Ex vivo comparative immunogenicity assessment (EVCIA) to determine relative immunogenicity in chronic plaque psoriasis in participants receiving Humira® or undergoing repeated switches between Humira® and AVT02.

Immunogenicity against biologic medicines is ubiquitous, and it is traditionally measured by the final humoral response. However, the onset of a sustained immunogenic response begins at the cellular level with activation of T cells and maturation of naïve B cells into plasma cells. Ex vivo comparative immunogenicity assessment (EVCIA) of cellular immunogenicity in participants with moderate-to-severe chronic plaque psoriasis in the AVT02-GL-302 study, who received either reference product (RP) alone (non-switching arm) or switched between RP and AVT02 (switching arm) after 1:1 randomization at week 12. Peripheral blood mononuclear cells (PBMCs) were collected and cryopreserved from 28 participants at: baseline (before treatment) (week 1); pre-randomization (week 12); and week 16 and week 28 in both switching and non-switching arms. PBMCs were thawed and re-exposed to either medium alone (negative control), RP, AVT02, keyhole limpet hemocyanin (KLH) (positive control), RP+KLH, or AVT02+KLH. Samples from 10 participants (predetermined average cell viability of 75% across all timepoints) from each arm were analyzed for cytokine release after 24 hours and for Th-cell proliferation, 6 days post-seeding. Until week 28, cytokine release and Th-cell proliferation was similar at all time points in both switching and non-switching arms. Overall cellular immune response was elevated post-KLH re-exposure at all timepoints. The comparable ex vivo cellular immunogenicity between switching and non-switching arms complements the confirmation of interchangeability in the main study. Given the sensitivity of novel EVCIA, detecting cellular immunogenicity could be a potential outcome in predicting the immunogenicity of biologic medicines.

Assessment of real-life patient handling experience of AVT02 administered subcutaneously via autoinjector in patients with moderate to severe active rheumatoid arthritis: an open-label, single-arm clinical trial, then an extension phase of AVT02 administered with a prefilled syringe.

BACKGROUND: This study investigated the ability of patients, naïve to adalimumab treatment and self-injection with an autoinjector (AI), to successfully self-administer AVT02, an adalimumab biosimilar, using a custom, ergonomic AI (Alvotech hf., Reykjavik, Iceland). RESEARCH DESIGN AND METHODS: This was a single-arm, open-label study, consisting of an 8-week active period and 48-week extension phase. Patients with moderate to severe rheumatoid arthritis (RA) self-administered 40 mg AVT02 subcutaneously via AI in the active period, followed by prefilled syringe in the extension phase. The primary endpoint was the percentage of successful self-injections up to Week 8. Usability and robustness of the AI were evaluated in the active period; safety, efficacy, pharmacokinetic and immunogenicity data were assessed throughout the study. RESULTS: The AI success rate was 100%. No handling events were noted up to Week 8. Both Ctrough measurements and immunogenicity profile were in line with expectations from previous studies, with no unexpected safety signals. CONCLUSIONS: This study demonstrated that AVT02-AI can be successfully and reliably used for repeated self-injections of AVT02 by moderate to severe RA patients, despite no previous experience of adalimumab self-administration. The extension phase provides long-term efficacy and safety data for AVT02 in RA. STUDY IDENTIFIER: NCT04224194.

A multicenter, randomized, open-label, 2-arm parallel study to compare the pharmacokinetics, safety and tolerability of AVT02 administered subcutaneously via prefilled syringe or autoinjector in healthy adults.

BACKGROUND: AVT02 is an adalimumab biosimilar, with bioequivalence previously established along with clinical similarity. This study assessed the pharmacokinetic (PK) similarity of a single dose of 100 mg/mL AVT02 administered via prefilled syringe (PFS) or autoinjector (AI). RESEARCH DESIGN AND METHODS: In this open-label, 2-arm, parallel-group study, healthy adults were randomized 1:1 to receive one 40 mg (100 mg/mL) dose of AVT02 subcutaneously via PFS (N = 102) or AI (N = 105). Primary PK parameters (Cmax, AUC0-t and AUC0-inf) were evaluated up to Day 64 of the study. Secondary PK parameters, safety, tolerability and immunogenicity were also assessed. RESULTS: The 90% CIs for the ratio of geometric least squares means were contained within the pre-specified 80-125% equivalence margins for the primary PK parameters, demonstrating bioequivalence of AVT02 when administered by PFS or AI. The incidence of treatment-emergent adverse events was comparable between the two groups, with a low frequency of injection site reactions observed. Immunogenicity profiles were also similar between the two groups. CONCLUSION: Bioequivalence was demonstrated for a single dose of AVT02 administered via PFS or AI. These results will help to increase availability of devices for patients, enabling treatment choice and flexibility.

