BIAcore Analysis Research Articles

Description of a non-competitive ELISA based on time course analysis of ligand binding at saturation, and a direct method for calculating the affinity of monoclonal antibodies

We present a time-course saturation ELISA for measuring the equilibrium constant of the monoclonal antibody (mAb) SIM 28 against horse radish peroxidase (HRP). The curves of HRP binding to a series of fixed mAb dilutions were plotted to completion, and the Kt (= Ks) value (time to occupy 50 % of the mAb paratopes) was determined for each mAb dilution and HRP concentration. Analysis of the kinetic mechanism of the reaction by Lineweaver-Burk and Hanes plots showed that the slope and y-intercept were affected, indicating that mAb ligand saturation follows non-competitive inhibition kinetics in this assay format. In this kinetics, the inhibition constant Ki (= Kd) is the time required to double the slope or halve the Vmax of the Lineweaver-Burk plot. The Kt values of the time courses were doubled (2 x Kt) and normalized by dividing by the total reaction time to obtain a unitless factor which, when multiplied by the concentration of HRP, gives the Ki. The affinity constant of mAb SIM 28 was determined from ELISA data (n = 16) by three methods: i) doubling of Kt, ii) Beatty equation (Kaff = (n-1)/2 (n [HRP’]t - [HRP]t), and iii) SPR (Biacore) analysis. The calculated affinities (mean ± 95 % confidence limits) were i) 4.6 ± 0.67 × 10−9 M, ii) Kaff = 0.23 ± 0.03 × 109 M−1 (Kd = 4.8 ± 0.81 × 10−9 M), and iii) 4.3 ± 0.57 × 10−9 M, respectively. The similar results obtained with the three different techniques indicate that this time-course saturation ELISA, combined with the double Kt method, is a repeatable and direct approach to mAb affinity determination.

GDH1 exacerbates renal fibrosis by inhibiting the transcriptional activity of peroxisome proliferator-activated receptor gamma.

Renal fibrosis is the common outcome of practically all progressive forms of chronic kidney disease (CKD), a significant societal health concern. Glutamate dehydrogenase (GDH) 1 is one of key enzymes in glutamine metabolism to catalyze the reversible conversion of glutamate to α-ketoglutarate and ammonia. However, its function in renal fibrosis has not yet been proven. In this study, GDH1 expression was significantly downregulated in kidney tissues of both children with kidney disease and animal models of CKD. In vivo, the use of R162 (a GDH1 inhibitor) significantly improved renal fibrosis, as indicated by Sirius red and Masson trichrome staining. These findings are consistent with the impaired expression of fibrosis indicators in kidneys from both the unilateral ureteral obstruction (UUO) and 5/6 nephrectomy (5/6 Nx) models. In vitro, silencing GDH1 or pretreatment with R162 inhibited the induction of fibrosis indicators in tissue kidney proximal tubular cells (TKPTS) treated with Transforming growth factor Beta 1 (TGF-β1), whereas activating GDH1 worsened TGF-β1's induction impact. Using RNA-sequence, luciferase reporter assays and Biacore analysis, we demonstrated that GDH1 interacts with Peroxisome proliferator-activated receptor gamma (PPARγ) and blocks its transcriptional activity, independent of the protein's expression. Additionally, R162 treatment boosted PPARγ transcriptional activity, and blocking of this signaling pathway reversed R162's protective effect. Finally, we discovered that R162 treatment or silencing GDH1 greatly lowered reactive oxygen species (ROS) and lipid accumulation. These findings concluded that suppressing GDH1 or R162 treatment could prevent renal fibrosis by augmenting PPARγ transcriptional activity to control lipid accumulation and redox balance.

Rupestonic Acid Derivative YZH-106 Promotes Lysosomal Degradation of HBV L- and M-HBsAg via Direct Interaction with PreS2 Domain.

Hepatitis B surface antigen (HBsAg) is not only the biomarker of hepatitis B virus (HBV) infection and expression activity in hepatocytes, but it also contributes to viral specific T cell exhaustion and HBV persistent infection. Therefore, anti-HBV therapies targeting HBsAg to achieve HBsAg loss are key approaches for an HBV functional cure. In this study, we found that YZH-106, a rupestonic acid derivative, inhibited HBsAg secretion and viral replication. Further investigation demonstrated that YZH-106 promoted the lysosomal degradation of viral L- and M-HBs proteins. A mechanistic study using Biacore and docking analysis revealed that YZH-106 bound directly to the PreS2 domain of L- and M-HBsAg, thereby blocking their entry into the endoplasmic reticulum (ER) and promoting their degradation in cytoplasm. Our work thereby provides the basis for the design of a novel compound therapy to target HBsAg against HBV infection.

