Lentiviral vector optimization enhances the expression and cytotoxicity of chimeric antigen receptors

Adoptive chimeric antigen receptor (CAR)-modified T or NK cells (CAR-T/NK) have emerged as a novel form of disease treatment. Lentiviral vectors (LVs) are commonly employed to engineer T/NK cells for the efficient expression of CARs. This study reported for the first time the influence of single-promoter and dual-promoter LVs on the CAR expression and cytotoxicity of engineered NK cells. Our results demonstrated that the selected CAR exhibits both a higher expression level and a higher coexpression concordance with the GFP reporter in HEK-293T or NK92 cells by utilizing the optimized single-promoter pCDHsp rather than the original dual-promoter pCDHdp. After puromycin selection, the pCDHsp produces robust CAR expression and enhanced in vitro cytotoxicity of engineered NK cells. Therefore, infection with a single-promoter pCDHsp lentivector is recommended to prepare CAR-engineered cells. This research will help to optimize the production of CAR-NK cells and improve their functional activity, to provide CAR-NK cell products with better and more uniform quality.

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RTID-07. HUMAN PLACENTAL HEMATOPOIETIC STEM CELL DERIVED NATURAL KILLER CELLS (CYNK-001) FOR TREATMENT OF RECURRENT GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa215.812 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii194-ii195

Author(s):

Nazanin Majd ◽

Maha Rizk ◽

Solveig Ericson ◽

Kris Grzegorzewski ◽

Sharmila Koppisetti ◽

...

Keyword(s):

Cell Therapy ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

In Vitro Cytotoxicity ◽

Orthotopic Mouse Model ◽

Hematopoietic Stem ◽

Dismal Prognosis

Abstract Glioblastoma (GBM) is the most aggressive primary brain tumor with dismal prognosis. Recent advances of immunotherapy in cancer have sparked interest in the use of cell therapy for treatment of GBM. Active transfer of Natural Killer (NK) cells is of particular interest in GBM because NK cells are capable of exerting anti-tumor cytotoxicity without the need for antigen presentation and sensitization, processes that are impaired in GBM. CYNK-001 is an allogeneic, off-the-shelf product enriched for CD56+/CD3- NK cells expanded from placental CD34+ cells manufactured by Celularity. Here, we demonstrate in vitro cytotoxicity of CYNK-001 against several GBM lines and its in vivo anti-tumor activity in a U87MG orthotopic mouse model via intracranial administration resulting in 94.5% maximum reduction in tumor volume. We have developed a phase I window-of-opportunity trial of CYNK-001 in recurrent GBM via intravenous (IV) and intratumoral (IT) routes. In the IV cohort, subjects receive cyclophosphamide for lymphodepletion followed by 3-doses of IV CYNK-001 weekly. In the IT cohort, subjects undergo placement of an IT catheter with an ommaya reservoir followed by 3-doses of IT CYNK-001 weekly. Patients are monitored for 28-days after last infusion for toxicity. Once maximum safe dose (MSD) is determined, patients undergo IV or IT treatments at MSD followed by surgical resection and the tumor tissue will be analyzed for NK cell engraftment and persistence. We will utilize a 3 + 3 dose de-escalation design (maximum n=36). Primary endpoint is safety and feasibility. Secondary endpoints are overall response rate, duration of response, time to progression, progression free survival and overall survival. Main eligibility criteria include age ≥18, KPS ≥60, GBM at first or second relapse with a measurable lesion on ≤2mg dexamethasone. This is the first clinical trial to investigate CYNK-001 in GBM and will lay the foundation for future NK cell therapy in solid tumors.

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Importance of T, NK, CAR T and CAR NK Cell Metabolic Fitness for Effective Anti-Cancer Therapy: A Continuous Learning Process Allowing the Optimization of T, NK and CAR-Based Anti-Cancer Therapies

Cancers ◽

10.3390/cancers14010183 ◽

2021 ◽

Vol 14 (1) ◽

pp. 183

Author(s):

