Onconase responsive genes in human mesothelioma cells: implications for an RNA damaging therapeutic agent.

BACKGROUND: Onconase represents a new class of RNA-damaging drugs. Mechanistically, Onconase is thought to internalize, where it degrades intracellular RNAs such as tRNA and double-stranded RNA, and thereby suppresses protein synthesis. However, there may be additional or alternative mechanism(s) of action. METHODS: In this study, microarray analysis was used to compare gene expression profiles in untreated human malignant mesothelioma (MM) cell lines and cells exposed to 5 microg/ml Onconase for 24 h. A total of 155 genes were found to be regulated by Onconase that were common to both epithelial and biphasic MM cell lines. Some of these genes are known to significantly affect apoptosis (IL-24, TNFAIP3), transcription (ATF3, DDIT3, MAFF, HDAC9, SNAPC1) or inflammation and the immune response (IL-6, COX-2). RT-PCR analysis of selected up- or down-regulated genes treated with varying doses and times of Onconase generally confirmed the expression array findings in four MM cell lines. RESULTS: Onconase treatment consistently resulted in up-regulation of IL-24, previously shown to have tumor suppressive activity, as well as ATF3 and IL-6. Induction of ATF3 and the pro-apoptotic factor IL-24 by Onconase was highest in the two most responsive MM cell lines, as defined by DNA fragmentation analysis. In addition to apoptosis, gene ontology analysis indicated that pathways impacted by Onconase include MAPK signaling, cytokine-cytokine-receptor interactions, and Jak-STAT signaling. CONCLUSIONS: These results provide a broad picture of gene activity after treatment with a drug that targets small non-coding RNAs and contribute to our overall understanding of MM cell response to Onconase as a therapeutic strategy. The findings provide insights regarding mechanisms that may contribute to the efficacy of this novel drug in clinical trials of MM patients who have failed first line chemotherapy or radiation treatment.

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-Hodgkin's 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.

Targeted therapeutic RNases (ImmunoRNases).

Immunotoxins combining antibody specificity with bacterial or plant toxins are limited by their strong immunogenicity and non-specific toxicity. Ribonucleases of the RNase A protein superfamily provide a solution to address these issues because they show potent antineoplastic activity on cell internalization but do not show appreciable immunogenicity or non-specific toxicity. Their therapeutic value is demonstrated by RNase derived from the frog (Rana pipiens), Onconase (ONC, Alfacell, Inc., New Jersey, USA), the first and only RNase being evaluated in clinical trials at present. Conjugation or fusion of RNases to tumor specific antibodies is a promising approach to further boost tumor cell killing of these compounds. This review focuses on 'targeted RNases/ImmunoRNases' as promising novel anticancer therapeutics.

Antibody-targeted RNase fusion proteins (immunoRNases) for cancer therapy.

Ribonucleases (RNases) of the superfamily A exhibit potent antineoplastic activity yet do not mediate appreciable immunogenicity or non-specific toxicity in both animal models and cancer patients. Ranpirnase (Onconase), the first ribonuclease being evaluated as a therapeutic in humans, has progressed to phase III clinical trials in patients with unresectable mesothelioma. Conjugation of RNases to internalizing tumor-targeting monoclonal antibodies was shown to enhance specific cell killing by several orders of magnitude both in vitro and in animal models. In this review we describe the development and current status of genetically engineered 2(nd) generation immunoRNases as promising novel anti-cancer therapeutics.

Cross-reactive HIV-1 neutralizing monoclonal antibodies selected by screening of an immune human phage library against an envelope glycoprotein (gp140) isolated from a patient (R2) with broadly HIV-1 neutralizing antibodies.

