scholarly journals Anti-Emicizumab Antibodies Do Not Cross-React with Mim8 in Vitro

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3193-3193
Author(s):  
Roberta Gualtierotti ◽  
Carla Valsecchi ◽  
Chiara Suffritti ◽  
Eugenia Biguzzi ◽  
Sara Arcudi ◽  
...  
Keyword(s):  
Observational Studies ◽  
Bispecific Antibody ◽  
Hemophilia A ◽  
Detection Method ◽  
Cross Reactivity ◽  
Pharmaceutical Companies ◽  
Steady State Levels ◽  
The Cross

Abstract Background Patients with severe hemophilia A may develop inhibitors against factor VIII (FVIII) in around 30% of cases. Recently, the introduction of non-replacement therapies such as emicizumab, a FVIII-mimicking agent administered as a subcutaneous injection, has revolutionized the treatment of patients with inhibitors. However, although rarely, some patients may develop antibodies against this drug. If neutralizing, these antibodies interfere with the activity of the drug, making it ineffective. Mim8 (Novo Nordisk®) is a novel experimental FVIII-mimetic human bispecific antibody that has a similar function as emicizumab, by bridging activated FIX (FIXa) and FX to activate FX, although with a different molecular structure compared to emicizumab. It is currently in phase II clinical trial for subcutaneous treatment of patients with hemophilia A with or without FVIII inhibitors (1, 2). It is currently unknown whether the antibodies developed against emicizumab by patients with hemophilia A could cross-react with Mim8. Aim Our aim was to study the cross-reactivity of anti-emicizumab antibodies developed by patients with hemophilia A against Mim8 with an in-house detection method. Methods We studied the serum of three patients who developed anti-emicizumab antibodies. Plasma from one patient with persistent inhibiting antibodies was collected both during the treatment (thus also containing emicizumab at steady-state levels) and two years after treatment discontinuation due to inefficacy (neutralizing persistent antibodies). Plasma from two patients who developed transient antibodies against emicizumab were also tested in the course of treatment with emicizumab (non-neutralizing transient antibodies). The plate was coated both with emicizumab and with Mim8 provided by the pharmaceutical companies for research purposes. Plasma samples, diluted 1/20, were loaded into the coated wells and incubated 90 min at 37°C. The cross-reactivity to Mim8 was evaluated also by using the affinity purified anti-emicizumab IgG, which was loaded at 5 ug/mL. A properly adapted ELISA method already described (3) was used as reference assay. Then, biotinylated-emicizumab (1.5 ug/mL) or biotinylated-Mim8 (at 2 and 4 ug/mL) were added and the plate incubated 1 hour at 37°C. Moreover, a competition test was performed by using a mixture of biotinylated-emicizumab (1.5 ug/mL) and an excess of Mim8 (at 4, 8 and 40 ug/mL) in the detection phase. Results The Mim8 molecule - either alone, or matched with emicizumab - used both in the capture phase and in the detection phase did not bind to neither patient's plasma antibodies nor to anti-emicizumab purified IgG, which were instead revealed with the reference assay. The binding of the anti-emicizumab antibodies to biotinylated-emicizumab was not inhibited by the addition of Mim8, even at 40 ug/mL. Conclusions Our in-house method showed that anti-emicizumab antibodies do not react with Mim8 in vitro. Observational studies to test whether Mim8 can be used safely in patients with anti-emicizumab antibodies are needed to confirm our findings in vivo as well. References 1. Østergaard et al. A FVIIIa-mimetic bispecific antibody (Mim8) ameliorates bleeding upon severe vascular challenge in hemophilia A mice. Blood. 2021;blood.2020010331. doi:10.1182/blood.2020010331. 2. Valsecchi C et al. J Thromb Haemost. 2021;19(3):711-718. Disclosures Peyvandi: Roche: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Sobi: Consultancy, Honoraria; Takeda: Consultancy, Honoraria.

scholarly journals Pharmacology and Pharmacokinetics of NXT007; Emicizumab-Based Engineered Fixa/Fx Bispecific Antibody with Improved Properties

