Generation of Natural Killer Cells with Enhanced Function from a CRISPR/Cas12a-Edited Induced Pluripotent Stem Cell Line

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 8-8
Author(s):  
Jung-Il Moon ◽  
Melissa S Chin ◽  
Andrew T Burden ◽  
Steven Sexton ◽  
Kevin Wasko ◽  
...  
Keyword(s):  
Natural Killer Cells ◽  
Cell Therapy ◽  
Nk Cells ◽  
Natural Killer ◽  
Tumor Cells ◽  
Effector Function ◽  
Publicly Traded ◽  
Killer Cells ◽  
Killing Assay ◽  
Induced Pluripotent

Adoptive cell therapy using T cells to treat cancer is efficacious in a number of hematologic malignancies. Recently, natural killer (NK) cells have emerged as an alternative cell type for clinical utility given the low propensity for graft-versus-host disease, thereby making NK cells a potential off-the-shelf cell therapy. NK cells distinguish tumor from healthy tissue via multiple mechanisms, including recognition of stress ligands and loss of MHC class I expression. For instance, KIR mismatching enables allogenic NK cells to kill MHC-positive tumor cells similar to MHC-negative tumor cells. Effector function of allogeneic NK cells are typically diminished by limited functional persistence, as well as tumor-intrinsic immunosuppressive mechanisms, such as TGF-β, a pleiotropic cytokine that inhibits immune effector function. Gene editing, however, can overcome these biological limitations. We hypothesized that knockout of CISH, a negative regulator of IL-2/IL-15 signaling, would improve NK cell effector function, while knockout of the TGF-β receptor gene 2, TGFBR2, would render NK cells resistant to TGF-β mediated suppression. NK cells are typically isolated from either cord blood or peripheral blood of healthy donors but recent advances with induced pluripotent stem cells (iPSCs) allows a nearly unlimited supply of iPSC-derived natural killer cells (iNK). In this study, we used CRISPR/Cas12a to generate edited iPSC lines that were differentiated into CD56+ iNK cells. Specifically, we generated TGFβR2-/-, CISH-/-, and TGFβR2-/-/CISH-/- iPSC clones with bi-allelic gene disruption confirmed by next generation sequencing. Importantly, we also confirmed that the edited clones were pluripotent. In particular, a minimum of 3 clones from each genotype were differentiated to CD56+ iNK cells. After differentiation, >90% of the cells expressed CD56 for all genotypes. Additionally, we observed the expression of canonical natural killer cell markers such as CD16, NKG2A, KIRs, NKp46, NKp44, and NKp30 within this CD56+ population. We tested the effector function of TGFβR2-/-, CISH-/-, and TGFβR2-/-/CISH-/- iNKs in a variety of molecular and functional assays, including a spheroid killing assay and an in vitro serial killing assay. For example, we utilized a SK-OV-3 spheroid killing assay to determine the intrinsic ability for the iNK cells to kill tumor targets following the differentiation process. TGFβR2-/-, CISH-/-, and TGFβR2-/-/CISH-/- iNKs reduce the size of SK-OV-3 ovarian tumor spheroids more effectively than control iNK cells in the presence of exogenous TGF-β. In conclusion, we have established an iPSC editing platform that can generate a near infinite supply of natural killer cells with enhanced tumor killing function, paving the way for future off-the-shelf cell therapies for application to broad oncology indications. Disclosures Moon: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Chin:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Burden:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Sexton:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wasko:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Nasser:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Antony:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wong:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Borges:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Morgan:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Welstead:Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

scholarly journals The Role of Natural Killer Cells as a Platform for Immunotherapy in Pediatric Cancers

2019 ◽  
Vol 21 (10) ◽  
Author(s):  
Miriam Santiago Kimpo ◽  
Bernice Oh ◽  
Shawn Lee
Keyword(s):  
Natural Killer Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Tumor Cells ◽  
Cell Function ◽  
Nk Cell ◽  
Childhood Cancers ◽  
Killer Cells ◽  
Cell Immunotherapy

