Human IL-2 Research Articles

Structure and assembly of the human IL-12 signaling complex

Interleukin (IL)-12 is a heterodimeric pro-inflammatory cytokine. Our cryoelectron microscopy structure determination of human IL-12 in complex with IL-12Rβ1 and IL-12Rβ2 at a resolution of 3.75Å reveals that IL-12Rβ2 primarily interacts with the IL-12p35 subunit via its N-terminal Ig-like domain, while IL-12Rβ1 binds to the p40 subunit with its N-terminal fibronectin III domain. This binding mode of IL-12 with its receptors is similar to that of IL-23 but shows notable differences with other cytokines. Through structural information and biochemical assays, we identified Y62, Y189, and K192 as key residues in IL-12p35, which bind to IL-12Rβ2 with high affinity and mediate IL-12 signal transduction. Furthermore, structural comparisons reveal two distinctive conformational states and structural plasticity of the heterodimeric interface in IL-12. As a result, our study advances our understanding of IL-12 signal initiation and opens up new opportunities for the engineering and therapeutic targeting of IL-12.

Prophylactic IL-23 blockade uncouples efficacy and toxicity in dual CTLA-4 and PD-1 immunotherapy

BackgroundImmune-related adverse events (irAEs), characterized by targeted inflammation, occur in up to 60% of patients with melanoma treated with immune checkpoint inhibitors (ICIs). Evidence proved that the baseline peripheral blood...

IL-10-modulated dendritic cells from birch pollen- and hazelnut-allergic patients facilitate Treg-mediated allergen-specific and cross-reactive tolerance.

Approximately 70% of individuals allergic to birch pollen (Bet v 1.01 [Bet]) develop a secondary food allergy (e.g., hazelnut: Cor a 1.04 [Cor]), due to allergen cross-reactivity. However, standard immunotherapy for type I allergies often does not improve the food allergy sufficiently. We analyzed the allergen-specific and cross-reactive suppressive capacity of primary human regulatory T cells (Treg) induced by autologous IL-10-modulated dendritic cells (IL-10 DC) invitro and invivo. CD4+ T cells of patients with birch pollen and associated hazelnut allergies were differentiated into Bet-specific or non-specific induced Treg (iTreg). After Bet- or Cor-specific restimulation the phenotype, proliferation, and suppressive capacity of iTreg subsets were analyzed. iTreg function was further investigated in humanized mouse models of airway and intestinal allergy, generated by engraftment of peripheral blood mononuclear cells from allergic donors into immunodeficient animals. After IL-10 DC priming and allergen-specific restimulation (Bet or Cor), non-specific control iTreg remained anergic, whereas Bet-specific iTreg proliferated extensively and exhibited a regulatory phenotype (enhanced expression of CTLA-4, PD-1, TNFR2, IL-10). Accordingly, activated Bet-specific iTreg displayed a high capacity to suppress Bet- and Cor-induced responder Th2 cell responses invitro, indicating induction of both allergen-specific (birch) and cross-reactive tolerance (hazelnut). Invivo, the beneficial effect of Bet-specific iTreg was verified in humanized mouse models of allergic airway and intestinal inflammation, resulting in reduced allergen-induced clinical symptoms, and immune responses. Human IL-10 DC-induced iTreg facilitate allergen-specific and cross-reactive tolerance. Therefore, they are potential candidates for regulatory cell therapy in allergic and autoimmune diseases.

IL-22/IL-22RA1 Promotes Human Tenon's Capsule Fibroblasts Proliferation and Regulates Fibrosis Through STAT3 Signaling Pathway.

Purpose: Although it is now understood that most antiglaucoma surgeries fail because of scarring of the filtering tract, the underlying mechanism remains to be elucidated. The present study investigated the mechanism by which the interleukin (IL)-22/IL-22 receptor alpha 1 (IL-22RA1) signaling pathway regulates scar formation in glaucoma patients. Method: A total of 31 glaucoma patients who underwent trabeculectomy surgery with uncontrollable intraocular pressure because of scarring and 19 strabismus patients as the control patient group were included in the present study. ELISA was performed to measure the content of IL-22 in peripheral blood. Serum from patients was used to incubate human Tenon's capsule fibroblasts (HTFs) cells and IL-22 antibody rescued the effect of IL-22 on the biological functions. qPCR and Western blot were performed to determine IL-22RA1 mRNA and protein expression levels. Flow cytometry was performed to assess the cell cycle distribution and the Cell Counting Kit-8 assay was used to analyze cell proliferation. Results: The ELISA assay revealed that the serum IL-22 level of glaucoma patients was significantly higher than the healthy group (29.80 ± 5.1 ng/µL vs. 5.21 ± 0.9 ng/µL). After incubation with patient serum, the proliferation and activation of human Tenon fibroblasts (HTFs) were promoted. IL-22 mediated the biological function of HTFs via directly binding IL-22RA1. Moreover, transfection of the siR-IL-22RA1 or IL-22RA1 gene resulted in significant antifibrosis or profibrosis in HTFs. When a signal transducer and activator of transcription (STAT) 3 inhibitor (BAY) was introduced to the IL-22RA1 overexpression group, IL-22-induced proliferation was reduced in HTFs. Additionally, glaucoma patients had increased levels of IL-22 expression following surgery. Conclusions: The IL-22/IL-22RA1/STAT3 signaling pathway promoted fibroblast cell proliferation and alpha-smooth muscle actin, potentially regulating fibrosis in glaucoma filtration tracts. Our results provide hitherto undocumented insights into the pathophysiology of postoperative scarring.