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 19-19
Author(s):  
Kazuki Yamaguchi ◽  
Tetsuhiro Soeda ◽  
Motohiko Sato ◽  
Norihito Shibahara ◽  
Hikaru Koga ◽  
...  
Keyword(s):  
Ternary Complex ◽  
Thrombin Generation ◽  
Turnover Rate ◽  
Bispecific Antibody ◽  
Hemophilia A ◽  
Minimal Impact ◽  
Kd Values ◽  
Cofactor Activity

Background Emicizumab (HEMLIBRA®) is a factor (F) VIII function-mimetic therapeutic bispecific antibody (BsAb) to FIXa and FX able to prevent bleeding in persons with hemophilia A (PwHA) when injected subcutaneously once every 1, 2 or 4 weeks. To develop a next generation version, we sought an agent able to keep hemostatic potential in non-hemophilic range with more convenient dosing regimen (dosing frequency/volume). We successfully created the emicizumab-based engineered four-chain BsAbs, NXT series. Among these, we selected NXT007 as a clinical candidate. Objectives The aim of this study is to clarify the in vitro and in vivo properties of NXT007 and predict its therapeutic potency non-clinically. Methods We evaluated the pharmacological activities of NXT007 in vitro using a thrombin generation assay (TGA) with FVIII-deficient patient plasma, and in vivo by inducing bleeding in FVIII-neutralizing antibody-treated acquired hemophilia A cynomolgus monkey (cyno) model. To clarify the FVIII-cofactor activity of NXT007, we performed an enzymatic kinetics analysis of FIXa-catalyzed FX activation with and without NXT007, as well as surface plasmon resonance analysis to determine the dissociation constant (KD) of NXT007 to FIX, FIXa, FX and FXa. We obtained its pharmacokinetic (PK) profile in non-human primates in a single dose SC/IV study. Results In vitro addition of NXT007 at 30 μg/mL increased the peak height of TGA in FVIII-deficient plasma to the same levels achieved by recombinant human FVIII at 40-100 IU/dL (FXIa-triggering) or 100-150 IU/dL (tissue factor-triggering). A single bolus intravenous injection of NXT007 (0.075 mg/kg) ameliorated bleeding symptoms in the cyno model to similar as a twice daily intravenous injection of recombinant porcine FVIII (20 U/kg). The in vitro and in vivo results were roughly concordant. NXT007 increased the turnover rate (kcat) of FIXa-catalyzed FX activation by approximately 4,000-folds compared to the condition without cofactor. The impact of NXT007 on the kcat was similar to that of emicizumab. As for binding affinities, the KD values of NXT007 to FIX, FIXa, FX and FXa were 1.08, 0.728, 0.0538 and 0.0231 μM, respectively in buffer solution. Compared to emicizumab, NXT007 bound more strongly to FX/FXa and with similar affinity to FIX/FIXa. This means that NXT007 would have an ability to form more FIX-BsAb-FX ternary complex than emicizumab. Calculated using the above KD values, at 30 μg/mL of BsAb the estimated concentration of FIX-NXT007-FX ternary complex in plasma is approximately 10-fold higher than that of the FIX-emicizumab-FX ternary complex which is roughly concordant with the difference in their FVIII equivalent thrombin generation activity. Prothrombin time (PT) was not clearly prolonged suggesting minimal impact on FX function by in vitro addition of NXT007 at up to 30 μg/mL, which was enough to induce sufficient thrombin burst in FVIII-deficient plasma as described above. A half-life of NXT007 was 19.6 to 24.4 days (0.02-2 mg/kg, SC) and SC bioavailability was 84.4% (2 mg/kg) in the in vivo cyno PK study, in which no obvious change in plasma FIX or FX levels was observed after 0.02-2 mg/kg single SC administration. Conclusions Based on the nonclinical results, NXT007, delivered in every-4-week SC injections, will keep a non-hemophilic range of equivalent FVIII thrombin generation in PwHA, Compared with emicizumab, NXT007's improved cofactor activity may be attributed to its more efficient ternary complex formation while keeping turnover rate with minimal impact on FX function suggested by PT value and antigen accumulation. A phase 1/2 clinical study of NXT007 is now on-going (NXTAGE; JapicCTI-194919). Disclosures Yamaguchi: Chugai Pharmaceutical Co., Ltd: Current Employment. Soeda:Chugai Pharmaceutical Co., Ltd.: Current Employment. Sato:Chugai Pharmaceutical Co., Ltd.: Current Employment. Shibahara:Chugai Pharmaceutical Co., Ltd.: Current Employment. Koga:Chugai Pharmaceutical Co., Ltd.: Current Employment. Ichiki:Chugai Pharmaceutical Co., Ltd.: Current Employment. Joyashiki:Chugai Pharmaceutical Co., Ltd.: Current Employment. Teranishi:Chugai Pharmaceutical Co., Ltd.: Current Employment. Nishimura:Chugai Pharmaceutical Co., Ltd.: Current Employment. Shiraiwa:Chugai Pharmaceutical Co., Ltd.: Current Employment. Kitamura:Chugai Pharmaceutical Co., Ltd.: Current Employment. Igawa:Chugai Pharmaceutical Co., Ltd.: Current Employment. Konishi:Chugai Pharmaceutical Co., Ltd.: Current Employment. Kitazawa:Chugai Pharmaceutical Co., Ltd.: Current Employment.

