Cell Cytotoxicity Assay Research Articles

Preclinical Development of an Actinium-225-Labeled Antibody Radio-Conjugate Directed Against CD45 for Targeted Conditioning and Radioimmunotherapy

Introduction Radiolabeled CD45 antibodies have demonstrated clinical promise as targeted conditioning agents prior to bone marrow transplant (BMT) as a better tolerated and potentially more effective alternative to chemotherapy and/or total body irradiation myeloablative conditioning regimens. CD45 is expressed on all immune cells, including hematopoietic stem cells and both precursor and mature lymphoid and myeloid cells, making it an ideal antigen for directing targeted radiation for conditioning. It is also highly expressed on most hematological cancers, offering the potential for a substantial anti-tumor effect. We describe the preclinical development of a novel anti-CD45 antibody radio-conjugate that consists of the pan-CD45 antibody BC8 labeled with the potent alpha emitter Actinium-225 (225Ac). Methods and Results The monoclonal antibody BC8 (apamistamab) was first conjugated with the DOTA chelator and then labeled with 225Ac, achieving a labeling efficiency typically greater than 80%. Furthermore, 225Ac-BC8 was shown to be stable and retain selective immunoreactivity to CD45+ cells following conjugation and labeling. To evaluate the anti-tumor potency of 225Ac-BC8, two CD45+ human multiple myeloma (MM) cell lines with high or low relative CD45 antigen density, U266 and H929, respectively, (Collette, et al. 2007 Eur. Cytokine Netw. 18:120-126) were tested by cell cytotoxicity assay and achieved greater than 90% cell killing of both lines. To assess the therapeutic potential of 225Ac-BC8, its anti-tumor activity was tested in xenograft models of H929 and U266. NOD-SCID mice bearing established tumors received a single dose of 300 nCi of 225Ac-BC8, 300 nCi of an irrelevant radio-conjugate 225Ac-18B7, naked BC8 antibody, or no treatment. In both models, 225Ac-BC8 exerted complete and durable tumor control for the duration of study. None of the control groups exhibited any anti-tumor effect. Favorable biodistribution results were obtained with SPECT/CT imaging using an 111In-labeled mouse pan CD45 surrogate antibody 30F11 (111Ac-30F11), since BC8 does not cross react with mouse CD45. 225Ac-30F11 is a useful surrogate molecule to study the tolerability and myeloablative properties of 225Ac-anti-CD45 antibodies for targeted conditioning prior to donor BMT, using B6-Ly5b donor cells in B6-Ly5a mice. Results of ongoing conditioning studies will be presented. Conclusions The pan-CD45 antibody BC8 was shown to be efficiently conjugated and labeled with the potent alpha emitter 225Ac to mediate robust and selective killing of CD45+ MM cells and xenografts. Biodistribution analysis confirmed selective uptake into organs of the immune system including homing to BM. These preclinical data and ongoing myeloablative/BM reconstitution studies support the development of 225Ac-BC8 as a novel potent and safe targeted conditioning approach for BMT.

Identification, formation and control of polar brominated disinfection byproducts during cooking with edible salt, organic matter and simulated tap water

Edible salt is essential to the health of humans and serves as a seasoning universally. Besides chloride, edible salt also contains other anions such as bromide, fluoride, sulfate, and carbonate due to incomplete removal during raw salt refinement. In a household cooking (e.g., soup making) process, a chlorine/monochloramine residual in tap water could react with bromide in edible salt and organic matter in food (e.g., rice, wheat) to form numerous brominated disinfection byproducts (Br-DBPs) at significant levels, which might induce adverse health effects to human beings. In this study, we solicited 20 edible salts of different types (i.e., sea salts, well and rock salts, lake salts, and bamboo salts) from nine countries and determined their bromide levels to be 67–375 mg/kg, with an average level of 173 mg/kg. A total of 25 polar Br-DBPs were detected and identified with structures/formulae in cooking water samples using ultra performance liquid chromatography/electrospray ionization-triple quadruple mass spectrometry (UPLC/ESI-tqMS) and high-resolution mass spectrometry. Effects of cooking conditions (e.g., disinfectant type and level, edible salt dose, organic matter type and dose, sequence and time interval of adding organic matter and salt, etc.) on the formation of polar Br-DBPs were investigated, and optimized cooking conditions with minimized formation of polar Br-DBPs were determined. Further aided with an Hep G2 cell cytotoxicity assay, it was found that the overall cytotoxicity of chlorinated and chloraminated cooking water samples prepared after cooking condition optimization was reduced by 57% and 22%, respectively, compared with those prepared before cooking condition optimization.

Assessing the potential of boronic acid/chitosan/bioglass composite materials for tissue engineering applications.

