Human Jurkat Cells Research Articles

In vitro antiplasmodial and anticancer analyses of endophytic fungal extracts isolated from selected Nigerian medicinal plants

Ethnomedicinal plants are thought to have better prospects of harboring endophytes that produce natural products with pharmacological activities. This study aimed to investigate the antiplasmodial and anticancer properties of secondary metabolites of endophytic fungi from three medicinal plants. The endophytic fungi included Lasiodiplodia theobromae isolated from Cola acuminata, Curvularia lunata Bv4 isolated from Bambusa vulgaris, and Curvularia lunata Eg7 isolated from Elaeis guineensis. The identification of the fungi was based on the internal transcribed spacer (ITS-rDNA) sequence. The fungi were subjected to solid-state fermentation and the secondary metabolites were extracted with ethyl acetate. In vitro antiplasmodial screening of extracts was performed using the SYBR green I-based fluorescence assay on the chloroquine-resistant Plasmodium falciparum strain DD2. The cytotoxicity of the extracts on human red blood cells and Jurkat (leukemia) cells was assessed using the tetrazolium-based colorimetric MTT assay. Gas chromatography-mass spectrometry (GC–MS) analysis was used to identify the constituents of the fungal extracts. The extract of L. theobromae showed the best antiplasmodial activity against chloroquine-resistant P. falciparum (IC50 = 5.4 µg/mL) and was not harmful to erythrocytes (CC50 > 100 µg/mL). All three fungal extracts showed a weak cytotoxic effect against Jukart cell lines (CC50 > 100 µg/mL). GC–MS analysis of the three endophytic fungal extracts revealed the presence of forty major bioactive compounds, including: oxalic acid, isobutyl nonyl ester, 2,4-di-tert-butylphenol, and hexadecanoic acid, among others. The endophytic fungi from the medicinal plants in this study were promising sources of bioactive compounds that could be further evaluated as novel drugs for the treatment of malaria caused by P. falciparum-resistant strains.

The octapeptide-repeat surface protein of Entamoeba nuttalli is a novel virulence factor that promotes adherence to host cells

Entamoeba nuttalli is genetically the closest to Entamoeba histolytica, the causative agent of human amebiasis, and its natural host is Macaca species. A unique E. nuttalli specific surface protein (PTORS) containing 42 repeats of octapeptide was identified by comparative genomic analysis of Entamoeba species. We aimed to elucidate the function of this protein. When trophozoites from various E. nuttalli strains were examined by immunofluorescence microscopy and flow cytometry using a PTORS-specific monoclonal antibody, only a limited proportion of trophozoites were stained, indicating that the protein was not commonly expressed in all E. nuttalli trophozoite. The proportion of trophozoites expressing PTORS increased after passage in hamster livers, suggesting that the protein functions in the virulence of trophozoites in the liver tissue. Single-cell analysis revealed that in the cluster including trophozoites with PTORS gene expression, genes of virulence-related proteins were also upregulated. Trophozoites of E. histolytica transfected with PTORS showed enhanced adherence and subsequent phagocytic activity towards human Jurkat cells, independent of the lectin. E. histolytica trophozoites expressing PTORS formed larger liver abscesses in hamsters. These results demonstrate that PTORS is a novel virulence factor in Entamoeba species.

Spleen Tyrosine Kinase (SYK) negatively regulates ITAM-mediated human NK cell signaling and CD19-CAR NK cell efficacy.

NK cells express activating receptors that signal through ITAM-bearing adapter proteins. The phosphorylation of each ITAM creates binding sites for SYK and ZAP70 protein tyrosine kinases to propagate downstream signaling including the induction of Ca 2+ influx. While all immature and mature human NK cells co-express SYK and ZAP70, clonally driven memory or adaptive NK cells can methylate SYK genes and signaling is mediated exclusively using ZAP70. Here, we examined the role of SYK and ZAP70 in a clonal human NK cell line KHYG1 by CRISPR-based deletion using a combination of experiments and mechanistic computational modeling. Elimination of SYK resulted in more robust Ca ++ influx after cross-linking of the CD16 and NKp30 receptors and enhanced phosphorylation of downstream proteins, whereas ZAP70 deletion diminished these responses. By contrast, ZAP70 depletion increased proliferation of the NK cells. As immature T cells express both SYK and ZAP70 but mature T cells often express only ZAP70, we transduced the human Jurkat cell line with SYK and found that expression of SYK increased proliferation but diminished TCR-induced Ca 2+ flux and activation. We performed transcriptional analysis of the matched sets of variant Jurkat and KHYG1 cells and observed profound alterations caused by SYK expression. As depletion of SYK in NK cells increased their activation, primary human NK cells were transduced with a CD19-targeting CAR and were CRISPR edited to ablate SYK or ZAP70 . Deletion of SYK resulted in more robust cytotoxic activity and cytokine production, providing a new therapeutic strategy of NK cell engineering for cancer immunotherapy.