Joint analysis of PK and immunogenicity outcomes using factorization model - a powerful approach for PK similarity study.

Biological products, whether they are innovator products or biosimilars, can incite an immunogenic response ensuing in the development of anti-drug antibodies (ADA). The presence of ADA's often affects the drug clearance, resulting in an increase in the variability of pharmacokinetic (PK) analysis and challenges in the design and analysis of PK similarity studies. Immunogenic response is a complex process which may be manifested by product and non-product-related factors. Potential imbalances in non-product-related factors between treatment groups may lead to differences in antibodies formation and thus in PK outcome. The current standard statistical approaches dismiss any associations between immunogenicity and PK outcomes. However, we consider PK and immunogenicity as the two correlated outcomes of the study treatment. In this research, we propose a factorization model for the simultaneous analysis of PK parameters (normal variable after taking log-transformation) and immunogenic response subgroup (binary variable). The central principle of the factorization model is to describe the likelihood function as the product of the marginal distribution of one outcome and the conditional distribution of the second outcome given the previous one. Factorization model captures the additional information contained in the correlation between the outcomes, it is more efficient than models that ignore potential dependencies between the outcomes. In our context, factorization model accounts for variability in PK data by considering the influence of immunogenicity. Based on our simulation studies, the factorization model provides more accurate and efficient estimates of the treatment effect in the PK data by taking into account the impact of immunogenicity. These findings are supported by two PK similarity clinical studies with a highly immunogenic biologic.

A randomized, adaptive design, double-blind, 3-arm, parallel study assessing the pharmacokinetics and safety of AVT02, a high-concentration (100 mg/mL) Adalimumab biosimilar, in healthy adult subjects (ALVOPAD FIRST).

BACKGROUND: This study (ALVOPAD FIRST) assessed bioequivalence, safety, and immunogenicity of AVT02, an adalimumab biosimilar, compared with reference product adalimumab (EU- and US-approved Humira®). METHODS: Healthy subjects (N = 392) were randomized 1:1:1 to receive one 40 mg dose of AVT02, EU-reference product, or US-reference product subcutaneously. An interim analysis was planned when ~30 subjects per arm had completed the study, to optimize final sample size. The primary PK parameters were Cmax, AUC0-t, and AUC0-inf. Bioequivalence was demonstrated if the 90% confidence intervals (CI) for the ratio of geometric means for the primary pharmacokinetic (PK) parameters were all contained within the prespecified margins of 80% and 125%. Safety and immunogenicity were assessed until Day 64. RESULTS: The 90% CI for the ratio of geometric means for the primary PK parameters, based on Fisher's Combination test analysis, were all contained within the prespecified bioequivalence margins of 80% and 125%, supporting the demonstration of bioequivalence between AVT02 and both EU- and US-reference product. The safety and immunogenicity profiles were comparable across all three treatment arms. CONCLUSION: PK bioequivalence was supported between AVT02, US-licensed- and EU-approved-reference product adalimumab. Similar safety and immunogenicity were also demonstrated. TRIAL REGISTRATION: The trial is registered at ClinicalTrials.gov (CT.gov identifier: NCT03849313).

Efficacy, Safety and Immunogenicity of AVT02 Versus Originator Adalimumab in Subjects with Moderate to Severe Chronic Plaque Psoriasis: A Multicentre, Double-Blind, Randomised, Parallel Group, Active Control, Phase III Study.

