Moon‐Tasson, Laurie L.

Abstract Background: Patients with SMM/IMM are at high risk for progression to active MM and are appropriate candidates for chemoprevention trials. High IL-1beta levels are a useful surrogate marker for progression from SMM/IMM to active myeloma. Methods: We carried out a Phase II clinical trial of IL-1Ra (Anakinra) in patients with SMM/IMM to determine the biologic activity, progression-free rate, and toxicity of IL-1Ra. Since IL-1beta induces paracrine IL-6, we hypothesized that IL-1Ra would inhibit IL-6 production and myeloma cell growth. Patients that had ≥ 10% bone marrow plasma cells and/or an IgG or IgA M-spike ≥ 3 g/dL and did not require immediate chemotherapy were eligible. Patients received 100 mg of Anakinra (IL-1Ra) SQ qd for a total duration of 6 months. Non-progressors were allowed to continue on therapy with IL-1Ra until they converted to active myeloma. IL-1beta levels were measured in a bioassay with a read out of IL-1 inducible IL-6 production. CRP levels served as a surrogate for IL-6 production. Results: Thirty-six patients are included for analysis based on intent to treat at diagnosis. At baseline, twenty-nine patients had elevated functional IL-1beta levels consistent with progressive disease and 12 patients had radiologic evidence of disease on bone survey. The median TTP for the entire group was 1 year and 2 patients exhibited a minor response based on M-protein reduction. A proportional hazards analysis was performed and the variables examined were: % bone marrow plasma cells, plasma cell labeling index (PCLI), circulating plasma cells, albumin, M-protein level, IgA subtype, CRP, CRP % reduction ≥ one-third, beta-2 microglobulin, creatinine, calcium, hemoglobin, urine total protein, presence of lytic bone disease, and IL-1 level. Univariate Cox model results showed that % BMPC, PCLI, circulating plasma cells, CRP % reduction, presence of lytic disease, and IL-1 level were all statistically significant (p < .05) variables. However, only the CRP % reduction from baseline remained significant in the multivariate analysis (p < 0.01). The median TTP for patients without (n=18) and with (n=18) a decrease in CRP was 6 months and > 2 years, respectively (p < .0001). Toxicities included injection site reactions during the first month of therapy and asymptomatic neutropenia necessitating a reduction in therapy to every other day. Five patients in whom the CRP decreased and the baseline PCLI was > 0 also demonstrated a parallel decrease in the PCLI. Several of the patients with progressive disease on IL-1Ra alone could later be rescued with the addition of low dose dexamethasone (20 mg once a week) suggesting that the endogenous IL-1beta levels were too high to inhibit with IL-1Ra alone. Conclusion: In summary, IL-1Ra can prolong the TTP to active myeloma in responsive SMM/IMM patients through inhibition of IL-6 production (decrease in CRP) and myeloma cell growth. Figure Figure

Abstract Background: Multiple myeloma (MM) is an incurable hematological malignancy characterized by the expansion of a plasma cell clone that localizes to the bone marrow. Stromal cells residing in the bone marrow respond to signals from MM cells and other cell types by producing cytokines and other proteins that stimulate tumor cell growth, survival, adhesion, migration, and drug resistance. We have examined the proteins produced by stromal cells in response to stimulation by bone marrow from patients diagnosed with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and MM. Furthermore, we have begun analyzing the properties of one of these proteins, the pro-angiogenic chemokine IL-8, in MM. Methods: Bone marrow aspirates from patients with MGUS (n=3), SMM (n=7), and MM (n=6) were cultured for 48 hours, and the culture supernatants were incubated with stromal cells for an additional 48 hours. Protein levels were analyzed using antibody array and ELISA. Microvessel density (MVD) was determined as a measure of angiogenesis in patient bone marrow samples using CD34 staining. Flow cytometry analysis of MM cell lines and patient bone marrow samples was performed using monoclonal antibodies against IL-8 receptors CXCR1 and CXCR2. Results and Conclusion: We observed a significant increase in stromal cell IL-8 production stimulated by bone marrow cells from patients with active myeloma and a subset of SMM patients (16.67 ± 9.82 ng/ml) in comparison to bone marrow of patients with MGUS and all other SMM patients (0.55 ± 0.17 ng/ml; P=0.0004). Use of an IL-1 inhibitor and recombinant IL-1β demonstrated that IL-8 production was dependent upon IL-1β signaling. Increased BM microvessel density correlated with stimulation of stromal cell IL-8 production (P=0.0005). Furthermore, the majority of MM cell lines (7/9) and MM patient plasma cells were found to express IL-8 receptors CXCR1 and CXCR2. In addition to its function as a pro-angiogenic factor, IL-8 may directly influence MM cells through its CXCR1 and CXCR2 receptors. We conclude that stromal cell IL-8 production parallels MM disease activity, is IL-1β induced, correlates with bone marrow angiogenesis, and may influence MM disease via impact upon both the microenvironment and tumor cells.

