Recombinant Human Protein Research Articles

YKL-40 Promotes the Expression of Inflammatory Factors in Type Ⅱ Alveolar Epithelial Cell Model of A549 Cell Line

YKL-40, also known as chitinase-3-like-1 (CHI3L1), is a human cartilage glycoprotein-39, with its N-terminus consisting of tyrosine (Y), lysine (K), and leucine (L), hence the name YKL-40. In this study, we explored whether YKL-40 could promote the expression of inflammatory factors in type Ⅱ alveolar epithelial cells. A549 cells were cultured in vitro with interleukin (IL)-1β (20 ng/mL), IL-6 (20 ng/mL), tumor necrosis factor-alpha (TNF-α) (20 ng/mL), and interferon-gamma (IFN-γ) (20 ng/mL). The expression of YKL-40 transcription was determined by RT-qPCR. A549 cells were cultured with IL-1β at 5, 10, and 20 ng/mL and the expression of YKL-40 protein was determined by Western blot. A549 cells were cultured with recombinant YKL-40 protein at 0, 100, 500, and 1 000 ng/mL and the expression levels of IL-6 and IL-8 were measured by RT-qPCR. Three pairs of small interfering RNAs targeting YKL-40 (si- YKL-40-1/2/3) and the negative control (NC) were designed and used to transfect A549 cells, respectively, and the expression of YKL-40 was determined by RT-qPCR and Western blot. si- YKL-40-3 was screened out for subsequent experiments. In A549 cells, si- YKL-40-3 and si-NC were transfected and, then, IL-1β (20 ng/mL) was added in for culturing. The expression of YKL-40, IL-6, and IL-8 was determined by RT-qPCR and the expression of multiple factors in the supernatant was measured with the QAH-INF-1 kit. RT-qPCR results showed that IL-1β could up-regulate YKL-40 protein transcription level compared with that of the control group and the difference was statistically significant ( P<0.01), but IL-6, TNF-α, and IFN-γ could not up-regulate YKL-40 protein transcription level. Western blot results showed that IL-1β (20 ng/mL) could significantly promote the expression of YKL-40 and, compared with that of the control group, the differences showed by groups treated with different concentrations of IL-1β were all statistical significant ( P<0.01). After adding human recombinant YKL-40 protein to A549 cells, the results showed that the expression of inflammatory factors IL-6 and IL-8 was significantly increased and the difference was statistically significant compared with that of the control group ( P<0.05). After the expression of YKL-40 was decreased by si- YKL-40-3 transfection, the expression of IL-6 ( P<0.05), IL-8 ( P<0.05), and other inflammatory factors was inhibited compared with that of the control group. YKL-40 can promote the expression and secretion of IL-6, IL-8, and other acute inflammatory factors in A549 cell line, a type Ⅱ alveolar epithelial cell model, thus aggravating the inflammatory response. Targeted inhibition of YKL-40 expression may effectively inhibit inflammatory response.

Efficacy and safety of tripterygium wilfordii Hook F plus TNF inhibitor for active rheumatoid arthritis: A multicentre, randomized, double-blind, triple-dummy controlled trial

An investigator-initiated, multicentre, randomized, double-blind, triple-dummy, controlled trial was conducted at 14 tertiary rheumatology centers in China to evaluate the efficacy and safety of Tripterygium wilfordii Hook F (TwHF) with recombinant human TNF receptor IgGFc fusion protein (rhTNFR-Fc) in active Rheumatoid Arthritis (RA). Primary endpoint was the proportion of patients achieved a 50% improvement of American College of Rheumatology criteria (ACR50) in TwHF+rhTNFR-Fc vs. methotrexate (MTX) group at week 12. ACR50 was achieved in 57.1% (72/126), 41.3% (52/126), 23.0% (29/126), and 26.2% (33/126) patients receiving TwHF+rhTNFR-Fc, MTX + rhTNFR-Fc, TwHF and MTX monotherapy, respectively, at week 12 (TwHF+rhTNFR-Fc vs. other three groups, all p < 0.05). No statistical difference in serious adverse events or adverse events leading to discontinuation of study across all groups was documented. TwHF+rhTNFR-Fc was superior to MTX for active RA, and was more effective than MTX + rhTNFR-Fc on ACR50, with a similar safety profile.Trial registration:ClinicalTrials.govNCT03589833.

Biological activity of recombinant human PDX1 protein produced from Escherichia coli.

