Human Immunoglobin Research Articles

Production of therapeutic levels of human FIX-R338L by engineered B cells using GMP-compatible medium

B cells can differentiate into plasmablast and plasma cells, capable of producing antibodies for decades. Gene editing using zinc-finger nucleases (ZFN) enables the engineering of B cells capable of secreting sustained and high levels of therapeutic proteins. In this study, we established an advanced invitro good manufacturing practice-compatible culturing system characterized by robust and consistent expansion rate, high viability, and efficient B cell differentiation. Using this process, an optimized B cell editing protocol was developed by combining ZFN/adeno-associated virus 6 technology to achieve site-specific insertion of the human factor IX R338L Padua into the silent TRAC locus. Invitro analysis revealed high levels of secreted human immunoglobulins and human factor IX-Padua. Following intravenous infusion in a mouse model, human plasma cells were detected in spleen and bone marrow, indicating successful and potentially long-term engraftment invivo. Moreover, high levels of human immunoglobin and therapeutic levels of human factor IX-Padua were detected in mouse plasma, correlating with 15% of normal human factor IX activity. These data suggest that the proposed process promotes the production of functional and differentiated engineered B cells. In conclusion, this study represents an important step toward the development of a manufacturing platform for potential B cell-derived therapeutic products.

A Review of the Immunologic Pathways Involved in Bullous Pemphigoid and Novel Therapeutic Targets.

Bullous pemphigoid (BP) is a rare, chronic antibody-mediated autoimmune blistering disease primarily affecting the elderly, with an age of onset over 60. Current treatment options are limited and involve the use of corticosteroids and immunosuppressants, but their long-term use is associated with significant morbidity and mortality. In Japan, human intravenous immunoglobin is approved for the treatment of corticosteroid-refractory BP. However, no treatment option is approved by the Food and Drug Administration for the management of BP. Therefore, developing effective therapies free of debilitating side effects is imperative. In this review, we summarize the main immunologic pathways involved in the pathogenesis of BP, with an emphasis on the role of eosinophils, immunoglobulins, cytokines such as the interleukin (IL)-4 and IL-5, and complements. We further discuss the latest advances with novel therapeutic targets tested for the management of BP. Ongoing efforts are needed to run well-designed controlled trials and test the efficacy and safety of investigational drugs while providing much-needed access to these medications for refractory patients who will not otherwise be able to afford them as off-label prescriptions.

A novel and unique ATP hydrolysis to AMP by a human Hsp70 Binding immunoglobin protein (BiP).

Hsp70s are ubiquitous and highly conserved molecular chaperones. They play crucial roles in maintaining cellular protein homeostasis. It is well established that Hsp70s use the energy of ATP hydrolysis to ADP to power the chaperone activity regardless of the cellular locations and isoforms. Binding immunoglobin protein (BiP), the major member of Hsp70s in the endoplasmic reticulum, is essential for protein folding and quality control. Unexpectedly, our structural analysis of BiP demonstrated a novel ATP hydrolysis to AMP during crystallization under the acidic conditions. Our biochemical studies confirmed this newly discovered ATP to AMP hydrolysis in solutions. Unlike the canonical ATP to ADP hydrolysis observed for Hsp70s, this ATP hydrolysis to AMP depends on the substrate-binding domain of BiP and is inhibited by the binding of a peptide substrate. Intriguingly, this ATP to AMP hydrolysis is unique to BiP, not shared by two representative Hsp70 proteins from the cytosol. Taken together, this novel and unique ATP to AMP hydrolysis may provide a potentially new direction for understanding the activity and cellular function of BiP.

Dose escalation and expansion (phase Ia/Ib) study of GLS-010, a recombinant fully human antiprogrammed death-1 monoclonal antibody for advanced solid tumors or lymphoma

