Review of ADC bioconjugation technology and research progress




Review of ADC bioconjugation technology and research progress

The design of clinically successful ADCs depends not only on the effectiveness of payloads and their junctions, the stability of linker and effective drug release, but also on the selection of antibodies and bioconjugation technologies. In the past 10 years, all FDA-approved ADCs have been in the form of heterogeneous mixtures of ADCs, with different amounts of drugs attached to different positions of monoclonal antibody. The conjugating sites have significant effects on ADC stability and pharmacokinetics. High DAR (drug-antibody ratio) often leads to rapid plasma clearance, while ADCs with low DAR show weaker activity. In ADC drugs, the presence of nude monoclonal antibody is an effective competitive inhibitor. Therefore, a large number of new conjugating strategies have been developed over the past decade to control the location and number of small molecule drugs while maintaining structural integrity and homogeneity.

Chemical-based modification of specific in situ antibodies

The natural structure of monoclonal antibodies provides a variety of possibilities for biological conjugating, and chemical-based and specific natural (non-engineering) antibody conjugating has some advantages. It can avoid the complexity of antibody specific site mutation and the possible challenges in the amplification and optimization of cell culture. According to the antibody sequence, the junctions of endogenous amino acids such as lysine, histidine, tyrosine and cysteine between disulfide bonds are very attractive. All FDA-approved ADCs were conjugated using these endogenous amino acids until 2021. However, the antibody scaffold also contains glycans, which are caused by the post-translational modification of FC region during the monoclonal antibody production process. Several studies have reported new strategies for glycoengineering, which seems to be an interesting alternative to bioconjugation.

  • Conjugating with endogenous amino acids

One of the most common conjugating methods is using the lysine residue of antibody, the amino acid nucleophilic NH2 group reacts with the electrophilic N-hydroxysuccinimide (NHS) group on the payload. Although the reaction is simple, the high abundance of available lysine residues leads to the formation of heterogeneous mixtures of many ADCs under random distribution. DAR is controlled by drug/antibody stoichiometric ratio and this method has been widely used, including approved ADCs such as Besponsa, Mylotarg, and Kadcyla.

Recently, specific modifications of lysine sites and residues have also been reported. Through computer-aided design, sulfonyl acrylate was used as an intermediate reagent to modify single lysine residue on natural protein sequences. The reaction regioselectivity is attributed to the design of sulfonyl acrylates and the unique local microenvironment around each lysine. The calculation indicated that the lysine with the lowest pKa was easily preferentially reacted at weak alkaline pH in a site-specific manner. This reaction was observed even in the presence of other nucleophilic residues such as cysteine. This technology has been applied to five different proteins and trastuzumab, all of which retain the original secondary structure and protein function after conjugating.

In 2018, Rai et al. reported another site-specific modification using reversible intermolecular reactions of key chemical proteins. This reagent carries a variety of functional groups that reversibly form imine moieties on all available lysine residues. Then, the key protein reacts with proximal histidine residues through epoxide in the reagent. Therefore, the key protein is separated from the lysine under physiological conditions, and the aldehyde is regenerated so that antibodies can be labeled by oxime binding. The targeted modification technology of this key protein was later developed into monolysine residue labeling technology, which has unquestionable selectivity even in the presence of N-terminal amine.

Merlul et al. recently reported a different binding strategy to effectively target histidine residues on natural antibodies. They introduced a linker based on cationic organometallic platinum (II) (labeled as Lx). This technique is based on two steps of complexation and conjugating. N-heterocyclic ligands such as piperidine coordinate with Lx to form a complex precursor. The stable intermediate contains a payload and a chloride ion on the ligand. The complex contains Pt (II) centers with positive charge, which improves the water solubility of linker and payload and minimizes antibody aggregation. This method is also extended to similar iodine complexes. In a recent report, the use of sodium iodide has been proved to significantly improve the conjugating yield and selectivity of this technology. The exchange of chlorine ligand residues on the Cl-Lx-payload complex with iodide produces more active I-Lx- payload, resulting in higher conjugating yields. This technology has been applied to large-scale production of ADC drugs.

