Recombinant expression of the beta-subunit of HLA-DR10 for the selection of novel lymphoma targeting molecules.

Abstract

Selective high-affinity ligands (SHALs) were selected as substitutes for monoclonal antibodies (mAbs) to deliver radioisotopes to malignant tumors. Because a SHAL (5 KD) is considerably smaller in comparison to an antibody (150 KD), a significant therapeutic index (TI) enhancement for radioimmunotherapy (RIT) is anticipated. The antibody-antigen (Ab-Ag) model system chosen for the development of SHALs consists of Lym-1, a MAb with proven selectivity in non-Hodgkin's lymphoma (NHL) patients and its well-characterized Ag, the beta subunit of HLA DR10. Whereas Lym-1 is readily available, the subunit of HLA-DR10 is not. Native, heterodimeric (alpha and beta subunits) HLA-DR10 can be purified from Raji cells, which are known to overexpress this Ag. Inconsistent homogeneity between preparations of HLA-DR10 solubilized in the presence of detergents prompted us to express a recombinant form of the beta subunit of HLA-DR10 in Escherichia coli. Negligible production yields (<or=50 microg/L) were achieved by the expression of the full-length protein in a soluble form. By contrast, yields of 240 mg/L were obtained by expressing only the extracellular domain (ED) of the beta subunit of HLA-DR10 in an insoluble form (inclusion bodies). The recovery yield of refolded protein was 75%. Circular dichroism (CD) and Lym-1 binding studies indicated that the recombinant ED of the beta subunit of HLA-DR10 was properly folded. Therefore, this recombinant protein can be used as a surrogate for native heterodimeric HLA DR10 for the in vitro selection of SHALs and related targeting molecules.

• Authors: Huguette Albrecht, Monique Cosman, Maria Ngu-Schwemlein, Michele Corzett, Kena W Curran, Cheryl Dolan, Xiangming Fang, Sally J DeNardo, Gerald L DeNardo, Rod Balhorn
• Journal: Cancer biotherapy & radiopharmaceuticals (Cancer Biother Radiopharm) Vol. 22 Issue 4 Pg. 531-42 (Aug 2007) ISSN: 1084-9785 [Print] United States
• PMID: 17803448 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, U.S. Gov't, Non-P.H.S.)

Chemical References

• Antibodies, Monoclonal
• Antibodies, Monoclonal, Murine-Derived
• Epitopes
• HLA-DR Antigens
• HLA-DR Serological Subtypes
• HLA-DR10 antigen
• Ligands
• Lym-1 monoclonal antibody
• Protein Subunits
• Recombinant Proteins

Topics

• Amino Acid Sequence
• Antibodies, Monoclonal (immunology)
• Antibodies, Monoclonal, Murine-Derived
• Cell Line, Tumor
• Circular Dichroism
• Cloning, Molecular
• Epitopes (immunology)
• Escherichia coli
• Gene Expression
• HLA-DR Antigens (chemistry, genetics, immunology, metabolism)
• HLA-DR Serological Subtypes
• Humans
• Ligands
• Lymphoma (immunology, metabolism)
• Models, Molecular
• Molecular Sequence Data
• Protein Conformation
• Protein Subunits (chemistry, genetics, immunology, metabolism)
• Radioimmunotherapy (methods)
• Recombinant Proteins (chemistry, genetics, immunology, metabolism)
• Sensitivity and Specificity
• Structural Homology, Protein
• Substrate Specificity
• Surface Plasmon Resonance