NOXXON Pharma AG Research Articles

CTNI-43. CXCL12 INHIBITION IN MGMT UNMETHYLATED GLIOBLASTOMA – RESULTS OF AN EARLY PROOF-OF-CONCEPT ASSESSMENT IN THE MULTICENTRIC PHASE I/II GLORIA TRIAL (NCT04121455)

Abstract BACKGROUND Preclinical studies showed that CXCL12-mediated influx of highly angiogenic monocytes/macrophages is a key driver of tumor re-vascularization and re-growth after radiotherapy (RT) of glioblastoma (GBM). We report findings from a phase I/II proof-of concept (PoC) study on CXCL12 inhibition during and after RT of GBM. METHODS Patients ≥18years with incompletely or unresected GBM without MGMT promoter hypermethylation and ECOG≤2 were eligible to participate. Patients received continuous (24/7) i.v. infusions of 200mg/week (n=3), 400mg/week (n=3) or 600mg/week (n=3) of the CXCL12 inhibitor olaptesed pegol (OLA) for 26 weeks during and after normo- or hypofractionated RT (60Gy/40.05Gy). The primary endpoint was safety as per the incidence of treatment-related adverse events. The study was accompanied by PoC-research including multiparametric MRI biomarkers (relative cerebral blood volume, rCBV; fractional tumor burden with high perfusion, FTBhigh; apparent diffusion coefficient, ADC) and of multiplexed immunofluorescence imaging (CODEX®) of reference and patient samples. Initial results of these analyses are reported for the first six patients enrolled. RESULTS Five of six (83%) patients assessed with advanced MRI showed response under OLA in rCBV/FTBhigh and ADC. Maximum reduction in perfusion (rCBV) from baseline was 55%, maximum reduction of FTBhigh was 55% and maximum increase in ADC was 77%. Furthermore, five of six (83%) patients analyzed showed reduction of enhancing tissue volumes in at least one scan under OLA therapy. In both one patient and two reference samples CXCL12 co-localized with endothelial cells of the microvascular proliferation zone. In a paired sample (before/during OLA) of one patient, endothelial cells stained positive for CXCL12 before but not during treatment and almost all GBM cells were negative in Ki67 staining in the sample obtained under OLA therapy. CONCLUSIONS Advanced MRI and multiplexed immunofluorescence suggest efficacy of combined radiotherapy and CXCL12 inhibition in unmethylated GBM. Funded by NOXXON Pharma AG; ClinicalTrials.gov number, NCT04121455.

Impact of the Novel Contrast Agent Gadopiclenol on Decision Making in Patients With Brain Metastases

To evaluate the impact on decision making and radiotherapy (RT) treatment planning of brain metastases (BM) using gadopiclenol, a new gadolinium-based contrast agent (GBCA).Patients who underwent two separate magnetic resonance imaging (MRI) examinations, one with gadopiclenol and one with gadobenate dimeglumine, both at 0.1 mmol/kg were included in the analysis. Those with ≥1 BM detected in any of both scans were subjected to a blinded reader analysis and contouring. For each patient, treatment plans (stereotactic radiosurgery [SRS] or whole-brain radiotherapy [WBRT]) were determined for both MRIs, with the gross target volume (GTV) indicating the contrast-enhancing aspects of the tumor. Mean GTVs and normal tissue volumes receiving 12 Gy (as well as the Dice similarity coefficient (DSC) were obtained for the paired contours. The Spearman´s rank (ρ) correlation was additionally calculated. Furthermore, 3 experts blindly of each lesion for contouring purposes and subjectively qualified them as "better", in detriment of the counterpart, or "equal".A total of 13 adult patients (31% females) presenting with at least one BM were analyzed. Gadopiclenol depicted additional BM as compared with gadobenate dimeglumine in 7 patients (54%). Treatment indication was changed in 2 (15%) patients, from no treatment to SRS and SRS to WBRT. The mean GTVs and V12 were comparable between gadopiclenol and gadobenate dimeglumine (P = 0.694, P = 1.974). The mean DSC was 0.70 (± 0.14, ρ 0.82). From a total 36 answers, an improvement in enhancement was qualified in 58.3% (n = 21) with g, while no difference was obtained in 19.4% (n = 7).Gadopiclenol improved BM detection and characterization with impact on RT treatment decisions. Further trials are warranted to determine the impact of this novel MRI contrast agent on a clinical setting.G.R. Sarria: Honoraria; Roche Pharma AG. Travel Expenses; Carl Zeiss Meditec AG. Volunteer; Rayos Contra Cancer Inc. J. Fleckenstein: None. M. Eckl: None. F. Stieler: None. M. Bendszus: None. L.C. Schmeel: None. D. Koch: None. A. Feißt: None. M. Essig: None. F. Wenz: Honoraria; Carl Zeiss Meditec AG, Celgene GmbH, Roche Pharma AG, Eli Lilly and Co., Ipsen Pharma GmbH, Elekta AB.F.A. Giordano: Research Grant; Carl Zeiss Meditec AG, NOXXON Pharma AG. Honoraria; Implacit GmbH, Oncare GmbH, Roche Pharma AG, AstraZeneca GmbH.

613P - Phase I/II study with CXCL12 inhibitor NOX-A12 and pembrolizumab in patients with microsatellite-stable, metastatic colorectal or pancreatic cancer

BackgroundNOX-A12 is an inhibitor of the chemokine CXCL12 which prevents receptor engagement and blocks the formation of chemotactic CXCL12 concentration gradients. The Opera study (NCT03168139) is a Phase 1/2 study to evaluate pharmacodynamic effects and safety of monotherapy with NOX-A12 as well as safety and efficacy of a combination with pembrolizumab in metastatic microsatellite-stable (MSS) colorectal (CRC) and pancreatic (PaC) cancer where PD-1 inhibition alone has not shown clinical benefit. MethodsPatients received 300mg NOX-A12 twice weekly during a 2-week monotherapy phase. Biopsies were taken from liver metastases before and after NOX-A12 monotherapy for analysis of immune cell infiltration and cytokine signature. In a combination phase, patients received repeated 21-day cycles of 300mg NOX-A12 and 200mg pembrolizumab until progression or intolerable toxicity. Results11 of the patients had CRC and 9 PaC. All patients were heavily pretreated with 5 (CRC) and 3 median lines (PaC) of prior treatment. Best responses to last prior treatment was progressive disease for 95% of patients. The AE profile was comparable with the pembrolizumab profile or typical for the underlying diseases. At baseline, mean T cell density at the invasive margin was 327 cells/mm², clearly below the 600 cells/mm² that are predictive for a good prognosis. 2 weeks of NOX-A12 monotherapy induced Th1 cytokines (IFNγ, IL-2, IL-16) in approx. half of the patients. Although there were no objective responses, 25% of patients achieved stable disease; 7 patients showed prolonged time on treatment vs. the prior line of therapy. Median progression-free survival was 1.87 months, overall survival was 42% at 6 months and 19% at 12 months. ConclusionsIn patients with MSS metastatic PaC and CRC cancer with impaired immune systems and a high tumor load that have failed multiple prior lines of therapy, NOX-A12 plus pembrolizumab shows induction of immune response, stable disease in 25% of patients, and prolonged time on treatment vs. prior therapy for 35% of patients. The safety profile of the combination therapy was consistent with that of pembrolizumab in advanced cancer patients. Clinical trial identificationNCT03168139. Legal entity responsible for the studyThe authors. FundingNOXXON Pharma AG. DisclosureN. Halama: Research grant / Funding (institution): NOXXON Pharma AG; Shareholder / Stockholder / Stock options, Officer / Board of Directors: Navitect Bio. A. Froemming: Full / Part-time employment: NOXXON Pharma AG. D. Beyer: Full / Part-time employment: NOXXON Pharma AG. D. Eulberg: Full / Part-time employment, Officer / Board of Directors: NOXXON Pharma AG. J.U. Jungnelius: Full / Part-time employment, Officer / Board of Directors: NOXXON Pharma AG. A. Mangasarian: Full / Part-time employment, Officer / Board of Directors: NOXXON Pharma AG. All other authors have declared no conflicts of interest.

CXCL12 Inhibition By Nox-A12 (Olaptesed Pegol) Synergizes with the ADCC Activity of CD20 Antibodies By Increasing NK Cell Infiltration in a 3D Lymphoma Model

Hematological malignancies are characterized by the expansion of malignant cells in the peripheral blood and in stroma-rich niches such as the bone marrow or lymphoid tissues. Anti-CD20 monoclonal antibodies (mAbs) are highly effective in eliminating malignant cells in the peripheral blood with the help of immune effector cells, e.g. NK cells mediating antibody-dependent cellular cytotoxicity (ADCC). However, residual malignant cells often continue to persist in protective stromal niches. These compartments have similarities to the solid tumor microenvironment (TME) where mAb therapy is restricted by poor tissue penetration and low effector cell infiltration.The CXCL12-neutralizing L-RNA aptamer NOX-A12 (olaptesed pegol) has been shown to mobilize malignant cells from the bone marrow into the peripheral blood, thereby sensitizing them to the action of standard therapy such as the anti-CD20 mAb rituximab (Blood. 2014;124(21):1996). In addition to malignant cells, CXCR4 expressing immune cells are effectively mobilized by NOX-A12 (Clin Pharmacol Ther. 2013;94(1):150-157). Recently we have shown that NOX-A12 increases lymphocyte infiltration into solid tumor-stroma spheroids, thereby synergizing with anti-PD-1 checkpoint blockade (Cancer Res 2016;76(14 Suppl): 1473). Here we established 3D lymphoid spheroidal microtissues mimicking the stroma-rich and CXCL12-abundant TME of lymphoid malignancies. We investigated the effect of NOX-A12 on NK effector cell infiltration into lymphoma spheroids and tested the combination with anti-CD20 mAbs.Spheroids were generated by co-culturing of CXCL12-expressing murine stromal MS-5 cells and CD20-expressing lymphoma cells in ultra-low attachment plates for 24 hours. Primary human NK cells, isolated from healthy donors, were added to the spheroids in the presence of various concentrations of NOX-A12 and an anti-CD20 mAb, either rituximab or obinutuzumab. The next day, spheroids were washed and dissociated for NK cell quantification and lymphoma cell viability determination by flow cytometry.We found that the ADCC efficacy of anti-CD20 mAbs is lower in 3D spheroids compared to conventional 2D assays due to low NK cell infiltration into the microtissues. Interestingly, NOX-A12 increased the amount of NK cells in the lymphoma-stroma spheroids up to 8-fold in a dose-dependent manner (Figure A), likely by forming de novo CXCL12 gradients into the dense microtissue due to the particular penetration characteristic of L-RNA aptamers. Of note, the NOX-A12-mediated increase of NK cells in the spheroids synergized with both anti-CD20 mAbs tested in terms of NK cell-mediated killing of lymphoma cells (Figure B).The present work complements the mechanism of action data of NOX-A12 by adding enhancement of NK cell infiltration into stroma-rich tumor compartments to the already established effects of mobilizing malignant and immune effector cells into the peripheral blood. These data as well as the good toxicity profile and the promising data in phase 2a clinical trials in patients with CLL and MM justify further clinical trials in patients with hematological malignancies to verify the greater efficacy of combination treatment using ADCC-inducing mAbs and NOX-A12. [Display omitted] DisclosuresZboralski:NOXXON Pharma AG: Employment. Kruschinski:NOXXON Pharma AG: Employment. Vater:NOXXON Pharma AG: Employment.