N-terminal truncated phospholipase A1 accessory protein PlaS from Serratia marcescens alleviates inhibitory on host cell growth and enhances PlaA1 enzymatic activity

Phospholipase A1 (PLA1) is a kind of specific phospholipid hydrolase widely used in food, medical, textile. However, limitations in its expression and enzymatic activity have prompted the investigation of the phospholipase-assisting protein PlaS. In this study, we elucidate the role of PlaS in enhancing the expression and activity of PlaA1 through N-terminal truncation. Our research demonstrates that truncating the N-terminal region of PlaS effectively overcomes its inhibitory effect on host cells, resulting in improved cell growth and increased protein solubility of the protein. The yeast two-hybrid assay confirms the interaction between PlaA1 and N-terminal truncated PlaS (∆N27 PlaS), highlighting their binding capabilities. Furthermore, in vitro studies using Biacore analysis reveal a concentration-dependent and specific binding between PlaA1 and ∆N27 PlaS, exhibiting high affinity. Molecular docking analysis provides insights into the hydrogen bond interactions between ∆N27 PlaS and PlaA1, identifying key amino acid residues crucial for their binding. Finally, the enzyme activity of PLA1 was boost to 8.4 U/mL by orthogonal test. Study significantly contributes to the understanding of the interaction mechanism between PlaS and PlaA1, offering potential strategies for enhancing PlaA1 activity through protein engineering approaches.

Demethylzeylasteral exerts potent efficacy against non-small-cell lung cancer via the P53 signaling pathway

Lung cancer has one of the highest mortality rates worldwide, with non-small-cell lung cancer (NSCLC) constituting approximately 85% of all cases. Demethylzeylasteral (DEM), extracted from Tripterygium wilfordii Hook F, exhibits notable anti-tumor properties. In this study, we revealed that DEM could effectively induce NSCLC cell apoptosis. Specifically, DEM can dose-dependently suppress the viability and migration of human NSCLC cells. RNA-seq analysis revealed that DEM regulates the P53-signaling pathway, which was further validated by assessing crucial proteins involved in this pathway. Biacore analysis indicated that DEM has high affinity with the P53 protein. The CDX model demonstrated DEM's anti-tumor actions. This work provided evidence that DEM-P53 interaction stabilizes P53 protein and triggers downstream anti-tumor activities. These findings indicate that DEM treatment holds promise as a potential therapeutic approach for NSCLC, which warrants further clinical assessment in patients with NSCLC.

Abstract 2606: Targeting mesothelin on tumor cells with a novel, completely human fusion construct delivering the serine protease granzyme B

Abstract Mesothelin is a cell-surface antigen identified as an excellent cancer-associated target upregulated on numerous tumors, including pancreatic and ovarian cancers, as well as mesotheliomas and lung cancers. Granzyme B is a highly cytotoxic serine protease nominally delivered by immune effector cells to target cells, resulting in induction of cell death pathways that are both caspase-dependent and -independent. We developed a fusion protein (GrB-Fc-SD1) composed of GrB and containing SD1, a novel human single-domain antibody that uniquely targets Region III of the mesothelin glycoprotein. The SD1 and GrB domains are tethered by a human IgG heavy chain fragment which enables dimerization. The fusion protein was transiently expressed in HEK-293E cells in serum-free conditions with a final yield of 5 mg/L. Western blot analysis confirmed the expression of this construct as a homodimer. The GrB enzymatic activity in GrB-Fc-SD1 was similar to that of commercially available human granzyme B. ELISA studies confirmed the specific recognition of the SD1 antibody to mesothelin extra-cellular domain. Biacore analysis confirmed binding of GrB-Fc-SD1 with a dissociation constant between 10−7 and 10−8 M. In vitro cytotoxicity against a limited panel of lung, pancreatic and ovarian cancer cell lines expressing surface mesothelin indicate cytotoxicity in the nanomolar range, while cytotoxicity against mesothelin-negative cells was at micromolar levels. Ex vivo serum stability studies showed excellent stability of GrB-Fc-SD1 at both 4°C and -20°C over ninety days and a half-life of approximately three days at elevated temperatures (37°C). In vivo efficacy studies against Capan-2 xenografts in nude mice using both weekly x 5 and QOD x 5 doses of GrB-Fc-SD1 (130 mg/mg total dose, delivered IP) indicated highly significant efficacy in both dose regimens, with p < 0.001 compared to vehicle-treated mice. Mouse body weights were not impacted at the efficacy doses used in this study, indicating that this dose was non-toxic. Histopathology studies revealed a significant decrease in the percentage of Ki-67+ nuclei in treated tumors compared to controls. These data warrant continued investigation of the protein as a therapeutic candidate. Research supported by H2Biologics. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Mihai G. Iurascu, Louis R. DePalatis, Claire Thuning-Roberson, Michael G. Rosenblum. Targeting mesothelin on tumor cells with a novel, completely human fusion construct delivering the serine protease granzyme B [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 2606.

Structural insight into subunit F of respiratory chain complex I from Xanthomonas oryzae pv. oryzae inhibition by parthenolide.

Bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most serious diseases of rice, and there is a lack of bactericides for controlling this disease. We previously found parthenolide (PTL) is a potential lead for developing bactericides against Xoo, and subunit F of respiratory chain complex I (NuoF) is an important target protein of PTL. However, the binding modes of PTL with NuoF need further elucidation. In this study, we obtained the crystal structure of Xoo NuoEF (complex of subunit E and F of respiratory chain complex I) with a resolution of 2.36 Å, which is the first report on the protein structure of NuoEF in plant-pathogenic bacteria. The possible binding sites of PTL with NuoF (Cys105 and Cys187) were predicted with molecular docking and mutated into alanine using a base mismatch method. The mutated proteins were expressed in Escherichia coli and purified with affinity chromatography. The binding abilities of PTL with mutated proteins were investigated via pull-down assay and BIAcore analysis, which revealed that double mutation of Cys105 and Cys187 in NuoF severely affected the binding ability of PTL with NuoF. In addition, the binding modes were further simulated with combined quantum mechanical/molecular mechanical calculations, and the results indicated that PTL may have a stronger binding with Cys105 than Cys187. NuoEF protein structure of Xoo was resolved, and Cys105 and Cys187 in NuoF are important binding sites of PTL. This study further clarified the action mechanism of PTL against Xoo, and will promote the innovation of bactericides targeting Xoo complex I. © 2024 Society of Chemical Industry.

Licochalcone A inhibits the assembly function of β-barrel assembly machinery in Escherichia coli

Antimicrobial resistance (AMR) crisis urges the development of new antibiotics. In the present work, we for the first time used bio-affinity ultrafiltration combined with HPLC-MS (UF-HPLC-MS) to examine the interaction between the outer membrane β-barrel proteins and natural products. Our results showed that natural product licochalcone A from licorice interacts with BamA and BamD with the enrichment factor of 6.38 ± 1.46 and 4.80 ± 1.23, respectively. The interaction was further confirmed by use of biacore analysis, which demonstrated that the Kd value between BamA/D and licochalcone was 6.63/28.27 μM, suggesting a good affinity. To examine the effect of licochalcone A on BamA/D function, the developed versatile in vitro reconstitution assay was used and the results showed that 128 μg/mL licochalcone A could reduce the outer membrane protein A integration efficiency to 20%. Although licochalcone A alone can not inhibit the growth of E. coli, but it can affect the membrane permeability, suggesting that licochalcone A holds the potential to be used as a sensitizer to combat AMR.

Abstract 6316: In vitro and in vivo studies of human granzyme B fusion constructs targeting mesothelin

Abstract Mesothelin is highly expressed on tumor cells and promotes tumor growth and drug resistance. Pancreatic, ovarian, mesothelioma, and lung cancers highly express mesothelin and are cancers of unmet needs with ≤16% relative 5-year survival using standard of care. When delivered to tumor cells by immune effector cells, granzyme B (GrB) is a highly cytotoxic serine protease which induces apoptosis through both caspase-dependent and -independent mechanisms. We developed a fusion protein (GrB-Fc-SD1) composed of human GrB and containing SD1, a novel human single-domain antibody that uniquely targets Region III of the glycoprotein, mesothelin. The SD1 and GrB domains are connected by a human IgG heavy chain fragment which enables dimerization. Thus, the increase in total construct molecular weight leads to an increased in vivo circulation time and anti-tumor efficacy. GrB-Fc-SD1 was transiently expressed in HEK-293E cells under serum-free conditions and purified to homogeneity to a final yield of approximately 5 mg/L. Anti-GrB western blot analysis confirmed the expression of GrB-Fc-SD1 exclusively as a dimer. Comparison of GrB-Fc-SD1 with its expected molecular weight suggests significant glycosylation when produced in HEK-293E cells. The enzymatic activity of granzyme B in GrB-Fc-SD1 was comparable to that of commercially available human granzyme B. In vitro cytotoxicity against a limited panel of lung, pancreatic and ovarian cancer cell lines expressing surface mesothelin indicate cytotoxicity in the nanomolar range, while cytotoxicity against mesothelin-negative cells was at micromolar levels. GrB-Fc-SD1 bound specifically to mesothelin extra-cellular domain compared to a non-specific control, as determined by ELISA. Biacore analysis confirmed binding with a dissociation constant between 10−7 to 10−8 M. These data warrant continued investigation of the protein as a therapeutic candidate. Accordingly, ex vivo serum stability studies are underway. In addition, screening of an expanded panel of tumor cell lines for in vitro cytotoxicity is currently in progress and will be reported. In vivo efficacy studies against nude mice bearing Capan-2 xenograft models are currently underway and will also be reported. Research supported by H2Biologics. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Louis R. DePalatis, Claire Thuning-Roberson, Michael G. Rosenblum. In vitro and in vivo studies of human granzyme B fusion constructs targeting mesothelin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6316.