Abstract 7532: NB005.1, a fully humanized anti-KLRG1 blocking mAb enhances effector function of human CD8 T-cells

Abstract Introduction: Immune checkpoint blockers (ICBs) have revolutionized treatment, enhancing survival and benefiting many patients. However, many patients fail to respond to the treatment, necessitating research for new targets and improved approaches. The Killer cell Lectin-like Receptor G1 (KLRG1), expressed on a subset of NK and T-cells harbors an immune receptor tyrosine-based inhibitory motif (ITIM) within its cytoplasmic domain, indicating its inhibitory function. On CD8+ T-cells, KLRG1 is expressed on highly differentiated antigen-specific effector memory T-cells (TEMs) and CD45RA+ effector memory T cells (TEMRA). These cells are known to exhibit strong cytotoxicity potential and display developmental plasticity following KLRG1 blockade. And 16-48% of CD8+ T-cells that infiltrate the tumor microenvironment (TME) are KLRG1 positive. We hypothesize that the blockade of KLRG1 should enhance the effector function of KLRG1+ CD8 T-cells in the TME and provide therapeutic benefit as a standalone or in combination with existing ICBs. Methods: Monoclonal antibodies (mAbs) targeting the human KLRG1 extracellular domain (ECD) were generated through mouse immunization and hybridoma technology. Purified mAbs from positive clones were screened using CHO-K1 and Jurkat cells overexpressing human or cynomolgus KLRG1 and human primary CD8 T-cells (n=3) to determine EC50 values. Epitope binning was performed, and high-affinity clones from each bin with cyno cross-reactivity were humanized to IgG1-Fc LALA variant. The Abs were evaluated for ligand blocking and enhancement of CD8 T-cell effector function through multiple functional assays. The assay measured enhancement of IFNγ or TNFα by KLRG1+ human CD8+ T-cells co-cultured with CHO-K1 expressing OKT3 and E-cadherin as artificial antigen-presenting cells (aAPCs) or PBMCs stimulated in with anti-CD3 and anti-CD28 Abs in presence of soluble E-cadherin. A human IgG1-LALA antibody (Ab) served as a negative control. PD-1 and KLRG1 synergy was evaluated in hPBMC assays. Results: NB005.1 demonstrated single digit nanomolar (nM) binding potency on human primary CD8 T-cells and Jurkat cells expressing human KLRG1 as identified by its EC50 value. Blocking of KLRG1 with NB005.1 on purified human CD8 T-cells in co-culture assay with aAPC expressing E-cad and OKT3 led to CD8 T-cell activation and increase in IFNγ production confirming inhibitory function of KLRG1. Similar result was obtained when the Ab was tested on hPBMCs stimulated with anti-CD3 and anti-CD28 Abs in presence of soluble E-cadherin. Conclusion: NB005.1, a fully humanized mAb, binds to human KLRG1 in the single digit nM range and effectively blocks the interaction with its ligand. This blockade enhances T-cell effector function as measured by IFNγ production, supporting the advancement of NB005.1 for preclinical testing, either as a standalone therapeutic or in combination with existing ICBs. Citation Format: Bishnu P. Nayak, Meiguang Xiong, Yajuan Xue, Binbin Wang, Shahrzad Lighvani, Shilpi Sharma, Dong Wang, Haojai Wang, Baiyang Wang, Yu Zhang, Xin Dong. NB005.1, a fully humanized anti-KLRG1 blocking mAb enhances effector function of human CD8 T-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7532.

RG7774 (Vicasinabin), an orally bioavailable cannabinoid receptor 2 (CB2R) agonist, decreases retinal vascular permeability, leukocyte adhesion, and ocular inflammation in animal models.