A Combined Molecular Docking/Dynamics Approach to Study Selectivity and Binding Affinity of L-Stereoisomer RNA Aptamer Towards CCL2 and Related Chemokines

CC chemokine ligand 2 (CCL2)- also known as Monocyte Chemoattractant Protein 1- is a primary chemoattractant in monocytes. It has a high-affinity interaction with chemokine receptor 2 (CCR2) and causes calcium mobilization and chemotaxis. In inflammatory processes and cancer, CCL2 is highly expressed. Preventing CCL2 function by interfering with CCL2-CCR2 binding can alleviate complications seen in many inflammatory diseases. Many oligonucleotide-based aptamers that target proteins are currently being developed to block the interaction of the target protein with other molecules. An L-stereoisomer oligonucleotide aptamer (emapticap pegol), targeting CCL2 was identified by Noxxon Pharma AG using an in vitro process known as “systematic evolution of ligands by exponential enrichment” (SELEX). This L-RNA aptamer was used to treat patients with diabetic nephropathy and completed Phase IIa of clinical trials. The mode of action of the drug was deciphered using a combination of binding and structural data; however, this data could not fully explain the observed selectivity profile of the L-aptamer. In this work, using molecular dynamics simulation approach, we were able to understand the dynamic behaviour of L-aptamer in free form and in complex with various chemokines, including CCL2. Furthermore, using calculated binding free energy, we were able to explain the experimentally observed binding affinity profile. Our present work provides a complete structural understanding of L-aptamer with various chemokines and also provides inputs to optimise the design of the L-aptamers which will further improve binding specificity towards CCL2.

Effects of CCL2/CCR2 Blockade in Acute Myeloid Leukemia

Background: A role for the Chemokine (C-C motif) ligand 2 (CCL2) in attracting tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSC) and infiltrating monocytes has been described for many solid tumors in which they play an essential role in modifying the adaptive immune response, ultimately favoring tumor progression. Unfortunately, little is known about the importance of this mechanism for the progression of AML. We recently identified CCL2 as the most prominent chemokine produced by bone marrow (BM) mesenchymal stromal cells (BM-MSC) in response to the interaction with myeloid leukemia cells (PMID: 24599548). In addition, elevated CCL2 plasma levels have been reported in patients of AML (PMID: 17822317), ALL (PMID: 21298741) and CLL (PMID: 22397722) when compared to normal controls. In this study we assessed the effects of blocking the CCR2-CCL2 axis on the migration and signaling of hematopoietic cells as well as on the infiltration of immune-suppressive cells in leukemia-bearing mice.Results: We first studied the efficacy and potency of agents at inhibiting CCL2-mediated migration, using the human monocytic leukemia cell line THP-1. Migration towards human recombinant CCL2 (5 ng/ml) was significantly inhibited by as little as 1 nM of NOX-E36, a human-specific CCL2 Spiegelmer (NOXXON Pharma, Berlin). Spiegelmers are RNA-like molecules built from L-ribose units that are able to bind molecules such as peptides and proteins with an affinity in the pico-to nanomolar range. Similar results were obtained with a CCR2 antagonist (100 ng/ml; Santa Cruz). In anticipation of in vivo studies in mice, we next confirmed the ability of a mouse-specific CCL2 Spiegelmer (mNOX-E36) to inhibit migration and signaling pathway activation in murine hematopoietic cells. For this purpose, we cloned and overexpressed via lentiviral transduction the murine CCL2 receptor (CCR2) in Ba/F3 cells (a murine pro-B cell line). Stimulation of Ba/F3-CCR2 cells with 5 ng/ml of mouse recombinant CCL2 induced a ~2000 fold increase in migration of Ba/F3-CCR2 cells and was successfully blocked with mNOX-E36 in a concentration-dependent manner. Western blot analysis of protein lysates from mCCL2-stimulated cells (30 minutes treatment) indicated activation of AKT, ERK and p38-MAPK. The CCL2-induced phosphorylation of these molecules was completely abrogated by pre-treatment with mNOX-E36.Subsequently, we determined whether the expression of CCL2 by stromal cells in leukemia-resident organs triggers the infiltration of TAMs and possibly other immune-suppressive cells into those organs. We conducted preliminary in vivo studies in non-irradiated immunocompetent C57BL/6 mice (n=5 per group) injected with syngeneic AML1/ETO9a-expressing primary murine leukemia cells (PMID: 19339691). After confirmation of leukemia engraftment by IVIS imaging, mice were treated with mNOX-E36 (14.4 mg/kg, s.c., three times per week) or vehicle control for 3 weeks. At this point, all animals were sacrificed and their tissues (spleens and BM from femurs) were collected for analysis. Although we did not observe differences in leukemia burden by imaging between vehicle and mNOX-E36 treated groups, flow cytometry analysis revealed an increase in the frequency of CD11b+ Ly6Clow MHC IIlow macrophages (2 to 7 fold increase) in spleens of mice engrafted with leukemia (vehicle-treated group) when compared to spleens collected from healthy mice. These MHC IIlow macrophages were previously identified as immunosuppressive M2-like macrophages as opposed to MHC IIhi macrophages which show a pro-inflammatory M1-like phenotype (PMID: 20570887). Importantly, CCL2 inhibitor mNOX-E36 abrogated this macrophage infiltration within the leukemia microenvironment.Conclusions: Our results indicate that blockade of the CCR2-CCL2 axis not only affects migration and signaling of treated cells in vitro, but also interferes with the infiltration of M2-like macrophages into spleens of leukemia-bearing mice. Current in vivo experiments using a combination of standard chemotherapy with mNOX-E36 in AML immunocompetent models are undergoing. We expect that in vivo modulation of CCL2 will improve response to chemotherapy of AML by reducing the marrow infiltration of infiltrating monocytes and tumor-associated macrophages, which would facilitate translation of this novel concept into clinical trials in AML. DisclosuresZuber:Boehringer Ingelheim: Research Funding; Mirimus Inc.: Consultancy, Other: Stock holder. Eulberg:NOXXON Pharma AG: Employment. Kruschinski:NOXXON Pharma AG: Employment.

Final Results from the Phase IIa Study of the Anti-CXCL12 Spiegelmer® Olaptesed Pegol (NOX-A12) in Combination with Bortezomib and Dexamethasone in Patients with Multiple Myeloma

BackgroundOlaptesed, an L-stereo-isomer RNA aptamer, binds and neutralizes the chemokine CXCL12. By interaction with the chemokine receptors CXCR4 and CXCR7, CXCL12 is responsible for trafficking and homing of normal and malignant blood cells to the bone marrow. Preclinical studies have shown synergistic activity of CXCL12-targeting and anti-myeloma agents, specifically bortezomib (BTZ). Thus, targeting the myeloma niche may increase treatment efficacy.AimsThis open label single arm study was conducted to assess the activity and safety of olaptesed when added to the combination of BTZ and dexamethasone (DEX) in patients with relapsed / refractory multiple myeloma (MM).Patients and MethodsTwenty-eight relapsed or refractory MM patients (males:females 14:14) were enrolled and treated according to a dose titration design. Olaptesed was administered intravenously at doses increasing from 1 mg/kg to 2 mg/kg and 4 mg/kg in cycles 1, 2 and 3, respectively, at 1 hour prior to bortezomib administration. During cycles 4 to 8, olaptesed was dosed at the highest individually titrated dose. BTZ (1.3 mg/m2) was given on days 1, 4, 8 and 11 as intravenous injection. Oral DEX (20 mg) was added on the day of and on the day after BTZ administration. Response was evaluated based on the uniform IMWG response criteria (Rajkumar SV et. al. Blood 2011; 117: 4691-5). Plasma cell mobilization was studied after a pilot dose of 1 to 4 mg/kg olaptesed administered to the initial 10 patients before start of the regular treatment regimen.ResultsFrom Aug 2012 to Feb 2014 we enrolled 28 patients who had received a median of 2 (range 1-6) lines of prior therapy. Pretreatments were lenalidomide (LEN) in 20, BTZ in 14 and carfilzomib in 1 patient. Ten patients had autologous stem cell transplantations prior to entering this study.The patient population enrolled presented predominantly with advanced disease and with adverse outcome predictors. Ten patients had ISS stage III. High-risk cytogenetics were identified in 9 of the 20 patients (45%) with FISH testing available for t(4;14), t(14;16) and/or del17p. Eleven patients were refractory to their last prior treatment, which contained BTZ in 8 cases.After two early withdrawals, 26 patients were available for outcome evaluations. The median number of completed cycles was 8. Progression led to treatment termination in 8 patients. The dose of olaptesed was titrated to 4 mg/kg in all 18 patients treated for 3 or more cycles.The single dose of olaptesed administered to 10 pilot-patients effectively mobilized plasma cells, which increased by approximately 200% for up to 3 days.Based on “best response” of the 26 evaluable patients, the overall response rate was 73%: Two patients (8%) achieved a complete response (CR), 6 patients (23%) a very good partial response (VGPR) and 11 patients (42%) a partial response (PR). Minimal response was recorded in 2 patients (8%), 4 patients (15%) had stable disease and 1 patient (4%) progressive disease.In the 9 evaluable patients with high-risk cytogenetics, the clinical responses were similar. The ORR was 67% with VGPR in 3 (33%) and PR in 3 (33%) patients. Of the 14 patients pre-treated with BTZ, 1 had a CR and 8 a PR (ORR 64%).M-protein decreased rapidly from treatment cycle 1 to cycle 4 with a decrease of ≥50% being observed in 15 of the 26 evaluable patients. Figure 1 shows a waterfall plot of the maximum observed decrease in M-protein. [Display omitted] Median progression-free survival (PFS) of the evaluable population was 6.5 months. It was also 6.5 months in the 9 patients with high-risk cytogenetics and 6.3 months in the 14 patients pre-treated with BTZ (Figure 2). The median follow-up was 6.3 months. [Display omitted] Treatment with olaptesed in combination with BTZ-DEX was safe and well tolerated without any appreciable increase in adverse events.ConclusionsA single dose of olaptesed effectively mobilized plasma cells. Olaptesed in combination with BTZ and DEX resulted in an ORR rate of 73% and PFS of 6.5 months. Response rates and PFS were similar in patients with or without high risk cytogenetic features or with or without previous exposure to BTZ. The combination regimen was well tolerated. These findings merit further exploration of this strategy in randomized trials. DisclosuresWeisel:NOXXON Pharma AG: Consultancy. Petrucci:Celgene: Honoraria; Jannsen-Cilag: Honoraria; Sanofi: Honoraria; Bristol-Myers Squibb: Honoraria. Leleu:Janssen, Celgene, leopharma, Takeda, Amgen, Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Laurent:Bristol-Myers Squibb: Honoraria. Kruschinski:NOXXON Pharma AG: Employment. Dümmler:NOXXON Pharma AG: Employment. Riecke:NOXXON Pharma AG: Employment. Engelhardt:NOXXON Pharma AG: Consultancy.

