Antibody-drug conjugate library prepared by scanning insertion of the aldehyde tag into IgG1 constant regions

Abstract

The advantages of site-specific over stochastic bioconjugation technologies include homogeneity of product, minimal perturbation of protein structure/function, and – increasingly – the ability to perform structure activity relationship studies at the conjugate level. When selecting the optimal location for site-specific payload placement, many researchers turn to in silico modeling of protein structure to identify regions predicted to offer solvent-exposed conjugatable sites while conserving protein function. Here, using the aldehyde tag as our site-specific technology platform and human IgG1 antibody as our target protein, we demonstrate the power of taking an unbiased scanning approach instead. Scanning insertion of the human formylglycine generating enzyme (FGE) recognition sequence, LCTPSR, at each of the 436 positions in the light and heavy chain antibody constant regions followed by co-expression with FGE yielded a library of antibodies bearing an aldehyde functional group ready for conjugation. Each of the variants was expressed, purified, and conjugated to a cytotoxic payload using the Hydrazinyl Iso-Pictet-Spengler ligation to generate an antibody-drug conjugate (ADC), which was analyzed in terms of conjugatability (assessed by drug-to-antibody ratio, DAR) and percent aggregate. We searched for insertion sites that could generate manufacturable ADCs, defined as those variants yielding reasonable antibody titers, DARs of ≥ 1.3, and ≥ 95% monomeric species. Through this process, we discovered 58 tag insertion sites that met these metrics, including 14 sites in the light chain, a location that had proved refractory to the placement of manufacturable tag sites using in silico modeling/rational approaches.

More than Toxins—Current Prospects in Designing the Next Generation of Antibody Drug Conjugates

“…In a second reaction step the aldehyde serves as a bioorthogonal handle to attach the payload of choice [101,102]. Initially used at the C-terminal position, recently an internal recognition sequence (LCTPSR) was inserted at 436 positions throughout the antibody sequence and screened for suitability of toxin conjugation [103].…”

High-Throughput Platform to Identify Antibody Conjugation Sites from Antibody–Drug Conjugate Libraries

“…Typically, engineered antibodies are individually expressed and purified, followed by conjugation and characterization to determine conjugatability and DAR. High throughput conjugation assays to identify optimal location for drug conjugation have been described; however those methods require individual evaluation of large numbers of variants and the need for expensive robotics. , …”

Hydrophilic Sequence-Defined Cross-Linkers for Antibody–Drug Conjugates

“…Trastuzumab was modified with an aldehyde tag on the C-terminal of the heavy chain via FGE as previously reported. Catalent Biologics’ HIPS ligation technology has been applied to perform unbiased screening of conjugation sites throughout the antibody backbone . Therefore, the system presented here is capable of independently modulating both conjugation site and cross-linker structure.…”