Pluripotent stem cell-derived NK cells with high-affinity noncleavable CD16a mediate improved antitumor activity

Abstract

Antibody-dependent cellular cytotoxicity (ADCC) is a key effector mechanism of natural killer (NK) cells that is mediated by therapeutic monoclonal antibodies (mAbs). This process is facilitated by the Fc receptor CD16a on human NK cells. CD16a appears to be the only activating receptor on NK cells that is cleaved by the metalloprotease a disintegrin and metalloproteinase-17 upon stimulation. We previously demonstrated that a point mutation of CD16a prevents this activation-induced surface cleavage. This noncleavable CD16a variant is now further modified to include the high-affinity noncleavable variant of CD16a (hnCD16) and was engineered into human induced pluripotent stem cells (iPSCs) to create a renewable source for human induced pluripotent stem cell-derived NK (hnCD16-iNK) cells. Compared with unmodified iNK cells and peripheral blood-derived NK (PB-NK) cells, hnCD16-iNK cells proved to be highly resistant to activation-induced cleavage of CD16a. We found that hnCD16-iNK cells were functionally mature and exhibited enhanced ADCC against multiple tumor targets. In vivo xenograft studies using a human B-cell lymphoma demonstrated that treatment with hnCD16-iNK cells and anti-CD20 mAb led to significantly improved regression of B-cell lymphoma compared with treatment utilizing anti-CD20 mAb with PB-NK cells or unmodified iNK cells. hnCD16-iNK cells, combined with anti-HER2 mAb, also mediated improved survival in an ovarian cancer xenograft model. Together, these findings show that hnCD16-iNK cells combined with mAbs are highly effective against hematologic malignancies and solid tumors that are typically resistant to NK cell-mediated killing, demonstrating the feasibility of producing a standardized off-the-shelf engineered NK cell therapy with improved ADCC properties to treat malignancies that are otherwise refractory.

Conflict of interest statement

Conflict-of-interest disclosure: R.B., S.G., P.R., T.T.L., R.A., G.B.B., P.-F.T., and B.V. are employees of Fate Therapeutics with stock holdings and options. J.S.M. consults for and hold stock options in Fate Therapeutics, a company which may commercially benefit from the results of this research project. These interests have been reviewed and managed by the University of Minnesota in accordance with its conflict of interest policy. B.W. collaborates with Fate Therapuetics with a sponsored research agreement. D.S.K. is a consultant for Fate Therapeutics, has equity and receives income. The terms of this arrangement 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 financial interests.

© 2020 by The American Society of Hematology.

Figures

Graphical abstract

Figure 1.

hnCD16-iPSC–derived NK cells are functionally mature and do not downregulate CD16 expression upon activation. (A) Unmodified iNK cells, hnCD16-iNK cells, and adult PB-NK cells were stained and analyzed by flow cytometry for CD56 and CD16 and the indicated NK cell surface receptors. In each panel, red line: isotype control; blue line: stained sample. Data were repeated independently in 3 separate experiments. (B) hnCD16-iNK cells, unmodified iNK cells, or PB-NK cells were stimulated as indicated for 4 hours, and CD16 expression was determined by flow cytometry (n = 4-6 per group). (C) Representative flow cytometric analysis of CD16 and TNF-α expression on unmodified iNK cells, hnCD16-iNK cells, and PB-NK cells that were left unstimulated or stimulated with K562 cells or PMA/ionomycin. (D) Representative flow cytometric analysis of intracellular TNF-α and IFN-γ production after a 4-hour incubation with culture media only (unstimulated), with P815 cells, or with P815 cells + anti-CD16 antibody. Data in panels C-D were repeated in 3 separate experiments.