Results from a Phase IIa Study of the Anti-CXCL12 Spiegelmer Olaptesed Pegol (NOX-A12) in Combination with Bendamustine/Rituximab in Patients with Chronic Lymphocytic Leukemia

BackgroundOlaptesed pegol (NOX-A12) is a novel, potent, L-stereoisomer RNA aptamer that binds and neutralizes CXCL12/SDF-1, a chemokine which attracts and activates immune- and non-immune cells via interaction with the receptors CXCR4 and CXCR7. Signaling of CXCL12 is pivotal to the interactions of leukemic cells with bone marrow microenvironment. The therapeutic concept of olaptesed is to inhibit such tumor-supporting pathways and thereby mobilizing and sensitizing CLL cells to therapy.Here we aim to assess the activity and safety of olaptesed in combination with bendamustine and rituximab (BR) in patients with relapsed / refractory CLL.MethodsTwenty-eight relapsed or refractory CLL patients were enrolled and treated in this open-label, single-arm Phase IIa study. To investigate PK/PD, a pilot dose of 1 to 4 mg/kg olaptesed alone was administered to 3 patients per dose group (plus one additional replacement pt) before start of the regular treatment regimen (pilot group). Patients were treated using a dose titration design with intravenous (IV) olaptesed at doses increasing from 1 mg/kg to 2 mg/kg and 4 mg/kg at cycles 1, 2 and 3, respectively, at 1 hour before rituximab treatment. During cycles 4 to 6, olaptesed was dosed at the highest individually titrated dose. Rituximab was administered IV at doses of 375 mg/m² on day 1 of the 1st28-day cycle and 500 mg/m² on day 1 of subsequent cycles. Bendamustine (70 - 100 mg/m²) was given IV on days 2-3 (cycle 1) or days 1-2 (cycles 2-6) of each 28-day cycle following administration of rituximab. Clinical response was assessed according to NCI-WG Guidelines (Hallek M et al. Blood 111; 2008: 5446-56).ResultsTo date, 24 patients completed treatment (12 women, 12 men) with a median age of 68.5 years (range 52 to 79). At screening 5, 9 and 10 patients presented with Binet stage A, B and C, respectively. The median number of prior treatment lines was 1 (range 1-2). Seven high-risk patients presented an unfavorable disease state being relapsed within 24 months after fludarabine/bendamustine treatment (5 pts) and/or presenting a deletion/mutation of the TP53 gene (3 pts). Most patients (19 of 24) were previously treated with fludarabine or bendamustine.A flow cytometric analysis of CD19+/CD5+CLL cells showed a rapid mobilization into the peripheral blood by a single dose of olaptesed which lasted throughout the observational time of 72h. Interestingly, CXCR4 expression levels increased on CLL cell surface in the periphery after olaptesed treatment. This increase, which peaked at 24h, likely reflects the extended circulation of CLL cells in the periphery due to the sustained blockade of CXCL12 by olaptesed. Reduction of lymphadenopathy by ≥ 50% was achieved in 14 out of 21 evaluable patients with reported enlarged lymph nodes by the end of treatment. Concomitantly, rapid reduction of lymphocytosis in peripheral blood with normalization by treatment cycle 2 – 3 was observed and the CLL to leukocyte ratio significantly improved.Efficacy was assessed at the end of cycles 3 and 6. In the full-analysis-set, which excludes two non-evaluable patients (drop-out after the 1st cycle due to adverse events), the overall response rate was 96%: Three patients (14%) achieved a complete response at end of cycle 6 (2 confirmed, 1 investigator reported) and eighteen patients (82%) achieved a partial response (fifteen at end of cycle 6 and three at end of cycle 3). Notably, all seven high-risk patients (defined as relapsed within 24 months after fludarabine/bendamustine treatment and/or presenting a deletion/mutation of the TP53 gene) responded to treatment with olaptesed + BR with a partial response. One patient had a progressive disease. Olaptesed at 1, 2 and 4 mg/kg at a single dose and in combination with BR was safe and well tolerated. The observed adverse reactions were qualitatively and quantitatively as expected for patients treated with BR.ConclusionOlaptesed in combination with BR was safe and well tolerated. Compared with historical data, olaptesed showed superiority over baseline therapy with regards to overall response rate and increasing rates of complete remission, warranting further development of this Spiegelmer in CLL. DisclosuresMontillo:Janssen: Honoraria. Kruschinski:NOXXON Pharma AG: Employment. Dümmler:NOXXON Pharma AG: Employment. Riecke:NOXXON Pharma AG: Employment.

Comparison of Ibrutinib, Idelalisib and Olaptesed Pegol on the Immune Effector Function Mediated By Rituximab

Olaptesed pegol (NOX-A12), an SDF-1 binding Spiegelmer® was found to detach SDF-1 from the surface of BMSCs (Hoellenriegel et al., Blood 2013) and to mobilize CXCR4 expressing, malignant cells from protective niches in the bone marrow or other secondary lymphoid tissues, thereby sensitizing them to the action of standard therapy. In addition to malignant cells CXCR4 expressing immune cells, e.g. T cells, neutrophils and monocytes are effectively mobilized by olaptesed (Vater et al., Clin Pharmacol Ther 2013). Here we investigated the combination of olaptesed with an immunotherapeutic agent, in particular rituximab (RTX). Several mechanisms of action (MoA) of RTX have been described including antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). The aim was to analyze whether olaptesed influences the immune effector cell activation by RTX as has been shown for the Btk or PI3Kd inhibitors ibrutinib and idelalisib, respectively.ADCC activity was measured by the calcein release assay. Raji lymphoma cells were labeled with calcein, treated with 10 µg/mL RTX and co-cultured for 4 hours with various ratios of PBMCs as effector cells, which have been incubated with either 10 µM olaptesed, 1 µM ibrutinib or 1 µM idelalisib, respectively, overnight. In order to analyze the impact on phagocyte function, ADCP assays with neutrophils and monocytes were performed. Phagocytes were preincubated with either 10 µM olaptesed, 1 µM ibrutinib or 1 µM idelalisib, respectively, for 6 hours, and cocultured overnight with DiD-labeled primary B-cells or Raji cells which were opsonized with different concentrations of RTX. ADCP was quantified in a guava easyCyte flow cytometer by counting the CD15+ (or CD14+) and DiD+ population (gated on CD19-negative cells) and normalizing to total phagocyte counts. To analyze B-cell depletion, heparinized whole blood from healthy volunteers was diluted with HBSS (1:2.5) and incubated with either 10 µM olaptesed, 1 µM ibrutinib or 1µM idelalisib, respectively, for 6 hours followed by the addition of different RTX concentrations. After incubation for 16 hours B-cell depletion was evaluated by determining the CD19+ B-cell and CD3+ T-cell populations.We found that preincubation of PBMCs with 1 µM ibrutinib significantly decreased calcein release. 1 µM idelalisib led to a slight decrease of calcein release, while 10 µM olaptesed did not influence ADCC activity (Figure 1A). Preliminary results from ADCP assays point to a decreased phagocytic activity in terms of maximal ADCP for neutrophils after preincubation with ibrutinib or idelalisib (Figure 1B). Importantly, although olaptesed is being endocytosed by phagocytes, the uptake of the drug did not inhibit ADCP. In accordance, the whole blood assay showed that olaptesed had no negative impact on RTX-mediated B-cell depletion, while ibrutinib and idelalisib were found to decrease B-cell depletion regarding EC50 and maximal killing (Figure 1C). Taken together, we found that olaptesed retains the immune functional repertoire of RTX, whereas ibrutinib and idelalisib decrease the immune effector function. These small molecule kinase inhibitors possess low specificity and are inhibiting also other kinases which may be important for immune cell activation, such as ITK which is blocked by ibrutinib. The MoA of olaptesed is partially overlapping with kinase inhibitors with regard to lymphoid cell mobilization; however, olaptesed is highly selective and therefore does not impair immune-cell mediated contribution to monoclonal antibody efficacy. Instead, olaptesed may further enhance the contribution of immune effector cells through mobilization of T cells from the bone marrow which are described to be exhausted in the peripheral blood of hematological malignancies like CLL. Furthermore, neutrophils and monocytes which contribute to ADCP are effectively mobilized with olaptesed.Based on their complementary mechanisms of action involving direct and immune-mediated cell death (RTX) or long-term mobilization of not only the malignant cells but also effector immune cells (olaptesed), the combination has the potential for greater efficacy in treating B lymphoid malignancies without impairing immune effector activity. Olaptesed is currently being tested in a Phase 2a clinical trial in combination with RTX and bendamustine in patients with relapsed/refractory CLL (#NCT01486797). [Display omitted] DisclosuresZboralski:NOXXON Pharma AG: Employment. Vater:NOXXON Pharma AG: Employment. Kruschinski:NOXXON Pharma AG: Employment.

