuresWe used descriptive statistics to characterize the clinical trials and the statistical power for the available studies. For the late-phase trials (i.e., phase 3 and 2/3 studies), we compared the geographic distribution of the clinical trials with the geographic distribution of people diagnosed with COVID-19.ResultsWe identified 200 randomized controlled trials of treatments for people with COVID-19. Across all trials, 87 (43.5%) were single-center, 64 (32.0%) were unblinded, and 80 (40.0%) were sponsored by industry. The most common treatments included monoclonal antibodies (N=46 trials), small molecule immunomodulators (N=28), antiviral medications (N=24 trials), and hydroxychloroquine (N=20 trials). Of the 9 trials completed by August 2020, the median sample size was 450 (IQR 67–1113); of the 191 ongoing trials, the median planned sample size was 150 (IQR 60–400). Of the late-phase trials (N=54), the most common primary outcome was a severity scale (N=23, 42.6%), followed by a composite of mortality and ventilation (N=10, 18.5%), and mortality alone (N=6, 11.1%). Among these late-phase trials, all trials of antivirals, monoclonal antibodies, or chloroquine/hydroxychloroquine had a power of less than 25% to detect a 20% relative risk reduction in mortality. Had the individual trials for a given class of treatments instead formed a single trial, the power to detect that same reduction in mortality would have been greater than 98%. There was large variability in access to trials with the highest number of trials per capita in the Northeast and the lowest in the Midwest.Conclusions and RelevanceA large number of randomized trials were launched early in the pandemic to evaluate treatments for COVID-19. However, many trials were underpowered for important clinical endpoints and substantial geographic disparities were observed, highlighting the importance of improving national clinical trial infrastructure.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11606-021-07167-9.

In silico screening and molecular dynamic simulation studies of potential small molecule immuno-modulators of the KIR2DS2 receptor

Cancer is reported to be one of the most common causes of global deaths and it accounted for an estimated 9.6 million fatalities in 2018. Immunotherapy destroys cancer cells with mechanisms of action considered to be safer and with greater precision than chemotherapy or radiotherapy. The killer cell immunoglobulin-like receptor 2DS2 (KIR2DS2) expressed on Natural Killer (NK) cells are involved in signal transduction processes that produce pro-inflammatory cytokines and directly destroy cancer and virally infected cells. The aim of this study is to identify small molecules from natural products that have strong binding affinity to KIR2DS2 and to predict possible nuclear receptor ligand bioactivity. A library of small molecule natural compounds obtained from edible African plants was used for in silico molecular docking simulations of the human natural killer cell activator receptor KIR2DS2 (PDBID: 1m4k) using PyRx. Only compounds with binding affinity stronger than that elicited by the target and the standard, N-acetyl-D-glucosamine were selected. Screening for drug-likeness and ligand efficiency was based on the molecular descriptors of the compounds as provided by PubChem. Further screening for saturation, molar refractivity, promiscuity, pharmacokinetic properties, and bioactivity was done using SWISSADME, PKCSM, and Molinspiration webservers respectively. The molecular dynamic simulation and analyses was done using the Galaxy webserver which uses the GROMACS software. Analyses of molecular dynamic simulation were done using Galaxy and MDWEB webservers. Gibberellin A20 and A29 were obtained as the lead compounds, and they show better promise as drug candidates for KIR2DS2 than the standard. It is recommended that the immuno-stimulatory effect of the lead compounds on KIR2DS2 be further investigated.

Second Near-Infrared Light-Activatable Polymeric Nanoantagonist for Photothermal Immunometabolic Cancer Therapy.

