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1.
《Bioscience, biotechnology, and biochemistry》2013,77(1):144-151
We have recently established the monoclonal antibodies (mAbs) specific to the major food allergen, ovomucoid, as mAb 7D, recognizing the carbohydrate moiety of ovomucoid, and mAb 6H, the peptide moiety (Biosci. Biothechnol. Biochem., 68, 2490–2497, (2004)). Using these mAbs, we found commercially available ovalbumin preparations contaminated with a considerable amount of ovomucoid together with other glycoproteins. To examine the contaminants, egg white was subjected to cation-exchange chromatography. An unidentified protein was found in egg white that reacted with mAb 7D but not with mAb 6H, having a molecular size of about 52 kDa and a blocked N-terminus. Two internal amino acid sequences of the fragments obtained after a lysyl endopeptidase and a hydroxylamine treatment revealed the protein to be ovalbumin Y (ovalbumin-related gene Y protein). We conclude that ovalbumin Y is a unique chimeric glycoprotein having an amino acid sequence similar to that of ovalbumin, but having a carbohydrate moiety similar to that of ovomucoid. 相似文献
2.
Hirose J Doi Y Kitabatake N Narita H 《Bioscience, biotechnology, and biochemistry》2006,70(1):144-151
We have recently established the monoclonal antibodies (mAbs) specific to the major food allergen, ovomucoid, as mAb 7D, recognizing the carbohydrate moiety of ovomucoid, and mAb 6H, the peptide moiety (Biosci. Biothechnol. Biochem., 68, 2490-2497, (2004)). Using these mAbs, we found commercially available ovalbumin preparations contaminated with a considerable amount of ovomucoid together with other glycoproteins. To examine the contaminants, egg white was subjected to cation-exchange chromatography. An unidentified protein was found in egg white that reacted with mAb 7D but not with mAb 6H, having a molecular size of about 52 kDa and a blocked N-terminus. Two internal amino acid sequences of the fragments obtained after a lysyl endopeptidase and a hydroxylamine treatment revealed the protein to be ovalbumin Y (ovalbumin-related gene Y protein). We conclude that ovalbumin Y is a unique chimeric glycoprotein having an amino acid sequence similar to that of ovalbumin, but having a carbohydrate moiety similar to that of ovomucoid. 相似文献
3.
Zhang Q Zhang SH Su MQ Bao GQ Liu JY Yi J Shen JJ Hao XK 《Cancer immunology, immunotherapy : CII》2007,56(4):477-489
Background The HAMA response is a major challenge when murine antibodies are repeatedly administered for antibody directed enzyme prodrug
therapy in vivo. In this study we have achieved humanization of the anti-γ-seminoprotein E4B7 murine mAb by guided selection.
Methods Using optimal Ig Fab primers, human Fd and CL gene repertoires were amplified by RT-PCR from PBMCs of prostate cancer patients.
The human Lc gene repertoire was first paired with the murine Fd gene of E4B7 mAb to construct a pComb3X hybrid Fab display library. This hybrid library was screened with purified γ-seminoprotein antigen.
The human Fd gene repertoire was then paired with the selected human Lc to construct a fully human Fab library. After four
more rounds of panning, completely human Fab antibodies specific for γ-seminoprotein were selected and further identified.
Results First, using the E4B7 Fd gene as a template, light chain shuffling was achieved by panning the hybrid library. Then, using the selected Lc as a
template, a human Fab antibody against γ-seminoprotein was produced through heavy chain Fd shuffling. Western blotting, ELISA,
and flow cytometry results demonstrated that the resulting human Fab antibody resembled the parental E4B7 mAb in that they both recognized the same epitope with similar affinities. Fluorescent cell staining and immunohistochemistry
analysis further confirmed that this newly constructed human anti-γ-seminoprotein Fab antibody indeed specifically bound prostate
cancer cells and tissue.
Conclusions Through guided-selection, we successfully produced a human anti-γ-seminoprotein Fab antibody. This work lays the foundation
for optimal antibody-directed enzyme prodrug therapy of prostate cancer using a fully human Fab antibody.
Zhang Qing and Zhang Si-He are co-first authors on the publication. 相似文献
4.
