anti-IL6 Antibody Research Articles

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Diagnosis, Natural History and Treatment of Eosinophilic Enteritis: a Review.

To review recent findings regarding eosinophilic enteritis, including epidemiology, pathogenesis, natural history, and treatment. A 2017 population-based study using a US healthcare system database identified 1820 patients with a diagnosis of eosinophilic enteritis among 35,826,830 individuals. The majority of patients with eosinophilic enteritis in this study were women (57.7%), Caucasian (77.5%), and adults (> 18years of age) (83.5%). The overall prevalence of eosinophilic enteritis was estimated at 5.1/100,000 persons. Eosinophilic enteritis, also known as eosinophilic gastroenteritis, is a rare primary eosinophilic gastrointestinal disorder (EGID) of unknown etiology characterized by the presence of an intense eosinophilic infiltrate on histopathological examination of the intestinal mucosa. The etiology of eosinophilic enteritis remains unknown. However, there is evidence to support the role of allergens in the pathogenesis of this disorder, as children and adults with EGIDs often have positive skin testing to food allergens and a family history of allergic diseases. Recent studies unraveling the role of IgE-mediated but also delayed Th2-type responses have provided insight into the pathogenesis of this disease. Eosinophilic enteritis causes a wide array of gastrointestinal symptoms such as abdominal pain, diarrhea, nausea, vomiting, bloating, or ascites, and its diagnosis requires a high degree of clinical likelihood, given the nonspecific clinical presentation and physical examination findings. Oral corticosteroids are considered to be the mainstay of treatment and are generally used for a short period with good response rates. Antihistamine drugs and sodium cromoglycate have also been used to treat patients with eosinophilic enteritis. Preliminary studies have demonstrated the potential benefit of biological therapies targeting the eosinophilic pathway such as mepolizumab, an anti-IL5 antibody, or omalizumab, an anti-IgE monoclonal antibody. Eosinophilic enteritis is generally considered to be a benign disease without relapse, but up to 50% of patients may present a more complex natural history characterized by unpredictable relapses and a chronic course.

Abstract 1537: Anti-CD19 ARTEMIS™ T cells prevent excessive inflammatory cytokine release, including IL-6, in a co-culture model of CRS

Abstract We designed the ARTEMISTM T-cell signaling platform to be a safer ACT therapy by relying on endogenous T-cell signaling factors. We show evidence that the ARTEMISTM receptor disentangles efficacy from CRS and holds the potential to be a clinically safer therapy by preventing ACT-triggered CRS. Despite the remarkable curative potential of anti-CD19-CAR T cells the therapy continues to trigger a potentially life threatening cytokine release syndrome (CRS) that is characterized by excessive concentrations of inflammatory cytokines. We reasoned that by redesigning the T-cell activating antigen receptor to require signaling through the endogenous CD3 complex the T-cell could better regulate its cellular responses and thus reduce the risk of CRS when used as an adoptive T-cell transfer (ACT) therapy. Through protein engineering, we developed the ARTEMIS™ signaling platform which when coupled with Eureka's human anti-CD19 antibody, ET190L1, and expressed on primary T-cells, results in a significant reduction of cytokine release during antigen specific T-cell activation. When CAR vs. ARTEMIS™ comparisons were tested in vitro, ET190L1-ARTEMISTM T-cells and ET190L1-CD28z-CAR T-cells specifically lysed multiple CD19+ leukemia and lymphoma cell lines with similar potencies. However, during the 16 hour in vitro killing assays, ARTEMIS™ T-cells secreted demonstrably less IL-2, IFN-γ, GM-CSF, and TNFα as compared to CAR T-cells. ARTEMISTM T-cells also accumulated less PD-1, LAG3, and TIM3 on their surface during culturing and following in vitro killing, indicating a diminished propensity for exhaustion compared to corresponding CAR T-cells. The ability of the anti-IL6 antibody tocilizumab to mitigate toxicity in patients that develop severe CRS in the clinic suggests that limiting IL-6 expression could improve the safety of adoptive T-cell therapies. However, T-cells are not efficient expressors of IL-6 and the vast majority of elevated IL-6 is produced by monocytes in response to activated CAR T-cells. To assay the effects of T-cell cytotoxicity on upregulation of cytokine expression within monocytes as a model for CRS, we co-cultured T-cells with CD19-positive ALL tumor cells on one side of a transwell and monocytes on the other side. After tumor cell lysis, RNA expression levels were measured from both T-cells and monocytes. Using ET190L1-ARTEMIS™ T-cells to kill NALM-6 tumor cells resulted in dramatically reduced levels of IL-6 and IL-5 mRNA upregulation within monocytes compared to that observed with CAR transduced T-cells. When tested in vivo against CD19+ xenografts mouse models, intravenous administration of ET190L1-ARTEMISTM T-cells caused efficient tumor regression. In agreement with the in vitro data, mice treated with ARTEMISTM T-cells released significantly lower levels of cytokines in their blood at 24 hours post dosing than mice treated with CAR T-cells. Citation Format: Hong Liu, Lucas H. Horan, Stephan A. Grupp, David M. Barrett, Cheng Liu. Anti-CD19 ARTEMIS™ T cells prevent excessive inflammatory cytokine release, including IL-6, in a co-culture model of CRS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1537.