Pancreatic and duodenum homeobox 1 (PDX1) is considered as a pivotal transcription factor that acts as a "master regulator" in pancreatogenesis and maintenance of β-cells. Earlier study has reported that PDX1 also functions as a tumor suppressor in human gastric cancer cells by inhibiting cell growth. Here, we report the bioactivity of the purified human PDX1 fusion protein using various assays like cell migration, proliferation, cell cycle analysis, and gene expression. In cancer cells, recombinant PDX1 protein reduced cell migration and proliferation, and arrested cell growth by inducing apoptosis in gastric cancer cells. In pancreatic ductal cancer cells, the application of the PDX1 protein resulted in the induction of insulin gene expression. The results of these experiments demonstrate the biological activity imparted by recombinant human PDX1 fusion protein on gastric and pancreatic cancer cells and its usefulness as a biological tool to elucidate its function in various cellular processes.

Ruxolitinib altered IFN-β induced necroptosis of human dental pulp stem cells during osteoblast differentiation

ObjectiveThis study aimed to evaluate the role of ruxolitinib in the interferon beta (IFN-β) mediated osteoblast differentiation using human dental pulp stem cells (hDPSCs). DesignhDPSCs from five deciduous teeth of healthy patients were stimulated by adding human recombinant IFN-β protein (1 or 2 ng/ml) to the osteogenic differentiation induction medium. Substrate formation was determined using Alizarin Red staining, calcium concentration, and osteoblast marker expression levels. Ruxolitinib was used to inhibit the Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathway. Apoptosis was detected using terminal deoxynucleotidyl nick-end labeling (TUNEL) staining, and necroptosis was detected using propidium iodide staining and phosphorylated mixed lineage kinase domain-like protein (pMLKL) expression. ResultsIn the IFN-β-treated group, substrate formation was inhibited by a reduction in alkaline phosphatase (ALP) expression in a concentration-dependent manner. Although the proliferation potency was unchanged between the IFN-β-treated and control groups, the cell number was significantly reduced in the experimental group. TUNEL-positive cell number was not significantly different; however, the protein level of necroptosis markers, interleukin-6 (IL-6) and pMLKL were significantly increased in the substrate formation. Cell number and ALP expression level were improved in the group administered ruxolitinib, a JAK-STAT inhibitor. Additionally, ruxolitinib significantly suppressed IL-6 and pMLKL levels. ConclusionRuxolitinib interfered with the IFN-β-mediated necroptosis and osteogenic differentiation via the JAK-STAT pathway.

Optimisation of recombinant TNFα production in Escherichia coli using GFP fusions and flow cytometry.

Escherichia coli is commonly used industrially to manufacture recombinant proteins for biopharmaceutical applications, as well as in academic and industrial settings for R&D purposes. Optimisation of recombinant protein production remains problematic as many proteins are difficult to make, and process conditions must be optimised for each individual protein. An approach to accelerate process development is the use of a green fluorescent protein (GFP) fusions, which can be used to rapidly and simply measure the quantity and folding state of the protein of interest. In this study, we used GFP fusions to optimise production of recombinant human protein tumour necrosis factor (rhTNFα) using a T7 expression system. Flow cytometry was used to measure fluorescence and cell viability on a single cell level to determine culture heterogeneity. Fluorescence measurements were found to be comparable to data generated by subcellular fractionation and SDS-PAGE, a far more time-intensive technique. We compared production of rhTNFα-GFP with that of GFP alone to determine the impact of rhTNFα on expression levels. Optimised shakeflask conditions were then transferred to fed-batch high cell density bioreactor cultures. Finally, the expression of GFP from a paraBAD expression vector was compared to the T7 system. We highlight the utility of GFP fusions and flow cytometry for rapid process development.

