Modification with IL-21 and CCL19 enhances killing efficiency and tumor infiltration of NKP30 CAR-T cells in lung cancer

doi:10.12122/j.issn.1673-4254.2024.10.11

Abstract

Abstract:

Objective To investigate whether modification with IL-21 and CCL19 enhances killing and tumor-infiltrating efficiency of NKP30 CAR-T cells in lung cancer. Methods The modified IL-21-CCL19 NKP30 CAR-T cells expressing IL-21 and CCL19 fusion gene was constructed based on NKP30 CAR-T cells and stimulated with CD3CD28 antibodies and IL-2. The immunophenotype and migration of the cells in the presence of IL-21 were investigated using flow cytometry and migration experiments. Lactate dehydrogenase (LDH) release and sphere formation assays were used to assess the killing and infiltration capabilities of CAR-T cells, and the secretion levels of IFN‑γ, IL-21 and CCL19 were determined with enzyme-linked immunospot assay (ELISPOT) and ELISA. A zebrafish model bearing HCG-27 cell xenograft was established by microinjection of the tumor cells into the yolk sac followed 24 h later by injection of the immune cells at the same site, and the fluorescence signals were captured using a fluorescent microscopy. Results The NKP30 ligand B7H6, which was almost undetectable in normal tissues and blood cells, was highly expressed (over 90%) in lung cancer cells. Compared with NKP30 CAR-T cells and conventional T cells, IL-21-CCL19 NKP30 CAR-T cells exhibited stronger proliferative and migration capabilities with the formation of central memory T cells. The reduced expressions of CTLA4 and PD1 in the constructed cells resulted in enhanced killing efficiency against lung cancer cells accompanied by significantly increased production of IFN‑γ, IL-21 and CCL19. In the zebrafish models, CAR-T cells exhibited stronger cytotoxicity and proliferative abilities than typical T cells, but these differences were not statistically significant between the two CAR-T cells. Conclusion Modification of NKP30 CAR-T cells with IL-21 and CCL19 facilitates their access into solid tumors for more effective tumor cell killing while producing a large number of memory T cells.

Zhifeng ZHOU, Shuoyan LIU, Jieyu LI, Mingqiu CHEN, Hui LIN, Yujie CHEN, Weijie CHEN, Junpeng LIN, Hang ZHOU, Qinfeng ZHENG. Modification with IL-21 and CCL19 enhances killing efficiency and tumor infiltration of NKP30 CAR-T cells in lung cancer[J]. Journal of Southern Medical University, 2024, 44(10): 1926-1936.

Figures/Tables 7

Fig.1 Expression of the NKP30 ligand B7H6 on the cells. A: Analysis of the Human Proteome Map database showing negative B7H6 expression in normal tissues. B: B7H6 is expressed in most of the tumor tissues. C: B7H6 is highly expressed in different lung cancer cell lines.

Fig.2 Construction of CAR and transfection of T cells. A: Schematic diagram of NKP30 CAR and IL-21-CCL19 NKP30 CAR. B: Fluorescence appeared in T cells 72 h after transfection (Original magnification: ×100). C: Results of CAR virus transfection in peripheral blood mononuclear cells from 3 donors, in which both Conv.CAR and 21×19 CAR achieved over 50% transfection after 7 days. D: Transfection efficiency of CAR-T cells on CD4+ and CD8+ T cells at different time points.

Fig.3 21×19 CAR-T promotes T cell proliferation and migration. A: Histograms of the numbers of cell divisions. The numbers in the donut charts represent percentages of each gated fraction in the cultured cells. B: Number of cells in each group on the 5th and 7th day. C: Anti-IL-21R monoclonal antibody down-regulates the formation of memory cells in 21×19 CAR-T group. D: Flow cytometry for analyzing chemotactic ability of the cells toward T cells in each group. E: Anti-CCR7 monoclonal antibody down-regulates the chemotactic ability of cells in 21×19 CAR-T group. **P<0.01, ***P<0.001, ****P<0.0001 by one-way ANOVA with multiple comparisons.

Fig.4 Phenotype of cultured CAR-T cells. 21×19 CAR-T cells expressed significantly higher levels of activation markers CD3/CD25 and central memory markers CD62L/CD45RO with lower CTLA4 and PD1 expressions than other cells. The red box indicates the positive region. *P<0.05, **P<0.01.

