Name: advances in human induced pluripotent stem cell-derived chimeric antigen receptor-expressing natural killer cells
Uploaded: 2025-02-14T14:10:00.000+00:00
Duration: 11 min 8 s
Description: Natural killer (NK) cells are innate immune cells that play a crucial role in the body's defense against tumors and viral infections. The generation of ...

We thank all Kaufman lab members for their support, scientific insights and discussions. These studies were supported by the NIH/NCI grants U01CA217885, P30CA023100 (administrative supplement) and the Sanford Stem Cell Institute at UCSD. JT: writing and revision of manuscript. DSK: reviewed and edited the manuscript.

DSK is a co-founder and advisor to Shoreline Biosciences and has an equity interest in the company. DSK also consults for Therabest and RedC Bio for which he receives income and/or equity. The terms of these arrangements have been reviewed and approved by the University of California, San Diego in accordance with its conflict-of-interest policies. The remaining authors declare no competing interests.

This protocol outlines a standardized and reproducible approach for generating CAR-expressing, iPSC-derived NK cells from a consistent cell source aimed at facilitating targeted &#34;off-the-shelf&#34; cancer immunotherapy. Multiple preclinical and clinical studies have shown the efficacy of adoptive NK cell-based immunotherapy in treating cancers while minimizing toxicities, like graft versus host diseases (GvHD) or cytokine-release syndrome (CRS)23,42, 49,50,51,52,53,54,55,56,57. This approach utilizes an efficient and well-defined system to produce homogeneous and well-characterized CAR iPSC-derived NK cells can be scaled for clinical application. Additionally, iPSC-derived NK cells engineered to express NK-optimized CAR retain typical NK cell phenotypes and cytolytic functions 23,39,42,50,51,52,58.
Using human iPSCs for CAR iPSC-derived NK cell production offers a more efficient method for genetic modification, including CAR expression, compared to primary NK cells isolated from peripheral blood31,59,60. In addition to CAR expression, further enhancements to the antitumor activity of these cells can be achieved through modifications such as the deletion of inhibitory receptors or the introduction of cytokine expression41,39,61. This can be achieved through a single genetic modification event, eliminating the need for patient-specific modifications as seen in current CAR-T cell therapies31,62,63. Additionally, NK cell-based therapies with novel CAR-expressing cells can potentially be used in treating chronic infectious diseases30,64,65,66,67,68.
This protocol collectively demonstrates the potential of using human iPSCs to create homogeneous populations of CAR-expressing NK cells to enhance in vitro and in vivo antitumor activity. NK cell-optimized CARs enable antigen-specific activation of signaling pathways, improving the function of these cells. Modifications and troubleshooting are essential for optimizing the yield and functionality of CAR NK cells. Adjusting the cytokine cocktail and timing can improve differentiation efficiency. Ensuring the stability and expression of the CAR construct in NK cells may require optimizing transfection methods or vector design. Additional troubleshooting could address issues such as low transduction efficiency, cell viability, and functional activity of CAR iPSC derived NK cells, and may involve iterative testing of different culture conditions or genetic modifications to enhance persistence, cytotoxicity, and safety.
The significance of our method lies in its ability to generate a potentially unlimited supply of CAR NK cells from a standardized iPSC source. iPSC-derived CAR NK cells offer a stable platform for multiple gene edits, reducing variability and improving the consistency of the engineered cells. This method also enables the creation of &#34;off-the-shelf&#34; NK cell therapies that are ready for immediate use, bypassing the need for individualized cell sourcing and processing.
In addition, this protocol has certain limitations in maintaining the stability of CAR expression when using piggyBac vectors, as the transient nature of transgene integration may result in variable expression levels over time. Furthermore, the reliance on aAPCs and specific cytokines for expansion can complicate the scalability of these processes for clinical applications. Lastly, potential immune responses against the piggyBac elements may pose risks in therapeutic settings.
In conclusion, the differentiation and expansion of CAR-NK cells from human iPSCs hold significant promise for treating hepatocellular carcinoma (HCC) and other malignancies. This innovative approach leverages the regenerative potential of iPSCs and the innate immune properties of NK cells to create a potent and targeted cancer immunotherapy. Advances in this field could lead to more effective and widely available NK cell-based therapies, offering new hope for patients with HCC and other hard-to-treat diseases.

