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1.
Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding. 总被引:3,自引:0,他引:3
Single chain variable fragments (scFv) of the murine monoclonal antibody HD37 specific to human B-cell antigen CD19 were constructed by joining the VH and VL domains with linkers of 18, 10, 1 and 0 residues. ScFv-18 formed monomers, dimers and small amounts of tetramers; scFv-10 formed dimers and small amounts of tetramers; scFv-1 formed exclusively tetramers; scFv-0 formed exclusively trimers. The affinities of the scFv-10 (diabody) and scFv-1 (tetrabody) were approximately 1.5- and 2.5-fold higher, respectively, than that of the scFv-0 (triabody). The tetrabody displayed a significantly prolonged association with cell-bound antigen (t1/2 cell surface retention at 37 degrees C of 26.6 min) compared to both the diabody (13.3 min) and triabody (6.7 min). This increase in avidity of the tetrabody combined with its larger size could prove to be particularly advantageous for imaging and the immunotherapy of B-cell malignancies. 相似文献
2.
Saurabh Jain Luca Aresu Stefano Comazzi Jianguo Shi Erin Worrall John Clayton William Humphries Sandra Hemmington Paul Davis Euan Murray Asmare A. Limeneh Kathryn Ball Eva Ruckova Petr Muller Borek Vojtesek Robin Fahraeus David Argyle Ted R. Hupp 《PloS one》2016,11(2)
Monoclonal antibodies are leading agents for therapeutic treatment of human diseases, but are limited in use by the paucity of clinically relevant models for validation. Sporadic canine tumours mimic the features of some human equivalents. Developing canine immunotherapeutics can be an approach for modeling human disease responses. Rituximab is a pioneering agent used to treat human hematological malignancies. Biologic mimics that target canine CD20 are just being developed by the biotechnology industry. Towards a comparative canine-human model system, we have developed a novel anti-CD20 monoclonal antibody (NCD1.2) that binds both human and canine CD20. NCD1.2 has a sub-nanomolar Kd as defined by an octet red binding assay. Using FACS, NCD1.2 binds to clinically derived canine cells including B-cells in peripheral blood and in different histotypes of B-cell lymphoma. Immunohistochemical staining of canine tissues indicates that the NCD1.2 binds to membrane localized cells in Diffuse Large B-cell lymphoma, Marginal Zone Lymphoma, and other canine B-cell lymphomas. We cloned the heavy and light chains of NCD1.2 from hybridomas to determine whether active scaffolds can be acquired as future biologics tools. The VH and VL genes from the hybridomas were cloned using degenerate primers and packaged as single chains (scFv) into a phage-display library. Surprisingly, we identified two scFv (scFv-3 and scFv-7) isolated from the hybridoma with bioactivity towards CD20. The two scFv had identical VH genes but different VL genes and identical CDR3s, indicating that at least two light chain mRNAs are encoded by NCD1.2 hybridoma cells. Both scFv-3 and scFv-7 were cloned into mammalian vectors for secretion in CHO cells and the antibodies were bioactive towards recombinant CD20 protein or peptide. The scFv-3 and scFv-7 were cloned into an ADEPT-CPG2 bioconjugate vector where bioactivity was retained when expressed in bacterial systems. These data identify a recombinant anti-CD20 scFv that might form a useful tool for evaluation in bioconjugate-directed anti-CD20 immunotherapies in comparative medicine. 相似文献
3.
Dr Jean-Charles Cailliez Cristina Cantelli Nathalie Séguy Stefania Conti Mara Gerloni Giulia Morace Luciano Polonelli 《Mycopathologia》1994,126(3):173-177
A secreted killer toxin was detected through the cell wall ofPichia anomala cells by ultrastructural immunodetection with a specific monoclonal antibody (MAb KT4). MAb KT4 was successively detected by colloidal gold labeled streptavidin and biotinylated anti-mouse F(ab')2 antibodies. The antigenic determinants of the toxin were localized throughout the cytoplasm and the cell wall of killer yeast cells. The Lowicryl K4M-immunogold method gave very satisfactory results and showed that the killer toxin was somewhat concentrated in the yeast cell wall layers before being exported into the medium. In agreement with previous reports, the binding of MAb KT4 suggested that theP. anomala killer toxin secretion did not result from a homogeneous diffusion across the yeast cell wall.Abbreviations G15
gold particles of 15 nm
- IEM
immunoelectron microscopy
- IFA
immunofluorescence assay
- MAb
monoclonal antibody
- PBS
phosphate buffered saline
- SAM/F(ab)2
sheep antibodies anti-mouse F(ab)2
- SBB
Sabouraud buffered broth 相似文献
4.
