Michael S. Lebowitz

A multifunctional cytoprotective agent that reduces neurodegeneration after ischemia

Cellular and molecular pathways underlying ischemic neurotoxicity are multifaceted and complex. Although many potentially neuroprotective agents have been investigated, the simplicity of their protective mechanisms has often resulted in insufficient clinical utility. We describe a previously uncharacterized class of potent neuroprotective compounds, represented by PAN-811, that effectively block both ischemic and hypoxic neurotoxicity. PAN-811 disrupts neurotoxic pathways by at least two modes of action. It causes a reduction of intracellular-free calcium as well as free radical scavenging resulting in a significant decrease in necrotic and apoptotic cell death. In a rat model of ischemic stroke, administration of PAN-811 i.c.v. 1 h after middle cerebral artery occlusion resulted in a 59% reduction in the volume of infarction. Human trials of PAN-811 for an unrelated indication have established a favorable safety and pharmacodynamic profile within the dose range required for neuroprotection warranting its clinical trial as a neuroprotective drug.

PAN-811 inhibits oxidative stress-induced cell death of human Alzheimer's disease-derived and age-matched olfactory neuroepithelial cells via suppression of intracellular reactive oxygen species.

Oxidative stress plays a significant role in neurotoxicity associated with a variety of neurodegenerative diseases including Alzheimers disease (AD). Increased oxidative stress has been shown to be a prominent and early feature of vulnerable neurons in AD. Olfactory neuroepithelial cells are affected at an early stage. Exposure to oxidative stress induces the accumulation of intracellular reactive oxygen species (ROS), which in turn causes cell damage in the form of protein, lipid, and DNA oxidations. Elevated ROS levels are also associated with increased deposition of amyloid-beta and formation of senile plaques, a hallmark of the AD brain. If enhanced ROS exceeds the basal level of cellular protective mechanisms, oxidative damage and cell death will result. Therefore, substances that can reduce oxidative stress are sought as potential drug candidates for treatment or preventative therapy of neurodegenerative diseases such as AD. PAN-811, also known as 3-aminopyridine-2-carboxaldehyde thiosemicarbazone or Triapine, is a small lipophilic compound that is currently being investigated in several Phase II clinical trials for cancer therapy due to its inhibition of ribonucleotide reductase activity. Here we show PAN-811 to be effective in preventing or reducing ROS accumulation and the resulting oxidative damages in both AD-derived and age-matched olfactory neuroepithelial cells.

A novel neuroprotectant PAN-811 protects neurons from oxidative stress

Hydrogen peroxide (H2O2), a major non-radical reactive oxygen species (ROS) could elicit intracellular oxidative damage and/or cause extracellular free calcium influx by activating the NMDA receptor or through calcium channels. In the present study, NMDA receptor antagonist MK-801 fully blocked H2O2-induced neuronal cell death, whereas green tea (GT) extract containing-antioxidants only partially suppressed the neurotoxicity of H2O2. These suggest that majority of ROS overproduction is downstream of H2O2-induced calcium influx. A novel neuroprotectant PAN-811 was previously demonstrated to efficiently attenuate ischemic neurotoxicity. PAN-811 hereby fully blocks H2O2-elicited neuronal cell death with a more advanced neuroprotective profile than that of GT extract. PAN-811 was also shown to protect against CaCl2-elicited neurotoxicity. Efficient protection against oxidative stress-induced neurotoxicity by PAN-811 indicates its potential application in treatment of ROS-mediated neurodegenerative diseases.

Lambda phage-based vaccine induces antitumor immunity in hepatocellular carcinoma

Background and aims Hepatocellular carcinoma (HCC) is a difficult to treat tumor with a poor prognosis. Aspartate β-hydroxylase (ASPH) is a highly conserved enzyme overexpressed on the cell surface of both murine and human HCC cells. Methods We evaluated therapeutic effects of nanoparticle lambda (λ) phage vaccine constructs against ASPH expressing murine liver tumors. Mice were immunized before and after subcutaneous implantation of a syngeneic BNL HCC cell line. Antitumor actively was assessed by generation of antigen specific cellular immune responses and the identification of tumor infiltrating lymphocytes. Results Prophylactic and therapeutic immunization significantly delayed HCC growth and progression. ASPH-antigen specific CD4+ and CD8+ lymphocytes were identified in the spleen of tumor bearing mice and cytotoxicity was directed against ASPH expressing BNL HCC cells. Furthermore, vaccination generated antigen specific Th1 and Th2 cytokine secretion by immune cells. There was widespread necrosis with infiltration of CD3+ and CD8+ T cells in HCC tumors of λ phage vaccinated mice compared to controls. Moreover, further confirmation of anti-tumor effects on ASPH expressing tumor cell growth were obtained in another murine syngeneic vaccine model with pulmonary metastases. Conclusions These observations suggest that ASPH may serve as a highly antigenic target for immunotherapy.