Composite materials aimed at bone tissue engineering need to have suitable bioactivity in order to promote cell proliferation and adhesion. In this article, we study the potential of boronic acids to improve the bioactivity of chitosan-based composite materials. Samples were prepared using boronic acid functionalised chitosan and Bioglass 45S5. These composite materials, prepared by freeze-drying, exhibit 3D interconnected porosity. The materials were fully characterized using scanning electron microscopy (SEM), FT-IR and x-ray diffraction. Their bioactivity was assessed by immersion in simulated body fluid and cell cytotoxicity assays. Composite materials containing boronic acid show no toxicity for Mouse Sertoli (TM4), Human embryonic kidney 293 (Hek293) and Human bone marrow/stroma (HS-5) cells, as opposed to composites containing non-functionalised chitosan.

Tutuilamides A-C: Vinyl-Chloride-Containing Cyclodepsipeptides from Marine Cyanobacteria with Potent Elastase Inhibitory Properties.

Marine cyanobacteria (blue-green algae) have been shown to possess an enormous capacity to produce structurally diverse natural products that exhibit a broad spectrum of potent biological activities, including cytotoxic, antifungal, antiparasitic, antiviral, and antibacterial activities. Using mass-spectrometry-guided fractionation together with molecular networking, cyanobacterial field collections from American Samoa and Palmyra Atoll yielded three new cyclic peptides, tutuilamides A-C. Their structures were established by spectroscopic techniques including 1D and 2D NMR, HR-MS, and chemical derivatization. Structure elucidation was facilitated by employing advanced NMR techniques including nonuniform sampling in combination with the 1,1-ADEQUATE experiment. These cyclic peptides are characterized by the presence of several unusual residues including 3-amino-6-hydroxy-2-piperidone and 2-amino-2-butenoic acid, together with a novel vinyl chloride-containing residue. Tutuilamides A-C show potent elastase inhibitory activity together with moderate potency in H-460 lung cancer cell cytotoxicity assays. The binding mode to elastase was analyzed by X-ray crystallography revealing a reversible binding mode similar to the natural product lyngbyastatin 7. The presence of an additional hydrogen bond with the amino acid backbone of the flexible side chain of tutuilamide A, compared to lyngbyastatin 7, facilitates its stabilization in the elastase binding pocket and possibly explains its enhanced inhibitory potency.

No Cytotoxic and Inflammatory Effects of Empagliflozin and Dapagliflozin on Primary Renal Proximal Tubular Epithelial Cells under Diabetic Conditions In Vitro.

Gliflozins are inhibitors of the renal proximal tubular sodium-glucose co-transporter-2 (SGLT-2), that inhibit reabsorption of urinary glucose and they are able to reduce hyperglycemia in patients with type 2 diabetes. A renoprotective function of gliflozins has been proven in diabetic nephropathy, but harmful side effects on the kidney have also been described. In the current project, primary highly purified human renal proximal tubular epithelial cells (PTCs) have been shown to express functional SGLT-2, and were used as an in vitro model to study possible cellular damage induced by two therapeutically used gliflozins: empagliflozin and dapagliflozin. Cell viability, proliferation, and cytotoxicity assays revealed that neither empagliflozin nor dapagliflozin induce effects in PTCs cultured in a hyperglycemic environment, or in co-medication with ramipril or hydro-chloro-thiazide. Oxidative stress was significantly lowered by dapagliflozin but not by empagliflozin. No effect of either inhibitor could be detected on mRNA and protein expression of the pro-inflammatory cytokine interleukin-6 and the renal injury markers KIM-1 and NGAL. In conclusion, empa- and dapagliflozin in therapeutic concentrations were shown to induce no direct cell injury in cultured primary renal PTCs in hyperglycemic conditions.

VCAM-1 Upregulation Contributes to Insensitivity of Vemurafenib in BRAF-Mutant Thyroid Cancer

Vemurafenib, an inhibitor of mutant BRAF activity, is a promising anticancer agent for patients with BRAF-mutant metastatic melanoma. However, it is less effective in BRAF-mutant thyroid cancer, and the reason for this discrepancy is not yet fully elucidated. By RNA sequencing analysis, we identified vascular cell adhesion molecular-1 (VCAM-1) to be highly upregulated in both time- and dose-dependent manners during BRAF inhibition (BRAFi) in a BRAF-mutant papillary thyroid cancer cell line (BCPAP). Cell cytotoxicity and apoptosis assays showed that knockdown of the induced VCAM-1 in BCPAP cells augmented the antitumor effects of vemurafenib, with decreased IC50 values of 1.4 to 0.8 μM. Meanwhile, overexpression of VCAM-1 in a BRAF-mutant anaplastic thyroid cancer cell line (FRO) reduced the sensitivity to vemurafenib, with increased IC50 values of 1.9 to 5.8 μM. Further investigation showed that PI3K-Akt-mTOR pathway was activated during BRAFi. Co-treatment with Akt signaling inhibitor MK2206 decreased the induced expression of VCAM-1 during BRAFi. This combination further improved the efficacy of vemurafenib. Moreover, VCAM-1 promoted migration and invasion in thyroid cancer cells in vitro, which was also indicated in thyroid cancer patients. The present study is the first to demonstrate that VCAM-1 is upregulated in thyroid cancer cells treated with vemurafenib and contributes to vemurafenib resistance in BRAF-mutant thyroid cancer cells. Targeting the PI3K-Akt-mTOR pathway–mediated VCAM-1 response may be an alternative strategy to sensitize BRAF-mutant thyroid cancers to vemurafenib.