Pharmacokinetics and biodistribution of a light-chain-shuffled CC49 single-chain Fv antibody construct 总被引:5,自引:0,他引:5
Pavlinkova G Colcher D Booth BJ Goel A Batra SK 《Cancer immunology, immunotherapy : CII》2000,49(4-5):267-275
Murine monoclonal antibodies to tumor-associated glycoprotein 72 (anti-TAG-72 mAb B72.3 and CC49) are among the most extensively
studied mAb for immunotherapy of adenocarcinomas. They have been used clinically to localize primary and metastatic tumor
sites; however, murine mAb generally induce potent human anti-(mouse antibody) responses. The immunogenicity of murine mAb
can be minimized by genetic humanization of these antibodies, where non-human regions are replaced by the corresponding human
sequences or complementary determining regions are grafted into the human framework regions. We have developed a humanized
CC49 single-chain antibody construct (hu/muCC49 scFv) by replacing the murine CC49 variable light chain with the human subgroup
IV germline variable light chain (Hum4 VL). The major advantages of scFv molecules are their excellent penetration into the tumor tissue, rapid clearance rate, and
much lower exposure to normal organs, especially bone marrow, than occur with intact antibody. The biochemical properties
of hu/muCC49 scFv were compared to those of the murine CC49 scFv (muCC49 scFv). The association constants (K
a) for hu/muCC49 and muCC49 constructs were 1.1 × 106 M−1 and 1.4 × 106 M−1 respectively. Pharmacokinetic studies in mice showed similar rapid blood and whole-body clearance with a half-life of 6 min
for both scFv. The biodistribution studies demonstrated equivalent tumor targeting to human colon carcinoma xenografts for
muCC49 and hu/muCC49 scFv. These results indicate that the human variable light-chain subgroup IV can be used for the development
of humanized or human immunoglobulin molecules potentially useful in both diagnostic and therapeutic applications with TAG-72-positive
tumors.
Received: 29 December 1999 / Accepted: 4 February 2000 相似文献
5.
Wongi Min Jin-Kyoo Kim Hyun Soon Lillehoj Eun Jung Sohn Jae Yong Han Ki Duk Song Erik Peter Lillehoj 《Biotechnology letters》2001,23(12):949-955
The chicken monoclonal antibody (mAb), 6D-12-G10, reacts with an apical complex protein at the anterior tip of E. acervulina sporozoites that inhibits parasite invasion in vitro. Because this mAb was produced at low amount from the original hybridoma cells, an scFv antibody was constructed by amplification of the corresponding V
H and V
L genes and expressed in E. coli. The scFv antibody was produced at a minimum of 7 mg l–1 and exhibited virtually identical antigen reactivity as the original mAb. 相似文献
6.
Simultaneous measurements of chlorophyll (Chl) fluorescence and CO2 assimilation (A) in Vicia faba leaves were taken during the first weeks of growth to evaluate the protective effect of 24-epibrassinolide (EBR) against
damage caused by the application of the herbicide terbutryn (Terb) at pre-emergence. V. faba seeds were incubated for 24 h in EBR solutions (2 × 10−6 or 2 × 10−5 mM) and immediately sown. Terb was applied at recommended doses (1.47 or 1.96 kg ha−1) at pre-emergence. The highest dose of Terb strongly decreased CO2 assimilation, the maximum quantum yield of PSII photochemistry in the dark-adapted state (F
V/F
M), the nonphotochemical quenching (NPQ), and the effective quantum yield (ΔF/F′M) during the first 3–4 weeks after plant emergence. Moreover, Terb increased the basal quantum yield of nonphotochemical processes
(F
0/F
M), the degree of reaction center closure (1 − q
p), and the fraction of light absorbed in PSII antennae that was dissipated via thermal energy dissipation in the antennae
(1 − F′V/F′M). The herbicide also significantly reduced plant growth at the end of the experiment as well as plant length, dry weight,
and number of leaves. The application of EBR to V. faba seeds before sowing strongly diminished the effect of Terb on fluorescence parameters and CO2 assimilation, which recovered 13 days after plant emergence and showed values similar to those of control plants. The protective
effect of EBR on CO2 assimilation was detected at a photosynthetic photon flux density (PFD) of 650 μmol m−2 s−1 and the effect on ΔF/F′M and photosynthetic electron transport (J) was detected under actinic lightings up to 1750 μmol m−2 s−1. The highest dose of EBR also counteracted the decrease in plant growth caused by Terb, and plants registered the same growth
values as controls. 相似文献
7.
