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Idiopathic multicentric Castleman disease (iMCD) is a poorly understood hematologic disorder involving cytokine-induced polyclonal lymphoproliferation, systemic inflammation, and potentially fatal multiorgan failure. Although the etiology of iMCD is unknown, interleukin-6 (IL-6) is an established disease driver in approximately one-third of patients. Anti–IL-6 therapy, siltuximab, is the only US Food and Drug Administration–approved treatment. Few options exist for siltuximab nonresponders, and no validated tests are available to predict likelihood of response. We procured and analyzed the largest-to-date cohort of iMCD samples, which enabled classification of iMCD into disease categories, discovery of siltuximab response biomarkers, and identification of therapeutic targets for siltuximab nonresponders. Proteomic quantification of 1178 analytes was performed on serum of 88 iMCD patients, 60 patients with clinico-pathologically overlapping diseases (human herpesvirus-8–associated MCD, N = 20; Hodgkin lymphoma, N = 20; rheumatoid arthritis, N = 20), and 42 healthy controls. Unsupervised clustering revealed iMCD patients have heterogeneous serum proteomes that did not cluster with clinico-pathologically overlapping diseases. Clustering of iMCD patients identified a novel subgroup with superior response to siltuximab, which was validated using a 7-analyte panel (apolipoprotein E, amphiregulin, serum amyloid P-component, inactivated complement C3b, immunoglobulin E, IL-6, erythropoietin) in an independent cohort. Enrichment analyses and immunohistochemistry identified Janus kinase (JAK)/signal transducer and activator of transcription 3 signaling as a candidate therapeutic target that could potentially be targeted with JAK inhibitors in siltuximab nonresponders. Our discoveries demonstrate the potential for accelerating discoveries for rare diseases through multistakeholder collaboration.

Because interleukin-6 (IL-6) is considered important in the proliferation of early multiple myeloma (MM), we hypothesized that the addition of the anti-IL-6 monoclonal antibody siltuximab to the bortezomib-melphalan-prednisone (VMP) regimen would improve outcomes in transplant-ineligible patients with newly diagnosed MM. One hundred and six patients were randomized to receive 9 cycles of VMP or VMP plus siltuximab (11 mg/kg every 3 weeks) followed by siltuximab maintenance. Baseline characteristics were well balanced except for immunoglobulin A subtype and 17p deletions. With a complete response (CR) rate of 27% on siltuximab plus VMP (S+VMP) and 22% on VMP, the study did not confirm its hypothesis that the addition of siltuximab would increase the CR rate by at least 10%. Overall response rate was 88% on S+VMP and 80% on VMP, and at least very good partial response rates were 71% and 51% (P = .0382), respectively. Median progression-free survival (17 months) and 1-year overall survival (88%) were identical in the 2 arms. Grade ≥3 adverse-event incidence was 92% on S+VMP and 81% on VMP (P = .09), with trends toward more hematologic events and infections on S+VMP. Maintenance therapy with siltuximab was well tolerated. In conclusion, the addition of siltuximab to VMP did not improve the CR rate or long-term outcomes. This study was registered at http://clinicaltrials.gov as #NCT00911859.

Because interleukin-6 (IL-6) is considered important in the proliferation of early multiple myeloma (MM), we hypothesized that the addition of the anti-IL-6 monoclonal antibody siltuximab to the bortezomib-melphalan-prednisone (VMP) regimen would improve outcomes in transplant-ineligible patients with newly diagnosed MM. One hundred and six patients were randomized to receive 9 cycles of VMP or VMP plus siltuximab (11 mg/kg every 3 weeks) followed by siltuximab maintenance. Baseline characteristics were well balanced except for immunoglobulin A subtype and 17p deletions. With a complete response (CR) rate of 27% on siltuximab plus VMP (S+VMP) and 22% on VMP, the study did not confirm its hypothesis that the addition of siltuximab would increase the CR rate by at least 10%. Overall response rate was 88% on S+VMP and 80% on VMP, and at least very good partial response rates were 71% and 51% (P= .0382), respectively. Median progression-free survival (17 months) and 1-year overall survival (88%) were identical in the 2 arms. Grade ≥3 adverse-event incidence was 92% on S+VMP and 81% on VMP (P= .09), with trends toward more hematologic events and infections on S+VMP. Maintenance therapy with siltuximab was well tolerated. In conclusion, the addition of siltuximab to VMP did not improve the CR rate or long-term outcomes. This study was registered at http://clinicaltrials.govas #NCT00911859.

