DOCSLIB.ORG

Von Willebrand Factor Synthesized by Endothelial Cells from a Patient With

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Von Willebrand Factor Synthesized by Endothelial Cells from a Patient With Proc. Natl. Acad. Sci. USA Vol. 86, pp. 3793-3797, May 1989 Medical Sciences von Willebrand factor synthesized by endothelial cells from a patient with type IIB von Willebrand disease supports platelet adhesion normally but has an increased affinity for platelets PHILIP G. DE GROOT*t, AUGUSTO B. FEDERICIt, HETTY C. DE BOER*, PATRIZIA D'ALESSIO*, PIER M. MANNUCCIt, AND JAN J. SIXMA* *Department of Haematology, University Hospital Utrecht, Catharijnesingel 101, 3511 GV Utrecht, The Netherlands; and tA. Bianchi Bonomi Hemophilia and Thrombosis Center and Institute of Internal Medicine, University of Milano, Milan, Italy Communicated by Kenneth M. Brinkhaus, December 12, 1988 ABSTRACT Endothelial cells were isolated from the um- vWF is synthesized by endothelial cells and megakaryo- bilical vein of a patient with subtype IIB von Willebrand cytes (5, 6). The vWF protein is synthesized with a signal disease, and the biosynthesis and function of von Willebrand peptide (22 amino acids), a precursor part (vWF antigen II, factor (vWF) synthesized by these cells were compared with 741 amino acids), and the mature protein (2050 amino acids) those of vWF synthesized by endothelial cells from normal (7, 8). There are three possible destinations of newly syn- individuals. The patient's endothelial cells synthesized, stored, thesized vWF: secretion into the culture medium ofthe cells, and secreted vWF indistinguishably from normal endothelial incorporation into the extracellular matrix, and storage inside cells: it was synthesized as a prepolypeptide of Mr 270,000 and the cell in the Weibel-Palade bodies (5, 9, 10). The secretion had a mature form of 220,000; the full spectrum of of vWF from the cells is a continuous process through Mr constitutive release, but there is also regulated release, multimers was found both inside the cells and in the culture induced by stimuli, from the Weibel-Palade bodies (11). medium; it was stored normally, in the Weibel-Palade bodies; Multimer assembly takes place during the processing of and similar amounts of vWF were secreted into the medium vWF in the cell (12). Constitutively released vWF consists of and deposited in the extracellular matrix. In a perfusion set-up, intermediate multimerized forms, whereas vWF present in- the extracellular matrix from JIB cells supported platelet side the Weibel-Palade bodies is highly multimerized (13). A adhesion similarly to the matrix from normal cells. vWF number of steps in the multimerization of vWF have been secreted constitutively by JIB cells into the culture medium characterized (14, 15), and abnormalities in posttranslational bound to platelets at concentrations of ristocetin lower than processing could play a role in the pathogenesis of type II those necessary for vWF from normal cells. vWF stored in the vWD. However, to understand the molecular defects under- Weibel-Palade bodies of type JIB cells was released upon lying the different type II vWD subtypes, culture of mutant stimulation with phorbol ester and bound almost completely to endothelial cells from IIB vWD patients is necessary. Levene platelets even in the absence of ristocetin. Moreover, sponta- et al. (16) isolated endothelial cells from a patient with type neous platelet aggregation was induced by vWF synthesized by IIA vWD and showed that this phenotype is caused by type IIB cells. These data support the hypothesis that the increased proteolytic sensitivity ofthe vWF protein. We have absence of highly multimeric forms of vWF in plasma of type isolated endothelial cells from the umbilical cord ofa type IIB IIB von Willebrand disease patients is due to specific removal vWD patient and studied the biosynthesis of vWF by these of these multimers by platelets. cells. The mutant cells synthesized an abnormal vWF mol- ecule with an increased affinity for platelets, but this protein von Willebrand factor (vWF) is a glycoprotein necessary for was normally synthesized, processed, and multimerized by