Koji Ikura Jun-ichi Fujimoto Kosuke Kubonishi Shunji Natsuka Hiroyuki Hashimoto Tetsuya Ito Koki Fujita 《Cytotechnology》2002,40(1-3):23-29
Cyclomaltoheptaose (β-cyclodextrin, β-CD) is a promising compound for application in various industrial fields because of
its ability to entrap various compounds into its hydrophobic cavity. A monoclonal antibody (A7) to β-CD was generated by using
a conjugate of glucosaminylmaltosyl-β-CD and bovine serum albumin as an antigen. The A7 monoclonal antibody was IgM/κ and
reacted with β-CD with high specificity. The epitope recognized by the A7 monoclonal antibody seemed to be located on the
secondary hydroxyl groups of the rim side of the β-CD molecule. The dissociation constant of the complex of β-CD and the immobilized
A7 monoclonal antibody was determined to be 1.2 × 10-4 M. A competitive ELISA using the A7 monoclonal antibody enabled determination of β-CD and its derivatives with a detection
limit of 0.05 μM. This immunoassay was useful to determine β-CD in biological fluids such as human plasma and urine after
appropriate pretreatment of the samples.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
5.
S W Bright T Y Chen L M Flebbe M G Lei D C Morrison 《Journal of immunology (Baltimore, Md. : 1950)》1990,145(1):1-7
Experiments are described for the partial purification of the 80-kDa LPS binding protein expressed on macrophages and lymphocytes. This partially purified Ag was used to immunize adult Armenian hamsters and splenocytes from immunized animals were fused with murine myeloma cell lines. Hybridoma cell culture supernatants containing mAb were screened by ELISA for positive binding to the immunizing Ag, murine splenocytes and the murine 70Z/3 pre B cell and for an absence of binding to sheep E. Positive clones were further screened for reciprocal competitive binding with LPS on spleen cells and ability to modulate B lymphocyte mitogenic activity. Two hybridoma cell lines secreting IgM monoclonals, termed mAb3D7 and mAb5D3, were identified that satisfied all of the selection criteria. These hybridoma cell lines were subcloned and expanded. Binding of one (mAb3D7) was abrogated by treatment of Ag with mild periodate; binding of the second (mAb5D3) was destroyed by digestion of Ag with proteinase K. Binding specificity for mAb5D3 has been confirmed by ELISA using highly purified 80-kDa protein. These mAb have been of value in establishing that the 80-kDa LPS binding protein previously identified may serve as a specific functional receptor for LPS. 相似文献
6.
T. Sakaguchi Y. Takamiya Michael Edidin Kiyoshi Nokihara Kiyoshi Miwa Christian Schönbach M. Takiguchi 《Immunogenetics》1997,47(2):149-158
The monoclonal antibody (mAb) 4D12 specific for the HLA-B5, -B35 cross-reacting group (CREG) bound to a fraction of HLA-B*3501 and HLA-B*5101 molecules carrying self-peptides. Analysis of the binding of mAb 4D12 to HLA-B*3501 and -B*5101 molecules pulsed with chemically synthesized peptides revealed that this mAb recognizes a restricted number of peptides
and that P1 of the bound peptides critically influences its binding. The 4D12 mAb bound only to HLA-B*3501 molecules carrying peptides with Asn, Asp, Glu, Ser, and Val at P1. Analysis using an HLA-B*3501 crystallographic model suggested that 4D12 may recognize the side chain of the P1 residue that is pointing to the solvent.
On the other hand, 4D12 bound only to HLA-B*5101 molecules carrying peptides with Asn or Asp at P1, suggesting that the 4D12 epitope formed by Glu, Ser, or Val at P1
and the A-pocket was changed by the substitution of His for Tyr at residue 171 of HLA-B*3501 molecules. This was confirmed by testing the binding of mAb 4D12 to HLA-B*3501 mutant molecules at residue 171 carrying these peptides. These results together suggest that the conformation of the
A-pocket and its hydrogen bound network with the P1 residue is also critical for the binding of mAb 4D12. The present study
shows the molecular basis of the specificity of 4D12 for the peptide-HLA class I complex.
Received: 19 June 1997 / Revised: 27 August 1997 相似文献
7.