Preclinical studies suggest that cannabinoid receptor type 2 (CB2R) activation has a therapeutic effect in animal models on chronic inflammation and vascular permeability, which are key pathological features of diabetic retinopathy (DR). A novel CB2R agonist, triazolopyrimidine RG7774, was generated through lead optimization of a high-throughput screening hit. The aim of this study was to characterize the pharmacology, absorption, distribution, metabolism, elimination, and toxicity (ADMET) profile of RG7774, and to explore its potential for managing the key pathological features associated with retinal disease in rodents. The in vitro pharmacology of RG7774 was investigated for CB2R binding and receptor activation using recombinant human and mouse CB2R expression in Chinese hamster ovary cells, and endogenous CB2R expression in human Jurkat cells, and rat and mouse spleen cells. The ADMET profile was evaluated and the effects of RG7774 on retinal permeability, leukocyte adhesion, and choroidal neovascularization (CNV) were investigated in rodent models of retinal disease. Pharmacokinetic (PK) parameters and the exposure-response relationship were characterized in healthy animals and in animals with laser-induced CNV. RG7774 was found to be a potent (EC50: 2.8nM and Ki: 51.3nM), selective, and full CB2R agonist with no signs of cannabinoid receptor type 1 (CB1R) binding or activation. The ligand showed a favorable ADMET profile and exhibited systemic and ocular exposure after oral delivery. Functional potency in vitro translated from recombinant to endogenous expression systems. In vivo, orally administered RG7774 reduced retinal permeability and leukocyte adhesion in rodents with lipopolysaccharide (LPS)-induced uveitis and streptozotocin (STZ)-induced DR, and reduced lesion areas in rats with laser-induced CNV with an ED50 of 0.32mg/kg. Anatomically, RG7774 reduced the migration of retinal microglia to retinal lesions. RG7774 is a novel, highly selective, and orally bioavailable CB2R agonist, with an acceptable systemic and ocular PK profile, and beneficial effects on retinal vascular permeability, leukocyte adhesion, and ocular inflammation in rodent animal models. Results support the development of RG7774 as a potential treatment for retinal diseases with similar pathophysiologies as addressed by the animal models.

Label-free detection of the cytotoxicity effect of cisplatin in human leukemic cells using Raman spectroscopy in conjunction with multivariate analysis.

The single-cell Raman spectra of human leukemic Jurkat cells can be obtained by confocal microscopy Raman spectroscopy, including cell groups treated with different doses of cisplatin (0, 3.5, 7, 10.5 and 14 μmol L-1) for 24 hours and those treated with 10.5 μmol L-1 cisplatin for different times (0, 6, 12, 24 and 36 hours). The structure and amount of protein, nucleic acid and other major molecules from different cell groups show special changes in the percentage of biochemical constituents. Compared with the control group, the two protein Raman bands (1449 and 1659 cm-1) and two DNA bands (1303 and 1338 cm-1) in the treatment groups decrease in intensity with the increase of the drug dose and treatment time of cisplatin. Partial least squares combined with support vector machines was used to develop diagnostic algorithms for distinguishing between control and treatment groups. The support vector machines for classification between the control group (0 μmol L-1) and cell groups treated with 10.5 and 14 μmol L-1 cisplatin for 24 hours have achieved good diagnostic results with a high sensitivity of 100%, specificity of 100% and accuracy of 100%, respectively, indicating that 10.5 μmol L-1 can be used as an appropriate therapeutic dose. Using the same method, the diagnostic sensitivity, specificity and accuracy between the control group (0 hours) and cell groups treated with 10.5 μmol L-1 cisplatin for 24 and 36 hours are all 100%, showing that 24 hours can be used as an appropriate therapeutic time. These results showed that Raman spectroscopy in conjunction with multivariate statistical analysis could be a useful tool for evaluating the cytotoxicity induced by cisplatin in human leukemic cells.

Evaluating the cytotoxicity mechanism of the cell-penetrating peptide TP10 on Jurkat cells

TP10, a classic cell-penetrating peptide, shows a high degree of similarity to AMPs in structure. Although TP10 has been widely used in drug delivery, the mechanism underlying its cytotoxicity is yet to be elucidated. Herein, we explored the cell-killing mechanism of TP10 against human leukemia Jurkat cells. TP10 induced necrosis in Jurkat cells via rapid disruption of cell membranes, particularly at high concentrations. Although mitochondria in Jurkat cells were damaged by TP10, mitochondria-mediated apoptosis did not occur, possibly due to intracellular ATP depletion. Necroptosis in TP10-treated Jurkat cells became an alternative route of apoptosis. Our results demonstrate that necrosis and necroptosis rather than apoptosis are involved in the cell-killing mechanism of TP10, which contributes to the understanding of its toxicity.

Radiosensitizing Effects of Lithium Ascorbate on Normal and Tumor Lymphoid Cells under X-ray Irradiation

