Oncolytic and immunostimulatory efficacy of a targeted oncolytic poxvirus expressing human GM-CSF following intravenous administration in a rabbit tumor model.

Targeted oncolytic poxviruses hold promise for the treatment of cancer. Arming these agents with immunostimulatory cytokines (for example, granulocyte-monocyte colony-stimulating factor; GM-CSF) can potentially increase their efficacy and/or alter their safety. However, due to species-specific differences in both human GM-CSF (hGM-CSF) activity and poxviruses immune avoidance proteins, the impact of hGM-CSF expression from an oncolytic poxvirus cannot be adequately assessed in murine or rat tumor models. We developed a rabbit tumor model to assess toxicology, pharmacodynamics, oncolytic efficacy and tumor-specific immunity of hGM-CSF expressed from a targeted oncolytic poxvirus JX-963. Recombinant purified hGM-CSF protein stimulated a leukocyte response in this model that paralleled effects of the protein in humans. JX-963 replication and targeting was highly tumor-selective after i.v. administration, and intratumoral replication led to recurrent, delayed systemic viremia. Likewise, hGM-CSF was expressed and released into the blood during JX-963 replication in tumors, but not in tumor-free animals. hGM-CSF expression from JX-963 was associated with significant increases in neutrophil, monocyte and basophil concentrations in the peripheral blood. Finally, tumor-specific cytotoxic T lymphocytes (CTL) were induced by the oncolytic poxvirus, and expression of hGM-CSF from the virus enhanced both tumor-specific CTL and antitumoral efficacy. JX-963 had significant efficacy against both the primary liver tumor as well as metastases; no significant organ toxicity was noted. This model holds promise for the evaluation of immunostimulatory transgene-armed oncolytic poxviruses, and potentially other viral species.

Gene delivery from the E3 region of replicating human adenovirus: evaluation of the 6.7 K/gp19 K region.

The use of genetically engineered, replication-selective viruses to treat cancer is being realized with viruses such as ONYX-015, a human adenovirus that selectively destroys p53 mutant cancer cells. To enhance further the clinical efficacy of ONYX-015 and viruses like it, we have developed a novel gene delivery system for replicating adenoviruses. This system has two unique features. First, it uses the endogenous adenoviral gene expression machinery (promoter, splicing, polyadenylation) to drive transgene expression. Second, a single region or gene in the multi-gene E3 transcription unit is selectively substituted for by the therapeutic transgene(s). Analyzing various transgene substitutions for the 6.7 K/gp19 K region of E3, we demonstrate the following: (1) transgene expression in this system is predictable and mimics the substituted endogenous gene expression pattern, (2) expression of surrounding E3 genes can be retained, (3) the insertion site choice can effect both the transgene expression level and the viral life cycle, and, (4) expression levels from this system are superior to those generated from a replication-defective virus using the HCMV enhancer-promoter and this is dependent on viral DNA replication. This unique methodology has broad application to the rapidly evolving field of replicating virus-based therapies.

Inflammatory bowel disease is associated with increased mucosal levels of bactericidal/permeability-increasing protein.

BACKGROUND & AIMS: Clinical sepsis seldom accompanies inflammatory bowel disease. The aim of this study was to measure colonic mucosal levels of the neutrophil product bactericidal/permeability-increasing protein (BPI), which kills gram-negative bacteria in addition to inactivating endotoxin. METHODS: Enzyme-linked immunosorbent assay and immunohistochemistry for BPI were performed on homogenates and tissue secretions of biopsy specimens from patients with ulcerative colitis (n=11) and Crohn's disease (n=5) and from normal controls (n=5). RESULTS: Mucosal neutrophil content (144 +/- 23 vs. 35 +/- 9 neutrophils/mg protein; P<0.007) and BPI content (2.07 +/- 0.75 vs. 0.12 +/- 0.02 ng/mg protein; P<0.002) were greater in the colitis groups and correlated closely (r=0.68; P<0.001). This relationship held for both ulcerative colitis (P<0.002) and Crohn's disease (P<0.01) with a trend towards greater levels in Crohn's disease. There was a trend towards higher BPI levels with an increasing endoscopic inflammation score (grade I, 1.32 +/- 0.6 ng/mg protein; grade II, 2.82 +/- 1.4 ng/mg protein). Immunohistochemistry and the biopsy culture showed BPI to be both intracellular and extracellular, to be present in the crypt lumen, and to be released into incubating medium. CONCLUSIONS: Mucosal levels of BPI are increased in colitis. Such localization may ameliorate mucosal responses to gram-negative bacteria and their products.

