title: Understanding How the Stability of the Thiol-Maleimide Linkage Impacts the Pharmacokinetics of Lysine-Linked Antibody-Maytansinoid Conjugates.
abstract: Antibody-drug conjugates (ADCs) have become a widely investigated modality for cancer therapy, in part due to the clinical findings with ado-trastuzumab emtansine (Kadcyla). Ado-trastuzumab emtansine utilizes the Ab-SMCC-DM1 format, in which the thiol-functionalized maytansinoid cytotoxic agent, DM1, is linked to the antibody (Ab) via the maleimide moiety of the heterobifunctional SMCC linker. The pharmacokinetic (PK) data for ado-trastuzumab emtansine point to a faster clearance for the ADC than for total antibody. Cytotoxic agent release in plasma has been reported with nonmaytansinoid, cysteine-linked ADCs via thiol-maleimide exchange, for example, brentuximab vedotin. For Ab-SMCC-DM1 ADCs, however, the main catabolite reported is lysine-SMCC-DM1, the expected product of intracellular antibody proteolysis. To understand these observations better, we conducted a series of studies to examine the stability of the thiol-maleimide linkage, utilizing the EGFR-targeting conjugate, J2898A-SMCC-DM1, and comparing it with a control ADC made with a noncleavable linker that lacked a thiol-maleimide adduct (J2898A-(CH2)3-DM). We employed radiolabeled ADCs to directly measure both the antibody and the ADC components in plasma. The PK properties of the conjugated antibody moiety of the two conjugates, J2898A-SMCC-DM1 and J2898A-(CH2)3-DM (each with an average of 3.0 to 3.4 maytansinoid molecules per antibody), appear to be similar to that of the unconjugated antibody. Clearance values of the intact conjugates were slightly faster than those of the Ab components. Furthermore, J2898A-SMCC-DM1 clears slightly faster than J2898A-(CH2)3-DM, suggesting that there is a fraction of maytansinoid loss from the SMCC-DM1 ADC, possibly through a thiol-maleimide dependent mechanism. Experiments on ex vivo stability confirm that some loss of maytansinoid from Ab-SMCC-DM1 conjugates can occur via thiol elimination, but at a slower rate than the corresponding rate of loss reported for thiol-maleimide links formed at thiols derived by reduction of endogenous cysteine residues in antibodies, consistent with expected differences in thiol-maleimide stability related to thiol pKa. These findings inform the design strategy for future ADCs.
authors.affiliation: ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451-1477, United States.
authors.forename: Jose F
authors.initials: JF
authors.lastname: Ponte
authors.affiliation: ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451-1477, United States.
authors.forename: Xiuxia
authors.initials: X
authors.lastname: Sun
authors.affiliation: ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451-1477, United States.
authors.forename: Nicholas C
authors.initials: NC
authors.lastname: Yoder
authors.affiliation: ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451-1477, United States.
authors.forename: Nathan
authors.initials: N
authors.lastname: Fishkin
authors.affiliation: ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451-1477, United States.
authors.forename: Rassol
authors.initials: R
authors.lastname: Laleau
authors.affiliation: ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451-1477, United States.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Coccia
authors.affiliation: ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451-1477, United States.
authors.forename: Leanne
authors.initials: L
authors.lastname: Lanieri
authors.affiliation: ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451-1477, United States.
authors.forename: Megan
authors.initials: M
authors.lastname: Bogalhas
authors.affiliation: ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451-1477, United States.
authors.forename: Lintao
authors.initials: L
authors.lastname: Wang
authors.affiliation: ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451-1477, United States.
authors.forename: Sharon
authors.initials: S
authors.lastname: Wilhelm
authors.affiliation: ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451-1477, United States.
authors.forename: Wayne
authors.initials: W
authors.lastname: Widdison
authors.affiliation: ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451-1477, United States.
authors.forename: Jan
authors.initials: J
authors.lastname: Pinkas
authors.affiliation: ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451-1477, United States.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Keating
authors.affiliation: ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451-1477, United States.
authors.forename: Ravi
authors.initials: R
authors.lastname: Chari
authors.affiliation: ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451-1477, United States.
authors.forename: Hans K
authors.initials: HK
authors.lastname: Erickson
authors.affiliation: ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451-1477, United States.
authors.forename: John M
authors.initials: JM
authors.lastname: Lambert
mesh_terms: D000818:Animals; D004355:Drug Stability; D018796:Immunoconjugates / Q000737:chemistry* / Q000493:pharmacokinetics*; D008239:Lysine / Q000737:chemistry*; D008301:Maleimides / Q000737:chemistry*; D008453:Maytansine / Q000737:chemistry*; D051379:Mice; D013329:Structure-Activity Relationship
chemical_list: D018796:Immunoconjugates; D008301:Maleimides; D008453:Maytansine; C043592:maleimide; D008239:Lysine