Abstract: The study aimed to assess the radiosensitizing effect of lithium ascorbate on tumor cells. Background: Cancer cells radioresistance is an important factor restraining the success of X-ray therapy. Radiosensitizing drugs make tumor cells more sensitive to ionizing radiation and improve the effectiveness of radiotherapy. Although many chemical substances can potentiate the cytotoxic effects of X-ray radiation, their clinical applications are limited due to possible adverse reactions. Recently, several approaches have been proposed to develop new radiosensitizers that are highly effective and feature low toxicity. Among new enhancers of X-ray therapy, ascorbic acid, and its derivates demonstrate very low toxicity along with a wide therapeutic range. Lithium ascorbate is a promising X-ray therapy enhancer, but its mechanism of action is unknown. This research focuses on the radiosensitizing properties of lithium ascorbate and its effects on both tumor and normal irradiated cells. Methods: The viability of the radiosensitized cells was evaluated by fluorescence flow cytometry using Annexin V-FITC Apoptosis Detection Kit and Cellular ROS Assay Kit (Abcam, UK). The test cell cultures included normal human mononuclear and Jurkat cells. Results: Lithium ascorbate sensitizes normal human mononuclear and Jurkat cells towards ionizing radiation. The combined cytotoxic effect of X-ray irradiation (3 Gy) and lithium ascorbate (1,2 mmol/L) substantially exceeds the effects of the individual factors, i.e. synergetic action appears. The major types of cell death were late apoptosis and necrosis caused by excessive production of reactive oxygen species. Conclusion: Lithium ascorbate in combination with X-ray irradiation exhibited the cytotoxic effect on both normal and cancer lymphoid cells by activating reactive oxygen species (ROS)-induced apoptosis. These findings indicate that lithium ascorbate is a promising substance to develop a new radiosensitizing drug.

Bio-based surfactants derived from pectin

Surfactants derived from renewable resources and synthesized using renewable feedstock and sustainable methods have become a major research focus over the past decade in the surfactant industry. This research presents an approach for rapidly converting readily available polysaccharides, like pectin derived from fruit waste, into safely biodegradable surface-active polymers. Commercially available pectin was modified with n-alkyl amines having different alkyl chain lengths using potassium carbonate as a catalyst. The effect of pectin molecular weight, alkyl chain length and degree of substitution (DS) on surface-active properties of the modified pectin derivatives was studied. Surface tension decreased slightly with lowering molecular weight, whereas interfacial tension decreased dramatically. Cytotoxicity evaluations using human dermal fibroblast, HepG2 and Jurkat cells demonstrated that these polysaccharide-based surfactants exhibit lower cytotoxicity compared to the conventional surfactants such as octyl phenol ethoxylates (i.e., Triton™ X-100), and therefore are more environmentally friendly. Biodegradation studies show that all modified pectins are “ultimately biodegradable” except for Pectin-amide C8 (1:10).

Local blockade of tacrolimus promotes T-cell-mediated tumor regression in systemically immunosuppressed hosts

BackgroundImmunosuppressive drugs such as tacrolimus have revolutionized our ability to transplant organs between individuals. Tacrolimus acts systemically to suppress the activity of T-cells within and around transplanted organs. However, tacrolimus...

Exploring the Anticancer Potential of Premna resinosa (Hochst.) Leaf Surface Extract: Discovering New Diterpenes as Heat Shock Protein 70 (Hsp70) Binding Agents.

Premna, a genus consisting of approximately 200 species, predominantly thrives in tropical and subtropical areas. Many of these species have been utilized in ethnopharmacology for diverse medicinal applications. In Saudi Arabia, Premna resinosa (Hochst.) Schauer (Lamiaceae) grows wildly, and its slightly viscid leaves are attributed to the production of leaf accession. In this study, we aimed to extract the surface accession from fresh leaves using dichloromethane to evaluate the anticancer potential. The plant exudate yielded two previously unknown labdane diterpenes, Premnaresone A and B, in addition to three already described congeners and four known flavonoids. The isolation process was accomplished using a combination of silica gel column chromatography and semi-preparative HPLC, the structures of which were identified by NMR and HRESIMS analyses and a comparison with the literature data of associated compounds. Furthermore, we employed a density functional theory (DFT)/NMR approach to suggest the relative configuration of different compounds. Consequently, we investigated the possibility of developing new chaperone inhibitors by subjecting diterpenes 1-5 to a Surface Plasmon Resonance-screening, based on the knowledge that oridonin, a diterpene, interacts with Heat Shock Protein 70 (Hsp70) 1A in cancer cells. Additionally, we studied the anti-proliferative activity of compounds 1-5 on human Jurkat (human T-cell lymphoma) and HeLa (epithelial carcinoma) cell lines, where diterpene 3 exhibited activity in Jurkat cell lines after 48 h, with an IC50 of 15.21 ± 1.0 µM. Molecular docking and dynamic simulations revealed a robust interaction between compound 3 and Hsp70 key residues.

A Quenched Size-Expanded Nucleotide Reports Activity of the Leukemia Biomarker Terminal Deoxynucleotidyl Transferase (TdT).

Terminal deoxynucleotidyl Transferase (TdT) is a template-independent DNA polymerase that plays an essential role in the human adaptive immune system and is upregulated in several types of leukemia. It has therefore gained interest as a leukemia biomarker and potential therapeutic target. Herein, we describe a FRET-quenched fluorogenic probe based on a size-expanded deoxyadenosine that reports directly on TdT enzymatic activity. The probe enables real-time detection of primer extension and de novo synthesis activity of TdT and displays selectivity over other polymerase and phosphatase enzymes. Importantly, TdT activity and its response to treatment with a promiscuous polymerase inhibitor could be monitored in human T-lymphocyte cell extract and Jurkat cells using a simple fluorescence assay. Finally, employing the probe in a high-throughput assay resulted in the identification of a non-nucleoside TdT inhibitor.

