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1.
Longlong Luo Qun Luo Leiming Guo Ming Lv Zhou Lin Jing Geng 《Journal of biomolecular structure & dynamics》2013,31(3):416-423
Ricin is a highly lethal toxin. Anti-ricin chimeric monoclonal antibody (mAb) C4C13 was prepared in our lab; however, its binding affinity was much weaker than that of the parent antibody 4C13. In this study, based on the computer-guided homology modeling and conformational optimization methods, the 3-D structure of C4C13 variable regions Fv was constructed and optimized. Using molecular docking and dynamics simulation methods, the 3-D complex structure of ricin and C4C13 Fv was obtained. Considering the orientation property, surface electrostatic distribution, residues chemical andphysical character and intermolecular hydrogen bond, the binding mode and key residues were predicted. According to C4C13 Fv fragment and ricin complementary binding surface, electrostatic attraction periphery and van der Waals interaction interface, three mutants (i.e., M1 (NH102F, WH103Y); M2 (WH103Y) and M3 (RL90G)) were designed, in which M1 and M2 were predicted to possess higher antigen-binding activity than C4C13, while M3 was weaker. The relative affinity assays by ELISA showed that M1 and M2 mutations had higher affinity (9.6 and 18.3?nmol/L) than C4C13 (130?nmol/L) and M3 had weaker affinity (234.5?nmol/L) than C4C13. The results showed that the modeling complex structure of the antigen (ricin) and antibody (C4C13) is reasonable. Our work offered affinity maturated antibodies by site mutations, which were beneficial for valuable anti-ricin antibody design and preparation in future. 相似文献
2.
Preclinical studies with the murine anti-CD48 antibody, mHuLym3 (IgG2a) have shown it to be a potentially useful therapeutic
reagent in the treatment of human leukaemia and lymphoma. For clinical use, humanised antibodies can have a number of advantages
over their original murine version, including mediation of higher effector cell function with human cells, longer serum half-life
and lower immunogenicity. In this study, we have produced a mouse/human chimeric HuLym3 antibody (cHuLym3) where the murine
antibody constant regions have been replaced with human constant regions. We report the production and preclinical characterisation
of the antibody, cHuLym3, with potent in vitro and in vivo antitumour activity. The genes encoding the variable heavy and
light chains were amplified by the polymerase chain reaction, sequenced and cloned into eukaryotic expression vectors containing
the human light- and heavy-chain constant regions (κ and IgG1). The chimeric and murine HuLym3 antibodies had similar cell-binding
specificity and affinity. In the human Raji cell severe combined immunodeficient mouse model the i.v. injection of cHuLym3
and mHuLym3 produced similar antitumour responses. Doses of cHuLym3 and mHuLym3 (100 μg) on days 1, 2 and 4 after i.v. Raji
cell injection produced a 40% longer time to hind-leg paralysis than when a control antibody was used. cHuLym3 had more potent
activity than mHuLym3 in antibody-dependent cellular cytotoxicity (ADCC) assays in vitro, with human peripheral blood mononuclear
cells as effectors. Up to 60% specific cell lysis was observed with cHuLym3 in ADCC assays. These properties suggest that
anti-CD48 antibodies may be useful in the treatment of a number of diseases including lymphoid leukaemias and lymphoma.
Received: 5 May 1999 / Accepted: 12 August 1999 相似文献
3.
Neutralizing antibodies are recognized to be one of the essential elements of the adaptive immune response that must be induced
by an effective vaccine against HIV. However, only a limited number of antibodies have been identified to neutralize a broad
range of primary isolates of HIV-1 and attempts to induce such antibodies by immunization were unsuccessful. The difficulties
to generate such antibodies are mainly due to intrinsic properties of HIV-1 envelope spikes, such as high sequence diversity,
heavy glycosylation, and inducible and transient nature of certain epitopes. In vitro neutralizing antibodies are identified using “conventional” neutralization assay which uses phytohe-magglutinin (PHA)-stimulated
human PBMCs as target cells. Thus, in essence the assay evaluates HIV-1 replication in CD4+ T cells. Recently, several laboratories including us demonstrated that some monoclonal antibodies and HIV-1-specific polyclonal
IgG purified from patient sera, although they do not have neutralizing activity when tested by the “conventional” neutralization
assay, do exhibit potent and broad neutralizing activity in “unconventional” ways. The neutralizing activity of these antibodies
and IgG fractions is acquired through post-translational modifications, through opsonization of virus particles into macrophages
and immature dendritic cells (iDCs), or through expression of antibodies on the surface of HIV-1-susceptible cells. This review
will focus on recent findings of this area and point out their potential applications in the development of preventive strategies
against HIV.