Figure 2.

hnCD16-iNK cells demonstrate improved in vitro ADCC against multiple tumor types. (A) hnCD16-iNK cells and unmodified iNK cells produce CD107α and IFN-γ in response to Raji cells with or without anti-CD20 antibody, in response to SKOV-3 cells with or without anti-HER2, and in response to Cal27 cells with or without anti-EGFR. hnCD16-iNK cells or unmodified iNK cells were left unstimulated or were stimulated with a 1:1 ratio of target cells with or without antibody and stained for CD107a and IFN-γ 4 hours later. (B) Quantification of CD107a (left panel) and IFN-γ (right panel) expression by cells in panel A. An increase in CD107a+ or IFNγ+ positive cells in the antibody group was normalized to the without-antibody group (fold increase: antibody/without antibody). Studies were repeated independently 3 times, and data are mean ± standard deviation. (C) Quantification of flow cytometric analysis of TNF-α and IFN-γ production and CD107a surface expression after a 4-hour incubation with culture media only (unstimulated) or with the indicated stimuli. Heat maps quantify the frequency of NK cells that are positive for IFN-γ, TNF-α, or CD107a and are scaled from 0% (black) to 30% (yellow), with background expression subtracted such that unstimulated = 0. (D) ADCC against Raji cells was analyzed using a caspase-3/7 green flow cytometry assay. Raji cells were incubated with NK cells, with or without anti-CD20 antibody, for 4 hours. (E) ADCC against Raji cells was analyzed over a 24-hour period using an IncuCyte real-time imaging system. Anti-CD20 was titrated from 0.001 μg/mL to 20 μg/mL. (F-G) Long-term (66-hour) ADCC assays using the IncuCyte real-time imaging system. ADCC against the lung cancer cell line A549 with and without anti-EGFR mAb (F) and against the ovarian cancer cell line SKOV-3 with and without anti-HER2 mAb (G). Data in panels F-G are presented as the normalized frequency of target cells remaining, where target cells without NK effectors = 100%. Data in panels D-G were repeated independently in 3 separate experiments. ***P < .001, 2-tailed Student t test.

Figure 3.

A single dose of hnCD16-iNK cells effectively mediates in vivo ADCC against human B-cell lymphoma. (A) Schema of single-dose NK infusion in vivo study. NSG mice were inoculated intraperitoneally with 2 × 105 Luc-expressing Raji cells, and tumor engraftment was assessed by IVIS imaging 3 days later for a baseline pretreatment reading. On day 4 after transplant, mice were left untreated or were treated with 1 × 107 PB-NK cells, unmodified iNK cells, or hnCD16-iNK cells, alone or in combination with 300 μg of anti-CD20 antibody. Mice were treated with IL-15 for the first week and with IL-2 for 3 weeks, and IVIS imaging was performed to track tumor progression. (B) Tumor burden was determined by BLI. (C) Quantification of IVIS imaging time course. Data are mean ± SEM for the mice in panel B. Data were not significant for anti-CD20 alone vs unmodified iNK cells + anti-CD20 or for PB-NK cells + anti-CD20 at all time points. (D) Kaplan-Meier curve demonstrating survival of the experimental groups. The median survival for the untreated group and the groups treated with anti-CD20, PB-NK cells + anti-CD20, unmodified iNK cells + anti-CD20, and hnCD16-iNK cells + anti-CD20 was 27, 38, 43, 44, and 52 days, respectively. Anti-CD20 vs PB-NK+anti-CD20, P = .0047; anti-CD20 vs unmodified-iNK+anti-CD20, P = .0067; anti-CD20 vs hnCD16-iNK+anti-CD20, P = .0027; PB-NK+anti-CD20 vs hnCD16-iNK+anti-CD20, P = .1098; unmodified-iNK+anti-CD20 vs hnCD16-iNK+anti-CD20, P = .3127; 2-tailed log-rank test. *P < .05, **P < .01, 2-tailed Student t test, anti-CD20 alone vs hnCD16-iNK cells + anti-CD20.

Figure 4.

