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An HIV-1-based expression vector has been constructed that produces protective genes tightly regulated by HIV-1 Tat and Rev proteins. The vector contains either a single protective gene (HIV-1 gag dominant negative mutant (delta-gag)) or a combination of two different protective genes (delta-gag and eosinophil-derived neurotoxin (EDN), a human ribonuclease) which are expressed from a dicistronic mRNA. After stable transfection of CEM T cells and following challenge with HIV-1, viral production was completely inhibited in cells transduced with the vector producing both delta-gag and EDN and delayed in cells producing delta-gag alone. In addition, cotransfection of HeLa-Tat cells with an infectious HIV-1 molecular clone and either protective vector demonstrated that the HIV-1 packaging signals present in the constructs were functional and allowed the efficient assembly of the protective RNAs into HIV-1 virions, thus potentially transmitting protection to the HIV-1 target cells. [ABSTRACT FROM AUTHOR]

Ribonucleases degrade RNA, now considered an important drug target. The parent member of this protein superfamily is bovine pancreatic RNase A that functions as a digestive enzyme. Other physiological roles and activities have been ascribed to more recently discovered members of this superfamily. Angiogenin was isolated by following angiogenic activity from cell culture media conditioned by colon cancer cells. ONCONASE kills tumor cells in vitro and in vivo and has advanced to a phase IIIb confirmatory clinical trial for the treatment of unresectable malignant mesothelioma. All three of these RNA degrading enzymes have been used to generate immunoRNases; chemical conjugates and ligand-RNase fusion proteins, for cancer therapy. The properties of each of these RNases are described along with the increasingly sophisticated construction of recombinant immunoRNases. The advantages of using RNase as an antibody payload is compared to using plant or bacterial toxins in the construction of immunotoxins, a related strategy for specifically killing malignant cells.

A number of different immunotoxins composed of cell-specific targeting structures coupled to plant or bacterial toxins have increasingly been evaluated for immunotherapy. Because these foreign proteins are highly immunogenic in humans, we have developed a new CD30 ligand-based fusion toxin (Ang-CD30L) using the human RNase angiogenin. The completely human fusion gene was inserted into a pET-based expression plasmid. Transformed Escherichia coli BL21(DE3) were grown under osmotic stress conditions in the presence of compatible solutes. After isopropyl beta-D-thiogalactoside induction, the M(r) 37,000 His(10)-tagged Ang-CD30L was directed into the periplasmic space and functionally purified by a combination of metal ion affinity followed by enterokinase cleavage of the His(10)-Tag and molecular size chromatography. The characteristics of the recombinant protein were assessed by ELISA, flow cytometry, and toxicity assays showing specific activity against CD30(+) Hodgkin-derived cells. Specific binding activity of Ang-CD30L was verified by competition with anti-CD30 monoclonal antibody Ki-4 and commercially available CD30L-CD8 chimeric protein. Ang-CD30L showed RNase activity in vitro. The human recombinant immunotoxin showed significant toxicity toward several CD30-positive cell lines (HDLM-2, L1236, KM-H2, and L540Cy) and exhibited highest cytotoxicity against L540 cells (IC(50) = 8 ng/ml) as determined by cell proliferation assays. CD30 specificity was confirmed by competitive toxicity assays. This is the first report on the specific cytotoxicity of a recombinant completely human fusion toxin with possibly largely reduced immunogenicity for the treatment of CD30-positive malignancies.

LL2, an anti-CD22 monoclonal antibody against B-cell lymphoma, was covalently linked to the amphibian ribonuclease, onconase, a member of the pancreatic RNase A superfamily. LL2 increased in vitro potency (10 000-fold) and specificity against human Daudi Burkitt lymphoma cells while decreasing systemic toxicity of onconase. Monensin further increased potency of LL2-onconase on Daudi cells (IC(50), 20 and 1.5 pM, absence and presence of monensin, respectively). A 1-hour exposure to LL2-onconase was sufficient to kill Daudi cells in culture. These favorable in vitro properties translated to significant antitumor activity against disseminated Daudi lymphoma in mice with severe combined immunodeficiency disease. In mice inoculated with tumor cells intraperitoneally (ip), LL2-onconase (100 microg 5 times ip every day) increased the life span of animals with minimal disease 200%. The life span of mice with advanced disseminated Daudi lymphoma (tumor cells inoculated intravenously) was increased 135%. Mice injected with LL2-onconase tolerated a dose as high as 300 mg/kg. Because both onconase and LL2 are in clinical trials as cancer therapeutics, the covalently linked agents should be considered for treatment of non-Hodgkin lymphoma.

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 (IC(50) = 0.3-1.0 microM) while (-4)rhEDN was more effective on immortalized HUVEC cell lines (IC(50) = 0.02-0.06 microM) than on primary HUVECs (IC(50) > 0.1 microM). 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.