Three-dimensional, label-free cell viability measurements in tissue engineering scaffolds using optical coherence tomography.

In the field of tissue engineering, 3D scaffolds and cells are often combined to yield constructs that are used as therapeutics to repair or restore tissue function in patients. Viable cells are often required to achieve the intended mechanism of action for the therapy, where the live cells may build new tissue or may release factors that induce tissue regeneration. Thus, there is a need to reliably measure cell viability in 3D scaffolds as a quality attribute of a tissue-engineered medical product. Here, we developed a noninvasive, label-free, 3D optical coherence tomography (OCT) method to rapidly (2.5min) image large sample volumes (1 mm3 ) to assess cell viability and distribution within scaffolds. OCT imaging was assessed using a model scaffold-cell system consisting of a polysaccharide-based hydrogel seeded with human Jurkat cells. Four test systems were used: hydrogel seeded with live cells, hydrogel seeded with heat-shocked or fixed dead cells and hydrogel without any cells. Time series OCT images demonstrated changes in the time-dependent speckle patterns due to refractive index (RI) variations within live cells that were not observed for pure hydrogel samples or hydrogels with dead cells. The changes in speckle patterns were used to generate live-cell contrast by image subtraction. In this way, objects with large changes in RI were binned as live cells. Using this approach, on average, OCT imaging measurements counted 326 ± 52 live cells per 0.288 mm3 for hydrogels that were seeded with 288 live cells (as determined by the acridine orange-propidium iodide cell counting method prior to seeding cells in gels). Considering the substantial uncertainties in fabricating the scaffold-cell constructs, such as the error from pipetting and counting cells, a 13% difference in the live-cell count is reasonable. Additionally, the 3D distribution of live cells was mapped within a hydrogel scaffold to assess the uniformity of their distribution across the volume. Our results demonstrate a real-time, noninvasive method to rapidly assess the spatial distribution of live cells within a 3D scaffold that could be useful for assessing tissue-engineered medical products.

Expression of the H2O2 Biosensor roGFP-Tpx1.C169S in Fission and Budding Yeasts and Jurkat Cells to Compare Intracellular H2O2 Levels, Transmembrane Gradients, and Response to Metals

Intracellular hydrogen peroxide (H2O2) levels can oscillate from low, physiological concentrations, to intermediate, signaling ones, and can participate in toxic reactions when overcoming certain thresholds. Fluorescent protein-based reporters to measure intracellular H2O2 have been developed in recent decades. In particular, the redox-sensitive green fluorescent protein (roGFP)-based proteins fused to peroxiredoxins are among the most sensitive H2O2 biosensors. Using fission yeast as a model system, we recently demonstrated that the gradient of extracellular-to-intracellular peroxides through the plasma membrane is around 300:1, and that the concentration of physiological H2O2 is in the low nanomolar range. Here, we have expressed the very sensitive probe roGFP2-Tpx1.C169S in two other model systems, budding yeast and human Jurkat cells. As in fission yeast, the biosensor is ~40-50% oxidized in these cell types, suggesting similar peroxide steady-state levels. Furthermore, probe oxidation upon the addition of extracellular peroxides is also quantitatively similar, suggesting comparable plasma membrane H2O2 gradients. Finally, as a proof of concept, we have applied different concentrations of zinc to all three model systems and have detected probe oxidation, demonstrating that an excess of this metal can cause fluctuations of peroxides, which are moderate in yeasts and severe in mammalian cells. We conclude that the principles governing H2O2 fluxes are very similar in different model organisms.

MiR-708 regulates the biological behavior of childhood leukemia cells by binding to the 3'utr end of the target gene and reducing the level of the target gene.

This study was performed to analyze the biological behavior of childhood leukemia cells regulated by miR-708 by binding to the 3&apos; UTR end of the target gene and reducing the level of the target gene. In this regard, human leukemia Jurkat cell lines were selected and divided into a control group, miR-708 overexpression group and miR-708 inhibition group. MTT assay was used to detect the cell proliferation inhibition rate, flow cytometry was used to detect the apoptosis rate and cell cycle change, the scratch test was used to detect the cell migration capacity, and Western Blot assay was used to detect the expression of CNTFR, apoptosis and JAK/STAT pathway related proteins. To verify the binding site of miR-708 and target gene CNTFR. The results showed that the cell proliferation inhibition rate, apoptosis rate, G1 phase ratio, Bax protein, and CNTFR protein in the miR-708 overexpression group were significantly lower than those in the control group at each time point, while the S phase ratio, Bcl-2 protein, cell migration ability, JAK3 and STAT3 protein were significantly higher than those in the control group (P<0.05). The results of the miR-708 inhibition group were contrary to those of the miR-708 overexpression group. The binding sites of miR-708 and CNTFR were predicted by TargetScan bioinformatics software. It was found that there were two binding sites of miR-708 and CNTFR, 394-400 bp and 497-503 bp respectively. In conclusion, miR-708 can reduce the expression of CNTFR by binding to the target gene CNTFR3&apos; UTR, activate the JAK/STAT pathway to regulate apoptosis-related proteins, reduce apoptosis, and enhance the migration ability of leukemia cells.

