Purpose: Durvalumab in combination with gemcitabine/cisplatin has shown a favorable benefit-risk profile in the TOPAZ-1 study for advanced biliary tract cancers (BTC). This analysis evaluated the population pharmacokinetics (PopPK) of durvalumab, and exposure-response for efficacy and safety (ERES) of TOPAZ-1. Methods: The PopPK model for durvalumab was updated using data from 5 previously analysed studies and TOPAZ-1. Individual exposure metrics were derived from the individual empirical Bayes estimates as drivers for exposure-response (ER) analysis related to efficacy and safety. Results: Consistent with previous analyses, the durvalumab pharmacokinetics in BTC followed a 2-compartment model with time-dependent clearance. The final population parameters were: CL, 0.298 L/day; V1, 3.42 L; V2, 1.99 L; Q, 0.452 L/day; and the time dependent clearance suggests that the clearance could decrease up to 39% over the time course of treatment. There were 111 patients (3.53%) with treatment-emergent ADA positive in the pooled group of 6 studies, and the exposure was comparable for ADA positive and negative patients. Covariates had minimal clinical impact on PopPK parameters. No significant associations were found between exposure and overall survival (OS), progression-free survival (PFS), using Cox proportional analysis (CPH). Logistic regression analysis indicated no significant relationship between the exposure and relevant adverse events measures of Grade 3 and higher treatment-related AE, Grade 3 and higher treatment-related AESI (AEs of special interest), or AE leading to treatment discontinuation. Conclusions: No dose adjustment for durvalumab is needed based on PopPK and ERES analyses. The analysis supports the TOPAZ-1 regimen for patients with advanced BTC.

Objectives: Dato-DXd, a TROP2-targeting antibody-drug conjugate, has demonstrated clinically meaningful efficacy and a manageable safety profile in multiple tumour types1. A population pharmacokinetic (PopPK) model describing PK profiles of Dato-DXd and the released drug (DXd) was developed based on data from the TROPION-PanTumor01, TROPION-Lung05, and TROPION-Lung01 studies, including 644 NSCLC patients and 85 breast cancer patients2. Data from patients in TROPION-Breast01 was added to the previously developed PopPK model to examine the impact of intrinsic and extrinsic covariates on Dato-DXd and DXd PK, and to evaluate if Dato-DXd dose adjustment is needed for HR-positive, HER2-negative breast cancer patients. Methods: The previously-developed model served as a starting point for the model refinement. From the full covariate model, backward elimination was conducted to find a statistically parsimonious model. After backward elimination, prespecified covariates of interest (e.g., tumor type) were tested by including them in a step-wise addition (forward selection). The adequacy of the final model was evaluated by prediction-corrected visual prediction check (pcVPC) and non-parametric bootstrap method. The impact of covariates of interest were investigated in post-hoc analyses Results: PK data from 1081 patients, including 437 breast cancer patients (40% of the total patients), were included in the PopPK analysis dataset. Consistent with prior results, Dato DXd PK was well-characterized by a two compartmental distribution model with parallel Dato DXd linear clearance (CLlinDatoDXd) and Dato DXd nonlinear Michaelis Menten clearance (CLnonlinDatoDXd) from the central compartment. DXd PK was well-characterized by a one compartment model with first order elimination kinetics from the central compartment. The release of DXd from intact Dato-DXd was equal to the total (linear + nonlinear) elimination rate of Dato DXd. The payload:intact ratio (PIR) decreased with time both within the dosing cycle and between cycles (cycle 1 vs later cycles). The evaluated covariates of clinical interest were not associated with a clinically significant impact on Dato-DXd or DXd PK. At 6 mg/kg, the difference in Dato-DXd AUC at the steady state (cycle 3 AUC, or AUC3) between lung cancer and breast cancer patients was 1.51%. DXd AUC3 of breast cancer patients was 1.74% higher than lung cancer patients. Neither difference was considered to be clinically relevant. Conclusions: The current PopPK analysis suggested no Dato-DXd dose adjustment is needed for HR-positive, HER2-negative breast cancer patients, further justifying the Dato-DXd 6 mg/kg Q3W dose in this patient population.Citations: 1Lu, Yasong, et al. CPT: Pharmacometrics & Systems Pharmacology 13.1 (2024): 23-28 2PAGE 2024

