Thiol-Based Conjugation Methods for ADC

The prevalence of thiols in antibody-drug conjugate (ADC) preparation can be attributed to three factors: (a) Thiols possess the strongest nucleophilicity among amino acid side chain functional groups (much stronger than that of amines); (b) The relatively low number of interchain cysteines in antibodies suitable for conjugation (8 for IgG1 and IgG4), hence they do not significantly affect antibody stability, allowing for relatively controlled customization of the drug-to-antibody ratio (DAR); (c) Interchain cysteines are located in the hinge region, connecting the heavy chain (HC) and light chain (LC), which appears advantageous for concealing hydrophobic payloads post-conjugation. It is essential to note that a reducing step is always required before conjugation to release free thiols. The majority of thiol-conjugated ADCs are based on maleimide chemistry. Alkylation occurs at native interchain cysteines or engineered cysteines, or disulfide exchange.

Reaction with Maleimides

Maleimides readily undergo alkylation with thiol groups via nucleophilic Michael addition reactions, forming stable thioether bonds. Maleimide reaction with thiols is specific within the pH range of 6.5-7.5, with a reaction rate towards thiols 1000 times faster at pH 7.0 than towards amines, hence requiring only a slight excess of reagent for complete conversion. The primary 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 linked. This reverse Michael reaction may lead to premature release of the linker payload in circulation and potential adverse reactions, driving the development of more stable maleimide variants.

Reduction steps are required for cysteine reduction before maleimide functionalization of payloads in monoclonal antibodies. Therefore, by carefully optimizing the amount of reducing agent (usually TCEP or DTT), a certain number of free thiol groups can be released before incorporating maleimide-functionalized payloads. ADCs conjugated with cysteines prepared this way include Adcetris, Polivy, Padcev, Enhertu, Blenrep, Trodelvy, Zynlonta, etc. Due to the lack of site specificity, these ADCs are typically produced as random mixtures with an average DAR of 2.3-4.

It is noteworthy that maleimide-activated groups are used in the vast majority of cases; however, some ADCs employ poorer electron β-aminoethyl or ethylene glycol spacers. It has been reported that some phenyl-substituted maleimides are more stable and are currently under preclinical development.

Hydrolysis of the initial thioether-succinimide adduct to provide maleamic acid derivatives can inhibit the reverse Michael reaction. This hydrolysis can be achieved by long-term treatment of ADCs at pH 9; however, such conditions may evidently lead to additional post-translational modifications of antibodies, such as deamidation or aspartate formation.

Cysteine Alkylation Reagents

The high reactivity of α-halogenated acetamide reagents is well known, with 2-iodoacetamide being an undisputed choice for blocking free cysteines in proteins or protein mixtures (e.g., cell lysates). Importantly, the thioether bonds formed are entirely irreversible, thus superior in stability to thiol-maleimide ether. However, despite the stability difference between bromoacetamide and maleimide-based ADCs, there are no apparent differences in potency, activity, and toxicity profiles. The surprising lack of distinction is likely attributed to the similarity in the half-lives of the linker of such specific maleimide-based ADCs and the half-life of ADC clearance: as ADC concentrations decrease over time, prolonging the linker half-life beyond the pharmacokinetic half-life is unlikely to have a significant impact on drug exposure.

Methylsulfonyl benzothiazole (MSBT) is a selective thiol-blocking agent, and researchers have developed a series of aromatic methylsulfonyl ketone reagents for thiol alkylation in thiol-containing proteins, primarily imidazoles and benzothiazoles. Similarly, methylsulfonyl pyridines react with thiols via nucleophilic aromatic substitution mechanisms, a concept applied in SKB264/BT001035.

The alkylation ability of maleimide is also applied in various crosslinking bis-maleimide reagents. Each reagent can capture two free cysteine thiol groups, thus forming DAR4 ADCs after complete reduction of all eight interchain disulfide bonds.

Conjugation through Dual Bromomethyl Aromatic Reagents

The C-lock technology combines thiol nucleophilicity with the tendency for nucleophilic SN2 substitution reactions of benzyl bromide groups. Thus, through reduction of interchain disulfide bonds followed by reaction with dual bromomethyl aromatic reagents (e.g., dibromomethylquinoline), irreversible crosslinking reactions occur, forming thioethers. Interestingly, clinically developed ADCs (CD38-ADC) prepared using C-lock technology are reported to be DAR3 (average) rather than DAR4.

Conjugation with Dibromopyridine-2,6-diones

Inspired by the rebridging of reduced disulfide bonds using dibromomaleimide groups, Chudasama et al. developed a rebridging conjugation technology based on pyridine-2,6-diones (PD) core structure. Unlike maleimide derivatives, the pyridine-2,6-dione ring is more stable and does not undergo ring opening via hydrolysis, leading to more uniform conjugates. The authors prepared mono-bromo-PD (MBPD) and dibromo-PD (DBPD) derivatives, demonstrating their ability to specifically react with thiol groups even in the presence of amines at pH 8.0. DBPD reactive groups were used to develop a range of crosslinking and modification reagents, integrating other reaction sites such as alkynes for click chemistry reactions, and even derivatives containing reducing agents for one-step antibody reduction and conjugation. The use of DBPD thiol rebridging chemistry with antibodies has been applied to create highly site-specific ADCs, such as the DAR4 MMAE-conjugated trastuzumab.

Disulfide Bond Conjugation

Disulfide bonds have been used in ADCs since early on, e.g., in MLN2704, CMD-401, BIWI-1, and IMGN242. Linker designs containing disulfide bonds located somewhere between the cytotoxic payload and the antibody can facilitate a reversible linkage, which can be cleaved by intracellular glutathione (GSH) in tumor cells to release the toxin. However, the reality is that premature cleavage of disulfide bonds in circulation often occurs due to the presence of free cysteines, leading to payload release before reaching tumor cells. Adding protecting groups, such as methyl or dimethyl groups, on carbons adjacent to disulfide bonds has been used to increase stability in circulation by creating steric hindrance around the disulfide bond, but in many cases, they also reduce the rate of toxin release within tumor cells. Moreover, even with increased stability provided by added hindrance, the efficacy of these ADCs is not as effective as engineering the drug to be conjugated to the optimal position in the antibody sequence, such as engineered cysteine residues.

Pillow et al. were the first to directly conjugate small molecule drugs onto engineered cysteine antibodies at different positions, generating ADCs with unobstructed disulfide bonds (ADCs with DAR4). Results were compared with ADCs containing disulfide bonds with different sterically hindered linker positions using lysine residues. The authors identified the engineered cysteine at LC-K149C as the most stable binding site for the drug attachment containing disulfide bonds. ADCs created using the LC-K149C site demonstrated excellent circulation stability while allowing for effective glutathione (GSH)-mediated cleavage within tumor cells, resulting in the highest efficacy among all evaluated mutants in a mouse-human non-Hodgkin's lymphoma xenograft.