Onconase, an anticancer ribonuclease, damages cellular tRNA and causes caspase-dependent apoptosis in targeted cells (M. S. Iordanov, O. P. Ryabinina, J. Wong, T. H. Dinh, D. L. Newton, S. M. Rybak, and B. E. Magun. Cancer Res. 60, 1983-1994, 2000). The proapoptotic action of onconase depends on its RNase activity, but the molecular mechanisms leading to RNA damage-induced caspase activation are completely unknown. In this study, we have investigated whether onconase activates two signal-transduction pathways commonly stimulated by conventional chemo- and radiotherapy, namely the stress-activated protein kinase (SAPK) cascade and the pathway leading to the activation of nuclear factor-kappa B (NF-kappaB). We found that, in all cell types tested, onconase is a potent activator of SAPK1 (JNK1 and JNK2) and SAPK2 (p38 MAP kinase), but that it is incapable of activating NF-kappaB. Inhibition of p38 MAP kinase activity with a pharmacological inhibitor, SB203580, demonstrated that p38 MAP kinase is not required for onconase cytotoxicity. Using explanted fibroblasts from mice that contain targeted disruption of both jnk1 and jnk2 alleles, we found that JNKs are important mediators of onconase-induced cytotoxicity. Surprisingly, following the immortalization of these same cells with human papilloma virus (HPV16) gene products E6 and E7, additional proapoptotic pathways (exclusive of JNK) were provoked by onconase. Our results demonstrate that onconase may activate proapoptotic pathways in tumor cells that are not able to be accessed in normal cells. These results present the possibility that the cytotoxic activity of onconase in normal cells may be reduced by blocking the activity of JNKs., (Copyright 2000 Academic Press.)

Cytotoxic endoribonucleases (RNases) possess a potential for use in cancer therapy. However, the molecular determinants of RNase-induced cell death are not well understood. In this work, we identify such determinants of the cytotoxicity induced by onconase, an amphibian cytotoxic RNase. Onconase displayed a remarkable specificity for tRNA in vivo, leaving rRNA and mRNA apparently undamaged. Onconase-treated cells displayed apoptosis-associated cell blebbing, nuclear pyknosis and fragmentation (karyorrhexis), DNA fragmentation, and activation of caspase-3-like activity. The cytotoxic action of onconase correlated with inhibition of protein synthesis; however, we present evidence for the existence of a mechanism of onconase-induced apoptosis that is independent of inhibition of protein synthesis. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethyl ketone (zVADfmk), at concentrations that completely prevent apoptosis and caspase activation induced by ligation of the death receptor Fas, had only a partial protective effect on onconase-induced cell death. The proapoptotic activity of the p53 tumor suppressor protein and the Fas ligand/Fas/Fas-associating protein with death domain (FADD)/caspase-8 proapoptotic cascade were not required for onconase-induced apoptosis. Procaspases-9, -3, and -7 were processed in onconase-treated cells, suggesting the involvement of the mitochondrial apoptotic machinery in onconase-induced apoptosis. However, the onconase-induced activation of the caspase-9/caspase-3 cascade correlated with atypically little release of cytochrome c from mitochondria. In turn, the low levels of cytochrome c released from mitochondria correlated with a lack of detectable translocation of proapoptotic Bax from the cytosol onto mitochondria in response to onconase. This suggests the possibility of involvement of a different, potentially Bax- and cytochrome c-independent mechanism of caspase-9 activation in onconase-treated cells. As one possible mechanism, we demonstrate that procaspase-9 is released from mitochondria in onconase-treated cells. A detailed understanding of the molecular determinants of the cytotoxic action of onconase could provide means of positive or negative therapeutic modulation of the activity of this potent anticancer agent.

We describe the use of phage libraries to derive new antibodies against p21Ras to be used for intracellular expression in mammalian cells. A panel of single-chain antibody fragments, binding to Ras, were analyzed and characterized for their capacity to interfere in vitro with (a) the intrinsic GTPase activity of Ras and (b) the binding of Ras to its effector Raf, and were found not to neutralize its function, according to these biochemical criteria. When expressed intracellularly in mouse 3T3 K-Ras transformed cells all the anti-Ras single-chain variable fragments (scFv) tested inhibited cell proliferation, as assessed by bromodeoxyuridine incorporation. Double immunofluorescence analysis of transfected cells using confocal microscopy confirmed that anti-Ras antibody fragments colocalize with endogenous Ras, at subcellular locations where the protein Ras is not normally found. These data suggest that the ability of phage-derived anti-Ras scFv fragments to inhibit the function of Ras in vivo is a rather general and frequent property and that the range of antibodies that can be successfully used for intracellular inhibition studies is much greater than anticipated, exploiting the mode of action of diverting protein traffic.

Selective cytotoxicity is an important goal of specific drug targeting. Toward this end, toxins isolated primarily from higher plants and bacteria have been coupled to monoclonal antibodies (MAbs) and evaluated for their clinical efficacy in cancer, AIDS, and immunological diseases (1,2). Immune responses against murine monoclonal antibodies MAbs (3,4) and antitoxin antibodies have been detected in both animals and humans treated with immunotoxins (ITs) (5-7) and present a major obstacle to the successful application of this technology. Although development of humanized antibodies have alleviated some of these effects (8, and references therein), the toxins themselves remain a problem. Consequently, the identification of human proteins to be used as components of immunoconjugates is highly desirable.

Immunotoxins based on human and humanized ribonuclease may have potential for cancer therapy while exhibiting less toxic side effects and stimulating less of an immune response in humans than immunotoxins based on plant and bacterial toxins (1). Both recombinant RNase fusion proteins (2-4 see also Chapter 6 , this volume) and chemical RNase conjugates have been made and characterized. The cytotoxic potential of targeted ribonuclease was first demonstrated with bovine RNase conjugated to transferrin or an antibody directed against the human transferrin receptor (5). Antibody RNase conjugates have also been shown to have potent anti-tumor activity against human glioma cells in athymic mice (6) and to enhance the activity of vincristine in mdr1 multidrug-resistant colon cancer cells in vitro and in vivo (7). Recently, RNase chemically conjugated to an antibody against CD22 was found to specifically kill Daudi lymphoma cells in cell culture at picomolar concentrations (IC(50), 10-50 pM) and to exhibit potent antitumor activity in SCID mice with disseminated Daudi lymphoma (unpublished data). Methods for linking RNase to specific cell binding ligands are described.

