Recombinant expression of the beta-subunit of HLA-DR10 for the selection of novel lymphoma targeting molecules.
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.
Albrecht
Huguette
H
Cancer Center, University of California, Davis Medical Center, Sacramento, CA 95816, USA. huguette.albrecht@ucdmc.ucdavis.edu
Cosman
Monique
M
Ngu-Schwemlein
Maria
M
Corzett
Michele
M
Curran
Kena W
KW
Dolan
Cheryl
C
Fang
Xiangming
X
DeNardo
Sally J
SJ
DeNardo
Gerald L
GL
Balhorn
Rod
R
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D058846:Antibodies, Monoclonal, Murine-Derived; D045744:Cell Line, Tumor; D002942:Circular Dichroism; D003001:Cloning, Molecular; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli; D015870:Gene Expression; D006684:HLA-DR Antigens / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D059810:HLA-DR Serological Subtypes; D006801:Humans; D008024:Ligands; D008223:Lymphoma / Q000276:immunology* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D016499:Radioimmunotherapy / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012680:Sensitivity and Specificity; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance