Ian J. Harmer

Rearrangement of the human heavy chain variable region gene V3-23 in transgenic mice generates antibodies reactive with a range of antigens on the basis of VHCDR3 and residues intrinsic to the heavy chain variable region.

To formulate a ‘logic’ for how a single immunoglobulin variable region gene generates antibodies with different antigen specificity and polyreactivity, we analysed chimeric antibodies produced in transgenic mice carrying the germ‐line human V3–23 gene, multiple diversity (D) and joining (J) gene segments. Hybridomas producing antibodies encoded by the V3–23 gene in combination with different mouse Vκ genes were obtained by fusion of splenocytes from transgenic mice. All antibodies had human μ‐chains and mouse light chains, were multimeric in structure and expressed the human V3–23 gene. Nucleotide sequence analyses of genes encoding the heavy and light chains of 12 antibodies in relation to antigen specificity highlighted the importance of heavy chain variable region CDR3 in determining reactivity with different antigens. However, the results also suggest that non‐CDR3 sequences intrinsic to the V3–23 gene itself may be involved in, or determine, the binding of the chimeric antibodies to some of the antigens tested in the current study.

Definition of novel GP6 polymorphisms and major difference in haplotype frequencies between populations by a combination of in-depth exon resequencing and genotyping with tag single nucleotide polymorphisms

Summary.  Background: Common genetic variants of cell surface receptors contribute to differences in functional responses and disease susceptibility. We have previously shown that single nucleotide polymorphisms (SNPs) in platelet glycoprotein VI (GP6) determine the extent of response to agonist. In addition, SNPs in the GP6 gene have been proposed as risk factors for coronary artery disease. Methods: To completely characterize genetic variation in the GP6 gene we generated a high‐resolution SNP map by sequencing the promoter, exons and consensus splice sequences in 94 non‐related Caucasoids. In addition, we sequenced DNA encoding the ligand‐binding domains of GP6 from non‐human primates to determine the level of evolutionary conservation. Results: Eighteen SNPs were identified, six of which encoded amino acid substitutions in the mature form of the protein. The single non‐synonymous SNP identified in the exons encoding the ligand‐binding domains, encoding for a 103Leu > Val substitution, resulted in reduced ligand binding. Two common protein isoforms were confirmed in Caucasoid with frequencies of 0.82 and 0.15. Variation at the GP6 locus was characterized further by determining SNP frequency in over 2000 individuals from different ethnic backgrounds. Conclusions: The SNPs were polymorphic in all populations studied although significant differences in allele frequencies were observed. Twelve additional GP6 protein isoforms were identified from the genotyping results and, despite extensive variation in GP6, the sequence of the ligand‐binding domains is conserved. Sequences from non‐human primates confirmed this observation. These data provide valuable information for the optimal selection of genetic variants for use in future association studies.

The FITC-anti-FITC system is a sensitive alternative to biotin-streptavidin in ELISA.

In a model ELISA system alkaline phosphatase (AP) absorbed onto microtitre wells was employed as the target antigen. The antigen was then reacted with a monoclonal antibody to AP either unlabelled or labelled with (a) FITC and (b) biotin. The bound anti-AP was then detected with horseradish peroxidase (HRP) conjugates of polyvalent anti-mouse IgG, and the FITC and biotin anti-AP conjugates with HRP conjugates of a monoclonal anti-FITC or streptavidin respectively. The FITC-anti-FITC system proved to be of similar sensitivity to the biotin-streptavidin system detecting 140 amol compared to 350 amol of antigen. Both these methods of antigen detection were superior to the anti-mouse IgG reagent (2100 amol). In contrast to biotinylated antibodies, FITC-labelled antibodies are highly coloured and fluorescent. These features aid the preparation, purification and characterisation of conjugates. In addition, very low non-specific binding is encountered with enzyme conjugates of anti-FITC and this may confer an advantage over enzyme conjugates of avidin/streptavidin reagents.

