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356 Generation of GD2-CAR neutrophils from hPSCs for targeted cancer immunotherapy of solid tumors
  1. Asiri Majumder1,
  2. Ho Sun Jung1,
  3. Jue Zhang2,
  4. Kran Suknuntha1,
  5. James Thomson2 and
  6. Igor Slukvin1
  1. 1University of Wisconsin, Madison, WI, USA
  2. 2Morgridge Institute for Research, Madison, WI, USA


Background Chimeric antigen receptor (CAR) T cell and NK cell therapies already been successful in the eradication of lymphoid malignancies. However, many challenges remain in the applying CAR therapies for solid tumors, and responses with CAR-T cells have been limited to isolated exceptional cases. Thus, opportunities exist for new immunotherapies for specific targeting of solid tumors using CAR-weaponized neutrophils which are capable of cytotoxicity and migration into solid tumors. Human pluripotent stem cells (hPSCs) are a logical alternative for large-scale production of CAR neutrophils due to their renewability and uniform quality. In this study we explored a feasibility of generation GD2-CAR neutrophils from hPSCs with superior cytotoxic activities against GD2-expressing tumors in vitro and in vivo.

Methods We used CRISPR-Cas9 gene editing method to integrate a third generation GD2-CAR (anti-GD2-14g2A-CD28-OX40-CD3z) into AAVS1 locus of IISH2i-BM9 hiPSCs. GD2-CAR-hiPSCs differentiated into neutrophils in defined serum- and feeder-free conditions using ETV2 modified mRNA. The in vitro antitumor activity of CAR-M was evaluated by co-culture with GD2-expressing CHLA-20 neuroblastoma and WM266-4 melanoma and GD2-negative SKOV3 ovarian carcinoma and SK-BR3 breast carcinoma. To assess in vivo potential of GD2 CAR neutrophils, NSG mice were inoculated intraperitoneally (IP) with 3x105 Luc2-eGFP+ WM266-4 melanoma cells and engraftment was assessed by IVIS bioluminescent imaging. On day 4 post WM266-4 injection, mice were either treated with 107 WT or GD2-CAR neutrophils via IP injection every 7 days.

Results GD2-CAR hiPSCs differentiated into CAR-neutrophils with the same efficiency as unmodified hiPSCs. CAR-neutrophils demonstrated typical neutrophil morphology and phenotype, including expression CD15, lactoferrin and MPO. Neutrophils generated from GD2-CAR hiPSCs, as compared to unmodified neutrophils, demonstrated superior cytotoxicity in vitro against GD2+ WM266-4 melanoma and CHLA20 neuroblastoma, while minimal differences were observed in cytotoxicity against GD2-negative SKOV3 ovarian and SK-BR3 breast cancer cells between unmodified and CAR-neutrophils. Upon assessment of anti-tumor activities of GD2-CAR neutrophils in mice engrafted with WM266-4 melanoma over 30 days (figure 1), CAR neutrophil-treated mice showed significantly reduced tumor burden (figure 2) and prolonged survival (figure 3) compared to untreated mice or mice treated with unmodified iPSC-derived neutrophils.

Conclusions Our studies demonstrate that hiPSCs can be used to efficiently generate CAR-neutrophils with potent activity against solid tumors. Thus, hiPSCs provide a novel approach for generation CAR-neutrophil off-the-shelf product for targeted immunotherapy of solid tumors.

Acknowledgements This work is supported by funds from National Institute of Health, United States (R01HL142665, and P51 OD011106).


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  2. Suknuntha K, Tao L, Brok-Volchanskaya V, D’Souza SS, Kumar A, Slukvin I. Optimization of synthetic mRNA for highly efficient translation and Its application in the generation of endothelial and hematopoietic cells from human and primate pluripotent stem cells. Stem Cell Rev. 2018; 14: 525–534.

  3. Brok-Volchanskaya V, Bennin DA, Suknuntha K, Klemm L, Huttenlocher A, Slukvin I. Effective and Rapid Generation of Functional Neutrophils from Induced Pluripotent Stem Cells Using ETV2-Modified mRNA. Stem Cell Reports. 2019; 13:1099–1110.

Ethics Approval The animal experiments were performed under approval from UW-Madison, Institutional Review Board.

Abstract 356 Figure 1

Schematic of the in vivo cytotoxicity assay.NSG mice inoculated IP with Luc2-GFP WM266-4 melanoma cells. On day 4 after melanoma injection, mice were left untreated, or treated with unmodified or CAR-N injected intraperitoneally every 7 days.

Abstract 356 Figure 2

In vivo evaluation of CAR-N cytotoxicity.Tumor burden was determined by bioluminescent imaging using IVIS imager showing total flux of the ventral axis.

Abstract 356 Figure 3

Kaplan-Meier curve for survivalKaplan-Meier curve representing the percent survival of the experimental groups.

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