Fully Myeloablative Antibody-Drug Conjugates Condition for Hematopoietic Stem Cell Transplantation and Provide Durable Antileukemia Benefit

INTRODUCTION: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) holds the potential for cure of numerous blood diseases and provides the best chance for durable control of acute myeloid leukemia (AML). Successful allo-HSCT requires that host hematopoietic stem cells (HSCs) are depleted to make bone marrow (BM) space for the incoming donor HSCs, and that host T and NK cell mediated immunity is suppressed to prevent donor HSC rejection. To meet these requirements, conditioning regimens comprised of chemotherapy and/or irradiation are used, which carry risks of severe toxicities that may prevent patients from undergoing this potentially curative therapy. Thus, there is an unmet clinical need for novel conditioning regimens that minimize toxicity without sacrificing therapeutic efficacy. Previously, we showed in mouse models that saporin-based antibody-drug conjugates (ADCs) targeting CD45 or cKit combined with Janus kinase 1/2 (JAK1/2) inhibitors enabled stable, high-level engraftment in fully mismatched HSCT with ≥99% donor myeloid lineage chimerism. However, saporin ADCs are not fully myeloablative, which limited their antitumor efficacy in prior studies and would likely limit the ability of these conditioning agents to provide protection against AML relapse. We hypothesized that alternative toxic payloads to saporin would enable generation of fully myeloablative ADCs capable of enhanced antileukemia benefit. METHODS: We conjugated anti-mouse CD45.2 (clone 104), anti-mouse cKit (clone 2B8), and their respective isotype control antibodies with pyrrolobenzodiazepine (PBD) dimers via maleimide-thiol chemistry. PBD-conjugated ADCs were tested for cytotoxicity in vitro against the murine YAC-1 (CD45.2+) or P815 (cKit+) cell lines, as well as the human Jurkat (CD45.2-cKit-) cell line using XTT viability assays. Cytotoxicity against BM HSCs from B6 mice and AML1 primary leukemia cells (B6-derived, Dnmt3aR878H/+ and FLT3-ITD; provided by Dr. Timothy Ley) was tested using methylcellulose assays. HSC depletion in vivo was assessed 7 days after treatment with a single dose of CD45- or cKit-PBD. Syngeneic and fully mismatched HSCT studies with CD45-PBD were done by conditioning B6 mice with 50 µg ADC 7 days before transplant (d-7), then infusing 10x106 B6-GFP (syngeneic) or 20x106 BALB/c (allogeneic) BM cells on d0. For in vivo leukemia killing studies, B6 mice were infused with 5x105 AML1 cells on d-6, treated with 50 mg CD45-PBD or 75 mg CD45-saporin on d-2, then transplanted on d0 with 10x106 T cell depleted BM cells from 129/Sv mice. In allo-HSCT studies, mice received oral ruxolitinib (2 mg/g chow) starting on d-3. RESULTS: CD45-PBD targeted murine YAC-1 cells and cKit-PBD targeted P815 cells in vitro with picomolar-range potencies; neither ADC was effective against human Jurkat cells. Target-specific killing was confirmed with blocking studies using excess unconjugated anti-CD45.2 or anti-cKit. While CD45-PBD and cKit-PBD showed similarly high efficacy and picomolar-range potencies at targeting AML1 leukemia cells in vitro, cKit-PBD was approximately 40-fold more potent at depleting B6 HSCs in vitro than CD45-PBD (IC50 values: 243 fM for cKit-PBD versus 10 pM for CD45-PBD). In vivo, a single 50 µg dose of CD45-PBD or 25 µg dose of cKit-PBD completely ablated the HSC niche in B6 mice. Initial transplantation studies done with CD45-PBD confirm that this ADC enables both syngeneic and allogeneic HSCT with near complete donor chimerism in all recipient mice (Fig. 1). Finally, a single CD45-PBD dose provided durable protection against AML1 leukemia, whereas the nonmyeloablative CD45-saporin was only minimally protective (Fig. 2). CONCLUSIONS: We successfully generated fully myeloablative CD45- and cKit-ADCs using PBD as the toxic payload. Notably, CD45-PBD enabled HSCT with near total conversion to donor hematopoiesis and was highly protective against the aggressive AML1 primary leukemia. Studies thus far with cKit-PBD have shown superior killing of murine HSCs in vitro and in vivo compared to CD45-PBD; testing of cKit-PBD in HSCT models and in vivo AML1 leukemia killing studies are in progress. Given the high potency of the PBD payload, we are conducting extensive histopathologic studies and serum chemistry testing to evaluate the acute and chronic organ toxicities of conditioning with PBD-based conjugates. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Streptavidin-Drug Conjugates Streamline Identification of Optimal Toxic Payloads for Antibody-Based Hematopoietic Stem Cell Transplantation Conditioning

