Anti-SEMA3A Antibody: A Novel Therapeutic Agent to Suppress Glioblastoma Tumor Growth / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Glioblastoma (GBM) is classified as one of the most aggressive and lethal brain tumor. Great strides have been made in understanding the genomic and molecular underpinnings of GBM, which translated into development of new therapeutic approaches to combat such deadly disease. However, there are only few therapeutic agents that can effectively inhibit GBM invasion in a clinical framework. In an effort to address such challenges, we have generated anti-SEMA3A monoclonal antibody as a potential therapeutic antibody against GBM progression. MATERIALS AND METHODS: We employed public glioma datasets, Repository of Molecular Brain Neoplasia Data and The Cancer Genome Atlas, to analyze SEMA3A mRNA expression in human GBM specimens. We also evaluated for protein expression level of SEMA3A via tissue microarray (TMA) analysis. Cell migration and proliferation kinetics were assessed in various GBM patient-derived cells (PDCs) and U87-MG cell-line for SEMA3A antibody efficacy. GBM patient-derived xenograft (PDX) models were generated to evaluate tumor inhibitory effect of anti-SEMA3A antibody in vivo. RESULTS: By combining bioinformatics and TMA analysis, we discovered that SEMA3A is highly expressed in human GBM specimens compared to non-neoplastic tissues. We developed three different anti-SEMA3A antibodies, in fully human IgG form, through screening phage-displayed synthetic antibody library using a classical panning method. Neutralization of SEMA3A significantly reduced migration and proliferation capabilities of PDCs and U87-MG cell line in vitro. In PDX models, treatment with anti-SEMA3A antibody exhibited notable tumor inhibitory effect through down-regulation of cellular proliferative kinetics and tumor-associated macrophages recruitment. CONCLUSION: In present study, we demonstrated tumor inhibitory effect of SEMA3A antibody in GBM progression and present its potential relevance as a therapeutic agent in a clinical framework.

Screening, expression and characterization of single chain Fv (scFv) against basic fibroblast growth factor / 中华微生物学和免疫学杂志

Objective To obtain the specific human scFv basic fibroblast growth factor(bFGF)using phage antibody library technology. Methods The library was panned with human recombinant bFGF for 4 rounds. The antigen binding activities of random clones were tested by ELISA in order to select specific antibodies, which were then examined by DNA sequence analysis. Results The positive clone selected from the 104 random clones was able to bind bFGF specifically, while not able to bind other growth factors,such as aFGF, VEGF(vascular endothelial growth factor). By competition ELISA assay we found one clone 44 could inhibit bFGF binding to FGFR1. Conclusion Seven specific human phage antibody against bFGF was obtained by phage display technique, one clone could inhibit bFGF binding to its high affinity receptor FGFR1.

Present Status and Perspectives of Antibody Therapy against Cancer / 日本補完代替医療学会誌

The purpose of this article is to review the current status and future perspectives of antibody therapy against cancer. Eight antibody drugs against cancer are now commercially and clinically available for treatment of cancer in the United States and two of them are also available in Japan. Current data suggest that antibodies or their genes against cancer can be used in order to increase the tumor specificity of various new immunotherapeutic or gene therapeutic approaches against cancer, thereby enhancing the tumoricidal effect of each treatment while reducing the side effects.<br>

Isolation of Mouse Ig Heavy and Light Chain Genomic DNA Clones, and Construction of Gene Knockout Vector for the Generation of Humanized Xenomouse

BACKGROUND: Monoclonal antibodies (mAb) of rodent origin are produced with ease by hybridoma fusion technique, and have been successfully used as therapeutic reagents for humans after humanization by genetic engineering. However, utilization of these antibodies for therapeutic purpose has been limited by the fact that they act as immunogens in human body causing undesired side effects. So far, there have been several attempts to produce human mAbs for effective in vivo diagnostic or therapeutic reagents including the use of humanized xenomouse that is generated by mating knockout mice which lost Ig heavy and light chain genes by homologous recombination and transgenic mice having both human Ig heavy and light gene loci in their genome. METHODS: Genomic DNA fragments of mouse Ig heavy and light chain were obtained from a mouse brain lamda genomic library by PCR screening and cloned into a targeting vector with ultimate goal of generating Ig knockout mouse. RESULTS: Through PCR screening of the genomic library, three heavy chain and three light chain Ig gene fragments were identified, and restriction map of one of the heavy chain gene fragments was determined. Then heavy chain Ig gene fragments were subcloned into a targeting vector. The resulting construct was introduced into embryonic stem cells. Antibiotic selection of transfected cells is under the progress. CONCLUSION: Generation of xenomouse is particularly important in medical biotechnology. However, this goal is not easily achieved due to the technical difficulties as well as huge financial expenses. Although we are in the early stage of a long-term project, our results, at least, partially contribute the successful generation of humanized xenomouse in Korea.

