Basically, the druggability of the cysteine-coupling-based ADCs can be improved by increasing the stability of the chemical bond and the homogeneity of the ADC after coupling. In the review from Zhang group (CPU) and WuXi Biologics1, the authors summarize the methods addressing these two aspects.
Each ADC R&D project is its own challenge due to the varieties in the assembly of ADC molecules. With this concept in mind, Medicilon promises careful planning, meticulous execution and accurate results through years of practical experience and effective communication with our clients.
Use the Appropriate Combination of Linker and Drug to Make the DAR8 Products.
Optimize the Coupling Process to Improve the Homogeneity of ADC Products.
The DAR of Adcetris has a 0, 2, 4, 6, and 8 distribution by hydrophobic interaction chromatography (HIC-HPLC) analysis, although the average DAR is around 4.1 (Figure 3). Such heterogeneous DAR brings certain difficulties to the quality control, also studies have shown four drugs per antibody have the strongest combined effect. Therefore methods to increase the percentage of DAR4 species in an ADC are actively sought. For example, DS-1062, a DXd-conjugated ADC targeting Trop2 protein, during the conjugation process, the reduction temperature was reduced to around 0°C and the reduction time was extended; the proportion of the DAR4 product has increased from 37.7% to 53.3% as compared to room temperature reduction. Compared with the physical cooling method, WuXi Biologics uses zinc ions to form chemical coordination bonds with the sulfhydryl groups in the hinge area of antibody. This shielding effect makes only the reduced sulfhydryl in the Fab region accept toxin drugs. This method increases the proportion of DAR4 components in ADCs from 38.4% to 70.4%, and almost all small molecule toxins are coupled to the Fab area (Figure 4).
Improve the Homogeneity of ADC Products through Antibody Engineering.
Antibody engineering technology provides another way to produce homogeneous site-specific ADCs. The first systematic study was initiated by Junutula and co-workers at Genentech (Figure 5). These site-specific ADCs, called THIOMAB antibody−drug conjugates (TDCs), were produced by the introduction of a “hot” cysteine residue, followed by a global reduction of “hot” cysteine and interchain disulfides and subsequent oxidation in the presence of CuSO4 to regenerate interchain disulfides. The TDCs exhibited improved in vivo efficacy in a mouse xenograft model of ovarian cancer. Importantly, TDCs have improved therapeutic index, higher dose tolerance and increased serum stability in rats and monkeys. However, the THIOMAB technology still faces some challenges in its implementation.
Accelerate the Hydrolysis of succinimide after thioether Bond Formation.
Currently, cysteine−maleimide coupling methods are used for most of approved ADCs. However, in PBS buffer and plasma stability tests, it was found that the conjugate could undergo a retro-Michael reaction, causing the toxins to fall off from the antibody and increasing toxic side effects. Studies have demonstrated that opening the succinimide ring by hydrolysis after coupling can produce a derivative that is resistant to the elimination reaction and improve the stability of ADC. In addition, the substituents on the adjacent sites of maleimide greatly affect the hydrolysis rate of succinimide ring after conjugation. For example, the introduction of −CH2−NH2 at this site can greatly accelerate the hydrolysis of the five-membered lactam ring thus improve the stability of ADC product (Figure 6).
Use Other Functional Groups for Coupling with Cysteine.
Since the retro-Michael reaction is the main factor for the instability of the cysteine−maleimide ADCs. Therefore, alternative functional groups reacting with cysteine that are not prone to reverse Michael reaction will greatly improve the stability of ADCs. As disclosed in US2021101906A2, 2-methylsulfonyl pyrimidine provides a possible option. By using this structure as the reactive functional group on the linker, the ADC product SKB264 (Figure 7) was produced by SnAr substitution to the thioether bond. Such ADC has better stability and efficacy than the approved maleimide linked ADC Trodelvy.
Re-bridging the Opened Disulfide Bonds.
The thiobridge technology uses the reduced disulfide pair to attach (bridge) one drug molecule to overcome the thio-exchange instability. Uniform DAR4 product can be obtained if all 4 interchain disulfide bonds are reduced and reacted with the linker−drug. The key to re-bridging is that the linker needs to have a functional group that can react with the sulfhydryl group twice, and with similar reactivity, otherwise instead of forming the bridge, each sulfhydryl will still react with the more reactive group on the linker and two drugs will be attached to the opened disulfide pair. In 1990, the Smith and Lawton group reported the use of bis-sulfone for specific modification of antibodies (Figure 8a). Bis-sulfone requires activation, by eliminating a sulfinate to yield the active mono-sulfone species that reacts with one of the free thiols generated by disulfide reduction. A second Michael acceptor is generated by elimination of the other sulfinate, and the remaining thiol then undergoes the second Michael addition to yield the bis-thioether with a three-carbon linkage. Other moieties, such as dibromomaleimide and dibromo-pyridazinedione, also allow bridged drug formation at the interchain disulfide region. All these linkers can quickly react with the sulfhydryl group twice to obtain an ADC with DAR4 and enhanced stability in plasma (Figure 8b). Furthermore, different derivatizations of the substituted pyridazinedione linker can also allow the introduction of different drugs on one linker. C-Lock technology provides another strategy for re-bridging. It introduces a dialkylated bromomethyl functional group on a heterocyclic nucleus to form a stable thioether structure with four carbons bridge backbone after conjugation (Figure 8c). In 2019, Walsh et al. reported a new type of bis-Michael acceptor, divinylpyrimidine (Figure 8d), it also offered the re-bridging product in good yields.
Despite extensive studies on thiobridge technology to facilitate the site-specific functionalization of interchain disulfides in native antibodies, certain pitfalls and limitations still exist.
1) Disulfide scrambling is unavoidable, affecting product efficacy, yield, and scalability.
2) The re-bridging reagents cannot distinguish between reduced disulfides and free thiols under reducing condition, limiting the use of disulfide re-bridging to a subset of antibodies lacking free cysteines.
3) Re-bridging reagents exhibit poor water solubility and usually require cosolvent to carry out the reaction. This increases the chance of denaturing the antibody.
In conclusion, the cysteine-based conjugation occupies a dominant position in both approved and clinically developed ADCs. The choice of conjugation method plays the key role in the synthesis of such ADCs and directly affects the success of ADC. Although various methods are developed to improve the stability and the homogeneity of the ADC, it still has much areas to further improve such conjugation.
Medicilon’s ADC team can offer all the payloads and linkers used in approved ADCs. We are eager to use our expertise, to help our client on the area of developing new linker-payloads to improve the properties of ADCs.
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