anti-IL6 Receptor Antibody Tocilizumab Research Articles

Angiopathic activity of LRG1 is induced by the IL-6/STAT3 pathway

Leucine-rich α-2-glycoprotein 1 (LRG1) is a secreted glycoprotein that under physiological conditions is produced predominantly by the liver. In disease, its local induction promotes pathogenic neovascularisation while its inhibition leads to reduced dysfunctional angiogenesis. Here we examine the role of interleukin-6 (IL-6) in defective angiogenesis mediated by LRG1. IL-6 treatment induced LRG1 expression in endothelial cells and ex vivo angiogenesis cultures and promoted vascular growth with reduced mural cell coverage. In Lrg1−/− explants, however, IL-6 failed to stimulate angiogenesis and vessels exhibited improved mural cell coverage. IL-6 activated LRG1 transcription through the phosphorylation and binding of STAT3 to a conserved consensus site in the LRG1 promoter, the deletion of which abolished activation. Blocking IL-6 signalling in human lung endothelial cells, using the anti-IL6 receptor antibody Tocilizumab, significantly reduced LRG1 expression. Our data demonstrate that IL-6, through STAT3 phosphorylation, activates LRG1 transcription resulting in vascular destabilisation. This observation is especially timely in light of the potential role of IL-6 in COVID-19 patients with severe pulmonary microvascular complications, where targeting IL-6 has been beneficial. However, our data suggest that a therapy directed towards blocking the downstream angiopathic effector molecule LRG1 may be of greater utility.

Difficulties of biological therapy in the patient with active rheumatoid arthritis and secondary renal amyloidosis: A report of clinical case

A clinical case of a patient with active rheumatoid arthritis (RA) resistant to standard basic therapy is presented, which served as the reason for the appointment of the target drug – Janus kinase, tofacitinib (Jaquinus) and then biological therapy using anti-IL6 receptor antibody tocilizumab (Actemra). This clinical example demonstrates the patient with the presence of several complications, both the course of the disease – amyloid nephropathy with the development of nephrotic syndrome (NS) as a manifestation of secondary amyloidosis with kidney damage, as well as basic therapy – the presence of comorbid infections with hospital pneumonia and infectious (septic) knee arthritis.An additional contribution of NS to the development of infectious complications in patients with RA receiving immunosuppressive therapy is supposed. Current treatment options for resistant RA and the feasibility of early use of biologics before the development of irreversible complications, as well as the difficulties of therapy and the complications associated with immunosuppression are discussed. Preventive measures for immunization with the anti-pneumococcal vaccine and the need to correct hemostatic disorders in patients with RA and NS are important.

Potent repression of C-reactive protein (CRP) expression by the JAK1/2 inhibitor ruxolitinib in inflammatory human hepatocytes.

To determine whether inflammatory hepatocytes may constitute primary targets for ruxolitinib, a Janus kinase (JAK) inhibitor, its effects towards expression of hepatic acute-phase proteins, especially C-reactive protein (CRP), were assessed. Ruxolitinib effects were analysed in primary human hepatocytes and human hepatoma HepaRG cells exposed to various inflammatory stimuli. Ruxolitinib was found to fully inhibit lipopolysaccharide (LPS)-induced CRP secretion and mRNA expression, at concentrations (IC50 = 12.9nM) achievable in human blood. It similarly repressed CRP up-regulation due to several Toll-like receptor agonists or pro-inflammatory cytokines [interleukin (IL) 1β, IL6 and tumour necrosis factor α] and counteracted LPS-mediated induction of serum amyloid A, fibrinogen, haptoglobin and serpin. Ruxolitinib was additionally found to block the activation of the IL6/JAK/signal transducer and activator of transcription (STAT) pathway triggered by LPS and whose inhibition by the neutralizing anti-IL6 receptor antibody tocilizumab prevented CRP induction. Ruxolitinib can potently repress induction of CRP in inflammatory human hepatocytes, most likely through targeting the IL6/JAK/STAT signalling cascade. Hepatic production of acute-phase proteins during liver inflammation may, therefore, constitute a target for ruxolitinib.

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.

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.

