An anti-transferrin receptor-avidin fusion protein exhibits both strong proapoptotic activity and the ability to deliver various molecules into cancer cells

Abstract

We have developed an antibody fusion protein (anti-rat TfR IgG3-Av) with the ability to deliver different molecules into cancer cells. It consists of avidin genetically fused to the C(H)3 region of a human IgG3 specific for the rat transferrin receptor. It forms strong, noncovalent interactions with biotinylated molecules such as glucose oxidase and beta-galactosidase, and delivers them into the rat myeloma cell line Y3-Ag1.2.3 through receptor-mediated endocytosis. Importantly, the beta-galactosidase retains activity after internalization. Furthermore, we have unexpectedly discovered that anti-rat TfR IgG3-Av, but not a recombinant anti-rat TfR IgG3 or a nonspecific IgG3-Av, possesses proapoptotic activities against Y3-Ag1.2.3 and the rat T cell lymphoma cell line C58 (NT) D.1.G.OVAR.1. These activities were not observed in two rat cell lines of nonhematopoietic lineage (bladder carcinoma BC47 and gliosarcoma 9L). Anti-human TfR IgG3-Av also demonstrated proapoptotic activity against the human erythroleukemia cell line K562. Studies showed that anti-rat TfR IgG3-Av exists as a dimer, suggesting that cross-linking of the surface transferrin receptor may be responsible for the cytotoxic activity. These findings demonstrate that it is possible to transform an antibody specific for a growth factor receptor that does not exhibit inhibitory activity into a drug with significant intrinsic cytotoxic activity against selected cells by fusing it with avidin. The antitumor activity may be enhanced by delivering biotinylated therapeutics into cancer cells. Further development of this technology may lead to effective therapeutics for in vivo eradication of hematological malignancies, and ex vivo purging of cancer cells in autologous transplantation.

Fig 1.

Flow cytometry demonstrating the internalization of biotinylated FITC complexed with anti-rat TfR IgG3-Av. Eight micrograms of anti-dansyl IgG3-Av (A–D) or anti-rat TfR IgG3-Av (E–H) was complexed with b-FITC and incubated with Y3-Ag1.2.3 cells for 2.5 h on ice (A, B, E, and F), or at 37°C (C, D, G, and H). Cells were then analyzed by flow cytometry either directly (A, C, E, and G) or following protease treatment (B, D, F, and H).

Fig 2.

Internalization of biotinylated FITC complexed with anti-rat TfR IgG3-Av detected by confocal microscopy. Y3-Ag1.2.3 cells incubated with anti-rat TfR IgG3-Av-b-FITC conjugates on ice (A) and at 37°C (B) were analyzed by confocal microscopy. Representative cells are shown. (Scale bar, 2 μm.)

Fig 3.

Internalization of biotinylated β-gal complexed with anti-rat TfR IgG3-Av. Y3-Ag1.2.3 cells were incubated at 37°C, for 3 h with 0.1 μg of anti-dansyl IgG3-Av (narrow line) or anti-rat TfR IgG3-Av (bold line) bound to biotinylated β-gal (1:1 molar ratio). After washes, intracellular β-gal activity was detected using a membrane permeable, fluorogenic β-gal substrate, and flow cytometry.

Fig 4.

Antiproliferative effect of antibody fusion proteins on selected rat cancer cell lines. (A) Y3-Ag 1.2.3 cells were treated with anti-rat TfR IgG3-Av (▪), anti-dansyl IgG3-Av (□), anti-rat TfR IgG2a (▵), anti-rat TfR IgG3 (•), or anti-dansyl IgG3 (○) at various concentrations for 24 h. The cells were then cultured in the presence of [³H]thymidine for an additional 24 h, harvested, and [³H]thymidine incorporation was determined. Each value is the mean of quadruplicate assays expressed as the % control mean (controls are cells treated with buffer alone). (B) Y3-Ag 1.2.3 (▪), BC47 (•), and 9L (▵) cells were treated with various concentrations of anti-rat TfR IgG3-Av for 24 h and processed as described in A.

Fig 5.

Anti-rat TfR IgG3-Av induces apoptosis in selected rat cancer cell lines. Y3-Ag1.2.3 (A and B), C58 (NT) D.1.G.OVAR.1 (C and D), BC47 (E and F), and 9L (G and H) cells were incubated with buffer alone (A, C, E, and G), or 9 nM of anti-rat TfR IgG3-Av (B, D, F, and H) for 48 h. The cells were then washed, stained with Alexa Fluor 488 Annexin V and PI, and analyzed by flow cytometry. The percentage of cells located in each quadrant is shown at the corner.

Fig 6.

Noncovalent association of anti-rat TfR IgG3-Av results in a dimeric structure. Anti-rat TfR IgG3 (173 kDa) and anti-rat TfR IgG3-Av (200 kDa for monomer) were analyzed by FPLC. As a standard the profile of dimeric IgA (360 kDa) and monomeric IgG (150 kDa) separated under identical conditions is shown. Fraction size is 1 ml.

Fig 7.

Antiproliferative effect of anti-human TfR IgG3-Av on K562 cells. K562 cells were treated with buffer (A), 25.9 nM (B), 51.9 nM (C), or 104 nM (D) of anti-human TfR IgG3-Av for 72 h. The cells were then cultured in the presence of [³H]thymidine for another 24 h before being harvested. The amount of [³H]thymidine incorporation is expressed as the % the incorporation seen when cells were treated with buffer.