The popularity of thiol coupling in ADC preparation can be attributed to three factors:
- thiol is the most nucleophilic functional group among amino acid side chains (much stronger than amine);
- the number of interchain cysteine residues in suitable antibodies for coupling (IgG1 and IgG4 have 8) is relatively low, and stability of the antibody is not crucial, allowing for relatively controlled customization of Drug-to-Antibody Ratio (DAR);
- interchain cysteine residues located in the hinge region connecting heavy chain (HC) and light chain (LC) seem advantageous for “hiding” hydrophobic payload after coupling.
It is essential to note that a reduction step is always required before coupling to release free thiol groups. The majority of thiol coupling in ADCs is based on maleimide chemistry. Alkylation occurs at native interchain cysteine residues or engineered cysteine residues. An exception is ADCs prepared through nucleophilic aromatic substitution. Other chemical methods, such as disulfide exchange, are also under development for some preclinical assets.
Reactions with Maleimides
Maleimides readily undergo alkylation reactions with thiol groups through Michael addition, forming stable thioether bonds. Maleimide reactions with thiols are highly specific within the pH range of 6.5-7.5, with reaction rates towards thiols being 1000 times faster at pH 7.0 than towards amines. Hence, complete conversion can be achieved with a small excess of reagent. The main drawback of maleimide alkylation is the reversibility of the Michael addition reaction, which is highly dependent on the pKa of the specific cysteine residue being connected. This reverse Michael reaction may lead to premature release of linker payload in circulation, potentially causing adverse reactions. This is a driving force for developing more stable maleimide variants.
As mentioned earlier, a reduction step is needed for interchain cysteines in monoclonal antibodies before coupling to release thiol groups. Therefore, careful optimization of the amount of reducing agent (usually TCEP or DTT) is performed to release a certain number of free thiol groups (up to 8 in IgG1 and IgG4) before introducing the payload with maleimide functionality. ADCs prepared by conjugating with cysteine residues include Adcetris, Polivy, Padcev, Enhertu, Blenrep, Trodelvy, Zynlonta, etc. Due to the lack of site specificity, these ADCs are usually produced as random mixtures with average DAR ranging from 2.3 to 4. However, Enhertu and Trodelvy are exceptions, achieving a higher DAR (7.7 and 7.6, respectively) due to their lower-toxicity maytansinoid payloads and close to quantitative conjugation of all interchain cysteine residues, termed as site-specific conjugation.
It is noteworthy that most cases use maleimide conjugates (mc); however, some ADCs use less electron-rich β-aminoethyl or ethylene glycol spacers. It has been reported that some phenyl-substituted maleimides are more stable, and clinical development is ongoing.
In 2008, it was reported that site-specific conjugation of cytotoxic linker payload with monoclonal antibodies carrying specific cysteines may enhance the therapeutic index compared to random ADCs. Since then, various site-specific ADCs have entered clinical development, such as SGNCD19B, CD123A, and CD352A (all involving HC-S239C mutation); RG7861/DSTA4637S (LC-V205C mutation); IMGN632 (S442C mutation); and BAT8003 (A114C mutation), and even ADCs based on double cysteine mutations, such as PF-06804103 (LC-K183C+HC-K290C). Additionally, two complementary technologies have been developed, namely cysteine insertion (e.g., MEDI2228 with HC-i239C) and fusion of HC C-terminal peptide tag (e.g., ALT-P7 with C-terminal ACGHAACGHA). Unfortunately, engineering antibodies with cysteines do not guarantee clinical success, as some assets have been discontinued (e.g., BAT8003 and all three Seagen ADCs mentioned above).
To inhibit reverse Michael reactions, hydrolyzing the initial thioether-succinimide adduct to provide maleamic acid derivatives has been proposed. Prolonged treatment of ADCs under pH 9 conditions can achieve this hydrolysis; however, this condition may lead to additional post-translational modifications of antibodies, such as deamidation or aspartic acid formation. Seagen has developed an effective solution to this issue by introducing an α-aminoethyl maleimide based on 2,3-diaminopropanoic acid (DPR technology) at the α-position of the N-alkyl chain. The amino group induces spontaneous hydrolysis of the succinimide conjugate, thus blocking the reverse Michael reaction. ADC product SGN-CD48A (now discontinued) was the first to use DPR technology, and SGN-CD228A also employs this technology.