Phylactic role of anti-lipoarabinomannan IgM directed against mannan core during mycobacterial infection in macrophages

Mycobacteria enter host phagocytes, such as macrophages by binding to several receptors on phagocytes. Several mycobacterial species, including Mycobacterium tuberculosis have evolved systems to evade host bactericidal pathways. Lipoarabinomannan (LAM) is an essential mycobacterial molecule for both binding to phagocytes and escaping from bactericidal pathways. Integrin CD11b plays critical roles as a phagocytic receptor and contributes to host defense by mediating both nonopsonic and opsonic phagocytosis. However, the mechanisms by which CD11b-mediated phagocytosis associates with LAM and drives the phagocytic process of mycobacteria remain to be fully elucidated. We recently identified TMDU3 as anti-LAM IgM antibody against the mannan core of LAM. The present study investigated the roles of CD11b and TMDU3 in macrophage phagocytosis of mycobacteria and subsequent bactericidal lysosomal fusion to phagosomes. CD11b knockout cells generated by a CRISPR/Cas9 system showed significant attenuation of the ability to phagocytose non-opsonized mycobacteria and LAM-conjugated beads. Moreover, recombinant human CD11b protein was found to bind to LAM. TMDU3 markedly inhibited macrophage phagocytosis of non-opsonized mycobacteria. This antibody slightly increased the phagocytosis of mycobacteria under opsonized conditions, whereas it significantly enhanced CD11b-mediated bactericidal functions. Taken together, these results show a novel phylactic role of anti-LAM IgM during mycobacterial infection in macrophages.

Expression of paraoxonase 1 in HEK293 cells and its effect on lactonase and arylesterase activity in samples containing human serum and/or prooxidant or antioxidant agents - a preliminary study

Expression of recombinant paraoxonase 1 (PON1) in the human embryonic kidney (HEK293F) cell line was evaluated. Two recombinant human PON1 proteins were investigated: PON1-Fc, which has an Fc-tag on its C-terminal, and His-PON1-Fc, which has an Fc-tag on its C-terminal and histidine on its N-terminal. The influence of high and low-density lipoproteins, copper ions, iron ions, and thiolactone homocysteine, on arylesterase and lactonase activities of PON1 in human serum was examined. Two activities of PON1, arylesterase and lactonase, were measured using earlier elaborated kinetic spectrophotometric method with temperature control at 37°C using the water thermostated cuvette holder. It was demonstrated that the HEK293F expression platform is useful for obtaining recombinant human PON1 protein. Also, addition of high-density lipoprotein increased serum arylesterase and lactonase activity of PON1 to a greater extent than the addition of PON1-Fc or HisPON1-Fc recombinant proteins. In contrast, the addition of high-density lipoprotein to samples containing PON1-Fc or His-PON1-Fc did not increase PON1 activities, while low-density lipoprotein, copper ions, and iron ions, depressed PON1 arylesterase and lactonase activity. Obtained results indicated that it may be advantageous to stimulate PON1 activities rather than supplement with endogenous PON1 protein and HDL, which could be an interesting direction for further study. Furthermore, the work has highlighted some potential research directions for the use of PON1 as a therapeutic molecules. Nevertheless, the study had some limitations and should therefore be treated as a preliminary study.

PTEN Overexpression Alters Autophagy Levels and Slows Sodium Arsenite-Induced Hepatic Stellate Cell Fibrosis.

Exposure to inorganic arsenic remains a global public health problem. The liver is the main target organ, leading to arsenic-induced liver fibrosis. Phosphatase and tensin homology deleted on chromosome ten (PTEN) may participate in arsenic-induced liver fibrosis by regulating autophagy, but the exact mechanisms remain unclear. We established a mouse model of arsenic poisoning through their drinking water and a fibrosis model using the human hepatic stellate cell line LX-2 through NaAsO2 exposure for 24 h. Masson staining measured liver fibrosis. The cells were transfected with a PTEN overexpression plasmid. Western blot and qRT-PCR determined the levels of protein/mRNA expression. Fibrosis was evident in both the mouse model and arsenic-exposed LX-2 cells. NaAsO2 upregulated expression of autophagic markers microtubule-associated protein light chain A/B (LC3), recombinant human autophagy effector protein (Beclin-1), and hairy and enhancer of split homolog-1 (HES1), but downregulated PTEN. Alongside this, α-smooth muscle actin (α-SMA) expression was significantly upregulated by NaAsO2. PTEN overexpression altered NaAsO2-induced autophagy and downregulated LC3 and Beclin-1. While Notch1, HES1, α-SMA, and collagen I expression were all downregulated in the NaAsO2 groups. Therefore, PTEN overexpression might decrease autophagy and inhibit fibrosis progression caused by arsenic, and the NOTCH1/HES1 pathway is likely involved.