Anti-CXCL12/SDF-1 Spiegelmer® Nox-A12 Alone and In Combination With Bortezomib and Dexamethasone In Patients With Relapsed Multiple Myeloma: Results From A Phase IIa Study

BackgroundNOX-A12 is a novel, potent, L-stereoisomer RNA aptamer (Spiegelmer®) that binds and neutralizes CXCL12/SDF-1, a chemokine which attracts and activates immune- and non-immune cells via interaction with its receptors, CXCR4 and CXCR7. CXCL12 plays an important role in homing and trafficking of multiple myeloma (MM) cells to the bone marrow (BM). Here, we evaluate the capacity of NOX-A12 to mobilize plasma cells into the circulation. Furthermore, we analyze a possible therapeutic impact of NOX-A12 which disrupts the interaction of MM cells with the BM stroma resulting in enhanced chemosensitivity to established therapies such as bortezomib and dexamethasone (VD). MethodsTo date, 21/28 planned patients have been enrolled into a multicenter Phase IIa study of NOX-A12 alone and in combination with VD in relapsed MM patients. Here we report interim data on PK, PD and preliminary efficacy of a pilot group consisting of 3 cohorts of 3 patients each. In the pilot phase, cohorts received single doses of 1, 2 or 4 mg/kg NOX-A12 alone, respectively, two weeks prior to 8 planned cycles of combined treatment with NOX-A12 and VD repeated every 21 days. During combination therapy, NOX-A12 was administered 1-2 hours prior to bortezomib following a dose titration design for all patients: NOX-A12 doses were increased from 1 mg/kg to 2 mg/kg and 4mg/kg at cycles 1, 2 and 3, respectively. During cycles 4-6, doses of NOX-A12 were kept at the highest individually titrated dose. Bortezomib (1.3 mg/m2) was given on days 1, 4, 8 and 11 as intravenous injection. Oral dexamethasone (20 mg) was added on the day of and the day after bortezomib administration. Response was evaluated by applying the uniform response criteria of the IMWG 2011. ResultsIn total, 10 patients were enrolled into the pilot group (one patient was replaced after withdrawal of consent). The median age was 63 years (range 56-78) with 7 women and 3 men being included. Median prior therapies were 2 (range 1-5), with 9 patients having been pretreated with dexamethasone and 3 with bortezomib prior to enrollment. At screening 1, 7 and 2 patients presented with ISS stage I, II and III, respectively. IgG, IgA and LC M-components were present in 6, 3 and 1 patients, respectively. Six patients had cytogenetic aberrations, which were high risk (t(4;14) or del17p) in 2 patients. Plasma profiles of NOX-A12 in the patient population of the pilot group (Figure 1) were similar to healthy volunteers in which a plasma half-life of approximately 38 hours was observed. After single doses of NOX-A12, a dose-linear exposure with peak plasma levels of 2.1, 3.9, and 6.7 µM was found in the corresponding cohorts. Furthermore, an increase of plasma cells in peripheral blood by approximately 200% was detected which lasted throughout the observation period of 3 days (Figure 2). NOX-A12 as single agent was safe and very well tolerated.Of nine evaluable patients who received the combination treatment of NOX-A12 and VD, 2 (22%) achieved a VGPR and 4 (44%) PR, resulting in an ORR (≥ PR) of 67%. In combination with VD, NOX-A12 was equally safe and well tolerated. [Display omitted] [Display omitted] ConclusionProof of principle was achieved as single doses of NOX-A12 reached the expected plasma exposure and translated into an effective and prolonged mobilization of plasma cells into the peripheral blood. In addition, responses (PR or better) were noted in 6/9 (67%) patients of this pilot group. Single agent NOX-A12 was not associated with any relevant toxicity and the combination of VD with NOX-A12 was well tolerated and not associated with additional toxicity on top of VD. Provided that these promising findings will be confirmed in the total sample of 28 patients, further development of this novel anti-CXCL12/SDF-1 Spiegelmer® seems warranted. Disclosures:Ludwig:Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Munipharma: Honoraria, Research Funding. Weisel:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria. Engelhardt:MSD, Janssen-Cilag: Research Funding. Greil:NOXXON Pharma AG: Research Funding. Dümmler:NOXXON Pharma AG: Employment. Zöllner:NOXXON Pharma AG: Employment. Zeitler:NOXXON Pharma AG: Employment. Riecke:NOXXON Pharma AG: Employment. Kruschinski:Noxxon: Employment.

Anti-CXCL12/SDF-1 Spiegelmer® Nox-A12 Alone and In Combination With Bendamustine and Rituximab In Patients With Relapsed Chronic Lymphocytic Leukemia (CLL): Results From A Phase IIa Study

BackgroundNOX-A12 is a novel, potent, L-stereoisomer RNA aptamer (Spiegelmer(®)) that binds and neutralizes CXCL12/SDF-1, a chemokine which attracts and activates immune- and non-immune cells via interaction with its receptors, CXCR4 and CXCR7. The signaling of CXCL12 has been shown to play an important role in the pathophysiology of chronic lymphocytic leukemia (CLL), especially in the interaction of leukemic cells with tissue microenvironment. The therapeutic concept of NOX-A12 is to mobilize these bone marrow- and tissue-resident CLL cells into the blood, thereby removing CLL cells from the nurturing milieu and sensitizing them to cytotoxic drugs such as bendamustine and rituximab (BR). MethodsTo date, 19/33 planned patients have been enrolled into a multicenter Phase IIa study of NOX-A12 alone and in combination with BR in relapsed CLL patients. Here we report interim data on PK, PD and preliminary efficacy of a pilot group consisting of 3 cohorts of 3 patients each. In the pilot phase, cohorts received single doses of 1, 2 or 4 mg/kg NOX-A12 alone, respectively, two weeks prior to 6 cycles of combined treatment of NOX-A12 with BR repeated every 28 days. During combination therapy, NOX-A12 was administered 1-2 hours prior to rituximab following a dose titration design for all patients: NOX-A12 doses were increased from 1 mg/kg to 2 mg/kg and 4mg/kg at cycles 1, 2 and 3, respectively. During cycles 4-6, doses of NOX-A12 were kept at the highest individually titrated dose. Bendamustine (70 - 100 mg/m², according to SPC) was given on day 2 and 3 (cycle 1) or 1 and 2 (cycle 2-6), combined with 375 mg/m2 rituximab on day 1 for the first cycle and 500 mg/m2 for subsequent cycles. Tumor response was assessed according to NCI-WG 1996 criteria (updated by iwCLL 2008). ResultsIn total, 10 patients were enrolled in the pilot group (one additional patient was enrolled due to one patients' under dosing). The median age was 69 years (range 57-77) with 8 women and 2 men being included. Median prior therapies were 2 (range 1-2), whereby all patients were bendamustine naïve and 6 patients had received rituximab treatment prior to enrolment. 2, 4 and 4 patients presented at screening with Binet stage A, B, and C, respectively. 8 patients showed at least 1 cytogenetic aberration at the beginning of treatment. Tumor assessments before enrolment and at end of cycles 3 and 6 were evaluated.Plasma profiles of NOX-A12 in the patient population of the pilot group (Figure 1) were similar to those of healthy volunteers in which a plasma half-life of approximately 38 h was observed. After single doses of NOX-A12, the exposure was dose-linear with peak plasma concentrations of 1.7, 3.5, and 6.7 µM in the corresponding cohorts. CLL cells in the peripheral blood were found to be increased throughout the observational period of 3 days (Figure 2). In all patients presenting with lymphadenopathy, the lymph node size decreased markedly. NOX-A12 as single agent was safe and very well tolerated. [Display omitted] [Display omitted] All patients responded to the combination treatment of NOX-A12 and BR (ORR 100%); one patient had to be withdrawn from treatment due to multiple infections during cycle 4 having achieved a partial response (PR). At the end of cycle 6, seven patients (78%) showed a PR and two patients (22%) achieved a complete remission (CR). In combination with BR, NOX-A12 was equally safe and well tolerated. ConclusionsProof of principle was achieved as single doses of NOX-A12 reached the expected plasma exposure which translated into an effective and prolonged mobilization of CLL cells into the peripheral blood. In addition, the 100% ORR and 22% CR as well as the virtual absence of additional toxicity on top of BR observed in this pilot group compares very favorably with historical controls. Provided that this promising clinical picture will be maintained in the total sample of 33 patients, further development of this novel anti-CXCL12/SDF-1 Spiegelmer(®) seems warranted. Disclosures:Montillo:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Dümmler:NOXXON Pharma AG: Employment. Zöllner:NOXXON Pharma AG: Employment. Zeitler:NOXXON Pharma AG: Employment. Riecke:NOXXON Pharma AG: Employment. Kruschinski:NOXXON Pharma AG: Employment.

