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Dual-targeting CAR-T cells with optimal co-stimulation and metabolic fitness enhance antitumor activity and prevent escape in solid tumors

Nature Cancer volume 2, pages 904–918 (2021)Cite this article

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Abstract

Chimeric antigen receptor (CAR)-T cells showed great activity in hematologic malignancies. However, heterogeneous antigen expression in tumor cells and suboptimal CAR-T-cell persistence remain critical aspects to achieve clinical responses in patients with solid tumors. Here we show that CAR-T cells targeting simultaneously two tumor-associated antigens and providing trans-acting CD28 and 4-1BB co-stimulation, while sharing the same CD3ζ-chain cause rapid antitumor effects in in vivo stress conditions, protection from tumor re-challenge and prevention of tumor escape due to low antigen density. Molecular and signaling studies indicate that T cells engineered with the proposed CAR design demonstrate sustained phosphorylation of T-cell-receptor-associated signaling molecules and a molecular signature supporting CAR-T-cell proliferation and long-term survival. Furthermore, metabolic profiling of CAR-T cells displayed induction of glycolysis that sustains rapid effector T-cell function, but also preservation of oxidative functions, which are critical for T-cell long-term persistence.

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Fig. 1: Single- or dual-antigen targeting and single or dual CD28 or 4-1BB co-stimulation do not eradicate tumors in stress conditions.
Fig. 2: One single shared CD3ζ chain is sufficient for transducing the activation signal in dual specific CAR-T cells.
Fig. 3: CD3ζ sharing in dual CAR relies on CD8α-mediated dimerization.
Fig. 4: Dual targeting with split co-stimulation and shared single CD3ζ promotes sustained antitumor activity.
Fig. 5: MSLN and CSPG4 dual-targeting CAR-T cells with split co-stimulation and shared CD3ζ show sustained T-cell activation and proliferation in vitro and in vivo.
Fig. 6: Dual targeting with split co-stimulation and shared D3ζ promote TCR tonic signaling.
Fig. 7: Dual targeting with split co-stimulation and shared CD3ζ promote CAR-T-cell proliferation and glycolytic and oxidative metabolism.
Fig. 8: Dual targeting, split signaling and one single CD3ζ endodomain prevent tumor escape due to antigen loss.

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Data availability

Source data for this study have been provided as source data files. RNA-seq datasets generated and analyzed during the current study are not publicly available (the genetic information from primary human T cells in this study was not consented to be published in the public domain) and will be available from corresponding authors upon request. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported in part by R01-CA193140-03 (G.D.) and R01-CA243543-01 (G.D.) from the National Cancer Institute (NCI). H.D. was supported by W81XWH-18-1-0441 from the Department of Defense (USA) and the Vicky Amidon Innovation Grant in Lung Cancer Research from the Lung Cancer Initiative of North Carolina (USA). The UNC Small Animal Imaging Facility at the Biomedical Imaging Research Center, the Microscopy Services Laboratory at Department of Pathology and Laboratory Medicine and the Flow Cytometry Core Facilities are supported in part by an NCI Cancer Center Core Support Grant to the UNC Lineberger Comprehensive Cancer Center (P30-CA016086-40) USA.

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Author notes

  1. These authors contributed equally: Koichi Hirabayashi, Hongwei Du.

Authors and Affiliations

  1. Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Koichi Hirabayashi, Hongwei Du, Yang Xu, Peishun Shou, Xin Zhou, Giovanni Fucá, Elisa Landoni, Chuang Sun, Yuhui Chen, Barbara Savoldo & Gianpietro Dotti

  2. Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Barbara Savoldo

  3. Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Gianpietro Dotti

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Contributions

Conceptualization was carried out by H.D. and G.D. Methodology was performed by K.H., H.D., Y.X., P.S., X.Z., B.S. and G.D. Investigations were conducted by K.H., H.D., Y.X., X.Z., P.S., G.F., E.L., C.S., Y.C., B.S. and G.D. Writing of the original draft was carried out by K.H., H.D., X.Y. and G.D. Review and editing was conducted by all authors. Supervision was carried by H.D. and G.D.

Corresponding authors

Correspondence to Hongwei Du or Gianpietro Dotti.

