Mari Lowe Center for Comparative Oncology Research

Ex vivo expansion and genetic manipulation of T cells for adoptive immunotherapy of canine cancer

Adoptive immunotherapy (AI) using autologous, genetically modified T lymphocytes to recognize and kill malignant cells holds great promise for cancer therapy in humans and in dogs with spontaneously occurring cancer. Highly efficient retroviral gene transfer into peripheral T cells requires robust expansion of functional T cells ex vivo. Previously, non-physiologic methods that require exogenous T cell growth factors such as IL-2 to expand canine T cells ex-vivo have been reported as potential systems for AI. Using these methods, retroviral transduction frequencies of canine T cells have not exceeded 40%. Researchers in Dr. Mason’s laboratory at PennVet have developed an artificial antigen presenting cell (aAPC) that supports ex-vivo expansion and high frequency retroviral transduction of canine T cells, providing the first building blocks required to make AI for the treatment of canine cancer a reality. In this system, the human erythroleukemic K562 cell line has been stably transduced to express canine co-stimulatory molecules that are required for physiologic activation of T cells. White blood cells taken from small volumes of peripheral blood from healthy client-owned volunteer dogs can be expanded in vitro using this system, without the requirement for exogenous IL-2. Furthermore, these expanded cells can be stably transduced with retroviral vectors allowing the introduction of genetic constructs that optimize the T cells ability to recognize and kill transformed cells. One strategy used to circumvent immune evasion by neoplastic cells is to genetically modify T cells to express chimeric immunoreceptors (CIRs) that consist of a cell surface expressed single chain Fv (scFv), composed of the VH and VL domains of an antigen-specific immunoglobulin, that is linked in turn to a flexible hinge, transmembrane domain and intracellular signaling domains that lie within the cytosolic tail of the CIR (Fig.2). These surrogate CIRs can bestow primary T cells with MHC independent antigen recognition capabilities and antigen-dependent T cell activation, cytokine production and cytotoxic killing in the absence of co-stimulatory ligands. Adoptive transfer of ex-vivo expanded, tumor-specific re-directed T cells have shown promising anti-tumor effects in phase I clinical trials in human oncology patients. Thus, with the design of optimal CIR for the canine T cells, the ability to achieve high frequency gene transfer into peripheral dog T cells and refined ex-vivo canine T cell expansion techniques large numbers of autologous re-directed tumor-specific T cells can be produced, offering exciting possibilities for the treatment of primary and metastatic neoplasia in the dog. This project is currently funded through a University of Pennsylvania’s Research Foundation Grant.