Abstract Background: Hypusine is a unique amino acid present in all eukaryotes but found in only one highly conserved protein, eukaryotic translation initiation factor 5A. It is formed during a two-step post-translational modification of eIF5A. eIF5A has been identified as a critical factor for cell proliferation in yeast, as a modulator of p53, and recent studies indicate that the unhypusinated form of eIF5A is a pro-apoptotic protein. Because myeloma remains incurable, we hypothesized that induction of apoptosis by modulation of eIF5A may be a useful therapeutic approach. In this study we utilized PEI nanoparticles to deliver plasmid DNA and siRNA to myeloma tumor cells in vitro and in vivo. Methods: An siRNA was used to suppress levels of endogenous hypusinated eIF5A in tumors while an RNAi-resistant plasmid expressing a mutant of eIF5A (eIF5AK50R), that cannot be hypusinated, was used to increase levels of unhypusinated eIF5A which is proapoptotic. In vitro, KAS-6/1 myeloma cells were incubated with control siRNA/control plasmid, eIF5A siRNA alone, or eIF5A siRNA/eIF5AK50R for 72 hours in the presence or absence of IL-6. Apoptosis was quantitated using flow cytometry to detect annexin V/propidium iodide stained cells; results were corrected for percent apoptosis of the mock control to enable comparison between experiments. In vivo, SCID mice (3/group) were injected with 10 million KAS-6/1 cells SQ in the right flank and approximately 6 weeks later injected intratumorally twice weekly as follows: Group 1 control mice received 20 μg empty vector and 10 μg control siRNA. Group 2 received 20 μg eIF5AK50R plasmid and 10 μg control siRNA. Group 3 received 20 μg eIF5AK50R plasmid and 10 μg eIF5A siRNA. Tumor volumes were quantitated in mm3 using a2b/2 where a is the smaller dimension. Results: The in vitro results showed the following % apoptosis: Absence of IL-6 - Control: 6.5%, eIF5A siRNA/eIF5AK50R 30.3%. Presence of IL-6–Control:8.2%; eIF5A siRNA alone 36.5%; eIF5A siRNA/eIF5AK50R 45.2%. Western analysis demonstrated that the siRNA successfully inhibited the production of endogenous eIF5A. In vivo, after 24 days of monitoring tumor volume: group 1 mice showed that the KAS-6/1 cells grew from a mean tumor volume of 7±1 up to 102±16 mm3; tumors in group 2 mice remained relatively stable in size from 21±9 to 26±10 mm3. Group 3 mice showed a mean 92% tumor regression from 99±21 down to 7±1 mm3. Conclusions: Delivery of eIF5A siRNA and eIF5AK50R plasmid by PEI nanoparticles results in significant anti-tumoral reponses in vitro and in vivo. These studies may provide a novel therapy for myeloma patients with end stage disease that have failed standard treatments.