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Competing interests

G. Dotti is a paid consultant for Bellicum Pharmaceuticals, Tessa Therapeutics and Catamaran and reports receiving commercial research grants from Cell Medica and Bluebird Bio; B. Savoldo is a paid consultant for Tessa Therapeutics; G. Dotti and H. Du filed a patent for the CAR targeting B7-H3. No other competing interests were disclosed by the other authors.

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Peer review information Nature Cancer thanks John Anderson and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 GD2-specific CAR-T cells and B7-H3-specific CAR-T cells target neuroblastoma in vitro.

(a) Flow cytometry histogram showing the expression of GD2 and B7-H3 in two human NB cell lines, CHLA-255 and LAN-1. Representative of three independent experiments. (b) Representative flow cytometry histograms showing the expression of CARs in human T cells transduced with retroviral vectors encoding CD19.28ζ, GD2.28ζ, GD2.BBζ, B7-H3.28ζ, and B7-H3.BBζ CARs. (c-e) Representative flow cytometry plots (c) and quantification of residual CHLA-255 (d) and LAN-1 (e) cells labelled with GFP and co-cultured with CAR-T cells at the T cell to tumor cell ratio of 1 to 5. On day 5, NB cells (GFP+) and CAR-T cells (CD3+) were enumerated by flow cytometry. Data are shown as individual values and the mean + SD, n = 6 independent co-cultures using CAR-T cells generated from 6 different donors. (f, g) Summary of IFN-γ (f) and IL-2 (g) released by CAR-T cells in the culture supernatant after 24 h of co-culture with NB cells as measured by ELISA. Data are shown as individual values and the mean + SD, n = 6 independent co-cultures using CAR-T cells generated from 6 different donors. (h) Representative CFSE dilution of CSFE-labeled CAR-T cells co-cultured with NB cells for 5 days at the T cell to tumor cell ratio of 1 to 1 (red histogram). CFSE-labeled CAR-T cell alone (grey histogram) was used as negative control. Representative of three independent experiments.

Source data

Extended Data Fig. 2 The antitumor activity of GD2-specific CAR-T cells and B7-H3-specific CAR-T cells with either CD28 or 4-1BB costimulation in vivo.

(a) Schema of the CHLA-255 metastatic xenograft NB model using NSG mice inoculated via tail vein injection with 2 × 106 of FFluc-CHLA-255 cells and 14 days later received high doses of CAR-T cells (6 × 106 cells/mouse) intravenously. (b, c) Representative tumor bioluminescence (BLI) images (b) and tumor BLI kinetics (c) of FFluc-CHLA-255 tumor growth (n = 3 mice for the CD19.28ζ group, n = 5 mice for the other four groups) in the metastatic xenograft NB models shown in (a). (d) Kaplan-Meier survival curve of mice in (b, c), n = 3 mice for CD19.28ζ group, n = 5 mice for other 4 groups, comparisons of survival curves were determined by Log-rank test, **p = 0.0042 for CD19.28ζ versus other 4 groups. (e) Schema of the LAN-1 metastatic xenograft NB model using NSG mice inoculated via tail vein injection with FFLuc-LAN-1 cells and treated 21 days later with low doses CD19.28ζ, GD2.28ζ, GD2.BBζ, B7-H3.28ζ or B7-H3.BBζ CAR-T cells intravenously. (f, g) Representative tumor BLI images (f) and tumor BLI kinetics (g) of FFLuc-LAN-1 tumor growth (n = 3 mice/group). (h) Kaplan-Meier survival curve of mice in (f, g), n = 3 mice/group, comparisons of survival curves were determined by Log-rank test, *p = 0.0253 for CD19.28ζ versus GD2.28ζ, GD2.BBζ and B7-H3.BBζ groups, *p = 0.0295 for GD2.28ζ versus GD2.BBζ, *p = 0.0246 for GD2.28ζ versus B7-H3.BBζ.

Source data

Extended Data Fig. 3 Addition of 4-1BB in tandem to the GD2.28ζ CAR and co-expression of both GD2.28ζ and B7-H3.BBζ CARs do not improve antitumor activity in vitro.

