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Background B7H3 is a tumor associated antigen (TAA), found on numerous malignancies including prostate, lung, and breast cancers. High levels of B7H3 expression are correlated with late stage disease and poor prognosis. Furthermore, B7H3 is minimally expressed on normal tissue, making it an ideal TAA for broad cancer treatment strategy. We developed a tri-specific killer engager (TriKETM) consisting of a nanobody anti-CD16, IL-15, and nanobody anti-B7H3 joined by flexible linkers (camB7H3 TriKE) (figure 1A). The combination of B7H3 TriKE with an off-the-shelf NK cell therapy presents an appealing therapeutic strategy for the treatment of solid tumors with decreased risk of toxicity in allogeneic settings compared to T-cell derived products. Methods An anti-B7H3 nanobody was developed via biopanning and cloned into a TriKE vector. TriKE was produced in Expi293 cells and affinity purified using poly-His tag. NK cells were co-incubated with cell lines exhibiting a range of B7H3 expression and with 3nM of camelid B7H3 TriKE or control. We have previously derived NK cells expressing high affinity non-cleavable hnCD16, CD38 KO, and IL-15/IL-15R fusion from clonal master engineered iPSC lines. Engineered iNK cells were tested in conjunction with the TriKE. A repeated measures ANOVA was used for statistical comparisons as noted in figure legends Results Engineered iNK cells co-incubated with camB7H3 TriKE and C4-2 prostate cells significantly increased degranulation (CD107a) and cytokine production (IFN-gamma) compared to controls (figure 1B/C, P Conclusions camB7H3 TriKE dramatically increases function and activation on endogenous NK cells as well as engineered iNK cell, which can be adoptively transferred to patients with a broad range of cancers, including prostate cancer. TriKE activity was potent across a broad concentration spectrum and corresponded directly with B7H3 target expression. These studies represent the proof-of-concept of a novel pairing of off-the-shelf, engineered iNK cells with B7H3-directed pan-cancer engager molecules (TriKEs and CARs) to enhance specificity, persistence and anti-tumor function.

Background Gliomas represent the most common brain tumors within the central nervous system, with glioblastoma being the most aggressive type.1 Conventional treatment combines several approaches including surgery, chemotherapy, and radiation.2 However, the prognosis for glioblastoma remains unfavorable, with only 5% of patients surviving more than 5 years post-diagnosis.3 Thus, new treatment approaches are urgently needed. Natural killer (NK) cells directly lyse malignantly transformed or virally infected cells and secrete inflammatory cytokines that polarize cytotoxic immunity. Allogeneic NK cell adoptive transfer has shown clinical benefit in patients with advanced cancer.4–7 However, limitations of this approach include relatively low numbers of donor NK cells that can be isolated during an apheresis and variability in the quality of NK cells between donors. To overcome these limitations, we have developed a GMP manufacturing strategy to mass produce NK cells from induced pluripotent stem cells (iPSCs) as an approach to off-the-shelf cancer immunotherapy. We refer to these cells as ‘iNK’ (iPSC-derived NK) cells. Here, we provide preclinical data demonstrating the efficacy of iNK cells for immunotherapy against glioblastoma. Methods We generated iNK cells using previously published methods.8–10 iNK cells were used as effectors against an array of patient-derived glioblastoma lines in 2-dimensional live imaging IncuCyte assays where iNK cell-mediated killing was observed over the course of 48 hours. To investigate iNK cell infiltration and cytotoxicity in a more physiological context that accounts for the 3-dimensional architecture of the tumor, we also performed live imaging IncuCyte assays using iNK cells as effectors against glioblastoma spheroids. To test the anti-tumor function of iNK cells in vivo, we implanted patient-derived glioblastoma cells into mice via intracranial injection. Seven days later, 5 mice received intratumoral injections of iNK cells, and 5 mice received vehicle alone (as a control; figure 1A). All mice were monitored for weight and survival over 100 days. Results iNK cells exhibited strong and sustained cytotoxicity against 6 primary patient-derived mesenchymal glioblastoma lines in 2-dimensional IncuCyte assays and complete infiltration and destruction of glioblastoma spheroids in 3-dimensional IncuCyte assays. In xenogeneic adoptive transfer experiments, all mice receiving intratumoral injections of iNK cells survived out to day 100, while all mice in the vehicle group became moribund and had to be sacrificed by day 60 (figure 1B). Conclusions iNK cells are highly cytotoxic against glioblastoma cells, and our preclinical in vivo data provides proof-of-concept for future clinical trials. Ethics Approval This project has been approved by the University of Minnesota IACUC. Approval ID: 1812-36595A References Louis D N, Perry A, Reifenberger G, von Deimling A, Figarella-Branger D, Cavenee W K, Ohgaki H, Wiestler O D, Kleihues P, Ellison D W. The 2016 world health organization classification of tumors of the central nervous system: a summary. Acta Neuropathol 2016;131:803–820. Stupp R, Mason W P, van den Bent M J, Weller M, Fisher B, Taphoorn M J B, Belanger K, Brandes A A, Marosi C, Bogdahn U, Curschmann J, Janzer R C, Ludwin S K, Gorlia T, Allgeier A, Lacombe D, Cairncross J G, Eisenhauer E, Mirimanoff R O, European Organization for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups; National Cancer Institute of Canada Clinical Trials Group. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 2005;352:987–996. Thakkar JP, Dolecek TA, Horbinski C, Ostrom QT, Lightner DD, Barnholz-Sloan JS, Villano JL. Epidemiologic and molecular prognostic review of glioblastoma. Cancer Epidemiol Biomarkers Prev 2017;23:1985–1996. Miller J S, Soignier Y, Panoskaltsis-Mortari A, McNearney S A, Yun G H, Fautsch S K, McKenna D, Le C, Defor T E, Burns L J, Orchard P J, Blazar B R, Wagner J E, Slungaard A, Weisdorf D J, Okazaki J, McGlave P B. Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood 2005;105:3051–3057. Bachanova V, Cooley S, Defor T E, Verneris M R, Zhang B, McKenna D H, Curtsinger J, Panoskaltsis-Mortari A, Lewis D, Hippen K, McGlave P, Weisdorf D J, Blazar B R, Miller J S. Clearance of acute myeloid leukemia by haploidentical natural killer cells is improved using IL-2 diphtheria toxin fusion protein. Blood 2014;123:3855. Ciurea S O, Schafer J R, Bassett R, Denman C J, Cao K, Willis D, Rondon G, Chen J, Soebbing D, Kaur I, Gulbis A, Ahmed S, Rezvani K, Scpall E J, Lee D A, Champlin R E. Phase 1 clinical trial using mbIL21 ex vivo-expanded donor-derived NK cells after haploidentical transplant. Blood 2017;130:1857–1868. Romee R, Rosario M, Berrien-Elliott M M, Wagner J A, Jewell B A, Schappe T, Leong J W, Abdel-Latif S, Schneider S E, Willey S, Neal C C, Yu L, Oh T, Lee S, Mulder A, Cooper M A, Fehniger T A. Cytokine-induced memory-like natural killer cells exhibit enhanced responses against myeloid leukiemia. Sci Transl Med2016:8;375ra123. Valamehr B, Abujarour R, Robinson M, Le T, Robbins D, Shoemaker D, Flynn P. A novel platform to enable the high-throughput derivation and characterization of feeder-free human iPSCs. Sci Rep2012:2;213. Valamehr B, Robinson M, Abujarour R, Rezner B, Vranceanu F, Le T, Medcalf A, Lee T T, Fitch M, Robbins D, Flynn P. Platform for induction and maintenance of transgene-free hiPSCs resembling ground state pluripotent stem cells. Stem Cell Reports 2014;2:366–381. Zhu H, Blum R H, Bjordahl R, Gaidarova S, Rogers P, Lee T T, Abujarour R, Bonello G B, Wu J, Tsai P-F, Miller J S, Walcheck B, Valamehr B, Kaufman D S. Pluripotent stem cell-derived NK cells with high-affinity noncleavable CD16a mediate improved antitumor immunity. Blood 2020;135:399–410.