Foundation item: Chinese Ministry of Science and Technology 973 program grant awarded to Paul Zhou (2006CB504308). 相似文献
4.
Kenya Shitara Yoshihisa Kuwana Kazuyasu Nakamura Yuko Tokutake So Ohta Hiromasa Miyaji Mamoru Hasegawa Nobuo Hanai 《Cancer immunology, immunotherapy : CII》1993,36(6):373-380
Ganglioside GD3, which is one of the major gangliosides expressed on the cell surface of human tumors of neuroectodermal origin has been focused on as a target molecule for passive immunotherapy. We have cloned the cDNA encoding the immunoglobulin light and heavy chains of an anti-GD3 monoclonal antibody KM641 (murine IgG3, ), and constructed the chimeric genes by linking the cDNA fragments of the murine light and heavy variable regions to cDNA fragments of the human and 1 constant regions, respectively. The transfer of these cDNA constructs into SP2/0 mouse myeloma cells resulted in the production of the chimeric antibody, designated KM871, that retained specific binding activity to GD3. Indirect immunofluorescence revealed the same staining pattern for chimeric KM871 and the mouse counterpart KM641 on GD3-expressing melanoma cells. When human serum and human peripheral blood mononuclear cells were used as effectors in complement-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity respectively, the chimeric KM871 was more effective in killing GD3-expressing tumor cells than was the mouse counterpart KM641. Intravenous injection of chimeric KM871 markedly suppressed tumor growth in nude mice. The chimeric KM871, having enhanced antitumor activities and less immunogenicity than the mouse counterpart, would be a useful agent for passive immunotherapy of human cancer. 相似文献
5.
Geoffrey A. Pietersz Li Wenjun Vivien R. Sutton Jane Burgess Ian F. C. McKenzie Heddy Zola Joseph A. Trapani 《Cancer immunology, immunotherapy : CII》1995,41(1):53-60
Mouse monoclonal antibodies to CD19 detect an antigenic determinant expressed exclusively on the surface of B lymphocytes, and have previously been shown to be potentially useful therapeutic reagents for human B cell lymphoma. We report the production and characterization of a mouse/human chimeric antibody, cCD19, with potent in vivo antitumour activity. The genes encoding the variable domains for heavy (VH) and light (VL) chains were subcloned into eukaryotic expression vectors containing human constant region genes (IgG1 and ), and co-transfected into non-secreting Sp2/0 mouse myeloma cells. Intraperitoneal administration of cCD19 produced inhibition of growth of subcutaneous CD19+ Sultan human B lymphoma tumours inscid/scid mice. When the antibody was administered 18 and 20 days after subcutaneous tumour inoculation, an approximately 30% reduction in tumour size was noted by day 29. cCD19 faithfully mimicked the in vitro binding characteristics of mCD19 as (a) the chimeric antibody was shown by flow cytometry to bind exclusively to cell lines that expressed CD19, (b) cCD19 was able to inhibit the binding of mCD19 on CD19+ cells completely and (c) the affinity of binding of the two antibodies was not significantly different [K
a=(2.03±1.5)×108]. In biodistribution studies, up to 14.8% of the total injected antibody dose per gram of tissue was localized in CD19+ Sultan tumours at 24 h approximately, 14.4% was present in the tumors at 48 h and about 13.7% at 72 h. These levels were comparable to mCD19 administered in the same fashion. cCD19 conjugated to idarubicin was specifically and strongly cytotoxic to CD19+ cells cultured in vitro, and demonstrated an IC50 of 0.17 M, similar to that of mCD19 (0.32 M) and approximately 14-fold greater than the IC50 of free idarubicin. The specific cytotoxic capacity of cCD19 and its likely reduced immunogenicity suggest that it may potentially be of use in the treatment of refractory B cell lymphoma in humans. 相似文献
6.
