Immune-modulating Activities Research Articles

Abstract PD10-07: Chemokine12 (CK12) tertiary lymphoid gene expression signature as a predictor of response in 3 immunotherapy arms of the neoadjuvant ISPY 2 TRIAL - pembrolizumab with and without SD101, and durvalumab combined with olaparib - and in 9 other arms of the trial including platinum-based and dual-anti-HER2 therapies

Abstract Background: The CK12 expression signature consists of genes CCL2, CCL3, CCL4, CCL5, CCL8, CCL18, CCL19, CCL21, CXCL9, CXCL10, CXCL11, CXCL13 and was previously shown to associate with the presence of T and B cell rich tertiary lymphoid structures in melanoma and other cancers, and with better patient survival independent of tumor staging and treatment. I-SPY 2 is a biomarker-rich, phase II neoadjuvant platform trial for high risk early stage breast cancer. Here we leverage the I-SPY 2 biomarker program to test the hypothesis that this signature associates with sensitivity to neoadjuvant immunotherapies and potentially other classes cancer therapeutics in breast cancer. Methods: Data from 1130 patients across 12 arms of I-SPY2 (control (ctr): 210; veliparib/carboplatin (VC): 71; neratinib (N): 114; MK2206: 93; ganitumab: 106; ganetespib: 93; AMG386: 134; TDM1/pertuzumab(P): 52; H/P: 44; pembrolizumab (pembro): 69; durvalumab/olaparib (durva/olap): 71; pembro/SD101: 72) were available for analysis. Pre-treatment FF (n=987) or FFPE (n=143) biopsies were assayed using Agilent gene expression arrays. Signature scores were calculated as the average expression level across the 12 genes, after z-score normalization. We used logistic modeling to assess association with pCR in each arm in a model adjusting for HR and HER2 (likelihood ratio test, p<0.05). This analysis was also performed within HR/HER2 receptor subsets, numbers permitting. We also assessed differences in levels across HR/HER2 subsets using ANOVA and Tukey post-hoc testing. Our statistics are descriptive rather than inferential and do not adjust for multiplicities of other biomarkers outside this study. Results: CK12 levels associate with HR/HER2 status (ANOVA p=1.07E-14), with higher levels in TN and HR-HER2+ subsets and lower levels in HR+ groups. Overall, patients with higher levels of CK12 were significantly more likely to achieve pCR in all 3 IO arms: pembro (OR=3.4/1SD), pembro/SD101 (OR=4/1SD), and durva/olaparib (OR=2.5/1SD) (LR p<0.05), in a model adjusting for HR status. The CK12 performed favorably in predicting response to pembro/SD101 compared to several other genomic signatures measuring intratumoral immune response. Higher CK12 also associates with response to the ANG1/2 inhibitor AMG386, an agent known to have immune modulatory activity. Higher CK12 was moderately associated with pCR in the control (OR=2.0/1SD), neratinib (OR=1.7/1SD), veliparib/carboplatin (OR=2.0/1SD), ganitumab (OR= 1.7/1SD) and TDM1/P arms (OR=2.1/1SD). Within the HR+HER2- subset, CK12 associated with pCR in all three IO arms, and in the control, AMG386, ganitumab, and ganetespib arms. Within the smaller TN subset, it associated with response in pembro and pembro/SD101 arms but not in durva/olaparib, and in the neratinib and AMG386 arms. Chemokine12 mostly did not associate with pCR in HER2+ subsets, except for HR+HER2+ patients treated with neratinib, and HR-HER2+ patients in the original control arm (trastuzumab). Conclusion: The CK12 signature is highly predictive of complete pathologic response to immuno-oncology agents and other therapeutics supporting the role of the crosstalk within the tumor immune microenvironment in predicting response across subtypes. This gene expression signature can be readily obtained from microarrays and warrants further investigation in future arms of ISPY2 as a predictive biomarker. Citation Format: Hatem Soliman, Denise Wolf, Jo Chien, Christina Yau, Michael Campbell, Mark Magbanua, Ruixiao Lu, Nicholas O'Grady, Lamorna Brown-Swigart, Gillian Hirst, Beverly Parker, Laura Sit, Smita Asare, Doug Yee, Angie DeMichele, Rita Nanda, Lajos Pusztai, Don Berry, Laura Esserman, Laura Van't Veer. Chemokine12 (CK12) tertiary lymphoid gene expression signature as a predictor of response in 3 immunotherapy arms of the neoadjuvant ISPY 2 TRIAL - pembrolizumab with and without SD101, and durvalumab combined with olaparib - and in 9 other arms of the trial including platinum-based and dual-anti-HER2 therapies [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD10-07.

Hepatitis C Virus NS3/4A Inhibition and Host Immunomodulation by Tannins from Terminalia chebula: A Structural Perspective.

