NALM-6 Leukemia Cells Research Articles

AMPK agonism optimizes the in vivo persistence and anti-leukemia efficacy of chimeric antigen receptor T cells.

Chimeric antigen receptor T cell (CART) therapy has seen great clinical success. However, up to 50% of leukemia patients relapse and long-term survivor data indicate that CART cell persistence is key to enforcing relapse-free survival. Unfortunately, ex vivo expansion protocols often drive metabolic and functional exhaustion, reducing in vivo efficacy. Preclinical models have demonstrated that redirecting metabolism ex vivo can improve in vivo T cell function and we hypothesized that exposure to an agonist targeting the metabolic regulator AMP-activated protein kinase (AMPK), would create CARTs capable of both efficient leukemia clearance and increased in vivo persistence. CART cells were generated from healthy human via lentiviral transduction. Following activation, cells were exposed to either Compound 991 or DMSO for 96 hours, followed by a 48-hour washout. During and after agonist treatment, T cells were harvested for metabolic and functional assessments. To test in vivo efficacy, immunodeficient mice were injected with luciferase+ NALM6 leukemia cells, followed one week later by either 991- or DMSO-expanded CARTs. Leukemia burden and anti-leukemia efficacy was assessed via radiance imaging and overall survival. Human T cells expanded in Compound 991 activated AMPK without limiting cellular expansion and gained both mitochondrial density and improved handling of reactive oxygen species (ROS). Importantly, receipt of 991-exposed CARTs significantly improved in vivo leukemia clearance, prolonged recipient survival, and increased CD4+ T cell yields at early times post-injection. Ex vivo, 991 agonist treatment mimicked nutrient starvation, increased autophagic flux, and promoted generation of mitochondrially-protective metabolites. Ex vivo expansion processes are necessary to generate sufficient cell numbers, but often promote sustained activation and differentiation, negatively impacting in vivo persistence and function. Here, we demonstrate that promoting AMPK activity during CART expansion metabolically reprograms cells without limiting T cell yield, enhances in vivo anti-leukemia efficacy, and improves CD4+ in vivo persistence. Importantly, AMPK agonism achieves these results without further modifying the expansion media, changing the CART construct, or genetically altering the cells. Altogether, these data highlight AMPK agonism as a potent and readily translatable approach to improve the metabolic profile and overall efficacy of cancer-targeting T cells.

Towards Automated Engineering of Donor-Derived T Lymphocytes into CRISPR/Cas9-Mediated CAR T Cells in a Closed-System

Chimeric antigen receptor (CAR)-expressing T cells have revolutionized immunotherapy of cancer. However, some patients derive no benefit from approved CARTcell therapies due to limitations such as toxicity and use of autologous cells which are sometimes unfit and risk manufacturing failures. Additional or more effective CARTcell therapies are needed. Harnessing the endogenous homology-directed DNA repair pathway via precision genome editing based on Cas9RNP/rAAV6, we and others have demonstrated the feasibility of targeting transgene(s) integration into specific gene site(s) including their incorporation under endogenous regulatory elements. Using this strategy, we aimed to engineer healthy donor-derived CAR T cells from αβ+ T lymphocytes. For enhanced safety, we metabolically reprogrammed the CAR T cells by inactivating the UMPS gene needed for uridine biosynthesis. The UMPS-/-CAR T cells become dependent on exogenous uridine thereby serving as a cell control mechanism. We hypothesized that donor-derived αβ+ T lymphocytes can be engineered using the Cas9RNP-rAAV6 strategy in an automated closed-system into controllable CAR T cells. We inactivated TRAC and UMPS gene loci in αβ+T lymphocytes using Lonza4D nucleofector. Electroporated cells were simultaneously transduced with CD19CAR-containing rAAV6 DNA vector. We obtained TRAC gene knockout up to 97% with knock-in up to 89%. Disruption of the UMPS gene would transform the cells to being auxotrophic and depend on uridine supply for viability and growth. To evaluate the CART cells' antitumor potency, we cocultured the CD19CART cells with GFP-expressing Nalm6 leukemia cell lines. The CD19CART cells eradicated the leukemia cells within 72 hours. To mimic relapse, we re-challenged cocultured CART with Nalm6 cells and the tumor cells were again depleted within 72 hours. Expectedly, IL-2 production was elevated in CD19CART cells challenged with Nalm6. Next, we tested the ability to control the cytotoxicity of the CD19CART cells by coculturing them with Nalm6 cells in separate groups with/without exogenous uridine. Interestingly, the CD19CART cells cocultured without uridine eradicated the target cells within 72 hours but lost their potency upon subsequent challenge (Figure 1). In contrast, CD19CAR T cells supplied with uridine remained potent and repeatedly eradicated target cells indicating the ability to turn off the potency of CART cells with uridine supply/withdrawal. To establish an automated closed-system for CART cells, we adopted the Miltenyi CliniMACS Prodigy with in-line electroporator (Elpo) for process engineering. First, we determined optimized parameters for electroporating αβ+ T lymphocytes on Elpo(Table1). Cells electroporated with these settings were (>80% viability) comparable to untreated cells. We first engineered CART cells with Elpo/Miltenyi reagents in an open system(i.e.cultivated Elpo-electroporated cells in culture vessels/incubators). We obtained 95% of TRAC and UMPS gene knockouts and up to 90% CD19CAR transgene knock-in to the TRAC locus - an efficiency slightly higher than using Lonza 4D nucleofector. To scale up the Cas9-based T cell engineering into a single automated process on the CliniMACS Prodigy, the Cas9 and guide RNA reagents would be scaled up 1000fold. To make the process cost-effective, we tested AZD7468, a small molecule DNAPKcs inhibitor. We optimized/reduced the amounts of reagents upto 90% less of Cas9RNP used without any significant reduction in gene editing outcomes. AZD7648 reduced the MOI needed for viral transduction by 50% while maintaining 80% or higher levels of transgene knock-in efficiency. Next, we commenced pilot studies to automate the CAR T cell engineering process in closed-system. Currently, we have achieved an automated TRAC gene knockout of over 90% and CAR transgene knock-in of slightly >10%. Further optimization studies are underway to improve automated gene knock-in efficiency. We have engineered donor-derived αβ+ T lymphocytes into potent/controllable CD19CART cells. The UMPS-deficient CD19CART cells lose antileukemia activity without uridine. We show that metabolic reprogramming can be combined with precision genome editing to generate controllable CART cells. Finally, our data demonstrate the feasibility of automating CRISPR/Cas9-mediated engineering of CAR T cells within closed-system.

