Anti-tumor mAbs are the most widely used and characterized cancer immunotherapy. Despite having a significant impact on some malignancies, most cancer patients respond poorly or develop resistance to this therapy. A known mechanism of action of these therapeutic mAbs is antibody-dependent cell-mediated cytotoxicity (ADCC), a key effector function of human NK cells. CD16A on human NK cells has an exclusive role in binding to tumor-bound IgG antibodies. Though CD16A is a potent activating receptor, it is also a low affinity IgG Fc receptor (FcγR) that undergoes a rapid downregulation in expression by a proteolytic process involving ADAM17 upon NK cell activation. These regulatory processes are likely to limit the efficacy of tumor-targeting therapeutic mAbs in the tumor environment. We sought to enhance NK cell binding to anti-tumor mAbs by engineering these cells with a recombinant FcγR consisting of the extracellular region of CD64, the highest affinity FcγR expressed by leukocytes, and the transmembrane and cytoplasmic regions of CD16A. This novel recombinant FcγR (CD64/16A) was expressed in the human NK cell line NK92 and in induced pluripotent stem cells from which primary NK cells were derived. CD64/16A lacked the ADAM17 cleavage region in CD16A and it was not rapidly downregulated in expression following NK cell activation during ADCC. CD64/16A on NK cells facilitated conjugation to antibody-treated tumor cells, ADCC, and cytokine production, demonstrating functional activity by its two components. Unlike NK cells expressing CD16A, CD64/16A captured soluble therapeutic mAbs and the modified NK cells mediated tumor cell killing. Hence, CD64/16A could potentially be used as a docking platform on engineered NK cells for therapeutic mAbs and IgG Fc chimeric proteins, allowing for switchable targeting elements and a novel cancer cellular therapy.

Introduction: Cancer survivors are increasingly diagnosed with a syndrome of neurocognitive dysfunction termed cancer-related cognitive impairment (CRCI). Chemotherapy and radiation therapy have been implicated in CRCI; however, its underlying pathogenesis remains unclear, hindering effective prevention or treatment.Methods: We used the hairless strain SKH1 (11–12-week-old) and treated the mice with radiation to the right hindlimb, doxorubicin (a chemotherapy agent), concurrent radiation, and doxorubicin, or no treatment (control). Neurocognition was evaluated via standardized behavioral testing following treatment. Mice were subsequently humanely euthanized, and plasma and brains were collected to identify inflammatory changes.Results: Mice treated with radiation, doxorubicin, or both radiation and doxorubicin demonstrated equivalent hippocampal dependent memory deficits and significant increases in activated microglia and astrocytes compared to control mice. Doxorubicin-treated mice had significantly increased plasma IL-6 and failed to gain weight compared to control mice over the study period.Discussion: This study demonstrates that non-brain directed radiation induces both gliosis and neurocognitive deficits. Moreover, this work presents the first characterization of SKH1 mice as a relevant and facile animal model of CRCI. This study provides a platform from which to build further studies to identify potential key targets that contribute to CRCI such that strategies can be developed to mitigate unintended neuropathologic consequences associated with anticancer treatment.

22Anti-tumor mAbs are the most widely used and characterized cancer immunotherapy agents. 23 Despite having a significant impact on some malignancies, most cancer patients respond poorly 24 or develop resistance to this therapy. A known mechanism of action of these therapeutic mAbs is 25 antibody-dependent cell-mediated cytotoxicity (ADCC), which is a primarily effector function of 26 NK cells. CD16A on human NK cells has an exclusive role in binding to tumor-bound IgG 27antibodies. Though CD16A is a potent activating receptor, it is a low affinity FcγR and its cell 28 surface levels can be rapidly downregulated by a proteolytic process involving ADAM17 upon 29 NK cell activation, which are likely to limit the efficacy of tumor-targeting therapeutic mAbs in 30 the tumor environment. We sought to enhance NK cell binding to anti-tumor mAbs by 31 engineering these cells with a recombinant FcγR consisting of the extracellular region of CD64, 32 the highest affinity IgG Fc receptor expressed by leukocytes, and the transmembrane and 33 cytoplasmic regions of CD16A. This novel recombinant FcγR (CD64/16A) was expressed in the 34 human NK cell line NK92 and in induced pluripotent stem cells from which primary NK cells 35were derived. CD64/16A also lacked the ADAM17 cleavage region in CD16A and it was not 36 rapidly downregulated in expression following NK cell activation during ADCC.

Natural killer (NK) cells are cytotoxic lymphocytes that can rapidly kill tumor cells with exquisite specificity by antibody-dependent cell-mediated cytotoxicity (ADCC), which is exclusively mediated by the IgG Fcγ receptor CD16A. Many clinically successful therapeutic monoclonal antibodies (mAbs) utilize ADCC as a mechanism of action. However, CD16A binds IgG with low affinity and is rapidly cleaved by a proteolytic process from the cell surface upon activation, limiting therapeutic mAb efficacy. We generated NK cells expressing engineered high affinity FcγRs. Here, we investigated CD64/16A, a recombinant receptor consisting of the extracellular region of CD64, the highest affinity IgG FcγR, and the transmembrane and intracellular regions of CD16A. We expressed CD64/16A in human NK92 cells, which lack endogenous FcγRs but mediates ADCC upon expression of CD16A, and in induced pluripotent stem cells differentiated into NK (iNK) cells. We determined that CD64/16A was functional in vitro, facilitated ADCC and the production of cytokines, did not undergo rapid downregulation in expression upon NK cell activation, and facilitated conjugation to antibody-opsonized target cells. We are currently exploring an in vivo xenograft model using NSG immunocompromised mice engrafted with a HER2+ ovarian cancer cells to further examine the ADCC potency of CD64/16A iNK cells. Our findings suggest that CD64/16A may be utilized by engineered NK cells, leading to the formation of an “off-the-shelf” cellular therapy that can be combined with therapeutic mAbs for the treatment of various tumor types.