Significant expertise and specialised cell and animal facilities are all needed for the successful creation of traditional hybridomas, usually over a 2-9 month period. This process can also be unpredictable, failing to yield results if the target molecules are either toxic or poorly immunogenic. Traditionally produced monoclonals are usually of unknown sequence and remain fixed in specificity and isotype. In addition, hybridoma output varies over time, and sometimes there is a loss of antibody-producing cell lines.
Recombinant human antibody technology has been developed to address these issues. It has been successfully used in human therapeutics and a wide range of research applications to produce over 11000 monoclonal antibodies. Originally, recombinant monoclonal antibodies were generated by isolating the relevant genetic material from a hybridoma cell line, or by PCR (polymerase chain reaction) amplification of a pool of antibody-producing cells obtained from an immunised animal. Today, they are generated instead from highly sophisticated libraries of antibody genes which represent the complexity of the human antibody repertoire, optimised for expression in E. coli.
One example of a sophisticated recombinant antibody library is HuCAL PLATINUM, with more than 45 billion specificities, which was developed by MorphoSys for the generation of therapeutic antibodies. It was constructed with sequence information obtained by bioinformatic analysis of the human immune system, and contains a modular system of 49 framework genes which represent the diversity in immunoglobulin heavy and light chains. Variable genetic cassettes with 6 CDR (Complementarity Determining Regions) are then superimposed on the frameworks to mimic the natural variability represented in typical human antibodies1, (Fig. 1).
For this library, the antibodies have been created in a monovalent Fab format, since the Fc region is not necessary for antigen binding. The monoclonal antibody fragments generated with this process are fully functional. They include the complete antigen binding site and display the same intrinsic affinity as their full-length counterparts. Since the library is modular, all antibodies produced are adaptable to divalent or full-length immunoglobulin molecules in various isotypes, or with epitope tags and fusions proteins.
Since the library is so large, the antibodies are identified by selection rather than screening. This is carried out using phage display, which is a popular and well-established selection method. It is achieved by genetically fusing the antibody gene with one of the phage coat proteins. Thus, each member of the library is used to create a phage which displays the corresponding antibody molecule on its surface (Fig. 2). As a result, phage biology links the antibody binding properties to the corresponding genetic information, which can be captured and used to generate a monoclonal cell line. It can be sequenced and retained for future use.
Desired antibodies are selected by 'phage panning', which is somewhat similar to solid-phase immunoassay2. In this process, the few phage that display an antibody with binding affinity to the target molecule are captured as shown in Fig. 3. At this stage, other substances such as molecules closely related to the antigen can be added to the solution to effectively block undesired antibody binding sites, or to deplete the library of unwanted specificities.
After washing to remove all non-specific material, the bound phage are eluted and amplified by replication in new host cells. This procedure is repeated several times, resulting in a phage population that is highly enriched with members that express the desired antibodies. Finally, the antibody genes are isolated as a pool and inserted into an antibody expression vector.
Following introduction into new host cells, the transformed cells are isolated as single colonies, each producing a uniquely defined monoclonal antibody. Cells are subsequently lysed and tested by ELISA for binding to the antigen. The selection, expression, and screening process is complete in about 4-6 weeks, and typically results in the identification of multiple unique antigen-specific monoclonal antibody fragments. These antibodies are then grown in larger scale for purification.
Since selection takes place in vitro, it is possible to manipulate the process in order to identify antibodies with carefully chosen binding properties. The process has been used to identify single amino acid modifications, such as phosphorylation or oxidation3 specific antibodies directly, or to select antibodies in the presence of buffers required for a particular assay. In addition, it can identify antibodies that do not cross-react with closely related antigens by use of a subtraction strategy which pre-absorbs the unwanted specificities, driving selection towards unique epitopes on a particular target molecule. For example, HuCAL antibodies were developed to specifically recognise two 10 amino acid peptides, but not the 20 amino acid full length peptide.
Recombinant monoclonal antibody technology offers numerous advantages over traditional hybridomas. Primary among these is the ease of genetic manipulation that accompanies a fully defined modular system allowing for simplified downstream alterations and further optimisation. Furthermore, since the process occurs in vitro, there is greater flexibility in choosing the conditions used for the initial generation of monoclonal antibodies, allowing for toxic antigens, special buffers, and directed selection strategies. As a result, the use of recombinant DNA technology increases efficiency and flexibility in identifying monoclonal antibodies for research and diagnostics.
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Claire Moore is a Technical Writer. AbD Serotec is based in Kidlington, Oxford, UK. www.abdserotec.com
1. Knappik et al, (2000) Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomised with trinucleotides. J Mol Biol. 296:57-86;
2. Barbas, C.F. and Lerner, R.A. (1991) Combinatorial immunoglobulin libraries on the surface of phage (phabs): Rapid selection of antigen-specific Fabs. METHODS: A Companion to Methods in Enzymology 2: 119-124;
3. Ooe et al, (2006) Establishment of specific antibodies that recognize C106-oxidized DJ-1. Neurosci Lett. 404:166-169.