X-ray crystallographic analysis of pokeweed antiviral protein-II after reductive methylation of lysine residues.

Pokeweed antiviral protein II (PAP-II) is a naturally occurring protein isolated from early summer leaves of the pokeweed plant (Phytolacca americana). PAP-II belongs to a family of ribosome-inactivating proteins which catalytically deadenylate ribosomal and viral RNA. The chemical modification of PAP-II by reductive methylation of its lysine residues significantly improved the crystal quality for X-ray diffraction studies. Hexagonal crystals of the modified PAP-II, with unit cell parameters a = b = 92.51 A, c = 79.05 A, were obtained using 1.8 M Na/K phosphate as the precipitant. These crystals contained one enzyme molecule per asymmetric unit and diffracted up to 2.4 A, when exposed to a synchroton source.

X-ray crystallographic analysis of the structural basis for the interactions of pokeweed antiviral protein with its active site inhibitor and ribosomal RNA substrate analogs.

The pokeweed antiviral protein (PAP) belongs to a family of ribosome-inactivating proteins (RIP), which depurinate ribosomal RNA through their site-specific N-glycosidase activity. We report low temperature, three-dimensional structures of PAP co-crystallized with adenyl-guanosine (ApG) and adenyl-cytosine-cytosine (ApCpC). Crystal structures of 2.0-2.1 A resolution revealed that both ApG or ApCpC nucleotides are cleaved by PAP, leaving only the adenine base clearly visible in the active site pocket of PAP. ApCpC does not resemble any known natural substrate for any ribosome-inactivating proteins and its cleavage by PAP provides unprecedented evidence for a broad spectrum N-glycosidase activity of PAP toward adenine-containing single stranded RNA. We also report the analysis of a 2.1 A crystal structure of PAP complexed with the RIP inhibitor pteoric acid. The pterin ring is strongly bound in the active site, forming four hydrogen bonds with active site residues and one hydrogen bond with the coordinated water molecule. The second 180 degrees rotation conformation of pterin ring can form only three hydrogen bonds in the active site and is less energetically favorable. The benzoate moiety is parallel to the protein surface of PAP and forms only one hydrogen bond with the guanido group of Arg135.

Pokeweed antiviral protein isoforms PAP-I, PAP-II, and PAP-III depurinate RNA of human immunodeficiency virus (HIV)-1.

Pokeweed antiviral protein (PAP) is a naturally occurring broad-spectrum antiviral agent with potent anti-human immunodeficiency virus (HIV)-1 activity by an as yet undeciphered molecular mechanism. In the present study, we sought to determine if PAP is capable of recognizing and depurinating viral RNA. Depurination of viral RNA was monitored by directly measuring the amount of the adenine base released from the viral RNA species using quantitative high-performance liquid chromatography. Our findings presented herein provide direct evidence that three different PAP isoforms from Phytolacca americana (PAP-I from spring leaves, PAP-II from early summer leaves, and PAP-III from late summer leaves) cause concentration-dependent depurination of genomic RNA (63 to 400 pmols of adenine released per micrograms of RNA) purified from human immunodeficiency virus type-I (HIV-I), plant virus (tobacco mosaic virus (TMV), and bacteriophage (MS 2). In contrast to the three PAP isoforms, ricin A chain (RTA) failed to cause detectable depurination of viral RNA even at 5 microM, although it was as effective as PAP in inhibiting protein synthesis in cell-free translation assays. PAP-I, PAP-II, and PAP-III (but not RTA) inhibited the replication of HIV-1 in human peripheral blood mononuclear cells with IC(50) values of 17 nM, 25 nM, and 16 nM, respectively. These findings indicate that the highly conserved active site residues responsible for the depurination of rRNA by PAP or RTA are not sufficient for the recognition and depurination of viral RNA. Our study prompts the hypothesis that the potent antiviral activity of PAP may in part be due to its unique ability to extensively depurinate viral RNA, including HIV-1 RNA.

TXU (anti-CD7)-pokeweed antiviral protein as a potent inhibitor of human immunodeficiency virus.