Adrien Krug ◽

Adriana Martinez-Turtos ◽

Els Verhoeyen

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Building Blocks ◽

Cancer Therapies ◽

Cell Therapies ◽

Anti Cancer ◽

Car T ◽

Metabolic Fitness

Chimeric antigen receptor (CAR) T and CAR NK cell therapies opened new avenues for cancer treatment. Although original successes of CAR T and CAR NK cells for the treatment of hematological malignancies were extraordinary, several obstacles have since been revealed, in particular their use for the treatment of solid cancers. The tumor microenvironment (TME) is competing for nutrients with T and NK cells and their CAR-expressing counterparts, paralyzing their metabolic effective and active states. Consequently, this can lead to alterations in their anti-tumoral capacity and persistence in vivo. High glucose uptake and the depletion of key amino acids by the TME can deprive T and NK cells of energy and building blocks, which turns them into a state of anergy, where they are unable to exert cytotoxic activity against cancer cells. This is especially true in the context of an immune-suppressive TME. In order to re-invigorate the T, NK, CAR T and CAR NK cell-mediated antitumor response, the field is now attempting to understand how metabolic pathways might change T and NK responses and functions, as well as those from their CAR-expressing partners. This revealed ways to metabolically rewire these cells by using metabolic enhancers or optimizing pre-infusion in vitro cultures of these cells. Importantly, next-generation CAR T and CAR NK products might include in the future the necessary metabolic requirements by improving their design, manufacturing process and other parameters. This will allow the overcoming of current limitations due to their interaction with the suppressive TME. In a clinical setting, this might improve their anti-cancer effector activity in synergy with immunotherapies. In this review, we discuss how the tumor cells and TME interfere with T and NK cell metabolic requirements. This may potentially lead to therapeutic approaches that enhance the metabolic fitness of CAR T and CAR NK cells, with the objective to improve their anti-cancer capacity.

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Expanded Natural Killer (NK) Cells Transfected with Anti-CD20 Chimeric Antigen Receptor (CAR) mRNA Have Significant Cytotoxicity Against Poor Risk B-Cell (CD20+) Leukemia/Lymphoma (B-L/L).

Blood ◽

10.1182/blood.v120.21.3007.3007 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3007-3007 ◽

Cited By ~ 2

Author(s):

Yaya Chu ◽

Janet Ayello ◽

Lowrence Lo ◽

Jared Katz ◽

Ashlin Yahr ◽

...

Keyword(s):

Flow Cytometry ◽

Nk Cells ◽

Natural Killer ◽

Chimeric Antigen Receptor ◽

Nk Cell ◽

In Vitro Cytotoxicity ◽

Activating Receptors ◽

Significant Difference ◽

Anti Cd20

Abstract Abstract 3007 Background: The outcome for children and adolescents with B-L/L has improved significantly but for patients who relapse or progress, the prognosis is dismal due to chemo-immuno-radiotherapy resistance (Cairo, et al, J Clin Oncol, 2012). Novel, non-chemotherapy-based therapies are desperately needed for this specific poor risk population. Natural Killer (NK) cells play an important role in tumor surveillance post allogeneic stem cell transplantation (Ruggeri L et al., Science 2002) but cell number and tumor specific recognition limit adoptive NK cell therapy (Shereck/Cairo, PBC 2007). Genetically engineered and expanded NK cells with an anti-CD19 CAR have been previously reported by Campana et al (Li L et al, Cancer Gene Ther. 2010). Anti-CD20 CAR transduced primary NK cells by retrovirus were reported by our group (Chu & Cairo, et al, ASH, 2011). Objective: To generate large-scale, efficiently modified NK cells with low cost, clinical applicable and a non-virus method, we investigated the functional activities of anti-CD20 chimeric antigen receptor (CAR+) modified PBNK cells following mRNA nucleofection against CD20+ B-L/L. Methods: PBMC were expanded with mitomycin C treated K562-mbIL15–41BBL cells for 7 or 14 days. CD56+CD3− expanded PBNK (exPBNK) cells were isolated using Miltenyi NK cell isolation kit. CD56, CD3 and receptor expression were evaluated by flow cytometry. Anti-CD20-4-1BB-CD3ζ was subcloned into a pcDNA3 vector. Anti-CD20-4-1BB-CD3ζ mRNA (CAR mRNA) was produced using the mMESSAGE mMACHINE T7 Ultra kit from T7 promoter. CAR mRNA was nucleofected into exPBNK using Amaxa nucleofector. CAR expression was detected using FITC-conjugated goat anti-mouse IgG, F(ab')2 fragment-specific antibody. exPBNK cytotoxicity was assessed by europium release assay at different E:T ratios against CD20+ B-L/L. CD107a degranulation and intracellular IFNgƒnproduction in exPBNK were measured by flow cytometry after stimulation with medium, K562-mbIL15–41BBL, CD20+ Ramos, CD20+Daudi, or CD20− RS4;11 for 4–6 hrs. Results: CD56+CD3− exPBNK cells were significantly expanded by mitomycin C treated K562-mbIL15–41BBL cells at day7. exPBNK cells were selected with more than 96% purity of CD56+CD3−. 50 to 95% exPBNK cells were detected to express CAR at 16 hrs after CAR mRNA nucleofection. CAR mRNA nucleofection did not affect the expression of exPBNK activating receptors (CD16, CD69, NKG2D, CD244, NKp30, NKp44, NKp46) or inhibitory receptors (NKG2A, KIR2DS4, CD94, CD158a, CD158b, CD158e). exPBNK in vitro cytotoxicity was significantly enhanced by CAR+ exPBNK compared to CAR− exPBNK against CD20+ B-L/L at 10:1 (n>3): Ramos (97.25+ 2.61% vs 82.5+ 4.058%, p<0.05), Daudi (71.5+ 3.26% vs 36.34+ 6.31%, p<0.001), Raji (21.45+ 1.98% vs 6.94+ 5.64%, p<0.05), Raji-2R (a Rituximab resistant cell line) (96.39+ 1.03% vs 86.3+ 1.52%, p<0.01), and U-698-M (82.84+ 1.17% vs 26.2+ 0.776%, p<0.001). However, there was no significant difference against CD20− RS4;11 or Jurkat cells. Consistently, CD107a degranulation was enhanced in CAR+ exPBNK compared to CAR− exPBNK in response to CD20+ Ramos (31.47+ 1.74% vs 15.2+ 0.26%, p<0.001, n=3) and Daudi (38.9+ 2.7% vs 19.73+ 0.58%, p<0.001, n=3) stimulation, however, there was no significant difference in response to RS4;11 or medium. Intracellular IFNγ production was also enhanced in CAR+ exPBNK compared to CAR− exPBNK in response to CD20+ Ramos and Daudi specific stimulation. We also observed that the expression of exPBNK activating receptors (CD69, NKp44 and NKG2D) were enhanced similarly and inhibitory receptors (CD94 and CD158b) were unchanged in mock exPBNK and CAR+ exPBNK cells after incubation with U-698-M compared to medium, implying CAR+PBNK directed enhanced cytotoxicity is mainly mediated by engineered CAR but not by endogenous NK receptors. Conclusion: Anti-CD20 CAR expression in exPBNK cells by mRNA nucleofection was associated with a significant increase in CD107 degradulation and INF-g production after stimulated with CD20+ BL/L compared to mock exPBNK cells. Consequently, Anti-CD20 CAR expression in exPBNK cells results in significant and specific exPBNK in vitro cytotoxicity against CD20+ B-L/L. Future directions include examining CAR+ exPBNK cytotoxic activity against CD20+ primary B-L/L tumor cells isolated from patients and testing the anti-tumor activity of CAR+ exPBNK against B-L/L and animal survival in xenograft mice. Disclosures: No relevant conflicts of interest to declare.

