Development of Efficient Chemistry to Generate Site-Specific Disulfide-Linked Protein– and Peptide–Payload Conjugates: Application to THIOMAB Antibody–Drug Conjugates

Abstract

Conjugation of small molecule payloads to cysteine residues on proteins via a disulfide bond represents an attractive strategy to generate redox-sensitive bioconjugates, which have value as potential diagnostic reagents or therapeutics. Advancement of such "direct-disulfide" bioconjugates to the clinic necessitates chemical methods to form disulfide connections efficiently, without byproducts. The disulfide connection must also be resistant to premature cleavage by thiols prior to arrival at the targeted tissue. We show here that commonly employed methods to generate direct disulfide-linked bioconjugates are inadequate for addressing these challenges. We describe our efforts to optimize direct-disulfide conjugation chemistry, focusing on the generation of conjugates between cytotoxic payloads and cysteine-engineered antibodies (i.e., THIOMAB antibody-drug conjugates, or TDCs). This work culminates in the development of novel, high-yielding conjugation chemistry for creating direct payload disulfide connections to any of several Cys mutation sites in THIOMAB antibodies or to Cys sites in other biomolecules (e.g., human serum albumin and cell-penetrating peptides). We conclude by demonstrating that hindered direct disulfide TDCs with two methyl groups adjacent to the disulfide, which have heretofore not been described for any bioconjugate, are more stable and more efficacious in mouse tumor xenograft studies than less hindered analogs.

Tetravalent biepitopic targeting enables intrinsic antibody agonism of tumor necrosis factor receptor superfamily members

“…ThioMab TM (K149C) antibodies were recombinantly expressed in CHO cells and purified as described previously. 42 Fabs contained a C-terminally extended variant hinge sequence on the heavy chain (DKTHTSPPC) that provided the free C-terminal cysteine for conjugation (referred to as Fab-C). Fab-C were expressed in E. coli and purified as previously described.…”

“…To remove the adduct, the antibody was reduced and reoxidized as previously described. 42 The final deprotected ThioMab TM (KiH, K149C) antibody was formulated into conjugation buffer and stored at 4°C.…”

Synthesis of poly(disulfide)s with narrow molecular weight distributionsvialactone ring-opening polymerization

“…10 Various pharmaceuticals can be conjugated to polymers or proteins via disulde bonds for drug delivery. 11,12 When appropriate signals are applied to the disulde-based materials in a spatiotemporal manner, the disulde bonds degrade either to disintegrate the polymer network structure or to release the active pharmaceuticals from the delivery carrier. UV-responsive self-healing gels, 13 nanogels, 14 and antibody-drug conjugates 12 responding to high glutathione concentrations around cancerous tissues or in the cytosol are all on the frontier of materials and pharmaceutical sciences.…”

Exposure-Efficacy Analysis of Antibody-Drug Conjugates Delivering an Excessive Level of Payload to Tissues

“…Anti-HER2 conjugates on light chain K149C, heavy chain A140C of non-methyl-and methyl-disulfide-linked PBD-dimer (A1: aHER2-heavy chain-H-SS-PBD, A2:aHER2-LC-H-SS-PBD, A3:aHER2-heavy ADCs Can Deliver Excess Payload to Tumors chain-Me-SS-PBD, and A4:aHER2-light chain-Me-SS-PBD) used in Groups A1-A4 were prepared as described previously (Zhang et al, 2016a(Zhang et al, , 2018. Anti-CD22 conjugates of disulfide and peptide-linked monomethyl auristatin E, MMAE (B1:aCD22-Me-SS-MMAE, B2:aCD22-Me-SS-PAB-MMAE, B3:aCD22-DiMe-SS-MMAE, and B4:aCD22-va-cit-PAB-MMAE) used in Groups B1-B4, variously linked maytansinoids DM1, DM3, and DM4 (C1:CD22-SS-DM1, C2:aCD22-SS-DM3, C3:aCD22-MPEO-DM1, C4: aCD22-SPDB-DM4, C5:aCD22-MBT-DM4, C6:aCD22-SS-DM4, C7: aCD22-Fc-SS-DM4, C8:aCD22-Fc-SS-DM3) used in Groups C1-C8, and aHER2-A118C-Me-SS-PBD (D1), aCD22-val-cit-PAB-MMAE (D2), and aCD22-LC-K149C-MCC-DM1 (D3) as well as the corresponding control conjugates were prepared at Genentech as described previously (Pillow et al, 2014;Sadowsky et al, 2017). The structures of these ADCs and associated names, structural elements, and doses are shown in Fig.…”