Site-Specific Conjugation of the Indolinobenzodiazepine DGN549 to Antibodies Affords Antibody–Drug Conjugates with an Improved Therapeutic Index as Compared with Lysine Conjugation

Abstract

Antibody−drug conjugates have elicited great interest recently as targeted chemotherapies for cancer. Recent preclinical and clinical data have continued to raise questions about optimizing the design of these complex therapeutics. Biochemical methods for site-specific antibody conjugation have been a design feature of recent clinical ADCs, and preclinical reports suggest that site-specifically conjugated ADCs generically offer improved therapeutic indices (i.e., the fold difference between efficacious and maximum tolerated doses). Here we present the results of a systematic preclinical comparison of ADCs embodying the DNA-alkylating linker-payload DGN549 generated with both heterogeneous lysine-directed and site-specific cysteine-directed conjugation chemistries. Importantly, the catabolites generated by each ADC are the same regardless of the conjugation format. In two different model systems evaluated, the site-specific ADC showed a therapeutic index benefit. However, the therapeutic index benefit is different in each case: both show evidence of improved tolerability, though with different magnitudes, and in one case significant efficacy improvement is also observed. These results support our contention that conjugation chemistry of ADCs is best evaluated in the context of a particular antibody, target, and linker-payload, and ideally across multiple disease models.

Chemical Diversification of Simple Synthetic Antibodies

“…32,[35][36][37][38] Numerous approaches to antibody chemical diversification have been explored extensively within the context of antibody-drug conjugates (ADCs). [39][40][41][42][43][44][45][46] However, most ADC development strategies focus on modification sites located in antibody constant regions that have little or no effect on antigen binding; [46][47][48][49][50][51][52] the result is modular addition of new chemistries that leave antibody binding function unchanged. In contrast, only sparse studies exist that examine the effects of adding chemical functionality near antibody complementarity determining regions (CDRs).…”

Site-Specific 89Zr- and 111In-Radiolabeling and In Vivo Evaluation of Glycan-free Antibodies by Azide–Alkyne Cycloaddition with a Non-natural Amino Acid

“…However, the inherent lack of selectivity in the number and location of conjugation of this method leads to concerns regarding heterogeneity, stability, and target affinity of the ensuing conjugate . To address this problem, many groups have developed methods to produce site-specific ADCs, via substituted cysteines, enzymatic modifications of engineered glutamine residues, enzymatic modification of N-glycans, or site-specific incorporation of non-natural amino acids with residues that can be exploited as chemical handles. − …”

“…Ideally, these site-specific ADCs will display improved stability and pharmacokinetics compared to the FDA-approved, stochastically conjugated ADCs. In many reported preclinical studies, comparison of site-specific ADCs against heterogeneous lysine-linked conjugates has shown equal or improved therapeutic efficacy and decreased toxicity of the site-specific ADCs. ,,− However, while it is generally accepted that the site-specific ADCs display improved in vivo properties compared to the conventional immunoconjugates, the degree of improvement is dependent on various factors such as the antibody, linker molecule, targeting antigen, payload, and disease model. ,− Thus, the results obtained for one particular site-specific conjugate system cannot be used to predict the outcomes of another, and each new site-specific ADC has to be evaluated independently.…”

Genetically-encoded discovery of proteolytically stable bicyclic inhibitors for morphogen NODAL

“…51,52 This method has been used for PEGylation of clinically relevant growth factors, 53 for improving the stability of cytokines in preclinical studies, 54 and for the synthesis of antibody-drug conjugates. 55 Libraries with N-terminal Ser have been previously converted to peptide-aldehydes and modied by oximes and hydrazines, 56 benzamidoxime, 57 or Wittig reaction, 58 and used for the selection of diverse chemically-modied peptide ligands. [59][60][61][62][63] Our group has previously demonstrated that the bicyclic topology akin to the one described in Fig.…”