NK cells, a type of lymphocyte within the innate immune system, are pivotal in the early defense against tumors and virally infected cells1,2,3. Unlike T cells, NK cells do not require antigen presentation via major histocompatibility complex (MHC) molecules. Instead, NK cells have a repertoire of activating and inhibitory receptors that regulate their activity3. NK cell-mediated cytotoxic activity utilizes various mechanisms, including the release of perforin and granzymes, engagement of death receptors, and the production of pro-inflammatory cytokines such as IFN-&#947; and TNF-&#945;. This unique mode of action positions NK cells as an attractive candidate for cancer immunotherapy, particularly in the treatment of solid cancers where immune evasion is a significant hurdle1,2,3,4.
Hepatocellular carcinoma (HCC) is one of the most common and deadly forms of liver cancer worldwide5. Traditional therapeutic approaches, including surgery, chemotherapy, and radiotherapy, typically provide limited clinical benefit and result in high recurrence rates6,7. Recent advances in immunotherapy and targeted therapy have significantly impacted the treatment of HCC8,9. Immune checkpoint inhibitors, like nivolumab and pembrolizumab, have shown promising results by enhancing the immune cell response against cancer cells10,11. These therapies have led to improved survival rates and better quality of life for some patients. However, they can also cause immune-related adverse effects, which may limit their use in certain patients12. Targeted therapies, such as sorafenib and lenvatinib, specifically inhibit pathways that promote cancer cell growth and angiogenesis13. These treatments have known efficacy to control disease progression and prolong survival8,14. Nonetheless, resistance to targeted therapies typically develops, and side effects of treatment are common15,16. Among the promising avenues in cancer immunotherapeutic strategies, the advent of chimeric antigen receptor (CAR) CAR T cells has revolutionized cancer treatment, especially in the treatment of hematologic malignancies such as lymphoma and multiple myeloma17,18,19.
CAR-expressing NK cells, combining the innate cytotoxic activity of NK cells with the precision targeting of CAR technology, represent an innovative and potentially transformative approach for solid tumors such as HCC20,21,22,23,24. CAR-engineered cells can specifically recognize and kill tumor cells expressing the target antigen while sparing normal tissues, thereby reducing the risk of off-target effects associated with conventional therapies25,26,27. CAR-NK cells produced from NK cells isolated from peripheral blood or cord blood are typically produced by transducing NK cells with CAR constructs that consist of an extracellular antigen-recognition domain, a transmembrane domain, and intracellular signaling domains necessary for activation and proliferation28,29,30.
The challenge of engineering a stable and homogenous population of functionally engineered NK cells for clinical treatment can be addressed by employing induced pluripotent stem cells (iPSCs)31,32. Engineering of human iPSCs with NK cell-optimized CARs provides an improved NK cell activation and proliferation signal and provides an inexhaustible and homogenous population of CAR-expressing NK cells as a standardized, &#34;off-the-shelf therapy&#34;20,33. In this protocol, genetic modification of iPSCs to generate CAR-expressing iPSC-derived NK cells involves integrating an NK cell optimized CAR construct that includes NK cell-derived transmembrane and signaling domains (NKG2D-2B4-CD3&#950;) and an anti-glypican-3 (GPC3) scFv as described in our previous studies23. The genetically engineered iPSCs are then differentiated and expanded using an NK cell differentiation protocol developed previously34. These engineered CAR-expressing iPSC-derived NK cells have the ability to recognize and eliminate HCC and other tumor cells expressing specific tumor-associated antigens, such as Glypican 3 (GPC3), which is overexpressed in HCC and other malignancies35,36,37,38.
The application of iPSC-derived CAR-NK cells for the treatment of diverse cancers holds significant promise30,39,40; many of these iPSC-derived CAR-NK cells are currently in clinical trials41,42,43. To facilitate advancement in this area, this protocol enables efficient production of engineered iPSC-derived CAR-NK cells, from cell engineering to differentiation into mature NK cells and in vitro expansion.