BACKGROUND: AVT02 (adalimumab) is a proposed biosimilar to Humira®. AVT02 is produced at a 100 mg/mL concentration with a citrate-free formulation. OBJECTIVES: The aim of this study was to compare the efficacy, safety and immunogenicity of AVT02 versus Humira® in subjects with moderate to severe chronic plaque psoriasis. METHODS: This double-blind, randomised, parallel group, active control study of adult subjects compared (at a 1:1 ratio) AVT02 with originator adalimumab 80 mg subcutaneously in Week 1, then 40 mg every other week. At Week 16, subjects who had received originator adalimumab were re-randomised at a 1:1 ratio to continue receiving originator adalimumab, or to switch to AVT02, every other week until Week 48, with final efficacy endpoint at Week 50. Subjects who initially received AVT02 continued to receive AVT02 from Week 16 to Week 48. The primary endpoint was percentage improvement in Psoriasis Area and Severity Index (PASI) score at Week 16. Secondary efficacy endpoints included percentage improvement in PASI score at additional timepoints, change from baseline in Dermatology Life Quality Index (DLQI) score and number and percentage of subjects achieving static Physician's Global Assessment (sPGA) responses of 'clear' or 'almost clear'. Additional secondary endpoints included comparison of adverse event profiles, anti-drug antibodies and neutralising antibodies, and serum trough levels of adalimumab at steady state. RESULTS: A total of 413 subjects were randomised (205 to AVT02 and 208 to originator). The percentage improvement in PASI score at Week 16 was 91.6% for AVT02-treated subjects and 89.6% for originator adalimumab. The 90% confidence intervals for the primary endpoint were within the pre-defined equivalence margin of ±10% (90% CI - 0.76 to 5.29; 95% CI - 1.34 to 5.88), and a comparable pattern for DLQI score (11.4-point and 10.6-point improvement in AVT02-treated and originator adalimumab-treated groups, respectively) and sPGA (90.5% in both groups achieving 'clear' or 'almost clear') at Week 16 supported the assessment. Efficacy persisted through Week 50 of the study in all treatment groups, including those who switched from originator adalimumab to AVT02, for percent improvement in PASI score, quality-of-life assessment and sPGA. The safety, tolerability and immunogenicity profiles between AVT02 and originator adalimumab were similar at Week 16, and this persisted in the switched and continued groups through Week 50. CONCLUSION: Objective and subjective measures of efficacy supported the evaluation of biosimilarity between AVT02 and originator adalimumab at Week 16 and until Week 50, in switched and continued treatment groups. AVT02 was safe and well tolerated, with a safety and immunogenicity profile similar to that observed in originator adalimumab with no clinically meaningful difference between the two. CLINICAL TRIAL REGISTRATION: EudraCT: 2017-003367-35; ClinicalTrials.gov: NCT03849404.

Essential protective role of tumor necrosis factor receptor 2 in neurodegeneration.

Despite the recognized role of tumor necrosis factor (TNF) in inflammation and neuronal degeneration, anti-TNF therapeutics failed to treat neurodegenerative diseases. Animal disease models had revealed the antithetic effects of the two TNF receptors (TNFR) in the central nervous system, whereby TNFR1 has been associated with inflammatory degeneration and TNFR2 with neuroprotection. We here show the therapeutic potential of selective inhibition of TNFR1 and activation of TNFR2 by ATROSAB, a TNFR1-selective antagonistic antibody, and EHD2-scTNFR2, an agonistic TNFR2-selective TNF, respectively, in a mouse model of NMDA-induced acute neurodegeneration. Coadministration of either ATROSAB or EHD2-scTNFR2 into the magnocellular nucleus basalis significantly protected cholinergic neurons and their cortical projections against cell death, and reverted the neurodegeneration-associated memory impairment in a passive avoidance paradigm. Simultaneous blocking of TNFR1 and TNFR2 signaling, however, abrogated the therapeutic effect. Our results uncover an essential role of TNFR2 in neuroprotection. Accordingly, the therapeutic activity of ATROSAB is mediated by shifting the balance of the antithetic activity of endogenous TNF toward TNFR2, which appears essential for neuroprotection. Our data also explain earlier results showing that complete blocking of TNF activity by anti-TNF drugs was detrimental rather than protective and argue for the use of next-generation TNFR-selective TNF therapeutics as an effective approach in treating neurodegenerative diseases.

Antagonistic TNF receptor one-specific antibody (ATROSAB): receptor binding and in vitro bioactivity.

