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Substantial numbers of B cell leukemia and lymphoma patients relapse due to antigen loss or heterogeneity after anti-CD19 chimeric antigen receptor (CAR) T cell therapy. To overcome antigen escape and address antigen heterogeneity, we engineered induced pluripotent stem cell-derived NK cells to express both an NK cell-optimized anti-CD19 CAR for direct targeting and a high affinity, non-cleavable CD16 to augment antibody-dependent cellular cytotoxicity. In addition, we introduced a membrane-bound IL-15/IL-15R fusion protein to promote in vivo persistence. These engineered cells, termed iDuo NK cells, displayed robust CAR-mediated cytotoxic activity that could be further enhanced with therapeutic antibodies targeting B cell malignancies. In multiple in vitro and xenogeneic adoptive transfer models, iDuo NK cells exhibited robust anti-lymphoma activity. Furthermore, iDuo NK cells effectively eliminated both CD19+ and CD19− lymphoma cells and displayed a unique propensity for targeting malignant cells over healthy cells that expressed CD19, features not achievable with anti-CAR19 T cells. iDuo NK cells combined with therapeutic antibodies represent a promising approach to prevent relapse due to antigen loss and tumor heterogeneity in patients with B cell malignancies.

The production of autologous T cells expressing a chimaeric antigen receptor (CAR) is time-consuming, costly and occasionally unsuccessful. T-cell-derived induced pluripotent stem cells (TiPS) are a promising source for the generation of ‘off-the-shelf’ CAR T cells, but the in vitro differentiation of TiPS often yields T cells with suboptimal features. Here we show that the premature expression of the T-cell receptor (TCR) or a constitutively expressed CAR in TiPS promotes the acquisition of an innate phenotype, which can be averted by disabling the TCR and relying on the CAR to drive differentiation. Delaying CAR expression and calibrating its signalling strength in TiPS enabled the generation of human TCR– CD8αβ+ CAR T cells that perform similarly to CD8αβ+ CAR T cells from peripheral blood, achieving effective tumour control on systemic administration in a mouse model of leukaemia and without causing graft-versus-host disease. Driving T-cell maturation in TiPS in the absence of a TCR by taking advantage of a CAR may facilitate the large-scale development of potent allogeneic CD8αβ+ T cells for a broad range of immunotherapies.

This supplementary file contains Supplemental Figures 1-6. Supplemental Figure 1 shows that 7-day ex vivo culture of NK cells with IL-15 and 5 μM CHIR99021 leads to an increase in the frequencies of NK cells with heterogeneous adaptive NK cell phenotypes (defined by expression of CD57, PLZF, SYK and FcεR1γ) relative to DMSO controls. Supplemental Figure 2 contains a detailed phenotypic characterization of receptor expression and cytotoxic granule component levels in sorted CD3-CD56dimCD57- and CD3-CD56dimCD57+ NK cells cultured for 7 days with IL-15 and either DMSO or 5 μM CHIR99021. Supplementary Figure 3 shows an analysis of NK cell phenotype, viability and proliferation after 7-day culture with IL-15 and either DMSO or CHIR99021 at several concentrations (1 μM, 3 μM and 5 μM). Supplemental Figure 4 shows an analysis of NK cell function (CD107a and IFN-γ) against K562 cells. NK cells were cultured for 7 days in IL-15 and either DMSO or 5 μM CHIR99021. Function was determined for individual CD3-CD56+ NK cell subsets gated on CD57 and NKG2C. Supplemental Figure 5 shows an analysis of NK cell function (CD107a and IFN-γ) against K562 cells. NK cells were cultured for 7 days in IL-15 and either DMSO or 5 μM CHIR99021. Function was determined for individual CD3-CD56+ NK cell subsets gated on CD57 and KIR. Supplemental Figure 6 shows the frequency of ex vivo expanded NK cells expressing CD57 and/or NKG2C 14 days after adoptive transfer into NSG mice.

Maturation of human natural killer (NK) cells as defined by accumulation of cell-surface expression of CD57 is associated with increased cytotoxic character and TNF and IFNγ production upon target-cell recognition. Notably, multiple studies point to a unique role for CD57+ NK cells in cancer immunosurveillance, yet there is scant information about how they mature. In this study, we show that pharmacologic inhibition of GSK3 kinase in peripheral blood NK cells expanded ex vivo with IL15 greatly enhances CD57 upregulation and late-stage maturation. GSK3 inhibition elevated the expression of several transcription factors associated with late-stage NK-cell maturation including T-BET, ZEB2, and BLIMP-1 without affecting viability or proliferation. When exposed to human cancer cells, NK cell expanded ex vivo in the presence of a GSK3 inhibitor exhibited significantly higher production of TNF and IFNγ, elevated natural cytotoxicity, and increased antibody-dependent cellular cytotoxicity. In an established mouse xenograft model of ovarian cancer, adoptive transfer of NK cells conditioned in the same way also displayed more robust and durable tumor control. Our findings show how GSK3 kinase inhibition can greatly enhance the mature character of NK cells most desired for effective cancer immunotherapy. Cancer Res; 77(20); 5664–75. ©2017 AACR.

Natural killer (NK) cells mediate the cytolysis of transformed cells and are currently used as an adoptive cellular therapy to treat cancer. Infection with human cytomegalovirus has been shown to expand a subset of "adaptive" NK cells expressing the activation receptor NKG2C that have preferred functional attributes distinct from conventional NK cells. Because NKG2C delivers a strong activating signal to NK cells, we hypothesized that NKG2C could specifically trigger NK-cell-mediated antitumor responses. To elicit a tumor-directed response from NKG2C+ NK cells, we created an anti-NKG2C/IL-15/anti-CD33 killer engager called NKG2C-KE that directs NKG2C+ cells to target CD33+ cells and tumor-associated antigen expressed by acute myelogenous leukemia cells. The NKG2C-KE induced specific degranulation, interferon-γ production, and proliferation of NKG2C-expressing NK cells from patients who reactivated cytomegalovirus after allogeneic transplantation. The NKG2C-KE was also tested in a more homogeneous system using induced pluripotent stem cell (iPSC)-derived NK (iNK) cells that have been engineered to express NKG2C at high levels. The NKG2C-KE triggered iNK-cell-mediated cytotoxicity against CD33+ cells and primary AML blasts. The NKG2C-KE-specific interaction with adaptive NK and NKG2C+ iNK cells represents a new immunotherapeutic paradigm that uniquely engages highly active NK cells to induce cytotoxicity against AML through redirected targeting.