Transiently boosting Vγ9+Vδ2+ γδ T cells early in Mtb coinfection of SIV-infected juvenile macaques does not improve Mtb host resistance.

Children living with HIV have a higher risk of developing tuberculosis (TB), a disease caused by the bacterium Mycobacterium tuberculosis (Mtb). Gamma delta (γδ) T cells in the context of HIV/Mtb coinfection have been understudied in children, despite in vitro evidence suggesting γδ T cells assist with Mtb control. We investigated whether boosting a specific subset of γδ T cells, phosphoantigen-reactive Vγ9+Vδ2+ cells, could improve TB outcome using a nonhuman primate model of pediatric HIV/Mtb coinfection. Juvenile Mauritian cynomolgus macaques (MCM), equivalent to 4-8-year-old children, were infected intravenously (i.v.) with SIV. After 6 months, MCM were coinfected with a low dose of Mtb and then randomized to receive zoledronate (ZOL), a drug that increases phosphoantigen levels, (n=5; i.v.) at 3- and 17- days after Mtb accompanied by recombinant human IL-2 (s.c.) for 5 days following each ZOL injection. A similarly coinfected MCM group (n=5) was injected with saline as a control. Vγ9+Vδ2+ γδ T cell frequencies spiked in the blood, but not airways, of ZOL+IL-2-treated MCM following the first dose, however, were refractory to the second dose. At necropsy eight weeks after Mtb, ZOL+IL-2 treatment did not reduce pathology or bacterial burden. γδ T cell subset frequencies in granulomas did not differ between treatment groups. These data show that transiently boosting peripheral γδ T cells with ZOL+IL-2 soon after Mtb coinfection of SIV-infected MCM did not improve Mtb host defense.

Arecoline promotes fibroblast activation and M2-macrophage polarization by up-regulating the expression of IL-4

ObjectiveTo determine the biological effects of arecoline on oral submucous fibrosis (OSF). DesignThe differential genes between OSF tissue and normal oral tissue were collected form GSE64216 dataset, analyzed by Gene Expression Omnibus (GEO) database. Real-time PCR and immunohistochemistry were used to analyze the expression of IL-4 gene and protein in oral tissue. Enzyme-linked immunosorbent assay (ELISA) was used to analyze the expression of exocrine IL-4 protein in human oral fibroblasts (HOF) pre-treated by arecoline. Cell Counting Kit-8 (CCK-8) and transwell assays were used to analyze the proliferation and migration of HOF cells, respectively. After IL-4 was knocked down by short hairpin (sh) plasmid, the proliferation and migration of HOF cells were detected. Flow cytometry was used to analyze the proportion of M2-macrophages. Real-time PCR and immunohistochemistry were used to verify the expression of biomarker proteins of macrophages in OSF tissues. ResultsThe expression of IL-4 gene and protein were both up-regulated in OSF tissue. Arecoline could enhance the expression of IL-4 gene and exocrine protein in HOF cells, and promote the proliferation and migration of HOF cells. While knockdown of IL-4 could inhibit arecoline-induced proliferation and migration in HOF cells. The results of flow cytometry showed that recombinant human IL-4 (rhIL-4) protein could increase the proportion of M2-macrophages. Similarly, the results of real-time PCR and immunohistochemistry showed the expression of ARG1 (Biomarker proteins of M2-macrophage) was up-regulated in OSF tissues. ConclusionArecoline promotes activation of fibroblasts and polarization of M2-macrophages by up-regulating the expression of IL-4.

A Critical Role of Culture Medium Selection in Maximizing the Purity and Expansion of Natural Killer Cells.