Terminalia chebula Retz. forms a key component of traditional folk medicine and is also reported to possess antihepatitis C virus (HCV) and immunomodulatory activities. However, information on the intermolecular interactions of phytochemicals from this plant with HCV and human proteins are yet to be established. Thus, by this current study, we investigated the HCV NS3/4A inhibitory and host immune-modulatory activity of phytocompounds from T. chebula through in silico strategies involving network pharmacology and structural bioinformatics techniques. To start with, the phytochemical dataset of T. chebula was curated from biological databases and the published literature. Further, the target ability of the phytocompounds was predicted using BindingDB for both HCV NS3/4A and other probable host targets involved in the immune system. Further, the identified targets were docked to the phytochemical dataset using AutoDock Vina executed through the POAP pipeline. The resultant docked complexes with significant binding energy were subjected to 50 ns molecular dynamics (MD) simulation in order to infer the stability of complex formation. During network pharmacology analysis, the gene set pathway enrichment of host targets was performed using the STRING and Reactome pathway databases. Further, the biological network among compounds, proteins, and pathways was constructed using Cytoscape 3.6.1. Furthermore, the druglikeness, side effects, and toxicity of the phytocompounds were also predicted using the MolSoft, ADVERpred, and PreADMET methods, respectively. Out of 41 selected compounds, 10 were predicted to target HCV NS3/4A and also to possess druglike and nontoxic properties. Among these 10 molecules, Chebulagic acid and 1,2,3,4,6-Pentagalloyl glucose exhibited potent HCV NS3/4A inhibitory activity, as these scored a lowest binding energy (BE) of −8.6 kcal/mol and −7.7 kcal/mol with 11 and 20 intermolecular interactions with active site residues, respectively. These findings are highly comparable with Asunaprevir (known inhibitor of HCV NS3/4A), which scored a BE of −7.4 kcal/mol with 20 key intermolecular interactions. MD studies also strongly suggest that chebulagic acid and 1,2,3,4,6-Pentagalloyl glucose as promising leads, as these molecules showed stable binding during 50 ns of production run. Further, the gene set enrichment and network analysis of 18 protein targets prioritized 10 compounds and were predicted to potentially modulate the host immune system, hemostasis, cytokine levels, interleukins signaling pathways, and platelet aggregation. On overall analysis, this present study predicts that tannins from T. chebula have a potential HCV NS3/4A inhibitory and host immune-modulatory activity. However, further experimental studies are required to confirm the efficacies.

Phase II trial of cabozantinib (Cabo) plus durvalumab (Durva) in chemotherapy refractory patients with advanced mismatch repair proficient/microsatellite stable (pMMR/MSS) colorectal cancer (CRC): CAMILLA CRC cohort results.

135 Background: Cabo is an anti-VEGFR2/MET/AXL drug with broad multi-kinase inhibitory spectrum. Preclinical and clinical studies in various solid tumors demonstrated favorable immune modulatory activity of Cabo with clinical synergy seen when combined with PD-1/ PD-L1 inhibitors like Durva. Upon completion of phase Ib gastrointestinal (GI) basket CAMILLA trial evaluating Cabo + Durva in 30 patients (pts) demonstrating favorable safety & efficacy, the trial was expanded to phase 2 multi-cohort, multi-center trial of 117 pts. Herein, we report results of the phase 2 CRC cohort, the first evaluation of cabo + IO in this population. Methods: Pts enrolled in this cohort were administered Cabo + Durva at the RP2D of 40mg QD and 1500mg IV Q4W respectively. Enrolled pts must have progressed on 2 or more lines of therapy. Primary outcome measure was investigator assessed overall response rate (ORR) and secondary outcomes were rate of treatment related adverse events (TRAE), investigator assessed disease control rate (DCR), progression free survival (PFS), and overall survival (OS). Subgroup analysis was done in pts with RAS wild type tumors. Exploratory analysis of pathogenic molecular tumor alterations using next generation sequencing (NGS) was done in pts who achieved confirmed partial response (PRc)/ stable disease (SD) > 6 months. Results: Of the 36 pts enrolled, 29 (16F, 13M) were evaluable for efficacy. Median age 57 years (27-76). 90% (26) had ECOG of 1. All had pMMR/MSS, 41% (12) had RAS wild type tumors, and 6.9% (2) had HER2 amplification. 52% (15) had received ≥ 3 lines of therapy. All had metastases at ≥ 3 sites and 79% (23) metastases in the liver. Among 36 pts evaluable for safety, treatment related serious adverse events (SAEs) occurred in 31% (11/36). Most common TRAEs were grade 1-2 fatigue (53%), nausea (42%), diarrhea (36%), anorexia (31%), hand-foot syndrome (22%), & hypertension (16%). Grade ≥ 3 immune-related adverse events (IRAE) occurred in 16.6% (6/36). Efficacy analysis revealed an ORR 27.6% (8/29); PRc 21% (6/29); DCR 86.2% (25/29); median DOR was not reached (NR); median PFS 4.4 months; 6-month PFS 28% & median OS 9.1 months. In the RAS wild type subgroup, ORR (PRc) was 50.0%; DCR 83.3%; median PFS 6.3 months and median OS was NR. Of the 7 pts who achieved PRc/SD > 6 months, one had KRAS G12V tumor mutation along with mutations in ARID1A & IDH1. Remaining pts had RAS wild type tumors & among those, the following NGS alterations were detected: 1 HER-2 amplification, 1 MET amplification, & 2 alterations in ATM. Conclusions: Cabo + Durva demonstrated promising efficacy and was fairly tolerated without new safety signals in heavily treated pMMR/MSS CRC pts. These encouraging results warrant further evaluation of this regimen in a randomized setting as salvage therapy in pMMR/MSS CRC. Clinical trial information: NCT03539822.