BackgroundGLS-010, a novel engineered fully human immunoglobin G4 monoclonal antibody, can specially block the PD-1/PD-L1/2 axis and reactivate the antitumor immunity. AimThis phase Ia/Ib study was carried out to evaluate the safety, recommended phase II dose (R2PD), and primary antitumor effects of GLS-010 in patients with advanced, refractory lymphoma and solid tumors. MethodsIn phase Ia study, patients with refractory solid tumors and lymphoma enrolled and received GLS-010 at a dose of 1, 4, or 10 mg/kg Q2W; 240 mg Q3W or Q2W. The primary objective was to assess the dose-limiting toxicity (DLT). In phase Ib study, doses were expanded in 9 specific tumors to ensure the R2PD and explore the efficacy. Tumor mutation burden level and PD-L1 expression were also assessed with whole-exome sequencing and immunohistochemistry (SP263), respectively. ResultsUp to April 18, 2020, a total of 289 patients (n = 24, phase Ia; n = 265, phase Ib) were enrolled. DLT was not observed in phase Ia part. The T1/2, CLss, and Vd were similar among all dose groups and different tumors. The most common treatment-emergent adverse events (TEAEs) were anemia, leukopenia, elevated alanine aminotransaminase/asparate aminotransferase (ALT/AST), and elevated bilirubin. And hypothyroidism was the most common immune-related adverse event (irAE). The incidence of grade ≥3 TEAE was 39.8%, while grade ≥3 irAE was only 4.5%. Based on safety studies, pharmacokinetics/pharmacodynamics, and preclinical data, 240-mg Q2W was recommended as the expansion dose. The overall objective response rate was 23.6%, with 10 patients achieving complete response. Patients with a high PD-L1 expression level (31.3% Versus. 13.7%, p = 0.012) or t-issue tumor mutation burden level (31.3% Versus. 5.6%, p = 0.009) showed a significantly better response. ConclusionGLS-010 showed acceptable safety profile and favorable clinical response. The dose of 240 mg Q2W was an optimal recommended dose as monotherapy.

Evaluating the clinical impact of a shortened infusion duration for ramucirumab: A population pharmacokinetic model-based approach.

191 Background: Ramucirumab is a human recombinant immunoglobin G1 monoclonal antibody (mAb) antagonist of vascular endothelial growth factor receptor-2. Ramucirumab dosed at 8 mg/kg every 2 weeks or 10 mg/kg every 3 weeks, either as monotherapy or in combination with chemotherapy, was initially studied with as an intravenous infusion over 60 minutes following premedication with a histamine-1 receptor antagonist. Lengthy intravenous infusions are inconvenient for patients and increase the workloads of nursing and administrative staff. Shortening the infusion duration of ramucirumab could therefore benefit both patients and healthcare professionals. The current analysis determined the impact such a change could have on the pharmacokinetic (PK) profile of ramucirumab. Additionally, the relationship between infusion rate and incidence of immediate infusion-related reactions (IRRs; occurring on the day of administration), common adverse events associated with mAb infusions, was assessed. Methods: A population pharmacokinetic model was established using concentration–time data collected from 2522 patients who received one of five different ramucirumab regimens involving an intravenous infusion over ~60 minutes in 17 clinical studies. The final PK model was used to simulate concentration–time profiles and exposure parameters following ramucirumab infusion durations of 30 vs 60 min. Phase II/III clinical study data from patients receiving ramucirumab were pooled to assess the association between ramucirumab infusion rate and incidence of immediate IRRs using multivariate logistic regression analysis. Results: Ramucirumab infusions of 30- and 60-min durations resulted in equivalent concentration–time profiles and, hence, equivalent systemic exposure to ramucirumab. Among 3216 patients receiving ramucirumab in phase II/III studies, 254 (7.9%) had at least one immediate any-grade IRR; 17 (0.5%) experienced grade ≥3 immediate IRRs. The incidence of immediate IRRs (any grade or grade ≥3) was similar across infusion rate quartiles. Under multivariate logistic analysis, infusion rate was not significantly associated with an increased risk of an immediate IRR (odds ratio per 1 mg/min increase 1.014, 95% confidence interval 0.999, 1.030; p=0.071). Conclusions: Administering ramucirumab using different infusion durations (30 vs 60 min) did not affect ramucirumab exposure. Analysis of clinical study data showed a faster infusion rate was not associated with an increased risk of immediate IRRs. It is considered unlikely that shortening the infusion duration of ramucirumab will impact its clinical efficacy or overall safety profile, and is now an option for administration in the U.S.

CS1001-304: A phase III study of fluorouracil and cisplatin (FP) with CS1001, an anti-PD-L1 antibody, or placebo in unresectable locally advanced, recurrent or metastatic esophageal squamous cell carcinoma (ESCC).