Abstract IA07: Chimeric antigen receptor T cells for cancer immunotherapy

Abstract Background: T cells can be genetically modified to target tumor antigens through the expression of a chimeric antigen receptor (CAR). CAR structure consists of two fundamental domains – the antigen binding portion (most commonly single chain variable fragment (scFv) derived from a monoclonal antibody) joined to one or more intracellular T cell signaling domains. To be effective CAR T cells must expand, persist, exhibit enduring anti-tumor cytotoxicity, withstand and/or counteract an immunosuppressive tumor microenvironment, and overcome targeted tumor antigen escape following infusion. T cells targeting the CD19 antigen is a novel therapeutic approach for patients with hematologic malignancies. To this end, we have previously demonstrated that CAR T cells have a significant clinical benefit in adult patients with relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). We now extend this novel treatment approach by testing of our CD19-specific CAR in a multicenter trial for children and young adults with relapsed CD19+ B-ALL. Patients and Methods: Pediatric and young-adult patients with very high risk (VHR) or R/R CD19+ B-ALL were eligible for enrollment and leukapheresis. Patients with R/R B-ALL were eligible for infusion if they demonstrated residual disease following salvage chemotherapy. Once eligibility was determined isolation and transduction of T cells was performed using a retroviral vector encoding a CD19-specific CAR comprised of a CD19-specific scFv and the CD28 and CD3ζ signaling domains (termed 19-28z). All patients received conditioning chemotherapy followed 2 days later by 1x106 3x106 19-28z CAR T cells/kg. The primary objective of the study was to evaluate the safety and anti-tumor activity of 19-28z CAR T cells. Post-treatment response was assessed at day 14-28 bone marrow aspirate including multiparameter flow cytometry for minimal residual disease (MRD). Results: To date, 28 patients with have been enrolled on protocol with a median age of 11.5 years (range 9 months to 20 years) at time of T cell collection. We have treated 11 patients with 19-28z CAR T cells with a median age 14.9 years (range 3-22 years). Patients received a dose of 1-3 x 10^6 CAR T cells/kg and complete response (complete remission or complete remission with incomplete count recovery) occurred in 7/11 (64%) patients. Significantly, correlations with response included lower disease burden (as assessed by bone marrow cellularity; p&amp;lt;0.004) and fold expansion following CD3/28 bead activation during the generation of CAR T cells (p&amp;lt;0.03). Specifically, BM cellularity ≤50% and fold expansion (average &amp;gt;135 vs &amp;lt;40 fold expansion) was seen in responders compared to non-responders. Pre-collection peripheral blood absolute lymphocyte count, lymphocyte proliferative response (phytohemagglutinin proliferation assay), and CD3 absolute count did not correlate with response in this small sample size. Development of fever and cytokine release syndrome (CRS) occurred in responders including grade I-II (n=3) and grade III-IV (n=4). Systemic immunosuppressants (corticosteroids or anti-IL6 receptor antibody tocilizumab) abrogated clinical symptoms of CRS. Elevated serum cytokines (notably IFN-γ and IL-6) was noted in patients with CRS. Monitoring of bone marrow demonstrated peak 19-28z CAR T cell detection within 1-2 weeks following infusion with gradual contracture over 1-2 months. Conclusions: These early results demonstrate the feasibility and significant clinical impact of CAR T cells in patients with R/R B-ALL. In an effort to more rapidly generate statistically relevant data, demonstrate the exportability of this technology between academic institutions, and offer this therapeutic option to a broader number of pediatric patients with chemo-refractory B-ALL we have expanded this trial to include a collaborating institution. The objective of our trial is not to provide an intent-to-treat cohort, but rather demonstrate the tolerability of this technology in patients with relapsed B-ALL. Furthermore, patients meeting disease eligibility were not pre-screened for lymphocyte function prior to collection and/or treatment. Subsequent cohorts of patients will receive 19-28z CAR T cells and will be evaluated for toxicity, persistence of CAR T cells, and for anti-leukemic efficacy. Citation Format: Kevin J. Curran, Lewis B. Silverman, Rachel Kobos, Nancy A. Kernan, Steven P. Margossian, Jae H. Park, Craig S. Sauter, Victoria Szenes, Xiuyan Wang, Richard J. O'Reilly, Michel Sadelain, Isabelle Riviere, Reiner J. Brentjens. Chimeric antigen receptor T cells for cancer immunotherapy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr IA07.

