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European Biopharmaceutical Review

Antibody Discovery

The mammalian adaptive immune system evolved to effi ciently recognise ‘non-self’ antigens. Upon exposure to a foreign antigen (such as an infection), antibodies are selected that specifi cally interact with (and help eliminate) the antigen in question. The generation of such antibodies relies on B cells: in the human body, approximately 109 different B cells are present, each producing one unique antibody. When a B cell encounters an antigen that interacts with the antibody it produces, it is triggered to divide, resulting in many similar B cells (clonal expansion). Generally, a number of different B cells expressing antibodies with overlapping specifi city are ‘activated’, generating a pool of antibodies that more or less specifi cally interact with different parts of the antigen.

Antibody characteristics can be expressed in two complementary ways: specifi city describes the exclusivity of the interaction towards (a part of ) the antigen, while affi nity describes the ‘stickiness’ towards the antigen.

In the Laboratory

This naturally occurring in vivo antibody selection process can be transposed to an appropriately equipped laboratory. B cells isolated from the blood of healthy individuals can be selected on an antigen of interest. Next, the genetic material encoding antibodies (cDNA) can be isolated from these antigen-specifi c B cells; the obtained populations of antibodyencoding cDNA are strongly enriched for antibodies with the desired selectivity and affi nity towards the antigen.

Yet another level of antibody-improvement can be achieved by using B cells from immunised individuals, or from certain patient populations. Repeated or prolonged exposure to the antigen in such individuals triggers the immune system to select for ‘improved’ antibodies with higher affi nity towards the antigen; a process called affi nity maturation.

Phage Display Technology

An (alternative) approach, synergistic to selecting antibodyproducing B cells, is the selection of antibodies that are artifi cially generated in the laboratory. Phage display is a well-established methodology in which naturally-occurring antibody repertoires are reproduced in vitro. Phage display antibody repertoires can be enriched for desired binding characteristics towards a given antigen, in a process analogous to B cell selection in vivo.

The highly controllable laboratory setting, in which many selection variables can be modified, enables researchers to target the specificity and affinity of the antibodies as desired. Depending on the design of a phage display experiment, panels of antibodies can be selected with a broad range of antigen selectivity, useful in diagnostic applications, or with a narrow specificity, as desirable for therapeutic applications.

A Powerful Combination

Combining B cell selection and phage display yields a highly efficient platform for antibody discovery. Building on antibody repertoires from B cell selections, which are highly enriched for antigen binding, phage display allows for the targeted selection of lead-antibodies with desired antigen binding characteristics.

In addition, a process called directed evolution can be applied to further optimise antibody specificity and/or affinity towards the antigen. Directed evolution encompasses an iterative series of modifications to the leadantibody, followed by very stringent phage display selections that mimic in vivo affinity maturation.

Alternatively, phage display allows for the selection of antibodies against ‘self’ antigens, a process which is difficult or impossible using in vivo antibody selection. Large repertoires of antibody-encoding cDNAs are isolated from healthy volunteers or from dedicated patient groups, and used to generate phage display repertoires. Such antibody repertoires can also be synthetically modified, expanding on the naturally occurring antibody variability and creating additional possibilities to isolate antibodies against a large range of antigens, including ‘self’ antigens.

cDNA encoding of a lead-antibody that is thus selected and optimised can be re-formatted into virtually any antibody species and/or isotype by means of recombinant DNA technology. Next, large amounts of this antibody can be produced in mammalian cell lines, purified and finished for use in diagnostic and therapeutic applications.

In Practice: Case Studies

Malaria Parasite Plasmodium falciparum
Two antibody phage display repertoires have been generated from blood obtained from malaria patients (1). By selecting from these patient-derived repertoires, antibodies were selected against specific antigens on the surface of malaria parasites (the Pfs48/45 protein). Such antibodies may be used for blocking the transmission of the parasite from man to mosquito, preventing the spread of the disease.

Rheumatoid Arthritis
Antibody phage display repertoires were generated from bone marrow and from blood obtained from patients with rheumatoid arthritis (RA) (2). From these repertoires, antibodies were selected that specifically interact with unique modifications that occur in the inflamed joints of RA patients. Because of the unique characteristics of these auto-antibodies, their presence in the blood of a given individual may indicate a genetic predisposition to develop RA. Such antibodies significantly facilitate research into the inflammatory process and may prove to be of therapeutic use in targeting the inflammatory cascade in RA-affected joints.

Tumour Vasculature and Tumour Cells
Starting with an antibody phage display repertoire from llama, highly specific antibodies were selected against plexin (3). Plexin is a protein that is present in early stages of the development of neural tissue and blood vessels. Using these antibodies, it was shown that plexin reoccurs in the vasculature of brain tumours (angiogenesis), as well as in cells of various tumour types. This occurrence pattern of plexin renders these anti-plexin antibodies clinically relevant markers for tumours and potential candidates for antivascular and anti-tumour therapies.

Disease Markers
A series of subtractive and selective phage display selections has been performed on antibody phage display repertoires from patient populations with various autoimmune diseases (4). A series of antibodies was selected that specifically interact with auto-antigens with relevance for the disease in question.

This method offers an attractive high-throughput platform for the discovery of potential diagnostic and therapeutic lead-antibodies.

Conclusion

Individually, B cell selection and phage display are powerful tools to select antibodies against a given antigen. Combining B cell selection and phage display technology generates a highly efficient platform for antibody discovery. This platform can be expanded with a directed evolution approach for optimising the antigen-specificity and affinity of a given lead antibody. When compared to classic hybridoma technology, this combined in vitro approach has many advantages, most notably a significantly increased efficiency of lead-antibody selection and additional possibilities for engineering the antigen binding characteristics of such lead-antibodies.


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Jos Raats is President and CEO of both ModiQuest Research and its sister company, ModiQuest BV. He has been involved in scientific research for more than 15 years, with over 45 publications and holds a guest appointment as Assistant Professor of Biomolecular Chemistry at the University of Nijmegen (The Netherlands). Jos has initiated and managed various combined projects of university research groups and commercial enterprises, including a new diagnostic target for rheumatoid arthritis (CCP).

Head of Recombinant Antibody Discovery, Guido Jenniskens currently supervises phage display and recombinant antibody projects. He has been active in the fields of antibody-phage display, antibody-yeast display and antibody engineering since 1997. His research work has included the investigation of the role of glycosaminoglycans by the use of phage displayderived antibodies. As an MIT postdoctoral researcher, he deployed antibody yeast display to select and affinitymature antibodies against clinical formulations of Heparin, to provide better quality control of heparinoids and their derivates.

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Jos Raats of ModiQuest Research
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Guido Jenniskens of ModiQuest Research
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