Jeffrey A. Mattis

Immunization of mice with the non-toxic HC50 domain of botulinum neurotoxin presented by rabies virus particles induces a strong immune response affording protection against high-dose botulinum neurotoxin challenge.

We previously showed that rabies virus (RABV) virions are excellent vehicles for antigen presentation. Here, a reverse genetic approach was applied to generate recombinant RABV that express a chimeric protein composed of the heavy chain carboxyterminal half (HC50) of botulinum neurotoxin type A (BoNT/A) and RABV glycoprotein (G). To promote surface expression and incorporation of HC50/A into RABV virions, the RABV glycoprotein (G) ER translocation sequence, various fragments of RABV ectodomain (ED) and cytoplasmic domain were fused to HC50/A. The HC50/A chimeric proteins were expressed on the surface of cells infected with all of the recombinant RABVs, however, the highest level of surface expression was detected by utilizing 30 amino acids of the RABV G ED (HV50/A-E30). Our results also indicated that this chimeric protein was effectively incorporated into RABV virions. Immunization of mice with inactivated RABV-HC50/A-E30 virions induced a robust anti-HC50/A IgG antibody response that efficiently neutralized circulating BoNT/A in vivo, and protected mice against 1000 fold the lethal dose of BoNT/A.

Determining efficacy of breast cancer therapy by PET imaging of HER2 mRNA

INTRODUCTION Monitoring the effectiveness of therapy early and accurately continues to be challenging. We hypothesize that determination of Human Epidermal Growth Factor Receptor 2 (HER2) mRNA in malignant breast cancer (BC) cells by positron emission tomography (PET) imaging, before and after treatment, would reflect therapeutic efficacy. METHOD WT4340, a peptide nucleic acid (PNA) 12-mer complementary to HER2 mRNA was synthesized together with -CSKC, a cyclic peptide, which facilitated internalization of the PNA via IGFR expressed on BC cells, and DOTA that chelated Cu-64. Mice (n = 8) with BT474 ER+/HER2+ human BC received doxorubicin (DOX, 1.5mg/kg) i.p. once a week for six weeks. Mice (n = 8) without DOX served as controls. All mice were PET imaged with F-18-FDG and 48 h later with Cu-64-WT4340. PET imaging were performed before and 72 h after each treatment. Standardized uptake values (SUVs) were determined and percent change calculated. Animal body weight (BW) and tumor volume (TV) were measured. RESULTS SUVs for Cu-64-WT4340 after DOX treatment declined by 54% ± 17% after the second dose, 41% ± 15% after the fourth dose, and 29% ± 7% after the sixth dose, compared with 42% ± 22%, 31% ± 18%, and 13% ± 9% (p<0.05) for F-18-FDG. In untreated mice, the corresponding percent SUVs for Cu-64-WT4340 were 145% ± 82%, 165% ± 39%, and 212% ± 105% of pretreatment SUV, compared with 108% ± 28%, 151% ± 8%, and 152% ± 35.5%, (p<0.08) for F-18-FDG. TV in mice after second dose was 114.15% ± 61.83%, compared with 144.7% ± 64.4% for control mice. BW of DOX-treated mice was 103.4% ± 7.6% of pretreatment, vs. 100.1% ± 4.3% for control mice. CONCLUSION Therapeutic efficacy was apparent sooner by molecular PET imaging than by determination of reduction in TV.

Recombinant rabies virus particles presenting botulinum neurotoxin antigens elicit a protective humoral response in vivo.

Botulinum neurotoxins are one of the most potent toxins found in nature, with broad medical applications from cosmetics to the treatment of various neuropathies. Additionally, these toxins are classified as Category A-Tier 1 agents, with human lethal doses calculated at as little as 90 ng depending upon the route of administration. Of the eight distinct botulinum neurotoxin serotypes, the most common causes of human illness are from serotypes /A, /B, and /E. Protection can be achieved by eliciting antibody responses against the receptor-binding domain of the neurotoxin. Our previous research has shown that recombinant rabies virus–based particles can effectively present heterologous antigens. Here, we describe a novel strategy using recombinant rabies virus particles that elicits a durable humoral immune response against the botulinum neurotoxin receptor binding domains from serotypes /A, /B, and /E. Following intramuscular administration of β-propiolactone-inactivated rabies virus particles, mice elicited specific immune responses against the cognate antigen. Administration of a combination of these vectors also demonstrated antibody responses against all three serotypes based on enzyme-linked immunosorbent assay (ELISA) measurements, with minimal decay within the study timeline. Complete protection was achieved against toxin challenge from the serotypes /A and /B and partial protection for /E, indicating that a multivalent approach is feasible.

