Comprehensive Overview of Antibody-Drug Conjugates (ADCs)

To completely avoid periodate treatment, some research groups have performed enzymatic modifications of oligosaccharides by introducing sugar residues with bioorthogonal groups. Bogman et al. demonstrated that using a mutant β1,4-galactosyltransferase GalT (Y289L) can incorporate galactose containing a ketone handle at position C2 into the antibody oligosaccharides. Selective coupling can then occur between the ketone handle and derivatives containing aminooxy functional groups (biotin or fluorescent dyes). However, the conversion rate and efficiency of this process need further improvement. Zeglis et al. applied enzyme-mediated glycan engineering and strain-promoted azide-alkyne cycloaddition reactions to achieve site-specific radioactive labeling of PSMA antibody J591 (see below image). They used β1,4-galactosidase to remove terminal galactose, followed by GalT (Y289L) to introduce azide-modified galactose as the terminal residue of the glycan. They then used chelators containing desferrioxamine (DFO) and employed strain-promoted azide-alkyne cycloaddition reactions to connect to the antibody. Finally, by mixing the chelator-modified antibody with 89Zr, they generated 89Zr-labeled ADCs. The 89Zr-labeled antibodies produced by this method exhibited selective uptake by tumors and demonstrated excellent anti-tumor activity in mouse models of prostate tumors expressing PSMA.

As an alternative strategy, Li and colleagues used Gal T to remodel the antibody glycosylation, transforming existing glycoforms into G2 glycoforms (see image E). They then incorporated sialic acid derivatives containing azide at position C9 into the N-glycans using sialyltransferase ST6GalI. Subsequently, biotin, fluorescent groups, or cytotoxic drugs modified with dibenzocyclooctyne (DIBO) reacted with the azide-containing antibodies, forming antibody conjugates with DAR values of 3.5-4.5. This glycosylation remodeling strategy provides an attractive alternative for preparing homodimeric ADCs with unchanged FcγRIIIa binding. For pharmacokinetic reasons, to achieve a lower DAR, van Delft and colleagues reported a new chemical enzyme coupling strategy to generate ADCs with a DAR of 2 (see image F). After deglycosylation and separately introducing GalNAz through endo-sialidase Endo S2 and galactosyltransferase GalT (Y289L), the modified antibodies can be copper-clicked to effective payloads, resulting in highly stable and homogeneous ADCs with a DAR of approximately 2.

In addition to using enzymatic and chemical strategies for the engineering of antibody oligosaccharides in vitro, it is also possible to introduce sugar analogs containing bioorthogonal reaction groups into ADCs through metabolic engineering of oligosaccharide residues. Okeley and colleagues first reported the use of fucosyltransferase VIII to incorporate fucose analog 6-thiofucose at the terminal N-glycans (see below image). Specifically, 6-thiofucose, screened from about 200 synthetic fucose analogs, can replace fucose in the antibody oligosaccharides with an efficiency of 60-70%. Subsequently, the 6-thiofucose moiety can be coupled with MMAE via maleimide, producing ADCs with an average DAR of 1.3. Compared to ADCs prepared through interchain disulfide/maleimide conjugation chemistry, the ADCs obtained using this strategy maintained improved plasma stability and in vitro anti-tumor activity.

Summary: Despite significant progress in obtaining IgGs with bioorthogonal groups on their glycans, most enzyme-mediated glycosylation engineering techniques remain in the exploratory phase at the laboratory scale. Furthermore, to produce ADCs, some non-natural components must always be introduced into the antibody glycans. Some subtly modified glycans have been reported to have immunogenicity in humans. Therefore, the in vivo stability and immunogenicity of glycan-engineered antibody conjugates still require thorough evaluation.

Conceptually, performing chemical reactions to directly modify specific amino acid residues based on chemoselectivity is the most straightforward method for modifying native antibodies. However, slight differences in the reactivity of different nucleophilic amino acids make it challenging to achieve the desired specificity in chemical modifications. Additionally, due to the heterogeneity of residues between structurally similar antibodies, chemical and regioselective modification methods are typically only effective in specific cases, limiting the generalizability of this approach. A widely used concept is to target lysines with the lowest pKa by precisely controlling the reaction pH; for example, the basicity of the N-terminal amine is weaker than that of other lysines, thus it can be targeted to react under conditions of pH 7.7 ±0.5. Below is a summary of the latest developments in site-specific antibody modifications based on chemoselectivity.