Measurement of bactericidal/permeability-increasing protein in human body fluids by sandwich ELISA.

A sensitive sandwich ELISA has been developed to measure levels of native bactericidal/permeability-increasing protein (BPI) as well as two recombinant forms of BPI (rBPI and rBPI23) in human body fluids. The linear range for the rBPI and rBPI23 standard curves were 100-6000 pg/ml and 25-800 pg/ml respectively. Recovery of different concentrations of rBPI spiked into pooled human plasma samples averaged 83% and ranged from 65% at 300 ng/ml to 97% at 3 ng/ml. Recovery of rBPI23 averaged 56% and ranged from 30% at 0.5 ng/ml to 90% at 50,000 ng/ml. Because LBP is present in normal human plasma and shares sequence homology with BPI, the effects of rLBP on the BPI ELISA were also evaluated. Under standard assay conditions, rLBP caused minimal interference with BPI detection. At 100 micrograms/ml, rLBP generated a signal equivalent to 3 ng/ml of rBPI and 0.6 ng/ml of rBPI23. Matched serum and plasma samples were collected from 20 healthy adults to measure endogenous levels of BPI. The range of BPI concentrations was < 0.2-2.1 ng/ml in plasma and 4.9-72.1 ng/ml in serum. Western blot analysis indicated that the BPI ELISA immunoreactivity in plasma and serum correlated with the presence of a protein doublet (M(r) approximately 60,000), which comigrated with native BPI extracted from human neutrophils. These data demonstrate that low levels of holo-BPI are present in plasma, and suggest that additional quantities of BPI were released from neutrophils during the process of coagulation.

Reactivity of monoclonal antibody E5 with endotoxin. I. Binding to lipid A and rough lipopolysaccharides.

The murine IgM monoclonal antibody (mAb) E5 was produced by a hybridoma derived from spleen cells of a mouse immunized with the J5 rough mutant of Escherichia coli O111:B4. In a multicenter randomized placebo-controlled clinical trial, E5 has been shown to reduce significantly the mortality and morbidity of patients with Gram-negative sepsis. The characteristics of E5 binding to endotoxin were studied in vitro. We report here the results of binding to an extensive panel of rough lipopolysaccharide (LPS) and lipid A preparations. Using standard immunologic techniques, including enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), as well as an antibody capture assay using immobilized antibody and a chromogenic Limulus amebocyte lysate (LAL) detection system, E5 was shown to bind to all rough LPS (chemotypes Ra through Re from Salmonella minnesota and E. coli J5) and lipid A preparations tested. E5 displayed a Kd for Ra LPS of approximately 6.5 nM. These results confirm and extend those reported previously and provide evidence that E5 binds specifically to lipid A and to the lipid A moiety of rough LPS.

Reactivity of monoclonal antibody E5 with endotoxin. II. Binding to short- and long-chain smooth lipopolysaccharides.

The murine monoclonal IgM antibody E5 has been shown to significantly reduce the mortality and morbidity of patients with Gram-negative sepsis in a multicenter randomized placebo-controlled clinical trial. The in vitro binding characteristics of monoclonal antibody (mAb) E5 were studied using highly purified smooth lipopolysaccharide (LPS) isolated from a variety of clinically relevant, wild-type Gram-negative bacteria. Using a sensitive antibody-capture assay which involves immobilized mAb E5 and a chromogenic Limulus amebocyte lysate (LAL) LPS-detection system, mAb E5 was shown to bind to all 15 smooth LPS preparations tested, including LPS isolated from Escherichia, Klebsiella, Proteus, Pseudomonas, Salmonella, Serratia and Yersinia species. When LPS was fractionated according to size by size-exclusion chromatography, mAb E5 was shown to bind to smooth LPS molecules that have long as well as short O-polysaccharide chains. These results confirm and extend those reported previously and demonstrate that the anti-lipid A mAb E5 binds specifically to a diverse spectrum of smooth LPS isolated from wild-type Gram-negative bacteria.