Anti-Proliferative and Pro-Apoptotic Effects of Calligonum comosum (L'Her.) Methanolic Extract in Human Triple-Negative MDA-MB-231 Breast Cancer Cells.

Triple-negative breast cancer (TNBC), the most aggressive subtype, does not respond to targeted therapy due to the lack of hormone receptors. There is an urgent need for alternative therapies, including natural product-based anti-cancer drugs, at lower cost. We investigated the impact of a Calligonum comosum L’Hér. methanolic extract (CcME) on the TNBC MDA-MB-231 cell line proliferation and related cell death mechanisms performing cell viability and cytotoxicity assays, flow cytometry to detect apoptosis and cell cycle analysis. The apoptosis-related protein array and cellular reactive oxygen species (ROS) assay were also carried out. We showed that the CcME inhibited the TNBC cell viability, in a dose-dependent manner, with low cytotoxic effects. The CcME-treated TNBC cells underwent apoptosis, associated with a concomitant increase of apoptosis-related protein expression, including cytochrome c, cleaved caspase-3, cyclin-dependent kinase inhibitor p21, and the anti-oxidant enzyme catalase, compared with the untreated cells. The CcME also enhanced the mitochondrial transition pore opening activity and induced G0/G1 cell growth arrest, which confirmed the cytochrome c release and the increase of the p21 expression detected in the CcME-treated TNBC cells. The CcME-treated TNBC cells resulted in intracellular ROS production, which, when blocked with a ROS scavenger, did not reduce the CcME-induced apoptosis. In conclusion, CcME exerts anti-proliferative effects against TNBC cells through the induction of apoptosis and cell growth arrest. In vivo studies are justified to verify the CcME anti-proliferative activities and to investigate any potential anti-metastatic activities of CcME against TNBC development and progression.

Truncated HDAC9 identified by integrated genome-wide screen as the key modulator for paclitaxel resistance in triple-negative breast cancer.

Rationale: Paclitaxel resistance is a major concern when treating triple-negative breast cancer (TNBC) patients. We aimed to identify candidates causing paclitaxel resistance and explore their significance in TNBC therapeutics.Methods: A genome-wide CRISPR screening, integrated with transcriptome analyses, was performed to identify candidates involved in paclitaxel-resistant TNBCs. Cell proliferation, cytotoxicity, immunofluorescent staining, and xenograft assays were conducted to verify the phenotypes of paclitaxel resistance induced by candidate genes, both in vitro and in vivo. RNA sequencing, Western blotting, and chromatin immunoprecipitation assays were used to explore the underlying mechanisms.Results: MEF2-interacting transcriptional repressor (MITR), the truncated isoform of histone deacetylase 9 (HDAC9) lacking the deacetylation domain, was enriched in paclitaxel-resistant cells. Elevated MITR expression resulted in increased interleukin-11 (IL11) expression and activation of downstream JAK/STAT3 signaling. Mechanistically, MITR counteracted MEF2A-induced transcriptional suppression of IL11, ultimately causing paclitaxel resistance. By contrast, pharmacological inhibition of JAK1/2 by ruxolitinib reversed paclitaxel resistance both in vitro and in vivo.Conclusion: Our in vitro and in vivo genetic and cellular analyses elucidated the pivotal role of MITR/MEF2A/IL11 axis in paclitaxel resistance and provided a novel therapeutic strategy for TNBC patients to overcome poor chemotherapy responses.

Conjugation of new DNA vaccine with polyethylenimine induces cellular immune response and tumor regression in neuroblastoma mouse model.

