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Potent suppression of neuroendocrine tumors and gastrointestinal cancers by CDH17CAR T cells without toxicity to normal tissues

Nature Cancer volume 3, pages 581–594 (2022)Cite this article

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Abstract

Gastrointestinal cancers (GICs) and neuroendocrine tumors (NETs) are often refractory to therapy after metastasis. Adoptive cell therapy using chimeric antigen receptor (CAR) T cells, though remarkably efficacious for treating leukemia, is yet to be developed for solid tumors such as GICs and NETs. Here we isolated a llama-derived nanobody, VHH1, and found that it bound cell surface adhesion protein CDH17 upregulated in GICs and NETs. VHH1-CAR T cells (CDH17CARTs) killed both human and mouse tumor cells in a CDH17-dependent manner. CDH17CARTs eradicated CDH17-expressing NETs and gastric, pancreatic and colorectal cancers in either tumor xenograft or autochthonous mouse models. Notably, CDH17CARTs do not attack normal intestinal epithelial cells, which also express CDH17, to cause toxicity, likely because CDH17 is localized only at the tight junction between normal intestinal epithelial cells. Thus, CDH17 represents a class of previously unappreciated tumor-associated antigens that is ‘masked’ in healthy tissues from attack by CAR T cells for developing safer cancer immunotherapy.

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Fig. 1: Isolating VHH1 and demonstrating its specific binding to CDH17 highly expressed in NETs.
Fig. 2: Optimized CDH17CARTs specifically killed NET cells.
Fig. 3: CDH17CARTs eliminated CDH17-expressing SKOV3 solid tumors in vivo.
Fig. 4: Elimination of NET NT-3 tumors in vivo by CDH17CARTs.
Fig. 5: VHH1-28BBz CARTs kill gastrointestinal cancer cells in vitro and in vivo.
Fig. 6: CDH17CARTs specifically recognized and killed mouse tumor cells in vitro and in vivo.
Fig. 7: CDH17CARTs significantly reduced the tumor burden of the mice with primary CRCs induced with AOM/DSS in WT C57BL/6J mice.
Fig. 8: CDH17CARTs markedly infiltrated into NT-3 tumors, but spared healthy mouse tissues with Cdh17 expression.

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Source data have been provided as Source Data files. 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

We thank J. Schrader at the University Medical Center Hamburg, C. Townsend at the University of Texas Medical Branch, G. Koretzky and B. Stanger at the University of Pennsylvania Perelman School of Medicine for kindly providing the NT-3, BON, NB4 and MH6694C2 cell lines, respectively. We acknowledge the following support: Care for Carcinoid Foundation Research Grant and Neuroendocrine Tumor Research Foundation Accelerator Grant.

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Author notes

  1. These authors contributed equally: Zijie Feng, Xin He.

Authors and Affiliations

  1. Department of Cancer Biology, Abramson Family Cancer Research Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

    Zijie Feng, Xin He, Xuyao Zhang, Yuan Wu, Bowen Xing, Alison Knowles, Qiaonan Shan, Samuel Miller, Taylor Hojnacki, Jian Ma, Bryson W. Katona & Xianxin Hua

  2. Division of Gastroenterology and Hepatology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

    Bryson W. Katona & David C. Metz

  3. Department of Radiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

    Terence P. F. Gade

  4. Center for Cellular Immunotherapies, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

    Don L. Siegel & Carl H. June

  5. Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

    Don L. Siegel & Carl H. June

  6. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    Jörg Schrader

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Contributions

Z.F. and X. Hua designed and generated VHH phage library and CDH17CAR constructs. Z.F. and X. Hua analyzed and interpreted the results. Z.F. and X. He performed most of the experiments and generated Figures. X.Z., Y.W., B.X., A.K., Q.S., S.M., T.H., J.M. and B.W.K. performed certain experiments. J.S. provided the NT-3 cell line. D.L.S. helped with some of the phage display methods. T.P.G., B.W.K. and D.C.M. provided the human NET samples. C.H.J. analyzed and interpreted data and provided reagents. Z.F. and X. Hua analyzed data. X. Hua conceived and supervised the project. Z.F. and X. Hua wrote the manuscript. All authors commented and revised on the manuscript and approved the paper.