Improved pharmacokinetics and tumor localization of immunotoxins constructed with the Mr 30,000 form of ricin A chain.

The two naturally occurring forms of ricin A chain, Mr 33,000 and Mr 30,000 (RTA33 and RTA30) have been purified, and their chemical compositions, toxicities, and tissue distributions have been determined. As reported previously, the in vitro and in vivo toxicities of RTA30 and RTA33 are similar. However, RTA30, which contains less carbohydrate with a lower mannose content than RTA33, accumulated less in the liver than did RTA33. Monoconjugate immunotoxins (i.e., containing one RTA per monoclonal antibody molecule) were constructed between RTA30 or RTA33 and the antitumor monoclonal antibody 791T/36, which recognizes a Mr 72,000 antigen on osteosarcoma and colon carcinoma cells. The two immunotoxins had similar cytoxicities in vitro but differed substantially in their pharmacokinetics and tissue distributions in vivo in nude mice bearing C170 human colorectal carcinoma xenografts. The immunotoxin derived from RTA30 (IT30) accumulated less in the liver than the immunotoxin derived from RTA33 (IT33) and cleared more slowly from the blood; the alpha and beta half-lives for IT30 and IT33 were 0.50 and 20.5 versus 0.17 and 14.6 h, respectively. As a probable consequence, IT30 accumulated to approximately 3-fold higher levels in the C170 xenografts than IT33. The reduced clearance of IT30 by the reticuloendothelial system thus resulted in prolonged survival in the blood and enhanced tumor localization relative to IT33.

Efficacy and toxicity elicited by recombinant interferons alpha and gamma when administered in combination to tumor-bearing mice.

Administration of rHuIFN-alpha A/D and rMuIFN-gamma as single agents to tumor-bearing mice resulted in a dose-related antitumor effect in each of the six models studied. When the IFNs were given in combination, the effects varied between the tumor systems. No increase in efficacy was seen in mice bearing B16-F10 melanoma or M5076 reticulum cell sarcoma while additive antitumor activity was shown in the KA31 fibrosarcoma and P388 leukemia systems. Mice inoculated with L1210 lymphoma or colon 38 carcinoma, however, revealed enhanced efficacy which was greater than additive. The data also reveal that combination of IFNs alpha and gamma administered to normal and tumor-bearing mice resulted in toxicity which was not predicted by the appropriate doses of the single agents. These studies suggest that combination of IFNs alpha and gamma may provide greater therapeutic utility than the single agents and underscore the need for additional, carefully designed preclinical and clinical efforts.

The detection of antibodies to recombinant interferon alfa-2a in human serum.

Three different procedures have been used for detecting antibodies to Roferon-A (recombinant human interferon alfa-2a, rHuIFN alpha-2a) in the serum of patients who received this interferon as part of ongoing clinical trials: an antiviral neutralization bioassay (ANB), the standard method recommended by the World Health Organization (WHO), and the more recently developed radioimmunoassay (RIA) and enzymeimmunoassay (EIA). Although the three tests are based on different principles, the correlation among them was excellent. The assays show differences in sensitivities with the ANB being the least sensitive of the three. The EIA equals the RIA in sensitivity, reproducibility, accuracy and labor and provides the advantage of safety and convenience in the use of non-radioactive materials. Therefore, the EIA has been selected as the most suitable assay for initial screening of the sera of patients receiving Roferon-A for the presence of antibodies to this interferon. EIA positive sera are then tested in the ANB to determine whether or not neutralizing activities are present.

Incidence and clinical significance of neutralizing antibodies in patients receiving recombinant interferon alfa-2a by intramuscular injection.