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.

Microinjection of Onconase or RNase A into NIH/3T3 cells was used to study the intracellular actions of these two proteins. Onconase preferentially killed actively growing cells in both microinjection and cell culture experiments. Moreover, agents that increased the number of cells in S phase such as serum or microinjected signal transduction mediators (Ras, protein kinase C, and mitogen-activated protein kinase) enhanced Onconase cytotoxicity. Conversely, agents that decreased these proliferative pathways (dibutyryl cAMP and protein kinase A) correspondingly diminished Onconase cytotoxicity in microinjection experiments. These results were also mimicked in cell culture experiments since log-phase v-ras-transformed NIH/3T3 cells were more sensitive to Onconase (IC50 of 7 microg/ml) than parental NIH/3T3 fibroblasts (IC50 of 40 microg/ml). Based on those data we postulated that Onconase-mediated cell death in NIH/3T3 cells was related to events occurring at two or more points in the cell cycle preferentially associated with late G1/S and S phases. In contrast, quiescent NIH/3T3 cells were more sensitive to microinjected RNase A than log phase cells and positive mediators of proliferative signal transduction did not enhance RNase A-mediated cytotoxicity. Taken together, these results demonstrate that these two RNases use different pathways and/or mechanisms to elicit cytotoxic responses in NIH/3T3 cells. Predictions formulated from these studies can be tested for relevance to RNase actions in different target tumor cells., (Copyright 1999 Academic Press.)

Background: Preparations of human chorionic gonadotropin (hCG) have been shown to exhibit anti-Kaposi's sarcoma (KS) activity, but the identity of the responsible agent(s) remains controversial. One candidate agent is an eosinophil-derived neurotoxin (EDN)-like polypeptide that contaminates preparations of hCG. We have genetically engineered a unique form of hEDN, which is a ribonuclease, and have evaluated the cytotoxic effects of the recombinant protein on KS Y-1 cells and on cells of other cancer types., Methods: The amino-terminus of hEDN was extended by four amino acid residues, corresponding to the proximal part of the hEDN signal peptide (serine, leucine, histidine, and valine; positions -4 to -1, respectively), by use of the polymerase chain reaction and an hEDN complementary DNA. The recombinant protein was isolated from bacterial inclusion bodies. The cytotoxic activity of this hEDN variant, (-4)rhEDN, was tested on KS Y-1 cells and human glioma, melanoma, breast carcinoma, and renal carcinoma cells., Results: Approximately half of the anti-KS activity in a crude commercial preparation of hCG was associated with a polypeptide that reacted with anti-recombinant-hEDN (rhEDN) polyclonal antibodies. Although rhEDN protein displayed little cytotoxicity against KS Y-1 cells (IC50 [50% inhibition concentration] = >100 microg/mL), (-4)rhEDN markedly inhibited cell viability (IC50 = 6 microg/mL). Neither version of rhEDN inhibited the viability of other tested human cancer cell types., Conclusions: A four amino acid extension of the amino-terminus of rhEDN confers cytotoxicity against KS Y-1 cells in vitro. Design of the (-4)rhEDN variant was based on the sequence of a natural human protein associated with hCG. Our results suggest that (-4)rhEDN is one of the agents in hCG responsible for anti-KS activity. A purified molecule is thus available for in vitro and in vivo mechanistic and, possibly, future clinical studies.

Onconase is a cytotoxic ribonuclease with antitumor properties. A semisynthetic gene encoding the entire protein sequence was constructed by fusing oligonucleotides coding for the first 15 and the last 6 of the 104 amino acids to a genomic clone that encoded the remaining amino acid residues [Newton, D. L., et al. (1997) Protein Eng. 10, 463-470]. The resulting protein product expressed in Escherichia coli exhibited little enzymatic or cytotoxic activity due to the unprocessed N-terminal Met amino acid residue. In this study, we demonstrate that modification of the 5'-region of the gene to encode [Met-(-1)]Ser or [Met-(-1)]Tyr instead of the native pyroglutamate results in recombinant onconase derivatives with restored activities. [Met-(-1)]rOnc(E1S) was more active than [Met-(-1)]rOnc(E1Y) in all assays tested. Consistent with the action of native onconase, [Met-(-1)]rOnc(E1S) was a potent inhibitor of protein synthesis in the cell-free rabbit reticulocyte lysate assay, degrading tRNA at concentrations that correlated with inhibition of protein synthesis. An interesting difference between the recombinant onconase derivatives and the native protein was their susceptibility to inhibition by the major intracellular RNase inhibitor, PRI (onconase is refractory to PRI inhibition). [Met-(-1)]rOnc(E1S) and [Met-(-1)]rOnc(E1Y) inhibited protein synthesis in intact SF539 neuroblastoma cells with IC50's very similar to that of onconase (IC50 3.5, 10, and 10 microg/mL after 1 day and 0.16, 0.35, and 2.5 microg/mL after 5 days for onconase, [Met-(-1)]rOnc(E1S), and [Met-(-1)]rOnc(E1Y), respectively). Similar to that of onconase, cytotoxic activity of the recombinant derivatives was potentiated by monensin, NH4Cl, and retinoic acid. Brefeldin A completely blocked the enhancement of cytotoxicity caused by retinoic acid with all three proteins. Thus, drug-induced alterations of the intracellular trafficking of the recombinant derivatives also resembles that of onconase. Stability studies as assessed in serum-containing medium in the presence or absence of cells at 37 degreesC showed that the recombinant proteins were as stable to temperature and cell culture conditions as the native protein. Therefore, exchanging the Glu amino acid residue at the amino terminus of onconase with an amino acid residue containing a hydroxyl group produces recombinant proteins with ribonuclease and cytotoxic properties similar to native onconase.

