Efficacy and safety of romiplostim in refractory aplastic anaemia: a Phase II/III, multicentre, open-label study.

A previous dose-finding study has suggested that romiplostim is effective in patients with refractory aplastic anaemia (AA) and 10 µg/kg once weekly was recommended as a starting dose. In this Phase II/III, multicentre, open-label study, romiplostim was administered subcutaneously at a fixed dose of 10 µg/kg once weekly for 4 weeks (weeks 1-4) followed by weekly doses (5, 10, 15 and 20 µg/kg) titrated by platelet response for up to 52 weeks (weeks 5-52). A total of 31 patients with AA who were refractory to immunosuppressive therapy (IST) and thrombocytopenia (platelet count of ≤30 × 109 /l) were enrolled. The primary efficacy endpoint of the proportion of patients achieving any haematological (platelet, neutrophil and erythrocyte) response at week 27 was 84% [95% confidence interval (CI) 66-95%]. Trilineage response was 39% (95% CI 22-58%) at week 53. The most common treatment-related adverse events (AEs) were headache and muscle spasms (each 13%). All AEs were mild or moderate except for three patients with Grade 3 hepatic AEs; no AEs necessitated romiplostim discontinuation. Two patients developed cytogenetic abnormalities, of whom one returned to normal karyotype at last follow-up. High-dose romiplostim is effective and well tolerated in the treatment of patients with AA refractory to IST.

Serum IgA Fc effector functions in infectious disease and cancer.

Immunoglobulin (Ig) A is the most abundant antibody isotype present at mucosal surfaces and the second most abundant in human serum. In addition to preventing pathogen entry at mucosal surfaces, IgA can control and eradicate bacterial and viral infections through a variety of antibody-mediated innate effector cell mechanisms. The role of mucosal IgA in infection (e.g. neutralization) and in inflammatory homeostasis (e.g. allergy and autoimmunity) has been extensively investigated; by contrast, serum IgA is comparatively understudied. IgA binding to fragment crystallizable alpha receptor plays a dual role in the activation and inhibition of innate effector cell functions. Mounting evidence suggests that serum IgA induces potent effector functions against various bacterial and some viral infections including Neisseria meningitidis and rotavirus. Furthermore, in the era of immunotherapy, serum IgA provides an interesting alternative to classical IgG monoclonal antibodies to treat cancer and infectious pathogens. Here we discuss the role of serum IgA in infectious diseases with reference to bacterial and viral infections and the potential for IgA as a monoclonal antibody therapy.

Circulating CD89-IgA complex does not predict deterioration of kidney function in Korean patients with IgA nephropathy.

BACKGROUND: Soluble CD89 (sCD89)-IgA complex plays a key role in the pathogenesis of IgA nephropathy (IgAN). However, there is a lack of evidence supporting this complex as a good biomarker for disease progression. This study aimed to evaluate the usefulness of sCD89-IgA complex for risk stratification of IgAN. METHODS: A total of 326 patients with biopsy-proven IgAN were included. sCD89-IgA complex was measured by sandwich-enzyme-linked immunosorbent assay. The study endpoints were a 30% decline in estimated glomerular filtration rate (eGFR). RESULTS: sCD89-IgA complex levels were inversely and weakly associated with eGFR at the time of biopsy (r=-0.12, p=0.03). However, the significance between the two factors was lost in the multivariate linear regression after adjustment of clinical factors (ß=0.35, p=0.75). In a multivariate Cox model, the highest (hazard ratio [HR], 0.75; 95% confidence interval [CI], 0.35-1.61; p=0.45) and middle (HR, 0.93; 95% CI, 0.46-1.89; p=0.84) tertiles of sCD89-IgA complex levels were not associated with an increased risk of developing a 30% decrease in eGFR. Furthermore, the decline rates in eGFR did not differ between groups and C-statistics revealed that the sCD89-IgA complex were not superior to clinical factors in predicting disease progression. CONCLUSIONS: This study found no association between sCD89-IgA complex levels and disease progression in IgAN. Although sCD89 can contribute to the formation of immune complexes, our findings suggest that the sCD89-IgA level is not a good predictor of adverse outcomes and has limited clinical utility as a biomarker for risk stratification in IgAN.