Lotus (Nelumbo nucifera Gaertn.) and Its Bioactive Phytocompounds: A Tribute to Cancer Prevention and Intervention.

Simple SummaryThe plant Nelumbo nucifera (Gaertn.), commonly known as lotus, sacred lotus, Indian lotus, water lily, or Chinese water lily, is an aquatic perennial crop belonging to the family of Nelumbonaceae. N. nucifera has traditionally been used as an herbal medicine and functional food in many parts of Asia. It has been found that different parts of this plant consist of various bioactive phytocompounds. Within the past few decades, N. nucifera and its phytochemicals have been subjected to intense cancer research. In this review, we critically evaluate the potential of N. nucifera phytoconstituents in cancer prevention and therapy with related mechanisms of action. Cancer is one of the major leading causes of death worldwide. Accumulating evidence suggests a strong relationship between specific dietary habits and cancer development. In recent years, a food-based approach for cancer prevention and intervention has been gaining tremendous attention. Among diverse dietary and medicinal plants, lotus (Nelumbo nucifera Gaertn., family Nymphaeaceae), also known as Indian lotus, sacred lotus or Chinese water lily, has the ability to effectively combat this disease. Various parts of N. nucifera have been utilized as a vegetable as well as an herbal medicine for more than 2000 years in the Asian continent. The rhizome and seeds of N. nucifera represent the main edible parts. Different parts of N. nucifera have been traditionally used to manage different disorders, such as fever, inflammation, insomnia, nervous disorders, epilepsy, hypertension, cardiovascular diseases, obesity, and hyperlipidemia. It is believed that numerous bioactive components, including alkaloids, polyphenols, terpenoids, steroids, and glycosides, are responsible for its various biological and pharmacological activities, such as antioxidant, anti-inflammatory, immune-modulatory, antiviral, hepatoprotective, cardioprotective, and hypoglycemic activities. Nevertheless, there is no comprehensive review with an exclusive focus on the anticancer attributes of diverse phytochemicals from different parts of N. nucifera. In this review, we have analyzed the effects of N. nucifera extracts, fractions and pure compounds on various organ-specific cancer cells and tumor models to understand the cancer-preventive and therapeutic potential and underlying cellular and molecular mechanisms of action of this interesting medicinal and dietary plant. In addition, the bioavailability, pharmacokinetics, and possible toxicity of N. nucifera-derived phytochemicals, as well as current limitations, challenges and future research directions, are also presented.

Preclinical toxicity test results of a new antiviral–immune-modulator compound consisting of flavonoid molecules (COVID-19 clinical trial preliminary data)

Aim: Isolated specific glycone–aglycone conjugated flavonoids which are investigated for their effect of bioavailability and molecular concentrations. The specific formula is then tested via in vitro and in vivo cytotoxicity tests. Methods: Considering the higher affinity for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), quercetin, quercetin 3-sambubioside-3’-glucoside, luteolin, apigenin-7-4’alloside, kaempferol-7-O-glucoside, epicatechin-epigallocatechin-3-O-gallate, and hesperetin were selected to investigate the effects of a new combination of the formula. Specific chemical analyses, such as high-performance liquid chromatography (HPLC), liquid chromatography–mass spectrometry (LC–MS), quadrupole time of flight mass spectrometry (QTOF–MS) analysis and ultraviolet–visible (UV–VIS) spectrophotometry, were performed for molecular qualification and quantification. Results: In silico molecular docking analyses have shown that flavonoids can bind strongly to the spike protein and main protease of the SARS-CoV-2 virus. Flavonoids also have anti-inflammatory and immune-modulating activity by inhibiting cytokines. Although flavonoids may be a treatment alternative for coronavirus disease 2019 (COVID-19), an effective flavonoid compound has yet to be developed. The main problem here is that the absorption rate of flavonoids is very low (2–10%) in the intestines, and these compounds are metabolized rapidly. In contrast, according to recent literature, a conjugated flavonoid mixture is better absorbed in the small intestine, and its toxic effects are relatively fewer. Conclusions: It is found that the new formula has no cytotoxic or genotoxic effects. Furthermore, oral administrations of the new compound did not produce any toxicity symptoms or any mortality in male and female rats. The pre-clinical in vitro and in vivo toxicity test results indicated that the new flavonoid formula can be safely used for clinical trials.

