Conjugation of anti-HIV gp41 monoclonal antibody to a drug capable of targeting resting lymphocytes produces an effective cytotoxic anti-HIV immunoconjugate.

HIV-infected cells persisting in the face of suppressive antiretroviral therapy are the barrier to curing infection. Cytotoxic immunoconjugates targeted to HIV antigens on the cell surface may clear these cells. We showed efficacy in mouse and macaque models using immunotoxins, but immunogenicity blunted the effect. As an alternative, we propose antibody drug conjugates (ADCs), as used in cancer immunotherapy. In cancer, the target is a dividing cell, whereas it may not be in HIV. We screened cytotoxic drugs on human primary cells and cell lines. An anthracycline derivative, PNU-159682 (PNU), was highly cytotoxic to both proliferating and resting cells. Human anti-gp41 mAb 7B2 was conjugated to ricin A chain or PNU. The conjugates were tested in vitro for cytotoxic efficacy and anti-viral effect, and in vivo for tolerability. The specificity of killing for both conjugates was demonstrated on Env+ and Env- cells. The toxin conjugate was more potent and killed more rapidly, but 7B2-PNU was effective at levels achievable in patients. The ricin conjugate was well tolerated in mice; 7B2-PNU was toxic when administered intraperitoneally but was tolerated intravenously. We have produced an ADC with potential to target the persistent HIV reservoir in both dividing and non-dividing cells while avoiding immunogenicity. Cytotoxic anti-HIV immunoconjugates may have greatest utility as part of an "activate and purge" regimen, involving viral activation in the reservoir. This is a unique comparison of an immunotoxin and ADC targeted by the same antibody and tested in the same systems.IMPORTANCEHIV infection can be controlled with anti-retroviral therapy, but it cannot be cured. Despite years of therapy that suppresses HIV, patients again become viremic shortly after discontinuing treatment. A long-lived population of memory T cells retain the genes encoding HIV, and these cells secrete infectious HIV when no longer suppressed by therapy. This is the persistent reservoir of HIV infection. The therapies described here use anti-HIV antibodies conjugated to poisons to kill the cells in this reservoir. These poisons may be of several types, including protein toxins (immunotoxins) or anti-cancer drugs (antibody drug conjugates, ADCs). We have previously shown that an anti-HIV immunotoxin had therapeutic effects in animal models, but it elicited an anti-drug immune response. Here, we have prepared an anti-HIV ADC, which would be less likely to provoke an immune response, and show its potential for use in eliminating the persistent reservoir of HIV infection.

Distinct Metabolic States Are Observed in Hypoglycemia Induced in Mice by Ricin Toxin or by Fasting.

Hypoglycemia may be induced by a variety of physiologic and pathologic stimuli and can result in life-threatening consequences if untreated. However, hypoglycemia may also play a role in the purported health benefits of intermittent fasting and caloric restriction. Previously, we demonstrated that systemic administration of ricin toxin induced fatal hypoglycemia in mice. Here, we examine the metabolic landscape of the hypoglycemic state induced in the liver of mice by two different stimuli: systemic ricin administration and fasting. Each stimulus produced the same decrease in blood glucose and weight loss. The polar metabolome was studied using 1H NMR, quantifying 59 specific metabolites, and untargeted LC-MS on approximately 5000 features. Results were analyzed by multivariate analyses, using both principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA), to identify global metabolic patterns, and by univariate analyses (ANOVA) to assess individual metabolites. The results demonstrated that while there were some similarities in the responses to the two stimuli including decreased glucose, ADP, and glutathione, they elicited distinct metabolic states. The metabolite showing the greatest difference was O-phosphocholine, elevated in ricin-treated animals and known to be affected by the pro-inflammatory cytokine TNF-α. Another difference was the alternative fuel source utilized, with fasting-induced hypoglycemia primarily ketotic, while the response to ricin-induced hypoglycemia involves protein and amino acid catabolism.

Parenteral Exposure of Mice to Ricin Toxin Induces Fatal Hypoglycemia by Cytokine-Mediated Suppression of Hepatic Glucose-6-Phosphatase Expression.

Ricin toxin is an agent of biodefense concern and we have been developing countermeasures for ricin threats. In doing so, we sought biomarkers of ricin toxicosis and found that in mice parenteral injection of ricin toxin causes profound hypoglycemia, in the absence of other clinical laboratory abnormalities. We now seek to identify the mechanisms underlying this hypoglycemia. Within the first hours following injection, while still normoglycemic, lymphopenia and pro-inflammatory cytokine secretion were observed, particularly tumor necrosis factor (TNF)-α. The cytokine response evolved over the next day into a complex storm of both pro- and anti-inflammatory cytokines. Evaluation of pancreatic function and histology demonstrated marked islet hypertrophy involving predominantly ß-cells, but only mildly elevated levels of insulin secretion, and diminished hepatic insulin signaling. Drops in blood glucose were observed even after destruction of ß-cells with streptozotocin. In the liver, we observed a rapid and persistent decrease in the expression of glucose-6-phosphatase (G6Pase) RNA and protein levels, accompanied by a drop in glucose-6-phosphate and increase in glycogen. TNF-α has previously been reported to suppress G6Pase expression. In humans, a genetic deficiency of G6Pase results in glycogen storage disease, type-I (GSD-1), a hallmark of which is potentially fatal hypoglycemia.

A modified SCID mouse model of HIV infection with utility for testing anti-HIV therapies.

Using human tumor cells we have developed a mouse model of active HIV infection that may be used for testing antiviral agents, although it does not reflect the pathogenesis of human infection. Irradiated SCID/NOD mice are injected with a tumor of human CD4+ lymphoma cells susceptible to infection and at a separate site, tumor cells persistently infected with either primary or T cell line-adapted strains of HIV. The spread of infection from the infected to the susceptible tumor is monitored as plasma p24 and the presence of HIV-infected cells in the spleen. We have used this model to examine the relative efficacy of neutralizing anti-HIV antibodies to halt the spread of infection. We have found that the tetrameric CD4-antibody fusion protein, CD4-IgG2, is highly effective compared to an anti-V3 loop antibody. This animal model, while not replicating the human disease, allows for the simultaneous testing of efficacy, toxicity, and pharmacokinetics of potential new antiviral therapies. The model can easily be powered to enable comparisons between different therapeutic agents and dosing regimens.

In vivo efficacy of anti-glycoprotein 41, but not anti-glycoprotein 120, immunotoxins in a mouse model of HIV infection.

Immunotoxins (ITs) targeting the HIV envelope protein are among the most efficacious antiviral therapies when tested in vitro. Yet a first-generation IT targeted to gp120, CD4-PE40 (chimeric immunotoxin using CD4 and the translocation and enzymatic domains of Pseudomonas exotoxin A), showed limited promise in initial clinical testing, highlighting the need for improved ITs. We have used a new mouse model of HIV infection to test the comparative efficacy of anti-HIV ITs targeted to gp120 or to gp41. Irradiated SCID/nonobese diabetic mice are injected with a tumor of human CD4(+) cells susceptible to infection and at a separate site persistently HIV-infected cells. The spread of infection from infected to susceptible tumor is monitored by plasma p24 and the presence of HIV-infected cells in the spleen. Anti-gp41 ITs in combination with tetrameric CD4-human Ig fusion protein have pronounced anti-HIV effects. Little if any anti-HIV efficacy was found with either CD4-PE40 or an Ab-targeted anti-gp120 IT. These data support continued exploration of the utility of ITs for HIV infection, particularly the use of anti-gp41 ITs in combination with soluble CD4 derivatives.