We have evaluated the clinical potential of TXU (anti-CD7)-pokeweed antiviral protein (PAP) immunoconjugate (TXU-PAP) as a new biotherapeutic anti-human immunodeficiency virus (anti-HIV) agent by evaluating its anti-HIV type 1 (anti-HIV-1) activity in vitro, as well as in a surrogate human peripheral blood lymphocyte-severe combined immunodeficient (Hu-PBL-SCID) mouse model of human AIDS. The present report documents in a side-by-side comparison the superior in vitro anti-HIV-1 activity of TXU-PAP compared to the activities of zidovudine, 2',3'-didehydro-2',3'-dideoxythymidine, unconjugated PAP, and B53-PAP, an anti-CD4-PAP immunoconjugate. Notably, TXU-PAP elicited potent anti-HIV activity in the Hu-PBL-SCID mouse model of human AIDS without any side effects and at doses that were very well tolerated by cynomolgus monkeys. Furthermore, plasma samples from TXU-PAP-treated cynomolgus monkeys showed potent anti-HIV-1 activity in vitro.

In vivo toxicity, pharmacokinetics, and antileukemic activity of TXU (anti-CD7)-pokeweed antiviral protein immunotoxin.

We evaluated the TXU (anti-CD7)-pokeweed antiviral protein (PAP) immunotoxin in both murine and nonhuman primate models. TXU-PAP caused dose-limiting cardiac toxicity in BALB/c mice. In a SCID mouse model of invariably fatal human T-lineage acute lymphoblastic leukemia (ALL), TXU-PAP therapy resulted in a marked improvement of leukemia-free survival without any side effects. Whereas 100% of control mice treated with PBS, unconjugated TXU antibody, or B43-PAP (an immunotoxin that does not react with T-lineage ALL cells) died of disseminated human leukemia within 80 days (median survival, 37 days), 80 +/- 13% of SCID mice treated with 15 microgram of TXU-PAP (median survival, >120 days) and 100% of mice treated with 30 microgram of TXU-PAP (median survival, > 120 days) remained alive and free of leukemia for >120 days. In cynomolgus monkeys, TXU-PAP showed favorable pharmacokinetics with an elimination half-life of 8.1-8.7 h. The monkeys treated with TXU-PAP at dose levels of 0.05 mg/kg/day x 5 days and 0.10 mg/kg/day x 5 days tolerated the therapy very well, without any significant clinical compromise or side effects, and at necropsy, no gross or microscopic lesions were found. This study provides a basis for further evaluation of TXU-PAP as an investigational biotherapeutic agent in the treatment of T-lineage ALL.

Pharmacokinetic features, immunogenicity, and toxicity of B43(anti-CD19)-pokeweed antiviral protein immunotoxin in cynomolgus monkeys.

We studied the pharmacokinetic features, immunogenicity, and toxicity of B43-pokeweed antiviral protein (PAP) immunotoxin in 13 cynomolgus monkeys. The disposition of B43-PAP in two monkeys, when administered as a single i.v. bolus dose, was characterized by a slow clearance (1-2 ml/h/kg) with a very discrete peripheral distribution. B43-PAP was retained and distributed largely in the blood as the sole compartment with no significant equilibration with the extravascular compartment. The circulating B43-PAP immunotoxin detected in monkey plasma samples by ELISA and protein immunoblotting was both immunoreactive with, and active against, human leukemic cells in vitro. In systemic immunogenicity and toxicity studies, which involved 11 cynomolgus monkeys, each monkey received a total of seven i.v. doses of B43-PAP at a specific dose level of the dose escalation schedule. B43-PAP-treated monkeys mounted a dose-dependent humoral immune response against both the mouse IgG and PAP moieties of the immunotoxin. When administered i.v. either on an every-day or every-other-day schedule, B43-PAP was very well tolerated, with no significant clinical or laboratory signs of toxicity at total dose levels ranging from 0.007 to 0.7 mg/kg. A transient episode of a mild capillary leak with a grade 2 hypoalbuminemia and 2+ proteinuria was observed at total dose levels equal to or higher than 0.35 mg/kg. At total dose levels of 3.5 and 7.0 mg/kg, B43-PAP caused dose-limiting renal toxicity due to severe renal tubular necrosis. The present study completes the preclinical evaluation of B43-PAP and provides the basis for its clinical evaluation in children with therapy-refractory B-lineage acute lymphoblastic leukemia.