adhesion of platelets to subendothelium after vascular injury the IIB vWD endothelial cells. These data support previous (1). vWF also plays an important role in the interaction suggestions that the absence of high molecular weight mul- between endothelial cells and their matrix (2). Qualitative and timers in plasma may have been caused by selective removal quantitative deficiencies of vWF are associated with an of these multimers by platelets in vivo. autosomal hemorrhagic disorder, von Willebrand disease (vWD) (3). vWD is a bleeding disorder that is heterogeneous MATERIALS AND METHODS in its genetic transmission, its clinical and laboratory mani- festations, and its underlying pathogenetic mechanisms. Description of the Patient. The mother of the newborn vWF is present in plasma as a set ofmultimers with molecular patient was originally diagnosed as type IIB together with x several other affected members ofa large family (17). She has weights of 2-20 106. Analysis of the multimeric structure a severe bleeding disorder characterized by a markedly of vWF with high-resolution agarose gel electrophoretic prolonged bleeding time [25 min; Simplate (General Diag- systems has been used for the differentiation of vWD into nostics, Morris Plains, NJ)], decreased levels ofvWF antigen subtypes. Type II vWD is characterized by the lack of large (45 units/dl) and ristocetin cofactor activity (12 units/dl), vWF multimers in plasma. In a variant form of type II vWD, normal clotting assay (factor VIII procoagulant activity, 65 subtype IIB, there is hyperresponsiveness of platelet-rich units/dl), the absence of high and intermediate vWF multi- plasma to low doses of ristocetin. Patients suffering from mers in plasma, and the presence ofall multimers in platelets. subtype IIB vWD may develop thrombocytopenia, especially Her platelets show an enhanced responsiveness to ristocetin after treatment with 1-desamino-[8-D-arginine]vasopressin. (0.5 mg/ml induced 30% platelet aggregation). In the course In contrast to subtype IIA, in JIB vWD the full spectrum of of her pregnancy the mother developed a thrombocytopenia vWF multimers is present in the platelets of the patients (4). (down to 66,000 platelets per ,l) and spontaneous aggrega- The publication costs of this article were defrayed in part by page charge Abbreviations: vWD, von Willebrand disease; vWF, von Willebrand payment. This article must therefore be hereby marked "advertisement" factor; PMA, phorbol 12-myristate 13-acetate. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. Downloaded by guest on September 29, 2021 3793 3794 Medical Sciences: de Groot et al. Proc. Natl. Acad Sci. USA 86 (1989) tion; spontaneous aggregation and thrombocytopenia disap- Perfusion Studies. After isolation of the endothelial cells peared after delivery. Her child, a girl born by caesarean from the umbilical vein, the arteries were prepared from the delivery, also expressed only the low multimer forms of vWF umbilical cord for studies with an annular perfusion chamber (Fig. 1). as described by Baumgartner (25) with some modifications Cell Culture. Human umbilical vein endothelial cells from (26). Perfusions with steady flow were carried out with this normal individuals were isolated and cultured as described annular perfusion chamber. A rectangular perfusion chamber (18). The cells were grown on culture plastics (Nunc), pre- as described by Sakariassen et al. (27) was used for studies coated with fibronectin, in a culture medium consisting of with glass coverslips coated with the endothelial cell extra- RPMI-1640 (GIBCO) supplemented with 10% human serum, cellular matrix. Perfusions were carried out with washed acidic fibroblast growth factor (19) at 150 heparin human platelets resuspended in human albumin solution, to Ag/ml, which washed and packed erythrocytes were added to a (Organon) at 5 units/ml, and antibiotics. To study platelet hematocrit of 0.4 (26). Platelets were washed as described interaction with endothelial matrices, the cells were subcul- (26). The final platelet count was 120,000 per ,ul. This tured on gelatin-coated glass coverslips. When the cells were perfusate (prewarmed for 5 min at 37°C) was