Michael Braunagel

Effect of Domain Order on the Activity of Bacterially Produced Bispecific Single-chain Fv Antibodies

Bispecific single-chain Fv antibodies comprise four covalently linked immunoglobulin variable (VH and VL) domains of two different specificities. Depending on the order of the VH and VL domains and on the length of peptides separating them, the single-chain molecule either forms two single-chain Fv (scFv) modules from the adjacent domains of the same specificity, a so-called scFv-scFv tandem [(scFv)(2)], or folds head-to-tail with the formation of a diabody-like structure, a so-called bispecific single-chain diabody (scBsDb). We generated a number of four-domain constructs composed of the same VH and VL domains specific either for human CD19 or CD3, but arranged in different orders. When expressed in bacteria, all (scFv)(2) variants appeared to be only half-functional, binding to CD19 and demonstrating no CD3-binding activity. Only the diabody-like scBsDb could bind both antigens. Comparison of the scBsDb with a structurally similar non-covalent dimer (diabody) demonstrated a stabilizing effect of the linker in the middle of the scBsDb molecule. We demonstrated that the mechanism of inactivation of CD19xCD3 diabody under physiological conditions is initiated by a dissociation of the weaker (anti-CD3) VH/VL interface followed by domain swapping with the formation of non-active homodimers. The instability of one homodimer makes the process of diabody dissociation/reassociation irreversible, thus gradually decreasing the fraction of active molecules. The structural parameters influencing the formation of functional bispecific single-chain antibodies are indicated and ways of making relatively stable bispecific molecules are proposed.

Selection and characterisation of recombinant single-chain antibodies to the hapten Aflatoxin-B1 from naive recombinant antibody libraries.

Selection of antibodies from large repertoire phage display libraries has become a common technique for isolation of specific antibodies to antigens. Many of these libraries are shown to contain antibodies specific to haptens, but only when these haptens are derivatised or conjugated to an immobilising molecule, such as bovine serum albumin (BSA). There has been little demonstration of the suitability of naive recombinant antibody libraries for isolating antibodies that bind low molecular weight haptens in the absence of a carrier molecule and few have addressed the problems associated with selecting antibodies that only recognize the combination of hapten and the carrier molecule. We have panned two-phage antibody libraries against AflatoxinB1-BSA and screened single-chain antibody fragments for binding to AflatoxinB1-BSA and Aflatoxin-B1. Many of the antibodies isolated specifically bound AflatoxinB1-BSA, but not soluble Aflatoxin-B1 or BSA. Modification of the protocol led to isolation of single-chain fragment variable antibody domain (scFv) antibodies that specifically bound soluble Aflatoxin-B1 with an affinity of 6x10(-9) M.

Generation of a large complex antibody library from multiple donors

We have generated a large complex library of single chain antibodies based on four individual libraries from each of 50 donors. DNA coding for the heavy and light chain variable domains of the IgM and IgG repertoires was amplified by PCR using two different sets of primers. Each individual library was composed of approximately 1-5x10(7) independent clones giving a final combined library of 4x10(9) members. Screening this library by phage display of single chain antibodies with small haptens, peptides and proteins yielded specific antibodies for each class of antigen.

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).

Identification of scFv antibody fragments that specifically recognise the heroin metabolite 6-monoacetylmorphine but not morphine

Use of phage display of recombinant antibodies and large repertoire naïve antibody libraries for identifying antibodies of high specificity has been extensively reported. Nevertheless, there have been few reported antibodies to haptens that have originated from naïve antibody libraries with potential use in diagnostics. We have used chain shuffling of lead single-chain fragment variable (scFv) antibodies, isolated from a naïve antibody library, to screen for antibodies that specifically recognise the major metabolite of heroin, 6-monoacetylmorphine (6MAM). The antibodies were identified by screening high-density colonies of Escherichia coli expressing soluble scFv antibody fragments without prior expression on bacteriophage (phage display). The antibodies recognise 6MAM with affinities of 1-3x10(-7) M with no crossreactivity to morphine. These antibodies can potentially be used for developing a rapid immunoassay in drug-testing programs. To our knowledge, this is the first report of an antibody that distinguishes 6MAM from its de-acetylated form, morphine.

Antibodies to steroids from a small human naive IgM library

Human antibodies specific for digoxigenin, estradiol, testosterone and progesterone have been isolated from a small combinatorial IgM repertoire (4×107) of single chain antibodies (scFv). The affinities of both the anti‐estradiol and anti‐progesterone scFv were approximately 108 M−1. Naive IgM genes appeared to be highly represented, since only the heavy chain variable domain of the anti estradiol antibody contained differences to corresponding germline sequences. The light chain variable domain of the progesterone receptor was also identical to a germline sequence, showing that it is possible for completely naive antibodies to bind steroids with affinities comparable to those obtained after a secondary immune response.

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.

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.

Screening of phage-displayed antibody libraries.

The problem of amplifying a specific antibody in a population of millions of other antibodies has been solved by the immune system using the process of clonal selection Binding of an antigen to an IgM receptor on the surface of B-lymphocytes stimulates the proliferation and differentiation of the lymphocyte until it matures to an IgG-producing plasma cell. To mimic the first step of this process in bacteria, vectors have been constructed for the expression of antibodies on the surface of bacteria and phages (for review see Chapter 32 ).