The clonal analysis of anticardiolipin antibodies in a single patient with primary antiphospholipid syndrome reveals an extreme antibody heterogeneity

Abstract

The mechanism underlying the prothrombotic state that characterizes the primary antiphospholipid syndrome proves to be difficult to define mainly because of the variety of the phospholipid and protein targets of antiphospholipid antibodies that have been described. Much of the debate is related to the use of polyclonal antibodies during the different antiphospholipid assays. To better describe the antiphospholipid antibodies, a strategy was designed to analyze the reactivity of each one antibody making up the polyclonal anticardiolipin activity, breaking down this reactivity at the clonal level. This was performed in a single patient with primary antiphospholipid syndrome by combining (1) the antigen-specific selection of single cells sorted by flow cytometry using structurally bilayered labeled anionic phospholipids and (2) IntroductionAntiphospholipid autoantibodies (aPLs) 1 are associated with thrombosis, recurrent fetal loss, and thrombocytopenia in patients with antiphospholipid syndrome (APS). [2][3][4] The mechanism of the characteristic prothrombotic state related to aPLs during APS is still under debate. 5 Much of it is related to the definition of the precise nature of aPL specificity, which remains unclear because of the number of possible antigenic targets discovered over the last few years. It is now accepted that plasma proteins play an important role in aPL reactivity. 6,7 The most common protein cofactors are ␤2GP1 8,9 and prothrombin. [10][11][12] Other phospholipid (PL)-binding proteins such as protein C9, protein S9, and annexin V 13 have also been found to have cofactor properties. On one hand, studies have shown that the binding of aPLs to cardiolipin (CL) is enhanced by the addition of one cofactor (␤2GP1, prothombin, protein C, protein S), which suggests that aPLs react to a complex compounded of anionic PLs and a cofactor 8,10,11 or to an epitope that becomes exposed when a protein is bound to a PL. [14][15][16] On the other hand, other studies have shown that aPLs are able to bind directly to immobilized ␤2GP1 [17][18][19] or PT 20,21 in the absence of PL. In addition, some studies have shown that anti-␤2GP1 antibodies (Abs) are primarily associated with thrombosis in APS 22-24 although other reports have involved others Abs as anti-PT 25 or lupus anticoagulant. 26 It appears that most of these difficulties are related to the use of polyclonal Abs (serum or purified immunoglobulin [Ig]) 7,8,10,11 or a few monoclonal IgM 15,27-29 or IgG 30-33 aPLs originating from different patients. We therefore considered it necessary to concentrate our attention on aPL reactivity of each Ab making up the polyclonal aPL reactivity, breaking down this reactivity at the clonal level in a single patient.The major methods currently in use for the production of human monoclonal antibodies (mAbs) are Epstein-Barr virus transformation, 34 mouse-human hybridomas, 35 or human-human hybridomas 36 and fusion of Epstein-Barr virus-transformed B lymphocytes with a malignant cell line. 37 These methods have their dr...

Receptors involved in cell activation by antiphospholipid antibodies

“…The diagnosis of APS is complicated by the polyclonal nature of the autoantibodies and the variety of target antigens. Moreover, heterogeneous antibodies may co-exist in the same patient [2]. The main antigenic target of aPLA is the plasma phospholipid binding protein β2-glycoprotein 1 (β2GP1).…”

Beta-2 glycoprotein I and its role in antiphospholipid syndrome—lessons from knockout mice

“…The model has the considerable benefit of allowing the role of this molecule to be interrogated against the complexity of the in vivo environment. In contrast to use of aPL to neutralize h 2 GPI, the model eliminates the confounding effects of marked heterogeneity in aPL antigen and epitope specificity [88]. Furthermore, null mutant mice permit experiments to strategically address questions relating to the mechanisms of action underlying the pathogenesis of APS.…”

Lepidopteran cells, an alternative for the production of recombinant antibodies?

“…The baculovirus expression system has been used for basic research to analyze the molecular basis of specificity, 21,22,41,52 autoimmunity 32,33,36,39,41,42 or the function of intact antibodies or of isolated immunoglobulin domains. 18,40,[43][44][45][46] It is very easy and fast to generate recombinant viruses expressing H or L bearing specific mutations and to determine the impact of these mutations on the specificity or activity of a given antibody.…”

“…21,22,41 This strategy has been successfully used to analyze the catalytic activity of the 6D9 monoclonal antibody and the specificity of the PAC1 antibody that binds to the integrin αII b β3 on activated platelets. 18,52 Universal expression cassettes have also been designed either to directly clone the VH and VL domains of antibodies expressed by murine cells or by human B-cells isolated from patients 26,32,33,36,39 or to convert scFv or Fab fragments selected from phage-display libraries into an intact fully human or chimeric IgG. 29,34,35,37,38,42 The aim of many of these experiments was to isolate, characterize and validate antibodies for further therapeutic applications in the treatment of human diseases such as hemolytic disease of newborn, 31 cancer 23,27,[56][57][58] and infectious diseases, with the selection of neutralizing antibodies against hemorrhagic fever viruses (e.g., hantaan, Puumala viruses), 34,35,72 human cytomegalovirus, 37 influenza virus 38 or prion protein.…”

“…Several immunoglobulin isotypes from many different species, including murine IgG, 2,3,[21][22][23][24][25] chimeric IgG, [26][27][28][29][30] human IgG, [31][32][33][34][35][36][37][38][39] IgA, [40][41][42] IgM 41 and IgE, [43][44][45] as well as pig Igs 30,45 have been successfully expressed in insect cells. These molecules are all correctly processed (signal peptide cleaved), glycosylated and assembled (H2L2) with a production rate varying from 0.75 to 100 mg/l, depending on the antibody ( …”