<table><tbody><tr><td>aAPC&amp;nbsp;</td><td>Dean A. 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Kaufman lab</td><td>N/A</td><td></td></tr><tr><td>Human: iPS cells&amp;nbsp;</td><td>Dan S. Kaufman lab&amp;nbsp;</td><td>N/A</td><td></td></tr><tr><td>Human: SNU-449 cells&amp;nbsp;</td><td>ATCC</td><td>Cat#CRL-2234</td><td></td></tr><tr><td>Human: SNU-449-td-tomato-luc cells&amp;nbsp;</td><td>Dan S. Kaufman lab</td><td>N/A</td><td></td></tr><tr><td>IncuCyte Caspase-3/7 Green Apoptosis Assay&amp;nbsp;</td><td>Essenbioscience&amp;nbsp;</td><td>Cat#4440</td><td></td></tr><tr><td>L-Ascorbic acid</td><td>Sigma Aldrich</td><td>A5960</td><td></td></tr><tr><td>MP Biomedicals&amp;nbsp;Human Serum, Type AB</td><td>MP Biomedicals</td><td>ICN2938249</td><td></td></tr><tr><td>mTeSR plus</td><td>StemCell Technologies, Inc.&amp;nbsp;</td><td>100-0276</td><td></td></tr><tr><td>NovoExpress software&amp;nbsp;</td><td>ACEA Biosciences&amp;nbsp;</td><td>https://www.agilent.com/en/product/research-flow-cytometry/flow-cytometry-software/novocyte-novoexpress-software-1320805</td><td></td></tr><tr><td>PE/Cy7 anti-human SSEA-4 Antibody&amp;nbsp;</td><td>Biolegend</td><td>330420</td><td></td></tr><tr><td>PE-CD16&amp;nbsp;</td><td>BD Biosciences&amp;nbsp;</td><td>Cat#560995&amp;nbsp;</td><td></td></tr><tr><td>PE-CD226</td><td>BD Biosciences&amp;nbsp;</td><td>Cat#559789</td><td></td></tr><tr><td>PE-CD34&amp;nbsp;</td><td>BD Biosciences</td><td>Cat# 555822&amp;nbsp;</td><td></td></tr><tr><td>PE-CD45&amp;nbsp;</td><td>BD Biosciences&amp;nbsp;</td><td>Cat# 555483&amp;nbsp;</td><td></td></tr><tr><td>PE-CD94&amp;nbsp;</td><td>BD Biosciences&amp;nbsp;</td><td>Cat#555888&amp;nbsp;</td><td></td></tr><tr><td>PE-cy7-CD56&amp;nbsp;</td><td>BioLegend</td><td>Cat# 318318&amp;nbsp;</td><td></td></tr><tr><td>PE-FAS Ligand&amp;nbsp;</td><td>BD Biosciences&amp;nbsp;</td><td>Cat#564261&amp;nbsp;</td><td></td></tr><tr><td>PE-NKp30</td><td>BD Biosciences</td><td>Cat# 558407</td><td></td></tr><tr><td>PE-NKp44&amp;nbsp;</td><td>BD Biosciences&amp;nbsp;</td><td>Cat#558563&amp;nbsp;</td><td></td></tr><tr><td>PE-NKp46</td><td>BD Biosciences&amp;nbsp;</td><td>Cat#331908</td><td></td></tr><tr><td>PE-NKp46&amp;nbsp;</td><td>BD Biosciences&amp;nbsp;</td><td>Cat#557991&amp;nbsp;</td><td></td></tr><tr><td>Peripheral blood buffy coat&amp;nbsp;</td><td>San Diego Blood Bank (https://www. sandiegobloodbank.org/)&amp;nbsp;</td><td>N/A</td><td></td></tr><tr><td>PE-TRAIL&amp;nbsp;</td><td>BD