CAR T cells targeting tumor endothelial marker CLEC14A inhibit tumor growth
Xiaodong Zhuang, … , Roy Bicknell, Steven P. Lee
Xiaodong Zhuang, … , Roy Bicknell, Steven P. Lee
Published October 2, 2020
Citation Information: JCI Insight. 2020;5(19):e138808. https://doi.org/10.1172/jci.insight.138808.
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Research Article Immunology Oncology

CAR T cells targeting tumor endothelial marker CLEC14A inhibit tumor growth

Abstract

Engineering T cells to express chimeric antigen receptors (CARs) specific for antigens on hematological cancers has yielded remarkable clinical responses, but with solid tumors, benefit has been more limited. This may reflect lack of suitable target antigens, immune evasion mechanisms in malignant cells, and/or lack of T cell infiltration into tumors. An alternative approach, to circumvent these problems, is targeting the tumor vasculature rather than the malignant cells directly. CLEC14A is a glycoprotein selectively overexpressed on the vasculature of many solid human cancers and is, therefore, of considerable interest as a target antigen. Here, we generated CARs from 2 CLEC14A-specific antibodies and expressed them in T cells. In vitro studies demonstrated that, when exposed to their target antigen, these engineered T cells proliferate, release IFN-γ, and mediate cytotoxicity. Infusing CAR engineered T cells into healthy mice showed no signs of toxicity, yet these T cells targeted tumor tissue and significantly inhibited tumor growth in 3 mouse models of cancer (Rip-Tag2, mPDAC, and Lewis lung carcinoma). Reduced tumor burden also correlated with significant loss of CLEC14A expression and reduced vascular density within malignant tissues. These data suggest the tumor vasculature can be safely and effectively targeted with CLEC14A-specific CAR T cells, offering a potent and widely applicable therapy for cancer.

Authors

Xiaodong Zhuang, Federica Maione, Joseph Robinson, Michael Bentley, Baksho Kaul, Katharine Whitworth, Neeraj Jumbu, Elizabeth Jinks, Jonas Bystrom, Pietro Gabriele, Elisabetta Garibaldi, Elena Delmastro, Zsuzsanna Nagy, David Gilham, Enrico Giraudo, Roy Bicknell, Steven P. Lee

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Figure 1

CLEC14A-specific CAR design, expression, and function.

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CLEC14A-specific CAR design, expression, and function. (A) Schematic rep...
(A) Schematic representation of a recombinant retroviral vector encoding CLEC14A-specific CARs. Retroviral CAR vector (pMP71) coexpresses a truncated CD34 marker gene and an scFv fragment/CD3ζ chain chimeric receptor with a CD28 costimulatory domain. Expression is driven from the LTR promoter, and the 2A peptide linker ensures equimolar expression of both molecules. (B) Recombinant retroviral expression vectors encoding CARs CAR3.28z and CAR5.28z (with scFv fragments from CLEC14A-specific monoclonal antibodies CRT3 or CRT5, respectively) were used to transduce human T cells. CAR expression was detected by staining for the CD34 marker. Percentage values show proportion of cells stained for CD34 compared with mock-transduced T cells. (C) Transduced T cells stained for expression of CAR using CLEC14A-Fc (percentage values show specific binding of CLEC14A-Fc having subtracted background staining with Fc alone). (D–F) Human T cells engineered to express putative CLEC14A-specific CARs (or mock-transduced T cell controls) were tested using an ELISA for IFN-γ production for response to plate-bound recombinant CLEC14A-Fc (extracellular domain) fusion protein (or Fc alone) (D), CHO cells engineered to express full-length human CLEC14A (or CHO transduced with vector alone) (responder/stimulator [R/S] ratio = 6:1) (E), and HUVECs naturally expressing CLEC14A (or medium alone). (R/S ratio = 1:1) (F). In all cases, the different CAR T cell lines were diluted with mock T cells to equalize for transduction efficiency. Cells were stimulated for 18 hours before testing for IFN-γ production. Results of ELISAs show data from 6–7 repeat experiments, having subtracted background responses of T cells alone. All P values shown were calculated using a Wilcoxon matched-pairs signed rank test.