Multi-Center Clinical Trial of CAR T Cells in Pediatric/Young Adult Patients with Relapsed B-Cell ALL

T cells can be genetically modified to target tumor antigens through the expression of a chimeric antigen receptor (CAR). CAR T cells targeting the CD19 antigen is a novel therapeutic approach for patients with relapsed B cell acute lymphoblastic leukemia (B-ALL). We have previously demonstrated that CAR T cells have a significant clinical benefit in adult patients with relapsed B-ALL. The primary objective of this study (NCT01860937) is to extend the use and test the safety of CD19 specific CAR T cells in a multicenter trial for children and young adults with relapsed CD19+ B-ALL. To date, 24 patients with very high risk (VHR) or relapsed B-ALL have been enrolled on protocol with a median age of 12 years (range 2-20 years) at time of T cell collection. We have treated 9 patients with relapsed B-ALL with a median age 15 years (range 3-22 years) using patient derived T cells expressing a CD19 specific CAR (19-28z). Patients received a dose of 1-3 x 10^6 CAR T cells/kg and complete response (complete remission or complete remission with incomplete count recovery) occurred in 5/9 (55%) patients. Significantly, correlations with response included lower disease burden (as assessed by bone marrow cellularity; p<0.02) and fold expansion following CD3/28 bead activation during the generation of CAR T cells (p<0.02). Specifically, BM cellularity ≤50% and fold expansion (average >150 vs <40 fold expansion) was seen in responders compared to non-responders. Pre-collection peripheral blood absolute lymphocyte count, lymphocyte proliferative response (phytohemagglutinin proliferation assay), and CD3 absolute count did not correlate with response in this small sample size. Development of fever and cytokine release syndrome (CRS) occurred in responders including grade I-II (n=2) and grade III-IV (n=3). Systemic immunosuppressants (corticosteroids or anti-IL6 receptor antibody tocilizumab) abrogated clinical symptoms of CRS. Elevated serum cytokines of IFN-g (>20 fold), fractalkine (>20 fold), Flt-3L (>55 fold), IL-5 (>15 fold), IL-6 (>100 fold), and IL-10 (>15 fold) were demonstrated in patients with CRS. Monitoring of bone marrow demonstrated peak 19-28z CAR T cell detection within 1-2 weeks following infusion with gradual contracture over 1-2 months. These early results demonstrate the feasibility and significant clinical impact of this approach in patients with relapsed B-ALL. In an effort to more rapidly generate statistically relevant data, demonstrate the "exportability" of this technology between academic institutions, and offer this therapeutic option to a broader number of pediatric patients with chemo-refractory B-ALL we have expanded this trial to include a collaborating institution. The objective of our trial is not to provide an intent-to-treat cohort, but rather demonstrate the tolerability of this technology in patients with relapsed B-ALL. Furthermore, patients meeting disease eligibility were not pre-screened for lymphocyte function prior to collection and/or treatment. Subsequent cohorts of patients will receive 19-28z CAR T cells and will be evaluated for toxicity, persistence of CAR T cells, and for anti-leukemic efficacy. DisclosuresCurran:Juno Therapeutics: Consultancy. Off Label Use: CAR T cells for B-ALL. Riviere:Juno Therapeutics: Other: Co-founder, stockholder and consultant. Prockop:Atara Biotherapeutics: Other: I have no financial disclosures, but Atara Biotherapeutics has exercised a licensing agreement with Memorial Sloan Kettering Cancer Center and MSKCC and some investigators at MSKCC have a financial interest in Atara.. Park:Actinium Pharmaceuticals, Inc.: Research Funding; Juno Therapeutics: Consultancy. O'Reilly:Atara Biotherapeutics: Research Funding. Sadelain:Juno Therapeutics: Other: Co-founder, stockholder and consultant. Brentjens:Juno Therapeutics: Other: Co-founder, stockholder and consultant.

