Affinity-matured recombinant immunotoxin targeting gangliosides 3'-isoLM1 and 3',6'-isoLD1 on malignant gliomas.

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 KD of DmAb14m-IT for gangliosides 3'-isoLM1 and 3',6'-isoLD1 was 2.6 × 10(-9)M. Also, DmAb14m-IT showed a significantly higher internalization rate in cells expressing 3'-isoLM1 and 3',6'-isoLD1. The DmAb14m-IT IC 50 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.

• Authors: Hailan Piao, Chien-Tsun Kuan, Vidya Chandramohan, Stephen T Keir, Charles N Pegram, Xuhui Bao, Jan-Eric Månsson, Ira H Pastan, Darell D Bigner
• Journal: mAbs (MAbs) 2013 Sep-Oct Vol. 5 Issue 5 Pg. 748-62 ISSN: 1942-0870 [Electronic] United States
• PMID: 23924792 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• Complementarity Determining Regions
• Gangliosides
• Immunotoxins
• LM1 ganglioside
• Recombinant Proteins

Topics

• Amino Acid Sequence
• Animals
• Antibody Affinity (immunology)
• Antibody Specificity (immunology)
• Brain Neoplasms (immunology, pathology, therapy)
• Cell Line, Tumor
• Cell Survival (immunology)
• Complementarity Determining Regions (genetics, immunology)
• Flow Cytometry
• Gangliosides (immunology)
• Glioma (immunology, pathology, therapy)
• HEK293 Cells
• Heterografts
• Humans
• Immunohistochemistry
• Immunotherapy (methods)
• Immunotoxins (genetics, immunology, therapeutic use)
• Mice
• Molecular Sequence Data
• Mutation
• Recombinant Proteins (immunology, therapeutic use)
• Sequence Homology, Amino Acid
• Surface Plasmon Resonance