Phagocytosis of Protein-Modified Polymer Microparticles by Immune Cells

The ability of three model green proteins to covalently bind to microparticles (MP) based on poly(D,L-lactic acid) (PLA). Green fluorescent protein (sfGFP), recombinant human beta2-microglobulin-sfGFP fusion protein (β2M-sfGFP), and recombinant human amylin-sfGFP fusion protein (IAPP-sfGFP) were isolated by affinity chromatography. The double emulsion method was used to form PLA-MPs. The modification of PLA MPs by proteins was testified using laser scanning microscopy (LSM). Phagocytosis of PLA-MPs modified with different proteins and free model proteins by macrophages was also studied using LSM. Recombinant sfGFP has been shown to bind to particle surfaces at lower levels compared to β2M-sfGFP and IAPP-sfGFP. Presumably, this is due to the fact that amino groups that could potentially react with activated carboxyl groups on particle surfaces, are spatially unavailable for this reaction due to the structure of sfGFP. β2M and IAPP within the corresponding recombinant proteins are spacer structures between the surface of spherical particles and sfGFP. It was also found that increasing the protein/particle ratio by a factor of three did not lead to an increase in the amount of bound protein per unit mass of particles, which may indicate that the amount of protein that can be bound per unit mass of particles is limited by the capacity of the particles themselves. The study of phagocytosis of PLA-MPs modified with model proteins revealed that MPs bearing β2M-sfGFP and IAPP-sfGFP were captured by macrophages and, therefore, contribute to the activation of the cellular immune response, which is important in the fight against various viral infections. In addition, model proteins (β2M-sfGFP, IAPP-sfGFP) appeared to be also capable of phagocytosis. This may be due to the fact that both β2M and IAPP are amyloidogenic and aggregation prone proteins. Apparently, the aggregates of these proteins are also able to be absorbed by macrophages due to the increase in size compared to their monomeric forms.

Parathyroid Hormone-Related Protein Promotes the Proliferation of Patient-Derived Glioblastoma Stem Cells via Activating cAMP/PKA Signaling Pathway.

Glioblastoma (GBM) is an aggressive primary brain tumor characterized by its heterogeneity and high recurrence and lethality rates. Glioblastoma stem cells (GSCs) play a crucial role in therapy resistance and tumor recurrence. Therefore, targeting GSCs is a key objective in developing effective treatments for GBM. The role of Parathyroid hormone-related peptide (PTHrP) in GBM and its impact on GSCs remains unclear. This study aimed to investigate the effect of PTHrP on GSCs and its potential as a therapeutic target for GBM. Using the Cancer Genome Atlas (TCGA) database, we found higher expression of PTHrP in GBM, which correlated inversely with survival. GSCs were established from three human GBM samples obtained after surgical resection. Exposure to recombinant human PTHrP protein (rPTHrP) at different concentrations significantly enhanced GSCs viability. Knockdown of PTHrP using target-specific siRNA (siPTHrP) inhibited tumorsphere formation and reduced the number of BrdU-positive cells. In an orthotopic xenograft mouse model, suppression of PTHrP expression led to significant inhibition of tumor growth. The addition of rPTHrP in the growth medium counteracted the antiproliferative effect of siPTHrP. Further investigation revealed that PTHrP increased cAMP concentration and activated the PKA signaling pathway. Treatment with forskolin, an adenylyl cyclase activator, nullified the antiproliferative effect of siPTHrP. Our findings demonstrate that PTHrP promotes the proliferation of patient-derived GSCs by activating the cAMP/PKA signaling pathway. These results uncover a novel role for PTHrP and suggest its potential as a therapeutic target for GBM treatment.

Lung Surfactant Protein B Peptide Mimics Interact with the Human ACE2 Receptor

Lung surfactant is a complex mixture of phospholipids and surfactant proteins that is produced in alveolar type 2 cells. It prevents lung collapse by reducing surface tension and is involved in innate immunity. Exogenous animal-derived and, more recently, synthetic lung surfactant has shown clinical efficacy in surfactant-deficient premature infants and in critically ill patients with acute respiratory distress syndrome (ARDS), such as those with severe COVID-19 disease. COVID-19 pneumonia is initiated by the binding of the viral receptor-binding domain (RBD) of SARS-CoV-2 to the cellular receptor angiotensin-converting enzyme 2 (ACE2). Inflammation and tissue damage then lead to loss and dysfunction of surface activity that can be relieved by treatment with an exogenous lung surfactant. Surfactant protein B (SP-B) is pivotal for surfactant activity and has anti-inflammatory effects. Here, we study the binding of two synthetic SP-B peptide mimics, Super Mini-B (SMB) and B-YL, to a recombinant human ACE2 receptor protein construct using molecular docking and surface plasmon resonance (SPR) to evaluate their potential as antiviral drugs. The SPR measurements confirmed that both the SMB and B-YL peptides bind to the rhACE2 receptor with affinities like that of the viral RBD–ACE2 complex. These findings suggest that synthetic lung surfactant peptide mimics can act as competitive inhibitors of the binding of viral RBD to the ACE2 receptor.

