B7-H3 and CSPG4-targeted CAR T cells as potent effectors in anaplastic thyroid cancer

Abstract

Background: Anaplastic thyroid cancer (ATC) is a rare and aggressive malignancy with poor survival and no available effective therapy. This unmet clinical need led us to investigate chimeric antigen receptor (CAR) T cells s as potential treatment option for this malignant disease. As target tumor antigens of our CAR T cell therapy, we selected the chondroitin sulfate proteoglycan 4 (CSPG4) and the B7-homolog 3 (B7-H3), as they are both highly and homogeneously expressed on different types of thyroid carcinoma cell lines and tissues, including ATC. Importantly, both CSPG4 and B7-H3 have a low distribution on normal tissues, thus limiting 'on-target off-tumor' CAR T-related toxicities.

Methods: We generated CSPG4-specific and B7-H3-specific CAR T cells by utilizing a second-generation CAR construct comprised of a CD28 costimulatory domain and tested their antitumor activity in vitro and in an orthotopic xenograft murine model of ATC.

Results: We demonstrated that thyroid cancer cells are specifically recognized and effectively eradicated in vitro by CSPG4-targeted and B7-H3-targeted CAR T cells. Additionally, both CAR T cell types were able to mediate significant control or complete eradication of primary ATC tumors when mice were treated with CSPG4 CAR T cells or B7-H3 CAR T cells, respectively.

Conclusion: Overall, in this study we identified CSPG4 and B7-H3 as valuable target antigens in thyroid cancer and demonstrated that CAR T cell immunotherapy can be a valuable therapeutic option for ATC patients. Our findings provide the translational basis for exploring CAR T cell immunotherapies targeting CSPG4 and B7-H3 with ATC patients who do not respond or relapse after first line treatment.

Keywords: Anaplastic thyroid cancer; B7-H3; CAR T; CSPG4; Immunotherapy.

Fig. 1

CSPG4 is highly and homogeneously expressed in thyroid cancers. (A) Box plot showing CSPG4 mRNA expression level in patients with anaplastic thyroid cancer (ATC), follicular thyroid carcinoma (FTC), papillary thyroid carcinoma (PTC), and in normal thyroid tissues (NT). Data were analyzed by utilizing a publicly available database (accession number GSE126698). (B) Representative images of primary thyroid tumors demonstrating high expression of CSPG4, assessed by IHC staining performed with the CSPG4-specific mAb 763.74 (4 µg/mL) (400x, scale bar 50 μm). (C) Box plot summarizing the overall score of CSPG4 expression on primary thyroid tumors, assessed by quantifying the percentage of positive stained cells and the intensity of expression. (D) Membrane expression of CSPG4 on human thyroid cancer cell lines assessed by flow cytometry by utilizing the CSPG4-specific 763.74 mAb; the MK2.23 mAb was utilized as isotype control

Fig. 2

CSPG4 CAR T cells specifically recognize and effective eradicate thyroid cancer cells in vitro. A) Schematic representation of the CSPG4-specific CAR construct. B) Representative flow cytometric histogram demonstrating CSPG4 CAR T cell transduction efficiency. C) Cumulative curves demonstrating the percentage of cancer cell killing mediated by CSPG4 CAR T cells against 4 thyroid cancer cell lines and the pancreatic cancer cell line PDAC6 after a 72-hour coculture period. D) CD19 CAR T cell-mediated antitumor activity against thyroid cancer cell lines. E) Levels of effector cytokines released in the cell culture supernatant by CAR T cells after a 24-hour coculture with 8505c and SW1736 cells (CSPG4 positive) or Jurkat and PDAC6 cells (CSPG4 negative). Results are shown as mean ± SD of n = 3 independent experiments. Statistical analysis was performed by one-way ANOVA multiple comparison test. ***p-value ≤ 0.001, ****p-value ≤ 0.0001

Fig. 3

CSPG4 CAR T cells effectively control ATC tumor growth and prevent the development of lung metastases in mice. (A) Schematic timeline of the in vivo experiment. (B) Representative images of mice sacrificed 15 days after CAR T cell administration and macroscopically examined for the presence of ATC tumors (n = 3 mice/group). (C) Weight of ATC tumors collected from mice of each treatment group at the time of sacrifice. (D) Representative BLI images of lungs collected from mice of each treatment group at the time of sacrifice. (E) Weight of lungs collected from mice of each treatment group at the time of sacrifice. Statistical analysis was performed by one-way ANOVA multiple comparison test. *p-value ≤ 0.05, **p ≤ 0.01, ***p-value ≤ 0.001

Fig. 4

B7-H3-targeted CAR T cells effectively eradicate thyroid cancer cells in vitro. (A) Box plot showing CD276 (B7-H3) mRNA expression level in patients with ATC, FTC, PTC, and in normal thyroid tissues (NT). Data were analyzed by utilizing a publicly available database (accession number GSE126698). (B) and (C) Percentage and intensity of membrane B7-H3 expression on human thyroid cancer cell lines assessed by flow cytometry. B7-H3 expression was determined by utilizing the B7-H3-specific 376.96 mAb; the F3C.25 mAb was utilized as isotype control. (D) Schematic representation of the B7-H3-specific CAR construct. (E) Representative flow cytometric histogram showing B7-H3 CAR T cell transduction efficiency. (F) Cumulative curves demonstrating the percentage of cancer cell killing mediated by B7-H3 CAR T cells against 4 thyroid cancer cell lines and the MDA-MB-231 B7-H3^-/- or MDA-MB-231 B7-H3^wt cell line after a 72-hour coculture period. (G) Levels of effector cytokines released in the cell culture supernatant by CAR T cells after a 24-hour coculture with 8505c and SW1736 cells (B7-H3 positive) or Jurkat and MDA-MB-231 B7-H3^-/- cells (B7-H3 negative). (H) Live monitoring of 8505c cell growth (phase image) cocultured with either B7-H3-specific, CSPG4-specific, or CD19-specific CAR T cells (PKH26-labeled, red) for a 96 h-coculture period at the 1:1 E: T ratio. Results are shown as mean ± SD of n = 3 independent experiments. Statistical analysis was performed by unpaired t-test or one-way ANOVA multiple comparison test. *p-value ≤ 0.05, **p-value ≤ 0.01, ***p-value ≤ 0.001, ****p-value ≤ 0.0001

Fig. 5

B7-H3 CAR T cells demonstrate superior antitumor activity compared to CSPG4 CAR T cells in an orthotopic murine model of ATC. (A) Schematic timeline of the in vivo experiment. (B) BLI monitoring of tumor growth in each group treated as indicated (n = 5 mice/group). ‘X’ denotes mice dead of disease. (C) Total photon flux as a function of time after CAR T cell administration. (D) Kaplan-Meier survival curves of mice in each treatment group. (E) CAR T cell (CD3 + CD45+) expansion in peripheral blood (50µL) collected from tumor-bearing mice. Statistical analysis was performed using one-way ANOVA multiple comparison test (C, E), and log-rank test (D). ns = not significant, *p-value ≤ 0.05, ****p-value ≤ 0.0001