Construction and primary identification of a naive Fab fragment phage library / 中国免疫学杂志

Objective:To construct a human Fab fragment phage display library and provide a platform for human antibody preparation.Methods:Peripheral blood lymphocytes were collected from healthy donor.The heavy chain Fd fragment and light chain of human immunoglobulin's genes were amplified by RT-PCR,and then cloned into phagemid pComb3XSS to generate human phage antibody library.Cutting with endonucleases such as SacⅠ,XbaⅠ,XhoⅠand SpeⅠto identify the insertion of the light chain or heavy chain Fd genes.IL-2 and digoxin as the antigen was used to scan the phage antibody library.Results:A phage antibody library of Fab had 8.4?107 members and it's recombinant rate was 70%.Through DNA sequencing of one positive clone,it was showed that its heavy chain belonged to IgG subvariety and its light chain to ? family.Conclusion:The success of constructing a nave human phage antibody library proves the useful of phage display system in human antibody preparation,and it can be used to select,purify and express of amylin Fab antibody.

Cloning of anti-MMP-2 human antibodies from semisynthetic phage antibody library / 中国免疫学杂志

Objective:To clone human anti MMP 2 antibodies from semisynthetic phage antibody library.Methods:Panning of semisynthetic phage antibody library against recombinant human MMP 2 was conducted to select specific antibodies.The antigen binding characterastics were analyzed by ELISAs.Results:MMP 2 binding phage antibody clones were obtanied after four rounds of panning,but they all showed binding activity to unrelated ags tested.One clone,AD20,was chosen for further analysis by competitive ELISA.It was found that the binding of AD20 to MMP 2 could be inhibited only by free MMP 2 but not unrelated Ags,while the binding to other Ags could not be inhibited by Ags tested,including MMP 2.Neutralization test demonstrated that AD20 could neutralize the enzymatic activity of MMP 2.Conclusion:A neutralizing human antibody against MMP 2 was obtained from a semisynthetic phage antibody library,which shows bindings with unrelated Ags nonspecifically that may be caused by different binding modes,a phenomenon termed polyreactivity.

Screening,identification and sequencing of human phage engineered antibody against HBsAg / 解放军医学杂志

Objective To screen and identify the human phage engineered antibodies against HBsAg, and to analyze their gene sequence. Methods The phage antibody library was constructed by phage surface display techniques, and then the antibody genes were isolated and amplified from human peripheral blood lymphocyte. The human phage engineered antibody against HBsAg was selected through bio-panning from the phage antibody library. The affinity and specificity of the phage antibody was identified by ELISA and inhibition ELISA assay. The genes of heavy chain and light chain of the phage antibody against HBsAg were analyzed by sequencing. Results 30 colonies were obtained after three rounds of bio-panning. One of them had the highest A490 (A490=1.47?0.08) with ELISA, and the inhibition rate was 76% with inhibition assay (A490=0.35?0.10), implying that the phage antibody against HBsAg was of high specificity. The findings of enzyme digestion demonstrated that the phage antibody included genes of heavy chain and light chain. Sequencing results indicated that the VH region of heavy chain belonged to VHI subgroup and the VL region of light chain belonged to V? Ⅰ and V? Ⅲ subgroup. Conclusions The specific human phage engineered antibody against HBsAg was selected successfully from the phage antibody library. These results suggest that screening and identification of human phage engineered antibody against HBsAg through the phage antibody library are technologically feasible. It lays a foundation for application of human engineered antibody against HBsAg to design new targeted therapy strategy for HBV.