scholarly journals Mim8 - a Next-Generation FVIII Mimetic Bi-Specific Antibody - Potently Restores the Hemostatic Capacity in Hemophilia a Settings in Vitro and In Vivo

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 96-96 ◽  
Author(s):  
Stine L Kjellev ◽  
Henrik Østergaard ◽  
Per Jr Greisen ◽  
Mette B Hermit ◽  
Karina Thorn ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Bispecific Antibody ◽  
Hemophilia A ◽  
Next Generation ◽  
Employment Equity ◽  
Thrombin Generation Assay ◽  
Equity Ownership ◽  
Target Binding

The treatment of hemophilia A (HA) is primarily based on replacement of factor VIII (FVIII), and in people with HA with inhibitors (HAwI) on the use of by-passing agents. Recently, a FVIII mimetic bispecific antibody emicizumab (Hemlibra®) was approved for treatment of HA and HAwI, offering a subcutaneous, prophylactic treatment opportunity with potential for significantly reducing the treatment burden. We describe the development and pre-clinical characterization of Mim8, a novel, next-generation FVIII mimetic human bispecific antibody. Mim8 is a highly potent molecule bridging factor IXa (FIXa) and factor X (FX) in development for subcutaneous treatment of people with HA and HAwI. Development of Mim8 utilized the Duobody® platform to initially screen for compatible anti-FIXa and anti-FX antibodies followed by several iterations of systematic mutational optimization. In total, more than 30,000 bispecific antibodies were analyzed. The optimization process aimed for efficient Mim8-mediated activation of FX by FIXa in the presence of procoagulant membrane, low target binding in solution, low immunogenicity risk, and for desirable biophysical parameters such as low viscosity. In vitro characterization demonstrated that Mim8 efficiently localizes FIXa and FX to the phospholipid surface and enhances FXa activation. The monovalent anti-FIXa arm alone stimulates the proteolytic activity of FIXa in the range of 15,000-fold and is an important contributor to the activity of the bispecific antibody. The dissociation constants (Kd) of Mim8 for FIXa and FX is in the micromolar range, minimizing target binding in the blood. Using thrombin generation assay in congenital HA plasma and thrombelastography (TEG) in whole blood from healthy volunteers spiked with anti-FVIII antibodies, Mim8 was capable of normalizing thrombin generation and blood clot formation, respectively, with approximately 15 times greater potency than emicizumab (Figure 1). A similar potency improvement was demonstrated in a tail vein transection bleeding model in FVIII-deficient mice co-dosed with human FIX and FX to circumvent lack of Mim8 cross reactivity to murine FIX and FX. The terminal half-life of Mim8 was estimated to 14 days (range 10-17 days) in cynomolgus monkeys and the subcutaneous bioavailability to 97%. In conclusion, Mim8 is a novel, next-generation FVIII mimetic bispecific antibody with anti-FIXa and anti-FX arms that potently stimulates FX activation resulting in efficacious haemostasis in vitro and in vivo. Mim8 has a high potency allowing for administration of small volumes in a pen device, good PK parameters, minimal target binding in the blood, and good biophysical properties. Collectively, these properties support clinical development of Mim8 as a potentially improved next-generation FVIII-mimetic prophylactic treatment option for persons with hemophilia A regardless of inhibitor status. Figure 1: Left: FXI-triggered thrombin generation assay in congenital HA plasma (mean and SD of n = 5). Right: thromboelastography in whole blood from healthy donors spiked with polyclonal anti-FVIII antibody (mean