Application of Biacore Analysis to Measure the Affinity of Neurotrophin-3 with Glycosylated and Deglycosylated p75<sup>NTR</sup>

Application of Biacore Analysis to Measure the Affinity of Neurotrophin-3 with Glycosylated and Deglycosylated p75<sup>NTR</sup>

Conformational alteration in glycan induces phospholipase Cβ1 activation and angiogenesis

BackgroundIn endothelial cells, phospholipase C (PLC) β1-activated Ca2+ is a crucial second messenger for the signaling pathways governing angiogenesis. PLCβ1 is inactivated by complexing with an intracellular protein called translin-associated factor X (TRAX). This study demonstrates specific interactions between Globo H ceramide (GHCer) and TRAX, which highlight a new angiogenic control through PLCβ1 activation.MethodsGlobo-series glycosphingolipids (GSLs), including GHCer and stage-specific embryonic antigen-3 ceramide (SSEA3Cer), were analyzed using enzyme-linked immunosorbent assay (ELISA) and Biacore for their binding with TRAX. Angiogenic activities of GSLs in human umbilical vein endothelial cells (HUVECs) were evaluated. Molecular dynamics (MD) simulation was used to study conformations of GSLs and their molecular interactions with TRAX. Fluorescence resonance energy transfer (FRET) analysis of HUVECs by confocal microscopy was used to validate the release of PLCβ1 from TRAX. Furthermore, the in vivo angiogenic activity of extracellular vesicles (EVs) containing GHCer was confirmed using subcutaneous Matrigel plug assay in mice.ResultsThe results of ELISA and Biacore analysis showed a stable complex between recombinant TRAX and synthetic GHCer with Kd of 40.9 nM. In contrast, SSEA3Cer lacking a fucose residue of GHCer at the terminal showed ~ 1000-fold decrease in the binding affinity. These results were consistent with their angiogenic activities in HUVECs. The MD simulation indicated that TRAX interacted with the glycan moiety of GHCer at amino acid Q223, Q219, L142, S141, and E216. At equilibrium the stable complex maintained 4.6 ± 1.3 H-bonds. TRAX containing double mutations with Q223A and Q219A lost its ability to interact with GHCer in both MD simulation and Biacore assays. Removal of the terminal fucose from GHCer to become SSEA3Cer resulted in decreased H-bonding to 1.2 ± 1.0 by the MD simulation. Such specific H-bonding was due to the conformational alteration in the whole glycan which was affected by the presence or absence of the fucose moiety. In addition, ELISA, Biacore, and in-cell FRET assays confirmed the competition between GHCer and PLCβ1 for binding to TRAX. Furthermore, the Matrigel plug assay showed robust vessel formation in the plug containing tumor-secreted EVs or synthetic GHCer, but not in the plug with SSEA3Cer. The FRET analysis also indicated the disruption of colocalization of TRAX and PLCβ1 in cells by GHCer derived from EVs.ConclusionsOverall, the fucose residue in GHCer dictated the glycan conformation for its complexing with TRAX to release TRAX-sequestered PLCβ1, leading to Ca2+ mobilization in endothelial cells and enhancing angiogenesis in tumor microenvironments.

Ginsenoside Rd ameliorates muscle wasting by suppressing the signal transducer and activator of transcription 3 pathway.

The effects of some drugs, aging, cancers, and other diseases can cause muscle wasting. Currently, there are no effective drugs for treating muscle wasting. In this study, the effects of ginsenoside Rd (GRd) on muscle wasting were studied. Tumour necrosis factor-alpha (TNF-α)/interferon-gamma (IFN-γ)-induced myotube atrophy in mouse C2C12 and human skeletal myoblasts (HSkM) was evaluated based on cell thickness. Atrophy-related signalling, reactive oxygen species (ROS) level, mitochondrial membrane potential, and mitochondrial number were assessed. GRd (10mg/kg body weight) was orally administered to aged mice (23-24months old) and tumour-bearing (Lewis lung carcinoma [LLC1] or CT26) mice for 5weeks and 16days, respectively. Body weight, grip strength, inverted hanging time, and muscle weight were assessed. Histological analysis was also performed to assess the effects of GRd. The evolutionary chemical binding similarity (ECBS) approach, molecular docking, Biacore assay, and signal transducer and activator of transcription (STAT) 3 reporter assay were used to identify targets of GRd. GRd significantly induced hypertrophy in the C2C12 and HSkM myotubes (average diameter 50.8±2.6% and 49.9%±3.7% higher at 100nM, vs. control, P≤0.001). GRd treatment ameliorated aging- and cancer-induced (LLC1 or CT26) muscle atrophy in mice, which was evidenced by significant increases in grip strength, hanging time, muscle mass, and muscle tissue cross-sectional area (1.3-fold to 4.6-fold, vs. vehicle, P≤0.05; P≤0.01; P≤0.001). STAT3 was found to be a possible target of GRd by the ECBS approach and molecular docking assay. Validation of direct interaction between GRd and STAT3 was confirmed through Biacore analysis. GRd also inhibited STAT3 phosphorylation and STAT3 reporter activity, which led to the inhibition of STAT3 nuclear translocation and the suppression of downstream targets of STAT3, such as atrogin-1, muscle-specific RING finger protein (MuRF-1), and myostatin (MSTN) (29.0±11.2% to 84.3±30.5%, vs. vehicle, P≤0.05; P≤0.01; P≤0.001). Additionally, GRd scavenged ROS (91.7±1.4% reduction at 1nM, vs. vehicle, P≤0.001), inhibited TNF-α-induced dysregulation of ROS level, and improved mitochondrial integrity (P≤0.05; P≤0.01; P≤0.001). GRd ameliorates aging- and cancer-induced muscle wasting. Our findings suggest that GRd may be a novel therapeutic agent or adjuvant for reversing muscle wasting.

