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

Cancer Biother Radiopharm. 2007 Aug;22(4):531-42. doi: 10.1089/cbr.2007.375A.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • 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
  • HLA-DR Antigens / genetics*
  • HLA-DR Antigens / immunology
  • HLA-DR Antigens / metabolism*
  • HLA-DR Serological Subtypes
  • Humans
  • Ligands
  • Lymphoma / immunology*
  • Lymphoma / metabolism*
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Conformation
  • Protein Subunits / chemistry
  • Protein Subunits / genetics
  • Protein Subunits / immunology
  • Protein Subunits / metabolism
  • Radioimmunotherapy / methods*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • Recombinant Proteins / metabolism
  • Sensitivity and Specificity
  • Structural Homology, Protein
  • Substrate Specificity
  • Surface Plasmon Resonance

Substances

  • 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