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3R-Project 84-02

Direct cloning of human antibodies from purified specific B-cells

Urs Wirthmueller and Clemens Dahinden
Institute of Immunology, University Hospital, 3010 Bern, Switzerland
urs.wirthmueller@insel.ch,, clemens.dahinden@iib.unibe.ch

Keywords: human; b-cell; cell cultures: hybridomas; molecular biology: pcr; reduction; replacement; diagnostic approaches: monoclonal antibodies

Duration: 2 years Project Completion: 2010

Background and Aim
The classical method of producing diagnostic and therapeutic monoclonal antibodies (mAb’s) is to fuse Ab-producing B-cells with cancerous mouse cells (hybridoma technology). The product of this cellular fusion serves as a source for the production of mAb’s in vitro or in vivo. In either case large numbers of animals (i.e. syngeneic mice for in vivo production) or large amounts of animal products (fetal calf serum for in vitro production) are required. In addition, the syngeneic mice are, depending on the treatment and the type of adjuvants applied, often exposed to high physical stress. In addition, if these mAb’ would be used in humans they would have to be engineered to contain only human DNA-sequences in their framework (humanization). We would like to replace these cumbersome and - in terms of animals sacrificed or fetal calf serum consumed - very intense procedures by a technology which starts directly with the immunological profile of human antigen-specific B-cells to produce pure human mAb’s in vitro using recombinant technology based on bacterial or yeast cells. This method would not only replace the use of animals for production but also would provide a more direct and less expensive source of human mAb’s.

Method and Results
in progress (present status)
Starting material is whole blood from healthy tetanus-vaccinated donors. Antibodies against the tetanus toxoid will be used as model mAb to establish the feasibility of the system, since they are well characterized at both the desoxyribonucleic acid (DNA) and protein level. The toxoid protein itself is readily available and sequences of hybridomas producing Ab’s against Tetanus toxoid will be used for comparison of our recombinant Ab’s..
In a first step lymphocytes are isolated using density centrifugation. B-cell enriched fractions will be generated by negative selection using magnetic bead separation technology. Antigen-specific B-cells will be isolated by tagging them with antigen (Ag) and by subsequent positive selection using a fluorescence activated cell sorter FACS sorter. Individual Ag-specific B-cells will serve as the starting material for single cell polymerase-chain reaction (PCR).
Ribonucleic acid (RNA) isolated from these cells will be first reverse transcribed and then amplified using primers specific for the variable regions of the Ag-binding sites of immunoglobulin G (IgG)-chains. Heavy and light chains will be amplified separately. In order to prevent any contamination –by unspecific DNA - which would compromise the sensitivity of single cell PCR - all laboratory procedures will be performed under an accredited quality-management-system in a laboratory designed to perform molecular diagnostic tests.
The resulting amplification products will be isolated and sequenced using standard methods. The sequences obtained will be compared to those published for the already known anti-Tetanus toxoid hybridomas. These sequences will then be cloned into specific expression vectors already containing the constant regions of IgG. The proteins will be expressed in either bacteria or yeast (Pichia pastoris). The choice of the corresponding expression vector will generate different IgG subclasses such as IgG1 or IgG4. The resulting IgG’s will be examined for their specificity for Tetanus toxoid and will be compared to already characterized antisera to verify their correct expression.
To validate the procedure the next antigen expressed will be proteinase 3, a well characterized Ag and one of the most frequent autoantigens known. This antigen was chosen since it is notorious for its low antigenicity. If we are able to find B-cells directed against this antigen and clone their specificity using the procedure described above, it will be a good indication of the validity of our system (proof of principle).

Conclusions and Relevance for 3R
The general strategic directions of the project are according to 3R’s aims refine and replace:
Refine:
Qualitative improvement of mAb production (light and heavy IgG-chains correspond to the situation in vivo);and production of these molecules in bacteria or yeast instead mice or cell culture (refine).
Replace:
Simplified production of human mAb’s without the use of difficult-to-get or non-available hybridomas or humanization strategies. Replacement of the use of large numbers of test animals (replace).
Replacement of animal antisera (e.g. from horse) which are still in high use for therapy and diagnostics (i.e. anti-lymphocyte Ab, anti-snake venom Ab, anti-diphtheria Ab etc.) (replace).



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