ABSTRACTIntroduction: Antitumor immune responses are postulated to initiate paraneoplastic neurological disorders when proteins that are normally restricted to neural cells are expressed as oncoproteins. Mutated oncopeptides could bypass self‐tolerant T cells to activate cytotoxic effector T lymphocytes and requisite helper T lymphocytes to stimulate autoantibody production by B lymphocytes. Methods: We investigated muscle‐type nicotinic acetylcholine receptor (AChR) antigen expression at transcriptional and protein levels in a small‐cell lung cancer line (SCLC) established from a patient with AChR‐immunoglobulin G (IgG)‐positive myasthenia gravis. Results: We identified messenger RNA transcripts encoding the 2 AChR α1‐subunit isoforms and 7 alternative‐splicing products, 3 of which yielded premature stop codons. Despite detecting native muscle‐type AChR pentamers in the tumor, we did not identify mutant α1‐peptides. However, we found α1‐subunit‐derived peptides bound to tumor major histocompatibility complex (MHC)1‐protein. In a control SCLC from an antineuronal nuclear autoantibody, type 1 (anti‐Hu)‐IgG‐positive patient, we identified MHC1‐complexed Hu protein‐derived peptides but not AChR peptides. Discussion: Our findings support onconeural protein products as pertinent immunogens initiating paraneoplastic neurological autoimmunity. Muscle Nerve 58: 600–604, 2018

ABSTRACT We are developing oncolytic vesicular stomatitis viruses (VSVs) for systemic treatment of multiple myeloma, an incurable malignancy of antibody-secreting plasma cells that are specifically localized in the bone marrow. One of the presumed advantages for using VSV as an oncolytic virus is that human infections are rare and preexisting anti-VSV immunity is typically lacking in cancer patients, which is very important for clinical success. However, our studies show that nonimmune human and mouse serum can neutralize clinical-grade VSV, reducing the titer by up to 4 log units in 60 min. In addition, we show that neutralizing anti-VSV antibodies negate the antitumor efficacy of VSV, a concern for repeat VSV administration. We have investigated the potential use of covalent modification of VSV with polyethylene glycol (PEG) or a function-spacer-lipid (FSL)–PEG construct to inhibit serum neutralization and to limit hepatosplenic sequestration of systemically delivered VSV. We report that in mice passively immunized with neutralizing anti-VSV antibodies, PEGylation of VSV improved the persistence of VSV in the blood circulation, maintaining a more than 1-log-unit increase in VSV genome copies for up to 1 h compared to the genome copy numbers for the non-PEGylated virus, which was mostly cleared within 10 min after intravenous injection. We are currently investigating if this increase in PEGylated VSV circulating half-life can translate to increased virus delivery and better efficacy in mouse models of multiple myeloma.

Neutralizing antibodies directed against measles virus (MV) surface glycoproteins prevent viral attachment and entry through the natural receptors. H protein specific IgG can enhance MV infectivity in macrophages via Fcγ receptor (FcγR)-dependent mechanism. H-specific IgM, anti-F antibodies and complement cascade activation are protective against antibody-mediated enhancement of MV infection. However, protective role of anti-H IgG against antibody-enhanced infection is not well understood. Here we designed a set of experiments to test the protective effect of H-specific IgG against FcγR-mediated infection in microglial cells. Microglial cells are also potential target of the antibody-mediated enhancement and spread of MV infection in the central nervous system. A partially neutralizing IgG monoclonal antibody (MAb) CL55, specific for MV H protein, at 10 μg/ml enhanced MV infection in mouse microglial cells by 13–14-fold. Infection-enhancing antibody concentrations induced large multinucleated syncytia formation 48–72 h post-inoculation. We generated anti-H IgG MAb 20H6 with a strong neutralization capacity >1:80,000 at 1 mg/ml concentration in MV plaque-reduction neutralization assay. In contrast to the partially protective MAb CL55, enhancement of MV infectivity by MAb 20H6 required dilutions below the 1:120 serum titer considered protective against measles infection in humans. At a concentration of 10 μg/ml MAb 20H6 exhibited a dominant protective effect and prevented MAb CL55-mediated enhancement of MV infection and virus-mediated fusion. These results indicate that neutralization capacity of the H-specific IgG determines the balance between antibody enhancement and protection against MV infection in microglial cells.