A number of selection systems have been developed for direct selection of recombinant plasmids in cloning experiments (positive selection). In this study, the commonly used LacZ-based α-complementation plasmid vectors have been used for designing a positive selection system for the selection of recombinants. The basis for the strategy is the phenomenon of galactose sensitivity exhibited by galactose epimerase (galE) mutants of Escherichia coli. It is known that lacZ+galE, but not LacZ−galE cells are killed upon addition of lactose due to the accumulation of a toxic intermediate, UDP-galactose, by hydrolysis of lactose. Using a galE mutant strain of E. coli that carries the lacZΔM15 allele, various α-complementation plasmids that vary in their copy number were examined for their ability to be killed following addition of lactose. The results show that some plasmids that exhibit relatively high β-galactosidase enzyme activity can be used effectively for positive selection. This selection would be extremely useful during primary cloning experiments such as construction of genomic or cDNA libraries and also in instances involving selection for rare recombinants.

Keliximab, a Primatized IgG1 CD4 mAb, was reconfigured to an IgG4 antibody. The γ4 constant region was further modified by substituting glutamic acid for serine at position 235 in the CH2 domain (IgG4-E), to remove residual binding to Fcγ receptors, and substitution of serine with proline at position 228 in the hinge region (IgG4-PE) for greater stability. Pharmacokinetic analysis in rats gave a t1/2of approximately 4 days for IgG4-E and 9 days for IgG4-PE, consistent with a greater stability of the IgG4-PE molecule. The effects on T cell subsets were assessed in chimpanzees given escalating doses of IgG4-PE: 0.05 mg/kg on Day 16, 1.5 mg/kg dose on Day 43, and 15 mg/kg on Day 85. Receptor modulation was observed at the two highest doses, but no depletion of T cells at any dose. The in vitroand in vivoresults demonstrate the potential of this IgG4-PE mAb for use in human trials.

TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines which induces apoptotic cell death in a variety of tumor cell lines. It mediates its apoptotic effects through one of two receptors, DR4 and DR5, which are members of of the TNF receptor family, and whose cytoplasmic regions contain death domains. In addition, TRAIL also binds to 3 “decoy” receptors, DcR2, a receptor with a truncated death domain, DcR1, a glycosylphosphatidylinositol-anchored receptor, and OPG a secreted protein which is also known to bind to another member of the TNF family, RANKL. However, although apoptosis depends on the expression of one or both of the death domain containing receptors DR4 and/or DR5, resistance to TRAIL-induced apoptosis does not correlate with the expression of the “decoy” receptors. Previously, TRAIL has been described to bind to all its receptors with equivalent high affinities. In the present work, we show, by isothermal titration calorimetry and competitive enzyme-linked immunosorbent assay, that the rank order of affinities of TRAIL for the recombinant soluble forms of its receptors is strongly temperature dependent. Although DR4, DR5, DcR1, and OPG show similar affinities for TRAIL at 4 °C, their rank-ordered affinities are substantially different at 37 °C, with DR5 having the highest affinity (KD≤ 2 nm) and OPG having the weakest (KD= 400 nm). Preferentially enhanced binding of TRAIL to DR5 was also observed at the cell surface. These results reveal that the rank ordering of affinities for protein-protein interactions in general can be a strong function of temperature, and indicate that sizeable, but hitherto unobserved, TRAIL affinity differences exist at physiological temperature, and should be taken into account in order to understand the complex physiological and/or pathological roles of TRAIL.