BACKGROUND: Selective inhibition of TNFR1 signaling holds the potential to greatly reduce the pro-inflammatory activity of TNF, while leaving TNFR2 untouched, thus allowing for cell survival and tissue homeostasis. ATROSAB is a humanized antagonistic anti-TNFR1 antibody developed for the treatment of inflammatory diseases. METHODOLOGY/PRINCIPAL FINDINGS: The epitope of ATROSAB resides in the N-terminal region of TNFR1 covering parts of CRD1 and CRD2. By site-directed mutagenesis, we identified Arg68 and His69 of TNFR1 as important residues for ATROSAB binding. ATROSAB inhibited binding of (125)I-labeled TNF to HT1080 in the subnanomolar range. Furthermore, ATROSAB inhibited release of IL-6 and IL-8 from HeLa and HT1080 cells, respectively, induced by TNF or lymphotoxin alpha (LTα). Different from an agonistic antibody (Htr-9), which binds to a region close to the ATROSAB epitope but elicits strong TNFR1 activation, ATROSAB showed a negligible induction of IL-6 and IL-8 production over a broad concentration range. We further verified that ATROSAB, comprising mutations within the Fc region known to abrogate complement fixation and antibody-mediated cellular effector functions, indeed lacks binding activity for C1q, FcγRI (CD64), FcγRIIB (CD32b), and FcγRIII (CD16) disabling ADCC and CDC. CONCLUSIONS/SIGNIFICANCE: The data corroborate ATROSAB's unique function as a TNFR1-selective antagonist efficiently blocking both TNF and LTα action. In agreement with recent studies of TNFR1 complex formation and activation, we suggest a model of the underlying mechanism of TNFR1 inhibition by ATROSAB.

In vivo imaging of choroidal angiogenesis using fluorescence-labeled cationic liposomes.

PURPOSE: Precise monitoring of active angiogenesis in neovascular eye diseases such as age-related macular degeneration (AMD) enables sensitive use of antiangiogenic drugs and reduces adverse side effects. So far, no in vivo imaging methods are available to specifically label active angiogenesis. Here, we report such a technique using fluorophore-labeled cationic liposomes (CL) detected with a standard clinical in vivo scanning laser ophthalmoscope (SLO). METHODS: C57Bl/6 mice underwent laser coagulations at day 0 (d0) to induce choroidal neovascularization (CNV). Liposomes labeled with Oregon green, rhodamine (Rh), or indocyanine green (ICG) were injected into the tail vein at various time points after laser coagulation, and their fluorescence was observed in vivo 60 min later using an SLO, or afterwards in choroidal flatmounts or cryosections. RESULTS: SLO detected accumulated fluorescence only in active CNV lesions with insignificant background noise. The best signal was obtained with CL-ICG. Choroidal flatmounts and cryosections of the eye confirmed the location of retained CL in CNV lesions. Neutral liposomes, in contrast, showed no accumulation. CONCLUSIONS: These results establish fluorophore-labeled CL as high affinity markers to selectively stain active CNV. This novel, non-invasive SLO imaging technique could improve risk assessment and indication for current intraocular antiangiogenic drugs in neovascular eye diseases, as well as monitor therapeutic outcomes. Labeling of angiogenic vessels using CL can be of interest not only for functional imaging in ophthalmology but also for other conditions where localization of active angiogenesis is desirable.

Comparison of lung accumulation of cationic liposomes in normal rats and LPS-treated rats.

OBJECTIVE: Cationic liposomes have been shown to target angiogenic endothelial cells in lungs and joints with evidence of chronic inflammation. We sought to determine whether cationic liposomes accumulate in acutely inflamed lung tissue. SUBJECTS, TREATMENT AND METHODS: Acute lung injury was induced by intratracheal instillation of lipopolysaccharide (LPS) in Sprague Dawley rats. The controls received saline. Following instillation, the rats were ventilated for 5 h. Four hours after LPS-instillation each rat received rhodamine-labeled, cationic liposomes intravenously. The liposomes were allowed to circulate for 1 h. Thereafter, a bronchoalveolar lavage (BAL) was done and the lungs were perfused with saline and formalin. Accumulation of liposomes was assessed by quantitative confocal microscopy and determination of rhodamine-content in lung tissue. RESULTS: LPS induced a significant increase in BAL white blood cell count (3,444 ± 1,420 vs. 1,314 ± 906*10(3)/µl) and cytokines (IL-1ß: 145.57 vs. 51.94 pg/ml; TNF-α: 3,467.5 vs. 42.1 pg/ml) as compared to controls. Cationic liposomes exhibited an accumulation up to twofold in the inflamed lung tissue as compared to healthy lungs (fluorescent pixels 2.93 vs. 1.90(%)). CONCLUSIONS: Our findings indicate that cationic liposomes accumulate in the acutely inflamed lung tissue. This uptake raises the possibility of using cationic liposomes to direct diagnostic/therapeutic agents selectively to the sites of acute inflammation in the lung.

Vascular targeting by EndoTAG-1 enhances therapeutic efficacy of conventional chemotherapy in lung and pancreatic cancer.

