Mouse Interleukin Research Articles

Abstract 1122: Selinexor synergizes with IL-12 to inhibit tumor growth in syngeneic mouse models of melanoma

Abstract Background: Melanoma is the most deadly skin cancer and represents 4.6% of all cancers diagnosed within the US (American Cancer Society). Risk factors for melanoma are almost entirely related to pale skin color and sunburns. Selinexor is a clinically approved oral inhibitor of nuclear exportation protein Exportin 1 (XPO1/CRM1). In preclinical studies, selinexor significantly inhibited growth of A375 and CHL-1 human melanoma cell lines in vivo at well tolerated doses (Yang et al. 2014). Interleukin 12 (IL-12) is a cytokine produced by dendritic cells, macrophages, neutrophils, and human B-lymphoblastoid cells in response to antigenic stimulation. IL-12 drives the differentiation of T-cells into T helper (Th1) cells, in addition to stimulating T-cell and NK cell activity, specifically through the activation of interferon-γ (IFN-γ). IL-12 exerts antitumor activity through IFN-γ-dependent and independent mechanisms, which include modulation of the immune system and anti-angiogenesis (Zhang et al. 2016). The objective of this study was to compare the impact and scheduling of recombinant mouse IL-12 alone and in combination with selinexor on the growth of murine B16F10 melanoma cells grown as subcutaneous tumors in C57B/6 mice. Methods: Sixty, 6-7 weeks old female C57B/6 mice were inoculated subcutaneously in the left flank with 1 x 105 B16F10 cells. Treatment was initiated when the tumors reached a mean volume of 100 mm3 (standard deviation ± 6.3 mm3). Mice were allocated to 6 groups of 10 such that mean tumor volume in each group was within the range of 11.8 to 13.4 mm3, then treated with: oral selinexor (10mg/kg, twice weekly on day 0 and 2) combined with a pre-dose of subcutaneous IL-12 (1µg/kg, once weekly on day -2); selinexor with post-dose IL-12 (1µg/kg, once weekly on day 3); selinexor alone; IL-12 alone (day -2); IL-12 alone (day 3); or vehicle. Animal weight was recorded daily, and tumors were measured 3 days a week to record percent of tumor growth inhibition (%TGI). Results: Both selinexor and IL-12 demonstrated single agent activity against B16F10 tumor in vivo. All treatment groups showed significant tumor growth inhibition as compared to vehicle. The group receiving selinexor with pre-dose IL-12 showed the largest tumor inhibition (%TGI=92%). Significant weight loss compared to vehicle occurred only in the single agent selinexor group (p=0.039). Conclusions: Synergistic anti-cancer activity of selinexor and IL-12 warrants further clinical investigation. Further mechanistic studies to understand the effects of IL-12 priming on increasing the effectiveness of selinexor will be undertaken. Citation Format: Shira Orr, Leah Henegar, Christopher J. Walker, Feng Wang, Trinayan Kashyap, Marie Maloof, Kathleen Martyn, Michael G. Kauffman, Sharon Shacham, Yosef Landesman. Selinexor synergizes with IL-12 to inhibit tumor growth in syngeneic mouse models of melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1122.

Abstract CT565: Clinical and translational results of a phase I study, tocilizumab plus gemcitabine/nab-paclitaxel in patient with gemcitabine/nab-paclitaxel-refractory metastatic pancreatic cancer

Abstract Background: Interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) pathway inhibition may overcome chemoresistance of metastatic pancreatic cancer (MPC) refractory to gemcitabine + nab-paclitaxel (GN) due to suppression of cancer associated fibroblast (CAF) and increase of drug infiltration in tumor. We sought to determine the safety, recommended dose of tocilizumab (TCZ), an IL-6 receptor monoclonal antibody, and biological correlates of tumor shrinkage in patients with GN-refractory MPC. Methods: This phase 1, dose finding trial was conducted following preclinical study of IL-6 signaling blockade in genetically engineered mouse model of pancreatic cancer (pGEMM) and patients-derived CAF, enrolled 10 patients with MPC that had progressed after GN. During the dose-finding part to determine dose-limiting toxicity (DLT), patients initially received TCZ 8 mg/kg on Day 1 and nab-paclitaxel 100 mg/m2 + gemcitabine 750 mg/m2 on Days 2, 9, and 16. The subsequent dose-expansion part used the TCZ dose identified in the dose-finding study. Before Cycle 1 and at the end of Cycle 1, biopsy of liver metastasis was performed 3 to 5 hours after levofloxacin (LVXF) administration to measure LVFX infiltration in tumor using matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI). Results: In pGEMM tumor, mouse IL-6 receptor antibody plus GN led pathological response. The growth of CAFs from patients with pancreatic cancer was inhibited by TCZ. In this phase I study, no DLTs occurred and the recommended dosage of TCZ was determined to be 8 mg/kg. Treatment-emergent adverse events (TEAEs) occurred in 80% of patients (Grade ≥3 in 60%) with decreased neutrophil count (n=5, 7 events) being the most common TEAE. Disease control rate was 80·0% (95%CI: 44·4-97·5) and tumor reduction during cycle 1 was observed in 4 patients who were defined as responder. In paired-biopsied samples, decrease of phosphorylated (p) STAT3 expression in tumor was observed in 7 of 8 patients (88%). Responder-related biological activities were increase of cleaved PARP expression of tumor nuclei (P = 0.01), decrease of proliferative CAF (P = 0.08), and increase of LVFX infiltration in tumor (P = 0.04). Decrease of pSTAT3 expression (P = 0.02) was favor to increase of LVFX infiltration against increase of gamma-H2AX, an index of gemcitabine exposure (P = 0.20). Conclusion: TCZ+GN-rechallenge had a manageable safety profile and showed preliminary activity via inhibition of CAF and gain of intratumoral drug infiltration in MPC. Citation Format: Shuichi Mitsunaga, Masafumi Ikeda, Kazunori Aoki, Kyoko Yamaguchi, Noriaki Sawada, Etsuko Fujii, Masanobu Nishidate, Takashi Fujitomo, Hideaki Mizuno, Yoko Kayukawa, Atsuhiko Kato, Mayu Makikawa, Hiroshi Imaoka, Mitsuhito Sasaki, Kazuo Watanabe, Hiroyuki Tsunoda, Kimio Terao, Atsushi Ochiai. Clinical and translational results of a phase I study, tocilizumab plus gemcitabine/nab-paclitaxel in patient with gemcitabine/nab-paclitaxel-refractory metastatic pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT565.