Natural killer (NK) cells hold great promise as a source for allogeneic cell therapy against hematological malignancies, including acute myeloid leukemia (AML). Current treatments are hampered by variability in NK cell subset responses, a limitation which could be circumvented by specific expansion of highly potent single killer immunoglobulin-like receptor (KIR)We developed a GMP-compliant protocol to expand adaptive NK cells from cryopreserved cells derived from select third-party superdonors, that is, donors harboring large adaptive NK cell subsets with desired KIR specificities at baseline. We studied the adaptive state of the cell product (ADAPT-NK) by flow cytometry and mass cytometry as well as cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq). We investigated the functional responses of ADAPT-NK cells against a wide range of tumor target cell lines and primary AML samples using flow cytometry and IncuCyte as well as in a mouse model of AML.ADAPT-NK cells were90% pure with a homogeneous expression of a single self-HLA specific KIR and expanded a median of 470-fold. The ADAPT-NK cells largely retained their adaptive transcriptional signature with activation of effector programs without signs of exhaustion. ADAPT-NK cells showed high degranulation capacity and efficient killing of HLA-C/KIR mismatched tumor cell lines as well as primary leukemic blasts from AML patients. Finally, the expanded adaptive NK cells had preserved robust antibody-dependent cellular cytotoxicity potential and combination of ADAPT-NK cells with an anti-CD16/IL-15/anti-CD33 tri-specific engager led to near-complete killing of resistant CD45These preclinical data demonstrate the feasibility of off-the-shelf therapy with a non-engineered, yet highly specific, NK cell population with full missing-self recognition capability.

Immune cell therapies derived from induced pluripotent stem cells (iPSC) provide a novel opportunity for the treatment of multiple cancer types. Assessment of the persistence and biodistribution of these product candidates requires specific and sensitive methods to detect engineered cells in both liquid and solid biopsies. Here, we present the development and validation of two complimentary nucleic acid-based detection assays for iPSC-derived natural killer (iNK) cell product candidates containing Fate’s proprietary high-affinity, non-cleavable CD16 transgene (hnCD16). The first assay is a droplet digital PCR (ddPCR) method to detect and quantify hnCD16 transgene copies present in a pool of genomic DNA (gDNA). The primers and probe were designed to recognize the optimized codons of hnCD16. Assay linearity and accuracy were assessed through titration studies using 0.024 to 1 ng of hnCD16-containing DNA spiked into different amounts of hnCD16-negative gDNA. Precision was determined through multiple assay runs by different operators on two instruments. The second assay is an in situ hybridization based method utilizing RNAscope࣪ technology to detect cells expressing hnCD16 in fixed tissue. Probes targeting hnCD16 were used to optimize signal specificity. Cells expressing hnCD16 and tissues from in vivo studies treated with iNK products served as positive controls. For the ddPCR assay, absolute limit of detection (aLoD) was determined to be 4.9 copies of hnCD16 per 20 µL reaction, regardless of total genomic mass input. Absolute limit of quantification (aLoQ) was 12 copies per 20 µL reaction with a %CV ≤30. Relative limit of quantification (rLoQ), assessing transgene to total DNA ratio, is affected by the background gDNA input and is less sensitive with lower input mass. rLoQ for total mass of 70 - 250 ng was 97 - 22 copies/µg gDNA (0.064% - 0.015%) with a %CV ≤30. The sensitivity of this input range allows evaluation of clinical samples with low cellularity. While ddPCR provides robust quantification of the hnCD16 transcript, the RNAscope࣪ assay informs localization of the iNK product. Specificity of the probe was established by confirming its lack of affinity for endogenous CD16 using a variety of human normal and tumor tissues and by staining hnCD16-positive fixed cell pellets and tissues from in vivo studies. In cell pellets, positive RNAscope࣪ signal correlated with the known ratio of transgene positive cells. In murine tissues previously confirmed to contain iNK cell product, the RNAscope࣪ positive staining correlated with NKG2A immunohistochemistry staining, confirming the presence of product NK cells. The combined use of both the ddPCR and RNAscope࣪ assays targeted to hnCD16 allows for detection and quantification of transgene-bearing iNK cells in a wide variety of patient samples including tumor biopsies. Both assays are being utilized for cell detection and quantification in our ongoing clinical trials. Citation Format: Cara E. Bickers, Judy L. Martin, Steven Castro, Jason Zhang, Thomas Dailey, Eric Sung, Suzanna Gasparian, Jason O'Rourke, Moyar Ge, Tom T. Lee, Janel Huffman, Jode Goodridge, Ryan Bjordahl, Bahram Valamehr, Peter M. Szabo, Lilly Wong, Sarah Cooley. Detection of genetically engineered iPSC-derived natural killer cells in blood and tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1138.

Adoptive T-cell therapy with chimeric antigen receptor (CAR) has shown promising results in cancer treatment, however, antigen escape and tumor heterogeneity are major causes for disease relapse. While CARs are known to trigger an effective immune response through surface antigen recognition many solid tumor cancer antigens are intracellular and presented by MHC molecules recognized by T cell receptors (TCRs). In addition, many therapeutic antibodies have shown clinical efficacy in solid tumor settings. However, antibody-dependent cellular cytotoxicity (ADCC) is mediated by the CD16 Fc receptor naturally expressed on NK cells although its application in T cells is yet not fully appreciated. Utilizing our proprietary induced pluripotent stem cell (iPSC) platform to engineer multiple modalities into a clonal iPSC line, which can serve as the starting cell source for mass production of off-the-shelf, iPSC-derived CAR-T cells (CAR-iT cells), we aimed to study the combination of these three targeting modalities, CAR, TCR, and CD16, to determine whether challenges associated with the treatment of solid tumors, which are heterogeneous and challenging to treat, may be overcome.To test the base line activity of CAR-iT cells in the solid tumor setting, we selected our anti-MICA/B CAR, previously shown to effectively target stress ligands found on transformed cells, to demonstrate effective anti-tumor activity against multiple solid tumor cell lines (72 hrs cytotoxicity: A2058 = 99%; 786-O = 98%; versus non-specific CAR-iT cells: A258 = 13%; 786-O = 17%). To test compatibility of TCR in our iT cell platform, we engineered MR1-TCR in iT cells to show increased cytokine release and degranulation upon stimulated with MR1 positive lung carcinoma epithelial cells line A549 (fold change compared to un-stimulated: IFNg = 210, p = 0.0032; TNFa = 76.9, p = 0.0005; CD107ab = 115.0, p=0.0013). Notably, with the engineering of tumor antigen specific TCR in TCR-less CAR-iT cells, CD3 complex can be re-established to provide an opportunity to combine with bispecific T cell engagers. Lastly, combining CAR-iT cells with MR1-TCR and hnCD16 uniquely demonstrated synergistic tumor growth inhibition and validated our approach to target multiple antigens at once for an effective anti-tumor response (A549 cytotoxicity: tumor only = 3.68±2.04%; effector+TCR = 41.31±2.27%; effector+TCR+ADCC = 90.28±1.87%). In summary, using the unique approach to engineer iPSCs at the clonal level to create a distinct population of engineered iT cells, we successfully demonstrated the compatibility between CAR, TCR, and hnCD16 to mitigate tumor heterogeneity. This approach is an ideal strategy to create off-the-shelf cellular immunotherapy for a promising therapeutic approach to combat heterogeneous and difficult to treat solid tumors, including those that are resistant due to antigen escape. Citation Format: Chia-Wei Chang, Bi-Huei Yang, Eason Lin, Soheila Shirinbak, Wen-I Yeh, Mochtar Pribadi, Helen Chu, Alma Gutierrez, Earl Avramis, Jason ORourke, Tom Lee, Alec Witty, Eigen Peralta, Martin Hosking, Bahram Valamehr. Multiplexed-engineered, iPSC-derived T cells expressing three unique targeting modalities address tumor heterogeneity and antigen escape [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2756.

