Expression and Characterization of a Single-Chain Variable Fragment

Total Page:16

File Type:pdf, Size:1020Kb

Expression and Characterization of a Single-Chain Variable Fragment J. Microbiol. Biotechnol. (2017), 27(5), 965–974 https://doi.org/10.4014/jmb.1702.02007 Research Article Review jmb Expression and Characterization of a Single-Chain Variable Fragment against Human LOX-1 in Escherichia coli and Brevibacillus choshinensis Wei Hu1†, Jun-Yan Xiang1†, Ping Kong1, Ling Liu1, Qiuhong Xie1,2,3*, and Hongyu Xiang1,2,3* 1School of Life Science, 2Key Laboratory for Molecular Enzymology and Engineering, Ministry of Education, 3National Engineering Laboratory for AIDS Vaccine, School of Life Science, Jilin University, Changchun, Jilin 130012, P.R. China Received: February 6, 2017 Revised: February 28, 2017 The single-chain variable fragment (scFv) against lectin-like oxidized low-density lipoprotein Accepted: March 9, 2017 receptor-1 (LOX-1) is a promising molecule for its potential use in the diagnosis and immunotherapy of atherosclerosis. Producing this scFv in several milligram amounts could be First published online the starting point for further engineering and application of the scFv. In this study, the March 9, 2017 abundant expression of the anti-LOX-1 scFv was attempted using Escherichia coli (E. coli) and *Corresponding authors Brevibacillus choshinensis (B. choshinensis). The scFv had limited soluble yield in E. coli, but it Q.X. Phone: +86-431-85153832; was efficiently secreted by B. choshinensis. The optimized fermentation was determined using Fax: +86-431-85153832; the Plackett-Burman screening design and response surface methodology, under which the E-mail: [email protected] yield reached up to 1.5 g/l in a 5-L fermentor. Moreover, the properties of the scFvs obtained H.X. Phone: +86-431-85153832; from the two expression systems were different. The antigen affinity, transition temperature, Fax: +86-431-85153832; and particle diameter size were 1.01E-07 M, 55.2 ± 0.3ºC, and 9.388 nm for the scFv expressed E-mail: [email protected] by B. choshinensis, and 4.53E-07 M, 52.5 ± 0.3ºC, and 13.54 nm for the scFv expressed by E. coli. †These authors contributed This study established an efficient scale-up production methodology for the anti-LOX-1 scFv, equally to this work. which will boost its use in LOX-1-based therapy. pISSN 1017-7825, eISSN 1738-8872 Copyright© 2017 by Keywords: Lectin-like oxidized low-density lipoprotein receptor-1, single-chain variable The Korean Society for Microbiology fragment antibody, Brevibacillus choshinensis, Escherichia coli, production methodology and Biotechnology Introduction (MAbs) [5]. Moreover, LOX-1 can be used for plaque imaging using MAbs and selective delivery of anti-atherosclerotic Lectin-like oxidized low-density lipoprotein receptor-1 agents [6]. (LOX-1), a multi-ligand scavenger receptor that was originally MAbs occupy a large portion of biopharmaceutical identified as the primary receptor for oxLDL, mediates a proteins [7]. There are mainly two disadvantages of using series of proatherogenic cellular responses implicated in the MAb molecules; one is the large molecular size, which the pathogenesis of atherosclerosis-related diseases [1, 2]. limits their penetration into diseased areas, and the other is These diseases continue to be a major cause of morbidity the binding of the crystallizable fragment (Fc) domain of and mortality in the world. LOX-1 basal expression is the MAbs to the cell surface Fc receptor, which limits their relatively low, and it is substantially up-regulated in circulation and mobility [8]. As a substitute for large intact multiple disease states [3]. LOX-1 expressed on the cell MAbs, scFvs have tremendous potential for use in surface can be proteolytically cleaved and released into the diagnosis and targeted therapy because of their specific circulation in a soluble form (sLOX-1). sLOX-1 has proved binding affinity to the antigen, their small size, superior to be a sensitive diagnostic biomarker of vascular pathology biodistribution, and blood clearance; additionally, they can [4]. In addition, recent evidence indicates that targeting be easily engineered or modified [9, 10]. Recently, an anti- LOX-1 is a reliable strategy for the therapy of vascular LOX-1 scFv has been developed for targeting LOX-1 [11], disease and that LOX-1-mediated proatherogenic effects which we believe has rich potential as a LOX-1 targeting can be inhibited by anti-LOX-1 monoclonal antibodies vector for immunodiagnostics, drug delivery, and potential May 2017 ⎪ Vol. 27⎪ No. 5 966 Hu et al. immunotherapy, that requires further research. However, extract, 0.5% beef extract, 0.001% FeSO4·7H2O, 0.001% MnSO4·4H2O, the minor yield of this scFv produced using the Free Style and 0.0001% ZnSO4·7H2O), and LB. 293 expression system limited its further engineering and application. Therefore, it is urgent to develop efficient Plasmid Construction methods for producing this scFv. The anti-LOX-1 scFv was designed on