Biosciences&amp;nbsp;</td><td>Cat#565499&amp;nbsp;</td><td></td></tr><tr><td>pKT2-mCAG-IRES-GFP-ZEO&amp;nbsp;</td><td>Branden Moriarity lab&amp;nbsp;</td><td>N/A</td><td></td></tr><tr><td>pMAX-GFP plasmid</td><td>Lonza</td><td>N/A</td><td>GFP positive control&amp;nbsp;</td></tr><tr><td>Prism 9</td><td>Graphpad&amp;nbsp;</td><td>Version 9</td><td></td></tr><tr><td>pSpCas9&amp;nbsp;</td><td>GenScript</td><td>PX165</td><td></td></tr><tr><td>RBC Lysis Buffer (10x)</td><td>Biolegend</td><td>Cat#420301</td><td></td></tr><tr><td>Recombinant human bFGF basic&amp;nbsp;</td><td>R&amp;amp;D Systems&amp;nbsp;</td><td>Cat#4114-TC</td><td></td></tr><tr><td>Recombinant human BMP-4&amp;nbsp;</td><td>PeproTech</td><td>Cat#120-05</td><td></td></tr><tr><td>Recombinant human FLT-3 Ligand&amp;nbsp;</td><td>PeproTech</td><td>Cat# 300-19</td><td></td></tr><tr><td>Recombinant human IL-15&amp;nbsp;</td><td>PeproTech</td><td>Cat# 200-15</td><td></td></tr><tr><td>Recombinant human IL-2&amp;nbsp;</td><td>PeproTech</td><td>Cat# 200-02</td><td></td></tr><tr><td>Recombinant human IL-3&amp;nbsp;</td><td>PeproTech</td><td>Cat#200-03</td><td></td></tr><tr><td>Recombinant human IL-7&amp;nbsp;</td><td>PeproTech</td><td>Cat# 200-07</td><td></td></tr><tr><td>Recombinant Human Nodal Protein</td><td>R&amp;amp;D Systems&amp;nbsp;</td><td>Cat#3218-ND-025</td><td></td></tr><tr><td>Recombinant human SCF&amp;nbsp;</td><td>PeproTech</td><td>Cat# 300-07</td><td></td></tr><tr><td>Recombinant Human TGF-&amp;beta;1</td><td>PeproTech</td><td>Cat#100-21</td><td></td></tr><tr><td>Recombinant human VEGF&amp;nbsp;</td><td>PeproTech</td><td>Cat# 100-20</td><td></td></tr><tr><td>RPMI1640</td><td>Gibco</td><td>11875093</td><td></td></tr><tr><td>Sodium selenite</td><td>Sigma Aldrich</td><td>214485</td><td></td></tr><tr><td>STEMdiff APEL 2 Medium</td><td>StemCell Technologies, Inc.&amp;nbsp;</td><td>5270</td><td>EB formation medium</td></tr><tr><td>STEMdiff APEL2 Medium&amp;nbsp;</td><td>StemCell Technologies, Inc.&amp;nbsp;</td><td>Cat#05270</td><td></td></tr><tr><td>Super piggyBac Transposase expression vector</td><td>SBI</td><td>Cat#PB210PA-1</td><td></td></tr><tr><td>SYTOX AADvanced Dead Cell Stain Kit</td><td>ThermoFisher Scientific</td><td>S10274, S10349</td><td>Dead cell staining solution kit</td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Gibco</td><td>21985023</td><td></td></tr></tbody></table>