recirculated confluent, the cultures were exposed to 0.1 M NH40H for 30 through the perfusion chamber for the indicated period at the min to isolate the extracellular matrix (20). The matrix-coated indicated shear rate. coverslips were stored in phosphate-buffered saline (PBS) at After perfusion, the artery segments were fixed in glutar- 40C and used for experiments the same day. dialdehyde and embedded in Epon for microscopic evalua- Endothelial cells were metabolically labeled with [31S]me- tion (28). Sections =1 ,um thick were stained with methylene thionine (DuPont/NEN). Cell lysate and medium fractions blue and basic fuchsin. The coverslips with matrix were were prepared and, after preclearing of the fractions with washed and fixed with 0.5% glutardialdehyde and directly gelatin coupled to Sepharose to remove fibronectin, vWF stained with May-Grunwald/Giemsa reagent. Platelet adhe- was immunoprecipitated with an anti-vWF monoclonal an- sion was expressed as percentage ofthe surface covered with tibody coupled to Sepharose (21). (The monoclonal antibody platelets. This was evaluated by en face light microscopy. against vWF was a generous gift ofJ. A. van Mourik, CLB, The light microscope was interfaced with an image analyzer Amsterdam.) To isolate vWF stored inside the cells, endo- (AMS, Saffron Walden, U.K.). thelial cells were stimulated for 1 hr with phorbol 12- Binding Studies. Human platelets were washed (26) and myristate 13-acetate (PMA; Sigma) at 20 ng/ml in RPMI- resuspended in Tyrode buffer (137 mM NaCI/2 mM MgCl2/ 1640. This treatment of the cells released all vWF present 0.42 mM NaH2PO4/11.9 mM NaHCO3/2.9 mM KCI/5.5 mM inside the Weibel-Palade bodies (22). glucose/10 mM Hepes, pH 7.35). Endothelial cell culture Assays. vWF antigen was determined by ELISA (21). The medium (0.25 ml) from normal or IIB cells (untreated or amount of vWF and fibronectin in the extracellular matrix PMA-stimulated) or control or IIB plasma was incubated was determined as described (20).
Load more

Recommended publications
  • Mannose Binding Protein (MBP) Enhances Mononuclear Phagocyte Function Via a Receptor That Contains the 126,000 M, Component of the Clq Receptor
    Immunity, Vol. 3, 485-493, October, 1995, Copyright 0 1995 by Cell Press Mannose Binding Protein (MBP) Enhances Mononuclear Phagocyte Function via a Receptor that Contains the 126,000 M, Component of the Clq Receptor Andrea J. Tenner,’ Susan L. Robinson,7 amino acid sequence. The carboxy half of the MBP mole- and R. Alan B. EzekowitzS cule constitutes a carbohydrate recognition domain (CRD) ‘Department of Molecular Biology and Biochemistry and has features of an ever-growing family of animal C-type University of California lectins (Drickamer, 1987). Irvine, California 92717 The presence of a collagen-like domain in a nonmatrix tAmerican Red Cross polypeptide, while previously somewhat unusual, is now J. H. Holland Laboratory being discovered in an, as of yet, small family of proteins Rockville, Maryland 20855 called collectins (Malhotra et al., 1992; Sastry and Ezeko- *Division of Hematology/Oncology witz, 1993), which contain a region of collagen-like sequence Children’s Hospital (Gly-X-Y) contiguous with recognition domains composed Dana-Farber Cancer Institute of noncollagen-like amino acid sequences (Drickamer et Department of Pediatrics al., 1966; Thiel and Reid, 1989), usually with lectin-like Harvard Medical School properties. In addition to MBP, the other members of this Boston, Massachusetts 02115 family are the pulmonary surfactant proteins, SP-A and SP-D (Shimizu et al., 1992), the classical complement component, Clq, and conglutinin, a protein that binds to Summary the iC3b fragment of complement C3 (Lee et al., 1991; Lachmann, 1967). MBP, SP-A, and Clq all have an inter- Mannose-binding protein (MBP), Clq, the recognition ruption in the Gly-X-Y repeat pattern of the collagen-like component of the classical complement pathway, and sequence (Drickamer et al., 1988), hydroxylated amino pulmonary surfactant protein A (SP-A) are members acids, and short NH,-terminal domains containing in- of a faniily of molecules containing a collagen-like se- terchain disulfide bonds.