Molecular docking and in vivo immunomodulatory activity of Albizia procera bark on doxorubicin induced immunosuppressive rats

ObjectiveTo study the immunomodulatory potential of Albizia procera (AP) bark using in vivo models and by in silico approach. MethodsIn silico models involved to study binding affinity of AP bioactive molecules on immune modified proteins such as Human NF-kappa B p52 (NFkB P52), human tumor necrosis factor-alpha (TNF-α). In vivo studies to evaluated immunomodulatory activity of ethanolic extract AP bark (EEAP) Doxorubicin (DOX) induced immunosuppressive rats. ResultsDocking results showed AP bioactive molecules 3-O-[α-L-arabinopyranosyl-(12)-β- → D fucopyranosyl - (16) - 2 - acetamido - 2 - deoxy- β - → Dglucopyranosyl] echinocystic acid (Compound 1), 3-O-[α-L-arabinopyranosyl-(12)-β- → D fucopyranosyl - (16) - 2 - acetamido - 2 - deoxy- β - → Dglucopyranosyl] acacic acid lactone (Compound 2), Catechin, Quercetin, Isoquercetin were showed immune modulatory activity due to high binding affinity and H bonding interaction with active sites of NFkB P52, TNF-α, without H bonding on anti-inflammatory cytokines IL 10. Based on docking Compound 1, Compound 2, Catechin, Quercetin, Isoquercetin were concluded as immunomodulatory potential candidate. EEAP exhibited a dose related incline in cell count of total leukocyte, neutrophils, and lymphocytes. The suppressive outcome of DOX on these cells was not reflected in EEAP treated rats. It enhanced the rate of clearance of the carbon particles in dose dependent manner from the blood circulation in both normal rats and in the immunosuppressive rats. Delayed type of hypersensitivity test (DTH) results showed an increase in footpad thickness of paw significantly in response to antigen, as an impact of EEAP treatment stimulatory response is observed on lymphocytes along with other essential cells of reaction and thus increased the cell mediated immunity. ConclusionAP improves the immune function in DOX induced immunosuppressive rats.

Mining for Active Molecules in Probiotic Supernatant by Combining Non-Targeted Metabolomics and Immunoregulation Testing.

Chronic respiratory diseases such as asthma are highly prevalent in industrialized countries. As cases are expected to rise, there is a growing demand for alternative therapies. Our recent research on the potential benefits of probiotics suggests that they could prevent and reduce the symptoms of many diseases by modulating the host immune system with secreted metabolites. This article presents the first steps of the research that led us to identify the immunoregulatory bioactivity of the amino acid d-Trp reported in our previous study. Here we analyzed the cell culture metabolic footprinting of 25 commercially available probiotic strains to associate metabolic pathway activity information with their respective immune modulatory activity observed in vitro. Crude probiotic supernatant samples were processed in three different ways prior to untargeted analysis in positive and negative ionization mode by direct infusion ESI-FT-ICR-MS: protein precipitation and solid phase extraction (SPE) using HLB and CN-E sorbent cartridges. The data obtained were submitted to multivariate statistical analyses to distinguish supernatant samples into the bioactive and non-bioactive group. Pathway analysis using discriminant molecular features showed an overrepresentation of the tryptophan metabolic pathway for the bioactive supernatant class, suggesting that molecules taking part in that pathway may be involved in the immunomodulatory activity observed in vitro. This work showcases the potential of metabolomics to drive product development and novel bioactive compound discovery out of complex biological samples in a top-down manner.

Bee Products: An Emblematic Example of Underutilized Sources of Bioactive Compounds.

Beside honey, honeybees (Apis mellifera L.) are able to produce many byproducts, including bee pollen, propolis, bee bread, royal jelly, and beeswax. Even if the medicinal properties of these byproducts have been recognized for thousands of years by the ancient civilizations, in the modern era, they have a limited use, essentially as nutritional supplements or health products. However, these natural products are excellent sources of bioactive compounds, macro- and micronutrients, that, in a synergistic way, confer multiple biological activities to these byproducts, such as, for example, antimicrobial, antioxidant, and anti-inflammatory properties. This work aims to update the chemical and phytochemical composition of bee pollen, propolis, bee bread, royal jelly, and beeswax and to summarize the main effects exerted by these byproducts on human health, from the anticancer and immune-modulatory activities to the antidiabetic, hypolipidemic, hypotensive, and anti-allergic properties.

Characterization of Alpinia officinarum Hance polysaccharide and its immune modulatory activity in mice.

This study aimed to characterize the structural features of a novel water-soluble polysaccharide (AOHP) extracted from Alpinia officinarum Hance and to verify its regulating effect on mouse immunity. Cellulose DEAE-52 and Sephadex G-100 columns were used to obtain purified AOHP. Techniques including NMR, methylation, monosaccharide composition, FT-IR, and molecular weight determination were applied to investigate the physicochemical properties and structural characterization of AOHP. Then, the influence of AOHP on mice was studied. After oral administration of AOHP, organ indexes, serum biochemistry indexes, and cytokines in the spleens of the mice were analysed. The results showed that AOHP was composed of T-α-D-Glcp, (1,4)-α-D-Glcp and (1,4,6)-α-D-Glcp with a number-average molecular weight of 26.0 kDa and a weight-average molecular weight of 52.8 kDa. Additionally, the innate immune statuses of the mice were improved by treatment with AOHP, while no obvious damage was identified. To conclude, the immunomodulatory activity and biological safety make AOHP a viable candidate as an ingredient for healthcare drugs.

