Development and Properties of β-Glucuronide Linkers for Monoclonal Antibody−Drug Conjugates

Abstract

A beta-glucuronide-based linker for attaching cytotoxic agents to monoclonal antibodies (mAbs) was designed and evaluated. We employed the cytotoxic auristatin derivatives MMAE (1a) and MMAF (1b) and doxorubicin propyloxazoline (DPO, 2) to give the beta-glucuronide drug-linkers 9a, 9b, and 17, respectively. Cysteine-quenched derivatives of 9b and 17 were determined to be substrates for E. coli beta-glucuronidase, resulting in facile drug release. The beta-glucuronide MMAF drug-linker 9b was highly stable in rat plasma with an extrapolated half-life of 81 days. Each drug-linker when conjugated to mAbs c1F6 (anti-CD70) and cAC10 (anti-CD30) gave monomeric antibody-drug conjugates (ADCs) with as many as eight drugs per mAb and had high levels of immunologically specific cytotoxic activity on cancer cell lines. cAC10-9a displayed pronounced antitumor activity in a subcutaneous Karpas 299 lymphoma tumor model. A single dose treatment led to cures in all animals at the 0.5 mg/kg dose level and above, and the conjugate was well tolerated at 100 mg/kg. In mice with subcutaneous renal cell carcinoma xenografts, the MMAF conjugate c1F6-9b was tolerated at 25 mg/kg and efficacious at 0.75 mg/kg. These results demonstrate that the beta-glucuronide linker system is an effective strategy for targeting cytotoxic agents providing ADCs with high degrees of efficacy at well-tolerated doses.

Development of Novel Quaternary Ammonium Linkers for Antibody–Drug Conjugates

“…S1) over this time period, indicating a high degree of linker stability in biologic matrices. These results are comparable with what was previously reported for a carbamate-linked glucuronide-MMAE linker, which also displayed stability in rat plasma (18).…”

“…2 were synthesized from previously described intermediates (18,19) and commercially available reagents as described in the Supplementary Information. ADCs loaded at 8 drugs/Ab were prepared by treating the mAb at approximately 10 mg/mL in PBS (pH 7.4) containing 1 mmol/L EDTA with 12 equivalents of tris(2-carboxyethyl)-phosphine (TCEP) to achieve full reduction of the four native interchain disulfides.…”

“…2) were prepared by alkylation of tertiary amines with a glucuronide benzyl bromide linker intermediate, forming the quaternary ammonium linkage. The analogous carbamate-based drug-linkers 7 and 9 were prepared using established linker chemistries (18,19).…”

Intracellular Released Payload Influences Potency and Bystander-Killing Effects of Antibody-Drug Conjugates in Preclinical Models

“…ADCs were prepared using valine-citrulline (vc)-PAB linkers with an average drug loading of 4 (15), unless specified otherwise. The glucuronide linker and PBD linker conjugation has been described elsewhere (16,17).…”

Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry

“…Once the sugar is cleaved from the phenolic backbone, self-immolation of the PAB group releases the free drug. Initially, this linker was used to conjugate MMAE, MMAF, and doxorubicin propyloxazoline to various antibodies to create ADCs (61). In a subsequent study, glucuronideand Val-Cit-PAB-linked ADCs were evaluated side by side for aggregation and efficacy.…”