Adjuvant non-bacteriolytic and anti-inflammatory combination therapy in pneumococcal meningitis: an investigation in a mouse model

ObjectivesTherapy with antibiotics, dexamethasone, and supportive intensive care has improved the prognosis of pneumococcal meningitis, but mortality remains high. Here, we investigated an adjunctive combination therapy of the non-bacteriolytic antibiotic daptomycin plus several anti-inflammatory agents to identify the currently most promising adjunctive combination therapy for pneumococcal meningitis. MethodsC57BL/6 mice were infected by injection of pneumococci into the cisterna magna. Treatment was begun 21 h after infection, and consisted of ceftriaxone plus (a) dexamethasone, (b) dexamethasone plus daptomycin, (c) daptomycin, (d) daptomycin plus an anti-IL1 antibody, (e) daptomycin plus roscovitine, or (f) daptomycin plus an anti-C5 antibody. Animals were followed until 45 h after infection. Furthermore, adjunctive daptomycin plus anti-C5 antibodies were assessed in a long-term follow-up. ResultsAdjunctive treatment with daptomycin and an anti-C5 antibody was superior to adjunctive dexamethasone and reduced disease symptoms (clinical score 1.1 ± 1.1 versus 5.0 ± 2.7, p < 0.0083), improved explorative activity (open field test 17.8 ± 8.2 versus 7.4 ± 4.3 crossed fields/2 minutes, p < 0.0083), and reduced hearing impairment (thresholds for click stimulus 96.1 ± 14.7 versus 114.8 ± 9.3 dB SPL, p < 0.0083) in the acute stage. Furthermore, explorative activity (14.4 ± 7.3 crossed fields/2 minutes versus 6.3 ± 7.2, p < 0.05) and cognitive function (t-maze test, exploration time previously unknown alley 72.4 ± 14.3 versus 48.7 ± 25.6%, p < 0.05) was improved at 2 weeks after infection. Treatment with daptomycin plus an anti-IL-1β antibody or roscovitine was not of significant benefit in comparison to adjunctive therapy with dexamethasone. ConclusionsAn adjunctive combination of the non-lytic antibiotic daptomycin plus an anti-C5 antibody was superior to standard therapy with adjunctive dexamethasone in the treatment of pneumococcal meningitis.

Inhibitory effect of anti interleukin-8 monoclonal antibodies on cervical cancer

Objective To investigate the inhibitory effect of anti interleukin (IL)-8 monoclonal antibodies on the growth and metastasis of cervical cancer. Methods Involved cervical cells included CaSki cells with high expression of IL-8 and SiHa cell lines with IL-8 plasmid transfected (pcDNA3.1-IL-8-SiHa). Cervical cancer animal model was established on nude mice. Boyden method was used in vitro study to observe the effects of anti IL-8 antibodies on the chemotaxis of high-expressed IL-8 cervical cancer cells. The effect of antiIL-8 antibodies on the growth of cervical cancer cells and nude mice transplantation tumor was observed by the experiment in vivo through reverse transcription-polymerase chain reaction (RT-PCR), enzyme linked immunosorbent assay (ELISA), TUNEL method. Cell line (CaSki and pcDNA3.1-IL-8-SiHa) modeled nude mice were divided into 5 groups with 5 animals in each group. The blank control group (group Ⅰ) was given the equal volume of phosphate buffer solution (PBS). Negative control group (group Ⅱ) was injected with IgG at the same volume of IgG. Treatment group (group Ⅲ) was injected with anti IL-8 antibodies at dose of 100 μg for once and intervals for once 2 days. Treatment group (group Ⅳ) was injected with anti IL-8 antibodies at dose of 500 μg for once and intervals for once 3 days. Treatment group (group V) was injected with anti IL-8 antibodies at dose of 1 000 μg for once and intervals for once 1 week. Results Experiments in vitro showed that the cell chemotaxis ability of anti IL-8 antibody in CaSki cells and pcDNA3.1-IL-8-SiHa cells was lower than that in the blank control group (CaSki cells: F = 289.6, P = 0.000; pcDNA3.1-IL-8-SiHa cells: F = 79.0, P = 0.005). Group Ⅳ was taken as the example for its best anti-tumor effect in experiments in vivo. The tumor weight in group Ⅳ was lower than that in group Ⅰ [CaSki cells: (0.172±0.031) g vs. (0.735±0.015) g, P < 0.05; pcDNA3.1-IL-8-SiHa cells: (0.400±0.029) g vs. (1.430±0.199) g, P < 0.05]. The tumor volume in group Ⅳ was less than that in group Ⅰ [CaSki cells: (0.049±0.028) cm3 vs. (0.214±0.016) cm3, P < 0.05; pcDNA3.1-IL-8-SiHa cells: (0.063±0.022) cm3 vs. (0.600±0.072) cm3, P < 0.05]. The tumor growth curve also showed that tumor growth was slow, and the time of tumor formation as well as survival time was prolonged in anti IL-8 antibody treated group. The expression of mRNA in IL-8 in group IV was lower than that in group Ⅰ (CaSki cells: 0.58±0.06 vs. 1.15±0.13, P < 0.05; pcDNA3.1-IL-8-SiHa cells: 0.69±0.08 vs. 1.16±0.13, P < 0.05). The protein expression of IL-8 in group Ⅳ was lower than that in group Ⅰ (CaSki cells: 126±29 vs. 411±112, P < 0.05; pcDNA3.1-IL-8-SiHa cells: 134±47 vs. 327±69, P < 0.05). Apoptotic index in group Ⅳ was higher than that in group Ⅰ (CaSki cells: 81.8±3.0 vs. 26.0±5.6, P < 0.05; pcDNA3.1-IL-8-SiHa cells: 84.4±3.6 vs. 32.0±4.9, P < 0.05). Conclusion Anti IL-8 antibody can inhibit cell migration of human cervical cancer in vitro, inhibit growth and metastasis of transplantation tumor in vivo, and promote apoptosis and necrosis with a dose-dependent way in vivo. Key words: Uterine cervical neoplasms; Interleukin-8; Antibodies, monoclonal