Magnetic Relaxation Switching Assay Using IFNα-2b-Conjugated Superparamagnetic Nanoparticles for Anti-Interferon Antibody Detection

Type I interferons, particularly IFNα-2b, play essential roles in eliciting adaptive and innate immune responses, being implicated in the pathogenesis of various diseases, including cancer, and autoimmune and infectious diseases. Therefore, the development of a highly sensitive platform for analysis of either IFNα-2b or anti-IFNα-2b antibodies is of high importance to improve the diagnosis of various pathologies associated with the IFNα-2b disbalance. For evaluation of the anti-IFNα-2b antibody level, we have synthesized superparamagnetic iron oxide nanoparticles (SPIONs) coupled with the recombinant human IFNα-2b protein (SPIONs@IFNα-2b). Employing a magnetic relaxation switching assay (MRSw)-based nanosensor, we detected picomolar concentrations (0.36 pg/mL) of anti-INFα-2b antibodies. The high sensitivity of the real-time antibodies’ detection was ensured by the specificity of immune responses and the maintenance of resonance conditions for water spins by choosing a high-frequency filling of short radio-frequency pulses of the generator. The formation of a complex of the SPIONs@IFNα-2b nanoparticles with the anti-INFα-2b antibodies led to a cascade process of the formation of nanoparticle clusters, which was further enhanced by exposure to a strong (7.1 T) homogenous magnetic field. Obtained magnetic conjugates exhibited high negative MR contrast-enhancing properties (as shown by NMR studies) that were also preserved when particles were administered in vivo. Thus, we observed a 1.2-fold decrease of the T2 relaxation time in the liver following administration of magnetic conjugates as compared to the control. In conclusion, the developed MRSw assay based on SPIONs@IFNα-2b nanoparticles represents an alternative immunological probe for the estimation of anti-IFNα-2b antibodies that could be further employed in clinical studies.

Enhanced soluble expression of active recombinant human interleukin-29 using champion pET SUMO system.

Current research focuses on the soluble and high-level expression of biologically active recombinant human IL-29 protein inEscherichia coli. The codon-optimized IL-29 gene was cloned into the Champion™ pET SUMO expression system downstream of the SUMO tag under the influence of the T7 lac promoter. The expression of SUMO-fused IL-29 protein was compared in E. coli Rosetta 2(DE3), Rosetta 2(DE3) pLysS, and Rosetta-gami 2(DE3). The release of the SUMO fusion partner resulted in approximately 98mg of native rhIL-29 protein with a purity of 99% from 1l of fermentation culture. Purified rhIL-29 was found to be biologically active, as evaluated by its anti-proliferation assay. It was found that Champion™ pET SUMO expression system can be used to obtained high yield of biologically active soluble recombinant human protein compared to other expression vector.

Klotho enhances bone regenerative function of hPDLSCs via modulating immunoregulatory function and cell autophagy

BackgroundHuman periodontal ligament stem cells (hPDLSCs) have a superior ability to promote the formation of new bones and achieve tissue regeneration. However, mesenchymal stem cells (MSCs) are placed in harsh environments after transplantation, and the hostile microenvironment reduces their stemness and hinders their therapeutic effects. Klotho is an antiaging protein that participates in the regulation of stress resistance. In our previous study, we demonstrated the protective ability of Klotho in hPDLSCs.MethodsA cranial bone defect model of rats was constructed, and the hPDLSCs with or without Klotho pretreatment were transplanted into the defects. Histochemical staining and micro-computed tomography were used to detect cell survival, osteogenesis, and immunoregulatory effects of hPDLSCs after transplantation. The in vitro capacity of hPDLSCs was measured by a macrophage polarization test and the inflammatory level of macrophages. Furthermore, we explored autophagy activity in hPDLSCs, which may be affected by Klotho to regulate cell homeostasis.ResultsPretreatment with the recombinant human Klotho protein improved cell survival after hPDLSC transplantation and enhanced their ability to promote bone regeneration. Furthermore, Klotho pretreatment can promote stem cell immunomodulatory effects in macrophages and modulate cell autophagy activity, in vivo and in vitro.ConclusionThese findings suggest that the Klotho protein protects hPDLSCs from stress after transplantation to maintain stem cell function via enhancing the immunomodulatory ability of hPDLSCs and inhibiting cell autophagy.

