Identification of Highly Reactive Cysteine Residues at Less Exposed Positions in the Fab Constant Region for Site-Specific Conjugation

Abstract

Engineered cysteine residues are currently used for the site-specific conjugation of antibody-drug conjugates (ADC). In general, positions on the protein surface have been selected for substituting a cysteine as a conjugation site; however, less exposed positions (with less than 20% of accessible surface area [ASA]) have not yet been evaluated. In this study, we engineered original cysteine positional variants of a Fab fragment, with less than 20% of ASA, and evaluated their thiol reactivities through conjugation with various kinds of payloads. As a result, we have identified three original cysteine positional variants (heavy chain: Hc-A140C, light chain: Lc-Q124C and Lc-L201C), which exhibited similar monomer content, thermal stability, and antigen binding affinity in comparison to the wild-type Fab. In addition, the presence of cysteine in these positions made it possible for the Fab variants to react with variable-sized molecules with high efficiency. The favorable physical properties of the cysteine positional variants selected in our study suggest that less exposed positions, with less than 20% of ASA, provide an alternative for creating conjugation sites.

Efficient Preparation of Site-Specific Antibody–Drug Conjugates Using Cysteine Insertion

“…One approach to overcome ADCs heterogeneity is to use site-specific conjugation, a method in which drug load and site of conjugation is controlled . Moreover, site-specific conjugation at defined sites in the antibody results in stable ADCs in vivo. − Three methods are commonly used to prepare site-specific ADCs: (a) cysteine-mutagenesis at specific residues, − (b) replacement of residues with unnatural amino acids with bio-orthogonal reactivity, − and (c) enzymatic ligation approaches. − …”

Attachment Site Cysteine Thiol pKa Is a Key Driver for Site-Dependent Stability of THIOMAB Antibody–Drug Conjugates

“…We have adopted a cysteine-engineering strategy called THIOMAB antibody technology that allows the straightforward conjugation of novel payloads to yield homogeneous THIOMAB antibody–drug conjugates (TDCs). ,, Native amino acids in the heavy chain (HC) or light chain (LC) of the antibody are mutated to cysteine for conjugation to small molecules, peptides or proteins using standard thiol-reactive coupling chemistries. The homogeneous TDCs produced using an appropriately selected stable attachment site have markedly improved pharmacokinetics, efficacy, and safety profiles in preclinical animal models compared to the heterogeneous ADCs prepared via conjugation to endogenous lysines and cysteines. − , Several other cysteine engineering strategies have been reported − as well as other site-specific conjugation strategies . Candidates utilizing cysteine engineering to produce site-specific conjugates have advanced to clinical evaluation ,, highlighting the importance of studies to further the understanding of this technology.…”

A Simple and Efficient Method to Generate Dual Site-Specific Conjugation ADCs with Cysteine Residue and an Unnatural Amino Acid

“…Another reason for choosing trastuzumab is that it is also a component of some antibody-drug conjugates, such as trastuzumab-emtansine and trastuzumab-deruxtecan, indicating its feasibility for further manipulation . Q124C on the light chain (LC) of trastuzumab-Fab (Tra-Fab, Kabat numbering) , was selected as one conjugate site, A121 on the heavy chain of CH1, which showed minimal perturbation on antibody expression and binding affinity in previous studies, was selected as another residue for substitution by p -acetyl-phenylalanine (pAcF). In Figure A, the position of the 2 Tra-Fab mutants and their association with HER2 are depicted on the crystal structure of the extracellular domain of human HER2 (PDB code 1N8Z).…”

“…The Tra-Fab strain was designed to coexpress the combination of pET21a trastuzumab-Fab-VL-CL and pET26b trastuzumab-Fab-VH–CH1. The Tra-ThioFab (thiol functional group site-specific insertion Tra-Fab) strain was designed to coexpress the combination of pET21a trastuzumab-Fab-LC-Q124C ,, (LC: mutant light chain) and pET26b trastuzumab-Fab-VH–CH1. The Tra-UAAFab (unnatural amino acid-pAcF site-specific insertion Tra-Fab) strain was designed to coexpress the combination of pET21a trastuzumab-Fab-VL-CL and pET26b trastuzumab-Fab-HC-A121X (HC: mutant heavy chain.…”