Natural killer (NK) cells hold promise in cancer treatment due to their ability to spontaneously lyse cancer cells. For clinical use, high quantities of pure, functional NK cells are necessary. Combining adherence-based isolation with specialized media showed the unreliability of the isolation method, but demonstrated the superiority of the NK MACS® medium, particularly in suboptimal conditions. Neither human pooled serum, fetal calf serum (FCS), human platelet lysate, nor chemically defined serum replacement could substitute human AB serum. Interleukin (IL-)2, IL-15, IL-21, and combined CD2/NKp46 stimulation were assessed. IL-21 and CD2/NKp46 stimulation increased cytotoxicity, but reduced NK cell proliferation. IL-15 stimulation alone achieved the highest proliferation, but the more affordable IL-2 performed similarly. The RosetteSep™ human NK cell enrichment kit was effective for isolation, but the presence of peripheral blood mononuclear cells (PBMCs) in the culture enhanced NK cell proliferation, despite similar expression levels of CD16, NKp46, NKG2D, and ICAM-1. In line with this, purified NK cells cultured in NK MACS® medium with human AB serum and IL-2 demonstrated high cytotoxicity against primary glioblastoma stem cells.

A “Prime and Expand” strategy using the multifunctional fusion proteins to generate memory-like NK cells for cell therapy

Adoptive cellular therapy (ACT) using memory-like (ML) natural killer (NK) cells, generated through overnight ex vivo activation with IL-12, IL-15, and IL-18, has shown promise for treating hematologic malignancies. We recently reported that a multifunctional fusion molecule, HCW9201, comprising IL-12, IL-15, and IL-18 domains could replace individual cytokines for priming human ML NK cell programming (“Prime” step). However, this approach does not include ex vivo expansion, thereby limiting the ability to test different doses and schedules. Here, we report the design and generation of a multifunctional fusion molecule, HCW9206, consisting of human IL-7, IL-15, and IL-21 cytokines. We observed > 300-fold expansion for HCW9201-primed human NK cells cultured for 14 days with HCW9206 and HCW9101, an IgG1 antibody, recognizing the scaffold domain of HCW9206 (“Expand” step). This expansion was dependent on both HCW9206 cytokines and interactions of the IgG1 mAb with CD16 receptors on NK cells. The resulting “Prime and Expand” ML NK cells exhibited elevated metabolic capacity, stable epigenetic IFNG promoter demethylation, enhanced antitumor activity in vitro and in vivo, and superior persistence in NSG mice. Thus, the “Prime and Expand” strategy represents a simple feeder cell-free approach to streamline manufacturing of clinical-grade ML NK cells to support multidose and off-the-shelf ACT.

A humanized mouse that mounts mature class-switched, hypermutated and neutralizing antibody responses

Humanized mice are limited in terms of modeling human immunity, particularly with regards to antibody responses. Here we constructed a humanized (THX) mouse by grafting non-γ-irradiated, genetically myeloablated KitW-41J mutant immunodeficient pups with human cord blood CD34+ cells, followed by 17β-estradiol conditioning to promote immune cell differentiation. THX mice reconstitute a human lymphoid and myeloid immune system, including marginal zone B cells, germinal center B cells, follicular helper T cells and neutrophils, and develop well-formed lymph nodes and intestinal lymphoid tissue, including Peyer’s patches, and human thymic epithelial cells. These mice have diverse human B cell and T cell antigen receptor repertoires and can mount mature T cell-dependent and T cell-independent antibody responses, entailing somatic hypermutation, class-switch recombination, and plasma cell and memory B cell differentiation. Upon flagellin or a Pfizer-BioNTech coronavirus disease 2019 (COVID-19) mRNA vaccination, THX mice mount neutralizing antibody responses to Salmonella or severe acute respiratory syndrome coronavirus 2 Spike S1 receptor-binding domain, with blood incretion of human cytokines, including APRIL, BAFF, TGF-β, IL-4 and IFN-γ, all at physiological levels. These mice can also develop lupus autoimmunity after pristane injection. By leveraging estrogen activity to support human immune cell differentiation and maturation of antibody responses, THX mice provide a platform to study the human immune system and to develop human vaccines and therapeutics.

IL-6 Inhibitory Compounds from the Aerial Parts of Piper attenuatum and Their Anticancer Activities on Ovarian Cancer Cell Lines.