Despite success in hematologic malignancies, chimeric antigen receptor (CAR) T cells have been less effective in solid tumors, in part because of the heterogeneous expression of the CAR-specific target antigen (1° Ag) found within the tumor mass. In combination with a CAR, bispecific T cell engagers (BiTEs) can target additional tumor antigens (2° Ag) while engaging CD3 signaling molecules on T cells, providing a unique way to address tumor heterogeneity, mitigate antigen escape and further potentiate durable effector function. However, in generating off-the-shelf universal CAR-T and NK cells, a combination strategy is not feasible as neither modified cell product is compatible to specifically engage with a BiTE. In developing allogeneic CAR-T cells, the T cell receptor (TCR) surface expression must be eliminated to prevent graft versus host disease, but the absence of surface TCR expression leads to loss of surface CD3 expression and BiTE compatibility. Similarly, NK cells naturally lack TCR expression and have no surface CD3 molecules for BiTE engagement. Here we discuss the development of a novel chimeric CD3ε fusion receptor (CD3-CFR) with a modified transmembrane and endodomain enabling surface expression in TCR-less T or NK cells, allowing for a novel combinatorial solution between universal CAR-T or NK cells with BiTEs in an allogeneic setting. CD3-CFR signal transduction was initially investigated in TRAC knockout NFAT reporter (T-KO) Jurkat cells engineered with CD3-CFR and co-cultured with EpCAM+ target cells. When soluble EpCAM-BiTE was added to the co-culture, the observed 6.9-fold increase in NFAT activity indicated successful engagement associated with the interaction of CD3-CFR, target cells and the BiTE. CD3-CFR T-KO Jurkat cells were further modified to secrete the EpCAM-BiTE and showed increased NFAT activity, demonstrating the feasibility of self-secreting BiTE-mediated targeting through CD3-CFR signaling. We next engineered CD3-CFR expressing iPSC-derived CAR-T (iT) cells to show preserved T cell differentiation kinetics and maintained CAR activity against 1° Ag positive targets (>90% cytolysis). Importantly, while CAR-iT cells showed only background lysis of 1° Ag negative tumor cells, CD3-CFR+ CAR-iT cells were able to lyse ~99% of 1° Ag negative tumor cells with the addition of soluble EpCAM-BiTE targeting the 2° Ag. When CD3-CFR+ CAR-iT cells were further engineered to secrete the EpCAM-BiTE, CD3-CFR+ BiTE+ CAR-iT cells demonstrated superior killing of heterogenous tumors compared to CD3-CFR+ BiTE- CAR-iT cells (70% vs. 5% cytolysis). Taken together these data demonstrate for the first time that CD3 expression and BiTE engagement can be combined in a universal iPSC-derived CAR-iT cell product to overcome antigen heterogeneity and enhance efficacy in solid tumor settings. Citation Format: Eigen Peralta, Dan Lu, Mark Landon, Hui-Yi Chu, Amit Mehta, Philip Chu, Alec Witty, Tom Lee, Bahram Valamehr. Chimeric CD3 fusion receptors expressed on iPSC-derived universal TCR-less CAR-T and -NK cells synergize with bispecific engagers to enhance antitumor activity and limit antigen escape [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5513.

Chimeric antigen receptor (CAR) T-cell therapies have revolutionized the treatment of hematologic malignancies and have shown significant potential in solid tumor indications. However, logistical complexities associated with patient-specific CAR T-cell therapies often limit broad accessibility. Many of these challenges can be overcome with an allogeneic cellular product, but immune cell-mediated rejection of allogeneic cellular therapies remains a significant concern. Both allogeneic and autologous cell therapies currently rely on lymphodepleting conditioning to modulate the immune system and create greater access to homeostatic cytokines. However, protracted lympho-conditioning has been associated with poor immune reconstitution and increased susceptibility to opportunistic infections. Therefore, an ideal allogeneic cell therapy would be able to avoid immune rejection while reducing or eliminating the need for chemotherapeutic conditioning to deplete host lymphocytes. To address many of these challenges, we engineered our novel alloimmune defense receptor (ADR) that targets 41BB+ alloreactive immune cells while providing a CD3z signaling boost into our off-the-shelf iPSC derived NK cells expressing anti-CD19 CAR (CAR iNK). The ability of ADR+ CAR iNK cells to resist alloimmune rejection was tested by co-culturing ADR+ CAR iNK cells with allogeneic PBMCs from ten donors in a mixed lymphocyte reaction assay. Notably, ADR+ CAR iNK cells co-cultured with allogeneic PBMCs persisted to similar levels as the PBMC-free culture while ADR negative CAR iNK cells were eliminated when co-cultured with allogeneic PBMCs. Furthermore, all PBMC donors screened in ADR+ CAR iNK cell co-cultures showed ablation of reactive 41BB+ NK and T cells, with non-activated T cells remaining intact. CAR iNK cells +/- ADR were then compared in a Nalm6 disseminated in vivo model for anti-tumor efficacy. ADR+ CAR iNK cells and their ADR negative counterparts were found to equivalently control tumor. Building on this tumor model, we co-infused allogeneic T cells to mimic an immuno-competent setting. The data demonstrated that ADR negative CAR iNK cells were depleted and were unable to control tumor growth while significant levels of allogeneic T cells persisted. In contrast, ADR+ CAR iNK cells were able to resist allogeneic T cell attack, control tumor, and persist when compared to the ADR negative control. Collectively, our preliminary data suggest that ADR-armed CAR iNK cells withstand immune cell-mediated rejection with uncompromised effector function. We are actively developing models to confirm our initial finding that ADR+ effector cells also benefit from their engagement with alloreactive cells in immuno-competent settings to promote enhanced anti-tumor responses, proliferation, and persistence. Citation Format: Alan M. Williams, Ken Hayama, Yijia Pan, Brian Groff, Rina Mbofung, Lauren Fong, Nicholas Brookhouser, Berhan Mandefro, Ramzey Abujarour, Tom Lee, Quirin Hammer, Karl-Johan Malmberg, Maksim Mamonkin, Ryan Bjordahl, Jode Goodridge, Bahram Valamehr. A novel synthetic stealth receptor that redirects host immune cell alloreactivity and potentiates functional persistence of adoptively transferred off-the-shelf cell-based cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2828.