Background: Immunotoxins based on plant and bacterial proteins are usually very immunogenic. Human ribonucleases could provide an alternative basis for the construction of less immunogenic reagents. Two members of the human RNase family, angiogenin and eosinophil-derived neurotoxin (EDN), have been fused to a single chain antibody against the transferrin receptor, which is known to be internalised by endocytosis. The fusion proteins proved to be very efficient inhibitors of protein synthesis using various cell lines. It is not yet known whether the side effects of angiogenin and EDN will compromise their potential use as immunotoxins., Objectives: The goal of this work was to construct a human immunotoxin with no harmful side effects. Bovine pancreatic ribonuclease has been shown to be as potent as ricin at abolishing protein synthesis on injection into oocytes. We therefore decided to clone its human analogue, which is fairly ubiquitous and per se non-toxic. An immunofusion of human pancreatic RNase with a single chain antibody against the transferrin receptor was tested for its ability to inhibit protein synthesis in three different human tumor cell lines., Study Design: DNA coding for the human pancreatic RNase was cloned partially from a human fetal brain cDNA library and then completed by PCR using a human placental cDNA library as a template. The RNase gene was then fused with a DNA coding for an single chain antibody against the transferrin receptor (CD71). After expressing the fusion protein in E. coli, the gene product was isolated from inclusion bodies and tested for cytotoxicity., Results: This fusion protein inhibited the protein synthesis of three human tumor cell lines derived from a melanoma, a renal carcinoma and a breast carcinoma, with IC50s of 8, 5 and 10 nM, respectively. These values were comparable with those using a similar fusion protein constructed with eosinophil derived neurotoxin (EDN) as the toxic moiety (IC50s of 8, 1.2 and 3 nM, respectively). The slightly lower activities of the human pancreatic RNase-scFv (pancRNase-scFv) with two of the cell lines suggests that fewer molecules are reaching the cytoplasmic compartment, since it was twice as active as EDN-scFv in inhibiting the protein synthesis of a rabbit reticulocyte lysate., Conclusion: These results demonstrate that the human pancreatic RNase, which is expected to have a very low immunogenic potential in humans with no inherent toxicity, may be a potent cytotoxin for tumor cells after antibody targeting.

Background: Onconase, a protein isolated from oocytes and early embryos of the frog Rana pipiens, shares extensive homology with bovine pancreatic ribonuclease (RNase A) and possesses similar enzyme activity. Onconase is cytotoxic toward cancer cells in vitro and exhibits antitumor activity in animal models. In addition, Onconase has been shown to enhance the cytotoxic activity of some chemotherapeutic agents in vitro., Purpose: We studied interactions between the cytotoxic effects of Onconase and the chemotherapeutic agent vincristine (VCR) in the treatment of drug-sensitive and multidrug-resistant human colon carcinoma cells in vitro and in mice., Methods: Transplantable human colon carcinoma cells (HT-29par cells) were infected with a retrovirus containing human mdr1 (also known as MDR1 and PGY1) complementary DNA (encoding P-glycoprotein [P-gp]), and clones that were cross-resistant to colchicine, doxorubicin, and vinblastine were selected (HT-29mdr1 cells). Drug-resistant HT-29mdr1 cells and drug-sensitive HT-29par parental cells were treated with Onconase and/or VCR in vitro at varying concentrations to measure the effects on protein synthesis and cell viability. The impact of Onconase on VCR accumulation in both types of cells was determined in the presence or absence of MRK-16, an anti-P-gp monoclonal antibody capable of reversing the multidrug-resistant phenotype. The antitumor effects of Onconase and/or VCR treatment were assessed in nude mice bearing established HT-29par or HT-29mdr1 intraperitoneal tumors. IC50 values (drug concentrations resulting in 50% inhibition of protein synthesis or cell viability) for Onconase and VCR were determined from semilogarithmic dose-response curves; interactions between the cytotoxic effects of these two agents were evaluated using data from protein synthesis inhibition experiments and a two-way analysis of variance. Survival distributions from in vivo experiments were compared using Cox proportional hazards models., Results: The combination of Onconase and VCR yielded enhanced cytotoxicity in vitro that was independent of P-gp expression. Evaluation of the effects of these two compounds on protein synthesis over a wide range of drug concentrations indicated possible synergistic interactions (i.e., greater than additive effects) in both drug-resistant and drug-sensitive cells. The enhancement of VCR cytotoxicity was dependent on Onconase enzyme activity and was not associated with increased intracellular levels of VCR. Simultaneous treatment of mice bearing HT-29par tumors with Onconase and VCR did not extend their median survival time (MST) significantly (MST with VCR = 66 days; MST with VCR plus Onconase = 69 days; two-tailed P = .57); however, the MST of mice with HT-29mdr1 tumors was extended significantly by this treatment (MST with VCR = 44 days; MST with VCR plus Onconase = 66 days; two-tailed P<.001)., Conclusion: Combined administration of Onconase and VCR yields enhanced cytotoxicity in vitro and in vivo against human colon carcinoma cells that overexpress the mdr1 gene.