(a) Representative flow cytometry plots showing the CAR expression in human T cells transduced with retroviral vectors encoding CD19.28ζ, GD2.28ζ, GD2.28.BBζ, or GD2.28ζ/B7-H3.28ζ CARs. Representative of six independent experiments. (b, c) Representative flow cytometry plots (b) and quantification of residual CHLA-255 cells (c) labelled with GFP co-cultured with CAR-T cells at the T cell to tumor cell ratio of 1 to 5. Data are shown as individual values and the mean + SD, n = 6 or 8 independent co-cultures using CAR-T cells generated from 6 or 8 different donors. (d, e) Summary of IFN-γ (d) and IL-2 (e) released by CAR-T cells in the culture supernatant after 24 h of co-culture with NB cells as measured by ELISA. Data are shown as individual values and the mean + SD, n = 6 or 8 independent co-cultures using CAR-T cells generated from 6 or 8 different donors; **p = 0.0011, ****p <0.0001 by one-way ANOVA with Tukey’s multiple comparison test adjusted p value.

Source data

Extended Data Fig. 4 Cytotoxic activity of the double CAR-T cells with shared CD3ζ is antigen dependent.

(a) Flow cytometry plots showing the expression of B7-H3 and GD2 in Raji cells wild type and B7-H3 expression in Raji cells transduced with a retroviral vector encoding B7-H3 (Raji-B7-H3). Representative of three independent experiments. (b–d) CAR-T cells (B7-H3.BB, B7-H3.BBζ, GD2.28ζ, GD2.28ζ/B7-H3.BB, dNGFR.28ζ/B7-H3.BB and 28ζ/B7-H3.BB) were co-cultured with Raji-B7-H3 cell at 1 to 1 ratio, and 5 days later tumor cells (CD19+) and T cells (CD3+) were collected and enumerated by flow cytometry (b). Supernatants of the co-cultures were collected 24 h later, and IFN-γ (c) and IL-2 (d) released by CAR-T cells were measured by ELISA. Data are shown as individual values and the mean + SD, n = 3 independent co-cultures using CAR-T cells generated from 3 different donors for dNGFR.28ζ/B7-H3.BB group, and n = 5 independent co-cultures using CAR-T cells generated from 5 different donors for all the other groups; *p <0.05 (0.0228 in c, 0.0141 in d), **p <0.01 (0.0025 in c, 0.0015 in d), ***p = 0.0005, ****p <0.0001 by one-way ANOVA with Tukey’s multiple comparison test adjusted p value. (e-g) CAR-T cells (CD19.28ζ, GD2.28ζ/B7-H3.BB, dNGFR.28ζ/B7-H3.BB and 28ζ/B7-H3.BB) were co-cultured with Raji cell wild type at 1 to 1 ratio, and 5 days later tumor cells (CD19+) and T cells (CD3+) were collected and enumerated by flow cytometry (e). Supernatants of the co-cultures were collected 24 h later, and IFN-γ (f) and IL-2 (g) released by CAR-T cells were measured by ELISA. Data are shown as individual values and the mean + SD, n = 4 independent co-cultures using CAR-T cells generated from 4 different donors for each group; ****p <0.0001 by one-way ANOVA with Tukey’s multiple comparison test adjusted p value.

Source data

Extended Data Fig. 5 CAR clustering and aggregation in CAR-T cells after CAR engagement.

Representative confocal microscopy imaging showing CAR molecule clustering in T cells expressing GFP-tagged GD2.28ζ (green) and B7-H3.BB (red) with and without engagement of the CARs using either the anti-14g2a idiotype antibody (1A7) or the B7-H3.Fc protein. Blue staining indicates the DAPI. Shown are representative cells of a single field (Magnification 63X). Data are representative of three independent validations. Shown in white are the scale bars that correspond to 20 µm.

Extended Data Fig. 6 Phenotypic analysis of CAR-T cells in vitro and in vivo.

