Patrick Fuchs

A family of vectors for surface display and production of antibodies.

Expression vectors for surface display and production of single-chain (Fv) antibodies (scAb) have been constructed based on the phagemid pSEX, which expresses DNA encoding a scAb fused to the gene III product of filamentous phage [Breitling et al., Gene 104 (1991) 147-153]. A smaller version of this phagemid, pSEX20, was made by removing an unnecessary cat. To produce a vector for the surface display of other proteins and peptides, the scAb of pSEX20 was substituted by a polycloning site (MCS) to give pSEX40. For the presentation of Ab on the surface of Escherichia coli, phagemid pAP10 was derived from pSEX20 by substituting gene III with a gene encoding the peptidoglycan-associated lipoprotein (PAL). Vectors for producing scAb that can be purified by antibody and metal affinity chromatography were constructed by substituting gene III in the vector pSEX20 with DNA encoding a peptide with a C-terminal epitope recognised by a monoclonal antibody (phagemid pOPE40) or with five C-terminal histidines (pOPE 90).

Isolation of IgG antibody Fv-DNA from various mouse and rat hybridoma cell lines using the polymerase chain reaction with a simple set of primers

To facilitate the isolation of IgG antibody Fv-DNA sequences from hybridoma cell lines, we have established a polymerase chain reaction (PCR) procedure requiring only a small number of primers. The sense primers homologous to DNA coding for the first framework sequences were designed to hybridize to all the known antibody sequences under conditions that permit a high number of mismatches. The antisense primers were homologous to DNA coding for the beginning of the constant regions of the gamma and kappa chains. Restriction sites introduced by the primers enable the DNA to be cloned into bacterial expression vectors. Only three sense VH primers and two sense VL primers paired with one backward primer for the heavy and light chains, respectively, were necessary for the amplification of Fv-DNA from a total of 17 rodent cell lines that we have so far worked with. These consisted of 12 mouse cell lines and five rat cell lines. This procedure will therefore probably be sufficient to isolate the Fv-DNA from most mouse cell lines and possibly also from most rat cell lines.

Human antibody libraries in Escherichia coli

A potentially vast pool of human antibodies with novel specificities for diagnostic and therapeutic purposes can be generated in Escherichia coli. Antibodies to infectious agents have already been isolated by amplifying the heavy and light chain repertoires of donor lymphocytes and they have even been rescued many years after the initial infection from memory cells cultivated in SCID mice. Eventually, however, the creation of extremely large and diverse libraries from the naive antibody repertoire of unactivated B lymphocytes or by gene synthesis using random oligonucleotides for the hypervariable regions could provide a rapid means of obtaining human antibodies to any particular antigen. An important breakthrough for exploiting the potential size and diversity of these libraries has been the development of systems for the surface display of antibodies that are physically linked to their own genes. This allows large numbers of clones to be screened simultaneously and antibodies with affinities of up to 10(8) M-1 have already been obtained using these vectors. It seems quite feasible, therefore, that antibodies with affinities approaching those obtained in the secondary immune response can be obtained by systematically optimizing the strategies for making antibody libraries. Furthermore, it might be possible to establish extremely large antibody repertoires in E. coli by the in vivo recombination of phage and plasmid antibody libraries. The affinity of the selected antibodies could be increased by chain shuffling or random mutagenesis followed by several rounds of selection under increasingly stringent conditions.

Separation of E. coli expressing functional cell-wall bound antibody fragments by FACS.

BACKGROUND The rapid development of recombinant antibody technology in the last few years has facilitated the generation of antibody libraries in bacteria. Recombinant antibodies against various antigens have been selected from these libraries by presenting each antibody on the surface of a phagemid particle that contains the antibodys gene. An alternative screening system is the display of antibody fragments on bacteria. A major advantage is the possibility to select single cells directly from a large number of bacteria by using fluorescently labeled antigens and fluorescence assisted cell sorting (FACS). OBJECTIVES pAP is an expression vector for the bacterial display of antibody fragments. E. coli transformed with pAP express a single chain antibody (scFv) fused to the peptidoglycan-associated-lipoprotein (PAL). This fusion protein binds strongly to the cell wall. To employ this system for screening, we have investigated the possibility of selecting antigen-specific clones by FACS. STUDY DESIGN AND RESULTS Several DNA fragments coding for various scFvs were inserted into the pAP expression vector. E. coli were transformed with these plasmids and immunostained with fluorescent antigens under given conditions. We were able to select stained E. coli expressing a specific scFv from unstained E. coli expressing a non-binding scFv by FACS. The specific selection of the bacteria was demonstrated by amplifying their genes by PCR. CONCLUSIONS Conditions are described for separating E. coli containing scFv bound to their cell wall by FACS using fluorescently labeled antigens. These studies provide a basis for screening libraries of scFv antibodies.

A single expression system for the display, purification and conjugation of single-chain antibodies.

To facilitate the purification and conjugation of single-chain antibodies (scFv) selected from a phage display library, we have incorporated His6, an amber stop codon and a C-terminal Cys into a surface expression vector. The vector also contains a lacIq gene for improving the efficiency of regulation and a sequence coding for a marker peptide.

Recombinant human monoclonal antibodies: basic principles of the immune system transferred to E. coli

To produce human monoclonal antibodies in bacteria, a gene repertoire of IgM variable regions was isolated from human peripheral B lymphocytes by the polymerase chain reaction. Alternatively, synthetic antibody genes with random hypervariable regions are being generated that may provide libraries of even higher complexity. For the selection of specific monoclonal antibodies from these libraries, we have developed twoE. coli vector systems that facilitate the surface display of an antibody physically linked to its own gene. The phagemid pSEX encodes a fusion protein of an antigen binding domain (Fv-antibody) with the docking protein (pIII) of filamentous phages. Specific antibody genes can therefore be enriched by antigen affinity chromatography. The plasmid pAP1 encodes a fusion protein of an Fv-antibody with a bacterial cell-wall protein. Bacteria carrying this plasmid express functional Fv-antibodies tightly bound to their surface. This should enable the selection of single cells with a fluorescence-assisted cell sorter (FACS) using labeled antigen or by adsorption to immobilized antigen.These vectors permit three major principles of the antibody response to be mimicked inE. coli:1.Generation of a highly complex antibody repertoire;2.Clonal selection procedures for library screening; and3.The possibility of increasing a given affinity by repeated rounds of mutation and selection.