The gene for human angiogenin (Ang), a member of the ribonuclease superfamily, was fused to a gene encoding a single-chain antibody (sFv) against the human transferrin receptor. Three Ang single-chain immunofusion proteins (AngsFvs) were constructed with variations in the type of linker connecting the VL and VH chain [EGKSSGSGSESKEF, L1 or (GGGGS)3, L2] as well as with or without a spacer (FB) connecting the Ang and sFv (AngFBsFvL1 or L2; AngsFv(L2)]. Although the nature of the linker did not affect the enzymatic activity of the FB-containing fusion proteins, the fusion protein containing the L2 linker was 2.3-fold more effective than the L1 linker in competing with the labeled monoclonal IgG1 antibody for binding to the transferrin receptor. The fusion protein containing the L2 linker without the FB spacer exhibited a 13-fold decrease in binding to the transferrin receptor as well as a decrease in its capacity to degrade tRNA and to inhibit translation in the rabbit reticulocyte lysate compared to its counterpart containing the FB spacer. Binding of placental ribonuclease inhibitor (PRI) to Ang also was affected by the nature of the linker and by the presence or absence of a spacer. PRI bound to Ang and AngFBsFv(L2) and inhibited their ribonuclease activity. A 3-fold greater concentration of PRI, however, did not affect the activity of AngFBsFv(L1) or AngsFv(L2), suggesting that the conformation of these fusion proteins was altered. Binding of monoclonal and polyclonal anti-Ang antibodies to AngsFvs was also used to investigate conformational alterations of the fusion proteins. AngFBsFv(L2) was the least altered while AngFBsFv(L1) exhibited the greatest change in structure. Yet maximal concentrations of all AngsFvs elicited angiogenesis in the chick chorioallantoic membrane assay, demonstrating that Ang in all three fusion proteins remained functionally active. Consistent with all the activities, the fusion protein containing the FB spacer and L2 linker was the most cytotoxic to three different human tumor cell lines. The fusion protein lacking the FB spacer exhibited the least cytotoxicity. These data demonstrate that the linker connecting the VH-VL chains can affect the binding and cellular cytotoxicity of Ang immunofusions and that placement of a spacer between the antibody binding domains and Ang is necessary for optimal activity. Thus, a new class of targeted therapeutic agents containing Ang as the toxic moiety can be designed that potentially will be less immunogenic and less toxic than immunotoxins available currently.

Onconase, a protein with anti-tumor activity, causes potent inhibition of protein synthesis in the rabbit reticulocyte lysate (IC50 10(-11) M) and when microinjected into Xenopus oocytes (IC50 10(-10) M). Onconase is a member of the RNase A superfamily; however, unlike RNase A, the mechanism of protein synthesis inhibition does not involve apparent degradation of lysate or cellular ribosomal RNAs. Rather, reticulocyte and oocyte tRNA is hydrolyzed after Onconase treatment. Furthermore, re-addition of tRNA to Onconase pretreated lysates or oocytes restores the translational capacity of the system. Taken together these results suggest that Onconase causes potent protein synthesis inhibition by a mechanism involving inactivation of cellular tRNA.

GAP 31 is an anti-HIV plant protein that we have identified and purified to homogeneity from Gelonium multiflorum. It is the first reported example of an anti-HIV agent capable of acting against multiple stages of the viral life cycle, on viral infection and viral replication. GAP 31 is a unique paragon of multi-functional protein. In addition to anti-HIV activity, it also exhibits anti-tumor action, DNA binding, RNA binding and ribosome inactivation. The present crystals diffract up to 2.0 A resolution and belong to monoclinic space group P2(1). The cell dimensions are a = 49.30(2) A, b = 44.57(2) A, c = 137.78(7) A and beta = 98.32(3) degrees. There are two molecules of molecular weight 31 kDa in an asymmetric unit with a solvent content of 49%.

A monoclonal antibody (mAb) to human angiogenin, a protein that induces formation of new blood vessels, was produced by somatic cell fusion techniques and designated as 26-2F. It is an IgGl kappa whose binding affinity, expressed as an IC50, is (1.6 +/- 0.1) x 10(-9) M as determined by a competition radioimmunoassay. mAb 26-2F neutralizes the ribonucleolytic activity of angiogenin as assessed by in vitro protein synthesis and tRNA degradation assays. It also effectively inhibits neovascularization induced by angiogenin on the chick chorioallantoic membrane. Epitope mapping indicates that the binding region of angiogenin recognized by mAb 26-2F is discontinuous and involves both Trp-89 and residues in the segment 38-41. This epitope is formed by two surface loops which are juxtaposed in the three-dimensional structure of human angiogenin recently determined by X-ray crystallography. Thus mAb 26-2F, along with similar antibodies under investigation, will facilitate structure/function studies of angiogenin, help define its physiological role, and lead to an understanding of the consequences of its inhibition in pathological situations in which angiogenin may be involved.