Introduction: B7-H3 (CD276) has gained significant clinical interest as a pan-tumor target antigen for development of various immuno-oncology agents. Due to its broad expression on a wide variety of solid tumors and minimal expression on normal tissues, B7-H3 is an ideal tumor antigen target. Additionally, high levels of B7-H3 are found on “immunologically cold” tumors such as glioblastoma multiforme, prostate cancer, head and neck cancer and soft tissue sarcomas, which typically have poor response to approved immune therapies. To effectively target B7-H3 with an off-the-shelf cellular therapy, we describe here the development of camB7-H3 CAR-NK cell utilizing our iPSC platform to engineer multiple modalities into a clonal iPSC line, which can serve as the starting cell source for mass production of off-the-shelf, iPSC-derived CAR-NK cells (CAR-iNK cells). Methods: A camelid nanobody specific for human B7-H3 (camB7-H3) was discovered using a phage display library and validated in functional assays. camB7-H3 CAR-iNK cells were designed to 1) express membrane-bound IL-15/IL-15 receptor fusion for enhanced persistence, 2) have a CD38 knockout to improve metabolic fitness, 3) express a high-affinity non-cleavable CD16 to maximize ADCC when combined with a therapeutic antibody, and 4) express an anti-camB7-H3 CAR optimized for NK cell signaling. As the initial preclinical study, camB7-H3 CAR-iNK cells were assessed using flow cytometry-based functional assays evaluating CD107a and IFNγ or xCelligence target killing assays against B7-H3 transgenic or naturally expressing tumor cell lines. Results: The camelid single domain B7-H3 initially tested in CAR-T exhibited B7-H3 specific binding and specific activity against several solid tumor cell lines (breast, ovarian, prostate, lung). We next produced camB7-H3 CAR-iNK cells and demonstrated superior B7-H3-specific target elimination compared to untransduced iNK cells. These results were seen across multiple tumor lines representing various solid tumor indications. Further enhancement of anti-tumor efficacy was seen when combined with therapeutic antibodies, including trastuzumab and cetuximab. Conclusions: We have successfully produced and validated the specificity and function of an engineered camB7-H3 CAR-iNK cell exhibiting robust target killing and on-target specificity. To our knowledge, this is the first camelid nanobody antigen recognition domain reported in a CAR-NK cell to be used as an off-the-shelf immunotherapy. The combining camB7-H3 CAR-NK with monoclonal antibodies targeting HER2 and EGFR as a dual targeting approach will add additional tumor specificity, further increase the efficacy of tumor cell elimination and prevent antigen escape. Additional camB7-H3 CAR-iNK cell preclinical studies are in process and will be discussed. Citation Format: Nicholas Zorko, Frank Cichocki, John Goulding, Bryan Hancock, Robert Blum, Mochtar Pribadi, Bjoern Gaertner, Tom Lee, Martin Felices, Ryan Bjordahl, Bahram Valamehr, Jeffrey S. Miller. FT573: Preclinical development of multiplexed-engineered iPSC-derived NK cells expressing a novel camelid nanobody chimeric antigen receptor (CAR) targeting pan-cancer antigen B7-H3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2761.

Resistance to CAR-T cell therapy through antigen loss and clonal heterogeneity is a major limiting factor in achieving durable responses, even in highly persistent cell therapies. Simultaneous targeting of multiple tumor antigens with a single therapeutic modality offers the potential to treat heterogeneous tumor populations, prevent antigen escape, and induce durable clinical remission. Here, we demonstrate the application of a unique dual-CAR approach simultaneously targeting two tumor associated antigens (TAA) for the treatment of multiple myeloma (MM) using an off-the-shelf induced pluripotent stem cell (iPSC) derived NK cell platform. The iPSC background has been functionally enhanced and can be combined with therapeutic antibodies. A CAR targeting B cell maturation antigen (BCMA), a well-defined TAA in MM, was designed utilizing a previously published high-affinity binding sequence shown to exhibit high selectivity to BCMA with enhanced recognition of low-BCMA expressing myeloma cells (Bluhm et al., Molec Ther 2018). This CAR was combined with a companion CAR targeting the pan-TAAs, MICA and MICB. The CAR binding sequence targets the conserved α3 domain of MICA/MICB, which we have previously shown to inhibit MICA/B shedding by blocking the putative cleavage site (Andrade et al., Science 2018). The designed anti-MICA/B-α3 CAR exhibits selective targeting potential against a broad range of tumor types. To determine the suitability of co-targeting BCMA and MICA/B in MM, we surveyed surface expression of BCMA and MICA/B on a variety of MM cancer cell lines and observed a complimentary pattern of co-expression compatible with a dual-CAR to broaden the targeting approach of malignant plasma cells. In a reductionist approach, dual CAR-iNK cells exhibited antigen-specific activation, degranulation and cytotoxicity against a Nalm6 target line constitutively expressing the BCMA and MICA/B. Similar trends were observed in a series of long-term cytotoxicity assays against several MM lines, consistent with antibody staining on target cells, illustrating that co-targeting of MICA/B and BCMA expands the breadth of coverage against MM. Known modulators of antigen expression were tested for their ability to provide further depth of response, and therapeutic antibodies such as anti-CD38 were tested in combination to exploit the non-cleavable CD16 and CD38KO edits in the iPSC backbone. These combinations were further tested in vivo against a disseminated model of multiple myeloma where BCMA/MICA dual CAR-iNK cells demonstrated superior tumor control relative to single-CAR controls, and TGI was augmented with the addition of Daratumamab. These data highlight the applicability of a multi-targeted approach in MM patients, whereby MM dual-CAR NK and/or T cells maintain responsiveness to malignant cells that shed or downregulate tumor antigens to evade treatment. Citation Format: Ketan Mathavan, John Reiser, Sajid Mahmood, Yijia Pan, Bryan Hancock, Robert Blum, Wen-I Yeh, Andrew Houk, Chia-Wei Chang, Tom Lee, Bobby Goulding, Jode Goodridge, Ryan Bjordahl, Bahram Valamehr, Uta Hoepken, Armin Rehm, Kai Wucherpfennig. Combining dual CAR iPSC-derived immune cells with antibody for multi-antigen targeting to overcome clonal resistance in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4190.

Select subsets of immune effector cells have the greatest propensity to mediate antitumor responses. However, procuring these subsets is challenging, and cell-based immunotherapy is hampered by limited effector-cell persistence and lack of on-demand availability. To address these limitations, we generated a triple-gene-edited induced pluripotent stem cell (iPSC). The clonal iPSC line was engineered to express a high affinity, non-cleavable version of the Fc receptor CD16a and a membrane-bound interleukin (IL)-15/IL-15R fusion protein. The third edit was a knockout of the ecto-enzyme CD38, which hydrolyzes NAD+. Natural killer (NK) cells derived from these uniformly engineered iPSCs, termed iADAPT, displayed metabolic features and gene expression profiles mirroring those of cytomegalovirus-induced adaptive NK cells. iADAPT NK cells persisted in vivo in the absence of exogenous cytokine and elicited superior antitumor activity. Our findings suggest that unique subsets of the immune system can be modeled through iPSC technology for effective treatment of patients with advanced cancer. acceptedVersion

Natural killer (NK) cells mediate the cytolysis of transformed cells and are currently used as an adoptive cellular therapy to treat cancer. Infection with human cytomegalovirus has been shown to expand a subset of “adaptive” NK cells expressing the activation receptor NKG2C that have preferred functional attributes distinct from conventional NK cells. Because NKG2C delivers a strong activating signal to NK cells, we hypothesized that NKG2C could specifically trigger NK-cell-mediated antitumor responses. To elicit a tumor-directed response from NKG2C(+) NK cells, we created an anti-NKG2C/IL-15/anti-CD33 killer engager called NKG2C-KE that directs NKG2C(+) cells to target CD33(+) cells and tumor-associated antigen expressed by acute myelogenous leukemia cells. The NKG2C-KE induced specific degranulation, interferon-γ production, and proliferation of NKG2C-expressing NK cells from patients who reactivated cytomegalovirus after allogeneic transplantation. The NKG2C-KE was also tested in a more homogeneous system using induced pluripotent stem cell (iPSC)-derived NK (iNK) cells that have been engineered to express NKG2C at high levels. The NKG2C-KE triggered iNK-cell-mediated cytotoxicity against CD33(+) cells and primary AML blasts. The NKG2C-KE-specific interaction with adaptive NK and NKG2C(+) iNK cells represents a new immunotherapeutic paradigm that uniquely engages highly active NK cells to induce cytotoxicity against AML through redirected targeting.