Inhibition of tumor growth in vivo by a nanoparticle-based therapeutic cancer vaccine targeting HAAH

We have designed, developed and produced a lambda-phage based therapeutic anti-cancer vaccine (nanoparticle) targeting human aspartyl (asparaginyl) β-hydroxylase (HAAH). This protein is over-expressed on the surface of cancer cells and plays a central role in cancer etiology that affects cancer cell growth, motility and invasiveness. To overcome the self-antigen tolerance of the molecule, we have designed a vaccine entity that contains an immunostimulant and presents HAAH in a manner that is unfamiliar to the body. We have expressed three portions of the HAAH protein, sequences from the N-terminus, middle portion and C-terminus, as fusion proteins (with the gpD bacteriophage antigen) on the surface of bacteriophage lambda, generating 200-300 copies per phage. All three entities display highly significant, dose-dependent immunogenicity as assessed by antibody ELISAs. To evaluate the therapeutic effect of the nanoparticle vaccine, we initiated tumor formation in BALB/c mice using a mouse hepatocellulular carcinoma cell line, BNLT3. This cell line, a highly tumorigenic subclone of the ATCC cell line, BNL 1ME A.7R.1, was developed by J. Wands at the Liver Research Center, Rhode Island Hospital by three serial subcutaneous passages of the parental cell line. On Day 0 of the tumor challenge study, 4 groups of 5 mice each were administered 5E03 BNLT3 cells subcutaneously. On the same day, the animals received the first of three weekly subcutaneous doses (i.e., Days 0, 7 and 14) of nanoparticle vaccine as monovalent vaccines of each of the three bacteriophage constructs (10E10 pfu/dose) or a buffer control. The mice were then observed for tumor growth and tumor volume was determined. After 4 weeks (Study Day 28), 3 of 5 mice in the control group had measurable tumor growth, while in the HAAH N-terminus, middle portion and C-terminus construct vaccine groups, 0/5, 2/5 and 0/5 animals, respectively, had measurable tumor growth. The mean tumor volumes of the 5 animals in each of the two groups with tumor growth were 85.8 mm3 for the control group and 24.9 mm3 (29 % of control tumor volume) for the HAAH middle portion group. Overall, the vaccinated groups had growth in 2/15 animals and mean tumor growth of 8.3 mm3 (> 10 % of the control group tumor size). We are continuing to evaluate the HAAH nanoparticle vaccines in the mouse tumor model under various conditions and combinations of constructs. We are also assessing study samples for cell-based immunity as we attempt to elicit the mechanism of action for this vaccine candidate. We are also taking all the required steps in preparation for an IND submission to FDA.

Immunogenicity of a lambda phage-based anti-cancer vaccine targeting HAAH

Meeting abstracts We have designed, developed and produced a lambda-phage based anti-cancer vaccine (nano-particle) targeting human aspartyl (asparaginyl) β-hydroxylase (HAAH). This follows accumulated evidence that HAAH meets requirements of a good target for anti-cancer immunotherapy. The

Abstract 2650: Radioimmunotherapy for acute myeloid leukemia targeting human aspartyl (asparaginyl) β-hydroxylase

Treatment options for acute myeloid leukemia (AML) are limited and have modest impact on 5-year survival rates in patients Citation Format: Michael S. Lebowitz, Ekaterina Revskaya, Kanam Malhotra, Amir Shahlaee, Steven Fuller, Maria R. Baer, Noa G. Holtzman, Ashkan Emadi, Ekaterina Dadachova, Hossein A. Ghanbari. Radioimmunotherapy for acute myeloid leukemia targeting human aspartyl (asparaginyl) β-hydroxylase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2650. doi:10.1158/1538-7445.AM2017-2650

Abstract 723: Improved detection of cancer specific serum exosomal aspartyl (asparaginyl) beta hydroxylase (HAAH)

