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. 2013 Feb 21:11:46.
doi: 10.1186/1479-5876-11-46.

Anti-KIT designer T cells for the treatment of gastrointestinal stromal tumor

Steven C Katz  1 , Rachel A Burga, Seema Naheed, Lauren A Licata, Mitchell Thorn, Doreen Osgood, Cang T Nguyen, N Joseph Espat, Jonathan A Fletcher, Richard P Junghans
Affiliations

Anti-KIT designer T cells for the treatment of gastrointestinal stromal tumor

Steven C Katz et al. J Transl Med. .

Abstract

Background: Imatinib mesylate is an effective treatment for metastatic gastrointestinal stromal tumor (GIST). However, most patients eventually develop resistance and there are few other treatment options VSports手机版. Immunotherapy using genetically modified or designer T cells (dTc) has gained increased attention for several malignancies in recent years. The aims of this study were to develop and test novel anti-KIT dTc engineered to target GIST cells. .

Methods: Human anti-KIT dTc were created by retroviral transduction with novel chimeric immune receptors (CIR). The gene for stem cell factor (SCF), the natural ligand for KIT, was cloned into 1st generation (SCF-CD3ζ, 1st gen) and 2nd generation (SCF-CD28-CD3ζ, 2nd gen) CIR constructs. In vitro dTc proliferation and tumoricidal capacity in the presence of KIT+ tumor cells were measured. In vivo assessment of dTc anti-tumor efficacy was performed by treating immunodeficient mice harboring subcutaneous GIST xenografts with dTc tail vein infusions. V体育安卓版.

Results: We successfully produced the 1st and 2nd gen anti-KIT CIR and transduced murine and human T cells. Average transduction efficiencies for human 1st and 2nd gen dTc were 50% and 42%. When co-cultured with KIT+ tumor cells, both 1st and 2nd gen dTc proliferated and produced IFNγ. Human anti-KIT dTc were efficient at lysing GIST in vitro compared to untransduced T cells. In mice with established GIST xenografts, treatment with either 1st or 2nd gen human anti-KIT dTc led to significant reductions in tumor growth rates. V体育ios版.

Conclusions: We have constructed a novel anti-KIT CIR for production of dTc that possess specific activity against KIT+ GIST in vitro and in vivo VSports最新版本. Further studies are warranted to evaluate the therapeutic potential and safety of anti-KIT dTc. .

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Figures

Figure 1
Figure 1
Structure of anti-KIT chimeric immune receptor. (A) Schematic diagram of 1st and 2nd generation CIR genetic constructs. (B) Structure of 1st and 2nd generation CIR. Anti-KIT CIRs were re-engineered from anti-CEA retroviral vector constructs, whereby the extracellular domain of kit ligand (KL) was amplified and cloned to replace the anti-CEA extracellular domain.
Figure 2
Figure 2
Transduction efficiency and phenotype of human anti-KIT designer T cells (dTc). (A) PBMC were isolated and primary human T cells activated and transduced with retrovirus expressing KIT-specific CIRs. Shaded histograms represent dTc and open histograms represent untransduced cells. (B) Flow cytometric analysis of the phenotype of 1st and 2nd generation dTc demonstrated that T cells (CD4+ and CD8+ T cells) of a central memory phenotype (CD45RO+CD62L+CCR7+) were in the majority. Naïve T cells were defined as (CD45RO-CD62L+). Data are representative of three or more repetitions.
Figure 3
Figure 3
Human anti-KIT dTc retain proliferative ability in vitro. 1st and 2nd gen dTc were stimulated with human KIT+ GIST cell lines, GIST 882 and GIST 48. T cells were stained with CFSE and proliferation assessed by gating on CD3+ cells to measure CFSE dilution (A-C) and KIT- cells were used as a control. CIR- T cells did not proliferate in the presence of KIT+ tumor (not shown). (D) To confirm dTc activation by KIT+ GIST cell lines, IFNγ production was measured by ELISA, and was found to be in the range of 462-475 pg/ml, while that of untransduced CIR- T cells (CTRL) was negligible. Data are representative of three or more repetitions.
Figure 4
Figure 4
Ability of human anti-KIT dTc to effectively lyse KIT+ tumor cells. (A) LDH assay to evaluate enzymatic release following tumor cell lysis was performed on supernatant from co-culturing of 1st and 2nd generation dTc with irradiated GIST 882 cells. Maximal release was defined by the highest experimental values. (B-C) To confirm the cytotoxic ability of dTc, irradiated GIST 882 or GIST 48 cells were labeled with CFSE and cultured with unlabelled T cells. Tumor cell death was quantified by measuring the decrease in CFSE fluorescence by gating on remaining live cells. Data are representative of three or more repetitions.
Figure 5
Figure 5
In vivo testing of human anti-KIT designer T cells. We used a subcutaneous xenograft model whereby GIST 882 cells were injected subcutaneously into immunodeficient mice prior to treatment with human anti-KIT dTc. Median results from a single representative experiment (A) and pooled data from 3 experiments (B) are shown. Activated CIR- T cells were used as negative controls. (C) Median tumor sizes at Day 5, Day 10, and Day 15 post-infusion are indicated by horizontal bars along with individual tumor measurements represented by the data points. (D) We performed immunohistochemistry (top row 10x, bottom row 40x) to detect tumor infiltrating CD3+ T cells (arrows). (E) Routine H&E was used to assess the degree of tumor necrosis (asterisks) in mice treated with 1st or 2nd gen dTc, both with and without supplemental IL2 (left column 10x, right column 40x).
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