Abstract Background: It has been hypothesized that multiple myeloma (MM) remains incurable due to a stemcell/proliferative component that responds only partially to standard treatment regimens. We have shown that abnormal production of IL-1beta in the myeloma microenvironment stimulates the generation of paracrine IL-6, the central myeloma growth factor, and that IL-1Ra will inhibit paracrine IL-6 production in vitro. Based on these preclinical studies, we have developed a Phase II trial using IL-1Ra. Purpose: To determine the biologic and clinical activity of IL-1Ra (Anakinra) in a subgroup of smoldering MM (SMM) patients that present with an elevated plasma cell labeling index (PCLI; a measure of myeloma cell proliferation). Methods: Patients that had ≥ 10% bone marrow plasma cells and/or an IgG or IgA M-spike ≥ 3 g/dL and did not require immediate chemotherapy were eligible. Patients received 100 mg of Anakinra (IL-1Ra) SQ qd for a total duration of 6 months. Results: Eleven of 29 patients enrolled on the protocol had an on-study PCLI > 0 and have completed six months of therapy with IL-1Ra. Seven of the 11 patients had a decrease in the PCLI of 75–100% (see Table), three had only modest changes (≤ 50%), and one had an increase. The decrease in the PCLI paralleled a decrease in the high sensitivity C-reactive protein (CRP) in all cases (43–90% decrease). The above results suggested that IL-1Ra inhibited IL-6 production in the myeloma microenvironment, as evidenced by a reduction in the CRP, resulting in suppression of myeloma cell growth. However, there was little effect on the M-protein. To investigate these clinical observations, we co-cultured IL-1beta transduced +/− myeloma cells with stromal cells +/− dexamethasone (DEX), IL-1Ra, or both for 48 hours and quantitated the percent apoptotic cells by flow cytometry and IL-6 production by ELISA. The results showed: 1) IL-1Ra was superior at inhibition of IL-6 but caused no increase in apoptosis; 2) the DEX apoptotic effect was eliminated by IL-6 3) DEX and IL-1Ra combined induced maximal IL-6 inhibition and apoptosis of myeloma cells. Based on these in vitro results, 5 of the 11 patients have been advanced to IL-1Ra + low dose DEX (20 mg/week) resulting in 4/5 minor responses (25–50% decrease M-spike) and 1/5 with stable disease. Conclusion: The use of IL-1Ra is a novel targeted therapeutic strategy that interferes with myeloma cell growth. By inhibiting IL-1beta induced IL-6 production, IL-1Ra specifically targets the proliferative myeloma fraction and also complements DEX induced apoptosis. Preliminary studies on the use of IL-1 inhibitors in SMM patients to delay/prevent progression to active MM and to minimize toxicity appear encouraging, however, more patients need to be studied. Patient # On Study 6 months % Change 1 PCLI 0.8% 0% −100% CRP (mg/L) 2.01 0.58 − 71% M-spike (g/dL) 2.5 2.4 − 4% 2 PCLI 0.2% 0% − 100% CRP 0.89 0.51 − 43% M-spike 2.8 2.7 − 3% 3 PCLI 0.2% 0% − 100% CRP 5.7 1.02 − 82% M-spike 2.9 2.9 0% 4 PCLI 4.1% 1% − 75% CRP 3.39 1.51 − 55% M-spike 3.8 3.6 − 5% 5 PCLI 0.4% 0% − 100% CRP 3.46 0.34 − 90% M-spike 2.7 3.1 15% 6 PCLI 0.3% 0% − 100% CRP 17 5.35 − 68% M-spike 4.4 3.9 − 11% 7 PCLI 1.2% 0% − 100% CRP 1.06 0.32 − 70% M-spike 3.1 2.9 − 6%

Abstract Abstract 2945 Background: We have previously reported on a Phase II trial using IL-1Ra and dexamethasone, in patients with SMM/IMM, showing that IL-1Ra targets the myeloma proliferative component which parallels a decrease in the C-reactive protein, a surrogate for IL-6 production. (Mayo Clinic Proceedings 84 :114 (Feb. 2009)). In early stage myeloma, IL-6 is a central myeloma growth factor and we have shown that abnormal production of IL-1 β in the myeloma microenvironment stimulates the generation of paracrine IL-6. These patients are the individuals most likely to benefit from anti-cytokine therapy in an attempt to delay/prevent the development of active myeloma. We now present updated results of this study including survival data. Methods: Patients that had ≥ 10% bone marrow plasma cells and/or an IgG or IgA M-spike ≥ 3 g/dL and did not require immediate chemotherapy were eligible. All patients received 100 mg of Anakinra (IL-1Ra) SQ qd for 6 months. Patients with evidence of reduction in M-protein levels continued receiving IL-1Ra alone. Patients with stable disease at 6 months or those with a rising M-protein before 6 months received low dose dexamethasone (20 mg qweek) in addition; the dose was adjusted based