Dinesh Simkhada Ph D He Him

Lead scientist responsible for the advancement of platform development of Listeria-based immunotherapies by directing the Microbiology/Molecular Biology group, a division of Research. - Plan and execute molecular biology studies essential for preclinical and clinical development of Listeria-based immunotherapies. - Participate alongside Regulatory with the writing/refining of Phase I IND filings. - Streamline and standardize all molecular/micro laboratory procedures. - Facilitate the enhancement of protein expression from recombinant DNA within Listeria. - Support Process Development, Manufacturing, and Quality, through GMP analytical assays, tech transfer, and general cross functional interactions. -Consulting CRO’s for QC assays required to fulfill FDA commitments. - Biomarker data analysis: T cell and T-cell memory subsets

• Developed novel gene knockout and knock in system in Listeria monocytogens. Engineered strains into fluorescent strains • Cloning using several techniques: PCR based, Gibson assembly, Golden gate etc. of antigen to develop the vaccine • Cell culture development and preparation of cell implant in mice. • Developed and modified SOPs and Batch records and worked as a SME to speed up the manufacturing process in vaccine development. • Development of vector that successfully expresses two antigens (or two separate HOT/NEO constructs) independently, Useful platform to express two different antigens in same vector system in low budget and time. • Run western blot analysis for verification of expression of antigens • Optimized Fermentation for vaccine development • Attenuated and developed Bacillus subtilis as a platform to express antigen

Attenuated Listeria monocytogenes (Lm) bacteria to stimulate cancer-fighting T-cells directed against cancer antigens • Developed a method that allows for stable, site-specific (tRNA) integration of antigen constructs for use in Listeria monocytogenes vaccine development. • Worked on Protein expression, Cell free protein extraction, western blotting, flow cytometry, Promoter strength study, DNA assembly, gene replacement, novel and rapid gene knockout technologies.

Metabolically engineered soil microbes, Streptomyces for the production of antibiotics for human being and animals in industrial scale, which worth huge benefits in outcome for the Pfizer for long term run. • Work co-operatively with the senior research advisor and managers • Supervised and trained interdisciplinary groups of people and worked collaboratively to attain specific goals • Presented and explained diverse scientific topics • Manipulation of primary metabolic pathway to scale up the production of acetyl co-A (starter unit of most of the polyketide unit) for the overproduction of antibiotics. • Biosynthetic pathway engineering, Regulation and Identification of resistance mechanism of microorganism, worked with Process development and Down-stream group to develop fermentation and purification method for natural product isolated from engineered strains.

Nucleoside Catabolism from Uropathogenic Bacteria Screening and identification of enzyme responsible for Heme catabolism • Screened microorganism responsible for Heme catabolism from soil samples collected from Nepal, USA and Switzerland. • Screened and Constructed genomic library from pathogenic bacteria responsible for thiamin catabolism. • Established and set-up protein facility lab for protein engineering • Mentored graduate students for their research in genomic library construction, screening, gene knockout and heterologous expression of enzyme and enzyme assays.

Unraveling of Azinomycin Biosynthesis-An antitumor compound • Established genetic engineering technique: transformation (protoplast/conjugation), gene knock out system in Streptomyces sahachiroi for the first time in laboratory. • Developed E. coli as a plat form for the heterologous expression and characterization of azinomycin biosynthetic genes. • Optimized and routinely operated fermenter (10L) to isolate antitumor compound Azinomycin B. • Mentored graduate students and worked as a consultant genetic engineering and enzymology for other lab. • Discovered and activated cryptic metabolites from Streptomyces sahachiroi - Identification of gene cluster for the biosynthesis of ficellomycin from Streptomyces ficellus. - Performed time studies on production equipment to successfully establish production bottlenecks. - Developed research strategy to apply fund for PI and graduate student for the research.

Production of glycosylated natural products from Microorganism • Established heterologous expression system of glycosyltransferase gene from Arabidopsis thaliana into E. coli. • Developed a heterologous expression system for a second generation antibiotic production project. • Established the genetically engineering strain for the production of novel plant based antioxidant (Xylosyl, rhamnosyl, allosyl and glucuronidated flavonoid from E. coli. • Established research platform of glycosylation of natural products, is major project of lab running by 15 graduate students in Institute of Laboratory Reconstruction (iBR) funded by National R&D Program for Cancer Control , Ministry of Health and Welfare and Ministry of Science and Technology, S Korea. • Successfully setup lab for strain isolation, characterization and natural product isolation via running small scale (1L) and large scale (10 L) fermentation and analysis by HPLC, Prep HPLC, LC-MS, NMR techniques. • Received the excellent research award (KRF) of $30,000 from Korean government.

• Conduct and identified in allelopathy chemicals from Eupatorium sp. in growth of rice weeds. • Research conduct Impacts of weeds on nutrients of wetland ecosystem • Identified ethno-medicine from plant used for treatment of diarrhea, dysentery, asthma for people in local community and plant based medicine used for pesticides in locals.