Where Did the Linker-Payload Go? A Quantitative Investigation on the Destination of the Released Linker-Payload from an Antibody-Drug Conjugate with a Maleimide Linker in Plasma

Abstract

The reactive thiol of cysteine is often used for coupling maleimide-containing linker-payloads to antibodies resulting in the generation of antibody drug conjugates (ADCs). Currently, a numbers of ADCs in drug development are made by coupling a linker-payload to native or engineered cysteine residues on the antibody. An ADC conjugated via hinge-cysteines to an auristatin payload was used as a model in this study to understand the impact of the maleimide linkers on ADC stability. The payload was conjugated to trastuzumab by a protease-cleavable linker, maleimido-caproyl-valine-citruline-p-amino-benzyloxy carbonyl (mcVC-PABC). In plasma stability assays, when the ADC (Trastuzumab-mcVC-PABC-Auristatin-0101) was incubated with plasma over a 144-h time-course, a discrepancy was observed between the measured released free payload concentration and the measured loss of drug-to-antibody ratio (DAR), as measured by liquid chromatography-mass spectrometry (LC-MS). We found that an enzymatic release of payload from ADC-depleted human plasma at 144 h was able to account for almost 100% of the DAR loss. Intact protein mass analysis showed that at the 144 h time point, the mass of the major protein in ADC-depleted human plasma had an additional 1347 Da over the native albumin extracted from human plasma, exactly matching the mass of the linker-payload. In addition, protein gel electrophoresis showed that there was only one enriched protein in the 144 h ADC-depleted and antipayload immunoprecipitated plasma sample, as compared to the 0 h plasma immunoprecipitated sample, and the mass of this enriched protein was slightly heavier than the mass of serum albumin. Furthermore, the albumin adduct was also identified in 96 h and 168 h postdose in vivo cynomolgus monkey plasma. These results strongly suggest that the majority of the deconjugated mc-VC-PABC-auristatin ultimately is transferred to serum albumin, forming a long-lived albumin-linker-payload adduct. To our knowledge, this is the first report quantitatively characterizing the extent of linker-payload transfer to serum albumin and the first clear example of in vivo formation of an albumin-linker-payload adduct.

Ethynylphosphonamidates for the Rapid and Cysteine‐Selective Generation of Efficacious Antibody–Drug Conjugates

“…[11] In our experiment, we observed that 90 %of the phosphonamidate-linked MMAE was still connected to brentuximab after 7days of incubation in rat serum at 37 8 8C, as measured by intact-protein MS after pulldown of the ADC from serum. [31] Although hydrolysis of maleimides was shown to improve conjugate stability and (Karpas299, top) and acontrol (HL60, bottom)f or brentuximab-10,Adcetris , and brentuximab alone. (Figure 3c and Figure S9 in the supporting information).…”

Bispecific Antibodies and Antibody–Drug Conjugates for Cancer Therapy: Technological Considerations

“…Brentuximab vedotin, trastuzumab deruxtecan, polatuzumab vedotin, rovalpituzumab tesirine, and many other ADCs are conjugated to drugs via cysteine residues. The linker-payloads are, in most cases, conjugated by maleimide chemistry, although the reaction is highly efficient and cysteine-specific, the thiosuccinimide linkage is somewhat unstable in plasma and undergoes maleimide exchange with free thiols of albumin, cysteine, and glutathione [157], which react with the maleimide liberated from ADC by a retro-Michael reaction [158,159]. Introducing a primary amino group near the thiosuccinimide ring has been reported to alleviate this problem by intramolecularly catalyzed hydrolysis of the ring [160].…”

Antibody-Drug Conjugates for the Therapy of Thoracic Malignancies

“…However, these efforts are often offset by deconjugation, fast clearance of conjugates, and aggregation and competition with unconjugated antibody. 40 All the components of ADCs contribute to their absorption, distribution, metabolism, and excretion (ADME). 41 Different bioanalytical methods have been adopted to study ADME of the ADCs compared to traditional anticancer agents.…”