Patients with pancreatic ductal adenocarcinoma (PDAC) invariably succumb to metastatic disease, but the underlying mechanisms that regulate PDAC cell movement and metastasis remain little understood. In this study, we investigated the effects of the chemokine gene CXCL12, which is silenced in PDAC tumors yet is sufficient to suppress growth and metastasis when re-expressed. Chemokines like CXCL12 regulate cell movement in a biphasic pattern, with peak migration typically in the low nanomolar concentration range. Herein, we tested the hypothesis that the biphasic cell migration pattern induced by CXCL12 reflected a bias of agonist bioenergetic signaling that might be exploited to interfere with PDAC metastasis. In human and murine PDAC cell models, we observed that non-migratory doses of CXCL12 were sufficient to decrease oxidative phosphorylation and glycolytic capacity and to increase levels of phosphorylated forms of the master metabolic kinase AMPK. Those same doses of CXCL12 locked myosin light chain into a phosphorylated state, thereby decreasing F-actin polymerization and preventing cell migration in a manner dependent upon AMPK and the calcium-dependent kinase CAMKII. Notably, at elevated concentrations of CXCL12 that were insufficient to trigger chemotaxis of PDAC cells, AMPK blockade resulted in increased cell movement. In two preclinical mouse models of PDAC, administration of CXCL12 decreased tumor dissemination, supporting our hypothesis that chemokine-biased agonist signaling may offer a useful therapeutic strategy. Our results offer a mechanistic rationale for further investigation of CXCL12 as a potential therapy to prevent or treat PDAC metastasis.

Tumor-targeting monoclonal antibodies (mAbs) are the most widely used and characterized immunotherapy for hematologic and solid tumors. The significance of this therapy is their direct and indirect effects on tumor cells, facilitated by the antibody’s antigen-binding fragment (Fab) and fragment crystallizable region (Fc region), respectively. The Fab can modulate the function of cell surface markers on tumor cells in an agonistic or antagonistic manner, whereas the Fc region can be recognized by an Fc receptor (FcR) on leukocytes through which various effector functions, including antibody-dependent cell-mediated cytotoxicity (ADCC), can be elicited. This process is a key cytolytic mechanism of natural killer (NK) cells. These innate lymphocytes in the human body recognize tumor-bound antibodies exclusively by the IgG Fc receptor CD16A (FcγRIIIA). Two allelic versions of CD16A bind IgG with either lower or higher affinity. Cancer patients homozygous for the higher affinity allele of CD16A have been reported to respond significantly better to mAb therapies for various malignancies. These studies revealed that mAb therapy efficacy positively correlates with higher affinity binding to CD16A. Approaches to enhance tumor antigen targeting by NK cells by modifying the Fc portion of antibodies or the FcR on NK cells are the focus of this review.

Natural killer (NK) cells are innate cytotoxic lymphocytes that can recognize assorted determinants on tumor cells and rapidly kill these cells. Due to their anti-tumor effector functions and potential for allogeneic use, various NK cell platforms are being examined for adoptive cell therapies. However, their limited in vivo persistence is a current challenge. Cytokine-mediated activation of these cells is under extensive investigation and interleukin-15 (IL-15) is a particular focus since it drives their activation and proliferation. IL-15 efficacy though is limited in part by its induction of regulatory checkpoints. A disintegrin and metalloproteinase-17 (ADAM17) is broadly expressed by leukocytes, including NK cells, and it plays a central role in cleaving cell surface receptors, a process that regulates cell activation and cell-cell interactions. We report that ADAM17 blockade with a monoclonal antibody markedly increased human NK cell proliferation by IL-15 both in vitro and in a xenograft mouse model. Blocking ADAM17 resulted in a significant increase in surface levels of the homing receptor CD62L on proliferating NK cells. We show that NK cell proliferation in vivo by IL-15 and the augmentation of this process upon blocking ADAM17 are dependent on CD62L. Hence, our findings reveal for the first time that ADAM17 activation in NK cells by IL-15 limits their proliferation, presumably functioning as a feedback system, and that its substrate CD62L has a key role in this process in vivo. ADAM17 blockade in combination with IL-15 may provide a new approach to improve NK cell persistence and function in cancer patients.

