Preparation Of Two Novel Mouse Anti-human CD133-2 Monoclonal Antibodies And The Expression And Clinical Significance Of CD133-2 In Tumour Cells

Prominin-1(alias CD133) is a membrane glycoprotein specifically associated with plasma membrane protrusions.In 1997,Yin et al first reported AC133-a new surface antigen of hematopoietic stem and progenitor cells(HSPCs),then prepared the monoclonal antibody recognized the AC133 antigen.In June 2000,AC133 was denominated as CD133 at the meeting of international leucocyte differentiation antigen work(ILDAW).CD133 cDNA encodes a 5-transmembrane(5-TM) domain molecule with and extracellular N-terminus and a cytoplasmic C-terminus,and two large extracellular loops with eight consensus sites for N-linked glycosylation with a molecular weight of 120 kDa.Different from the members of the 4-TM and 7-TM receptor family,CD133 was believed as the first in a class of novel 5-TM receptors. According to the molecular structure and protein expression CD 133 maybe function as growth factor receptors.Thereafter,12 splice variants of prominin-1 of rodents and primates have been identified one after another.There are two isoforms of human CD133:CD133-1 and CD133-2.Yu et al cloned and identified CD133-2 following the cloning and identification of CD133-1 by Yin. CD133-2 differs from the previously identified isoform CD133-1 by the absence of exon 3.As a consequence,AC133-2 encodes 856 amino acid residues with a deletion of nine amino acids in the N-terminal extracellular domain.CD 133-2 was predominant in the fetal tissues such as liver,skeletal muscle,kidney,and heart,and also in the adult tissues such as kidney,pancreas and placenta.Additionally,CD133-2 was also detected in poorly differentiated human cell lines of lung,prostatic,pancreatic,colonic and breast carcinoma.Different from CD133-2,CD133-1 was predominant in the fetal brain and skeletal muscle.Because of the different expression of CD133 isoforms in different human tumours,providing a useful tool to separate stem cell subpopulation specifically. Whether the two isoforms are functionally redundant or serve distinct functions remains unclear,because little is known regarding their biological functions.The CD133 isoforms may be of significance in the analysis and isolation of stem and progenitor cells from specific tissues and thereby facilitate functional characterization and application to tissue-engineering and regenerative medicine.Expanding evidence highlights the roles of CD133 as a marker of cancer stem cells (CSCs) in various human tumours,because CD133 expressed in leukemic cell,glioma, colon carcinoma,prostatic carcinoma,hepatoma and pancreatic cancer except for in normal tissue.These CD133⁺ tumour cells were capable of self-renewal,proliferation, and multi-lineage differentiation in vitro to recapitulate the original tumour phenotype, consistent with CSC properties.Many studies showed that CD133⁺ tumour cells were associated with the tumor immune escape,chemoresistance and radioresistance,indicating CD133⁺ tumour cells maybe a potential target molecule for immunotherapy.Since its identification 10 years ago,CD133 molecule has been the focus in medicine research,but its exact biological function remains unclear and the ligand of this molecule is still unknown.Furthermore,the signaling and relevant function of CD133 need to be explored.Because the widely used CD133 mAbs have their limitations(due to the restricted binding to a glycosylated epitope of CD133,the cell's glycosylation state can influence the CD133 expression detected with these mAbs). Thus,to establish functional anti-human CD133-2 monoclonal antibodies might be useful to outline new prospects for functional characteristics and to provide a novel clue in CSC study and tumor biological therapies.Conclusion:In this study,we firstly cloned human CD133-2 full length gene, established transgenic cell line expressing stably human CD133-2 molecule.Two anti-human CD133-2 monoclonal antibodies recognizing different epitopes were obtained,providing tools to study the biological characteristics of CD133-2.To estimate the value of CD133-2,we further analyzed the expression and clinical significance in non-small cell lung cancer(NSCLC). Partâ… Establishment of transgenic cell line of human CD133-2 gene and preliminary studying of the biological function of the transfectantsObjective:Cloning human CD 133-2 full length cDNA,establishing transgenic cell line L929/CD133-2 expressing stably human CD133-2 molecule,and study the transfectant cells' biological function on T cells in vitro.Methods:The full length human CD133-2 cDNA was amplified by overlap PCR from the human fetal liver cDNA library.Cloned into cloning vector pMDT-18,after double digested with restriction endonuclease Hindâ…¢and BamHâ… ,the CD 133-2 gene was inserted into eukaryotic expressing vector pIRES2-EGFP.The recombinant vector was transfected into L929 cells with LipofectAMINE 2000^TM.After being selected with G418,transgenic cell line expressing stably human CD133-2 molecule was established. Then the transfected cell line was analyzed and identified by RT-PCR,Western-blot and FCM.The preliminary biological functions of CD133-2 for proliferation and IL-10, IFN-γsecretion of T cells were studied by MTT and ELISA respectively.Results:The full length human CD133-2 cDNA was successfully cloned,and the recombinant eukaryotic expression vector pIRES2-EGFP/CD133-2 was constructed. After confirmation by flow cytometry,RT-PCR and Western blot,the transfectants stably expressing human CD133-2 was established successfully.Analysis of biological functions suggested CD133-2 play