Purkinje cell toxicity is one of the characteristic features of the Gordon phenomenon, a syndrome manifested by ataxia, muscular rigidity, paralysis, and tremor that may lead to death (Gordon, 1933). Two members of the RNase superfamily found in humans, EDN (eosinophil-derived neurotoxin) and ECP (eosinophil cationic protein), cause the Gordon phenomenon when injected intraventricularly into guinea pigs or rabbits. We have found that another member of the RNase superfamily, an antitumor protein called onconase, isolated from Rana pipiens oocytes and early embryos, will also cause the Gordon phenomenon when injected into the cerebrospinal fluid of guinea pigs at a dose similar to that of EDN (LD50, 3-4 micrograms). Neurologic abnormalities of onconase-treated animals were indistinguishable from those of EDN-treated animals, and histology showed dramatic Purkinje cell loss in the brains of onconase-treated animals. The neurotoxic activity of onconase correlates with ribonuclease activity. Onconase modified by iodoacetic acid to eliminate 70% and 98% of the ribonuclease activity of the native enzyme displays a similar decrease in ability to cause the Gordon phenomenon. In contrast, the homologous bovine pancreatic RNase A injected intraventricularly at a dose 5000 times greater than the LD50 dose of EDN or onconase is not toxic and does not cause the Gordon phenomenon. A comparison of the RNase activities of EDN, onconase, and bovine pancreatic RNase A using three pancreatic RNA substrates demonstrates that onconase is orders of magnitude less active enzymatically than EDN and RNase A. Thus, another member of the RNase superfamily in addition to EDN and ECP can cause the Gordon phenomenon.(ABSTRACT TRUNCATED AT 250 WORDS)

Malignant tumors of the central nervous system can result from metastatic dissemination of a variety of cancers. Percutaneous intracisternal injection of an anti-idiotype monoclonal antibody (M6) ricin immunotoxin was shown to be moderately effective in prolonging the survival of tumor bearing animals supporting the use of immunotoxins for the treatment of central nervous system neoplasia (Zovickian J and Youle R.J. J. Neurosurg 68: 767, 1988). This report describes a method that significantly improves the survival of immunotoxin treated Strain 2 guinea pigs in a syngeneic animal model of leptomeningeal neoplasia. Strain 2 guinea pigs, implanted with subarachnoid catheters, received three courses of treatment with an (M6)-intract ricin immunotoxin following intracisternal inoculation of L2C leukemia tumor cells. Animals were treated with three to four micrograms of immunotoxin in three divided doses. This was found to be less toxic and more effective than single bolus administration of immunotoxin. These results demonstrate that a permanent indwelling catheter in this animal model facilitates multiple dose delivery of immunotoxin therapy allowing the assessment of various treatment schedules and the achievement of enhanced therapeutic effect. Furthermore, these results support the continued evaluation of immunotoxins for the treatment of central nervous system neoplasia.

Onconase, or P-30, is a protein initially purified from extracts of Rana pipiens oocytes and early embryos based upon its anticancer activity both in vitro and in vivo. It is a basic single-chain protein with an apparent molecular mass of 12,000 daltons and is homologous to RNase A. In cultured 9L glioma cells, onconase inhibits protein synthesis with an IC50 of about 10(-7) M. The inhibition of protein synthesis correlates with cell death determined by clonogenic assays. 125I-Labeled onconase binds to specific sites on cultured 9L glioma cells. Scatchard analysis of the binding data shows that onconase appears to bind to cells with two different affinities, one with a Kd of 6.2 x 10(-8) and another of 2.5 x 10(-7) M. Each cell could bind about 3 x 10(5) molecules of onconase at each of the two affinity sites. The low affinity Kd is similar to the IC50 for onconase toxicity. Onconase also demonstrates a saturability of cytotoxicity at a concentration that would saturate the low affinity binding site. Incubation at 4 degrees C increased the binding of onconase to cells relative to 37 degrees C binding and also increased the sensitivity of cells to onconase toxicity, indicating that receptor binding may be an initial step in cell toxicity. Onconase cytotoxicity can be blocked by metabolic inhibitors, NaN3 and 2-deoxyglucose, and cytotoxicity is potentiated 10-fold by monensin. Ribonuclease activity appears necessary for onconase toxicity because alkylated onconase, which only retains 2% of the ribonuclease activity, was at least 100-fold less potent in inhibiting protein synthesis in cells. Onconase inhibition of protein synthesis in 9L cells coincides with the degradation of cellular 28 S and 18 S rRNA. In contrast to RNase A, onconase is resistant to two RNase inhibitors, placental ribonuclease inhibitor and Inhibit-Ace. Northern hybridization with placental ribonuclease inhibitor cDNA probe indicates that 9L glioma cells contain endogenous placental ribonuclease inhibitor mRNA. Based on these results, we propose that onconase toxicity results from onconase binding to cell surface receptors, internalization to the cell cytosol where it degrades ribosomal RNA, inhibiting protein synthesis and causing cell death.

Ribonucleases serve as cytotoxic agents during host defense and physiological cell death pathways. In bacteria, higher plants, and mammals, ribonucleases appear to bind cells, enter the cytosol where they degrade RNA, and kill the target cell. This process functions in interstrain competition in bacteria, in the death of incompatible pollen in higher plants, and likely plays a role in the antiparasitic and anticancer activity of eosinophils in man. One can alter the target cell specificity of RNases by coupling them to new cell-binding domains. Chemically coupling RNases to new binding moieties or fusing RNase genes to antibody genes results in chimeric molecules with specified cell-type cytotoxicity. Thus, one can target one's own host defense cytotoxins to select cell populations. This allows the use of human proteins, instead of plant and bacterial toxins, in the construction of immunotoxins. RNases also can be engineered to kill cells by cytosolic expression or to kill viruses by packaging into viruses. Engineering RNases into cell-type-specific cytotoxins may result in a new class of therapeutic reagents. We review a number of interesting physiological cell cytotoxicity pathways utilizing RNases and then describe the recent results on engineering RNases for therapeutic use.

