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Abstract
Background Glioblastoma multiforme (GBM) is the major malignant primary tumor of brain with extremely poor prognosis and the most undifferentiated type of tumor. Median survival of GBM patients is just a year from diagnosis. Patients with GBM have limited natural cytotoxicity and inactivity of their natural killer (NK) cells, which correlate with the tumor progression. NK cells are equipped with chemokine receptors that promote their infiltration into solid tissues, including the solid tumors, therefore making them attractive cell-therapy agents. CD276 (B7-H3) is a transmembrane immune checkpoint protein which is widely expressed in GBM tumors, however limited expression is found in normal tissues.
Methods In our efforts to develop an off-the-shelf and allogeneic immunotherapy product for human GBM, we have generated CD276 CAR-NK cells from peripheral blood (PB) of healthy donors (figure 1). We have selected high-affinity human CD276 binders by screening in-house yeast and phase binder libraries for the relevant single-chain variable fragments (scFvs) and then cloning them into a lentiviral vector. The chimeric antigen receptor (CAR)-NK cells were generated through baboon envelop virus (BaEV) pseudotyped lentiviral transduction for high NK cell transduction.
Results We have observed an efficient and robust transduction (up to 80%) of primary NK cells from the peripheral blood (PB) of healthy donors using BaEV-pseudotyped lentivirus system. The integrated proviral copy numbers per CAR-NK cell were estimated to be less than five. The expansion efficiency of CAR-NK cells in vitro was comparable to the non-transduced (NTD) NK cells. We have observed enhanced cytotoxicity of the GBM cell lines and secreted effector cytokine levels by CAR-NK cells even at a low effector to target (E:T) ratio.
Conclusions CD276 CAR-NK cell is a potential therapeutic agent to treat human GBM disease. We will discuss generation of non-clinical data to characterize the CD276 binders.
Generation of CD276 CAR-NK cells
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