Piper attenuatum Buch-Ham, a perennial woody vine belonging to the Piperaceae family, is traditionally used in Southeast Asia for treating various ailments such as malaria, headache, and hepatitis. This study described the isolation and identification of three new compounds, piperamides I-III (1-3), which belong to the maleimide-type alkaloid skeletons, along with fifteen known compounds (4-18) from the methanol extract of the aerial parts of P. attnuatum. Their chemical structures were elucidated using spectroscopic methods (UV, IR, ESI-Q-TOF-MS, and 1D/2D NMR). All the isolates were evaluated for their ability to inhibit IL-6 activity in the human embryonic kidney-Blue™ IL-6 cell line and their cytotoxic activity against ovarian cancer cell lines (SKOV3/SKOV3-TR) and chemotherapy-resistant variants (cisplatin-resistant A2780/paclitaxel-resistant SKOV3). The compounds 3, 4, 11, 12, 17, and 18 exhibited IL-6 inhibition comparable to that of the positive control bazedoxifene. Notably, compound 12 displayed the most potent anticancer effect against all the tested cancer cell lines. These findings highlight the importance of researching the diverse activities of both known and newly discovered natural products to fully unlock their potential therapeutic benefits.

LPS-related muscle loss is associated with the alteration of Bacteroidetes abundance, systemic inflammation, and mitochondrial morphology in a weaned piglet model.

We previously demonstrated that lipopolysaccharide (LPS) injection-induced immune stress could impair muscle growth in weaned piglets, but the precise mechanisms behind this remain elusive. Here, we found that chronic immune stress induced by LPS resulted in a significant reduction of 36.86% in the total muscle mass of piglets at 5 d post-treatment compared with the control group. At 1 d, prior to muscle mass loss, multiple alterations were noted in response to LPS treatment. These included a reduction in the abundance of Bacteroidetes, an increase in serum concentrations of pro-inflammatory cytokines, compromised mitochondrial morphology, and an upregulation in the expression of dynamin-related protein 1 (Drp1), a critical protein involved in mitochondrial fission. We highlight a strong negative correlation between Bacteroidetes abundance and the levels of serum pro-inflammatory cytokines, corroborated by in vivo intervention strategies in the musculature of both pig and mouse models. Mechanistically, the effects of Bacteroidetes on inflammation and muscle mass loss may involve the signaling pathway of the tauro-β-muricholic acid-fibroblast growth factor 15. Furthermore, the induction of overexpression of inflammatory cytokines, achieved without LPS treatment through oral administration of recombinant human IL-6 (rhIL-6), led to increased levels of circulating cytokines, subsequently causing a decrease in muscle mass. Notably, pre-treatment with Mdivi-1, an inhibitor of Drp-1, markedly attenuated the LPS-induced elevation in reactive oxygen species levels and rescued the associated decline in muscle mass. Collectively, these data indicate that LPS-induced muscle mass loss was linked to the reduction of Bacteroidetes abundance, increased inflammation, and the disruption of mitochondrial morphology. These insights offer promising avenues for the identification of potential therapeutic targets aimed at mitigating muscle mass loss.

Role of secreted frizzled-related protein 4 in prediabetes and type 2 diabetes: a cross sectional study

BackgroundType 2 diabetes (T2D) has become an epidemic. Delays in diagnosis and as a consequent late treatment has resulted in high prevalence of complications and mortality. Secreted frizzled-related protein 4 (SFRP4), has been recently identified as a potential early biomarker of T2D related to obesity, due to its association with low grade inflammation in adipose tissue and impaired glucose metabolism. We aimed to evaluate the role of SFRP4 in prediabetes and T2D in a Mexican population.MethodsThis was a cross-sectional study that included 80 subjects with T2D, 50 subjects with prediabetes and 50 healthy individuals. Fasting SFRP4 and insulin concentrations were measured by ELISA. Human serum IL-10, IL-6, IL-1β and IL-8 levels were quantified by flow cytometry. Genotyping was performed by TaqMan® probes.ResultsPrediabetes and T2D patients had significantly higher SFRP4 levels than controls (P < 0.05). In turn, prediabetes subjects had higher SFRP4 concentrations than control subjects (P < 0.05). Additionally, the prediabetes and T2D groups had higher concentrations of proinflammatory molecules such as IL-6, IL-1β and IL-8, and lower concentrations of IL-10, an anti-inflammatory cytokine, than controls (P < 0.001). The serum SFRP4 concentrations were positively correlated with parameters that are elevated in prediabetes and T2D states, such as, HbA1c and homeostasis model assessment insulin resistance (HOMA-IR), (r = 0.168 and 0.248, respectively, P < 0.05). Also, serum SFRP4 concentrations were positively correlated with concentrations of pro-inflammatory molecules (CRP, IL-6, IL-1β and IL-8) and negatively correlated with the anti-inflammatory molecule IL-10, even after adjusting for body mass index and age (P < 0.001). The genetic variant rs4720265 was correlated with low HDL concentrations in T2D (P < 0.05).ConclusionsSFRP4 correlates positively with the stage of prediabetes, suggesting that it may be an early biomarker to predict the risk of developing diabetes in people with high serum concentrations of SFRP4, although further longitudinal studies are required.

