EpCAM-targeting CAR-T cell immunotherapy is safe and efficacious for epithelial tumors

Abstract

The efficacy of CAR-T cells for solid tumors is unsatisfactory. EpCAM is a biomarker of epithelial tumors, but the clinical feasibility of CAR-T therapy targeting EpCAM is lacking. Here, we report pre- and clinical investigations of EpCAM-CAR-T cells for solid tumors. We demonstrated that EpCAM-CAR-T cells costimulated by Dectin-1 exhibited robust antitumor activity without adverse effects in xenograft mouse models and EpCAM-humanized mice. Notably, in clinical trials for epithelial tumors (NCT02915445), 6 (50%) of the 12 enrolled patients experienced self-remitted grade 1/2 toxicities, 1 patient (8.3%) experienced reversible grade 3 leukopenia, and no higher-grade toxicity reported. Efficacy analysis determined two patients as partial response. Three patients showed >23 months of progression-free survival, among whom one patient experienced 2-year progress-free survival with detectable CAR-T cells 200 days after infusion. These data demonstrate the feasibility and tolerability of EpCAM-CAR-T therapy.

Fig. 1.. The Dectin-1–stimulated EpCAM-specific CAR has a distinct effect on T cell in self-activation, differentiation, and exhaustion compared with that of CARs with the CD28 or 4-1BB costimulatory domain.

(A) Schematic of EpCAM-specific CARs carrying CD28, 4-1BB, or Dectin-1 costimulatory domain. (B) Expression of different EpCAM-specific CARs on T cells, respectively. (C and D) Expression of activation-related markers on CAR-T cells after 9 days of CAR transduction. n = 5 different donors. Cells were pregated for the CD3⁺CAR⁺ subset. (E) Expression of exhaustion-associated receptors on CAR-T cells after 9 days of lentivirus transduction. The color lines are represented as mean. (F) Fold change of EpCAM–CAR-T cells differentiation in central memory and effector phenotypes of five different donors. The data are displayed as mean. (G) Schematic of the xenograft model. B-NDG mice were intraperitoneally injected with 2 × 10⁵ HT-29–Luc cells on day −7. After that, the mice were intraperitoneally infused with three doses of Mock-T or CAR-T cells. n = 6 mice for each group. After 21 days of tumor inoculation, mice were euthanized to analyze CAR-T cell persistence. (H and I) Bioluminescence images and statistical results of tumor burden. (J and K) The percent of splenetic Mock-T cells and CAR-T cells after 21 days of adoptive T cell therapy. (L) Differentiation of splenetic CD4⁺ and CD8⁺ CAR-T cells in central memory and effector phenotypes in the xenograft model. (M and N) Exhaustion-associated receptors expression on splenetic CAR-T cells after 21 days of adoptively T cells administration. The data are presented as mean ± SD. *P < 0.05 and **P < 0.01. P < 0.05 was considered statistically significant. ns, no significance. Wilcoxon test was used for (D) to (F). Two-tailed unpaired Student’s t test was used for differences analysis in (K), (L), and (N). TM, transmembrane domain; Teff, effector T cell; Tcm, central memory T cells.

Fig. 2.. Intraperitoneal and intravenous infusion of EpCAM–CAR-T cells induced colon cancer remission in a xenograft mouse model.

