Background Despite aggressive treatments and care, the median survival for GBM patients is 14.6 months, which has only modestly improved over the past several decades, highlighting the need for new therapeutic approaches. NK cells, innate cytotoxic effectors, are showing potential for cancer immunotherapy for GBM. 1–3 However, tumor antigen heterogeneity and a severely immunosuppressive tumor microenvironment (TME) have rendered GBM highly resistant to most single antigen-based NK monotherapies. 4–6 Methods To overcome these challenges, our solution has been to develop a first multifunctional immunotherapy for GBM based on genetically-engineered NK cells bearing multiple simultaneous anti-tumor functions, including local tumor responsiveness and the ability to avoid antigen escape. The activity of these lentivirally-transduced multi-functional NK (E-pNK) cells were evaluated against patient-derived GBM cells both in vitro and in vivo. Results We have designed and synthesized a multifunctional CAR construct that expresses an anti-CD73 scFv which is cleavable by GBM-associated proteases, and a dual CAR redirected against ligands for NKG2D and GBM-associated GD2 receptors (figure 1A-B). We have isolated primary NK cells (figure 1C) and genetically manipulated them to express NKG2D, anti-GD2 scFv and anti-CD73 scFv (figure 1D-E). E-pNK cells showed a significantly higher in vitro antitumor activity towards GBM43 targets, patient-derived GBM cells, including increased percentage of tumor killing, degranulation and IFN-γ production (figure 1F-G). E-pNK cells lacking the anti-CD73 scFv following uPA treatment displayed significantly decreased killing ability of target GBM43 cells after co-culture at E/T ratios of 2.5 and 5 for 4 h (figure 1H). In addition, after treatment with cleaved anti-CD73 scFv, GBM43 cells showed a significantly reduced ability to produce adenosine due to the inhibition of CD73 enzyme activity (figure 1I). Furthermore, E-pNK cells showed potent anti-GBM activity in subcutaneously GBM43 xenografts (figure 1J-L). In vivo-adoptively transferred E-pNK cells also showed superior intratumoral infiltration into GBM43 tumors when analyzed by IHC (data not shown). Abstract 134 Figure 1 Multifunctional genetically-engineered NK cells for immunotherapy of GBM. (A) Schematic representation of transgene representing the complete multi-functional construct: tumor-responsive anti-CD73 scFv-secreting dual-specific CAR targeting NKG2DL and GD2. (B) Schematic representation of tumor-responsive anti-CD73 scFv secreting dual-specific CARs. (C) Flow cytometry data showing the purity of isolated peripheral blood-derived NK (pNK) cells (CD56 CD3-). (D) NKG2D expression on engineered pNK cells determined by flow cytometry after two rounds of lentiviral transduction. (E) Expression of anti-CD73 scFv and anti-GD2 scFv on pNK cells determined by flow cytometry after two rounds of lentiviral transduction. (F) In vitro cytotoxicity of pNK and E-pNK cells against different GBM43 at indicated E/T ratios over 4 h. (G) Degranulation (% CD107) and IFN-γ production of pNK and E-pNK cells (% IFN-γ) after 4 h coculture with GBM43 cells (E/T ratio, 5:1). (H) In vitro cytotoxicity of pNK and E-pNK (following aCD73 scFv cleavage) cells against GBM43 cells at indicated E/T ratios over 4 h. (I) CD73 activity of GBM43 cells after incubation with cleaved aCD73 scFv following cleavage from uPA-treated E-pNK cells. (J) Tumor growth of individual treatment groups, including PBS, pNK cells and E-pNK cells. Tumor size was determined by caliper measurements. (K) Average tumor weight of the mice in each treatment group after necropsy on day 28 post-start of treatment. (L) Changes in the body weight of the mice in each group during the treatment period. Note: the data shown in this study is for isolated pNK cells from one representative donor. Data are shown as mean ± SEM. *P??0.05, **P 0.01 Conclusions We have generated E-pNK cells showing improved antitumor activity against GBM through increased resistance to the immunosuppressive TME via adenosinergic CD73 blockade and the simultaneous ability to specifically target GBM cells via dual CARs. Based on these results, we are currently building the orthotopic GBM mouse model to further evaluate their in vivo therapeutic effects. Acknowledgements This work is supported, in part, by a Ralph W. and Grace M. Showalter Research Trust award and a Walther Cancer Foundation Embedding Grant. Ethics Approval Primary human NK (pNK) cells used in this study were obtained using Purdue University’s Institutional Review Board (IRB)-approved consent forms (IRB-approved protocol #1804020540).
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