Natural killer (NK) cells are innate lymphocytes that play essential roles in mediating antitumor immunity. NK cells respond to various inflammatory stimuli including cytokines and stress-induced cellular ligands which activate germline-encoded activation receptors (NKRs), such as NKG2D. The signaling molecules activated downstream of NKRs are well defined; however, the mechanisms that regulate these pathways are not fully understood. IQ domain-containing GTPase-activating protein 1 (IQGAP1) is a ubiquitously expressed scaffold protein. It regulates diverse cellular signaling programs in various physiological contexts, including immune cell activation and function. Therefore, we sought to investigate the role of IQGAP1 in NK cells. Development and maturation of NK cells from mice lacking IQGAP1 (Iqgap1−/−) were mostly intact; however, the absolute number of splenic NK cells was significantly reduced. Phenotypic and functional characterization revealed a significant reduction in the egression of NK cells from the bone marrow of Iqagp1−/− mice altering their peripheral homeostasis. Lack of IQGAP1 resulted in reduced NK cell motility and their ability to mediate antitumor immunity in vivo. Activation of Iqgap1−/− NK cells via NKRs, including NKG2D, resulted in significantly reduced levels of inflammatory cytokines compared with wild-type (WT). This reduction in Iqgap1−/− NK cells is neither due to an impaired membrane proximal signaling nor a defect in gene transcription. The levels of Ifng transcripts were comparable between WT and Iqgap1−/−, suggesting that IQGAP1-dependent regulation of cytokine production is regulated by a post-transcriptional mechanism. To this end, Iqgap1−/− NK cells failed to fully induce S6 phosphorylation and showed significantly reduced protein translation following NKG2D-mediated activation, revealing a previously undefined regulatory function of IQGAP1 via the mechanistic target of rapamycin complex 1. Together, these results implicate IQGAP1 as an essential scaffold for NK cell homeostasis and function and provide novel mechanistic insights to the post-transcriptional regulation of inflammatory cytokine production.

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Natural killer (NK) cells are innate cytotoxic lymphocytes. They target malignant cells via non-clonotypic receptors to induce natural cytotoxicity and also recognize tumor-bound antibodies to induce antibody-dependent cell-mediated cytotoxicity (ADCC). While ADCC by NK cells is a key mechanism of several clinically successful therapeutic monoclonal antibodies (mAbs), most patients exhibit or acquire resistance to mAb therapies. ADCC by human NK cells is exclusively mediated by the IgG Fc receptor, CD16A (FcγRIIIA). Studies have demonstrated that increasing the binding affinity between CD16A and therapeutic mAbs can augment their clinical efficacy. Given the exquisite specificity and diverse antigen detection of anti-tumor mAbs, we are interested in enhancing the ADCC potency of NK cell-based therapies for various malignancies. CD64 is the only high affinity FcγR family member and binds to the same IgG isotypes as CD16A (IgG1 and IgG3) but with > 30-fold higher affinity. CD64 (FcγRI) is normally expressed by certain myeloid cells but not by NK cells. We generated a recombinant version of this receptor consisting of the extracellular region of CD64 and the transmembrane and intracellular regions of human CD16A, referred to as CD64/16A (figure 1A). An important feature of CD64/16A is that due to its high affinity state, soluble monomeric anti-tumor mAbs can be pre-adsorbed to engineered NK cells expressing the recombinant FcγR, and these pre-absorbed mAbs can be switched or mixed for universal tumor antigen targeting (figure 1B). The engineered NK cells used in our study were derived from genetically edited and clonally derived induced pluripotent stem cells (iPSCs) through a series of stepwise differentiation stages (figure 2). Engineered iPSC-derived NK (iNK) cells can be produced in a uniform and clinically scalable manner (figure 2). In Figure 3, using an in vitro Delfia® ADCC assay, we show that iNK-CD64/16A cells mediated ADCC against SKOV3 cells, an ovarian adenocarcinoma cell line, in the presence of the anti-HER2 therapeutic mAb trastuzumab (Herceptin) or anti-EGFR1 therapeutic mAb cetuximab (Erbitux), when either added to the assay or pre-adsorbed to the iNK cells (figure 3). Considering the high affinity state of CD64, we examined the effects of free IgG in human serum on ADCC by iNK-CD64/16A cells. Using an IncuCyte® Live Cell Analysis System, ADCC was evaluated in the presence or absence of 5% human AB serum, in which free IgG was approximately 50-fold higher than the IgG saturation level of the CD64/16A receptors on iNK cells (data not shown). Despite the high levels of excess free IgG, iNK-CD64/16A cells mediated efficient ADCC when Herceptin was either added to the assay or pre-adsorbed to the cells (figure 4). ADCC assays were also performed with Raji cells, a Burkitt lymphoma cell line, as target cells and the therapeutic mAb rituximab (Rituxan). iNK-CD64/16A cells were added with or without pre-adsorbed Rituxan and the assay was performed in 10% AB serum. Again, iNK-CD64/16A cells mediated effective target cell killing in the presence of serum IgG (figure 5), demonstrating that saturating levels of free IgG did not prevent ADCC. To determine if we can further optimize the function of recombinant CD64, we engineered CD64 with the transmembrane regions of CD16A or NKG2D and signaling/co-signaling domain from CD28, 2B4 (CD244), 4-1BB (CD137), and CD3ζ (figure 6). CD64/16A signals by non-covalent association with the immunoreceptor tyrosine-based activation motif (ITAM)-containing signaling adapters CD3ζ and FcRγ found in the cell membrane, whereas the other recombinant CD64 constructs use ITAM and non-ITAM regions to mediate their signaling. The various recombinant CD64 constructs were initially expressed in NK92 cells (lacks expression of endogenous FcγRs) (figure 7). Using the Delfia® ADCC assay system, we examined the function of each recombinant CD64 construct and found all combinations are able to effectively induce ADCC (figure 8). We are in the process of generating iNK cells with these constructs and testing their ability to kill hematologic and solid tumors in vitro and in vivo. Our goal is to utilize this docking approach to pre-absorb mAbs to iNK cells for adoptive cell therapy. The mAbs would thus provide tumor-targeting elements that could be exchanged as a means of preventing tumor cell escape by selectively and easily altering NK cell specificity for tumor antigens. Figure Disclosures Lee: Fate Therapeutics, Inc.: Current Employment. Chu:Fate Therapeutics: Current Employment. Abujarour:Fate Therapeutics, Inc: Current Employment. Dinella:Fate Therapeutics: Current Employment. Rogers:Fate Therapeutics, Inc: Current Employment. Bjordahl:Fate Therapeutics: Current Employment. Miller:Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Vycellix: Consultancy; GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Onkimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company. Walcheck:Fate Therapeutics: Consultancy, Research Funding.