Background Although CAR T cells have been shown to be effective and potent in treating several hematologic malignancies, engineered T-cell therapies have had limited success in addressing solid tumors. Unlike liquid tumors where uniformly expressed antigens are accessible and can be effectively targeted, tumor access and antigen heterogeneity are a significant barrier to the successful development of CAR-T cells in solid tumors. Methods Here we demonstrate that the combination of a bi-specific T-cell engager (BiTE) targeting EpCAM with a CAR T cell targeting HER2 enhances the in vitro and in vivo anti-tumor activity against heterogenous solid tumors. Results We observed a dose-dependent enhancement of cytolytic activity when EpCAM-specific BiTEs were titrated alongside 4D5-based HER2-specific CAR T cells against HER2low tumors, enhancing maximal cytolysis by two-fold compared to CAR T cells alone (figure 1). Moreover, the escape of HER2low tumor cells in mixed heterogenous culture systems was circumvented by the combination of HER2-specific CAR T cells and EpCAM-specific BiTEs. The enhancement of efficacy was further demonstrated in an established HER2low MDA-MB-231 xenografts. HER2-specific CAR T cells were unable to contain Her2low tumors, whereas tumor growth was effectively controlled in mice receiving both EpCAM-specific BiTEs and HER2-specific CAR T cells. Conclusions Collectively, these data demonstrate that multi-antigen targeting mediated by BiTEs and CARs extends overall anti-tumor efficacy in preclinical models of heterogenous solid tumors. Fate Therapeutics is currently using its proprietary induced pluripotent stem cell (iPSC) product platform to generate iPSC-derived CAR T cells and iPSC-derived CAR NK cells that secrete BiTEs for the treatment of solid tumors. Ethics Approval These studies were approved by Fate Therapeutics Institutional Animal Care and Use Committee and were carried out in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals.

Background FT500 is an investigational, off-the-shelf NK cell cancer immunotherapy derived from a human clonal master iPSC line, a renewable cell source from which innate effector cells can be mass produced and made available off-the-shelf for broad patient access and multiple dose administration. FT500 has potent innate cellular cytotoxicity as compared to NK cells sourced from healthy donors and has been shown to synergize with T cells and anti-PD-1 blockade in preclinical studies.1 Methods FT500 is being investigated in a Phase I clinical trial as monotherapy and in combination with immune checkpoint inhibitors (ICIs) in patients with advanced solid tumors and lymphomas (ClinicalTrials.gov: NCT03841110). Treatment consists of 2 days lympho conditioning (fludarabine 25 mg/m2 and cyclophosphamide 300 mg/m2) followed by 2 cycles of 3 once weekly doses of FT500 as monotherapy or combined with 1 of 3 approved ICIs (nivolumab, pembrolizumab, or atezolizumab) in patients who have failed prior ICI therapy. Key clinical and translational readouts include FT500 safety and tolerability, including immune mediated toxicities and anti-product immunogenicity. Results 15 patients with relapsed/refractory disease following a median of 4 prior therapies were treated in dose escalation, including 9 with FT500 monotherapy (3 with 1×108 cells, 6 with 3×108 cells) and 6 with FT500 (3 each with 1×108 and 3×108 cells) combined with ICI. No dose limiting toxicities, Grade ³3 related adverse events (AEs), Grade ³3 related serious AEs, or related AEs leading to treatment discontinuation were reported. No graft-versus-host disease (GvHD), cytokine release syndrome (CRS), or neurotoxicity (NT) was observed. The most common treatment-emergent AEs in >3 patients were nausea (9), fatigue (7), constipation, decreased appetite, decreased lymphocyte count, decreased white blood cell count (5 each), anemia, and decreased neutrophil count (4 each). Nine of 13 efficacy-evaluable solid tumor patients had best response of stable disease by iRECIST. One patient with classical Hodgkin lymphoma (cHL) refractory to prior experimental anti-PD-1 therapy had a 58% reduction in target lesions size following FT500 plus ICI. No evidence of robust B- or T-cell mediated anti product responses was observed despite endogenous immune cell recovery following lympho-conditioning. Conclusions Administration of 6 doses of up to 3×108 FT500 cells is safe and tolerable without evidence of GvHD, CRS, NT, or host immune rejection. Enrollment of advanced non-small cell lung cancer and cHL patients at 3×108 FT500 cells per dose combined with ICI is ongoing. Ethics Approval This study is being conducted in accordance with the Declaration of Helsinki and was approved by all Institutional Review Boards from each clinical site participating in the study. Specific approval numbers can be provided upon request. Reference Cichocki F, Bjordahl R, Gaidarova S, Mahmood S, Rogers P, Ge MQ, Kaufman DS, Cooley S, Valamehr B, Miller JS. iPSC-derived NK cells and anti-PD-1 antibody synergize to enhance T cell cytokine and cytolytic responses against multiple tumors. Blood 2018;132(Supplement 1):730.

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.

The development of immunotherapeutic monoclonal antibodies targeting checkpoint inhibitory receptors, such as programmed cell death 1 (PD-1), or their ligands, such as PD-L1, has transformed the oncology landscape. However, durable tumor regression is limited to a minority of patients. Therefore, combining immunotherapies with those targeting checkpoint inhibitory receptors is a promising strategy to bolster antitumor responses and improve response rates. Natural killer (NK) cells have the potential to augment checkpoint inhibition therapies, such as PD-L1/PD-1 blockade, because NK cells mediate both direct tumor lysis and T cell activation and recruitment. However, sourcing donor-derived NK cells for adoptive cell therapy has been limited by both cell number and quality. Thus, we developed a robust and efficient manufacturing system for the differentiation and expansion of high-quality NK cells derived from induced pluripotent stem cells (iPSCs). iPSC-derived NK (iNK) cells produced inflammatory cytokines and exerted strong cytotoxicity against an array of hematologic and solid tumors. Furthermore, we showed that iNK cells recruit T cells and cooperate with T cells and anti-PD-1 antibody, further enhancing inflammatory cytokine production and tumor lysis. Because the iNK cell derivation process uses a renewable starting material and enables the manufacturing of large numbers of doses from a single manufacture, iNK cells represent an "off-the-shelf" source of cells for immunotherapy with the capacity to target tumors and engage the adaptive arm of the immune system to make a "cold" tumor "hot" by promoting the influx of activated T cells to augment checkpoint inhibitor therapies.