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A Bicistronic Vector Expressing CD16 and a Membrane Bound IL-15 Construct in iPSC Derived NK Cells Increased Cytotoxicity and Persistence

Blood ◽

10.1182/blood-2021-153258 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4809-4809

Author(s):

Alexander G Allen ◽

Rithu Pattali ◽

Kaitlyn M Izzo ◽

Jared A Getgano ◽

Kevin M Wasko ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Nk Cell ◽

Constitutive Expression ◽

Publicly Traded ◽

Cell Therapies ◽

Car T Cells ◽

Car T ◽

Membrane Bound

Abstract Current cell and gene therapy medicines for oncology have reshaped how cancer is treated. Specifically, chimeric antigen receptor (CAR)-T cells have demonstrated that cell therapy can achieve durable remissions in hematologic malignancies. However, CAR-T cell therapies have limited efficacy in solid tumors and are often associated with severe toxicity, highlighting the need for novel cell therapies that are safer and more efficacious. With their intrinsic killing capacity of tumor cells and few, if any, treatment related toxicities, natural killer (NK) cell therapies represent an attractive alternative therapy option to CAR-T cells. In addition, NK cells can be generated from allogeneic donors and given to patients off-the-shelf without causing graft versus host disease. Of the various sources of donor types to generate NK cells from, induced pluripotent stem cells (iPSCs) have the unique advantage of being a renewable source. A clone with any desired edits to enhance the effector function of NK cells can be derived, fully characterized, and expanded indefinitely, to generate large quantities of a naturally allogeneic medicine, therefore streamlining the manufacturing process and increasing scalability. Here, a bicistronic cargo encoding CD16 and a membrane-bound IL-15 (mbIL-15) was knocked into iPSCs at the GAPDH locus using an engineered and highly active AsCas12a. The promoter at the GAPDH locus drives robust constitutive expression of inserted cargos and avoids the promoter silencing that often occurs during differentiation with other strategies. CD16 and mbIL-15 were selected as Knock-Ins (KI) to specifically enhance NK cell therapy in two areas, namely NK cell deactivation caused by CD16 downregulation, and the reliance of co-administration of cytokines such as IL-15 or IL-2 for persistence. CD16 (FcRyIII) can bind the Fc portion of IgG antibodies triggering the lysis of targeted cells. This mechanism of cytotoxicity is known as antibody dependent cellular cytotoxicity (ADCC), and is an innate immune response largely mediated by NK cells through CD16. ADCC is severely impaired when surface CD16 is cleaved by a metalloprotease known as ADAM17. By having CD16 expressed from the GAPDH locus, there is consistent CD16 protein expression to replace what is shed. This hypothesis was demonstrated by performing flow cytometry before and after a cytotoxicity assay. WT cells showed a marked reduction in the surface level expression of CD16 compared to CD16 KI cells after tumor cell exposure. Using a lactate dehydrogenase (LDH) release assay as a measure of cytotoxicity, only the iNK cells expressing the CD16 construct showed statistically significant increases in cytotoxicity when trastuzumab was added. Furthermore, to better model a solid tumor, a 3D tumor spheroid killing assay was utilized where CD16 KI cells showed an increase in ADCC capacity. The benefit of increased effector function via CD16 KI cannot be fully realized without iNK cells persisting. IL-2 or IL-15 is needed for NK maintenance but the administration of either cytokine is associated with acute clinical toxicities. mbIL-15 allows NK cells to survive for a prolonged period without the support of homeostatic cytokines. An in vitro persistence assay was performed that demonstrated IL-15 KI cells showed an increase in persistence compared to WT cells. Specifically, during the three-week in vitro assay, WT cells became undetectable by Day 14 while IL-15 KI NK cells remained stable over time. In summary, to overcome two shortfalls of NK cell therapies, a bicistronic construct encoding CD16 and a mbIL-15 was knocked into the GAPDH locus of iPSCs. The strong GAPDH promoter drove constitutive expression of CD16 that mitigated CD16 shedding, enhanced ADCC of iNK cells, which can be used in combination with any ADCC enabling IgG1 and IgG3 antibodies, such as trastuzumab and rituximab, for tumor-specific targeting. In addition, mbIL-15 KI allowed iNK cells to persist without exogenous cytokine administration and thus can circumvent exogeneous cytokine-induced clinical toxicities. CD16 and mbIL-15 double KI iNKs, with enhanced ADCC and increased cytokine-independent persistence, can potentially be developed into a safe and efficacious therapy for the treatment of a variety of liquid and solid tumors with high unmet medical needs. Disclosures Allen: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Pattali: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Izzo: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Getgano: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wasko: Editas Medicine: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Blaha: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zuris: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zhang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Shearman: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Chang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

