Design and validation of a novel generic platform for the production of tetravalent IgG1-like bispecific antibodies

J. Golay , S. Choblet (1) , J. Iwaszkiewicz (1) , P. Cérutti (1) , A. Ozil (1) , S. Loisel (2) , M. Pugnière (3) , G. Ubiali , V. Zoete , O. Michielin , C. Berthou (4) , J. Kadouche , J.-P. Mach , M. Duonor-Cérutti (1)

1 Baculovirus et Thérapie
2 UBO UFR MSS - Université de Bretagne Occidentale - UFR Médecine et Sciences de la Santé
3 IRCM - U1194 Inserm - UM - Institut de Recherche en Cancérologie de Montpellier
4 CHRU Brest - Centre Hospitalier Régional Universitaire de Brest

We have designed and validated a novel generic platform for production of tetravalent IgG1-like chimeric bispecific Abs. The VHCH1-hinge domains of mAb2 are fused through a peptidic linker to the N terminus of mAb1 H chain, and paired mutations at the CH1-CL interface mAb1 are introduced that force the correct pairing of the two different free L chains. Two different sets of these CH1-CL interface mutations, called CR3 and MUT4, were designed and tested, and prototypic bispecific Abs directed against CD5 and HLA-DR were produced (CD5xDR). Two different hinge sequences between mAb1 and mAb2 were also tested in the CD5xDRCR3 or-MUT4 background, leading to bispecific Ab (BsAbs) with a more rigid or flexible structure. All four Abs produced bound with good specificity and affinity to CD5 and HLA-DR present either on the same target or on different cells. Indeed, the BsAbs were able to efficiently redirect killing of HLA-DR+ leukemic cells by human CD5+ cytokine-induced killer T cells. Finally, all BsAbs had a functional Fc, as shown by their capacity to activate human complement and NK cells and to mediate phagocytosis. CD5xDRCR3 was chosen as the best format because it had overall the highest functional activity and was very stable in vitro in both neutral buffer and in serum. In vivo, CD5xDR-CR3 was shown to have significant therapeutic activity in a xenograft model of human leukemia. Copyright © 2016 by The American Association of Immunologists, Inc.

Mots clés: antibody production; antibody structure; antigen binding; antigen expression; Article; binding affinity; binding site; controlled study; cytotoxicity; degranulation; dimerization; human; human cell; hydrophobicity; immunoglobulin production; macrophage; mouse; natural killer cell; nonhuman; phagocytosis; priority journal; target cell; animal; Baculoviridae; biosynthesis; chemical structure; chemistry; gene vector; genetics; immunology; isolation and purification; molecular genetics; Amino Acid Sequence; Animals; Antibodies; Bispecific; Antigens; Cell Line; Drug Design; Gene Expression; Genetic Vectors; Humans; Immunoglobulin G; Models; Molecular; Molecular Sequence Data; Mutation; Protein Binding; Protein Conformation; Protein Stability; Recombinant Fusion Proteins; Sequence Alignment; Surface Plasmon Resonance; complementary DNA; HLA DR antigen; immunoglobulin G1 antibody; antigen; bispecific antibody; hybrid protein; animal cell; animal experiment

Domaines: Cancer

J. Golay, S. Choblet, J. Iwaszkiewicz, P. Cérutti, A. Ozil, et al.. Design and validation of a novel generic platform for the production of tetravalent IgG1-like bispecific antibodies. Journal of Immunology, 2016, 196, pp.3199--3211. ⟨10.4049/jimmunol.1501592⟩. ⟨hal-01392208⟩