  • Rebridging conjugation strategy of disulfide

The IgG antibody contains four interchain disulfide bonds, two light and heavy chains and two hinge regions connecting two heavy chains. They maintain the integrity of monoclonal antibody. Another classical biological conjugating approach explores the role of these cysteines as payload junctions.  The reduction of four disulfide bonds usually produces eight thiols, which can react with the maleimide linker to produce ADC with DAR of 8.

Doronina et al. reported an ADC example with DAR=8, that is, chimeric anti-CD30 monoclonal antibody conjugating MMAE. Compared with the classical lysine conjugation, this payload loading method is better controlled. However, it is reported that higher drug load increases the risk of aggregation, resulting in higher plasma clearance and lower in vivo efficacy. Badescu and al reported a new site-specific rebridging conjugation strategy in 2014, which was the first to prove that the new bis-sulfone could alkylate two thiols from the reduced disulfide in the antibody and antibody fragments with minimal impact on antigen binding. Later, Wang and al described a new water-soluble allyl sulfone, which improved the reaction activity without in-situ activation. It showed high stability, high water solubility and site specificity.

Chudasama et al. introduced a new rebridging reagent in 2015, that is, dibromopyridazinediones. They proved that it could be effectively inserted into disulfide bonds, and the resulting structure showed excellent hydrolysis stability even at high temperature. However, heterogeneity was also observed with the increase of reduction temperature and this structure also allowed the selective introduction of different functional groups. Divinylpyrimidine is another effective rebridging reagent that can produce stable ADC with DAR=4. Spring et al. studied the effect of vinyl heteroaryl scaffold on cysteine rebridging. They believed that substituted pyridine with pyrimidine could make heteroaromatic become better electron acceptor, thereby improving the crosslinking efficiency. Their work was extended to divinyltriazines, where rebridging showed higher efficiency at high temperatures.

To avoid the disadvantages of in vivo instability associated with classical maleimide conjugation, Barbas et al. studied methylsulfonyl phenyl oxadiazole which had specific reactions to cysteine. They are more stable than cysteine-maleimide conjugates in plasma. Inspired by this, Zeglis designed the DiPODS reagent, which contains two oxadiazolyl methyl sulfone moieties linked by a phenyl group. DiPODS forms a covalent bond with two sulfate radicals through rebridging manner. This way has superior in vitro stability and in vivo performance comparing with maleimide conjugating.

  • Polysaccharide conjugating

Since IgG is a glycoprotein, it contains an N-glycan at N297 position of each heavy chain CH2 domain in Fc fragment. This glycosylation can be used as a junction for the payload. The long-distance localization between polysaccharide and Fab region reduces the risk of damaging the antigen binding ability after coupling. In addition, their chemical composition is different from the peptide chain of antibody, which allows site-specific modification so that they become appropriate coupling sites. Glycan bioconjugation can be distinguished according to the technology used to target carbohydrates, that is, glycanmetabolism engineering, glycotransferase treatment after glycan oxidation, endoglycosidase and glycosyl-transferase treatment and azide tagging.

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Neri et al. reported the site-specific modification of fucose at the N-glycosylation site of IgG antibody. This glucose contains a cis-diol portion that is suitable for selective oxidation. They oxidized fucose residues with sodium metaperiodate to generate aldehyde group that can react with hydrazine-containing linker. In this way, the antibody is linked to the drug through hydrazone bond.  Senter et al. added sulfur analogues to the cell culture medium to bring 6-thiofucose into the antibody modification by metabolism. They believe that substitution is accomplished by hijacking the fucosylation pathway, so chemical sites are introduced to achieve site-specific binding. Compared with classical cysteine conjugates, this method significantly reduces the heterogeneity level and produces conjugates with more predictable pharmacokinetic and pharmacodynamic properties.