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Enhancing Human NK Cell Function and Specificity for Cancer Immunotherapy

Blood ◽

10.1182/blood-2018-99-120173 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2044-2044

Author(s):

Pomeroy Emily ◽

Hunzeker John ◽

Kluesner Mitchell ◽

Crosby Margaret ◽

Laura Bendzick ◽

...

Keyword(s):

Nk Cells ◽

Cancer Immunotherapy ◽

Peripheral Blood ◽

High Efficiency ◽

Cell Function ◽

Nk Cell ◽

Negative Regulator ◽

Car T

Abstract Natural Killer (NK) cells are cytotoxic lymphocytes capable of immune surveillance and represent an excellent source of cells for cancer immunotherapy for numerous reasons: 1) they mediate direct killing of transformed cells with reduced or absent MHC expression, 2) they can carryout antibody-dependent cell-mediated cytotoxicity (ADCC) on cells bound by appropriate antibodies via CD16, 3) they are readily available and easy to isolate from peripheral blood, 4) they can be expanded to clinically relevant numbers in vitro. Moreover, as NK cells do not cause graft versus host disease, they are inherently an off-the-shelf cellular product, precluding the need to use a patient's own NK cells to treat their cancer. In light of these attributes, NK cells have been used in many clinical trials to treat a number of cancer types; however, the results have not been as successful as other cellular based immunotherapies, such as CAR-T. In light of this, many groups have taken approaches to augment NK cell function, such as high dose IL15, CARs and Bi- or Tri-specific killer engagers. A synergistic or even alternative approach to these technologies is the use of CRISPR/Cas9-based genome editing to disrupt or manipulate the function of NK genes to improve their utility as an immunotherapeutic agent. In order to enhance the immunotherapeutic efficacy of NK cells we have implemented the CRISPR/Cas9 system to edit genes and deliver CARs. To this end, we have developed methods for high efficiency nucleic acid delivery to NK cells using electroporation. First, primary human NK cells are immunomagnetically isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors. Purified NK cells are then activated and expanded using artificial antigen presenting cells (aAPCs) expressing membrane bound IL21 and 41BB for 7 days and subsequently electroporated (Figure 1A). Using this approach with EGFP encoding mRNA, we achieve high rates of transfection (>90%) and high viability (>90%) (Figure 1B). We next developed gRNAs targeting PD1, CISH, and ADAM17. PD1 is a negative regulator of NK cell function and its cognate receptor, PD-L1, is upregulated in a number of cancers. ADAM17 mediates CD16 cleavage on NK cells to negatively regulate their ability to perform ADCC. CISH is a recently described negative regulator of NK cell activation and integrates cytokine signals, including IL-15. We consistently achieved high rates (up to 90%) of gene inactivation in primary human NK cells across multiple donors (Figure 1C). Importantly, these gene edits do not affect expansion potential and are stable over several rounds of expansion (Figure 1D, E). Moreover, ADAM17 KO NK cells are highly resistant to CD16 cleavage upon activation (Figure 2A-E) and PD1 KO NK cells demonstrate significantly enhanced function against PD-L1 expressing cancer cell lines in vitro and in vivo (Figure 2F-J). These data demonstrate that high efficiency gene editing of NK cells can significantly enhance their function while maintaining in vitro expansion. In an effort to engineer NK cell specificity for cancer immunotherapy, we recently developed CAR molecules designed for use in NK cells (Li et al., 2018, Cell Stem Cell 23, 1-12). To this end, we engineered and tested 10 mesothelin CAR molecules with NK specific transmembrane domains (CD16, NKp44, NKp46, or NKG2D) and intracellular signaling domains (2B4, DAP10, DAP12, CD3ζ, and/or CD137). Utilizing several cancer models, we identified an architecture that significantly enhanced NK activation compared to T-CAR architectures (CAR4: scFv-NKG2D-2B4-CD3ζ). Moreover, NK-CAR4 cells demonstrated increased in vivo expansion, improved activity, and reduced toxicity compared to CAR-T cell therapy. In our studies to develop novel NK CARs, CARs were delivered to iPSC derived test NK cells (iNKs) using the PiggyBac transposon system. In order to deliver NK-CAR4 to peripheral blood NK cells we developed methods for high frequency, site specific integration. To this end, we utilized CRISPR/Cas9 combined with non-integrating recombinant Adeno-Associated Virus (rAAV) DNA donor for homologous recombination. Using an EGFP reporter we were able to optimize this process and deliver EGFP reporter to the AAVS1 safe harbor site with efficiencies >80% in NK cells. We are now utilizing our optimized gene editing approaches to generate multiplex edited CAR-NK cells and results from these studies will be presented. Disclosures Webber: BEAM Therapeutics: Consultancy; B-MoGen Biotechnologies: Employment, Equity Ownership. Felices:GT Biopharma: Research Funding. Moriarity:BEAM Therapeutics: Consultancy; B-MoGen Biotechnologies: Employment, Equity Ownership.