The purpose of our study was to validate the ability of pinhole micro-single-photon emission computed tomography/computed tomography (SPECT/CT) to: 1) accurately resolve the intratumoral dispersion pattern and 2) quantify the infection percentage in solid tumors of an oncolytic measles virus encoding the human sodium iodide symporter (MV-NIS). Sodium iodide symporter (NIS) RNA level and dispersion pattern were determined in control and MV-NIS-infected BxPC-3 pancreatic tumor cells and mouse xenografts using quantitative, real-time, reverse transcriptase, polymerase chain reaction, autoradiography and immunohistochemistry (IHC). Mice with BxPC-3 xenografts were imaged with 123I or 99TcO4 micro-SPECT/CT. Tumor dimensions and radionuclide localization were determined with imaging software. Linear regression and correlation analyses were performed to determine the relationship between tumor infection percentage and radionuclide uptake (% injected dose per gram) above background and a highly significant correlation was observed (r2=0.947). A detection threshold of 1.5-fold above the control tumor uptake (background) yielded a sensitivity of 2.7% MV-NIS-infected tumor cells. We reliably resolved multiple distinct intratumoral zones of infection from non-infected regions. Pinhole micro-SPECT/CT imaging using the NIS reporter demonstrated precise localization and quantitation of oncolytic MV-NIS infection, and can replace more time-consuming and expensive analyses (for example, autoradiography and IHC) that require animal killing.

This study was undertaken to determine concentrations for eight metals in human liver and investigate correlations among these metals as well as subject age. Autopsy specimens from 32 males, ranging from 38-88 years of age, were analyzed for calcium (Ca), cadmium (Cd), copper (Cu), iron (Fe), mercury (Hg), magnesium (Mg), lead (Pb), and zinc (Zn). Respective mean concentrations were 49.6, 2.1, 4.9, 318.7, 0.72, 135.2, 0.19, and 53.4 microg/g wet wt., and 209.2, 8.8, 20.6, 1342.2, 0.81, 569.4, 3.1 and 228.8 microg/g dry wt. Variables, including age and metal concentrations, were subjected to simple and multiple correlation analysis. Four metal pairs Zn-Cd, Fe-Pb, Mg-Hg, and Mg-Zn gave significant positive correlations. Age and Cu correlated negatively. Multiple correlations were found for Mg with Hg and Zn, Pb with Fe and age, Pb with Fe and Cu, and Zn with Mg and Cd. Results are discussed within the context of relevant literature.

Toxicology studies were performed in rats and rhesus macaques to establish a safe starting dose for intratumoral injection of an oncolytic vesicular stomatitis virus expressing human interferon-beta (VSV-hIFNbeta) in patients with hepatocellular carcinoma (HCC). No adverse events were observed after administration of 7.59 x 10(9) TCID(50) (50% tissue culture infective dose) of VSV-hIFNbeta into the left lateral hepatic lobe of Harlan Sprague Dawley rats. Plasma alanine aminotransferase and alkaline phosphatase levels increased and platelet counts decreased in the virus-treated animals on days 1 and 2 but returned to pretreatment levels by day 4. VSV-hIFNbeta was also injected into normal livers or an intrahepatic McA-RH7777 HCC xenograft established in Buffalo rats. Buffalo rats were more sensitive to neurotoxic effects of VSV; the no observable adverse event level (NOAEL) of VSV-hIFNbeta in Buffalo rats was 10(7) TCID(50). Higher doses were associated with fatal neurotoxicity and infectious virus was recovered from tumor and brain. Compared with VSV-hIFNbeta, toxicity of VSV-rIFNbeta (recombinant VSV expressing rat IFN-beta) was greatly diminished in Buffalo rats (NOAEL, >10(10) TCID(50)). Two groups of two adult male rhesus macaques received 10(9) or 10(10) TCID(50) of VSV-hIFNbeta injected directly into the left hepatic lobe under computed tomographic guidance. No neurological signs were observed at any time point. No abnormalities (hematology, clinical chemistry, body weights, behavior) were seen and all macaques developed neutralizing anti-VSV antibodies. Plasma interleukin-6, tumor necrosis factor-alpha, and hIFN-beta remained below detection levels by ELISA. On the basis of these studies, we will be proposing a cautious approach to dose escalation in a phase I clinical trial among patients with HCC.

