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

Mason-Osann E, Hollevoet K, Niederfellner G, Pastan I - Sci Rep (2015)

Bottom Line: Solid tumors present challenges for delivery of protein therapeutics; current methods cannot quantify the functional effects of these agents.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.

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.

No MeSH data available.


Related in: MedlinePlus

Quantification of RG7787 molecules per tumor cell.A. Representative dot plots of KLM-1 xenografts (120-140 mm3) excised after treatment with 2.5 mg/kg i.v. RG7787-Alexa Fluor 647. Line is drawn above untreated tumor. The number in the top right corner of each frame is the MFI of Alexa Fluor 647 for KLM-1 (Sytox negative, CD71 R-PE positive). B. MFI values translated into average RG7787-Alexa Fluor 647 molecules per cell at each time point.
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f2: Quantification of RG7787 molecules per tumor cell.A. Representative dot plots of KLM-1 xenografts (120-140 mm3) excised after treatment with 2.5 mg/kg i.v. RG7787-Alexa Fluor 647. Line is drawn above untreated tumor. The number in the top right corner of each frame is the MFI of Alexa Fluor 647 for KLM-1 (Sytox negative, CD71 R-PE positive). B. MFI values translated into average RG7787-Alexa Fluor 647 molecules per cell at each time point.

Mentions: Previously, we measured tumor penetration by quantifying the percentage of KLM-1 cells internalizing RG778713. In the current study, we measured the amount of RIT internalized per tumor cell, which allowed us to understand the functional effects of internalized RIT in vivo. We used Alexa Fluor 647 Molecules of Equivalent Soluble Fluorochrome beads (MESF; Bangs Laboratories) to establish a standard curve, and interpolated the geometric mean fluorescence intensity (MFI) of KLM-1 tumor cell populations on this curve using Bangs Laboratories QuickCal software (Supporting Information Fig. 2) to calculate RG7787 molecules per cell. While the previously reported method estimated the number of internalized molecules based on saturation staining19, calibrated beads increase accuracy over a wider range of values by generating a standard curve. We subtracted the MESF value of an untreated tumor to correct for the auto-fluorescence of cells. Figure 2A shows representative dot plots of the tumor cell population after treatment with 2.5 mg/kg i.v. RG7787-Alexa Fluor 647. The MFI of the tumor cell population increased over time, indicating increased amounts of RIT internalized. MFI was converted to molecules per cell in Fig. 2B, showing that KLM-1 tumor cells internalized an average of 1,620 ± 340 RG7787 molecules per cell 6 hours after treatment. This is the time when maximal internalization occurs in this tumor13.


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

Mason-Osann E, Hollevoet K, Niederfellner G, Pastan I - Sci Rep (2015)

Quantification of RG7787 molecules per tumor cell.A. Representative dot plots of KLM-1 xenografts (120-140 mm3) excised after treatment with 2.5 mg/kg i.v. RG7787-Alexa Fluor 647. Line is drawn above untreated tumor. The number in the top right corner of each frame is the MFI of Alexa Fluor 647 for KLM-1 (Sytox negative, CD71 R-PE positive). B. MFI values translated into average RG7787-Alexa Fluor 647 molecules per cell at each time point.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482048&req=5

f2: Quantification of RG7787 molecules per tumor cell.A. Representative dot plots of KLM-1 xenografts (120-140 mm3) excised after treatment with 2.5 mg/kg i.v. RG7787-Alexa Fluor 647. Line is drawn above untreated tumor. The number in the top right corner of each frame is the MFI of Alexa Fluor 647 for KLM-1 (Sytox negative, CD71 R-PE positive). B. MFI values translated into average RG7787-Alexa Fluor 647 molecules per cell at each time point.
Mentions: Previously, we measured tumor penetration by quantifying the percentage of KLM-1 cells internalizing RG778713. In the current study, we measured the amount of RIT internalized per tumor cell, which allowed us to understand the functional effects of internalized RIT in vivo. We used Alexa Fluor 647 Molecules of Equivalent Soluble Fluorochrome beads (MESF; Bangs Laboratories) to establish a standard curve, and interpolated the geometric mean fluorescence intensity (MFI) of KLM-1 tumor cell populations on this curve using Bangs Laboratories QuickCal software (Supporting Information Fig. 2) to calculate RG7787 molecules per cell. While the previously reported method estimated the number of internalized molecules based on saturation staining19, calibrated beads increase accuracy over a wider range of values by generating a standard curve. We subtracted the MESF value of an untreated tumor to correct for the auto-fluorescence of cells. Figure 2A shows representative dot plots of the tumor cell population after treatment with 2.5 mg/kg i.v. RG7787-Alexa Fluor 647. The MFI of the tumor cell population increased over time, indicating increased amounts of RIT internalized. MFI was converted to molecules per cell in Fig. 2B, showing that KLM-1 tumor cells internalized an average of 1,620 ± 340 RG7787 molecules per cell 6 hours after treatment. This is the time when maximal internalization occurs in this tumor13.

Bottom Line: Solid tumors present challenges for delivery of protein therapeutics; current methods cannot quantify the functional effects of these agents.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.

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.

No MeSH data available.


Related in: MedlinePlus