Héla Kallel Hind Zaïri Samia Rourou Makram Essafi Ridha Barbouche Koussay Dellagi Dahmani M. Fathallah 《Cytotechnology》2002,39(1):9-14
Taguchi’s methods were used for the design of an experimental strategy aimed at optimizing cell density and monoclonal antibody
(mAb) production from a spinner flask hybridoma culture. 23G11 is an antibody to the human leukocyte adhesion molecule, CR3
or β 2 integrin (CD11b/CD18). It recognizes specifically the A-domain of the α subunit CD11b. Anti β 2 integrin monoclonal
antibodies hold a great potential for preventing inflammation mediated tissue injuries. An L8 orthogonal experimental design
was used to investigate four different culture components: stirring speed, nature of serum, concentration of serum and nature
of media (RPMI 1640 or RPMI 1640 supplemented with glucose and glutamine). The experiments were conducted using two levels
for each factor studied and a direct ELISA test was used to estimate the level of antibody production. Statistical analysis
of the collected data pointed to the stirring speed and serum concentration, and the interaction between these parameters,
as the components that affected cell growth. Antibody production was affected by these factors and by the nature of medium
but also by the following two interactions: stirring speed/nature of serum and stirring speed/concentration of serum. This
study emphasizes the value of using Taguchi’s methods as a basis for optimization of mAb production from a hybridoma culture,
in cost effective and significantly less labor intensive ways.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
8.
T cells are crucial for the anti-metastatic effect of anti-epidermal growth factor receptor antibodies 总被引:2,自引:0,他引:2
Garrido G Lorenzano P Sánchez B Beausoleil I Alonso DF Pérez R Fernández LE 《Cancer immunology, immunotherapy : CII》2007,56(11):1701-1710
Experimental evidences supporting the epidermal growth factor receptor (EGFR) as an important molecule for tumor metastasis
had been accumulated. Currently, anti-EGFR monoclonal antibodies (mAbs) constitute a promising approach for the treatment
of patients with metastatic tumors. However, the mechanisms associated with the potent anti-metastatic effect of these mAbs
have not been completely elucidated due to the lack of appropriate syngeneic preclinical models. In this paper, we have investigated
the effects of 7A7, an antibody specific to murine EGFR, on the metastatic properties of D122 murine lung carcinoma. 7A7 mAb
significantly impaired metastatic spread of D122 cells in C57BL/6 mice by direct anti-proliferative and pro-apoptotic effects
on tumor metastasis. 7A7 mAb capacity to inhibit EGFR activation on D122 cells could contribute to its anti-metastatic effect.
In addition, 7A7 mAb was able to induce in vitro antibody-dependent cell-mediated cytotoxicity on D122 cells. Interestingly,
7A7 mAb treatment increased the number of natural killer cells, T lymphocytes and dendritic cells infiltrating the metastatic
sites. More strikingly, depletion of CD8+ and CD4+ T cells in vivo completely abrogated the 7A7 mAb anti-metastatic activity whereas function of natural killer cells was irrelevant.
This study supports an in vivo role for T cell response in the mechanism of action of anti-EGFR mAbs, suggesting the induction
of an adjuvant effect.
This work was supported by the Cuban Government. 相似文献
9.
Wu Gang Chen LiHua Zhong Shan Li Qi Song ChaoJun Jin BoQuan Zhu ZuoYan 《中国科学C辑(英文版)》2008,51(2):157-163
The aim of the present study was to purify the common native carp growth hormone (ncGH), produce monoclonal antibodies (mAbs)
to common native carp growth hormone (ncGH), and further enhance the sensitivity of enzyme-linked immunosorbent assays (ELISA)
for ncGH. Additionally, we investigated changes in serum ncGH levels in carps raised in different environmental conditions.
The recombinant grass carp (Ctenopharyngodon idella) growth hormone was purified and used as antigen to immunize the rabbit. The natural ncGH was isolated from the pituitaries
of common carp. SDS-PAGE and Western blot utilizing the polyclonal anti-rgcGH antibody confirmed the purification of ncGH
from pituitaries. Purified ncGH was then used as an immunogen in the B lymphocyte hybridoma technique. A total of 14 hybridoma
cell lines (FMU-cGH 1–14) were established that were able to stably secrete mAbs against ncGH. Among them, eight clones (FMU-cGH1–6,
12 and 13) were successfully used for Western blot while nine clones (FMU-cGH 1–7, 9 and 10) were used in fluorescent staining
and immunohistochemistry. Epitope mapping by competitive ELISA demonstrated that these mAbs recognized five different epitopes.