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KIR Expression on inVitro-Derived Natural Killer Cells Does Not Regulate Killing of Allogeneic Targets

Blood ◽

10.1182/blood-2018-99-117151 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3705-3705

Author(s):

Yunzu Michele Wang ◽

Huang Zhu ◽

Alessa Ruiz-Cisneros ◽

Naveen Heragu ◽

Eivind Heggernes Ask ◽

...

Keyword(s):

Stem Cells ◽

Nk Cells ◽

Genetic Background ◽

Research Funding ◽

Caspase 3 ◽

Fas Ligand ◽

Nk Cell ◽

In Vitro Cytotoxicity ◽

Hematopoietic Stem

Abstract Natural killer (NK) cells play an essential role in early innate killing of virally-infected and tumor targets. NK cell-mediated activity is regulated by a repertoire of activating and inhibitory receptors that recognize ligands on diseased, stressed, or tumor targets. Killer cell immunoglobulin-like receptors (KIRs) are a family of polymorphic receptors that can be inhibitory or activating based on their intracellular signaling motifs. Expression of certain KIR haplotypes plays a key role in survival and relapse prevention for patients with acute myelogenous leukemia (AML) who receive allogeneic hematopoietic cell transplantation. Therefore, KIR haplotypes are an important consideration in selecting allogeneic donors for patients with AML. However, it is unclear if KIRs play a role in adoptive transfer of NK cells that are becoming more routinely utilized to treat refractory AML and other malignancies. To better address this question we used umbilical cord blood to isolate both CD34+ hematopoietic stem cells and CD45+CD56+ NK cells (UCB-56-NK) from the same umbilical cord blood unit. The CD34+ hematopoietic stem cells were then differentiated in vitro into CD56+ NK cells (UCB-34-NK). Despite originating from the same donor and sharing the same genetic background, as well as comparable expression of Fas ligand, TRAIL, NKp46, NKp44, NKG2A, and NKG2D, the UCB-34-NK cells have characteristically low KIR expression, whereas the UCB-56-NK cells have high KIR expression. This phenotype was further confirmed by mass cytometric (CyTOF) analysis of UCB-56-NK cells and UCB-34-NKcells with a panel of 36 phenotypic and functional NK cell markers. This unique system allows us to study the role of KIR expression independent of any other variations in donor or cell characteristics. The cytotoxicity and NK cell activation of UCB-34-NK cells and UCB-56-NK cells are compared to control NK cells isolated from peripheral blood (PB-NK cells) with standard in vitro cytotoxicity assays against neuroblastoma lines with varying HLA genotypes and a control K562 leukemic targets. Our data demonstrates that there is no statistical difference in NK cytotoxicity and activation of UCB-34-NK cells and UCB-56-NK cells across a spectrum of target cell HLA types, despite the differences in KIR expression. For example, at effector to target (E:T) ratios of 1:5 and 1:20 against neuroblastoma line IMR32, UCB-34-NK cells (KIR-low) demonstrated 68.5% and 84.1% maximal Caspase 3/7 activation, compared to 81.3% and 89.6% by UCB-56-NK cells (KIR-high). Additionally, we have used human induced pluripotent stem cells to derive NK cells (iPSC-NK cells) that vary in KIR expression levels. These CD45+CD56+ iPSC-NKs are differentiated from the same well-established iPSC line in the laboratory and therefore again share the same genetic background, and they have similar NK cell surface receptor expression of Fas ligand, TRAIL, NKp46, NKp44, and NKG2D, but differ in levels of KIR expression. Again in vitro cytotoxicity against hematopoietic tumor targets such as K562 and MOLM13 do not demonstrate a significant difference in killing, despite these KIR differences. For example, in targeting erythroleukemia K562 cells, iPSC-NK cells with high levels of KIR expression at E:T ratios of 1:2.5, 1:5, and 1: 10 have Caspase 3/7 activation of 21.1%, 28.2%, and 41.0%, compared to 20.1%, 22.0%, and 31.2% by iPSC-NK cells with low KIR expression. Together, these studies demonstrate that in vitro-derived NK cells do not require KIR expression to become licensed for anti-tumor activity and these cells are able to kill tumor targets whether or not they express KIRs. These studies better enable use of these allogeneic NK cell populations for off-the-shelf NK cell-based therapies without the need to optimize for KIR profiles for patients of differing HLA haplotypes. Disclosures Malmberg: Fate Therapeutics Inc.: Consultancy, Research Funding. Kaufman:Fate Therapeutics: Consultancy, Research Funding.

