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Resistance to targeted tyrosine kinase inhibitors (TKI) remains a challenge for the treatment of myeloid leukemias. Following treatment with TKIs, the bone marrow microenvironment has been found to harbor a small pool of surviving leukemic CD34+ progenitor cells. The long-term survival of these leukemic cells has been attributed, at least in part, to the protective effects of bone marrow stroma. We found that the NOX-A12 'Spiegelmer', an L-enantiomeric RNA oligonucleotide that inhibits SDF-1α, showed in vitro and in vivo activity against BCR-ABL- and FLT3-ITD-dependent leukemia cells. NOX-A12 was sufficient to suppress SDF-1-induced migration in vitro. The combination of NOX-A12 with TKIs reduced cell migration in the same in vitro model of SDF-1-induced chemotaxis to a greater extent than either drug alone, suggesting positive cooperativity as a result of the SDF-1 blocking function of NOX-A12 and cytotoxicity resulting from targeted oncogenic kinase inhibition. These results are consistent with our in vivo findings using a functional pre-clinical mouse model of chronic myeloid leukemia (CML), whereby we demonstrated the ability of NOX-A12, combined with the ABL kinase inhibitor, nilotinib, to reduce the leukemia burden in mice to a greater extent than either agent alone. Overall, the data support the idea of using SDF-1 inhibition in combination with targeted kinase inhibition to override drug resistance in oncogene-driven leukemia to significantly diminish or eradicate residual leukemic disease.

The CXC chemokine ligand (CXCL12, or stromal cell-derived factor-1 as previously known) plays a critical role for homing and retention of chronic lymphocytic leukemia (CLL) cells in tissues such as the bone marrow (BM). In tissues, stromal cells constitutively secrete and present CXCL12 via cell-surface-bound glycosaminoglycans (GAGs), thereby attracting CLL cells and protecting them from cytotoxic drugs, a mechanism that may account for residual disease after conventional CLL therapy. NOX-A12, an RNA oligonucleotide in L-configuration (Spiegelmer) that binds and neutralizes CXCL12, was developed for interference with CXCL12 in the tumor microenvironment and for cell mobilization. Here, we examined effects of NOX-A12 on CLL cell migration and drug sensitivity. We found that NOX-A12 effectively inhibited CXCL12-induced chemotaxis of CLL cells. In contrast, NOX-A12 increased CLL migration underneath a confluent layer of BM stromal cells (BMSCs) due to interference with the CXCL12 gradient established by BMSCs. In particular, NOX-A12 competes with GAGs such as heparin for CXCL12 binding, leading to the release of CXCL12 from stromal cell-surface-bound GAGs, and thereby to neutralization of the chemokine. Furthermore, NOX-A12 sensitizes CLL cells toward bendamustine and fludarabine in BMSC cocultures. These data demonstrate that NOX-A12 effectively interferes with CLL cell migration and BMSC-mediated drug resistance, and establishes a rationale for clinical development of NOX-A12 in combination with conventional agents in CLL.

Mobilized bone marrow-derived stem cells (BMSC) have been discussed as an alternative strategy for endogenous repair. Thereby, different approaches for BMSC mobilization have been pursued. Herein, the role of a newly discovered oligonucleotide for retinal homing and regeneration capability of BMSCs was investigated in the sodium iodate (NaIO3) model of retinal degeneration. Mobilization was achieved in GFP-chimera with NOX-A12, a CXC-motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 (SDF-1)-neutralizing L-aptamer. BMSC homing was directed by intravitreal SDF-1 injection. Visual acuity was measured using the optokinetic reflex. Paraffin cross sections were stained with hematoxylin and eosin for retinal thickness measurements. Immunohistochemistry was performed to investigate the expression of cell-specific markers after mobilization. A single dose of NOX-A12 induced significant mobilization of GFP(+) cells which were found in all layers within the degenerating retina. An additional intravitreal injection of SDF-1 increased migration towards the site of injury. Thereby, the number of BMSCs (Sca-1(+)) found in the damaged retina increased whereas a decrease of activated microglia (Iba-1(+)) was found. The mobilization led to significantly increased visual acuity. However, no significant changes in retinal thickness or differentiation towards retinal cell types were detected. Systemic mobilization by a single dose of NOX-A12 showed increased homing of BMSCs into the degenerated retina, which was associated with improved visual function when injection of SDF-1 was additionally performed. The redistribution of the cells to the site of injury combined with their observed beneficial effects support the endogenous therapeutic strategy for retinal repair.