Background: MV-NIS is an Edmonston-lineage measles virus that expresses the human sodium-iodide symporter (hNIS). The virus is oncolytic, and its activity can be monitored by noninvasive imaging of radioiodine uptake by hNIS. The receptor is CD46, a membrane regulator of complement activation that is overexpressed on myeloma cells.

Antibodies to viral surface glycoproteins play a crucial role in immunity to measles by blocking both virus attachment and subsequent fusion with the host cell membrane. Here, we demonstrate that certain immunoglobulin G (IgG) antibodies can also enhance the entry of measles virus (MV) into monocytes and macrophages. Antibody-dependent enhancement of infectivity was observed in mouse and human macrophages using virions opsonized by a murine monoclonal antibody against the MV hemagglutinin (H) glycoprotein, polyclonal mouse anti-MV IgG, or diluted measles-immune human sera. Neither H-specific Fab fragments nor H-specific IgM could enhance MV entry in monocytes or macrophages, indicating involvement of a Fc gamma receptor (FcgammaR)-mediated mechanism. Preincubation with an anti-fusion protein (anti-F) monoclonal antibody or a fusion-inhibitory peptide blocked infection, indicating that a functional F protein was required for viral internalization. Classical complement pathway activation did not promote infection through complement receptors and inhibited anti-H IgG-mediated enhancement. In vivo, antibody-enhanced infection allowed MV to overcome a highly protective systemic immune response in preimmunized IfnarKo-Ge46 transgenic mice. These data demonstrate a previously unidentified mechanism that may contribute to morbillivirus pathogenesis where H-specific IgG antibodies promote the spread of MV infection among FcgammaR-expressing host cells. The findings point to a new model for the pathogenesis of atypical MV infection observed after immunization with formalin-inactivated MV vaccine and underscore the importance of the anti-F response after vaccination.

Attenuated measles virus (MV) propagates selectively in human tumor cells and phase I clinical trials are currently underway to test their oncolytic activity. However, rapid antibody-mediated neutralization of the therapeutic viruses in blood or peritoneal fluid poses a major threat to the success of the approach. Classical and alternative complement pathway activation also contributes to complete virus neutralization in the plasma. Viremia during natural measles infection is exclusively cell-associated such that live MV can be isolated only from infected PBMC and not as cell-free virions from serum samples. We therefore hypothesized that autologous measles infected cells might be a more reliable vehicle than cell free virions to deliver the infection to tumor cells in subjects with neutralizing titers of anti-measles antibodies. Our in vitro studies, using a dual color fluorescent model, demonstrated the efficiency of cell-to-cell transfer of infection. In contrast to naked virions, heterofusion between infected monocytic line and tumor cells was 16-32 times more resistant to antibody neutralization. Systemically and intraperitoneally (i.p.) administered infected monocytes or stimulated peripheral blood cells successfully delivered oncolytic MV to the tumor lesions. Repeated i.p. injections of cell carriers significantly improved survival in an ovarian cancer xenograft model. MV survived antibody and complement neutralization within infected cells. In contrast to the naked virus, cell delivered MV and heterofusion escaped from the neutralizing antibodies after systemic or i.p. injection in a lymphoma metastatic model, and an i.p. hepatocellular cancer model. In addition, we demonstrated antibody-enhanced infectivity in monocytes/macrophages and subsequent transfer of MV infection to the target cells. These results suggest a novel strategy for systemic delivery of oncolytic virotherapy in cancer patients that can “by-pass” the pre-existing humoral immunity against MV.