on response/toxicity. Results: Data were available on 47 patients based on intent to treat, and pts were classified as smoldering (72%) vs. indolent (28%). All SMM patients were in Group 1 or 2 and thirty-eight patients (81%) had greater than 20% bone marrow plasma cells. All 47 patients received IL-1Ra initially and 25/47 subsequently received IL-1Ra/Dex. Seven patients had a decrease in the plasma cell labeling index (PCLI) on IL-1Ra alone which paralleled a decrease in the high sensitivity C-reactive protein (CRP) in all cases. Three patients achieved a minor response to IL-1Ra alone and 9 patients achieved a PR/MR after addition of dexamethasone. For the group of 47 patients, the median progression-free survival was 45 months and the median overall survival has not been reached. When patients were grouped into whether they exhibited a reduction in the C-reactive protein from baseline after 6 months of therapy, the median PFS for patients without (21 patients) or with (26 patients) a greater than one-third reduction in baseline CRP was 1 year vs more than 8 years (p<.01). Similarly, the median OS for patients without or with a reduction in baseline CRP was 7.9 years vs. median not reached (p=.02; see Figure). Toxicity included injection site reactions during the first month of therapy in 86% of patients. Grade 3–4 adverse events consisted mainly of asymptomatic neutropenia (17% of patients) and infection (18% of patients). Patients that progressed to active MM have been treated with standard therapies including lenalidomide, bortezomib, transplant, and novel therapies. In contrast, seven of the 26 patients with a CRP decrease continue with stable disease and minimal toxicity on IL-1Ra alone or in combination with standard agents. Conclusion: The above results suggest that agents such as IL-1Ra that specifically inhibit paracrine IL-6 production are effective at targeting the proliferative myeloma component and warrant further investigation in combination with standard myeloma therapies. Combination therapy that effectively targets both the proliferative and the IL-1 producing myeloma components may be useful in patients with active myeloma that have an elevated growth rate at diagnosis. Disclosures: Lust: Mayo Clinic: Patents & Royalties. Off Label Use: IL-1Ra use in smoldering myeloma. Donovan:Mayo Clinic: Patents & Royalties.

Abstract Introduction: Features of multiple myeloma (MM) include a proliferative clonal plasma cell population, bone resorption, and neovascularization. Cytokines and chemokines represent two families of molecules that are capable of propagating and enhancing these disease features. In this study, we have utilized antibody array technology to assess the contributions of cytokines and chemokines to the progression of disease, and evaluated the contribution of stromal cells (SCs) in their production. Methods: Wild-type and IL-1beta transduced KAS 6/1 myeloma cell lines or bone marrow cells isolated from patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering myeloma (SMM), and MM were cultured for 48 hrs. Culture supernatants were either analyzed directly, or co-cultured with normal SCs for an additional 48 hrs +/− IL-1 inhibitors, after which the supernatants were removed and analyzed using antibody arrays. SCs alone were also cultured with recombinant IL-1beta +/− IL-1 receptor antagonist (IL-1Ra) to define IL-1 dependent effects. IL-6 and IL-8 ELISAs were utilized to quantify IL-6 and IL-8 levels, and validate antibody array findings. Results: Antibody array analysis of IL-1 effects on stromal cell cultures using recombinant IL-1beta and supernatants from an IL-1beta transduced myeloma cell line demonstrated that stimulation of IL-6, MCP-1 and IL-8 were induced in an IL-1 and stromal cell dependent manner. Although levels of TIMP-2 varied in these cultures, they appeared unrelated to an IL-1 effect. Studies utilizing supernatants from patient bone marrow cells co-cultured with SCs resulted in levels of IL-6, MCP-1, and IL-8 higher than those seen with patient supernatants alone or SC cultures alone. More interestingly, the IL-8 levels appeared to correlate with diagnosis; MGUS samples generated low levels and MM samples stimulated high levels. Furthermore, this stimulation was reduced by the addition of IL-1 inhibitors, demonstrating a dependence on IL-1. To confirm the relationship between diagnosis and IL-8 production, the levels of IL-8 produced by the bone marrow supernatants were quantified directly