Angiogenin is a 14.4-kDa human plasma protein with 65% homology to RNase A that retains the key active site residues and three of the four RNase A disulfide bonds. We demonstrate that recombinant angiogenin functions as a cytotoxic tRNA-specific RNase in cell-free lysates and when injected into Xenopus oocytes. Inhibition of protein synthesis by angiogenin correlates with degradation of endogenous oocyte tRNA. Exogenous, radiolabeled tRNA is also hydrolyzed by angiogenin, whereas oocyte rRNA and mRNA are not detectably degraded by angiogenin. Protein synthesis was restored to angiogenin-injected oocytes by injecting the RNase inhibitor RNasin plus total Xenopus or calf liver tRNAs, thereby demonstrating that the tRNA degradation induced by angiogenin was the sole cause of cytotoxicity. A similar tRNA-reversible inhibition of protein synthesis was seen in rabbit reticulocyte lysates. Angiogenin therefore appears to be a specific cellular tRNase, whereas five homologues in the RNase A superfamily lack angiogenin's specificity for tRNA. One of these homologues purified from human eosinophils, eosinophil-derived neurotoxin, nonspecifically degrades oocyte RNA similar to RNase A and is also cytotoxic at very low concentrations.

Monoclonal antibodies to the transferrin receptor or to the T cell antigen, CD5, were chemically linked to mammalian RNase A and found to specifically inhibit protein synthesis in antigen-positive cells. Antibody-mediated specificity of these cytotoxic ribonuclease chimeras (CRCs) was demonstrated in three ways. 1) Toxicity was due to the chemical linkage of RNase to antibody, as the individual components added separately or in combination did not inhibit protein synthesis; 2) the anti-transferrin receptor CRCs inhibited protein synthesis in those cells expressing the human transferrin receptor (K562, U251, Jurkat cells) but had no detectable toxicity to cells lacking the human transferrin receptor (Vero or NIH 3T3 cells); 3) free antibody to either the human transferrin receptor (454A12 or 5E-9) or to the T cell antigen, CD5 (T101), blocked the cytotoxicity of the respective CRC. Two CRC species, designated P1 and P2, that differed in size and stoichiometry of RNase A to antibody, were purified by size-exclusion high performance liquid chromatography. The higher molecular weight P1 conjugate had an IC50 of 20-30 nM, whereas the P2 conjugate had a higher IC50 of 300-500 nM. Bioactivity could be reversibly increased more than 10-fold by freezing. The cytotoxicity of the CRCs was examined in vivo in a solid tumor animal model. Intratumoral injections of an anti-transferrin receptor CRC into established U251 human glioblastoma tumors grown in the flanks of nude mice prevented tumor growth, whereas RNase A mixed with antibody was ineffective. CRCs, therefore, express cytotoxicity in vitro and in vivo. Mammalian nucleases coupled to antibodies may be utilized as cell type-selective cytotoxins and have potential as pharmacologic reagents. The systemic toxicity and immunogenicity observed with mammalian derived cytotoxins may be significantly less than that of the currently employed plant- and bacterial-derived immunotoxins.

Members of the pancreatic ribonuclease (RNase) family have diverse activities toward RNA that could cause them to function during host defense and physiological cell death pathways. This activity could be harnessed by coupling RNases to cell binding ligands for the purpose of engineering them into cell-type specific cytotoxins. Therefore, the cytotoxic potential of RNase was explored by linking bovine pancreatic ribonuclease A via a disulfide bond to human transferrin or antibodies to the transferrin receptor. The RNase hybrid proteins were cytotoxic to K562 human erythroleukemia cells in vitro with an IC50 around 10(-7) M, whereas > 10(-4) M of native RNase was required to inhibit protein synthesis. Cytotoxicity required both components of the conjugate since excess transferrin or ribonuclease inhibitors added to the medium protected the cells from the transferrin-RNase toxicity. Importantly, the RNase conjugates were found to have potent antitumor effects in vivo. Chimeric RNase fusion proteins were also developed. F(ab')2-like antibody-enzyme fusions were prepared by linking the gene for human RNase to a chimeric antitransferrin receptor heavy chain gene. The antibody enzyme fusion gene was introduced into a transfectoma that secreted the chimeric light chain of the same antibody, and cell lines were cloned that synthesized and secreted the antibody-enzyme fusion protein of the expected size at a concentration of 1-5 ng/mL. Culture supernatants from clones secreting the fusion protein caused inhibition of growth and protein synthesis toward K562 cells that express the human transferrin receptor but not toward a nonhuman derived cell line. Since human ribonucleases coupled to antibodies also exhibited receptor mediated toxicities, a new approach to selective cell killing is provided. This may allow the development of new therapeutics for cancer treatment that exhibit less systemic toxicity and, importantly, less immunogenicity than the currently employed ligand-toxin conjugates.