To estimate immunogenicity and antitumor effect of new DNA vaccine against neuroblastoma using tyrosine hydroxylase as an antigen and linear polyethylenimine (PEI) 20kDa as a synthetic DNA carrier in syngeneic mouse tumor model. DNA vaccine was made by cloning the tyrosine hydroxylase minigene fused to the potato virus X coat protein gene into the expression vector. The A/J mice were vaccinated by three intramuscular injections. For immunogenicity study, immune response was estimated by target cells cytotoxicity assay, interferon-gamma production in enzyme-linked immunospot assay and antigen-specific antibodies in 14days after the final vaccination. Antitumor effect was assessed by measurement of tumor volume and event-free survival rate in mice with engrafted NB41A3murine neuroblastoma cells following three intramuscular injections of the vaccine: 7days before, 5and 10days after tumor engraftment. The immune response was also assessed on the 30th day after tumor engraftment. The immunogenicity and antitumor effect of the vaccine in the form of aqueous solution of DNA and DNA-PEI conjugate were compared. Splenocytes cytotoxicity was the highest in the group of DNA-PEI vaccines (37.3 ± 6.9% lysis of target cells) compared with the unconjugated DNA vaccine (26.2 ± 4.0%) and placebo control (21.9 ± 3.7%). The production of interferon-gamma in the enzyme-linked immunospot assay was about ten times higher in the DNA-PEI group than in the other groups. The vaccine slowed or prevented the growth of the tumor. Mice vaccinated with the DNA-PEI vaccine had significantly better survival compared to control group (p < 0.0003). DNA vaccine against tyrosine hydroxylase, administered as a DNA-PEI 20kDa conjugate, slows down the growth of neuroblastoma cells engrafted to mice.

The Effects of Astaxanthin on Proliferation and Differentiation of MG-63 Osteosarcoma Cells via Aryl Hydrocarbon Receptor (AhR) Pathway: A Comparison with AhR Endogenous Ligand

Background: Osteosarcoma (OS) is the most prevalent bone-related malignancy with a high mortality rate among children and adolescents. In the present study, first we explored the effects of astaxanthin (AST) on proliferation and differentiation of the MG-63 osteosarcoma cell line, and then compared its effects with AhR endogenous ligand (FICZ).Methods: Cell proliferation and cytotoxicity assay were performed using MTT. To identify possible mechanisms underlying AST-induced changes in osteogenic metabolism via the AHR pathway, we defined changes in CYP1A1, osteocalcin, osteopontin, type I collagen, and Runx2 gene expression using RT-PCR.Results: AST upregulated CYP1A1, osteocalcin, osteopontin, type I collagen, and Runx2 expression in trends of increasing its concentration. FICZ showed a biphasic effect on MG-63 cell proliferation. At high concentrations, it significantly decreased the cell viability, while at lower concentrations it was increased as compared to the control. Increasing FICZ concentrations from 1 nm to 1 μM, down-regulated the expression of Runx2, osteopontin, osteocalcin and collagen type 1 at the transcriptional levels. It seems that AST can augment the proliferation and differentiation of MG-63 via the AhR-dependent pathway, while FICZ suppresses the proliferation and differentiation of MG-63.Conclusion: We concluded that various AhR ligands show different behaviors in the modulation of MG-63 cells.

Ciguatoxin Occurrence in Food-Web Components of a Cuban Coral Reef Ecosystem: Risk-Assessment Implications.

In Cuba, ciguatera poisoning associated with fish consumption is the most commonly occurring non-bacterial seafood-borne illness. Risk management through fish market regulation has existed in Cuba for decades and consists of bans on selected species above a certain weight; however, the actual occurrence of ciguatoxins (CTXs) in seafood has never been verified. From this food safety risk management perspective, a study site locally known to be at risk for ciguatera was selected. Analysis of the epiphytic dinoflagellate community identified the microalga Gambierdiscus. Gambierdiscus species included six of the seven species known to be present in Cuba (G. caribaeus, G. belizeanus, G. carpenteri, G. carolinianus, G. silvae, and F. ruetzleri). CTX-like activity in invertebrates, herbivorous and carnivorous fishes were analyzed with a radioligand receptor-binding assay and, for selected samples, with the N2A cell cytotoxicity assay. CTX activity was found in 80% of the organisms sampled, with toxin values ranging from 2 to 8 ng CTX3C equivalents g−1 tissue. Data analysis further confirmed CTXs trophic magnification. This study constitutes the first finding of CTX-like activity in marine organisms in Cuba and in herbivorous fish in the Caribbean. Elucidating the structure–activity relationship and toxicology of CTX from the Caribbean is needed before conclusions may be drawn about risk exposure in Cuba and the wider Caribbean.

A novel wound dressing consisting of PVA-SA hybrid hydrogel membrane for topical delivery of bacteriophages and antibiotics.