CLASH of the Titans: How CAR-T Cells Can Triumph Over Tumors.

CLASH of the Titans: How CAR-T Cells Can Triumph Over Tumors.

An organometallic analogue of combretastatin A-4 and its apoptosis-inducing effects on lymphoma, leukemia and other tumor cells in vitro.

Hexacarbonyl[1,3-dimethoxy-5-((4'-methoxyphenyl)ethynyl)benzene]dicobalt (NAHO27), an organometallic analogue of combretastatin A-4, has been synthesized and its activity against lymphoma, leukemia, breast cancer and melanoma cells has been investigated. It was shown that NAHO27 specifically induces apoptosis in BJAB lymphoma and Nalm-6 leukemia cells at low micromolar concentration and does not affect normal leukocytes in vitro. It also proved to be active against vincristine and daunorubicin resistant leukemia cell lines with p-glycoprotein-caused multidrug resistance and showed a pronounced (550%) synergistic effect when co-applied with vincristine at very low concentrations. Mechanistic investigations revealed NAHO27 to induce apoptosis via the mitochondrial (intrinsic) pathway as reflected by the processing of caspases 3 and 9, the involvement of Bcl-2 and smac/DIABLO, and the reduction of mitochondrial membrane potential. Gene expression analysis and protein expression analysis via western blot showed an upregulation of the proapoptotic protein harakiri by 9%.

Off-the-Shelf, Multiplexed-Engineered iPSC-Derived NK Cells Mediate Potent Multi-Antigen Targeting of B-Cell Malignancies with Reduced Cytotoxicity Against Healthy B Cells

Off-the-Shelf, Multiplexed-Engineered iPSC-Derived NK Cells Mediate Potent Multi-Antigen Targeting of B-Cell Malignancies with Reduced Cytotoxicity Against Healthy B Cells

Clinical Manufacture of FT819: Use of a Clonal Multiplexed-Engineered Master Induced Pluripotent Stem Cell Line to Mass Produce Off-the-Shelf CAR T-Cell Therapy

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 DisclosuresYuan: 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.

Combination of Three Unique Anti-Tumor Modalities Engineered into iPSC-Derived T Cells Demonstrate a Synergistic Effect in Overcoming Tumor Heterogeneity and Cancer Escape

