Development and properties of beta-glucuronide linkers for monoclonal antibody-drug conjugates.

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.

Dipeptide-based highly potent doxorubicin antibody conjugates.

Highly potent and novel derivatives of doxorubicin were linked to monoclonal antibodies (mAbs) for site-specific drug delivery. Drug linker 5 consisted of a dipeptide linker attached directly to the daunosamine nitrogen of the n-butyldiacetate doxorubicin derivative 2a. Upon hydrolysis of the peptide linker and acetate groups, the free daunosamine nitrogen is able to form the highly potent 2-pyrrolinodoxorubicin (3a). The second approach involved the use of an oxazolidine carbamate (13) to mask an activating aldehyde group until proteolytic hydrolysis releases 3a. Both drug linkers were shown to be substrates for the lysosomal enzyme cathepsin B. Each molecule was conjugated to the mAbs c1F6 (anti-CD70) and cAC10 (anti-CD30) to give potent drug conjugates against renal cell carcinoma and anaplastic large cell lymphoma cell lines, respectively. The activities were immunologically selective, since antigen negative cell lines were much less sensitive to treatment with the drug conjugates. The approaches described here for attaching highly potent doxorubicin derivatives to mAbs are novel and allow for control of drug stability while covalently bound to the delivery agent.

Design, synthesis, and in vitro evaluation of dipeptide-based antibody minor groove binder conjugates.

Antibody-drug conjugates (ADCs) were prepared consisting of DNA minor groove binder drugs (MGBs) attached to monoclonal antibodies (mAbs) through peptide linkers designed to release drugs inside the lysosomes of target cells. The site of linker attachment on the MGB was at the 5-position on the B-ring, since model studies showed that attachment of an electron-withdrawing group (i.e., acyl, carbamoyl) at this position increased the stability of the molecule. Because of the hydrophobic nature of the MGBs, several measures were required to overcome their tendencies to induce mAb aggregation upon conjugation. This is exemplified in the series of ADCs containing the amino-CBI drug 1. Initial adducts were prepared using the peptide sequence valine-citrulline, attached to a self-immolative para-aminobenzyl carbamate spacer. The resulting ADCs were completely aggregated. Removal of the self-immolative spacer, introduction of a more hydrophilic valine-lysine sequence, and incorporation of a tetraethyleneglycol unit between the mAb and the peptide resulted in conjugates that were nonaggregated, even with as many as eight drugs per mAb. These results were extended to include the hydroxy aza-CBI drug 2, which was linked to the valine-lysine sequence through a para-aminobenzyl ether self-immolative spacer. The resulting mAb conjugates were monomeric and released the hydroxy aza-CBI drug upon treatment with human cathepsin B. In vitro cytotoxicity assays established that the mAb-MGB drug conjugates were highly cytotoxic and effected immunologically specific cell kill at subsaturating doses. The results provide a general strategy for MGB prodrug design and illustrate the importance of linker hydrophilicity in making nonaggregated, active mAb-MGB conjugates.