Highly Potent, Anthracycline-based Antibody–Drug Conjugates Generated by Enzymatic, Site-specific Conjugation

Abstract

Antibody-drug conjugates (ADC) are highly potent and specific antitumor drugs, combining the specific targeting of mAbs with the potency of small-molecule toxic payloads. ADCs generated by conventional chemical conjugation yield heterogeneous mixtures with variable pharmacokinetics, stability, safety, and efficacy profiles. To address these issues, numerous site-specific conjugation technologies are currently being developed allowing the manufacturing of homogeneous ADCs with predetermined drug-to-antibody ratios. Here, we used sortase-mediated antibody conjugation (SMAC) technology to generate homogeneous ADCs based on a derivative of the highly potent anthracycline toxin PNU-159682 and a noncleavable peptide linker, using the anti-HER2 antibody trastuzumab (part of Kadcyla) and the anti-CD30 antibody cAC10 (part of Adcetris). Characterization of the resulting ADCs in vitro and in vivo showed that they were highly stable and exhibited potencies exceeding those of ADCs based on conventional tubulin-targeting payloads, such as Kadcyla and Adcetris. The data presented here suggest that such novel and highly potent ADC formats may help to increase the number of targets available to ADC approaches, by reducing the threshold levels of target expression required.

Fc-Mediated Anomalous Biodistribution of Therapeutic Antibodies in Immunodeficient Mouse Models

“…This in itself could dampen the in vivo performance of antibody-based radiotracers and the efficacy of therapeutic antibodies by impacting their bioavailability for the intended target expressed by the tumor. More recently, the limited efficacy of antibody-drug conjugates tested in the NSG mouse background has been reported (32). …”

Harnessing a catalytic lysine residue for the one-step preparation of homogeneous antibody-drug conjugates

“…A chemical approach that does not rely on engineered carbohydrates or engineered amino acids implements native Cys rebridging for selective conjugation 25 – 27 . Enzymatic approaches that rebuild glycans 28 , 29 or utilize engineered peptide motifs 30 – 34 for generating site-specific ADCs have also been reported. Although existing site-specific ADC technologies have shown clear advantages over heterogeneous ADCs, an important consideration is that most of these strategies introduce mutations and it remains unclear if these will be problematic in immunocompetent patients.…”

Novel Antibody Drug Conjugates Targeting Tumor-Associated Receptor Tyrosine Kinase ROR2 by Functional Screening of Fully Human Antibody Libraries Using Transpo-mAb Display on Progenitor B Cells

“…Human V H and V L sequences recovered from L11 cell clones were obtained by sequence recovery as previously described and assembled into the pCB14b or pCB14g vector with the respective constant domains ( 36 ). The resulting antibody sequences contained a sortase A recognition motif and a Twin-Strep-tag as described previously ( 38 ) for subsequent conjugation to toxin. These tag sequences were: IgH chain, (LPETG-G-WSHPQFEK(G 3 S) 3 AWSHPQFEKGS); Igκ chain, G 4 S-LPETG-G-WSHPQFEK(G 3 S) 3 AWSHPQFEKGS).…”

“…LPETG-tagged antibodies were site-specifically conjugated to glycine-modified toxins Gly 3 -EDA-PNU or Gly 5 -EDA-PNU using sortase-enzyme mediated antibody conjugation (SMAC-technology TM ), as previously described ( 38 , 39 ). hROR2-specific ADCs were formulated in PBS.…”

“…hROR2-specific ADCs were formulated in PBS. The drug-to-antibody-ratio (DAR) of the final ADCs ranged between 3 and 4, as determined by HPLC ( 38 , 39 ).…”