Sylvie Jorieux

Specificity and affinity of human Fcγ receptors and their polymorphic variants for human IgG subclasses

Distinct genes encode 6 human receptors for IgG (hFcgammaRs), 3 of which have 2 or 3 polymorphic variants. The specificity and affinity of individual hFcgammaRs for the 4 human IgG subclasses is unknown. This information is critical for antibody-based immunotherapy which has been increasingly used in the clinics. We investigated the binding of polyclonal and monoclonal IgG1, IgG2, IgG3, and IgG4 to FcgammaRI; FcgammaRIIA, IIB, and IIC; FcgammaRIIIA and IIIB; and all known polymorphic variants. Wild-type and low-fucosylated IgG1 anti-CD20 and anti-RhD mAbs were also examined. We found that (1) IgG1 and IgG3 bind to all hFcgammaRs; (2) IgG2 bind not only to FcgammaRIIA(H131), but also, with a lower affinity, to FcgammaRIIA(R131) and FcgammaRIIIA(V158); (3) IgG4 bind to FcgammaRI, FcgammaRIIA, IIB and IIC and FcgammaRIIIA(V158); and (4) the inhibitory receptor FcgammaRIIB has a lower affinity for IgG1, IgG2, and IgG3 than all other hFcgammaRs. We also identified parameters that determine the specificity and affinity of hFcgammaRs for IgG subclasses. These results document how hFcgammaR specificity and affinity may account for the biological activities of antibodies. They therefore highlight the role of specific hFcgammaRs in the therapeutic and pathogenic effects of antibodies in disease.

Chronic lymphocytic leukaemia cells are efficiently killed by an anti‐CD20 monoclonal antibody selected for improved engagement of FcγRIIIA/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 Fcγ receptor IIIA (FcγRIIIA)/CD16 binding and FcγRIIIA‐dependent effector functions. It induced a higher in vitro antibody‐dependent cellular cytotoxicity against CLL cells and a higher FcγRIIIA‐mediated interleukin‐2 production by FcγRIIIA+ 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.

Biological effect of desmopressin in eight patients with type 2n (‘Normandy’) von Willebrand disease

Summary It is generally thought that the plasma increase in factor VIII (FVIII) after desmopressin (dDAVP) infusion is related to the plasma increase in von Willebrand factor (vWF), which is the plasma carrier for FVIII. The aim of this study was to evaluate the FVIII and vWF responses in patients with type 2N vWD, characterized by the mild FVIII deficiency related to markedly decreased affinity of vWF for FVIII. At different times after one intravenous dose of dDAVP (0.3 or 0.4 μg/kg) we measured the FVIII coagulant activity, FVIII antigen, vWF antigen and ristocetin cofactor activity, in eight patients with either Arg91Gln or Arg53Trp amino acid substitution in mature vWF. In all the patients, whatever their mutation, the dDAVP infusion resulted in a 2.3. ± 0.7 ‐fold increase of vWF and a variable rise (9.5 ± 7.7 times) of FVIII, whereas the vWF capacity to bind FVIII was not improved. The FVIII response was more transient than vWF response, and FVIII half disappearance time was evaluated to the approximately 3h. The data indicate that the stabilizing effect of vWF on FVIII is not responsible for the FVIII increase induced by dDAVP. The clinical implication of this study is that, in the 2N vWD patients, dDAVP may be a useful prophylactic or curative treatment when the test dose has been shown to be effective to reach a haemostatic FVIII level.

Evidence for a von Willebrand factor defect in factor VIII binding in three members of a family previously misdiagnosed mild haemophilia A and haemophilia A carriers: consequences for therapy and genetic counselling

A plasma von Willebrand factor (vWf) defect limited to its failure to bind factor VIII (FVIII) was previously characterized in a woman with FVIII deficiency and normal primary haemostasis. By using in vitro tests we found a similar pattern in three siblings of another family previously thought to be affected with mild haemophilia A. Furthermore, a decrease in vWf ability to bind FVIII was found in the parents and the brother of the three patients. This decrease was consistent with heterozygous expression of a recessive vWf gene abnormality. FVIII deficiency was corrected by infusion with a vWf concentrate almost devoid of FVIII coagulant activity. FVIII recovery and half‐life thus obtained showed that this treatment was more effective than a FVIII infusion performed by way of comparison. These results indicate that this vWf defect may account for FVIII deficiency in patients without the usual laboratory and clinical features of von Willebrands disease. Changes in therapy and genetic counselling following the new diagnosis in this family emphasize the need to search for such a vWf defect in patients in whom FVIII deficiency is not obviously X‐linked.

