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Objective To evaluate the safety and potential efficacy of AMG 557, a fully human antibody directed against the inducible T cell costimulator ligand (ICOSL) in patients with systemic lupus erythematosus (SLE) with arthritis. Methods In this phase Ib, randomized, double-blind, placebo-controlled study, patients received AMG 557 210 mg (n = 10) or placebo (n = 10) weekly for 3 weeks, then every other week for 10 additional doses. The corticosteroid dosage was tapered to ≤7.5 mg/day by day 85, and immunosuppressants were discontinued by day 29. Primary end points on day 169 were safety, immunogenicity, the Lupus Arthritis Response Index (LARI; defined by a reduction in the tender and swollen joint counts), ≥1-letter improvement in the musculoskeletal domain of the British Isles Lupus Assessment Group (BILAG) index, and medication discontinuation. The secondary/exploratory end points were changes in the tender and swollen joint counts, BILAG index scores (musculoskeletal, global), and the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). Results The incidence of adverse events, most of which were mild, was similar between groups. LARI responses occurred in 3 of 10 patients receiving AMG 557 and 1 of 10 patients receiving placebo (P = 0.58). More patients in the AMG 557 group achieved a ≥4-point improvement in the SLEDAI score on day 169 (7 of 10 patients) compared with the placebo group (2 of 10 patients) (P = 0.07). Patients treated with AMG 557 (versus placebo) had greater improvements from baseline in the global BILAG index scores (-36.3% versus -24.7%) and the SLEDAI score (-47.8% versus -10.7%) and in tender (-22.8% versus -13.5%) and swollen (-62.1% versus -7.8%) joint counts on day 169. Conclusion AMG 557 showed safety and potential efficacy, supporting further evaluation of the clinical efficacy of ICOSL blockade in patients with SLE.

In this study we compared the pharmacokinetic profile of four unrelated antibodies, which do not bind to mammalian antigens, in IgG1 and IgG2 frameworks in both rats and non-human primates (NHP). This allowed for extensive cross comparison of the impact of antibody isotype, complementarity determining regions (CDR) and model species on pharmacokinetics without the confounding influence of antigen binding in the hosts. While antibody isotype had no significant impact on the pharmacokinetics, the CDRs do alter the profile, and there is an inverse correlation between the neonatal Fc receptor (FcRn) affinity and pharmacokinetic performance. Faster clearance rates were also associated with higher isoelectric points; however, although this panel of antibodies all possess basic isoelectric points, ranging from 8.44 to 9.18, they also have exceptional in vivo half-lives, averaging 369 hours, and low clearance rates, averaging 0.18 ml/h/kg in NHPs. This pattern of pharmacokinetic characteristics was conserved between rats and NHPs.

Introduction: While minimal residual disease (MRD) negativity is not yet an established regulatory surrogate for a clinical endpoint in multiple myeloma (MM), it does have value as a prognostic biomarker and in assessing disease status. Previously we reported results from a phase 1b trial that described the safety and efficacy of the triplet regimen carfilzomib (given weekly), lenalidomide, and dexamethasone (KRd) in patients with relapsed and/or refractory MM (RRMM) or newly diagnosed MM (NDMM) (Biran et al, Am J Hematol 2019;94:794-802; Alsina et al, Clin Lymphoma Myeloma Leuk 2019;19:Suppl E52). Here, we evaluate MRD status by flow cytometry after treatment with weekly KRd in this trial. Methods: A total of 56 patients with RRMM and 51 with NDMM were enrolled and treated with weekly KRd. Treatment was given in 28-day cycles. Carfilzomib was given on days 1, 8, and 15; lenalidomide 25 mg on days 1-21; and dexamethasone 40 mg on days 1, 8, and 15 (also day 22 for cycle 1-8). In the NDMM cohort, patients were enrolled regardless of transplant eligibility, and treatment interruption for mobilization and collection with or without autologous stem cell transplant was allowed after cycle 4. Per the protocol, aspirate was collected for flow cytometry evaluation at two separate endpoints: at cycle 8 day 1 (C8D1); and at the