and SD of n = 3). Coagulation was triggered with low concentration (∼30 fM) of tissue factor (Innovin® 1:200,000). Shaded areas: standard deviation of controls. Blue circles: Mim8. Grey squares: a sequence identical analogue (SIA) to emicizumab (comparable data were obtained with a commercially available batch of Hemlibra®). Disclosures Kjellev: Novo Nordisk A/S: Employment, Equity Ownership. Østergaard:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Greisen:Novo Nordisk A/S: Equity Ownership, Patents & Royalties: Patents. Hermit:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Thorn:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Hansen:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Zhou:Novo Nordisk A/S: Equity Ownership, Other: Previous employment, Patents & Royalties: Patents. Bjelke:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Kjalke:Novo Nordisk A/S: Employment, Honoraria. Lund:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Holm:Novo Nordisk A/S: Equity Ownership, Other: Previous employment. Ley:Novo Nordisk A/S: Employment, Equity Ownership. Elenius:Novo Nordisk A/S: Equity Ownership, Other: Previous employment; Leo Pharma A/S: Employment, Equity Ownership. Thygesen:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Loftager:Novo Nordisk A/S: Employment, Equity Ownership. Rasch:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Lorenzen:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Gandhi:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Lamberth:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Egebjerg:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Lund:Novo Nordisk A/S: Employment, Equity Ownership. Henriksen:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Rahbek-Nielsen:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Yang:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Hilden:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties.

scholarly journals A bispecific antibody agonist of the IL-2 heterodimeric receptor preferentially promotes in vivo expansion of CD8 and NK cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Katherine E. Harris ◽  
Kyle J. Lorentsen ◽  
Harbani K. Malik-Chaudhry ◽  
Kaitlyn Loughlin ◽  
Harish Medlari Basappa ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Bispecific Antibody ◽  
T Regulatory Cells ◽  
Tumor Regression ◽  
Interleukin 2 ◽  
In Vivo Studies ◽  
Preferential Binding

AbstractThe use of recombinant interleukin-2 (IL-2) as a therapeutic protein has been limited by significant toxicities despite its demonstrated ability to induce durable tumor-regression in cancer patients. The adverse events and limited efficacy of IL-2 treatment are due to the preferential binding of IL-2 to cells that express the high-affinity, trimeric receptor, IL-2Rαβγ such as endothelial cells and T-regulatory cells, respectively. Here, we describe a novel bispecific heavy-chain only antibody which binds to and activates signaling through the heterodimeric IL-2Rβγ receptor complex that is expressed on resting T-cells and NK cells. By avoiding binding to IL-2Rα, this molecule circumvents the preferential T-reg activation of native IL-2, while maintaining the robust stimulatory effects on T-cells and NK-cells in vitro. In vivo studies in both mice and cynomolgus monkeys confirm the molecule’s in vivo biological activity, extended pharmacodynamics due to the Fc portion of the molecule, and enhanced safety profile. Together, these results demonstrate that the bispecific antibody is a safe and effective IL-2R agonist that harnesses the benefits of the IL-2 signaling pathway as a potential anti-cancer therapy.

