(T1030-02-09) Evaluating the Effects of Differently Charged Linker-Drugs on Pharmacokinetics and Tissue Distribution of Antibody Drug Conjugates

Purpose: The emergence of antibody-drug conjugates (ADCs) has led to increased efforts to identify new payloads and develop improved linker-drug technologies. Most antibody payloads impart significant hydrophobicity to the ADC, resulting in accelerated plasma clearance and potential non-specific toxicities [1]. While modifications such as the addition of hydrophilic PEGs have been explored to improve circulation stability [2], the impact of linker-drug molecular charge on ADC pharmacokinetics (PK) and biodistribution remains unclear. Here, we evaluate two charged linker-drug payloads conjugated to a tumor-targeting antibody with charge variants of +3 (ADC 1) and -3 (ADC 2), in which each linker-drug is conjugated at a drug to antibody ratio (DAR) of 6, relative to the naked antibody (mAb). Single Photon Emission Computed Tomography (SPECT) imaging of ¹¹¹In-labeled ADCs was performed to assess longitudinal tissue uptake in tumor-bearing SCID mice. We hypothesize that the presence of net negative charge on ADCs will lead to slower systemic clearance and higher target tissue uptake.

Methods: To evaluate stability of both ADCs, DARs were calculated via liquid chromatography – mass spectrometry (LC/MS) from plasma samples obtained from non-tumor SCID mice dosed with ADCs at 5 mg/kg. SPECT-CT imaging was used to evaluate whole-body distribution of molecules labeled with ¹¹¹In. Radiosynthesis of ¹¹¹In-labeled antibodies was achieved through incubation of ¹¹¹InCl₃ (BWXT) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA, Macrocyclics) modified mAbs (conjugated randomly through lysines) in 0.3M ammonium acetate pH 7 at 37°C for 1 hour. Purification of all radioimmunoconjugates was achieved using NAP5 columns (GE Healthcare) equilibrated with PBS and confirmed by radio-size exclusion chromatography (Phenomenex, Agilent). In vivo SPECT-CT imaging was performed on severe combined immunodeficient (SCID) beige mice bearing HCC515-flank tumors (n = 3/group) up to 7 days post administration. All tumor-bearing mice received doses of 200 µCi of ¹¹¹In-DOTA-mAb/ADC at a final dose of 5 mg/kg. Antibody uptake was calculated by drawing 3-dimensional regions of interest around tissues or whole heart (as a surrogate for blood) in acquired SPECT data using VivoQuant image analysis software and are reported as percent injected dose per gram (%ID/g) for whole tissue or percent injected dose per milliliter (%ID/mL) for blood.

Results: PK and stability experiments on plasma samples from non-tumor mice demonstrated that ADC 1 had greater clearance and deconjugation with a mean DAR of 2 by 21 days compared to ADC 2 with a DAR of 5.6 (Fig. 1). Longitudinal SPECT-CT imaging of tumor-bearing mice (Fig. 2A) similarly revealed that ADC 1 had a ~2-fold lower systemic exposure with greater clearance through the liver (p < 0.05) than both ADC 2 and unconjugated antibody 2 hours post administration (Fig. 2B). The rapid clearance likely attributed to the reduced tumor uptake of ADC 1 (8.0 ± 1.9 %ID/g) compared to ADC 2 (17.2 ± 2.3 %ID/g) and control antibody (19.4 ± 3.2 %ID/g) by Day 7. No differences in uptake among the three molecules were observed in non-target tissue such as muscle (Fig. 2B).

Conclusion: Here, we assessed the effects of varying linker-drug charge on the stability and clearance of ADCs. The presence of a negatively charged linker-drug led to slower clearance of the ADC and lower hepatic uptake, likely leading to reduced deconjugation of the ADC and enhanced tumor uptake. The in vivo data shown here demonstrates that linker-drug charge modification may serve as another potential avenue to maximize the potency and therapeutic window of ADCs.

References: 1. Buecheler JW, Winzer M, Tonillo J, Weber C, Gieseler H (2018) Impact of Payload Hydrophobicity on the Stability of Antibody-Drug Conjugates. Mol Pharm 15:2656-2664.
2. Burke PJ, Hamilton JZ, Jeffrey SC, et al. (2017) Optimization of a PEGylated Glucuronide-Monomethylauristatin E Linker for Antibody-Drug Conjugates. Mol Cancer Ther 16:116-123.

Influence of charge on pharmacokinetics and stability of ADCs. A) Plasma concentrations of control antibody (mAb) and charge variants over 21 days in non-tumor SCID mice. B) Drug to Antibody Ratios of different ADCs calculated via LC/MS. All graphs show mean ±SD error bars of each group with n = 3.

Influence of charge on tissue distribution of ADCs. A) Representative maximum intensity projection SPECT-CT (111In) images of control antibody and charge variants 7 days post administration. ‘H’ refers to heart; ‘L’ refers to liver; ‘TM’ refers to tumor. B) 3D regions of interest were generated via VivoQuant software and reported as %ID/mL for blood and %ID/g for muscle, liver and tumor. All graphs show mean ±SD error bars of each group with n = 3.