More than 1600 patients with neoplastic disorders have received recombinant human interferon alfa-2a (Roferon-A, Hoffmann-La Roche, Nutley, NJ) as part of ongoing or completed clinical trials. In this report, the efficacy of interferon alfa-2a therapy was compared with the incidence of antibodies to this interferon in 617 patients who received the drug by intramuscular administration. Antibody measurements were performed using a highly sensitive enzyme immunoassay, and an interferon antiviral neutralization bioassay. Partial or complete remission occurred in 28% (43 of 152) of the antibody-positive patients, and in 24% (112 of 465) of the antibody-negative patients (P = 0.33). The highest incidence of antibody formation occurred among patients with renal cell carcinoma and acquired immune deficiency syndrome (AIDS)-related Kaposi's sarcoma (44% and 34%, respectively). Both the duration of treatment and length of survival were significantly longer for antibody-positive than for antibody-negative patients. No significant intergroup differences emerged for response rates or for time to onset or duration of therapeutic response. When results from the above assays were compared to those used for the detection of antibodies to recombinant interferon alfa-2b (Intron A, Schering-Plough Inc., Kenilworth, NJ), the immunoradiometric assay method was determined to be seriously deficient for determination of antibody incidence. This decreased assay sensitivity may account for the reportedly lower incidence of antibodies to recombinant alfa-2b interferon.

A sensitive radioimmunoassay for detection of antibodies to recombinant human interferon-alpha A.

A radioimmunoassay (RIA) for the detection of antibodies to recombinant human leukocyte interferon A (rHuIFN-alpha A) in human serum has been developed and validated against the standard antiviral neutralization bioassay (ANB). The assay measures the binding of 125I-labeled rHuIFN-alpha A to immunoglobulins in serum. Aliquots of patients' sera are incubated with 125I-rHuIFN-alpha A and the complexes formed between antibodies in the sera and the 125I-rHuIFN-alpha A are precipitated with goat anti-human IgG serum. The radioactivity in the immune precipitate is a measure of the quantity of antibody (if present) in the serum. The sensitivity of this RIA is 5 ng of IgG/ml of serum.

Pharmacokinetics and tissue distribution of recombinant human alpha A, D, A/D(Bgl), and I interferons and mouse alpha-interferon in mice.

The pharmacokinetics and tissue distribution in mice of several recombinant human alpha-interferons [rHuIFN-alpha A, D, I, and A/D(Bgl)] as well as natural mouse alpha-interferon (MuIFN-alpha) were assessed following single intravenous injections. The serum profiles of rHuIFN-alpha A, rHuIFN-alpha D, rHuIFN-alpha A/D(Bgl), and MuIFN-alpha were similar, whereas those following rHuIFN-alpha I showed a much longer terminal elimination phase. Differences in elimination half-life, volume of distribution, and total body clearance between these IFNs were observed. There was appreciable uptake of IFN in the kidney: the amount of each interferon per gram of tissue in the kidney ranges from 1 to 9 times the amount found in the serum. The greatest uptake appeared with rHuIFN-alpha D, followed by rHuIFN-alpha A, rHuIFN-alpha A/D(Bgl), and MuIFN-alpha. The only exception was rHuIFN-alpha I which showed no uptake into the kidney.

The preclinical development of Roferon-A.

Interferon alfa-2a (Roferon-A, Hoffmann-La Roche Inc., Nutley, NJ) is identical to one of approximately 15 subtypes of interferon alpha made by human leukocytes and is produced in bacteria using recombinant DNA techniques. In its antiviral, antiproliferative, and immunomodulatory activities it is similar to leukocyte interferon alpha. These activities are species-restricted and have been demonstrable, thus far, only in humans, certain other primates, bovines, and guinea pigs or cells derived therefrom. The possibility that the toxicity of interferon alfa-2a would also be species-restricted appears to have been confirmed by results obtained thus far. Toxicological studies in rats, mice and several species of monkeys have failed to indicate the side effects that have been observed in humans. However, studies in species in which interferon alfa-2a is active and in others in which it is not, have revealed similar pharmacokinetics and elimination mechanisms.

Antitumor activity of recombinant-derived interferon alpha in metastatic renal cell carcinoma.

Fifty-six patients with metastatic renal cell carcinoma (RCC) were treated with recombinant DNA-derived interferon alpha (rIFN alpha A). The first 30 patients were randomized between doses of 2 X 10(6) U/m2 and 20 X 10(6) U/m2 intramuscularly daily. No complete (CR) or partial (PR) remissions were achieved in 15 patients receiving the low dose. In contrast, 27% of those receiving the high dose achieved CR or PR. Subsequently, 26 additional patients were given the high dose and achieved a 31% response rate. Remissions lasted from 1 to more than 12 months (median, 3 months). Responses occurred predominantly in lung parenchyma or mediastinal node metastases. Toxicity of the high dose required dose reduction in 50% of the patients. Neutralizing antibodies to rIFN alpha A developed in seven of 12 responsive (58%) and nine of 29 (31%) nonresponsive patients (P = greater than .5). The median duration of remission among the antibody-positive and antibody-negative patients were 2 and 10 months, respectively (P = .009). The clinical significance of the antibodies to rIFN alpha A remains unclear, but the coincidence between the detection of antibodies and the early relapse of the disease in some responsive patients suggests that these antibodies may abrogate the biologic activity of rIFN alpha A. This effect, however, was not associated with adverse clinical sequelae.