scholarly journals Previous Humoral Immunity to the Endemic Seasonal Alphacoronaviruses NL63 and 229E Is Associated with Worse Clinical Outcome in COVID-19 and Suggests Original Antigenic Sin

Life ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 298
Author(s):  
Daniele Focosi ◽  
Angelo Genoni ◽  
Ersilia Lucenteforte ◽  
Silvia Tillati ◽  
Antonio Tamborini ◽  
...  
Keyword(s):  
Humoral Immunity ◽  
Cross Reactivity ◽  
Clinical Severity ◽  
World Health ◽  
Laboratory Findings ◽  
Cellular And Humoral Immunity ◽  
Rate Ratios ◽  
Original Antigenic Sin

Antibody-dependent enhancement (ADE) of severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) infection has been hypothesized. However, to date, there has been no in vitro or in vivo evidence supporting this. Cross-reactivity exists between SARS CoV-2 and other Coronaviridae for both cellular and humoral immunity. We show here that IgG against nucleocapsid protein of alphacoronavirus NL63 and 229E correlate with the World Health Organization’s (WHO) clinical severity score ≥ 5 (incidence rate ratios was 1.87 and 1.80, respectively, and 1.94 for the combination). These laboratory findings suggest possible ADE of SARS CoV-2 infection by previous alphacoronavirus immunity.

scholarly journals Allergenic Characterization of New Mutant Forms of Pru p 3 as New Immunotherapy Vaccines

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
C. Gómez-Casado ◽  
M. Garrido-Arandia ◽  
P. Gamboa ◽  
N. Blanca-López ◽  
G. Canto ◽  
...  
Keyword(s):  
Food Allergy ◽  
Ex Vivo ◽  
Binding Capacity ◽  
Cross Reactivity ◽  
T Cell Epitopes ◽  
Native Form ◽  
Ige Binding ◽  
Pru P 3

Nowadays, treatment of food allergy only considered the avoidance of the specific food. However, the possibility of cross-reactivity makes this practice not very effective. Immunotherapy may exhibit as a good alternative to food allergy treatment. The use of hypoallergenic molecules with reduced IgE binding capacity but with ability to stimulate the immune system is a promising tool which could be developed for immunotherapy. In this study, three mutants of Pru p 3, the principal allergen of peach, were produced based on the described mimotope and T cell epitopes, by changing the specific residues to alanine, named asPru p 3.01, Pru p 3.02, andPru p 3.03.Pru p 3.01showed very similar allergenic activity as the wild type byin vitroassays. However,Pru p 3.02andPru p 3.03presented reduced IgE binding with respect to the native form, byin vitro,ex vivo,and in vivo assays. In addition,Pru p 3.03had affected the IgG4 binding capacity and presented a random circular dichroism, which was reflected in the nonrecognition by specific antibodies anti-Pru p 3. Nevertheless, bothPru p 3.02andPru p 3.03maintained the binding to IgG1 and their ability to activate T lymphocytes. Thus,Pru p 3.02andPru p 3.03could be good candidates for potential immunotherapy in peach-allergic patients.

scholarly journals The Effect of Oxidized Dopamine on the Structure and Molecular Chaperone Function of the Small Heat-Shock Proteins, αB-Crystallin and Hsp27

2021 ◽  
Vol 22 (7) ◽  
pp. 3700
Author(s):  
Junna Hayashi ◽  
Jennifer Ton ◽  
Sparsh Negi ◽  
Daniel E. K. M. Stephens ◽  
Dean L. Pountney ◽  
...  
Keyword(s):  
Heat Shock ◽  
Protein Aggregation ◽  
Molecular Chaperone ◽  
Cross Linking ◽  
Αb Crystallin ◽  
The Cross ◽  
Shock Proteins ◽  
And Function