Chimeric antigen receptor (CAR) is known to trigger an effective immune response through surface antigen recognition enhanced by T-cell activation signal one (ex. CD3) and signal two (ex. CD28); however, targeting neoantigens and intracellular antigens remains a challenge. On the other hand, the T-cell receptor (TCR) can target neo/intracellular antigens presented by MHC molecules, but often the response is not as potent. The CD16 Fc receptor, which is naturally expressed on NK cells, mediates antibody-dependent cellular cytotoxicity (ADCC), but 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 (tri-modal), to determine whether challenges associated with the treatment of heterogeneous tumors may be overcome.To evaluate the benefit of the three distinct targeting modalities, we tested the functional activity of individual and various combinations of i) anti-CD19 CAR, anti-MICA/B CAR, and anti-BCMA CAR, ii) high-affinity, non-cleavable CD16 (hnCD16), and iii) MR1 and NYESO1 TCR modalities in iT cells. All tested combinations successfully expressed the designated edits and differentiated into iT cells (T-lymphocytes > 95%). Initially, we tested CD19 CAR and MR1 TCR in a 9-day serial killing assay of Nalm6 leukemia cells (CD19 high, MR1 +), where we observed CD19 CAR-iT cells induce prompt CAR-mediated tumor growth inhibition (TGI), saw similar effective killing by MR1 TCR-iT cells but with a 24-hr delay, and observed the most effective response of tumor cell elimination when both where combined in the same iT cell population (relative tumor counts; Day 1, no stim: 2.6, CAR: 0.28, TCR: 1.02, CAR+TCR: 0.02; Day 2, no stim: 5.36, CAR: 0.25, TCR: 0.05, CAR+TCR: 0.02). All conditions (CAR, TCR, and CAR+TCR) reached and maintained complete TGI by Day 9 of the assay (relative tumor count, Day 9, no stim: 50.97, all other stim conditions: <0.01). In line with killing kinetics, the time for the activation marker CD25 upregulation differed between CAR and TCR (peak time and percentage of CD25 +, CAR: Day 1, 49.9%; TCR: Day 5, 74.6%). Co-triggering of CAR and TCR in combination revealed quickest, highest, and sustained CD25 upregulation levels (CD25 +, CAR+TCR Day 1: 62.5%, Days 3 to 9: >90%), indicating a synergistic effect and compatibility between CAR and TCR.Assessing anti-MICA/B CAR and hnCD16, we confirmed the hnCD16-mediated response in iT cells in the presence of anti-MICA/B CAR when crosslinking hnCD16 via biotinylated anti-CD16 antibody with streptavidin (phosphorylated CD3zeta peaked at 10 min upon ADCC triggering), indicating the compatibility between a CAR-iT cell and the hnCD16 motif.Lastly, combining iT cells expressing anti-BCMA CAR + MR1 TCR + hnCD16 with daratumumab (anti-CD38 mAb) in a 9-day serial killing assay demonstrated the best TGI among the groups with a near-elimination of transgenic Nalm6 cells (area under curve, no stim: 30.29, TCR: 1.564, CAR: 0.7087, hnCD16+mAb: 1.452, trimodal+mAb: 0.5824). To assess the function of the tri-modal iT cells in vivo, we used a disseminated xenograft model of B-cell leukemia where a heterogenous mixture of transgenic Nalm6 leukemia cells was used to mimic tumor heterogeneity. Assessment of the bone marrow revealed the unique capacity of each target modality to eliminate its target designated Nalm6 leukemia group, with tri-modal iT cells effectively clearing all populations (Figure 1).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. [Display omitted] DisclosuresYang: Fate Therapeutics, Inc.: Current Employment. Lin: Fate Therapeutics, Inc.: Current Employment. Shirinbak: Fate Therapeutics, Inc.: Current Employment. Pribadi: Fate Therapeutics, Inc.: Current Employment. Chu: Fate Therapeutics, Inc.: Current Employment. Gutierrez: Fate Therapeutics, Inc.: Current Employment. Mehta: Fate Therapeutics, Inc.: Current Employment. Avramis: Fate Therapeutics, Inc.: Current Employment. Whitlock: Fate Therapeutics, Inc.: Current Employment. ORourke: Fate Therapeutics, Inc.: Current Employment. van der Stegen: Fate Therapeutics, Inc.: Current Employment. Lee: Fate Therapeutics, Inc.: Current Employment. Witty: Fate Therapeutics, Inc.: Current Employment. Peralta: Fate Therapeutics, Inc.: Current Employment. Hosking: Fate Therapeutics: Current Employment. Chang: Fate Therapeutics, Inc.: Current Employment. Valamehr: Fate Therapeutics, Inc.: Current Employment.

Viscum album (L) extracts in cancer treatment: a systematic review of in vitro and in vivo studies

Background: Several reviews about phytotherapy and homeopathy have been published in the last years, including Viscum album (VA.L). VA is a parasite plant whose extract has anti-cancer proprieties and is used alone or in combination with conventional chemotherapy. &#x0D; Methods: We performed a systematic review about the in vivo and in vitro models described in the literature, including veterinary clinical trials. The literature was consulted from Pubmed database.&#x0D; Results: There are several kinds of pharmaceutical preparations about VA and their active principles used in experimental studies, lectin being frequently studied (alone or as an extract compound). More than 50% of available literature about VA is related to the lectin effects. On the other hand, the effects of viscotoxins are less studied. Among the in vivo experimental studies about VA and its compounds, the B16 murine melanoma is the most used model, followed by Ehrlich, Walker and Dalton tumors. The results point to the apoptotic effects, metastasis control and tumor regression. Some veterinary clinical studies about the use of VA in the treatment of sarcoid, fibrosarcoma and neuroblastoma are quoted in literature too, with interesting results. Considering the in vitro models, our review revealed that NALM6 leukemia cells, B16 melanoma and NC1-H460 lung carcinoma were the most studied tumor models, apoptosis signals being the most important findings. Only one study verified immunoglobulin and interleukin production. All consulted papers were related to phytotherapy preparations only.&#x0D; Conclusions: Although the literature about the anti-cancer activity of VA extract and its lectins is enough, there is a marked lack of information about viscotoxin activities and about the effects of homeopathic preparations of this plant on animal tumors and on in vitro cultivated tumor cells.

Leishmanicidal and cytotoxic activities and 4D-QSAR of 2-arylidene indan-1,3-diones.

The indan-1,3-dione and its derivatives are important building blocks in organic synthesis and present important biological activities. Herein, the leishmanicidal and cytotoxicity evaluation of 16 2-arylidene indan-1,3-diones is described. The compounds were evaluated against the leukemia cell lines HL60 and Nalm6, and the most effective ones were 2-(4-nitrobenzylidene)-1H-indene-1,3(2H)-dione (4) and 4-[(1,3-dioxo-1H-inden-2(3H)-ylidene)methyl]benzonitrile (10), presenting IC50 values of around 30 µmol/L against Nalm6. The leishmanicidal activity was assessed on Leishmania amazonensis, with derivative 4 (IC50 = 16.6 µmol/L) being the most active. A four-dimensional quantitative structure-activity analysis (4D-QSAR) was applied to the indandione derivatives, through partial least-squares regression. The statistics presented by the regression models built with the selected field descriptors of Coulomb (C) and Lennard-Jones (L) nature, considering the activities against L. amazonensis, HL60, and Nalm6 leukemia cells, were, respectively, R2 = 0.88, 0.92, and 0.98; Q2 = 0.83, 0.88, and 0.97. The presence of positive Coulomb descriptors near the carbonyl groups indicates that these polar groups are related to the activities. Besides, the presence of positive Lennard-Jones descriptors close to substituents R3 or R1 indicates that bulky nonpolar substituents in these positions tend to increase the activities. This study provides useful insights into the mode of action of indandione derivatives for each biological activity involved.

