The random conjugation of toxins, dyes, peptides, or other payloads to monoclonal antibodies often targets free thiol groups generated by partial reduction methods or lysine residues using succinimide- or isothiocyanate-based chemistry. There remains a need for conjugation technologies targeting specific amino acid residues as a way to produce a homogeneous antibody-drug conjugate (ADC) product with a defined drug-to-antibody ratio. Our REsidue-SPEcific Conjugation TechnologyTM (RESPECTTM) utilizes two methods by which payloads can be conjugated to specific residues in an antibody:
Our cysteine-specific conjugation method exploits a unique intra-chain disulfide bond in the light chain of oryctolagus cuniculus-derived antibodies between residues 80 and 171 of the variable and constant domains, respectively. Our humanization strategy allows retention of the cysteine at position 80 with a free thiol group that is both amenable for residue-specific conjugation and compatible with optimal antibody biophysical properties, including antigen binding and structural stability. This platform has been optimized via antibody engineering using in-silico modeling, extensive mutagenesis and crystallographic studies that have defined optimal adjacent residues to the retention of a reactive thiol group, as well as the desired humanized antibody’s properties.
Our C-terminal lysine-specific linkage method employs the transglutaminase enzyme that catalyzes the formation of a stable isopeptide bond between the γ-carboxyamide group (acyl donor) of a glutamine and the ε-amino group (acyl acceptor) of a lysine. While no acyl acceptor sites are present in recombinant wild-type IgG, mAbs lack the C-terminal Lys447 due to cleavage by carboxypeptidase B in the host production cell line. Blocking the cleavage of Lys447 by addition of a C-terminal amino acid results in transamidation of Lys447 by a variety of acyl donor substrates in the presence of non-acidic, non-proline amino acid residue at position 448.
RESPECTTM technologies can be combined to allow for the conjugation of up to two cytotoxic payloads with different mechanisms of action (MOAs), or conjugation of four of the same payload.
Starting RESPECTTM-L (LC) Ab vs. Conjugated RESPECTTM-L (LC+) Ab
Starting RESPECTTM-H (HC) Ab vs. Conjugated RESPECTTM-H (HC+) Ab
* Any antibody drug conjugate (ADC) employing eribulin as a payload is investigational and has not been approved by regulatory authorities. For information on Halaven® (eribulin mesylate), please see the package insert or contact Eisai’s Medical Information toll-free number 1.888.274.2378.