Lactoferrin modulates gut microbiota and Toll-like receptors (TLRs) in mice with dysbiosis induced by antibiotics.

Background: Antibiotic administration can result in gut microbiota and immune system alterations that impact health. Bovine lactoferrin is a milk protein with anticancer, anti-inflammatory, antimicrobial and immune modulatory activities. The aim was to study the ability of native and iron-saturated lactoferrin to reverse the effects of clindamycin on gut microbiota and intestinal Toll-like receptor (TLR) expression in a murine model. Methods: Male C57BL/6 mice were treated with vehicle, clindamycin (Clin), native bovine lactoferrin (nLf), nLf + clindamycin (nLf_Clin), iron-saturated bovine lactoferrin (sLf) and sLf + clindamycin (sLf_Clin). Fecal samples of each group were collected, and bacterial DNA was extracted. Sequencing of 16s rRNA V4 hypervariable gene regions was conducted to assess the microbial composition. mRNA expression levels of TLRs (1-9) were determined in mouse colon by qPCR. Pearson's correlation test was carried out between bacteria showing differences in abundance among samples and TLR2, TLR8 and TLR9. Results: Beta-diversity analysis showed that the microbial community of the vehicle was different from the communities of Clin, nLf_Clin and sLf_Clin. At the family level, Bacteroidaceae, Prevotellaceae and Rikenellaceae decreased in the Clin group, and treatment with nLf or sLf reverted these effects. Clin reduced the expression of TLR2, TLR8 and TLR9 and sLf reverted the decrease in the expression of these receptors. Finally, TLR8 was positively correlated with Rikenellaceae abundance. Conclusion: In a situation of intestinal dysbiosis induced by clindamycin, lactoferrin restores the normal levels of some anti-inflammatory bacteria and TLRs and, therefore, could be a good ingredient to be added to functional foods.

A review on the functional properties of fermented soymilk

Fermented soybean products have been used for several centuries. Fermented soymilk is one of the known soy products with several health benefits. The current literature review recapitulates the various health benefits of fermented soymilk. In vitro and in vivo studies have shown that the consumption of fermented soymilk protects one from cancer, diabetes, hyperlipidemia, etc. It has also been reported that fermented soymilk has immune-modulatory, memory improvement, anti-colitis, and wound healing activities. However, there are few studies to confirm the health benefits of fermented soymilk for human subjects. The dose and duration of the intervention may vary between the animal model and humans. So, additional studies are obligatory to authorize the proposed health benefits of fermented soymilk.

Comparative effects of free doxorubicin, liposome encapsulated doxorubicin and liposome co-encapsulated alendronate and doxorubicin (PLAD) on the tumor immunologic milieu in a mouse fibrosarcoma model.

Background: We have previously shown that alendronate, an amino-bisphosphonate, when reformulated in liposomes, can significantly enhance the efficacy of cytotoxic chemotherapies and help remodel the immunosuppressive tumor microenvironment towards an immune-permissive milieu resulting in increased anticancer efficacy. In addition, we have previously shown that the strong metal-chelating properties of alendronate can be exploited for nuclear imaging of liposomal biodistribution. To further improve anticancer efficacy, a pegylated liposome formulation co-encapsulating alendronate and doxorubicin (PLAD) has been developed. In this study, we examined the effects of PLAD on the tumor immunologic milieu in a mouse fibrosarcoma model in which the tumor microenvironment is heavily infiltrated with tumor-associated macrophages (TAM) that are associated with poor prognosis and treatment resistance.Methods: Doxorubicin biodistribution, characterization of the tumor immunologic milieu, cellular doxorubicin uptake, and tumor growth studies were performed in Balb/c mice bearing subcutaneously implanted WEHI-164 fibrosarcoma cells treated intravenously with PLAD, pegylated liposomal doxorubicin (PLD), free doxorubicin, or vehicle.Results: PLAD delivery resulted in a high level of tumor doxorubicin that was 20 to 30-fold greater than in free doxorubicin treated mice, and non-significantly higher than in PLD treated mice. PLAD also resulted in increased uptake in spleen and slightly lower plasma levels as compared to PLD. Importantly, our results showed that PLAD, and to a lesser extent PLD, shifted cellular drug uptake to TAM and to monocytic myeloid-derived suppressor cells (MDSC), while there was no drug uptake in neutrophilic MDSC or lymphoid cells. Free doxorubicin cellular drug uptake was below detectable levels. PLAD, and to a lesser extent PLD, also induced significant changes in number and functionality of tumor-infiltrating TAM, MDSC, Treg, NKT, and NK cells that are consistent with enhanced antitumor immune responses in the tumor microenvironment. In contrast, free doxorubicin induced moderate changes in the tumor microenvironment that could promote (decreased Treg) or be detrimental to antitumor immune responses (decreased M1 TAM and NK cells). These immune modulatory effects are reflected in the therapeutic study which showed that PLAD and PLD inhibited tumor growth and significantly prolonged survival, while free doxorubicin showed little or no anticancer activity.Conclusion: We show that liposomal delivery of doxorubicin not only alters pharmacokinetics, but also dramatically changes the immune modulatory activity of the drug cargo. In addition, our data support that the PLAD nanotheranostic platform further enhances some immune changes that may act in synergy with its cytotoxic chemotherapy effects.