Identification of two point mutations in the von Willebrand factor gene of three families with the ‘Normandy’variant of von Willebrand disease

Summary Plasma von Willebrand factor (vWf) is a multidomain multimerized glycoprotein which has a dual role in haemostasis: it promotes platelet adhesion to subendothelium and is the carrier of blood coagulation factor VIII (FVIII). We previously characterized a functional defect of vWf, limited to its ability to bind FVIII, in two families whose affected members have the same phenotype that mimics mild haemophilia A and was tentatively named von Willebrands disease (vWD) ‘Normandy’. A homozygous point mutation C T converting Thr 28 to Met in mature vWf subunit was identified in one of these patients who was born of third‐cousin parents. In the present studies we report two unrelated new cases of vWD ‘Normandy’and characterize, using the analysis of the vWf gene intron 40 region containing a variable number of tandem repeats, the recessive inheritance of the disease in two affected families without known consanguinity. Exons 18–24 of the vWf gene encoding for the first 311 amino acids of mature vWf subunit were amplified by the polymerase chain reaction method and sequenced. Two new missense mutations, both corresponding to a C T transition and predicting respectively an Arg 53 Trp and an Arg 91 Gln substitution, were characterized. The three patients from family 1 were homozygous for the first‐mentioned mutation while the patient from family 3 was homozygous for the second. The patient from family 2 was found a compound heterozygote for the two mutations. None of the two point mutations reported, both destroying a MspI restriction site, could be detected in DNA from 50 normal controls screened by restriction endonuclease analysis. Our data show that different mutations may be found in patients with the ‘Normandy’phenotype. The mutations characterized so far are all localized on the N‐terminal region of mature vWf subunit, within or near the major FVIII binding domain, and some of them occur within the epitope of monoclonal antibodies inhibiting the vWf/FVIII interaction. These observations suggest a causal relationship between these mutations and the vWD ‘Normandy’phenotype.

Two novel mutations, Q1053H and C1060R, located in the D3 domain of von Willebrand factor, are responsible for decreased FVIII-binding capacity

Summary. In type 2N von Willebrand disease (VWD), von Willebrand factor (VWF) is characterized by a markedly decreased affinity for Factor VIII (FVIII), and the mutations responsible are essentially located in the D′ domain of VWF. We report the identification, in seven unrelated French families, of two novel type 2N VWD mutations, Q1053H and C1060R (Gln290His and Cys297Arg in mature VWF sequence), in exon 24 of the VWF gene. These missense mutations have been identified in the heterozygous, homozygous or hemizygous states. Using site‐directed mutagenesis and transient expression in COS‐7 cells, we showed that both mutations, although located in the D3 domain of VWF, outside the tryptic fragment containing the FVIII domain, dramatically decrease the binding of VWF to FVIII. In contrast, the R924Q substitution, which was identified in a patient who was heterozygous for C1060R, was shown to be a polymorphism.

Substitution of Arg527 and Arg531 in factor VIII associated with mild haemophilia A: characterization in terms of subunit interaction and cofactor function

The functional defect caused by substitution of Arg527 (→ Trp) and Arg531 (→ Gly, His) in factor VIII (FVIII), was explored by employing FVIII derived from patient plasma and recombinant FVIII variants. Mutation of these residues is associated with mild haemophilia A. For both FVIII‐R527W and FVIII‐R531H, activity was lower than antigen, indicating a functional defect for both variants. In contrast to FVIII‐R527W, the amount of FVIII‐R531H heterodimer present in plasma was reduced compared to heavy and light chain levels. Factor X (FX) activation experiments employing recombinant FVIII‐R531G revealed that the activated FVIII‐R531G heterotrimer was less stable than normal FVIIIa, apparently due to rapid dissociation of the A2 domain. These findings suggest that Arg531 is involved in maintaining the stability of both the heterodimer and the activated FVIII heterotrimer. Recombinant FVIII‐R527W displayed reduced stimulation of FX activation, suggesting a defect in interaction with factor IXa (FIXa). The contribution of Arg527 in the interaction with FIXa was supported by the observation that FVIII‐derived synthetic peptide Tyr511‐Leu530 was able to inhibit FX activation and that this inhibition could be overcome by addition of increasing concentrations of FIXa. Furthermore, in the three‐dimensional FVIII model residues Val517‐Arg527 are located near the FIXa binding site Ser558‐Gln565. Therefore we propose that Arg527 is part of an extended FIXa binding site, comprising residues Ser558‐Gln565 and Val517‐Arg527.