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FT576: A Novel Multiplexed Engineered Off-the-Shelf Natural Killer Cell Immunotherapy for the Dual-Targeting of CD38 and Bcma for the Treatment of Multiple Myeloma

Blood ◽

10.1182/blood-2019-131373 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3214-3214 ◽

Cited By ~ 2

Author(s):

Ryan Bjordahl ◽

Svetlana Gaidarova ◽

Jode P Goodridge ◽

Sajid Mahmood ◽

Greg Bonello ◽

...

Keyword(s):

Nk Cells ◽

Cell Line ◽

Pluripotent Stem Cell ◽

Nk Cell ◽

Treatment Strategies ◽

Preclinical Studies ◽

Stem Cell Line ◽

Cell Immunotherapy ◽

Car T

Multiple myeloma (MM) is a B cell neoplasm that originates from the malignant transformation of plasma cells, with treatment strategies that include chemotherapeutic agents and immunomodulatory drugs. Recently, significant effort has been applied to the development of monoclonal antibody (mAb) and chimeric antigen receptor (CAR) T cell therapies for the treatment of advanced MM. Anti-CD38 mAb therapy is at the forefront of these efforts, with clearly demonstrated clinical benefit and availability of a FDA-approved mAb in daratumumab. Antibody-dependent cellular cytotoxicity (ADCC) is a key mechanism of action of CD38-targeted mAbs; however, high CD38 expression on natural killer (NK) cells results in fratricide, which depletes the NK cells necessary for ADCC. In addition to CD38, targeting of other MM-associated cell-surface proteins has been explored. Of these antigens, the TNF-superfamily member BCMA is among the most researched and is under development by multiple groups as a CAR target. Several clinical trials in MM have shown promising initial results targeting BCMA with CAR T cells, however there remains significant opportunity to improve both relapse rates and treatment of relapsed patients. Collectively, clinical data would suggest that combinatorial targeting of both CD38 and BCMA may improve clinical efficacy compared with targeting either antigen alone. We have developed a multiple-target, adoptive NK cell immunotherapy approach for the treatment of MM. The strategy utilizes our off-the-shelf NK cell platform with four engineered attributes: 1) an anti-BCMA CAR for direct MM targeting, 2) high affinity non-cleavable CD16 (hnCD16) for enhanced ADCC in combination with anti-CD38 mAbs, 3) CD38 deletion for resistance to anti-CD38 mAb induced NK cell depletion, and 4) IL-15/IL-15 receptor α fusion protein (IL-15RF; IL-15 fused to IL-15Rα) for enhanced NK cell persistence. The anti-BCMA CAR consists of a unique single chain variable fragment (scFv) targeting domain with a BCMA binding affinity in the low nanomolar range, providing high functional avidity and efficacy in disease settings where BCMA antigen density is low. Our approach utilizes NK cells derived from a genetically engineered, clonally-derived master pluripotent stem cell line with uniform expression of anti-BCMA CAR, IL-15RF, hnCD16, and CD38 bi-allelic knockout. The engineered master pluripotent stem cell line serves as the starting material for consistent and repeatable manufacture of off-the-shelf NK cells that contain all described attributes in a homogenous manner (termed FT576) and that can be produced at a scale to support multi-dose treatment strategies and on-demand dose availability. In preclinical studies, FT576 NK cells exhibited uniform expression of CD16, CAR, and IL15-RF and did not express CD38 (<1%). In an in vitro fratricide assay, FT576 NK cells were entirely resistant to daratumumab-induced fratricide, with no detectable specific cytotoxicity when exposed to increasing concentrations of daratumumab. Conversely, peripheral blood NK cells were sensitive to daratumumab-induced fratricide (up to 33% cytotoxicity within 3 hrs of daratumumab exposure). FT576 NK cells demonstrated enhanced cytotoxicity against the MM1.S MM cell line during a long-term cytotoxicity assay compared with control NK cells that lacked CAR expression (62% cytotoxicity for FT576 vs 26% for control). In addition, cellular persistence was greater than NK