IL-1 and TNF mediates IL-6 signaling at the maternal-fetal interface during intrauterine inflammation.

IL6 signaling plays an important role in triggering labor and IL6 is an established biomarker of intrauterine infection/inflammation (IUI) driven preterm labor (PTL). The biology of IL6 during IUI at the maternal-fetal interface was investigated in samples from human subjects and non-human primates (NHP). Pregnant women with histologic chorioamnionitis diagnosed by placenta histology were recruited (n=28 term, n=43 for preterm pregnancies from 26-36 completed weeks of gestation). IUI was induced in Rhesus macaque by intraamniotic injection of lipopolysachharide (LPS, n=23). IL1 signaling was blocked using Anakinra (human IL-1 receptor antagonist, n=13), and Tumor necrosis factor (TNF) signaling was blocked by anti TNF-antibody (Adalimumab n=14). The blockers were given before LPS. All animals including controls (intraamniotic injection of saline n=27), were delivered 16h after LPS/saline exposure at about 80% gestation. IUI induced a robust expression of IL6 mRNAs in the fetal membranes (chorion-amnion-decidua tissue) both in humans (term and preterm) and NHP. The major sources of IL6 mRNA expression were the amnion mesenchymal cells (AMC) and decidua stroma cells. Additionally, during IUI in the NHP, ADAM17 (a protease that cleaves membrane bound IL6 receptor (IL6R) to release a soluble form) and IL6R mRNA increased in the fetal membranes, and the ratio of IL6 and soluble forms of IL6R, gp130 increased in the amniotic fluid signifying upregulation of IL6 trans-signaling. Both IL1 and TNF blockade suppressed LPS-induced IL6 mRNAs in the AMC and variably decreased elements of IL6 trans-signaling. These data suggest that IL1 and TNF blockers may be useful anti-inflammatory agents via suppression of IL6 signaling at the maternal-fetal interface.

A phase 1/1b, first-in-human, multi-part study of DF6215, an engineered IL-2Rα-active agonist, to investigate the safety, tolerability, pharmacokinetics, and biological and clinical activity in patients with advanced solid tumors.

TPS2704 Background: IL-2 is a pleiotropic cytokine that stimulates strong anti-tumor responses by promoting expansion and activation of cytotoxic tumor-infiltrating immune cells including CD8 + T cells and NK cells (1). Aldesleukin is an approved rhIL-2; however, it has clinical limitations due to short half-life, requirement for 5 days of hospitalization per cycle, and high toxicity [eg, cytokine-related toxicity, capillary leak syndrome (CLS)] that limits its use [aldesleukin USPI]. DF6215 is a modified monovalent human IL-2 that binds to the IL2R. Preclinically, DF6215 administration results in sustained and modulated IL-2 pharmacology, leading to protracted pharmacodynamic (PD) effects and enhanced anti-tumor activity without observation of CLS in murine and primate toxicology studies. In response to the limitations of previous IL-2 therapeutics, DF6215 was designed as an IL-2Rα-active agonist with increased IL-2Rβγ stimulation to improve the benefit-to-risk ratio of historic IL-2 drugs. An additional design element is a prolonged elimination half-life due to Fc fusion, allowing for less frequent dosing. Methods: This is a first-in-human, open-label, multipart, Phase 1/1b clinical trial to characterize the initial safety, tolerability, pharmacokinetics, and preliminary efficacy of DF6215 in patients with advanced solid tumors. The Phase 1 dose escalation (DE) starting dose is 10 ug/kg IV every 2 weeks (up to 42 subjects expected to be enrolled), and the Safety/PK/PD will be explored at doses already characterized as safe in dose escalation and potentially efficacious (max of 40 subjects). Phase 1b includes an efficacy expansion (EE) cohort in subjects with advanced melanoma who have received prior anti-PD-1 agents and, if the tumor harbors a BRAF-activating mutation, a BRAF inhibitor. Key inclusion criteria include ≥ 18 years of age; ECOG PS of 0 or 1; adequate organ function, and locally advanced or metastatic solid tumors for which standard therapy options have been exhausted; and availability of baseline tumor biopsy. Primary objectives include determination of the MTD of DF6215 in DE and demonstration of clinical activity of DF6215 in the EE. Secondary objectives include assessment of safety and tolerability, pharmacokinetics, and immunogenicity of DF6215. This study plans to enroll up to 102 subjects across ~30 global centers in the US, European, and the Asia/Pacific regions, and is currently open to enrollment. 1. Arenas-Ramirez N, et al. 2015. Trends Immunol. Clinical trial information: NCT06108479 .

