pMINERVA: A donor–acceptor system for the in vivo recombineering of scFv into IgG molecules

Abstract

Phage display is the most widely used method for selecting binding molecules from recombinant antibody libraries. However, validation of the phage antibodies often requires early production of the cognate full-length immunoglobulin G (IgG). The conversion of phage library outputs to a full immunoglobulin via standard subcloning is time-consuming and limits the number of clones that can be evaluated. We have developed a novel system to convert scFvs from a phage display vector directly into IgGs without any in vitro subcloning steps. This new vector system, named pMINERVA, makes clever use of site-specific bacteriophage integrases that are expressed in E. coli and intron splicing that occurs within mammalian cells. Using this system, a phage display vector contains both bacterial and mammalian regulatory regions that support antibody expression in E. coli and mammalian cells. A single-chain variable fragment (scFv) antibody is expressed on the surface of bacteriophage M13 as a genetic fusion to the gpIII coat protein. The scFv is converted to an IgG that can be expressed in mammalian cells by transducing a second E. coli strain. In that strain, the phiC31 recombinase fuses the heavy chain constant domain from an acceptor plasmid to the heavy chain variable domain and introduces controlling elements upstream of the light chain variable domain. Splicing in mammalian cells removes a synthetic intron containing the M13 gpIII gene to produce the fusion of the light chain variable domain to the constant domain. We show that phage displaying a scFv and recombinant IgGs generated using this system are expressed at wild-type levels and retain normal function. Use of the pMINERVA completely eliminates the labor-intensive subcloning and DNA sequence confirmation steps currently needed to convert a scFv into a functional IgG Ab.

A high-throughput platform for population reformatting and mammalian expression of phage display libraries to enable functional screening as full-length IgG

“…For example, we and others have repeatedly found that, during reformatting of scFv to IgG, there is not only significant attrition, but also changes in properties of the molecules such as affinity and activity [14][15][16] (unpublished results). We and others [17][18][19][20][21][22] therefore suggest that screening of selected phage display libraries directly as IgG would be a preferred approach for antibody discovery compared with surrogate approaches using scFv or scFv.Fc fusion proteins.…”

“…This approach is appropriate for limited germlines because restriction sites cannot be added to the V-domain-Linker boundaries without introducing mutations in the V-genes. Using a model scFv, Batonick et al 22 reported batch reformatting of scFv to IgG via the phiC31 phage integrase system in E.coli and expression of IgG through the mammalian trans-splicing machinery. It is not known if this technology can be applied in a library setting because the (Gly 4 Ser) 3 scFv linker 23 used for making scFv must be substituted by the 13 amino acid long attP site.…”

Advances in the Production and Batch Reformatting of Phage Antibody Libraries

“…The method produced 1 × 10 4 IgGs from a panning sub-library and the diversity and relative abundance of clones correlated between the original scFv library and the IgG resultant library. Batonick et al [58] developed a system to reformat scFv into IgG that takes advantage of both bacterial integrases and also intron splicing in mammalian cells. Their system, termed pMINERVA, uses a phagemid ‘donor’ that contains mammalian and bacterial promoters and an scFv with a linker between the VH and VL genes that is also the substrate for phiC31 integrase.…”

“…2a. Alternatively, the pMINERVA system [58] utilises both bacterial integrases and also intron splicing in mammalian cells (a). A phagemid ‘donor’ vector contains mammalian (grey font) and bacterial (black font) promoters and a leader sequence compatible with both cell types (symbols are the same as in Fig.…”

Current Challenges and Limitations in Antibody-Based Detection of Citrullinated Histones

“…Increased efforts to isolate monoclonal mouse or rabbit Ab, including possibly by recombinant methods, should be promoted. Notably, advances in phage display technology have led to the discovery of antibodies that are specific for various post-translational protein modifications, including acetylation, phosphorylation, methylation, and citrullination (50) and that allow their efficient conversion into IgG molecules (51). …”