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 CH3 region of a human IgG3 specific for the rat transferrin receptor. It forms strong, noncovalent interactions with biotinylated molecules such as glucose oxidase and ␤-galactosidase, and delivers them into the rat myeloma cell line Y3-Ag1.2.3 through receptor-mediated endocytosis. Importantly, the ␤-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 gliosarcoma9L).Anti-humanTfRIgG3-Avalsodemonstratedproapoptotic 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.

A neutralizing monoclonal antibody (mAb A24) directed against the transferrin receptor induces apoptosis of tumor T lymphocytes from ATL patients

“…It was recently shown that a nonblocking antirat TfR engineered with avidin (anti-TfR-Av) also triggers TfR endocytosis, inhibits cell proliferation, and induces apoptosis against myeloma cell line Y3-Ag1.2.3 and rat T-cell lymphoma cell line C58 (NT) D1.G.OVAR.1 cells, but only when aggregated. 25 Interestingly, A24 did not require aggregation to induce apoptosis.…”

“…[23][24][25] It has been shown that neutralizing anti-TfR antibodies can block the interaction between transferrin and TfR, and consequently iron uptake, leading to iron deprivation and negative regulation of cell growth. In this work, we have investigated the level of expression of TfR in tumor cells from acute and chronic ATL forms and the in vitro impact of a new neutralizing anti-TfR monoclonal antibody (mAb), A24, 26 on cell proliferation and survival.…”

“…It was recently shown that a nonblocking antirat TfR engineered with avidin (anti-TfR-Av) also triggers TfR endocytosis, inhibits cell proliferation, and induces apoptosis against myeloma cell line Y3-Ag1.2.3 and rat T-cell lymphoma cell line C58 (NT) D1.G.OVAR.1 cells, but only when aggregated. 25 Interestingly, A24 did not require aggregation to induce apoptosis.A24 is highly efficient, although its affinity for TfR is lower than that of Tf. Interestingly, we demonstrated that, in contrast to Tf, the apparent affinity (avidity) of A24 for TfR significantly increased with the density of the receptor, a consequence of the bivalency of the mAb.…”

High-level expression of tamavidin 2 in human cells by codon-usage optimization

“…The cells of mammals such as human, mouse, rat, and Chinese hamster serve as hosts for the expression of fusion proteins of biotin-binding proteins and antibodies or other pharmaceutical proteins. [11][12][13][14][15][16][17][18] Given that the codon preferences among these mammals are similar, there is a good chance that mam-tam2A and mam-tam2B would also be highly expressed in the cells of these other mammals. One or two amino acids of mam-tam2A and mam-tam2B could be changed to create derivatives with a lower isoelectric point, 7) reversible biotin-binding capability, 8) or extremely high thermal stability.…”

“…In many cases, the fusion proteins were expressed in mammalian cells so that the characteristics of the fusion partners, which are quite often mammalian proteins, would be fully retained. [11][12][13][14][15][16][17][18] Monoclonal antibodies against targets such as receptors and surface proteins of cancer cells are popular fusion partners, because these fusion proteins can attach to the cancer cells to deliver the biotinylated therapeutics.…”