Chimaeric monoclonal antibodies encoded by the human VH26 gene from naive transgenic mice display a wide range of antigen binding specificities

To elucidate the molecular basis for the ability of antibodies encoded by the human VH26 heavy‐chain variable region gene to react with diverse antigens, we have generated 34 hybridomas secreting chimaeric monoclonal antibodies (human μ heavy chain/mouse light chains) from transgenic mice. The transgenic mice carry an immunoglobulin minilocus containing the human VH26 gene, human DH and JH gene segments, and genes encoding the human Cμ region. The minilocus in these animals undergoes functional rearrangement resulting in the production of chimaeric antibodies in which human μ heavy chains utilizing the VH26 gene are paired with mouse k or Λ light chains. The hybridomas described in this study were generated from naïve animals and were selected solely on the basis of human μ‐chain expression. The antibodies described have covalently attached mouse light chains and are multimeric in structure. The binding properties of the antibodies were examined using a panel of both self‐ and foreign antigens using enzyme‐linked immunosorbent assays, agglutination or radio‐immunoprecipitation assays and immunofluorescence. Chimaeric immunoglobulins from 21 of the 34 hybridoma clones (61·7%) reacted with one or more antigens, of which 13 (38·2%) reacted with more than two antigens. These studies demonstrate that the VH26 gene, in combination with human DH and JH gene segments, and mouse light‐chain genes, is able to encode antibodies with a wide range of ligand‐binding specificities. These findings have important implications in the context of the possible origins of autoantibodies encoded by VH26 which may play a role in the pathogenesis of a number of autoimmune conditions.

Phospholipid binding specificities and idiotype expression of hybridoma derived monoclonal autoantibodies from splenic cells of patients with systemic lupus erythematosus.

OBJECTIVE--To analyse the phospholipid binding specificity, functional characteristics and idiotype expression of human hybridoma derived monoclonal autoantibodies (MAb) derived from the spleens of two patients with active systemic lupus erythematosus (SLE). METHODS--The IgM MAbs binding to phospholipids were generated from spleen cells of two patients (RSP and RT) with active SLE and their specificity of binding to neutral phospholipids (phosphatidyl ethanolamine, phosphatidyl choline, platelet activating factor, sphingomyelin) and negatively charged phospholipids (phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, phosphatidyl inositol and cardiolipin (CL)) analysed. Binding specificity of cross reactive antibodies (those binding to CL and DNA) was confirmed by fluid phase inhibition assays. Lupus anticoagulant activity and beta 2-glycoprotein-1 (beta 2 GP-1) requirement for the antigen binding of these MAbs were detected using the modified dilute Russells viper venom test and modified anti-CL enzyme linked immunosorbent assay (ELISA), respectively. Expression of idiotypes (Id) Id RT-84 and Id H3 was analysed using rabbit polyclonal and murine monoclonal anti-idiotype reagents, respectively. RESULTS--Twelve clones from the patient RSP and eight clones from patient RT were reactive with phospholipids. Marked differences in phospholipid binding of these MAbs were noted, varying from truly polyreactive (RT-72 bound to most phospholipids tested) to monospecific (RT-84 bound only to CL). Furthermore, MAbs RT-84, RT-129, and RSP-57 had lupus anticoagulant activity and required beta 2 GP-1 for CL binding. It was found that 75% of phospholipid binding antibodies from RT clones expressed RT-84 Id, but none from RSP clones did so, and that Id H3 was expressed only by the RT-83 antibody. CONCLUSION--These results show that human anti-phospholipid MAbs are heterogeneous with respect to phospholipid binding, functional characteristics, and Id expression.

The role of antigen in the selection of the human V3‐23 immunoglobulin heavy chain variable region gene

The immune system mounts antibody responses using few of the available immunoglobulin variable region (IgV) genes with some, such as the V3‐23 heavy chain gene, regularly over‐represented in responses to many antigens. The reasons for the over‐representation of some V genes have not been established; the process could be either stochastic or selective. We demonstrated previously that the V3‐23 gene, which is over‐represented in the primary B lymphocyte repertoire in humans, encodes antibodies with differing antigen‐binding reactivities in transgenic mice that express the human V3‐23 gene. The aim of the current study was to assess if V3‐23 gene over‐representation is stochastic or could be influenced by antigen exposure. Transgenic mice were immunized with human IgG‐Fc (hIgG‐Fc), bovine collagen type II (bCII) or tetanus toxoid (TT), and hybridomas secreting human µ chain‐containing  antibodies  generated.  These  were  tested  for  binding  to  the  immunogens  and  a  panel  of self‐ and exogenous antigens. In hybridomas derived from hIgG‐Fc‐immunized mice, 53% secreted antibodies specific for hIgG‐Fc. A similar proportion (54%) of hybridomas from bCII‐immunized mice secreted antibody that bound to collagen. By contrast, only 21% of hybridomas from mice immunized with TT bound to tetanus toxoid. Intriguingly, chimaeric antibodies generated from mice immunized with bCII or TT were mainly polyreactive, similar to antibodies generated from naive transgenic mice. However, hybridomas generated from mice immunized with hIgG‐Fc were mainly specific, reacting exclusively with hIgG‐Fc. These results suggest that selection and eventual expansion of B lymphocytes expressing the V3‐23 gene are likely to be determined by exposure to self‐ and/or environmental antigens.