Development of Romiplostim for Treatment of Primary Immune Thrombocytopenia From a Pharmacokinetic and Pharmacodynamic Perspective.

Romiplostim is a novel thrombopoiesis-stimulating peptibody consisting of a carrier Fc domain and a peptide domain that binds to the thrombopoietin receptor (TPOR) on platelets and platelet precursors. Similar to endogenous thrombopoietin, romiplostim activates the TPOR to stimulate the growth and maturation of megakaryocytes, resulting in increased production of platelets in the circulation. Binding of romiplostim to TPOR on the platelets and megakaryocytes presumably triggers subsequent internalization and degradation. Therefore, increased platelet counts following romiplostim treatment results in increased elimination of the drug. The TPOR target-mediated process is saturable, resulting in nonlinear volume of distribution and clearance of romiplostim. Therefore, target-mediated disposition plays a decreasing role in drug elimination with increasing romiplostim serum concentration. Conversely, nonspecific elimination processes such as renal clearance play an increasing role with increasing romiplostim serum concentration. Limited pharmacokinetics data demonstrated that the exposure to romiplostim was lower after multiple dose administrations than after the first dose, although large inter-subject variability was observed. Large inter- and intra-subject variability in the platelet response was also observed at a given dose. These findings suggest considerable heterogeneity of disease in patients with primary immune thrombocytopenia and support the need for individual dose adjustments based on platelet counts.

Recurrent IgA nephropathy is predicted by altered glycosylated IgA, autoantibodies and soluble CD89 complexes.

IgA nephropathy (IgAN), the most common primary glomerulonephritis worldwide, frequently leads to end-stage renal disease and kidney transplantation. However, disease recurrence often occurs after transplantation. Here we evaluated the predictive value of three markers for IgAN recurrence: the presence of galactose-deficient IgA1, IgG anti-IgA autoantibodies, and IgA-soluble (s) CD89 complexes. This was analyzed in 38 kidney transplant recipients with IgAN recurrence and compared with 22 patients transplanted for IgAN but without recurrence and with 17 healthy controls. Pre-transplantation galactose-deficient IgA1 serum levels were significantly higher in the recurrence compared with the no recurrence or control groups. IgA-IgG complexes were significantly elevated in the recurrence group. Both the recurrence and no recurrence groups had increased values of IgA-sCD89 complexes compared with healthy controls, but values were significantly lower in patients with recurrence compared with no recurrence. Areas under the receiver operating curve of the markers in pre-transplantation sera were 0.86 for galactose-deficient-IgA, 0.82 for IgA-IgG, and 0.78 for sCD89-IgA; all significant. Disease recurrence was associated with decreased serum galactose-deficient IgA1 and appearance of mesangial-galactose-deficient IgA1 deposits, whereas increased serum IgA-sCD89 complexes were associated with mesangial sCD89 deposits. Thus, galactose-deficient-IgA1, IgG autoantibodies, and IgA-sCD89 complexes are valuable biomarkers to predict disease recurrence, highlighting major pathogenic mechanisms in IgAN.

Expression Profile of Human Fc Receptor-Like 1, 2, and 4 Molecules in Peripheral Blood Mononuclear Cells of Patients with Hashimoto's Thyroiditis and Graves' Disease.

Recently identified Fc receptor-like (FCRL) molecules are new members of the immunoglobulin superfamily dominantly expressed by B cells. Although FCRL expression patterns have been studied in normal and malignant cells, their biological functions and roles remain to be clearly identified in humans. Research has particularly focused on FCRL gene polymorphisms in autoimmune diseases, however, their involvement in the pathogenesis of autoimmune diseases is an interesting field for investigation. In the present study, we have investigated the gene expression profiles of FCRL1, 2, and 4 in 2 common thyroid diseases, Hashimoto's thyroiditis (HT) and Graves' disease (GD). FCRL1, 2, and 4 expressions were determined in peripheral blood samples of 55 HT patients, 40 GD patients and equal numbers of normal subjects by quantitative real-time PCR. Our results showed downregulation of FCRL1 and upregulation of FCRL2 transcripts in both HT and GD groups compared to healthy counterparts. Overexpression of FCRL4 was observed only in GD patients compared to controls. A significant correlation was observed between all FCRL gene expression levels in HT patients. Only FCRL2 and 4 had a correlation in GD patients. In addition, FCRL1, 2, and 4 gene expressions showed no correlations with the level of anti-thyroid peroxidase antibody (anti-TPO) or anti-thyroglobulin (anti-Tg) antibody from patients' sera. In conclusion, expressions of activating or inhibitory FCRL1, 2, and 4 showed significant alterations in HT and GD patients compared to healthy subjects.

