Cancer patient T cells genetically targeted to prostate-specific membrane antigen specifically lyse prostate cancer cells and release cytokines in response to prostate-specific membrane antigen

Abstract

The expression of immunoglobulin-based artificial receptors in normal T lymphocytes provides a means to target lymphocytes to cell surface antigens independently of major histocompatibility complex restriction. Such artificial receptors have been previously shown to confer antigen-specific tumoricidal properties in murine T cells. We constructed a novel zeta chain fusion receptor specific for prostate-specific membrane antigen (PSMA) termed Pz-1. PSMA is a cell-surface glycoprotein expressed on prostate cancer cells and the neovascular endothelium of multiple carcinomas. We show that primary T cells harvested from five of five patients with different stages of prostate cancer and transduced with the Pz-1 receptor readily lyse prostate cancer cells. Having established a culture system using fibroblasts that express PSMA, we next show that T cells expressing the Pz-1 receptor release cytokines in response to cell-bound PSMA. Furthermore, we show that the cytokine release is greatly augmented by B7.1-mediated costimulation. Thus, our findings support the feasibility of adoptive cell therapy by using genetically engineered T cells in prostate cancer patients and suggest that both CD4+ and CD8+ T lymphocyte functions can be synergistically targeted against tumor cells.

Figure 1

Pz-1 vector structure and expression in primary human T cells. (A) Schematic diagram of the Pz-1 retroviral vector (Gong et al., manuscript in preparation). J591-scFv: PSMA-specific scFv derived from the monoclonal antibody (mAb) J591 [8]; CD8: CD8 hinge and transmembrane domains; zeta: T-cell receptor ζ chain cytoplasmic domain; LTR: long terminal repeat; hatched box: CD8 leader sequence; arrows indicate start of transcription. (B) FACS (Becton Dickinson & Co) analysis of transduced primary T cells. PBL harvested from cancer patients were transduced as previously described [9] and stained 3 days after retroviral infection to enumerate the fraction of Pz-1⁺ CD4⁺ and Pz-1⁺ CD8⁺ lymphocytes. Lymphocytes were incubated with an FITC-conjugated idiotype-specific antiserum, which recognizes the J591-derived scFv. After washing and incubation with 10% normal mouse serum, lymphocytes were stained with a PE-conjugated anti-CD8 mAb. Y axis: CD8^PE, X axis: Pz-1 signal. Left panel: Mock transduction; right panel: Pz-1 transduction. The percentage of cells present in each quadrant is indicated.

Figure 2

Pz-1-transduced prostate cancer patient T lymphocytes lyse LNCaP cells. (A) LNCaP cell expression of PSMA. FACS (Becton Dickinson & Co) analysis of wild type LNCaP cells. Cells were stained with mAb J591 followed by FITC-conjugated goat anti-mouse (GAM-FITC) antibody. Y axis: cell count, X axis: fluorescence (515 nm). PSMA: PSMA signal, GAM^F: GAM-FITC antibody alone signal. (B) Pz-1 directed cytotoxicity against LNCaP cells. PBL from three patients (Tables 1 and 2) were transduced with either Pz-1 or NTP [9]. Transduced PBL were used in CTL assays with LNCaP target cells. Percentage of specific lysis is represented on the Y axis, effector to target ratio on the X axis.

Figure 3

Pz-1-transduced prostate cancer patient T lymphocytes specifically lyse target cells expressing PSMA. (A) Left panel: FACS (Becton Dickinson & Co) analysis of wild type PC-3 cells (PSMA^-) and PSMA-transduced PC-3 (PSMA⁺); PSMA^- cells (dotted line) coincide with PC-3 cells stained with the secondary GAM-FITC antibody alone (data not shown). Cells were stained with mAb J591 [8], followed by GAM-FITC antibody. Y axis: cell count; X axis: fluorescence(515 nm). Right panel: FACS (Becton Dickinson & Co) analysis of wild type EL-4 murine thymoma cells (PSMA^-) and PSMA-transduced EL-4 cells (PSMA⁺). (B) Pz-1-transduced PBL specifically lyse PSMA⁺ target cells. PBL from three patients(Tables 1 and 2) were transduced with either Pz-1 or NTP [9]. Percentage of specific lysis is represented on the Y axis, effector to target ratio on the X axis. Targets are PC3-PSMA⁺ and PC3-PSMA^- (wild type), as described in (A, left panel). (C) Pz-1-transduced PBL specifically lyse PSMAq EL-4 cells. Pz-1q-transduced PBL of all patients effectively lysed PSMA⁺ targets. Background activity was variable on human target cells and uniformly low on murine target cells.

Figure 4

NIH3T3 fibroblast feeder cells coexpressing PSMA and B7.1 (CD80) direct cytokine release by Pz-1-transduced T cells. (A) Transduced NIH3T3 feeder cells were stained with mAb J591 as described in Figure 2 or with FITC-labeled mAb BB1 specific for B7.1. Y axis: cell count; X axis: PSMA or B7.1 signal. GAM^F: GAM-FITC antibody alone. PSMA and B7.1 indicate the fluorescence obtained for each stain. (B) PSMA induced IL-2 secretion by Pz-1⁺ T cells. Pz-1-transduced T cells were cocultured with NIH3T3 fibroblast feeder cells. Supernatants collected 24, 48, and 72 hours after the start of the coculture were assayed for IL- 2 by ELISA. B7 + PSMA, PSMA, B7 refer to the molecules expressed by the different fibroblast monolayers. Results are from quadruplicate experiments. Error bars represent 2 standard deviations.