Immunometabolic modulation offers new opportunities to treat cancers as it is highly associated with cancer progression and immunosuppressive microenvironment. However, traditional regimens using nonselective small-molecule immunomodulators lead to the off-target adverse effects and insufficient therapeutic outcomes. Herein a second near-infrared (NIR-II) photothermally activatable semiconducting polymeric nanoantagonist (ASPA) for synergistic photothermal immunometabolic therapy of cancer is reported. ASPA backbone is obtained by conjugating vipadenant, an antagonist to adenosine A2A receptor, onto NIR-II light-absorbing semiconducting polymer via an azo-based thermolabile linker. Under deep-penetrating NIR-II photoirradiation, ASPA induces tumor thermal ablation and subsequently immunogenic cell death, triggers the cleavage of thermolabile linker, and releases the antagonist to block the immunosuppressive adenosinergic pathway. Such a remotely controlled immunometabolic regulation potentiates cytotoxic T cell functions while suppresses regulatory T cell activities, leading to efficient primary tumor inhibition, pulmonary metastasis prevention, and long-term immunological memory. Thereby, this work provides a generic polymeric approach for precise spatiotemporal regulation of cancer immunometabolism.

Abstract 1720: Ax-101, an immunostimulatory small molecule, markedly enhances the antitumor activity of PD1 immune checkpoint blockade in multiple preclinical solid tumor models

Abstract Ax-101, a small-molecule immunomodulator with a yet-to-be elucidated MoA, enhances lymphocyte proliferation, induces a Th1 profile, increases T-cell cytotoxicity, and improves the M1/M2 ratio. Specific Ax-101 effects include upregulated cell-surface TNFalpha (sTNF) on human T cells and monocytes, increased (>7.5x) T-cell cytotoxicity, upregulation by human PBMC of antitumor cytokines (e.g., IL-2, IFN-gamma, TNFalpha) and downregulation of cytokines that mediate immune evasion (e.g., IL-10), and decreased immune-suppressive myeloid cells. Syngeneic mouse cancer models including bladder (MBT-2), colorectal (CT-26), melanoma (Cloudman), and lymphoma (A20) show the addition of Ax-101 to anti-PD1 to improve outcomes. For example, neither Ax-101 nor anti-PD1 monotherapy had significant effects on mean tumor volume (MTV) in MBT-2 mice, while an Ax-101/anti-PD1 (same doses/regimen) combo resulted in up to 44% reduction in MTV c/w anti-PD1 alone (p < 0.05). Further, 30% (3/10) of MBT-2 mice achieved CR with the combo, whereas no CRs were seen with anti-PD1 alone. In CT-26 mice, 25% (2/8) achieved CR, whereas no CRs occurred with anti-PD1 only; subset analysis of mice exhibiting at least some antitumor response to anti-PD1 alone (6/8) or the Ax-101/anti-PD1 combo (5/8) revealed a 40% CR rate and MTV reduction of up to 75% in combo-treated animals. In Cloudman melanomas, all (5/5) mice receiving Ax-101/anti-PD1 throughout their treatment achieved CR. Importantly, in mice with rapidly growing melanomas refractory to anti-PD1 for 12 d, Ax-101 addition reversed growth, with 4/5 mice achieving CR. Of note, 9 Cloudman mice achieving CR were re-injected with 3x the melanoma cells they initially received and followed with no therapy. Only 1/9 mice developed tumor over 73 d; thus, Ax-101/anti-PD1 may elicit a vaccine-like effect. Anti-PD1 alone and the Ax-101/antiPD1 combo in A20 mice resulted in 23% and 40% lower MTVs c/w control. Ax-101 was previously in Ph 1/2 monotherapy studies in follicular lymphoma (FL) and myeloma (M) (CT.gov IDs: NCT00006466, NCT00007839). Thirty-two patients received 2 µg SQ Ax-101 q2 wkly or wkly for up to 18 months. No Grade 3 or higher toxicities were observed. Five of 14 FL patients showed tumor reduction from 16-64%, while 1/17 M patients showed 50% reduced M-protein; maximum responses occurred between 43 days and over 1 year. Increased PBMC sTNF was seen in all patients tested (8 with FL, 6 with M) (p = 0.0006). Consistent with Ax-101 activity depending on a functional immune system, delayed type hypersensitivity (DTH) testing showed all responders to be DTH+ (5 tumor reduction, 4 SD); 5 anergic (DTH-) patients had SD at best (2 SD, 3 worse). Current Ax-101 clinical development will leverage its beneficial immunostimulatory properties to augment the efficacy of anti-PD1 inhibitors such as pembrolizumab, nivolumab and cemiplimab. Citation Format: Stephan W. Morris, Betsy Fisher, Kent Murphy. Ax-101, an immunostimulatory small molecule, markedly enhances the antitumor activity of PD1 immune checkpoint blockade in multiple preclinical solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1720.