The emergence of antibiotic-resistant pathogens has made the treatment of infected burn wounds even more problematical than the pre-antibiotic era. Phage therapy is now being considered a promising treatment options to fight against antibiotic resistant pathogens. Hence, we introduce a novel PVA-SA hydrogel based wound dressing system for topical delivery of bacteriophages and antibiotic to treat infected burn injuries. Hydrogel membrane provides wound healing environment while surface absorbed bacteriophages/antibiotic takes care of the local infection. Different blends of PVA and SA were crosslinked by boric acid and calcium ions to form the hydrogel membrane and assessed for ideal wound dressing properties. 1:2 blended PVA: SA membrane displayed highest swelling index, gel fraction, protein adsorption, hemocompatibility and best mechanical properties among the 3 blends studied in this study. The selected membrane was further characterised by FTIR, FESEM and TGA. Overall, self-adherent, antibacterial and biocompatible membrane was obtained as revealed by the in-vitro antibacterial assays, elution assays and cell cytotoxicity assays. The in-vivo potential was evaluated using MRSA-infected murine burn wound model revealing significant bacterial reduction, wound contraction and reduced inflammation in membrane treated groups in comparison to control group. The dual coated hydrogel membrane delivering both MR10 phage and minocycline proved to be better treatment strategy to treat the resistant burn wound infection rather than phage and antibiotic alone.

Targeting CD38high Acute Myeloid Leukaemia with “Affinity Optimized” Chimeric Antigen Receptor and Membrane Bound TRAIL Expressing Natural Killer Cells

Targeting CD38high Acute Myeloid Leukaemia with “Affinity Optimized” Chimeric Antigen Receptor and Membrane Bound TRAIL Expressing Natural Killer Cells

Preclinical Development of an Actinium-225-Labeled Antibody Radio-Conjugate Directed Against CD45 for Targeted Conditioning and Radioimmunotherapy

Introduction: Radiolabeled CD45 antibodies have demonstrated clinical promise as targeted conditioning treatments prior to bone marrow transplant (BMT) as a better tolerated and potentially more effective alternative to harsh chemotherapy and/or total body irradiation regimens for myeloablative conditioning. CD45 is expressed on all immune cells, including hematopoietic stem cells and both precursor and mature lymphoid and myeloid cells, with an average copy number up to 200,000 molecules per cell, making it an ideal antigen for directing targeted radiation for conditioning. It is also highly expressed on most lymphomas and leukemias, and approximately 50% of multiple myeloma, further offering the potential for mediating a substantial anti-tumor effect in treated patients. We describe the preclinical development of a novel anti-CD45 antibody radio-conjugate (ARC) that harnesses the power of alpha radiation by arming the pan-CD45 antibody BC8 with Actinium-225 (225Ac). 225Ac is a potent radionuclide with high linear energy transfer (80-100 keV/μm) yet short path length (50-80 μm) to deliver significantly more lethal double strand DNA breaks per alpha track than beta particles. In addition, the use of 225Ac for targeted conditioning has advantages of safe handling and ease of use and a long 10 day half-life that enables flexibility in preparation and use. Methods and Results: To generate 225Ac-BC8, the monoclonal antibody was first conjugated with the DOTA chelator, then labeled with 225Ac, achieving a labeling efficiency typically greater than 80%. 225Ac-BC8 was subsequently purified to high radiochemical purity by column chromatography. Using human CD45+ Ramos lymphoma cell line and EL4 as negative control, 225Ac-BC8 was shown to be stable and retain selective immunoreactivity following conjugation and labeling. To evaluate the anti-tumor potency of 225Ac-BC8, two CD45+ human multiple myeloma (MM) cell lines with high or low relative CD45 antigen density, U266 and H929, respectively, (Collette, et al. 2007) were tested by cell cytotoxicity assay. Following transient exposure (4 or 12 hours) to 225Ac-BC8, the ARC demonstrated potent dose-dependent MM cell killing 72 hours post-treatment, achieving greater than 90% cytotoxicity of both cell lines. To further assess the therapeutic potential of 225Ac-BC8, its anti-tumor activity was tested in subcutaneous (s.c.) xenograft models of H929 and U266. NOD-SCID mice bearing established s.c. tumors were randomized to receive a single dose of 300 nCi of 225Ac-BC8, 300 nCi of an irrelevant radio-conjugate 225Ac-18B7, naked BC8 antibody, or no treatment control. In both models, 225Ac-BC8 exerted complete and durable tumor control for the duration of study. None of the control groups exhibited any anti-tumor effect. Studies to evaluate 225Ac-BC8 in models of CD45+ lymphoma are ongoing. Since BC8 does not cross-react with mouse CD45 and only weakly with macaque CD45, biodistribution, dosimetry and imaging analysis was performed in mice using radio-labeled anti-mouse pan-CD45 surrogate antibody 30F11. SPECT/CT imaging and biodistribution was performed with 111In-labeled 30F11. Following IV administration, 111In-CD45 (30F11) rapidly cleared from the blood and accumulated in immune system organs including spleen, bone marrow, and lymph node. Dosimetry calculations for 225Ac-CD45 (30F11) were determined for absorbed dose to target organs. Only modest uptake was noted in the kidney, but significant uptake occurred in liver, the major site for catabolism of full-length antibodies. 225Ac-CD45 (30F11) is a useful molecule to study the tolerability and myeloablative properties of 225Ac-CD45 antibody for targeted conditioning prior to donor BMT, using B6-Ly5b donor cells in B6-Ly5a mice. Results of ongoing studies will also be presented. Conclusions: The pan-CD45 antibody BC8 was shown to be efficiently conjugated and labeled with the potent alpha emitter 225Ac to mediate robust and selective killing of CD45+ MM cells and xenografts. Biodistribution analysis confirmed selective uptake into organs of the immune system including homing to BM. Supported by ongoing myeloablative/BM reconstitution studies, these preclinical data support the development of 225Ac-BC8 as a novel potent and safe targeted conditioning approach for BMT. Reference: Collette, et al., (2007). Eur. Cytokine Netw. 18:120-126. Disclosures Ludwig: Actinium Pharmaceuticals: Employment, Equity Ownership. Dawicki:Actinium Pharmaceuticals: Research Funding. Geoghegan:Actinium Pharmaceuticals: Employment. Liang:Actinium Pharmaceuticals: Employment. Seth:Actinium Pharmaceuticals: Employment, Equity Ownership. Gokhale:Actinium Pharmaceuticals: Employment. Berger:Actinium Pharmaceuticals, Inc: Employment, Equity Ownership. Reddy:Actinium Pharmaceuticals: Employment. Dadachova:Actinium Pharmaceuticals: Consultancy, Research Funding; RadImmune Therapeutics: Consultancy, Research Funding.

