Rene Hoet

Engineering Antibody Heavy Chain CDR3 to Create a Phage Display Fab Library Rich in Antibodies That Bind Charged Carbohydrates

A number of small charged carbohydrate moieties have been associated with inflammation and cancer. However, the development of therapeutic Abs targeting these moieties has been hampered by their low immunogenicity and their structural relationship to self-Ag. We report the design of an Ab repertoire enriched in Abs binding to small charged carbohydrates and the construction of a human Fab phagemid library, “FAB-CCHO.” This library combines L chain Ig sequences from human donors and H chain synthetic diversity constructed in key Ag contact sites in CDRs 1, 2, and 3 of the human framework VH3–23. The H chain CDR3 has been engineered to enrich the library in Abs that bind charged carbohydrates by the introduction of basic residues at specific amino acid locations. These residues were selected on the basis of anti-carbohydrate Ab sequence alignment. The success of this design is demonstrated by the isolation of phage Abs against charged carbohydrate therapeutic target Ags such as sulfated sialyl-Lewis X glycan and heparan sulfate.

Screening isolates from antibody phage-display libraries

Antibody phage display, coupled with automated screening, facilitates and potentiates the mining of complex combinatorial libraries and the identification of potent drug leads. In managing phage screening data, the behavior of individual phage isolates in binding assays must be linked to their antibody identities as deduced from DNA sequencing. Reviewed here are recently reported approaches for high-throughput screening of clones isolated from phage antibody libraries after selection on a defined antigen. Specific information management challenges, and possible solutions, are described for organizing screening data to enable rapid lead discovery using these antibody libraries.

Isolation and Characterization of Single Anti‐U1A‐specific B Cells from Autoimmune Patients

Patients with systemic lupus erythematosus (SLE) or SLE-overlap syndromes often produce autoantibodies directed to UlRNA-binding proteins.] Previously, we isolated and characterized autoantigen-binding Ab fragments directed to the U1 RNAassociated A protein (UlA) from several variable (V) gene combinatorial phage libraries.2 Although these libraries have proven to be very successful, heavy (VH) and light (V,) chains are randomly combined during construction of such libraries. Because we were interested in the utilization of the original VHNL pairings of the encoding autoantibodies, we developed a single cell culture system for B cells using mouse thymoma EL-4 B5 cells.3 In combination with a preselection via recombinant antigen and single cell sorting using fluorescence-activated cell sorting (FACS), we were able to enrich for autoantigen-specific B cells. VH and VL genes originating from single U1A-specific B cells were cloned in a phage display vector for expression of single-chain variable fragments (scFv). Human mononuclear cells were isolated from SLE patients and selected against U 1 A-coated plates or biotinylated U1 A coated to streptavidin-coupled superparamagnetic microbeads (MACS). Adhering cells were collected from the plates via trypsin treatment or were directly used in the case of U1A-bound MACS (FIGURE 1). The selected lymphocytes were plated as single CD 19/CDZO-positive cells using FACS with an automatic cell deposit unit. Single selected B cells were seeded on 96-well plates containing 20,000 irradiated EL4 B5 cells and 10% supernatant of phorbol myristate acetate (PMA)and phytohemagglutinin (PHA)-activated human T cells (FIGURE 2). Cultures were grown for 16-11 days and tested in ELISA for antigen (Ag)-specific antibodies and total Ig production. Cell cultures that were positive in the Ag-specific ELISA screening were used for RNA isolation. An RT reaction was performed with an oligo-dT primer and the cDNA was used in a first PCR with VH family-specific primers or a mixture of V-kappa or V-lambda primers (in the case of the light chains) as in Marks et aL4 These first PCR products were used in a second

Analysis of autoimmune bone marrow by antibody-phage display: Somatic mutations and third complementarity-determining region arginines in anti-DNA γ and κ V genes

Objective To examine anti–double-stranded DNA (anti-dsDNA) IgG autoantibodies from the bone marrow of individuals with systemic lupus erythematosus (SLE). Methods A library of single-chain variable fragments (scFv) was constructed from SLE bone marrow complementary DNA of γ, κ, and λ isotype by cloning into the pHENIX phagemid vector. The library was screened with dsDNA in solution, and 2 anti-DNA phage, DNA1 and DNA4, were isolated and their Ig V genes sequenced. Soluble scFv corresponding to DNA1 and DNA4, and their heavy (H)– and light (L)–chain recombinants, were prepared, purified, and analyzed for binding to DNA by enzyme-linked immunosorbent assay. Results DNA1 and DNA4 used different Ig H-chain (3-30 and 5-51, respectively) and L-chain (DPK15 and DPK22, respectively) V genes. The ratios of replacement mutations to silent mutations in DNA1 and DNA4 suggest that their V genes were selected for improved antigen binding in vivo. The recombinant between DNA4VH and DNA1VL showed the highest relative affinity for both single-stranded DNA and dsDNA. These 2 Ig subunits contained third complementarity-determining region arginines and had acquired the majority of replacement mutations. Conclusion Anti-dsDNA IgG autoantibodies from the bone marrow of SLE patients exploit diverse V genes and cationic V–D–J and V–J junctions for DNA binding, and accumulate replacement mutations that enhance binding.