Design, Synthesis, and in Vitro Evaluation of Dipeptide-Based Antibody Minor Groove Binder Conjugates

Abstract

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.

Preclinical Profile of the HER2-Targeting ADC SYD983/SYD985: Introduction of a New Duocarmycin-Based Linker-Drug Platform

“…The duocarmycin class of compounds has been explored by several others for application in ADCs (38)(39)(40). The anti-CD70 ADC MDX-1203 is the only duocarmycin-based ADC; however, that has been taken into the clinic.…”

Lymphocyte Activation Antigen CD70 Expressed by Renal Cell Carcinoma Is a Potential Therapeutic Target for Anti-CD70 Antibody-Drug Conjugates

“…A comparison of IC 50 of free MMAF and c1F6-vcMMAF conjugate revealed that the molar concentrations of free MMAF required to elicit 50% killing in the RCC lines examined in this study was at least 10-fold higher than that delivered by 1F6-vcMMAF (Table 2), further illustrating the efficiency of drug delivery via CD70 targeting. Besides auristatinbased 1F6 conjugates, DNA-binding cytotoxic drugs have also shown potent, target-selective cytotoxic activities (46). This drug delivery capability is also not limited only to mAb 1F6.…”

Oxidizing potential of endosomes and lysosomes limits intracellular cleavage of disulfide-based antibody–drug conjugates

“…Such linkers include hydrazone linkers, designed to hydrolyze upon internalization into acidic endosomes and lysosomes (1-3); peptide linkers optimized for cleavage by certain lysosomal proteases (3)(4)(5); and disulfide linkers, thought to be cleaved by the reducing environment within the endocytic pathway (6)(7)(8)(9)(10). In the latter category, the monoclonal antibody C242 against CanAg (a glycotope on the mucin1 (MUC1) colorectal tumor antigen) has shown efficacy against colorectal xenograft models in vivo when disulfide-linked to the ribosomal inhibitor ricin A chain via a 4-succinimdyloxycarbonyl-methyl-␣-[2-pyridyldithio]-toluene (SMPT) linker (11) and to the maytansinoid-derived microtubule active drug DM1 via an N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) linker (12).…”