11 results on '"Lundberg, Dianna"'
Search Results
2. Recombinant human complement component C2 produced in a human cell line restores the classical complement pathway activity in-vitro: an alternative treatment for C2 deficiency diseases
- Author
-
Martini Paolo GV, Cook Lynette C, Alderucci Scott, Norton Angela W, Lundberg Dianna M, Fish Susan M, Langsetmo Knut, Jönsson Göran, Lood Christian, Gullstrand Birgitta, Zaleski Kate J, Savioli Nancy, Lottherand Jason, Bedard Charles, Gill John, Concino Michael F, Heartlein Michael W, Truedsson Lennart, Powell Jan L, and Tzianabos Arthur O
- Subjects
Immunologic diseases. Allergy ,RC581-607 - Abstract
Abstract Background Complement C2 deficiency is the most common genetically determined complete complement deficiency and is associated with a number of diseases. Most prominent are the associations with recurrent serious infections in young children and the development of systemic lupus erythematosus (SLE) in adults. The links with these diseases reflect the important role complement C2 plays in both innate immunity and immune tolerance. Infusions with normal fresh frozen plasma for the treatment of associated disease have demonstrated therapeutic effects but so far protein replacement therapy has not been evaluated. Results Human complement C2 was cloned and expressed in a mammalian cell line. The purity of recombinant human C2 (rhC2) was greater than 95% and it was characterized for stability and activity. It was sensitive to C1s cleavage and restored classical complement pathway activity in C2-deficient serum both in a complement activation ELISA and a hemolytic assay. Furthermore, rhC2 could increase C3 fragment deposition on the human pathogen Streptococcus pneumoniae in C2-deficient serum to levels equal to those with normal serum. Conclusions Taken together these data suggest that recombinant human C2 can restore classical complement pathway activity and may serve as a potential therapeutic for recurring bacterial infections or SLE in C2-deficient patients.
- Published
- 2010
- Full Text
- View/download PDF
3. Generation of FX−/− and Gmds−/−CHOZN host cell lines for the production of afucosylated therapeutic antibodies.
- Author
-
Liu, Weiyi, Padmashali, Roshan, Monzon, Omar Quintero, Lundberg, Dianna, Jin, Shan, Dwyer, Brian, Lee, Yun‐Jung, Korde, Anisha, Park, Sophia, Pan, Clark, and Zhang, Bohong
- Subjects
CHO cell ,CELL lines ,ANTIBODY-dependent cell cytotoxicity ,KILLER cells ,MANUFACTURING cells ,IMMUNOGLOBULINS - Abstract
Antibody‐dependent cellular cytotoxicity (ADCC) is the primary mechanism of actions for several marketed therapeutic antibodies (mAbs) and for many more in clinical trials. The ADCC efficacy is highly dependent on the ability of therapeutic mAbs to recruit effector cells such as natural killer cells, which induce the apoptosis of targeted cells. The recruitment of effector cells by mAbs is negatively affected by fucose modification of N‐Glycans on the Fc; thus, utilization of afucosylated mAbs has been a trend for enhanced ADCC therapeutics. Most of afucosylated mAbs in clinical or commercial manufacturing were produced from Fut8−/− Chinese hamster ovary cells (CHO) host cells, generally generating low yields compared to wildtype CHO host. This study details the generation and characterization of two engineered CHOZN® cell lines, in which the enzyme involved in guanosine diphosphate (GDP)‐fucose synthesis, GDP mannose‐4,6‐dehydratase (Gmds) and GDP‐L‐fucose synthase (FX), was knocked out. The top host cell lines for each of the knockouts, FX−/− and Gmds−/−, were selected based on growth robustness, bulk MSX selection tolerance, production titer, fucosylation level, and cell stability. We tested the production of two proprietary IgG1 mAbs in the engineered host cells, and found that the titers were comparable to CHOZN® cells. The mAbs generated from either KO cell line exhibited loss of fucose modification, leading to significantly boosted FcγRIIIa binding and ADCC effects. Our data demonstrated that both FX−/− and Gmds−/− host cells could replace Fut8−/− CHO cells for clinical manufacturing of antibody therapeutics. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
4. In vitro characterization of an acetylcholine receptor-transferrin fusion protein for the treatment of myasthenia gravis.