Recombinant IgG rarely contain s sialic acid. However, it has been proved that glycine can be modified enzymatically by using galactosyl and sialic acid transferase. Galactose was added through an enzyme reaction to obtain G2 glycans, and then terminal sialic acid was added. This modification generates an aldehyde group through periodic acid oxidation, which can be conjugated to linker-payload with hydroxylamine group. The obtained conjugate has high targeting selectivity and good anti-tumor activity in vivo. Periodic acid can also oxidize sensitive amino acids such as methionine and affect the binding to FcRn.

In addition to these conjugation strategies, galactose residues can also be used as modification sites. Several studies have reported the use of mutant β-1,4-galactosyl transferase to replace galactose with a galactose containing ketone or azide functional groups. This galactose derivative with biorthogonal functional groups has opened up a way for efficient conjugating. These technologies have been developed for imaging and anti-cancer applications.

Site-specific bioconjugation of engineered antibodies

The progress in bioorthogonal chemistry and protein engineering contributes to produce more uniform ADC. Although there are many available attachment methods for natural monoclonal antibodies, site-specific bioconjugation on engineered antibodies can more effectively control DAR and avoid changing the affinity of antigen binding. In this way, natural or non-natural amino acids are added at certain positions to obtain homogeneous products with excellent pharmacokinetic and pharmacodynamic characteristics.

  • Enzymatic method

The attachment of the payload can be achieved in a very selective manner by inserting specific amino acid tags in the antibody sequence. These tags are recognized by specific enzymes, such as formylglycine-generating enzyme (FGE), microbial transglutaminase (MTG), transpeptidase or tyrosinase, which enables site-specific conjugating.

Aaron et al. explored a new site-specific conjugating of aldehyde-labeled proteins. This technology utilizes the gene-coded pentapeptide sequence (Cys-X-Pro-X-Arg), in which the cysteine residues are recognized by FGE and co-translated to formylglycine during protein expression in cells. In this way, the engineering antibody is selectively conjugated with the aldehyde-specific linker by HIPS (hydrazino-Pictet-Spengler) chemical method. Microbial transglutaminase (MTGase) strategy is also often developed for localization-specific conjugating. MTGase catalyzes the formation of peptide bond between the glutamine side chain at 295-position of deglycosylation antibody and primary amine of substrate. Compared with other enzyme strategies, MTG is a flexible technology that does not require peptide donors to achieve conjugating. As long as the acyl receptor contains a primary amine, there is no structural limitation. Glutamine residues naturally exist in the Fc region of each heavy chain of monoclonal antibodies. After deglycosylation at 295-position, glutamine residues are conjugated via MTGase-mediated reaction to produce uniform ADC with DAR=2. In order to improve efficiency, the linker with branched chain can be conjugated to double the DAR. The mutation of asparagine at 297-position to glutamine can also increase the DAR.

NBE Therapeutics developed transpeptidase A-mediated conjugating based on S. aureus. Their strategy is to use transpeptidase A (SrtA) to cleave the amide bond between threonine and glycine residues in the motif of LPXTG (X=any amino acid) pentapeptide. Then, it catalyzes the conjugating of glycine-related payload with the newly generated C-terminal to generate peptide bonds at physiological temperature and pH. This method is applied to different antibodies (such as anti-CD30 and anti-Her2) and Maytansine and MMAE are conjugated using 5-glycine-labeled linker. Both ADCs show similar cytotoxicity to classical conjugates in vitro. The trastuzumab-Maytansine produced by enzymatic method completely matches Kadcyla in vivo. In another example, the ADC of highly efficient anthracycline toxin derivative PNU-159682 was generated by transpeptidase method. Interestingly, the conjugating efficiency is even higher than that of Adcetris and Kadcyla analogues with this technology. In addition, the prepared PNU-159682 ADC has high stability in vitro and in vivo and shows more potency than ADC containing tubulin targeted payload.

