Synthesis of Rh Fv phage-antibodies using VH and VL germline genes.

Antibodies to the D antigen of the Rh system use a restricted set of immunoglobulin V and J gene segments, especially VH DP50 and DP63, JH6, Vlambda DPL16 and Jlambda 2/3. These gene segments may confer a natural affinity on the antibodies for the D antigen and this hypothesis has been tested by constructing two single-chain Fv phage-antibody libraries based on the germline gene segments DP50 and DP63; structural variability was obtained by insertion of 11 amino acids in random sequence in the VHCDR3. 10 anti-D antibodies were selected from these libraries, each with a unique VHCDR3. In contrast, selections with the CcEe antigens produced antibodies reacting with the Rh polypeptide molecules but without strict blood group specificity. One of these latter DP50-based antibodies was converted into 12 different antibodies with specificity for E by replacing the original germline light chain with chains from a rearranged L chain library. The CDR1 and CDR2 sequences of the DP50-based antibodies were common to both anti-D and anti-E molecules; differentiation between D and E specificity was dependent on VHCDR3 sequences and their correct pairing with an appropriate L chain.

Host response to EBV infection in X-linked lymphoproliferative disease results from mutations in an SH2-domain encoding gene.

X-linked lymphoproliferative syndrome (XLP or Duncan disease) is characterized by extreme sensitivity to Epstein-Barr virus (EBV), resulting in a complex phenotype manifested by severe or fatal infectious mononucleosis, acquired hypogammaglobulinemia and malignant lymphoma. We have identified a gene, SH2D1A, that is mutated in XLP patients and encodes a novel protein composed of a single SH2 domain. SH2D1A is expressed in many tissues involved in the immune system. The identification of SH2D1A will allow the determination of its mechanism of action as a possible regulator of the EBV-induced immune response.

Human recombinant antibodies specific for hepatitis C virus core and envelope E2 peptides from an immune phage display library.

Hepatitis C virus (HCV) is the aetiological agent responsible for most cases of non-A non-B hepatitis. Hepatitis C is a disease of clinical importance because of its high infection rate in blood donors and its persistence as chronic infections which may lead to cirrhosis and hepatocellular carcinoma in the long term. The variability of the HCV genome has posed difficulties in serological detection and vaccine design. The recent advance in phage technology offers a means of cloning human anti-HCV antibodies of a defined specificity that may have potential therapeutic use. We now report the generation of a phage display library using the V(H) genes of a HCV-infected patient and the V(L) genes of two non-immune individuals. From this library we were able to obtain specific IgG single-chain Fvs (scFvs) that recognize viral core and envelope proteins by selection on synthetic peptides derived from the core sequence PKARRPEGRTWAQPG and the envelope E2 sequence RPIDDFDQGWGPITY. The specificity of the scFvs was demonstrated by their specific reactions with homologous peptides in ELISA and the specific blocking of scFv binding by homologous peptides, in a dose-dependent manner, in inhibition ELISA. The binding of the anticore 4c2 to homologous peptide was blocked by HCV-positive human sera in an antibody-concentration-dependent manner, suggesting that the scFv recognizes a similar if not identical epitope to those of one or more of the polyclonal antibodies present in the sera.

Molecular characteristics of anti-self antibody fragments against neutrophil cytoplasmic antigens from human V gene phage display libraries.

Recently it has been demonstrated that human antibody fragments with binding activities against self antigens can be isolated from repertoires of rearranged V genes from non-immunized humans. We have applied phage display technology to study the B cell repertoire for antibody activity against neutrophil cytoplasmic antigens. These antibodies may play an important role in Wegener's granulomatosis (WG) and related forms of vasculitides. Autoantibodies in patients with WG are directed against proteinase 3. The immunodominant antigen in other forms of vasculitis is myeloperoxidase, but the B cell response can also be directed against other neutrophil enzymes, e.g. lysozyme, human neutrophil elastase, lactoferrin and cathepsin G. We show here that anti-self reactivity against neutrophil cytoplasmic antigens can be detected in the rearranged V gene repertoire of healthy individuals and that the reactivity can be directed against structural related epitopes which are present on different neutrophil cytoplasmic antigens. The scFv with binding activities were sequenced and the V gene usage, the level of somatic mutations and the immunoserological characteristics of the antibody fragments are discussed. Further evidence is presented that antibody fragments consisting only of a heavy chain variable domain can recognize neutrophil cytoplasmic antigens in a specific manner. These single-domain antibody fragments were used in experiments designed to establish the relative role of the light chain variable domains in antigen binding.

