Affinity-matured recombinant immunotoxin targeting gangliosides 3'-isoLM1 and 3',6'-isoLD1 on malignant gliomas.
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.
Preston Robert Tisch Brain Tumor Center at Duke and Department of Pathology; Duke University Medical Center; Durham, NC USA.
Hailan
H
Piao
Chien-Tsun
CT
Kuan
Vidya
V
Chandramohan
Stephen T
ST
Keir
Charles N
CN
Pegram
Xuhui
X
Bao
Jan-Eric
JE
Månsson
Ira H
IH
Pastan
Darell D
DD
Bigner
D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D001932:Brain Neoplasms / Q000276:immunology* / Q000473:pathology / Q000628:therapy; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000276:immunology; D022801:Complementarity Determining Regions / Q000235:genetics / Q000276:immunology; D005434:Flow Cytometry; D005732:Gangliosides / Q000276:immunology*; D005910:Glioma / Q000276:immunology* / Q000473:pathology / Q000628:therapy; D057809:HEK293 Cells; D064593:Heterografts; D006801:Humans; D007150:Immunohistochemistry; D007167:Immunotherapy / Q000379:methods; D000922:Immunotoxins / Q000235:genetics / Q000276:immunology* / Q000627:therapeutic use; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000276:immunology / Q000627:therapeutic use; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance
D022801:Complementarity Determining Regions; D005732:Gangliosides; D000922:Immunotoxins; C036208:LM1 ganglioside; D011994:Recombinant Proteins
affinity maturation; ganglioside; glioblastoma; recombinant immunotoxin; single-chain variable fragment