Antibody-dependent cellular cytotoxicity (ADCC) is a key effector mechanism of natural killer (NK) cells that is mediated by therapeutic monoclonal antibodies (mAbs). This process is facilitated by the Fc receptor CD16a on human NK cells. CD16a appears to be the only activating receptor on NK cells that is cleaved by the metalloprotease a disintegrin and metalloproteinase-17 upon stimulation. We previously demonstrated that a point mutation of CD16a prevents this activation-induced surface cleavage. This noncleavable CD16a variant is now further modified to include the high-affinity noncleavable variant of CD16a (hnCD16) and was engineered into human induced pluripotent stem cells (iPSCs) to create a renewable source for human induced pluripotent stem cell–derived NK (hnCD16-iNK) cells. Compared with unmodified iNK cells and peripheral blood–derived NK (PB-NK) cells, hnCD16-iNK cells proved to be highly resistant to activation-induced cleavage of CD16a. We found that hnCD16-iNK cells were functionally mature and exhibited enhanced ADCC against multiple tumor targets. In vivo xenograft studies using a human B-cell lymphoma demonstrated that treatment with hnCD16-iNK cells and anti-CD20 mAb led to significantly improved regression of B-cell lymphoma compared with treatment utilizing anti-CD20 mAb with PB-NK cells or unmodified iNK cells. hnCD16-iNK cells, combined with anti-HER2 mAb, also mediated improved survival in an ovarian cancer xenograft model. Together, these findings show that hnCD16-iNK cells combined with mAbs are highly effective against hematologic malignancies and solid tumors that are typically resistant to NK cell–mediated killing, demonstrating the feasibility of producing a standardized off-the-shelf engineered NK cell therapy with improved ADCC properties to treat malignancies that are otherwise refractory.

Cell therapy is emerging as a very promising therapeutic modality against cancer, spearheaded by the clinical success of chimeric antigen receptor (CAR) modified T cells for B cell malignancies. Currently, FDA-approved CAR-T cell products are based on engineering of autologous T cells harvested from the patient, typically using a central manufacturing facility for gene editing before the product can be delivered to the clinic and infused to the patients. For a broader implementation of advanced cell therapy and to reduce costs, it would be advantageous to use allogeneic “universal” cell therapy products that can be stored in cell banks and provided upon request, in a manner analogous to biopharmaceutical drug products. In this review, we outline a roadmap for development of off-the-shelf cell therapy based on natural killer (NK) cells derived from induced pluripotent stem cells (iPSCs). We discuss strategies to engineer iPSC-derived NK (iPSC-NK) cells for enhanced functional potential, persistence, and homing.

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.

Chimeric antigen receptor (CAR) T-cell therapy has proven highly effective in patients with hematological malignancies. However, resistance to CAR-T cell therapy arising from target protein shedding and other forms of antigen downregulation can lead to CAR-resistant disease relapse. Tumor escape may be successfully prevented through the simultaneous targeting of multiple tumor antigens. The ability to target multiple antigens with a single therapeutic modality offers the potential for anti-tumor responses, broader coverage of heterogeneous tumor populations, and the potential to prevent antigen escape, potentially inducing durable clinical remission. Multiple myeloma (MM) presents an ideal case to employ a dual-CAR approach, as BCMA-targeting cell therapies have shown impressive efficacy to date, but curative treatment remains elusive. Additionally, the oligoclonal nature of MM may contribute to antigen escape and clonal resistance. Here, we demonstrate the application of a unique dual-CAR approach simultaneously targeting two tumor associated antigens (TAA) for the treatment of MM. We further demonstrate the efficacy in an induced pluripotent stem cell (iPSC) platform, where a master engineered iPSC line is used as the starting material for mass production of off-the-shelf, dual-CAR immune effector cells. We selected B cell maturation antigen (BCMA), a well-defined TAA in MM, as the first antigen target. To develop the CAR-BCMA motif, we utilized our previously published high-affinity binding sequence shown to exhibit high selectivity to BCMA with enhanced recognition of low-BCMA expressing myeloma cells (Bluhm et al., Molec Ther 2018). As shown previously, the designed CAR-BCMA demonstrates potent and selective cellular cytotoxicity against MM (Figure 1a, left panel). BCMA has been observed to be actively cleaved from the surface of MM cells though, resulting in reduced efficacy and clinical relapse. To circumvent BCMA antigen escape, we developed a companion CAR targeting the pan-TAAs, MICA and MICB, which are expressed on MM plasma cells as well as monoclonal gammopathy of undetermined significance (MGUS) plasma cells. The CAR binding sequence targets the conserved α3 domain of MICA/MICB, which we have previously shown to inhibit MICA/B shedding and drive anti-tumor immunity (Andrade et al., Science 2018). The designed anti-MICA/B-α3 CAR exhibits selective targeting potential against an array of cancers, including the MM.1S cancer cell line (Figure 1a, right panel). To determine the suitability of co-targeting BCMA and MICA/B in MM, we surveyed surface expression patterns of BCMA and MICA/B antigens on a variety of MM cancer cell lines and observed a complimentary pattern of co-expression compatible with a dual-CAR to broaden targeting approach of malignant plasma cells (Figure 1b). Initial studies to evaluate the dual CAR approach in MM were performed by generating anti-BCMA and anti-MICA/B-α3 dual-CAR (MM dual-CAR) T-cells. MM dual-CAR T cells showed antigen-specific activation, degranulation and cytotoxicity against both antigens in an additive manner, consistent with the initial antibody staining on target cells and illustrating that co-targeting MICA/B and BCMA may increase the activity against MM (Figure 1c). Similar trends were observed in a series of cytotoxicity assays against several MM lines. Preliminary studies are ongoing in induced pluripotent stem cell (iPSC)-derived NK (iNK) cells expressing MM dual-CARs as a unique off-the-shelf cell therapy targeting both BCMA and MICA/B. Since MM dual-CAR iNK cells also express CD16, which mediates antibody-dependent cellular cytotoxicity, combination with therapeutic antibodies, such as anti-CD38 antibodies, can be deployed to target three TAAs for a complete therapeutic approach in MM. The data highlights the applicability of a multi-targeted approach in MM patients, whereby MM dual-CAR NK and/or T cells maintain responsiveness to malignant cells that shed or downregulate tumor antigens to evade treatment. Figure 1 Figure 1. Disclosures Lee: Fate Therapeutics, Inc.: Current Employment. Wucherpfennig: Novartis: Research Funding; SQZ Biotech: Membership on an entity's Board of Directors or advisory committees; TScan Therapeutics: Membership on an entity's Board of Directors or advisory committees; Immunitas Therapeutics: Current holder of individual stocks in a privately-held company; Nextechinvest: Membership on an entity's Board of Directors or advisory committees; TCR2 Therapeutics: Membership on an entity's Board of Directors or advisory committees. Bjordahl: Fate Therapeutics: Current Employment. Valamehr: Fate Therapeutics, Inc.: Current Employment.