BackgroundChimeric antigen receptor (CAR)-T cell therapy has revolutionized cancer treatment, but it is associated with significant dose-limiting toxicities, restricted tumor targeting (limited by specific antigen expression), and, notably, a lack of multi-antigen targeting capability to mitigate tumor associated immune evasion and heterogeneity. Furthermore, dysfunctional starting material, product inconsistency, and small manufacturing lot size limits the application and on-demand availability of CAR-T cell therapy.MethodsTo overcome these considerable limitations, we have developed FT536, a first-of-kind, induced pluripotent stem cell (iPSC)-derived NK (iNK) cell with a novel CAR that ubiquitously targets cancer cells through canonical stress ligand recognition. We have previously reported FT536 recognizes the conserved α3 domain of the pan-tumor associated antigens MICA and MICB (MICA/B), and is derived from a renewable master iPSC line that contains multiplexed genetic edits to enhance effector cell functionality, persistence, and multi-antigen targeting capabilities via high affinity non cleavable CD16 (hnCD16) mediated antibody dependent cellular cytotoxicity (ADCC). Here we preview the nonclinical study for the investigational new drug (IND) application for FT536.ResultsUtilizing a manufacturing process analogous to pharmaceutical drug product development, we demonstrate FT536 can be consistently and uniformly produced with a greater than 4x10E7 fold cellular expansion per manufacturing campaign. Furthermore, FT536 can be cryopreserved at clinical scale to support off-the-shelf clinical application, with rapid product thaw and immediate patient infusion in an out-patient setting. Functional evaluation demonstrated that FT536 uniquely possesses potent and persistent antigen specific cytolytic activity against an array of solid and hematological tumor lines. Through its hnCD16 modality, FT536 can be utilized in combination with monoclonal antibodies to provide multi-antigen targeting capabilities and in conjunction with chemotherapeutics and/or radiation that augment surface MICA/B expression. In addition, directly thawed and infused FT536 demonstrated significant tumor growth inhibition in multiple solid and liquid in vivo xenograft models, in which tumor control was further enhanced in combination with a therapeutic antibody (figure 1). Finally, ongoing studies utilizing a lung adenocarcinoma model have highlighted the sustained persistence of FT536 in lung tissue up to 33 days following a single dose infusion without the need for exogenous cytokine support.Abstract 117 Figure 1FT536 provides statistically significant in vivo anti-tumor activity which is enhanced in combination with ADCC active monoclonal antibody therapy. (A-B) FT536 significantly reduced the number of lung and liver (not shown) metastases compared to CAR negative iNK control cells in a murine metastatic melanoma model using B16-F10 cells engineered to overexpress human MICA. (C-D) FT536 alone, and in combination with Herceptin, demonstrate significant tumor growth inhibition (TGI) compared to Herceptin alone in an orthotopic xenograft model of human lung adenocarcinoma.ConclusionsCollectively, these studies demonstrate that FT536 is a highly potent, multi-tumor targeting CAR-iNK cell product that is uniform in composition and can be effectively and safely used off-the-shelf for on-demand treatment of multiple solid and hematological malignancies. An IND submission is planned for 2021, with an initial Phase 1 clinical trial to follow.

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.