OBJECTIVES/SPECIFIC AIMS: (1) To delineate the function of theheavy-chain receptor binding domain (HCR), a portion of botulinum neurotoxintype A (BoNT/A) and synaptic vesicle 2 (SV2) signaling pathway, whichprovide a novel multipurpose biologic with potential clinical applications intumor detection/imaging, inhibition of tumor progression, andreduction of bioactive hormone secretion in metastatic neuroendocrine (NE)cancers. (2) To evaluate the expression pattern of SV2 receptors in NE cancerpatient-derived tissues for prediction of patient response for recombinant HCR(rHCR) treatment. (3) To assess the in vivo efficacy and toxicity of rHCR in aNE cancer liver metastasis mouse models and in the NE patient-derived 3DMicroTumor system. (4) To collect preclinical data to design and conduct aclinical trial with NE cancer patients, a major goal toward translating ourdiscoveries into much needed therapies. METHODS/STUDY POPULATION:Recombinant botulinum heavy chain (rHCR) was produced using an IPTG-inducibleexpression vector in E. coli BL21. The rHCR was His-Tagpurified and stored in PBS buffer before usage. Cytotoxicity: H727, TT, and MZcells were plated at a density of 5000 cells/well in 96-well platesand incubated under standard conditions overnight. The next day, cells weretreated with 10, 100, or 500 nmol/L of rHCR and incubated for 72hours. Following incubation, cell viability was assessed by ATP quantificationusing the CellTiter-Glo (Promega) assay. Fresh NE tumors were dissociated andinjected into polydimethylsiloxane bioreactors in a matrigel and collagensuspension for 3D culture experiments. The viability of 3D cultures incubatedwith various doses of rHCR was assessed by measuring the uptake of thenear-infrared dye IR-783 using an IVIS imaging system. Western blot: H727, TT,and MZ cells were seeded in 6-well plates at a density of 3×105cells/well for 24 hours followed by treatment with 100nmol/L for 72 hours. Total cellular proteins were isolated andanalyzed to assess the level of SV2A expression and the effect of rHCR on theexpression levels of NET marker proteins. Immunohistochemistry: Deparaffinizedtissue culture slides were incubated with SV2A primary antibody in 1%BSA and incubated overnight at 4°C. Slides were rinsed twice with TBScontaining 0.025% Triton, followed by 0.3%H2O2 for 15 minutes. Slides were then incubated withHRP-conjugated secondary antibody for 1 hour at room temperature. Detection ofprotein-protein interaction: Precleared cell lysate was incubated withglutathione-agarose beads in the presence of 10 μg of GST-tagged rHCRfor 2 h at 4°C with end-over end mixing. Samples were thencentrifuged at 10,000 g for 2 minutes and the supernatant was analyzed bySDS-PAGE. Preclinical models: To allow rHCR testing on NET patient derived cellsin a very novel 3D surrogates, sterile collected fresh NET tissues will beobtained from the UAB Tissue Procurement, dissociated into a single cellsuspension and injected into a polydimethylsiloxane bioreactors containingextracellular matrix composed of bovine collagen and matrigel. Such 3D cellculture will be maintained in bioreactors with constant supply of media throughthe microchannels and treated with rHCR at concentrations ranging from 10 nM to1 mM. Following histologic confirmation of growth and morphology of NETpatient-derived 3D surrogates we will test the anticancer activity of rHCR inthis system using the standard cytotoxicity assays as well as we will validatethe NET hormone expression using immunohistochemistry assay. To create an animalmodel of NE cancer progression, we will perform intrasplenic injection of NETcell lines. In approximately 4 weeks, the animals should develop NE livermetastases based upon our previous experience. rHCR-iFPs accumulation in thetumor mass: rHCR-iFPs will be injected to the tumor bearing mice after 4 weeksof cells implantation in 1 week interval for total of 4 treatments at theconcentrations of 0.125, 1.25, and 12.5 mg/kg.RESULTS/ANTICIPATED RESULTS: Based on the preliminary data, we expectto detect rHCR-iFPs in NE cancer xenografts and in patient derived 3D explants.Our preliminary data revealed that treating NETcells with rHCR significantlyreduced NE peptide expression in 3 days. Thus, we expect to see the decrease ofNE tumor markers even if the fluorescent detection method is not sensitiveenough to monitor the signal. The reduction of the NET markers can be used as anindicator of the rHCR-iFPs uptake by the tumor mass. If HCR exhibit high bindingaffinity to SV2 receptors in NET models but moderate anticancer efficacy, weplan to use rHCR peptide to conjugate with the nanocarrier for targeted drugdelivery. In this case rHCR peptide can be used as a ligand that specificallybinds to NE cancer cells and delivers anticancer drug. In 3D NET patient derivedexplants we expect significant reduction of NET markers and hormones (serotoninand calcitonin) in NE cancer cells upon long-term rHCR-iFPs treatment. Inaddition, we will perform multiplex protein quantification assay using Luminexto assess the various hormones, cytokines, and growth factors to be repurposedinto a diagnostic and detection reagent, or a drug delivery ligand for targetedtherapies. DISCUSSION/SIGNIFICANCE OF IMPACT: NE cancers are highlymetastatic: NE cancers such as carcinoid, islet cell tumors, and medullarythyroid cancer frequently metastasize to the liver. They are the second mostprevalent GI malignancy. Ninety percent of patients with pancreatic carcinoidtumors and 50% of patients with islet cell tumors develop isolatedhepatic metastases. Patients with untreated, isolated NE liver metastases have<30% 5-year survival. Thus, there is a critical need fornew therapies for NE cancers. Surgery is the only curative therapy available forpatients with NE cancers but most cannot be cured: surgical removal is the mosteffective treatment for NE cancers; however, a very high percentage of patientspresent with metastatic disease. While surgical resection can be potentiallycurative, many patients are not candidates for operative intervention due towidespread metastases or the degree of hepatic involvement by the NE cancers.Moreover, other forms of therapy including chemoembolization, radioembolization,cryoablation, and chemotherapy have had limited efficacy. We hope that our invitro data on rHCR toxicity and specificity to NET, validated in thepre-clinical models will allow the first in-human application of this technologyin clinical studies.