Targeting of CD19 By Tafasitamab Does Not Impair CD19 Directed Chimeric Antigen Receptor T Cell Activity in Vitro

CD19 directed chimeric antigen receptor T cell (CART) therapy has shown remarkable activity in B cell lymphoma and acute lymphoblastic leukemia leading to the approval of two CART therapies. With the emergence of therapeutic anti-CD19 antibodies for the treatment of B cell malignancies, it remains to be elucidated whether such antibodies would interfere with the ability of CD19 targeting CARTs to exert their anti-tumor effect in a subsequent therapy. To address a part of this question, we investigated the potential for functional interference between the monoclonal anti-CD19 antibody tafasitamab (MOR208) and CD19 directed CART cells (CART19). CART19 cells were generated through lentiviral transduction of healthy donor T cells with a second generation CD19 CAR construct (FMC63-CD8h-CD8TM-41BBζ) which is similar to the construct used for the FDA-approved CART tisagenlecleucel. Tafasitamab, is an Fc-enhanced humanized monoclonal antibody which mediates antibody-dependent cellular toxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and direct cytotoxicity. It is currently being studied in phase 2 and 3 clinical trials in diffuse large B-cell lymphoma (DLBCL) in combination with the immunomodulatory agent lenalidomide (L-MIND) and the chemotherapeutic drug bendamustine (B-MIND). As a first step we confirmed the relevance of the tested CD19-positive target cell lines, JEKO (mantel cell lymphoma), Ly7 (DLBCL) and NALM-6 (ALL) based on functional activity of tafasitamab and CART19. In a 24 hours ADCC (tafasitamab titration plus natural killer (NK) cells; Figure 1A) and T cell cytotoxicity assays (CART19, E:T titrations; data not shown) distinct activity was observed for both therapies on all tested cell lines. Secondly, we studied whether the observed CART19 activity may be influenced by tafasitamab in case of a direct CD19 binding competition between tafasitamab and the CAR. To test for such binding competition we incubated the CD19+ cell lines NALM6 or JEKO with up to 100 µg/ml tafasitamab, to saturate the receptors. Subsequent flow cytometry analysis using the FMC63 antibody (carrying the same CD19 binding domain as contained in CART19) failed to detect CD19 expression, indicating a direct binding competition between FMC63 and tafasitamab (Figure 1B). Next, to investigate the potential impact of such binding competition on CART19 cell effector functions, we co-cultured tafasitamab CD19+ JEKO cell line at increasing concentrations of up to 100µg/ml, and then added CART19 cells at different effector to target ratios to the cell culture. The presence of tafasitamab, binding to the CD19 antigen, did not affect important CART cell effector functions such as antigen specific killing (Figure 1C), degranulation (Figure 1D), cytokine production or proliferation of CART19 (Figure 1E). In summary, our studies indicate that CART19 continue to exhibit potent antigen specific effector functions despite presence of tafasitamab and the related competition for CD19 binding. Besides the presented in vitro work the questions of therapeutic sequencing of tafasitamab and CART19 is being studied in xenograft models and will be presented at the meeting. Disclosures Sakemura: Humanigen: Patents &amp; Royalties. Cox:Humanigen: Patents &amp; Royalties. Schanzer:MorphoSys AG: Employment. Endell:MorphoSys AG: Employment, Patents &amp; Royalties. Nowakowski:Selvita: Membership on an entity's Board of Directors or advisory committees; NanoString: Research Funding; MorphoSys: Consultancy, Research Funding; Genentech, Inc.: Research Funding; F. Hoffmann-La Roche Ltd: Research Funding; Curis: Research Funding; Bayer: Consultancy, Research Funding; Celgene: Consultancy, Research Funding. Kay:MorphoSys: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Agios: Other: DSMB. Kenderian:Novartis: Patents &amp; Royalties, Research Funding; Tolero: Research Funding; Humanigen: Other: Scientific advisory board , Patents &amp; Royalties, Research Funding; Lentigen: Research Funding; Morphosys: Research Funding; Kite/Gilead: Research Funding.

