Effect of zinc on human IgG1 and its FcγR interactions.

In the present study, we show that histidines 310 and 435 at the CH2-CH3 interface of the Fc portion of human IgG1 can coordinate a Zn2+ and participate in the control of the CH2-CH2 interdomain opening. Structures obtained in the absence of Zn2+ have a reduced interdomain gap that likely hamper FcγR binding. This closed conformation of the Fc is stabilized by inter-CH2 domain sugar contacts. Zinc appears to counteract the sugar mediated constriction, suggesting that zinc could be an important control factor in IgG1/FcγR interactions. The results of binding studies performed in the presence of EDTA on FcγR expressing cells supports this hypothesis. When a mutated Fc fragment, in which histidines 310 and 435 have been substituted by lysines (Fc H/K), was compared with the wild-type Fc in crystallographic studies, we found that the mutations leave the interface unaltered but have a long-range effect on the CH2 interdomain separation. Moreover, these substitutions have a differential effect on the binding of IgG1 to Fcγ receptors and their functions. Interaction with the inhibitory FcγRIIB is strongly perturbed by the mutations and mutant IgG1 H/K only weakly engages this receptor. By contrast, higher affinity FcγR are mostly unaffected.

A novel subset of NK cells expressing high levels of inhibitory FcgammaRIIB modulating antibody-dependent function.

NK cells can kill antibody-coated target cells following engagement of FcgammaRIIIA, the major activating FcgammaR expressed by these cells. The presence of FcgammaRIIC (CD32C) has also been reported, but its contribution to the FcgammaR-dependent effector functions of NK cells remains debated. We demonstrate here that inhibitory FcgammaRIIB is also expressed by a small subset of CD56+/NKp46+ NK cells and can efficiently down-modulate their FcgammaR-dependent effector function. Immunofluorescence analyses of NK cells from 52 healthy donors showed the presence of CD56bright/FcgammaRII(-) (5.2%+/-3.4), CD56dim/FcgammaRII(lo/-) (94.1%+/-3.4), and CD56dim/FcgammaRIIbright (0.64%+/-0.72) cells. QRT-PCR and protein analyses performed on isolated FcgammaRIIbright NK cells indicated that FcgammaRIIB is strongly expressed by these cells but not by FcgammaRII(lo/-) cells. In addition, FcgammaRIIbright cells showed a weaker antibody-dependent degranulation when incubated with IgG-coated target cells compared with FcgammaRII(lo/-) NK cells, although a strong FcgammaRIIIA expression was detected in both cells. Furthermore, the addition of anti-FcgammaRII Fab paralleled a higher degranulation of FcgammaRIIbright NK cells, indicating a direct role for FcgammaRIIB in this down-modulating effect. Thus, it is proposed that FcgammaRIIBbright NK cells represent a new NK cell compartment able to down-modulate NK cell functions triggered by the engagement of activating FcgammaR.

A human anti-D monoclonal antibody selected for enhanced FcgammaRIII engagement clears RhD+ autologous red cells in human volunteers as efficiently as polyclonal anti-D antibodies.

A human anti-RhD immunoglobulin G1 monoclonal antibody (mAb), R297, was tested in a phase I study to assess its ability to induce the clearance of antibody-coated autologous RhD+ red blood cells (RBCs) in healthy male volunteers. The clearance potency of R297 was compared with that of a marketed human polyclonal anti-D product (Rhophylac). This mAb has been selected for its ability to strongly engage Fc-gamma receptor IIIA and to mediate a potent antibody-dependent cell cytotoxicity (ADCC) against RhD+ RBCs. Autologous RhD+ RBCs were sensitized with either Rhophylac or R297 at three different coating percentages (25, 12.5 and 6.25%), before re-infusion. This phase I study showed that the human R297 mAb promoted rapid and complete clearance of RBCs, and showed activity that was at least as potent as the human polyclonal anti-D antibody preparation. Clearance of RBCs could still be observed when the percentage of R297 used to coat the RBCs was reduced to 6.25%. Finally, none of the adverse events was severe or considered to be related to R297. Thus, R297 is a promising candidate for the prevention of allo-immunization and represents a new generation of Fc-modified monoclonal antibodies with increased FcgammaRIII binding and increased ADCC.

Chronic lymphocytic leukaemia cells are efficiently killed by an anti-CD20 monoclonal antibody selected for improved engagement of FcgammaRIIIA/CD16.

