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331 Functional enhancement of T cells by the co-expression of IL-7 and dominant-negative TGFβRII
  1. Zhihong Huang1,
  2. Mingyu Liu1,
  3. Deping Han1,
  4. Jean Paul Thiery1,2 and
  5. Xi Zhang1
  1. 1Biosyngen Pte Ltd, Singapore, Singapore
  2. 2Guangzhou Laboratory, Bioisland, Guangzhou, China
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background The effect of transforming growth factor β (TGF-β) is cell type- and context-dependent. In the tumor microenvironment, TGF-β restricts T cells’ expansion, survival, and antitumor efficacy.1 2 Blocking of the TGF-β signaling enhanced the infiltration and antitumor responses of chimeric antigen receptor-T (CAR-T) cells and improved the cytotoxicity of tumor-infiltrating lymphocytes.3 4 However, TGF-β signaling is also required for IL-7Rα expression and the abrogation of TGF-β receptor expression led to the failed maintenance of peripheral CD4+ T cells,5 since IL-7 signaling is essential for homeostasis, persistence, and survival of naïve and memory T cells.6 7 Therefore we tested whether co-expression of IL-7 and dominant-negative TGFβRII (dnTGFβRII) could enhance the proliferation and antitumor efficacy of CAR-T cells and TILs isolated from primary hepatocellular carcinoma (HCC) tissues.

Methods T cells isolated from peripheral blood mononuclear cells or primary resected HCC tissues were activated by CD3/CD28 stimulation, lentivirally transduced with IL-7 and dnTGFβRII-expressing cassettes, and expanded ex vivo. For CAR-T cell preparation, anti-CD133 CAR was additionally transduced. Transgene expression was assessed by flow cytometry. In vitro, antitumor efficacy was analyzed by IFN-γ ELISA and real-time quantitative cytotoxicity assay with an IncuCyte instrument. The antitumor efficacy of the engineered T cells was further evaluated in a xenograft immunocompromised mouse model.

Results The expression of dnTGFβRII was analyzed with flow cytometry, and ELISA confirmed the secretion of IL-7 in TILs and anti-CD133 CAR-T cells (figure 1A-C). The genetically modified TILs revealed an increased activation level compared to unmodified TILs and the dnTGFβRII-expressing TILs as determined by IFN-γ ELISA (figure 1D). These engineered TILs also showed more potent and sustained killing efficacy against HCC cell line SK-Hep1 in the presence of TGF-β (figure 1E) in vitro and significantly higher tumor control efficacy in vivo (figure 1G). Similarly, IL-7 and dnTGFβRII co-expressing CAR-T cells also exhibited increased cytotoxicity against colorectal cancer cells HCT116 and HT-29 in the presence of TGF-β (figure 1F) in vitro and higher tumor control efficacy in vivo (figure 1H). Interestingly, a membrane-bound fusion protein, IL7:dnTGFβRII, linking IL-7 to the N terminus of dnTGFβRII further improved CAR-T cell proliferation when co-cultured with HT-29 cells. This finding prompts further investigation on this novel chimeric construct.

Conclusions A robust co-expression of IL-7 and dnTGFβRII on both TILs and CAR-T cells has been achieved. The combination of IL-7 signaling enhancement and TGF-β inhibition can synergistically improve T cells’ anti-tumor efficacy and their resistance to immunosuppression.


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Ethics Approval All animal studies were conducted at the Ruiye model animal (Guangzhou) Biotechnology Co., Ltd facility as approved by its Experimental Animal Care and Use Committee (approval No. RYEth20220516–1).

Abstract 331 Figure 1

Co-expression of IL-7 and dnTGFßRII enhanced the antitumor activity of TILs and CAR-T cells. (A) Schematic diagrams of transgene cassettes in lentiviral vectors. (B) Flow cytometry analysis of the surface expression of TGFßRII on TILs transduced by lentiviral vectors with a dnTGFßRII-expressing cassette or with an IL-7 and dnTGFßRII-expressing cassette. (C) IL-7 secretion. Control and engineered TILs were cultured at a density of 1 x 106 cells/ml for 96 h and the level of IL-7 released in the medium was measured by ELISA. (D) IFN-y release. Control and engineered TILs were co-cultured with SK-Hep1 cells for 16 h at an E:T of 1:1 in the presence of 10 ng/ml TGF-ß. The level of IFN-y released in the medium was measured by ELISA. (E) IncuCyte real-time quantitative cytotoxicity assay. Control and engineered TILs were co-cultured with mCherry-expressing SK-Hep1 cells at an initial E:T ratio of in the presence of 10 ng/ml TGF-ß. Arrows, time- points of target cell addition for repetitive stimulation of TILs. (F) IncuCyte real-time quantitative cytotoxicity assay. Control and enhanced CAR-T cells were co-cultured with mCherry-expressing HCT 116 or HT-29 cells at an initial E:T ratio of 2:1 in the presence of 10 ng/ml TGF-ß. Arrows, time-points Of target cell addition for repetitive stimulation of T cells. (G) In vivo antitumor efficacy engineered TILs. SK-Hep1 cells were subcutaneously inoculated in NOG immunocompromised mice, and TILs were intravenously infused. (H) In vivo antitumor efficacy enhanced CAR-T cells. SW620 cells were subcutaneously inoculated in NOG mice, and T cells were intravenously infused.

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