title: Design and Validation of a Novel Generic Platform for the Production of Tetravalent IgG1-like Bispecific Antibodies.
abstract: We have designed and validated a novel generic platform for production of tetravalent IgG1-like chimeric bispecific Abs. The VH-CH1-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 CD5xDR-CR3 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. CD5xDR-CR3 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.
authors.affiliation: Centro di Terapia Cellulare "G. Lanzani," Divisione di Ematologia, Azienda Ospedaliera Papa Giovanni XXIII, 24122 Bergamo, Italy; Jean-Pierre.Mach@unil.ch jgolay@asst-pg23.it.
authors.forename: Josée
authors.initials: J
authors.lastname: Golay
authors.affiliation: Centre National de la Recherche Scientifique UPS3044 "Baculovirus et Thérapie," F-30380 Saint-Christol-Lèz Alès, France;
authors.forename: Sylvie
authors.initials: S
authors.lastname: Choblet
authors.affiliation: Molecular Modeling Group, Swiss Institute of Bioinformatics, University of Lausanne, 1015 Lausanne, Switzerland;
authors.forename: Justyna
authors.initials: J
authors.lastname: Iwaszkiewicz
authors.affiliation: Centre National de la Recherche Scientifique UPS3044 "Baculovirus et Thérapie," F-30380 Saint-Christol-Lèz Alès, France;
authors.forename: Pierre
authors.initials: P
authors.lastname: Cérutti
authors.affiliation: Centre National de la Recherche Scientifique UPS3044 "Baculovirus et Thérapie," F-30380 Saint-Christol-Lèz Alès, France;
authors.forename: Annick
authors.initials: A
authors.lastname: Ozil
authors.affiliation: Animalerie, Faculté de Médecine, Université de Bretagne Occidentale-Université Européenne de Bretagne, 29238 Brest, France;
authors.forename: Séverine
authors.initials: S
authors.lastname: Loisel
authors.affiliation: INSERM, U1194, Institut de Recherche en Cancérologie de Montpellier, Université de Montpellier, Institut du Cancer de Montpellier, Institut Régional du Cancer, 34298 Montpellier, France;
authors.forename: Martine
authors.initials: M
authors.lastname: Pugnière
authors.affiliation: Centro di Terapia Cellulare "G. Lanzani," Divisione di Ematologia, Azienda Ospedaliera Papa Giovanni XXIII, 24122 Bergamo, Italy;
authors.forename: Greta
authors.initials: G
authors.lastname: Ubiali
authors.affiliation: Molecular Modeling Group, Swiss Institute of Bioinformatics, University of Lausanne, 1015 Lausanne, Switzerland;
authors.forename: Vincent
authors.initials: V
authors.lastname: Zoete
authors.affiliation: Molecular Modeling Group, Swiss Institute of Bioinformatics, University of Lausanne, 1015 Lausanne, Switzerland; Ludwig Center for Cancer Research, University of Lausanne, CH-1011 Lausanne, Switzerland; Département d'oncologie, Université de Lausanne-Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland;
authors.forename: Olivier
authors.initials: O
authors.lastname: Michielin
authors.affiliation: Centre Hospitalier Universitaire Regional, 29200 Brest, France;
authors.forename: Christian
authors.initials: C
authors.lastname: Berthou
authors.affiliation: MAT Biopharma, 91030 Evry, France; Immune Pharmaceuticals Inc., New York, NY 10016;
authors.forename: Jean
authors.initials: J
authors.lastname: Kadouche
authors.affiliation: Department of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland; and Biomunex Pharmaceuticals, 75006 Paris, France Jean-Pierre.Mach@unil.ch jgolay@asst-pg23.it.
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Mach
authors.affiliation: Centre National de la Recherche Scientifique UPS3044 "Baculovirus et Thérapie," F-30380 Saint-Christol-Lèz Alès, France;
authors.forename: Martine
authors.initials: M
authors.lastname: Duonor-Cérutti
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018033:Antibodies, Bispecific / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D000941:Antigens / Q000276:immunology; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D015195:Drug Design; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding / Q000276:immunology; D011487:Protein Conformation; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance
chemical_list: D018033:Antibodies, Bispecific; D000941:Antigens; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins