iPSC-derived NK cells maintain high cytotoxicity and enhance in vivo tumor control in concert with T cells and anti-PD-1 therapy

Abstract

The development of immunotherapeutic monoclonal antibodies targeting checkpoint inhibitory receptors, such as programmed cell death 1 (PD-1), or their ligands, such as PD-L1, has transformed the oncology landscape. However, durable tumor regression is limited to a minority of patients. Therefore, combining immunotherapies with those targeting checkpoint inhibitory receptors is a promising strategy to bolster antitumor responses and improve response rates. Natural killer (NK) cells have the potential to augment checkpoint inhibition therapies, such as PD-L1/PD-1 blockade, because NK cells mediate both direct tumor lysis and T cell activation and recruitment. However, sourcing donor-derived NK cells for adoptive cell therapy has been limited by both cell number and quality. Thus, we developed a robust and efficient manufacturing system for the differentiation and expansion of high-quality NK cells derived from induced pluripotent stem cells (iPSCs). iPSC-derived NK (iNK) cells produced inflammatory cytokines and exerted strong cytotoxicity against an array of hematologic and solid tumors. Furthermore, we showed that iNK cells recruit T cells and cooperate with T cells and anti-PD-1 antibody, further enhancing inflammatory cytokine production and tumor lysis. Because the iNK cell derivation process uses a renewable starting material and enables the manufacturing of large numbers of doses from a single manufacture, iNK cells represent an "off-the-shelf" source of cells for immunotherapy with the capacity to target tumors and engage the adaptive arm of the immune system to make a "cold" tumor "hot" by promoting the influx of activated T cells to augment checkpoint inhibitor therapies.

Fig. 1.. iNK cells efficiently differentiate and expand in culture and are phenotypically and transcriptionally similar to primary peripheral blood NK cells.

(A) Overview of the steps leading from human fibroblasts to expanded iNK cells. (B) The percentages of cells in culture with a CD3⁻CD56⁺ phenotype at the indicated days after the initiation of iNK cell differentiation and expansion from iCD34⁺ HPCs (N = 6 independent expansions from 1 iPSC clone). (C) iNK cells were differentiated and expanded from iCD34⁺ HPCs over the course of 42 days. Shown are calculated fold expansion values for total cells in culture relative to day 0 (N = 6). (D) The percentage of CD3⁻CD56⁺ iNK cells positive for each surface receptor or intracellular granule component at the end of culture (n = 7). (E) Principal component analysis of microarray gene expression analyses of the indicated primary lymphocyte and iNK cell populations. PB, peripheral blood. (F) Unsupervised hierarchical clustering of RNA transcripts associated with NK cell identity, as determined by microarray analysis, for the indicated cell populations. CD4⁺ Tn, naïve CD4⁺ T cells from peripheral blood; CD4⁺ Tcm, CD4⁺ central memory T cells from peripheral blood; CD8⁺ Tn, naïve CD8⁺ T cells from peripheral blood; CD8⁺ Tcm, central memory CD8⁺ T cells from peripheral blood; CD4⁺ Teff, effector CD4⁺ T cells from peripheral blood; CD8⁺ Teff, effector CD8⁺ T cells from peripheral blood; Exp PNBK, expanded NK cells from peripheral blood; Exp iNK, expanded iNK cells; PB NK, NK cells from peripheral blood (not expanded); iNK, iNK cells not expanded. Genes shown in the boxes represent signature genes for each cell type.

Fig. 2.. iNK cells are functional against multiple hematopoietic and solid tumor cell lines.

iNK cells were used as effectors at the indicated effector cell: tumor cell (E:T) ratios in 66-hour IncuCyte-based function assays with A549 lung carcinoma cells, HepG2 hepatocyte carcinoma cells, SKOV-3 ovarian adenocarcinoma cells, K562 myeloid leukemia cells, and SK-MEL2 melanoma cells. Data are representative of at least two independent experiments with each tumor cell line and are shown as mean ± SD (n = 3 technical replicates).

Fig. 3.. iNK cells infiltrate and destroy solid tumor spheroids.

SKOV-3 cells were seeded into tissue culture plate wells where they formed spheroids over the course of 72 hours. iNK cells were then added as effectors at the indicated E:T ratios. (A) Representative images are shown from wells containing SKOV-3 spheroids alone or with the addition of iNK cells at each day of the assay. SKOV-3 cells are labeled red, and iNK cells are labeled green. (B) Cumulative data are shown of SKOV-3 spheroid size throughout the assay for each condition. (C) Quantification of iNK cell infiltration of SKOV-3 spheroids as indicated by integrated green signal within each spheroid. Data in B and C are representative of two independent experiments and are shown as mean ± SD (n = 3 technical replicates).

