H. Perry Fell

Identification of serum components that inhibit the tumoricidal activity of amphiphilic alpha helical peptides

Antimicrobial peptides that can form amphiphilic alpha helices were tested for their ability to lyse various human tumor cell lines in vitro. These peptides include C18G, whose sequence is a derivative of the carboxyl terminus of human platelet factor IV, and 399, an idealized amphiphilic alpha helix. Both peptides exhibited potent antitumor activity against all cell lines tested, unlike magainin 2, a naturally occurring antimicrobial peptide of similar structure, which was relatively inactive under the same conditions. Also, the lytic activity of C18G is specific for tumor cells versus human red blood cells. The effects of serum can be important when evaluating the potency of lytic peptides, since other tumoricidal peptides have been shown to be completely inactivated by low serum levels. Experiments with C18G and 399 revealed that their activity was indeed reduced in the presence of human serum, but that significant lytic activity remained even at relatively high serum concentrations. Various serum components were tested for their inhibitory activity. Whereas albumin and high-density lipoprotein had only slight inhibitory properties, low-density lipoprotein was found to be a potent inhibitor of peptide-mediated cell lysis. The peptide 399, which is more sensitive to serum inhibition than C18G, also binds more extensively to all serum components tested.

Surface Expression of CD28 Single Chain Fv for Costimulation by Tumor Cells

Single-chain antibody binding fragments (sFv) (Bird et al. 1988) can be directed to distinct cellular compartments where they can cause specific functional alterations based on their binding specificity. For example, intracellular expression of an sFv to the human immunodeficiency virus type-1 (HIV-l) rev regulatory protein prevented viral replication (Duan et al. 1994), and another sFv to the HIV envelope protein reduced infectivity of virus particles (Marasco et al. 1993). This approach has also been used with specific sFv to protect plants from intracellular pathogens (Tavladoraki et al. 1993). To explore the possible uses of sFv as artificial adhesion receptors, we expressed the 2E12 sFv to human CD28 with a transmembrane region or a glycosylphosphatidylinositol (GPI) anchor to generate tumor cells capable of activating the CD28 receptor during adhesion with T cells. In comparison with CD80, a natural adhesion receptor for CD28, the 2E12 sFv showed increased binding affinity for CD28 and was efficient in activating T cells during coculture. These results suggest that cell surface expression of sFv may offer advantages over natural ligands for binding and activation of adhesion receptors. An sFv to the CD28 receptor was chosen for these studies because of the recent experiments showing that tumor cells are not immunogenic when they do not express natural ligands (CD80 or CD86) for CD28 (Baskar et al. 1993). Expression of CD80 or CD86 in experimental murine tumors resulted in the generation of antigen-specific cytotoxic T lymphocytes (CTL) in vivo and allowed acti-

Construction, Expression, and Characterization of Anticarcinoma sFv Fused to IL-2 or GM-CSF

Local production of cytokines by genetically engineered tumor cells decreases their tumorigenicity and elicits protective immune responses against the parental tumor cells. An alternative approach to elicit a therapeutic immune response is to use fusion proteins that can target tumor cells and simultaneously activate effector cells. Fusion proteins between human IL-2, murine or human GM-CSF, and sFv of antihuman carcinoma antibody L6 have been constructed, expressed in both COS and Chinese hamster ovary (CHO) cells, and purified by affinity chromatography. The biologic activity of L6 sFV-hIL-2, L6 sFv-mGM-CSF, and L6 sFv-hGM-CSF was tested on human T cell blasts, factor-dependent FDCP-1, and TF-1 cells, respectively. The ability of soluble L6 sFv-hIL-2, L6 sFv-mGM-CSF, and L6 sFv-hGM-CSF to stimulate the proliferation of the indicator cells was found to be comparable to that of recombinant hIL-2, mGM-CSF, or hGM-CSF. Tumor cells coated with L6 sFV-mGM-CSF or L6 sFv-hGM-CSF were also tested in this way and were found to be potent stimulators, indicating that the cytokines were functionally active when bound to the tumor cell surface. This work demonstrates the feasibility of targeting sFv-cytokine fusion proteins for the activation of effector cells as an alternative to cytokine gene therapy.