Abstract LB105: In vivo CAR T therapy: targeted in vivo gene delivery of a CAR using a CD8-specific fusogen results in tumor eradication

Abstract CAR T cells are highly effective at inducing remissions in patients with refractory B cell malignancies. The ex vivo process used to manufacture these therapies limits patient access and exposes T cells to non-physiologic conditions in culture. Viral vectors are efficient at genetically modifying T cells and are used in the ex vivo manufacture of most CAR T cell products but lack target-cell specificity. Here we describe how viral envelope glycoproteins, termed fusogens, engineered to target the CD8 co-receptor can efficiently and specifically deliver a chimeric antigen receptor (CAR) to human T cells in vivo that support target cell killing and tumor eradication. Modification of viral envelope proteins to include antibody-based binding domains (CD8 binders) can produce fusogen-viral vector compositions that deliver genetic payloads to CD8 T cells with therapeutic effect (1, 2). Screening CD8-targeted binders identified fusogens with a range of on-target cell transduction efficiencies and off-target cell line specificities. CD8-targeted fusogens with the highest efficiency were comparable to VSVg-pseudotyped lentiviruses at transducing CD8 T cells, but unlike VSVg showed CD8 T cell specific transduction. Interestingly, CD8-targeted fusogens were superior to VSVg-pseudotyped lentivirus at transducing non-activated human T cells. In vivo fusogen-mediated delivery showed efficient and specific CD8 T cell transduction and expression of a GFP reporter gene. Second-generation CARs containing a CD19 binding domain and a 41BB costimulatory domain delivered by targeted fusogen demonstrated CD8 specific expression and functional activity against non-malignant B cells and CD19+ Nalm6 leukemia cells. To demonstrate targeted fusogen-mediated in vivo CAR delivery and activity, we established Nalm6 xenografts in mice into which unmodified activated human PBMCs were infused. Activated human PBMCs alone were unable to control tumor growth. A single intravenous delivery of a CD8 fusogen containing a second-generation CD19 CAR transgene across a range of functional titer doses resulted in the generation of CD8 CAR Ts that eradicated the CD19+ tumor xenografts. A high percentage of T cells demonstrated CAR expression after fusogen delivery with clear specificity for the CD8+ cells. Importantly, the fusogen was able to generate a functional CAR response regardless of prior activation status of the T cells. Targeted in vivo delivery of CAR into T cell subsets may represent a novel therapeutic approach for human B cell malignancies. The ability to specifically deliver a CAR gene to a T cell in vivo would be transformative in terms of patient access and is likely to generate a qualitatively superior therapeutic T cell.

Development of a Novel MICA/B-Specific CAR As a Pan-Tumor Targeting Strategy for Off-the-Shelf, Cell-Based Cancer Immunotherapy

