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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

Anti-tumor activity and corresponding quantification of RG7787 molecules per cell.A. Tumor volumes of mice with KLM-1 xenografts (120-140 mm3) treated with a single dose of D-PBS 0.2% HSA (control, black), 2.5 mg/kg RG7787 (white) or 7.5 mg/kg RG7787 (gray) i.v. B. Average RG7787-Alexa Fluor 647 molecules per cell in KLM-1 tumor cell population (CD71 R-PE +) 6 hrs after treatment with 1.25 mg/kg (n = 3), 2.5 mg/kg (n = 7) or 7.5 mg/kg (n = 4) RG7787-Alexa Fluor 647 i.v.
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f3: Anti-tumor activity and corresponding quantification of RG7787 molecules per cell.A. Tumor volumes of mice with KLM-1 xenografts (120-140 mm3) treated with a single dose of D-PBS 0.2% HSA (control, black), 2.5 mg/kg RG7787 (white) or 7.5 mg/kg RG7787 (gray) i.v. B. Average RG7787-Alexa Fluor 647 molecules per cell in KLM-1 tumor cell population (CD71 R-PE +) 6 hrs after treatment with 1.25 mg/kg (n = 3), 2.5 mg/kg (n = 7) or 7.5 mg/kg (n = 4) RG7787-Alexa Fluor 647 i.v.

Mentions: The maximum dose of RG7787 that can be given safely to mice is 2.5 mg/kg every other day x3; however 7.5 mg/kg can be given safely as a single dose. We assessed the functional effect of increased RIT delivery following treatment with a higher dose by measuring the volume change of KLM-1 xenografts (Fig. 3A). A single 2.5 mg/kg dose arrested tumor growth, but did not cause tumor regression. In contrast, a single 7.5 mg/kg dose induced a 22% reduction in tumor volume measured on day 3 (100 ± 8 mm3 compared to 128 ± 7 mm3 starting volume, p = 0.0004). Starting on day 3 there was a statistically significant difference between tumor volumes from mice treated with 2.5 mg/kg and 7.5 mg/kg (131 ± 10 mm3 and 100 ± 8 mm3, respectively, p = 0.047). Treatment with a 7.5 mg/kg dose also increased the amount of internalized RG7787 to 3,710 ± 610 RG7787 molecules per cell from 1620 ± 340 when measured 6 hours following treatment (Fig. 3B), consistent with the better anti-tumor response. We then analyzed the relationship between the amount of immunotoxin internalized and the tumor response by determining how much RG7787 needs to be internalized in order to kill cells in vitro, and how that amount relates to the amount of RG7787 internalized in tumors in the mice.


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)

Anti-tumor activity and corresponding quantification of RG7787 molecules per cell.A. Tumor volumes of mice with KLM-1 xenografts (120-140 mm3) treated with a single dose of D-PBS 0.2% HSA (control, black), 2.5 mg/kg RG7787 (white) or 7.5 mg/kg RG7787 (gray) i.v. B. Average RG7787-Alexa Fluor 647 molecules per cell in KLM-1 tumor cell population (CD71 R-PE +) 6 hrs after treatment with 1.25 mg/kg (n = 3), 2.5 mg/kg (n = 7) or 7.5 mg/kg (n = 4) RG7787-Alexa Fluor 647 i.v.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Anti-tumor activity and corresponding quantification of RG7787 molecules per cell.A. Tumor volumes of mice with KLM-1 xenografts (120-140 mm3) treated with a single dose of D-PBS 0.2% HSA (control, black), 2.5 mg/kg RG7787 (white) or 7.5 mg/kg RG7787 (gray) i.v. B. Average RG7787-Alexa Fluor 647 molecules per cell in KLM-1 tumor cell population (CD71 R-PE +) 6 hrs after treatment with 1.25 mg/kg (n = 3), 2.5 mg/kg (n = 7) or 7.5 mg/kg (n = 4) RG7787-Alexa Fluor 647 i.v.
Mentions: The maximum dose of RG7787 that can be given safely to mice is 2.5 mg/kg every other day x3; however 7.5 mg/kg can be given safely as a single dose. We assessed the functional effect of increased RIT delivery following treatment with a higher dose by measuring the volume change of KLM-1 xenografts (Fig. 3A). A single 2.5 mg/kg dose arrested tumor growth, but did not cause tumor regression. In contrast, a single 7.5 mg/kg dose induced a 22% reduction in tumor volume measured on day 3 (100 ± 8 mm3 compared to 128 ± 7 mm3 starting volume, p = 0.0004). Starting on day 3 there was a statistically significant difference between tumor volumes from mice treated with 2.5 mg/kg and 7.5 mg/kg (131 ± 10 mm3 and 100 ± 8 mm3, respectively, p = 0.047). Treatment with a 7.5 mg/kg dose also increased the amount of internalized RG7787 to 3,710 ± 610 RG7787 molecules per cell from 1620 ± 340 when measured 6 hours following treatment (Fig. 3B), consistent with the better anti-tumor response. We then analyzed the relationship between the amount of immunotoxin internalized and the tumor response by determining how much RG7787 needs to be internalized in order to kill cells in vitro, and how that amount relates to the amount of RG7787 internalized in tumors in the mice.

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