A Primatized anti-CD4 monoclonal antibody (MAb), CE9.1, with V-domain from cynomolgus macaque (showing 92% homology with human consensus sequence V-domains), and a human IgG1 constant region, was characterizedin vitroandin vivoin chimpanzees. This MAb binds human CD4 withKdof 1.0 nMand was also able to bind to human IgG Fc receptors (FcγR). However, despite being of the IgG1 subclass, CE9.1 did not bind to complement component C1q, nor did it mediate complement-dependent cytotoxicity. Examination of T cells from a number of species showed restricted reactivity for CE9.1, recognizing only human and chimpanzee CD4. In both human and chimpanzee MLRs, it had an IC50of about 10.0 ng/mL. Therefore, a chimpanzeein vivomodel was used to characterize CE9.1. CE9.1 caused transient decrease in the number of lymphocytes bearing the CD4 receptor starting at doses of 0.3 mg/kg in anin vivodose ranging study in one chimpanzee. This effect was reversed within approximately 7 days. In a multiple high-dose study in which 10.0 mg/kg of CE9.1 was administered at intervals of 1–3 months, there was a dramatic loss of CD4 marker with a reciprocal increase in the number of CD3+CD8−CD4−cells. The CD4 receptor was totally undetectable on these lymphocytes for 1–2 weeks, with a gradual, but complete, reversal within 4 weeks. We interpret these observations as receptor modulation because, although there was apparent loss of CD4+lymphocytes, an equivalent number of CD3+CD8−T lymphocytes were present in circulation in all four chimpanzees treated with 10.0 mg/kg CE9.1. Even at this high dose, only limited reduction of CD4+T lymphocytes was observed in these animals. These observations are in sharp contrast to what has been reported in rodents or in human clinical studies using other IgG1 mAbs to human CD4. CD8 counts, although variable, remained unaffected by CE9.1 treatment. No adverse events were observed following administration of CE9.1 to chimpanzees, and there was no detectable host immune responses to the Primatized MAb.

Idiopathic multicentric Castleman disease (iMCD) is a poorly-understood hematologic disorder involving cytokine-induced polyclonal lymphoproliferation, systemic inflammation, and potentially-fatal multi-organ failure. Though the etiology of iMCD is unknown, interleukin-6 (IL-6) is an established disease driver in approximately one-third of patients. Anti-IL-6 therapy, siltuximab, is the only FDA-approved treatment. Few options exist for siltuximab non-responders, and no validated tests are available to predict likelihood of response. We procured and analyzed the largest-to-date cohort of iMCD samples, which enabled classification of iMCD into disease categories, discoveries of siltuximab response biomarkers, and identification of therapeutic targets for siltuximab non-responders. Proteomic quantification of 1,178 analytes was performed on serum of 88 iMCD patients, 60 patients with clinico-pathologically overlapping diseases(human herpesvirus-8(HHV8)-associated MCD, N=20; Hodgkin lymphoma, N=20; rheumatoid arthritis, N=20), and 44 healthy controls. Unsupervised clustering revealediMCD patients have heterogeneous serum proteomes that did not cluster with clinico-pathologically overlapping diseases. Clustering of iMCD patients identified a novel subgroup with superior responseto siltuximab, which wasvalidated using a 7-analyte panel (apolipoprotein E, amphiregulin, serum amyloid P-component,inactivatedcomplement C3b, immunoglobulin E, IL-6, erythropoietin)in an independent cohort. Enrichment analyses and immunohistochemistry identified JAK-STAT3 signaling as a candidate therapeutic targetthatcould potentially be targetedwith JAK inhibitorsin siltuximab non-responders. Ourdiscoveries demonstrate the potentialfor accelerating discoveries for rare diseases through multi-stakeholder collaboration.

Katz: Medidata Solutions: Employment. Nabel:Thermo Fisher: Patents & Royalties: royalty income. Diamond:Brigham and Women's Hospital and Harvard Medical School's Department of Medicine, Division of Genetics, Genomes2People Translational Genomics Research Program: Consultancy. Karvir:Medidata Solutions: Employment. Zhang:Medidata Solutions: Employment. Reddy:Janssen Pharmaceuticals: Employment, Equity Ownership. Guilfoyle:Janssen Pharmaceuticals: Employment, Equity Ownership. Tendler:Janssen Pharmaceuticals: Employment, Equity Ownership. van Rhee:Takeda: Consultancy; Sanofi Genzyme: Consultancy; Castleman Disease Collaborative Network: Consultancy; EUSA: Consultancy; Adicet Bio: Consultancy; Kite Pharma: Consultancy; Karyopharm Therapeutics: Consultancy. Beineke:Medidata Solutions: Employment. Oromendia:Medidata Solutions: Employment, Equity Ownership. Fajgenbaum:Janssen Pharmaceuticals: Research Funding.

