The use of recombinant antibodies in proteomics.

Recombinant antibodies are becoming increasingly important in the field of proteomics. Recent advances include the development of large phage-antibody libraries that contain high-affinity binders to almost any target protein, and new methods for high-throughput selection of antibody-antigen interactions. Coupled with a range of new screening technologies that use high-density antibody arrays to identify differentially expressed proteins, these antibody libraries can be applied to whole proteome analysis.

Antibody arrays for high-throughput screening of antibody-antigen interactions.

We have developed a novel technique for high-throughput screening of recombinant antibodies, based on the creation of antibody arrays. Our method uses robotic picking and high-density gridding of bacteria containing antibody genes followed by filter-based enzyme-linked immunosorbent assay (ELISA) screening to identify clones that express binding antibody fragments. By eliminating the need for liquid handling, we can thereby screen up to 18,342 different antibody clones at a time and, because the clones are arrayed from master stocks, the same antibodies can be double spotted and screened simultaneously against 15 different antigens. We have used our technique in several different applications, including isolating antibodies against impure proteins and complex antigens, where several rounds of phage display often fail. Our results indicate that antibody arrays can be used to identify differentially expressed proteins.

Comparable heavy and light chain pairings in normal and systemic lupus erythematosus IgG(+) B cells.

Systemic lupus erythematosus (SLE) is an autoimmune disease that is characterized by the presence of high immunoglobulin serum titers, but the mechanism by which these arise remains unclear. It has been suggested that the disease is associated with specific antibody features, including variable gene use, the presence of charged complementarity-determining region residues and/or an aberrant process of secondary light chain rearrangement. To study this in more detail, we compared variable, diversity and joining gene segment use, somatic mutation, and heavy and light chain pairings in single peripheral IgG(+) B cells between one normal (209 B cells) and two SLE (156 B cells) donors. In contrast to others, we found no systematic differences, indicating that the memory B cell repertoires in normal and SLE donors are shaped in a similar way.

Somatic insertions and deletions shape the human antibody repertoire.

We have sequenced the heavy and light chain genes from 365 IgG(+) B cells and found that 24 (6.5 %) contain somatically introduced insertions or deletions. These insertions and deletions are clustered at "hot-spots" in the antigen-binding site and frequently result in the creation of new combinations of canonical loop structures or entirely new loops that are not present in the human germline repertoire, but are similar to those seen in other species. Somatic insertion and deletion therefore provides a further mechanism for introducing structural diversity into antibodies in addition to somatic point mutation and receptor editing, which have small (single amino acid changes) and large (chain replacement) impacts on structural diversity, respectively.

Analysis of heavy and light chain pairings indicates that receptor editing shapes the human antibody repertoire.

In the bone marrow, diversity in the primary antibody repertoire is created by the combinatorial rearrangement of different gene segments and by the association of different heavy and light chains. During the secondary response in the germinal centres, antibodies are diversified by somatic mutation and possibly by further rearrangements, or "receptor editing". Here, we have analysed the pairings of heavy and light chain variable domains (VH and VL) in 365 human IgG+ B cells from peripheral blood, and established that these pairings are largely random. The repertoire is dominated by a limited number of pairings of segments and folds. Among these pairings we identified two identical mutated heavy chains in combination with two different mutated light chains (one kappa and one lambda). This shows that receptor editing occurs in the human periphery and that the same antibody lineage can be subjected to both receptor editing and somatic hypermutation. This suggests that receptor editing may be used together with somatic mutation for the affinity maturation of antibodies. We also propose that receptor editing has shaped variable gene segment use and the evolution of V gene families.

A new method for the analysis and production of monoclonal antibody fragments originating from single human B cells.

The phage display approach has proven to be a major step forward in studies on the human autoimmune repertoire. However, it remains doubtful whether the heavy and light chains of the antibodies obtained from these libraries resemble original in vivo pairings. Here we describe a novel, simple method for the immortalization of the variable heavy and light chain regions originating from individual, nonboosted, autoantigen-specific human B cells. Our method is based on the clonal expansion of B cells in which cell-cell interactions (CD40-CD40L) as well as soluble factors were shown to be essential. This B cell culture system combined with a selection on antigen (the U1A protein, a frequent autoantigenic target in patients with systemic lupus erythematosus) and single cell sorting resulted in the isolation of U1A-specific human B cells and the subsequent expression of an U1A-specific single chain variable fragment (scFv). Our method circumvents laborious plating and screening and has the advantage that original heavy/light chain pairings can be isolated. Due to the high growth efficiency of single cultured B cells (50-70% outgrowth) even rare B cell activities can be studied using this system.

Heavy chain CDR3 optimization of a germline encoded recombinant antibody fragment predisposed to bind the U1A protein.

Previously, we described a DP-65 encoded heavy chain variable (VH) gene restriction in anti-U1A antibodies. The U1A protein (a component of the U1 ribonucleoprotein particle) is an important autoantigenic target in certain systemic lupus erythematosus (SLE) patients. Here we examined the effect of randomizing amino acids in the heavy chain complementarity determining region 3 (CDR3) of this germline encoded recombinant antibody fragment on binding to the U1A protein. A phage display library was constructed using the DP-65 VH domain with four randomized CDR3 residues and our results showed that a high frequency (10%) of the randomized mutants in the unselected library were able to bind the U1A protein. This corroborates our previous finding that this VH domain provides an appropriate structure for U1A binding, although the nature of the CDR3 residues appears crucial in determining whether or not this VH domain binds U1A. After two rounds of selection U1A binders show a consensus sequence in their randomized CDR3 residues i.e. S(K,R,S)XG, in which X is an uncharged residue. This consensus is partially present in an antibody which was derived from an SLE patient indicating that this consensus, to some extent, is also followed in vivo. Clones which match the consensus sequence obtained up to 25-fold higher affinities compared with the original clones, illustrating the importance of the VH CDR3 residues in determining the affinity of these antibodies.

Characterization of human variable domain antibody fragments against the U1 RNA-associated A protein, selected from a synthetic and patient-derived combinatorial V gene library.

This is the first study describing recombinant human antibody fragments directed to the U1 RNA-associated A protein (U1A). Three anti-U1A antibody fragments (Fab) were isolated from a semi-synthetic human Fab library and one anti-U1A single-chain variable fragment (scFv) was isolated from a library which was derived from the IgG-positive splenic lymphocytes of an autoimmune patient. Competition studies with autoantibodies against the U1 small nuclear ribonucleoprotein (snRNP) particle from patients with systemic lupus erythematosus (SLE) and SLE-overlap syndromes revealed that U1A binding of these antibody fragments can be inhibited by about 40% of the patient sera. All antibody fragments recognized the native U1 snRNP in immunoprecipitation assays. Two of three Fab clones as well as the scFv clone derived from the repertoire of an autoimmune patient use the same heavy chain germ-line gene DP-65. Epitope mapping revealed that these three clones appear to recognize an identical epitope domain present on the C-terminal RNP motif of the U1A protein. The DP-65 heavy chain gene is used in less than 1% of the B cells in healthy individuals, while three out of four anti-U1A antibody fragments use this gene. This points to a restricted VH gene usage in the case of U1A, suggesting that the DP-65 heavy chain has a natural shape complementarity to the U1A protein.