by ELISA. Correlating with the antibody array data, background production of IL-8 from the cultures of patient cells alone was lower than the corresponding co-culture value. Supernatants from MM patients and a subset of SMM patients stimulated high levels of SC IL-8 secretion in contrast to bone marrow cell supernatants from MGUS patients and most SMM patients. This activity was inhibited by IL-1 inhibitors (see Figure). The IL-8 levels closely parallel the IL-1beta induced IL-6 levels in the same samples. Conclusion: These data indicate that the concentration of IL-8 may be relevant to the pathogenesis of MM. IL-8 production is largely dependent on SCs, and production appears to be at least partially dependent on IL-1 function. IL-8 is a chemokine with activities including chemotaxis of neutrophils, increased vascular permeability and angiogenesis. IL-8 expression has been implicated in multiple tumor types and may play an important role in the stimulation of angiogenesis during the progression from MGUS to active MM. Figure Figure

Abstract Abstract 1845 Inflammation is a driving factor in the pathogenesis of many cancers including multiple myeloma (MM). The active role that cytokines play, IL-1β in particular, during the early stages of MM has been extensively characterized. Myeloma is a plasma cell tumor whose early stages are characterized by cytokine dependent growth, predominantly driven by IL-6. We have demonstrated that IL-1β is a key inducer of IL-6 expressed by bone marrow stromal cells in a paracrine fashion during the progression of multiple myeloma from smoldering myeloma (SMM) to active disease (Mayo Clinic Proc 84:114 (Feb. 2009)). Elevated ATP levels have been documented in vivo in tumor micro-environments and is a potential contributing factor to IL-1β induced inflammation. The P2X7 receptor (P2X7R) is an ATP-gated ion channel expressed by cells of the hematopoetic lineage that has been shown to play a critical role in IL-1b processing and secretion. We investigated the role this receptor may have in the release of IL-1β in the bone marrow microenvironment using bone marrow samples from both chemotherapy treated and untreated patients. Myeloma cell lines were evaluated by RT-PCR for the expression of P2X7R mRNA. Expression was detected and confirmed by sequencing in ANBL-6, MM1.S, U266, RPMI-8226 and KAS-6/1 and was comparable to U937, a previously characterized P2X7R positive cell line. A newly derived plasmacytoma cell line, PCYT3, showed very low levels of message expression. The ability of ATP, the P2X7R agonist, to trigger the processing and release of IL-1β by activation of the P2X7 channel was studied using KAS-Pro, a myeloma cell line stably transfected with a pro- IL-1β gene. IL-1β, measured by ELISA, was released in a dose dependent fashion in response to increasing amounts of ATP ranging from.6mM to 5mM. Four P2X7R specific antagonists were tested for their effect on ATP stimulated IL- 1β release from KAS-Pro; all 4 compounds demonstrated inhibition in a dose dependent manner with varying degrees of potency correlating with P2X7R inhibition. In addition fresh patient samples (8 MGUS/7SMM/9 newly diagnosed MM as well as 12 Treated MM) were evaluated for receptor expression and function. All stages of disease expressed the receptor by RT-PCR. Analysis of samples from all stages of MM, as measured directly by IL-1β ELISA as well as indirectly by measurement of IL-1β induced IL-6 production from normal bone marrow stromal cells, showed functional receptor activation in response to ATP stimulation. IL-6 production detected from MGUS patient samples had a mean result of 2,253 pg/ml, the mean for SMM was 5,495 pg/ml, MM were 6,875 pg/ml while treated MM samples showed a mean result of 15,219 pg/m. Greater than 90% inhibition of IL-1β release by the P2X7R antagonists was seen for all patients who demonstrated a positive ATP induced IL-1β release (see figure). We observed a greater than 2 fold increase in IL-1β release between the treated MM samples and the newly diagnosed MM samples, suggesting a chemotherapy induced inflammation process may be ongoing in these patients. These results confirm the activity of functional P2X7 receptors on myeloma cell lines and in fresh patient samples and suggest a role for the use of P2X7 receptor antagonists in the therapy of myeloma, particularly in patients undergoing chemotherapy. Disclosures: Hromockyj: Pfizer Corporation: Employment. Meyer:Pfizer Corporation: Employment.