BackgroundOvarian cancer is a leading cause of cancer-related deaths among women due to the development of therapeutic resistance. Natural killer (NK) cells are cytotoxic lymphocytes that can kill neoplastic cells without prior sensitization. A key anti-tumor function of human NK cells is antibody-dependent cell-mediated cytotoxicity (ADCC), mediated exclusively by the IgG Fcy (FcyR) receptor CD16A. The mechanism of action of several clinically successful antitumor therapeutic monoclonal antibodies (mAbs) involves ADCC; however, their therapeutic efficacy is reduced due to regulatory checkpoints of CD16A, which include its low IgG binding affinity and rapid downregulation upon NK cell activation by the membrane metalloprotease ADAM17.1–3 CD64, the highest affinity FcyR, is expressed on myeloid-derived cells and not lymphocytes and is also not cleaved by ADAM17. To enhance ADCC, we generated CD64/16A, a novel recombinant FcyR consisting of extracellular CD64 and intracellular and transmembrane CD16A to mediate high affinity IgG binding and potent cell signaling.4 5MethodsEngineered NK cells expressing CD64/16A were derived from human induced pluripotent stem-cells (iPSCs), referred to here as iNKs, which are clonal and clinically scalable NK cells. ADCC and natural cytotoxicity of three ovarian cancer cell lines were measured in vitro using Delfia EuTDA and IncuCyte cytotoxicity assays, and production of anti-tumor cytokines was determined via flow cytometry. Finally, tumor cell killing was assessed in vivo using a human xenograft mouse model of peritoneal metastatic ovarian cancer.ResultsOur data show that iNK-CD64/16A cells uniquely facilitate mAb absorption, robustly produce IFN-y and TNF-alpha, and kill several ovarian cancer cell lines in the presence of the therapeutic mAbs trastuzumab or cetuximab, which target HER2 or EGFR, respectively. We found that iNK-CD64/16A cells can capture soluble mAbs and retain antibody arming and ADCC efficacy after cryopreservation. Additionally, iNK-CD64/16A cells robustly mediate ADCC and reduce overall tumor burden of HER2+ tumor cells in vivo in the described metastatic ovarian cancer xenograft model.ConclusionsOur findings provide new insights into using high affinity Fc receptor-based adoptive NK cell therapies and lay the preclinical foundation necessary for developing an ‘off-the-shelf’ cellular therapy that can be combined with therapeutic tumor-targeting mAbs for the treatment of ovarian cancer. Importantly, iNK-CD64/16A cells can serve as a docking platform for therapeutic antibodies that can be switched and mixed for universal tumor antigen targeting to treat multiple malignancies.AcknowledgementsGrant supportNIH R01CA203348, HHMI Medical Research Fellows Program/Burroughs Wellcome FundReferencesJing Y, Ni Z, Wu J, et al. Identification of an ADAM17 cleavage region in human CD16 (FcgammaRIII) and the engineering of a non-cleavable version of the receptor in NK cells. PLoS One 2015;10(3):e0121788.Wu J, Mishra HK, Walcheck B. Role of ADAM17 as a regulatory checkpoint of CD16A in NK cells and as a potential target for cancer immunotherapy. J Leukoc Biol 2019;105(6):1297–303.Dixon KJ, Wu J, Walcheck B. Engineering Anti-Tumor Monoclonal Antibodies and Fc Receptors to Enhance ADCC by Human NK Cells. Cancers (Basel) 2021;13(2).Snyder KM, Hullsiek R, Mishra HK, et al. Expression of a Recombinant High Affinity IgG Fc Receptor by Engineered NK Cells as a Docking Platform for Therapeutic mAbs to Target Cancer Cells. Front Immunol 2018;9:2873.Walcheck B, Wu J. iNK-CD64/16A cells: a promising approach for ADCC? Expert Opin Biol Ther 2019;19(12):1229–32.Ethics ApprovalThis study was approved by the University of Minnesota’s Institutional Animal Care and Use Committee, protocol number 1902–36768A.

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.