ADC (antibody-drug conjugate) has emerged as a promising class of anti-cancer drugs, with over a dozen ADC drugs currently approved for the treatment of various types of cancer patients. Traditional ADCs utilize the thiol group of cysteine obtained from antibody lysine or cleaved disulfide bonds for coupling. However, they suffer from poor homogeneity and low stability, which impacts their efficacy and therapeutic window.
To address these issues, researchers have developed various site-specific conjugation methods. These methods link cytotoxic agents or chemotherapeutic drugs to specific locations on antibody molecules, such as cysteine, glutamine, non-natural amino acids, short peptide tags, and polysaccharides, resulting in ADCs with high homogeneity, improved stability, and better pharmacological properties.
Achieving site-specific conjugation through specific amino acids
Several natural or engineered amino acids, including cysteine and glutamine, are chosen as site-specific conjugation points.
THIOMAB technology is the first method to modify natural amino acids, specifically unpaired cysteine residues, for site-specific conjugation. This method involves the insertion of cysteine residues at different positions in the heavy chain (HC) or light chain (LC) of antibodies for conjugation. Since engineered cysteines are susceptible to being covered by glutathione or other caps during expression, the antibodies need partial reduction to remove the caps. Subsequently, an unblocked cysteine is reacted with a thiol-containing linker using a thiol-maleimide chemistry approach. Research has shown that ADCs produced through conjugation with the drug linker via the cysteine residue (HC-A114C) display nearly homogeneous conjugates and improved therapeutic indices. For instance, the anti-MUC16 TDC (THIOMAB-drug conjugate) exhibited twice the efficacy in a mouse ovarian cancer xenograft model compared to ADCs prepared using the traditional cysteine method at an equivalent drug dose. Rats and cynomolgus monkeys also showed higher tolerable doses for TDC compared to conventional ADCs.
Subsequent researchers have developed ADCs that involve site-specific conjugation of the drug linker to cysteine residues specifically engineered in the antibody. Currently, several site-specific ADCs have entered clinical trials, such as SGN-CD19B, CD123A, and CD352A (all with HC-S239C mutation); RG7861/DSTA4637S (LC-V205C mutation); IMGN632 (S442C mutation); BAT8003 (A114C mutation); and ADCs based on dual cysteine mutations, such as PF-06804103 (LC-K183C HC-K290C). Two other techniques have also been developed: cysteine insertion, as seen in MEDI2228 (HC-i239C), and HC-terminal peptide fusion, exemplified by ALT-P7 (C-terminal ACGHAACGHA fusion).
However, the use of engineered cysteine antibodies does not guarantee clinical success, and some have been abandoned in development, such as BAT8003 and the three Seagen ADCs mentioned above.
Apart from unpaired cysteine conjugation, thiol-bridging methods have also been developed. Each bifunctional drug linker can capture two free cysteine thiol groups, resulting in DAR4 ADCs with reduced heterogeneity after a complete reduction of all eight interchain disulfide bonds.
Glutamine has also been reported for site-specific conjugation. This method does not involve reducing and oxidizing agents but utilizes glutamine transaminase (MTGase) to transfer amine-containing drug linkers or reactive spacers onto the HC-Q295 deglycosylated antibody. An ADC using this technology, Innate ADC (IPH43), is currently in the preclinical stage.
Achieving site-specific conjugation through non-natural amino acids
Conjugation of drug linkers with non-natural amino acids in antibodies is another novel method for generating homogeneous ADCs at specific sites. Non-natural amino acids introduced into antibodies typically include p-Acetylphenylalanine (pAF), p-Azidomethyl-L-phenylalanine (pAMF), and p-Azidonorleucine. Antibodies with introduced non-natural amino acids can achieve site-specific and quantifiable conjugation with drug linkers, resulting in ADCs with uniform DAR (drug-to-antibody ratio), high efficacy, good stability, and low immunogenicity. However, this approach comes with challenges, such as difficulties in antibody expression and the potential for immunogenicity.
ARX788 by Ambrx is the first antibody-drug conjugate developed using non-natural amino acids and is currently in clinical research. ARX788 utilizes the non-natural amino acid p-Acetylphenylalanine (pAF), where the ketone group on pAF forms an oxime bond with the hydroxylamine group on the payload AS269, enabling specific site conjugation and producing a homogeneous ADC.
Sutro Biophma has developed a cell-free protein expression system that incorporates p-Azidomethyl-L-phenylalanine (pAMF) at specific positions, suitable for subsequent click chemistry conjugation with drug linkers to prepare ADCs. Due to the efficiency of cell-free protein expression, site scanning has been performed to determine the optimal conjugation sites, and this method has been applied to various ADC projects. For example, STRO-001 (DAR2 ADC with conjugation at HC-F404) and STRO-002 (DAR4 ADC with conjugation at HC-Y180 and HC-F404).