Conjugation Methods at BOC Sciences
| Name | Description |
| Lys Conjugation | Currently, lysine has been extensively studied in many bioconjugation applications, including the synthesis of antibody-drug conjugates (ADCs), drug delivery, and antimicrobial vaccines. |
| Cys Conjugation | The cysteine conjugation strategy is based on the reaction between cysteine residues of antibodies and specific thio groups anchored on linkers. |
| Unnatural Amino Acid Conjugation | Unnatural amino acid (uAA) conjugation refers to the process of chemically linking or conjugating unnatural amino acids to antibody, protein or peptide sequences. |
| Site-Specific Conjugation | Site-specific conjugation of antibody-drug conjugates (ADCs) refers to the process of attaching drug molecules to specific sites on antibody molecules. |
| Disulfide Conjugation | Disulfide conjugation is a chemical process that involves the formation of a covalent bond between two molecules through a disulfide bridge. |
| Enzymatic Conjugation | Enzymatic conjugation involves modification of an antibody or linker molecule to introduce site-specific attachment points for enzymes. |
Other Cysteine Alkylation Reagents
For instance, the high reactivity of α-haloacetamide reagents is well-known. Taking 2-iodoacetamide as an example, it is an undisputed choice for blocking free cysteines in proteins or protein mixtures (e.g., cell lysates). Importantly, the thioether bond formed is completely irreversible, making it superior in stability compared to thiol-maleimide ether. In fact, Alley et al. reported in 2008 that using α-bromoacetamide hexanoyl linker can provide a highly stable thioether ADC with excellent plasma stability, showing no measurable systemic drug release in mice for two weeks after administration and a 25% increase in tumor drug exposure within seven days. However, despite differences in stability between bromoacetamide and maleimide-based ADCs, there is no significant difference in potency, activity, and toxicity characteristics. The surprising lack of distinction is likely because, for this particular maleimide-based ADC, the half-life of the linker of the ADC and the half-life of ADC clearance are similar: since the concentration of ADC decreases over time, extending the linker half-life beyond the pharmacokinetic half-life is unlikely to have a significant effect on drug exposure.
On the basis of the discovery that methylsulfonylbenzothiazole (MSBT) is a selective thiol-blocking agent, Barbas et al. developed a series of aromatic methylsulfonyl ketone reagents for protein alkylation with thiols, mainly imidazole and benzothiazole. Similarly, methylsulfonylpyrimidine reacts with thiols through nucleophilic aromatic substitution mechanism (see figure below), a concept applied in SKB264/BT001035.
Researchers at Glythera reported highly stable thioether ADCs obtained by incubating free cysteine thiols with 4-vinylpyridine, a technique known as PermaLinks. The resulting ADCs exhibit significant stability and are not easily degraded.
A noteworthy technology in development is the phosphoramidate coupling technology developed by the University of Berlin, currently in early stages but already commercially applied by Tubulis. Studies indicate that arylphosphoramidate salts with electron-deficient triple bonds show excellent selectivity in reacting with cysteines compared to traditional maleimide linkage. The resulting (Z)-vinyl sulfides also exhibit excellent stability.
Double Maleimide or Double Maleimide Acid Alkylation/Crosslinking
The ability of maleimide alkylation has also been applied to various crosslinking double maleimide reagents. Each reagent can capture two free cysteine thiol groups, so after fully reducing all eight interchain disulfide bonds, a DAR4 ADC can be formed. It is worth noting that although interchain crosslinking (e.g., from HC-226C to HC-229C) may produce mixtures, the formed ADCs still have a relatively narrow DAR distribution. NewBio’s NBT828 uses double maleimide crosslinkers. Conjugates of double succinimide after nucleophilic addition with thiol groups may also hydrolyze into a formylamine derivative, providing a conjugate that cannot undergo reverse Michael degradation.