Abstract Purpose of Review We aim to review the most recent findings in the use of NK cells in childhood cancers. Recent Findings Natural killer cells are cytotoxic to tumor cells. In pediatric leukemias, adoptive transfer of NK cells can bridge children not in remission to transplant. Interleukins (IL2, IL15) can enhance NK cell function. NK cell-CAR therapy has advantages of shorter life span that lessens chronic toxicities, lower risk of graft versus host disease when using allogeneic cells, ability of NK cells to recognize tumor cells that have downregulated MHC to escape T cells, and possibly less likelihood of cytokine storm. Cytotoxicity to solid tumors (rhabdomyosarcoma, Ewing’s sarcoma, neuroblastoma) is seen with graft versus tumor effect in transplant and in combination with antibodies. Challenges lie in the microenvironment which is suppressive for NK cells. Summary NK cell immunotherapy in childhood cancers is promising and recent works aim to overcome challenges.

scholarly journals Analysis of sphingosine kinase activity in single natural killer cells from peripheral blood

2015 ◽  
Vol 7 (4) ◽  
pp. 392-401 ◽  
Author(s):  
Alexandra J. Dickinson ◽  
Megan Meyer ◽  
Erica A. Pawlak ◽  
Shawn Gomez ◽  
Ilona Jaspers ◽  
...  
Keyword(s):  
Natural Killer Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Tumor Cells ◽  
Peripheral Blood ◽  
Kinase Activity ◽  
Sphingosine Kinase ◽  
Killer Cells ◽  
Pathway Activity

Heterogeneity of sphingosine kinase (SK) pathway activity in natural killer (NK) cells may enable cells to respond effectively to a diverse array of pathogens as well as incipient tumor cells.

scholarly journals Off-the-shelf cell therapy with induced pluripotent stem cell-derived natural killer cells

2018 ◽  
Vol 41 (1) ◽  
pp. 59-68 ◽  
Author(s):  
Michelle L. Saetersmoen ◽  
Quirin Hammer ◽  
Bahram Valamehr ◽  
Dan S. Kaufman ◽  
Karl-Johan Malmberg
Keyword(s):  
Stem Cell ◽  
Natural Killer Cells ◽  
Cell Therapy ◽  
Natural Killer ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Killer Cells ◽  
Induced Pluripotent

Natural Killer cells and monoclonal antibodies: Two partners for successful antibody dependent cytotoxicity against tumor cells

2021 ◽  
Vol 160 ◽  
pp. 103261
Author(s):  
Mélanie Gauthier ◽  
Caroline Laroye ◽  
Danièle Bensoussan ◽  
Cédric Boura ◽  
Véronique Decot
Keyword(s):  
Monoclonal Antibodies ◽  
Natural Killer Cells ◽  
Natural Killer ◽  
Tumor Cells ◽  
Killer Cells

scholarly journals 799 Neoantigen-cytokine-chemokine multifunctional natural killer cell engager for immunotherapy of solid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A834-A834
Author(s):  
Xue Yao ◽  
Sandro Matosevic
Keyword(s):  
Tumor Microenvironment ◽  
Cell Therapy ◽  
Nk Cells ◽  
Natural Killer ◽  
Solid Tumors ◽  
Tumor Cells ◽  
Nk Cell ◽  
Killer Cell ◽  
Nk Cell Therapy