Effects of fermented soybean meal on innate immunity-related gene expressions in nursery pigs acutely challenged with lipopolysaccharides.

This experiment was to determine if replacing soybean meal with fermented soybean meal (FSBM) would reduce the innate immune response after lipopolysaccharide challenge and the changes of gene expression profiles associated with this response. Forty-eight 21 day-old pigs were housed individually and fed three diets for 15 days: CON (a diet without FSBM or spray-dried plasma protein; SDPP), PP7 (a diet with 7% SDPP), and FS10 (a diet with 10% FSBM). Pigs were fitted with a jugular vein catheters receiving lipopolysaccharide challenge (25 µg/kg body weight (BW)) on day 15. Blood was collected for 5 h at 30-min intervals to measure cortisol. Expressions of gene transcripts in total RNA from leukocytes were compared using an oligonucleotide microarray at 210 min after lipopolysaccharides injection. Cortisol of FS10 was lower (P < 0.05) than CON after lipopolysaccharides challenge. The expression levels of 17 transcripts, including cytosolic glutathione peroxidase and glutathione S-transferase A4-4 were increased (P < 0.05), whereas 23 genes including adiponectin, neonatal Fc receptor and tumor necrosis factor ligand superfamily member 5 were decreased (P < 0.05) in FS10. This study suggests that FSBM-fed pigs can modulate expression of genes related to inflammatory response and anti-oxidant activity which can be a potential reason for reduced serum cortisol.

Circulating pro-surfactant protein B as a risk biomarker for lung cancer.

BACKGROUND: Our prior studies of lung cancer suggested that a novel biomarker (pro-surfactant protein B or pro-SFTPB) might serve as a predictive marker for this disease. We aimed to determine the potential use of pro-SFTPB for distinguishing lung cancer cases from matched controls as a risk marker. METHODS: Study subjects were drawn from the longitudinal Physicians' Health Study (PHS). Cases (n = 188) included individuals who were cancer-free at study enrollment but developed lung cancer during follow-up. Controls (n = 337) were subjects who did not develop lung cancer. Cases and controls were matched on date of study enrollment, age at enrollment, and smoking status and amount. Baseline plasma samples drawn at enrollment were analyzed for pro-SFTPB using ELISA to detect differences in protein expression levels for cases and controls. RESULTS: Pro-SFTPB nondetectable status was significantly associated with lung cancer risk [OR = 5.88; 95% confidence interval (CI) 1.24-27.48]. Among subjects with detectable levels of the protein, increasing plasma concentration of pro-SFTPB was associated with higher lung cancer risk (OR = 1.41 per unit increase in log pro-SFTPB; 95% CI 1.08-1.84). CONCLUSION: These results suggest a nonlinear, J-shaped association between plasma pro-SFTPB levels and lung cancer risk, with both nondetectable and higher levels of the marker being associated with lung cancer. IMPACT: These results show promise of a risk marker that could contribute to predicting risk for lung cancer development and to narrowing the high-risk population for low-dose computed tomography screening.

Pharmacokinetic and pharmacodynamic modeling of romiplostim in animals.

PURPOSE: Romiplostim is a novel thrombopoiesis-stimulating peptibody that targets the thrombopoietin c-Mpl receptor, resulting in increased platelet production. The pharmacodynamic-mediated disposition (PDMDD) and its stimulatory effect on platelet production in Sprague-Dawley rats, rhesus monkeys, and cynomolgus monkeys following IV bolus and SC administration at various dose levels were determined. METHODS: The pharmacokinetic (PK) profile was described by a PDMDD model that accounts for romiplostim binding to the c-Mpl receptor. The PD model contained a series of aging compartments for precursor cells in bone marrow and platelets. The stimulatory function was described by an on-and-off function operating on the fractional receptor occupancy (RO). The threshold effect, RO(thr), and K(D) parameters were determinants of drug potency, whereas S(max) reflected drug efficacy. RESULTS: The model implicated that receptor-mediated clearance was negligible. RO(thr) estimated occupancies were 0.288, 0.385, 0.771 for rats, rhesus, and cynomolgus monkeys, respectively. The analogous estimated values of K(D) were 4.05, 2320, and 429 ng/mL, implying that romiplostim was much more potent in rats, which was confirmed by a dose-response (ratio of peak platelet count to baseline) relationship. CONCLUSIONS: The model adequately described romiplostim serum concentrations and platelet counts in rats, rhesus monkeys, and cynomolgus monkeys, and quantified linear clearance, PDMDD, and potency of romiplostim.