Fig.5 21×19 CAR show enhanced cytotoxic activity in vitro. A: LDH assay for assessing cytotoxic activity of immune cells from each group co-cultured with lung cancer cells at effector-to-target (E:T) ratios of 1:1, 5:1, 10:1, and 20:1. B: Immune cells from each group co-cultured with the target tumor cell spheroids at different time points (E:T=1:1). Green fluorescence represents the effector cells, and red fluorescence represent the tumor cells (×100). C: ELISPOT for measuring the amount of IFN-γ produced by the effector cells in response to lung cancer cells. **P<0.01, ****P<0.0001.

Fig.6 21×19 CAR-T cells can effectively secrete IL-21 and CCL19 cytokines. A, B: Compared with Mock cells and conventional CAR-T cells, 21×19 CAR-T cells secreted a large amount of IL-21 (A) and CCL-19 (B). C: Both CAR-T and 21×19 CAR-T cells produce higher IL-6 level than cells in the Mock group, there is no significant difference among the 3 groups. ***P<0.001, ****P<0.0001.

Fig. 7 CAR-T cells eliminate lung cancer cells in zebrafish. A: Fluorescence microscopy of the tumor-bearing zebrafish at 0 and 24 hpi time points (×30). The tumor cells A549 were stained with red fluorescent Dil, and the effector cells carry green fluorescent protein (GFP). B: Tumor volume increases in the zebrafish models 24 h after PBS injection. C, D: No significant area expansion of T cells and increased tumor volume in the Mock group after 24 h. E, F: In the conv. group, the area of CAR-T cells shows partial expansion with reduced tumor volume after 24 hours. G: In the 21×19 CAR-T group, the area of CAR-T cells significantly expands and the tumor volume is significantly reduced after 24 h. *P<0.05, **P<0.01.

