The use of IVIg in fetal and neonatal alloimmune thrombocytopenia— Principles and mechanisms

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a rare neonatal disorder that is caused by alloimmunization against platelet antigens during pregnancy. Although rare, affecting only 1 in 1000 live births, it can cause intracranial hemorrhage and other bleeding complications that can lead to miscarriage, stillbirth and life-long neurological complications. One of the gold-standard therapies for at risk pregnancies is the administration of IVIg. Although IVIg has been used in a variety of different disorders for over 40 years, its exact mechanism of action is still unknown. In FNAIT, the majority of its therapeutic effect is thought the be mediated through the neonatal Fc receptor, however other mechanisms cannot be excluded. Due to safety, supply and other concerns that are associated with IVIg use, alternative therapies that could replace IVIg are additionally being investigated. This includes the possibility of a prophylaxis regimen for FNAIT, similarly to what has been successfully used in hemolytic disease of the fetus and newborn for over 50 years.     

In vivo metabolism of diallyl disulphide in the rat: identification of two new metabolites

1. Diallyl disulphide (DADS), a compound formed from the organosulphur compounds present in garlic, is known for its anticarcinogenic effects in animal models. 2. The aim was to identify and analyse the metabolites produced in vivo after a single oral administration of 200?mg kg -1 DADS to rats. The organic sulphur metabolites present in the stomach, liver, plasma and urine were measured by gas chromatography coupled with mass spectrometry over 15 days. 3. Data indicate that DADS is absorbed and transformed into allyl mercaptan, allyl methyl sulphide, allyl methyl sulphoxide (AMSO) and allyl methyl sulphone (AMSO 2), which are detected throughout the excretion period. Overall, the highest amounts of metabolites were measured 48-72h after the DADS administration. AMSO 2 is the most abundant and persistent of these compounds. The levels of all the sulphur compounds rapidly decline within the first week after administration and disappear during the second week. Only AMSO and AMSO 2 are significantly excreted in urine. 4. These potential metabolites are thought to be active in the target tissues. Our data warrant further studies to check this hypothesis.     

Hybrid IgA2/IgG1 antibodies with tailor-made effector functions

Immunoglobulin (Ig) A and IgG are the principal immune effector molecules at mucosal surfaces and in blood, respectively. Mucosal IgA is polymeric and bound to secretory component, whereas serum IgG is monomeric. We have now produced IgA2/IgG1 hybrid antibodies that combine the properties of IgA and IgG. Antibodies with Calpha3 at the end of the IgG H chain resemble IgA and form polymers with J chain that bind the polymeric Ig receptor. Like IgG, the hybrid proteins activated complement and bound FcgammaRI and protein A. Though the hybrid proteins contained both Cgamma2 and Cgamma3, they have a short in vivo half-life. Surprisingly, this decreased half-life correlated with a higher avidity than that of IgG for murine FcRn. Interestingly, antibodies with Calpha1 replacing Cgamma1 were resistant to extremes of pH, suggesting that Calpha1 increases antibody stability. These results provide insights into engineering antibodies with novel combinations of effector functions.     

Effect of Peroxide- Versus Alkoxyl-Induced Chemical Oxidation on the Structure,Stability, Aggregation,and Function of a Therapeutic Monoclonal Antibody

Current guidelines indicate that the effects of oxidation should be included as part of forced degradation studies on protein drugs. We probed the effect of 3 commonly used oxidants, hydrogen peroxide, tert-butyl hydroperoxide, and 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH), on a therapeutic monoclonal IgG1 antibody (mAb8). Upon oxidation, mAb8 did not show noticeable changes in its secondary structure but showed minor changes in tertiary structure. Significant decrease in conformational stability was observed for all the 3 oxidized forms. Both hydrogen peroxide and tert-butyl hydroperoxide destabilized mainly the C_H2 domain, whereas AAPH destabilized the variable domain in addition to C_H2. Increased aggregation was found for AAPH-oxidized mAb8. In addition, a significant decrease in Fc receptor binding was observed for all 3 oxidized forms. Antibody dependent cell-mediated cytotoxicity, binding to target protein receptor, and cell proliferation activity were significantly reduced in the case of AAPH-oxidized mAb8. The presence of free methionine in the formulation buffer seems to alleviate the effect of oxidation. The results of this study show that the 3 oxidants differ in terms of their effects on the structure and function of mAb8 because of chemical modification of different sets of residues located in Fab versus Fc.     