Abstract PD6-06: Understanding the complexity of macrophage and mesenchymal stem cell interactions to improve treatment outcome for IBC patients

Abstract Cells within the tumor microenvironment, including but not limited to macrophages and mesenchymal stem cells (MSCs), can promote the phenotype and aggressiveness of inflammatory breast cancer (IBC). For example, co-injection of MSCs with SUM149 IBC cells significantly increased the clinical features of IBC such as skin invasion and metastasis. Our preliminary work showed that MSCs can be educated by co-culture with M1 polarized (anti-tumor) or M2 polarized (pro-tumor) mouse Raw macrophages. Such education of MSCs by M2 Raw macrophages leads to increased IL6 secretion by MSCs, relative to M1-educated or uneducated MSCs. M2-educated MSCs also have increased migration toward IBC cell lines SUM149 and IBC3, effects that can be blocked by an anti-IL6 antibody. Co-culture with M2-educated MSCs also enhances migration and mammosphere formation of IBC cells. Radiation response of IBC cells upon interactions with cells from the tumor microenvironment was also analyzed. Preliminary work shows that co-culture of IBC cells (SUM149 and KPL4) with M2-polarized human THP1 macrophages, prior to ionizing radiation, mediates radiation resistance of IBC cells, and this effect can be decreased by either adding HDL lipoproteins during co-culture period or by STAT6 inhibitors that block IL4/IL13-mediated phosphorylation of STAT6 and M2-polarization in THP1 macrophages. MSCs can also be polarized into either a MSC1 phenotype or a MSC2 phenotype by exposure to toll-like receptor (TLR) ligands TLR4 or TLR3, respectively. Indoleamine-pyrrole 2,3-dioxygenase (IDO) expression in MSCs is a marker of MSC2 polarization that is induced after exposure with TLR3 ligand (PolyIC) relative to MSC1 (TLR4 stimulated; LPS-treated) or parental MSCs. Similar to macrophage polarization, while MSC1 mediates anti-tumor effects, MSC2 are immunosuppressive and thus contribute to tumor growth. Preliminary work also shows that co-culture of IBC cells with MSC2 mediates radioresistance and this can be decreased as well by exposure to HDL during co-culture period prior to radiation. HDL protective effects, in part, can be explained by decreased expression of TLR3-induced IDO mRNA levels in MSC2. In the present work, we extended the above mentioned observations regarding the crosstalk between mouse Raw macrophages and MSCs by analyzing the effect of co-culture of human THP1 macrophages (parental designated as M0, M1- or M2-polarized) with MSCs on the IDO mRNA expression in MSCs, a marker of MSC2 polarization. Surprisingly, co-culture of M1-polarized THP1 with MSCs resulted in a robust increased expression of IDO mRNA in MSC relative to parental MSC (uneducated) or MSCs co-cultured with M2-THP1. Further studies are needed to determine the effects of increased IDO expression in MSC, upon M1-THP1 co-culture, on the aggressive behavior of IBC cells and whether this could be altered with IDO inhibitors. Our results suggest that there could be inter-species differences between mouse and human macrophages on the education of human MSCs. Based on our findings we propose testing a combination of STAT6 inhibitors that reverse M2-polarization of macrophages and IDO inhibitors that can decrease MSC2 phenotype mediated by TLR3 exposure and/or M1-THP1 education. Citation Format: Rahal OM, Wolfe AR, Mandal PK, Larson R, Tin S, Reuben JM, McMurray JS, Woodward WA. Understanding the complexity of macrophage and mesenchymal stem cell interactions to improve treatment outcome for IBC patients [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD6-06.

A mathematical model for IL-6-mediated, stem cell driven tumor growth and targeted treatment.

Targeting key regulators of the cancer stem cell phenotype to overcome their critical influence on tumor growth is a promising new strategy for cancer treatment. Here we present a modeling framework that operates at both the cellular and molecular levels, for investigating IL-6 mediated, cancer stem cell driven tumor growth and targeted treatment with anti-IL6 antibodies. Our immediate goal is to quantify the influence of IL-6 on cancer stem cell self-renewal and survival, and to characterize the subsequent impact on tumor growth dynamics. By including the molecular details of IL-6 binding, we are able to quantify the temporal changes in fractional occupancies of bound receptors and their influence on tumor volume. There is a strong correlation between the model output and experimental data for primary tumor xenografts. We also used the model to predict tumor response to administration of the humanized IL-6R monoclonal antibody, tocilizumab (TCZ), and we found that as little as 1mg/kg of TCZ administered weekly for 7 weeks is sufficient to result in tumor reduction and a sustained deceleration of tumor growth.

Monoclonal antibody sequence assessment using a hybrid quadrupole-Orbitrap mass spectrometer

A multi-faceted method to verify anti-IL8 monoclonal antibody protein sequence using hybrid Orbitrap mass spectrometer.

The Immune-microenvironment Confers Chemoresistance of Colorectal Cancer through Macrophage-Derived IL6.