A sensitive sandwich ELISA for detection of ncGH was developed using FMU-cGH12 as the coating mAb and FMU-cGH6 as the enzyme
labeled mAb. This detection system was found to be highly stable and sensitive, with detection levels of 70 pg/mL. Additionally,
we found that serum ncGH levels in restricted food group and in the net cage group increased 6.9-and 5.8-fold, respectively,
when compared to controls, demonstrating differences in the GH stress response in common carp under different living conditions. 相似文献
10.
Ovomucoid-trypsin association complex was prepared by incubating chicken egg white ovomucoid with bovine trypsin. The reactivity of ovomucoid-trypsin complex was investigated by immunodiffusion, quantitative precipitation and enzyme-linked immunosorbent assay (ELISA). It was demonstrated that the association of trypsin with ovomucoid hindered the binding of the specific antibody at some antigenic sites of ovomucoid by lowering the antibody-binding affinity of these sites. The anti-ovomucoid antiserum was absorbed with ovomucoid-trypsin complex, and non-absorbed antibody was collected by immunoaffinity chromatography of ovomucoid-coupled Sepharose 4B. The antibody blocked the trypsin-inhibitory activity of ovomucoid in a molar ratio (antibody/ovomucoid) of about 1.2:1. The findings suggested that at least one antigenic site is located near the reactive site of trypsin inhibition (Arg89 decreases Ala90) of ovomucoid. 相似文献
11.
We previously reported that a conformational epitope-specific monoclonal antibody (mAb; #1-46-12) neutralized the rabies virus by binding only a small number (less than 20) of the antibody molecules per virion, while a linear epitope-specific mAb (#7-1-9) required more than 250 IgG molecules for the neutralization. We also isolated both the epitope-negative (R-31) and-positive (R-61) escape mutants that resisted mAb #1-46-12. Co-infection studies with wild type (wt) and R-61 mutant have shown that although the infectivity of R-61 mutant was not affected by the binding of about 300 IgG molecules per virion, incorporation of a small number of wt G protein into the R-61 virion resulted in dramatic loss of the resistance. In this study, we further investigated properties of the mutant G proteins. The R-61 G protein lost reactivity to the mAb when solubilized, even keeping a trimer form, suggesting that membrane-anchorage is essential for the maintenance of its epitope-positive conformation. On the other hand, incorporation of wt G proteins into the R-31 virions did not affect their resistance to the mAb very much. Although we have not so far found the presumed conformational changes induced by the mAb-binding, we think that these results are not inconsistent with our previously proposed novel model (referred to as a domino effect model) for the virus neutralization by mAb #1-46-12 other than a classical spike-blocking model, which implicates successive spreading of the postulated antibody-induced conformational changes of G protein to the neighboring spikes until abolishing the host cell-binding ability of the virion. 相似文献
12.
A monoclonal antibody against the glutaraldehyde-conjugated polyamine, putrescine: application to immunocytochemistry 总被引:1,自引:1,他引:0
Kunio Fujiwara Toshio Tanabe Masahiko Yabuuchi Ryuichi Ueoka Daisuke Tsuru 《Histochemistry and cell biology》2001,115(6):471-477
We developed a mouse monoclonal antibody (mAb; APUT-32, IgG1 subisotype mAb) against putrescine (Put) conjugated to bovine serum albumin using a glutaraldehyde (GA)-sodium borohydride procedure, for applications in immunocytochemistry (ICC). The antibody specificity was evaluated by an ELISA binding test, simulating the ICC of tissue sections. APUT-32 mAb was highly specific to Put, and distinguished alterations in the chemical structure of other polyamine (PA) analogs, showing 3.8% crossreaction with cadaverine, 3.3% with spermidine (Spd), and 2.3% with 1,3-diaminopropane. Comparable results in immunoreactivity of APUT-32 mAb were obtained with the ELISA inhibition test. By the indirect immunoperoxidase method using the APUT-32 mAb, Put-like immunoreactivities were observed in the cytoplasm of HeLa and MCF-7 cell lines fixed with GA in combination with NaBH4 reduction, but almost no immunoreaction was seen in the cytoplasm of the human melanoma BD cell line. On the other hand, the same method but using a previously prepared ASPM-29 mAb, specific for spermine (Spm) and Spd, produced intense immunostaining in the cytoplasm of all the three cell types. The Put-like immunoreaction was completely abolished by absorption of the APUT-32 mAb with 10 microg/ml Put-human serum albumin conjugate prepared using GA and NaBH4. HPLC analysis was also performed for the levels of each of the PAs in the three types of cell, showing that the levels of Put detected were much lower than those of Spm and Spd, and were strikingly different in the three cell lines among which the human melanoma BD cell line contained the lowest levels of Put. These results strongly suggest that APUT-32 mAb reacts specifically with Put in the tumor cells and therefore has the potential as a new tool for elucidating the biological roles of Put in cells and tissues. 相似文献
13.
