Production of a human single-chain variable fragment antibody against esophageal carcinoma

Xu, Mingyan; Xu, Xiuli; Chen, Gengzhen; Deng, Xiaoling; Li, Jonathan; Yu, Xiuchong; Chen, Meizhen

doi:10.3748/wjg.v10.i18.2619

Cited by 20 publications

(16 citation statements)

References 17 publications

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“…The absorbance (490 nm) was then determined on a PowerWave XS Microplate spectrophotometer (Bio-Tek; ref. 16). Selectivity is determined using the following formula:…”

Section: In Vivo Panningmentioning

confidence: 99%

Targeted Drug Delivery to Hepatocarcinoma In vivo by Phage-Displayed Specific Binding Peptide

Han

Wang

et al. 2010

Molecular Cancer Research

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Hepatocellular carcinoma is one of the deadliest cancers in the world. In this study, a hepatocarcinoma-specific binding peptide, which could be used for drug delivery in targeting therapy, was obtained by in vivo phage display technology. After three rounds of panning, only the potential motif Pro-Ser was found in 80 sequenced phage clones. Phage A54 (sequence AGKGTPSLETTP) was shown to be the most effective and specific to the liver cancer cells by cell-based ELISA in all 130 tested clones. After phage A54 was injected i.v. into the xenograft-bearing mice for in vivo distribution, phage enrichment was found in tumor tissues compared with control phage C10 and normal liver tissues through phage titering and immunohistochemical staining. Next, the specific binding ability of synthesized peptide A54 was further confirmed by fluorescence microscopy, competition binding, and fluorescence-activated cell sorting assay. A54 and A54M (sequence AGKGTAALETTP) were synthesized and coupled to doxorubicin (DOX) to do the preliminary targeting therapy. After the treatment, the proliferation of liver cancer cells treated with A54-DOX was restrained significantly in vitro when compared with A54M-DOX-treated group. Reduction in tumor size and prolongation of long-term survival were also found in xenograft-bearing models compared with free DOX-treated group. In conclusion, the specific binding peptide A54, which was screened from phage display library, represents a promising approach for the development of novel target therapy strategies against hepatocellular carcinoma. Mol Cancer Res; 8(2); 135-44. ©2010 AACR.

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“…The absorbance (490 nm) was then determined on a PowerWave XS Microplate spectrophotometer (Bio-Tek; ref. 16). Selectivity is determined using the following formula:…”

Section: In Vivo Panningmentioning

confidence: 99%

Targeted Drug Delivery to Hepatocarcinoma In vivo by Phage-Displayed Specific Binding Peptide

Han

Wang

et al. 2010

Molecular Cancer Research

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“…Although most previous studies have used non-fixed living cells as the targets to screen for cancer cell antibodies (7,19,20), some researchers still prefer to use PFA fixed cells to screen for antibodies. Fixed cells ensure the integrity of the cell membrane during the entire process (21,22), decrease cell breakage and antigen degradation and avoid the inhibition of bacterial growth caused by hydrolytic enzymes and other harmful substances released by the breakdown of residual cancer cells.…”

Section: Discussionmentioning

confidence: 99%

“…Three human esophageal cancer cell lines, KYSE-180, KYSE-170 and EC0156, all in good growth status, were digested with trypsin and washed twice in PBS. The intact cells were equally mixed to form a cell pool and then divided into 2 equal portions of approximately 4-5x10 7 cells each. The screen was performed in two rounds, PF and NF, according to the treatment method of the cells (fixed with 2% PFA or not).…”

Section: Solid-phase Antigen Screening Of the Phage Antibody Librarymentioning

confidence: 99%

“…Currently, the strategy for anti-tumor antibody screening involves making a phage antibody library through the amplification of peripheral blood B lymphocytes derived from immunized animals or cancer patients (7). However, because of immune escape, cancer cells often fail to evoke a normal immune response and may even cause immune suppression, which results in B cell population changes and reduced antibody diversity.…”

Section: Introductionmentioning

confidence: 99%

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Screening for antibodies against intact cancer cells with a naï¿½ve large phage antibody library

