Maternofetal transplacental transport of recombinant IgG antibodies lacking effector functions.

The neonatal Fc receptor (FcRn) directs the transfer of maternal immunoglobulin G (IgG) antibodies across the placenta and thus provides the fetus and newborn with passive protective humoral immunity. Pathogenic maternal IgG antibodies will also be delivered via the placenta and can cause alloimmunity, which may be lethal. A novel strategy to control pathogenic antibodies would be administration of a nondestructive IgG antibody blocking antigen binding while retaining binding to FcRn. We report on 2 human IgG3 antibodies with a hinge deletion and a C131S point mutation (IgG3ΔHinge) that eliminate complement activation and binding to all classical Fcγ receptors (FcγRs) and to C1q while binding to FcRn is retained. Additionally, 1 of the antibodies has a single point mutation in the Fc (R435H) at the binding site for FcRn (IgG3ΔHinge:R435H). We compared transplacental transport with wild-type IgG1 and IgG3, and found transport across trophoblast-derived BeWo cells and ex vivo placenta perfusions with hierarchies as follows: IgG3ΔHinge:R435H>wild-type IgG1≥IgG3ΔHinge and IgG3ΔHinge:R435H=wild-type IgG1=wild-type IgG3>>>IgG3ΔHinge, respectively. Collectively, IgG3ΔHinge:R435H was transported efficiently from the maternal to the fetal placental compartment. Thus, IgG3ΔHinge:R435H may be a good candidate for transplacental delivery of a nondestructive antibody to the fetus to combat pathogenic antibodies.

Pneumatic tube transport of blood samples affects global hemostasis and platelet function assays.

INTRODUCTION: Pneumatic tube systems (PTS) are frequently used for rapid and cost-effective transportation of blood samples to the clinical laboratory. The impact of PTS transport on platelet function measured by the Multiplate system and global hemostasis measured by the TEG 5000 was evaluated. METHODS: Paired samples from healthy adult individuals were obtained at two study sites: Rigshospitalet (RH) and Nordsjaellands Hospital (NOH). One sample was transported by PTS and one manually (non-PTS). Platelet function was assessed by platelet aggregation (Multiplate) and global hemostasis was assessed by a variety of thrombelastography (TEG) assays. Multiplate (n = 39) and TEG (n = 32) analysis was performed at site RH, and Multiplate (n = 28) analysis was performed at site NOH. RESULTS: A significant higher agonist-induced platelet aggregation was found for PTS samples compared to manual transport at site NOH (P < .02, all agonists). No significant difference was found at site RH (P > .05, all agonists). For Kaolin TEG, samples transported by PTS showed a significant lower R-time and higher Angle (P < .001). No significant differences in MA and LY30 was found (P > .05). ACT of RapidTEG was significantly reduced (P = .001) and MA of Functional Fibrinogen TEG was significantly increased (P < .001) after PTS transport. No significant impact of PTS was observed for TEG assays with heparinase (P > .05). CONCLUSIONS: Depending on the type of PTS, transportation by PTS affected platelet aggregation measured by Multiplate. Furthermore, PTS alters TEG parameters possibly reflecting coagulation factors. Clinical laboratories should evaluate the effect of the local PTS on Multiplate and TEG results.

Recombinant human immunoglobulin (Ig)A1 and IgA2 anti-D used for detection of IgA deficiency and anti-IgA.

BACKGROUND: To avoid anaphylactic reactions, immunoglobulin (Ig)A-deficient patients with anti-IgA should be transfused with IgA-deficient blood components. There is a need for fast and robust assays for demonstration of IgA deficiency and for detection of anti-IgA. STUDY DESIGN AND METHODS: Recombinant human IgA1 and IgA2 anti-D molecules were constructed, expressed in Chinese hamster ovary cells, and purified. These antibodies were used to sensitize group O D+ red blood cells (RBCs) for use as indicator cells, either in the format of a passive hemagglutination inhibition assay for detection of IgA deficiency or in a passive hemagglutination assay for detection of anti-IgA. Both assays were performed in gel card. RESULTS: The sensitivity for IgA detection was adjusted to approximately 100 ng per mL. The assay for demonstration of IgA deficiency correlated with an enzyme-linked immunosorbent assay for quantification of IgA. Anti-IgA were easily detected, and the reactivity with IgA anti-D-sensitized RBCs could be inhibited by purified IgA1 and/or IgA2 and by normal plasma containing IgA but not by IgA-deficient plasma. Anti-IgA was found in 64 percent of IgA-deficient donors with less than 3 ng of IgA per mL. CONCLUSION: The assays for detection of IgA and anti-IgA described in this article are fast and robust. Furthermore, they are applicable in all standard blood typing laboratories and are therefore well suited for immediate investigation of transfusion reactions.

In vitro assessment of recombinant, mutant immunoglobulin G anti-D devoid of hemolytic activity for treatment of ongoing hemolytic disease of the fetus and newborn.

BACKGROUND: A specific treatment for ongoing hemolytic disease of the fetus and newborn (HDFN) due to anti-D would be very attractive. One approach could be administration to the mother of nonhemolytic anti-D, which by crossing the placenta can block the binding of hemolytic maternal anti-D. STUDY DESIGN AND METHODS: Two anti-D immunoglobulin G3 (IgG3) heavy-chain mutants were expressed in Chinese hamster ovary cells. To investigate whether these anti-D IgG3 mutants could inhibit the red blood cell-destructive activity of recombinant human (rHu)IgG1 with identical antigen-binding region as well as polyclonal anti-D having multiple D epitope specificities, two assays were used, antibody-dependent cell-mediated cytotoxicity (ADCC) and a chemiluminescence (CL)-based method for detection of respiratory burst in peripheral blood monocytes. RESULTS: The two IgG3 anti-D heavy-chain mutants inhibited the ADCC and CL responses mediated by a rHuIgG1 anti-D with identical antigen-binding region as the mutant antibodies, as well as the destructive activity mediated by a polyclonal anti-D. CONCLUSION: The use of nonhemolytic anti-D may be an effective countermeasure against hemolysis in HDFN due to anti-D.

A novel antibody-dependent cellular cytotoxicity mechanism involved in defense against malaria requires costimulation of monocytes FcgammaRII and FcgammaRIII.

Clinical experiments have shown that the Ab-dependent cell-mediated inhibition of Plasmodium falciparum is a major mechanism controlling malaria parasitemia and thereby symptoms. In this study, we demonstrate that a single merozoite per monocyte (MN) is sufficient to trigger optimal antiparasitic activity. Using particulate Ag as pseudomerozoites, we show that only Ags, and no other parasite-derived factor, are required to trigger MN activation and that a single Ag is as potent as the complex combination of Ags constituting the merozoite surface. Moreover, we found that soluble Ags binding at least two Abs are as effective as the parasite at stimulating MN and that nonmalarial Ags are as efficient provided they are targeted by cytophilic Abs. Indeed, only cytophilic IgGs are potent and, in agreement with immunoepidemiological findings, IgG3 is superior to IgG1. Very low Ab concentrations (>700 pM), i.e., in the range of molecules having a hormonal effect, are effective, in contrast to Abs having a direct, neutralizing effect. Finally, Ab-dependent cell-mediated inhibition proved to require the synergistic activation of both FcgammaRIIa and FcgammaRIIIa which both distinguish it from other Ab-dependent cellular cytotoxicity and implies that all MN are not equally effective. These findings have both fundamental and practical implications, particularly for vaccine discovery.