TPS255 Background: ESCC is the predominant histological subtype of esophageal cancer, particularly in Asian countries. Platinum-based chemotherapy is the first-line standard therapy for patients with unresectable, locally advanced, recurrent or metastatic ESCC. The FP regimen is recommended as the preferred treatment by guidelines. However, the survival benefit conferred by this therapy leaves considerable space for improvement, with median OS being less than 1 year. Blockade of the immune checkpoint receptors has shown clinical benefits in multiple tumor types. Recent studies combining standard treatments with checkpoint inhibitors have shown encouraging efficacy and favorable safety profile in patients with unresectable, locally advanced, recurrent or metastatic ESCC. CS1001 (sugemalimab) is the first full-length, fully human immunoglobin G4 (IgG4, s228p) anti-programmed death-ligand 1 (PD-L1) monoclonal antibody developed by the OMT transgenic rat platform. In an ongoing Phase Ib trial, CS1001 in combination with FP regimen demonstrated an ORR of 67.6% (25/37) and an mPFS of 9.0 months with a manageable safety profile in unresectable, locally advanced or distantly metastatic ESCC (19 Feb 2020 data cutoff; Shen, L., et al, ESMO 2020). Methods: CS1001-304 is a randomized, double-blind Phase III study to compare the efficacy and safety of FP regimen with CS1001 or placebo as first-line treatment in ESCC. The study enrolls patients with histologically or cytologically confirmed unresectable locally advanced, recurrent or metastatic ESCC who have ECOG PS of 0-1, patients are not eligible for curative therapy (curative surgery or definitive chemoradiotherapy), and have not received any prior systemic anti-tumor therapy for locally advanced or metastatic disease. Approximately 540 patients will be randomized at 2:1 into CS1001 + FP and placebo + FP arms respectively, stratified by PD-L1 expression status (PD-L1 expression < 1% vs ≥ 1% and < 10% vs ≥10%), ECOG PS (0 vs 1) and distant metastasis (no vs yes). Patients randomized to either arm will receive FP regimen (fluorouracil: 800 mg/m2/day, continuous intravenous infusion [IV], D1-4 of each cycle; cisplatin: 80 mg/m2, IV, D1 of each cycle), Q3W for up to 6 cycles in combination with CS1001 1200 mg or placebo (IV, D1 of each cycle), Q3W for up to 24 months. AEs will be monitored throughout the study and graded per NCI CTCAE v5.0. Tumor response will be assessed by RECIST v1.1 every 6 weeks in the first 12 months, and every 12 weeks thereafter. The primary endpoints are blinded independent central review (BICR)-assessed PFS and OS. Secondary endpoints include investigator-assessed PFS, BICR and investigator-assessed ORR and DoR, safety, PK profile, and immunogenicity. The study is actively enrolling patients in over 60 sites in China. Clinical trial information: NCT04187352.

GEMSTONE-301: a phase III clinical trial of CS1001 as consolidation therapy in patients with locally advanced/unresectable (stage III) non-small cell lung cancer (NSCLC) who did not have disease progression after prior concurrent/sequential chemoradiotherapy.

BackgroundIn China, platinum-based doublet chemotherapy is the standard treatment for patients who have unresectable stage III non-small cell lung cancer (NSCLC), administered with radiotherapy on either a concurrent or sequential basis. However, NSCLC patients who undergo this treatment can expect poor median progression-free survival (PFS) of around 8–10 months and a dismal 5-year overall survival (OS) rate of about 15%. In the recent PACIFIC trial, durvalumab was demonstrated to hold significant clinical benefit for patients with locally advanced/unresectable NSCLC who experienced no disease progression after definitive concurrent chemoradiotherapy (cCRT). CS1001 is the first full-length, fully human immunoglobin G4 (IgG4) monoclonal antibody (mAb) that targets programmed death ligand-1 (PD-L1) created through the OMT transgenic rat platform. The phase Ia/Ib study indicated CS1001 was well tolerated and exhibited anti-tumor potential with a range of tumors. GEMSTONE-301 is a phase III randomized, double-blind, study to explore the efficacy and safety of CS1001 compared with a placebo as consolidation therapy for stage III unresectable NSCLC patients.MethodsIn this trial, eligible patients will be randomized to receive CS1001 1,200 mg or placebo, every 3 weeks (Q3W). The primary endpoint will be investigator-assessed PFS, based on the Response Evaluation Criteria in Solid Tumors (RECIST) v1.1. The secondary endpoints will include OS, PFS assessment based on Blinded Independent Center Review (BICR), objective response rate (ORR), other efficacy measurements, safety, and tolerability.DiscussionThis phase III trial will determine the efficacy and safety of CS1001 as consolidation therapy in patients with locally advanced/unresectable (stage III) NSCLC who did not have disease progression after prior concurrent/sequential chemoradiotherapy (cCRT or sCRT), and is the first phase III trial on an anti-PD-L1 mAb initiated in China for this indication.Protocol versionVersion 3.0/September 12, 2019.