(a, b) Frequency of CD45RA+CCR7+, CD45RA-CCR7+, CCR7-CD28+CD27+, CCR7-CD28+CD27-, CCR7-CD28-CD27+, and CCR7-CD28-CD27- in CD4+ (a) and CD8+ (b) T cells on day 13 after retroviral vector transduction and expansion in vitro. Data are shown as individual values and the mean + SD, n = 4 independent experiments using CAR-T cells generated from 4 different donors; *p = 0.0299, two-tailed p value determined by unpaired t test. (c–f) Tumor-baring mice infused with CAR-T cells were bled at day 14 and CAR-T cells in the peripheral blood were analyzed by flow cytometry. (c, d) Frequency of CD45RA+CCR7+, CD45RA-CCR7+, CCR7-CD28+CD27+, CCR7-CD28+CD27-, CCR7-CD28-CD27+, and CCR7-CD28-CD27- in CD4+ (c) and CD8+ (d) T cells. Data are shown as individual values and the mean + SD, n = 5 samples from 5 mice, *p < 0.05, two-tailed p value determined by unpaired t test. (e, f) Mean Fluorescence Intensity (MFI) of PD-1 (e) and TIM-3 (f) in T cells. Data are shown as individual values and the mean + SD, n = 5 samples from 5 mice; *p = 0.0109, ***p = 0.0008, two-tailed p value determined by unpaired t test.

Source data

Extended Data Fig. 7 Inverting the orientation of the B7-H3-specifc CAR and GD2-specific CAR does not alter the beneficial effects of dual targeting CAR-T cells with split costimulation and shared CD3ζ in vitro.

(a) Schematic representation of retroviral vectors encoding B7-H3.28ζ, GD2.BB and B7-H3.28ζ/GD2.BB CARs. (b) Representative flow cytometry plots of 5 independent experiments showing the expression of CARs. (c) Summary of the transduction efficiency of the CARs. Data are shown as individual values and the mean + SD, n = 5 or 7 independent experiments using CAR-T cells generated from 5 or 7 different donors; ***p = 0.002, ****p <0.0001 by one-way ANOVA with Tukey’s multiple comparison test adjusted p value. (d–f) CAR-T cells were co-cultured with CHLA-255-GFP at T cell to tumor cell ratio of 1 to 5. IFN-γ (e) and IL-2 (f) released by CAR-T cells were measured by ELISA. On day 5, tumor cells (GFP+) and CAR-T cells (CD3+) number were measured by flow cytometry (d). Data are shown as individual values and the mean + SD, n = 3 independent co-cultures using CAR-T cells generated from 3 different donors ; *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001 by one-way ANOVA with Tukey’s multiple comparison test adjusted p value, the full list of p values can be found in the source data. (g) Schema of the repetitive multi-round co-culture experiments. Tumor cells were seeded in 24-well plates one day prior to the addition of T cells. At day 0, CAR-T cells were added at T cell to tumor cell ratio of 1 to 5. At day 4, 7, and 10, all T cells were collected and transferred into a new well in which 5 × 105 NB cells were seeded one day before. T cells and tumor cells, and cytokine were quantified at each cycle. (h–o) Multi-round co-culture with NB cell lines CHLA-255 (h–k) and LAN-1 (l–o) cells as described in (g). Summary of percentage of residual CHLA-255 (h) and LAN-1 (l) cells and number of T cells (i, m) at the end of each round of co-culture. Summary of IFN-γ (j, n) and IL-2 (k, o) released by CAR-T cells in the culture supernatant after 24 h of co-culture with CHLA-255 (j, k) and LAN-1 (n, o) cells. Data are shown as individual values and the mean + SD, n = 6 independent co-cultures using CAR-T cells generated from 6 different donors; *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001 by one-way ANOVA with Tukey’s multiple comparison test adjusted p value, the full list of p values can be found in the source data.

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Extended Data Fig. 8 Dual targeting with split co-stimulation and shared CD3ζ provide superior antitumor activity and better T cell persistence in NB model when mice are treated with inverted B7-H3-specifc CAR and GD2-specific CAR.

(a) Schema of the CHLA-255 metastatic xenograft NB model in NSG mice. Eight week old female NSG mice were inoculated with 2 x 106 FFLuc-labelled CHLA-255 cells via tail vein injection, and 14 days later mice were treated with 2 x 106 CD19.28ζ, B7-H3.28ζ or B7-H3.28ζ/GD2.BB CAR-T cells via tail vein injection. (b, c) Representative tumor bioluminescence (BLI) images (b) and tumor BLI kinetics (c) of FFLuc-CHLA-255 tumor growth in the metastatic xenograft NB model shown in (a) (n = 3 mice for the CD19.28ζ group, n = 5 mice for the other two groups). (d) Kaplan-Meier survival curve of mice in (b, c) (n = 3 for the CD19.28ζ group, n = 5 for the other two groups); **p = 0.0016 (B7-H3.28ζ vs. B7-H3.28ζ/GD2.BB) by Log-rank test. (e) Detection of circulating CAR-T cells (CD45+CD3+) in mice 14 days after CAR-T cell treatment by flow cytometry. Data are shown as individual values and the mean + SD, (n = 3 samples from 3 mice for the CD19.28ζ group, n = 5 samples from 5 mice for the other two groups); *p = 0.0144, **p = 0.0042 by one-way ANOVA with Tukey’s multiple comparison test adjusted p value.