Elicitation of broadly cross-reactive neutralizing antibodies (bcnAbs) in HIV infections is rare. To test the hypothesis that such antibodies could be elicited by HIV envelope glycoproteins (Envs) with unusual immunogenic properties and to identify novel bcnAbs, we used a soluble Env ectodomain (gp140) from a donor (R2) with high level of bcnAbs as an antigen for panning of an immune phage-displayed antibody library. The panning with the R2 Env resulted in significantly higher number of cross-reactive antibody clones than by using Envs from two other isolates (89.6 and IIIB). Two of the identified human monoclonal antibodies (hmAbs), m22 and m24, had sequences, neutralizing and binding activities similar or identical to those of the gp120-specific bcnAbs m18 and m14. The use of the R2 Env but not other Envs for panning resulted in the identification of a novel gp41-specific hmAb, m46. For several of the tested HIV-1 primary isolates its potency on molar basis was comparable to that of T20. It inhibited entry of primary isolates from different clades with an increased activity for cell lines with low CCR5 surface concentrations. The m46 neutralizing activity against a panel of clade C isolates was significantly higher in an assay based on peripheral blood mononuclear cells (4 out of 5 isolates were neutralized with an IC(50) in the range from 1.5 to 25 microg/ml) than in an assay based on a cell line with relatively high concentration of cell-surface-associated CCR5. In contrast to 2F5 and Z13, this antibody did not bind to denatured gp140 and gp41-derived peptides indicating a conformational nature of its epitope. It bound to a 5-helix bundle but not to N-heptad repeat coiled coils and a 6-helix bundle construct indicating contribution of both gp41 heptad repeats to its epitope and to a possible mechanism of neutralization. These results indicate that the R2 Env may contain unique exposed conserved epitopes that could contribute to its ability to elicit broadly cross-reactive antibodies in animals and humans; the newly identified antibodies may help in the development of novel vaccine immunogens and therapeutics.

Proteomic analysis of plasma membrane from hypoxia-adapted malignant melanoma.

Hypoxic conditions often persist within poorly vascularized tumors. At the cellular level constitutive activation of transcriptional regulators of the hypoxic response leads to the emergence of clones with aggressive phenotypes. The primary interface between the cell and the hypoxic environment is the plasma membrane. A detailed investigation of this organelle is expected to yield further targets for therapeutic perturbation of the response to hypoxia. In the present study, quantitative proteomic analysis of plasma membrane from hypoxia-adapted murine B16F10 melanoma was performed using differential 16O/18O stable isotopic labeling and multidimensional liquid chromatography-tandem mass spectrometry. The analysis resulted in the identification of 24,853 tryptic peptides, providing quantitative information for 2,433 proteins. For a subset of plasma membrane and secreted proteins, quantitative RT-PCR was used to gain further insight into the genomic regulatory events underlying the response to hypoxia. Consistent increases at the proteomic and transcriptomic levels were observed for aminopeptidase N (CD13), carbonic anhydrase IX, potassium-transporting ATPase, matrix metalloproteinase 9, and stromal cell derived factor I (SDF-1). Antibody-based analysis of a panel of human melanoma cell lines confirmed that CD13 and SDF-1 were consistently upregulated during hypoxia. This study provides the basis for the discovery of novel hypoxia-induced membrane proteins.

Efficient killing of CD22+ tumor cells by a humanized diabody-RNase fusion protein.

We report on the generation of a dimeric immunoenzyme capable of simultaneously delivering two ribonuclease (RNase) effector domains on one molecule to CD22(+) tumor cells. As targeting moiety a diabody derived from the previously humanized scFv SGIII with grafted specificity of the murine anti-CD22 mAb RFB4 was constructed. Further engineering the interface of this construct (V(L)36(Leu-->Tyr)) resulted in a highly robust bivalent molecule that retained the same high affinity as the murine mAb RFB4 (K(D)=0.2 nM). A dimeric immunoenzyme comprising this diabody and Rana pipiens liver ribonuclease I (rapLRI) was generated, expressed as soluble protein in bacteria, and purified to homogeneity. The dimeric fusion protein killed several CD22(+) tumor cell lines with high efficacy (IC(50)=3-20 nM) and exhibited 9- to 48-fold stronger cytotoxicity than a monovalent rapLRI-scFv counterpart. Our results demonstrate that engineering of dimeric antibody-ribonuclease fusion proteins can markedly enhance their biological efficacy.