A novel dextran-grafted tetrapeptide resin for antibody purification.

Short peptide biomimetic affinity chromatography as a novel antibody separation chromatography is a potential alternative to protein A chromatography. However, if directly attaching ligand to matrix, the adsorption capacity and mass transfer rate would be affected by pore blockage and steric effect. Grafting resin is an effective method to solve this problem by using polymer as a bridge between matrix and ligand. In this work, a novel resin was prepared by grafting a tetrapeptide to the dextran-grafted matrix. Then, the adsorption properties for human immunoglobin G and BSA were determined. The results showed the saturation adsorption capacity could reach to 133mg/g resin at pH 8.9 with a significantly low dissociation constant (0.03mg/mL). The influence of flow rates to dynamic binding capacity of this resin was less than that of the non-grafted resin. The separation performance of the resin showed monoclonal antibody could be well isolated from the Chinese hamster ovary culture supernatant at pH 9.0 with the purity of 93.0% and yield of 84.7% by one step. Overall, this resin could achieve higher binding capacity by the possible of gaining higher ligand density, indicating its potential significance for separation in larger scale systems.

Generation and Characterisation of KY1066, a Fully Human Antibody Targeting the Enzymatic Activity of Matriptase-2 for the Treatment of Iron Overload in Beta Thalassemia

Beta thalassemia is an inherited hemoglobinopathy caused by a genetic defect in beta-globin gene and characterised by ineffective erythropoiesis, iron overload, splenomegaly and anemia. Excessive EPO production, resulting from the anemia, has a suppressive effect on the iron regulator hepcidin leading to increased iron absorption from the gut and release from internal stores. This in turn leads to tissue iron overload in transfusion independent (NTDT) patients and exacerbates the situation in transfusion dependent (TDT) patients. Primary standard of care treatment for TDT patients involves regular blood transfusions resulting in secondary iron overload and toxic tissue damage caused by non-transferrin bound iron (NTBI), which requires iron chelator treatment. Although transfusions and iron chelation have improved TDT patient prognosis, the iron overload experienced by some patients today still represents an unmet clinical need. The role of TMPRSS6 in iron regulation is already well established and reducing Tmprss6 expression was shown to increase hepcidin expression, correcting the iron overload, splenomegaly and anemia in Hbbth3/+ mice (Guo et al, JCI, 2013). Here we describe a first-in-class antibody targeting MTP-2, termed KY1066, for the treatment of diseases of iron overload, such as beta thalassemia. Through immunising mice transgenic for human immunoglobin heavy and light chain variable regions with purified human MTP-2 extra-cellular domain (ECD) and full-length human MTP-2 expressed on cells, we were able to obtain cross-reactive, MTP-2-specific monoclonal antibodies (mAbs). Our lead molecule, KY1066, was found to be a cross-reactive neutraliser of MTP-2 enzymatic activity both in vitro and in vivo and mechanistically blocks MTP-2 through binding to the serine protease (SP) domain active site, preventing cleavage of substrates. As a result, it was seen to increase BMP/SMAD signalling and elevate levels of hepcidin expression from hepatocyte cells following a single I.P. dose (10 mg/kg) in C57BL/6J mice. The elevation of hepcidin decreased serum iron and transferrin saturation through increased internalisation and degradation of ferroportin. In Hbbth3/+ mice, a single I.P. dose (10 mg/kg) showed a decrease of 52% and 47% in serum iron and transferrin saturation (TSAT) respectively, at the 2-week timepoint. Furthermore, with repeat dosing, we were able to show consistent iron restriction in a Hbbth3/+ mice over multiple weeks. Together this provides evidence KY1066 has the potential to treat iron overloaded patients, such as in beta thalassemia, to improve the anemia and reduce the transfusion and iron chelation need. Disclosures Wake: Kymab Ltd: Employment, Equity Ownership. Papworth:Kymab Ltd: Employment, Equity Ownership. Bayliss:Kymab Ltd: Employment, Equity Ownership. Grimshaw:Kymab Ltd: Employment, Equity Ownership. Rynkiewicz:Kymab Ltd: Employment, Equity Ownership. Paterson:Kymab Ltd: Employment, Equity Ownership. Poindron:Kymab Ltd: Employment, Equity Ownership. Carter:Kymab Ltd: Employment. Hudson:Kymab Ltd: Employment, Equity Ownership. Theurl:Kymab Ltd: Consultancy, Equity Ownership. Germaschewski:Kymab Ltd.: Employment, Equity Ownership. Meynard:Kymab Ltd: Research Funding.