Patients with chronic lymphocytic leukaemia (CLL) treated with a combination of fludarabine, cyclophosphamide and rituximab show a high response rate. However, only a poor response is observed following rituximab monotherapy. The use of chemo-immunotherapy is often associated with haematological and infectious complications. Thus, an antibody with an enhanced ability to kill CLL cells could lead to better clinical responses to antibody monotherapy and the possibility of lowering drug doses during chemo-immunotherapy. We generated a chimeric anti-CD20 monoclonal antibody (mAb), EMAB-6, which has a low fucose content. Apoptosis and complement activities for EMAB-6 were similar to those seen for rituximab. By contrast, EMAB-6 mAb showed improved Fcgamma receptor IIIA (FcgammaRIIIA)/CD16 binding and FcgammaRIIIA-dependent effector functions. It induced a higher in vitro antibody-dependent cellular cytotoxicity against CLL cells and a higher FcgammaRIIIA-mediated interleukin-2 production by FcgammaRIIIA(+) Jurkat cells in the presence of CLL cells at both low and maximally saturating concentrations. Comparative studies between CLL and lymphoma cells coated with EMAB-6 or rituximab indicated that the difference of efficacy was more pronounced at low doses and when target cells expressed fewer CD20 molecules. Thus, EMAB-6 mAb represents a promising drug candidate for the treatment of CLL by inducing a strong cytotoxicity against tumour cells that express low CD20 levels.

[Monoclonal antibodies: technology around the clock for new therapeutic hopes]. / Anticorps monoclonaux: tours et détours technologiques pour de nouveaux espoirs thérapeutiques.

Engineering monoclonal antibodies, now widely used in the clinic, has made it possible to develop a new generation of antibodies with optimized functional properties. These antibodies should allow a significant improvement of the treatment of diseases where only few drugs are available, if any. However, the cost of treatments with monoclonal antibodies requires further improvements in production and purification technologies, and raises the question of generic antibodies. The present review summarizes some of the technological past and present challenges in the field.

Selection of a human anti-RhD monoclonal antibody for therapeutic use: impact of IgG glycosylation on activating and inhibitory Fc gamma R functions.

The substitution of plasmatic anti-RhD polyclonal antibodies by a monoclonal antibody (mAb) for preventing the hemolytic disease of the newborn (HDN) is an important issue due to supply and safety concerns. Since it has been suggested that FcgammaR are involved in the prevention of HDN, the in vitro functional properties of two anti-RhD mAbs differing through their glycosylation profiles were compared using FcgammaR-based assays to select a candidate mAb. T125(YB2/0), a low fucosylated antibody, bound strongly to both activating FcgammaRIII and inhibitory FcgammaRII, as opposed to its highly fucosylated counterpart. It also exerted a strong ADCC against RhD+ RBCs and a potent FcgammaRIIB-mediated inhibition of cytokine release. Moreover, an in vivo RhD+ red blood cells (RBCs) clearance assay showed that this antibody exhibits a RhD+ RBCs clearance as potent as polyclonal anti-RhD antibodies in NOD-SCID mice. Thus, T125(YB2/O) has been selected to be tested for the prevention of anti-RhD allo-immunization.

Polyreactive autoantibodies purified from human intravenous immunoglobulins prevent the development of experimental autoimmune diseases.

Intravenous immunoglobulins (IVIg) are therapeutic preparations of normal human polyclonal Ig G (IgG) that exert immunomodulatory effects in patients with autoimmune or systemic inflammatory diseases. Two different IgG subfractions were evaluated for their respective immunomodulatory effects in the treatment of experimental autoimmune diseases: a fraction enriched in antibodies that recognize the F(ab')(2) portion of IVIg and a fraction of natural polyreactive autoantibodies purified on a dinitrophenyl (DNP)-Affiprep immunoadsorbent. A very small fraction of IgG interacting with DNP but not with F(ab')(2) fragments expressed an increased ability to bind to self-antigens. The anti-DNP fraction, but not the anti-idiotype fraction, protected against inflammation observed in collagen-induced arthritis and experimental autoimmune encephalomyelitis in rats. Furthermore, it was able to reduce the occurrence of spontaneous diabetes mellitus in nonobese diabetic mice at lower concentrations than unfractionated IVIg. The therapeutic benefit of the anti-DNP fraction was associated with the inhibition of secretion of proinflammatory cytokines and stimulation of secretion of IL-1 receptor antagonist. Our results provide evidence that polyreactive autoantibodies play a role in the protective effect of IVIg in experimental models of autoimmune diseases in which inflammatory reactions are part of the disease process.