cells lacking the IL-15RF protein, and FT576 NK cells demonstrated the unique ability to expand in vitro absent of exogenous cytokine support (61-fold expansion vs. 4-fold for IL-15RF negative). Importantly, FT576 NK cells remained ADCC competent, as combination with daratumumab enhanced cytotoxicity against MM cell lines in a 2D cytotoxicity assay. Additionally, FT576 mediated direct cytotoxicity against RPMI-8226 MM spheroids, leading to >99% cytotoxicity in a 3D-spheroid culture model. Preclinical studies are ongoing to support the advancement of FT576 as the first-of-kind cellular therapeutic for the combination of anti-BCMA CAR and mAb-directed targeting of MM. Disclosures Bjordahl: Fate Therapeutics, Inc.: Employment. Gaidarova:Fate Therapeutics, Inc: Employment. Goodridge:FATE THERAPEUTICS: Employment. Mahmood:Fate Therapeutics, Inc: Employment. Bonello:Fate Therapeutics, Inc.: Employment. Robinson:Fate Therapeutics, Inc.: Employment. Ruller:Fate Therapeutics, Inc.: Employment. Pribadi:Fate Therapeutics, Inc.: Employment. Lee:Fate Therapeutics, Inc.: Employment. Abujarour:Fate Therapeutics, Inc.: Employment. Dinella:Fate Therapeutics, Inc.: Employment. Huffman:Fate Therapeutics, Inc.: Employment. Chu:FATE THERAPEUTICS: Employment. Valamehr:Fate Therapeutics, Inc: Employment.

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Assessment of antitumor function of NK cells expanded with exosomes from K562.mb21 cells.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.7_suppl.132 ◽

2017 ◽

Vol 35 (7_suppl) ◽

pp. 132-132 ◽

Cited By ~ 1

Author(s):

Jeremiah Oyer ◽

Sarah B. Gitto ◽

Sara Khederzadeh ◽

Kari Shaver ◽

Dean A. Lee ◽

...

Keyword(s):

Nk Cells ◽

Cell Expansion ◽

Nk Cell ◽

Ex Vivo ◽

K562 Cells ◽

In Vitro Cytotoxicity ◽

Feeder Cell ◽

Nsg Mice

132 Background: NK cells can kill malignant cells to provide innate immunity against tumors. Due to their low abundance in blood, a focus is to expand NK cells ex vivo having enhanced anti-tumor cytotoxicity to be used as a treatment. Our group has pioneered a cell-free method using plasma membrane (PM) particles derived from K562 cells expressing 41BBL and membrane-bound IL-21 (K562.mb21) which were developed for NK cell expansion. Compared to feeder cell based methods for NK cell expansion, PM21-particles improve safety and allow for potential wide-spread dissemination, and also allows direct in vivo use. Exosomes, vesicles naturally secreted by cells, may yet be another novel feeder cell free way for NK cell expansion and may have further advantageous therapeutic dimensions. Methods: EX21-exosomes and PM21-particles were prepared from K562.mb21 cells and characterized by Nanosight and Western blot analysis. CD3-depleted PBMCs were cultured with EX21 for 14 days, NK cell amounts were monitored and media changed every 2-3 days. In vitro cytotoxicity against K562 cells were comparatively assessed for EX21-NK cells and PM21-NK cells. In vivo anti-tumor efficacy of EX21- and PM21-NK cells was assessed in NSG mice implanted ip with SKOV3_luc ovarian tumor cells (1 x 106 cells seeded for 4 days). SKOV3-bearing mice were treated with vehicle, or two doses of EX21-NK cells or PM21-NK cells (1 x 107, in 5 day intervals), and with or without in vivo administration of EX21 (10 µg, 3x/week) or PM21-particles (600 µg, 3x/week). All groups were injected ip with IL-2 (10 KU, 3x/week). Survival analysis was performed with a Log-rank (Mantel-Cox) test. Results: NK cells cultured with EX21 expanded 530 fold (344-710) over 14 days compared to 735 fold (667-802) in presence of PM21-particles. Treatment of SKOV3 engrafted NSG mice with NK cells, expanded with either EX21 or with PM21, allowed significant ( < 0.0001) increase in survival compared to untreated animals (41-44 vs 29 days post treatment). Ip delivery of EX21 to SKOV3 bearing mice had no effect on survival in either untreated control or EX21-NK cell treated groups. Conclusions: EX21 efficiently expands NK cells and EX21-NK cells have equal anti-tumor effect as PM21-NK cells, both in vitro and in vivo.