The Spiegelmer® Nox-A12 Abrogates Homing Of Human CLL Cells To Bone Marrow and Mobilizes Murine CLL Cells In The Eμ-TCL1 Transgenic Mouse Model Of CLL

▪NOX-A12 is a novel mirror-image oligonucleotide (Spiegelmer®) that specifically antagonizes the chemokine stromal cell-derived factor 1 (SDF-1, CXCL12). SDF-1 is highly expressed by stromal and endothelial cells, and binding to its classical receptor CXCR4 leads to the activation of multiple signaling pathways regulating cell migration, retention and survival. SDF-1-induced inside-out activation of the integrin VLA-4 (a4ß1, CD49d/CD29) via CXCR4 is essential for homing of various normal and malignant hematopoietic cells to bone marrow (BM). We previously found that VLA-4 is indispensable for BM homing of chronic lymphocytic leukemia (CLL) cells. Hence, CXCR4 binding to SDF-1 may be a prerequisite for VLA-4 mediated trafficking of CLL cells into the protective BM microenvironment and for their retention in lymphoid organs, favoring their survival and chemotherapy-resistance.In this project we compared the efficacy of NOX-A12 and the CXCR4 antagonist AMD3100 to mobilize CLL cells from their protective niches to the peripheral blood, thereby sensitizing them to cytotoxic agents such as fludarabine, and to prevent their recirculation into the protective lymphoid microenvironment.To assess the contribution of SDF-1/CXCR4 signals to homing of CLL cells, we performed in vivo short term adoptive transfers of human VLA-4+ CLL cells into immune deficient mice. CLL cells (5-10 x 106) were injected into the tail vein of NOD/SCID mice that were treated either with NOX-A12 (20 mg/kg) or AMD3100 (5 mg/kg). After a homing period of 3 hours lymphatic organs were analyzed for human CLL cell count. We found that for the majority of investigated CLL samples (except patient samples bearing the chromosomal aberration trisomy 12), CXCR4/SDF-1 inhibition reduced CLL cell recirculation into BM, thereby redirecting them into the spleen. The mean (± SD) relative homing rate (homed human CLL cells relative to injected cells per 1 x 106 acquired murine cells) of CLL cells to BM decreased upon NOX-A12 treatment from 137 (± 87) to 30 (± 17) (p = 0.047), and upon AMD3100 treatment from 137 (± 71) to 38 (± 24) (p = ns). Further, we determined CXCR4 surface levels of CLL cells before injection and after cells had homed to BM. We noticed that homed CLL cells expressed elevated CXCR4 surface levels as mean MFIR (± SD) rose from 25 (± 13) to 93 (± 11) and that CXCR4 levels were further increased upon CXCR4/SDF-1 inhibition with NOX-A12 to MFIR 225 (± 2) and with AMD3100 to MFIR 213 (± 7).Next, we used the Tcl1 transgenic transplantation model that mimics human CLL to test NOX-A12 and AMD3100-induced CLL cell mobilization. C57BL/6J wild type mice engrafted with splenocytes (1 x 107) from Tcl1tg mice with established disease were treated with separate doses of NOX-A12 (0, 10, 20 and 50 mg/kg) by single intravenous injections. Separate doses of AMD3100 (0, 3, 5, and 10 mg/kg) were administered subcutaneously. Blood was collected from the tail vein before treatment, 1 hr, 3 hrs, 6 hrs, and 24 hrs post treatment and analyzed for absolute leukocyte counts and numbers of CD5+/CD19+cells. NOX-A12 demonstrated a considerable potential to mobilize CLL cells and other lymphocytes as the relative CLL cell and absolute lymphocyte blood count raised within 1 hour by 50% when using mice with high tumor load and by 200% when using mice with lower tumor load. Mobilization reached a peak after 6 hours with 100% and 220% relative increase to 0 hrs value. Compared to untreated control mice, CLL cell blood count raised from 80% to 120% within 6 hours. Even low doses (10 mg/kg) of NOX-A12 showed a high degree of mobilization of CLL cells and total lymphocytes. Higher doses lead to a prolonged mobilization duration. AMD3100 treatment did not lead to a significant increase in CLL cell or total lymphocyte blood count in peripheral blood.In summary our data demonstrate a high efficiency of NOX-A12 to mobilize CLL cells and to prevent their recirculation into protective lymphatic organs. Abrogating SDF-1/CXCR4 signaling in combination with chemotherapy may provide an attractive approach for treatment of CLL. Currently, the effect of combined NOX-A12 and fludarabine treatment on progression and overall survival is evaluated using the Tcl1 transgenic transplantation model. Disclosures:Kruschinski:Noxxon: Employment. Greil:NOXXON Pharma AG: Research Funding. Hartmann:NOXXON Pharma AG: Research Funding.

Chronic Lymphocytic Leukemia Cells With Trisomy 12 Home To The Bone Marrow In a CXCR4-Independent Manner and Are Prone To Proliferate In Vitro

Trisomy 12 (tri12) is a frequent chromosomal aberration in chronic lymphocytic leukemia (CLL) associated with atypical cell morphology, high in vivo tumor proliferation activity and a predisposition to Richter's transformation. Tri12 harboring CLL cells express increased levels of the negative prognostic marker CD49d, the α4 subunit of the integrin very late antigen 4 (VLA-4), which we previously identified as a key regulator of CLL cell homing to bone marrow (BM). During this process, inside-out activation of VLA-4 upon CXCR4 binding to endothelially displayed CXCL12 is thought to upregulate the adhesive properties of VLA-4 and augment the arrest of CLL cells on the VCAM-1 presenting vessels.Here, we investigated the functional interplay of VLA-4 and CXCR4 in CLL carrying tri12. We first found that the upregulation of CD49d expression in this subset (MFIR CD49d 9.8±5.3 (n=22) vs. 2.7±3.9 (n=126), p<0.0001) was paralleled by their reduced CXCR4 expression (MFIR CXCR4 11.8±7.2 (n=22) vs. 22.7±14.2 (n=126), p=0.0003). Using short term adoptive transfers, we compared the ability of tri12 and no tri12 CLL cells to home to the BM of NOD/SCID mice. 5-10x106 CLL cells were injected into tail vein and homing was evaluated after 3 hours. Based on their more frequent CD49d high phenotype, we observed increased homing rates (homed human CLL cells per 106 injected cells per 106 acquired murine cells) of tri12 compared to no tri12 CLL (225±160 (n=7) vs. 90±117 (n=20), p=0.025). However, when comparing CD49d+ tri12 and CD49d+ no tri12 subsets, we did not observe any significant differences in their homing capacity. To further study CXCL12/CXCR4 function in BM homing, we pretreated mice with either the novel CXCL12 antagonist NOX-A12 or the CXCR4 inhibitor AMD3100 prior to CLL cell injection. While homing of no tri12 CLL cells (n=3, in duplicates) was reduced by both pretreatments (homing rates 137 vs 38 vs 30), the homing capacity of tri12 CLL cells (n=3, in duplicates) was not affected.We next tested whether VLA-4 expressed on these cells was able to undergo CXCL12-induced activation and support cell arrest under shear conditions. To this end, we perfused CLL cells over VCAM-1 or VCAM-1/CXCL12 substrates and analyzed rates and categories of cell tethering at a single cell level by videomicroscopy. CXCL12 induced the arrests of no tri12 CLL cells (n=3) on VCAM-1 under shear flow in a CXCR4 and VLA-4 dependent manner. In contrast, tri12 CLL cells (n=3) robustly tethered to VCAM-1 in the absence of the chemokine, and interactions could not be further enhanced by additional CXCL12 nor could they be abrogated by use of AMD3100. This failure of CXCR4-induced adhesion was not based on a general defect in CXCR4 functionality as in vitro chemotaxis of tri12 CLL cells (n=5) towards CXCL12 was fully maintained. To detect potential differences in VLA-4 affinity regulation, we used a conformationally sensitive antibody that recognizes epitopes induced by VLA-4 ligation, and an LDV-containing VLA-4 specific ligand to probe resting integrin affinity. Also, we used a small fluorescent ligand to study rapid VLA-4 affinity changes during inside-out chemokine induced activation. On resting tri12 CLL, VLA-4 exhibited an affinity state similar to that observed on circulating lymphocytes, and tri12 CLL cells failed to undergo the rapid affinity up-regulation triggered by CXCL12 pretreatment, in keeping with tethering experiments.Next, we investigated whether the tumor microenvironment has a different influence on the behavior of the tri12 subset. Therefore we subjected the cells to in vitro co-cultures mimicking the lymphoid proliferation centers. Basal levels of the early activation marker CD69 were similar in tri12 CLL compared to no tri12 cases. Tri12 CLL, however, underwent stronger activation when cultured in presence of accessory cells (%CD69+ cells 60.0±18.5 (n=4) vs. 17.7±20.1 (n=19), p=0.008). Moreover, in several setups, proliferation rates of these cells were increased, irrespective of the proliferative stimulus and detection method used.In summary, our results provide a mechanistical basis at least in part explaining the peculiar and clinical features of the tri12 CLL subset. In light of the specific migratory and proliferative properties of tri12 cells and novel agents targeting particularly these functions, our findings may also imply therapeutical consequences. Disclosures:Greil:NOXXON Pharma AG: Research Funding. Hartmann:NOXXON Pharma AG: Research Funding.