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 homogeneously 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.

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 IC(50) of 56 nmol/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.

Antigen binding and stability properties of non-covalently linked anti-CD22 single-chain Fv dimers.

By varying linker length and domain orientation three multivalent derivatives of a monovalent anti-CD22 single-chain fragment variable (scFv) antibody were generated. Shortening the linker of the V(H)-V(L) oriented scFv to 5 or 0 residues resulted in the formation of diabodies or a mixture of tetramers and trimers, respectively. Unexpectedly, a V(L)-0-V(H) scFv assembled to homogenous dimers, remained substantially more stable than the V(H)-5-V(L) diabody when incubated in human serum at 37 degrees C, and retained its dimeric state when concentrated up to 4 mg/ml. These properties suggest the V(L)-0-V(H) scFv could become an attractive vehicle for the selective delivery of multiple effector molecules to CD22(+) tumor cells.

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-alpha, 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.

Specificity grafting of human antibody frameworks selected from a phage display library: generation of a highly stable humanized anti-CD22 single-chain Fv fragment.

A prerequisite for the enrichment of antibodies screened from phage display libraries is their stable expression on a phage during multiple selection rounds. Thus, if stringent panning procedures are employed, selection is simultaneously driven by antigen affinity, stability and solubility. To take advantage of robust pre-selected scaffolds of such molecules, we grafted single-chain Fv (scFv) antibodies, previously isolated from a human phage display library after multiple rounds of in vitro panning on tumor cells, with the specificity of the clinically established murine monoclonal anti-CD22 antibody RFB4. We show that a panel of grafted scFvs retained the specificity of the murine monoclonal antibody, bound to the target antigen with high affinity (6.4-9.6 nM), and exhibited exceptional biophysical stability with retention of 89-93% of the initial binding activity after 6 days of incubation in human serum at 37 degrees C. Selection of stable human scaffolds with high sequence identity to both the human germline and the rodent frameworks required only a small number of murine residues to be retained within the human frameworks in order to maintain the structural integrity of the antigen binding site. We expect this approach may be applicable for the rapid generation of highly stable humanized antibodies with low immunogenic potential.

Generation of a highly stable, internalizing anti-CD22 single-chain Fv fragment for targeting non-Hodgkin's lymphoma.

The generation of a single chain Fv (scFv) fragment derived from the anti-CD22 monoclonal antibody LL2 resulted in a molecule with good antigen binding but very poor stability properties, thus hampering its clinical applicability. Here we report on the construction of an engineered LL2 scFv fragment by rational mutagenesis. The contribution of uncommon wild-type sequence residues for providing stability to the conserved common core structure of immunoglobulins was examined. Aided by computer homology modeling, 3 destabilizing residues within the core of the wild-type VH domain were identified. Owing to the conserved nature of the buried core structure, mutagenesis of these sites to respective consensus residues markedly stabilized the molecule but did not influence its antigen binding properties: the engineered scFv MJ-7 exhibited exceptional biophysical stability with a half-life not reached after 6 days of incubation in human serum at 37 degrees C, while fully retaining the epitope specificity of the monoclonal antibody, and antigen binding affinity of the wild-type scFv. Furthermore, both the monoclonal antibody LL2 and the engineered scFv fragment became fully internalized after only 30 min of incubation at 37 degrees C with CD22+ tumor cells. These properties predict scFv MJ-7 could become a novel powerful tool to selectively deliver cytotoxic agents to malignant CD22+ cells.

"Vasocrine" formation of tumor cell-lined vascular spaces: implications for rational design of antiangiogenic therapies.