Kanazawa J Ohta S Shitara K Fujita F Fujita M Hanai N Akinaga S Okabe M 《Cancer immunology, immunotherapy : CII》2000,49(4-5):253-258
KM871 is a chimeric antibody recognizing ganglioside GD3, which is one of the major gangliosides expressed on the cell surface
of human tumors of neuroectodermal origin. This study demonstrates the antitumor activity of KM871 against human melanoma
xenografts in nude mice, and analyzes the effector function operating in mice. In a well-established tumor model, KM871 showed
antitumor activity against H-15 and SK-MEL-28 human melanoma but not against H-187 and G361 human melanoma when administered
intravenously 5 days/week for 2 weeks. The G361 tumor became sensitive when KM871 was first administered on the day of tumor
inoculation. In this assay, it was observed that almost all the mice were tumor-free, but a few mice developed tumors. Therefore,
we examined the amount and expression pattern of GD3 antigen on G361 tumors escaping from KM871 treatment, but no change was
observed. Next we examined the optimal administration schedule for KM871 in mice, using H-15 melanoma. KM871 showed antitumor
activity when administered intravenously either 5 days/week for 2 weeks or three biweekly doses. However, the effect of the
former schedule was stronger than three biweekly doses. To compare the effector function in humans and mice, we studied the
complement-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity and antibody-dependent macrophage-mediated
cytotoxicity of KM871 using complement or effector cells prepared from humans and mice. It was found that the antibody-dependent
cell-mediated cytotoxicity exerted by polymorphonuclear cells and antibody-dependent macrophage-mediated cytotoxicity were
the only antitumor mechanism of KM871 in mice. However their action was very weak compared with that in humans, and complement-mediated
cytotoxicity, which was strong in humans, was not observed in mice. Therefore, the antitumor activity of KM871 against human
melanomas evaluated by the nude mouse model might be underestimated. These results indicate that KM871 shows good antitumor
activity against GD3-positive human melanoma and the antitumor activity expected in humans might be superior to that of the
nude mouse model.
Received: 10 July 1999 / Accepted: 21 January 2000 相似文献
7.
Tong Lin Jing Li Jun-jun Shao Guo-zheng Cong Jun-zheng Du Shan-dian Gao Hui-yun Chang 《中国病毒学》2011,26(4):273-278
In order to develop an anti-FMDV A Type monoclonal antibo by (mAb),BABL/c mice were immunized with FMDV A type.Monoclonal antibodies (mAbs) 7B11 and 8H4 against Foot-and-mouth disease virus (FMDV) serotype A were produced by fusing SP2/O myeloma cells with splenocyte from the mouse immunized with A/AV88.The microneutralization titer of the mAbs 7B11 and 8H4 were 1024 and 512,respectively.Both mAbs contain kappa light chains,the mAbs were IgG1.In order to define the mAbs binding epitopes,the reactivity of these mAbs against A Type FMDV,were examined using indirect ELISA,the result showed that both mAbs reacted with A Type FMDV.These mAbs may be used for further vaccine studies,diagnostic methods,prophylaxis,etiological and immunological research on FMDV.Characterization of these ncindicated that prepared anti-FMDV A mAbs had no cross-reactivity with Swine Vesicular Disease (SVD) or FMDV O,Asial and C Type antigens.Their titers in abdomen liquor were 1:5×106 and 1:2×106,respectively.7B11 was found to be of subtype IgG1,8H4 was classified as IgG2b subtype.The mAbs prepared in this study,are specific for detection of FMDV serotype A,and is potentially useful for pen-side diagnosis. 相似文献
8.
