Antibody-Drug Conjugate Sacituzumab Govitecan Drives Efficient Tissue Penetration and Rapid Intracellular Drug Release

Abstract

Antibody-drug conjugates (ADC) are a rapidly growing class of targeted cancer treatments, but the field has experienced significant challenges from their complex design. This study examined the multiscale distribution of sacituzumab govitecan (SG; Trodelvy), a recently clinically approved ADC, to clarify the mechanism(s) of efficacy given its unique design strategy. We employed a multiscale quantitative pharmacokinetic approach, including near-infrared fluorescence imaging, single-cell flow cytometry measurements, payload distribution via γH2AX pharmacodynamic staining, and a novel dual-labeled fluorescent technique to track the ADC and payload in a high trophoblast cell-surface antigen 2 expression xenograft model of gastric cancer (NCI-N87). We found that rapid release of the SN-38 payload from the hydrolysable linker inside cells imparts more DNA damage in vitro and in vivo than an ADC with a more stable enzyme cleavable linker. With SG, little to no extracellular payload release in the tumor was observed using a dual-labeled fluorescence technique, although bystander effects were detected. The high dosing regimen allowed the clinical dose to reach the majority of cancer cells, which has been linked to improved efficacy. In addition, the impact of multiple doses (day 1 and day 8) of a 21-day cycle was found to further improve tissue penetration despite not changing tumor uptake [percent injected dose per gram (%ID/g)] of the ADC. These results show increased ADC efficacy with SG can be attributed to efficient tumor penetration and intracellular linker cleavage after ADC internalization. This quantitative approach to study multiscale delivery can be used to inform the design of next-generation ADCs and prodrugs for other targets.

Figure 1.

Multiscale pharmacokinetics of ADCs. A, ADCs in systemic circulation distribute to both tumor and healthy tissue, where they can bind their target and release the payload. In addition, deconjugation in the systemic circulation releases payload, lowering the DAR. B, Within the tumor, ADCs extravasate, diffuse through the tissue, bind their target, internalize, and release the payload via hydrolysis or protease cleavage for CL2A and CL2E linkers, respectively. Linker hydrolysis can also occur extracellularly, and released payload can diffuse to nearby cells (bystander effect).

Figure 2.

Organ, tissue, and cellular biodistribution. Tissue penetration of hRS7 is dose-dependent, with a 5-mg/kg dose showing heterogeneous distribution after 24 hours (A), while the clinical dose of 10 mg/kg penetrates deeper into the tumor (B). Blood vessels are imaged with anti-CD31 stain (red), intravenous Hoechst 33342 is shown in blue, and hRS7-AlexaFluor680 is in green. The high expression and rapid internalization results in efficient tumor uptake (n = 3; C). Flow cytometry of single-cell suspensions at 24 hours from three different tumors at each dose confirms a greater proportion of cells are hRS7-AlexaFluor680 positive at the higher dose (D). Data are shown as the mean and SD.

Figure 3.

In vitro Payload delivery and DNA damage. NCI-N87 cells were pulsed for 8 hours with SG (CL2A linker), the enzyme-cleavable (CL2E) linker ADC (structures shown in A), hydrolyzable (CL2A) linker nonspecific ADC, or left untreated and stained for DNA damage using γH2AX (B). Data show the median fluorescence intensity (MFI) and SD of three or four separate experiments. SG showed rapid and significant DNA damage that decreased over time, whereas the enzyme-cleavable CL2E linker ADC released the payload more slowly for lower signal that increased over 3 days. The hydrolyzable CL2A linker nonspecific control ADC showed some signal at 24 hours but lower than SG, highlighting the need for Trop-2–mediated targeting. Microscopy of NCI-N87 cells at the 24-hour time point showing nuclei (blue, Hoechst 33342) of cells treated with SG (C) with higher γH2AX signal (red) than the CL2E ADC (D) and nonspecific ADC (E).

Figure 4.

In vivo imaging of ADC distribution and payload-mediated DNA damage. Forty-eight hours following a 10-mg/kg dose of SG (A), nonspecific CL2A-SN38 ADC (B), hRS7-CL2E-SN38 (C), or uninjected mice bearing NCI-N87 xenografts, tissue was excised and imaged using an anti-Fc stain (green) or γH2AX DNA damage marker (red). SG shows the highest signal including cells lacking ADC targeting (bystander effects). Signal from the other ADCs was lower but above background.

Figure 5.

Dual-labeled ADC for quantifying extracellular release. By labeling hRS7 antibody with two residualizing dyes, AF488 (green star) attached directly to antibody via a stable amide linkage and SN38-AF680 (red star) connected via the CL2A hydrolysable linker, the location of payload release could be quantified. Extracellular released SN38-AF680 dye (red star) is unable to enter cells and washes out of the tumor, while intracellularly released payload is trapped (A). Biodistribution (% injected dose/gram) of the dual-labeled ADC (as measured by SN38-AF680 signal) shows high tumor uptake, consistent with intracellular release and residualization of the dye (B). The ratio of AF680 to AF488 indicates little extracellular release of the payload in NCI-N87 tumors. There is a significant drop in the AF680 to AF488 ratio between the ex vivo control and the ratio at 48 hours (P < 0.05; C). The decreased ratio at 48 hours is less than the positive control cells labeled with ADC following 50% SN38-AF680 release. This lower ratio can be attributed to the loss of SN38-AF680 in systemic circulation, where AF680 signal decreases faster than AF488 in the plasma due to deconjugation (D). Data are shown as the mean and SD for n = 3 mice at each timepoint. An outlying point at 24 hours in the lung (15.9, 16.5, and 73%ID/g), likely from blood clotting during processing, resulted in a large standard deviation at 24 hours.

Figure 6.

Impact of Dosing Schedule. SG is dosed on D1 and D8 of a 21-day cycle. To examine the impact on distribution and uptake, 10 mg/kg SG was administered to mice bearing NCI-N87 xenografts, followed 7 days later by 5 mg/kg of fluorescent hRS7. The total tumor uptake (fluorescent %ID/g) was similar following SG treatment (mean and SD, n = 3; A). However, the distribution within the tumor was greater following SG treatment (B) than fluorescent hRS7 alone (C). CD31, red; intravenous Hoechst, blue; hRS7-AF680, green. Image quantification showing the mean and SEM using a Euclidean distance map confirmed deeper tissue penetration (D), which could be due to antibody from the SG treatment contributing to increased tissue penetration.