advances in human induced pluripotent stem cell-derived chimeric antigen receptor-expressing natural killer cells

Natural killer (NK) cells are innate immune cells that play a crucial role in the body's defense against tumors and viral infections. The generation of human induced pluripotent stem cell (iPSC)-derived chimeric antigen receptor (CAR) expressing NK cells has emerged as a promising avenue for "off the shelf" cancer immunotherapy. Here, we utilized an NK cell-optimized CAR construct that includes the transmembrane domain of NKG2D, the 2B4 co-stimulatory domain, and the CD3&#950; signaling domain, which has been demonstrated to stimulate robust antigen-specific NK cell-mediated antitumor activity. The use of iPSCs for CAR NK cell generation offers several advantages, including homogenous CAR expression, scalability, reproducibility, and the potential for clinical application. This detailed step-by-step protocol from cell engineering to differentiation enables the generation of NK cell-optimized iPSC-derived CAR-expressing NK cells, providing a standardized and targeted cancer immunotherapy with improved antitumor activity and highlighting their potential as a promising treatment option for various malignancies.

Schematic diagram of anti-GPC3-CAR iPSC derived NK cells differentiation and expansion 
The schematic diagram illustrates the in vitro differentiation of anti-GPC3-CAR engineered NK cells derived from human induced pluripotent stem cells (iPSCs) and a schematic representation of piggyBac vector carrying GPC3 CAR construct. Initially, unmodified iPSCs are transfected with a piggyBac vector encoding the GPC3 CAR (chimeric antigen receptor). Following transfection, these GPC3 CAR-expressing iPSCs are clonally expanded and then differentiated into functional CAR iPSC-derived NK cells. These engineered NK cells are subsequently subjected to in vitro and in vivo functional assays to evaluate their antitumor activity against various tumor cell lines (Figure 1).
Microscopic analysis of the NK differentiation process of WT and anti-GPC3-CAR engineered iPSCs at various stages 
The GPC3 CAR-engineered iPSCs are differentiated into anti-GPC3-CAR-engineered iPSC NK cells. The formation of spin embryoid bodies (EBs) from GPC3 CAR engineered iPSCs, to the differentiation of GPC3-CAR iPSC NK cells were presented as a series of microscopic images documenting the differentiation stages of anti-GPC3-CAR engineered iPSCs into functional GPC3 CAR iPSC NK cells. The undifferentiated wild-type (WT) and GPC3 CAR iPSCs were cultured in mTeSR Plus medium (Figure 2A). These WT and anti-GPC3-CAR iPSCs are then dissociated into single cells and cultured in STEMdiff APEL medium containing SCF (40 ng/mL), BMP4 (20 ng/mL), VEGF (20 ng/mL) and 10 &#181;M ROCK inhibitor for 6 days in an ultra-low attachment U-bottom 96 well plate. The formation of spin EBs was captured on day 6 from both WT and GPC3 CAR iPSCs (Figure 2B). These spin EBs are further transferred to 6 well plate containing NK differentiation medium with the following cytokines IL-3 (5 ng/mL), SCF (20 ng/mL), IL-7 (20 ng/mL), and IL-15 (10 ng/mL). The differentiation process of WT and GPC3 CAR iPSC-derived NK cells at various time points from day 3 to day 28, were captured using a microscope at 100x magnification (Figure 2C).
Phenotype of WT and anti-GPC3 CAR iPSC derived EBs (day 6) and WT and GPC3 CAR iPSC derived NK cells (day 35) 
The WT and GPC3 CAR iPSC-derived cells are phenotypically characterized at different stages of NK differentiation process. Panel A shows the expression of typical hematopoietic antigens CD34, CD31, CD43, and CD45 from the spin EBs of WT and GPC3 CAR iPSCs collected on day 6 were measured by flow cytometry (Figure 3A). Panel b presents the phenotype of WT and GPC3 CAR iPSC-derived NK cells harvested from the NK differentiation medium on day 35, highlighting the NK-specific markers (CD45, CD56, CD16, and NKG2D) associated with mature NK cells were measured by flow cytometry (Figure 3B).
Phenotype of expanded WT and anti-GPC3-CAR iPSC derived NK cells 
After the Differentiation of WT and anti-GPC3 CAR iPSC-derived NK cells, the differentiated NK cells are harvested and expanded using irradiated aAPCs in a Gibco NK Xpander medium containing IL-2 (100 U/mL) and IL-15 (10 ng/mL). The expanded WT and anti-GPC3 CAR iPSC NK cells activation receptors such as CD94, CD16, NKp30, NKp44, NKp46, NKG2D, CD226, FasL, and TRAIL were measured by using flow cytometry (Figure 4A). After confirming the NK activation phenotype on the expanded WT and anti-GPC3 CAR iPSC NK cells, we evaluated the surface expression of GPC3 antigen on various tumor cell lines, including HepG2, SNU-449, SKOV3, and CAL27, via flow cytometry (Figure 4B). We have also confirmed the CAR expression on expanded anti- GPC3 CAR iPSC-derived NK cells by flow cytometric analysis.