本文旨在对发热伴血小板减少综合征(severe fever with thrombocytopenia syndrome,SFTS)患者中和抗体进行定性和效价评估,建立中和抗体酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)。用96孔微量培养板培养非洲绿猴肾细胞(Vero-E6)并接种发热伴血小板减少综合征病毒(severe fever with thrombocytopenia syndrome virus,SFTSV),以抗核衣壳蛋白(nucleocapsid protein,NP)单克隆抗体为一抗,使用间接ELISA检测SFTSV NP,根据光密度(optical density,OD)判断阳性孔数,采用ReedMuench方法计算病毒半数组织培养感染剂量(50%tissue culture infective dose,TCID_(50)),以反映SFTSV在Vero-E6细胞中的复制水平。ELISA检测中和抗体作用后的病毒残余量,可间接反映中和抗体的作用效果并进行定量。应用以上建立的微量中和-ELISA对10例SFTS患者的双份血清进行中和抗体效价测定,8例患者恢复期血清效价较急性期增高4倍以上,7份患者恢复期血清效价达1∶1 280,急性期血清效价最高为1∶640。结果提示,本研究建立的ELISA操作简便,结果判定客观,所需时间短,可用于临床血清抗体诊断,也可用于血清流行病学调查和疫苗效果临床评价等。 相似文献
9.
J. Schüpbach H. Günthard M. S. C. Fung R. -S. Liou L. Botta P. Gowland W. Gordon D. Gygax N. T. Chang T. W. Chang R. Lüthy D. G. Braun 《Biotherapy》1993,6(3):205-215
The pharmacokinetics of mouse V/human C (1,) chimeric monoclonal antibody CGP47 439 specific for the principal neutralizing determinant of human immunodeficiency virus type 1 (HIV-1) was studied in patients with stage IV HIV-1 disease in an open-labeled phase I/IIA trial. Twelve male patients were enrolled and nine completed the study. Patients were divided into three groups according to the extent of CGP 47 439 to bind to gp120 from their viral isolates: undetectable for group 1, modestly reactive for group 2, and strongly reactive for group 3. A first dose of 1, 10, or 25 mg was administered by intravenous infusion to group 1, group 2 and group 3 patients, respectively. The patients then received seven doses of 50, 100, or 200 mg, respectively, every three weeks. CGP 47 439 serum concentrations were determined by an ELISA using monoclonal antibody AB19-4 specific for the idiotope of CGP 47 439. Half an hour after infusion only 25.5–36.1% of the administered antibody was found in the serum, reflecting its rapid distribution in the extravascular space and possibly binding to gp120 antigen in some of the patients. The terminal elimination half-life (T1/2) was 16.2 days in group 1 patients, 9.7 days in group 2 and in group 3 patients 7.5 days and 9.1 days. An antibody response to CGP 47 439 was not a factor in determining elimination rates, because only very low and transient responses were found in three patients. These results suggest that the reactivity of CGP 47 439 with HIV-1 gp120 contributed to its elimination in HIV-1 infected patients.Abbreviations AIDS
aquired immune deficiency syndrome
- ARC
AIDS-related complex
- HIV-1
human immune deficiency virus type 1
- gp120
envelope glycoprotein with 120 KD molecular weight
- V3
variable domain of gp120
- PND
principle neutralizing determinant of gp120
- IgG
immunoglobulin G
- CD4+ lymphocytes: lymphocytes expressing the CD4 marker
VH and VL variable heavy and variable light chain region of an antibody
- C1 and CK
constant heavy chain region of gamma l and constant K light chain region of an antibody
- anti-id
anti-idiotypic
- AUC
area under curve
- T1/2
terminal elimination half-life
- ELISA
enzyme-linked imuno sorbent assay
- PBS
phosphate buffered saline
- NP-40
detergent
- CDC
center of disease control
- GMP
good manufacturing practice 相似文献
10.