Recombinant measles viruses are being evaluated as oncolytic virotherapy agents in various pre-clinical models and a human phase I clinical trial for ovarian cancer. To support these studies, we have grown and purified measles viruses, including pre-clinical and clinical grade lots using various excipients. For clinical trials using viruses, maintenance of the agent's infectious titer is critical. Therefore, a detailed understanding of the effects of temperature and excipients on the stability of the virus during production, short-term storage, and long-term storage is required. We evaluated the biological activity of measles virus stocks by measuring the infectious titer of samples stored frozen (<-70|[deg]|C), refrigerated (1-6|[deg]|C), or diluted into normal saline at room temperature as necessary for administration to cancer patients.

Recombinant measles viruses have shown potent oncolytic effects against tumors in animal models. In order to test the potential of recombinant measles viruses as virotherapy agents in humans, a large quantity of high-titer purified virus is needed. The characteristics of measles virus particles add to the difficulty of scalable purification for clinical administration: large pleomorphic size (100–300 nm), enveloped, inefficient budding from the infected cell, and cytotoxicity to the producer cell. The size and pleomorphic nature of the measles virus particles prevents a final sterile filtration step, requiring aseptic processing conditions to be maintained throughout the entire production process. In support of a Phase I clinical trial using a measles virus expressing a soluble form of carcinoembryonic antigen (MV-CEA) for treatment of ovarian cancer, a process was developed to manufacture a high-titer, large-scale, clinical-grade product using current Good Manufacturing Practices.

The Lambert-Eaton myasthenic syndrome (LES) is an autoimmune presynaptic disorder of peripheral cholinergic neurotransmission in which there is often an associated small cell lung carcinoma (SCC). SCC lines established from patients with and without LES exhibit a Ca2+ influx response to depolarization by K+ that is consistent with the presence of voltage-gated Ca2+ channels. Autoantibodies antagonistic to SCC Ca2+ channel activity were found exclusively in patients with LES, independent of cancer status. Depolarization-induced uptake of 45Ca2+ by SCC lines was reduced maximally after 3-4 days of exposure to serum immunoglobulins from 14 of 19 LES patients, while 53 control immunoglobulins (including patients with SCC, other tumors, other paraneoplastic syndromes, and other neurological and autoimmune diseases) were without effect. The snail neurotoxin omega-conotoxin of subtype GVIA, which is a specific antagonist of presynaptic Ca2+ channels, inhibited K+-stimulated Ca2+ uptake in a dose-dependent manner that was essentially irreversible. Adenosine, reported to be a specific antagonist of neuronal Ca2+ channels, also impaired voltage-stimulated Ca2+ influx in SCC. Use of LES patients' IgG and omega-conotoxin in further studies of SCC may facilitate identification and purification of the LES antigen(s) and yield a quantitative serological test for diagnosing this autoimmune paraneoplastic syndrome.