Generation of antagonistic monoclonal antibodies against the neoepitope of active mouse interleukin (IL)-18 cleaved by inflammatory caspases

Interleukin 18 (IL-18) is a member of the IL-1 family and plays an important role in both the innate and acquired immune systems. It is constitutively expressed as an inactive precursor (24 kDa) in various cell types, and the mature IL-18 (18 kDa) cleaved by inflammatory caspase-1/4 binds to the interleukin-18 receptor, thereby activating downstream signaling pathways. We previously generated anti-human IL-18 antibodies that specifically recognize the human IL-18 neoepitope cleaved by inflammatory caspase-1/4. Because the N-terminal amino acid sequences of the neoepitopes are different between human IL-18 and mouse IL-18, the anti-human IL-18 neoepitope antibodies do not recognize mouse mature IL-18. We have now generated novel anti-mouse IL-18 neoepitope antibodies. We also confirmed CXCL2 secretion from P-815 mouse cells by mouse IL-18 stimulation, and established a simple assay to evaluate the activity of mouse IL-18. Using this evaluation system, we confirmed that the anti-mouse IL-18 neoepitope antibodies could inhibit mouse IL-18. By demonstrating the therapeutic efficacy of the anti-mouse IL-18 neoepitope and function-blocking mAbs established in the present study in mouse models, corresponding to human inflammatory diseases in which IL-18 may be involved, such as inflammatory bowel diseases, we can provide the proof-of-concept that the previously established anti-human IL-18 neoepitope and function-blocking mAbs work in human inflammatory disorders corresponding to mouse models.

Identification of G-quadruplex anti-Interleukin-2 aptamer with high specificity through SELEX stringency

Aptamers are short single-stranded oligonucleotides capable of binding to various targets with high specificity and affinity. This study aimed to identify an aptamer against mouse interleukin-2 (mIL-2) as one of the most important cytokines in autoimmune diseases for diagnostic and therapeutic purposes. For this purpose, 14 SELEX rounds were performed on recombinant mIL-2 with high stringency. The dot blot and flow cytometry techniques were conducted to determine affinity, dissociation constant (Kd), specificity, and SELEX rounds screening. The stringency of rounds was considered based on aptamer/target incubation time, washing steps, and target proteins. Finally, the aptamer's structure was mapped and predicted by M-fold and QGRS Mapper web-based software. After 14 rounds, the flow cytometry analysis revealed that the 11th round was a proper round. The high-affinity aptamers M20 and M15 were chosen for their ability to bind mIL-2. According to DNA folding software, M20 and M15 aptamers had G-quadruplex and stem-loop structures, respectively. The M20 aptamer affinity was greater than M15, and its predicted Kd was 91 nM. A simple SELEX protocol with round stringency was explained to identify DNA aptamers against protein targets. The reported G-quadruplex aptamer might have potential diagnostic or therapeutic application in IL-2–related disorders.

Target-Mediated Drug Disposition Affects the Pharmacokinetics of Interleukin-10 Fragment Crystallizable Fusion Proteins at Pharmacologically Active Doses.