Achieving site-specific conjugation through glycoengineering
Conjugation through glycoengineering provides a unique site-specific conjugation method by attaching the drug linker to the N297 glycan located in the CH2 domain. Due to the presence of several different monosaccharides at the non-reducing end of the glycan chain, various methods have been developed to attach the drug linker to these sugars, including fucose, galactose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), and sialic acid (SA).
Okeley and colleagues reported that 6-thiofucose (a fucose analog) can be metabolically incorporated into anti-CD30 or anti-CD70 antibodies. The thiofucose in the antibody is then conjugated to a maleimide containing MMAE drug linker, resulting in a DAR of 1.3. The ADC generated with thiofucose maintains good plasma stability and exhibits strong anti-tumor activity.
Galactose or galactose analogs have also been introduced through galactosyltransferases. Bacterial-promoted click chemistry reactions between alkynes and azides have been used in GlycoConnect, a technology developed by Synaffix. The core of this technology is the enzymatic introduction of azido sugars onto the native antibody’s polysaccharides. The antibody is first subjected to the action of endoglycosidases and GalNAc-T (GalNAc transferase) in the presence of UDP 6-azido-GalNAc to convert natural sugars into homogeneous, truncated, and azido-labeled trisaccharides. Subsequently, the azido-labeled antibody is conjugated with the drug linker to produce a homogeneous ADC. GlycoConnect technology is currently used in three clinical ADC drugs: ADCT-601 (ADC Therapeutics), XMT-1592 (Mersana Therapeutics), and MRG004A (Miracogen).
Furthermore, methods for site-specific conjugation using sialic acid (SA) have also been developed. SA is first transferred onto the antibody and then connected to the amino-modified drug linker through periodate oxidation. Antibodies conjugated using this method have exhibited strong in vitro and in vivo anti-tumor activity, especially in the case of anti-HER2 ADCs.
Another similar approach involves transferring C9-azido sialic acid onto the antibody using copper-free click chemistry, followed by conjugation with a cytotoxic drug containing DBCO (dibenzocyclooctyne).
The uniqueness of glycoengineering methods lies in the conjugation of the drug linker to glycans without the need to design amino acid sequences, and they connect at sites that are distant from amino acid residues. However, it’s important to note that this method does require special reagents and enzymes for glycoengineering.
Achieving site-specific conjugation through short peptide tags
Currently, there are several site-specific conjugation methods that utilize specific short peptide tags containing four to six amino acid residues to conjugate cytotoxic agents.
In a study by Strop and colleagues, they designed a glutamine tag (LLQG) as part of the antibody molecule, where the glutamine in the tag could be recognized by MTG (microbial transglutaminase) for transferring amine-containing drugs. The research demonstrated that the drug linker MMAD could be efficiently transferred to the glutamine tag, including LLQGA on the C-terminus of the heavy chain or GGLLQGA on the C-terminus of the light chain. This ADC exhibited a homogeneous DAR of 2 and showed potent anti-tumor activity.
Another study achieved site-specific conjugation of ADCs through the use of transpeptidase-mediated transpeptidation reactions. Transpeptidases are enzymes that assist in attaching proteins to the bacterial cell wall and assembling pili. These enzymes act on secreted proteins with C-terminal cell wall-sorting signals containing a pentapeptide recognition motif, such as LPXTG, which can undergo an acyl transfer reaction with a specific oligoglycine receptor substrate, replacing the terminal glycine of LPXTG with a receptor’s oligoglycine fragment. This method has been applied to generate ADCs. For instance, NBE Therapeutics used this approach to develop the clinical-stage ADC drug NBE-002 by fusing an LPETG tag to the C-terminus of the heavy chain and then connecting it with the five-glycine-modified cytotoxic payload PNU-159,682. Similarly, GeneQuantum developed the drug GQ-1001 based on the heavy chain C-terminal tag LPGTG.
These methods rely on introducing unique short peptide tags into antibodies for enzymatic modification. While these methods are relatively straightforward, the potential immunogenicity of these short peptide tags is currently not fully understood and requires further clinical validation.
By conjugating cytotoxic agents or chemotherapeutic drugs with engineered specific amino acids, non-natural amino acids, short peptide tags, and N297 glycans, researchers have developed the next generation of site-specific conjugation technologies. ADCs produced using these methods exhibit high homogeneity, ensuring reproducibility between production batches, and have a higher therapeutic index compared to traditional conjugates. Undoubtedly, these novel technologies are poised to play a significant role in further breakthroughs and successes in ADC drug development in the future.