- Author
-
Keefe, Dennis, Parng, Chuenlei, Lundberg, Dianna, Ray, Satyajit, Martineau-Bosco, Jennifer, Leng, Charan, Tzartos, Socrates, Powell, Jan, Concino, Michael, Heartlein, Michael, Lamsa, Justin, and Josiah, Serene
- Subjects
MYASTHENIA gravis treatment ,ACETYLCHOLINE ,AUTOANTIBODIES ,NEUROMUSCULAR diseases ,SERUM - Abstract
SHG2210, a fusion protein containing the N-terminus of human nicotinic acetylcholine receptor α (AchR-α; aa1-210) and human transferrin (TF), was characterized as a potential therapeutic for myasthenia gravis (MG) caused predominately by α subunit autoantibodies. SHG2210 was shown to be able to bind to α subunit autoantibodies and the TF receptor (TFR). SHG2210 and SHG2210-anti-AchR antibody complex are internalized through TFR-mediated endocytosis. The SHG2210 and SHG2210-anti-AchR antibody complex is present in Lamp1-positive lysosomal compartments after internalization; however, neither SHG2210 nor SHG2210-antibody complex is present in Rab11-positive recycling endosomes. SHG2210 bound to α subunit of AChR autoantibodies may be cleared by the lysosome, resulting in short cellular half-life relative to SHG2210. SHG2210 is shown to have a protective effect on antigenic modulation of the AChR induced by serum from select patients with MG, suggesting that a fusion protein approach may be an effective therapeutic for treating MG. [ABSTRACT FROM AUTHOR]
- Published
- 2010
- Full Text
- View/download PDF
5. Expression, purification, and characterization of human mannose-6-phosphate receptor – Extra cellular domain from a stable cell line utilizing a small molecule biomimetic of the mannose-6-phosphate moiety.
- Author
-
Dwyer, Brian, Lundberg, Dianna, Iskenderian, Andrea, Strack-Logue, Bettina, Pescatore, Brian, Norton, Angela W., Xu, Jin, Meiyappan, Muthuraman, Concino, Michael F., and Zhang, Bohong
- Subjects
- *
SMALL molecules , *LYSOSOMES , *BIOMIMETIC materials , *INSULIN-like growth factor receptors , *SOMATOMEDIN A , *CELL lines , *SOMATOMEDIN C , *MEMBRANE proteins - Abstract
The cation-independent mannose-6-phosphate receptor (CI-M6PR, aka insulin-like growth factor II receptor or IGFIIR) is a membrane protein that plays a central role in the trafficking of lysosomal acid hydrolases into lysosomes via mannose-6-phosphate (M6P) binding domains. In order to maintain cellular metabolic/catabolic homeostasis, newly synthesized lysosomal acid hydrolases are required to bind to M6PR for transit. Acid hydrolases secreted by cells can also be internalized via M6PR residing on the cell membrane and are transported to the lysosomes, a feature that enables enzyme replacement therapy for the treatment of several lysosomal storage disorders. Therefore, a thorough characterization of this receptor is critical to the development of lysosomal enzyme-based therapeutics that utilize M6PR for drug delivery to the lysosome. However, the extracellular domain (ECD) of M6PR is highly complex, containing 15-mannose receptor homology (MRH) domains. In addition, homodimerization of the receptor can occur at the membrane, making its characterization challenging. In this study, a novel human M6PR (hM6PR)-overexpressing cell line originally established for hM6PR cellular uptake assay was utilized for production of hM6PR-ECD, and a novel small molecule biomimetic (aminophenyl-M6P) affinity resin was developed for the purification of M6PR-ECD. The affinity-purified hM6PR-ECD was monomeric, contained 14 intact MRH domains (1–14) and a partial MRH domain 15, and was successfully employed in ELISA-based and surface plasmon resonance-based binding assays to demonstrate its ligand-binding functionality, making it suitable for the evaluation of biotherapeutics that utilize M6PR for cellular internalization. • A novel hM6PR expressing cell line expresses a soluble form of hM6PR, hM6PR-ECD. • A novel small molecule biomimetic, aminophenyl-M6P, affinity purifies hM6PR-ECD. • The hM6PR-ECD contains 14 intact MRT domains and a partial MRT domain 15 and demonstrated expected M6P binding affinity. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
6. Structural characterization of the α-N-acetylglucosaminidase, a key enzyme in the pathogenesis of Sanfilippo syndrome B.