PF338 DEVELOPMENT AND OPTIMISATION OF A FULLY HUMAN FVIII MIMETIC BISPECIFIC ANTIBODY FOR PATIENTS WITH HAEMOPHILIA A

Background:Recently a range of alternative novel therapies have been developed to improve treatment options for patients with Hemophilia A. One approach is to generate a Factor VIII (F.VIII) mimetic molecule using a humanised bispecific antibody, as was done for Hemlibra. Taking advantage of Kymab's fully human antibody discovery platform, we describe the selection and optimisation of an FVIII mimetic common light chain (CLC) bispecific antibody which can similarly catalyse the generation of Factor Xa (FXase) and normalise the activated partial thromboplastin time (aPTT).Aims:To generate a functionally active F.VIII mimetic bispecific antibody for Haemophilia A treatmentMethods:F.IX and F.X. binding antibodies were generated by immunizing Kymouse™, which contain the full human immunoglobin repertoire, with F.IX or F.X, respectively. Isolated F.IX and F.X specific arms were co‐expressed as 2‐heavy‐2‐light‐chain (2H2L) bispecific antibodies. Purified 2H2L bispecifics were screened using a high‐throughput chromogenic FXase assay. The light chain of a promiscuous F.IX arm was chosen to generate transgenic mice expressing this bespoke common light chain (CLC) in the Kymouse™ background. By immunizing these transgenic mice with F.X, F.X binding antibodies containing the CLC were recovered. The heavy chains of these F.X binding antibodies were co‐expressed with the heavy/light chains of the chosen F.IX arm as CLC bispecific antibodies. One biologically active CLC bispecific antibody was identified by functional assays, and chosen for further optimization. The optimization of the lead bispecific antibody, KY1049, was achieved by data mining of next generation sequencing information using Kymab's IntelliSelect™ bioinformatic platform, coupled with site‐specific mutagenesis.Results:More than 8,000 2H2L bispecifics were screened by a chromogenic FXase assay to select the most active and versatile F.IX arm (Figure 1A). More than 400 F.X heavy chains subsequently isolated from the bespoke CLC Kymouse™ were screened to identify a highly active CLC bispecific (Figure 1B). Further optimisation of the molecule was carried out to iteratively increase FVIII mimetic activity by deep data‐mining of heavy chain NGS sequence data, or site‐specific mutagenesis. The combinatorial optimization process resulted in a highly functional CLC bispecific, KY1049 with comparable F.VIII mimetic activities to a sequence‐identical analogue of Hemlibra (Figure 1C).Summary/Conclusion:Our bispecific antibody discovery platform consisting of four‐chain matrix screening, common light chain transgenic mouse technology, B cell network analysis and site‐specific mutagenesis was applied to develop KY1049, a potent FV.III mimetic bispecific antibody, which shows equivalent activity to Hemlibra in vitro and holds promise as a future therapeutic in Haemophilia A.image

Anti-TNF Therapy Induces CD4+ T-Cell Production of IL-22 and Promotes Epithelial Repairs in Patients With Crohn’s Disease