time when laboratory data supported a response of complete response (CR) or better (on the basis of negative immunofixation on serum and urine). Sample quality was assessed by several measures (including the presence of mast cells, erythroid precursors, and immature B cells), and viability was determined using a flow assay. Adequate samples were tested for MRD using 8-color flow cytometry in two tubes, with a sensitivity of 10-5. MRD was reported as positive when a minimum of 2 x 106 CD138+ cells per tube were evaluated, and ≥ 20 abnormal events detected. Results: Overall, 58 of 79 (73.4%) study-specified samples were obtained and evaluated by flow cytometry. Of the NDMM patients, 15 had bone marrow aspirate evaluable for MRD at C8D1 (in total 22 patients were treated to C8D1), and 12 had bone marrow aspirate evaluable for MRD at the time of CR (14 patients achieved CR). At C8D1, 53% (8/15) of NDMM patients with flow data achieved MRD negativity, and at time of CR, 83% (10/12) of NDMM patients were MRD negative (the remaining patients with flow data were determined to be MRD positive). Among the 12 NDMM patients evaluated for MRD at time of CR, 17% had high-risk cytogenetics, 58% had standard-risk cytogenetics, and 25% had unknown cytogenetic risk status. Of the RRMM patients, 26 had bone marrow aspirate evaluable for MRD at C8D1 (30 patients were treated to C8D1), and five had bone marrow aspirate evaluable for MRD at the time of suspected CR or better (in total 13 patients achieved CR). Of RRMM patients with available flow data at time of CR, 40% (2/5) achieved MRD negativity, and of those with flow data at C8D1, 50% (13/26) achieved MRD negativity. Of all patient samples evaluated for MRD, 67% of NDMM and 48% of RRMM samples were MRD negative. The results reported here are directionally comparable to the rates of MRD-negative CRs observed in previous studies of twice-weekly KRd in NDMM (Jasielec J et al, Blood 2014;124:2127; Kazandjian D et al, JAMA Oncol 2018;4:1781-1783; Zimmerman T et al, Blood 2016;128:675). Our findings are limited by incomplete acquisition of samples to support a full MRD analysis per protocol (approximately 73% of the intended time point samples were acquired). Additionally, we reported MRD status without censoring for missing MRD data, as would be required to analyze MRD for a randomized controlled trial to eliminate acquisition bias. A robust comparison of MRD-negative CR rates between regimens would require an RCT with full MRD sampling, and sensitivity analyses that treat missing MRD data as MRD-positive (Chari A et al, Blood 2017;130:974-981; Voorhees PM et al, Blood 2020). Conclusions: We have previously shown that once-weekly KRd is active and has acceptable toxicity in both the RRMM and NDMM settings. We found that among MRD-evaluable patients who had a CR or better, MRD negativity rates were impressive in both the NDMM setting (83%) and in the RRMM setting (40%), suggesting that the weekly KRd regimen can induce MRD-negative CRs in both settings. Disclosures Landgren: Adaptive: Consultancy, Honoraria; Takeda: Other: Independent Data Monitoring Committees for clinical trials, Research Funding; Janssen: Consultancy, Honoraria, Other: Independent Data Monitoring Committees for clinical trials, Research Funding; Juno: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Independent Data Monitoring Committees for clinical trials, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria; Cellectis: Consultancy, Honoraria; Merck: Other; Cellectis: Consultancy, Honoraria; Binding Site: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Merck: Other; BMS: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Seattle Genetics: Research Funding; Glenmark: Consultancy, Honoraria, Research Funding; Binding Site: Consultancy, Honoraria; Karyopharma: Research Funding; Takeda: Other: Independent Data Monitoring Committees for clinical trials, Research Funding; Juno: Consultancy, Honoraria; Glenmark: Consultancy, Honoraria, Research Funding; Karyopharma: Research Funding; Seattle Genetics: Research Funding. Alsina:Janssen: Honoraria, Speakers Bureau; BMS: Consultancy, Research Funding; Celgene: Consultancy, Honoraria; Amgen: Honoraria, Speakers Bureau. Biran:Janssen: Consultancy, Honoraria, Other: reimbursement of