Surface expression of the HLA-I related molecules MICA and MICB (MICA/B) in response to oncogenic and cellular stress acts as a natural anti-cancer immunosurveillance mechanism. The recognition of MICA/B by the activating immunoreceptor NKG2D, which is expressed by natural killer (NK) and T cell subsets, is responsible for the removal of many transformed and virally infected cells. However, tumors frequently evade NKG2D-mediated immunosurveillance by proteolytic shedding of MICA/B, which can inhibit NKG2D function and promote tumor immune escape. Recently, we demonstrated that monoclonal antibodies targeting the conserved, membrane-proximal α3 domain of MICA/B can prevent MICA/B shedding and enhance NK cell anti-tumor efficacy. With the goal of leveraging the ubiquity of MICA/B expression on malignant cells, we have developed a novel chimeric antigen receptor targeting the α3 domain of MICA/B (CAR-MICA/B) and are currently evaluating application of CAR-MICA/B in an off-the-shelf NK cell immunotherapy platform for both solid and hematopoietic tumor indications. Optimization of CAR-MICA/B design was performed by primary T cell transduction using a matrix of CAR spacers and ScFv heavy and light chain orientations. Six candidate CAR-MICA/B designs were screened in vitro against a panel of tumor cell lines and in vivo against the Nalm6 leukemia cell line engineered to express MICA (Nalm6-MICA). All tested constructs demonstrated MICA-specific in vitro activation and cytotoxicity as well as in vivo tumor control (Figure 1A). Additional studies utilizing the optimal CAR-MICA/B configuration demonstrated MICA/B-specific reactivity against a panel of solid and hematopoietic tumor cell lines in vitro, including melanoma, renal cell carcinoma, and lung cancer lines (Figure 1B). Further, CAR-MICA/B T cells were superior to NKG2D-CAR T cells in clearing A2058 melanoma cells in an in vivo xenograft metastasis model (Figure 1C). Although MICA/B expression has primarily been studied in the context of solid tumors, moderate MICA/B mRNA expression was identified in a number of hematopoietic tumor cell lines, including acute myeloid leukemia (AML) and multiple myeloma (MM) lines. Following the confirmation of surface MICA/B protein expression on a selection of MM and AML cell lines, we utilized MICA/B CAR primary T cells to further demonstrate MICA/B-specific activation and cytotoxicity and to confirm CAR-MICA/B targeting of hematological malignancies (Figure 1D). To further advance CAR-MICA/B development, we introduced the CAR-MICA/B construct into an induced pluripotent stem cell (iPSC) line designed for production of off-the-shelf natural killer (NK) cell immunotherapies. Using a panel of tumor cell lines expressing MICA/B, CAR-MICA/B iPSC-derived NK (iNK) cells displayed specific MICA reactivity, resulting in enhanced cytokine production, degranulation, and CAR-mediated cytotoxicity compared to CAR-negative iNK control cells (Figure 1E). In addition to MICA/B-specific cytotoxicity mediated by CAR, iNK cells also mediated innate cytotoxicity against cancer cells through endogenous NKG2D and other NK cell activating receptors, highlighting the multifaceted targeting capacity of CAR iNK cells. In order to isolate CAR-directed cytotoxicity from the iNK cells' innate anti-tumor capacity, an in vivo proof of concept study was performed using mouse B16-F10 melanoma cells engineered to express human MICA. In this model, iNK expressing CAR-MICA/B significantly reduced B16-F10-MICA liver and lung metastases from CAR-MICA/B iNK cells compared to CAR negative control cells, with reductions of the number of metastases by 87% in the lung (p&amp;lt;0.0001) and 93% in the liver (p&amp;lt;0.006) for CAR-MICA/B iNK cells vs non-CAR controls (Figure 1F). Additionally, CAR-MICA/B iNK cells were effective at controlling Nalm6-MICA progression in a disseminated leukemia model, suggesting potential application against both hematopoietic and solid tumors. Ongoing work is focused on extending these studies into disease-specific models of endogenous MICA/B expression to further advance CAR-MICA/B iNK cells in both solid and hematologic cancers. In summary, these preclinical data support the development and translation of an off-the-shelf NK cell immunotherapy targeting the conserved α3 domain of MICA/B with potential therapeutic application to multiple hematopoietic and solid tumor types. Figure 1 Disclosures Bjordahl: Fate Therapeutics: Current Employment. Goulding:Fate Therapeutics: Current Employment. Blum:Fate Therapeutics: Current Employment. Chang:Fate Therapeutics: Current Employment. Wucherpfennig:Fate Therapeutics: Research Funding. Chu:Fate Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company; Roche Holding AG: Current equity holder in publicly-traded company. Chu:Fate Therapeutics, Inc: Current Employment. Gaidarova:Fate Therapeutics, Inc: Current Employment. Liu:Fate Therapeutics: Current Employment. Sikaroodi:Fate Therapeutics: Current Employment. Fong:Fate Therapeutics: Current Employment. Huffman:Fate Therapeutics: Current Employment. Lee:Fate Therapeutics, Inc.: Current Employment. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company.

Discovery of a cobalt (III) salen complex that induces apoptosis in Burkitt like lymphoma and leukemia cells, overcoming multidrug resistance in vitro

A very small number of cobalt complexes is examined in oncology research. In this work, we investigate the cobalt (III) salen complex MBR-60 that turns out to be a promising anticancer drug. It induces apoptosis in Nalm6 leukemia and BJAB lymphoma cells and overcomes multidrug resistances by blocking the drug efflux pump P-glycoprotein. It further develops the apoptotic effects over the intrinsic pathway. An activation of caspase-3, caspase-8 and caspase-9 can be detected by western blot analysis. The independence of CD95 is shown by similar apoptotic inductions in BJAB and BJAB FADDdn cells. MBR-60 displays synergistic effects with daunorubicin and vincristine and has a selectivity to tumor cells. In comparison to the apoptotic effects of MBR-60 in BJAB lymphoma cells, the cobalt-free ligand 5 does not influence these cells. The research highlights that a cobalt complex has a therapeutic potential for cancer treating with a focus on drug-resistant tumors.

Novel allosteric PARP1 inhibitors for the treatment of BRCA-deficient leukemia

The successful use of PARP1 inhibitors like olaparib (Loparza®) in the treatment of BRCA1/2- deficient breast cancer has provided clinical proof of concept for applying personalized medicine based on synthetic lethality to the treatment of cancer. Unfortunately, all marketed PARP1 inhibitors act by competing with the cofactor NAD+ and resistance is already developing to this anti-cancer mechanism. Allosteric PARP1 inhibitors could provide a means of overcoming this resistance. A high throughput screen performed by Tulin et al. identified 5F02 as an allosteric PARP inhibitor that acts by preventing the enzymatic activation of PARP1 by histone H4. 5F02 demonstrated anti-cancer activity in several cancer cell lines and was more potent than olaparib and synergistic with olaparib in these assays. In the present study we explored the structure-activity relationship of 5F02 by preparing analogs that possessed structural variation in four regions of the chemical scaffold. Our efforts led to lead molecule 7, which demonstrated potent anti-clonogenic activity against BRCA-deficient NALM6 leukemia cells in culture and a therapeutic index for the BRCA-deficient cells over their BRCA-proficient isogenic counterparts.

