An immunologically relevant rodent model demonstrates safety of therapy using a tumour-specific IgE.

BACKGROUND: Designing biologically informative models for assessing the safety of novel agents, especially for cancer immunotherapy, carries substantial challenges. The choice of an in vivo system for studies on IgE antibodies represents a major impediment to their clinical translation, especially with respect to class-specific immunological functions and safety. Fcε receptor expression and structure are different in humans and mice, so that the murine system is not informative when studying human IgE biology. By contrast, FcεRI expression and cellular distribution in rats mirror that of humans. METHODS: We are developing MOv18 IgE, a human chimeric antibody recognizing the tumour-associated antigen folate receptor alpha. We created an immunologically congruent surrogate rat model likely to recapitulate human IgE-FcεR interactions and engineered a surrogate rat IgE equivalent to MOv18. Employing this model, we examined in vivo safety and efficacy of antitumour IgE antibodies. RESULTS: In immunocompetent rats, rodent IgE restricted growth of syngeneic tumours in the absence of clinical, histopathological or metabolic signs associated with obvious toxicity. No physiological or immunological evidence of a "cytokine storm" or allergic response was seen, even at 50 mg/kg weekly doses. IgE treatment was associated with elevated serum concentrations of TNFα, a mediator previously linked with IgE-mediated antitumour and antiparasitic functions, alongside evidence of substantially elevated tumoural immune cell infiltration and immunological pathway activation in tumour-bearing lungs. CONCLUSION: Our findings indicate safety of MOv18 IgE, in conjunction with efficacy and immune activation, supporting the translation of this therapeutic approach to the clinical arena.

In vivo occupancy of D2 dopamine receptors by 7-OH-DPAT.

7-OH-DPAT has been shown to exhibit selectivity between D2 and D3 dopamine receptors in a variety of in vitro assay systems. Although the drug has been used in a variety of studies to evaluate the behavioral effects of D3 receptor stimulation, the in vivo D2/D3 selectivity of the compound has not been determined. In this study, protection against inactivation by EEDQ was used as a measure of in vivo occupancy of D2 receptors by behaviorally relevant doses of 7-OH-DPAT (0.03-10 mg/kg, s.c.) in adult, male Sprague-Dawley rats. Ex vivo [3H]spiperone binding was then determined in striatal membranes. 7-OH-DPAT protected against inactivation of D2 receptors with an ED50 value of 11 mg/kg. Significant protection of D2 receptors was observed at doses of 1 mg/kg and above. These data suggest that stimulation of D2 receptors contributes to the pharmacological effects of 7-OH-DPat when administered at doses above 0.3 mg/kg (s.c.).

PAC 1: an epitope associated with two novel glycoprotein components of isolated postsynaptic densities and a novel cytoskeleton-associated polypeptide.

A monoclonal antibody has been raised which recognizes an epitope, PAC 1 (postsynaptic density and cytoskeleton enriched), which is specifically associated with two novel glycoprotein components of forebrain postsynaptic density preparations and a novel neuronal cytoskeletal-associated polypeptide. The monoclonal antibody has been used to study the cellular and subcellular localization of these molecules and for the partial characterization of all three PAC 1 antigens in the rat. The PAC 1 epitope is present on two concanavalin A binding glycoproteins of apparent molecular weights 130,000 (pgp130) and 117,000 (pgp117). Both species are enriched in preparations of rat forebrain postsynaptic densities and to a lesser extent in synaptic membranes. The epitope is also expressed by a polypeptide of 155,000 mol. wt, cp155. This molecule is highly enriched in cytoskeleton rather than membrane preparations. Enzymic removal of N-linked carbohydrate lowers the molecular weights of the PAC 1 glycoproteins pgp130 and pgp117 by 11,000 and 14,000 respectively, and suggests that cp155 is not glycosylated. Detergent, alkaline and salt extractions of postsynaptic densities and synaptic membranes indicate that pgp130 and pgp117 are integral membrane glycoproteins and are tightly bound components of postsynaptic density preparations. Immunocytochemical studies of adult rat forebrain show prominent staining of pyramidal cell dendrites and perikarya. There is no evidence of glial staining. Electron microscope studies show staining of microtubules together with punctate deposits of plasma membrane-associated reaction product. Several criteria have been used to show that pgp130 and pgp117 do not correspond to other known neuronal glycoproteins of similar molecular weight. We conclude that the PAC 1 epitope is expressed by two novel synaptic glycoproteins which are very probably integral components of the postsynaptic density and by a novel neuronal cytoskeleton-associated protein.

Characterization of novel glycoprotein components of synaptic membranes and postsynaptic densities, gp65 and gp55, with a monoclonal antibody.

A monoclonal antibody, mab SMgp65, which recognises two major glycoprotein components of isolated forebrain synaptic subfractions has been raised. The mab has been used to study the cellular and subcellular localisation of these novel glycoproteins and for the partial characterisation of both molecular species. Western blots show that the mab reacts with two diffuse glycoprotein bands (gp) of apparent Mr 65,000, gp65, and 55,000, gp55. Both glycoproteins are membrane-bound, only detectable in CNS tissue and exist solely in a concanavalin A (con A) binding form. Digestion with endoglycosidase H lowers the Mr of both glycoproteins by some 5-7 kDa. Gp65 and gp55 are enriched in synaptic membrane (SM), light membrane (LM) and microsomal fractions. However, whilst gp65 is enriched in isolated postsynaptic densities (psds) gp55 is conspicuously absent from this fraction. Regional distribution studies show a marked variation in the level of gp65. Gp65 is concentrated in several forebrain regions notably cerebral cortex, hippocampus and striatum, is present only in low levels in cerebellum and is barely detectable in pons and medulla. In contrast gp55 is present in all regions studied, but is most concentrated in cerebellum. Immunocytochemical studies show intense staining of regions rich in gp65, but no staining of regions deficient in this glycoprotein. This suggests that the mab recognises gp65, but not gp55 in fixed tissue sections. Exposure of tissue sections to Triton X-100 increases the intensity of gp65-like immunoreactivity, but does not alter its pattern of subcellular distribution. Higher resolution studies show the immunoreactivity to be localised to subsets of neurites, many being axonal. The reaction deposits also extend into the synaptic region of the immunoreactive neurones. Cultured cerebellar granule cells, but not astrocytes express gp55. The results are discussed in terms of the molecular properties and localisation of these two novel glycoproteins.