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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

Partitioning of RIT internalization in KLM-1 tumors and relating in vivo RIT internalization to in vitro internalization to determine in vitro dose equivalents.Representative histogram of RG7787-Alexa Fluor 647 signal intensity of KLM-1 tumor cell population 6 hrs after i.v. treatment with 7.5 mg/kg (A) or 2.5 mg/kg (C) showing tumor cell population is divided into five equal quintiles based on MFI. MFI for each quintile is shown, and translated into average RG7787 molecules per cell for the quintile. Dose equivalents for each quintile calculated by interpolating average number of RG7787 molecules per cell for each quintile on to Fig. 4A. Dose equivalents calculated for amount of RIT internalized 6 hrs following i.v. treatment with 7.5 mg/kg (B) and 2.5 mg/kg (D). Each in vivo measurement is the mean of at least triplicate experiments.
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f5: Partitioning of RIT internalization in KLM-1 tumors and relating in vivo RIT internalization to in vitro internalization to determine in vitro dose equivalents.Representative histogram of RG7787-Alexa Fluor 647 signal intensity of KLM-1 tumor cell population 6 hrs after i.v. treatment with 7.5 mg/kg (A) or 2.5 mg/kg (C) showing tumor cell population is divided into five equal quintiles based on MFI. MFI for each quintile is shown, and translated into average RG7787 molecules per cell for the quintile. Dose equivalents for each quintile calculated by interpolating average number of RG7787 molecules per cell for each quintile on to Fig. 4A. Dose equivalents calculated for amount of RIT internalized 6 hrs following i.v. treatment with 7.5 mg/kg (B) and 2.5 mg/kg (D). Each in vivo measurement is the mean of at least triplicate experiments.

Mentions: The data in Fig. 2A demonstrate that there is a large range of Alexa Fluor 647 signal intensity 6 hours after treatment, indicating that some KLM-1 cells internalized much more RG7787 than others. To assess these differences, we divided the KLM-1 tumor cells into 20% quintiles according to the average amount of RG7787-Alexa Fluor 647 internalized, ranging from low (Quintile 1) to high (Quintile 5). Figure 5A shows the fluorescence intensity of each quintile of the tumor cell population 6 hours following a single 7.5 mg/kg treatment, and the corresponding number of RG7787 molecules internalized. Quintile 5 has an MFI of 1150, which is equivalent to 20,300 molecules of RG7787 per cell. Quintile 1 internalized an average of 460 immunotoxins per cell.


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)

Partitioning of RIT internalization in KLM-1 tumors and relating in vivo RIT internalization to in vitro internalization to determine in vitro dose equivalents.Representative histogram of RG7787-Alexa Fluor 647 signal intensity of KLM-1 tumor cell population 6 hrs after i.v. treatment with 7.5 mg/kg (A) or 2.5 mg/kg (C) showing tumor cell population is divided into five equal quintiles based on MFI. MFI for each quintile is shown, and translated into average RG7787 molecules per cell for the quintile. Dose equivalents for each quintile calculated by interpolating average number of RG7787 molecules per cell for each quintile on to Fig. 4A. Dose equivalents calculated for amount of RIT internalized 6 hrs following i.v. treatment with 7.5 mg/kg (B) and 2.5 mg/kg (D). Each in vivo measurement is the mean of at least triplicate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Partitioning of RIT internalization in KLM-1 tumors and relating in vivo RIT internalization to in vitro internalization to determine in vitro dose equivalents.Representative histogram of RG7787-Alexa Fluor 647 signal intensity of KLM-1 tumor cell population 6 hrs after i.v. treatment with 7.5 mg/kg (A) or 2.5 mg/kg (C) showing tumor cell population is divided into five equal quintiles based on MFI. MFI for each quintile is shown, and translated into average RG7787 molecules per cell for the quintile. Dose equivalents for each quintile calculated by interpolating average number of RG7787 molecules per cell for each quintile on to Fig. 4A. Dose equivalents calculated for amount of RIT internalized 6 hrs following i.v. treatment with 7.5 mg/kg (B) and 2.5 mg/kg (D). Each in vivo measurement is the mean of at least triplicate experiments.
Mentions: The data in Fig. 2A demonstrate that there is a large range of Alexa Fluor 647 signal intensity 6 hours after treatment, indicating that some KLM-1 cells internalized much more RG7787 than others. To assess these differences, we divided the KLM-1 tumor cells into 20% quintiles according to the average amount of RG7787-Alexa Fluor 647 internalized, ranging from low (Quintile 1) to high (Quintile 5). Figure 5A shows the fluorescence intensity of each quintile of the tumor cell population 6 hours following a single 7.5 mg/kg treatment, and the corresponding number of RG7787 molecules internalized. Quintile 5 has an MFI of 1150, which is equivalent to 20,300 molecules of RG7787 per cell. Quintile 1 internalized an average of 460 immunotoxins per cell.

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