travel and accomodation, Research Funding, Speakers Bureau; KAryopharma: Research Funding; Sanofi: Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Other: reimbursement of travel and accomodation, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Other: reimbursement of travel and accommodation, Research Funding, Speakers Bureau. Vesole:Amgen: Speakers Bureau; Takeda: Speakers Bureau; Sanofi: Speakers Bureau; Janssen: Speakers Bureau; BMS: Speakers Bureau. Fang:Amgen: Current Employment, Current equity holder in publicly-traded company. Arnold:Amgen: Current Employment, Current equity holder in publicly-traded company. Kimball:Amgen: Current Employment, Current equity holder in publicly-traded company; WindMIL Therapeutics: Current equity holder in private company. Siegel:Karyopharma: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Merck: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau; Celulatiry: Consultancy. OffLabel Disclosure: Carfilzomib is a proteasome inhibitor that can be used for the treatment of relapses/refractory multiple myeloma

Objective To assess the safety and immunologic impact of inhibiting interferon-γ (IFNγ) with AMG 811, a human IgG1 monoclonal antibody against IFNγ, in patients with systemic lupus erythematosus (SLE). Methods Twenty-six patients with mild-to-moderate, stable SLE were administered placebo or a single dose of AMG 811, ranging from 2 mg to 180 mg subcutaneously or 60 mg intravenously. Results Similar to results previously reported following inhibition of type I IFNs, treatment of SLE patients with AMG 811 led to a dose-dependent modulation of the expression of genes associated with IFN signaling, as assessed by microarray analysis of the whole blood. The list of impacted genes overlapped with that identified by stimulating human whole blood with IFNγ and with those gene sets reported in the literature to be differentially expressed in SLE patients. Serum levels of IFNγ-induced chemokines, including IFNγ-inducible protein 10 (IP-10), were found to be elevated at baseline in SLE patients as compared to healthy volunteers. In contrast to previously reported results from studies using type I IFN–blocking agents, treatment with AMG 811 led to dose-related reductions in the serum levels of CXCL10 (IP-10). Conclusion The scope and nature of the biomarkers impacted by AMG 811 support targeting of IFNγ as a therapeutic strategy for SLE.

Objectives To evaluate the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of single-dose and multiple-dose administration of AMG 557, a human anti-inducible T cell co-stimulator ligand (ICOSL) monoclonal antibody, in subjects with systemic lupus erythematosus (SLE). Methods Patients with mild, stable SLE (n=112) were enrolled in two clinical trials to evaluate the effects of single (1.8–210 mg subcutaneous or 18 mg intravenous) and multiple (6 –210 mg subcutaneous every other week (Q2W)×7) doses of AMG 557. Subjects received two 1 mg intradermal injections 28 days apart of keyhole limpet haemocyanin (KLH), a neoantigen, to assess PD effects of AMG 557. Safety, PK, target occupancy, anti-KLH antibody responses, lymphocyte subset analyses and SLE-associated biomarkers and clinical outcomes were assessed. Results AMG 557 demonstrated an acceptable safety profile. The PK properties were consistent with an antibody directed against a cell surface target, with non-linear PK observed at lower concentrations and linear PK at higher concentrations. Target occupancy by AMG 557 was dose dependent and reversible, and maximal occupancy was achieved in the setting of this trial. Anti-AMG 557 antibodies were observed, but none were neutralising and without impact on drug levels. A significant reduction in the anti-KLH IgG response was observed with AMG 557 administration without discernible changes in the anti-KLH IgM response or on the overall IgG levels. No discernible changes were seen in lymphocyte subsets or in SLE-related biomarkers and clinical measures. Conclusions The selective reduction in anti-KLH IgG demonstrates a PD effect of AMG 557 in subjects with SLE consistent with the biology of the ICOS pathway and supports further studies of AMG 557 as a potential therapeutic for autoimmune diseases. Trial registration numbers NCT02391259 and NCT00774943.