A phase I study to evaluate the safety and tolerability of JCXH-211 (a self-replicating mRNA encoding IL-12) intratumoral injection in patients with malignant solid tumors: Results from the phase Ia dose escalation.

2539 Background: Although interleukin (IL)-12 demonstrated strong preclinical antitumor activity and potent immune-stimulating potentials clinically, systemic administration of IL-12 protein was associated with poor clinical safety profiles. IL-12 functions locally through paracrine and autocrine mechanisms, thus maximizing local concentration of IL-12 in the tumors is important. JCXH-211 is a self-replicating ribonucleic acid (srRNA) encoding human IL-12 encapsulated in lipid nanoparticles. Intratumoral (IT) administration of JCXH-211 is expected to produce the desired spatiotemporal distribution of IL-12 in the cancer lesions. srRNA features enhanced translatability in immunosuppressed tumor microenvironment (TME) than in normal tissues further limiting off-target toxicity. Additionally, the self-replicating nature of JCXH-211 engages multiple immune effector mechanisms and helps inflame TME. We present interim results of the dose escalation part of JCXH-211 IT injection as monotherapy in patients (pts) with malignant solid tumors. Enrollment for the 100 μg cohort is ongoing and updated data will be presented at the meeting. Methods: Pts with advanced solid tumors suitable for IT injection are enrolled. JCXH-211 at 5, 25, 50, and 100 μg are given every 4 weeks (Q4W). Dose Limiting Toxicities (DLT) are monitored for 28 days after 1st dose. Dose escalation follows “3+3” principle. Tumor assessment is performed Q6W using RECIST v1.1. Results: Ten pts with advanced cancers have been enrolled in 5 μg, 25 μg, and 50 μg cohorts: 3 melanoma (MEL), 3 breast cancer (BC), 2 head and neck cancer (HNSC), 1 nasopharyngeal cancer and 1 sarcoma. Nine pts completed DLT observation without DLT; 1 pt withdrew early. No drug-related SAE was reported. Most drug-related AEs were Grade 1/2 and recovered quickly. Only 1 pt in the 25 μg cohort reported 3 Grade 3 AEs possibly related to drug: lymphocytopenia (2 events) and anemia. Seven pts completed at least one post-dose tumor assessment. Three pts experienced shrinkages of the treated lesions by 13.0%, 33.3% and 43.0%, corresponding to BC, HNSC and MEL in the 5 μg, 25 μg, and 50 μg cohorts, respectively. Tumor shrinkage of 31% was also observed in a non-injected lesion of an HNSC pt receiving 25μg JCXH-211 suggesting abscopal effects. Histopathological analysis of the treated lesions demonstrated increased T and NK cell infiltration (as high as 138 folds) post study drug administration. Conclusions: JCXH-211 IT administration with doses of 5μg, 25μg, 50μg Q4W demonstrated good safety profile. Antitumor activities were observed in the heavily pretreated late-stage pts. Significant increases of T and NK cell infiltration were observed. These data support continued evaluation of JCXH-211 IT. Clinical trial information: NCT05727839 .

Preliminary safety, antitumor activity, and pharmacodynamics of intratumoral ABO2011 (IL-12 mRNA) in patients with advanced solid tumors.