    [Show full text]
  • Three-Step Monoclonal Antibody Purification Processes Using Modern Chromatography Media
    Three-step monoclonal antibody purification processes using modern chromatography media Intellectual Property Notice: The Biopharma business of GE Healthcare was acquired by Danaher on 31 March 2020 and now operates under the Cytiva™ brand. Certain collateral materials (such as application notes, scientific posters, and white papers) were created prior to the Danaher acquisition and contain various GE owned trademarks and font designs. In order to maintain the familiarity of those materials for long-serving customers and to preserve the integrity of those scientific documents, those GE owned trademarks and font designs remain in place, it being specifically acknowledged by Danaher and the Cytiva business that GE owns such GE trademarks and font designs. cytiva.com GE and the GE Monogram are trademarks of General Electric Company. Other trademarks listed as being owned by General Electric Company contained in materials that pre-date the Danaher acquisition and relate to products within Cytiva’s portfolio are now trademarks of Global Life Sciences Solutions USA LLC or an affiliate doing business as Cytiva. Cytiva and the Drop logo are trademarks of Global Life Sciences IP Holdco LLC or an affiliate. All other third-party trademarks are the property of their respective owners. © 2020 Cytiva All goods and services are sold subject to the terms and conditions of sale of the supplying company operating within the Cytiva business. A copy of those terms and conditions is available on request. Contact your local Cytiva representative for the most current information. For local office contact information, visit cytiva.com/contact CY13645-21May20-AN GE Healthcare Three-step monoclonal antibody purification processes using modern chromatography media This application note describes monoclonal antibody (MAb) Capto S ImpAct is a strong CIEX medium designed for purification processes using the expanded MAb purification MAb polishing.
    [Show full text]
  • TECH TIP #34 Binding Characteristics of Antibody-Binding Proteins
    TECH TIP #34 Binding characteristics of antibody-binding proteins: Protein A, Protein G, Protein A/G and Protein L TR0034.4 Protein A, G, A/G and L are native and recombinant proteins of microbial origin that bind to mammalian immunoglobulins. Binding specificities and affinities of these proteins differ between source species and antibody subclass. Use the following table to select the antibody-binding protein that is best for your application. Consult the Thermo Scientific Pierce Product catalog or web site for more information and a listing of the many available products based on these proteins. Species Antibody Class Protein A Protein G Protein A/G Protein L† Human Total IgG S S S S† IgG1 S S S S† Binding Strength: IgG2 S S S S† IgG3 W S S S† W = weak IgG4 S S S S† M = medium IgM W NB W S† S = strong IgD NB NB NB S† NB = no binding IgA W NB W S† ? = unknown Fab W W W S† ScFv W NB W S† Mouse Total IgG S S S S† IgM NB NB NB S† IgG1 W M M S† IgG2a S S S S† IgG2b S S S S† IgG3 S S S S† Rat Total IgG W M M S† IgG1 W M M S† IgG2a NB S S S† IgG2b NB W W S† IgG2c S S S S† Cow Total IgG W S S NB IgG1 W S S NB IgG2 S S S NB Goat Total IgG W S S NB IgG1 W S S NB IgG2 S S S NB Sheep Total IgG W S S NB IgG1 W S S NB IgG2 S S S NB Horse Total IgG W S S ? IgG(ab) W NB W ? IgG(c) W NB W ? IgG(T) NB S S ? Rabbit Total IgG S S S W† Guinea Pig Total IgG S W S ? Hamster Total IgG M M M S† Donkey Total IgG M S S ? Monkey Total IgG S S S ? Pig Total IgG S W S S† Dog Total IgG S W S ? Cat Total IgG S W S ? Chicken Total IgY NB NB NB NB † The stated binding strengths for Protein L refer only to antibody species and subtypes with appropriate kappa light chains.