Purpose: Tumor-associated macrophages (TAMs) are frequently associated with poor prognosis in human cancers. However, the effects of TAMs in colorectal cancer are contradictory. We therefore investigated the functions, mechanisms, and clinical significance of TAMs in colorectal cancer.Experimental Design: We measured the macrophage infiltration (CD68), P-gp, and Bcl2 expression in colorectal cancer tissues using IHC staining. Coculture of TAMs and colorectal cancer cells both in vitro and in vivo models was used to evaluate the effects of TAMs on colorectal cancer chemoresistance. Cytokine antibody arrays, ELISA, neutralizing antibody, and luciferase reporter assay were performed to uncover the underlying mechanism.Results: TAM infiltration was associated with chemoresistance in patients with colorectal cancer. Colorectal cancer-conditioned macrophages increased colorectal cancer chemoresistance and reduced drug-induced apoptosis by secreting IL6, which could be blocked by a neutralizing anti-IL6 antibody. Macrophage-derived IL6 activated the IL6R/STAT3 pathway in colorectal cancer cells, and activated STAT3 transcriptionally inhibited the tumor suppressor miR-204-5p. Rescue experiment confirmed that miR-204-5p is a functional target mediating the TAM-induced colorectal cancer chemoresistance. miR-155-5p, a key miRNA regulating C/EBPβ, was frequently downregulated in TAMs, resulting in increased C/EBPβ expression. C/EBPβ transcriptionally activated IL6 in TAMs, and TAM-secreted IL6 then induced chemoresistance by activating the IL6R/STAT3/miR-204-5p pathway in colorectal cancer cells.Conclusions: Our data indicate that the maladjusted miR-155-5p/C/EBPβ/IL6 signaling in TAMs could induce chemoresistance in colorectal cancer cells by regulating the IL6R/STAT3/miR-204-5p axis, revealing a new cross-talk between immune cells and tumor cells in colorectal cancer microenvironment. Clin Cancer Res; 23(23); 7375-87. ©2017 AACR.

Combination of PTH (1-34) with anti-IL17 prevents bone loss by inhibiting IL-17/N-cadherin mediated disruption of PTHR1/LRP-6 interaction

Combinations of anabolic and anti-resorptive agents have potential to improve bone density more than either agent alone. In this study, we determine the combining effect of anti-IL17 antibody and PTH (1-34) in mitigation of ovariectomy induced bone loss. Ovariectomized BALB/c female mice were treated with anti-IL17 and iPTH monotherapies and their combination. Combination of iPTH and anti-IL17 has synergistic effect in the restoration of skeletal and immune parameters compared to mono-therapies. Immunofluorescence analysis shows decreased expression of PTHR1 in iPTH+anti-IL17 treated bone sections. Our studies show that IL-17 up regulates N-cadherin which disrupts PTHR1/LRP-6 interaction thereby inhibiting wnt signaling and promoting bone loss. Our studies advocate use of iPTH and anti-IL17 combination therapy for post-menopausal osteoporosis.

P020 Increased IL-6 signaling in DSA positive renal transplant patients predicts graft failure

Aim IL-6 is associated with chronic inflammation seen in allograft rejection. Clinical trials using anti-IL6 antibodies have found a significant benefit in kidney allograft survival and a reduction in donor-specific anti-HLA antibodies (DSA). We developed a new cell-based IL-6 assay and tested transplant patient sera to determine the temporal relationship between IL-6 and DSA. Methods Serial samples from 45 deceased-donor kidney transplant (Tx) recipients (189 samples total) transplanted between 2011 and 2013 at East Carolina University were tested for both DSA and IL-6. Patients were categorized as DSA-, DSA + stable graft function (DSAstable) or DSA + eventual graft loss (DSAfail). In all patients, samples were tested post-transplant. In the case of the DSA positive cases, the samples were collected pre-DSA appearance and at DSA onset. Similar time points post-transplant in the DSA- patients were used. Results Post-transplant, DSA negative patient samples had significantly lower IL-6-fold-induction (n = 58, mean = 0.83, SE = 0.02) than DSAfail patients (n = 10, mean = 0.98, SE = 0.05; ANOVA, p Conclusions We detected significantly lower levels of IL-6 in DSA negative and DSA stable patients compared to DSA + patients who eventually failed. This testing was done pre-DSA or at the time of DSA onset suggesting IL-6 signaling may be important in the progression to failure in renal transplant patient. Therefore, treating DSA + patients with high IL-6 with an anti-IL6 may prove to be beneficial. Download : Download high-res image (114KB) Download : Download full-size image

IL33-mediated ILC2 activation and neutrophil IL5 production in the lung response after severe trauma: A reverse translation study from a human cohort to a mouse trauma model.

BackgroundThe immunosuppression and immune dysregulation that follows severe injury includes type 2 immune responses manifested by elevations in interleukin (IL) 4, IL5, and IL13 early after injury. We hypothesized that IL33, an alarmin released early after tissue injury and a known regulator of type 2 immunity, contributes to the early type 2 immune responses after systemic injury.Methods and findingsBlunt trauma patients admitted to the trauma intensive care unit of a level I trauma center were enrolled in an observational study that included frequent blood sampling. Dynamic changes in IL33 and soluble suppression of tumorigenicity 2 (sST2) levels were measured in the plasma and correlated with levels of the type 2 cytokines and nosocomial infection. Based on the observations in humans, mechanistic experiments were designed in a mouse model of resuscitated hemorrhagic shock and tissue trauma (HS/T). These experiments utilized wild-type C57BL/6 mice, IL33-/- mice, B6.C3(Cg)-Rorasg/sg mice deficient in group 2 innate lymphoid cells (ILC2), and C57BL/6 wild-type mice treated with anti-IL5 antibody.Severely injured human blunt trauma patients (n = 472, average injury severity score [ISS] = 20.2) exhibited elevations in plasma IL33 levels upon admission and over time that correlated positively with increases in IL4, IL5, and IL13 (P < 0.0001). sST2 levels also increased after injury but in a delayed manner compared with IL33. The increases in IL33 and sST2 were significantly greater in patients that developed nosocomial infection and organ dysfunction than similarly injured patients that did not (P < 0.05). Mechanistic studies were carried out in a mouse model of HS/T that recapitulated the early increase in IL33 and delayed increase in sST2 in the plasma (P < 0.005). These studies identified a pathway where IL33 induces ILC2 activation in the lung within hours of HS/T. ILC2 IL5 up-regulation induces further IL5 expression by CXCR2+ lung neutrophils, culminating in early lung injury. The major limitations of this study are the descriptive nature of the human study component and the impact of the potential differences between human and mouse immune responses to polytrauma. Also, the studies performed did not permit us to make conclusions about the impact of IL33 on pulmonary function.ConclusionsThese results suggest that IL33 may initiate early detrimental type 2 immune responses after trauma through ILC2 regulation of neutrophil IL5 production. This IL33–ILC2–IL5–neutrophil axis defines a novel regulatory role for ILC2 in acute lung injury that could be targeted in trauma patients prone to early lung dysfunction.