Anti-tumor necrosis factor (TNF) therapy appears to be effective in the treatment of Crohn's disease (CD), a chronic inflammatory disease of the gastrointestinal tract. However, the mechanisms involved are not completely understood. Fifty-seven active CD patients were enrolled, and cytokine profiles in colonic biopsies of patients with active CD receiving anti-TNF monoclonal antibody (mAb) (infliximab [IFX]) treatment were determined using quantitative real-time polymerase chain reaction (qRT-PCR). Colonic biopsies of active CD patients and healthy donors were cultured with IFX in vitro, and cytokine profiles were measured by qRT-PCR. Peripheral blood (PB)-CD4+ T cells were stimulated with anti-CD3 and anti-CD28 mAbs in the presence of human immunoglobin (HIg), IFX, recombinant human TNF-α converting enzyme (rhTACE), and aryl hydrocarbon receptor (AhR) inhibitor (CH-223191), respectively, to determine interleukin (IL)-22 expression by CD4+ T cells. Caco2 cells were also utilized to study their potential role in modulating epithelial cell barrier repairs in vitro. IFX therapy markedly upregulated IL-22 mRNA expression in the gut mucosa of CD patients. In vitro treatment with IFX greatly promoted CD CD4+ T cells to express IL-22, which was inhibited by rhTACE, indicating that reverse signaling through binding to membrane-bound TNF mediates anti-TNF-induced IL-22 expression of CD CD4+ T cells. However, blockade of AhR markedly inhibited anti-TNF-induced IL-22+CD4+ T (Th22) cell differentiation in CD patients. Moreover, treatment with IL-22 induced intestinal epithelial cell expression of tight junction proteins (eg, claudin1 and ZO-1) and facilitated transepithelial resistance, indicating that IL-22 protects intestinal mucosa from inflammation via maintenance of epithelial barrier integrity. Our results uncover a novel mechanism whereby anti-TNF therapy upregulates IL-22 production in CD patients through promoting Th22 cell differentiation and contributes to intestinal epithelial barrier repairs.

A targeted transforming growth factor-beta (TGF-β) blocker, TTB, inhibits tumor growth and metastasis.

Transforming growth factor beta (TGF-β) promotes cancer growth in late stage cancers. To inhibit the TGF-β pathway, we investigated a tumor-targeting TGF-β receptor blocker, TTB, and its role in tumor progress. The targeted TTB comprised of the extracellular domain of the TGF-β receptor II, the endoglin domain of TGF-β receptor III, and the human immuno-globin IgG1 constant fragment (Fc). To enhance tumor microenvironment targeting, a RGD peptide was fused at the N-terminal of TTB. The targeted TTB exhibited potent TGF-β neutralization activities, and inhibited cancer cell migration and invasion as well as colony formation. In xenograft models, the TTB had potent tumor inhibition activities. The TTB also attenuated the TGF-β1-induced Smad2 phosphorylation and epithelial to mesenchymal transformation (EMT), and suppressed breast cancer metastasis. Thus, the TTB is an effective TGF-β blocker with a potential for blocking excessive TGF-β induced pathogenesis in vivo.

A Review on Pharmacological Management of Generalised Tetanus

Background: Tetanus is a major public concern in the developing world and is still encountered in the developed world. The evidence regarding the management of the tetanus is limited making the protocol based treatment of tetanus an unmet goal especially in low to middle-income countries. This article was our endeavour to review the management of the patient.Methods: Two authors extensively searched electronic databases like MEDLINE/Pub Med and Google Scholar, searched with Mesh (Medical Subject Headings) terms like “tetanus”, “treatment”, “benzodiazepines”, “ Tetanus immunoglobin” from the earliest possible date of 1966 to January 2018.Conclusion: We found that diazepam was the drug of choice for controlling muscle spasm, magnesium sulphate controls the autonomic instability, human tetanus immunoglobin bind the unbound toxin, and proper wound care is a must.

Patterned Photonic Nitrocellulose for Pseudo-Paper Microfluidics.

We report a pseudo-paper microfluidic chip based on patterned photonic nitrocellulose. The photonic nitrocellulose is fabricated using self-assembled monodisperse SiO2 nanoparticles as template. The SiO2 nanoparticles form a photonic crystal having a close-packed hexagonal structure in the microchannels, so the resulting nitrocellulose has a complementary inverse-opal structure. After lamination, a hollow channel is obtained that is partially filled with the photonic nitrocellulose. Owing to the highly ordered photonic structure of the pseudo-paper chip, the flow profile of aqueous solution wicking through the channel is more uniform than conventional paper microfluidic chip. It is also found that the wicking rate of aqueous solution can be easily manipulated by changing the diameter of the self-assembled monodisperse SiO2 nanoparticles, which determines the pore size of the photonic nitrocellulose. The fluorescent enhancement property of the photonic nitrocellulose is used to increase the fluorescent intensity for multiplex detection of two cancer biomarkers. Label-free detection of human immunoglobin G based on the structure color of the photonic nitrocellulose is also demonstrated.