    [Show full text]
  • Tumor Necrosis Factor-A in Human Milk'
    003 1-399819213101-0029$03.00/0 PEDIATRIC RESEARCH Vol. 31, No. 1, 1992 Copyright O 199 1 International Pediatric Research Foundation, Inc. Printed in U S.A. Tumor Necrosis Factor-a in Human Milk' H. ELIZABETH RUDLOFF, FRANK C. SCHMALSTIEG, JR., AKRAM A. MUSHTAHA, KIMBERLY H. PALKOWETZ, STEPHEN K. LIU, AND ARMOND S. GOLDMAN Departments of Pediatrics, Human Biological Chemistry and Genetics, and Microbiology, University of Texas Medical Branch, Galveston, Texas 77555 ABSTRACT. We previously demonstrated that certain inducing factors in human milk. When blood mononuclear cells biologic activities in human milk were partially blocked by were incubated in whole human colostrum or its whey protein antibodies directed against human tumor necrosis factor-a fraction, the motility of the blood monocytes increased to that (TNF-a). In this study, immunochemical methods were of milk macrophages (8). The activity was abrogated by trypsin used to verify the presence of TNF-a in human milk and considerably decreased by polyclonal antibodies to rhTNF- obtained during the first few days of lactation. Gel filtration a. Three peaks of chemokinetic activity, corresponding to 50- revealed the presence of TNF-a by RIA in molecular 55, 25, and 10-17 kD, were demonstrated by gel filtration weight fractions between 80 and 195 kD. TNF-a could not chromatography. The chemokinetic activity peaks in human be detected consistently by conventional Western blotting milk were blocked by antibodies to rhTNF-a. In addition, the or cytotoxic assays. Although immunoreactive bands were studies suggested that some of the limited cytotoxic activity of detected by a Western blot-lZ5I protein A technique in human milk against murine L-929 cells was blocked by antibod- TNF-a-positive fractions from gel filtration, those bands ies to rhTNF-a (8).
    [Show full text]
  • Purification of Igg Using Protein A- Or Protein G-Agarose
    Purification of IgG Using Protein A- or Protein G-Agarose The following protocol is a simple, reliable method 2. Column and Resin Preparation for purifying total IgG from crude protein mixtures a. Pour 20% Ethanol in the bottom of a petri dish or in a flat bottom container. Float the frit on such as serum or ascites fluid. Protein A binds to the top of the ethanol. Using the large round end of Fc portion of IgG. Protein G binds preferentially to a 1 mL pipette tip, press the frit firmly into the the Fc portion of IgG, but can also bind to the Fab ethanol to force air out. Repeat this step until the frit is completely wet. region, making it useful for purification fo F(ab’)2 b. Push the frit into the barrel of the column until fragments of IgG1. Some species and IgG subtypes bind differentially to Protein A or Protein G. Refer it rests firmly on the bottom. c. With the cap removed, clip the end of the col- to Table 2 for recommendations on which resin to umn to create a hole to allow liquid to flow use. through. d. Wash the frit with 5 column volumes of 1X Procedure: Purification of IgG Molecules Wash/Binding Buffer. e. Prepare a 1/1 suspension of resin in 1X Wash/ 1. Buffer Preparation: Binding buffer. The required amount of agarose Prepare all buffers necessary for this procedure prior per mg immunoglobulin to be purified can be to starting. estimated by the binding capacity. a. Wash/Binding Buffer: Dilute the wash/binding buffer 1:5 in dH2O in a clean container (e.g.