BackgroundThe effectiveness of natural killer (NK) cell-based immunotherapy against solid tumors is limited by the lack of specific antigens and the immunosuppressive tumor microenvironment (TME). Glioblastoma multiforme (GBM) is one such heavily immunosuppressive tumor that has been particularly hard to target and remains without a viable treatment. The development of novel approaches to enhance the efficacy of NK cells against GBM is urgently needed. NK cell engagers (NKCE) have been developed to enhance the efficacy of NK cell therapy.MethodsTo improve the clinical efficacy of NK cell therapy, we are developing a new generation of multi-specific killer engagers, which consists of a neoantigen-targeting moiety, together with cytokine and chemokine-producing domains. Neoantigens are new antigens formed specifically in tumor cells due to genome mutations, making them highly specific tools to target tumor cells. Our engager has been designed to target Wilms' tumor-1 (WT-1), a highly specific antigen overexpressed in GBM among other solid tumors. This is done through the generation of an scFv specific targeting the complex of WT-1126-134/HLA-A*02:01 on the surface of GBM. On the NK cell side, the engager is designed to target the activating receptor NKp46. Incorporation of the cytokine IL-15 within the engager supports the maturation, persistence, and expansion of NK cells in vivo while favoring their proliferation and survival in the tumor microenvironment. Additionally, our data indicated that the chemokine CXCL10 plays an important role in the infiltration of NK cells into GBM, however, GBM tumors produce low levels of this chemokine. Incorporation of a CXCL10-producing function into our engager supports intratumoral NK cell trafficking by promoting, through their synthetic production, increased levels of CXCL10 locally in the tumor microenvironment.ResultsCollectively, this has resulted in a novel multifunctional NK cell engager, combining neoantigen-cytokine-chemokine elements fused to an activating domain-specific to NK cells, and we have investigated its ability to support and enhance NK cell-mediated cytotoxicity against solid tumors in vitro and in vivo against patient-derived GBM models. The multi-specific engager shows both high tumor specificity, as well as the ability to overcome NK cell dysfunction encountered in the GBM TME.ConclusionsWe hypothesize that taking advantage of our multi-functional engager, NK cells will exhibit superior ex vivo expansion, infiltration, and antitumor activity in the treatment of GBM and other solid tumors.

scholarly journals In Vivo Survival and Homeostatic Proliferation of Natural Killer Cells

2003 ◽  
Vol 197 (8) ◽  
pp. 967-976 ◽  
Author(s):  
Martin Prlic ◽  
Bruce R. Blazar ◽  
Michael A. Farrar ◽  
Stephen C. Jameson
Keyword(s):  
T Cells ◽  
Natural Killer Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Sorting ◽  
Functional Activity ◽  
Homeostatic Proliferation ◽  
Killer Cells ◽  
Bystander Cells

While the specificity and development of natural killer (NK) cells have been intensely studied, little is known about homeostasis of the mature NK population. Here we show that mouse NK cells undergo homeostatic proliferation when transferred into NK-deficient Rag−/− γC−/− hosts. Normal NK functional activity is maintained during this process, although there are some changes in NK phenotype. Using cell sorting, we demonstrate that mature (Mac-1hi) NK cells undergo homeostatic proliferation in an NK-deficient environment, yet immature (Mac-1lo) NK cells also proliferate in such hosts. We find that mature NK cells survive but do not proliferate in hosts which possess an endogenous NK pool. However, we go on to show that mature NK survival is critically dependent on interleukin (IL)-15. Surprisingly, NK survival is also compromised after transfer of cells into IL-15Rα−/− mice, implying that IL-15 responsiveness by bystander cells is critical for NK maintenance. These data imply that, similar to T cells, homeostasis of the NK pool is much more dynamic than previously appreciated and this may be relevant to manipulation of NK cells for therapeutic purposes.

scholarly journals Placentation and antitumor immunity regulated by a scaffolding protein in NK cells

2019 ◽  
Vol 4 (38) ◽  
pp. eaax9589
Author(s):  
Francesco Colucci
Keyword(s):  
Natural Killer Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Tumor Cells ◽  
Antitumor Immunity ◽  
Adaptor Protein ◽  
Scaffolding Protein ◽  
Trophoblast Invasion ◽  
Related Research ◽  
Research Article

Natural killer cells use the Gab3 adaptor protein to limit trophoblast invasion during pregnancy and to reject tumor cells. See the related Research Article by Sliz et al.

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