Treatments for B-cell malignancies have improved over the past several decades with clinical application of the CD20-specific antibody rituximab and chimeric antigen receptor (CAR) T cells targeting CD19. Despite the success of these therapies, loss of CD20 after rituximab treatment has been reported in leukemia and lymphoma patients. Additionally, up to 50% of all patients receiving anti-CD19 CAR T-cell therapy relapse within the first year with many of those patients exhibiting CD19 loss. Thus, new therapeutic approaches are needed to address tumor antigen escape. Accordingly, we generated triple gene-modified iPSC-derived NK (iNK) cells, termed "iDuo" NK cells, tailored to facilitate multi-antigen targeting. The iPSC line was clonally engineered to express high-affinity, non-cleavable CD16a (hnCD16), an anti-CD19 CAR optimized for NK cell signaling, and a membrane-bound IL-15/IL-15R fusion (IL-15RF) molecule to enhance NK cell persistence (Fig. 1A). To model antigen escape, we generated CD19 knockout AHR77 lymphoma cells alongside wild type AHR77 cells (both CD20 +) as targets in cytotoxicity assays. Activated peripheral blood NK (PBNK) cells, non-transduced iNK cells, and iDuo NK cells were tested as effectors. Unlike PBNK cells or non-transduced iNK cells, iDuo NK cells efficiently eliminated wild type AHR77 cells with or without the addition of rituximab at all tested E:T ratios. Similarly, iDuo NK cells in combination with rituximab were uniquely able to efficiently eliminate CD19 KO AHR77 cells due to enhanced antibody-dependent cellular cytotoxicity (ADCC) driven by hnCD16 (Fig. 1B-E). Cytotoxicity mediated by iDuo NK cells was also evaluated using primary chronic lymphocytic leukemia (CLL) cells. Compared to expanded PBNK cells and non-transduced iNK cells, only iDuo NK cells (in the absence of rituximab) were able to kill primary CLL cells (Fig. 1F). Expression of IL-15RF by iDuo NK cells uniquely supports in vitro expansion without the need for cytokine supplementation. To determine whether IL-15RF supports in vivo persistence of iDuo NK cells, CD19 CAR iNK cells (lacking IL-15RF) and iDuo NK cells were injected into NSG mice without the addition of cytokines or CD19 antigen availability. iDuo NK cell numbers peaked within a week after injection and persisted at measurable levels for ~5 weeks, in marked contrast to CD19 CAR iNK cell numbers that were undetectable throughout (Fig. 1G). To evaluate the in vivo function of iDuo NK cells, NALM6 leukemia cells were engrafted into NSG mice. Groups of mice received tumor alone or were treated with 3 doses of thawed iDuo NK cells. iDuo NK cells alone were highly effective in this model as evidenced by complete survival of mice in the treatment group (Fig. 1H). To assess iDuo NK cells in a more aggressive model, Raji lymphoma cells were engrafted, and groups of mice received rituximab alone, iDuo NK cells alone, or iDuo NK cells plus rituximab. Mice given the combination of iDuo NK cells and rituximab provided extended survival compared to all other arms in the aggressive disseminated Raji lymphoma xenograft model (Fig. 1I). One disadvantage of anti-CD19 CAR T cells is their inability to discriminate between healthy and malignant B cells. Because NK cells express inhibitory receptors that enable "self" versus "non-self" discrimination, we reasoned that iDuo NK cells could have higher cytotoxicity against tumor cells relative to healthy B cells. To address this, we labeled Raji cells, CD19 + B cells from healthy donor peripheral blood mononuclear cells (PBMCs) and CD19 - PBMCs. Labeled populations of cells were co-cultured with iDuo NK cells, and specific killing was analyzed. As expected, iDuo NK cells did not target CD19 - PBMCs. Intriguingly, iDuo NK cells had much higher cytotoxic activity against Raji cells compared to primary CD19 + B cells, suggesting a preferential targeting of malignant B cells compared to healthy B cells. Together, these results demonstrate the potent multi-antigen targeting capability and in vivo antitumor function of iDuo NK cells. Further, these data suggest that iDuo NK cells may have an additional advantage over anti-CD19 CAR T cells by discriminating between healthy and malignant B cells. The first iDuo NK cell, FT596, is currently being tested in a Phase I clinical trial (NCT04245722) for the treatment of B-cell lymphoma. Figure 1 Figure 1. Disclosures Cichocki: Gamida Cell: Research Funding; Fate Therapeutics, Inc: Patents & Royalties, Research Funding. Bjordahl: Fate Therapeutics: Current Employment. Gaidarova: Fate Therapeutics, Inc: Current Employment. Abujarour: Fate Therapeutics, Inc.: Current Employment. Rogers: Fate Therapeutics, Inc: Current Employment. Huffman: Fate Therapeutics, Inc: Current Employment. Lee: Fate Therapeutics, Inc: Current Employment. Szabo: Fate Therapeutics, Inc: Current Employment. Wong: BMS: Current equity holder in publicly-traded company; Fate Therapeutics, Inc: Current Employment. Cooley: Fate Therapeutics, Inc: Current Employment. Valamehr: Fate Therapeutics, Inc.: Current Employment. Miller: Magenta: Membership on an entity's Board of Directors or advisory committees; ONK Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Vycellix: Consultancy; GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees; Wugen: Membership on an entity's Board of Directors or advisory committees.