- Author
-
Birrane, Gabriel, Dassier, Anne-Laure, Romashko, Alla, Lundberg, Dianna, Holmes, Kevin, Cottle, Thomas, Norton, Angela W., Zhang, Bohong, Concino, Michael F., and Meiyappan, Muthuraman
- Subjects
- *
ENZYMES , *CRYSTAL structure , *BLOOD coagulation factor XIII - Abstract
Graphical abstract Highlights • The active site of NAGLU is more open compared to the homolog, CpGH89. • NAGLU has a trimeric arrangement of molecules with outwardly facing active sites. • The structure details a molecular basis for mutations giving rise to MPS IIIB. Abstract Mucopolysaccharidosis III B (MPS III-B) is a rare lysosomal storage disorder caused by deficiencies in Alpha- N -acetylglucosaminidase (NAGLU) for which there is currently no cure, and present treatment is largely supportive. Understanding the structure of NAGLU may allow for identification of novel therapeutic targets for MPS III-B. Here we describe the first crystal structure of human NAGLU, determined to a resolution of 2.3 Å. The crystal structure reveals a novel homotrimeric configuration, maintained primarily by hydrophobic and electrostatic interactions via domain II of three contiguous domains from the N- to C-terminus. The active site cleft is located between domains II and III. Catalytic glutamate residues, E316 and E446, are located at the top of the (α/β) 8 barrel structure in domain II. We utilized the three-dimensional structure of NAGLU to map several MPS III-B mutations, and hypothesize their functional consequences. Revealing atomic level structural information about this critical lysosomal enzyme paves the way for the design of novel therapeutics to target the underlying causes of MPS III-B. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
7. Affinity purification of human alpha galactosidase utilizing a novel small molecule biomimetic of alpha-D-galactose.
- Author
-
Dwyer, Brian, Hu, Jun, Madduri, Ameya, Lundberg, Dianna, Miller, Brian, Gill, John, Meiyappan, Muthuraman, Pan, Clark, Miller, Tom, and Zhang, Bohong
- Subjects
- *
SMALL molecules , *GALACTOSIDASES , *ANGIOKERATOMA corporis diffusum , *MOIETIES (Chemistry) , *LYSOSOMAL storage diseases , *THERMAL stability - Abstract
Alpha galactosidase (a-Gal) is an acidic hydrolase that plays a critical role in hydrolyzing the terminal alpha-galactoyl moiety from glycolipids and glycoproteins. There are over a hundred mutations reported for the GLA gene that encodes a-Gal that result in reduced protein synthesis, protein instability, and reduction in function. The deficiencies of a-Gal can cause Fabry disease, a rare lysosomal storage disorder (LSD) caused by the failure to catabolize alpha- d -galactoyl glycolipid moieties. The current standard of care for Fabry disease is enzyme replacement therapy (ERT) where the purified recombinant form of human a-Gal is given to patients. The manufacture of a-Gal is currently performed utilizing traditional large-scale chromatography processes. Developing an affinity resin for the purification of a-Gal would reduce the complexity of the manufacturing process, reduce costs, and potentially produce a higher quality a-Gal. After the evaluation of many small molecules, a commercially available small molecule biomimetic, N-5-Carboxypentyl-1-deoxygalactonojirimycin (N5C-DGJ), was utilized for the development of a novel small molecule biomimetic affinity resin for a-Gal. Affinity purified a-Gal demonstrated a purity greater than 90%, exhibited expected thermal stability and specific activity. Complementing this affinity purification is the development of an elution buffer system that confers an increased thermal stability to a-Gal. The N5C-DGJ affinity resin tolerated sodium hydroxide sanitization with no loss of binding capacity, making it amenable to large scale purification processes and potential use in manufacturing. This novel method for purifying the challenging a-Gal enzyme can be extended to other enzyme replacement therapies. • A novel small molecule biomimetic, N5C-DGJ, affinity purifies human a-Gal. • Elution buffer system effectively elutes a-Gal while conferring increased thermal stability. • The N5C-DGJ affinity resin is amenable to large scale purification processes and potential use in manufacturing. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
8. VEGFR-1/Flt-1 inhibition increases angiogenesis and improves muscle function in a mouse model of Duchenne muscular dystrophy.