Functional activity of WT and anti-GPC3 CAR iPSC derived NK cells against HCC and other tumor cell lines 
After confirming the phenotype of WT and anti-GPC3 CAR iPSC-derived NK cells, we evaluated the functional antitumor activity against various tumor cell lines. Caspase3/7-based killing assays were conducted to assess the cytotoxic activity of WT and anti-GPC3 CAR iPSC-derived NK cells against HepG2, SNU-449, CAL27, and SKOV3 cell lines at various effector-to-target ratios. The results indicate the enhanced antitumor efficacy of anti-GPC3 CAR iPSC-derived NK cells compared to WT iPSC NK cells, showcasing their potential in targeting GPC3-expressing tumor cells via CAR specificity and other NK activation mechanisms (Figure 5A-D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67438/67438fig01.jpg" /> 
Figure 1: Schematic diagram of anti-GPC3-CAR iPSC derived NK differentiation, expansion and clinical application. (A) Overview of the process for generating anti-GPC3 CAR iPSC-derived NK cells for preclinical and clinical use. Unmodified iPSCs are transfected with a piggyBac vector carrying anti-GPC3-CAR gene. After successful transfection, anti-GPC3-CAR-expressing iPSCs are clonally expanded and differentiated into functional NK cells. These anti-GPC3 CAR iPSC-derived NK cells are subjected to both in vitro and in vivo functional assays to assess their cytotoxic activity against various GPC3-expressing tumor cell lines39. Upon validation of their efficacy and safety, these CAR iPSC-derived NK cells are further developed as a potential &#34;off-the-shelf&#34; therapy for clinical applications in cancer treatment. (B) Schematic representation of the piggyBac vector containing the anti-GPC3 CAR construct used for transfecting iPSCs. (C) Typical timeline of iPSC-derived NK cell differentiation, expansion, harvesting, and performing functional assays. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67438/67438fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67438/67438fig02.jpg" /> 
Figure 2: Microscopic images of CAR iPSC NK differentiation from anti-GPC3-CAR engineered iPSCs at different stages. (A) Undifferentiated WT and GPC3 CAR iPSC cultured in mTeSR plus medium. (B) WT and GPC3 CAR iPSC spin embryoid body on day 6. (C) WT and GPC3 CAR iPSC derived NK cell differentiation at different days from D3 to D28. Magnification of 100x for a to c. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67438/67438fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67438/67438fig03.jpg" /> 
Figure 3: Phenotype of WT and anti-GPC3 CAR iPSC derived day 6 EB and differentiated WT and GPC3 CAR iPSC derived NK cells on Day 35. (A) Typical hematopoietic antigens expressed on WT and GPC3 CAR iPSCs on day 6 of Differentiation of hematopoietic Differentiation. (B) Phenotype of WT and GPC3 CAR iPSC derived NK cells are harvested from NK differentiation medium on day 35. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67438/67438fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67438/67438fig04.jpg" /> 
Figure 4: Phenotype of WT and anti-GPC3-CAR iPSC derived NK cells after expansion. (A) WT and GPC3 CAR iPSC derived NK cells are expanded in Gibco NK Xpander medium, and NK maturation markers were assessed by using flow cytometry. (B) Surface expression of GPC3 antigen on various tumor cell lines including HepG2, SNU-449, SKOV3 and CAL27 were measured by using flow cytometry. (C) Representative histogram showing the CAR expression on the GPC3 CAR iPSC derived NK cells after expansion. The CAR construct incorporated with FLAG tag and GFP to measure the CAR by flow cytometry. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67438/67438fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67438/67438fig05.jpg" /> 
Figure 5: Functional activity of WT and anti-GPC3 CAR iPSC derived NK cells against HCC and other tumor cell lines. (A-D) Antitumor activity of WT and GPC3 CAR iPSC-derived NK cells were tested against various tumor cell lines, including HepG2, SNU-449, CAL27, and SKOV3. The X-axis represents the E:T ratios of the WT and anti-GPC3 CAR iPSC-derived NK cells tested against HepG2, SNU-449, CAL27, and SKOV3. The specific E: T ratios indicated on the x-axis are 10:1, 5:1, 2.5:1, 1:1, 0.5:1, and 0.25:1. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67438/67438fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Here, we present a method to differentiate and expand human iPSC-derived chimeric antigen receptor (CAR)-expressing natural killer cells with improved killing against various malignancies. This protocol demonstrates the differentiation and expansion of natural killer (NK) optimized iPSC-derived CAR-NK cells and the measurement of antitumor activity against various tumor cell lines.

