Generation and testing of clinical-grade exosomes for pancreatic cancer
Mayela Mendt, Sushrut Kamerkar, Hikaru Sugimoto, Kathleen M. McAndrews, Chia-Chin Wu, Mihai Gagea, Sujuan Yang, Elena V. Rodriges Blanko, Qian Peng, Xiaoyan Ma, Joseph R. Marszalek, Anirban Maitra, Cassian Yee, Katayoun Rezvani, Elizabeth Shpall, Valerie S. LeBleu, Raghu Kalluri
Mayela Mendt, Sushrut Kamerkar, Hikaru Sugimoto, Kathleen M. McAndrews, Chia-Chin Wu, Mihai Gagea, Sujuan Yang, Elena V. Rodriges Blanko, Qian Peng, Xiaoyan Ma, Joseph R. Marszalek, Anirban Maitra, Cassian Yee, Katayoun Rezvani, Elizabeth Shpall, Valerie S. LeBleu, Raghu Kalluri
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Research Article Oncology

Generation and testing of clinical-grade exosomes for pancreatic cancer

Abstract

Exosomes are extracellular vesicles produced by all cells with a remarkable ability to efficiently transfer genetic material, including exogenously loaded siRNA, to cancer cells. Here, we report on a bioreactor-based, large-scale production of clinical-grade exosomes employing good manufacturing practice (GMP) standards. A standard operating procedure was established to generate engineered exosomes with the ability to target oncogenic Kras (iExosomes). The clinical-grade GMP iExosomes were tested in multiple in vitro and in vivo studies to confirm suppression of oncogenic Kras and an increase in the survival of several mouse models with pancreatic cancer. We perform studies to determine the shelf life, biodistribution, toxicology profile, and efficacy in combination with chemotherapy to inform future clinical testing of GMP iExosomes. Collectively, this report illustrates the process and feasibility of generating clinical-grade exosomes for various therapies of human diseases.

Authors

Mayela Mendt, Sushrut Kamerkar, Hikaru Sugimoto, Kathleen M. McAndrews, Chia-Chin Wu, Mihai Gagea, Sujuan Yang, Elena V. Rodriges Blanko, Qian Peng, Xiaoyan Ma, Joseph R. Marszalek, Anirban Maitra, Cassian Yee, Katayoun Rezvani, Elizabeth Shpall, Valerie S. LeBleu, Raghu Kalluri

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Figure 8

GMP-grade iExosomes stability.

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GMP-grade iExosomes stability. (A) Comparison of the number of MSC exoso...
(A) Comparison of the number of MSC exosomes, quantified by NanoSight, prior to freezing and after thawing of frozen exosomes (n = 4 distinct exosomes aliquots). The times listed refer to the times that exosomes were stored for at –80°C. (B) Particle size distribution analysis of fresh and frozen (45 days) and then thawed MSC exosomes by NanoSight. (C) TEM of MSC exosomes, prior to freezing (fresh) and after freezing (45 days) and thawing. Scale bar: 100 nm. (D) Representative histogram of flow cytometry analyses of exosomal markers (CD9, CD63, CD81, CD47) on fresh versus freeze (45 days) and thaw MSC exosomes. Numbers represent the percentage of positive beads (gray, isotype control). (E and F) Representative dot plot of flow cytometry analyses (E) and quantification (F) of apoptosis in Panc-1 cells induced by MSCs siKrasG12D iExo (48 hours following iExo treatment), comparing the efficacy of freeze (3 months) and thaw iExosomes that were allowed to incubate for 3, 6, and 24 hours and 2, 3, 4, and 5 days at room temperature (RT) or 4°C (n = 2–3 independent experiments). Numbers represent the percentage of positive cells. One-way ANOVA compared with fresh exosomes. (G) KRASG12D transcript levels in Panc-1 cells treated with MSCs siKrasG12D iExo after 3 hours, comparing the efficacy of freeze (3 months) and thaw of iExosomes, under the listed conditions (n = 3 independent experiments, 1-tailed unpaired t test). The mean ± SEM is depicted. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. See Supplemental Source Data 1 and 2.