Jonathan Hurtado Jake Stahnke Anja Fuchs Erin Mehlhop Sergey Gorlatov Andreas Loos Michael S. Diamond Qiang Chen 《Plant biotechnology journal》2014,12(8):1098-1107
Previously, our group engineered a plant‐derived monoclonal antibody (MAb pE16) that efficiently treated West Nile virus (WNV) infection in mice. In this study, we developed a pE16 variant consisting of a single‐chain variable fragment (scFv) fused to the heavy chain constant domains (CH) of human IgG (pE16scFv‐CH). pE16 and pE16scFv‐CH were expressed and assembled efficiently in Nicotiana benthamiana ?XF plants, a glycosylation mutant lacking plant‐specific N‐glycan residues. Glycan analysis revealed that ?XF plant‐derived pE16scFv‐CH (?XFpE16scFv‐CH) and pE16 (?XFpE16) both displayed a mammalian glycosylation profile. ?XFpE16 and ?XFpE16scFv‐CH demonstrated equivalent antigen‐binding affinity and kinetics, and slightly enhanced neutralization of WNV in vitro compared with the parent mammalian cell‐produced E16 (mE16). A single dose of ?XFpE16 or ?XFpE16scFv‐CH protected mice against WNV‐induced mortality even 4 days after infection at equivalent rates as mE16. This study provides a detailed tandem comparison of the expression, structure and function of a therapeutic MAb and its single‐chain variant produced in glycoengineered plants. Moreover, it demonstrates the development of anti‐WNV MAb therapeutic variants that are equivalent in efficacy to pE16, simpler to produce, and likely safer to use as therapeutics due to their mammalian N‐glycosylation. This platform may lead to a more robust and cost‐effective production of antibody‐based therapeutics against WNV infection and other infectious, inflammatory or neoplastic diseases. 相似文献
8.
Activation of a Refolded, Berberine-specific, Single-chain Fv Fragment by Addition of Free Berberine
A single-chain variable fragment (scFv) specific for berberine was produced in Escherichia coli. The anti-berberine scFv gene was cloned from hybridoma 1D5-3B-7 producing the monoclonal antibody. The variable regions of the heavy (VH) and light chain (VL) genes were connected with a flexible linker using an assembly PCR. The VH-linker-VL gene was inserted into a plasmid, pET28a (+), then overexpressed in E. coli BL21 (DE3). The active of the scFv by refolding based on stepwise dialysis methods and an artificial chaperone was determined by direct and competitive enzyme-linked immunosorbent assay (ELISA). The results of direct ELISA showed that the anti-berberine scFv retained specific binding activity to berberine. In competitive ELISA, however, activity was increased depending on the concentration of berberine. 相似文献
9.