Advances in Human Induced Pluripotent Stem Cell-Derived Chimeric Antigen Receptor-Expressing Natural Killer Cells

Natural killer (NK) cells are innate immune cells that play a crucial role in the body's defense against tumors and viral infections. The generation of ...

advances-human-induced-pluripotent-stem-cell-derived-chimeric-antigen

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

616 Views.  University of California, San Diego. De reikwijdte van ons onderzoek richt zich voornamelijk op de ontwikkeling van menselijke iPSC CAR NK-cellen met verbeterde antitumoractiviteit en in-vivo persistentie om diverse kwaadaardige aandoeningen beter te behandelen. Met dit nieuwe CAR iPSC NK-generatieprotocol willen we antwoord geven op de vraag hoe deze cellen de micro-omgevingsresistentie van tumoren kunnen overwinnen en de werkzaamheid kunnen verbeteren en een veilige en schaalbare productie kun...

Video: Vooruitgang in door mensen geïnduceerde pluripotente stamcel-afgeleide chimere antigeenreceptor-expressie natuurlijke killercellen

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells 

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and manufacturing optimizations. One challenge for these development efforts, however, has been the establishment of in vitro assays that can robustly inform selection of the optimal CAR T cell products for in vivo therapeutic success. Standard in vitro tumor-lysis assays often fail to reflect the true antitumor potential of the CAR T cells due to the relatively short co-culture time and high T cell to tumor ratio. Here, we describe an in vitro co-culture method to evaluate CAR T cell recursive killing potential at high tumor cell loads. In this assay, long-term cytotoxic function and proliferative capacity of CAR T cells is examined in vitro over 7 days with additional tumor targets administered to the co-culture every other day. This assay can be coupled with profiling T cell activation, exhaustion and memory phenotypes. Using this assay, we have successfully distinguished the functional and phenotypic differences between CD4+ and CD8+ CAR T cells against glioblastoma (GBM) cells, reflecting their differential in vivo antitumor activity in orthotopic xenograft models. This method provides a facile approach to assess CAR T cell potency and to elucidate the functional variations across different CAR T cell products.

Here, we describe an in vitro co-culture method to recursively challenge tumor-targeted T cells, which allows for phenotypic and functional analysis of antitumor T cell activity.

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and ...

Cancer Research

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Natural Killer (NK) and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line

Chimeric antigen receptor (CAR)-modified immune cell therapy has become an emerging treatment for cancers and infectious diseases. NK-based immunotherapy, particularly CAR-NK cell, is one of the most promising 'off-the-shelf' development without severe life-threatening toxicity. However, the bottleneck for developing a successful CAR-NK therapy is achieving sufficient numbers of non-exhaustive, long-lived, 'off-the-shelf' CAR-NK cells from a third party. Here, we developed a new CAR-NK expansion method using an Epstein-Barr virus- (EBV) transformed B cell line expressing a genetically modified membrane form of interleukin-21 (IL-21). In this protocol, step-by-step procedures are provided to expand NK and CAR-NK cells from cord blood and peripheral blood, as well as solid organ tissues. This work will significantly enhance the clinical development of CAR-NK immunotherapy.

Natural Killer NK and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line

Here, we present a method to expand peripheral blood natural killer (PBNK), NK cells from liver tissues, and chimeric antigen receptor (CAR)-NK cells derived from peripheral blood mononuclear cells (PBMCs) or cord blood (CB). This protocol demonstrates the expansion of NK and CAR-NK cells using 221-mIL-21 feeder cells in addition to the optimized purity of expanded NK cells.