Molecular characterization of the variable domains of an αIIbβ3‐specific immunoglobulin M κ platelet cold agglutinin in a follicular lymphoma patient with treatment refractory autoimmune thrombocytopenia: idiotypic overlap between αIIbβ3 integrin antibodies

BACKGROUND: Cold hemagglutinins are generally immunoglobulin M (IgM) κ antibodies reactive at temperatures below 37°C and if of high titer may cause hemolysis. Platelet (PLT) cold agglutinins (CAs) are rare and poorly characterized. A detailed molecular characterization of the variable domains of a pathologic, PLT‐reactive, CA is presented.

Platelet integrin α 2 I-domain specific antibodies produced via domain specific DNA vaccination combined with variable gene phage display

Antibodies are a powerful tool for structure/function studies of platelet proteins. However, classic immunisation frequently elicits antibody responses against domains of minor functional interest. Robust strategies to generate antibodies against defined domains would be of significant interest in post-genome research. In this study, we report a new strategy using a combination of DNA vaccination and V gene phage display that allows the rapid generation of domain specific single-chain Fv antibodies (scFvs). This system was validated using the I-domain of alpha2 integrin as a model. The alpha2beta1 integrin, which is expressed on many cell types, is the dominant collagen attachment receptor on platelets, functioning in close interplay with the collagen signalling receptor glycoprotein VI. A novel set of I-domain specific antibodies was obtained by a DNA vaccination/V gene repertoire cloning approach. Mice were first immunised with a DNA vaccine in which the alpha2 I-domain is expressed as a fusion protein with fragment C of tetanus toxoid (FrC-TT). Then the heavy and kappa light chain variable gene repertoires were rescued from immune splenocytes using antibody phage display. A total of four alpha2 I-domain specific scFvs were isolated by selection on recombinant I-domain or native platelet alpha2beta1 integrin. Characterisation of the scFvs indicated that they recognised distinct epitopes that had profound differences in accessibility between native and recombinant I-domain. Our data suggest DNA immunisation and phage display represent versatile alternatives to protein immunisation and hybridoma-fusion techniques for the isolation of recombinant antibody reagents. This approach will be particularly useful for the generation of domain or splice-variant specific antibodies that recognise native protein.

P48 Molecular Characterisation of the Variable Domains of an ΑIIbβ3 Specific IgM κ Platelet Cold Agglutinin in a Follicular Lymphoma Patient with Treatment Refractory Thrombocytopenia

Cold agglutinins (CA) were originally defined as IgM κ antibodies capable of agglutinating erythrocytes at temperatures below 37°C and cold haemagglutinin disease may ensue in patients with high titre cold agglutinins at low environmental temperature. Platelet CA are rare and have been poorly characterised. We present the first molecular characterisation of a pathological, platelet reactive, CA. This antibody was identified in a patient with follicular lymphoma (FL) who had profound, refractory, thrombocytopenia that resolved in response to Rituximab. Serological and molecular studies of this IgM κ that recognises αIIbβ3 shows that binding is temperature dependent but independent of the integrin activation state. The VH1‐02 and Vλ3‐20 encoded variable domains of the heavy and light chains have characteristics typical of a low affinity autoantibody. Maldi‐Tof analysis of the platelet reactive IgM showed unequivocally that the IgM was derived from the FL clone. Interestingly we observed idiotypic cross‐reactivity between this IgM paraprotein, several other FL idiotypes and our β3‐leucine 33 (HPA‐1a) specific human recombinant antibody. Our results suggest that binding of the latter and other αIIbβ3 specific monoclonal antibodies induces a subtle conformational change that enhances platelet binding of the patient IgM.