Site-Specific Conjugation of Monomethyl Auristatin E to Anti-CD30 Antibodies Improves Their Pharmacokinetics and Therapeutic Index in Rodent Models

Abstract

Antibody-drug conjugates (ADCs) have demonstrated clinical benefits that have led to the recent FDA approval of KADCYLA and ADCETRIS. Most ADCs that are currently in clinical use or development, including ADCETRIS, are produced by chemical conjugation of a toxin via either lysine or cysteine residues, inevitably leading to heterogeneous products with variable drug-to-antibody ratios (DARs). Here, we describe the in vitro and in vivo characterization of four novel ADCs that are based on the anti-CD30 antibody cAC10, which has the same polypeptide backbone as ADCETRIS, and compare the results with the latter. Bacterial transglutaminase (BTG) was exploited to site-specifically conjugate derivatives of monomethyl auristatin E (all comprising a cleavable linker) to the glutamine at positions 295 and 297 of cAC10, thereby yielding homogeneous ADCs with a DAR of 4. In vitro cell toxicity experiments using two different CD30-positive cell lines (Karpas 299 and Raji-CD30(+)) revealed comparable EC50 values for ADCETRIS (1.8 ± 0.4 and 3.6 ± 0.6 ng/mL, respectively) and the four cAC10-based ADCs (2.0 ± 0.4 to 4.9 ± 1.0 ng/mL). Quantitative time-dependent in vivo biodistribution studies (3-96 h p.i.) in normal and xenografted (Karpas 299 cells) SCID mice were performed with a selected (125)I-radioiodinated cAC10 ADC and compared with that of (125)I-ADCETRIS. The chemo-enzymatically conjugated, radioiodinated ADC showed higher tumor uptake (17.84 ± 2.2% ID/g 24 h p.i.) than (125)I-ADCETRIS (10.5 ± 1.8% ID/g 24 h p.i.). Moreover, (125)I-ADCETRIS exhibited higher nontargeted liver and spleen uptake. In line with these results, the maximum tolerated dose of the BTG-coupled ADC (>60 mg/kg) was significantly higher than that of ADCETRIS (18 mg/kg) in rats. These results suggest that homogeneous ADCs display improved pharmacokinetics and better therapeutic indexes compared to those of chemically modified ADCs with variable DARs.

Improving theranostics in pancreatic cancer

“…Assuming each amine is equivalent, this would lead to 128 different combinations (eg, no conjugates, seven single conjugates, twenty one double conjugates, etc…). While T‐DM1 is in the clinic and proven to provide clinical benefit, it is widely acknowledged that the heterogeneous distribution affects the PK and PD …”

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“…The introduction of a N297Q mutation enabled them to express an aglycosylated antibody variant that displays four potential acyl-donor sites for MTGase, thereby doubling the number of payloads that can be attached to the antibody [136]. This concept has recently been used to generate ADCs that carry a defined number of toxins per antibody [137,138].…”

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“…38 Innate Pharma has introduced the mutation Asn297Gln, which eliminates the glycosylation site and leads to the production of ADCs with two drugs per heavy chain, as BTGase also recognizes Gln297 as a substrate. 39 Strop and coworkers at Rinat/Pfizer (South San Francisco, USA) have developed a four-residue glutamine tag sequence (LLQG) that can be introduced in any flexible and surface-accessible position in the primary sequence ( Fig. 2A).…”