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. Figure Figure. Disclosures Zboralski: NOXXON Pharma AG: Employment. Kruschinski:NOXXON Pharma AG: Employment. Vater:NOXXON Pharma AG: Employment.

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. Disclosures Zuber: Boehringer Ingelheim: Research Funding; Mirimus Inc.: Consultancy, Other: Stock holder. Eulberg:NOXXON Pharma AG: Employment. Kruschinski:NOXXON Pharma AG: Employment.

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 (SDF1) may represent a target for preventing transition from MGUS (micrometastatic-stage) to active-MM (macrometastatic-stage). We therefore evaluated SDF1 expression in the BM of patients with MGUS, MM, compared to healthy individuals; and tested NOX-A12, a high affinity L-oligonucleotide binder to SDF1, looking at its ability to modulate MM cell tumor growth and homing to the BM in vivo and in vitro. Methods. SDF1 levels were evaluated by immunohistochemistry on BM specimens of patients with MGUS, active-MM, or healthy individuals; and confirmed by ELISA, using conditioned-medium of BM-mesenchymal stromal cells (BM-MSCs) from MGUS, active-MM and healthy individuals. BM metastatic lesions from primary epithelial tumors were also evaluated. Co-localization of SDF1 with MM cell (MM.1S-GFP+)-enriched BM niches was evaluated using in vivo confocal microscopy. Effect of NOX-A12 on modulating MM cell dissemination was tested in vivo, by using in vivo MM metastasis model. In vivo homing and in vivo tumor growth of MM cells (MM.1S-GFP+/luc+) was assessed by using in vivo confocal microscopy and bioluminescence, in mice treated with 1) vehicle; 2) NOX-A12; 3) bortezomib; 4) NOX-A12+bortezomib. Effects of drug combination on dissemination of MM cells to distant BM niches was evaluated ex vivo by immunofluorescence on explanted femurs. DNA synthesis and adhesion of MM cells in the context of NOX-A12 treated primary MM BM-MSCs in presence or absence of bortezomib were tested by thymidine uptake and adhesion in vitro assay, respectively. Synergism was calculated by using CalcuSyn software. Results. Patients with active-MM present with higher BM SDF1 expression vs MGUS patients and healthy individuals. Similarly, BM presenting with metastasis from epithelial primary tumors had higher SDF1 levels compared to healthy subjects, thus suggesting the importance of SDF1 in favoring tumor cell metastasis to BM niches. SDF1 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. Importantly, NOX-A12 induced MM cell mobilization from the BM to the peripheral blood (PB) as shown ex vivo by reduction of MM cells in the BM and increased number of MM cells within the PB compared to control mice (P Conclusion. SDF-1 represents a valid target for inhibiting MM cell dissemination to distant BM niches, thus providing the evidence for using the SDF1 inhibiting spiegelmer NOX-A12 to target MM cells at the stage of micrometastasis (MGUS), thus preventing development of macrometastatic MM. This abstract is also presented as Poster B80. Citation Format: Aldo M. Roccaro, Antonio Sacco, Michele Moschetta, Patricia Maiso, Yuji Mishima, Yosra Aljawai, Fabio Facchetti, Anna Kruschinski, Giuseppe Rossi, Irene M. Ghobrial. In vivo targeting of stromal-derived factor-1 as a strategy to prevent myeloma cell dissemination to distant bone marrow niches. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR14. doi:10.1158/1538-7445.CHTME14-PR14

Background Olaptesed 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. Methods Twenty-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). Results To 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. Conclusion Olaptesed 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. Disclosures Montillo: Janssen: Honoraria. Kruschinski:NOXXON Pharma AG: Employment. Dümmler:NOXXON Pharma AG: Employment. Riecke:NOXXON Pharma AG: Employment.

Background NOX-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). Methods To 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. Results In 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. Conclusion Proof 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.

