Promising results have been obtained by cell-mediated immunotherapy but several obstacles remain to improve the success rate. Among these challenges, tumor cells can escape T cell-mediated killing by expressing proteins that inactivate the T-cells. Several receptor/ligand pairs have been identified to mediate these pathways including PD-1/PD-L1 or, more recently, CD200R/CD200. Fusion proteins combining the extracellular portion of an immunosuppressive receptor with a stimulatory signaling domain of different origin can potentially be employed as a novel approach to maintain T cell activation despite inhibitory ligand expression.
One such strategy was recently described in the journal Blood. Dr. Shannon Oda, a research associate in the group of Dr. Philip Greenberg (Clinical Research Division) reports the development of a new CD200R-based chimeric protein to improve in vivo anti-tumoral T cell function against acute myeloid leukemia (AML). CD200 protein expression is abnormally increased in AML and other cancer cells and has been associated with poor prognoses. CD200 engages its receptor, CD200R, on the T cell surface and thereby prevents T cell activation. To maintain T cell stimulation following this interaction, the authors created a chimeric protein combining the extracellular domain of CD200R with the intracellular co-stimulatory signaling domain of CD28. The new chimeric protein was called CD200R-CD28 IFP (immunomodulatory fusion protein).
Dr. Oda explained: “We are treating patients by adoptive immunotherapy with T cells engineered to express a cancer-targeting T cell receptor (TCR). Such T cells can encounter inhibitory signals in the tumor microenvironment as well as diminished costimulatory signals, and both can interfere with the activation of T cells specifically recognizing cancer. Our colleagues in the chimeric antigen receptor field demonstrated that adding synthetic costimulation to T cells was requisite for efficacy, and we are excited to have developed a new synthetic costimulatory receptor that further improves anti-cancer function by targeting a second tumor-associated inhibitory molecule”.
Several forms of CD200R-CD28 were cloned and tested in vitro following retroviral transduction of mouse T lymphocytes. The optimal construct was selected to continue the study based on expression level as well as T cell stimulation and proliferation in a CD200-dependent manner. The function of the modified CD200R-CD28-expressing T cells was evaluated by quantifying their cytolytic activity, release of the cytokines interferon gamma, interleukin-2, and tumor necrosis factor alpha. Dr. Oda also used a new assay she developed to document CD28 signaling by measuring phosphorylation of residue Y394. CD200R-CD28 expressing T cells were specifically activated in the presence of target leukemic cells expressing the CD200 ligand unlike control T cells.
The modified T cells were used to treat immunocompetent mice with disseminated leukemia. Impressively, mouse survival was significantly improved to 89% when treated with CD200R-CD28 modified T cells relative to 40% survival with control T cells. Overall, these experiments demonstrate that the CD200R-CD28-expressing T cells are indeed activated and their ability to block CD200+ tumor cells progression is improved.
Finally, the authors evaluated CD200R-CD28 in human T cells for future clinical trials. CD200 expression was not detected in healthy donors but significantly increased in human primary AML cells. The human version of the CD200R-CD28 IFP was generated and tested in vitro in cells expressing an anti-WT1 TCR already being evaluated in clinical trials for AML patients. The CD200R-CD28 expressing T cells proliferated in the presence of CD200+ and WT1+ tumor cells, and showed increased lysis of primary AML cells.
This study paves the way for future clinical trials. Dr. Oda concluded: “we are expanding our initial findings to engineer new IFPs to target other inhibitory molecules on cancer cells and creating fusions with different costimulatory signaling domains. We are examining how IFPs can impact the persistence, function, metabolism, serial killing ability and trafficking of T cells, as well as the impact on immunotherapy of liquid and solid tumors. The latest IFP we have engineered combines a death receptor ectodomain, Fas, with the pro-survival costimulatory 4-1BB signaling domain, converting a death signal to an activation boost for T cells. We have preliminary data with the Fas-41BB IFP supporting improved survival in murine models of AML, ovarian cancer (in collaboration with a postdoctoral fellow in the lab, Kristin Anderson) and pancreatic cancer. We are excited to determine the tumor microenvironment obstacles that IFPs can overcome and extend these findings to create effective tumor immunotherapies”.
Funding for this study was provided by the National Institutes of Health/National Cancer Institute, the Leukemia & Lymphoma society and Juno Therapeutics.
Oda SK,Daman AW,Garcia NM,Wagener F,Schmitt TM,Tan X,Chapuis AG,Greenberg PD. 2017. A CD200R-CD28 fusion protein appropriates an inhibitory signal to enhance T cell function and therapy of murine leukemia. Blood.