Fragment crystallizable (Fc) fusion is commonly used for extending the half-life of biotherapeutics such as cytokines. In this work, we studied the pharmacokinetics of Fc-fused interleukin-10 (IL-10) proteins that exhibited potent antitumor activity in mouse syngeneic tumor models. At pharmacologically active doses of ≥0.1 mg/kg, both mouse Fc-mouse IL-10 and human Fc-human IL-10, constructed as the C terminus of the Fc domain fused with IL-10 via a glycine-serine polypeptide linker, exhibited nonlinear pharmacokinetics after intravenous administration to mice at the doses of 0.05, 0.5, and 5 mg/kg. With a nominal dose ratio of 1:10:100; the ratio of the area under the curve for mouse Fc-mouse IL-10 and human Fc-human IL-10 was 1:181:1830 and 1:75:633, respectively. In contrast, recombinant mouse or human IL-10 proteins exhibited linear pharmacokinetics in mice. Compartmental analysis, using the Michaelis-Menten equation with the in vitro IL-10 receptor alpha binding affinity inputted as the Km, unified the pharmacokinetic data across the dose range. Additionally, nontarget-mediated clearance estimated for fusion proteins was ∼200-fold slower than that for cytokines, causing the manifestation of target-mediated drug disposition (TMDD) in the fusion protein pharmacokinetics. The experimental data generated with a mouse IL-10 receptor alpha-blocking antibody and a human Fc-human IL-10 mutant with a reduced receptor binding affinity showed significant improvements in pharmacokinetics, supporting TMDD as the cause of nonlinearity. Target expression and its effect on pharmacokinetics must be determined when considering using Fc as a half-life extension strategy, and pharmacokinetic evaluations need to be performed at a range of doses covering pharmacological activity. SIGNIFICANCE STATEMENT: Target-mediated drug disposition can manifest to affect the pharmacokinetics of a fragment crystallizable (Fc)-fused cytokine when the nontarget-mediated clearance of the cytokine is decreased due to neonatal Fc receptor-mediated recycling and molecular weight increases that reduce the renal clearance. The phenomenon was demonstrated with interleukin-10 Fc-fusion proteins in mice at pharmacologically active doses. Future drug designs using Fc as a half-life extension approach for cytokines need to consider target expression and its effect on pharmacokinetics at relevant doses.

Maintenance and gene electrotransfer efficiency of antibiotic resistance gene-free plasmids encoding mouse, canine and human interleukin-12 orthologues

Interleukin 12 (IL-12) is a cytokine used as a therapeutic molecule in cancer immunotherapy. Gene electrotransfer mediated delivery of IL-12 gene has reached clinical evaluation in the USA using a plasmid that in addition to IL-12 gene also carry an antibiotic resistance gene needed for its production in bacteria. In Europe however, European Medicines Agency recommends against the use of antibiotics during the production of clinical grade plasmids. We have prepared several antibiotic resistance gene-free plasmids using an antibiotic-free selection strategy called operator-repressor titration, including plasmids encoding mouse, canine and human IL-12 orthologues. The aim of this study was to evaluate the maintenance of these plasmids in bacterial culture and test their transfection efficiency using gene electrotransfer. Plasmid maintenance was evaluated by determining plasmid yields and topologies after subculturing transformed bacteria. Transfection efficiency was evaluated by determining the plasmid copy number, expression and cytotoxicity after gene electrotransfer to mouse, canine and human melanoma cells. The results demonstrated that our IL-12 plasmids without an antibiotic resistance gene are stably maintained in bacteria and provide sufficient IL-12 expression after in vitro gene electrotransfer; therefore, they have the potential to proceed to further in vivo evaluation studies.

A human orthogonal IL-2 and IL-2Rβ system enhances CAR T cell expansion and antitumor activity in a murine model of leukemia.

Interleukin-2 (IL-2) is a central T cell cytokine that promotes T cell proliferation and effector function; however, toxicity due to its pluripotency limits its application to enhance CAR T cell immunotherapy. Previously, mouse IL-2 and its cognate receptor were engineered to create an orthogonal (ortho) cytokine-cytokine receptor pair capable of delivering an IL-2 signal without toxicity. Here, we engineered a human orthogonal IL-2 (ortho-hIL-2) and human orthogonal IL-2Rβ (ortho-hIL-2Rβ) pair, containing human-specific mutations. Ortho-hIL-2 is selective toward ortho-hIL-2Rβ–expressing cells with no appreciable signaling on wild-type T cells. Ortho-hIL-2 induces IL-2 receptor signaling and supports proliferation of both an IL-2–dependent cell line and primary T cells transduced to express the ortho-hIL-2Rβ. Using CD19-specific chimeric antigen receptor (CAR) T cells, we show that ortho-hIL-2 induces a dose-dependent increase in ortho-hIL-2Rβ+ CAR T cell expansion in vivo by as much as 1000-fold at 2 weeks after adoptive transfer into immunodeficient mice bearing CD19+ Nalm6 leukemia xenografts. Ortho-hIL-2 can rescue the antileukemic effect of an otherwise suboptimal CAR T cell dose. In addition, ortho-hIL-2 administration initiated at the time of leukemic relapse after CAR T cell therapy can rescue an otherwise failed antileukemic response. These data highlight the potential of combining an orthogonal cytokine approach with T cell–based immunotherapies to augment the antitumor efficacy of engineered T cells.