Chimeric Antigen Receptor (CAR) T Cells Targeting the CD19 Antigen for the Treatment of Pediatric Relapsed B Cell ALL

T cells can be genetically modified to target tumor antigens through the expression of a chimeric antigen receptor (CAR). CAR T cells targeting the CD19 antigen is a novel therapeutic approach for patients with relapsed B cell acute lymphoblastic leukemia (B-ALL). We have previously demonstrated that CAR T cells have a significant clinical benefit in adult patients with relapsed B-ALL. The primary objective of this study (NCT01860937) is to extend the use and test the safety of CD19 specific CAR T cells in children with relapsed CD19+ B-ALL. To date, 11 pediatric patients with very high risk (VHR) or relapsed B-ALL have been enrolled on protocol with a median age of 13 years (range 2-23 years) at time of T cell collection. We have treated 4 pediatric patients with relapsed B-ALL (ages 13, 14, 19, and 22 years) using patient derived T cells expressing a CD19 specific CAR (19-28z). Complete response (complete remission or complete remission with incomplete count recovery) occurred in 2/4 (50%) patients. Severe cytokine release syndrome (sCRS) defined by fever for ≥3 consecutive days, elevated serum cytokine levels, and one clinical sign of toxicity (hypotension, hypoxia, neurologic disorder including altered mental status, obtundation, and/or seizure) occurred in both patients who responded to CAR T cells. Morphologic disease (≥5% bone marrow blasts) at time of treatment was present in three patients including both patients with sCRS. Systemic immunosuppressants (corticosteroids or anti-IL6 receptor antibody tocilizumab) abrogated clinical symptoms of sCRS. Elevated serum cytokines of IFN-g (>20 fold), fractalkine (>20 fold), Flt-3L (>55 fold), IL-5 (>15 fold), IL-6 (>100 fold), and IL-10 (>15 fold) were demonstrated in patients with sCRS. Monitoring of bone marrow demonstrated peak 19-28z CAR T-cell detection within 1-2 weeks following infusion with gradual contracture over 2-3 months. These early results demonstrate the feasibility and significant clinical impact of this approach in patients with relapsed B-ALL. To more rapidly generate statistically relevant data, demonstrate the “exportability” of this technology between academic institutions, and offer this therapeutic option to a broader number of pediatric patients with chemo-refractory B-ALL our trial will expand into a phase I multicenter clinical trial with a collaborating institution. Subsequent cohorts of patients will receive 19-28z CAR T cells and will be evaluated for toxicity, persistence of CAR T cells, and for anti-leukemic efficacy. DisclosuresOff Label Use: CAR T cells for relapsed B-ALL. Riviere:Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other. Boulad:Genzyme Sanofi: Trials partially funded by Genzyme Sanofi Other. Sadelain:Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other. Brentjens:Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other.

Abstract 336: IL-6 primes melanoma cells from early stages to statin induced apoptosis

Abstract Introduction: Statins trigger apoptosis in tumour cells in vitro and, particularly, melanoma cells are susceptible to statin induced apoptosis. Growth of normal melanocytes and early stage melanoma can be inhibited by IL-6, whereas metastatic melanoma cells are mostly resistant to the anti-proliferative effects of IL-6. It has been shown that nearly 50% of metastatic melanoma secrete IL-6 into the supernatant. Nevertheless until now the stage dependant effects of IL-6 on melanoma cells are not fully understood. Methods: The secretion of IL-6 by A375, 518A2 human metastatic melanoma cells was compared with WM 35 cells which derive from an early lesion (radial growth phase) and investigated with ELISA. The sensitivity toward statin induced caspase 3 activation was investigated in these cells in the absence and presence of IL-6 or an anti IL-6 receptor antibody (tocilizumab). Apoptosis was also confirmed by FACS analyses for AnnexinV/PI staining and proliferation by cell cycle analysis using PI staining in such treated cells. Phosphorylation of Stat3 and the regulation of pro- (Bax, Bak) and anti-apoptotic proteins (Bcl-2, Bxl-XL) were studied by Western blot. Results: Melanoma cells derived from late stage lesions (A375, 518A2) secrete high amounts of IL-6 in contrast to WM35 cells. IL-6 signalling is intact in A375, 518A2 and WM 35 cells indicated by IL-6 triggered phosphorylation of Stat-3. Most interestingly, the A375 and 518A2 cells show a high sensitivity to simvastatin induced apoptosis with EC50 of 0,52 μM (518A2) and 1,7 μM (A375) in contrast to WM35 cells which are more resistant (EC50 16 μM). Co-treatment with IL-6 leads to an augmentation of the statin induced apoptosis in WM35 cells. Statin induced apoptosis can be mediated by the intrinsic apoptotic pathway which requires the loss of mitochondrial membrane potential. This is often alleviated by a downregulation of antiapoptotic Bcl-2 family members. Interestingly IL-6 treatment led to a marked decrease in the amount of Bcl-2 and Bcl-XL in WM35 cells which may explain the increased activation of apoptosis. Since A375 and 518A2 secrete high amounts of IL-6 we investigated the effects of the anti IL-6 receptor antibody tocilizumab. Conversely interruption of the IL-6 signalling with tocilizumab had no effect on the proliferation of 518A2 and A375 cells. Moreover the simvastatin induced caspase 3 activation was not decreased by blockage of the IL-6 signalling. Conclusion: Taken together our data prove different sensitivities of melanoma cells from various stages to statin induced apoptosis. Moreover, IL-6 facilitates apoptosis in early stage melanoma cells by the down-regulation of anti-apoptotic proteins. Nevertheless, these pro-apoptotic effects of IL-6 are not observed in metastatic melanoma cells indicating a stage specific role of IL-6 in the development and tumorigenesis of melanoma. Citation Format: Christoph Minichsdorfer, Christine Wasinger, Martin Hohenegger. IL-6 primes melanoma cells from early stages to statin induced apoptosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 336. doi:10.1158/1538-7445.AM2014-336