Corresponding author

Correspondence to Xianxin Hua.

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Competing interests

X. Hua and Z.F. are inventors of the following patent that develops the VHH1-CAR T cells targeting CDH17: Compositions and Methods for Retrieving Tumor-related Antibodies and Antigens, International application no. PCT/US2019/029333. Part of the patent was licensed to Chimeric Therapeutics. X. Hua is a consultant to Chimeric Therapeutics. The remaining authors declare no competing interests.

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Nature Cancer thanks J. Ignacio Casal, Andras Heczey and Aurel Perren for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 CDH17 as the antigen of VHH1 nanobody is expressed in NET BON cells.

(a) Flow cytometry analysis of VHH1 nanobody binding to various cell lines. Representative of n = 2 independent experiments with similar results. (b) Western Blot analysis of CDH17 protein expression in various cell lines. ACTIN as a control. Experiments were performed two independent times with similar results. (c) 293 T cells were transfected with full length CDH17 or truncation mutant plasmids, followed by Western Blot analysis of CDH17 protein expression by using VHH1 or commercial anti-CDH17 antibody (Santa Cruz, clone H-1) which binds to the C-terminal domain of CDH17. ACTIN as a control. Experiments were performed two independent times with similar results. (d-e) SPR detection of the binding affinity of the CDH17 protein to the VHH1 nanobody. The equilibrium dissociation constant KD of the CDH17 protein to the VHH1 nanobody was 4.79 × 10-7 M.

Source data

Extended Data Fig. 2 CDH17 expression in human NETs.

(a-d) Representative micrographs of the CDH17 expression in hNETs assessed by staining with anti-CDH17 antibody (Santa Cruz, clone H-1). Staining ranked as low (+), moderate (++), or high (+++). Scale bars are 100 or 20 µm. (e) Summary of the CDH17 expression in hNETs (n = 35 patients). (f) Representative micrographs of the heterogeneous CDH17 expression in hNETs. Scale bars are 100 or 20 µm. (g) Summary of the CDH17 positivity in CDH17 positive hNETs (n = 19 patients).

Extended Data Fig. 3 Protein levels of various VHH1-CARs.

(a) Western blot analysis of non-reduced or reduced protein levels of CARs in VHH1-CAR JRT3 cells using anti-CD3zeta antibody. Ponceau S (PS) as a control. Experiments were performed two independent times with similar results. (b) Western blot analysis of non-reduced or reduced protein levels of CARs in primary VHH1-CARTs using anti-CD3zeta antibody. Experiments were performed two independent times with similar results.

Source data

Extended Data Fig. 4 CDH17CARTs eliminated CDH17-expressing NB4 tumors, but not the control CDH17-negative NB4 tumor in vivo.

