A general approach to site-specific antibody drug conjugates

Abstract

Using an expanded genetic code, antibodies with site-specifically incorporated nonnative amino acids were produced in stable cell lines derived from a CHO cell line with titers over 1 g/L. Using anti-5T4 and anti-Her2 antibodies as model systems, site-specific antibody drug conjugates (NDCs) were produced, via oxime bond formation between ketones on the side chain of the incorporated nonnative amino acid and hydroxylamine functionalized monomethyl auristatin D with either protease-cleavable or noncleavable linkers. When noncleavable linkers were used, these conjugates were highly stable and displayed improved in vitro efficacy as well as in vivo efficacy and pharmacokinetic stability in rodent models relative to conventional antibody drug conjugates conjugated through either engineered surface-exposed or reduced interchain disulfide bond cysteine residues. The advantages of the oxime-bonded, site-specific NDCs were even more apparent when low-antigen-expressing (2+) target cell lines were used in the comparative studies. NDCs generated with protease-cleavable linkers demonstrated that the site of conjugation had a significant impact on the stability of these rationally designed prodrug linkers. In a single-dose rat toxicology study, a site-specific anti-Her2 NDC was well tolerated at dose levels up to 90 mg/kg. These experiments support the notion that chemically defined antibody conjugates can be synthesized in commercially relevant yields and can lead to antibody drug conjugates with improved properties relative to the heterogeneous conjugates formed by nonspecific chemical modification.

Fig. 1.

Fed-batch cell culture results of A1 HC-S115pAF stable cell line. (A) Growth profile in shake flask and 5-L bioreactor. (B) Titer (in milligrams per liter) in shake flask and 5-L bioreactor.

Scheme 1.

Drug and linkers used in efficacy studies. A noncleavable linker with MMAD (NC-D1) was used in synthesizing site-specific NDCs (A1 HC-S115pAF-NC-D1 and Her HC-A114pAF-NC-D1). Linker mc-D1 was used to synthesize conventional anti-5T4 ADC (A1 mc-D1) by conjugation through interchain cysteines. mNC-D1 was used to synthesize anti-Her2 Ab conventional drug conjugate (Her mNC-D1) and a site-specifically engineered cysteine-containing ADC (Her HC-A114C-mNC-D1). The resulting drug loading per Ab was around 4:1 and 2:1 for conventional conjugations and site-specific conjugates, respectively.

Fig. 2.

Nonnative amino acid site-specific conjugates display superior in vivo efficacy compared with conventional and site-specific cysteine conjugates dosed on days 1, 5, 9, and 13. (A and B) In vivo efficacy determination of A1 mc-D1 and A1HC-S115pAF-NC-D1 in 5T4-positive tumor models of MDAMB435/5T4 at 1, 3, and 10 mg/kg. (C and D) Comparison of Her HC-A114pAF-NC-D1 conjugate with Her mNC-D1 and Her HC-A114C-mNC-D1 in two Her2-positive tumor models of MDAMB361DYT2 and HCC1954 at 1 mg/kg.

Fig. 3.

Pharmacokinetics of Ab A1 and its conjugates A1 HC-S115pAF-NC-D1 and A1-mc-D1 over 14 d in mice dosed i.v. with 3 mg Ab/kg (single dose). For the conjugates, the total Ab (Ab with or without drug, A1 HC-S115pAF-NC-D1_Ab, or A1-mc-D1_Ab), NDC (A1 HC-S115pAF-NC-D1), and ADC (A1-mc-D1_ADC) were determined.