Abstract
CAR-T (chimeric antigen receptor T cells) immunotherapy is effective in many hematological cancers; however, efficacy in solid tumors is disappointing. Doublecortin-like kinase 1 (DCLK1) labels tumor stem cells (TSCs) in genetic mouse models of colorectal cancer (CRC). Here, we describe a novel CAR-T targeting DCLK1 (CBT-511; with our proprietary DCLK1 single-chain antibody variable fragment) as a treatment strategy to eradicate CRC TSCs. The cell surface expression of DCLK1 and cytotoxicity of CBT-511 were assessed in CRC cells (HT29, HCT116, and LoVo). LoVo-derived tumor xenografts in NOD Scid gamma (NSG™) mice were treated with CBT-511 or mock CAR-T cells. Adherent CRC cells express surface DCLK1 (two-dimensional, 2D). A 4.5-fold increase in surface DCLK1 was observed when HT29 cells were grown as spheroids (three-dimensional, 3D). CBT-511 induced cytotoxicity (2D; p < 0.0001), and increased Interferon gamma (IFN-γ) release in CRC cells (2D) compared to mock CAR-T (p < 0.0001). Moreover, an even greater increase in IFN-γ release was observed when cells were grown in 3D. CBT-511 reduced tumor growth by approximately 50 percent compared to mock CAR-T. These data suggest that CRC cells with increased clonogenic capacity express increased surface DCLK1. A DCLK1-targeted CAR-T can induce cytotoxicity in vitro and inhibit xenograft growth in vivo.
Highlights
Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in the U.S [1].For individuals diagnosed with stage IV of the disease, the five-year survival rate is just 12% [1].A growing body of evidence suggests that cancer stem cells (CSCs) play a critical role in the initiation, progression, and metastatic spread of cancer [2,3]
We demonstrate that HT29 cells grown in three-dimensional (3D) matrices exhibit a 4.5-fold increase in cell surface Doublecortin-like kinase 1 (DCLK1) expression compared to cells grown in two-dimensional (2D)
We previously reported that targeting renal cell carcinoma tumor stem cells (TSCs) with CBT-15 resulted in xenograft growth arrest and epithelial-mesenchymal transition (EMT) inhibition [47]
Summary
Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in the U.S [1].For individuals diagnosed with stage IV of the disease, the five-year survival rate is just 12% [1].A growing body of evidence suggests that cancer stem cells (CSCs) play a critical role in the initiation, progression, and metastatic spread of cancer [2,3]. Immunotherapy has been remarkably successful for treating solid tumor cancers due to its unique ability to inhibit immune system checkpoint proteins (e.g., PD-1, PD-L1, CTLA4) and reactivate the host’s anti-tumor immunity [6]. These checkpoint inhibitors enable cytotoxic CD8+ T cells to attack and destroy cancer cells [6]. Infiltrating T lymphocytes that invade the TME and exert cytotoxic effects within the tumor have been shown to correlate with increased efficacy and improved survival rate in several cancers [8,9]. The efficacy again varies greatly between patient populations and the type of tumor being targeted
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