e14583 Background: Interleukin (IL)-12 is a potent immunomodulatory cytokine demonstrating robust antitumor activity in multiple preclinical studies and potent immune-stimulating potential in humans. However, systemic delivery of IL-12 can result in life-threatening toxicity and therefore has shown limited efficacy at doses that can be safely administered. ABO2011 is a human IL-12 mRNA encapsulated in lipid nanoparticle and has been developed to be more effective and less toxic through the use of novel delivery technology and intratumoral injection. Methods: This phase I trial enrolled adults with advanced solid tumors who failed available standard treatments (NCT05392699). The trial was designed to primarily evaluate the safety of ABO2011 in single-dose cohorts and multiple-dose cohorts using “3+3” dose escalation approach. Patients (pts) would receive intratumoral ABO2011 at either a single dose, QW with 3 doses overall, every 3 weeks (Q3W) or QW followed by Q3W. Results: As of 09 December 2023, a total of 20 pts were enrolled and received at least one dose of ABO2011 at 4 μg single dose, 4 μg QW for 3 times, 12 μg single dose, 12 μg QW for 3 times followed by Q3W, 24 μg Q3W and 48 μg Q3W. Seventeen (85%) pts experienced treatment-related adverse events (TRAEs), and all were grade 1 or 2, including pyrexia (35%), pain (25%), and gamma-glutamyl transferase increased (25%). There were no dose-limiting toxicities or treatment-related SAEs reported and no TRAEs leading to treatment discontinuation. Of the 17 evaluable pts per RECISIT v1.1, 5 maintained stable disease, and the progression free survivals achieved 6.0 months, 5.6 months and 4.1 months for pts with colorectal cancer (6th line), non-small cell lung cancer (5th line) and pancreatic cancer (4th line), respectively. Peripheral blood IL-12 and IFN-γ levels increased and peaked at 24 hours and 48 hours after dosing, respectively, indicating expression of IL-12 and subsequent induction of IFN-γ after ABO2011 administration. Dose-dependent elevation of CXCL9, CXCL10, CXCL11 in peripheral blood was observed, consistent with the activation of downstream pathway of IFN-γ. CD8 T cells infiltration and PD-L1 expression were increased in tumor microenvironment (TME) at day 7, and further RNA sequencing analysis revealed increase of activated CD8 T cell and antigen processing and presentation after study treatment, suggesting the regulation of TME by ABO2011 and supporting the combination with PD-1(L1) inhibitor. Conclusions: Intratumoral ABO2011 was safe and well-tolerated in pts with advanced solid tumors. The pharmacodynamic changes were as expected. Promising preliminary antitumor activities of the single agent and the pharmacodynamic profiles support further investigation of ABO2011 in combination with immune checkpoint inhibitor. Clinical trial information: NCT05392699 .

Evaluation of Immunological Levels of IL-37, IL-38, and IL-17A in Iraqi Patients with Diabetic Foot Ulcers

t Diabetes mellitus, a significant cause of mortality around the globe, can result in several secondary complications, including diabetic foot syndrome, which is brought on by diabetic neuropathy and ischemia. Approximately 15% of diabetic patients suffer from diabetic foot complications, which results in high rates of morbidity and mortality of people with Diabetes mellitus. The study's objective is to measure serum levels of IL-37, IL-38 &amp; IL-17A in diabetic mellitus (type 1 &amp; 2) patients with and without diabetic foot ulcer complications and in the control group as well as the possibility of using them as early biomarkers for diagnosis of diabetic mellitus complications &amp; future prevention. Overall, of 193 participants included in this case-control study, they were divided into three groups: the first one contains patients with type 1 diabetes mellitus (29 with diabetic foot ulcer DFU+ 35 non-diabetic foot), the second group includes type 2 diabetes mellitus patients (41 with DFU + 38 Non). The third group includes (50) as apparently healthy controls. Serological techniques of sandwich ELISA did laboratory tests for specific serum human IL-37, IL-38, and IL- 17A. The study revealed that IL-37 and IL-17A levels were significantly high (P&lt;0.01) in all diabetic groups compared to the control healthy group. The results of IL-38 show substantially higher levels of T1DM and T2DM (P&lt;0.05) compared to the control group. In addition, the DFU group of T2DM illustrated higher levels of IL-37, IL-38, and IL-17A compared with other diabetic groups. In conclusion, Iraqi DM subjects with and without complications had higher values of interleukins, IL-37, IL-38, and IL-17A, than healthy controls, which suggests an inflammatory state in these patients. In addition, the DFU of T2DM patients expressed higher levels of interleukins than other diabetic groups. This can be focused on using them as novel therapeutic targets for preventing and treating DM complications. As well as the possibility of using them as markers of inflammation and progression for complications in DM patients.

#2324 Do urinary tract infections trigger chronic inflammation in the kidney allograft? Interleukin 17 levels in the urine of kidney recipients