The 18th Workshop on Recent Issues in Bioanalysis (18th WRIB) took place in San Antonio, TX, USA on May 6-10, 2024. Over 1100 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 18th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines.Moreover, in-depth workshops on "IVDR Implementation in EU & Changes for LDT in the US" and on "Harmonization of Vaccine Clinical Assays Validation" were the special features of the 18th edition.As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and Regulatory Agencies experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues.This 2024 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2024 edition of this comprehensive White Paper has been divided into three parts for editorial reasons.This publication (Part 3) covers in the Part 3A the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity and in Part 3B the Regulatory Inputs on these topics. Part 1 (Mass Spectrometry Assays and Regulated Bioanalysis/BMV) and Part 2 (Biomarkers/BAV, IVD/CDx, LBA and Cell-Based Assays) are published in volume 17 of Bioanalysis, issues 4 and 5 (2025), respectively.

A novel single-dose regimen of 300 mg tremelimumab in combination with durvalumab (STRIDE) has demonstrated a favorable benefit-risk profile in the phase 1/2 Study 22 trial (in patients with unresectable hepatocellular carcinoma, uHCC) and in the phase 3 HIMALAYA study. The current analysis evaluated the population pharmacokinetics (PopPK) of tremelimumab and durvalumab, and the exposure-response (ER) relationship for efficacy and safety of STRIDE in patients with uHCC. Previous PopPK models for tremelimumab and durvalumab were updated using data from previous studies in various cancers combined with data from Study 22 and HIMALAYA. Typical population mean parameters and associated inter- and intra-individual variability were assessed, as was the influence of covariates. Individual exposure metrics were derived from the individual empirical Bayes estimates as drivers for ER analysis related to efficacy and safety from HIMALAYA. The observed pharmacokinetics of tremelimumab in uHCC were well described by a 2-compartment model with both linear and time-dependent clearance. All identified covariates changed tremelimumab PK parameters by <25%, and thus had minimal clinical relevance; similar results were obtained from durvalumab PopPK analysis. None of tremelimumab or durvalumab exposure metrics were significantly associated with overall survival (OS), progression-free survival (PFS), or adverse events. Baseline aspartate aminotransferase and neutrophil-to-lymphocyte ratio (NLR) were associated with OS (P < .001) by the Cox proportional hazards model. No covariate was identified as a significant factor for PFS. No dose adjustment for tremelimumab or durvalumab is needed based on PopPK covariate analyses or ER analyses. Our findings support the novel STRIDE dosing regimen in patients with uHCC.

Blockade of CTLA‐4 by tremelimumab combined with anti‐PD‐L1 durvalumab and chemotherapy provided increased antitumor activity and long‐term survival benefits in first‐line metastatic non‐small cell lung cancer (mNSCLC) in the phase III POSEIDON study. We performed population pharmacokinetic modeling for tremelimumab using data from 1,605 patients across 6 studies (including POSEIDON) in multiple tumors (lung cancer, bladder cancer, malignant mesothelioma, and other solid tumors), and identified a 2‐compartment model with linear and time‐varying clearance for tremelimumab. Cox proportional hazard regression models were applied to 326 patients with mNSCLC from POSEIDON to evaluate the association between exposure metrics and efficacy end points, adjusting for baseline prognostic covariates. Improved progression‐free survival (PFS) and overall survival (OS) in the tremelimumab arm (in combination with durvalumab and chemotherapy) was associated with higher tremelimumab exposure (e.g., minimum concentration at 5th dose ( C min,dose5 ) and area under the curve at 5th dose (AUC dose5 )). However, further case‐matching analyses yielded hazard ratios for the comparison of tremelimumab‐treated patients in the C min,dose5 quartile 1 (Q1) subgroup with matched chemotherapy‐treated patients of 1.04 (95% confidence interval (CI): 0.76–1.44) for OS and 0.99 (95% CI: 0.72–1.36) for PFS, suggesting that the observed apparent exposure–response relationship might be confounded. No relationship between tremelimumab exposure and safety (grade ≥3 treatment‐emergent adverse events [AEs], AEs of special interest, or discontinuation due to AEs) was identified. These results support the consistent benefit observed with tremelimumab 75 mg every 3 weeks for up to 5 doses in combination with durvalumab and chemotherapy in POSEIDON as first‐line therapy for mNSCLC.