    [Show full text]
  • DNA Unwinding by Replication Protein a Is a Property of the 32 Kda Subunit
    1993 Oxford University Press Nucleic Acids Research, 1993, Vol. 21, No. 16 3659-3665 DNA unwinding by replication protein A is a property of the 70 kDa subunit and is facilitated by phosphorylation of the 32 kDa subunit Anthi Georgaki and Ulrich Hubscher* Department of Veterinary Biochemistry, University of Zurich-lrchel, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland Received May 28, 1993; Revised and Accepted July 1, 1993 ABSTRACT Replication protein A (RP-A) is a heterotrimeric single- three subunits of 70 kDa, 32-34 kDa and 11-14 kDa and binds stranded DNA binding protein with important functions preferentially to ssDNA through its 70 kDa subunit. RP-A in DNA replication, DNA repair and DNA recombination. appears to have an important role in the T antigen dependent We have found that RP-A from calf thymus can unwind generation of a ss region at the SV40 origin prior to initiation DNA in the absence of ATP and MgCI2, two essential of DNA synthesis. This protein is highly conserved since it has cofactors for bona fide DNA helicases (Georgaki, A., been found to have a similar subunit structure and function in Strack, B., Podust, V. and Hubscher, U. FEBS Lett. 308, Saccharomyces cerevisiae [3]. RP-A has a preference to bind 240 - 244, 1992). DNA unwinding by RP-A was found to ss pyrimidine stretches in a stoichiometry of 1 molecule to to be sensitive to MgCI2, ATP, heating and freezing/ 30 bases [2]. In addition, it has direct effects on DNA thawing. Escherichia coil single stranded DNA binding polymerases a and a [4].
    [Show full text]
  • Protein a Product Number P 4931 Storage
    Protein A Product Number P 4931 Storage Temperature 2-8 °C Product Description This product is purified by affinity chromatography pI: 5.11,2 from the culture medium of a Protein-A secreting strain of Staphlococcus aureus. The strain used for Protein A is a highly stable cell surface receptor production of this product is derived from the original produced by several strains of Staphylococcus aureus. strain NCTC 8325.9 Proteases are removed from the It consists of a single polypeptide chain with a supernatant prior to purification on an IgG-agarose molecular weight of 42 kDa, containing four repetitive affinity resin at pH 7.5. All the Protein A in the medium domains rich in aspartic and glutamic acids, but devoid binds to the IgG-agarose affinity resin at this pH. The of cysteine. It contains little or no carbohydrate.1,3 column is washed several times and the Protein A is However, Protein A runs anomalously on SDS-PAGE then eluted from the column with 0.1 M acetic acid, analysis with an observed molecular weight of 55-56 pH 2.4. kDa.1 The powdered product is aseptically filled. Protein A is capable of binding to the Fc portion of immunoglobulins, especially IgG, from a large number Precautions and Disclaimer of species.3 One Protein A molecule has been shown For Laboratory Use Only. Not for drug, household or to bind at least 2 molecules of IgG simultaneously.4 other uses. The IgG binding domain of Protein A consists of three anti-parallel α-helicies, the third of which is disrupted Preparation Instructions when the protein is complexed with the Fc region of This product is soluble in water (1 mg/ml), yielding a the immunoglobulins.
    [Show full text]
  • Protein a from Staphylococcus Aureus
    Protein A from Staphylococcus aureus Product Number P 3963 Storage Temperature 2-8 °C Product Description Protein A also participates in a number of different pI: 5.11,2 protective biological functions including anti-tumor, toxic, and carcinogenic activities. In addition to acting Protein A is a highly stable cell surface receptor as an immunomodulator, it also has antifungal and produced by several strains of Staphylococcus aureus. antiparasitic properties. It consists of a single polypeptide chain with a molecular weight of 42 kDa, containing four repetitive Precautions and Disclaimer domains rich in aspartic and glutamic acids, but devoid For Laboratory Use Only. Not for drug, household or of cysteine. It contains little or no carbohydrate.1,3 other uses. However, Protein A runs anomalously on SDS-PAGE analysis with an observed molecular weight of Preparation Instructions 55-56 kDa.1 This product is soluble in water (1 mg/ml). A pH greater than 8 will denature the Protein A, and the Protein A is capable of binding to the Fc portion of solution stability will be poor. Good solution stability is immunoglobulins, especially IgG, from a large number seen at neutral pH when stored frozen in working of species.3 One Protein A molecule has been shown aliquots. to bind at least 2 molecules of IgG simultaneously.4 The IgG binding domain of Protein A consists of three Storage/Stability anti-parallel α-helicies, the third of which is disrupted Protein A is very stable to heat and denaturing agents, when the protein is complexed with the Fc region of and binds to IgG even after treatment with 4 M urea, the immunoglobulins.