Abstract Background and Aims Urinary tract infections (UTI) represent the most common complication following kidney transplantation. Their role in triggering immune phenomena in kidney allograft remains unclear. Interleukin 17 (IL17) represents the significant branch in antimicrobial immunity. At the same time IL17 has been found associated with autoimmune inflammatory disorders. The triggering role of infections in autoimmunity as well as the similarities between autoimmunity and post-transplant immunity have been postulated. The aim of the study was to analyze the levels of urine IL 17A levels in relation to prior UTI history and the timeframe after the last UTI episode. Method The group of 91 kidney recipients who received kidney transplantation between 2016 and 2019 was analyzed. The set of 233 urine samples (109 from females and 124 from males) obtained within first 24 months post-transplant was investigated, among these 144 samples from the patients with no previous UTI history and 89 samples from the patients with previous UTI history (1-22 months after the last episode, average 6.7 ± SD 5.7). Furthermore, 18 samples obtained at the time of an UTI episode and 29 samples from healthy controls were included. Urine IL 17A levels were measured with Human IL 17 ELISA Kit (Sigma Aldrich) and urine IL/creatinine concentrations ratio was calculated for each value. Szapiro-Wilk test was used to assess data distribution, Kruskal-Wallis test was used to assess the differences between independent groups and two proportions Z test was used to compare the groups compositions. Results Interleukin 17A appeared detectable in 105 out of the 233 investigated samples (45%), with significantly higher frequency in samples from the recipients with previous UTI history (p = 0.02). The difference was not significant in the group who underwent recurrent UTI episodes vs. these after a single UTI episode. Among the samples in which IL 17A was detected, the period between the last UTI episode and sample collection was divided into three timeframes: 1-3 months, 4-12 months and 12-24 months. Urine IL17A levels appeared significantly higher independently in each timeframe post-UTI when compared to the samples from the recipients without UTI history and controls (p = 0.0001). Fig. 1 It was apparent within the whole group but also in males and females separately. Further, urine IL17A levels were significantly higher in males 1-3 months after the last UTI episode than in females in the analogous period (p = 0.001). Conclusion Interleukin 17A was more frequently detected in urine samples from kidney recipients with previous UTI history. UTI history impacted IL17A urine levels in the analyzed kidney recipients. Within the first 24 months post-transplant, these were higher in the recipients with UTI history prior to the sample collection and it was independent on the timeframe between the last UTI episode and the sample collection. The factors other than UTI history which influence urine IL17A detectability are not fully recognized and remain under investigation.

In vitro identification of single-stranded DNA aptamers targeting human IL-23 using the protein-SELEX strategy

Interleukin (IL)-23 inhibitor monoclonal antibodies shown significant efficacy in treating autoimmune diseases. DNA or RNA aptamers exhibit comparable specificity to antibodies, are cost-effective, non-immunogenic, and do not have batch to batch variation. This study aimed to characterize a single-stranded DNA (ssDNA) aptamer targeting human IL-23. The alpha subunit of IL-23 (P19) and intact IL-23 were cloned, expressed, and the proteins finally were purified through Ni2+-iminodiacetic acid affinity chromatography. The selection and characterization of ssDNA aptamer against P19 were conducted using the protein-systematic evolution of ligands by exponential enrichment (SELEX). Dot blot assay was carried out to monitor binding of the aptamer output of SELEX rounds, to P19 protein. The dissociation constant (Kd) of aptamers with positive results in dot blot assay, determined based on their binding to IL-23 using an ELISA method. Recombinant P19 and IL-23 proteins were 26 and 72 kDa, respectively, observed on SDS-PAGE .12 %. The aptamers output from 7, 8, 9, 10, 11, and 12 rounds of the SELEX was monitored by dot blot assay, revealing that the aptamer from the round 8 has stronger luminescent signal and was selected for TA-cloning. After analyzing the biotinylated aptamers from clones, positive clones in dot blot assay and ELISA were sequenced. Finally, the Kd calculation revealed three aptamers with high affinity, named A23P3, A23P6, and A23P15 with Kd values of 1.37, 2.139, and 2.88 nM, respectively. Results of this study introduced three specific anti-IL-23 ssDNA aptamers with high affinity, which could be utilized for therapeutic and diagnostic purposes.

Lung Transplant Immunomodulation with Genetically Engineered Mesenchymal Stromal Cells-Therapeutic Window for Interleukin-10.

Lung transplantation results are compromised by ischemia-reperfusion injury and alloimmune responses. Ex vivo lung perfusion (EVLP) is used to assess marginal donor lungs before transplantation but is also an excellent platform to apply novel therapeutics. We investigated donor lung immunomodulation using genetically engineered mesenchymal stromal cells with augmented production of human anti-inflammatory hIL-10 (MSCsIL-10). Pig lungs were placed on EVLP for 6 h and randomized to control (n = 7), intravascular delivery of 20 × 106 (n = 5, low dose) or 40 × 106 human MSCs IL-10 (n = 6, high dose). Subsequently, single-lung transplantation was performed, and recipient pigs were monitored for 3 days. hIL-10 secretion was measured during EVLP and after transplantation, and immunological effects were assessed by cytokine profile, T and myeloid cell characterization and mixed lymphocyte reaction. MSCIL-10 therapy rapidly increased hIL-10 during EVLP and resulted in transient hIL-10 elevation after lung transplantation. MSCIL-10 delivery did not affect lung function but was associated with dose-related immunomodulatory effects, with the low dose resulting in a beneficial decrease in apoptosis and lower macrophage activation, but the high MSCIL-10 dose resulting in inflammation and cytotoxic CD8+ T cell activation. MSCIL-10 therapy during EVLP results in a rapid and transient perioperative hIL-10 increase and has a therapeutic window for its immunomodulatory effects.