Bone marrow (BM) mesenchymal stromal cells (MSC) protect leukemia cells from chemotherapy-induced apoptosis. CXCR4, the receptor for the stromal-derived factor 1 (SDF-1/CXCL12), is involved in modulating leukemic progenitor cell homing and it is essential for the migration of these cells to the BM niche. As the BM niche imparts favorable survival advantage to leukemia cells, a strategy to disrupt leukemic cell homing to the BM microenvironment may represent a novel way to effectively kill these malignant cells. Strategies to disrupt the SDF-1/CXCR4 axis have relied on inhibition of the CXCR4 receptor using various drugs including plerixafor, which is potent at mobilizing normal progenitor cells. We previously found that plerixafor antagonized SDF-1 and MSC-induced migration of leukemic cells, inhibited SDF-1-induced AKT and ERK activation, and decreased stroma-mediated protection from AraC-induced apoptosis in 11 out of 18 primary AML samples (Zeng et al Blood 2009; 113: 6215). We also found that the inhibition of CXCR4 induces miRNA let-7a expression via the transcription factor Yin Yang1 (Chen et al, JCI, 2013; 123:2395), and induces chemosensitization irrespective of cell mobilization. While inhibition of CXCR4 has shown promise for use in AML therapy (Andreeff, et al ASH, Blood 120(21) (#142), 11/2012), a more effective strategy might be to target the SDF-1 ligand itself since SDF-1 can bind to other receptors (i.e. CXCR7) that are likely not recognized by CXCR4 antibodies. NOXXON (Berlin, Germany) has recently identified a potent inhibitor to SDF-1 that is based on a so-called Spiegelmer®. The Spiegelmer NOX-A12 is a structured l-RNA oligonucleotide, PEGylated at the 5'-end to improve the pharmacokinetics. In healthy volunteers, NOX-A12 displays a half-life of∼38 hours and has demonstrated a long-term and dose-dependent mobilization of WBCs and CD34+ cells. The drug is currently tested in clinical trials in multiple myeloma and chronic lymphocytic leukemia patients (Gobbi et al., ASH 2012 120: 4593). In the current study, the ability of NOX-A12 to suppress survival signaling in leukemic and MSC cells was examined. In addition, we tested the ability of the Spiegelmer to mobilize leukemia cells in a murine xenograft model. NOX-A12 was found to suppress the phosphorylation of ERK in AML cells, and suppress the phosphorylation of ERK, FAK, and STAT3 in MSC. These results suggested that SDF-1 affected diverse survival signaling pathways in the leukemic cells and the critical supporting stromal niche. In vivo activity was examined in NOD/SCID/-g- mice injected intravenously with murine leukemia Baf/ITDluc/GFP cells and monitored by a non-invasive bioluminescent imaging. The in vivo mobilization assay was assessed when the majority of mice had approximately 10% tumor burden. Plerixafor, NOX-A12, or a combination of both agents was subcutaneously administrated and leukemic GFP+ cells were detected by analytical flow cytometry. Plerixafor and NOX-A12 administered separately increased total WBC count by 2- to 3-fold and concomitantly increased the percentage of circulating GFP+ cells in all mice (to 14.2% and 18.5%, respectively). Importantly, the combination of both targeted agents further increased the percentage of circulating GFP+ cells in an approximately additive fashion (to 26.4%). Conclusion we demonstrate that the novel SDF-1 Spiegelmer NOX-A12 can suppress survival signaling in both leukemia and supportive MSC cells. Furthermore, increased mobilization of leukemic cells in vivo was observed when NOX-A12 and plerixafor were used in combination. These findings suggest the possibility that targeted disruption of leukemia/stroma interactions by optimized blockade of the pro-survival SDF-1/CXCR4 axis will not only increase leukemia cell mobilization, but also enhance the sensitivity to chemotherapy of both mobilized and non-mobilized the leukemic cells and leukemic stem cells. Experiments are ongoing to further validate this hypothesis. Disclosures: Kruschinski: Noxxon: Employment.

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.

Background NOX-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). Methods To 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). Results In 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. 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. Conclusions Proof 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.

Abstract 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 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.

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 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.

Abstract 3878 NOX-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. Disclosures: Zboralski: NOXXON: Employment. Kruschinski:Noxxon Pharma AG: Employment. Burger:NOXXON: Consultancy, Research Funding.

