(M1430-06-39) A Comprehensive Array of Bioanalytical Assays to Assess the Pharmacokinetic Properties of Antibody-Drug Conjugates In Vivo and In Vitro

Purpose: Antibody-drug conjugates (ADCs) are complex biologics, which have received an increasing amount of attention in the pharmaceutical field in recent years. To further understand the in vivo pharmacology of ADC drug candidates, several advanced modeling approaches have been developed, such as PK/PD, QSP and PBPK. To support these efforts, advanced bioanalytical methods to characterize and quantify ADC in various matrices are required. Here we report a comprehensive array of bioanalytical assays for the quantification of an ADC drug candidate ADC-X and the key metabolites from the in vivo and in vitro systems. These data enhanced the understanding of ADC pharmacology, gave insight into targeted ADC delivery, and informed modeling efforts.

Methods: The in vivo and in vitro study: For an in vivo mouse study, ADC-X and isotype control ADC were dosed in tumor-bearing mice. Post dose, both plasma and fresh-frozen tumor samples were collected at multiple timepoints. For an in vitro cell study, cells were incubated with 100 nM ADC-X or 100 nM payload, followed by either continuous exposure or buffer washout after 2 hours. Both cell media and cell pellet samples were collected at various timepoints post incubation. Sample preparation: RIPA buffer was added to tumor and cell pellet for homogenate and lysis, and the supernatant from centrifugation was proceed to further analysis. Plasma samples and cell media samples were directly used for bioanalysis. Bioanalytical Assays

Overview: Multifaceted bioanalytical assays were applied to samples in various matrices. For plasma and media samples, total antibody, ADC and unconjugated payload were quantified. To provide further normalization of the data, total protein analysis for cell lysate and tumor was performed with BCA, in addition to the cell count and tumor weight. Assay Procedure: Total antibody and ADC quantification was done via a hybrid LBA-LCMS/MS method using immunocapture of ADC-X followed by trypsin digestion (total antibody assay) or papain enzymatic cleavage (ADC assay). Unconjugated payload assay used protein precipitation and was analyzed by LCMS. All LCMS based assays data were acquired on a SCIEX 6500+ triple quadrupole mass spectrometer coupled with a Shimadzu Sil-30 UHPLC system. Data were processed using MultiQuant with 1/x² weighing. Total protein assay was conducted using Thermo Fisher Scientific Pierce BCA Protein Assay Kits.

Results: All assays were successfully qualified and applied to sample analysis. For in vivo study plasma samples, the PK profiles resulting from total antibody, ADC and unconjugated payload bioanalysis assay were similar between ADC-X and isotype control ADC. For tumor homogenate samples, BCA normalized results demonstrated that ADC-X ADC and unconjugated payload concentrations were up to 3-fold and 9-fold higher than isotype control ADC at the same dose level, showing higher tumor exposure by targeted delivery of ADC-X. In vitro and in vivo samples were analyzed similarly. Cell media samples in vitro incubation results were comparable among cell lines for continuous incubation and 2 hours incubation followed by buffer wash. For cell lysate, the exposure in high expression cell lines was higher than the low expressing cell lines, including both continuous exposure and buffer washout samples.

Conclusion: The comprehensive array of LCMS-based bioanalytical assays provided quantitative information on ADC-X and its major metabolites in a variety of key matrices. From the results, the targeted delivery of the cytotoxic payload to the tumor or high expressing cell lines was observed. The rich PK data of the time related target delivery can be further applied to modeling efforts and provide a deeper mechanistic understanding of ADC in both in vivo and in vitro system, which can provide useful information on TI calculation of ADC molecules.

References: Singh, Aman P., and Dhaval K. Shah. "Measurement and mathematical characterization of cell-level pharmacokinetics of antibody-drug conjugates: a case study with trastuzumab-vc-MMAE" Drug Metabolism and Disposition 2017, 45, 1120-1132.

Acknowledgements: The authors declare the following competing financial interest(s): authors are employees of, and hold or may hold stock in, AstraZeneca.

Figure 1. Overview of assays developed for in vivo and in vitro studies in various matrices.

Figure 2. ADC and unconjugated payload assay pharmacokinetic results of ADC-X and isotype control ADC in mouse study. A) plasma PK profile. B) BCA normalized tumor homogenate ADC assay PK profile. C) BCA normalized tumor homogenate unconjugated payload assay PK profile.