In Situ Vaccination with IL-12Fc and TLR Agonist - a Crucial Role for B Cells in Generating Anti-Tumor T Cell Immunity

Interleukin 12 (IL-12) is a potent proinflammatory cytokine that plays a central role in regulating both innate and adaptive immune responses. This cytokine has been tested as a cancer immunotherapy agent based on its impressive anti-tumor effects in animal models. Clinically, systemic delivery of IL-12 incurred dose-limiting toxicities because of its pharmaco-kinetic extremes of peaks and troughs, a result of its short serum half-life. Here, we evaluated mDF6006, a mouse IL-12 Fc-fusion protein which was designed to have a prolonged half-life with equivalent potency to recombinant mouse IL-12.We hypothesized that local intra-tumoral (IT) delivery of low doses IL-12Fc would lead to immune-therapeutic potential.To screen candidates for in situ vaccination, we have employed a preclinical strategy whereby the same syngeneic tumor is implanted at two separate sites in the body. One tumor is then injected with the test agents and the resulting immune response is detected by the regression of the distant, untreated tumor.Using this assay for abscopal therapeutic effects we have tested a variety of immune stimulatory ligands and antibodies against immune checkpoints and T cell co-stimulatory targets, alone and in multiple combinations.Among these, the combination of unmethylated CG-enriched oligodeoxynucleotide (CpG), a TLR9 ligand, and IL-12Fc has provided the most impressive results. TLRs, expressed in antigen-presenting cells and are components of the innate immune system that recognize molecular patterns on bacterial, fungal, or viral pathogens.In a mouse model of lymphoma, IT injection of CpG and IL-12Fc, combined to cause tumor eradication at both the injected site and the untreated tumor. All these results were dependent on T cells in the animal.During this therapy we observed a significant increase in activated B cells within the treated tumor and especially in its draining lymph nodes (dLN). This B cell stimulation was contributed by both the CpG and the IL12-Fc. Surprisingly, depletion of the animals of B cells by an anti-CD20 antibody completely ablated the abscopal therapeutic effect.B cells function as antigen-presenting cells, secrete cytokines and are precursors of antibody producing plasma cells. To evaluate which of these roles B cells play in the therapeutic efficacy of CpG and IL-12Fc we isolated B cells from the LN of the treated mice and co-cultured them with T cells. These B cells activated T cells in an antigen-specific manner that was MHC dependent. No such T cell activation occurred when MHC molecules were blocked with anti-MHC antibodies or when the cells were physically separated by a semi-permeable membrane. Thus, local CpG and IL12-Fc directly activated B cells to present tumor antigens to specific T cells in the tumor microenvironment, resulting ultimately in an abscopal tumor response.IL-12Fc and CpG are both currently in phase-1/2 trials as single agents and in combinations with checkpoint antibodies. Our results provide strong rationale for using low doses of IL-12Fc with CpG as an in situ therapeutic vaccine for lymphoma. DisclosuresLevy: Virsti: Membership on an entity's Board of Directors or advisory committees; Khloris: Membership on an entity's Board of Directors or advisory committees; Abintus Bio: Membership on an entity's Board of Directors or advisory committees; Kira: Membership on an entity's Board of Directors or advisory committees; Walking Fish: Membership on an entity's Board of Directors or advisory committees; Immunocore: Membership on an entity's Board of Directors or advisory committees; Spotlight: Membership on an entity's Board of Directors or advisory committees; Viracta: Membership on an entity's Board of Directors or advisory committees; Apexigen: Membership on an entity's Board of Directors or advisory committees; Dragonfly: Membership on an entity's Board of Directors or advisory committees; Nurix: Membership on an entity's Board of Directors or advisory committees; Teneobio: Membership on an entity's Board of Directors or advisory committees; GigaGen: Membership on an entity's Board of Directors or advisory committees; BeiGene: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; Quadriga: Membership on an entity's Board of Directors or advisory committees.

Mesenchymal Stem Cell Derived Extracellular Vesicles Reverse Radiation-Induced Cytokine Storm

Cytokine storm or cytokine release syndrome is a systemic inflammatory response to different triggers which leads to excessive activation of immune cells with the release of a large amount of pro-inflammatory cytokines. These uncontrolled and excessive releases of cytokines accompany multisystem organ failure and death. Acute radiation exposure leads to acute hematopoietic and acute gastrointestinal syndromes with early mortality. Cytokine storm accompanies these syndromes and significantly complicates the clinical outcomes.Recent data has suggested that the inflammasome and associate cytokine storm may play a major role in various injuries and tissue problems after irradiation. Inflammasome activates caspase-1 which give rise to the proinflammatory cytokines such as interleukin-18 (IL-18) and interleukin-1 beta (IL-1β) into their biologically active forms and further induce other cytokines release.Extracellular vesicles (EVs) are nanosized lipid bilayer vesicles, released from cells in the whole body. EVs play an important role in intercellular communication. EVs have been shown to transfer protein, lipid, DNA, mRNAs, and microRNAs to recipient cells, thereby mediating a variety of biological responses. We have been investigating the capacity of marrow-derived mesenchymal stem cell extracellular vesicles (MSC-EV) to reverse radiation damage to murine bone marrow stem/progenitor cells.In this study, we have evaluated the effect of MSC-EVs on reversal of high dose radiation injury to bone marrow cells. C57BL/6 mice received 0, 700, and 950-1000cGy WBI (either single or split dose). After 24 hrs. post-irradiation, mice received an IV infusion of 2X10 9 hMSC EVs daily for three days with a control group receiving vehicle only. Marrow was harvested from 700 cGy exposed mice which were vesicle treated or not. These marrow cells were evaluated for engraftment into 950 cGy exposed mice. The engraftment rates in mice transplanted with marrow from irradiated EV injected animals was 34.66±14.19% at 4 months post transplantation and marrow from the group not treated with EV gave an engraftment rate of 6.92±3.53%. We further evaluated the effect of MSCs-EVs on decreasing the mortality in mice exposed to 950cGy WBI. The MSC-EV untreated mice were dead between 12-18 days post radiation, but MSC-EV treated mice maintained a 60% survival rate at 130 days post radiation, suggesting that MSC-EV treatment could significantly extend the survival rate of mice after exposure to lethal radiation. We evaluated the circulating inflammatory markers in 1000 cGy whole body irradiated mice with/without MSC-EV treatment. Twenty-four hours post-irradiation, mice received an IV infusion of 2x10 9 hMSC EVs every day for three days with a control group receiving vehicle only. The serum was collected after 8 days post radiation for inflammatory response analysis by ProcartaPlex multiplex immunoassays. There were dramatic increases in cytokines secretion in serum including IFN gamma, IL-12p70, IL-13, IL-1 beta, IL-2, IL-5, IL-6, TNF alpha, GM-CSF and IL-18 in irradiated mice compared to non-irradiated mice and a significant reduction in cytokine levels after MSC-EVs treatment. We further found significantly higher levels of IL-1 beta and activated caspase-1 p20 protein expression in the spleen after 1000cGy radiation compared to non-radiated mice. Moreover, we found that with MSC-EV treatment, the expression level of IL-1 beta and active caspase-1 p20 was significantly inhibited in spleen compared to untreated irradiated mice, indicating that the inhibition of cytokine storm post-radiation by MSC-EVs might be mediated by inhibition of inflammasome activation. Our study suggests that EV Inhibition of an inflammasome mediated cytokine storm could be therapeutically important in many disease entities. DisclosuresNo relevant conflicts of interest to declare.