INTRODUCTION: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) provides the best and possibly only chance for cure for acute myeloid leukemia (AML). In preparation for allo-HSCT, patients undergo conditioning with chemotherapy and/or irradiation to deplete host hematopoietic stem cells (HSCs), suppress the host immune system, and destroy residual leukemia cells. However, conditioning associated toxicities impose a formidable barrier to patients undergoing this potentially curative therapy. Antibody-drug conjugates (ADCs) have emerged as a promising and potentially safer alternative for allo-HSCT conditioning by allowing targeted depletion of the host HSC niche. Previously, we used saporin-conjugated CD45- or cKit-ADCs as the backbone of a novel conditioning regimen which enabled engraftment between fully mismatched mice. However, saporin ADCs were nonmyeloablative and showed modest antitumor activity. We hypothesized that fully myeloablative ADCs capable of enhanced antileukemia activity could be produced using alternative toxic payloads to saporin. However, direct conjugation of multiple candidate payloads and/or antibodies is costly, time-consuming, and laborious. Here, we describe the production of a panel of streptavidin (SAv)-drug conjugates which allows efficient ADC generation from any biotinylated protein. This flexible platform enabled the rapid production of CD45-ADCs conjugated to multiple toxic payloads, facilitating in vitro screening of their ability to target HSCs and leukemia cells. METHODS: Azide-alkyne cycloaddition ("Click" chemistry) was used to covalently link SAv-azide (Protein Mods, Inc) with dibenzocyloocytne (DBCO)-coupled drug payloads (Levena Biopharma) (Fig. 1). We conjugated three toxic payloads to SAv for these studies: monomethyl auristatin E (MMAE), Duocarmycin SA (Duo), and PNU-159682 (PNU). All of these payloads contain cleavable valine-citrulline linkers for payload release following ADC internalization. For the conjugation reactions, SAv-azide was combined with DBCO-coupled payload and incubated 2 hours at 20oC, with 20% DMSO added as cosolvent. Free payload and DMSO were removed using size exclusion chromatography and dialysis. ADCs were freshly prepared before use by mixing SAv-drug conjugates in a 1:1 molar ratio for 15 minutes with biotinylated antibodies. For our initial studies, we evaluated murine anti-CD45.2 (Clone 104) and human anti-CD45 (Clones BC8 and HI30). Non-biotinylated antibodies combined with SAv-drug conjugates were used as negative controls. All ADCs were tested for cytotoxicity in vitro against 1) murine YAC-1 (mCD45.2+) and human Jurkat (mCD45.2-) cell lines using XTT viability assays, 2) primary HSCs from B6 mice using methylcellulose assays, and 3) AML1 primary murine leukemia cells (Dnmt3aR878H/+ and FLT3-ITD; provided by Dr. Tim Ley) using methylcellulose assays. IC50 values for each assay were derived with nonlinear regression of dose response data. RESULTS: Mouse CD45-Duo and CD45-PNU were highly effective at killing AML1 cells (IC50 values: 39.2pM and 61.1pM, respectively; Fig. 2), as well as B6 HSCs (IC50 values: 0.85nM and 0.13nM, respectively) in vitro. Notably, mouse CD45-MMAE was ineffective against YAC-1 cells, B6 HSCs, and AML1 cells, suggesting that MMAE is not an effective payload for ADC-based conditioning in the mouse. Importantly, nonbiotinylated antibodies mixed with SAv-drug conjugates were largely ineffective, indicating the need for the antibody to bind the SAv-drug conjugate to achieve specific toxicity. Preliminary studies using human CD45-MMAE and CD45-Duo demonstrated that while both payloads could yield ADCs able to kill Jurkat cells, ADCs made using human antibody clone BC8 were considerably more effective than ADCs made with clone HI30. CONCLUSIONS: Our novel SAv-drug conjugate successfully enabled rapid generation of ADCs with a number of potential toxic payloads. This system identified multiple effective candidate payloads for use in mouse and human ADCs for targeting HSCs and leukemia cells in vitro and in vivo. Our ongoing and future studies with this system are focused in three areas: 1) production and testing of mouse and human cKit ADCs, 2) using mouse ADCs to condition for murine HSCT and target AML1 cells in vivo, and 3) evaluating killing of human cord blood-derived HSCs and patient-derived AML cells with human ADCs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