Blocking Tumor-Educated MSC Paracrine Activity Halts Osteosarcoma Progression.

Purpose: Human osteosarcoma is a genetically heterogeneous bone malignancy with poor prognosis despite the employment of aggressive chemotherapy regimens. Because druggable driver mutations have not been established, dissecting the interactions between osteosarcoma cells and supporting stroma may provide insights into novel therapeutic targets.Experimental Design: By using a bioluminescent orthotopic xenograft mouse model of osteosarcoma, we evaluated the effect of tumor extracellular vesicle (EV)-educated mesenchymal stem cells (TEMSC) on osteosarcoma progression. Characterization and functional studies were designed to assess the mechanisms underlying MSC education. Independent series of tissue specimens were analyzed to corroborate the preclinical findings, and the composition of patient serum EVs was analyzed after isolation with size-exclusion chromatography.Results: We show that EVs secreted by highly malignant osteosarcoma cells selectively incorporate a membrane-associated form of TGFβ, which induces proinflammatory IL6 production by MSCs. TEMSCs promote tumor growth, accompanied with intratumor STAT3 activation and lung metastasis formation, which was not observed with control MSCs. Importantly, intravenous administration of the anti-IL6 receptor antibody tocilizumab abrogated the tumor-promoting effects of TEMSCs. RNA-seq analysis of human osteosarcoma tissues revealed a distinct TGFβ-induced prometastatic gene signature. Tissue microarray immunostaining indicated active STAT3 signaling in human osteosarcoma, consistent with the observations in TEMSC-treated mice. Finally, we isolated pure populations of EVs from serum and demonstrated that circulating levels of EV-associated TGFβ are increased in osteosarcoma patients.Conclusions: Collectively, our findings suggest that TEMSCs promote osteosarcoma progression and provide the basis for testing IL6- and TGFβ-blocking agents as new therapeutic options for osteosarcoma patients. Clin Cancer Res; 23(14); 3721-33. ©2017 AACR.

Assessment of neutralizing interleukin-4 effect on CD133 gene expression in colon cancer cell line

Colorectal cancer may be maintained by cancer stem-like cells (CSCs) that express the cell surface marker CD133. CSCs (CD133+cells) exhibits greater resistance to the chemotherapy and this resistance may be mediated in part by an autocrine response to IL4.The aim of the study was to assess the effect of anti-IL4 antibody alone or in combination with chemotherapy on the CD133 expression andthe tumor growth.We used Caco cell line in our experiments and the samples were as the following; untreated colorectal cell line, cells treated by chemotherapy, cells treated by anti-IL4 antibody in 3doses (2.5, 5, 10μg/ml), cells treatedby combination of chemotherapy and anti-IL4 antibody in 3 doses.Results of our in vitro studies demonstrated that anti-IL4 inhibited growth of Caco cell line in a dose-dependent manner revealing a 32.11% inhibition at the highest concentration (10µg/ml). There was further significant inhibition by combination of anti IL4 and chemotherapy in a dose response manner when compared to group treated by chemotherapy only. These effects were associated with decreased expression of CD133 in tumor cells also. Lastly, anti-IL4 antibody stimulated apoptosis.Our study suggested that neutralizing of IL4 by anti IL4 antibody affect the CD133+ cells may be by increasing their apoptosis. The effects of anti IL4 antibody either, alone or in combination with chemotherapy, inhibited the tumor growth and decreased the viable tumor cells. Furthermore, neutralizing of IL4 increased the efficacy of chemotherapy treatment.

Abstract TMEM-030: INTERLEUKIN–6 (IL6) AND LEUKEMIA INHIBITORY FACTOR (LIF) ARE FUNCTIONALLY REDUNDANT IN PROMOTING OVARIAN CANCER GROWTH AND REQUIRE DUAL BLOCKADE FOR THERAPEUTIC EFFECT

