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269 CAR T cell production expands endogenous T cell repertoire recognizing additional tumor antigen(s)
  1. Alicia Hou1,
  2. Linchun Jin2,
  3. Haipeng Tao1,
  4. Duane A Mitchell1 and
  5. Jianping Huang1
  1. 1University of Florida, Gainesville, FL, USA
  2. 2Zhejiang University, Hangzhou, Zhejiang Province, 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 Glioblastoma (GBM) is an aggressive and heterogeneous brain tumor that currently has no curative treatment. Our group identified CD70, which is overexpressed by low-/high-grade gliomas and associated with poor patient survival.1 2 We established a CAR-T cell therapy platform targeting CD70-expressing gliomas, which showed efficient antitumor activity preclinically. Modification of CD70CAR to express IL-8 receptors (8R-70CAR) enhanced tumor trafficking and persistence, resulting in improved antitumor efficacy and long-lasting immunity to tumor rechallenge.3 These findings culminated in a phase-I trial of 8R-70CAR T cells (NCT05353530) for patients with newly diagnosed GBM that will begin at UF soon. However, CAR T cell therapy currently faces obstacles such as single antigen targeting in heterogeneous tumors and maintenance of activation of CAR T cells. Cytomegalovirus pp65 protein is a tumor-specific target in GBM.4 5 A combinatorial approach against CD70 and pp65 may result in enhanced effect against heterogeneous GBM.

Methods Healthy donor peripheral blood mononuclear cells were stimulated with anti-CD3/CD28 Dynabeads and retrovirally transduced with 8R-70CAR. Frequencies of 8R-70CAR- and pp65-specific T cells were evaluated by flow cytometry.

Results The pp65-specific CD8+ T cell population was expanded post DynaBeads activation to a frequency tenfold over baseline (figure 1). Single-specific (CD70 CAR+ only and pp65-specific only) and double-specific (CD70 CAR+ and pp65-specific) T cells were successfully generated. Importantly, these cells secreted greater IFN-g when tumor cells expressed both targets (figure 2).

Conclusions The CAR T cell production process significantly expands pp65-specific T cells, resulting in enhanced antitumor efficacy through recognition of both tumor targets. Our CAR T cell product may contain T cell repertoires that effectively target multiple tumor antigens.


  1. Wang QJ, Yu Z, Hanada K-i, et al. Pre-clinical Evaluation of Chimeric Antigen Receptors Targeting CD70-Expressing Cancers. Clin Cancer Res. 2016;23(9):2267–2276

  2. Ge H, Mu L, Jin L, et al. Tumor associated CD70 expression is involved in promoting tumor migration and macrophage infiltration in GBM. Int J Cancer. 2017;141(7):1434–44.

  3. Jin L, Tao H, Karachi A, et al. CXCR1- or CXCR2-modified CAR T cells co-opt IL-8 for maximal antitumor efficacy in solid tumors. Nat Commun. 2019;10(1):4016.

  4. Scheurer ME, Bondy ML, Aldape KD, et al. Detection of human cytomegalovirus in different histological types of gliomas. Acta Neuropathol. 2008;116(1):79–86.

  5. Schuessler A, Smith C, Beagley L, et al. Autologous T-cell therapy for cytomegalovirus as a consolidative treatment for recurrent glioblastoma. Cancer Res. 2014;74(13):3466–76.

Abstract 269 Figure 1

pp65-specific T cell frequency was assessed via flow cytometry using HLA-matched pp65 tetramer at baseline (’Non-Activated’) and after CAR T cell production (‘Activated/CAR-transduced’). These are representative flow cytometry data for three healthy donors (HD1–3).

Abstract 269 Figure 2

IFN-y-secreting cells as percentages of the total CD3+ populations after tumor/T-cell coculture. U87 (CD70+/pp65-), U87/pp65 (CD70+/pp65+), and L0 (CD70-/pp65-) GBM lines were used as target cells for coculture with CAR- and vector-transduced T cells. The U87/pp65 line is U87 transduced to overexpress pp65. Cytostim was used as positive control.

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