Background AZD7442 is a combination of extended half-life, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific neutralizing monoclonal antibodies (tixagevimab and cilgavimab). Methods This phase 1, first-in-human, randomized, double-blind, placebo-controlled, dose-escalation study evaluated AZD7442 administered intramuscularly (300 mg) or intravenously (300, 1000, or 3000 mg) in healthy adults (aged 18-55 years). The primary end point was safety and tolerability. Secondary end points included pharmacokinetics and antidrug antibodies. Results Between 18 August and 16 October 2020, a total of 60 participants were enrolled; 50 received AZD7442, and 10 received placebo. Adverse events (all of mild or moderate intensity) occurred in 26 participants (52.0%) in the AZD7442 groups and 8 (80.0%) in the placebo group. No infusion or injection site or hypersensitivity reactions occurred. Tixagevimab and cilgavimab had mean half-lives of approximately 90 days (range, 87.0-95.3 days for tixagevimab and 79.8--91.1 days for cilgavimab) and similar pharmacokinetic profiles over the 361-day study period. SARS-CoV-2-specific neutralizing antibody titers provided by AZD7442 were maintained above those in plasma from convalescent patients with coronavirus disease 2019 (COVID-19). Conclusions AZD7442 was well tolerated in healthy adults, showing a favorable safety profile across all doses. Depending on the SARS-CoV-2 variant, pharmacokinetic analyses suggest the AZD7442 could offer protection for >= 6 months against symptomatic COVID-19 after a single 300-mg intramuscular administration. Lay Summary Antibodies are proteins produced by the body in response to infections caused by microbes, including viruses. AZD7442 is a combination of 2 human antibodies, with an extended duration of effect, sourced from people who had recovered from coronavirus disease 2019 (COVID-19). These antibodies recognize a specific part (spike protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, and prevent the virus from infecting cells in the body. The current study evaluated the safety of AZD7442 in healthy volunteers. Sixty adults were given AZD7442 or placebo (salt solution) as injections into the muscle (300-mg dose) or infusions into a vein (300-3000-mg doses). The study did not find any safety issues with AZD7442, including at the highest dose. AZD7442 was measured in the blood 12 months after dosing, suggesting a long duration of protection. Following this study, AZD7442 was tested in larger clinical trials to investigate its potential in preventing and treating COVID-19. AZD7442 is currently authorized as treatment for outpatients with COVID-19 and as a preventive drug in people who may not respond well to COVID-19 vaccines and need additional protection (eg, those taking medications that dampen the immune system). A single 300-mg intramuscular AZD7442 dose (150-mg each of tixagevimab/cilgavimab) exhibited an approximate 90-day half-life, was well tolerated in healthy adults (phase 1 study), and could offer protection for >= 6 months against some severe acute respiratory syndrome coronavirus 2 variants.