ADAMTS4 as an enzyme cleaving at APP669 site

AbstractBackgroundWe have previously identified APP669‐711 (a.k.a. Aβ(‐3)‐40) in human plasma using immunoprecipitation combined with matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (IP‐MALDI‐MS) (Kaneko et al., Proc Jpn Acad Ser B Phys Biol Sci. 2014). Our assay has revealed that the composite biomarker, which is a combination of APP669‐711/Aβ1‐42 ratio and Aβ1‐40/ Aβ1‐42 ratio in human plasma, correlates with amyloid PET status (Nakamura et al., Nature 2018). Our previous research using cultured cells has suggested that A Disintegrin and Metalloproteinase with a Thrombospondin type 1 motif, type 4 (ADAMTS4) is involved in APP669‐711 production. Here, we present further evidence on the involvement of ADAMTS4 in the APP669 site‐cleavage.MethodWe prepared a recombinant APP substrate, which is APP81 corresponding to APP619‐699 sequence with a V5/His tag at the C‐terminus and a FLAG tag at the N‐terminus (FLAG‐APP81‐V5/His) for in vitro cleavage assay. Recombinant human ADAMTS4 protein and the tagged APP81 were incubated either alone or together at 37°C. The incubated samples were desalted and then analyzed by MALDI‐TOF MS. In vivo evaluation of ADAMTS4, we analyzed plasma samples from Adamts4‐/‐ mice and WT mice using IP‐MALDI‐MS.ResultIn vitro cleavage assay, N‐ and C‐terminal fragments cleaved at APP669, Aβ4th, Aβ12th site were detected in the mixture of the recombinant APP81 and ADAMTS4 proteins but not in the sample of substrate or enzyme alone. Furthermore, a peptide derived from double digestion by APP669 and Aβ12th site‐cleavages (i.e., APP669‐Aβ11) was also identified by MS/MS analysis. The analysis of mouse plasma showed that murine APP669‐711 level was decreased in Adamts4‐/‐ mice compared to WT mice.ConclusionThese results indicated that ADAMTS4 is one of the enzymes that can cleave the APP669 site directly and contribute to the generation of APP669‐711 in vivo.

Novel peptide binder to Glypican-3 for targeted radiopharmaceutical therapy of hepatocellular carcinoma.

e16131 Background: Glypican-3 (GPC3) is a membrane-associated heparan sulfate proteoglycan primarily involved in embryonic development, and is barely detectable in normal adult tissues. Significant upregulation of GPC3 protein in hepatocellular carcinomas (HCC) has been observed in multiple immunohistochemistry (IHC) studies with up to 75% positivity rate, and is associated with poor prognosis. GPC3 is not expressed in healthy or non-malignant liver tissue. Targeting GPC3 could fulfill an unmet medical need for HCC, a leading cause of cancer-related deaths worldwide for which efficacious therapies are lacking. The differential expression of GPC3 between tumor and normal tissues provides an opportunity for GPC3-targeted radiopharmaceutical therapy (RPT) to treat HCC. Methods: RAYZ-8009 is comprised of a novel macrocyclic peptide binder to GPC3, a linker, and chelator DOTA that can be complexed with different radioisotopes. The affinity of peptide binders to GPC3 was determined by surface plasma resonance (SPR), as well as a radioligand binding assay in human HCC cell line HepG2. The cross-species binding was assessed by radioligand binding using recombinant mouse, cynomolgus monkey, and human GPC3 proteins. Target-mediated internalization in HepG2 cells was measured using Microbeta at various time points. In vivo biodistribution with 177Lu, and anti-tumor efficacy studies using 225Ac were performed in HepG2 tumor-bearing athymic nude mice. Results: RAYZ-8009 showed high binding affinity to human GPC3 with a KD of 0.7 nM as determined by SPR. Binding affinity was maintained across mouse, cynomolgus monkey and human GPC3. Potent cellular binding was confirmed in GPC3+ HepG2 cells, and was independent of choice of isotope. 177Lu-RAYZ-8009 showed fast and efficient internalization with 42% internalized by 20 minutes in HepG2 cells. In vivo biodistribution of 177Lu-RAYZ-8009 showed tumor uptake of 19.8, 16.6, 16.4, and 8.8 %ID/g at 2, 24, 48, and 96 hours, respectively. Renal uptake was 16.1, 4.7, 1.6, and 0.7 %ID/g at the same timepoints, with tumor/kidney ratios of 1.3, 3.7, 11.3, and 15.0, respectively. Minimal uptake was observed in other normal tissues. Tumor-specific uptake and retention were also observed when tumors were implanted orthotopically with no uptake in non-malignant liver tissue. Furthermore, significant tumor growth inhibition and survival benefit were achieved with 225Ac-RAYZ-8009 in GPC3+ HCC xenografts. Conclusions: Preclinical pharmacodynamic, pharmacokinetic, biodistribution and efficacy data demonstrate the potential of RAYZ-8009 as a RPT agent for the treatment of patients with GPC3-positive HCC.

