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. 2022 Aug 16;8:80. doi: 10.1038/s41421-022-00433-y

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Design of the anti-hIL-5Rα CAR-T cells using the scFv derived from human IL-5Rα mAb as the antigen-binding domain. b Cytotoxic activity of anti-hIL-5Rα CAR-T cells as determined by a bioluminescence assay using luciferase-expressing hIL-5Rα⁺ or hIL-5Rα^– U2OS cells as target cells. Differences between the CAR-T cell-treated and control group were examined by the two-way ANOVA, ^****P < 0.0001. c Design of the hIL-5-anchored CCAR-T cells using human IL-5 as the antigen-binding domain. d Cytotoxic activity of hIL-5-anchored CCAR-T cells. Two-way ANOVA, ^****P < 0.0001. e Design of the hCCL11-anchored CCAR-T cells. f Cytotoxic activity of the CCL11-anchored CCAR-T cells against target cells. Two-way ANOVA, ^****P < 0.0001. g Design of the hCCL24-anchored CCAR-T cells. h Cytotoxic activity of the CCL24-anchored CCAR-T cells to target cells. Two-way ANOVA, ^****P < 0.0001. i Cytotoxic activity of anti-hIL-5Rα CAR-T cells or hIL-5-anchored CCAR-T cells from healthy human donors against hIL-5Rα⁺ U2OS cells. Two-way ANOVA, ^****P < 0.0001. UTD-T, un-transduced T cells. j, k The production of interferon-γ (IFN-γ) in the supernatant of anti-hIL-5Rα CAR-T cells (j) or hIL-5-anchored CCAR-T cells (k) from healthy human donors after coculture with target cells for 24 h was determined by ELISA kit. Two-way ANOVA, ^****P < 0.0001. l Schematic diagram of the bio-membrane force probe assay for the adhesion frequency assay. m Binding specificities (P_a) of anti-hIL-5Rα CAR/hIL-5-anchored CCAR and hIL-5Rα as measured by adhesion frequency with different contact duration. n In-situ effective affinity (A_ck_a) of the interaction between anti-hIL-5Rα CAR-hIL-5Rα or hIL-5-anchored CCAR-hIL-5Rα and hIL-5Rα. N.D., not detected. o In-situ effective on-rate (A_ck_on) of the anti-hIL-5Rα CAR-hIL-5Rα bond and the hIL-5-anchored CCAR-hIL-5Rα bond. p Average off-rate (k_off) of the anti-hIL-5Rα CAR-hIL-5Rα bond and the hIL-5-anchored CCAR-hIL-5Rα bond. Two-tailed t-test, ^*P < 0.05.