Background Durvalumab, an immune checkpoint inhibitor (ICI) targeting PD-L1, has demonstrated clinical activity with or without tremelimumab, a CTLA-4 inhibitor, in Phase 3 studies in multiple tumor types.1-3 The occurrence of anti-drug antibodies (ADA) could potentially negatively impact ICI safety and efficacy.4 This analysis assessed the immunogenicity of durvalumab using pooled pan-tumor data. Methods Durvalumab immunogenicity was assessed using pooled data from 18 clinical studies of 7826 participants with lung cancer (MYSTIC, ATLANTIC, NEPTUNE, Study 6, ARCTIC, PACIFIC), hepatocellular carcinoma (HIMALAYA, Study 22), bladder cancer (DANUBE, Study 10), head and neck cancer (KESTREL, HAWK, CONDOR, EAGLE, Study 11), gastric or gastroesophageal junction adenocarcinoma (Study 21) or advanced solid tumors (Study 1108, Japan Study 2). ADA and neutralizing ADA (nAb) to durvalumab were detected using validated solution-phase bridging electrochemiluminescence immunoassays. Impact of durvalumab ADA on safety was assessed using pan-tumor data from the pool of 18 studies. Impact of ADA on durvalumab pharmacokinetics was assessed using a pooled dataset from Study 1108, PACIFIC, ATLANTIC, CASPIAN, POSEIDON, HIMALAYA, and Study 22. Due to differences in cancer settings of the studies, the impact of durvalumab ADA on efficacy was not assessed. Results The proportion of durvalumab ADA-positive participants at any visit (ADA prevalence) was 6.2% with durvalumab monotherapy (D), 6.8% with T75+D, and 7.3% with T300+D (STRIDE) (table 1). The proportion of treatment-emergent ADA–positive participants (ADA incidence) was low and similar across data pools: 2.7% with D, 3.4% with T75+D, and 2.8% with T300+D (table 1). Median ADA titers were ≤16 across all ADA categories in all pools (table 1). The proportion of transiently ADA-positive participants out of those with ADA prevalence was 32/191 with D, 22/146 with T75+D, and 3/26 with T300+D. The proportion of participants who tested positive for durvalumab nAb at any visit was 0.5% with D, 0.6% with T75+D, and 1.4% with T300+D (table 1). Incidence of infusion-related reactions was low in ADA-positive participants across treatment regimens (1% with D, and 0% with T75+D and T300+D); there was no marked impact of durvalumab ADA presence on categorical AE data (table 2). Durvalumab ADA presence did not have a clinically meaningful impact on durvalumab exposure metrics. Conclusions Although incidences of ADA vary greatly among ICI 4, analysis of this large, multi-study, pooled pan-tumor dataset demonstrates that durvalumab has a low-risk immunogenicity profile as monotherapy or in combination with tremelimumab. Immunogenicity of durvalumab appeared to have no marked impact on safety or exposure. Acknowledgements Medical writing support, under the direction of the authors, was provided by Claire Tinderholm, PhD, of CMC Connect, McCann Health Medical Communications, with funding from AstraZeneca, in accordance with Good Publications Practice (GPP3) guidelines. Trial Registration MYSTIC: NCT02453282 ATLANTIC: NCT02087423 NEPTUNE: NCT02542293 Study 6: NCT02000947 ARCTIC: NCT02352948 PACIFIC: NCT02125461 HIMALAYA: NCT03298451 Study 22: NCT02519348 DANUBE: NCT02516241 Study 10: NCT02261220 KESTREL: NCT02551159 HAWK: NCT02207530 CONDOR: NCT02319044 EAGLE: NCT02369874 Study 11: NCT02262741 Study 21: NCT02340975 Study 1108: NCT01693562 Japan Study 2: NCT01938612 CASPIAN: NCT03043872 POSEIDON: NCT03164616 References Abou-Alfa GK, Lau G, Kudo M, et al. Tremelimumab plus durvalumab in unresectable hepatocellular carcinoma. NEJM Evid. 2022. Doi: https://doi.org/10.1056/EVIDoa2100070. Oh D-Y, He AR, Qin S, et al. Durvalumab plus gemcitabine and cisplatin in advanced biliary trance cancer. NEJM Evid. 2022. Doi: https://doi.org/10.1056/EVIDoa2200015. Goldman JW, Dvorkin M, Chen Y, et al. Durvalumab, with or without tremelimumab, plus platinum-etoposide versus platinum-etoposide alone in first-line treatment of extensive-stage small-cell lung cancer (CASPIAN): updated results from a randomised, controlled, open-label, phase 3 trial. Lancet Oncol. 2021;22:51–65. Enrico D, Paci A, Chaput N, Karamouza E, Besse B. Antidrug antibodies against immune checkpoint blockers: impairment of drug efficacy or indication of immune activation? Clin Cancer Res. 2020;26:787–792. Ethics Approval The trial protocol was approved by local institutional review boards.

This Phase 1 study evaluated the absolute bioavailability, pharmacokinetics (PK), tolerability, and safety of belimumab 200 mg/mL administered subcutaneously (SC) to healthy subjects as a single dose and as multiple doses up to 240 mg. In all, 118 subjects (age range 18-55 years; body weight 51-115 kg) were enrolled. Seventy-eight subjects received a single dose of belimumab 240 mg intravenously, or 2 x 120, 1 x 240, or 1 x 200 mg SC. Forty subjects received 4 weekly injections of belimumab 2 x 120 or 1 x 200 mg SC. Randomization was stratified by weight (<75 kg vs. >= 75 kg) and injection site (abdomen vs. thigh). Following single belimumab SC doses, bioavailability was 74-82%, indicating that belimumab SC was well absorbed, and bioavailability was similar among the three SC groups. Following 4 weekly belimumab SC doses, bioavailability was similar to that following single SC administration. Four subjects had persistent positive immune responses; neutralizing antibodies in these subjects were not detected and there was no apparent impact on PK. Belimumab was generally well tolerated after single and multiple SC dosing, and 200 mg SC weekly dosing is expected to provide an exposure similar to 10 mg/kg intravenously every 28 days.

