High level prokaryotic expression of anti-Müllerian inhibiting substance type II receptor diabody, a new recombinant antibody for in vivo ovarian cancer imaging.
Prescription of therapeutic antibodies has radically modified the prognosis of some important diseases. However, the very high cost of these new drugs is a problem for public health organizations, which require assessment of the effectiveness of the antibody for each patient before beginning or during the treatment. In vivo immunoimaging is particularly well adapted to meet this demand. However, full-length antibodies are unsuitable for in vivo imaging due to their persistence in the serum and must be engineered in smaller formats to improve their pharmacokinetic properties without modifying their affinity and specificity. The small bivalent antibody fragment called diabody perfectly meets these in vivo imaging requirements. However, obtaining diabodies is laborious, time-consuming and sometimes unsuccessful. Using a diabody derived from a monoclonal antibody (12G4) directed against the human anti-Müllerian hormone receptor, a biomarker of ovarian cancers for which therapeutic antibodies are already undergoing clinical trials, we describe here a new diabody refolding protocol with various reducing conditions. Diabody functionality was checked in vitro and ex vivo with, respectively, a new immunoassay involving the epitopic peptide as a tracer and flow cytometry experiments with cells expressing recombinant anti-Müllerian hormone receptors. Our optimized protocol allows us to find the best refolding conditions for each diabody and to obtain large amounts of functional diabodies.
CEA, iBiTecS, SPI, Laboratoire d'Ingénierie des Anticorps pour la Santé, Bt 136, CEA Saclay, F-91191 Gif sur Yvette, France.
Céline
C
Ortega
Amaury
A
Herbet
Sophie
S
Richard
Nathalie
N
Kersual
Narciso
N
Costa
André
A
Pèlegrin
Frédéric
F
Ducancel
Jean-Yves
JY
Couraud
Isabelle
I
Navarro-Teulon
Didier
D
Boquet
D000595:Amino Acid Sequence; D018033:Antibodies, Bispecific / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000918:Antibody Specificity / Q000276:immunology; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000276:immunology / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D015870:Gene Expression; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D008969:Molecular Sequence Data; D010051:Ovarian Neoplasms / Q000175:diagnosis / Q000276:immunology* / Q000628:therapy; D011485:Protein Binding / Q000276:immunology; D058849:Protein Refolding; D018000:Receptors, Peptide / Q000276:immunology*; D018125:Receptors, Transforming Growth Factor beta / Q000276:immunology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism
D018033:Antibodies, Bispecific; D000911:Antibodies, Monoclonal; D000939:Epitopes; D018000:Receptors, Peptide; D018125:Receptors, Transforming Growth Factor beta; D011994:Recombinant Proteins; C084621:anti-Mullerian hormone receptor