Susanna M. Rybak

Human Ribonuclease A Superfamily Members, Eosinophil-Derived Neurotoxin and Pancreatic Ribonuclease, Induce Dendritic Cell Maturation and Activation

A number of mammalian antimicrobial proteins produced by neutrophils and cells of epithelial origin have chemotactic and activating effects on host cells, including cells of the immune system. Eosinophil granules contain an antimicrobial protein known as eosinophil-derived neurotoxin (EDN), which belongs to the RNase A superfamily. EDN has antiviral and chemotactic activities in vitro. In this study, we show that EDN, and to a lesser extent human pancreatic RNase (hPR), another RNase A superfamily member, activates human dendritic cells (DCs), leading to the production of a variety of inflammatory cytokines, chemokines, growth factors, and soluble receptors. Human angiogenin, a RNase evolutionarily more distant to EDN and hPR, did not display such activating effects. Additionally, EDN and hPR also induced phenotypic and functional maturation DCs. These RNases were as efficacious as TNF-α, but induced a different set of cytokine mediators. Furthermore, EDN production by human macrophages could be induced by proinflammatory stimuli. The results reveal the DC-activating activity of EDN and hPR and suggest that they are likely participants of inflammatory and immune responses. A number of endogenous mediators in addition to EDN have been reported to have both chemotactic and activating effects on APCs, and can thus amplify innate and Ag-specific immune responses to danger signals. We therefore propose these mediators be considered as endogenous multifunctional immune alarmins.

RNA Cleavage and Inhibition of Protein Synthesis by Bleomycin

Bleomycin is a clinically used antitumor antibiotic long thought to function therapeutically at the level of DNA cleavage. Recently, it has become clear that bleomycin can also cleave selected members of all major classes of RNA. Using the computer program COMPARE to search the database established by the Anticancer Drug Screening Program of the National Cancer Institute, a possible mechanism-based correlation was found between onconase, an antitumor ribonuclease currently being evaluated in phase III clinical trials, and the chemotherapeutic agent bleomycin. Following these observations, experimentation revealed that bleomycin caused tRNA cleavage and DNA-independent protein synthesis inhibition in rabbit reticulocyte lysate and when microinjected into Xenopus oocytes. The correlation of protein synthesis inhibition to the previously reported site-specific RNA cleavage caused by bleomycin supports the thesis that RNA cleavage may constitute an important element of the mechanism of action of bleomycin.

Growth effects of lithium chloride in BALB/c 3T3 fibroblasts and Madin-Darby canine kidney epithelial cells.

Abstract The ability of LiCl to initiate DNA synthesis was studied in Madin-Darby canine kidney (MDCK) cells, and mouse BALB/c 3T3 fibroblasts. In a defined culture medium lacking serum, LiCl increased DNA synthesis in BALB/c 3T3 cells 100–200% over control values. Maximum DNA synthesis was observed with concentrations of LiCl between 10 and 25 mM and increases from 40–50% over control were observed with concentrations as low as 1 mM. Exposure of BALB/c 3T3 cultures to LiCl resulted in an increase in the percentage of cells initiating DNA synthesis, total DNA content and cell number. Lithium chloride, in combination with insulin or epidermal growth factor (EGF), had either an additive or synergistic effect upon the growth of BALB/c 3T3 fibroblasts. MDCK cells proved refractory to the growth actions of LiCl, although they responded to EGF and insulin with increased DNA synthesis. Lithium chloride appears to have a direct effect on cell proliferation in some but not all cell types.

Antitransferrin receptor antibody-RNase fusion protein expressed in the mammary gland of transgenic mice.

Antibodies fused to human enzymes offer an alternative to specifically targeting tumors with antibodies linked to plant or bacterial toxins. Since large amounts of these reagents can be administered without eliciting non-specific toxicities, efficient methods of production are needed. The goal of this work was to express a complex immunoenzyme fusion protein (immunotoxin) in the mammary gland of transgenic mice. A chimeric mouse/human antibody directed against the human transferrin receptor (E6) was fused at its CH2 domain to the gene for a human angiogenic ribonuclease, angiogenin (Ang). It was expressed in the mammary gland of mice and secreted into mouse milk. Expression levels in milk were approximately 0.8 g/l. The chimeric protein retained antibody binding activity and protein synthesis inhibitory activity equivalent to that of free Ang. It was specifically cytotoxic to human tumor cells in vitro.

Specifically Targeting the CD22 Receptor of Human B-cell Lymphomas with RNA Damaging Agents: A New Generation of Therapeutics

Targeting CD22 on human B-cells with a monoclonal antibody conjugated to a cytotoxic RNAse causes potent and specific killing of the lymphoma cells in vitro. This translates to anti-tumor effects in human lymphoma models in SCID mice. RNA damage caused by RNAses could be an important alternative to standard DNA-damaging chemotherapeutics. A second generation construct with an improved recombinant cytotoxic RNAse is described. Targeted RNAses may overcome problems of toxicity and immunogenicity associated with plant or bacterial toxin-containing immunoconjugates.

Targeting malignant B-cell lymphoma with a humanized anti-CD22 scFv-angiogenin immunoenzyme‡

We report on the generation and functional characterization of a humanized immunoenzyme comprising a stable humanized single chain Fv (scFv) with grafted specificity of the anti‐CD22 murine monoclonal antibody RFB4 and the human ribonuclease angiogenin (ANG). The fusion protein produced from transiently transfected mammalian Chinese hamster ovary cells could easily be purified to homogeneity, retained full ribonucleolytic activity, and efficiently killed CD22+ tumour cells with an IC50 of 56u2003nmol/l. In contrast, incubation of tumour cells with either ANG or scFv alone did not result in any cytotoxicity. Potent receptor‐mediated killing of target cells, expected lack of extracellular toxicity, predictable low immunogenic potential, and ease of production, suggest that this novel immunoenzyme has potential for the immunotherapy of CD22+ malignancies.

A dimeric angiogenin immunofusion protein mediates selective toxicity toward CD22+ tumor cells.

To improve selective cytotoxicity and pharmacokinetics of an anti-CD22 antibody single chain Fv (scFv)-ribonuclease fusion protein, a dimeric derivative was generated. Human angiogenin was fused via a (G4S)3 spacer peptide to the carboxy-terminal end of the stable dimeric anti-CD22 VL-VH zero-linker scFv MLT-7. The dimeric fusion protein and a monovalent counterpart were produced as soluble proteins in the periplasm of Escherichia coli. Comparative studies with homogenously purified fusion proteins revealed that both constructs specifically bound to the target antigen and retained ribonucleolytic activity. However, they exhibited a markedly different capability for killing CD22+ tumor cells. The monomeric construct inhibited protein synthesis of target cells in a dose-dependent manner, but 50% inhibition (IC50) could be achieved only at the highest tested concentration (>350 nM). In contrast, the dimeric fusion protein efficiently killed CD22+ Raji and Daudi tumor cell lines with IC50 values of 74 nM and 118 nM, respectively. These results show that the therapeutic potential of scFv-ANG fusion proteins can be markedly enhanced by engineering dimeric derivatives.

Antibody targeted therapeutics for lymphoma: new focus on the CD22 antigen and RNA

The approval of antibodies for cancer treatment has provoked increased interest in the development of new and improved antibody-mediated therapies. This emerging approach centres on targeting CD22 on human B-cells with a monoclonal antibody (mAb). Anti-CD22 antibodies conjugated to a cytotoxic RNAse elicits potent and specific killing of the lymphoma cells in vitro and in human lymphoma models in severe combined immune deficiency (SCID) mice. RNA damage caused by RNAses could be an important alternative to standard DNA damaging chemotherapeutics. Moreover, targeted RNAses may overcome problems of toxicity and immunogenicity associated with plant- or bacterial toxin-containing immunotoxins.

RNA Damage and Inhibition of Neoplastic Endothelial Cell Growth: Effects of Human and Amphibian Ribonucleases

Abstract Newton, D. L., Kaur, G., Rhim, J. S., Sausville, E. A. and Rybak, S. M. RNA Damage and Inhibition of Neoplastic Endothelial Cell Growth: Effects of Human and Amphibian Ribonucleases. Angiogenesis defines the many steps involved in the growth and migration of endothelial cell-derived blood vessels. This process is necessary for the growth and metastasis of tumors, and considerable effort is being expended to find inhibitors of tumor angiogenesis. This usually involves screening of potential anti-angiogenic compounds on endothelial cells. To this end, two candidate anti-angiogenic RNA-damaging agents, onconase and (-4)rhEDN, were screened for their effects on endothelial cell proliferation using three distinct types of endothelial cells in culture: HPV-16 E6/E7-immortalized human umbilical vein endothelial cells (HUVECs), a Kras-transformed HPV-16 E6/E7 HUVEC (Rhim et al., Carcinogenesis 4, 673–681, 1998), and primary HUVECs. Onconase similarly inhibited proliferation in all three cell lines (IC50 = 0.3–1.0 μM) while (-4)rhEDN was more effective on immortalized HUVEC cell lines (IC50 = 0.02–0.06 μM) than on primary HUVECs (IC50 > 0.1 μM). Differential sensitivity to these agents implies that more than one endothelial cell type must be used in proliferation assays to screen for novel anti-angiogenic compounds.

Anti-CD22 Onconase: Preparation and Characterization

Antibodies can be conjugated to effector molecules to derive targeted therapeutics with properties such as cell-specific cytotoxicity. The murine anti-CD22 antibody RFB4 linked to a member of the ribonuclease A superfamily, Onconase (Onc), becomes a potential drug candidate for non-Hodgkins lymphoma. Onc is currently in Phase III clinical trials for unresectable malignant mesothelioma but conjugation to RFB4 considerably enhances its specificity for CD22+ lymphomas. RFB4-targeted Onc is effective in preclinical models, causes little non-specific toxicities in mice, and has favorable formulation properties. Derivatization and conjugation of RFB4 and Onc have been optimized.