K. Kissel S. Hamm Martina Schulz Annunciata Vecchi Cecilia Garlanda B. Engelhardt 《Histochemistry and cell biology》1998,110(1):63-72
A novel monoclonal antibody (mAb), 8D3 (IgG2a), that specifically recognizes the murine transferrin receptor (TfR) was produced
by immunizing a Lewis rat with a polyoma middle T oncogene-transformed endothelioma cell line. The 8D3 mAb was obtained by
immunohistochemical screening for exclusive staining of vessels forming a blood–brain barrier (BBB), but not of other vessels.
The anti-TfR mAb 8D3 recognizes the TfR also in FACS analysis and in western blots and should prove to be useful for affinity
purification of the TfR. Whereas 8D3 brightly stains BBB-forming vessels in the central nervous system of mice, it does not
stain the fenestrated capillaries within the choroid plexus and the circumventricular organs. In testis, where the blood–tissue
barrier is located at the level of the Sertoli cells, the 8D3 mAb specifically stains Sertoli cells but not endothelial cells.
Finally, in vitro, 8D3 does not interfere with iron uptake of lymphocytes as it does not influence their proliferation. Taken
together, 8D3 represents a versatile new tool to study the tissue distribution of the murine TfR and TfR-mediated transcytosis
across tissue barriers in the mouse.
Accepted: 7 January 1998 相似文献
14.
Neutrophils and mononuclear cells (MNC) can mediate antibody-dependent cellular cytotoxicity (ADCC) against cancer cells.
To study cytotoxicity and growth inhibition of neuroblastoma cells by neutrophils and MNC with chimeric anti-disialoganglioside
(GD2) monoclonal antibody (mAb) ch14.18, we developed digital image microscopy scanning (DIMSCAN) assays that measure fluorescence
of target cells in 96-well plates after 6–18 h (cytotoxicity assay) or 7 days (growth assay). Neuroblastoma cell lines (GD2-positive:
SMS-KCN, SMS-LHN, LA-N-1; GD2-negative: SK-N-SH) were preloaded with calcein acetoxymethyl ester for the cytotoxicity assay
or labeled in situ after 7 days of culture with fluorescein diacetate in the growth assay. Fluorescence, as quantified by
DIMSCAN, was correlated with neuroblastoma cell number in both assays (100–2000 cells/well). In the cytotoxicity test, both
neutrophils and MNC effectively mediated ADCC of GD2-positive but not GD2-negative neuroblastoma cell lines. Cytotoxicity
of both neutrophils and MNC increased with effector to target cell (E:T) ratio (5–50:1) and mAb ch.14.18 dose (0.1–10 μg/ml).
ADCC of neutrophils, but not MNC, increased with addition of GM-CSF. Neutrophils, especially with rhGM-CSF, significantly
suppressed growth of GD2-positive cell lines at a high E:T ratio (50:1) and mAb dose (10 μg/ml). Without antibody, neutrophils
inhibited growth of one cell line (LA-N-1) but stimulated growth of two others (SMS-KCN, SMS-LHN). If neuroblastoma cells
did not express GD2 (SK-N-SH), neutrophils stimulated growth whether or not antibody was present. Neutrophil culture supernatants
increased growth of SK-N-SH, LA-N-1, and SMS-KCN cells, and MNC culture supernatants increased growth of SK-N-SH. In conclusion,
neutrophils can mediate cytotoxicity and growth inhibition with a chimeric anti-GD2 antibody but also can promote tumor cell
growth if antibody is not present or if GD2 is not expressed.
Received: 18 November 1998 / Accepted: 24 September 1999 相似文献
15.
