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. 2023 Oct;72(10):3191-3202.
doi: 10.1007/s00262-023-03491-7. Epub 2023 Jul 7.

CD24 is a novel target of chimeric antigen receptor T cells for the treatment of triple negative breast cancer

Peiwei Yang #  1   2 , Fan Yu #  1   3 , Zheng Yao  1   2 , Xu Ding  4 , Hanmei Xu  5   6 , Juan Zhang  7   8
Affiliations

CD24 is a novel target of chimeric antigen receptor T cells for the treatment of triple negative breast cancer

Peiwei Yang et al. Cancer Immunol Immunother. 2023 Oct.

Abstract

Triple negative breast cancer (TNBC) is a subtype of breast cancer with the highest degree of malignancy and the worst prognosis. The application of immunotherapy for TNBC is limited. This study was to verify the potential application of chimeric antigen receptor-T cells (CAR-T cells) targeting CD24 named as 24BBz in treatment of TNBC. 24BBz was constructed by lentivirus infection and then was co-culture with breast cancer cell lines to evaluate the activation, proliferation and cytotoxicity of engineered T cells. The anti-tumor activity of 24BBz was verified in the subcutaneous xenograft model of nude mice. We found that CD24 gene was significantly up-regulated in breast cancer (BRCA), especially in TNBC. 24BBz showed antigen-specific activation and dose-dependent cytotoxicity against CD24-positive BRCA tumor cells in vitro. Furthermore, 24BBz showed significant anti-tumor effect in CD24-positive TNBC xenografts and T cells infiltration in tumor tissues, while some T cells exhibited exhaustion. No pathological damage of major organs was found during the treatment. This study proved that CD24-specific CAR-T cells have potent anti-tumor activity and potential application value in treatment of TNBC.

Keywords: CAR-T cell therapy; CD24 antigen; Solid tumor; TNBC.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
Expression of CD24 in breast cancer a CD24 gene expression profile across tumor samples and paired normal tissues. b CD24 expression level of tumor or normal tissues in BRCA was analyzed. c CD24 expression level in different BRCA types was analyzed. d Kaplan–Meier analysis indicated that patients with high CD24 expression level suffered significantly worse outcomes (OS, RFS and DMFS). e A single-cell clustering plot was generated, and a total of 89471 cells were analyzed. f Dot plots showing the expression of major cell markers in each cell type. g Violin plots depicting the expression of CD24 in malignant epithelial cells across different subtypes of tumor patients. h Violin plots depicting the expression of CD24 in different cell subtypes were generated specifically for TNBC patients. i The expression of CD24 was detected in several kinds of human breast cancer cells by flow cytometry analysis
Fig. 2
Fig. 2
Construction and characterization of 24BBz a Schematic diagram of the CAR. b Expansion curve of engineered T cells cultured in vitro after stimulation. c Flow cytometry detection of EGFRt expression in engineered T cells to evaluate their transfection rate. d CD4+ and CD8+ engineered T cells were assessed by flow cytometry. e T cell memory phenotypic analysis, TCM (CD45RA−/CD62L+central memory T cells), TEM (CD45RA−/CD62L− effector memory T cells). f The apoptosis level of T cells after 7 days of culture in vitro was detected by flow cytometry. (PI+/Annexin V+ late apoptosis, PI−/Annexin V+ early apoptosis)
Fig. 3
Fig. 3
Antigen-specific activation and dose-dependent cytotoxicity of 24BBz against CD24 expressing tumor cell lines in vitro a lactate dehydrogenase (LDH) levels in the supernatants of different E: T co-culture systems were detected to characterize the lysis effect of 24BBz and Mock T on target cells. The expression of CD25 b and CD69 c on the surface of T cells was detected by flow cytometry after co-incubating with target cells for 24 h. d The engineered T cells were stained with Cell Trace™ CFSE, and after three days of co-incubation, the proliferation ability was detected by flow cytometry. e 24 h after incubation of effector cells and target cells at ratio of 2:1, levels of IFN-γ and IL-2 secreted by T cells were measured. f Flow cytometry analysis of the expression of PD-1on the T cells after three days co-incubation. (n = 3, error bars denote standard deviation, ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)
Fig. 4
Fig. 4
In vivo anti-tumor activities of 24BBz in TNBC bearing mice a, d The imaging of tumors in MDA-MB-231 bearing mice or MDA-MB-468 bearing mice treated with 24BBz/i.v., 24BBz/p.t. and Mock T. b, e Tumor volume quantification was monitored weekly. c, f The weight of the tumors in each group was measured. g HE staining was performed to evaluate the pathological changes of heart, lung, liver, kidney and spleen tissues from each group (× 100). (n = 5, error bars denote standard deviation, **P < 0.01, ***P < 0.001, ****P < 0.0001)
Fig. 5
Fig. 5
In vivo effector function of 24BBz a, b The proportion of CD3+ T cells in peripheral blood of mice was detected by flow cytometry. c After intravenous and peritumoral administration of the treated cells, T cells infiltration in tumor tissue was analyzed by IHC. d Detection of the level of granzyme B secreted by T cells in tumor tissues from i.v. group. e Detection of the level of IFN-γ secreted by T cells in tumor tissues from i.v. group. f IHC analysis of the expression of PD-1 on the surface of infiltrating T cells in tumor tissues from i.v. group. (n = 5, error bars denote standard deviation, *P < 0.05, **P < 0.01, ****P < 0.0001)
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