The population pharmacokinetics (PK) of belimumab were characterized in 1,603 patients with systemic lupus erythematosus receiving belimumab 1, 4, 10, or 20mg/kg doses in Phase 1-3 trials. Belimumab PK were well described with a linear two-compartment model, with clearance from the central compartment (CL). Belimumab exposure was approximately dose-proportional. The estimated population terminal half-life was 19.4 days and steady-state volume of distribution (Vss) was 5.29L for the currently approved 10mg/kg dose used in the Phase 3 trials, with an estimated CL of 215mL/day. No effects of age, sex, race, disease activity, co-medications, or baseline characteristics on belimumab PK were found to alter exposure in a manner requiring dose adjustment. An association observed between increasing baseline proteinuria and increasing CL may be clinically relevant in nephropathy with very high proteinuria levels. No evidence of target-mediated clearance was observed. Clinically relevant effects of body size (increased CL and V1 with increased body weight, and reduced V1 with increased body mass index) have been accounted for in current weight-normalized belimumab dosing.

Inhaled Bacillus anthracis spores germinate and the subsequent vegetative growth results in bacteremia and toxin production. Anthrax toxin is tripartite: the lethal factor and edema factor are enzymatic moieties, while the protective antigen (PA) binds to cell receptors and the enzymatic moieties. Antibiotics can control B. anthracis bacteremia, whereas raxibacumab binds PA and blocks lethal toxin effects. This study assessed plasma PA kinetics in rabbits following an inhaled B. anthracis spore challenge. Additionally, at 84 h post-challenge, 42% of challenged rabbits that had survived were treated with either levofloxacin/placebo or levofloxacin/raxibacumab. The profiles were modeled using a modified Gompertz/second exponential growth phase model in untreated rabbits, with added monoexponential PA elimination in treated rabbits. Shorter survival times were related to a higher plateau and a faster increase in PA levels. PA elimination half-lives were 10 and 19 h for the levofloxacin/placebo and levofloxacin/raxibacumab groups, respectively, with the difference attributable to persistent circulating PA-raxibacumab complex. PA kinetics were similar between untreated and treated rabbits, with one exception: treated rabbits had a plateau phase nearly twice as long as that for untreated rabbits. Treated rabbits that succumbed to disease had higher plateau PA levels and shorter plateau duration than surviving treated rabbits.

BACKGROUND:HGS004 is a fully human immunoglobulin (Ig) G4 monoclonal antibody against CC chemokine receptor 5 (CCR5) with robust in vitro activity against a diverse panel of CCR5-tropic human immunodeficiency virus type 1 (HIV-1) isolates.METHODS:A single-blind, randomized, placebo-controlled study was conducted in patients infected with CCR5-tropic HIV-1 to evaluate the safety, pharmacokinetics, and antiviral activity of HGS004. Sixty-three subjects were randomized into 5 dose cohorts (0.4, 2, 8, 20, and 40 mg/kg) and received a single intravenous dose of HGS004 or placebo.RESULTS:HGS004 was well tolerated, and no dose-limiting toxicities were observed. Pharmacokinetics were nonlinear across the 0.4-40-mg/kg dose range, with dose-proportional increases in maximum concentration, although the area under the curve increased more than proportionally to dose. High levels of receptor occupancy were observed for up to 28 days in the higher-dose cohorts. Plasma HIV-1 RNA reductions of >1 log(10) at day 14 were observed in 14 (54%) of 26 subjects in the 8-, 20-, and 40-mg/kg cohorts. In the 40-mg/kg cohort, 4 of 10 subjects had a >1 log(10) HIV-1 RNA reduction at day 28. Drug concentrations relative to isolate sensitivity (the ratio of the concentration at day 14 to IC(90)) predicted antiviral response on day 14.CONCLUSIONS:HGS004 is safe and well tolerated and demonstrates meaningful antiviral activity when administered to patients infected with CCR5-tropic HIV-1.

The primary therapeutic goal for the treatment of diabetes is maintenance of a long-term, near-normoglycemic condition and prevention of the onset or progression of the complications associated with the disease. Although several analogs of human insulin have been developed, the currently prescribed long-acting insulin analogs do not provide a stable basal glycemia for more than a few hours. Here, we report the development of Albulin, a long-acting insulin analog obtained by direct gene fusion of a single-chain human insulin to human serum albumin. Albulin showed an elimination t(1/2) of approximately 7 h in normoglycemic mice. In vitro pharmacodynamic profiles for Albulin characterized by receptor binding, inhibition of gluconeogenesis, induction of glucose uptake, and global regulation of gene expression in relevant cell types showed that Albulin produced similar activity profiles compared with that of recombinant human insulin. A single Albulin administration in vivo normalized blood glucose level in diabetic mice in a relatively peakless and sustained (24-h) fashion. A further reduction in glucose levels was achieved by administering a recombinant human insulin a few hours after Albulin injection in mice, indicating the potential for Albulin therapy in combination with available fast-acting insulin derivatives. In summary, Albulin displays characteristics of a potent long-acting insulin analog that can be evaluated for use as a novel insulin therapy for patients with insulin-dependent diabetes.