Preclinical evaluation of novel HER3-targeting radioconjugates for the imaging and treatment of HER3-expressing cancers.

e15114 Background: HER3 overexpression in tumors is associated with poor survival and is implicated in drug resistance mechanisms of HER1 (EGFR) and HER2 targeted therapies. Although antibodies against HER3 have been developed and evaluated in clinical trials, no HER3 targeted therapy has been approved, underscoring the unmet need for novel HER3 therapeutic approaches. We hypothesized that HER3 radioligand targeted therapies could provide enhanced therapeutic efficacy against HER3-positive cancers through the energy deposited by alpha or beta emitting radionuclides in tumor cells. Here we describe the development and preclinical evaluation of novel anti-HER3 antibody radioconjugates (ARCs), 225Ac-HER3 and 177Lu-HER3, using the alpha-emitting Actinium-225 (225Ac) and beta-emitting Lutetium-177 (177Lu) radioisotopes, respectively. Methods: ARCs were prepared by radiolabeling HER3 antibody with 225Ac or 177Lu using S-2-(4-Isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA) to yield 225Ac or 177Lu-HER3-ARCs. DOTA modified antibodies were characterized by HPLC and MALDI-TOF and HER3-ARCs were fully characterized by instant thin-layer chromatography (iTLC) and HPLC. The in vitro binding properties of the HER3-ARCs were evaluated using HER3 recombinant proteins and cells expressing HER3 in ELISA and flow cytometry. The in vivo pharmacological properties of the HER3-ARCs were evaluated by molecular imaging using Zirconium-89 (89Zr) Positron Emission Tomography (PET), biodistribution and antitumor efficacy studies. Results: HER3 antibody was successfully modified with p-SCN-Bn-DOTA to yield a conjugate with approximately 8 DOTAs per antibody molecule. Results from the in vitro binding assays of ARCs to either HER3 human recombinant protein or cells expressing HER3 demonstrated similar binding properties of 225Ac-HER3 and 177Lu-HER3 to unmodified HER3 antibody. The biodistribution study in NCI-H1975 tumor bearing mice showed that both 225Ac-HER3 and 177Lu-HER3-ARCs specifically accumulate in HER3-expressing tumors relative to other tissues. In agreement with the biodistribution studies, PET images revealed specific uptake of 89Zr-HER3 in NCI-H1975 tumors. Furthermore, 225Ac and 177Lu-HER3-ARCs showed significantly more potent NCI-H1975 tumor growth inhibition compared to controls. Conclusions: Our findings suggest that a HER3 targeted radiotherapy approach can enhance therapeutic response and overcome the limitations of previously explored HER3 targeted drug modalities. Thus, further preclinical evaluation of 225Ac-HER3 and 177Lu-HER3 is warranted, including in settings of tumor drug resistance, which can provide the basis for investigating HER3-ARCs as an effective treatment approach in future clinical trials.

