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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

Optimization of flow cytometry method for measuring RIT internalization.A. Structural model of anti-mesothelin immunotoxin RG7787. Model is hypothetical and previously described in detail13. RG7787 contains a humanized SS1 Fab, shown on the left, linked to a small portion of domain II (processing) and all of domain III (catalytic) of Pseudomonas Exotoxin A through a GGS linker containing a furin cleavage site. Domain III contains 7-point mutations to silence B-cell epitopes. B-C, A431/H9 tumor cell population (anti-EGFR R-PE+) 3 hrs after treatment with 5.85 mg/kg RG7787-Alexa Fluor 488 (B) or RG7787-Alexa Fluor 647 (C). Line is drawn above level at which untreated control tumor cell population has 2-5% positivity (data not shown). D. Histogram showing unstained KLM-1 cells (black, unfilled), stained with anti-EGFR R-PE (gray line, filled), or anti-CD71 R-PE (black line, filled). E. Triple negative breast cancer, HCC70 cells stained with anti-CD71 R-PE. F. Mesothelin transfected epidermoid cancer cell, A431/H9 cells stained with anti-CD71 R-PE.
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f1: Optimization of flow cytometry method for measuring RIT internalization.A. Structural model of anti-mesothelin immunotoxin RG7787. Model is hypothetical and previously described in detail13. RG7787 contains a humanized SS1 Fab, shown on the left, linked to a small portion of domain II (processing) and all of domain III (catalytic) of Pseudomonas Exotoxin A through a GGS linker containing a furin cleavage site. Domain III contains 7-point mutations to silence B-cell epitopes. B-C, A431/H9 tumor cell population (anti-EGFR R-PE+) 3 hrs after treatment with 5.85 mg/kg RG7787-Alexa Fluor 488 (B) or RG7787-Alexa Fluor 647 (C). Line is drawn above level at which untreated control tumor cell population has 2-5% positivity (data not shown). D. Histogram showing unstained KLM-1 cells (black, unfilled), stained with anti-EGFR R-PE (gray line, filled), or anti-CD71 R-PE (black line, filled). E. Triple negative breast cancer, HCC70 cells stained with anti-CD71 R-PE. F. Mesothelin transfected epidermoid cancer cell, A431/H9 cells stained with anti-CD71 R-PE.

Mentions: We have been studying a RIT named SS1P that targets mesothelin, a cell surface glycoprotein highly expressed on many malignancies, including mesothelioma, ovarian cancer, triple negative breast cancer and pancreatic cancer13. While SS1P had very modest anti-tumor effect as a single agent in clinical trials, it produced striking responses in a subset of patients when combined with immune-suppressive therapy, which prevented anti-drug antibody formation, and allowed more doses to be given14. To decrease immunogenicity and side effects that limit SS1P therapy1516, we have developed a clinically-optimized anti-mesothelin RIT (RG7787) in collaboration with Roche Pharma Research and Early Development (Fig. 1A)131718. RG7787 is highly active in vitro against several pancreatic ductal adenocarcinoma (PDAC) cell lines, including KLM-1. When tested on KLM-1 tumors in mice, RG7787 produced minor tumor regressions as a single agent, and profound tumor regressions when combined with paclitaxel13. One possible explanation for RG7787’s failure to produce profound regressions as a single agent is that insufficient concentrations of RIT reach tumor cells. Because current methods are insufficient for quantifying amounts of RIT or other antibody based agents that are delivered in vivo to tumor cells, we have developed a method to do this and applied it to a pancreatic cancer.


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)

Optimization of flow cytometry method for measuring RIT internalization.A. Structural model of anti-mesothelin immunotoxin RG7787. Model is hypothetical and previously described in detail13. RG7787 contains a humanized SS1 Fab, shown on the left, linked to a small portion of domain II (processing) and all of domain III (catalytic) of Pseudomonas Exotoxin A through a GGS linker containing a furin cleavage site. Domain III contains 7-point mutations to silence B-cell epitopes. B-C, A431/H9 tumor cell population (anti-EGFR R-PE+) 3 hrs after treatment with 5.85 mg/kg RG7787-Alexa Fluor 488 (B) or RG7787-Alexa Fluor 647 (C). Line is drawn above level at which untreated control tumor cell population has 2-5% positivity (data not shown). D. Histogram showing unstained KLM-1 cells (black, unfilled), stained with anti-EGFR R-PE (gray line, filled), or anti-CD71 R-PE (black line, filled). E. Triple negative breast cancer, HCC70 cells stained with anti-CD71 R-PE. F. Mesothelin transfected epidermoid cancer cell, A431/H9 cells stained with anti-CD71 R-PE.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Optimization of flow cytometry method for measuring RIT internalization.A. Structural model of anti-mesothelin immunotoxin RG7787. Model is hypothetical and previously described in detail13. RG7787 contains a humanized SS1 Fab, shown on the left, linked to a small portion of domain II (processing) and all of domain III (catalytic) of Pseudomonas Exotoxin A through a GGS linker containing a furin cleavage site. Domain III contains 7-point mutations to silence B-cell epitopes. B-C, A431/H9 tumor cell population (anti-EGFR R-PE+) 3 hrs after treatment with 5.85 mg/kg RG7787-Alexa Fluor 488 (B) or RG7787-Alexa Fluor 647 (C). Line is drawn above level at which untreated control tumor cell population has 2-5% positivity (data not shown). D. Histogram showing unstained KLM-1 cells (black, unfilled), stained with anti-EGFR R-PE (gray line, filled), or anti-CD71 R-PE (black line, filled). E. Triple negative breast cancer, HCC70 cells stained with anti-CD71 R-PE. F. Mesothelin transfected epidermoid cancer cell, A431/H9 cells stained with anti-CD71 R-PE.
Mentions: We have been studying a RIT named SS1P that targets mesothelin, a cell surface glycoprotein highly expressed on many malignancies, including mesothelioma, ovarian cancer, triple negative breast cancer and pancreatic cancer13. While SS1P had very modest anti-tumor effect as a single agent in clinical trials, it produced striking responses in a subset of patients when combined with immune-suppressive therapy, which prevented anti-drug antibody formation, and allowed more doses to be given14. To decrease immunogenicity and side effects that limit SS1P therapy1516, we have developed a clinically-optimized anti-mesothelin RIT (RG7787) in collaboration with Roche Pharma Research and Early Development (Fig. 1A)131718. RG7787 is highly active in vitro against several pancreatic ductal adenocarcinoma (PDAC) cell lines, including KLM-1. When tested on KLM-1 tumors in mice, RG7787 produced minor tumor regressions as a single agent, and profound tumor regressions when combined with paclitaxel13. One possible explanation for RG7787’s failure to produce profound regressions as a single agent is that insufficient concentrations of RIT reach tumor cells. Because current methods are insufficient for quantifying amounts of RIT or other antibody based agents that are delivered in vivo to tumor cells, we have developed a method to do this and applied it to a pancreatic cancer.

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