Background

Prc, also known previously as Tsp, is the founding member of the carboxyl-terminal processing protease (CTP) family of PDZ domain-containing proteases that include CtpA and CtpB. The substrate-binding PDZ domain is responsible for regulating the protease activity of CTP proteases; however, the regulatory role of PDZ domain is stimulatory in Prc but inhibitory in CtpA/B. By determining a series of crystal structures of Prc in the unliganded resting state, this study presents the structural basis for PDZ-dependent activation of Prc, the results of which explain the contrasting roles of the PDZ domain in the regulation of the protease activity of CTPs.

Guilfoyle: Janssen Pharmaceuticals, Inc.: Employment. Tendler:Janssen Pharmaceuticals, Inc.: Employment, Equity Ownership. Reddy:Janssen Pharmaceuticals, Inc.: Employment, Equity Ownership. Weinblatt:Amgen: Consultancy, Research Funding; Sanofi/Regeneron: Consultancy, Research Funding; Crescendo Bioscience: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; UCB Pharmaceuticals: Consultancy, Research Funding. Bower:Merck: Honoraria; ViiV: Honoraria; Gilead: Honoraria; Janssen Pharmaceuticals: Honoraria. Masaki:Ono: Research Funding; Phizer: Research Funding; Astellas: Research Funding; Eisai: Research Funding; Kyowa Hakko Kirin: Research Funding. Beineke:Medidata Solutions: Employment, Equity Ownership. Miljovska:Medidata Solutions: Employment. Katz:Medidata Solutions: Employment. Shenoy:Medidata Solutions: Consultancy. Oromendia:Medidata Solutions: Employment, Equity Ownership. Mezey:Medidata Solutions: Consultancy. Wiser:Medidata Solutions: Employment, Equity Ownership. Benbanaste:Medidata Solutions: Employment, Equity Ownership. Lee:Medidata Solutions: Employment. Fosså:Janssen Pharmaceuticals, Inc.: Honoraria. Fajgenbaum:Janssen Pharmaceuticals, Inc.: Research Funding.