Covalent Small Molecule Immunomodulators Targeting the Protease Active Site.

Cells of the immune system utilize multiple proteases to regulate cell functions and orchestrate innate and adaptive immune responses. Dysregulated protease activities are implicated in many immune-related disorders; thus, protease inhibitors have been actively investigated for pharmaceutical development. Although historically considered challenging with concerns about toxicity, compounds that covalently modify the protease active site represent an important class of agents, emerging not only as chemical probes but also as approved drugs. Here, we provide an overview of technologies useful for the study of proteases with the focus on recent advances in chemoproteomic methods and screening platforms. By highlighting covalent inhibitors that have been designed to target immunomodulatory proteases, we identify opportunities for the development of small molecule immunomodulators.

Novel Selective IDO1 Inhibitors with Isoxazolo[5,4-d]pyrimidin-4(5H)-one Scaffold.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a promising target in immunomodulation of several pathological conditions, especially cancers. Here we present the synthesis of a series of IDO1 inhibitors with the novel isoxazolo[5,4-d]pyrimidin-4(5H)-one scaffold. A focused library was prepared using a 6- or 7-step synthetic procedure to allow a systematic investigation of the structure-activity relationships of the described scaffold. Chemistry-driven modifications lead us to the discovery of our best-in-class inhibitors possessing p-trifluoromethyl (23), p-cyclohexyl (32), or p-methoxycarbonyl (20, 39) substituted aniline moieties with IC50 values in the low micromolar range. In addition to hIDO1, compounds were tested for their inhibition of indoleamine 2,3-dioxygenase 2 and tryptophan dioxygenase, and found to be selective for hIDO1. Our results thus demonstrate a successful study on IDO1-selective isoxazolo[5,4-d]pyrimidin-4(5H)-one inhibitors, defining promising chemical probes with a novel scaffold for further development of potent small-molecule immunomodulators.

Impact of Excipients on Stability of Polymer Microparticles for Autoimmune Therapy.

Therapies for autoimmune diseases such as multiple sclerosis and diabetes are not curative and cause significant challenges for patients. These include frequent, continued treatments required throughout the lifetime of the patient, as well as increased vulnerability to infection due to the non-specific action of therapies. Biomaterials have enabled progress in antigen-specific immunotherapies as carriers and delivery vehicles for immunomodulatory cargo. However, most of this work is in the preclinical stage, where small dosing requirements allow for on-demand preparation of immunotherapies. For clinical translation of these potential immunotherapies, manufacturing, preservation, storage, and stability are critical parameters that require greater attention. Here, we tested the stabilizing effects of excipients on the lyophilization of polymeric microparticles (MPs) designed for autoimmune therapy; these MPs are loaded with peptide self-antigen and a small molecule immunomodulator. We synthesized and lyophilized particles with three clinically relevant excipients: mannitol, trehalose, and sucrose. The biophysical properties of the formulations were assessed as a function of excipient formulation and stage of addition, then formulations were evaluated in primary immune cell culture. From a manufacturing perspective, excipients improved caking of lyophilized product, enabled more complete resuspension, increased product recovery, and led to smaller changes in MP size and size distribution over time. Cocultures of antigen-presenting cells and self-reactive T cells revealed that MPs lyophilized with excipients maintained tolerance-inducing function, even after significant storage times without refrigeration. These data demonstrate that excipients can be selected to drive favorable manufacturing properties without impacting the immunologic properties of the tolerogenic MPs.