Abstract PURPOSE: We have previously isolated mesenchymal stem cells from the microenvironment of tumors from ovarian cancer patients (cancer-associated MSC, CA-MSC). We found that CA-MSC promote tumorigenesis in vitro and in vivo by increasing cancer stem cells (CSC) in the epithelial tumor cells. Microarray analysis demonstrates the upregulation of the cytokines IL6 and LIF in CA-MSC versus control MSC. The purpose of this work is to characterize the role of cytokines interleukin-6 (IL6) and leukemia inhibitory factor (LIF) secreted by CA-MSC in ovarian cancer tumorigenesis. EXPERIMENTAL PROCEDURES: We have isolated and established cultures of CA-MSC. Healthy donor adipose-derived MSC were used as controls (Invitrogen). Ovarian cancer cells were treated with MSC-conditioned media or recombinant IL6 and/or LIF (EBioscience). Cytokine function was inhibited with IL6 blocking antibody, LIF blocking antibody, both antibodies or the JAK2 inhibitor ruxolitinib for dual blockade. Phosphorylated STAT3 and total STAT3 immunoblotting was performed by standard methods. Sphere assays were performed using low-adherence plates and X-Vivo20 serum free media (Lonza). Flow cytometry was carried out using the Aldefluor assay for aldehyde dehydrogenase activity (Stem Cell Technologies). For in vivo studies, NSG mice were injected with OVCAR3 ovarian cancer cells without or with CA-MSC, and then either mock treated or treated with IL6 blocking antibody (tocilizumab, Genentech), LIF blocking antibody or ruxolitinib. Tumor size was monitored both during treatment and at sacrifice, and tumors were analyzed. RESULTS: Treatment of ovarian cancer cells with either recombinant IL6 or LIF alone leads to STAT3 phosphorylation, suggesting functional redundancy in stimulation of this pathway. Neither LIF nor IL6 blocking antibodies alone can prevent upregulation of phospho-STAT3, but combined therapy with both LIF and IL6 blocking antibodies prevents STAT3 phosphorylation. The JAK2 inhibitor ruxolitinib inhibits cytokine-mediated upregulation of phospho-STAT3. Either LIF or IL6 treatment results in an increase the percentage of cancer stem cells in ovarian cancer cell lines, as measured using both sphere assays and aldehyde dehydrogenase activity. Ruxolitinib blocks both LIF and IL6 mediated increase in cancer stem cells. In vivo, the pro-tumorigenic effect of CA-MSC is abrogated by dual blockade with ruxolitinib but not by treatment with anti-IL6 antibody alone or anti-LIF antibody alone. Ruxolitinib treated tumors demonstrate decreased phospho-STAT3, indicating on-target activity. CONCLUSIONS: IL6 and LIF are secreted by CA-MSC in the ovarian cancer tumor microenvironment. Both cytokines upregulate phospho-STAT3 and increase the percentage of cancer stem cells in epithelial tumor cells. Functional inhibition of these effects cannot be achieved in vitro or in vivo with blockade of either cytokine individually; dual blockade with compounds such as the JAK2 inhibitor ruxolitinib is necessary. Thus the shared downstream signaling molecules of the JAK/STAT pathway may be preferred targets for novel anticancer therapeutics for ovarian cancer. Citation Format: Karen McLean, Lijun Tan and Ronald J. Buckanovich. INTERLEUKIN–6 (IL6) AND LEUKEMIA INHIBITORY FACTOR (LIF) ARE FUNCTIONALLY REDUNDANT IN PROMOTING OVARIAN CANCER GROWTH AND REQUIRE DUAL BLOCKADE FOR THERAPEUTIC EFFECT [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-030.

Combinatorial treatment with polyI:C and anti-IL6 enhances apoptosis and suppresses metastasis of lung cancer cells.

Activation of TLR3 stimulates cancer cell apoptosis and triggers secretion of inflammatory cytokines. PolyI:C, a TLR3 agonist, activates immune cells and regresses metastatic lung cancer in vivo. Although polyI:C reportedly kills lung carcinomas, the mechanism remains elusive. Here, we demonstrated that polyI:C suppressed the proliferation and survival of metastatic (NCI-H358 and NCI-H292) and non-metastatic (A549) lung cancer cells. Notably, A549, NCI-H292 and NCI-H358 which are inducible by polyI:C, expressed low-to-medium level of TLR3 protein, and were susceptible to polyI:C treatment. By contrast, NCI-H1299, which endogenously expresses high level of TLR3 protein, was insensitive to polyI:C. We showed that polyI:C stimulated pro-inflammatory cytokines associated with survival and metastasis in a cell type-specific manner. While A549 and NCI-H292 released high levels of IL6, IL8 and GRO, the NCI-H358 cells endogenously secretes abundant levels of these cytokines, and was not further induced by polyI:C. Thus, NCI-H358 was resistant to the inhibition of cytokine-dependent metastasis. NCI-H1299, which was unresponsive to polyI:C, did not produce any of the pro-inflammatory cytokines. Treatment of A549 with a combination of polyI:C and anti-IL6 antibody significantly decreased IL6 production, and enhanced polyI:C-mediated killing and suppression of oncogenicity and metastasis. While polyI:C stimulated the phosphorylation of STAT3 and JAK2, blockade of these proteins enhanced polyI:C-mediated suppression of survival and metastasis. Taken together, polyI:C alone provoked apoptosis of lung cancer cells that express low-to-medium levels of functional TLR3 protein. The combinatorial treatment with polyI:C and anti-IL6 enhanced polyI:C-mediated anticancer activities through IL6/JAK2/STAT3 signalling, and apoptosis via TLR3-mediated caspase 3/8 pathway.