Polydimethylsiloxane microfluidic chemiluminescence immunodevice with the signal amplification strategy for sensitive detection of human immunoglobin G

A polydimethylsiloxane (PDMS) microfluidic chemiluminescence (CL) immunodevice for sensitive detection of human immunoglobin G (IgG) with the signal amplification strategy was developed in this work. The immunodevice was prepared by covalently immobilizing capture antibodies (Abs) on the silanized microchannel of microfluidic chip. Gold nanoparticles (AuNPs) functionalized with a high molar ratio of horseradish peroxidase (HRP) were used as an Ab label for signal amplification. Using a sandwich immunoassay, the multi-HRP conjugated AuNPs can catalyze the luminol-H2O2 CL system to achieve the high sensitivity. In addition, the double spiral flow-channel was adopted here, which can still contribute to the high sensitivity. Based on signal amplification strategy, the performance of human IgG tests revealed a lower detection limit (DL) of 0.03ng/mL and showed an increase of 7.4-fold in detection sensitivity compared to a commercial Ab-HRP conjugation. This microfluidic immunodevice can provide an alternative approach for sensitive detection of human IgG in the field of clinic diagnostic and therapeutic.

Detection of Human Ig G Using Photoluminescent Porous Silicon Interferometer.

Photoluminescent porous silicon (PSi) interferometers having dual optical properties, both Fabry-Pérot fringe and photolumincence (PL), have been developed and used as biosensors for detection of Human Immunoglobin G (Ig G). PSi samples were prepared by electrochemical etching of p-type silicon under white light exposure. The surface of PSi was characterized using a cold field emission scanning electron microscope. The sensor system studied consisted of a single layer of porous silicon modified with Protein A. The system was probed with various fragments of aqueous human immunoglobin G (Ig G) analyte. Both reflectivity and PL were simultaneously measured under the exposure of human Ig G. An increase of optical thickness and decrease of PL were obtained under the exposure of human Ig G. Detection limit of 500 fM was observed for the human Ig G.

Toxic shock syndrome following inguinal hernia repair: a rare condition

A 25-year-old man developed fulminant multisystem failure 28 hours after elective repair of an inguinal hernia. Toxic shock syndrome (TSS) was diagnosed. The patient recovered fully with supportive care in ICU, antibiotics, and IV human immunoglobin . To the best of our knowledge, only one case of TSS following inguinal hernia repair have ever been previously published. Journal of College of Medical Sciences-Nepal, 2013, Vol-9, No-2, 57-59 DOI: http://dx.doi.org/10.3126/jcmsn.v9i2.9689

Gold Nanoparticle-Based Immuno Dual Probes for Targeting Proteomics

Immunoprecipitation combined with mass spectrometry (MS) is a promising technique for targeting proteomics in characterizing submicrograms of target protein and interacting proteins in living cells. This method, however, is limited by interference arising from nonspecific binding. We report a novel gold nanoparticle (AuNP)-based immuno probe approach for immunoprecipitation. By cross-linking the antibody Fc domain to protein G covalently modified on AuNPs, the probe was fabricated and characterized to have 60 protein G and 30 immunoglobins per AuNP. We used human immunoglobin against the target and mouse immunoglobin with the same isotype (IgG) to fabricate the target and preclear probe, respectively, and termed it as the dual probe approach. Our results showed that the preclear probe (AuNP-IgG) and the target probe (AuNP-anti-ERα) share a similar panel of nonspecific binders but dramatic different specificity toward the target. Thus, using the dual probe method, we showed major nonspecific binders in the cell lysate could be largely removed without sacrificing the target protein. Compared to the conventional agarose gel-chromatography, the AuNP-based probe exhibited less nonspecific interference and higher recovery yield for ERα. Moreover, the AuNP-based probe is more inert than the agarose gel under harsh conditions and does not induce dissociation of the cross-linked IgG that could interfere with target identification. Using AuNP-based dual probes, ERα was shown to be purified from MCF-7 cells with minimum nonspecific binding. Moreover, the identity and phosphorylation sites on the C-terminus of the purified ERα could be positively confirmed by MS using only 1 mg of cellular protein.