Zhou²,

Wang³

et al. 2011

Int J Mol Med

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Abstract. Large phage antibody libraries are expected to be an efficient technology for obtaining humanized anti-tumor antibodies. However, few reports have been concerned about the selection strategies for intact cancer cells as targets. In this study, a 2x1011 large naïve scFv library from blood B lymphocytes of normal adult donors and neonatal cord blood was constructed using the LoxP-cre system. Three human esophageal cancer cell lines were equally mixed for use as the target. These intact cells were divided into two groups, PF and NF, according to the treatment method of the cells (fixed with 2% PFA or not, respectively). Positive phage antibodies were identified following 4 panning cycles of adhesion, elution and amplification. Using a cell ELISA assay, it was found that the additional procedure of directly infecting XL1-Blue bacteria with the cell pellet following washing with an acidic solution can effectively decrease the loss of positive phage, yielding a positive screening rate of 11.6% (61/525). Most of the phage antibodies displayed binding activity with all three esophageal cancer cell lines. Moreover, other phages (such as NFc53a and NFc70a) appeared to be specific for certain cell lines. Regarding the method used to treat the target cells, both the positive screening rate and the genetic diversity of the antibody variable region were significantly higher in the NF group (12.9 and 71.4%, respectively) than in the PF group (10.5 and 14.2%, respectively). Immunohistochemical analysis demonstrated that the scFv phages have good specificity for esophageal cancer. This technology is helpful for developing small molecular tracers and targeted therapies for malignant tumors.

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“…The EphA2 KD cell was used in the subtraction biopanning step. This step depletes the phage library of phage antibodies that bind to the host cell membrane surface antigens [305,306]. This should theoretically remove or minimise the number binders to common, shared epitopes between the two cell lines, and allowing the selection of phage antibodies that specifically bind the target protein upon biopanning with antigen positive cells [183,187,191,192,198].…”

Section: Monoclonal Phage Elisa For Human Epha2 Scfv-phage Bindersmentioning

confidence: 99%

Isolation and characterisation of antibodies against human and canine EphA2_ targets for comparative studies in brain cancer

Hagh¹

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Glioblastoma multiforme (GBM) is one of the high-grade gliomas, with a patient median survival of around 15 months from the time of diagnosis. Current treatments for this brain cancer initially comprise of surgery and radiotherapy, followed by administration of Temozolomide. Despite many advances in preclinical studies of GBM in mice, the outcomes have not been translated to the clinical level for humans. This is because the mouse model of GBM does not recapitulate the human disease and is not a suitable model for comparative studies of spontaneous tumours in humans. The canine model represents a powerful, large animal model of gliomas, since canine brain tumours occur spontaneously, with an incidence rate and patient age profile quite similar to human populations. EphA2 is one of the multi-domain receptors of the most significant tyrosine kinase family of receptors, and is highly expressed in both human and canine glioblastoma. Overexpression of EphA2 has been shown to correlate with tumour stage, progression and patient survival. Since EphA2 is not expressed in normal brain tissue, but is over-expressed on GBM cells, it is potentially a highly useful receptor for antibody-based therapy of brain cancer in both humans and dogs. Thus, monoclonal antibodies (mAbs) which cross-react with both human and canine EphA2 would be valuable molecular entities with diagnostic and therapeutic potential. To generate mAbs specific for EphA2 with cross-reactivity to both canine and human EphA2 receptors, phage display technology was employed using various strategies for the presentation of the EphA2 antigen to the phage antibody library. This included biopanning against firstly recombinant whole extracellular domain (ECD) of both human and canine EphA2, secondly recombinant ligandbinding domain (LBD) of human and canine EphA2, and lastly against cells expressing the native EphA2. Biopanning against recombinant EphA2-ECD generated one promising antibody (mAb AGH001) which showed cross-reactivity to both recombinant human and canine EphA2-ECD. However, mAb AGH001 was shown to also bind to EphA2 Knock Down (KD) cells, suggesting mAb AGH001 may bind to a domain within EphA2-ECD, which is conserved among EphA family receptors, other than the unique LBD of EphA2. To isolate specific anti-EphA2 antibodies for the LBD on EphA2-ECD, the human and canine LBD was utilised as an antigen for a biopanning campaign. Utilising this strategy of biopanning against the LBD of EpahA2, mAb HuB1 was isolated and showed binding to both human and canine EphA2-LBD and EphA2-ECD as well; however, mAb HuB1 also showed binding to the negative EphA2 cell line. This could be due to partially overlapping epitopes on other Eph receptors. mAb AGH001 and a positive control antibody, murine mAb 4B3 did not bind to EphA2-ECD subdomains. It can be concluded that the recombinant ECD sub-domains did not recapitulate the native conformation of EphA2. Furthermore, we demonstrated that mAb AGH001 has a conformational III epitope, therefore relies on a structural conform...

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Production of a human single-chain variable fragment antibody against esophageal carcinoma

Abstract: A human scFv phage display library can be constructed from the metastatic lymph nodes of esophageal cancer patients. A whole human scFv against esophageal cancer shows some bioactivity.

Cited by 20 publications

References 17 publications

Targeted Drug Delivery to Hepatocarcinoma In vivo by Phage-Displayed Specific Binding Peptide

Targeted Drug Delivery to Hepatocarcinoma In vivo by Phage-Displayed Specific Binding Peptide

Screening for antibodies against intact cancer cells with a naï¿½ve large phage antibody library

Isolation and characterisation of antibodies against human and canine EphA2_ targets for comparative studies in brain cancer

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