By means of an ELISA, we have found the pan-cancer biomarker HAAH to be closely associated with serum exosomes primarily in cancers of the breast, lung, colon, and prostate. This new understanding of the HAAH target has led to a dramatically improved serological detection assay, and in turn diagnostic reagent kit. In keeping with the multivalent particulate nature of exosomes, appropriately reformatting the ELISA assay has yielded a simplified so-called simultaneous-homologous format. Currently in this format, a single monoclonal anti-HAAH antibody (FB50) serves both as microplate capture and as a biotinylated detector. All reagents, including peroxidase-streptavidin, are co-incubated in the FB50- coated microplate simultaneously with serum samples, in the absence of intervening sequential steps. This considerably shorter exosome-enabled ELISA format requires half the time and yields generally on average a two-fold or higher increase in detection of HAAH. This format was evaluated with over 100 banked serum samples from several early stage cancers. While increasing the sensitivity for HAAH in cancer samples, the improved detection and performance has not caused any undesirable increases in false positives among more than 30 normal serum samples from healthy subjects. The reagent kit components include recombinant HAAH calibrators, positive /negative controls, vialed detection reagents and a Mylar packaged FB50 pre-coated microplate. The simplified reagent kit format has yielded close inter-operator and day to day trending consistency with a limit of detection (LOD) of 3 ng/mL. Recognizing the exosomal nature of the HAAH target has resulted in some changes in blood sample procurement, sample processing, and sample shipping. Such field testing of the HAAH reagent kit was recently done with serum samples from 48 high risk or mildly symptomatic volunteers with general concerns about cancer. Upon testing these samples 9 had an HAAH level greater than 3.0 ng/ mL (range 4.2 to 116.7) and 39 had less than 3.0 ng/ mL. Based upon the post-analysis diagnostic determination, there were two false negatives and one false positive, hence an overall accuracy of 93.8%. Citation Format: Mark A. Semenuk, Anokhi S. Cifuentes, Eleanor R. Ghanbari, Michael S. Lebowitz, Hossein A. Ghanbari. Improved detection of cancer specific serum exosomal aspartyl (asparaginyl) beta hydroxylase (HAAH) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 723. doi:10.1158/1538-7445.AM2017-723

Enhanced immunogenicity of a nanoparticle therapeutic cancer vaccine targeting HAAH delivered intradermally using 3M's hollow microstructured transdermal system (hMTS)

Background We are evaluating the immunogenicity and efficacy of a nanoparticle vaccine (NPV) targeting the tumor marker human aspartyl (asparaginyl) b-hydroxylase (HAAH). HAAH is an embryonic protein that is over-expressed on the surface of cancer cells, is demonstrated to be responsible for cell proliferation, motility and invasiveness, processes which can be inhibited by anti-HAAH antibodies in vitro. We have developed novel anticancer NPVs in which portions of the HAAH molecule are expressed on l-phage (200-300 copies per NPV). These NPVs produce high-titer anti-HAAH polyclonal antibodies in mice despite the fact that the HAAH protein is highly conserved between mammalian species. We have shown that the NPV inhibits tumor growth and metastasis in mouse liver and breast cancer models and inhibits metastasis in a rat prostate cancer model, inducing both humoral and cellular responses. We report here the use of 3M’s hollow microstructured transdermal system (hMTS) to determine if this intradermal (ID) delivery device further enhances immunogenicity of the NPV.

A nanoparticle therapeutic vaccine targeting HAAH stimulates cellular immunity.

Background We are evaluating the immunogenicity and efficacy of a nanoparticle vaccine (NPV) targeting the tumor marker human aspartyl (asparaginyl) b-hydroxylase (HAAH). HAAH is an embryonic protein that is over-expressed on the surface of cancer cells, is demonstrated to be responsible for cell proliferation, motility and invasiveness, processes which can be inhibited by anti-HAAH antibodies in vitro. We have developed novel anticancer NPVs in which portions of the HAAH molecule are expressed on l-phage (200-300 copies per NPV). These NPVs are immunogenic; producing high-titer antiHAAH polyclonal antibodies in mice despite the fact that the HAAH protein is highly conserved between mammalian species. We have further shown that the NPVs inhibit tumor growth and metastasis and extends survival in mouse models of liver and breast cancer and in a rat model of prostate cancer. It is generally understood that for a vaccine to provide lasting protective immunity it must also elicit strong cellular immune responses. Here we demonstrate that these HAAH-targeted NPVs can induce antigen specific cytotoxic T cell (CTL) responses.