Role of exogenous interleukin 4 in the dynamics of acute liver injury

BackgroundMortality in acute liver failure is very high, and the only reliable treatment is transplantation. Interleukin 4 (IL4) has pleiotropic effects on a network of cells directing repair, regeneration, and fibrosis. Pretreatment with subcutaneous IL4 complexed with anti-IL4 antibody (IL4c) promotes repair of carbon tetrachloride-mediated murine liver injury by stimulating hepatocyte proliferation. We hypothesised that IL4c could be administered therapeutically and act by a variety of mechanisms beyond engagement of hepatocytes. MethodsC57Bl/6 mice were given carbon tetrachloride intraperitoneally to induce acute liver injury. Preinjury and postinjury dosing regimens of IL4c were compared. The role of IL4 receptor α-chain (IL4Rα) signalling in bone-marrow-derived cells was investigated using tissue-protected chimeras generated with wild type or IL4Rα−/– donor bone marrow. Liver was analysed by immunohistochemistry and fluorescence-activated cell sorting (FACS). FindingsWhen compared with vehicle, administration of IL4c after injury produced a significant reduction in mean alanine aminotransferase (242 U/L [61·9] vs 131 [30·8], p<0·0001) and necrotic cell area (11·6% [7·04] vs 0·36 [0·6], p<0·0001). Although necrosis was reduced, the necrotic area in IL4c-treated liver was not replaced with hepatocytes but with a cellular infiltrate characterised by high F4/80 expression. FACs analysis showed a reduced proportion and number of Ly6chi MHCIIlo monocytes and a concomitant increase in the number of Ly6clo MHCIIhi F4/80hi macrophages in injured livers treated with IL4c. This phenotypic change was dependent on IL4c signalling to Ly6chi MHCIIlo monocytes, since these cells exhibited developmental arrest when derived from IL4Rα-deficient bone marrow in chimeric mice. InterpretationWe demonstrate that IL4c can be administered therapeutically after carbon tetrachloride injury to improve indices of hepatic injury. Furthermore, IL4c treatment mediates a change in the monocyte–macrophage compartment which is consistent with a switch from a proinflammatory (Ly6chi) to a proreparative (Ly6clo) macrophage phenotype. We postulate that these Ly6clo macrophages mediate clearance of necrotic debris and facilitate hepatic regeneration. FundingWellcome Trust.

Lipids and Their Effects in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is an incurable common B-cell malignancy with a spectrum of clinical outcomes. Over the past decade, our increasing understanding of the drivers of CLL progression has led to the development and use of novel therapeutics. For example, B-cell receptor (BCR) signaling was shown to be overactive in CLL, and subsequently the kinase inhibitors ibrutinib (BTK inhibitor) and idelalisib (PI3K delta inhibitor) were found to have clinical efficacy in this malignancy (Byrd et al., 2013, Furman et al., 2014). Despite these new treatments, CLL remains incurable and there remains a need to identify new therapeutic targets.

Kinase Inhibitor Ibrutinib Prevents Cytokine-Release Syndrome after Anti-CD19 Chimeric Antigen Receptor T Cells (CART) for B Cell Neoplasms

Chimeric antigen receptor T cells (CART) hold great promise for the treatment of B cell neoplasms as multiple clinical studies show high complete response rates with anti-CD19 CART. However, widespread clinical use of this novel immunotherapy is limited by cytokine-release syndrome (CRS). CRS is characterized by high fevers and a systemic inflammatory response that is at times fatal. CRS is associated with elevation of multiple inflammatory cytokines (IFNγ, TNFα, IL-6 and others) and may be treated with the anti-IL6 receptor antibody tocilizumab and/or steroids. Importantly, approaches to prevent CRS are currently lacking. The Bruton’s tyrosine kinase inhibitor ibrutinib has been approved for relapsing chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) and is now extensively used in B cell neoplasms. Our group has recently demonstrated that the rational combination of ibrutinib with CART19 leads to enhanced anti-tumor responses in preclinical model of MCL, CLL and B-cell acute lymphoblastic leukemia (ALL). In addition ibrutinib has been shown to modulate T cell cytokine production. We therefore hypothesized that ibrutinib would reduce CART19-mediated CRS without impairing the anti-tumor efficacy of these cells.We developed a novel xenogeneic model of CRS by intravenously injecting NOD/SCID/gamma-chain deficient (NSG) mice with primary human MCL cells. Since CRS has been associated with high tumor burden we allowed the tumor to grow for up to 50-60 days, at which time the mice developed palpable splenomegaly. We then injected i.v. 1x106 human CART19 cells or medium (PBS). By day 2 after the infusion, mice that received CART19, but not mice receiving PBS alone, began to display clinical signs of distress (reduced mobility, hyperventilation, generalized weakness, emaciation, hunched bodies, withdrawal and poor motor response) and experienced early death, as compared to controls (p<0.05), clinically resembling CRS. At day 4 after CART19 infusion, we collected blood and measured serum concentration of 30 cytokines by Luminex assay. Importantly, CART19-treated mice, but not control mice, showed significantly elevated serum concentrations of several human cytokines, including IL-6, IFNγ, TNFα, IL-2 and GM-CSF (p<0.05). Having established a clinically relevant model of CRS in the context of B cell neoplasms treated with CART19, we sought to understand if the addition of ibrutinib to CART19 would reduce this early toxicity. We treated high-tumor burden mice with either CART19 plus ibrutinib or CART19 plus vehicle. Of note, mice treated with the CART19/ibrutinib combination experienced prolonged overall survival (OS) as compared to mice receiving CART19 alone (median OS 17.5 days vs. 5 days, p<0.05) (Figure 1 A) Importantly, serum cytokines evaluated on day 4 were significantly reduced in ibrutinib-treated mice, including IL-6, IFNγ, TNFα, IL-2 and GM-CSF (Figure 1 B). In vitro studies revealed that ibrutinib reduced cytokine production by CART cells as well as by MCL cells, leading us to postulate that both CRS and its successful prevention involve cross-talk between immune cells and cancer cells. Studies to dissect the pathogenic pathway are in progress.In summary, CRS is a major obstacle to the widespread applicability of CART therapy. We here report a novel, clinically relevant xenograft model of CRS developing in B cell neoplasms as a result of CART19 therapy. Furthermore, we show that ibrutinib reduces CART19-mediated CRS and prolongs survival by inhibiting the production of inflammatory cytokines from both CART and tumor cells. Having previously shown that ibrutinib does not impair T cell expansion in vivo and indeed enhances the anti-tumor effect, we suggest that the CART19-ibrutinib combination could be a novel strategy to prevent CRS in B-cell lymphomas as well as in B-cell acute lymphoblastic leukemia. Our institution recently opened clinical trial (NCT02640209) where CART19 (CTL019) is added to ibrutinib in CLL patients who have not achieved a complete response after 6 months. [Display omitted] DisclosuresRuella:Novartis: Patents & Royalties. Kenderian:Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Patents & Royalties, Research Funding. Wasik:Gilead Sciences: Equity Ownership; Seattle Genetics: Honoraria; Novartis: Research Funding; University of Pennsylvania: Patents & Royalties: NPM-ALK as an omncogene; University of Pennsylvania: Patents & Royalties: CAR T-cells; Gilead Sciences: Research Funding; Pharmacyclics: Research Funding. Lacey:Novartis: Research Funding. June:Novartis: Honoraria, Patents & Royalties, Research Funding; Celldex: Consultancy, Equity Ownership; Tmunity Therapeutics: Equity Ownership; Immune Design: Consultancy, Equity Ownership; Pfizer: Honoraria; Novartis: Honoraria, Patents & Royalties, Research Funding; Johnson & Johnson: Honoraria. Gill:Novartis: Patents & Royalties, Research Funding.