Strategic Decoy Peptides Interfere with MSI1/AGO2 Interaction to Elicit Tumor Suppression Effects.

Simple SummaryPeptide drugs that can specifically target undesirable protein–protein interactions that lead to oncogenic developments have emerged as the next era of future medicine for cancers. To combat GBM tumor progression, our study offers an alternative therapeutic strategy via targeting the protein–protein interaction between MSI1 and AGO2 with synthetic peptides identified from the C-terminus of MSI1 in peptide arrays. Our present data revealed for the first time that peptidic disruption to the MSI1/AGO2 complex known for promoting cancer stemness and progression could lead to encouraging therapeutic efficacy at both in vitro and in vivo levels. The significantly suppressed tumor growth and prolonged survival rates in PDX tumor models by decoy peptides evidently provided a new rationale for stratifying patients with MSI1/AGO2-targeted therapeutics.Peptide drugs that target protein–protein interactions have attracted mounting research efforts towards clinical developments over the past decades. Increasing reports have indicated that expression of Musashi 1 (MSI1) is tightly correlated to high grade of cancers as well as enrichment of cancer stem cells. Treatment failure in malignant tumors glioblastoma multiform (GBM) had also been correlated to CSC-regulating properties of MSI1. It is thus imperative to develop new therapeutics that could effectively improve current regimens used in clinics. MSI1 and AGO2 are two emerging oncogenic molecules that both contribute to GBM tumorigenesis through mRNA regulation of targets involved in apoptosis and cell cycle. In this study, we designed peptide arrays covering the C-terminus of MSI1 and identified two peptides (Pep#11 and Pep#26) that could specifically interfere with the binding with AGO2. Our Biacore analyses ascertained binding between the identified peptides and AGO2. Recombinant reporter system Gaussian luciferase and fluorescent bioconjugate techniques were employed to determine biological functions and pharmacokinetic characteristics of these two peptides. Our data suggested that Pep#11 and Pep#26 could function as decoy peptides by mimicking the interaction function of MSI1 with its binding partner AGO2 in vitro and in vivo. Further experiments using GMB animal models corroborated the ability of Pep#11 and Pep#26 in disrupting MSI1/AGO2 interaction and consequently anti-tumorigenicity and prolonged survival rates. These striking therapeutic efficacies orchestrated by the synthetic peptides were attributed to the decoy function to C-terminal MSI1, especially in malignant brain tumors and glioblastoma.

Synergistic Therapy of Doxorubicin with Cationic Anticancer Peptide L-K6 Reverses Multidrug Resistance in MCF-7/ADR Cancer Cells In Vitro via P-glycoprotein Inhibition

Multidrug resistance (MDR) is one of the major obstacles to efficient chemotherapy against cancers, resulting from the overexpression of drug efflux transporters such as P-glycoprotein (P-gP). In the present study, we aimed to evaluate the MDR reversal activity and synergistic therapeutic potential of cationic anticancer peptide L-K6 with doxorubicin (DOX) on P-gP-overexpressing and DOX-resistant MCF-7/ADR human breast cancer cells. Flow cytometry and confocal laser scanning microscopy were used to determine the intracellular accumulation of DOX and another P-gP substrate, Rho123. P-gP-Glo assay, Western blot and Biacore analysis were further performed to evaluate the P-gP function and expression. The cytotoxicity in MCF-7 or MCF-7/ADR cells was measured by MTT assay. Flow cytometry assay and confocal laser scanning microscopy observation clearly revealed an increased intracellular accumulation of DOX and Rho123 in MCF-7/ADR cells treated with L-K6, suggesting a P-gP inhibiting potential. Biacore analysis, P-gP-Glo assay and Western blot further confirmed that L-K6 could directly interact with P-gP, inhibit P-gP function and decrease P-gP expression in MCF-7/ADR cells. In addition, as expected, the data from MTT assay indicated that L-K6 restored the sensitivity of MCF-7/ADR cells to DOX, indicating a MDR reversal potential and a promising synergistic anticancer activity. All these findings may provide experimental evidence to support the promising applications and synergistic therapeutic potential of peptidic P-gP inhibitors against MDR cancer.

Luteolin stimulates the NGF-induced neurite outgrowth in cultured PC12 cells through binding with NGF and potentiating its receptor signaling.

Luteolin, a flavonoid in fruits and vegetables, has neurotrophic functions without a well-characterized mechanism. Here, we hypothesize a direct interaction of luteolin with nerve growth factor (NGF); as such, the functionality of the NGF could be potentiated. The direct binding of luteolin with NGF was validated by ultra-filtration, Biacore, and docking analyses. In cultured PC12 cells, application of luteolin in combination with a low dose of NGF potentiated the NGF-induced differentiation of neurons by an increase of the differentiated cell number to 25.4 ± 4.8% (p < 0.01), as well as the increased expression of neurofilaments by 119 ± 32.1% (p < 0.05), 191 ± 12.6% (p < 0.01), and 110 ± 23.4% (p < 0.05) for NF68, NF160 and NF200, respectively. The co-treatment induced the phosphorylations of tropomyosin receptor kinase A (TrkA), extracellular signal-regulated kinase 1/2 (ERK1/2), protein kinase B (Akt), phospholipase C-γ1 (PLCγ1), and cAMP response element-binding protein (CREB) by 2 to 3 fold: these induced phosphorylations were mimicking that of a high dose of NGF. Moreover, the application of the TrkA inhibitor, K252a, blocked the luteolin-mediated induction of neurofilament expression and neurite outgrowth in cultured PC12 cells, suggesting the target specificity. The result supports the development of luteolin as a therapeutic, or preventive, agent for NGF insufficiency-associated neurodegenerative diseases.

