ALBERT

Description: We investigated whether interleukin-1β (IL-1β) is differentially expressed in plasma cells from monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) patients because IL-1β appears to play a major role in the development of lytic bone lesions, the major clinical feature distinguishing MGUS from myeloma. In situ hybridization (ISH) for IL-1β was performed using bone marrow aspirates from 51 MM, 7 smoldering MM, 21 MGUS, and 5 normal control samples. Using the ISH technique IL-1β mRNA was detectable in the plasma cells from 49 of 51 patients with active myeloma and 7 of 7 patients with smoldering myeloma. In contrast, 5 of 21 patients with MGUS and 0 of 5 normal controls had detectable IL-1β message. Bone lesions were present in 40 of the 51 MM patients analyzed, and all 40 patients had IL-1β mRNA by ISH. These results show that greater than 95% of MM patients but less than 25% of MGUS patients are positive for IL-1β production. In the future, continued follow-up of IL-1β positive and negative MGUS patients should determine whether aberrant expression of plasma cell IL-1β is predictive of those MGUS patients that will eventually progress to active myeloma.

Description: 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%

Description: 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

Description: 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 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.

Description: 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.

Description: Live attenuated measles virus (MV-Edm) has potent oncolytic activity against myeloma xenografts in mice. Therapy of multiple myeloma, a disseminated plasma cell malignancy, would require systemic administration of the virus. Thus, the virus should ideally be targeted to infect only myeloma cells to minimize collateral damage to normal tissues: viral binding to its natural receptors must be ablated and a new specificity domain that targets entry into myeloma cells be added. This study covers 2 critical steps toward generating such a retargeted virus: (1) a new specificity domain against the plasma cell marker CD38 was constructed in the form of a single-chain antibody (scFv) and (2) display of that scFv on the measles viral envelope glycoprotein successfully redirected virus entry through CD38 expressed on target cells devoid of the natural MV receptors. The anti-CD38 scFv was tethered to the C-terminus of the hemagglutinin (H) glycoprotein of MV-Edm through a Factor Xa protease cleavable linker. Immunoblot analysis demonstrated that the scFv was efficiently incorporated into recombinant viral particles. Replication of MV-αCD38 was not hindered by the scFv, reaching titers comparable to MV-Edm. Chinese hamster ovary (CHO) cells were resistant to infection by MV-Edm and MV-αCD38. In contrast, CHO cells expressing CD38 became susceptible to infection by MV-αCD38 but not MV-Edm. Removal of the displayed scFv rendered MV-αCD38 noninfectious on CHO-CD38 cells. Tumorigenicity of CHO-CD38 cells in immunocompromised mice was significantly attenuated by MV-αCD38, resulting in enhanced survival of these mice compared with the control group.

Description: 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

Description: Abstract 1831 Poster Board I-857 IL-1 Antagonists Are More Effective at Inhibiting IL-6 Production than Apoptosis Inducing Agents: Implications for Targeting the Myeloma Proliferative Component. John A. Lust, MD,PhD1, Steven R. Zeldenrust, MD, PhD1, Laurie L. Moon-Tasson1*, and Kathleen A. Donovan, PhD1*. 1Division of Hematology, Mayo Clinic, Rochester, MN, United States, 55905. Background Multiple myeloma patients with an elevated growth rate have a shortened duration of response and survival. IL-6 is a central myeloma growth factor and we have shown that abnormal production of IL-1beta in the myeloma microenvironment stimulates the generation of paracrine IL-6 and myeloma cell growth in vivo. Dexamethasone and IL-1Ra both have the ability to inhibit IL-1 induced IL-6 production in vitro however their ability to inhibit IL-6 production and myeloma cell growth have not been investigated in a comparative fashion. Methods In vitro, IL-1 (100, 10, 1 pg/ml) was added to stromal cell cultures in the presence or absence of IL-1Ra (1 and 0.1 μg/ml) or dexamethasone (100 and 10 μM). IL-6 levels were quantitated by ELISA. In vivo, a patient with smoldering myeloma (≥ 10% bone marrow plasma cells) received 100 mg of IL-1Ra SQ qd for 6 months, low dose dexamethasone (20 mg qweek) for 6 months, followed by the combination of IL-1Ra and dexamethasone for 6 months. The bone marrow plasma cell percentage (BMPC), serum IgG, the plasma cell labeling index (marker of myeloma cell proliferation) and the C-reactive protein (marker of IL-6 production) were monitored serially. Results The effects of IL-1Ra and dexamethasone on IL-1 induced IL-6 production by marrow stromal cells are detailed in Figure 1. The results showed that IL-1Ra at 1 μg/ml was able to inhibit IL-1 induced IL-6 production back to baseline at all IL-1 concentrations tested. The inhibitory effect was less pronounced at 0.1 μg/ml IL-1Ra but still superior to dexamethasone. Dexamethasone was less effective at IL-6 inhibition compared to IL-1Ra using 100 and 10 pg/ml of IL-1 but similar at 1 pg/ml of IL-1. Of interest, the patient treated with IL-1Ra alone, dexamethasone alone, and the combination appeared to mimic these results in vivo. Anakinra alone induced a reduction of the PCLI and CRP. Dexamethasone alone decreased the M-protein; however the PCLI and CRP values increased. The PCLI increased from 0% to 0.8% and the CRP increased from 0.18 up to 1.48 indicating increased levels of IL-6 and a more active proliferative component of the disease. Subsequently, the combination of dexamethasone and IL-1Ra led to a further decrease in the IgG and the PCLI and CRP decreased again; PCLI decreased from 0.8% to 0.2% and CRP decreased from 1.48 down to 0.40. Conclusion The above results suggest that agents such as IL-1Ra are more effective at IL-6 inhibition and targeting the proliferative myeloma component than apoptosis inducing agents such as dexamethasone. Combination therapy with IL-1 inhibitors and apoptosis inducing agents may be useful in patients with active myeloma that have an elevated PCLI at diagnosis. Disclosures No relevant conflicts of interest to declare.