Abstract B64: Coexpression of a CD19-OX40-CD3ζ CAR with membrane-bound IL-15 enhances natural killer cell function

Abstract Introduction: Chimeric antigen receptors (CARs) have been used successfully to retarget T cells in patients with hematologic malignancies. Natural killer (NK) cells offer an alternative to T cells for cellular immunotherapy and are suitable for allogeneic use as they are not HLA-restricted and patients who receive NK cell treatment do not develop graft-versus-host disease (GVHD). Therefore, NK cells can provide an attractive alternative for “off-the-shelf” cellular therapy. Here, we investigated multiple approaches to modify and enhance CD19 CAR expression on NK cells. It has been previously reported that costimulatory domains play an important role in proliferation, efficacy, and persistence of CAR-T cells both in vitro and in vivo. To understand how CAR structure effects the functional behavior of NK cells, we assessed the capability of various costimulatory signaling domains, including CD28, OX40, CD28-41BB, etc., all coupled to CD3ζ, to enhance CD19 CAR signaling and cytotoxicity in NK cells. We demonstrated that NK activity and persistence can be elevated by simultaneous expression of chimeric constructs directing the expression of a CD19 CAR and a membrane-bound form of IL-15 (mbIL-15). Methods: NK cells were generated by coculture of peripheral blood mononuclear cells (PBMC) with genetically modified irradiated K562 feeder cells. NK cells were transduced at a MOI of 1-2 with a gamma-retrovirus encoding a CD19 CAR and mbIL-15. NK expansion and CAR expression were evaluated by flow cytometry. In vitro cytotoxicity of transduced NK cells was measured using both flow cytometry and the IncuCyte S3 live cell analysis system. The in vivo activity of engineered NK cells was further assessed in a xenograft tumor model in NSG mice, using a Nalm6 leukemia cell line. Results: CD19 CAR constructs containing various costimulatory domains were all expressed in expanded and transduced NK cells. CAR expression in multiple donor NK cells was typically between 60-90%. In cytotoxicity assays, CD19 CAR constructs containing the costimulatory domains of OX40, CD28, or CD27 generally exhibited the greatest cytotoxic activity against Nalm6 and Raji tumor cell lines in vitro. High expression of these three CD19 CAR constructs was also maintained over the course of 4 weeks in addition to sustained NK proliferation and cell numbers, indicating an increase in NK cell persistence. In comparing these constructs in an in vivo Nalm6 tumor model, all three constructs demonstrated greater antitumor efficacy relative to unmodified NK cells. The CD19 CAR construct containing the OX40 costimulatory domain showed moderately enhanced efficacy than the CD28 or CD27 variants in vivo. Conclusion: Transduction of NK cells with CD19-OX40-CD3ζ CAR and mbIL15 increases their cytotoxic activity and persistence. Based on these data, further development of NK CD19 CAR for clinical use will be pursued. Citation Format: Luxuan G. Buren, Chao Guo, Yanying Fan, Alexander Aronov, Xiumin Wu, Daofeng Liu, Ming-Hong Xie, Sasha Lazetic, Ivan H. Chan, James B. Trager. Coexpression of a CD19-OX40-CD3ζ CAR with membrane-bound IL-15 enhances natural killer cell function [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B64.

A Preclinical Embryonic Zebrafish Xenograft Model to Investigate CAR T Cells In Vivo.

Chimeric antigen receptor (CAR) T cells have proven to be a powerful cellular therapy for B cell malignancies. Massive efforts are now being undertaken to reproduce the high efficacy of CAR T cells in the treatment of other malignancies. Here, predictive preclinical model systems are important, and the current gold standard for preclinical evaluation of CAR T cells are mouse xenografts. However, mouse xenograft assays are expensive and slow. Therefore, an additional vertebrate in vivo assay would be beneficial to bridge the gap from in vitro to mouse xenografts. Here, we present a novel assay based on embryonic zebrafish xenografts to investigate CAR T cell-mediated killing of human cancer cells. Using a CD19-specific CAR and Nalm-6 leukemia cells, we show that live observation of killing of Nalm-6 cells by CAR T cells is possible in zebrafish embryos. Furthermore, we applied Fiji macros enabling automated quantification of Nalm-6 cells and CAR T cells over time. In conclusion, we provide a proof-of-principle study that embryonic zebrafish xenografts can be used to investigate CAR T cell-mediated killing of tumor cells. This assay is cost-effective, fast, and offers live imaging possibilities to directly investigate CAR T cell migration, engagement, and killing of effector cells.

Patient's T Cell Functionality Determines Blinatumomab-Mediated Killing of B-ALL Leukemia Cells

