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| Production of recombinant antibodies |
Applicant:
Prof. Dr. Pierre Cosson
Centre Medical Universitaire, Dpt of Cell Physiology and Metabolism
1 rue Michel Servet, 1211 Geneva 4
Switzerland
tel. +41-22-379 5293; fax +41-22-379 5338
e-mail:Pierre.Cosson@unige.ch |
The aim of this project is to develop the use of recombinant antibodies in the scientific community. For this it has been planned:
1. to acquire the necessary techniques in our laboratory,
2. to develop collaborations with interested laboratories, and
3. to develop a core facility and an annual course to spread these alternative methods as widely as possible in the scientific community.
1. Aims and problems
Animals are frequently used to produce specific antibodies destined to a variety of protocols in fundamental research laboratories (e.g. immunoflurescence, histology, etc...). Over the last 15 years, new systems have been developed to select and produce recombinant antibodies with the help of phage display. This approach is now well established, and has the potential to virtually completely replace the use of animals for the isolation of specific antibodies. However, this technology is spreading extremely slowly, if at all, in academic research laboratories. Rabbits and mice remain the usual method for developing antibodies destined to research uses. In principle, private companies might fill this gap by providing a service-for-fee. Indeed at least one company(Morphosys/ABD/Serotec) is proposing the development of custom recombinant antibodies for a fee. However there are serious limitations to this offer. First the cost is very high (10'000 euros for the basic service, higher for more specific projects). Second the company only provides the purified protein to researchers, and not the DNA encoding it. This means that any additional antibody must be bought from the company at a high cost. This also prohibits any further development for the researchers (e.g. expression in transfected cells). Finally specific projects (e.g. developing antibodies against complex mixtures of proteins or against whole cells) are difficult to envisage. It seems highly unlikely that this approach could become a standard approach for academic scientists trying to develop antibodies to specific proteins.
In this project I proposed to address this issue by acquiring the technical know-how to generate recombinant antibodies, then generalizing its use in other laboratories. Ultimately this project will result in the transfer of the know-how to numerous laboratories, in the creation of an academic core facility and in an annual practical course. To acquire this technology we will develop a project making use of recombinant antibodies, focused on the in vitro analysis of bacterial virulence. Although this project is of high interest to us, I will only evoke it briefly, since it only represents a secondary aim for this project in the perspective of 3R research. The development of recombinant antibody production would virtually fully replace the use of animals to obtain specific antibody, and in particular the use of rabbits and rodents to obtain polyclonal antibodies. Virtually every biology research laboratory uses a few rabbits each year to produce polyclonal antibodies, suggesting that thousands of rabbits are used each year for this purpose by Swiss laboratories alone. It is however very difficult to evaluate the total number of rabbits used by Swiss laboratories for antibody production, because rabbit antisera are mostly obtained from private companies outside of Switzerland (mostly in Europe). According to the Swiss FVO, approximately 6000 rabbits are used each year in Switzerland for animal experiments, most of them (60%) in category one. According to official statistics, in the European Union 312'000 rabbits were used in 2005, and it is likely that a significant fraction was used for the production of antibodies.
In summary, it is extremely difficult to know how many animals, mostly rabbits would be spared by the use of recombinant antibodies. A very approximate estimate would be several thousand rabbits a year in Switzerland alone, and at least twenty times more in the European Union.
2. Scientific background
The main objective of this project is to acquire the know-how to generate and produce recombinant antibodies. The principles as well as the techniques necessary to obtain recombinant antibodies have been extensively described in many excellent reviews (for example [1,2]). Phage display allows the selection of specific recombinant antibodies in a large initial library. It is a field actively evolving, partly because recombinant antibodies may represent promising new therapeutic tools [3]. The libraries available are being optimized, as well as the techniques for selection and production of recombinant antibodies. This technique can now be mastered in standard biology laboratories. However few laboratories are ready to invest the amount of efforts needed to acquire this know-how and the necessary tools. One of the key elements is to be introduced to the details of these techniques by a competent laboratory. We have established a collaboration with the laboratory of Dr. Franck Perez at the Institut Curie (Paris, France), who is a recognized expert in this field. He has already developed numerous recombinant antibodies. The person in charge of this project in our laboratory will be hosted in his laboratory for the time necessary to learn these techniques.
The main protocols that are of potential interest to standard biology laboratories, and that would represent an alternative to the use of animals are:
1. The selection of recombinant antibodies against a purified protein.
2. The selection of recombinant antibodies against synthetic peptides.
3. The selection of recombinant antibodies against a complex mixture, for example a whole cell, or a purified cellular compartment.