A novel magnetic bead-based immunoprecipitation method for anti-dense fine speckled 70 antibodies

The indirect immunofluorescence assay (IIFA) on HEp-2 cells has been widely used for screening anti-nuclear antibodies (ANA) that are associated with systemic autoimmune rheumatic diseases (SARD). Sera containing ANA display multiple distinct fluorescence patterns on HEp-2 cells. Among them, a dense fine speckled (DFS) pattern caused by anti-DFS70 antibodies has been reported to have higher prevalence in healthy individuals than in patients with SARD. This DFS pattern is often difficult to distinguish amongst other SARD-associated ANA patterns, in particular a mixed homogeneous and speckled pattern. Furthermore, a strong DFS pattern can mask other SARD-associated patterns. Hence, we developed a novel immunoprecipitation method using magnetic beads to remove anti-DFS70 antibodies in serum prior to running IIFA. We also aimed to confirm the presence of anti-DFS70 and to uncover any SARD-associated ANA patterns masked by a strong DFS pattern. The sera used in our study were from 70 individuals who had routine ANA screen, of which 35 sera had an isolated DFS pattern with monospecific anti-DFS70 antibodies confirmed by a complementary assay, and 35 were control sera without a DFS pattern. An immunoprecipitation method using magnetic beads coated with recombinant human full length DFS70 protein was developed. The performance of this new method was evaluated in comparison to an immunoadsorption method using the same DFS70 protein. Our newly developed immunoprecipitation method demonstrated excellent sensitivity (91.4%) and specificity (100%) in confirming the DFS pattern associated with anti-DFS70 in sera. Additionally, our method was able to remove anti-DFS70 and uncover SARD-associated ANA patterns masked by a strong DFS pattern. It also showed a clearer background on IIFA than that of the immunoadsorption method. The novel magnetic bead-based immunoprecipitation method demonstrated satisfactory performance in removing anti-DFS70 without interfering with the detection of other antibodies. It can be easily integrated with IIFA to confirm anti-DFS70 associated DFS pattern. Additionally, it can simultaneously unmask other ANA patterns, which cannot be achieved by a conventional protocol that requires a complementary anti-DFS70 specific assay to be performed. Therefore, the novel method provides a more effective and accurate solution for ANA screening.

The stability and function of human cochaperone Hsp40/DNAJA1 are affected by zinc removal and partially restored by copper

The imbalance in metal homeostasis can be associated with several human diseases, and exposure to increasing concentrations of metals promotes cell stress and toxicity. Therefore, understanding the cytotoxic effect of metal imbalance is important to unravel the biochemical mechanism of homeostasis and the action of potential protective proteins against metal toxicity. Several studies, including gene deletion in yeast, provide evidence indicating the possible indirect involvement of cochaperones from the Hsp40/DNAJA family in metal homeostasis, possibly through modulating the activity of Hsp 70.This work first investigated the effect of zinc and copper on the conformation and function of the human Hsp40 cochaperone DNAJA1, a zinc-binding protein. DNAJA1 was capable to complement the phenotype of a yeast strain deleted of the ydj1 gene, which was more sensitive to the presence of zinc and copper than the wild-type strain. To gain further insight about the role of the DNAJA family in metal binding, the recombinant human DNAJA1 protein was studied. Zinc removal from DNAJA1 affected both its stability and ability to act as a chaperone, i.e., to protect other proteins from aggregation. The reintroduction of zinc restored the native properties of DNAJA1 and, surprisingly, the addition of copper partially restored the native properties.

Enhancement of cancer-type specific cytotoxicity of natural killer cells via pre-conditioning with cancer cell culture medium

Recently, we have established a method for culturing NK cell-enriched lymphocytes (NKELs) with high anti-cancer activity. These NKELs are found to be highly cytotoxic against various types of cancer cells, and even the extracellular vesicles derived from those NKELs were also demonstrated high anti-cancer activity. In the present study, to find a way to enhance cancer type specificity of these NKELs, the impact of tumor cell-priming of these NKELs on their cytotoxic effect against particular type of cancer cells was investigated. According to our data, when these NKELs were primed with conditioned media (CM) from three different lines of liver cancer cell lines, their cytotoxicity against liver cancer cell lines was significantly increased compared to that of the NKELs without priming. Based on cytokine arrays of the tumor CM and subsequent network analysis, 6 proteins were selected as a potential candidate factors that facilitated the liver cancer cell-specific priming of those NKELs. When these 6 candidate proteins (human recombinant proteins) were separately applied to prime NKELs, 2 out of 6 proteins (DKK1 and THBS1) showed enhanced liver cancer-specific cytotoxicity. With further optimization and elucidation of the underlying mechanisms, it may be possible to prime NKELs for learned cancer type specificity.