Here we report that B16F10 murine melanoma cells mimic endothelial cell behavior and the angiogenic process in vitro and in vivo. Cord formation in vitro by tumor cells is stimulated by hypoxia and vascular endothelial growth factor (VEGF) and inhibited by antibodies against VEGF and the VEGF KDR receptor (VEGF receptor 2). We define regulation of tumor cell-derived vascular space formation by these vasoactive compounds as "vasocrine" stimulation. ICRF 159 (Razoxane; NSC 129943) prevents tumor cell but not endothelial cell cord formation in vitro, and the antiangiogenic drug TNP-470 (NSC 642492) inhibits endothelial but not tumor cell cord formation in vitro. Both drugs inhibit formation of blood-filled vascular spaces in vivo. These results bear on the anticipated action of ICRF 159 in human clinical trials and novel strategies for targeting tumor blood supplies.

Ribonuclease is partly responsible for the HIV-1 inhibitory effect activated by HLA alloantigen recognition.

OBJECTIVE: This study was performed to determine whether ribonucleases (RNases) contribute to the soluble HIV-1 inhibitory activity that results from the recognition of HLA alloantigens. DESIGN AND METHODS: Supernatants from mixed lymphocyte reactions of peripheral blood mononuclear cells from healthy HLA-discordant individuals exhibited HIV-1 inhibitory activity (alloantigen-stimulated factors; ASF). These supernatants were tested for their sensitivity to heating (90 degrees C for 3 min), and for the presence of three RNases belonging to the RNase A superfamily: eosinophil-derived neurotoxin (EDN); RNase A; and angiogenin. Polyclonal antibodies specific for these RNases were used for Western blot analysis of the ASF, as well as for blocking the HIV-1 inhibitory activity of ASF. In addition, an RNase inhibitor (RI) was used to determine whether the anti-viral activity of ASF was due to RNase activity. RESULTS: HIV-1 inhibitory activity of ASF was: (i). resistant to heat treatment; (ii). blocked by 58% with an antibody specific for EDN, but not with antibodies against RNase A or angiogenin; and (iii) blocked by 65-100% with an RI. Moreover, Western blot analysis with an anti-EDN antibody detected EDN in the ASF. CONCLUSION: These findings indicate that the majority of the soluble HIV-1 inhibitory activity contained in the supernatants of mixed lymphocyte reactions is due to EDN or a closely related RNase.

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.

Crystallographic and functional studies of a modified form of eosinophil-derived neurotoxin (EDN) with novel biological activities.

The crystal structure of a post-translationally modified form of eosinophil-derived neurotoxin (EDN) with four extra residues on its N terminus ((-4)EDN) has been solved and refined at atomic resolution (1 A). Two of the extra residues can be placed unambiguously, while the density corresponding to two others is poor. The modified N terminus appears to influence the position of the catalytically important His129, possibly explaining the diminished catalytic activity of this variant. However, (-4)EDN has been shown to be cytotoxic to a Kaposi's sarcoma tumor cell line and other endothelial cell lines. Analysis of the structure and function suggests that the reason for cytotoxicity is most likely due to cellular recognition by the N-terminal extension, since the intrinsic activity of the enzyme is not sufficient for cytotoxicity and the N-terminal extension does not affect the conformation of EDN.

Preparation of recombinant RNase single-chain antibody fusion proteins.

This article describes the construction, expression, and purification of RNase single-chain antibody fusion proteins. To construct a fusion protein, the gene for each moiety, the RNase and the binding ligand, is modified separately to contain complementary DNA encoding a 13 amino acid spacer that separates the RNase from the binding moiety. Appropriate restriction enzyme sites for cloning into the vector are also added. The modified DNA is combined and fused using the PCR technique of splicing by overlap extension (1). The resulting DNA construct is expressed in inclusion bodies in BL21(DE3) bacteria that are specifically engineered for the expression of toxic proteins (2). After isolation and purification of the inclusion bodies, the fusion protein is solubilized, denatured, and renatured. The renatured RNase fusion protein mixture is purified to homogeneity by two chromatography steps. The first column, a CM-Sephadex C-50 or a heparin Sepharose column, eliminates the majority of contaminating proteins while the second column, an affinity column (Ni2+-NTA agarose), results in the final purification of the RNase fusion protein.