  • Cysteine engineering: thiomonoclonal antibody technology

Random cysteine conjugating and rebridging are technologies that utilize the natural cysteine residues in the antibody structure. However, the heterogeneity of random cysteine method and the fragmentation of monoclonal antibodies in rebridging strategy need to be considered in ADC synthesis, especially when hydrophobic drugs are conjugated. Different from them, the thiomonoclonal antibody technology achieves selective and uniform modification of the desired sites on the antibody by using engineering reactive cysteine that does not involve the structural disulfide bond. In general, cysteine mutations are designed to promote cytotoxic payload conjugating while maintaining the stability and affinity of monoclonal antibodies and minimizing ADC aggregation. In order to determine the optimal location of mutations, several techniques are usually used, including computational modeling, model system screening and high-throughput scanning.

Junutula et al. first reported a thiomonoclonal antibody strategy in which an engineered cysteine residue was substituted for the alanine at position 114 (HC-A114) of the heavy chain of the anti-MUC16 antibody, and reactive thiols in the engineered position can react with Maleimide-payload linkers. The synthesized anti-MUC16 ADC showed potency in xenograft mouse model and high dose tolerance in rats and cynomolgus monkeys, which established a general method for the thiomonoclonal antibody conjugating strategy.

In addition, succinimide can undergo two parallel reactions in the cytoplasm, that is, the reverse Michael reaction leads to the loss of linker-payload and the hydrolysis of succinimide. Both reactions have significant effects on the ADC activity in vivo. To improve stability, Lyon and his collaborators designed a linker in which the basic amino group adjacent to the maleimide was integrated. The addition of diaminopropionic acid (DPR) in the linker promoted the rapid quantitative hydrolysis of thiosuccinimide at neutral pH and room temperature, so that nonspecific deconjugating effect was prevented, thereby improving the stability in vivo. In addition to the commonly used maleimides, different cysteine reagents were also explored, such as iodoacetamide, bromoformamide, carbonyl acrylate, and N-alkylvinylpyridine salt.

Bioconjugation with engineering unnatural amino acids

In addition to thiomonoclonal antibody technology, the addition of non-standard amino acids (ncAA) provides another possibility for site-specific conjugating. This technology uses amino acids with unique chemical structures, so that linker-payload complexes can be introduced in a chemoselective manner. This technique requires the recombination of antibody sequences and use all endogenous tRNAs and aminoacyl tRNA synthetases (aaRS) in host cells to respond to unvalued codons to bring ncAA into proteins. Usually, ncAA is added to the medium during fermentation. Choosing unnatural amino acids is important because they may stimulate immunogenicity. Commonly used ncAAs are analogues of natural amino acids with unique groups, such as ketones, azides, cyclopropenes or dienes.

Studies have successfully integrated p-acetylphenylalanine (pAcF) into anti-CXCR4 antibodies.  The payload Auristin was effectively coupled to the antibody through oxime linker, so as to generate chemically uniform ADC. The ADC showed good in vitro activity in mice and completely eliminated lung tumors. Due to the acidic conditions required for oxime connection and the sustained release kinetics of ADC, another option is to add azides containing ncAA. The widely used p-azidopiperidine (pAzF) can rapidly react with CuAAC or SPAAC under physiological conditions. This strategy is used to successfully conjugate the glucocorticoid payload to the anti-CD74 antibody. In addition to the pAcF technology, lysine analogues containing azide (AzK) were successfully introduced into the antibody to produce site-specific ADCs with Auristin, PBD dimer or tubulin payload. Furthermore, cyclopropene derivatives of lysine (CypK) and naturally occurring atypical amino acids such as selenocysteine (Sec) were successfully integrated into the antibody. The resulting ADC showed good stability, selectivity, and in vitro and in vivo activities.

Overview of ADC clinical development

At present, the rapid development of payloads, linkers and conjugating technologies have enabled ADCs to have higher uniformity, stability and generation efficiency. This has provided the impetus for the clinical development of ADC drugs. The following three tables summarize the ADCs in the late stage of clinical development as of February 2021 according to the classification of small molecules, biological macromolecules, and radioisotopes.

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  1. The Chemistry Behind ADCs. Pharmaceuticals (Basel). 2021 May; 14(5): 442.
* Only for research. Not suitable for any diagnostic or therapeutic use.


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