Half-life extended biotherapeutics

ABSTRACT

Introduction: Many of the biotherapeutics approved or under development suffer from a short half-life necessitating frequent applications in order to maintain a therapeutic concentration over an extended period of time. The implementation of half-life extension strategies allows the generation of long-lasting therapeutics with improved pharmacokinetic and pharmacodynamic properties.

Areas covered: This review gives an overview of the different half-life extension strategies developed over the past years and their application to generate next-generation biotherapeutics. It focuses on srategies already used in approved drugs and drugs that are in clinical development. These strategies include those aimed at increasing the hydrodynamic radius of the biotherapeutic and strategies which further implement recycling by the neonatal Fc receptor (FcRn).

Expert opinion: Half-life extension strategies have become an integral part of development for many biotherapeutics. A diverse set of these strategies is available for the fine-tuning of half-life and adaption to the intended treatment modality and disease. Currently, half-life extension is dominated by strategies utilizing albumin binding or fusion, fusion to an immunoglobulin Fc? region and PEGylation. However, a variety of alternative strategies, such as fusion of flexible polypeptide chains as PEG mimetic substitute, have reached advanced stages and offer further alternatives for half-life extension.     

A standardized conversion of IgG antibody to bispecific form with inversely altered affinities for Fcγ-receptors II and III

This work aims to enhance killing of antibody-coated human tumor cells by altering the antibody's affinity for two effector-cell Fcγ-receptors (FcγR). Affinity for the activating FcγRIII is raised, affinity for the inhibitory FcγRIIB is reduced, with the ratio between the two association constants increasing >1000-fold. We use as a standard tool the Fab′γ from a monoclonal antibody specific for human FcγRIII. This Fab′γ module is bonded to an IgG antibody by a tandem thioether link running between cysteine residues in the hinge vicinity of each protein, thus forming a bispecific FabIgG construct. Simultaneously, effector function of the IgG module is adjusted by leaving its hinge open and adding negative charges. FabIgG constructs derived from the chimeric IgG1 antibody rituximab show the following properties. (1) The titer for antibody-dependent cellular cytotoxicity is enhanced by 12-100-fold, reflecting the affinity of the Fab′γ module for effector-cell FcγRIII. (2) Two functions of the construct's Fcγ, activation of complement and prolonged metabolic survival, are moderately reduced. (3) In contrast, affinities of the Fcγ for all FcγR are severely reduced, with two anticipated consequences. First, attacks by macrophages on antibody-coated cells are favored by reduced engagement of the inhibitory FcγRIIB. Second, reduced engagement of activating FcγR by the Fcγ lowers the probability of untoward crosslinkings of FcγR, which have been shown to provoke toxicity. If the Fab′γ module possesses human constant regions, the linkage strategy requires prior genetic deletion of at least one cysteine residue. With both Fab′γ and IgG modules available, FabIgG can be prepared in 35 h.     

Improvement of pharmacokinetic properties of therapeutic antibodies by antibody engineering

Monoclonal antibodies (mAbs) have become an important therapeutic option for several diseases. Since several mAbs have shown promising efficacy in clinic, the competition to develop mAbs has become severe. In efforts to gain a competitive advantage over other mAbs and provide significant benefits to patients, innovations in antibody engineering have aimed at improving the pharmacokinetic properties of mAbs. Because engineering can provide therapeutics that are more convenient, safer, and more efficacious for patients in several disease areas, it is an attractive approach to provide significant benefits to patients. Further advances in engineering mAbs to modulate their pharmacokinetics were driven by the increase of total soluble target antigen concentration that is often observed after injecting a mAb, which then requires a high dosage to antagonize. To decrease the required dosage, several antibody engineering techniques have been invented that reduce the total concentration of soluble target antigen. Here, we review the various ways that antibody engineering can improve the pharmacokinetic properties of mAbs.