    [Show full text]
  • A Novel Phage-Library-Selected Peptide Inhibits Human TNF-Α Binding to Its Receptors
    Molecules 2014, 19, 7255-7268; doi:10.3390/molecules19067255 OPEN ACCESS molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Article A Novel Phage-Library-Selected Peptide Inhibits Human TNF-α Binding to Its Receptors Jlenia Brunetti 1, Barbara Lelli 1, Silvia Scali 1, Chiara Falciani 2, Luisa Bracci 1,3 and Alessandro Pini 1,3,* 1 Department of Medical Biotechnologies, University of Siena, Via A. Moro 2, 53100 Siena, Italy; E-Mails: [email protected] (J.B.); [email protected] (B.L.); [email protected] (S.S.); [email protected] (L.B.) 2 SetLance srl, via Fiorentina 1, 53100 Siena, Italy; E-Mail: [email protected] 3 Laboratorio di Patologia Clinica, Azienda Ospedaliera Universitaria Senese, 53100 Siena, Italy * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +39-0577-234921. Received: 10 April 2014; in revised form: 22 May 2014 / Accepted: 27 May 2014 / Published: 3 June 2014 Abstract: We report the identification of a new human tumor necrosis factor-alpha (TNF-α) specific peptide selected by competitive panning of a phage library. Competitive elution of phages was obtained using the monoclonal antibody adalimumab, which neutralizes pro-inflammatory processes caused by over-production of TNF-α in vivo, and is used to treat severe symptoms of rheumatoid arthritis. The selected peptide was synthesized in monomeric and branched form and analyzed for binding to TNF-α and competition with adalimumab and TNF-α receptors. Results of competition with TNF-α receptors in surface plasmon resonance and melanoma cells expressing both TNF receptors make the peptide a candidate compound for the development of a novel anti-TNF-α drug.
    [Show full text]
  • Design and Production of Bispecific Antibodies
    antibodies Review Design and Production of Bispecific Antibodies Qiong Wang, Yiqun Chen , Jaeyoung Park , Xiao Liu, Yifeng Hu, Tiexin Wang, Kevin McFarland and Michael J. Betenbaugh * Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA * Correspondence: [email protected] Received: 12 June 2019; Accepted: 31 July 2019; Published: 2 August 2019 Abstract: With the current biotherapeutic market dominated by antibody molecules, bispecific antibodies represent a key component of the next-generation of antibody therapy. Bispecific antibodies can target two different antigens at the same time, such as simultaneously binding tumor cell receptors and recruiting cytotoxic immune cells. Structural diversity has been fast-growing in the bispecific antibody field, creating a plethora of novel bispecific antibody scaffolds, which provide great functional variety. Two common formats of bispecific antibodies on the market are the single-chain variable fragment (scFv)-based (no Fc fragment) antibody and the full-length IgG-like asymmetric antibody. Unlike the conventional monoclonal antibodies, great production challenges with respect to the quantity, quality, and stability of bispecific antibodies have hampered their wider clinical application and acceptance. In this review, we focus on these two major bispecific types and describe recent advances in the design, production, and quality of these molecules, which will enable this important class of biologics to reach their therapeutic potential. Keywords: single-chain variable fragment (scFv); bispecific antibody; quadroma technology; knobs-into-holes; CrossMAb; bispecific T-cell engager (BiTE) 1. Introduction Over recent decades, immunotherapies, including checkpoint inhibitors, adoptive cell transfer, monoclonal antibodies, and vaccine treatments, have become efficient and highly specific treatments to fight cancer by boosting a patient’s immune system.
