2003; baxter - Supplements - Haematologica

IV International Workshop on Immune Tolerance in Hemophilia53induction, animal models have been developed.A first model, the FVIII-knockout mouse, 9 inwhich there is no circulating FVIII molecule,allowed us to show an immune response uponinjection of human FVIII without the possibleinterference due to the cross-reactivity betweenmouse and human FVIII. A second model, calledthe severe combined immunodeficiency (SCID)mouse 9,10 lacks the enzyme required to rearrangeDNA for immunoglobulin synthesis and antigenreceptor T-cells, can be used to reconstitute animmune response de novo. For example, it wasshown that by reconstituting mice with cellsfrom hemophilia A patients, followed by FVIIIinjection, it is possible to reproduce a humananti-FVIII immune response in this strain ofmice.Based on the results of idiotypic interactionsobtained in healthy donors, hemophilia Apatients and animal models, passive and activetherapies can be envisaged. Passive therapy couldbe helpful in the case of emergency and involvethe infusion of anti-Id Abs in patients withinhibitors. Such anti-Id Abs are present in intravenousimmunoglobulin (IVIg) solution 11 orcould be obtained from enriched or purifiedpreparations from a healthy donor plasma pool,since it has been demonstrated that plasma fromhealthy donors contains significant amounts ofanti-Id Abs. In this context, one can perhaps considerthat the efficacy of IVIg infusion in patientswith auto-immune inhibitors 12 is related to itscontent of anti-Ids Abs, even though anti-Id levelsare usually found to be very low. Anti-Id Abenrichedpreparations might represent a suitablealternative for passive immunotherapy. Activeimmunization with idiotype has already beensuccessfully attempted in man in the case oftumor-specific non-Hodgkin’s lymphomas. 13 Wealso observed that classical desensitizationtowards FVIII inhibitors through infusion of highdoses FVIII induced an increased production ofanti-Id Abs, a result also achievable at the lowestcost by injection of autologous FVIII/anti-FVIIIAbs complexes. 8The key question is to define what should beinjected to enhance a specific anti-Id immuneresponse. This question is particularly importantwith FVIII, with regard to the size of the FVIIImolecule and the number of recognized epitopes.In an attempt to address this major question, theanti-FVIII Abs repertoire is being explored at theclonal level. In our laboratory, we started producinghigh-affinity human monoclonal Abstowards FVIII using a method involving cellimmortalization with the Epstein-Barr virus. 14,15Peripheral blood lymphocytes are used to this endto ensure that the Abs obtained are part of thepatient’s antibody repertoire. Such monoclonalantibodies have then been sequenced and relevantidiotypes identified.Thus, Bo2C11, a human monoclonal antibodyto FVIII was injected into Balb/c mice to produceanti-Id mAbs. Eight clones were selected on theRestoration of FVIII activityAnti-Id mouse mAb conc. (µg/mL)Figure 1. The figure illustrates the capacity of anti-Id mousemonoclonal Abs 14C12 and 10E9 to neutralize the inhibitingactivity of the human monoclonal anti-FVIII in a chromogenicfunctional assay. At an anti-Id Abs/anti-FVIII Absmolar ratio from 10/1 to 100/1, for 14C12 and 10E9,respectively, 100% of the FVIII coagulation activity isrestored.basis of strong recognition of Bo2C11 insolubilizedon a ELISA plate; 2 of such clones (14C12and 10E9) also inhibited Bo2C11 binding toinsolubilized FVIII in ELISA. More interestingly,in a chromogenic assay 14C12 and 10E9 neutralizedup to 100% of Bo2C11 anti-FVIII activityat molar ratios varying from 10/1 to 100/1, asshown in Figure 1. Bo2C11 is representative ofantibodies inhibiting the binding of FVIII tophospholipids. We, therefore, tested the capacityof the two anti-Id mAbs to neutralize the anti-FVIII activity of purified polyclonal antibodiesisolated from the plasma of the Bo2C11 patient.14C12 and 10E9 neutralized 70% and 20% ofthe anti-FVIII activity of the polyclonal antibodies,respectively.Based on these data indicating that anti-Id Abscould be of high specificity and affinity we used14C12 in a FVIII knockout mouse model, in anattempt to restore hemostasis after successiveinjections of FVIII and Bo2C11. Once again,administration of anti-Id Abs was able to neutralizethe anti-FVIII inhibiting activity and tofully restore hemostasis.These data demonstrate that anti-Ids havefunctional properties that would be useful in caseof emergency situations for neutralizing anti-FVIII antibodies. It also indicates that increasingthe production of such anti-Ids by active immunizationcould be of interest in the regulation ofthe anti-FVIII immune response. To this latterend, further experiments in animal modelshaematologica vol. 88(supplement n. 12):september 2003