Several toxins abolish cellular protein synthesis by attacking specific sites in 28 S RNA. One of these toxins, alpha-sarcin, is an RNase that also cleaves nonspecifically on the 3' side of purines in deproteinized RNA. Several other RNases were injected into Xenopus oocytes, examined for their ability to abolish protein synthesis, and compared with alpha-sarcin and ricin. Surprisingly, pancreatic RNase A or B abolished oocyte protein synthesis at concentrations (approximately 0.03 nM) comparable to, or lower than, the amount of alpha-sarcin (approximately 2 nM) or ricin (approximately 0.07 nM) required to abolish protein synthesis. RNases S and T1 only inhibited oocyte protein synthesis when used at concentrations approximately 10 x higher than RNase A whereas RNases C, T2, U2, and nuclease P1 required concentrations approximately 100 times higher than RNase A to abolish protein synthesis. There was a direct correlation between the degradation of oocyte RNA and the inhibition of protein synthesis. The RNase inhibitors RNasin and Inhibit-Ace injected into the oocyte both prevented RNase A from hydrolyzing oocyte rRNA and abolishing protein synthesis. Enzymatically inactive oxidized RNase A did not inhibit protein synthesis when injected into the oocyte. None of the RNases or alpha-sarcin abolished protein synthesis when added to oocyte extracellular medium. Angiogenin is a human plasma protein that induces blood vessel formation in chick embryos, has 35% amino acid identity with RNase A, and cleaves 18 S and 28 S RNA in rabbit reticulocyte lysates (St. Clair, D. K., Rybak, S. M., Riordan, J. F. & Vallee, B. L. (1988) Biochemistry 27, 7263-7268, and references therein). Recombinant angiogenin injected into oocytes abolished protein synthesis, and this toxic effect was inhibited by RNasin but was not inhibited by Inhibit-Ace. Unlike RNase A and the other nucleases that hydrolyzed cellular rRNA, no cleavage of 18 or 28 S RNA by recombinant angiogenin was seen at concentrations 100 x greater than necessary to abolish protein synthesis. Recombinant angiogenin must selectively attack specific RNA(s) or another target in the cell.

Pancreatic RNase injected into Xenopus oocytes abolishes protein synthesis at concentrations comparable to the toxin ricin yet has no effect on oocyte protein synthesis when added to the extracellular medium. Therefore RNase behaves like a potent toxin when directed into a cell. To explore the cytotoxic potential of RNase toward mammalian cells, bovine pancreatic ribonuclease A was coupled via a disulfide bond to human transferrin or antibodies to the transferrin receptor. The RNase hybrid proteins were cytotoxic to K562 human erythroleukemia cells in vitro with an IC50 around 10(-7) M whereas greater than 10(-5) M native RNase was required to inhibit protein synthesis. Cytotoxicity requires both components of the conjugate since excess transferrin or ribonuclease inhibitors added to the medium protected the cells from the transferrin-RNase toxicity. Compounds that interfere with transferrin receptor cycling and compartmentalization such as ammonium chloride decreased the cytotoxicity of transferrin-RNase. After a dose-dependent lag period inactivation of protein synthesis by transferrin-RNase followed a first-order decay constant. In a clonogenic assay that measures the extent of cell death 1 x 10(-6) M transferrin-RNase killed at least 4 logs or 99.99% of the cells whereas 70 x 10(-6) M RNase was nontoxic. These results show that RNase coupled to a ligand can be cytotoxic. Human ribonucleases coupled to antibodies also may exhibit receptor-mediated toxicities providing a new approach to selective cell killing possibly with less systemic toxicity and importantly less immunogenicity than the currently employed ligand-toxin conjugates.

Biological effects of class 1 or class 2 heparin-binding growth factors (HBGFs) were compared in BALB/c-3T3 cells. Changes in protein synthesis, as monitored by two-dimensional gel electrophoresis, reveal that while both HBGFs induce the same changes in the synthesis of intracellular proteins, class 2 HBGF selectively increases the synthesis of a 43-kD extracellular protein. Heparin, which potentiates the mitogenic activity of class 1 but not class 2 HBGF, does not potentiate the changes in protein synthesis elicited by HBGF-1. Since each HBGF increases actin synthesis, regulation of actin mRNA expression was examined. Actin mRNA levels increase rapidly and transiently in response to either HBGF, and similar superinduction responses are observed in the presence of HBGF and cycloheximide. Although the maximum increase in actin mRNA stimulated by either HBGF is similar, the levels of mRNA induced by class 2 HBGF remain elevated up to 48 hours compared to the level induced by class 1 HBGF. These results imply that in the same cell type class 1 and class 2 HBGFs may modulate some biological effects differently.

The translational capacity of a rabbit reticulocyte lysate is rapidly abolished on treatment with angiogenin, an effect that is due to cleavage of rRNA [St. Clair, D. K., Rybak, S. M., Riordan, J. F., & Vallee, B. L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8330-8334]. The same time course of inhibition is seen when isolated ribosomes are treated with angiogenin prior to being added to a ribosome-dependent lysate system. In both cases, the onset of inhibition occurs at a rate similar to that seen on addition of puromycin, a known inhibitor of elongation, suggesting that this is the step in the protein synthesis machinery that is inactivated by angiogenin. The action of angiogenin on ribosomes is quite specific: both 28S and 18S rRNAs are cleaved whereas 5.8S and 5S rRNAs are not. Moreover, 28S and 18S rRNAs are affected differently. Prolonged incubation with angiogenin degrades 28S rRNA extensively but only causes limited cleavage of 18S rRNA. Remarkably, it is the effect of angiogenin on 18S rRNA that seems to be responsible for the inhibition of protein synthesis rather than the nucleolytic degradation of 28S rRNA. This has been demonstrated by separating the isolated ribosomes into their 40S and 60S subunits and treating them individually with angiogenin. The pattern of rRNA cleavage is the same with the separated subunits as with intact ribosomes, but translation is abolished only on treatment of the 40S, not the 60S, subunit with angiogenin. These results confirm our previous observations on the effect of angiogenin on the rabbit reticulocyte cell-free translation system and extend the understanding of its mechanism of action on the ribosome.