Enhanced anticancer effect of doxorubicin by TPGS-coated liposomes with Bcl-2 siRNA-corona for dual suppression of drug resistance

Multiple drug resistance (MDR) is a tough problem in developing hepatocellular carcinoma (HCC) therapy. Here, we developed TPGS-coated cationic liposomes with Bcl-2 siRNA corona to load doxorubicin (Dox) i.e., Bcl-2 siRNA/Dox-TPGS-LPs, to enhance anticancer effect of Dox in HCC-MDR. TPGS i.e., d-α-tocopheryl polyethylene glycol 1000 succinate, inhibited P-glycoprotein (P-gp) efflux pump and Bcl-2 siRNA suppressed anti-apoptotic Bcl-2 protein. The Bcl-2 siRNA loaded in the liposomal corona was observed under transmission electron microscopy. The stability and hemolysis evaluation demonstrated Bcl-2 siRNA/Dox-TPGS-LPs had good biocompatibility and siRNA-corona could protect the liposomal core to avoid the attachment of fetal bovine serum. In drug-resistant cells, TPGS effectively prolonged intracellular Dox retention time and siRNA-corona did improve the internalization of Dox from liposomes. In vitro and in vivo anticancer effect of this dual-functional nanostructure was examined in HCC-MDR Bel7402/5-FU tumor model. MTT assay confirmed the IC50 value of Dox was 20–50 fold higher in Bel7402/5-FU MDR cells than that in sensitive Bel7402 cells. Bcl-2 siRNA corona successfully entered the cytosol of Bel7402/5-FU MDR cells to downregulate Bcl-2 protein levels in vitro and in vivo. Bcl-2 siRNA/Dox-TPGS-LPs showed superior to TPGS- (or siRNA-) linked Dox liposomes in cell apoptosis and cytotoxicity assay in Bel7402/5-FU MDR cells, and 7-fold greater effect than free Dox in tumor growth inhibition of Bel7402/5-FU xenograft nude mice. In conclusion, TPGS-coated cationic liposomes with Bcl-2 siRNA corona had the capacity to inhibit MDR dual-pathways and subsequently improved the anti-tumor activity of the chemotherapeutic agent co-delivered to a level that cannot be achieved by inhibiting a MDR single way.

STEM-16. DUAL INHIBITION OF PROTEIN ARGININE METHYLTRANSFERASE 5 AND PROTEIN PHOSPHATASE 2A ENHANCES THE ANTI-TUMOR EFFICACY IN PRIMARY GLIOBLASTOMA NEUROSPHERES

Abstract INTRODUCTION Glioblastoma (GBM) is the most common malignant primary brain tumor and has a heterogeneous tumor cell population. The median survival of GBM patients is less than two years with current multi-model therapy of maximal surgical resection followed by chemotherapy, radiation and tumor treating fields. Protein Arginine Methyltransferase 5 (PRMT5) regulates cellular functions through symmetric di-methylation of arginine residues of histone and non-histone proteins. Our recent findings show that PRMT5 is overexpressed in GBM; its inhibition causes apoptosis and senescence of mature and immature GBM tumor cells respectively. Protein Phosphatase 2A (PP2A), a serine-threonine phosphatase, is associated with senescent tumor cells that contribute to therapy resistance; LB100 is a first-in-class small molecule inhibitor of PP2A that can sensitize tumor cells that are chemo- and radiation-therapy resistant. In this study, using patient-derived primary GBM neurospheres (GBMNS), we tested the anti-GBM effect of concurrent PRMT5 and PP2A inhibition. METHODS Patient-derived primary GBM neurospheres transfected with PRMT5 target-specific siRNA were treated with LB100 and subjected to in vitro assays such as proliferation assay, cell cycle analysis, cytotoxicity assay, PP2A activity and western blot. RESULTS LB100 treatment significantly reduced the viability of PRMT5-depleted GBM cells as compared to PRMT5 intact cells. LB100 caused G1 cell cycle arrest through inactivation of Rb protein; this effect is further enhanced in combination with PRMT5-depletion. Combination therapy also increased the expression of phospho-MLKL; Necrostatin-1 rescued PRMT5-depleted cells from the cytotoxic effects of LB100 indicating that necroptosis caused the enhanced cytotoxicity in combination therapy. We also found that PRMT5-depletion increased the PP2A activity in GBM neurospheres. CONCLUSION Combining the PRMT5 inhibition and PP2A inhibition produced greater antitumor effects than with PRMT5 inhibition alone.