Enhanced levels of both the membrane-bound and soluble forms of IgM Fc receptor (FcµR) in patients with chronic lymphocytic leukemia.

The association of an IgM-Fc receptor (FcµR) with chronic lymphocytic leukemia (CLL) was suggested more than 30 years ago, but its authenticity has never been formally addressed. We examined the expression of the recently identified FcµR by B and T cells in CLL patients using receptor-specific monoclonal antibodies. CLL B cells (CD5(+)/CD19(+)) expressed much higher levels of FcµR on their cell surface than B cells from healthy donors. Such enhanced expression was more evident in immunoglobulin heavy chain variable region (IGHV)-mutated, CD38(-) or early Rai-stage CLL than in IGHV-unmutated, CD38(+), or advanced Rai-stage CLL. Intriguingly, surface FcµR levels also were significantly elevated in the non-CLL B cells (CD5(-)/CD19(+)) and T cells (CD5(+)/CD19(-)), especially in IGHV-mutated CLL. CLL patients also had high serum titers of FcµR compared with healthy donors, and serum FcµR levels correlated significantly with circulating lymphocyte numbers but not with the IGHV mutation status or Rai stage. The serum FcµR was resolved as an ∼ 40-kDa protein, distinct from the cell surface FcµR of ∼ 60 kDa, and it was produced by both CLL B and non-CLL B cells. Mass spectrometric analysis revealed that the serum FcµR is a soluble form of the receptor encoded by an alternatively spliced FcµR transcript. These findings indicate enhanced levels of both membrane-bound and soluble forms of FcµR in CLL patients.

Both IgA nephropathy and alcoholic cirrhosis feature abnormally glycosylated IgA1 and soluble CD89-IgA and IgG-IgA complexes: common mechanisms for distinct diseases.

Abnormalities of IgA arise in alcoholic cirrhosis, including mesangial IgA deposits with possible development of secondary IgA nephropathy (IgAN). Since little is known about circulating immune complexes in cases of secondary IgAN, we analyzed IgA-associated parameters in the serum of 32 patients with compensated or advanced alcoholic cirrhosis. Galactose deficiency and decreased sialylation of IgA1, as well as increased amounts of abnormally glycosylated polymeric IgA1, were detected in the serum of patients with advanced alcoholic cirrhosis. Moreover, aberrant IgA1 formed complexes with IgG and soluble CD89 in serum of patients with advanced alcoholic cirrhosis, similar to those found in primary IgAN. The IgA1 of alcoholic cirrhosis, however, had a modified N-glycosylation, not found in primary IgAN. In patients with alcoholic cirrhosis and IgAN, IgA deposits were associated with CD71 overexpression in mesangial areas, suggesting that CD71 might be involved in deposit formation. Although the IgA1 found in alcoholic cirrhosis bound more extensively to human mesangial cells than control IgA1, they differ from primary IgAN by not inducing mesangial cell proliferation. Thus, abnormally glycosylated IgA1 and soluble CD89-IgA and IgA-IgG complexes, features of primary IgAN, are also present in alcoholic cirrhosis. Hence, common mechanisms appear to be shared by diseases of distinct origins, indicating that common environmental factors may influence the development of IgAN.

Investigation of the pharmacokinetics of romiplostim in rodents with a focus on the clearance mechanism.