Objective Interferon-γ (IFNγ) is implicated in the pathogenesis of discoid lupus erythematosus (DLE). This study sought to evaluate a single dose of AMG 811, an anti-IFNγ antibody, in patients with DLE. Methods The study was designed as a phase I randomized, double-blind, placebo-controlled crossover study of the pharmacodynamics, safety, and clinical efficacy of AMG 811 in patients with DLE. Patients received a single subcutaneous dose of AMG 811 (180 mg) or placebo. The patients in sequence 1 received AMG 811 followed by placebo, while those in sequence 2 received placebo followed by AMG 811. Pharmacodynamic end points included global transcriptional analyses of lesional and nonlesional skin, IFNγ blockade signature (IGBS) transcriptional scores in the skin and blood, keratinocyte IFNγ RNA scores, and serum levels of CXCL10 protein. Additional end points were efficacy outcome measures, including the Cutaneous Lupus Erythematosus Disease Area and Severity Index, and safety outcome measures. Results Sixteen patients with DLE were enrolled in the study (9 in sequence 1 and 7 in sequence 2). AMG 811 treatment reduced the IGBS score (which was elevated in DLE patients at baseline) in both the blood and lesional skin. The keratinocyte IFNγ RNA score was not affected by administration of AMG 811. Serum CXCL10 protein levels (which were elevated in the blood of DLE patients) were reduced with AMG 811 treatment. The AMG 811 treatment was well tolerated but did not lead to statistically significant improvements in any of the efficacy outcome measures. Conclusion AMG 811 treatment led to changes in IFNγ-associated biomarkers and was well tolerated, but no significant clinical benefit was observed in patients with DLE.

Objective To evaluate safety, pharmacokinetics and pharmacodynamics of anti-interferon (IFN)-γ monoclonal antibody AMG 811 in subjects with SLE without or with lupus nephritis (LN). Methods In this phase Ib, randomised, multiple-dose escalation study (NCT00818948), subjects without LN were randomised to subcutaneous AMG 811 (6, 20 or 60 mg) or placebo and subjects with LN were randomised to subcutaneous AMG 811 (20, 60 or 120 mg) or placebo every four weeks for three total doses. Outcomes included incidence of adverse events (AEs); pharmacokinetics; levels of serum proteins (CXCL-10, interleukin 18, monocyte chemotactic protein-1); changes in gene transcript profiles and clinical parameters (Safety of Estrogen in Lupus Erythematosus National Assessment-Systemic Lupus Erythematosus Disease Activity Index (SELENA-SLEDAI) scores, proteinuria, anti-double-stranded DNA (anti-dsDNA) antibodies, C3 complement, C4 complement). Results Fifty-six subjects enrolled (28 SLE without LN; 28 with LN). Baseline mean SELENA-SLEDAI scores were 2.2 and 12.0 for SLE subjects without and with LN, respectively. Most subjects reported an AE; no meaningful imbalances were observed between AMG 811 and placebo. Pharmacokinetic profiles were similar and mostly dose-proportional in subjects without or with LN. AMG 811 treatment reduced CXCL-10 protein levels and blood-based RNA IFN-γ Blockade Signature compared with placebo. Reductions were less pronounced and not sustained in subjects with LN, even at the highest dose tested, compared with subjects without LN. No effect on SELENA-SLEDAI scores, proteinuria, C3 or C4 complement levels, or anti-dsDNA antibodies was observed. Conclusion AMG 811 demonstrated favourable pharmacokinetics and acceptable safety profile but no evidence of clinical impact. IFN-γ-associated biomarkers decreased with AMG 811; effects were less pronounced and not sustained in LN subjects. Trial registration number NCT00818948; results.