Introduction B cell acute lymphoblastic leukemia (B-ALL) is a severe disease caused by malignant transformation and uncontrolled proliferation of immature B lymphocytes. Blinatumomab, a Bi-specific T cell engager (BiTE®) combining the VH and VL domains of two antibodies against human CD19 and CD3, has been approved by U.S. Food and Drug Administration (FDA) for the treatment of Philadelphia chromosome negative (Ph-) relapsed or refractory B-ALL in 2014, and recently Philadelphia chromosome-positive (Ph+) B-ALL. Blinatumomab brings killer T and target B cells into close proximity, activating patients' autologous T cells to kill both normal and malignant B cells via mechanisms such as cytolytic immune synapse formation and inflammatory cytokine production. Clinical trials have shown that there are still patients refractory to blinatumomab. It is thus of great importance to understand the resistance mechanisms and search for biomarkers for patient selection.Methods and Results In this study, we used Mass cytometry-based immunophenotyping and Luminex based multiple protein quantitation, to search for biomarkers correlated with the killing capacity of blinatumomab. First, we collected PBMCs from 12 B-ALL patients for an in vitro killing assay, focusing on the quantity and quality profiling of T cells. PBMCs were used as the effector cells and NALM-6 as the target cells. The final effector to target (E/T) ratio is 10:1. Blinatumomab were added to the co-cultured cells (final concentration 10 ng/mL) for 18hrs. A 5% specific lysis of NALM-6 was used as cut-off threshold. Samples with specific killing above 5% were classified as “positive” for blinatumomab response, and below 5% as “negative”. Among the patients analyzed, 4 out of 12 patients (33.3%) showed apparent and specific lysis of NALM6 leukemia cells, while the remaining samples showed no obvious effect. This divergent killing effect of blinatumomab with the presence of PBMCs from different donors may reflect the differentiated treatment response in B-ALL patients.Concurrently we immunophenotyped each patient's PBMCs by Mass cytometry with a panel of antibodies for surface lineage markers and intracellular effector molecules and cytokines /chemokines. Unsupervised clustering showed that a panel of T-cell associated biomarkers highly significantly correlated with B-ALL cell response to blinatumomab (p=0.001, R=0.839). This panel of biomarkers included T cell markers of CD45, CD3 and CD4; cytolytic proteins of Perforin and GranzymeB; cytokines of IL-2, INFγ and TNFα; and chemokine CCL4 (also known as MIP-1b).In a Luminex analysis examining multiple proteins accumulating in the culture supernatant from the in vitro killing assay, the top biomarkers identified by Mass cytometry also showed significant response to blinatumomab at different time points (12, 24 and 48 hrs). They included TNF-α, CCL4, IL-2, INFγ and Granzyme B. Furthermore, released CCL4 at 24 and 48 hrs and INFγ at 48 hrs significantly correlated with blinatumomab mediated cytotoxicity.Conclusion In summary, the immunophenotyping and multiplexed protein measurement by Mass cytometry and Luminex assay identified key biomarkers that may correlate with blinatumomab-mediated killing of B-ALL leukemia cells. The biomarker panel reflected the importance of primed T cell activation, associated key cytokine/chemokine as well as the consequent cytolytic protein release in the overall response to blinatumomab. Thus, in vitro biomarker profiling focusing on T cell status with these key molecules may facilitate patient selection and predict the response to the immunotherapy. DisclosuresFu:Amgen: Research Funding.

Synthesis of Nerol Derivatives Containing a 1,2,3-Triazole Moiety and Evaluation of Their Activities against Cancer Cell Lines

In the present investigation, a collection of twenty two nerol derivatives, containing 1,2,3-triazolic appendages, was synthesized and screened in vitro for their cytotoxic activity against HL60, Nalm6, and Jurkat human leukemia cells as well as against B16F10 (melanoma cell line). In most cases, derivatives were able to reduce cell viability. The most potent compound (Z)-4-(((3,7-dimethylocta-2,6-dien-1-yl)oxy)methyl)-1-(4-(trifluoromethoxy)benzyl)-1H-1,2,3 triazole showed antiproliferative activity against Jurkat cells and reduced B16F10 cell migration. Physicochemical properties of the compounds were calculated in order to evaluate their potential for drug development. Most of the evaluated physicochemical parameters seemed to be favorable for drug development. In addition, for a better understanding of the biological activity results, 3D quantitative structure-activity relationship (QSAR) studies were carried out. 3D-QSAR studies indicate that the anticancer activities observed for the cell lines HL60 and Jurkat may occur by a similar mechanism of action and the same was found for the Nalm6 and B16F10 cell lines.

Gold(I) NHC Complexes: Antiproliferative Activity, Cellular Uptake, Inhibition of Mammalian and Bacterial Thioredoxin Reductases, and Gram-Positive Directed Antibacterial Effects.

Gold complexes with N-heterocyclic carbene (NHC) ligands represent a promising class of metallodrugs for the treatment of cancer or infectious diseases. In this report, the synthesis and the biological evaluation of halogen-containing NHC-AuI -Cl complexes are described. The complexes 1 and 5 a-5 f displayed good cytotoxic activity against tumor cells, and cellular uptake studies suggested that an intact Au-NHC fragment is essential for the accumulation of high amounts of both the metal and the NHC ligand. However, the bioavailability was negatively affected by serum components of the cell culture media and was influenced by likely transformations of the complex. One example (5 d) efficiently induced apoptosis in vincristine- and daunorubicin-resistant P-glycoprotein overexpressing Nalm-6 leukemia cells. Cellular uptake studies with this compound showed that both the wild-type and resistant Nalm-6 cells accumulated comparable amounts of gold, indicating that the gold drug was not excreted by P-glycoprotein or other efflux transporters. The effective inhibition of mammalian and bacterial thioredoxin reductases (TrxR) was confirmed for all of the gold complexes. Antibacterial screening of the gold complexes showed a particularly high activity against Gram-positive strains, reflecting their high dependence on an intact Trx/TrxR system. This result is of particular interest as the inhibition of bacterial TrxR represents a relatively little explored mechanism of new anti-infectives.