Huston James S. Adams Gregory P. McCartney John E. Tai Mei-Sheng Hudziak Robert M. Oppermann Hermann Stafford Walter F. Liu Sen Fand Irwin Apell Gerald Laminet Axel Bookman Michael A. Houston L. L. Weiner Louis M. 《Cell biochemistry and biophysics》1994,24(1-3):267-278
This investigation has utilized novel forms of the single-chain Fv (sFv), wherein a cysteine-containing peptide has been fused
to the sFv carboxyl terminus to facilitate disulfide bonding or specific crosslinking of this sFv′ to make divalent (sFv′)2. The 741F8 anti-c-erbB-2 monoclonal antibody was used as the basis for construction of 741F8 sFv, from which the sFv′ and (sFv′)2 derivatives were prepared. Recombinant c-erbB-2 extracellular domain (ECD) was prepared in CHO cells and the bivalency of 741F8 (sFv′)2 demonstrated by its complex formation with ECD. The tumor binding properties of125I-labeled anti-c-erbB-2 741F8 sFv, sFv′, and (sFv′)2 were compared with radiolabeled antidigoxin 26-10 sFv′ and (sFv′)2 controls. Following intravenous administration of radiolabeled species to severe combined immune-deficient (SCID) mice bearing
SK-OV-3 tumors (which overexpress c-erbB-2), blood and organ samples were obtained as a function of time over 24 h. Comparative analysis of biodistribution and tumor-to-organ
ratios demonstrated the 741F8 sFv, sFv′, and (sFv′)2 had excellent specificity for tumors, which improved with time after injection. This contrasted with nonspecific interstitial
pooling in tumors observed with the 26-10 sFv, sFv′, and (sFv′)2, which decreased with time after administration. Tumor localization was significantly better for disulfide or peptide crosslinked
741F8 (sFv′)2 having Gly4Cys tails than for monovalent 741F8 sFv′ or Fab. The superior properties of the 741F8 (sFv′)2 in targeting SK-OV-3 tumors in SCID mice suggests the importance of further investigations of divalent sFv analogs for immunotargeting. 相似文献
10.
A UDP-glucose: anthocyanin 3′,5′-O-glucosyltransferase (UA3′5′GT) (EC 2.4.1.-) was purified from the petals of Clitoria ternatea L. (Phaseoleae), which accumulate polyacylated anthocyanins named ternatins. In the biosynthesis of ternatins, delphinidin
3-O-(6″-O-malonyl)-β-glucoside (1) is first converted to delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′-O-β-glucoside (2). Then 2 is converted to ternatin C5 (3), which is delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′,5′-di-O-β-glucoside. UA3′5′GT is responsible for these two steps by transferring two glucosyl groups in a stepwise manner. Its substrate
specificity revealed the regioselectivity to the anthocyanin′s 3′- or 5′-OH groups. Its kinetic properties showed comparable
k
cat values for 1 and 2, suggesting the subequality of these anthocyanins as substrates. However, the apparent K
m value for 1 (3.89 × 10−5 M), which is lower than that for 2 (1.38 × 10−4 M), renders the k
cat/K
m value for 1 smaller, making 1 catalytically more efficient than 2. Although the apparent K
m value for UDP-glucose (6.18 × 10−3 M) with saturated 2 is larger than that for UDP-glucose (1.49 × 10−3 M) with saturated 1, the k
cat values are almost the same, suggesting the UDP-glucose binding inhibition by 2 as a product. UA3′5′GT turns the product 2 into a substrate possibly by reversing the B-ring of 2 along the C2-C1′ single bond axis so that the 5′-OH group of 2 can point toward the catalytic center.
K. Kogawa, N. Kato, K. Kazuma, and N. Noda contributed equally to this work. 相似文献
11.
Han-Lin Liu Wei-Fang Lin Wen-Chi Hu Yung-An Lee Ya-Chun Chang 《Applied and environmental microbiology》2015,81(19):6839-6849
Potyviruses are major pathogens that often cause mixed infection in calla lilies. To reduce the time and cost of virus indexing, a detection method for the simultaneous targeting of multiple potyviruses was developed by generating a broad-spectrum monoclonal antibody (MAb) for detecting the greatest possible number of potyviruses. The conserved 121-amino-acid core regions of the capsid proteins of Dasheen mosaic potyvirus (DsMV), Konjak mosaic potyvirus (KoMV), and Zantedeschia mild mosaic potyvirus (ZaMMV) were sequentially concatenated and expressed as a recombinant protein for immunization. After hybridoma cell fusion and selection, one stable cell line that secreted a group-specific antibody, named C4 MAb, was selected. In the reaction spectrum test, the C4 MAb detected at least 14 potyviruses by indirect enzyme-linked immunosorbent assay (I-ELISA) and Western blot analysis. Furthermore, the variable regions of the heavy (VH) and light (VL) chains of the C4 MAb were separately cloned and constructed as single-chain variable fragments (scFvs) for expression in Escherichia coli. Moreover, the pectate lyase E (PelE) signal peptide of Erwinia chrysanthemi S3-1 was added to promote the secretion of C4 scFvs into the medium. According to Western blot analysis and I-ELISA, the soluble C4 scFv (VL-VH) fragment showed a binding specificity similar to that of the C4 MAb. Our results demonstrate that a recombinant protein derived from fusion of the conserved regions of viral proteins has the potential to produce a broad-spectrum MAb against a large group of viruses and that the PelE signal peptide can improve the secretion of scFvs in E. coli. 相似文献
12.