Human herpesvirus-8(HHV-8)-negative/idiopathic multicentric Castleman disease (iMCD) is a rare and poorly understood disorder diagnosed in ~1,000 individuals in the USA each year. It involves polyclonal lymphoproliferation, constitutional symptoms, systemic inflammation and an uncontrollable cytokine storm resulting in life-threatening multi-organ failure. Diagnosis and treatment can be difficult due to limited etiological understanding and heterogeneous presentation - clinical, laboratory, and histopathological abnormalities overlap with infectious, autoimmune and oncological diseases. iMCD symptoms and disease progression are largely believed to be driven by interleukin-6 (IL-6). However, approximately 66% of iMCD patients did not respond to anti-IL-6 therapy, siltuximab, the only FDA-approved iMCD therapy, in its phase II study (NCT01024036). Few treatment options exist for anti-IL-6 refractory patients because alternative driver cytokines and signaling pathways are not known. Herein we report the largest study to-date of iMCD serum proteomes with correlative anti-IL6 response data from 92 iMCD patients in disease flare (n=75 of which were collected as part of NCT01024036), in order to: (1) molecularly define iMCD, (2) identify predictors of response to anti-IL6 therapy, and (3) gain insights into the pathogenesis of iMCD. Proteomes of HHV8-positive MCD (n=20), Hodgkin lymphoma (n=20), rheumatoid arthritis (n=20) and healthy individuals (n=44) were also analyzed. Of the ~1,300 analytes measured using SomaLogic SOMAscan, 1,178 passed QC and were included in analyses. Each analyte was log2transformed and capped at the 2.5thand 97.5thpercentiles. Clinical and laboratory data collected at the time of sample draw were used to calculate disease activity following a modified CHAP scale: C-reactive protein, hemoglobin and albumin; missing performance status. Response to siltuximab was determined in NCT01024036. Data analysis was performed using the Medidata Rave Omics machine learning platform and R v3.4.4. Clustering of baseline proteomic data for iMCD patients identified six clusters that ranged in size from seven to 27 subjects. No associations with race, site, sex, age, or batch were found. Analytes identified among the strongest differentiators include cytokines, chemokines and inflammatory molecules. Interestingly, the largest cluster was associated with response to siltuximab (p<0.05; 65% (11/17) vs 19% (5/27) in all others), higher disease activity (p<0.01), and higher IL-6 levels (p<0.01). Analysis of data for the entire study population separated HHV-8-positive MCD, Hodgkin lymphoma, and rheumatoid arthritis into distinct clusters. Of note, iMCD patients did not form a single or unique cluster, reinforcing the heterogeneity of the disease, and a subset of iMCD patients demonstrated similar, but not overlapping, proteomic profiles to those of Hodgkin lymphoma. These results indicate that previously undiscovered proteomically-distinct iMCD subtypes or disease states exist, which can be used to inform treatment options. Given that iMCD may have a sudden and severe onset, predictive markers of anti-IL6 therapy response are critical for timely administration of the correct treatment. Additionally, overlapping proteomic profiles of a subset of iMCD patients with Hodgkin lymphoma provide etiological insights. More broadly, this study represents the first use of high-quality serum proteomics data to study a rare non-malignant lymphoproliferative disorder and to assist with molecularly defining a heterogeneous disease, developing candidate diagnostic biomarkers, predicting response or failure to therapy, and identifying novel candidate therapeutic targets.

Siltuximab (CNTO 328) is a chimeric, murine-human, monoclonal antibody that specifically binds human interleukin (IL)-6 with high affinity. C-reactive protein (CRP) can be a pharmacodynamic (PD) marker of IL-6 bioactivity, i.e., reductions in CRP suggest inhibition of systemic IL-6. A population mechanistic pharmacokinetic (PK)/PD model was developed to describe the relationship between siltuximab serum concentrations and CRP suppression in patients with B-cell non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), or Castleman's disease (CD). Simulation was used to support the dose selection in the CD registration study and future clinical studies.PK/PD data were obtained from a phase 1 clinical study examining multiple dosing regimens of siltuximab administered intravenously in patients with NHL, MM, or CD. Dosing regimens included siltuximab 2.8, 5.5, or 11 mg/kg every 2 weeks; 11 mg/kg every 3 weeks; or 5.5 mg/kg every week. Serial samples to determine serum concentration of siltuximab and serial CRP samples were collected following the first dose. NONMEM 7 was used to simultaneously fit a two-compartment PK model and an inhibitory indirect-response PD model to the observed data. Simulation of 1000 replications was then used to identify siltuximab dosage regimens that would maintain CRP suppression below the lower limit of quantification (LLOQ) of 1 mg/L.The mechanistic PK/PD model was able to describe the serum siltuximab and CRP concentration-time profiles. Volume of distribution and systemic clearance rate constant of siltuximab were estimated at 68.42 mL/kg and 0.0584/day, respectively. The PD parameter estimates (Kin and Kout of CRP) were 5.03 mg/L/day and 0.457/day, respectively, and were similar between the three disease types in this study. IC50was estimated at 9.73 μg/mL and was also similar between disease types. For all disease types, simulations showed that siltuximab 11 mg/kg every 3 weeks or 15 mg/kg every 4 weeks after the second dose would reduce serum CRP to below the LLOQ throughout the entire treatment period. However, lower dose intensive schedules, including a dose of 5.5 mg/kg every 2 weeks, would not reduce CRP to below the LLOQ at any time point during the treatment period.The population PK/PD modeling and simulation support using a siltuximab dose of 11 mg/kg every 3 weeks or 15 mg/kg every 4 weeks in future clinical development studies. This dosing recommendation is supported by the observed efficacy dose-response relationship in patients with CD (J Clin Oncol 2010;28:3701–8).Xie: Johnson & Johnson: Employment, Equity Ownership. Li:Johnson & Johnson: Employment, Equity Ownership. Kurzrock:Johnson & Johnson: Honoraria, Research Funding. van Rhee:Johnson & Johnson: Research Funding. Qin:Johnson & Johnson: Employment, Equity Ownership. Reddy:Johnson & Johnson: Employment, Equity Ownership. Qi:Johnson & Johnson: Employment, Equity Ownership. Davis:Johnson & Johnson: Employment, Equity Ownership. Zhou:Johnson & Johnson: Employment, Equity Ownership. Puchalski:Johnson & Johnson: Employment, Equity Ownership.