Characterization of human blood group scFv antibodies derived from a V gene phage-display library.

We previously reported the initial characterization of five human single-chain Fv (scFv) antibody fragments specific for the blood group antigens B, D(Rh), E(Rh), Kpb and HI. The scFvs were isolated from a phage-antibody library constructed from the variable region genes of two non-immunized donors. In this paper we report the specificity, affinity and kinetics of antigen binding of these scFv fragments. All five scFvs agglutinated the appropriate red cell phenotype following the addition of a monoclonal antibody which recognizes a peptide tag incorporated into the scFv. The anti-B and anti-HI scFv molecules, which recognize high density carbohydrate antigens, spontaneously polymerized and agglutinated red cells directly. None of the antibody fragments showed cross-reactivity with other red cell antigens, with the exception of the anti-E which reacted weakly with E-negative cells. Specific scFv binding was confirmed by ELISA, flow cytometry and radioactive labelling. The anti-D scFv recognized 17,600 sites on cDE/cDE red cells with an association constant (Ka), of 5.2 x 10(7) M-1 and a rate constant for dissociation (koff) of 1.9 x 10(-2) s-1. The anti-E scFv recognized 29,800 and 39,800 sites on cDE/cDE red cells in two experiments with Kas of 8.4 x 10(6) and 4.4 x 10(7) M-1. The koff for this antibody was 2.7 x 10(-2) s-1. The results demonstrate that scFv antibody fragments specific for cell surface antigens and possessing affinities typical of the primary immune response can be obtained from a phage-display library.

Human antibody fragments specific for human blood group antigens from a phage display library.

We have isolated human antibody fragments with binding specificities against the blood group antigens of the ABO and I blood group systems (B and HI), of the Rh system (D and E) and of the Kell system (Kpb), by selecting a phage-antibody library on human red cells. The fragments, expressed in bacteria, were antigen-specific and effective in assays including agglutination and immunohistochemistry. Selection of phage-antibody libraries using intact cells seems to offer a promising alternative to hybridoma technology for the production of antibodies against cell surface molecules.

Human anti-self antibodies with high specificity from phage display libraries.

Recently we demonstrated that human antibody fragments with binding activities against foreign antigens can be isolated from repertoires of rearranged V-genes derived from the mRNA of peripheral blood lymphocytes (PBLs) from unimmunized humans. The heavy and light chain V-genes were shuffled at random and cloned for display as single-chain Fv (scFv) fragments on the surface of filamentous phage, and the fragments selected by binding of the phage to antigen. Here we show that from the same phage library we can make scFv fragments encoded by both unmutated and mutated V-genes, with high specificities of binding to human self-antigens. Several of the affinity purified scFv fragments were shown to be a mixture of monomers and dimers in solution by FPLC gel filtration and the binding kinetics of the dimers were determined using surface plasmon resonance (k(on) = 10(5)-10(6) M-1s-1, k(off) = 10(-2)s-1 and Ka = 10(7) M-1). The kinetics of association are typical of known Ab-protein interactions, but the kinetics of dissociation are relatively fast. For therapeutic application, the binding affinities of such antibodies could be improved in vitro by mutation and selection for slower dissociation kinetics.

Germline variable region gene segment derivation of human monoclonal anti-Rh(D) antibodies. Evidence for affinity maturation by somatic hypermutation and repertoire shift.

To date, there has been no systematic study of the process of affinity maturation of human antibodies. We therefore sequenced the variable region genes (V genes) of 14 human monoclonal antibodies specific for the erythrocyte Rh(D) alloantigen and determined the germline gene segments of origin and extent of somatic hypermutation. These data were correlated with determinations of antibody affinity. The four IgM antibodies (low affinity) appear to be derived from two germline heavy chain variable region gene segments and one or two germline light chain variable region gene segments and were not extensively mutated. The 10 IgG antibodies (higher affinity) appear to be derived from somatic hypermutation of these V gene segments and by use of new V gene segments or V gene segment combinations (repertoire shift). Affinity generally increased with increasing somatic hypermutation; on average, there were 8.9 point mutations in the V gene segments of the four IgM antibodies (Ka = 1-4 x 10(7)/M-1) compared with 19 point mutations in the V gene segments of the 10 IgG antibodies. The four highest affinity antibodies (Ka = 0.9-3 x 10(9)/M-1) averaged 25.5 point mutations. The use of repertoire shift and somatic hypermutation in affinity maturation of human alloantibodies is similar to data obtained in inbred mice immunized with haptens.