Selection of IgG Variants with Increased FcRn Binding Using Random and Directed Mutagenesis: Impact on Effector Functions

Despite the reasonably long half-life of immunoglogulin G (IgGs), market pressure for higher patient convenience while conserving efficacy continues to drive IgG half-life improvement. IgG half-life is dependent on the neonatal Fc receptor (FcRn), which among other functions, protects IgG from catabolism. FcRn binds the Fc domain of IgG at an acidic pH ensuring that endocytosed IgG will not be degraded in lysosomal compartments and will then be released into the bloodstream. Consistent with this mechanism of action, several Fc-engineered IgG with increased FcRn affinity and conserved pH dependency were designed and resulted in longer half-life in vivo in human FcRn-transgenic mice (hFcRn), cynomolgus monkeys, and recently in healthy humans. These IgG variants were usually obtained by in silico approaches or directed mutagenesis in the FcRn-binding site. Using random mutagenesis, combined with a pH-dependent phage display selection process, we isolated IgG variants with improved FcRn-binding, which exhibited longer in vivo half-life in hFcRn mice. Interestingly, many mutations enhancing Fc/FcRn interaction were located at a distance from the FcRn-binding site validating our random molecular approach. Directed mutagenesis was then applied to generate new variants to further characterize our IgG variants and the effect of the mutations selected. Since these mutations are distributed over the whole Fc sequence, binding to other Fc effectors, such as complement C1q and FcγRs, was dramatically modified, even by mutations distant from these effectors’ binding sites. Hence, we obtained numerous IgG variants with increased FcRn-binding and different binding patterns to other Fc effectors, including variants without any effector function, providing distinct “fit-for-purpose” Fc molecules. We therefore provide evidence that half-life and effector functions should be optimized simultaneously as mutations can have unexpected effects on all Fc receptors that are critical for IgG therapeutic efficacy.

Combined glyco- and protein-Fc engineering simultaneously enhance cytotoxicity and half-life of a therapeutic antibody

While glyco-engineered monoclonal antibodies (mAbs) with improved antibody-dependent cell-mediated cytotoxicity (ADCC) are reaching the market, extensive efforts have also been made to improve their pharmacokinetic properties to generate biologically superior molecules. Most therapeutic mAbs are human or humanized IgG molecules whose half-life is dependent on the neonatal Fc receptor FcRn. FcRn reduces IgG catabolism by binding to the Fc domain of endocytosed IgG in acidic lysosomal compartments, allowing them to be recycled into the blood. Fc-engineered mAbs with increased FcRn affinity resulted in longer in vivo half-life in animal models, but also in healthy humans. These Fc-engineered mAbs were obtained by alanine scanning, directed mutagenesis or in silico approach of the FcRn binding site. In our approach, we applied a random mutagenesis technology (MutaGenTM) to generate mutations evenly distributed over the whole Fc sequence of human IgG1. IgG variants with improved FcRn-binding were then isolated from these Fc-libraries using a pH-dependent phage display selection process. Two successive rounds of mutagenesis and selection were performed to identify several mutations that dramatically improve FcRn binding. Notably, many of these mutations were unpredictable by rational design as they were located distantly from the FcRn binding site, validating our random molecular approach. When produced on the EMABling® platform allowing effector function increase, our IgG variants retained both higher ADCC and higher FcRn binding. Moreover, these IgG variants exhibited longer half-life in human FcRn transgenic mice. These results clearly demonstrate that glyco-engineering to improve cytotoxicity and protein-engineering to increase half-life can be combined to further optimize therapeutic mAbs.

In vitro Evaluation of a Very‐High‐Purity, Solvent/Detergent‐Treated, Von Willebrand Factor Concentrate

Abstract. A very highly purified von Willebrand factor (vWF) concentrate was analyzed in order to evaluate its suitability for the treatment of von Willebrands disease (vWD). The functional activity of vWF assessed by its ristocetin cofactor activity (vWF:RCo) correlated with the level of vWF antigen (vWF:Ag), with the vWF:RCo/vWF:Ag ratios ranging from 0.56 to 1.02, and the specific activity being always > 50 IU vWF:RCo/mg protein. Electrophoretic analysis showed a normal pattern of high, intermediate and low‐molecular‐weight multimers of vWF. The biological activity of vWF was also evaluated by studying its ability to bind to collagen and to platelet receptors in the presence of either ristocetin or thrombin. Furthermore, these functional activities of vWF were confirmed by the capacity of this concentrate to induce platelet adhesion to collagen in a perfusion system. Moreover, the vWF present in this preparation was able to bind factor VIII. All these in vitro data suggest that this preparation is likely to be effective in the treatment of vWD patients.