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Extended Data Fig. 9 MSLN and CSPG4 dual targeting CAR-T cells with split co-stimulation and shared CD3ζ show sustained T cell activation and proliferation in vitro.

(a) Representative flow cytometry plots showing the expression of CARs. (b) Summary of the transduction efficiency of the CARs (n = 7 or 9 independent experiments using CAR-T cells generated from 7 or 9 different donors). Data are shown as individual values and the mean + SD. (c-e) CAR-T cells co-cultured with GFP labeled H2052 cell at T cell to tumor cell ratio of 1 to 5. IFN-γ (d) and IL-2 (e) released by CAR-T cells. On day 5, tumor cells (GFP+) and CAR-T cells (CD3+) were measured by flow cytometry (c). Data are shown as individual values and the mean + SD, n = 3 independent co-cultures using CAR-T cells generated from 3 different donors for the CSPG4.BBζ group, n = 5 independent co-cultures using CAR-T cells generated from 5 different donors for the other groups; *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001 by one-way ANOVA with Tukey’s multiple comparison test adjusted p value, the full list of p values can be found in the source data. (f) Schema of the multi-round co-culture experiments of CAR-T cells and H2052. Tumor cells were seeded one day prior to the addition of T cells. At day 0, CAR-T cells were added at T cell to tumor cell ratio of 1 to 5. At the end of each round of co-culture, which are at days 5, 9, 13 and 17, one third of T cells were collected and transferred into a new well with 2.5 × 105 H2052 cells that were seeded one day before. T cells and tumor cells and cytokine released by CAR-T cells were quantified at each round of co-culture. (g, h) Summary of IFN-γ (g) and IL-2 (h) released by CAR-T cells in the multi-round co-culture with H2052 as described in (f). Data are shown as individual values and the mean + SD, n = 3 independent co-cultures using CAR-T cells generated from 3 different donors for the CSPG4.BBζ group, n = 4 independent co-cultures using CAR-T cells generated from 4 different donors for the other groups; *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001 by one-way ANOVA with Tukey’s multiple comparison test adjusted p value, the full list of p values can be found in the source data.

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Extended Data Fig. 10 Dual specific GD2 and B7-H3 CAR-T cells with split costimulation and shared CD3z have superior antitumor activity and prevent antigen escape in high tumor burden xenograft model with neuroblastoma cells showing heterogeneous GD2 expression.

(a) Schema of the high tumor burden SH-SY5Y metastatic xenograft NB model using NSG mice inoculated via tail vein injection with FFLuc-SH-SY5Y cells (1 × 106 cell/mouse) and treated 7 days later with CD19.28ζ, GD2.28ζ or GD2.28ζ/B7-H3BB CAR-T cells (1 × 107 cells/mouse) intravenously. (b,c) Representative tumor bioluminescence (BLI) images (b), and tumor BLI kinetics (c) of FFLuc-SH-SY5Y tumor growth (n = 5 mice/group) in the metastatic xenograft NB models shown in (a). (d) Kaplan-Meier survival curve of mice in (b, c), n = 5 mice/group, comparisons of survival curves were determined by Log-rank test, **p = 0.0023 for CD19.28ζ vs. GD2.28ζ, **p = 0.0027 for GD2.28ζ vs. GD2.28ζ/B7-H3.BB.

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Hirabayashi, K., Du, H., Xu, Y. et al. Dual-targeting CAR-T cells with optimal co-stimulation and metabolic fitness enhance antitumor activity and prevent escape in solid tumors. Nat Cancer 2, 904–918 (2021). https://doi.org/10.1038/s43018-021-00244-2

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