A Platform for the Generation of Site-Specific Antibody–Drug Conjugates That Allows for Selective Reduction of Engineered Cysteines

Abstract

Engineering cysteines at specific sites in antibodies to create well-defined ADCs for the treatment of cancer is a promising approach to increase the therapeutic index and helps to streamline the manufacturing process. Here, we report the development of an in silico screening procedure to select for optimal sites in an antibody to which a hydrophobic linker−drug can be conjugated. Sites were identified inside the cavity that is naturally present in the Fab part of the antibody. Conjugating a linker−drug to these sites demonstrated the ability of the antibody to shield the hydrophobic character of the linker−drug while resulting ADCs maintained their cytotoxic potency in vitro. Comparison of site-specific ADCs versus randomly conjugated ADCs in an in vivo xenograft model revealed improved efficacy and exposure. We also report a selective reducing agent that is able to reduce the engineered cysteines while leaving the interchain disulfides in the oxidized state. This enables us to manufacture site-specific ADCs without introducing impurities associated with the conventional reduction/oxidation procedure for site-specific conjugation.

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).…”

“…Several strategies for expanding the chemistries within antibodies are feasible, , including post-translational modification of antibody structures containing canonical amino acids − and the introduction of genetically encoded noncanonical amino acids (ncAAs). , Numerous approaches to antibody chemical diversification have been explored extensively within the context of antibody–drug conjugates (ADCs). − However, most ADC development strategies focus on modification sites located in antibody constant regions that have little or no effect on antigen binding; − 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 CDRs.…”

Dual Site-Specific Chemoenzymatic Antibody Fragment Conjugation Using CRISPR-Based Hybridoma Engineering

“…More recently, several groups have reported disulfide bond reduction and rebridging strategies to modify native proteins. 30 − 33 While elegant, such strategies could lead to affinity loss following rebridging and influence antibody rigidity and flexibility, 34 , 35 which has recently been linked to therapeutic efficacy. 36 By targeting the C-termini of both HC and LC, we do not expect that the conjugation strategy applied in our approach will affect structure or flexibility of the antibody fragment, which is supported by the fact the target binding capacity was not compromised.…”

Cancer nanotechnology: current status and perspectives

“…There are currently clinical trials underway with therapeutics targeting the 5T4 antigen (ClinicalTrials.gov Identifier: NCT04202705). SYD1875 is a next generation ADC, comprised of a humanized IgG1 monoclonal antibody targeting the 5T4 oncofetal antigen, and a cleavable linker-drug called valine-citrulline-seco-DUocarmycin-hydroxyBenzamide-Azaindole (vc-seco-DUBA), employing site specific conjugation that improves efficacy, exposure, and manufacturing process [ 164 ]. This proprietary ADC utilizes an inactivated synthetic duocarmycin-based cytotoxin that rapidly decomposes if released prematurely, further demonstrating its specificity and stability [ 155 ].…”

“…The results of that trial demonstrated iNKT cell expansion, CD4 + T cell responses against the 118-143 peptide in 7/8 patients, and CD8 + T cell responses against the 79-116 peptide in 3/8 patients [201]. Here, an additional short peptide (157)(158)(159)(160)(161)(162)(163)(164)(165) is included, which is presented by the highly prevalent HLA-A2.1 molecule. Hence, higher CD8 + T cell responses against this epitope and superior activation of human iNKT cells by IMM60 are expected due to co-encapsulation [199,202].…”