Abstract 1365

CNTO328 is a chimeric monoclonal antibody against the inflammatory cytokine, IL-6, which is currently being studied in hematologic and solid malignancies. IL-6 is reported to play a key role in the etiology and symptoms of Anemia of Cancer (AOC). Increased IL-6 during cancer associated inflammation up-regulates the hepatic production of hepcidin, which is the iron-regulatory hormone that may be responsible for most of the features of this disorder. CNTO328 treatment has previously been shown to be associated with Hb increases in Castleman's Disease (a disorder caused by deregulated IL-6 production) and Renal Cell Carcinoma (RCC). We retrospectively undertook this assessment in the RCC patients, to assess whether there was an associated decrease in hepcidin.Serum Hb, IL-6 and CRP (a surrogate for IL-6 activity) levels were prospectively studied in RCC patients selected from two 6 mg/kg CNTO328 treatment cohorts (IV Q2W or Q3W) in a phase 1/2 study. Free and total IL-6 could not be measured due to interference of the drug with assay performance which prevents accurate measurement. Serum hepcidin levels were retrospectively measured using a hepcidin C-ELISA. The change from baseline in Hb, hepcidin and CRP levels was calculated at multiple time points. The association between Hb response (defined as max Hb increase of ≥1 g/dL) and change in hepcidin and CRP levels was evaluated by Pearson Correlation Coefficient (r).38 RCC patients with a median baseline Hb of 13.2 g/dL (10.1-17.1) were studied. All patients had normal renal function throughout the study. Two patients were excluded from analysis because of blood transfusions at baseline. None of the patients studied received ESAs or blood transfusions during screening or treatment. Treatment with CNTO328 resulted in an Hb increase during study in 35 (92%) of the 38 patients starting on Day 8, with 25 (66%) achieving a max Hb increase of 31 g/dL (median 2.0; range 1.0-3.5). Hb responses were not due to tumor response, were independent of dosing schedule (Q2W vs Q3W) and were even seen in 6 (86%) out of 7 patients with a baseline Hb <12 g/dL. As expected, a marked reduction in day 8 hepcidin was noted (median decrease of 61.1%, range -90%-53.9%). A moderate correlation was found between Hb response and the Day 8 percent change in hepcidin (r = -0.56, n=19) but not between baseline hepcidin and Hb response (r = 0.056, n=21). A sustained suppression of serum CRP levels from baseline was evident in all patients and Hb responses were moderately correlated with the Day 8 absolute change in CRP (r = -0.41, n=24) and less well correlated with baseline CRP (r = 0.31, n=25) levels. Max Hb levels (median 14.9 g/dl, range 11.1-17.7) did not exceed the upper limit of normal for any patient, and no thromboembolic events were observed.CNTO328 was associated with normalization of Hb in this moderately anemic RCC population, presumably due to reduction of hepcidin via IL-6 blockade. This mechanism may provide a physiologic alternative to transfusions in AOC and warrants further evaluation with prospective mechanistic studies in more anemic patients. A possible Hb response correlation with hepcidin and CRP levels is of particular interest and will be actively pursued in prospective trials.Rijnbeek: Johnson & Johnson: Employment. Reddy:Johnson & Johnson: Employment. Qin:Johnson & Johnson: Employment. Cornfeld:Johnson & Johnson: Employment, Equity Ownership.

Abstract 4045