In the course of our project, we will select antibodies against whole Dictyostelium cells. We will also select antibodies against known cell surface proteins in Dictyostelium, by producing the corresponding recombinant proteins in bacteria, or by using synthetic peptides. Concerning the specific project planned in Dictyostelium, the scientific background and planned experiments are described in the research plan.
3. Methods
This project makes only use of a collection of in vitro techniques and specific biological materials: mammalian and Dictyostelium cell culture, molecular biology tools, phage display libraries. All these tools are now available in our laboratory or in the collaborating laboratory of Dr. F. Perez (Institut Curie, Paris, France) who has a proven record as an expert in developing recombinant antibodies.
4. Research plan
This section is focused on the specific project in our laboratory for which recombinant antibodies will initially be produced. Recombinant antibodies will be key reagents allowing progress in our understanding of an alternative host to study bacterial infections.
Alternative hosts to study bacterial infections
Introduction
Pathogenic bacteria use a vast array of virulence mechanisms to mount infections in hosts. These bacterial strategies include for example the secretion of toxins, the ability to escape the host immune system, or the ability to replicate inside host cells. Many laboratories are studying the various facets of bacterial virulence, in order to understand the mechanisms determining the outcome of a bacteria infection. In order to study bacterial virulence it is essential at some stage to infect a host, and to monitor the progress of the infection. Rodents (mice and rats) are typically used as hosts, and these experiments inflict significant suffering to these animals. For both ethical and practical reasons, many non-mammalian models have been developed to study bacterial infections, replacing mammalian hosts with Drosophila flies, C. elegans nematodes, or even unicellular amoebae [4,5]. Our laboratory has been actively engaged in this line of research. We have notably developed new tools to study bacterial pathogenicity against Dictyostelium amoebae, a model that has proven both extremely simple and very similar to mammalian models [6,7]. Our laboratory is currently participating to the NEMO network (supported by the 3R Research Foundation, project 99-05) dedicated to the use of non-mammalian models for the study of bacterial infections. Many aspects of the interaction of pathogenic bacteria with amoebae remain un-elucidated, and this is one of the main limitations today to extrapolate results obtained using amoebae to mammalian models. Among the key unanswered questions, it is not known what receptors at the host cell surface are used by bacteria to invade amoebal cells. Similarly, although it seems likely that Dictyostelium is capable of recognizing pathogenic bacteria, nothing is known about the putative receptors involved in this recognition.
Production of recombinant antibodies
Our aim is to obtain a collection of reagents to characterize the composition of the Dictyostelium cell surface. We have previously developed monoclonal antibodies against Dictyostelium membranes [8]. Although these reagents have proven valuable for the characterization of Dictyostelium biology, they were all directed against a very limited set of immuno-dominant epitopes present on three distinct cell surface proteins. In order to avoid this heavy bias, we are planning to use phage display to obtain recombinant antibodies recognizing a collection of proteins at the cell surface. For this we will adsorb phages at the cell surface following procedures already well established [2,9,10]. After appropriate washes and selection, we will obtain single chain antibodies recognizing various elements of the Dictyostelium cell surface. These will be modified by classical molecular biology techniques to add the Fc portions of immunoglobulins. This increases the avidity of antibodies for their specific epitopes compared to monovalent variable fragments. In addition, such antibodies can be recognized by classical secondary reagents, and are thus experimentally similar to classical monoclonal antibodies. These antibodies will be produced in transfected CHO cells. In parallel, we will also develop recombinant antibodies against a few known cell surface proteins in Dictyostelium, by selecting them against recombinant proteins produced in bacteria (GST fusion proteins or His-tagged proteins) or synthetic peptides. Following a classical protocol, the proteins/peptides will be biotinylated and adsorbed to magnetic avidin-coated beads to achieve the selection of specific phages. We notably want to produce antibodies against three surface proteins that have previously been implicated in adhesion of Dictyostelium cells to their substrate, the Phg1 [11], SadA [12] and SibA [13] proteins.