Treatment of human B-cell precursor leukemia in SCID mice using a combination of the investigational biotherapeutic agent B43-PAP with cytosine arabinoside.

Combined immunochemotherapy regimens using the investigational biotherapeutic agent B43(anti-CD19)-poke-weed antiviral protein (PAP) immunotoxin may offer an effective treatment for refractory B-cell precursor leukemias. The purpose of the present study was to explore and identify effective combinations of B43-PAP with standard chemotherapeutic drugs, including the anthracyclin doxorubicin, the epipodophyllotoxin etoposide, the nitrosurea carmustine, and the antimetabolite cytosine arabinoside. Here, we report that the B43-PAP plus cytosine arabinoside combination has potent antileukemic activity against human B-cell precursor leukemia in SCID mice and leads to 100% long-term event-free survival from an otherwise invariably fatal leukemia. Surprisingly, none of the other treatment protocols tested, including combinations of B43-PAP with carmustine, doxorubicin, or etoposide, proved more effective than B43-PAP alone.

Toxicity profile of the investigational new biotherapeutic agent, B43 (anti-CD19)-pokeweed antiviral protein immunotoxin.

The investigational biotherapeutic agent, B43(anti-CD19)-pokeweed antiviral protein (PAP) immunotoxin, has shown substantial anti-leukemic activity in SCID mouse models of human B-lineage leukemia and lymphoma. In this report, we describe the results of a comprehensive preclinical toxicity study which determined the toxicity profile of B43-PAP in BALB/c mice. Administration of unconjugated B43 monoclonal antibody was not associated with any toxicity, whereas B43-PAP caused dose-limiting and cardiac and renal toxicities which were fatal. In addition, B43-PAP also caused multifocal skeletal myofiber necrosis, which was associated with abnormal gait and lethargy. Notably, parenteral administrations of methylprednisolone, pentoxyphylline, or dopamine were able to markedly reduce B43-PAP related toxicity. This study provides a basis for further evaluation of the toxicity of B43-PAP in monkeys and humans.

In vitro and in vivo antileukemic activity of B43-pokeweed antiviral protein against radiation-resistant human B-cell precursor leukemia cells.

B-cell precursor (BCP) leukemia is the most common form of childhood cancer and represents one of the most radiation-resistant forms of human malignancy. In this study, we examined the antileukemic efficacy of the B43 (anti-CD19)-pokeweed antiviral protein (B43-PAP) immunotoxin against radiation-resistant BCP leukemia cells. B43-PAP caused apoptosis of radiation-resistant primary BCP leukemia cells, killed greater than 99% of radiation-resistant primary leukemic progenitor cells from BCP leukemia patients, and conferred extended survival to severe combined immunodeficiency (SCID) mice xenografted with radiation-resistant human BCP leukemia. Furthermore, the combination of B43-PAP and total body irradiation (TBI) was more effective than TBI alone in two SCID mouse bone marrow transplantation models of radiation-resistant human BCP leukemia. Thus, B43-PAP may prove useful in the treatment of radiation-resistant BCP leukemia.

Favorable pharmacodynamic features and superior anti-leukemic activity of B43 (anti-CD19) immunotoxins containing two pokeweed antiviral protein molecules covalently linked to each monoclonal antibody molecule.

Standard immunotoxin production procedures using whole IgG as the MoAb moiety yield a heterogeneous mixture of 180 kDa, 210 kDa, and 240 kDa immunotoxin species with 1 to 1, 1 to 2, and 1 to 3 MoAb to toxin ratios. This heterogeneity makes it impossible to precisely deliver a predetermined immunotoxin dose to target cells and impairs the accuracy of pharmacologic studies. In this report, we describe the preparation and characterization of B43(anti-CD19)-pokeweed antiviral protein (PAP) immunotoxins containing either one or two 30 kDa PAP toxin molecules covalently linked to each 150 kDa B43 monoclonal antibody molecule. Compared to the 180 kDa immunotoxin, the 210 kDa immunotoxin displayed greater in vitro chemical stability, resulted in higher systemic exposure levels in vivo, and was a more effective anti-leukemic agent in a SCID mouse model of human B-lineage acute lymphoblastic leukemia. Taken together, the results of this study recommend the clinical evaluation of 210 kDa B43-PAP as a potentially more effective immunotoxin against relapsed B-lineage ALL.