A Potent Inhibitor of IL-10/STAT3 Axis Signaling Modulates Anti-Inflammatory Responses and Boosts Anti-Tuberculosis Immunity in rBCG30 Immunized Mice

Aims and Objectives: In the present study we tried to improve anti-TB immunity offered by 2nd generation rBCG30 vaccine. We attempted to bolster rBCG30 invoked anti-TB immunity in mice through modulation of the IL-10/STAT3 interaction mediated anti-inflammatory program. The study was aimed towards generating optimum activated APCs, early either after vaccination (with rBCG30) or infection (with Mtb), which can process and present mycobacterial antigens more efficiently to T cells. It was envisage that downmodulating IL-10/STAT3 signaling may improve immunity and protection offered by rBCG30 vaccine. Methods: A small molecule immunomodulator aimed at disrupting anti-inflammatory signaling orchestrated by IL-10/STAT3 axis was administered in rBCG30 immunized mice. The comparative expansion of pathogen permissive Alternatively Activated Monocytes/Macrophages/DCs (AAMs/AADCs) and Classically Activated Monocytes/Macrophages/DCs (CAMs/CADCs), the cells that resist establishment of successful Mtb infection, was profiled in both peritoneal and splenic compartment. To assess innate and adaptive T cell responses, splenocytes cytokine production was checked ex vivo using sandwich ELISA. We also checked polyfunctional T cell response, T cell memory response and comparative Th17/Treg cells expansion using flow cytometry. Finally, to assess the real time efficacy of the employed immunotherapeutic strategy, in vivo protection study was undertaken in BALB/c mice challenge with aerosolized Mtb (H37 Rv). Results: The small molecule mediated inhibition of IL-10/STAT3 signaling was resulted in improved pro inflammatory immune responses in both post-vaccination and post-infection immunotherapy schedules. The improved immune profile in rBCG30 vaccinated and immunotherapy administered mice was accompanied with enhanced expansion of CAMs/CADCs, elevated production of polyfunctional T cells producing signature Th1 cytokines, long lasting CD4+ and CD8+ memory T cell response and balanced Th17/Treg cells ratio. Interestingly, post-infection, and not post-vaccination immunotherapy, was able to significantly reduce mycobacterial loads in spleen and lungs of experimentally infected mice. Conclusions: Small molecule mediated transient disruption of anti-inflammatory effectors, such as IL-10/STAT3, during vaccination or post infection with Mtb, may serve to enhance the efficacy of existing and forthcoming TB vaccines. The strategy can also be tested concomitantly with standard chemotherapy (either in vaccinated or unvaccinated host) to enhance the efficacy of therapeutic regimen and to reduce the treatment length.

Abstract 5327: KPG-121, a novel CRBN modulator, potently inhibits growth of metastatic castration resistant prostate cancer as a single agent or in combination with androgen receptor signaling inhibitors both in vitro and in vivo

Abstract Prostate cancer is the common cancer in men and the second leading cause of cancer-related deaths in men. This disease depends on dihydrotestosterone (DHT) which activates the androgen receptor (AR) signal. Endocrine therapy (including castration surgery, anti-androgen deprivation therapy or combined androgen blockade therapy) is a preferred treatment for prostate cancer. The treatment is very effective for the majority of patients at the initial stage, but after a median period of treatment for 14 to 30 months, most patients will gradually develop metastatic castration resistant prostate cancer (mCRPC) with a median survival of less than 20 months, and urgently need a further treatment. Androgen receptor antagonists in combination with anti-androgen drugs are currently the most effective drug treatment to manage mCRPC where castration has failed. Enzalutamide, Abiraterone Acetate, Apalutamide and Darolutamide belong to this class of androgen receptor or androgen synthesis inhibitors. Here we show that KPG-121, a small molecule immunomodulator, significantly enhances the therapeutic effects of Enzalutamide, Abiraterone Acetate, Apalutamide and Darolutamide in pre-clinical mCRPC tumor models. The phase 1 trials were initiated to evaluate the safety, PK profile and preliminary efficacy of the combinatorial therapy in the patients with metastatic castration resistant prostate cancer in the US (IND 134979). KPG-121 is a novel generation of Lenalidomide analogue. KPG-121 is structurally similar to lenalidomide but with stronger binding with CRBN target, enhanced immunomodulatory activity, better anti-proliferative and anti-angiogenic properties compared to lenalidomide. Results from in vivo metastatic castration resistant prostate cancer xenograft models demonstrated that KPG-121 at tested daily dose levels ranging from 1.0 mg/kg to 10 mg/kg in combination with Enzalutamide, Abiraterone Acetate, Apalutamide or Darolutamide significantly improved anti-tumor efficacy compared to the AR signaling inhibitors alone. In addition, KPG-121 showed favorable DMPK profiles and potentially manageable toxicity in pre-clinical IND enabling study. Citation Format: Chuansheng Ge, Lei Zhang, Baisong Liao. KPG-121, a novel CRBN modulator, potently inhibits growth of metastatic castration resistant prostate cancer as a single agent or in combination with androgen receptor signaling inhibitors both in vitro and in vivo [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5327.