Effect of recombinant antibodies on host-pathogen interactions
Once a panel of antibodies recognizing distinct cell surface antigens has been obtained, we will test the effect of these antibodies as either activators or inhibitors of various cellular functions representing the various facets of host-pathogens' interactions. We will notably test their effect on the entry of various pathogens, or on activation of the cell by various pathogens. One can expect such reagents to function as inhibitors of the protein that they are recognizing (by blocking its active site). In other cases antibodies can activate the protein to which they are binding, particularly when they are aggregated by the addition of a secondary antibody. We have already developed assays to measure the phagocytosis of various bacteria by amoebae [13]. Our previous results indicated clearly that the known proteins involved in cellular adhesion in Dictyostelium only participate in the phagocytosis of a small subset of bacteria, indicating that there are other, as yet unidentified receptors at teh cell surface that are used for the phagocytosis of many other bacteria. In order to test the ability of various recombinant antibodies to inhibit the phagocytosis of various bacterial species, Dictyostelium cells will be preincubated with recombinant antibodies (either only one, or a defined mixture), then with fluorescently-labeled bacteria. Phagocytosed bacteria will be detected by FACS as previously described [13]. Several publications have shown that gene expression is modified in Dictyostelium cells when they are exposed to various bacteria [14,15], suggesting that Dictyostelium cells recognize bacteria and modify their physiology in response. In order to test if various recombinant antibodies bind proteins involved in recognition of bacteria, we will incubate Dictyostelium cells with recombinant antibodies (either alone or in the presence of secondary cross-linking antibodies), and test if the cells respond to the presence of bacteria by testing the expression of adequate genes. For this, first we will use RT-PCR, a technique commonly used in our laboratory, to identify genes whose expression is modified in the presence of pathogens. The candidates will be chosen among the genes differentially expressed in Dictyostelium cells exposed to various bacteria [14,15], or in Dictyostelium cells with immune-like activities [16]. Once a few genes are chosen, their expression will be tested in cells exposed to recombinant antibodies. Since these tests are technically demanding, we will test initially mixtures of 10 to 50 antibodies, in order to identify as simply as possible the antibodies inducing a cellular response.
Identification of proteins recognized by recombinant antibodies
The final step of this project will be to select a few (approximately six) particularly interesting antibodies, and to identify the proteins that they are recognizing. For this we will immunoprecipitate cell lysates with selected antibodies, and identify by mass spectrometry the cellular proteins recognized by various antibodies. A core facility in our institute will perform the mass spectrometry (Mald-tof) experiments.
Overall this project will:
1. produce much-needed recombinant antibodies recognizing a panel of proteins at the Dictyostelium cell surface. This will enormously facilitate work on many facets of Dictyostelium biology, since very few antibodies recognizing Dictyostelium membrane proteins are available to date.
2. Allow identification of cell surface receptors involved in entry of bacterial pathogens or in recognition of pathogens by the cells.
This project will represent a significant step forward in our understanding of Dictyostelium biology, and will enormously facilitate work on host-pathogen interactions using Dictyostelium as an alternative non-mammalian host. I am however stressing again the fact that, in the perspective of this request, the main interest of this project is to acquire and develop the recombinant antibodies techniques.
5. Milestones
In December 2008, P. Cosson visited for one week the laboratory of Dr. F. Perez (Institut Curie, Paris) to learn the techniques involved in the production of recombinant antibodies. Since Jan 1st 2009, a postdoctoral student (Dr. C. Blanc) of our laboratory is working on the production of recombinant antibodies. We expect that all the necessary techniques will be running in our laboratory in Sept 2009. This will be a perfect time to initiate collaborative projects leading to the development of a core facility. It is for the development of these collaborative projects and of a core facility that we are requesting the support of the Doerenkamp-Zbinden Foundation. The funding that we are requesting from the Doerenkamp-Zbinden Foundation is destined to hire one post-doctoral student who will start working in October 2009. Besides his participation in our laboratory's projects, the role of this person will be to develop collaborations with all interested academic laboratories, in order to create a core facility. The main milestones specifically assigned to this person are underlined below.
Oct 2009-Dec 2009
-Learn recombinant antibody techniques in our laboratory
Jan 2010-Dec 2010
-Collaborations with at least six distinct groups in the Geneva University to develop recombinant antibodies.
Jan 2011- October 2012:
-Initiate annual practical course
-Develop a core facility dedicated to recombinant antibodies at the Faculty of Medicine.
After completion of the project, the annual course and the core facility will remain. Indeed several core facilities have been created in this manner in our Faculty, and persist after their creation by using the fees paid by the academic users. There are currently more than 12 core facilities at the Faculty of Medicine. We will reevaluate more precisely how such a core facility should be organized and financed as this project progresses.
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