FT819 is a first-of-kind, allogeneic, off-the-shelf CAR T-cell therapy derived from a clonal master induced pluripotent stem cell (iPSC) line precisely engineered to insert a novel 1XX anti-CD19 chimeric antigen receptor (CAR) under the regulation of the T-cell receptor alpha constant (TRAC) locus for optimized control of anti-tumor activity and to completely delete T-cell receptor (TCR) expression to eliminate the potential of graft-versus-host disease (GvHD). Unlike conventional allogeneic CAR T-cell therapies which require repeatedly sourcing of T cells from various donors as the starting material, the use of a clonal master engineered iPSC line serves as a renewable starting cell source and ensures routine mass production of a uniformly engineered, homogenous CAR T-cell product for broad patient access. T cell-derived iPSCs were generated using a proprietary non-integrating cellular reprogramming system and genetically modified to integrate a novel anti-CD19 1XX CAR into both alleles of the TRAC gene. After single cell subcloning, each engineered iPSC clone was screened for multiple critical quality attributes including pluripotency, identity, genomic stability, cassette integration, on/off-target integration, T-cell differentiation propensity, and CAR T-cell function. Accordingly, the ideal single cell-derived engineered iPSC clone was selected as the clonal master iPSC line for FT819 and was converted into a master cell bank (MCB). The iPSC MCB serves as a renewable source for the routine GMP manufacture of FT819 drug product. The FT819 production process consists of three stages: 1) generation of CD34-expressing hematopoietic progenitor cells from iPSCs (>90% CD34+ cells post enrichment); 2) lineage-specification to T cells followed by T-cell expansion (>5e5 fold expansion); and 3) fill/finish and cryopreservation of the drug product. As an example, in an initial small-scale manufacturing campaign, a total of 2.5 × 10 10 FT819 CAR T-cells were generated and filled and finished starting from one vial of the MCB. The FT819 drug product was tested on safety, identity, purity, and potency. The final product was comprised of CD45+CD7+ lymphocytes (>99%), with homogeneous CAR expression (>99% CAR+) and lacking expression of TCRαβ (not detected) on the cell surface. Importantly, there were no residual iPSCs detected in the FT819 drug product. The FT819 drug product exhibited potent and consistent effector function against NALM6 leukemia cells. The FT819 drug product is currently being used in a landmark Phase I study (NCT04629729), the first-ever iPSC-derived T-cell therapy to undergo clinical investigation, for the treatment of patients with relapsed/refractory B-cell lymphoma, chronic lymphocytic leukemia and precursor B-cell acute lymphoblastic leukemia. In summary, FT819 is a first-of-kind, off-the-shelf, CAR T-cell therapy uniquely derived from a clonal multiplexed-engineered master iPSC line. The novel manufacturing paradigm enables mass production of a uniformly engineered, homogenous cell therapy product that is available on-demand for broad patient access. A multi-center Phase 1 study of FT819 is currently ongoing for the treatment of B-cell malignancies. Key Words: cancer immunotherapy, cell therapy, CAR-T, CD19, allogeneic, induced pluripotent stem cell, iPSC, clonal master iPSC line, engineered, off-the-shelf, cGMP, production, manufacturing, FT819 Disclosures Yuan: Fate Therapeutics, Inc.: Current Employment. Clarke: Fate Therapeutics, Inc.: Current Employment. Lai: Fate Therapeutics, Inc.: Current Employment. Chang: Fate Therapeutics, Inc.: Current Employment. Yang: Fate Therapeutics, Inc.: Current Employment. Hsia: Fate Therapeutics, Inc.: Current Employment. Abujarour: Fate Therapeutics, Inc.: Current Employment. Lee: Fate Therapeutics, Inc.: Current Employment. van der Stegen: Fate Therapeutics, Inc.: Current Employment. Shaked: Fate Therapeutics, Inc.: Current Employment. Jalloh: Fate Therapeutics, Inc.: Current Employment. Moreno: Fate Therapeutics, Inc.: Current Employment. ORourke: Fate Therapeutics, Inc.: Current Employment. Sung: Fate Therapeutics, Inc.: Current Employment. Gutierrez: Fate Therapeutics, Inc.: Current Employment. Rezner: Fate Therapeutics, Inc.: Current Employment. Eberhart: Fate Therapeutics, Inc.: Current Employment. Magdaleno: Fate Therapeutics, Inc.: Current Employment. Farnan: Fate Therapeutics, Inc.: Current Employment. Plavsic: Fate Therapeutics, Inc.: Current Employment. Bressi: Fate Therapeutics, Inc.: Current Employment. Rivière: Centre for Commercialization of Cancer Immunotherapy: Other: Provision of Services; Fate Therapeutics: Other: Provision of Services, Patents & Royalties; The Georgia Tech Research Corporation (GTRC): Other: Provision of Services (uncompensated); FloDesign Sonics: Other: Provision of Services; Juno Therapeutics: Patents & Royalties. Valamehr: Fate Therapeutics, Inc.: Current Employment.

Chimeric antigen receptor (CAR) T-cell therapies have revolutionized the treatment of hematologic malignancies, however, logistical complexities associated with patient-specific CAR T-cell therapies often limit broad accessibility to patients. Many of these challenges can be overcome with an allogeneic cellular product that is available off-the-shelf, and overcoming immune cell-mediated rejection of allogeneic cell therapy is an area of significant research. Conditioning chemotherapies, which are commonly administered with CAR T-cell therapy, suppress a patient's immune system and may create a suitable window of activity for allogeneic cell therapies to elicit clinical response. However, protracted lympho-conditioning has been associated with poor immune reconstitution and increased susceptibility to opportunistic infections. Deletion of human leukocyte antigen (HLA) surface expression is known to abrogate T-cell alloreactivity, but deletion of class I HLA must be combined with other immune-modulating strategies to avoid NK cell-mediated recognition. To this end, allogeneic models combining class I HLA deletion with NK cell inhibitory molecules, such as HLA-E and CD47, have been shown to abrogate NK cell reactivity in mouse models. However, since HLA-E is the canonical activator of NKG2C, a dominant activating receptor found on human NK cells, and since the ligand for CD47, SIRPα, is known to be expressed on macrophages and dendritic cells and not on human NK cells, the observed effects of these immune-modulating strategies may not translate into the patient-treatment setting. In assessment of their protective effects in a defined human system, we found that HLA-E or CD47 overexpression on class I HLA null human cells offer only partial protection in evading various human NK cell compartments. We found that class I HLA-null K562 cells engineered to over-express CD47 were ineffective in inhibiting NK cells (0 to 7% inhibition). Separately, K562 cells engineered to over-express HLA-E, while effective in inhibiting NKG2A+ NK cells (90.2% +/- 3.7% inhibition), were unable to completely inhibit CD56 dim NK cells (33.2% +/- 29.6% inhibition) and not only failed, but instead activated, NKG2C+ NK cells (167% +/- 69% activation). Our data highlight the limitations of engineered CD47 and HLA-E modalities in suppressing broad populations of NK cells in clinically relevant settings. We therefore evaluated expression of the alloimmune defense receptor (ADR) that uniquely targets alloreactive immune cells (Mo et al. Nat Biotechnol 2021). We have shown that the expression of ADR has the potential to evade host immune cells without the need for further genetic editing such as class I HLA deletion. To determine its applicability for off-the-shelf cell therapy, ADR expression was engineered into induced pluripotent stem cells (iPSCs) and iPSC-derived CAR-NK (CAR-iNK) cells were generated. CAR-iNK cells carrying the ADR modality (ADR+ CAR-iNK cells) showed normal patterns of differentiation (>99% CD56+ with co-expression of NK cell receptors such as NKG2D, NKp30 and NKp46), suggesting that ADR expression did not disrupt hematopoiesis or the expansion of iNK cells. Additionally, ADR+ and ADR-negative CAR-iNK cells produced similar cytotoxicity against tumor cells. We next tested the ability of ADR to provide resistance to alloimmune rejection by coculturing ADR+ CAR-iNK cells with allogeneic pBMCs in a mixed lymphocyte reaction (MLR) assay. Notably, ADR+ CAR-iNK cells maintained durable persistence throughout the entire duration of the MLR assay and suppressed the expansion of alloreactive T- and NK-cells in comparison to the control arm (Figure 1). Collectively, initial preclinical studies suggest that ADR-modified CAR-iNK cells resist host immune cell rejection, while eliciting a durable anti-tumor response. Our preliminary data show evidence toward a promising off-the-shelf solution for elimination of broad pools of alloreactive T- and NK- effector cells in the clinical setting without the need for lympho-depleting conditioning or genetic editing strategies. Figure 1 Figure 1. Disclosures Williams: Fate Therapeutics: Current Employment. Abujarour: Fate Therapeutics, Inc.: Current Employment. Lee: Fate Therapeutics, Inc.: Current Employment. Malmberg: Merck: Research Funding; Vycellix: Consultancy; Fate Therapeutics: Consultancy, Research Funding. Mamonkin: Beam Therapeutics: Other: Licensing payments; Fate Therapeutics: Other: Licensing payments; Allogene Therapeutics: Consultancy, Other: Licensing payments; Xenetic Biosciences: Consultancy, Membership on an entity's Board of Directors or advisory committees. Bjordahl: Fate Therapeutics: Current Employment. Valamehr: Fate Therapeutics, Inc.: Current Employment.