We have earlier described a haemagglutination-based assay for on-site detection of antibodies to HIV using whole blood. The reagent in this assay comprises of monovalent Fab fragment of an anti-human RBC antibody fused to immunodominant antigens of HIV-1 and HIV-2. In the present work, we describe a rational and systematic method for directed evolution of scFv and Fab antihuman RBC antibody fragments. Based on homology modeling and germline sequence alignments of antibodies, target residues in the anti-RBC MAb B6 sequence were identified for mutagenesis. A combinatorial library of 107 clones was constructed and subjected to selection on whole RBC under competitive binding conditions to identify several phage-displayed B6 scFv clones with improved binding as determined in an agglutination assay. Selected VL and VH sequences were shuffled to generate a second generation phage-displayed Fab library which on panning yielded Fab clones with several fold better binding than wild type. The mutants with better binding also displayed more Fab molecules per phage particle indicating improved in vivo folding which was also confirmed by their increased periplasmic secretion compared to the wild type. The mutant Fab molecules also showed superior characteristics in large scale production by in vitro folding of LC and Fd. The biophysical measurements involving thermal and chemical denaturation and renaturation kinetics clearly showed that two of the mutant Fab molecules possessed significantly improved characteristics as compared to the wild type B6 Fab. Structural modelling revealed that B6 Fab mutants had increased hydrogen bonding resulting in increased stability. Our approach provides a novel and useful strategy to obtain recombinant antibodies with improved characteristics.Key words: phage display, antibody maturation, Fab, antibody folding, scFv, agglutination 相似文献
13.
J. J. Joensuu K. D. Brown A. J. Conley A. Clavijo R. Menassa J. E. Brandle 《Transgenic research》2009,18(5):685-696
Low-cost recombinant antibodies could provide a new strategy to control Foot-and-mouth disease virus (FMDV) outbreaks by passive
immunization of susceptible animals. In this study, a single chain variable antibody fragment (scFv) recognizing FMDV coat
protein VP1 was expressed in transgenic tobacco plants. To enhance the accumulation of scFv protein, the codon-usage of a
murine hybridoma-derived scFv gene was adjusted to mimic highly expressed tobacco genes and fused to an elastin-like polypeptide
(ELP) tag. This scFv–ELP fusion accumulated up to 0.8% of total soluble leaf protein in transgenic tobacco. To recover scFv–ELP
protein from the leaf extract, a simple and scalable purification strategy was established. Purified scFv–ELP fusion was cleaved
to separate the scFv portion. Finally, it was shown that the purified scFv proteins retained their capacity to bind the FMDV
in the absence or presence of ELP fusion.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
14.
Denton G Brady K Lo BK Murray A Graves CR Hughes OD Tendler SJ Laughton CA Price MR 《Cancer immunology, immunotherapy : CII》1999,48(1):29-38
A recombinant diabody fragment based on the anti-MUC1 monoclonal antibody, C595 has been produced in a bacterial expression
system. Substitution of a 7-amino-acid linker sequence (Gly6Ser) for the original single-chain (sc)Fv 15-amino-acid linker (Gly4Ser)3, using polymerase-chain-reaction-based strategies, forces variable heavy (VH) and light (VL) domains to pair with complementary domains on neighbouring scFv molecules, forming a scFv dimer (diabody). This recombinant
protein shows similar binding characteristics to the parental C595 monoclonal antibody. The ability to bind to MUC1 mucin
on carcinoma cell surfaces will allow its potential as a diagnostic and therapeutic reagent of clinical utility to be investigated.
