Ex vivo expanded antigen-specific cytotoxic T lymphocytes (CTL) are able to kill both virally infected cells and tumor cells. Our preliminary data indicates that HLA-Ig complexes coupled to beads can induce and expand both CMV-specific and Mart-1 specific T cells. This raises the possibility that HLA-Ig can be used to replace standard dendritic cell based ex vivo expansion of antigen-specific CTL. Therefore the potential exists to use HLA-Ig complexes as a commercially viable method for induction, expansion and activation of targeted T cells for therapeutic use. Initially we propose to demonstrate functional efficacy of HLA-Ig complexes conjugated to beads as artificial Antigen Presenting Cells (aAPC) for inducing and expanding anti-CMV and anti-Mart-1 specific CTL.
Three specific aims are proposed. 1) We will optimize aAPC structure and duration of stimulation, T cell activation requires delivery of a combination of signals through the T cell receptor (Signal 1) and through costimulatory molecules (Signal 2) such as engagement of CD28 by B7. Using aAPC it is easy to vary the ratio of signal 1 to signal 2 as well as the type of signal 2 delivered. The efficacy of the various formulations will be determined by analyzing aAPC-mediated cell expansion, acquisition of in vitro effector functions (effector cytokine release and lysis of target cells), and analysis of off-rates and expression of homing receptors. 2) We will analyze the in vivo function of aAPC induced CTL. This will be studied in two models a human/SCID model and the murine A2-transgenic mice using adoptively transferred CTL. 3) We will analyze the ability to induce/expand antigen specific CTL from lysate containing a diverse repertoire of peptides. Experiments to study this will include """"""""spiking"""""""" experiments where a defined antigenic peptide is added to cell lysates. In addition the use of lysates of cells expressing the antigens endogenously pulsed on to aAPC to generate antigen specific CTL will be explored. Since adoptive immunotherapy will probably not consist of transfer of CTL with a single defined antigenic-specificity, these studies will explore the potential of using aAPC in a complex peptide mixture. These studies will further define the potential uses of HLA-Ig in replacing normal cellular dendritic cells (DC) and enhancing clinical approaches for adoptive immunotherapy.