Cationic lipid complexed paclitaxel (EndoTAG-1) is a novel vascular targeting agent for the treatment of cancer. Here, the aim was to investigate intratumoral drug distribution after EndoTAG-1 therapy and analyze the impact of EndoTAG-1 scheduling on antitumoral efficacy. The therapeutic effect of EndoTAG-1 in combination with conventional gemcitabine or cisplatin therapy was evaluated in L3.6pl orthotopic pancreatic cancer and a subcutaneous Lewis lung (LLC-1) carcinoma model. Oregon Green paclitaxel encapsulated in cationic liposomes in combination with intravital fluorescence microscopy clearly exhibited delivery of the drug by EndoTAG-1 to the tumor endothelium, whereas Oregon Green paclitaxel dissolved in cremophor displayed an interstitial distribution pattern. The therapeutic efficacy of EndoTAG-1 was critically dependent on the application schedule with best therapeutic results using a metronomic rather than a maximum tolerated dose application sequence. The combination of EndoTAG-1 therapy and cytotoxic chemotherapy significantly enhanced antitumoral efficacy in both tumor models. Interestingly, only EndoTAG-1 in combination with gemcitabine was able to inhibit the incidence of metastasis in pancreatic cancer. In conclusion, vascular targeting tumor therapy by EndoTAG-1 combined with standard small molecular chemotherapy results in markedly enhanced antitumoral efficacy. Therefore, this combination represents a promising novel strategy for clinical cancer therapy.

Maternal HIV type 1 infection suppresses MMP-1 expression in endothelial cells of uninfected newborns: nonviral vertical transmission of HIV type 1-related effects.

HIV-1 infection is associated with vascular alterations. This is accompanied by an increased risk of cardiovascular diseases and Kaposi's sarcoma, an endothelial cell-derived tumor. We investigated the impact of maternal HIV-1 infection on phenotype and gene expression of endothelial cells in newborns. For this reason endothelial precursor cells and differentiated endothelial cells were isolated from cord blood as well as from umbilical veins and arteries of noninfected infants born to HIV-1-infected (H-group) and noninfected (Ngroup) mothers. No apparent differences in proliferation, capillary formation, and expression of endothelial cell markers were detected in these cells. Interestingly, the expression of matrix metalloproteinase was repressed significantly (X2 analysis, p < 0.002) and consistently at the RNA, the protein, and the secretory levels in the H-group as compared to the N-group. Neither treatment with zidovudine (AZT), mutations in the matrix metalloproteinase-1 (MMP-1) promoter, nor epigenetic changes in the promoter methylation pattern were responsible for the repression of MMP-1 expression in H-group endothelial cells. The reduced MMP-1 expression may contribute to the impaired cardiac function that has been observed in children of HIV-1-infected women. Most interestingly, our findings indicate that HIV-1-related effects can be transferred from mother to child in the absence of HIV-1 transmission.

EBV latent membrane protein-1 protects B cells from apoptosis by inhibition of BAX.

Latent membrane protein 1 (LMP-1) of Epstein-Barr virus (EBV) promotes tumorigenesis by inhibiting apoptosis. We show that an important antiapoptotic activity of LMP-1 is the inhibition of Bcl2-associated protein X (Bax), a potent proapoptotic protein. BAX expression was regulated by LMP-1 activation of nuclear factor kappaB (NF-kappaB) via the C-terminal activation region 1 (CTAR-1) and CTAR-2. Interestingly, p65/p50 inhibited, whereas p50/p50 increased, BAX promoter activity as demonstrated by overexpression and selective inhibition of these NF-kappaB isoforms. Electrophoretic mobility shift analysis revealed that LMP-1 activates 2 of the 3 NF-kappaB binding sites (kappaB1-kappaB3) in the BAX promoter. LMP-1 induced binding of the NF-kappaB heterodimer p65/p50 to the kappaB2 site and of the p50/p50 homodimer to the kappaB3 site. Promoter mutation analysis revealed that the kappaB2 site is necessary for inhibition of BAX promoter activity and the kappaB3 site, for its activation. However, the activation of the BAX promoter by LMP-1 was observed only in the presence of specific inhibitors of p65/p50. In all other cases, LMP-1 inhibited BAX promoter activity. Most importantly, the antiapoptotic activity of LMP-1 was considerably decreased in cells deficient for BAX. These results indicate that the inhibition of Bax may be an important antiapoptotic activity of LMP-1.

Nuclear factor-kappaB motif and interferon-alpha-stimulated response element co-operate in the activation of guanylate-binding protein-1 expression by inflammatory cytokines in endothelial cells.

