Identification of a novel monoclonal antibody recognizing CD133
Human CD133 (prominin-1), a cell surface glycoprotein, is used as a marker of hematopoietic and neural stem cells. Antibodies that recognize a glycosylation-dependent CD133 epitope have been extensively used for enrichment of tumor initiating cells in a variety of cancers. These currently available antibodies are restricted for use in only a subset of biological assays. We have generated a novel anti-human CD133 monoclonal antibody, using a recombinant protein consisting of highly immunogenic amino acid residues selected from the native CD133 protein as an immunogen. The antibody (identified as clone 7) specifically recognizes the CD133 protein in a variety of immunological applications including Western blot, immunofluorescence, flow cytometry and immunohistochemistry. Further, clone 7 specifically recognizes an unmodified CD133 extracellular domain, and not its glycosylated epitope. In conclusion, the specificity and usefulness in a wide range of applications suggest that clone 7 could be a valuable tool to identify CD133 positive cells as well as to target them for therapy.
[Show abstract] [Hide abstract] ABSTRACT: Somatic stem cells are rare cells with unique properties residing in many organs and tissues. They are undifferentiated cells responsible for tissue regeneration and homeostasis, and contain both the capacity to self-renew in order to maintain their stem cell potential and to differentiate towards tissue-specific, specialized cells. However, the knowledge about the mechanisms controlling somatic stem cell fate decisions remains sparse. One mechanism which has been described to control daughter cell fates in selected somatic stem cell systems is the process of asymmetric cell division (ACD). ACD is a tightly regulated and evolutionary conserved process allowing a single stem or progenitor cell to produce two differently specified daughter cells. In this concise review, we will summarize and discuss current concepts about the process of ACD as well as different ACD modes. Finally, we will recapitulate the current knowledge and our recent findings about ACD in human hematopoiesis.0Comments 1Citation
- "Additionally, both AC133 and AC141 have been reported to recognize glycosylation-dependent residues on the extracellular domains of CD133 [60,71]. When we tested a novel anti-CD133 antibody (HC7) reported in 2010 to bind glycosylation-independent protein residues of CD133 and being insensitive to fixation , we found clear asymmetric HC7 epitope distributions in approximately 30% of the mitotic human HSPCs . Using fluorescence-labeled anti-CD133 (HC7) antibodies combined with time-lapse fluorescence microscopy, we developed single cell assays to study the mitotic distribution of CD133 on viable, dividing human MPPs, and the cell fate of both paired daughter cells, respectively. "
- "Subcellular Distribution of HC7 and AC133 Epitopes on Polarized and Mitotic Human HSPCs Recently, a novel anti-CD133 antibody has been described whose epitopes, unlike those of classical AC133 and AC141 anti-CD133 antibodies, were claimed to be insensitive to fixation (Swaminathan et al., 2010). Because of our interest in analyzing the subcellular distribution of CD133 on polarized and mitotic HSPCs, we decided to test for the usability of the HC7 antibody in human hematopoietic stem cell research. "
[Show abstract] [Hide abstract] ABSTRACT: The cancer stem cell (CSC) marker CD133 is an attractive target to improve antitumor therapy. We have used photochemical internalization (PCI) for the endosomal escape of the novel CD133-targeting immunotoxin AC133–saporin (PCIAC133–saporin). PCI employs an endocytic vesicle-localizing photosensitizer, which generates reactive oxygen species upon light-activation causing a rupture of the vesicle membranes and endosomal escape of entrapped drugs. Here we show that AC133–saporin co-localizes with the PCI-photosensitizer TPCS2a, which upon light exposure induces cytosolic release of AC133–saporin. PCI of picomolar levels of AC133–saporin in colorectal adenocarcinoma WiDr cells blocked cell proliferation and induced 100% inhibition of cell viability and colony forming ability at the highest light doses, whereas no cytotoxicity was obtained in the absence of light. Efficient PCI-based CD133-targeting was in addition demonstrated in the stem-cell-like, triple negative breast cancer cell line MDA-MB-231 and in the aggressive malignant melanoma cell line FEMX-1, whereas no enhanced targeting was obtained in the CD133-negative breast cancer cell line MCF-7. PCIAC133–saporin induced mainly necrosis and a minimal apoptotic response based on assessing cleavage of caspase-3 and PARP, and the TUNEL assay. PCIAC133–saporin resulted in S phase arrest and reduced LC3-II conversion compared to control treatments. Notably, co-treatment with Bafilomycin A1 and PCIAC133–saporin blocked LC3-II conversion, indicating a termination of the autophagic flux in WiDr cells. For the first time, we demonstrate laser-controlled targeting of CD133 in vivo. After only one systemic injection of AC133–saporin and TPCS2a, a strong anti-tumor response was observed after PCIAC133–saporin. The present PCI-based endosomal escape technology represents a minimally invasive strategy for spatio-temporal, light-controlled targeting of CD133 + cells in localized primary tumors or metastasis.0Comments 8Citations
- "An AC133– saporin-dose dependency was indicated for the PCI AC133–saporin group. CD133 is a glycosylated protein and changes in the glycosylation status may have an effect on tumor growth and recurrence, as well as the ability of AC133–saporin to target all of the CD133-expressing CSC within the tumor535455. Hence, down-regulation of the AC133-epitope due to differentiation  or reduced plasma membrane expression of CD133 due to endocytosis of the receptor  is a potential explanation of the low anti-tumor effects seen in this study. "