Synergistic activity of combinations of recombinant human alpha interferon and acyclovir, administered concomitantly and in sequence, against a lethal herpes simplex virus type 1 infection in mice.

The nucleoside analog acyclovir [9-(2-hydroxyethoxymethyl)guanine] and the hybrid recombinant human alpha interferon (rHuIFN-alpha A/D) were evaluated in weanling mice for their efficacy alone and in combination against a lethal systemic infection with herpes simplex virus type 1. Simultaneous parenteral treatment with combinations of both agents at various doses resulted in a higher percentage of survival than when either agent was administered alone, with a synergistic interaction demonstrated at certain dose combinations. Sequential administration of parenteral rHuIFN-alpha A/D and oral acyclovir, administered by gavage or supplied ad libitum in drinking water, resulted in a synergistic interaction at all dose combinations tested. These results suggest that combinations of interferon and acyclovir may be useful in treating primary herpes simplex virus infections in humans.

Synergistic interaction between interferon-alpha and acyclovir in the treatment of herpes simplex virus type 1 infection in mice.

Hairless mice were infected intracutaneously with HSV-1 and treated with rHuIFN-alpha A/D, a recombinant DNA-derived hybrid human interferon-alpha that is active on mouse cells in vitro and in vivo. When given alone (1 or 2 X 10(5) units/dose) at times soon after infection, interferon showed some efficacy, reducing disease severity by 20-30% compared to control. Oral acyclovir was also effective in reducing disease severity in a dose-dependent manner, even when treatment was begun 72 h post-infection after herpetic vesicles had become apparent. When used in combination with acyclovir (400 mg/kg/day beginning 72 h post-infection), rHuIFN-alpha A/D (beginning 4 h post-infection) greatly enhanced the therapeutic effect of the nucleoside, giving a 64% reduction in disease severity score relative to control (compared to 14% for acyclovir alone). Furthermore, although interferon treatment alone was ineffective if begun after disease was apparent, it nonetheless potentiated the activity of acyclovir when co-administered with the nucleoside beginning 72 h post-infection. Combination therapy markedly reduced disease severity, limited the progression of the infection to the vesicular stage in 50% of recipient mice and promoted a more rapid onset of healing than was obtained by treatment with acyclovir alone.

Plasma and cerebrospinal fluid pharmacokinetics of recombinant interferon alpha A in monkeys: comparison of intravenous, intramuscular, and intraventricular delivery.

Interferons are currently undergoing clinical testing in patients with cancer and other diseases. A variety of routes of administration are being utilized, and there is particular interest in delivery of interferon to the central nervous system. A biphasic decline in plasma concentrations was observed in monkeys following an i.v. bolus, with initial half-times of 15 to 33 min and terminal half-times of 1.7 to 4.6 hours. Total body clearance ranged from 24 to 39 ml/sq. m/min and steady-state volume of distribution was similar to extracellular space. CSF exposure was 1% or less than that of plasma. Intramuscular injections produced lower peak concentrations and more sustained levels, but there was substantial variation in bioavailability (range 19-103%). Levels in the CSF were not detectable for the i.m. route. For intraventricular doses, CSF exposure was 3,000-fold greater than for i.v. doses, despite a 20-fold lower dose.

Effects of cloned human leukocyte interferons in the human tumor stem cell assay.