By-passing immunization: building high affinity human antibodies by chain shuffling.

Diverse antibody libraries can be displayed on the surface of filamentous bacteriophage, and selected by panning of the phage with antigen. This allows human antibodies to be made directly in vitro without prior immunization, thus mimicking the primary immune response. Here we have improved the affinity of one such "primary" antibody by sequentially replacing the heavy and light chain variable (V) region genes with repertoires of V-genes (chain shuffling) obtained from unimmunized donors. For a human phage antibody for the hapten 2-phenyloxazol-5-one (phOx) (Kd = 3.2 x 10(-7) M), we shuffled the light chains and isolated an antibody with a 20 fold improved affinity. By shuffling the first two hypervariable loops of the heavy chain, we isolated an antibody with a further 15-fold improved affinity. The reshuffled antibody differed in five of the six hypervariable loops from the original antibody and the affinity for phOx (Kd = 1.1 x 10(-9) M) was comparable to that of mouse hybridomas from the tertiary immune response. Reshuffling offers an alternative to random point mutation for affinity maturation of human antibodies in vitro.

Disulfide bond formation during the folding of influenza virus hemagglutinin.

To study the importance of individual sulfhydryl residues during the folding and assembly in vivo of influenza virus hemagglutinin (HA), we have constructed and expressed a series of mutant HA proteins in which cysteines involved in three disulfide bonds have been substituted by serine residues. Investigations of the structure and intracellular transport of the mutant proteins indicate that (a) cysteine residues in the ectodomain are essential both for efficient folding of HA and for stabilization of the folded molecule; (b) cysteine residues in the globular portion of the ectodomain are likely to form native disulfide bonds rapidly and directly, without involvement of intermediate, nonnative linkages; and (c) cysteine residues in the stalk portion of the ectodomain also appear not to form intermediate disulfide bonds, even though they have the opportunity to do so, being separated from their correct partners by hundreds of amino acids including two or more other sulfhydryl residues. We propose a role for the cellular protein BiP in shielding the cysteine residues of the stalk domain during the folding process, thus preventing them from forming intermediate, nonnative disulfide bonds.

Cloning and sequencing of human lambda immunoglobulin genes by the polymerase chain reaction.

Universal oligonucleotide primers, designed for amplifying and sequencing genes encoding the rearranged human lambda immunoglobulin variable region, were validated by amplification of the lambda light chain genes from four human heterohybridoma cell lines and in the generation of a cDNA library of human V lambda sequences from Epstein-Barr virus-transformed human peripheral blood lymphocytes. This technique allows rapid cloning and sequencing of human immunoglobulin genes, and has potential applications in the rescue of unstable human antibody-producing cell lines and in the production of human monoclonal antibodies.

Nucleotide sequences and three-dimensional modelling of the VH and VL domains of two human monoclonal antibodies specific for the D antigen of the human Rh-blood-group system.

The nucleotide sequences were determined for the VH and VL domains of two human IgG1 antibodies, Pag-1 and Fog-B, specific for the D antigen of the Rh-blood-group system. The VH-region genes of the two antibodies were derived from separate germ-line genes within the VH-IV gene family, but both antibodies used the same JH6 gene. The D-region genes differed from each other, and no similarity was found to known D regions. The light chain of Fog-B belongs to the V lambda-I subgroup and that of Pag-1 probably belongs to the V lambda-V subgroup; both light chains used the J2/3 gene. Three-dimensional models of the variable domains were made, based on those of known crystallographic structure. The surface contours at the combining sites are clearly different, consistent with the evidence that the antibodies recognize different but overlapping epitopes. Some details of the molecular modelling of hypervariable regions have been deposited as Supplementary Publication SUP 50155 (6 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1990) 265, 5.