Design, Synthesis, and Biological Evaluation of Antibody−Drug Conjugates Comprised of Potent Camptothecin Analogues

Abstract

Antibody-drug conjugates (ADCs) were prepared with potent camptothecin analogues attached to monoclonal antibodies (mAbs) via dipeptide or glucuronide-based linkers. Aniline-containing camptothecin analogues were employed to provide a site of linker attachment via carbamate bonds that would be stable in circulation. The camptothecin analogues, 7-butyl-10-amino-camptothecin and 7-butyl-9-amino-10,11-methylenedioxy-camptothecin, are generally 10-1000 times more potent than camptothecin. Dipeptide and glucuronide drug linkers were employed containing self-immolative spacers that release drug following lysosomal degradation upon ADC internalization into antigen-positive cell lines. The camptothecin drug linkers were conjugated to three antibodies: chimeric BR96, chimeric AC10, and humanized 1F6, which bind to the Lewis-Y antigen on carcinomas, CD30 on hematologic malignancies, and CD70 present on hematologic malignancies and renal cell carcinoma, respectively. ADCs bearing the potent camptothecin analogue, 7-butyl-9-amino-10,11-methylenedioxy-camptothecin, were highly potent and immunologically specific on a panel of cancer cell lines in vitro, and efficacious at well-tolerated doses in a renal cell carcinoma xenograft model.

Optimization of a PEGylated Glucuronide-Monomethylauristatin E Linker for Antibody–Drug Conjugates

“…As shown in Fig. 4D, the surviving fraction increased as pharmacokinetic exposure increased, with 100% survival for ADCs bearing PEGs sufficient to preserve exposure (PEG 8 , PEG 12 , and PEG 24 ).…”

“…As shown in Fig. 4C, ADCs bearing PEG sufficient in size to optimize pharmacokinetic exposure (PEG 8 , PEG 12 , and PEG 24 ) were well tolerated with minimal, transient weight loss observed over the course of 9 days. In contrast, ADCs bearing PEGs smaller than PEG 8 (drug-linkers 7 and 8) or lacking PEG altogether (druglinkers 9 and 10) were not tolerated, with some members in each of the four groups euthanized with greater than 20% weight loss.…”

“…A single intraperitoneal dose at 3 mg/kg was given once the average tumor volume reached 100 mm 3 . ADCs bearing PEG side chains sufficient in size to preserve parental antibody pharmacokinetic properties (PEG 8 , PEG 12 , and PEG 24 ) led to much more pronounced activity than faster clearing conjugates without PEG (drug-linker 10) or with the PEG 4 (drug-linker 7)-containing ADC. For example, aCD19-10 displayed the most rapid clearance in the pharmacokinetic experiment and had minimal activity, whereas almost all of the animals treated with PEG 8 , PEG 12 , and PEG 24 -containing ADCs experienced cures or complete regressions of their tumors.…”

“…ADCs bearing PEG side chains sufficient in size to preserve parental antibody pharmacokinetic properties (PEG 8 , PEG 12 , and PEG 24 ) led to much more pronounced activity than faster clearing conjugates without PEG (drug-linker 10) or with the PEG 4 (drug-linker 7)-containing ADC. For example, aCD19-10 displayed the most rapid clearance in the pharmacokinetic experiment and had minimal activity, whereas almost all of the animals treated with PEG 8 , PEG 12 , and PEG 24 -containing ADCs experienced cures or complete regressions of their tumors. Thus, as in the tolerability experiment, in vivo activity correlated with conjugate pharmacokinetic properties, with the highest activity observed for ADCs bearing PEGs sufficient to maximize exposure.…”

“…The glucuronide linker system represents a hydrophilic alternative to the val-cit dipeptide, and exploits lysosomal b-glucuronidase (10) to initiate drug release. The glucuronide linker has been used to mitigate aggregation issues with lipophilic payloads like minor groove binders (11) and camptothecins (12). Glucuronide-MMAE linker 2 was found to provide ADCs at 4-drugs/Ab with in vitro and in vivo properties similar to val-cit-MMAE linker 1 conjugates (13).…”