Detection of Myocardial Ischemia-Reperfusion Injury Using a Fluorescent Near-Infrared Zinc(II)-Dipicolylamine Probe and 99mTc Glucarate

A fluorescent zinc 2,2′-dipicolylamine coordination complex PSVue®794 (probe 1) is known to selectively bind to phosphatidylserine exposed on the surface of apoptotic and necrotic cells. In this study, we investigated the cell death targeting properties of probe 1 in myocardial ischemia-reperfusion injury. A rat heart model of ischemia-reperfusion was used. Probe 1, control dye, or 99mTc glucarate was intravenously injected in rats subjected to 30-minute and 5-minute myocardial ischemia followed by 2-hour reperfusion. At 90 minutes or 20 hours postinjection, myocardial uptake was evaluated ex vivo by fluorescence imaging and autoradiography. Hematoxylin-eosin and cleaved caspase-3 staining was performed on myocardial sections to demonstrate the presence of ischemia-reperfusion injury and apoptosis. Selective accumulation of probe 1 could be detected in the area at risk up to 20 hours postinjection. Similar topography and extent of uptake of probe 1 and 99mTc glucarate were observed at 90 minutes postinjection. Histologic analysis demonstrated the presence of necrosis, but only a few apoptotic cells could be detected. Probe 1 selectively accumulates in myocardial ischemia-reperfusion injury and is a promising cell death imaging tool.

Molecular Imaging of Apoptosis in Cancer Therapy-Related Cardiac Dysfunction Before LVEF Reduction

Detecting chemotherapy-related cardiac dysfunction (CRCD) before a decrease in left ventricular ejection fraction (LVEF) or an enzyme leak may help employ strategies to preserve ventricular function. Although the mechanisms of CRCD are not fully elucidated, myocyte apoptosis has been proposed to

Abstract B55: Hypoxanthine wobble base multimutant KRAS2 mRNA PET imaging agent in G12D mice

Activating mutations in KRAS2 oncogene lead to constitutive K-Ras-GTP signals regardless of EGFR signaling, enabling resistance to EGFR-targeted therapies. Some cancers are activated by mutation of the KRAS2 12th codon from the wild type GGU that encodes glycine (G) to a mutant GAU that encodes aspartate (D) or a mutant GUU that encodes valine (V). For this reason, we hypothesize that determining multiple KRAS2 mutations in vivo by external mRNA PET imaging will enable physicians to decide on alternatives to EGFR-directed therapy. We previously pioneered tumor mRNA PET imaging in pancreatic cancer and breast cancer xenografts [Paudyal, et al. (2013) Nuclear Medicine and Biology40(8):994-9] with peptide nucleic acid (PNA) 12-mers coupled to receptor-targeting peptides and imaging reporter moieties, showing single mismatch specificity. Thus, we designed and synthesized a 12-mer PNA hybridization agent with a hypoxanthine wobble base opposite the middle base of the KRAS2 mRNA 12th codon. At the C-terminus, we included a cyclized tetrapeptide based on insulin-like growth factor 1 (IGF1) to enable IGF1R-mediated cellular uptake. We included an N-terminal N2S2 chelator in order to bind 64Cu. Molecular dynamics and circular dichroism analysis of the wobble base PNA H-bonded to KRAS2 RNA showed preference for mutant A or U over wild type G [Sanders, et al. (2013) Journal of Physical Chemistry B 117(39):11584-95]. We then administered 200 μCi of the [64Cu]PNA wobble base agent, or the complementary G12D agent, or the wild type agent, by tail vein into 3-month-old G12D transgenic mice that developed spontaneous lung tumors. 4 hr and 24 hr later, mice were imaged in a Siemens Inveon microPET/CT scanner. The PET standard uptake value (SUV) with the G12D agent was 1.4, while SUV was 1.3 with the G12DVA wobble base agent, vs. 0.04 with the G12 wild type sequence. The wobble base approach enables detection of three different KRAS2 mutations, G12D, G12V, or G12A, with a single agent. mRNA PET imaging also enables external monitoring of therapeutic efficacy. Supported by NIH CA148565; IP owned by EW/MLT, licensed to MTTI. Citation Format: Eric Wickstrom, Chang-Po Chen, Matthew E. Wampole, Kaijun Zhang, Bishnuhari Paudyal, Antican Wang, Sushil Tripathi, Pardeep Kumar, Edith P. Mitchell, Bo Lu, Mathew L. Thakur, Brian D. Gray, Jeffrey A. Mattis. Hypoxanthine wobble base multimutant KRAS2 mRNA PET imaging agent in G12D mice. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B55. doi: 10.1158/1557-3125.RASONC14-B55