Recently, researchers at MedImmune (AstraZeneca’s research division) also reported coupling engineered cysteine antibodies with double maleimide-modified payload PBD to obtain a DAR1 ADC.
THIObridget Coupling
A novel coupling reagent initially reported by Del Rosario et al. and later developed by Poly-Therics (now called Abzena; utilizing THIObridget technology) utilizes the high nucleophilicity of free thiols and the easily occurring Michael addition reaction. This rebridging technology enhances the stability of the reduced antibody, effectively maintaining the conformational integrity of the final conjugate. Double thiol reaction groups, positioned close at the molecular scale, are suitable for this purpose as they can seamlessly integrate into the protein structure without disrupting interactions between subunits. Applying α-tosylmethyl-a,b-unsaturated ketone reagents to reduced antibodies, where the reagent is generated in situ from double α,α-tosylmethyl ketone precursors, triggers a series of reactions, including Michael addition, b-elimination of tosylate, and subsequent Michael addition, resulting in a stable conjugate.
Alkylation/Coupling through Double Bromomethyl Aromatic Reagents
Concortis (now Sorrento) developed a third method for DAR4 ADCs, named C-lockt technology. C-lockt technology combines the nucleophilicity of thiols with the tendency of benzyl bromide groups to undergo nucleophilic SN2 substitution reactions. Thus, by reducing interchain disulfide bonds and then reacting with double bromomethyl aromatic reagents (e.g., dibromomethyl quinoxaline), an irreversible crosslinking reaction occurs, forming a thioether. Interestingly, ADCs prepared using C-lockt technology (CD38-ADC) are reported to have a DAR3 (on average), even though interchain disulfide crosslinking (e.g., from HC-226C to HC-229C) may produce mixtures.
Using dibromo pyridine-2,6-dione for coupling
Inspired by the rebridging and reduction of disulfide bonds using dibromo maleimide groups, Chudasama and colleagues developed a rebridging coupling technique based on the pyridine-2,6-dione (PD) core structure. Unlike maleimide derivatives, the pyridine-2,6-dione ring is more stable and does not undergo ring-opening through hydrolysis, resulting in more uniform conjugates. The authors prepared mono-bromo PD (MBPD) and di-bromo PD (DBPD) derivatives and demonstrated their ability to selectively react with thiol groups even in the presence of amines at pH 8.0. The reactive group of DBPD was used to develop a series of crosslinking and modification reagents that integrated other reaction sites, such as alkynes for click chemistry reactions and derivatives containing reducing agents for one-step antibody reduction and coupling. The rebridging chemistry using DBPD for thiol rebridging has been applied to create highly site-specific ADCs, such as trastuzumab-deruxtecan with a DAR4 MMAE payload.
Disulfide bond coupling
Disulfide bonds have been used in ADCs since the early days, for example, in MLN2704, CMD-401, BIWI-1, and IMGN242. The design of linkers containing disulfide bonds between the cytotoxic payload and the antibody allows for a reversible connection that can be cleaved by intracellular glutathione in tumor cells, releasing the toxin. However, premature cleavage of the disulfide bond often occurs due to the presence of free cysteines, leading to payload release before the ADC enters tumor cells. Adding protective groups, such as methyl or dimethyl groups, on carbons adjacent to the disulfide bond has been used to increase stability in circulation by creating steric hindrance around the disulfide bond. However, in many cases, they also decrease the rate of toxin release inside tumor cells. Furthermore, even with increased stability due to added hindrance, these ADCs are not as effective as engineering the drug at the optimal position within the antibody sequence.
Pillow et al. first directly coupled small molecule drugs to engineered cysteine residues in antibodies at different positions, generating ADCs with unobstructed disulfide bonds. The results were compared with ADCs having disulfide bonds with different hindrances coupled to lysine residues. The authors identified the engineered cysteine residue at LC-K149C as the most stable binding site for attaching disulfide bond drugs. ADCs created using the LC-K149C site exhibited excellent circulatory stability while allowing efficient intracellular glutathione (GSH)-mediated cleavage, making them the most potent among all evaluated mutants for a mouse-human non-Hodgkin’s lymphoma xenograft.