A novel murine IgM-type anti-human CD20 monoclonal antibody (mAb) 1-28 was prepared in our Lab, which can induce apoptosis and inhibit proliferation of Daudi and Raji cells. However, the efficacy of 1-28mAb in human cancer therapy is likely to be limited by human anti-mouse antibody responses. A chimeric antibody, C1-28, containing 1-28mAb variable region genes fused to human constant region genes (gamma 1, kappa) was constructed. However, C1-28 lost the antigen-binding activity. Here, using sequence similarity and known 3D structure of antibody variable regions as template, the spatial conformations of 1-28 variable regions (i.e. V(H) and V(L)) were analyzed with computer-guided homology modeling methods. According to the surface electrostatic distribution and interaction free energy analysis, the relationship between structure and stability of 1-28 variable regions was studied theoretically and a new chimeric anti-CD20 antibody scFv-Ig named 5S was designed. Expression level of 5S in the culture supernatant was determined to be around 50mug/mL using sandwich ELISA method with chimeric antibody Rituxan as reference. 5S retained its murine counterpart's binding activity by fluorescence-activated cell-sorting analysis. Furthermore, it could kill CD20 positive Daudi and Raji cells by complement-dependent cytotoxicity. For binding affinity often decreased even lost when IgM antibody was constructed into chimeric IgG1 form, our success give a hint about how to construct a IgG1-type chimeric antibody from IgM-type murine antibody to preserve its binding activity. 相似文献
16.
Kiyoshi Takamuku Kinya Baba Shinya Arinaga Jian Li Masaki Mori Tsuyoshi Akiyoshi 《Cancer immunology, immunotherapy : CII》1996,43(4):220-225
Antibody-dependent cell-mediated cytotoxicity (ADCC) has been considered to be one of the main effector mechanisms by which
unconjugated monoclonal antibody (mAb) 17-1A can exert an antitumor effect in vivo. 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 monocytes (ADMC) using mAb 17-1A as an antibody and the human colorectal carcinoma cell line,
COLO205, as target cells in vitro. The implications of the apoptosis during ADMC was demonstrated by means of both a DNA fragmentation
assay and a TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay. Furthermore, interferon γ (IFNγ) was also found to enhance
the induction of apoptosis significantly. The addition of superoxide dismutase did not reduce the level of the apoptosis,
although superoxide anion (O2
–) was observed to be produced. However, the release of tumor necrosis factor α (TNFα) was significantly enhanced during ADMC,
while, in addition, apoptosis was significantly inhibited by the addition of anti-TNFα antibody. These findings indicated
that apoptosis might be implicated in ADMC with mAb 17-1A, which was augmented by IFNγ, while, in addition, TNFα may also
be one of the major mediators of apoptosis.
Received: 1 August 1996 / Accepted: 27 August 1996 相似文献
17.
Phage-selected primate antibodies fused to superantigens for immunotherapy of malignant melanoma 总被引:7,自引:0,他引:7
Tordsson JM Ohlsson LG Abrahmsén LB Karlström PJ Lando PA Brodin TN 《Cancer immunology, immunotherapy : CII》2000,48(12):691-702
The high-molecular-weight melanoma-associated antigen, HMW-MAA, has been demonstrated to be of potential interest for diagnosis
and treatment of malignant melanoma. Murine monoclonal antibodies (mAb) generated in response to different epitopes of this
cell-surface molecule efficiently localise to metastatic lesions in patients with disseminated disease. In this work, phage-display-driven
selection for melanoma-reactive antibodies generated HMW-MAA specificities capable of targeting bacterial superantigens (SAg)
and cytotoxic T cells to melanoma cells. Cynomolgus monkeys were immunised with a crude suspension of metastatic melanoma.
A strong serological response towards HMW-MAA demonstrated its role as an immunodominant molecule in the primate. Several
clones producing monoclonal scFv antibody fragments that react with HMW-MAA were identified using melanoma cells and tissue
sections for phage selection of a recombinant antibody phage library generated from lymph node mRNA. One of these scFv fragments,
K305, was transferred and expressed as a Fab-SAg fusion protein and evaluated as the tumour-targeting moiety for superantigen-based
immunotherapy. It binds with high affinity to a unique human-specific epitope on the HMW-MAA, and demonstrates more restricted
crossreactivity with normal smooth-muscle cells than previously described murine mAb. The K305 Fab was fused to the superantigen
staphylococcal enterotoxin A (D227A) [SEA(D227A)], which had been mutated to reduce its intrinsic MHC class II binding affinity,
and the fusion protein was used to demonstrate redirection of T cell cytotoxicity to melanoma cells in vitro. In mice with
severe combined immunodeficiency, carrying human melanoma tumours, engraftment of human lymphoid cells followed by treatment
with the K305Fab-SEA(D227A) fusion protein, induced HMW-MAA-specific tumour growth reduction. The phage-selected K305 antibody
demonstrated high-affinity binding and selectivity, supporting its use for tumour therapy in conjunction with T-cell-activating
superantigens.