Extended-release felodipine in patients with mild to moderate hypertension. Felodipine ER Dose-Response Study Group.

Two hundred eighty-six patients with mild to moderate hypertension who had untreated diastolic blood pressure while seated of 95 to 115 mm Hg were randomized to receive placebo or once-daily doses of 2.5, 5, or 10 mg of the dihydropyridine calcium channel blocker felodipine extended release (ER). Blood pressure was measured 24 hours after dosing (at trough). Mean reductions in diastolic blood pressure after 8 weeks of double-blind treatment were significantly greater in each of the ER felodipine treatment groups (2.5, 5, and 10 mg ER felodipine: -7.8, -9.5, and -11.3 mm Hg, respectively) than in the placebo group (-5.3 mm Hg). The effect was dose dependent for both diastolic and systolic blood pressure. Moreover, much of the peak antihypertensive effect was still present at trough, confirming the 24-hour efficacy of the drug. Felodipine was well tolerated.

Pokeweed antiviral protein inactivates pokeweed ribosomes; implications for the antiviral mechanism.

Pokeweed antiviral protein (PAP) and other ribosome-inactivating proteins (RIPs) had previously been thought to be incapable of attacking conspecific ribosomes, thus having no effect on endogenous processes. This assertion conflicts with a model for PAP's in vivo antiviral mechanism in which PAP (a cell wall protein) selectively enters virus-infected cells and disrupts protein synthesis, thus causing local suicide and preventing virus replication. We show here that pokeweed (Phytolacca americana) ribosomes, as well as endod (Phytolacca dodecandra) ribosomes, are indeed highly sensitive to inactivation by conspecific RIPs. Ribosomes isolated from RIP-free pokeweed and endod suspension culture cells were found to be highly active in vitro, as measured by poly(U)-directed polyphenylalanine synthesis. Phytolacca ribosomes challenged with conspecific RIPs generated dose-response curves (IC50 of 1 nM PAP or dodecandrin) very similar to those from wheat germ ribosomes. To determine if Phytolacca cells produce a cytosolic 'anti-RIP' protective element, ribosomes were combined with Phytolacca postribosomal supernatant factors from culture cells, then challenged with conspecific RIPs. Resulting IC50 values of 3-7 nM PAP, PAP-II, PAP-S or dodecandrin indicate that supernatants from these Phytolacca cells lack a ribosomal protective element. This research demonstrates that PAP inactivates pokeweed ribosomes (and is therefore potentially toxic to pokeweed cells) and supports the local suicide model for PAP's in vivo antiviral mechanism. The importance of spatial separation between PAP and ribosomes of cells producing this RIP is emphasized, particularly if crop plants are transformed with the PAP gene to confer antiviral protection.

The 2.5 A structure of pokeweed antiviral protein.

The pokeweed antiviral protein (PAP), isolated from the leaves of Phytolacca americana, is one of a family of plant and bacterial ribosome-inhibiting proteins (RIPs) which act as specific N-glycosidases on rRNA. Here we report the three-dimensional structure of PAP determined to 2.5 A resolution by X-ray crystallography. After 14 rounds of refinement, the R factor is 0.17 for 5.0 to 2.5 A data. The protein is homologous with the A chain of ricin and exhibits a very similar folding pattern. The positions of key active site residues are also similar. We also report the 2.8 A structure of PAP complexed with a substrate analog, formycin 5'-monophosphate. As seen previously in ricin, the formycin ring is stacked between invariant tyrosines 72 and 123. Arg179 bonds to N-3 which is thought to be important in catalysis.

Effects of the intermolecular toxin-monoclonal antibody linkage on the in vivo stability, immunogenicity and anti-leukemic activity of B43 (anti-CD19) pokeweed antiviral protein immunotoxin.