Clonogenic tumor cells from fresh biopsies of human cancers were cultivated in vitro and tested for sensitivity by continuous exposure to pharmacologically achievable concentrations of either of two highly purified human leukocyte interferon subtypes (IFN-alpha A and IFN-alpha D) prepared by recombinant DNA methods. The interferons were compared on a weight basis at concentrations of 0.4 and 4.0 ng/ml (equivalent to 80 and 800 units of interferon activity for IFN-alpha A and 2.0 and 20 units for IFN-alpha D). Inhibition of tumor colony-forming units (50% of control or less) was observed in 38.1% of the 273 tumors tested against IFN-alpha A, and in 16% of the 71 tumors tested against IFN-alpha D. Of the tumor types with at least ten samples tested against IFN-alpha A, the percentage of cases exhibiting inhibition was as follows: melanoma (51.7%), lung cancer (50%), myeloma (33.4%), ovarian cancer (33.9%), sarcoma (33.3%), adenocarcinoma of unknown primary (30.4%), breast cancer (28%), acute leukemia (30.8%), and renal cancer (23%). More marked inhibition (30% of control or less) was observed in 18.7% of all tumors tested against IFN-alpha A. Of 60 melanomas tested, 18 (30%) exhibited marked in vitro inhibition of growth with IFN-alpha A. Although a smaller number of tumors (71) were tested against IFN-alpha D on a weight basis, it appeared, in general, to be slightly less active than IFN-alpha A (p less than 0.01), and only 8% of tumors tested exhibited marked inhibition over the same dosage range of interferon. Comparison of the dose-response curves for the 68 tumors tested simultaneously against both interferons did not reveal marked interpatient differences in the inhibition curves, although IFN-alpha D was slightly less active overall. Tumors exhibiting at least 50% inhibition of tumor colony formation also proved to be sensitive to a significantly larger number of cytotoxic drugs (tested simultaneously) than the tumors not inhibited with interferon (p less than 0.0001 for IFN-alpha A). We conclude that the in vitro clonogenic assay may aid in targeting tumor types most likely to exhibit interferon sensitivity and assist in case selection for entry into clinical trials with cloned interferons.

Cell and virus sensitivity studies with recombinant human alpha interferons.

The cell sensitivity of recombinant human alpha interferons (rIFN-alpha) of greater than 95% purity (A,D and the hybrid A/D) and crude nature (B and F) was studied in human (WISH, HeLa, AG1732), bovine (MDBK, BT), monkey (Vero), mouse (L), rabbit (RK-13), and hamster (BHK-21) cells. Based on an activity of 100% in WISH cells, the other cells responded to rIFN-alpha A as follows: AG1732 (90%), HeLa (94%), and MDBK and BT cells (170-190%). Rabbit, mouse, and hamster cells had a relative sensitivity of less than 1%. rIFN-alpha B and F were essentially equivalent to rIFN-alpha A in terms of cell sensitivity, but MDBK and BT cells were about 20 times more sensitive to rIFN-alpha D than were WISH cells and rIFN-alpha D was 1/5 to 1/10 as active on L cells as on WISH cells. The activity of the hybrid IFN A/D on bovine, mouse, and human cells was similar. The inhibitory dose50 (U/ml) of rIFN-alpha A, B, D, and F against virus infections in WISH cells were: vesicular stomatitis virus (1-4), rhinovirus types 1 and 42 (2-18), and herpes simplex virus (HSV) type 2 (45-70). Type 1 HSV, Semliki Forest (SFV) and encephalomyocarditis (EMC) viruses were tested against only rIFN-alpha A and D where SFV and EMC were the most sensitive to both IFNs (ID50-SFV, 0.2 U/ml, EMC, 1.2 U/ml) while 13 U/ml of rIFN-alpha A and D inhibited Type 1 HSV. The various rIFN-alpha s did not exhibit different antiviral spectra in vitro. When tested in mice rIFN-alpha A did not protect against infections with SFV, EMC, HSV, or influenza viruses. rIFN-alpha D and A/D protected mice infected with EMC, SFV, or HSV.

Antibodies to human leucocyte interferons in cancer patients.

During the course of clinical investigation of partly purified human leucocyte interferon (IFN) prepared at the Finnish Red Cross (PIF), neutralising IgG antibodies to human leucocyte IFN were detected in the sera of 3 patients with cancer. In 2 of these patients, the antibodies were detected in serum before treatment with PIF. In the third patient antibodies developed during the course of treatment. Antibody titres against six recombinant human leucocyte IFN sub-types and one recombinant hybrid human leucocyte IFN were different in the 3 patients.