Our results have several implications for the generation, expansion, and therapeutic use of T lymphocytes genetically targeted to a defined antigen. Using highly efficient retroviral-mediated gene transfer [9
], we were able to investigate Pz-1 function in human primary CD4+
First, we show that T cells of cancer patients can be genetically directed to recognize and lyse tumor cells that express the cell-surface antigen PSMA. The demonstration that an antigen-specific ζ chain fusion receptor is functional in cancer patient lymphocytes is an important step toward the implementation of adoptive cell therapies based on the genetic alteration of autologous polyclonal PBL. Indeed, defective TCR signaling has been observed in chronic tumor bearing mice and cancer patients [10,11
]. However, we found that the Pz-1 receptor was functional in T cells in five of five cancer patients, including three with advanced disease. These results suggest that T cell activation defects are either uncommon or bypassed by genetically modifying T cells with γ chain fusion receptors. These results, therefore, alleviate our earlier concerns that findings in normal T cells might not extend to cancer patient cells.
All reports to date describing ζ chain fusions have focused on the antigen-specific killing imparted by the receptor (reviewed in Ref. [4
]). However, the ability of ζ chain receptors to provide antigen specific helper functions has not been examined. We show that Pz-1 specifically elicited cytokine release when culturing the transduced lymphocytes on PSMA+
murine fibroblasts. This result indicates that the Pz-1 receptor provided a sufficient signal to trigger T-cell activation. However, the cytokine secretion was relatively weak and not sustained (). We therefore investigated whether providing costimulation to the transduced lymphocytes would increase their cytokine response. Indeed, whereas ζ chain signaling provides a powerful activation signal after engagement of the physiologic TCR, full T-cell activation typically requires a costimulatory signal [12
]. Costimulation could act by amplifying the activation signal or offsetting putative proapoptotic signaling by the Pz-1 receptor.
On a practical level, our results also suggest a strategy to expand cells expressing an artificial TCR. In vitro
, fibroblast monolayers that express antigen and B7 are very effective as shown here. Different approaches can be envisaged to provide costimulation in vivo
. One is to express B7.1 in tumor cells [13
], providing that B7.1 can be expressed in a sufficient fraction of the tumor cells. Another is to provide antigen-dependent costimulation [5
] by expressing in the T cells an antigen-specific CD28 fusion receptor. In this manner, contact with PSMA+
cells would provide CD28 signaling in the absence of the B7.1 or B7.2 ligands on tumor cells. Dendritic cells, which constitutively express B7.1, do not express native PSMA and thus could not appropriately stimulate the transduced T cells. To do so, the dendritic cells would have to be genetically engineered to express the intact cell-surface molecule [14
Retroviral-mediated gene transfer is efficient in both CD4+
subsets of human T lymphocytes (Ref. [9
]; ). As the Pz-1 receptor signals for both the activation of cytolysis ( and ) and cytokine secretion (), it becomes envisagable to combine the adoptive transfer of PSMA-targeted CD4+
T cells. Cytokine release by the former should sustain the activity of the latter, in addition to exerting either proinflammatory and/or antitumoral functions. The expression of an artificial T-cell receptor that is not restricted by either MHC class I or class II creates the intriguing situation in which both CD4+
T lymphocytes could be activated side by side at the tumor site.
PSMA is expressed by normal prostate epithelial cells, and its expression is increased in prostate cancer, including metastatic and hormone-deprived cells. It is also expressed on the neovasculature of multiple carcinomas, the brush border of duodenal epithelium and salivary glands ([6–8
] Chang et al., manuscript submitted). Thus, Pz-1 may be useful to target T cells to local and metastatic prostate cancer and tumor neovasculature of other solid tumors. Expression on normal tissues does not necessarily represent an obstacle to the therapeutic use of monoclonal antibodies [15,16
]. Clinical studies are underway to define the biodistribution of the PSMA-specific J591 antibody in vivo