Abstract Background: Myeloma remains incurable due to a stem cell/proliferative component that responds poorly to standard treatments. We have shown that aberrant IL-1beta stimulates the generation of paracrine IL-6, a central myeloma growth factor. A Phase II trial was completed using IL-1 receptor antagonist (IL-1Ra/Anakinra), which inhibits paracrine IL-6 production, and low dose Dexamethasone (Dex), which decreases IL-1 levels through myeloma cell apoptosis, in patients with SMM/IMM. SMM/IMM patients are most likely to benefit from anti-cytokine therapy in order to delay/prevent the development of active myeloma. Methods: Patients that had ≥ 10% bone marrow plasma cells and/or an IgG or IgA M-spike ≥ 3 g/dL and did not require immediate chemotherapy were eligible. All patients received 100 mg of IL-1Ra SQ qd for 6 months unless clinical progression occurred. Non-progressors were allowed to continue on therapy with IL-1Ra alone. Low dose Dex (20 mg qweek) was added after 6 months of IL-1Ra or for evidence of clinical progression; the dose was adjusted based on response/toxicity. Results: To investigate the effects of IL-1Ra and Dex alone and in combination in vitro, we co-cultured IL-1beta transduced +/− myeloma cells with stromal cells +/− dexamethasone, IL-1Ra, or both for 48 hours and quantitated the percent apoptotic cells by flow cytometry and IL-6 production by ELISA. The results showed that: 1) IL-1Ra was superior to Dex at inhibition of IL-6 but caused no myeloma apoptosis; 2) Dex induced apoptosis was inhibited by IL-6 3) Dex and IL-1Ra combined induced maximal IL-6 inhibition and apoptosis of myeloma cells. In the clinical trial, data were available on 47 patients; smoldering (79%) / indolent (21%). All patients received IL-1Ra initially and 20/47 subsequently received IL-1Ra/Dex. Seven patients had a decrease in the plasma cell labeling index (PCLI), a marker of cell proliferation, on IL-1Ra alone which paralleled a decrease in the high sensitivity C-reactive protein (CRP). Interestingly, in one patient treated with Dex alone for 6 months, Dex induced a decrease in the M-protein and IL-1 levels, however, the PCLI and CRP values increased. The IL-1Ra/Dex combination has resulted in stability of disease in the majority of the 47 patients; 28 continue on therapy with a median overall progression-free survival (PFS) of 37.5 months. Four of the 47 pts achieved a minor response (MR) with IL-1Ra alone, and an additional 6 pts achieved a MR/PR after addition of dex. The continuous measure of % reduction in CRP was significantly associated with time to progression (p=0.02). Similarly, those patients who had a >5% decrease in serum M protein also had a longer PFS (p<0.001). Of note, 2 patients with high PCLI values of 6.4 and 3.0, characteristic of poor prognosis, responded with a reduction in the PCLI values and continue on combination therapy for 3 and 1.5 years, respectively with stable disease. Conclusion: Agents such as IL-1Ra that specifically inhibit paracrine IL-6 production are more effective at targeting the proliferative component than apoptosis inducing agents such as Dex; the combination may be especially useful in patients that have an elevated PCLI at presentation.