References 30

1	Liu YJ, He YY. A narrative review of chimeric antigen receptor-T (CAR-T) cell therapy for lung cancer[J]. Ann Transl Med, 2021, 9(9): 808.
2	Schubert ML, Schmitt M, Wang L, et al. Side-effect management of chimeric antigen receptor (CAR) T-cell therapy[J]. Ann Oncol, 2021, 32(1): 34-48.
3	Cochrane RW, Fiorentino A, Allen E, et al. How to test human CAR T cells in solid tumors, the next frontier of CAR T cell therapy[M]// Cancer Immunotherapy. New York: Humana, 2024: 243-265.
4	Gutierrez-Silerio GY, Bueno-Topete MR, Vega-Magaña AN, et al. Non-fitness status of peripheral NK cells defined by decreased NKp30 and perforin, and increased soluble B7H6, in cervical cancer patients[J]. Immunology, 2023, 168(3): 538-53.
5	Mohammadi A, Najafi S, Amini M, et al. The potential of B7-H6 as a therapeutic target in cancer immunotherapy[J]. Life Sci, 2022, 304: 120709.
6	SHAEivary, Kheder RK, Najmaldin SK, et al. Implications of IL-21 in solid tumor therapy[J]. Med Oncol, 2023, 40(7): 191.
7	Ma MC, Xie YY, Liu JH, et al. Biological effects of IL-21 on immune cells and its potential for cancer treatment[J]. Int Immunopharmacol, 2024, 126: 111154.
8	Xu DH, Liu X, Ke SY, et al. CCL19/CCR7 drives regulatory T cell migration and indicates poor prognosis in gastric cancer[J]. BMC Cancer, 2023, 23(1): 464.
9	Gacerez AT, Hua CK, Ackerman ME, et al. Chimeric antigen receptors with human scFvs preferentially induce T cell anti-tumor activity against tumors with high B7H6 expression[J]. Cancer Immunol Immunother, 2018, 67(5): 749-59.
10	Silvestre RN, Eitler J, de Azevedo JTC, et al. Engineering NK-CAR.19 cells with the IL-15/IL-15Rα complex improved proliferation and anti-tumor effect in vivo [J]. Front Immunol, 2023, 14: 1226518.
11	Liu ZH, Zhou JY, Yang XZ, et al. Safety and antitumor activity of GD2-Specific 4SCAR-T cells in patients with glioblastoma[J]. Mol Cancer, 2023, 22(1): 3.
12	Morello A, Sadelain M, Adusumilli PS. Mesothelin-targeted CARs: driving T cells to solid tumors[J]. Cancer Discov, 2016, 6(2): 133-46.
13	Feng KC, Guo YL, Dai HR, et al. Chimeric antigen receptor-modified T cells for the immunotherapy of patients with EGFR-expressing advanced relapsed/refractory non-small cell lung cancer[J]. Sci China Life Sci, 2016, 59(5): 468-79.
14	Zeltsman M, Dozier J, McGee E, et al. CAR T-cell therapy for lung cancer and malignant pleural mesothelioma[J]. Transl Res, 2017, 187: 1-10.
15	Zeng R, Spolski R, Casas E, et al. The molecular basis of IL-21-mediated proliferation[J]. Blood, 2007, 109(10): 4135-42.
16	Ngai H, Tian GW, Courtney AN, et al. IL-21 selectively protects CD62L⁺ NKT cells and enhances their effector functions for adoptive immunotherapy[J]. J Immunol, 2018, 201(7): 2141-53.
17	Ptáčková P, Musil J, Štach M, et al. A new approach to CAR T-cell gene engineering and cultivation using piggyBac transposon in the presence of IL-4, IL-7 and IL-21[J]. Cytotherapy, 2018, 20(4): 507-20.
18	Singh H, Figliola MJ, Dawson MJ, et al. Reprogramming CD19-specific T cells with IL-21 signaling can improve adoptive immunotherapy of B-lineage malignancies[J]. Cancer Res, 2011, 71(10): 3516-27.
19	Pang NZ, Shi JX, Qin L, et al. IL-7 and CCL19-secreting CAR-T cell therapy for tumors with positive glypican-3 or mesothelin[J]. J Hematol Oncol, 2021, 14: 118.
20	Adachi K, Kano Y, Nagai T, et al. IL-7 and CCL19 expression in CAR-T cells improves immune cell infiltration and CAR-T cell survival in the tumor[J]. Nat Biotechnol, 2018, 36(4): 346-51.
21	Yao YX, Wang L, Wang X. Modeling of solid-tumor microenvironment in zebrafish (Danio rerio) larvae[M]// Tumor Microenvironment. Cham: Springer, 2020: 413-428.
22	Reinhardt F, Coen L, Rivandi M, et al. DanioCTC: analysis of circulating tumor cells from metastatic breast cancer patients in zebrafish xenografts[J]. Cancers, 2023, 15(22): 5411.
23	殷果, 李荣, 刘岳飞, 等. Notch信号通路抑制剂DAPT改善酒精诱导的斑马鱼神经元分化障碍[J]. 南方医科大学学报, 2023, 43(6): 889-99. DOI: 10.12122/j.issn.1673-4254.2023.06.03
24	Li Y, Liu WH, Xu J, et al. Chlorahololide D, a lindenane-type sesquiterpenoid dimer from Chloranthus holostegius suppressing breast cancer progression[J]. Molecules, 2023, 28(20): 7070.
25	Murali Shankar N, Ortiz-Montero P, Kurzyukova A, et al. Preclinical assessment of CAR-NK cell-mediated killing efficacy and pharmacokinetics in a rapid zebrafish xenograft model of metastatic breast cancer[J]. Front Immunol, 2023, 14: 1254821.
26	Barati M, Chahardehi AM, Hosseini Y. Finding integrative medication for neuroblastoma and glioblastoma through zebrafish as A model of organism[J]. Curr Top Med Chem, 2023, 23(30): 2807-20.
27	Pascoal S, Salzer B, Scheuringer E, et al. A preclinical embryonic zebrafish xenograft model to investigate CAR T cells in vivo [J]. Cancers, 2020, 12(3): 567.
28	Rejeski K, Subklewe M, Locke FL. Recognizing, defining, and managing CAR-T hematologic toxicities[J]. Hematology Am Soc Hematol Educ Program, 2023, 2023(1): 198-208.
29	Li XL, Gong NQ, Tian FL, et al. Suppression of cytokine release syndrome during CAR-T-cell therapy via a subcutaneously injected interleukin-6-adsorbing hydrogel[J]. Nat Biomed Eng, 2023, 7(9): 1129-41.
30	Stock S, Klüver AK, Fertig L, et al. Mechanisms and strategies for safe chimeric antigen receptor T-cell activity control[J]. Int J Cancer, 2023, 153(10): 1706-25.