Fabrication of Human IgG Sensors Based on Porous Silicon Interferometer Containing Bragg Structures

A simply modified biosensor based on protein A-modified distributed Bragg reflectors (DBR) porous silicon (PSi) chip for the detection of human immunoglobin G (IgG) are developed. The fabrication, optical characterization, and surface derivatization of DBR PSi are investigated. The sensor system studied consist of multi-layer of porous silicon modified with protein-A. The sensor is operated by the measurement of the reflection peak in the white light reflection spectrum. Molecular binding is detected as a shift in wavelength of reflection peaks.

The Effect of ICAM-1 Antibody Therapy in the SCID-Hu Mouse Model Using Primary Myeloma Cells

Abstract 2914 Background:Myeloma (MM) - stromal cell-cell interactions play a crucial role in MM cell growth, survival, and drug resistance. Intercellular adhesion molecule-1 (ICAM-1) is up-regulated in different cancers, including MM, and represents one of the major adhesion molecules mediating tumor-stromal cell-cell contacts. Previous investigations have shown that ICAM antibodies induce antitumor activity in SCID mice xenografted with MM cell lines, likely involving Fc :Fc gamma receptor dependent anti-tumor mechanisms. We have examined the anti-MM activity in the SCID-hu mouse model of a specific humanized ICAM1 antibody (BI-505, Bio-Invent, Sweden) using primary MM cells. Materials and Methods:The expression of ICAM1 was examined in plasma cells from 22 donors, 351 newly diagnosed MMs, and 9 MM cell lines using Affymetrix U133 Plus2 chips. Human fetal femurs and tibias, obtained at 17 to 22 gestational weeks, were cut into fragments and implanted subcutaneously in 16 SCID mice (SCID-hu) at age 6 to 8 weeks. Four weeks after bone implantation, approximately 5 ×106 CD138+ plasma cells from MM patients were injected directly into the human bone of the SCID-hu mice in final volume of 30 to 40μl of phosphate-buffered saline(PBS). Human immunoglobin (hIg) levels by ELISA were used as an indicator of myeloma cell growth. When hIg level reached 10μg/mL or higher in 2 consecutive samples after 4 weeks of injection of the tumor cells, the mice were divided into four groups (n=4), Control group (not injected MM cells and no drug treatment); the other 3 groups were injected with MM cells: the isotype control group (isotype IgG 2mg/kg, i.p., twice weekly), BI-505 group (2mg/kg, i.p., twice weekly) and bortezomib group (1mg/kg, i.p., twice weekly). Bone remolding was detected by X-radiography and by measuring bone mineral density (BMD). Tumor cells were detected by immunohistochemistry using the CD138 antibody. Results:High expression of ICAM1 was observed in normal plasma cells, primary MM cells and MM cell lines. With a follow- up of 10 weeks, the tumor burden in the mice treated with BI-505 or bortezomib was significantly lower compared with the isotype control group (p: BI505 < 0.01; p: bortezomib < 0.01). Also, the number of MM tumor cells stained with the CD138 antibody was significantly less in samples treated with either BI-505 or bortezomib than in the isotype control group (p < 0.01). In addition, 6 weeks after injection of tumor cells, X-rays showed severe bone resorption in the isotype-control group, while there was no obvious bone resorption in the fetal bones after treatment with BI-505 or bortezomib. The BMD (0.0715±0.006 g/cm2) of isotype control was significantly lower than that in other 3 groups including control: 0.1278±g/cm2, BI-505 group: 0.102±0.0064 g/cm2, and bortezomib group: 0.106±0.0059g/cm2. Importantly, the number of TRAP-positive cells was significantly higher in the isotype control group than in the other 3 groups (p < 0.01). Conclusion:ICAM1 expression presents in all subtypes of myeloma. The ICAM1 antibody BI-505 significantly inhibits primary MM cell growth and bone destruction in the SCID-hu mice to the same degree as bortezomib, indicating its clinical applicability in therapy of MM. Disclosures:No relevant conflicts of interest to declare.