Preclinical Assessment of CAR T-Cell Therapy Targeting the Tumor Antigen 5T4 in Ovarian Cancer

Abstract

Chimeric antigen receptor (CAR) T cells represent a novel targeted approach to overcome both quantitative and qualitative shortfalls of the host immune system relating to the detection and subsequent destruction of tumors. The identification of antigens expressed specifically on the surface of tumor cells is a critical first step in the ability to utilize CAR T cells for the treatment of cancer. The 5T4 is a tumor-associated antigen which is expressed on the cell surface of most solid tumors including ovarian cancer. Matched blood and tumor samples were collected from 12 patients with ovarian cancer; all tumors were positive for 5T4 expression by immunohistochemistry. Patient T cells were effectively transduced with 2 different anti-5T4 CAR constructs which differed in their affinity for the target antigen. Co-culture of CAR T cells with matched autologous tumor disaggregates resulted in antigen-specific secretion of IFN-gamma. Furthermore, assessment of the efficacy of anti-5T4 CAR T cells in a mouse model resulted in therapeutic benefit against established ovarian tumors. These results demonstrate proof of principle that 5T4 is an attractive target for immune intervention in ovarian cancer and that patient T cells engineered to express a 5T4-specific CAR can recognize and respond physiologically to autologous tumor cells.

FIGURE 1

5T4 CAR construct and transduction efficiency. A, Anti-5T4 CAR construct shown in the integrated form. B, Percentage of CD3 T cells from healthy donors and patients transduced with H8-CAR and 2E4-CAR. C, Percentage of patient-derived and healthy donor-derived CD4 and CD8 T cells transduced with H8-CAR and 2E4-CAR. The Student t test, *P<0.05; **P<0.01; ***P<0.001. CAR indicates chimeric antigen receptor, LTR, long terminal repeat; Neo, Neomycint; NS, not significant; SIN, self-inactivating; WPRE, Woodchuck Hepatitis Virus posttranscriptional regulatory element.

FIGURE 2

5T4 expression on FFPE sections of tumor biopsies from patients with ovarian cancer. A, Light microscopy (×20 magnification) of FFPE sections after staining with a mouse anti-human 5T4 monoclonal antibody and hematoxylin. B, Percentage of EpCAM+ 5T4+ cells in the tumor disaggregates as determined by flow cytometry. C, Percentage of EpCAM positive tumor cells expressing 5T4. D, 5T4 expression on tumor samples as determined by H-score and mean fluorescence intensity (MFI). FFPE indicates formalin-fixed and paraffin-embedded.

FIGURE 3

IFNγ production by 5T4 CAR T cells in response to immortalized ovarian cell lines expressing 5T4 and autologous tumor cells. Peripheral T cells were successfully transduced from 11 patients. T cells were transduced with the H8-CAR or 2E4-CAR or no CAR (Mock) vector. T cells (1×10⁵) were co-cultured for 24 hours with 1×10⁵ SKOV-3, OVCAR-3 (A) and primary autologous tumor cells (B). After 24 hours, supernatant was collected and IFNγ quantitified by enzyme-linked immunosorbent assay. Error bars represent the mean and SD of triplicate results. Two-way analysis of variance with Sidak’s correction; ^*P<0.05, ^**P<0.01, ^***P<0.001. CAR indicates chimeric antigen receptor; INFγ, interferon-γ; NS, not significant.

FIGURE 3

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FIGURE 4

Dose escalation of 5T4 CAR T cells in NSG ovarian cancer model. NSG mice were challenged with 2.5×10⁶ SKOV-3 tumor cells on day 0 and 7 days later were treated with either ascending doses of H8-CAR T cells or saline. A, In-life bioluminescence images of NSG mice treated with ascending doses of H8-CAR T cells are shown over time alongside control animals. B, Kaplan-Meier survival curves of NSG mice receiving ascending doses of H8-CAR T cells. CAR indicates chimeric antigen receptor.

FIGURE 5

Comparison of higher versus lower affinity CAR constructs. NSG mice were inoculated with ovarian cancer cell lines on day 0. Seven days later mice were treated with either the higher affinity H8-CAR or the lower affinity 2E4-CAR T cells. Kaplan-Meier survival curves of NSG mice bearing SKOV-3 tumors and treated with 1×10⁷ CAR T 5T4 cells. Log-rank (Mantel-Cox) test; *P<0.05, **P<0.01 compared with Mock T cells, ##P<0.01 compared with 2E4-CAR T cells. CAR indicates chimeric antigen receptor.