Background Interferon-gamma (IFN-g) is a pro-inflammatory cytokine that modulates the function of several important immune populations. Evidence from human and animal models suggests that increased levels of Type I and/or Type II IFN are associated with SLE. Objectives This study assessed the safety, tolerability, PK and pharmacodynamic (PD) data from AMG 811, an anti-IFNg mAb, in subjects with active class III or IV LN. Methods Subjects were enrolled if they had new onset or reactivation of biopsy-proven (within 18 months) class III or IV LN, UP/Cr >1 or 24 hr urine protein >1 g after at least 12 wks of treatment with MMF or AZA. Superimposed membranous changes were allowed. Subjects with rapidly progressive GN or significant chronicity were excluded. Subjects could receive oral prednisone at doses up to 20 mg/day. Subjects were randomized to placebo or ascending doses of AMG 811 (20, 60 or 120 mg given SC q4 wks for three doses at 3:1 allocation) in addition to MMF or AZA. The primary endpoints were safety, tolerability, and anti-drug antibodies. Serum AMG 811 concentrations, anti-AMG 811 antibodies and PD biomarkers (blood RNA and serum) were also assessed. Disease related assessments included reduction from baseline in proteinuria (24 hr urine and spot UP/Cr), SLEDAI scores and changes in SLE-related biomarkers. Results 28 subjects were enrolled: 7 in the PBO group and 21 in the AMG 811 treatment groups. The proportion of subjects reporting treatment emergent AEs, including serious AEs, was similar between PBO and AMG 811 groups although a numerically higher proportion of subjects treated with AMG 811 had infectious events. AMG 811 displayed linear PK with half-life of 11-21 days. Exposures after doses of 20 & 60 mg in LN subjects were similar to exposures in non-renal SLE subjects. Antibodies to AMG 811 were not observed at any time. Baseline serum CXCL10 (IP-10) levels and IFNgamma-modulated mRNAs were higher in LN compared to non-renal SLE; both populations had elevated levels compared to healthy controls. AMG 811 (60 & 120 mg cohorts) led to a reduction in these biomarkers, albeit incomplete and transient, suggesting that AMG 811 may have had reduced target coverage in LN relative to non-renal SLE, where levels were reduced into the healthy range. Although numerous subjects demonstrated improvement in proteinuria, no consistent differences between AMG 811 treatment cohorts and PBO were discernable at week 12 in renal outcome or SLE-related serum biomarkers. Conclusions AMG 811 demonstrated a favorable PK and immunogenicity profile in active LN subjects, and the overall safety profile was acceptable in this small study. Inhibition of pharmacodynamic biomarkers with doses up to 120 mg of AMG 811 appeared incomplete in LN subjects. There were no discernible effects of AMG 811 on clinical or SLE-related serologic outcome measures over the treatment period although interpretation is challenging given the small sample size. Disclosure of Interest D. Martin Employee of: Amgen, Z. Amoura: None declared, J. Romero-Diaz: None declared, Y. Chong: None declared, J. Sanchez-Guerrero: None declared, T. Chan: None declared, G. Arnold Employee of: Amgen, M. Damore Employee of: Amgen, W. Sohn Employee of: Amgen, N. Chirmule Employee of: Amgen, K. Chiu Employee of: Amgen, C. Wang Employee of: Amgen, M. Boedigheimer Employee of: Amgen, B. Sullivan Employee of: Amgen, A. Welcher Employee of: Amgen, B. Kotzin Employee of: Amgen, J. Chung Employee of: Amgen

Abstract 1303 Studies of the regulation of erythropoiesis by hypoxic induction of erythropoietin (EPO) transcription led to the discovery of oligonucleotide binding motifs, hypoxia responsive elements (HREs) essential for hypoxic upregulation of EPO. HREs bind hypoxia-inducible transcription factors (HIFs). HIFs are heterodimers of 3 α subunit isotypes; HIF-1α, HIF-2α, and HIF-3α, and a common β-subunit. EPO is expressed in diverse tissues, some regulated mainly by HIF-2 and others by HIF-1. Tissue-specific regulatory elements (i.e. Kidney Inducible Element KIE, Negative Regulatory Element NRE, and Negative Regulatory Liver specific Element NRLE) of the EPO gene spanning several kilobases were reported. Our in silico analysis of the human EPO genome found two additional canonical HIF-binding elements in the KIE and NRE regions, and