Immune Modulatory Effect of Outer Membrane Vesicles Derived from Salmonella on Mouse Bone Marrow-Derived Dendritic Cells

To study the effect of outer membrane vesicles (OMVs) derived from Salmonella typhimurium (ST) on the ultrastructural features and immune function of dendritic cells (DC). Mice bone marrow cells were collected aseptically, and myeloid DC were generated by the combined induction and amplification with recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) and recombinant mouse interleukin-4 (rm IL-4). Cell morphology was observed under inverted phase contrast microscope and the phenotype was identified with flow cytometry. ST-OMVs were isolated through ultracentrifugation. The survival rate of DC was assessed with CCK-8 assay, and the stimulus concentration of OMVs was henceforth determined. The ultrastructural characteristics of DC loaded with OMVs were observed with transmission electron microscopy. The cytokine secretion, surface molecule expression and phagocytic capacity of DC were examined with flow cytometry. The DC induced and amplified in vitro displayed typical DC phenotype in morphological analysis and the purity of DC exceeded 85%. Transmission electron microscopy showed that there were large numbers of protrusions on the cell surface. After stimulation with ST-OMVs, it was observed that the dendritic structures on the surface of DC were reduced and a large number of phagolysosomes were found in the cytoplasm. In addition, increased numbers of mitochondria, swelling and typical apoptosis were observed. After treatment with ST-OMVs at 5 μg/mL and 10 μg/mL, the secretion of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) of DC increased significantly ( P<0.05). Furthermore, the immature DC could differentiate into mature DCs after stimulation with ST-OMVs, which were characterized by a decrease in phagocytic capacity ( P<0.05) and an upregulation of phenotypic markers ( P<0.05). ST-OMVs can stimulate DC to produce TNF-α and IL-1β and promote DC maturation and antigen presentation.

High-dose IL-2/CD25 fusion protein amplifies vaccine-induced CD4+ and CD8+ neoantigen-specific T cells to promote antitumor immunity

BackgroundImmunization with tumor neoantigens is a promising vaccine approach to promote antitumor immunity due to their high immunogenicity, lack of expression in normal tissue, and preferential induction of tumor neoantigen-specific...

Reduced pannexin 1-IL-33 axis function in donor livers increases risk of MRSA infection in liver transplant recipients.

Liver transplantation patients are at increased risk for methicillin-resistant Staphylococcus aureus (MRSA) infection, but the molecular mechanism remains unclear. We found that genetic predisposition to low pannexin 1 (PANX1) expression in donor livers was associated with MRSA infection in human liver transplantation recipients. Using Panx1 and Il-33-knockout mice for liver transplantation models with MRSA tail vein injection, we demonstrated that Panx1 deficiency increased MRSA-induced liver injury and animal death. We found that decreased PANX1 expression in the liver led to reduced release of adenosine triphosphate (ATP) from hepatocytes, which further reduced the activation of P2X2, an ATP-activating P2X receptor. Reduced P2X2 function further decreased the NLRP3-mediated release of interleukin-33 (IL-33), reducing hepatic recruitment of macrophages and neutrophils. Administration of mouse IL-33 to Panx1-/- mice significantly (P = 0.011) ameliorated MRSA infection and animal death. Reduced human hepatic IL-33 protein abundance also associated with increased predisposition to MRSA infection. Our findings reveal that genetic predisposition to reduced PANX1 function increases risk for MRSA infection after liver transplantation by decreasing hepatic host innate immune defense, which can be attenuated by IL-33 treatment.

Generation and Characterization of Torudokimab (LY3375880): A Monoclonal Antibody That Neutralizes Interleukin-33.

