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Natural killer (NK) cells are innate lymphocytes that target malignant cells via non-clonotypic receptors to induce natural cytotoxicity and that recognize tumor-bound antibodies to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Human NK cells exclusively mediate ADCC through the IgG Fc receptor, CD16A, and studies have demonstrated that increasing the binding affinity between CD16A and therapeutic monoclonal antibodies (mAbs), mediated by the high-affinity 158V polymorphism, can augment clinical efficacy. Given the exquisite specificity and diverse antigen detection of anti-tumor mAbs, we sought to arm iPSC-derived NK (iNK) cells expressing a high-affinity recombinant FcγR with various mAbs as unique tumor-targeting strategy for various malignancies. As a member of the FcγR family, CD64 (FcγRI) possesses the highest affinity and can uniquely facilitate antibody preabsorption but it is normally expressed by myeloid cells. To leverage CD64 in NK cells, we developed a novel FcγR recombinant fusion comprising the extracellular region of CD64 with the transmembrane and intracellular regions of other NK cell activating receptors, including CD16A (CD64/16A) (figure 1A). The recombinant CD64/16A engineered into a clonal master induced pluripotent stem cell (iPSC) line for mass production of off-the-shelf iPSC-derived CD64/16A NK (iNK-CD64/16A) cells, can be armed with mAbs, including various combinations thereof to enable multi-antigen targeting and to address tumor heterogeneity (figures 1B and 2). To determine optimal binding and FcR saturation of iNK-CD64/16 cells, rituximab (anti-CD20 therapeutic mAb) was added in a two-hour preabsorbtion assay (figure 3A). Using an in vitro Delfia® ADCC assay, we show that iNK-CD64/16A cells mediated ADCC against Raji cells, a Burkitt Lymphoma cell line, when the iNKs were preabsorbed and armed with rituximab (figure 3B). Considering the high-affinity state of CD64, we examined the effects of free IgG on ADCC by iNK-CD64/16A cells. Using an IncuCyte® Live Cell Analysis, ADCC was evaluated in the presence of purified human IgG. Despite the high levels of excess IgG, iNK-CD64/16A cells mediated efficient ADCC when rituximab was either added to the assay (figure 4A) or preabsorbed to the cells (figure 4B), demonstrating that saturating levels of free IgG did not prevent ADCC in either setting. To determine the ability of preabsorbed and armed iNK-CD64/16 cells to retain rituximab and perform serial killing, we performed a sequential killing assay using an IncuCyte® Live Cell Analysis where preabsorbed iNK-CD64/16A cells were thawed and co-cultured with or without Raji cells for 48 hours, followed by a second round of co-culture. As shown in figure 5, iNK-CD64/16A cells armed with rituximab retain ADCC capacity and perform serial killing for an extended time. To establish that iNK-CD64/16A cells can be armed with assorted therapeutic mAbs to target other tumor-associated antigens, we next determined the ability of iNK-CD64/16A cells preabsorbed and armed with anti-HER2 mAb, trastuzumab, to target the adenocarcinoma ovarian cancer cell line SKOV-3. Indeed iNK-CD64/16A cells armed with preabsorbed trastuzumab were able to effectively kill SKOV-3 cells via in vitro ADCC by IncuCyte® Live Cell Analysis (figure 6). We next investigated in vivo ADCC using NSG mice implanted with 3x10 5 SKOV-3 cells expressing firefly luciferase intraperitoneally (IP). 10 million iNK-CD64/16A with or without preabsorbed trastuzumab were injected IP (figure 7A), and a significant reduction in tumor volume in animals treated with iNK-CD64/16A cells armed with trastuzumab compared to unarmed iNK-CD64/16A cells (figure 7B). Collectively, our data show that iNK-CD64/16A cells can be armed with various therapeutic mAbs through a unique preabsorption strategy to mediate a potent and durable ADCC activity. The versatility of mAb-armed iNK-CD64/16A cells is being further investigated in various preclinical models to further elucidate the potential of this approach to overcome antigen escape and address tumor heterogeneity. Figure 1 Figure 1. Disclosures Lee: Fate Therapeutics, Inc: Current Employment. Chu: Fate Therapeutics: Current Employment. Rogers: Fate Therapeutics: Current Employment. Bjordahl: Fate Therapeutics: Current Employment. Hosking: Fate Therapeutics: Current Employment. Shirinbak: Fate Therapeutics, Inc.: Current Employment. Miller: Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Vycellix: Consultancy; ONK Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Magenta: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Wugen: Membership on an entity's Board of Directors or advisory committees. Valamehr: Fate Therapeutics, Inc.: Current Employment. Walcheck: Fate Therapeutics: Research Funding.

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.