(A) Schematic of the animal experiments. B-NDG mice were intraperitoneally injected with 2 × 10⁵ Luc-expressing HT-29–Luc cells on day −7. After that, the mice were randomly assigned to different groups and were intraperitoneally or intravenously infused with three doses of Mock-T or CAR-T cells. n = 5 mice for each group. (B and C) Bioluminescence images and statistical result of tumor burden after intravenous administration of CAR-T cells. (D and E) Tumor weight at the end point of the experiment and the survival of mice who received intravenous CAR-T infusion. (F and G) Bioluminescence images and statistical result of tumor burden after intraperitoneal administration of CAR-T cells. (H and I) Tumor weight at the end point of the experiment and the survival of mice who received intraperitoneal CAR-T infusion. (J to M) Flow cytometry analysis of EpCAM–CAR-T cell persistence in the spleens of mice with intravenous (J) and (K) or intraperitoneal (L) and (M) T cell infusion. The Mock-T cell group was examined on day 21, and the CAR-T cell groups were examined when euthanized. n = 4 mice in the 1 × 10⁷ CAR-T cells intravenously treated group and n = 5 mice in the other group. (N and O) EpCAM–CAR-T cells persisted in the peripheral blood of mice that received intravenous infusion of T cells. (P and Q) Representative results showing tumor-infiltrated human CD3⁺ T cells in mice that received intravenous injection of T cells, as determined by immunohistochemistry (P) and statistical results shown in (Q). Scale bars, 200 μm (white) and 50 μm (black). The data are expressed as the means ± SD. Log-rank test for (E) and (I) and Mann-Whitney U test for (C), (D), (G), (H), (K), (M), and (O). *P < 0.05, **P < 0.01, and ****P < 0.0001. P < 0.05 was considered statistically significant.

Fig. 3.. EpCAM–CAR-T cells were well tolerated by EpCAM-humanized mice.

(A) Genetic engineering strategy for EpCAM-humanized C57BL/6 mice. The murine EpCAM gene locus was truncated, and a homologous human EpCAM gene locus was inserted from exon 2 to exon 7. Purple, exon of mouse EpCAM; green, exon of human EpCAM; E, exon. (B) Experimental schematic showing the safety evaluation in the C57BL/6-EpCAM^{tml EpCAM} mouse model. The mice were randomly grouped; n = 3 per group. All mice were preconditioned with cyclophosphamide (100 mg/kg). Two days later, a single dose of Mock-T cells (1 × 10⁶) or EpCAM–CAR-T cells (1 × 10⁶, 5 × 10⁶, and 1 × 10⁷) was intravenously administered. (C) Percentage of body weight change was normalized to that on day 0 after T cell infusion. (D) Serum cytokine levels in peripheral blood at the indicated points after T cell infusion (means ± SD). (E and F) Representative immunohistochemical (E) and quantitative real-time PCR (F) analysis of mouse CD3⁺ cells (E) and EpCAM-CAR gene (F) distribution in the spleen, lung, and other organs 7 days after T cell transfer. Scale bars, 50 μm. (G to J) Organ damage-related serum biomarkers were measured in the liver (G), kidney (H), heart (I), and pancreas (J) on day 7 after T cell transfer. The data are presented as the means ± SD, and data analysis was performed by one-way analysis of variance (ANOVA). P < 0.05 was considered statistically significant. AST, aspartate aminotransferase; ALT, alanine aminotransferase; CRE, creatinine; BUN, blood urea nitrogen; LDH, lactate dehydrogenase; CK-MB, creatine kinase-MB; AMY, amylase.

Fig. 4.. EpCAM–CAR-T cell clinical protocol design and cohort diagram.

(A) Clinical protocol schematic for patient screening, CAR-T cell manufacture, and time points of the clinical study. Cy, cyclophosphamide; Flu, fludarabine; CT c/a/p, computed tomography of chest/abdomen/pelvis. (B) Number of patients screened, enrolled, and treated with autologous EpCAM–CAR-T cells in the clinical study. (C) Waterfall plot summarizing the adverse effects of CAR-T therapy in the clinical study. (D) Swimming plot showing patients’ disease development and substantial therapy and present status. IMRT, intensity-modulated radiotherapy.

Fig. 5.. EpCAM–CAR-T cell persistence, cytokine secretion and radiologic evaluation of patients after EpCAM–CAR-T cell infusion.

(A) EpCAM-CAR gene copies in genomic DNA of peripheral blood from patients at the indicated times following CAR-T cell transfer. (B to D) Representative serum IL-6 (B), TNF-α (C), and CRP (D) levels in patients treated with EpCAM–CAR-T cells. Cohort 1: violet; cohort 2: blue; cohort 3: orange. (E) Computed tomography scans showing the tumor response in patient 10 and patient 3 after EpCAM–CAR-T cell transfer. Red arrows indicate the tumor site.