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 5

Validation of GMP-grade iExosome efficacy in vivo.

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Validation of GMP-grade iExosome efficacy in vivo. (A) Kaplan-Meier curv...
(A) Kaplan-Meier curve indicating survival after tumor induction of mice with KPC689 orthotopic tumors in the listed treatment groups (Control Exo [n = 4], BJ siKrasG12D–1 iExo [n = 6], MSCs siKrasG12D–1 iExo [n = 6]; log-rank [Mantel-Cox] test). (B) Surface lung nodules of KPC689 mice (Control Exo [n = 4], BJ siKrasG12D–1 iExo [n = 6], MSCs siKrasG12D–1 iExo [n = 6]). (C) Representative H&E-stained lung sections from KPC689 mice. Tumor metastasis is indicated by a dashed yellow line. Scale bar: 100 μm. (D) Representative images of luciferase activity of KPC689 tumors at day 28 and day 51 after tumor induction (Control Exo [n = 4], BJ siKrasG12D–1 iExo [n = 6], MSCs siKrasG12D–1 iExo [n = 6]). (E) KPC689 orthotopic tumor growth (bioluminescence) and total flux at day 51 after tumor induction (Control Exo [n = 4], BJ siKrasG12D–1 iExo [n = 6], MSCs siKrasG12D–1 iExo [n = 6]). (F) KrasG12D transcript levels in KPC689 tumors at endpoint in the listed experimental groups (Control Exo [n = 4], BJ siKrasG12D–1 iExo [n = 5], MSCs siKrasG12D–1 iExo [n = 5]; 1-tailed unpaired t test). (G) Correlation between survival and 1/dCT for KrasG12D transcript levels in KPC689 tumors (Pearson correlation test) (black dots, Control Exo [n = 4]; red dots, BJ siKrasG12D–1 iExo [n = 5]; blue dots, MSCs siKrasG12D–1 iExo [n = 5]). (H) Kaplan-Meier curve indicating the survival of Panc-1 tumor-bearing mice after tumor induction in the listed treatment groups (Control Exo [n = 5], MSCs siKrasG12D–1 iExo, CB [n = 5], MSCs siKrasG12D–1 iExo, RB [n = 4], BJ siKrasG12D–1 iExo, RB [n = 5]; log-rank (Mantel-Cox) test). #, pancreas was normal and mice were not moribund (see Supplemental Figure 7A for details). (I) Representative H&E-stained sections of tumors from Panc-1 tumor-bearing mice (Control Exo [n = 5], MSCs siKrasG12D–1 iExo, CB [n = 5], MSCs siKrasG12D–1 iExo, RB [n = 4], BJ siKrasG12D–1 iExo, RB [n = 5]). Scale bar: 100 μm. Data are also depicted in Supplemental Figure 7B. The mean ± SEM is depicted. Unless stated otherwise, 1-way ANOVA, comparing experimental groups to the control group, was used to determine statistical significance. Unless the P value is specified on the figure, *P < 0.05, **P < 0.01, ***P < 0.001. See Supplemental Source Data 1 and 2.