Fabian Richter Kirstin A. Zettlitz Oliver Seifert Andreas Herrmann Peter Scheurich Klaus Pfizenmaier 《MABS-AUSTIN》2019,11(1):166-177
Selective inhibition of tumor necrosis factor (TNF) signaling through the proinflammatory axis of TNF-receptor 1 (TNFR1) while leaving pro-survival and regeneration-promoting signals via TNFR2 unaffected is a promising strategy to circumvent limitations of complete inhibition of TNF action by the approved anti-TNF drugs. A previously developed humanized antagonistic TNFR1-specific antibody, ATROSAB, showed potent inhibition of TNFR1-mediated cellular responses. Because the parental mouse antibody H398 possesses even stronger inhibitory potential, we scrutinized the specific binding parameters of the two molecules and revealed a faster dissociation of ATROSAB compared to H398. Applying affinity maturation and re-engineering of humanized variable domains, we generated a monovalent Fab derivative (13.7) of ATROSAB that exhibited increased binding to TNFR1 and superior inhibition of TNF-mediated TNFR1 activation, while lacking any agonistic activity even in the presence of cross-linking antibodies. In order to improve its pharmacokinetic properties, several Fab13.7-derived molecules were generated, including a PEGylated Fab, a mouse serum albumin fusion protein, a half-IgG with a dimerization-deficient Fc, and a newly designed Fv-Fc format, employing the knobs-into-holes technology. Among these derivatives, the Fv13.7-Fc displayed the best combination of improved pharmacokinetic properties and antagonistic activity, thus representing a promising candidate for further clinical development. 相似文献
11.
12.
Takafumi Yamamoto Fumiko Arakawa Ken Nakamura Tarumi Senba Yoshihiro Tomita Seiyo Ikeda Masahide Kuroki 《Cancer immunology, immunotherapy : CII》1999,48(4):165-171
Mouse monoclonal antibody FU-MK-1, raised against a human gastric adenocarcinoma, recognizes a glycoprotein antigen (termed
MK-1 antigen) present on most carcinomas and seems to be valuable in immunodiagnosis and immunotherapy of various cancers.
In a recent study, we constructed a mouse/human chimeric antibody, designated Ch FU-MK-1, by fusing the FU-MK-1 VH and Vκ genes to the human Cγ1 and Cκ genes, respectively. In the present study, we tested combination immunotherapy of Ch
FU-MK-1 with human lymphokine-activated killer (LAK) cells in vitro and in mice with severe combined immunodeficiency (SCID)
bearing human MK-1-expressing tumors. In in vitro experiments, Ch FU-MK-1 effectively mediated antibody-dependent cell-mediated
cytotoxicity (ADCC) against MK-1-expressing MKN-74 cells, which was completely blocked by an anti-FcR antibody. Since the
apoptotic pathway as well as the necrotic pathway have been shown to be utilized in various cytotoxic effector mechanisms,
we investigated the role of apoptosis in ADCC mediated by LAK cells and Ch FU-MK-1 against MKN-74 cells. The implication of
the apoptosis during ADCC was demonstrated by means of both a terminal-deoxynucleotidyltransferase-mediated dUTP-biotin nick-end-labeling
assay and a propidium iodide staining method. In vivo antitumor activity of combination treatment with LAK cells and Ch FU-MK-1
was estimated using SCID mice inoculated s.c. with MKN-74 cells. The i.v. administration of LAK cells and i.p. administration
of Ch FU-MK-1 and interleukin-2 (IL-2) produced a marked growth inhibition of MKN-74 tumors in SCID mice. When the actual
tumor weights were measured 16 days after initiation of treatment, more than 70% reduction was observed in the group receiving
LAK cells plus Ch FU-MK-1 plus IL-2 as compared to the control untreated group. Together these results suggest that Ch FU-MK-1
may serve as a potentially useful immunotherapeutic reagent for human MK-1-expressing tumors.
Received: 27 November 1998 / Accepted: 23 February 1999 相似文献
13.
Zhi Qi Hong Lu Lin Li Xiaojuan Li Shuwen Liu 《Biochemical and biophysical research communications》2010,398(3):506-1133
HIV-1 gp41 prehairpin fusion intermediate (PFI) composed of three N-terminal heptad repeats (NHR) plays a crucial role in viral fusion and entry and represents an attractive target for anti-HIV therapeutics (e.g., enfuvirtide) and vaccines. In present study, we constructed and expressed two recombinant gp41 PFI mimetics, designated N46Fd and N46FdFc. N46Fd consists of N46 (residues 536-581) in gp41 NHR and foldon (Fd), a trimerization motif. N46FdFc is composed of N46Fd fused with human IgG Fc fragment as an immunoenhancer. We immunized mice with N46 peptide, N46Fd and N46FdFc, respectively, and found that only N46FdFc elicited neutralizing antibody response in mice against infection by HIV-1 strains IIIB (clade B, X4), 92US657 (clade B, R5), and 94UG103 (clade A, X4R5). Anti-N46FdFc antibodies inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines. 相似文献
14.
