Technical noteA method for rapid, ligation-independent reformatting of recombinant monoclonal antibodies
Introduction
Over the past 10–15 years there has been a surge of interest in the use of recombinant monoclonal antibodies (mAbs) as therapeutic agents. In 2007, mAb sales in the USA alone exceeded $14 billion, with a year on year growth rate of 22% (Aggarwal, 2008). With the number of approved mAbs approaching 30 and hundreds of new candidates in the pipeline, this trend shows no signs of slowing. Most therapeutic recombinant mAbs are members of the IgG family and owing to their large size and complex glycosylation patterns, these molecules are currently produced in mammalian cells, with the vast majority utilizing Chinese Hamster Ovary (CHO) cells as the production host (Wurm, 2004).
The path from discovery to the clinic for a therapeutic, recombinant mAb can be a long and tedious process, often taking several years. The first step of this process involves identification of a high-affinity binder to a target molecule, such as a surface antigen over-expressed during tumourigenesis. Considerable effort has been dedicated to elucidating methods that facilitate isolation of binding moieties to an antigen of interest. The first mAbs were produced utilizing hybridoma technology, however the resultant murine antibodies are not suitable for therapeutic applications (Berger et al., 2002). Subsequently, methods such as CDR grafting, phage, yeast and ribosome display were developed (for review see: Hoogenboom (2005)). Phage display is the most commonly used method. This technique identifies single chain variable fragment (scFv) or fragment antigen binding (Fab) elements, that bind to the target molecule isolated from libraries of high-complexity, emulating the naïve immune repertoire. This library may contain murine or human sequences and more recently, completely synthetic libraries have been created. Crucially, since these fragments contain antibody variable regions, they require “reformatting” into an expression vector containing both the requisite constant region sequences and the elements for high-level expression in mammalian cells. This reformatting step can be a protracted and complicated process since the sequences of the isolated fragments are by nature variable. This makes traditional PCR and/or restriction endonuclease cloning problematic. For example an anti-TNF antibody isolated from a naïve Fab immunoglobulin gene library was rebuilt as a complete antibody by a tripartite ligation; a fragment containing the leader sequence and the amino terminus of the V (variable) domain, a second fragment containing the remainder of the V domain and Cλ constant region, and the expression vector. The reformatting required PCR using fragment specific primers and appendage of compatible restriction sites (Mahler et al., 1997). Existing antibody reformatting vectors exhibit limited flexibility and the codons formed by restriction endonuclease recognition sequences result in the addition of several “foreign” amino acids into the primary sequence (Coloma et al., 1992, Persic et al., 1997, Jostock et al., 2004). These legacy vectors are also no longer commercially available and are difficult to source. In this paper we describe a set of novel vectors that will facilitate a high-throughput-compatible and sequence-independent method for rapid antibody reformatting. We demonstrate the effectiveness of this system by creating a functional, fully human anti-human CD83 mAb.
Section snippets
Expression vector design
mAbXpress vectors were assembled using publically available human constant region heavy (IgG1 and IgG4 subtypes) and light chain (κ) sequences. Required DNA was synthesized and codon-optimized for mammalian expression by Geneart AG (Germany). These cassettes were then placed into mammalian expression vectors containing sequences for expression, selection and amplification in mammalian cells (Acyte Biotech, Australia) (Fig. 1). A single SacI site was included in the expression vector to
Results and discussion
The vectors described here (Fig. 1) overcome several major challenges confronting the reformatting of antibody fragments with regards to the insertion of variable sequences into a constant region backbone. Firstly, this method is sequence-independent. Since scFv constructs contain semi-conserved framework adjacent to hypervariable regions, the semi-conserved framework sequence can be used as template for PCR. This potentially allows the use of a single primer set to construct the complete,
Acknowledgments
This work is supported through funding to the NCRIS Biologics Facility at the University of Queensland, an Early Career Research Grant to TPM and NHMRC Project Grant #434513 to DH, Prof Ken Bradstock and DM. TS is supported by funding from the Leukaemia Foundation. Special thanks to Ben Hughes, Karen Hughes, Michael Song and Cherrine Chan for technical assistance.
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These Authors contributed equally.