A RANTES-antibody fusion protein retains antigen specificity and chemokine function.

The successful eradication of cancer cells in the setting of minimal residual disease may require targeting of metastatic tumor deposits that evade the immune system. We combined the targeting flexibility and specificity of mAbs with the immune effector function of the chemokine RANTES to target established tumor deposits. We describe the construction of an Ab fusion molecule with variable domains directed against the tumor-associated Ag HER2/neu, linked to sequences encoding the chemokine RANTES (RANTES.her2.IgG3). RANTES is a potent chemoattractant of T cells, NK cells, monocytes, and dendritic cells, and expression of RANTES has been shown to enhance immune responses against tumors in murine models. RANTES.her2.IgG3 fusion protein bound specifically to HER2/neu Ag expressed on EL4 cells and on SKBR3 breast cancer cells as assayed by flow cytometry. RANTES.her2.IgG3 could elicit actin polymerization of THP-1 cells and transendothelial migration of primary T lymphocytes. RANTES.her2.IgG3 prebound to SKBR3 cells also facilitated migration of T cells. RANTES.her2.IgG3 bound specifically to the CCR5 chemokine receptor, as demonstrated by flow cytometry, and inhibited HIV-1 infection via the CCR5 coreceptor. RANTES.her2.IgG3, alone or in combination with other chemokine or cytokine fusion Abs, may be a suitable reagent for recruitment and activation of an expanded repertoire of effector cells to tumor deposits.

A B7.1-antibody fusion protein retains antibody specificity and ability to activate via the T cell costimulatory pathway.

We describe the construction and characterization of an Ab fusion protein specific for the tumor-associated Ag HER2/neu linked to sequences encoding the extracellular domain of the B7.1 T cell costimulatory ligand. The Ab domain of the fusion molecule will specifically target HER2/neu-expressing tumor cells, while the B7.1 domain is designed to activate a specific immune response. We show that the B7.1 fusion Ab retained ability to selectively bind to the HER2/neu Ag and to the CTLA4/CD28 counter-receptors for B7.1. Specific T cell activation was observed when the B7.1 Ab fusion protein was bound to HER2/neu-expressing cells. The use of the B7.1 Ab fusion protein may overcome limitations of gene transfer and/or standard Ab therapy and represents a novel approach to the eradication of minimal residual disease.

Immortalized cerebellar cells can be induced to display mature neuronal characteristics.

We have established and characterized a cell line (designated Cb-E1A) that can be induced to display a variety of neuronal characteristics under simple culture conditions. This cell line was generated by retroviral-mediated gene transfer of the adenovirus 12S E1A-immortalizing gene in cerebellar cells isolated from one-week-old rats. Actively dividing cells express the E1A adenovirus protein, and exhibit minimal expression of glial cell markers and low level expression of neuronal cell markers. The immortalized cells can be induced to differentiate by culture in an alternative depolarizing medium or calcium ionophore-containing medium. This caused the expression of neuronal markers to increase rapidly, while glial markers remain unchanged. Under these culture conditions, the Cb-E1A cells also display a variety of other characteristics which suggest that they may provide a good model system for differentiated cerebellar granule neurons. Such neuronal characteristics include a reduction or cessation of mitosis and an increased susceptibility to glutamate toxicity. We think that this novel cell line and differentiation strategy will facilitate future studies of the cellular mechanisms involved in a wide variety of neuronal functions, including development and neurodegenerative disease.