Antibody-mediated endocytosis of G250 tumor-associated antigen allows targeted gene transfer to human renal cell carcinoma in vitro

Specific gene transfer into targeted tumor cells remains a critical issue f or the development of systemic gene therapy protocols. With this end in vie w, we have tested the possibility of selectively directing genes to tumor c ells through the recognition of tumor-associated antigens (TAA). This was a pproached in vitro on four human renal cell carcinoma (RCC) lines by means of the highly specific mouse G250 monoclonal antibody (mAb) chemically conj ugated to a plasmid DNA conveying a reporter activity. This mAb directed to a TAA that is present on 95% of primary RCCs and on 60% of metastatic huma n RCCs was extensively characterized, including during clinical trials. Epi fluorescence microscopy analysis indicated that upon specific binding to G2 50 TAA, G250 mAb alone or conjugated to plasmid DNA was internalized by an active endocytic process and colocalized with the transferrin concentrated in the late recycling perinuclear compartment. We also observed that both u nconjugated G250 mAb or G250 mAb conjugated to plasmid DNA remained in the perinuclear region of the cells for greater than or equal to 20 hours and w ere not rapidly translocated to lysosomes or recycled to the plasma membran e. In contrast, unconjugated plasmid DNA was not internalized. After transf ection of G250 TAA-positive RCC lines with G250 mAb conjugated to a plasmid cDNA encoding mouse interleukin-2, a significant and sustained production of mouse interleukin-2 protein was detected from days 5-15 and was abrogate d by inhibiting the internalization process. Altogether, our data showed th at endocytosis of G250 TAA should be the basis of gene transfer to RCC, sug gesting that targeting of TAA capable of internalization may be the basis o f new approaches for designing alternative cancer gene therapy procedures.