Randomized Double Blind Placebo Controlled PK/PD Study On the Effects of a Single Intravenous Dose of the Anti-Hepcidin Spiegelmer Nox-H94 On Serum Iron During Experimental Human Endotoxemia

AbstractAbstract 3452 ObjectiveAnemia of chronic disease (ACD) is generally attributed to increased hepcidin production. The first-in-class hepcidin antagonist NOX-H94, a PEGylated anti-hepcidin L-RNA oligonucleotide, is in development for targeted treatment of ACD. We investigated whether NOX-H94 prevents the inflammation-induced serum iron decrease during experimental human endotoxemia. Effects on the innate immune response were studied as secondary endpoints. Materials and MethodsThe study protocol (ClinicalTrials.gov identifier NCT01522794) was approved by the local ethics committee of the Radboud University Nijmegen Medical Centre and was conducted in accordance with the Declaration of Helsinki (2000) of the World Medical Association.This randomized, double-blind, placebo-controlled trial was carried out in 24 healthy young men. At T=0 hours (h), 2 ng/kg E. coli endotoxin was administered intravenously (i.v.), followed by 1.2 mg/kg NOX-H94 or placebo i.v. at T=0.5h. Blood was drawn serially for 24h and at day 3, 8 and 15 after endotoxin administration for measurements of inflammatory parameters, cytokines, NOX-H94 pharmacokinetics, total hepcidin-25 (free and NOX-H94-bound), and iron parameters. The difference of serum iron change from baseline at T=9h was defined as primary endpoint. Data are expressed as mean±SD. ResultsEndotoxin administration led to flu-like symptoms peaking at T=1.5h, irrespective of the treatment group. Body temperature rose by 1.9±0.5 °C in both groups. Peak CRP at T=24h was also similar in NOX-H94 and placebo treated groups (34.3±18.0 vs. 36.8±8.7 mg/L) as was the rise in leucocytes at T=6 h (12.1±2.2 vs. 12.1±2.3 ×109/L). Plasma levels of TNF-α, IL-6, IL-10, and IL-1RA peaked markedly and similarly in both treatment groups.NOX-H94 was well tolerated. Plasma concentrations peaked at 0.7±0.4 h after the start of administration, after which they declined according to a two-compartment model, with rapid initial elimination followed by a slower elimination phase with a plasma half-life of 22.5 ± 4.28h. Hepcidin-25 rose to a peak of 23.0±5.2 nM at T=5.5±0.05 h in control subjects and normalized at about T=24 h. In the NOX-H94 treated group, total hepcidin-25 concentrations, which represent the sum of free hepcidin-25 and hepcidin-25 bound to NOX-H94, rose to 559±112 nM at T=9.5±1.5 h and normalized slowly until day 15.The observed serum iron concentrations are shown in the table. In the placebo group, serum iron increased from baseline to a peak at T=3h, returned close to baseline at T=6h and decreased under the baseline concentration at T=9h reaching its lowest point at T=12h. In the NOX-H94 group, serum iron concentrations rose until T=6h and then slowly declined until T=24h. From 6 to 12 h post LPS, the serum iron concentrations in NOX-H94 treated subjects were significantly higher than in placebo treated subjects (P<0.0001, ANCOVA). At T=24 h and 48 h, serum iron was lower in NOX-H94 treated subjects than in placebo controls.TableMean serum iron concentrations (μmol/L, ± SD) in subjects treated with LPS at T= 0h and with NOX-H94 or Placebo at T=0.5hDayTime (h)Placebo (n=12)1.2 mg/kg NOX-H94 (n=12)10 (baseline)19.0 ± 7.6418.0 ± 6.821332.6 ± 6.7133.1 ± 6.291622.8 ± 6.3138.3 ± 8.09 *1910.7 ± 5.6033.9 ± 9.04 *1128.0 ± 2.9528.7 ± 9.62 *22413.7 ± 5.969.3 ± 5.8334817.4 ± 3.9610.1 ± 4.17821.2 ± 4.3221.6 ± 5.761520.8 ± 7.8523.1 ± 8.03*p<0.0001, ANCOVA. ConclusionExperimental human endotoxemia induced a robust inflammatory response with hepcidin release and a subsequent decrease in serum iron. Treatment with NOX-H94 had no effect on innate immunity, but effectively prevented the inflammation-induced drop in serum iron concentrations. These findings demonstrate the clinical potential of the anti-hepcidin drug NOX-H94 for further development to treat patients with ACD. Disclosures:van Eijk:NOXXON Pharma AG: Research Funding. Swinkels:NOXXON Pharma AG: Research Funding. Schwoebel:NOXXON Pharma AG: Employment. Fliegert:NOXXON Pharma AG: Employment. Summo:NOXXON Pharma AG: Employment. Stéphanie:NOXXON Pharma AG: Employment. Riecke:NOXXON Pharma AG: Employment. Pikkers:NOXXON Pharma AG: Research Funding.

Single and Repeated Dose First-in-Human Study with the Anti-Hepcidin Spiegelmer Nox-H94.

AbstractAbstract 2342 Background:NOX-H94, the first-in-class hepcidin inhibitor in development for treatment of anemia of chronic disease (ACD), is a PEGylated anti-hepcidin L-RNA oligonucleotide. ACD is caused by iron sequestration in the reticulo-endothelial macrophages with subsequent iron restricted erythropoiesis due to high hepcidin production and subsequent ferroportin degradation. The treatment of ACD is challenging: a significant number of ACD patients do not respond to erythropoiesis stimulating agents (ESAs), while repeated intravenous iron administrations bear a risk of iron overload. Targeting hepcidin may provide more efficacious and well tolerated treatment alternatives. Methods:This First-in-Human study investigated the safety and tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of escalating single and repeated doses of intravenous (i.v.) NOX-H94 in healthy men and women. The study protocol (ClinicalTrials.gov identifier NCT01372137) was approved by an independent ethics committee and conducted in accordance with the Declaration of Helsinki. Five successive cohorts of 8 healthy subjects, at least 3 men and women, were randomly assigned to i.v. doses of 0.3, 0.6, 1.2, 2.4, and 4.8 mg/kg of NOX-H94 (n=6) or placebo (n=2). Similarly, 2 cohorts of 8 male subjects randomly received 5 doses of either 0.6 or 1.2 mg/kg NOX-H94 or placebo every other day (q2d). Safety parameters, iron parameters, total hepcidin-25 (sum of free circulating hepcidin-25 and hepcidin-25 bound to NOX-H94), and PK were assessed during treatment and follow-up periods of ≥3 weeks. Data are given as means±SD. Results:One man, assigned to 5 i.v. doses of 0.6 mg/kg, withdrew consent after 4 administrations; all other 55 subjects completed the study as scheduled. Safety: Treatment with NOX-H94 was generally safe and well tolerated. No serious adverse event occurred; headache and fatigue were the only treatment related events that occurred more than once. Mild and transient increases in transaminases (<2×ULN) were noted in subjects treated with NOX-H94 at single doses ≥2.4 mg/kg or with repeated doses of 1.2 mg/kg (4.2 mg/kg weekly). PK: After escalating single i.v. administrations of 0.3 to 4.8 mg/kg of NOX-H94, peak plasma concentrations of NOX-H94 (Cmax) and systemic exposure (AUC) increased dose-proportionally. The elimination was bi-phasic with a terminal plasma half-life (t ½) in the range of 17 to 26 h. The systemic clearance (CL) was low (Table 1). After repeated q2d i.v. administrations, no appreciable plasma accumulation was found based on Cmax and AUC. No obvious gender-difference was observed. PD: The plasma concentration of total hepcidin-25 increased dose-dependently upon NOX-H94 treatment, without ever exceeding the plasma concentration of NOX-H94. The PD effects were assessed by analysis of the area under the data time curve above baseline (AUD) of various iron parameters. Single and repeated doses of NOX-H94 up to 0.6 mg/kg had no effect on serum iron, serum ferritin, and transferrin saturation (TSAT) in the healthy subjects studied. At doses ≥1.2 mg/kg NOX-H94, serum iron, serum ferritin, and TSAT increased dose dependently.Table 1:Single dose PK of NOX-H94Dose Group [mg/kg]nCmax [μM]AUC 0-tlast [μM×h]t ½ [h]CL [L/h]0.360.524±0.0985.63±3.4316.5±7.50.34±0.220.661.09±0.36013.0±4.5518.0±7.50.24±0.101.262.17±0.26130.4±6.2423.9±9.90.18±0.032.464.75±0.67980.0±12.023.5±4.40.15±0.034.869.85±1.53213±26.526.3±3.20.12±0.03Table 2:Serum iron parameters after single dose treatment with NOX-H94 analyzed based on the area under the data time curve above baseline (means±SD)Dose Group [mg/kg]nSerum iron [μmol/L×h]Serum ferritin [μg/L×h]TSAT [%×h]Placebo1044.6±28.429.5±45.362.5±41.00.3647.6±51.915.5±31.382.2±76.90.6667.0±40.344.4±53.2111±60.11.26193±53.7125±205321±1372.46134±45.0371±290239±97.84.86225±138644±670422±334 Conclusions:Treatment with NOX-H94 was generally safe and well tolerated over all dose levels and schedules studied; for subsequent phase II studies in patients, twice weekly i.v. doses of 1.2 mg/kg are recommended. PK analyses showed a dose-linear systemic exposure. In these healthy subjects, only mild dose-dependent increases in iron parameters were observed which likely underestimate the effects that may be obtained in patients with iron-restricted anemia. Disclosures:Riecke:NOXXON Pharma AG: Employment. Zöllner:NOXXON Pharma AG: Employment. Vauléon:NOXXON Pharma AG: Employment. Swinkels:NOXXON Pharma AG: Research Funding. Dümmler:NOXXON Pharma AG: Employment. Summo:NOXXON Pharma AG: Employment. Schwoebel:NOXXON Pharma AG: Employment. Fliegert:NOXXON Pharma AG: Employment.

Phase IIa Study of the Anti-CXCL12/SDF-1 Spiegelmer® Nox-A12 Alone and Combined with Bendamustine/Rituximab in Patients with Relapsed Chronic Lymphocytic Leukemia (CLL)