The large GTPase GBP-1 (guanylate-binding protein-1) is a major IFN-gamma (interferon-gamma)-induced protein with potent anti-angiogenic activity in endothelial cells. An ISRE (IFN-alpha-stimulated response element) is necessary and sufficient for the induction of GBP-1 expression by IFN-gamma. Recently, we have shown that in vivo GBP-1 expression is strongly endothelial-cell-associated and is, in addition to IFN-gamma, also activated by interleukin-1beta and tumour necrosis factor-alpha, both in vitro and in vivo [Lubeseder-Martellato, Guenzi, Jörg, Töpolt, Naschberger, Kremmer, Zietz, Tschachler, Hutzler, Schwemmle et al. (2002) Am. J. Pathol. 161, 1749-1759; Guenzi, Töpolt, Cornali, Lubeseder-Martellato, Jörg, Matzen, Zietz, Kremmer, Nappi, Schwemmle et al. (2001) EMBO J. 20, 5568-5577]. In the present study, we identified a NF-kappaB (nuclear factor kappaB)-binding motif that, together with ISRE, is required for the induction of GBP-1 expression by interleukin-1beta and tumour necrosis factor-alpha. Deactivation of the NF-kappaB motif reduced the additive effects of combinations of these cytokines with IFN-gamma by more than 50%. Importantly, NF-kappaB p50 rather than p65 activated the GBP-1 promoter. The NF-kappaB motif and ISRE were detected in an almost identical spatial organization, as in the GBP-1 promoter, in the promoter regions of various inflammation-associated genes. Therefore both motifs may constitute a cooperative inflammatory cytokine response module that regulates GBP-1 expression. Our findings may open new perspectives for the use of NF-kappaB inhibitors to support angiogenesis in inflammatory diseases including ischaemia.

The guanylate binding protein-1 GTPase controls the invasive and angiogenic capability of endothelial cells through inhibition of MMP-1 expression.

Expression of the large GTPase guanylate binding protein-1 (GBP-1) is induced by inflammatory cytokines (ICs) in endothelial cells (ECs), and the helical domain of the molecule mediates the repression of EC proliferation by ICs. Here we show that the expression of GBP-1 and of the matrix metalloproteinase-1 (MMP-1) are inversely related in vitro and in vivo, and that GBP-1 selectively inhibits the expression of MMP-1 in ECs, but not the expression of other proteases. The GTPase activity of GBP-1 was necessary for this effect, which inhibited invasiveness and tube-forming capability of ECs in three-dimensional collagen-I matrices. A GTPase-deficient mutant (D184N-GBP-1) operated as a transdominant inhibitor of wild-type GBP-1 and rescued MMP-1 expression in the presence of ICs. Expression of D184N-GBP-1, as well as paracrine supplementation of MMP-1, restored the tube-forming capability of ECs in the presence of wild-type GBP-1. The latter finding indicated that the inhibition of capillary formation is specifically due to the repression of MMP-1 expression by GBP-1, and is not affected by the anti-proliferative activity of the helical domain of GBP-1. These findings substantiate the role of GBP-1 as a major regulator of the anti-angiogenic response of ECs to ICs.

Guanylate-binding protein-1 expression is selectively induced by inflammatory cytokines and is an activation marker of endothelial cells during inflammatory diseases.

During angiogenesis and inflammatory processes, endothelial cells acquire different activation phenotypes, whose identification may help in understanding the complex network of angiogenic and inflammatory interactions in vivo. To this goal we investigated the expression of the human guanylate-binding protein (GBP)-1 that is highly induced by inflammatory cytokines (ICs) and, therefore, may characterize IC-activated cells. Using a new rat monoclonal antibody raised against GBP-1, we show that GBP-1 is a cytoplasmic protein and that its expression in endothelial cells is selectively induced by interferon-gamma, interleukin-1alpha, interleukin-1beta, or tumor necrosis factor-alpha, but not by other cytokines, chemokines, or growth factors. Moreover, we found that GBP-1 expression is highly associated with vascular endothelial cells as confirmed by the simultaneous detection of GBP-1 and the endothelial cell-associated marker CD31 in a broad range of human tissues. Notably, GBP-1 expression was undetectable in the skin, but it was highly induced in vessels of skin diseases with a high-inflammatory component including psoriasis, adverse drug reactions, and Kaposi's sarcoma. These results indicate that GBP-1 is a novel cellular activation marker that characterizes the IC-activated phenotype of endothelial cells.