Anti-mucin 1 chimeric antigen receptor T cells for adoptive T cell therapy of cholangiocarcinoma

Abstract

Current treatments for cholangiocarcinoma (CCA) are largely unsuccessful due to late diagnosis at advanced stage, leading to high mortality rate. Consequently, improved therapeutic approaches are urgently needed. Chimeric antigen receptor (CAR) T cell therapy is a newly potential therapy that can recognize specific surface antigen without major histocompatibility complex (MHC) restriction. Mucin 1 (MUC1) is an attractive candidate antigen as it is highly expressed and associated with poor prognosis and survival in CCA. We, therefore, set forth to create the fourth-generation CAR (CAR4) construct containing anti-MUC1-single-chain variable fragment (scFv) and three co-stimulatory domains (CD28, CD137, and CD27) linked to CD3ζ and evaluate anti-MUC1-CAR4 T cells in CCA models. Compared to untransduced T cells, anti-MUC1-CAR4 T cells produced increased levels of TNF-α, IFN-γ and granzyme B when exposed to MUC1-expressing KKU-100 and KKU-213A CCA cells (all p < 0.05). Anti-MUC1-CAR4 T cells demonstrated specific killing activity against KKU-100 (45.88 ± 7.45%, p < 0.05) and KKU-213A cells (66.03 ± 3.14%, p < 0.001) at an effector to target ratio of 5:1, but demonstrated negligible cytolytic activity against immortal cholangiocytes. Furthermore, the anti-MUC1-CAR4 T cells could effectively disrupt KKU-213A spheroids. These activities of anti-MUC1-CAR4 T cells supports the development of this approach as an adoptive T cell therapeutic strategy for CCA.

Figure 1

Expression of MUC1 protein in cholangiocarcinoma tissue samples. MUC1 protein in CCA was detected by immunohistochemistry (IHC). (a) Normal bile duct tissue. (b) The adjacent area of normal (liver) and cancerous (CCA) tissues (indicated by blue and black arrow, respectively). (c–f) Four tissue samples from four individual patients with CCA. In the positive areas, MUC1 were stained in both cytoplasm and on cell surface.

Figure 2

Expression of MUC1 protein in cholangiocarcinoma cell lines. (a) Representative immunoblot analysis showing total protein expression of MUC1 in cholangiocytes (MMNK-1), CCA cell lines (KKU-055, KKU-100, and KKU-213A), and a breast cancer cell line (MCF-7). The blots of cell lysate samples were cropped from different parts of the same gel. Full-length blots were presented in Supplementary Fig. 1. The MUC1 protein expression levels were normalized with β-actin. The data obtained from three independent experiments were plotted as bar graphs (b). Expression of MUC1 in cells was detected by immunofluorescence assay (IFA) (c), and cell surface expression of MUC1 was stained without permeabilization and examined by flow cytometry (d), which was plotted as bar graphs (e). All data with error bars (in b and e) represents the mean ± SD of three independent experiments. Staining with isotype control antibody was used as a negative control for IFA (data not shown) and flow cytometry. All data was analyzed by Student t-test (asterisks indicate statistically significant differences: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001).

Figure 3

Anti-MUC1-CAR constructs, anti-MUC1-CAR T cell generation and their anti-tumor responses. (a) A schematic representation of the anti-MUC1-CAR2 (top) and anti-MUC1-CAR4 (bottom) lentiviral constructs. H represents hinge (spacer) domain. (b) Representative transduction efficiency of the untransduced T cells (UTD, gray), anti-MUC1-CAR2 T cells (red), and anti-MUC1-CAR4 T cells (green) detected by flow cytometry. (c) The transduction efficiency summarized from 4 independent experiments. (d) The bar graph represented percentages of KKU-213A cell lysis from 3 independent experiments. (e) T cell expansion after exposure to either MMNK-1 or KKU-213A cells. All data with error bars represents mean ± SD. Statistical differences were analyzed by Student t-test (asterisks indicate p-values: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001).

Figure 4

Phenotypes, activation and exhaustion markers, and memory subsets of anti-MUC1-CAR4 T cells analyzed by flow cytometry. The phenotypic analyses of unactivated PBMCs (green), PHA-activated lymphocyte (yellow), and anti-MUC1-CAR4 T cells (red) were summarized: (a) cellular phenotypes (n = 4, mean ± SD); (b) activation markers (CD25 and CD69) (n = 3, mean ± SD); (c) exhaustion markers (LAG-3, TIM-3, and PD-1) (n = 3, mean ± SD); and (d) memory subsets (n = 5, mean ± SEM). The statistical differences were analyzed by Student t-test (asterisks represent p-values: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 5

TNF-α and IFN-γ production by anti-MUC1-CAR4 T cells following exposure to CCA cells. The representative cell gating data of (a) TNF-α and (b) IFN-γ cytokine production in untransduced (UTD) T cells, UTD T cells plus target cells, anti-MUC1-CAR4 T cells alone, and anti-MUC1-CAR4 T cells co-cultured with MMNK-1, KKU100, and KKU-213A. The percentages of TNF-α (c) and IFN-γ (d) producing cells plotted as bar graphs were individually averaged from 3 independent experiments (mean ± SD). All data were analyzed by Student t-test (asterisks represent p-values: *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 6

Cytotoxic function of anti-MUC1-CAR4 T cells on MUC1-expressing CCA cells. (a) The remaining target cells detected by fluorescence microscopic method after co-culturing with anti-MUC1-CAR4 T cells. (b) Specific cell lyses of MMNK-1, KKU-100, and KKU-213A cells after co-culture with the anti-MUC1-CAR4 T cells, examined by luciferase assay at E:T ratios of 1:1, 2.5:1, and 5:1, respectively. Four independent experiments were conducted and summarized (mean ± SEM). (c) The representative cell gating to detect granzyme B in UTD T and anti-MUC1-CAR4 T cells. (d) The bar graphs showed percentages of the granzyme B-producing cells after exposure to MMNK-1, KKU-100, and KKU-213A. The data were summarized from 3 independent experiments (mean ± SD). The statistical differences were analyzed by Student t-test (asterisks represent p-values: *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 7

Anti-tumor effect of anti-MUC1-CAR4 T cells against MUC1-expressing KKU-213A spheroids. (a) Representative results of KKU-213A spheroids expressing a green fluorescence protein; left panel: no treatment (NT); middle panel: spheroids co-cultured with untransduced T cells (UTD T); right panel: spheroids co-cultured with anti-MUC1-CAR4 T cells (E:T ratio of 10:1). The spheroids were captured under a fluorescence microscope at 10 × objective lens. (b) The bar graphs represent summarized data of corrected total cell fluorescence (CTCF) from 4 independent donors (mean ± SD). The statistical differences were analyzed by Student t-test (asterisks indicate p-values: *p < 0.05, **p < 0.01, ***p < 0.001).