Received: 9 September 1999 / Accepted: 21 October 1999 相似文献
18.
Collin Jugler Haiyan Sun Katherine Nguyen Roman Palt Mitchell Felder Herta Steinkellner Qiang Chen 《Plant biotechnology journal》2023,21(3):549-559
This study describes a novel, neutralizing monoclonal antibody (mAb), 11D7, discovered by mouse immunization and hybridoma generation, against the parental Wuhan-Hu-1 RBD of SARS-CoV-2. We further developed this mAb into a chimeric human IgG and recombinantly expressed it in plants to produce a mAb with human-like, highly homogenous N-linked glycans that has potential to impart greater potency and safety as a therapeutic. The epitope of 11D7 was mapped by competitive binding with well-characterized mAbs, suggesting that it is a Class 4 RBD-binding mAb that binds to the RBD outside the ACE2 binding site. Of note, 11D7 maintains recognition against the B.1.1.529 (Omicron) RBD, as well neutralizing activity. We also provide evidence that this novel mAb may be useful in providing additional synergy to established antibody cocktails, such as Evusheld™ containing the antibodies tixagevimab and cilgavimab, against the Omicron variant. Taken together, 11D7 is a unique mAb that neutralizes SARS-CoV-2 through a mechanism that is not typical among developed therapeutic mAbs and by being produced in ΔXFT Nicotiana benthamiana plants, highlights the potential of plants to be an economic and safety-friendly alternative platform for generating mAbs to address the evolving SARS-CoV-2 crisis. 相似文献
19.
Kimura N Kawai S Kinoshita Y Ishiguro T Azuma Y Ozaki S Abe M Sugimoto M Hirata Y Orita T Okabe H Matsumoto T Tsuchiya M 《Biochemical and biophysical research communications》2004,325(4):1201-1209
A mouse monoclonal antibody (2D7 mAb), which specifically bound to the alpha2 domain of HLA class I, rapidly induces cell aggregation accompanied by weak cytotoxicity against ARH-77 cells, suggesting that 2D7 mAb had a potential for agonist antibody. In order to enhance this cytotoxicity, 2D7 mAb was engineered to be a small bivalent antibody fragment, 2D7 diabody. The resultant 2D7 diabody showed a strong cytotoxicity against ARH-77 cells. As a notable characteristic feature, the lethal effect of 2D7 diabody was quite rapid, mediated by a caspase-independent death pathway. Furthermore, 2D7 diabody also showed cytotoxicity against several leukemia and lymphoma cell lines, and mitogen-activated peripheral blood mononuclear cells (PBMC), but not for normal resting PBMC and adherent cell lines such as HUVEC. These results suggest that 2D7 diabody could be expected as a novel therapeutic antibody for hematological malignancies as well as inflammatory diseases. 相似文献
20.
Huang J Liang J Tang Q Wang Z Chen L Zhu J Feng Z 《Applied microbiology and biotechnology》2011,91(5):1341-1351
Many recombinant murine monoclonal antibodies (mAbs) were studied under pre-clinical or clinical development and became one
of the most prolific drug classes in oncology. Vascular endothelial growth factors receptor 2 (VEGFR2) has been implicated
to play an important role in tumors. We have established a murine anti-VEGFR2 mAb. To reduce the shortcoming of the mAb, a
murine–human chimeric Fab (cFab) named FA8H1 was constructed with gene engineering techniques and expressed as a soluble and
functional protein in Escherichia coli Top10F′. Several immunological methods were used to characterize the cFab, including ELISA, affinity and kinetics assay,
IP, IF, FACS, and IHC. The results illuminated that cFab maintained the specificity for the VEGFR2 antigen. The active cFab
also effectively identified VEGFR2 over-expressing cells in a number of archived human cancer tissues, compared to its parental
antibody. The FA8H1 provided the basis for potential therapy research against over-expressing VEGFR2 human solid tumors. 相似文献