Generation of DAR1 Antibody-Drug Conjugates for Ultrapotent Payloads Using Tailored GlycoConnect Technology

Abstract

GlycoConnect technology can be readily adapted to provide different drug-to-antibody ratios (DARs) and is currently also evaluated in various clinical programs, including ADCT-601 (DAR2), MRG004a (DAR4), and XMT-1660 (DAR6). While antibody-drug conjugates (ADCs) typically feature a DAR2–8, it has become clear that ADCs with ultrapotent payloads (e.g., PBD dimers and calicheamicin) can only be administered to patients at low doses (<0.5 mg/kg), which may compromise effective biodistribution and may be insufficient to reach target receptor saturation in the tumor. Here, we show that GlycoConnect technology can be readily extended to DAR1 ADCs without the need of antibody re-engineering. We demonstrate that various ultrapotent, cytotoxic payloads are amenable to this methodology. In a follow-up experiment, HCC-1954 tumor spheroids were treated with either an AlexaFluor647-labeled DAR1 or DAR2 PBD-based ADC to study the effect on tumor penetration. Significant improvement of tumor spheroid penetration was observed for the DAR1 ADC compared to the DAR2 ADC at an equal payload dose, underlining the potential of a lower DAR for ADCs bearing ultrapotent payloads.

Chemical technology principles for selective bioconjugation of proteins and antibodies

“…289 These reagents can also be designed to engage the glycan motif from both the heavy chains in the antibody ( 102a ). 290 The N -glycan remodelling can be coupled with other antibody engineering approaches. For example, its implementation to conjugate LacNAc-based drug-linker oxazoline can drive Fc-selective peptide-guided acylation of Lys ( 103a ).…”

Disulfide re-bridging reagents for single-payload antibody-drug conjugates

“…4,12 However, there are very few methods for the accurate synthesis of ADCs bearing a single payload. 5–11…”

“…3 However, due to the inherent 2-fold symmetry of antibodies, current site-selective conjugation methods are mostly limited to the synthesis of ADCs with even-integer DAR. 4 While there have been rare reports of the generation of homogeneous ADCs with odd DARs, [5][6][7][8][9][10][11] the applied methods tend to rely on engineered antibodies or bespoke payloads, or have poor efficiency, which greatly limits their widespread applicability. Methods which expand the repertoire of available drug loading, such as for the generic synthesis of ADCs with odd-integer DAR, would enable the synthesis of more finely tuned therapeutics.…”

“…4,12 However, there are very few methods for the accurate synthesis of ADCs bearing a single payload. [5][6][7][8][9][10][11] Our group recently reported the first generation of all-in-one disulfide bridging crosslinking reagents, termed TetraDVPs, which use four divinylpyrimidine (DVP) moieties to simultaneously re-bridge all four interchain disulfides of an IgG1 antibody (Fig. 1).…”

Rapid Access to Potent Bispecific T Cell Engagers Using Biogenic Tyrosine Click Chemistry

“…Most bsAbs are produced via protein engineering of the native mAb framework. − However, the evolution of bio-orthogonal (click) chemistry has facilitated the generation of novel synthetic antibody conjugates, such as ADCs. , At the moment, strain-promoted azide alkyne cycloaddition (SPAAC) and the inverse electron-demand Diels–Alder (IEDDA) reactions between strained unsaturated carbon–carbon systems and tetrazine , or ortho -quinone have been successfully applied for antibody modification …”