In 2007, Scheck et al. reported the use of pyridoxal-5′-phosphate (PLP, vitamin B6) to convert the N-terminal amine of immunoglobulin light chains into ketones through an amine transfer reaction. The ketones can then be easily functionalized via oxime ligation. Compared to other side chains of lysine residues, the basic N-terminal amine (imine formation) and the more acidic N-terminal α-proton (tautomerization) provide the basis for the N-terminal amine’s chemical selectivity. Moderate conversion rates (47%) were achieved under elevated temperatures (50°C). Based on the same transamination mechanism, Witus et al. selectively converted the N-terminal amine of immunoglobulin heavy chains into ketones using Rapoport salt (RS) under much milder conditions (37°C), achieving an improved yield of 67% (see below image). These amine transfer reagents exhibit some residue preferences; for example, in mouse IgG, PLP prefers light chain aspartic acid over heavy chain glutamine, while RS is more inclined toward heavy chain glutamic acid rather than light chain aspartic acid. Further experiments indicated that the optimal reaction partners for PLP and RS are the tripeptides H-Ala-Lys-Thr and H-Glu-Glu-Ser. Since these N-terminal sequences do not exist in native antibodies, genetic engineering is required to achieve optimal reactivity.

Several attempts have been made to selectively modify certain antibody lysine residues based on controlling reaction kinetics, protein site specificity, or targeting highly reactive lysines. However, due to the unclear mechanism of lysine selectivity, these methods have failed to achieve high yields. Therefore, it has been challenging to apply them to the modification of antibodies or other proteins. To address this issue, Matos et al. reported the use of reagents based on acrylamide sulfonate, combined with pH control, achieving selective and high-yield modifications (>95%) of a single lysine with the lowest pKa (see below image). The sulfonyl oxygen stabilizes the positive charge produced by the amine through a chair-like transition state addition. The smaller polarity of the cysteine S-H group reduces hydrogen bonding interactions, thus explaining the chemical selectivity of lysine over cysteine. The regioselectivity is mainly based on pKa control, as lysines with the lowest pKa are most likely to be reactive under neutral to slightly alkaline conditions. Lysine residues modified with acrylamide can further bind with amine-containing compounds through nitrogen-hetero-Michael addition. The universality of this modification strategy has been successfully demonstrated with five model proteins, including a humanized full-length trastuzumab antibody. Flow cytometry experiments showed that the site-specific modification of trastuzumab with the anticancer drug crizotinib did not affect its binding affinity and specificity towards human HER2. However, if the modified lysine is crucial for antibody function, the conjugation may fail.

Adusumalli et al. developed another reactivity-based method called key-directed modification (LDM). LDM utilizes fast and reversible amine-reactive groups to guide a slow irreversible reaction between epoxy groups and protein histidine residues (see below image). While a rapid switch equilibrium was established between hydroxymethylbenzaldehyde and lysine residues, the attached epoxide portion would only react with histidine located within a certain distance from the lysine residues. After the irreversible linkage between histidine and the epoxide occurs, the free aldehyde can undergo oxime ligation to conjugate other molecules of interest. Two ADCs, trastuzumab-doxorubicin and trastuzumab-emtansine, have been produced using this technology, exhibiting anti-tumor activity in vitro. To expand the substrate range of LDM, Adusumalli et al. reported another LDM reagent for modifying lysines instead of histidines. By converting the leaving group from the epoxide to 2,6-dibromo-4-(morpholine-4-carbonyl)phenyl ester, more options for substrate optimization are provided.

Summary: The selective reactions between probes and specific natural residues (e.g., lysine, cysteine, serine) are becoming an important class of antibody conjugation methods. These strategies enable the preparation of site-specific antibody conjugates without additional antibody engineering or modifications. Currently, efforts based on proximity conjugation are mainly focused on new proximity-inducing chemistries and antibody conjugates that cause minimal disruption to antibody structure and function.

Due to the high therapeutic index and excellent biochemical properties of site-specific ADCs, many efforts are currently focused on site-specific conjugation strategies for antibodies. Numerous methods have been developed for the specific covalent connection of functional molecules to antibodies, most of which require antibody engineering to install unique reactive portions at specific sites, followed by the use of bioorthogonal chemistry to selectively modify these portions. While these technologies provide excellent control over conjugation sites, they have drawbacks such as technical challenges and a lack of generalizability in antibody engineering strategies. Therefore, methods for site-specific conjugation of natural antibodies are more attractive.