Quantification of recombinant immunotoxin delivery to solid tumors allows for direct comparison of in vivo and in vitro results

Abstract

Solid tumors present challenges for delivery of protein therapeutics; current methods cannot quantify the functional effects of these agents. RG7787 (anti-mesothelin recombinant immunotoxin) is highly cytotoxic to pancreatic cancer cell lines, but with limited activity in vivo. To investigate this discrepancy, we developed a flow cytometry method to quantify the amount of RG7787 internalized per cell in tumors and used it to analyze tumor responses by determining the number of molecules of RG7787 internalized per cell in vivo and comparing it to that needed to kill cells in vitro. At a maximum tolerated dose of 7.5 mg/kg, tumor cells in vivo internalized a wide range of RG7787 with the average amount equivalent to the amount that induced growth arrest in vitro. However, 20% of cells accumulated 20,300 ITs per cell, sufficient to kill cells in vitro. At 2.5 mg/kg the top 20% of cells internalized enough RG7787 to only induce growth arrest. These data are in agreement with tumor responses; 22% regression following a 7.5 mg/kg dose and growth stabilization following 2.5 mg/kg. Comparing amounts of RIT delivered in vivo and in vitro can explain tumor responses and should facilitate the development of more active immunotoxins and other antibody based agents.

Actinomycin D enhances killing of cancer cells by immunotoxin RG7787 through activation of the extrinsic pathway of apoptosis

“…Because immunotoxin RG7787 has a relatively short half-life in the circulation, it is important that cells are killed after a short exposure to RG7787 (20). To evaluate if Act D treatment shortens the time needed for RG7787 to kill cells, KLM1 cells were treated with low doses of RG7787 in combination with Act D for 6, 24, or 48 h, and the cells were transferred to drug-free medium and followed.…”

Wide Variability in the Time Required for Immunotoxins to Kill B Lineage Acute Lymphoblastic Leukemia Cells: Implications for Trial Design

“…Surface bound molecules were stripped for 10 minutes in 0.2 M Glycine pH 2.5, cells washed twice with PBS and analyzed by flow (FACS Calibur, Becton Dickinson). Internalized rIT-molecules were quantified with Alexa647-Quantum beads (Bangs Laboratories, Fishers, IN) as described (29). Beads were used to create a standard curve for conversion of MFI into absolute molecule number.…”

Reduced Shedding of Surface Mesothelin Improves Efficacy of Mesothelin-Targeting Recombinant Immunotoxins

“…Surface staining using labeled RG7787 was described previously (32). Briefly, 2.5×10 5 cells were plated in 6 well Falcon tissue culture plates in 2 ml media.…”

“…Cells were then trypsinized, washed with cold internalization-FACS buffer (PBS with 1% FBS and 0.1% NaN3) twice, then resuspended in 500 μl cold FACS buffer and analyzed using FACSCanto II. Quantification of internalized RG7787 molecules was performed as described previously (32). For surface staining, cells were either detached using trypsin or citric saline for comparison.…”