Abstract P040: Harness the immune-modulatory activities of Toxoplasma gondii to improve lymphocyte infiltration into brain tumors

Abstract While checkpoint inhibitors carry great promises for T-cells-based immunotherapy for brain tumors, the presence of abundant T cells supported by the tumor microenvironment (TME) is a prerequisite for it to be effective. Our lab and others' work using mouse models and patient samples demonstrated that, Shh-subtype medulloblastoma, a well-studied pediatric brain tumor, lacks both T cell infiltration and a supportive TME, posing a critical barrier for immunotherapy. I envision that the protozoans Toxoplasma gondii (T.gondii) as a biological agent could provide a unique solution, because T.gondii can naturally disseminate to the brain and induce significant infiltration and activation of T cells asymptomatically. We hypothesize that T.gondii could attract T cells into brain tumors while at the same time convert the non-supportive TME into one that is conducive for T cells. Using a mouse model for medulloblastoma based on a genetic system called Mosaic Analysis with Double Markers (MADM) that recapitulates the tumorigenic process in human by producing few and precisely labeled mutant cells, I was able to assess the immuno-modulatory potential of T.gondii in the context of an intact immune system and endogenous TME. Our preliminary data revealed three exciting facets about T.gondii's immunotherapeutic potential. Via histological assessment and flow cytometry, we found that at 24 days post infection, abundant T cells are recruited into TME. Correspondingly, we detected significant elevation of IFNg and IFNg-driven genes in the tumors, suggesting Th-1 immunity presence in TME. Lastly, we found that T.gondii challenge is associated with reduced tumor incidence. These results indicates T.gondii holds potent immuno-modulatory capability and potentially anti-tumoral functions. My follow-up studies with dive more deeply into three aspects: 1) determine whether T. gondii can sustainably recruit functional T cells into the tumor mass; 2) determine whether T. gondii can help shape a T cell-supportive TME, especially through activating Th1-immune response in innate immune cells; and 3) identify the underlying mechanisms for T.gondii to exert tumor-suppressing activities. Citation Format: Yen Nguyen, Xiaoyu Zhao, Sarah Ewald, Tajie Harris, Hui Zong. Harness the immune-modulatory activities of Toxoplasma gondii to improve lymphocyte infiltration into brain tumors [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P040.

Preclinical toxicity test results of a new antiviral–immune-modulator compound consisting of flavonoid molecules (COVID-19 clinical trial preliminary data)

Aim: Isolated specific glycone–aglycone conjugated flavonoids which are investigated for their effect of bioavailability and molecular concentrations. The specific formula is then tested via in vitro and in vivo cytotoxicity tests. Methods: Considering the higher affinity for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), quercetin, quercetin 3-sambubioside-3’-glucoside, luteolin, apigenin-7-4’alloside, kaempferol-7-O-glucoside, epicatechin-epigallocatechin-3-O-gallate, and hesperetin were selected to investigate the effects of a new combination of the formula. Specific chemical analyses, such as high-performance liquid chromatography (HPLC), liquid chromatography–mass spectrometry (LC–MS), quadrupole time of flight mass spectrometry (QTOF–MS) analysis and ultraviolet–visible (UV–VIS) spectrophotometry, were performed for molecular qualification and quantification. Results: In silico molecular docking analyses have shown that flavonoids can bind strongly to the spike protein and main protease of the SARS-CoV-2 virus. Flavonoids also have anti-inflammatory and immune-modulating activity by inhibiting cytokines. Although flavonoids may be a treatment alternative for coronavirus disease 2019 (COVID-19), an effective flavonoid compound has yet to be developed. The main problem here is that the absorption rate of flavonoids is very low (2–10%) in the intestines, and these compounds are metabolized rapidly. In contrast, according to recent literature, a conjugated flavonoid mixture is better absorbed in the small intestine, and its toxic effects are relatively fewer. Conclusions: It is found that the new formula has no cytotoxic or genotoxic effects. Furthermore, oral administrations of the new compound did not produce any toxicity symptoms or any mortality in male and female rats. The pre-clinical in vitro and in vivo toxicity test results indicated that the new flavonoid formula can be safely used for clinical trials.

