Synthesis and Biological Evaluation of Antibody Conjugates of Phosphate Prodrugs of Cytotoxic DNA Alkylators for the Targeted Treatment of Cancer

Abstract

The synthesis and biological evaluation of phosphate prodrugs of analogues of 1 (CC-1065) and their conjugates with antibodies are described. The phosphate group on the 1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI) portion of the compounds confers enhanced solubility and stability in aqueous solutions. In the presence of phosphatases, these compounds convert into active DNA-alkylating agents. The synthesis of the prodrugs was achieved sequentially through coupling of CBI with a bis-indolyl moiety, followed by attachment of a thiol-containing linker, and conversion of the hydroxyl group of CBI into a phosphate prodrug. The linkers incorporated into the prodrugs enable conjugation to an antibody via either a stable disulfide or thioether bond, in aqueous buffer solutions containing as little as 5% organic cosolvent, resulting in exclusively monomeric and stable antibody-cytotoxic prodrug conjugates. Two disulfide-containing linkers differing in the degree of steric hindrance were used in antibody conjugates to test the effect of different rates of intracellular disulfide cleavage and effector release on biological activity. The prodrugs can be converted to the active cytotoxic compounds through the action of endogenous phosphatases. Antibody-prodrug conjugates displayed potent antigen-selective cytotoxic activity in vitro and antitumor activity in vivo.

A New Class of Antibody–Drug Conjugates with Potent DNA Alkylating Activity

“…However, developing ADCs that meet the criteria for clinical advancement using potent DNA-interacting agents has been a challenge owing to at least two factors: (i) a paucity of DNA-interacting compounds that meet the requirement of high potency, stability, and solubility in aqueous formulation and (ii) difficulty in achieving a sufficient therapeutic index in vivo (30). The duocarmycins and CC-1065 analogues are potent DNA minor groove binders and alkylators that are being evaluated in ADCs, yet compounds from these two classes suffer the major drawbacks of low aqueous stability and solubility, in turn making them poorly suited for use in ADCs.…”

In Vivo Biotransformations of Antibody–Drug Conjugates

“…The ADC designed by ImmunoGen, huB4-DC1, employs a CBI scaffold protected by a phosphate prodrug [68]. The noncleavable linker SMCC or a disulfide bond containing SPP linker was used for the conjugation to lysine residues on the antibody.…”

Antikörper‐Wirkstoff‐Konjugate: ein neues Konzept in der Krebstherapie

“…So entstanden die Verbindungen DC4 und DC44 (Abbildung 30) mit deutlich besserer Lçslichkeit (3000-mal besser als DC1) und stark erhçhter Stabilität unter den herrschenden wässri-gen Konjugationsbedingungen. [103] Der Einbau der PhosphatVorstufe änderte weder die Aktivität noch die Spezifität der Abbildung 27. Strukturen der Duocarmycine A und SA und des Cyclopropabenzindolrestes (CBI).…”