Ruxolitinib Prevents Cytokine Release Syndrome after CART Cell Therapy without Impairing the Anti-Tumor Effect in a Xenograft Model

Chimeric antigen receptor T (CART) cell therapy results in impressively high remission rates in B cell neoplasms but is limited by the development of cytokine release syndrome (CRS). CRS is characterized by the development of high-grade fevers, hypotension, fluid overload and respiratory compromise, coincides with T cell expansion and is associated with marked elevation of interleukin-6, interferon-γ and other inflammatory cytokines. Severe CRS is seen in 25-80% of patients treated with CD19 directed CART cell therapy and mortality has been reported. While the use of the anti-IL6 receptor antibody tocilizumab with or without steroids can usually reverse this syndrome, there is concern that the early introduction of immunosuppressive medications could impair the anti-tumor activity and therefore most investigators currently reserve tocilizumab as therapy for severe (grade 3-4) CRS.Ruxolitinib is a JAK/STAT pathway inhibitor that is FDA approved for myelofibrosis and polycythemia vera and has resulted in a significant reduction of inflammatory cytokines in clinical studies. In preclinical models, treatment with ruxolitinib ameliorates cytokine production and manifestations of hemophagocytic lymphohistiocytosis. Therefore, there is a compelling rationale to investigate and develop ruxolitinib as a modality to prevent CRS after CART cell therapy. To our knowledge, there are no relevant models for CRS after human CART therapy, thus severely limiting the development of CRS prevention modalities that would in turn enhance the clinical feasibility of CART therapy.In this study, we established a novel acute myeloid leukemia (AML) xenograft model to study the development of CRS. Here, NSG-S (Non Obese Diabetic, SCID γ-/- mice that are additionally transgenic for human stem cell factor, IL-3 and GM-CSF) are engrafted with blasts from AML patients and treated with 1x106 CD123-directed CART cells (CART123), ten-fold higher than we previously used in our primary AML xenograft models. These animals develop an illness characterized by progressive weight loss, generalized weakness, emaciation, hunched bodies, withdrawal and poor motor response. This illness starts within one week of CART cell injection, correlates with T cell expansion and rapidly evolves and results in the death of the animals in 7-14 days (Figure 1A). Serum from these mice seven days after CART123 shows an extreme elevation of human IL-6, Interferon-γ, tumor necrosis factor-α, and other inflammatory cytokines (Figure 1B), resembling human CRS after CART cell therapy.NSG-S mice bearing primary AML were treated with CART123 and randomized to receive different doses of ruxolitinib (30, 60, or 90 mg/kg) or vehicle by oral gavage twice a day. Treatment started on the day of CART123 injection and continued for a week. Mice treated with ruxolitinib 60 or 90 mg/kg exhibited less severe clinical illness as manifested by attenuated weight loss when compared with mice treated with CART123 alone (Figure 2A). With the exception of ruxolitinib-only treated mice, all other groups exhibited an equivalent anti-leukemic effect (Figure 2B). We therefore established ruxolitinib 60 mg/kg for further experimental use. Most importantly, ruxolitinib treatment attenuated inflammatory cytokines (figure 2C) and led to long-term survival (figure 2D).Here we have described for the first time a clinically relevant animal model of human CRS and demonstrated that the JAK/STAT inhibitor ruxolitinib can prevent the development of severe CRS without impairing the anti-tumor effect of CART cells. These findings provide a useful platform for the future study of CRS prevention and treatment modalities. These experiments indicate that ruxolitinib could also be combined with CART cell therapy for the prevention of CRS in patients identified to be at high risk for the development of CRS. [Display omitted] [Display omitted] DisclosuresKenderian:Novartis: Patents & Royalties, Research Funding. Ruella:novartis: Patents & Royalties: Novartis, Research Funding. Lacey:Novartis: Research Funding. Melenhorst:Novartis: Patents & Royalties, Research Funding. June:Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; Pfizer: Honoraria; Johnson & Johnson: Research Funding; University of Pennsylvania: Patents & Royalties; Tmunity: Equity Ownership, Other: Founder, stockholder ; Celldex: Consultancy, Equity Ownership; Immune Design: Consultancy, Equity Ownership. Gill:Novartis: Patents & Royalties, Resear\\ch Funding.