Abstract Introduction eIF5A is the only known protein to be modified by hypusination and is highly conserved across species. Hypusinated eIF5A, the predominant form in normal and cancer cells, is involved in cell survival and inflammatory pathway activation. siRNAs targeting eIF5A inhibit NF-kB activation and reduce pro-inflammatory cytokine production. Accumulation of the unhypusinated lysine form of eIF5A is associated with apoptosis. Mutants of eIF5A that cannot be hypusinated (e.g. eIF5AK50R) are pro-apoptotic in vitro and have anti-tumoral activity in vivo in multiple cancer types including melanoma and lung cancer. SNS01-T is a novel therapeutic with a dual mechanism of eIF5A modulation: inducing cell death via siRNA-mediated inhibition of hypusinated eIF5A while simultaneously causing over-expression of pro-apoptotic eIF5AK50R via a DNA plasmid with a B-cell promoter to induce tumor cell death. SNS01-T significantly inhibited tumor growth and increased survival in mouse models of myeloma (MM), mantle cell and diffuse large B-cell lymphoma. The phase 1-2 study of SNS01-T has completed 4 planned dosing cohorts 0.0125, 0.05, 0.2 and 0.375 mg/kg twice weekly IV for 6 weeks in pts with refractory B-cell cancers. Methods PK and PD secondary endpoints included characterization of PK by measuring pExp5A plasmid DNA and eIF5A siRNA in blood and bone marrow (BM), assessing potential immunogenicity of SNS01-T by measuring serum concentrations of antibodies against SNS01-T nanoparticles, and measuring serum concentrations of select proinflammatory cytokines by enzyme-linked immunosorbent assay in serum and plasma samples. Blood PK timepoints were 30 minutes before the first infusion and at 30 minutes, 2, 6, and 24 hours after the first infusions on Week 1, Week 3, and Week 6 and at the 4, 8, and 12 week visits after the last infusion; BM samples were collected 1 day after the final infusion. Serum and plasma PD sampling timepoints were 30 minutes before and at 2, 6, and 24 hours after the first infusion on Week 1, Week 3, and Week 6, and at 4 weeks after the final infusion. Cytokines assayed included TNF-α, IFN-α, IFN-ß, IFN-g, CXCL1, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, and IL-12. Results Table 1 shows data available for interpretation as of August 2014. The remaining samples are under analysis and will be presented. Abstract 2121. Table 1:Data available for interpretation (number of patients)SNS01-T DoseOverall(n=18)0.0125 mg/kg(n=6)0.05 mg/kg(n=4)0.2 mg/kg(n=4)0.375 mg/kg(n=4)Blood PK DNA and RNA156441Bone marrow PK82321Serum antibodies to SNS01-T nanoparticles186444Serum and plasma cytokines156441 Plasmid and siRNA blood levels generally peaked 30 minutes post-dosing at weeks 1, 3 and 6 of dosing. Both plasmid and siRNA exhibited rapid clearance from the blood, with levels dropping to near pre-dosing levels within 24 hours of administration. pExp5A plasmid DNA was detectable in the bone marrow of 2 pts at cohort 1, 2 at cohort 2, 1 at cohort 3 and 1 at cohort 4. eIF5A siRNA was not detectable in bone marrow. No antibodies to SNS01-T nanoparticles were detected at any timepoint at any dose level. Cytokines remained within the expected range of inter-patient variability, similar to baseline across all timepoints at the first 2 dose levels. At dose level 3, levels of IL-6, IL-8 and TNF-α in particular increased at the 2 and 6 hour timepoints but had recovered to baseline levels 24-hours post dosing. This effect was more pronounced at the first infusion. Conclusions PCR analysis demonstrated the presence of both plasmid DNA and siRNA components of SNS01-T in blood at all dose levels, with a dose-dependent increase in plasmid copy number. Plasmid DNA was also detected in bone marrow collected 24 hours after the final infusion of SNS01-T. Pro-inflammatory cytokines did increase within hours of infusion but returned to baseline within 24 hours, synchronous with the clinical infusion reactions (see Abstract 70148). No evidence of an anti-SNS01-T antibody response was observed in any subject. Phase 2 trials are planned. Disclosures Bexon: Senesco: Consultancy. Craig:Senesco: PI Other. Siegel:Senesco: PI Other. Bensinger:Senesco: PI Other. Novitzky:Senesco: PI Other. McDonald:Senesco: PI Other. Gutierrez:Senesco: PI Other. Libby:Senesco: PI Other. van Rhee:Senesco: PI Other. Heidel:Senesco: Consultancy. Thompson:Senesco: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Barranco:Senesco: Consultancy. Taylor:Senesco: Research Funding. Browne:Senesco: Employment. Kurman:Senesco: Consultancy. Lust:Senesco: PI Other. Dondero:Senesco: Employment.