A hybridoma secreting human monoclonal antibody (MAB) against Pseudomonas aeruginosa exotoxin A (PEA) was constructed by fusing Epstein-Barr virus-transformed peripheral blood lymphocytes with human B lymphoblastoid cell line TAW-925. The human-human hybridoma stably produced human IgG2 MAB at the rate of 0.4–0.5 g/ml per 106 cells per day for more than six months, and the MAB was capable of neutralizing the in vitro cytotoxic and in vivo lethal effects of PEA with approximately 100-and 70-fold, respectively, higher activity than serum polyclonal antibody preparations.Abbreviations MAB
Monoclonal Antibody
- PEA
Pseudomonas aeruginosa exotoxin A
- LPS
Lipopolysaccharides
- OMP
Outer Membrane Proteins
-
P.
Pseudomonas
- EBV
Epstein-Barr Virus
- PEG
Polyethylene Glycol 相似文献
15.
Gary E. Goodman Ingegerd Hellstrom Dale E. Yelton James L. Murray Sarah O'Hara Elaine Meaker Lane Zeigler Paulette Palazollo Claude Nicaise J. Usakewicz Karl Erik Hellstrom 《Cancer immunology, immunotherapy : CII》1993,36(4):267-273
We report a single institution phase I trial of chimeric (mouse-human) monoclonal antibody (chL6) directed against a tumor-associated cell surface antigen expressed in non-small cell lung, colon, and breast cancer. The results of the study were contrasted with a previous trial of murine L6. ChL6 was administered intravenously to 18 patients with advanced cancer as a single, 4–16 infusion in doses ranging from 350 mg/m2 to 700 mg/m2. One patient received four weekly doses of 350 mg/m2. Patients were followed for side effects, localization of antibody to tumor cells, pharmacokinetics and the development of antibodies against chL6. Side effects associated with treatment were chills, fever, and nausea, which lasted 24–48 hours. Platelet count and absolute leukocyte count fell immediately after treatment, but returned to pretreatment levels by day 7. Localization of chL6 to tumor cells in vivo was seen at 350 mg/m2 and saturation at 700 mg/m2 and 350 mg/m2 per week×4. The pharmacokinetics of this antibody appeared similar to its murine analogue. Human antibodies against chL6 were detected in only 4 of 18 patients. These antibodies were directed against murine variable regent and their titers were lower than those occurring in most patients who received murine L6 in an earlier trial. No tumor reductions were seen. Chimeric L6 appears to be a suitable antibody for delivering anti-tumor agents because of its low immunogenicity and favorable in vivo tumor binding characteristics. 相似文献
16.
17.
《MABS-AUSTIN》2013,5(6):1585-1597
Recombinant Secretory IgA (SIgA) complexes have the potential to improve antibody-based passive immunotherapeutic approaches to combat many mucosal pathogens. In this report, we describe the expression, purification and characterization of a human SIgA format of the broadly neutralizing anti-HIV monoclonal antibody (mAb) 2G12, using both transgenic tobacco plants and transient expression in Nicotiana benthamiana as expression hosts (P2G12 SIgA). The resulting heterodecameric complexes accumulated in intracellular compartments in leaf tissue, including the vacuole. SIgA complexes could not be detected in the apoplast. Maximum yields of antibody were 15.2 μg/g leaf fresh mass (LFM) in transgenic tobacco and 25 μg/g LFM after transient expression, and assembly of SIgA complexes was superior in transgenic tobacco. Protein L purified antibody specifically bound HIV gp140 and neutralised tier 2 and tier 3 HIV isolates. Glycoanalysis revealed predominantly high mannose structures present on most N-glycosylation sites, with limited evidence for complex glycosylation or processing to paucimannosidic forms. O-glycan structures were not identified. Functionally, P2G12 SIgA, but not IgG, effectively aggregated HIV virions. Binding of P2G12 SIgA was observed to CD209 / DC-SIGN, but not to CD89 / FcalphaR on a monocyte cell line. Furthermore, P2G12 SIgA demonstrated enhanced stability in mucosal secretions in comparison to P2G12 IgG mAb. 相似文献
18.