- Author
-
Bosco J, Zhou Z, Gabriëls S, Verma M, Liu N, Miller BK, Gu S, Lundberg DM, Huang Y, Brown E, Josiah S, Meiyappan M, Traylor MJ, Chen N, Asakura A, De Jonge N, Blanchetot C, de Haard H, Duffy HS, and Keefe D
- Abstract
Duchenne muscular dystrophy is characterized by structural degeneration of muscle, which is exacerbated by localized functional ischemia due to loss of nitric oxide synthase-induced vasodilation. Treatment strategies aimed at increasing vascular perfusion have been proposed. Toward this end, we have developed monoclonal antibodies (mAbs) that bind to the vascular endothelial growth factor (VEGF) receptor VEGFR-1 (Flt-1) and its soluble splice variant isoform (sFlt-1) leading to increased levels of free VEGF and proangiogenic signaling. The lead chimeric mAb, 21B3, had high affinity and specificity for both human and mouse sFlt-1 and inhibited VEGF binding to sFlt-1 in a competitive manner. Proof-of-concept studies in the mdx mouse model of Duchenne muscular dystrophy showed that intravenous administration of 21B3 led to elevated VEGF levels, increased vascularization and blood flow to muscles, and decreased fibrosis after 6-12 weeks of treatment. Greater muscle strength was also observed after 4 weeks of treatment. A humanized form of the mAb, 27H6, was engineered and demonstrated a comparable pharmacologic effect. Overall, administration of anti-Flt-1 mAbs in mdx mice inhibited the VEGF:Flt-1 interaction, promoted angiogenesis, and improved muscle function. These studies suggest a potential therapeutic benefit of Flt-1 inhibition for patients with Duchenne muscular dystrophy., Competing Interests: J.B., Z.Z., N.L., B.K.M., S. Gu, D.M.L., Y.H., E.B., M.M., N.C., and H.S.D. are full-time employees of Shire Human Genetic Therapies, a Takeda company, and own stock/options in Takeda. S.J., M.J.T., and D.K. were employees of Shire Human Genetic Therapies, a Takeda company, at the time of these studies. S. Gabriëls, N.D.J., C.B., and H.d.H. are full-time employees of and own stock/options in argenx. A.A. received research support from Shire Human Genetic Therapies. M.V. declares no competing interests., (© 2021 Shire Human Genetic Therapies.)
- Published
- 2021
- Full Text
- View/download PDF
9. Generation of FX -/- and Gmds -/- CHOZN host cell lines for the production of afucosylated therapeutic antibodies.
- Author
-
Liu W, Padmashali R, Monzon OQ, Lundberg D, Jin S, Dwyer B, Lee YJ, Korde A, Park S, Pan C, and Zhang B
- Subjects
- Amino Acid Sequence, Animals, Antibodies, Monoclonal immunology, Antibody-Dependent Cell Cytotoxicity, Base Sequence, CHO Cells, CRISPR-Cas Systems, Carbohydrate Epimerases genetics, Carbohydrate Epimerases metabolism, Cricetinae, Cricetulus, Glycosylation, Humans, Hydro-Lyases genetics, Hydro-Lyases metabolism, Immunoglobulin G immunology, Ketone Oxidoreductases genetics, Ketone Oxidoreductases metabolism, Receptors, IgG metabolism, Antibodies, Monoclonal biosynthesis, Carbohydrate Epimerases antagonists & inhibitors, Fucose metabolism, Guanosine Diphosphate metabolism, Hydro-Lyases antagonists & inhibitors, Ketone Oxidoreductases antagonists & inhibitors
- Abstract
Antibody-dependent cellular cytotoxicity (ADCC) is the primary mechanism of actions for several marketed therapeutic antibodies (mAbs) and for many more in clinical trials. The ADCC efficacy is highly dependent on the ability of therapeutic mAbs to recruit effector cells such as natural killer cells, which induce the apoptosis of targeted cells. The recruitment of effector cells by mAbs is negatively affected by fucose modification of N-Glycans on the Fc; thus, utilization of afucosylated mAbs has been a trend for enhanced ADCC therapeutics. Most of afucosylated mAbs in clinical or commercial manufacturing were produced from Fut8
-/- Chinese hamster ovary cells (CHO) host cells, generally generating low yields compared to wildtype CHO host. This study details the generation and characterization of two engineered CHOZN® cell lines, in which the enzyme involved in guanosine diphosphate (GDP)-fucose synthesis, GDP mannose-4,6-dehydratase (Gmds) and GDP-L-fucose synthase (FX), was knocked out. The top host cell lines for each of the knockouts, FX-/- and Gmds-/-, were selected based on growth robustness, bulk MSX selection tolerance, production titer, fucosylation level, and cell stability. We tested the production of two proprietary IgG1 mAbs in the engineered host cells, and found that the titers were comparable to CHOZN® cells. The mAbs generated from either KO cell line exhibited loss of fucose modification, leading to significantly boosted FcγRIIIa binding and ADCC effects. Our data demonstrated that both FX-/- and Gmds-/- host cells could replace Fut8-/- CHO cells for clinical manufacturing of antibody therapeutics., (© 2020 Takeda Pharmaceutical Company Limited. Biotechnology Progress published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)- Published
- 2021
- Full Text
- View/download PDF
10. Protein Engineering on Human Recombinant Follistatin: Enhancing Pharmacokinetic Characteristics for Therapeutic Application.