(a) Flow cytometry analysis of VHH1 binding to WT or sorted CDH17-expressing NB4 cells. Experiments were performed three independent times with similar results. (b) A diagram of the 2nd generation VHH1-BBz CAR structure. (c) Flow cytometry analysis of VHH1-BBz CAR transduction efficiency in human primary T cells by detecting GFP expression. Experiments were performed three independent times with similar results. (d) In vitro killing of WT or CDH17-expressing NB4 cells by control UTD T cells or VHH1-CARTs, using LDH release assay 20 hours after co-culture (n = 3 independent co-culture). Data are presented as the mean ± SD. (e) The flowchart of establishing WT or CDH17-expressing NB4 xenograft models and transfusion of control UTD T cells or VHH1-CARTs to the NSG mice. (f) Summary of growth curve of control or CDH17-expressing NB4 tumors from mice treated with UTD T cells or VHH1-CARTs (n = 4 tumors/group). Data are presented as the mean ± SD. Statistical comparisons of tumor volumes were conducted using two-way ANOVA, *** p < 0.0001. (g-j) Tumor growth curves for each of the indicated groups of the tumors (n = 4 tumors/group). (k) Kaplan-Meier survival curve of mice in (Fig. 3i) (n = 4 mice/group). Of note, presentation of the results was based on conversion of the tumor xenograft volume > or = 1 cm^3, a common mark for tumor burden recommended for sacrificing the tumor bearing mice, to mortality. Comparisons of survival curves were determined by log-rank test, ** p = 0.0084 for CDH17-UTD vs CDH17-VHH1-28BBz, ** p = 0.0084 for CDH17-VHH1-28BBz vs WT-VHH1-28BBz. (l) Kaplan-Meier survival curve of mice in (Fig. 4g) (n = 4 mice for UTD and VHH1-28BBz groups; n = 3 mice for VHH1-BBz group). Comparisons of survival curves were determined by log-rank test, ** p = 0.0084 for UTD vs VHH1-BBz, ** p = 0.0084 for UTD vs VHH1-28BBz.

Source data

Extended Data Fig. 5 Infiltration of CDH17CARTs in SKOV3 and NT-3 tumors.

(a-h) SKOV3 tumors were harvested and fixed at day 14 after the first T cell injection, followed by IF staining with anti-CDH17, anti-CD3 and DAPI. Experiments were performed two independent times with similar results. Scale Bar: 100 μM. Representative of n = 3 tumors per group. (i-l) Histological analysis of SKOV3 tumor sections from control UTD T cell or VHH1-CART-treated mice by H&E staining at day 14 after the first T cell injection. Experiments were performed two independent times with similar results. Scale bars: 100 μM. Representative of n = 3 tumors per group. (m-p) IHC staining of the SKOV3 tumors from UTD T cell or VHH1-CART treated mice with anti-CD31 antibody. Experiments were performed two independent times with similar results. Scale Bar: 500 or 100 µM. Representative of n = 3 tumors per group. (q-v) H&E staining of NT-3 tumor sections from the control UTD T cell or the VHH1-CART-treated mice at day 10 after the first T cell injection. Experiments were performed two independent times with similar results. Scale bars: 200 μM and 50 μM. Representative of n = 3 tumors per group. (w-y) IHC staining of the NT-3 tumors from UTD T cell or VHH1-CART treated mice with anti-CD31 antibody. Experiments were performed two independent times with similar results. Scale Bar: 500 or 100 µM. Representative of n = 3 tumors per group.

Extended Data Fig. 6 Single-time and low-dose CDH17CARTs are potent enough to eliminate NT-3 tumors in vivo.

(a) The flowchart of establishing NT-3 tumor xenograft model and transfusion of the control UTD T cells or the indicated VHH1-CARTs into the NSG mice. (b) Summary of tumor growth following treatment with either control UTD T cells or the VHH1-CARTs (n = 8 tumors/group). Data are presented as the mean ± SD. Statistical comparisons of tumor volumes were conducted using two-way ANOVA, *** p < 0.0001. (c-d) Tumor growth curve for each tumor group (n = 8 tumors/group). (e-f) Body weight of the control UTD T cell or theVHH1-CART-treated mice bearing NT-3 tumor (n = 4 mice/group). (g-h) Circulating T cells (CD3+) in control UTD T cell (g) or VHH1-CART (h) treated mice 10 days after the T cell injection, shown by flow cytometry (n = 4 mice/group). (i-j) Random blood glucose of the mice bearing NT-3 tumors 30 days after treatment of either control UTD T cells (i) or VHH1-CARTs (j) (n = 4 mice/group). (k) Kaplan-Meier survival curve of mice (n = 4 mice/group). Comparisons of survival curves were determined by log-rank test, ** p = 0.0062. (l) NT-3 tumors were harvested and fixed, followed by IF staining with anti-insulin antibody and DAPI. Experiments were performed two independent times with similar results. Scale Bar: 20 µM. (m) Flowchart of establishing BON tumor xenograft model in NSG mice and transfusion with UTD T cells or VHH1-CARTs. (n) BON tumor growth curve following treatment with VHH1-CARTs (n = 6 tumors/group). Data are presented as the mean ± SD. Statistical comparisons of tumor volumes were conducted using two-way ANOVA (** p = 0.0074). (o) Kaplan-Meier survival curve of mice (n = 3 mice/group). Comparisons of survival curves were determined by log-rank test, * p = 0.0295. (p) Circulating T cells (CD3+) from peripheral blood of mice 14 days after the 1st CART injection shown by flow (n = 3 mice/group). The data are presented as the mean ± SD (Two-tailed unpaired Student’s t-test; ** p = 0.0437).

