NEW ways of making antibodies have recently been demonstrated using gene technology. Immunoglobulm variable (V) genes are amplified from hybridomas or B cells using the polymerase chain reaction, and cloned into expression vectors. Soluble antibody fragments secreted from bacteria are then screened for binding activities (see ref. 1 for review). Screening of V genes would, however, be revolutionized if they could be expressed on the surface of bacteriophage. Phage carrying V genes that encode binding activities could then be selected directly with antigen. Here we show that complete antibody V domains can be displayed on the surface of fd bacteriophage, that the phage bind specifically to antigen and that rare phage (one in a million) can be isolated after affinity chromatography.

Phage antibodies: filamentous phage displaying antibody variable domains

Random association of VL and VH repertoires contributes considerably to antibody diversity. The diversity and the affinity are then increased by hypermutation in B cells located in germinal centres. Except in the case of 'heavy chain' disease, naturally occurring heavy-chain antibodies have not been described, although antigen binding has been demonstrated for separated heavy chains or cloned VH domains. Here we investigate the presence of considerable amounts of IgG-like material of M(r) 100K in the serum of the camel (Camelus dromedarius). These molecules are composed of heavy-chain dimers and are devoid of light chains, but nevertheless have an extensive antigen-binding repertoire, a finding that calls into question the role of light chains in the camel. Camel heavy-chain IgGs lack CH1, which in one IgG class might be structurally replaced by an extended hinge. Heavy-chain IgGs are a feature of all camelids. These findings open new perspectives in the engineering of antibodies.

Naturally occurring antibodies devoid of light chains

A member of a specific binding pair (sbp) is identified by expressing DNA encoding a genetically diverse population of such sbp members in recombinant host cells in which the sbp members are displayed in functional form at the surface of a secreted recombinant genetic display package (rgdp) containing DNA encoding the sbp member or a polypeptide component thereof, by virtue of the sbp member or a polypeptide component thereof being expressed as a fusion with a capsid component of the rgdp. The displayed sbps may be selected by affinity with a complementary sbp member, and the DNA recovered from selected rgdps for expression of the selected sbp members. Antibody sbp members may be thus obtained, with the different chains thereof expressed, one fused to the capsid component and the other in free form for association with the fusion partner polypeptide. A phagemid may be used as an expression vector, with said capsid fusion helping to package the phagemid DNA. Using this method libraries of DNA encoding respective chains of such multimeric sbp members may be combined, thereby obtaining a much greater genetic diversity in the sbp members than could easily be obtained by conventional methods.

Methods for producing members of specific binding pairs

http://theory.bio.uu.nl/immbio/Marks91.pdf

By-passing immunization: Human antibodies from V-gene libraries displayed on phage

The murine monoclonal antibody mumAb4D5, directed against human epidermal growth factor receptor 2 (p185HER2), specifically inhibits proliferation of human tumor cells overexpressing p185HER2. However, the efficacy of mumAb4D5 in human cancer therapy is likely to be limited by a human anti-mouse antibody response and lack of effector functions. A "humanized" antibody, humAb4D5-1, containing only the antigen binding loops from mumAb4D5 and human variable region framework residues plus IgG1 constant domains was constructed. Light- and heavy-chain variable regions were simultaneously humanized in one step by "gene conversion mutagenesis" using 311-mer and 361-mer preassembled oligonucleotides, respectively. The humAb4D5-1 variant does not block the proliferation of human breast carcinoma SK-BR-3 cells, which overexpress p185HER2, despite tight antigen binding (Kd = 25 nM). One of seven additional humanized variants designed by molecular modeling (humAb4D5-8) binds the p185HER2 antigen 250-fold and 3-fold more tightly than humAb4D5-1 and mumAb4D5, respectively. In addition, humAb4D5-8 has potency comparable to the murine antibody in blocking SK-BR-3 cell proliferation. Furthermore, humAb4D5-8 is much more efficient in supporting antibody-dependent cellular cytotoxicity against SK-BR-3 cells than mumAb4D5, but it does not efficiently kill WI-38 cells, which express p185HER2 at lower levels.

https://www.pnas.org/content/pnas/89/10/4285.full.pdf

Humanization of an anti-p185HER2 antibody for human cancer therapy.

IN antibodies, a heavy and a light chain variable domain, VH and VL, respectively, pack together and the hypervariable loops on each domain contribute to binding antigen1–4. We find, however, that isolated VH domains with good antigen-binding affinities can also be prepared. Using the polymerase chain reaction5, diverse libraries of VH genes were cloned from the spleen genomic DNA of mice immunized with either lysozyme or keyhole-limpet haemocyanin. From these libraries, VH domains were expressed and secreted from Escherichia coli. Binding activities were detected against both antigens, and two VH domains were characterized with affinities for lysozyme in the 20 nM range. Isolated variable domains may offer an alternative to monoclonal antibodies and serve as the key to building high-affinity human antibodies. We suggest the name 'single domain antibodies (dAbs)' for these antigen binding demands.

Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli

We have designed a set of oligonucleotide primers to amplify the cDNA of mouse immunoglobulin heavy and light chain variable domains by the polymerase chain reaction. The primers incorporate restriction sites that allow the cDNA of the variable domains to be force-cloned for sequencing and expression. Here we have applied the technique to clone and sequence the variable domains of five hybridoma antibodies and to express a mouse-human chimeric antibody that binds to the human mammary carcinoma line MCF-7. The technique should also lead to the cloning of antigen-binding specificities directly from immunoglobulin genes.

https://www.pnas.org/content/pnas/86/10/3833.full.pdf

Cloning immunoglobulin variable domains for expression by the polymerase chain reaction.

Generating binding activities from Escherichia coli by expression of a repertoire of immunoglobulin variable domains.

Cloning immunoglobulin variable domains for expression by the polymerase chain reaction. 1989.

The production of monoclonal antibodies using hybridoma technology (Kohler and Milstein, 1975) represents a landmark for the use of antibodies as reagents for medicine and biology It is now possible to use recombinant DNA methods to isolate and genetically manipulate antibody genes, and the resulting immunoglobulins or immunoglobulin fragments can be efficiently expressed in mammalian or bacterial hosts (for a review, see Morrison et al, 1988)The immunoglobulin molecule consists of a string of domains, each domain consisting of about 100 amino acid residues The constant domains carry the effector functions, such as complement mediated lysis and ADCC The antigen binding site is fashioned by both heavy (VH) and light (VL; Vκ or Vλ) chain variable domains, as demonstrated by the solved crystallographic structures of antibody in association with antigen (Amit et al, 1986; Sheriff et al, 1987; Colman et al,1987) or hapten (Satow et al, 1986) Variable domains have been pasted onto constant domains (Morrison et ah, 1984; Boulianne et al, 1984; Neuberger et al,1985) and hypervariable loops (CDRs) onto the underlying (β-sheet framework of variable domains (Jones et al,1986; Verhoeyen et al,1988; Riechmann et al,1988a) Grafting CDRs has been used to humanise rodent antibodies and one such antibody used in the successful treatment of 2 patients with non- Hodgkins lymphoma (Hale et al,1988; Riechmann et al,1988a)

Detlef Güssow

Papers

Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli

Cloning immunoglobulin variable domains for expression by the polymerase chain reaction.

Generating binding activities from Escherichia coli by expression of a repertoire of immunoglobulin variable domains.

Cloning immunoglobulin variable domains for expression by the polymerase chain reaction. 1989.

Expression and Secretion of Repertoires of VH Domains in Escherichia Coli : Isolation of Antigen Binding Activities