2003; baxter - Supplements - Haematologica

20J. Voorberg et al.immunoglobulin repertoires are enriched foranti-factor VIII antibodies. A further simplificationinvolves the use of a immunoglobulin lightchain repertoire that has been amplified fromperipheral blood lymphocytes of healthy individuals.19,20 Different immunoglobulin light chainscan pair to a single heavy chain and therefore itis anticipated that VH domains derived frompatients with hemophilia A will pair with a suitablelight chain from the available non-immunerepertoire. Because of our focus on the variablepart of the heavy chain we could only determinethe characteristics of this part of immunoglobulinmolecules that bind to factor VIII. The variableheavy chain locus encodes 51 functionalvariable heavy chain segments that can be classifiedinto seven families (see Figure 1). Somefamilies, such as VH2, 5, 6 and 7 contain onlyone or a few members whereas others (VH1, VH3and VH4) harbor more then 10 members. Usageof the different families of VH germline gene segmentsin the peripheral repertoire roughly correspondsto the number of VH gene segments presentwithin a single family (see Figure 1; based ondata in ref. 21). It has been shown that the variableheavy chain gene segments contribute to theoverall fold of the VH domain of immunoglobulins.22 Fusion of a VH gene with a D and a J segment,a process that involves addition and deletionof nucleotides at the sites of junction, createsantigen-independent diversity on this initialfold. Suitably assembled IgG molecules make upthe naïve B cell repertoire. The presence of antigenthen stimulates selective outgrowth and maturationof B cells that express surface immunoglobulinsthat can interact with antigen. We haveassessed the characteristics of the variable heavychain domains of human antibodies directedagainst factor VIII. At present we have isolatedhuman antibodies directed against majorinhibitor epitopes in the A2, A3-C1 and C2domains of factor VIII. 23-27 In the following paragraphsan initial analysis of the characteristics ofthe variable heavy chain (VH) domains of theseantibodies will be presented.Characteristics of anti-A2 and anti-A3-C1antibodies obtained by phage displaySince the characteristics of anti-A2 and anti-A3antibodies display some common features we willfirst discuss this subset of human anti-factor VIIIantibodies. Four different human antibodiesdirected against the A2 domain have been isolatedof which three bind to the residues Arg 484 -Ile 508 , an immunodominant region on factorVIII. 24,27 One anti-A2 antibody, designated VK41,bound to the acidic region that follows the A2domain. Two out of four antibodies were derivedfrom VH segments DP-47 (3-23) whereas theother ones were encoded by DP-10 (1-69) andDP-58 (gene segment not yet mapped). Amongthe six human antibodies directed against theA3-C1 domains of factor VIII that have been isolated5, were directed against residues Gln 1778 -Met 1823 , a major binding site for factor VIIIinhibitors in the A3 domain of factor VIII. 5,6,25Figure 1. (A) Schematic representation of a phylogenetic tree of human variable heavy (VH) gene segments. Based on sequencehomology human VH gene segments can be divided into seven families. The number of individual gene segments present withineach family is given in brackets (adapted from ref. #16). (B) Variable heavy chain segments found in peripheral IgG-positiveB-cells. VH gene segments are classified into seven families (x-axis). On the y-axis the percentage of IgG-positive B cells expressingan IgG with a VH gene segments from one of these families is depicted (data drawn from ref. #21).haematologica vol. 88(supplement n. 12):september 2003