Efficacy of herbal anti-microbial soap in Tinea corporis: A randomized controlled study

Ethnopharmacological relevanceCurcuma longa L., Azadirachta indica A Juss. Cassia tora L. has been used in Unani medicine for various skin ailments. Several researches have been conducted on these plants which have shown anti-microbial, anti-bacterial, anti-fungal, antiviral, anti-oxidant, wound healing, anti-inflammatory, and immune modulation activities. Skin diseases and the use of these drugs are mentioned in classical Unani literature like The Canon of medicine, Continens Liber, Hippocratic treatments, The Complete Book of the Medical Art etc. AimThe aim of the study was to formulate anti-microbial soap and to evaluate its clinical efficacy of in the management of Tinea corporis. Materials and methodsThe anti-microbial soap was prepared by hydroalcholic extracts of Curcuma longa L., Azadirachta indica A Juss. and Cassia tora L. The prepared soap was evaluated for various physicochemical parameters, microbiological evaluation, stability study, skin irritation, In-vitro anti-microbial activity, GCMS analysis, and a clinical trial was carried out to evaluate its efficacy. A Single Blind Randomized Placebo Controlled trail on 30 patients aged between 18 and 60 years of either gender was carried out. The participants were randomly allocated to receive either anti-microbial soap or Placebo soap for 4 weeks. Subjective parameters including erythema, pruritis and desquamation were assessed weekly while as objective parameter including Photograph of lesion, Total Symptom Score (TSS) and KOH mount was assessed at baseline and at the end of the trial. ResultsThe improvement in subjective parameters was found significant in test group. Erythema, scaling, and desquamation was completely relieved by 70%, 80% and 25% patients respectively in test group while as none of the patients got complete relief in control group. There was statistically significant reduction in average TSS 8.65 ± 0.6708 to 3.05 ± 1.35 p < 0.001. KOH mount turned negative in 80% patients in test group while as only 20% turned negative in control group. ConclusionIt is concluded that Unani drugs can be utilized in better way by modifying into a convenient dosage form. Anti-microbial soap was formulated by adding minimal additives to achieve effectiveness, with cost effective benefits and less or no side effects. Anti-microbial soap was effective in management of management of Tinea corporis. Moreover further studies on large sample size are required to fine-tune these observations.

Cysteamine with In Vitro Antiviral Activity and Immunomodulatory Effects Has the Potential to Be a Repurposing Drug Candidate for COVID-19 Therapy.

The ongoing pandemic of coronavirus disease-2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), needs better treatment options both at antiviral and anti-inflammatory levels. It has been demonstrated that the aminothiol cysteamine, an already human applied drug, and its disulfide product of oxidation, cystamine, have anti-infective properties targeting viruses, bacteria, and parasites. To determine whether these compounds exert antiviral effects against SARS-CoV-2, we used different in vitro viral infected cell-based assays. Moreover, since cysteamine has also immune-modulatory activity, we investigated its ability to modulate SARS-CoV-2-specific immune response in vitro in blood samples from COVID-19 patients. We found that cysteamine and cystamine decreased SARS-CoV-2-induced cytopathic effects (CPE) in Vero E6 cells. Interestingly, the antiviral action was independent of the treatment time respect to SARS-CoV-2 infection. Moreover, cysteamine and cystamine significantly decreased viral production in Vero E6 and Calu-3 cells. Finally, cysteamine and cystamine have an anti-inflammatory effect, as they significantly decrease the SARS-CoV-2 specific IFN-γ production in vitro in blood samples from COVID-19 patients. Overall, our findings suggest that cysteamine and cystamine exert direct antiviral actions against SARS-CoV-2 and have in vitro immunomodulatory effects, thus providing a rational to test these compounds as a novel therapy for COVID-19.

Bifidobacterium longum subsp. longum 51A attenuates intestinal injury against irinotecan-induced mucositis in mice

Intestinal mucositis (IM) is a critical side-effect associated with antineoplastic therapy. Treatment available is only palliative and often not effective. However, alternative therapeutic strategies, such as probiotics, have attracted significant attention due to their immune-modulatory action in several diseases. Thus, the present study aims to elucidate the therapeutic potential of the probiotic strain Bifidobacterium longum 51A in a murine model of mucositis induced by irinotecan. Due to the scarcity of studies on dose-response and viability (probiotic vs paraprobiotic), we first evaluated which dose and cell viability would be most effective in treating mucositis. In this study, the oral pretreatment with viable B. longum 51A at a concentration of 1 × 109 CFU/mL reduced the daily disease activity index (p < 0.01), protected the intestinal architecture, preserved the length of the intestine (p < 0.05), and reduced intestinal permeability (p < 0.01), inflammation, and oxidative damage (p < 0.01) induced by irinotecan. Also, treatment with B. longum 51A increased the production of secretory immunoglobulin A (p < 0.05) in the intestinal fluid of mice with mucositis. Furthermore, B. longum 51A reversed the mucositis-induced increase in Enterobacteriaceae bacterial group in the gut (p < 0.01). In conclusion, these results showed that oral administration of B. longum 51A protects mice against intestinal damage caused by irinotecan, suggesting its use as a potential probiotic in therapy during mucositis.

Recent Insights Into the Prognostic and Therapeutic Applications of Lysozymes.