We have successfully constructed highly potent and selective anti-CD19 PAP immunotoxins using each of the three crosslinking agents, SPDP, LC-SPDP, or SMPT, to generate an intermolecular bridge between the B43 MoAb and PAP toxin moieties. These immunotoxins were selectively immunoreactive with and cytotoxic against CD19+ B-lineage ALL cells. In this report, we compared (a) in vivo chemical, immunological, and biological stability, (b) in vivo immunogenicity, and (c) in vivo anti-leukemic activity of various B43-PAP immunotoxin constructs. Our data recommend the use of SPDP and SMPT rather than LC-SPDP for generation of B43(anti-CD19)-PAP immunotoxins as clinical anti-leukemic agents. To our knowledge, this is the first comparative analysis of the in vivo pharmacokinetic features, immunogenicity, and anti-leukemic activity of anti-CD19 PAP immunotoxins that were prepared with different heterobifunctional crosslinking agents.

Anti-human immunodeficiency virus type 1 activity of an anti-CD4 immunoconjugate containing pokeweed antiviral protein.

The ability of an alpha CD4-pokeweed antiviral protein (PAP) immunoconjugate to inhibit replication of human immunodeficiency virus type 1 (HIV-1) was evaluated in vitro with 22 clinical HIV-1 strains obtained from four seropositive asymptomatic individuals, three patients with AIDS-related complex, and four patients with AIDS. Fifteen isolates were from zidovudine-untreated individuals, whereas seven isolates were obtained after 24 to 104 weeks of therapy with zidovudine, alone or alternating with zalcitabine. Mean zidovudine 50% inhibitory concentrations (IC50s) were 126 nM (range, 1 to 607 nM) for isolates from zidovudine-untreated individuals and 2,498 nM (range, 14 to 6,497 nM) for strains from patients treated with antiretroviral agents. Mean alpha CD4-PAP IC50s were 48 x 10(-3) nM (range, 0.02 x 10(-3) to 212 x 10(-3) nM) for isolates from zidovudine-untreated individuals, and 16 x 10(-3) nM (range, 2 x 10(-3) to 28 x 10(-3) nM) for isolates from treated patients. Overall, higher concentrations of alpha CD4-PAP were necessary to inhibit HIV-1 strains from untreated individuals at more advanced stages of disease. Seventeen isolates were susceptible to zidovudine (mean IC50, 117 nM), and five were resistant to zidovudine (mean IC50, 3,724 nM). Mean alpha CD4-PAP IC50s were 43 x 10(-3) nM for zidovudine-susceptible isolates and 19 x 10(-3) nM for isolates resistant to zidovudine. All HIV-1 strains had IC50s greater than 0.5 nM for unconjugated PAP, the alpha CD19-PAP immunoconjugate, and monoclonal antibody alpha CD4. At concentrations as high as 5,000 nM, alphaCD4-PAP did not inhibit colony formation by normal bone marrow progenitor cells(BFU-E, CFU-GM , and CFU-GEMM) or myeloid cell lines (KG-1 and HL-60) and did not decrease cell viabilities of T-cell (Jurkat) or B-cell (FL-112 and Raji) precursor lines. Overall, alphaCD4-PAP demonstrated more potent anti-HIV-1 activity than zidovudine and inhibited replication of zidovudine-susceptible and zidovudine-resistant viruses at concentrations that were not toxic to lymphohematopoietic cell populations.

In vivo anti-leukemic efficacy of anti-CD7-pokeweed antiviral protein immunotoxin against human T-lineage acute lymphoblastic leukemia/lymphoma in mice with severe combined immunodeficiency.