The Central Nervous System Microenvironment Influences the Leukemia Transcriptome and Enhances Leukemia Chemo-Resistance

Central nervous system (CNS) relapse is a significant cause of treatment failure among patients with acute lymphoblastic leukemia (ALL). Isolated CNS relapse occurs in ~3-8% of children with leukemia and accounts for 30-40% of initial relapses in some clinical trials. In addition, CNS-directed therapies are associated with seizures, secondary neoplasms, encephalopathy, and long-term endocrine, developmental, neurovascular, and cognitive deficits. While many studies have demonstrated that interactions between leukemia cells and components of the bone marrow microenvironment influence leukemia development, maintenance, and chemo-resistance, the role of the CNS microenvironment in leukemia is less well studied.To investigate the influence of the CNS niche in leukemia, we asked whether the CNS niche could impart unique and functionally important gene expression changes in leukemia cells. We transplanted NALM-6 human, pre-B leukemia cells into NSG mice without prior irradiation to avoid perturbing the bone marrow and CNS niches. After systemic leukemia development the mice were euthanized and leukemia cells were isolated from the bone marrow and CNS microenvironments. Gene expression profiling of ~700 cancer-associated genes (NanoString® Technology) identified 36 leukemia genes differentially expressed (30 up-regulated and 6 down-regulated; fold change ≥ 2 and FDR<0.05) in leukemia cells in the CNS microenvironment relative to the bone marrow. Furthermore, functional annotation revealed the up-regulated genes were involved in known leukemia and cancer pathways including MAPK, RAS, and apoptosis.We elected to further examine the gene PBX1 as it is a transcription factor with known roles in hematopoiesis, leukemia, and cancer biology. PBX1 contributes to Evi-1 mediated leukemia development in a murine model of leukemia and is a partner with TCF3 in the t(1;19) translocation that occurs in ~5% of pre-B ALL. Interestingly, this translocation is also associated with an increased risk for CNS relapse. We confirmed PBX1 mRNA up-regulation in leukemia cells either isolated from the CNS or in leukemia cells co-cultured with CNS-derived, murine choroid plexus cells by quantitative PCR. Supporting the generalizability of these results, additional human B-cell leukemia lines SEM and REH also exhibited PBX1 mRNA up-regulation in leukemia cells isolated from the murine CNS niche. Western blots of NALM-6 and SEM leukemia cells isolated from the mouse CNS as well as from leukemia cells co-cultured ex vivo with choroid plexus cells also showed PBX1 up-regulation at the protein level. Finally, culture of leukemia cells in either choroid plexus cell conditioned media or human cerebral spinal fluid (CSF) had minimal effects on PBX1 protein levels, suggesting that direct cell contact, rather than a soluble factor(s), contributes to PBX1 up-regulation in leukemia cells.Following confirmation of PBX1 up-regulation in leukemia cells within the CNS microenvironment, we next ectopically expressed PBX1, or GFP as a control, in leukemia cells to identify functional consequences of PBX1 expression in leukemia cells. Leukemia cells expressing PBX1 exhibited decreased sensitivity to cytarabine relative to control cells as measured by both proliferation and apoptosis assays. Furthermore, shRNA targeting PBX1, but not control shRNA, prevented PBX1 up-regulation in leukemia cells when co-cultured with choroid plexus cells and modestly, but significantly, attenuated the ability of choroid plexus cells to protect leukemia cells from cyatabine-induced apoptosis. Finally, although PBX1 had no effect on leukemia proliferation, it caused enhanced colony-forming ability in semi-solid media, consistent with increased leukemia self-renewal properties. Together these results suggest PBX1 up-regulation in leukemia cells may contribute to both leukemia self-renewal properties and chemo-resistance in the CNS niche.In summary, we believe this work illustrates the unique and functionally important effect that the CNS microenvironment has on leukemia cells. More comprehensive analyses of the leukemia transcriptome, genome, and proteome in the CNS niche will build upon this foundation and provide a more detailed understanding of the role of the CNS niche in leukemia biology. Finally, this approach may identify targetable CNS niche-mediated mechanisms of leukemia chemo-resistance and self-renewal. DisclosuresNo relevant conflicts of interest to declare.

Further drimane sesquiterpenes from Drimys brasiliensis stem barks with cytotoxic potential.

Drimys brasiliensis Miers (Winteraceae) is used in folk medicine for the treatment of cancer. Its anti-tumor activity has been demonstrated in vitro models using extracts and isolated compounds. This study investigates the cytotoxic effects of stem bark extracts of D. brasiliensis as well as isolated compounds that may be responsible for the activitys and evaluates them in leukemia cells. The stem bark extract were subjected to column chromatography, and the structures of compounds were elucidated based on spectroscopic methods by using NMR and infrared spectroscopy and GC/MS. The cytotoxicity of the isolated compounds was evaluated in chronic myeloid (K562) and acute B lymphoblastic (Nalm6) leukemia cells using tetrazolium assay (MTT). Two new compounds were isolated 1β-O-p-methoxy-E-cinnamoyl-5α-keto-11α-enol-albicanol (1a) and the isomer 1β-O-p-methoxy-E-cinnamoyl-5α-keto-11β-enol-albicanol (1b) and 1β-O-p-methoxy-E-cinnamoyl-isodrimeninol (2). The known compounds polygonal acid (3a) and the isomer isopolygonal acid (3b), fuegin (4a) and the isomer epifuegin (4b), the mixture drimanial (5) and 1β-O-(p-methoxy-E-cinnamoyl)-6α-hydroxypolygodial (6) were also isolated. The drimanes (1-4) and drimanial (5), 1β-(p-coumaroyloxy)-polygodial (7), 1β-(p-methoxycinnamoyl)-polygodial (8), and polygodial (9) isolated previously were assessed in tumor cells. The IC50 values were between 3.56 and 128.91 μM. 1-β-(p-cumaroiloxi)-polygodial showed the best result with IC50 8.18 and 3.56 μM by K562 and Nalm6, respectively. The chloroform extract of the stem bark of D. brasiliensis is a great source of drimane sesquiterpenes. Our experimental data suggest that drimanes are responsible for cytotoxicity activity demonstrated by this species, especially those with the aldehyde group linked to carbons C-11 and C-12.