title: Engineered antibody-drug conjugates with defined sites and stoichiometries of drug attachment.
abstract: The chimeric anti-CD30 IgG1, cAC10, conjugated to eight equivalents of monomethyl auristatin E (MMAE) was previously shown to have potent antitumor activity against CD30-expressing tumors xenografts in mice. Moreover, the therapeutic index was increased by lowering the stoichiometry from 8 drugs/antibody down to 2 or 4. Limitations of such 'partially-loaded' conjugates are low yield (10-30%) as they are purified from mixtures with variable stoichiometry (0-8 drugs/antibody), and heterogeneity as the 2 or 4 drugs are distributed over eight possible cysteine conjugation sites. Here, the solvent-accessible cysteines that form the interchain disulfide bonds in cAC10 were replaced with serine, to reduce the eight potential conjugation sites down to 4 or 2. These Cys-->Ser antibody variants were conjugated to MMAE in near quantitative yield (89-96%) with defined stoichiometries (2 or 4 drugs/antibody) and sites of drug attachment. The engineered antibody-drug conjugates have comparable antigen-binding affinities and in vitro cytotoxic activities with corresponding purified parental antibody-drug conjugates. Additionally, the engineered and parental antibody-drug conjugates have similar in vivo properties including antitumor activity, pharmacokinetics and maximum tolerated dose. Our strategy for generating antibody-drug conjugates with defined sites and stoichiometries of drug loading is potentially broadly applicable to other antibodies as it involves engineering of constant domains.
authors.affiliation: Seattle Genetics, Inc. 21823 30th Drive SE, Bothell, WA 98021, USA. cmcdonagh@seagen.com
authors.forename: Charlotte F
authors.initials: CF
authors.lastname: McDonagh
authors.forename: Eileen
authors.initials: E
authors.lastname: Turcott
authors.forename: Lori
authors.initials: L
authors.lastname: Westendorf
authors.forename: Jennifer B
authors.initials: JB
authors.lastname: Webster
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Alley
authors.forename: Kristine
authors.initials: K
authors.lastname: Kim
authors.forename: Jamie
authors.initials: J
authors.lastname: Andreyka
authors.forename: Ivan
authors.initials: I
authors.lastname: Stone
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Hamblett
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Francisco
authors.forename: Paul
authors.initials: P
authors.lastname: Carter
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D000970:Antineoplastic Agents / Q000737:chemistry / Q000493:pharmacokinetics*; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D018796:Immunoconjugates / Q000276:immunology / Q000493:pharmacokinetics*; D017730:Ki-1 Antigen / Q000276:immunology; D008024:Ligands; D020714:Maximum Tolerated Dose; D051379:Mice; D009842:Oligopeptides / Q000737:chemistry / Q000493:pharmacokinetics*; D015202:Protein Engineering; D012694:Serine / Q000737:chemistry; D012997:Solvents / Q000737:chemistry; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D004220:Disulfides; D018796:Immunoconjugates; D017730:Ki-1 Antigen; D008024:Ligands; D009842:Oligopeptides; D012997:Solvents; D012694:Serine; D003545:Cysteine; C495575:monomethyl auristatin E