BackgroundInterleukin-33 (IL-33) is an alarmin that is released following cellular damage, mechanical injury, or necrosis. It is a member of the IL-1 family and binds to a heterodimer receptor consisting of ST2 and IL-1RAP to induce the production of a wide range of cellular mediators, including the type 2 cytokines IL-4, IL-5 and IL-13. This relationship has led to the hypothesis that the IL-33/ST2 pathway is a driver of allergic disease and inhibition of the IL-33 and ST2 association could have therapeutic benefit.MethodsIn this paper, we describe the selection of a phage antibody through the ability to bind human IL-33 and block IL-33/ST2 interaction. This hit antibody was then affinity matured by site-directed mutagenesis of the antibody complementarity-determining regions (CDRs). Further characterization of a fully human monoclonal antibody (mAb), torudokimab (LY3375880) included demonstration of human IL-33 neutralization activity in vitro with an NFκB reporter assay and IL-33 induced mast cell cytokine secretion assay, followed by an in vivo IL-33-induced pharmacodynamic inhibition assay in mice that used IL-5 production as the endpoint.ResultsTorudokimab is highly specific to IL-33 and does not bind any of the other IL-1 family members. Furthermore, torudokimab binds human and cynomolgus monkey IL-33 with higher affinity than the binding affinity of IL-33 to ST2, but does not bind mouse, rat, or rabbit IL-33. Torudokimab’s half-life in cynomolgous monkey projects monthly dosing in the clinic.ConclusionDue to torudokimab’s high affinity, its ability to completely neutralize IL-33 activity in vitro and in vivo, and the observed cynomolgus monkey pharmacokinetic properties, this molecule was selected for clinical development.

Interleukin-18: A Novel Participant in the Occurrence, Development, and Drug Therapy of Obliterative Bronchiolitis Postlung Transplantation.

Background Obliterative bronchiolitis (OB) was a main cause of deterioration of long-term prognosis in lung transplant recipients after the first posttransplant year. Proinflammatory cytokine interleukin-18 (IL-18) strengthened both the natural immunity and acquired immunity and played an important role in organ transplantation. The roles of IL-18 in the occurrence, development, and drug treatment of OB remained unclear. Methods Small interfering RNA (siRNA) against mouse IL-18 (siRNA-IL-18) was used to silence IL-18 expression. Mouse heterotopic tracheal transplantation model was used to simulate OB. Recipient mice were divided into 5 groups (n = 12) according to donor mouse strains and drug treatment: isograft group, allograft group, allograft+tacrolimus group, allograft+azithromycin group, and allograft+tacrolimus+azithromycin group. The luminal obliteration rates were pathological evaluation. Expressions of cytokines and MMPs were detected by real-time PCR, western blot, and enzyme chain immunosorbent assay (ELISA). Results The luminal obliteration rates of IL-18 of the siRNA-IL-18 group were significantly lower than those of the negative control group (p < 0.0001) and the blank control group (p = 0.0002). mRNA expressions of IFN-γ, EMMPRIN, MMP-8, and MMP-9 of the siRNA-IL-18 group were significantly lower than those of the negative and blank control groups. No tracheal occlusion occurred in grafts of the isograft group. The rates of tracheal occlusion of the allograft group, allograft+tacrolimus group, allograft+azithromycin group, and allograft+tacrolimus+azithromycin group were 72.17 ± 4.66%, 40.33 ± 3.00%, 38.50 ± 2.08%, and 23.33 ± 3.24%, respectively. There were significant differences between the 4 groups (p < 0.001). Serum protein expressions of IL-17 (p = 0.0017), IL-18 (p = 0.0036), IFN-γ (p = 0.0102), and MMP-9 (p = 0.0194) were significantly decreased in the allograft+tacrolimus+azithromycin group compared to the allograft group. Conclusions IL-18 could be a novel molecular involved in the occurrence, development, and drug treatment of OB.

Abstract 1714: Preclinical characterization of DF6002/BMS-986415, a novel differentiated IL-12 Fc-fusion protein with robust antitumor activity as monotherapy or in combination with anti-PD-1