Mice with severe combined immunodeficiency (SCID) were injected with 1 x 10(7) MOLT-3 human T-lineage acute lymphoblastic leukemia cells to provide a model for the evaluation of anti-CD7-pokeweed antiviral protein (PAP) immunotoxin directed against the human CD7 antigen. Of control SCID mice (treated with phosphate-buffered saline, PBS) challenged intravenously with 1 x 10(7) MOLT-3 cells, 5/5 died at 29 to 35 days after inoculation, with a median event-free survival of 33 days. Similarly, 6/6 anti-CD19-PAP treated control SCID mice died of MOLT-3 leukemia at a median of 36 days. In contrast, treatment with anti-CD7-PAP (15 micrograms total dose in 5 micrograms intraperitoneal injections on days 1-3) significantly improved event-free survival of SCID mice challenged with 1 x 10(7) MOLT-3 cells. Of nine SCID mice treated with anti-CD7-PAP, four died at 54-149 days and five remained alive for > 172 days without clinical evidence of leukemia (median event-free survival > 172 days). When long-term survivors among the anti-CD7-PAP treated SCID mice were electively killed at 173 days to assess their leukemia burden, histopathologic examination and polymerase chain reaction provided evidence of disseminated leukemia in some of these mice. Intriguingly, marked differences in morphology, tissue distribution, and histologic pattern of organ invasion existed between leukemic blasts killing 100% of PBS-treated control mice at a median of 33 days and 'therapy-refractory' leukemic blasts detected in anti-CD7-PAP-treated long-term survivors. This novel SCID mouse model of disseminated human T-lineage ALL provides a unique in vivo system to investigate the therapeutic potential of new treatment strategies and to study possible mechanisms of in vivo immunotoxin resistance.

Effective immunochemotherapy of CALLA+C mu+ human pre-B acute lymphoblastic leukemia in mice with severe combined immunodeficiency using B43 (anti-CD19) pokeweed antiviral protein immunotoxin plus cyclophosphamide.

A highly aggressive human CALLA+C mu+ pre-B acute lymphoblastic leukemia (ALL) cell line (NALM-6-UM1) causes disseminated and invariably fatal leukemia in CB.17 mice with severe combined immunodeficiency (SCID). We used this SCID mouse model of human pre-B ALL to evaluate and compare, in a total of 434 SCID mice, the antileukemic efficacy of B43 (anti-CD19)-pokeweed antiviral protein (PAP) immunotoxin and cyclophosphamide (CPA) as individual reagents and as combined immunochemotherapeutic regimens. B43-PAP plus CPA was superior to either the immunotoxin or drug alone, and combined immunochemotherapy markedly improved the event-free survival (EFS) of SCID mice challenged with NALM-6-UM1 pre-B ALL cells. Notably, 90% to 100% of SCID mice challenged with 1 x 10(6) leukemia cells and then treated with B43-PAP plus CPA combined immunochemotherapy regimens became long-term survivors, a result not achieved with B43-PAP alone or CPA alone. The advantage was particularly evident in mice inoculated with 5 x 10(6) leukemia cells. While neither 15 micrograms B43-PAP (median survival, 58 days) nor 1 mg CPA (median survival, 49 days) resulted in long-term EFS of SCID mice challenged with 5 x 10(6) NALM-6-UM1 pre-B ALL cells, the probability of EFS at 6 months was 50% +/- 16% for SCID mice treated with 15 micrograms B43-PAP plus 1 mg CPA (median survival, greater than 180 days) (P less than .0001). The probability of long-term EFS was only 14% +/- 7% for mice treated with 30 micrograms B43-PAP and 0% +/- 0% for mice treated with 1 mg CPA, but 40% +/- 16% for mice treated with 30 micrograms B43-PAP plus 1 mg CPA (P less than .0001). Similarly, the probability of EFS at 6 months was 40% +/- 16% for mice treated with 2 mg CPA alone, 70% +/- 15% for mice treated with 2 mg CPA plus 15 micrograms B43-PAP, and 70% +/- 15% for mice treated with 2 mg CPA plus 30 micrograms B43-PAP. Ten SCID mice in the B43-PAP plus CPA combined immunochemotherapy arms surviving long term after the inoculation of 5 x 10(6) NALM-6-UM1 pre-B ALL cells were electively killed at 174 to 181 days to assess their leukemia burden. We found no evidence of leukemia in any of the bone marrow specimens by two-color immunofluorescence and multiparameter flow cytometry.(ABSTRACT TRUNCATED AT 400 WORDS)