‘Transfersome-Embedded-Gel’ for Dual-Mechanistic Delivery of Anti-psoriatic Drugs to Dermal Lymphocytes

Aim Develop a platform for co-delivering clobetasol propionate (CP) and cyclosporine (CyA) to the epidermis and dermis to treat psoriasis. Methods The transfersomes were prepared by thin-film hydration method. Transfersomes were characterized by dynamic light scattering (DLS) and transmission electron microscope (TEM). Then, the gel stability, viscosity, pH, and spreadability were measured. Cytotoxicity of the CyA-loaded transfersome embedded in CP dispersed gel (TEG-CyA-CP) was assessed on both human keratinocyte cell line (HaCaT) and Jurkat cells. In-vitro cellular uptake and ex-vivo dermal distribution was measured. The expression of inflammatory markers was assessed by Reverse-transcription PCR (RT-PCR). Results Nanoscale (<150 nm) transferosomes with high CyA encapsulation efficiency (> 86%) were made. TEG-CyA-CP demonstrated higher viscosity (4808.8 ± 12.01 mPas), which may help control dual drug release. Ex-vivo results showed TEG-CyA-CP ability to deliver CyA in the dermis and CP in the epidermis. RT-PCR studies showed the optimized formulation helps reduce the tumor necrosis factor (TNF-α) and Interleukin-1 (IL-1) levels to relieve psoriasis symptoms. Conclusion The developed TEG-CyA-CP represents a promising fit-to-purpose delivery platform for the dual-site co-delivery of CyA and CP in treating psoriasis.

Chlamydia trachomatis L2/434/Bu Favors Hypoxia for its Growth in Human Lymphoid Jurkat Cells While Maintaining Production of Proinflammatory Cytokines.

The role of lymphocytes as a cornerstone of the inflammatory response in the invasive pathogenesis of Chlamydia trachomatis (Ct) LGV (L1-3) infection is unclear. Therefore, we assessed whether the adaptation of CtL2 to immortal lymphoid Jurkat cells under hypoxic conditions occurred through proinflammatory cytokine profile modification. The quantities of inclusion-forming units with chlamydial 16S rDNA confirmed that CtL2 grew well under hypoxic rather than normoxic conditions in the cells. Confocal microscopic imaging and transmission electron microscopy revealed the presence of bacterial progeny in the inclusions and showed that the inclusions were larger under hypoxic rather than normoxic conditions; this was supported by the results of 3D image construction. Furthermore, PCR-based analysis of proinflammatory cytokines revealed that the gene expression levels under hypoxic conditions were significantly higher than those under normoxic conditions. In particular, the expression of two genes (CXCL8 and CXCR3) was significantly diminished under normoxic conditions. Taken together, the results indicated that hypoxia promoted CtL2 growth in Jurkat cells while maintaining the levels of proinflammatory cytokines. Thus, Ct LGV infection in lymphocytes under hypoxic conditions might be crucial to a complete understanding of the invasive pathogenesis.

Improved Therapy of B-Cell Non-Hodgkin Lymphoma by Obinutuzumab-Dianthin Conjugates in Combination with the Endosomal Escape Enhancer SO1861.

Immunotoxins do not only bind to cancer-specific receptors to mediate the elimination of tumor cells through the innate immune system, but also increase target cytotoxicity by the intrinsic toxin activity. The plant glycoside SO1861 was previously reported to enhance the endolysosomal escape of antibody-toxin conjugates in non-hematopoietic cells, thus increasing their cytotoxicity manifold. Here we tested this technology for the first time in a lymphoma in vivo model. First, the therapeutic CD20 antibody obinutuzumab was chemically conjugated to the ribosome-inactivating protein dianthin. The cytotoxicity of obinutuzumab-dianthin (ObiDi) was evaluated on human B-lymphocyte Burkitt’s lymphoma Raji cells and compared to human T-cell leukemia off-target Jurkat cells. When tested in combination with SO1861, the cytotoxicity for target cells was 131-fold greater than for off-target cells. In vivo imaging in a xenograft model of B-cell lymphoma in mice revealed that ObiDi/SO1861 efficiently prevents tumor growth (51.4% response rate) compared to the monotherapy with ObiDi (25.9%) and non-conjugated obinutuzumab (20.7%). The reduction of tumor volume and overall survival was also improved. Taken together, our results substantially contribute to the development of a combination therapy with SO1861 as a platform technology to enhance the efficacy of therapeutic antibody-toxin conjugates in lymphoma and leukemia.