Abstract Background: Interleukin 12 (IL-12) is a proinflammatory cytokine that plays a central role in regulating both innate and adaptive antitumor immune responses. Clinically, administration of recombinant human IL-12 (rhIL-12) has been limited by a short half-life (t1/2) requiring frequent dosing, causing tachyphylaxis as well as toxic side effects. DF6002/BMS-986415 is an IL-12 Fc-fusion protein designed to have a prolonged t1/2 while retaining equivalent potency to rhIL-12 and promoting sustained IFN-γ response with reduced toxicity. Here we present the preclinical evaluation of DF6002/BMS-986415 ± anti–PD-1. Methods: DF6002/BMS-986415 and rhIL-12 stimulation of IFN-γ release from human and cynomolgus macaque (Cyno) peripheral blood mononuclear cells (PBMCs) and in vivo exposure in Cynos was assessed. As rhIL-12 is inactive in mice, pharmacokinetic, pharmacodynamic, and antitumor effects were evaluated with mDF6006, a mouse surrogate of DF6002/BMS-986415, and recombinant mouse IL-12 (rmIL-12). Tumor growth was evaluated in inflamed (CT26 and MC38) and immunosuppressed (4T1 and B16F10) mouse tumor models (staged ≥ 200 mm3). Results: DF6002/BMS-986415 induced IFN-γ production in human (EC50, 3.35 pM) and Cyno (EC50, 3.09 pM) PBMCs comparable to rhIL-12. DF6002/BMS-986415 was well tolerated at doses yielding greater exposure vs equimolar rhIL-12 in Cynos. In mice, the mouse surrogate mDF6006 vs rmIL-12 yielded a 5-fold increase in t1/2 and a 40-fold greater serum IFN-γ exposure. In contrast to rmIL-12, mDF6006 achieved protracted IFN-γ levels without peak effects associated with increased toxicity; serum IFN-γ Cmax occurred after day 6 and subsided by day 8. mDF6006 was efficacious across mouse tumor models tested. In the CT26 tumor model, mDF6006 (1 µg IP QW) monotherapy showed greater antitumor activity vs equimolar doses of rmIL-12 and led to complete regression (CR) in mice with large tumors (&amp;gt; 800 mm3); immunological memory response was observed in responders rechallenged with fresh tumor cells. In the B16F10 tumor model, mDF6006 (0.5 µg equivalent rmIL-12 SC QW) + anti–PD-1 (10 mg/kg IP Q2W) yielded a higher CR rate (mDF6006 + anti-PD-1, 4 of 10 CR; mDF6006, 1 of 10 CR) and increased survival vs either agent alone. An increase in the number and activation of CD4+ and CD8+ T cells as well as NK cells was observed 4 days after administration of mDF6006 (0.5 µg) in the B16F10 model. Conclusions: DF6002/BMS-986415, a novel IL-12 Fc-fusion protein, retained the full potency of rhIL-12 and showed improved exposure and tolerability; mDF6006 exhibited greater antitumor activity than rmIL-12. These data support the ongoing first-in-human phase 1/2 study evaluating DF6002/BMS-986415 as a novel therapeutic for the treatment of solid tumors in combination with nivolumab (NCT04423029). Citation Format: Eva Gutierrez, Mitchell Bigelow, Colin LaCroix, Patrick Kirby, Lynn Markowitz, Michael Naill, Steve O'Neil, Philip Ansumana Hull, John Engelhardt, Jean-Marie Cuillerott, Ann Cheung, Asya Grinberg, Nicolai Wagtmann. Preclinical characterization of DF6002/BMS-986415, a novel differentiated IL-12 Fc-fusion protein with robust antitumor activity as monotherapy or in combination with anti-PD-1 [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 1714.

Obinutuzumab Activates CD64 More Potently Than Other Anti-CD20 Antibodies in Chronic Lymphocytic Leukemia (CLL)

IntroductionTreatment with monoclonal antibodies has revolutionized clinical medicine, especially in the fields of cancer and immunology. One of the oldest antibodies, which is widely used for the treatment of lymphomas and autoimmune diseases, is the anti-CD20 antibody rituximab. In recent years, new antibodies against CD20 have been developed, notably ofatumumab and obinutuzumab. Both antibodies are currently used for the treatment of chronic lymphocytic leukemia (CLL), with a possible clinical advantage of obinutuzumab over rituximab. In addition to CLL, obinutuzumab was recently approved for the treatment of relapsed/refractory follicular lymphoma in patients who had previously received a rituximab-containing regimen.An important mechanism of action of therapeutic monoclonal antibodies is activation of immune cells via Fc receptors (FcγRs). Surprisingly, however, the interactions of various therapeutic antibodies with various Fc receptors have not been fully elucidated. Moreover, the available data on the interaction of different anti-CD20 antibodies and human Fc receptors is based on affinity measurements, mainly surface plasmon resonance (SPR), and not on functional assays. Although primary immune cells can help us gain information regarding the activation of Fc receptors, these cells are difficult to handle and usually express several Fc receptors.MethodsHere we established a functional assay to study how different anti-CD20 antibodies activate different human Fc receptors. To this aim, we stably transfected mouse BW5147 cells (BW cells) with chimeric FcγRs which included the extracellular part of a given FcγR fused to the transmembrane and cytoplasmic segments of the mouse CD3ζ chain. Activation of a specific FcγR results in secretion of mouse interleukin-2 (mIL-2) which can be detected by enzyme-linked immunosorbent assay (ELISA). We prepared BW cells which express the following human FcγR-CD3ζ chimeras: CD16A (FcγRIIIA, low and high affinity variants 158F and 158V respectively), CD32A (FcγRIIA, low and high affinity variants, 131R and 131H respectively), and CD64 (FcγRI). The advantages of this system are that it is a functional assay, easy to use, sensitive (was used to detect anti-viral antibodies in patients) and enables to study the activation of a single Fc receptor. Additionally, the activation of a specific Fc receptor by this reporter system is studied in the context of the natural ligand of the antibody, which better reflects the activity of the antibody in vivo .We assessed the activation of Fc receptors by different anti-CD20 antibodies which were incubated with chronic lymphocytic leukemia (CLL) cells obtained from patients.ResultsUsing this functional assay we corroborated previous reports regarding the higher affinity of obinutuzumab to CD16A. Importantly, we discovered that obinutuzumab activates CD64 significantly more efficiently than rituximab or ofatumumab. We show that the advantage of obinutuzumab in activating CD64 translates into better elimination of CLL cells by primary macrophages. Mechanistically, we found that CD64 is activated more potently by obinutuzumab since obinutuzumab does not internalize when bound to its target protein CD20, in contrast to rituximab and ofatumumab. We show that the internalization of rituximab and ofatumumab is mediated by binding to the FcγR CD32B, which is expressed on the surface of CLL cells. Indeed, blocking of CD32B led to increased activation of CD64 by rituximab and ofatumumab.DiscussionUsing a functional assay we found that obinutuzumab activates CD64 more potently than other anti-CD20 antibodies. Is it possible that this advantage is relevant only to tumor cells with high expression of CD32B. This could be applied clinically by incorporating obinutuzumab instead of rituximab in certain lymphoproliferative diseases (e.g., CLL) or specifically in patients with high expression of CD32B. Furthermore, the BW reporter assay can be applied to other therapeutic antibodies and may assist in developing antibodies with improved immunological properties. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Dual vaccination against IL-4 and IL-13 protects against chronic allergic asthma in mice