Received: 16 September 1998 / Accepted: 2 December 1998 相似文献
15.
Kabir ME Krishnaswamy S Miyamoto M Furuichi Y Komiyama T 《Applied microbiology and biotechnology》2011,90(2):553-564
Phage-display and competitive panning elution leads to the identification of minimum-sized antigen binders together with conventional
antibodies from a mouse cDNA library constructed from HM-1 killer toxin neutralizing monoclonal antibody (nmAb-KT). Antigen-specific
altered camelid-like single-domain heavy chain antibody (scFv K2) and a conventional antibody (scFv K1) have been isolated
against the idiotypic antigen nmAb-KT. The objectives of the study were to examine (1) their properties as compared to conventional
antibodies and also (2) their antifungal activity against different pathogenic and non-pathogenic fungal species. The alternative
small antigen-binder, i.e., the single-domain heavy chain antibody, was originated from a conventional mouse scFv phage library
through somatic hyper-mutation while selection against antigen. This single-domain antibody fragment was well expressed in
bacteria and specifically bound with the idiotypic antigen nmAb-KT and had a high stability and solubility. Experimental data
showed that the binding affinity for this single-domain antibody was 272-fold higher (K
d = 1.07 × 10−10 M) and antifungal activity was three- to fivefold more efficient (IC50 = 0.46 × 10−6 to 1.17 × 10−6 M) than that for the conventional antibody (K
d = 2.91 × 10−8 M and IC50 = 2.14 × 10−6 to 3.78 × 10−6 M). The derived single-domain antibody might be an ideal scaffold for anti-idiotypic antibody therapy and the development
of smaller peptides or peptide mimetic drugs due to their less complex antigen-binding site. We expect that such single-domain
synthetic antibodies will find their way into a number of biotechnological or medical applications. 相似文献
16.
Xiang-sheng Zhou Dian-xing Wu Sheng-quan Shen Jun-wei Sun Qing-yao Shu 《Photosynthetica》2006,44(2):316-319
Comparative analysis revealed that a xantha rice mutant (cv. Huangyu B) had higher ratios of chlorophyll (Chl) a/b and carotenoids/Chl, and higher photosynthetic efficiency than its wild type parent (cv. II32 B). Unexpectedly, the mutant
had higher net photosynthetic rate (P
N) than II32 B. This might have resulted from its lower non-photochemical quenching (qN) but higher maximal photochemical efficiency (FV/FM), higher excitation energy capture efficiency of photosystem 2 (PS2) reaction centres (FV′/FM′), higher photochemical quenching (qP), higher effective PS2 quantum yield (ΦPS2), and higher non-cyclic electron transport rate (ETR). This is the first report of a chlorophyll mutant that has higher photosynthetic
efficiency and main Chl fluorescence parameters than its wild type. This mutant could become a unique material both for the
basic research on photosynthesis and for the development of high yielding rice cultivars. 相似文献
17.
Production and validation of the pharmacokinetics of a single-chain Fv fragment of the MGR6 antibody for targeting of tumors expressing HER-2 总被引:4,自引:0,他引:4
Turatti F Mezzanzanica D Nardini E Luison E Maffioli L Bambardieri E de Lalla C Canevari S Figini M 《Cancer immunology, immunotherapy : CII》2001,49(12):679-686
The HER-2 antigen, which is overexpressed in many breast carcinomas, is an ideal target for monoclonal antibodies due to
its low expression in normal tissue and its homogeneous distribution in the tumor mass. We have developed and characterized
the murine MAb MGR6 against HER-2, which is able to inhibit proliferation of tumor cells overexpressing HER-2. On the basis
of these preclinical results, phase I studies in breast carcinoma patients were conducted and radiolocalization data indicated
an antibody half life which directly paralleled that of other whole antibodies and thus resulting in a limited in vivo diagnostic
capacity. To obtain a smaller reagent with possibly improved in vivo properties, a single chain variable fragment (scFv) of
the original MGR6-producing hybridoma was generated by phage display technology. Biologically active MGR6 scFv was purified
rapidly and at high yield by metal affinity chromatography. Competition FACS and ELISA analyses identified an epitope on the
HER-2 extracellular domain that was shared by the scFv and the parental MAb. BIAcore analysis indicated a Koff of 9.3 × 10−4 s−1, similar to that of the intact MGR6 MAb. Distribution and elimination half-lives of MGR6 scFv, calculated from in vivo preclinical
evaluations, were much faster (13 min and 6.2 h, respectively) than previously published results for the intact MAb (mean
t1/2β of 46 h). This represents a theoretical improvement in pharmacokinetics with respect to the parental murine MAb and
points to the potential for utilizing this fragment in redirecting therapeutic agents, such as radioisotopes, to different
human carcinomas overexpressing HER-2.