PURPOSE: Romiplostim, a treatment for adults with immune thrombocytopenia (ITP), is a novel thrombopoietin mimetic agent with a similar mechanism of action as thrombopoietin with no sequence homology. Structurally, it is a peptibody containing thrombopoietin mimetic peptides and the Fc portion of human IgG(1). We investigated romiplostim pharmacokinetics in rodents with a focus on the clearance mechanism. METHODS: Studies with appropriate controls were conducted in four models: FcRn knockout mice, thrombocytopenic mice, splenectomized rats, and bilateral nephrectomized rats. Catabolic breakdown of romiplostim was investigated in normal rats. The primary analytical method determines the intact/active romiplostim concentration, and the secondary method determines the sum of romiplostim and its catabolic degradants. RESULTS: FcRn interaction results in prolonged exposure. Platelets are involved in the target-mediated elimination, a saturable process and more prominent at low dose. Splenectomy does not affect the romiplostim pharmacokinetics in rats, an observation not unexpected. Nephrectomy in rats results in a greater increase of romiplostim exposure at a higher romiplostim dose, a nonlinearity likely due to saturation of competing pathway. Catabolism plays a major role in romiplostim elimination. CONCLUSION: Romiplostim clearance involves multiple mechanisms, including a nonlinear pathway. Consequently, the relative contribution of different mechanisms appears to be dose dependent.

Tuning the serum persistence of human serum albumin domain III:diabody fusion proteins.

The long circulation persistence of human serum albumin (HSA) is enabled by its domain III (DIII) interaction with the neonatal Fc receptor (FcRn). A protein scaffold based on HSA DIII was designed. To modify the serum half life of the scaffold, residues H535, H510, and H464 were individually mutated to alanine. HSA DIII wild type (WT) and variants were fused to the anti-carcinoembryonic antigen (CEA) T84.66 diabody (Db), radiolabeled with (124)I and injected into xenografted athymic mice for serial PET/CT imaging. All proteins targeted the CEA-positive tumor. The mean residence times (MRT) of the proteins, calculated by quantifying blood activity from the PET images, were: Db-DIII WT (56.7 h), H535A (25 h), H510A (20 h), H464A (17 h), compared with Db (2.9 h). Biodistribution confirmed the order of blood clearance from slow to fast: Db-DIII WT > H535A > H510A > H464A > Db with 4.0, 2.0, 1.8, 1.6 and 0.08 %ID/g of remaining blood activity at 51 h, respectively. This study demonstrates that attenuating the DIII-FcRn interaction provides a way of controlling the pharmacokinetics of the entire Db-DIII fusion protein without compromising tumor targeting. H464 appears to be most crucial for FcRn binding (greatest reduction in MRT), followed by H510 and H535. By mutating the DIII scaffold, we can dial serum kinetics for imaging or therapy applications.

Ligand-binding mass spectrometry to study biotransformation of fusion protein drugs and guide immunoassay development: strategic approach and application to peptibodies targeting the thrombopoietin receptor.

The knowledge of in vivo biotransformation (e.g., proteolysis) of protein therapeutic candidates reveals structural liabilities that impact stability. This information aids the development and confirmation of ligand-binding assays with the required specificity for bioactive moieties (including intact molecule and metabolites) for appropriate PK profiling. Furthermore, the information can be used for re-engineering of constructs to remove in vivo liabilities in order to design the most stable candidates. We have developed a strategic approach of ligand-binding mass spectrometry (LBMS) to study biotransformation of fusion proteins of peptides fused to human Fc ("peptibodies") using anti-human Fc immunoaffinity capture followed by tiered mass spectrometric interrogation. LBMS offers the combined power of selectivity of ligand capture with the specificity and detailed molecular-level information of mass spectrometry. In this paper, we demonstrate the preclinical application of LBMS to three peptibodies, AMG531 (romiplostim), AMG195(linear), and AMG195(loop), that target the thrombopoietin receptor. The data show that ligand capture offers excellent sample cleanup and concentration of intact peptibodies and metabolites for subsequent query by matrix-assisted laser desorption ionization time-of-flight mass spectrometry for identification of in vivo proteolytic points. Additional higher-resolution analysis by nanoscale liquid chromatography interfaced with electrospray ionization mass spectrometry is required for identification of heterogeneous metabolites. Five proteolytic points are accurately identified for AMG531 and two for AMG195(linear), while AMG195(loop) is the most stable construct in rats. We recommend the use of LBMS to assess biotransformation and in vivo stability during early preclinical phase development for all novel fusion proteins.

Immature neutrophils mediate tumor cell killing via IgA but not IgG Fc receptors.

