Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system

Abstract

A prerequisite for the use of recombinant antibody technologies starting from hybridomas or immune repertoires is the reliable cloning of functional immunoglobulin genes. For this purpose, a standard phage display system was optimized for robustness, vector stability, tight control of scFv-ΔgeneIII expression, primer usage for PCR amplification of variable region genes, scFv assembly strategy and subsequent directional cloning using a single rare cutting restriction enzyme. This integrated cloning, screening and selection system allowed us to rapidly obtain antigen binding scFvs derived from spleen-cell repertoires of mice immunized with ampicillin as well as from all hybridoma cell lines tested to date. As representative examples, cloning of monoclonal antibodies against a his tag, leucine zippers, the tumor marker EGP-2 and the insecticide DDT is presented. Several hybridomas whose genes could not be cloned in previous experimental setups, but were successfully obtained with the present system, expressed high amounts of aberrant heavy and light chain mRNAs, which were amplified by PCR and greatly exceeded the amount of binding antibody sequences. These contaminating variable region genes were successfully eliminated by employing the optimized phage display system, thus avoiding time consuming sequencing of non-binding scFv genes. To maximize soluble expression of functional scFvs subsequent to cloning, a compatible vector series to simplify modification, detection, multimerization and rapid purification of recombinant antibody fragments was constructed.

Introduction

Molecular cloning and sequencing of antibody variable domains forms the basis of antibody modelling (Rees et al., 1994), antibody engineering (Plückthun, 1994; Nilsson, 1995) and experimental structure determination by NMR (Freund et al., 1994) or X-ray crystallography at high resolution (Ostermeier et al., 1995). Moreover, once the variable region genes have been cloned, the antibody domains can be further engineered in a multitude of ways to produce antibody variants with lower immunogenicity (Güssow and Seemann, 1991), higher affinity (Marks et al., 1992; Riechmann and Weill, 1993; Deng et al., 1994), altered antigenic specificity (Ohlin et al., 1996), or enhanced stability (Glockshuber et al., 1990; Reiter et al., 1994). Furthermore, genetic fusions of scFv fragments to effector proteins and toxins are powerful tools in the fields of medicine and diagnostics (Huston et al., 1993).

In all application areas, the demand for efficient generation of functional antibody fragments increases continuously. Although large prefabricated antibody libraries are gradually becoming a source of recombinant antibody fragments that cover a wide range of useful affinities (Vaughan et al., 1996), it may still be necessary to use the diversity of the immune system to create the most extensive panel of different antibodies against a given target possible. Furthermore, it is often of great interest and importance to clone V_H and V_L domains of the natural antibody response to a given antigen. In cases in which a large amount of experimental or clinical data is available on a given monoclonal antibody (mAb), it is frequently useful to base new constructs on this work and to determine its specific sequence and binding mode. Cloning and sequencing retains and immortalizes the unique and extensively characterized specificity of mAbs, which can be crucial for the rescue of unstable hybridoma cell lines.

One major problem in rapidly and simply obtaining sequence information about mAbs stems from the occurrence of aberrant mRNAs which are transcribed from rearranged, but non-functional, heavy and light chain genes in the hybridoma (Cabilly and Riggs, 1985; Strohal et al., 1987; Carroll et al., 1988; Kaluza et al., 1992; Kütemeier et al., 1992; Nicholls et al., 1993; Duan and Pomerantz, 1994; Yamanaka et al., 1995; Ostermeier and Michel, 1996). These non-productive chains are frequently preferentially amplified over the productive ones by sets of primers specific for the variable regions of antibody genes. The aberrant chains may greatly dilute the desired antibody sequences, which are the only ones binding the antigen in a pool of non-productive antibody-like sequences. Several attempts have been reported to overcome this problem, such as ribozyme cleavage of a known aberrant κ chain sequence (Duan and Pomerantz, 1994), treatment of aberrant mRNA/DNA hybrids with RNAseH (Ostermeier and Michel, 1996), or functional screening for full length scFv products in an in vitro transcription/translation system (Nicholls et al., 1993). Each of these methods is time consuming, depends on prior sequence information of the contaminating gene and fails to enrich binding molecules by selection procedures. Since antibody genes are usually amplified by PCR using degenerate sets of primers, mismatches and PCR errors will lead to point mutations or out-of-frame clones, which can also contribute to a background of non-functional scFv molecules. Therefore, it is absolutely vital, but often neglected (Miller et al., 1995; Kwak et al., 1996), that the binding specificity of the recombinant antibody sequence is demonstrated to be comparable with the binding characteristics of the parental monoclonal antibody, even when the deduced antibody sequence seems reasonable.

The inherent advantage of phage display is its direct link of DNA sequence to protein function (McCafferty et al., 1990; Winter et al., 1994). Thus, single clones can be rapidly screened for antigen binding and, even more importantly, selected from pools in the same experimental setup. This obviates the use of sequence specific methods to eliminate undesired sequences and leads to a more generally applicable procedure for hybridoma cloning.

However, phage display suffers from the fact that non-productive, aberrant chains are often very well expressed and non-toxic to the bacterial cell, whereas cells expressing functional scFv-geneIII fusions have a growth disadvantage and are selected against. The scFv-geneIII fusion protein can cause vector instability, creating deletions in the antibody fusion genes as occasionally observed (Courtney et al., 1995; Dziegiel et al., 1995; A. Krebber, unpublished observations; footnote 1). Thus, it is highly recommended to use a regulatable vector system allowing tight product suppression during all propagation steps as well as controlled expression of low amounts of scFv-geneIII fusion protein for phage display. Since a variety of serious technical problems concerning hybridoma cloning and enrichment of binding antibody fragments from phage display libraries have been reported¹, we have developed the reengineered phage display system described in this work. In order to provide a robust and straightforward methodology which ensures fast and reliable cloning, not only of hybridomas but also of larger antibody libraries, each step in the process was optimized. To illustrate the utility of our improved phage display system we report in detail several case studies of successfully cloned scFvs derived from monoclonal antibodies as well as enrichment of binding scFv sequences from cloned B cell repertoires.