Abstract 197: Molecular interactions of Globo H ceramide with translin-associated factor X (TRAX) underlie its proangiogenic activities

Abstract Glycosphingolipids (GSLs) are ubiquitous glycolipids in eukaryotic plasma membranes that consist of a polar glycan chain and a hydrophobic sphingosine- containing ceramide tail. Although GSLs display various diversities and functions, the mechanistic details of GSL interactions with membranous or intracellular components are poorly understood. Globo H ceramide (GHCer), a tumor-associated GSL overexpressed in most types of epithelial cancers, is a potential target of cancer immunotherapy. Previously, we showed that GHCer is shed by cancer cells to mediate immune suppression and promote angiogenesis in the tumor microenvironment. At the cellular level, GHCer from cancer cells binds to translin-associated factor (TRAX), with consequent release of PLCB1 from TRAX to trigger Ca2+ mobilization. Herein, we found that Gb5-ceramide (Gb5Cer), the pentasaccharide precursor of GHCer, could not promote angiogenesis nor Ca2+ mobilization. At the molecular level, ELISA and biacore analysis revealed that GHCer binds to TRAX with a distinct dissociation constant of 4.09×10-8 M, while its precursors, galactosyl-, lactosyl-, Gb4-, and Gb5Cer- showed negligible binding to TRAX. Molecular docking and molecular dynamics indicated that the fucose residue of Globo H is an important contributor to a favorable glycan conformation for interaction with TRAX, consistent with the finding that Gb5Cer had no proangiogenic activities. In addition, GHCer competed with recombinant C-terminus region of phospholipase C β1 (PLCB1) for binding to TRAX, thus providing a molecular mechanism for the proangiogenic effect of GHCer on tumor microenvironment. This study provides the first evidence for interaction of TRAX with a non-protein GSL, deciphered distinct regions of TRAX for interacting with glycan and lipid portions of GHCer, and illustrated the crucial contribution of fucose moiety on GHCer for its interaction with TRAX and angiogenic activity. Citation Format: John Yu, Sheng-Hung Wang, Jing-Yan Cheng, Jung-Tung Hung, Chun-Cheng Lin, Fei-Yun Lo, Chien-Wei Lee, Alice L. Yu. Molecular interactions of Globo H ceramide with translin-associated factor X (TRAX) underlie its proangiogenic activities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 197.

MDNA109: Effect of an interleukin-2 superkine on CD8 T-cell properties in the tumor microenvironment.

e14220 Background: Proleukin (a cysteine-modified variant of interleukin (IL)-2) is the only common-γc cytokine approved for the treatment of metastatic melanoma and renal cell carcinoma. Its therapeutic use is however hampered by its short half-life and severe toxicity. At low doses, Proleukin administration leads to preferential activation of regulatory T cells (Tregs) due to their expression of the high affinity trimeric IL-2 receptor (IL-2R) consisting of CD25, CD122 and CD132 instead of cytotoxic CD8 T cells expressing the intermediate affinity IL-2R (CD122 and CD132). Methods: To bypass these limitations, we evaluated engineered variants of IL-2 (MDNA109 superkine) exhibiting enhanced affinity towards CD122 including their long-acting and bispecific superkine fusions. This approach allows preferential activation of CD8 T cells while displaying reduced adverse effects in vivo. Results: Both Biacore analysis and signaling studies on human peripheral blood mononuclear cells confirmed enhanced binding of MDNA109 to CD122 and STAT5 activation, respectively. When tested in vivo, MDNA109 co-administration with the immune-checkpoint blockers anti-programmed cell death (PD)-1 or anti-cytotoxic T-Lymphocyte-Associated Protein (CTLA)4 cured mice with pre-established MC38 or CT26 colon cancers respectively. In addition, bi-weekly administration of the long-acting MDNA109-Fc fusion led to similar therapeutic outcomes in the B16F10 model when compared to MDNA109 administered daily. Finally, in vitro and in-vivo results using (a) MDNA109 muteins with completely impaired CD25 binding activity and (b) novel bispecific superkine fusions consisting of MDNA109 and a dual IL-4/IL-13 super-antagonist capable of selective CD8 T-cell activation while mitigating the suppressive functions of myeloid-derived suppressor cells and tumor-associated macrophages will be presented. Conclusions: Altogether, these data demonstrate that both MDNA109 and MDNA109-Fc are therapeutically superior to Proleukin. Furthermore, use of MDNA109-based strategies will not only ensure complete abrogation of adverse effects, but will in addition eliminate immunosuppression caused by both Tregs and myeloid cells.