the basis of sequences of the variable heavy (V ) and light (V ) chains of the anti-human ScFv can be produced using a wide range of platforms, H L LOX-1 antibody [11], in the form of V −(Gly Ser) −V with a C- including production in the periplasm of prokaryotes [12]; H 4 3 L terminal peptide encoding the myc (myc) epitope and a in the endoplasmic reticulum of eukaryotes [13]; and in polyhistidine (6×His) metal-binding tag to facilitate the purification cell-free expression systems [14]. Of these expression and immunodetection of the gene product. The gene fragment systems, Escherichia coli is a widely used host that is was chemically synthesized and cloned into a pUC19 vector by suitable for expressing small non-glycosylated recombinant Beijing Genomic Institute (BGI, China), and the plasmid was antibody fragments, including scFv [15, 16]. In recent years, named pUC19-scFv. a promising host, Brevibacillus choshinensis, was developed Plasmid pUC19-scFv was used as the template for the PCR. To for antibody fragment production. Using B. choshinensis, we append the EcoRI and SalI restriction sites for cloning into the have successfully obtained secreted matrix metalloproteinase vectors pET-21a(+) and pET-23a(+), the primer sets 5’-CGG 26 [17], which expressed as inclusion bodies in E. coli hosts. AATTCGAAGTCAAACTGCTGGAATCTGG-3’ (forward) and 5’- In this study, the abundant expression of the anti-LOX-1 GCGTCGACTCATTAGTGGTGGTGATGATGGTGAGC-3’ (reverse) were used. For cloning into the vectors pET-20b(+) and pET- scFv was attempted using E. coli and B. choshinensis. The 27b(+), primer sets 5’-CGGAATTCCGAAGTCAAACTGCTGGAA optimization study was performed using Plackett-Burman TCTGG-3’ (forward) and the same reverse primer as mentioned screening design and the response surface methodology above were used. For cloning into the vector pNCMO2, primer (RSM). After that, the scFvs obtained from the two sets 5’-GCGTCGACGAAGTCAAACTGCTGGAATCTGG-3’ (forward) expression systems were purified using a two-step column and 5’-CGGAATTCTCATTAGTGGTGGTGATGATGGTGAGC-3’ chromatography purification progress. Finally, properties (reverse) were used. The resulting PCR products were digested of the purified anti-LOX-1 scFv proteins were studied. As with EcoRI and SalI, and then they were cloned into the vectors an ideal antibody for use in therapeutic purposes, it must digested using the corresponding restriction enzymes to form the have good pharmaceutical properties, such as antigen affinity expression plasmids. These expression plasmids were correspondingly and thermal stability. Results showed that B. choshinensis named pET21-scFv, pET23-scFv, pET20-scFv, pET27-scFv, and can act as an efficient host for the secreted expression of the pNC-scFv. anti-LOX-1 scFv in abundance. Expression of the Anti-LOX-1 scFv in E. coli Hosts E. coli strain BL21(DE3), Origami, Rosetta-gami 2(DE3), and Materials and Methods Rosetta-gami 2(DE3)pLysS were respectively transformed with the expression plasmids pET21-scFv, pET23-scFv, pET20-scFv, Bacterial Strains, Vectors, and Media and pET27-scFv, and then the transformants were cultured in LB The E. coli strains (Novagen, USA) BL21(DE3), Origami, Rosetta- medium containing the required antibiotic(s). The culture was gami 2(DE3), and Rosetta-gami 2(DE3)pLysS, and B. choshinensis shaken at 37ºC until the cell density (OD600) reached 0.4-0.6. Then, SP3 (Takara, Japan) were used for scFv expression. The vector the induction was conducted by adding 0.5 mM isopropyl-1-thio- pNCMO2, a B. choshinensis-E. coli shuttle vector obtained from β-D-galactopyranoside (IPTG). For optimization, cultivations were Takara, was used for the expression in B. choshinensis. The vectors performed using different IPTG concentrations (0.25, 0.5, 1, or (Novagen) pET-20b(+), pET-21a(+), pET-23a(+), and pET-27b(+) 2 mM) and temperatures (18ºC, 25ºC, 30ºC, or 37ºC). The cells were were used for expressing the scFv in E. coli. harvested by centrifugation, and the cell pellet was resuspended LB medium was used to culture E. coli. B. choshinensis strains in 50 mM sodium phosphate buffer (PBS, pH 7.4) containing 0.2 M used for the expression and secretion of anti-LOX-1 scFv was NaCl. For cell lysis, the cells were homogenized and disrupted by respectively grown in medium 5YC (3% glucose, 3% polypeptone, sonication. The lysates were centrifuged at 27,000 ×g for 30 min at 0.2% yeast extract, 0.01% MgCl ·6H O, 0.01% CaCl ·2H O, 0.001% 2 2 2 2 4ºC. FeSO4·7H2O, 0.001% MnSO4·4H2O, and 0.0001% ZnSO4·7H2O), B2 (0.5% glucose, 1% soytone, 2.5% yeast extract, 2.5% NaCl, and Secreted Expression of Anti-LOX-1 scFv in the B. choshinensis Host 0.1% K HPO ), 2SY (2% glucose, 4% soytone, 0.5% yeast extract, 2 4 B. choshinensis SP3 cells harboring the plasmid pNC-scFv were and 0.015% CaCl ·2H O), BTY (1% glucose, 1% polypeptone, 0.5% 2 2 cultured overnight at 37ºC in 2SY, BTY, LB, 5YC, B2, and/or TM yeast extract, 0.001% FeSO ·7H O, 0.001% MnSO ·4H O, and 0.
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]