Human osteoarthritic joint explant model reflects diverse disease-relevant changes in inflammation and degradation

Purpose: Immune cell-mediated inflammation has been implicated in the pathogenesis and progression of osteoarthritis (OA). However, our current understanding of the phenotype and interactions between joint resident and infiltrating immune cells is not comprehensive enough to appropriately guide the design of new inflammation-targeting OA therapeutics. We have developed a human OA joint explant model containing osteoarthritic synovium and cartilage that more accurately represents the human immune condition. The explants contain joint cell types within their endogenous matrix, including resident synovial macrophages and fibroblasts that are drivers of inflammation and fibrosis. The purpose of this study is to optimize a human OA joint explant model to be representative of the human condition by maintaining in vivo morphology, viability, and responsiveness to inflammation-mediated changes in gene expression and extracellular matrix (ECM) dynamics. Methods: Cartilage and synovium are obtained from late-stage OA knee replacement. Full-depth 1.5 mm diameter punch-cut cartilage and minced (fat removed) synovial tissue are randomly distributed by wet weight into 24-wells or transwells inserts for 48 hours before co-culture. Pro-inflammatory cytokine conditions are used as positive controls (oncostatin M + interleukin 1B). Treatment conditions or other cell types can be co-cultured with the joint explants for 2-day or 7-day time points. Explant conditions are evaluated by cartilage, synovium, and conditioned medium harvest: viability (resazurin), tissue gene expression (RT-qPCR), secreted cytokine/protease (immunoassay) and proteoglycan (GAG) content (dimethylmethylene blue), and morphological (H&E) and ECM (Safranin-O) histology. Synovium tissue is digested for flow cytometry of fibroblast and polarized monocyte/macrophage surface markers. Conditions are compared by principal component analysis for gene panels and analysis of variance for single dependent variable assays. Results: 48-hour cartilage gene expression demonstrates distinct clusters between the pro-inflammatory cytokine control and cartilage alone control conditions within a 26-gene panel capturing catabolic, anabolic, and inflammatory/chemotactic genes (N=12; ΔΔCT values relative to cartilage alone control in PCA plot). The pro-inflammatory control upregulates genes such as IL6, CCL2, MMP1, MMP13, ADAMTS4 while downregulating ACAN, PRG4, COL2A1, TIMP1. The synovium-cartilage co-culture cluster overlaps with the control group, but a subset of this condition is shifted towards the pro-inflammatory control cluster. GAG loss into the medium was significantly increased with the pro-inflammatory positive control (N=18, P<0.0001). The mean GAG loss is higher in the synovium-cartilage co-culture but is not statistically significant. These changes are observable by GAG staining of histological sections. Synovium digest reveals presence of CD90+ fibroblasts and CD45+CD14+ monocytes/macrophage before and after explant co-culture. Conclusions: Our human OA joint explant model is viable and demonstrates changes relevant to inflammation and cartilage degradation in response to pro-inflammatory insult. Co-culture of synovium and cartilage as our baseline OA joint condition shows a shift towards the pro-inflammatory response. Increasing the sample size will allow us to annotate patient subsets based on their clinical outcomes to identify subsets with different levels of synovium-derived inflammatory involvement in their disease, and therefore understand the scope of our model. We position our explant model as a useful complement to in vivo animal models in understanding immune contribution to OA progression and for developing new OA treatment strategies ranging from small molecule immunomodulators to cell-based therapies.