AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics 2017:
- Poster:MORAb-202: a Folate Receptor-α (FRA)-targeting antibody-drug conjugate, exhibiting targeted antitumor activity and bystander elimination of cancer-associated fibroplasts
World ADC 2017:
- Poster: Eribulin, a novel microtubule-targeting payload for antibody-drug-conjugate development
- Poster: Site-Specific Conjugation to Native & Engineered Lysines in IgG by Transglutaminase
- Poster: MORAb-202, a novel antibody-drug conjugates (ADC) comprised of farletuzumab conjugated with eribulin, exhibits long-lasting targeted antitumor activity and payload-mediated bystander effects on the tumor microenvironment.
- Site-Specific Conjugation: The user of microbial transglutaminase (MTG) to produce site-specific-antibody-drug conjugates (ADCs) has thus far focused on the transamidation of engineered acyl donor glutamine residues in an antibody based on the hypothesis that the lower specificity of MTG for acyl acceptor lysines may result in the transamidation of multiple native lysine residues, thereby yielding heterogeneous products. We investigated the utilization of native lgG lysines as acyl acceptor sites for glutamine-based acyl donor substrates. Of the approximately 80 lysines in multiple recombinant lgG monoclonal antibodies (mAbs), none were transamidated. Because recombinant mAbs lack the C-terminal Lys447 due to cleavage by carboxypeptidase B in the production cell host, we explored whether blocking the cleavage of Lys447 by the addition of a C-terminal amino acid culd result in transamidation of Lys447 by a variety of acyl donor substrates. MTG efficiently transamidated Lys447 in the presence of any nonacidic, nonproline amino acid residue at position 448. Lysine scanning mutagenesis throughout the anibody further revealed several transamidation sites in both the heavy- and light-chain constant regions. Additionally, scanning mutagenesis of the hinge region in a Fab' fragment revealed sites of transamidation that were not reactive in the context of the full-length mAb. Here, we demonstrate the utility of single lysine substitutions and the C-terminal Lys447 for engineering efficient acyl acceptor sites suitable for site-specific conjugation to a range of glutamine-based acyl donor substrates.
- mAbs: The prevailing techniques used to generate antibody-drug conjugates (ADCs) involve random conjugation of the linker-drug to multiple lysines or cysteines in the antibody. Engineering natural and non-natural amino acids into an antibody has been demonstrated to be an effective strategy to produce homogeneous ADC products with defined drug-to-antibody ratios. We recently reported efficient residue-specific conjugation technology (RESPECT) where thiol-reactive payloads can be efficiently conjugated to a native unpaired cysteine in position 80 (C80) of rabbit light chains. Deimmunizing the rabbit variable domains through humanization is necessary to reduce the risk of anti-drug antibody responses in patients. However, we found that first-generation humanized RESPECT ADCs showed high levels of aggregation and low conjugation efficiency. We correlated these negative properties to the phenylalanine at position 83 present in most human variable kappa frameworks. When position 83 was substituted with selected amino acids, conjugation was restored and aggregation was reduced to levels similar to the chimeric ADC. This engineering strategy allows for the development of second-generation humanized RESPECT ADCs with desirable biopharmaceutical properties.
- Poster: Site-Specific Conjugation to a Native Lysine in IgG by Microbial Transglutaminase
- Cancer Biology & Therapy Journal Article: The conjugation of toxins, dyes, peptides, or proteins to monoclonal antibodies is often performed via free thiol groups generated by either partial reduction methods or engineering free cysteine residues into the antibody sequence. Antibodies from the rabbit Oryctolagus cuniculus have an additional intrachain disulfide bond, whereby the light chain variable kappa domain is bridged to the constant kappa region between cysteine residues at positions 80 and 171, respectively. Chimerization of rabbit antibodies with human constant domains allows for the generation of a free thiol group at the light chain position 80 (C80) that can be utilized for site-specific conjugation. An efficient process for the purification of highly conjugatable antibody was developed. The unpaired C80 was shown to be efficiently conjugated using a number of different maleimido-based ligands. REsidue SPEcific Conjugation Technology (RESPECT) antibody-drug conjugates prepared using rabbit-human chimeric anti-human mesothelin rabbit antibodies and maleimido-PEG2-auristatin conjugated to C80 were shown to be highly potent and specific in vitro and effective in vivo in reduction of tumor growth in a highly aggressive mesothelin-expressing xenograft tumor model.
- Poster: RESPECT (REsidue-SPEcific Conjugation Technology): Platform technologies utilizing native cysteine and lysine residues for the generation of homogeneous antibody-drug conjugates
World ADC 2016:
- Abstract and Poster: Residue-Specific Conjugation Technology (ReSpeCT) utilizing a native cysteine residue in the light chain of Oryctolagus cuniculus immunoglobulins
- Abstract and Poster: Site-specific Conjugation to a Native Lysine in IgG by Microbial Transglutaminase