Hassan Chaabihi

Design of cassette baculovirus vectors for the production of therapeutic antibodies in insect cells

BACKGROUND Various systems have been described for the expression of recombinant monoclonal antibodies for therapeutical applications. Insect cells offer great advantages with respect to post-translational modifications, stability, yields and applications. OBJECTIVES To construct plasmid cassette transfer vectors in order to express chimeric, humanized or human antibodies in insect cells using baculovirus expression system. STUDY DESIGN Two transfer vectors, pBHuC kappa and pBHuC gamma 1, were designed. They contain a viral promoter (polyhedrin or p10 promoters, respectively), a signal peptide sequence and a human immunoglobulin light chain C kappa gene or heavy chain C gamma 1 sequence, respectively. Restriction sites have been introduced to allow insertion of rearranged variable genes, after amplification by polymerase chain reaction. RESULTS Recombinant baculoviruses expressing complete immunoglobulins have been generated by a double-recombination event between baculovirus DNA and the loaded cassette transfer vectors. CONCLUSION Our genetic cassette approach makes this system a very flexible and convenient one for the rapid production of therapeutic monoclonal antibodies with heavy and light chains of any human isotype. Specific variable regions selected by the antibody phage display technology can be easily transferred in these vectors to obtain a complete antibody.

Anti-digoxin scFv fragments expressed in bacteria and in insect cells have different antigen binding properties

A gene encoding a single‐chain antibody fragment directed against digoxin (named 1C10 scFv) was cloned in two expression systems. For this purpose, a new baculovirus transfer cassette fully compatible with the procaryotic pHEN vector was constructed. Baculovirus production led to higher yield than did Escherichia coli expression. The procaryotic fragment showed variations in the fine specificity profile but an affinity constant nearly identical to that of the 1C10 Fab, whereas the eucaryotic scFv fragment had a lower affinity with a specificity profile identical to original mAb. The half‐lives of the digoxin:scFv complexes and the global specificity are compatible with therapeutic use of this antibody fragment.