Abstract Abstract 1874 In early stage myeloma, IL-6 is a central myeloma growth factor and we have shown that abnormal production of IL-1 in the myeloma microenvironment stimulates the generation of IL-6 in a paracrine fashion. IL-1 has also been shown to be a crucial factor in the induction of IL-17 producing T-cells in vivo. IL-1Ra is a specific blocker of IL-1 activity. We have previously reported on a Phase II trial using IL-1Ra and dexamethasone, in patients with smoldering/indolent MM (SMM/IMM), showing that IL-1Ra targets the myeloma proliferative component which parallels a decrease in the C-reactive protein (CRP), a surrogate for IL-6 production. These patients are the individuals most likely to benefit from anti-cytokine therapy in an attempt to delay/prevent the development of active myeloma. Patients that had > 10% bone marrow plasma cells and/or an IgG or IgA M-spike > 3 g/dL and did not require immediate chemotherapy were eligible. All patients received 100 mg of Anakinra (IL-1Ra) SQ qd for 6 months. Patients with evidence of reduction in M-protein levels continued receiving IL-1Ra alone. Patients with stable disease at 6 months or those with a rising M-protein before 6 months received low dose dexamethasone (20 mg qweek) in addition; the dose was adjusted based on response/toxicity. Data were available on 47 patients based on intent to treat, and patients were classified as smoldering (72%) vs. indolent (28%). All 47 patients received IL-1Ra initially and 25/47 subsequently received IL-1Ra/Dex. Myeloma cell growth rate (PCLI), C-reactive protein (an in vivo marker of IL-6 levels) and IL-17 were measured in patients on trial. Seven patients had a decrease in the plasma cell labeling index (PCLI) on IL-1Ra alone which paralleled a decrease in the C-reactive protein in all cases. Three patients achieved a minor response to IL-1Ra alone and 9 patients achieved a PR/MR after addition of dexamethasone. When patients were grouped into whether they exhibited a reduction in the C-reactive protein from baseline after 6 months of therapy, the median PFS for patients without (21 patients) or with (26 patients) a greater than one-third reduction in baseline CRP was 1 year vs more than 8 years (p<.01). Analyses of biomarkers suggest that patients with elevated IL-17 levels may be less likely to respond to IL-1Ra treatment. Only 25% of the responders with a decrease in CRP had IL-17 levels > 10 pg/ml versus 60% of those without a CRP decrease. Although not statistically significant do to the small sample size, the median PFS in the IL-17 < 10 pg/ml group was 2047days vs 1367 days in the IL-17 > 10 pg/ml group. In conclusion, the above results suggest that agents such as IL-1Ra that specifically inhibit IL-1 induced paracrine IL-6 production are effective at targeting the proliferative myeloma component and warrant further investigation in combination with standard myeloma therapies. Elevated IL-17 levels may suggest that the inflammatory process is too far advanced in some individuals to respond to IL-1 blockade. Biomarkers such as CRP and IL-17 may be useful to predict those patients that are most likely to benefit from IL-1 treatment. Disclosures: Off Label Use: IL-1Ra in myeloma.