Allergic asthma is characterized by elevated levels of IgE antibodies, type 2 cytokines such as interleukin-4 (IL-4) and IL-13, airway hyperresponsiveness (AHR), mucus hypersecretion and eosinophilia. Approved therapeutic monoclonal antibodies targeting IgE or IL-4/IL-13 reduce asthma symptoms but require costly lifelong administrations. Here, we develop conjugate vaccines against mouse IL-4 and IL-13, and demonstrate their prophylactic and therapeutic efficacy in reducing IgE levels, AHR, eosinophilia and mucus production in mouse models of asthma analyzed up to 15 weeks after initial vaccination. More importantly, we also test similar vaccines specific for human IL-4/IL-13 in mice expressing human IL-4/IL-13 and the related receptor, IL-4Rα, to find efficient neutralization of both cytokines and reduced IgE levels for at least 11 weeks post-vaccination. Our results imply that dual IL-4/IL-13 vaccination may represent a cost-effective, long-term therapeutic strategy for the treatment of allergic asthma as demonstrated in mouse models, although additional studies are warranted to assess its safety and feasibility.

Production of an active Mus musculus IL-3 using updated silkworm-based baculovirus expression vector system

• • Recombinant MmIL-3 was purified from the silkworm-BEVS. • • Silkworm-derived rMmIL-3 was N -glycosylated. • • Active rMmIL-3 has been verified in Ba/F3 cells. Due to the biological significance and therapeutic potential of Interleukin-3 (IL-3) secreted mainly by activated T cells, various protein expression systems have been challenged to produce recombinant IL3 to meet the increasing demands worldwide. Recently, we established an updated silkworm-based baculovirus expression vector system (silkworm-BEVS), which in most cases, produces eukaryotic proteins in biological or enzymatical active forms with considerable amounts. We attempted to reconstruct and express a recombinant mouse IL-3 (rMmIL-3) with C-terminal His8-Strep tags in silkworm-BEVS in the current study. From our results, we gained an active glycosylated rMmIL-3 protein in a substantial amount and quality. As compared with the E. coli expression system, silkworm-BEVS is a better choice regarding the glycosylations attached in rMmIL-3 and up-scalable system in case that a commercial amount is required in the future. Collectively, our method shares an excellent model to produce interleukin molecular for approaching pharmaceutical applications.

Interleukin-10 suppression enhances T-cell antitumor immunity and responses to checkpoint blockade in chronic lymphocytic leukemia.

T-cell dysfunction is a hallmark of B-cell Chronic Lymphocytic Leukemia (CLL), where CLL cells downregulate T-cell responses through regulatory molecules including programmed death ligand-1 (PD-L1) and Interleukin-10 (IL-10). Immune checkpoint blockade (ICB) aims to restore T-cell function by preventing the ligation of inhibitory receptors like PD-1. However, most CLL patients do not respond well to this therapy. Thus, we investigated whether IL-10 suppression could enhance antitumor T-cell activity and responses to ICB. Since CLL IL-10 expression depends on Sp1, we utilized a novel, better tolerated analogue of the Sp1 inhibitor mithramycin (MTMox32E) to suppress CLL IL-10. MTMox32E treatment inhibited mouse and human CLL IL-10 production and maintained T-cell effector function in vitro. In the Eμ-Tcl1 mouse model, treatment reduced plasma IL-10 and CLL burden and increased CD8+ T-cell proliferation, effector and memory cell prevalence, and interferon-γ production. When combined with ICB, suppression of IL-10 improved responses to anti-PD-L1 as shown by a 4.5-fold decrease in CLL cell burden compared to anti-PD-L1 alone. Combination therapy also produced more interferon-γ+, cytotoxic effector KLRG1+, and memory CD8+ T-cells, and fewer exhausted T-cells. Since current therapies for CLL do not target IL-10, this provides a novel strategy to improve immunotherapies.

Maintenance and Gene Electrotransfer Efficiency of Antibiotic Resistance Gene-Free Plasmids Encoding Mouse, Canine and Human Interleukin-12 Orthologues

Maintenance and Gene Electrotransfer Efficiency of Antibiotic Resistance Gene-Free Plasmids Encoding Mouse, Canine and Human Interleukin-12 Orthologues