The Recombinant Antibody Pages
- Introduction

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Antibodies are a modular weapon system of our body created by evolution to identify any foreign intruder. By combining a set of variant gene cassettes with additional mutation mechanisms, a variety of more than a billion different sequences can be achieved. The genetic material for this huge " library " of different antibodies is stored in the B-cell pool of our lymphatic tissue.
The antigen binding region of antibodies is located at the "upper" tips of the Y-shaped immunglobulin-molecule. It is composed of the two variable regions of heavy and light chain, which include 6 hypervariable loops (CDRs). These CDR-loops create most of the antigen binding surface of the molecule.

Schematic drawing of IgG, Fab and scFv (click for full size, 51k). (c) S. Dübel

In general, people use the term "recombinant antibodies" for the antigen binding fragments (Fab or Fv) of an antibody produced from a heterologous source (i.e. these fragments do not contain the Fc parts and thus the effector functions mediated by these part, e.g. complement activation, Fc-receptor binding etc.). The Fig. above shows schematic drawings of the organisation of a natural IgG and derived recombinant fragments. The Fv fragment is shown as a "Single Chain Fv Fragment". In this type of recombinant fusion protein, the two antigen binding regions of the light and heavy chain (Vh and Vl) are connected by a 15-18 amino acid peptide. This stabilizes the protein and ensures the equal expression of both regions in heterologous organisms, e.g. E. coli .

3Dc view of a Fv region showing the CDRs (click for full size, 77k) (c) S. Dübel

Recent advances in gene technology have greatly facilitated the genetic manipulation , production, identification and conjugation of recombinant antibody fragments. The antigen binding domains of valuable monoclonal antibodies can be rescued from the hybridoma cell lines and produced in heterologous systems for "Hybridoma Immortalizaion". Easy genetic manipulation of recombinant antibodies has improved our knowledge about the structure and functional organisation of immunoglobulins. Genetic fusion and recombinant expression has led to the development of a zoo of new heterologous fusion proteins for research, diagnosis and therapy.

"Hybridoma Immortalization"(click for full size,189k) (c) S. Dübel

Example: The scFv antibody derived from Myc1-9E10 to a peptide epitope of the human oncogene c-myc (well known for his use as a tag (click onto the model for details).

New fascinating perspectives, too, have been opened up by developments to screen for specific monoclonal antibodies outside the human body . To do this, one first has to construct huge antibody gene libraries. This is usually achieved by PCR-amplification from B-lymphocyte cDNA (useful Primer sets for mouse see: [Ref.] or man see: [Ref.] . Alternatively, antibody genes can be constructed in vitro by gene synthesis using "randomized wobble"-primers [Ref.] or a combination of both methods.

Generation of an antibody library for phage display (jpeg, 336k) (c) S. Dübel

To screen these antibody libraries that contain many millions of different clones, a selection system is required with an efficiency comparable to that of the immune system. This can be achieved by displaying antibodies on the surface of microorganisms containing the antibody´s gene, in analogy to the expression of the IgM antigen receptor on the surface of unactivated B-lymphocytes. Examples of these procaryontic organisms are filamentous bacteriophage ( M13 , [Ref.] )or bacteria [Ref.] .

Life Cycle of Filamentous Phage ( jpeg 102k)

This surface display generates a particle which mediates a physical link between the antigen binding function and the antibody genes. Using the affinity to the antigen, the whole organism can be identified out of billions of nonspecific others. Specific clones binding to an antigen can then be amplified and used to produce the antibody fragment in E.coli or other suitable organisms. A picture of this process showing filamentous phage display as an example is given below:

Screening Selection of a scFv from a phagemid library (jpeg 231k) (c) S. Dübel

These new screening procedures provide the power to select one out of more than ten to the nine different expression clones in solution. The screened libraries can contain synthetic sequences, e.g. randomised antigen binding regions, or new combinations of light/heavy chains, thus creating the potential for generating human antibodies which could never be obtained from the blood stream . For example, antibodies to highly toxic substances or antigens which our immune system tolerates may be developed. By random or designed mutations, affinity or specificity of the antigen binding can be changed, reaching e.g. affinities never observed with natural antibodies. This process can be regarded as an in vitro imitation of somatic hypermutation during an immune response in our body.
These methods opened the way to a new chapter for the use of antibodies in research, diagnosis and therapy. For example, human anti mouse immune respone (HAMA), a major obstacle in patient treatment or in vivo diagnosis with conventional mouse monoclonal antibodies, can be avoided using recombinant human antibody fragments.

Furthermore, genetic coupling of the antibody to heterologous protein by fusion of gene fragments generates new possibilities for immunotargeting . Immunotargeting utilises the affinity of the antibody part of the fusion protein to increase the concentration/activity of the heterologous fusion part at sites where antigen is present. This can be e.g. used to construct immunotoxins [Ref.] or to generate bispecific and /or multifunctional proteins, e.g. by fusion to the tetramerising biotin binding protein streptavidin [Ref.] . A large variety of other fusions have been constructed and demonstrate the potential of antibody gene engineering for generating new therapeutic and diagnostic agents.

The commercialization of the technology has been promoted by patents and resulted in an exploding number of companies using this technology e.g. for novel tumour therapy approaches or in other prospering markets (see the Company links section).  In summer 1999, about 150 different clinical trials were under way employing recombinant antibodies, mostly for the treatment of cancer. Keeping in mind the delay between research and even first clinical trials and the approval as a drug, it can be expected that recombinant antibody based therapies will be a widespread and acknowledged tool in the hands of the physicians of the year 2010. The rise of antibody-based therapeutics further illustrates the substantial change in the paradigms of pharmaceutical development, by utilising the body's own capabilities as a source for a drug rather than the chemists reagents vessel.

While the first recombinant therapeutic antibodies reach the market, the technological development of antibody engineering is still far from reaching an endpoint. After the first decade, second generation libraries and screening technologies are emerging, and things to come will change the scientific goals which can be reached as well as the affordability of the technology. Read a Meeting report on recent developments here .

A n extensive introduction as well as detailed information about all aspects of recombinant antibody technology can be found in the textbook " Recombinant Antibodies " (John Wiley & Sons, NY, 1999), german version by: Spektrum Akademischer Verlag, Heidelberg). A comprehensive collection of detailed antibody engineering lab Protocols is available from Springer Verlag, Heidelberg/New York (book in print, Find Info on Contents here ).

F urther interesting information can be found on this page

I would be grateful for every suggestion to increase the quality of the page. Please send comments to Prof. Dr. Stefan Dübel lst update 25.10.2003

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