Sortase Enzyme-Mediated Generation of Site-Specifically Conjugated Antibody Drug Conjugates with High In Vitro and In Vivo Potency

Abstract

Antibody drug conjugates (ADCs) have recently been proven to be highly potent anti-tumor drugs, typically exceeding the efficacy of conventional monoclonal antibodies (mAbs). ADCs are currently produced by chemical conjugation of a small-molecule toxin to the mAb through lysine or cysteine side chains. This leads to heterogeneous mixtures of ADCs in which variable numbers of drugs are conjugated to individual antibodies and in which the site of conjugation cannot be defined. Consequently, there is currently significant interest in further development of drug conjugation technologies, with a particular focus on site-specific payload conjugation. Here, we present an enzymatic conjugation platform based on the S. aureus sortase A-mediated transpeptidation reaction, allowing the efficient generation of ADCs with toxins conjugated to pre-defined sites at pre-defined drug-to-antibody ratios. For this, two modifications were introduced: first, immunoglobulin heavy (IgH) and light (IgL) chains were modified at their C-termini by addition of the sortase A recognition motif LPETG, and second, the small molecule tubulin polymerization inhibitors monomethylauristatin E (MMAE) and maytansine were modified by addition of a pentaglycine peptide, thus making them suitable substrates for sortase A-mediated transpeptidation. We demonstrate efficient generation and characterization of the anti-CD30 ADC Ac10-vcPAB-MMAE, an enzymatically conjugated counterpart of brentuximab vedotin (Adcetris), as well as several anti-HER-2 ADCs including trastuzumab-maytansine, the counterpart of trastuzumab emtansine (Kadcyla). ADCs generated in this manner were found to display in vitro cell killing activities indistinguishable from the classic conjugates. Further, when tested in vivo in a HER-2-overexpressing ovarian cancer xenograft mouse model, enzymatically generated trastuzumab-maytansine was found to lead to complete regression of established tumors, similar to Kadcyla.

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

“…Cell killing assays Cytotoxicity of ADCs was investigated in cell killing assays essentially as described previously (24). Briefly, cells were plated on 96-well plates in 75 mL growth medium and grown at 37 C in a humidified incubator in a 7.5% CO 2 atmosphere.…”

“…These strategies not only include the introduction of unique handles for chemical conjugation, such as by incorporation of unnatural amino acids (20) or by site-directed mutagenesis (21), but also various enzymatic approaches, including the use of bacterial transglutaminase (22), formylglycine-generating enzyme (FGE; ref. 23), and S. aureus sortase A (24). Using such novel approaches, highly homogeneous, site-specifically conjugated "next-generation" ADCs can now be prepared (1).…”

“…Site-specifically conjugated and homogeneous ADCs based on a derivative of the anthracycline PNU-159682 (34) were generated using SMAC technology (24), and their in vitro and in vivo activities were investigated.…”

RN927C, a Site-Specific Trop-2 Antibody–Drug Conjugate (ADC) with Enhanced Stability, Is Highly Efficacious in Preclinical Solid Tumor Models

“…One approach that can potentially overcome these limitations is to employ site-specific conjugation of antibodies, and many experimental approaches have been developed, including conjugation through engineered cysteine residues, nonnatural amino acids, antibody carbohydrates, aldehyde tags, and utilizing enzymes such as sortases and phosphopantetheinyl transferases (2,(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). A site specifically conjugated anti-CD33 ADC, SGN-CD33A, has shown encouraging activities in targeting drug-resistant acute myeloid leukemia (37).…”

Antibody-Drug Conjugates in Head and Neck Cancer

“…The resulting conjugate retained epitope generation ability upon binding to dendritic cells and enabled monitoring of intracellular processes in vitro and in vivo. Beerli and co-workers demonstrated the potential of this powerful approach for stoichiometric site-specific ADC conjugation (Beerli et al, 2015) (Fig. 6A).…”