Bone morphogenetic proteins (BMPs), a subset of the transforming growth factor (TGF)-beta superfamily, regulate a diverse array of cellular functions during development and in the adult. BMP-9 (also known as growth and differentiation factor (GDF)-2) potently induces osteogenesis and chondrogenesis, has been implicated in the differentiation of cholinergic neurons, and may help regulate glucose metabolism. We have determined the structure of BMP-9 to 2.3 angstrom and examined the differences between our model and existing crystal structures of other BMPs, both in isolation and in complex with their receptors. TGF-beta ligands are translated as precursors, with pro-regions that generally dissociate after cleavage from the ligand, but in some cases (including GDF-8 and TGF-beta 1, -2, and -3), the pro-region remains associated after secretion from the cell and inhibits binding of the ligand to its receptor. Although the pro-region of BMP-9 remains tightly associated after secretion, we find, in several cell-based assays, that the activities of BMP-9 and BMP-9(.)pro-region complex were equivalent. Activin receptor-like kinase 1 (ALK-1), an orphan receptor in the TGF-beta family, was also identified as a potential receptor for BMP-9 based on surface plasmon resonance studies (BIAcore) and the ability of soluble ALK-1 to block the activity of BMP-9(.)pro-region complex in cell-based assays.

A coordinated effort combining bioinformatic tools with high-throughput cell-based screening assays was implemented to identify novel factors involved in T-cell biology. We generated a unique library of cDNAs encoding predicted secreted and transmembrane domain–containing proteins generated by analyzing the Human Genome Sciences cDNA database with a combination of two algorithms that predict signal peptides. Supernatants from mammalian cells transiently transfected with this library were incubated with primary T cells and T-cell lines in several high-throughput assays. Here we describe the discovery of a T cell factor, TIP (T cell immunomodulatory protein), which does not show any homology to proteins with known function. Treatment of primary human and murine T cells with TIP in vitro resulted in the secretion of IFN-γ, TNF-α, and IL-10, whereas in vivo TIP had a protective effect in a mouse acute graft- versus -host disease (GVHD) model. Therefore, combining functional genomics with high-throughput cell-based screening is a valuable and efficient approach to identifying immunomodulatory activities for novel proteins.

A coordinated functional genomics program was implemented to identify secreted polypeptides with therapeutic applications in the treatment of diabetes. Secreted factors were predicted from a diverse expressed-sequence tags (EST) database, representing >1,000 cDNA libraries, using a combination of bioinformatic algorithms. Subsequently, ∼8,000 human proteins were screened in high-throughput cell-based assays designed to monitor key physiological transitions known to be centrally involved in the physiology of type 2 diabetes. Bone morphogenetic protein-9 (BMP-9) gave a positive response in two independent assays: reducing phosphoenolpyruvate carboxykinase (PEPCK) expression in hepatocytes and activating Akt kinase in differentiated myotubes. Purified recombinant BMP-9 potently inhibited hepatic glucose production and activated expression of key enzymes of lipid metabolism. In freely fed diabetic mice, a single subcutaneous injection of BMP-9 reduced glycemia to near-normal levels, with maximal reduction observed 30 hours after treatment. BMP-9 represents the first hepatic factor shown to regulate blood glucose concentration. Using a combination of bioinformatic and high-throughput functional analyses, we have identified a factor that may be exploited for the treatment of diabetes.

DR3 is a death domain-containing receptor that is upregulated during T cell activation and whose overexpression induces apoptosis and NF-kappaB activation in cell lines. Here we show that an endothelial cell-derived TNF-like factor, TL1A, is a ligand for DR3 and decoy receptor TR6/DcR3 and that its expression is inducible by TNF and IL-1alpha. TL1A induces NF-kappaB activation and apoptosis in DR3-expressing cell lines, while TR6-Fc protein antagonizes these signaling events. Interestingly, in T cells, TL1A acts as a costimulator that increases IL-2 responsiveness and secretion of proinflammatory cytokines both in vitro and in vivo. Our data suggest that interaction of TL1A with DR3 promotes T cell expansion during an immune response, whereas TR6 has an opposing effect.