Site-specific conjugation leads to more homogeneous conjugates and allows control of the site of drug attachment. This approach frequently relies on antibody engineering to incorporate rare or non-natural amino acids into the primary sequence of the protein.

A number of novel technologies couple cytotoxins or chemotherapeutic drugs to specifically defined sites in antibody molecules including cysteine, glutamine, unnatural amino acids, short peptide tags, and glycans.

The ADCs produced showed high homogeneity, increased therapeutic index, and strong antitumor activities in vitro and in vivo. Moreover, there are recent trends in using these next generation technologies beyond the cytotoxin-conjugated ADC. These site-specific conjugations have been applied for the generation of many different immunoconjugates including bispecific Fab or small molecule-antibody conjugates, immunosuppressive antibodies, and antibody-antibiotic conjugates.

However, when the primary sequence is unknown or when antibody engineering is not feasible, there are very limited options for site-specific protein modification.

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A group of researchers has developed a transglutaminase-mediated conjugation that incorporates a thiol at a “privileged” location on deglycosylated antibodies (Q295).

This conjugation method employs a reported transglutaminase inhibitor, cystamine, as the key enzyme substrate. The chemical incorporation of a thiol at the Q295 site allows for the site-specific attachment of a plethora of commonly used and commercially available payloads via maleimide chemistry.

The scientists demonstrated the utility of this method by comparing the conjugatability, plasma stability, and in vitro potency of these site-specific ADCs with analogous endogenous-cysteine conjugates. They found that ADCs with a cytotoxic payload prepared using this methodology exhibit comparable in vitro efficacy to stochastic cysteine conjugates while displaying dramatically improved plasma stability and conjugatability.

They also noted that this technique appears to be useful for the incorporation of highly hydrophobic linker-payload combinations without the addition of PEG modifiers.

We postulate a possible mechanism for this feature by probing the local environment of the Q295 site with two fluorescent probes that are known to be sensitive to the local hydrophobic environment.

In summary, we describe a highly practical method for the site-specific conjugation of genetically non-engineered antibodies which results in plasma-stable ADCs with low intrinsic hydrophobicity. We believe that this technology will find broad utility in the ADC community.

Benjamin SR, Jackson CP, Fang S, Carlson DP, Guo Z, Tumey LN. Thiolation of Q295: Site-specific conjugation of hydrophobic payloads without the need for genetic engineering.Mol Pharm. 2019 May 8. doi: 10.1021/acs.molpharmaceut.9b00323. [Epub ahead of print]

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