Chimeric antigen receptor (CAR)-modified immune cell therapy has become an emerging treatment for cancers and infectious diseases. NK-based immunotherapy, ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Generation of Natural Killer Cells from Human Expanded Potential Stem Cells

The differentiation of natural killer (NK) cells from human pluripotent stem cells allows for research on and the manufacture of clinical-grade cellular products for immunotherapy. Described here is a two-phase protocol that uses a serum-free commercial medium and a cocktail of cytokines (interleukin [IL]-3, IL-7, IL-15, stem cell factor [SCF], and FMS-like tyrosine kinase 3 ligand [Ftl3L]) to differentiate human expanded potential stem cells (hEPSCs) into cells that possess NK cell properties in vitro with both 3-dimensional (3D) and 2-dimensional (2D) culture technology. Following this protocol, CD3&#8722;CD56+ or CD45+CD56+ NK cells are consistently generated. When cocultured with tumor targets for 3 h, the differentiated products display mild cytotoxicity as compared to an IL-2-independent permanent cell line, NK92mi cells. The protocol preserves the complexity of the differentiation microenvironment by the generation of 3D structures, thus facilitating the study of the spatial relationships between immune cells and their niches. Meanwhile, the 2D culture system enables the routine phenotypical validation of cell differentiation without harming the delicate differentiation niche.

The present protocol shows how to differentiate CD3&#8722;/CD45+CD56+ cells with mild cytotoxicity from human expanded potential stem cells (hEPSCs) under both 3D and 2D culture conditions. This allows for routine phenotypical validation without the destruction of the complex microenvironment.

The differentiation of natural killer (NK) cells from human pluripotent stem cells allows for research on and the manufacture of clinical-grade cellular ...

Developmental Biology

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological malignancies such as childhood acute lymphocytic leukemia. Efforts are currently underway to extend this promising therapy to solid tumors in addition to other hematological cancers. Here, we describe the development and production of potent CAR T cells targeting antigens with unique or preferential expression on solid and liquid tumor cells. The in vitro potency of these CAR T cells is then evaluated in real-time using the highly sensitive impedance-based xCELLigence assay. Specifically, the impact of different costimulatory signaling domains, such as glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR), on the in vitro potency of CAR T cells is examined. This report includes protocols for: generating CAR T cells for preclinical studies using lentiviral gene transduction, expanding CAR T cells, validating CAR expression, and running and analyzing xCELLigence potency assays.

We describe a quantitative real-time in vitro cytolysis assay system to evaluate the potency of chimeric antigen receptor T cells targeting liquid and solid tumor cells. This protocol can be extended to assess other immune effector cells, as well as combination treatments.

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological ...

Production of Human CRISPR-Engineered CAR-T Cells

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.

Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with ...

Sleeping Beauty Approach to Obtain CAR-NK Cells for Targeting Fungal Pathogens

Engineering Chimeric Antigen Receptor-Natural Killer Cells Targeting Fungal Infections Using the Non-viral Sleeping Beauty Transposon System

Chimeric antigen receptor (CAR)-based cell therapies have shown impressive efficacy in the treatment of hematological malignancies. Recently, these therapies are being developed for infectious diseases, yet studies targeting fungal infections remain scarce. To identify optimal targets and optimize cellular products, we developed a method to engineer chimeric antigen receptor-natural killer (CAR-NK) cells and evaluated their response to stimulation by fungi. This paper describes a straightforward and robust method for generating CAR-NK cells tailored to fungal targets using the non-viral Sleeping Beauty transposon system. NK-92 cells are transfected with the hyperactive transposase SB100X vectorized as minicircle DNA (MC) along with a plasmid-encoded CAR transposon. Transfection efficiency is assessed 1 week later using flow cytometric analysis. Prior to functional testing, the cells expressing the transgene are enriched using magnetic-activated cell sorting and cultured for 1 more week. To evaluate antigen-specific activation, the engineered cells are co-cultured with Aspergillus fumigatus germ tubes for at least 6 h. Subsequently, the concentration of the secreted interferon-gamma (IFN-&#947;) is measured using an enzyme-linked immunosorbent assay.

We outline a transfection protocol for producing chimeric antigen receptor-natural killer (CAR-NK) cells targeting fungal pathogens using the non-viral Sleeping Beauty transposon system. To assess antigen-specific activation, we co-cultured the engineered cells with Aspergillus fumigatus germ tubes and measured IFN-&#947; secretion.

Chimeric antigen receptor (CAR)-based cell therapies have shown impressive efficacy in the treatment of hematological malignancies. Recently, these ...

Advances in Human Induced Pluripotent Stem Cell-Derived Chimeric Antigen Receptor-Expressing Natural Killer Cells

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