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Combinatorial Natural Killer Cell–based Immunotherapy Approaches Selectively Target Chordoma Cancer Stem Cells

Cancer Research Communications ◽

10.1158/2767-9764.crc-21-0020 ◽

2021 ◽

Vol 1 (3) ◽

pp. 127-139

Author(s):

Austin T.K. Hoke ◽

Michelle R. Padget ◽

Kellsye P. Fabian ◽

Anjali Nandal ◽

Gary L. Gallia ◽

...

Keyword(s):

Stem Cells ◽

Cancer Stem Cells ◽

Nk Cells ◽

Nk Cell ◽

Killer Cell ◽

Flow Cytometric Analysis ◽

In Vitro Cytotoxicity ◽

Programmed Death ◽

Tumor Types

Chordoma is a rare tumor derived from notochord remnants that has a propensity to recur and metastasize despite conventional multimodal treatment. Cancer stem cells (CSC) are implicated in chordoma's resistant and recurrent behavior; thus, strategies that target CSCs are of particular interest. Using in vitro cytotoxicity models, we demonstrated that anti-programmed death ligand 1 (anti–PD-L1; N-601) and anti-EGFR (cetuximab) antibodies enhanced lysis of chordoma cells by healthy donor and chordoma patient NK cells through antibody-dependent cellular cytotoxicity (ADCC). Treatment of NK cells with an IL15 superagonist complex (N-803) increased their cytotoxicity against chordoma cells, which was further enhanced by treatment with N-601 and/or cetuximab. PD-L1–targeted chimeric antigen receptor NK cells (PD-L1 t-haNKs) were also effective against chordoma cells. CSCs were preferentially vulnerable to NK-cell killing in the presence of N-601 and N-803. Flow cytometric analysis of a chordoma CSC population showed that CSCs expressed significantly more NK-activating ligand B7-H6 and PD-L1 than non-CSCs, thus explaining a potential mechanism of selective targeting. These data suggest that chordoma may be effectively targeted by combinatorial NK cell–mediated immunotherapeutic approaches and that the efficacy of these approaches in chordoma and other CSC-driven tumor types should be investigated further in clinical studies. Significance: Combinatory immunotherapy using NK-mediated approaches demonstrates robust antitumor activity in preclinical models of chordoma and selectively targets chordoma CSCs.

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NKG2D-CAR-transduced natural killer cells efficiently target multiple myeloma

Blood Cancer Journal ◽

10.1038/s41408-021-00537-w ◽

2021 ◽

Vol 11 (8) ◽

Author(s):

Alejandra Leivas ◽

Antonio Valeri ◽

Laura Córdoba ◽

Almudena García-Ortiz ◽

Alejandra Ortiz ◽

...

Keyword(s):

T Cells ◽

Cell Therapy ◽

Nk Cells ◽

Nk Cell ◽

In Vitro Cytotoxicity ◽

T Cell Therapy ◽

Car T Cell Therapy ◽

Wide Range

AbstractCAR-T-cell therapy against MM currently shows promising results, but usually with serious toxicities. CAR-NK cells may exert less toxicity when redirected against resistant myeloma cells. CARs can be designed through the use of receptors, such as NKG2D, which recognizes a wide range of ligands to provide broad target specificity. Here, we test this approach by analyzing the antitumor activity of activated and expanded NK cells (NKAE) and CD45RA− T cells from MM patients that were engineered to express an NKG2D-based CAR. NKAE cells were cultured with irradiated Clone9.mbIL21 cells. Then, cells were transduced with an NKG2D-4-1BB-CD3z-CAR. CAR-NKAE cells exhibited no evidence of genetic abnormalities. Although memory T cells were more stably transduced, CAR-NKAE cells exhibited greater in vitro cytotoxicity against MM cells, while showing minimal activity against healthy cells. In vivo, CAR-NKAE cells mediated highly efficient abrogation of MM growth, and 25% of the treated mice remained disease free. Overall, these results demonstrate that it is feasible to modify autologous NKAE cells from MM patients to safely express a NKG2D-CAR. Additionally, autologous CAR-NKAE cells display enhanced antimyeloma activity demonstrating that they could be an effective strategy against MM supporting the development of NKG2D-CAR-NK-cell therapy for MM.

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