Matthew Paul Rajko Reljic Katja Klein Pascal MW Drake Craig van Dolleweerd Martin Pabst Markus Windwarder Elsa Arcalis Eva Stoger Friedrich Altmann Catherine Cosgrove Angela Bartolf Susan Baden Julian K-C Ma 《MABS-AUSTIN》2014,6(6):1585-1597
Recombinant Secretory IgA (SIgA) complexes have the potential to improve antibody-based passive immunotherapeutic approaches to combat many mucosal pathogens. In this report, we describe the expression, purification and characterization of a human SIgA format of the broadly neutralizing anti-HIV monoclonal antibody (mAb) 2G12, using both transgenic tobacco plants and transient expression in Nicotiana benthamiana as expression hosts (P2G12 SIgA). The resulting heterodecameric complexes accumulated in intracellular compartments in leaf tissue, including the vacuole. SIgA complexes could not be detected in the apoplast. Maximum yields of antibody were 15.2 μg/g leaf fresh mass (LFM) in transgenic tobacco and 25 μg/g LFM after transient expression, and assembly of SIgA complexes was superior in transgenic tobacco. Protein L purified antibody specifically bound HIV gp140 and neutralised tier 2 and tier 3 HIV isolates. Glycoanalysis revealed predominantly high mannose structures present on most N-glycosylation sites, with limited evidence for complex glycosylation or processing to paucimannosidic forms. O-glycan structures were not identified. Functionally, P2G12 SIgA, but not IgG, effectively aggregated HIV virions. Binding of P2G12 SIgA was observed to CD209 / DC-SIGN, but not to CD89 / FcalphaR on a monocyte cell line. Furthermore, P2G12 SIgA demonstrated enhanced stability in mucosal secretions in comparison to P2G12 IgG mAb. 相似文献
19.
猪瘟(Classical swine fever,CSF)是严重危害养猪业的一种烈性传染病,常造成巨大的经济损失,是世界动物卫生组织要求必须申报的动物疫病之一。猪瘟的病原是猪瘟病毒(Classical swine fever virus,CSFV),CSFV的结构蛋白由衣壳蛋白(C)和囊膜糖蛋白(E~(rns)、E1、E2)构成。E2蛋白是CSFV主要的保护性抗原,可以诱导机体产生中和抗体,从而抵抗CSFV的感染。此前,本团队制备了一株针对CSFV E2蛋白的鼠源单克隆抗体HQ06。文中将HQ06抗体重链和轻链可变区基因与猪源恒定区基因嵌合后克隆至真核表达载体,利用中国仓鼠卵巢(CHO)细胞制备一株针对CSFV E2蛋白的嵌合猪源化单克隆抗体c HQ06。应用ELISA、Western blotting试验证实了c HQ06与CSFV E2蛋白具有良好的反应性;中和试验结果表明c HQ06可以中和CSFV。综上所述,本研究应用CHO细胞稳定表达了具有良好反应性和中和活性的针对CSFV E2蛋白的嵌合猪源化单克隆抗体c HQ06,为研究CSFV E2蛋白结构、功能以及开发新型的CSFV诊断和治疗制剂奠定基础。 相似文献
20.
Induction of neutralizing antibody against human cytomegalovirus (HCMV) with DNA-mediated immunization of HCMV glycoprotein B in mice. 总被引:1,自引:0,他引:1
E S Hwang K B Kwon J W Park D J Kim C G Park C Y Cha 《Microbiology and immunology》1999,43(3):307-310
Immunization was accomplished by inoculating pcGB containing human cytomegalovirus (HCMV) glycoprotein B (gB) gene into BALB/c mice intramuscularly. IgM antibody was detected in all the immunized group. IgG antibody was also found in all the tested mice with a mean peak antibody titer of 1:262 in three-times immunized groups. IgG antibody appeared at 2 weeks postinoculation, raised peak levels at 7 weeks postinoculation and persisted over 6 months. Neutralizing antibody was developed, and the percent reduction of input infectivity in 1:100 diluted sera was 74.5 % in three-times immunized groups. This study suggested that DNA vaccine using the gene encoding HCMV gB is a candidate method for developing immunity to HCMV. 相似文献