| 1. |
- Piao, H. L., et al.
(författare)
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Affinity-matured recombinant immunotoxin targeting gangliosides 3 '-isoLM1 and 3 ', 6 '-isoLD1 on malignant gliomas
- 2013
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Ingår i: Mabs. - : Informa UK Limited. - 1942-0862 .- 1942-0870. ; 5:5, s. 748-762
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Tidskriftsartikel (refereegranskat)abstract
- About 60 percent of glioblastomas highly express the gangliosides 3-isoLM1 and 3,6-isoLD1 on the cell surface, providing ideal targets for brain tumor immunotherapy. A novel recombinant immunotoxin, DmAb14m-(scFv)-PE38KDEL (DmAb14m-IT), specific for the gangliosides 3-isoLM1 and 3,6-isoLD1, was constructed with improved affinity and increased cytotoxicity for immunotherapeutic targeting of glioblastoma. We isolated an scFv parental clone from a previously established murine hybridoma, DmAb14, that is specific to both 3-isoLM1 and 3,6-isoLD1. We then performed in vitro affinity maturation by CDR hotspot random mutagenesis. The binding affinity and specificity of affinity-matured DmAb14m-IT were measured by surface-plasmon resonance, flow cytometry, and immunohistochemical analysis. In vitro cytotoxicity of DmAb14m-IT was measured by protein synthesis inhibition and cell death assays in human cell lines expressing gangliosides 3-isoLM1 and 3,6-isoLD1 (D54MG and D336MG) and xenograft-derived cells (D2224MG). As a result, the K-D of DmAb14m-IT for gangliosides 3-isoLM1 and 3,6-isoLD1 was 2.6 x 10(-9)M. Also, DmAb14m-IT showed a significantly higher internalization rate in cells expressing 3-isoLM1 and 3,6-isoLD1. The DmAb14m-IT IC50 was 80 ng/mL (1194 pM) on the D54MG cell line, 5 ng/ml (75 pM) on the D336MG cell line, and 0.5 ng/ml (7.5 pM) on the D2224MG xenograft-derived cells. There was no cytotoxicity on ganglioside-negative HEK293 cells. Immunohistochemical analysis confirmed the specific apparent affinity of DmAb14m-IT with 3-isoLM1 and 3,6-isoLD1. In conclusion, DmAb14m-IT showed specific binding affinity, a significantly high internalization rate, and selective cytotoxicity on glioma cell lines and xenograft-derived cells expressing 3-isoLM1 and 3,6-isoLD1, thereby displaying robust therapeutic potential for testing the antitumor efficacy of DmAb14m-IT at the preclinical level and eventually in the clinical setting.
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| 2. |
- Kato, Yukinari, et al.
(författare)
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GMab-1, a high-affinity anti-3'-isoLM1/3',6'-isoLD1 IgG monoclonal antibody, raised in lacto-series ganglioside-defective knockout mice
- 2010
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Ingår i: Biochemical and biophysical research communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 391:1, s. 750-5
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Tidskriftsartikel (refereegranskat)abstract
- The lacto-series gangliosides 3'-isoLM1 and 3',6'-isoLD1 have been identified as tumor-associated antigens whose formation is initiated by the Lc3-synthase. Until now, high-affinity IgG monoclonal antibodies (mAbs) against 3'-isoLM1 and 3',6'-isoLD1, which are highly expressed in gliomas, have not been developed, although mAbs against lacto-series gangliosides are powerful tools for functional studies. We previously produced the Lc3-synthase gene beta3Gn-T5 knockout mice. In this study, we immunized beta3Gn-T5 knockout mice with 3'-isoLM1/3',6'-isoLD1 and produced the anti-3'-isoLM1/3',6'-isoLD1 mAb GMab-1, of the IgG(3) subclass, which should be useful for functional analysis of lacto-series gangliosides and for antibody-based therapy of gliomas.
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