Summary: Bone marrow (BM) metastasis remains one of the main causes of death associated with solid tumors as well as multiple myeloma (MM). Targeting the BM niche to prevent or modulate metastasis has not been successful to date. Here, we show that stromal cell-derived factor-1 (SDF-1/CXCL12) is highly expressed in active MM, as well as in BM sites of tumor metastasis and report on the discovery of the high-affinity anti-SDF-1 PEGylated mirror-image l-oligonucleotide (olaptesed-pegol). In vivo confocal imaging showed that SDF-1 levels are increased within MM cell-colonized BM areas. Using in vivo murine and xenograft mouse models, we document that in vivo SDF-1 neutralization within BM niches leads to a microenvironment that is less receptive for MM cells and reduces MM cell homing and growth, thereby inhibiting MM disease progression. Targeting of SDF-1 represents a valid strategy for preventing or disrupting colonization of the BM by MM cells. : Roccaro et al. show that stromal-cell-derived factor-1 (SDF-1) is highly expressed in active multiple myeloma (MM), as well as in bone marrow (BM) sites of tumor metastasis, and report on a high-affinity PEGylated mirror-image l-oligonucleotide (olaptesed pegol) that specifically binds and neutralizes SDF-1 in vitro and in vivo. Using in vivo murine and xenograft mouse models, the authors document that in vivo SDF-1 neutralization within BM niches leads to a microenvironment that is less receptive for MM cells and reduces clonal plasma cell homing and growth, thereby inhibiting MM disease progression.

Olaptesed pegol (olaptesed, NOX-A12) is a pegylated l-oligoribonucleotide that binds and neutralizes CXCL12, a chemokine which signals through CXCR4 and CXCR7 regulating a variety of processes during multiple myeloma (MM) development.1 CXCL12 inhibition reduces the myeloma-supportive activity of the bone marrow microenvironment and mobilizes myeloma cells to the circulation.2 In addition, CXCL12α levels were reported to correlate with osteolytic bone lesions and with increased bone marrow angiogenesis.3 Targeting CXCL12, therefore, is a promising strategy for disrupting myeloma-stroma interactions and inhibiting myeloma growth and survival. Here, we report pharmacokinetic, pharmacodynamic, safety and efficacy data of olaptesed in patients with relapsed/refractory MM and present data on baseline CXCL12α levels in this patient cohort. This first-in-patient Phase IIa study aims to translate the novel concept of combining proteasome and CXCL12 inhibition into the clinic and builds on a preclinical proof-of-concept regarding the significance of CXCL12 blockade in MM2 and on Phase I data in healthy subjects.4 Combining CXCL12 inhibition with bortezomib and dexamethasone (VD) was investigated in 28 patients with relapsed/refractory MM, who were either bortezomib-naive or considered not refractory to bortezomib. The median number of prior lines of therapy was 2 (range: 1–5), 39 and 14% of the patients presented with 3 or 4 prior lines, respectively. Cytogenetics were tested in 21 patients and high risk features were found in 36% of them. 54% of patients had prior treatment with bortezomib, 39% had a prior stem cell transplant and 57% were refractory to prior treatment. Further details on patient characteristics (Supplementary Table S1) and inclusion and exclusion criteria are available in the Supplementary Information. In the pilot phase, three cohorts of three patients each (four patients in the 1 mg/kg cohort due to patient replacement) were administered single doses of 1, 2 or 4 mg/kg of olaptesed alone by slow intravenous bolus injection 2 weeks prior to starting the combination treatment. Combination treatment was administered for eight cycles of 21 days. An intra-patient escalation was applied for safety reasons as olaptesed was combined for the first time with VD in cancer patients. Olaptesed was given 1–2 h prior to bortezomib at doses of 1 mg/kg in cycle 1, 2 mg/kg in cycle 2 and 4 mg/kg in cycles 3–8. Bortezomib was given on days 1, 4, 8 and 11 of each 21-day treatment cycle as intravenous injection of 1.3 mg/m2. Oral dexamethasone (20 mg) was added on the day of and the day after bortezomib administration. An outline of the study and details of patients’ flow are given in Supplementary Figures S1 and S2.