Persistent Polyfunctional Chimeric Antigen Receptor T Cells That Target Glypican 3 Eliminate Orthotopic Hepatocellular Carcinomas in Mice

Abstract

Background and aims: Glypican 3 (GPC3) is an oncofetal antigen involved in Wnt-dependent cell proliferation that is highly expressed in hepatocellular carcinoma (HCC). We investigated whether the functions of chimeric antigen receptors (CARs) that target GPC3 are affected by their antibody-binding properties.

Methods: We collected peripheral blood mononuclear cells from healthy donors and patients with HCC and used them to create CAR T cells, based on the humanized YP7 (hYP7) and HN3 antibodies, which have high affinities for the C-lobe and N-lobe of GPC3, respectively. NOD/SCID/IL-2Rgc^null (NSG) mice were given intraperitoneal injections of luciferase-expressing (Luc) Hep3B or HepG2 cells and after xenograft tumors formed, mice were given injections of saline or untransduced T cells (mock control), or CAR (HN3) T cells or CAR (hYP7) T cells. In other NOD/SCID/IL-2Rgc^null (NSG) mice, HepG2-Luc or Hep3B-Luc cells were injected into liver, and after orthotopic tumors formed, mice were given 1 injection of CAR (hYP7) T cells or CD19 CAR T cells (control). We developed droplet digital polymerase chain reaction and genome sequencing methods to analyze persistent CAR T cells in mice.

Results: Injections of CAR (hYP7) T cells eliminated tumors in 66% of mice by week 3, whereas CAR (HN3) T cells did not reduce tumor burden. Mice given CAR (hYP7) T cells remained tumor free after re-challenge with additional Hep3B cells. The CAR T cells induced perforin- and granzyme-mediated apoptosis and reduced levels of active β-catenin in HCC cells. Mice injected with CAR (hYP7) T cells had persistent expansion of T cells and subsets of polyfunctional CAR T cells via antigen-induced selection. These T cells were observed in the tumor microenvironment and spleen for up to 7 weeks after CAR T-cell administration. Integration sites in pre-infusion CAR (HN3) and CAR (hYP7) T cells were randomly distributed, whereas integration into NUPL1 was detected in 3.9% of CAR (hYP7) T cells 5 weeks after injection into tumor-bearing mice and 18.1% of CAR (hYP7) T cells at week 7. There was no common site of integration in CAR (HN3) or CD19 CAR T cells from tumor-bearing mice.

Conclusions: In mice with xenograft or orthoptic liver tumors, CAR (hYP7) T cells eliminate GPC3-positive HCC cells, possibly by inducing perforin- and granzyme-mediated apoptosis or reducing Wnt signaling in tumor cells. GPC3-targeted CAR T cells might be developed for treatment of patients with HCC.

Keywords: Hepatic; Immunotherapy; Lymphocyte; Tumor-Specific T Cells.

Figure 1.

GPC3-targeted CAR T cells kill GPC3-positive HCC cells in vitro. (A) The binding of two antibodies to GPC3. HN3 binds to the N-lobe of GPC3 around residue 41 (phenylalanine). YP7 binds to the C-lobe of GPC3 (residue 521–530). (B) Schematic of CAR T cell production and evaluation in mouse models. (C) Schematic of the 2^nd generation (2G) and 3^rd generation (3G) CAR constructs. (D) CD4⁺ and CD8⁺ T cell analysis of CAR (hYP7) T cells from 6 healthy donors and 6 HCC patients. (E-F) Cytolytic activity of CAR (HN3) T cells and CAR (hYP7) T cells from healthy donors (E) and HCC patients (F) after 24 hours of co-culture with Hep3B cells. (G) GPC3-targeted CAR T cells-mediated killing of HepG2 cells as determined by using IncuCyte zoom. HepG2 cells were incubated with CAR T cells at the E:T ratio of 2:1 up to 140 hours. GFP, green fluorescent protein. Values represent mean ± SEM. ***P < .001, ns, not significant.

Figure 2.

Cytokine/chemokine profiles of polyfunctionality of T cells redirected with GPC3. (A) The validated 32-plex panel including five groups of cytokines: effector, stimulatory, chemoattractive, regulator and inflammatory. (B-C) PSI computed for healthy donor-derived GPC3-targeted CAR T cells co-cultured with Hep3B (B) and G1/A431 (C) cells for 20 hours at the single-cell level. (D-E) Polyfunctional heat map displaying major functional cytokines/chemokines secreted across GPC3-specific CAR T cells from healthy donor upon Hep3B (D) and G1/A431 (E) cell stimulation. (F) Polyfunctional heat map of HCC patient-derived GPC3-specific CAR T cells upon Hep3B and GPC3 knockout Hep3B cell stimulation for 20 hours.

Figure 3.

Inhibition of Wnt/β-catenin and YAP signaling by CAR (hYP7) T cell administration. (A) CAR (hYP7) T cells from healthy donor suppressed the expression of β-catenin in Hep3B cells after 6 hours of administration. (B) CAR (hYP7) T cells from healthy donor and HCC patient inhibited the expression of β-catenin in Hep3B cells in a time-dependent manner. (C-D) CAR (hYP7) T cells from healthy donor suppressed the expression of β-catenin in HepG2 cells (C), but not in A431 cells (D). (E) CAR (hYP7) T cells from HCC patient inhibited YAP signaling as evidenced by the increase of phospho-YAP expression and decrease of total-YAP expression after co-culture with Hep3B cells.