A novel interleukin-13 receptor alpha 2-targeted hybrid peptide for effective glioblastoma therapy.

We previously designed and reported a novel class of drugs, namely hybrid peptides, which are chemically synthesized and composed of a targeted binding peptide and a lytic-type peptide containing cationic amino acid residues that cause cancer cell death. In the present study, we screened for peptides that bind to interleukin-13 receptor alpha 2 (IL-13Rα2) by using a T7 random peptide phage display library system and isolated several positive phage clones. The A2b11 peptide, which was one of the positive clones, was shown to bind to IL-13Rα2 protein by Biacore analysis and a binding assay using glioblastoma (GB) cell lines. This peptide was linked with a lytic peptide containing a linker sequence to form the IL-13Rα2-lytic hybrid peptide. The IL-13Rα2-lytic hybrid peptide showed cytotoxic activity against GB cell lines in vitro. The IL-13Rα2-lytic hybrid peptide also affected Akt and Erk1/2 activation following treatment with interleukin-13 and induced rapid ATP dynamics in GB cells. Anti-tumor activity of the IL-13Rα2-lytic hybrid peptide was observed in vivo after intratumoral injection in a mouse xenograft model of human GB cells. These results suggest that the IL-13Rα2-lytic hybrid peptide might be a potent therapeutic option for patients with GB.

14-3-3E, a new alarmin candidate, elicits a catabolic and proinflammatory effect involving innate immunity through TLR signaling in osteoarthritis

Purpose: Osteoarthritis (OA) is now considered as a whole-joint disease characterized by cartilage degradation, synovial inflammation and bone remodeling. Recent studies have shown the implication of the innate immune system and low-grade inflammation in OA progression. Our team identified 14-3-3ε as a novel soluble mediator critical in the communication between subchondral bone and cartilage in OA. This protein acts as a potent MMP-3 and MMP-13-stimulatory factor in chondrocytes leading to catabolic phenotype. 14-3-3ε seems to share common characteristics with alarmins, endogenous molecules with intracellular functions that are released in the extracellular media after infection or tissue damage. Alarmins (such as S100 and HMGB1 involved in cartilage degradation) bind to receptors expressed on the surface of cells engaged in host defense and tissue repair. These receptors include the Toll-like receptors, TLR2 and TLR4. Moreover, innate alarmins can stimulate the activation of resident immune cells of the synovium, particularly macrophages leading to synovitis. As well as alarmins, 14-3-3ε could determine macrophage polarization. Our aim is to investigate the interactions between 14-3-3ε and its potential receptors, TLR2 and TLR4. Methods: Primary cultures of chondrocytes were performed from murine cartilage of the C57BL/6J mice (5 days old), and from human cartilage samples, obtained from OA patients undergoing total joint replacement surgery. The receptors TLR2 and TLR4 were blocked by monoclonal antibodies and pharmacologic inhibitors in chondrocytes (15mn) and then stimulated by recombinant 14-3-3ε during 24h. The stimulation by 14-3-3ε was also done on primary cultures of chondrocytes from TLR4 KO or TLR2 KO mice pups. Stimulation by 14-3-3ε has been performed with addition of Polymyxin B to avoid endotoxin effects. Immunocytochemistry was realized on murine chondrocytes with receptors antibodies. Chondrocyte gene expression and release of MMP-3, MMP-13 and IL-6 were evaluated by RT-PCR and ELISA. The direct interaction of 14-3-3ε with TLR2 was measured using Surface plasmonic resonance (SPR) on a Biacore 3000 instrument. Their molecular interaction was modeled in silico using GrammX and Rosetta programs to model the interaction and optimize the predicted conformations, respectively. The best complexes of 14-3-3ε with TLR2 were visually analyzed using PyMOL. M1 polarization of macrophages was performed by activating THP1 cells (ATCC) with PMA during 24h and and followed by 24h stimulation with LPS or 14-3-3ε. Different markers of the M1 type were analysed by ELISA and RT-qPCR to identify the polarisation of macrophages (M1 : TNF, IL-6, IL-1β, MCP1, Nos2). Results: TLR2 and TLR4 receptors were found on murine articular chondrocytes by immunocytochemistry using monoclonal antibodies. Invalidation of TLR2 and TLR4 receptors by blocking antibody in articular chondrocytes reduced significantly mRNA expression and protein release of MMP-3, MMP-13 and IL-6 induced by 14-3-3ε. For MMP3 mRNA expression, the inhibition rate reached 78% with blocking antibodies and 98% with OxPAPC, a TLR2/TLR4 inhibitor. The inhibition rate reached 65% (blocking antibodies), 98% (OxPAPC) for MMP13 and 74% (blocking antibodies) and 93% (OxPAPC) for IL-6. ELISA experiments showed an inhibition rate of 84% (blocking antibodies) and 97% (OxPAPC) for MMP3. Similar rates were observed for IL-6 secretion (83% and 97%). Moreover, the effect of 14-3-3ε on MMP3, MMP13 and IL6 expression was reduced in TLR4-KO and abolished in TLR2-KO chondrocytes. In the Biacore analysis, human recombinant TLR2 and TLR4 were covalently immobilized on CM5 chip. Results demonstrated a direct interaction between recombinant TLR2 and 14-3-3ε (83 RU) and between TLR4 and 14-3-3ε (41 RU). Computational structural analysis identified in silico a putative region of interaction, centered on a phosphorylation site in TLR2. This region of binding is consistent as 14-3-3ε is known to accommodate specifically phosphorylated residues. The affinity measurements between 14-3-3ε and peptides of TLR2 designed accordingly to this phospho-site are currently in progress. Finally, polarization of macrophages stimulated by recombinant 14-3-3ε seems to polarize them toward a M1 inflammatory phenotype. Conclusions: We have discovered 14-3-3ε as a new catabolic factor, produced by the subchondral bone and able to induce a catabolic phenotype on chondrocytes. These effects are mediated, at least in part, by TLR2 or TLR4 signalling, leading to the activation of innate immunity. Taken together, our results designate 14-3-3ε as a novel alarmin in OA, triggering catabolic and inflammatory effects through TLRs signaling. This new alarmin candidate could represent a new target either for therapeutic and/or prognostic purposes.