STEM-13. CANCER STEM CELL CYTOTOXICITY ASSAY-GUIDED TREATMENT IN RECURRENT GLIOBLASTOMA AND PROGRESSIVE ANAPLASTIC GLIOMA

Abstract BACKGROUND The presence of chemotherapy-resistant cancer stem cells (CSC) leading to tumor recurrence is postulated to be one of the major factors in the poor response to the current standard of care treatments in progressive high-grade gliomas. Unfortunately, in spite of several new drug discoveries for other tumor types, not many advances have been reported for this disease and therefore recurrent and progressive high-grade glioma survival remains dismal. METHODS We have used a CLIA and CAP accredited Cancer Stem Cell Cytotoxicity Assay(ChemoID) to guide most effective chemotherapy treatments from a panel of FDA approved drugs or their combinations for 12 recurrent glioblastoma (GBM) patients and 2 progressive anaplastic gliomas, all eligible to receive a stereotactic biopsy. Patients were evaluated by MRI scans and response was assessed according to RANO criteria. RESULTS The median age of our patient cohort was 49 years (21–63), with 11 males (79%) and 3 females (21%). We observed 6 complete responses (CR) 43%, 6 partial responses (PR) 43%, and 2 progressive diseases (PD) 14%. Of note, the two PD were observed in patients that could not be treated with assay recommended therapy due to their health status. Patients treated with ChemoID assay-directed therapy had a longer median overall survival (OS) of 13.3 months (5.4-NA) compared to the historical median OS of 9.0 months (8.0–10.8 months) previously reported (1). Notably, our recurrent and progressive high-grade glioma patient cohort treated with assay-guided therapy had a 57% probability to survive at 12 months, compared to the 27% historical probability of survival at 12 months observed in previous studies (1). CONCLUSIONS The data suggests that the ChemoID Cancer Stem Cell Cytotoxicity Assay has the potential to help guide individualized chemotherapy choices to improve recurrent and progressive high-grade glioma patient outcome. 1) Friedman, H.S. et. al. JCO2009 27:28, 4733–4740.

Proscillaridin A induces apoptosis and inhibits the metastasis of osteosarcoma in vitro and in vivo

The side effects of chemotherapy, drug resistance, and tumor metastasis hinder the development of treatment for osteosarcoma, leading to poor prognosis of patients with the disease. Proscillaridin A, a kind of cardiac glycoside, has been proven to have anti-proliferative properties in many malignant tumors, but the efficacy of the drug in treating osteosarcoma is unclear. In the present study, we assessed the effects of Proscillaridin A on osteosarcoma and investigated its underlying action mechanism. The cell cytotoxicity assay showed that Proscillaridin A significantly inhibited the proliferation of 143B cells in a dose- and time-dependent manner. Also, flow cytometry and invasion assay revealed that Proscillaridin A induced apoptosis and reduced 143B cell motility. Western blotting and PCR were used to detect the expressions of Bcl-xl and MMP2 and showed that mRNA/protein expression levels decreased significantly in Proscillaridin A-treated osteosarcoma cells. Using a mouse xenograft model, we found that Proscillaridin A treatment significantly inhibited tumor growth and lung metastasis in vivo and decreased the expression levels of Bcl-xl and MMP2. No noticeable side effect was observed in the liver, kidney, and hematological functions. Conclusively, Proscillaridin A suppressed proliferation, induced apoptosis, and inhibited 143B cell metastasis in vitro and in vivo, and these effects could be mediated by downregulating the expressions of Bcl-xl and MMP2.

Improvement of platelet aggregation and rapid induction of hemostasis in chitosan dressing using silver nanoparticles

There is an increasing demand for high-performance hemostatic agents with good antibacterial efficiency and biocompatibility. In this study, a chitosan/gelatin/sodium hyaluronate hemostatic dressing with silver nanoparticles (CGSH/Ag50) was designed and characterized. Our results suggest that CGSH/Ag50 has high blood absorption and promotes platelet aggregation by the activation of positively charged surface and stimulation of silver nanoparticles. Owing to these excellent properties, CGSH/Ag50 could rapidly control hemorrhage in rabbit ear and liver injury models. CGSH/Ag50 also shows good antibacterial activity, good liquid absorption, and significantly promotes full-thickness wound healing. In the hemolysis and L929 cell cytotoxicity assays, CGSH/Ag50 exhibited no cytotoxicity, suggesting good hemocompatibility and biocompatibility. These observations suggest that CGSH/Ag50 can be an efficient hemostatic material with enough biocompatibility to be used for controlling hemorrhage and promoting wound healing after hemostasis surgery.