an inhibitory effect on the proliferation and IL-10, IFN-γsecretion of T cells stimulated by anti-CD3 mAb with or without anti-CD28 mAb.Conclusion:The transgenic cell line L929/CD133-2 stably expressing human CD133-2 molecule on the cell surface has been obtained,which is an effective immunogen for preparing mouse anti-human CD133-2 monoclonal antibody,as well as the valuable tool for the following studies of CD133-2. Partâ…¡Preparation and characterization of the monoclonal antibodies against human CD133-2Objective:To prepare mouse anti-human CD133-2 monoclonal antibodies,which were used to analyze the expression pattern of CD133-2 on various human tumor cell lines and fetal tissues;to observe the effects of anti-CD133-2 mAbs on CD133-2 expressing tumor cell lines U937 and SW480 in vitro;to provide necessary material and experimental basis for the further study of tumor stem cells.Methods:BALB/c mice were immunized with human CD133-2 transfectant L929/CD133-2 as immunogen.Mouse spleen B cells were fused with mouse plasmocytoma cells SP2/0.Hybridoma cells were screened with human L929/CD133-2, L929/mock and CD133 expressing cell lines WERI-Rb1 and Y79.After multiple screening and clone culture,two hybridoma cells secreting specifically anti-human monoclonal antibodies were obtained.Fast-strip analysis was performed to identify Ig subclass of the generated monoclonal antibodies.The methods of indirect immunofluorescence and western blot were designed to identify the specificity of generated mAbs.The epitopes recognized by mAbs were analyzed by competition assay using FCM.The expression of CD133-2 on human tumor cell lines was detected by FCM.The immunogenicity of CD133-2 on human fetal tissues and human healthy placenta tissue was detected by immunohistochemical staining assay.To analyze the effects of anti-CD133-2 mAbs on U937 and SW480 cells,we carried out experiments as following:The viable cell number counting,CCK-8,and colony formation were performed to analyze cell proliferation;DNA distribution was examined by PI staining and FCM.Results:After multiple screening and subcloning,two monoclonal antibodies recognizeing CD133-2 specially(thereafter named as 6B3 and 9G4) were obtained.The isotype of the two mAbs were proved to be IgG2b withκlight chain.The mAb competition test conducted among the two mAbs evidenced that these two mAbs were completely specific to different antigen binding sites.Positive immunostaining was observed using mAb 6B3,CD133-2⁺ cells were found in pallium,pronephros and liver of 6W embryo.CD133-2⁺ cells were also detected in villus of human placenta.In addition,CD133-2 was also found on the several cancer cell lines,such as U937,THP-1, SW480,WERI-Rb1,and Y79 and so on.Viable cell counting and CCK-8 assay showed that mAb 6B3 could enhance the growth of human myelogenous leukemic cell line U937 and human colon adenocarcinoma cell line SW480 in a dose-dependent manner. Soft agar assays were performed to determine the role of mAb 6B3 on the colony formation of U937 cells.The result showed that mAb 6B3 could enhance the colony formation of U937 cells.In contrast,mAb 9G4 has no significant effect on cell growth, which indicated mAb 6B3 but not 9G4 was a functional mAb.Conclusion:Two mouse anti-human CD133-2 mAbs were generated,which recognize different epitopes from commercially available CD133-2 mAb AC141. Immunofluorescence and flow cytometry demonstrated that several tumor cell lines expressing CD133-2.CD133-2⁺ cells were found in embryonic tissue and placenta by immunohistochemistry analysis.Furthermore,mAb 6B3 has been revealed to profoundly enhance the growth of human myelogenous leukemic cell line U937 and human colon adenocarcinoma cell line SW480 in vitro.The successful preparation of mouse anti-human CD133-2 mAbs also provided materials to reveal the expression characteristics and function of human CD133-2 molecule on tumor stem cells.Partâ…¢The expression and clinical significance of CD133-2 in NSCLCObjective:To investigate the expression and functional relevance of stem cell marker CD133-2,co-stimulatory molecule B7 homolog 4(B7-H4),and clinical significance in NSCLC.Methods:The expressions of CD133-2 and BT-H4 in NSCLC were studied by SP immunohistochemistry.103 specimens from non-small cell lung cancer,25 from adjacent non-cancer tissue and 24 from inflammatory pseudotumor were analyzed. Furthermore,we examined the correlation between CD133-2 expression and clinical parameters,the correlation between CD133-2 and B7-H4 in NSCLC was also studied.Results:The expression of CD133-2 in NSCLC tissues was in accordance with the cell differentiation degree,and also the 3-year overall survival rates in NSCLC. CD133-2 also has positive relation with the expression of co-stimulatory molecule B7-H4.The multivariate analysis revealed that CD133-2 expression was an independent prognostic factor(P＜0.01) for NSCLC.Conclusion:In this study,we found CD133-2 expression in NSCLC was significantly associated with cell differentiation degree,3-year overall survival,and B7-H4 expression.The results suggested that CD133-2 was a poor prognosis factor for NSCLC.In summary,this study has successfully accomplished investigations as following: the human CD133-2 full length gene has been cloned;the transgenic cell line L929/CD133-2 has been obtained;two specific anti-human CD133-2 monoclonal antibodies 6B3 and 9G4 were generated;further investigations indicated that mAb 6B3 could enhance growth of CD133-2 expression cells U937 and SW480 in vitro;Besides, CD133-2 expressions of NSCLC tissues were explored.It was proved that CD133-2 was a poor prognosis factor for NSCLC.