Non-clinical efficacy, safety and stable clinical cell processing of induced pluripotent stem cell-derived anti-glypican-3 chimeric antigen receptor-expressing natural killer/innate lymphoid cells

Abstract

The use of allogeneic, pluripotent stem-cell-derived immune cells for cancer immunotherapy has been the subject of recent clinical trials. In Japan, investigator-initiated clinical trials will soon begin for ovarian cancer treatment using human leukocyte antigen (HLA)-homozygous-induced pluripotent stem cell (iPSC)-derived anti-glypican-3 (GPC3) chimeric antigen receptor (CAR)-expressing natural killer/innate lymphoid cells (NK/ILC). Using pluripotent stem cells as the source for allogeneic immune cells facilitates stringent quality control of the final product, in terms of efficacy, safety and producibility. In this paper, we describe our methods for the stable, feeder-free production of CAR-expressing NK/ILC cells from CAR-transduced iPSC with clinically relevant scale and materials. The average number of cells that could be differentiated from 1.8-3.6 × 10⁶ iPSC within 7 weeks was 1.8-4.0 × 10⁹ . These cells showed stable CD45/CD7/CAR expression, effector functions of cytotoxicity and interferon gamma (IFN-γ) production against GPC3-expressing tumor cells. When the CAR-NK/ILC cells were injected into a GPC3-positive, ovarian-tumor-bearing, immunodeficient mouse model, we observed a significant therapeutic effect that prolonged the survival of the animals. When the cells were injected into immunodeficient mice during non-clinical safety tests, no acute systemic toxicity or tumorigenicity of the final product or residual iPSC was observed. In addition, our test results for the CAR-NK/ILC cells generated with clinical manufacturing standards are encouraging, and these methods should accelerate the development of allogeneic pluripotent stem cell-based immune cell cancer therapies.

Keywords: GPC3; ILC; NK; chimeric antigen receptor; iPSC; immunotherapy.

Figure 1

Characterization of third generation chimeric antigen receptor (CAR) with a novel scFv that efficiently binds to the GPC3 N‐terminus. A, Schematic representation of the lentiviral vector expressing G2 CAR. B‐C, Binding specificity of anti–GPC3 antibody B. Anti–GPC3 antibody was bound to GPC3 full‐length and N‐terminus fragment but not C‐terminus domain expressing 293T cells. C, Binding affinity was analysis by multi‐cycle method of SPR measurement. The antibody fixed on CM5 sensor chip was bound to GPC3. D, Cytolytic activity of anti–GPC3 CAR‐T cells specific to SK‐Hep‐1‐GPC3 cells. Anti–GPC3 CAR‐T cells or non–transduced T cells were co–cultured with GPC3‐positive or GPC3‐negative SK‐Hep‐1 cells at 1:1 or 1:3 CAR‐T to target ratios for 48 h. The cultured cells were harvested and analyzed by flow cytometry to detect CD45, a marker expressed on T cells but not target tumor cells

Figure 2

Clinically relevant manipulation of anti–GPC3 28bbz QHJI‐iPSC #22 to generate iCAR‐NK/ILC. A, Schematic for differentiation of anti–GPC3 28bbz QHJI‐iPSC #22 into iCAR‐NK/ILC. B, Flow‐cytometric analysis of differentiated cells at the end of the first step. C, Flow‐cytometric analysis of differentiated cells at the end of the second step. D, Cell number of differentiated cells during the second step. E, Flow‐cytometric analysis of differentiated cells at the end of the third step. F, Cell number of iCAR‐NK/ILC during the third step

Figure 3

iCAR‐NK/ILC cells generated from anti–GPC3 28bbz QHJI‐iPSC #22 effectively suppress GPC3‐expressing tumor growth in vitro and in vivo. A, In vitro 51Cr‐release assay of iCAR‐NK/ILC co–cultured with SK‐Hep‐GPC3 or SK‐Hep‐Vector. n = 5 per point. B, Expression of NK‐related surface antigens was analyzed with flow cytometry. C, Intracellular IFN‐γ of iCAR‐NK/ILC was measured after co–culturing with SK‐Hep‐GPC3, SK‐Hep‐Vector or no target cells. D, Comparison of IFN‐γ production of iCAR‐NK/ILC to SK‐Hep‐GPC3, SK‐Hep‐Vector or no target cells. E, F, G, In vivo analysis of iCAR‐NK/ILC in NOD‐SCID IL2Rγc^null (NSG) xenograft model. A batch of 5 × 10⁵ KOC7c were inoculated into the intraperitoneal cavity of NSG mice on day 0. Then, 5 × 10⁶ iCAR‐NK/ILC was administered into the intraperitoneal cavity on days 3, 7, 10, 14, 17 and 21. Tumor burden was analyzed with in vivo imaging. E, Luciferase imaging of mice from each group. F, Summary of bioluminescence from each group. G, Kaplan‐Meier analysis of mouse survival. *p < .05, **p < .01, ***p < .001, ****p < .0001

Figure 4

No evidence of ICAR‐NK/ILC‐mediated toxicity or tumorigenicity in non–clinical in vivo tests. A, Schematic of general toxicity test. B, Formalin‐fixed and paraffin‐embedded omentum sections stained with H&E for histologic analysis. Anti–human nuclear antibody staining was performed to distinguish human from mouse cells. C, Summary table of histological analyses

Figure 5

Differentiation of ICAR‐NK/ILC cells within cell processing facility using clinically relevant materials and manipulation. A, Cell processing chart of iCAR‐NK/ILC. B, Quality check of differentiated cells during cell processing