Antitumor Abs are promising therapeutics for cancer. Currently, most Ab-based therapies focus on IgG Ab, which interact with IgG FcR (FcgammaR) on effector cells. In this study, we examined human and mouse neutrophil-mediated tumor cell lysis via targeting the IgA FcR, FcalphaRI (CD89), in more detail. FcalphaRI was the most effective FcR in triggering tumor cell killing, and initiated enhanced migration of neutrophils into tumor colonies. Importantly, immature neutrophils that are mobilized from the bone marrow upon G-CSF treatment efficiently triggered tumor cell lysis via FcalphaRI, but proved incapable of initiating tumor cell killing via FcgammaR. This may provide a rationale for the disappointing results observed in some earlier clinical trials in which patients were treated with G-CSF and antitumor Ab-targeting FcgammaR.

Fc alpha RI/CD89 circulates in human serum covalently linked to IgA in a polymeric state.

The FcR for IgA CD89/FcalphaRI, is a type I receptor glycoprotein, expressed on myeloid cells, with important immune effector functions. In vitro CD89 can be released from CD89-expressing cells upon activation. Little information is available on the existence of this soluble molecule in vivo. Using specific and sensitive ELISA techniques (detection limit 50 pg/ml), we were not able to detect circulating CD89 in human sera. However, using Western blotting, a 30-kDa soluble CD89 molecule was demonstrated in both serum and plasma. Moreover, using a specific semiquantitative dot-blot system, we found CD89 in all human sera tested (mean concentration 1900 ng/ml). Size fractionation of human serum using gel filtration chromatography showed that the CD89 molecule was predominantly present in larger molecular mass fractions. Direct complexes between IgA and CD89 were demonstrated by anti-IgA affinity purification, and when analyzed under nonreducing conditions appeared to be covalently linked. Size fractionation of affinity-purified IgA showed the presence of soluble CD89 only in the high molecular mass fractions of IgA, but not in monomeric IgA. High molecular mass complexes of CD89-IgA could be distinguished from J chain containing dimeric IgA. These data show that CD89 circulates in complex with IgA, and suggest that CD89 might contribute to the formation of polymeric serum IgA.

Enhanced expression of Fc alpha receptor I on blood phagocytes of patients with gram-negative bacteremia is associated with tyrosine phosphorylation of the FcR-gamma subunit.

Sepsis caused by gram-negative bacteria is a common finding having high incidence and mortality. Fc alpha RI (CD89), a receptor for immunoglobulin A (IgA), has been shown to mediate bacterial phagocytosis, which might play a role in the pathogenesis of sepsis. In this study the expression and function of Fc alpha RI were analyzed on blood monocytes and neutrophils of patients with bacteremia. We found a marked increased in expression of the alpha- and gamma-subunits of the Fc alpha RI on both types of cells in patients with gram-negative bacteremia, but not in patients with gram-positive bacteremia. This increase was independent of serum IgA levels. Fc alpha RI M(r) was lower on cells from gram-negative patients than on cells from controls (50-65 kDa versus 55-75 kDa), despite a similar 32-kDa backbone, indicating altered glycosylation. Increased levels of Fc alpha RI on blood phagocytes correlated with enhanced serum IL-6 levels, but not with IFN gamma or TNF-alpha. FcR-gamma chain associated with Fc alpha RI was phosphorylated in patients neutrophils, indicating functional engagement of this receptor during gram-negative sepsis. Increased expression and activation of Fc alpha RI-gamma 2 complexes following gram-negative infections suggests its involvement in host defense against bacteria.

Establishment and characterisation of ovine blood monocyte-derived cell lines.

Studies of the important functions in host defense assured by macrophages, both as functional elements and as potential targets for intracellular pathogens, are often inhibited by the lack of a source of large numbers of uniform, well-characterised cells. To address this lack for ovine studies, we have established cell lines from spontaneously-proliferating adherent mononuclear cells from sheep blood. Eight such lines which have been continuously cultured for over 400 passages have phagocytic activities and cytochemical characteristics indicating that they retain the nature of mononuclear phagocytes. They display typical functional membrane proteins such as CD14, Fc receptors and MHC class II. Such cells can facilitate in vitro studies of pathogen-monocyte interactions and can furnish copious amounts of cells for transfer experiments.