    [Show full text]
  • Protein L—More Than Just an Affinity Ligand
    processes Review Protein L—More Than Just an Affinity Ligand Stefan Kittler 1,2, Mihail Besleaga 1, Julian Ebner 1,2 and Oliver Spadiut 1,* 1 Research Division Integrated Bioprocess Development, Institute of Chemical, Environmental and Bioscience Engineering, TU Wien, Gumpendorfer Strasse 1a, 1060 Vienna, Austria; [email protected] (S.K.); [email protected] (M.B.); [email protected] (J.E.) 2 Alfred Gruber GmbH, Nordstrasse 6, 5301 Eugendorf, Austria * Correspondence: [email protected]; Tel.: +43-1-58801-166473 Abstract: In the past 30 years, highly specific drugs, known as antibodies, have conquered the biopharmaceutical market. In addition to monoclonal antibodies (mAbs), antibody fragments are successfully applied. However, recombinant production faces challenges. Process analytical tools for monitoring and controlling production processes are scarce and time-intensive. In the downstream process (DSP), affinity ligands are established as the primary and most important step, while the application of other methods is challenging. The use of these affinity ligands as monitoring tools would enable a platform technology to monitor process steps in the USP and DSP. In this review, we highlight the current applications of affinity ligands (proteins A, G, and L) and discuss further applications as process analytical tools. Keywords: monoclonal antibodies; antibody fragments; affinity ligands; process analytical technol- ogy; protein A; protein G; protein L Citation: Kittler, S.; Besleaga, M.; Ebner, J.; Spadiut, O. Protein L—More 1. Introduction Than Just an Affinity Ligand. Processes 2021, 9, 874. https:// In 1986, a new type of drug was approved and introduced to the biopharmaceutical doi.org/10.3390/pr9050874 market—the first monoclonal antibody (mAb) with the trade name Orthoclone (OKT3), preventing rejection after kidney transplantation [1].
    [Show full text]
  • Protein a Chromatography in Monoclonal Antibody Purification By
    Protein A Chromatography in Monoclonal Antibody Purification by Michael Curtis B.S., Arizona State University, 1997 A Report submitted in partial fulfillment of the requirements for the degree MASTER OF SCIENCE Department of Chemical Engineering College of Engineering KANSAS STATE UNIVERSITY Manhattan, Kansas Approved by: Major Professor Dr. James Edgar Copyright © Michael Curtis 2018. Abstract The bio-pharmaceutical industry began over 30 years ago with the production of human insulin and has shown incredible growth ever since. With forecasted annual worldwide sales of over $450B in 2025 for biopharmaceuticals, they are expected to be at least 25% of the entire pharmaceutical market. Therapeutics based on monoclonal antibodies (mAbs) make up roughly a third of all biopharmaceutical sales with indications from asthma, to cancer, to Parkinson’s disease. The recent approval of the first biosimilar mAb products in the US and Europe has exposed up to 20 of the top grossing biologic products to competition for the first time, while 75% of the US market is expected to lose patent exclusivity by 2020. With the increased competition from biosimilars, the costs associated with producing mAb based therapeutics will be a constraint on maintaining market share going forward. The majority of the total manufacturing costs for mAbs resides in the downstream processing where Protein A chromatography is the predominantly employed technology for the primary capture step. With Protein A’s high unit cost of up to $15,000 per liter and susceptibility to deamidification when exposed to high pH cleaning and sanitization chemicals, it is no surprise that many mAb manufacturers are considering alternatives.
    [Show full text]