Multiple doses of hnCD16-iNK cells effectively mediate improved ADCC in vivo against B-cell lymphoma. (A) Schema of multiple NK cell dosing study. NSG mice were inoculated intraperitoneally with 2 × 105 Luc-expressing Raji cells, and tumor engraftment was assessed by IVIS imaging 3 days later for a baseline pretreatment reading. On day 4 after transplant, mice were left untreated or were treated with 1 × 107 PB-NK cells or hnCD16-iNK cells, alone or in combination with 300 μg of rituximab weekly for 4 weeks. NK cells were supported by injection of IL-15 for the first week and by injection of IL-2 for 3 weeks. IVIS imaging was done weekly to monitor tumor progression. (B) Tumor burden was determined by BLI over the first 28 days. (C) Time course of IVIS imaging. Data are mean ± SEM for the mice in panel B. Anti-CD20 vs PB-NK+anti-CD20 not significant for all data points, 2-tailed Student t test. (D) Kaplan-Meier curve representing the percent survival of the experimental groups. The median survival for the untreated group and the groups treated with Anti-CD20, PB-NK+anti-CD20, and hnCD16-iNK+anti-CD20 are 25, 47, 61, and 76 days, respectively. Anti-CD20 vs PB-NK+anti-CD20, P = .0185; anti-CD20 vs hnCD16-iNK+anti-CD20, P = .0065; PB-NK+anti-CD20 vs hnCD16-iNK+anti-CD20, P = .0485; 2-tailed log-rank test. *P < .05, **P < .01, anti-CD20 vs hnCD16-iNK+anti-CD20, 2-tailed Student t test.

Figure 5.

hnCD16-iNK cells effectively mediate ADCC in a human lymphoma systemic tumor model. (A) Flow scheme of IV NK cell infusion in vivo study. NSG mice were inoculated IV with 2 × 105 Luc-expressing Raji cells. On day 1 after transplant, mice were left untreated or were treated with 1 × 107 PB-NK cells, unmodified iNK cells, or hnCD16-iNK cells, alone or in combination with 300 μg of anti-CD20 antibody. NK cells were supported by injection of IL-15 for the first week and by injection of IL-2 for 3 weeks; IVIS imaging was performed weekly to track tumor progression. (B) Tumor burden was determined by BLI over the first 35 days. (C) IVIS imaging time course. Data are mean ± SEM for the mice in panel B. (D) Kaplan-Meier curve representing the percent survival of the experimental groups. The median survival was not reached in the hnCD16-iNK + anti-CD20 group. Anti-CD20 vs untreated, P = .0021; anti-CD20 vs hnCD16-iNK+anti-CD20, P = .0269; hnCD16-iNK+anti-CD20 vs PB-NK+anti-CD20, P = .0342; hnCD16-iNK+anti-CD20 vs umnodified-iNK+anti-CD20, P = .0350; 2-tailed log-rank test. *P < .05, **P < .01, ***P < .001, hnCD16-iNK+anti-CD20 vs umnodified-iNK+anti-CD20, 2-tailed Student t test.

Figure 6.

hnCD16-iNK cells mediate improved ADCC in vivo against ovarian cancer. NSG mice were inoculated intraperitoneally with 1 × 105 Luc-expressing SKOV-3 cells, and tumor engraftment was assessed by IVIS imaging 4 days later. On day 5 after tumor transplant, mice were left untreated or were treated with 100 μg of anti-HER2 alone or in combination with 5 × 106 hnCD16 iNK cells. NK cells were supported by twice weekly injections of IL-2, and IVIS imaging was done weekly to track tumor load. (A) IVIS imaging. (B) Quantification of geometric mean ± standard deviation for the mice in panel A. (C) Kaplan-Meier curve representing the percent survival of the experimental groups. Untreated vs anti-HER2, P = .0140; anti-HER2 vs anti-HER2 + hnCD16 iNK+, P = .004; 2-tailed log-rank test. (D) Mice injected with Luc-expressing SKOV-3 cells were treated with 1 × 107 (1e7) or 2 × 107 (2e7) cryopreserved hnCD16-iNK cells or with 2 × 107 fresh hnCD16-iNK cells + anti-HER2 antibody. (D) IVIS imaging. (E) Quantification of the geometric mean ± standard deviation for the mice in panel D.