Oxidation of the neurotransmitter, dopamine (DA), is a pathological hallmark of Parkinson’s disease (PD). Oxidized DA forms adducts with proteins which can alter their functionality. αB-crystallin and Hsp27 are intracellular, small heat-shock molecular chaperone proteins (sHsps) which form the first line of defense to prevent protein aggregation under conditions of cellular stress. In vitro, the effects of oxidized DA on the structure and function of αB-crystallin and Hsp27 were investigated. Oxidized DA promoted the cross-linking of αB-crystallin and Hsp27 to form well-defined dimer, trimer, tetramer, etc., species, as monitored by SDS-PAGE. Lysine residues were involved in the cross-links. The secondary structure of the sHsps was not altered significantly upon cross-linking with oxidized DA but their oligomeric size was increased. When modified with a molar equivalent of DA, sHsp chaperone functionality was largely retained in preventing both amorphous and amyloid fibrillar aggregation, including fibril formation of mutant (A53T) α-synuclein, a protein whose aggregation is associated with autosomal PD. In the main, higher levels of sHsp modification with DA led to a reduction in chaperone effectiveness. In vivo, DA is sequestered into acidic vesicles to prevent its oxidation and, intracellularly, oxidation is minimized by mM levels of the antioxidant, glutathione. In vitro, acidic pH and glutathione prevented the formation of oxidized DA-induced cross-linking of the sHsps. Oxidized DA-modified αB-crystallin and Hsp27 were not cytotoxic. In a cellular context, retention of significant chaperone functionality by mildly oxidized DA-modified sHsps would contribute to proteostasis by preventing protein aggregation (particularly of α-synuclein) that is associated with PD.

The ADP/ATP carrier is the 32-kilodalton receptor for an NH2-terminally myristylated src peptide but not for pp60src polypeptide

1993 ◽  
Vol 13 (5) ◽  
pp. 3084-3092
Author(s):  
C T Sigal ◽  
M D Resh
Keyword(s):  
Binding Capacity ◽  
Membrane Binding ◽  
Mitochondrial Fraction ◽  
Fatty Acyl ◽  
Affinity Tag ◽  
Peptide Probe ◽  
The Cross ◽  
Docking Protein

Membrane binding of pp60src is initiated via its myristylated NH2 terminus. To identify a candidate pp60src docking protein or receptor in the membrane, a radiolabelled peptide corresponding to the pp60src NH2-terminal membrane binding domain was cross-linked to fibroblast membranes and found to specifically label a 32-kDa protein. This protein was purified by appending an affinity tag to the peptide probe so that the cross-linked complex could be isolated via affinity chromatography. Microsequencing indicated that the 32-kDa protein was the mitochondrial ADP/ATP carrier (AAC). This result was further confirmed by the ability of an antibody to the AAC to immunoprecipitate the cross-linked complex, by the ability of certain inhibitors of the AAC to block cross-linking, and by membrane fractionation to show that complex formation occurred essentially exclusively in the mitochondrial fraction. While the AAC bound the myristyl-src peptide in a specific manner both in vitro and in vivo, its localization to the inner membrane of the mitochondrion precludes its being a pp60src binding protein. An analysis of pp60v-src binding in vitro was consistent with this expectation. Thus, use of a myristyl-src peptide revealed an unexpected and previously unidentified binding capacity of the AAC, most likely related to the ability of long-chain fatty acyl coenzyme As to serve as AAC inhibitors. The amphipathic nature of the pp60src NH2 terminus suggests alternative strategies for uncovering pp60src membrane binding species.

scholarly journals Active immunization against the proregions of GDF9 or BMP15 alters ovulation rate and litter size in mice

Reproduction ◽  
2012 ◽  
Vol 143 (2) ◽  
pp. 195-201 ◽  
Author(s):  
C Joy McIntosh ◽  
Steve Lawrence ◽  
Peter Smith ◽  
Jennifer L Juengel ◽  
Kenneth P McNatty
Keyword(s):  
Litter Size ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Cross Reactivity ◽  
Full Length ◽  
Ovulation Rate ◽  
Corpora Lutea ◽  
Immunization Group

