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

Abstract

Specific gene transfer into targeted tumor cells remains a critical issue for the development of systemic gene therapy protocols. With this end in view, we have tested the possibility of selectively directing genes to tumor cells through the recognition of tumor-associated antigens (TAA). This was approached in vitro on four human renal cell carcinoma (RCC) lines by means of the highly specific mouse G250 monoclonal antibody (mAb) chemically conjugated 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 human RCCs was extensively characterized, including during clinical trials. Epifluorescence microscopy analysis indicated that upon specific binding to G250 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 unconjugated G250 mAb or G250 mAb conjugated to plasmid DNA remained in the perinuclear region of the cells for Ն20 hours and were not rapidly translocated to lysosomes or recycled to the plasma membrane. In contrast, unconjugated plasmid DNA was not internalized. After transfection 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 abrogated by inhibiting the internalization process. Altogether, our data showed that endocytosis of G250 TAA should be the basis of gene transfer to RCC, suggesting that targeting of TAA capable of internalization may be the basis of new approaches for designing alternative cancer gene therapy procedures.Key words: Renal cell carcinoma; tumor-associated antigen; endocytosis; antifection; gene therapy.M ost ongoing clinical trials in cancer gene therapy are based primarily on direct intratumoral administration of recombinant vectors and/or on ex vivo cell manipulations. 1 To date, only a few procedures permit cell-or tissue-specific targeting in vivo. This can be achieved with specific viral-based gene delivery systems 2,3 and nonviral approaches through DNA complexed to a ligand capable of binding the surface molecules expressed on the targeted cells. 4 -6 We have developed an approach named antifection that allows specific gene targeting. This technique is based on the use of antibodies (Abs) directed toward cell-specific receptors, which are chemically conjugated to plasmid DNA. This was first validated by using various reporter genes expressed in lymphoid cells, both in vitro and in vivo. 7,8 Recently, we have also reported the successful transfer of interleukin-3 (IL-3)-encoding plasmid DNA linked to anti-CD117 Abs into cultured human primary hemopoietic progenitors. 9 Although most of the ligands examined so far continue to be expressed on nontumor cells, limiting the tumor targeting, several Abs directed to tumor-associated antigen (TAA) present a widely restricted...

Chemosensitization by erythropoietin through inhibition of the NF-κB rescue pathway

“…Human renal carcinoma cell lines (RCC, kindly obtained from E Angevin, IGR, France, were maintained in culture as described (Durrbach et al, 1999). The presence of EPO receptors was determined by cytofluorometry using a specific mouse antihuman EPO receptor mAb IgG2b (R&D Systems) or a control isotypic IgG2b mAb (Caltag Laboratories), both revealed with an FITC-labeled goat anti-mouse IgG2b (Jackson Immuno Research Laboratories).…”

Generation of RAGE‐1 and MAGE‐9 peptide‐specific cytotoxic T‐Lymphocyte lines for transfer in patients with renal cell carcinoma

“…Besides application of chemokines, application of antibodies, particularly anti-G250, 30 which is expressed by >95% of RCCs, 31 should be mentioned. The antibody becomes internalized, 32 and application of 125 I-coupled G250 has been reported to retard tumor progression. [33][34][35] Cell-mediated immunotherapeutic protocols in RCC have been based on tumor lysate-loaded DCs.…”

Fabrication of DNA-antibody–apatite composite layers for cell-targeted gene transfer

“…Antibody's facilitating effects on gene transfer have also been observed for conventional gene transfer systems using particulate complexes of an antibody and DNA [19][20][21] or those further combined with viruses [22], cationic polymers [23], lipids [24] and/or calcium phosphates [25]. For example, Kozlova et al showed that the covalent attachment of a CD11c antibody onto calcium phosphate-based nanoparticles increased gene transfer efficiency for the CD11c-positive cells [25].…”

“…The role of the immobilized antibody in gene transfer has not yet been fully clarified, but we believe that antigen-antibody binding at the cell-layer interface could be responsible for the increased gene transfer capability of the DA-Ap layer compared with that of the D-Ap layer. Antigen-antibody binding can facilitate gene transfer by enhancing the adhesion of cells to the DA-Ap layer and/or by promoting endocytosis by the cell [20,41].…”

“…Cell-targeted gene transfer utilizing an antibody has been extensively studied in the past, with attention to gene therapy applications [19][20][21][22][23][24][25]. However, these previous studies were carried out on conventional gene transfer systems using particulate complexes of antibody and plasmid DNA [19][20][21] or those further combined with viruses [22], cationic polymers [23], lipids [24] or calcium phosphates [25]. To the best of our knowledge, no attempts have been made to create a cell-targeted gene transfer system using apatite-based composite layers.…”