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Extended Data Fig. 7 No obvious structural damage of CDH17CARTs to various normal mouse tissues.

(a) A/J mice were immunized three times with human CDH17 protein. Splenocytes from the immunized mice were fused into hybridoma cells. Single clones of fused hybridoma cells were generated in 96 well plates. Binding of the culture medium from fused hybridoma cells to coated human CDH17 protein were determined by ELISA and positive clones were further analyzed by flow cytometry. WT, human CDH17 or mouse Cdh17-expressing SKOV3 cells were incubated with culture medium from #3 and #29 clones of the positive clones and secondary anti-mouse-FITC antibody, followed by flow cytometry analysis. The results showed that #3 Ab only bound to human CDH17, not mouse Cdh17. The other clone, #29 bound both human (hCDH17) and mouse (mCdh17) CDH17. Representative of n = 2 independent experiments with similar results. (b-c) IHC staining of the colons from murine UTD T cell (mUTD) or murine VHH1-28BBz CART (mCAR) treated mice (Fig. 7) with anti-Foxp3 antibody. Scale Bar: 100 µM. Representative of n = 2 independent experiments with similar results. (d) Counting of the Foxp3+ Treg cells from (b-c) in the tumors (n = 6 mice/group). The data are presented as the mean ± SD (Two-tailed unpaired Student’s t-test). (e) Mouse small intestine, colon, pancreas, stomach, heart, liver and kidney were collected and fixed from control UTD T cell or the indicated VHH1-CART-treated NSG mice bearing NT-3 tumors at day 10 after the first T cell injection, followed by H&E staining. Experiments were performed two independent times with similar results. Scale Bar: 500 µM.

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Extended Data Fig. 8 Increased T cell infiltration into colon following acute DSS administration.

(a) Diagram of the DSS-induced colitis mouse model. Immunocompetent C57B6J mice were infused with murine VHH1-28BBz CARTs (mCART) one day after the leukapheresis with CTX (cyclophosphamide), followed by normal water or 3% DSS in drinking water. (b) Images of the colons of the murine VHH1-28BBz CART infused mice treated with water (left panel) or DSS (right panel) (n = 5 mice/group). (c) Colon lengths of the mice from (b) were measured (n = 5 mice/group). The data are presented as the mean ± SD (Two-tailed unpaired Student’s t-test; *** p < 0.0001). (d) IHC staining of the colons from water or DSS treated mice with anti-CD3 antibody. Experiments were performed two independent times with similar results. Scale Bar: 100 µM. (e) Counting of the infiltrated CD3+ T cells from (d) in the colons (n = 5 mice/group). The data are presented as the mean ± SD (Two-tailed unpaired Student’s t-test; *** p < 0.0001).

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Feng, Z., He, X., Zhang, X. et al. Potent suppression of neuroendocrine tumors and gastrointestinal cancers by CDH17CAR T cells without toxicity to normal tissues. Nat Cancer 3, 581–594 (2022). https://doi.org/10.1038/s43018-022-00344-7

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