The transforming growth factor β (TGFB) superfamily proteins bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), are essential for mammalian fertility. Recent in vitro evidence suggests that the proregions of mouse BMP15 and GDF9 interact with their mature proteins after secretion. In this study, we have actively immunized mice against these proregions to test the potential in vivo roles on fertility. Mice were immunized with either N- or C-terminus proregion peptides of BMP15 or GDF9, or a full-length GDF9 proregion protein, each conjugated to keyhole limpet hemocyanin (KLH). For each immunization group, ovaries were collected from ten mice for histology after immunization, while a further 20 mice were allowed to breed and litter sizes were counted. To link the ovulation and fertility data of these two experimental end points, mice were joined during the time period identified by histology as being the ovulatory period resulting in to the corpora lutea (CL) counted. Antibody titers in sera increased throughout the study period, with no cross-reactivity observed between BMP15 and GDF9 sera and antigens. Compared with KLH controls, mice immunized with the N-terminus BMP15 proregion peptide had ovaries with fewer CL (P<0.05) and produced smaller litters (P<0.05). In contrast, mice immunized with the full-length GDF9 proregion not only had more CL (P<0.01) but also had significantly smaller litter sizes (P<0.01). None of the treatments affected the number of antral follicles per ovary. These findings are consistent with the hypothesis that the proregions of BMP15 and GDF9, after secretion by the oocyte, have physiologically important roles in regulating ovulation rate and litter size in mice.

scholarly journals Factor VIII Inhibitors: Von Willebrand Factor Makes A Difference In Vitro and In Vivo

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 709-709
Author(s):  
Qizhen Shi ◽  
Erin L. Kuether ◽  
Jocelyn A. Schroeder ◽  
Crystal L. Perry ◽  
Scot A. Fahs ◽  
...  
Keyword(s):  
Protective Effect ◽  
Hemophilia A ◽  
Inhibitory Antibody ◽  
Chromogenic Assay ◽  
Fviii Activity ◽  
Inhibitory Antibodies ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 709 The important association between von Willebrand factor (VWF) and factor VIII (FVIII) has been investigated for decades, but the effect of VWF on FVIII inhibitors is still controversial. Studies have demonstrated that some anti-FVIII inhibitory antibodies inhibit VWF-FVIII interaction, while others rely on the presence of VWF to inhibit FVIII activities. The influence of VWF on the Bethesda assay, which is routinely used in the clinic to determine the titer of FVIII-neutralizing inhibitors, is still uncertain because the plasma from hemophilia A patients with inhibitors contains normal levels of VWF. To explore the effect of VWF on the reactivity of FVIII inhibitors, we immunized VWF and FVIII double knockout (VWFnullFVIIInull) mice with recombinant human B-domain deleted FVIII (rhFVIII) to induce anti-FVIII inhibitory antibody development. Inhibitory plasma was collected and the titer of inhibitors was determined by Bethesda assay. Murine plasma-derived VWF (from FVIIInull mice) or recombinant human VWF (rhVWF) was used to study the influence of VWF on inhibitor inactivation of FVIII activity (FVIII:C). The remaining FVIII:C after inactivation was determined by chromogenic assay. When inhibitory plasma was incubated with rhFVIII in the presence of 1 U/ml VWF, the residual FVIII activity recovered was higher than in the absence of VWF, resulting in 6.82 ± 1.12 (n = 27) fold lower apparent inhibitor titers. This protective effect is VWF dose dependent. The source of VWF (plasma-derived murine VWF vs. rhVWF) did not affect its protection of FVIII from inhibitor inactivation and VWF does not affect FVIII:C measured in the chromogenic assay in the absence of inhibitors. Interestingly, we found that inhibitor inactivation of FVIII:C in the absence of VWF occurred much faster than in its presence. When the usual 2 hr. incubation at 37°C was omitted from the Bethesda assay, adding rhVWF to rFVIII before mixing with inhibitory plasma resulted in 67.29 ± 20.18 (n = 5) fold lower apparent inhibitor titers than without added VWF. In contrast, if VWF was added to inhibitory plasma first and then mixed with rhFVIII, the inhibitor titers were only 11.04 ± 3.56 (n = 5) fold lower than without added VWF. These results indicate that rhFVIII present in a preformed VWF-FVIII complex is protected from inhibitory antibody inactivation. Conversely, when VWF and inhibitory plasma are added to rhFVIII at the same time, the VWF and inhibitors appear to compete to bind to rhFVIII. Inhibitor titers were lower than in the absence of VWF, but the protective effect is not as efficient as when VWF and rhFVIII were already associated with one another before encountering inhibitors. To confirm the protective effect of VWF on FVIII from inhibitor inactivation, we infused FVIIInull or VWFnullFVIIInull mice with inhibitory plasma and rhFVIII followed by a tail clip survival test. When rhFVIII was infused into FVIIInull mice to 2% followed by inhibitory plasma infusion, all mice with inhibitor titer of 2.5 BU/ml (n = 4) survived tail clipping, and 2 of 4 survived with either 25 BU/ml or 250 BU/ml. If inhibitory plasma was infused first followed by rhFVIII infusion, then only 2 of 6 mice with inhibitor titers of 2.5 BU/ml survived tail clip challenge and none survived with 25 BU/ml and 250 BU/ml. In the first set of mice the infused FVIII was able to form a protective complex with endogenous VWF before encountering inhibitors, while in the second set FVIII is exposed to VWF and pre-infused inhibitory antibodies at the same time, a competitive binding that appears to reduce VWF's protective effect. In contrast, when rhFVIII was infused into VWFnullFVIIInull mice followed by inhibitory plasma infusion, no animals (n = 4 for each group) survived tail clipping with inhibitor titers of 2.5 BU/ml or higher. In summary, our studies demonstrate that VWF exerts a protective effect, reducing inhibitor inactivation of FVIII, both in vitro and in vivo. While the role of VWF in stabilizing plasma FVIII in a milieu rich in proteases has been appreciated for decades, our results indicate that treatment utilizing products containing a complex of FVIII with VWF may be especially beneficial in hemophilia A patients with inhibitors. Disclosures: No relevant conflicts of interest to declare.