- Author
-
Shen C, Iskenderian A, Lundberg D, He T, Palmieri K, Crooker R, Deng Q, Traylor M, Gu S, Rong H, Ehmann D, Pescatore B, Strack-Logue B, Romashko A, Baviello G, Gill J, Zhang B, Meiyappan M, Pan C, and Norton AW
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Animals, Female, Follistatin metabolism, Follistatin therapeutic use, Glycosylation, Heparin metabolism, Humans, Mice, Mutation, Recombinant Proteins metabolism, Recombinant Proteins therapeutic use, Tissue Distribution, Follistatin genetics, Follistatin pharmacokinetics, Protein Engineering, Recombinant Proteins genetics, Recombinant Proteins pharmacokinetics
- Abstract
Follistatin (FS) is an important regulatory protein, a natural antagonist for transforming growth factor-β family members activin and myostatin. The diverse biologic roles of the activin and myostatin signaling pathways make FS a promising therapeutic target for treating human diseases exhibiting inflammation, fibrosis, and muscle disorders, such as Duchenne muscular dystrophy. However, rapid heparin-mediated hepatic clearance of FS limits its therapeutic potential. We targeted the heparin-binding loop of FS for site-directed mutagenesis to improve clearance parameters. By generating a series of FS variants with one, two, or three negative amino acid substitutions, we demonstrated a direct and proportional relationship between the degree of heparin-binding affinity in vitro and the exposure in vivo. The triple mutation K(76,81,82)E abolished heparin-binding affinity, resulting in ∼20-fold improved in vivo exposure. This triple mutant retains full functional activity and an antibody-like pharmacokinetic profile, and shows a superior developability profile in physical stability and cell productivity compared with FS variants, which substitute the entire heparin-binding loop with alternative sequences. Our surgical approach to mutagenesis should also reduce the immunogenicity risk. To further lower this risk, we introduced a novel glycosylation site into the heparin-binding loop. This hyperglycosylated variant showed a 10-fold improved exposure and decreased clearance in mice compared with an IgG1 Fc fusion protein containing the native FS sequence. Collectively, our data highlight the importance of improving pharmacokinetic properties by manipulating heparin-binding affinity and glycosylation content and provide a valuable guideline to design desirable therapeutic FS molecules., (Copyright © 2018 The Author(s).)
- Published
- 2018
- Full Text
- View/download PDF
11. Expression and purification methods for the production of recombinant human complement component C2.
- Author
-
Norton AW, Martini PG, Cook LC, Alderucci S, Lundberg DM, Fish SM, Bedard C, Gill J, Tzianabos AO, and Concino MF
- Subjects
- Cell Line, Chromatography, High Pressure Liquid, Complement C2 chemistry, Complement C2 isolation & purification, Electrophoresis, Polyacrylamide Gel, Enzyme-Linked Immunosorbent Assay, Humans, Complement C2 biosynthesis, Complement C2 genetics, Gene Expression, Recombinant Proteins
- Abstract
Human complement component C2 is a critical factor of the classical complement pathway. Here we provide a method for the production of recombinant human C2 (rhC2) protein for research purposes. The human complement component C2 (hC2) is cloned from a human cDNA library by polymerase chain reaction and inserted in a mammalian expression vector (Martini et al., BMC Immunol 11:43, 2010). Transient transfection is utilized to express hC2 in a mammalian cell line, and the expressed C2 is harvested from the conditioned media. rhC2 is purified from the conditioned media by sequential steps of cation exchange and affinity column chromatography. The purified hC2 is characterized for protein purity, stability, and enzymatic activity. The recombinant hC2 activity is tested in a complement activation ELISA assay that measures classical, alternative, and lectin complement pathway activity in C2-depleted serum.
- Published
- 2014
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.