Abstract A076: DSP107—a novel SIRPα-4-1BBL dual signaling protein (DSP) for cancer immunotherapy

Abstract Background: The (re)activation of anticancer innate and adaptive immunity is at the forefront of developments in cancer therapy. Here, we report on a new immunotherapeutic fusion protein, termed Dual Signaling Protein 107 (DSP107). DSP107 was designed to combine activation of innate and adaptive immunity, by both blocking CD47/SIRPα interaction and activating 4-1BB. CD47 is overexpressed on cancer cells and upon binding to SIRPα on phagocytes transmits a “don’t eat me” signal, thereby suppressing innate immunity. 4-1BB is a costimulatory receptor that is transiently upregulated on tumor-infiltrating T-cells and is considered a surrogate marker for the tumor-reactive T-cell population. Activation of 4-1BB using its ligand or by agonistic antibodies reactivates anti-cancer T-cell immunity. In DSP107, the extracellular domains of SIRPα and 41BBL have been fused, yielding a dual function protein. DSP107 is produced as a homotrimer due to the trimerization property of 41BBL, an essential element for activating the 41BB receptor, a member of the TNF super-family of receptors. DSP107 was designed to bind to CD47 on tumor cells, thereby removing the inhibitory signal delivered to phagocytes. Simultaneously, CD47-mediated surface immobilization of DSP107 enables delivery of the 41BBL-4-1BB costimulatory signal to tumor localized T-cells. This dual immunomodulatory effect of DSP107 is designed to unleash both innate and adaptive immune responses targeted to the tumor site. Methods and Results: Trimeric DSP107 was successfully produced in a mammalian expression system. Both sides of DSP107 bound their cognate counterparts in kinetic Blitz binding assays and on human tumor and immune cell surfaces. The binding affinity of DSP107 was 1.6 nM for human CD47 and 0.69 nM for human 4-1BB as determined using BIAcore analysis. DSP107 blocked the interaction of SIRPα with CD47 in an ELISA-based competition assay (EC50 of 0.03 nM). DSP107 induced granulocyte- and macrophage-mediated phagocytosis of several lymphoma, leukemia and carcinoma cell lines in vitro. Further, DSP107 treatment triggered phagocytosis of primary AML cells by autologous macrophages. Co-treatment with DSP107 and therapeutic tumor-targeting antibodies, i.e., rituximab or cetuximab, resulted in enhanced phagocytosis of lymphoma or carcinoma cells, respectively. In a reporter assay measuring IL-8 secretion upon binding to/activation of 4-1BB, DSP107 activated 4-1BB signaling only in the presence of CD47-expressing cells. Further, DSP107 augmented the activation of purified T-cells activated by suboptimal concentrations of αCD3 + IL2 or αCD3/αCD28 Dynabeads in CD47 coated plates, as measured by percentage of CD25 expressing cells (up to 3-fold). When PBMCs were co-cultured with or without CD47-expressing cancer cells and stimulated with suboptimal concentrations of αCD3 + IL2, DSP107 treatment resulted in increased secretion of IFNg up to 2-fold), and increased T-cell proliferation (up to 2-fold). Conclusions: Here we demonstrate the feasibility and functional activity of DSP107, a novel therapeutic protein that combines activation of both the innate and adaptive immune system. Dual targeting, by the two functional sides of DSP107, offers multiple functionalities that act simultaneously and may result in a synergistic effect. DSP107 is now in IND-enabling studies and CMC development. The DSP platform can be designed for selective tumor site or microenvironment targeting and is adaptable to most checkpoint targets. Citation Format: Yosi M. Gozlan, Susan Hilgendorf, Alexandra Aronin, Yehudith Sagiv, Liat Ben-gigi-Tamir, Shira Amsili, Ami Tamir, Iris Pecker, Shirley Greenwald, Ayelet Chajut, Adam Foley-Comer, Yaron Pereg, Amnon Peled, Michal Dranitzki-Elhalel, Edwin Bremer. DSP107—a novel SIRPα-4-1BBL dual signaling protein (DSP) for cancer immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A076.