AbstractAbstract 4593 BackgroundNOX-A12 is a novel, potent, L-aptamer inhibitor of CXCL12/SDF-1, a chemokine which attracts and activates immune- and non-immune cells. The signaling of CXCL12 has been shown to play an important role in the pathophysiology of chronic lymphocytic leukemia (CLL), especially in the interaction of leukemic cells with their tissue microenvironment. The therapeutic concept of NOX-A12 is to inhibit such tumor-supporting pathways and thereby sensitizing the CLL cells towards chemotherapy. MethodsThe purpose of this phase IIa study is to evaluate the safety and efficacy of NOX-A12 in combination with background chemo-immunotherapy of bendamustine and rituximab (BR) in patients with relapsed CLL. The described study is being performed in compliance with ethical principles based on the Declaration of Helsinki and ICH-GCP guidelines. The study population was split into a pilot and expansion group. In the pilot group, 3 cohorts of 3 patients each received escalating doses of single agent NOX-A12 two weeks prior to the combined treatment of NOX-A12 and BR. Interim data from these patients are reported. Based on previous Phase I studies in healthy volunteers, pilot patients received a dose of 1, 2 or 4 mg/kg body weight (BW) single agent NOX-A12 on day -14, followed by a 2-weeks period of safety, PK and PD assessments prior to the combined treatment with NOX-A12 and BR. To date, the first cohort of the pilot group already progressed to the 2nd cycle of combined treatment. Evaluation criteria included adverse events according to CTCAE V4, flow cytometry of peripheral blood CD34+ cells and CLL cells, pharmacokinetics of NOX-A12, plasma concentration of CXCL12 and tumor response (NCI-WG 1996 criteria, updated 2008). ResultsTo date 3 patients (age range: 58 – 65 years) have been enrolled in the pilot group of this study. They had received 1 or 2 prior therapies, but no bendamustine. Single i.v. doses of 1 mg/kg BW NOX-A12 had no clinically relevant effects on vital signs, 12-lead ECG parameters and laboratory parameters. One patient reported grade 1 pain in the lower limbs two days after treatment with NOX-A12. This event was not dose-limiting and resolved spontaneously on the same day. Flow cytometry of CD34+ cells and CLL cells (CD19+/CD5+high) showed a rapid mobilization of these cells into the peripheral blood on day 1. Interestingly, return to baseline was not complete at the last assessment on day 3 (for details see Figure 1). The NOX-A12 pharmacokinetics in these 3 patients (for concentration-time profile see Figure 2) is very comparable to healthy volunteers receiving i.v. NOX-A12, with a maximum plasma concentration of 1.52 ± 0.14 μM after 1 h (tmax) and a plasma elimination half-life of about 50 h. As seen in healthy volunteers the plasma concentration of CXCL12 increased upon NOX-A12 treatment and reached a maximum of 0.434 ± 0.076 μM at 24 to 72 h p.a. without ever approaching the plasma concentration of NOX-A12 (Figure 2). ConclusionSingle i.v. doses of NOX-A12 at 1 mg/kg BW were safe and well tolerated; the maximum tolerated dose was not reached. NOX-A12 induced a long-lasting mobilization of CD34+ cells and leukemic cells in patients with relapsed CLL, consistent with a mechanism of action based on CXCL12 inhibition. Patient accrual and identification of an optimal chemosensitization regimen of NOX-A12 combined with BR is being continued. [Display omitted] [Display omitted] Disclosures:Vauléon:NOXXON Pharma AG: Employment. Zöllner:NOXXON Pharma AG: Employment. Dümmler:NOXXON Pharma AG: Employment. Kruschinski:NOXXON Pharma AG: Employment. Fliegert:NOXXON Pharma AG: Employment.

Mode of Action of the SDF-1/CXCL12 Inhibiting Spiegelmer® Nox-A12 and Its Impact On Chronic Lymphocytic Leukemia (CLL) Cell Motility and Chemosensitization

Abstract 318NOX-A12 is a novel Spiegelmer®-based antagonist of SDF-1/CXCL12, a chemokine involved in the regulation of chronic lymphocytic leukemia (CLL) cell trafficking. Spiegelmers® are mirror-image oligonucleotides that are identified to specifically bind to proteins in a manner conceptually similar to antibodies. Unlike aptamers, however, Spiegelmers® are built from the non-natural L-isomer form of nucleotides which confers resistance to the action of nucleases and avoids potential immunogenicity.CXCL12 is constitutively secreted and presented by bone marrow stromal cells (BMSC) via glycosaminoglycans (GAG) and acts as a homing factor for normal and malignant hematopoietic cells to the bone marrow (BM) and secondary lymphoid tissues via CXCR4 receptors that are expressed at high levels on circulating CLL cells. The microenvironment in the BM and secondary lymphoid tissues, in particular the CXCL12-CXCR4 axis, favors survival and chemotherapy-resistance of leukemic cells. We therefore investigated the effects of NOX-A12 in an in vitro co-culture system to model the interaction of CLL cells with their microenvironment.Surprisingly we observed that NOX-A12 increased pseudoemperipolesis in vitro, i.e. spontaneous leukemia cell migration beneath BMSC. Interestingly, this NOX-A12 induced trans-migration of CLL cells was completely inhibited by the CXCR4 antagonist AMD3100, suggesting a CXCL12/CXCR4 dependent mechanism. We postulated that this observation might result from a direct effect of NOX-A12 on CXCL12 release by the stromal cells. Therefore, we investigated this hypothesis in different BMSC lines (MS-5, R15C, and TSt-4) and we found that NOX-A12 induced a significant CXCL12 release in all three tested cell lines.We asked whether this NOX-A12 dependent increase of CXCL12 of BMSCs is due to release from either intracellular or extracellular storages. Intracellular staining of CXCL12 using flow cytometry did not reveal significant changes when BMSCs were incubated with NOX-A12. Furthermore, the transcription of CXCL12 was not found to be altered after NOX-A12 incubation over a period of three days as shown by quantitative RT-PCR. Rather, CXCL12 is released from extracellular storages of BMSCs. First hints were obtained through a rapid CXCL12 release within five minutes of incubation with NOX-A12. To confirm that CXCL12 is bound to the extracellular surface (by GAGs like heparin) and is being detached by NOX-A12 we first incubated BMSCs with NOX-A12, followed by a wash step and the addition of recombinant CXCL12. Recombinant CXCL12 was bound by BMSCs that were pre-incubated with NOX-A12 but not with a non-functional control (revNOX-A12), indicating that NOX-A12 strips off CXCL12. To corroborate the findings we incubated the BMSCs with heparin which also led to the release of CXCL12 in a dose dependent manner. Of note, the EC50 of heparin regarding CXCL12 release was much higher compared to the EC50 of NOX-A12 (≈ 12 μM vs. 5 nM) revealing the high affinity of NOX-A12 to CXCL12. The competition of NOX-A12 with heparin regarding CXCL12 binding was confirmed by Biacore experiments.Based on these findings, we developed a novel adapted co-culture approach to examine the ability of NOX-A12 to chemosensitize CLL cells. In this setting, we first strip off CXCL12 from BMSCs by NOX-A12 and subsequently add CLL cells which will be either non-treated or treated with chemotherapy (fludarabine combined with bendamustine). We found that NOX-A12 slightly decreased CLL cell viability. As expected, a strong viability decrease was observed with chemotherapy, which could be even further decreased by the combination with NOX-A12, suggesting synergistic effects.In conclusion, we propose that NOX-A12's mode of action is the release of extracellular bound CXCL12 and its subsequent inhibition. Since CXCL12 induces leukemia cell trafficking and homing to tissue microenvironment and also favors leukemia cell survival, we believe that targeting CXCL12 is an attractive approach to remove the protective effects of CXCL12-secreting BMSCs in order to sensitize CLL cells for subsequent chemotherapy. Thus, NOX-A12 represents a very promising agent to significantly improve the treatment of CLL. The compound is currently being tested in a Phase IIa study in relapsed CLL patients. Disclosures:Zboralski:NOXXON Pharma AG, Berlin, Germany: Employment. Maasch:NOXXON Pharma AG: Employment. Kruschinski:NOXXON Pharma AG: Employment. Burger:NOXXON Pharma AG: Consultancy, Research Funding.

In Vivo Targeting of Stromal-Derived Factor-1 As a Strategy to Prevent Myeloma Cell Dissemination to Distant Bone Marrow Niches

AbstractAbstract 440 Background.Multiple myeloma (MM) patients present with multiple lytic lesions at diagnosis, indicating the presence of continuous dissemination of MM cells from the primary site of tumor development to multiple distant bone marrow (BM) niches. We hypothesized that stromal-derived factor-1 (SDF-1) may represent a target for preventing transition from MGUS (micrometastatic stage) to active-MM (macrometastatic stage); thus resulting in inhibition of MM progression. We therefore evaluated SDF-1 expression in the BM of patients with MGUS, MM, compared to healthy individuals; and tested NOX-A12, a high affinity l-oligonucleotide (Spiegelmer) binder to SDF-1 in MM, looking at its ability to modulate MM cell tumor growth and MM cell homing to the BM in vitro and in vivo . Methods.SDF-1 levels were evaluated by immunohistochemistry on BM specimens obtained from patients with MGUS, active-MM, or healthy individuals; and confirmed by ELISA, using conditioned-medium of BM-mesenchymal stromal cells obtained from MGUS, active-MM and healthy individuals. BM metastatic lesions from primary epithelial tumors were also considered. Co-localization of MM tumor cells (MM.1S-GFP+) with SDF-1 was tested in vivo by in vivo confocal microscopy, using both AlexaFluor633-conjugated-anti-SDF-1 monoclonal antibody and AlexaFluor647-conjugated-NOX-A12 oligonucleotide. Effect of NOX-A12 on modulating MM cell dissemination was tested in vivo, by using in vivo confocal microscopy. In vivo homing and in vivo tumor growth of MM cells (MM.1S-GFP+/luc+) were assessed by using in vivo confocal microscopy and in vivo bioluminescence, in SCID mice treated with 1) vehicle; 2) NOX-A12; 3) bortezomib; 4) NOX-A12+bortezomib. Detection of mobilized MM-GFP+ cells ex vivo was performed by flow cytometry. Effects of drug combination on dissemination of MM cells to distant BM niches was evaluated ex vivo by immunofluorescence on femurs obtained from each cohort of mice. DNA synthesis and adhesion of MM cells in the context of NOX-A12 (50–100nM) treated primary MM BM stromal cells (BMSCs) in presence or absence of bortezomib (2.5–5nM) were tested by thymidine uptake and adhesion in vitro assay, respectively. Synergism was calculated by using CalcuSyn software. NOX-A12-dependent-modulation of signaling was evaluated by western blot on MM cells exposed or not to primary BM-MSCs. Results.Patients with active-MM present with higher BM SDF-1 expression vs. MGUS patients and healthy individuals. Similarly, BM presenting with metastasis from epithelial primary malignancies had higher SDF-1 levels compared to healthy subjects, thus suggesting the importance of SDF-1 in favoring tumor cell metastasis to BM niches. SDF-1 co-localized at BM level with MM tumor cells in vivo. In vitro, NOX-A12 induced a dose-dependent de-adhesion of MM cells from the BMSCs supported by inhibition of BM-MSC-mediated phosphorylation of ERK1/2 and cofilin. These findings were corroborated and validated in vivo: NOX-A12 induced MM cell mobilization from the BM to the peripheral blood as shown ex vivo, by reduced percentage of MM cells in the BM and increased number of MM cells within the peripheral blood of mice treated with NOX-A12 vs. control (BM: 57% vs. 45%; PB: 2.7% vs. 15%). This was supported by inhibited homing of MM cells to the BM of those mice pre-treated with NOX-A12. We next showed that NOX-A12-dependent de-adhesion of MM cells from BMSCs lead to enhanced MM cell sensitivity to bortezomib, as shown in vitro, where a synergistic effect between NOX-A12 and bortezomib was observed (C.I.: .57-.76). These findings were validated in vivo: tumor burden was similar between NOX-A12- and control mice whereas bortezomib-treated mice showed significant reduction in tumor progression compared to the control (P<.05); importantly, significant reduction of tumor burden in those mice treated with sequential administration of NOX-A12 and bortezomib was observed, compared to bortezomib alone-treated mice (P <.05). Similarly, NOX-A12+bortezomib combination induced significant inhibition of MM cell homing, as shown by in vivo confocal microscopy. Conclusion.SDF-1 represents a valid target for inhibiting MM cell dissemination to distant BM niches, thus providing the evidence for using the SDF-1 inhibiting Spiegelmer NOX-A12 to target MM cells at the stage of micrometastasis (MGUS), thus preventing development of symptomatic macrometastatic MM. Disclosures:Zboralski:NOXXON Pharma AG, Berlin, Germany: Employment. Kruschinski:NOXXON Pharma AG, Berlin, Germany: Employment. Ghobrial:Novartis: Advisory Board Other; Onyx: Advisory Board, Advisory Board Other; Millennium: Advisory Board, Advisory Board Other; Bristol Myers Squibb: Advisory Board, Advisory Board Other.