Annals of the New York Academy of SciencesVolume 540, Issue 1 p. 369-371 Voltage-Dependent Ca2+ Channels in Small Cell Carcinomas Are Blocked by Autoantibodies from Patients with Lambert-Eaton Myasthenic Syndromea HENRY J. DE AIZPURUA, HENRY J. DE AIZPURUA Neuroimmunology Laboratory Mayo Clinic Rochester, Minnesota 55905Search for more papers by this authorGUY E. GRIESMANN, GUY E. GRIESMANN Neuroimmunology Laboratory Mayo Clinic Rochester, Minnesota 55905Search for more papers by this authorEDWARD H. LAMBERT, EDWARD H. LAMBERT Department of Neurology University of Minnesota Minneapolis, Minnesota 55455Search for more papers by this authorVANDA A. LENNON, VANDA A. LENNON Neuroimmunology Laboratory Mayo Clinic Rochester, Minnesota 55905Search for more papers by this author HENRY J. DE AIZPURUA, HENRY J. DE AIZPURUA Neuroimmunology Laboratory Mayo Clinic Rochester, Minnesota 55905Search for more papers by this authorGUY E. GRIESMANN, GUY E. GRIESMANN Neuroimmunology Laboratory Mayo Clinic Rochester, Minnesota 55905Search for more papers by this authorEDWARD H. LAMBERT, EDWARD H. LAMBERT Department of Neurology University of Minnesota Minneapolis, Minnesota 55455Search for more papers by this authorVANDA A. LENNON, VANDA A. LENNON Neuroimmunology Laboratory Mayo Clinic Rochester, Minnesota 55905Search for more papers by this author First published: November 1988 https://doi.org/10.1111/j.1749-6632.1988.tb27102.xCitations: 3 a This work was supported by Grants CA 37343 (VAL) and NS 23691 (EHL) from the National Institutes of Health. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume540, Issue1Advances in NeuroimmunologyNovember 1988Pages 369-371 RelatedInformation

Annals of the New York Academy of SciencesVolume 540, Issue 1 p. 516-519 Synthetic Peptide of Human Acetylcholine Receptor α-Subunit Sequence 125-147 (Methionine 144), a More Potent Autoantigen Than Its Norleucine 144 Analoga VANDA A. LENNON, VANDA A. LENNON Departments of Immunology, Neurology, Mayo Clinic Rochester, Minnesota 55905Search for more papers by this authorZHONG-XIAN HUANG, ZHONG-XIAN HUANG Department of Neurology, University of Minnesota Minneapolis, Minnesota 55455Search for more papers by this authorDANIEL J. McCORMICK, DANIEL J. McCORMICK Departments Biochemistry and Molecular Biology, Mayo Clinic Rochester, Minnesota 55905Search for more papers by this authorGUY E. GRIESMANN, GUY E. GRIESMANN Departments of Immunology, Neurology, Mayo Clinic Rochester, Minnesota 55905Search for more papers by this authorNAOKI FUJII, NAOKI FUJII Departments of Immunology, Neurology, Mayo Clinic Rochester, Minnesota 55905Search for more papers by this authorEDWARD H. LAMBERT, EDWARD H. LAMBERT Department of Neurology, University of Minnesota Minneapolis, Minnesota 55455Search for more papers by this author VANDA A. LENNON, VANDA A. LENNON Departments of Immunology, Neurology, Mayo Clinic Rochester, Minnesota 55905Search for more papers by this authorZHONG-XIAN HUANG, ZHONG-XIAN HUANG Department of Neurology, University of Minnesota Minneapolis, Minnesota 55455Search for more papers by this authorDANIEL J. McCORMICK, DANIEL J. McCORMICK Departments Biochemistry and Molecular Biology, Mayo Clinic Rochester, Minnesota 55905Search for more papers by this authorGUY E. GRIESMANN, GUY E. GRIESMANN Departments of Immunology, Neurology, Mayo Clinic Rochester, Minnesota 55905Search for more papers by this authorNAOKI FUJII, NAOKI FUJII Departments of Immunology, Neurology, Mayo Clinic Rochester, Minnesota 55905Search for more papers by this authorEDWARD H. LAMBERT, EDWARD H. LAMBERT Department of Neurology, University of Minnesota Minneapolis, Minnesota 55455Search for more papers by this author First published: November 1988 https://doi.org/10.1111/j.1749-6632.1988.tb27156.xCitations: 2 a This work was supported by grants from the National Institutes of Health (NS 15057, V.A.L.; NS 24694, D.J.M.; NS 23691, E.H.L.) and The William K. Warren Foundation. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume540, Issue1Advances in NeuroimmunologyNovember 1988Pages 516-519 RelatedInformation