Received: 10 August 2000 / Accepted: 19 October 2000 相似文献
18.
《Gene》1997,189(1):83-88
Increased levels of plasminogen activator inhibitor-1 (PAI-1) are a well-known risk for cardiovascular diseases. A significant number of investigations are aimed at lowering plasma levels of PAI-1 to enhance endogenous fibrinolysis. We have recently generated monoclonal antibodies that neutralize PAI-1 activity by switching the inhibitory conformation to a substrate conformation. However, intact murine antibodies have quite some disadvantages for therapeutic use in man. In the current study, we describe the construction of a smaller antibody fragment derived from a monoclonal antibody (MA-8H9D4) with PAI-1 neutralizing properties. The cDNAs encoding the variable domains of the heavy and light chain were amplified, linked and cloned into a phagemid vector. Resulting clones were expressed as a single-chain variable fragment (scFv, VH-(Gly4Ser)3-VL) on the surface of a phage and selected for binding to PAI-1. Subsequently, a positive phage was used for the production of soluble scFv-8H9D4. Following purification, the characteristics of the scFv-8H9D4 were compared to those of the original MA-8H9D4. The scFv inhibited PAI-1 activity to a similar extent as MA-8H9D4 and by a similar mechanism, i.e., induction of a conformational switch. Thus, this smaller antibody fragment, exhibiting the same properties as the parent molecule may constitute a useful starting point for the design of PAI-1 neutralizing therapeutics. © 1997 Elsevier Science B.V. All rights reserved. 相似文献
19.
Net photosynthetic (P
N) and dark respiration (R
D) rate, stomatal (rs′) and internal (ri′) resistances to carbon dioxide were measured by gas exchange methods on leaves of different ages, expressed in leaf plastochron
index units (LPI) for a fast growing poplar cultivar Unal 2. Although the optimal leaf age differs slightly for the different
gas exchange parameters, leaf ontogeny is reflected in the same way in these different parameters. MaximalP
N and minimalrs′ and ri′ values were found at LPI between 6 and 10. Chlorophyll concentrations were lowest at LPI lower than 10 although an
increase in two steps was found, when leaf age increases up to maturity. 相似文献
20.
Maneerat Koohapitagtam Suang Rungpragayphan Ratchanee Hongprayoon Wichai Kositratana Theerapol Sirinarumitr 《Molecular biology reports》2010,37(4):1677-1683
Non-immune phage scFv library is one of the most attractive resources for therapeutics, diagnostics and basic research. As
a matter of fact, quality of the library is limited by inefficient PCR cloning of antibody genes using degenerated primers.
PCR using this type of primers is difficult to optimize conditions for efficient amplification, and therefore causes loss
of antibody diversities. To overcome this problem, we described a novel two-step amplification of Vκ and VH genes with newly designed primer sets. Initially, we amplified Vκ and VH genes from their signal sequences to the joining region to keep antibody diversity as large as possible. Thereafter, highly
degenerated primers were used to amplify the Vκ and VH genes from the framework region 1 to the joining region. The Vκ and VH genes from the second PCR then were linked by PCR overlapping extension to generate the scFv library. Fifteen clones from
the library were randomly picked and sequenced, and the diversity of full-length scFvs was confirmed. Expression capability
of clones in the library was 80% after confirmation using colony hybridization. The results demonstrated the efficiency of
this strategy and the primer sets for construction of the scFv library. 相似文献