Efficacy of the anti–IL-6 receptor antibody tocilizumab in neuromyelitis optica

Objective:To evaluate the safety and efficacy of a humanized anti–interleukin-6 receptor antibody, tocilizumab (TCZ), in patients with neuromyelitis optica (NMO).Methods:Seven patients with anti–aquaporin-4 antibody (AQP4-Ab)-positive NMO or NMO spectrum disorders were recruited on the basis of their limited responsiveness to their current treatment. They were given a monthly injection of TCZ (8 mg/kg) with their current therapy for a year. We evaluated the annualized relapse rate, the Expanded Disability Status Scale score, and numerical rating scales for neurogenic pain and fatigue. Serum levels of anti-AQP4-Ab were measured with AQP4-transfected cells.Results:Six females and one male with NMO were enrolled. After a year of TCZ treatment, the annualized relapse rate decreased from 2.9 ± 1.1 to 0.4 ± 0.8 (p < 0.005). The Expanded Disability Status Scale score, neuropathic pain, and general fatigue also declined significantly. The ameliorating effects on intractable pain exceeded expectations.Conclusion:Interleukin-6 receptor blockade is a promising therapeutic option for NMO.Classification of evidence:This study provides Class IV evidence that in patients with NMO, TCZ reduces relapse rate, neuropathic pain, and fatigue.

A Case of AA Amyloidosis Associated with Rheumatoid Arthritis Successfully Treated with Anti IL-6 Receptor Antibody Tocilizumab.

A Case of AA Amyloidosis Associated with Rheumatoid Arthritis Successfully Treated with Anti IL-6 Receptor Antibody Tocilizumab.

The recombinant humanized anti-IL-6 receptor antibody tocilizumab, an innovative drug for the treatment of rheumatoid arthritis

Background: IL-6 is a pro-inflammatory cytokine with multiple roles in the pathogenesis of rheumatoid arthritis (RA). Targeting IL-6 with the humanized anti IL-6 receptor antibody tocilizumab was effective in several placebo-controlled clinical studies in RA. Objectives: To address how clinically efficacious blockade of IL-6 signalling with inteleukin-6 receptor antibody is in RA patients and what the potential mode of action explaining tocilizumab activity in RA treatment could be. Results/conclusion: IL-6 induces autoantibody-producing plasma cells and effector T cells and is implicated in the development of clinical signs and symptoms, including increased synthesis of acute phase reactants, fatigue, anaemia and anorexia. Its effects also included significant improvements in American College of Rheumatology (ACR)20, ACR50 and ACR70 values, as well as in health-related quality of life measures, compared with controls. Tocilizumab also prevents radiographic progression of joint damage. Tocilizumab is generally well tolerated and efficacious in patients refractive to conventional DMARD therapies.