N6-methyladenosine residues in an intron-specific region of prolactin pre-mRNA

1990 ◽  
Vol 10 (9) ◽  
pp. 4456-4465
Author(s):  
S M Carroll ◽  
P Narayan ◽  
F M Rottman
Keyword(s):  
Consensus Sequence ◽  
Specific Sequence ◽  
Free System ◽  
Consensus Sequences ◽  
Neplanocin A ◽  
A Cell ◽  
Steady State Levels ◽  
Precursor Rna

N6-methyladenosine (m6A) residues occur at internal positions in most cellular and viral RNAs; both heterogeneous nuclear RNA and mRNA are involved. This modification arises by enzymatic transfer of a methyl group from S-adenosylmethionine to the central adenosine residue in the canonical sequence G/AAC. Thus far, m6A has been mapped to specific locations in eucaryotic mRNA and viral genomic RNA. We have now examined an intron-specific sequence of a modified bovine prolactin precursor RNA for the presence of this methylated nucleotide by using both transfected-cell systems and a cell-free system capable of methylating mRNA transcripts in vitro. The results indicate the final intron-specific sequence (intron D) of a prolactin RNA molecule does indeed possess m6A residues. When mapped to specific T1 oligonucleotides, the predominant site of methylation was found to be within the consensus sequence AGm6ACU. The level of m6A at this site is nonstoichiometric; approximately 24% of the molecules are modified in vivo. Methylation was detected at markedly reduced levels at other consensus sites within the intron but not in T1 oligonucleotides which do not contain either AAC or GAC consensus sequences. In an attempt to correlate mRNA methylation with processing, stably transfected CHO cells expressing augmented levels of bovine prolactin were treated with neplanocin A, an inhibitor of methylation. Under these conditions, the relative steady-state levels of the intron-containing nuclear precursor increased four to six times that found in control cells.

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