Fig. 4.. Adoptive transfer of iNK cells delays tumor progression in a xenogeneic model of ovarian cancer.

(A) Schematic of the experimental design to test in vivo antitumor function of iNK cells immediately post-culture. (B) Bioluminescence images of groups of mice that were not engrafted with tumor (IVIS control, n = 3), were engrafted with OVCAR8 cells without treatment (n = 7), or were engrafted with OVCAR8 cells and treated with iNK cells and IL-2 (n =7). Images were taken at the indicated days as defined in A. (C) Cumulative radiance for the OVCAR8 alone and OVCAR8 + iNK groups over the first 21 days of the experiment. Statistical significance was determined by two-way ANOVA. ***p ≤ 0.001. (D) Kaplan Meier analysis of overall survival. Statistical significance was determined by Log-rank (Mantel-Cox) tests. *p ≤ 0.05. (E) Schematic of the experimental design to test in vivo antitumor function with multiple doses of thawed iNK cells. (F) Bioluminescence images of groups of mice that were not engrafted with tumor (IVIS control, n = 2), were engrafted with MA-148 cells without treatment (n = 8), or were engrafted with MA-148 cells and treated with three doses of cryopreserved iNK cells and IL-2 (n = 8). Images were taken at the indicated days as defined in E. (G) Cumulative radiance for each group over the first 35 days of the experiment. Statistical significance was determined by two-way ANOVA. ****p ≤ 0.0001. (H) Kaplan Meier analysis of overall survival. Statistical significance was determined by Log-rank (Mantel-Cox) tests. ***p ≤ 0.001. BLI, bioluminescence imaging

Fig. 5.. iNK cells recruit peripheral blood T cells both in vitro and in vivo.

(A) CD3- and CD28-stimulated CD3⁺ T cells were seeded into transwells with either control media or conditioned media from K562 cell cultures, iNK cell cultures, or iNK and K562 cell co-cultures. Cumulative data of the numbers of CD3⁺ T cells that migrated across the transwell in each condition are shown. Statistical significance was determined by paired t tests (n = 4). *p ≤ 0.05, **p ≤ 0.01, n.s., not significant. (B) Schematic of cell injections to test T cell recruitment from the circulating blood to the peritoneum. (C) CD3- and CD28-stimulated CD3⁺ T cells were injected intravenously (i.v.) into mice with or without additional intraperitoneal (i.p.) injections of either K562 cells, iNK cells, or a 1:1 mixture of iNK cells and K562 cells. Mice were euthanized after 5 days, and the numbers of total CD3⁺ T cells, CD3⁺CD4⁻CD8⁺ T cells, and CD3⁺CD4⁺CD8⁻ T cells in the peritoneal lavages were counted. Shown are cumulative data from 2 independent experiments. Statistical significance was determined by unpaired t tests corrected for multiple comparisons (n = 8). *p ≤ 0.05, **p ≤ 0.01

Fig. 6.. iNK cells cooperate with T cells and anti-PD-1 antibody to eliminate tumor spheroids and release inflammatory cytokines.

SKOV-3 cells were seeded into 96-well tissue culture wells where they formed spheroids over the course of 72 hours. The indicated combinations of iNK cells, CD3-and CD28-activated CD3⁺CD4⁺ or CD3⁺CD8⁺ T cells, and anti-PD-1 antibody were then added to the wells. Experiments were performed with T cells from 3 different peripheral blood donors. (A) Quantification is shown for the indicated chemokines and cytokines collected from individual wells at the end of the assay. (B) Representative images are shown of wells containing SKOV-3 spheroids and combinations of iNK cells, CD3⁺ T cells, and anti-PD-1 antibody. SKOV-3 cells are labeled red, and CD3⁺ T cells are labeled green. (C) Cumulative data are shown from a representative experiment showing SKOV-3 viability, quantified by red intensity, in each coculture condition over the course of 150 hours. Data are shown as mean ± SD of 3 images per timepoint. Flow cytometry was used to count the number of remaining SKOV-3 cells in each well at the end of each IncuCyte assay. Shown are cumulative data from 2 independent experiments. Statistical significance was determined by unpaired t tests corrected for multiple comparisons (n = 3–19). **p ≤ 0.01, ***p≤ 0.001****, p ≤ 0.0001

Fig. 7.. Combining iNK cells with T cells and anti-PD-1 antibody promotes durable tumor control in vivo.

(A) Schematic of the experimental design to test the effectiveness of iNK cells, CD3- and CD28-activated CD3⁺ T cells, and anti-PD-1 antibody treatment in vivo. (B) Cumulative radiance for the OVCAR8 group and each treatment group at the indicated time points. Data are from 3 independent experiments. Statistical significance was determined using unpaired t tests corrected for multiple comparisons (n = 4–20). * p ≤ 0.5, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001