comparative analysis of phylogenically conserved sequences of Epo genes refined these HIF-binding elements from several kbs to a few hundred nucleotides and further delineated them as mKIE, mNRE1, mNRE2, and mNRLE2. To further define the structure and function of these elements in vivo, we exposed mice to 8% O2 hypoxia and monitored hypoxic induction of Epo mRNA levels in the liver, kidney, brain, and spleen. Spleen, kidney, and brain had a peak induction of the Epo transcript at 3 hours of hypoxia treatment, while liver showed peak induction at 6 hours. At the end of the hypoxia treatment, the mice were sacrificed and organs were harvested, and in vivo chromatin immunoprecipitation (ChIPs) was performed with antibodies against HIF-1α and HIF-2α. The results from these studies are summarized below. “+” denotes presence and “−” absence of binding of HIF-1 and HIF-2. “Norm” - normoxia, “Hyp” - hypoxia. To further define the role of these elements in tissue-specific Epo expression, we cloned and mutated these elements with reporter constructs, and generated transgenic mouse strains. The reporter gene was expressed in renal tubules, the red pulp area of the spleen, and liver hepatocytes. By creating a designed point mutation to disrupt the function of KIE, we suppressed the hypoxia response in the kidney, while mutations of NRE1 and NRE2 abolished reporter expression in the liver. Studies of HREs in the brain are ongoing. In conclusion, we have refined the previously reported HREs to a few hundred bases (particularly, KIE is a 6 nucleotide bearing motif) and now demonstrate tissue-specific differential hypoxia-induced binding of HIF-1 and HIF-2 and their functional roles in EPO induction. This provides a background to examine the molecular basis of these elements in patients with aberrant EPO expression. Disclosures: Elliott: Amgen, Inc: Employment.

Hypoxia regulates erythropoiesis and other essential processes via hypoxia-inducible transcription factors (HIFs). HIFs are heterodimers that consist of an α subunit (3 isotypes with significant homology; HIF-1α, HIF-2α, HIF-3α), and a common b-subunit; HIF-1 and HIF-2, in some instances exhibiting tissue- and gene-specific gene regulation. Erythropoietin (EPO) was the first identified HIF-1 target gene with the defined HIF-1 binding sequence. However, subsequent works suggested that HIF-2 also regulates EPO transcription and that there are other regulatory elements of EPO gene (i.e. Kidney Inducible Element KIE, Negative Regulatory Element NRE, and Negative Regulatory Liver specific Element NRLE). In silico analysis of the human EPO genome found two additional potential HIF-binding elements in the KIE and NRE regions. The comparative analysis of phylogenically conserved sequences of human, mouse, dog, and rat Epo genes further refined these mouse Epo gene HIF-binding elements as mKIE, mNRE1, mNRE2, and mNRLE2. We treated mice in hypoxia chamber (8% O2) and monitored changes of Epo mRNA levels in liver, kidney, brain, spleen, and bone marrow. All tested tissues increased Epo transcription during hypoxia. Bone marrow, spleen, kidney, and brain showed a peak of induction of Epo transcript at 3 hours of hypoxia treatment, while liver reached the highest level at 6 hours. Mice were sacrificed and organs were harvested, and in vivo chromatin immunoprecipitation (ChIPs) was performed with antibodies against HIF-1α and HIF- 2α and tissue-specific binding regions were defined. The results from these studies are summarized below. HIF-1 mKIE rnNRE mNRE2 mNRLE2 Norm Hyp Norm Hyp Norm Hyp Norm Hyp Liver − + − − + − ? ? Kidney − + − − + − + − Brain − + − − − + − + BM − + − − − − − + Splsen − + − − − − − + HIF-2 mKIE mNRE mNRE2 mNRLE2 Norm Hyp Norm Hyp Norm Hyp Norm Hyp Liver − + − − − + − + Kidney + − − − + − ? ? Brain − − − − − − − + BM − − − − − − + − Spleen − + − − − − − + “+” denotes presence and “-” absence of binding of HIF-1 and HIF-2, “?” – indicates inconclusive results. “Norm” - normoxia, “Hyp” - hypoxia. In conclusion, we demonstrate the differential hypoxia-induced binding of HIF-1 and HIF-2 at different HIF binding elements in the tissues known to express Epo. Further studies will be required to define the function of these HIF-1 and HIF-2 binding elements in tissue specific Epo expression and their role in health and disease.