L-Stereoisomer RNA Oligonucleotide Anti-SDF-1 (Nox-A12) Disrupts the Interaction of Multiple Myeloma Cells with the Bone Marrow Milieu In Vivo, Leading to Enhanced Sensitivity to Bortezomib

Abstract 887 Background.Stomal-cell-derived factor 1 (SDF-1) is known to be involved in bone marrow (BM) engrafment for malignant tumor cells, including CXCR4 expressing multiple myeloma (MM) cells. We hypothesized that de-adhesion of MM cells from the surrounding BM milieu through SDF-1 inhibition will enhance MM sensitivity to therapeutic agents. We therefore tested NOX-A12, a high affinity l-oligonucleotide (Spiegelmer) binder to SDF-1in MM, looking at its ability to modulate MM cell tumor growth and MM cell homing to the BM in vivo and in vitro. Methods.Bone marrow (BM) co-localization of MM tumor cells with SDF-1 expressing BM niches has been tested in vivo by using immunoimaging and in vivo confocal microscopy. MM.1S/GFP+ cells and AlexaFluor633-conjugated anti-SDF-1 monoclonal antibody were used. Detection of mobilized MM-GFP+ cells ex vivo has been performed by flow cytometry. In vivo homing and in vivo tumor growth of MM cells (MM.1S-GFP+/luc+) were assessed by using in vivo confocal microscopy and in vivo bioluminescence detection, in SCID mice treated with 1) vehicle; 2) NOX-A12; 3) bortezomib; 4) NOX-A12 followed by bortezomib. DNA synthesis and adhesion of MM cells in the context of NOX-A12 (50–200nM) treated primary MM BM stromal cells (BMSCs), in presence or absence of bortezomib (2.5–5nM), were tested by thymidine uptake and adhesion in vitro assay, respectively. Synergism was calculated by using CalcuSyn software (combination index: C.I. according to Chou-Talalay method). Results.We first showed that SDF-1 co-localizes in the same bone marrow niches of growth of MM tumor cells in vivo. NOX-A12 induced a dose-dependent de-adhesion of MM cells from the BM stromal cells in vitro. These findings were corroborated and validated in vivo: NOX-A12 induced MM cell mobilization from the BM to the peripheral blood (PB) as shown ex vivo, by reduced percentage of MM cells in the BM and increased number of MM cells within the PB of mice treated with NOX-A12 vs. control (BM: 57% vs. 45%; PB: 2.7% vs. 15%). We next showed that NOX-A12-dependent de-adhesion of MM cells from BMSCs lead to enhanced MM cell sensitivity to bortezomib, as shown in vitro, where a synergistic effect between NOX-A12 (50–100 nM) and bortezomib (2.5–5 nM) was observed (C.I.: all between 0.57 and 0.76). These findings were validated in vivo: tumor burden detected by BLI was similar between NOX-A12- and control mice whereas bortezomib-treated mice showed significant reduction in tumor progression compared to the control (P<.05); importantly significant reduction of tumor burden in those mice treated with sequential administration of NOX-A12 followed by bortezomib was observed as compared to bortezomib alone treated mice (P <.05). Similarly, NOX-A12 + bortezomib combination induced significant inhibition of MM cell homing in vivo, as shown by in vivo confocal microscopy, as compared to bortezomib used as single agent. Conclusion.Our data demonstrate that the SDF-1 inhibiting Spiegelmer NOX-A12 disrupts the interaction of MM cells with the BM milieu both in vitro and in vivo, thus resulting in enhanced sensitivity to bortezomib. Disclosures:Roccaro:Roche:. Kruschinski:Noxxon Pharma AG: Employment. Ghobrial:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Advisory Board, Research Funding.

The Spiegelmer Nox-A12, a Novel SDF-1 (CXCL12) Inhibitor, and Its Effects on Chronic Lymphocytic Leukemia (CLL) Cell Migration,

Abstract 3878NOX-A12 is a PEGylated mirror-image oligonucleotide (a so-called Spiegelmer), that binds the chemokine SDF-1/CXCL12 with high affinity (approx. 200 pM), thereby efficiently inhibiting its activity. CXCL12 is constitutively secreted by bone marrow stromal cells (BMSC) and acts as a homing factor for normal and malignant hematopoietic cells to the marrow. It induces leukemia cell trafficking and homing to tissue mircroenvironments (i.e. the marrow and secondary lymphatic tissues) via CXCR4 receptors that are expressed at high levels on circulating CLL cells. The microenvironments in the bone marrow and secondary lymphoid tissues favor survival and chemotherapy-resistance of CLL cells. Therefore, targeting the CXCR4-CXCL12 axis is an attractive approach for disrupting the protective effects of CXCL12-secreting stromal cells and for sensitizing CLL cells towards subsequent therapy with conventional agents (chemotherapy, antibodies).We investigated the effect of NOX-A12 on CLL chemotaxis towards CXCL12 and CLL cell migration in BMSC co-cultures. To determine the effect of NOX-A12 on CLL cell chemotaxis, we performed chemotaxis assays across polycarbonate transwell inserts towards the chemokine CXCL12 (200 ng/mL, corresponds to 25 nM). CLL cell chemotaxis was significantly reduced in a dose-dependent manner in samples from 12 patients. Even when a very low NOX-A12 concentration of 3 nM was used, the mean (±SEM) number of CLL cells migrating towards CXCL12 decreased from 1556 (±303) to 640 (±211). Higher concentrations of NOX-A12 reduced CLL cell chemotaxis as significant as AMD3100, e.g. NOX-A12 at a concentration of 100 nM reduced the chemotaxis to 340 (±73) and AMD3100 at a concentration of 10 μg/mL (12.6 μM) to 384 (±156) migrated cells, respectively. Moreover, in other leukemia cell types like the T cell leukemic cell line Jurkat as well as the acute lymphoid leukemia (ALL) cell line Nalm-6, which are very sensitive to CXCL12, NOX-A12 mediated inhibition of chemotaxis was observed with a sub-nanomolar IC50.Next we quantified CLL cell pseudoemperipolesis, i.e. spontaneous leukemia cell migration beneath BMSC, which mimics in vivo migration and homing to stromal cells in the tissues. Surprisingly and in contrast to the inhibited chemotaxis, our results showed increased migration of CLL cells beneath BMSC after NOX-A12 treatment. After pre-treatment with 100 nM NOX-A12, the mean (±SEM) number of migrating cells increased from 10382 (±1604) to 12488 (±6965) using R-15C cells, and from 17888 (±2808) to 22677 (±11492) using TSt-4 cells (n=6). In comparison, the CXCR4 antagonists AMD3100 and BKT14O inhibited the migration beneath BMSC significantly. In accordance with these findings, NOX-A12 also promoted pseudoemperipolesis of Jurkat and Nalm-6 cells beneath MS-5 BMSC layers. Interestingly, this NOX-A12-induced migration could be completely inhibited by AMD3100.We hypothesized that this increased migration of CLL cells as well as Jurkat and Nalm-6 cells beneath BMSC might be the result of a stimulating effect of NOX-A12 on CXCL12 release by the stromal cells. Therefore, we investigated the effect of NOX-A12 on the CXCL12 release in three different BMSC lines (MS-5, R15C, and TSt-4). We found that NOX-A12 induced a rapid CXCL12 release into the medium in all three cell lines. The increase of the CXCL12 concentration was approximately sevenfold in MS-5 cells and approximately twofold in R15C and TSt-4 cells.Given the fact that NOX-A12 induces leukocyte- and hemapopoiectic stem cell mobilization in vivo, the enhanced migration of CLL cells in BMSC co-cultures suggests that NOX-A12 has a unique effect on leukemia cell trafficking that is distinct from small molecule (AMD3100) or peptide (BTK140) CXCR4 antagonists. Future dissection in an in vivo CLL model and in a first clinical trial will help us to better define the effect of NOX-A12 on CLL cells migration.In summary, the CXCL12 inhibitor NOX-A12 is a novel, targeted agent for interference with CLL cell migration and homing to CXCL12-secreting stromal cells. Its effects on CLL cell chemotaxis and migration beneath BMSC make NOX-A12 distinct from CXCR4 antagonists. In vivo experiments and ultimately a clinical trial will help defining how effectively NOX-A12 can mobilize and sensitize CLL cells. [Display omitted] Disclosures:Zboralski:NOXXON: Employment. Kruschinski:Noxxon Pharma AG: Employment. Burger:NOXXON: Consultancy, Research Funding.