Lysozymes are naturally occurring enzymes present in a variety of biological organisms, such as bacteria, fungi, and animal bodily secretions and tissues. It is also the main ingredient of many ethnomedicines. It is well known that lysozymes and lysozyme-like enzymes can be used as anti-bacterial agents by degrading bacterial cell wall peptidoglycan that leads to cell death, and can also inhibit fungi, yeasts, and viruses. In addition to its direct antimicrobial activity, lysozyme is also an important component of the innate immune system in most mammals. Increasing evidence has shown the immune-modulatory effects of lysozymes against infection and inflammation. More recently, studies have revealed the anti-cancer activities of lysozyme in multiple types of tumors, potentially through its immune-modulatory activities. In this review, we summarized the major functions and underlying mechanisms of lysozymes derived from animal and plant sources. We highlighted the therapeutic applications and recent advances of lysozymes in cancers, hypertension, and viral diseases, aiming toseeking alternative therapies for standard medical treatment bypassing side effects. We also evaluated the role of lysozyme as a promising cancer marker for prognosis to indicate the outcomes recurrence for patients.

Abstract P107: PSGL-1 blocking antibodies repolarize tumor associated macrophages, reduce suppressive myeloid populations and induce inflammation in the tumor microenvironment, leading to suppression of tumor growth

Abstract Suppressive myeloid populations in the tumor microenvironment are associated with worse survival of cancer patients and low effectiveness of T cell checkpoint inhibitors. Recently, several early clinical studies have produced positive data for therapies aimed at repolarizing suppressive myeloid populations in the tumor microenvironment. One new macrophage repolarizing target, PSGL-1, is expressed at high levels on immuno-suppressive TAMs and differentiated M2 macrophages. PSGL-1 has been shown to have an immune-modulatory activity, which includes its role in maintaining a suppressive functional macrophage state. To assess the ability of PSGL-1 antibodies to convert macrophages and the tumor microenvironment from an immuno-suppressive toward a pro-inflammatory state, we employed in vitro primary macrophage and multi-cellular assays, ex vivo patient-derived tumor cultures, and a humanized mouse PDX model. We have determined that our lead anti-PSGL-1 antibody repolarized M2-like macrophages to a more M1-like state both phenotypically and functionally as assessed in primary in vitro macrophage assays. Transcriptomics profiling of M2c macrophages showed that the anti-PSGL-1 antibody upregulated TNF-a/NF-kB and chemokine-mediated signaling, while downregulating oxidative phosphorylation, fatty acid metabolism and Myc signaling pathways, consistent with a broad M2-to-M1 shift of the macrophage state. Furthermore, these repolarized M1-like macrophages enhanced the inflammatory response in complex multi-cellular assays. The PSGL-1 antibody also showed efficacy in a humanized mouse PDX model of melanoma. The antibody suppressed tumor growth to a significantly greater degree compared to anti-PD-1. At the cellular and molecular levels, the anti-PSGL-1 treatment led to a more enhanced inflammatory microenvironment, including a reduced M2:M1 macrophage ratio, and an increase in systemic pro-inflammatory mediators. Compared to anti-PD-1 monotherapy, anti-PSGL-1 alone and in combination with anti-PD-1 increased the fraction of effector CD8+ T cells among the infiltrating T cells. Significant combination effects of anti-PSGL-1 plus anti-PD-1 were seen at the cellular and molecular levels within the tumor tissue, the spleen, and peripheral blood. Lastly, pre-clinical efficacy of our lead anti-PSGL-1 antibody was demonstrated using ex vivo cultures of fresh patient-derived tumors that preserve the cellular heterogeneity of the TME. Anti-PSGL-1 increased production of inflammatory cytokines and chemokines involved in immune activation of the TME and T cell recruitment. The data presented here provide biological and mechanistic support for clinical testing of antibodies targeting PSGL-1 for the treatment of cancer. Citation Format: Ani Nguyen, Jessica Ritter, Mohammad Zafari, Denise Manfra, Veronica Komoroski, Brian O'Nuallain, Ryan Phennicie, Kevin Kauffman, Dominika Nowakowska, Joe Wahle, Steve Sazinsky, Michael Brehm, Igor Feldman, Tatiana Novobrantseva. PSGL-1 blocking antibodies repolarize tumor associated macrophages, reduce suppressive myeloid populations and induce inflammation in the tumor microenvironment, leading to suppression of tumor growth [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P107.

Safety Evaluation of Weissella cibaria JW15 by Phenotypic and Genotypic Property Analysis.

Weissella cibaria is one of the bacteria in charge of the initial fermentation of kimchi and has beneficial effects such as immune-modulating, antagonistic, and antioxidant activities. In our study, we aimed to estimate the safety of W. cibaria JW15 for the use of probiotics according to international standards based on phenotypic (antibiotic resistance, hemolysis, and toxic metabolite production) and genotypic analysis (virulence genes including antibiotic resistance genes). The results of the safety assessment on W. cibaria JW15 were as follows; (1) antibiotic resistance genes (ARGs) (kanamycin and vancomycin etc.) were intrinsic characteristics; (2) There were no acquired virulence genes including Cytolysin (cylA), aggregation substance (asa1), Hyaluronidase (hyl), and Gelatinase (gelE); (3) this strain also lacked β-hemolysis and the production of toxic metabolites (D-lactate and bile salt deconjugation). Consequently, W. cibaria JW15 is expected to be applied as a functional food ingredient in the food market.