Figure 4.

CAR (hYP7) T cells eradicate tumors in the peritoneal Hep3B xenograft mouse model. (A) Experimental schematic. Hep3B tumor-bearing NSG mice were i.p. injected with mock T cells (mock), 5 million CAR (HN3) T cells (HN3–5M), and 5, 10 and 20 million CAR (hYP7) T cells (hYP7–5M, hYP7–10M, hYP7–20M). (B) CAR (hYP7) T cells regressed established Hep3B xenografts at high dose (20M) and inhibited tumor growth at low doses (5M and 10M), whereas CAR (HN3) T cells did not inhibit tumor growth. Symbol ‘ƗƗƗ’ indicated that mice were euthanized in advance for analysis. (C) Tumor bioluminescence in mice treated in Figure 4B. (D) Kaplan–Meier survival curve of mice after infusion. (E) AFP levels in serum collected from groups shown in Figure 4B after 2 weeks of injection. (F) Detection of CAR vector-positive cells in xenograft tumor tissues after 3 weeks of injection. (G) CAR (hYP7) T cells caused the reduction of active- and total-β-catenin levels as compared with mock T cell-treated mice. (H) Representative pictures of mouse from mock and hYP7–10M group. Values represent mean ± SEM. *P < .05, **P < .01, ***P < .001, ns, not significant.

Figure 5.

CAR (hYP7) T cells eliminate tumor cells in the HepG2 xenograft mouse models. (A) Experimental schematic of the peritoneal HepG2 xenograft mouse model. HepG2 tumor-bearing male NSG mice were i.p. injected with 20 million mock T cells or CAR (hYP7) T cells. (B) CAR (hYP7) T cells demonstrated potent antitumor activity and mediated eradication of HepG2 xenograft tumors. (C) Tumor bioluminescence in mice treated in Figure 5B. (D) Detection of CAR vector-positive cells in tumor and spleen from mice after 5 weeks of injection. (E) Representative pictures of mouse from mock and hYP7 groups. (F) Experimental schematic of the orthotopic HepG2 xenograft model. HepG2 tumor-bearing female NSG mice were infused with 10 million CD19 CAR T cells and CAR (hYP7) T cells. (G) CAR (hYP7) T cells regressed growth of orthotopic HepG2 tumors. (H) Tumor bioluminescence in mice treated in Figure 5G. Values represent mean ± SEM. **P < .01.

Figure 6.

Tumor eradication in the orthotopic Hep3B xenograft mouse model by CAR (hYP7) T cells from a healthy donor. (A) Experimental schematic. The orthotopic Hep3B tumor-bearing NSG mice were i.p. or i.v. injected with 20 million CAR (hYP7) T cells. (B) Mice treated with CAR (hYP7) T cells via tail vein (hYP7 IV) demonstrated tumor eradication, while intraperitoneal infusion (hYP7 IP) resulted in tumor growth inhibition. (C) Experimental schematic. The orthotopic Hep3B tumor-bearing NSG mice were i.v. injected with 10 million CD19 CAR, CAR (HN3) and CAR (hYP7) T cells. (D) CAR (hYP7) T cell administration regressed Hep3B tumor growth in mice, while CAR (HN3) T cells failed to inhibit tumor growth. (E) Experimental schematic of Hep3B tumor re-challenge. CAR (hYP7)-treated mice that showed no detectable tumor were i.p. implanted with 0.5 million Hep3B cells. As control, naïve mice were implanted with Hep3B cells following an injection of mock T cells. (F) CAR (hYP7)-treated mice resisted Hep3B tumor re-challenge. (G) Frequency of integrated genes in spleen (S) of mice treated with CAR (HN3) and CAR (hYP7) T cells at 3 weeks, 5 weeks and 7 weeks post-infusion. The top twenty genes were listed. (H) Distribution of integration sites in spleen of mice treated with CD19 CAR, CAR (HN3) and CAR (hYP7) T cells over 3–7 weeks. The shared integrated genes in mice after 5 to 7 weeks of CAR (hYP7) T cell injection was highlighted in a separate heatmap. Values represent mean ± SEM. *P < .05, **P < .01.

Figure 7.

Persistent polyfunctional CAR (hYP7) T cells from a HCC patient eradicate orthotopic Hep3B xenograft tumors. (A) Experimental schematic. The orthotopic Hep3B tumor-bearing NSG mice were i.v. injected with 5 million CD19 CAR and CAR (hYP7) T cells. (B) CAR (hYP7) T cells regressed Hep3B tumor growth in mice. (C) CAR (hYP7) T cells recovered from mouse spleen after 4 weeks of injection named as mCAR (hYP7) T cells were co-cultured with Hep3B cells, and cytolytic activity was analyzed 24 hours after co-culture. (D) mCAR (hYP7) T cells showed profound increase of polyfunctionality by the stimulation of Hep3B cells, but not the GPC3 knockout Hep3B cells. (E) Distribution of integration sites in individual mouse treated with CAR (hYP7) T cells for 3–7 weeks. The shared integrated genes in mice after 5 to 7 weeks of CAR (hYP7) T cell injection was highlighted in a separate heatmap. S, T and L represent spleen, tumor and liver, respectively. (F) Frequency of integrated genes in spleen and liver/tumor of mice treated with CAR (hYP7) T cells from 5 weeks to 7 weeks post-infusion. The top twenty genes were listed. Values represent mean ± SEM.